POLY(AMINO ACID) TARGETING MOIETIES
The present invention generally relates to polymers and macromolecules, in particular, to polymers useful in particles such as nanoparticles. One aspect of the invention is directed to a method of developing nanoparticles with desired properties. In one set of embodiments, the method includes producing libraries of nanoparticles having highly controlled properties, which can be formed by mixing together two or more macromolecules in different ratios. One or more of the macromolecules may be a polymeric conjugate of a moiety to a biocompatible polymer. In some cases, the nanoparticle may contain a drug. Other aspects of the invention are directed to methods using nanoparticle libraries.
Latest MASSACHUSETTS INSTITUTE OF TECHNOLOGY Patents:
This application claims priority to U.S. Provisional Application No. 60/910,097, Attorney Docket No. BBZ-011-1, filed Apr. 4, 2007, titled “Amphiphilic compound assisted polymeric particles for targeted delivery;” U.S. Provisional Application No. 60/985,104, Attorney Docket No. BBZ-011-2, filed Nov. 2, 2007, titled “Lipid-Stabilized Polymeric Nanoparticles for Targeted Drug Delivery;” U.S. Provisional Application No. 60/938,590, Attorney Docket No. BBZ-012-1, filed May 17, 2007, titled “Poly(Amino Acid)-Targeted Drug Delivery;” U.S. Provisional Application No. 60/986,202, Attorney Docket No. BBZ-012-2, filed Nov. 7, 2007, titled “Poly(Amino Acid)-Targeted Drug Delivery;” and U.S. Provisional Application No. 60/990,250, Attorney Docket No. BBZ-012-3, filed Nov. 26, 2007, titled “Poly(Amino Acid)-Targeted Drug Delivery;” all of which are incorporated herein by reference in their entirety. Additionally, the contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.
FIELD OF INVENTIONThe present invention generally relates to targeted nanoparticles that target tissue basement membrane.
BACKGROUNDThe delivery of a drug to a patient with controlled-release of the active ingredient has been an active area of research for decades and has been fueled by the many recent developments in polymer science. In addition, controlled release polymer systems can be designed to provide a drug level in the optimum range over a longer period of time than other drug delivery methods, thus increasing the efficacy of the drug and minimizing problems with patient compliance.
Nanoparticles have been developed as vehicles used in the administration for the delivery of small molecule drugs as well as proteins, peptide drugs and nucleic acids. The drugs are typically encapsulated or conjugated in a polymer matrix which is biodegradable and biocompatible. As the polymer is degraded and/or as the drug diffuses out of the polymer, the drug is released into the body. Typically, polymers used in preparing these particles are polyesters such as poly(lactide-co-glycolide) (PLGA), polyglycolic acid, poly-beta-hydroxybutyrate, polyacrylic acid ester, polymetacrylate, polyglutamate, etc. These particles can also protect the drug from degradation by the body. Furthermore, these particles can be administered using a wide variety of administration routes.
Targeting controlled release polymer systems (e.g., targeted to a particular tissue or cell type or targeted to a specific diseased tissue but not normal tissue) is desirable because it reduces the amount of a drug present in tissues of the body that are not targeted. This is particularly important when treating a condition such as cancer where it is desirable that a cytotoxic dose of the drug is delivered to cancer cells without killing the surrounding non-cancerous tissue. Effective drug targeting should reduce the undesirable and sometimes life threatening side effects common in anticancer therapy.
Targeted delivery for diagnosis and therapeutic applications has until recently largely been limited to receptor ligands such as antibodies, modified-antibodies and nucleic acids. Antibodies are the most widely used type of targeting agent today. The large size of antibody molecules can be advantageous for bimodal binding mechanisms but it may also lead to poor solid penetration and slow elimination from the blood circulation. Unfortunately, slow elimination kinetics can cause myelotoxicity. In addition, its in vivo application has been proven more challenging because of cost and potential immunogenicity after repeat injections of such formulations. To avoid these problems, Fab's and scFv have successfully been made but are still too large. The molecular weight of fragments has been shown to be a major factor of capillary permeability, so fragments can reach the interstitial spaces more easily than whole antibody. However, the effects of the increased permeability are offset by the more rapid excretion of the antibody fragments which decreases the ability of the antibody to cross membranes resulting in lower absolute tumor levels as well as lower blood and tissue levels.
Accordingly, there is a need for developing new and alternative targeting controlled release polymer systems, especially those useful in the treatment of diseases, e.g., cancer, restenosis, and vulnerable plaques.
SUMMARY OF THE INVENTIONThere remains a need for compositions useful in the treatment or prevention or amelioration of one or more symptoms of cancer and cardiovascular disease. There also remains a need for compositions useful in the treatment or prevention or amelioration of one or more symptoms of vulnerable plaques. In one aspect, the invention provides a controlled-release system, comprising a plurality of target-specific stealth nanoparticles; wherein said nanoparticles contain targeting moieties attached thereto, wherein the targeting moiety targets the tissue basement membrane, such as the vascular basement membrane.
In one aspect, the invention provides a controlled-release system, comprising a plurality of target-specific stealth nanoparticles; wherein said nanoparticles contain targeting moieties attached thereto, wherein the targeting moiety is a poly(amino acid) that targets the tissue basement membrane, such as the vascular basement membrane. In one embodiment, the nanoparticle has an amount of targeting moiety effective for the treatment of vulnerable plaque in a subject in need thereof. In another embodiment, the nanoparticle has an amount of targeting moiety effective for the treatment of cancer in a subject in need thereof. As discussed below, the nanoparticle has an amount of targeting moiety effective for the treatment of restinosis in a subject in need thereof. In one embodiment, the cancer to be treated is selected from the group consisting of breast cancer, ovarian cancer, brain cancer, colon cancer, renal cancer, lung cancer, bladder cancer, prostate cancer and melanoma. In still another embodiment, the cancer is breast cancer.
In one embodiment, the nanoparticles of the invention can be used to treat ovarian cancer, breast cancer, and human pancreatic cancer in a subject in need thereof.
In one embodiment of the controlled-release system of the invention, the poly(amino acid) comprises natural amino acids, unnatural amino acids, modified amino acids, protected amino acids or mimetic of amino acids. In still another embodiment, the poly(amino acid) is selected from the group consisting of a glycoprotein, protein, peptidomimetic, affibody or peptide. In yet another embodiment, the poly(amino acid) binds to the basement membrane of tissues, such as the vascular basement of a blood vessel. In another embodiment, the poly(amino acid) binds to collagen. In still another embodiment, the poly(amino acid) binds to collagen IV.
In one embodiment, the poly(amino acid) is an affibody, wherein the affibody is an anti-HER2 affibody. In another embodiment, the poly(amino acid) is a peptide, wherein the peptide comprises a sequence selected from the group consisting of AKERC, CREKA, ARYLQKLN and AXYLZZLN, wherein X and Z are variable amino acids.
In one embodiment of the controlled-release system of the invention, the nanoparticle comprises a polymeric matrix. In one embodiment, the polymeric matrix comprises two or more synthetic or natural polymers. In a particular embodiment, the polymeric matrix comprises polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, or polyamines, polyglutamate, dextran, or combinations thereof.
In another embodiment, the polymeric matrix comprises one or more polyesters, polyanhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates or polycyanoacrylates. In another embodiment, at least one polymer is a polyalkylene glycol. In still another embodiment, the polyalkylene glycol is polyethylene glycol. In yet another embodiment, at least one polymer is a polyester. In one embodiment, the polyester is selected from the group consisting of poly-lactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactones. In still another embodiment, the polyester is PLGA or PLA.
In one embodiment of the controlled-release system of the invention, the nanoparticle comprises a polymeric matrix, wherein the polymeric matrix comprises a copolymer of two or more polymers. In another embodiment, the copolymer is a copolymer of a polyalkylene glycol and a polyester. In still another embodiment, the copolymer is a copolymer of PLGA and PEG. In yet another embodiment, the polymeric matrix comprises PLGA and a copolymer of PLGA and PEG.
In another embodiment of the controlled-release system of the invention, the nanoparticle comprises a polymeric matrix, wherein the polymeric matrix comprises a lipid-terminated polyalkylene glycol and a polyester. In one embodiment, the polymeric matrix comprises lipid-terminated PEG and PLGA. In one embodiment, the lipid is of the Formula V, and salts thereof. In one one embodiment, the lipid is 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.
In one embodiment of the controlled-release system, a portion of the polymer matrix is covalently bound to the poly(amino acid). In another embodiment, the polymer matrix is covalently bound to the poly(amino acid) via the free terminus of PEG. In still another embodiment, the polymer matrix is covalently bound to the poly(amino acid) via a carboxyl group at the free terminus of PEG. In yet another embodiment, the polymer matrix is covalently bound to the poly(amino acid) via a maleimide functional group at the free terminus of PEG.
In one embodiment of the controlled-release system, the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of cancer. In another embodiment, the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of a vulnerable plaque. In still another embodiment, the polymers of the polymer matrix have a molecular weight effective for the treatment of cancer. In another embodiment, the polymers of the polymer matrix have a molecular weight effective for the treatment of vulnerable plaque.
In another embodiment of the controlled-release system, the nanoparticle has a surface charge effective for the treatment of cancer. In still another embodiment, the nanoparticle has a surface charge effective for the treatment of vulnerable plaque. In yet another embodiment, said system is suitable for target-specific treatment of a disease or disorder and delivery of a therapeutic agent.
In another embodiment of the controlled-release system, the nanoparticle further comprises a therapeutic agent. In one embodiment, the therapeutic agent is associated with the surface of, encapsulated within, surrounded by, or dispersed throughout the nanoparticle. In another embodiment, the therapeutic agent is encapsulated within the hydrophobic core of the nanoparticle. In still another embodiment, the therapeutic agent is selected from the group consisting of mitoxantrone, platin and docetaxel. In yet another embodiment, the therapeutic agent is selected from the group consisting of VEGF, fibroblast growth factors, monocyte chemoatractant protein 1 (MCP-1), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), DEL-1, insulin like growth factors (IGF), placental growth factor (PLGF), hepatocyte growth factor (HGF), prostaglandin E1 (PG-E1), prostaglandin E2 (PG-E2), tumor necrosis factor alpha (THF-alpha), granulocyte stimulating growth factor (G-CSF), granulocyte macrophage colony-stimulating growth factor (GM-CSF), angiogenin, follistatin, and proliferin, PR39, PR11, nicotine, hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, statins, niacin, bile acid resins, fibrates, antioxidants, extracellular matrix synthesis promoters, inhibitors of plaque inflammation and extracellular degradation, and estradiol.
In another aspect, the invention provides a method of treating breast cancer in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention. In one embodiment for the treatment of breast cancer, the controlled-release system is administered systemically. In still another embodiment, the controlled-release system is administered directly to breast cancer cells. In another embodiment, the controlled-release system is administered directly to breast cancer cells by injection into tissue comprising the breast cancer cells. In another embodiment, the controlled-release system is administered to the subject by implantation of nanoparticles at or near breast cancer cells during surgical removal of a tumor. In still another embodiment, the controlled-release system is administered via intravenous administration.
In another aspect, the invention provides a method of treating vulnerable plaque in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention. In one embodiment, the controlled-release system is locally administered to a designated region of the blood vessel where the vulnerable plaque occurs. In still another embodiment, the controlled-release system is administered via a medical device. In yet another embodiment, the medical device is a drug eluding stent, needle catheter, or stent graft.
In another aspect, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention. In one embodiment, the controlled-release system is locally administered to a designated region of the blood vessel where the restenosis occurs. In still another embodiment, the controlled-release system is administered via a medical device. In yet another embodiment, the medical device is a drug eluding stent, needle catheter, or stent graft. In another embodiment, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention wherein the controlled release system contains a drug suitable for the treatment of restenosis. In another embodiment, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention wherein the controlled release system contains at least two drugs suitable for the treatment of restenosis. In another embodiment, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention wherein the controlled release system contains zotarolimus and dexamethasone.
In one embodiment, the nanoparticles of this invention are delivered locally to the coronary arteries, central arteries, peripheral arteries, veins, and bile ducts. In another embodiment, the nanoparticles of this invention are delivered locally to the coronary arteries, central arteries, peripheral arteries, veins, and bile ducts after the implantation of a stent in such tissue in a patient for the treatment of restenosis. In another embodiment, the nanoparticles of this invention are administered to a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis. In another embodiment, the nanoparticles of this invention are administered within 12 hours of a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis. In another embodiment, the nanoparticles of this invention are administered locally to a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis.
In one embodiment, the nanoparticles of this invention pass through the endothelial layer of a blood vessel due to plaque damage of the endothelial tissue and bind to collage 4 of the basement membrane.
In another aspect, the invention provides a method of preparing a stealth nanoparticle, wherein the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of a disease, comprising: providing a therapeutic agent; providing a polymer; providing a poly(amino acid) ligand; mixing the polymer with the therapeutic agent to prepare particles; and associating the particles with the poly(amino acid) ligand. In one embodiment of the method, the polymer comprises a copolymer of two or more polymers. In another embodiment, the copolymer is a copolymer of PLGA and PEG.
In another aspect, the invention provides a method of preparing a stealth nanoparticle, wherein the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of a disease, comprising: providing a therapeutic agent; providing a first polymer; providing a poly(amino acid) ligand; reacting the first polymer with the poly(amino acid) ligand to prepare a ligand-bound polymer; and mixing the ligand-bound polymer with a second, non-functionalized polymer, and the therapeutic agent; such that the stealth nanoparticle is formed. In one embodiment of this method, the first polymer comprises a copolymer of two or more polymers. In another embodiment, the second, non-functionalized polymer comprises a copolymer of two or more polymers. In another embodiment of this method, the first polymer is first reacted with a lipid, to form a polymer/lipid conjugate, which is then reacted with the poly(amino acid). In still another embodiment, the lipid is 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof. In yet another embodiment, the copolymer is a copolymer of PLGA and PEG. In still another embodiment, the first polymer is a copolymer of PLGA and PEG, wherein the PEG has a carboxyl group at the free terminus.
In one embodiment of the aforementioned methods, the disease to be treated is cancer, vulnerable plaque, or restenosis.
In another aspect, the invention provides a stealth nanoparticle, comprising a copolymer of PLGA and PEG; and a therapeutic agent comprising mitoxantrone, platin or docetaxel; wherein said nanoparticle contains targeting moieties attached thereto, wherein the targeting moiety is an anti-HER2 affibody.
In yet another aspect, the invention provides a stealth nanoparticle, comprising a copolymer of PLGA and PEG; and a therapeutic agent; wherein said nanoparticle contains targeting moieties attached thereto, wherein the targeting moiety comprises AKERC or CREKA.
In another aspect, the invention provides a stealth nanoparticle, comprising a polymeric matrix comprising a complex of a phospholipid bound-PEG and PLGA; and a therapeutic agent; wherein said nanoparticle contains targeting moieties attached thereto, wherein the targeting moiety is a poly(amino acid).
In still another aspect, the invention provides a controlled-release system, comprising a plurality of target-specific stealth nanoparticles; wherein said nanoparticles contain targeting moieties attached thereto, wherein the targeting moiety is a basement membrane-targeting moiety. In one embodiment, the basement membrane-targeting moiety is AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids.
In another embodiment of the controlled-release system of the invention, the polymeric matrix is surrounded by a lipid monolayer shell. In one embodiment, the lipid monolayer shell comprises an amphiphilic compound. In another embodiment, the amphiphilic compound is lecithin. In another embodiment, the lipid monolayer is stabilized.
In another aspect, the invention provides a controlled-release system, comprising a plurality of target-specific stealth nanoparticles; wherein said nanoparticles contain targeting moieties attached thereto, wherein the targeting moiety is a poly(amino acid). In one embodiment, the nanoparticles target antigen presenting cells and elicit an immunomodulatory response.
The present invention generally relates to particles, and, in particular, nanoparticles, wherein the nanoparticles comprise a drug delivery system for the targeted delivery of a therapeutic agent.
In one embodiment, the nanoparticle of the controlled release system has an amount of targeting moiety (i.e., a poly(amino acid)) effective for the targeting of tissue basement membrane. In one embodiment, the nanoparticle of the controlled release system has an amount of targeting moiety (i.e., a poly(amino acid)) effective for the targeting of vascular basement membrane. In certain embodiments, the poly(amino acid) is conjugated to a polymer, and the nanoparticle comprises a certain ratio of ligand-conjugated polymer to non-functionalized polymer. The nanoparticle can have an optimized ratio of these two polymers, such that an effective amount of ligand is associated with the nanoparticle for treatment of a disease, e.g., cancer (e.g., breast cancer), vulnerable plaque, restenosis. For example, increased ligand density (e.g., on a PLGA-PEG copolymer) will increase target binding (cell binding/target uptake), making the nanoparticle “target specific.” Alternatively, a certain concentration of non-functionalized polymer (e.g., non-functionalized PLGA-PEG copolymer) in the nanoparticle can control inflammation and/or immunogenicity (i.e., the ability to provoke an immune response), and allow the nanoparticle to have a circulation half-life that is therapeutically effective for the treatment of, e.g., cancer, vulnerable plaque, or restenosis. Furthermore, the non-functionalized polymer can lower the rate of clearance from the circulatory system via the reticuloendothelial system. Thus, the non-functionalized polymer gives the nanoparticle “stealth” characteristics. Additionally, the non-functionalized polymer balances an otherwise high concentration of ligands, which can otherwise accelerate clearance by the subject, resulting in less delivery to the target cells.
Target-Specific Stealth Nanoparticles Comprising PolymersIn preferred embodiments, the nanoparticles of the invention comprise a matrix of polymers. In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In some embodiments, a therapeutic agent and/or targeting moiety (i.e., a poly(amino acid)) can be associated with the polymeric matrix. In some embodiments, the targeting moiety can be covalently associated with the surface of a polymeric matrix. In some embodiments, covalent association is mediated by a linker. In some embodiments, the therapeutic agent can be associated with the surface of, encapsulated within, surrounded by, and/or dispersed throughout the polymeric matrix.
A wide variety of polymers and methods for forming particles therefrom are known in the art of drug delivery. In some embodiments of the invention, the matrix of a particle comprises one or more polymers. Any polymer may be used in accordance with the present invention. Polymers may be natural or unnatural (synthetic) polymers. Polymers may be homopolymers or copolymers comprising two or more monomers. In terms of sequence, copolymers may be random, block, or comprise a combination of random and block sequences. Typically, polymers in accordance with the present invention are organic polymers.
A “polymer,” as used herein, is given its ordinary meaning as used in the art, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units may all be identical, or in some cases, there may be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer. Non-limiting examples include peptides or proteins (i.e., polymers of various amino acids), or nucleic acids such as DNA or RNA. In some cases, additional moieties may also be present in the polymer, for example biological moieties such as those described below.
If more than one type of repeat unit is present within the polymer, then the polymer is said to be a “copolymer.” It is to be understood that in any embodiment employing a polymer, the polymer being employed may be a copolymer in some cases. The repeat units forming the copolymer may be arranged in any fashion. For example, the repeat units may be arranged in a random order, in an alternating order, or as a “block” copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers may have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
It should be understood that, although the terms “first,” “second,” etc. may be used herein to describe various elements, including polymeric components, these terms should not be construed as being limiting (e.g., describing a particular order or number of elements), but rather, as being merely descriptive, i.e., labels that distinguish one element from another, as is commonly used within the field of patent law. Thus, for example, although one embodiment of the invention may be described as having a “first” element present and a “second” element present, other embodiments of the invention may have a “first” element present but no “second” element present, a “second” element present but no “first” element present, two (or more) “first” elements present, and/or two (or more) “second” elements present, etc., and/or additional elements such as a “first” element, a “second” element, and a “third” element, without departing from the scope of the present invention.
Various embodiments of the present invention are directed to copolymers, which, in particular embodiments, describes two or more polymers (such as those described herein) that have been associated with each other, usually by covalent bonding of the two or more polymers together. Thus, a copolymer may comprise a first polymer and a second polymer, which have been conjugated together to form a block copolymer where the first polymer is a first block of the block copolymer and the second polymer is a second block of the block copolymer. Of course, those of ordinary skill in the art will understand that a block copolymer may, in some cases, contain multiple blocks of polymer, and that a “block copolymer,” as used herein, is not limited to only block copolymers having only a single first block and a single second block. For instance, a block copolymer may comprise a first block comprising a first polymer, a second block comprising a second polymer, and a third block comprising a third polymer or the first polymer, etc. In some cases, block copolymers can contain any number of first blocks of a first polymer and second blocks of a second polymer (and in certain cases, third blocks, fourth blocks, etc.). In addition, it should be noted that block copolymers can also be formed, in some instances, from other block copolymers.
For example, a first block copolymer may be conjugated to another polymer (which may be a homopolymer, a biopolymer, another block copolymer, etc.), to form a new block copolymer containing multiple types of blocks, and/or to other moieties (e.g., to non-polymeric moieties).
In some embodiments, the polymer (e.g., copolymer, e.g., block copolymer) is amphiphilic, i.e., having a hydrophilic portion and a hydrophobic portion, or a relatively hydrophilic portion and a relatively hydrophobic portion. A hydrophilic polymer is one generally that attracts water and a hydrophobic polymer is one that generally repels water. A hydrophilic or a hydrophobic polymer can be identified, for example, by preparing a sample of the polymer and measuring its contact angle with water (typically, the polymer will have a contact angle of less than 60°, while a hydrophobic polymer will have a contact angle of greater than about 60°). In some cases, the hydrophilicity of two or more polymers may be measured relative to each other, i.e., a first polymer may be more hydrophilic than a second polymer. For instance, the first polymer may have a smaller contact angle than the second polymer.
In one set of embodiments, a polymer (e.g., copolymer, e.g., block copolymer) of the present invention includes a biocompatible polymer, i.e., the polymer that does not typically induce an adverse response when inserted or injected into a living subject, for example, without significant inflammation and/or acute rejection of the polymer by the immune system, for instance, via a T-cell response. Accordingly, the nanoparticles of the present invention can be “non-immunogenic.” The term “non-immunogenic” as used herein refers to endogenous growth factor in its native state which normally elicits no, or only minimal levels of, circulating antibodies, T-cells, or reactive immune cells, and which normally does not elicit in the individual an immune response against itself.
It will be recognized, of course, that “biocompatibility” is a relative term, and some degree of immune response is to be expected even for polymers that are highly compatible with living tissue. However, as used herein, “biocompatibility” refers to the acute rejection of material by at least a portion of the immune system, i.e., a non-biocompatible material implanted into a subject provokes an immune response in the subject that is severe enough such that the rejection of the material by the immune system cannot be adequately controlled, and often is of a degree such that the material must be removed from the subject. One simple test to determine biocompatibility is to expose a polymer to cells in vitro; biocompatible polymers are polymers that typically will not result in significant cell death at moderate concentrations, e.g., at concentrations of 50 micrograms/106 cells. For instance, a biocompatible polymer may cause less than about 20% cell death when exposed to cells such as fibroblasts or epithelial cells, even if phagocytosed or otherwise uptaken by such cells. Non-limiting examples of biocompatible polymers that may be useful in various embodiments of the present invention include polydioxanone (PDO), polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide, polylactide, PLGA, polycaprolactone, or copolymers or derivatives including these and/or other polymers.
In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. As used herein, “biodegradable” polymers are those that, when introduced into cells, are broken down by the cellular machinery (biologically degradable) and/or by a chemical process, such as hydrolysis, (chemically degradable) into components that the cells can either reuse or dispose of without significant toxic effect on the cells.
In a preferred embodiment, the biodegradable polymer and their degradation byproducts are biocompatible.
For instance, the polymer may be one that hydrolyzes spontaneously upon exposure to water (e.g., within a subject), the polymer may degrade upon exposure to heat (e.g., at temperatures of about 37° C.). Degradation of a polymer may occur at varying rates, depending on the polymer or copolymer used. For example, the half-life of the polymer (the time at which 50% of the polymer is degraded into monomers and/or other nonpolymeric moieties) may be on the order of days, weeks, months, or years, depending on the polymer. The polymers may be biologically degraded, e.g., by enzymatic activity or cellular machinery, in some cases, for example, through exposure to a lysozyme (e.g., having relatively low pH). In some cases, the polymers may be broken down into monomers and/or other nonpolymeric moieties that cells can either reuse or dispose of without significant toxic effect on the cells (for example, polylactide may be hydrolyzed to form lactic acid, polyglycolide may be hydrolyzed to form glycolic acid, etc.).
In some embodiments, polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as “PLGA”; and homopolymers comprising glycolic acid units, referred to herein as “PGA,” and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA.” In some embodiments, exemplary polyesters include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA, and derivatives thereof. In some embodiments, polyesters include, for example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylated polylysine, poly(ethylene inline), PEGylated poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.
In some embodiments, a polymer may be PLGA. PLGA is a biocompatible and biodegradable co-polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by altering the lactic acid-.glycolic acid ratio. In some embodiments, PLGA to be used in accordance with the present invention is characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.
In particular embodiments, by optimizing the ratio of lactic acid to glycolic acid monomers in the polymer of the nanoparticle (e.g., the PLGA block copolymer or PLGA-PEG block copolymer), nanoparticle parameters such as water uptake, therapeutic agent release (e.g., “controlled release”) and polymer degradation kinetics can be optimized.
In some embodiments, polymers may be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymers, polycyanoacrylates, and combinations comprising one or more of the foregoing polymers. The acrylic polymer may comprise fully-polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.
In some embodiments, polymers can be cationic polymers. In general, cationic polymers are able to condense and/or protect negatively charged strands of nucleic acids (e.g. DNA, RNA, or derivatives thereof). Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), polyethylene imine) (PEI; Boussif et al, 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively-charged at physiological pH, form ion pairs with nucleic acids, and mediate transfection in a variety of cell lines.
In some embodiments, polymers can be degradable polyesters bearing cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwonef al, 1999, Macromolecules, 22325Q-, Urn et al., 1999, J. Am. Chem. Soc., 121:5633; and Zhou et al, 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-co-L-lysine) (Barrera et al, 1993, J. Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al, 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc., 121:5633). Poly(4-hydroxy-L-proline ester) was demonstrated to condense plasmid DNA through electrostatic interactions, and to mediate gene transfer (Putnam et al, 1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc., 121:5633). These new polymers are less toxic than poly(lysine) and PEI, and they degrade into non-toxic metabolites.
A polymer (e.g., copolymer, e.g., block copolymer) containing poly(ethylene glycol) repeat units is also referred to as a “PEGylated” polymer. Such polymers can control inflammation and/or immunogenicity (i.e., the ability to provoke an immune response) and/or lower the rate of clearance from the circulatory system via the reticuloendothelial system (RES), due to the presence of the poly(ethylene glycol) groups.
PEGylation may also be used, in some cases, to decrease charge interaction between a polymer and a biological moiety, e.g., by creating a hydrophilic layer on the surface of the polymer, which may shield the polymer from interacting with the biological moiety. In some cases, the addition of poly(ethylene glycol) repeat units may increase plasma half-life of the polymer (e.g., copolymer, e.g., block copolymer), for instance, by decreasing the uptake of the polymer by the phagocytic system while decreasing transfection/uptake efficiency by cells. Those of ordinary skill in the art will know of methods and techniques for PEGylating a polymer, for example, by using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to react a polymer to a PEG group terminating in an amine, by ring opening polymerization techniques (ROMP), or the like.
In another embodiment, the nanoparticle of the invention does not contain PEG.
In addition, certain embodiments of the invention are directed towards copolymers containing poly(ester-ether)s, e.g., polymers having repeat units joined by ester bonds (e.g., R—C(O)—O—R′ bonds) and ether bonds (e.g., R—O—R′ bonds). In some embodiments of the invention, a biodegradable polymer, such as a hydrolyzable polymer, containing carboxylic acid groups, may be conjugated with poly(ethylene glycol) repeat units to form a poly(ester-ether).
In a particular embodiment, the molecular weight of the polymers of the nanoparticles of the invention are optimized for effective treatment of cancer, e.g., breast cancer. For example, the molecular weight of the polymer influences nanoparticle degradation rate (particularly when the molecular weight of a biodegradable polymer is adjusted), solubility, water uptake, and drug release kinetics (e.g. “controlled release”). As a further example, the molecular weight of the polymer can be adjusted such that the nanoparticle biodegrades in the subject being treated within a reasonable period of time (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.). In particular embodiments of a nanoparticle comprising a copolymer of PEG and PLGA, the PEG has a molecular weight of 1,000-20,000, e.g., 5,000-20,000, e.g., 10,000-20,000, and the PLGA has a molecular weight of 5,000-100,000, e.g., 20,000-70,000, e.g., 20,000-50,000.
In certain embodiments, the polymers of the nanoparticles may be conjugated to a lipid. The polymer may be, for example, a lipid-terminated PEG. As described below, the lipid portion of the polymer can be used for self assembly with another polymer, facilitating the formation of a nanoparticle. For example, a hydrophilic polymer could be conjugated to a lipid that will self assemble with a hydrophobic polymer.
In some embodiments, lipids are oils. In general, any oil known in the art can be conjugated to the polymers used in the invention. In some embodiments, an oil may comprise one or more fatty acid groups or salts thereof. In some embodiments, a fatty acid group may comprise digestible, long chain (e.g., C8-C50), substituted or unsubstituted hydrocarbons. In some embodiments, a fatty acid group may be a C10-C20 fatty acid or salt thereof. In some embodiments, a fatty acid group may be a C15-C20 fatty acid or salt thereof. In some embodiments, a fatty acid may be unsaturated. In some embodiments, a fatty acid group may be monounsaturated. In some embodiments, a fatty acid group may be polyunsaturated. In some embodiments, a double bond of an unsaturated fatty acid group may be in the cis conformation. In some embodiments, a double bond of an unsaturated fatty acid may be in the trans conformation.
In some embodiments, a fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, a fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
In a particular embodiment, the lipid is of the Formula V:
and salts thereof, wherein each R is, independently, C1-30 alkyl. In one embodiment of Formula V, the lipid is 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof, e.g., the sodium salt.
The properties of these and other polymers and methods for preparing them are well known in the art (see, for example, U.S. Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al, 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Ace. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, a variety of methods for synthesizing suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al, 1997, Nature, 390:386; and in U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
In still another set of embodiments, a particle (comprising, e.g., a copolymer, e.g., a block copolymer) of the present invention includes a therapeutic moiety, i.e., a moiety that has a therapeutic or prophylactic effect when given to a subject. Examples of therapeutic moieties to be used with the nanoparticles of the present invention include antineoplastic or cytostatic agents or other agents with anticancer properties, or a combination thereof.
Thus, in certain embodiments, a library of such particles may be created, as discussed herein.
In some cases, the particle is a nanoparticle, i.e., the particle has a characteristic dimension of less than about 1 micrometer, where the characteristic dimension of a particle is the diameter of a perfect sphere having the same volume as the particle. For example, the particle may have a characteristic dimension of the particle may be less than about 300 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 3 nm, or less than about 1 nm in some cases. In particular embodiments, the nanoparticle of the present invention has a diameter of 50 nm-200 nm.
In some cases, a population of particles may be present. For example, a population of particles may include at least 20 particles, at least 50 particles, at least 100 particles, at least 300 particles, at least 1,000 particles, at least 3,000 particles, or at least 10,000 particles. Various embodiments of the present invention are directed to such populations of particles. For instance, in some embodiments, the particles may each be substantially the same shape and/or size (“monodisperse”). For example, the particles may have a distribution of characteristic dimensions such that no more than about 5% or about 10% of the particles have a characteristic dimension greater than about 10% greater than the average characteristic dimension of the particles, and in some cases, such that no more than about 8%, about 5%, about 3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01% have a characteristic dimension greater than about 10% greater man the average characteristic dimension of the particles. In some cases, no more than about 5% of the particles have a characteristic dimension greater than about 5%, about 3%, about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01% greater than the average characteristic dimension of the particles.
In one set of embodiments, the particles may have an interior and a surface, where the surface has a composition different from the interior, i.e., there may be at least one compound present in the interior but not present on the surface (or vice versa), and/or at least one compound is present in the interior and on the surface at differing concentrations. For example, in one embodiment, a compound, such as a targeting moiety (i.e., a poly(amino acid)) of a polymeric conjugate of the present invention, may be present in both the interior and the surface of the particle, but at a higher concentration on the surface than in the interior of the particle, although in some cases, the concentration in the interior of the particle may be essentially nonzero, i.e., there is a detectable amount of the compound present in the interior of the particle.
In some cases, the interior of the particle is more hydrophobic than the surface of the particle. For instance, the interior of the particle may be relatively hydrophobic with respect to the surface of the particle, and a drug or other pay load may be hydrophobic, and readily associates with the relatively hydrophobic center of the particle. The drug or other payload may thus be contained within the interior of the particle, which may thus shelter it from the external environment surrounding the particle (or vice versa). For instance, a drug or other payload contained within a particle administered to a subject will be protected from a subject's body, and the body will also be isolated from the drug. A targeting moiety present on the surface of the particle may allow the particle to become localized at a particular targeting site, for instance, a tumor, a disease site, a tissue, an organ, a type of cell, etc. The drug or other payload may then, in some cases, be released from the particle and allowed to interact locally with the particular targeting site. Yet another aspect of the invention is directed to polymer particles having more than one polymer or macromolecule present, and libraries involving such polymers or macromolecules. For example, in one set of embodiments, particles may contain more than one distinguishable polymers (e.g., copolymers, e.g., block copolymers), and the ratios of the two (or more) polymers may be independently controlled, which allows for the control of properties of the particle. For instance, a first polymer may be a polymeric conjugate comprising a targeting moiety and a biocompatible portion, and a second polymer may comprise a biocompatible portion but not contain the targeting moiety, or the second polymer may contain a distinguishable biocompatible portion from the first polymer. Control of the amounts of these polymers within the polymeric particle may thus be used to control various physical, biological, or chemical properties of the particle, for instance, the size of the particle (e.g., by varying the molecular weights of one or both polymers), the surface charge (e.g., by controlling the ratios of the polymers if the polymers have different charges or terminal groups), the surface hydrophilicity (e.g., if the polymers have different molecular weights and/or hydrophilicities), the surface density of the targeting moiety (e.g., by controlling the ratios of the two or more polymers), etc.
As a specific example, a particle may comprise a first polymer comprising a poly(ethylene glycol) and a targeting moiety conjugated to the poly(ethylene glycol), and a second polymer comprising the poly(ethylene glycol) but not the targeting moiety, or comprising both the poly(ethylene glycol) and the targeting moiety, where the poly(ethylene glycol) of the second polymer has a different length (or number of repeat units) than the poly(ethylene glycol) of the first polymer. As another example, a particle may comprise a first polymer comprising a first biocompatible portion and a targeting moiety, and a second polymer comprising a second biocompatible portion different from the first biocompatible portion (e.g., having a different composition, a substantially different number of repeat units, etc.) and the targeting moiety. As yet another example, a first polymer may comprise a biocompatible portion and a first targeting moiety, and a second polymer may comprise a biocompatible portion and a second targeting moiety different from the first targeting moiety.
Libraries of such particles may also be formed. For example, by varying the ratios of the two (or more) polymers within the particle, libraries of particles may be formed, which may be useful, for example, for screening tests, high-throughput assays, or the like. Entities within the library may vary by properties such as those described above, and in some cases, more than one property of the particles may be varied within the library. Accordingly, one embodiment of the invention is directed to a library of nanoparticles having different ratios of polymers with differing properties. The library may include any suitable ratio(s) of the polymers.
In another embodiment, the nanoparticle is associated with (e.g., surrounded by) a small molecule amphiphilic compound, giving the “amphiphilic-nanoparticle” three main components: 1) a biodegradable polymeric material that forms the core of the particle, which can carry bioactive drugs and release them at a sustained rate after cutaneous, subcutaneous, mucosal, intramuscular, ocular, systemic, oral or pulmonary administration; 2) a small molecule amphiphilic compound that surrounds the polymeric material forming a shell for the particle; and 3) a stealth material that can allow the particles to evade recognition by immune system components and increase particle circulation half life. This embodiment may also include a fourth component: 4) a targeting molecule that can bind to a unique molecular signature on cells, tissues, or organs of the body. In a preferred embodiment, these particles would be useful in drug delivery for therapeutic applications. In an alternative preferred embodiment, these particles would be useful for molecular imaging, for diagnostic applications, or for a combination thereof.
In another embodiment, the amphiphilic-nanoparticle of the invention comprises a: 1) a biodegradable polymeric core which can carry bioactive drugs and release them at a sustained rate; 2) a lipid monolayer shell which can prevent the carried agents from freely diffusing out of the nanoparticle and reduce water penetration rate into the nanoparticle, thereby enhancing drug encapsulation efficiency and slowing drug release; 3) a stealth material that can allow the particles to evade recognition by immune system components and increase particle circulation half life; and 4) a targeting molecule that can bind to a unique molecular signature on cells, tissues, or organs of the body.
In a preferred embodiment of the amphiphilic-nanoparticle, a poly(amino acid) targeting molecule is first chemically conjugated to the hydrophilic region of a small molecule amphiphilic compound. This conjugate is then mixed with a certain ratio of unconjugated small molecule amphiphilic compounds in an aqueous solution containing one or more water-miscible solvents. In a preferred embodiment, the poly(amino acid) targeting molecule is one or a plurality of antibodies, aptamers, peptides, small molecules, or combinations thereof. The amphiphilic compound can be, but is not limited to, one or a plurality of the following: naturally derived lipids, surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. The water miscible solvent can be, but is not limited to: acetone, ethanol, methanol, and isopropyl alcohol. Separately, a biodegradable polymeric material is mixed with the agent or agents to be encapsulated in a water miscible or partially water miscible organic solvent. In a preferred embodiment, the biodegradable polymer can be any of the biodegradable polymers disclosed herein, for example, poly(D,L-lactic acid), poly(D,L-glycolic acid), poly(ε-caprolactone), or their copolymers at various molar ratios. The carried agent can be, but is not limited to, one or a plurality of the following therapeutic agents discussed below, including, for example, therapeutic drugs, imaging probes, or hydrophobic or lipophobic molecules for medical use. The water miscible organic solvent can be but is not limited to: acetone, ethanol, methanol, or isopropyl alcohol. The partially water miscible organic solvent can be, but is not limited to: acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate, or dimethylformamide. The resulting polymer solution is then added to the aqueous solution of conjugated and unconjugated amphiphilic compound to yield nanoparticles by the rapid diffusion of the organic solvent into the water and evaporation of the organic solvent.
As used herein, the term “amphiphilic” refers to a property where a molecule has both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the invention, the amphiphilic compound can be, but is not limited to, one or a plurality of the following: naturally derived lipids, surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
Specific examples of amphiphilic compounds include, but are not limited to, phospholipids, such as 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of between 0.01-60 (weight lipid/w polymer), most preferably between 0.1-30 (weight lipid/w polymer). Phospholipids which may be used include, but are not limited to, phosphatidic acids, phosphatidyl cholines with both saturated and unsaturated lipids, phosphatidyl ethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, and β-acyl-y-alkyl phospholipids. Examples of phospholipids include, but are not limited to, phosphatidylcholines such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcho-line (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines such as dioleoylphosphatidylethanolamine or 1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Synthetic phospholipids with asymmetric acyl chains (e.g., with one acyl chain of 6 carbons and another acyl chain of 12 carbons) may also be used.
In a particular embodiment, an amphiphilic component that can be used to form an amphiphilic layer is lecithin, and, in particular, phosphatidylcholine. Lecithin is an amphiphilic lipid and, as such, forms a phospholipid bilayer having the hydrophilic (polar) heads facing their surroundings, which are oftentimes aqueous, and the hydrophobic tails facing each other. Lecithin has an advantage of being a natural lipid that is available from, e.g., soybean, and already has FDA approval for use in other delivery devices. In addition, a mixture of lipids such as lethicin is more advantageous than one single pure lipid.
In certain embodiments of the invention, the amphiphilic layer of the nanoparticle, e.g., the layer of lecithin, is a monolayer, meaning the layer is not a phospholipid bilayer, but exists as a single continuous or discontinuous layer around, or within, the nanoparticle. A monolayer has the advantage of allowing the nanoparticles to be smaller in size, which makes them easier to prepare. The amphiphilic layer is “associated with” the nanoparticle of the invention, meaning it is positioned in some proximity to the polymeric matrix, such as surrounding the outside of the polymeric matrix (e.g., PLGA), or dispersed within the polymers that make up the nanoparticle.
By covering the polymeric nanoparticles with a thin film of small molecule amphiphilic compounds and conjugating poly(amino acid) targeting molecules to the amphiphilic compounds before formulating nanoparticles, the disclosed invention has merits of both polymer- and lipid-based nanoparticles, while excluding some of their limitations. The amphiphilic compounds form a tightly assembled monolayer around the polymeric core. This monolayer effectively prevents the carried agents from freely diffusing out of the nanoparticle, thereby enhancing the encapsulation yield and slowing drug release. Moreover, the amphiphilic monolayer also reduces water penetration rate into the nanoparticle, which slows hydrolysis rate of the biodegradable polymers, thereby increasing particle stability and lifetime. In addition, by conjugating targeting ligands to the amphiphilic component prior to incorporating them into the nanoparticle, the composition of the nanoparticle and its surface properties can be more accurately quantified.
In one embodiment, upon being administered to a subject, the amphipilic layer of the nanoparticle of the invention can degrade, such that the polymer core is eventually “unshielded.” Such a process, particularly when occurring after penetration into target tissue, can lead to more efficient delivery of the therapeutic agent, thereby affording an enhanced therapeutic effect. Without being bound by theory, in the case of basement membrane targeting, the nanoparticle may aggregate at the first collagen IV at the surface of the basement membrane. By “shedding” the lipid shell, the drug/polymer core can more deeply penetrate into the basement membrane,
The surface of the nanoparticles of the invention can also be modified to enhance their arterial uptake. Nanoparticle surface modifying agents include, but are not limited to, heparin, L-R-phosphatidylethanolamine, cyanoacrylate, epoxide, fibronectin, fibrinogen, ferritin, lipofectin, didodecyldimethylammonium bromide, and DEAE-Dextran, and any other surface modifying agent disclosed in J Pharm Sci. 1998 October; 87(10):1229-34, which is incorporated herein by reference in it entirety. The nanoparticulate system of the invention can also be manipulated to have better compatibility with a drug delivery device, e.g., stent. For example, viscosity can be adjusted to adjust the drag force of the nanoparticulate system.
In general, the nanoparticles of the present invention are about 40 nm to about 500 nm in size. In one embodiment, the nanoparticles of the invention are less than or equal to about 90 nm in size, e.g., about 40 nm to about 80 nm, e.g., about 40 nm to about 60 nm. Because the nanoparticles of the invention can be less than 90 nm in size, liver uptake by the subject is reduced, thereby allowing longer circulation in the bloodstream.
In one embodiment, a nanoparticle of this invention is between 40 nm and 80 nm in diameter and contains an amphiphilic component to polymer ration of between 14:1 to 34:1. In one embodiment, a nanoparticle will have approximately 10% to 40% lipid (by weight). In another embodiment, the nanoparticle will have a size of about 90 nm to about 40 nm. In one embodiment, a nanoparticle that is approximately 10% to 40% lipid (by weight) will have a corresponding size of about 90 nm to about 40 nm.
The nanoparticles of the invention also have a surface zeta potential ranging from about −80 mV to 50 mV. Zeta potential is a measurement of surface potential of a particle. In some embodiments, the particles have a zeta potential ranging between 0 mV and −50 mV, e.g., between −1 mV and −50 mV. In some embodiments, the particles have a zeta potential ranging between −1 mV and −25 mV. In some embodiments, the particles have a zeta potential ranging between −1.1 mV and −10 mV.
In other embodiments, the nanoparticles of the invention are liposomes, liposome polymer combinations, dendrimers, and albumin particles that are functionalized with a poly(amino acid) ligand. These nanoparticles can be used to deliver a therapeutic agent to a subject, such as an anti-cancer agent like mitoxantrone or docetaxel.
As used herein, the term “liposome” refers to a generally spherical vesicle or capsid generally comprised of amphipathic molecules (e.g., having both a hydrophobic (nonpolar) portion and a hydrophilic (polar) portion). Typically, the liposome can be produced as a single (unilamellar) closed bilayer or a multicompartment (multilamellar) closed bilayer. The liposome can be formed by natural lipids, synthetic lipids, or a combination thereof. In a preferred embodiment, the liposome comprises one or more phospholipids. Lipids known in the art for forming liposomes include, but are not limited to, lecithin (soy or egg; phosphatidylcholine), dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dicetylphosphate, phosphatidylglycerol, hydrogenated phosphatidylcholine, phosphatidic acid, cholesterol, phosphatidylinositol, a glycolipid, phosphatidylethanolamine, phosphatidylserine, a maleimidyl-derivatized phospholipid (e.g., N-[4(p-malei-midophenyl)butyryl] phosphatidylethanolamine), dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidic acid, and a combination thereof. Liposomes have been used to deliver therapeutic agents to cells.
The nanoparticles of the invention can also be “stealth liposomes,” which comprise lipids wherein the head group is modified with PEG. This results in extended circulating half life in the subject.
Dendritic polymers (otherwise known as “dendrimers”) are uniform polymers, variously referred to in the literature as hyperbranched dendrimers, arborols, fractal polymers and starburst dendrimers, having a central core, an interior dendritic (hyperbranched) structure and an exterior surface with end groups. These polymers differ from the classical linear polymers both in form and function. Dendrimer chemistry constructs macromolecules with tight control of size, shape topology, flexibility and surface groups (e.g., a poly(amino acid) ligand). In what is known as divergent synthesis, these macromolecules start by reacting an initiator core in high-yield iterative reaction sequences to build symmetrical branches radiating from the core with well-defined surface groups. Alternatively, in what is known as convergent synthesis, dendritic wedges are constructed from the periphery inwards towards a focal point and then several dendritic wedges are coupled at the focal points with a polyfunctional core. Dendritic syntheses form concentric layers, known as generations, with each generation doubling the molecular mass and the number of reactive groups at the branch ends so that the end generation dendrimer is a highly pure, uniform monodisperse macromolecule that solubilizes readily over a range of conditions. For the reasons discussed below, dendrimer molecular weights range from 300 to 700,000 daltons and the number of surface groups (e.g., reactive sites for coupling) range significantly.
“Albumin particles” (also referred to as “albumin microspheres”) have been reported as carriers of pharmacological or diagnostic agents (see, e.g., U.S. Pat. Nos. 5,439,686; 5,498,421; 5,560,933; 5,665,382; 6,096,331; 6,506,405; 6,537,579; 6,749,868; and 6,753,006; all of which are incorporated herein by reference). Microspheres of albumin have been prepared by either heat denaturation or chemical crosslinking. Heat denatured microspheres are produced from an emulsified mixture (e.g., albumin, the agent to be incorporated, and a suitable oil) at temperatures between 100° C. and 150° C. The microspheres are then washed with a suitable solvent and stored. Leucuta et al. (International Journal of Pharmaceutics 41:213-217 (1988)) describe the method of preparation of heat denatured microspheres.
Poly(Amino Acid) Targeting MoietiesIn yet another set of embodiments, the nanoparticles of the present invention includes a poly(amino acid) targeting moiety, i.e., a moiety able to bind to or otherwise associate with a biological entity, for example, a membrane component, a cell surface receptor, Her-2, the basement membrane of a blood vessel, basement membrane proteins, collagen, collagen IV or the like. In the case of the instant invention, the targeting moiety is a poly(amino acid) ligand. The term “bind” or “binding,” as used herein, refers to the interaction between a corresponding pair of molecules or portions thereof that exhibit mutual affinity or binding capacity, typically due to specific or non-specific binding or interaction, including, but not limited to, biochemical, physiological, and/or chemical interactions. “Biological binding” defines a type of interaction that occurs between pairs of molecules including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, or the like. The term “binding partner” refers to a molecule that can undergo binding with a particular molecule. “Specific binding” refers to molecules, such as polynucleotides, that are able to bind to or recognize a binding partner (or a limited number of binding partners) to a substantially higher degree than to other, similar biological entities. In one set of embodiments, the targeting moiety has an affinity (as measured via a disassociation constant) of less than about 1 micromolar, at least about 10 micromolar, or at least about 100 micromolar.
The term “poly(amino acid)” as used herein, refers to a protein, affibody, peptide, or peptidomimetic containing natural and unnatural amino acids, modified amino acids or protected amino acids. The agents to be incorporated in the polymeric nanocarrier and delivered to a target cell or tissue by a conjugate of the present invention may be therapeutic, diagnostic, prophylactic or prognostic agents. Any chemical compound to be administered to an individual may be delivered using the conjugates of the invention. The agent may be a small molecule, organometallic compound, radionuclides, nucleic acid, protein, peptide, polynucleotide, metal, an isotopically labeled chemical compound, drug, vaccine, immunological agent, etc.
The term “affibody” (see, e.g., U.S. Pat. No. 5,831,012, incorporated herein by reference) refers to highly specific affinity proteins that can be designed to bind to any desired target molecule. These antibody mimics can be manufactured to have the desired properties (specificity and affinity), while also being highly robust to withstand a broad range of analytical conditions, including pH and elevated temperature. The specific binding properties that can be engineered into each protein allow it to have very high specificity and the desired affinity for a corresponding target protein. A specific target protein will thus bind only to its corresponding capture protein.
The present invention further provides a nanoparticle conjugated to a poly(amino acid) that selectively targets tumor vasculature and selectively binds collagen, such as non-helical collagen. In another embodiment, the invention provides a nanoparticle conjugated to a poly(amino acid) that selectively targets breast tumor vasculature and that selectively binds collagen, e.g., collagen IV, e.g., denatured collagen IV or native collagen IV. In a one embodiment, the invention provides a nanoparticle conjugated to a poly(amino acid) that selectively targets tumor vasculature and that selectively binds the alpha 2 chain of collagen IV.
In preferred embodiments, the poly(amino acid) targeting moiety targets tissue basement membrane, such as the basement membrane of a blood vessel. A “basement membrane” refers to a thin membrane upon which is posed a single layer of cells. The basement membrane is made up of proteins held together by type IV collagen. The epithelial cells are anchored with hemidesmosome to the basement membrane. The end result resembles a layer of tiles attached to a thin sheet. As discussed below, in cases where the endothelium is disrupted (by disease or trauma), the basement membrane may be exposed and accessible to particles.
A variety of poly(amino acids) that selectively target tumor vasculature are useful targeting moieties for the nanoparticles of the invention. Such poly(amino acids) include, without limitation, targeting peptides and peptidomimetics. In one embodiment, the targeting peptide or peptidomimetic portion of the nanoparticle has a length of at most 200 residues. In another embodiment, the targeting peptide or peptidomimetic portion of the nanoparticle has a length of at most 50 residues. In a further embodiment, a nanoparticle of the invention contains a targeting peptide or peptidomimetic that includes the amino acid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids, or conservative variants or peptidomimetics thereof. In particular embodiments, the poly(amino acid) targeting moiety is a peptide that includes the amino acid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids, and has a length of less than 20, 50 or 100 residues. The CREKA peptide is known in the art, and is described in U.S. Patent Application No. 2005/0048063, which is incorporated herein by reference in its entirety. The octapeptide AXYLZZLN is described in Dinkla et al., The Journal of Biological Chemistry, Vol. 282, No. 26, pp. 18686-18693, which is incorporated herein by reference in its entirety.
Moreover, the authors of The Journal of Biological Chemistry, Vol. 282, No. 26, pp. 18686-18693 describe a binding motif in streptococci that forms an autoantigenic complex with human collagen IV. Accordingly, any peptide, or conservative variants or peptidomimetics thereof, that binds or forms a complex with collagen IV, or the basement membrane of a blood vessel, can be used as a targeting moiety for the nanoparticles of the invention.
In one embodiment, the targeting moiety is an isolated peptide or peptidomimetic that has a length of less than 100 residues and includes the amino acid sequence CREKA (Cys Arg Glu Lys Ala) or a peptidomimetic thereof. Such an isolated peptide- or peptidomimetic can have, for example, a length of less than 50 residues or a length of less than 20 residues. In particular embodiments, the invention provides a peptide that includes the amino acid sequence CREKA and has a length of less than 20, 50 or 100 residues.
As used herein in reference to a specified amino acid sequence, a “conservative variant” is a sequence in which a first amino acid is replaced by another amino acid or amino acid analog having at least one biochemical property similar to that of the first amino acid; similar properties include, for example, similar size, charge, hydrophobicity or hydrogen-bonding capacity.
The peptides and peptidomimetics of the invention to be used as poly(amino acid) targeting moieties are provided in isolated form. As used herein in reference to a peptide or peptidomimetic of the invention, the term “isolated” means a peptide or peptidomimetic that is in a form that is relatively free from material such as contaminating polypeptides, lipids, nucleic acids and other cellular material that normally is associated with the peptide or peptidomimetic in a cell or that is associated with the peptide or peptidomimetic in a library or in a crude preparation.
The peptides and peptidomimetics of the invention to be used as poly(amino acid) targeting moieties, including the bifunctional, multivalent and targeting peptides and peptidomimetics discussed below, can have a variety of lengths. A peptide or peptidomimetic of the invention can have, for example, a relatively short length of less than six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35 or 40 residues. A peptide or peptidomimetic of the invention also can be useful in the context of a significantly longer sequence. In another embodiment, a peptide or peptidomimetic of the invention can have, for example, a length of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000 residues. In particular embodiments, a peptide or peptidomimetic of the invention has a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 200 residues. In further embodiments, a peptide or peptidomimetic of the invention has a length of 5 to 200 residues, 5 to 100 residues, 5 to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60 residues, 5 to 50 residues, 5 to 40 residues, 5 to 30 residues, 5 to 20 residues, 5 to 15 residues, 5 to 10 residues, 10 to 200 residues, 10 to 100 residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to 60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10 to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90 residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to 50 residues, 20 to 40 residues or 20 to 30 residues. As used herein, the term “residue” refers to an amino acid or amino acid analog.
As used herein, the term “peptide” is used broadly to mean peptides, proteins, fragments of proteins and the like. The term “peptidomimetic,” as used herein, means a peptide-like molecule that has the activity of the peptide upon which it is structurally based. Such peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, and peptoids and have an activity such as selective targeting activity of the peptide upon which the peptidomimetic is derived (see, for example, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages 803-861).
In another embodiment, the poly(amino acid) targeting moiety targets Her-2. In a particular embodiment, the poly(amino acid) targeting moiety is an affibody that is an anti-HER2 affibody.
A polymeric conjugate to be used in the preparation of a nanoparticle of the present invention may be formed using any suitable conjugation technique. For instance, two components such as a targeting moiety and a biocompatible polymer, a biocompatible polymer and a poly(ethylene glycol), etc., may be conjugated together using techniques such as EDC-NHS chemistry (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide) or a reaction involving a maleimide or a carboxylic acid, which can be conjugated to one end of a thiol, an amine, or a similarly functionalized polyether. The conjugation of such polymers, for instance, the conjugation of a poly(ester) and a poly(ether) to form a poly(ester-ether), can be performed in an organic solvent, such as, but not limited to, dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, acetone, or the like. Specific reaction conditions can be determined by those of ordinary skill in the art using no more than routine experimentation.
In another set of embodiments, a conjugation reaction may be performed by reacting a polymer that comprises a carboxylic acid functional group (e.g., a poly(ester-ether) compound) with a polymer or other moiety (such as a targeting moiety) comprising an amine. For instance, a targeting moiety, such as a poly(amino-acid) ligand, may be reacted with an amine to form an amine-containing moiety, which can then be conjugated to the carboxylic acid of the polymer. Such a reaction may occur as a single-step reaction, i.e., the conjugation is performed without using intermediates such as N-hydroxysuccinimide or a maleimide. The conjugation reaction between the amine-containing moiety and the carboxylic acid-terminated polymer (such as a poly(ester-ether) compound) may be achieved, in one set of embodiments, by adding the amine-containing moiety, solubilized in an organic solvent such as (but not limited to) dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines, dioxane, or dimethysulfoxide, to a solution containing the carboxylic acid-terminated polymer. The carboxylic acid-terminated polymer may be contained within an organic solvent such as, but not limited to, dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, or acetone. Reaction between the amine-containing moiety and the carboxylic acid-terminated polymer may occur spontaneously, in some cases. Unconjugated reactants may be washed away after such reactions, and the polymer may be precipitated in solvents such as, for instance, ethyl ether, hexane, methanol, or ethanol.
As a specific example, a poly(amino acid) ligand may be prepared as a targeting moiety in a particle as follows. Carboxylic acid modified poly(lactide-co-glycolide) (PLGA-COOH) may be conjugated to an amine-modified heterobifunctional poly(ethylene glycol) (NH2—PEG-COOH) to form a copolymer of PLGA-PEG-COOH. By using an amine-containing poly(amino acid) ligand (NH2-Lig), a triblock polymer of PLGA-PEG-Lig may be formed by conjugating the carboxylic acid end of the PEG to the amine functional group on the ligand. The multiblock polymer can then be used, for instance, as discussed below, e.g., for therapeutic applications.
Another aspect of the invention is directed to particles that include polymer conjugates such as the ones described above. The particles may have a substantially spherical (i.e., the particles generally appear to be spherical), or non-spherical configuration. For instance, the particles, upon swelling or shrinkage, may adopt a non-spherical configuration. In some cases, the particles may include polymeric blends. For instance, a polymer blend may be formed that includes a first polymer comprising a targeting moiety (i.e., a poly(amino acid) ligand) and a biocompatible polymer, and a second polymer comprising a biocompatible polymer but not comprising the targeting moiety. By controlling the ratio of the first and second polymers in the final polymer, the concentration and location of targeting moiety in the final polymer may be readily controlled to any suitable degree.
Accordingly, the present invention provides poly(amino acid) targeting moieties bound to a polymer. For example, the invention provides CREKA bound to PEG (CREKA-PEG), CREKA bound to PEG that is bound to a lipid (e.g., CREKA-PEG-DSPE), and CREKA bound to PEG-PLGA (CREKA-PEG-PLGA). The invention also provides the following conjugates:
wherein n is 20 to 1720; and
wherein R7 is an alkyl group, R8 is an ester or amide linkage, X=0 to 1 mole fraction, Y=0 to 0.5 mole fraction, X+Y=20 to 1720, and Z=25 to 455.
Preparation of Target-Specific Stealth NanoparticlesAnother aspect of the invention is directed to systems and methods of producing such target-specific stealth nanoparticles. In some embodiments, a solution containing a polymer is contacted with a liquid, such as an immiscible liquid, to form nanoparticles containing the polymeric conjugate. Other aspects of the invention are directed to methods of using such libraries, methods of using or administering such polymeric conjugates, methods of promoting the use of such polymeric conjugates, kits involving such polymeric conjugates, or the like.
As mentioned, one aspect of the invention is directed to a method of developing nanoparticles with desired properties, such as desired chemical, biological, or physical properties. In one set of embodiments, the method includes producing libraries of nanoparticles having highly controlled properties, which can be formed by mixing together two or more polymers in different ratios. By mixing together two or more different polymers (e.g., copolymers, e.g., block copolymers) in different ratios and producing particles from the polymers (e.g., copolymers, e.g., block copolymers), particles having highly controlled properties may be formed. For example, one polymer (e.g., copolymers, e.g., block copolymers) may include a poly(amino acid) ligand, while another polymer (e.g., copolymers, e.g., block copolymers) may be chosen for its biocompatibility and/or its ability to control immunogenicity of the resultant particle.
Another aspect of the invention is directed to systems and methods of making such particles. In one set of embodiments, the particles are formed by providing a solution comprising one or more polymers, and contacting the solution with a polymer nonsolvent to produce the particle. The solution may be miscible or immiscible with the polymer nonsolvent. For example, a water-miscible liquid such as acetonitrile may contain the polymers, and particles are formed as the acetonitrile is contacted with water, a polymer nonsolvent, e.g., by pouring the acetonitrile into the water at a controlled rate. The polymer contained within the solution, upon contact with the polymer nonsolvent, may then precipitate to form particles such as nanoparticles. Two liquids are said to be “immiscible” or not miscible, with each other when one is not soluble in the other to a level of at least 10% by weight at ambient temperature and pressure. Typically, an organic solution (e.g., dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines, dioxane, dimethysulfoxide, etc.) and an aqueous liquid (e.g., water, or water containing dissolved salts or other species, cell or biological media, ethanol, etc.) are immiscible with respect to each other. For example, the first solution may be poured into the second solution (at a suitable rate or speed). In some cases, particles such as nanoparticles may be formed as the first solution contacts the immiscible second liquid, e.g., precipitation of the polymer upon contact causes the polymer to form nanoparticles while the first solution poured into the second liquid, and in some cases, for example, when the rate of introduction is carefully controlled and kept at a relatively slow rate, nanoparticles may form. The control of such particle formation can be readily optimized by one of ordinary skill in the art using only routine experimentation.
By creating a library of such particles, particles having any desirable properties may be identified. For example, properties such as surface functionality, surface charge, size, zeta (ζ) potential, hydrophobicity, ability to control immunogenicity, and the like, may be highly controlled. For instance, a library of particles may be synthesized, and screened to identify the particles having a particular ratio of polymers that allows the particles to have a specific density of moieties (e.g., poly(amino acid) ligands) present on the surface of the particle. This allows particles having one or more specific properties to be prepared, for example, a specific size and a specific surface density of moieties, without an undue degree of effort. Accordingly, certain embodiments of the invention are directed to screening techniques using such libraries, as well as any particles identified using such libraries. In addition, identification may occur by any suitable method. For instance, the identification may be direct or indirect, or proceed quantitatively or qualitatively.
In some embodiments, already-formed nanoparticles are functionalized with a targeting moiety using procedures analogous to those described for producing ligand-functionalized polymeric conjugates. As a specific, non-limiting example, this embodiment is exemplified schematically in
As a specific, non-limiting example, another embodiment is shown schematically in
In another embodiment, the invention provides a method of preparing a stealth nanoparticle wherein the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of breast cancer, wherein the hydrophilic, ligand-bound polymer is conjugated to a lipid that will self assemble with the hydrophobic polymer, such that the hydrophobic and hydrophilic polymers that constitute the nanoparticle are not covalently bound. “Self-assembly” refers to a process of spontaneous assembly of a higher order structure that relies on the natural attraction of the components of the higher order structure (e.g., molecules) for each other. It typically occurs through random movements of the molecules and formation of bonds based on size, shape, composition, or chemical properties. For example, such a method comprises providing a first polymer that is reacted with a lipid, to form a polymer/lipid conjugate. The polymer/lipid conjugate is then reacted with the poly(amino acid) ligand to prepare a ligand-bound polymer/lipid conjugate; and mixing the ligand-bound polymer/lipid conjugate with a second, non-functionalized polymer, and the therapeutic agent; such that the stealth nanoparticle is formed. In certain embodiments, the first polymer is PEG, such that a lipid-terminated PEG is formed. In one embodiment, the lipid is of the Formula V, e.g., 2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof, e.g., the sodium salt. The lipid-terminated PEG can then, for example, be mixed with PLGA to form a nanoparticle.
More generally, the polymers chosen to be used to create the library of particles may be any of a wide variety of polymers, such as described in detail below. Generally, two, three, four, or more polymers are mixed, in a wide range of ratios (e.g., each ranging from 0% to 100%), to form particles such as nanoparticles having different ratios of each of the polymers. The two or more polymers may be distinguishable in some fashion, e.g., having different polymeric groups, having the same polymeric groups but with different molecular weights, having some polymeric groups in common but having others that are different (e.g., one may have a polymeric group that the other does not have), having the same polymeric groups but in different orders, etc. The library of particles may have any number of members, for example, the library may have 2, 3, 5, 10, 30, 100, 300, 1000, 3000, 10,000, 30,000, 100,000, etc. members, which can be identified in some fashion. In some cases, the library may exist contemporaneously; for example, the library may be contained in one or more microtiter plates, vials, etc., or in some embodiments, the library may have include members created at different times.
The library of particles can then be screened in some fashion to identify those particles having one or more desired properties, for example, surface functionality, surface charge, size, zeta (ζ) potential, hydrophobicity, ability to control immunogenicity, and the like. One or more of the macromolecules within the particles may include one or more polymers chosen to be biocompatible or biodegradable, one or more polymers chosen to reduce immunogenicity, and/or one or more poly(amino acid) ligands. These are discussed in detail below. The macromolecules within the library may comprise some or all of these polymers, in any suitable combination (including, but not limited to, combinations in which a first polymer comprises a poly(amino acid) ligand and a second polymer does not contain any of these species).
As a specific example, in one embodiment, the particles may include a first macromolecule comprising a biocompatible polymer, and a poly(amino acid) ligand, and a second macromolecule comprising a biocompatible polymer, which may or may not be the same as that of the first macromolecule. As another example, a first macromolecule may be a block copolymer comprising a biocompatible hydrophobic polymer, a biocompatible hydrophilic polymer, and a poly(amino acid) ligand; and a second macromolecule distinguishable from the first macromolecule in some fashion. For instance, the second macromolecule may comprise the same (or a different) biocompatible hydrophobic polymer and the same (or a different) biocompatible hydrophilic polymer, but a different poly(amino acid) ligand (or no ligand at all) than the first macromolecule.
The nanoparticle of the invention may also be comprised of, as another example, a first macromolecule comprising a biocompatible hydrophobic polymer, a biocompatible hydrophilic polymer, and a poly(amino acid) ligand, and a second macromolecule that is distinguishable from the first macromolecule. For instance, the second macromolecule may contain none of the polymers of the first macromolecule, the second macromolecule may contain one or more polymers of the first macromolecule and one or more polymers not present in the first macromolecule, the second macromolecule may lack one or more of the polymers of the first macromolecule, the second macromolecule may contain all of the polymers of the first macromolecule, but in a different order and/or with one or more of the polymers having different molecular weights, etc.
As yet another example, the first macromolecule may comprise a biocompatible hydrophobic polymer, a biocompatible hydrophilic polymer, and a poly(amino acid) ligand, and the second macromolecule may comprise the biocompatible hydrophobic polymer and the biocompatible hydrophilic polymer, and be distinguishable from the first macromolecule in some fashion. As still another example, the first macromolecule may comprise a biocompatible hydrophobic polymer and a biocompatible hydrophilic polymer, and the second macromolecule may comprise the biocompatible hydrophobic polymer and a poly(amino acid) ligand, where the second macromolecule is distinguishable from the first macromolecule in some fashion.
The nanoparticles described above may also contain therapeutic agents. Examples of therapeutic agents include, but are not limited to, a chemotherapeutic agent, a radioactive agent, a nucleic acid-based agent, a lipid-based agent, a carbohydrate based agent, a natural small molecule, or a synthetic small molecule.
The polymers or macromolecules may then be formed into a particle, using techniques such as those discussed in detail below. The geometry formed by the particle from the polymer or macromolecule may depend on factors such as the polymers that form the particle.
Such a library may be useful in achieving particles having any number of desirable properties, for instance properties such as surface functionality, surface charge, size, zeta (ζ) potential, hydrophobicity, ability to control immunogenicity, or the like. In
For instance, as shown in
As specific examples, in some embodiments of the present invention, the library includes particles comprising polymeric conjugates of a biocompatible polymer and a poly(amino acid) ligand, as discussed herein. Referring now to
As a specific example, particle 10 may contain polymers including a relatively hydrophobic biocompatible polymer and a relatively hydrophilic targeting moiety 15, such that, during particle formation, a greater concentration of the hydrophilic targeting moiety is exposed on the surface and a greater concentration of the hydrophobic biocompatible polymer is present within the interior of the particle.
In some embodiments, the biocompatible polymer is a hydrophobic polymer. Non-limiting examples of biocompatible polymers include polylactide, polyglycolide, and/or poly(lactide-co-glycolide).
In some cases, the polymeric conjugate is part of a controlled release system. A “controlled release system,” as used herein, is a polymer combined with an active agent or a drug or other payload, such as a therapeutic agent, a diagnostic agent, a prognostic, a prophylactic agent, etc., and the active agent is released from the controlled release system in a predesigned or controlled manner. For example, the active agent may be released in a constant manner over a predetermined period of time, the active agent may be released in a cyclic manner over a predetermined period of time, or an environmental condition or external event may trigger the release of the active agent. The controlled release polymer system may include a polymer that is biocompatible, and in some cases, the polymer is biodegradable.
Therapeutic AgentsAnother aspect of the present invention is directed to a therapeutic “payload,” or a species (or more than one species) contained within a particle, such as those described above. For instance, the targeting moiety may target or cause the particle to become localized at specific portions within a subject, and the payload may be delivered to those portions. In a particular embodiment, the drug or other payload is released in a controlled release manner from the particle and allowed to interact locally with the particular targeting site (e.g., a tumor). The term “controlled release” (and variants of that term) as used herein (e.g., in the context of “controlled-release system”) is generally meant to encompass release of a substance (e.g., a drug) at a selected site or otherwise controllable in rate, interval, and/or amount. Controlled release encompasses, but is not necessarily limited to, substantially continuous delivery, patterned delivery (e.g., intermittent delivery over a period of time that is interrupted by regular or irregular time intervals), and delivery of a bolus of a selected substance (e.g., as a predetermined, discrete amount if a substance over a relatively short period of time (e.g., a few seconds or minutes)).
For example, a targeting portion may cause the particles to become localized to a tumor, a disease site, a tissue, an organ, a type of cell, etc. within the body of a subject, depending on the targeting moiety used. For example, a poly(amino acid) ligand may become localized to Her-2, the basement membrane of a blood vessel, collagen, collagen IV or the like. The subject may be a human or non-human animal. Examples of subjects include, but are not limited to, a mammal such as a dog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat, a mouse, a guinea pig, a hamster, a primate, a human or the like.
In one set of embodiments, the payload is a drug or a combination of more than one drug. Such particles may be useful, for example, in embodiments where a targeting moiety may be used to direct a particle containing a drug to a particular localized location within a subject, e.g., to allow localized delivery of the drug to occur. Exemplary therapeutic agents include chemotherapeutic agents such as doxorubicin (adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, paltin derivatives, cladribine, camptothecin, CPT-1,10-hydroxy-7-ethylcamptothecin (SN38), dacarbazine, S—I capecitabine, ftorafur, 5′deoxyfluorouridine, UFT, eniluracil, deoxycytidine, 5-azacyto sine, 5-azadeoxycyto sine, allopurinol, 2-chloroadeno sine, trimetrexate, aminopterin, methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin, satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, and analogs thereof, epirubicin, etoposide phosphate, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide, perfosfamide, trophosphamide carmustine, semustine, epothilones A-E, tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposide phosphate, karenitecin, acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine, lamivudine, zidovudine, bevacizumab, trastuzumab, rituximab, 5-Fluorouracil, and combinations thereof.
Suitable non-genetic therapeutic agents for use in connection with the present invention may be selected, for example, from one or more of the following: (a) anti-thrombotic agents such as heparin, heparin derivatives, urokinase, clopidogrel, and PPack (dextrophenylalanine proline arginine chloromethylketone); (b) anti-inflammatory agents such as dexamethasone, prednisolone, corticosterone, budesonide, estrogen, sulfasalazine and mesalamine; (c) antineoplastic/antiproliferative/anti-miotic agents such as paclitaxel, 5-fluorouracil, cisplatin, vinblastine, vincristine, epothilones, endostatin, angiostatin, angiopeptin, monoclonal antibodies capable of blocking smooth muscle cell proliferation, and thymidine kinase inhibitors; (d) anesthetic agents such as lidocaine, bupivacaine and ropivacaine; (e) anti-coagulants such as D-Phe-Pro-Arg chloromethyl ketone, an RGD peptide-containing compound, heparin, hirudin, antithrombin compounds, platelet receptor antagonists, anti-thrombin antibodies, anti-platelet receptor antibodies, aspirin, prostaglandin inhibitors, platelet inhibitors and tick antiplatelet peptides; (f) vascular cell growth promoters such as growth factors, transcriptional activators, and translational promotors; (g) vascular cell growth inhibitors such as growth factor inhibitors, growth factor receptor antagonists, transcriptional repressors, translational repressors, replication inhibitors, inhibitory antibodies, antibodies directed against growth factors, bifunctional molecules consisting of a growth factor and a cytotoxin, bifunctional molecules consisting of an antibody and a cytotoxin; (h) protein kinase and tyrosine kinase inhibitors (e.g., tyrphostins, genistein, quinoxalines); (i) prostacyclin analogs; (j) cholesterol-lowering agents; (k) angiopoietins; (l) antimicrobial agents such as triclosan, cephalosporins, aminoglycosides and nitrofurantoin; (m) cytotoxic agents, cytostatic agents and cell proliferation affectors; (n) vasodilating agents; (o) agents that interfere with endogenous vasoactive mechanisms; (p) inhibitors of leukocyte recruitment, such as monoclonal antibodies; (q) cytokines; (r) hormones; (s) inhibitors of HSP 90 protein (i.e., Heat Shock Protein, which is a molecular chaperone or housekeeping protein and is needed for the stability and function of other client proteins/signal transduction proteins responsible for growth and survival of cells) including geldanamycin, (t) smooth muscle relaxants such as alpha receptor antagonists (e.g., doxazosin, tamsulosin, terazosin, prazosin and alfuzosin), calcium channel blockers (e.g., verapimil, diltiazem, nifedipine, nicardipine, nimodipine and bepridil), beta receptor agonists (e.g., dobutamine and salmeterol), beta receptor antagonists (e.g., atenolol, metaprolol and butoxamine), angiotensin-II receptor antagonists (e.g., losartan, valsartan, irbesartan, candesartan, eprosartan and telmisartan), and antispasmodic/anticholinergic drugs (e.g., oxybutynin chloride, flavoxate, tolterodine, hyoscyamine sulfate, diclomine), (u) bARKct inhibitors, (v) phospholamban inhibitors, (w) Serca 2 gene/protein, (x) immune response modifiers including aminoquizolines, for instance, imidazoquinolines such as resiquimod and imiquimod, (y) human apolioproteins (e.g., AI, AII, AIII, AIV, AV, etc.), (z) selective estrogen receptor modulators (SERMs) such as raloxifene, lasofoxifene, arzoxifene, miproxifene, ospemifene, PKS 3741, MF 101 and SR 16234, (aa) PPAR agonists such as rosiglitazone, pioglitazone, netoglitazone, fenofibrate, bexaotene, metaglidasen, rivoglitazone and tesaglitazar, (bb) prostaglandin E agonists such as alprostadil or ONO 8815Ly, (cc) thrombin receptor activating peptide (TRAP), (dd) vasopeptidase inhibitors including benazepril, fosinopril, lisinopril, quinapril, ramipril, imidapril, delapril, moexipril and spirapril, (ee) thymosin beta 4, and (ff) phospholipids including phosphorylcholine, phosphatidylinositol and phosphatidylcholine.
Preferred non-genetic therapeutic agents include taxanes such as paclitaxel (including particulate forms thereof, for instance, protein-bound paclitaxel particles such as albumin-bound paclitaxel nanoparticles, e.g., ABRAXANE), sirolimus, everolimus, tacrolimus, zotarolimus, Epo D, dexamethasone, estradiol, halofuginone, cilostazole, geldanamycin, ABT-578 (Abbott Laboratories), trapidil, liprostin, Actinomcin D, Resten-NG, Ap-17, abciximab, clopidogrel, Ridogrel, beta-blockers, bARKct inhibitors, phospholamban inhibitors, Serca 2 gene/protein, imiquimod, human apolioproteins (e.g., AI-AV), growth factors (e.g., VEGF-2), as well derivatives of the forgoing, among others.
Suitable genetic therapeutic agents for use in connection with the present invention include anti-sense DNA and RNA as well as DNA coding for the various proteins (as well as the proteins themselves) and may be selected, for example, from one or more of the following: (a) anti-sense RNA, (b) tRNA or rRNA to replace defective or deficient endogenous molecules, (c) angiogenic and other factors including growth factors such as acidic and basic fibroblast growth factors, vascular endothelial growth factor, endothelial mitogenic growth factors, epidermal growth factor, transforming growth factor α and β, platelet-derived endothelial growth factor, platelet-derived growth factor, tumor necrosis factor α, hepatocyte growth factor and insulin-like growth factor, (d) cell cycle inhibitors including CD inhibitors, and (e) thymidine kinase (“TK”) and other agents useful for interfering with cell proliferation. Also of interest is DNA encoding for the family of bone morphogenic proteins (“BMP's”), including BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and BMP-16. Currently preferred BMP's are any of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. These dimeric proteins can be provided as homodimers, heterodimers, or combinations thereof, alone or together with other molecules. Alternatively, or in addition, molecules capable of inducing an upstream or downstream effect of a BMP can be provided. Such molecules include any of the “hedgehog” proteins, or the DNA's encoding them.
Vectors for delivery of genetic therapeutic agents include viral vectors such as adenoviruses, gutted adenoviruses, adeno-associated virus, retroviruses, alpha virus (Semliki Forest, Sindbis, etc.), lentiviruses, herpes simplex virus, replication competent viruses (e.g., ONYX-015) and hybrid vectors; and non-viral vectors such as artificial chromosomes and mini-chromosomes, plasmid DNA vectors (e.g., pCOR), cationic polymers (e.g., polyethyleneimine, polyethyleneimine (PEI)), graft copolymers (e.g., polyether-PEI and polyethylene oxide-PEI), neutral polymers such as polyvinylpyrrolidone (PVP), SP1017 (SUPRATEK), lipids such as cationic lipids, liposomes, lipoplexes, nanoparticles, or microparticles, with and without targeting sequences such as the protein transduction domain (PTD).
Cells for use in conjunction with the present invention include cells of human origin (autologous or allogeneic), including whole bone marrow, bone marrow derived mono-nuclear cells, progenitor cells (e.g., endothelial progenitor cells), stem cells (e.g., mesenchymal, hematopoietic, neuronal), pluripotent stem cells, fibroblasts, myoblasts, satellite cells, pericytes, cardiomyocytes, skeletal myocytes or macrophage, or from an animal, bacterial or fungal source (xenogeneic), which can be genetically engineered, if desired, to deliver proteins of interest.
Further therapeutic agents, not necessarily exclusive of those listed above, have been identified as candidates for vascular treatment regimens, for example, as agents targeting restenosis (anti-restenotic agents). Suitable agents may be selected, for example, from one or more of the following: (a) Ca-channel blockers including benzothiazapines such as diltiazem and clentiazem, dihydropyridines such as nifedipine, amlodipine and nicardapine, and phenylalkylamines such as verapamil, (b) serotonin pathway modulators including: 5-HT antagonists such as ketanserin and naftidrofuryl, as well as 5-HT uptake inhibitors such as fluoxetine, (c) cyclic nucleotide pathway agents including phosphodiesterase inhibitors such as cilostazole and dipyridamole, adenylate/Guanylate cyclase stimulants such as forskolin, as well as adenosine analogs, (d) catecholamine modulators including α-antagonists such as prazosin and bunazosine, β-antagonists such as propranolol and α/β-antagonists such as labetalol and carvedilol, (e) endothelin receptor antagonists such as bosentan, sitaxsentan sodium, atrasentan, endonentan, (f) nitric oxide donors/releasing molecules including organic nitrates/nitrites such as nitroglycerin, isosorbide dinitrate and amyl nitrite, inorganic nitroso compounds such as sodium nitroprusside, sydnonimines such as molsidomine and linsidomine, nonoates such as diazenium diolates and NO adducts of alkanediamines, S-nitroso compounds including low molecular weight compounds (e.g., S-nitroso derivatives of captopril, glutathione and N-acetyl penicillamine) and high molecular weight compounds (e.g., S-nitroso derivatives of proteins, peptides, oligosaccharides, polysaccharides, synthetic polymers/oligomers and natural polymers/oligomers), as well as C-nitroso-compounds, O-nitroso-compounds, N-nitroso-compounds and L-arginine, (g) Angiotensin Converting Enzyme (ACE) inhibitors such as cilazapril, fosinopril and enalapril, (h) ATII-receptor antagonists such as saralasin and losartin, (i) platelet adhesion inhibitors such as albumin and polyethylene oxide, (j) platelet aggregation inhibitors including cilostazole, aspirin and thienopyridine (ticlopidine, clopidogrel) and GP IIb/IIIa inhibitors such as abciximab, epitifibatide and tirofiban, (k) coagulation pathway modulators including heparinoids such as heparin, low molecular weight heparin, dextran sulfate and β-cyclodextrin tetradecasulfate, thrombin inhibitors such as hirudin, hirulog, PPACK(D-phe-L-propyl-L-arg-chloromethylketone) and argatroban, FXa inhibitors such as antistatin and TAP (tick anticoagulant peptide), Vitamin K inhibitors such as warfarin, as well as activated protein C, (l) cyclooxygenase pathway inhibitors such as aspirin, ibuprofen, flurbiprofen, indomethacin and sulfinpyrazone, (m) natural and synthetic corticosteroids such as dexamethasone, prednisolone, methprednisolone and hydrocortisone, (n) lipoxygenase pathway inhibitors such as nordihydroguairetic acid and caffeic acid, (o) leukotriene receptor antagonists, (p) antagonists of E- and P-selectins, (q) inhibitors of VCAM-1 and ICAM-1 interactions, (r) prostaglandins and analogs thereof including prostaglandins such as PGE1 and PGI2 and prostacyclin analogs such as ciprostene, epoprostenol, carbacyclin, iloprost and beraprost, (s) macrophage activation preventers including bisphosphonates, (t) HMG-CoA reductase inhibitors such as lovastatin, pravastatin, atorvastatin, fluvastatin, simvastatin and cerivastatin, (u) fish oils and omega-3-fatty acids, (v) free-radical scavengers/antioxidants such as probucol, vitamins C and E, ebselen, trans-retinoic acid and SOD (orgotein), SOD mimics, verteporfin, rostaporfin, AGI 1067, and M 40419, (w) agents affecting various growth factors including FGF pathway agents such as bFGF antibodies and chimeric fusion proteins, PDGF receptor antagonists such as trapidil, IGF pathway agents including somatostatin analogs such as angiopeptin and ocreotide, TGF-β pathway agents such as polyanionic agents (heparin, fucoidin), decorin, and TGF-β antibodies, EGF pathway agents such as EGF antibodies, receptor antagonists and chimeric fusion proteins, TNF-α pathway agents such as thalidomide and analogs thereof, Thromboxane A2 (TXA2) pathway modulators such as sulotroban, vapiprost, dazoxiben and ridogrel, as well as protein tyrosine kinase inhibitors such as tyrphostin, genistein and quinoxaline derivatives, (x) matrix metalloprotease (MMP) pathway inhibitors such as marimastat, ilomastat, metastat, batimastat, pentosan polysulfate, rebimastat, incyclinide, apratastat, PG 116800, RO 1130830 or ABT 518, (y) cell motility inhibitors such as cytochalasin B, (z) antiproliferative/antineoplastic agents including antimetabolites such as purine analogs (e.g., 6-mercaptopurine or cladribine, which is a chlorinated purine nucleoside analog), pyrimidine analogs (e.g., cytarabine and 5-fluorouracil) and methotrexate, nitrogen mustards, alkyl sulfonates, ethylenimines, antibiotics (e.g., daunorubicin, doxorubicin), nitrosoureas, cisplatin, agents affecting microtubule dynamics (e.g., vinblastine, vincristine, colchicine, Epo D, paclitaxel and epothilone), caspase activators, proteasome inhibitors, angiogenesis inhibitors (e.g., endostatin, angiostatin and squalamine), rapamycin (sirolimus) and its analogs (e.g., everolimus, tacrolimus, zotarolimus, etc.), cerivastatin, flavopiridol and suramin, (aa) matrix deposition/organization pathway inhibitors such as halofuginone or other quinazolinone derivatives, pirfenidone and tranilast, (bb) endothelialization facilitators such as VEGF and RGD peptide, (cc) blood rheology modulators such as pentoxifylline and (dd) glucose cross-link breakers such as alagebrium chloride (ALT-711).
Numerous additional therapeutics for the practice of the present invention may be selected from suitable therapeutic agents disclosed in U.S. Pat. No. 5,733,925 to Kunz.
Non-limiting examples of potentially suitable drugs include anti-cancer agents, including, for example, docetaxel, mitoxantrone, and mitoxantrone hydrochloride. In another embodiment, the payload may be an anti-cancer drug such as 20-epi-1, 25 dihydroxyvitamin D3,4-ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfiilvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine, aminoglutethimide, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizdng morphogenetic protein-1, antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin, baccatin III derivatives, balanol, batimastat, benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride, bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene A, bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists, breflate, brequinar sodium, bropirimine, budotitane, busulfan, buthionine sulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone, camptothecin derivatives, canarypox IL-2, capecitabine, caraceraide, carbetimer, carboplatin, carboxamide-amino-triazole, carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derived inhibitor, carubicin hydrochloride, carzelesin, casein kinase inhibitors, castanosperrnine, cecropin B, cedefingol, cetrorelix, chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost, cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatol mesylate, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cyclophosphamide, cycloplatam, cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin, dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate, diaziquone, didemnin B, didox, diethyhiorspermine, dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel, docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, dronabinol, duazomycin, duocannycin SA, ebselen, ecomustine, edatrexate, edelfosine, edrecolomab, eflomithine, eflomithine hydrochloride, elemene, elsarnitrucin, emitefur, enloplatin, enpromate, epipropidine, epirubicin, epirubicin hydrochloride, epristeride, erbulozole, erythrocyte gene therapy vector system, esorubicin hydrochloride, estramustine, estramustine analog, estramustine phosphate sodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide, etoposide phosphate, etoprine, exemestane, fadrozole, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine, fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil, fluorocitabine, forfenimex, formestane, fosquidone, fostriecin, fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabine hydrochloride, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid, idarubicin, idarubicin hydrochloride, idoxifene, idramantone, ifosfamide, ihnofosine, ilomastat, imidazoacridones, imiquimod, immunostimulant peptides, insulin-like growth factor-1 receptor inhibitor, interferon agonists, interferon alpha-2A, interferon alpha-2B, interferon alpha-N1, interferon alpha-N3, interferon beta-IA, interferon gamma-IB, interferons, interleukins, iobenguane, iododoxorubicin, iproplatm, irinotecan, irinotecan hydrochloride, iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, lanreotide acetate, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alpha interferon, leuprolide acetate, leuprolide/estrogen/progesterone, leuprorelin, levamisole, liarozole, liarozole hydrochloride, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compounds, lissoclinamide, lobaplatin, lombricine, lometrexol, lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantrone hydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin lysofylline, lytic peptides, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, merbarone, mercaptopurine, meterelin, methioninase, methotrexate, methotrexate sodium, metoclopramide, metoprine, meturedepa, microalgal protein kinase C uihibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, mycophenolic acid, myriaporone, n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, nocodazole, nogalamycin, n-substituted benzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfate sodium, pentostatin, pentrozole, peplomycin sulfate, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pipobroman, piposulfan, pirarubicin, piritrexim, piroxantrone hydrochloride, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compounds, platinum-triamine complex, plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2, prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-based immune modulator, protein kinase C inhibitor, protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, puromycin, puromycin hydrochloride, purpurins, pyrazorurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, RAF antagonists, raltitrexed, ramosetron, RAS farnesyl protein transferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptine demethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes, RH retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, safingol hydrochloride, saintopin, sarcnu, sarcophytol A, sargramostim, SDI1 mimetics, semustine, senescence derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, signal transduction modulators, simtrazene, single chain antigen binding protein, sizofuran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosafe sodium, sparfosic acid, sparsomycin, spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride, temoporfin, temozolomide, teniposide, teroxirone, testolactone, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan hydrochloride, topsentin, toremifene, toremifene citrate, totipotent stem cell factor, translation inhibitors, trestolone acetate, tretinoin, triacetyluridine, triciribine, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tropisetron, tubulozole hydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, velaresol, veramine, verdins, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine, vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidine sulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, zinostatin, zinostatin stimalamer, or zorubicin hydrochloride.
In another embodiment, the nanoparticles of the invention can be used to treat vulnerable plaque in a subject in need thereof. In particular, the payload associated with, i.e., encapsulated within, the nanoparticle of the invention is a biologically active agent used to stabilize a vulnerable plaque. Such agents are described in U.S. Pat. No. 7,008,411, which is incorporated herein by reference in its entirety.
In another embodiment, the nanoparticles of the invention can be used to treat restenosis or atherosclerosis in a subject in need thereof. Restenosis is the reobstruction of an artery following interventional procedures such as balloon angioplasty or stenting.
Additional examples of potentially suitable drugs include for delivery by the nanoparticles of the invention include doxorubicin, 2-aminochromone (U-86983, Upjohn and Pharmacia) (U-86), cytarabine, vincristine, dalteparin sodium, cyclosporine A, colchicines, etoposide, sirolimus, paclitaxel, ceramide, cilostazol, clodronate, pamidronate, alendronate, ISA-13-1, AG-1295, AGL-2043, dexamethasone, everolimus, ABT-578, tacrolimus (FK506), estradiol, lantrunculinD, cytochalasin A, dexamethasone, zotarolimus, angiopeptin, bisphosphonates, estrogen, angiopeptin, ROCK inhibitors, PDGF inhibitors, MMP inhibitors, statins, and well as combinations of these therapies (e.g., a combination of zotarolimus and dexamethasone), as well as any therapeutic disclosed in Circ Res 2003 Apr. 18; 92(7):e62-9. Epub 2003 Mar. 27; J Pharm Sci 1998 October; 87(10):1229-34; Int J Nanomedicine 2007; 2(2):143-61; and Atherosclerosis 2002 February; 160(2):259-71, which are incorporated herein by reference in their entirety.
In one embodiment, therapeutic or biologically active agents may be released by the nanoparticles of the invention to induce therapeutic angiogenesis, which refers to the processes of causing or inducing angiogenesis and arteriogenesis, either downstream, or away from the vulnerable plaque. Arteriogenesis is the enlargement of pre-existing collateral vessels. Collateral vessels allow blood to flow from a well-perfused region of the vessel into an ischemic region (from above an occlusion to downstream from the occlusion). Angiogenesis is the promotion or causation of the formation of new blood vessels downstream from the ischemic region. Having more blood vessels (e.g., capillaries) below the occlusion may provide for less pressure drop to perfuse areas with severe narrowing caused by a thrombus. In the event that an occlusive thrombus occurs in a vulnerable plaque, the myocardium perfused by the affected artery is salvaged. Representative therapeutic or biologically active agents include, but are not limited to, proteins such as vascular endothelial growth factor (VEGF) in any of its multiple isoforms, fibroblast growth factors, monocyte chemoatractant protein 1 (MCP-1), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta) in any of its multiple isoforms, DEL-1, insulin like growth factors (IGF), placental growth factor (PLGF), hepatocyte growth factor (HGF), prostaglandin E1 (PG-E1), prostaglandin E2 (PG-E2), tumor necrosis factor alpha (THF-alpha), granulocyte stimulating growth factor (G-CSF), granulocyte macrophage colony-stimulating growth factor (GM-CSF), angiogenin, follistatin, and proliferin, genes encoding these proteins, cells transfected with these genes, pro-angiogenic peptides such as PR39 and PR11, and pro-angiogenic small molecules such as nicotine. The nanoparticles of the invention may also include lipid lowering agents (e.g., hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, statins, niacin, bile acid resins, and fibrates), antioxidants (e.g., vitamin E (α-tocopherol), vitamin C, and β-carotene supplements), extracellular matrix synthesis promoters, inhibitors of plaque inflammation and extracellular degradation, estradiol drug classes and its derivatives.
Other therapeutic agents to be delivered in accordance with the present invention include, but are not limited to, nucleic acids (e.g., siRNA, RNAi, and microRNA agents), proteins (e.g. antibodies), peptides, lipids, carbohydrates, hormones, metals, radioactive elements and compounds, vaccines, immunological agents, etc., and/or combinations thereof. In some embodiments, the agent to be delivered is an agent useful in the treatment of cancer (e.g., prostate cancer).
In one embodiment, the nanoparticles of this invention will contain nucleic acids such as siRNA. Preferably, the siRNA molecule has a length from about 10-50 or more nucleotides. More preferably, the siRNA molecule has a length from about 15-45 nucleotides. Even more preferably, the siRNA molecule has a length from about 19-40 nucleotides. Even more preferably, the siRNA molecule has a length of from about 21-23 nucleotides.
The siRNA of the invention preferably mediates RNAi against a target mRNA. The siRNA molecule can be designed such that every residue is complementary to a residue in the target molecule. Alternatively, one or more substitutions can be made within the molecule to increase stability and/or enhance processing activity of said molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand.
The target mRNA cleavage reaction guided by siRNAs is sequence specific. In general, siRNA containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition. However, 100% sequence identity between the siRNA and the target gene is not required to practice the present invention. Sequence variations can be tolerated including those that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
Moreover, not all positions of an siRNA contribute equally to target recognition. Mismatches in the center of the siRNA are most critical and essentially abolish target RNA cleavage. In contrast, the 3′ nucleotides of the siRNA do not contribute significantly to specificity of the target recognition. Generally, residues at the 3′ end of the siRNA sequence which is complementary to the target RNA (e.g., the guide sequence) are not critical for target RNA cleavage.
Sequence identity may readily be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. NatL Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. NatL Acad. Sci. USA 90:5873. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the siRNA and the portion of the target mRNA is preferred. Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target mRNA transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6 (log10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. The length of the identical nucleotide sequences may be at least about or about equal to 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.
In one embodiment, the siRNA molecules of the present invention are modified to improve stability in serum or in growth medium for cell cultures. In order to enhance the stability, the 3′-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2′-deoxythymidine is tolerated and does not affect the efficiency of RNA interference. For example, the absence of a 2′hydroxyl may significantly enhance the nuclease resistance of the siRNAs in tissue culture medium.
In another embodiment of the present invention the siRNA molecule may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the target-specific activity, e.g., the RNAi mediating activity is not substantially effected, e.g., in a region at the 5′-end and/or the 3′-end of the RNA molecule. Particularly, the ends may be stabilized by incorporating modified nucleotide analogues.
Nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In preferred backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In preferred sugar modified ribonucleotides, the 2′OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or NO2, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
Nucleotide analogues also include nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.
RNA may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. In one embodiment, an siRNA is prepared chemically. Methods of synthesizing RNA molecules are known in the art, in particular, the chemical synthesis methods as described in Verina and Eckstein (1998), Annul Rev. Biochem. 67:99. In another embodiment, an siRNA is prepared enzymatically. For example, an siRNA can be prepared by enzymatic processing of a long, double-stranded RNA having sufficient complementarity to the desired target mRNA. Processing of long RNA can be accomplished in vitro, for example, using appropriate cellular lysates and siRNAs can be subsequently purified by gel electrophoresis or gel filtration. siRNA can then be denatured according to art-recognized methodologies. In an exemplary embodiment, siRNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the siRNA may be used with no or a minimum of purification to avoid losses due to sample processing.
Alternatively, the siRNAs can also be prepared by enzymatic transcription from synthetic DNA templates or from DNA plasmids isolated from recombinant bacteria. Typically, phage RNA polymerases are used such as T7, T3 or SP6 RNA polyimerase (Milligan and Uhlenbeck (1989) Methods EnzynioL 180:51-62). The RNA may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to inhibit annealing, and/or promote stabilization of the double strands.
Commercially available design tools and kits, such as those available from Ambion, Inc. (Austin, Tex.), and the Whitehead Institute of Biomedical Research at MIT (Cambridge, Mass.) allow for the design and production of siRNA. By way of example, a desired mRNA sequence can be entered into a sequence program that will generate sense and antisense target strand sequences. These sequences can then be entered into a program that determines the sense and antisense siRNA oligonucleotide templates. The programs can also be used to add, e.g., hairpin inserts or Ti promoter primer sequences. Kits also can then be employed to build siRNA expression cassettes.
In various embodiments, siRNAs are synthesized in vivo, in situ, and in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo or in situ, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from a transgene in vivo or an expression construct, a regulatory region (e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation) may be used to transcribe the siRNAs. Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age. A transgenic organism that expresses siRNAs from a recombinant construct may be produced by introducing the construct into a zygote, an embryonic stem cell, or another multipotent cell derived from the appropriate organism.
In one embodiment, the target mRNA of the invention specifies the amino acid sequence of at least one protein such as a cellular protein (e.g., a nuclear, cytoplasmic, transmembrane, or membrane-associated protein). In another embodiment, the target mRNA of the invention specifies the amino acid sequence of an extracellular protein (e.g., an extracellular matrix protein or secreted protein). As used herein, the phrase “specifies the amino acid sequence” of a protein means that the mRNA sequence is translated into the amino acid sequence according to the rules of the genetic code. The following classes of proteins are listed for illustrative purposes: developmental proteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2. CBL, CSFIR, ERBA, ERBB, EBRB2, ERBB2, ERBB3, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADPglucose pyrophorylases, acetylases and deacetylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases), proteins involved in tumor growth (including vascularization) or in metastatic activity or potential, including cell surface receptors and ligands as well as secreted proteins, cell cycle regulatory, gene regulatory, and apoptosis regulatory proteins, immune response, inflammation, complement, or clotting regulatory proteins.
As used herein, the term “oncogene” refers to a gene which stimulates cell growth and, when its level of expression in the cell is reduced, the rate of cell growth is reduced or the cell becomes quiescent. In the context of the present invention, oncogenes include intracellular proteins, as well as extracellular growth factors which may stimulate cell proliferation through autocrine or paracrine function. Examples of human oncogenes against which siRNA and morpholino constructs can designed include c-myc, c-myb, mdm2, PKA-I (protein kinase A type I), Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclin dependent kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos, and src, to name but a few. In the context of the present invention, oncogenes also include a fusion gene resulted from chromosomal translocation, for example, the Bcr/Abl fusion oncogene.
Further proteins include cyclin dependent kinases, c-myb, c-myc, proliferating cell nuclear antigen (PCNA), transforming growth factor-beta (TGF-beta), and transcription factors nuclear factor kappaB (NF-.kappa.B), E2F, HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR, P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin, transferrin receptor, IRE, C-fos, HSP27, C-raf and metallothionein genes.
The siRNA employed in the present invention can be directed against the synthesis of one or more proteins. Additionally or alternatively, there can be more than one siRNA directed against a protein, e.g., duplicate siRNA or siRNA that correspond to overlapping or non-overlapping target sequences against the same target protein. Accordingly, in one embodiment two, three, four or any plurality of siRNAs against the same target mRNA can be included in the nanoparticles of the invention. Additionally, several siRNAs directed against several proteins can be employed. Alternatively, the siRNA can be directed against structural or regulatory RNA molecules that do not code for proteins.
In a preferred aspect of the invention, the target mRNA molecule of the invention specifies the amino acid sequence of a protein associated with a pathological condition. For example, the protein may be a pathogen-associated protein (e.g., a viral protein involved in immunosuppression or immunoavoidance of the host, replication of the pathogen, transmission of the pathogen, or maintenance of the infection), or a host protein which facilitates entry of the pathogen into the host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of infection in the host, or assembly of the next generation of pathogen. Alternatively, the protein may be a tumor-associated protein or an autoimmune disease-associated protein.
In one embodiment, the target mRNA molecule of the invention specifies the amino acid sequence of an endogenous protein (i.e. a protein present in the genome of a cell or organism). In another embodiment, the target mRNA molecule of the invention specifies the amino acid sequence of a heterologous protein expressed in a recombinant cell or a genetically altered organism. In another embodiment, the target mRNA molecule of the invention specifies the amino acid sequence of a protein encoded by a transgene (i.e., a gene construct inserted at an ectopic site in the genome of the cell). In yet another embodiment, the target mRNA molecule of the invention specifies the amino acid sequence of a protein encoded by a pathogen genome which is capable of infecting a cell or an organism from which the cell is derived.
By inhibiting the expression of such proteins, valuable information regarding the function of said proteins and therapeutic benefits which may be obtained from said inhibition may be obtained.
In one embodiment, the nanoparticles of this invention comprises one or more siRNA molecules to silence a PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, INK gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYB gene, JIJN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, Skp2 gene, kinesin spindle protein gene, Bcr-Abl gene, Stat3 gene, cSrc gene, PKC gene, Bax gene, Bcl-2 gene, EGFR gene, VEGF gene, myc gene, NFκB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, PLK1 gene, protein kinase 3 gene, CD31 gene, IGF-1 gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21 (WAF 1/CIP 1) gene, mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, mutations in the p53 tumor suppressor gene, mutations in the p53 family member DN-p63, mutations in the pRb tumor suppressor gene, mutations in the APC1 tumor suppressor gene, mutations in the BRCA1 tumor suppressor gene, mutations in the PTEN tumor suppressor gene, mLL fusiongene, BCRIABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene, alpha v-integrin gene, Fit-i receptor gene, tubulin gene, Human Papilloma Virus gene, a gene required for Human Papilloma Virus replication, Human Immunodeficiency Virus gene, a gene required for Human Immunodeficiency Virus replication, Hepatitis A Virus gene, a gene required for Hepatitis A Virus replication, Hepatitis B Virus gene, a gene required for Hepatitis B Virus replication, Hepatitis C Virus gene, a gene required for Hepatitis C Virus replication, Hepatitis D Virus gene, a gene required for Hepatitis D Virus replication, Hepatitis E Virus gene, a gene required for Hepatitis B Virus replication, Hepatitis F Virus gene, a gene required for Hepatitis F Virus replication, Hepatitis G Virus gene, a gene required for Hepatitis G Virus replication, Hepatitis H Virus gene, a gene required for Hepatitis H Virus replication, Respiratory Syncytial Virus gene, a gene that is required for Respiratory Syncytial Virus replication, Herpes Simplex Virus gene, a gene that is required for Herpes Simplex Virus replication, herpes Cytomegalovirus gene, a gene that is required for herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene, a gene that is required for herpes Epstein Barr Virus replication, Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is required for Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene, human gene that is required for JC Virus replication, myxovirus gene, a gene that is required for myxovirus gene replication, rhinovirus gene, a gene that is required for rhinovirus replication, coronavirus gene, a gene that is required for coronavirus replication, West Nile Virus gene, a gene that is required for West Nile Virus replication, St. Louis Encephalitis gene, a gene that is required for St. Louis Encephalitis replication, Tick-borne encephalitis virus gene, a gene that is required for Tick-borne encephalitis virus replication, Murray Valley encephalitis virus gene, a gene that is required for Murray Valley encephalitis virus replication, dengue virus gene, a gene that is required for dengue virus gene replication, Simian Virus 40 gene, a gene that is required for Simian Virus 40 replication, Human T Cell Lymphotropic Virus gene, a gene that is required for Human T Cell Lymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, a gene that is required for Moloney-Murine Leukemia Virus replication, encephalomyocarditis virus gene, a gene that is required for encephalomyocarditis virus replication, measles virus gene, a gene that is required for measles virus replication, Vericella zoster virus gene, a gene that is required for Vericella zoster virus replication, adenovirus gene, a gene that is required for adenovirus replication, yellow fever virus gene, a gene that is required for yellow fever virus replication, poliovirus gene, a gene that is required for poliovirus replication, poxvirus gene, a gene that is required for poxvirus replication, plasmodium gene, a gene that is required for plasmodium gene replication, Mycobacterium ulcerans gene, a gene that is required for Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene, a gene that is required for Mycobacterium tuberculosis replication, Mycobacterium leprae gene,-185-a gene that is required for Mycobacterium leprae replication, Staphylococcus aureus gene, a gene that is required for Staphylococcus aureus replication, Streptococcus pneumoniae gene, a gene that is required for Streptococcus pneumoniae replication, Streptococcus pyogenes gene, a gene that is required for Streptococcus pyogenes replication, Chlamydia pneumoniae gene, a gene that is required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a gene that is required for Mycoplasma pneumoniae replication, an integrin gene, a selectin gene, complement system gene, chemokine gene, chemokine receptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene, Pro-Platelet Basic Protein gene, MIP-11 gene, MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene, CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309 gene, a gene to a component of an ion channel, a gene to a neurotransmitter receptor, a gene to a neurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, allele gene found in LOH cells, or one allele gene of a polymorphic gene. Examples of relevant siRNA molecules to silence genes and methods of making siRNA molecules can be found from commercial sources such as Dharmacon or from the following patent applications: US2005017667, WO2006066158, WO2006078278, U.S. Pat. No. 7,056,704, U.S. Pat. No. 7,078,196, U.S. Pat. No. 5,898,031, U.S. Pat. No. 6,107,094, EP 1144623, and EU 1144623, all of which are incorporated by reference in their entireties. While a number of specific gene silencing targets are listed, this list is merely illustrative and other siRNA molecules could also be used with the nanoparticles of this invention.
In one embodiment, the nanoparticles of this invention comprise an siRNA molecule having RNAi activity against an RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having coding or non-encoding sequence, such as those sequences referred to by GenBank Accession Nos. described in Table V of PCT/US03/05028 (International PCT Publication No. WO 03/4654) or otherwise known in the art.
In one embodiment, the nanoparticles of this invention comprise an siRNA molecule which silences the vascular endothelial growth factor gene. In another embodiment, the nanoparticles of this invention comprise an siRNA molecule which silences the vascular endothelial growth factor receptor gene.
In another embodiment, the nanoparticles of this invention comprise an siRNA molecule, wherein the sequence of the siRNA molecule is complementary to tumor-related targets, including, but not limited to, hypoxia-inducible factor-1 (HIF-1), which is found in human metastatic prostate PC3-M cancer cells (Mol. Carcinog. 2008 Jan. 31 [Epub ahead of print]); the HIF-1 downstream target gene (Mol. Carcinog. 2008 Jan. 31 [Epub ahead of print]), mitogen-activated protein kinases (MAPKs), hepatocyte growth factor (HGF), interleukin 12p70 (IL12), glucocorticoid-induced tumor necrosis factor receptor (GITR), intercellular adhesion molecule 1 (ICAM-1), neurotrophin-3 (NT-3), interleukin 17 (IL17), interleukin 18 binding protein a (IL18 Bpa) and epithelial-neutrophil activating peptide (ENA78) (see, e.g., “Cytokine profiling of prostatic fluid from cancerous prostate glands identifies cytokines associated with extent of tumor and inflammation”, The Prostate Early view Published Online: 24 Mar. 2008); PSMA (see, e.g., “Cell-Surface labeling and internalization by a fluorescent inhibitor of prostate-specific membrane antigen” The Prostate Early view Published Online: 24 Mar. 2008); Androgen receptor (AR), keratin, epithelial membrane antigen, EGF receptor, and E cadherin (see, e.g., “Characterization of PacMetUT1, a recently isolated human prostate cancer cell line”); peroxisomes proliferators-activated receptor γ (PPARγ; see e.g., The Prostate Volume 68, Issue 6, Date: 1 May 2008, Pages: 588-598); the receptor for advanced glycation end products (RAGE) and the advanced glycation end products (AGE), (see, e.g., “V domain of RAGE interacts with AGEs on prostate carcinoma cells” The Prostate Early view Published Online: 26 Feb. 2008); the receptor tyrosine kinase erb-B2 (Her2/neu), hepatocyte growth factor receptor (Met), transforming growth factor-beta 1 receptor (TGFβR1), nuclear factor kappa B (NFκB), Jagged-1, Sonic hedgehog (Shh), Matrix metalloproteinases (MMPs, esp. MMP-7), Endothelin receptor type A (ETA), Endothelin-1 (ET-1), Nuclear receptor subfamily 3, group C, member 1 (NR3C1), Nuclear receptor co-activator 1 (NCOA1), NCOA2, NCOA3, E1A binding protein p300 (EP300), CREB binding protein (CREBBP), Cyclin G associated kinase (GAK), Gelsolin(GSN), Aldo-keto reductase family 1, member C1 (AKR1C1), AKR1C2, AKR1C3, Neurotensin(NTS), Enolase 2(ENO2), Chromogranin B (CHGB, secretogranin 1), Secretagogin (SCGN, or EF-hand calcium binding protein), Dopa decarboxylase(DDC, or aromatic L-amino acid decarboxylase), steroid receptor co-activator-1 (SRC-1), SRC-2 (a.k.a. TIF2), SRC-3 (a.k.a. AIB-1) (see, e.g., “Longitudinal analysis of androgen deprivation of prostate cancer cells identifies pathways to androgen independence” The Prostate Early view Published Online: 26 Feb. 2008); estrogen receptors (ERα, ERβ or GPR30) (see, e.g., The Prostate Volume 68, Issue 5, Pages 508-516); the melanoma cell adhesion molecule (MCAM) (see, e.g., The Prostate Volume 68, Issue 4, Pages 418-426; angiogenic factors (such as vascular endothelial growth factor (VEGF) and erythropoietin), glucose transporters (such as GLUT1), BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (see, e.g., The Prostate Volume 68, Issue 3, Pages 336-343); types 1 and 2 5α-reductase (see, e.g., The Journal of Urology Volume 179, Issue 4, Pages 1235-1242); ERG and ETV1, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), α-Methylacyl coenzyme A racemase (AMACR), PCA3DD3 glutathione-S-transferase, pi 1 (GSTP1), p16, ADP-ribosylation factor (ARF), O-6-methylguanine-DNA methyltransferase (MGMT), human telomerase reverse transcriptase (hTERT), early prostate cancer antigen (EPCA), human kallikrein 2 (HK2) and hepsin (see, e.g., The Journal of Urology Volume 178, Issue 6, Pages 2252-2259); bromodomain containing 2 (BRD2), eukaryotic translation initiation factor 4 gamma, 1 (eIF4G1), ribosomal protein L13a (RPL13a), and ribosomal protein L22 (RPL22) (see, e.g., N Engl J Med 353 (2005), p. 1224); HER2/neu, Derlin-1, ERBB2, AKT, cyclooxygenase-2 (COX-2), PSMD3, CRKRS, PERLD1, and C17ORF37, PPP4C, PARN, ATP6V0C, C16orf14, GBL, HAGH, ITFG3, MGC13114, MRPS34, NDUFB10, NMRAL1, NTHL1, NUBP2, POLR3K, RNPS1, STUB1, TBL3, and USP7. All of the references described herein are incorporated herein by reference in their entireties.
Thus, in one embodiment, the invention comprises a nanoparticle comprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradable polymer, a stealth polymer, and an siRNA molecule. In one embodiment, the invention comprises a nanoparticle comprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradable polymer, a stealth component, and an siRNA molecule that silences the vascular endothelial growth factor gene. In one embodiment, the invention comprises a nanoparticle comprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradable polymer, a stealth component, and an siRNA molecule that silences the vascular endothelial growth factor receptor gene. In another embodiment, the invention comprises a nanoparticle comprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), PLGA, polyethylene glycol, and an siRNA molecule. In one embodiment, the invention comprises a nanoparticle comprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradable polymer, a stealth component, and an siRNA molecule wherein the nanoparticle can selectively accumulate in the prostate or in the vascular endothelial tissue surrounding a cancer. In one embodiment, the invention comprises a nanoparticle comprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradable polymer, a stealth component, and an siRNA molecule wherein the nanoparticle can selectively accumulate in the prostate or in the vascular endothelial tissue surrounding a cancer and wherein the nanoparticle can be endocytosed by a PSMA expressing cell.
In another embodiment, the siRNA that is incorporated into the nanoparticle of the invention are those that treat prostate cancer, such as those disclosed in U.S. application Ser. No. 11/021,159 (siRNA sequence is complementary to SEQ ID No.8: gaaggccagu uguauggac), and U.S. application Ser. No. 11/349,473 (discloses siRNAs that bind to a region from nucleotide 3023 to 3727 of SEQ ID No. 1). Both of these references are incorporated herein by reference in their entirety.
In another embodiment, the therapeutic agents of the nanoparticles of the invention include RNAs that can be used to treat cancer, such as anti-sense mRNAs and microRNAs. Examples of microRNAs that can be used as therapeutic agents for the treatment of cancer include those disclosed in Nature 435 (7043): 828-833; Nature 435 (7043): 839-843; and Nature 435 (7043): 834-838, all of which are incorporated herein by reference in their entireties.
In one embodiment, the invention specifically excludes nanoparticles containing iron oxide. In one embodiment, blood clotting does not occur at the location where the nanoparticle accumulates.
In one embodiment, the therapeutic agents used in conjunction with the nanoparticles of the invention include one or more agents useful for the treatment of restenosis. Examples of such agents include, but are not limited to, everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653, rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus, biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCK inhibitors, MMP inhibitors, and 2-CdA. In a certain embodiment, the therapeutic agents useful for the treatment of restenosis are zotarolimus and dexamethasone, including combinations of zotarolimus and dexamethasone.
In a preferred embodiment, the nanoparticles of the invention can be delivered to or near a vulnerable plaque using a medical device such as a needle catheter, drug eluding stent or stent graft. Such devices are well known in the art, and are described, for example, in U.S. Pat. No. 7,008,411, which is incorporated herein by reference in its entirety. In one embodiment, a drug eluting stent and/or needle catheter may be implanted at the region of vessel occlusion that may be upstream from a vulnerable plaque region. A medical device, such as a drug eluting stent, needle catheter, or stent graft may be used to treat the occlusive atherosclerosis (i.e., non-vulnerable plaque) while releasing the nanoparticle of the invention to treat a vulnerable plaque region distal or downstream to the occlusive plaque. The nanoparticle may be released slowly over time.
The nanoparticles of the invention can also be delivered to a subject in need thereof using the Genie™ balloon catheter available from Acrostak (http://www.acrostak.com/genie_en.htm). The nanoparticles of the invention can also be delivered to a subject in need thereof using delivery devices that have been developed for endovascular local gene transfer such as passive diffusion devices (e.g., double-occlusion balloon, spiral balloon), pressure-driven diffusion devices (e.g., microporous balloon, balloon-in-balloon devices, double-layer channeled perfusion balloon devices, infusion-sleeve catheters, hydrogel-coated balloons), and mechanically or electrically enhanced devices (e.g., needle injection catheter, iontophoretic electric current-enhanced balloons, stent-based system), or any other delivery system disclosed in Radiology 2003; 228:36-49, or Int J Nanomedicine 2007; 2(2):143-61, which are incorporated herein by reference in their entirety.
Once the inventive conjugates have been prepared, they may be combined with pharmaceutical acceptable carriers to form a pharmaceutical composition, according to another aspect of the invention. As would be appreciated by one of skill in this art, the carriers may be chosen based on the route of administration as described below, the location of the target issue, the drug being delivered, the time course of delivery of the drug, etc.
Methods of TreatmentIn some embodiments, targeted particles in accordance with the present invention may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, inventive targeted particles may be used to treat cancer, e.g., breast cancer, and/or cancer cells, e.g., breast cancer cells.
The term “cancer” includes pre-malignant as well as malignant cancers. Cancers include, but are not limited to, prostate, gastric cancer, colorectal cancer, skin cancer, e.g., melanomas or basal cell carcinomas, lung cancer, cancers of the head and neck, bronchus cancer, pancreatic cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. “Cancer cells” can be in the form of a tumor, exist alone within a subject (e.g., leukemia cells), or be cell lines derived from a cancer.
Cancer can be associated with a variety of physical symptoms. Symptoms of cancer generally depend on the type and location of the tumor. For example, lung cancer can cause coughing, shortness of breath, and chest pain, while colon cancer often causes diarrhea, constipation, and blood in the stool. However, to give but a few examples, the following symptoms are often generally associated with many cancers: fever, chills, night sweats, cough, dyspnea, weight loss, loss of appetite, anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly, hemoptysis, fatigue, malaise, cognitive dysfunction, depression, hormonal disturbances, neutropenia, pain, non-healing sores, enlarged lymph nodes, peripheral neuropathy, and sexual dysfunction.
In one aspect of the invention, a method for the treatment of cancer (e.g. breast cancer) is provided. In some embodiments, the treatment of cancer comprises administering a therapeutically effective amount of inventive targeted particles to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention a “therapeutically effective amount” of an inventive targeted particle is that amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of cancer.
In one aspect of the invention, a method for administering inventive compositions to a subject suffering from cancer (e.g. breast cancer) is provided. In some embodiments, particles to a subject in such amounts and for such time as is necessary to achieve the desired result (i.e., treatment of cancer). In certain embodiments of the present invention a “therapeutically effective amount” of an inventive targeted particle is that amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of cancer.
In other embodiments, the nanoparticles of the present invention can be used to inhibit the growth of cancer cells, e.g., breast cancer cells. As used herein, the term “inhibits growth of cancer cells” or “inhibiting growth of cancer cells” refers to any slowing of the rate of cancer cell proliferation and/or migration, arrest of cancer cell proliferation and/or migration, or killing of cancer cells, such that the rate of cancer cell growth is reduced in comparison with the observed or predicted rate of growth of an untreated control cancer cell. The term “inhibits growth” can also refer to a reduction in size or disappearance of a cancer cell or tumor, as well as to a reduction in its metastatic potential. Preferably, such an inhibition at the cellular level may reduce the size, deter the growth, reduce the aggressiveness, or prevent or inhibit metastasis of a cancer in a patient. Those skilled in the art can readily determine, by any of a variety of suitable indicia, whether cancer cell growth is inhibited.
Inhibition of cancer cell growth may be evidenced, for example, by arrest of cancer cells in a particular phase of the cell cycle, e.g., arrest at the G2/M phase of the cell cycle. Inhibition of cancer cell growth can also be evidenced by direct or indirect measurement of cancer cell or tumor size. In human cancer patients, such measurements generally are made using well known imaging methods such as magnetic resonance imaging, computerized axial tomography and X-rays. Cancer cell growth can also be determined indirectly, such as by determining the levels of circulating carcinoembryonic antigen, prostate specific antigen or other cancer-specific antigens that are correlated with cancer cell growth. Inhibition of cancer growth is also generally correlated with prolonged survival and/or increased health and well-being of the subject.
The present invention is directed, in part, to the discovery that a collagen IV alpha-2 chain related polypeptide can act as a receptor for the CREKA tumor targeting peptide. Collagens are a major component of the extracellular matrix (ECM), an interconnected molecular network providing mechanical support for cells and tissues and regulating biochemical and cellular processes such as adhesion, migration, gene expression and differentiation (see, e.g., U.S. Patent Application No. 2005/0048063, which is incorporated herein by reference in its entirety). In higher animals, at least 19 distinct collagen types differing in their higher order structures and functions have been identified based on the presence of the characteristic collagen triple-helix structure. The collagens are sometimes categorized into the fibrillar and nonfibrillar collagens. The fibrillar (interstitial) collagens include types I, II, III, V and XI, while the nonfibrillar collagens include types IV, VI, IX, X, XI, XII, XIV and XIII.
As a non-limiting example, a method of the invention for treating cancer can be useful for treating breast cancer. Targeting poly(amio acids) useful in the invention include those which selectively target tumor vasculature and selectively bind non-helical collagen. Targeting poly(amio acids) useful in the invention also include those which selectively target to tumor vasculature and selectively bind collagen IV, and those which selectively target tumor vasculature and selectively bind denatured collagen IV in preference to native collagen IV.
Inventive therapeutic protocols involve administering a therapeutically effective amount of an inventive targeted particle to a healthy individual (i.e., a subject who does not display any symptoms of cancer and/or who has not been diagnosed with cancer). For example, healthy individuals may be “immunized” with an inventive targeted particle prior to development of cancer and/or onset of symptoms of cancer; at risk individuals (e.g., patients who have a family history of cancer; patients carrying one or more genetic mutations associated with development of cancer; patients having a genetic polymorphism associated with development of cancer; patients infected by a virus associated with development of cancer; patients with habits and/or lifestyles associated with development of cancer; etc.) can be treated substantially contemporaneously with (e.g., within 48 hours, within 24 hours, or within 12 hours of) the onset of symptoms of cancer. Of course individuals known to have cancer may receive inventive treatment at any time.
In another aspect, the invention provides a method of treating cardiovascular conditions in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention. Such cardiovascular conditions include, but are not limited to, restenosis and vulnerable plaque. In one embodiment, the nanoparticles of this invention are delivered locally to the coronary arteries, central arteries, peripheral arteries, veins, and bile ducts. In another embodiment, the nanoparticles of this invention are delivered locally to the coronary arteries, central arteries, peripheral arteries, veins, and bile ducts after the implantation of a stent in such tissue in a patient for the treatment of restenosis. In another embodiment, the nanoparticles of this invention are administered to a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis.
In one embodiment, the nanoparticles of this invention pass through the endothelial layer of a blood vessel due to plaque damage of the endothelial tissue and bind to the basement membrane.
In another aspect, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention. In one embodiment, the controlled-release system is locally administered to a designated region of the blood vessel where the restenosis occurs. In still another embodiment, the controlled-release system is administered via a medical device. In yet another embodiment, the medical device is a drug eluding stent, needle catheter, or stent graft. In another embodiment, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention wherein the controlled release system contains a drug selected from the group consisting of everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653, rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus, biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCK inhibitors, MMP inhibitors, and 2-CdA. In another embodiment, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention wherein the controlled release system contains two drugs selected from everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653, rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus, biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCK inhibitors, MMP inhibitors, and 2-CdA. In another embodiment, the invention provides a method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of the invention wherein the controlled release system contains zotarolimus and dexamethasone.
In one embodiment, the nanoparticles of this invention are delivered locally to the coronary arteries, central arteries, peripheral arteries, veins, and bile ducts. In another embodiment, the nanoparticles of this invention are delivered locally to the coronary arteries, central arteries, peripheral arteries, veins, and bile ducts after the implantation of a stent in such tissue in a patient for the treatment of restenosis. In another embodiment, the nanoparticles of this invention are administered to a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis. In another embodiment, the nanoparticles of this invention are administered within 12 hours of a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis. In another embodiment, the nanoparticles of this invention are administered locally to a patient undergoing a coronary angioplasty, a peripheral angioplasty, a renal artery angioplasty, or a carotid angioplasty in order to prevent resenosis.
Pharmaceutical CompositionsAs used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Remington's Pharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as TWEEN™ 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. If filtration or other terminal sterilization methods are not feasible, the formulations can be manufactured under aseptic conditions.
The pharmaceutical compositions of this invention can be administered to a patient by any means known in the art including oral and parenteral routes. In certain embodiments parenteral routes are desirable since they avoid contact with the digestive enzymes that are found in the alimentary canal. According to such embodiments, inventive compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays).
In one embodiment, the nanoparticles of the present invention are administered to a subject in need thereof systemically, e.g., by IV infusion or injection. In a particular embodiment, the nanoparticles of the present invention are locally administered to a subject in need thereof. As used herein, “local administration” is when nanoparticles of the invention are brought into contact with the blood vessel wall or vascular tissue through a device (e.g., a stent).
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In one embodiment, the inventive conjugate is suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™ 80. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Compositions for rectal or vaginal administration may be suppositories which can be prepared by mixing the inventive conjugate with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the inventive conjugate.
Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The inventive conjugate is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of this invention. The ointments, pastes, creams, and gels may contain, in addition to the inventive conjugates of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the inventive conjugates in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the inventive conjugates in a polymer matrix or gel.
Powders and sprays can contain, in addition to the inventive conjugates of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures thereof. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
When administered orally, the inventive nanoparticles can be, but are not necessarily, encapsulated. A variety of suitable encapsulation systems are known in the art (“Microcapsules and Nanoparticles in Medicine and Pharmacy,” Edited by Doubrow, M., CRC Press, Boca Raton, 1992; Mathiowitz and Langer J. Control. Release 5:13, 1987; Mathiowitz et al. Reactive Polymers 6:275, 1987; Mathiowitz et al. J. Appl. Polymer Sci. 35:755, 1988; Langer Ace. Chem. Res. 33:94, 2000; Langer J. Control. Release 62:7, 1999; Uhrich et al. Chem. Rev. 99:3181, 1999; Zhou et al. J. Control. Release 75:27, 2001; and Hanes et al. Pharm. Biotechnol. 6:389, 1995). The inventive conjugates may be encapsulated within biodegradable polymeric microspheres or liposomes. Examples of natural and synthetic polymers useful in the preparation of biodegradable microspheres include carbohydrates such as alginate, cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes, polypropylfumarates, polyethers, polyacetals, polycyanoacry lates, biodegradable polyurethanes, polycarbonates, polyanhydrides, polyhydroxyacids, poly(ortho esters), and other biodegradable polyesters. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
Pharmaceutical compositions for oral administration can be liquid or solid. Liquid dosage forms suitable for oral administration of inventive compositions include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to an encapsulated or unencapsulated conjugate, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. As used herein, the term “adjuvant” refers to any compound which is a nonspecific modulator of the immune response. In certain embodiments, the adjuvant stimulates the immune response. Any adjuvant may be used in accordance with the present invention. A large number of adjuvant compounds is known in the art (Allison Dev. Biol. Stand. 92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al. Vaccine 10:151-158, 1992).
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or unencapsulated conjugate is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
It will be appreciated that the exact dosage of the targeted particle is chosen by the individual physician in view of the patient to be treated, in general, dosage and administration are adjusted to provide an effective amount of the targeted particle to the patient being treated. As used herein, the “effective amount” of an targeted particle refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of targeted particle may vary depending on such factors as the desired biological endpoint, the drug to be delivered, the target tissue, the route of administration, etc. For example, the effective amount of targeted particle containing an anti-cancer drug might be the amount that results in a reduction in tumor size by a desired amount over a desired period of time. Additional factors which may be taken into account include the severity of the disease state; age, weight and gender of the patient being treated; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy.
The nanoparticles of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of nanoparticle appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. For any nanoparticle, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of nanoparticles can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.
The present invention also provides any of the above-mentioned compositions in kits, optionally with instructions for administering any of the compositions described herein by any suitable technique as previously described, for example, orally, intravenously, pump or implantable delivery device, or via another known route of drug delivery. “Instructions” can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
The “kit” typically defines a package including any one or a combination of the compositions of the invention and the instructions, but can also include the composition of the invention and instructions of any form that are provided in connection with the composition in a manner such that a clinical professional will clearly recognize that the instructions are to be associated with the specific composition.
The kits described herein may also contain one or more containers, which may contain the inventive composition and other ingredients as previously described. The kits also may contain instructions for mixing, diluting, and/or administrating the compositions of the invention in some cases. The kits also can include other containers with one or more solvents, surfactants, preservative and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the components in a sample or to a subject in need of such treatment.
The compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the composition provided is a dry powder, the composition may be reconstituted by the addition of a suitable solvent, which may also be provided. In embodiments where liquid forms of the composition are used, the liquid form may be concentrated or ready to use. The solvent will depend on the nanoparticle and the mode of use or administration. Suitable solvents for drug compositions are well known, for example as previously described, and are available in the literature. The solvent will depend on the nanoparticle and the mode of use or administration.
The invention also involves, in another aspect, promotion of the administration of any of the nanoparticle described herein. In some embodiments, one or more compositions of the invention are promoted for the prevention or treatment of various diseases such as those described herein via administration of any one of the compositions of the present invention. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention.
The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.
EXAMPLESThe invention is further illustrated by the following examples. The examples should not be construed as further limiting.
Example 1 Amphiphilic Nanoparticle with AptamerIn one embodiment, the A10 RNA aptamer which binds to the Prostate Specific Membrane Antigen (PSMA) on the surface of prostate cancer cells is conjugated to DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine)-PEG-COOH using EDC/NHS chemistry with a conjugate concentration of 0.7 mg/mL. 0.21 mg of this DSPE-PEG-aptamer bioconjugate is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mL tetrahydrofuran (THF) solvent, to which 5% docetaxel of the mass of PLGA is added. This PLGA solution is then mixed with the aqueous solution of lecithin/DSPE-PEG-Aptamer. These mixtures are vortexed for 3 minutes, followed by stirring for 2 hours. In order to remove all organic solvents, these mixtures are then dialyzed for another 4 hours against PBS buffer. This procedure would yield nanoparticles targeting to prostate cancer cells expressing PSMA antigens.
In a second embodiment, poly(D,L-lactic-co-glycolic acid) (PLGA) is used as a polymeric core, lecithin monolayer (˜2.5 nm) as a lipid shell, poly(ethylene glycol) (PEG) as a stealth material, and the A10 RNA aptamer were used to develop targeted PLGA-Lecithin-PEG nanoparticles (NPs). Particle size could be tuned within the range from 40 nm to 500 nm, accompanied with a surface zeta potential ranging from −80 mV to −30 mV. Using docetaxel (a widely used chemotherapeutics for cancers) as a model small molecule hydrophobic drug, the PLGA-Lecithin-PEG NP had drug encapsulation efficiency around 65% as contrast to 19% for the conventional PLGA-b-PEG diblock copolymer NP. In addition, less than 20% drugs were released from the NP during the first 6 hours, which holds broad promise for clinical applications. Both in vitro and in vivo results demonstrated that the attached RNA aptamer effectively targeted PLGA-Lecithin-PEG NPs to prostate cancer cells which express PSMA antigen on their plasma membrane, such as LNCaP cells.
Encapsulation efficiency is determined by taking a known amount of DNA, encapsulating it into a nanoparticle, removing any unencapsulated DNA by filtration, lysing the nanoparticle, then detecting the amount of DNA that was encapsulated by measuring its absorbance of light at 260 nm. The encapsulation efficiency is calculated by taking the amount of DNA that was encapsulated, then dividing it by the amount of DNA that we began with. Stated alternatively, it is the fraction of initial DNA that is successfully encapsulated.
Zeta potential is determined by Quasi-elastic laser light scattering with a ZetaPALS dynamic light scattering detector (Brookhaven Instruments Corporation, Holtsville, N.Y.; 15 mW laser, incident beam=676 nm).
Example 2 Amphiphilic Nanoparticle with CREKAThe peptide CREKA is conjugated to DSPE-PEG-Maleimide before formulating nanoparticles using the protocol of Example 1. This peptide will target the delivery and uptake of the nanoparticles to extracellular basement membranes which are exposed under the leaky endothelial layer covering atherosclerotic plaques.
Example 3 Amphiphilic Nanoparticle with AXYLZZLNThe peptide AXYLZZLN, or conservative variants or peptidomimetics thereof, wherein X and Z are variable amino acids, can be conjugated to DSPE-PEG-Maleimide before formulating nanoparticles using the protocol of Example 1. This peptide will target the delivery and uptake of the nanoparticles to extracellular basement membranes which are exposed under the leaky endothelial layer covering atherosclerotic plaques.
Example 4 Anti-Her2/AKERC-Targeted Nanoparticle Triblock Polymer SynthesisMaleimide terminal poly(D,L-lactide)-block-poly(ethylene glycol) (PLA-PEG-MAL) (Mw˜10 kDa determined by GPC) was synthesized by ring opening polymerization. Carboxylic acid terminal poly(D,L-lactide) and/or poly(lactic-co-glycolic acid) was purchased from the DURECT corporation (Pelham, Ala.). Bifunctional PEG (HO-PEG-MAL) was purchased from Nektar Therapeutics (San Carlos, Calif.). Cysteine end terminal affibody was purchased from Affibody® (Sweden). All other reagents were purchased from Sigma Aldrich.
Maleimide-poly(ethylene glycol)-block-poly(D,L-lactic acid) (MAL-PEG-PLA), COOH-poly(ethylene glycol)-block-poly(D,L-lactic acid) (COOH-PEG-PLA), and methoxypoly(ethylene glycol)-block-poly(D,L-lactic acid) (mPEG-PLA) were synthesized by ring opening polymerization in anhydrous toluene using tin(II) 2-ethylhexanoate as catalyst. General procedure for syntheses of the copolymers is as follows: D,L-Lactide (1.6 g, 11.1 mmol) and MAL-PEG3500-OH (0.085 mmol) or COOH-PEG3500-OH (0.085 mmol) in anhydrous toluene (10 mL) was heated to reflux temperature (ca. 120° C.), after which the polymerization was initiated by adding tin(II) 2-ethylhexanoate (20 mg). After stirring for 9 h with reflux, the reaction mixture was cooled to room temperature. To this solution was added cold water (10 mL) and then resulting suspension was stirred vigorously at room temperature for 30 min to hydrolyze unreacted lactide monomers. The resulting mixture was transferred to separate funnel containing CHCL3 (50 mL) and water (30 mL). After layer separation, organic layer was collected, dried using anhydrous MgSO4, filtered, and concentrated under reduced vacuum. Then, hexane was added to the concentrated solution to precipitate polymer product. Pure MAL-PEG3500-PLA or COOH-PEG3500-PLA was collected as a white solid. 111PEG2000-PLA was also prepared by same procedure above. Both copolymers were characterized by ′H-NMR (400 MHz, Bruker Advance DPX 400) and gel permeation chromatography (GPC) (Waters Co, Milford, Mass., USA). Alternatively, the conjugation of PLGA or PLA and PEG was achieved in the presence of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NHS). Briefly, PLGA particles were dissolved in acetonitrile. The carboxylic end of PLGA was activated by mixing with NHS and EDC at a molar ratio of COOH to EDC and NHS and stir overnight at room temperature. The excess EDC and NHS in the solution were quenched by adding 2-mercaptoethanol. The NHS activated PLGA was purified by precipitation in a solution containing ethyl ether and methanol, and followed by centrifugation at 3000 g for 10 minutes. To conjugate the amine end of NH2-PEG-MAL with the NHS-activated PLGA, both polymers mixed at a molar ratio of 1:1.3 (PLGA-NHS:NH2-PEG-MAL) at room temperature overnight. The resulting PLGA-PEG-MAL copolymer was purified by precipitation in ethyl ether-methanol solution. The conjugation of the maleimide end of MAL-PEG-PLGA and the free end thiol of affibody.
Nanoparticles were formed by precipitating the triblock copolymer in water. Briefly, the triblock polymer was dissolved in acetonitrile, and then mixed slowly with water. The nanoparticles formed instantly upon mixing. The residual acetonitrile in the suspension was evaporated by continuously stirring the suspension at room temperature for 4 hrs. Alternatively, polymeric nanoparticles were formed in a first step as above and subsequently functionalized with affibody in aqueous solution.
Anti-Her2 Affibody Targeted Polymeric NanoparticlesThe synthesis of a multi-block polymer is initiated by conjugation of functionalized biodegradable polyesters with chemical groups such as, but not limited to, maleimide or carboxylic acid for easy conjugation to one end of thiol, amine or similarly functionalized polyethers. The conjugation of polymer to the affibody will be performed in organic solvents such as but not limited to dimethyl sulfoxide, dichloromethane, acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, and acetone or in aqueous buffer including phosphate buffers and Tris buffers. The other free end of the polyether would be functionalized with chemical groups for conjugation to a library of targeting molecules such as affibodies, and its derivatives. The affibody may be conjugated through functional group including but not limited to thiol, amine, carboxylates, hydroxyls, aldehydes, ketones and photoreactions. The conjugation reaction between the targeting molecules and the poly-ester-ether copolymer is achieved by adding the affibody molecules solublized in an organic solvent or aqueous solution. Following each of the two conjugation reactions, unconjugated macromers are washed away by precipitating the polymer of interest in solvents such as but not limited to ethyl ether, hexane, methanol and ethanol. Alternatively, the nanopartciles conjugated to affibody in aqueous solution are washed using distilled water and ultracentrifuge membranes. Biodegradable and biocompatible polymer poly(lactide-co-glycolide) (PLGA)/PLA and polyethylene glycol (PEG) can be used as a model for the block copolymer of poly(ester-ether). In a representative embodiment, the human epidermal growth factor receptor 2 (HER-2/neu, also known as erbB-2) can be used for breast or ovarian specific targeting using an anti-HER-2 Affibody as the targeting molecule to cancer cells. Carboxylic acid modified PLGA (PLGA-COOH) or PLA can be conjugated to the amine modified heterobifunctional PEG (NH2-PEG-Maleimide) and form a copolymer of PLGA-PEG-COOH. By using a C-end terminal cysteine modified Anti-Her2 affibody (HS-Affibody). A triblock copolymer of PLGA/PLA-PEG-Affibody can be obtained by conjugating the maleimide end of PEG and free thiol functional group on the affibody. The multiblock polymer can also be useful for imaging and diagnostic applications. In such embodiment, a photo-sensitive or environmental-responsible compound will be linked to the multiblock polymer.
The targeted nanoparticles are formed by precipitation of the multi-block polymer in an aqueous environment. The nanoparticle formulation system described here is compatible with high throughput biological assays in order to test the nanoparticles generated from the multi-block polymer. Alternatively, polymeric nanoparticles can be formed by nanoprecipitation and subsequently functionalized with the affibody in aqueous solution. It is possible to control the density of affibody on the surface and to optimize the formulation polymer/affibody for therapeutic application.
AKERC Peptide Targeted Lipid-Polymer NanoparticlesThe peptide is first chemically conjugated to the hydrophilic region of a lipid molecule. This conjugate is then mixed with a certain ratio of unconjugated lipid molecule in an aqueous solution containing one or more water-miscible solvents. In a preferred embodiment, the amphiphilic lipid can be, but is not limited to, one or a plurality of the following: phosphatidylcholine, lipid A, cholesterol, dolichol, shingosine, sphingomyelin, ceramide, cerebroside, sulfatide, glycosylceramide, phytosphingosine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, phophatidic acid, and lysophophatides. The water miscible solvent can be, but is not limited to: acetone, ethanol, methanol, and isopropyl alcohol. Separately, a biodegradable polymeric material is mixed with the agent or agents to be encapsulated in a water miscible or partially water miscible organic solvent. In a preferred embodiment, the biodegradable polymer can be, but is not limited to one or a plurality of the following: poly(D,L-lactic acid), poly(D,L-glycolic acid), poly(s-caprolactone), or their copolymers at various molar ratios. The carried agent can be, but is not limited to, one or a plurality of the following: therapeutic drugs, imaging probes, or hydrophobic or lipophobic molecules for medical use. The water miscible organic solvent can be but is not limited to: acetone, ethanol, methanol, or isopropyl alcohol. The partially water miscible organic solvent can be, but is not limited to: acetonitrile, tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate, or dimethylformamide. The resulting polymer solution is then added to the aqueous solution of conjugated and unconjugated amphiphilic lipid to yield nanoparticles by the rapid diffusion of the organic solvent into the water and evaporation of the organic solvent.
In a preferred embodiment, the peptide AKERC, which binds to collagen IV in the extracellular basement membranes, is conjugated to DSPE-PEG-Maleimide (DSPE: 1,2 distearoyl-sn-glycero-3-phosphoethanolamine sodium salt) using EDC/NHS chemistry with a conjugate concentration of 0.7 mg/mL. 0.21 mg of this DSPE-PEG-KAERC bioconjugate is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mL acetonitrile (ACN) solvent, to which 5% docetaxel of the mass of PLGA is added. This PLGA solution is then mixed with the aqueous solution of lecithin/DSPE-PEG-KAERC. These mixtures are vortexed for 3 minutes, followed by stirring for 2 hours. In order to remove all organic solvents, these mixtures are then washed three times using copious PBS buffer. This peptide will target the delivery and uptake of the nanoparticles to extracellular basement membranes which are exposed under the leaky endothelial layer covering atherosclerotic plaques.
Example 5 CREKA-Targeted Nanoparticle Lipid-PEG-CREKA Synthesis:1 mg CREKA peptide is dissolved in 0.2 mL PBS buffer containing 10 mg dithiothreitol (DTT). The solution is incubated at room temperature for 30 minutes before mixed with 1 mg DSPE-PEG-Maleimide (DSPE: 1,2 distearoyl-sn-glycero-3-phosphoethanolamine sodium salt). These mixtures are incubated at 4° C. for 24 hr. In order to remove DTT agent and the extra CREKA, these mixtures are then dialyzed against PBS buffer for 48 hr.
CREKA-Targeted Nanoparticle Synthesis:0.03 mg of the DSPE-PEG-CREKA bioconjugate is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mL acetonitrile (ACN). This PLGA solution is then mixed with the aqueous solution of lecithin/DSPE-PEG-CREKA. These mixtures are vortexed for 3 minutes, followed by stirring for 2 hours. In order to remove all organic solvents, these mixtures are then washed three times using copious PBS buffer. For fluorescence imaging purposes, 10% of the PLGA polymer is labeled with a fluorescent probe such as Alexa Fluor 647.
CREKA-Targeted Nanoparticle with Therapeutic Agent:
For example, 0.03 mg DSPE-PEG bioconjugate is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mL acetonitrile solvent, to which 5% docetaxel of the mass of PLGA is added. The lecithin/DSPE-PEG solution is first heated up to 65° C. for 3 minutes. Then the PLGA solution is added to the aqueous solution of lecithin/DSPE-PEG dropwise under gentle stirring. These mixtures are vortexed for 3 minutes, followed by stirring for 2 hours. In order to remove all organic solvents, these mixtures are then dialyzed for another 3 hours against PBS buffer. These procedures would yield lipid-polymer hybrid nanoparticles with a diameter of about 50-60 nm and a zeta potential of about −40 mV.
In another method, 0.036 mg DSPE-PEG-CREKA triblock compound is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mL acetonitrile solvent, to which 5% docetaxel of the mass of PLGA is added. For fluorescence imaging purpose, 10% of the PLGA polymer is labeled with a fluorescent probe such as Alexa Fluor 647. The lecithin/DSPE-PEG solution is first heated up to 65° C. for 3 minutes. Then the PLGA solution is added to the aqueous solution of lecithin/DSPE-PEG dropwise under gentle stirring. These mixtures are vortexed for 3 minutes, followed by stirring for 2 hours. In order to remove all organic solvents, these mixtures are then dialyzed for another 3 hours against PBS buffer. Alternatively, these mixtures can be washed three times using copious PBS buffer to remove organic solvents and any free molecules. These procedures would yield CREKA-targeted lipid-polymer nanoparticles with specific a binding affinity to extracellular basement membrane.
In another method, 0.03 mg DSPE-PEG-Maleimide bioconjugate is mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mL acetonitrile solvent, to which 5% docetaxel of the mass of PLGA is added. For fluorescence imaging purpose, 10% of the PLGA polymer is labeled with a fluorescent probe such as Alexa Fluor 647. The Lecithin/DSPE-PEG solution is first heated up to 65° C. for 3 minutes. Then the PLGA solution is added to the aqueous solution of lecithin/DSPE-PEG dropwise under gentle stirring. These mixtures are vortexed for 3 minutes, followed by stirring for 2 hours. In order to remove all organic solvents, these mixtures are then dialyzed for another 3 hours against PBS buffer. Alternatively, these mixtures can be washed three times using copious PBS buffer to remove organic solvents and any free molecules. 0.03 mg CREKA peptide is dissolved in 0.2 mL PBS buffer containing 1 mg dithiothreitol (DTT). The solution is incubated at room temperature for 30 minutes before mixed with 1 mg PLGA-Lipid-PEG-Maleimide nanoparticle. These mixtures are incubated at 4° C. for 24 hr. In order to remove DTT agent and the extra CREKA, these mixtures washed three times using PBS buffer. These procedures would yield CREKA-targeted lipid-polymer nanoparticles with specific a binding affinity to extracellular basement membrane.
In another method, 0.036 mg DSPE-PEG-CREKA triblock compound can be mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) can be dissolved in 1 mL acetonitrile solvent, to which 5% of a combination of dexamethasone and zotarolimus of the mass of PLGA can be added.
In another method, 0.036 mg DSPE-PEG-CREKA triblock compound can be mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) can be dissolved in 1 mL acetonitrile solvent, to which 5% of everolimus of the mass of PLGA can be added.
In another method, 0.036 mg DSPE-PEG-CREKA triblock compound can be mixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) can be dissolved in 1 mL acetonitrile solvent, to which 5% of paclitaxel of the mass of PLGA can be added.
ARYLQKLN-Targeted Nanoparticle Synthesis:For ease of conjugation, a cystin amino acid is added to the C-terminal or N-terminal of ARYLQKLN. The additional procedures/methods follow those of CREKA-targeted nanoparticles given immediately above.
CREKA-Targeted Nanoparticle Binding to Collagen-Coated SurfaceTo prepare a collagen IV-coated surface, 0.5 mL Collagen IV acetic acid solution (10 μg/mL) is spread to completely cover the bottom of a glass well. After 1 hr incubation at 37° C., the extra collagen is removed by washing the surface using copious water. The quality of the coating is checked by atomic force microscopy (AFM). The collagen coated surface is then incubated with 1 mL CREKA-targeted nanoparticle aqueous solution (0.25 mg/mL) for 30 minutes at room temperature. The extra nanoparticles are washed by copious PBS buffer. Fluorescence microscopy is used to image the sample, thereby identifying the binding efficiency of CREKA-targeted nanoparticle to collagen coated surface.
CREKA-Targeted Nanoparticle Binding to Rat Basement Membrane Ex Vivo:A rat is sacrificed and its abdominal aorta is exposed in situ. After washing the aorta with copious PBS buffer, a balloon catheter is placed in the aorta. The balloon is inflated with 0.2 mL air and dragged through the aorta five times to injure the endothelia layer. The proper amount of CREKA-targeted nanoparticles are injected into the aorta and incubated for 30 minutes under constant pressure. The extra nanoparticles are washed away using copious PBS buffer. 10 mm abdominal aorta is sectioned and kept in 4% formaldehyde solution for histological imaging use.
Other peptides such as d-CREKA and CEAKR (a scrambled sequence) are used as negative control to investigate the binding specificity of CREKA-targeted nanoparticles to basement membrane. d-CREKA and CEAKR replace CREKA and repeated in the above experiments.
Example 6 Prophetic Example of Synthesis of PLGA-PEG-CREKA Macromolecule for Preparation of CREKA-Targeted NanoparticleAs described herein, the nanoparticles of the invention can be delivered to a subject using a variety of methods, such as intravenous or local administration using, e.g., a balloon stent. The following example demonstrates how to test the advantages of locally delivering the nanoparticles of the invention.
Pre-Interventional ProceduresAnimal Monitoring and Examination
Upon arrival at the Testing Facility and until sacrifice, the animals will be monitored and observed at least once a day. A physical examination of all animals entered in the study will be done by the Facility Veterinarian or a trained employee during acclimation, as per Testing Facility SOPs.
Fasting
Fasting will be conducted prior to induction of anesthesia. Food, including any dietary supplements, will be withheld the morning of the procedure. Water will not be withheld.
Anesthesia
Animals will be tranquilized with acepromazine administered subcutaneously [SC]. Animal weight will be recorded. Anesthesia induction will be achieved with propofol injected intravenously [IV]. Upon induction of light anesthesia, the subject animal will be intubated and supported with passive balloon ventilation. Isoflurane in oxygen will be administered to maintain a surgical plane of anesthesia. Fluid therapy will be achieved by saline injections before surgery. Intravenous saline injection may be performed to replace blood loss or to correct low systemic blood pressure.
Anticoagulant TherapyDuring the procedure an initial bolus of heparin (˜70 IU/kg) will be given following cannulation of the carotid artery. Additional heparin may be given if needed.
Animal Preparation
The animal will be placed in dorsal recumbency, and the hair will be removed from the access areas. A rectal temperature probe will be inserted, and the temperature will be monitored regularly. The access site will be prepared with topical application of chlorhexidine, 70% isopropyl alcohol and proviodine. The area will then be appropriately draped to maintain a sterile field.
Denudation ProceduresVascular Access
After induction of anesthesia, the left or right carotid artery will be accessed with an incision made in the throat region. An application of bupivacain on the carotid access site will be performed to achieve local anesthesia and manage pain after surgery. An arterial sheath will be introduced and advanced into the artery.
Vessel Angiography
Before the first angiogram, 1 ml of nitroglycerine (0.5 mg/ml) IV will be given. For subsequent angiograms, more nitro can be given if slow flow or spams are observed, as per operator judgment and animal condition. The iliac artery will be circumscribed (from the femoral to the internal iliac branch) and Quantitative Angiography (QA) will be performed to document the vessel size. Extra angiograms may be recorded at this point or later on in the procedure at the discretion of the operator. In such case, the operator will select a suitable angiogram for analysis.
Balloon Injury
The appropriate balloon will be advanced over the guidewire to traverse the distal portion of the pre-selected injury site. The inflated balloon will then be retracted from the femoral artery back into the aorta to enable denudation of the target vasculature. The balloon will be deflated and re-advanced to traverse the target injury site. At the operator's discretion, the inflation pressure will be adjusted for each subsequent denudation pass, based on the amount of force required to pull the balloon. If there is little resistance to the pulling, the balloon inflation pressure will be increased by an increment of one Atm. If there is too much resistance, the balloon inflation pressure will be decreased by an increment of one ATM. The third denudation will not be performed if resistance is still present. The balloon will be deflated while it is in the terminal descending aorta and the denudation procedure repeated one more time (total 3 times denudation). Post-denudation angiogram will be performed and TIMI flow will be assessed. Animals with post-TIMI flow of zero or 1 will receive intra-arterial infusion of nitroglycerine (at the discretion of the interventionalist) to restore the flow to 2 or 3.
Test Article DeliveryThe delivery catheter will be introduced into the artery over the guide wire. The Genie balloon catheter will be continuously inflated at a low pressure of 2 atm that allows for distal and proximal occlusion of the vessel while simultaneously forming a central drug depot. A continuous pressure of 2 atm is maintained throughout application of the contrast agent. The total volume injected will be recorded. Following treatment of the vessel a final angiography will be taken and recorded, TIMI flow and QCA will also be performed and documented.
Monitoring ProceduresParameters including isoflurane level, blood oxygen saturation, pulse rate, and temperature will be regularly monitored and manually recorded and noted in the raw data for each animal.
ClosureFollowing successful delivery and completion of angiography, all catheters and the sheath will be removed from the animals and the carotid artery will be ligated.
NecropsyUpon completion of follow-up angiography the animals will be kept deeply anesthetised before euthanasia with a rapid bolus of pentobarbital.
Stented arteries along with proximal and distal non-stented segments will then be dissected out rinsed and immersion-fixed in neutral-buffered formalin and processed for histology.
HistopathologyThe treated iliacs will be cut in 4 sections that will be embedded in paraffin 4 separate blocks. For each block 2 adjacent sections will be prepared and then 2 other adjacent section ˜100μ deeper in the block. One section from each adjacent pair will be stained with hematoxylin and eosin (H&E). The remaining section of each adjacent pair will be left unstained and sent for fluorescence analyses. The H&E stained section will be analyzed by the Study Pathologist for the extent of vessel injury, the presence of endothelium and other relevant observations. The analysis will be reported as a narrative text including representative images, with scoring of some parameters if deemed appropriate.
Example 8 HER-2 Targeted Drug Encapsulated NanoParticles (FIGS. 21-24)To develop HER-2 targeted drug encapsulated NPs, the anti-HER-2 Affibody was conjugated to the thiol-reactive maleimide end group of the PLA-PEG-Maleimide (PLA-PEG-Mal) copolymer through a stable thioether bond and the targeting specificity and efficacy was evaluated using fluorescent microscopy. Subsequently, we encapsulated paclitaxel into the targeted polymeric NPs and examined whether this system could increase the drug cytotoxicity in HER-2 positive cell lines: SK-BR-3 and SK-OV-3.
Materials and MethodsConjugation and Characterization of nanoparticle-Affibody bioconjugates: PLA-PEG-Mal polymeric NPs were incubated under stirring conditions with the Anti-HER-2 Affibody molecules (15 kDa) at a molar ratio of Affibody:PLA-PEG-Mal of 5% to form a stable bioconjugate. The NP-Affibody bioconjugates were purified to remove free Affibody molecules using Amicon Ultra centrifuge device (100 kDa molecular weight size exclusion). Subsequently, the thioether bond formation between the PLA-PEG-Mal NPs and the Affibody molecules was characterized using proton nuclear magnetic resonance (1H-NMR, 600 MHz, Bruker Advance). Additionally, the chemical attachment of the fluorescent Affibody was confirmed using Ultra Violet Imaging system (Kodak Electrophoresis Documentation and Analysis System 120). The Affibody molecule was fluorescently labeled with a red fluorescent probe, Alexa Fluor 532 (Invitrogen), purified and subsequently conjugated to PLA-PEG-Mal polymeric NPs at different molar ratios of Affibody:PLA-PEG-Mal ranging from 0 to 20% (molar ratio). Then the purified NP-Affibody bioconjugates suspensions were imaged using a UV Kodak camera assisted with a red filter to show the visible effect of the fluorescent Affibody conjugated on non-fluorescent polymeric NPs.
Uptake assays of targeted and untargeted nanoparticles: The human ovarian adenocarcinoma (SK-OV-3; ATCC), human breast adenocarcinoma (SK-BR-3; ATCC), and human pancreatic adenocarcinoma (Capan-1, ATCC) were the HER-2 positive cell lines used for cytotoxicity and uptake efficacy studies of the NP-Affibody bioconjugates. HER-2 positive cell lines were grown in chamber slides (Cab-TekII, 8 wells; Nunc) within their growth medium (Modified McCoy's 5a (ATCC) supplemented with 100 units/ml aqueous penicillin G, 100 ug/ml streptomycin, and 10% FBS) to 70% confluence in 24 h (i.e., 50,000 cells/cm2) in 5% CO2 incubator. On the day of the experiment, cells were washed with pre-warmed PBS and incubated with pre-warmed phenol-red-reduced OptiMEM media for 30 minutes, before adding 50 μg of NPs or NP-Affibody bioconjugates loaded with same amount of fluorescent NBD dye. NP formulations were incubated with cells for 2 hours at 37° C., washed with PBS three times, fixed with 4% paraformaldehyde, blocked for 30 minutes at room temperature with 1% BSA/PBS, permeabilized with 0.01% Triton-X for 3 minutes, counterstained with Alexa-Fluor Phalloidin-Rhodamine (cytoskeleton staining), 4′,6-diamidino-2-phenylindole (DAPI, nucleus staining), mounted in fluorescence protecting imaging solution, and visualized using fluorescent microscopy (DeltaVision system; Olympus IX71). Digital images of green, red and blue fluorescence were acquired along the z-axis at 0.2 μm intervals using 60× oil immersion objective and DAPI, FITC and Rhodamine filters respectively. Images were overlaid, deconvoluted and 3D reconstruction was performed using Softwork software for acquisition and analysis.
In vitro cellular toxicity assay of paclitaxel encapsulated into targeted and untargeted NPs: SK-BR-3 and SK-OV-3 were grown in 96-well plates at concentrations leading to 70% confluence in 24 h (i.e., 50,000 cells/cm2). Defined amounts of paclitaxel were encapsulated into targeted and non-targeted nanoparticles and incubated with cell lines (5 ug Paclitaxel/well) in OptiMEM for two hours. Next, cells were washed and fresh media was supplemented. The cells were then allowed to grow for 72 hours and cell viability was assessed colorimetrically with MTS reagents (Invitrogen).
ResultsDevelopment of targeted, controlled release drug delivering NP-Affibody bioconjugates. We first synthesized a copolymer comprised of a hydrophobic block, poly(D,Llactic acid), and a hydrophilic block, poly(ethylene glycol) with a maleimide terminal group (PLA-PEG-Mal). Then the copolymers form negatively charged NPs with a core-shell structure in aqueous environment via the nanoprecipitation method. The hydrophobic core of the NPs is capable of carrying pharmaceuticals, especially poorly soluble drugs. The hydrophilic shell not only provides a “stealth” layer, together with the surface charge property (Zeta potential)=−10 mV±5 mV), to improve the stability and the circulation half-time of these drug delivering NPs, but also functional maleimide groups for Affibody conjugation. Lack of protein adsorption in solutions including 10%, 20% and 100% serum demonstrated the stability of NP size (<100 nm). We also evaluated the freeze-drying process for storing the nanoparticles in a dry state, as described previously. We were able to reconstitute nanoparticles with a similar original size after lyophilization, confirming the stability of this type of carrier to this process.
The anti-HER-2 Affibody molecule was previously selected against the extracellular domain of the HER-2 protein and further modified by affinity maturation and dimerization. The anti-HER-2 Affibody is commercially available and has been shown to have high binding specificity and affinity in vitro and in vivo as a targeted imaging agent. Particle size and surface charge (Zeta potential) of PLA-PEG-Mal NPs both with and without Affibody were characterized using laser light scattering, ZetaPALS system and electron microscopy (
Efficient and specific receptor mediated internalization of NP-Affibody bioconjugates. We next demonstrated the efficient binding and internalization of targeted NP-Affibody bioconjugates to HER-2 positive cancer cells using three cell lines: Capan-1, SK-BR-3, and SK-OV-3 (see
In vitro cellular cytotoxicity assays using breast cancer and ovarian cancer cells (MTS assays). We prepared targeted and untargeted NPs (with and without paclitaxel) to evaluate their differential cytotoxicity using in vitro cell viability assay (MTS assays) with breast cancer and ovarian cancer cells (SK-BR-3; SK-OV-3), which over-express the HER-2 cell membrane receptors. In this study, we incubated various NP formulations with SK-BR-3 and SK-OV-3 cancer cells for 2 hours in optimem, washed cells with PBS to remove excess of NPs, and supplemented with fresh cell growth medium. We further incubated the cells for 3 days before using MTS assay to quantify cell viability which was normalized to that of the cells in the absence of NPs. The results showed that drug encapsulated targeted NPs had the highest cytotoxicity to both SK-BR-3 and SK-OV-3 cell lines; cell viability was 70±5% and 59±5%, respectively (
For
Claims
1. A controlled-release system, comprising
- a plurality of target-specific stealth nanoparticles;
- wherein said nanoparticles contain targeting moieties attached thereto, wherein the targeting moiety is a poly(amino acid) that targets the basement membrane of a blood vessel.
2. The controlled-release system of claim 1, wherein the nanoparticle has an amount of targeting moiety effective for the treatment of vulnerable plaque in a subject in need thereof.
3. The controlled-release system of claim 1, wherein the nanoparticle has an amount of targeting moiety effective for the treatment of restenosis.
4. The controlled-release system of claim 1, wherein the nanoparticle has an amount of targeting moiety effective for the treatment of cancer in a subject in need thereof.
5-6. (canceled)
7. The controlled-release system of claim 1, wherein the poly(amino acid) comprises natural amino acids, unnatural amino acids, modified amino acids, or protected amino acids.
8. The controlled-release system of claim 1, wherein the poly(amino acid) is selected from the group consisting of a protein, peptidomimetic, affibody or peptide.
9. The controlled-release system of claim 1, wherein the poly(amino acid) binds to the basement membrane of a blood vessel.
10. The controlled-release system of claim 1, wherein the poly(amino acid) binds to collagen.
11. The controlled-release system of claim 1, wherein the poly(amino acid) binds to collagen IV.
12. (canceled)
13. The controlled-release system of claim 8, wherein the peptide comprises a sequence selected from the group consisting of AKERC, CREKA, ARYLQKLN and AXYLZZLN, wherein X and Z are variable amino acids.
14. The controlled-release system of claim 1, wherein the nanoparticle comprises a polymeric matrix.
15. The controlled-release system of claim 14, wherein the polymeric matrix comprises two or more polymers.
16. The controlled-release system of claim 13, wherein the polymeric matrix comprises polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, dextran or polyamines, or combinations thereof.
17-19. (canceled)
20. The controlled-release system of claim 14, wherein at least one polymer is a polyester.
21. The controlled-release system of claim 20, wherein the polyester is selected from the group consisting of PLGA, PLA, PGA, and polycaprolactones.
22. The controlled-release system of claim 20, wherein the polyester is PLGA or PLA.
23. The controlled-release system of claim 14, wherein the polymeric matrix comprises a copolymer of two or more polymers.
24. The controlled-release system of claim 23, wherein the copolymer is a copolymer of a polyalkylene glycol and a polyester.
25. The controlled-release system of claim 24, wherein the copolymer is a copolymer of PLGA and PEG.
26. The controlled-release system of claim 24, wherein the polymeric matrix comprises PLGA and a copolymer of PLGA and PEG.
27. (canceled)
28. The controlled-release system of claim 14, wherein the polymeric matrix comprises lipid-terminated PEG and PLGA.
29-34. (canceled)
35. The controlled-release system of claim 14, wherein the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of cancer.
36. The controlled-release system of claim 14, wherein the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of a vulnerable plaque.
37. The controlled-release system of claim 14, wherein the polymers of the polymer matrix have a molecular weight effective for the treatment of cancer.
38. The controlled-release system of claim 14, wherein the polymers of the polymer matrix have a molecular weight effective for the treatment of vulnerable plaque.
39-42. (canceled)
43. The controlled-release system of claim 1, wherein the nanoparticle further comprises a therapeutic agent.
44-45. (canceled)
46. The controlled-release system of claim 43, wherein the therapeutic agent is selected from the group consisting of mitoxantrone and docetaxel.
47. The controlled-release system of claim 43, wherein the therapeutic agent is selected from the group consisting of VEGF, fibroblast growth factors, monocyte chemoatractant protein 1 (MCP-1), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-beta), DEL-1, insulin like growth factors (IGF), placental growth factor (PLGF), hepatocyte growth factor (HGF), prostaglandin E1 (PG-E1), prostaglandin E2 (PG-E2), tumor necrosis factor alpha (THF-alpha), granulocyte stimulating growth factor (G-CSF), granulocyte macrophage colony-stimulating growth factor (GM-CSF), angiogenin, follistatin, and proliferin, PR39, PR11, nicotine, hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, statins, niacin, bile acid resins, fibrates, antioxidants, extracellular matrix synthesis promoters, inhibitors of plaque inflammation and extracellular degradation, and estradiol.
48. The controlled-release system of claim 43, wherein the therapeutic agent is selected from the group consisting of everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653, rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus, biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCK inhibitors, MMP inhibitors, 2-CdA, zotarolimus and dexamethasone.
49. A method of treating breast cancer in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of claim 1.
50-54. (canceled)
55. A method of treating vulnerable plaque in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of claim 1.
56. A method of treating restenosis in a subject in need thereof, comprising administering to the subject an effective amount of the controlled-release system of claim 1.
57. The method of claim 55, wherein the controlled-release system is locally administered to a designated region of the blood vessel where the vulnerable plaque occurs.
58. The method of claim 55, wherein the controlled-release system is administered via a medical device.
59. The method of claim 58, wherein the medical device is a drug eluding stent, needle catheter, or stent graft.
60-62. (canceled)
63. A method of preparing a stealth nanoparticle, wherein the nanoparticle has a ratio of ligand-bound polymer to non-functionalized polymer effective for the treatment of a disease, comprising:
- providing a therapeutic agent;
- providing a first polymer;
- providing a poly(amino acid) ligand;
- reacting the first polymer with the poly(amino acid) ligand to prepare a ligand-bound polymer; and
- mixing the ligand-bound polymer with a second, non-functionalized polymer, and the therapeutic agent;
- such that the stealth nanoparticle is formed.
64-71. (canceled)
72. A stealth nanoparticle, comprising
- a copolymer of PLGA and PEG; and
- a therapeutic agent;
- wherein said nanoparticle contains targeting moieties attached thereto, wherein the targeting moiety comprises AKERC or CREKA.
73. A stealth nanoparticle, comprising
- a polymeric matrix comprising a complex of a phospholipid bound-PEG and PLGA; and
- a therapeutic agent;
- wherein said nanoparticle contains targeting moieties attached thereto, wherein the targeting moiety is a poly(amino acid).
74-78. (canceled)
79. A controlled-release system, comprising a plurality of target-specific stealth nanoparticles;
- wherein said nanoparticles contain targeting moieties attached thereto, wherein the targeting moiety is a poly(amino acid).
80. (canceled)
81. The compounds: wherein n is 20 to 1720; and wherein R7 is an alkyl groups, R8 is an ester or amide linkage, X=0 to 1 mole fraction, Y=0 to 0.5 mole fraction, X+Y=20 to 1720, and Z=25 to 455.
Type: Application
Filed: Apr 4, 2008
Publication Date: Mar 19, 2009
Applicant: MASSACHUSETTS INSTITUTE OF TECHNOLOGY (Cambridge, MA)
Inventors: Frank Alexis (Brighton, MA), Liangfang Zhang (San Diego, CA), Aleksandar F. Radovic-Moreno (Cambridge, MA), Frank X. Gu (Waterloo), Pamela Basto (Somerville, MA), Etgar Levy-Nissenbaum (Tel-Aviv), Juliana Chan (Cambridge, MA), Robert S. Langer (Newton, MA), Omid C. Farokhzad (Chestnut Hill, MA)
Application Number: 12/098,354
International Classification: A61K 9/14 (20060101); A61K 47/42 (20060101); A61K 31/135 (20060101); A61K 31/337 (20060101); A61K 38/16 (20060101); A61K 31/4353 (20060101); A61K 31/56 (20060101); A61K 9/00 (20060101); C07K 5/10 (20060101); A61P 9/00 (20060101);
