Chlorotoxin-labeled nanoparticle compositions and methods for targeting primary brain tumors
Chlorotoxin-labeled nanoparticles that target primary brain tumors, compositions that include the nanoparticles, methods of imaging tissues using the nanoparticles, and methods for treating tissues using the nanoparticles.
The present application is a continuation of U.S. patent application Ser. No. 11/385,985, filed Mar. 20, 2006, which claims the benefit of U.S. Patent Application Ser. No. 60/674,280, filed Apr. 22, 2005, each application which is expressly incorporated herein by reference in its entirety.
STATEMENT OF GOVERNMENT LICENSE RIGHTSThis invention was made with government support under Grant No. N01-C037122 awarded by National Institutes of Health—National Cancer Institute. The government has certain rights in the invention.
BACKGROUND OF THE INVENTIONGliomas are currently the most common and lethal type of primary brain tumor and are one of the leading causes of cancer-related deaths. Treating malignant gliomas remains a formidable challenge due to the difficulty in differentiating between tumor and healthy brain tissue; the rapid growth rate of invasive gliomas; the sensitivity of normal brain tissue to current therapies; the intrinsic cellular resistance of gliomas to drugs; and the blood brain barrier's ability to prevent the passage of substances including drugs and contrast agents.
Use of magnetic nanoparticles conjugated with targeting or therapeutic agents, combined with magnetic resonance imaging (MRI), has been recognized as a potential approach for early cancer detection and treatment through real-time assessment of therapeutic and surgical efficacy.
When used as drug carriers, enhanced permeation and retention of the circulating nanoparticles yields significant accumulation in tumors versus healthy tissue. Various bioactive peptides have been conjugated to nanoparticles. However, none have been shown to specifically target the vast majority of glioma tumors. Chlorotoxin (Cltx), a 36-amino acid peptide, has been recently shown to have a strong affinity for gliomas, but not non-neoplastic brain tissue. Cltx selectively binds to the membrane-bound matrix metalloproteinase 2 (MMP-2) protein complex highly expressed in gliomas and other primary tumors of the neuroectodermal origin.
The effectiveness of tumor resection in neurosurgery is severely limited by the poor visual contrast between neoplastic and normal brain tissue. Gross tumor resection restricts both patient quality of life and patient survival, while limited resection leaves residual glioma cells that have the ability to proliferate and migrate rapidly. Various targeting biomolecules have been labeled with fluorophores to delineate tumor margins to improve surgical outcome. However, using these probes, preoperative diagnostic images cannot be correlated with intraoperative pathology. This has sparked new research geared towards the development of multimodal probes that can be detected with both magnetic resonance imaging (MRI) and intraoperative optical devices. Currently, the major limitation of the multimodal probes is their low specificity and limited internalization by glioma cells.
There exists a need for a magnetic nanoparticle conjugated with targeting or therapeutic agents for early cancer detection and treatment through real-time assessment of therapeutic and surgical efficacy. A need also exists for a probe that allows for improved effectiveness of tumor resection in neurosurgery by providing a visual contrast between neoplastic and normal brain tissue. A further need exists for a multifunctional probe that can be detected with both magnetic resonance imaging (MRI) and intraoperative optical devices for correlating preoperative diagnosis with intraoperative pathology, and that can kill and impede cancer cells. The present invention seeks to fulfill these needs and provides further related advantages.
SUMMARY OF THE INVENTIONIn one aspect, the invention provides a chlorotoxin-labeled particle comprising:
(a) a core having a surface, the core comprising a material having magnetic resonance imaging activity;
(b) a chlorotoxin; and
(c) a linker covalently coupling the chlorotoxin to the surface.
In one embodiment, the nanoparticle comprises:
(a) a core comprising a magnetic material and having a surface; and
(b) a plurality of silane moieties, wherein each silane moiety is covalently coupled to the surface, wherein at least one terminus of the plurality of silane moieties comprises a chlorotoxin, and wherein the silane moieties comprise a polyalkylene oxide moiety intermediate the surface and the chlorotoxin.
In one embodiment, the magnetic nanoparticle including the chlorotoxin comprises:
(a) a core comprising a magnetic material and having a surface; and
(b) a plurality of silane moieties, wherein each silane moiety is covalently coupled to the core, and wherein at least one silane moiety has the formula:
wherein,
n is 1, 2, 3, 4, 5, or 6;
m is an integer from about 10 to about 1000;
L is a direct bond or a linker; and
T is a chlorotoxin.
In another aspect of the invention, compositions that include the particles of the invention are provided. In one embodiment, the composition includes a nanoparticle suitable for administration to a human. The composition can include an acceptable carrier.
In other aspects, the invention provides methods for using nanoparticles.
In one embodiment, the invention provides a method for differentiating neuroectodermal-derived tumor cells from non-neoplastic brain tissue, comprising:
(a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for neuroectodermal-derived tumor cells; and
(b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
In one embodiment, the invention provides a method for detecting neuroectodermal-derived tumor cells, comprising:
(a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for neuroectodermal-derived tumor cells; and
(b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is a glioma.
In one embodiment, the invention provides a method for detecting a tissue expressing membrane-bound matrix metalloproteinase (MMP-2) protein complex, comprising:
(a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for membrane-bound matrix metalloproteinase (MMP-2) protein complex; and
(b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of a tumor expressing membrane-bound matrix metalloproteinase (MMP-2) protein complex.
In one embodiment, the invention provides a method for determining the location of glioma cells in a patient pre-operatively, intra-operatively, and post-operatively, comprising:
(a) administering a pharmaceutical composition to a patient, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an amount of a fluorophore/chlorotoxin-labeled nanoparticle sufficient to image glioma cells in vivo;
(b) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by magnetic resonance imaging pre-operatively to determine the location of glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioma cells;
(c) surgically removing from the patient at least some glioma cells located by magnetic resonance imaging;
(d) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by fluorescence imaging intra-operatively to determine the location of residual glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of residual glioma cells;
(e) surgically removing from the patient at least some residual glioma cells located by fluorescence imaging; and
(f) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by magnetic resonance imaging post-operatively to determine the location of glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioma cells.
The invention provides methods for treating a tissue using the nanoparticles.
In one embodiment, the invention provides a method for treating a glioma in a patient, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
In one embodiment, the invention provides a method for treating a neuroectodermal tumor, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
In one embodiment, the invention provides a method for treating a tumor expressing matrix metalloproteinase (MMP-2) protein complex, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
In one embodiment, the invention provides a method for inhibiting invasive activity of neoplastic cells, comprising administering to neoplastic cells an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
Methods for making the chlorotoxin-labeled particles are also provided.
DESCRIPTION OF THE DRAWINGSThe foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:
The present invention provides chlorotoxin-labeled nanoparticles capable of targeting primary brain tumors, compositions that include the nanoparticles, methods of imaging tissues using the nanoparticles, and methods for treating cells expressing chlorotoxin binding sites using the nanoparticles.
In one aspect, the invention provides a chlorotoxin-labeled particle comprising:
(a) a core having a surface, the core comprising a material having magnetic resonance imaging activity;
(b) a chlorotoxin; and
(c) a linker covalently coupling the chlorotoxin to the surface.
The particle includes a core having a surface that can be reacted with the silane compounds of the invention. The particles can be core-shell particles in which the core is a material different from the shell. In one embodiment, the surface or shell comprises hydroxyl groups that are reactive toward the silane compounds.
The core includes a material having magnetic resonance imaging activity. Suitable materials having magnetic resonance imaging activity include metal oxides, such as ferrous oxide, ferric oxide, silicon oxide, polycrystalline silicon oxide, aluminum oxide, germanium oxide, zinc selenide, tin dioxide, titanium dioxide, indium tin oxide, and gadolinium oxide. Mixtures of one or more metal oxide can be used.
In addition to magnetic materials, the core can include non-magnetic materials, such as silicon nitride, stainless steel, titanium, and nickel titanium. Mixtures of one or more non-magnetic materials can also be used.
The core of the particles useful in making the particles of the invention have a diameter of from about 2 nm to about 25 nm.
The particles of the invention can be nanoparticles having particle diameter of from about 15 nm to about 200 nm.
The chlorotoxin of the particles of the invention can be native chlorotoxin, synthetic chlorotoxin, recombinant chlorotoxin, and fragments and variants thereof having chlorotoxin binding activity.
The particles of the invention include from about 1 to about 50 chlorotoxins/particle. In one embodiment, the particles include from about 10 to about 50 chlorotoxins/particle. In one embodiment, the particles include about 10 chlorotoxins/particle.
As noted above, the magnetic nanoparticle of the invention includes a chlorotoxin that serves as a targeting moiety that is effective to direct the nanoparticle to cells expressing chlorotoxin binding sites where the nanoparticle is bound. Primary brain tumor cells (e.g., neuroectodermal-derived tumor cells and glioma cells) include chlorotoxin binding sites. In one embodiment, the nanoparticle comprises:
(a) a core comprising a magnetic material and having a surface; and
(b) a plurality of silane moieties, wherein each silane moiety is covalently coupled to the surface, wherein at least one terminus of the plurality of silane moieties comprises a chlorotoxin, and wherein the silane moieties comprise a polyalkylene oxide moiety intermediate the surface and the chlorotoxin.
In one embodiment, the particles of the invention include a plurality of silane moieties covalently coupled to the core's surface. In one embodiment, the covalently coupled silane moieties provide a layer surrounding the core. In one embodiment, the covalently coupled silane moieties provide a monolayer on the core's surface.
Each silane moiety includes a polyalkylene oxide moiety. In one embodiment, the polyalkylene oxide is a polyethylene oxide. Suitable polyethylene oxides have molecular weights of from about 100 to about 100,000 g/mole.
In one embodiment, the nanoparticle including a plurality of silane moieties useful for reaction with the chlorotoxin to provide the chlorotoxin-labeled particles of the invention is prepared as described in Zhang et al., J. Am. Chem. Soc. 2004, 126:7206-7211, expressly incorporated herein by reference in its entirety.
In one embodiment, the magnetic nanoparticle including the chlorotoxin comprises:
(a) a core comprising a magnetic material and having a surface; and
(b) a plurality of silane moieties, wherein each silane moiety is covalently coupled to the core, and wherein at least one silane moiety has the formula:
wherein,
n is 1, 2, 3, 4, 5, or 6;
m is an integer from about 10 to about 1000;
L is a direct bond or a linker; and
T is a chlorotoxin.
For this embodiment, the linker may be one or more atoms that link the chlorotoxin to the polyoxyethylene moiety covalently coupled to the core surface.
The chlorotoxin-labeled nanoparticles can further include other useful agents. Other useful agents include diagnostic agents.
Suitable diagnostic agents include agents that provide for the detection of the nanoparticle by methods other than magnetic resonance imaging.
Suitable diagnostic agents include light-emitting compounds (e.g., fluorophores, phosphors, and luminophors). Suitable fluorophores include fluorophores that can be covalently coupled to silane moiety and that emit fluorescence in the visible and near-infrared region of the spectrum. Representative fluorophores include ALEXA FLUOR, AMCA, BODIPY, CASCADE BLUE, CASCADE YELLOW, coumarins, fluoresceins, eosins, erythrosins, rhodamines, OREGON GREEN, PACIFIC BLUE, and TEXAS RED dyes commercially available in suitable reactive forms from Molecular Probes, Inc. (Eugene, Oreg.) and cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7) CYDYE fluors commercially available in suitable reactive forms from Amersham Pharmacia Biotech (Piscataway, N.J., now GE Healthcare).
In one embodiment, the chlorotoxin-labeled particle further comprises a fluorescent moiety. The particles of the invention include from about 1 to about 10 fluorescent moieties/particle. In one embodiment, the particles include from about 1 to about 2 fluorescent moieties/particle. In one embodiment, the particles include about 1.5 fluorescent moieties/particle.
In one embodiment, the fluorescent moiety is selected from red and near infrared emitting fluorescent moieties (i.e., fluorescent moieties having emission maxima greater than about 600 nm). In one embodiment, the fluorescent moiety is a cyanine moiety. In one embodiment, the fluorescent moiety is a Cy5.5 moiety.
Other suitable diagnostic agents include radiolabels (e.g., radio isotopically labeled compounds) such as 125I, 14C, and 31P, among others.
In embodiments that include a diagnostic agent, a portion of the plurality of silane moieties comprise a diagnostic agent (e.g., a fluorescent agent or fluorophore, a radiolabel).
In another aspect of the invention, compositions that include the particles of the invention are provided. In one embodiment, the composition includes a nanoparticle suitable for administration to a human or an animal subject. The composition can include an acceptable carrier. In one embodiment, the composition is a pharmaceutically acceptable composition and includes a pharmaceutically acceptable carrier. As used herein the term “carrier” refers to a diluent (e.g., saline) to facilitate the delivery of the particles.
In other aspects, the invention provides methods for using nanoparticles.
In one embodiment, the invention provides a method for differentiating neuroectodermal-derived tumor cells from non-neoplastic brain tissue. In the method, neuroectodermal-derived tumor cells are differentiated from non-neoplastic brain tissue by:
(a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for neuroectodermal-derived tumor cells; and
(b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
In one embodiment, the invention provides a method for detecting neuroectodermal-derived tumor cells. In the method, neuroectodermal-derived tumor cells are detected by:
(a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for neuroectodermal-derived tumor cells; and
(b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
The above methods are useful in differentiating and detecting glioma cells.
In one embodiment, the invention provides a method for detecting a tissue expressing matrix metalloproteinase (MMP-2) protein complex. In the method, a tissue expressing metalloproteinase (MMP-2) protein complex is detected by:
(a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for matrix metalloproteinase (MMP-2) protein complex; and
(b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of a tumor expressing matrix metalloproteinase (MMP-2) protein complex.
The matrix metalloproteinase (MMP-2) protein complex may be membrane bound.
In the methods above, measuring the level of binding of the chlorotoxin-labeled nanoparticle comprises magnetic resonance imaging.
In certain embodiments of the methods above, the chlorotoxin-labeled nanoparticle further comprises a fluorescent moiety. In these embodiments, measuring the level of binding of the chlorotoxin-labeled nanoparticle can include fluorescence imaging.
In one embodiment, the invention provides a method for determining the location of glioma cells in a patient pre-operatively, intra-operatively, and post-operatively. The methods includes the steps of:
(a) administering a pharmaceutical composition to a patient, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an amount of a fluorophore/chlorotoxin-labeled nanoparticle sufficient to image glioma cells in vivo;
(b) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by magnetic resonance imaging pre-operatively to determine the location of glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioma cells;
(c) surgically removing from the patient at least some glioma cells located by magnetic resonance imaging;
(d) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by fluorescence imaging intra-operatively to determine the location of residual glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of residual glioma cells;
(e) surgically removing from the patient at least some residual glioma cells located by fluorescence imaging; and
(f) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by magnetic resonance imaging post-operatively to determine the location of glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioma cells.
In the method, an amount of a fluorophore/chlorotoxin-labeled nanoparticle sufficient to image glioma cells in vivo is an amount from about 1-20 mg Fe/kg body weight (“Fe” refers to iron present in particle core.
In the above method, steps (d) and (e) may be repeated.
The above method includes pre-operative, intra-operative, and post-operative imaging. It will be appreciated that variations of the above method are within the scope of the invention. Other variations of the method include, for example, (1) pre-operative imaging only; (2) intra-operative imaging only; (3) post-operative imaging only; (4) pre-operative and intra-operative imaging only; (5) pre-operative and post-operative imaging only; and (6) intra-operative and post-operative imaging only.
The invention provides methods for treating a tissue using the nanoparticles.
In one embodiment, the invention provides a method for treating a glioma in a patient, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
In one embodiment, the invention provides a method for treating a neuroectodermal tumor, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
In one embodiment, the invention provides a method for treating a tumor expressing matrix metalloproteinase (MMP-2) protein complex, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
In one embodiment, the invention provides a method for inhibiting invasive activity of neoplastic cells, comprising administering to neoplastic cells an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle and a pharmaceutically acceptable carrier.
The chlorotoxin-labeled nanoparticles of the invention can be used to detect and treat various cancers (e.g., prostate cancer, sarcomas, hematological malignancies, and leukemias) and various neuroectodermal tumors (e.g., glioma, meningioma, ependymonas, medulloblastoma, neuroblastoma, glioblastoma, ganglioma, pheochromocytoma, melanoma, Ewing's sarcoma, small cell lung carcinoma, and metastatic brain tumors.)
Based on current literature, virtually every type of malignant cancer showing MMP-2 expression is expected to specifically bind the chlorotoxin-labeled nanoparticle of the invention (e.g., CTX:Cy5.5). These malignant cancers include gliomas, astrocytomas medulloblastomas, choroids plexus carcinomas, ependymomas, other brain tumors, neuroblastoma, head and neck cancer, lung cancer, breast cancer, intestinal cancer, pancreatic cancer, liver cancer, kidney cancer, sarcomas (over 30 types), osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma, carcinomas, melanomas, ovarian cancer, cervical cancer, lymphoma, thyroid cancer, anal cancer, colo-rectal cancer, endometrial cancer, germ cell tumors, laryngeal cancer, multiple myeloma, prostate cancer, retinoblastoma, gastric cancer, testicular cancer, and Wilm's tumor.
The methods of the invention are applicable to human and animal subjects.
As noted above, the present invention provides a chlorotoxin-labeled nanoparticle capable of targeting glioma cells detectable by magnetic resonance (MR) imaging. The nanoparticle can be used to correlate pre-operative and post-operative diagnostic images.
The nanoparticle was synthesized by coating iron oxide nanoparticles with covalently-bound bifunctional polyethylene glycol (PEG) that were subsequently functionalized with chlorotoxin.
The chlorotoxin-labeled nanoparticles of the invention include a polyoxyethylene moiety intermediate the particle and chlorotoxin. The polyoxyethylene moiety is formed covalently coupling a polyethylene glycol to the nanoparticle via self-assembly. The use of the polyoxyethylene linker (PEG) improves cellular internalization, prevents nanoparticle agglomeration, increases blood circulation time in vivo, and offers binding sites for chlorotoxin and fluorescent label. The siloxane bond coupling the nanoparticle with the short PEG chains confers the stability of the PEG self-assembled monolayers (SAMS) and increases PEG packing density on nanoparticles by establishing covalent bonds between PEG interchains. PEG-coated nanoparticles used in this study had an overall size less than 15 nm as shown in
The delineation of the tumor margins with targeting-magnetic nanoparticles plays a crucial role in the detection and characterization of lesions in situ to guide treatment and determine surgical resection effectiveness. Currently, dextran-coated nanoparticles are being studied for intravascular administration in intraoperative MRI examinations to determine and remove tumors with a high degree of certainty. With this technique, however, tumor margins are inferred by labeling macrophages situated at the tumor margins, and consequently detected by identifying adjacent microglial cells and not the tumor cells directly. This limits its application for detecting highly invasive brain tumors such as gliomas whose cells may quickly infiltrate surrounding healthy tissue. The effectiveness of tumor delineation is also limited because macrophages are situated partly as activated microglial cells in the central nervous system and only take up a limited amount of nanoparticles. MRI results shown in
The ability of chlorotoxin to attach to, and inhibit the activity of, surface-bound MMP-2 protease limits the invasive nature of tumor cells, lending a therapeutic facet to the NPC system. It has been demonstrated that chlorotoxin inhibits the upregulation of MMP-2, further decreasing the invasive nature of the glioma cells. The matrigel invasion assay confirmed that the bioactivity of chlorotoxin molecules was retained after their conjugation to nanoparticles via the chemical scheme introduced herein. Interestingly, the same therapeutic effect was observed with a lower concentration of chlorotoxin on NPCs versus free chlorotoxin. One possible explanation of this therapeutic enhancement is that the PEG on nanoparticles facilitated cellular uptake of NPC and the nanoparticles helped localize the short peptides in the target cells. Limiting tumor cell invasion can prevent further metastasis of the tumor cells to healthy brain tissue and improve surgical outcome.
The chlorotoxin-labeled nanoparticle of the invention is a magnetic nanoparticle that serves as both an MRI contrast enhancement agent and a drug carrier to inhibit brain tumor cell invasion. A high degree of internalization by targeted glioma cells was observed making the probe ideally suited for further modification with cytotoxic agents. The chlorotoxin-labeled nanoparticle of the invention demonstrated a strong affinity for gliomas, but not healthy brain cells. The high sensitivity of the nanoparticle of the invention provides an effective approach to the early detection, real-time monitoring, and treatment of brain tumors.
The therapeutic effect of free chlorotoxin is due to its ability to limit further expression of the surface-bound MMP-2 and inhibit its enzymatic activity, subsequently inhibiting tumor migration and invasion.
Thus, in one aspect, the present invention provides a chlorotoxin-labeled nanoparticle for targeting gliomas and inhibiting tumor migration. The chlorotoxin-labeled nanoparticle is an iron oxide superparamagnetic nanoparticle coated with a chlorotoxin. The preferential binding of the chlorotoxin-labeled nanoparticle to glioma cells, as compared to dextran-coated nanoparticles, was evaluated qualitatively by magnetic resonance imaging (MRI) and quantitatively by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The specificity of glioma-targeting was evaluated through a comparison of the nanoparticle binding to glioma cells that express matrix metalloproteinase 2 (MMP-2) versus healthy cells that do not express detectable MMP-2. The ability of the nanoparticle to inhibit cell migration was demonstrated by the matrigel invasion test in which the chlorotoxin-labeled nanoparticle showed similar efficacy to free chlorotoxin at a concentration of 4 times lower than that of free chlorotoxin peptide. This indicates that chlorotoxin retains its bioactivity after conjugation to the nanoparticle and that the nanoparticle enhanced the cellular uptake and retention of chlorotoxin in the target cells. The chlorotoxin-labeled nanoparticle can be used for highly sensitive detection of glioma tumors and targeted therapy in the central nervous system.
In another aspect, the present invention provides a multifunctional nanoparticle that includes a chlorotoxin and a fluorescent moiety. The chlorotoxin/fluorophore-labeled nanoparticle is capable of targeting glioma cells, detectable by both magnetic resonance imaging and fluorescence imaging. Significant preferential uptake of the chlorotoxin/fluorophore-labeled nanoparticle by glioma cells was identified by both MRI and fluorescence imaging. This multifunctional nanoprobe can be used to image resections of glioma brain tumors in real time and to correlate preoperative diagnostic images with intraoperative pathology at cellular-level resolution.
The probe was fabricated by coating iron oxide nanoparticles (NPs) with covalently-bound bifunctional polyethylene glycol (PEG) polymers that were subsequently functionalized with (a) a chlorotoxin (Cltx), a glioma tumor targeting molecule, and (b) a near infrared fluorescing (NIRF) molecule.
Unlike other targeting ligands that are specific only to certain types of glioma cells (e.g., epidermal grow factor receptor and vascular endothelial growth factor ligands), Cltx is a unique peptide shown to specifically target the vast majority of glioma tumors. Cltx is a small 36-amino acid peptide purified from the venom of the giant Israeli scorpion (Leiurus quinquestriatus). This peptide has been shown to bind with high affinity to the membrane-bound matrix metalloproteinase-2 (MMP-2) endopeptidase, which is preferentially upregulated in gliomas, medulloblastomas, and other tumors of the neuroectodermal origin. Use of small peptides such as Cltx overcomes the limitations of widely-used antibodies which are bulky and exhibit limited tissue penetration and cellular uptake when introduced in vivo. Conjugating small peptides on NPs can also overcome the limitation of the short half-life in blood and poor tissue retention generally associated with the peptides.
Use of an NIRF molecule minimizes autofluorescence interference from healthy brain tissue and allows visualization of tissues millimeters in depth due to the efficient penetration of photons in the near infrared range. PEG coatings were used to prevent nanoparticles from agglomeration and protein adsorption, and thus would increase particle blood circulation time and the efficiency of their internalization by targeted cells when introduced in vivo.
One of the major challenges in targeting brain tumors is the blood brain barrier (BBB) formed by intercellular tight junctions of the endothelial cells of the brain capillaries, which limits the access of drugs or targeting agents to the brain tissue. It has been demonstrated that the BBB may be overcome by particles with a size dimension smaller than about 50 nm or by lipid-mediated or receptor-mediated and paracellular transport processes, or combination of these processes. Thus, the PEG-linked nanoprobe of the present invention having a diameter of about 15 nm may promise the penetration of the nanoprobe across the BBB. Compared to dextran-coated nanoparticles currently used in MRI intraoperative examination for glioma resection, which label macrophages situated at the tumor boundary rather than the tumor cells themselves, the nanoparticle of the invention, by virtue of its covalently coupled chlorotoxin, directly targets tumor cells and thus, can potentially “follow” the cell migration to delineate the tumor boundary in real time. This is particularly beneficial for monitoring high-grade glioma cells that are highly invasive and quickly infiltrate the surrounding healthy tissue.
In one aspect, the present invention provides iron oxide nanoparticles (NPs) that have been conjugated with a chlorotoxin (Cltx) and a fluorescent compound (e.g., Cy.5.5) to create a multifunctional nanoprobe that specifically targets glioma cells and that is detectable both magnetically and optically. MRI and confocal fluorescence analysis show strong preferential uptake of the nanoparticles of the invention (e.g., NPC-Cy5.5) by glioma cells over control nanoparticles. A significantly higher degree of internalization of the nanoparticles of the invention by glioma cells versus control cells was observed indicating the preferential targeting abilities of the nanoparticles for gliomas. The high stability and prolonged retention (at least 24 hrs) of the nanoparticles of the invention within targeted cells as demonstrated by confocal imaging are particularly advantageous in intraoperative imaging applications as compared to conventional optical fluorophores conjugated to targeting biomolecules. The cellular-level resolution provided by the nanoparticles of the invention provide accurate delineation of otherwise poorly defined glioma interfaces resulting from their highly invasive morphology. The application of the nanoparticles of the invention for preoperative and postoperative diagnostic imaging with MRI and real-time intraoperative visualization of tumor margins with optical devices is a novel approach that will improve the effectiveness of diagnostic and therapeutic modalities available for brain tumor patients.
The following examples are provided for the purpose of illustrating, not limiting, the invention.
EXAMPLES Example 1 The Preparation of Representative Chlorotoxin-Labeled NanoparticlesIn this example, the preparation of representative chlorotoxin-labeled nanoparticles (e.g., a chlorotoxin-labeled nanoparticle referred to herein as “NPC”) of the invention is described.
Materials. All chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) and all reagents for cell culture were obtained from Invitrogen (Carlsbad, Calif.) unless otherwise noted. Chlorotoxin (Cltx) was purchased from Alomone (Jerusalem, Israel).
Synthesis of nanoparticle-PEG-chlorotoxin conjugates (NPC). A schematic illustration of the preparation is shown in
Dextran-coated NPs were synthesized as described in Molday et al., J. Immunol. Methods 1982, 52:353-367, expressly incorporated herein by reference in its entirety.
Cell culture. Rat gliosarcoma (9L, ATCC, Manassas, Va.) and rat cardiomyocytes (rCM, Cell Applications, San Diego, Calif.) cells were grown in DMEM and rCM medium respectively, supplemented with 1% streptomycin/penicillin and 10% fetal bovine serum FBS. On the day of experiment, cells were incubated with pre-warmed cell culture medium containing NPs. Following incubation, cells were washed twice with cell culture medium and twice with PBS, then trypsinized and suspended in a 4:1 (v/v) solution of PBS and cell culture medium.
Quantification of nanoparticle uptake. Nanoparticle uptake by cells was determined through the measurement of iron concentrations by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Cell samples were prepared by dissolving the cell pellets in 200 μl of concentrated hydrochloric acid (HC1) for 1 hr at 60° C. and then analyzed on a Jarrell Ash 955 ICP-AES spectrophotometer.
Magnetic resonance imaging of brain tumor cells. To examine the effectiveness of NPCs in targeting brain tumor cells, the internalization of NPCs by 9L cells, as compared to dextran coated nanoparticles, was evaluated first over a range of NP concentrations and then for various incubation times.
Phantom samples were prepared by suspending 106 cells in 50 μl of 1% low-melting agarose (BioRad, Hercules, Calif.). Cell suspensions were then loaded into a pre-fabricated 12-well agarose sample holder and allowed to solidify at 4° C. MR images were acquired using a 4.7-T Varian MR Spectrometer (Varian, Inc., Palo Alto, Calif.) and a Bruker magnet (Bruker Medical Systems, Karlsruhe, Germany) equipped with a 5 cm volume coil. A spin-echo multisection pulse sequence was selected. T2 values were obtained using a built-in Varian macro, “t2” fit program, to generate a T2 map of the acquired images. Then, the transverse relaxation rate R2 or 1/T2 values were calculated using the obtained T2 values.
MR images of 9L cells incubated with fixed nanoparticle concentrations over time (over a time period of 1-4 hrs) are shown in
Specificity of nanoparticle binding. To evaluate the specificity of NPC for tumor tissue as opposed to healthy tissue, the uptake of NPC by both 9L and freshly-isolated rCM cells that do not over-express MMP-2, was compared. The MR phantom image of 9L and rCM cells incubated with NPCs shown in
The therapeutic effect of free chlorotoxin is due to its ability to limit further expression of the surface-bound MMP-2 and inhibit its enzymatic activity, subsequently inhibiting tumor migration and invasion.
To evaluate the therapeutic potential of NPCs and retention of chlorotoxin therapeutic functionality after nanoparticle conjugation, a transwell migration assay was performed.
Transwell migration assay. The assay was performed by a method reported previously to assess the bioactivity of chlorotoxin. See Deshane et al., J. Biol. Chem. 2003, 278:4135-4144, expressly incorporated herein in its entirety. Prior to the experiment, matrigel invasion chamber inserts (BD Biosciences, Bedford, Mass.) of 8 μm pore size were rehydrated. The lower surfaces of the chamber inserts were immersed in 5 μg/ml solution of vitronectin for 8 hours. 9L cells were then plated at a density of 2.5×105 cells per chamber. Cells were treated with solutions of 4 μg/ml of chlorotoxin, and 10 μg Fe/ml of NPCs, respectively. The chambers were then incubated in a 37° C. humidified incubator maintained at 5% CO2 for 24 hrs. The cells on the upper inserts were scrubbed off, and the invaded cells were fixed and stained with Diff-Quik stain kit (IMEB INC., Chicago, Ill.). The filters were then detached from the inserts and mounted on glass slides using immersion oil and imaged on a Nikon E800 upright microscope. Relative inhibition of cell invasion was determined through reduction of migrating cells in presence of chlorotoxin and NPCs. Triplicates of each sample were prepared and cell counts were obtained from five random spots on each filter. The data was then averaged and presented as a relative number of invasive cells per treatment.
In this example, the preparation of representative chlorotoxin/fluorophore-labeled nanoparticles (e.g., a chlorotoxin/cyanine-labeled nanoparticle referred to herein as “NPC-Cy5.5”) of the invention is described.
Synthesis of representative iron oxide nanoparticles. Iron oxide (Fe3O4) nanoparticles were synthesized via a co-precipitation process of iron chlorides and sodium hydroxide. A 1.5 M sodium hydroxide solution was added dropwise to a deoxygenated solution of iron chloride with a Fe(II)/Fe(III) molar ratio of 0.5 under mechanical stirring and ultrasonication. The precipitation of nanoparticles occurred at pH of 12. The resulting black precipitate was then isolated with a rare-earth magnet and washed with deionized water until a pH of 10.5 was reached. The morphology and size distribution of nanoparticles were examined by transmission electron microscopy (Philips CM 100 TEM) at an accelerating voltage of 80 kV. TEM sample grids were prepared by dipping 300 mesh silicon-monoxide support films in the aqueous suspension of Fe3O4 nanoparticles. The samples were air-dried for 24 h prior to analysis.
Synthesis of representative chlorotoxin/fluorophore-labeled nanoparticles. A schematic illustration of the preparation is shown in
Referring to
The remaining terminal amines of the PEG coated nanoparticles (see
To functionalize chlorotoxin with sulfhydryl groups, a stock solution of chlorotoxin was prepared by dissolving 200 mg chlorotoxin in 200 μl of 50 mM bicarbonate buffer at pH 8.5. A solution of SATA (Molecular Bioscience, Boulder, Colo.) in anhydrous DMSO was prepared at a concentration of 1 mg/ml. Eight μl of the SATA solution was then added to the stock chlorotoxin solution, and the mixture was allowed to react for 3-4 hrs at 4° C. Following the reaction of SATA with chlorotoxin, the excess SATA was removed by dialysis against 50 mM bicarbonate buffer at pH 8.0 using regenerated cellulose membranes (3500 MWCO) in an equilibrium dialyzer (Spectrum Laboratories, Los Angeles, Calif.). Following dialysis the thiol groups of SATA-modified chlorotoxin were deprotected by addition of 30 μl of 25 mM hydroxylamine and 10 mM EDTA solution. The chlorotoxin solution was then incubated for 1 hr at room temperature. The resulting sulfhydryl modified chlorotoxin was then added to the iodoacetate modified particles at a molecular ratio of 50 to 1 and shaken overnight in an ice bath. Unreacted chlorotoxin was removed from the suspension through gel filtration chromatography using PD10desalting columns equilibrated with 20 mM sodium citrate, 0.15 M NaCl buffer at pH 8.0.
The degree of Cy5.5 labeling of NPs was controlled through stoichiometry and reaction conditions, and quantified by fluorescence spectroscopy. Emission intensity of a dilute sample of nanoparticle-Cy5.5 (50 μg of Fe/ml) at 689 nm was compared to a linear standard prepared using various concentrations of Cy5.5. The number of NPs was calculated on the assumption that each 10 nm NP (as determined from TEM analysis,
Similarly, the number of chlorotoxin peptides linked to each NP was quantified using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, Ill.), and NP concentrations were determined by inductive couple plasma atomic emission spectroscopy. From this analysis, the average number of Cltx molecules per NP was determined to be 10.2.
Example 5 Cellular Uptake of Representative Chlorotoxin/Fluorophore-Labeled NanoparticlesIn this example, the cellular uptake of representative chlorotoxin/fluorophore-labeled nanoparticles of the invention is described.
Confocal fluorescence microscopy and MR imaging were utilized to monitor the cellular uptake of the nanoparticles. Rat cardiomyocytes (rCM, Cell Applications, San Diego, Calif.) were grown in rat cardiomyocyte cell culture media. 9L glioma cells (American Type Culture Collection, Manassas, Va.) were grown in DMEM medium formulation with high glucose supplemented with sodium pyruvate, 1% streptomycin/penicillin and 10% FBS (Invitrogen, Carlsbad, Calif.). Trypan Blue staining was used to determine cell density and viability, and cell counts were obtained using a hemocytometer.
For confocal imaging experiments, 2×105 cells were seeded on cover slips 24 hrs prior to labeling and staining. Cells were cultured with nanoparticle conjugates for 1 hr in a 37° C. humidified incubator maintained at 5% CO2. Following labeling, the cover slips were washed twice with cell culture medium and twice with PBS buffer. After washes, cellular membranes were stained with FM 1-43FX (Molecular Probes, Eugene, Oreg.). Cells were incubated in a 1 μM solution of FM 1-43FX for 20 min at room temperature and washed twice with PBS. The cells were then fixed using a 4% paraformaldehyde solution. Following fixation, cellular nuclei were stained with 4′, 6-diamidino-2-phenyindole (DAPI) according to the manufacturers' instructions (Sigma Aldrich, St. Louis, Mo.). Confocal images were acquired using a DeltaVision SA3.1 Wide-field Deconvolution Microscope (Applied Precision, Inc., Issaquah, Wash.) equipped with DAPI, TRITC, and Cy5 filters. Image processing was performed using SoftWoRX (Applied Precision, Inc., Issaquah, Wash.). Using this software the fluorescence intensity of the images was normalized.
For MR phantom imaging, samples were prepared by suspending 106 cells in 50 μl of 1% low-melting agarose (BioRad, Hercules, Calif.). Cell suspensions were loaded into a pre-fabricated 12-well agarose sample holder and allowed to solidify at 4° C. MR imaging was performed using a 4.7-T Bruker imager (Bruker Medical Systems, Karlsruhe, Germany) equipped with a 5 cm volume coil. A spin-echo multisection pulse sequence was selected to acquire MR phantom images. Repetition time (TR) of 3000 msec and variable echo times (TE) of 15-90 msec were used. The spatial resolution parameters were as follows: an acquisition matrix of 256×128, field of view of 4×4 cm, section thickness of 1 mm, and 2 averages. Regions of interest (ROIs) of 5.0 mm in diameter were placed in the center of each sample image to obtain signal intensity measurements using NIH ImageJ. T2 values were obtained using VnmrJ “t2” fit program to generate a T2 map of the acquired images.
To evaluate the specificity of NPC for 9L cells, 9L rat glioma cells were cultured with NP-Cy5.5 and NPC-Cy5.5, and their fluorescence confocal images are shown in
To evaluate the specificity of NPC for 9L cells versus non-cancerous freshly isolated cells (control cells) that lack MMP-2 expression, NPCs at a concentration of 100 μg Fe/mL were incubated with 2×1059L and rat cardiomyocyte (rCM) cells, respectively.
To ascertain that NPC-Cy5.5 was indeed internalized by cells rather than binding to cellular membranes, 9L cells cultured with NPC-Cy5.5 were examined sectionally by confocal fluorescence microscopy at different depths.
While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
Claims
1. A chlorotoxin-labeled particle, comprising:
- (a) a core having a surface, the core comprising a material having magnetic resonance imaging activity;
- (b) a chlorotoxin; and
- (c) a linker covalently coupling the chlorotoxin to the surface.
2. The particle of claim 1, wherein the material having magnetic resonance imaging activity comprises a metal oxide selected from the group consisting of ferrous oxide, ferric oxide, silicon oxide, polycrystalline silicon oxide, aluminum oxide, germanium oxide, zinc selenide, tin dioxide, titanium dioxide, indium tin oxide, gadolinium oxide, and mixtures thereof.
3. The particle of claim 1, wherein the core comprises a material selected from the group consisting of silicon nitride, stainless steel, titanium, nickel titanium, and mixtures thereof.
4. The particle of claim 1, wherein the chlorotoxin is selected from the group consisting of native chlorotoxin, synthetic chlorotoxin, recombinant chlorotoxin, and fragments and variants thereof having chlorotoxin binding activity.
5. The particle of claim 1, wherein the chlorotoxin-labeled particle has from about 1 to about 50 chlorotoxins/particle.
6. The particle of claim 1, wherein the linker comprises a polyalkylene oxide moiety.
7. The particle of claim 1, wherein the linker comprises a polyethylene oxide moiety.
8. The particle of claim 1, wherein the chlorotoxin-labeled particle further comprises a fluorescent moiety.
9. The particle of claim 8, wherein the chlorotoxin-labeled particle has from about 1 to about 10 fluorescent moieties/particle.
10. The particle of claim 8, wherein the fluorescent moiety is selected from the group consisting of near infrared emitting fluorescent moiety.
11. The particle of claim 8, wherein the fluorescent moiety is a cyanine moiety.
12. The particle of claim 8, wherein the fluorescent moiety is a Cy5.5 moiety.
13. The particle of claim 1, wherein the core has a diameter of from about 2 mn to about 25 nm.
14. The particle of claim 1, wherein labeled particle has a diameter of from about 15 nm to about 200 nm.
15. A pharmaceutical composition, comprising a chlorotoxin-labeled particle of claim 1 and a pharmaceutically acceptable carrier.
16. A pharmaceutical composition, comprising a pharmacologically effective amount of a chlorotoxin-labeled particle of claim 1 to treat a patient suffering from a glioma.
17. A method for differentiating neuroectodermal-derived tumor cells from non-neoplastic brain tissue, comprising:
- (a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for neuroectodermal-derived tumor cells; and
- (b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
18. A method for detecting neuroectodermal-derived tumor cells in a patient, comprising:
- (a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for neuroectodermal-derived tumor cells; and
- (b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative that the tissue is neoplastic.
19. A method for detecting a tissue expressing matrix metalloproteinase (MMP-2) protein complex, comprising:
- (a) contacting a tissue of interest with a chlorotoxin-labeled nanoparticle having affinity and specificity for matrix metalloproteinase (MMP-2) protein complex; and
- (b) measuring the level of binding of the chlorotoxin-labeled nanoparticle, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of a tumor expressing matrix metalloproteinase (MMP-2) protein complex.
20. The method of claim 17, wherein measuring the level of binding of the chlorotoxin-labeled nanoparticle comprises magnetic resonance imaging.
21. The method of claim 17, wherein the chlorotoxin-labeled nanoparticle further comprises a fluorescent moiety.
22. The method of claim 21, wherein measuring the level of binding of the chlorotoxin-labeled nanoparticle comprises fluorescence imaging.
23. A method for determining the location of glioma cells in a patient preoperatively, intraoperatively, and postoperatively, comprising:
- (a) administering a pharmaceutical composition to a patient, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an amount of a fluorophore/chlorotoxin-labeled nanoparticle sufficient to image glioma cells in vivo;
- (b) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by magnetic resonance imaging pre-operatively to determine the location of glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioma cells;
- (c) surgically removing from the patient at least some glioma cells located by magnetic resonance imaging;
- (d) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by fluorescence imaging intra-operatively to determine the location of residual glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of residual glioma cells;
- (e) surgically removing from the patient at least some residual glioma cells located by fluorescence imaging; and
- (f) measuring the level of binding of the fluorophore/chlorotoxin-labeled nanoparticle by magnetic resonance imaging post-operatively to determine the location of glioma cells, wherein an elevated level of binding, relative to normal tissue, is indicative of the presence of glioma cells.
24. A method for treating a glioma in a patient, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle of claim 1 and a pharmaceutically acceptable carrier.
25. A method for treating a neuroectodermal tumor, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle of claim 1 and a pharmaceutically acceptable carrier.
26. A method for treating a tumor expressing matrix metalloproteinase (MMP-2) protein complex, comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle of claim 1 and a pharmaceutically acceptable carrier.
27. A method for inhibiting invasive activity of neoplastic cells, comprising administering to neoplastic cells an effective amount of a pharmaceutical composition comprising a chlorotoxin-labeled nanoparticle of claim 1 and a pharmaceutically acceptable carrier.
Type: Application
Filed: Nov 14, 2006
Publication Date: Jul 5, 2007
Inventors: Miqin Zhang (Bothell, WA), James Olson (Seattle, WA), Raymond Sze (Mercer Island, WA), Richard Ellenbogen (Seattle, WA), Omid Veiseh (Kirkland, WA), Conroy Sun (Kirkland, WA), Jonathan Gunn (Seattle, WA)
Application Number: 11/600,004
International Classification: G01N 33/574 (20060101);