A METHOD FOR DELIVERING A SUBSTANCE INTO CELLS

Abstract

A method for delivering a substance into cells, wherein the method comprises the following steps: a) providing the substance, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group, b) providing cells; and c) contacting the substance with the cells; wherein the method comprises the additional step of—adjusting a p H in an extracellular fluid to a p H of at least 7.7.

Description

This invention was made with United States Government support under Grant No. R01-GM086801 awarded by the National Institutes of Health to the Rensselaer Polytechnic Institute. The United States Government has certain rights in this invention.

The invention relates to a method for delivering a substance into cells; a composition comprising a substance to be delivered to cells, particularly a skin treatment composition, more particularly in form of a cream, a gel, an unguent, a lotion, a spray, a solution, in an emulsion, in liposomes or in microcapsules; a use of the composition for treatment and/or prevention of a disease, diagnosis of diseases, as a research tool, as a targeting system, as a pharmaceutical composition or as a cosmetic composition; a use of the composition in the preparation of a medicament for treatment, prevention and/or diagnosis of a disease; a method for manufacturing the composition; and a kit comprising the composition.

Cell-penetrating peptides (CPP) consist of short amino acid sequences, usually rich in arginine amino acids, able to penetrate into almost any cell type, carrying with them cargoes such as proteins, oligonucleotides, and drugs. These short sequences are capable of directly crossing the cell membrane in a seemingly energy-independent manner. A fundamental question since the discovery of these peptides has been how they do cross the plasma membrane to reach freely the interior of cells. There is abundant evidence suggesting two main pathways: direct translocation across the plasma membrane and endocytosis. Both pathways are described in general terms with fundamental questions left unanswered. The most fundamental questions, at the heart of either pathway, are (a) how do these highly cationic peptides cross the membrane hydrophobic barrier and (b) what mechanism breaks the CPPs-, particularly RRPs-membrane strong ionic binding, once in the cytosol, detaching these peptides from the plasma or endosomal membrane.

Arginine-rich peptides (RRPs) are highly cationic and therefore hydrophilic. To directly cross the plasma membrane, or the endosomal membrane, and diffuse freely in the cytosol these peptides must somehow overcome the strong barrier imposed by biological membranes. This membrane barrier has two distinctive components for RRPs, on one hand the peptides need to cross the hydrophobic barrier and on the other hand they need to detach from membranes overcoming their strong electrostatic binding affinity for membrane components. Recently, a mechanism on how these peptides could directly cross biological membranes and transport other molecules was proposed. This mechanism suggests that RRPs induce the nucleation of small transient toroidal pores and then diffuse through these pores towards the cytosolic side of the membrane. A transient pore formation explains how these peptides are able to directly cross into cells carrying along a wide range of cargoes.

However, a critical step for the nucleation of a pore in the known model involves the membrane insertion of arginine amino acids to initiate the nucleation of a toroidal pore. The cost of insertion of these arginine-rich peptides into model phospholipid bilayers seems too high (˜200 KJ/Mol). Therefore, this event might be too rare to be effective unless other factors help reduce this energetic cost. Furthermore, this mechanism does not explain how the peptides detach from the membrane after the RRPs cross towards the cytosolic side of the membranes.

Early in vitro studies have suggested how RRPs could be absorbed into a hydrophobic environment. These studies have shown that arginine-rich peptides can be absorbed into a hydrophobic environment by binding to negatively charged amphiphilic molecules forming a less polar or more hydrophobic complex. It has been shown that some of the molecules that can complex with guanidinium groups in this way are amphiphilic anions with sulfur, phosphate or carboxyl groups, which are able to form bidentate hydrogen bonding with guanidinium groups. It has been shown that these molecules can drive arginine-rich peptides into a hydrophobic environment such as chloroform and octanol. Using a typical, yet elegant, “U-tube” experiment with chloroform as a hydrophobic barrier between two buffer compartments, it has been shown that these complexes can mediate the transfer of reporter hydrophilic dyes across the hydrophobic phase from the cis buffer to the trans buffer. A similar flux of reporter dyes across vesicles containing amphiphilic anions was also observed. This suggests that these peptides might bind to amphiphilic anions forming complexes, which resemble inverted micelles, and that this complex can diffuse within hydrophobic environments. Consequently, the interior of these inverted micelles being hydrophilic might capture and transport dyes across the hydrophobic barrier. An important aspect is that these experiments report the flux of dyes between two buffers separated by a hydrophobic barrier, not the flux of the RRPs themselves, which most of them are likely to just remain trapped in the hydrophobic environment. Another important aspect of this in vitro phenomenon is that it occurs spontaneously without the requirement of an external electrostatic potential.

In cells, on the other hand, it has been shown that the transmembrane potential is required for the cellular uptake of cell-penetrating peptides. This membrane potential might be critical to reduce the plasma membrane barrier. However, since the internal side of the bilayer should be even more negative that the external side to maintain a negative potential this should increase even further the affinity of the peptides towards the bilayer. This opens the question of what mechanism allows the peptides to get released from the plasma membrane after crossing to the cytosolic side.

Therefore, even if the peptides are somehow able to reach the cytosolic side of membranes there should be in place a mechanism to release the peptides from the membranes. A distinctive aspect of these peptides is their extremely high affinity for negatively charged membrane components. It has been shown that this ionic binding is so strong that regular cell washes cannot remove the plasma membrane bound peptides. Removal of these peptides from the external side of the plasma membrane requires degrading them, a step usually done by trypsinization. Moreover, it has been suggested that not even degradation by trypsinization is effective enough and that the addition of strong counterions such as heparin to the wash solution is required to competitively bind to guanidinium groups and remove the membrane bound degraded peptides. The transmembrane potential would in principle favor an even tighter peptide-membrane binding in the cytosolic side of the plasma membrane than in the exterior of the cell. Therefore, a fundamental step in the uptake pathway is the mechanism under which the peptides detach from the cell plasma membrane or the endosomal membrane to freely diffuse into the cytosol and reach the nucleus after reaching the cytosolic side of these membranes.

With respect to the described limitations of delivering substances such as peptides into cells, it is an object of the present invention to provide a method for delivering efficiently various substances into cells.

These and other problems are solved by the subject matter of the attached independent claims.

The above objects of the invention are achieved by a method for delivering a substance into cells according to claim 1, a composition comprising a substance to be delivered to the cells according to claim 19, a use of the composition according to claim 25, a use of the composition according to claim 26, a method for manufacturing the composition according to claim 27, and a kit according to claim 28.

Preferred embodiments may be taken from the dependent claims.

The present inventors have surprisingly found how to use the pH gradient across the plasma membrane of a target cell and thereby to increase the efficiency of delivery of a substance into target cells. Particularly, providing a high extracellular pH advantageously allows binding of compounds having at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue to extracellular cell-penetrating compounds (particularly CPPs), which comprise a basic amino functional group, (particularly at least one guanidinium group), and efficiently mediate the membrane transport of the cell-penetrating compounds and their release into the lower-pH environment of the cytosol. In the context of the present invention it is important to understand that the basic amino functional group of the cell-penetrating compound may particularly be part of a guanidinium functional group, for example of RRPs. Thereby, the compounds with the carboxyl or carboxylate functional group may particularly be represented by the deprotonated fatty acids of the target cell's membrane and/or, optionally, added as a mediator compound, which allows further enhancement of the efficiency of delivery. The cell-penetrating compound thereby may particularly comprise various cargoes such as small molecules, nucleic acids, peptide nucleic acids, proteins, oligonucleotides, inorganic particles, and liposomes, biomarkers, drugs and/or medically, pharmaceutically or cosmetically active substances.

According to the object of the invention, the present invention provides a method for delivering a substance into cells comprising the features of claim 1. Said method comprises at least the following steps:

• • a) providing the substance, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group,
  • b) providing cells; and
  • c) contacting the substance with the cells;
    wherein the method comprises the additional step of
  • adjusting a pH in an extracellular fluid to a pH of at least 7.7, preferably of at least 7.8.

Thereby it is preferred that the extracellular fluid contains the substance, and particularly the cell-penetrating compound. Preferably, a step a) of providing the substance comprises providing the substance in an extracellular fluid.

Preferably, in step c) of contacting the substance with the cells, said contacting comprises delivering the substance into the cell. In other words, the method of the present invention preferably provides a pH in the extracellular fluid, which is advantageously enhancing the delivery of the substance into the cell, if the substance is contacted with the cells.

In some embodiments of the method according to the present invention, the method further comprises a step of applying the substance to a subject, wherein the method is a cosmetic or therapeutic method.

In some embodiments of the method, step b) of providing cells is consisting of providing a suspension of cells, and in step c) contacting the substance with the cells is consisting of creating a cell-substance-suspension by combining the substance with the suspension of cells, and wherein the pH in an extracellular fluid is an extracellular pH in the cell-substance-suspension. Preferably, the extracellular fluid is the cell-substance suspension.

In an embodiment the method may further comprise in step a) providing a suspension or solution of the substance in a pH buffer, particularly in an extracellular fluid being a pH buffer.

In connection with the present invention it is to be understood that the substance to be delivered may be advantageously delivered to nearly any type of cell. The cell may be a eukaryotic or a prokaryotic cell. The cell may be selected from the group comprising animal or human cells, mammalian cells, plant cells, bacterial cells or insect cells. Particularly a cell may also constitute a part of a multi-cellular organism, in other words, a transgenic or non-transgenic organism comprising at least one such cell. Particularly, some embodiments of the present invention comprise the use of the composition and/or methods according to the present invention for treatment and/or prevention of a disease, diagnosis of diseases, and/or as a pharmaceutical composition or as a cosmetic composition. In such embodiments the cell may also be part of a multi-cellular organism represented by a subject subjected to said method or use, particularly a mammal, more particularly a human.

In some embodiments the method comprises creating a cell-substance-suspension by combining the substance with the cells, particularly the suspension of cells.

According to the invention, the substance comprises a cell-penetrating compound comprising a basic amino functional group, particularly at least one guanidinium group and/or at least one amino group. Preferably, the cell-penetrating compound comprises at least one guanidinium group and, optionally, at least one amino group. Preferably, the cell-penetrating compound is selected from peptides, proteins, carbohydrates, lipids and nucleic acids, oligonucleotides, peptide nucleic acids, inorganic particles, biomarkers, drugs, silica nanoparticles, particularly silica nanoparticles having a cubic structure, wherein amino groups are provided on the vertices of the cube. It is believed that at high extracellular pH the cell-penetrating compound binds to plasma membrane molecules containing acidic groups such as e.g. fatty acids already present in the cell membrane thereby enhancing uptake significantly. It is to be understood that in most mammalian cells the extracellular pH is about 7.4.

According to the invention, the extracellular pH is the pH in the extracellular fluid (ECF) outside of cells or in general terms the pH which is measurable outside of cells, such as e.g. the pH of the substance or composition, the pH outside of cells of the skin, particularly the pH of the skin of humans. The pH within cells is called intracellular pH.

The substance can be or be provided as a homogeneous or heterogeneous solution of various cell-penetrating compounds, particularly guanidinium-rich compounds selected from peptides, proteins, carbohydrates, lipids and nucleic acids oligonucleotides, peptide nucleic acids, inorganic particles, biomarkers and drugs, silica nanoparticles, particularly silica nanoparticles having a cubic structure, wherein amino groups are provided on the vertices of the cube. It will be understood that the method can also be used to deliver any combination of the aforementioned compounds into cells. The solution may be a fluid, preferably a liquid or a gel such as an aqueous buffer solution. A pH buffer solution is an aqueous solution containing a mixture of a weak acid and its conjugate base, or vice versa.

According to the invention, a higher pH is preferred. According to the invention, the pH of the composition and/or the extracellular fluid and/or the suspension of cells is adjusted to a pH of at least 7.7, preferably of at least 7.8, still preferably of at least 8.0, still preferably of at least 8.5, more preferably of at least 8.75, most preferably of at least 9.0. Preferably, the pH is at maximum 9.5, preferably at maximum 10.0, more preferably at maximum 11.0, still more preferably at maximum 12.0. Any suitable buffer may be used for this purpose. Preferably, a pH buffer and/or a buffering agent may be added to the suspension of cells to adjust the pH. A buffering agent can particularly be either a weak acid or weak base or a chemical formulation such as magnesium oxide or calcium carbonate that is added to the cells, particularly the suspension of cells, to form a buffered solution with the desired pH. In some embodiments, the method and/or composition of the present invention is for treatment and/or prevention of a disease, diagnosis of diseases, as a pharmaceutical composition or as a cosmetic composition. Such methods may particularly in a step c) of contacting the substance with the cells, comprise a step of application of the composition and/or substance to a subject. Thereby it is preferred that the substance and/or the composition and particularly a buffer is non-irritant and not toxic.

According to the invention, the cell-penetrating compound comprises a basic amino functional group. Amino functional groups contain a basic nitrogen atom with a lone electron pair. Preferably, the basic amino functional group of the cell-penetrating compound is part of a guanidinium functional group. It is to be understood that the present inventors have found that a less number of —NH₂ groups can be compensated by an increased number of guanidinium functional groups. Accordingly, the term “basic amino functional group” as used herein, preferably comprises —NH₂ group or guanidinium functional group, or mixtures thereof. In a preferred embodiment, the cell-penetrating compound is a peptide or a protein comprising a basic amino acid selected from arginine and lysine. Preferably, the number of basic amino functional groups is at least 1, still preferably at least 2, more preferably at least 5, at least 6, at least 7, most preferably at least 8 per 10 nm³ molecule of the cell-penetrating compound. Preferably, the number of basic amino functional groups is at least 1, still preferably at least 2, more preferably at least 5, at least 6, at least 7, most preferably at least 8 per molecule of the cell-penetrating compound. The cell-penetrating compounds of the substance that are ought to be delivered into cells can also be described as biological oligomers. Particularly, the cell-penetrating compound can be a peptide, particularly a CPP, more particularly an RRP. The peptide may comprise a D-isomer or an L-isomer. Peptides which comprise polypeptides and oligopeptides are distinguished from proteins on the basis of size, and as an arbitrary benchmark can be understood to contain approximately 50 or less amino acids, However, also other length of peptides are considered to be in the scope of the present invention. Preferably, a cell-penetrating compound according to the present invention in the form of a peptide comprises 150 or less amino acid residues, 125 or less amino acid residues, more preferably 105 or less amino acid residues. Preferably, a cell-penetrating compound according to the present invention in the form of a peptide comprises at least 85 amino acid residues. In an embodiment the cell-penetrating compound is or comprises a cyclic peptide. The use of a cyclic peptide may advantageously enhance the cellular uptake. Proteins consist of one or more polypeptides arranged in a biologically functional way. Either proteins or peptides can be bound to ligands such as coenzymes and cofactors, or to another protein or macromolecule (DNA, RNA, polysaccharides), or to complex macromolecular assemblies.

Another preferred cell-penetrating compound comprises a DNA repair enzyme comprising a basic amino functional group. A particularly preferred DNA repair enzyme is photolyase that is useful for repairing damage caused by exposure of the cells to ultraviolet light. It is believed that photolyase is rich in guanidinium and amino groups.

According to another preferred embodiment, the cell-penetrating compound comprises a capping agent, and/or the method further comprises the step of adding a capping agent, preferably to the cell-penetrating compound. Such capping agent may particularly comprise a guanidinium group when using a cell-penetrating compound comprising a peptide or a protein. The capping agent binds covalently or non-covalently to the N-terminus of the peptide or the protein. The additional guanidinium group provided by the capping agent enhances the affinity of the peptide or the protein for the cell membrane and therefore facilitates cellular uptake. A preferred capping agent is 4-guanidinobutyric acid.

The method may also employ cell-penetrating compounds such as carbohydrates, lipids and nucleic acids. Nucleic acids are for example DNA or RNA, including messenger RNA, which DNA or RNA may be single stranded or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. Also a combination of peptides, proteins carbohydrates, lipids and/or nucleic acids can be described as a substance to be delivered into cells.

According to a further aspect of the present invention, the method comprises the step of adding to the substance or cell-substance mix, particularly to the cell-substance-suspension- and/or to an extracellular fluid, preferably containing the substance, a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue.

The term “hydrophobic residue” as used herein, preferably means a carbon containing backbone, preferably of at least 8 C-atoms. Preferably, a carbon containing backbone means a carbon-chain of at least 8 C-atoms. Thereby, said carbon containing backbone and/or carbon chain, may be partially or completely saturated or unsaturated.

It is believed that at high extracellular pH the mediator compound comprising a carboxylate functional group binds the cell-penetrating compound comprising a basic amino functional group, mediates their membrane-transport across the hydrophobic core of the cell membrane and releases into the lower pH environment of the cytosol. This results in a very efficient way of delivering the substance into virtually any cell. It is immediately understood by a person skilled in the art that also the cell to which the substance is to be delivered may comprise compounds comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue, for example, in the form of fatty acids of the cell membrane. However, the addition of a mediator compound can advantageously increase the efficiency of the delivery.

The mediator compound comprising a carboxyl/carboxylate functional group attracts and binds arginine and/or lysine rich cell-penetrating peptides or proteins and carries these peptides or proteins across the cell membrane into the cytosol.

It is assumed that the cell-penetrating compound interacts with the cell membrane, thereby reducing the energetic cost of insertion of the substance into the plasma membrane. The mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue which is added to the cell-substance-suspension is preferably an anionic amphiphilic molecule that electrostatically binds to the substance to be delivered into the cells.

Typical mediator compounds display high solubility in 1-octanol and low solubility in water. Preferably, the distribution of a mediator compound in a mixture of water and 1-octanol is more than 50 mol-% in the 1-octanol phase, most preferred more than 80 mol-% in the 1-octanol phase. The hydrophobicity of the mediator compound may be enhanced by coupling a functional group present in the mediator compound, such as an amine group, to a protective group such as Fmoc, e.g. 12 Ado-OH:

Preferred mediator compounds are compounds that are naturally occurring in cell membranes, such as fatty acids. Fatty acids increase the fluidity of membranes and can be both negatively charged and neutral within a physiological pH range. Particularly preferred mediator compounds are saturated and non-saturated fatty acids, saturated and non-saturated hydroxy fatty acids, dicarboxy acids, aromatic acids and salts thereof. A mediator compound preferably has a pKa of 2 or more, 3 or more, 4 or more, 5 or more, more preferably of 7 or more, most preferably of 8 or more. The pKa is preferably at most 12, more preferred at most 11, most preferred at most 10.

Particularly, the molar ratio of mediator compound per cell-penetrating compound depends on the desired mediator compound and the particular cell-penetrating compound. In a preferred embodiment the molar ratio of mediator compound to cell-penetrating compound is at least 1.0, i.e. one mediator per cell penetrating compound. In other words, the ratio of mediator compound to cell-penetrating compound preferably is 1 or more, still preferably 1.5 or more, 2.0 or more, 2.5 or more, more preferably 3.0 or more, 4.0 or more, most preferably 5.0 or more.

In some embodiments of the method and the composition of the present invention various cargoes such as small molecules, nucleic acids, peptide nucleic acids, proteins, oligonucleotides, inorganic particles, and liposomes are to be delivered to the cell to mediate a desired effect, e.g. a therapeutic, medical or cosmetic effect, as a biomarker, drug and/or a medically, pharmaceutically or cosmetically active substance. Such cargoes as used herein may particularly be comprised in the cell-penetrating compound and/or added as a, or a part of a mediator compound.

Most preferred mediator compounds, which particularly may be useful cargoes, are compounds having a desired effect, e.g. a therapeutic, medical or cosmetic effect, in the cell, such as artemisinin and derivatives, such as artesunate, folic acid, levomefolic acid, levothyroxine, curcumin, (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, atorvastatin, (3R,5R)-7-[2-(4-Fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid, S-Adenosyl methionine, (2S)-2-Amino-4-[[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl-methylsulfonio]butanoate, salicylic acid, 2-Hydroxybenzoic acid, lipoic acid, (R)-5-(1,2-Dithiolan-3-yl)pentanoic acid, 5,10-methylenetetrahydrofolate, D-α-tocopherol succinate, a tocopheryl acid succinate, ibuprofen, dexibuprofen, and salts thereof. Further preferred mediator compounds are peptidomimetics comprising a hydrophobic backbone and a carboxyl functional group, biphenyl-4-carboxylic acid, benzoic acid, ricinoleic acid, (2E)-18-hydroxyoctadec-2-enoic acid 18-hydroxyoctadec-9-enoic acid, 6-hydroxyhex-3-enoic acid, trans-3-hydroxyhex-4-enoic acid, (E)-10-hydroxydec-2-enoic acid, (2E)-9-hydroxydec-2-enoic acid, 8-hydroxyoctanoic acid, 9-hydroxynonanoic acid, 11-hydroxyundecanoic acid, 12-hydroxydodecanoic acid, 15-hydroxypentadecanoic acid, 16-hydroxyhexadecanoic acid, 17-hydroxyheptadecanoic acid, 2-hydroxyheptadecanoate, (5Z,8Z,11Z,14Z)-19-hydroxynonadeca-5,8,11,14-tetraenoic acid, (10E,12E)-14-hydroxy-10,12-nonadecadienoic acid, 20-hydroxyeicosatetraenoic acid, (5Z,8Z,10E,14Z)-12-hydroxyicosa-5,8,10,14-tetraenoic acid, 16-Hydroxyhenicosa-2,4,6-trienoic acid, 18-bromo-16-hydroxytricosa-8,17,19-trien-4,6-diynoic acid, 12-Ado-OH, ascorbic acid, azelaic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, (6aR,11aS,11bR)-10-Acetyl-11-hydroxy-7,7-dimethyl-2,6,6a,7,11a,11b-hexahydro-9H-pyrrolo[1′,2′:2,3]isoindolo[4,5,6-cd]indol-9-one and salts thereof.

Generally, the interior of cells is maintained at a lower pH than the exterior. This pH gradient across the cell plasma membrane can probably increase the deprotonation of above-mentioned acids in the extracellular layer of the plasma membrane at higher pH. Presumably, the mediator compound neutralizes the charge of the substance and reduces the energetic cost of the insertion of the substance into the hydrophobic core of the lipid bilayer of the cell plasma membrane. This insertion leads to the formation of a channel across the cell plasma membrane facilitating the transport of the substance across the cell plasma membrane. It was surprisingly found that the binding affinity between the mediator compound and the substance to be delivered into cells is reduced when the substance has reached the cytosolic side of the membrane. It is believed that in contact with the lower cytosolic pH, the mediator compound becomes protonated and neutrally charged, the substance is released from the plasma membrane into the cytosol and the channel closes. Experiments suggest that also the mediator compound is released into the cytosol to a significant extent, thus enabling the transport of therapeutically effective mediator compounds into the cytosol.

In a particularly preferred embodiment, the cells are pre-incubated with the mediator compound before the cell-substance-suspension is created. It was surprisingly observed that cells enriched with the mediator compound displayed enhanced substance uptake efficiency. It is assumed that this effect is due to enrichment of the plasma membrane with the mediator compound. Alternatively or additionally, the substance can be pre-incubated with the mediator compound before the cell-substance-suspension is created.

The substance that is to be delivered into cells, particularly the cell-penetrating compound, can also be a cell-penetrating peptide, particularly an RRP. Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various additional agents, particularly cargo molecules, such as nanosize particles, small chemical molecules and fragments of DNA. The additional agent is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions. Cell-penetrating peptides typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of cell-penetrating peptides are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups.

In a preferred embodiment, in order to use the substance for transporting an additional agent, for example DNA, RNA or a peptide into cells, the cell-penetrating compound comprises a linker moiety. The linker moiety is preferably a peptide or a nucleotide linker that is able to conjugate the additional molecule covalently or non-covalently conjugated to the cell-penetrating compound. Typical additional agents are selected from small molecules, nucleic acids, peptide nucleic acids, peptides, proteins, nucleotides, oligonucleotides, inorganic particles and liposomes. Preferably, the additional agent comprises at least one carboxyl functional group or carboxylate functional group. In case of peptides or proteins, the additional agent comprises at least one acidic amino acid selected from aspartate/aspartic acid and glutamate/glutamic acid. Particularly, the additional agent can be selected from the group comprising biomarker, drug, medically, pharmaceutically and cosmetically active substances.

In another aspect, the invention relates to a composition comprising a substance to be delivered to the cells, according to claim 19. In connection therewith it is to be understood that any feature described herein with regard to the method of the method according to the present invention may also be a feature of the composition of the present invention and vice versa.

In a composition according to the present invention the substance comprises a cell-penetrating compound comprising a basic amino functional group, and wherein the pH of the composition is at least 7.7, preferably at least 7.8. In an embodiment the cell-penetrating compound is selected from the group comprising peptides, proteins, carbohydrates, lipids and nucleic acids. In some embodiments the composition comprises a suspension of the substance in pH-buffer, particularly in an extracellular fluid being a pH buffer. The composition may also comprise a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue. Particularly, the composition can be provided in the form of a cream, a gel, an unguent, a lotion, a spray, an aerosol, a solution, in an emulsion, in liposomes or in microcapsules. Particularly the composition provided for skin treatment. Particularly, the present invention provides a skin treatment composition in a form of a cream, a gel, an unguent, a lotion, a spray, an aerosol, a solution, in an emulsion, in liposomes or in microcapsules, wherein the composition comprises a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue, a substance to be delivered to the cells of the skin and a pH buffer, wherein the pH of the composition is at least 7.7, preferably at least 7.8. The skin treatment composition allows for uptake of substances and preferably additional active agents bound to the substance into the skin cells thereby providing a desired effect.

The composition of the present invention may be used for the treatment and/or prevention of a disease, diagnosis of diseases, as a research tool, as a targeting system, as a pharmaceutical or cosmetic composition. Preferably, the substance to be delivered to the cells comprises a pharmaceutically active compound. However, the substance can also be used as a carrier for delivering an additional pharmaceutical active agent, such as DNA, a protein, a peptide or another biomolecule to the cells. In this case, the additional active agent is covalently or non-covalently linked to the substance as described above. A pharmaceutical active agent is a compound contained in a pharmaceutical drug that is biologically active.

Furthermore, it is preferred that the composition comprises a pharmaceutical acceptable carrier, filler, bulking agent, disintegrant, stabilizer, binder, humectant, extender, emulsifying agent, dissolution retarder, absorption enhancer, preservative, antioxidant, wetting agent, adsorbent, lubricant or a combination thereof. The term “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a mammal in need of the active compound. The term “pharmaceutically acceptable carrier” as used herein means any material or substance present in a formulation in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness.

As a result, the composition can be adjusted to the specific requirement of multiple biologically different diseases. Particularly, the concentration or amount of cell-penetrating compound can be adjusted to the desired needs and in order to achieve the desired effect. For example, the concentration or amount of cell-penetrating compound can be adjusted to allow for efficient transport into skin cells.

In some embodiments the composition of the present invention is for the use in the application to a subject, e.g. for the treatment and/or prevention of a disease, diagnosis of diseases, as a pharmaceutical or cosmetic composition. In such embodiments, the composition may be administered to the subject by applying the composition of the present invention to the subject. Particularly, with regard to the application to the skin, the composition may be applied to the skin and thus to the skin cells. However, other forms of application are also considered herein, for example, the composition may be locally injected at a tumor site. Accordingly, a method of the present invention may comprise a step of applying the composition to a subject. Particularly, said application step may be part of step c) of contacting the substance with the cells—depending on the desired application. For example, a cosmetic treatment of the skin may comprise application of the composition to the skin, in the form of a cream, a gel, an unguent, a lotion, a spray, an aerosol, a solution, in an emulsion, in liposomes or in microcapsules, and thereby contacting the composition, particularly the substance, with the skin cells.

The active compound can thereby be delivered specifically to the desired locations, for example the skin of a subject. Examples of types of active compounds that may be transported to the target cells, particularly skin cells, by the composition comprise analgesics, anesthetics, antianginals, antifungals, antibiotics, anticancer drugs, anti-inflammatories, anthelmintics, antidepressants, antidotes, antiemetics, antihistamines, antihypertensives, antimalarials, antimicrotubule agents, antimigraine agents, antimicrobials, antiphsychotics, antipyretics, antiseptics, antisignaling agents, antiarthritics, antithrombin agents, antituberculotics, antitussives, antivirals, appetite suppressants, cardioactive drugs, chemical dependency, drugs, cathartics, chemotherapeutic agents, coronary, cerebral or peripheral vasodilators, contraceptive agents, depressants, diuretics, expectorants, growth factors, hormonal agents, hypnotics, immunosuppression agents, narcotic antagonists, parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins and tranquilizers.

In another aspect, the present invention is related to a use of the composition according to the present invention in the preparation of a medicament for treatment, prevention and/or diagnosis of a disease.

In another aspect, the present invention is related to a method for manufacturing

• • the composition according to the present invention. Said method for manufacturing comprises at least the following steps:
  • providing a substance comprising a cell-penetrating compound comprising a basic amino functional group, and
  • adjusting the pH of the composition to at least 7.7, preferably at least 7.8.

In another aspect, the present invention is related to a kit comprising in (a) suitable container(s) at least a composition according to the present invention, and optionally a package insert. Preferably, the composition is contained in a ready-to-use form.

The invention is illustrated but not limited by the following examples.

EXAMPLES

Example 1: Uptake of TAMRA Labeled HIV 1 TAT-Peptide in HeLa Living Cells

HeLa cells where seeded at 60% confluence in a tissue culture treated 6 channel μ-Slide VI (from Ibidi GmbH, Germany) 24 hr before peptide treatment. The uptake imaging at different pHs was done by washing two times with HEPES buffers (140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mM glycine, pH adjusted with NaOH or HCl) at the pH of interest. The substance, here the HIV 1 TAT-peptide was provided in HEPES buffer. The buffer solution was replaced with said suspension, comprising the HEPES buffer with the peptide added at a final concentration of 2 μM, and adjusted to the desired pH value. The sample was taken to the microscope and imaged at equally spaced intervals of 2 min. This was done simultaneously for pH 6, 7.5 and 9 to compare the relative peptide uptake side by side. HeLa cells were imaged by swapping at each time point between two objectives, an ×60 and an ×20 immersion oil. This was done to be able to perform two types of time-lapse analysis over the same sample to compute the cellular uptake of the TAT peptide. With the ×20 objective is possible to simultaneously visualize several cells but is not easy to separate the fluorescence intensity from internalized peptide from the fluorescent intensity of membrane bound peptide. With the ×60 objective a fewer number of cells are visualized but the relative intensity of free peptide can be computed by measuring the fluorescence intensity of peptides accumulated at the nucleolus, since peptide bound to the cell plasma membrane or trapped in endosomes cannot reach the nucleolus. Using the ×20 images, the uptake was computed by measuring the average background fluorescence intensity in an area of the images without cells and subtracting this value from the average intensity of the whole image. Using the ×60 images, the uptake was computed by measuring the average background fluorescence intensity in an area without cells and subtracting this value from the average intensity in the nucleus. The nucleus area was obtained using the DIC channel. This experiment was repeated 3 times and the average and the standard error plotted. In each experiment, after 30 min the cells were washed with DMEM cell culture media and calcein was added to detect cell viability at a final concentration of 5 μM. In live cells, the non-fluorescent calcein AM is converted to green-fluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. This was incubated for 30 min and then imaged. Cell viability was also assessed using the DIC images used to detect the cell morphology along the experiments. To further evaluate the viability of cells after uptake of the TAT at pH 9, cell division was monitored for 16 hours.

The results are shown in FIG. 1. FIG. 1 shows the fluorescence intensity of TAT (2 μM) uptake in living cells at pH 6, 7.5 and 9 vs. time. Accordingly, at the concentration of 2 μM of the TAT peptide there is no uptake at pH 6 and 7.5, most cells kept at pH 9 display a significant uptake within this time interval (30 min).

Example 2: Comparison of Different Mediator Compounds on the Uptake Amino-Rich and Guanidinium-Rich Cell-Penetrating Compounds in Living Cells

A comparative test of cellular penetration of different cell-penetrating compounds was performed in the presence and absence of different mediator compounds at low and high pH. The cell-penetrating compounds (2 μM) and the mediator compounds (6 μM) were mixed in Hepes buffers at low pH (6.5) and high pH (8.5).

The cell media (DMEM) was removed and exchanged by these solutions and incubated for 30 min. Then the solutions were removed and the cells were embedded back in cell culture media (DMEM) and imaged. The efficiency of uptake in the different conditions was determined.

The results are shown in Table 1 below. The determined efficacy is depicted with increasing efficacy from “−” no uptake detectable, “+” low efficiency of uptake detectable, “++” moderate efficiency of uptake detectable, “+++” strong de efficiency of uptake detectable, “++++” very strong efficiency of uptake detectable.

TABLE 1
Efficiency of uptake in different conditions.
		Mediator 1:	Mediator 2:
	no mediator	Curcumin	Levothyroxine
	low pH	high pH	low pH	high pH	low pH	high pH
	of 6.5	of 8.5	of 6.5	of 8.5	of 6.5	of 8.5
Peptide 1: TAT peptide	−	+	−	++	−	++
labeled with TAMRA
Peptide 2: R10	−	+	−	+++	−	+++
labeled with FITC
cyclic Peptide: cR10	−	++	−	++++	−	++++
labeled with FITC
DNA repair enzyme	−	−	−	++	−	++
Nanoparticle with amino	+	++	+	+++	+	+++
functional groups:
Cube octameric
silsesquioxanes (COSS)
labeled with FITC.
Comparative example peptide	−	−	−	−	−	−
without amino functional
groups: TAMRA-Ahx-
LGQQQPFPPQQPY

Example 3: Cellular Uptake in Fatty Acid Enriched Cells

A HEPES buffer at pH 7.5 was mixed with 0.2% volume of oleic acid. Cells were washed twice and incubated for 15 min with this buffer. Next, cells were washed once with a HEPES buffer at pH 7.5 (without oleic acid) and the TAT peptide mixed with this last buffer was added at different concentrations. Cells were washed after 5 min with DMEM cell culture media two times, media plus calcein was added to monitor for enzymatic activity and the cells were imaged.

The results are shown in FIG. 5. It may immediately taken therefrom that fatty acid enriched cells display a much higher uptake efficiency than the control cells and most cells are viable as indicated by their morphology and their enzymatic activity. Therefore, enriching the plasma membrane with fatty acids enhances the binding and uptake of arginine-rich peptides.

Example 4: Peptides Absorption into a Hydrophobic Phase as a Function of pH

TAMRA labeled TAT peptides were diluted in HEPES buffer (140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mM glycine, pH adjusted with NaOH or HCl) to a final concentration of 10 μM at each pH. 150 μl of the buffer-peptide mix was added to 146 μl of octanol plus 4 μl of a compound selected from octanol, oleic acid, acetic acid, mono-N-dodecylphosphate, sodium dodecylsulfate, lithocholic acid, sunflower oil, castor oil and olive oil. Each pH mix was vortexed for 5 min and centrifuged for 2 min with a centripetal force of 2200 g to separate the octanol from aqueous phases. The octanol phase in contact with each pH buffer was then extracted with a pipette and mixed with a buffer at pH 4, which was also vortexed and centrifuged. In this way the fraction of peptide previously absorbed in the octanol phase was reabsorbed in the pH 4 buffer and in this case the octanol phase (with little or no traces of the TAT peptides) was again removed with a pipette and discarded. The peptide in each buffer solution was measured using a fluorescent spectrometer, using as a reference a buffer solution of 10 μM peptide at pH 4, measuring the relative fluorescent intensity emission between the reference and solution and the solution of interest, exciting with a laser wavelength of 543 nm and measuring the emission wavelength of 575 nm. Similarly the peptides that remained in the aqueous phase were compared to a reference solution of 10 μM peptide at each pH without having been in contact with the octanol phase.

The results are shown in FIG. 2. In the left column of FIG. 2 are shown snapshots of microcentrifuge tubes containing the different hydrophobic phases in contact with the aqueous buffers at different pHs. FIG. 2a shows that in the absence of oleic acid as well and in the presence of acetic acid TAT does not enter the hydrophobic phase. FIG. 2b shows that phosphate and sulfur groups containing compounds remain bounded to the TAT peptide and partition into the hydrophobic phase at every pH. Lithocholic acid displays a similar behavior as oleic acid although the deprotonation in this case is shifted to a higher pH. FIG. 2c shows partition of the TAT peptide into three distinct types of natural vegetable oils: sunflower oil, castor oil and olive oil. Sunflower oil displays a behavior consistent with a composition of only triglycerides displaying no absorption of the TAT peptide in the hydrophobic phase. Castor oil behaves as having also free fatty acids showing an absorption behavior similar to oleic acid. Olive oil displays absorption of the TAT peptide at the interface for every pH revealing the presence of phospholipids.

The results shown in FIG. 2c open a new approach for detecting if natural oil has expired or if it has been adulterated by mixing it with a different oil.

It is well known that oxidized or aged olive oil can be detected by determining the content and nature of free fatty acids. Therefore, the absorption of a peptide at high pHs into a hydrophobic phase would change.

An olive oil that is adulterated with tea tree oil can be detected by two significant changes depending on the percentage of tea tree oil added. Firstly, the absorption of peptides at the interface between the oil and the aqueous buffer changes, since the concentration of phospholipids naturally present in olive oil changes, and secondly, the amount of peptide absorbed at high pH also changes, since the mix reduces the amount of free fatty acids.

Using a proper calibration, the above methods offer a simple, immediate and cheap way to detect oil that has been adulterated or that is expired. Particularly, the present application in a further aspect is related to a method for determining the state, particularly the quality, of an oil. Such method particularly comprises at least the following steps: providing a sample of the oil, applying an aqueous buffer to the oil sample, adding a peptide according to the present invention to the sample, and determining the amount of peptide absorbed at high pH and/or determining the absorption of peptides at the interface between the oil and the aqueous buffer. The absorption of the peptides in olive oil and canola oil or other oils is very different. Therefore, knowing how the peptide is absorbed at different pHs allows a person skilled in the art to conclude on the quality of the oil, and even if the oil has been adulterated.

As apparent from FIG. 2, the peptides can be easily used to detect different kind of oils and compositions within the oils.

Example 5: Efficacy of the Composition of the Present Invention as a Skin Treatment

For determining the efficacy of the composition of the present invention, a composition was manufactured comprising a buffer solution at a pH higher than 7.8 an RRP at a concentration of 10 micro Molars labeled with a fluorescent die.

The composition is tested in a clinical study, wherein volunteers donate 30 mm² of skin extracted by a punch biopsy. The skin sample is placed in a tube with the stratum corneum exposed to air while the other kin layers are submerged into cell culture media. A drop of the buffer solution containing the cell-penetrating compound is applied on top of the stratum corneum for 1 hr. Then the area is washed 3 times with PBS, the skin is sectioned with a scalp and imaged at a confocal microscope. It can be seen that the cell-penetrating substance is able to cross the stratum corneum and get inserted in cells within several layers below the stratum corneum.

The efficacy can be clearly seen as the fluorescently labeled guanidinium-rich peptide is able to cross the stratum corneoum and reach the interior of cells. In the cells the nucleolus is also labeled indicating the peptide is not entrapped in endosomes and is consequently immediately bioavailable.

Example 6: Uptake of Compounds at Different pHs

FIG. 3 shows the uptake of other compounds at different pHs, showing that the pH enhances penetration of guanidinium and amino groups into the cells.

It can be immediately taken therefrom that increasing the extracellular pH consistently increases the transduction efficiency of arginine rich peptides with different structures, lengths and chirality. Fluorescence images show the uptake of the peptides listed in Table 1 (2 μM) in living cells (HeLa) at pH 6, 7.5 and 9 after 30 min. For each pH is shown the fluorescent confocal image of the peptides (green) and an image composed of an overlay of the DIC, the peptides and the nucleus (red). Cells permanently expressing Proliferating Cell Nuclear Antigen (PCNA) labeled with Cherry or GFP were used to facilitate the detection and visualization of confocal planes across the nucleus. This helped to easily classify and count cells containing transduced peptides from the cells that only contain membrane bound peptides. Although, this is clear when the peptides are in D form (R10 or cR10) since they are stable and distinctly label the nucleolus, the peptides in L form are being actively degraded and the signal quickly redistributes more homogeneously within the cell making more challenging to distinguish membrane bound peptides form internalized peptides. Cells were counted as positive when the peptide signal co-localized with the PCNA signal. The percentage of cells counted with intracellular peptide at pH 9 was consistently larger relative to pH 6 and 7.5 for all arginine rich peptides, while the poly-lysine peptide K10 displays no uptake at all pHs. The uptake efficiency also increases with the number of arginine amino acids and by cyclization. The images where acquired with a ×60 objective magnification. Each experiment was repeated 3 times and the percentage represents the average over more than 400 cells in each case. Scale bar 15 μm.

Example 7: Delivery of an Arginine and Amino Rich Peptide

FIG. 4 shows the delivery of an arginine and amino rich peptide, here the TAT peptide, transporting a florescent molecule across the skin.

A skin sample was obtained from a healthy individual and a drop of the compound in a buffer at pH 9 mixed with ricinoliec acid (the compound and the fatty acid at a concentration of 20 μM) was immediately applied on the skin surface for 60 min. The dermis was kept permanently in contact with cell media supplemented with nutrients to keep the tissue alive. The living sample was then washed, fractioned with a scalp and imaged with a confocal microscope specially designed to image living tissue.

It can be immediately taken from FIG. 4 that the fluorescently labeled molecule is transported across the stratum corneum and reaches the epidermis and dermis layers and the cells interior (distributed in the cytosol and the cell nucleus). The different layers are schematically shown as a reference. The distribution gradient of the drug accumulation is immediately evident, as it goes deeper into the skin layers, naturally with higher concentration in the upper layers.

One with ordinary skill in the art will recognize from the provided description and examples that modifications and changes can be made to the various embodiments of the invention without departing from the scope of the invention defined by the claims and their equivalents.

The features of the present invention disclosed in the specification, the claims, examples and/or the figures may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

1. A method for delivering a substance into cells, wherein the method comprises the following steps: wherein the method comprises the additional step of

a) providing the substance, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group,

b) providing cells; and

c) contacting the substance with the cells;

adjusting a pH in an extracellular fluid to a pH of at least 7.7.

2. The method according to claim 1, wherein the cell-penetrating compound is selected from the group comprising peptides, proteins, carbohydrates, lipids and nucleic acids.

3. The method according to claim 1, wherein the cells are chosen from the group comprising mammalian cells, plant cells, bacterial cells or insect cells.

4. The method according to claim 1, wherein in step b) providing cells is consisting of providing a suspension of cells, and wherein in step c) contacting the substance with the cells is consisting of creating a cell-substance-suspension by combining the substance with the suspension of cells, and wherein the pH in an extracellular fluid is an extracellular pH in the cell-substance-suspension.

5. The method according to claim 1, further comprising in step a) providing a suspension or solution of the substance in a pH buffer, particularly in an extracellular fluid being a pH buffer.

6. The method according to claim 1, wherein the method further comprises adding to the substance and/or to an extracellular fluid and/or the cell-substance-suspension, a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue.

7. The method according to claim 1, wherein the basic amino functional group of the cell-penetrating compound are part of a guanidinium functional group.

8. The method according to claim 1, wherein the cell-penetrating compound is a peptide or a protein comprising a basic amino acid selected from arginine and lysine.

9. The method according to claim 1, wherein the cell-penetrating compound is a peptide or a protein and the method further comprises the step of adding a capping agent comprising a guanidinium group.

10. The method according to claim 1, wherein the cell-penetrating compound comprises a DNA repair enzyme comprising a basic amino functional group.

11. The method according to claim 1, wherein the cell-penetrating compound comprises a linker moiety.

12. The method according to claim 11, wherein the linker moiety is a peptide linker or a nucleotide linker.

13. The method according to claim 1, wherein an additional agent is covalently or non-covalently conjugated to the cell-penetrating compound, the additional agent being selected from small molecules, nucleic acids, peptide nucleic acids, peptides, proteins, nucleotides, oligonucleotides, inorganic particles and liposomes.

14. The method according to claim 13, wherein the additional agent is selected from the group comprising biomarker, drug, medically, pharmaceutically and cosmetically active substance.

15. The method according to claim 13, wherein the additional agent comprises at least one carboxyl functional group or carboxylate functional group.

16. The method according to claim 15, wherein the additional agent is a peptide or a protein comprising an acidic amino acid selected from aspartate/aspartic acid and glutamate/glutamic acid.

17. The method according to claim 1, wherein the mediator compound is selected from the group consisting of saturated and non-saturated fatty acids, saturated and non-saturated hydroxy fatty acids, dicarboxy acids, aromatic acids, and salts thereof.

18. The method according to claim 1, wherein the cells and/or the substance are pre-incubated with the mediator compound before the cell-substance-suspension is created and/or before in step c) the substance is contacted with the cells.

19. The method according to claim 1, wherein step c) of contacting the substance with the cells, comprises delivering the substance into the cell.

20. The composition according to claim 19, wherein the cell-penetrating compound is selected from the group comprising peptides, proteins, carbohydrates, lipids and nucleic acids.

21. The composition according to claim 19, wherein the composition comprises a suspension of the substance in pH-buffer, particularly in an extracellular fluid being a pH buffer.

22. The composition according to claim 19, wherein the composition comprises a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue.

23. The composition according to claim 19, wherein the composition is in a form of a cream, a gel, an unguent, a lotion, a spray, an aerosol, a solution, in an emulsion, in liposomes or in microcapsules, wherein preferably the composition is for skin treatment.

24. The composition according to claim 19, further comprising a pharmaceutical acceptable carrier, filler, bulking agent, disintegrant, stabilizer, binder, humectant, extender, emulsifying agent, dissolution retarder, absorption enhancer, preservative, antioxidant, wetting agent, adsorbent, lubricant or a combination thereof.

25. Use of the composition according to claim 31 for treatment and/or prevention of a disease, diagnosis of diseases, as a research tool, as a targeting system, as a pharmaceutical composition or as a cosmetic composition.

26. Use of the composition according to claim 31 in the preparation of a medicament for treatment, prevention and/or diagnosis of a disease.

27. A method for manufacturing the composition according to claim 31, comprising at least the following steps:

providing a substance comprising a cell-penetrating compound comprising a basic amino functional group, and

adjusting the pH of the composition to at least 7.7.

28. A kit comprising in (a) suitable container(s) at least a composition according to claim 31, and optionally a package insert.

29. The kit according to claim 28, wherein the composition is contained in a ready-to-use form.

30. The method according to claim 1, further comprising a step of applying the substance to a subject, wherein the method is a cosmetic or therapeutic method.

31. A composition comprising a substance to be delivered to cells, wherein

the substance comprises a cell-penetrating compound comprising a basic amino functional group, and wherein

the pH of the composition is at least 7.7.