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.

What is claimed is: 
     
         1 . 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 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.