Transfer of molecules into the cytosol of cells

Abstract

The present invention relates to a method for introducing molecules in cells by disrupting endosomal and lysosomal membranes using photodynamic treatment, without killing the majority of the cells by the photodynamic treatment. More particularly, this invention includes a method for transferring DNA and/or RNA, such as genes, to cells by photochemically inducing the disruption of endosomes and lysosomes.

What is claimed is:  
     
         1 . A composition comprising a molecule to be released into a cytosol, a photosensitizing compound, and a carrier molecule; the photosensitizing compound being effective to rupture an intracellular compartment upon activation by light; the carrier molecule being effective for facilitating translocation into the intracellular compartment.  
     
     
         2 . The composition of  claim 1 , comprising a carrier attached both to the molecule to be released into the cytosol and to the photosensitizing compound.  
     
     
         3 . The composition of  claim 1 , comprising several carriers wherein at least one carrier has attached thereto the molecule to be released into the cytosol and another carrier has attached thereto the photosensitizing compound.  
     
     
         4 . The composition of  claim 1 , comprising a mixture of a carrier bound to the molecule to be released into the cytosol and a carrier bound to the photosensitizing compound.  
     
     
         5 . The composition of  claim 1 , wherein the composition is effective for modification of neoplastic and other cellular processes in a warm-blooded animal or in a cell cultures.  
     
     
         6 . The composition of  claim 1 , wherein the intracellular compartment is an endosome or a lysosome.  
     
     
         7 . The composition of  claim 1 , wherein the molecule to be released into the cytosol is an antineoplastic compound.  
     
     
         8 . The composition of  claim 1 , wherein the antineoplastic compound is gelonin.  
     
     
         9 . The composition of  claim 1 , wherein the molecule to be released into the cytosol comprises a DNA, an oligo(deoxy)nucleotide, an mRNA, an antisense DNA, a sugar, a protein, a peptide, a membrane impermeable drug or other membrane impermeable molecule, or a covalently or noncovalently bonded combination thereof.  
     
     
         10 . The composition of  claim 1 , wherein the molecule to be released into the cytosol comprises DNA, RNA, or a combination thereof.  
     
     
         11 . The composition of  claim 10 , wherein the DNA or RNA comprises a plasmid, a ribozyme, an antisense oligonucleotide, an aptamer, a triplex forming oligonucleotide, a peptide nucleic acid, or a combination thereof.  
     
     
         12 . The composition of  claim 10 , wherein the DNA or RNA encodes a ribozyme, an antisense oligonucleotide, an intracellular recombinant antibody, an antiangiogenetic factors, an angiogenetic factor, an anti-inflammatory molecule, a marker protein, a therapeutic protein, or a combination thereof.  
     
     
         13 . The composition of  claim 12 , wherein the therapeutic protein comprises a suicide enzyme, a toxin or a part of a toxin, a recombinant immunotoxin, a cytokine, an immunostimulatory molecule, or a combination thereof.  
     
     
         14 . The composition of  claim 13 , wherein the suicide enzyme comprises a Herpes Simplex Virus thymidine kinase, a cytidine deaminase, a D-amino acid oxidase, or a combination thereof.  
     
     
         15 . The composition of  claim 13 , wherein the toxin or a part of a toxin comprises all or part of a diphtheria toxin, a diphtheria toxin A-chain, a ricin, a gelonin, or a combination thereof.  
     
     
         16 . The composition of  claim 13 , wherein the cytokine comprises tumor necrosis factor-α, transforming growth factor-β, interleukin-12 an interleukin other than interleukin 12, a colony-stimulating factor, a chemokine, or a combination thereof.  
     
     
         17 . The composition of  claim 13 , wherein the immunostimulatory molecule comprises an HLA-B7, a B7.1, a costimulatory protein, or a combination thereof.  
     
     
         18 . The composition of  claim 12 , wherein the marker protein comprises green fluorescent protein.  
     
     
         19 . The composition of  claim 12 , wherein the ribozyme comprises a synthetic ribozyme designed against mRNA of the metastasis associated protein capl.  
     
     
         20 . The composition of  claim 1 , wherein the photosensitizing compound is a porphyrin, a phtalocyanine, a purpurin, a chlorin, a benzoporphyrin, a napthalocyanine, a cationic dye, a tetracycline, or a lysosomotropic weak base or derivative thereof.  
     
     
         21 . The composition of  claim 1 , wherein the photosensitizing compound is a porphyrin, a phtalocyanine, a purpurin, a benzoporphyrin, a napthalocyanine, a cationic dye, a tetracycline, or a lysosomotropic weak base or derivative thereof.  
     
     
         22 . The composition of  claim 21 , wherein the photosensitizing compound is tetraphenylporphine with two sulfonate groups on adjacent phenyl groups.  
     
     
         23 . The composition of  claim 21 , wherein the photosensitizing compound is a tetraphenyl porphine with 2 sulfonate groups on adjacent phenyl groups (TPPS 2a ), a meso-tetraphenyl porphine with 4 sulfonate groups (TPPS 4 ), an aluminum phthalocyanine with 2 sulfonate groups on adjacent phenyl rings (AlPcS 2a ), or a combination thereof.  
     
     
         24 . The composition of  claim 1 , wherein the carrier is a site-directing molecule which facilitates the uptake of the photosensitizing compound(s) or the molecule to be released into a cytosol.  
     
     
         25 . A method for introducing a molecule of interest into a cytosol of a living cell, comprising: 
 a) delivering a photosensitizing compound and the molecule of interest to the cell, wherein each are taken up into an intracellular compartment of the cell;    b) irradiating the cell with light of a suitable wavelength to activate the photosensitizing compound so that the membrane surrounding the intracellular compartment is disrupted, releasing the molecule of interest into the cytosol of the cell without killing the cell.    
     
     
         26 . The method according to  claim 25 , wherein the molecule of interest is DNA, an oligo(deoxy)nucleotide, mRNA, antisense DNA, a sugar, a protein, a peptide, a membrane impermeable molecule, or a covalently or noncovalently bonded combination thereof.  
     
     
         27 . The composition of  claim 25 , wherein the molecule to be released into the cytosol comprises DNA, RNA, or a combination thereof.  
     
     
         28 . The composition of  claim 27 , wherein the DNA or RNA comprises a plasmid, a ribozyme, an antisense oligonucleotide, an aptamer, a triplex forming oligonucleotide, a peptide nucleic acid, or a combination thereof.  
     
     
         29 . The composition of  claim 27 , wherein the DNA or RNA encodes a ribozyme, an antisense oligonucleotide, an intracellular recombinant antibody, an antiangiogenetic factors, an angiogenetic factor, an anti-inflammatory molecule, a marker protein, a therapeutic protein, or a combination thereof.  
     
     
         30 . The composition of  claim 29 , wherein the therapeutic protein comprises a suicide enzyme, a toxin or a part of a toxin, a recombinant immunotoxin, a cytokine, an immunostimulatory molecule, or a combination thereof.  
     
     
         31 . The composition of  claim 30 , wherein the suicide enzyme comprises a Herpes Simplex Virus thymidine kinase, a cytidine deaminase, a D-amino acid oxidase, or a combination thereof.  
     
     
         32 . The composition of  claim 30 , wherein the toxin or a part of a toxin comprises all or part of a diphtheria toxin, a diphtheria toxin A-chain, a ricin, a gelonin, or a combination thereof.  
     
     
         33 . The composition of  claim 30 , wherein the cytokine comprises tumor necrosis factor-α, transforming growth factor-β, interleukin-12 an interleukin other than interleukin 12, a colony-stimulating factor, a chemokine, or a combination thereof.  
     
     
         34 . The composition of  claim 30 , wherein the immunostimulatory molecule comprises an HLA-B7, a B7.1, a costimulatory protein, or a combination thereof.  
     
     
         35 . The composition of  claim 29 , wherein the marker protein comprises green fluorescent protein.  
     
     
         36 . The composition of  claim 29 , wherein the ribozyme comprises a synthetic ribozyme designed against mRNA of the metastasis associated protein capl.  
     
     
         37 . The method according to  claim 25 , wherein the molecule of interest is gelonin, saporin, agrostin, or a combination thereof.  
     
     
         38 . The method according to  claim 25 , wherein the photosensitizing compound is a porphyrin, a phthalocyanine, a purpurin, a chlorin, a benzoporphyrin, a napthalocyanine, a cationic dye, a tetracycline, or a lysosomotropic weak base or derivative thereof.  
     
     
         39 . The method according to  claim 25 , wherein the photosensitizing compound is a porphyrin, a phthalocyanine, a purpurin, a benzoporphyrin, a napthalocyanine, a cationic dye, a tetracycline, or a lysosomotropic weak base or derivative thereof.  
     
     
         40 . The method according to  claim 39 , wherein the photosensitizing compound is a tetraphenyl porphine with 2 sulfonate groups on adjacent phenyl groups (TPPS 2a ), a meso-tetraphenyl porphine with 4 sulfonate groups (TPPS 4 ), an aluminum phthalocyanine with 2 sulfonate groups on adjacent phenyl rings (AlPcS 2a ), or a combination thereof.  
     
     
         41 . The method according to  claim 25 , further comprising a vector molecule which facilitates the uptake of either the photosensitizing compound or the molecule which is to be released into the cytosol.  
     
     
         42 . The method according to  claim 25 , wherein the step of irradiating includes selecting a light dose and wavelength and a photosensitizing compound so that after the irradiation, a portion of the living cells are killed.  
     
     
         43 . The method of  claim 25 , wherein delivering further comprises delivering a carrier molecule to the cell.