Cell membrane penetrating conjugates

Abstract

A cell penetrating conjugate comprising a recombinant β helical protein linked to a functional molecule wherein the β helical protein length is in the range of from 5 nm to 25 nm, suitably, from 10 nm to 15 nm and width is in the range of from 1 nm to 5 nm, suitably, from 1 nm to 3 nm. Processes for preparing said conjugates and uses thereof are also disclosed.

1 . A cell penetrating conjugate comprising a recombinant β helical protein linked to a functional molecule, wherein the β helical protein length is in the range of from 5 nm to 25 nm, and width is in the range of from 1 nm to 5 nm. 
     
     
         2 . The conjugate as claimed in  claim 1 , wherein the β helical protein length is in the range of from 10 nm to 15 nm, and width is in the range of from 1 nm to 3 nm. 
     
     
         3 . The conjugate as claimed in  claim 1 , wherein the β helical protein comprises one or more amino acid ladder structures selected from the group consisting of an arginine ladder; a lysine ladder; an asparagine ladder; an aspartic acid ladder; and a glutamic acid ladder. 
     
     
         4 . The conjugate as claimed in  claim 3 , wherein when present, the arginine ladder comprises from 10 to 20 arginine residues; the lysine ladder comprises from 10 to 30 lysine residues; the asparagine ladder comprises from 10 to 40 asparagine residues; the aspartic acid ladder comprises from 10 to 40 aspartic acid residues; and the glutamic acid ladder comprises from 10 to 40 glutamic acid residues. 
     
     
         5 . The conjugate as claimed in  claim 1 , wherein the β helical protein has a total charge that is less than zero. 
     
     
         6 . The conjugate as claimed in  claim 5 , wherein the β helical protein has a total charge of from −20 to −60. 
     
     
         7 . The conjugate as claimed in  claim 1 , wherein the β helical protein has a β helical structure with a stiffness parameter K (beta helix) 0.2 to 12 N/m 2 , as measured by atomic force microscopy. 
     
     
         8 . The conjugate as claimed in  claim 1 , wherein the β helical protein is a pentapeptide-repeat protein. 
     
     
         9 . The conjugate as claimed in  claim 8 , wherein the β helical protein comprises tandemly repeated pentapeptide with consensus sequence (STAV) 1 (DN) 2 (LF) 3 (STR) 4 (G) 5 . 
     
     
         10 . The cell penetrating conjugate as claimed in  claim 1 , wherein the β helical protein is represented by a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and combinations thereof. 
     
     
         11 . The cell penetrating conjugate as claimed in  claim 1 , wherein the recombinant β helical protein is linked to the functional molecule via a linker molecule selected from the group consisting of: polyethyleneglycol (PEG); peptide; metal conjugate, drug-metal conjugate, DNA binding domain, nucleic acid intercalating molecule and combinations thereof. 
     
     
         12 . The cell penetrating conjugate as claimed in  claim 11 , wherein when the linker molecule is a peptide, the peptide comprises amino acids selected from the group consisting of: aliphatic amino acids; aromatic amino acids; and combinations thereof. 
     
     
         13 . The cell penetrating conjugate as claimed in  claim 1 , wherein the linker and the recombinant β helical protein are linked by one or more of the group selected consisting of: covalent bonds; non-covalent bonds; and combinations thereof. 
     
     
         14 . The cell penetrating conjugate as claimed in  claim 1 , wherein the recombinant β helical protein is linked to a functional molecule via by a linkage selected from the group consisting of: an ester linkage; and an amide linkage. 
     
     
         15 . The cell penetrating conjugate as claimed in  claim 1 , wherein the functional molecule is selected from the group consisting of dyes, drugs, metal, drug-metal conjugate, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localizing signals, nanoparticles, and combinations thereof. 
     
     
         16 . The cell penetrating conjugate as claimed in  claim 1 , wherein the conjugate further comprises a signal sequence wherein the signal sequence directs the conjugate to a particular cell or part of a cell. 
     
     
         17 . The cell penetrating conjugate as claimed in  claim 9 , wherein the signal sequence is selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. 
     
     
         18 . The cell penetrating conjugate as claimed in  claim 1 , wherein the conjugate further comprises a phosphatidyl choline molecule. 
     
     
         19 . The cell penetrating conjugate as claimed in  claim 9 , wherein the conjugate transfers the functional molecule to a location selected from the group consisting of: cell organelles; nucleus; endoplasmic reticulum; mitochondria; cell membrane; and P-cadherin overexpressing breast cancer cells. 
     
     
         20 . The cell penetrating conjugate as claimed in  claim 12 , wherein the cell organelles are selected from the group comprising actin filaments, golgi bodies, cell membrane, microtubulins. 
     
     
         21 . A process for transferring a functional molecule into a cell, said process comprising:
 a) linking the functional molecule to a recombinant β helical protein to obtain a conjugate;   b) contacting the conjugate with at least one cell;   
       wherein contacting the conjugate of step (b) transfers the functional molecule into the cell; and wherein the β helical protein length is in the range of from 5 nm to 25 nm and width is in the range of from 1 nm to 5 nm. 
     
     
         22 . The process as claimed in  claim 21 , wherein the process comprises after step (b):
 c) detecting the transfer of the conjugate inside the cell.   
     
     
         23 . The process as claimed in  claim 21 , wherein the cell is selected from the group consisting of eukaryotic cells, prokaryotic cells, and combinations thereof.