1. Field of the Invention
The present invention relates generally to the fields of copolymer chemistry and gene delivery systems. More specifically, the present invention relates to the design and synthesis of a novel biodegradable polymer, for example, reducible linear L-lysine-modified copolymers (LLC).
2. Description of the Related Art
During the last decade, there has been a rapid development of nonviral gene delivery systems based on cationic polymers for the treatment of both inherited and acquired diseases. This is due to the dissemination of many disease pathways in which the modulation of expressed proteins or genes via gene therapy has the potential of significantly improving the treatment options of patients. Natural cationic polymers such as chitosan (1) and atelocollagen (2) or synthetic cationic polymers including poly(L-lysine) (PLL) (3), poly(ethylenimine) (PEI) (4-6), and dendrimers (4,7) have been widely explored as a means of delivering therapeutic nucleic acids to target cells. However, even though cationic polymers have produced modest gene expression, their translation into a clinical setting has been severely mired by carrier-mediated cytotoxicities associated with their high charge densities and high molecular weights. Thus, biodegradable carriers have been designed to overcome these hurdles as well as to increase transfection efficiencies by facilitating the unpacking of the polymer/pDNA polyplexes after cellular uptake (8).
Previously, biodegradable polymers such as polyurethanes (PUs) that contain tertiary amines on the backbone and primary, secondary, and tertiary amines on the side chains have been synthesized as nonviral gene delivery vectors (9). To improve the solubility and biocompatibility of the polymers, glycidol was conjugated into the structure. These backbone modifications resulted in higher transfection efficiencies comparable to the well-known non-degradable gene carrier poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA) and lower cytotoxicities. Similarly, hydrolytically degradable poly(_-amino esters) were developed as cationic polymers for gene transfer, which produced about four times higher gene expression in human embryonic stem cells with minimal toxicity (10). In addition, many other hydrolysable polymers such as poly(ester amines) (11-13), poly(esters) (14), ketalized PEI (15-17), chitosans (18), dendrimers (4,7), and polyphosphazenes (19-21) have been developed as alternatives to non-degradable polymers for gene delivery.
Recently, reducible disulfide-containing cationic polymers also have been extensively explored as alternatives to non-degradable gene delivery systems due to the difference in redox potential between the reducing cytoplasm and the oxidizing extracellular space (22). Thus, the inclusion of disulfide bonds within the polymeric carriers would render the polymers biodegradable as a result of the reduction of the bonds to free thiols in the cytosol followed by the concomitant release of the nucleic acid cargo. It was demonstrated that triggered release of pDNA following reduction of disulfide-containing poly(amido ethylenimines) (SS-PAEIs) within the cytosol of several cell lines increases transfection efficiency 20-fold compared with PEI (23).
Similarly, other studies have focused on developing reducible forms of PEI including disulfide cross-linked low molecular weight PEI (24-25), poly(amido ethylenediamine) polymer with multiple disulfide bonds (SS-PAEDs) (26-27), reducible poly(amido amine) (poly(DAH/CBA)) (8,28) and bioreducible cationic arginine-conjugated poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH-R)) (29). However, apart from PEI, there have been limited studies focused on developing other forms of reducible cationic polymers, which could potentially produce additional significant improvements over current nonviral delivery systems. Poly(L-lysine) and its derivatives have been shown to be very effective gene delivery carriers with much less cytotoxicity compared with PEI (30-36). However, these carriers also pose a significant problem of prolonged cytotoxicity in clinical applications due to their high molecular weight.
Thus, there is a recognized need in the art for improved biodegradable, biocompatible and reducible copolymers for useful in a biommolecule delivery system. More specifically, the prior art is deficient in disulfide-reducible linear L-lysine-modified copolymers. The present invention fulfills this longstanding need and desire in the art.
USPTO Backgrounds
USPTO Backgrounds