Investigation of in vitro biocompatibility of novel pentablock copolymers for gene delivery.
Novel pentablock copolymers of poly(diethylaminoethylmethacrylate) (PDEAEM), poly(ethylene oxide) (PEO), and poly(propylene oxide) (PPO), (PDEAEM-b-PEO-b-PPO-b-PEO-b-PDEAEM), were synthesized as vectors for gene delivery, and were tested for their biocompatibility on SKOV3 (human ovarian carcinoma) and A431 (human epidermoid cancer) cell lines under different in vitro conditions using various assays to elucidate the mechanism of cell death. These copolymers form micelles in aqueous solutions and can be tuned for their cytotoxicity by tailoring the weight percentage of their cationic component, PDEAEM. Copolymers with higher PDEAEM content were found to be more cytotoxic, though their polyplexes were less toxic than the polycations alone. Pentablock copolymers displayed higher cell viability than commercially available ExGen 500 at similar N:P ratios. While cell death with ExGen was found to be accompanied by an early loss of cell membrane integrity, pentablock copolymers caused very little membrane leakage. Caspase-3/7 assay confirmed that none of these polymers induced apoptosis in the cells. These pentablock copolymers form thermo-reversible gels at physiological temperatures, thereby enabling controlled gene delivery. Toxicity of the polymer gels was tested using an agarose-matrix, simulating an in vivo tumor model where injected polyplex gels would dissolve to release polyplexes, diffusing through tumor mass to reach the target cells. Twenty five weight percent of copolymer gels were found to be nontoxic or mildly cytotoxic after 24 h incubation. Transfection efficiency of the copolymers was found to be critically correlated to cytotoxicity and depended on DNA dose, polymer concentration, and N:P ratios. Transgene expression obtained was comparable to that of ExGen, but ExGen exhibited greater cell death.
Agarwal
Ankit
A
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA.
Unfer
Robert
R
Mallapragada
Surya K
SK
D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D018014:Gene Transfer Techniques; D006801:Humans; D008422:Materials Testing; D008689:Methacrylates / Q000138:chemical synthesis / Q000737:chemistry*; D008823:Micelles; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D011409:Propylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*
D008689:Methacrylates; D008823:Micelles; D011108:Polymers; D011409:Propylene Glycols; C012504:polypropylene glycol; D011092:Polyethylene Glycols; D004247:DNA; D053148:Caspase 3; D053179:Caspase 7