A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery

Abstract

CONSPECTUSThe potential of gene therapy to benefit human health is tremendous because almost all human diseases have a genetic component, from untreatable monogenic disorders to cancer and heart disease. Unfortunately, a method for gene therapy that is both effective and safe has remained elusive. It has been said that "there are only three problems in gene therapy -delivery, delivery, and delivery." (quote from I. M. Verma in Jaroff, L. TIME, 1999; Jan 11).This Account describes an alternative strategy to viral gene delivery: the design of biodegradable polymers that are able to deliver DNA like a synthetic virus. Using high-throughput synthesis and screening techniques, we have created libraries of over 2000 structurally unique poly(β-amino esters) (PBAEs). PBAEs are formed by the conjugate addition of amines to diacrylates. These biomaterials are promising for nonviral gene delivery due to their ability to condense plasmid DNA into small and stable nanoparticles and their ability to promote cellular uptake and endosomal escape. Our laboratory has iteratively improved PBAE nanoparticles through polymer end modifications and nanoparticle coatings. Lead PBAEs have high gene delivery efficacy and low cytotoxicity both in vitro and in vivo.Certain polymer structural characteristics are important for effective gene delivery. The best PBAEs are linear polymers of ~10 kDa that contain hydroxyl side chains and primary amine end groups. These polymers bind DNA to form nanoparticles that are small (<200 nm) and stable and have near-neutral ζpotential in the presence of serum-containing media. Lead PBAEs also contain tertiary amines that can buffer the low pH environment of endosomes and facilitate escape of polymer/DNA particles into the cytoplasm.Diamine end-modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers (C32) bind DNA more tightly and form smaller nanoparticles than other PBAEs. These nanoparticles also have higher cellular uptake and the best gene expression of all gene delivery polymers in the library. These polymers are more effective for gene delivery than top commercially available nonviral vectors including jet-PEI and Lipofectamine 2000 and are comparable to adenovirus for in vitro gene delivery to human primary cells. In vivo, these PBAE/ DNA particles are promising as cancer therapeutics. This Account summarizes the results of our laboratory in using a combinatorial polymer library approach to elucidate polymer structure/function relationships and enable the development of polymeric gene delivery nanoparticles with viral-like efficacy. Nonviral gene delivery systems make use of physical methods or a synthetic chemical vector or both to deliver the gene of interest. Limitations with viral approaches, including small cargo capacity, resistance to repeated infection, difficulty in production and quality control, and low safety, can be potentially overcome with a nonviral approach. Originally, nonviral gene delivery was simply delivery of naked plasmid DNA. To improve transfection, physical str...

A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system

“…It is suggested that positively charged nanoparticles are generally taken up into the cells through endocytosis mechanism. 33 Our TEM experiments also support a similar mechanism and the high transfection of RSV-F gene via PCNSs, due to the high endocytosis efficiency of PCNSs (through cell membranes).…”

“…Since the cells could not be cultured beyond a few days, we could 33 showed identical result in Cos-7 cells. The superior transfection capability of PCNSs may be attributed to the cationic nature of chitosan shell that can interact with negatively charged cell membranes, allowing it to be taken up by cells via endocytosis.…”

“…Hu et al prepared a hydrophilic shell of aminoethyl methacrylate that surrounded a pH-sensitive core of diethylamino ethyl methacrylate, to efficiently deliver small molecules or proteins into dendritic cells in vitro. In a similar study, Green et al 33 demonstrated a nonviral polymeric gene delivery system that employed poly(B-amino ester) as a proton sponge, facilitating endosomal escape for high-throughput gene transfection. Also, Guo et al 9 proposed a similar endosomal escape mechanism leading to enhanced intracytoplasmic uptake of GFP gene and siRNA by exploiting gold nanoparticles functionalized with charge-reversal polyelectrolyte.…”

Synthetic poly(ester amine) and poly(amido amine) nanoparticles for efficient DNA and siRNA delivery to human endothelial cells

“…PEAs were synthesized as previously reported. 34,37,38 Briefly, one diacrylate backbone monomer (B) was mixed with one amine-containing sidechain (S) at 1.2:1 or 1.05:1 ratio and stirred overnight at 90°C. Acrylate-terminated B-S base polymers resulted from Michael addition of amines to the acrylate groups.…”

“…Natural and synthetic cationic polymers have been previously studied because they are potentially safer and easier to manufacture, 27,28 but nondegradable polymers like poly(ethyleneimine) suffer from high toxicity, 29 which correlates with high molecular weight. 30 Although polymeric gene delivery has been traditionally less efficient than virusmediated gene transfer, 31 members of one class of polymers, ie, poly(ester amine)s, have been found to be very effective in DNA delivery to a number of cell types while causing less toxicity than leading commercially available transfection reagents, [32][33][34] likely due to the ability of polymers to degrade under physiological conditions and their theoretically unlimited variation in structure and properties, providing the flexibility to choose a polymer structure based on the particular purpose for which it is best suited.…”