The gene delivery system is one of the threecomponents of a gene medicine, which is the bottle neck ofcurrent gene therapy. Nonviral vectors offer advantages overthe viral system of safety, ease of manufacturing, etc. As importantnonviral vectors, polymer gene delivery systemshave gained increasing attention and have begun to showincreasing promising. In this review, the fundamental andrecent progress of polymer-based gene delivery vectors isreviewed.

Polyaspartamide-based oligo-ethylenimine brushes (PASP-EDA, PASP-TEPA, PASP-PEHA, and PASP-PEI 423)were synthesized from polysuccinimide (PSI) via a ring-opening reaction with N-Boc protected ethylenediamine,tetraethylenepentamine, pentaethylenehexamine, and linear polyethylenimine (Mn 423), respectively. PASP-TEPA,PASP-PEHA, and PASP-PEI 423 possess high buffer capacity between pH 5 and pH 7, which is comparable tothat of branched PEI 25000. The cytotoxicity assay indicated that they all are less toxic than PEI 25000. At anN/P ratio of above 2, all of the four synthetic polycation brushes can condense plasmid DNA to form small sized(160-400nm) polyelectrolyte complexes with positive surface charge. The transfection of HEK 293 cells witholigo-ethylenimine brush/pRE Luc polyplexes indicated that the transfection efficiencies increased with increasingthe length of oligo-ethylenimine side chains. The luciferase expression with PASP-PEHA and PASP-PEI 423were as high as or even a little higher than that of PEI 25000. The results demonstrate that polyaspartamidebasedoligo-ethylenimine brushes are a very promising class of novel polycations for highly efficient and lesstoxic gene delivery.

Three hydrolytically degradable poly(_-aminoester)s containing ester bonds in the main chain and primary aminesin the side chain, synthesized by Michael polyaddition, were applied to deliver foreign DNA into cells in Vitro.These linear polycations can condense DNA into small-sized particles with positive surface charge at high N/Pratios. Their high buffer capacity at pH 5-7 facilitated the escape of DNA from the endosome and resulted inefficient gene expression. Under the optimal conditions, poly(_-aminoester)s with a pendant aminoethyl group(1a) showed higher transfection efficiencies than branched poly(ethylenimine) (PEI) 25KDa in 293T cells. Theeffect of side chain structure of the poly(_-aminoester) on transfection efficiency has been investigated, whichindicated that the poly(_-aminoester) containing the pendant aminoethyl group was the most efficient carrier forboth of 293T cells and COS-7 cells. The combination of hydrolytical degradation, high buffer capacity, relativelylow cytotoxicity, and high transfection efficiency suggested that this kind of poly(_-aminoester)s are novel promisingnonviral gene carriers.

Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. Toinvestigate the effect of the core structure and generation of dendrimers on the complex formation andtransfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations(DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived frompentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer hasenough amino group density to effectively condense DNA was higher (generation 6) than those of DPs andDIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameterranging from 100 to 300nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severeaggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generationnumbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPsand DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression inCOS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimersas evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 >DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. Theacid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pKa-6).This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfectionefficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. TheLD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 íg/mL, respectively, which were higherthan that of poly(ethyleneimide) (18 íg/mL) and poly(L-lysine) (28 íg/mL) in the same assay. With a lowercytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrantfurther investigation for nonviral gene delivery.

Cationic polyphosphoesters (PPEs) with different side-chain charge groups were designed and synthesized as biodegradablegene carriers. Poly(N-methyl-2-aminoethyl propylene phosphate) (PPE-MEA), with a secondary amino group (-CH2CH2NHCH3)side chain released DNA in several hours at N/P (amino group of polymer to phosphate group of DNA) ratios from 0.5 to 5;whereas PPE-HA, bearing-CH2(CH2)4CH2NH2 groups in the side chain, did not release DNA at the same ratio range for 30days. Hydrolytic degradation and DNA binding results suggested that side chain cleavage, besides the polymer degradation,was the predominant factor affected the DNA release and transfection efficiencies. The side chain of PPE-MEA was cleavedfaster than that of PPE-HA, resulting poor cellular uptake and no transgene expression for PPE-MEA/DNA complexes in COS-7cells at charge ratios from 4 to 12. In contrast, PPE-HA/DNA complexes were stable enough to be internalized by cells andeffected gene transfection (3400 folds higher than background at a charge ratio of 12). Interestingly, gene expression levelsmediated by PPE-MEA and PPE-HA in mouse muscle following intramuscular injection of complexes showed a reversed order:PPE-MEA/DNA complexes mediated a 1.5–2-fold higher luciferase expression in mouse muscle as compared with naked DNAinjection, while PPE-HA/DNA complexes induced delayed and lowered luciferase expression than naked DNA. These resultssuggested that the side chain structure is a crucial factor determining the mechanism and kinetics of hydrolytic degradation ofPPE carriers, which in turn influenced the kinetics of DNA release from PPE/DNA complexes and their transfection abilities invitro and in vivo.