A nonionic and water-soluble polyphosphoester, poly(2-hydroxyethyl propylene phosphate) (PPE3), wassynthesized by chlorination of poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane), followed by esterificationwith 2-benzyloxyethanol and deprotection of the hydroxyl group by catalytic hydrogenation in thepresence of Pd-C. PPE3 degraded rapidly in PBS 7.4 at 37℃. The cytotoxicity and tissue compatibilityassays suggested good biocompatibility of PPE3 in vitro and in vivo. The expression of pVR1255 Lucplasmid in mouse muscle after intramuscular (i.m.) injection of DNA formulated with PPE3 solution insaline was enhanced up to 4-fold compared with that of naked DNA. These results suggest the potential ofthis polyphosphoester for naked DNA-based gene therapy. The advantages of this polymer design includethe biodegradability of the polyphosphoester and its structural versatility, which allows the fine-tuning ofthe physicochemical properties to optimize the enhancement of gene expression in muscle.

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.

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.

To design successful polymeric gene delivery vehicles with good biocompatibility and highly efficientgene transfer ability is one of the great scientific challenges in modern gene therapy. Poly(amidoamine)with pendant aminobutyl group (PAA-BA) has been proved to exhibit high transfection efficiency againstbone marrow stromal cells (BMSCs) in vitro. In this work, based on previous research, PAA-BA'sbiocompatibility including in vitro cytotoxicity determined by effect on BMSCs' morphology, viability,membrane damage and apoptosis/necrosis, and in vivo tissue compatibility determined by muscular andhepatic tissue response were further investigated in comparison to branched polyethylenimine (PEI)25 kDa. The results demonstrated that PAA-BA possess much better cytocompatibility than PEI, yieldingslight cell morphological change, high cell viability and mild effect on cell membrane damage as well asinducing less apoptotic/necrotic cells at optimal N/P ratio. PAA-BA also exhibited better tissue compatibility,reflected by no or less inflammatory response in the site of muscle injection at the same (0.03% w/v)or higher concentration (0.1% w/v) and no hepatic tissue morphological change with normal hepatocytes.We concluded that PAA-BA was promising and safe candidate for in vitro BMSCs gene delivery and hadpotential for in future in vivo gene therapy.

Polyamidoamine dendrimers using inositol as a core were designed, synthesized andcharacterized. The electrostatics interaction of PAMAM dendrimer with DNA causes the condensationof DNA to form DNA/dendrimer complexes. The sizes of complexes prepared from dendrimerG6 and pRE Luc at N/P ratio 10︰1 range from 80 to 300nm. Dendrimers G3 G6 are able toefficiently deliver pRE Luc and pSV-gal gene into HeLa and HEK 293 cells. The transfectionefficiencies of G5 and G6 are much higher than that of poly L-lysine (PLL) and comparable to that ofthe commercial branched polyethylenimine (PEI, Mw 25000), whereas the cytoctoxicities of G5 andG6 are lower than that of PLL.