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.

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.

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.

Poly(amidoamine)s with pendant primary amine (polymer 1a-1c) were evaluated as in vitro non-viralgene delivery vectors for bone marrow stromal cells (BMSCs). The cytotoxicity of these poly(amidoamine)s, measured by MTT assay, increased with increasing length of side chain, however, they were lesstoxic than branched polyethylenimine (PEI) 25 kDa. Using pGL-3 and pEGFP-C1 as luciferase gene andgreen fluorescent protein (GFP) gene, among all polycations including polymer 1a-1c and PEI, polymer1b at optimal N/P ratio showed highest luciferase expression (1.92×108 RLU/mg protein) as well aspercentage of cells expressing GFP (29.01±2.33%). For all polycations, intracellular trafficking of Cy3-labelled plasmid DNA (pDNA) was similar. Fluorescent particles attached to cell membrane at 0.5h afteradding the polycation/DNA complexes, aggregated in cytoplasm after 2h, and then stayed around theperinuclear region after 4h. pDNA nuclear localization appeared at 4h post-transfection, but much morepDNA entered into nucleus at 24h. At high N/P ratio, polymer 1a-1c could deliver pDNA into 70-80% ofBMSCs after 24h transfection, however, labelled pDNA was observed in only 4-25% of cells at the sametime. Compared to PEI, polymer 1b showed comparable or even higher percentage of pDNA uptake andnuclear localization. We concluded that poly(amidoamine)s with pendant primary amine, especiallypolymer 1b, are new kind of promising candidates of less toxic and highly efficient non-viral genedelivery vectors for BMSCs.

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.