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.

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.

An amphiphilic diblock copolymer of poly(acrylic acid-b-DL-lactide) (PAAc-b-PDLLA) was synthesized byring-opening polymerization of DL-lactide initiated by hydroxyl-terminated polyacrylic acid (PAAc-OH).The critical micelle concentration (CMC) of PAAc-b-PDLLA in aqueous solution, determined by fluorescencespectroscopy using pyrene as a probe, was found about 80 mg L_1. A solution of PAAc-b-PDLLA intetrahydrofuran (THF) was dialyzed against pure water to form pH-responsive micelles. Transmissionelectron microscopy (TEM) measurement showed that the micelles exhibited regular sphericalmorphology and the diameters of particles were in the range from 40 to 90 nm. The micelles were stableat a pH above 3 or at an ionic strength below 1.0, however, they aggregated and precipitated in thesolutions when further decreasing pH or increasing ionic strength. Prednisone acetate, as a modelhydrophobic drug, was loaded into the polymeric micelles. In vitro release of prednisone acetate frompolymeric micelles showed that the release kinetics was strongly pH-dependent. Hydrophobic drugdisplayed "burst" release at pH 7.4, while only a small part of loaded drug released at pH 1.4. Thisprovides a new choice to design delivery system for the gastrointestinal tract (GI tract), where the pHenvironment is strongly acidic in stomach and basic in intestine. The cytotoxicity measurement by MTTassay indicated that PAAc-b-PDLLA was low toxic in HeLa cells with an IC50 value of 2.8 mg mL_1, whichsuggests that PAAc-b-PDLLA could be used as a safe candidate for pH-responsive drug delivery.

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.