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We synthesized a novel arginine-grafted dendritic block copolymer, R-PAMAM-PEG-PAMAM-R G5 (PPP5-R) for gene delivery systems. Its Mw was measured as 2.74 x 104 Da by MALDI-TOF, and approximately 36 arginine residues are found to be grafted to the polymer by 1H NMR. PPP5-R was able to form polyplexes with plasmid DNA, the average size of which was about 200 nm. Positive zeta-potential values (+22 to +28 mV) of PPP5-R polyplex indicate the formation of positively charged stable polyplex particles and suggest that large dendritic blocks with high positive charge may not be fully shielded by PEG chains even after PEG-coated complex formation. PPP5-R polyplex shows enhanced water solubility due to the polymer's PEG core and also shows low cytotoxicity, representing the potential for in vivo application. We identified the greatly enhanced transfection efficiency of PPP5-R in comparison with that of native PPP5 on various cell lines. Moreover, in view of the result of various cellular uptake inhibitor treatments during a transfection step, the cellular uptake of PPP5-R polyplex leading to effective transfection is thought to be not dependent on one exclusive pathway and to have the possibility of multiple pathways (caveolae-, clathrin-, and macropinocytosis-mediated pathways), contrary to the caveolae-dependent uptake of the PPP5 polyplex lacking arginine residues.

A lot of non-viral gene delivery carriers including cationic lipids and polymers have been developed due to their advantages such as easy introduction of specific bio-functionalities, convenient handling, non-immunogenicity and unlimited capacity of genes delivered. Poly(ethylenimine) (PEI) and poly(amido amine) (PAMAM) dendrimer are representative and commercialized polymeric gene delivery carriers. PEI shows very high transfection efficiency caused by its ‘endosome buffering’ capacity but its severe cytotoxicity limited its application for gene delivery and led its chemical modification for reducing the cytotoxicity. Recently, interesting strategies based on the preincubation of potent glucocorticoid, dexamethasone before transfection and the conjugation of mitochondrial leader peptide to PEI have been also tried in order to enhance the transfection efficiency of PEI by glucocorticoid ligand-receptor interaction and to perform selective delivery of genes to mitochondria, respectively. The transfection efficiency of PAMAM dendrimer is lower than that of PEI. However, its controllable multivalent functionality, defined structure and biocompatibility is still arising many scientists’ interest for gene delivery systems. So, we intended to design a novel hybrid dendrimer, poly(amido ethylenimine) (PAMEIM) dendrimer possessing the endosome buffering capacity of PEI and the defined structure, biocompatibility of PAMAM dendrimer in order to combine their advantages. Here, we report synthesis of a novel PAMEIM dendrimer and characterization of its self-assembly with plasmid DNA in order to identify its potential as a gene delivery carrier.

In recent years, various cationic polymers constructed using ester bonds, phosphoester bonds or disulfide bonds have been synthesized to create environmentally-responsible biodegradable delivery systems. Among them, a crosslinked type polymeric gene delivery carrier was reported to show enhanced transfection efficiency due to its threedimensional structure leading endosome buffering effect, and self-assemble with plasmid DNA giving more stable polyplexes due to its slow degradation in aqueous media, in contrast to linear type carriers. Several quaternary amine-based polymeric gene delivery carriers were reported and they showed considerable gene delivery potencies. Moreover, a primary amine-based biodegradable polymeric carrier was known to have potentially rapid self-destructive tendency due to the attack of its own primary amines. Because quaternary amines have no nucleophilicity, quaternary amine-based biodegradable polymeric carriers have no possibility of ‘self-destruction’. So, we designed a novel quaternary amine-based crosslinked poly(β-amino ester) polymer (Q-CLPAE). Here, we report the synthesis, characterization of QCLPAE. In addition, the self-assembly of polymers with plasmid DNA and in vitro disassembly of plasmid DNA from polyplexes are presented. Q-CLPAE was synthesized by using a Michael reaction of a quaternary amine monomer and a triacrylate monomer according to Lynn et al. First, a quaternized amine monomer was synthesized through the following steps (Scheme 1). Fmoc blocking reaction of N,N-dimethylethylenediamine (DMEDA). Fmoc-OSu (1 g, 2.96 mmol, Novabiochem, Laufelfingen, Switzerland) was dissolved in 1,4dioxane. DMEDA (0.488 mL, 4.45 mmol, Sigma-Aldrich, St. Louis, MO) was dissolved in 1,4-dioxane and added to an Fmoc-OSu solution dropwise, keeping it at r.t. overnight. Then, the reaction mixture was poured into diethyl ether and extracted with water to remove the residual DMEDA. The ether layer was extracted again with 0.1 M HCl solution. At this time, the acidified product was transferred to the HCl layer and the residual Fmoc-OSu was left in the ether layer. The HCl layer was basified to ~ pH 11 with 1 N NaOH solution and the hydrophobic product in the aqueous layer was extracted with diethyl ether. Finally, after evaporation of the ether layer, a purified product, N-(9-Fluorenylmethoxycarbornyl)-N',N'-dimethylethylenediamine (Fmoc-DMEDA) was left as a white solid. Methylation of Fmoc-DMEDA. Fmoc-DMEDA (195 mg, 0.598 mmol) was dissolved in DMF. 3.72 mL of iodomethane (d = 2.28, 59.8 mmol, Sigma-Aldrich) was added to an Fmoc-DMEDA solution, keeping it at r.t. overnight. The product was precipitated with diethyl ether three times to remove the unreacted materials, leaving a light yellow solid, N-(9-Fluorenylmethoxycarbornyl)-N',N',N'trimethylethylenediamine (Fmoc-TMEDA). Fmoc deblocking of Fmoc-TMEDA. Fmoc-TMEDA (101 mg, 0.296 mmol) was dissolved in DMF and mixed with an equal volume of 30% piperidine solution (DMF). After stirring at room temperature, the product was pre

We synthesized arginine-conjugated polypropylenimine dendrimer G2 (DAB-8), PPI2-R for gene delivery systems. Synthesized PPI2-R could retard plasmid DNA at a weight ratio of 4 completely and PPI2-R polyplexes showed a fluorescence of less than 10% over a charge ratio of 2 by PicoGreen reagent assay, suggesting its good DNA condensing ability. The size of PPI2-R polyplex was measured to about 200nm at a charge ratio of 150. PPI2-R displayed 80-90% cell viability at even a 150microg/mL concentration. Transfection efficiency of PPI2-R was found to be high comparable to that of PEI25kD and to be 8-214 times higher than that of unmodified PPI2 on HeLa and 293 cells. Moreover, PPI2-R showed 4 times higher transfection efficiency than PEI25kD, treating with 10microg pDNA because of its low cytotoxicity on HeLa cells. Finally, PPI2-R showed a transfection efficiency 2-3 times higher than PEI25kD on HUVECs, showing its potency as a gene delivery carrier for primary cells. These results demonstrate that arginine-conjugation of PPI2 is successful in developing a low toxic and highly transfection efficient gene delivery carrier.

Biodegradable cross-linked poly(beta-amino ester) (CLPAE) was synthesized by Michael addition of pentaerythritol triacrylate and N,N-dimethylethylenediamine and modified with aminohexanoic acid and lysine to CLPAE-Ahx and CLPAE-Lys, respectively, for a gene delivery system. They could self-assemble with plasmid DNA, forming nanosized polyplexes, and CLPAE-Ahx polyplex released plasmid DNA slowly during a week through stepwise degradation. The polymers showed minimal cytotoxicity on 293 cells due to their biodegradability and biocompatibility. Transfection efficiencies of CLPAE-Ahx and CLPAE-Lys were comparable to that of PEI in 293 cells and C2C12 cells. Additionally, high transfection of CLPAE-Ahx on primary rat aorta vascular smooth muscle cells (SMC) and primary mouse embryonic fibroblast cells (MEF) shows a potential for a gene delivery system on primary cells, restenosis treatment of human SMC, and MEF cell function research. In conclusion, CLPAE-Ahx could be used as a nontoxic and highly efficient gene delivery system.