A Study Of Enhanced Gene Transfection And Serum Stability Of Pdmaema-based Polyplexes

In this work, we synthesized a star cationic polymer(s-PDMAEMA) consisting of cleavable poly[N, N-bis(acryloyl) cystamine](PBAC) crosslinked core and poly(N, N-dimethyl-ethylamine methacrylate)(PDMAEMA) arms by atomic transfer radical polymerization using one-pot‘‘arm first’’method. The s-PDMAEMA that was degradable in a mimic intracellular redox environment was more efficient in condensing DNA. It was shown that s-PDMAEMA achieved higher gene transfection levels relative to their linear precursors and s-PDMAEMA200 with longer and more arms exhibited superior transfection efficiencies and lower cytotoxicity compared to PEI25K. The buffer capacities of polymers were examined by acid-base titration; the pH-dependent morphological evolution and enzyme stability of PDMAEMA/DNA complexes were investigated by atomic force microscopy(AFM) and time-resolved fluorescence spectroscopy, respectively. The results indicated that the star polymers exhibited a stronger buffering ability than their linear precursors due to the increased inner osmotic pressure. Decreasing the pH from 7.4 to 5.0, the linear PDMEMA/DNA complexes became more compact; in contrast, s-PDMAEMA200/DNA complex adopted a loose morphology due to the steric barrier of inter-arms and outward extension of positively charged arms. Analysis of the fluorescence life times of free and intercalated ethidium bromide unveiled more effective protection of DNA afforded by s-PDMAEMA. The effect of medium pH on the star PDMAEMA system was smaller owing to the ability of densely tertiary amino groups along multiple arms to absorb more protons, which was favorable for endosomolytic escape of complexes.A polysulfobetaine-cationic methacrylate copolymer: 2-(dimethylamino) ethyl methacrylate-block-(N-(3-(methacryloylamino) propyl)-N, N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide) (PDMAEMA-b-PMPDSAH) diblock copolymer was synthesized via atomic transfer radical polymerization method and investigated as a new non-viral vector for gene delivery. Incorporation of polysulfobetaine into cationic methacrylate retained a better DNA condensation capability. MTT assays revealed that the cytotoxicity of PDMAEMA200-PMPDSAHn copolymer was lower than that of PDMAEMA200. PDMAEMA200-PMPDSAH80 which was much superior to its homopolymer in mediating gene transfection demonstrated comparable efficiency to PEI25 kDa at a weight ratio of 8 in the presence of 10% serum. At higher serum contents, the transfection of PDMAEMA200 and PEI25 kDa was deteriorated, whereas PDMAEMA200-PMPDSAH80 still retained better transfection efficiency, 4-5 fold more effective than PEI25 kDa. For the sake of comparative study, we synthesized structurally similar copolymer from DMAEMA and 2-methacryloyloxyethyl phosphorylcholine, PDMAEMA200-PMPC80. PDMAEMA200-PMPDSAH80 exhibited much higher gene transfer levels than PDMAEMA200-PMPC80 under the same conditions. The results of flow cytometry indicated that highly hydrophilic MPC block profoundly impeded the cellular internalization of nanocomplexes; in contrast, incorporation of polysulfobetaine remained the increased cellular uptake. Differential scanning calorimetry assay of thermodynamic phase transition of dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) induced by polymer vectors demonstrated that MPC only marginally contributed to the perturbation of DPPC; polysulfobetaine facilitated more evident perturbation of DPPC bilayer instead, an indication that polysulfobetaine units could aid in the endocytosis of nanocomplexes.