Abstract：The clinical application of cationic polymer carriers was limited because the adsorption of protein in serum environment led to low transfection efficiency. In this work, we synthesized carboxylic Dextran (Dex-COOH) and water-soluble carboxylic fullerene (C60-Dex-COOH) anionic polymers, coated on the pullulan-spermine and siRNA complex (PS/siRNA) to reduce the adsorption of serum protein. The particle size and Zeta potential were analyzed by DLS/ELS, respectively. The formation of anion coating and bovine serum albumin (BSA) adsorption of complexes were tested by QCM-D. Cellular uptake, cytotoxicity on the Hela cells and RNAi efficiency on the Hela-EGFP cells were examined by cck-8 reagent and flow cytometry. The results showed that Dex-COOH and C60-Dex-COOH could coat on PS/siRNA to form stable electronegative complexes, prevent the adsorption of serum effectively. Cytotoxicity of complexes was reduced in the absence of serum. On the other hand, in the serum environment, anionic polymer coated complexes could help complexes enter cells and PS/siRNA@C60-Dex-COOH with visible light irradiation would escape from the lysosome, increased the interference efficiency by 20% compared with PS/siRNA. In summary, this coating strategy provided an effective solution of increasing RNAi efficiency mediated by cationic polymers.
[1] Maguire AM, High KA, Auricchio A, et al. Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial [J]. Lancet,2009, 374(9701):1597-1605. [2] Cartier N, Hacein-Bey-Abina S, Bartholomae CC, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy [J]. Science, 2009, 326 (5954):818-823. [3] Dong Jie, Li Song, Mo Jinglin, et al. Nurr1-based therapies for Parkinson's disease [J]. CNS Neurosci Ther, 2016, 22(5):351-359. [4] Fan Xiaohui, Zhao Yanli, Xu Wei, et al. Linear-dendritic block copolymer for drug and gene delivery [J]. Mater Sci Eng C Mater Biol Appl, 2016, 62:943-959. [5] Collins M, Thrasher A. Gene therapy:progress and predictions [J]. Proc Biol Sci, 2015, 282(1821): 2014-3003. [6] Pack DW, Putnam D, LangerR. Design of imidazole-containing endosomolytic biopolymers for gene delivery [J]. Biotechnol Bioeng, 2000, 67(2):217-223. [7] Moreira C, Oliveira H, Pires LR, et al. Improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone [J]. Acta Biomater, 2009, 5:2995-3006. [8] Hunter AC. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity [J]. Adv Drug Deliv Rev, 2006, 58:1523-1531. [9] Zhang Jingshi, Liu Feng, Huang L. Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity [J]. Adv Drug Deliv Rev, 2005, 57:689-698. [10] Reischl D, Zimmer A. Drug delivery of siRNA therapeutics: potentials and limits of nanosystems [J]. Nanomedicine, 2009, 5:8-20. [11] Ogris M, Brunner S, Schüller S, et al. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery [J]. Gene Ther, 1999, 6:595-605. [12] Rekha MR, Sharma CP. Blood compatibility and in vitro transfection studies on cationically modified pullulan for liver cell targeted gene delivery [J]. Biomaterials, 2009, 30:6655-6664. [13] Sun Jingjing, Luo Ting, Sheng Ruilong, et al. Preparation of functional water-soluble low-cytotoxic poly (methacrylate)s with pendant cationic L-lysines for efficient gene delivery [J]. Macromol Biosci, 2013, 13(1):35-47. [14] Liu Jian, Tabata Y. Effect of modification manner on the photodynamic antitumor activity of C60 modified with pullulan [J]. J Biomater Sci Polym Ed, 2011, 22(16):2147-2163. [15] Tiscornia G, Singer O, Ikawa M, et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA [J]. Proc Natl Acad Sci USA, 2003, 100:1844-1848. [16] Villares GJ, Zigler M, Wang Hua, et al. Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA [J]. Cancer Res, 2008, 68:9078-9086. [17] Ma Wenjuan, Liu Jian, XieJundan, et al. Modulating the growth and imatinib sensitivity of chronic myeloid leukemia stem/progenitor cells with pullulan/microRNA nanoparticles in vitro [J]. J Biomed Nanotechnol,2015, 11(11):1961-1974. [18] Glomm WR, Halskau ? Jr, Hanneseth AM, et al. Adsorption behavior of acidic and basic proteins onto citrate-coated Au surfaces correlated to their native fold, stability, and PI [J]. J PhysChem B, 2007, 111(51):14329-14345. [19] Briand E, Gu C, Boujday S, et al. Functionalisation of gold surfaces with thiolate SAMs: Topography/bioactivity relationship—A combined FT-RAIRS, AFM and QCM investigation [J]. Surf Sci, 2007, 601(18):3850-3855. [20] Zhang Weiqi, Liu Jian, Tabata Y, et al. The effect of serum in culture on RNAi efficacy through modulation of polyplexes size [J]. Biomaterials, 2014, 35(1):567-577.
