pH-Responsive PEGylated Gene Delivery System†

doi:10.7503/cjcu20150907

Abstract

Abstract:

The pH-sensitive polyethylene glycol-grafted polyethylenimine(mPEG-CH═N-PEI) was synthesized via benzoic imine linker according to the acidic environment of endosomal. The corresponding stable PEGylated polyethylenimine(mPEG-PEI) without pH-sensitivity was synthesized as control. The results showed that both PEGylated polycations could condense DNA into tight spherical nanoparticles about 80 nm in size and 10 mV in ξ-potential, which showed excellent stability under physiological conditions. The benzoic imine linkers were easily cleaved under the pH of 5, which was similar with the endosomal pH. Human hepatoblastoma cell line(HepG2) cell culture results indicated that the cytotoxicity and cellular uptake efficiency of both PEGylated gene nanoparticles decreased remarkably because of the shielding effect of PEG shell. Due to the cleavage of benzoic imine linker at acidic environment of endosomal, the PEG shell of mPEG-CH═N-PEI/DNA polyplexes was detachable and lead to quick endosomal escape. So mPEG-CH═N-PEI/DNA polyplexes showed higher gene expression than the mPEG-PEI/DNA polyplexes. Based on these results, we concluded that PEGylated polyplexes via benzoic imine linkers showed high extracellular stability, intracellular DNA release and effective gene transfection.

Key words: Non-viral vector, Polyethylenimine, pH-Sensitive, PEGylation

CLC Number:

O631.3
Q782

TrendMD:

LIU Yajie, ZHANG Peng, DU Jianwei, WANG Youxiang. pH-Responsive PEGylated Gene Delivery System^†[J]. Chem. J. Chinese Universities, 2016, 37(5): 1003.

Figures/Tables 8

Fig.1 1H NMR spectra of mPEG-CHO in CDCl3(a) and mPEG-CH═N-PEI in D2O(b)

Fig.2 Agarose gel electrophoresis assay of mPEG-CH═N-PEI/DNA(A), mPEG-PEI/DNA(B), PEI/DNA(C) in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl) with various N/P ratios

Fig.3 Particle sizes(A) and ζ-potentials(B) of different polyplexes in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl)Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.4 Typical TEM images of gene polyplexes at different N/P ratiosAll the polyplexes were prepared in 20 mmol/L Hepes buffer solution(pH=7.4, 20 mmol/L NaCl). (A) mPEG-CH═N-PEI/DNA, N/P=10; (B) mPEG-CH═N-PEI/DNA, N/P=20; (C) mPEG-PEI/DNA, N/P=10; (D) mPEG-PEI/DNA, N/P=20.

Fig.5 Time-dependent change of the particle sizes under physiological salt(150 mmol/L NaCl) conditions(A) or culture media with 10% FBS(B)(t=0 means at prepared condition) and ξ-potentials of various polyplexes at pH=5.0 and 7.4(C)(A) a. PEI/DNA, pH=7.4; b. mPEG-CH═N-PEI/DNA, pH=7.4; c. mPEG-PEI/DNA, pH=7.4; d. mPEG-CH═N-PEI/DNA, pH=5.0; e. mPEG-PEI/DNA, pH=5.0. (B) a. mPEG-CH═N-PEI/DNA in DMEM with 10%(volume fraction) FBS; b. mPEG-PEI/DNA in DMEM with 10%(volume fraction) FBS; c. PEI/DNA in DMEM with 10%FBS. (C) a. mPEG-CH═N-PEI/DNA; b. mPEG-PEI/DNA. Error bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.6 Cell cytotoxicity of HepG2 cells exposed to different nanoparticles with 1 μg DNA after incubation for 48 h(A) and in vitro transfection of the luciferase gene into HepG2 cells mediated by different nanoparticles for 48 h(B)(A) Error bars represent mean± SD for n=5; (B) error bars represent mean ±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.7 Cellular uptake efficiency of different nanoparticles by HepG2 cells for incubation of 4 hError bars represent mean±SD for n=3. * Denotes statistically significant difference at p<0.05.

Fig.8 Intracellular distribution of Cy3-DNA polyplexed with mPEG-CHN-PEI(A1—A3), mPEG-PEI(B1—B3) and PEI(C1—C3) at an N/P ratio of 10(A1—C1) Cy3; (A2—C2) Lyso-Tracker; (A3—C3) Cy3+Lyso-Tracker.

References 22

[1]	Wang Y. X., Chen P., Hu Q. L., Shen J. C., Chem. J. Chinese Universities,2008, 29(11), 2289—2293
	(王幽香, 陈平, 胡巧玲, 沈家骢. 高等学校化学学报,2008, 29(11), 2289—2293)
[2]	Liu M., Chen B., Xue Y. N., Huang J., Zhang L. M., Huang S. W., Li Q. W., Zhang Z. J., Bioconjugate Chemistry,2011, 22(11), 2237—2243
[3]	Ke F. Y., Mo X. L., Yang R. M., Wang Y. M., Liang D. H., Electrophoresis,2010, 31(3), 520—527
[4]	He W. T., Xue Y. N., Peng N., Liu W. M., Zhuo R. X., Huang S. W., J. Mater. Chem., 2011, 21(28), 10496—10503
[5]	Boussif O., Lezoualc’ h F., Zanta M. A., Mergny M. D., Scherman D., Demeneix B., Behr J. P., Proceedings of the National Academy of Sciences of the United States of America,1995, 92(16), 7297—7301
[6]	Zhang N., Li J. H., Jiang W. F., Ren C. H., Li J. S., Xin J. Y., Li K., International Journal of Pharmaceutics,2010, 393(1), 213—219
[7]	Li W. Y., Liu Y. J., Du J.W., Ren K. F., Wang Y. X., Nanoscale,2015, 7(18), 8476—8484
[8]	Wu W., Zhang Q. J., Wang J. T., Chen M., Li S., Lin Z. F., Li J. S., Polym. Chem., 2014, 5(19), 5668—5679
[9]	Li W. Y., Zhang P., Zheng K. F., Hu Q. L., Wang Y. X., J. Mater. Chem. B,2013, 1(46), 6418—6426
[10]	Mindell J. A., Annual Review of Physiology, 2012, 74(1), 69—86
[11]	Xiong M. P., Bae Y., Fukushima S., Forrest M. L., Nishiyama N., Kataoka K., Kwon G. S., Chem. Med. Chem., 2007, 2(9), 1321—1327
[12]	Cai X. J., Dong C. Y., Dong H. Q., Wang G. M., Pauletti G. M., Pan X. J., Wen H. Y., Mehl I., Li Y. Y., Shi D. L., Biomacromolecules,2012, 13(4), 1024—1034
[13]	Akinc A., Bioconjugate Chemistry, 2003, 14(5), 979—988
[14]	Ding C. X., Gu J. X., Qu X. Z., Yang Z. Z., Bioconjugate Chemistry,2009, 20(6), 1163—1170
[15]	Krämer M., Türk H., Krause S., Komp A., Delineau L., Prokhorova S., Kautz H., Haag R., Angewandte Chemie International Edition,2002, 41(22), 4252—4256
[16]	Li W. Y., Chen L. N., Huang Z. X., Wu X. F., Zhang Y. F., Hu Q. L., Wang Y. X., Org.Biomol.Chem., 2011, 9(22), 7799—7806
[17]	Chen L. N., Wang H. B., Zhang Y. F., Wang Y. X., Hu Q. L., Ji J., Colloids Surf. B Biointerfaces,2013, 111(6), 297—305
[18]	Li W. Y., Wang Y. X., Chen L. N., Huang Z. X., Hu Q. L., Ji J., Chemical Communications,2012, 48(81), 10126—10128
[19]	Petersen H., Fechner P. M., Martin A. L., Kunath K., Stolnik S., Roberts C. J., Fischer D., Davies M. C., Kissel T., Bioconjugate Chemistry,2002, 13(4), 845—854
[20]	Lee R. J., Biochimica Et Biophysica Acta, 1995, 1233(2), 134—144
[21]	Moghimi S. M., Symonds P., Murray J. C., Hunter A. C., Debska G., Szewczyk A., Molecular Therapy the Journal of the American Society of Gene Therapy,2005, 11(6), 990—995
[22]	Li Y. Y., Hua S. H., Xiao W., Wang H. Y., Luo X. H., Li C., Cheng S. X., Zhang X. Z., Zhuo R. X., Journal of Materials Chemistry,2011, 21(9), 3100—3106