Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA conjugate

Junxiao Ye; Xing Pei; Hui Cui; Zhili Yu; Hyukjin Lee; Jianxin Wang; Xu Wang; Lu Sun; Huining He; Victor C. Yang

Journal of Industrial and Engineering Chemistry, Vol.63, 103-111, July, 2018

DOI10.1016/j.jiec.2018.02.005 Export Citation

Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA conjugate

Junxiao Ye^{1, 2}, Xing Pei¹, Hui Cui¹, Zhili Yu¹, Hyukjin Lee³, Jianxin Wang⁴, Xu Wang¹, Lu Sun^{1, †}, Huining He^{1, †}, and Victor C. Yang^{1, 5}

¹Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
²College of Pharmacy, Tsinghua University, Beijing 100084, China
³College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 13760, Republic of Korea
⁴Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, China
⁵Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA

E-mail:,

The covalent attachment of CPPs to siRNA molecules offers great potential for CPP-mediated siRNA delivery. We recently reported a concise and high-yield synthesis strategy of the cell-permeable, cytosoldissociable LMWP-siRNA covalent conjugate. Herein, cell uptake mechanism and cellular toxicity studies of this conjugate were performed to evaluate the potential of LMWP-siRNA conjugate for clinical translation. Cellular uptake mechanism study indicated that the conjugate could be taken up by cells via multiple pathways, including direct penetration of the plasma membrane and clathrin- and caveolae- independent endocytosis. In vitro cytotoxicity study revealed that the conjugation promoted internalization in a low-toxic fashion.

Keywords:Cell penetrating peptide;siRNA delivery;Covalent CPP-siRNA chemical conjugate;Cell uptake mechanism;Cytotoxicity

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[Referenced By]

[1] van Rij RP, Andino R, Trends Biotechnol., 24, 186 (2006)

[2] Draz MS, Fang BA, Zhang P, Hu Z, Gu S, Weng KC, Gray JW, Chen FF, Theranostics, 4, 872 (2014)

[3] Thi EP, Mire CE, Lee ACH, Geisbert JB, Zhou JZ, Agans KN, Snead NM, Deer DJ, Barnard TR, Fenton KA, MacLachlan I, Geisbert TW, Nature, 521(7552), 362 (2015)

[4] Shen J, Kim HC, Su H, Wang F, Wolfram J, Kirui D, Mai J, Mu C, Ji LN, Mao ZW, Shen H, Theranostics, 4, 487 (2014)

[5] Choi YS, Lee JY, Suh JS, Kwon YM, Lee SJ, Chung JK, Lee DS, Yang VC, Chung CP, Park YJ, Biomaterials, 31, 1429 (2010)

[6] Guo P, Yang J, Jia D, Moses MA, Auguste DT, Theranostics, 6, 1 (2016)

[7] Lee J, Saw PE, Gujrati V, Lee Y, Kim H, Kang S, Choi M, Kim JI, Jon S, Theranostics, 6, 192 (2016)

[8] Dowdy SF, Nat. Biotechnol., 35, 222 (2017)

[9] Ye J, Shin MC, Liang Q, He H, Yang VC, J. Control. Release, 205, 58 (2015)

[10] Huang YZ, Jiang YF, Wang HY, Wang JX, Shin MC, Byun Y, He HN, Liang YQ, Yang VC, Adv. Drug Deliv. Rev., 65, 1299 (2013)

[11] Mae M, Langel U, Curr. Opin. Pharmacol., 6, 509 (2006)

[12] Copolovici DM, Langel K, Eriste E, Langel U, ACS Nano, 8, 1972 (2014)

[13] Meade BR, Dowdy SF, Adv. Drug Deliv. Rev., 59, 134 (2007)

[14] Reischl D, Zimmer A, Nanomedicine, 5, 8 (2009)

[15] Shi NQ, Qi XR, Xiang B, Zhang Y, J. Control. Release, 194, 53 (2014)

[16] Sawant R, Torchilin V, Mol. Biosyst., 6, 628 (2010)

[17] Freire JM, de Figueiredo IR, Valle J, Veiga AS, Andreu D, Enguita FJ, Castanho MARB, J. Control. Release, 245, 127 (2017)

[18] Park YJ, Liang JF, Ko KS, Kim SW, Yang VC, J. Gene Med., 5, 700 (2003)

[19] Park YJ, Chang LC, Liang JF, Moon C, Chung CP, Yang VC, Faseb J., 19, 1555 (2005)

[20] Ye J, Liu E, Gong J, Wang J, Huang Y, He H, Yang VC, Theranostics, 7, 2495 (2017)

[21] El-Andaloussi S, Jarver P, Johansson HJ, Langel U, Biochem. J., 407, 285 (2007)

[22] Godbey WT, Wu KK, Mikos AG, J. Control. Release, 60, 149 (1999)

[23] Nabzdyk CS, Chun MC, Oliver-Allen HS, Pathan SG, Phaneuf MD, You JO, Pradhan-Nabzdyk LK, LoGerfo FW, Biomaterials, 35, 3071 (2014)

[24] Kong L, Qiu J, Sun W, Yang J, Shen M, Wang L, Shi X, Biomater. Sci., 5, 258 (2017)

[25] Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A, Gene Ther., 12, 461 (2005)

[26] Spagnou S, Miller AD, Keller M, Biochemistry, 43, 13348 (2004)

[27] Filion MC, Phillips NC, Biochim. Biophys. Acta, 1329, 345 (1997)

[28] Kim SW, Ogawa T, Tabata Y, Nishimura I, Biomed J, Mater. Res. A, 71a, 308 (2004)

[29] Gunther M, Lipka J, Malek A, Gutsch D, Kreyling W, Aigner A, Eur. J. Pharm. Biopharm., 77, 438 (2011)

[30] Schafer J, Hobel S, Bakowsky U, Aigner A, Biomaterials, 31, 6892 (2010)

[31] Israel LL, Lellouche E, Ostrovsky S, Yarmiayev V, Bechor M, Michaeli S, Lellouche JP, ACS Appl. Mater. Interfaces, 7, 15240 (2015)

[32] Patel LN, Zaro JL, Shen WC, Pharm. Res., 24, 1977 (2007)

[33] Ye JX, Liu EG, Yu ZL, Pei X, Chen SH, Zhang PW, Shin MC, Gong JB, He HN, Yang VC, Int. J. Mol. Sci., 17, 1892 (2016)

[34] Zaro JL, Shen WC, Exp. Cell Res., 307, 164 (2005)