Multifunctional peptide conjugated amphiphilic cationic copolymer for enhancing ECs targeting, penetrating and nuclear accumulation

doi:10.1007/s11705-020-1919-8

Front. Chem. Sci. Eng.

2020, Vol. 14

Issue (5) : 889-901 https://doi.org/10.1007/s11705-020-1919-8

RESEARCH ARTICLE

Multifunctional peptide conjugated amphiphilic cationic copolymer for enhancing ECs targeting, penetrating and nuclear accumulation

Xinghong Duo^1,², Lingchuang Bai¹, Jun Wang¹, Jintang Guo¹, Xiangkui Ren^1,^3,⁴, Shihai Xia⁵, Wencheng Zhang⁶, Abraham Domb⁷, Yakai Feng^1,^3,⁴(

)

¹. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China
². School of Chemistry and Chemical Engineering, Qinghai University for Nationalities, Xining 810007, China
³. Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Tianjin 300350, China
⁴. Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China
⁵. Department of Hepatopancreatobiliary and Splenic Medicine, Affiliated Hospital, Logistics University of People’s Armed Police Force, Tianjin 300162, China
⁶. Department of Physiology and Pathophysiology, Logistics University of People’s Armed Police Force, Tianjin 300162, China
⁷. Institute of Drug Research (IDR), School of Pharmacy-Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91905, Israel

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Abstract

Gene therapy has drawn great attention in the treatments of many diseases, especially for cardiovascular diseases. However, the development of gene carriers with low cytotoxicity and multitargeting function is still a challenge. Herein, the multitargeting REDV-G-TAT-G-NLS peptide was conjugated to amphiphilic cationic copolymer poly(ε-caprolactone-co-3(S)-methyl-morpholine-2,5-dione)-g-polyethyleneimine (PCLMD-g-PEI) via a heterobifunctional orthopyridyl disulfide-poly(ethylene glycol)-N-hydroxysuccinimide (OPSS-PEG-NHS) linker to prepare PCLMD-g-PEI-PEG-REDV-G-TAT-G-NLS copolymers with the aim to develop the gene carriers with low cytotoxicity and high transfection efficiency. The multitargeting micelles were prepared from PCLMD-g-PEI-PEG-REDV-G-TAT-G-NLS copolymers by self-assembly method and used to load pEGFP-ZNF580 plasmids (pDNA) to form gene complexes for enhancing the proliferation and migration of endothelial cells (ECs). The loading pDNA capacity was proved by agarose gel electrophoresis assay. These multitargeting gene complexes exhibited low cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The high internalization efficiency of these gene complexes was confirmed by flow cytometry. The results of in vitro transfection demonstrated that these multitargeting gene complexes possessed relatively high transfection efficiency. The rapid migration of ECs transfected by these gene complexes was verified by wound healing assay. Owing to ECs-targeting ability, cell-penetrating ability and nuclear targeting capacity of REDV-G-TAT-G-NLS peptide, the multitargeting polycationic gene carrier with low cytotoxicity and high transfection efficiency has great potential in gene therapy.

Keywords gene carriers multitargeting function ECs transfection efficiency

Corresponding Author(s): Yakai Feng

Just Accepted Date: 30 December 2019 Online First Date: 09 April 2020 Issue Date: 25 May 2020

Cite this article:

Xinghong Duo,Lingchuang Bai,Jun Wang, et al. Multifunctional peptide conjugated amphiphilic cationic copolymer for enhancing ECs targeting, penetrating and nuclear accumulation[J]. Front. Chem. Sci. Eng., 2020, 14(5): 889-901.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-020-1919-8
https://academic.hep.com.cn/fcse/EN/Y2020/V14/I5/889

Fig.1 Scheme 1 Synthesis route of CP-CREDVW and CP-REDV-G-TAT-G-NLS copolymers.

Fig.2 ¹H NMR spectra of amphiphilic copolymers in CDCl₃ and DMSO-d₆. (A(a)) PCLMD-OH, (A(b)) PCLMD-COOH, and (A(c)) PCLMD-g-PEI in CDCl₃. (B) CP-CREDVW and (C) CP-REDV-G-TAT-G-NLS in DMSO-d₆.

Fig.3 Fluorescence emission spectra of (A) CP-CREDVW and (B) CP-REDV-G-TAT-G-NLS copolymers after dialysis. The inserted figures were the standard curve of CREDVW and REDV-G-TAT-G-NLS peptides. The curve in the upper right of Fig. 2(A) was the standard curve of CREDVW peptide, y = 526.48x– 42.67; the curve in the upper right of Fig. 2(B) was the standard curve of REDV-G-TAT-G-NLS peptide, y = 1480.56x + 20.99.

Tab.1 Size and zeta potential of CP, CP-CREDVW, CP-CREVDW and CP-REDV-G-TAT-G-NLS copolymer micelles

Fig.4 (A) Size and (B) zeta potential of CP/pDNA, CP-CREDVW/pDNA, CP-CREVDW/pDNA, and CP-REDV-G-TAT-G-NLS/pDNA gene complexes at different N/P molar ratios.

Fig.5 Agarose gel electrophoresis of gene complexes at different N/P molar ratios. (A) CP/pDNA gene complexes, (B) CP-CREDVW/pDNA gene complexes, (C) CP-CREVDW/pDNA gene complexes, and (D) CP-REDV-G-TAT-G-NLS/pDNA gene complexes.

Fig.6 Relative cell viability of EA.hy926 cells after 48 h of treatment with different PEI concentrations of micelles and complexes at N/P molar ratio of 10. (n = 3, mean±SD, *statistically different from PEI group (p<0 .05), ^#statistically different from PEI/pDNA group (p<0.05)).

Fig.7 (A) Mean fluorescence intensity (the column chart) and cellular uptake (%) (the line chart) and (B) Intracellular fluorescence intensity of different gene complexes in ECs measured by flow cytometry. (a) cells treated with CP/Cy5-oligonucleotide complexes, (b) cells treated with CP-CREDVW/Cy5-oligonucleotide complexes, (c) cells treated with CP-REDV-G-TAT-G-NLS/Cy5-oligonucleotide complexes, (d) cells treated with PEI (10 kDa)/Cy5-oligonucleotide complexes as the positive control, (e) treated with Cy5-oligonucleotide as the negative control. (n = 3, mean±SD, *statistically different from d group (p<0.05)).

Fig.8 (A) Fluorescence images (image below) and corresponding bright-field image (image above) of EA. hy926 cells transfected by different gene complexes for 24 h, and (B) the cell transfection efficiency at 24 h. (a) cell treated with CP/pDNA gene complexes, (b) cell treated with CP-CREDVW/pDNA gene complexes, (c) cell treated with CP-CREVDW/pDNA gene complexes, (d) cell treated with CP-REDV-G-TAT-G-NLS/pDNA gene complexes, (e) cell treated with PEI (10 kDa)/pDNA gene complexes as a control. (n = 3, mean±SD, *statistically different from e group (p<0.05))

Fig.9 (A) Wound recovery of EA.hy926 cells at 0 and 12 h time points, and (B) the relative recovered area after 12 h. (a) cells treated with CP/pDNA gene complexes, (a) cells treated with CP-CREDVW/pDNA gene complexes, (c) cells treated with CP-CREVDW/pDNA gene complexes, (d) cells treated with CP-REDV-G-TAT-G-NLS/pDNA gene complexes, (e) cells treated with PEI (10 kDa)/pDNA gene complexes as a control. (n = 3, mean±SD, *statistically different from e group (p<0.05)).

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