Fabrication of Multifunctional Gene Delivery Systems Responsible to Intracellular Microenvironments Through in situ Polymerization

doi:10.19894/j.issn.1000-0518.220080

Chinese Journal of Applied Chemistry ›› 2022, Vol. 39 ›› Issue (10): 1510-1522.DOI: 10.19894/j.issn.1000-0518.220080

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Fabrication of Multifunctional Gene Delivery Systems Responsible to Intracellular Microenvironments Through in situ Polymerization

Yuan-Hao JIAO, Hong-Yan CUI, Liu-Wei ZHANG, Shuang ZENG, Hao WANG, Ming ZHANG, Jing-Yun WANG(), Qi-Xian CHEN()

School of Bioengineering，Dalian University of Technology，Dalian 116024，China

Received:2022-03-17 Accepted:2022-07-03 Published:2022-10-01 Online:2022-10-05
Contact: Jing-Yun WANG,Qi-Xian CHEN
About author:wangjingyun67@dlut.edu.cn
qixian@dlut.edu.cn；
Supported by:
the National Natural Science Foundation of China(21878041);the Fundamental Research Funds for the Central Universities （Nos.DUT17RC（3）059, DUT20YG126）,and the Dalian Science and Technology Innovation Foundation Funding(2020JJ26SN050)

Abstract

Abstract:

The convention non-viral gene delivery systems are basically formulated based on electrostatic interactions between negatively charge nucleic acids and cationic materials， whose colloidal stabilities are not adequate in the harsh biological environment and subjected to structural disassembly. To resolve this drawback， we attempt to employ in situ polymerization onto plasmid DNA （pDNA） with a variety of monomers including N?（3-aminopropyl）methacrylamide hydrochloride，1-vinylimidazole，2-methacryloyloxyethyl phosphorylcholine and N，N'-bis（acryloyl） cystamine） for overcoming sequential biological barriers encountered in the journey of transcellular gene delivery. Overall， our proposed in situ reaction of functional monomers into covalent network on top of pDNA could afford excellent structural stabilities. Of note， the appreciable proton buffering capacities of 1-vinylimidazole due to its responsiveness to the acidic and digestive endosomal environment could facilitate the escape of the pDNA payloads from endosomal entrapment. Redox-mediated cleavage of disulfide bond in N，N'-bis（acryloyl）cystamine allows selective breakage of covalent linked network in the cytosolic microenvironment due to the striking high intracellular level of glutathione， thereby promoting liberation of the pDNA payloads in the cell interiors. The subsequent investigations determined that the proposed polymerization strategies result in well-defined nanoscaled pDNA delivery systems （135 nm in hydrodynamic radius， -6.5 mV in ζ potential and uniform spherical structures by TEM measurement）. Excellent colloidal stabilities are validated despite incubation in presence of exceeding high concentration of heparin （10 mg/mL）. In contrast， ready liberation of pDNA is confirmed when incubated in presence of glutathione by mimicking intracellular microenvironment. Cell transfection experiments verify its efficient cellular uptake and gene expression activities in the hard-transfected MCF-7 cells. To this end， the obtained results indicate the tempting potential of polymerization strategy in fabrication of covalent linked non-viral gene delivery systems， which could shed novel avenues in engineering a variety of high-performance gene delivery systems with diverse functionalities.

Key words: Gene delivery, In situ polymerization, Cross-linked polymers, Lysosomal escape, Glutathione response

CLC Number:

O648

Yuan-Hao JIAO, Hong-Yan CUI, Liu-Wei ZHANG, Shuang ZENG, Hao WANG, Ming ZHANG, Jing-Yun WANG, Qi-Xian CHEN. Fabrication of Multifunctional Gene Delivery Systems Responsible to Intracellular Microenvironments Through in situ Polymerization[J]. Chinese Journal of Applied Chemistry, 2022, 39(10): 1510-1522.

Figures/Tables 13

Fig.1 Schematic illustration of fabrication of the vector-pDNA complex and the release of pDNA in response to sequential biological microenvironments

Fig.2 Gel electrophoresis analysis of the synthesized NC at varied N/P ratios

Table 1 Monomer compositions of a number of NCs

	N?（3?氨丙基）甲基丙烯酰胺盐酸盐 APMA	1?乙烯基咪唑 VI	2?甲基丙烯酰氧乙基磷酸胆碱 MPC	交联剂 Crosslinker	质粒 Plasmid
non?NC₄₀	+	+	+	?	RFP
NC（ss）₄₀	+	+	+	BACA（+）	RFP
NC（cc）₄₀	+	+	+	MBA（+）	RFP
NC（MPC+）₄₀	+	+	+	BACA（+）	?
NC（MPC-）₄₀	+	+	?	BACA（+）	?
NC（CAG?LUC）₄₀	+	+	+	BACA（+）	CAG?LUC

Fig.3 Particle size analysis of NC（ss）40. （A） Hydrodynamic radius of NC（ss）40 by DLS measurement；（B） Microscopic morphologies of NC（ss）40 by TEM measurement

Fig.4 Agarose gel electrophoresis for insight into the structural stabilities of NC（PEI）（A）， non-NC40 （B） and NC（ss）40 （C）

Fig.5 Chemical structures of （A） N，N′-bis（acryloyl）cystamine and （B） N，N′-methylenebisacryloyl. （C） Electropherograms of two pDNA-loaded complexes after various DTT and heparin treatments

Table 2 Samples and reaction conditions in each lane in Fig.5

泳道 Line	质粒/复合物 pDNA/NC	二硫苏糖醇 DTT	肝素 Heparin
1	pDNA	?	?
2	NC（ss）₄₀	?	?
3	NC（cc）₄₀	?	?
4	NC（ss）₄₀	?	+
5	NC（cc）₄₀	?	+
6	NC（ss）₄₀	+	?
7	NC（cc）₄₀	+	?
8	NC（ss）₄₀	+	+
9	NC（cc）₄₀	+	+

Fig.6 Remaining intact DNA under treatment of DNase I of naked DNA， NC（SS）40 and non-NC40

Fig.7 Erythrocyte hemolysis rate of red blood cells upon incubation in presence of varied concentrated NC（MPC+）40 and NC（MPC-）40Note： “**” indicates P<0.01 in one-way ANOVA， “***” indicates P<0.001 in one-way ANOVA

Fig.8 Cell viabilities of MCF-7 cells under treatment of varied concentrated NC（ss）40

Fig.9 Flow cytometry analysis of endocytosis results of non-NC40 and NC（ss）40

Fig.10 Quantification of gene expression of plasmid released from NC（CAG-LUC）40

Fig.11 Quantification of gene expression of NC（CAG-LUC）40 in MCF-7 cellsNote： “**” indicates P<0.01 in one-way ANOVA

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Fabrication of Multifunctional Gene Delivery Systems Responsible to Intracellular Microenvironments Through in situ Polymerization

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