Nanocarrier-mediated PiggyBac transposon system for preparation of CAR-NK cells
10.3872/j.issn.1007-385x.2020.02.002
- VernacularTitle:纳米载体介导的PiggyBac转座子制备CAR-NK细胞
- Author:
YUE Ran
1
,
2
,
3
;
LIU Ziyang
3
,
4
;
ZHENG Yan
3
,
4
;
LU Xiaodan
3
,
4
;
HU Shanshan
3
,
4
;
ZHANG Bingyong
3
,
4
;
LI Xiuling
3
,
5
,
6
;
LI Jingguo
3
,
4
;
HAN Shuangyin
3
,
4
Author Information
1. (1. Postgraduate Division, Xinxiang Medical College, Xinxiang 453003, Henan, China
2. 2. Department of Gastroenterology, Zhengzhou University People&rsquo
3. s Hospital, Zhengzhou 450003, Henan, China
4. 2. Department of Gastroenterology, Zhengzhou University People&rsquo
5. 3. Henan Provincial Eye Hospital &
6. Eye Center of Henan Provincial People&rsquo
- Publication Type:Journal Article
- Keywords:
nanocarrier;
PiggyBac transposon;
chimeric antigen receptor (CAR);
NK cell;
tumor immunotherapy
- From:
Chinese Journal of Cancer Biotherapy
2020;27(2):109-114
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.
- Full text:20200202.pdf
