Study on the Delivery of RGD Modified Virus-Like Particles to ICG Targeted Tumors

doi:10.13523/j.cb.2001065

China Biotechnology

2020, Vol. 40

Issue (7): 22-29 DOI: 10.13523/j.cb.2001065

Study on the Delivery of RGD Modified Virus-Like Particles to ICG Targeted Tumors

JIANG Dan-dan¹,WANG Yun-long^2,^**(

),LI Yu-lin²,Zhang Yi-qing²

1 Henan Normal University, Xinxiang 453000, China
2 Henan Bioengineering Technology Research Center, Zhengzhou 450000, China

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Abstract

Objective: To obtain hepatitis B core virus-like particles containing RGD targeting peptide and provide a new carrier for drug targeting nanometer delivery system. Methods: RGD-modified recombinant plasmid of hepatitis B virus was transformed into E. coli BL21 (DE3), and the optimal expression conditions of recombinant protein were investigated by single factor analysis and orthogonal test. Under the optimum expression conditions, the bacteria were cultured, collected after ultrasonic crushing, centrifuged, purified by Gel filtration chromatography, ion exchange and sucrose density gradient centrifugation, and the morphology and stability of RGD-HBc VLPs were identified by transmission electron microscopy.The purified RGD-HBc VLPs loaded the photosensitizer ICG into the inner part of the particle and injected it intravenously into 4T1 tumor-bearing mice of breast cancer to explore the targeting of recombinant RGD-HBc VLPs as a nano-delivery system. Results: RGD-HBc VLPs was efficiently expressed in the form of soluble protein at 32℃ and IPTG at 0.5mmol/L for 4h.After centrifugation with sucrose density gradient, the purity reached above 95%.The purified RGD-HBc VLPs were observed under transmission electron microscopy in uniform shape and size, with a diameter of about 32nm. Near-infrared fluorescence in vivo imaging confirmed the targeting of RGD-HBc VLPs as a nano-carrier. Conclusion: After expression and purification, RGD-HBc VLPs has a high expression level and uniform appearance, and near-infrared fluorescence in vivo imaging has a good targeting property, which not only provides a fast, accurate and convenient method for the visual diagnosis of tumor, but also provides a new carrier for future targeted immunotherapy.

Key words： RGD-HBc VLPs Expression purification Targeting Near infrared fluorescence in vivo imaging

Received: 03 February 2020 Published: 13 August 2020

ZTFLH:

Q819

Corresponding Authors: Yun-long WANG E-mail: biojdd@126.com

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	Dan-dan JIANG
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Cite this article:

JIANG Dan-dan,WANG Yun-long,LI Yu-lin,Zhang Yi-qing. Study on the Delivery of RGD Modified Virus-Like Particles to ICG Targeted Tumors. China Biotechnology, 2020, 40(7): 22-29.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2001065 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I7/22

Table 1 Process in parameters and their levels in the orthogonal experiment

Fig.1 Growth curve of RGD-HBc recombinant strain

Fig.2 RGD-HBc recombinant protein expression under different conditions (a) RGD-HBc recombinant protein expression at different pH values M: Protein marker; 1-4: Medium pH is 4,6,7,9 in turn (b) RGD-HBc recombinant protein expression at different induction temperatures M:Protein marker; 1-4: Induction temperatures 20,26,32,42℃ (c) RGD-HBc recombinant protein expression at different induction times M: Protein marker; 1-4: Induction 2,4,8,10,20h (d) RGD-HBc recombinant bacterial protein expression at different concentrations of inducers M: Protein marker; 1-4:0.2,0.5,1.0,2.0mmol/L

Fig.3 Induced expression of RGD-HBc recombinant bacteria under different conditions 1: Induction for 2h,20℃,IPTG 0.2mmol/L; 2: Induction for 4h,26℃,IPTG 0.2mmol/L; 3: Induction for 8h,32℃,IPTG 0.2mmol/L; 4: Induction for 2h,26℃,IPTG 0.5mmol/L; 5: Induction for 4h,32℃,IPTG 0.5mmol/L; 6: Induction for 8h,20℃,IPTG 0.5mmol/L; 7: Induction for 2h,32℃,IPTG 1mmol/L; 8: Induction for 4h,20℃,IPTG 1mmol/L; 9: Induction for 8h,26℃,IPTG 1mmol/L

Table 2 The evaluation parameter of the orthogonal experimental

Fig.4 SDS-PAGE Analysis of RGD-HBc recombinant protein expression M:Protein marker;1:Hyperacoustic supernatat; 2:Hyperacoustic precitation

Fig.5 Purification of RGD-HBc recombinant protein

Fig.6 Stability of RGD-HBc VLPs (a)TEM image after purification at 4℃ for 30 days (b)TEM image after purification at -20℃ for 30 days

Fig.7 TEM and particle size detection of RGD-HBc /ICG VLPs (a)RGD-HBc /ICG VLPs TEM image (b)RGD-HBc /ICG VLPs particle size

Fig.8 Fluorescence imaging and bio-distribution of 4T1 breast cancer bearing mice (a)Fluorescence images in vivo of RGD-HBc VLPs,RGD-HBc /ICG VLPs and free ICG at different time points (b)Fluorescence images of in vitro organs and tumors


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