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. 2013 Apr 9;8(1):161.
doi: 10.1186/1556-276X-8-161.

Surface modification of TPGS-b-(PCL-ran-PGA) nanoparticles with polyethyleneimine as a co-delivery system of TRAIL and endostatin for cervical cancer gene therapy

Yi Zheng  1 , Hongbo Chen, Xiaowei Zeng, Zhigang Liu, Xiaojun Xiao, Yongqiang Zhu, Dayong Gu, Lin Mei
Affiliations

Surface modification of TPGS-b-(PCL-ran-PGA) nanoparticles with polyethyleneimine as a co-delivery system of TRAIL and endostatin for cervical cancer gene therapy

Yi Zheng et al. Nanoscale Res Lett. .

Abstract

The efficient delivery of therapeutic genes into cells of interest is a critical challenge to broad application of non-viral vector systems. In this research, a novel TPGS-b-(PCL-ran-PGA) nanoparticle modified with polyethyleneimine was applied to be a vector of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and endostatin for cervical cancer gene therapy. Firstly, a novel biodegradable copolymer, TPGS-b-(PCL-ran-PGA), was synthesized and characterized. The nanoparticles were fabricated by an emulsion/solvent evaporation method and then further modified with polyethyleneimine (PEI) carrying TRAIL and/or endostatin genes. The uptake of pIRES2-EGFP and/or pDsRED nanoparticles by HeLa cells were observed by fluorescence microscopy and confocal laser scanning microscopy. The cell viability of TRAIL/endostatin-loaded nanoparticles in HeLa cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Severe combined immunodeficient mice carrying HeLa tumor xenografts were treated in groups of six including phosphate-buffered saline control, blank TPGS-b-(PCL-ran-PGA) nanoparticles, blank TPGS-b-(PCL-ran-PGA)/PEI nanoparticles, and three types of gene nanoparticles. The activity was assessed using average increase in survival time, body weight, and solid tumor volume. All the specimens were then prepared as formalin-fixed and paraffin-embedded tissue sections for hematoxylin-eosin staining. The data showed that the nanoparticles could efficiently deliver plasmids into HeLa cells. The cytotoxicity of the HeLa cells was significantly increased by TRAIL/endostatin-loaded nanoparticles when compared with control groups. The use of TPGS in combination with TRAIL and endostatin had synergistic antitumor effects. In conclusion, the TRAIL/endostatin-loaded nanoparticles offer considerable potential as an ideal candidate for in vivo cancer gene delivery.

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Figures

Figure 1
Figure 1
FT-IR spectra of TPGS and TPGS-b-(PCL-ran-PGA) copolymer.
Figure 2
Figure 2
Chemical structure (A) and typical 1H NMR spectra (B) of TPGS-b-(PCL-ran-PGA) copolymer.
Figure 3
Figure 3
Western blot analysis of recombined pShuttle2-endostatin and pShuttle2-TRAIL expression in 293 T cells. Control: 293 T cells transfected by pShuttle2. rE: 293 T cells transfected by pShuttle2-endostatin. rT: 293 T cells transfected by pShuttle2-TRAIL.
Figure 4
Figure 4
Effects of PEI modification, binding of pDNA with TPGS-b-(PCL-ran-PGA)/PEI nanoparticles, and FESEM image of HNP. (A) The effects of PEI modification on particle size. (B) The effects of PEI modification on surface charge. (C) The binding of pDNA with TPGS-b-(PCL-ran-PGA)/PEI nanoparticles determined by agarose gel electrophoresis. A series of different weight ratios (w/w) of pDNA to TPGS-b-(PCL-ran-PGA)/PEI nanoparticles was loaded on the agarose gel (a, pDNA/NPs = 1:0; b, pDNA/NPs = 1:4; c, pDNA/NPs = 1:10; d, pDNA/NPs = 1:20; e, pDNA/NPs = 1:20; f, pDNA/NPs = 1:20). (D) FESEM image of TRAIL- and endostatin-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles (HNP).
Figure 5
Figure 5
In vitro release profile of TRAIL- and endostatin-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles at pH 7.4 and 5.0.
Figure 6
Figure 6
Fluorescence and confocal laser scanning microscopy images of HeLa cells after incubation. (A to C) The fluorescence microscopy images of HeLa cells after incubation with pIRES2-EGFP-loaded and pDsRED-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles. (D to F) Confocal laser scanning microscopy images of HeLa cells after incubation with pIRES2-EGFP-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles at 37.0°C. The cells were stained by DAPI (blue), and the pIRES2-EGFP-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles are in green. The cellular uptake was visualized by overlaying images obtained using DAPI filter and FITC filter: (D) from DAPI channel, (E) from FITC channel, (F) from combined DAPI channel and FITC channel.
Figure 7
Figure 7
Viability of HeLa cells cultured with various nanoparticles in comparison with that of PBS. After 24- and 48-h incubation. (n = 5).
Figure 8
Figure 8
Antitumor effect of various nanoparticles in comparison with that of PBS.
Figure 9
Figure 9
Representative H&E staining of tumors. Treated with PBS (A), TRAIL-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles (B), endostatin-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles (C), and TRAIL and endostatin-loaded TPGS-b-(PCL-ran-PGA)/PEI nanoparticles (D).
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