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. 2014 May 21:7:779-87.
doi: 10.2147/OTT.S59872. eCollection 2014.

Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

Jingjing Yang  1 , Xiaoping Huang  1 , Fanghong Luo  2 , Xiaofeng Cheng  3 , Lianna Cheng  3 , Bin Liu  4 , Lihong Chen  5 , Ruyi Hu  1 , Chunyan Shi  1 , Guohong Zhuang  1 , Ping Yin  5
Affiliations

Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

Jingjing Yang et al. Onco Targets Ther. .

Abstract

Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action.

Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors.

Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected following treatment. The average weight gain, tumor weight, and mean tumor volume of the protein and protein-loaded hydroxyethyl chitosan nanoparticle groups were significantly different (P<0.05) compared with the phosphate-buffered saline group.

Conclusion: The protein-loaded hydroxyethyl chitosan nanoparticles effectively suppressed tumor growth, indicating that nanotechnology has the potential for broad application in cancer therapy.

Keywords: DR5; GCS-aDR5ScFv; H22; anticancer effect.

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Figures

Figure 1
Figure 1
(A–F) Purification and identification of recombinant aDR5ScFv protein. Notes: (A) Purified aDR5-ScFv proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Lane M, molecular weight markers; lanes 1–4, three batches of proteins were purified using a nickel-affinity chromatography column. (B) eDR5 combination with aDR5ScFv by Coomassie Brilliant Blue R-250 dyeing. Lane M, molecular weight markers; lane 1, 0.315 μg aDR5ScFv; lane 2, 10 μg eDR5 reaction with 0.315 μg aDR5ScFv; lane 3, 10 μg eDR5 reaction with 0.625 μg aDR5ScFv; lane 4, 10 μg eDR5 reaction with 1.25 μg aDR5ScFv; lane 5, 10 μg eDR5 reaction with 2.5 μg aDR5ScFv; lane 6, 10 μg eDR5 reaction with 5 μg aDR5ScFv; lane 7, 10 μg eDR5 reaction with 10 μg aDR5ScFv; lane 8, 10 μg of eDR5. (C) Fluorescein isothiocyanate aDR5ScFv was used for immunofluorescence toward SW480. (D) The fixed SW480 was dyed by Hoechst 33342. (E) Overlay of (C and D) to assess cells stained by fluorescein isothiocyanate aDR5ScFv and Hoechst 33342. (F) The titers of aDR5scFv and aDR5mAb. Abbreviations: mAb, monoclonal antibody; aDR5, anti-human DR5; eDR5, extracellular domain constructed DR5; scFv, single-chain fragment variable.
Figure 2
Figure 2
(A–D) Distribution and morphological properties of glycol chitosan (GCS). Notes: (A) Shape of GCS detected by scanning electron microscopy (SEM). (B) Shape of GCS-aDR5 single-chain fragment variable (scFv) tested by SEM. (C) Analysis the fluorescence labeled aDR5ScFv and GCS-aDR5ScFv tumor targeting by in vivo imaging system. (D) Detection of the toxic effects of H22 cells treated with GCS by CellTiter-Blue ®(Promega, Fitchburg, WI, USA). Abbreviations: aDR5, anti-human DR5; min, minutes; h, hours.
Figure 3
Figure 3
(A–D) In vivo and in vivo studies on the inhibition effects of glycol chitosan (GCS) aDR5 single-chain fragment variable (scFv). Notes: (A) Detection of hepatocellular H22 cytotoxic cells by CellTiter-Blue ®(Promega, Fitchburg, WI, USA) after treatment with aDR5 scFv and GCS-aDR5 scFv. (B) Flow cytometry detected aDR5ScFv, GCS-aDR5ScFv and GCS induce H22 cells apoptosis. (C) H22 cells treated with GCS-aDR5ScFv and aDR5ScFv (0.136mg/ml) for 24 hours, stained with Hoechst33342 and analyzed by Fluorescence microscopy. (D) Hematoxylin and eosin staining of tumor visualization. Abbreviations: FITC, fluorescein isothiocyanate; h, hours; aDR5, anti-human DR5.
Figure 4
Figure 4
The mechanism of cell apoptosis. Notes: Western blot analysis of active caspase 8, active caspase 3, and BAX expressions in a GCS-aDR5 single-chain fragment variable (scFv)-treated mouse H22 tumor model. Abbreviations: GCS, glycol chitosan; aDR5, anti-human DR5.
Figure 5
Figure 5
Analysis of toxicity. Comparison of body weight in H22 tumor-bearing mice of each group. Abbreviations: GCS, glycol chitosan; scFv, single-chain fragment variable; aDR5, anti-human DR5.
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