Preparation of Nanovesicle via Oxidative Stress and Extrusion for Cancer Vaccine Application

• Main Authors: Hsueh-Fang Huang, 黃雪芳
• Other Authors: Meng-Jiy Wang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/68qqbf

碩士 === 國立臺灣科技大學 === 化學工程系 === 107 === The Ministry of Health and Welfare of Taiwan reported that, cancer was the first leading cause of death in Taiwan at 2017, which was also the second important cause of death globally. The most common types of cancers was lung cancer which caused 1.76 million deaths in 2018. Therefore, cancer therapies are of great importance. This study aims to prepare cancer vaccine by oxidative stress treatment and extrusion. This study applied three different kinds of oxidative stress sources to treat Lewis lung carcinoma (LLC) cell: (i) 30 μM H2O2 (H2O2 vaccine), (ii) photo-dynamic therapy (PDT), keep the medium to collect nanovesicles as Vs vaccine, and extruded LLC after PDT treatment as Ve vaccine, and (iii) atmospheric pressure plasma jet (APPJ), which can get APPJ vaccine. The cancer cells became immunogenic cancer cells after exposure to oxidative stresses, and the cells were induced to apoptosis cells. Immunogenic cancer cells would then express damage-associated molecular patterns (DAMPs) and antigens on the surfaces of cell membranes followed by releasing spontaneous vesicles, which could activate the antigen presenting cells. In order to promote the yield of vesicles, the apoptosis cancer cells were extruded by with 80 psi dry air of filter extrusion, followed by centrifugation. After H2O2 treatment and filter extrusion, the average diameter of vesicles was 207 nm and concentration of vesicles was 3.84  1010 NVs/2  107 cells, analyzed by nanoparticle tracking analysis (NTA). The efficiency of activating reticulum cell sarcoma (J774A.1) by different oxidative stress treated vesicles was determined by Griess assay. The results showed that the highest concentration of NO was 16.87  2.46 μM, by H2O2 treatment on vaccine for the activation of J774A1. The cytotoxicity of the J774A.1 primed with various vaccines towards LLC cells was evaluated in vitro by co-culturing LLC cells and J774A.1 to understand the potential of vesicles as cancer vaccine. The LLC/J774A.1 ratio by co-culture of LLC and J774A.1 activated by F/T, H2O2, Vs, Ve, and APPJ vaccine was 3.48  0.64, 3.44  0.59, 2.00  0.48, 1.42  0.15, and 1.81  0.11, respectively. The results showed that the incorporation of oxidative stress and extrusion method possessed good efficacy and reproducibility for the preparation of cancer vaccine For the polyethylene oxide (PEO) electrospinning nanofibers, RT4-D6P2T cells were cultivated on the nanofibers as scaffolds to evaluate the potential for nerve repair. The effects of the applied voltages for electrospinning on the average diameter of the PEO nanofibers were evaluated by scanning electron microscopy (SEM). In order to prevent the dissolution of PEO nanofibers in aqueous solutions, 2.5 wt% of pentaerythritol triacrylate (PETA) was used as a chemical cross-linker. The correlations between the diameter of nanofibers and the cell responses were determined by MTT assay. The results of SEM observation indicated that the average diameter of the as-electrospun PEO nanofibers was 418 ± 134 nm, 286 ± 89 nm, and 328 ± 95 nm at the applied voltages of 10, 15, and 20 kV, respectively. To promote the biocompatibility of the electrospun PEO nanofibers, 2.0 wt% of apatite was incorporated into the polymer solution during electrospinning processes. With the addition of apatite, the average diameter of PEO nanofibers increased from 286 ± 89 to 426 ± 133 nm. MTT assay showed that PEO nanofibers containing 2 wt% apatite can significantly promote the cell growth comparing with the pristine PEO fibers, such that the cell density increased from 0.082 to 2.26 with the prolonged incubation time from 1 to 7 days. The results suggested that the as-prepared PEO nanofibers incorporated with 2.0 wt% of apatite displayed great potential for the applications in nerve tissue repair.