文獻(xiàn)：Exosomal Vaccine Loading T Cell Epitope Peptides of SARS-CoV-2 Induces Robust CD8+ T Cell Response in HLA-A Transgenic Mice

作者：An-Ran Shen 1, Xiao-Xiao Jin 2, Tao-Tao Tang 1, Yan Ding 2, Xiao-Tao Liu 2, Xin Zhong 1, Yan-Dan Wu 2, Xue-Lian Han 3, Guang-Yu Zhao 3, Chuan-Lai Shen 2, Lin-Li Lv 1,?, Bi-Cheng Liu 1

文獻(xiàn)鏈接：https://pmc.ncbi.nlm.nih.gov/articles/PMC9346304/

摘要：

The feasibility of this novel approach in generating peptides-PEG-lipid-exosome complexes was first tested by preparing BSA-PEG-lipid-exosome complexes. The molar ratio between BSA and DPME-PEG-NHS was optimized by testing 1:1, 1:1.5, 1:3, and 1:4 ratios as shown in Table 1. In order to calculate the BSA loading capacity and loading efficiency in the BSA-PEG-micelles, the conjugated BSA with DMPE in BSA-PEG micelle solution and the free BSA in the filtered solution was quantified using BCA kit, respectively. The results showed that the BSA loading capacity was highest (85 μg BSA was conjugated with 2 mg DMPE-PEG-NHS) at 1:3 molar ratio of DMPE-PEG-NHS with BSA (Figure 3A). The loading efficiency was 91.84% at 1:3 molar ratio and then decreased rapidly when the BSA input increased (Figure 3B). Then, the RBC-exosomes were mixed with BSA-PEG-micelles in a 1:1 ratio based on the protein concentrations. The mixture was incubated in a water bath for 2 h at 40℃. The BSA-PEG-lipid-exosomes solution was then cooled to 4℃ and instantly purified using size-exclusion chromatography.

通過制備BSA-PEG脂質(zhì)外泌體復(fù)合物，首次測(cè)試了這種新方法產(chǎn)生肽-PEG-脂質(zhì)外泌物復(fù)合物的可行性。如表1所示，通過測(cè)試1:1、1:1.5、1:3和1:4的比例，優(yōu)化了BSA和DPME-PEG-NHS之間的摩爾比。

為了計(jì)算BSA-PEG膠束中的BSA負(fù)載能力和負(fù)載效率，分別使用BCA試劑盒定量BSA-PEG膠束溶液中與DMPE偶聯(lián)的BSA和過濾溶液中的游離BSA。

結(jié)果表明，當(dāng)DMPE-PEG-NHS與BSA的摩爾比為1:3時(shí)，BSA的負(fù)載能力最高（85μg BSA與2mg DMPE-PEG-NaHS偶聯(lián)）（圖3A）。在1:3摩爾比下，負(fù)載效率為91.84%，然后隨著BSA輸入的增加而迅速降低。

然后，根據(jù)蛋白質(zhì)濃度，將RBC外泌體與BSA-PEG膠束以1:1的比例混合。將混合物在40℃的水浴中孵育2小時(shí)。然后將BSA-PEG脂質(zhì)外泌體溶液冷卻至4℃，并立即使用尺寸排阻色譜法純化。

BSA-PEG脂質(zhì)外泌體的純化與鑒定。

首先將一系列量的BSA與2mg DMPE-PEG-NHS偶聯(lián)，然后與DSPE-PEG混合形成BSA-PEG膠束。對(duì)游離BSA進(jìn)行定量，并分別計(jì)算BSA-PEG膠束中的BSA負(fù)載能力（A）和負(fù)載效率（B）。

此外，BSA-PEG膠束以DMPE-PEG-NHS與BSA的1:3摩爾比再生，并在40℃下與RBC外泌體一起孵育2小時(shí)，以產(chǎn)生BSA-PEG脂質(zhì)外泌體，并使用尺寸排阻色譜法立即純化，F(xiàn)PLC圖譜顯示三個(gè)主峰（C）。

用抗BSA和抗CD63對(duì)三個(gè)峰的組分進(jìn)行蛋白質(zhì)印跡，以鑒定每個(gè)峰中的BSA和外泌體（D），并對(duì)每個(gè)峰中PEG、水解NHS和DMPE元素進(jìn)行液體NMR鑒定（E）。

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