摘要（一）

血小板膜融合的凋亡囊泡纳米递送平台构建

背景：

近年来研究表明，干细胞来源的细胞外囊泡对于治疗心肌缺血再灌注损伤（ischemia/reperfusion injury，I/RI）具有转化应用潜力。作为细胞外囊泡的亚型之一，凋亡囊泡（apoptotic vesicles，apoMV）因其独特的来源及制备方式，已在创伤修复等领域显示有效，但对心血管疾病的干预作用尚未得到充分证实。另外，apoMV通过静脉注射给药后，心肌损伤局部滞留率过低，极大地限制了apoMV的干预效果与转化潜力。

目的：

本研究基于血小板对损伤血管及炎症部位的天然趋化作用，拟构建血小板膜仿生纳米递送平台，提高骨髓间充质干细胞（Mesenchymal stem cell，MSC）来源的apoMV靶向干预损伤心肌的效果。

方法：

选择4-6代MSC在含有0.5μmol/L星形孢菌素（STS）无血清培养基中孵育12h后，收集上清液，并采用高速离心法反复离心，以收集凋亡MSC分泌的apoMVs。将反复冻融后的血小板通过超声破碎机处理15分钟后，依次通过1μm与400nm孔径的聚碳酸酯多孔膜，以构建直径均一的血小板膜囊泡。随后，将血小板膜囊泡与凋亡囊泡混合，通过400nm与200nm的聚碳酸酯多孔膜反复挤压，构建血小板膜融合的凋亡囊泡（P-apoMV）。最后，通过免疫蛋白印迹（Western blt, WB）、粒径分析（Nanoparticle tracking analysis,，NTA）及透射电镜（Transmission electron microscopy，TEM）等方法评估P-apoMV的形态变化及融合情况。

结果：

TEM显示，P-apoMV与传统apoMV形态相似，均为球形纳米结构；NTA结果表明，相较于apoMV（239±129nm），P-apoMV展现出小而均一的直径分布（181.9±82.5nm）。此外，囊泡稳定性试验显示，在10% 胎牛血清（Fetal bovine serum，FBS）中孵育apoMV与P-apoMV，两组于不同时间点对应的吸光度值没有显著差异；DiI标记的apoMV（红色）与DiO标记的血小板膜囊泡（绿色）融合后的共聚焦荧光定位图像表明P-apoMV成功融合。WB结果显示，P-apoMV可同时表达血小板膜的特异性蛋白（CD 62P、CD 42b与CD 47）和apoMV标志性蛋白（TSG 101与caspase 3）。

结论：

本研究基于血小板膜的天然趋化作用，将血小板膜与apoMV融合，使之同时具备二者的生物学功能，进而成功构建出血小板膜融合apoMV纳米递送平台。

摘要（二）

血小板膜融合的凋亡囊泡纳米递送平台修复心肌缺血再灌注损伤

研究背景：

心肌I/RI是经皮冠状动脉介入治疗（Percutaneous coronary intervention, PCI）术中常见且严重的并发症。越来越多研究表明，以干细胞衍生产品为核心的无细胞疗法在心肌I/RI的防治方面具备很好的转化前景。鉴于干细胞来源的apoMV在多种组织和器官修复中展现出的优越治疗效果，其在心肌I/RI中的治疗潜力值得进一步探索。

研究目的：

本研究旨在探究P-apoMV对I/RI心肌部位的靶向能力和生物学功能。

方法：

体外实验验证血小板膜递送技术在体外对多种细胞的靶向效果与功能。使用共聚焦显微镜观察各类细胞摄取情况；采用流式细胞术评估P-apoMV对原代乳鼠心肌细胞（Neonatal rat cardiomyocyte，NRCM）凋亡的影响；通过定量聚合酶链式反应（Quantitative polymerase chain reaction，q-PCR）检测骨髓来源巨噬细胞（Bone marrow-derived macrophage, BMDM）中炎症基因表达水平；利用划痕、Transwell及成管实验评估人脐静脉内皮细胞（Human umbilical vein endothelial cell，HUVEC）迁移和成管能力。此外，应用RNA测序技术（RNA-sequencing）分析apoMV和P-apoMV内含miRNA基因表达差异及功能差异。

在体外实验中，选取48只6-7周龄的健康雄性SD大鼠，随机分为4组：假手术组（sham组）、I/RI组、apoMV+I/RI组、P-apoMV+I/RI组。对左冠状动脉前降支（Left anterior descending artery，LAD）进行结扎/松解构建大鼠I/RI模型：每只大鼠结扎LAD 15分钟后，于再灌注前经尾静脉分别注射磷酸盐缓冲液（PBS）、apoMV或P-apoM后，进行再灌注干预。使用活体成像系统（In Vivo Imaging System，IVIS）检测囊泡在损伤心肌局部的聚集情况；通过三苯基四氮唑盐染色（Triphenyltetrazolium Chloride，TTC）和天狼星红染色分别评估心肌梗死面积和纤维化程度；免疫荧光染色（CD 31、α- SMA、Ki 67）用于评估血管新生及增殖情况。使用超声心动图检测大鼠的心脏结构和功能，记录左室射血分数（Left ventricular ejection fraction，LVEF）、左室短轴缩短率（Left ventricular fractional shortening，LVFS）、左室收缩末期内径（Left centricular end-systolic dimension，LVESD）与左室舒张末期内径（Left ventricular end-diastolic dimension，LVEDD）

结果：

体外实验表明，P-apoMV显著提高了NRCM、BMDM和HUVEC对囊泡的摄取能力（P < 0.001）。相较于apoMV组，P-apoMV组显著降低了糖氧剥夺/再灌注（Oxygen glucose deprivation/reperfusion，ORG/D）处理后NRCM凋亡率（P < 0.05），上调抗炎因子精氨酸酶 - 1（Arginase-1，Arg-1）（P < 0.05）和转化生长因子 -β（Transforming Growth Factor-β，TGF-β）表达（P < 0.05），下调促炎因子诱导型一氧化氮合酶（Inducible Nitric Oxide Synthase，iNOS）（P < 0.001）和白细胞介素 - 6（Interleukin-6，IL-6）（P < 0.05）表达。此外，P-apoMV还显著增强了HUVEC的迁移和成管能力（P < 0.05）。

在I/RI的SD大鼠中，与apoMV相比，尾静脉注射DiD标记的P-apoMV在缺血心肌局部具有更强的靶向积聚（apoMV 7044309±646453 vs P-apoMV 30840975±7484446，P < 0.001），局部新生血管增殖信号更强（Ki 67⁺ 细胞数：apoMV 11.8±2.5 vs P-apoMV 18.4±1.2，P < 0.05），且心功能改善更显著（LVEF：apoMV 42.8±4.6% vs P-apoMV 71.75±1.8%，P < 0.01）、心梗面积减少（apoMV 27.0±0.7 vs P-apoMV 19.8±2.2，P < 0.05）。此外，与PBS组相比，P-apoMV对肝肾功能具有潜在改善作用，血脂相关指标（如甘油三酯（apoMV 0.7±0.1 mmol/L vs P-apoMV 0.4±0.03 mmol/L，P < 0.05）等）显著下降，且并未观察到各组主要器官结构异常，血常规和血生化指标显著差异。

结论：

血小板膜融合后的骨髓MSC来源的apoMV能够显著提升心肌损伤部位的囊泡富集浓度，进一步减轻心肌缺血再灌注损伤，为心肌缺血再灌注损伤的修复提供了新的方向。

Abstract 1

Construction of platelet membrane-fusing apoptotic vesicle nanodelivery platform

Background

In recent years, stem cell-derived extracellular vesicles have shown great therapeutic potential for the treatment of myocardial ischemia/reperfusion injury (I/RI). Apoptotic microvesicles (ApoMVs), as a subtype of EVs, have an unclear role in cardiovascular disease because of their unique biological characters. After intravenous injection in regional myocardial injury, the low accumulation of apoMVs limits their therapeutic efficacy and clinical translation.

Objective

This study aims to construct a platelet membrane coated nano-drug delivery platform based on apoMVs. By utilizing the natural adhesion and chemotactic properties of platelets to injured blood vessels and inflammatory areas, the platform enhances the targeting ability of apoMV from bone marrow derived mesenchymal stem cells (MSCs) in vitro and vivo.

Methods

After MSCs were cultured in serum-free medium with 0.5 μmol/L STS for 12 h,  the supernatant from apoptotic MSCs were collected and the apoMVs were isolated by repeated high-speed centrifugation. Platelets were subjected to repeated freeze-thaw cycles, then treated with a ultrasonic disruptor for 15 min followed by successive extrusions through 1 μm and 400 nm polycarbonate membranes for platelet vesicles. Then, the apoMVs and platelet vesicles were mixed and co-extruded through 400 nm and 200 nm membranes to construct the platelet-based apoMV delivery platform (P-apoMV). The morphology, fusion efficiency, stability and particle size were identified by transmission electron microscopy (TEM), confocal immunofluorescence, western blot and nanoparticle tracking analysis（NTA）, respectively.

Result:

This study constructed the platelet-based apoMV delivery platform successfully. The TEM showed that P-apoMV exhibited the similar ring-shaped nanoparticle structure in morphology compared to apoMV. The results of NTA demonstrated that, compared to apoMV (239±129 nm), the P-apoMV exhibited smaller and more uniform diameter distribution (181.9±82.5 nm). Additionally, the stability assay showed that there is no difference in 10% fetal bovine serum (FBS) at absorbance of 590 nm between apoMV and P-apoMV. Confocal fluorescence images of DiD-labeled apoMV (red) and DiO-labeled platelet membrane vesicles (green) demonstrated the successful fusion of P-apoMV. Western blotting results showed that P-apoMV expressed both specific proteins of the platelet membrane (CD62P, CD42b, and CD 47) and apoptotic vesicles (TSG 101 and caspase-3).

Conclusion:

Utilizing the natural chemotactic characters of platelet membranes towards injured sites, this study successfully conducted platelet-based apoMV delivery platform by the fusion of both apoMV and platelet membrane.

Abstract 2

Platelet membrane-fused apoptotic vesicles derived from bone marrow mesenchymal stem cells promote myocardial repair after cardiac ischemia-reperfusion

Background:

Myocardial ischemia/reperfusion injury is an unavoidable issue following percutaneous coronary interventions (PCI) for myocardial infarction, and the effects of traditional treatment are limited. Increasing researches demonstrated that cell-free therapy based stem cells exhibited excellent clinical translational potential for alleviating myocardial ischemia/reperfusion injury. Given the superior tissue regenerative effects of MSC-derived apoMV in various other tissues, they may hold potential therapeutic benefits for myocardial ischemia/reperfusion injury.

Objective

This study aims to explore the targeting ability and biological functions of platelet based apoMV delivery platform towards injured myocardium in myocardial ischemia/reperfusion.

Methods

In vitro, the confocal fluorescence microscopy was employed to examine the uptake of vesicles by various cell types. Flow cytometry was used to evaluate the effect of P-apoMV on apoptosis in neonatal rat cardiomyocytes (NRCMs), while Quantitative polymerase chain reaction (q-PCR) was utilized to measure the expression of inflammatory genes in bone marrow-derived macrophages (BMDMs). The scratch tests, transwell assays, and tube formation assays were conducted to evaluate the migration and tube-forming abilities of human umbilical vein endothelial cells (HUVECs) under Oxygen glucose deprivation/reperfusion (OGD/R) stress. RNA-seq analysis was performed to analyze differences in miRNA gene expression between apoMV and P-apoMV group.

In animal experiments, 48 healthy male SD rats aged 6-7 weeks were randomly divided into four groups: sham group, I/RI group, apoMV+I/RI group, and P-apoMV+I/RI group. A myocardial I/RI model was established by ligating the left anterior descending coronary artery (LAD) for 15 minutes followed by reperfusion. Rats in each group received intravenous injections of phosphate buffered saline (PBS), apoMV, or P-apoMV before reperfusion. The in vivo imaging system (IVIS) was used to detect the aggregation of vesicles in the damaged myocardium of different groups. The triphenyltetrazolium chloride (TTC) staining and sirius red staining were used to measure the infarct size and fibrosis areas, respectively. Immunofluorescence staining for CD 31, α-SMA, and Ki 67 was performed to assess angiogenesis and proliferation. Echocardiography was conducted to assess cardiac functions in rats of each group, recording left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left centricular end-systolic dimension (LVESD) and left Ventricular end-diastolic dimension (LVEDD).

Results

This study successfully prepared platelet membrane-fused apoptotic vesicles (P-apoMV) and validated their functions in both cellular and animal experiments. In cellular experiments, P-apoMV significantly enhanced the uptake of vesicles by neonatal rat cardiomyocytes (NRCMs), bone marrow-derived macrophages (BMDMs), and human umbilical vein endothelial cells (HUVECs) (P<0.001). Compared to the apoMV group, P-apoMV significantly reduced apoptosis of cardiomyocytes (P<0.05), upregulated the expression of anti-inflammatory molecules including arginase-1 (ARG-1) (P<0.05) and transforming growth factor-β (TGF-β) (P<0.05), and downregulated the expression of pro-inflammatory molecules including inducible nitric oxide synthase (iNOS) (P<0.001) and interleukin-6 (IL-6) (P<0.05). Additionally, P-apoMV significantly enhanced the migration and tube formation abilities of HUVECs (P<0.05).

In vivo experiments demonstrated that compared to apoMV group, P-apoMV exhibited stronger targeting fluorescence accumulation in ischemic myocardial tissue (apoMV 7044309±646453 vs P-apoMV 30840975±7484446，P<0.001) and improved myocardial function (LVEF：apoMV 42.8±4.6% vs P-apoMV 71.75±1.8%，P<0.01) in rats with myocardial infarction by increasing local neovascularization-related proliferation signal (the number of Ki 67⁺ cells ：apoMV 11.8±2.5 vs P-apoMV 18.4±1.2，P < 0.05) and reduced infarct size (apoMV 27.0±0.7 vs P-apoMV 19.8±2.2，P < 0.05). Furthermore, P-apoMV showed potential benefits for hepatic and renal functions with significant reductions in lipid metabolism-related indicators (including triglycerides (0.7±0.1 mmol/L vs P-apoMV 0.4±0.03 mmol/L，P < 0.05)). No significant damage to other organs was observed, and complete blood count and biochemical indicators showed no significant differences. 

Conclusion:

Apoptotic vesicles derived from bone marrow mesenchymal stem cells fused with platelet membranes can effectively enhance accumulation at the site of ischemia-reperfusion injury in the myocardium, thereby further improving the therapeutic efficacy of MSCs and effectively alleviating myocardial ischemia-reperfusion injury. This study provides an innovative therapeutic direction for the treatment of myocardial ischemia-reperfusion injury.