Nemidkanam Variya, Banlunara Wijit, Chaichanawongsaroj Nuntaree
Department of Clinical Chemistry, Graduate Program in Clinical Biochemistry and Molecular Medicine, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Int J Nanomedicine. 2024 Feb 27;19:1967-1983. doi: 10.2147/IJN.S444686. eCollection 2024.
extracellular vesicles (KPEVs) have been reported as promising nanovesicles for drug delivery. This study aimed to load clarithromycin (CLA) into KPEVs (KPEVS-CLA) and determine the physical properties, drug-releasing efficiency, gastric cell uptake, anti- activities, and anti-inflammatory responses in comparison with free CLA and KPEVs.
The size and surface charge of KPEVs-CLA were evaluated using dynamic light scattering and visualized using a transmission electron microscope. The encapsulation efficiency (EE%), loading capacity (LC%), and drug release of KPEVs-CLA were examined using HPLC. Anti- growth and anti-adhesion were evaluated. gene expression, NF-κB signaling proteins, and anti-inflammatory profiles were examined using qRT-PCR, Western blotting, and Bio-Plex immunoassay, respectively. Anti-chemotaxis was then examined using a Transwell assay.
KPEVs-CLA were intact and showed a negative surface charge similar to that of KPEVs. However, slightly enlarged KPEVs were observed. CLA was successfully loaded into KPEVs with EE of 93.45% ± 2.43%, LC of 9.3% ± 3.02%. CLA release in the PBS and gastric mimic buffer with Fickian diffusion ( ≤ 0.43) according to Korsmeyer-Peppas kinetic model (R=0.98). KPEVs-CLA was localized in the gastric cells' cytoplasm and perinuclear region. Anti- growth and anti- adhesion of KPEVs-CLA were compared with those of free CLA with no cytotoxicity to adenocarcinoma gastric cells. KPEVs-CLA significantly reduced IL-8, G-CSF, MIP-1α, and MIP-1β levels. Moreover, KPEVs-CLA showed a superior effect over CLA in reducing G-CSF, MIP-1α, and NF-κB phosphorylation and monocyte chemotactic activities.
KPEVs serve as potential carriers of CLA. They exhibited a higher efficiency in inhibiting gastric cell inflammation mediated by infection than free CLA. The establishment of KPEVs-CLA as a nanodrug delivery model for treatment could be applied to other plant extracellular vesicles or loaded with other cancer drugs for gastric cancer treatment.
细胞外囊泡（KPEVs）已被报道为有前景的药物递送纳米囊泡。本研究旨在将克拉霉素（CLA）载入KPEVs（KPEVs-CLA），并与游离CLA和KPEVs比较，确定其物理性质、药物释放效率、胃细胞摄取、抗菌活性及抗炎反应。
使用动态光散射评估KPEVs-CLA的大小和表面电荷，并用透射电子显微镜观察其形态。采用高效液相色谱法检测KPEVs-CLA的包封率（EE%）、载药量（LC%）及药物释放情况。评估其抗菌生长和抗黏附能力。分别采用实时定量聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法和生物芯片免疫分析法检测基因表达、核因子κB（NF-κB）信号蛋白及抗炎谱。然后采用Transwell实验检测抗趋化性。
KPEVs-CLA完整，表面电荷为负，与KPEVs相似。然而，观察到KPEVs略有增大。CLA成功载入KPEVs，包封率为93.45%±2.43%，载药量为9.3%±3.02%。根据Korsmeyer-Peppas动力学模型（R=0.98），CLA在磷酸盐缓冲液（PBS）和胃模拟缓冲液中的释放符合菲克扩散（≤0.43）。KPEVs-CLA定位于胃细胞的细胞质和核周区域。将KPEVs-CLA的抗菌生长和抗黏附能力与游离CLA进行比较，其对胃腺癌细胞无细胞毒性。KPEVs-CLA显著降低白细胞介素-8（IL-8）、粒细胞集落刺激因子（G-CSF）、巨噬细胞炎性蛋白-1α（MIP-1α）和巨噬细胞炎性蛋白-1β（MIP-1β）水平。此外，在降低G-CSF、MIP-1α和NF-κB磷酸化及单核细胞趋化活性方面，KPEVs-CLA比CLA表现出更优的效果。
KPEVs可作为CLA的潜在载体。与游离CLA相比，它们在抑制幽门螺杆菌感染介导的胃细胞炎症方面表现出更高的效率。将KPEVs-CLA建立为幽门螺杆菌治疗的纳米药物递送模型，可应用于其他植物细胞外囊泡或负载其他抗癌药物用于胃癌治疗。
