Polyethylene glycol-functionalized poly (Lactic Acid-co-Glycolic Acid) and graphene oxide nanoparticles induce pro-inflammatory and apoptotic responses in Candida albicans-infected vaginal epithelial cells

Mucous-penetrating nanoparticles consisting of poly lactic acid-co-glycolic acid (PLGA)-polyethylene glycol (PEG) could improve targeting of microbicidal drugs for sexually transmitted diseases by intravaginal inoculation. Nanoparticles can induce inflammatory responses, which may exacerbate the inflammation that occurs in the vaginal tracts of women with yeast infections. This study evaluated the effects of these drug-delivery nanoparticles on VK2(E6/E7) vaginal epithelial cell proinflammatory responses to Candida albicans yeast infections. Vaginal epithelial cell monolayers were infected with C. albicans and exposed to 100 μg/ml 49.5 nm PLGA-PEG nanospheres or 20 μg/ml 1.1 x 500 nm PEG-functionalized graphene oxide (GO-PEG) sheets. The cells were assessed for changes in mRNA and protein expression of inflammation-related genes by RT-qPCR and physiological markers of cell stress using high content analysis and flow cytometry. C. albicans exposure suppressed apoptotic gene expression, but induced oxidative stress in the cells. The nanomaterials induced cytotoxicity and programmed cell death responses alone and with C. albicans. PLGA-PEG nanoparticles induced mRNA expression of apoptosis-related genes and induced poly (ADP-ribose) polymerase (PARP) cleavage, increased BAX/BCL2 ratios, and chromatin condensation indicative of apoptosis. They also induced autophagy, endoplasmic reticulum stress, and DNA damage. They caused the cells to excrete inflammatory recruitment molecules chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-1α (IL1A), interleukin-1β (IL1B), calprotectin (S100A8), and tumor necrosis factor α (TNF). GO-PEG nanoparticles induced expression of necrosis-related genes and cytotoxicity. They reduced autophagy and endoplasmic reticulum stress, and apoptotic gene expression responses. The results show that stealth nanoparticle drug-delivery vehicles may cause intracellular damage to vaginal epithelial cells by several mechanisms and that their use for intravaginal drug delivery may exacerbate inflammation in active yeast infections by increased inflammatory recruitment.由聚乳酸-羟基乙酸共聚物（poly lactic acid-co-glycolic acid, PLGA）-聚乙二醇（polyethylene glycol, PEG）构建的黏液穿透型纳米颗粒，可通过阴道接种途径提升杀微生物药物对性传播疾病的靶向递送效率。不过纳米颗粒可诱导炎症反应，可能会加重念珠菌感染女性阴道的炎症负荷。本研究旨在评估此类药物递送纳米颗粒对VK2(E6/E7)阴道上皮细胞在白念珠菌感染状态下的促炎反应的影响。研究人员将阴道上皮细胞单层接种白念珠菌，随后分别暴露于100 μg/ml、粒径49.5 nm的PLGA-PEG纳米球，以及20 μg/ml、尺寸为1.1×500 nm的聚乙二醇功能化氧化石墨烯（PEG-functionalized graphene oxide, GO-PEG）薄片。通过实时荧光定量聚合酶链式反应（RT-qPCR）检测炎症相关基因的mRNA与蛋白表达变化，并采用高内涵分析与流式细胞术检测细胞应激的生理标志物。白念珠菌暴露处理可抑制细胞凋亡相关基因的表达，但会诱导细胞产生氧化应激。纳米材料无论是单独处理还是与白念珠菌联合处理，均可诱导细胞毒性与程序性死亡反应。PLGA-PEG纳米颗粒可上调凋亡相关基因的mRNA表达，触发聚(ADP-核糖)聚合酶（poly (ADP-ribose) polymerase, PARP）的切割活化，提升BAX/BCL2比值，并出现提示细胞凋亡的染色质凝聚现象；此外，该纳米颗粒还可诱导自噬、内质网应激与DNA损伤，并促使细胞分泌炎性趋化招募分子：C-X-C基序趋化因子配体1（chemokine (C-X-C motif) ligand 1, CXCL1）、白细胞介素-1α（interleukin-1α, IL1A）、白细胞介素-1β（interleukin-1β, IL1B）、钙卫蛋白（calprotectin, S100A8）以及肿瘤坏死因子α（tumor necrosis factor α, TNF）。GO-PEG纳米颗粒则可诱导坏死相关基因的表达并引发细胞毒性，同时抑制自噬、内质网应激以及凋亡相关基因的表达响应。本研究结果表明，隐形纳米颗粒药物递送载体可通过多种机制对阴道上皮细胞造成细胞内损伤；将其用于阴道内药物递送时，还可能通过增强炎性招募作用，加重活动性念珠菌感染的炎症状态。