Supporting data for thesis entitled "A dual pronged strategy to treat fungal superinfections in oral mucositis"

Medical advances have substantially improved cancer survival rates, yet post-cancer care remains challenging due to debilitating chemotherapy side effects such as oral mucositis. Beyond causing severe pain, and reduced quality of life, mucositis creates epithelial ulcerations that serve as niches for opportunistic pathogen colonization. Current microbiome studies demonstrate that fungi, particularly Candida albicans, play critical roles in developing and perpetuating inflammation in mucositis. Mucosal candidiasis affects 50-75% of cancer patients undergoing chemotherapy, resulting in two critical challenges: (1) it facilitates the epithelial breach and invasion of what is usually a superficial infection and (2) it sustains mucosal inflammation contributing to persistent mucositis. Taken together, this serves as a contributing factor to disseminated fungal infections, with increased morbidity and mortality.We first investigated whether a truncated LL-37 version could demonstrate potent antifungal activity under physiomimetic conditions that typically abrogate peptide biological effects. Through comprehensive testing against reference and patient-derived multidrug-resistant C. albicans strains, we discovered GK17, a 17-amino acid sequence within LL-37 showing potent antifungal activity, biocompatibility, and stability in human saliva and serum. To improve antibiofilm activity, we engineered an enantiomeric peptide, D-GK17, demonstrating exceptional antifungal activity, unprecedented stability, and disassembled established biofilms. D-GK17 was also active against non-albicans Candida strains and Nakaseomyces glabratus (formerly Candida glabrata). Noteworthily, both GK17 and D-GK17 exhibited excellent anti-inflammatory activity. We hypothesized that GK17 and D-GK17 could effectively treat mucositis lesions through multimodal antifungal and anti-inflammatory activities. To test this hypothesis, an optimal model was required. Therefore, we created an organotypic oral mucosa model with keratinocytes, macrophages, and fibroblasts, simulating chemotherapy-induced mucositis using 5-Fluorouracil application. This model reproduced macroscopic and microscopic features observed in clinical mucositis. Testing demonstrated that GK17, D-GK17, and nystatin potently eradicated infection and reduced pro-inflammatory cytokine release. However, only GK17 and D-GK17 restored epithelial barrier integrity. Given these very promising findings, we encapsulated both peptides in mucoadhesive hydrogels and evaluated their effects in murine chemotherapy-induced mucositis models with superimposed mucosal candidiasis. Our preclinical studies demonstrated that peptide-loaded hydrogels eradicated fungal infection, prevented oral mucosal invasion, maintained epithelial barrier integrity, and significantly reduced NF-κB-mediated inflammation. Taken together, this body of work substantially advances a novel peptide-therapeutic for the treatment of oral candidiasis in models of epithelial injury such as those observed in cancer chemotherapy.医学进步已大幅提升了癌症患者的生存率，但癌症后续护理仍面临诸多挑战，这源于化疗引发的衰弱性不良反应，例如口腔黏膜炎（oral mucositis）。口腔黏膜炎不仅会引发剧烈疼痛、降低患者生活质量，还会造成上皮溃疡，为机会性病原体定植提供微环境。当前的微生物组研究表明，真菌尤其是白色念珠菌（Candida albicans）在口腔黏膜炎的炎症发生与持续进程中发挥关键作用。黏膜念珠菌病会影响50%-75%接受化疗的癌症患者，并带来两大核心难题：其一，它会促使原本属于浅表感染的病原体突破并侵袭上皮组织；其二，它会持续引发黏膜炎症，进而导致黏膜炎迁延不愈。综上，这会成为播散性真菌感染的诱因之一，提升患者的发病率与死亡率。我们首先探究了截短型LL-37（LL-37）是否能在通常会抵消肽类生物活性的模拟生理条件下，展现出强效抗真菌活性。通过针对参考菌株及患者来源的多重耐药白色念珠菌菌株开展全面测试，我们发现了LL-37内部一段含17个氨基酸的序列GK17，该序列在人类唾液与血清中展现出强效抗真菌活性、良好生物相容性与稳定性。为提升抗生物膜活性，我们设计了对映体肽D-GK17，该肽展现出优异的抗真菌活性、前所未有的稳定性，并能解离已形成的生物膜。D-GK17对非白色念珠菌菌株及光滑假丝酵母菌（Nakaseomyces glabratus，旧称光滑念珠菌Candida glabrata）同样具有抗菌活性。值得注意的是，GK17与D-GK17均展现出优异的抗炎活性。我们推测，GK17与D-GK17可通过多模式的抗真菌与抗炎活性，有效治疗黏膜炎病灶。为验证这一假说，我们需要构建最优的实验模型。为此，我们利用角质形成细胞、巨噬细胞与成纤维细胞构建了器官型口腔黏膜模型，并通过施加5-氟尿嘧啶（5-Fluorouracil）模拟化疗诱导的口腔黏膜炎。该模型重现了临床黏膜炎中观察到的宏观与微观特征。实验测试表明，GK17、D-GK17与制霉菌素（nystatin）均能有效根除感染，并减少促炎细胞因子的释放。但仅有GK17与D-GK17能够恢复上皮屏障的完整性。基于上述极具前景的研究结果，我们将两种肽封装于黏膜黏附性水凝胶中，并在叠加了黏膜念珠菌病的小鼠化疗诱导黏膜炎模型中评估了其疗效。我们的临床前研究表明，载肽水凝胶能够根除真菌感染、阻止口腔黏膜侵袭、维持上皮屏障完整性，并显著降低核因子κB（NF-κB）介导的炎症反应。综上，本研究成果极大推动了一种新型肽类疗法的发展，该疗法可用于治疗上皮损伤模型（如癌症化疗中观察到的上皮损伤）中的口腔念珠菌病。