壳聚糖纳米颗粒介导miR-30c-5p经黏膜递送用于口腔鳞状细胞癌

Transmucosal Delivery of miR-30c-5p by Chitosan Nanoparticles for Oral Squamous Cell Carcinoma.

作者信息

Fang Yi-Ping, Lin Yu-Chih, Lin Chien-Yu, Wang Po-Jen, Chang Ting-Yu, Hsieh Ya-Ju

机构信息

School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.

Regeneration Medicine and Cell Therapy Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

出版信息

Int J Nanomedicine. 2025 Jul 19;20:9179-9194. doi: 10.2147/IJN.S524558. eCollection 2025.

DOI:10.2147/IJN.S524558

PMID:40703474

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12285890/

Abstract

BACKGROUND

Oral squamous cell carcinoma (OSCC) remains difficult to treat with current modalities. miR-30c-5p, a tumor-suppressive microRNA frequently downregulated in OSCC, inhibits cancer cell proliferation and migration. However, its clinical application is limited by poor stability and inefficient uptake. To address these issues, miR-30c-5p was encapsulated into chitosan nanoparticles (CS-NPs) using ionic gelation to enhance delivery and protect against degradation.

METHODS

miR-30c-5p-loaded CS-NPs were characterized for particle size, zeta potential, morphology, and encapsulation efficiency. HSC-3 and OEC-M1 cells were treated with free miRNA, CS-NPs, or CS-miRNA-NPs at final concentrations of 5%, 10%, 25%, and 50% (v/v) in culture medium. Cellular uptake was assessed by confocal microscopy. Ex vivo porcine buccal membrane studies evaluated mucosal penetration. Cytotoxicity was determined using MTT assays, while gene regulation was analyzed via quantitative polymerase chain reaction and Western blotting.

RESULTS

The prepared CS-NPs had particle sizes ranging from 434 to 452 nm and encapsulation efficiencies between 79% and 87%. Confocal imaging revealed significantly greater cytoplasmic uptake of CS-miRNA-FAM NPs versus free miRNA. Ex vivo studies showed that CS-miR-30c-5p-FAM NPs penetrated mucosa up to 80-160 μm with a 5.42-fold higher fluorescence intensity than free miR-30c-5p-FAM. Cytotoxicity testing showed high cell viability (>93%) for all treatments at concentrations ≤25% (v/v). At 50% (v/v) nanoparticle suspension, viability significantly decreased in OEC-M1 cells (84.41% for naked miRNA, 54.52% for CS-NPs, 61.10% for CS-miRNA-NPs; P < 0.001). After 48 h, greater reductions were observed at 50% (v/v), with cell viability in HSC-3 cells decreasing to 85.55% (free miRNA), 42.72% (CS-NPs), and 51.82% (CS-miRNA-NPs), and in OEC-M1 cells to 73.98%, 33.00%, and 39.89%, respectively. Functional assays showed vimentin mRNA reductions of 85% in HSC-3 and 30% in OEC-M1, with protein decreases confirmed by Western blot.

CONCLUSION

CS-NPs enhance miRNA delivery and gene-silencing efficacy in OSCC cells. These findings support CS-based systems for miRNA therapeutics in oral cancer.

摘要

背景

口腔鳞状细胞癌（OSCC）目前的治疗方式仍存在困难。miR - 30c - 5p是一种在OSCC中经常下调的肿瘤抑制性微小RNA，可抑制癌细胞增殖和迁移。然而，其临床应用受到稳定性差和摄取效率低的限制。为了解决这些问题，通过离子凝胶法将miR - 30c - 5p封装到壳聚糖纳米颗粒（CS - NPs）中，以增强递送并防止降解。

方法

对负载miR - 30c - 5p的CS - NPs进行粒径、zeta电位、形态和包封率表征。在培养基中以5%、10%、25%和50%（v/v）的终浓度用游离miRNA、CS - NPs或CS - miRNA - NPs处理HSC - 3和OEC - M1细胞。通过共聚焦显微镜评估细胞摄取。体外猪颊黏膜研究评估黏膜渗透。使用MTT法测定细胞毒性，同时通过定量聚合酶链反应和蛋白质印迹分析基因调控。

结果

制备的CS - NPs粒径范围为434至452 nm，包封率在79%至87%之间。共聚焦成像显示，与游离miRNA相比，CS - miRNA - FAM NPs的细胞质摄取明显更多。体外研究表明，CS - miR - 30c - 5p - FAM NPs穿透黏膜达80 - 160μm，荧光强度比游离miR - 30c - 5p - FAM高5.42倍。细胞毒性测试表明，在浓度≤25%（v/v）时，所有处理的细胞活力都很高（>93%）。在50%（v/v）纳米颗粒悬浮液中，OEC - M1细胞的活力显著下降（裸miRNA为84.41%，CS - NPs为54.52%，CS - miRNA - NPs为61.10%；P < 0.001）。48小时后，在50%（v/v）时观察到更大程度的降低，HSC - 3细胞的细胞活力降至85.55%（游离miRNA）、42.72%（CS - NPs）和51.82%（CS - miRNA - NPs），OEC - M1细胞分别降至73.98%、33.00%和39.89%。功能分析显示，HSC - 3中波形蛋白mRNA减少85%，OEC - M1中减少30%，蛋白质印迹证实蛋白质减少。