頭頸部鱗狀細胞癌 (HNSCC) 常表現出內在或後天獲得的放射抗性，因此亟需開發有效的放射增敏劑。儘管 miR-148a-3p 具備已證實的腫瘤抑制特性，但由於缺乏安全且高效的遞送系統，其直接的臨床應用受到嚴重阻礙。本研究探討了利用源自牛乳與 HEK293 細胞、具生物相容性之細胞外囊泡 (EVs) 作為 miR-148a-3p 奈米載體，以提升體外放射治療療效之可行性。 臨床資料庫分析顯示，高水平的 miR-148a-3p 表現與多種惡性腫瘤的整體存活率提升呈正相關；而 CRISPR 基因剔除數據 (DepMap) 則指出，癌細胞對 miR-148a-3p 的已知下游標靶基因 ITGA5 具有廣泛的依賴性。初步的體外篩選證實，異位表現 miR-148a-3p 可作為廣效型放射增敏劑，顯著削弱多種癌細胞株（包括 HNSCC 細胞株 FaDu）的聚落形成存活率 (劑量增敏比1.24)。 為開發生物遞送平台，透過對公開 miRNA-seq 數據集進行生物資訊學分析，並經 RT-qPCR 驗證，確認 miR-148a-3p 在天然的牛乳 EVs 及 HEK293-EVs 中高度富集。分離出的奈米囊泡依據 MISEV 指南進行了嚴格的鑑定。流式細胞儀分析證實 FaDu 細胞能大量攝取這些未經修飾的 EVs。關鍵的是，基線功能評估表明，未經修飾的牛乳與 HEK293-EVs 在生物學上呈現惰性；它們不會實質改變細胞增殖、基礎遷移或對放射線的反應，從而確立了其作為安全載體候選者的地位。 隨後，透過轉染技術將 miR-148a-3p 積極裝載至 EVs 中。結合 RNase A 處理與絕對定量分析，證實其成功封裝於囊泡腔內。功能性評估顯示，在 2 Gy 放射線照射下，與裝載對照組 (scramble) 相比，經 miR-148a-3p 裝載之 EVs 處理的 FaDu 細胞，其聚落形成能力顯著受損且傷口癒合延遲。總結而言，這些發現提供了強而有力的概念驗證，證明以 EV 為介導的遞送策略—特別是利用具量產潛力且高度生物相容的牛乳 EVs—在 HNSCC 應用 miRNA 進行放射增敏是一項極具可行性且高效的策略。;Head and neck squamous cell carcinoma (HNSCC) frequently presents with intrinsic or acquired radioresistance， necessitating the development of effective radiosensitizers. While miR-148a-3p possesses established tumor-suppressive properties， its direct clinical application is severely hindered by the lack of safe and efficient delivery systems. This study investigated the feasibility of utilizing biocompatible extracellular vesicles (EVs) derived from bovine milk and HEK293 cells as nanocarriers for miR-148a-3p to enhance radiotherapy efficacy in vitro. Clinical database analyses revealed that high miR-148a-3p expression correlates with improved OS across multiple malignancies， while CRISPR knockout data (DepMap) indicated a broad dependency of cancer cells on ITGA5， a known downstream target of miR-148a-3p. Initial in vitro screening confirmed that ectopic miR-148a-3p expression acts as a potent broad-spectrum radiosensitizer， significantly impairing clonogenic survival in diverse cancer lineages， including the HNSCC cell line FaDu (Dose Enhancement Ratio approximately 1.24). To develop a biological delivery platform， bioinformatic profiling of public miRNA-seq datasets—subsequently validated by RT-qPCR—identified miR-148a-3p as highly enriched in native Milk-EVs and HEK293-EVs. The isolated nanovesicles were rigorously authenticated according to MISEV guidelines. Flow cytometric analysis confirmed robust cellular uptake of these naive EVs by FaDu cells. Crucially， baseline functional assessments demonstrated that naive Milk- and HEK293-EVs in low concentration， as 20 μg， did not significantly alter cell proliferation， baseline migration， or radiation response， establishing them as safe vehicle candidates. Subsequently， miR-148a-3p was actively loaded into the EVs using a transfection-based approach. RNase A treatment coupled with absolute quantification confirmed successful luminal encapsulation. Functional evaluations revealed that treatment with miR-148a-3p-loaded EVs significantly impaired colony formation capacity and delayed wound closure in FaDu cells under 2 Gy irradiation compared to scramble-loaded controls. Ultimately， these findings provide a robust proof-of-concept that EV-mediated delivery—particularly utilizing scalable and highly biocompatible bovine milk-EVs—is a highly viable and effective strategy for miRNA-based radiosensitization in HNSCC.
