Objectives: Drug-loaded mucoadhesive silk fibroin (SF) microneedle patch can overcome the limitations of low bioavailability and significant pain associated with traditional treatment methods, such as topical application or injection of triamcinolone for oral submucous fibrosis (OSF). However, these systems release the drug too quickly, failing to meet the clinical requirements. This study aims to construct a mucoadhesive SF microneedle patch pre-assembled with silk fibroin nanospheres (SFN) and explore its ability to sustain the release of triamcinolone in the treatment of OSF.
Methods: SFN was pre-assembled via precipitation reaction and characterized by scanning electron microscope (SEM) for the morphology. The particle size and ζ-potential were measured by dynamic light scattering (DLS). Triamcinolone was loaded onto SFN using a diffusional post-loading method. The effective loading of triamcinolone was confirmed using Fourier-transform infrared spectroscopy (FTIR). The concentration of unloaded triamcinolone was quantified by high-performance liquid chromatography. Drug encapsulation efficiency and loading capacity of SFN were then calculated to determine the optimal amount of drug loading. The SFN suspension was pre-mixed with SF solution to prepare the microneedle under-layer. The microneedle morphology was observed by SEM. Compression mechanical tests were performed to evaluate the fracture force of microneedles at different nanosphere contents (5%, 10%, and 20%), determining the optimal pre-mixing ratio. Ex-vivo mouse oral mucosa permeation studies were performed to ascertain the insertion depth of the microneedles via histological sections. The adhesive top layer was synthesized using SF and tannic acid, with FTIR confirming its successful synthesis. Its viscoelasticity was characterized by a rheometer, and differential scanning calorimetry analyzed thermal properties. Tensile tests evaluated the interfacial bonding strength between the adhesive layer and microneedle base to ensure no detachment during use. Adhesion to wet oral mucosal tissues was tested and compared to commercial oral patches.Under the optimized conditions, the double-layered mucoadhesive microneedle patch with pre-assembled nanospheres was prepared. Its cell compatibility was evaluated by cell counting kit-8 (CCK-8), live/dead staining, and phalloidin staining after co-culturing with fibroblasts. The drug release experiment was conducted to demonstrate its sustained release efficacy.
Results: SFN (mean diameter 46.25 nm) was successfully prepared. The maximum drug encapsulation efficiency was (63.88±1.09)% (corresponding loading capacity of SFN was (27.41±3.06)% when the weight ratio of triamcinolone/SFN was 0.5. The corporation of SFN did not affect microneedle morphology. The mechanical properties of microneedles decreased with increasing nanosphere amount. Only the fracture force of the group with 5% SFN [(0.07±0.01) N/needle] exceeded the minimum force required for mucosal penetration, thus selected as the optimal pre-mixing ratio. Histological sections confirmed that the SFN microneedles could penetrate the epithelial layer and deliver drugs to OSF-affected areas. Adhesion strength between the microneedle base and top layer was (94.8±6.89) kPa, confirming strong bonding with no detachment during use. The wet adhesive strength of the double-layered mucoadhesive microneedle patch [(41.28±7.43) kPa] was significantly enhanced compared to commercial oral patches (4.5 kPa, P<0.01). CCK-8 and live/dead staining results confirmed no significant cytotoxicity. Drug release experiment showed the double-layered mucoadhesive microneedle patch with pre-assembled SFN enabled sustained release time of triamcinolone from 4 days to 14 days.
Conclusions: Pre-assembling nanospheres in mucoadhesive SF microneedle patches can extend triamcinolone release time, meeting clinical requirements for sustained drug delivery.
目的: 载药湿态黏附蚕丝蛋白(silk fibroin，SF)微针贴片能克服涂抹或注射曲安奈德等传统口腔黏膜下纤维性变(oral submucous fibrosis，OSF)治疗方式中存在的药物生物利用度低和注射疼痛明显的缺陷，但其释放药物速率过快，无法满足临床需要。本研究旨在构建预组装SF纳米球(silk fibroin nanosphere，SFN)结构单元的湿态黏附SF微针贴片，探究其缓释曲安奈德治疗OSF的能力。方法: 通过沉淀反应法制备SFN，采用扫描电镜(scanning electron microscope，SEM)观察其形貌，动态光散射(dynamic light scattering，DLS)测量其粒径及表面ζ-电位；通过后载法加载曲安奈德，傅里叶变换红外光谱(Fourier-transform infrared spectroscopy，FTIR)验证药物是否成功加载，高效液相色谱测定未加载上的药物浓度以间接计算药物包封率和SFN的载药率，确定最佳载药浓度比。将SFN分散成悬液，与SF溶液预混后制备微针底层，采用SEM观察微针形貌及针尖形态，通过压缩力学测试检测不同SFN含量(5%、10%、20%)制备的微针的机械强度以确定最佳预混比例，随后将其应用于离体小鼠口腔黏膜后进行组织切片，确定其插入深度。制备蚕丝-丹宁酸湿态黏附顶层，通过FTIR验证其是否成功合成，流变仪表征其黏弹性，差示扫描量热法分析其热效应特性，拉伸力学测试测量其与微针底层之间的界面结合力以确保不发生界面脱离，进一步检测其与离体湿润口腔黏膜组织间的湿态黏附性，并与现有商业口腔贴片对比。使用前述优化后的条件制备双层湿态黏附微针贴片，与成纤维细胞共培养，通过细胞计数试剂盒-8(cell counting kit-8，CCK-8)、活死染色及鬼笔环肽染色检测其细胞相容性；通过释药实验检测湿态黏附微针贴片的药物缓释性能。结果: 成功制备SFN(平均粒径46.25 nm)，在曲安奈德与SFN质量比为0.5时，达到最大药物包封率[(63.88±1.09)%]，此时SF载药率为(27.41±3.06)%。预组装SFN的微针形貌无明显改变，但机械性能随着SFN含量的增加而下降，SFN相对含量为5%时断裂强度[(0.07±0.01) N/微针]超过穿透黏膜所需最小力，因此选定为最佳预混比。小鼠黏膜组织切片表明预组装SFN的微针可穿透上皮层，确保药物递送至OSF病变部位。微针底层与湿态黏附顶层间黏接力为(94.80±6.89) kPa，证实双层湿态黏附微针贴片界面间结合牢固，使用过程中不会发生脱离。双层湿态黏附微针贴片的湿态黏附性能相较于商业口腔贴片明显增强[(41.28±7.43) kPa vs 4.5 kPa，P<0.01]。CCK-8和活死染色结果证实预组装SFN的双层湿态黏附微针贴片无明显细胞毒性。释药实验结果显示：预组装SFN的双层湿态黏附微针贴片可实现曲安奈德的缓释，释药时间从4 d延长至14 d。结论: 在SF湿态黏附微针贴片中预组装SFN结构单元可延长曲安奈德的释放时间，可以满足临床释药时长的需求。.
Keywords: mucoadhesive microneedle patch; oral disease; oral mucosal disease; oral submucous fibrosis.