低能近红外响应水凝胶技术调控免疫因子时序给药在肌腱粘连中的应用研究

Immunotherapy is widely concerned in the treatment of cancer, but it is not reported in the prevention of postoperative tendon adhesion. In our previous study, we found that PD-L1 was significantly up-regulated in tendon adhesion after operation, and the application of PD-L1 antibody could significantly inhibit the formation of tendon adhesion. However, adhesion occurs at the middle and late stage of healing after sugery, and mainly occurs between damaged tissues and surrounding tissues. After injection, antibody is easy to degrade, deactivate and other defects, which affect the curative effect. Therefore, how to sequentially delivery drug in time has been a challenge. In this study, we used near infrared light triggered hydrogel system containing PD-L1 antibody and the engineered microRNA-based plasmid-loaded nanoparticles for preventing of adhesion formation. The low-energy near infrared signal was used to control drug release timing. On the one hand, a release of plasmid miRNA nanoparticles can inhibit the expression of PD-L1, on the other hand, the release of the antibody can inhibit PD-L1 function to enhance the immune response. The adhesion was inhibited with maximum degree by synergistic therapy. At the same time, we explored the up-regulated mechanism of PD-L1 from the epigenetics and tendon adhesion immune escape mechanism. This study can provide a new method and theoretical basis for the prevention of postoperative adhesion.

免疫疗法在肿瘤治疗中受到广泛关注，但在抑制术后肌腱粘连中未见报道。我们前期研究发现PD-L1在术后肌腱粘连组织中显著上调，且应用PD-L1抗体能显著抑制粘连形成。但因粘连在愈合的中后期形成，且主要发生在损伤组织与周围组织之间，同时抗体注射后易降解，易失活等缺点影响疗效，所以如何时序给药成为研究难点。本研究在前期成功制备低能近红外响应水凝胶基础上，合成载PD-L1工程化miRNA表达质粒纳米粒，同PD-L1抗体一起复合到水凝胶中，形成低能近红外响应的载PD-L1抗体和质粒纳米粒水凝胶。通过本项目实施，采用低能近红外信号控制药物时序释放，一方面释放miRNA质粒纳米粒来沉默PD-L1表达，另一方面释放PD-L1抗体可抑制PD-L1功能来增强免疫应答，通过协同治疗达到最大程度抑制粘连。同时从表观遗传学角度探讨PD-L1上调机制及粘连免疫逃逸机制。本研究可为抑制术后粘连提供新的治疗方法和理论依据。

粘连严重影响肌腱滑动功能的恢复。我们研究小组先前发现，与粘附形成密切相关的TGF-β1的抑制对抑制粘连是有效的，但并不能完全消除它们，这表明粘连的形成可能存在其他机制。在这项研究中，我们认为不受控制和过度增殖的成纤维细胞发生了免疫逃逸，这加剧了粘连形成过程中细胞外基质的沉积。我们发现，损伤后免疫检查点PD-L1的表达显著升高，并可能参与粘附形成。因此，我们打算沉默TGF-β1和PD-L1，以改善屈肌肌腱损伤后微环境中的免疫优势，进而减少粘连。我们构建了载TGF-β1 siRNA纳米球或/和载PD-L1 siRNA纳米球复合物，并验证了其高生物相容性和高转染效率。我们发现CD8+T细胞对转染载TGF-β1 siRNA纳米球或/和载PD-L1 siRNA纳米球复合物的过度增殖细胞具有更大的杀伤作用。在损伤的大鼠FDL肌腱和鸡FDP肌腱模型中，载TGF-β1 siRNA纳米球或/和载PD-L1 siRNA纳米球水凝胶系统可以有效地改善肌腱的滑动功能，且不会削弱力学性能。此外，我们观察到CD8+T细胞有包围肌腱表面粘附细胞的潜力，这导致细胞凋亡水平的增加。因此，我们的研究证实，联合敲除TGF-β1和PD-L1可以激活屈肌肌腱修复后的免疫优势，提供了限制粘连形成和改善滑动功能的潜在治疗方法。