Abstract：
Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of -3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.
摘要：
糖尿病足溃疡(DFU),其特征是伤口周围的微小血管或毛细血管受损，是糖尿病最严重和最可怕的并发症之一。修复慢性非愈合DFU伤口是具有挑战性的。血管内皮生长因子（VEGF）在DFU血管生成中起重要作用，并促进伤口愈合。然而,由于其稳定性差和容易降解,难以将VEGF持续地递送至创伤部位。为了克服这一挑战，已经开发了封装编码VEGF-A的环状RNA(circRNA)的脂质纳米颗粒(LNP),以连续产生和释放VEGF-A并加速糖尿病伤口愈合。首先,使用I组内含子自催化策略合成VEGF-AcircRNA，并通过酶消化证实，聚合酶链反应，和测序测定。使用微流体技术将VEGF-AcircRNA封装在可电离的脂质U-105衍生的LNP(U-LNP)中以制造U-LNP/VEGF-AcircRNA。为了比较，使用封装circRNA(A-LNP/circRNA)的商业可电离脂质ALC-0315衍生的LNP(A-LNP)。动态光散射和透射电子显微镜表征表明，U-LNP/circRNA具有球形结构，平均直径为108.5nm，多分散指数为0.22,ζ电位为-3.31mV。U-LNP的信使RNA(mRNA)包封率(EE%)为87.12%。体外转染数据证实，与线性mRNA相比，circRNA的稳定性和长期VEGF-A表达更好。细胞毒性和先天免疫的评估进一步揭示U-LNP/circRNA是生物相容的并且诱导弱的先天性免疫应答。细胞划痕和血管生成测试表明U-LNP/VEGF-AcircRNA由于其VEGF-A表达而具有生物活性。萤火虫荧光素酶(F-Luc)探针的原位生物发光成像和酶联免疫吸附试验表明，circRNA在第一周内具有VEGF-A的长期强表达，并且在接下来的一周内伤口部位和周围区域逐渐减少。最后,糖尿病小鼠模型用于验证U-LNP/VEGF-AcircRNA制剂的愈合效果.结果表明，通过滴注给予单剂量的U-LNP/VEGF-AcircRNA在第12天导致几乎完全的伤口恢复，其明显优于U-LNP/VEGF-A线性mRNA，它也优于重组人血管内皮生长因子（rhVEGF）注射和A-LNP/circRNA滴注。组织学分析证实了U-LNP/VEGF-AcircRNA制剂的愈合效率和低毒性。一起,由U-105衍生的LNP递送的VEGF-AcircRNA在伤口愈合中显示出良好的性能，这归因于VEGF-A的长期表达和持续释放，对糖尿病足溃疡创面的治疗具有潜在的应用价值。
