Abstract:
For decades, animal models have been the standard approach in drug research and development, as they are required by regulations in the transition from preclinical to clinical trials. However, there is growing ethical and scientific concern regarding these trials, as 80 % of the therapeutic potential observed in pre-clinical studies are often unable to be replicated, despite demonstrating efficacy and safety. In response to this, Tissue Engineering has emerged as a promising alternative that enables the treatment of various diseases through the production of biological models for advanced biological assays or through the direct development of tissue repairs or replacements. One of the promising applications of Tissue Engineering is the development of three-dimensional (3D) models for in vitro tests, replacing the need for in vivo animal models. In this study, 3D skin equivalents (TSE) were produced and used as an in vitro model to test photobiostimulation using curcumin-loaded nanocapsules. Photodynamic biostimulation therapy uses photodynamic processes to generate small amounts of reactive oxygen species (ROS), which can activate important biological effects such as cell differentiation, modulation of inflammatory processes and contribution to cell regeneration. The PLGA nanocapsules (NC) used in the study were synthesized through a preformed polymer deposition method, exhibiting particle size <200 nm, Zeta potential >|30| and polydispersity index between 0.5 and 0.3. Atomic force microscopy analyzes confirmed that the particle size was <200 nm, with a spherical morphology and a predominantly smooth and uniform surface. The NC biocompatibility assay did not demonstrate cytotoxicity for the concentrations tested (2.5-25 μg mL-1).The in vitro release assay showed a slow and sustained release characteristic of the nanocapsules, and cellular uptake assays indicated a significant increase in cellular internalization of the curcumin-loaded nanostructure. Monolayer photobiostimulation studies revealed an increase in cell viability of the HDFn cell line (viability 134 %-228 %) for all LED fluences employed at λ = 450 nm (150, 300, and 450 mJ cm-2). Additionally, the scratch assays, monitoring in vitro scar injury, demonstrated more effective effects on cell proliferation with the fluence of 300 mJ cm-2. Staining of TSE with hematoxylin and eosin showed the presence of cells with different morphologies, confirming the presence of fibroblasts and keratinocytes. Immunohistochemistry using KI-67 revealed the presence of proliferating cells in TSE after irradiation with LED λ = 450 nm (150, 300, and 450 mJ cm-2).
摘要:
几十年来,动物模型一直是药物研发的标准方法,因为从临床前到临床试验过渡的法规要求它们。然而,关于这些试验的伦理和科学问题越来越多,因为在临床前研究中观察到的80%的治疗潜力往往无法复制,尽管证明了疗效和安全性。对此,组织工程已成为一种有前途的替代方案,可通过生产用于高级生物测定的生物模型或通过直接开发组织修复或替代来治疗各种疾病。组织工程的有前途的应用之一是开发用于体外测试的三维(3D)模型,取代体内动物模型的需要。在这项研究中,产生3D皮肤等效物(TSE)并将其用作体外模型以测试使用加载姜黄素的纳米胶囊的光生物刺激。光动力生物刺激疗法利用光动力过程产生少量的活性氧(ROS),可以激活重要的生物学效应,如细胞分化,炎症过程的调节和对细胞再生的贡献。研究中使用的PLGA纳米胶囊(NC)是通过预制聚合物沉积方法合成的,显示粒径<200nm,Zeta电位>|30|和多分散指数在0.5和0.3之间。原子力显微镜分析证实,粒径<200nm,具有球形形态和主要光滑均匀的表面。NC生物相容性测定未显示所测试浓度(2.5-25μgmL-1)的细胞毒性。体外释放试验显示了纳米胶囊的缓慢和持续释放特性,和细胞摄取测定表明负载姜黄素的纳米结构的细胞内化显著增加。单层光生物刺激研究显示,对于在λ=450nm(150、300和450mJcm-2)下使用的所有LED注量,HDFn细胞系的细胞活力(活力134%-228%)均增加。此外,划痕试验,体外监测瘢痕损伤,在300mJcm-2的注量下对细胞增殖表现出更有效的作用。用苏木精和伊红染色TSE显示存在不同形态的细胞,确认成纤维细胞和角质形成细胞的存在。使用KI-67的免疫组织化学显示,在LEDλ=450nm(150、300和450mJcm-2)照射后,TSE中存在增殖细胞。