构建新型纳米光敏剂及光动力作用下治疗瘢痕的实验研究

Photodynamic theraypy(PDT) mediated fibroblasts cell apoptosis/death is a peculiar treatment for the pathological scar tissue.Currently, the topical delivery of photosensitizer is an optional application of PDT. However,the hydrophilic and lipophilic photosensitizer shows lower penetrative characteristics in scar dermis according to the specific structure of the tissue, thus,inefficient concentration of photosensitizer results the limited effect of the PDT. It was stated that the photosensitizer with high penetrating capability leads to an impactful cytotoxic action to the fibroblasts cell.Based on our primary research finding, nano-ethosomes and nano-deformable liposomes as the topical drug delivery carrier presented high ability of the penetration and high deposition within the hypertrophic scar tissue. Therefore, further study on depicting the penetrating features of these nanolipsomes encapsulated with photosensitizer and its interfering to the metabilism of the fibroblast cell, will advance a key step in PDT.. This study is a sequential investigation on the penetration behavior of the nanoliposomes encapsulated with photosensitizer as the topical drug carrier, as well as its vito/vivo effects on the cell growth,cell sub-structure and protein synthesizing following PDT. The aim of this study will explores its capability and mechanisim of penetrating into the fibroblast cell, constrcut a novel photosensitizer with a high penetrative features and improve the therapeutic effects on pathological scar.

诱导成纤维细胞凋亡是光动力治疗（photodynamic therapy，PDT)病理性瘢痕的重要作用机制。经皮给药是活性光敏剂进入组织细胞的主要途径，然而瘢痕组织特殊病理结构阻碍了水溶性或脂溶性光敏剂渗透入瘢痕深部组织，无法在局部形成有效滞留浓度，限制了PDT对瘢痕的治疗效果。因此促光敏剂顺利"渗入"瘢痕组织并"进入"相关细胞，产生细胞毒性，是PDT治疗瘢痕的关键。我们前期实验发现，纳米级醇脂体和传递体都具备较强透增生性瘢痕能力，所以，进一步研究其能否携带光敏剂高效"进入"成纤维细胞并干预其代谢，将解决光动力治疗中的关键问题。.本研究在原有基础上构建并筛选透瘢痕纳米级脂质体载体，通过体内、外实验，继续研究纳米级光敏剂在细胞内分布及对细胞生长、超微结构、蛋白分泌等功能影响，将阐明纳米级光敏剂进入瘢痕成纤维细胞的能力和机制，合成新的具有高渗透能力的光敏剂药物并提高PDT治疗病理性瘢痕的效果。

本研究利用新型透皮药物载体—醇脂体包载光敏剂5-氨基酮戊酸（ALA），用于局部瘢痕给药治疗。考察其在离体增生性瘢痕组织的渗透能力，阐述渗透机制，并观察其在体外培养的增生性瘢痕成纤维细胞内向原卟啉IX（PpIX）的转化效率，以及在光动力治疗下在成纤维细胞内产生细胞毒物质活性氧簇（ROS）的水平、线粒体的损伤情况以及对成纤维细胞的杀伤作用，并考察了新型纳米光敏剂醇脂体（ALA-ES）对兔耳增生性瘢痕的渗透和治疗作用，为最终构建新型纳米光敏剂，提高ALA的瘢痕渗透性能和在成纤维细胞内的聚集，以达到减少给药剂量和缩短给药时间，从而减少副作用发生率的目的提供相关实验依据。本研究首次采用跨膜pH梯度法制备的ALA-ES包封率高，粒径小、形态理想。ALA-ES体系的pH值为7.4，和呈酸性的ALA水溶液相比，可减少皮肤的刺激性。ALA-ES具有较好的瘢痕渗透性能，可显著促进ALA的渗透速度和滞留量，局部给药时可达到缩短给药时间，降低给药浓度的目的。ALA-ES能增强细胞内PpIX的积聚，在光动力作用下通过生成ROS，造成线粒体损伤来抑制成纤维细胞的活性。通过在动物实验表明，ALA-ES介导的光动力治疗能够有效抑制兔耳增生性瘢痕，减少瘢痕内胶原纤维沉积，使胶原排列趋于有序。