高响应性闪烁体纳米晶介导的X线触发光动力治疗侵袭性皮肤鳞癌实验研究

Photodynamic therapy (PDT) is a very effective method of treatment for non-invasive squamous cell carcinoma of skin in clinic. However, the effect of PDT on invasive squamous cell carcinoma of skin is not so good due to the short penetration depth of exciting light of photosensitizers in tissues. Therefore, we plan to establish a “Conversion Platform for Light Wave Energy” converting X-ray with strong penetration capacity to effective exciting light for a photosensitizer based on highly responsive scintillator for X-ray synthesized by ourselves in the previous experimental work. So X-ray indirectly generating reactive oxygen species can excite the photosensitizer. We design this novel model of X-ray excited PDT (XE-PDT) to increase action depth. The scintillators synthesized in previous studies can emit high-intensitive 544 nm lights under irradiation with low-dose X-ray because of their high luminescence efficiency. We will further optimize the luminescence property of scintillators and build an efficient “Conversion Nano-platform for Energy” with good safety by using scintillators as the energy donors, merocyanine 540 as energy acceptors and PEGylated amphiphilic polymeric liposomes as nanocarriers. And then a XE-PDT with strong penetrability in tissues, potent anti-tumor effect, little adverse effect and independent intellectual property right will be established based on low-dose X-ray irradiation. Finally, a series of researches on cell and animal level will be done to verify and optimize the anti-tumor effects and safety of XE-PDT for invasive squamous cell carcinoma of skin. This research program will provide the scientific basis for improving clinical efficacy of PDT in the treatment of invasive squamous cell carcinoma of skin.

临床光动力治疗非侵袭性皮肤鳞癌卓有成效，但因光敏剂激发光组织穿透深度浅，治疗深部侵袭性皮肤鳞癌疗效差。本项目采用全新思路，在前期自行合成的X线高响应性闪烁体纳米晶基础上，构建“光波能量转换平台”，将穿透力强的X线有效转换成光敏剂激发光以活化毗邻光敏剂产生活性氧簇，创建新型X线触发光动力治疗模式提高作用深度。前期所合成闪烁体发光效率高，低剂量X线便可激发出高强度544 nm发射光。本项目拟进一步优化闪烁体性能并以其为能量供体，光敏剂“部花青540”为能量受体，功能化聚乙二醇修饰的双亲性脂质体为载体基质，构建光能量转换效率高、安全性好的“肿瘤原位能量转换纳米平台”，结合低剂量X线照射，创建出组织穿透力强、抗肿瘤活性强、不良反应小、具有自主知识产权的X线触发光动力治疗体系。并通过一系列动物和细胞水平研究验证和优化其抗侵袭性皮肤鳞癌活性及安全性，为提高临床光动力治疗侵袭性皮肤鳞癌疗效提供科学依据。

研究背景 临床光动力治疗非侵袭性皮肤鳞癌卓有成效，但因光敏剂激发光组织穿透深度浅，治疗深部侵袭性皮肤鳞癌疗效差。本项目拟构建X线高响应的闪烁体-光敏剂纳米粒，创建新型X线触发光动力治疗模式提高作用深度。.研究目的 本课题拟达到以下研究目的：1）构建X线触发光动力治疗所需的新型X线高响应性闪烁体-光敏剂纳米粒；2）阐明这一新型X线高响应性闪烁体-光敏剂纳米粒介导X线触发光动力治疗侵袭性皮肤鳞癌的疗效、安全性、作用机制。 .研究方法 1）采用油相法制备磷酸镧铽闪烁体纳米晶。进而采用溶胶-凝胶合成法制备闪烁体-部花青540介孔硅纳米粒（ScMM）。2）采用CCK-8法研究ScMM的体外暗毒性、体外杀伤鳞癌细胞能力，观察治疗后瘤体增殖情况和小鼠生存时间评估抗侵袭性皮肤鳞癌在体疗效。通过组织病理学和TUNEL染色评估治疗后深部鳞癌细胞的死亡情况。采用q-PCR和蛋白免疫印迹杂交法研究INFα、IL-4、IL-12A和IL-10细胞因子表达量，采用免疫组化方法观察CD4+、CD8+T细胞浸润情况，评估抗肿瘤免疫激活作用。采用CD31免疫组化染色评估肿瘤血管损伤情况。观察皮肤毒性，脏器损伤及一般情况以评价其安全性。 .研究结果 1）成功制得ScMM，水合粒径为20.01±4.3 nm，部花青载药量为17.67%，在X线激发下可生成大量单态氧。2）ScMM无体外暗毒性，ScMM介导X线触发光动力可显著杀伤鳞癌细胞（p<0.05），可显著抑制小鼠侵袭性皮肤鳞癌增殖（p<0.05），小鼠生存时间明显长于对照组（p<0.05）。可直接杀伤大量深部鳞癌组织内的肿瘤细胞。X线触发光动力治疗后肿瘤组织内INFα、IL-4、IL-12A、IL-10细胞因子表达增高，肿瘤组织内CD4+、CD8+T细胞浸润增多，抗肿瘤免疫激活作用显著。X线触发光动力治疗后肿瘤组织CD31染色提示可抑制肿瘤血管生成。未见皮肤毒性反应发生。心脏、肝脏、脾脏、肺脏和肾脏未见明显损伤，小鼠体重、摄食量和摄水量亦未见异常。.结论 新型ScMM介导X线触发光动力治疗侵袭性皮肤鳞癌疗效显著、作用机制明确，未见明显不良反应，值得进一步研究以推进其临床转化。