光照触发霍乱毒素B链蛋白介导的视网膜母细胞瘤靶向D型多肽药物递送

Retinoblastoma, a tumor arising from the retina, is the most common primary intraocular tumor of childhood. It often causes blindness and even death of patients. In clinic, systemic chemotherapy in combination with local treatments (i.e. photocoagulation and cryotherapy) is the primary protocol for the management of retinoblastoma. The intraocular biodistribution of ordinary chemotherapeutic agents is low after systemic administration. Elevating the dose may cause severe side effects. The oncoproteins, MDM2 and MDMX, are implicated in the origination and progression of retinoblastoma, and have been recognized as new targets for molecularly targeted therapy of retinoblastoma. We have identified a D-peptide antagonist that is resistant to proteolytic degradation and can specifically bind to MDM2 and MDMX with high affinities. However, this D-peptide is impermeable to cell membrane to interact with intracellular oncoproteins. Here we aim to develop a photoactivatable derivative of cholera toxin subunit B (CTB) and conjugate it on the surface of D-peptide loaded liposomes. After systemic administration, the derivative of CTB would not bind to the widely expressed receptor (ganglioside GM1) in normal organs. Irradiation with visible LED light on the eye can rapidly activate CTB and recover its bioactivity in the posterior segment of the eye, leading to targeted delivery of D-peptide into retinoblastoma. The intracellular D-peptide can specifically antagonize MDM2 and MDMX, activate the p53 pathway and kill the retinoblastoma cells. Since the visible light is able to penetrate the eyeball efficiently, the photoactivatable derivative of CTB designed in this project would be also useful for the therapy of other diseases in the posterior segment of the eye.

视网膜母细胞瘤是最常见的儿童眼内恶性肿瘤，极易导致失明甚至威胁生命。临床上采用全身化疗辅助局部治疗（如激光光凝和冷冻治疗等）方案，但普通化疗药眼内分布低，提高剂量易加剧毒副作用。已知致癌蛋白MDM2和MDMX是视网膜母细胞瘤分子靶向治疗新靶点，申请人前期设计获得的D型多肽类p53蛋白激活剂可双效抑制MDM2和MDMX，且生物稳定性高，但因无法入胞，难以发挥抗肿瘤活性。本课题拟设计并合成光敏霍乱毒素B链蛋白衍生物，修饰于载D型多肽的脂质体表面，静脉给药后光敏霍乱毒素B链蛋白衍生物与全身广泛表达的受体无结合，在眼部经蓝光LED快速激活后，生成的原形霍乱毒素B链蛋白可介导脂质体高效、快速跨血-视网膜屏障，并将D型多肽药物靶向递送至视网膜母细胞瘤内，激活p53通路而发挥抗肿瘤活性。本课题拟利用可见光触发蛋白功能恢复，实现眼底（可见光通透性极佳）靶向递药，该思路对其他眼底疾病的治疗有借鉴作用。

视网膜母细胞瘤是最常见的儿童眼内恶性肿瘤，极易导致失明甚至威胁生命。临床上采用全身化疗辅助局部治疗（如激光光凝和冷冻治疗等）方案，但普通化疗药眼内分布低，提高剂量易加剧毒副作用。研制可跨越血-视网膜屏障的递送系统，将药物经全身给药后高效靶向递送至视网膜母细胞瘤细胞内，将对该类疾病的临床药物治疗现状发挥积极作用。本团队研究发现霍乱毒素B链蛋白（CTB）与血-视网膜屏障组成细胞（如：视网膜色素上皮细胞和视网膜内皮细胞）和视网膜母细胞瘤细胞具有高亲和力，可有效跨越体外血-视网膜屏障，以及靶向视网膜母细胞瘤细胞。但CTB蛋白对应的受体GM1在体内广泛表达，静脉注射后CTB蛋白在全身主要脏器均有较高分布，且可以突破血-脑屏障入脑。本课题利用分子模拟手段，将光敏分子DEACM连接到CTB蛋白Lys91侧链氨基上，测算其与受体GM1的结合活性降低3个数量级，为46.8 μM，表明Lys91侧链氨基修饰后可有效调控CTB蛋白的活性。利用本位化学拼接技术，成功合成获得Lys91侧链残基修饰的CTB-DEACM。较CTB蛋白，CTB-DEACM蛋白体内免疫原性显著下降，静脉注射后其在视网膜内分布具有较强的光控特征，当利用蓝光照射小鼠眼部，视网膜内的蛋白分布显著增加，表明CTB-DEACM光照后可快速脱去光敏基团并恢复CTB蛋白活性。CTB-DEACM生物安全性高，与视网膜内皮细胞和视网膜色素上皮细胞孵育后，未发现显著的细胞毒性。CTB-DEACM蛋白修饰在包载D型p53蛋白激活肽的脂质体表面，在血-视网膜屏障体外模型上，可实现激活肽的光控跨血-视网膜屏障，即：无光照时脂质体无法跨体外血-视网膜屏障，光照后跨膜转运效率显著提升。静脉注射后脂质体表面的CTB-DEACM蛋白与全身广泛表达的受体GM1无结合， 当在眼部经蓝光LED照射后，生成的原形CTB蛋白可介导脂质体高效、快速跨血-视网膜屏障，实现眼底视网膜母细胞瘤药物靶向递送。本课题结合CTB蛋白的生物活性和结构特点，以及眼部的生理特征，利用可见光触发蛋白功能恢复，实现眼底（可见光通透性极佳）靶向递药，该思路对其他眼底疾病的治疗有借鉴作用。

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