【作者】 高敏；

【导师】 孙耕耘；

【作者基本信息】 安徽医科大学 ， 内科学（呼吸系病）， 2023， 博士

【摘要】 背景肺癌是全球最常见的癌症。肺癌的常规治疗策略包括手术,化疗和放疗等,但手术治疗仅能消除局部肿瘤。放疗和化疗都存在一定毒副作用,多次治疗后易产生耐药性,一定程度上影响了治疗效果。由于光动力疗法（Photodynamic therapy,PDT）是一种高度选择性的方法,侵入性小,对周围健康组织无损伤,通过活性氧（Reactive oxygen species,ROS）的生成来破坏癌细胞。PDT已成为许多临床研究的热点,并已被证明是癌症治疗的有效策略。然而,即使药物能够安全到达肿瘤微空间,也难以克服一些生理屏障,导致药物外排,抗肿瘤药物生物利用度低。既往已报道细胞膜是有效治疗的最佳靶点,细胞膜的损伤会诱导高水平的细胞毒性;但细胞膜靶向肺癌治疗的方法非常有限,目前很少有光敏剂可以定位于细胞膜发挥抗肺癌作用。因此,制备多功能纳米囊泡锚定光敏剂靶向肺癌治疗的递送平台至关重要。此外,PDT中使用的抗肿瘤光敏剂在实体肿瘤中的渗透也是纳米药物递送系统在肺癌治疗中的主要障碍之一,且用于肺癌治疗的常用光敏剂对癌细胞是非特异性的,应该进一步开发更加特异性地靶向肺癌细胞,以及可以穿透深层肺癌组织的高效光敏剂,为增强抗肿瘤药物的穿透提供一种新的策略。同时,一些研究表明,PDT不仅能对肿瘤细胞产生光细胞毒性,还能诱导免疫原性细胞死亡（Immunogenic cell death,ICD）。其中,探讨PDT增强肿瘤免疫原性的机制已成为近年来的研究热点。结合目前的研究发现细胞焦亡是一种溶解性程序性细胞死亡,其特征是细胞不断膨胀,形成巨大的气泡,直到细胞膜破裂,促进细胞内容物和炎症因子的免疫刺激性释放,具有激活抗肿瘤免疫应答的潜力。因此,我们还进一步探讨本研究中设计的多功能纳米囊泡锚定光敏剂靶向PDT是否诱导肺癌细胞焦亡并揭示其潜在机制。总之,一种有效、安全的特异性诱导肿瘤细胞死亡的治疗方式在肺癌的临床PDT中具有广阔的应用前景。本实验将围绕如何利用细胞锚定纳米光敏剂实现抗肺癌的高效治疗开展研究。第一部分细胞膜锚定纳米光敏剂用于光控钙超载和肺癌特异性协同治疗目的研究细胞膜锚定纳米光敏剂（Cell membrane anchored nano photosensitizers,CMA-nPS）在Ca²⁺超载和肺癌特异性协同治疗中的作用。方法1.细胞膜锚定纳米光敏剂的制备与表征本研究首先制备了叠氮化修饰的巨噬细胞膜和具有膜融合功能水疱性口炎病毒糖蛋白（Vesicular stomatitis virus glycoprotein,VSV-G）修饰的细胞膜;随后将两种功能的细胞膜通过不同孔径大小的聚碳酸酯膜进行挤压得到多功能纳米囊泡;接着受生物正交反应的启发,将二苯并环辛炔基团修饰的4-氨基苯基卟啉（DBCO conjugated meso-Tetra（4-aminophenyl）porphyrin,DBCO-TAPP）与多功能纳米囊泡反应,成功制备了细胞膜锚定纳米光敏剂。最后,通过透射电镜（Transmission electron microscope,TEM）,纳米颗粒跟踪分析仪（Nanoparticle tracking analyzer,NTA）和动态光散射仪（Dynamic light scattering,DLS）等进行表征。2.细胞实验首先,我们抑制细胞的内吞途径,激光扫描共聚焦显微镜（Confocal laser scanning microscope,CLSM）研究CMA-nPS的是通过细胞膜融合途径摄取,从而进一步将光敏剂锚定在肺癌细胞膜上;其次,3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide,MTT）方法验证CMA-nPS的暗毒性,并研究细胞内ROS的产生,细胞内Ca²⁺的变化,细胞膜损伤,线粒体功能障碍,细胞凋亡,细胞活性/毒性实验证实CMA-nPS在光照下体外杀伤肺癌细胞。3.动物实验在研究体内肺癌消融前,我们先利用小鼠活体成像观察CMA-nPS的富集情况,并在治疗期间监测肺癌荷瘤小鼠的体重,肿瘤体积;治疗结束后,解剖小鼠对主要脏器（如心、肝、脾、肺、肾）进行苏木精和伊红（Hematoxylin-eosin staining,H&E）染色和进行血液相关分析评估生物安全性,肿瘤组织予以H&E,末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记法（Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling,TUNEL）,Ki67细胞增殖实验评估CMS-nPS的体内治疗效果。结果1.TEM观察CMA-nPS呈现为囊泡形态,可见脂质层。NTA和DLS分析表明,CMA-nPS的平均直径约为200 nm,PDI值显示出较窄的粒径分布。2.CMA-nPS的摄取主要通过非内吞机制发生。MTT实验证实CMA-nPS无光照下对细胞活力没有影响,但在光照下,肺癌细胞的存活率降低,且细胞内ROS的生成明显增加,引起肺癌细胞膜的损伤,细胞内的Ca²⁺浓度升高,加重线粒体功能障碍,导致肺癌细胞明显凋亡。3.小鼠活体成像的结果表明CMA-nPS能在肿瘤部位较好的富集,并通过尾静脉注射至小鼠体内后,对肿瘤部位予以光照,实现了光控钙超载和肺癌特异性协同治疗,明显抑制肺癌的生长甚至完全消融肺癌。结论本研究制备的CMA-nPS主要将光敏剂锚定在肺癌细胞膜上,并在光照射下进一步引起肺癌细胞膜损伤、线粒体功能障碍以及细胞内Ca²⁺超载。细胞和动物实验验证协同增强的抗肺癌效率。本研究为基于Ca²⁺超载的癌症治疗提供新的协同策略,以及将光敏剂锚定在细胞膜上的策略,为肺癌的治疗提供广阔的应用前景。第二部分穿透深层肿瘤组织的纳米光敏剂增强抗肺癌疗效目的研究多功能纳米囊泡制备的光敏剂实现肺癌组织深层穿透,增强抗肺癌疗效。方法1.适配体共轭细胞膜锚定纳米光敏剂（Aptamer conjugate cell membrane anchored nano photosensitizers,AVnPS）的制备与表征首先,制备多功能细胞膜锚定纳米光敏剂,其次适配体AS1411采用脂质体插入法成功构建AVnPS。最后,通过琼脂糖凝胶电泳、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium lauryl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）和Western blotting进行表征。2.细胞实验通过CLSM验证AVnPS的摄取途径后,接着对AVnPS的肿瘤靶向性进行研究并且体外构建肺癌多细胞肿瘤球体来评价纳米光敏剂穿透肿瘤组织的能力。此外,采用transwell模型研究AVnPS的迁移,进一步验证细胞外囊泡在肿瘤穿透中的意义。最后,一系列的相关细胞试验被用来验证AVnPS对肺癌细胞的杀伤情况。3.动物实验首先,通过小鼠活体成像对AVnPS的肿瘤靶向性进行研究;然后,在予以AVnPS治疗期间检测肺癌荷瘤小鼠的体重,肿瘤生长体积变化;最后,治疗结束时,解剖小鼠,取主要脏器（如心、肝、脾、肺、肾）进行HE染色评估生物安全性,并且肿瘤组织予以HE,TUNEL,Ki67细胞增殖实验评估AVnPS的体内治疗效果。结果1.琼脂糖凝胶电泳检测结果表明AS1411适配体成功组装于AVnPS中,AVnPS成功制备;而且SDS-PAGE和Western blotting验证多功能纳米囊泡具有母细胞来源的功能蛋白,其特征蛋白在AVnPS中保留良好。2.CLSM结果表明AVnPS具有肿瘤靶向性,同时体外模拟肺癌球体,观察到VSV-G的修饰促进光敏剂对多细胞肿瘤球体的渗透。为进一步研究肿瘤穿透机制,transwell模型研究AVnPS的迁移,结果表明VSV-G修饰的细胞膜锚定纳米光敏剂更容易在细胞之间迁移。此外,我们还收集肿瘤细胞分泌内源性囊泡（endogenous vesicles secreted by tumor cells,TCEVs）进行实验发现,纳米光敏剂会转移到TCEVs中,随着TCEVs的介导纳米光敏剂会逐层穿透至肺癌组织的深处。3.体内的小鼠活体成像结果再一次证实AVnPS的肿瘤靶向性,AVnPS在肿瘤部位富集明显;治疗期间检测肺癌小鼠的体重无明显变化,AVnPS处理后的小鼠经肿瘤部位光照后,明显抑制肿瘤的生长;治疗结束后的主要脏器进行HE染色未见损伤,表明AVnPS的生物安全性好。结论多功能纳米囊泡构建光敏剂首先在VSV-G膜融合的功能下,将光敏剂转移到肺癌细胞膜上,随后通过肺癌细胞的分泌的细胞外囊泡,纳米光敏剂会与TCEVs融合,并且随着TCEVs的迁移,光敏剂可以逐层转移到肺癌组织深处。通过TCEVs可导致光敏剂深入肺癌组织内部,增强光动力疗法的疗效,为肺癌的治疗提供广阔的应用前景。第三部分生物合成纳米光敏剂激活Caspase-3/GSDME通路诱导肺癌细胞焦亡目的探讨光动力疗法诱导肺癌细胞死亡的另一方式-细胞焦亡及潜在机制。方法1.光触发光动力疗法纳米平台（Photoinitiated photodynamic therapy nanoplatform,HCNP）的制备与表征结合第一,第二部分的相关实验方法制备HCNP并且通过TEM观察形貌,DLS和NTA测定粒径和浓度分布。2.细胞实验首先,我们通过CLSM观察HCNP的摄取途径,其次,细胞活性/毒性、MTT实验对肺癌细胞的存活率进行分析;此外,肺癌细胞焦亡的机制我们通过细胞内ROS、线粒体损伤进一步研究,而且Western blotting验证线粒体损伤诱导细胞Caspase-3/GSDME裂解的相关焦亡蛋白表达。结果1.TEM发现HCNP的结构为球形,DLS和NTA表明HCNP的平均直径约为200nm,且具有良好的尺寸均匀性。2.通过CLSM的观察结果验证我们制备的HCNP是通过细胞膜融合的方式进行摄取。在660nm,1.4 W cm^-2,予以5分钟光照后,肺癌细胞的存活率明显下降,细胞内的ROS生成进一步加重线粒体的损伤,且Western blotting实验结果表明膜靶向PDT能有效激活Caspase-3并裂解GSDME,可通过焦亡途径诱导细胞死亡。结论HCNP在激光照射下肺癌细胞通过产生ROS有效地破坏细胞线粒体功能;同时,GSDME被激活的Caspase-3裂解并促进肺癌细胞的焦亡,为光激活PDT诱导肺癌细胞焦亡提供一种有效的干预策略,也证明细胞膜锚定光敏剂在通过激活焦亡途径治疗肺癌方面的潜力。更多还原

【Abstract】 BackgroundLung cancer is the most common cancer in the worldwide.Conventional treatment strategies for lung cancer include surgery,chemotherapy,and radiotherapy.However,surgery can only eliminate local tumors.Both radiotherapy and chemotherapy have certain toxic side effects,and drug resistance is easy to develop after multiple treatments,which affects the treatment effect to a certain extent.Photodynamic therapy（PDT）is a highly selective method that is less invasive and does not damage the surrounding healthy tissue.It destroys cancer cells through the production of reactive oxygen species（ROS）.PDT has become a hotspot in many clinical studies and has proven to be an effective strategy for cancer treatment.But,even if the drug can safely reach the tumor microspace,it is difficult to overcome some physiological barriers,resulting in drug efflux and low bioavailability of antitumor drugs.Cell membranes have been previously reported to be the best target for effective therapeutic,and damage to cell membranes can induce high levels of cytotoxicity;However,cell membrane-targeted lung cancer treatment methods are very limited,and few photosensitizers can be localized to cell membranes to exert anti-lung cancer effects.Therefore,it is of great importance to prepare a delivery platform of multifunctional nanovesicles anchored photosensitizers for targeted lung cancer therapy.In addition,the penetration of anti-tumor photosensitizers used in PDT in solid tumors is also one of the main obstacles of nanodrug delivery systems in lung cancer treatment,and the commonly used photosensitizers for lung cancer treatment are non-specific to cancer cells,and more specific targeting of lung cancer cells should be further developed,as well as high-efficiency photosensitizers that can penetrate deep lung cancer tissues,providing a new strategy for enhancing the penetration of anti-tumor drugs.At the same time,some studies have shown that PDT can not only produce phototoxicity to tumor cells,but also induce immunogenic cell death（ICD）.Among them,the mechanism of PDT enhancing tumor immunogenicity has become a research hotspot in recent years.Combined with current research findings,pyroptosis is a lytic programmed cell death characterized by cells expanding to form huge bubbles until the cell membrane ruptures,promoting the immunostimulatory release of cell contents and inflammatory factors,with the potential to activate anti-tumor immune responses.Therefore,we also further explore whether the multifunctional nanovesicle anchored photosensitizer designed in this study targets PDT to induce pyroptosis in lung cancer cells and reveal its underlying mechanism.In conclusion,an effective and safe treatment that specifically induces tumor cell death has a broad application prospect in clinical PDT of lung cancer.This study focues on how to use cell-anchored nanophotosensitizer to achieve efficient treatment of lung cancer.Part 1 Membrane-anchored nano photosensitizers for photocontrolled calcium overload and lung cancer specific synergistic therapyObjectiveTo investigate the role of cell membrane anchored nano photosensitizers（CMA-nPS）in Ca²⁺overload and lung cancer specific synergic therapy.Methods1.Preparation and characterization of cell membrane-anchored nano photosensitizersIn this study,azide-modified macrophage membranes and cell membrane modified with vesicular stomatitis virus glycoprotein（VSV-G）with membrane fusion function were first prepared.Subsequently,the cell membranes of the two functions were extruded through polycarbonate membranes of different pore sizes to obtain multifunctional nanovesicles.Then,inspired by the bioorthogonal reaction,dibenzoctynemodified4-aminophenyl-porphyrin（DBCO-conjugated meso-Tetra（4-aminophenyl）porphyrin,DBCO-TAPP）was reacted with multifunctional nanovesicles to successfully prepare the cell membrane-anchored nano photosensitizers.Finally,characterization is performed by transmission electron microscope（TEM）,nanoparticle tracking analyzer（NTA）,dynamic light scattering（DLS）,etc.2.Cell experimentsFirst,we inhibited endocytosis,and confocal laser scanning microscope（CLSM）studied the uptake of CMA-nPS through the cell membrane fusion pathway,thereby further anchoring the photosensitizers to the membrane of lung cancer cells;Secondly,the MTT method verified the dark toxicity of CMA-nPS,and studied the production of intracellular ROS,changes in intracellular Ca²⁺,cell membrane damage,mitochondrial dysfunction,apoptosis,and cell viability/cytotoxicity experiments confirmed that CMA-nPS kills lung cancer cells in vitro under light.3.Animal experimentsBefore studying in vivo lung cancer ablation,we first used mouse imaging in vivo to observe the enrichment of CMA-nPS,and monitored the weight and tumor volume of tumor-bearing mice with lung cancer during treatment.After the treatment,the dissected mice were subjected to hematoxylin-eosin staining（H&E）of the main organs（such as heart,liver,spleen,lung,kidney）and blood-related analysis to evaluate the biological safety,and the tumor tissues were subjected to H&E,Terminal Deoxynucleotidyl Transferase mediated dUTP Nick End Labeling（TUNEL）,Ki67cell proliferation experiments to evaluate the therapeutic effect of CMS-nPS in vivo.Results1.TEM observation CMA-nPS presents as vesicle morphology,and the lipid layer can be seen.NTA and DLS analysis showed that the average diameter of CMA-nPS was about 200 nm,and the PDI value showed a narrow particle size distribution.2.Ingestion of CMA-nPS occurs primarily through non-endocytic mechanisms.MTT experiments confirmed that CMA-nPS had no effect on cell viability under no light,but under light,the survival rate of lung cancer cells decreased,and the production of intracellular ROS increased significantly,causing damage to the membrane of lung cancer cells,and the concentration of Ca²⁺in cells increased,aggravating mitochondrial dysfunction and leading to obvious apoptosis of lung cancer cells.3.The results of in vivo imaging of mouse showed that CMA-nPS could be well enriched at the tumor site,and after being injected into mice through the tail vein,the tumor site was illuminated,which realized the photocontrolled calcium overload and lung cancer-specific synergistic treatment,which significantly inhibited the growth of lung cancer or even completely ablated lung cancer.ConclusionThe CMA-nPS prepared in this study mainly anchors the photosensitizers to the membrane of lung cancer cells,and further causes membrane damage,mitochondrial dysfunction and intracellular Ca²⁺overload under light irradiation.Cell and animal experiments verified synergistic enhanced anti-lung cancer efficiency.This study provides a new synergistic strategy for the treatment of cancer based on Ca²⁺overload,as well as a strategy for anchoring photosensitizers to the cell membrane,which provides broad application prospects for the treatment of lung cancer.Part 2 Nano photosensitizers penetrate deep tumor tissue to enhance anti-lung cancer efficacyObjectiveTo study the deep penetration of lung cancer tissue by multifunctional nanovesicle photosensitizer and enhance the anti-lung cancer effect.Methods1.Preparation and characterization of aptamer conjugate cell membrane anchored nano photosensitizers（AVnPS）Firstly,multifunctional cell membrane-anchored nano photosensitizers were prepared;secondly,the aptamer AS1411 successfully constructed AVnPS by liposome insertion.Finally,characterization was performed by agarose gel electrophoresis,sodium lauryl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）,and Western blotting.2.Cell experimentsAfter verifying the uptake pathway of AVnPS by CLSM,the tumor targeting of AVnPS was studied and lung cancer multicellular tumor spheroids were constructed in vitro to evaluate the ability of nano photosensitizers to penetrate tumors.In addition,the transwell model was used to study the migration of AVnPS,further verifying the significance of extracellular vesicles in tumor penetration.Finally,a series of related cell tests were used to verify AVnPS’s killing of lung cancer cells.3.Animal experimentsFirstly,the tumor targeting of AVnPS was studied by in vivo imaging of mouse.Then,the body weight and tumor growth volume changes of tumor-bearing mice with lung cancer were detected during AVnPS treatment.Finally,at the end of treatment,mice were dissected,major organs（such as heart,liver,spleen,lung,kidney）were taken for HE staining to assess biological safety,and tumor tissues were subjected to HE,TUNEL,Ki67 experiments to evaluate the therapeutic effect of AVnPS in vivo.Results1.The results of agarose gel electrophoresis showed that AS1411 aptamers were successfully assembled in AVnPS,and AVnPS was successfully prepared.Moreover,SDS-PAGE and Western blotting verified that multifunctional nanovesicles have functional proteins derived from mother cells,and their characteristic proteins are well preserved in AVnPS.2.CLSM results showed that AVnPS had tumor targeting,and at the same time simulated lung cancer spheroids in vitro,and it was observed that the modification of VSV-G promoted the penetration of photosensitizers into multicellular tumor spheroids.To further investigate the tumor penetration mechanism,the transwell model investigated the migration of AVnPS,and the results showed that VSV-G-modified cell membrane-anchored nano photosensitizers are more likely to migrate between cells.In addition,we also collected endogenous vesicles secreted by tumor cells（TCEVs）for experiments,and found that nano photosensitizers are transferred to TCEVs,and with the mediation of TCEVs,nano photosensitizers penetrate layer by layer deep into lung cancer.3.In vivo imaging results of mouse again confirmed the tumor targeting of AVnPS,and AVnPS was significantly enriched at the tumor site.There was no significant change in the weight of mice with lung cancer during treatment,and the AVnPS-treated mice were exposed to light at the tumor site,which significantly inhibited the growth of tumors.After the end of treatment,HE staining of major organs showed no damage,indicating that AVnPS had good biological safety.ConclusionThe multifunctional nanovesicle construction photosensitizers first metastasizes the photosensitizers to the lung cancer cell membrane under the function of VSV-G membrane fusion,and then through the secreted extracellular vesicles of lung cancer cells,the nano photosensitizers will fuse with TCEVs,and with the migration of TCEVs,the photosensitizers can be transferred layer by layer to the depths of tumor tissue.TCEVs can cause photosensitizers to penetrate into tumor tissue,enhance the efficacy of photodynamic therapy,and providing broad application prospects for the treatment of lung cancer.Part 3 Biosynthetic Nano Photosensitizers Activated Caspase-3/GSDME Pathway Induces Pyroptosis in Lung Cancer CellsObjectiveTo explore another way of photodynamic therapy induced lung cancer cell death-cell pyroptosis and its potential mechanism.Methods1.Preparation and characterization of photoinitiated photodynamic therapy nanoplatform（HCNP）HCNP was prepared by combining the relevant experimental methods of the first and second parts and the morphology,DLS and NTA were observed by TEM,and the particle size and concentration distribution were determined.2.Cell experimentsFirstly,we observed the uptake pathway of HCNP through CLSM,and secondly,the survival rate of lung cancer cells was analyzed by cell viability/cytotoxicity and MTT experiments.In addition,the mechanism of pyroptosis in lung cancer cells was further investigated by intracellular ROS and mitochondrial damage,and Western blotting verified the expression of proteins related to mitochondrial damage inducing cleavage of Caspase-3/GSDME in cells.Results1.TEM found that the structure of HCNP is spherical,DLS and NTA show that the average diameter of HCNP is about 200 nm,and it has good dimensional uniformity.2.The observation results of CLSM verify that the HCNP prepared by us is uptaken by cell membrane fusion.At 660 nm,1.4 W cm^-2,after 5 min illumination,the survival rate of lung cancer cells decreased significantly,and the ROS production in the cells further aggravated the damage of mitochondria,and the Western blotting experimental results showed that membrane-targeted PDT could effectively activate Caspase-3 and lyse GSDME,which could induce cell death through the pyroptosis pathway.ConclusionHCNP effectively destroys cellular mitochondrial function by producing ROS under laser irradiation;At the same time,GSDME is lysed by activated Caspase-3 and promotes pyroptosis of lung cancer cells,which provides an effective intervention strategy for photoactivated PDT to induce pyroptosis in lung cancer cells,and also demonstrates the potential of cell membrane-anchored photosensitizers in the treatment of lung cancer by activating the pyroptosis pathway.更多还原