仿生生物自发光纳米材料用于肿瘤光动力治疗

Biomimetic biological self-luminous nanosystem for photodynamic therapy

光动力疗法(PDT)具有高效的肿瘤细胞杀伤能力,因此被用于多种肿瘤的治疗,但是光在组织中穿透性较差,限制了光动力疗法的进一步发展.为解决肿瘤光动力治疗中外部照射光组织穿透力弱的问题,本文仿生萤火虫生物发光特性,构建了可在组织内部进行生物自发光的纳米材料.选择ZIF-8对生物发光系统进行封装,并选择Mn O2对ZIF-8进行包裹以此来减少纳米粒子的毒性.该纳米粒子能够实现发光系统在肿瘤部位响应释放.同时,选择激发波长与生物发光波长重叠的光敏剂直接与发光系统相连,保证了高效的生物发光共振能量转移.本文研究证实了该纳米材料具有层次分明的核壳结构,以及良好的细胞生物相容性,可被肿瘤细胞大量摄取.进一步研究证明该纳米材料可在肿瘤细胞高谷胱甘肽的微环境中降解,释放催化酶及发光底物,并在腺嘌呤核苷三磷酸(ATP)的作用下进行生物自发光.同时,细胞实验证实了发光底物可通过生物发光共振能量转移效应激发光敏剂产生活性氧,进行光动力治疗,对耐药肿瘤细胞具有良好的细胞杀伤效果.本文构建的生物自发光纳米材料具有在生物深层组织进行生物自发光激活光敏剂光动力的潜力,有望解决肿瘤光动力治疗中外部照射光组织穿透力弱的缺点,有效杀伤深位组织耐药肿瘤.

Photodynamic therapy(PDT)has been used for the treatment of a variety of tumor species due to its excellent tumor cell killing ability,but the tissue penetration ability of external irradiation light limits its further development.As an internal excitation light source,biological self-luminous feature has the potential to solve the weak tissue penetration ability of external irradiation light in photodynamic therapy.Here,we successfully synthesized the biomimetic firefly bioluminescence nanosystem of ZIF-8@D@Luc-RB@Mn O2.Scanning electron microscope(SEM)and transmission electron microscope(TEM)revealed that ZIF-8@D@Luc-RB@Mn O2has regular morphology and core-shell structure.The uniform distribution of major elements(Zn,N,Mn,and K)in nanosystem was demonstrated by high angle annular dark field scanning transmission electron microscopy.It was also proved that the content of D-Luciferin in nanosystem is about 25%by detecting the UVabsorption spectrum of the supernatant after preparation of nanosystem.By co-incubating with glutathione(GSH)and adenosine triphosphate(ATP),it was found that the synthesized nanosystem could be degraded by GSH to release the loaded luciferase and the luciferase substrate of D-luciferin.With the participation of ATP,firefly luciferase catalyzes the oxidation reaction of D-luciferin for bioluminescence.The ability of nanosystem for bioluminescence was evaluated by chemiluminescence instrument.The results proved that ZIF-8@D@Luc-RB@Mn O2can produce obvious bioluminescence in aqueous solution.It was also proved that the resonance energy transfer occurs between the bioluminescence system and Bengal Rose,and causes part of the energy consumption.Because the excitation wavelength of Bengal Rose is similar to the emission wavelength of bioluminescence system,the energy consumption during resonance energy transfer is less.Further incubating with reactive oxygen species fluorescence probe of 2′,7′-dichlorofluorescein diacetate(DCFH-DA)to evaluate the reactive oxygen species(ROS)generation,the result showed that the bioluminescence produced by the nanosystem can activate the photosensitizers of RB to produce reactive oxygen species.The presence of GSH could significantly improve the production ability of ROS due to the rapid degradation of Mn O2shell.At the same time,highly effective bioimaging property of different concentrations of nanosystem was achieved.By co-incubating with 4T1 tumor cell,we found that 4T1 tumor cells can internalize the synthetic nanosystem in high content.DCFH-DA was further used to detect the change of intracellular ROS level.According to the results of inverted microscope observation,the intracellular ROS level of cells increased significantly after treating with nanosystem.These results confirmed that the nanosystem can induce bioluminescence and resonance energy transfer to effectively activate photosensitizer to produce ROS in tumor cell.3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide(MTT)colorimetric assay and live/dead cell staining detected high cytotoxicity of the nanosystem towards drug-resistant 4T1tumor cells,demonstrated that the high content of intracellular ROS finally caused tumor cell death.In summary,the biomimetic biological self-luminous nanosystem can activate photosensitizers to produce reactive oxygen species for photodynamic therapy,which has the potential to solve the limitation of tissue penetration depth of external irradiation light in photodynamic therapy.