Synthesis Of Self-oxygen Supported Nanoparticles And Their Antitumor Efficacy On Breast Cancer In Photodynamic Therapy

With the improvement of people's living standard,the morbidity and mortality of cancer also increased year by year,its incidence has exceeded heart desease and become the disease with maximum mortality in the world,and seriously threaten the human life and health.The most common methods of cancer treatment are chemotherapy,radiotherapy and surgical treatment.Photodynamic therapy?PDT?is widely concerned as a non-invasive and selective treatment method.Oxygen,photosensitizer?PS?and specific light source are the three factors that cause photodynamic effects.Although PDT has many advantages,it is also limited by the above three factors.On the one hand,PDT is a treatment that relies on oxygen.However,due to enhanced tumor proliferation activity and abnormal structures of tumor vessels,tumor tissue usually produces a certain hypoxia microenvironment.This is an extremely negative factor for PDT in the cancer treatment.A large amount of H₂O₂ will also be produced during the process of cancer formation.Recently,various micro-vehicles aiming to carry oxygen to the tumor area have been developed to overcome the anoxic environment,such as use perfluocarbon,hemoglobin?Hb?and catalase?CAT?and so on as oxygen donors,showing improved effect of PDT.On the other hand,the clinical approved PSs are only able to effectively absorb light in the visible light region below700 nm,and this light source can only be used for superficial treatment of lesions near the surface of the skin.An improved method is to apply two-photon technology in PDT.Two-photon excitation light source has deeper penetration depth,and the application of two-photon technology to photodynamic therapy is expected to extend the treatment from superficial to deeper tissue.In addition,some of the work,including ours,reported the fluorescence resonance energy transfer?FRET?technology used in photodynamic therapy of two-photon excitation?TPE-PDT?.Under the two-photon excitation,TPE-PDT linkage effect occured.But FRET in particle requires careful design.This is a research field that has just arisen in recent years.So far,there are only a few research groups in the world have studied and obtained preliminary research progress.In conclusion,we analysed that if the oxygen carrier,photosensitizer and two-photon dye were assembled together in a system,not only it could provided rich O₂ in PDT,but also could take advantage of the characteristics of two-photon dye and increase treatment depth in the process of PDT.The rich nano-carriers of drug and various co-assembly technologies that our research team has been working on will provide the potential for this solution.Vaterite CaCO₃ nanoparticles with good biological compatibility and biodegradable properties,and liposome complex with good biocompatibility and tumor targeted effect,making it possible for successful assembled particle.Breast cancer has the highest incidence of malignancies in women,which seriously affects their health,and there is a rich folate receptor expression on the surface of human breast cancer MCF-7 cell,which is suitable for the targeted research of drugs.We selected the MCF-7 cell as the study subject and evaluated the therapeutic effects of the assembled particles.The study is divided into four parts:Part 1.Preparation and characterization of CAT-based self-oxygen supported systemThe synthesized CaCO₃ nanoparticles with vaterite structure in our recent work has presented good biological compatibility and biodegradable properties,they are rich in nanometer pores and have extremely mild preparation condition without introducing any organic reagents.The fluorescent dye FITC,photosensitizer RB and oxygen carrier CAT were assembled in CaCO₃ nanoparticles by a co-deposition method.Such a complex in tumor tissue,on the one hand,can remove the large amount of H₂O₂ generated in the canceration process,which will slow down the invasion process of cancer.On the other hand,the aprticle produced a large amount of O₂ in the process of the removal of H₂O₂,improving the efficiency of PDT.The FRET effect occurs between the two photon dye and RB in the case of one/two-photon excitation,and it is possible to extend the PDT treatment from superficial to deep tissue.The results showed that the composite nanoparticles?F/R-CAT-VN?were uniform and well dispersed.FITC and RB were successfully bond with CAT,and the FRET effect can occur under one/two-photon excitation.The CAT in the particle has good biological activity.The performance of the synthesized nanoparticles with intraparticle FRET is obviously improved,which is beneficial to further study.Part 2.Biological effect assesment of CAT-based self-oxygen supported systemIn order to further study the composite nanoparticles,its biological correlation effect was evalulated.The endocytosis experiment showed that the particle size of CaCO₃ nanoparticles based on CAT was appropriate,biocompatible,and easy to be captured by cells.After incubation with red blood cells,the nanoparticles showed no obvious hemolysis,which demonstrated the good blood compatibility and the property suitable for in vivo study.Nanoparticles show good performance of ROS generation in cells,and the F/R-CAT-VN particle based on FRET effect produced more siglet oxygen than photosensitizer.Nanoparticle under the condition of one/two-photon irradiation could effectively kill cells,dark toxicity was very low.F/R-CAT-VN based on FRET effct showed higher cytotoxicity than the same concentration of photosensitizer on MCF-7 cell line.The CAT doped F/R-CAT-VN showed higher cytotoxicity than D-F/R-CAT-VN without CAT.Nanoparticles exhibited good vascular blocking effects in mice.The distribution in vivo was mainly concentrated in the liver and tumor.Part 3.Preparation and characterization of Hb-based self-oxygen supported systemHb has been widely used in the synthesis of artificial oxygen carriers due to its excellent oxygen carrying capacity.And liposome has been applied to encapsulate anti-tumor drugs for its good biocompatibility.In this part,the fluorescence dye BP with a large absorption cross-section was introduced,which was mixed with Hb,photosensitizer and liposome.A one-step ultrasonic method was used for the synthesis of folate targeted nanoparticles?BP@RB-Hb?.The synthesized particles showed good dispersibility and uniform particle size,and Hb exhibited effective oxygen carrying function.FRET validation experiments showed exact FRET process intraparticle,and the detection depth was extened to 2000mm under two-photon excitation.The ROS production rate of the particle in solution is higher than that of the pure photosensitizer and the nanoparticle without oxygen carrier.Part 4.Biological effect assesment of Hb-based self-oxygen supported systemThe hemoglobin based nanoparticle was further studied by biological experiments.The results showed that the liposome particle size was suitable and easy to be swallowed by cells.The particle produced more singlet oxygen than the particle without Hb and pure photosensitizer in MCF-7 cell.In the case of one/two-photon irradiation,nanoparticles show good anti-tumor effect,and the cytotoxity of BP@RB-Hb particle is higher than that of pure photosensitizer.The nanoparticles were incubated with red blood cells and showed good biocompatibility.In vivo distribution experiment showed that nanoparticles were mainly concentrated in the liver and tumor in mice.Due to the addition of folate liposome,nanoparticles showed excellent tumor targeted effect.The experimental results showed that the complex had good anti-tumor effect in vivo,and their anti-tumor activity was significantly enhanced compared with the particle without oxygen donor.In conclusion,through simple assembly method,oxygen donor,two-photon fluorescence dye and photosensitizer were assembled in one system,which was used to solve the problems of treatment depth and efficiency in the antitumor photodynamic therapy.The treatment effect was evaluated with breast cancer MCF-7cells.The fluorescence dye in the system could be excited by two-photon,and the treatment depth can be improved under the help of FRET in the particle.The assembled oxygen carrier could overcome the hypoxia environment of tumor and improve the anti-tumor efficiency of PDT.In vivo and in vitro experiments had confirmed that the FRET effect intraparticle could occur.Then the obtained particles possessed good stability,biocompatibility,deeper penetration depth and strong ability of carrying oxygen.The multifunctional nanocomposites exhibited good therapeutic effects and were of great significance for the clinical application of one/two-photon anti-tumor photodynamic therapy.