Multifunctional Nano-liposomes For Synergistic Phototherapy And Hypoxia-activated Bioreductive Therapy

Photodynamic therapy（PDT）is a kind of tumor therapy that uses photosensitizers（PSs）mediated light source radiation to produce high levels of reactive oxygen species（ROS）or singlet oxygen（¹O₂）to effectively kill cells.It has become one of the main strategies for the treatment of solid cancer due to its high selectivity,non invasiveness and low systemic toxicity.However,the natural hypoxic tumor micro-environment and PDT mediated oxygen consumption limit their wide clinical application.Anoxic response drug tirapazamine（TPZ）has specific cytotoxicity to hypoxic tumor cells.However,when applied alone,it can not produce enough toxic free radicals for relatively oxygen-enriched cells in tumor tissues.Therefore,the combination of PDT and hypoxia responsive drugs can turn their disadvantages into advantages through PDT mediated hypoxia activated chemotherapy,which provides a new and effective strategy for the treatment of hypoxia tumor.In this project,a novel nano-liposome Lipid（Cy I-TPZ）（LCT）was developed by combining the near infrared dye Cy I as photosensitizer with TPZ as a biological reducing precursor.Cy I,a kind of iodinated cyanine dye synthesized in our group,which could generate stronger reactive oxygen species yield,higher fluorescence quantum yield and photothermal conversion ability than other traditional cyanine dyes.It can be used as an efficient photosensitizer/photothermal reagent and provide fluorescence signal for accurate real-time imaging.TPZ is a novel bioreductive drug,which has been proved to have highly selective cytotoxicity to hypoxic cells,and can metabolize toxic free radicals under hypoxia conditions,leading to DNA damage and cell death.Upon specific near infrared light（NIR）irradiation,LCT could simultaneously produce ROS and heat for synergistic PDT and photothermal therapy（PTT）.Meanwhile,the continuous consumption of oxygen would result in the hypoxia micro-environment,further activating TPZ to produce free radicals for photo-chemotherapy.In addition,LCT can also induce acute immune response and further eliminate residual tumors through photodynamic therapy.The encapsulation and fluorescence characteristics of LCT were identified by UV-Vis-NIR absorption spectrum and fluorescence spectrum;the release characteristics of LCT were studied by dialysis method;the singlet oxygen production rate and tumor hypoxia were investigated by singlet oxygen fluorescence probe SOSG and hypoxia kit;the photothermal effect was analyzed by near infrared imager and thermocouple thermometer;the tumor killing ability,apoptosis and necrosis were evaluated by cytotoxicity test and flow cytometry;the distribution of LCT in vivo was observed through a animal imaging system;the subcutaneous tumor model of 4T1 in mice was established to evaluate the tumor therapeutic effect and immune response of LCT.The results show that LCT prepared by membrane hydration had no precipitation or phase separation within 5 weeks,and had good biological stability.The results of UV absorption and fluorescence spectra show that Cy I and TPZ were successfully encapsulated in liposomes and kept the fluorescence characteristics of Cy I,which could be excited in near infrared region and used for real-time monitoring and imaging of drugs in vivo.According to the results of in vitro photodynamic study,the ROS yield of LCT is time and power density dependent,which can produce a large number of ROS under certain conditions,and play a good role in photodynamic therapy.The release experiment show that the LCT can melt lipid and break down gradually under the laser irradiation.Thus,it is used as a near infrared light triggered nano-liposome to control the release of drugs.In vitro cell studies show that LCT could be effectively absorbed by 4T1 cells at 4 h,and a large amount of reactive oxygen species and heat were produced under 0.96 W/cm²near infrared light irradiation.At the same time,oxygen was consumed to produce hypoxic environment,thus activating TPZ to play the antitumor role of PDT/PTT/chemotherapy.MTT results further show that the combined application of Cy I and TPZ significantly reduced the IC₅₀of LCT to 30.69μg/m L,and its combined treatment index was 0.83,which proved that Cy I and TPZ actually produced a synergistic effect,not just a simple superposition of therapeutic effects.The results of synergetic therapy and immune evaluation in mice show that LCT had the better tumor inhibition effect,and could induce immune response in vivo through photodynamic therapy,so as to produce a marked effect of clearing residual tumor.Compared with traditional PDT or single chemotherapy,LCT can destroy cancer cells more effectively by PDT/PTT/chemo/immunotherapy,which has better therapeutic effects.This nano-liposome based on iodinated cyanine dye is expected to provide new ideas and strategies for tumor targeted phototherapy and hypoxia-induced chemotherapy.