A ROS-Responsive Nanoparticle System Combines Photodynamic/Photothermal Therapy And Chemotherapy For Hepatoma Treatment

ObjectiveHepatocellular carcinoma（HCC）is one of the most common and deadly malignant tumors both in China and worldwide.Currently,the clinical treatment of hepatoma mainly includes surgery,chemotherapy,radiotherapy and immunotherapy,However,any single clinic treatment has its limitation.Phototherapy,which mainly includes photodynamic therpay（PDT）and photothermal therapy（PTT）,is a novel non-invasive clinic treatment in recent years.PDT/PTT combined with chemotherapy possesses synergistic antitumor effects and has shown great potential for cancer treatment.Cancer cells often exhibit a persistent high level of reactive oxygen species（ROS）due to their abnormal metabolism.Besides,some treatments such as PDT and chemotherapy can induce the generation of intracellular ROS,thus further enhancing the ROS levels in cancer cells.In this study,we synthesized a ROS-responsive poly（β-amino ester）(PBAE_ROS)and used it as a carrier material to prepare nanoparticles for efficient co-loading photosensitizer IR780 and chemotherapeutic drug doxorubicin（DOX）.The obtained nanosystem（named PPID nanoparticles）is composed of a hydrophobic nanocore and a hydrophilic propylene glycol alginate sodium sulfate（PSS）nanoshell which could exert the effect of anti-angiogenesis to develop a nanotherpeutic system named as PSS/PBAE_ROS/IR780/DOX（PPID）nanoparticles.Here,we also evaluated the antitumor effects of PPID nanoparticles in vitro and preliminarily explored their functional mechanisms.ContentsThe main content of this paper included the following three parts.The first section is the preparation and characterization of PPID nanoparticles,including the synthesis and structure confirmation of PBAE_ROS,and the preparation and characterization of PPID nanoparticles.The second section is the in vitro study of PPID nanoparticles,including the evaluation of ROS-responsiveness of PBAE_ROS and PPID nanoparticles,in vitro drug release behavior and in vitro PDT and PTT efficiencies of PPID nanoparticles.The third section is the in vitro study of PPID nanoparticles on cellular level and in vivo targeting capability,including the assessment of cellular internalization of PPID nanoparticles,the synergistic cytotoxicity of PPID nanoparticles-mediated combination treatment of PDT/PTT and chemotherapy in Hep1-6 cells.Furthermore,the biodistribution of PPID nanoparticles in tumor-bearing mice was studied.Methods1.Preparation and characterization of PPID nanoparticles.Diacetoxyl thioketal（DCT）,diethanol thioketal（DET）,and diacrylate thioketal（DAT）were synthesized and then used to synthesize PBAE_ROS via Michael addition reaction.The apparent molecular weight of PBAE_ROS was determined by gel permeation chromatography（GPC）.The chemical structures of DET,DCT,DAT,and PBAE_ROS were determined by IR and ¹H-NMR.PPID nanoparticles were prepared using nanoprecipitation.Size-zeta potential analyzer was used to measure the particle size,particle size distribution and average zeta potential of PPID nanoparticles.The morphology of PPID nanoparticles was observed using a transmission electron microscopy（TEM）.The loading content（DC）and encapsulation efficiency（EE）of DOX and IR780 in PPID nanoparticles were detected using the fluorescence and UV-visible spectrophotometric method to evaluate the drug carrying capacity of this nanosystem.2.In vitro properties of PPID nanoparticles.The structural and morphological changes of PPID nanoparticles in the H₂O₂ solution were investigated by the¹H-NMR and TEM,thus to evaluate the ROS-response ability of PPID nanoparticles.The in vitro release characteristics of DOX from PPID nanoparticles was evaluated using the dynamic dialysis method.The temperature change of the PPID nanoparticle solution under laser irradiation was detected by the infrared thermal imager to evaluate the PTT efficacy of PPID nanoparticles.Singlet oxygen sensor green（SOSG）was used as a green ROS fluorescent probe to detect the generation of ¹O₂in the PPID nanoparticle solution,thus to evaluate the PDT efficacy of PPID nanoparticles.3.In vitro pharmacodynamics and in vivo tumor-targeting capability of PPID nanoparticles.The uptake of PPID nanoparticles by mouse Hep1-6 hepatocellular carcinoma cells was first investigated by laser confocal microscopy and flow cytometry.The synergistic cytotoxicity of PTT/PDT and chemotherapy mediated by PPID nanoparticles was assessed using the MTT and LIVE/DEAD cell staining methods in Hep1-6 cells.2’,7’-Dichlorodihydrofluorescein diacetate（DCFH-DA）was used as a specific fluorescent probe to detect the ROS generation in Hep1-6 cells for evatulating the PDT efficacy of PPID nanoparticles after laser irradiation.A transplantation mouse model of hepatoma was constructed through subcutaneously injecting Hep1-6 cells into Balb/c mice.The biodistribution of PPID nanoparticles was studied using the fluorescence signal of IR780 in Hep1-6 tumor-bearing mice.Results1.PBAE_ROS was successfully synthesized and its chemical structure was confirmed by the IR,¹H-NMR and GPC methods.PPID nanoparticles prepared by a simple nanoprecipitaion method had a clear“core-shell”structure and a small size about 198 nm with a polydispersity index of 0.162,and their zeta potential was?43.3m V.Furthermore,PPID nanoparticles displayed a very good storage stability.In PPID nanoparticles,the loading contents of IR780 and DOX were 12.6%and 3.44%respectively,and correspondingly their encapsulation efficiencies were high to 89.6%and 97.5%.2.Under the 808 nm laser irradiation,the temperature of PPID nanoparticle solution was raised to about 60°C,and PPID nanoparticles exhibited a significantly higher PTT efficiency than IR780.Meanwhile,the fluorescence signal of SOSG in the PPID nanoparticle solution was obviously stronger than the solution of IR780,demonstrating that PPID nanoparticles had a higher PDT efficiency than IR780 and could induce the generation of a large amount of ROS.In PBS solutions with H₂O₂,the thioketal linker in the monomer structure of PBAE_ROS could be efficiently ruptured,hence resulting to the disintegration of PPID nanoparticles and the subsequent rapid release of DOX,e.g.more than 60%of DOX was released from PPID nanoparticles after 120 h.All these results demonstrated that PPID nanoparticles had an obvious ROS-responsiveness.3.PPID nanoparticles notably improved the entry of IR780 into mouse hepatoma Hep1-6 cells and showed excellent PTT/PDT efficacies.Under the 808 nm laser irradiation,PPID nanoparticles exerted the direct cell-killing effects by rising the temperature and triggering the ROS generation.Subseqently,DOX was released efficiently from PPID nanoparticles through the ROS-responsive rupture of thioketal and then entered the cell nucleus to exert cytotoxic activity.Hence,PPID nanoparticles showed significant synergistic anti-hepatoma effects of PTT/PDT and chemotherapy in vitro.A transplantation mouse model of hepatoma was successfully constructed through subcutaneously injecting Hep1-6 cells into Balb/c mice.After intravenous injection,PPID nanoparticles were mainly located in the tumor in Hep1-6tumor-bearing mice,indicating their good hepatoma-targeting ability in vivo.ConclusionsIn this study,a ROS-responsive nanocarrier material named PBAE_ROS was successfully synthesized,and also confirmed its chemical structure.After that,PBAE_ROS and PSS were used as the carrier materials to prepare PPID nanoparticles with co-loading of photosensitizer IR780 and chemotherapeutic drug DOX.PPID nanoparticles showed a regular spherical shape and a clear“core-shell”nanostructure.Combining the irradiation of near-infrared laser,the photosensitizer produced ROS and heat which can exert the PDT and PTT efficiencies.In Hep1-6 cancer cells,PPID nanoparticles notably improved the cell entry of IR780,and thus exhibited the higher PTT/PDT efficacies than free IR780 after laser irradiation.By responding to the intracellular ROS,DOX could be efficiently released from PPID nanoparticles to exert the cytotoxic activity.Therefore,PPID nanoparticles displayed significant synergistic in vitro anti-hepatoma effects through combining PTT/PDT and chemotherapy.Altogether,we provide a novel nanocarrier system for the combination of pototherapy and chemotherapy to treat hepatoma.