| Background and Objective:Hepatocellular carcinoma（HCC）is the most common type of primary hepatocellular carcinoma.Because of its high heterogeneity,insidious occurrence,and lack of specific markers,HCC is often diagnosed at a late stage of development.At this time,effective treatment strategies for HCC are insufficient,and the 5-year survival probability of HCC patients is difficult to meet expectations.Based on the above characteristics,HCC has been one of the malignant tumors of great concern,and there is an urgent need to propose effective treatment strategies for it.Photodynamic therapy（PDT）,an emerging disease treatment modality,has demonstrated efficient and minimally invasive treatment characteristics in diverse tumor treatments and is expected to open a new blueprint for tumor treatment.As one of the three major elements of PDT,photosensitizers play an irreplaceable role in tumor treatment.Chlorin e6（Ce6）,a rapidly developing photosensitizer,has been successfully commercialized.Ce6 has been used in several basic experiments for the exploration of tumor photodynamic therapy,showing good anti-tumor ability.However,disadvantages such as poor biocompatibility and weak hydrophilicity have also emerged.Regorafenib（RGF）,as a new angiogenesis-inhibiting targeted drug,plays a crucial role in the second-line treatment of advanced HCC;However,it is gradually facing problems of tumor resistance and obvious adverse effects in clinical utility.In this study,we aim to prepare a biocompatible RGF-Ce6 nanoparticles（RGF/Ce6-NPs）to combine molecular targeting therapy and photodynamic therapy,and then investigate its role in the treatment of HCC.Methods:In this study,nanoparticles of RGF/Ce6-NPs were prepared by nanoprecipitation method with a carrier of amphiphilic block copolymer poly（ethylene glycol）-block-poly（D,L lactic acid）（PEG-PLA）.The drug loading capacity（DLS）and encapsulation efficiency（EE）of RGF and Ce6 in the nanoparticles were measured by UV spectrophotometer,and the drug release performance in vitro was observed by dialysis method.Cellular uptake behavior and intracellular reactive oxygen species（ROS）release were observed with laser confocal microscopy and fluorescence microscopy;the antitumor effect in vitro and apoptosis-inducing ability of RGF/Ce6-NPs on two HCC cell lines（Hep3B and SMMC-7721）were explored by MTT method,live-dead cell staining and Annexin V-FITC/PI double staining;finally,laser confocal microscopy was used to observe the effect of sorafenib-resistant HCC cell line（SMMC-7721S）on the uptake effect and intracellular ROS release level of RGF/Ce6-NPs,and explored the killing effect of RGF/Ce6-NPs on SMMC-7721S with MTT method.Results:（1）RGF/Ce6-NPs nanoparticles were self-assembled by wrapping RGF and Ce6with PEG-PLA as the carrier.The morphology and particle size were observed by transmission electron microscopy and dynamic light scattering analyzer,which showed that the nanoparticles exhibited a monodisperse spherical structure with uniform size.The dynamic light scattering results showed that the RGF/Ce6-NPs had a particle size of 63.68±2.65 nm,a PDI of 0.269±0.058,and a zeta potential of-31.931±0.275 m V,and showed good stability in water and under physiological conditions.（2）The physicochemical properties of the nanoparticles RGF/Ce6-NPs were further explored based on various characterization tools,and the RGF/Ce6-NPs exhibited both specific UV absorption peaks of RGF and Ce6.Among the RGF/Ce6-NPs,the DLS and EE of regorafenib were 12.31±0.682%and 24.53±2.19%,respectively;and the DLS and EE of Ce6 were 15.28±0.365%and 31.16±1.63%,respectively.The results of drug release assay confirmed that the release of RGF/Ce6-NPs showed gradient release and time dependence of regorafenib in physiological environment;the results of ROS release assay showed that RGF/Ce6-NPs could produce ROS under 660 nm laser irradiation with great yield.（3）Cellular uptake experiments and photodynamic effects of RGF/Ce6-NPs were carried out at the cellular level.Laser confocal microscopy results confirmed that HCC cells could successfully uptake RGF/Ce6-NPs,and laser microscopy results confirmed that more ROS were produced by intracellular RGF/Ce6-NPs than by free Ce6 under 660 nm laser irradiation.（4）Cytotoxicity experiments demonstrated that the toxicity of RGF/Ce6-NPs to HCC cells showed a drug concentration dependence.The IC50 of RGF/Ce6-NPs with laser were 1.161μg/m L and 1.714μg/m L for Hep3B cells and SMMC-7721 cells,respectively;while the IC50 of free RGF and free Ce6 with laser were 2.989μg/m L and 2.127μg/m L for Hep3B cells,respectively,and the IC50s of free RGF and The IC50 of free Ce6 with laser for SMMC-7721 cells were 3.566μg/m L and 2.789μg/m L,respectively.The results confirmed that the killing effect of nanoparticle RGF/Ce6-NPs on HCC cells under 660 nm laser irradiation were significantly stronger than that of free RGF and free Ce6 under light;live-dead cell staining assays also observed similar results.Meanwhile,the results of apoptosis detection by flow cytometry showed that RGF/Ce6-NPs significantly induced apoptosis in Hep3B cells and SMMC-7721 cells under light conditions.（5）Based on the results of sorafenib-resistant HCC cells SMMC-7721S,RGF/Ce6-NPs could be successfully taken up by this drug-resistant cells and produced ROS intracellularly.The results of cytotoxicity assay showed that the IC50values of RGF/Ce6-NPs under light conditions were 9.78μg/m L for SMMC-7721S cells,while the IC50 values of free RGF and free Ce6 under light conditions were14.17μg/m L and 13.66μg/mL for SMMC-7721S cells,respectively.Conclusions:In this study,we designed and synthesized a functional nanoparticle RGF/Ce6-NPs encapsulating RGF and Ce6.This nanoparticle exhibited good physicochemical properties,improved the biocompatibility of the two drugs,and reduced the dose of regorafenib.By effectively co-delivering RGF and Ce6 to tumor tissues,this functional nanoparticle achieves synergy between molecular targeting therapy and photodynamic therapy,providing a new direction for the clinical treatment of liver cancer. |
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