A Multifunctional Nanoparticle System Combines Sonodynamic Therapy And Chemotherapy To Treat Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma?HCC?is one of the most common and deadly malignancies worldwide.To date,the survival of patients with HCC has not improved because of the insensitivity of HCC to conventional treatments.Recently,Sonodynamic therapy?SDT?has been attempted in clinical practice to combat cancers.During SDT treatment,sonosensitizer is accumulated in tumor and then activated by a specific wavelength of ultrasound which will lead to the creation of the sonodynamic reaction and reactive oxygen species?ROS?.SDT is a promising new approach that shows remarkable potential in the treatment of HCC.Here,we designed a simple,biocompatible,and multifunctional nanosystem that combines SDT and chemotherapy to treat HCC.This nanosystem,called HPDF nanoparticles,had a core-shell structure in which hematoporphyrin?HP?was complexed with doxorubicin?DOX?to form the hydrophobic core and the surface was coated with Pluronic F68 to form the hydrophilic shell.Its function in coloading of HP and DOX,tumor targeted drug delivery,synergistic anti-tumor effect will be studied in vitro and in vivo.Methods:1)HP was complexed with DOX to form the hydrophobic core HPD and the surface was coated with Pluronic F68 to form the hydrophilic shell.The structure,morphology,diameter and zeta potential,colloidal stability of HPDF nanoparticle were analyzed with transmission electronic microscopy?TEM?,atomic force microscope?AFM?and dynamic light scattering?DLS?,respectively.High Performance Liquid Chromatography?HPLC?was applied to investigate the drug release behavior of HPDF.2)Cells were treated with free DOX,free HP,HPF,and HPDF nanoparticles at different DOX and/or HP concentrations.The cell viability and 50%inhibition concentration of various treatments and the proliferations of cells were evaluated using the CCK-8 assay.Cellular uptake was detected via flow cytometry.Intracellular location and endo/lysosomal escape of HPDF was detected by living cell imaging and confocal microscopy.The expressions of stem cell-related genes,such as Nanog,Oct4,and Sox2 in Nanog^Pos CSCs and Nanog^Neg cells were detected using RT-q PCR assay.3)Apoptotic cell percentage was evaluated by Anexin V-APC/7AAD staining.Cell cycle evaluated by PI/RNase staining.ROS production in Hep G2 was detected via confocal microscopy and flow cytometry using DCFH-DA as a fluorescent probe.Cells were treated with Rh123 to detect mitochondrial trans-membrane potential or cytochrome c antibody to study its subcellular distribution,respectively.Important components of mitochondrial apoptosis pathway protein and nuclear damage related protein expression were detected by western blotting.4)Subcutaneous Hep G2 xenograft mouse model was estabilished to evaluate the targeting property of HPDF nanoparticles via the in vivo imaging technique.Distribution of HPDF in tumor bearing mice were measured by tail-vein administration of HPDF/Cy5.5 nanoparticles or Cy5.5 alone,respectively.The tissue accumulations of HPDF/Cy5.5 nanoparticles were also evaluated in Hep G2 tumor-bearing mice within 2-10 h after administration.Cryosections obtained were stained with DAPI,and then the DOX accumulations in the heart and tumor were visualized using a confocal microscope.5)Hep G2 tumor-bearing nude mice were treated with normal saline,ultrasound radiation alone,free DOX,HPF nanoparticles in combination with ultrasound radiation,and HPDF nanoparticles in combination with and without ultrasonic irradiation via administration of tail vein injection,the tumor growth was detected by measuring the tumor size.Heart,liver,spleen,lung,kidney and tumor were collected after treatment.The histopathological changes of the organs and tissues of tumor-bearing mice were observed using hematoxylin&eosin staining,the intratumoral collagen deposition were detected using Masson's trichrome staining and the inhibitory effect against angiogenesis was assessed by using immunohistochemical CD31 staining.Results:1)HPD had a regular spherical shape and an average size<50 nm.Pluronic F68was coated on the surfaces of HPD to form HPDF nanoparticles.The HPDF nanoparticles had a clear“core–shell”structure and excellent in vitro stability,the mean diameter of the HPDF nanoparticles was 87.9±10.8 nm.HPDF exhibited a significant p H-sensitive release property,DOX's release rate distinctly increased with the decrease in the p H value of the released media.2)On combination with ultrasonic irradiation,HPDF nanoparticles increased the intracellular ROS production in a time dependent manner;the ROS level reached the highest value in Hep G2 cells at 12 h after treatment with ultrasonic.Compared with the control,both free DOX and HPDF nanoparticles caused mitochondrial membrane damage and release of Cyt c from mitochondria into the cytoplasm.The treatment with HPDF nanoparticles in combination with ultrasonic irradiation showed the highest expression levels of cleaved caspase-9,caspase-3,p53 and Rad51.3)HPDF nanoparticles were easily taken up by Hep G2 cells and began to transfer from the cytoplasm to the nuclei via escaping endocytosis.Ultrasonic irradiation further enhanced the cytotoxicity of HPDF nanoparticles,resulting in an approximately 5.5-fold decrease in the IC₅₀ value compared to that of free DOX.Compared with free DOX,the combined treatment further promoted the apoptosis and cell cycle arrests in the S-phase of Hep G2 cells.4)Nanog^Pos CSCs exhibited significantly higher expression levels of several stem cell-related genes?Nanog,Oct4,and Sox2?,compared to Nanog^Neg cells.Compared with free DOX,ultrasonic irradiation further increased the cytotoxicity of HPDF nanoparticles,resulting in an approximately 10-fold decrease in the IC₅₀ value.HPDF nanoparticles remarkably enhanced DOX accumulation in Nanog^Pos CSCs and the red fluorescence was mostly distributed in the cell nucleus.5)HPDF/Cy5.5 nanoparticles began to accumulate in the tumor at 4 h and reached the highest level during 6–8 h,nearly only observed in the tumor at 24 h.Compared to free DOX,DOX delivered by HPDF nanoparticles exhibited a markedly reduced heart distribution and a dramatically increased tumor accumulation.6)Compared to free DOX,ultrasonic irradiation combined with HPDF nanoparticles almost completely inhibited the tumor growth,no detectable pathological change or injury was observed in the main organs of tumor-bearing mice.The mice receiving the combined treatment with HPDF nanoparticles and ultrasonic irradiation showed the smallest tumor microvessel and considerably stronger inhibitory effects on intratumoral collagen deposition compared with the other treatments.Conslusion:In this study,we designed a simple,biocompatible,and multifunctional nanoparticle system HPDF.The results showed that HPDF nanoparticles can effectively combine SDT and chemotherapy to treat hepatomas,and this nanoparticle system exhibits considerable potential for clinical applications in tumor treatment.