Targeted Nanosystem Combined With Chemo-Photothermal Therapy For Hepatocellular Carcinoma Treatment

Poor tumor treatment is a major problem in current clinical research.At present,the common clinical methods used for tumor treatment mainly include chemotherapy,radiotherapy and surgical treatment.However,it is difficult for a single treatment to prolong the survival time of patients.Multi-modality cooperative treatment is still the research focus and development trend for a long time in the future.Nanobiotechnology,which integrates various technologies of chemistry,biology,materials science,and medicine,is considered to have inestimable development potential in clinical tumor treatment.Therefore,in recent years,relying on nanobiotechnology,the strategy to develop a nano drug-loaded composite system using nano-materials as its carrier,combined with one or more therapeutic methods for tumor treatment,has been highly valued in the scientific community and the biomedical community.In this thesis,we aim to construct a drug-loaded nanocomposite system using graphene oxide as a biological nanocarrier,and verify the system’s specific ability and chemo-photothermal therapy performance to liver cancer,which lays the foundation for the development of targeted drugs for the treatment of hepatocellular carcinoma.In this study,we first prepared graphene oxide（GO）sheet nanomaterials using a classic method,and characterized by transmission electron microscopy（TEM）and atomic force electron microscopy（AFM）,showing that the prepared GO has standard appearance and suitable size.Then,we modified GO with the aminoated polyethylene glycol（NH₂-PEG-NH₂）and lactobionic acid（LA）.LA is a targeting molecule of asialoglycoprotein receptor（ASGPR）specifically expressed by liver cells.The above modifications were verified by ultraviolet-visible spectrophotometer,fourier infrared spectrometer,and malvern particle size analyzer.Finally,the hydrophobic drug curcumin（CUR）was loaded by?-?stacking,and the graphene oxide drug-loaded nanocomposite（GO-PEG/LA-CUR）was finally obtained with a hydrodynamic size of 257.5±4.5 nm and a zeta potential of-29.8±1.19 mv.The drug loading rate was 56.82±1.38%and encapsulation rate was 56.34±2.05%.On the basis,we mainly carried out related research work in the following three aspects:（1）Drug release and photothermal experiment:Under the presence or absence of near-infrared irradiation（NIR）,GO-PEG/LA-CUR was placed in different pH buffers to conduct the drug release study.The results showed that in neutral（pH 7.4）and weakly acidic（pH 5.6）buffers,the drug release in 72 hours was 27%and 68%,respectively;after near-infrared irradiation,the drug release in 72 hours was significantly improved,reaching39%and 95%,respectively,indicating that the composite has better drug release ability under the acidic condition and NIR condition.Through the photothermal experiment（near-infrared light irradiation for 5 min）,we found that the temperature changes of the composite solution were positively correlated with the solution concentrations and the intensities of near infrared laser;the final product solution with a concentration of 50?g/mL increased its temperature by about 8℃after 1.5 W/cm² irradiation for 5 min,demonstrating good photo-thermal conversion performance.（2）In vitro targeting and therapeutic effect:Human liver cancer cell lines HepG2 and Huh7 were used as the experimental groups,human normal liver cell L02 and human embryonic kidney cell HEK293T were used as the control groups.Flow cytometry and laser confocal technology were used to verify the targeting performance of the drug-loaded nanocomposite.The experimental results showed that the fluorescence intensities of GO-PEG/LA-CUR in the experimental groups were much stronger than those in the control groups;and after the competitive inhibition of ASGPR by galactose（GAL）,the fluorescence intensities in the hepatoma cell lines were significantly weakened.The results indicated that GO-PEG/LA-CUR has the ASGPR-mediated specifically targeting delivery performance to hepatoma cell lines.In the In vitro treatment experiments,the vitality of liver cancer cells is about 80%,55%,and 40%,respectively,among the hyperthermia-only group,the GO-PEG/LA-CUR group with a drug concentration of 30?mol/L,and the GO-PEG/LA-CUR with a drug concentration of 30?mol/L plus near-infrared irradiation for 5 minutes.The results showed that our targeted drug delivery system（GO-PEG/LA-CUR）exhibited significant cytotoxic effects compared to pure photothermal therapy or chemotherapy.（3）In vivo distribution and therapeutic effect:We modified the fluorescent dye Rhodamine B（RhB）on the surface of non-targeted carrier（GO-PEG）and targeted carrier（GO-PEG/LA）,and used the small animal in vivo imaging technology to investigate the distribution of nanocarriers in mice with subcutaneously implanted tumors.It was found that after injection of targeted nanocarriers,fluorescence signals appeared in the liver,tumor,and kidney parts one after another.The fluorescence intensity of the tumor part reached the strongest at 12 h after injection,and was still visible at 24 h.The distribution in vivo indicated that the nanocarriers have the properties of targeting liver tumors and long-term retention.Through a 14-day treatment experiment,the in vivo integration efficacy of the nano-drug-loaded composites was evaluated.The results showed that the tumor growth of mice treated with GO-PEG/LA-CUR in combination with chemo-photothermal therapy was the slowest,and the tumor suppression rate was 86.2%,indicating that GO-PEG/LA-CUR can be used synergistic chemo-photothermal therapy for hepatocellular carcinoma.The graphene oxide nanocomposite system proposed in this study maintains the excellent photothermal properties of graphene oxide.The drug release exhibits acid response and photothermal response,and the nanocomposite system has a good targeted delivery capability to liver cancer cell lines both in vivo and in vitro.In addition,the graphene oxide nanocomposite system can synergize the two methods of chemotherapy and photothermal therapy for the treatment of hepatocellular carcinoma.The therapeutic dose not cause obvious biological toxic side effects in vivo and in vitro,and significantly improve the treatment effect,and achieve the goal of precise treatment and combined treatment of hepatocellular carcinoma.Therefore,the research of this topic is expected to provide a meaningful reference for the future construction of targeted drug delivery system and combined treatment of hepatocellular carcinoma.