Curcumin-and Resveratrol-co-loaded Nanoparticles In Synergistic Treatment Of Hepatocellular Carcinoma

BackgroundIn recent years,the incidence of hepatocellular carcinoma has been increasing year by year all over the world,especially in our country,which brings a great burden to the medical environment.Due to the lack of specific clinical indications of HCC,most patients were in the middle and late stage when they had relevant symptoms.At this time,They have lost the opportunity of surgery and can only receive conservative treatment,mainly chemotherapy.However,the existing treatment regimens are still not perfect.Taking Sorafenib as an example,no more than 30%of cases respond to this first-line treatment regimens,and most patients have serious side effects,such as hair loss,nausea,vomiting,weight loss,loss of appetite,rash,etc.Even if the survival period of some patients is prolonged,its application is limited due to the reduced living quality of patients and high cost.Therefore,a new therapeutic regimen is urgently needed to effectively treat patients with hepatocellular carcinoma,reduce the adverse reactions and improve the prognosis of patients.Curcumin（CUR）is an active substance extracted from plant turmeric,which has antioxidant and anti-inflammatory effects,such as inhibiting the proliferation of various liver cancer cell lines and inducing apoptosis of Hep G2 cells.Resveratrol（RSV）is a natural antioxidant polyphenol compound found in grapes and berries.Recent studies have demonstrated that RSV can prevent or inhibit the development of various tumors,including inhibiting proliferation and inducing apoptosis of Hep G2 and Hep3B cells.The reason may be that RSV induces phase II drug metabolizing enzymes,mediates anti-inflammatory effects,reduces cyclooxygenase and inhibits hydroperoxidase.Since both CUR and RSV have anti-HCC effects,the potential synergies of the drugs make combination therapy an important line of research,as combination therapy can further reduce the drug dose and improve its therapeutic potential.However,the poor bioavailability of these two drugs limits the clinical application.These problems can be solved by delivering drugs with nanomaterials.In recent years,nanomedicine has developed rapidly,and its easy preparation,stable and directional delivery has attracted wide attention.By using its amphiphilic properties,it is possible to encase fat-soluble substances,thereby increasing their solubility.Moreover,recent studies have confirmed that particles less than 200nm in diameter preferentially accumulate in the tumor area,thereby increasing the probability of uptake by tumor cells and reducing the impact on other normal tissues.By modifying the surface of nanoparticles,tumor targeting can be further enhanced.Therefore,nanomaterial encapsulation of CUR+RSV can be used to treat hepatocellular carcinoma to improve its bioavailability,and nanomaterial surface modification can further reduce drug dosage and side effects.This scheme can also be used to explore other drugs,which is of great significance for the treatment of liver cancer.ObjectiveThe aim is to load drugs with poor bioavailability with nanomaterials,and modify the surface of nanoparticles to take advantage of their inherent property that tends to accumulate at the tumor site,so as to delivery drugs to the tumor site directly.Under the premise of evaluating the safety of the therapy,the efficacy of the drug can be improved,the drug consumption and the side effects of the drug can be greatly reduced.Methods（1）DSPE-PEG₂₀₀₀-SP94 was prepared by coupling reaction between maleimide group on DSPE-PEG₂₀₀₀-Mal and cysteine residue in SP94.Polymer NPs loaded with CUR+RSV were prepared by nano-precipitation method.NPs were prepared from RSV,CUR,DSPE-PEG₂₀₀₀or DSPE-PEG₂₀₀₀-SP94,DSPC and cholesterol.After hydration,it was ultrasonic treated and extruded through a 200nm polycarbonate membrane.（2）Morphological examination of NPs was performed by transmission electron microscope.The particle size and zeta potential of NPs were measured by Malvern instrument.The instrument was used to measure changes in particle size over a week.Then the concentration curve was generated by ultraviolet spectrophotometer,and then the encapsulation rate of NPs was measured.It was placed in phosphate buffer and released in vitro by dialysis.Small animal imager was used to detect the change of solubility intuitively.（3）Laser confocal microscopy was used to detect the brightness of CUR in cells,so as to judge the uptake efficiency of NPs by Hep G2 cells.The changes in cell viability under different treatment regimens were measured by MTT method to explore whether there is synergy between CUR and RSV,the optimal treatment time,and whether NP_S-supported drugs are more effective.Then,the mechanism of NPs was explored,and the intracellular ROS production was detected with DCFH-DA.The cells were treated with NAC,an inhibitor of ROS,and tested again.At the same time,Caspase-3activity was detected,and then treated with caspase inhibitor Z-VAD-FMK.（4）To evaluate the antitumor effect of NPs in human hepatoma tumor models.The drug was administered through the tail vein,and the concentration of the drug in important organs in the body was observed by fluorescence imaging in mice.The tumor size,body weight changes,serum and tissue samples of mice in different groups were recorded,so as to determine the effectiveness and safety of NPs.TUNEL and Ki-67 were used to determine the apoptosis and proliferation of tumor tissues.Results（1）SP94 peptide ligand further improved the targeting ability of NPs.The results of transmission electron microscopy showed that the NPs were uniformly circular at different magnifications,and the particle size of NPs remained within 200nm.The zeta potential also proved that the NP_Shad good dispersion.The long-term stability of NPs was also demonstrated when it was stored at room temperature for one week.These results indicated that NPs were monodisperse spherical nanostructures.The drug encapsulation rate was more than 80%,so it had a good carrying capacity.The drug release kinetics showed that NPs had a stable and slow release rate,while the solubility experiment showed that NPs greatly increased the solubility of the CUR+RSV to improve its bioavailability.（2）Cell uptake experiment results showed that NPs modified with SP94 targeted peptide could more effectively target hepatocellular carcinoma cells,thus enabling effective drug accumulation.The results of IC₅₀and ci showed that CUR and RSV had synergistic effect,which could increase the antitumor efficiency and reduce the dosage.However,blank NPs had little toxicity to cells,indicating that the nanomaterials themselves were safe.Hep G2 cells were ROS detected by DCFH-DA after NPs treatment and showed bright green fluorescence.The green fluorescence of NAC pretreated cells decreased significantly.NPs treatment significantly increased caspase-3,and the caspase-3 inhibitor Z-VAD-FMK inhibited apoptosis.These results suggest that NPs may contribute to tumor cell apoptosis by influencing ROS production and caspase-3 activation in vivo.（3）Further in vivo studies confirmed that SP94-modified NPs preferentially concentrated drugs in tumor and liver of tumor mouse models,and the fluorescence signal tended to converge gradually over time.This demonstrates the ability of NPs to target tumors in vivo.Tumor growth was significantly inhibited and body weight was not significantly changed in the NPs treatment group.Blood samples of each group showed no significant changes in ALP,BUN and LDH,and hematoxyn-eosin staining also confirmed that NPs had no significant effects on liver,kidney and heart function,and was safe in mouse models.Ki-67 and TUNEL results were consistent,suggesting that SP94-targeted NPs achieve therapeutic effects by increasing tumor apoptosis and inhibiting tumor proliferation.ConclusionsThe SP94-modified NPs allow large amounts of CUR+RSV to accumulate in the tumor in a safe and efficient manner.It provides a new strategy for innovative HCC treatment.The nanoplatform also provides an idea for exploring other potential chemotherapy approaches.