Dual-targeted Nanoparticles Transporting Curcumin And 5-fluorouracil For The Treatment Of Hepatocarcinoma

Objective:To improve the utilization of 5-fluorouracil（5-FU）chemotherapeutic drugs in the treatment of liver tumors,increase the accumulation of drugs in tumor sites and reduce the side effects of drugs on the systemic system,a novel targeting nanocarrier was constructed to transport the drug to the liver tumor site.The novel targeted nanocarriers have significantly improved drug utilization,prolonged blood circulation time,improved drug stability,enhanced tumor tissue targeting and retention.Drug combination therapy increased the bioavailability of 5-FU to reduce systemic toxicity and improve the therapeutic effect of liver tumors.In this study,an amphiphilic triblock PEG-PLGA-PEG copolymer was coupled with two targeting ligands（BIO and LAC）to prepare dual-targeted nanoparticles（BLPPNPs）,which encapsulated curcumin（CUR）and 5-FU.And the mechanism of synergistic anti-hepatocarcinogenesis of the two drugs was studied.Methods:1.PLGA,PEG,BIO and LAC were synthesized stepwise into the targeted nanomaterials by classical esterification reaction,detecting the nuclear magnetic resonance spectrum of the material.Nano-precipitation methods are used to prepare no-load or drug-loaded dual-targeted nanoparticles,single-targeted nanoparticles,and untargeted nanoparticles.Detecting the particle size and surface charge of nanoparticles and observing the morphology by transmission electron microscopy.The indirect method was used to calculate the drug loading and encapsulation efficiency,and in vitro release characteristics and degradation rate of the nanoparticles were studied.2.MTT assay was used to detect the cytotoxicity of unloaded dual-targeted nanoparticles,single-targeted nanoparticles and untargeted nanoparticles in vitro.The IC₅₀ value was calculated to study the optimal ratio of cytotoxicity of the combination of the two drugs to HepG2 cells.The cytotoxicity of the dual targeting nanoparticles against HepG2 cells and HL7702 cells was compared.Fluorescence intensity was measured by flow cytometry to quantitatively analyze the targeting of nanoparticles.Laser confocal microscopy further observed and compared targeted nanoparticle targeting in vitro.3.H&E pathology sections were used to detect the biocompatibility of empty ual-targeted nanoparticles,single-targeted nanoparticles and untargeted nanoparticles to the main organs of mice.Ultraviolet spectrophotometry was used to detect nanoparticles blood circulation time in vivo.The in vivo imager measures the organs distribution of nanoparticles in mice.The tumor-bearing nude mouse model explores the therapeutic effect of double-drug combined nanoparticles.4.Western blotting to investigate the effects of dual-targeted nanoparticles,single-targeted nanoparticles and untargeted nanoparticles on apoptosis,and to validate the synergistic anti-tumor mechanism.Result:1.The results of nuclear magnetic resonance showed that the double-targeted PEG-PLGA-PEG material was successfully synthesized.The prepared dual-targeted nanoparticles have a unique blue opacity of nanoparticles,which is spherical by electron microscopy,and the particle size of BLPPNPs is about187.4±2.5 nm.In summary,the preparation of nanoparticles was successful.The surface has an appropriate amount of negative charge to give the nanoparticles good stability.Double-targeted nanoparticles have a good sustained release and a moderate degradation rate.2.The results of MTT assay showed that the safety of blank dual-targeted nanoparticles（BLPPNPs/blank）on HepG2 cells and HL7702 cells was a Class 1standard according to USP standards.So nanoparticles have good biocompatibility.The cytotoxicity experiment showed that the combined feeding ratio was 1:2.5（5-FU:CUR）,and the synergistic effect was the best.Due to the combination therapy,the drug utilization rate is significantly improved,and the reduced 5-FU concentration avoids systemic toxicity.The targeted experiments of quantitative and qualitative analysis demonstrated that HepG2 cells showed strong uptake of BLPPNPs,indicating that dual targeting promoted cellular uptake of nanoparticles.HL7702 cells have no apparent enhanced endocytosis.3.The acute toxicity test of empty nanoparticles in mice showed that there was no obvious organ damage and inflammatory reaction.Nanoparticles as a drug carrier also showed good biosafety and biocompatibility in vivo.The experimental results show that the combined two free drugs are faster than nanoparticles,and the basic clearance rate is 80%.AUC_t0–t values in vivo indicate that nanoparticles have good blood circulation time due to the addition of PEG.However,the AUC_t0–_t values of dual-targeted nanoparticles were lower than either single or no-target,indicating that dual-targeted nanoparticles enter tissues or organs faster than other nanoparticles.Biodistribution experiments have shown that double-targeted nanoparticles enter the tumor tissue more and a little enter normal tissues or organs than single or no-target nanoparticles.BLPPNPs were confirmed to have high tumor targeting.In vivo anti-tumor activity experiments verify that the combination of two drugs increases their anti-tumor effect under nanoparticles transport.4.Western blotting results showed that the synergistic mechanism of the two drugs may be that CUR down-regulates the expression of DPYP protein by up-regulating the expression of p53 protein,increasing the cytotoxicity of 5-FU and enhancing the anti-tumor effect.At the same time,CUR may decrease the expression of Bcl-2 protein,increase the release of cyt c,and promote the apoptosis of hepatoma cells.Conclusion:The BLPP polymer material was synthesized and the double-targeted drug-loaded nanoparticles BLPPNPs/C+F were prepared.The spherical shape with a particle size of 187.4±2.5nm has good stability.5-FU and CUR in BLPPNPs have good sustained release properties.The no-load dual-targeted nanoparticles have better biocompatibility.Compared with single-targeted or non-targeted nanoparticles,BLPPNPs/C+F has a higher specificity and therapeutic effect on HepG2 tumor cells in a two-drug mass ratio of 1:2.5（5-FU:CUR）.BLPPNPs/C+F is more effective than single-targeted and non-targeted nanoparticles in the treatment of HepG2 nude mice,while reducing the side effects of chemotherapy drugs and effectively inhibiting tumor growth.The synergistic effect of the two drugs may be that CUR enhances the anticancer effect by promoting the expression of p53 protein to affect the decomposition of 5-FU,thereby promoting the apoptosis of liver cancer cells.