Preparation And Preliminary Evaluation Of Curcumin-loaded Nanocomplexes With Hepatitis B Virus-like Particles

Objective:This project uses the modified RGD-HBc VLPs to load the anti-cancer drug Cur to perform loading methods,loading efficiency,spatial conformation,size,uniformity,purification degree,molecular weight,etc,as well as different p H values,time,and temperature.The state,stability,and killing effect of NSCLC A549 cell line.Provide theoretical support and method reference for Cur to be able to make medicine.Methods:1.Four HBV core virus-like particles(different number)with different chimeric RGD motifs with a purity of more than 95% prepared in this laboratory are selected to complete Cur loading through protein depolymerization,drug binding,and protein recombination.After purification by sucrose density gradient ultracentrifugation,gel filtration chromatography,ion exchange chromatography,nanocomposites with higher purity are obtained.Ultraviolet spectrophotometry is used to identify the loading rate(LC%)of Cur in the nanocomposite.2.Physical characterization,particle size,distribution status and performance detection are carried out by transmission electron microscope detection(TEM),dynamic light scattering detection(DLS),HPLC detection,SDS-PAGE and other methods.The functional evaluation of the composite is carried out from the difference in Cur loading rate and the change in particle size range,stability and repeatability.3.The biocompatibility,pharmacological activity and targeting of RGD-HBc-Cur nanocomposite are verified through hemolysis experiment and cell killing experiment.Result1.The load rate of RGD-HBc VLPs composed of 14 is the highest.Without optimization,each VLP molecule contains 214 ± 26 Cur molecules,.The optimal conditions for the entire loading procedure that is the depolymerization time of the protein depolymerization solution and the recombination buffer is 2.5 h,the reassembly time is 24 h,the volume ratio of the solution to the protein particles is 1:2,and the mass ratio of Cur to the protein particles 2: 1,the combined reaction time of the two is 30 min,and the combined reaction temperature is 0 ℃.Purification conditions are sucrose density gradient centrifugation at 30,000 rpm for 3 h,gel filtration chromatography with 20 m MTris-HCl as buffer and agarose as filler.Get higher purity nanocomposites2.Morphological observation and electron microscopy show that the load RGD-HBc-Cur nanocomposite shows uniform morphology,moderate size,and no agglomeration in the whole,and good uniformity;the particle size range of the composite was 35.0 ± 12.0 nm after detection by a particle size analyzer PDI < 0.2;the nanocomposite in Na Cl solution with ionic strength 150 m M,p H 6.0-9.0,storage within 28 days,no significant change in particle size.The content of Cur in the composite is better than that of Cur alone when the p H is 5.9,6.8,7.4 and light environment.The experimental method of loading Cur with RGD-HBc VLPs has good repeatability and recovery rate.The nanocomposite in normal saline solution that is not freeze-thawed has no significant change in particle size and concentration within 28 days.3.The hemolysis rate of RGD-HBc-Cur nanocomposite is 1.89%.In vitro killing experiments of cancer cells A549,it is verified that RGD-HBc VLPs with concentrations of 10,20,30,40 mg/m L do not have the effect of killing cells,Good security.Under the conditions of Cur concentration of 10,20,30,40 m g/m L,the killing experiment was performed on NSCLC A549 cells,and Cur-load VLPs showsgood killing effect,which is significantly better than the original drug Cur.In the targete verification experiment,the ICG-loaded RGD-HBc-Cur nanocomposite and ICG-only VLPs shows targeting on NSCLC tumors of Balb/c nude mice,while the free ICG was free of the whole body without a target tropism.Conclusion:1.RGD-HBc VLPs are nanocarriers that can be used as Cur.The nanocomposite formed is stable in preparation method,good in purification effect,and accurate in loading rate detection method.2.The RGD-HBc-Cur nanocomposite has a good microscopic morphology and is characterized by homogeneity.The p H range,in a relatively long time,has better stability than the original drug Cur.3.The RGD-HBc-Cur nanocomposite has good biocompatibility and shows good killing and targeting effects on cancer cells.