| Prostate cancer is a malignant tumor that occurs on the prostate epithelium.It is the second serious cancer among men and one of the leading causes of cancer death in men worldwide.Melanoma,which is the most malignant tumor in skin cancer,is derived from melanocytes,and it is prone to distant metastasis.At present,the most effective treatments in clinical practice are still surgical treatment,radiotherapy and chemotherapy.Chemotherapy is currently one of the most effective means of treating cancer,but its therapeutic effect is not optimistic.The main reason is that chemotherapy drugs are mostly broad-spectrum.It has the disadvantages of poor targeting,serious side effects and short circulation time in the body,which limits its clinical application.Experimental purpose: Based on the overexpression of sigma receptor on the surface of prostate cancer and melanoma cells,and the ability to bind to its specific ligand anisamide(AA),a PEGylated targeted gold nanoparticle delivery system by electrostatic interaction was prepared through the exploration and optimization of preparation methods,which was capable of loading epirubicin(EPI).The method for detecting the content of the EPI was also established.The final formulation was characterized,and the anti-tumor effect in vitro was studied.Experimental methods:(1)to establish a method for the determination of EPI,the content of EPI was detected by UV spectrophotometry or HPLC,including drawing of the standard curve.(2)The preparation method of gold nanoparticles formulation loaded with EPI was explored and optimized,including different ratios of drug to lipid,incubation time and temperature,and finally determined according to its characterization and stability to obtain a mature dosage form.(3)The gold nanoparticles loaded with EPI,Au NP-PEG5000.EPI and Au NP-PEG5000-AA.EPI,were prepared and characterized,including particle size,PDI and zeta potential.The encapsulation efficiency of EPI was determined by centrifugation,and its stability in 0.01 M PBS at 4? and normal saline with 50% FBS at 37? was investigated;The p H-sensitive drug release of gold nanoparticles loaded with EPI in vitro was detected at 37?.(4)To investigate the anti-tumor effect of gold nanoparticles in vitro.The cytotoxic effects of targeted and non-targeted gold nanoparticles with their comparison groups were investigated on PC-3 and B16 cells using MTT assay;Fluorescence microscopy was used to investigate the cellular uptake of targeted and non-targeted gold nanoparticles in PC-3 and B16 cells;Flow cytometry was used to study the apoptosis induced by targeted and non-targeted gold nanoparticles in PC-3 and B16 cells,and the expression of the related apoptosis regulator Caspase 3,Bcl-2 and Bax were studied using western blot to determine the relevant apoptosis mechanism;The endosomal escape induced by targeted and non-targeted gold nanoparticles in PC-3 and B16 cells was studied by confocal microscopy.Experimental results:(1)a standard curve of EPI content was drawn and the linear relationship was good within the measured concentration range.(2)Through the exploration and optimization of the preparation method of gold nanoparticles,the final drug-to-lipid ratio was determined as EPI: Au NPs(w: w)=1:5,the incubation temperature and time was 45 °C and 12 h.At this time,the EPI encapsulation rate was the highest about 80%.(3)The targeted and non-targeted gold nanoparticles were characterized.The particle size was about 130-160 nm,PDI was about 0.22,even distribution.And the zeta potential was also corresponding changed after loading EPI because of positive charge of EPI.The encapsulation efficiency of EPI was measured to nearly 80%,and the stability of the formulation was good in 0.01 M PBS at 4? or normal saline with 50% FBS at 37?.The results of p H-sensitive drug release of EPI in vitro showed that gold nanoparticles carrier had a significant sustained release effect on EPI and a higher release rate at p H 5.5(simulated the p H of tumor microenvironment).(4)MTT results showed that the targeted gold nanoparticle showed higher killing effect on PC-3 and B16 cells;It could be observed under the bright field of fluorescence microscopy that the targeted gold nano preparation had higher uptake in PC-3 and B16 cells;Flow cytometry results showed that the targeted gold nanoparticle could induce apoptosis in PC-3 and B16 cells,and there was no significant difference in the expression of Caspase 3 among the related apoptosis regulators.But for Cleaved caspase 3,as well as the pro-apoptotic factor Bax,higher expression was observed in the targeted gold nanoparticle group,while for the inhibitor of the apoptotic factor Bcl-2,the lowest expression in targeted gold nanoparticle was observed;The results of confocal microscopy showed that more EPI escaped from the cell endosome in the targeted gold nanoparticle group,avoiding it being isolated after protonation in the acidic environment of the endosome,so that the drug could maximize its function.In this study,a PEGylated targeted gold nanoparticles delivery system was prepared,which was capable of efficiently loading EPI,and greatly reduced the toxic side effects of EPI to other normal tissues and organs in the body.Targeting can make the drug enriched in the tumor area and improve the killing effect of the drug on the tumor.Therefore,this study will provide a strong practical basis for the clinical application of targeted delivery of EPI. |
PDF Full Text Request