| Malignant tumor seriously endangers modern human health.Breast cancer is one of the most common malignant tumors of women,becoming one of the major diseases that threaten the health of women.Chemotherapy is a main method of tumor treatment at present,but its clinical application is greatly limited by its severe side effects.Therefore,it is very important to develop a method with low toxicity and high effect for tumor therapy.In order to achieve targeted and synergistic tumor therapy and improve the therapeutic effect,we designed a drug delivery system with MMP-2 sensitivity loading DOX and 5-ALA.This study consists of the following four parts: Firstly: Preparation and characterization of Ge-DOX-5-ALA/NPs.In this part,we synthesized Ge-DOX-5-ALA/NPs by a single-phase coacervation method,and the stability,size distribution,morphology,drug encapsulation,loading efficiency and the production of reactive oxygen of nanoparticles were detected.There was no obvious difference in the size distribution,encapsulation and loading efficiency after being stored in 4?,25?,and 37? conditions for one month indicating Ge-DOX-5-ALA/NPs appeared pretty good stability.The average size of the nanoparticles which was detected by Malvern Zetasizer Nano-ZS90 was about 160 nm.The nanoparticles were homogeneous,evenly rounded and smooth on the surface as observed by using TEM.The encapsulation efficiency and loading efficiency of DOX measured by ultrafiltration centrifuge method and ultraviolet-visible(UV-VIS)spectrophotometry were 92% and 6.7%,respectively.We cancluded that Ge-DOX-5-ALA/NPs can produce reactive oxygen upon laser radiation by observing the ultraviolet absorption spectra of DPA.Secondly: Investigation on anti-tumor activity and MMP-2 sensitivity of Ge-DOX-5-ALA/NPs in vitro.A gelatin zymography was performed to detect the expression of MMP-2 in MCF-7 and Hs578 Bst.And the result revealed a high-level expression of MMP-2 in the MCF-7 cell line and a low-level expression of MMP-2 in the Hs578 Bst.Then,these two cells were used to investigate the anti-tumor activity and MMP-2 sensitivity of Ge-DOX-5-ALA/NPs.The intracellular release drug test showed that the Ge-DOX-5-ALA/NPs could achieve a good sustained release effect in MCF-7 cells.Both the qualitative and quantitative cellular uptake experiments showed that the DOX in Ge-DOX-5-ALA/NPs can be more efficiently taken in MCF-7 cells under the mediated action of MMP-2.The result of the MTT assay indicated that each experimental group has different degree of inhibition effect on both cells and the inhibition efficiency was dose-dependent and time-dependent.However,the gelatin nanoparticles had a higher inhibition rate for MCF-7 cells and the Ge-DOX-5-ALA/NPs+Laser showed the best effect.All of the above experiment results demonstrated that the Ge-DOX-5-ALA/NPs is a MMP-2-responsive drug delivery system and can realize enzyme responsive release under the action of MMP-2 to achieve targeted therapy and synergistic therapeutic effect.Thirdly:Investigating on the pharmacokinetics of Ge-DOX-5-ALA/NPs in vivo.SD rats were chosen as animal model in this part.And the experimental animals were randomly divided into two groups—DOX group and Ge-DOX-5-ALA/NPs group.DOX and Ge-DOX-5-ALA/NPs were administered by injection into the tail veins.At predetermined times,blood samples were drawn from orbit.After a series of processing,the DOX concentration were detected by HPLC.The drug-time curve was generated and the pharmacokinetic parameters of the rat plasma samples were calculated using a noncompartmental statistical model with Pksolver.The pharmacokinetic properties of Ge-DOX-5-ALA/NPs were studied by comparing the pharmacokinetic parameters of different drug groups such as t1/2,AUC,MRT,and Cl and so on.The result showed that the average retention time of Ge-DOX-5-ALA/NPs was higher than that of the DOX group,and the half-life was 2.8 times higher than that of the DOX group.This result indicated that the nanoparticles have slower metabolism and sustained release effects.Furthermore,compared with that of DOX,the area under the curve(AUC)of the nanoparticles was 3 times higher than that of DOX,and the elimination rate of plasma was significantly reduced.This result indicated the nanoparticles prolonged the retention time of DOX,improved the effective drug concentration and enhanced the bioavailability.Fourthly:Investigating on the pharmacodynamics of Ge-DOX-5-ALA/NPs in vivo.S180 tumor-bearing mice were chosen as animal model to investigate the pharmacodynamics of Ge-DOX-5-ALA/NPs.After intravenous administration,The effects and side effects of the drugs were analyzed by comparing the changes in the relative weight,relative rumen volume,tumor morphology and the pathological slices of HE staining.Experimental results of antitumor activity in vitro showed Ge-DOX-5-ALA/NPs +Laser group has the best best anti-tumor activity,indicating Ge-DOX-5-ALA/NPs increased anti-tumor effect and achieved synergistic tumor therapy of chemotherapy and photodynamic therapy under the laser of 635 nm.The pathology slice suggested there was no obvious toxicity in the organs of the mice treated with Ge-DOX-5-ALA/NPs,suggesting Ge-DOX-5-ALA/NPs achieved targeted delivery and concentrated release and the side effect of adriamycin was greatly reduced.The drug delivery system designed in this study can improve targeting by EPR effect and transportate the drug successfully into the tumor site.Then,the gelatin was degraded by MMP-2 which is high expression in tumor tissues,leading the chemotherapy drug and photosensitizer were released.Afterwards,put the tumor site under the 635 nm laser irradiation to induce the 5-ALA to produce reactive oxygen species,so as to achieve the desired effect of tumor microenvironment responsive,targeted delivery,concentrated release and combined therapy produced by chemotherapy and photodynamic.Our study laid a theoretical and practical foundation for the research of targeted therapy and synergistic therapy of tumor. |
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