Redox-responsive Nanoparticles From Bioreducible Poly(Amido Amine)S Encapsulated Gold Nanoparticles As Drug Carries For Overcoming Multidrug Resistance In Cancer Cells

The multi-drug resistance to anti-cancer chemotherapeutic drugs occurred in cancer cells is one of the major obstacles in cancer chemotherapy. It is found that the mechanism of MDR is associated with the overexpression of several efflux pumps including P-glycoprotein(P-gp)、multidrug-resistant protein(MRP). The drugs taken up by cells via passive diffusion mechanism are recognized by P-gpcoprotein and then effluxed to the outside of cell membrane, lowering the intracellular drug concentration; thus the intracellular optimal dose is never achieved and leads to MDR. To overcome this problem, we designed a drug delivery system to avoid the recognition of efflux pump to drugs and circumvent MDR.Doxorubicin (DOX) which belongs to anthracyclines is a kind of anti-cancer drugs that acts on DNA molecules. It has been widely used in cancer therapy due to its wide antimicrobial spectrum. In this study, we chose DOX as the model drug for the research. During the past decades, kinds of delivery systems based on DOX have been developed such as liposome、micelles、polymer vehicles. However, the slow release rates of drugs limit their applications in cancer therapy. So we designed a GSH-trigged release drug delivery system to control drug release rates through responding to the stimulations of intracellular environments. In the system, gold nanoparticles were designed as the core to load drugs due to its unique properties such as homogeneous size distribution and excellent biocompatibility. Thiolated Doxorubicin (DOX-SH) was bounded to the surface of gold nanoparticles via Au-S covalent bond forming DOX-SH@Au nanoparticles. To make this nanoparticle internalize into cells easily,we introduced bioreducible polyamidoamines (ss-PAA) to coat DOX-SH@Au nanoparticles; finally the GSH-responsive DOX-SH@Au@ss-PAA drug delivery system was constructed.This study includes four parts. The first part introduced the preparations and characterizations of ss-PAA and gold nanoparticles. The results showed that the size of gold nanoparticle we prepared is about18±2.3nm and zeta potential is about-38±4.3mV. TEM images showed the gold nanoparticles is spherical and without adhesions.1H NMR shows that all the proton signals of ss-PAAs existed in the molecule structure indicating that the successful synthesis of ss-PAA.Secondly,the thiolation of DOX were modified through recations with SATA and the thiolated DOX were charecterized by MS-ESI、HPLC and NMR. It shows that the molecular weight of DOX increased from544to616and the retention time of DOX changed from7min to10min;new protone signal appeared in NMR spectrum.All the results demonstrated that DOX-SH were synthesized successfully.In this part,we also established the fluorescence spectroscopy and high performance chromatography to determin the content of DOX-SH.The results show that the concentration of DOX-SH had a good correlation with the smaple absorbance and chromatographic peak area in the range of0.25～16μg·mL-1(r=0.9997and0.9998,respectively).The linear regression equation was C=0.0066A-0.0211and C=0.0002A-0.2204, respectively.It has been validated that the linearity、 precision、accuracy and specificity accorded with the detective requirements.In the third part,we constructed the GSH-responsive drug delivery system and invesgated the drug-loading content、drug-loading efficience and drug release behavior.After DOX-SH and ss-PAA were adsorpted on the surface of gold nanopaticles,the size increased from18±2.3nm to80.5±6.8nm,zeta potential changed from-38±4.3mV to22.3±5.2mV. The maximum absorbance of UV spectra appeared red shift indicating that the system was successfully constructed.The drug-loading content and efficience were detected by fluorescence spectroscopy and the results showed that increasing the molar ratio of gold nanoparticles to DOX-SH,the drug-loading content and efficience increased.When the concentration of gold nanoparticles increased to7.5nM,the drug-loading content was10.84μM.After treated with GSH,DOX can be released from the system quickly.In the last part, cytotoxicity and cellular uptake of the drug delivery system were evaluated on both MCF-7sensitive cells and MCF-7/ADR resistant cells. The results of CCK8showed that,in MCF-7sensitive cells, both; DOX-SH@Au and DOX-SH@Au@ss-PAA nanoparticles exhibited decreased cytotqxicity compared to free DOX, however, in MCF-7/ADR resistant cells DOX-SH@Au@ss-PAA nanoparticles showed enhanced cytotoxicity than free DOX and DOX-SH@Au.Both the two kinds of cells were treated with GSH,the cytotoxicity increased than the non-treated groups.From Confocal images,red fluorescence could be observed in both cells, in MCF-7sensitive cells,the free DOX showed strongest fluoresence intensity; in MCF-7/ADR resistant cells,the fluoresence intensity of free DOX decreased.Both the cells showed enhanced fluoresence intensity than non-treated groups.The flow cytometry quantitative analysis was consistent with CLSM observations.In conclution,to overcome the multi-drug resistance exists in cancer chemotherapy,we designed a GSH-responsive based on gold nanopatticles drug delivery system could response to the GSH stimulation and released DOX from the system,incresed the intracellular DOX concentration and finally reverse the MDR.