Application Of Multifunctional Nano Drug Delivery System In Reversal Of Multidrug Resistance And Photothermal Therapy

ObjectivesThe research of this dissertation focuses on the synthesis,application and biological evaluation of functionalized and intelligent nano drug delivery systems.The nano drug delivery system mainly includes the following two parts:Part I:drug delivery and reversal of multidrug resistance of the tumor.Part II:drug delivery and imaging-guided photothermal therapy.We will conduct a series of in vitro and in vivo experiments to correlate the biocompatibility of functional nano drug delivery systems,in vivo drug distribution,transport metabolic pathways,and the efficacy,side effects,and in vivo targeting of breast cancer tumor models.Research provides a certain experimental basis for future clinical trials.MethodsIt is divided into two parts:Part I::TPGS functionalized mesoporous silica nanoparticles for anticancer drug delivery to overcome multidrug resistance:1)We designed TPGS-functionalized MSN to load DOX to overcome MDR.Doxorubicin hydrochloride（DOX）was studied as a model cancer drug.At the same time,the structure,particle size,stability,biocompatibility,drug loading rate,entrapment efficiency,drug sustained-release function under different pH conditions of the delivery system were studied to provide the following cytology and animal experiments.2)In vitro cytology experiment:The effects of the nanocarriers on proliferation of breast cancer MCF-7 and doxorubicin-resistant breast cancer MCF-7/ADR cells were studied by MTT assay.Confocal laser scanning microscope（CLSM）was used to observe the uptake ability and mechanism of nano drug delivery syatem on MCF-7 and MCF-7/ADR.We will verify the anti-tumor effect and mechanism of TPGS-modified nano-drug delivery system on MCF-7 and MCF-7/ADR,and discuss the possible mechanism.CLSM confirmed that high-efficiency and intelligent nanocarriers can be targeted to enter tumor cells.At the same time,we will demonstrated the endocytic pathways of nano drug delivery system by the use of specific inhibitors of endocytic pathways including 10μM filipin（to inhibit caveolae-mediated endocytosis）,3μM amiloride（to inhibit macropinocytosis）,30μM chlorpromazine（to inhibit clathrin-mediated endocytosis）,10μM nystatin（an inhibitor of lipid-raft mediated endocytosis）.3)We will study the in vivo pharmacodynamics and targeted localization of a functionally modified nano drug delivery system.Mice bearing tumor were at random divided into5 groups（n=9）.The treatment groups include PBS,MSNs-TPGS,free DOX（5mg/kg）,DOX@MSNs and DOX@MSNs-TPGS（5 mg DOX/kg）.The study focus on the distribution,in vivo circulation time,tumor targeting,in vivo imaging,tumor volume and survival time of mice at different groups.Finally,we evaluate the anti-tumor effect and mechanism of reversing multidrug resistance.Part II:Upconverting and persistent luminescent nanocarriers for accurately imaging-guided photothermal therapy:1)We first synthesized an up-conversion and long-lasting luminescent nanocarrier,and the physicochemical properties of the upconversion and long-lasting luminescent nanocarriers will be evaluated through the transmission electron microscope（TEM）,x-ray diffraction（XRD）and dynamic light scattering（DLS）.MTT and flow cytometry were used to detect the biocompatibility of nanoparticles.In addition,through a series of in vitro and in vivo experiments,we focused on the efficiency of continuous luminescence imaging and photothermal therapy for different samples.ResultsIt is divided into two parts:Part I:TPGS functionalized mesoporous silica nanoparticles for anticancer drug delivery to overcome multidrug resistance:DOX@MSNs-TPGS were characterized to have a single peak size distribution,high DOX loading efficiency and a pH dependent drug release profile.MSNs-TPGS were internalized via caveolae,clathrin-mediatedendocytosisandenergy-dependentcellularuptake.The DOX@MSNs-TPGS exhibited 10-fold enhanced cell killing potency compared to free DOX and DOX@MSNs.The enhanced MDR reversal effect was ascribed to the higher amount of cellular uptake of DOX@MSNs-TPGS in MCF-7/ADR cells than that of free DOX and DOX@MSNs,as a result of the inhibition of P-gp mediated drug efflux by TPGS.In vivo studies of NDDS in tumor-bearing mice showed that DOX@MSNs-TPGS displayed better efficacy against MDR tumors in mice and reached the tumor site more effectively than DOX and DOX@MSNs,with minimal toxicity.Part II:Upconverting and persistent luminescent nanocarriers for accurately imaging-guided photothermal therapy:We first prepared the upconversion and persistent luminescent nanocarriers.UPLNs@mSiO₂ and（ICG+UPLNs）@mSiO₂nanoparticles all have very good monodispersity.The average diameter is 200 nm and they all have a very clear outline.The XRD results also confirm that the UPLNs were successfully compounded.Flow cytometry and MTT experiments confirmed that the cytotoxicity of ICG and（ICG+UPLNs）@mSiO₂ nanoparticles on MDA-MB-231was small,indicating that（ICG+UPLNs）@mSiO₂ has good biocompatibility.In order to test whether the sample shows persistent luminescence,up-conversion luminescence and synergistic photothermal properties,different samples are injected into the subcutaneous tissue of the mouse.We set up four control groups and one experimental group,respectively.In vivo luminescence imaging systems and thermal radiometers verify long-lasting luminescence,up-conversion luminescence,and photothermal performance.Compared with the four control groups,long-lasting luminescence,up-conversion luminescence,and photothermal properties can be simultaneously achieved in the experimental group,which indicates that（UPLNs+ICG）@mSiO₂ nanoparticles can be used for imaging-guided PTT.A series of in vivo experiments confirmed that the tumor volume of（ICG+UPLNs）@mSiO₂ at 808 nm was significantly reduced,whereas PBS or free ICG group don not cause tumor destruction,and the median survival of mice treated with 808 nm laser（ICG+UPLNs）@mSiO₂ was significantly prolonged.HE staining analysis shows that tumor necrosis and structural damage were very severe with（ICG+UPLNs）@mSiO₂ and laser irradiation,whereas in the PBS and free ICG group there is no or very little necrosis and apoptosis,further demonstrating photothermal effects of（ICG+UPLNs）@mSiO₂.Furthermore,biocompatibility results showed that（ICG+UPLNs）@mSiO₂was well tolerated and had no apparent toxicity to the experimental mice.ConclusionsBased on the above research results,we can conclude that the functional nano drug delivery system have a very good application prospect in the field of reversal of tumor resistance and imaging guided photothermal therapy,and it is worth further research.