Construction Of Environment-responsive Microporous Nanocarriers For Drug Delivery And Its Application In Cancer Therapy

Background:As one of the major diseases,tumor threatens human health seriously.Various stratigies such as surgery,radiation,chemotherapy,immunotherapy has been employed for the treatment.Among them,chemotherapy is one of the most common method,which can effectively kill tumor cells and inhibit tumor growth.However,due to the lack of targeting ability,chemotherapy has unnecessary side effects,including damaging immune system,causing viscera disease,gastronintestinal reaction and bone marrow suppression.The employment of drug carriers provided great potential to effectively improve the accumulation of chemotherapy drugs at tumor sites and reduce the unnecessary side effects.Porous drug carriers have attracted great interest due to their high surface area,finely tunable pore size,high drug loading effects,excellent biocompatibility and biodegradability,which can be used for drugs loading,transporting and releasing.However,although loading chemotherapy drugs into porous drug carrier can effectively avoid the side effects,porous drug carrier can be easily cleared by the immune system together with non-specific adsorption by tissues and organs and unsatisfactory release,leading to compromise in the therapy outcomes.Herein,to solve above problems this thesis would focus on the design and synthesis of the biomimic membranes-encapsulated porous drug carriers with targeting properties.The drug carrier could be delivered to the tumor precisely without clearance by the immune system.Moreover,by taking advantage of tumor microenvironment,multimodal diaganosis and therapy of tumor have been realized in the animal level with the as-constructed drug carrier system.Objectives:By taking advantage of the merits of the porous materials,e.g,large surface area and higher drug loading,this thesis aims at the construction of two kinds of environmentally responsiveiron-based biomimic membranes-encapsulated porous drug carriers for the multimodal therapy and diaganosis materials with enhanced tumor therapy effect and improved biocompatibility.Part Ⅰ: Hyaluronic acid-modified mesoporous silica-coated Fe₃O₄ nanoparticles for targeted drug deliveryMethods:1.Construction and biological evaluation of Fe₃O₄-MS-HA NPsThe mesoporous silica-coated Fe₃O₄ nanoparticles（Fe₃O₄-MS NPs）were prepared using silica and Fe₃O₄ as the starting materials.The reaction between the amino group on the surface of Fe₃O₄-MS NPs and HA led to the formation of HA modified mesoporous silica-coated Fe₃O₄ nanoparticles（Fe₃O₄-MS-HA NPs）.Then the Fe₃O₄-MS-HA NPs were analyzed with TEM,FTIR and XPS.The biocompatibility of Fe₃O₄-MS-HA NPs was determined by CCK-8 assays.2.Biological functions of Fe₃O₄-MS-HA NPsThe enzyme-like property of Fe₃O₄-MS-HA NPs was evaluated by immersing Fe₃O₄-MS-HA NPs into the simulated TME in vitro,where the generated oxygen was detected with a dissolved oxygen detector.Meanwhile,the oxygen generation after the incubation of Fe₃O₄-MS-HA NPs with tumor cells was determined by using the oxygen probe RDPP,which was analyzed by CLSM.3.Drug loading and releasing behavior in vitroFe₃O₄-MS-HA NPs were mixed with DOX to form Fe₃O₄-MS/DOX-HA NPs.The loading effect of Fe₃O₄-MS-HA NPs was verified by detecting the fluorescence signal change before and after DOX loading.Fe₃O₄-MS/DOX-HA NPs were immersed into the simulated tumor microenvironment in vitro.The releasing behavior was evaluated by detecting the fluorescence signal of DOX.To determine the target ability,Fe₃O₄-MS/DOX-HA NPs were incubated with 4T1 cells and GES-1 cells.The cell viability was determined by the CCK-8 assay.4.In vivo treatment of subcutaneous xenograft tumorsSubcutaneously xenograft tumors were established by injected 1×10⁷ 4T1 cells into male mice.When the tumor volume reached approximately 50 mm³,tumor-bearing mice were randomized into 6 groups（n=6/group）and treated with PBS,DOX,MS/DOX-HA,Fe₃O₄-MS/DOX NP,Fe₃O₄-MS/DOX-HA NPs and Fe₃O₄-MS-HA NPs by tail vein injection every 3 days.After injection,a small magnet was placed on the tumor site of the mice for 0.5 h.The injections were repeated seven times over a 22-day treatment period.The body weights of the mice and tumor growth were monitored every day.The tumor volume was measured according to the following formula: Tumor volume = width²×length×0.5.After 22 days,all mice were sacrificed,and the tumors were excised and weighed.Results:1.Structural and biological functions of Fe₃O₄-MS-HA NPs1)Characterization of Fe₃O₄-MS-HA NPs: FTIR and XPS results shown Fe₃O₄-MSHA NPs was successfully constructed.Fe₃O₄-MS-HA NPs has uniform size with coreshell structure by TEM.2)The biological functions of Fe₃O₄-MS-HA NPs: Fe₃O₄-MS-HA NPs has enzymelike property,which can react with H₂O₂ to generate oxygen.GSH can reduce Fe³⁺ in Fe₃O₄-MS-HA NPs into Fe²⁺.2.Drug loading and releasing behavior of Fe₃O₄-MS-HA NPs.1)Drug loading effect of Fe₃O₄-MS-HA NPs: the DOX loading efficiency of Fe₃O₄-MS-HA NPs was about 65%,as determined by the DOX fluorescence signal before and after DOX loading.2)Environment-responsive Fe₃O₄-MS/DOX-HA NPs release: Less than 20% of the DOX was released when the Fe₃O₄-MS/DOX-HA NPs were incubated at pH 7.4 environment for 80 h,while as much as 60% of the DOX was released within 20 h at pH 5.5 encironment.3.The targeting delivery and anti-tumor effect of Fe₃O₄-MS/DOX-HA NPs1)Fe₃O₄-MS/DOX-HA NPs targeting ability: Fe₃O₄-MS/DOX-HA NPs can rapidly aggregate at the tumor tissue upon exposed under magnetic field,and effectively cross the cytoplasmic membrane via HA receptor-mediated transcytosis.2)The anti-tumor effect of Fe₃O₄-MS/DOX-HA NPs: Fe₃O₄-MS/DOX-HA NPs exhibited a high tumor targeting ability,leading to effective tumor inhibition without any apparent systemic side effects.Conclusions:1.We successfully fabricated pH-sensitive Fe₃O₄-MS-HA NPs with dual targeting ability.2.Fe₃O₄-MS-HA NPs has enzyme-mimic property,which can facilitate O₂ generation by reacting with H₂O₂.3.Fe₃O₄-MS-HA NPs not only has highly drug loading effects,but also can specifically uptake by cancer cells,thus improving the therapeutic efficacy.4.Fe₃O₄-MS-HA NPs can effectively accumulate at the tumor tissues under magnetic field,and effectively cross the cytoplasmic membrane via HA receptor-mediated transcytosis that can achieve enhanced therapeutic effects and reduced side-effects.Part Ⅱ: Folic acid-modified erythrocyte membrane cloaking iron porphyrin metal-organic framework for tumor synergistic therapyMethods:1.Construction of FA-EM@GO-MOFGO-MOF nanocarrier were prepared using GO,TCPP and Fe³⁺ as the element materials.Then,the GO-MOF was analyzed with BET,XRD and TEM.Gel electrophoresis was utilized to analyze the protein composition of FA-EM@GO-MOF.Immune elimination and targeting delivery ability of FA-EM@GO-MOF was observed by CLSM after incubated with macrophage cells and tumor cells.The biocompatibility of FA-EM@GO-MOF was determined by CCK-8 assays.2.Biological functions of FA-EM@GO-MOFThe enzyme-like property of FA-EM@GO-MOF was evaluated by immersing FAEM@GO-MOF into the simulated TME in vitro,where the generated oxygen was detected with oxygen probe RDPP,which was analyzed by fluorescence spectrometer.Meanwhile,the oxygen generation after the incubation of FA-EM@GO-MOF with tumor cells was determined by using the oxygen probe RDPP,which was analyzed by CLSM.The photothermal conversion of FA-EM@GO-MOF was monitored by thermal infrared camera under NIR laser irradiation.A clinical MRI scaneer and Prussian Blue staining were used to analyze the FA-EM@GO-MOF distribution in vivo.3.Drug loading and release of FA-EM@GO-MOF In vitro/vitro synergistic therapyGO-MOF were mixed with DOX to form GO-MOF/DOX.The loading effect of GO-MOF/DOX was verified by detecting the fluorescence signal change before and after DOX loading.FA-EM@GO-MOF/DOX were immersed into the simulated tumor microenvironment in vitro.The releasing behavior was evaluated by detecting the fluorescence signal of DOX.FA-EM@GO-MOF/DOX were incubated with 4T1 cells and GES-1 cells,respectively,and then exposed under NIR laser.The synergistic antitumor effect of FA-EM@GO-MOF/DOX was determined by the CCK-8.4.Tumor-bearing mouse model and systemic safety evaluation4T1 tumor-bearing BALB/c mice were randomized into seven groups and treated with PBS,PBS+NIR,DOX,FA-EM@GO-MOF/DOX,GO-MOF/DOX+NIR,FA-EM@GO-MOF+NIR and FA-EM@GO-MOF/DOX+NIR.All the major organs and tumors were fixed with 4% neutral buffered formalin and embedded in paraffin.Specimens were subjected to H&E staining along with a TUNEL assay.Tumor growth curve was used to evaluate CT/PDT/PTT synergistic antitumor efficacy.Results:1.Characterization of GO-MOF1)Characterization of GO-MOF: GO-MOF shows a particle size of about 100 nm and uniform distribution on the surface of GO.GO can quench the fluorescence signal of MOF.The surface area of GO-MOF was approximately 1010 m²/g obtained from BET analysis.2)Biological function of GO-MOF: high concentration of GSH can reduce GO into rGO,which weaked the interaction between GO and MOF,making fluorescence signal of MOF recovered.Meanwhile,GSH-triggered conversion of Fe³⁺ to Fe²⁺ caused the dissociation of MOF and promoted drugs precisely releasing at tumor tissues.FAEM@GO-MOF has excellent photothermal conversion behavior under NIR laser.2.Characterization of FA-EM@GO-MOF1)Characterization of FA-EM@GO-MOF: the protein profile of FA-EM@GOMOF was consistent well with pure EM by gel electrophoresis analysis.2)Biological function of FA-EM@GO-MOF: FA-EM endowed FA-EM@GO-MOF targeting delivery to cancer cells,while avoiding immune elimination.FA-EM@GOMOF could be targeted for delivery to tumor tissues after 24 h post-injection.FA-EM cloaking had no apparent influence on the photothermal conversion behavior of FAEM@GO-MOF.3.Synergistic antitumor therapy1)Drug loading and releasing behavior of FA-EM@GO-MOF nanocarriers: GOMOF has highly drug loading behavior of DOX.FA-EM@GO-MOF/DOX nanocarriers could efficiently delivery DOX to the tumor tissues,and gradually released DOX due to the unique properties of TME.2)FA-EM@GO-MOF/DOX antitumor therapy: FA-EM@GO-MOF/DOX nanocarrier can be specificly uptaken by tumor cells and effectively inhibit tumor growth under NIR irradiation.Conclusions:1.A novel kind of GO-MOF nanocarrier for cancer diagnoses and treatment was successfully fabricated.2.FA-EM@GO-MOF has excellent photothermal conversion behavior,and could be targetedly delivered to cancer cells while avoiding immune elimination.3.FA-EM@GO-MOF has enzyme-mimic activity,which can facilitate O₂ generation by reacting with H₂O₂,and supplied more O₂ for PDT;4.GSH can reduce GO into rGO,which weaked the intraction between GO and MOF,causing fluorescence recover of GO-MOF.Meanwhile,GSH-triggered conversion of Fe³⁺ to Fe²⁺ caused the dissociation of MOF and promoted drugs precisely releasing at tumor sites.5.FA-EM@GO-MOF/DOX nanocarrier can achieve chemotherapy-photodynamic therapy-photothermal therapy synergistic therapy under NIR irradiation,which can effectively inhibit tumor growth.