Construction Of Biomimetic Responsive Drug-loaded Nanoparticles And Its Application In Cancer Therapy

Background:Cancer is the second leading cause of death in the world and poses a serious threat to human health.Radiotherapy and chemotherapy are the main treatment modes at present.However,due to the non-specific,chemotherapy lack the ability to identify tumor cells and will affect normal cells and tissues as well as kill tumor cells.Radiotherapy also causes great damage to the surrounding tissues and often bringing serious toxic and side effects to the patients.The limitations of traditional treatment methods have promoted the modern research on new drugs for effective treatment of cancer which include nano-medicine.Nanotechnology is one of the innovative technologies to manufacture potential drug delivery system,which have the advantages of remarkable drug delivery ability,good drug loading performance,longer internal circulation time and so on.Owning to the heterogeneity,nanocarriers are usually limited by biological barriers such as mononuclear phagocytosis system,hemorheological force and endothelial vessel wall.To overcome the internal biological barrier,the biological cell membranes are used to wrap the nanocarriers and enhance their biological interface capability.This top-bottom technique is simple,highly versatile and has the potential to improve the potency and safety of existing nanocarriers greatly.Inspired by the above,the platelet membrane with tumor targeting function was used to coat the multifunctional nanocarrier and construct a biomimetic responsive nano drug loading system.Finally,the efficient aggregation and environmental responsive release of drug as well as multimodal treatment on tumor are realized.Objectives:Multifunctional responsive nanocarrier based on platelet membrane was designed and prepared to achieve the functions of targeted drug recruitment at tumor sites,improved tumor microenvironment（TME）,environmental responsive drug release as well as high effective anti-tumor effect with multimodal treatment modality.Part Ⅰ GOx-functionalized platelet membranes-camouflaging nanoreactors for enhanced multimodal tumor treatmentMethods:1.Construction of HMMS@PG nanoreactorHollow mesoporous copper sulfide nanoparticles（HMCu S NPs）were used as the drug carrier to design and prepare nanoreactors.The chemotherapy drug doxorubicin（DOX）was loaded in HMCu S NPs（HMCu S-DOX NPs）and manganese dioxide（MnO₂）was coated on the surface of HMCu S-DOX NPs as a nanoscale shell to form HMCu S-DOX-MnO₂ nanoplatform（HMMD）.The biotinylated glucose oxidase（GOx）was anchored on the surface of platelet membrane through the strong affinity between protein and biotin to form GOx functionalized platelet membranes（PG）.PG was used to wrapped the HMMD nanoplatform to form the nanoreactor（HMMD@PG）.The structure of HMMD@PG was characterized by transmission electron microscope（TEM）,X-ray photoelectron spectroscopy（XPS）,and confocal laser scanning microscope（CLSM）.2.Drug loading and releasing of HMMD@PGThe encapsulation rate of HMCuS NPs to DOX was calculated and optimized according to the intensity of the DOX absorption peak at 480 nm as measured by the uv-visible spectrophotometer（UV-vis）.The HMMD@PG was placed in PBS （Phosphate buffered saline）containing different pH and different concentrations of GSH to detect the responsive release efficiency of DOX in HMMD@PG.3.Biological function and cytotoxicity analysis of HMMD@PGThe catalytic activity of GOx in HMMD@PG was monitored according to the changes in the concentrations of glucose and hydrogen peroxide（H2O2）during glucose oxidation.A certain amount of HMMD@PG was placed in different concentrations of GSH solution,and the MnO₂ consumption in the HMMD@PG nanoplatform was monitored by UV-Vis.In addition,a certain concentration of HMMD@PG was exposed to 808 nm near-infrared（NIR）light,and the photothermal conversion efficiency of the HMMD@PG nanoplatform was verified by an infrared thermal imager.After HMMD@PG co-incubating with mouse breast cancer 4T1 cells,the fluorescence signal of the intracellular hypoxia probe was visualized by CLSM to examine the oxygen production capacity of HMMD@PG.Cell internalization of the nanoplatform was assessed by co-incubating HMMD@PG with 4T1 cells.The biocompatibility of HMCu S NPs was analyzed by incubating it with GES-1,4T1 cells.Various concentrations of HMMD@PG were co-incubated with 4T1 tumor cells and treated with 808 nm NIR of irradiation to verify their tumor cell-killing effect using the CCK-8 assay.4.Nude mouse subcutaneous graft tumor model was constructed to verify the tumor-targeting ability and anti-tumor effect of HMMD@PGThe mouse breast cancer（4T1）subcutaneous transplantation tumor model was constructed,and the tumor-bearing mice were randomly divided into 5 treatment groups.Five tumor-bearing mice in each group were treated with PBS,DOX,HMMD,HMMD@PG,and HMMD@PG + L（Laser）.Thereafter,ocular blood was collected from the tumor-bearing mice to examined the liver and kidney function.Heart,liver,spleen,lung,kidney and tumor tissues of tumor-bearing mice were dissected for H&E staining to evaluate the toxic side effects of the nanoplatform and its killing ability to the tumor cells.The antitumor effect of HMMD@PG + L combination therapy was evaluated by the tumor growth curves of tumour-bearing mice.Results:1.Structural characterization of the HMMD@PG1)The Characterization of the HMMD: The result of TEM showed that HMCu S NPs with a uniform size of about 150 nm was successfully prepared.The particle size of the prepared HMMD became larger when DOX was encapsulated inside the nano carrier and MnO₂ was coated on its surface.The results of the TEM element analysis showed that Cu,S,Mn,and O were evenly distributed in the nanocarrier.The formation of the MnO₂ was also further supported by the XPS analysis results.Moreover,the UV-vis results showed HMMD has a strong absorption in the NIR region which suggest that it has a good photothermal conversion performance under 808 nm NIR laser irradiation.2)Characterization of the HMMD@PG: TEM characterization result showed the successful preparation of platelet membranes.The CLSM validated that the biotinylated GOx could be successfully anchored onto the platelet surface.The optimal connection amount of GOx on the membrane surface of platelets was detected and optimized throughthe GOx quantification kit.Using ultrasound to wrap PG around the HMMD surface.Compared with HMMD,the zeta potential and hydrodynamic diameter of HMMD@PG also changed correspondingly.2.Drug encapsulation and releasing of HMMD@PG1)Encapsulation of DOX: The drug loading capacity of the nanocarrier was optimized.The encapsulation efficiency of DOX reached（73.67±2.44）% when the mass ratio of HMCu S NPs to DOX was 10:1.2)Released of DOX: The drug release efficiency was verified by placing HMMD@PG in solutions with different conditions.The results showed that the DOX release rate was reached（73.16 ± 2.23）% within 2 h when HMMD@PG in PBS（pH=5.5）containing 2 mmol/L GSH.3.Study of the biological function of HMMD@PG and its cytotoxicity analysis1)The biological functions of the HMMD@PG: The consumption of glucose was measured by a 3,5-dinitrosalicylic acid colorimetric method.The generated hydrogen peroxide was detected by the method of ammonium molybdate colorimetry after an additional of HMMD@PG to glucose solution.The results showed no significant difference in glucose consumption and hydrogen peroxide production of the HMMD@PG group as compared with the free GOx group which indicated that the GOx activity did not change in the HMMD@PG.The dissolved oxygen detector verified that MnO₂ nanoshells can catalyze hydrogen peroxide to generate O2.The condition of cell hypoxia was detected by a hypoxia probe after HMMD@PG co-incubated with 4T1 cells,and the results showed HMMD@PG improved cell hypoxia.2)Cytotoxicity analysis of HMMD@PG: Incubation of no-loaded HMCu S NPs with GES-1 and 4T1 cells,and the CCK8 results showed that the viability of cells was over 90 % even with the rise of HMCu S NPs up to 100 μg/m L.This result suggested that HMCu S NPs has good biocompatibility.Different concentrations of HMMD@PG were co-incubated with 4T1 tumor cells and then irradiated with 808 nm NIR irradiation,and the results of the CCK-8 experiments indicated that the prepared nanoplatforms had an efficient killing effect on tumor cells.4.Antitumour effect of HMMD@PGHMMD@PG can cluster to the tumor site efficiently,and shown good antitumor effect under combination therapy.The venous blood analysis of the tumor-bearing mice indicated that no liver and kidney damage in the tumor-bearing mice after treatment.Conclusion:1.Environment-responsive targeted nanoplatform was successfully constructed,and HMMD@PG also has excellent photothermal conversion efficiency;2.HMMD@PG can consume glucose and GSH,while generate oxygen,which is conducive to improving TME;3.The MnO₂ nanoshell on the surface of HMMD@PG serve as a "gatekeeper" of chemotherapy drug DOX,can achieve high efficiency and accurate release of DOX when it reacts with the high concentration of GSH in the tumor tissue;4.HMMD@PG achieved a triple-in-one multimodal treatment of chemotherapy/starvation therapy/photothermal therapy,which obtained good anti-tumor effect.Part Ⅱ Platelet membrane camouflage PLGA-based nanocarrier used for fluorescence imaging guidance and multimodal tumor treatmentMethods:1.Construction of the PDI@PM nanoplatformPLGA-based nanoparticles containing chemotherapy agent DOX and indocyanine green（ICG）were prepared（PLGA-DOX-ICG nanoparticles,PDI NPs）by the method of water in oil in water（W-O-W）.The platelet membrane（PM）which was obtained by repeated freeze and thawing method,was used to wrapped the PDI NPs and ultimately got PDI@PM.PDI NPs was characterized by TEM,SEM,DLS,and UV-vis.The expression of the surface-related proteins in prepared platelet membrane was analyzed by flow cytometry.2.Optimization of drug loading,responsive drug release of PDI@PM,and its cell assay in vitroThe encapsulation rate of PLGA NPs was optimized by changing the amount of ICG and DOX invested in the preparation process.The released efficiency of DOX was assessed by placing the prepared PDI@PM in PBS buffer（pH=5.5）and then treated with NIR irradiation at different time points.The biocompatibility of the nanocarrier was assessed by co-incubating empty-load PLGA@PM with GES-1 cells,4T1 cells,and B16 cells.The ability of PDI@PM to reduce immunogenic phagocytosis was verified by co-incubating PDI@PM with mouse monocyte macrophages（RAW 264.7）.The production of intracellular reactive oxygen species（ROS）under NIR illumination was verified.The cytotoxic effect of PDI@PM on 4T1 cells was verified by CCK-8 experiments.3.Fluorescence（FL）imaging of PDI@PM in vivoThe mouse breast cancer（4T1）subcutaneous transplantation tumor model was constructed.And free ICG,PDI NPs and PDI@PM were injected into the tail vein of tumor bearing mice respectively.The IVIS Spectrum live animal imaging system was used to verify the real-time FL imaging capability of PDI@PM.Also,the distribution of the nanoplatform and tumor targeted aggregation capability in vivo were tracked at different time points.4.Construction of the tumor-bearing mouse model and validation of the anti-tumor effect of PDI@PMAfter construction of the mouse breast cancer（4T1）subcutaneous transplantation tumor model,the tumor-bearing mice were randomly divided into 6 groups when the tumor volume was about 120 mm3.There were 6 mice in each group.Each group of mice were injected with PBS,ICG + L,DOX,PDI NPs PDI@PM and PDI@PM +L respectively through tail vein.The body weight and tumor volume of tumour-bearing mice were observed.The heart,liver,spleen,lung and kidney tissues of tumor bearing mice were stained with H&E to evaluate the toxic and side effects of naoplatform.The tumor tissues of tumor bearing mice were analyzed by H&E staining and TUNEL staining to evaluate the tumor killing ability.The anti-tumor effect of PDI@PM combined with NIR irradiation was evaluated by the tumor growth curves.Results1.Structural characterization of PDI@PM and its biological functionThe results of TEM shown uniformly dispersed PDI NPs and verified successful wrapping of PDI NPs by platelet membranes.Dynamic light scattering shows that the average particle size of PDI NPs is（171±15.39）nm.The results of flow cytometry validated that the prepared platelet membrane retained the key protein such as CD41 and CD61.Temperature was increased significantly at different concentrations of PDI@PM suspension by irradiation using 808 nm NIR which suggested the good photothermal conversion effect of PDI@PM.2.Drug loading,release of PDI@PM,and its cell toxicity in vitroThe encapsulation efficiency（EE）results of ICG and DOX show that the best EE of ICG is（74.79±1.35）%,and the maximum EE of DOX is（38.5±1.63）%.Drug release experiments showed that PDI@PM possessed excellent pH-responsive drug releasing characteristic.In addition,the DOX release efficiency was obviously improved and closed to 100% with an additional of 808 nm NIR irradiation,which indicate that the drug release of PDI@PM also can be controlled by photothermal.The dual responsive drug release of pH and NIR irradiation helps to ensure the effective release of PDI@PM at the tumor site.The results of the cellular uptake experiments showed that the amount of PDI@PM uptake by the 4T1 cells was significantly higher when compared to the PDI NPs.Conversely,PDI@PM is barely ingested by mouse mononuclear macrophage RAW264.7,which validated that the PDI@PM nanoplatform has not only tumor targeting but also low immunogenicity.After irradiation of 808 nm NIR,the presence of photodynamic therapy（PDT）was verified by the production of ROS in 4T1 cells.The CCK-8 experiment results showed that the final cell viability was only（12.62±1.92）% when 4T1 cells were treated with PDI@PM+L.This indicates that the combination treatment of chemotherapy/PTT/PDT has a strong killing ability to tumor cell.3.FL imaging of PDI@PM in vivoTo test the ability of FL imaging of PDI@PM,tumor-bearing mice were injected with free ICG,PDI NPs and PDI@PM respectively.The results show that PDI@PM can be highly enriched in the tumor site,and the fluorescence intensity of the tumor site reaches the peak at 12 h after injection.Compared with the free ICG group and PDI NPs group,the retention time of fluorescence was longer.4.Validation of anti-tumor effect of PDI@PMAfter treatment of tumor bearing mice,PDI@PM combined with PTT/PDT treatment showed high anti-tumor capacity and the tumor suppression rate was（92.31±5.36）%.The weight did not change significantly in the experimental group during treatment.Additionally,combined PDI@PM treatment with PTT/PDT treatment showed no obvious toxicity to the heart,liver,spleen,lung,and kidney in tumor-bearing mice.Furthermore,the results of H&E staining and TUNEL staining showed that the massive necrosis of the tumor cells in the tumor tissues of experimental groups.Conclusion:1.A platelet membrane biomimetic nanoplatform with the function of pH/photothermal dual response to drug release,FL imaging,and tumor therapy was constructed.Moreover,the real-time FL imaging capability of PDI@PM is beneficial to guide tumor treatment.2.PDI@PM not only combines the ability of targeting tumors actively and passively,but also can avoid phagocytosis by macrophages effectively,making it a nanoplatform integrating targeting and low immunogenicity.3.The combination treatment of PDI@PM and NIR light irradiation formed a multimodal anti-tumor pattern that integrates chemotherapy/PTT/PDT,which possess strong capability of anti-tumor.