Targeted Therapy Of Lymphoma With Biomimetic Nanoparticle Drug Delivery Systems

PART Ⅰ:Preparation and assessment of platelet membrane coated with amino-modified mesoporous silica nanoparticlesObjectives:To synthesize platelet membrane(PM)coated with amino-modified mesoporous silica nanoparticles(NH2-MSN)and systematically assess the stability,drug release performance,platelet membrane protein expressions,biocompatibility and safety in vitro.Methods:Firstly,nanoparticle drug delivery systems MSN-DOX,MSN-BLZ and MSNDOX/BLZ were prepared by loading Doxorubicin(DOX)or BLZ-945 on NH2-MSN by physisorption.PM was extracted from mouse blood,and then PM@MSN-DOX,PM@MSN-BLZ and PM@MSN-DOX/BLZ were obtained by repeated extrusion.Transmission electron microscopy(TEM)was used to observe and compare the morphology of nanoparticles.Dynamic light scattering(DLS)was used to measure the size and zeta potential of nanoparticles.The internal structure of the particles was investigated by infrared spectroscopy and ultraviolet and visible absorption spectrum.High-performance liquid chromatography(HPLC)or ultraviolet and visible absorption spectrum was used to determine the encapsulation efficiency.The dialysis method was used to investigate the drug release rate in vitro.The PM protein expression was determined using Coomassie brilliant blue staining and western blot.The biocompatibility was evaluated using hemolytic assays,and the in vitro safety was evaluated using cytotoxicity assays.Results:TEM showed that the membrane structure could be clearly observed on the surface of the PM@MSN-DOX,PM@MSN-BLZ and PM@MSN-DOX/BLZ nanoparticles.The particle sizes of PM@MSN-DOX,PM@MSN-BLZ and PM@MSN-DOX/BLZ were 152.4 nm,154.6 nm and 160.5 nm,respectively.The zeta potential of PM@MSN-DOX,PM@MSN-BLZand PM@MSN-DOX/BLZ were 17.2±3.2mV,-16.8±2.4mV and-15.9±2.1mV,respectively.The infrared spectroscopy and ultraviolet and visible absorption spectrum proved the drug successfully loaded on the nanoparticles.The cumulative release of DOX in PM@MSN-DOX and PM@MSN-DOX/BLZ in 96 h was above 20.8%and 18.6%at pH 7.4,and above 38.5%and 35.5%at pH 5.0.The cumulative release of BLZ-945 in PM@MSN-BLZ and PM@MSN-DOX/BLZ in 96 h was above 25%and 23.6%at pH 7.4,and above 61%and 60%at pH 5.0.The Coomassie brilliant blue staining and western blot showed the protein of PM@MSN-DOX,PM@MSN-BLZ and PM@MSN-DOX/BLZ was consistent with that of the platelet membrane.The expression levels of CD47 protein in the nanoparticles and platelet membranes were not significantly different.PM@MSN-DOX,PM@MSN-BLZ and PM@MSN-DOX/BLZ nanoparticles’ hemolysis rate was less than 5%.The CCK-8 assay showed that the vector nanoparticles have no obvious inhibitory effect on the growth of RAW264.7 cells and EL-4 cells.Conclusion:The author successfully synthesized platelet membranes coated with aminomodified mesoporous silica nanoparticles.The nanoparticles are safe and biocompatible and can release drugs in a pH-dependent way with a higher release rate in an acidic environment.PART Ⅱ:The anti-tumor effect of PM@MSN-BLZ/PM@MSN-DOX biomimetic nanoparticle drug delivery systems in vitroObjectives:To investigates the cellular uptake of nanoparticles,the effect of PM@MSNBLZ on the polarization,proliferation and apoptosis of tumor-associated macrophages(TAMs),and the effect of the combination of PM@MSN-DOX/PM@MSN-BLZ on the proliferation and apoptosis of EL-4 cells.Methods:Fluorescence microscopy was used to detect the EL-4 cells’ uptake of nanoparticles.PM@MSN-BLZ or PBS were subsequently added to RAW264.7 cells cultured using EL4 conditioned media(EL4-CM).Then,the culture supernatant and RAW264.7 cells were collected.The flow cytometry,the real-time polymerase chain reaction(RT-PCR),the enzyme-linked immunosorbent assay(ELISA)and the western blot were used to investigate the effect of nanoparticles on the polarization of TAMs.In addition,RAW264.7 cells were stimulated with EL4-CM to mimic the tumor microenvironment in the presence or absence of PM@MSN-BLZ.Then,the culture supernatant was collected and subsequently added to EL-4 cells.Cell apoptosis was evaluated using flow cytometry.Results:The cell uptake experiments indicated that the EL-4 cells can effectively uptake the nanoparticles.Flow cytometry showed that PM@MSN-BLZ significantly decreased the proportion of CD206+macrophages compared with the EL4-CM group.RT-PCR showed that PM@MSN-BLZ significantly lowered the expression of CD206,arginase-1(Arg-1)and interleukin-10(IL-10)mRNA in macrophages(P<0.0001).ELISA demonstrated that the IL-10 level in RAW264.7 cells treated with PM@MSN-BLZ was significantly lower than that of the EL4-CM group(52.7±2.2 pg/ml vs 32.6±2.1 pg/ml,P<0.001).The interleukin-12(IL-12)level in RAW264.7 cells treated with PM@MSN-BLZ was significantly higher than that of the EL4-CM group(192.8±1.9 pg/ml vs 145.3±1.7 pg/ml,P<0.001).In addition,the western blot showed that the level of CSF-1R protein in RAW264.7 cells treated with PM@MSN-BLZ was lower than that of the EL4-CM group.Finally,compared with the TAM-CM+PM@MSN-DOX group,the proportion of apoptotic EL-4 cells was increased significantly in the TAM-CM+PM@MSN-DOX/PM@MSN-BLZ group(34.7±0.37%vs 13.04±0.75%,P<0.001).Conclusion:The EL-4 cells can effectively uptake the nanoparticles.PM@MSNDOX/PM@MSN-BLZ enhance the anti-tumor effect of DOX in vitro by inhibiting the viability of M2 macrophages and blocking the interaction between EL-4 cells and M2 macrophages.PART Ⅲ:The anti-tumor effect of PM@MSN-DOX/BLZ bio mimetic nanoparticle drug delivery systems in vivoObjectives:To evaluate the tumor-targeting ability,the anti-tumor effect,the influence on the tumor immune microenvironment and the biological safety of the PM@MSNDOX/BLZ biomimetic nanoparticle drug delivery system in vivo.Methods:Firstly,the EL-4 subcutaneous tumor model was established.In vivo imaging system(IVIS)was used to evaluate the tumor-targeting ability of PM@MSN-DOX/BLZ.Mice were randomly assigned to the PBS group,the free DOX group,the PM@MSN-DOX group,the free DOX/BLZ group and the PM@MSN-DOX/BLZ group.The anti-tumor effects were evaluated according to the volume and weight of tumors and the survival of the mice.Flow cytometry was used to detect the immune cells in the tumors and spleens.Western blot was used to investigate the expression level of CSF-1R protein in the tumors.ELISA was used to quantify the cytokine levels in the serum of mice.The Hematoxylin-eosin(H&E)staining and biochemical testing were used to verify the biosafety of nanoparticles.Results:After injection of PM@MSN-DOX/BLZ,IVIS showed the PM@MSN-DOX/BLZ has excellent tumor-targeting property in mouse.Flow cytometry of TAMs showed that PM@MSN-DOX/BLZ group significantly increased the proportion of M1 macrophages of tumor tissue with the PBS group(67.4±5.47%vs 36.5±4.32%,P<0.01).ELISA demonstrated that the IL-10 level in PM@MSN-DOX/BLZ group was significantly lower than that of the PBS group(27.3 ± 1.0pg/ml vs 110.1 ± 4.4pg/ml,P<0.01).In addition,the IL12 level in PM@MSN-DOX/BLZ group was significantly higher than that of the PBS group(347.6±7.4 pg/ml vs 160.1 ±6.1pg/ml,P<0.0001).Flow cytometry of T cells showed that PM@MSN-DOX/BLZ group significantly increased the proportion of CD3+CD8+T lymphocytes in tumor tissue with the PBS group(1.60±0.12%vs 0.10±0.02%,P<0.0001),In addition,PM@MSN-DOX/BLZ group significantly increased the proportion of CD3+CD8+T lymphocytes in spleen tissue with the PBS group(12.5±0.8%vs 6.8±1.3%,P<0.0001).The anti-tumor study showed that PM@MSN-DOX/BLZ group significantly prolonged the survival time of mice compared with the PBS group(61.5±1.9 days vs 28.6±1.5 days,P<0.001);additionally,the tumor volume and tumor mass of mice in the PM@MSN-DOX/BLZ group were significantly reduced compared with the PBS group(P<0.05).The evaluation of the biological safety in vivo showed that PM@MSN-DOX/BLZ was not significantly toxic to all organs of mice and had a good in vivo biosafety.Conclusion:PM@MSN-DOX/BLZ can efficiently target tumor tissue and reshape the tumor immunosuppressive microenvironment to inhibit tumor growth effectively.PM@MSN-DOX/BLZ nanoparticles is a safe and effective drug delivery system which has a good in vivo biosafety.