Treatment Of Skin Transplant Rejection Using Drug Loaded Mesoporous Silica Nanoparticles Targeting Lymph Nodes

Nowadays,the anti-rejection management of patients after organ transplantation is still up against great challenge.On the one hand,the main clinical immunosuppressants such as calcium phosphatase inhibitors tacrolimus have low lymph node-targeting efficiency,and therefore cannot reach the maximum therapeutic effect.On the other hand,long-term oral or intravenous drug administration may induce some systemic side effects.This study is aimed to prepare a kind of drug-loaded nanoparticles targeting lymph nodes,explore their targeting efficiency to the lymph nodes,and evaluate their enhanced drug anti-rejection effect.The study consists of the following three sections:Part 1 Preparation and characterization of targeted drug-loaded mesoporous silica nanoparticlesObjective To prepare MSNs-FK506-MECA79 and evaluate its basic characterization.Methods(1)MSNs and MSNs-FK506 were prepared by means of soft template and solvent evaporating method.MSNs-FK506-MECA79 was prepared by ultrasonic water bath method,and then using thiol-maleimide chemistry to link the MECA79 antibody.(2)The particle size distribution,zeta-potential and polydispersity index of the three kinds of nanoparticles were measured by a Brookhaven Zeta PALS analyzer.(3)High performance liquid chromatography was used to evaluate the drug loading and encapsulation efficiency,and 3-day drug release profile in PBS at room temperature for MSNs-FK506 and MSNs-FK506-MECA79.The pore size change of MSNs before and after FK506 loading of MSNs was detected by nitrogen adsorption-desorption analyzer.The efficiency of MSNs encapsulated with phospholipid film and MECA79 antibody were detected by confocal microscopy and flow cytometry.Results(1)The mesoporous silica nanoparticles were successfully prepared and coated with phospholipid membrane to successfully link the MECA79 antibody.(2)The particle size of MSNs was 174.0 ± 2.0 nm,PDI was 0.155,zeta-potential was-16.22 ± 2.13 m V.The particle size of MSNs-FK506 was 179.0 ± 1.0 nm,PDI was 0.152,zeta-potential was-13.79 ± 2.02 m V.The particle size of MSNs-FK506-MECA79 was 191.8 ± 0.8 nm,PDI was 0.146,zeta-potential was-14.93 ± 1.51 m V.(3)The drug loading rate of MSNs-FK506-MECA79 was 76.27 ± 2.28 %,encapsulation rate was 74.46 ± 1.39 %.The cumulative FK506 release rate for 3 days in vitro was 72.92 ± 3.19 %.The pore size of MSNs was slightly smaller after drug loading.Confocal imaging showed that the phospholipid film and MECA79 antibody were conjugated successfully,and flow cytometry showed that 95.8 % and 81.3 % of MSNs were successfully coated with phospholipid film and conjugated with MECA79 antibody,respectively.Conclusion In this study,targeted nanoparticles with high drug payload(MSNs-FK506-MECA79)were successfully prepared,and the drug release was stable in vitro.Part 2 In vivo targeting efficiency and toxicity test of targeted drugloaded mesoporous silica nanoparticlesObjective To verify in vivo lymph node targeting of MSNs-MECA79 and to evaluate their biodistribution and safety in vivo.Methods(1)Di R labeled MSNs,MSNs-MECA79 were injected intravenously into mice,respectively.The fluorescence intensity in lymph nodes was quantified and compared by in vivo imaging after 2 h and 24 h.(2)FITC labeled MSNs,MSNs-MECA79 were injected intravenously into mice respectively.After 24 hours,lymph nodes were collected and stained with CD31 frozen sections.The density of nanoparticles in the two groups of lymph node sections were captured under microscope and compared by photoshop.(3)Di R labeled MSNs,MSNs-MECA79 were injected intravenously into mice,respectively.The fluorescence intensities in heart,liver,spleen,lung and kidney were quantified and compared by in vivo imaging after 2 h and 24 h.(4)The mice were injected with PBS,FK506,MSNs-FK506,MSNs-FK506-MECA79 respectively in the tail vein at 1,3,5,7,9 days,then blood was collected at 1 and 10 days after injection for routine blood test and blood biochemical test,and their heart,liver,spleen,lung,kidney were harvested for HE staining.Results(1)2 h after injection,there was no significant difference found in the fluorescence intensity of lymph nodes in the two groups.After 24 hours,the fluorescence intensity of lymph nodes in the two groups were significantly different.(2)After 24 hours,the amounts of nanoparticles in lymph node sections of the two groups observed under a microscope were significantly different.(3)2 h after injection,the fluorescence of nanoparticles in both groups were mainly concentrated in the lungs,while after 24 h,the nanoparticles were mainly concentrated in the livers and spleens.(4)After 1 day and 10 days of injection,there was no significant difference in the blood cells,liver and kidney functions among the four groups,and the HE staining of main organs showed no obvious injury in all four groups.Conclusion This study revealed that the MECA79 conjugated nanoparticles could be effectively targeted to lymph nodes within 24 hours after intravenously injection.And the nanoparticles caused no obvious side effects in mice.Part 3 Evaluation of targeted drug-loaded mesoporous silica nanoparticles in the treatment of skin graft rejection in miceObjective To explore the anti-rejection effect of lymph node-targeted drug-loaded nanoparticles in murine skin transplantation.Methods(1)A murine skin transplantation model was constructed by collecting donor skin grafts and anastomosing recipient skin regions.(2)PBS,FK506,MSNs-FK506,MSNs-FK506-MECA79 were administered to the mice intravenously at 1,3,5,7 and 9 days after skin transplantation.The condition of skin grafts was assessed and considered as graft failure when more than 90 % of skin became necrosis.(3)The FK506 concentration in the lymph nodes were measured and compared by liquid-mass spectrometry at 1 and 10 days after injection.(4)On the 10 th day,the skin grafts were collected and evaluated by HE staining and CD3 immunohistochemistry.The sections were observed under a microscope and the degree of rejection in the four groups were analyzed.Results(1)The murine skin transplantation model was successfully constructed in 64 mice.(2)The average survival time of the skin grafts in the MSNs-FK506-MECA79 group was 9.43 ± 3.05 days,which was statistically longer than that in the PBS group and the FK506 group.(3)1 day after injection,there was no significant difference for the average drug concentration in lymph nodes of the four groups.However,10 days after injection,the drug concentration in lymph nodes of each group varied vastly.In the MSNsFK506-MECA79 group it was 5569.33 ± 783.78 ng/g,which was statistically higher than that in the PBS group and the FK506 group.(4)After 10 days of injection,the skin grafts of the PBS group showed the highest degree of rejection and the most infiltrated inflammatory cells and CD3 positive T cells,while the MSNs-FK506-MECA79 group demonstrated the lowest degree of rejection and the least infiltrated inflammatory cells and CD3 positive T cells.Conclusion In this study,we proved that the lymph node-targeted drug-loaded nanoparticles had more FK506 concentrated in lymph nodes and could inhibit the rejection of skin transplantation in mice effectively.