Preparation Of T-cell Targeted And FK506-loaded Mesoporous Silica Nanoparticles And Its Effect On Cell In Vitro

Acute rejection is mainly treated with immunosuppressive drugs clinically.FK506 is an effective immunosuppressant for the prevention and treatment of allograft rejection.Its mechanism is to inhibit the activation and proliferation of T lymphocytes.After oral or intravenous administration of drugs,FK506 is distributed systemically,and cannot be effectively concentrated in the regions that T lymphocytes gather.Therefore it cannot exert its maximum efficacy.What is more,its side effects are inevitable during long-term administration.Therefore,to improve the distribution of drugs in the body and reduce the side effects while improving the efficacy is one of the key directions of the current treatment of acute rejection.Based on mesoporous silica nanoparticles,combined with CD3 specifically expressed on the surface of T lymphocytes,a safe and effective drug delivery system loading FK506 was constructed in this study.We evaluate the characteristics and investigate its ability to inhibit T lymphocytes proliferation and secretion of inflammatory factors.This study consists of the following two parts:Part 1 Preparation and characterization of targeted drug-loaded mesoporous silica nanoparticlesObjective To prepare FK506-loaded mesoporous silica nanoparticles conjugated with anti-CD3 antibody and evaluate their characterization.Methods Mesoporous silica nanoparticles（MSNs）were synthesised by template method.FK506 was loaded into MSNs by solvent evaporation method.The biotinylated phospholipids and MSNs were co-incubated to prepare lipid coated MSNs.Then the biotinylated CD3 antibody or isotype antibody were connected to the MSNs surface by the biotin-avidin-biotin linking system.In that way the FK506-loaded MSNs carrying CD3 antibody or isotype antibody(FK506-MSN_CD3 or FK506-MSNcon)were prepared.The morphology,particle size,zeta potential,stability and pore size of the nanoparticles were evaluated by DLS,Nitrogen absorption desorption,TEM and SEM.Fluorescence microscopy and flow cytometry were used to detect lipid coated and antibody conjugation efficiency.High performance liquid chromatography（HPLC）and thermo-gravimetric analysis（TGA）were used to evaluate drug loading and entrapment efficiency,and dialysis was used to analyze drug release in vitro.Result ⑴ MSNs was synthesized by template synthesis method and FK506-MSN_CD3 or FK506-MSNcon were also prepared.The results of DLS showed that the mean particle sizes of FK506-MSN_CD3 and FK506-MSNcon were 327.30 ± 7.60 nm and 322.70 ± 6.00 nm,respectively.And there was no significant difference in particle size,zeta potential and PDI.SEM and TEM showed that FK506-MSNs were spherical,evenly distributed and uniform in size.⑵ Flow cytometry results showed that more than 85% of MSNs were successfully coated with biotinylated lipid membrane.⑶ After Di I-MSN_CD3 and FITC-labeled secondary antibody were incubated,a large amount of green fluorescence was observed under the fluorescence microscope and colocated with the red fluorescence of Di I-labeled MSNs.⑷ The specific surface area and pore size analyzer revealed that the specific surface area,total pore volume and pore size of MSNs was 777.64 m2/g,0.94 cm3/g and 3.0 nm,respectively.After loading,the specific surface area,total pore volume and pore size of FK506-MSNs were 570.70 m2/g,0.49 cm3/g and 2.5 nm,respectively.⑸ The percentages of weigh loss in MSNs,FK506-MSNs and FK506-MSN_CD3 groups in the process of rising from 0℃ to 800℃ were 14%,78% and 86%,respectively.And the loading rate and encapsulation rate of FK506-MSN_CD3 were calculated to be 76.83% and 68%,respectively.⑹ The drug loading rate and encapsulation rate of FK506-MSN_CD3 detected by HPLC were about 75.86 ± 0.65% and 63.25 ± 2.85%,respectively,which were in consistent with the results obtained by TGA.In vitro drug release research,FK506-MSN_CD3 and FK506-MSNcon showed similar release behavior,reaching a plateau at 72 h,and the cumulative drug release was 58.07 ±6.89%,56.21 ± 3.50%,respectively.Conclusion In this study,FK506-MSN_CD3 were successfully prepared with a uniform size and good stability,and the drug could be slowly released in vitro.Part 2 The binding of nanoparticles to T cells and inhibition of T lymphocytes functionObjective To verify the binding of targeted drug-loaded mesoporous silica nanoparticles to T lymphocytes and their inhibitory effect on the proliferation of T lymphocytes and secretion of inflammatory factors.Methods Immunomagnetic isolation kit was used to isolate T lymphocytes from rats.And the purity of the obtained T lymphocytes was determined by flow cytometry analysis.Confocal laser scanning microscopy was used to observe the attachment ability of the Di I-MSN_CD3 and Di I-MSNcon to T lymphocytes,and flow cytometry analysis was used to quantitatively evaluate the attachment efficiency.The cytotoxicities of the MSN_CD3 or MSNcon to lymphocytes were evaluated by CCK-8 kit.In the mixed lymphocyte reaction（MLR）study,ELISA and CCK-8 kit were used to analyze the effect of FK506-MSN_CD3,FK506-MSNcon and FK506 on the secretion of IFN-γ and IL-2 and lymphocytes proliferation.Results ⑴ The suspension cells were isolated and purified by immunomagnetic isolation kit,and 96.07 ± 1.89% of them were T lymphocytes detected by flow cytometry.⑵ After incubation of Di I-MSN_CD3 and Di I-MSNcon with T lymphocytes under the same conditions,confocal laser scanning microscopy showed that a large number of Di I-MSN_CD3 attached to the surface of T lymphocytes,while Di I-MSNcon hardly bound to the surface of T lymphocytes.Flow cytology analysis revealed the binding rate of Di I-MSN_CD3 and Di I-MSNcon to T lymphocytes was about 82.3 ± 0.8 %,24.6 ± 0.7 %,separately（P < 0.05）.⑶ CCK-8 results showed that different amount of MSN_CD3 and MSNcon had no significant effect on T lymphocyte viabilities after incubation with T lymphocytes for 6 h,12 h or 24 h.⑷ CCK-8 assay showed tha in the MLR,after lymphocytes incubation with FK506,FK506-MSNcon,and FK506-MSN_CD3,the cell proliferation rate was 48 ± 16%,44 ± 4.0%,27 ± 7.0%,respectively.The cell viability of FK506-MSN_CD3 compareed with FK506,FK506-MSNcon has statistical difference（P < 0.05）.⑸ The results of ELISA showed that the contents of IL-2 in the supernatant of FK506,FK506-MSNcon,FK506-MSN_CD3 treatment group were 858.73 ± 46.52 pg/m L,831.53 ± 82.22 pg/m L,597.75 ± 65.56 pg/m L,respectively.The content of IFN-γ was 1231.27 ± 79.69 pg/m L,1345.96 ± 85.80 pg/m L,957.27 ± 92.98 pg/m L,respectively. The concentration of IL-2 and IFN-γ of FK506-MSN_CD3 has statistical difference with FK506 and FK506-MSNcon（P < 0.05）.Conclusion The MSN_CD3 prepared in this study could bind to T lymphocyte efficiently and had no significant effect on T lymphocyte viabilities.Moreover,FK506-MSN_CD3 could inhibit the proliferation of T lymphocytes and secretion of IL-2 and IFN-γ by specific binding to T lymphocytes.