Effect of Sirolimus and cyclospurin nanostructured lipid carriers (NLC) on T lymphocytes biology in recurrent pregnancy loss patients as compared with healthy pregnant

Abstract

Recurrent pregnancy loss (RPL) is defined as two or more pregnancy losses prior to 20 weeks of gestation which affects 1 to 2% of women trying to conceive. In this study for the first time, we developed and optimized nano-lipid carriers co-loaded with Sirolimus (SRL) and Cyclosporine (CsA) to increase their efficacy as immunosuppressive agents and reduce their toxicity effects. As well we explored the in vitro evaluation of prepared formulations on peripheral Th17, Treg, Th1, and Th2 cells response in RPL patients. Methods: In this study, 30 patients with RPL and 30 healthy pregnant women were selected as our study populations. Flow cytometry was used to determine the frequency of T cells. NLCs mono-delivery and co-delivery SRL and CsA were developed (S-NLCs, C-NLCs, S-C-NLCs) and characterized include size, zeta potential, morphology and also in-vitro cellular uptake, cell viability, and apoptosis of human T cells under the influence of nanodrugs were investigated. After determining the effective dose, nanodrugs were exposed to patients' T cells then the expression levels of RORγt, Foxp3, T-bet and GATA-3 with qPCR, their protein levels of these transcription factors with Western blot and cytokines secretion with ELISA were assessed. Results: The size of prepared nanoparticles was 34-143nm with negative zeta potential and encapsulation efficacy over 90% was observed for nanodrugs. Their cellular uptake was higher than that of free drugs. In addition, data analysis showed that combination therapy of T cells with concentrations of 0.5 μM CsA and 0.05 μM SRL in S-C-NLC resulted in 73% cell viability. Which were selected as optimal concentrations to exposure with patients' T cells. Regarding the results of cell apoptosis, exposure to S-NLC and C-NLC increased apoptosis, while no significant difference was observed in S-C-NLC. Flow cytometry results showed that the frequency of Th17 and Th1 cells in RPL patients was higher than in healthy pregnant women, while the frequency of Th2 and Treg cells was lower. After exposure to S-NLC and C-NLC, a decrease in the expression level of T-bet, RORγt was observed and these changes were significant as compared to healthy pregnant women. In association with Foxp3, the results showed increase. However, after exposure to S-C-NLC, no statistical difference was found in the expression of these genes compared to healthy pregnant women. The results of Western blotting confirm these findings. Additionally, after exposure with S-NLC and C-NLC, a decrease in the level of IFN-γ, TNF-α, IL-17 and IL-21 cytokines and an increase in IL-10 were observed, these changes were significant as compared to healthy pregnant women. While at TGF-β level a slight increase was observed which was not statistically significant. Also, no significantl changes were observed in the level of IL-4. On the other hand, after exposure to S-C-NLC, there was no statistical difference in the level of these cytokines in RPL compared to healthy pregnant women.