Metoclopramide nanoparticles modulate immune response in a diabetic rat model: association with regulatory T cells and proinflammatory cytokines

Abstract

Background: The inflammatory basis of diabetes mellitus directed the researchers' attention to the immune system for better management and prevention of complications. Metoclopramide (MCA; the only US Food and Drug Administration-approved for gastroparesis) has the ability to restore immune function through increasing prolactin secretion. This study aimed to test the effect of BSA/MCA nanoparticles (NPs) on modulating immune response.

Methods: BSA/MCA NPs were fabricated by desolvation and evaluated in vitro via measuring loading efficiency, particle size, and surface charge. The selected formula was further evaluated via differential scanning calorimetry and release behavior. Then, NPs were injected into rats (25 mg MCA/kg/week) for 3 weeks to be evaluated histopathologically and immunologically via measuring proinflammatory cytokines, such as IL1β, IL6, and TNFα, in addition to measuring regulatory T-cell frequency.

Results: MCA was successfully loaded on BSA, achieving high encapsulation efficiency reaching 63±2%, particles size of 120-130 nm with good polydispersity, and a negative surface charge indicating that entire positively charged drug was encapsulated inside NPs. Differential scanning calorimetry thermography of selected NPs showed an obvious interaction between components and cross-linking of BSA molecules using glutaraldehyde, resulting in sustained release of MCA (around 50% within 3 days). MCA NPs significantly restored the immune response via decreasing proinflammatory cytokines and increasing regulatory T-cell frequency when compared to control and free MCA (drug not loaded in NPs)-treated groups. Histopathological examination of this MCA NPs-treated group did not show the characteristic lesions of diabetes, and apoptosis nearly disappeared.

Conclusion: BSA/MCA NPs could be considered a new modality for treatment of gastro-paresis, in addition to management of diabetes itself and preventing its complications via an MCA-immunomodulatory effect.

Keywords: bovine serum albumin; diabetes; gastroparesis; immunity; metoclopramide; nanoparticles.

Figure 1

Flow cytometry detection of regulatory T (T_reg) cells. Notes: (A) Forward and side scatter histogram depicting lymphocyte population (R1). (B) The expression of CD4 was assessed in the lymphocyte population (R1) and CD4⁺ T cells were gated. (C) Then, the expression of CD25 and FOXP3⁺ cells on CD4⁺ T cells was assessed to detect CD4⁺CD25⁺FOXP3⁺ T_reg cells. Abbreviations: SSC, side scatter; FSC, forward scatter; FITC, fluorescein isothiocyanate.

Figure 2

Entrapment efficiency (EE) and drug loading (DL) of nanoparticles at different BSA:metoclopramide (MCA) weight ratios.

Figure 3

Particle size, polydispersity index (PDI), and Zeta-potential of nanoparticles at different BSA:metoclopramide (MCA) weight ratios.

Figure 4

(A) Transmission electron microscopy of selected BSA/metoclopramide nanoparticles (NPs). (B) Differential scanning calorimetry thermography of individual components of NPs, physical mixture, and selected NPs: metoclopramide HCl (a), BSA (b), physical mixture of components (c), and selected NPs (d; BSA:metoclopramide at weight ratio of 2:1).

Figure 5

In vitro release of metoclopramide from selected BSA nanoparticles (A) and release kinetics of metoclopramide from these nanoparticles (B).

Figure 6

Histopathological changes in diabetic rats. Notes: (A) Liver shows centrilobular hepatic necrosis (arrows); (B) liver shows clear nonstaining vacuoles in hepatic cells (notched arrows); (C) liver shows focal areas of Kupffer-cell proliferation in portal area (star); (D) liver shows dilatation and congestion of central veins and in hepatic sinusoids (arrows); (E) lymph nodes show apoptotic lymphocytes (arrow); (F) lymph nodes show subcapsular vascular congestion (notched arrow); (G) spleen shows accumulation of collagen deposition in red pulp (star); H&E. (H) Collagen fibers in spleen confirmed by van Gieson stain.

Figure 7

Histopathological changes in free metoclopramide-treated group. Notes: (A) Liver shows clear vacuoles in hepatic cells (arrow); (B) liver shows Kupffer-cell proliferation (notched arrow); (C) lymph nodes show congestion (arrow) and apoptosis in some lymphocytes in lymphoid follicles (notched arrow); (D) Spleen shows focal areas of hemorrhage (star) and hemosiderosis (arrow); H&E.

Figure 8

Histopathological changes in metoclopramide nanoparticle–treated group. Notes: (A) Liver shows normal hepatic cords; (B) lymph nodes show follicular hyperplasia; (C) lymphoid follicles heavily populated with small lymphocytes; (D) spleen shows extramedullary formation of hematopoietic cells (arrow) and hemosiderosis (notched arrow); H&E.