Cellular Uptake and Transport Mechanism of 6-Mercaptopurine Nanomedicines for Enhanced Oral Bioavailability

Abstract

Background: Nanomedicines have significant advantages in enhancing the oral bioavailability of drugs, but a deeper understanding of the underlying mechanisms remains to be interpreted. Hence, the present study aims to explain the uptake and trafficking mechanism for 6-MP nanomedicines we previously constructed.

Methods: 6-MP loaded poly(lactide-co-glycolide) (PLGA) nanomedicines (6-MPNs) were prepared by the multiple emulsion method. The transcytosis mechanism of 6-MPNs was investigated in Caco-2 cells, Caco-2 monolayers, follicle associated epithelium (FAE) monolayers and rats, including transmembrane pathway, intracellular trafficking, paracellular transport and the involvement of transporter.

Results: Pharmacokinetics in rats showed that the area under the curve (AUC) of 6-MP in the 6-MPNs group (147.3 ± 42.89 μg/L·h) was significantly higher than that in the 6-MP suspensions (6-MPCs) group (70.31 ± 18.24 μg/L·h). The uptake of 6-MPNs in Caco-2 cells was time-, concentration- and energy-dependent. The endocytosis of intact 6-MPNs was mediated mainly through caveolae/lipid raft, caveolin and micropinocytosis. The intracellular trafficking of 6-MPNs was affected by endoplasmic reticulum (ER)-Golgi complexes, late endosome-lysosome and microtubules. The multidrug resistance associated protein 4 (MRP4) transporter-mediated transport of free 6-MP played a vital role on the transmembrane of 6-MPNs. The trafficking of 6-MPNs from the apical (AP) side to the basolateral (BL) side in Caco-2 monolayers was obviously improved. Besides, 6-MPNs affected the distribution and expression of zona occludens-1 (ZO-1). The transport of 6-MPNs in FAE monolayers was concentration- and energy-dependent, while reaching saturation over time. 6-MPNs improved the absorption of the intestinal Peyer's patches (PPs) in rats.

Conclusion: 6-MPNs improve the oral bioavailability through multiple pathways, including active transport, paracellular transport, lymphatic delivery and MRP4 transporter. The findings of current study may shed light on the cellular uptake and transcellular trafficking mechanism of oral nanomedicines.

Keywords: cellular uptake; nanomedicines; oral bioavailability; transport mechanism.

Figure 1

Characterization of 6-MPNs in vitro and in vivo. The morphology of 6-MPNs measured by Transmission Electron Microscope (TEM) (A). (B) In vitro release of 6-MP from 6-MPNs and 6-MPCs in PBS containing 0.02% Tween 20. (C) In vivo plasma concentrations (ng/mL) of 6-MP vs Time (h) profiles after a single oral administration of 6-MPNs or 6-MPCs in SD rats.

Figure 2

Uptake mechanisms study of 6-MPNs in Caco-2 cells. (A) Cytotoxicity analysis of Caco-2 cells incubated with 6-MPNs for 4 h by the Cell Counting Kit-8 (CCK-8) method. (B) The effects of time on the uptake of 6-MPNs by Caco-2 cells (*P value < 0.05, **P value < 0.01). (C) The effects of concentration on the uptake of 6-MPNs by Caco-2 cells (*P value < 0.05). (D) Analysis of energy dependence of endocytosis of 6-MPNs in Caco-2 cells (*P value < 0.05).

Figure 3

Endocytosis and transport mechanism analysis of 6-MPNs in Caco-2 cells. (A) The effect of different endocytosis inhibitors on the internalization of 6-MPNs in Caco-2 cells (**P value < 0.01). (B) The effect of different intracellular transport inhibitors on the internalization of 6-MPNs in Caco-2 cells (**P value < 0.01, ***P value < 0.001, ****P value < 0.0001). (C) The effect of indomethacin on the transport of 6-MPNs in Caco-2 cells (*P value < 0.05, **P value < 0.01).

Figure 4

Immunofluorescence images of tight junction protein (ZO-1) in the untreated control group (A), 6-MPCs group (B) and 6-MPNs group (C), respectively (scale bar: 5 μm).

Figure 5

Effects of 6-MPNs on the expression of tight junction protein (ZO-1) in Caco-2 monolayers detected by Western blot. (A) Representative immunoblot images (cropped images). (B) Quantification analysis of ZO-1 protein expression (**P value < 0.01, ***P value < 0.001).

Figure 6

(A) Relationship between uptake amounts of 6-MPNs and concentration (*P value < 0.05). (B) Relationship between uptake amounts of 6-MPNs and time (*P value < 0.05). (C) Relationship between uptake amounts of 6-MPNs and temperature. (D) Relationship between cumulative transport of 6-MP and concentration (**P value < 0.01, ****P value < 0.0001). (E) Relationship between cumulative transport of 6-MP and time (**P value < 0.01). (F) Relationship between cumulative transport of 6-MP and temperature (*P value < 0.05, **P value < 0.01).

Figure 7

(A) The cumulative transport of 6-MP on follicle associated epithelium (FAE) monolayers and Caco-2 monolayers at 2 h, respectively (*P value < 0.05). (B) Absorption of 6-MPNs in Peyer’s patch and Peyer’s patch-free small intestine (*P value < 0.05).