Folic acid functionalized nanoparticles for enhanced oral drug delivery

Abstract

The oral absorption of drugs that have poor bioavailability can be enhanced by encapsulation in polymeric nanoparticles. Transcellular transport of nanoparticle-encapsulated drug, possibly through transcytosis, is likely the major mechanism through which nanoparticles improve drug absorption. We hypothesized that the cellular uptake and transport of nanoparticles can be further increased by targeting the folate receptors expressed on the intestinal epithelial cells. The objective of this research was to study the effect of folic acid functionalization on transcellular transport of nanoparticle-encapsulated paclitaxel, a chemotherapeutic with poor oral bioavailability. Surface-functionalized poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles loaded with paclitaxel were prepared by the interfacial activity assisted surface functionalization technique. Transport of paclitaxel-loaded nanoparticles was investigated using Caco-2 cell monolayers as an in vitro model. Caco-2 cells were found to express folate receptor and the drug efflux protein, p-glycoprotein, to high levels. Encapsulation of paclitaxel in PLGA nanoparticles resulted in a 5-fold increase in apparent permeability (Papp) across Caco-2 cells. Functionalization of nanoparticles with folic acid further increased the transport (8-fold higher transport compared to free paclitaxel). Confocal microscopic studies showed that folic acid functionalized nanoparticles were internalized by the cells and that nanoparticles did not have any gross effects on tight junction integrity. In conclusion, our studies indicate that folic acid functionalized nanoparticles have the potential to enhance the oral absorption of drugs with poor oral bioavailability.

Figure 1

Representative scanning electron micrograph of non–functionalized nanoparticles (A) and folic acid-functionalized nanoparticles (B).

Figure 2

Proton NMR of (a) free folic acid; (b) and (c) PLA-PEG-folic acid; and (d) PLA-PEG-folic acid functionalized NP. The NMR spectrum in (c) is expanded in (b) to show the peaks corresponding to folic acid. # indicate folic acid peaks; * indicate PEG peaks.

Figure 3

Characterization of Caco-2 cells for (A) P-gp expression and (B) folate receptor expression. (A) Western blotting was used to characterize P-gp expression in Caco-2 and MCF-7 (negative control) cells. β-actin served as the loading control. (B) Flow cytometry was used to characterize the folate receptor expression in Caco-2 cells. Cells were stained with either mouse anti-folate receptor antibody or mouse IgG2b k isotype control, followed by FITC-labeled goat–anti-mouse secondary antibody. Flow cytometric analysis was carried out with 488 Laser 530/30(FL-1) to enable FITC discrimination.

Figure 4

Apparent permeability coefficients (P_app) of different paclitaxel (Ptx) formulations: Ptx solution, Ptx-loaded non-functionalized nanoparticles (NP), Ptx-loaded, PEG functionalized nanoparticles (PEG-NP), Ptx-loaded, folic acid-PEG-functionalized nanoparticles (FA-NP). Transport study was conducted using Caco-2 monolayers as described in the Methods section (n=4; data shown is mean ± SD, * p<0.05 in comparison with Ptx solution, **p<0.05 in comparison with NP and PEG-NP).

Figure 5

Effect of different treatments on the TEER across the Caco-2 monolayers. Legend: paclitaxel solution - Ptx solution; Ptx-loaded non-functionalized nanoparticles - NP, Ptx-loaded, PEG-functionalized nanoparticles - PEG-NP, Ptx-loaded, folic acid-PEG-functionalized nanoparticles - FA-NP.

Figure 6

Effect of encapsulation in nanoparticles on paclitaxel uptake by Caco-2 cells (n=4; data showed is mean ± SD, * p<0.05 in comparison with Ptx solution). Legend: paclitaxel solution - Ptx solution; Ptx-loaded non-functionalized nanoparticles - NP, Ptx-loaded, PEG functionalized nanoparticles - PEG-NP, Ptx-loaded, folic acid-PEG-functionalized nanoparticles - FA-NP.

Figure 7

Nanoparticle uptake in Caco-2 cells after 6 h of incubation. (A) Apical side and (B) intracellular images. Blue channel represents nuclei stained with DAPI (column 1), green channel indicates 6-coumarin fluorescence (column 2), red channel denotes the cell perimeter highlighted by tight junctions labeled with antibody against ZO-1 coupled with Alexa fluor 594 (column 3). Overlay of the three channels are shown in column 4. Scale bar represents 150 μm.

Figure 8

Flow cytometric analysis showing that (A) folic acid functionalization increases the cellular internalization of nanoparticles and (B) presence of excess folic acid (20 μM) eliminates the enhancement in uptake provided by folic acid functionalization. (C) Effect of excess folic acid on the geometric mean of the fluorescence intensities in cells treated with different nanoparticle formulations. Caco-2 cells (250,000) were incubated with nanoparticle formulations (125 μg/mL) at 4° C for 30 min, followed by at 37° C for 15 min, and then analyzed on a flow cytometer.