Preparation, characterization, and transport of dexamethasone-loaded polymeric nanoparticles across a human placental in vitro model

Abstract

The purpose of this study was to prepare dexamethasone-loaded polymeric nanoparticles and evaluate their potential for transport across human placenta. Statistical modeling and factorial design was applied to investigate the influence of process parameters on the following nanoparticle characteristics: particle size, polydispersity index, zeta potential, and drug encapsulation efficiency. Dexamethasone and nanoparticle transport was subsequently investigated using the BeWo b30 cell line, an in vitro model of human placental trophoblast cells, which represent the rate-limiting barrier for maternal-fetal transfer. Encapsulation efficiency and drug transport were determined using a validated high performance liquid chromatography method. Nanoparticle morphology and drug encapsulation were further characterized by cryo-transmission electron microscopy and X-ray diffraction, respectively. Nanoparticles prepared from poly(lactic-co-glycolic acid) were spherical, with particle sizes ranging from 140 to 298 nm, and encapsulation efficiency ranging from 52 to 89%. Nanoencapsulation enhanced the apparent permeability of dexamethasone from the maternal compartment to the fetal compartment more than 10-fold in this model. Particle size was shown to be inversely correlated with drug and nanoparticle permeability, as confirmed with fluorescently labeled nanoparticles. These results highlight the feasibility of designing nanoparticles capable of delivering medication to the fetus, in particular, potential dexamethasone therapy for the prenatal treatment of congenital adrenal hyperplasia.

Keywords: BeWo cells; Congenital adrenal hyperplasia; Dexamethasone; Nanoparticles; Placenta; Pregnancy.

Figure 1

Response surface plots showing the effects of the following parameters on the properties of drug-loaded nanoparticles: A. Effects of PLGA concentration in acetone and dexamethasone (DEX) concentration in acetone on particle size (with sodium taurocholate held constant at 0.5% (w/v)); B. Effects of dexamethasone concentration in acetone and the aqueous concentration of sodium taurocholate (ST, % w/v) on nanoparticle zeta potential (ζ, with PLGA concentration in acetone held constant at 20 mg/mL); C. Effects of PLGA and dexamethasone concentrations in acetone on encapsulation efficiency (EE, with sodium taurocholate held constant at 0.5% (w/v)). P-values for the statistically significant effects of the parameters were as follows: [PLGA] on particle size, <0.0001; [ST] on ζ, 0.0015; [DEX] on EE, 0.0032; [DEX]² on EE, 0.0182; [PLGA]² on EE, 0.0203. These plots were generated by using Statgraphics Plus 5.1 software (SAS Inc., Minneapolis, MN).

Figure 2

Powder X-ray diffraction patterns of the following samples (bottom to top): bulk dexamethasone, bulk PLGA, bulk sodium taurocholate, a physical mixture of the aforementioned samples, unloaded PLGA nanoparticles (without dexamethasone, but prepared in the presence of sodium taurocholate), and dexamethasone-loaded PLGA nanoparticles. Curves were displaced along the ordinate for better visualization.

Figure 3

A representative cryo-TEM image of dexamethasone-loaded nanoparticles (Formulation #1 in Table 1).

Figure 4

Comparison of transepithelial electrical resistance (TEER) of BeWo b30 cell monolayers and the apparent permeability (P_e) of Lucifer yellow across the cell monolayers on days 3-6 post-seeding. P_e was calculated at the two-hour time point as described in the text; data are presented as the average ± standard deviation (n=4).

Figure 5

Influence of particle size on the apparent permeability (P_e) of PLGA-nanoparticles across BeWo b30 cell monolayers. These nanoparticles did not contain dexamethasone, but were loaded with the fluorescent compound coumarin-6; these nanoparticles were prepared in water (without sodium taurocholate). The 145-nm particles were prepared with 20 mg/mL PLGA in acetone, and the 196-nm particles were prepared with 40 mg/mL PLGA. Initial nanoparticle concentrations applied to the apical (maternal) chamber matched those applied for the experiments from the first formulation in Table 4. The transport medium (DMEM) was without phenol red. P_e was calculated at the two-hour time point as described in the text; data are presented as the average ± standard deviation (n=3).