Core-Shell PLGA Nanoparticles: In Vitro Evaluation of System Integrity

Abstract

The objective of this study was to compare the properties of core-shell nanoparticles with a PLGA core and shells composed of different types of polymers, focusing on their structural integrity. The core PLGA nanoparticles were prepared either through a high-pressure homogenization-solvent evaporation technique or nanoprecipitation, using poloxamer 188 (P188), a copolymer of divinyl ether with maleic anhydride (DIVEMA), and human serum albumin (HSA) as the shell-forming polymers. The shells were formed through adsorption, interfacial embedding, or conjugation. For dual fluorescent labeling, the core- and shell-forming polymers were conjugated with Cyanine5, Cyanine3, and rhodamine B. The nanoparticles had negative zeta potentials and sizes ranging from 100 to 250 nm (measured using DLS) depending on the shell structure and preparation technique. The core-shell structure was confirmed using TEM and fluorescence spectroscopy, with the appearance of FRET phenomena due to the donor-acceptor properties of the labels. All of the shells enhanced the cellular uptake of the nanoparticles in Gl261 murine glioma cells. The integrity of the core-shell structures upon their incubation with the cells was evidenced by intracellular colocalization of the fluorescent labels according to the Manders' colocalization coefficients. This comprehensive approach may be useful for the selection of the optimal preparation method even at the early stages of the core-shell nanoparticle development.

Keywords: DIVEMA; Gl261 cells; HSA; PLGA; confocal microscopy; core–shell nanoparticles; poloxamer 188.

Figure 1

Chemical structures of fluorescently labeled polymers used for the preparation of core–shell nanoparticles: (1) PLGA conjugate with Cyanine5 (PLGA-Cy5), (2) DIVEMA in anhydride form, (3) DIVEMA conjugate with Cyanine3 (DIVEMA-Cy3), (4) poloxamer 188 conjugate with rhodamine B (P188-RhB), and (5) HSA conjugate with rhodamine B (HSA-RhBITC).

Figure 2

Schematic representation of the types of dual-labeled core–shell PLGA nanoparticles.

Figure 3

TEM images: (a) PLGA/HSA-C; (b) PLGA/HSA-A cross-linked; (c) PLGA/HSA-IE cross-linked; (d) PLGA/DIVEMA-N; (e) PLGA without a shell (JEOL JEM-1400 electron microscope, negative staining with UranyLess stain).

Figure 4

Average diameters of core–shell nanoparticles during 6 h of incubation in model media (pH 7.4; 37 °C; mean ± SD; n = 3): (a) PLGA-Cy5/HSA-RhB incubation in PBS; (b) PLGA-Cy5/HSA-RhB incubation in PBS in the presence of 4.5% HSA; and (c) PLGA-Cy5/DIVEMA-Cy3 incubation in PBS in the presence or absence of 4.5% HSA. PLGA nanoparticles without a shell were used as a control. Size measurements were performed using DLS (volume distribution).

Figure 5

Fluorescence spectra of representative nanoparticle samples: (a) PLGA-Cy5/HSA-RhBITC (PLGA/HSA-IE cross-linked); (b) PLGA-Cy5/DIVEMA-Cy3 (PLGA/DIVEMA-N).

Figure 6

Evaluation of core–shell structure stability. Percentage of total shell-dye concentration remaining on the nanoparticles’ surface (based on relative fluorescence intensity values) after (a) 6 h of PLGA-Cy5/HSA-RhB incubation in PBS (pH 7.4); (b) 6 h of PLGA-Cy5/HSA-RhB incubation in PBS in the presence of 4.5% HSA (pH 7.4); and (c) 6 h of PLGA-Cy5/DIVEMA-Cy3 incubation in PBS in the presence or absence of 4.5% HSA (pH 7.4). Representative data.

Figure 7

Confocal images of Gl261 cells after incubation with core–shell nanoparticles. (a) Manders’ overlap coefficients (MOCs) showing colocalization between the core and the shell of the nanoparticles at different time points (15, 30, and 45 min; n = 3); (b) PLGA/HSA-C; (c) PLGA/HSA-A cross-linked; (d) PLGA/HSA-IE cross-linked; (e) PLGA-DIVEMA-N; (f) PLGA/P188. For each type of nanoparticles (b–f) were shown: (1) Merged images (green—lysosomes; cyan—shell; red—core); (2) shell (HSA-RhBITC, DIVEMA-Cy3, or P188-RhB); and (3) core (PLGA-Cy5). CLSM. Scale bar: 20 μm.

Figure 8

Core–shell nanoparticle uptake by Gl261 cells depending on the nanoparticle preparation technique and incubation time (15, 30, and 45 min of incubation). (a) % Cy5-positive cells and (b) % cells doubly positive for Cy5- and RhBITC/Cy3/RhB with PLGA-Cy5/HSA-RhBITC NPs, PLGA-Cy5/DIVEMA-Cy3 NPs, and PLGA-Cy5/P188-RhB NPs, respectively. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparison test (mean ± SD, n = 3); ns = not significant; p > 0.05.