Nanofunctionalized Microparticles for Glucose Delivery in Three-Dimensional Cell Assemblies

Abstract

Three-dimensional (3D) cell assemblies, such as multicellular spheroids, can be powerful biological tools to closely mimic the complexity of cell-cell and cell-matrix interactions in a native-like microenvironment. However, potential applications of large spheroids are limited by the insufficient diffusion of oxygen and nutrients through the spheroids and, thus, result in the formation of a necrotic core. To overcome this drawback, we present a new strategy based on nanoparticle-coated microparticles. In this study, microparticles function as synthetic centers to regulate the diffusion of small molecules, such as oxygen and nutrients, within human mesenchymal stem cell (hMSC) spheroids. The nanoparticle coating on the microparticle surface acts as a nutrient reservoir to release glucose locally within the spheroids. We first coated the surface of the poly(lactic-co-glycolic acid) (PLGA) microparticles with mesoporous silica nanoparticles (MSNs) based on electrostatic interactions and then formed cell-nanofunctionalized microparticle spheroids. Next, we investigated the stability of the MSN coating on the microparticles' surface during 14 days of incubation in cell culture medium at 37 °C. Then, we evaluated the influence of MSN-coated PLGA microparticles on spheroid aggregation and cell viability. Our results showed the formation of homogeneous spheroids with good cell viability. As a proof of concept, fluorescently labeled glucose (2-NBD glucose) was loaded into the MSNs at different concentrations, and the release behavior was monitored. For cell culture studies, glucose was loaded into the MSNs coated onto the PLGA microparticles to sustain local nutrient release within the hMSC spheroids. In vitro results demonstrated that the local delivery of glucose from MSNs enhanced the cell viability in spheroids during a short-term hypoxic culture. Taken together, the newly developed nanofunctionalized microparticle-based delivery system may offer a versatile platform for local delivery of small molecules within 3D cellular assemblies and, thus, improve cell viability in spheroids.

Keywords: 3D cell culture; drug delivery; glucose; mesoporous silica nanoparticles; microparticles; spheroids.

Figure 1

Schematic illustration of the hierarchal structure and components of the cell-nanofunctionalized microparticle spheroids to reduce spheroids’ inner necrotic cores. From the left: Glucose is loaded into the pores of MSNs, and PLGA microparticles are coated with glucose-loaded MSNs. The glucose-loaded PLGA-MSN microparticles are coaggregated with human mesenchymal stem cells (hMSCs) to form cell-nanofunctionalized microparticle spheroids. The PLGA-MSN microparticles are cell-organized within the spheroid and act as multipoint delivery centers to the cells. In this way, the cells within the spheroids are directly exposed to glucose released from the MSNs, which prevents the formation of an inner necrotic core.

Figure 2

Nano- and microparticle visualization. (A) TEM image of MSNs. Scale bar represents 100 nm. (B) SEM image of MSNs. Scale bar represents 4 μm. (C) SEM image of bare PLGA microparticles before oxygen plasma treatment. Scale bar represents 10 μm. (D) SEM image of oxygen plasma-treated, bare PLGA microparticles. Scale bar represents 10 μm.

Figure 3

Stability of PLGA-MSN microparticles in cell culture medium over the course of 14 days at 37 °C. SEM images of PLGA-MSN microparticles (A) before incubation (day 0) and after (B) 2, (C) 7, and (D) 14 days of incubation. Scale bars represent (top row) 10 μm and (bottom row) 2 μm.

Figure 4

Formation and characterization of hMSC-PLGA-MSN spheroids. (A) SEM images of hMSC and hMSC-(nanofunctionalized) microparticle spheroids after 3 days of culture. The spheroids are formed with about 3 μg of either PLGA (hMSC-PLGA) or MSN-coated PLGA (hMSC-PLGA-MSN). Spheroids formed from hMSCs only (hMSC) served as a control. Scale bar represents 100 μm and applies for all images of the subfigure. (B) Quantification of the projected area of hMSC-nanofunctionalized microparticle spheroids after 3 days of culture by quantitative analysis of confocal fluorescence microscopy images in CellProfiler (n = 3 spheroids). ‘n.s.’ stands for ‘not statistically significant’. Statistical method used is one-way ANOVA with Tukey’s post hoc test for multiple comparisons. (C) Maximum projection of a confocal microscopy image of a hMSC-PLGA-MSN spheroid after 24 h of culture. Cells are stained with phalloidin (in green) and DAPI (in blue) to label cytoskeletal F-actin and cell nuclei, respectively. MSNs are labeled with an ATTO647N-Maleimide (in red). Scale bar represents 50 μm. (D) Quantification of cell metabolic activity in spheroids after 3 days of culture by using the CellTiterGlo 3D Assay (n = 3). ‘A.U.’ stands for ‘arbitrary units’ and ‘n.s.’ stands for ‘not statistically significant’. The statistical method used is one-way ANOVA with Tukey’s post hoc test for multiple comparisons. (E) Maximum projection of confocal microscopy images of spheroids after (top row) 4 and (bottom row) 24 h. Cells are stained with an antivinculin antibody (in red) and DAPI (in blue) to label focal adhesion points and cell nuclei, respectively. Scale bar represents 100 μm and applies for all images of the subfigure.

Figure 5

Glu_F release from PLGA-MSNs and glucose effect on cell viability in hMSC-nanofunctionalized microparticle spheroids. (A) Glu_F release from PLGA-MSNs for 1 and 100 Mm Glu_F loading. Quantification of cell metabolic activity in spheroids in (B) normoxia and (C) hypoxia (5% pO₂) conditions after 1 day of culture in DPBS (n = 3) by using the CellTiterGlo Assay. ‘*’, ‘**’, ‘***’, and ‘****’ represent p values smaller than 0.05, 0.01, 0.001, and 0.0001, respectively. ‘A.U.’ stands for ‘arbitrary units’. Statistical method used is one-way ANOVA with Tukey’s post hoc test for multiple comparisons.

Figure 6

Glucose release effect on cell death in hMSC-nanofunctionalized microparticle spheroids. (A) Maximum projection of confocal microscopy images of spheroids after 24 h of culture. Cells are stained with Fixable Far Red Dead Cell Stain (in red), DAPI (in blue), and phalloidin additionally (in each case right column of the two double columns, in green) to label dead cells, cell nuclei, and F-actin, respectively. Scale bar represents 100 μm and applies for all images of the subfigure. (B) Quantification of cell viability in spheroids in normoxia and hypoxia conditions (5% pO₂) after 24 h of culture by quantitative analysis of confocal fluorescence microscopy images in CellProfiler (n = 4 spheroids). ‘*’ represents p values smaller than 0.05 and ‘n.s.’ stands for ‘not statistically significant’. Statistical method used is two-way ANOVA with Tukey’s post hoc test for multiple comparisons.

Figure 7

Glucose release effect on cell death in large hMSC-nanofunctionalized microparticle spheroids. (A) Maximum projection of confocal microscopy images of larger spheroids after 3 days of culture in normoxic condition. Cells are stained with Fixable Far Red Dead Cell Stain (in red), DAPI (in blue), and phalloidin (in each case right column of the two double columns, in green) to label dead cells, cell nuclei, and F-actin, respectively. Scale bar represents 100 μm and applies for all images of the subfigure. (B) Quantification of cell death in spheroids in normoxia after 3 days of culture by quantitative analysis of confocal fluorescence microscopy images in CellProfiler (n = 6 spheroids). ‘**’, ‘***’, and ‘****’ represent p values smaller than 0.05, 0.01, and 0.001, respectively. ‘n.s.’ stands for ‘not statistically significant’. Statistical method used is one-way ANOVA with Tukey’s post hoc test for multiple comparisons.