Gut Organoid as a New Platform to Study Alginate and Chitosan Mediated PLGA Nanoparticles for Drug Delivery

Abstract

Intestinal organoids can be used as an ex vivo epithelial model to study different drug delivery effects on epithelial cells' luminal surface. In this study, the impact of surface charge on the delivery of 5-ASA loaded PLGA nanoparticles into the lumen of organoids was investigated. Alginate and chitosan were used to coat the nanoparticles and provide negative and positive charges on the particles, respectively. The organoid growth and viability were not affected by the presence of either alginate- or chitosan-coated nanoparticles. It was shown that nanoparticles could be transported from the serosal side of the organoids to the lumen as the dye gradually accumulated in the lumen by day 2-3 after adding the nanoparticles to the Matrigel. By day 5, the dye was eliminated from the lumen of the organoids. It was concluded that the positively charged nanoparticles were more readily transported across the epithelium into the lumen. It may be attributed to the affinity of epithelial cells to the positive charge. Thus, the organoid can be utilized as an appropriate model to mimic the functions of the intestinal epithelium and can be used as a model to evaluate the benefits of nanoparticle-based drug delivery.

Keywords: PLGA; alginate; chitosan; nanoparticles; organoids.

Figure 1

(A) The mean size of the nanoparticles using different 5-ASA ratios for two other surfactants. Three separate batches of NPs were tested for each data point. (B) The mean zeta potential of the nanoparticles using different 5-ASA ratios for two other surfactants. Three separate batches of NPs were tested for each data point.

Figure 2

Encapsulation efficiency of 5-ASA loaded PLGA nanoparticles using alginate or chitosan as the surfactant. Three separate batches of NPs were tested for each data point. Differences between groups were considered significant if p-values were <0.05. (*) indicates p-values < 0.05, (**) indicates p-values < 0.01.

Figure 3

The trend of organoid’s growth made by Alginate surfactant and different ratios of 5-ASA using optical microscopy over 6-days (1:4 V/V Nanoparticle: Matrigel suspension). The magnification is 10×. The scale bar represents 200 µm.

Figure 4

The trend of organoid’s growth made by Chitosan surfactant and different ratios of 5-ASA using optical microscopy over 6-days (1:4 V/V Nanoparticle: Matrigel suspension). The magnification is 10×. The scale bar represents 200 µm.

Figure 5

The percent change in the area of the organoids relative to day-2 (1:4 V/V Nanoparticle: Matrigel suspension). More than nine organoids were tested for each sample. Differences between groups were considered significant if p-values were <0.05. (*) indicates p-values < 0.05, (**) indicates p-values < 0.01.

Figure 6

Bright field, DSU RFP, and merged pictures of the mixture of 10 µL 2.5% Rhodamine B loaded nanoparticles made by Alginate surfactant, Matrigel, and organoids using a confocal fluorescent microscope (1:4 V/V Nanoparticle: Matrigel suspension). The experiment was performed for six days after the passage. Red areas are the indicators of the Rhodamine B loaded inside the PLGA nanoparticles. The magnification is 10×. Scale bars represent 200 µm.

Figure 7

Bright field, DSU RFP, and merged pictures of the mixture of 10 µL 2.5% Rhodamine B loaded nanoparticles made by Chitosan surfactant, Matrigel, and Organoids using a confocal fluorescent microscope (1:4 V/V Nanoparticle: Matrigel suspension). The experiment was performed for six days after the passage. Red areas are the indicators of the Rhodamine B loaded inside the PLGA nanoparticles. The magnification is 10×. Scale bars represent 200 µm.

Figure 8

Quantitative analysis of cell fluorescent recorded by confocal fluorescent microscope using alginate or chitosan as the surfactants for PLGA nanoparticles loaded with 2.5% Rhodamine B. The nanoparticles were mixed with Matrigel and organoids (1:4 V/V Nanoparticle: Matrigel suspension). More than nine organoids were measured for each sample. Differences between groups were considered significant if p-values were <0.05. (*) indicates p-values < 0.05, (**) indicates p-values < 0.01, (***) indicates p-values < 0.001.

Figure 9

After adding 2.5% loaded PLGA nanoparticles coated with Alginate and Chitosan (1:4 Nanoparticle: Matrigel), laser microscopy of organoids was added. Green represents SYTO™ 9, magenta represents phalloidin, and red represents Rhodamine B. The magnification of images is 40× with a digital zoom of 1.8, and scale bars represent 20 μm.

Figure 10

Live (SYTO^® 10)/dead (DEAD REDTM) cytotoxicity test of organoids in contact with 10 μL PLGA nanoparticles coated with Chitosan and Alginate surfactant recorded by confocal fluorescent microscope (1:4 Nanoparticle: Matrigel). Green fluorescent nucleic acid labels all live and dead cells. Red fluorescent nucleic acid labels the dead cells with compromised membrane. The magnification is 20×, and the scale bar represents 100 μm.