Anisotropic, Hydrogel Microparticles as pH-Responsive Drug Carriers for Oral Administration of 5-FU

Abstract

In the last 20 years, the development of stimuli-responsive drug delivery systems (DDS) has received great attention. Hydrogel microparticles represent one of the candidates with the most potential. However, if the role of the cross-linking method, polymer composition, and concentration on their performance as DDS has been well-studied, still, a lot needs to be explained regarding the effect caused by the morphology. To investigate this, herein, we report the fabrication of PEGDA-ALMA-based microgels with spherical and asymmetric shapes for 5-fluorouracil (5-FU) on-demand loading and in vitro pH-triggered release. Due to anisotropic properties, the asymmetric particles showed an increased drug adsorption and higher pH responsiveness, which in turn led to a higher desorption efficacy at the target pH environment, making them an ideal candidate for oral administration of 5-FU in colorectal cancer. The cytotoxicity of empty spherical microgels was higher than the cytotoxicity of empty asymmetric microgels, suggesting that the gel network's mechanical proprieties of anisotropic particles were a better three-dimensional environment for the vital functions of cells. Upon treatment with drug-loaded microgels, the HeLa cells' viability was lower after incubation with asymmetric particles, confirming a minor release of 5-FU from spherical particles.

Keywords: 5-FU; cytotoxicity; drug loading; in vitro drug release; microfluidics; microgels; oral administration; pH responsiveness.

Figure 1

Schematic representation of the three-inlet microfluidic device used for the generation of asymmetric PEGDA–ALMA:dextran microgels. Droplet-in-droplet microparticles become asymmetric microgels after UV-photopolymerization.

Figure 2

PEGDA 30% w/w–ALMA 1.5% w/w asymmetric and spherical microparticles. (a,c) Bright field images: scale bar is 10 µm. (b,d) Cryo-SEM images: scale bar is 1 µm.

Figure 3

Cryo-SEM images of PEGDA 30% w/w–ALMA 1.5% w/w: dextran 20% w/w asymmetric microparticles in (a) pH 2.0 and (b) pH 7.4. Scale bar is 1 µm. (c) Storage modulus (G’) over time of PEGDA 30% w/w–ALMA 1.5% w/w hydrogel after treatment with different pH solutions.

Figure 4

(a) Shrinking and swelling measurements of spherical and asymmetric PEGDA–ALMA microgels. The 2R symbol represents the microparticles diameter at different pH and is expressed as mean ± standard deviation (n = 50). CV is the coefficient of variation. Bright-field images of PEGDA–ALMA: (b) asymmetric and (c) spherical microgels in Milli-Q. Scale bar is 20 µm.

Figure 5

(a) Shrinking and swelling measurements of asymmetric PEGDA–ALMA:dextran microgels at different pH. The 2R symbol represents the full diameter of the particle and 2r is the diameter of its respective cavity. Values are expressed as mean ± standard deviation (n = 50). CV is the coefficient of variation. (b) Percentage of the volume occupied by the cavity in the full volume of the PEGDA–ALMA:dextran microgels. Values are averaged over 50 particles.

Figure 6

Bright-field images of asymmetric (a–d) and spherical (e–h) PEGDA 30% w/w–ALMA 1.5% w/w microgels at different pH. Scale bar is 20 µm.

Figure 7

Top and side views of an PEGDA–ALMA:dextran asymmetric microparticle (a,d) bright-field and (b,e) fluorescence images. Scale bar is 10 µm. The fluorescence intensity expressed as grey values, shows the homogeneous distribution of ALMA in the microgel’s shell (c), while less ALMA is located at the interface (f), due to the presence of dextran.

Figure 8

5-FU cumulative loading (a) and cumulative percentage of release (b) of asymmetric and spherical PEGDA–ALMA hydrogel microparticles. Values are expressed as mean of three replicates (n = 3) ± the cumulative standard deviation at each time point.

Figure 9

(a) NIH/3T3 cells’ viability after 72 h of incubation with different concentrations of PEGDA 30% w/w–ALMA 1.5% w/w empty spherical and asymmetric particles. The cell viability values are statistically significant different at 500 µg/mL. Values are plotted as mean ± standard deviation (n = 5), for each concentration. Statistical significance: *** is established for p < 0.001. (b) HeLa cells’ viability after 72 h of incubation with different PEGDA 30% w/w–ALMA 1.5% w/w spherical and asymmetric particles loaded with different concentrations of 5-FU. The cell viability values are statistically significant different at 100 µM. Values are plotted as mean ± standard deviation (n = 3), for each concentration. Statistical significance: ** is established for p < 0.01.

Figure 10

Live/dead assay staining of HeLa cells with PEGDA 30% w/w–ALMA 1.5% w/w asymmetric microgels loaded with a final 5-FU concentration of (a) 10 µM, (b) 50 µM, and (c) 200 µM. Green cells are living cells and red ones are dead. Scale bar is 50 µm.