(W1030-01-06) Facile and Tunable Nanoparticle Delivery with Granular Hydrogels

Purpose: Integration of nanoparticles into hydrogels is of great interest in drug delivery, yielding nanocomposite hydrogels with enhanced properties. While incorporation by directly soaking hydrogels in nanoparticle solutions presents benefits such as easy clinical application and enhanced drug stability, achieving compatible porous structures and optimal nanoparticle affinity proves challenging with bulk hydrogels. (1) Macroporous granular hydrogels, composed of densely packed microgels, show promise in overcoming these hurdles due to their porosity and inherent modularity. Yet, current microgel fabrication methods face challenges such as polydisperse size distribution or reliance on complex microfluidics designs. (2) Here, we introduce a novel granular hydrogel scaffold designed for convenient and adaptable delivery of nanoparticle therapeutics. Utilizing hybrid lipid polymer nanoparticles known for their broad applications, we expand upon our previous work on glutathione-conjugated poly(ethylene glycol) (GSH-PEG) hydrogels, which have demonstrated efficacy in locally delivering small molecules and proteins. (3) We propose that loading cationic nanoparticles into GSH-PEG granular scaffolds can be achieved by exploiting electrostatic interactions between the nanoparticles and the hydrogel components, thereby enabling precise control over the delivery process.

Methods: For microgel fabrication, the aqueous phase, comprised of 40% w/w poly(ethylene glycol) diacrylate (PEGDA) MW 575 and photoinitiator, was mixed with the oil phase using a confined T-junction connector, with syringe pumps regulating the flow rates. Droplets were polymerized upon collection on a glass slide and UV exposure at 20mW/cm² intensity. Upon extensive washing steps, granular hydrogel formation was initiated by secondary crosslinking of microgels upon exposure to UV light, following the addition of photoinitiator. GSH-PEG microgels were synthesized using a similar approach, incorporating GSH into the prepolymer solution at 1 to 10 GSH to PEGDA molar ratios. The nanoparticles consisted of poly(lactic-co-glycolic acid) (PLGA), the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and DMG-PEG (2000), and were prepared using a one-step nanoprecipitation method with the T-junction connector. RhodamineB-PLGA was incorporated into the formulation for quantification purposes, while DiIC18(5) dye was encapsulated for confocal microscopy imaging. Nanoparticles at 800 μg/mL were incorporated into 35μL hydrogels via a single-step soaking method for 24 hours to ensure complete nanoparticle diffusion within the hydrogel. Soaking was carried out either in water or 1x phosphate-buffered saline (PBS) at pH 7.4. The distribution of nanoparticles within the hydrogel was monitored using confocal microscopy. Release studies were conducted in PBS at pH 7.4 and 37°C, with sample volumes periodically withdrawn for fluorescence measurements and replaced with fresh release media.

Results: Microgels had spherical shapes with tunable sizes ranging from 10μm to 500μm, achieving homogeneous distribution with values below 0.05 in polydispersity. The formed granular hydrogels could be stored in an aqueous solution at 4°C for at least one month, or alternatively, they could be dried and rehydrated before application, providing a long shelf-life option. The hybrid nanoparticles had an average diameter of 109.9 ±4.5nm and a surface zeta potential +30.7 ±1.5 mV, with no size changes following release from the hydrogel, as determined by dynamic light scattering. The conditions of the loading solution significantly influenced nanoparticle loading in the hydrogels. Loading in water resulted in approximately three times higher loading compared to PBS (p-value < 0.05), attributed to the screening of electrostatic interactions in the presence of ions. Notably, there was no significant difference in loading between PEG and GSH-PEG hydrogels. However, the release profiles differed between the two, with PEG hydrogels exhibiting a faster release reaching a plateau at 3 days, while GSH-PEG hydrogels demonstrated sustained release for up to 5 days. The release kinetics from PEG hydrogels were best described by a one-phase association model using nonlinear regression. In contrast, release from GSH-PEG hydrogels followed a two-phase association model, characterized by both fast and slow half-lives, suggesting that GSH-PEG involves two distinct mechanisms influencing release.

Conclusion: The development of an adaptable precipitation polymerization method for granular hydrogels, coupled with a straightforward loading approach, demonstrates the efficacy of GSH-PEG granular hydrogels in achieving controlled nanoparticle delivery. Our findings highlight the influence of both the loading solution and the hydrogel material on nanoparticle release, offering avenues for adjusting nanoparticle dosage and achieving controlled release. The proposed GSH-PEG granular scaffold holds great promise for the versatile delivery of nanoparticles, particularly for topical application.

References: 1. Wahid, Fazli, et al. "Nanocomposite hydrogels as multifunctional systems for biomedical applications: Current state and perspectives." Composites Part B: Engineering 200 (2020): 108208.
2. Daly, Andrew C., et al. "Hydrogel microparticles for biomedical applications." Nature Reviews Materials 5.1 (2020): 20-43.
3. Andrianopoulou, Angeliki, et al. "Assessment of antibiotic release and antibacterial efficacy from pendant glutathione hydrogels using ex vivo porcine skin." Journal of Controlled Release 365 (2024): 936-949.


Figure 1. Schematic illustration of the microgel fabrication method and nanoparticle loading process in the resulting granular hydrogel.


Figure 2. (A) Microgel diameter (μm) as a function of varying the continuous flow rate (QC) at constant disperse flow rate of 1μL/min. Inset: microscope image of produced PEGDA microgels. Scale bar: 300μm. (b) Microgel diameter (μm) and polydispersity (PDI) as a function of total flow rate (μL/min). Data presented for three replicates, with measurements obtained from the average of 10 particles per replicate.


Figure 3. (A) Confocal microscopy images of DiIC18(5)-labelled nanoparticles loaded in GSH-PEG granular hydrogel in PBS pH 7.4. Mosaic (left) and snapshot (right) images of a slice obtained from the center of the hydrogel. (B) Nanoparticle loading (μg) in PEG or GSH-PEG granular hydrogels upon loading in water or 1x PBS pH 7.4, as determined by fluorescence microplate reader. (C) Release of nanoparticles (μg) from PEG or GSH-PEG granular hydrogels in PBS. Data are presented as mean ± standard deviation of three independent experiments.