D10 - Polymer-Salt Aqueous Two-Phase Separation for the Fabrication of Biomaterial Scaffolds

Introduction: : Polymeric hydrogel microspheres have emerged as attractive biomaterials for a wide range of applications, including drug delivery and cell scaffolds. Hydrogel microsphere scaffolds are superior to bulk hydrogels due to increased porosity that enables nutrient transport and cell migration, improved mechanical properties and adhesivity due to a concentrated polymer phase, and modularity enabled by the ability to “click” various microspheres into designer scaffolds. As drug delivery vehicles, microspheres offer benefits as injectable depots with controlled release kinetics contingent on microsphere mesh size and overall diameter. Several techniques have been widely developed to fabricate hydrogel microspheres, including microfluidics, batch emulsion, electrospraying, and aqueous two-phase separation (ATPS). While the first three techniques typically rely on the immiscibility of aqueous polymers in non-polar solvents, ATPS allows for the fabrication of microspheres in a completely aqueous environment. As such, repetitive washing steps are avoided, allowing for mitigation of undesired release of encapsulated cargo and risk of nonpolar solvent contamination. Here, we used polyethylene glycol (PEG) crosslinked through Michael-type addition with incorporated two-dimensional nanosilicate particles as a representative drug delivery device, capable of achieving tunable and prolonged release of small molecules. We also demonstrated the use of UV-crosslinked gelatin methacrylate (GelMA) to form a one-pot macroporous granular scaffold for cellular encapsulation. These two applications serve to demonstrate the utility of ATPS to form small microspheres in a rapid, inexpensive, and accessible manner.

Materials and

Methods: : Nanocomposite PEG hydrogel microspheres for drug delivery were fabricated by first dissolving 4-arm PEG-Acrylate (10 kDa) and PEG-dithiol (3.4 kDa) separately in 1X phosphate buffered saline (PBS, pH 7.4) to achieve a stock concentration of 20% w/v. Two-dimensional nanosilicate particles (Laponite XLG, BYK Additives) were separately exfoliated in deionized water to achieve a stock concentration of 3% w/v. PEG solutions and nanosilicates (NS) were added to a 1.5 mL microcentrifuge tube to achieve the desired final concentrations, with typical concentrations of 2% w/v PEG and 1 mg/mL NS. Model small molecule acridine orange (AO) was added to achieve a final concentration of 4 µM. Sodium sulfate (1.25 M stock) was added to this solution at a final concentration of 0.375 M to begin phase separation and microsphere formation (Figure 1A). The effect of fabrication parameters on microsphere size and polydispersity was examined, including PEG concentration, NS concentration, temperature, and salt concentration. Small molecule release was followed for 20 days. GelMA microspheres were fabricated by dissolving GelMA in 1X PBS and mixing with sodium sulfate and Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator to achieve final concentrations of 2% or 10% w/v GelMA, 0.1% or 0.3% w/v LAP, and 0.25-1.0 M sodium sulfate. Phase separation and UV crosslinking of GelMA were carried out either sequentially or concurrently to observe the effect on microsphere diameter. U-87 glioblastoma or NIH 3T3 fibroblast cells were introduced to a precursor GelMA and photoinitiator solution, demonstrating cell encapsulation capabilities of the microsphere and the resulting scaffolds.

Results, Conclusions, and Discussions:: PEG-NS composite microspheres were successfully fabricated with diameters ranging from 5-25 µm. Salt concentration was optimized to ensure dispersible microspheres were formed, with elevated salt concentrations leading to droplet aggregation and bulk gel formation, while low salt concentrations did not lead to phase separation. Reaction temperature had the most profound effect on microsphere size, with cooler temperatures resulting in larger microspheres or a lack of phase separation, while a high temperature of 60 oC resulted in the smallest microspheres. As NS concentration increased, microsphere size increased marginally. NS and AO were shown to be successfully entrapped within PEG microspheres, leading to tunable release kinetics contingent on NS concentration (Figure 1B). At the highest tested NS concentration of 1 mg/mL, release was slowed by over 95% compared to PEG-only control. GelMA was also successfully used to fabricate hydrogel microspheres. Individual microspheres with a diameter ~2 µm were achieved by allowing for GelMA, LAP, and the salt mixture to react for 30 s under UV light. However, if this reaction time was increased to 10 minutes, microspheres “clicked” together, forming a macroporous bulk scaffold that conformed to the shape of the reaction vessel (Figure 1C). Microsphere size was tuned by modulating GelMA and LAP concentrations. Our results indicated a mechanically stable scaffold able to quickly imbibe water due to its interconnected macropores, leading to stiffer scaffolds than hydrogels of equivalent polymer concentration. Cells were added to the hydrogel precursor solution, allowing for facile encapsulation within the fabricated scaffolds without adverse effects on cell viability. Cells exhibited an elongated morphology at 24 hours and >90% viability over 21 days of culture (Figure 1C). Together, these results demonstrated the unique ability of ATPS to produce hydrogel microspheres in solely aqueous solutions, negating the need for excessive washing steps or introduction of non-biocompatible surfactants. As such, small molecules could be encapsulated within the microspheres without rapid burst releases during the washing process, and cells were not subjected to harmful solvents. Furthermore, the facile fabrication of high numbers of microspheres enabled the rapid assembly of macroscale scaffolds that may be used for many biomedical applications.

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