(G-268) Nanosilicate-Hydrogel Microspheres Formed by Aqueous Two-Phase Separation for Sustained Release of Small Molecules

Introduction:: Hydrogel microspheres are an attractive option for drug delivery applications due to their ease of injection and potential for tunable controlled delivery. However, their utility is limited due to high initial burst release and rapid overall release, which is especially pronounced for small molecules or small size microspheres. We and others have shown that the addition of two-dimensional nanosilicate (NS) particles to hydrogels can significantly prolong release kinetics from hydrogels while minimizing burst release. Small molecules are electrostatically adsorbed to the charged faces and edges of NS particles, significantly slowing their release. However, this effect is not well-studied in hydrogel microspheres, which are desirable for drug delivery due to their injectability and potential for systemic release but are also more susceptible to burst release. This study aimed to investigate whether NS particles could be incorporated into polyethylene-glycol hydrogel microspheres using Aqueous Two-Phase Separation (ATPS) to achieve sustained release of small molecules. The ATPS method relies on the immiscibility of a polymer/salt mixture beyond a certain temperature and is an attractive fabrication method because it allows for facile, high-yield, and scalable microsphere production, without the use of organic solvents or oils.


Materials and Methods::

For microsphere formation via ATPS, 29.8 μL of 20% 4-arm PEG-Acrylate and 20.2 μL of 20% PEG-diSH were allowed to pre-react via a Michael-type addition reaction. For PEG-NS nanocomposite microspheres, NS powder was dispersed in DI water and probe sonicated for 30 s in a 1 s on/1 s off pattern. NS stock solution and the model small molecule were then added to the PEG precursor solution for final NS concentrations ranging from 0.01 μg/mL to 1,000 μg/mL. Then 300 µL of 1X PBS and 150 µL of 1.25 M Na₂SO₄ solution were added to the PEG-NS mixture and incubated at 37°C for 45 min, allowing NS particles and small molecules to phase separate into the PEG-rich phase. After, 1,000 μL of fresh 1X PBS was added, and the microspheres were dispersed by vigorous pipetting. The solution was centrifuged for 30 s at ×2000 g to sediment the microspheres. The supernatant was removed, and the microspheres were resuspended in PBS to a final volume of 1.5 mL. The solution was then probe sonicated for 30 s in a 1 s on/1 s off pattern to disperse microsphere aggregates. Microspheres were characterized by measuring microsphere diameters (using ImageJ software), via mechanical testing with rheology, Fourier-transform infrared spectroscopy (FTIR), and bulk release studies to study small molecule release.


Results, Conclusions, and Discussions::
Nanocomposite hydrogel microspheres containing two-dimensional nanosilicate (NS) particles were successfully fabricated via ATPS (Fig. 1), representing an enhanced delivery device with improved mechanical and release properties, compared to PEG-only microspheres. The effects of PEG concentration, pre-reaction time, incubation temperature and pH on microsphere diameter and polydispersity were studied to optimize microsphere fabrication conditions to achieve microspheres < 10 μm and minimize polydispersity. The optimal fabrication conditions were determined to be a 2% PEG concentration, pH 7.4, 5 min pre-reaction time, and a 37 °C incubation temperature. To confirm NS and the model cationic small molecule, Acridine Orange (AO), were present in the PEG-rich phase and successfully encapsulated within the hydrogel microspheres, FTIR was utilized. Peaks representing NS were present in the PEG-NS and PEG-NS-AO samples but absent in PEG-only and PEG-AO samples, as expected. Peaks indicative of AO were present in the PEG-AO and PEG-NS-AO samples. This result confirmed that NS and AO phase separated into the PEG-rich phase and were encapsulated within the hydrogel microspheres. The release of the AO from PEG microspheres containing various NS concentrations was evaluated for short release times (up to 24 h) and longer release times (up to 20 days) (Fig. 2). In PEG-only microspheres, AO release was rapid, and a high initial burst release was observed as expected. Conversely, AO release was significantly reduced from PEG-NS microspheres, where an increase in NS concentration resulted in reduced initial burst release and slower release of AO over time. Furthermore, two other small molecules of varying net charge, the chemotherapeutic doxorubicin (DOX), which is positively charged at pH 7.4, and Brilliant Blue (BB), which exhibits a negative charge at physiological pH, all demonstrated sustained release from PEG-NS microspheres, further validating the robustness of this PEG-NS microsphere delivery device. Overall, this delivery platform offers an exciting opportunity to combine the benefits of hydrogel microsphere delivery devices with those of nanomaterials, offering enhanced delivery kinetics and device tunability. Future work will include cytotoxicity and blood clotting evaluation of NS particles, as well as the encapsulation and release of macromolecules such as proteins and enzymes.




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2. Stealey, S., Khachani, M., Zustiak, S.P. (2022). Adsorption and Sustained Delivery of Small Molecules from Nanosilicate Hydrogel Composites. Pharmaceuticals 15, 56. doi:10.3390 /ph15010056.