(A-37) Nanosilicate Hydrogel Composites for Sustained Delivery of Small Molecules and Proteins

Introduction:: Hydrogels are widely used for drug delivery applications due to the biocompatibility, biodegradability, and ability to control release kinetics by tuning hydrogel swelling and mechanical properties. However, traditional hydrogels are susceptible to high initial burst release and ineffectiveness of sustained delivery due to rapid drug diffusion out of the hydrogel, especially for small molecules (< 1000 Da). Two-dimensional (2D) nanosilicate (NS) particles such as Laponite have recently been incorporated into hydrogels to serve as an additional lever by which drug release can be controlled. Disc-shaped NS particles are dually charged with negatively charged faces and positively charged edges, providing for electrostatic interactions for molecules with a variety of surface charges. Small molecules, especially those that are cationic, can be intercalated into the NS interlayer space via cationic exchange, as well as adsorbed to the NS surface. Proteins, which are larger, form relatively big complexes with NS. However, the structure, stability, and reversibility of complexes formed between NS and proteins remains unexplored. Additionally, characterization of the adsorption and subsequent release of proteins of varying size and charge has not been studied in detail. Deeper understanding of the mechanisms of small molecule and protein interaction with NS can further improve tunability of NS-based hydrogel delivery devices and allow for a variety of release profiles, providing a “plug-and-play” delivery platform. To answer these questions, we fabricated and characterized NS-protein and NS-small molecules interactions in the presence and absence of polyethylene glycol (PEG) hydrogels.


Materials and Methods:: Three model proteins were used to study NS-protein interactions: lysozyme (Lys), bovine serum albumin (BSA), and ribonuclease A (RNase). Small molecules studied included acridine orange (AO), clinically relevant chemotherapeutic doxorubicin (DOX), and Brilliant Blue FCF (BB). NS, NS-protein, and NS-small molecule complex sizes and structure were measured using dynamic light scattering (DLS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), fluorescent correlation spectroscopy (FCS), and Fourier-transform infrared spectroscopy (FTIR). NS-protein and NS-small molecule complex stability was also measured as a function of solvent pH and salinity, as well as in the presence of a protein denaturant. Protein stability was also characterized using circular dichroism (CD) and protein activity assays. Next, nanocomposite hydrogels were fabricated by encapsulating NS in PEG hydrogel formed by Michael-type addition. Hydrogels were characterized by swelling measurements and rheology. DLS, bulk release experiments, and FCS were used to study NS-protein interaction and stability and protein release from hydrogels.

Results, Conclusions, and Discussions:: NS formed complexes with both positively charged (RNase and Lys) and negatively charged proteins (BSA, Fig. 1A). XRD and FTIR confirmed successful adsorption of positively charged proteins in the interlayer space of NS particles due to cationic exchange. FCS measurements showed protein diffusivity decreased as NS or protein concentration increased. RNase and Lys were able to bind NS at higher concentrations compared to BSA (Fig. 1B). These differences can be attributed to positively charged proteins interacting with the NS particles faces, while negatively charged BSA interacted primarily with the NS edges. Results indicated that proteins may unfold to some degree while interacting with NS but retain bioactivity following release. All proteins showed slowed release in PEG-NS hydrogels compared to PEG-only hydrogels (Fig. 1C). While protein release in PEG-only hydrogels was mostly dependent on protein size, release in PEG-NS hydrogels was dependent on protein charge, with negatively charged proteins being released faster than positively charged ones. Positively charged proteins interacted with NS particles more profoundly than negatively charged proteins, forming larger complexes and exhibiting slower release kinetics from PEG-NS hydrogels. Therefore, protein charge can be implicated as a major determinant of strength and stability of NS-protein interactions. Similarly, small molecule release from PEG-NS hydrogels was governed by small molecule charge. Cationic AO and DOX exhibited both surface adsorption and intercalation in the NS interlayer space leading to prolonged release, while negatively charged BB was released faster in comparison to cationic small molecules. Release was also shown to be governed by release buffer pH and salinity, with increasing pH and increasing salt concentration leading to faster release kinetics, presumably due to desorption of small molecules or proteins. Further characterization of NS-protein and NS-small molecule complexes in complex mediums such as blood are needed to assess their utility in physiological systems. Nonetheless, these PEG-NS nanocomposite hydrogels show great promise for improving sustained release kinetics from hydrogels and may be used for delivery of both small molecules and macromolecules such as proteins.


Acknowledgements (Optional): :

References (Optional): : [1] S.T. Stealey, A.K. Gaharwar, N. Pozzi, S.P. Zustiak, Development of Nanosilicate–Hydrogel Composites for Sustained Delivery of Charged Biopharmaceutics, ACS Applied Materials & Interfaces 13(24) (2021) 27880-27894.

[2] S. Stealey, M. Khachani, S.P. Zustiak, Adsorption and Sustained Delivery of Small Molecules from Nanosilicate Hydrogel Composites, Pharmaceuticals 15(1) (2022).

[3] M. Khachani, S. Stealey, E. Dharmesh, M.S. Kader, S.W. Buckner, P.A. Jelliss, S.P. Zustiak, Silicate Clay-Hydrogel Nanoscale Composites for Sustained Delivery of Small Molecules, ACS Applied Nano Materials 5(12) (2022) 18940-18954.