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Biomaterials

Biomaterials - Poster Session E

Poster N19 - Formulation of Dynamic Polymer-nanoparticle Hydrogels using Yeast Cell-derived Nanovesicles as a Supramolecular Crosslinker

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cell-derived nanovesicles (CDNs), are naturally-derived biomaterials that present a significant opportunity for developing targeted delivery systems with enhanced cellular signaling capabilities. Currently, synthetic nanomaterials such as liposomes face limitations in tissue targeting and uptake due to their non-specificity and rapid clearance by the reticuloendothelial system. By comparison, CDNs are less cytotoxic, more biocompatible, can better evade phagocytosis, and have longer blood half-lives [1]. Furthermore, since they are naturally-derived, CDNs possess a multitude of bioactive ligands that can enable tissue targeting and uptake, in addition to more advanced functions including stem cell proliferation and differentiation, blood brain barrier crossing, and immunomodulation [1-3].
To promote the retention and sustained release of CDNs at the site of administration, we have developed novel yeast cell-derived nanovesicle hydrogels. These polymer-nanoparticle (PNP) hydrogels are dynamically crosslinked by the CDNs through supramolecular interactions between the CDN membrane and carbon-chain modified hypromellose (HPMC) polymers [4]. This imparts tunable mechanical properties to the hydrogels, including shear thinning and self-healing characteristics that allow the gels to be injected through a needle and reform a depot at the site of administration. In contrast to previous hydrogels that entrap CDNs, usage of the CDNs as the crosslinker allows for orders of magnitude higher particle concentrations and more effectively sustained release that correlates with the material’s degradation in vivo. Future applications of this hydrogel material technology include tissue generation and wound healing, cancer vaccination and immunotherapy, and in situ gene editing and stem cell differentiation [1, 3].

Materials and

Methods:  Yeast cell-derived nanovesicles (yCDNs) were synthesized by first dissolving dried pellets of saccharomyces cerevisiae in 1M NaOH heated to 80°C and stirred for 1 hour. The yeast slurry was then cooled and centrifuged at 2000xg for 10min. The precipitate was washed with DI water, isopropanol, and acetone then resuspended in DI water. The resulting yeast particles were bath sonicated to break up remaining cell wall components then extruded by 0.22µm sterile filtration to produce nanoparticles. The yCDNs were lyophilized for storage then reconstituted in DI water at 80 mg/ml for hydrogel preparation. yCDN size and zeta potential was measured on a Malvern Zetasizer and nanoparticle tracking analysis was performed on a Particle Metrix ZetaView x30. Total protein analysis was conducted on the yCDNs using a Pierce BCA assay (Thermo Fischer) following the manufacturer protocol.
Octadecyl-modified hydroxypropylmethylcellulose (HPMC-C18) was prepared by the synthesis method described in Correa et. al. (2022) using octodecyl-isocyanate as the carbon chain reactant [4]. yCDN hydrogels were then formulated by loading yCDNs and HPMC-C18 in two 1mL Luer lock syringes and mixing them through an elbow joint until a homogenous hydrogel formed. Rheological characterization of the hydrogel was performed on a TA Instruments DHR-2 rheometer using an 8 mm diameter serrated stainless steel parallel plate geometry at 25°C.

Results, Conclusions, and Discussions: Yeast-derived materials have emerged as a promising vehicle for drug delivery due to their endogenous targeting ability gleaned from unique membrane proteins and polysaccharides such as β-glucans [5]. These components have been reported to activate DCs and macrophages which in turn can program T cells, lending yeast-derived materials specialized functions as immunomodulatory vehicles. We have demonstrated that yCDNs can crosslink biopolymers through reversible, supramolecular interactions, forming dynamic hydrogels with shear thinning and self-healing abilities.
Firstly, we have developed a synthesis process for yCDNs that produces nanoparticles of ~100nm in diameter and negative surface charge (Figure 1a, b). These yCDNs can be lyophilized for storage and maintain their size and surface charge when reconstituted at the high weight percentages (>8wt%) necessary to formulate hydrogels (Figure 1c). We confirmed that the yCDNs retain protein components of the yeast via BCA assay, which gave a total protein concentration of ~9.3mg/ml pre-lyophilization and are working to determine the particular protein composition via western blot. We then utilized the yCDNs to crosslink HPMC-C18 polymer through supramolecular interactions between the yCDN surface and lipophilic C18 chains on the polymer. Hydrogels were formulated at 3wt% HPMC-C18 and 4wt% yCDN (~10^12 particles/ml) by a syringe mixing procedure (Figure 2a). Rheological characterization of this hydrogel showed a higher storage (G’) than loss (G’’) modulus over a large frequency range, indicating solid-like behavior of the material (Figure 2b). Further, the hydrogel exhibited shear thinning over a ~3-order of magnitude shear rate range (Figure 2c) and was able to recover its viscosity after repeated high shearing conditions (Figure 2d), indicating its ability to be administered via syringe-needle.
In this work, we have developed novel PNP hydrogels crosslinked by yCDNs through supramolecular interactions. Utilizing yCDNs as the crosslinking agent itself enables high concentrations (>10^12 particles/ml) to be incorporated into the gel, which will be crucial for future applications of this material for drug delivery. We aim to explore the mechanical design space of these yCDN hydrogels further by varying the concentration and identity of the polymer.

Acknowledgements (Optional):  The authors would like to thank James Wang for assistance in figure creation.
S.N. would like to acknowledge the National Science Foundation Graduate Research Fellowship Program for financial support.

REFERENCES

1. Elkhoury K, Koçak P, Kang A, Arab-Tehrany E, Ellis Ward J, Shin SR. Engineering Smart Targeting Nanovesicles and Their Combination with Hydrogels for Controlled Drug Delivery. Pharmaceutics. 2020;12(9):849. doi:10.3390/pharmaceutics12090849
2. Liang Y, Iqbal Z, Lu J, et al. Cell-derived nanovesicle-mediated drug delivery to the brain: Principles and strategies for vesicle engineering. Mol Ther J Am Soc Gene Ther. 2023;31(5):1207-1224. doi:10.1016/j.ymthe.2022.10.008
3. Amengual-Tugores AM, Ráez-Meseguer C, Forteza-Genestra MA, Monjo M, Ramis JM. Extracellular Vesicle-Based Hydrogels for Wound Healing Applications. Int J Mol Sci. 2023;24(4):4104. doi:10.3390/ijms24044104
4. Correa S, Grosskopf AK, Klich JH, Lopez Hernandez H, Appel EA. Injectable liposome-based supramolecular hydrogels for the programmable release of multiple protein drugs. Matter. 2022;5(6):1816-1838. doi:10.1016/j.matt.2022.03.001
5. Xu J, Ma Q, Zhang Y, et al. Yeast-derived nanoparticles remodel the immunosuppressive microenvironment in tumor and tumor-draining lymph nodes to suppress tumor growth. Nat Commun. 2022;13(1):110. doi:10.1038/s41467-021-27750-2