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Biomaterials

Biomaterials - Poster Session B

Poster C5 - Engineering Lymph Node-Mimetic Polymer-Nanoparticle Hydrogels for Injectable Immunotherapeutic Delivery

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction: Polymer-nanoparticle (PNP) hydrogels are tunable shear-thinning biomaterials that are effective in controlling therapeutic delivery while maintaining minimal invasiveness. In these materials, nanoparticles interact with modified polymers to form dynamic noncovalent crosslinking, facilitating the formation of the hydrogel matrix. However, the binding motifs that dictate these cross-linkages are not well defined and are limiting in biomaterial design. These noncovalent interactions are necessary for PNP hydrogels to exhibit shear-thinning and self-healing properties, allowing for syringe injectability. Thus, understanding the physiochemical properties of the polymer and nanoparticle components is key to developing novel hydrogel technology. In previous work, we used dodecyl-modified hydroxypropyl methylcellulose (HPMC) as a synthetic polymer backbone to develop robust hydrogels capable of loading agonist antibodies and subunit vaccines. [1][2] Although this hydrogel was effective in functionalization, we look to build upon this work with hyaluronic acid (HA), a biopolymer whose versatility in potential modifications and biomimetic capabilities due to its natural occurrence as an essential component in the extracellular matrix makes it ideal for therapeutic delivery in lymphatic organs. We hypothesize that we can improve the functionality and tunability of PNP hydrogels using HA as the polymer backbone. By tuning the density and identity of crosslinking functional groups, we have modulated stiffness and viscosity to characterize the behavior of supramolecular binding motifs that define the material properties. We aim to implement these defined relationships towards PNP hydrogel designs for immunotherapeutic injection, expanding the functionality of HA.

Materials and

Methods: Modification of HA: HA of varying molecular weights (HA100K, HA200K, and HA700K) was modified with tetrabutylammonium (TBA) to enhance solubility in organic solvents.[3] HA-TBA was resuspended in DMSO under an inert atmosphere and dodecyl isocyanate (16.8 mol % reactive polymer:dodecyl isocyanate)[1] and NN-Diisopropylethylene (DIPEA) were subsequently added. Polymer Recovery: After 24 hours, we transferred the solution to 3.5 kDa dialysis tubing. Reactions were dialyzed against 2 L of H2O, adding 3 g/L NaCl to remove excess TBA. Following dialysis over 5 days, samples were lyophilized. The extent of polymer modification was analyzed using NMR spectroscopy (New York Structural Biology Center). Hydrogel Fabrication: HA-C12 was resuspended in deionized water and stir-mixed with carboxylate-modified latex (CML) for 10-15 minutes. We used 3 sizes of CML (20 nm, 40 nm, and 100 nm) and mixed at concentrations of 2 wt%, 5 wt%, and 10 wt%. HA-C12 was kept at 2 wt% for each gel and both modified polymers and CML were kept in 4o C. Rheology Testing: A TA Instrument Discovery HR 20 rheometer with an 8 mm parallel plate geometry was used to measure the stiffness at varying angular frequencies, self-healing viscosity and stress peaks, and shear thinning behavior.

Results, Conclusions, and Discussions: Compared to HPMC-C12 prepared using an analogous procedure, HA-C12 polymers had a lower extent of modification. This result was consistent across all molecular weights of HA tested. Despite HPMC having approximately equal molecular weight, HA100K produced softer gels. Thus, HA is less reactive to dodecyl isocyanate than HPMC in the current conditions we are modifying the biopolymer in. We observed that gel stiffness was positively correlated with nanoparticle concentration and negatively correlated with nanoparticle size. Gels with 2:10 wt% saw the stiffest properties across each modified polymer while 100 nm CML nanoparticles produced significantly softer gels. The stiffness range across HA gels of varying molecular weight encompassed the stiffness of a lymph node, a target injection site for immunotherapeutics. [2] These hydrogels also demonstrated shear thinning properties that were consistent with what was seen in stiffness. Lower viscosity was observed with the 100 nm CML nanoparticle and concurrently decreased with the smaller diameter gels while increasing the shear rate for a controlled concentration and polymer type. Similarly, gels across nanoparticle concentrations also concurrently decreased as the shear rate increased, supporting the correlations of previous stiffness observations. Self-healing of HPMC-C12 and HA100K-C12 reveals reliable syringe injectability. This supports that the material properties can be tuned without affecting the desired flow behavior of the PNP hydrogel.
This study highlights the modularity of HA and its efficacy as a crosslinking polymer for hydrogel formation. We demonstrated that the mechanical properties of HA PNP gels can be dynamically tuned through controlled modification of HA and nanoparticle formulation. Both HPMC-C12 and HA100K-C12 exhibited consistent stiffness and viscosity trends influenced by NP size and concentration, detailing HA’s lower reactivity. Moving forward, we will optimize polymer modification to further expand upon HA’s functionality, thereby advancing its application in immunotherapeutic delivery systems.

Acknowledgements (Optional): [1] Appel, E., Tibbitt, M., Webber, M. et al. (2015). Self-assembled hydrogels utilizing polymer–nanoparticle interactions. Nat Commun 6, 6295. https://doi.org/10.1038/ncomms7295
[2] Correa, S., Grosskopf, A. K., Klich, J. H., Hernandez, H. L., & Appel, E. A. (2022). Injectable Liposome-based Supramolecular Hydrogels for the Programmable Release of Multiple Protein Drugs. Matter, 5(6), 1816–1838. https://doi.org/10.1016/j.matt.2022.03.001
[3] Loebel, C., Rodell, C., Chen, M. et al. (2017). Shear-thinning and self-healing hydrogels as injectable therapeutics and for 3D printing. Nat Protoc 12, 1521–1541. https://doi.org/10.1038/nprot.2017.053
[4] Miyaji, K., Omata, S., et al. (2000). The stiffness of lymph nodes containing lung carcinoma metastases. Cancer, 80(10), 1920-1925. https://doi.org/10.1002/(SICI)1097-0142(19971115)80:10 < 1920:AID-CNCR8>3.0.CO;2-R
[5] Xu, X., Xinqiao, J., et al. (2012). Hyaluronic acid-based hydrogels: from a natural polysaccharide to complex networks. Soft Matter 8(12), 3280-3294. http://dx.doi.org/10.1039/C2SM06463D
[6] New York Structural Biology Center, NMR Department
[7] The Leong Lab for Nanotherapeutics and Stem Cell Engineering
[8] Figures made using Biorender.com