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    Tissue Engineering

    (H-311) Bioprinting PLGA nanoparticle-laden biomaterial inks for delivery of therapeutics via intrathecal injection

    Thursday, October 12, 2023
    2:00 PM – 3:00 PM PDT
    Location: Exhibit Hall - Row H - Poster # 311
    Introduction::

    Macromolecular therapeutics promise to combat debilitating neurological diseases such as Alzheimer's and multiple sclerosis. Despite their potential, the limited transport of larger molecules to deep tissue sites and inadequate targeting of the central nervous system (CNS) necessitates the development of novel therapeutic approaches [1]. These challenges are attributed, in part, to the semi-permeable nature of the Blood Brain Barrier (BBB) [2]. One potential approach to circumventing the BBB is an intrathecal (IT) injection into the Cerebral Spinal Fluid. For example, injectable hydrogels, loaded with PLGA nanoparticles and agarose, were developed to encapsulate desired macromolecules for IT injections, facilitating more effective delivery through the CNS [3]. In this context, our laboratory is dedicated to synthesizing and studying the delivery of macromolecular therapeutics from nanoparticle-laden hydrogels comprising gelatin methacryloyl (GelMA) and methacrylated hyaluronic acid (HAMA). As we are particularly interested in the injectability of this formulation and its potential to serve as a basis for a small tissue model, we sought to assess the novel material's performance in a bioprinting setting. We hypothesize that a biomaterial ink, designed to mimic the physical and chemical composition of the brain extracellular matrix (ECM) and loaded with PLGA nanoparticles, could be potent in treating neurological diseases via IT injections.



    Materials and Methods:: GelMA was synthesized using the previously published "One-Pot" method [4]. 1H-NMR determined the material's purity, and the degree of modification was assessed using TNBS and Fe(III)-hydroxamate assays. For the synthesis of HAMA, sodium hyaluronate was dissolved in ultrapure water. Methacrylic anhydride was then added to the aqueous solution, and the pH was discretely adjusted to 8.5-9 with NaOH. The resulting solution was dialyzed in DI water, lyophilized, and analyzed with 1H NMR. PLGA nanoparticles were synthesized via nanoprecipitation; PLGA was dissolved in acetone and added dropwise to a poly(vinyl alcohol) solution. GelMA (G), HAMA (H), and PLGA (P) were then rehydrated in phosphate-buffered saline (PBS) at % w/v of 10, 2, and 1, respectively, to form the final material. Scaffolds composed of G+H and G+H+P were fabricated with an Allevi 1 bioprinter. The extrudability of these two biomaterial inks was assessed through filament drop tests under various pressure-temperature conditions. Filament uniformity and fusion tests were performed and quantitatively analyzed with ImageJ to evaluate the fidelity of the printed structures. A separate Design of Experiments (DOE) measured the impact of temperature, pressure, needle length, print speed, and material type on pore viability, i.e., printability. ImageJ was used to assess the number of viable pores for this analysis, defining specific particle size and circularity ranges. "Viable" pores were designated as having an area of 50 mm2 to 600 mm2 and a circularity range of 0.45-1.0.

    Results, Conclusions, and Discussions:: GelMA and HAMA were successfully synthesized based on the characterization of the materials via 1H-NMR; GelMA by analyzing the degree of methylation and HAMA by calculating the grafting ratio of methacrylate. We found that the addition of PLGA nanoparticles did not have a statistically significant impact on filament uniformity or filament fusion, indicating that nanoparticle addition did not reduce the printability of the final formulation. Lattices had viable pores across various printing conditions at near-ambient temperatures. Our DOE indicated that several individual printing parameters and combinations of printing parameters were statistically significant. In order of decreasing influence, we found that the needle length, pneumatic pressure, and nozzle speed had the most significant influence on the printability of the final scaffold. We conclude that biomaterial inks of GelMA, HAMA, and PLGA nanoparticles have considerable potential as hydrogel carriers for macromolecular therapeutic delivery. We plan to evaluate how the addition of nanoparticles impacts the compressive modulus, viscosity, and swelling of the final hydrogel. We will then employ this in tissue culture and murine models with patient-derived glioblastoma cells to begin evaluating the therapeutic potential of the system.

    Acknowledgements (Optional): :

    The authors acknowledge the contributions of Jon Smart for his previous development of bioprinting protocols on the Allevi 1 bioprinter.


    Funding: This work was supported by the Robert A. Welch Foundation (AF-0005), the Sam Taylor Foundation, and a generous gift to Southwestern University from Bob and Annie Graham.



    References (Optional): :

    [1] Ajeeb, Rana and Clegg, John R. “Intrathecal delivery of macromolecules: Clinical status and emerging technologies”.  Advanced Drug Delivery Reviews (2023).


    [2] DeStefano, Jackson G., et al. "Benchmarking in vitro tissue-engineered blood–brain barrier models." Fluids and Barriers of the CNS 15 (2018).


    [3] Yang, Bo, et al. "Fabrication of 3D GelMA scaffolds using agarose microgel embedded printing." Micromachines 13.3 (2022).


    [4] Shirahama, Hitomi, et al. "Precise tuning of facile one-pot gelatin methacryloyl (GelMA) synthesis." Scientific Reports 6.1 (2016).