Assistant Professor Boston University, United States
Introduction: Local delivery of bioactive proteins has been explored as part of experimental treatments for central nervous system (CNS) disorders1, but clinical translation has been impeded by delivery challenges2,3. Biomaterial-based drug delivery may provide spatiotemporal controlled release of proteins that optimize therapeutic effects while minimizing off-target neurotoxicity but several challenges such as retention of bioactivity and minimizing neural tissue disruption remain to be addressed with current technologies4. In this work, we developed and tested a platform for protein delivery to the CNS using a trehalose-based complex coacervate system. Coacervates are small liquid droplets formed via ionic complexation-induced liquid-liquid phase separation5. We hypothesized that trehalose-based coacervates: (i) can encapsulate and stabilize high doses of proteins; (ii) enable tunable prolonged release of proteins; (iii) are readily dispersible within CNS tissue by non-invasive injection causing minimal disruption to healthy neural tissue; and (iv) would evoke a minimal foreign body response (FBR) such that proteins can be delivered predominantly to neurons and glia rather than infiltrating immune cells.
Materials and Methods: Trimethylolpropane tris(3-mercaptopropionate) (TMPTMP), 2-Carboxyethyl acrylate, 3-Sulfopropyl acrylate potassium salt, [2-(Acryloyloxy)ethyl] trimethylammonium chloride and model proteins were from Sigma-Aldrich. 2-(Dimethylamino)ethyl acrylate, N,N-Diisopropylethylamine (DIEA) and 2,2,2-Trifluoroethyl acrylate were from TCI Chemicals. Novozym 435 (N435) was a gift from Novozymes. Nerve growth factor (NGF) and ELISA kit were from Peprotech. C57BL/6J mice were from Jackson Laboratories. Trehalose diacrylate (tre-da) was synthesized by enzymatic transesterification using N435. Poly-cationic or -anionic branched oligomers were synthesized by DIEA catalyzed thiol-ene Michael addition of TMPTMP, tre-da and charged mono-acrylates as endcaps and characterized by NMR and FTIR. Coacervates were formed by mixing oppositely charged aqueous soluble oligomers in buffered saline. Coacervate size and loading was evaluated by light and fluorescence microscopy and microplate reader assays. NGF loading was measured by ELISA. Coacervate cytocompatibility was tested with neural progenitor cells in vitro while in vivo injection into mouse striatum enabled evaluation of FBR and bioactive delivery outcomes by immunohistochemistry.
Results & Conclusions: Tre-da was synthesized and purified by flash chromatography at high yields (Fig. 1a). Branched oligomers were synthesized with precise control over branching functionality and charge density through tuned stoichiometry of constituent monomers. Branching functionality and endcap determined pH- and salt-dependent solubility of oligomers, such that increasing the amount of charge with pH adjustment and/or decreased branching increased aqueous solubility. Mixing of soluble oppositely charged oligomers formed coacervates with composition-dependent variations in size and morphology (Fig. 1b, c). Soluble oligomers with the best trehalose incorporation and biocompatibility were used in coacervate protein studies. Coacervates encapsulated diverse model proteins at high efficiencies (50-99%) (Fig. 1d) and enabled tunable protein release over 3-14 days in vitro by adjusting formulation parameters. Coacervates stabilized a model enzyme under physiological conditions compared to its solution and were cytocompatible in vitro. Injection of NGF or biotinylated dextran amines-loaded coacervates into mouse striatum caused minimal FBR and retained bioactive cargo more locally around the injection site compared to cargo solution (Fig. 1e).
In conclusion, we synthesized trehalose-based coacervates using a library of oligomers with different branching functionalities and endcap chemical functionalities. Coacervates enable high loading and prolonged release of proteins while supporting enhanced protein bioactivity during release. In vitro assays and preliminary in vivo studies show that our coacervates are well tolerated in the CNS and support prolonged protein retention and bioactivity where injected.
Acknowledgements: We would like to thank Boston University Core Facilities. We are grateful to the National Science Foundation for the purchase of the NMR (CHE0619339). This work was supported by funding from Boston University (TMO) and the Wings for Life Foundation (TMO).
References: [1] Thorne, R.G. & Frey, W.H. Clin Pharmacokinet. (2001). 40(12): p. 907-946. [2] Arvidsson, A., et al. Neurobiol. Dis. (2003). 14(3): p. 542-556. [3] Luz, M., et al. Neurotoxicology (2016). 52: p. 46-56. [4] Kearney, C.J. & Mooney, D.J. Nat. Mater. (2013). 12: p. 1004. [5] Astoricchio, E., et al. Trends in Biochem. Sci. (2020). 45(8): p. 706-717.
