(F-239) Nanoparticle Shells for Enhanced Therapeutic Efficacy of Mesenchymal Stem Cell derived Extracellular Vesicles

Introduction:: Mesenchymal stem cells (MSCs) support tissue formation, blood vessel growth, release various growth factors, suppress inflammation, and prevent fibrosis [1]. Extracellular vesicles (EVs) are secreted by mammalian cells for intercellular communication [2]. They contain bioactive molecules such as lipids, proteins, mRNAs, and microRNAs unique to the cell of origin [3, 4]. EVs recapitulate the broad therapeutic effects attributed to MSCs [5, 6]. As an acellular MSC byproduct, EVs can readily circulate through organs, elicit a minimal immune response, avoid phagocytosis, and stimulate cellular responses [7]. The administration of EVs in vivo results in rapid clearance from blood circulation. This work's primary goal is to augment EVs' therapeutic effect by prolonging their retention at the injury site and preventing premature clearance. To achieve this goal, we focus on gelatin, a widely studied delivery vehicle for small biomolecules, because of its many biological advantages [8]. This work uses the desolvation method to prepare gelatin nanoparticles (GNPs) with different surface charges [9]. This work aims to electrostatically link EVs with GNPs so that the GNPs can form a protective shell around the EVs. Thus, the GNPs can shield the EVs from damage and prevent their rapid clearance.


Materials and Methods:: Gelatin nanoparticles were prepared using desolvation method and crosslinked using carbodiimide chemistry. Type A or type B gelatin in either acidic or alkaline conditions was subjected to desolvation to obtain GNPs of different surface charges. The GNPs were frozen, lyophilized, resuspended in deionized water, and sonicated prior to characterization. The GNPs were analyzed by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments) to determine their size and zeta potentialThe cytotoxicity of GNPs was assessed by a lactate dehydrogenase (LDH) cytotoxicity assay (Cayman Chemical) following 24-hour incubation with rat dermal fibroblasts. EVs were isolated from murine bone-marrow-derived MSCs using the ExoQuick Ultra method and analyzed for size using nanoparticle tracking analysis (NTA). GNPs were mixed with EVs in different ratios, and the zeta potential of the individual and combined solutions was measured.

Results, Conclusions, and Discussions:: GNPs derived from Type A gelatin under acidic pH conditions showed an average size of 74.6 nm and a zeta potential of 8.5 mV. GNPs derived from type B alkaline under alkaline pH conditions showed an average size of 60.9 nm and a zeta potential of -4.6 mV. Both types A and B GNPs did not cause cytotoxicity in fibroblasts. MSC-EVs were 181.3 nm in size with a zeta potential of -14.6 mV. The combined solution of GNP: EV (400:1) resulted in a complete inversion of the zeta potential to a positive value of 10.33 mV. This result suggests complete coating of EVs by GNPs. However, when GNP: EV ratio was (100:1), the zeta potential value only became less negative (i.e., -1.72), suggesting incomplete coating of negatively charged EVs with GNPs.


We have synthesized positively and negatively charged GNPs that can be electrostatically linked to oppositely charged biomolecules. These GNPs are non-cytotoxic. Ongoing studies are investigating the effect of GNP, EV, and GNP: EV conjugates on macrophage polarization in vitro. Future studies will determine the efficacy of GNP-conjugated EVs in modulating inflammation and fibrosis in a rodent model of muscle injury.


Acknowledgements (Optional): : This work is supported by Saint Louis University Institute for Drug and Biotherapeutic Innovation (SLU-IDBI).

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