Biocompatibility and biodistribution of matrix-bound nanovesicles in vitro and in vivo

Elsevier

Available online 22 November 2022

Acta BiomaterialiaAuthor links open overlay panelAbstract

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses.

Statement of Significance

Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications.

Section snippetsINTRODUCTION

Extensive research in the field of extracellular matrix (ECM) biology and the study of bioscaffolds composed of ECM, including structure-function relationships, has shown that ECM bioscaffolds promote a constructive tissue remodeling process following injury and play an important role in restoring and/or maintaining a healthy homeostatic state [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. It has long been known that the ECM is a rich repository of biologically active signaling

MBV isolation

MBV were isolated from porcine urinary bladder matrix (UBM) ECM by enzymatic digestion [13]. Enzymatic digestion was performed using Liberase TL (highly purified Collagenase I and Collagenase II, Roche, Basel, Switzerland) in buffer (50mM Tris pH 7.5, 5mM CaCl2, 150mM NaCl) for 24 hours at room temperature on an orbital rocker. Digested ECM was then subjected to centrifugation at 10,000 x g for 30 minutes at 4°C and filtered through a 0.22-µm filter. The clarified supernatant containing the

UBM MBV are not cytotoxic and do not have an adverse effect on cellular proliferation across various cell types in vitro

Across serial dilutions of MBV ranging from 1 × 1011 particles/mL to 3.3 × 109 particles/mL, there was no significant difference in cytotoxicity compared to media-only controls in murine NIH 3T3, human PVSCs, and human SAOS-2 cells (p<0.05, Figure 1. A.). In the human MSCs, a significant increase in metabolic activity was observed at the highest dose of MBV tested (p<0.05, Figure 1. A.). 5% DMSO-induced significant decreases in cell viability compared to media across all cell types and served

DISCUSSION

The results of the present study showed pre-clinical evidence that UBM-derived MBV in vitro and in vivo are well-tolerated and are not cytotoxic in cell culture, not toxic to the whole organism, nor immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide.

While limited to only four cell types, the in vitro toxicity data presented herein represents a compilation of data to support that a broad range of MBV doses in vitro may be used for cell-based studies as they do not

FUNDING DISCLOSURE

This study was sponsored by ECM Therapeutics Inc., Warrendale PA.

Declaration of Competing Interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interest: This study was sponsored by ECM Therapeutics Inc., Warrendale PA. GSH and SFB are employees of and own stock in ECM Therapeutics Inc. The other authors have no financial conflicts or competing interests to disclose.

Acknowledgments

The authors would like to acknowledge the contributing assistance of the University of Pittsburgh Division of Laboratory Animal Research staff for animal care and support.

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© 2022 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

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