An artificial membrane binding protein-polymer surfactant nanocomplex facilitates stem cell adhesion to the cartilage extracellular matrix

Rosalia Cuahtecontzi Delint, Graham J Day, William J P Macalester, Wael Kafienah, Wenjin Xiao, Adam W Perriman

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Abstract

One of the major challenges within the emerging field of injectable stem cell therapies for articular cartilage (AC) repair is the retention of sufficient viable cell numbers at the site of injury. Even when delivered via intra-articular injection, the number of stem cells retained at the target is often low and declines rapidly over time. To address this challenge, an artificial plasma membrane binding nanocomplex was rationally designed to provide human mesenchymal stem cells (hMSCs) with increased adhesion to articular cartilage tissue. The nanocomplex comprises the extracellular matrix (ECM) binding peptide of a placenta growth factor-2 (PlGF-2) fused to a supercharged green fluorescent protein (scGFP), which was electrostatically conjugated to anionic polymer surfactant chains to yield [S-]scGFP_PlGF2. The [S-]scGFP_PlGF2 nanocomplex spontaneously inserts into the plasma membrane of hMSCs, is not cytotoxic, and does not inhibit differentiation. The nanocomplex-modified hMSCs showed a significant increase in affinity for immobilised collagen II, a key ECM protein of cartilage, in both static and dynamic cell adhesion assays. Moreover, the cells adhered strongly to bovine ex vivo articular cartilage explants resulting in high cell numbers. These findings suggest that the re-engineering of hMSC membranes with [S-]scGFP_PlGF2 could improve the efficacy of injectable stem cell-based therapies for the treatment of damaged articular cartilage.

Original languageEnglish
Article number120996
Pages (from-to)120996
JournalBiomaterials
Volume276
DOIs
Publication statusPublished - Sep 2021

Bibliographical note

Funding Information:
We would like to acknowledge the Wolfson Bioimaging Centre (BBSRC Alert 13 capital grant BB/L014181/1 ) and Dr Andrew Herman at the Flow Cytometry Facility at the University of Bristol . We also would like to acknowledge the Diamond Light Source (UK) for access to Far-UV SR–CD at beamline B23 and SR–SAXS at beamline B21. We would like to thank Dr D. Frankel for assisting with the microfluidics, Dr Thomas I. P. Green and Dr Robert C. Deller for their support for protein purification techniques, Dr Benjamin Carter for his aid in Fig. 1 . We thank the EPSRC (Early Career Fellowship EP/K026720/1 ), the UKRI (Future Leaders Fellowship MR/S016430/1) for support for Professor Adam. W. Perriman, Consejo Nacional de Ciencia y Tecnologia (CONACyT) and the Bristol Centre for Functional Nanomaterials for support for Rosalia Cuahtecontzi Delint, and Defence science and technology laboratory (Dstl) for funding Graham J. Day. Data are available at the University of Bristol data repository, data.bris, at https://doi.org/10.5523/bris.9r3ofty1lipb2n7x1lnpt3e78 .

Funding Information:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: A.W.P. is the Founder, a Director and a shareholder of CytoSeek, a company engaged in the development of cell membrane reengineering. Work in the Perriman laboratories at the University of Bristol is supported in part by CytoSeek. The remaining authors declare no competing interests.

Funding Information:
In light of the retention of structure and function by scGFP_PlGF2, the anionic polymer surfactant, S? (H19C9-Ph?(CH2CH2O)39?O?CH?COO?), was used to generate an electrostatically-stabilised polymer surfactant corona on the surface of the supercharged fusion protein [35,37,43,44] (Fig. 1). UV?vis spectroscopy from the [S?]scGFP_PlGF2 nanohybrid dispersion revealed a protein positive charge:surfactant stoichiometric ratio of at 1.4:1 (Fig. 2d), which indicated a small excess of protein-bound surfactant chains. Deconvolution of the SR?CD spectra from [S?]scGFP_PlGF2 showed a high proportion of ?-sheet secondary structure, with similar levels to that found in scGFP and scGFP_PlGF2 (Fig. 2b and Supplementary Table 3) [41]. The preservation of the protein tertiary structure was supported by fluorescence spectroscopy measurements, which illustrated the retention of scGFP fluorophore geometry, with a maximum absorbance at 487 nm and excitation at 511 nm (Fig. 2e) [39,41].We would like to acknowledge the Wolfson Bioimaging Centre (BBSRC Alert 13 capital grant BB/L014181/1) and Dr Andrew Herman at the Flow Cytometry Facility at the University of Bristol. We also would like to acknowledge the Diamond Light Source (UK) for access to Far-UV SR?CD at beamline B23 and SR?SAXS at beamline B21. We would like to thank Dr D. Frankel for assisting with the microfluidics, Dr Thomas I. P. Green and Dr Robert C. Deller for their support for protein purification techniques, Dr Benjamin Carter for his aid in Fig. 1. We thank the EPSRC (Early Career Fellowship EP/K026720/1), the UKRI (Future Leaders Fellowship MR/S016430/1) for support for Professor Adam. W. Perriman, Consejo Nacional de Ciencia y Tecnologia (CONACyT) and the Bristol Centre for Functional Nanomaterials for support for Rosalia Cuahtecontzi Delint, and Defence science and technology laboratory (Dstl) for funding Graham J. Day. Data are available at the University of Bristol data repository, data.bris, at https://doi.org/10.5523/bris.9r3ofty1lipb2n7x1lnpt3e78.

Publisher Copyright:
© 2021 The Authors

Structured keywords

  • BrisSynBio
  • Bristol BioDesign Institute

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