Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration

Gerardo Cedillo-Servin; Ana Filipa Louro; Beatriz Gamelas; Ana Meliciano; Anne Zijl; Paula M. Alves; Jos Malda; Margarida Serra; Miguel Castilho

doi:10.1016/j.bioadv.2023.213692

Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration

Gerardo Cedillo-Servin, Ana Filipa Louro, Beatriz Gamelas, Ana Meliciano, Anne Zijl, Paula M. Alves, Jos Malda, Margarida Serra, Miguel Castilho (Corresponding author)

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Abstract

Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin methacryloyl (GelMA) hydrogels could serve as sustained delivery platforms for human induced pluripotent stem cell (hiPSC)-derived EV. EV with 50-200 nm size and typical morphology were isolated from hiPSC-conditioned culture media and tested negative for common co-isolated contaminants. hiPSC-EV were then incorporated into GelMA hydrogels with or without a melt electrowritten reinforcing mesh. EV release was found to increase with GelMA concentration, as 12 % (w/v) GelMA hydrogels provided higher release rate and total release over 14 days in vitro, compared to lower hydrogel concentrations. Release profile modelling identified diffusion as a predominant release mechanism based on a Peppas-Sahlin model. To study the effect of reinforcement-dependent hydrogel mechanics on EV release, stress relaxation was assessed. Reinforcement with highly porous microfiber meshes delayed EV release by prolonging hydrogel stress relaxation and reducing the swelling ratio, thus decreasing the initial burst and overall extent of release. After release from photocrosslinked reinforced hydrogels, EV remained internalizable by human umbilical vein endothelial cells (HUVEC) over 14 days, and increased migration was observed in the first 4 h. EV and RNA cargo stability was investigated at physiological temperature in vitro, showing a sharp decrease in total RNA levels, but a stable level of endothelial migration-associated small noncoding RNAs over 14 days. Our data show that hydrogel formulation and microfiber reinforcement are superimposable approaches to modulate EV release from hydrogels, thus depicting fiber-reinforced GelMA hydrogels as tunable hiPSC-EV vehicles for controlled release systems that promote endothelial cell migration.

Original language	English
Article number	213692
Number of pages	13
Journal	Biomaterials Advances
Volume	155
DOIs	https://doi.org/10.1016/j.bioadv.2023.213692
Publication status	Published - Dec 2023

Funding

The authors acknowledge Andrei Hrynevich for technical support in melt electrowriting, and A.L. Sousa and E.M. Tranfield from the Electron Microscopy Facility at the IGC for the transmission electron microscopy work. The authors acknowledge financial support from the European Union Horizon 2020 program through project BRAV3 (ID: 874827 ) and from Fundação para a Ciência e a Tecnologia (FCT) through project EXCELERATE ( DOI 10.54499/2022.10467.PTDC ). This work was funded by FCT /Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health ( UIDB/04462/2020 and UIDP/04462/2020 ) and the Associate Laboratory LS4FUTURE ( LA/P/0087/2020 ). A.F.L. and A.M. were financed by FCT under Grant No. PD/BD/139078/2018 and 2023.04995.BD , respectively. M.C. acknowledges financial support from the Reprint project ( OCENW.XS5.161 ) by The Netherlands Organization for Scientific Research .

Funders	Funder number
European Union's Horizon 2020 - Research and Innovation Framework Programme	874827
Portuguese Fundação para a Ciência e a Tecnologia
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Ministério da Ciência, Tecnologia e Ensino Superior	2023.04995, OCENW.XS5.161, UIDB/04462/2020, LA/P/0087/2020, PD/BD/139078/2018, UIDP/04462/2020

Keywords

Angiogenesis
Controlled release
Extracellular vesicles
Hydrogel encapsulation
Melt electrowriting

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Cedillo-Servin, G., Louro, A. F., Gamelas, B., Meliciano, A., Zijl, A., Alves, P. M., Malda, J., Serra, M., & Castilho, M. (2023). Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration. Biomaterials Advances, 155, Article 213692. https://doi.org/10.1016/j.bioadv.2023.213692

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title = "Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration",

abstract = "Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin methacryloyl (GelMA) hydrogels could serve as sustained delivery platforms for human induced pluripotent stem cell (hiPSC)-derived EV. EV with 50-200 nm size and typical morphology were isolated from hiPSC-conditioned culture media and tested negative for common co-isolated contaminants. hiPSC-EV were then incorporated into GelMA hydrogels with or without a melt electrowritten reinforcing mesh. EV release was found to increase with GelMA concentration, as 12 % (w/v) GelMA hydrogels provided higher release rate and total release over 14 days in vitro, compared to lower hydrogel concentrations. Release profile modelling identified diffusion as a predominant release mechanism based on a Peppas-Sahlin model. To study the effect of reinforcement-dependent hydrogel mechanics on EV release, stress relaxation was assessed. Reinforcement with highly porous microfiber meshes delayed EV release by prolonging hydrogel stress relaxation and reducing the swelling ratio, thus decreasing the initial burst and overall extent of release. After release from photocrosslinked reinforced hydrogels, EV remained internalizable by human umbilical vein endothelial cells (HUVEC) over 14 days, and increased migration was observed in the first 4 h. EV and RNA cargo stability was investigated at physiological temperature in vitro, showing a sharp decrease in total RNA levels, but a stable level of endothelial migration-associated small noncoding RNAs over 14 days. Our data show that hydrogel formulation and microfiber reinforcement are superimposable approaches to modulate EV release from hydrogels, thus depicting fiber-reinforced GelMA hydrogels as tunable hiPSC-EV vehicles for controlled release systems that promote endothelial cell migration.",

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AU - Louro, Ana Filipa

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AU - Meliciano, Ana

AU - Zijl, Anne

AU - Alves, Paula M.

AU - Malda, Jos

AU - Serra, Margarida

AU - Castilho, Miguel

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AB - Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin methacryloyl (GelMA) hydrogels could serve as sustained delivery platforms for human induced pluripotent stem cell (hiPSC)-derived EV. EV with 50-200 nm size and typical morphology were isolated from hiPSC-conditioned culture media and tested negative for common co-isolated contaminants. hiPSC-EV were then incorporated into GelMA hydrogels with or without a melt electrowritten reinforcing mesh. EV release was found to increase with GelMA concentration, as 12 % (w/v) GelMA hydrogels provided higher release rate and total release over 14 days in vitro, compared to lower hydrogel concentrations. Release profile modelling identified diffusion as a predominant release mechanism based on a Peppas-Sahlin model. To study the effect of reinforcement-dependent hydrogel mechanics on EV release, stress relaxation was assessed. Reinforcement with highly porous microfiber meshes delayed EV release by prolonging hydrogel stress relaxation and reducing the swelling ratio, thus decreasing the initial burst and overall extent of release. After release from photocrosslinked reinforced hydrogels, EV remained internalizable by human umbilical vein endothelial cells (HUVEC) over 14 days, and increased migration was observed in the first 4 h. EV and RNA cargo stability was investigated at physiological temperature in vitro, showing a sharp decrease in total RNA levels, but a stable level of endothelial migration-associated small noncoding RNAs over 14 days. Our data show that hydrogel formulation and microfiber reinforcement are superimposable approaches to modulate EV release from hydrogels, thus depicting fiber-reinforced GelMA hydrogels as tunable hiPSC-EV vehicles for controlled release systems that promote endothelial cell migration.

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Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration

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