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Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy

Nature Biomedical Engineering volume 7, pages 887–900 (2023)Cite this article

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Abstract

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.

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Fig. 1: CNP generates large quantities of EVs loaded with COL1A1 mRNA.
Fig. 2: In vivo kinetics of COL1A1-EV mRNA delivery and protein formation in murine skin.
Fig. 3: COL1A1-EV mRNA delivery reduces dermal wrinkles in a UV-irradiation photoaging model.
Fig. 4: Histologic analysis demonstrates collagen replacement in the dermis of photoaged mice treated with COL1A1-EV mRNA.
Fig. 5: A microneedle delivery system for improved EV distribution in tissue.
Fig. 6: COL1A1-EV delivery via COL1A1-EV microneedle improves long-term treatment of photoaged skin.
Fig. 7: COL1A1-EV mRNA delivery via COL1A1-EV MN results in long-term protein replacement in skin.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Source data for the figures are available from figshare at https://figshare.com/articles/dataset/SD_FIGS_xlsx/21514641. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request.

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Acknowledgements

We thank C. Wogan of the Division of Radiation Oncology, MD Anderson Cancer Center, for editorial assistance.

Author information

Author notes

  1. These authors contributed equally: Yi You, Yu Tian and Zhaogang Yang.

Authors and Affiliations

  1. Peking University Shenzhen Graduate School, Shenzhen, China

    Yi You, Yu Tian, Yuhao Tong, Andreanne Poppy Estania, Jianhong Cao, Wei-Hsiang Hsu, Yutong Liu & Andrew S. Lee

  2. Institute of Cancer Research, Shenzhen Bay Laboratory, Shenzhen, China

    Yi You, Yu Tian, Yuhao Tong, Andreanne Poppy Estania, Jianhong Cao, Wei-Hsiang Hsu, Yutong Liu & Andrew S. Lee

  3. Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Zhaogang Yang, Benjamin R. Schrank, JongHoon Ha, Shiyan Dong, Xuefeng Li, Yifan Wang & Wen Jiang

  4. School of Life Sciences, Jilin University, Changchun, China

    Zhaogang Yang, Ziwei Li, Yarong Zhao, Huan Zhang & Lesheng Teng

  5. Spot Biosystems Ltd., Palo Alto, CA, USA

    Junfeng Shi, Kwang Joo Kwak & Ly James Lee

  6. Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA

    Chi-Ling Chiang

  7. Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Kristin Huntoon, DaeYong Lee, Thomas D. Gallup & Betty Y. S. Kim

  8. The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital; State Key Laboratory of Respiratory Disease, Sino-French Hoffmann Institute, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China

    Xuefeng Li

  9. Fuwai Hospital Chinese Academy of Medical Sciences Shenzhen, Shenzhen Key Laboratory of Cardiovascular Disease, State Key Laboratory of Cardiovascular Disease and Peking Union Medical College, Shenzhen, China

    Wen-Jing Lu & Feng Lan

  10. Beijing Laboratory for Cardiovascular Precision Medicine, The Key Laboratory of Biomedical Engineering for Cardiovascular Disease Research, The Key Laboratory of Remodeling‐Related Cardiovascular Disease, Ministry of Education, Beijing Anzhen Hospital, Capital Medical University, Beijing, China

    Wen-Jing Lu

  11. Department of Dermatology, Vanderbilt University Medical Center, Nashville, TN, USA

    Eman Bahrani

  12. Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, USA

    Kristin Huntoon, DaeYong Lee, Thomas D. Gallup & Betty Y. S. Kim

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  1. Yi You
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Contributions

A.S.L. and F.L. conceived the work; A.S.L., W.J., F.L., Z.Y. and B.Y.S.K. supervised the research; A.S.L., J.S., L.J.L. and K.J.K. developed the technology; A.S.L., Y.Y., Y.T., F.L., W.J., Z.Y., L.T. and B.Y.S.K. designed the experiments; A.S.L., L.J.L., Z.Y., Y.T., Y.Y., W.J., F.L., B.Y.S.K., K.J.K., J.S., B.S., K.H., D.L., T.G., L.T., W.-J.L. and E.B. provided intellectual input; A.S.L., L.J.L., Z.Y., W.J., J.S., S.D., E.B. and B.Y.S.K. wrote the manuscript, with input from all authors; Y.Y., Y.T., J.S., K.J.K., Y.T., A.P.E., J.C., C.-L.C., W.-H.H. Y.L., Z.L., Y.Z., H.Z., X.L., Y.W. and J.H. conducted experiments; Y.Y., Y.T., Z.Y. and A.P.E. prepared figures and videos.

Corresponding authors

Correspondence to Feng Lan, Betty Y. S. Kim or Andrew S. Lee.

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Competing interests

A.S.L. and L.J.L. are consultants and shareholders of Spot Biosystems, Ltd. J.S. and K.J.K are employees of Spot Biosystems, Ltd.

Peer review

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Nature Biomedical Engineering thanks Sun Hwa Kim, Chuanbin Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 In vitro delivery of COL1A1 mRNA-containing EVs.

a, Fluorescence images of serum-starved nHDFs treated with COL1A1-GFP EVs and protein translated from delivered COL1A1-GFP mRNA after 48 h. Scale bar, 100 µm. b, Fluorescence intensity of cells treated with COL1A1-EVs (n = 3 for all groups, ***P < 0.001 Control vs COL1A1-EVs) in 48 h. c, RT-qPCR shows higher collagen mRNA transcript levels after in vitro delivery of COL1A1 mRNA from EVs (n = 3 for all groups, ***P < 0.001 Control vs COL1A1-EVs) in 48 h. d, Western blots show elevated COL1A1 protein in treated fibroblasts (n = 3 for all groups, **P = 0.001 Control vs COL1A1-EVs).e, Pro-collagen I collected from supernatant and detected by ELISA (n = 3 for all groups, ***P < 0.001 Control vs COL1A1-EVs) in 48 h. All data are from three independent experiments and are presented as means ± SEM; two-sided Student’s t tests were used for the comparisons in (b–e).

Extended Data Fig. 2 Skin plaster assessment of dorsal skin after COL1A1-EV treatment.

a, Microscopic observations of dorsal skin and skin replicas. Scale bar, 5 mm. b, Mean wrinkle depth in skin replicas (n = 4 for all groups, ※※※P < 0.001 COL1A1-EVs vs Saline; **P = 0.0025 COL1A1-LNPs vs Saline; +++P < 0.001 COL1A1-EVs vs COL1A1-LNPs). c, Mean wrinkle length analysed on skin replicas (n = 4 for all groups, #P = 0.0203 Unloaded EVs vs Saline; *P = 0.0405 COL1A1-LNPs vs Saline; ※※※P < 0.001 COL1A1-EVs vs Saline; ++P = 0.0015 COL1A1-EVs vs COL1A1-LNPs).All data are from three independent experiments and are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for the comparisons in (b, c). NS, not significant.

Extended Data Fig. 3 Assessment of in vivo immunogenicity of COL1A1-LNPs and COL1A1-EVs.

a, Skin samples from the mice injected with a single dose injection of 22E9 copy number COL1A1 mRNA in COL1A1-EVs and COL1A1-LNPs were harvested after 24 h. Skin samples of mice were analysed by flow cytometry, for b, leukocyte cell percentage, and c, neutrophil percentage (n = 3 for all groups, **P = 0.0034 COL1A1-LNPs vs sham for %CD45 + cells; ***P < 0.001 COL1A1-LNPs vs Sham for %neutrophils among CD45 + cells; NS, not significant). d, Protein quantification via ELISA for IFN-γ, IL-1β, IL-6 and TNF-α shows elevation of inflammatory cytokines in the COL1A1-LNPs group as compared to COL1A1-EVs (n = 3 for all groups, **P = 0.0074 COL1A1-LNPs vs Sham for IFN-γ; #P = 0.0333 COL1A1-EVs vs Sham and ***P < 0.001 COL1A1-LNPs vs Sham for IL-1β; ***P < 0.001 COL1A1-LNPs vs Sham for IL-6; *P = 0.0146 COL1A1-LNPs vs Sham for TNF-α; NS, not significant). e, Representative immunostaining images for TNF-α and (f) IL-6 after injected with COL1A1-EVs and COL1A1-LNPs. Scale bar, 100 µm. All data are from three independent experiments and are presented as means ± SEM. One-way ANOVA was used for the comparisons in (b–d).

Extended Data Fig. 4 Return of dermal wrinkles to baseline after treatment with low dose COL1A1-EVs.

a, Wrinkles were tracked on days 0, 4, 7, 14, 21, 28, 35, 42, 49, and 56 d after the indicated treatments (5 low-dose injections of COL1A1-EVs (2.7E9 copy number COL1A1 mRNA), COL1A1-LNPs (2.7E9 copy number COL1A1 mRNA), unloaded EVs, 0.05% retinoic acid [RA], saline). n = 4, Scale bar, 5 mm. Female nude mice that were not exposed to UV comprised the sham group. b, Numbers of wrinkles on the dorsal skin of the mice over time. (n = 4 for all groups; **P = 0.008 COL1A1-EVs vs Saline at day 7; **P = 0.004 COL1A1-EVs vs Saline at day 21, **P = 0.001 COL1A1-EVs vs Saline at day 35; ***P < 0.001 COL1A1-EVs vs Saline at days 14, 28, 42, and 49; †P = 0.025 RA vs Saline at day 21; ††P = 0.007 RA vs Saline at day 28; ##P = 0.0071 Unloaded EVs vs Saline at day 14; ##P = 0.004 Unloaded EVs vs Saline at day 21; ##P = 0.002 Unloaded EVs vs Saline at day 28; #P = 0.015 Unloaded EVs vs Saline at day 35; ※※P = 0.0053 COL1A1-LNPs vs Saline at day 14; ※※P = 0.0041 COL1A1-LNPs vs Saline at day 21; ※P = 0.017 COL1A1-LNPs vs Saline at day 28; ※P = 0.022 COL1A1-LNPs vs Saline at day 35). c, Total wrinkle area (n = 4 for all groups, *P = 0.012 COL1A1-EVs vs Saline at day 7; ***P < 0.001 COL1A1-EVs vs Saline at days 14, 21, 28 and 35; *P = 0.015 COL1A1-EVs vs Saline at day 42; ††P = 0.008 RA vs Saline at day 14; ††P = 0.007 RA vs Saline at days 21 and 35;††P = 0.005 RA vs Saline at day 28; #P = 0.012 Unloaded EVs vs Saline at day 14; #P = 0.035 Unloaded EVs vs Saline at day 21; ##P = 0.002 Unloaded EVs vs Saline at day 28; ※P = 0.062 COL1A1-LNPs vs Saline at day 14; ※P = 0.039 COL1A1-LNPs vs Saline at day 21; ※※P = 0.0027 COL1A1-LNPs vs Saline at day 28; ※※P = 0.046 COL1A1-LNPs vs Saline at day 35). All data are from three independent experiments and are presented as means ± SEM. Two-way ANOVA was used for the comparisons in (b, c).

Extended Data Fig. 5 Evaluation of long term COL1A1-EV MN dermal wrinkle treatment by skin replica plaster.

a–c, Microscopic observation of dorsal skin and skin replica at 1 month, 2 months, and 3 months after treatment. Scale bar, 5 mm. d, e Quantification of mean wrinkle length (n = 4 for all groups, ***P < 0.001 COL1A1-EV MN vs Saline at 1 month and 2 months; ###P < 0.001 Needle injection vs Saline at 1 month; ††P = 0.031 HA MN vs Saline at 1 month;※※P = 0.029 Unloaded EV MN vs Saline at 1 month) and mean wrinkle depth (n = 4 for all groups, ***P < 0.001 COL1A1-EV MN vs Saline at 1 month; **P = 0.001 COL1A1-EV MN vs Saline at 2 months; Needle injection vs Saline not significant at 1 month; HA MN vs Saline not significant at 1 month) from skin replicas. All data are from three independent experiments and are presented as means ± SEM. Two-way ANOVA was used for the comparisons in (d, e).

Extended Data Fig. 6 Maintenance of wrinkle treatment via serial injection of COL1A1-EVs and COL1A1-EV MN.

a, After 8 weeks of UV irradiated photoaging, wrinkles were tracked for mice treated every 30 days with 1) saline, 2) COL1A1-EVs, and 3) COL1A1-EV MN on days 0, 4, 7, 14, 21, 28, 49, 70, and 91 (COL1A1-EVs, COL1A1-EV MN, Saline). n = 4, Scale bar, 5 mm. b, Total wrinkle number (n = 4 for all groups; *P = 0.019 COL1A1-EV MN vs Saline at day 4; #P = 0.034 COL1A1-EVs vs Saline, *P = 0.031 COL1A1-EV MN vs Saline at day 7; #P = 0.030 COL1A1-EVs vs Saline, **P = 0.008 COL1A1-EV MN vs Saline at day 14; **P = 0.008 COL1A1-EV MN vs Saline at day 21; *P = 0.025 COL1A1-EV MN vs Saline at day 28; **P = 0.006 COL1A1-EV MN vs Saline at day 35; #P = 0.031 COL1A1-EVs vs Saline, *P = 0.030 COL1A1-EV MN vs Saline at day 42; #P = 0.044 COL1A1-EVs vs Saline, *P = 0.016 COL1A1-EV MN vs Saline at day 49; *P = 0.016 COL1A1-EV MN vs Saline at day 56; #P = 0.017 COL1A1-EVs vs Saline, *P = 0.020 COL1A1-EV MN vs Saline at day 63; *P = 0.018 COL1A1-EV MN vs Saline at day 70; #P = 0.011 COL1A1-EVs vs Saline, **P = 0.006 COL1A1-EV MN vs Saline at day 77; #P = 0.028 COL1A1-EVs vs Saline, *P = 0.037 COL1A1-EV MN vs Saline at day 84; #P = 0.043 COL1A1-EVs vs Saline, *P = 0.043 COL1A1-EV MN vs Saline at day 91) and c, wrinkle area on the dorsal skin of the mice during 90 day study window (n = 4 for all groups; #P = 0.012 COL1A1-EVs vs Saline, **P = 0.005 COL1A1-EV MN vs Saline at day7; #P = 0.010 COL1A1-EVs vs Saline, *P = 0.048 COL1A1-EV MN vs Saline at day 14; #P = 0.023 COL1A1-EVs vs Saline, *P = 0.021 COL1A1-EV MN vs Saline at day 21; *P = 0.022 COL1A1-EV MN vs Saline at day 28; *P = 0.046 COL1A1-EV MN vs Saline at day 35; #P = 0.019 COL1A1-EVs vs Saline, **P = 0.009 COL1A1-EV MN vs Saline at day 42; #P = 0.042 COL1A1-EVs vs Saline, *P = 0.030 COL1A1-EV MN vs Saline at day 49; *P = 0.030 COL1A1-EV MN vs Saline at day63; *P = 0.029 COL1A1-EV MN vs Saline at day 70; #P = 0.048 COL1A1-EVs vs Saline, *P = 0.022 COL1A1-EV MN vs Saline at day 77; #P = 0.040 COL1A1-EVs vs Saline, *P = 0.027 COL1A1-EV MN vs Saline at day 84; *P = 0.045 COL1A1-EV MN vs Saline at day 91). All data are from three independent experiments and are presented as means ± SEM. Two-way ANOVA was used for the comparisons in (b, c).

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Supplementary Video 1

Microneedles delivering HA EVs on mouse skin.

Supplementary Video 2

Ex vivo time course of the dissolution of the tips of microneedles delivering HA EVs into the skin.

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You, Y., Tian, Y., Yang, Z. et al. Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy. Nat. Biomed. Eng 7, 887–900 (2023). https://doi.org/10.1038/s41551-022-00989-w

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