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

nature biomedical engineering Article https://doi.org/10.1038/s41551-022-00989-w Intradermally delivered mRNAencapsulating extracellular vesicles for collagen-replacement therapy Received: 1 April 2022 Accepted: 18 November 2022 Published online: xx xx xxxx Check for updates Yi You1,2,13, Yu Tian1,2,13, Zhaogang Yang3,4,13, Junfeng Shi5, Kwang Joo Kwak5, Yuhao Tong1,2, Andreanne Poppy Estania1,2, Jianhong Cao1,2, Wei-Hsiang Hsu 1,2, Yutong Liu1,2, Chi-Ling Chiang6, Benjamin R. Schrank 3, Kristin Huntoon 7,12, DaeYong Lee7,12, Ziwei Li4, Yarong Zhao4, Huan Zhang4, Thomas D. Gallup 7,12, JongHoon Ha3, Shiyan Dong 3, Xuefeng Li 3,8, Yifan Wang 3, Wen-Jing Lu9,10, Eman Bahrani11, Ly James Lee 5, Lesheng Teng 4, Wen Jiang 3, Feng Lan9 , Betty Y. S. Kim 7,12 & Andrew S. Lee1,2 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. Recent developments in messenger RNA-modification techniques have enhanced the therapeutic efficiency of mRNA delivery and its potential for near-term clinical applications, including protein-replacement therapy and vaccination against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus1,2. However, the intrinsic inability and potential immunogenicity of mRNAs require that they be encapsulated within delivery vehicles. Current mRNA-delivery modalities centre on the usage of lipid nanoparticle (LNP) carriers for encapsulation and Peking University Shenzhen Graduate School, Shenzhen, China. 2Institute of Cancer Research, Shenzhen Bay Laboratory, Shenzhen, China. Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 4School of Life Sciences, Jilin University, Changchun, China. 5Spot Biosystems Ltd., Palo Alto, CA, USA. 6Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA. 7Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 8The 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. 9Fuwai 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. 10Beijing 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. 11Department of Dermatology, Vanderbilt University Medical Center, Nashville, TN, USA. 12Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, USA. 13These authors contributed equally: Yi You, Yu Tian and Zhaogang Yang. e-mail: ; bykim@ mdanderson.org; 1 3 Nature Biomedical Engineering Article transport3,4. However, LNPs pose several major challenges, including cytotoxicity, poor biodistribution, lack of target specificity and immunogenicity. These problems may be caused by the requirement for the surface PEGylation (PEG stands for poly(ethylene glycol)) of LNPs to improve their circulatory half-life and to reduce non-specific clearance5,6. Notably, the administration of LNPs in people has been linked to anaphylaxis, hypersensitivity and autoimmune adverse events7,8. Therefore, the identification of mRNA carriers that can overcome some of these LNP-associated challenges would be helpful for the further development of mRNA-based therapeutics. Extracellular vesicles (EVs), including exosomes and microvesicles, play a major role in the transport of biomolecules and nucleic acids, including mRNAs, within the human body9–11. As a result, in recent years, EVs have emerged as promising carriers for nucleic-acid-based therapeutics owing to their intrinsic biocompatibility, their ability to cross physiological barriers and their low immunogenicity12,13. Unlike LNPs, EVs, including exosomes, are endogenously produced by the body’s cells and lead to lower levels of inflammatory responses. Moreover, strategies to cheaply and easily produce large quantities of exosomes have been developed. We previously reported a cellular nanoporation (CNP) method in which transient nanometric pores were created on the surface of source cells to allow for the large-scale loading of full-transcript mRNAs into secreted EVs14. Here, by using a mouse model of acute photoaging that closely mimics the pathophysiological features of aging-damaged skin in humans15, we show the utility of exosome-based COL1A1 mRNA therapy to replace dermal collagen-protein loss as an anti-aging treatment for photoaged skin. To improve the efficiency of mRNA delivery and retention, we also show that the delivery of collagen mRNA via a hyaluronic acid (HA) microneedle (COL1A1-EV MN) patch allows for a more efficient distribution of mRNA in the dermis, resulting in durable collagen-protein engraftment and in an improved treatment of wrinkles in photoaged skin. Results Preparation and in vitro delivery of COL1A1 mRNA-containing EVs Dermal atrophy owing to irreversible loss of collagen is a hallmark of skin aging16,17. Numerous methods have aimed to restore loss of collagen protein in skin, ranging from over the counter and pharmaceutical approaches (antioxidants18–20, retinoids21, peptides22,23) to medical devices (that is, laser therapy24 and synthetic dermal fillers25,26). However, none of these existing technologies have been able to achieve long-term endogenous collagen replacement to maintain skin strength, firmness and elasticity over time27–29. Stimulating fibroblasts responsible for synthesizing collagen proteins can also be an effective way for short-term control of skin aging30. However, fibroblasts gradually lose their capacity to proliferate and synthesize collagen as they senesce, resulting in challenges for longer-term methods of collagen replacement for anti-aging treatment31. To overcome these limitations, we aimed to repla (...truncated)