Inhalation of ACE2-expressing lung exosomes provides prophylactic protection against SARS-CoV-2.

Continued emergence of SARS-CoV-2 variants of concern that are capable of escaping vaccine-induced immunity highlights the urgency of developing new COVID-19 therapeutics. An essential mechanism for SARS-CoV-2 infection begins with the viral spike protein binding to the human ACE2. Consequently, inhibiting this interaction becomes a highly promising therapeutic strategy against COVID-19. Herein, we demonstrate that ACE2-expressing human lung spheroid cells (LSC)-derived exosomes (LSC-Exo) could function as a prophylactic agent to bind and neutralize SARS-CoV-2, protecting the host against SARS-CoV-2 infection. Inhalation of LSC-Exo facilitates its deposition and biodistribution throughout the whole lung in a female mouse model. We show that LSC-Exo blocks the interaction of SARS-CoV-2 with host cells in vitro and in vivo by neutralizing the virus. LSC-Exo treatment protects hamsters from SARS-CoV-2-induced disease and reduced viral loads. Furthermore, LSC-Exo intercepts the entry of multiple SARS-CoV-2 variant pseudoviruses in female mice and shows comparable or equal potency against the wild-type strain, demonstrating that LSC-Exo may act as a broad-spectrum protectant against existing and emerging virus variants.

A SARS-CoV-2 and influenza double hit vaccine based on RBD-conjugated inactivated influenza A virus.

The circulating flu viruses merging with the ongoing COVID-19 pandemic raises a more severe threat that promotes the infectivity of SARS-CoV-2 associated with higher mortality rates. Here, we conjugated recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein onto inactivated influenza A virus (Flu) to develop a SARS-CoV-2 virus-like particle (VLP) vaccine with two-hit protection. This double-hit vaccine (Flu-RBD) not only induced protective immunities against SARS-CoV-2 but also remained functional as a flu vaccine. The Flu core improved the retention and distribution of Flu-RBD vaccine in the draining lymph nodes, with enhanced immunogenicity. In a hamster model of live SARS-CoV-2 infection, two doses of Flu-RBD efficiently protected animals against viral infection. Furthermore, Flu-RBD VLP elicited a strong neutralization activity against both SARS-CoV-2 Delta pseudovirus and wild-type influenza A H1N1 inactivated virus in mice. Overall, the Flu-RBD VLP vaccine is a promising candidate for combating COVID-19, influenza A, and coinfection.

Inhalable exosomes outperform liposomes as mRNA and protein drug carriers to the lung.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide, coupled with the ongoing coronavirus disease 2019 (COVID-19) pandemic. mRNA lipid nanoparticle (LNP) vaccines have been developed, but their intramuscular delivery limits pulmonary bioavailability. Inhalation of nanoparticle therapeutics offers localized drug delivery that minimizes off targeted adverse effects and has greater patient compliance. However, LNP platforms require extensive reformulation for inhaled delivery. Lung-derived extracellular vesicles (Lung-Exo) offer a biological nanoparticle alternative that is naturally optimized for mRNA translation and delivery to pulmonary cells. We compared the biodistribution of Lung-Exo against commercially standard biological extracellular vesicles (HEK-Exo) and LNPs (Lipo), where Lung-Exo exhibited superior mRNA and protein cargo distribution to and retention in the bronchioles and parenchyma following nebulization administration. This suggests that inhaled Lung-Exo can deliver mRNA and protein drugs with enhanced pulmonary bioavailability and therapeutic efficacy.

Exosomes decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine.

The first two mRNA vaccines against infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that were approved by regulators require a cold chain and were designed to elicit systemic immunity via intramuscular injection. Here we report the design and preclinical testing of an inhalable virus-like-particle as a COVID-19 vaccine that, after lyophilisation, is stable at room temperature for over three months. The vaccine consists of a recombinant SARS-CoV-2 receptor-binding domain (RBD) conjugated to lung-derived exosomes which, with respect to liposomes, enhance the retention of the RBD in both the mucus-lined respiratory airway and in lung parenchyma. In mice, the vaccine elicited RBD-specific IgG antibodies, mucosal IgA responses and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile in the animals' lungs, and cleared them of SARS-CoV-2 pseudovirus after a challenge. In hamsters, two doses of the vaccine attenuated severe pneumonia and reduced inflammatory infiltrates after a challenge with live SARS-CoV-2. Inhalable and room-temperature-stable virus-like particles may become promising vaccine candidates.

Inhalable dry powder mRNA vaccines based on extracellular vesicles.

Respiratory diseases are a global burden, with millions of deaths attributed to pulmonary illnesses and dysfunctions. Therapeutics have been developed, but they present major limitations regarding pulmonary bioavailability and product stability. To circumvent such limitations, we developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) as mRNA and protein drug carriers. Compared with standard synthetic nanoparticle liposomes (Lipos), Lung-Exos exhibited superior distribution to the bronchioles and parenchyma and are deliverable to the lungs of rodents and nonhuman primates (NHPs) by dry powder inhalation. In a vaccine application, severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein encoding mRNA-loaded Lung-Exos (S-Exos) elicited greater immunoglobulin G (IgG) and secretory IgA (SIgA) responses than its loaded liposome (S-Lipo) counterpart. Importantly, S-Exos remained functional at room-temperature storage for one month. Our results suggest that extracellular vesicles can serve as an inhaled mRNA drug-delivery system that is superior to synthetic liposomes.

Exosome therapeutics for COVID-19 and respiratory viruses.

Respiratory viral diseases are a leading cause of mortality in humans. They have proven to drive pandemic risk due to their complex transmission factors and viral evolution. However, the slow production of effective antiviral drugs and vaccines allows for outbreaks of these diseases, emphasizing a critical need for refined antiviral therapeutics. The delivery of exosomes, a naturally secreted extracellular vesicle, yields therapeutic effects for a variety of diseases, including viral infection. Exosomes and viruses utilize similar endosomal sorting pathways and mechanisms, providing exosomes with the potential to serve as a therapeutic that can target, bind, and suppress cellular uptake of various viruses including the novel severe acute respiratory syndrome coronavirus 2. Here, we review the relationship between exosomes and respiratory viruses, describe potential exosome therapeutics for viral infections, and summarize progress toward clinical translation for lung-derived exosome therapeutics.

Cell-mimicking nanodecoys neutralize SARS-CoV-2 and mitigate lung injury in a non-human primate model of COVID-19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has grown into a global pandemic, and only a few antiviral treatments have been approved to date. Angiotensin-converting enzyme 2 (ACE2) plays a fundamental role in SARS-CoV-2 pathogenesis because it allows viral entry into host cells. Here we show that ACE2 nanodecoys derived from human lung spheroid cells (LSCs) can bind and neutralize SARS-CoV-2 and protect the host lung cells from infection. In mice, these LSC-nanodecoys were delivered via inhalation therapy and resided in the lungs for over 72 h post-delivery. Furthermore, inhalation of the LSC-nanodecoys accelerated clearance of SARS-CoV-2 mimics from the lungs, with no observed toxicity. In cynomolgus macaques challenged with live SARS-CoV-2, four doses of these nanodecoys delivered by inhalation promoted viral clearance and reduced lung injury. Our results suggest that LSC-nanodecoys can serve as a potential therapeutic agent for treating COVID-19.

Exosome-eluting stents for vascular healing after ischaemic injury.

Drug-eluting stents implanted after ischaemic injury reduce the proliferation of endothelial cells and vascular smooth muscle cells and thus neointimal hyperplasia. However, the eluted drug also slows down the re-endothelialization process, delays arterial healing and can increase the risk of late restenosis. Here we show that stents releasing exosomes derived from mesenchymal stem cells in the presence of reactive oxygen species enhance vascular healing in rats with renal ischaemia-reperfusion injury, promoting endothelial cell tube formation and proliferation, and impairing the migration of smooth muscle cells. Compared with drug-eluting stents and bare-metal stents, the exosome-coated stents accelerated re-endothelialization and decreased in-stent restenosis 28 days after implantation. We also show that exosome-eluting stents implanted in the abdominal aorta of rats with unilateral hindlimb ischaemia regulated macrophage polarization, reduced local vascular and systemic inflammation, and promoted muscle tissue repair.

Advanced Nanobiomedical Approaches to Combat Coronavirus Disease of 2019.

New infectious diseases are making themselves known as the human population grows, expands into new regions, and becomes more dense, increasing contact with each other and animal populations. Ease of travel has also increased infectious disease transmission and has now culminated into a global pandemic. The emergence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 has already infected over 83.7 million people and caused over 1.8 million deaths. While there have been vaccine candidates produced and supportive care implemented, the world is impatiently waiting for a commercially approved vaccine and treatment for the coronavirus disease of 2019 (COVID-19). The different vaccine types investigated for the prevention of COVID-19 all have great promise but face safety obstacles that must be first addressed. Some vaccine candidates of key interest are whole inactivated viruses, adeno-associated viruses, virus-like particles, and lipid nanoparticles. This review examines nanobiomedical techniques for combatting COVID-19 in terms of vaccines and therapeutics.

Exosome therapeutics for lung regenerative medicine.

Exosomes are 30 to 100 nm extracellular vesicles that are secreted by many cell types. Initially viewed as cellular garbage with no biological functions, exosomes are now recognized for their therapeutic potential and used in regenerative medicine. Cell-derived exosomes are released into almost all biological fluids, making them abundant and accessible vesicles for a variety of diseases. These naturally occurring nanoparticles have a wide range of applications including drug delivery and regenerative medicine. Exosomes sourced from a specific tissue have been proven to provide greater therapeutic effects to their native tissue, expanding exosome sources beyond traditional cell lines such as mesenchymal stem cells. However, standardizing production and passing regulations remain obstacles, due to variations in methods and quantification techniques across studies. Additionally, obtaining pure exosomes at sufficient quantities remains difficult due to the heterogeneity of exosomes. In this review, we will underline the uses of exosomes as a therapy and their roles in lung regenerative medicine, as well as current challenges in exosome therapies.

Dermal exosomes containing miR-218-5p promote hair regeneration by regulating ß-catenin signaling.

The progression in the hair follicle cycle from the telogen to the anagen phase is the key to regulating hair regrowth. Dermal papilla (DP) cells support hair growth and regulate the hair cycle. However, they gradually lose key inductive properties upon culture. DP cells can partially restore their capacity to promote hair regrowth after being subjected to spheroid culture. In this study, results revealed that DP spheroids are effective at inducing the progression of the hair follicle cycle from telogen to anagen compared with just DP cell or minoxidil treatment. Because of the importance of paracrine signaling in this process, secretome and exosomes were isolated from DP cell culture, and their therapeutic efficacies were investigated. We demonstrated that miR-218-5p was notably up-regulated in DP spheroid-derived exosomes. Western blot and immunofluorescence imaging were used to demonstrate that DP spheroid-derived exosomes up-regulated ß-catenin, promoting the development of hair follicles.

A pre-investigational new drug study of lung spheroid cell therapy for treating pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a lethal interstitial lung disease with unknown etiology, no cure, and few treatment options. Herein, a therapy option is presented that makes use of a heterogeneous population of lung cells, including progenitor cells and supporting cells lines, cultured in adherent and suspension conditions, the latter of which induces spontaneous spheroid formation. Within these spheroids, progenitor marker expression is augmented. The cells, called lung spheroid cells, are isolated from fibrotic lungs, expanded, and delivered in single cell suspensions into rat models of pulmonary fibrosis via tail-vein injections. Two bleomycin-induced fibrotic rat models are used; a syngeneic Wistar-Kyoto rat model, treated with syngeneic cells, and a xenogeneic nude rat model, treated with human cells. The first objective was to study the differences in fibrotic progression in the two rat models after bleomycin injury. Nude rat fibrosis formed quickly and extended for 30 days with no self-resolution. Wistar-Kyoto rat fibrosis was more gradual and began to decrease in severity between days 14 and 30. The second goal was to find the minimum effective dose of cells that demonstrated safe and effective therapeutic value. The resultant minimum effective therapeutic dose, acquired from the nude rat model, was 3 × 106 human cells. Histological analysis revealed no evidence of tumorigenicity, increased local immunological activity in the lungs, or an increase in liver enzyme production. These data demonstrate the safety and efficacy of lung spheroid cells in their application as therapeutic agents for pulmonary fibrosis, as well as their potential for clinical translation.

Tumor cell-derived exosomes home to their cells of origin and can be used as Trojan horses to deliver cancer drugs.

Cancer is the second leading cause of death worldwide and patients are in urgent need of therapies that can effectively target cancer with minimal off-target side effects. Exosomes are extracellular nano-shuttles that facilitate intercellular communication between cells and organs. It has been established that tumor-derived exosomes contain a similar protein and lipid composition to that of the cells that secrete them, indicating that exosomes might be uniquely employed as carriers for anti-cancer therapeutics. Methods: We isolated exosomes from two cancer cell lines, then co-cultured each type of cancer cells with these two kinds of exosomes and quantified exosome. HT1080 or Hela exosomes were systemically injected to Nude mice bearing a subcutaneous HT1080 tumor to investigate their cancer-homing behavior. Moreover, cancer cell-derived exosomes were engineered to carry Doxil (a common chemotherapy drug), known as D-exo, were used to detect their target and therapeutic efficacy as anti-cancer drugs. Exosome proteome array analysis were used to reveal the mechanism underly this phenomenon. Results: Exosomes derived from cancer cells fuse preferentially with their parent cancer cells, in vitro. Systemically injected tumor-derived exosomes home to their original tumor tissues. Moreover, compared to Doxil alone, the drug-loaded exosomes showed enhanced therapeutic retention in tumor tissues and eradicated them more effectively in nude mice. Exosome proteome array analysis revealed distinct integrin expression patterns, which might shed light on the underlying mechanisms that explain the exosomal cancer-homing behavior. Conclusion: Here we demonstrate that the exosomes' ability to target the parent cancer is a phenomenon that opens up new ways to devise targeted therapies to deliver anti-tumor drugs.

Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal and incurable form of interstitial lung disease in which persistent injury results in scar tissue formation. As fibrosis thickens, the lung tissue loses the ability to facilitate gas exchange and provide cells with needed oxygen. Currently, IPF has few treatment options and no effective therapies, aside from lung transplant. Here we present a series of studies utilizing lung spheroid cell-secretome (LSC-Sec) and exosomes (LSC-Exo) by inhalation to treat different models of lung injury and fibrosis. Analysis reveals that LSC-Sec and LSC-Exo treatments could attenuate and resolve bleomycin- and silica-induced fibrosis by reestablishing normal alveolar structure and decreasing both collagen accumulation and myofibroblast proliferation. Additionally, LSC-Sec and LSC-Exo exhibit superior therapeutic benefits than their counterparts derived from mesenchymal stem cells in some measures. We showed that an inhalation treatment of secretome and exosome exhibited therapeutic potential for lung regeneration in two experimental models of pulmonary fibrosis.

Bispecific Antibody Inhalation Therapy for Redirecting Stem Cells from the Lungs to Repair Heart Injury.

Stem cell therapy is a promising strategy for cardiac repair. However, clinical efficacy is hampered by poor cell engraftment and the elusive repair mechanisms of the transplanted stem cells. The lung is a reservoir of hematopoietic stem cells (HSCs) and a major biogenesis site for platelets. A strategy is sought to redirect lung resident stem cells to the injured heart for therapeutic repair after myocardial infarction (MI). To achieve this goal, CD34-CD42b platelet-targeting bispecific antibodies (PT-BsAbs) are designed to simultaneously recognize HSCs (via CD34) and platelets (via CD42b). After inhalation delivery, PT-BsAbs reach the lungs and conjoined HSCs and platelets. Due to the innate injury-finding ability of platelets, PT-BsAbs guide lung HSCs to the injured heart after MI. The redirected HSCs promote endogenous repair, leading to increased cardiac function. The repair mechanism involves angiomyogenesis and inflammation modulation. In addition, the inhalation route is superior to the intravenous route to deliver PT-BsAbs in terms of the HSCs' homing ability and therapeutic benefits. This work demonstrates that this novel inhalable antibody therapy, which harnesses platelets derived from the lungs, contributes to potent stem cell redirection and heart repair. This strategy is safe and effective in a mouse model of MI.

Cardiac Stromal Cell Patch Integrated with Engineered Microvessels Improves Recovery from Myocardial Infarction in Rats and Pigs.

The vascularized cardiac patch strategy is promising for ischemic heart repair after myocardial infarction (MI), but current fabrication processes are quite complicated. Vascularized cardiac patches that can promote concurrent restoration of both the myocardium and vasculature at the injured site in a large animal model remain elusive. The safety and therapeutic benefits of a cardiac stromal cell patch integrated with engineered biomimetic microvessels (BMVs) were determined for treating MI. By leveraging a microfluidic method employing hydrodynamic focusing, we constructed the endothelialized microvessels and then encapsulated them together with therapeutic cardiosphere-derived stromal cells (CSCs) in a fibrin gel to generate a prevascularized cardiac stromal cell patch (BMV-CSC patch). We showed that BMV-CSC patch transplantation significantly promoted cardiac function, reduced scar size, increased viable myocardial tissue, promoted neovascularization, and suppressed inflammation in rat and porcine MI models, demonstrating enhanced therapeutic efficacy compared to conventional cardiac stromal cell patches. BMV-CSC patches did not increase renal and hepatic toxicity or exhibit immunogenicity. We noted a significant increase in endogenous progenitor cell recruitment to the peri-infarct region of the porcine hearts treated with BMV-CSC patch as compared to those that received control treatments. These findings establish the BMV-CSC patch as a novel engineered-tissue therapeutic for ischemic tissue repair.

Needle-Free Injection of Exosomes Derived from Human Dermal Fibroblast Spheroids Ameliorates Skin Photoaging.

Human dermal fibroblasts (HDFs), the main cell population of the dermis, gradually lose their ability to produce collagen and renew intercellular matrix with aging. One clinical application for the autologous trans-dermis injection of HDFs that has been approved by the Food and Drug Administration aims to refine facial contours and slow down skin aging. However, the autologous HDFs used vary in quality according to the state of patients and due to many passages they undergo during expansion. In this study, factors and exosomes derived from three-dimensional spheroids (3D HDF-XOs) and the monolayer culture of HDFs (2D HDF-XOs) were collected and compared. 3D HDF-XOs expressed a significantly higher level of tissue inhibitor of metalloproteinases-1 (TIMP-1) and differentially expressed miRNA cargos compared with 2D HDF-XOs. Next, the efficacy of 3D HDF-XOs in inducing collagen synthesis and antiaging was demonstrated in vitro and in a nude mouse photoaging model. A needle-free injector was used to administer exosome treatments. 3D HDF-XOs caused increased procollagen type I expression and a significant decrease in MMP-1 expression, mainly through the downregulation of tumor necrosis factor-alpha (TNF-α) and the upregulation of transforming growth factor beta (TGF-ß). In addition, the 3D-HDF-XOs group showed a higher level of dermal collagen deposition than bone marrow mesenchymal stem cell-derived exosomes. These results indicate that exosomes from 3D cultured HDF spheroids have anti-skin-aging properties and the potential to prevent and treat cutaneous aging.

Circulating tumor cells exit circulation while maintaining multicellularity, augmenting metastatic potential.

Metastasis accounts for the majority of all cancer deaths, yet the process remains poorly understood. A pivotal step in the metastasis process is the exiting of tumor cells from the circulation, a process known as extravasation. However, it is unclear how tumor cells extravasate and whether multicellular clusters of tumor cells possess the ability to exit as a whole or must first disassociate. In this study, we use in vivo zebrafish and mouse models to elucidate the mechanism tumor cells use to extravasate. We found that circulating tumor cells exit the circulation using the recently identified extravasation mechanism, angiopellosis, and do so as both clusters and individual cells. We further show that when melanoma and cervical cancer cells utilize this extravasation method to exit as clusters, they exhibit an increased ability to form tumors at distant sites through the expression of unique genetic profiles. Collectively, we present a new model for tumor cell extravasation of both individual and multicellular circulating tumor cells.This article has an associated First Person interview with the first author of the paper.

microRNA-21-5p dysregulation in exosomes derived from heart failure patients impairs regenerative potential.

Exosomes, as functional paracrine units of therapeutic cells, can partially reproduce the reparative properties of their parental cells. The constitution of exosomes, as well as their biological activity, largely depends on the cells that secrete them. We isolated exosomes from explant-derived cardiac stromal cells from patients with heart failure (FEXO) or from normal donor hearts (NEXO) and compared their regenerative activities in vitro and in vivo. Patients in the FEXO group exhibited an impaired ability to promote endothelial tube formation and cardiomyocyte proliferation in vitro. Intramyocardial injection of NEXO resulted in structural and functional improvements in a murine model of acute myocardial infarction. In contrast, FEXO therapy exacerbated cardiac function and left ventricular remodeling. microRNA array and PCR analysis revealed dysregulation of miR-21-5p in FEXO. Restoring miR-21-5p expression rescued FEXO's reparative function, whereas blunting miR-21-5p expression in NEXO diminished its therapeutic benefits. Further mechanistic studies revealed that miR-21-5p augmented Akt kinase activity through the inhibition of phosphatase and tensin homolog. Taken together, the heart failure pathological condition altered the miR cargos of cardiac-derived exosomes and impaired their regenerative activities. miR-21-5p contributes to exosome-mediated heart repair by enhancing angiogenesis and cardiomyocyte survival through the phosphatase and tensin homolog/Akt pathway.