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.

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Rationale: Cardiac stem cell-derived exosomes have been demonstrated to promote cardiac regeneration following myocardial infarction in preclinical studies. Recent studies have used intramyocardial injection in order to concentrate exosomes in the infarct. Though effective in a research setting, this method is not clinically appealing due to its invasive nature. We propose the use of a targeting peptide, cardiac homing peptide (CHP), to target intravenously-infused exosomes to the infarcted heart.Methods: Exosomes were conjugated with CHP through a DOPE-NHS linker. Ex vivo targeting was analyzed by incubating organ sections with the CHP exosomes and analyzing with fluorescence microscopy. In vitro assays were performed on neonatal rat cardiomyocytes and H9C2 cells. For the animal study, we utilized an ischemia/reperfusion rat model. Animals were treated with either saline, scramble peptide exosomes, or CHP exosomes 24 h after surgery. Echocardiography was performed 4 h after surgery and 21 d after surgery. At 21 d, animals were sacrificed, and organs were collected for analysis.Results: By conjugating the exosomes with CHP, we demonstrate increased retention of the exosomes within heart sections ex vivo and in vitro with neonatal rat cardiomyocytes. In vitro studies showed improved viability, reduced apoptosis and increased exosome uptake when using CHP-XOs. Using an animal model of ischemia/reperfusion injury, we measured the heart function, infarct size, cellular proliferation, and angiogenesis, with improved outcomes with the CHP exosomes.Conclusions: Our results demonstrate a novel method for increasing delivery of for treatment of myocardial infarction. By targeting exosomes to the infarcted heart, there was a significant improvement in outcomes with reduced fibrosis and scar size, and increased cellular proliferation and angiogenesis.

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Polymer micelles are one of the most investigated nanocarriers for drug delivery; many have entered clinical trials and some are in clinic use, but their delivery systems have not yet shown the expected high therapeutic efficacy in clinics. Further understanding their in vivo behaviors, particularly how quickly and by what mechanism polymer micelles are cleared ( i. e., via micelles or unimers) once injected, is key to solving this dilemma. Herein, we hope to answer these questions for the clinically relevant polyethylene glycol- block-poly(ε-caprolactone) (PEG-PCL) and PEG- block-poly(d,l-lactide) (PEG-PDLLA) micelles. A small fraction of the hydrophobic chain ends was conjugated with a pair of fluorescence resonance energy transfer (FRET) dyes, Cy5 and Cy5.5, and used to fabricate FRET micelles whose FRET efficiency was correlated to the percentage of polymer chains in the micelles, the micelle degree. In vitro, serum proteins induced PEG-PCL micelle dissociation to some extent; mouse serum or blood surprisingly did not induce micelle dissociation but once with shear applied by a microfluidic channel caused most PEG-PCL micelles dissociated. After intravenous administration in mice, the PEG-PCL or PEG-PDLLA micelles were quickly sequestered into the liver as unimers, and the micelle degree in the blood quickly decreased to about 20%. The FRET-imaging experiments showed that in blood vessels the micelles quickly dissociated into unimers, which were found associated with albumin in blood and in liver. Thus, it is concluded that, upon intravenous injection, the shear and the bloodborne proteins (particularly albumin) induced the most (∼80%) PEG-PCL and PEG-PDLLA micelles to quickly dissociate into unimers, which were sequestered by Kupffer cells, while intact micelles were difficult to clear. These micelles were able to penetrate tumors and were very stable with cell membranes, but dissociated gradually inside cells. These findings on in vivo micelle fate and the clearance mechanism are directional for the rational design of polymer micelles for improved therapeutics; particularly, improving micelle stability in blood is the prerequisite for surface functionalizations such as introducing targeting ligands.

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-β). 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.

Cell therapy has been a promising strategy for cardiac repair after injury or infarction; however, low retention and engraftment of transplanted cells limit potential therapeutic efficacy. Seeding scaffold material with cells to create cardiac patches that are transplanted onto the surface of the heart can overcome these limitations. However, because patches need to be freshly prepared to maintain cell viability, long-term storage is not feasible and limits clinical applicability. Here, we developed an off-the-shelf therapeutic cardiac patch composed of a decellularized porcine myocardial extracellular matrix scaffold and synthetic cardiac stromal cells (synCSCs) generated by encapsulating secreted factors from isolated human cardiac stromal cells. This fully acellular artificial cardiac patch (artCP) maintained its potency after long-term cryopreservation. In a rat model of acute myocardial infarction, transplantation of the artCP supported cardiac recovery by reducing scarring, promoting angiomyogenesis, and boosting cardiac function. The safety and efficacy of the artCP were further confirmed in a porcine model of myocardial infarction. The artCP is a clinically feasible, easy-to-store, and cell-free alternative to myocardial repair using cell-based cardiac patches.

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.