Background 1.1. New Coronavirus Infection and Current Medical Strategies The 2019
coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) has been raging for over three years, causing more than 5 million deaths
worldwide. The World Health Organization has declared COVID-19 pneumonia a global
pandemic and a public health emergency[1]. Although diagnostic efficiency and treatment
accuracy have improved, the overall treatment effect is still limited. The main causes
of death include severe pneumonia, acute respiratory distress syndrome (ARDS), pulmonary
edema, or multiple organ failure[2]. Among them, ARDS is the most severe. Pathological
studies have shown that the pathogenesis of ARDS is mainly due to the immune response
induced by the coronavirus attacking alveolar cells; immune cell infiltration leads to
destruction of the pulmonary capillary endothelial structure, causing plasma, plasma
proteins, and blood cells to enter the lung interstitium and alveolar cavity, forming
pulmonary edema. Due to the destruction of the alveolar epithelial tissue, a large
amount of edema fluid enters the alveoli, resulting in limited diffusion function of the
alveoli and inability to perform normal respiratory function. At the same time, with the
aggravation of tissue damage, cells further release various inflammatory factors to form
a so-called cytokine storm, which finally completely destroys most of the pulmonary
alveoli and bronchial respiratory structures, causing severe ventilation-perfusion
imbalance and ultimately death of the patient[3].
In this global emergency event of coronavirus, the main medical strategy to combat
COVID-19 is to use antibiotics and antiviral drugs to block the virus replication cycle
and suppress host inflammation. This strategy has led to effective supportive and
symptomatic treatment in some cases, even yielding promising results, but it is not the
ultimate treatment for this infection. For example, immune regulation interventions like
tocilizumab (IL-6 receptor blocker), adalimumab (anti-TNF antibody), and eculizumab
(anti-C5 antibody) can effectively relieve patient symptoms but cannot cure the disease
fundamentally. In fact, there are two major characteristics of COVID-19 pneumonia:
first, people with low immunity are more susceptible to COVID-19[4], and second, the
main target organ is the lungs[5]. In fact, respiratory failure has been reported as one
of the main causes of death from COVID-19[6], and autopsies have shown lung damage,
significant exudative reactions, and pulmonary embolism in many patients[7].
1.2. Mesenchymal Stem Cell Exosomes Mesenchymal stem cells (MSCs) have been shown to
have comprehensive and powerful immunomodulatory and regenerative functions[8]. MSCs can
combat cell death associated with the pathogenesis of chronic obstructive pulmonary
disease (COPD), idiopathic pulmonary fibrosis, asthma, ARDS, and pulmonary hypertension,
and promote cell regeneration[9,10]. Exosomes are one of the key paracrine effectors
secreted by MSCs and are considered attractive candidates for alternative MSC therapy
due to their biocompatibility similar to parent cells and their ability to maintain
healing properties[11]. Under physiological and pathological conditions, exosomes play a
key role in intercellular communication by transporting various biomolecules such as
miRNA and proteins to target cells[12]. Unlike apoptotic bodies and microvesicles
derived from the cell surface, exosomes are produced through the endocytic pathway and
load cytoplasmic content of the parent cell. Therefore, they are mini versions of parent
cells, mimicking some of their physiological characteristics. Compared to cell
counterparts, the non-toxicity, low immunogenicity, high stability, ease of storage, and
potential for mass production as ready-made products are several advantages of exosomes,
which have led to their expansion in clinical applications as new therapeutic
alternatives. In addition, the natural function of exosomes allows them to deliver their
membrane and cytoplasmic bioactive components from parent cells to target cells through
membrane fusion[13]. There are also other unique characteristics, including the natural
ability to cross biological barriers such as the blood-brain barrier (BBB). Furthermore,
biocompatibility is another characteristic. Due to their origin from biological sources
and inherent targeting ability, exosomes have been used as carriers of drug components
in preclinical studies[14].
1.3. Mechanism of Mesenchymal Stem Cell-derived Exosomes Treatment MSC-derived exosomes
inherit immune suppressive properties from their parent cells, and MSC-evs may use
various mechanisms to balance the immune system's function. One key mechanism is
reprogramming and altering the phenotype of various immune cells. For example, the
ability of MSC-derived exosomes to promote the survival of alveolar macrophages and
shift their phenotype from pro-inflammatory (M1) polarization to anti-inflammatory (M2)
polarization has been demonstrated in at least two studies. These findings suggest that
exosomes can serve as viable alternatives to their parent cells, and this ability has
also been reported to alter the Treg/Teff ratio to increase Treg and promote the
secretion of anti-inflammatory cytokines[15,16].
In multiple models, MSC-evs have similar therapeutic characteristics to MSCs, are easier
to prepare, store and transport to the bedside, and avoid some limitations of cell
therapy, such as the risk of pulmonary embolism and tumor formation. In recent years,
MSC-evs have received widespread attention as biomarkers of pathogenesis and therapeutic
drugs for a variety of diseases. In addition, MSC-secreted exosomes can regulate
immunity through interaction with immune cells and inhibit inflammatory responses
through cytokines[17,18]. Numerous studies have shown that MSC-secreted exosomes can be
used to treat immune deficiencies, inflammation, ARDS, and other pulmonary
diseases[19,20], so MSC-secreted exosomes may also be effective in treating the lung
inflammation caused by COVID-19.Exosomes are one of the main active ingredients secreted
by stem cells and are 30-150 nm in size. After nebulization, exosomes can directly reach
the bronchioles and alveoli, which is conducive to the maximum absorption of drugs[21].
Multiple clinical trials have shown that intravenous infusion of MSCs and MSC-evs for
the treatment of severe lung damage caused by SARS-CoV-2 is safe and effective[22], and
in the case of lung injury, the nebulization route provides a particularly effective
drug delivery method to target lung sites. Therefore, we speculate that nebulized
MSC-secreted exosomes may be an effective method for reducing COVID-19 lung damage and
promoting recovery.
1.4. Clinical Case Studies of Stem Cell-derived Exosomes Recently, the inhaled
anti-COVID-19 drug Exo-CD24, led by Israeli medical center expert Nadir Arber, which is
a combination of exosomes and CD24 protein, has shown promising results in early
clinical trials. The drug was able to cure 29 out of 30 severe patients within 5 days of
treatment and has the potential to treat COVID-19 patients within 3-5 days. Although
Exo-CD24 has not yet passed phase III testing, it has shown great potential in the
future[23]. The US FDA has approved the use of extracellular vesicles (EVS) for the
treatment of COVID-19 through a Phase I/II trial conducted by Direct Biologics. ExoFlo,
the EVS used, restores the health and vitality of patients by reducing sustained
inflammation, promoting the revascularization of damaged tissue, and reshaping scar
tissue. Domestic clinical studies have also been initiated, clinical research at the
Fifth People's Hospital of Wuxi City, Jiangsu Province has confirmed that nebulized
umbilical cord mesenchymal stem cell-derived extracellular vesicles are a safe and
viable treatment for COVID-19. The study was published in Stem Cell Reviews and Reports
in June of this year and included seven patients diagnosed with COVID-19 pneumonia,
including two severe cases (Patients 2 and 4) and five mild cases (Patients 1, 3, 5, 6,
and 7). No acute allergic reactions such as throat or tongue swelling, rash, shortness
of breath, dizziness, vomiting, or hypotension were observed in any of the patients
within two hours after nebulization treatment. There were also no reported adverse
events or secondary allergic reactions following treatment. Chest CT scans showed a
decrease in lung lobe nodule density and absorption of lung lesions in both severe and
mild patients after nebulized treatment with MSC-derived extracellular vesicles[21].
Joint research by Ruijin Hospital and Jinyintan Hospital is being conducted on nebulized
COVID-19 treatment using adipose-derived human allogeneic mesenchymal stem cell-derived
exosomes (HAMSCs-Exos). Seven critically ill COVID-19 patients were treated with
HAMSC-Exos via nebulization inhalation, and all patients tolerated the treatment well
without evidence of adverse events or clinical instability during or immediately after
nebulization. All patients showed an increase in serum lymphocyte count (median of
1.61×10^9/L vs 1.78×10^9/L), and lung lesions were reduced to varying degrees in all
patients after inhaling HAMSC-Exos aerosols, with four patients showing significant
improvement. Preliminary results suggest that participants' lung injuries were
significantly improved[21].
However, most clinical trials using stem cell exosomes for COVID-19 infection have the
following issues: (a) small sample size, making it difficult to effectively demonstrate
the advantages of stem cell exosomes in COVID-19 treatment; (b) lack of a control group;
(c) use of traditional two-dimensional production methods, making it difficult to scale
up production of large quantities of exosomes for clinical treatment; and (d) lack of
in-depth mechanism exploration for relevant phenomena. These problems hinder progress in
the use of stem cell exosomes for COVID-19 infection treatment. To address these issues,
this project proposes combining nebulized umbilical cord mesenchymal stem cell exosomes
with conventional treatment to treat middle to severe COVID-19 patients and evaluate its
safety and efficacy.