1. 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.

2. Research Objective This study aims to evaluate the safety and effectiveness of
umbilical cord mesenchymal stem cell-derived exosomes (nebulized) combined with
conventional therapy for the treatment of moderate, severe, and critically ill
COVID-19 patients through a multicenter, randomized controlled, single-blind
clinical trial.

3. Research Design This study adopts a multicenter, randomized controlled, single-blind
trial design. Based on the COVID-19 treatment regimen formulated by clinical
guidelines, nebulized umbilical cord mesenchymal stem cell-derived exosomes
(experimental group) or medically sterile saline solution (control group) will be
administered to treat moderate, severe, and critically ill COVID-19 patients. By
comparing the relief of symptoms and the occurrence of adverse events before and
after treatment, the safety and effectiveness of umbilical cord mesenchymal stem
cell-derived exosomes for the treatment of moderate, severe, and critically ill
COVID-19 patients will be evaluated.