Nebulization Therapy for COVID-19 Pneumonia with Embryonic Mesenchymal Stem Cells-derived Exosomes

Background: Scientist have been facing numerous challenges in the development of an effective therapeutic strategy for the treatment of COVID-19 pneumonia. Several studies have suggested that improving patient immunity and reducing lung injury induced by COVID -19 could be effective in treating the patients with COVID-19. Methods: A pilot trial of nebulization therapy for COVID-19 pneumonia with exosomes of MSCs was performed on seven patients with COVID-19 infected pneumonia. Exosomes were collected and puried from secretion of MSCs using multiple ultraltration. All patients was treated with nebulization of MSC-derived exosomes. The primary safety and ecacy outcome were observed. Results: Our clinical study demonstrated that nebulization mesenchymal stem cell (MSCs)-derived exosomes is a novel method that could be utilized in the treatment of COVID-19 pneumonia. Nebulization of MSC-derived exosomes did not induce acute allergic and secondary allergic reactions. It could promote the absorption of pulmonary lesions, and reduce the time of hospitalization for minor cases of COVID-19 pneumonia. Conclusions: Nebulization of MSC-derived exosomes is a safe, effective, and simple method. Nebulization of MSC-derived exosomes from the beginning of the treatment could be more benecial to the patients. We also demonstrated that MSC-derived can reduce the CRP level in different degrees of patients with COVID-19 pneumonia, which was similar to the ndings of a previous study Although there was a decrease in CRP level after the nebulization treatment, it did not achieve statistical signicancedue to the small number of cases and large standard deviation. The patients who received nebulization treatment at an earlier stage showed more benefecial effects in terms of the absorption of pulmonary inammation. Our results showed that nebulization of MSC-derived exosomes is also benecial to the absorption of pulmonary lesions in minor cases of COVID-19 pneumonia and the reduction of cellulose residues in severe cases of COVID-19.

Background COVID − 19 pneumonia has been declared as a global pandemic by the World Health Organization (WHO) and has become a matter of public health emergency (1). Although the diagnostic e ciency and accuracy of the treatment modalities have improved, the overall therapeutic effect remains poor (2). The major causes of death were severe pneumonia, pulmonary edema, ARDS or multiple organ failure (3).
The two major characteristics of COVID − 19 pneumonia are: 1. People with low immunity are more likely to be infected with COVID − 19 (4); 2. The major target organ of attack is the lung (5). Respiratory failure has been reported as one of the major causes of death due to COVID-19 (6). Autopsy revealed the occurrence of pulmonary injury, signi cant exudative reaction, and pulmonary embolism in many patients (7). Therefore,  induced lung injury could be reduced by improving the immunity of patients.
Mesenchymal stem cells (MSCs) have been shown to possess a comprehensive, powerful immunomodulatory function (8,9). The exosomes of MSCs can not only regulate human immunity through immune cells, but also inhibit in ammatory response through cytokines (10,11). Numerous studies have demonstrated that the exosomes of MSCs could be employed in the treatment of immune de ciency, in ammation, acute respiratory distress syndrome (ARDS), and other lung diseases (10,12,13). Therefore, exosomes of MSCs could also be employed in the treatment of COVID- 19 pneumonia.
For stem cell therapy, the routine methods of administration are intravenous injection (14,15). Exosomes are one of the major active components secreted by stem cells (16), with their size ranging from 30-200 nm (17). They can reach the bronchioles and alveoli directly after nebulization, which is conducive to the extent of drug absorption (13). Therefore, we hypothesized that nebulization MSC-derived exosomes could an effective treatment for COVID-19 pneumonia.

Study design
A pilot trial of nebulization therapy for COVID-19 pneumonia with exosomes of MSCs was performed on seven patients with COVID-19 infected pneumonia. The study was conducted in Wuxi No.5 people's Hospital, China. The safety and scienti c validity of this study "have been issued in Chinese Clinical Trial Registry (ChiCTR2000030261).

Inclusion criteria:
We initially enrolled patients with COVID-19 (age 18-65 years) according to the guidance of National Health and Health Commission of China (18).
We comprehensively considered the patient's epidemiological history, clinical symptoms, nucleic acid test. Informed consents were obtained from all the patients to participate in this clinical study.
Exclusion criteria: (1) Age < 18 or > 65; (2) patients with severe heart, brain, lung, kidney dysfunction, endocrine disease, hematopoiesis system disease or other serious diseases and psychosis; (3) pregnant and lactating women; (4) patients who were participating in other clinical trials or who have participated in other clinical trials in the last 3 months; (5) patients who were unwilling or unable to sign the informed consent due to illness.

Patients
The patients were enrolled from Feb 26, 2020 to April 30, 2020. All the enrolled patients were con rmed with COVID-19 via the real-time reverse transcription polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 RNA following the protocol outlined in a previous study. (3,19).
All patients were treated with Ritonavir oral, Abidol oral, Interferon nebulization, or Clerking phosphate oral (Fig. 1). The clinical, laboratory, and radiological outcomes of all patients were recorded and certi ed by a trained group of doctors. The detailed record included primary safety data (allergic reactions, secondary infection and life-threatening adverse events) and the primary e cacy data (the level of CRP in plasma and the oxygen saturation). The secondary e cacy outcomes mainly included the total white blood cell count, total lymphocyte count (tested by Hitachi 7600-020 automatic biochemical analyzer), SARS-CoV-2 nucleic acid detection (tested by RT-PCR protocol, DAAN GENE Co., Ltd, China), chest CT (tested by 320-slice spiral CT scanner, Aquilion One, Toshiba Medical System, Japan), respiratory rate, patient symptoms (especially the fever and shortness of breath), and time of hospitalization.

Preparation of MSCs
Clinical grade MSCs were supplied, by Cruilife Stem Cell co. LTD. The number of MSCs were calculated based on the weight of the patient (1 × 10 6 MSCs / kg body weight). We used passage 4-6 MSCs for collection of exosomes. MSCs were analyzed using ow cytometry for the appropriate markers before use (CD73, CD90, CD105, CD14, CD19, CD34, CD45 and HLA-DR). All procedures performed in this study involving human samples were in accordance with the ethical standards of the institutional research committee and the guidelines set by the Declaration of Helsinki.

Isolation and characterization of exosomes isolated from MSCs
Exosomes were collected and puri ed from secretion of MSCs using multiple ultra ltration ( Fig. 2A). The secretion was rst centrifuged at 4 ℃ at 3000 g for 20 min, and ltered through a 0.22 µm lter to remove any cells or cell debris. The ltered secretomes were then placed in a new sterile EP tube, followed by addition of 0.2 ml exosomes separation and puri cation solution (Shanghai Gefan Biotechnology Co., Ltd. Product No.: ex010). The contents were mixed well at 4 ℃ overnight, and centrifuged at 4 ℃ at 3000 g for 20 min, the following day. After the supernatant was aspirated, it was centrifuged at 4 ℃ for 1500 g 5 min to remove the residual solution. After adding 1000 µl sterile PBS to re suspend and precipitate exosomes, the solution was centrifuged for 1 hour at 100 000 g on a high-speed centrifuge, and this was repeated three times. Exosome samples were analyzed for proper size using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern), and for morphology using transmission electron microscopy (TEM; Tecnai G2 Spirit Bio TWIN). Additionally, successful exosome isolation was con rmed using immunoblotting for known exosome markers CD9 (ab92726, abcam), CD81 (ab109201, abcam), and Flotillin 2 (ab181988, abcam) (Fig. 2).
After nebulization, the MSCs exosomes were sprayed on a sterile glass plate, and a dish was placed under the glass plate for collecting the liquid containing exosomes. After nebulization, the size and markers of MSCs exosomes were evaluated again. No signi cant difference was observed in the size and markers of MSC-derived exosomes before and after nebulization ( Fig. 2C and D).

Nebulization of MSC-derived exosomes
After obtaining ethical approval, all patients diagnosed with COVID-19 pneumonia who provided an informed consent was treated with nebulization of MSC-derived exosomes. The extracted exosomes from MSCs were diluted to 5 ml with 0.9% sodium chloride, and added to the atomizer (Emedical, Excellentcare Medical Ltd. China). The nebulization was performed twice a day (am 8:30, pm 16:00), for 10 minutes each. The patients were assessed by the investigators after receiving the nebulization treatment.

Treatment procedure for MSC-derived exosomes and general patient information
This study was conducted from Feb 26, 2020 to Sep 4, 2020. Seven patients diagnosed with COVID-19 pneumonia, including 2 severe cases (patient 2 and 4) and 5 minor cases (patient 1, 3, 5, 6 and 7) were enrolled in the study (Table 1). Patient 1, 2, 3 and 4 received nebulization of MSCderived exosomes at the end of the after antiviral treatment for a period of time. Patient 1 was a minor case of COVID-19 and did not have any underlying disease conditions. Patient 2 was a severe case of COVID-19 with liver damage. Patient 3 was a minor case and patient 4 was a severe case, both without any underlying disease. Patient 5, 6 and 7 received nebulization of MSC-derived exosomes from the beginning of treatment. The information about all the treatment modalities of the patients were collected. The timepoint of the delivery of MSC-derived exosomes nebulization treatment for each patient is shown in Fig. 1. Table 1 The general information of the enrolled patients.

Statistical analysis
Data which were suitable for statistical analysis were analyzed using SPSS software (SPSS 22.0). Differences between two groups were assessed using unpaired two-tailed t tests or chi square test based on the type of the data. Data involving more than two groups were assessed using analysis of variance (ANOVA). P values < 0.05 indicated statistical signi cance.

Results
The primary safety outcome No acute allergic reactions, such as itchy rash, swelling of the throat or tongue, shortness of breath, vomiting, lightheadedness, and low blood pressure were observed within two hours after the nebulization treatment. Secondary allergic reaction was also not observed post treatment. No adverse events were reported.
MSCs therapy is considered safe for lung diseases, such as chronic obstructive pulmonary disease (COPD), Acute respiratory distress syndrome (ARDS), and Idiopathic pulmonary brosis (IPF) (20,21). Numerous completed phase I trials have demonstrated that no serious, acute, adverse events were reported in MSC therapy (22,23). Several studies have been conducted on the safety of MSCs exosomes therapy. The ndings of these studies suggested that MSC-derived exosomes could be safely and easily used in the treatment of lung diseases (24). However, the route of  Table 2). Although the CRP value before the nebulization treatment (23.00 ± 31.87) was comparatively higher than that after the treatment (5.92 ± 6.92), there was no signi cant difference between the two values (p = 0.178). Table 2 The e cacy outcomes (C-reactive protein, total white blood cell count, total lymphocyte count, the respiratory rate, the fever and shortness of breath) before and after the nebulization treatment. Before :Before nebulization treatment.
After : After nebulization treatment.
No: No shortness of breath.
Yes: Presence of shortness of breath.
Additionally there was no signi cant difference in the total white blood cell count, total lymphocyte count, fever and shortness of breath before and at the later stage of treatment. Compared to the patients who did not receive the nebulization treatment, patient 2 showed obvious absorption of pulmonary lesions. In patients who did not receive the nebulization treatment, there were presence of brous shadows in the lung lesions (Fig. 3).
Several studies have demonstrated that bone marrow-derived exosomes can reduce lung in ammation, alleviate pulmonary edema and postin ammatory complications in animal models of acute lung injury, ARDS, asthma and other in ammatory diseases. (25)(26)(27). MSC-derived exosomes usually contain bioactive substances such as mRNA, miRNA and protein (28). These substances have been shown to effectively reduce in ammatory processes and modulate airway remodeling (13,29). We also demonstrated that MSC-derived exosomes can reduce the CRP level in different degrees of patients with COVID-19 pneumonia, which was similar to the ndings of a previous study (18). Although there was a decrease in CRP level after the nebulization treatment, it did not achieve statistical signi cancedue to the small number of cases and large standard deviation. The patients who received nebulization treatment at an earlier stage showed more benefecial effects in terms of the absorption of pulmonary in ammation. Our results showed that nebulization of MSC-derived exosomes is also bene cial to the absorption of pulmonary lesions in minor cases of COVID-19 pneumonia and the reduction of cellulose residues in severe cases of COVID-19.

Time of hospitalization
The  Table 4). The largest difference was observed in case of IFN -γ post nebulization treatment. There was a two-fold increase in IFN -γ, IL-17A and TH19 after the nebulization treatment. However, NK cells showed a two-fold decrease after the treatment (Table 3). However, our research has obvious shortcomings. Due to the short duration of the outbreak in China, only seven COVID-19 pneumonia patients were included in this study. Additionally, only 3 patients underwent the nebulization of MSC-derived exosomes from the beginning of treatment. It also should be noted that we have not compared the exosomes nebulization treatment to various other administration methods in this study, such as intravenous injection.
In conclusion, nebulization MSCs exosomes is a novel method that could be used in the treatment of COVID-19 pneumonia. Our clinical study involving a small sample size, shows that this method is safe, effective and simple especially for minor cases of COVID-19 pneumonia. Nebulization of MSC-derived exosomes from the beginning of the treatment may bene t patients more effectively. Clinical data from patients were obtained after acquiring consent of patients in accordance with the protocol approved by the Ethics Committee of the ethics committee of Wuxi No.5 people's Hospital, China.

Consent for publication
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Availability of Data and Materials
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