key: cord-0984132-xxvrt41o authors: Ma, Qingle; Fan, Qin; Xu, Jialu; Bai, Jinyu; Han, Xiao; Dong, Ziliang; Zhou, Xiaozhong; Liu, Zhuang; Gu, Zhen; Wang, Chao title: Calming Cytokine Storm in Pneumonia by Targeted Delivery of TPCA-1 Using Platelet-derived Extracellular Vesicles date: 2020-05-22 journal: Matter DOI: 10.1016/j.matt.2020.05.017 sha: d3c88dbf8c201937db784f9cc2263a047c2e4723 doc_id: 984132 cord_uid: xxvrt41o Summary Pneumonia can cause high morbidity and mortality due to the uncontrolled inflammation in the lung tissue. Calming the cytokine storm may be one key to save the life of patients with severe pneumonia. Here, inspired by the intrinsic affinity of platelets to the site of inflammation, we have engineered platelet-derived extracellular vesicles (PEVs) for pneumonia-targeted drug delivery. It is demonstrated that PEVs that are easily generated from the activated platelets can selectively target pneumonia in the mice model with acute lung injury (ALI). By loading with [5-(p-Fluorophenyl)-2-ureido] thiophene-3-carboxamide (TPCA-1) that can inhibit the production of inflammatory factors, the PEVs significantly improve therapeutic benefits by inhibiting the pulmonary inflammatory cells infiltration, and calming local cytokine storm compared with the free drug-treated group. Furthermore, we find that PEVs could serve as a broad platform that can selectively target various inflammatory sites, including chronic atherosclerotic plaque, rheumatoid arthritis and wound associated with skin. Inflammation as a defense mechanism in the body is an immune response as the basis of many physiological and pathological processes. [1] [2] However, certain infections can also cause an overwhelming local/systemic inflammatory response, leading to life-threatening diseases such as the pneumonia. 3 For example, 2019 novel coronavirus (SARS-Cov-2) has infected more than 3,000,000 people and killed more than 200,000 of people in worldwide so far. 4 The mortality rate of severe patients is over 60%. 5 More and more evidences suggest that patients with severe pneumonia have cytokine storm syndrome (CSS), in which the body's immune response leads to an uncontrolled inflammation in the lung tissue. 6 As far as we know, inhibiting the cytokine storm may be one key to save the life of patients with severe pneumonia infected with highly pathogenic virus such as SARS-Cov-2. 7-10 Certain anti-inflammatory therapeutics have proven to be beneficial in the clinical treatment. [11] [12] [13] For example, Tocilizumab (IL-6 receptor blockade) and corticosteroids have been approved to treat patients with COVID-19 pneumonia in China. Although promising, current systemic treatment options for inflammation therapy in clinical often lead to high/frequent dosing and side effects. For instance, osteonecrosis often happened in severe acute respiratory syndrome (SARS) patients caused by corticosteroids usage. 14 Targeting the pneumonia to improve the efficacy, while reducing the dosage and side effects remains elusive in clinical treatment. Further efforts are demanded to develop targeting delivery systems for modulating and reducing the local inflammatory responses in pneumonia. [15] [16] [17] Cellular delivery systems have raised increasing attentions due to their excellent biocompatible and unique delivery behaviors. [18] [19] [20] [21] [22] Platelets are one kind of inherent cells in the body, which can serve as drug delivery systems are equipped with several advantages compared with other synthetic delivery systems. 19 For example, as a one role of immune cells, platelets have the intrinsic affinity to the site of inflammation. [23] [24] [25] They can bind to the activated/inflamed vascular walls through a range of receptor patterns, including CD40L, glycoproteins Ibα, αIIb and VI and P-selectin. [23] [24] [25] In light of this, we here leveraged platelet-derived extracellular vesicles (PEVs) to facilitate the delivery of anti-inflammatory agents to pneumonia upon the intravenous administration (Figure 1a) . A large number of PEVs can be readily obtained by activating the platelets in vitro. [26] [27] Interestingly, we found that the PEVs showed the excellent capacity of accumulating at the site of pneumonia. PEVs released limited inflammatory factors in the activate environment compared to platelets, enabling them as an anti-inflammatory drug carrier. By loading with anti-inflammation agents [5-(p-Fluorophenyl)-2-ureido] thiophene-3-carboxamide (TPCA-1), the TPCA-1-PEVs significantly reduced the inflammation and cytokine storm syndromes, as well as relieved symptom in the mice with acute lung injury (ALI) induced pneumonia. The therapeutic benefit of drug loaded PEVs was significant enhanced compared to that of drug alone in a mouse disease model. Furthermore, such a PEV-based platform could be made by mixing the activated platelets from the patient and anti-inflammation agents ex vivo, followed by isolation and re-infusion into the same patient for the personalized medication. Lung is a major source for cytokine storms in patients with pneumonia infected by highly pathogenic viral such as SARS-Cov-2, which can results in high morbidity and mortality. In this study, we first evaluated the cytokine storm in the lung following the acute lung injury (ALI) of mice. ALI can be caused by the pathogens, such as influenza A virus H5N1 and SARS-CoV-2, 28 which is characterized by excessive inflammatory response in the lungs that induce the dyspnea and terminal failure of the respiratory system and the acute respiratory distress syndrome (ARDS). 29 It is validated that overwhelming inflammatory reactions lead to varying degrees of lung injury. [30] [31] As a result, we confirmed that high CD45 immune cells infiltration (Figure S1a-b) and high pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β production (Figure S1c-e) in the lung tissue were induced in the ALI mice. Next, we prepared the targeted delivery platform using the platelet-derived extracellular vesicles (Figure 1a) . A large number of platelet-derived vesicles could be generated and released after platelet activation. 32-33 Here, we used thrombin to activate the platelets in vitro in this study. Preand post-activated platelets and PEVs were analyzed by the transmission electron microscopy (TEM) (Figure 1b-c) and dynamic light scattering (DLS) (Figure 1d-f, Figure S2a ). The activated platelets exhibited around 100-200 nm as shown in DLS data compared with platelets without activation which indicated the PEVs generation after activation (Figure 2e ). PEVs were then isolated from the platelet-activated supernatant by ultracentrifugation according to a standard protocol. 32 The purified PEVs showed spherical shape with sizes of about 100-150 nm (Figure 1c) compared with the non-activated platelets (Figure 1b) . To ensure the obtained vehicle was derived from platelets, protein profiles of platelets and PEVs were examined by SDS-PAGE and western blot. As expected, PEVs partially contained proteins from original platelets (Figure 1g) . The existence of CD41 on PEVs further confirmed the PEVs were derived from platelets, while some cytosolic proteins such as actin was lost in PEVs compared with platelets (Figure 1h) . Besides, PEVs were stored in various buffers at 4 o C for at least 4 days in vitro ( Figure S2b ). In the absence of platelet aggregation inhibitors PGE1, the size of PEVs did not change significantly, indicating the high stability of PEVs ex vivo. As pro-inflammatory cells, platelets may also accelerate the inflammation and progression by the release of inflammatory factors when binding to the disease site. 34 To determine whether PEVs also release pro-inflammatory cytokines upon the activation, an enzyme-linked immunosorbent assay (ELISA) was used to detect the interleukin -1β (IL-1β) and interleukin-6 (IL-6) in the supernatant with thrombin activation in PBS. Unlike platelets, PEVs did not release cytokines after thrombin treatment significantly (Figure 1i- injection at the current dosage (Figure 1k) . To mimic the inflammatory microenvironment, RAW264.7 cells were converted to activate macrophages by lipopolysaccharide (LPS) treatment. [36] [37] The activated macrophages were then incubated with platelets or PEVs labeled with 1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD) (Figure 2a-b) . Platelets were used as a positive control. It was observed by fluorescence imaging that similar to the naïve platelets, PEVs showed a higher affinity toward activated cells compared with the nonactivated macrophages (Figure 2a-b) . The targeting effect was further observed in activated endothelia, which also play a dominant role in initiating the process of inflammation (Figure 2c-d) . 38 Both platelets and PEVs could target human umbilical vein endothelial cells (HUVECs) induced by LPS. This binding ability was significantly weakened on resting activated cells or unactivated endothelial cells. Collectively, these results indicated that PEVs also had affinity with the major components of inflammation-associated cells in vitro. Nevertheless, receptors/proteins between the PEVs and inflamed cells as well as the mechanism of vesicle internalization by inflamed cell needs to be further investigated in detail. We then investigated whether PEVs could specifically target to inflammatory lungs in vivo (Figure 2e ), free DiD or DiD labeled PEVs (at the same dose of DiD determined by absorption, PEVs: 12.6 mg/kg) were injected in the acute lung injury mice intravenously. After injection for 2 h (Figure 2f -i), the lungs were collected and imaged by ex vivo imaging system. Interestingly, we observed that DiD-PEVs showed the strongest fluorescence intensity in the affected lungs compared to those in healthy mice or free dye treated ALI mice (Figure 2f-g) . Of note, in the DiD-PEVs-treated healthy mice or DiD treated ALI mice at 2 h point, most of the signals were located in the liver. In contrast, the major organ of DiD distribution was the lung in the DiD-PEVs-treated ALI mice (Figure 2h-i) , suggesting that excellent accumulation capacity of PEVs at the acute lung 6 inflammation site. The confocal images of lung tissue also showed that the enrichment of PEVs compared with free DiD treatment or in the normal lungs (Figure 2j) , further confirming the targeting capacity of PEVs to the injured lung tissue. These results can be explained by the intrinsic affinity of PEVs to the site of inflammation. They could bind to the activated/inflamed vascular walls through a range of receptor patterns, including CD40L, glycoproteins Ibα, αIIb and VI, P-selectin. [23] [24] [25] In addition, nano-sized PEVs can also passively target to the inflamed lung tissue -temporary dilated and leaky blood vessels caused by histamine in response to injury allowed the injected nano-sized PEVs to pass through the vasculature and reached the injured tissue. 39 We then hypothesized that PEVs were a platform delivery carrier for targeting drug delivery toward In vitro anti-inflammation therapeutic effect of TPCA-1-PEVs was also confirmed. All treatment could reduce the production of IL-6 and TNF-α (Figure 3e-f) . The IL-1β was not affected remarkably ( Figure S4 ), probably due to the negative regulation of NF-κB to the IL-1β secretion 43 . In addition, we studied the immune response of macrophages after the treatment of TPCA-1-PEVs. As expected, LPS-stimulated macrophages displayed a typical M1 phenotype. Addition of TPCA-1 or TPCA-1-PEVs into the LPS stimulated macrophages resulted in a lower level of CD80+, indicating that TPCA-1-PEVs inhibited the inflammatory response and M1-polarization induced by LPS (Figure 3g) . Moreover, treatment with TPCA-1-PEVs significantly reduced LPS-induced reactive oxygen species (ROS) production of macrophages (Figure 3h) . To demonstrate the therapeutic efficacy by targeting delivery, mice were challenged with LPS (8 mg/kg) to induce ALI. 30 Besides, we further measured the lung wet/dry weight ratios to observe the lung edema. Pathologic examinations revealed that the lungs of COVID-19 patients exhibited edema, which may due to the promoted mucus production and immune cells infiltration. Administration with TPCA-1-PEVs significantly reduced the lung edema compared with free drug treatment (Figure 4f , Figure S5 ). Moreover, histologic examination validated the existence of excessive pulmonary edema, alveolar inflammatory cell exudation/infiltration, and alveolar injury in the untreated ALI group (Figure 4g ). In the treatment groups, the inflammatory cell infiltration of mice receiving TPCA-1-PEVs was reduced significantly compared with the untreated ALI group and free drug treatment. As mentioned, preventing or inhibiting the cytokine storm may be one key to save the life of 8 patients with severe pneumonia. Therefore, we investigated whether the lung cytokine storm could be calmed by targeted delivery of TPCA-1 using platelet-derived extracellular vesicles. The levels of TNF-α, IL-6, and IL-1β in lung tissue homogenate were measured by enzyme linked immunosorbent assay (ELISA) following the treatments (Figure 4a) . Although the TPCA-1 has been demonstrated to decrease the cytokine storm in previous studies, the therapeutic efficacy was limited in our experimental mouse ALI model at the dosage of 1 mg/kg. The free TPCA-1 treatment moderately reduced the cytokine level. PEVs alone did not result in the reduced immune cell infiltration and cytokine concentration ( Figure S6) . Encouragingly, these inflammatory factors were significantly declined after treatment with TPCA-1-PEVs, indicating that cytokines storm was efficiently inhibited by the targeted delivery of TPCA-1 using the PEVs (Figure 5a-c) . Infiltration of immune cells is a key sign at pneumonia and associated with cytokine storm. In patients infected with SARS-CoV-2, it has been reported that the severity of pulmonary immune injury correlated with the great infiltration of neutrophils, macrophages and T cells in the lungs. 45 The persistence of immune cells in the lung after treatment was tested as well. It was observed that the total percentage of CD45+ cells at the site of lung tissue was reduced remarkably of the mice receiving the TPCA-1-PEVs compared to the TPCA-1 alone (Figure 5d-f) . We next measured macrophages in the lung. CD11b lo F4/80 hi resident macrophages, which play crucial roles in acute lung injury (ALI). In the acute phase of ALI/ARDS, resident macrophages shift into the classically activated phenotype (M1) and release various potent proinflammatory mediators. [46] [47] [48] We observed CD11b lo F4/80 hi macrophages cell subset with decreased level in mice treated with TPCA-1-PEVs compared with mice treated with free drugs (Figure 5g-h) . Furthermore, significantly higher percentage of CD14+CD45+ inflammatory infiltrating monocytes/macrophages (Figure 5i-j) 45, [49] [50] was found in the untreated mice, while the mice injected with TPCA-1-PEVs exhibited a remarkable decrease of CD14+CD45+ inflammatory immune cells. We further analyzed the CD3+CD45+ T cells in the lung (Figure 5k-l) which was significantly lower of the mice treated with TPCA-1-PEVs than that treated with TPCA-1 alone. In addition, H&E staining of other major tissues confirmed that PEVs have limited toxic effects on treated mice ( Figure S7 ). Inflammation is associated with a varying of human diseases that impact people's health and quality of life. Encouraged by our results associated with ALI, we hypothesized that PEVs could be also applied to target other inflammations. To test our hypothesis, several other inflammation disease models, including atherosclerotic plaque, rheumatoid arthritis as well as skin wound to potency of PEVs target to inflammatory site as a universal strategy. Interestingly, we found that PEVs could selectively target both chronic and/or acute inflammatory site in various disease models, including chronic atherosclerotic plaque (Figure 6a) , rheumatoid arthritis (Figure 6b ) as well as acute injured wound (Figure 6c ) compared to free dyes. Obvious accumulation of DiD-PEVs was noticed at the inflamed tissue. Our results indicated that the PEVs system may potentially provide a simple platform technique way that be useful for the inflammation disease detection and drug delivery. In summary, we have developed a pneumonia-targeting treatment strategy platform based on the Resource Availability Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Chao Wang (cwang@suda.edu.cn). This study did not generate new unique reagents. This study did not generate custom code, software, or algorithm. Lung wet/dry ratios. The tissue samples were weighed (wet weight) immediately after removal, and then dried in an oven at 45 ° C until a stable dry weight was reached after 48 hours. The ratio of wet weight to dry weight was then calculated to quantify the degree of pulmonary edema. 14 Analysis of lung tissue homogenate. The lung tissues was weighted and 10% tissue was homogenate with PBS as the homogenization medium. The MPO, MDA test followed the steps of the kit manufacturer. Inflammatory factors were detected by ELISA according to the manufacturer's protocol. All results are expressed as mean ± SD or mean ± SEM. Repeated groups were included in all experiments unless otherwise stated. When the data of the two groups were compared, the results were significant (P<0.05), multiple comparisons. Use Tukey's post hoc test to make statistical differences. Survival rates were determined using a log-rank test. All statistical analyses were performed using a Graph prism (5.0). *P<0.05, **P<0.01, ***P<0.005. 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Editorial: Alveolar Macrophages in Lung Inflammation and Resolution CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein CD14 contributes to pulmonary inflammation and mortality during murine tuberculosis (a) Photos of extracted unpurified platelets, purified platelets, and PEVs solution taken by iPhone. (b) Particle size of platelet-derived extracellular vesicles during incubation at 4 °C for 4 days in different buffers The size (a) and the zeta potential (b) of the PEVs and TPCA-1 loaded PEVs H Cell-based drug delivery systems have raised increasing attentions as biocompatible delivery vehicles. Among them, platelets have the intrinsic affinity to the site of inflammation, based on which many platelet-or platelet membrane-mediated therapeutic carriers have been developed. In this work, platelet-derived extracellular vesicles are engineered with loading of anti-inflammation therapeutics (TPCA-1) for targeted toward pneumonia treatment. We find that the PEVs showed the excellent capacity of accumulating at the site of pneumonia. These TPCA-1 loaded PEVs significantly inhibited the infiltration of pulmonary inflammatory cells and calmed local cytokine storm syndromes compared to the free drug-treated group. Furthermore, it is demonstrated that PEVs could selectively target various inflammatory sites, broadening theragnostic applications of this delivery strategy.