key: cord-0885827-oazlscz0 authors: Niemiec, Stephen M.; Hilton, Sarah A.; Wallbank, Alison; Azeltine, Mark; Louiselle, Amanda E.; Elajaili, Hanan; Allawzi, Ayed; Xu, Junwang; Mattson, Courtney; Dewberry, Lindel C.; Hu, Junyi; Singh, Sushant; Sakthivel, Tamil S; Seal, Sudipta; Nozik-Grayck, Eva; Smith, Bradford; Zgheib, Carlos; Liechty, Kenneth W. title: Cerium oxide nanoparticle delivery of microRNA-146a for local treatment of acute lung injury() date: 2021-03-20 journal: Nanomedicine DOI: 10.1016/j.nano.2021.102388 sha: 7bf077df77527713a954d8502994f9fecabf4d10 doc_id: 885827 cord_uid: oazlscz0 Acute respiratory distress syndrome (ARDS) is a devastating pulmonary disease with significant in-hospital mortality and is the leading cause of death in COVID-19 patients. Excessive leukocyte recruitment, unregulated inflammation, and resultant fibrosis contribute to poor ARDS outcomes. Nanoparticle technology with cerium oxide nanoparticles (CNP) offers a mechanism by which unstable therapeutics such as the anti-inflammatory microRNA-146a can be locally delivered to the injured lung without systemic uptake. In this study, we evaluated the potential of the radical scavenging CNP conjugated to microRNA-146a (termed CNP-miR146a) in preventing acute lung injury (ALI) following exposure to bleomycin. We have found that intratracheal delivery of CNP-miR146a increases pulmonary levels of miR146a without systemic increases, and prevents ALI by altering leukocyte recruitment, reducing inflammation and oxidative stress, and decreasing collagen deposition, ultimately improving pulmonary biomechanics. . CNP, when conjugated to miRNA, may protect the miRNA from oxidative damage while neutralizing its negative charge to promote cellular uptake (21) (22) (23) (24) . miR146a is an anti-inflammatory miRNA that inhibits tumor necrosis factor 6 (TRAF6) and interleukin-1 receptor associated kinase 1 (IRAK1), promotors of NFκB (25). Downstream, miR146a therefore plays an intrinsic role in downregulating pro-inflammatory mediators IL-6, IL-8, and TNFα (25). miR146a has been shown to be modestly upregulated in ALI to downregulate inflammatory pathways and correct injury, and upregulation of miR146a has shown promise in downregulating pro-inflammatory pathways in models of ALI (26, 27) . We have conjugated the anti-inflammatory miR146a to CNP, termed CNP-miR146a, to synergistically target the oxidative stress and inflammation seen in acute lung injury and ARDS. We hypothesize that intratracheal delivery of CNP-miR146a will result in local increases in miR146a levels without systemic exposure to the treatment. To evaluate the therapeutic effects on preventing lung injury with local delivery of CNP-miR146a, we will utilize a well-established murine model of ALI/ARDS with toxic exposure to bleomycin. This injury results in a significant inflammatory and oxidative stress response that later progresses to fibrosis, as is seen in ARDS. We hypothesize that intratracheal administration of CNP-miR146a after bleomycin-induced acute lung injury reduces the inflammatory infiltrate, decreases pro-inflammatory cytokine levels, and lowers oxidative stress, thereby preventing alveolar damage and improving pulmonary function. Cerium oxide nanoparticles were synthesized and oxidized, as previously described (28, 29) . Briefly, CNP was synthesize using a precipitation method. Ce(NO) 3 , 6H 2 O was dissolved in deionized in water and oxidized from cerium (III) ions to cerium (IV) oxide with the use of excess hydrogen peroxide. The solution is then maintained at a pH below 3.5 to maintain a suspension of ceria nanoparticle. Upon oxidation, the crystalline nanoparticles precipitate and were isolated by centrifugation at 8000 g. Five mM of nanoceria suspension was diluted in pure water to obtain a final nanoceria concentration of ten µM. Conjugation of CNP to miR146a was performed by utilizing the activated form of 1,1-carbonyldiimidazole (CDI) to couple the amino group of miR146a to the CNP hydroxyl group. The conjugated CNP-miR146a was diluted to 100ng/50µL in J o u r n a l P r e -p r o o f Journal Pre-proof phosphate buffered saline (PBS) for intratracheal dosing. An MTT assay for CNP-miR146a toxicity was performed in vitro, with a full description of MTT assay methods can be found in Supporting Information (SI). Eight-to ten-week-old male mice (C57BL/6, strain No. 000642, Jackson Laboratory) were used after allowing the animals to acclimate to our 1600 m altitude for 1 week. Care of the animals was in accordance with the guidelines defined in the NIH Guide for the Care and Use of Laboratory Animals. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at University of Colorado Denver -Anschutz Medical Campus (License #84-R-0059). Each measurement was taken from distinct samples. Uninjured mice were given intratracheal delivery of 100 ng CNP-miR146a in 50µL PBS (n = 5 for all timepoints). Animals were euthanized at 0, 3, 12, 24, 72, and 168 hours after treatment for whole lung harvest and blood collection. Mice harvested at 0, 12, and 24 hours had liver, kidneys, heart, and spleen harvested. Organ tissue was processed for total RNA extraction by homogenizing in Qiazol (Qiagen) per manufacturer's instructions. Isolated RNA samples were diluted to 5ng/µL by serial dilution and converted to U6 and miR146a cDNA (Applied Biosystems RT kit), which was amplified by reverse transcriptase amplification using the BioRad CFX-9600 thermal cycler. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for miR146a with the U6 housekeeper gene used for normalization. PCR analysis was performed in triplicate with the average of each triplicate used for normalization. Additional methods for comparative pathology of control and CNP-miR146a treated mice is included in SI. Prior to lung harvest in the above mice, blood was collected via right atrial puncture in an EDTA lined syringe and tube. Blood samples were centrifuged at 3000rpm for 15 minutes and the plasma layer was isolated. A second centrifugation at 3000rpm for 5 minutes was performed and the collected plasma was Bleomycin is a well-accepted model of acute lung injury that produces a short-term inflammatory response followed by long-term pulmonary fibrosis, a similar pattern seen in severe cases of ALI/ARDS (30) (31) (32) . Mice were injured with five units/kg bleomycin or an equivalent weight-based volume of PBS (Control group). The bleomycin-injured mice were further divided into four treatment groups. One group received no treatment (Bleo group). The remaining group received a concurrent treatment at time of injury of 100ng CNP-miR146a (Bleo + CNP-miR146a group), 100ng CNP alone (Bleo + CNP alone), or 20 ng miR146a alone (Bleo + miR146a), where CNP and miR146a are the component doses of the conjugate treatment, CNP-miR146a, which were determined by ultraviolet vis spectrophotometry and hydrodynamic size measurement. Lung tissue harvested 7 days following injury (n = 5, 10, 7, 7, 11 for control, bleo, bleo + CNP, bleo + miR146a, bleo + CNP-miR146a) was homogenized in sucrose buffer in a 1:6 ratio of lung weight to buffer volume (0.25M sucrose, 10mM TRIS-HCl, 1mM EDTA, pH = 7.4). 60μL of lung homogenate for each sample was mixed with 140μL of Krebs-HEPES buffer (KHB) consisting of 100μM DTPA and 0.2mM CMH. A blank sample of 200μL KHB was used for background signal subtraction. The samples were incubated for 1 hour at 37 o C and 150μL samples were loaded in PTFE tubing with rubber seal stoppers (33) . ROS production was measured by EPR spectrometry using the superoxide selective spin probe 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethylpyrrolidine (CMH). The Bruker EMX nano X-band spectrometer was used at 77 K with Bruker liquid nitrogen Finger Dewar to detect ROS as CM•. EPR acquisition parameters were: microwave frequency = 9.65 GHz; center field = 3438 G; modulation amplitude = 4.0 G; sweep width = 150 G; microwave power = 0.316 mW; total number of scans = 10; sweep time = 60 s; and time constant = 1.28 ms. CM• nitroxide radical concentration was obtained by double integration followed by Spin count (Bruker). Lung tissue used for RT-PCR was collected 3 days, 7 days, and 14 days following bleomycin-induced lung injury (n = 7 for all treatment groups, n = 14 at 7 days for the Bleo group). Harvested lung tissue was homogenized and processed for RT-qPCR as described above. Normalization was attained using the housekeeper genes GAPDH for mRNA. Expression analysis of pro-inflammatory TNFα, IL-6, and IL-8, and J o u r n a l P r e -p r o o f Journal Pre-proof pro-fibrotic TGFβ-1, Col1α2, and Col3α1 was performed. All samples were examined in triplicate and the average of each triplicate was used for statistical analysis of each sample. Lung tissue was harvested at time of euthanasia 14 days after injury. The left lung was inflated with melted agarose solution with care to not overinflate the lung. Once inflated, the lung was removed and placed in 4% Paraformaldehyde (PFA) in 10mL sterile PBS incubated on ice. The lung was kept in PFA at room temperature for 24 hours, then dehydrated in 70% EtOH, and embedded in paraffin prior to sectioning at 4μm. Slides were stained with Mason's trichrome, which stains collagen fibers blue. Twenty high-power fields at 400x total magnification were randomly imaged for each sample by an independent researcher. A second researcher then used an automated algorithm on NIS Elements -Advanced Research imaging software to quantify collagen deposition by area of blue staining. The average area per HPF was averaged for each sample prior to comparative analysis (n = 7 for all treatment groups). Slides used for immunohistochemistry were deparaffinized and placed in a citrate buffer (pH 6.0). Biocare Medical's Decloaker was used to retrieve the heat-induced epitope and slides were stained with Leica's Bond Rx instrument. Primary CD45 antibodies at 1:50 solutions (BD Biosciences) were applied to the slides, and the slides were developed with a Vectastain Elite ABC kit (Vector Laboratories). Slides were analyzed as described above for the number of CD45-positive cells per high-power field by evaluating twenty random high-power fields at 400x total magnification and averaging counts for each sample with the use of two independent researchers (n=7 for all treatment groups). Fourteen days after lung injury a cohort of mice were invasively ventilated to measure lung function (n = 12, 8, 6, 5, 11 for control, bleomycin, bleomycin + CNP, bleomycin + miR146a, bleomycin + CNP-miR146a groups). Mice were anesthetized with 100 mg/kg ketamine, 16 mg/kg xylazine, and 3 mg/kg acepromazine via intraperitoneal (IP) injection. Following tracheostomy with an 18-gauge metal cannula, the mice were ventilated with a computer-controlled rodent ventilator (SCIREQ flexiVent). Respiratory drive was suppressed with 0.8 mg/kg IP pancuronium bromide and the electrocardiogram was used to monitor anesthesia. A baseline ventilation with a tidal volume 10 ml/kg at 200 breaths per minute and PEEP = 3 cmH 2 0 was applied for 10 J o u r n a l P r e -p r o o f Journal Pre-proof lung 72 hours after administration compared to baseline levels ( Figure 1C , p = 0.0234), however there was no change in systemic plasma levels over the course of 168 hours. The maximum average plasma concentration was 0.9623 ng/µL 12 hours after CNP-miR146a administration, but this was not significantly different from baseline ( Figure 1D , p = 0.2859). Comparative pathology analysis of mice treated with saline or CNP-miR146a is included in SI. Histologic evidence of inflammatory cell infiltration was evaluated using CD45+ staining in lung tissue harvested 14 days following bleomycin injury (Figure 2A The concentration of CM• following oxidation of 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5tetramethylpyrrolidine (CMH), a superoxide selective nitroxide probe, was measured in lungs harvested at 14 days following injury using electron paramagnetic resonance (EPR) spectrometry. Bleomycin-injured lungs had higher nitroxide concentrations of 169μM than control concentrations of 126μM (p = 0.0239, 95% CI: [-82.05, -4.161]). All treatment groups had lower nitroxide concentrations than bleomycin-injured lungs as depicted in Figure 3 (p < 0.0001, F = 11.12, R 2 = 0.5596). To evaluate the effects of CNP-miR146a on pro-inflammatory gene expression, real-time quantitative polymerase chain reaction (RT-qPCR) was performed on right lung digests. Figure 4 RT-qPCR was performed on pro-fibrotic genes TGFβ-1, Col1α2, and Col3α1. TGFβ-1 expression was significantly elevated three days after injury in the bleomycin group compared to controls (p = 0.0464, 95% CI: Histologic evaluation of inflation-fixed lung tissue was performed on tissue harvested 14 days following injury to assess overall architectural change and the degree of fibrosis. 4μm sections were trichrome stained and area of collagen per HPF was quantified for each treatment group 14 days following bleomycin injury using NIS-Elements -Advanced Research computer software. Bleomycin injury increased collagen levels compared to controls per HPF (p = 0.0354, 95% CI: [33.24, 1313 ]. There was a significant reduction in area of collagen J o u r n a l P r e -p r o o f Journal Pre-proof per HPF with CNP-miR146a treatment (p = 0.0125, 95% CI: [-1408, -127.7]) compared to bleomycin-injured mice ( Figure 5F ). Pulmonary function was measured during invasive mechanical ventilation 14 days after bleomycin injury ( Figure 6 ). Quasi-static pressure-volume loop analysis shows that, compared to uninjured controls, the bleomycin-injured mice had significantly lower compliance at 5 cmH20 on the expiratory limb (p < 0.0001, 95% In this experiment, we have shown that intratracheal administration of CNP-miR146a localizes to the lung and prevents bleomycin-induced lung injury through downregulation of inflammation and oxidative stress. CNP-miR146a is the conjugation of multivalent CNP to the anti-inflammatory miR146a. The conjugate size on average is 190nm, which has previously been described in detail with Transmission Electron Microscopy (TEM) (35) . CNP has previously been shown to be taken up by endocytosis, and cellular toxicity in various cell lines, including a human lung cancer cell line A549, which showed no toxicity after 24 hours of CNP exposure at concentrations up to 2000 µg/mL, 1000 times the concentration of our tested IT dose, consistent with our data demonstrating no cellular toxicity with CNP or CNP-miR146a ( Figure S1 ) (36, 37) . The 100ng dose chosen for this study was based on our previous work with CNP-miR146a in diabetic wounds (38). A single IT application of 100ng/50µL CNP-miR146a successfully prevented bleomycin-induced lung dysfunction by down regulating inflammatory gene signaling, lowering oxidative stress, and preventing significant fibrotic changes. We plan to test optimal dosing in a future study where treatments are administered days after onset of injury, a more clinically relevant model. CNP conjugation to miR146a neutralizes the negative charge on the anti-inflammatory miRNA, allowing for stable uptake of miR146a in the lung at twelve hours after intratracheal administration without significant changes in pulmonary cerium concentration. The level of miR146a in the lung normalizes by 24 hours after intratracheal delivery, which would allow for repeat dosing in a clinical setting should there be ongoing injury. Importantly, there is no significant increase in miR146a in major organ systems outside of the lung or cerium within the plasma. Additionally, four weeks after therapy, there was no histopathologic injury difference between PBS and CNP-miR146a therapy in the brain, heart, lung, spleen, kidney, jejunum, or ileum (Table S1 ). The lack of systemic increase in cerium and miR146a, and lack of systemic toxicity, highlights the benefit of local administration directly to the site of injury. Bleomycin-induced lung injury results in a robust inflammatory response with increased inflammatory cell recruitment. Leukocytes, identified histologically using CD45+ staining, play a critical role in the development of ALI. Alveolar macrophages, for instance, play a critical role in both the development of ALI and its resolution as it transitions from a pro-inflammatory activation state to a pro-resolving/anti-inflammatory phenotype (39, 40) . Nanoceria has been shown to inhibit NFκB, which lowers important chemokines involved in leukocyte recruitment, such as IL-6 and IL-8 (16) . We see a robust infiltration of leukocytes on histologic analysis of lung tissue 14 days following injury, and treatment with CNP-miR146a reduced this leukocyte infiltrate, as did CNP alone ( Figure 2 ). Leukocytes play an important role in free radical production, and excess oxidative stress is intrinsic to the pathogenesis of ALI and ARDS, with higher levels of ROS promoting the release of pro-inflammatory cytokines (39) . CNP, miR146a, and CNP-miR146a all lowered CM• nitroxide production to control levels ( Figure 3 ). The reduction in ROS by CNP could be secondary to its multivalency, as has been seen in other disease models (41) , as well as its anti-inflammatory properties and reduction in leukocyte recruitment. We have previously shown that CNP-miR146a targets the NF-κB pro-inflammatory pathway by inhibiting TRAF6 and IRAK1 (38). Downregulation of these pathways by miR146a have been shown to lower ROS levels in models of sepsis-induced cardiac injury (42) . Furthermore, NF-κB is known to promote IL-6, IL-8, and TNFα J o u r n a l P r e -p r o o f cytokine production, which play significant roles in the pathogenesis of ALI (43, 44) . We found that treatment with CNP-miR146a at the time of injury is able to significantly reduce IL-6 gene expression three days following bleomycin injury. Furthermore, treatment with CNP-miR146a reduced IL-8 and TNFα gene expression seven days following injury. Preventing the early increase in IL-6, which is an early acute-phase reactant, may prevent an inflammatory cascade and prevent persistent elevation of IL-8 and TNFα at later timepoints ( Figure 4 ). Ultimately, the goal of treatment with CNP-miR146a is to improve long-term outcomes following ALI by decreasing fibrosis and improving pulmonary biomechanics, and long-term morbidity seen in ALI/ARDS is often driven by lung structural changes, such as interstitial fibrosis. CNP-miR146a lowers early TGFβ-1 signaling three days after injury and Col1α2 gene expression seven days following injury (Figures 4 and S3 ). This reduction in pro-fibrotic gene signaling correlated with reductions in collagen quantification on histologic analysis ( Figure 5 ). While trichrome staining is a pan-collagen stain, the reduction in Col1α2 gene expression seen with CNP-miR146a treatment supports that the early reduction of inflammatory gene expression may result in a reduction of ROS production and pathologic remodeling. The potential of CNP-miR146a in a clinical setting is highlighted by the prevention of pulmonary mechanical dysfunction with CNP-miR146a. While CNP alone and miR146a alone additionally lower ROS and inflammatory signaling, we found only the conjugate treatment to be effective in improving pulmonary mechanics ( Figure 6 ). Treatment with CNP-miR146a at the time of injury improved pulmonary elastance, inspiratory capacity, pulmonary compliance, and tissue resistance. While CNP alone lowered inflammatory cell infiltrate and ROS levels, and miR146a alone lowered pro-inflammatory gene signaling, only the conjugate treatment improved pulmonary function. By lowering inflammatory signaling and collagen deposition through the synergistic effects of CNP and miR146a, CNP-miR146a prevents acute lung injury and protects pulmonary function. This could prevent the need for mechanical ventilation or ICU admission, and prevent the need for long-term pulmonary rehabilitation. Despite advances in mechanical ventilation and critical care, the progression of acute lung injury to ARDS results in devastating morbidity and an in-hospital mortality of nearly 40% (45) . There is a pressing need to develop novel therapeutics that may prevent ALI and protect pulmonary function. We have shown that Relative gene expression of pro-inflammatory markers 3-(A-D), 7-(E-H), and 14-days (I-L) after bleomycin injury with comparison to GAPDH housekeeping gene expression. Bleomycin injury significantly raised IL-6, IL-8, TNFα, and TGFβ-1 gene expression compared to controls. IL-6 and TGFβ-1 relative gene expression were significantly reduced with CNP-miR146a 3 days following injury, while IL-8 and TNFα gene expression were significantly lowered with CNP-miR146a treatment 7 days following injury. CNP and miR146a mimetic also J o u r n a l P r e -p r o o f lowered IL-6 gene expression at day 3, while miR146a mimetic also lowered IL-8 and TNFα gene expression 7 days following injury. Bars indicate statistical significance with p < 0.05. Mean and standard deviation for n=7 samples is shown. Bleomycin-injured mice had significantly worse pulmonary compliance, tissue resistance, elastance, and inspiratory capacity compared to controls, while prevention treatment with CNP-miR146a improved pulmonary mechanics compared to untreated, bleomycin-injured lungs and returned function to control levels. Bars indicate statistical significance with p < 0.05. Mean and standard deviation for n = 12, 8, 6, 5, 11 for control, bleomycin, bleomycin + CNP, bleomycin + miR146a, bleomycin + CNP-miR146a samples are shown. J o u r n a l P r e -p r o o f Graphical Abstract: Bleomycin-induced lung injury produces a robust inflammatory response with increases in oxidative stress and pro-inflammatory markers such as IL-6. This leads to later remodeling with fibrosis and decreased lung function. Intratracheal delivery of cerium oxide nanoparticles (CNP) conjugated to microRNA-146a mitigates this initial inflammatory response, lowering later fibrosis and improving lung function. Diffuse alveolar damage--the role of oxygen, shock, and related factors. 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editing Data curation, investigation, methodology, project administration, writing -review & editing Ayed Allawzi: Data curation, investigation, methodology, project administration, writing -review & editing Junwang Xu: Conceptualization, resources, supervision, writing -review & editing Data curation, investigation, methodology, project administration, writing -review & editing Dewberry: Data curation, investigation, methodology, project administration, writing -review & editing Data curation, investigation, methodology, project administration, resources, writing -review & editing Data curation, investigation, methodology, project administration, resources, writing -review & editing Data curation, investigation, methodology, project administration, resources, writingreview & editing Conceptualization, funding acquisition Conceptualization, funding acquisition, project administration, supervision, validation, writing -review & editing Sudipta Seal: Conceptualization, funding acquisition, project administration, supervision, validation, writingreview & editing The authors would like to thank the University of Colorado Denver Histology Shared Resource Center for their contribution to this work.