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1.
Eur J Pharm Sci ; 176: 106234, 2022 Sep 01.
Article in English | MEDLINE | ID: covidwho-1881967

ABSTRACT

INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.


Subject(s)
COVID-19 , Nanoparticles , Animals , Green Fluorescent Proteins/genetics , Humans , Lipids , Liposomes , Male , Mice , RNA, Messenger/genetics , RNA, Small Interfering , RNA, Viral , Respiratory System/metabolism , SARS-CoV-2
2.
American Journal of Respiratory and Critical Care Medicine ; 203(9), 2021.
Article in English | EMBASE | ID: covidwho-1277065

ABSTRACT

RATIONALE: Coronavirus disease 2019 (COVID-19) is a global pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV2). SARS-CoV-2 uses the receptor angiotensin-converting enzyme 2 (ACE2) to gain entry to host cells in the respiratory tract epithelium. Clinical features post viral infection include symptoms of viral pneumonia, fever, cough, chest discomfort, and in severe cases dyspnea and bilateral lung infiltration. Adults with any age are at risk of getting COVID-19, however co-morbidities like hypertension, COPD, smoking, type 2 diabetes, asthma, obesity etc. increase infection susceptibility. Modern day therapeutics to control aforementioned health conditions include angiotensin II receptor blockers (ARBs), ACE inhibitors (ACEi), and anti-inflammatory drugs such as dexamethasone and hydroxychloroquine. Since binding of viral spike protein with receptor ACE2 initiates viral entry in the host, hence ACE2 has been one of the main candidates to understand the mechanism of viral infection. In this study our aim was to investigate if ACE2 expression levels can be modulated with therapeutic interventions. Methods: In an in vitro model of monolayer cultures, human airway epithelial (1HAEo-) cell lines were treated with losartan and telmisartan (ARB), captopril (ACEi), and hydroxychloroquine at half-log concentrations from 1 to 100 μM, and fluticasone, ciclesonide, and dexamethasone (steroids) at half-log concentrations from 0.3 to 10 μM. Whole cell lysates were obtained at 24hr post-treatment to quantify ACE2 expression via western blotting. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test. Results: The in vitro experiments have shown that total ACE2 protein expression in 1HAEo-cells is modulated in response to certain drugs. Compared to untreated control cells, losartan treatment showed ∼45 % significant increase in ACE2 expression at both 3 μM and 10 μM concentrations (p< 0.01), whereas dexamethasone showed ∼ 40% significant increase at 10 μM dose (p< 0.001). Captopril treatment showed ∼35% decrease at 30 and 100 μM (p<0.01), whereas ciclesonide treatment demonstrated statistically significant decrease in ACE2 expression with all tested concentrations. No difference in ACE2 expression was observed with telmisartan and hydroxychloroquine treatments. Conclusion: Our findings suggest that daily medications like losartan, captopril, dexamethasone and ciclesonide for certain pre-existing conditions may modulate ACE2 protein levels in airway epithelium hence possibly priming for COVID19 infections if exposed. This affect susceptibility for COVID19 infection and severity of illness in vulnerable populations. Hence basic understanding of mechanism of action for daily standard of care prophylactic therapies can reduce or prevent SAR-CoV-2 infection in at-risk individuals.

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