Rationale: Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19), a predominantly respiratory illness. The first step in SARS-CoV-2 infection is binding of the virus to ACE2 (angiotensin-converting enzyme 2) on the airway epithelium. Objectives: The objective was to gain insight into the expression of ACE2 in the human airway epithelium. Methods: Airway epithelia sampled by fiberoptic bronchoscopy of trachea, large airway epithelia (LAE), and small airway epithelia (SAE) of nonsmokers and smokers were analyzed for expression of ACE2 and other coronavirus infection–related genes using microarray, RNA sequencing, and 10x single-cell transcriptome analysis, with associated examination of ACE2 -related microRNA. Measurements and Main Results: 1 ) ACE2 is expressed similarly in the trachea and LAE, with lower expression in the SAE; 2 ) in the SAE, ACE2 is expressed in basal, intermediate, club, mucus, and ciliated cells; 3 ) ACE2 is upregulated in the SAE by smoking, significantly in men; 4 ) levels of miR-1246 expression could play a role in ACE2 upregulation in the SAE of smokers; and 5 ) ACE2 is expressed in airway epithelium differentiated in vitro on air–liquid interface cultures from primary airway basal stem/progenitor cells; this can be replicated using LAE and SAE immortalized basal cell lines derived from healthy nonsmokers. Conclusions: ACE2 , the gene encoding the receptor for SARS-CoV-2, is expressed in the human airway epithelium, with variations in expression relevant to the biology of initial steps in SARS-CoV-2 infection.
Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.
Folate and retinoic acid grafted/dextran (FA-RA/DEX) copolymers with different molecular weight of DEX were synthesized using carbonyldiimidazole and dimethylaminopyridine for targeted delivery of doxorubicin (DOX) in acute myelogenous leukemia (AML). The copolymers structure was confirmed by 1H NMR and FTIR. Critical micelle concentration (CMC) of each copolymer was determined using pyrene as a fluorescent probe. DOX was loaded in micelles by the direct dissolution method. Physical properties of micelles, including particle size, zeta potential, drug loading efficiency, and drug release profiles, were examined. The orientation of the folate ligand on the surface of the micelles was studied by X-ray photoelectron spectroscopy (XPS) technique. The cytotoxicity of micelles loaded with DOX at different concentrations was studied in KG1 cells using MTT assay and their cellular uptake by flow cytometry technique. FTIR and 1H NMR spectra confirmed successful production of the targeted micelles and XPS spectra showed the surface orientation of folate. R15D10F7 copolymer produced micelles with particle size of 82.86 nm, polydispersity index of 0.3, zeta potential of −4.68 mV, drug loading efficiency of 96%, and release efficiency of 63%. DOX loaded in folate-targeted micelles of RA/DEX was more toxic than that in nontargeted micelles and free drug and seems promising in reducing drug resistance in AML.
A water-insoluble anti-tumor agent, paclitaxel (PTX) was successfully incorporated into noveltargeted polymeric micelles based on tocopherol succinate-chitosan-polyethylene glycol-folic acid (PTX/TS-CS-PEG-FA). The aim of the present study was to evaluate the pharmacokinetics, tissue distribution and efficacy of PTX/TS-CS-PEG-FA in comparison to Anzatax Õ in tumor bearing mice. The micellar formulation showed higher in vitro cytotoxicity against mice breast cancer cell line, 4T1, due to the folate receptor-mediated endocytosis. The IC 50 value of PTX, a concentration at which 50% cells are killed, was 1.17 and 0.93 mM for Anzatax Õ and PTX/TS-CS-PEG-FA micelles, respectively. The in vivo anti-tumor efficacy of PTX/TS-CS-PEG-FA, as measured by reduction in tumor volume of 4T1 mouse breast cancer injected in Balb/c mice was significantly greater than that of Anzatax Õ . Pharmacokinetic study in tumor bearing mice revealed that the micellar formulation prolonged the systemic circulation time of PTX and the AUC of PTX/TS-CS-PEG-FA was obtained 0.83-fold lower than Anzatax Õ . Compared with Anzatax Õ , the V d , T 1/2ß and MRT of PTX/TS-CS-PEG-FA was increased by 2.76, 2.05 and 1.68-fold, respectively. As demonstrated by tissue distribution, the PTX/TS-CS-PEG-FA micelles increased accumulation of PTX in tumor, therefore, resulted in anti-tumor effects enhancement and drug concentration in the normal tissues reduction. Taken together, our evaluations show that PTX/TS-CS-PEG-FA micelle is a potential drug delivery system of PTX for the effective treatment of the tumor and systematic toxicity reduction, thus, the micellar formulation can provide a useful alternative dosage form for intravenous administration of PTX. KeywordsBiodistribution, in vivo anti-tumor effect, Paclitaxel, pharmacokinetics, targeted polymeric micelle History
The aim of this study was to develop chitosan derivative polymeric micelles for co-delivery of paclitaxel (PTX) and α-tocopherol succinate (α-TS) to the cancer cells to improve the therapeutic efficiency and reduce side effects of PTX. In this study, amphiphilic tocopheryl succinate-grafted chitosan oligosaccharide was synthesized and physically loaded by PTX and α-TS with entrapment efficiency of 67.9% and 73.2%, respectively. Physical incorporation of α-TS into the micelles increased the hydrophobic interaction between PTX and the micelles core, which improved micelle stability, reduced the micelle size and also sustained the PTX release from the micelles. The mean particle size and zeta potential of αTS/PTX-loaded micelles were about 133 nm and +25.2 mV, respectively, and PTX release was completed during 6-9 d from the micelles. Furthermore, the cytotoxicity of α-TS/PTX-loaded micelles against human ovarian cancer cell line cancer cell in vitro was higher than that of PTX-loaded micelles and the free drug solution. Half maximal inhibitory concentration values of PTX after 48-h exposure of the cells to the PTX-loaded micelles modified and unmodified with α-TS were 110 and 188 ng/ml, respectively.
The objective of this study was the synthesis of folic acid- (FA-) targeted polymeric micelles of Synperonic PE/F 127-cholesteryl hemisuccinate (PF127-Chol) for specific delivery of docetaxel (DTX). Targeted or nontargeted micelles loaded with DTX were prepared via dialysis method. The effects of processing variables on the physicochemical properties of targeted micelles were evaluated using a full factorial design. After the optimization of the polymer/drug ratio, the organic solvent type used for the preparation of the micelles, and the temperature of dialyzing medium, the in vitro cytotoxicity and cellular uptake of the optimized micelles were studied on B16F10 melanoma cells by flow cytometry and fluorescent microscopy. The anticancer efficacy of DTX-loaded FA-PF127-Chol was evaluated in mice bearing melanoma tumor. Optimized targeted micelles had the particle size of 171.3 nm, zeta potential of −7.8 mV, PDI of 0.325, and a high encapsulation efficiency that released the drug within 144 h. The MTT assay indicated that targeted micelles carrying DTX were significantly more cytotoxic, had higher cellular uptake, and reduced the tumor volume significantly more than the nontargeted micelles and the free drug. FA-PF127-Chol could be, therefore, a promising biomaterial for tumors overexpressing folate receptors.
Background: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. Methods: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. Results: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. Conclusions: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.
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