An Advanced Lyophilization Toward Intact Lipid Nanovesicles: Liquid-mediated Freezing With Cryoprotectant to Retain the Integrity of Lipid Nanovesicles

A novel lyophilization method using liquid-mediated freezing (LMF) with cryoprotectant (CPA) was designed to achieve intact lipid nanovesicles after lyophilization. During the freezing step, CPA prevented water supercooling, and the freezing rate was controlled by LMF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933°C/min) was the optimal medium for the LMF system. Lyophilized lipid nanovesicle using both LMF and CPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 135.5 nm, polydispersity index = 0.074), similar to intact vesicles (112.3 nm and 0.184, respectively), after rehydration. Only lyophilized lipid nanovesicle using both LMF and CPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs such as a mRNA vaccine, while retaining its original activity.

Integrative Metabolomic and Lipidomic Profiling of Lung Squamous Cell Carcinoma for Characterization of Metabolites and Intact Lipid Species Related to the Metastatic Potential

SQCC is a major type of NSCLC, which is a major cause of cancer-related deaths, and there were no reports regarding the prediction of metastatic potential of lung SQCC by metabolomic and lipidomic profiling. In this study, metabolomic and lipidomic profiling of lung SQCC were performed to predict its metastatic potential and to suggest potential therapeutic targets for the inhibition of lung SQCC metastasis. Human bronchial epithelial cells and four lung SQCC cell lines with different metastatic potentials were analyzed using gas chromatography–mass spectrometry and direct infusion-mass spectrometry. Based on the obtained metabolic and lipidomic profiles, we constructed models to predict the metastatic potential of lung SQCC; glycerol, putrescine, β-alanine, hypoxanthine, inosine, myo-inositol, phosphatidylinositol (PI) 18:1/18:1, and PI 18:1/20:4 were suggested as characteristic metabolites and intact lipid species associated with lung SQCC metastatic potential. In this study, we established predictive models for the metastatic potential of lung SQCC; furthermore, we identified metabolites and intact lipid species relevant to lung SQCC metastatic potential that may serve as potential therapeutic targets for the inhibition of lung SQCC metastasis.

Intact Lipid Rafts Regulate HIV-1 Tat Protein-Induced Activation of the Rho Signaling and Upregulation of P-Glycoprotein in Brain Endothelial Cells

The Rho signaling has an essential function in human immunodeficiency virus (HIV)-1-mediated disruption of the integrity of the blood–brain barrier (BBB). However, it is unknown how membrane domains, such as lipid rafts, can influence HIV-1-mediated activation of the Rho pathway and how these processes can affect the expression of the efflux transporters at the BBB level. This study is focused on the function of HIV-1 protein Tat in activation of the Rho signaling and upregulation of P-glycoprotein (P-gp) in human brain endothelial cells. Treatment with Tat markedly elevated GTP-RhoA levels and the potential downstream effectors, such as myosin phosphatase target subunit 1 and myosin light chain. In addition, Tat upregulated expression and promoter activity of P-gp as well as its efflux function. Inhibition of the Rho signaling cascade effectively blocked P-gp overexpression at the level of promoter activity. Disruption of lipid rafts by depletion of membrane cholesterol by methyl-beta-cyclodextrin, but not caveolin-1 silencing, also abolished Tat-mediated RhoA activation and P-gp upregulation. The present data indicate the critical function of intact lipid rafts and the Rho signaling in HIV-1-mediated upregulation of P-gp and potential development of drug resistance in brain endothelial cells.