Green Formulation Strategy for Preparing Oil-in-Water Emulsions via Lipase-Catalyzed Transesterification

Formulation of submicronic diglyceride-in-water emulsions was carried out without addition of synthetic surfactant in case of commercial caprylic/capric diglyceride. Sugar surfactant was prepared by contacting the oil with a concentrated aqueous solution of sorbitol (70 wt %) containing lipase AY. Interfacial lipase-catalyzed transesterification took place and led to limited but sufficient amounts of sorbitol ester and monoglyceride, which accumulated in the oil. The enzyme-treated oil could be easily separated from the aqueous phase and used for preparing oil-in-water emulsion without adding any other surfactant. A stable emulsion was obtained for at least 7 days and exhibited better stability at 60 °C than Tween 20-stabilized emulsion

Heat map display of cytokine and chemokine productions.

<p>The iDCs were treated with BCG-CS-NPS, UVI- DENV (1, 3 and 10 μg) and DNV (1, 3 and 10 μg). Supernatant was collected on day 1, 2 and 3 and cytokine production was measured by Bio-plex assay.</p

Immunogenic Properties of a BCG Adjuvanted Chitosan Nanoparticle-Based Dengue Vaccine in Human Dendritic Cells

<div><p>Dengue viruses (DENVs) are among the most rapidly and efficiently spreading arboviruses. WHO recently estimated that about half of the world’s population is now at risk for DENV infection. There is no specific treatment or vaccine available to treat or prevent DENV infections. Here, we report the development of a novel dengue nanovaccine (DNV) composed of UV-inactivated DENV-2 (UVI-DENV) and <i>Mycobacterium bovis</i> Bacillus Calmette-Guerin cell wall components (BCG-CWCs) loaded into chitosan nanoparticles (CS-NPs). CS-NPs were prepared by an emulsion polymerization method prior to loading of the BCG-CWCs and UVI-DENV components. Using a scanning electron microscope and a zetasizer, DNV was determined to be of spherical shape with a diameter of 372.0 ± 11.2 nm in average and cationic surface properties. The loading efficacies of BCG-CWCs and UVI-DENV into the CS-NPs and BCG-CS-NPs were up to 97.2 and 98.4%, respectively. THP-1 cellular uptake of UVI-DENV present in the DNV was higher than soluble UVI-DENV alone. DNV stimulation of immature dendritic cells (iDCs) resulted in a significantly higher expression of DCs maturation markers (CD80, CD86 and HLA-DR) and induction of various cytokine and chemokine productions than in UVI-DENV-treated iDCs, suggesting a potential use of BCG- CS-NPs as adjuvant and delivery system for dengue vaccines.</p></div

The expression levels of CD80 (A), CD86 (B) and HLA-DR (C) on various regimens treated iDCs.

<p>The iDCs were treated with mock, BCG-CS-NPs, UVI-DENV (1, 3 and 10 μg) and DNV (1, 3 and 10 μg) for 24, 48 and 72 h. The expressions of CD80, CD86 and HLA-DR were determined as the mean fluorescence intensity (MFI) by flow cytometry. ^a indicates significant difference between mock- and adjuvant-treated culture. *,** and *** indicate significant difference in MFI level of CD80, CD86 and HLA-DR between DNV and UVI-DENV antigen at 1 μg, 3 μg and 10 μg, respectively.</p

Morphology of DNV.

<p>Examination under SEM showed that DNV was of spherical shape and of approximate 300 nm in diameter (A = magnification at 20,000X and B = magnification at 40,000X).</p

Nanoparticle properties investigated by zetasizer.

<p>Ten microliters of CS-NPs, BCG-CS-NPs or DNV were added into 1,000 μl of 0.1 mM NaCl prior to measure their properties by zetasizer.</p

Identification of BCG-CWCs localization on BCG-CS-NPs observed with ConA induced aggregation.

<p>ConA solution was added into CS-NPs or BCG-CS-NPs to induce NPs aggregation whereas ConA+MDM was added as an inhibitor of ConA. Aggregation was measured by zetasizer at the designated time point starting from 0, 5, 10 and 20 minutes. * indicates a significant difference in particle sizeof BCG-CS-NPs in the presence or absence of ConA or ConA inhibitor. ** indicates a significant difference in particle size of CS-NPs in the presence or absence of ConA or ConA inhibitor. *** indicates a significant difference in particle size of BCG-CS-NPs and CS-NPs in the presence of ConA.</p

The presence of BCG-CWCs and UVI-DENV antigen in different NP preparations.

<p>NPs in each step of the DNV preparation were stained with anti-LAM (A) and anti-DENV (B) antibodies and the mean fluorescence intensity (MFI) were measured by flow cytometry.</p

The mean fluorescence intensity (MFI, A) and the frequency (B) of UVI-DENV positive THP-1 cells.

<p>THP1 cells were stimulated with BCG-CS-NPs (grey bar, white dots), UVI-DENV (white bar, black dots) or DNV (black grid bar) or left unstimulated (negative control, white bar) for 1 or 2 days prior to intracellularly staining with anti-DENV antibody and determined the MFI and the frequency of UVI-DENV positive cells by flow cytometry. * indicates significant differences (<i>p</i><0.05) between UVI-DENV and DNV.</p