Cytokine levels in J774A1 macrophages and BMDC supernatants.

<p><b>Cells were incubated along 14 and 48 h with UDA, UDL, ds<i>L</i>p-UDA, ds<i>L</i>p-UDL, ds<i>L</i>p alone and LPS (TLR4 agonist) as positive control.</b> The absorbance of the basal condition (non-stimulated cells, incubated with culture media as negative control) was subtracted to that of each cytokine concentration. Each point represents the media of n = 3 and its corresponding SD. ** denotes p < 0.01, *** denotes p < 0.001; n.s. not significant. (A) TNF- α levels in J774A1 supernatants. (B) IL-12p40 levels in J774A1 supernatants. (C) IL-6 levels in J774A1 supernatants. (D) IL-1β levels in J774A1 supernatants. (E) IL-12p40 levels in BMDC supernatants. (F) IL-6 levels in BMDC supernatants.</p

Characterization of nanovesicles.

<p>(A) Gradient (7.5%-15%) PAGE of whole-cell extract (5 to 30 μg, lanes 1 to 4) and ds<i>L</i>p (5 to 30 μg, lanes 6 to 9) from <i>L</i>. <i>braziliensis</i>, separated by molecular mass markers (lane 5). (B) Calibration curve PAGE for ds<i>L</i>p (5–30 μg, lanes 1 to 4); molecular mass marker (lane 5); ds<i>L</i>p-UDA (20 μl, lanes 6 and 7); ds<i>L</i>p-UDL (20 μl, lanes 8 and 9). (C) A plot of phospholipids from nanovesicles extruded across 50 nm pore size membranes versus time. Values represented mean ± SD. (D) Hydrodynamic diameter of ds<i>L</i>p and ds<i>L</i>p- nanovesicles measured by dynamic light scattering expressed in intensity, volume and number mode. The percentages indicated the proportion of each structure.</p

Uptake of Rhodamine–PE labeled UDA and Rhodamine–PE labeled UDL by BMDC, determined by flow cytometry.

<p>Values represent mean ± SD, ** denotes p < 0.01</p

Serum IgG titers after topical application of ds<i>L</i>p-nanovesicles and intramuscular application of ds<i>L</i>p adsorbed in alum.

<p>(A) IgG isotypes. (B) Values represent mean ± SD. ** denotes p < 0.01, *** denotes p < 0.001 vs ds<i>L</i>p-UDL and ds<i>L</i>p.</p

Cytotoxicity of empty and ds<i>L</i>p- nanovesicles.

<p>(A) J774 cells. (B) HaCaT cells. (C) Bone marrow derived dendritic cells (BMDC). Values represent mean ± SD (n = 5). Not significant differences were found between treatments and control cells.</p

Structural features of nanovesicles.

<p>Structural features of nanovesicles.</p

Ultradeformable Archaeosomes for Needle Free Nanovaccination with <i>Leishmania braziliensis</i> Antigens - Fig 7

<p><b>Scheme depicting the main structural sections of the skin: <i>stratum corneum</i> (SC), viable epidermis and dermis, and barriers to permeation-penetration (not a scale):</b> The diffusive pathway across the lipids of the SC is mediated by disordered bilayers, represented by the X-ray diffraction pattern corresponding to the lateral packing in liquid phase of bilayers shown in a) (distance between planes ∼0.46 nm). Bilayers b) and c) with more organized lateral packing (distance between planes 0.41 and 0.41–0.37 nm, respectively), not involved in diffusion across the skin. The interaction between ultradeformable nanovesicles (UDN), conventional liposomes, hydrophilic solutes and lipids at the SC surface is also represented. Ultradeformable nanovesicles and hydrophilic solutes penetrate across hydrophilic leads in the canyons in-between corneocyte clusters. A scheme of ultradeformable nanovesicles and associated cargo crossing an hydrophilic channel in the SC. The colloidal structure is lost below the surface. The associated cargo penetrates along with the lipid bilayers. No endocytic uptake occurs at I and II levels since the SC is made of dead corneocytes. Below 10 μm depth, endocytic uptake of material penetrating across hydrophilic channels accessing the viable epidermis may occur. Epidermis: <i>stratum basale</i> (SB), <i>stratum spinosum</i> (SS), <i>stratum granulosum</i> (SG). (Dermo epidermal basal membrane is represented as black line and the reticular capillary plexus in the dermis as red dots)</p

Morphology of nanovesicles.

<p>(A and B) ds<i>L</i>p-UDA. (C and D) ds<i>L</i>p-UDL. (A and C) TEM images (30000 X). (B and D) Three-dimensional AFM scans.</p

Immunization scheme.

<p>Immunization scheme.</p