RESUMO
The ultradeformable archaeosomes (UDA, made of total polar archaeolipids (TPA) extracted from the extreme halophile archaea Halorubrum tebenquichense:soybean phosphatidylcholine (SPC):sodium cholate (NaChol), 3:3:1 w:w), are promising topical adjuvants showing high deformability, an essential property for intact skin penetration up to the viable epidermis/dermis. To gain insights on UDA structure, the interactions between TPA, SPC and the edge activator NaChol, were assessed by electrospray ionization mass spectroscopy (ESI-MS) and confocal fluorescence microscopy of giant unilamellar vesicles (GUV). The non covalent heterodimers NaChol-SPC, NaChol-phosphatidylglycerophosphate methyl ether (PGPMe), NaChol-sulfated diglycosyl diphytanyl-glycerol diether (SDGD5) and SPC-PGPMe detected in the gas phase by ESI-MS after direct infusion of UDA, together with the homogeneous partition of FASTDiO and DiIC18 in GUV suggested that in these proportions, lipids and NaChol were miscible. We propose therefore, a model where in UDA the SPC diluted sufficient enough in the rich PGPMe TPA, so as to the low lateral mobility of molecules (typical of rich in PGPMe bilayers) was no longer experienced. We also found that 50µm deep within in vitro human skin canyons, the fluorescence of Alexa fluor 647-ovalbumin in UDL was â¼1.5 folds higher than in UDA, indicating a potential steric hindrance of the voluminous structure of PGPMe UDA bilayer, to the penetration of a particulate cargo such as the 7nm diameter ovalbumin. According to these observations, a further reduction in PGPMe - a lipid playing no immune role - content could help to improve the performance of UDA as topical adjuvants.
Assuntos
Sistemas de Liberação de Medicamentos , Halorubrum/química , Lipídeos/química , Lipossomos/química , Ovalbumina/administração & dosagem , Ovalbumina/farmacologia , Administração Cutânea , Adulto , Animais , Galinhas , Feminino , Humanos , Pessoa de Meia-Idade , Pele/citologia , Pele/efeitos dos fármacos , Colato de Sódio/química , Espectrometria de Massas por Ionização por Electrospray , Lipossomas Unilamelares/químicaRESUMO
Ultradeformable archaeosomes (UDA) are vesicles made of soybean phosphatidylcholine (SPC), sodium cholate (NaChol) and polar lipids from Halorubrum tebenquichense (3:1:3 wt/wt). Although ultradeformable liposomes (UDL, made of SPC and NaChol at 6:1 wt/wt) and UDA were neither captured nor caused cytotoxicity on keratinocytes, UDA was avidly captured by macrophages, their viability being reduced by 0.4-1.6 mg/mL phospholipids by 25 to 60%. Instead, UDL were poorly captured and caused no toxicity. Balb/C mice immunized by the topical route with four doses of ovalbumin (OVA)-loaded UDA, at 75 µg OVA/600 µg phospholipids (125 nm mean size and -42 mV zeta potential), induced IgG titers tenfold to 100-fold higher than those immunized with OVA-loaded UDL at the same dosage. Both matrices penetrate to the same skin depth (nearly 10 µm after 1 hour on excised human skin), being the higher topical adjuvancy and higher phagocytic uptake of UDA related to its glycolipid content. FROM THE CLINICAL EDITOR: This work summarizes key findings related to the development of ultradeformable archaeosomes as vehicles utilized in transdermal delivery systems with improved skin penetration.
Assuntos
Lipídeos , Lipossomos , Fosfatidilcolinas , Colato de Sódio , Administração Tópica , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Feminino , Halorubrum/química , Humanos , Queratinócitos/efeitos dos fármacos , Lipídeos/administração & dosagem , Lipídeos/química , Lipossomos/administração & dosagem , Camundongos , Fosfatidilcolinas/administração & dosagem , Fosfatidilcolinas/química , Absorção Cutânea/fisiologia , Colato de Sódio/administração & dosagem , Colato de Sódio/química , VacinaçãoRESUMO
The Belousov-Zhabotinsky reaction was studied under the influence of nanometric confinements induced by a complex polymer, the PAMAM-G4 dendrimers. They are well-defined in both molecular weight and architecture and are capable of molecular inclusion, making "unimolecular active micelles". The effect of such nanocompartments in the BZ reaction is analyzed by changing both the excitability and the concentration of the dendrimer, obtaining a wide range of behaviours, ranging from stationary Turing-like patterns to time dependent structures, such as jumping waves or packet waves.
Assuntos
Dendrímeros/química , Nylons/química , Bromatos/química , Compostos de Sódio/química , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 microg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 microg of bare BSA, were determined. RESULTS: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 +/- 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37 degrees C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. CONCLUSION: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.