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Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy.

ABSTRACT: 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.

SUBMITTER: Gonzalez RO

PROVIDER: S-EPMC2739508 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy.

Gonzalez Raul O RO Higa Leticia H LH Cutrullis Romina A RA Bilen Marcos M Morelli Irma I Roncaglia Diana I DI Corral Ricardo S RS Morilla Maria Jose MJ Petray Patricia B PB Romero Eder L EL

BMC biotechnology 20090813

<h4>Background</h4>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 ...[more]

PMID: 19678953

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Project description:Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from -48 mV at 37 °C to -16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid 'flip-flop'. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell's cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42-44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions.

Project description:Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular compartments.

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Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy.

Publications

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy.

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