Structural Characterization of Photopolymerizable Binary Liposomes Containing Diacetylenic and Saturated Phospholipids

The use of liposomes to encapsulate materials has received widespread attention for drug delivery, transfection, diagnostic reagent, and as immunoadjuvants. Phospholipid polymers form a new class of biomaterials with many potential applications in medicine and research. Of interest are polymeric phospholipids containing a diacetylene moiety along their acyl chain since these kinds of lipids can be polymerized by Ultra-Violet (UV) irradiation to form chains of covalently linked lipids in the bilayer. In particular the diacetylenic phosphatidylcholine 1,2-bis(10,12-tricosadiynoyl)- sn-glycero-3-phosphocholine (DC8,9PC) can form intermolecular cross-linking through the diacetylenic group to produce a conjugated polymer within the hydrocarbon region of the bilayer. As knowledge of liposome structures is certainly fundamental for system design improvement for new and better applications, this work focuses on the structural properties of polymerized DC8,9PC:1,2-dimyristoyl-sn-glycero-3-phusphocholine (DMPC) liposomes. Liposomes containing mixtures of DC8,9PC and DMPC, at different molar ratios, and exposed to different polymerization cycles, were studied through the analysis of the electron spin resonance (ESR) spectra of a spin label incorporated into the bilayer, and the calorimetric data obtained from differential scanning calorimetry (DSC) studies. Upon irradiation, if all lipids had been polymerized, no gel-fluid transition would be expected. However, even samples that went through 20 cycles of UV irradiation presented a DSC band, showing that around 80% of the DC8,9PC molecules were not polymerized. Both DSC and ESR indicated that the two different lipids scarcely mix at low temperatures, however few molecules of DMPC are present in DC8,9PC rich domains and vice versa. UV irradiation was found to affect the gel fluid transition of both DMPC and DC8,9PC rich regions, indicating the presence of polymeric units of DC8,9PC in both areas, A model explaining lipids rearrangement is proposed for this partially polymerized system.Universidade de São Paulo (USP)Universidade de São Paulo USPFAPESP[01/11721-3]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNQ-MPBioUNQ-MPBio[53/1001(PUNQ0396/07)]CONICETConsejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)MTL (CNPq)ELD (EAPESP)ELD (EAPESP)[06/55493-8]S. del VA (CONICET)Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET)Ministério da Ciência, Tecnologia e Inovação do Brasil (MCTI)MCT (UNQ)CET (CONICET)[3100/08]Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET)[PIP 5832/05

Rotavirus VP6 protein mucosally delivered by cell wall-derived particles from Lactococcus lactis induces protection against infection in a murine model.

Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery

Stoichiometry of the cationic lipopolymer/DNA complex.

<p>Percentage of plasmid DNA association as a function of cationic lipopolymer/pCH110 (a and b) or pDsRed (c and d) plasmid DNA ratios (mol of lipids: mol of base pairs) for the DMPC:DC_8,9PC:DOTAP (1:1:0.2), DMPC:DC_8,9PC:SA (1:1:0.2), and DMPC:DC_8,9PC:MCL (1:1:0.2) formulations.</p

Z-potential measurements.

<p>Z-potential measurements.</p

Serum nucleases digestion assay.

<p>The different cationic lipopolymers DMPC:DC_8,9PC:DOTAP (1:1:0.2) and DMPC:DC_8,9PC:MCL (1:1:0.2)/pDsRed plasmid DNA complexes (16:1 mol of lipids: mol of base pairs ratio) were formed in water, PBS or MEM (indicated on the right). Each lane was loaded with 1 μg of plasmid DNA. Lanes correspond to: (1) pDsRed alone with a 24-h incubation in the indicated medium, (2) pDsRed alone with a 24-h incubation in the presence of 50% v/v FBS, (3) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with a 24-h incubation in the indicated medium without FBS, and (4) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with a 24-h incubation in the presence of 50% v/v FBS. The two main topological plasmid conformations, relaxed and negatively supercoiled, are indicated with arrows on the left of the figure noted as relax and -supercoiled, respectively. Degraded DNA is also indicated with an arrow on the left of the figure.</p

Hemolysis percentage (% H).

<p>Hemolysis percentage (% H).</p

Polymerization confirmation.

<p>Absorbance as a function of wavelength (nm) for the DMPC:DC_8,9PC:DOTAP (1:1:0.2), DMPC:DC_8,9PC:MCL (1:1:0.2) and DMPC:DC_8,9PC:SA (1:1:0.2) mixtures, prepared with the copolymerization methodology, after 20 UV irradiation cycles. Peaks observed around 480 and 520 nm are indicative of polymer formation.</p

Light-scattering measurements.

<p>Light-scattering measurements.</p

Effect of different incubation media on the cationic lipopolymer/DNA interaction.

<p>The different cationic lipopolymers were incubated with the pDsRed plasmid DNA in a 16:1 (mol of lipids: mol of base pairs) ratio in the medium (water, PBS or MEM) indicated on the right. Each lane was loaded with 1 μg of plasmid DNA. Lanes correspond to: (1) pDsRed alone with an additional 10-min incubation in the indicated medium, (2) pDsRed alone with an additional 10-min incubation in the presence of 10% v/v FBS, (3) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation without FBS, (4) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation in the presence of 10% v/v FBS, (5) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with an additional 10-min incubation in the presence of 50% v/v FBS, (6) cationic lipopolymer (stated above the gel picture)/pDsRed plasmid DNA complex formed in the indicated medium with 10% v/v FBS and with an additional 10-min incubation in the presence of 50% v/v FBS. The two main topological plasmid conformations, relaxed and negatively supercoiled, are indicated with arrows on the left of the figure noted as relax and -supercoiled, respectively.</p

Optimization of the study of cationic lipopolymer/DNA interaction by flow cytometry.

<p>Flow cytometry analysis results for non-polymerized DMPC:DC_8,9PC:MCL (1:1:0.2) liposomes, used to set control values of (a) SSC-H values (related to particle complexity) versus FSC-H values (related to particle size) and (b) FL1 values (where SYBR^® Green I-labeled plasmid DNA fluorescence is detected) and FL2 values (where cationic lipopolymer fluorescence is detected). (c) and (d) show the results for polymerized DMPC:DC_8,9PC:MCL (1:1:0.2) for SSC-H versus FSC-H and FL2 versus FL1 values respectively. (e) and (f) show the results for non-polymerized DMPC:DC_8,9PC:MCL (1:1:0.2)/SYBR^® Green I-labeled pDsRed plasmid DNA complexes for SSC-H and FSC-H values and FL2 versus FL1 values respectively.</p