156 research outputs found
Release of outer membrane fragments by exponentially growing Brucella melitensis cells
Rough and smooth strains of Brucella melitensis released a membranous material that was devoid of detectable NADH oxidase and succinic dehydrogenase activity (cytoplasmic membrane markers) but that contained lipopolysaccharide, proteins, and phospholipids. This material was composed of two fractions that had similar chemical compositions but that were of different sizes which were separated by differential ultracentrifugation. Electron microscopy showed that both fractions are made of unit membrane structures. The membrane fragments were released during the exponential phase of growth, and no leakage of malic dehydrogenase activity (cytosol marker) was detected. Thus, the fragments were unlikely a result of cell lysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that, although group 2 Brucella outer membrane proteins and lipoprotein were not detected, the proteins in the membranous material were outer membrane proteins. Gas-liquid chromatography analysis showed a similar fatty acid profile for the cell envelope and the outer membrane fragments of the smooth strain B. melitensis 16M. In contrast, the outer membrane fragments from the rough 115 strain were enriched in palmitic and stearic acids. With respect to the unfractionated cell envelope, outer membrane fragments were enriched in phosphatidylcholine, a phospholipid that is unusual in bacterial membranes
Drug delivery systems for potential treatment of intracellular bacterial infections.
Despite the advent of a considerable number of new antibiotics, treatment of intracellular pathogens still represents a major pharmaceutical challenge. The antibiotic concentration in those specialized niches are often subtherapeutic, for which high doses of antibiotics must often be used. This is not only costly but may also increase localized or systemic side effects. There is therefore an urgent need for materials and methods to enable clinicians to achieve therapeutically effective intracellular concentration of those antibiotics which show good efficiency in vitro. In this setting, the possible use of drug delivery systems (DDS) loaded with antibiotics that exhibit a high in vitro bactericidal activity deserves to be considered. Entrapping or encapsulating the drug within a delivery system provides a greater control of the pharmacokinetic behavior of the active molecule. This more efficient use of antibiotics may diminish their drawbacks and provide the basis for shortening the current time required by classical treatments. This review will focus on the role of DDS as a potential tool against intracellular bacteria
Intracellular killing of Brucella melitensis in human macrophages with microspheres-encapsulated gentamicin
Objectives: Treatment of human brucellosis demands antibiotic targeting into the mononuclearphagocytic
system. The aim of this work was to prepare and characterize particulate carriers containing
gentamicin and to study their interactions with phagocytic cells and bactericidal activity against intracellular
Brucella melitensis.
Methods: Different poly(lactide-co-glycolide) (PLGA)polymers with free carboxylic end-group wereusedto
formulate micro- and nanoparticles containing gentamicin, by a water-oil-water solvent-evaporation technique.
PLGA 502H and 75:25H microparticles were selected because they showed the highest gentamicin
loadings as well as good physico-chemical properties and sustained release in vitro.
Results: Gentamicin-containing microspheres of both polymers were successfully phagocytosed by infected
THP-1 human monocytes, and immunocytochemistry studies revealed that the antibiotic reached
Brucella-specific compartments. A dose of 30 mg of encapsulated gentamicin was able to reduce intracellular
Brucella infection by 2.2 log.
Conclusions: Altogether, these results suggest that 502H and 75:25H microspheres are suitable carriers for
gentamicin targeting inside human macrophages and thus for brucellosis treatment
Determination of gentamicin in different matrices by a new sensitive high-performance liquid chromatography-mass spectrometric method
OBJECTIVES:
The aim of this work was to develop and validate an HPLC method for gentamicin quantification in different types of biological samples such as animal tissues and cellular material and also in pharmaceuticals.
METHODS:
Poly(lactide-co-glycolide) microparticles (MP) of gentamicin (PLGA 502H MP), THP-1 cells, and plasma and tissue samples of mice treated with the antibiotic either free or loaded into PLGA 502H MP were processed by a simple preparation procedure, subjected to chromatography on a reversed-phase column and measured by mass spectrometry detection. The developed method was compared with bioassay and fluorimetric assay methods previously used for gentamicin determination.
RESULTS:
The HPLC method was linear over the ranges 40-800 ng/mL and 0.1-100 microg/mL and showed good accuracy (average accuracy < 5.59%) and reproducibility (CV < 6.13%). Encapsulation of gentamicin in PLGA 502H MP was determined by the three methods. Good correlation was observed between bioassay (reference method) and HPLC. Extra- and intracellular in vitro antibiotic accumulation was determined by bioassay and chromatography. Both methods gave similar extracellular concentrations but the HPLC-MS technique demonstrated an improved accuracy (5.59% versus 14%) and precision (6.13% versus 15%) compared with bioassay. However, only the HPLC-MS method was sensitive enough to detect the drug, intracellularly and in tissues.
CONCLUSIONS:
All these data favour the use of chromatography-mass spectrometry as a versatile technique not only suitable for gentamicin quantification loaded in drug delivery systems, but also sensitive and specific enough for in vivo and intracellular studies
Gentamicin-loaded microspheres for reducing the intracellular Brucella abortus load in infected monocytes
Objectives: The intracellular antibiotic efficiency of gentamicin-loaded microspheres in the context of Brucella-infected murine monocytes was examined in vitro with a view to developing improved therapies for the treatment of brucellosis.
Methods: Biodegradable microspheres made of end-group capped and uncapped poly(lactide-co-glycolide) 50:50 (PLGA 50:50 and PLGA 50:50H) and containing gentamicin sulphate were used to target Brucella abortus-infected J774 monocyte-macrophages. The infected cells were treated with 15 µg of free or microencapsulated gentamicin and the efficacy of the treatments was measured after 24 h.
Results: The particle sizes were below 8 µm and in vitro release of gentamicin from the microspheres followed a continuous (PLGA 50:50H) or a multiphasic (PLGA 50:50) pattern over 50 days. Treatment with gentamicin microencapsulated into the end-group uncapped PLGA 50:50H microspheres, decreased significantly the number of intracellular bacteria (typically by 2 log10) in comparison with untreated infected cells. Addition of 2% poloxamer 188 to the microsphere dispersion medium further reduced the infection (3.5 log10). Opsonization of the particles with non-immune mouse serum had no effect on the antibacterial efficacy of the microspheres. End-group capped PLGA 50:50 type microspheres containing the antibiotic were less effective at reducing intracellular bacteria (∼1 log10 reduction), although addition of poloxamer 188 to the dispersion medium again enhanced their intracellular antibacterial activity. Placebo PLGA 50:50 and PLGA 50:50H microspheres had no bactericidal activity.
Conclusions: The results indicate that PLGA 50:50-microencapsulated gentamicin sulphate may be suitable for efficient drug targeting and delivery to reduce intracellular Brucella infections
Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation
The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride)
nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery
systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results
revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5),
triggering a Th1-profile cytokine release (gamma interferon [IFN- ], 478 pg/ml versus 39.6 pg/ml from
negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation
with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression.
Furthermore, in vivo studies suggest that NPs actively elicit a CD8 T-cell response. Immunization with empty
NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a
protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken
together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis
and immunotherapy through TLR exploitation
Development of a novel vaccine delivery system based on Gantrez nanoparticles.
The adjuvant capacity of a novel vaccine vector “Gantrez-nanoparticles” (NP)
towards coated or encapsulated ovalbumin (OVA) was investigated. OVA nanoparticles
were prepared by a solvent displacement method previously described. The protein was
incorporated during the manufacturing process (OVA-encapsulated nanoparticles) or
after the preparation (OVA-coated nanoparticles). The mean size of the different
nanoparticle formulations was lower than 300 nm, and the OVA content ranged
approximately from 67 μg/mg nanoparticles (for OVA-coated nanoparticles) to 30
μg/mg nanoparticles (for OVA-encapsulated nanoparticles). All the OVA-NP
formulations were capable of amplifying the antibodies titres (IgG1 and IgG2a) in mice
after a single subcutaneous inoculation with respect free OVA or OVA adsorbed to
Alum. Furthermore, the elicited response was, for some formulations, predominantly
Th1 subtype. Thus, the formulation that contained mainly the antigen inside, and with a
low concentration of cross-linking agent, displayed the best potential to induce a Th1
response after 35 days post-immunisation. These results are highly suggestive for the
use of Gantrez nanoparticles as an efficient antigen delivery system, especially when a
long lasting Th1 cytokine response is required
Poly(D,L-lactide-coglycolide) particles containing gentamicin: pharmacokinetics and pharmacodynamics in Brucella melitensis-infected mice
Drug delivery systems containing gentamicin were studied as a treatment against experimental brucellosis in mice. Micro- and nanoparticles prepared by using poly(D,L-lactide-coglycolide) (PLGA) 502H and microparticles made of PLGA 75:25H were successfully delivered to the liver and the spleen, the target organs for Brucella melitensis. Both polymers have the same molecular weight but have different lactic acid/glycolic acid ratios. Microparticles of PLGA 502H and 75:25H released their contents in a sustained manner, in contrast to PLGA 502H nanoparticles, which were degraded almost completely during the first week postadministration. The values of the pharmacokinetic parameters after administration of a single intravenous dose of 1.5 mg/kg of body weight of loaded gentamicin revealed higher areas under the curve (AUCs) for the liver and the spleen and increased mean retention times (MRTs) compared to those for the free drug, indicating the successful uptake by phagocytic cells in both organs and the controlled release of the antibiotic. Both gentamicin-loaded PLGA 502H and 75:25H microparticles presented similar pharmacokinetic parameter values for the liver, but those made of PLGA 75:25 H were more effective in targeting the antibiotic to the spleen (higher AUCs and MRTs). The administration of three doses of 1.5 mg/kg significantly reduced the load associated with the splenic B. melitensis infection. Thus, the formulation made with the 75:25H polymer was more effective than that made with 502H microspheres (1.45-log and 0.45-log reductions, respectively, at 3 weeks posttreatment). Therefore, both, pharmacokinetic and pharmacodynamic parameters showed the suitability of 75:25H microspheres to reduce the infection of experimentally infected mice with B. melitensis
Co-encapsulation of an antigen and CpG oligonucleotides into PLGA microparticles by TROMS technology.
It seems well established that CpG oligonucleotides Th1 biased adjuvant activity
can be improved when closely associated with a variety of antigens in, for example,
microparticles. In this context, we prepared 1-μm near non-charged PLGA 502 or
PLGA 756 microparticles that loaded with high efficiency an antigen (50% ovalbumin
(OVA), approximately) into their matrix and CpG-chitosan complexes (near to 20%)
onto their surface maintaining OVA and CpG integrity intact. In the intradermal
immunization studies, whereas OVA microencapsulated into PLGA 756 alone induced
a strong humoral immune response assisted by a very clear Th1 bias
(IgG2a/IgG1=0.875) that was decreased by CpG co-delivery (IgG2a/IgG1=0.55), the
co-encapsulation of CpG with OVA in PLGA 502 particles significantly improved the
antibody response and isotype shifting (IgG2a/IgG1=0.73) in comparison with mice
immunized with OVA loaded PLGA 502 (IgG2a/IgG1=0). This improvement was not
correlated with the cellular immune response where the effect of co-encapsulated CpG
was rather negative (2030.2 pg/mL and 335.3 pg/mL IFN-g for OVA PLGA 502 for
OVA CpG PLGA 502, respectively). These results underscore the critical role of
polymer nature and microparticle characteristics to show the benefits of coencapsulating
CpG motifs in close proximity with an antigen
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