Departments of 1 Microbiology and 3 Pharmacy and Pharmaceutical Technology, University of Navarra, 31008 Pamplona, Spain; 2 Institute of Pharmaceutical Sciences, ETH, 8057 Zürich, Switzerland
Received 20 November 2003; returned 15 January 2004; revised and accepted 8 March 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
Keywords: biodegradable microspheres, drug delivery systems, Brucella-infected monocytes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gentamicin sulphate is an aminoglycoside with a wide spectrum of antibacterial activity though important side effects mostly related to nephrotoxicity and ototoxicity restrict its use.3 It is a highly soluble drug but does not cross cell membranes efficiently, which is an important drawback for the therapy of intracellular infections such as brucellosis, due to the low antibiotic levels achievable inside infected cells. Several reports indicate that gentamicin is more active in vitro against clinical isolates of Brucella than streptomycin.4 In combined doxycyclineaminoglycoside regimens, gentamicin appears to be more cost-effective and less toxic given the duration of the administration (1421 days for streptomycin compared with 7 days for gentamicin), with no increases in clinical relapse or treatment failure.2 These properties make gentamicin an attractive candidate for the treatment of brucellosis provided that the antibiotic can be delivered intracellularly. Therefore, an optimum strategy to treat brucellosis should target a highly active drug to the intracellular compartment and prolong the release of that antibiotic, thereby reducing the number of doses to be administered and minimizing drug side effects.
Liposomes containing gentamicin are quite efficient for targeting Brucella-infected monocyte-macrophages.5 Liposomes have a membrane-like structure that favours a good cell interaction and their versatility in terms of structure and composition grant their main advantages. However, liposomes suffer from low encapsulation efficiency, stability problems, both during storage and upon injection, their therapeutic efficacy is not always optimal and cannot provide a controlled release of the encapsulated drug.6 Small biodegradable microspheres are attractive alternatives to liposomes for targeting drug in the monocyte-macrophagic system. Upon phagocytosis of antibiotic-loaded microspheres, intracellularly located and highly protected bacteria, such as Mycobacterium tuberculosis, Salmonella typhimurium, Listeria monocytogenes and Brucella spp., may become accessible to antibiotic treatment. Biodegradable microspheres made of poly(lactide) (PLA) and its copolymers with glycolide (PLGA) can release encapsulated drugs in a sustained manner, depending on the physicochemical properties of the polymer and drug, and the microencapsulation procedure.7 Microsphere technology has been widely used for a wide variety of therapeutic compounds including antibacterial agents.811 Thus, PLA/PLGA microspheres should be a potentially suitable and stable delivery system for antibiotic treatment of brucellosis that could provide controlled release of the encapsulated drug and minimize the need for multiple injections.
In this study, we examined the intracellular antibiotic efficiency of gentamicin-loaded microspheres in the context of Brucella-infected murine monocytes. Two microsphere types, made of end-group capped PLGA 50:50 and uncapped PLGA 50:50H, were used because they are phagocytosed efficiently by monocytes in vitro;12 the end-group uncapped PLGA 50:50H microspheres also promote cell activation, as measured by oxidative burst.12,13
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gentamicin was microencapsulated by spray-drying into poly(D,L-lactide-co-glycolide) carrying either capped, i.e. esterified end-groups, or uncapped, i.e. free hydroxyl and carboxyl end-groups,14 hereafter abbreviated as PLGA 50:50 and PLGA 50:50H, respectively. Briefly, 100 mg of gentamicin was dissolved in 1 mL of PBS (67 mM, pH 7.4 or 6.0), which was then dispersed in 20 g of a 5% (w/w) polymer solution in ethyl formate by ultrasonication under cooling on ice. The water-in-oil emulsion formed was spray-dried (Mini Spray Drier B-190; Büchi, Flawil, Switzerland) and the resulting microspheres were collected on a cellulose acetate filter of 0.8 µm pore size. Microspheres were washed with 0.1% (w/w) poloxamer 188 solution, rinsed with distilled water and dried under vacuum (10 mbar). Residual water was eliminated by dispersing the particles in n-hexane, followed by additional vacuum drying for 24 h. The final product was stored under dry conditions at 4°C. Placebo microspheres were produced accordingly, but without the gentamicin solution.
Determination of microsphere size and morphology
Microsphere size distribution was determined by laser light scattering (Mastersizer X; Malvern Instruments, Malvern, UK). The morphology of the microspheres was examined in a Zeiss DSM 940A scanning electron microscope.
Gentamicin content in the microspheres
Gentamicin content in the microspheres was determined by dissolving 40 mg of particles in 3 mL of dichloromethane and collecting the undissolved gentamicin on regenerated cellulose filters of 0.2 µm pore size. The dried filters were transferred into test tubes and the antibiotic was eluted with 2 mL of water and assayed photometrically (320350 nm) after derivation with o-phthalaldehyde.14 Gentamicin extraction from the microspheres was carried out in triplicate and the eluted gentamicin was determined from the replicates.
In vitro release of gentamicin from microspheres
Gentamicin release was determined by suspending 1030 mg of microspheres, weighed accurately, in 4 mL of PBS (67 mM, pH 7.4) containing 0.05% polysorbate 20 and 0.02% sodium azide, in borosilicate vials (Chromacol, Welwyn Garden City, UK). The vials were briefly sonicated to facilitate wetting of the particles and placed horizontally on a shaking platform at 37°C. At regular intervals, the vials were centrifuged at 2000g for 10 min to obtain a particle-free supernatant that was assayed fluorometrically with a Cytofluor 2300/2350 (Millipore)5 after derivation of the gentamicin with o-phthalaldehyde. At every sampling point, the release medium collected was replaced with fresh phosphate buffer. Burst release was described as the amount of drug released after 24 h. Gentamicin release from the microspheres was determined in triplicate for each microsphere preparation.
J774 murine monocyte-macrophage cell line
The J774.2 murine monocyte-macrophage cell line was obtained from the European Collection of Cell Cultures (ECACC No. 85011428). The cells were maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal calf serum and preserved with 0.1% antibiotic-antimycotic solution (complete medium) at 37°C, 5% CO2 and controlled humidity.
Brucella strain and preparation of bacteria for infection
B. melitensis is the more frequent cause of Brucella human infections. However, as it is a class three pathogen, B. abortus was chosen for these studies. B. abortus 2308 was isolated from spleens of infected mice by homogenization of the organ in saline solution (Stomacher 80 Lab Blender; Seward Medical, London, UK) and seeding onto trypticase soy agar (TSA) plates (bioMérieux, France). Further, the bacterial colonies were suspended on skimmed milk and maintained in cryovials at 85°C. For monocyte infection, the brucellae were thawed, seeded onto TSA plates and incubated for 23 days at 37°C. Two or three isolated colonies were transferred into 5 mL of trypticase soy broth (TSB) and incubated for 2024 h at 37°C under shaking, so that the bacteria reached exponential growth phase. The bacterial suspension was first diluted in TSB to an optical density of 0.12 (590 nm) and further diluted in saline solution to a concentration of 5x107 cfu/mL to infect the cultured monocytes.
Infection of monocytes with Brucella abortus 2308
J774 murine monocytes cultured in complete medium were washed, collected by centrifugation and cell viability was determined by Trypan Blue exclusion. Cell numbers were adjusted to 2 x 106 cells/mL and 500 µL of the cell suspension was added to each well of a 24-well culture plate (Nunc, Naperville, IL, USA). The cells were incubated in DMEM supplemented with L-glutamine and 10% (v/v) fetal calf serum for 23 h to allow cell adherence. Afterwards, the medium was withdrawn to remove non-adherent cells and replaced. For bacterium-specific opsonization, the B. abortus suspension, previously adjusted to an adequate bacterial concentration as described above, was shaken with a sub-agglutinating concentration of antiserum from B. abortus-infected mice for 30 min at 37°C. Then, 100 µL of opsonized B. abortus was added to the monocytes at a ratio of 5 bacteria per cell and further incubated at 37°C for 148 h. After incubation, the culture medium was discarded, the infected cells were carefully washed five times to remove extracellular bacteria and 500 µL of DMEM was added to each well. The number of remaining extracellular bacteria in each well was determined by diluting and seeding the added medium onto TSA plates. The total number of cfu per well was determined after lysing the monocytes with cold 0.2% Triton X-100 for 20 min at 37°C, thorough homogenization and plating of serial dilutions of the lysates onto TSA plates. This Triton X-100 treatment had no effect on viability of the bacteria. Intracellular bacteria were calculated by subtracting the number of extracellular Brucella from the total cfu counts in three wells.
Treatment of Brucella-infected monocytes with gentamicin-loaded microspheres
The infected J774 monocytes were treated with 15 µg of free gentamicin sulphate in solution, with placebo microspheres, or with gentamicin-loaded microspheres containing 15 µg of the antibiotic, and the effectiveness of the different treatments in reducing the number of intracellular bacteria was determined. The influences of prior opsonization of the particles in fresh mouse serum and the presence of 2% (w/v) of the non-ionic surfactant poloxamer 188 in the microsphere dispersion medium were also determined. For opsonization, the microspheres were dispersed in DMEM supplemented with L-glutamine and containing 8% (v/v) non-immune fresh mouse serum and incubated at 37°C under shaking for 30 min. In some experiments, the microspheres were simply dispersed in the 8% (v/v) serum solution without incubation.
The J774 monocytes-macrophages were infected by incubating the cells with the opsonized B. abortus for 15 h at a bacteria/cell ratio of 5:1 as described above. Afterwards, non-phagocytosed bacteria were removed by washing carefully five times and the effectiveness of the different treatments was studied by incubating the infected monocytes for 24 h at 37°C and 5% CO2 with 1 mL of DMEM/L-glutamine containing either free gentamicin in solution, gentamicin encapsulated in microspheres or placebo microspheres. DMEM/L-glutamine without fetal calf serum was used to avoid potential effects of the serum proteins on the particle uptake and to moderate the proliferation of monocytes and bacteria. Further, cells were washed twice with DMEM/L-glutamine and extracellular cfu and total cfu after cell lysis with Triton X-100 were determined by plating serial dilutions onto TSA plates as described previously. Intracellular brucellae were calculated by subtracting the number of extracellular bacteria from the total cfu counts in three wells. Results were expressed as log10 of intracellular Brucella surviving the treatments and the efficiency of the treatments was tested in one to six independent assays.
In vitro susceptibility of B. abortus to gentamicin and poloxamer
Gentamicin sulphate and poloxamer 188 as single agents or in combination were tested for their ability to inhibit the bacterial growth of B. abortus in broth culture. Gentamicin sulphate was added to the bacterial suspension to obtain final concentrations of 0.0070.960 µg/mL, alone or in combination with 0.5, 1 and 2% (w/v) poloxamer 188. Bacterial growth in TSB was measured turbidimetrically with an automated Labsystems Bioscreen (Finland) using Bioscreen microtitre plates (100-well honeycomb). Organisms were retrieved from suspensions stored at 85°C in cryoballs (Microbank, Biolab Diagnostics, Canada), plated on TSA and incubated at 37°C overnight. Several colonies were then subcultured in TSB at 37°C for 36 h to reach mid-log phase. The final inoculum suspension was prepared in TSB and adjusted to an optical density of 0.12 (590 nm) (108 cfu/mL). The real inoculum dose was estimated by plating appropriate dilutions of the stock suspension onto TSA plates. Finally, an inoculum of 105 cfu per well was added into Bioscreen plates containing serial dilutions of the agents (gentamicin and/or poloxamer 188). Appropriate positive and negative growth controls were also included. The optical density of each well was measured automatically at 30 min intervals using wide band irradiation (420580 nm) and bacterial growth curves were generated.
Statistical analysis
Comparisons were made between groups by a one-way analysis of variance (ANOVA) and post-hoc Tukeys HSD test. The statistical significance level was defined as P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The average diameter of the PLGA 50:50H and PLGA 50:50 microspheres was 4 µm, except for the placebo PLGA 50:50 microspheres which had an average diameter of 0.5 µm (Table 1). This size appeared adequate for cellular uptake and was mainly a result of the encapsulation method used. Scanning electron micrographs showed spherically shaped and regular particles with smooth surfaces for both the placebo and gentamicin-loaded PLGA 50:50 microspheres. Gentamicin-loaded PLGA 50:50H microspheres were, however, porous and the particles tended to aggregate.
|
|
Opsonized B. abortus were incubated from 1 to 48 h with J774 cells in complete medium without antibiotic-antimycotic supplement and afterwards, the number of extracellular and intracellular bacteria was determined. Incubation for 1 h was insufficient for significant phagocytosis of opsonized bacteria at a monocyte/Brucella ratio of 1:5. Incubation times between 15 and 48 h allowed an adequate number of intracellular brucellae to carry out our study (Figure 2). In this time, the bacteria reached the intracellular compartment and multiplied within the monocytes increasing by approx 1.3 log10 after 48 h. Incubation times above 48 h resulted in excessive cell and bacteria proliferation, as reflected by turbidity and diminished cell viability. Therefore, for all antibiotic activity assays, an infection time of 15 h was chosen. Thus, the cells were incubated with the bacteria for 15 h, non-phagocytosed bacteria were washed off and the different treatments were subsequently applied. Under these conditions, the percentage of intra- and extracellular Brucella at the time of the antibiotic treatments was 9395% and 57%, respectively.
|
Monocytes infected with Brucella were treated for 24 h with (i) free gentamicin in solution, (ii) gentamicin encapsulated in PLGA 50:50 or PLGA 50:50H microspheres, and (iii) placebo PLGA 50:50 or PLGA 50:50H microspheres. The amounts of microspheres used per well were 0.7 mg for PLGA 50:50 and 1.0 mg for PLGA 50:50H, which corresponded to a gentamicin dose of 15 µg/well for both microsphere types; corresponding amounts of placebo microspheres were used. A dose of 15 µg of gentamicin was used to ensure the intracellular delivery of the antibiotic at levels well above the MBC (0.06 mg/L, see below). Placebo microspheres had no bactericidal effect on intracellular Brucella counts. In contrast, treatment with gentamicin-loaded microspheres reduced intracellular bacteria, compared with untreated controls, by 1.0 and 2.0 log10 for PLGA 50:50 and PLGA 50:50H, respectively. Free gentamicin in solution exerted variable intracellular bactericidal activity in different experiments, varying from a 1.1 to 1.8 log10 reduction. However, treatment with gentamicin-loaded PLGA 50:50H microspheres was significantly more effective at reducing the intracellular infection than free gentamicin treatment (Figure 3).
|
Previous results in our laboratory showed that opsonization of gentamicin-loaded microspheres increased significantly the capacity of the particles to induce oxidative burst of monocytes,12 which may potentially increase the bactericidal activity of antibiotic treatments. With that hypothesis, the effect of opsonization on the antibacterial efficacy of the microsphere treatment was examined in this study. Our hypothesis was, however, not supported by our findings. The antibiotic activity of the gentamicin-loaded microspheres did not increase upon particle opsonization (Figure 3). In contrast, addition of 2% (w/v) of poloxamer 188 to the medium to improve the dispersion characteristics of the microspheres, enhanced significantly the intracellular antibiotic activity of the gentamicin-loaded PLGA 50:50H microspheres (Figure 4). In the presence of the surfactant, the total reduction of intracellular bacteria reached 3.5 log10 cfu, relative to the untreated monocytes, which represented a 1.2 log10 further reduction in intracellular cfu relative to the same treatment without poloxamer 188. A similar improvement, i.e. 1 log10 further reduction, was also observed with the gentamicin loaded end-group capped PLGA 50:50 microspheres when the poloxamer was added to the medium; however, in this case the increase was not statistically significant. Control wells demonstrated the lack of activity of both a poloxamer 188 solution and a suspension of placebo microspheres in poloxamer on the viability of the intracellular Brucella.
|
The effect of combining gentamicin sulphate and poloxamer 188 against B. abortus was studied in broth culture in vitro. When sub-MIC concentrations of gentamicin sulphate and non-inhibitory concentrations of poloxamer 188 were combined, a synergic effect was evidenced between the two agents (not shown). For example, the MBCs (3 log10 reduction) of gentamicin and poloxamer 188 tested alone were 0.06 mg/L and >2% (w/v), respectively. When gentamicin was combined with the poloxamer at 1% or 2% (w/v), the MBC of gentamicin was reduced to 0.03 mg/L (poloxamer 1%) and 0.015 mg/L (poloxamer 2%).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gentamicin-loaded PLGA 50:50H microspheres decreased significantly the intracellular bacterial levels (typically by 2 log10) compared with untreated monocytes and free gentamicin; the efficiency of PLGA 50:50 microspheres was lower (typically 1 log10 reduction) and normally no different to the free drug; placebo microspheres had no effect. Opsonization of gentamicin-loaded microspheres in fresh mouse serum did not improve the antibiotic efficiency of the treatment although, in previous work, opsonization increased the uptake of placebo microspheres and the cell oxidative metabolism.12 The release of oxygen metabolites can alter the bacterial membrane permeability and thereby increase drug efficiency.15 Because Brucella spp. alone only slightly increase the oxidative burst in blood leucocytes, we speculated that an enhanced production of oxidative metabolites triggered by the phagocytosis of microspheres should improve treatment efficacy. This hypothesis was supported by the observation that brucellicidal activity of human polymorphonuclear leucocyte granule lysates was enhanced by addition of hydrogen peroxide.16 In our experiments, no bactericidal effect was observed with placebo PLGA 50:50H microspheres. Nevertheless, the stimulation of oxygen derivatives within the cells may have increased the bacterial membrane permeability and made them more susceptible to the antibiotic.
Free gentamicin sulphate lowered the number of intracellular bacteria by 1 to 1.8 log10. Because gentamicin cannot diffuse through cellular membranes, its internalization and accumulation inside cells has been ascribed to pinocytosis.17 The observed variation in the reduction in bacterial counts might result from variations in the pinocytic capacity of the cells. In our investigation, microencapsulated gentamicin achieved bacterial reduction at levels superior or comparable to free drug. Considering that gentamicin release from the microspheres was prolonged over up to 50 days, antibiotic activity may be expected for prolonged periods of time. Incubation times were not prolonged in our studies due to limited cell viability and high bacterial proliferation. We hypothesize that this may provide intracellular bactericidal gentamicin concentrations over prolonged periods of time. Thus, this approach could reduce the multiple injections needed in the aminoglycoside component of current brucellosis therapies, and could ensure efficient clearance of the infection without increasing the possibility of ototoxic and nephrotoxic reactions. It would therefore be interesting to quantify the intracellular concentration of gentamicin after microsphere uptake to improve understanding of the mechanism and kinetics by which the encapsulated drug reduces intracellular Brucella. Nonetheless, in vivo processing of the microspheres is likely to affect the kinetics of drug release and polymer degradation. Intracellular reduction of Brucella was greater for PLGA 50:50H than for PLGA 50:50 microspheres, although in vitro burst release within the initial 24 h of incubation amounted to only 14% for the PLGA 50:50H as compared with 50% for the PLGA 50:50 microspheres. Two factors may explain the results. First, the pronounced stimulation of oxidative metabolites by the PLGA 50:50H particles may have enhanced the antibiotic activity of gentamicin. Second, a higher number of PLGA 50:50H particles might have been phagocytosed, because a larger amount of these particles had been added to the infected monocytes to achieve identical gentamicin doses (1 mg of PLGA 50:50H versus 0.7 mg of PLGA 50:50 microspheres, with both types having a similar phagocytosis rate of
50%).12 However, the present data are insufficient to conclude that a higher number of phagocytosed particles directly affect antimicrobial efficacy.
An interesting result of this study was the increased antibacterial activity (up to 1.2 log10) of microencapsulated gentamicin when the dispersion medium for the microspheres contained 2% (w/v) poloxamer 188. Several mechanisms may be responsible for this phenomenon: (i) adsorption of the surfactant on the microsphere surface thereby altering the surface polarity and the potential for cell adhesion; (ii) enhanced phagocytosis due to a higher degree of particle dispersion or another mechanism; (iii) non-specific cell activation by the surfactant; and (iv) synergic enhancement of antibiotic activity. Particle coating by poloxamer type surfactants is well known to suppress phagocytosis in vitro and alter the opsonization by serum proteins in vivo, slowing down the elimination of particles from the blood by the mononuclear phagocytic system.18 The efficiency of poloxamer types to reduce particle phagocytosis depends on the molecular weight and chain length of the propylene oxide (PO) and ethylene oxide (EO) blocks.18 Thin coatings conferring poor steric stabilization were obtained on highly polar surfaces19,20 where the PO blocks were adsorbed preferentially onto the particle surface, whereas the EO chains stretched out into the dispersion medium.18 On larger particles, such a thin layer was probably adsorbed flatly so that the hydrophobic PO units remained exposed to the medium. With the relatively polar and hydrophilic PLGA 50:50 and PLGA 50:50H microspheres studied herein, adsorption of poloxamer probably occurred through interaction with the EO chains, resulting in a flat arrangement of adsorbed poloxamer. As a result, the exposed PO chains possibly increased the hydrophobicity of the particle surface, leading to increased particle uptake by monocytes, as observed by optical microscopy (data not shown). Further, improved particle dispersion in 2% poloxamer 188 should have also contributed to the increased phagocytosis. Finally, mechanisms involving interaction between poloxamer and cell membrane21,22 or cell activation cannot be excluded. Some poloxamers have indeed increased phagocytic activity, and altered bacterial cell wall integrity and permeability yielding a synergic effect with antibiotic agents.2326 Our susceptibility studies in culture broth demonstrated that poloxamer can potentiate the antibacterial activity of gentamicin. Enhancement of antibiotic susceptibility was observed with subinhibitory concentrations of poloxamer. However, no bactericidal effect of 2% soluble poloxamer alone or 2% poloxamer incubated with placebo microspheres was observed on the intracellular bacterial counts. The results indicate that only non-inhibitory amounts of poloxamer may reach the intracellular bacteria. Nevertheless, non-inhibitory concentrations of the surfactant may have synergically enhanced the anti-Brucella activity of gentamicin delivered intracellularly from microspheres by as yet unknown mechanisms.
Finally, an important aspect for discussing the effectiveness of encapsulated gentamicin is the location of the Brucella inside the cell. Intracellular bacteria can evade the host defences by different mechanisms and prevention of phagosomelysosome fusion has been proposed for intracellular survival of virulent Brucella spp., e.g. B. abortus 2308.27 B. abortus is located in the perinuclear region within compartments resembling autophagosomes and is then delivered to the endoplasmic reticulum where intracellular bacterial replication takes place.2830 This special localization of B. abortus might prevent contact between bacteria and antibiotic-loaded microspheres. On the other hand, virulent Brucella can transit from early endosomes to autophagosomes, which apparently did not fuse with endosomes loaded with exogenous material.30 Nonetheless, fusion between phagosomes and other endocytic vesicles can generally occur, so that phagosomes loaded with particles or pinocytic endosomes have the potential to eliminate Brucella that reside in accessible phagosomes.17 Two localizations for B. abortus have been found at 24 h post-infection: accessible single membrane phagosomes and multimembranous autophagosomes,28 where the bacteria may escape from the antibiotics delivered to phagosomes.
In conclusion, we suggest that microsphere internalization is responsible for the significant reduction in the intracellular bacteria by gentamicin-loaded PLGA 50:50 and PLGA 50:50H microspheres within 24 h. Microencapsulated gentamicin was efficiently targeted to infected monocytes and reduced intracellular Brucella infections. In addition, gentamicin may still be available for further release from the microspheres for prolonged periods of time. Poloxamer 188 added to the dispersion medium for the microspheres significantly enhanced the efficiency of the antibiotic treatment. Further studies should focus on determining the mechanisms by which microencapsulated gentamicin is capable of reducing intracellular viable Brucella and investigate the efficacy of gentamicin-loaded microspheres in the treatment of brucellosis in vivo.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
Corresponding author. Tel: +34-948-425688; Fax: +34-948-425649; E-mail: cgamazo{at}unav.es
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Solera, J., Martínez-Alfaro, E. & Espinosa, A. (1997). Recognition and optimum treatment of brucellosis. Drugs 53, 24556.[ISI][Medline]
3 . Tulkens, P. M. (1986). Experimental studies on nephrotoxicity of aminoglycosides at low doses. Mechanisms and perspectives. American Journal of Medicine 80, 10514.[ISI][Medline]
4 . Gargani, G. & Pacetti, A. M. (1998). Sensitivity of 115 strains of the genus Brucella to some antibiotics (cephalosporins, ureidopenicillins and aminoglycosides). Chemioterapia 5, 713.
5 . Vitas, A. I., Díaz, R. & Gamazo, C. (1996). Effect of composition and method of preparation of liposomes on their stability and interaction with murine monocytes infected with Brucella abortus. Antimicrobial Agents and Chemotherapy 40, 14651.[Abstract]
6 . Codde, J. P., Lumsden, A. J., Napoli, S. et al. (1993). A comparative study of the anticancer efficacy of doxorubicin carrying microspheres and liposomes using a rat liver tumour model. Anticancer Research 13, 53943.[ISI][Medline]
7 . Thomasin, C., Corradin, G., Men, Y. et al. (1996). Tetanus toxoid and synthetic malaria antigen containing poly(lactide)/poly(lactide-co-glycolide) microspheres: importance of polymer degradation and antigen release for immune response. Journal of Controlled Release 41, 13145.[CrossRef][ISI]
8
.
Barrow, E. L. W., Winchester, G. A., Staas, J. K. et al. (1998). Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis-infected macrophages. Antimicrobial Agents and Chemotherapy 42, 26829.
9
.
Dutt, M. & Khuller, G. K. (2001). Therapeutic efficacy of poly(DL-lactide-co-glycolide)-encapsulated antitubercular drugs against Mycobacterium tuberculosis infection induced in mice. Antimicrobial Agents and Chemotherapy 45, 3636.
10 . Puisieux, F., Barrat, G., Couarraze, G. et al. (1994). Polymeric micro- and nanoparticles as drug carriers. In Polymeric Biomaterials (Dumitriu, S., Ed.), pp. 74994. Marcel Dekker, New York, Basle, Hong Kong.
11
.
Quenelle, D. C., Winchester, G. A., Staas, J. K. et al. (2001). Treatment of tuberculosis using a combination of sustained-release rifampicin-loaded microspheres and oral dosing with isoniazid. Antimicrobial Agents and Chemotherapy 45, 163744.
12 . Prior, S., Gander, B., Blarer, N. et al. (2002). In vitro phagocytosis and monocyte-macrophage activation with poly(lactide) and poly(lactide-co-glycolide) microspheres. European Journal of Pharmaceutical Sciences 15, 197207.[CrossRef][ISI][Medline]
13 . Murillo, M., Gamazo, C., Goñi, M. M. et al. (2002). Development of microparticles prepared by spray-drying as a vaccine delivery system against brucellosis. International Journal of Pharmaceutics 242, 3414.[CrossRef][ISI][Medline]
14 . Prior, S., Gamazo, C., Irache, J. M. et al. (2000). Gentamicin encapsulation in PLA/PLGA microspheres in view of treating Brucella infections. International Journal of Pharmaceutics 196, 11525.[CrossRef][ISI][Medline]
15 . Rastogi, N., Potar, M. C. & David, H. L. (1987). Intracellular growth of pathogenic mycobacteria in the continuous murine macrophage cell line J774: ultrastructure and drug-susceptibility studies. Current Microbiology 16, 7992.[ISI]
16 . Kreutzer, D. L., Dreyfus, L. A. & Robertson, D. C. (1979). Interaction of polymorphonuclear leukocytes with smooth and rough strains of Brucella abortus. Infection and Immunity 23, 73742.[ISI][Medline]
17 . Drevets, D. A., Canono, B. P., Leenen, P. J. M. et al. (1994). Gentamicin kills intracellular Listeria monocytogenes. Infection and Immunity 62, 22228.[Abstract]
18 . Rudt, S. & Müller, R. H. (1993). In vitro phagocytosis assay of nano- and microparticles chemiluminescence. II. Effect of surface modification by coating of particles with poloxamer on the phagocytic uptake. Journal of Controlled Release 25, 519.[CrossRef][ISI]
19 . Müller, R. H. (1991). Modification of drug carriers. In Colloidal Carriers for Controlled Drug Delivery and Targeting (Müller, R. H., Ed.), pp. 1941. Wissenschaftliche Verlagsgesellschaft, Stuttgart, CRC Press, Boca Raton, Ann Arbor, Boston, USA.
20 . Illum, L., Jacobsen, L. O., Müller, R. H. et al. (1987). Surface characteristics and the interaction of colloidal particles with mouse peritoneal macrophages. Biomaterials 8, 1137.[CrossRef][ISI][Medline]
21 . Batrakova, E., Lee, S., Li, S. et al. (1999). Fundamental relationships between the composition of pluronic block copolymers and their hypersensitisation effect in MDR cancer cells. Pharmaceutical Research 16, 13739.[CrossRef][ISI][Medline]
22 . Miller, D. W., Batrakova, E. V. & Kabanov, A. V. (1999). Inhibition of multidrug resistance associated protein (MRP) functional activity with pluronic block polymers. Pharmaceutical Research 16, 396401.[CrossRef][ISI][Medline]
23
.
Espuelas, S., Legrand, P., Loiseau, P. M. et al. (2000). In vitro reversion of amphotericin B resistance in Leishmania donovani by poloxamer 188. Antimicrobial Agents and Chemotherapy 44, 21902.
24 . Hunter, R. L., Jagannath, C., Tinkley, A. et al. (1995). Enhancement of antibiotic susceptibility and suppression of Mycobacterium avium complex growth by poloxamer 331. Antimicrobial Agents and Chemotherapy 39, 4359.[Abstract]
25 . Jagannath, C., Sepulveda, E., Actor, J. K. et al. (2000). Effect of poloxamer CRL-1072 on drug uptake and nitric-oxide-mediated killing of Mycobacterium avium by macrophages. Immunopharmacology 48, 18597.[CrossRef][ISI][Medline]
26 . Newman, M. J., Actor, J. K., Balussubramian, M. et al. (1998). Use of ionic block copolymers in vaccines and therapeutics. Critical Reviews in Therapeutic Drug Carrier Systems 15, 89142.[ISI][Medline]
27 . Frenchick, P. J., Markham, R. J. F. & Cochrane, A. H. (1985). Inhibition of phagosome-lysosome fusion in macrophages by soluble extracts of virulent Brucella abortus. American Journal of Veterinary Research 46, 3325.[ISI][Medline]
28
.
Pizarro-Cerdá, J., Moreno, E., Sanguedolce, V. et al. (1998). Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infection and Immunity 66, 238792.
29 . Detilleux, P. G., Deyoe, B. L. & Cheville, N. F. (1990). Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy. Veterinary Pathology 27, 31728.[Abstract]
30
.
Pizarro-Cerdá, J., Meresse, S., Parton, R. G. et al. (1998). Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infection and Immunity 66, 571124.