Editor: Anthony George
Targeting Brucella melitensis with polymeric nanoparticles containing streptomycin and doxycycline
Article first published online: 24 MAR 2009
© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 294, Issue 1, pages 24–31, May 2009
How to Cite
Seleem, M. N., Jain, N., Pothayee, N., Ranjan, A., Riffle, J. S. and Sriranganathan, N. (2009), Targeting Brucella melitensis with polymeric nanoparticles containing streptomycin and doxycycline. FEMS Microbiology Letters, 294: 24–31. doi: 10.1111/j.1574-6968.2009.01530.x
- Issue published online: 2 APR 2009
- Article first published online: 24 MAR 2009
- Received 19 September 2008; accepted 28 January 2009.First published online 24 March 2009.
- drug delivery;
Treatment and eradication of intracellular pathogens such as Brucella is difficult because infections are localized within phagocytic cells and most antibiotics, although highly active in vitro, do not actively pass through cellular membranes. Thus, an optimum strategy to treat these infections should address targeting of active drugs to the intracellular compartment where the bacteria replicate, and should prolong the release of the antibiotics so that the number of doses and associated toxicity can be reduced. We incorporated streptomycin and doxycycline into macromolecular nanoplexes with anionic homo- and block copolymers via cooperative electrostatic interactions among the cationic drugs and anionic polymers. The approach enabled simultaneous binding of both antibiotics into the nanoplexes, and their use resulted in an improvement in performance as compared with the free drugs. Administration of two doses of the nanoplexes significantly reduced the Brucella melitensis load in the spleens and livers of infected BALB/c mice. The nanoplexes were more effective than free drugs in the spleens (0.72-log and 0.51-log reductions, respectively) and in the livers (0.79-log and 0.42-log reductions, respectively) of the infected mice. Further research regarding the design of optimum nanoplex structures will be directed towards alterations in both the core and the shell properties to investigate the effects of the rates and pathways of entry into immune cells where the brucellae replicate.
Brucellosis, especially caused by Brucella melitensis, remains the most common zoonotic disease worldwide, with > 500 000 cases reported annually and an estimated 2.4 billion people at risk (Jennings et al., 2007). The bacterial pathogen is also classified by the CDC as a category B pathogen that has potential for development as a bio-weapon (Corbel, 1997; Pappas et al., 2006a, b). Although many successful vaccines are being used for immunization of animals, no satisfactory vaccine against human brucellosis is available (Corbel, 1997). Because of intracellular localization of Brucella and its ability to adapt to the environmental conditions encountered in its replicative niche (Seleem et al., 2008), treatment failure and relapse rates are high and depend on the drug combination and patient compliance. The optimal treatment for brucellosis is a combination regimen using two antibiotics, because monotherapies with single antibiotics have been associated with increased relapse rates (Solera et al., 1997; Pappas et al., 2005, 2006a, b). The combination of doxycycline with streptomycin (SD) is currently the best therapeutic option with less side effects, especially in cases of acute and localized forms of brucellosis (Ariza et al., 1992; Solera et al., 1995; Ersoy et al., 2005; Alp et al., 2006; Falagas & Bliziotis, 2006). Neither streptomycin nor doxycycline alone can prevent multiplication of intracellular brucellae (Shasha et al., 1994). Although the SD combination is accepted to be the most effective regimen, therapeutic failure and adverse drug reactions due to prolonged use and relapse related to the pharmacokinetic and pharmacodynamic properties of the antibiotics are still common among brucellosis patients (Alp et al., 2006). These problems have led to investigations of new drugs and improved drug carrier strategies for treating brucellosis, including antibiotics loaded into microspheres and poly(d,l-lactide-co-glycolide) nanoparticles and IFN-γ-loaded albumin nanoparticles (Lecaroz et al., 2006, 2007; Dizbay et al., 2007; Segura et al., 2007).
Nanocarriers, due to their small size and target-specific localization properties, are being actively investigated for preferential drug delivery to various disease sites in the body, including intracellular bacterial and viral infections (Shah & Amiji, 2006). Crosslinked poly(acrylic acid) (PAA) derivatives have been approved by the FDA and used as safe penetration enhancers for in vivo drug delivery (Verhoef, 1998; Weidner, 2001; Deutel et al., 2008). PAA–amoxicillin polyionic complexes also improved therapy for Helicobacter pylori infected patients (de la Torre et al., 2005). Trubetskoy et al. (2003) condensed cationic polyethyleneimine with anionic DNA with an excess of cation relative to anion, and then adjusted the charge ratio by adding the anionic salt form of PAA to form tertiary complexes. Complexation of the PAA reduced the toxicity of intravenously delivered complexes, prevented serum inhibition and enhanced in vivo gene transfer in mice. In this paper, we report an alternative approach for delivering drugs for Brucella treatment by incorporating streptomycin and doxycycline into macromolecular complexes with anionic homo- and block copolymers via cooperative electrostatic interactions among both cationic drugs and the anionic polymers. Block copolymers of poly(ethylene oxide-b-sodium acrylate) (PEO-b-PAA−+Na) and poly(sodium acrylate) (PAA−+Na) were complexed with cationic streptomycin and doxycycline. The new nanoplexes can incorporate these antibiotics simultaneously, and are more effective in treating murine brucellosis relative to the same combination of free drugs.
Materials and methods
All chemicals were purchased from Sigma-Aldrich unless otherwise noted. t-Butyl acrylate (tBA) was distilled from calcium hydride under vacuum before polymerization. Tetrahydrofuran (THF) was dried over sodium containing benzophenone as an indicator and distilled immediately before use. PEO-b-PAA was converted to the sodium salt by titrating with 1 N NaOH. The PAA homopolymer was obtained from Polyscience Inc. and its molecular weight (MW) was 6000 g mol−1. 9,10-Phenanthraquinone was dissolved in dimethylformamide at a concentration of 2 mg mL−1.
Synthesis of α-methoxy-ω-bromoisobutyrate poly(ethylene oxide) (PEO)
α-Methoxy-ω-hydroxy-PEO (6.0 g, 3.0 × 10−3 mol, Mn=2000 g mol−1) was dissolved in 50 mL of dry, freshly distilled tetrahydrofuran. Triethylamine (0.88 mL, 6.2 × 10−3 mol) was added to the solution. 2-Bromoisobutyryl bromide (0.75 mL, 6.0 × 10−3 mol) was added dropwise at room temperature. After 24 h, the reaction mixture was filtered and concentrated in vacuo. The polymer was isolated by precipitation into cold diethyl ether twice and dried under vacuum.
Synthesis of a PEO-b-PtBA diblock copolymer by atom transfer radical polymerization (ATRP)
PEO-b-PtBA diblock copolymers were synthesized according to a previously reported procedure (Sijian, 2003). Briefly, 1.0 g of α-methoxy-ω-bromoisobutyrate-PEO (4.65 × 10−4 mol), 4.2 mL of tBA (2.9 × 10−2 mol), 210 μL of pentamethyldiethylenetriamine (1 × 10−3 mol) and 8 mL dry toluene were added to a Schlenk flask. After degassing, 72 mg of cuprous bromide (CuBr, 5 × 10−4 mol) was added quickly under nitrogen. The reaction mixture was deoxygenated with three freeze–thaw cycles, and then maintained at 80 °C for 8 h. After the polymerization, the catalyst was removed by filtering the reaction mixture through basic alumina using dichloromethane as the eluent. The solvent was evaporated and the block copolymer was dried under vacuum at 50 °C overnight. Target=10 000 Mn. GPC showed 10 200 Mn and nuclear magnetic resonance (NMR) showed 9200 Mn.
Synthesis of PEO-b-PAA−+Na
A PEO–b–PtBA diblock copolymer (1.0 g, 6.1 × 10−3 eq of t-butyl ester groups) was dissolved in 25 mL of dichloromethane. Trifluoroacetic acid (5 mL, 6.5 × 10−2 mol) was slowly added and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated at 40 °C under vacuum and the residue was dissolved in deionized water with adjustment to a pH of 8 using 1 N NaOH. The copolymer was recovered by freeze-drying. In the acid form, 1H NMR showed the block MWs to be 2000 Mn PEO and 3600 Mn PAA.
Formation of polymer–antibiotic complexes
In a 100-mL round-bottom flask, PAA−+Na (25 mg, 3.2 × 10−6 mol, 2.70 × 10−4 eq of anions) and PEO-b-PAA−+Na (26 mg, 3.88 × 10−6 mol, 1.94 × 10−4 eq of anions) were dissolved in deionized water (50 mL) to prepare a polymer solution with a concentration of 1 mg mL−1. The solution was placed in a sonication bath and 5 mL of streptomycin solution (35 mg streptomycin, c. 1.8 × 10−4 eq cations) was added using a syringe. Any free streptomycin was removed by dialysis against 4 L of deionized water at 4 °C for 24 h and the nanoplex was recovered by freeze-drying. The dialyzed and isolated complex was found to contain 24 wt% of streptomycin. After freeze-drying, the complex (10 mg, c. 1.2 × 10−4 eq cations and c. 1.4 × 10−4 eq anions) was dissolved in 10 mL of deionized water and transferred to a dialysis bag with an MW cutoff of 3500 g mol−1. A solution of deionized water (40 mL) and 20 mg doxycycline hyclate (3.9 × 10−5 mol, 3.9 × 10−5 eq cations) was prepared. The dialysis bag containing the polymer–streptomycin complex was submerged in the doxycycline solution and the doxycycline was allowed to adsorb into the complex for 15 h. The mixture was then dialyzed against deionized water (2 L) at 4 °C for 24 h to remove any unbound doxycycline. The contents in the dialysis bag were freeze-dried. The final complex was found to contain 5 wt% of doxycycline. Thus, the ratio of streptomycin to doxycycline in the nanoplex was c. 5 : 1 w : w.
Characterization of the polymer–antibiotic nanoplexes
Determining the concentrations of antibiotics in the nanoplexes
The solute sizes and ζ potentials of the nanoplexes were characterized by dynamic light scattering with a Zetasizer 1000 HS with laser diffractometry (Malvern Instruments, Malvern, UK). Each nanoplex (1 mg) was dispersed in 1 mL of water and analyzed. Measurements were performed in triplicate for each batch of nanoplexes. The results were taken as the mean volume diameter of three measurements.
The release profile of streptomycin from the nanoplexes was obtained according to a previously reported procedure for assaying streptomycin (Belal et al., 2001). Before adding the doxycycline, free streptomycin (10 mg) or the nanoplex (42 mg, 10 mg streptomycin) was diluted with deionized water to 10 mL and the solution was placed in a dialysis bag with an MW cutoff of 3500 g mol−1. The solution was dialyzed against 300 mL of phosphate-buffered saline (PBS) at pH 7.4 and 37 °C for 24 h. At different times, 0.1-mL aliquots were withdrawn and stored at 4 °C before making the measurements. To determine the amount of streptomycin, the aliquots were mixed with 0.5 mL of 9,10-phenanthraquinone reagent (2 mg mL−1 in dimethylformamide) and 0.2 mL of NaOH (1.0 N). The reaction was conducted at room temperature for 30 min, and then 0.1 mL of concentrated hydrochloric acid was added. The final volume was adjusted to 3 mL with deionized water and the absorbance intensity was measured at 310 nm. The release rate of doxycycline was obtained by dissolving 4 mg of doxycycline hyclate or the nanoplex containing both streptomycin and doxycycline at a concentration corresponding to 4 mg doxycycline in 10 mL deionized water, and then transferring it to a dialysis bag with an MW cutoff of 3500 g mol−1. The solution was dialyzed against 300 mL of PBS as a receptor medium for 24 h. Aliquots (0.3 mL) were withdrawn over time and stored at 4 °C before analysis. To determine the amount of doxycycline, the aliquots were diluted to 3 mL with deionized water and the UV absorbance was measured at 350 nm.
Brucella melitensis 16M (DelVecchio et al., 2002) was routinely grown at 37 °C in tryptic soy broth (TSB) or on tryptic soy agar (TSA, Difco).
Cell culture: in vitro infection assay
To study the activities of the nanoplexes in vitro, murine macrophage-like cells J774A.1 were seeded at a density of c. 5 × 105 cells per well in 24-well plates (Corning Incorporated) 48 h before infection. The cells were routinely grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma Chemical Co.) in a humidified 5% CO2 atmosphere at 37 °C. The J774.A1 cells were infected with B. melitensis for 1 h at a 1 : 100 multiplicity of infection. The cells were washed three times with DMEM medium containing 50 μg mL−1 gentamicin (Sigma Chemical Co.) to kill and wash off nonphagocytized bacteria before incubation with DMEM medium supplemented with 10% FCS. At 24 h postinfection, the cells were washed two times with DMEM medium, and either free drug or nanoplexes (45 μg streptomycin and 9 μg doxycycline per well) resuspended into DMEM medium were added to the infected cells and incubated further for 18 h. Control polymer (no antibiotics) and nontreated control-infected cells were compared. Infected cells were lysed and the CFUs of Brucella in the lysates were determined by plating a series of 10-fold serial dilutions onto TSA and incubating the plates at 37 °C under 5% CO2.
Animal experiment: in vivo infection treatment assay
Female BALB/c mice (Charles River Laboratories, Wilmington, MA) that were 6–8 weeks old were used. A total of 20 mice were inoculated intraperitoneally with 2 × 104 CFUs of B. melitensis. After 2 weeks, one group of mice was kept untreated as controls and the remaining three groups (five mice each) were administered two doses of (1) SD nanoplex (180 μg streptomycin and 36 μg doxycycline), (2) free SD (180 μg streptomycin and 36 μg doxycycline) or (3) control polymer (no antibiotics) at days 14 and 16 postinfection. At the third day after administration of the last dose, the animals were euthanized. The spleens and livers were collected and bacterial CFUs per individual organ were determined by plating serial dilutions of the organ homogenates on TSA plates. The number of colonies was determined after incubation for 4 days at 37 °C and 5% CO2. The experimental procedures on mice and the facilities used to hold the experimental animals are in compliance with the Virginia Tech Institutional Animal Care and Use Committee (IACUC). All experiments were conducted according to CDC-approved standard operating protocols for the biosafety level 3 and animal biosafety level 3 facility at the Infectious Diseases Unit at Virginia Tech (CDC approval no. C20031120-0016).
All statistical analyses were performed using the Student two-tailed t-test using microsoft excel. P values≤0.05 were considered significant.
PEO-b-PtBA was synthesized with a tertiary bromoalkane-functionalized PEO oligomer to initiate the ATRP chain growth of tBA utilizing a previously reported procedure (Sijian, 2003). The narrow MW distribution and the control over the MW of the PtBA block signify the living nature of this ATRP polymerization. The t-butyl ester units were deprotected with trifluoroacetic acid under mild conditions and the resultant carboxylic acids were neutralized with base to form PEO-b-PAA−+Na. 1H NMR (Fig. 1) confirmed the expected copolymer structure. MW of the block copolymer derived from 1H NMR was 2000 g mol−1 PEO and 3600 g mol−1 PAA. Stable nanoplexes comprising streptomycin and doxycycline incorporated a homopolymer PAA−+Na in combination with the PEO-b-PAA−+Na block copolymer into complexes with both cationic drugs.
Properties of the nanoplexes
Dynamic light scattering showed that the PAA/PAA-b-PEO–streptomycin nanoplex had a mean intensity average diameter of 110 nm. The streptomycin and the doxycycline loading in the nanoplexes were 24% and 5%, respectively, by weight.
Drug release from the nanoplexes
The release of streptomycin from the nanoplexes at the physiological pH was relatively slow (Fig. 2). A burst release occurred during the first 5 h comprising c. 15–20% of the total amount of the drug, and 25–30% had been released at 24 h. Doxycycline was released significantly faster than streptomycin from the nanoplexes with 100% release after 15 h (Fig. 3).
In vitro infection assay
Intracellular B. melitensis reduction after incubation of the nanoplexes with the infected macrophage cell line J774.A1 was compared with the same concentrations of the free antibiotics. Both the nanoplexes and the free drugs were able to clear the infection completely (Fig. 4). Cells treated with empty nanoplexes had 4.27 ± 0.11 [intracellular CFU (log ± SD)] while the untreated cells had 4.56 ± 0.07 [intracellular CFU (log ± SD)].
In vivo infection assay
The efficacy of nanoplexes vs. the free form of the same SD drug combination was tested in BALB/c mice. The treatment of mice started 2 weeks after infection with B. melitensis. The mice received two doses of free drugs, antibiotic-loaded nanoplexes or empty nanoplexes. At 3 days after administration of the last dose, the animals were euthanized, and the CFUs in the livers and spleens were assayed. The results are summarized in Table 1. The combined free SD therapy induced a significant (P<0.05) reduction in the log CFUs of B. melitensis per spleen, but the reduction was not significant in the liver. The nanoplexes were more effective in reducing the infection than free drugs and induced a significant (P<0.05) reduction in the log CFUs of B. melitensis per spleen and liver. The empty nanoplexes did not produce a significant reduction of B. melitensis.
|Treatment||Log CFU/ spleen||Reduction (log)||Log CFU/ liver||Reduction (log)|
|Untreated||6.59 ± 0.06||0.00||4.32 ± 0.20||0.00|
|Polymer control||6.35 ± 0.02||0.24||4.16 ± 0.03||0.16|
|Polymer SD||5.87 ± 0.20||0.72*||3.53 ± 0.20||0.79*|
|Free SD||6.08 ± 0.17||0.51*||3.90 ± 0.36||0.42|
Doxycycline is one of the most widely used antibiotics for treating human brucellosis, but the relapse rates are very high when it is used as a monotherapy. For this reason, combination therapy with doxycycline and aminoglycosides is recommended to increase the efficacy of treatments and avoid relapses (Grillo et al., 2006). Aminoglycosides exhibit several characteristics that make them useful as a second antimicrobial agent for brucellosis. Among these are concentration-dependent bactericidal activity, postantibiotic effects, reasonably predictable pharmacokinetics and synergism with other antibiotics (Vakulenko & Mobashery, 2003). However, due to the polar nature of aminoglycosides, they exhibit only a low level of intracellular penetration. Thus, their incorporation into nanoparticle carriers might facilitate the entry of the drug into the intracellular replicative niche of brucellae. Moreover, sustained drug delivery could reduce long-term treatment, improve drug bioavailability, reduce dosing frequency, encourage patient compliance and eliminate some of the toxic side effects associated with the free drug (Lecaroz et al., 2007).
Nanotechnology has already contributed significantly to pharmacology and antimicrobial therapy through drug delivery systems targeting phagocytic cells that are infected by intracellular pathogens (Gelperina et al., 2005; Lecaroz et al., 2007; Gaspar et al., 2008). In the present study, we investigated the capability of a polymer-based nanoparticulate system (nanoplexes) as a carrier for combination therapy (streptomycin and doxycycline), and the efficacy of treatment compared with the free form of the same combination of drugs against murine brucellosis.
The fabrication method for these nanoplexes enabled multiple drugs to be incorporated, and this allowed for multiple treatment pathways to be implemented. The PAA−+Na and the polyanionic block of the PEO-b-PAA−+Na were first condensed with the polycationic streptomycin to form a core-corona structure with the polyanionic components and the streptomycin in the core. The PEO block of the copolymer formed the corona chains that protruded into the aqueous physiological medium. Because of the multiple-charged nature of both the polymers and the streptomycin, the drug was complexed through cooperative electrostatic interactions, and this enabled high concentrations of the drug to be incorporated into the cores. Nanoplexes obtained by this technique contained 20–25% by weight of streptomycin, 25-fold higher than the values reported earlier using liposomes (Khalil et al., 1996). The concentration of drug in these materials after extensive dialysis to remove any unbound species was determined through derivatization with 9,10-phenanthraquinone, and then UV analysis (Belal et al., 2001). This could not be measured in the presence of doxycycline due to overlapping absorptions. Thus, due to analytical methods, it was necessary to first form the complexes with streptomycin, isolate them and quantify the aminoglycoside, and then adsorb the doxycycline into these materials in a subsequent step. Doxycycline is also cationic, but has only one cation per molecule, so the electrostatic interaction with the anionic core is not as strong. Because of the fact that doxycycline was adsorbed in a second step where the core was somewhat shielded by the corona, together with the fact that the doxycycline only possesses one cation, lower concentrations of doxycycline were obtained relative to streptomycin. The doxycycline concentration was also measured by UV spectroscopy, but this could be done in the presence of the streptomycin because the peak at 350 nm was sufficiently resolved. If different concentration assays were available, it would be interesting to determine whether both drugs could be incorporated in one step simultaneously. It is reasoned that such an approach might afford higher concentrations of doxycycline.
The in vitro drug release profiles of the two drugs from the nanoplexes were distinctly different at pH 7.4. About 25% of the total streptomycin was released from the nanoplexes over 24 h in PBS at pH 7.4, while 100% release of doxycycline was achieved after 18 h (Figs 2 and 3). The faster rate of release of doxycycline is consistent with the monocationic chemical structure and the consequently lower binding energy relative to streptomycin. Both release rates were sufficiently slow, indicating that the nanoplexes remained intact under physiological conditions at pH 7.4 for short periods, and this could enhance phagocytosis by the reticulendothelial system and reduce ototoxic activity associated with the use of free streptomycin (Forge & Schacht, 2000). As the drugs begin to leave the nanoplexes, the net anionic nature of the complexes increases. As this occurs, greater electrostatic attraction between the polymer and drugs may slow the release. Therefore, we hypothesize that the nanoplexes may act as depot systems, releasing drugs slowly and continuously into the target organ of infection.
Brucella melitensis-infected macrophages and infected BALB/c mice were selected as the models for testing the therapeutic potentials of the nanoplexes. Comparing the efficiency of treatment of the nanoplexes with the free drugs with the J774.A1 macrophage-like cell line infected with B. melitensis was not possible because of the ability of free doxycycline to penetrate the cell membranes and clear the infection in the cell culture model (Reveneau et al., 2005). The results, however, do support the capability of the nanoplexes to penetrate the cell membranes and target intracellular B. melitensis.
Determination of the efficiency of the nanoplexes for elimination of Brucella from the spleens and livers of infected mice was assessed after treatment with two doses. The results indicated that the nanoplexes were more effective in clearing the infection in the spleen and liver compared with the same dose and combination of free drugs. The nanoplexes achieved a 0.72-log reduction in the spleens, while for the free drugs, the reduction was 0.51 log. Both of these values are statistically different (P<0.05) from the results achieved for the negative control. On the other hand, the nanoplexes achieved a statistically significant 0.79-log reduction in the livers (P<0.05), while for the free drugs the reduction was a nonsignificant value of 0.42 log from the results achieved for the control.
The present study demonstrates the feasibility and efficacy of targeting Brucella using two antibiotics incorporated into macromolecular complexes with anionic homo- and block copolymers via cooperative electrostatic interactions among cationic drugs and anionic polymers. Considerable research will be required to understand how efficiently such complexes localize within the immune cells where brucellae can replicate, as well as the effects of the rates of entry into the cells. Thus, one aspect of continuing research will be to investigate a systematic range of shell structures on these nanoplexes to tailor their rates of entry into macrophages. Moreover, the particular structures of the nanoplexes investigated herein do not allow for the concentrations of streptomycin relative to doxycycline within the nanoplex cores to be varied over the ranges that may be optimum. Thus, another aspect relative to the design of nanoplexes for treating Brucella will be to alter the core structures so that a larger range of drug (relative) concentrations can be studied.
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