The present study was aimed at evaluating the effectiveness of amoxicillin-bearing HSA (human serum albumin) and PLGA [poly(lactic-co-glycolic acid)] microparticles in combating Listeria monocytogenes infection in Swiss albino mice. Amoxicillin-bearing HSA microspheres were prepared by chemical cross-linking of a drug/albumin mixture with glutaraldehyde, and PLGA microspheres were prepared by the W/O/W (water-in-oil-in-water) emulsion technique. The microspheres were characterized for their size, ζ potential and entrapment efficiency using SEM (scanning electron microscopy) and a Zetasizer. Release kinetics was performed in a phosphate buffer (pH 7.4) at 37°C simulating physiological conditions. Bacterial burden in various vital organs and survival data established enhanced efficacy of PLGA and HSA microspheres as compared with free drug. Among the two delivery systems, PLGA microspheres, when compared with HSA microspheres, imparted better efficacy in terms of reduction in bacterial load as well as increase in survival. The results of the present study clearly demonstrate that microparticles successfully target the infected macrophages and the approach could be well exploited for targeting the intracellular pathogens as well.
Listeriosis is an invasive disease that affects immunocompromised patients and has the highest mortality rate among various food-borne illnesses [1–5]. Besides, it can also cause a severe form of disease in immunocompetent persons as well . Another form of Listeria related to human disease is perinatal infection, which is associated with a high rate of fetal loss (including full-term stillbirths) and serious neonatal disease [7,8]. Perinatal infection is caused by a ubiquitous pathogen Listeria monocyto-genes that inflicts both human beings and animals with equal propensity.
L. monocytogenes multiplies in the RES (reticuloendothelial system) and survives intracellularly after ingestion by the circulating macrophages. The intracellular parasitism opted by Listeria spp. not only protects them from antibody onslaught but also offers escape from various chemotherapeutic agents. Antimicrobial agents that are widely used for the treatment of listeriosis include cephalosporins, clindamycin, rifampicin, the fluoroquinolones and the aminoglycosides [9–11]. The reference treatment currently in practice involves high doses of aminopenicillin (ampicillin or amoxicillin) and gentamicin (intravenous) [12,13]. Unfortunately, despite the availability of potential antibiotics against L. monocytogenes, listeriosis continues to result in a high fatality rate, especially when it affects the central nervous system of the patient [14–17].
It is well known that amoxicillin, a β-lactam antibiotic, acts by inhibiting the cell wall formation of Gram-positive bacteria. L. monocytogenes located in the macrophages escapes from amoxicillin treatment as only a small proportion of antibiotic reaches the intracytoplasmic compartments of infected cells and offers weak and slow action against intracytoplasmic bacterial progression [18–20].
Improved delivery of active antibiotics specifically targeting pathogen-harbouring macrophages can be a useful strategy against intracellular pathogens [21–23]. In addition to specific targeting, sustained release of antimicrobial agents from particle-loaded macrophages for a prolonged period may also be advantageous in inhibiting the multiplication cycle of the actively dividing pathogen. Microsphere technology has the ability for accomplishing these goals by achieving intracellular delivery of entrapped drug molecules and, once allowed inside the macrophages, resulting in programmed controlled and sustained release over a prolonged period .
Microsphere-based drug and antigen delivery systems have been prepared from naturally occurring as well as chemically synthesized polymers . Microspheres composed of biodegradable polymer PVA (polyvinyl alcohol) and PLGA [poly(lactic-co-glycolic acid)] are among the primary delivery systems that can be used as carriers for sustained release of drugs and antigens administered by oral or parenteral routes [26,27]. The polymers used in the preparation of microspheres have demonstrated excellent tissue compatibility. Moreover, the restorable synthetic polymers are non-toxic and have already been used for various biochemical applications including drug delivery [28,29]. In fact, the broad range of physiochemical properties, degradation rates and biocompatibility of microspheres give them wide scope as a potential therapeutic delivery system.
Because of the chemical and physical stability and rapid clearance by phagocytic cells, HSA (human serum albumin) microspheres have also been successfully employed in the delivery of drug molecules to the RES .
In the present study, we have developed amoxicillin-bearing PLGA and HSA microsphere formulations and establish their role in the elimination of Listeria infection from the systemic circulation in model animals.
MATERIALS AND METHODS
All reagents used in the present study were of the highest purity available. Amoxicillin, PLGA, HSA, PVA and glutaraldehyde were purchased from Sigma. DCM (dichloromethane) and other chemicals used were of analytical grade of purity and procured locally.
Preparation of amoxicillin-loaded albumin microspheres
Microspheres containing amoxicillin were prepared by the emulsion polymerization method following the published protocol [31,32]. For amoxicillin-bearing HSA microsphere preparation, 125 mg of HSA (dissolved in distilled water) was mixed with 10 mg of amoxicillin (dissolved in a minimum volume of DMSO). The solution was mixed with cottonseed oil and sonicated to obtain a primary emulsion. The emulsion was added to cotton seed oil continuously stirred at 1400 rev./min maintaining the temperature of the solution at 25°C. The resulting microspheres were washed with anhydrous diethyl ether to remove the oil phase and cross-linked by suspending in 0.75 ml of glutaraldehyde solution (0.001%, v/v) for 15 min. The cross-linked microspheres were washed three times with sterile 20 mM PBS, freeze-dried and stored at 4°C until further use.
Preparation of amoxicillin-loaded PLGA microspheres
PLGA microparticles were prepared using W/O/W (water-in-oil-in-water) emulsion by the solvent evaporation technique as described previously and modified in our laboratory . For entrapment of amoxicillin, 30 mg of drug was dissolved in DMSO and was mixed with 190 mg of PLGA dissolved in DCM and sonicated in a bath-type sonicator to form a primary emulsion. The primary emulsion was added to 10% (w/v) PVA and homogenized using a Silverson L4RT homogenizer (Silverson Machines, East Longmeadow, MA, U.S.A.) and the resulting O/W (oil-in-water) emulsion was stirred at room temperature (25°C) for 18 h to allow solvent evaporation, leading to amoxicillin-loaded microparticles formation. The microparticles were centrifuged and washed with distilled water to remove unentrapped drug. The microparticles were freeze-dried and finally stored in a desiccator at 4°C.
Determination of entrapment efficiency
The amount of entrapped amoxicillin in the two microparticle formulations was determined by a published procedure . Briefly, 10 mg of freeze-dried microspheres was dissolved in 1.0 ml of a 0.1 M sodium hydroxide solution. The solution was vortex-mixed and centrifuged at 8832 g for 5 min at room temperature. The absorbance of the supernatant was read at 276 nm and the concentration was calculated from the amoxicillin standard prepared in 0.1 M NaOH.
Shape and surface morphology of microspheres by SEM (scanning electron microscopy)
SEM (using a Leo model 430 scanning electron microscope) of both amoxicillin-loaded microspheres was performed to characterize their size and surface morphology. A freeze-dried preparation of amoxicillin-loaded microspheres was suspended in 20 mM PBS (pH 7.0) and a drop was mounted on a clear glass stub, air-dried and coated with gold–palladium alloy using a sputter coater. An accelerating voltage of 29.34 kV was used for SEM imaging.
Determination of ζ potential
The ζ potential of microsphere formulation was measured by the instrument Zetasizer nano ZS using DTS software (Malvern Instruments) based on M3-PALS technology. The experimental formulation was freeze-dried in a 2 ml centrifuge tube and the samples were reconstituted in phosphate buffer (pH 7.4). This dispersion was then dispensed to the electrophoretic cell to measure the electrophoretic mobility and the results were used to determine ζ potential values. The experiment was repeated three times and the average ζ potential was calculated by taking the mean values obtained in each set of experiments.
In vitro release kinetics of amoxicillin microspheres
In order to determine the release kinetics of amoxicillin from the two microsphere formulations, multiple samples of each formulation were transferred into microvials. To each vial, 1.0 ml of 20 mM sterile PBS was added. A small amount of sodium azide was added to avoid the microbial growth and the vials were incubated at room temperature. PBS was separated from microspheres by centrifugation at 10000 g for 10 min and fresh buffer was exchanged at definite time intervals thereafter. The percentage of released amoxicillin was determined by measuring A276 and the concentration was calculated from a standard plot.
Female Swiss albino mice of weight 18±2 g were used in the whole study. The animals were given a standard pellet diet (Hindustan Lever) and water ad libitum. Animals were examined daily for their mortality and morbidity prior to commencement of the study, and only healthy animals were included in the experiment. Bleeding, injection and killing of animals was performed strictly following the mandates approved by the Animal Ethics Committee (Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India).
The two strains of L. monocytogenes used for the study were ATCC 19115 and MTCC 839. PMA agar/broth was used for growing L. monocytogenes.
Antibacterial susceptibility testing
The MIC (minimal inhibitory concentration) of amoxicillin for L. monocytogenes strains was determined by the broth microdilution method as described by National Committee for Clinical Laboratory Standards . The antibacterial agent was tested over the final concentration range of 0.02–5 μg/ml against two strains of L. monocytogenes. Testing was performed in 96-well round-bottomed microtitre plates. The cell suspension of L. monocytogenes was prepared to give a final inoculum concentration of 1×106 cfu (colony-forming units)/ml. The wells containing bacterial inoculum with different concentrations of drugs and proper controls were incubated for 48 h. The MIC was defined as the lowest concentration of drug at which there was complete inhibition of growth. The MIC was 1.25 for the L. monocytogenes strain MTCC 839 and 1.0 μg/ml for ATCC 19115 isolate. L. monocytogenes ATCC 19115 was used to examine the in vivo efficacy of amoxicillin microspheres.
Preparation of inoculum
The L. monocytogenes cells for infection were grown in tryptose phosphate broth at 37°C for 24 h. The cell suspension was centrifuged at 2208 g for 15 min at 4°C in refrigerated centrifuge followed by washing with normal saline. The number of cells was counted by measuring the attenuance against normal saline and the cell count was determined. Each animal was challenged with 1×106 cfu of L. monocytogenes suspended in 0.2 ml of normal saline through the intravenous route.
The efficacy of microsphere amoxicillin was determined against L. monocytogenes infection in the mouse model. Various dosage regimens were examined by administering different amounts of amoxicillin via the intraperitoneal route in Swiss albino mice. Our pilot studies showed that among various regimens (5–25 mg of free drug), the dose of 5 mg/kg of b.w. (body weight) behaved like an untreated control group, whereas the dose of 25 mg/kg of b.w. of free drug resulted in moderate survival (~40%) of the treated animals. Finally, this dose (25 mg/kg of b.w.) was selected for evaluating the efficacy of formulated amoxicillin microspheres. Interestingly, this dose is significantly less than that used in other reported studies [36,37]. Antibacterial treatment was started 24 h post-infection for a period of 7 days. Treatment was given once on days 2, 4 and 6 post-infection. The infected animals were treated with various microsphere formulations of amoxicillin and were divided into the following groups: (i) saline, (ii) sham-HSA-microsphere, (iii) sham-PLGA-microsphere, (iv) free drug (25 mg/kg of b.w.), (v) HSA-micro-amoxicillin (25 mg/kg of b.w.) and (vi) PLGA-micro-amoxicillin (25 mg/kg of b.w.).
Assessment of antibacterial load
Quantitative assessment of the bacterial burden in various vital organs was performed following the published procedure . The animals from different groups were killed on days 7, 15 and 21 post-infection and vital organs, namely kidney, spleen and brain, were removed aseptically. The organs were washed extensively with hypo-osmotic buffer, homogenized and serially diluted with normal saline. Various dilutions of each sample (200 μl) were dispersed on PMA agar plates. After incubation for 24 h at 37°C, the colonies were counted and the bacterial load was calculated by multiplying by the dilution factor. The efficacy of different microsphere amoxicillin formulations was assessed by monitoring the survival of the treated animals. For survival studies, the animals were observed twice for possible mortality each day during 30 days of observation.
Results were analysed using SigmaPlot 11.0 software by one-way ANOVA of mean values following a Student's t test. P<0.05 was considered statistically significant.
Entrapment efficiency and release kinetics of amoxicillin-bearing HSA and PLGA microspheres
Amoxicillin-bearing HSA and PLGA microspheres were prepared using procedures as described in the Material and methods section. Both amoxicillin-bearing microsphere formulations were characterized for their size, ζ potential and entrapment efficiency. The entrapment efficiency of drug was 70±5% in HSA microspheres and 42.0±5% in PLGA microspheres. The electron microscopy studies revealed the size of HSA microspheres as 0.1–0.5 μm, while that of PLGA microspheres was in the range of 0.2–0.5 μm (Figures 1A and 1B). The ζ potential of PLGA microspheres was −4.21±1.98 mV, while it was −0.27±0.8 mV for HSA microspheres. At 25°C, approx. 20% of the drug was released from HSA microspheres in 50 h and it was increased up to 38% in 100 h. As shown in Figure 2, during 7 days of incubation, HSA microspheres released 45% of its total entrapped drug content at 25°C, while this increased up to 76% of total drug content at 37°C. The drug release almost doubled when the temperature was raised from 25°C to 37°C. PLGA with the composition 50:50 degrades in approx. 1–2 months by hydrolysis of the ester backbone . The release profile of drug from in-house prepared PLGA microspheres increased from 4% to 8% up to 4 days at both 25°C and 37°C. After 4 days, release kinetics of amoxicillin from PLGA microspheres was much sustained at both 25°C and 37°C. Only 11.0% of active drug content was released at room temperature (25°C) on 7 days incubation, whereas it increased up to 16.0% when the incubation temperature was elevated to 37°C.
Scanning electron micrograph of (A) amoxicillin-bearing PLGA microspheres and (B) amoxicillin-bearing HSA microsphere
In vitro release kinetics of amoxicillin from HSA and PLGA microspheres in PBS
Effect of amoxicillin microspheres on the establishment of infection in animals challenged with
The efficacy of amoxicillin microspheres was evaluated against experimental murine listeriosis. The animals were challenged with 1×106 cells of L. monocytogenes and subsequently treated with various formulations of amoxicillin (25 mg/kg of b.w.). Animals treated with amoxicillin-bearing PLGA microspheres showed the most significant decrease in bacterial load in spleen of killed animals when compared with amoxicillin-bearing HSA microspheres (25 mg/kg of b.w.) or the free form of drug treatment (Figure 3A). HSA-based amoxicillin formulation was, however, more effective in elimination of bacterial load as compared with the free form of the drug. Animals were not available in control and sham microsphere-treated groups beyond the 5th day post-infection as none of them survived beyond this period. On day 21 post-infection, there was negligible bacterial load in animals treated with amoxicillin-bearing PLGA microspheres, whereas a modest load was observed in the case of amoxicillin-bearing HSA microspheres. The animals treated with the free form of the drug succumbed to the infection and were not available after day 21 post-infection.
Bacterial load in vital organs of animals treated with various formulations of amoxicillin
In the next set of experiments, we determined the residual bacterial burden in kidney of experimental animals. As shown in Figure 3(B), amoxicillin-bearing microsphere formulation of PLGA further cleared the bacterial burden in kidney of treated animals. Among animals that survived 21 days post-infection, those amoxicillin-bearing PLGA microspheres revealed very negligible bacterial burden in the amoxicillin-bearing PLGA microsphere group, whereas it was still significant in amoxicillin-bearing HSA microspheres. Mice given the free form of amoxicillin showed far high bacterial burden.
Figure 3(C) shows that amoxicillin-bearing PLGA microspheres cleared the pathogen burden from the brain more efficiently than amoxicillin-bearing HSA microspheres and the free form of the drug. Amoxicillin-bearing HSA microspheres were clearly more effective than the free form of the drug.
Increased survival of animals infected with
L. monocytogenes after treatment with microsphere-based formulations of amoxicillin
For survival studies, mice were challenged with 1×106L. monocytogenes cells per animal. The dose was effective in establishing a full-blown infection as animals receiving no treatment succumbed to death on day 5 post-infection. As shown in Figure 4 no animal survived in a group of animals treated with sham PLGA and HSA microspheres beyond day 5 post-infection. On the other hand, there was 50% survival in the free drug-treated animals, 70% survival in HSA-amoxicillin-treated animals, 90% survival in PLGA amoxicillin-treated animals on day 6 post-infection (P<0.05). Animals treated with PLGA amoxicillin microspheres (25 mg/kg of b.w.) showed maximum survival rate (80% beyond 40 days post-infection), whereas HSA-amoxicillin microspheres (25 mg/kg of b.w.) managed survival rate only to 60% beyond 40 days post-infection. The survival rate of mice receiving free drug was only 40% (Figure 5).
L. monocytogenes-infected animals after treatment with various formulations of amoxicillin
A comparison of the survival of mice infected with
Listeria and treated with various amoxicillin formulations on day 40 post-infection
Recent advancement in the field of drug delivery has led to the development of various promising novel drug delivery systems. For example, microsphere-based formulations have been used for sustained delivery of various biological components, including antigens, peptides, proteins, antibiotics, steroids and anticancer drugs [39–47]. Interestingly, such delivery systems are efficient in delivering their contents at targeted sites and are biodegradable as well .
In spite of early breakthroughs, the use of albumin microspheres to improve the efficacy of therapeutic drugs has not been exploited adequately. In the present study, we have developed HSA microspheres by chemical cross-linking of protein core material. Different concentrations of glutaraldehyde were used to obtain a range of cross-linking and porosity of HSA microspheres. The HSA microspheres treated with 5% (v/v) glutaraldehyde did not release appreciable quantities of entrapped amoxicillin (results not shown). Preparations obtained using lower concentration of glutaraldehyde resulted in relatively slow release of the drug from microspheres. Microspheres given 0.5% glutaraldehyde released drug from day 4 onwards, whereas microspheres prepared with 1.0% glutaraldehyde treatment showed no release of entrapped drug in 7 days (M. Farazuddin and M. Owais, unpublished work). Finally, we treated the HSA microspheres with 0.001% glutaraldehyde and examined the release pattern. As shown in Figure 2, 40% entrapped drug was released at room temperature, whereas elevation of incubation temperature to 37°C resulted in 60% release of total incorporated drug in 7 days.
As 50:50 poly(DL-lactide-co-glycolide) microspheres have been reported to degrade in approx. 1–2 months, we opted for the same composition . As shown in Figure 2, PLGA microspheres released 10% of the entrapped drug at room temperature in 7 days and the quantity of drug released increases moderately on incubation at 37°C. PLGA microspheres showed 4% release at room temperature and 6–8% release at 37°C. Finally, both types of amoxicillin microsphere formulations were examined for their efficacy in clearance of L. monocytogenes infection in model animals.
L. monocytogenes is a Gram-positive bacterium widely found in the environment . This facultative intracellular pathogen causes severe food-borne infections, septicaemia and central nervous system infections, primarily in elderly people and patients with impaired cellular immunity, and abortion [14,15]. Prospective clinical studies on the best antibiotic regimen are not available, as listeriosis is rare in humans [47,48]. Recalcitrance of listeriosis to therapy is mainly attributed to the failure of the administered antibiotic to reach the cytosol of the macrophage-harbouring pathogen. The use of high doses of the drug was, however, found to be effective in elimination [12,13,18–20]. The reported paradoxical effect of amoxicillin against listeriosis may be attributed to the susceptibilities of the bacteria released after cellular lysis, and the prevention of spread of infection to other cells [18–20,49]. The dosage regimen used in the present study was 25 mg of amoxicillin per kg of b.w. Amoxicillin-entrapped PLGA and HSA microsphere treatment led to maximum clearance of pathogen from vital organs on day 7 post-infection as compared with the free form of the drug (Figures 3A–3C). Similar results were obtained on day 14 post-infection. In the 3 weeks after treatment there was observed a significant decrease in bacterial count in HSA microsphere-treated animals, but the effect was rarely marked in mice receiving amoxicillin/PLGA microspheres. There were no survivors among the group receiving free amoxicillin.
No animal survived beyond 5 days in groups receiving no amoxicillin, sham HSA or PLGA microspheres. The survival rate at the end of the experiment in animals receiving free drug was 40%, whereas in those receiving HSA and PLGA microspheres it was 60 and 80% respectively (Figure 4).
In summary, the novel formulations of PLGA and HSA microsphere amoxicillin were highly effective in eliminating Listeria infection from the mice as compared with the free form of the drug. The effectiveness can be correlated well with sustained drug release profile of the two microsphere-based formulations. Being particulate in nature, the microsphere formulations were avidly taken up by macrophages. The microsphere-harbouring macrophages acted as secondary depots of drug and caused effective suppression of intracellular Listeria pathogen. The results suggest the remarkable potential of the microsphere combinations in the treatment of bacterial, fungal and other infectious diseases.
L. monocytogenes, a Gram-positive organism, is mainly associated with food-borne diseases but is also known to cause septicaemia and meningitis in elderly people and patients with impaired immunity. Although a range of antibiotics are available commercially for its treatment, mainly aminopenicillins are in use widely. As this organism resides intracellularly, it is not readily accessible to administered antibiotics. In the present study, we tried to develop amoxicillin-bearing HSA and PLGA microspheres with potential to deliver its content intracellularly. HSA microspheres were prepared using glutaraldehyde cross-linking and PLGA microspheres were prepared by the W/O/W emulsion technique. Both formulations were evaluated for their release pattern under simulated physiological conditions and evaluated for their anti-listeriosis activity in the mice model. PLGA microspheres were most effective in reducing the bacterial burden and mortalities in the animals challenged with Listeria. Amoxicillin-bearing HSA microspheres were less effective in this regard, but were superior in lowering bacterial load and promoting the survival of animals as compared with the free drug, whereas the bacterial burden remained very high in animals treated with the free form of drug, who also showed less survival. We conclude that PLGA microspheres are highly effective in the treatment of listeriosis in Swiss albino mice.
Mohammad Farazuddin and Arun Chauhan carried out all the experiments. Raza Khan helped in statistical analysis and also helped in carrying out certain experiments. Mohammad Owais designed the study and was a guide throughout the experiments.
This work was made possible by the kind support of Professor M. Saleemuddin, Co-ordinator, Interdisciplinary Biotechnology Unit, Aligarh Muslim University.
This work was supported by the University Grants Commission (UGC), Government of India (research fellowship to F.M.).