Anti Pneumococcal Activity of Azithromycin-Eudragit RS100 Nano-Formulations

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(3), 455-459 doi: 10.15171/apb.2016.059 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Azithromycin (AZI) is a semi-synthetic antibiotic from azalide class (14-membered ring macrolide) which was FDA approved for use in 1994.This antibiotic has been launched to pharmacy market as a result of notable limitations of former 14-membered ring macrolide i.e. erythromycin. 1AZI achieves high tissue concentrations and reach the sites of infection by direct uptake via phagocytes. 2 Consequently the obtained high tissue concentrations are retained for extended periods resulted in once-daily dosing for 3 or 5 days.AZI like other macrolides inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit of the bacterial 70S ribosome which inhibits peptidyl transferase activity and interferes with amino acid translocation during the translation process (Figure 1). 3 Remarkable microbiological features of make it a good candidate for the management of a wide range of bacterial infections especially those of bacterial pneumonia. 4evertheless, in line with the global antibiotic resistance trend, significant resistance among the formerly susceptible microorganisms like Streptococcus pneumoniae and Staphylococcus aureus has been detected.In fact the increasing numbers of reports on the failures of treatment of infections caused by macrolide resistant pneumococcal isolates in the last two decades has been a matter of concern. 4Three prominent mechanisms for Pneumococcal macrolide resistance can be summarized as the alteration of the ribosomal target site, the production of inactivating enzymes, and the production and utilization of active efflux mechanisms.The first two approach are shown to be more important in the macrolide resistance development. 1everal approaches to overcome bacterial resistance issue were developed from which the use of drug carriers such as nanoparticles seems to be one of the promising strategies.Nanoparticles drug delivery vehicles are generally in dimensions of about 5 -350 nm in diameter.][11][12]  Electrospinning is a promising method for nanoparticle production.A polymer-drug solution is injected into a capillary tube while applying electrical charge on the solution to create a liquid jet flying towards a grounded collector screen.By its movement to the collector, the solvent evaporates and a fine network of nanofibers or nanobeads would form on the collector. 13ZI loaded Eudragit RS100 nano-formulations were prepared using Electrospinning in our previous published work. 13Here we aimed to evaluate the activity of the selected nano-formulations against AZI resistant S. pneumonia and S. aureus strains in comparison with the untreated drug.

Preparation and characterization of the nanobeads and nanofibers
The fabrication of AZI-Eudragit RS100 nanobeads and nanofibers was conducted according to our previously published paper. 13

Nano-suspension preparation
The stock nano-suspension (containing equivalent of 2.62 mg/ml) was prepared by dispersing 157.2 mg of the AZI-Eudragit RS100 nano-formulations, in 10 ml distilled water by vortexing.In the next step, the vortexed suspension was entered the process of serial dilution in order to obtain a bunch of distinct concentrations to use them within the ongoing agar dilution process.Furthermore, the stock of AZI solution in the same concentration (2.62 mg/ml) was prepared in 5% ethanol in distilled water as negative control.

Antibacterial activity of AZI-Eudragit RS100 nanoformulations
Antibacterial properties of the nano-formulations were investigated against AZI resistant S. pneumonia and S. aureus standard strains.After activating of the freezed bacteria, the culture was maintained in the blood base agar and nutrient agar media for S. pneumonia and S. aureus respectively at 4°C and used as stock cultures.A single colony from the stock plate was transferred into the relevant agar media and incubated over night at 37°C.After incubation time the cells were collected from the surface of agar medium, washed twice, and resuspended in saline solution to provide an optical density equal to 0.5 McFarland or bacterial concentration around 10 8 CFU/ml.Antibacterial performance against S. pneumonia was evaluated by agar dilution method.Firstly, a series of concentrations of antibiotics were prepared by 1:2 diluting trend in order to be added into the agar medium.Two millilitres from each concentration of AZI nanoformulation as well as untreated AZI solution (concentrations ranging from 0.625 to 160 µg/ml) was added to the plate containing 30 ml Haemophillus agar supplemented with 5% sheep blood (temperature=45°C).Therefore, for each concentration from each formulation two plates were obtained.After preparing the plates, they were inoculated with a swab from the adjusted S. pneumoniae inoculum suspension and incubated for 2 days at 37°C.The MICs were recorded after 48 h of incubation on the basis of the first concentration that inhibit the bacterial growth.The antimicrobial activities of prepared nanoformulations against S. aureus were assessed by broth macrodilution method according to CLSI protocol.Briefly bacterial inoculum in Muller-Hinton Broth medium, added to serial diluted NPs suspension as well as drug solutions to reach the final concentration of 10 6 CFU/ml.After 24 h incubation at 37 °C , from the content of the tubes streak cultured onto Muller Hinton agar plates.MICs was designated as the first concentration of antibiotic with no sign of bacterial growth. 14

Characteristics of prepared nano-formulations
Nanofibers in the sizes about 100-300 nm in diameter and micro scale in length as well as nanobeads in the range of 100-500 nm in diameter were achieved.As concluded from the SEM results depicted in Figure 2 (a and b), application of higher concentrations of solution resulted in production of nanofibers with smooth surfaces.[17]  formulations compared to drug solution.According to the MIC test results, the tested S. pneumonia was shown to be in vitro resistant to AZI even at higher concentrations.In fact in all the tested concentrations (up to 160µg/ml) S. pneumonia colonies were observed on the surface of the cultured plates (Figure 3).While MIC values for sensitive strains were reported to be 0.5 to 2µg/ml in standard CLSI protocol that was significantly different from the MIC value obtained for our test strain.In spite of the obtained in vitro resistance pattern, it should be in vivo studies to extrapolate the data into clinical failures.In fact it has been shown that the hostdefense responses lead macrolides and azalides after absorption to the infection site and release them at the infection site.Also, after phagocytosis of the pathogens, expose them to high intracellular drug concentrations. 1,18On the other hand, the MIC values recorded for AZI-Eudragit RS100 nanofibers were 40 µg/ml, which was significantly lower than the MIC value of the untreated counterpart (Figure 4).This can be presumed as a remarkable improvement in the AZI performance.This improvement in the antibacterial performance of AZI in nano-form can be probably explained by some possible mechanisms.Overall, nanoparticle based antimicrobial delivery systems could interact with microorganisms via two mechanisms and consequently enhance the performance; by fusing with microbial cell wall or membrane and release the load within the cell wall or membrane; via adsorbing to cell wall and provide a antibiotic depot to continuously release drug molecules, which will diffuse into the interior of the microorganisms. 9Furthermore, it has been reported that antibiotic-loaded NPs can enter host cells and tissues through endocytosis, followed by releasing the loaded drug to eradicate intracellular microorganisms. 19In the case of AZI-Eudragit RS100 nanofiber the adsorption to the bacteria cell wall to provide an antibiotic depot on the bacterial surface seems to be more feasible due to the large surface area of the nanofibers with more interaction opportunity of the bacterial cell wall.Nevertheless, according to the fact that nano-fibres are nano size just in one dimension, they could not pass through absorption barriers and the fate of formulation in in vivo experiments could be different.Figure 4 shows the cellular structure of S. pneumonia which composed of cell wall and a rough polysaccharide capsule.Based on the capsule structure of S. pneumonia, it can be predictable that the adsorbing and trapping of the nanofibers within the complex capsule structure of the bacteria to provide an in situ antibiotic depot would not be farfetched. 20Nevertheless, based on the MIC value obtained for AZI-Eudragit RS100 nanobeads, the observed figures were >160 µg/ml, same as that of the untreated AZI solution indicating that the prepared nanobeads simply maintained the antibacterial efficiency of AZI.Still it would be valuable because of the potential benefits of prepared nanobeads which need to be evaluated in the upcoming in vivo or animal studies.In vitro antibacterial activity of the prepared nanosuspensions was assessed against S. aureus by broth macrodilution method and compared to untreated drug solution.

Antibacterial activity of the nanosuspensions S. pneumonia was cultured in
According to the MIC test results, the experimented strain of S. aureus was shown to be in vitro resistant to AZI.In fact in all the tested concentrations (0.25 to 128µg/ml) S. aureus colonies were observed on the surface of the streak cultured plates (MIC >128µg/ml).According to standard CLSI protocol MIC values for susceptible strains were reported to be 2 to 8µg/ml.This finding is in good agreement with the various reports indicating growing in vitro resistance trends described about S. aureus against macrolides.On the other hand, the MIC values recorded for both AZI-Eudragit RS100 nanofibers and nanobeads represented a resistant paradigm as well, with MIC values of >128µg/ml.Indeed, preparation of nanoformulation (nanofibers or nanobeads) could not improve the in vitro antibacterial activity of AZI in comparison with drug solution and just maintained the MIC values of AZI equal to untreated solution.Figure 5 (B) shows a schematic picture of a S. aureus cell wall structure.It seems that the adsorption and trapping opportunity of the nano-formulations to the capsule less cell wall structure of S. aureus would be lower than capsulated rough S. pneumonia cell wall.

Conclusion
In the current in vitro study we assessed the anti bacterial activity of formulated AZI-Eudragit RS100 nanobeads and nanofibers against S. pneumonia and S. aureus; two common cause of bacterial pneumonia.Herein, it was shown that AZI-Eudragit RS100 nanofibers with enhanced in vitro antimicrobial effect against S. pneumonia can be considered as a candidate for in vivo evaluations in antibiotic therapy of Pneumococcal infections.

Figure 1 .
Figure 1.Schematic representation for antibacterial mechanism of macrolides.

Figure 2 .
Figure 2. SEM images for AZI-Eudragit RS100 electrospuns.Samples with the drug: polymer ratio of 1:5 and solution concentrations of 20% (a) samples with the drug: polymer ratio of 1:5 and solution concentrations of 10% (b).

Figure 3 .
Figure 3. Haemophillus agar plates containing AZI-Eudragit RS100 nanofibers as well as untreated AZI solution with streak cultures of S. pneumonia.