Colloids and Surfaces B: Biointerfaces 80 (2010) 3439

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Development of azithromycinPLGA nanoparticles: Physicochemical

characterization and antibacterial effect against Salmonella typhi
Ghobad Mohammadi a,b,c , Hadi Valizadeh a,c , Mohammad Barzegar-Jalali a,d , Farzaneh Lotpour a ,
Khosro Adibkia a,c , Morteza Milani a , Morteza Azhdarzadeh a , Farhad Kiafar a,c , Ali Nokhodchi d,e,
a

Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
d
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
e
Medway School of Pharmacy, Universities of Kent and Greenwich, Central Ave., Anson Building, Chatham Maritime, Kent ME4 4TB, United Kingdom
b
c

a r t i c l e

i n f o

Article history:
Received 29 January 2010
Received in revised form 4 May 2010
Accepted 18 May 2010
Available online 24 May 2010
Keywords:
Nanoparticle
Azithromycin
PLGA
Antibacterial
Salmonella typhi

a b s t r a c t
The objective of the present research was to formulate poly(lactide-co-glycolide) nanoparticles
loaded with azithromycin with appropriate physicochemical properties and antimicrobial activity.
Azithromycin-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were prepared in three
different ratios of drug to polymer by nanoprecipitation technique. Antibacterial activity of these
nanoparticles was examined against gram-negative intra cellular microorganism Salmonella typhi. The
antibacterial effect was investigated using serial dilution technique to achieve the minimum inhibitory
concentration (MIC) of nanoparticles. The results showed that physicochemical properties were affected
by drug to polymer ratio. The results showed that nanoscale size particles ranging from 212 to 252 nm
were achieved. Physicochemical properties were affected by drug to polymer ratio. The highest entrapment efciency (78.5 4.2%) was obtained when the ratio of drug to polymer was 1:3. Zeta () potential of
the nanoparticles was fairly negative. The DSC thermograms and X-ray diffraction patterns revealed that
the drug in the nanoparticles was in amorphous state. FT-IR spectroscopy demonstrated no detectable
interactions between the drug and polymer in molecular level. In vitro release study showed two phases:
an initial burst for 4 h followed by a very slow release pattern during a period of 24 h. The results of
antimicrobial activity test showed that the nanoparticles were more effective than pure azithromycin
against S. typhi with the nanoparticles showing equal antibacterial effect at 1/8 concentration of the intact
drug. In conclusion, the azithromycin nanoparticle preparations showed appropriate physicochemical
and improved antimicrobial properties which can be useful for oral administration.
2010 Elsevier B.V. All rights reserved.

1. Introduction
The need for intracellular chemotherapy has been recognized
for many years. Despite the discovery of new antibiotics, the treatment of intracellular infections often fails completely to eradicate
the pathogens [1]. In order to eradicate the pathogens efciently,
an antibiotic should be carried in a form that is able to be endocytosed by phagocytic cells and then release it into these cells. By
loading antibiotics into the nanoparticles, one can expect improved
delivery to infected cells. Nanoparticles are the carriers developed
for these logistic targeting strategies and are colloidal in nature,
biodegradable and similar in behavior to intracellular pathogens.
These colloidal carriers, when administered intravenously, are

Corresponding author. Tel.: +44 1634 202947.

E-mail address: a.nokhodchi@kent.ac.uk (A. Nokhodchi).
0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.05.027

rapidly taken up by the cells of the mononuclear phagocyte system,

the very cells which may constitute a sanctuary for intracellular bacteria [2,3]. Therefore, the entrapment of antibiotics within
nanoparticles has been proposed for the treatment of intracellular
infections [2].
Ciprooxacin-loaded nanoparticles were more active in human
macrophages infected with Mycobacterium avium complex than
free drug [4]. For example, the efcacy of ampicillin was found to
be increased by 120-fold in murine salmonellosis by loading the
drug into the nanoparticles [5].
Due to intracellular polymeric over loading of polymers,
nanoparticles must be degraded in vivo to avoid side effects.
PLGA nanoparticles fulll such requirements (e.g. biocompatibility,
biodegradability, sustained release and safety) and were extensively studied by researchers [6]. The antibiotics ciprooxacin
and rifampicin and the antifungal uconazole and voriconazole
are examples of antimicrobial incorporated in PLGA preparations
recently [710].

G. Mohammadi et al. / Colloids and Surfaces B: Biointerfaces 80 (2010) 3439

35

the Silent Crusher M (Heidolph, Germany). The agitation speed

was 13,000 rpm and kept on an ice-water bath. Acetone was
removed at room temperature under stirring conditions for 12 h.
The nal nanosuspension was centrifuged (Beckman Centrifuge,
AvantiTMJ-25, USA) at 14,000 rpm for 30 min and the precipitated
nanoparticles were washed twice with water using the previously
described centrifugation approach and then lyophilized by using a
lyophilizer (Christ Alpha 1-4; Germany). The nal dry powder was
taken out for physicochemical and anti bacterial investigations.
2.3. Encapsulation efciency

Fig. 1. Chemical structure of azithromycin.

Azithromycin, AZI, is a new macrolide antibiotic (Fig. 1) that is

similar in structure to erythromycin. Compared with erythromycin,
AZI has increased activity against gram-negative organisms. AZI
has a broader gram-negative antibacterial spectrum than erythromycin, demonstrating activity against Salmonella spp. Its
pharmacokinetic prole is characterized by improved bioavailability, excellent tissue and intracellular distribution and extended
half-life [11].
Azithromycin nanoparticles have been made into nanosuspensions to overcome the poor bioavailability [12], but there are
no studies on formulation of polymeric azithromycin nanoparticles in order to improve the antibacterial activity of this drug.
Therefore, the purpose of the present study was to prepare
azithromycinPLGA 50:50 nanoparticles by means of a modied
quasi emulsion solvent diffusion technique [13] to improve the
antibacterial activity of azithromycin hence the side effects of the
drug. The antimicrobial activity of the nanoparticles was compared to pure azithromycin against gram-negative intracellular
bacterium Salmonella typhi. Typhoid fever is a clinical syndrome
caused by bacteraemic infection with S. typhi that is an important
cause of morbidity and mortality in resource-poor regions of the
world [14]. It has been reported that AZI is more effective than most
of the drugs in treatment of multidrug-resistant (MDR) S. typhi [14].
2. Materials and methods
2.1. Materials
Azithromycin powder was obtained from Dr Reddys Pharmaceutical Company, India. poly(d,l-lactide-co-glycolide) (PLGA)
(50:50 d,l-lactide:glycolide) with average molecular weight of
12,000 g/mol (Resomer RG 502), was purchased from Boehringer
Ingelheim, Germany. Poly vinyl alcohol, PVA, with molecular
weight of MW 95000 (Acros Organics, Geel, Belgium) and acetone
(Merck, Darmstadt, Germany) were used. All other materials used
were of analytical or HPLC grade.
2.2. Preparation of nanoparticles
Nanoparticles (nanospheres) with 1:3, 1:2 and 1:1 ratios of
the drug to PLGA were prepared by nanoprecipitation according
to the modied quasi emulsion solvent diffusion technique [13].
Azithromycin and PLGA 50:50 powders were dissolved in acetone
(2.5 mL) at room temperature (25 C). The resulting organic solution was injected at the constant rate of 0.5 mL/min in aqueous
phase (40 mL) containing PVA 95000 (2%, w/v), as a stabilizing
agent. Due to low solubility of AZI in higher pH [15], the aqueous phase pH was adjusted at 8 to increase drug loading. The
process was carried out under homogenization for 5 min using

Three samples from each preparation (equivalent to 5 mg drug)

were randomly chosen. Each sample was dissolved in 0.5 mL
acetone for the extraction of the drug from nanoparticles. After
evaporation of the acetone the drug was dissolved in 100 mL sterile
water. The mixture was then centrifuged and the supernatant was
drawn to measure their azithromycin contents microbiologically
after appropriate dilution. A microbiological assay was adapted
from the method described in the ofcial US Pharmacopoeia (USP
2004) monograph.
Briey, lyophilized Micrococcus luteus (PTCC 1169) purchased
from Persian Type Culture Collection, Iran, was activated in Tripticase Soy broth medium. Twenty four hours before drug assay,
50 L of the growth medium was transferred to the antibiotic agar
medium I and incubated at 35 C for 1 day. The growth bacterial
culture was diluted in 0.9% NaCl to reach 30% turbidity at 580 nm
wavelength. The resultant bacterial suspension was used as culturing inoculums. In the assay, plates containing 25 mL of antibiotic
agar I were incubated with the bacterial inoculums and 6 wells of
6 mm diameter were punched and lled with 100 L of AZI test or
reference samples. After 24 h incubation at 35 C, inhibition zone
diameter was measured. The method was validated by determination of linearity, precision and accuracy operational characteristics.
The linearity was evaluated by linear regression analysis, which was
calculated by the least squares regression method. 100 L AZI reference solutions in concentrations of 3000, 2000, 1000, 500 and
250 ng/mL were subjected to the well diffusion assay described
above.
Precision of the assay determined by repeatability (intra-assay)
and intermediate precision (inter-assay) was expressed as the relative standard deviation (RSD) of four quality control samples. The
accuracy was determined by adding the known amount of AZI reference substance (quality control samples) at the beginning of the
process. Encapsulation efciency was calculated using the equation
shown below:
encapsulation efciency =

drug content in NPs

100
drug amount used

2.4. Nanoparticle size, morphology and zeta potential

To assess the size of nanoparticles, they were dispersed in water
(pH 8) and the mean particle-size values were measured by using a
laser diffraction particle-size analyzer (Shimadzu, Japan) equipped
with Wing software (version 1201).
The morphology of the nanoparticle was investigated by scanning electron microscopy (SEM) with LEO 440i (Leo Electron
Microscopy Ltd, Cambridge, UK) at an accelerating voltage of 20 kV.
Prior to examination, samples were prepared on aluminum stubs
and coated with gold under argon atmosphere by means of a sputter
coater.
The particle surface charge was quantied as zeta potential (
potential) using a Zetassizer 4 (Malvern Instr., UK). Measurements
were performed in distilled water adjusted with sodium hydroxide
to a pH 8 and conductivity of 50 S/cm.

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G. Mohammadi et al. / Colloids and Surfaces B: Biointerfaces 80 (2010) 3439

2.5. X-ray powder diffraction

The X-ray powder diffraction (XRPD) of the drug, PLGA 50:50,
nanoparticles and physical mixture in the 1:2 drug to polymer ratio
were recorded using an automated X-ray diffractometer (Siemens
D5000, Munich, Germany). Cross-section of the samples were taken
and held in place on a quartz plate for exposure to Cu K radiation
of wavelength 1.5406 . The samples were then analyzed at room
temperature over a 2 range of 450 , with sampling intervals of
0.02 2 and a scanning rate of 0.6 /min.
2.6. Differential scanning calorimetry (DSC)
Thermograms of azithromycin, PLGA 50:50, nanoparticles
(drug:polymer 1:2 ratio) and physical mixture of drug with the
polymer (1:2 ratio) were recorded on a DSC-60 (Shimadzu, Kyoto,
Japan). Samples (5 mg weighed to a precision of 0.005 mg) were
placed in aluminium pans and the lids were crimped using a Shimadzu crimper. Thermal behavior of the samples was investigated
at a scanning rate of 40 C/min covering temperature range of
25260 C. The instrument was calibrated with an indium standard.

from Persian Type Culture Collection, Iran. The bacterium obtained

in lyophilized form was cultured in Luria Bertuni agar medium
(Scharlau, Spain) after suspending them in sterile distilled water.
The plate was incubated for 24 h at 37 C. Single colony from the
plate was transferred into 4 mL uid of LB medium and incubated
overnight at 37 C and 200 rpm in shaking incubator. The cells
were harvested by centrifugation at 3000 rpm (Behdad, Iran) for
15 min at 4 C. Subsequently, they were washed twice and resuspended in Ringer solution to provide the concentration range of
105106 CFU/mL for broth dilution method [18].
In order to evaluate the MIC of NPs the mentioned nanoparticle suspensions were diluted in decreasing concentrations. The
tubes were inoculated with a 1 mL of inoculums of tested bacterium
(nal concentration of 105106 CFU/mL). After 24 h of incubation at
37 C, the tubes were screened for any evidence of bacterial growth.
MIC was dened as the lowest concentration of AZI that completely
suppressed the bacterial growth [19]. Tubes of PLGA suspensions
as a negative control and AZIPLGA PMs in the corresponding NP
ratios as positive controls were also included.
3. Results

2.7. Fourier-transform infrared spectroscopy (FT-IR)

3.1. Encapsulation efciency

Fourier-transform infrared spectroscopy of intact AZI, PLGA

50:50, nanoparticles and physical mixture in the 1:2 drug to polymer ratio were obtained on a Bomem 2000 FT-IR system (Bomem,
Quebec, Canada) using the KBr disk method. Samples were mixed
with KBr powder and compressed to 10-mm discs by hydraulic
press at pressure of 150 bar for 30 s. The scanning range and resolution were 4504000 cm1 and 4 cm1 respectively.

Encapsulation efciencies of AZI are reported in the Table 1. It is

evident from Table 1 that the percentage encapsulation efciency
was affected by drug:polymer ratio. Higher encapsulation efciencies were obtained with decreasing the ratio of drug:polymer.

2.8. Dissolution study

Drug release was performed according to the pervious publication for piroxicam nanoparticles [15] with some modication.
Briey the prepared nanoparticles, azithromycin powder as well as
the physical mixtures of azithromycin: PLGA 50:50 (all containing
10 mg azithromycin) were placed in the vessels containing 250 mL
phosphate buffer (pH 6). The vessels were incubated at 37.8 C
with continuous orbital mixing (50 rpm). This mixing rate has been
previously used in the dissolution test of AZI capsules [16]. At specied time intervals, 3 mL of supernatant was removed using a glass
syringe tted to cellulose acetate membrane (Whatman, UK) with
25 mm diameter and 20 nm pore diameter. The dissolution medium
was replaced with fresh media passed through the same ltration
assembly. The drug concentrations in the aliquots were assayed
microbiologically according to the method described above for drug
content test. The cumulative percentage of azithromycin released
(considering the replaced volume of the dissolution medium) was
plotted versus time. The mean calculated values were obtained
from 3 to 4 replicates.
2.9. Nanosuspension preparation
The suspensions (containing 0.5 mg/mL AZI) were formulated by
the dispersing of AZI particles alone, AZI- PLGA 50:50 nanoparticles
at the ratios of 1:3, 1:2 and 1:1, their corresponding physical mixtures, and blank nanoparticles in physiological serum (0.9% NaCl),
supplemented with 0.005% (w/w) of HPMC as a dispersing and viscosity adjusting agent, as reported previously [17]. The pH of the
nal nano-formulations was adjusted at 7.4.

3.2. Characterization of nanoparticles: size, morphology and zeta

potential
As shown in Table 1, the submicron particles ranging from 212
to 252 nm were achieved. Narrow range of size distribution was
observed for all nanoparticles and all of the prepared particles
exhibited a relative monodisperse distribution (data not shown).
The same conclusion can be deduced from the SEM micrograph of
the AZI loaded nanoparticles (Fig. 2) which shows that the resultant
nanoparticles are in spherical shape with narrow particle-size distribution. All formulations appeared to be homogenous irrespective
of their compositions.
 potentials of the nanoparticles are also given in Table 1. In the
pH 8, -potential of the intact polymer and AZI were 21.42 2.84
and 2.79 2.31 mV respectively. As the drug molecules are neutral in charge at pH 8, therefore, it is expected that an increase in
the amount of PLGA in the nanoparticles increases negative surface
charge of the nanoparticles (Table 1).
3.3. X-ray powder diffraction (XRPD)
XRPD patterns are seen in Fig. 3. Sharp peaks at diffraction angles
of 2 10, 16.5, 18.6 and 19.8 are present in the XRPD of intact AZI
powder. The mentioned peaks are seen in the physical mixture
XRPD pattern but the peaks heights are smaller than that of intact
AZI powder. XRPD of nanoparticles containing AZI does not show
any distinctive peaks for AZI.
Table 1
Encapsulation efciency, mean particle diameter and  potential of various AZI
loaded PLGA nanoparticles (n = 3 SD).
Formulations

Encapsulation
efciency (%)

Mean particle
size (nm)

 potential (mV)

1:1 AZI:PLGA 50:50

1:2 AZI:PLGA 50:50
1:3 AZI:PLGA 50:50

50.5 3.4
66.8 2.8
78.5 4.2

252 5
230 7
212 4

5.6 2.15
11.10 1.87
15.56 2.53

2.10. Antibacterial activity of the nanoparticle suspensions

Antibacterial evaluation of the different nanoparticle suspensions (pH 8) was performed against S. typhi (PTCC 1609) purchased

G. Mohammadi et al. / Colloids and Surfaces B: Biointerfaces 80 (2010) 3439

37

Fig. 3. Powder X-ray diffraction pattern of the PLGA powder (PLGA), intact
azithromycin powder (AZI), physical mixtures (PM 1:2) and azithromycin-loaded
nanoparticles with 1:2 drug: polymer ratio (NANO 1: 2).

3.5. Fourier-transform infrared spectroscopy (FT-IR)

The FT-IR spectra of nanoparticles, PLGA, AZI and physical
mixtures of PLGAAZI are shown in Fig. 5. The AZI spectrum shows characteristic principal peaks at 1052 (symmetrical
aliphatic ether), 1721 (C O stretch of the ester form), 2950 (CH

Fig. 4. DSC curves of the PLGA powder (PLGA), intact azithromycin powder
(AZI), physical mixtures (PM 1:2) and azithromycin-loaded nanoparticles with 1:2
drug:polymer ratio (NANO 1: 2).

Fig. 2. SEM images of azithromycin-loaded nanoparticles with different ratios of

drug:polymer: (A) 1:3, (B) 1:2 and (C) 1:1.

3.4. Differential scanning calorimetry (DSC)

In order to investigate the effect of the process on thermal
behavior of formulations, DSC was carried out. The DSC scans of
samples and the melting point data are presented in Fig. 4. At
the mentioned scanning rate (40 C/min) pure AZI powder had an
endothermic peak at 149.3 C corresponding to its melting point
with an enthalpy of 142.61 J/g. In nanoparticle samples there were
an obvious decrease in melting point (131.62 C) and the enthalpy
(12.14 J/g) indicating the presence of amorphous state of AZI in
nanoparticles. The reduction in enthalpy of the physical mixture of
drugpolymer (89.06 J/g) is due to the presence of smaller amount
of drug in the mixture in comparison with pure drug.

Fig. 5. Infrared spectra of the PLGA powder (PLGA), intact azithromycin powder
(AZI), physical mixtures (PM 1:2) and azithromycin-loaded nanoparticles with 1:2
drug:polymer ratio (NANO 1:2).

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G. Mohammadi et al. / Colloids and Surfaces B: Biointerfaces 80 (2010) 3439

whereas the inhibitory concentration was about 25 g/mL for the

corresponding physical mixture suspensions. PLGA suspensions
indicated no antibacterial activity against S. typhi.
4. Discussion

Fig. 6. Dissolution proles of the intact azithromycin powder (AZI) and

azithromycin-loaded nanoparticles with different ratios of drug:polymer.

stretch), 3496 and 3561 cm1 (OH stretch). Peaks at wavenumber of 12001600 cm1 , 17001750 cm1 and 29003000 cm1 are
present in the PLGA spectrum. In the FT-IR spectrum of physical
mixture the AZI peaks with smaller intensity are seen. Nanoparticle spectrum shows a broad peak at about 3500 cm1 region that is
different from the corresponding physical mixture. But other peaks
that mentioned for AZI are present with smaller intensity.
3.6. Dissolution study
The dissolution proles of the AZI powder and nanoparticles
with different drug to polymer ratios are shown in Fig. 6. The time
required for 70% of drug dissolved (t70%) which is inversely related
to the dissolution rate, are: 28.2 min for AZI and 143.4, 193.2 and
262.2 min for nanoparticles with drug to polymer ratios of 1:1, 1:2
and 1:3 respectively. It is evident from Fig. 6 that nanoparticles had
lowest dissolution rates. An enhancement in the amount of polymer
in the nanoparticles led to a more reduction in release rate of AZI
from nanoparticles.
3.7. Antibacterial activity of the nanoparticle suspensions
Fig. 7 shows the mean MICs of the suspensions against S. typhi.
The lowest inhibitory concentration was about 3.12 g/mL regardless of the ratio of drug:polymer in nanoparticle suspensions,

Fig. 7. Minimum Inhibitory Concentrations (MICs) of the intact azithromycin (AZI),

physical mixtures (PMs) and azithromycin-loaded nanoparticle suspensions with
different drug:polymer ratios (n = 6 SD).

The present study showed that the PLGA 50:50 polymer could
be a useful nanocarrier for azithromycin. The results showed that,
indeed, nanoparticles with efcient loading of azithromycin were
obtained and the articles are in nanoscale range with narrow size
distribution. The results reported in Table 1 showed that maximum
AZI loading was 78.5% that could be due to washing procedure that
washes away some of the drug from nanoparticles. This could also
be the reason for the reduced burst release. An increase in the
amount of PLGA resulted in an increase in the drug entrapment.
This may be attributed to the higher viscosity of the internal organic
phase for higher PLGA ratios, which in turn would decrease the diffusion coefcient of the drug. The table also shows an increase in
the amount of PLGA could decrease the particle size. When the ratio
of drug:polymer was 1:3 the particle size of nanoparticles reduced
to 212 nm with a  potential of 15.56. It has been reported that
this size of nanoparticles with the mentioned  potential could
be suitable for I.V. administration [2022]. Size, surface property,
composition and other physicochemical properties of nanoparticles play a signicant role in the uptake by macrophages [23].
For both negatively and positively charged particles, it has been
proved that the extent of phagocytosis increases with increasing
zeta potentials, and is the lowest when zeta potential is zero [23].
As the surface charge of the vessels endothelial cells is negative
and the nanoparticles obtained in the present study has negative
charge, therefore the half-life of negatively charged NPs might be
increased in blood circulation [22].
It is reported that AZI has 13 crystalline forms [24]. Dominant
XRPD peaks that are shown in Fig. 3 for intact AZI are comparable to those that reported for crystalline form A (AZI dihydrate).
XRPD patterns of nanoparticles were characterized by the complete
absence of any diffraction peaks, suggesting a complete amorphization of AZI in the NPs [25]. The results obtained from DSC
thermograms (Fig. 4) are in consistent with those of XRPD patterns
and reveals that AZI intact powder is dihydrate form and the drug
in the nanoparticles is in amorphous state with no water and/or
solvent molecules in AZI crystal lattice [25].
FT-IR spectrums of AZI dihydrate and amorphous forms are
different (Fig. 5). The spectrum of dihydrate form shows two distinctive peaks at 3469 and 3561 cm1 which could be attributed to
the presence of water, that also reported for other drugs [26] but in
the amorphous form there is a broad band in the 3500 cm1 region
[25]. Fig. 5 reveals no appreciable interaction between the drug and
polymer in molecular levels during the preparation of nanoparticles, however, the crystallinity of the drug crystals was changed as
it is evident from XRPD patterns and DSC thermograms.
Fig. 6 shows that the release of AZI from NPs was slower and
more sustained than that of intact AZI. The rate of drug release from
any solid or semi solid delivery system is usually controlled by dissolution and/or diffusion [27]. In this study the control of the drug
release may be modied by the presence of insoluble polymer in
the NPs matrix body which in turn reduces the water penetration,
hence dissolution and diffusion. In the present study the release
proles of AZI from nanoparticle formulations could be divided
in two phases: an initial burst release for 4 h followed by a very
slow release pattern for the rest of release prole. However, the
release curves showed that the amount of initial burst drug release
depended upon the amount of PLGA in the nanoparticle samples.
The results showed that during the rst 4 h, an initial burst release
led to an early release of 68%, 79% and 85% of drug from the nanoparticles with 1:3, 1:2 and 1:1 drug to polymer ratios, respectively. The

G. Mohammadi et al. / Colloids and Surfaces B: Biointerfaces 80 (2010) 3439

burst release of AZI may be due to the dissolution and diffusion of

the drug that was poorly entrapped in the polymer matrix. The
poor entrapment of AZI could be because of the washing procedure
of nanoparticles that washes away the burst release of the drug,
while the slower and continuous release may be attributed to the
diffusion of the drug localized in the PLGA core of the NPs. Similar observations were reported by other researchers working on
paclitatel and enalapril PLGA nanoparticles [28,29].
Fig. 7 indicated that the antibacterial activity property was not
affected by the amount of polymer in the NPs. The azithromycinloaded NPs were about 8-fold more effective than the free drug
against S. typhi in in vitro model, since the MIC for all nanoparticle
formulations was approximately 8 times lower than that of free
drug (Fig. 7). The higher antibacterial effect of azithromycin NPs
may have resulted from higher diffusion of the NPs through the
bacteria. This indicates that the effective dose of this antibiotic can
be reduced against S. typhi hence the side effects of the drug.
It has been revealed that NPs are able to be endocytosed by
phagocytic cells and release the drug into the cells [14,23]. The
azithromycin-loaded NPs could be useful in the targeting of the
drug to the phagocytic cells to improve the treatment of intracellular infections compared to the treatment using free AZI. Other NPs
have been evaluated for ocular [30] and oral [31] administration,
and the formulated AZIPLGA nanoparticles could be useful for the
mentioned routes of administration.
Furthermore, the negatively charged azithromycin NPs can circulate for a longer time in blood rather than those of positively or
neutral charged [22] which may correspond to more effectiveness
of the azithromycin-loaded NPs in the treatment of intracellular
infections.
5. Conclusion
Azithromycin-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were successfully prepared by nanoprecipitation
technique. The results of antimicrobial activity test showed that
the nanoparticles were more effective than pure azithromycin
against S. typhi. Therefore, the clinical activity of azithromycin can
be enhanced if nanoparticle formulation of the drug is used and
this allows more efcient therapy compared to the conventional
formulation of azithromycin present in the market.
Acknowledgments
The authors wish to thank Tabriz University of Medical Sciences
and Drug Applied Research Center, Tabriz, Iran for the nancial
support of this work.

39

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