Colloids and Surfaces A: Physicochem. Eng.

Aspects 362 (2010) 135139

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Preparation of azithromycin microcapsules by a layer-by-layer self-assembly

approach and release behaviors of azithromycin
Zhen Zhang, Yihua Zhu , Xiaoling Yang, Chunzhong Li
Key Laboratory for Ultrane Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science
and Technology, Shanghai 200237, China

a r t i c l e

i n f o

Article history:
Received 13 January 2010
Received in revised form 17 March 2010
Accepted 5 April 2010
Available online 13 April 2010
Keywords:
Azithromycin
Microcapsule
Layer-by-layer self-assembly
Polyelectrolyte
Dissolution

a b s t r a c t
A novel preparation method of azithromycin (AZI) microcapsules based on hollow polyelectrolyte (PE)
microcapsules, which were prepared by layer-by-layer self-assembly onto the surface of silica microsphere (SiO2 ) followed by core dissolution has been investigated. The prepared AZI/PE microcapsules
with an average diameter 1.2 m possess homogeneous size and regular spherical shape. FTIR spectra
and XRD patterns indicated that AZI molecular structure was not changed and AZI crystal state changed
from monohydrate to dihydrate. The drug release experimental results showed an obvious improvement
in the dissolution rate of the prepared AZI/PE microcapsules in comparing with AZI raw material drug
powder.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Azithromycin (AZI) is a semi-synthetic acid-stable macrolide
antibiotic with a 15-membered azalactone ring. It shows a broad
spectrum of bacteriostatic activity, and is proved to be clinically
effective against not only Gram-positive but also Gram-negative
bacteria and atypical pathogens, which make up the deciency of
erythromycin, the rst macrolide used clinically as an antibiotic
[1]. Although AZI is derived from erythromycin, it differs by the
insertion of a methylsubstituted nitrogen on the lactone ring at
position 9-a of the large macrolactone ring. This modication produces an enhanced spectrum and potency against bacteria, and a
superior stability in acidic environment [2]. Besides, AZI is available in immediate oral or intravenous release, and has a longer
half-life period, fewer side-effects, and higher concentration in tissue than erythromycin, and can even be applied to children or
pregnant women [3]. So AZI and other newer macrolides, such as
larithromycin, dirithromycin and roxithromycin, are regarded as an
advanced-generation for erythromycin [4]. As for bacteriostatic
mechanism, like erythromycin, it appears to bind to the same receptor, 50 S ribosomal subunits of susceptible bacteria and suppresses
protein synthesis [1]. AZI plays a leading role in the treatment or
prophylaxis of common respiratory tract infections, skin structure
infection and several other clinical diseases, such as opportunistic

Corresponding authors. Tel.: +86 21 64252022; fax: +86 21 64250624.

E-mail addresses: yhzhu@ecust.edu.cn (Y. Zhu), czli@ecust.edu.cn (C. Li).
0927-7757/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2010.04.006

infections in AIDS, toxoplasmosis, pediatric infections, urethritis,

cervicits, among others [5,6].
However, the clinical application of AZI is limited by its low
bioavailability as a result of its poor solubility in water and poor
gastrointestinal response, as diarrhea for instance [5]. According
to NoyesWhitney equation [7], the dissolution rate of a given
drug particles is proportional to the particles specic surface area.
Therefore, one promising way to improve the solubility and dissolution behavior of AZI is to reduce the particle size, thus leading to an
increased specic surface area and an augmented dissolution rate
[5]. Particles size reduction methods include mechanical comminution, reprecipitation, high-pressure homogenization, ultrasonic
emulsication solvent diffusion method, spontaneous emulsication solvent diffusion method, etc. [8]. However, each of these
methods has its own drawback. In the process of mechanical comminution, impurities are brought in inevitably, and the distribution
of drug particles size is difcult to control. As for high-pressure
homogenization, ultrasonic or spontaneous emulsication solvent
diffusion method, although the AZI particles size can be reduced
to m or nm level, the resulting AZI particles are easily polluted
owning to the introduction of a surfactant or an emulsier. In addition, these methods do not deal much with the microstructure of
AZI particles and require a great deal of energy.
The present study introduces a novel method for the preparation of AZI/PE microcapsules. This method is based on hollow PE
microcapsules and difference of AZI solubility in water and in alcohol solution. Pharmaceutical microspheres and microcapsules are
highly favored in drug delivery and controlled release systems for

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their convenience for subsequent processing and the potential of

incorporating with other materials (i.e., vaccines, drugs, or inorganics) in the core or on the surface [9]. Hollow PE microcapsules
are prepared by sequential adsorption of oppositely charged polyelectrolyte, also known as layer-by-layer self-assembly, onto the
surface of a template core of 0.110 m in diameter followed by
the removal of the core [10]. The size of the template core is determinant for the size of the AZI/PE microcapsules, and some other
properties of AZI/PE microcapsules can be controlled according
to the need by choosing proper template core of different type
or diameter, such as SiO2 , melamine formaldehyde, polystyrene
latex, erythrocytes, and MnCO3 particles. Furthermore, owning to
the repulsive interaction of the external polyelectrolyte with the
same charge, the AZI/PE microcapsules will disperse easily in the
solution, resulting in a higher dissolution rate to same extent [11].
Then hollow PE microcapsules with AZI alcohol solution is slowly
added into water under mixing conditions provided by a magnetic
stirrer. As a result of the difference of AZI solubility in water and
alcohol solution, called good-poor solvent strategies, AZI is precipitated on the base of the hollow PE microcapsules [9], thus AZI/PE
microcapsules can be produced.
Basket method is adopted to compare the dissolution of commercial AZI raw material drug and AZI/PE microcapsules [12]. To
better represent the adsorption of AZI by blood or tissue, the screen
used to restrict the drug particle is substituted by a semi-permeable
membrane (the molecular weight cut-off of the membrane ranges
from 12,000 to 14,000), which simulate the partial pervasion properties of a cell membrane. Phosphate buffer solution (pH = 6.0) and
hydrochloric acid solution (pH = 2.0), which is analogous to the pH
of gastric acid, is used as the dissolution medium. The results of
dissolution show that the prepared AZI/PE microcapsules have an
apparent improvement in the dissolution velocity compared to AZI
raw material drug powder.
2. Experimental
2.1. Materials
AZI was purchased from Beijing Taiyang Pharmaceutical Co.,
Ltd. (Beijing, PR China). Silica microspheres (SiO2 ) used here had
an average diameter of 1.2 m, and were synthesized by Template Mechanism as described elsewhere [13]. Poly (allylamine
hydrochloride) (PAH, Mw = 70,000) and 4-poly (styrene sulfonate
sodium) (PSS, Mw = 70,000) were purchased from SigmaAldrich.
All other reagents used for buffer and standard solution preparation
were purchased from various commercial sources and were of analytical grade. The water used in all the experiments was prepared
in a three-stage Millipore Milli-Q Plus 185 purication system.
2.2. The preparation of hollow PE microcapsules and AZI/PE
microcapsules
Polyelectrolyte microcapsules were obtained by alternate
adsorption of three bilayers of PAH/PSS onto the surface of silica
microspheres via layer-by-layer self-assembly technique. Typical
adsorption conditions were 1 mg mL1 PAH in 0.5 mg mL1 NaCl,
and 2 mg mL1 PSS in 0.5 mg mL1 NaCl. The adsorption time was
15 min. After each adsorption step, the unadsorbed PE is removed
by repeated centrifugation and washing. The PAH/PSS multilayer
lm is formed by the alternate adsorption of oppositely charged
polyions, beginning with the deposition of positively charged
PAH onto the negatively charged silica particles. The 35 bilayers
PAH/PSS were then deposited on the silica microspheres. The silica microspheres template cores were removed by exposure to a
hydrouoric acid (HF)/ammonium uoride (NH4 F) buffer (pH = 5)

for 5 min. Following several washing cycles, the decomposition

products of the silica sphere were discarded, and then hollow PE
microcapsules were obtained [13].
The appropriate quantity of hollow PE microcapsules with certain concentration AZI alcohol solution were washed by alcohol
once to wash the AZI outside the PE microcapsules, then were
slowly added into appropriate volume of water under magnetic
stirring (1000 rpm). The mixed solution transformed from clear
to opalescent within a few seconds. AZI/PE microcapsules were
obtained after washing by water [9].
2.3. Dissolution test
The AZI concentration was measured by colorimetry after
adding the AZI solution to sulfuric acid solution (85 100) for
coloration.
Plot the AZI solution standard curve. First, a series of AZI
solutions at different concentrations, 40, 80, 120, 160, 200, 240,
300 and 360 g mL1 , was prepared, then in accordance with the
Chinese Pharmacopoeia, a precise volume of 5 mL of the abovementioned AZI solutions was mixed uniformly with exact volume
of 5 mL sulfuric acid solution (85 100). The mixture was cooled
at room temperature for 30 min, and then was scanned within
the ultraviolet to visible light spectrum by using an ultravioletvisible spectrophotometer. The maximum absorbance wavelength
of 482 nm and the corresponding average absorbance were adopted
for linear regression to draw the AZI solution standard curve [14].
Based on the relationship between the AZI standard solution concentration and the corresponding absorbance at 482 nm, a linear
equation for AZI standard curve was obtained: A = 0.00621C + 0.061
(C, g mL1 ), n = 8. The correlation coefcient calculated from the
linear equation was Adj. R-Square = 0.99792, which showed a good
linearity.
Dissolution test for AZI raw material drug powder and AZI/PE
microcapsules. The modication of basket method, which meant
screen was replaced by semi-permeable membrane, was introduced to test the dissolution of AZI raw material drug powder and
AZI/PE microcapsules. According to the Chinese Pharmacopoeia,
phosphate buffer (0.1 mol L1 disodium hydrogen phosphate with
appropriate volume HCl, pH = 6.0 (0.05)) is as dissolution medium
under magnetic stirring (100 rpm) at room temperature [14]. After
the addition of semi-permeable membrane carrying certain AZI
into the dissolution medium, a series of 5 mL dissolution medium
volumes were collected to measure the AZI concentrations in the
medium at proper intervals by using the ultraviolet-visible spectrophotometer method and the AZI solution standard curve to
determine the AZI dissolution rate. After each sampling, 5 mL fresh
dissolution medium was added to keep the dissolution volume
unchanged. The relation curve of AZI concentration released in dissolution medium and release time was obtained through the AZI
standard curve.
The release of AZI in gastric acid was also simulated by substituting the phosphate buffer by HCL solution (pH = 2). A similar method
to the one discussed above was used to test the AZI dissolution.
2.4. Characterization
The surface morphology of AZI was studied by using JSM6360LV scanning electron microscopy (SEM, JEOL, Japan). All the
transmission electron microscope (TEM) images were obtained
using a model JEM-100CX (JEOL, Japan) system operated at 120 kV.
AZI raw material drug size was analyzed by LS230 laser particle
size analyzer (Beckman Coulter, USA). The group and structure was
studied by IFS28 Fourier transform infrared spectroscopy (FTIR,
Bruker, Germany). The X-ray powder diffraction (XRD) data were
recorded on a Rigaku D/max 2550 VB/PC diffraction using nickel-

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137

3.2. Analyses of FTIR and XRD

Fig. 1. SEM image of AZI raw material drug powder.

ltered Cu K radiation with wavelength  = 1.5406 . To measure

the concentration of AZI, the UV-2102 PC spectrophotometer was
used.

3. Results and discussion

3.1. Preparation of AZI/PE microcapsules
The commercial AZI raw material drug was used to prepare the
AZI/PE microcapsules. Fig. 1 shows the SEM image of AZI raw material drug. The size of AZI raw material drug particles is relatively
large and inhomogeneous, and the shape of AZI raw material particles is irregular. Analysis of the AZI raw material drug size in AZI
aqueous solution by using laser particle size analyzer shows that
the average drug particles size is 359.8 m and the most frequent
value is 471.1 m.
Silica microspheres used here have a uniform and special shape
with an average particle diameter of approximately 1.2 m. Fig. 2a
and b shows the TEM images of SiO2 microspheres and the SiO2 /PE
composite microspheres. After the absorption of three bilayers of
PAH/PSS onto the surface of silica microspheres by layer-by-layer
self-assembly, the appearance of SiO2 /PE composite microspheres
almost does not change compared with silica microspheres. To
research the adsorption of PE on the SiO2 microspheres, we determined zeta-potential of SiO2 microspheres and the variation of
zeta-potential outside the microsphere after each polyelectrolyte
adsorption. Fig. 3 shows the relationship between zeta-potential
and adsorption layer number. The zeta-potential of SiO2 microspheres is about 5 mV. And after each adsorption of PAH or PSS,
the charges are changed to the opposite charges, which indicates
the polyelectrolyte multilayer are well deposited on the surface of
SiO2 microspheres.
After disposal with HF/NH4 F (pH = 5) for appropriate time and
washing cycles, SiO2 , the core of SiO2 /PE composite microspheres,
is decomposed and the decomposition products were discarded,
then the hollow polyelectrolyte microcapsules were obtained, as
shown in Fig. 2c. The hollow polyelectrolyte microcapsules keep
good integrity and monodispersity. The surface fold of the hollow
polyelectrolyte microcapsules is due to dehydration [10]. Fig. 2d
shows the TEM image of newly prepared AZI/PE microcapsules.
The AZI was precipitated on the surface of hollow polyelectrolyte
microcapsules as a result of good-poor solvent method. Due to the
lling in of hollow polyelectrolyte microcapsules inner space, the
AZI/PE microcapsules show spherical shape again. And the drug
content of AZI/PE is about 38.25 g/mg.

Fig. 4 displays the FTIR spectra of SiO2 (a), SiO2 /PE composite
microspheres (b), hollow PE microcapsules (c), AZI raw material
drug (d) and AZI/PE microcapsules (e). In the silica microspheres
spectra, the peak 1097.6 cm1 is the SiOSi anti-symmetric
stretching vibration absorption, the peaks 802.4 and 468.9 cm1
are related respectively to the symmetrical stretching vibration and
the bending vibration, and the peaks 1631.5 and 3451.4 cm1 represent respectively the characteristic water absorption peak and
the OH radicals of silica microspheres. However, the FTIR spectra
of hollow PE microcapsules, SiO2 /PE composite microspheres and
silica microspheres were nearly unchanged, which is owing to the
residues of SiO2 disposed by HF solution. Moreover, residues of SiO2
are the support materials to prevent microspheres collapsing. The
FTIR spectra of AZI raw material drug and AZI/PE microcapsules are
very similar, except part of the H2 O-correlated characteristic FTIR
peaks. So in the preparation process of AZI/PE microcapsules, the
basic groups and AZI intramolecular structures were unaltered.
However, the crystal state of the prepared AZI/PE microcapsules
has been changed. AZI is found to exhibit polymorphism, pseudopolymorphism and amorphism. Pseudopolymorph AZI can exist
as monohydrate and dihydrate [15]. The anhydrous form of AZI
seems to be unstable since it converts to dihydrate on storage at
room temperature, and the monohydrate form converts to the more
stable dihydrate form in the presence of moisture [16]. The structural change that occurred in the AZI/PE microcapsules during the
preparation process can be conrmed as follows:
On one hand, the FTIR spectra of AZI raw material drug and
AZI/PE microcapsules revealed distinct differences within the OH
stretching region (3450 cm1 ), which contributed to the change of
AZI crystal lattice structure. The presence of a sharp high frequency
peak at 3494 cm1 in the case of AZI raw material is indicative of the
presence of tightly bound water in the crystal lattice, while the
broad band at 33003600 cm1 in AZI/PE microcapsules is due to
the OH stretching for self associated water that may be loosely
bounded [16].
On the other hand, by comparing the X-ray diffraction (XRD)
patterns of AZI raw material drug and AZI/PE microcapsules, as
shown in Fig. 5, it is fair to say that the crystal state of AZI had
been changed during the preparation of AZI/PE microcapsules. The
XRD spectral characteristic of AZI raw material is very complex with
many diffraction peaks, while the XRD pattern of AZI/PE microcapsules basically corresponds with XRD spectra of AZI monohydrate.
Some studies show that AZI monohydrate has a faster dissolution
rate at the initial 48 h [16], which is one of the reasons that dissolution rate of AZI/PE microcapsules increases. Moreover, Zhao et al.
[9] have conrmed that the AZI monohydrate prepared by goodpoor solvent method demonstrate a much-improved antibacterial
activity.
3.3. Release behavior of AZI raw material drug and AZI/PE
microcapsules
The release curves of the AZI raw material drug and the
AZI/PE microcapsules prepared under the conditions of pH = 6.0 and
pH = 2.0 are presented in Fig. 6. As shown in this gure, AZI is lowly
soluble in water and highly soluble in acid solution. Thus, the pH
condition plays a very important role in the AZI dissolution rate,
as evidenced by the releasing results. For AZI raw material drug
and AZI/PE microcapsules, the dissolution rate in the dissolution
medium under pH = 2.0 is obviously better than it is under pH = 6.0,
respectively. Under the same pH condition, pH = 2 or pH = 6, the
prepared AZI/PE microcapsules show an apparent improvement
in dissolution rate compared with AZI raw material drug. AZI/PE
microcapsules cost about 4 h to release 80%, while AZI raw mate-

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Z. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 362 (2010) 135139

Fig. 2. TEM images of SiO2 microspheres (a), SiO2 /PE composite microspheres (b), hollow PE microcapsules (c), and AZI/PE microcapsules (d).

rial drug need almost 9 h to release 80% under pH = 6. When pH = 2,

3 h is needed for AZI/PE microcapsules releasing 80%, while AZI raw
material drug need 5 h to release 80%. According to the approximate
slope of AZI microcapsules release curve in the initial 5 h, the release

Fig. 3. The zeta-potential vs. layer number of PAH/PSS on SiO2 microspheres.

Fig. 4. FTIR spectra of SiO2 microspheres (a), SiO2 /PE composite microspheres (b),
hollow PE microcapsules (c), AZI raw material drug (d), and AZI/PE microcapsules
(e).

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139

The ubiquitous but miraculous self-assembly process constructs

much of our natural world at all scales. Building microspheres
articially with delicate three-dimensional (3D) architectures is a
valuable pursuit in pharmaceuticals because of the convenience
of such structures in subsequent processing and their potential
of incorporating other materials. Previous studies reported that
approximately 80% of the documented bioequivalence problems
identied by the Pure Food and Drug Administration (FDA) resulted
primarily from the failure of the drug product dissolution. Meanwhile, the goodpoor solvent method that leads to the precipitation
of solids can be adapted as a common method for a wide range of
chemicals. Therefore, the preparation method applied to produce
AZI/PE microcapsules can be generalized to many pharmaceuticals
with low bioavailability or poor dissolution. Besides, PAH and PSS
could be replaced by the polyglutamic acid (PGA) and polylactic
acid (PLA) to achieve a better biocompatibility.
Acknowledgements
Fig. 5. XRD patterns of AZI raw material drug and AZI/PE microcapsules.

This work was supported by the National Natural Science Foundation of China (20925621, 20976054), the Key Project of Science
and Technology for Ministry of Education (107045), the Innovation
Program of Shanghai Municipal Education Commission (09ZZ58),
the Program of Shanghai Subject Chief Scientist (08XD1401500)
and the Shanghai Leading Academic Discipline Project (project
number: B502).
References

Fig. 6. Releasing curves of AZI raw material drug and AZI/PE microcapsules in
pH = 6.0 and pH = 2.0, respectively.

of AZI can be regarded as uniform. In addition, it is worth noticing

that the AZI/PE microcapsules dissolution rate under pH = 6 is better
than the AZI raw material drug under pH = 2. So we may reasonably reach the conclusion that the prepared AZI/PE microcapsules
by the method introduced in the present study distinctly improve
the dissolution rate of AZI raw material drug.
4. Conclusion
This work reports a preparation method of AZI microcapsules
based on hollow polyelectrolyte microcapsules produced by layerby-layer self-assembly technology as well as by AZI good-poor
(alcoholwater) method. This preparation method ameliorates the
low bioavailability of AZI as a result of its poor solubility. The experimental results showed an obvious improvement in the dissolution
velocity of the prepared AZI/PE microcapsules in comparing with
the dissolution rate of azithromycin raw material drug powder.

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