Materials Science and Engineering C 29 (2009) 387–392

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Materials Science and Engineering C

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c

Evaluation of cross-linked chitosan microparticles containing acyclovir obtained

by spray-drying
Hellen Karine Stulzer a,b,c,⁎, Monika Piazzon Tagliari b, Alexandre Luis Parize a,
Marcos Antonio Segatto Silva b, Mauro Cesar Marghetti Laranjeira a
a
Laboratório Quitech, Departamento de Química, Universidade Federal de Santa Catarina, Brazil
b
Laboratório de Controle de Qualidade, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Brazil
c
Laboratorio de Controle de Qualidade, Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to obtain microparticles containing acyclovir (ACV) and chitosan cross-linked with
Received 21 May 2008 tripolyphosphate using the spray-drying technique. The resultant system was evaluated through loading
Received in revised form 23 July 2008 efficiency, differential scanning calorimetry (DSC), thermogravimetric analysis (TG), X-ray powder diffraction
Accepted 28 July 2008
(XRPD), scanning electron microscopy (SEM), in vitro release and stability studies. The results obtained
Available online 5 August 2008
indicated that the polymer/ACV ratio influenced the final properties of the microparticles, with higher ratios
giving the best encapsulation efficiency, dissolution profiles and stability. The DSC and XRPD analyses
Keywords:
Acyclovir
indicated that the ACV was transformed into amorphous form during the spray-drying process.
Chitosan © 2008 Elsevier B.V. All rights reserved.
Tripolyphosphate
Microparticles
Spray-drying

1. Introduction as multifunctional permeation enhancers to improve the permeation

of hydrophilic macromolecules in peroral drug delivery. Chitosan, a
Spray-drying is extensively applied in the pharmaceutical industry natural polyaminosaccharide obtained from the N-deacetylation of
to produce raw drugs or excipients or in the microencapsulation chitin, is a non-toxic, biocompatible and biodegradable polymer that
process. This technique transforms liquid feed into dry powder in one has been used in biomedical fields [3–5].
step and is feasible for the scaling-up of the microencapsulation in a Desai and Park [6] have demonstrated that tripolyphosphate (TPP)
continuous particle processing operation which can be used for a wide can act as a new stabilizing agent for the preparation of chitosan
variety of materials [1]. microspheres by spray-drying and it has been used with success to
The drug entrapped-particles can be prepared from a variety of prepare particles loaded with acetaminophen. Ionic interactions
both water-soluble and water-insoluble polymers, of synthetic, semi- between the negative charges of the cross-linker agent (TPP) and the
synthetic and natural origin. The dry powder particulates produced positively charged groups in the chitosan are the main interactions in the
can be processed for many practical purposes such as tablets or polymeric chain.
capsules and other convenient drug dosage forms. One of the most Acyclovir (ACV), previously known as acycloguanosine, has
important characteristic of the spray-drying is that it can be applied to potent inhibitory effects on viruses of the herpes group, particularly
both heat resistant and heat sensitive, as well as water-soluble and the Herpes simplex virus (HSV, I and II) and Varicella zoster virus. It
water-insoluble, drugs. This is important in the development of also combines inhibitory effects on the hepatitis B virus with very
pharmaceutical carriers specifically designed for the delivery of low toxicity to mammalian host cells [7]. Several reports have
hydrophobic drugs, which represents one of the major challenges in indicated that ACV is as effective as, or even superior to, other
the field of drug delivery [2]. antiviral agents with lower host toxicity and milder side effects [8].
In the past decade, biodegradable polymers such as chitosan and Since ACV has a short half-life (2–3 h), low solubility, and its oral
its quaternized derivatives have been studied extensively for their role dosage forms must be taken five times daily, there is a need to
develop different systems to improve the drug efficacy and therefore
the patient treatment.
⁎ Corresponding author. Laboratório Quitech, Departamento de Química, Universidade
The aim of this study was to develop a new system containing
Federal de Santa Catarina, Brazil. Tel.: +55 48 3721 5066. ACV/TPP/chitosan, obtained using the spray-drying technique, to
E-mail address: hellen.stulzer@gmail.com (H.K. Stulzer). investigate the process variables, particularly the influence of the

0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2008.07.030
388 H.K. Stulzer et al. / Materials Science and Engineering C 29 (2009) 387–392

Table 1 temperature 92 °C; feed rate 7 mL min− 1; airflow rate 500 cm3/h and
Formulations content aspirator set at 100% (Fig. 1).
Formulation Chitosan Cross-linker agent Acyclovir HCl 0.1
amount (tripolyphosphate amount mol L− 1 2.2.1. Characterization
(g) 0.2% v/v) (mL) (mg) (mL)
F1 1 1 100 100 2.2.1.1. Determination of loading efficiency. A sample of cross-linked
F2 1 1 150 100
microparticles loaded with 10 mg of ACV was accurately weighted and
F3 1 1 200 100
F4 2 2 100 100 dissolved in 10 mL of 90% ethanol in a 200 mL volumetric flask and
F5 2 2 150 100 stirred in an ultrasonic bath for 15 min to extract the drug from the
microparticles. The volume was completed with the mobile phase
constituted of water and acetonitrile (95:5 v/v). A volume (5 mL) of
this solution was diluted with mobile phase in a 50 mL volumetric
polymer/ACV ratio on the correlated characteristics of the particulate
flask (5 µg mL− 1). The HPLC analysis was performed on a Shimadzu LC-
formulations.
10 system (Kyoto, Japan) equipped with an LC-10AD pump, and SPD-
10AV UV detector (set at 254 nm). This assay was previously validated
2. Experimental
according to ICH, 2003 [9]. Experiments were performed in triplicate
(n = 3) and loading efficiencies were calculated using Eq. (1).
2.1. Materials and methods
Mass of drug present in microparticles
The ACV reference substance was received from Shenyang Fine % Loading efficiency ¼  100
Theoretical mass of acyclovir
Chemical Co. (China). Chitosan with a molecular weight of 122.740 Da
ð1Þ
and degree of deacetylation of 90% was purchased from Purifarma
(São Paulo, Brazil). All other materials were at least of analytical
2.2.1.2. Differential scanning calorimetry (DSC). DSC curves were
grade.
obtained with a Shimadzu DSC-50 cell using aluminum crucibles with
about 2 mg of the samples, under dynamic N2 atmosphere (100 mL
2.2. Preparation of spray-dried ACV/TPP/chitosan microparticles
min− 1) at a heating rate of 10 °C min− 1 in the temperature range of 25
to 500 °C. The DSC cell was calibrated with indium (mp 156.6 °C;
The ACV (100 to 200 mg) was dissolved in 0.1 mol L− 1 HCl solution.
ΔHfus = 28.54 J g− 1) and zinc (mp 419.6 °C).
The polymer in different concentrations and the cross-linked agent
tripolyphosphate were dissolved in a solution containing the drug and
2.2.1.3. Thermogravimetric analysis (TG). TG curves were obtained
homogenized for 1 h (Table 1). The mixture was stirred for 30 min and
with a Shimadzu thermobalance (model TGA-50) in the temperature
the resulting solutions were spray-dried to obtain microparticles
range of 25–600 °C, using platinum crucibles with 4.0 ± 0.1 mg of
containing ACV. Spray-drying of solutions was carried out using
sample, under dynamic N2 atmosphere (50 mL min− 1) with a heating
a laboratory-scale spray dryer Buchi (model B-191, Switzerland)
rate of 10 °C min− 1.
under the following set of conditions: inlet temperature 180 °C, outlet
2.2.1.4. X-ray powder diffraction (XRPD). X-ray diffraction patterns
were obtained on a Siemens X-ray diffractometer, model D 5000, with
Cu Kα radiation, voltage of 40 kW and current of 40 mA, in the range of
3–65 (2θ) with 1 s of scan time, using the powder XRD method.

2.2.1.5. Scanning electron microscopy (SEM). Samples were mounted

onto metal stubs using double-sided adhesive tape, vacuum-coated
with gold (350 Å) in a Polaron E-5000 and analyzed using a scanning
electron microscopy (Philips, Model XL 30) at an intensity of 10 kV,
using various magnifications.

2.2.1.6. In vitro release. The in vitro release of ACV was evaluated

using a dissolution methodology (Apparatus I, 100 rpm, 37 °C), in
500 mL of 0.1 mol L− 1 HCl at pH 1.2 and in phosphate buffer at pH 6.8
to simulate gastrointestinal fluid. An aliquot of the release medium
(5 mL) was withdrawn through a sampling syringe attached to a
0.22 µm membrane filter (Milipore, USA) at pre-determined time
intervals (5, 10, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and
360 min) and an equivalent amount of fresh dissolution media pre-

Table 2
Loading efficiency of formulations

Formulation Chitosan/ACV (mg) Loading efficiencya (%) R.S.D.b (%)

F1 1000:100 86.4 ±0.79
F2 1000:150 79.7 ±0.86
F3 1000:200 73.8 ±0.99
F4 1000:50 92.5 ±1.07
F5 1000:75 90.6 ±0.88
a
Fig. 1. Preparation process of cross-linked ACV/TPP/chitosan microparticles by spray- Mean of three determinations.
b
drying. Relative Standard Deviation.
 H.K. Stulzer et al. / Materials Science and Engineering C 29 (2009) 387–392 389

after the spray-drying process. The characteristic peak of ACV fusion

did not appear in all formulations, although a poorly defined
endothermic event was observed at 220 °C for F4 and F5 (Fig. 2).
The disappearance of the ACV fusion peak observed for the
microparticles may be related to a chemical interaction between the
drug and TPP/chitosan or the possible formation of an amorphous
system.
The TG curves (Fig. 3) for all formulations were identical, showing
thermal stability up to 221.3 °C. Above this temperature, mass loss
events with Δm = 22.13% (F1), 25.52% (F2), 24.52% (F3), 25.36% (F4) and
24.95% (F5) occurred. These results suggest that there was no
significant reduction in the thermal stability of the formulations in
relation to the isolated drug and chitosan cross-linked microparticles
without ACV (control).

3.3. X-ray powder diffraction (XRPD)

Fig. 2. DSC curves of ACV, F1, F2, F3, F4 and F5. Substances in solid state can present crystalline or amorphous
characteristics, and in some cases both. A crystal has an ordered
arrangement of molecules and atoms, maintained in contact through
non-covalent interactions. On the other hand, amorphous solids are
warmed to 37 °C was applied. The samples were centrifuged and
characterized by a random state. These characteristics are important
analyzed using a UV spectrophotometer at 254 nm.
in the absorption process. Amorphous solids are, in general, more
soluble than the crystalline form, due to free energies involved in
2.2.1.7. Stability studies. The stability studies were carried out in a
the dissolution process. Solids in amorphous state have randomly
climatic chamber at 40 ± 2 °C and 75 ± 5% of relative humidity (RH) as
arranged molecules and thus low energy is required to separate them
well as under ambient conditions (25 ± 2 °C and 75 ± 5% RH), during a
and, consequently, their dissolution is faster than when in the
period of 6 months [10]. Formulation samples were removed at time
crystalline form [11–13].
intervals of 0, 30, 60, 90, 120, 150 and 180 days and the drug was
The ACV has crystalline characteristics which are represented by
determined by high performance liquid chromatography (conditions
peaks in X-ray diffractograms, and the most evident peaks appear at
describe in Section 2.2.1.1).
2θ = 4.76, 19.51 and 23.44. The chitosan diffractogram did not have
peaks, which is characteristic of an amorphous compound (Fig. 4).
3. Results and discussion

3.1. Determination of loading efficiency

Comparisons of the ACV loading efficiencies for the different ACV/

TPP/chitosan microparticles are shown in Table 2. The results indicated
that this parameter ranged between 73.8% and 92.5%. Moreover, it could
be noted that 1 g of chitosan encapsulated different concentrations of
ACV. The best result was observed for formulation F4.

3.2. Differential scanning calorimetry (DSC) and thermogravimetry (TG)

The DSC curves of formulations F1, F2 and F3 showed an

endothermic event in the temperature range of 61 °C to 95 °C,
which is related to water and solvents that remained in the samples

Fig. 3. TG curves of ACV, microparticles without ACV, F1, F2, F3, F4 and F5. Fig. 4. X-ray diffraction spectra of ACV, chitosan, F1, F2, F3, F4 and F5.
390 H.K. Stulzer et al. / Materials Science and Engineering C 29 (2009) 387–392

The XRD diffractograms of F1, F2 and F3 did not show the same (28.9 µm ± 2.79), F2 (34.9 µm ± 2.89), F3 (29.9 µm ± 3.06), F4 (23.0 µm ±
peaks as ACV, indicating that the ACV underwent a transition from a 3.67) and F5 (18.7 µm ± 3.65). All formulations had heterogeneous
crystalline to an amorphous state (Fig. 4). In the formulations F4 and particle sizes and, due to the adhesive characteristics of chitosan,
F5 the diffraction peaks decreased in comparison with the ACV, the particles were aggregated. The formulation F5 had the smallest,
suggesting that in these formulations the drug was partially and F2 the largest, mean particle size. The photomicrography of
transformed into an amorphous form. In addition, in these formula- formulation F2 showed ACV crystals, indicating that the drug was
tions the amount of ACV was higher than in the others (data shown as probably not completely encapsulated.
loading efficiency).Therefore, these data confirm the results obtained
from the DSC assays. 3.5. In vitro release

3.4. Scanning electron microscopy (SEM) Release rates of the different ACV/TPP/chitosan microparticles are
presented in Figs. 6 and 7. The results clearly indicated that the
The crystalline structure of ACV was confirmed by SEM in formulations had a differentiated pattern of release. The formulation
photomicrography (Fig. 5A), where an orthorhombic crystal form F4 released ACV over a longer time period in both media. At acid pH
could be observed. The particle size was measured using this assay the NH2 group remaining from the cross-linking process was
(n = 3) (Fig. 5) and the results indicated different mean sizes for F1 protonated to NH3 and the polymer swelling led to the drug release.

Fig. 5. SEM of pure ACV (A) (magnification of 800×); F1 (B) (magnification of 1000×); F2 (C) (magnification of 1000×); F3 (D) (magnification of 1000×); F4 (E) (magnification of 1500×)
and F5 (F) (magnification of 2000×).
 H.K. Stulzer et al. / Materials Science and Engineering C 29 (2009) 387–392 391

Table 3
Analysis of release data from ACV/TPP/chitosan microparticles

Formulation pH 1.2 pH 6.8

−n −n
n r2 k (min ) n r2 k (min )
F1 0.48 0.9419 21.55 0.21 0.8973 23.55
F2 0.49 0.9356 32.34 0.29 0.9156 29.91
F3 0.60 0.9705 31.06 0.32 0.8921 30.89
F4 0.45 0.9936 8.28 0.22 0.9930 9.22
F5 0.46 0.9624 17.85 0.22 0.9852 14.22

to coupled diffusion/polymer relaxation) [17,18]. Occasionally, values

of n N 1 have been observed, which is regarded as Super Case II kinetics
[19,20]. The linear form of Eq. (1), plotting ln Mt/M∞ against ln t,
yielded the diffusion exponential (n), the Pearson coefficient (r2) and
the diffusion constant (k).
The results shown in Table 3 indicate that the formulations
presented different behaviors depending on the pH of the medium.
Fig. 6. Dissolution profiles of F1, F2, F3, F4 and F5 in chloridric acid (pH 1.2). Under acid conditions (pH 1.2) the mechanism involved in the ACV
release displayed non-Fickian kinetics, corresponding to coupled
diffusion/polymer relaxation related with the NH2 group present in
The best release times were obtained for the highest chitosan/ACV chitosan chain. On the other hand, the formulations in pH 6.8
ratio. demonstrated a Fickian release.
The mechanism of ACV release from the microparticles is The k values were related to the ACV release kinetics, a lower k
determined by different physical–chemical phenomena. According value indicating a slower release. The k values obtained for F4 were
to Nixon [14], three steps lead to drug release from microparticles in 8.28 and 9.22 for pH 1.2 and 6.8, respectively. The other values were
aqueous medium: (1) imbibition of the release medium by the higher, and the dissolution profiles showed that ACV was released
microparticles, (2) dissolution of the drug inside the microparticles, over a shorter time period.
and (3) drug release into the aqueous medium through a diffusion
process. 3.6. Stability studies
The Korsmeyer–Peppas model (Eq. (2)), a semi-empirical model
correlating drug release with time through a simple exponential In the preparation of a pharmaceutical system some excipients can
equation for a drug release fraction b0.6, has been used to evaluate be used to obtain products with the desired physical and chemical
drug release from controlled release polymeric devices. It is characteristics or to improve the appearance. Other substances can be
particularly useful when the drug release mechanism is unknown used to increase the stability of the drug, particularly in hydrolytic and
or when there is more than one release mechanism [15,16]. oxidative processes [21]. In relation to the polymers, they can increase
the stability of the final dry product [22].
Mt The results showed that all formulations maintained at ambient
¼ kdt n ð2Þ temperature and humidity (25 ± 2 °C; 75 ± 5%) had a lower degree
M∞
of degradation than the samples maintained in a climatic chamber
Mt/M∞ is the proportion of drug released at time t, k is the kinetic (40 ± 2 °C; 75 ± 5%) (Figs. 8 and 9). It has been reported in the
constant, and the exponent n has been proposed as indicative of the literature that temperature affects the drug stability with a two or
release mechanism. In this context, n ≤ 0.43 indicates Fickian release three-fold increase in the reaction velocity for each 10 °C increase in
and n = 0.85 indicates a purely relaxation-controlled delivery which is temperature. Higher humidity is another factor associated with
referred to as Case II transport. Intermediate values (0.43 b n b 0.85) reduced stability [23,24].
indicate an anomalous behavior (non-Fickian kinetics corresponding

Fig. 8. ACV concentrations of formulations maintained at ambient conditions (25 ± 2 °C;

Fig. 7. Dissolution profiles of F1, F2, F3, F4 and F5 in phosphate buffer (pH 6.8). 75 ± 5%).
392 H.K. Stulzer et al. / Materials Science and Engineering C 29 (2009) 387–392

Table 5
Values of velocity constant (k25) and t90%

Formulations k25 (days− 1) t90% (days)

F1 1.42 × 10− 5 78
F2 2.55 × 10− 5 43
F3 3.04 × 10− 5 36
F4 1.07 × 10− 5 103
F5 1.24 × 10− 5 89

obtain stable microparticles. Comparing all of the formulations

developed, F4 is the most promising for improved ACV release.

Acknowledgement

The authors acknowledge Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior (CAPES) for fellowships and Conselho
Fig. 9. ACV concentrations of formulations maintained at climatic chamber (40 ± 2 °C;
75 ± 5%). Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for
financial support.

The stability of the formulations kept under the two different References
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Kinetic equations for calculate ACV degradation

Reaction order k (days− 1) t90% (days)

Zero-order C0a − Cb / t 0.1. C0 / k
First-order 2.303 / t × log C0 / C 0.106 / k
Second-order 1 / t × (1 / C − 1 / C0) 1 / 9k × C0
a
ACV concentration in time zero.
b
ACV concentration after degradation for a time t.