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Evaluation of cross-linked chitosan microparticles containing acyclovir obtained by spray-drying
Materials Science and Engineering C 29 (2009) 387–392
Contents lists available at ScienceDirect
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 b c
Laboratório Quitech, Departamento de Química, Universidade Federal de Santa Catarina, Brazil Laboratório de Controle de Qualidade, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Brazil 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
Article history: Received 21 May 2008 Received in revised form 23 July 2008 Accepted 28 July 2008 Available online 5 August 2008 Keywords: Acyclovir Chitosan Tripolyphosphate Microparticles Spray-drying
a b s t r a c t The aim of this study was to obtain microparticles containing acyclovir (ACV) and chitosan cross-linked with tripolyphosphate using the spray-drying technique. The resultant system was evaluated through loading efficiency, differential scanning calorimetry (DSC), thermogravimetric analysis (TG), X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), in vitro release and stability studies. The results obtained 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 indicated that the ACV was transformed into amorphous form during the spray-drying process. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Spray-drying is extensively applied in the pharmaceutical industry to produce raw drugs or excipients or in the microencapsulation process. This technique transforms liquid feed into dry powder in one step and is feasible for the scaling-up of the microencapsulation in a continuous particle processing operation which can be used for a wide variety of materials [1]. The drug entrapped-particles can be prepared from a variety of both water-soluble and water-insoluble polymers, of synthetic, semisynthetic and natural origin. The dry powder particulates produced can be processed for many practical purposes such as tablets or capsules and other convenient drug dosage forms. One of the most important characteristic of the spray-drying is that it can be applied to both heat resistant and heat sensitive, as well as water-soluble and water-insoluble, drugs. This is important in the development of pharmaceutical carriers specifically designed for the delivery of hydrophobic drugs, which represents one of the major challenges in the field of drug delivery [2]. In the past decade, biodegradable polymers such as chitosan and its quaternized derivatives have been studied extensively for their role
⁎ Corresponding author. Laboratório Quitech, Departamento de Química, Universidade Federal de Santa Catarina, Brazil. Tel.: +55 48 3721 5066. E-mail address: [email protected] (H.K. Stulzer). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.07.030
as multifunctional permeation enhancers to improve the permeation of hydrophilic macromolecules in peroral drug delivery. Chitosan, a natural polyaminosaccharide obtained from the N-deacetylation of chitin, is a non-toxic, biocompatible and biodegradable polymer that has been used in biomedical fields [3–5]. Desai and Park [6] have demonstrated that tripolyphosphate (TPP) can act as a new stabilizing agent for the preparation of chitosan microspheres by spray-drying and it has been used with success to prepare particles loaded with acetaminophen. Ionic interactions between the negative charges of the cross-linker agent (TPP) and the positively charged groups in the chitosan are the main interactions in the polymeric chain. Acyclovir (ACV), previously known as acycloguanosine, has potent inhibitory effects on viruses of the herpes group, particularly the Herpes simplex virus (HSV, I and II) and Varicella zoster virus. It also combines inhibitory effects on the hepatitis B virus with very low toxicity to mammalian host cells [7]. Several reports have indicated that ACV is as effective as, or even superior to, other antiviral agents with lower host toxicity and milder side effects [8]. Since ACV has a short half-life (2–3 h), low solubility, and its oral 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. The aim of this study was to develop a new system containing ACV/TPP/chitosan, obtained using the spray-drying technique, to investigate the process variables, particularly the influence of the
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temperature 92 °C; feed rate 7 mL min− 1; airflow rate 500 cm3/h and aspirator set at 100% (Fig. 1).
Table 1 Formulations content Formulation
F1 F2 F3 F4 F5
Chitosan amount (g)
Cross-linker agent (tripolyphosphate 0.2% v/v) (mL)
Acyclovir amount (mg)
HCl 0.1 mol L− 1 (mL)
1 1 1 2 2
1 1 1 2 2
100 150 200 100 150
100 100 100 100 100
polymer/ACV ratio on the correlated characteristics of the particulate formulations. 2. Experimental 2.1. Materials and methods The ACV reference substance was received from Shenyang Fine Chemical Co. (China). Chitosan with a molecular weight of 122.740 Da and degree of deacetylation of 90% was purchased from Purifarma (São Paulo, Brazil). All other materials were at least of analytical grade. 2.2. Preparation of spray-dried ACV/TPP/chitosan microparticles The ACV (100 to 200 mg) was dissolved in 0.1 mol L− 1 HCl solution. The polymer in different concentrations and the cross-linked agent tripolyphosphate were dissolved in a solution containing the drug and homogenized for 1 h (Table 1). The mixture was stirred for 30 min and the resulting solutions were spray-dried to obtain microparticles containing ACV. Spray-drying of solutions was carried out using a laboratory-scale spray dryer Buchi (model B-191, Switzerland) under the following set of conditions: inlet temperature 180 °C, outlet
2.2.1. Characterization 2.2.1.1. Determination of loading efficiency. A sample of cross-linked microparticles loaded with 10 mg of ACV was accurately weighted and dissolved in 10 mL of 90% ethanol in a 200 mL volumetric flask and 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 flask (5 µg mL− 1). The HPLC analysis was performed on a Shimadzu LC10 system (Kyoto, Japan) equipped with an LC-10AD pump, and SPD10AV UV detector (set at 254 nm). This assay was previously validated according to ICH, 2003 [9]. Experiments were performed in triplicate (n = 3) and loading efficiencies were calculated using Eq. (1). % Loading efficiency ¼
Mass of drug present in microparticles 100 Theoretical mass of acyclovir ð1Þ
2.2.1.2. Differential scanning calorimetry (DSC). DSC curves were obtained with a Shimadzu DSC-50 cell using aluminum crucibles with about 2 mg of the samples, under dynamic N2 atmosphere (100 mL 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; ΔHfus = 28.54 J g− 1) and zinc (mp 419.6 °C). 2.2.1.3. Thermogravimetric analysis (TG). TG curves were obtained with a Shimadzu thermobalance (model TGA-50) in the temperature range of 25–600 °C, using platinum crucibles with 4.0 ± 0.1 mg of sample, under dynamic N2 atmosphere (50 mL min− 1) with a heating rate of 10 °C min− 1. 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
Fig. 1. Preparation process of cross-linked ACV/TPP/chitosan microparticles by spraydrying.
Formulation
Chitosan/ACV (mg)
Loading efficiencya (%)
R.S.D.b (%)
F1 F2 F3 F4 F5
1000:100 1000:150 1000:200 1000:50 1000:75
86.4 79.7 73.8 92.5 90.6
±0.79 ±0.86 ±0.99 ±1.07 ±0.88
a b
Mean of three determinations. Relative Standard Deviation.
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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.
warmed to 37 °C was applied. The samples were centrifuged and analyzed using a UV spectrophotometer at 254 nm. 2.2.1.7. Stability studies. The stability studies were carried out in a climatic chamber at 40 ± 2 °C and 75 ± 5% of relative humidity (RH) as well as under ambient conditions (25 ± 2 °C and 75 ± 5% RH), during a period of 6 months [10]. Formulation samples were removed at time intervals of 0, 30, 60, 90, 120, 150 and 180 days and the drug was determined by high performance liquid chromatography (conditions describe in Section 2.2.1.1). 3. Results and discussion
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 characterized by a random state. These characteristics are important in the absorption process. Amorphous solids are, in general, more soluble than the crystalline form, due to free energies involved in the dissolution process. Solids in amorphous state have randomly arranged molecules and thus low energy is required to separate them and, consequently, their dissolution is faster than when in the crystalline form [11–13]. The ACV has crystalline characteristics which are represented by peaks in X-ray diffractograms, and the most evident peaks appear at 2θ = 4.76, 19.51 and 23.44. The chitosan diffractogram did not have peaks, which is characteristic of an amorphous compound (Fig. 4).
3.1. 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.
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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 peaks as ACV, indicating that the ACV underwent a transition from a crystalline to an amorphous state (Fig. 4). In the formulations F4 and F5 the diffraction peaks decreased in comparison with the ACV, suggesting that in these formulations the drug was partially transformed into an amorphous form. In addition, in these formulations the amount of ACV was higher than in the others (data shown as loading efficiency).Therefore, these data confirm the results obtained from the DSC assays.
(28.9 µm ± 2.79), F2 (34.9 µm ± 2.89), F3 (29.9 µm ± 3.06), F4 (23.0 µm ± 3.67) and F5 (18.7 µm ± 3.65). All formulations had heterogeneous particle sizes and, due to the adhesive characteristics of chitosan, the particles were aggregated. The formulation F5 had the smallest, and F2 the largest, mean particle size. The photomicrography of formulation F2 showed ACV crystals, indicating that the drug was probably not completely encapsulated.
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 formulations had a differentiated pattern of release. The formulation F4 released ACV over a longer time period in both media. At acid pH the NH2 group remaining from the cross-linking process was protonated to NH3 and the polymer swelling led to the drug release.
The crystalline structure of ACV was confirmed by SEM in photomicrography (Fig. 5A), where an orthorhombic crystal form could be observed. The particle size was measured using this assay (n = 3) (Fig. 5) and the results indicated different mean sizes for F1
3.5. In vitro 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×).
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Table 3 Analysis of release data from ACV/TPP/chitosan microparticles Formulation
F1 F2 F3 F4 F5
Fig. 6. Dissolution profiles of F1, F2, F3, F4 and F5 in chloridric acid (pH 1.2).
The best release times were obtained for the highest chitosan/ACV ratio. The mechanism of ACV release from the microparticles is determined by different physical–chemical phenomena. According to Nixon [14], three steps lead to drug release from microparticles in aqueous medium: (1) imbibition of the release medium by the microparticles, (2) dissolution of the drug inside the microparticles, and (3) drug release into the aqueous medium through a diffusion process. The Korsmeyer–Peppas model (Eq. (2)), a semi-empirical model correlating drug release with time through a simple exponential equation for a drug release fraction b0.6, has been used to evaluate drug release from controlled release polymeric devices. It is particularly useful when the drug release mechanism is unknown or when there is more than one release mechanism [15,16]. Mt ¼ kdt n M∞
ð2Þ
Mt/M∞ is the proportion of drug released at time t, k is the kinetic constant, and the exponent n has been proposed as indicative of the release mechanism. In this context, n ≤ 0.43 indicates Fickian release and n = 0.85 indicates a purely relaxation-controlled delivery which is referred to as Case II transport. Intermediate values (0.43 b n b 0.85) indicate an anomalous behavior (non-Fickian kinetics corresponding
Fig. 7. Dissolution profiles of F1, F2, F3, F4 and F5 in phosphate buffer (pH 6.8).
pH 1.2
pH 6.8
n
r2
k (min
0.48 0.49 0.60 0.45 0.46
0.9419 0.9356 0.9705 0.9936 0.9624
21.55 32.34 31.06 8.28 17.85
−n
)
n
r2
k (min
0.21 0.29 0.32 0.22 0.22
0.8973 0.9156 0.8921 0.9930 0.9852
23.55 29.91 30.89 9.22 14.22
−n
)
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. 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 chitosan chain. On the other hand, the formulations in pH 6.8 demonstrated a Fickian release. The k values were related to the ACV release kinetics, a lower k value indicating a slower release. The k values obtained for F4 were 8.28 and 9.22 for pH 1.2 and 6.8, respectively. The other values were higher, and the dissolution profiles showed that ACV was released over a shorter time period. 3.6. Stability studies In the preparation of a pharmaceutical system some excipients can be used to obtain products with the desired physical and chemical characteristics or to improve the appearance. Other substances can be used to increase the stability of the drug, particularly in hydrolytic and oxidative processes [21]. In relation to the polymers, they can increase the stability of the final dry product [22]. The results showed that all formulations maintained at ambient temperature and humidity (25 ± 2 °C; 75 ± 5%) had a lower degree of degradation than the samples maintained in a climatic chamber (40 ± 2 °C; 75 ± 5%) (Figs. 8 and 9). It has been reported in the literature that temperature affects the drug stability with a two or three-fold increase in the reaction velocity for each 10 °C increase in temperature. Higher humidity is another factor associated with reduced stability [23,24].
Fig. 8. ACV concentrations of formulations maintained at ambient conditions (25 ± 2 °C; 75 ± 5%).
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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 F2 F3 F4 F5
1.42 × 10− 5 2.55 × 10− 5 3.04 × 10− 5 1.07 × 10− 5 1.24 × 10− 5
78 43 36 103 89
obtain stable microparticles. Comparing all of the formulations developed, F4 is the most promising for improved ACV release. Acknowledgement
Fig. 9. ACV concentrations of formulations maintained at climatic chamber (40 ± 2 °C; 75 ± 5%).
The stability of the formulations kept under the two different conditions of temperature and humidity was similar. For both, the formulations F4 and F5 had a lower lever of ACV degradation. The results indicated that the chitosan/ACV ratio also influenced the stability of the final formulations. This is because a higher chitosan-toACV ratio leads to greater ACV protection, hindering the direct action of temperature and humidity. The same behavior has been described by Stulzer and Silva for captopril granules produced with a fluid bed dryer using ethylcellulose and methylcellulose [25]. The final formulations demonstrated greater stability in relation to the isolated drug, when submitted to conditions of increased temperature, humidity and light. Scientific data obtained from stability studies has contributed to determining the shelf life of products, through the time period in which the formulations degraded 10% of the initial drug concentration in the formulations [26]. To calculate this parameter, it is necessary to define the order of the reaction (zero, first or second), through plotting the drug concentration as a function of time. The best correlation coefficient indicates the order of the reaction. For all formulations under both conditions, the degradation reactions occurred according to a second-order reaction. To calculate the velocity constant (k) the equations described in Table 4 were use, according to the calculated order of the reaction and the results are given in Table 5. Formulation F4 gave the highest values with 103 days of shelf life. 4. Conclusions In summary, the results show that ACV/TPP/chitosan microparticles increase the release time of the drug. The polymer/ACV ratio influences several parameters including loading efficiency, release profiles and stability. Another important aspect to note regarding the system developed was the fact that the drug was converted to amorphous form, which improved the ACV solubility. Thus, this study demonstrates the high potential of the spray-drying technique to
Table 4 Kinetic equations for calculate ACV degradation Reaction order
k (days− 1)
t90% (days)
Zero-order First-order Second-order
C0a − Cb / t 2.303 / t × log C0 / C 1 / t × (1 / C − 1 / C0)
0.1. C0 / k 0.106 / k 1 / 9k × C0
a b
ACV concentration in time zero. ACV concentration after degradation for a time t.
The authors acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowships and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. References [1] S. K Broadhead, E. Rouan, C.T. Rhodes, Drug Development and Industrial Pharmacy 18 (1992) 1169. [2] M. Aldén, M. Lydén, J. Tegenfeldt, International Journal of Pharmaceutics 110 (1994) 267. [3] V. Dodane, V.D. Vilivalam, Pharmaceutical Science and Technology Today 1 (1998) 246. [4] M. Asada, H. Takahashi, H. Okamoto, H. Tanino, K. Danjo, International Journal of Pharmaceutics 270 (2004) 167. [5] Y. Gao, L. Hen, H. Katsumi, T. Sakane, T. Fujita, A. Yamamoto, International Journal of Pharmaceutics 359 (2008) 70. [6] K.G. Desai, H.J. Park, Journal of Microencapsulation 22 (4) (2005) 377. [7] E. de Clerck, L. Naesens, L. de Bole, D. Schols, Y. Zhang, J. Neyts, Reviews in Medical Virology 11 (2001) 381. [8] Y. Gong, K.M. Raj, C.A. Luscombe, I. Gadawski, T. Tam, J. Chu, D. Gibson, R. Carlson, S.L. Sacks, Antiviral Research 64 (2004) 127. [9] ICH, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use: Guideline for the Industry Q2B Validation of Analytical Procedures Methodology, 2003. [10] ICH, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use: Guideline for the Industry Q1A Stability Testing of New Drug Substances and Products, 2003. [11] P. Dangprasirt, G.C. Ritthidej, Drug Development and Industrial Pharmacy 21 (1995) 2323. [12] S. Storpirts, P.G. Oliveira, D. Rodrigues, D. C. Maranhão Revista Brasileira de Ciências Farmacêuticas 35 (1) (1999) 1. [13] A.T. Florence, D. Attwood, Princípios físico-químicos em farmácia, Editora da Universidade de São Paulo, São Paulo, 2003. [14] J.R. Nixon, in: F. Lim (Ed.), Biomedical Applications of Microcapsulation, CRC Press, Boca Raton, 1983. [15] R.W. Korsmeyer, R. Gurny, E. Doelker, International Journal of Pharmaceutics 15 (1983) 25. [16] P. Costa, J.M. Lobo, European Journal of Pharmaceutical Sciences 13 (2001) 123. [17] P.L. Ritger, N.A. Peppas, Journal of Controlled Release 5 (1987) 23. [18] P.L. Ritger, N.A. Peppas, Journal of Controlled Release 5 (1987) 37. [19] R. Ranga, K.D. Devi, P. Buri, Drug Development and Industrial Pharmacy 14 (1988) 2299. [20] D.L. Munday, P.L. Cox, International Journal of Pharmaceutics 203 (2000) 179. [21] H.C. Ansel, N.G. Popovich, L.V. Allen, Formas Farmacêuticas e Sistemas de Liberação de Fármacos, Editorial Premier, São Paulo, 2000. [22] C. Leuner, J. Dressman, European Journal of Pharmaceutics and Biopharmaceutics 50 (2000) 47. [23] K.A. Connors, G.L. Amidon, V. Stella, Chemical Stability of Pharmaceuticals, John Wiley & Sons, New York, 1986. [24] L.A. Trissel, Trissel's Stability of Compounded Formulations, 2 ed.American Pharmaceutical Association, Washington, 2000. [25] H.K. Stulzer, M.A.S. Silva, Acta Farmaceutica Bonaerense 25 (2006) 497. [26] L. Lachman, H.A. Lieberman, J.L. Kaning, Teoria e Prática na Indústria Farmacêutica, Lisboa, Fundação Calouste, 2001, p. 1277.
Contents lists available at ScienceDirect
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 b c
Laboratório Quitech, Departamento de Química, Universidade Federal de Santa Catarina, Brazil Laboratório de Controle de Qualidade, Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Brazil 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
Article history: Received 21 May 2008 Received in revised form 23 July 2008 Accepted 28 July 2008 Available online 5 August 2008 Keywords: Acyclovir Chitosan Tripolyphosphate Microparticles Spray-drying
a b s t r a c t The aim of this study was to obtain microparticles containing acyclovir (ACV) and chitosan cross-linked with tripolyphosphate using the spray-drying technique. The resultant system was evaluated through loading efficiency, differential scanning calorimetry (DSC), thermogravimetric analysis (TG), X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), in vitro release and stability studies. The results obtained 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 indicated that the ACV was transformed into amorphous form during the spray-drying process. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Spray-drying is extensively applied in the pharmaceutical industry to produce raw drugs or excipients or in the microencapsulation process. This technique transforms liquid feed into dry powder in one step and is feasible for the scaling-up of the microencapsulation in a continuous particle processing operation which can be used for a wide variety of materials [1]. The drug entrapped-particles can be prepared from a variety of both water-soluble and water-insoluble polymers, of synthetic, semisynthetic and natural origin. The dry powder particulates produced can be processed for many practical purposes such as tablets or capsules and other convenient drug dosage forms. One of the most important characteristic of the spray-drying is that it can be applied to both heat resistant and heat sensitive, as well as water-soluble and water-insoluble, drugs. This is important in the development of pharmaceutical carriers specifically designed for the delivery of hydrophobic drugs, which represents one of the major challenges in the field of drug delivery [2]. In the past decade, biodegradable polymers such as chitosan and its quaternized derivatives have been studied extensively for their role
⁎ Corresponding author. Laboratório Quitech, Departamento de Química, Universidade Federal de Santa Catarina, Brazil. Tel.: +55 48 3721 5066. E-mail address: [email protected] (H.K. Stulzer). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.07.030
as multifunctional permeation enhancers to improve the permeation of hydrophilic macromolecules in peroral drug delivery. Chitosan, a natural polyaminosaccharide obtained from the N-deacetylation of chitin, is a non-toxic, biocompatible and biodegradable polymer that has been used in biomedical fields [3–5]. Desai and Park [6] have demonstrated that tripolyphosphate (TPP) can act as a new stabilizing agent for the preparation of chitosan microspheres by spray-drying and it has been used with success to prepare particles loaded with acetaminophen. Ionic interactions between the negative charges of the cross-linker agent (TPP) and the positively charged groups in the chitosan are the main interactions in the polymeric chain. Acyclovir (ACV), previously known as acycloguanosine, has potent inhibitory effects on viruses of the herpes group, particularly the Herpes simplex virus (HSV, I and II) and Varicella zoster virus. It also combines inhibitory effects on the hepatitis B virus with very low toxicity to mammalian host cells [7]. Several reports have indicated that ACV is as effective as, or even superior to, other antiviral agents with lower host toxicity and milder side effects [8]. Since ACV has a short half-life (2–3 h), low solubility, and its oral 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. The aim of this study was to develop a new system containing ACV/TPP/chitosan, obtained using the spray-drying technique, to investigate the process variables, particularly the influence of the
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H.K. Stulzer et al. / Materials Science and Engineering C 29 (2009) 387–392
temperature 92 °C; feed rate 7 mL min− 1; airflow rate 500 cm3/h and aspirator set at 100% (Fig. 1).
Table 1 Formulations content Formulation
F1 F2 F3 F4 F5
Chitosan amount (g)
Cross-linker agent (tripolyphosphate 0.2% v/v) (mL)
Acyclovir amount (mg)
HCl 0.1 mol L− 1 (mL)
1 1 1 2 2
1 1 1 2 2
100 150 200 100 150
100 100 100 100 100
polymer/ACV ratio on the correlated characteristics of the particulate formulations. 2. Experimental 2.1. Materials and methods The ACV reference substance was received from Shenyang Fine Chemical Co. (China). Chitosan with a molecular weight of 122.740 Da and degree of deacetylation of 90% was purchased from Purifarma (São Paulo, Brazil). All other materials were at least of analytical grade. 2.2. Preparation of spray-dried ACV/TPP/chitosan microparticles The ACV (100 to 200 mg) was dissolved in 0.1 mol L− 1 HCl solution. The polymer in different concentrations and the cross-linked agent tripolyphosphate were dissolved in a solution containing the drug and homogenized for 1 h (Table 1). The mixture was stirred for 30 min and the resulting solutions were spray-dried to obtain microparticles containing ACV. Spray-drying of solutions was carried out using a laboratory-scale spray dryer Buchi (model B-191, Switzerland) under the following set of conditions: inlet temperature 180 °C, outlet
2.2.1. Characterization 2.2.1.1. Determination of loading efficiency. A sample of cross-linked microparticles loaded with 10 mg of ACV was accurately weighted and dissolved in 10 mL of 90% ethanol in a 200 mL volumetric flask and 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 flask (5 µg mL− 1). The HPLC analysis was performed on a Shimadzu LC10 system (Kyoto, Japan) equipped with an LC-10AD pump, and SPD10AV UV detector (set at 254 nm). This assay was previously validated according to ICH, 2003 [9]. Experiments were performed in triplicate (n = 3) and loading efficiencies were calculated using Eq. (1). % Loading efficiency ¼
Mass of drug present in microparticles 100 Theoretical mass of acyclovir ð1Þ
2.2.1.2. Differential scanning calorimetry (DSC). DSC curves were obtained with a Shimadzu DSC-50 cell using aluminum crucibles with about 2 mg of the samples, under dynamic N2 atmosphere (100 mL 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; ΔHfus = 28.54 J g− 1) and zinc (mp 419.6 °C). 2.2.1.3. Thermogravimetric analysis (TG). TG curves were obtained with a Shimadzu thermobalance (model TGA-50) in the temperature range of 25–600 °C, using platinum crucibles with 4.0 ± 0.1 mg of sample, under dynamic N2 atmosphere (50 mL min− 1) with a heating rate of 10 °C min− 1. 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
Fig. 1. Preparation process of cross-linked ACV/TPP/chitosan microparticles by spraydrying.
Formulation
Chitosan/ACV (mg)
Loading efficiencya (%)
R.S.D.b (%)
F1 F2 F3 F4 F5
1000:100 1000:150 1000:200 1000:50 1000:75
86.4 79.7 73.8 92.5 90.6
±0.79 ±0.86 ±0.99 ±1.07 ±0.88
a b
Mean of three determinations. Relative Standard Deviation.
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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.
warmed to 37 °C was applied. The samples were centrifuged and analyzed using a UV spectrophotometer at 254 nm. 2.2.1.7. Stability studies. The stability studies were carried out in a climatic chamber at 40 ± 2 °C and 75 ± 5% of relative humidity (RH) as well as under ambient conditions (25 ± 2 °C and 75 ± 5% RH), during a period of 6 months [10]. Formulation samples were removed at time intervals of 0, 30, 60, 90, 120, 150 and 180 days and the drug was determined by high performance liquid chromatography (conditions describe in Section 2.2.1.1). 3. Results and discussion
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 characterized by a random state. These characteristics are important in the absorption process. Amorphous solids are, in general, more soluble than the crystalline form, due to free energies involved in the dissolution process. Solids in amorphous state have randomly arranged molecules and thus low energy is required to separate them and, consequently, their dissolution is faster than when in the crystalline form [11–13]. The ACV has crystalline characteristics which are represented by peaks in X-ray diffractograms, and the most evident peaks appear at 2θ = 4.76, 19.51 and 23.44. The chitosan diffractogram did not have peaks, which is characteristic of an amorphous compound (Fig. 4).
3.1. 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.
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The XRD diffractograms of F1, F2 and F3 did not show the same peaks as ACV, indicating that the ACV underwent a transition from a crystalline to an amorphous state (Fig. 4). In the formulations F4 and F5 the diffraction peaks decreased in comparison with the ACV, suggesting that in these formulations the drug was partially transformed into an amorphous form. In addition, in these formulations the amount of ACV was higher than in the others (data shown as loading efficiency).Therefore, these data confirm the results obtained from the DSC assays.
(28.9 µm ± 2.79), F2 (34.9 µm ± 2.89), F3 (29.9 µm ± 3.06), F4 (23.0 µm ± 3.67) and F5 (18.7 µm ± 3.65). All formulations had heterogeneous particle sizes and, due to the adhesive characteristics of chitosan, the particles were aggregated. The formulation F5 had the smallest, and F2 the largest, mean particle size. The photomicrography of formulation F2 showed ACV crystals, indicating that the drug was probably not completely encapsulated.
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 formulations had a differentiated pattern of release. The formulation F4 released ACV over a longer time period in both media. At acid pH the NH2 group remaining from the cross-linking process was protonated to NH3 and the polymer swelling led to the drug release.
The crystalline structure of ACV was confirmed by SEM in photomicrography (Fig. 5A), where an orthorhombic crystal form could be observed. The particle size was measured using this assay (n = 3) (Fig. 5) and the results indicated different mean sizes for F1
3.5. In vitro 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×).
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Table 3 Analysis of release data from ACV/TPP/chitosan microparticles Formulation
F1 F2 F3 F4 F5
Fig. 6. Dissolution profiles of F1, F2, F3, F4 and F5 in chloridric acid (pH 1.2).
The best release times were obtained for the highest chitosan/ACV ratio. The mechanism of ACV release from the microparticles is determined by different physical–chemical phenomena. According to Nixon [14], three steps lead to drug release from microparticles in aqueous medium: (1) imbibition of the release medium by the microparticles, (2) dissolution of the drug inside the microparticles, and (3) drug release into the aqueous medium through a diffusion process. The Korsmeyer–Peppas model (Eq. (2)), a semi-empirical model correlating drug release with time through a simple exponential equation for a drug release fraction b0.6, has been used to evaluate drug release from controlled release polymeric devices. It is particularly useful when the drug release mechanism is unknown or when there is more than one release mechanism [15,16]. Mt ¼ kdt n M∞
ð2Þ
Mt/M∞ is the proportion of drug released at time t, k is the kinetic constant, and the exponent n has been proposed as indicative of the release mechanism. In this context, n ≤ 0.43 indicates Fickian release and n = 0.85 indicates a purely relaxation-controlled delivery which is referred to as Case II transport. Intermediate values (0.43 b n b 0.85) indicate an anomalous behavior (non-Fickian kinetics corresponding
Fig. 7. Dissolution profiles of F1, F2, F3, F4 and F5 in phosphate buffer (pH 6.8).
pH 1.2
pH 6.8
n
r2
k (min
0.48 0.49 0.60 0.45 0.46
0.9419 0.9356 0.9705 0.9936 0.9624
21.55 32.34 31.06 8.28 17.85
−n
)
n
r2
k (min
0.21 0.29 0.32 0.22 0.22
0.8973 0.9156 0.8921 0.9930 0.9852
23.55 29.91 30.89 9.22 14.22
−n
)
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. 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 chitosan chain. On the other hand, the formulations in pH 6.8 demonstrated a Fickian release. The k values were related to the ACV release kinetics, a lower k value indicating a slower release. The k values obtained for F4 were 8.28 and 9.22 for pH 1.2 and 6.8, respectively. The other values were higher, and the dissolution profiles showed that ACV was released over a shorter time period. 3.6. Stability studies In the preparation of a pharmaceutical system some excipients can be used to obtain products with the desired physical and chemical characteristics or to improve the appearance. Other substances can be used to increase the stability of the drug, particularly in hydrolytic and oxidative processes [21]. In relation to the polymers, they can increase the stability of the final dry product [22]. The results showed that all formulations maintained at ambient temperature and humidity (25 ± 2 °C; 75 ± 5%) had a lower degree of degradation than the samples maintained in a climatic chamber (40 ± 2 °C; 75 ± 5%) (Figs. 8 and 9). It has been reported in the literature that temperature affects the drug stability with a two or three-fold increase in the reaction velocity for each 10 °C increase in temperature. Higher humidity is another factor associated with reduced stability [23,24].
Fig. 8. ACV concentrations of formulations maintained at ambient conditions (25 ± 2 °C; 75 ± 5%).
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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 F2 F3 F4 F5
1.42 × 10− 5 2.55 × 10− 5 3.04 × 10− 5 1.07 × 10− 5 1.24 × 10− 5
78 43 36 103 89
obtain stable microparticles. Comparing all of the formulations developed, F4 is the most promising for improved ACV release. Acknowledgement
Fig. 9. ACV concentrations of formulations maintained at climatic chamber (40 ± 2 °C; 75 ± 5%).
The stability of the formulations kept under the two different conditions of temperature and humidity was similar. For both, the formulations F4 and F5 had a lower lever of ACV degradation. The results indicated that the chitosan/ACV ratio also influenced the stability of the final formulations. This is because a higher chitosan-toACV ratio leads to greater ACV protection, hindering the direct action of temperature and humidity. The same behavior has been described by Stulzer and Silva for captopril granules produced with a fluid bed dryer using ethylcellulose and methylcellulose [25]. The final formulations demonstrated greater stability in relation to the isolated drug, when submitted to conditions of increased temperature, humidity and light. Scientific data obtained from stability studies has contributed to determining the shelf life of products, through the time period in which the formulations degraded 10% of the initial drug concentration in the formulations [26]. To calculate this parameter, it is necessary to define the order of the reaction (zero, first or second), through plotting the drug concentration as a function of time. The best correlation coefficient indicates the order of the reaction. For all formulations under both conditions, the degradation reactions occurred according to a second-order reaction. To calculate the velocity constant (k) the equations described in Table 4 were use, according to the calculated order of the reaction and the results are given in Table 5. Formulation F4 gave the highest values with 103 days of shelf life. 4. Conclusions In summary, the results show that ACV/TPP/chitosan microparticles increase the release time of the drug. The polymer/ACV ratio influences several parameters including loading efficiency, release profiles and stability. Another important aspect to note regarding the system developed was the fact that the drug was converted to amorphous form, which improved the ACV solubility. Thus, this study demonstrates the high potential of the spray-drying technique to
Table 4 Kinetic equations for calculate ACV degradation Reaction order
k (days− 1)
t90% (days)
Zero-order First-order Second-order
C0a − Cb / t 2.303 / t × log C0 / C 1 / t × (1 / C − 1 / C0)
0.1. C0 / k 0.106 / k 1 / 9k × C0
a b
ACV concentration in time zero. ACV concentration after degradation for a time t.
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