Development of albendazole sulfoxide-loaded Eudragit microparticles: A potential strategy to improve the drug bioavailability

Advanced Powder Technology 23 (2012) 801–807

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Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt

Original Research Paper

Development of albendazole sulfoxide-loaded Eudragit microparticles: A potential strategy to improve the drug bioavailability Marina Claro de Souza ⇑, Juliana Maldonado Marchetti Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 25 June 2011 Received in revised form 5 October 2011 Accepted 26 October 2011 Available online 10 November 2011 Keywords: Albendazole sulfoxide Polymeric microparticles Emulsification/solvent evaporation method Anthelmintic therapy

a b s t r a c t Albendazole sulfoxide (ABZSO), a broad spectrum anthelmintic drug extensively used in veterinary medicine, exhibits a low and erratic bioavailability due to its poor solubility in biological fluids. The aims of this study were the development, physicochemical characterization, and in vitro release profile evaluation of ABZSO-loaded Eudragit RS POÒ microparticles (MPs) in order to improve the rate of dissolution and the dissolved percentage of the drug in pH 7.4. MPs were successfully obtained by the emulsification/solvent evaporation method, achieving entrapment efficiency and process yield of about 60% and mean size of 254 nm. The in vitro release profile study showed that dissolution of ABZSO followed a pseudo-second order kinetics and MPs were able to increase significantly (p < 0.05) the rate of dissolution of ABZSO compared to the micronized and non-micronized free drug, what could lead to an improvement in bioavailability and, consequently, in the antiparasitic activity. Ó 2011 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.

1. Introduction Helminthes can be classified as cylindrical (nematodes) and plate (cestodes and trematodes) worms. Helminthic infections are found worldwide, especially in tropical areas [1], constituting a serious sanitary problem in human and veterinary medicine, besides causing great economical problems [2]. Several parasites are very important to public health due to their high zoonotic potential, like the nematodes Toxocara canis and Ancylostoma braziliensis and the protozoans Cryptosporidium, Leishmania, and Toxoplasma [3]. On the other hand, there are the cestodes of Taenia gender, especially Taenia solium, responsible for causing cysticercosis, a disease found in animals and human beings. Cysticercus cellulosae, the larval stage of T. solium, is responsible for neurocysticercosis which is a disease caused by the presence of cysts containing this larva in the central nervous systems and could cause serious illness to the patient, even death [4]. Benzimidazole compounds are especially effective against nematodes found in the gastrointestinal tube in combating adult worms besides their larvae and eggs [1]. They are also highly effective against lung worms [5]. Their mechanism of action is related to several biochemical changes, particularly the inhibition of

⇑ Corresponding author. Address: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Avenida do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil. Tel.: +55 (16) 36024302. E-mail address: [email protected] (M.C. Souza).

b-tubulin, leading to the suspension of cellular processes like mitotic division and transport of nutrients, and also the inhibition of the fumarate reductase enzyme in mitochondrial reactions blocking, in this manner, the metabolic route of the parasite [1,6]. Albendazole sulfoxide (ABZSO), a broad spectrum antiparasitic drug from the benzimidazole group, is used to control infections by nematodes, cestodes, and trematodes in big and small ruminants, besides companion animals. Apart from their broad spectrum, benzimidazole compounds have the advantage of causing relatively low toxicity to hosts compared to other antiparasitic drugs [7–9]. ABZSO is almost insoluble in water and sparingly soluble in water-miscible solvents like ethanol and propylene glycol. Usually, it is administered at a dose of 7.5 mg kg1 and its bioavailability varies from 36.8% to 40.5% [9]. Besides, this drug is well distributed in the body after intravenous administration, achieving volume of distribution values ranging from 0.67 to 1.2 L kg1 for cattle and sheep, respectively [10,11], due to its low and erratic bioavailability, when administered by other routes, a high dosage is required to achieve plasmatic therapeutic levels [12]. One of the advantages of micro- and nanoencapsulation processes is their ability to improve bioavailability of poorly water-soluble drugs [13,14]. According to Devalapally et al. [15], in general, conventional formulations show low and irregular bioavailability of poorly water-soluble drugs. Microencapsulation processes can be used to improve pharmacokinetic properties of these compounds. The reduced size of microparticles (MPs) and the consequent increase in surface area can substantially increase the rate

0921-8831/$ - see front matter Ó 2011 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved. doi:10.1016/j.apt.2011.10.009

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of dissolution, leading to higher maximum plasmatic concentration (Cmax) and area under the curve (AUC). Many studies have been performed using MPs as drug delivery systems in pharmaceutical technology area [16,17]. The veterinary pharmaceutical market has also been exploring several strategies to improve drug efficacy and to reduce administration frequency through the development of drug delivery systems [18]. One of these strategies is the use of MPs, which are very versatile systems that, according to their characteristics, can be administered through many varied routes like parenteral, oral, ocular, nasal, and pulmonary [19,14]. Several studies using EudragitÒ copolymers in microencapsulation processes have been done, like the one conducted by Al-Zoubi et al. [20], where EudragitÒ MPs for modified release of buspirone hydrochloride were successfully obtained, achieving an entrapment efficiency of about 100%. Also, Raffin et al. [21] prepared sodium pantoprazole–EudragitÒ MPs in a comparative study by emulsification/solvent evaporation and spray-drying techniques, in order to improve photo stability of the referred drug. The objective was satisfactorily achieved by both these methods. The aims of this work were to prepare, characterize physicochemically and evaluate the in vitro release profile of ABZSOloaded Eudragit RS POÒ MPs, applicable to veterinary medicine, in order to increase the rate of dissolution of the drug in biological fluids.

2. Material and methods 2.1. Material ABZSO (MW = 281,33) was kindly donated by Formil (Brazil) and Eudragit RS POÒ (Degusa Rohm Pharma Polymers, Germany) by Pfizer Laboratories; NaH2PO4H2O, NaCl, polysorbate 80 (Tween 80Ò), and diethyl amine (DEA), all of analytical grade, were purchased by Synth (Brazil); Na2HPO42H2O and benzyl alcohol (BA) (both analytical grade) were purchased by Vetec (Brazil); dichloromethane (DCM) analytical grade was purchased by Quimex (Brazil); HClO4 and H3PO4 (analytical grade) were purchased from Merck (Germany); polyvinyl alcohol (PVA) analytical grade was purchased from Sigma–Aldrich (USA); acetonitrile (ACN), absolute ethanol (EtOH), and methanol (MeOH), all of chromatographic grade, were purchased by J.T. Baker (USA).

2.2.1.3. Assay validation. Validation of the analytical method was performed for linearity, within-day and between-days accuracy and precision, and robustness, following the ICH Q2(R1) guideline [23]. Linearity was evaluated by the linear correlation coefficient (r), obtained by linear regression of the calibration curve described on item 2.2.1.1. Within-day and between-days accuracy and precision of the method were done at concentrations of 0.5, 7.5, and 12.5 lg mL1. Results were expressed as relative standard deviation or coefficient of variation (CV – %) and relative error (E – %). The robustness assay was tried at the concentrations of 0.5, 7.5, and 12.5 lg mL1. Each sample was analyzed three times and CV and E were calculated. Linear correlation coefficient was determined for each condition evaluated. The conditions analyzed were pH of mobile phase (2.1, 2.3, and 2.5), flow rate (0.7, 1.0, and 1.2 mL min1), and ACN:H2O ratio in mobile phase (10:90, 15:85, and 20:80). 2.2.2. Preparation of microparticles ABZSO-loaded MPs were prepared by emulsification/solvent evaporation, a classical methodology, based on the studies performed by Truong Cong et al. [24], Scholes et al. [25], adapted by Ricci-Júnior and Marchetti [26], and Hombreiro-Pérez et al. [27]. Briefly, the organic phase consisted of the polymer and the drug (dissolved or dispersed depending on the composition of the organic phase) in different ratios were added to a PVA aqueous solution stirred continuously at 13,000 rpm for 6 min, using a ultra-turrax homogenizer (IKA™ – Labortechnik model T25), resulting in an O/W emulsion. Some organic:aqueous phase ratios were tried (1:2, 1:5 and 1:10). DCM (where ABZO is partially soluble) and the solvent mixtures DCM:EtOH (9:1) and DCM:benzyl alcohol (BA) (10:0.25) were used as organic phase, in which the drug is completely soluble. Drug:polymer ratios evaluated were (1:4), (1:5) and (1:10). PVA solution concentration was tried ranging from 0.25% to 3.0% (w/v). Solvent evaporation step was performed by reduced pressure (30 min) and by magnetic stirring under exhaustion in order to set the process. After solvent evaporation, MPs were separated by centrifugation at 8460g (15 °C) for 1 h. The supernatant was discarded; MPs were washed with purified water and submitted to centrifugation again. This process was repeated twice in order to eliminate free ABZO and the surfactant (PVA), once non-encapsulated drug remains as a fine dispersion in supernatant. MPs were frozen in dry ice and freeze-dried (Liobras L101 lyophilizer) overnight. Dried samples were stored at 25 °C before analysis.

2.2. Methods 2.2.1. Quantification of ABZO 2.2.1.1. Standard solutions and chemicals. A stock solution containing 0.25 mg mL1 of ABZO in methanol was prepared and stored at 4 °C. From the stock solution, a standard curve was prepared at 0.5, 2.5, 5.0, 7.5, 10.0, and 12.5 lg mL1 concentrations by appropriate dilutions. 2.2.1.2. Apparatus and chromatographic conditions. Quantification of ABZSO was carried out based on the method described by Sarin et al. [22]. This study was performed using a Shimadzu liquid chromatography equipment composed of an LC-10AD model pump, a SPD-10A model ultraviolet (UV) detector, and a model CR6-A integrator. Separation was carried out on a Purospher StarÒ (Merck) C18 – 5 lm reversed phase column (250  4.6 mm I.D.) with a Merck C18 – 5 lm guard column (4  4 mm I.D.) at a controlled temperature (25 °C). A mixture composed of water, ACN, DEA and HClO4 was used as mobile phase in the ratio (85:15:0.2:0.08, v/v) (pH 2.3) at a flow rate of 1.0 mL min1. The injection volume was 20 lL and the detection wavelength 290 nm.

2.2.2.1. Process yield. The process yield was calculated according to Eq. (1):

Yð%Þ ¼ ðM MP =M T Þ  100

ð1Þ

where Y is the process yield, MMP is the mass of MPs recovered after freeze-drying step, and MT is the initial mass of Eudragit RS POÒ plus the mass of ABZSO. 2.2.2.2. Entrapment efficiency. The entrapment efficiency was determined by the direct method. MPs were dissolved in methanol under ultrasonic stirring and ABZSO was quantified by HPLC. All of the MPs were analyzed in triplicate. Entrapment efficiency was calculated by dimensional analysis using Eq. (2):

EEð%Þ ¼ ðAS =ASt Þ  ðMSt =DSt Þ  ðDS =MS Þ  100

ð2Þ

where EE is the entrapment efficiency, AS is the medium area of sample peaks, ASt is the medium area of standard peaks, MSt is the mass of standard, DSt is the dilution of standard, DS is the dilution of sample, and MS is the theoretical mass of drug in the sample.

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Table 1 Influence of the nature of organic phase and drug:polymer ratio on the entrapment efficiency and obtainment process yield of ABZSO-Eudragit RS POÒ MPs by emulsification/ solvent evaporation method. Aqueous phase

Organic phase

Drug:polymer ratio

Entrapment efficiency (%)

Yield of process (%)

20 mL 20 mL 20 mL 20 mL 20 mL

10 mL 10 mL 10 mL 10 mL 10 mL

1:4 1:10 1:4 1:10 1:10

6.67 25.61 20.02 61.52 6.78

27.32 36.64 52.98 61.79 32.65

PVA PVA PVA PVA PVA

1.0% 1.0% 1.0% 1.0% 1.0%

CH2Cl2:EtOH (9:1) CH2Cl2:EtOH (9:1) CH2Cl2 CH2Cl2 CH2Cl2 + 0.25 mL BA

2.2.3.3. X-ray diffraction analysis. X-ray diffraction analyses (Simenes X-ray Diffractometer, model D5005) were carried out using a sealed cooper tube (anode; 1.5406 Å CuK(a) radiation), at the followed conditions: 2–70° scanning, 0.02°/s. 2.2.4. In vitro release profile study In vitro release profile study was carried out using a dissolution equipment (SR8 Plus, Hanson Research). A solution composed of phosphate buffered saline (PBS) pH 7.4:EtOH (60:40) plus 5.0% Tween 80Ò was used as the dissolution medium in order to guarantee sink conditions. The dissolution medium was stirred at 100 rpm, using the apparatus mini paddles, and kept at 37 °C. A known amount of freeze-dried MPs, equivalent to 1.5 mg of ABZSO, were inserted into a dialysis tube (n = 5) and then introduced in the dissolution vessels containing 150 mL of dissolution medium. Samples of free drug, micronized (ball mill/24 h) and non-micronized, were analyzed in the same manner to make a comparison. The study was performed for 48 h. Samples (1.0 mL) were collected at 1, 2, 4, 8, 12, 24, 36, and 48 h, with replacement of the collected medium volume, and analyzed by HPLC. The amount of dissolved drug was calculated by dimensional analysis according to Eq. (3):

Q t ð%Þ ¼ ðAS =ASt Þ  C St  ðV=MS Þ þ ðQ t1  1=VÞ  100

ð3Þ

where Qt is the dissolved percentage in the referred time, AS is the medium area of sample peaks, ASt is the medium area of standard peaks, CSt is the concentration of standard, V is the volume of dissolution medium, MS is the mass of ABZSO in the sample, Qt1 is dissolved percentage in the immediately previous time and 1 is the volume of sample. The results were submitted to mathematical analysis to specify the release kinetics order.

Fig. 1. Photomicrographies of AZSO-Eudragit RS POÒ MPs and free drug sputtercoated with gold, obtained by SEM (Zeiss Scanning Electron Microscope, model EVO 50). A: ABZSO free drug (increased 7000); B: AZSO-Eudragit RS POÒ MPs (increased 50,000).

2.2.3. Physicochemical characterization of ABZSO-Eudragit RS POÒ microparticles 2.2.3.1. Particle size analysis. Particle size and size distribution of MPs were measured by laser light scattering using a particle analyzer (Zetasizer Malvern, model Nano SZ). To make a comparison, these analyses were also performed for non-micronized and micronized (prepared in our laboratory – ball mill/24 h) free drug (LS Particle Size Analyzer, Beckman Coulter, model LS 13 320). 2.2.3.2. Scanning electron microscopy (SEM). The shape and surface morphology of the MPs were analyzed by SEM (Zeiss Scanning Electron Microscope, model EVO 50). Free drug was also analyzed to make a comparison. Dried samples were prepared on a sample compartment and sputter-coated with gold 100 s (Sputter Coater Bal-Tec, model SCD 050) prior to the SEM analysis. The voltage ranged from 15 to 25 kV.

2.2.5. Statistical analysis Results obtained from the in vitro release profile study, performed by a completely randomized design, were statistically analyzed by one-way Analysis of Variance (ANOVA) with post hoc Tukey’s test. The objective was to evaluate significant differences related to dissolved percentages in function of time among MPs and free drug (micronized and non-micronized). Results were expressed as mean ± standard deviation (SD). 3. Results and discussion 3.1. Assay validation Analytical validation is a very important step as it is responsible for guaranteeing the veracity of the results. The linear coefficient correlation (r) obtained from linear regression of the calibration curve (concentrations varying from 0.5 to 12.5 lg mL1) was 0.9998. All of the mean values of coefficient of variation (CV) and relative error (E) obtained from within-day and between-days precision and accuracy tests were <5.0%. Concerning the robustness assay, all of the calibration curves, obtained from each analyzed parameter, exhibited values of r > 0.99 and CV and E values <5.0%. On the basis of this, it was possible to conclude that the proposed changes in analytical conditions did not influence the

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Fig. 2. Particle size distribution of ABZSO-Eudragit RS POÒ MPs, analyzed by laser light scattering (Zetasizer Malvern, model Nano SZ).

Fig. 3. Particle size distribution of micronized and non-micronized free ABZSO analyzed by laser light scattering (Beckman Coulter Particle Analyzer, model LS 13 320). A: non-micronized free ABZSO; B: micronized free ABZSO.

method linearity, accuracy and precision. So, it was concluded that the proposed method was able to analyze ABZSO in a reliable manner in the range of concentration evaluated. 3.2. Preparation and physicochemical characterization of MPs It was observed that the minimum concentration of PVA required to stabilize the emulsion was 1.0% and no advantages were achieved with higher concentrations of surfactant. Related to the organic: aqueous phase ratios, emulsion was properly formed with all those were tested. The (1:2) ratio was chosen in order to reduce the migration of ABZO to the external phase and, consequently, improve the entrapment efficiency. Drug:polymer ratio and composition of organic phase were also evaluated, considering entrapment efficiency and yield of the process (Table 1). With regard to the drug: polymer ratios tried, the best results were achieved using

the (1:10) one. Using these conditions, the entrapment efficiency and the yield of the process were both a little higher than 60%. Considering the solvent evaporation step, no differences relating to yield and entrapment efficiency were observed between the two studied processes. The influence of the organic phase nature was analyzed. When pure DCM was used, solubilization of ABZSO was not complete. So, the drug was microencapsulated in the form of a very fine dispersion. This was responsible for the imperfect spherical shape of the MPs observed in the SEM photomicrographies (Fig. 1). On the other hand, the use of EtOH or BA associated with DCM as organic phase allowed complete solubilization of the drug leading to spherical shaped MPs (not shown), although, with the use of a watermiscible solvent in the organic phase the entrapment efficiency decreased to less than 20% and the yield of the process to less than 35%. According to Birnbaum et al. [28] and Shenderova et al.

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(A)

3000 d=8,46716

2000

d=1,75043

d=1,71188

d=1,66963

d=1,89781

d=1,82747

d=2,02683

d=1,94558

d=2,18978

d=2,14056

d=2,28136 d=2,24360

d=2,54067

d=2,44889 d=2,40910

d=2,69930 d=2,63711

d=3,16925

d=4,01119

d=4,19493

d=4,59055

d=5,92762

d=5,49390 d=5,26350

d=6,65998 d=6,36667

d=7,56204

d=9,39317

d=16,77153

1000

d=3,32811

d=3,79911

d=3,50636

d=4,75583

Lin (Counts)

4000

0 2

10

20

30

40

50

60

70

2-Theta - Scale File: SA.raw - Type: 2Th/Th locked - Start: 2.000 ° - End: 70.000 ° - Step: 0.020 ° - Step time: 1. s - Temp.: 25 °C (Room) - Time Started: 13 s - 2-Thet a: 2.000 ° - Theta: 1.000 ° - Chi: 0.00 ° - Phi: 0. 00 ° - X: 0.0 mm - Y: 0.0 mm - Z: Operations: Smooth 0.150 | Strip kAlpha2 0.500 | Background 0.000,0.000 | Import

(B) 500

d=6,73635

Lin (Counts)

400

300

200

100

0 2

10

20

30

40

50

60

70

2-Theta - Scale sem pressão; com espalhamento - File: PI.raw - Type: 2Th/Th locked - Start: 2.000 ° - End: 70.000 ° - Step: 0.020 ° - Step time: 1. s - Temp.: 25 °C (Roo m) - Time Started: 13 s - 2-Theta: 2.000 ° - Theta: 1.000 ° - Chi: 0.00 ° - Phi: 0 Operations: Smooth 0.150 | Strip kAlpha2 0.500 | Background 0.000,0.000 | Import

(C)

500

d=4,80039

300 d=8,54012

Lin (Counts)

400

d=3,53674

200

100

0 2

10

20

30

40

50

60

70

2-Theta - Scale sem pressão; com espalhamento - File: PF.raw - Type: 2Th/Th locked - Start: 2.000 ° - End: 70.000 ° - Step: 0.020 ° - Step time: 1. s - Temp.: 25 °C (Roo m) - Time Started: 9 s - 2-Theta: 2.000 ° - Theta: 1.000 ° - Chi: 0.00 ° - Phi: 0. Operations: Smooth 0.150 | Strip kAlpha2 0.500 | Background 0.000,0.000 | Import

Fig. 4. X-ray diffraction analyses (Siemens X-ray Diffractometer, model D5005), conducted at a 2–70° scanning (0.02°/s). A: ABZSO free drug; B: placebo MPs; C: drug-loaded MPs.

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(Fig. 4) indicated the presence of the drug in the MPs surface once one can see the presence of peaks in the drug-loaded MPs diffractogram corresponding to the interatomic distances: 8.54, 4.80 and 3.53 Å, as also shown in the free drug diffractogram. 3.3. In vitro release profile study

Fig. 5. In vitro release profile study comparing ABZSO-Eudragit RS POÒ (1:10) MPs and free ABZSO (micronized and non-micronized). Dissolution medium: PBS pH 7.4: EtOH (60:40) plus 5.0% Tween 80Ò. Medium volume: 150 mL. Rotational speed: 100 rpm. Temperature: 37 °C. Sampling times: 1, 2, 4, 8, 12, 24, 36, and 48 h. Mean ± SD (n = 5).

[29], the use of water-miscible solvents are sometimes required to allow a suitable solubilization of the drug. At the same time, during the solvent evaporation step, the presence of these solvents may cause the migration of solubilized drug to the aqueous phase, resulting in a low entrapment efficiency level. It can also result in the deposition of drug crystals on the surface of the MPs and increased superficial porosity [30]. The process of microencapsulation of hydrophobic drugs by the emulsification/solvent evaporation method, in general, results in an entrapment efficiency level higher than 70% when the drugs are soluble in water-immiscible solvents such as DCM and chloroform. Although ABZSO is partially soluble in these solvents, several previous studies have shown that when it occurred, the entrapment efficiency was lower. Gupte and Ciftci [31] prepared PLGA microspheres containing paclitaxel, 5-fuoruracil (5-FU), and paclitaxel plus 5-FU by emulsification/solvent evaporation method. While paclitaxel was solubilized in the organic phase, 5-FU remained only dispersed. The entrapment efficiency of paclitaxel was about 90% (when microencapsulated alone as well as when associated with 5-FU). The entrapment efficiency of 5-FU was about 19% when microencapsulated alone and about 30% when it was associated with paclitaxel. Giunchedi et al. [32] prepared PLA MPs containing the hydrophobic drugs hydrocortisone and hydrocortisone 21-acetate by the emulsification/solvent evaporation technique. During the emulsification step, both drugs remained in suspension in the organic phase (DCM), the latter being a little more soluble in the solvent than the former. The entrapment efficiency values were 9.4% and 55.0%, respectively, which confirm that drug solubility in the organic phase has a direct impact on the entrapment efficiency rate. Benelli et al. [33] observed that the non-complete solubilization of the drug in the organic phase led to the presence of crystals in the MPs surfaces when clonazepam-loaded PLGA MPs were prepared by two different methods, namely spray-drying and emulsification/solvent evaporation. Beside the irregular shape verified by the SEM analysis, the MPs obtained exhibited a narrow range of size, with monomodal particle size distribution and low polidispersivity index (PdI). The mean size was 254 nm (Fig. 2). With regard to free drug, both micronized as well as non-micronized ones were submitted to particle size analysis and exhibited irregular particle size distribution (Fig. 3). The mean size was 7.13 and 27.4 lm for micronized and nonmicronized free drug, respectively. From these data, it is observed that microencapsulation process reduced particle size about 90 times compared to non-micronized drug and 20 times compared to micronized drug, which means that the process led to a significant improvement in the surface area. The results obtained by the X-ray diffraction analyses, performed with the free drug, placebo MPs and drug-loaded ones

As ABZSO is almost insoluble in PBS pH 7.4, an organic solvent and a surfactant were needed to guarantee its appropriate solubilization in the dissolution medium. The addition of an organic solvent was based on the study conducted by Montenegro et al. [34] where PBS:EtOH (50:50) was used as acceptor solution for an in vitro cutaneous permeation study of retinoic acid-loaded liposomes due to the very low solubility of this drug in aqueous medium. The results from the in vitro release profile study showed an increase in the rate of dissolution of microencapsulated drug compared to free drug, even after the micronization process, as shown in Fig. 5. This can be due to the lower dimensions of obtained MPs (<300 nm), which certainly promoted an improvement in surface area, increasing the rate of dissolution. The reduced dimensions of nano- and microparticles can lead to improved pharmacokinetics properties [15]. The reduction of size, and the consequent increase in surface area, may significantly improve the rate of dissolution of poorly water-soluble molecules, besides the small size may affect in vivo permeability, resulting in higher maximum plasmatic concentration (Cmax) and area under the curve (AUC). Depending on the nature of the delivery system (drug loading, type of polymer, additives, etc.), many different mass transport phenomena may be evolved (like drug dissolution, polymer erosion, drug diffusion by polymer pores, etc.), affecting the drug release profile. In addition, geometry (size and shape) may affect the release kinetics mechanism [35]. By the linear regression of the data from the in vitro release profile study, obtained by plotting t/Q against t [36], an r value of 0.9995 was obtained, showing the developed MPs followed a pseudo-second order kinetics drug release model, which is based on Noyes–Whitney equation, described in 1897 [37]:

dW=dt ¼ ðD  A  ðC s  CÞÞ=L

ð4Þ

where dW/dt is the rate of dissolution, D is the diffusion coefficient, A is de surface area, Cs is the concentration of the solid in the diffusion layer, C is the concentration of the solid in the bulk dissolution medium and L is the diffusion layer thickness. By the addiction of a surfactant and an organic solvent in the dissolution medium in order to achieve sink conditions, the drug is readily solubilized once it has diffused out of the MPs and across the diffusion layer surrounding the MPs. Therefore, an increase in surface area was achieved by the reduced size of MPs. These two factors lead to an increase in the rate of dissolution. The same data were also submitted to statistical analysis (ANOVA with post hoc Tukey’s test) in order to verify the significance of the results. By the results obtained, one can see that only in the first sampling time (1 h) no statistical difference (p > 0.05) was observed among the three groups. With regard to sampling times of 4, 8, and 12 h, the ABZSO mean dissolved percentage was significantly (p < 0.05) higher in MPs compared to micronized and nonmicronized free drug, what means that the microencapsulation process increased significantly the rate of dissolution. From sampling times of 24, 36, and 48 h, no statistical difference (p > 0.05) was noticed between MPs and micronized free drug as both achieved 100% of dissolution. Non-micronized free drug dissolution percentage showed statistical difference (p < 0.05), being lower than the other two samples, achieving only about 85% of the dissolved drug at the end of the experiment, due to its smaller superficial area compared to micronized free drug and MPs.

M.C. Souza, J.M. Marchetti / Advanced Powder Technology 23 (2012) 801–807

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