Microencapsulation of Metoprolol Tartrate into Chitosan for ... - UMF Iasi

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Microencapsulation of Metoprolol Tartrate into Chitosan for Improved Oral Administration and Patient Compliance Lăcrămioara Ochiuz,†,¶ Graţiela Popa,† Iulian Stoleriu,‡ Alina Maria Tomoiagă,*,§,¶ and Marcel Popa⊥ †

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and Pharmacy “Grigore T. Popa”, Universitatii Street, no. 16, 700115 Iasi, Romania ‡ The Faculty of Mathematics, “Alexandru Ioan Cuza” from Iasi, 11, Carol I Bd, 700506 Iasi, Romania § Department of Materials Chemistry and Chemical Technology, Faculty of Chemistry, University “Alexandru Ioan Cuza” from Iasi, 11, Carol I Bd, 700506 Iasi, Romania ⊥ ”Gh. Asachi” Technical University of Iasi, Department of Natural and Synthetic Polymers, D. Mangeron Bd., 71A, 700050 Iasi, Romania S Supporting Information *

ABSTRACT: Microparticles made from naturally occurring biopolymers, such as chitosan, appear to be promising carrier systems for the sustained release of orally administered drugs. In the current study, we followed a microencapsulation technique based on the spray-drying method to prepare metoprolol tartrate-containing chitosan microparticles with various compositions. The prepared microparticulate drug delivery systems were investigated for their morphological, structural, and thermal behavior by optical microscopy, infrared spectroscopy, and thermogravimetric analysis. Microencapsulation efficiency and drug content were assessed by a validated HPLC method. In vitro dissolution tests performed in simulated gastric fluid (SGF) (pH 1.2) and simulated intestinal fluid (SIF) (pH 6.8) revealed that the drug-to-polymer ratio is an important element in controlling the release features of microparticulate systems based on CHT. Also, the pH of the dissolution fluid plays an important role in the release of the drug substance from the microspheres. Additionally, the analysis of the release kinetic mechanism concluded that in SGF media as well as in SIF media, the MT from the chitosan-based microparticles is released by means of a Fickian diffusion process.

1. INTRODUCTION In the past decade, more and more people suffer from various forms of cardiovascular diseases, affecting the human longevity. Thus, tremendous efforts are made by chemists, pharmacists, and medical doctors worldwide for finding new delivery approaches, new modes of action, and new pharmaceutically active substances to treat these life-threatening illnesses. Metoprolol tartrate (MT) with the chemical formula (C15H25NO3)2C4H6O6 (Figure 1A) is a β1-selective andrenergic blocker agent widely used for the treatment of hypertension, angina, arrhythmia, hyperthyroidism, and other related diseases, as well as a prophylactic after myocardial infarction.1−3 According to the Biopharmaceutics Classification System, MT is classified as a class I drug because it is a highly watersoluble and permeable drug.4 Because the biological half-life of MT ranges to 3−4 h,1 multiple doses are needed to maintain a constant plasma concentration for a good therapeutic response. It has also been reported that MT absorption in the duodenum and jejunum is directly proportional to the dose availability.5,6 The conventional oral administration dosage form fails to maintain the drug plasma concentration over the extended period of time, resulting in frequent administration of the drug with a higher dose, causing unwanted toxic effects. Beside this, if the entire amount of drug is released at once, dose dumping occurs and a low therapeutic response is obtained. Therefore, the success of a drug delivery formulation depends on the ability to build a biocompatible carrier, capable of high drug © 2013 American Chemical Society

Figure 1. (A) Chemical structure of metoprolol and (B) chemical structural representation of chitin and chitosan depicting the copolymer character of the biopolymers.

Received: Revised: Accepted: Published: 17432

August 10, 2013 October 31, 2013 November 5, 2013 November 5, 2013 dx.doi.org/10.1021/ie402625h | Ind. Eng. Chem. Res. 2013, 52, 17432−17441

Industrial & Engineering Chemistry Research

Article

Table 1. Thermogravimetric Features of MT, CHT, and MT−CHT Microparticles (n = 3)a sample

stage

Tonset

Tpeak

Tendset

MT CHT

I I II III I II III I II III I II III I II III

200 61 272 494 55 152 270 59 140 210 52 140 216 60 140 222

284 85 299 565 82 174 289 75 155 268 77 155 290 78 152 294

378 114 328

Fb

F1

F2

F3

Wloss (%) Mean ± SD

Wres (%) Mean ± SD

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.01 ± 0.82 29.37 ± 0.38

91.98 8.77 46.21 15.65 11.85 5.84 51.17 7.22 7.64 64.77 8.32 7.64 61.40 11.52 8.05 56.12

108 200 399 100 210 451 98 210 412 104 222 470

0.84 0.18 0.71 0.32 0.25 0.14 0.19 0.32 0.80 0.94 1.02 0.80 0.87 0.72 0.53 0.79

Wtheo (%)

31.14 ± 0.21

20.37 ± 0.66

18.74

24.27 ± 8.84

22.19

24.31 ± 0.69

24.35

a

Tonset: degradation starting temperature at each stage. Tpeak: maximum degradation rate temperature. Tendset: degradation ending temperature at every stage. Wloss: mass loss at every stage. Wres: residual weight at 600 °C. Wtheo: theoretical residual weight at 600 °C.

was proven to be very efficient, and the resultant microspheres were used for intranasal delivery of MT. Spray drying is widely used method for the preparation of microparticulate drug delivery systems. In the past few years, this method has been intensively investigated mainly for aqueous polymeric formulations as an alternative to the conventional methods that generally use organic solvents, which involves a high risk of product contamination, toxicity, and explosion hazards, as well.38 It has several advantages including high reliability, reproducibility, and control of particle size.39 More, chitosan microparticles prepared by the spray-drying method are characterized by high sphericity and specific surface area, parameters that are very important for the research field dealing with drug development.40 Therefore we have chosen to prepare chitosan-based microparticles encapsulating metoprolol tartrate by means of the spray-drying method using different ratios of MT:CHT. The newly formulated CHT−MT therapeutic microsystems were investigated using SEM, FTIR, and TGA/DSC techniques in order to monitor the modifications occurred at microencapsulation of various amounts of MT into chitosan. Production yield, microencapsulation efficiency, and drug content were also assessed by means of gravimetric and HPLC measurements. Fully validated HPLC method was used (see Supporting Information for details). In vitro release studies were performed in order to establish the dissolution profiles of newly formulated microsystems.

loadings and controlled release, thus improving patient compliance and convenience. Biopolymer-based microparticles are good examples of modern therapeutic systems that offer many advantages such as decreased adverse secondary effects, controlled/sustained release of the active agent, resulting in reduced administration frequencies and optimization of the therapeutic effect.7−10 Thus, the formulation of MT as sustained release microparticulate drug delivery systems seems one of the most feasible ideas to overcome some of the problems concerning its pharmacological availability. Various polymers have been evaluated to develop therapeutic microsystems capable of delivering drugs in a controlled manner over a prolonged period. Among them, chitosan (CHT), a natural linear polysaccharide obtained from the deacetylation of chitin11 (Figure 1B), has many advantages that are highly important for the pharmaceutical development field, namely its biodegradability and biocompatibility,12−15 its ability to control the release of drug substance,12,16−19 its solubility in aqueous acidic solutions, thus avoiding the use of hazardous organic solvents while obtaining particles,20 its excellent surface chemistry based on a high number of free amino groups that are readily available for cross-linking,21−24 and its cationic nature that allows ionic cross-linking with multivalent anions.25 Over the years, CHT has been used in the preparation of mucoadhesive formulations26,27 in order to improve the dissolution rate of poorly soluble drugs,28 for drug targeting,19,29,30 for pH-stimulated drug delivery,31 for enhancement of poorly absorbable drug and peptide absorption,31−34 and as microcarriers for nanoparticle delivery.16 There are few previous reports on the preparation of metoprolol-encapsulated microparticles for various pharmaceutical applications. For example, Shabaraya reported the preparation of metoprolol tartrate-loaded microspheres by phase separation−emulsification technique.35 Palanisamy reported the preparation of metoprolol succinate chitosan-based microspheres by a cross-linking method.36 The encapsulation efficiency in this case was only 65−70%, and the size of the microspheres was of 330 − 527 nm. In 2013, Adi and collaborators reported the preparation of metoprolol tartrate chitosan-based microspheres by ionic precipitation and chemical cross-linking method.37 The preparation method

2. MATERIALS AND METHODS 2.1. Materials. Metoprolol tartrate, propranolol hydrochloridum, and practical-grade chitosan with a degree of deacetylation >85% were supplied by Sigma-Aldrich, Germany. Chromatographic-grade methylic alcohol and triethanolamine were purchased from Merck. Metoprolol tartrate CRS was acquired from EDQM, France. Ammonium acetate, glacial acetic acid, and phosphoric acid were kindly supplied by Chemical Company, Romania. Metoprolol tartrate tablets (Egilok 50 mg/tablet, Egis Pharmaceuticals, Hungary) were purchased from a local pharmacy. All chemicals were of analytical grade and used as received. 17433

dx.doi.org/10.1021/ie402625h | Ind. Eng. Chem. Res. 2013, 52, 17432−17441


Article

2.5. Calculations. Production yield (PY) was determined as follows (eq 1):

2.2. Preparation of Chitosan-Based Microparticles Containing Metoprolol Tartrate. Three different formulations of CHT−MT microparticles are prepared with various drug-to-polymer ratios (MT:CHT w/w), according to Table 1. In a typical synthesis procedure, CHT is dissolved in 4% (v/v) acetic acid solution to obtain a 1% (m/v) polymer solution. Then, proper amounts of MT are added under vigorous continuous stirring, resulting in solutions containing variable drug-to-polymer ratios. Further, the resulting solutions are sprayed through the nozzle of a Mini Spray Dryer B29 (Büchi, Switzerland) under the following conditions: inlet temperature, 150 ± 2 °C; outlet temperature, 100 ± 2 °C; aspirator rate, 100%; pump, 5 mL/min; air flow, 530 L/h (45 mm in the rotameter); nozzle cleaner, 3. A placebo microparticle formula was prepared under the same condition by spraying a CHT solution 1% (w/v). Through the whole procedure, temperature and humidity were controlled and maintained at 23 ± 1 °C and 55 ± 2% relative humidity. Each formulation was carried out in triplicate. 2.3. Characterization Methods and Equipment. The morphological characteristics of chitosan and MT-encapsulated chitosan microparticles were examined using a TESCAN VEGA II SBH scanning electron microscope (SEM). Each microparticulated sample was coated with a 15 nm layer of gold by means of cathode pulverization. Imaging parameters (kV, magnification, etc.) can be visualized on the registered micrographs. The mean microparticles diameter and the size distribution were assessed by laser diffraction using a SHIMADZU−SALD 7001 particle size analyzer. All measurements were carried out in triplicate on suspensions of particles in acetone after thorough sonication. Thermal stability of MT, CHT, and prepared microparticles was investigated with a Mettler Toledo TGA-SDTA 851e derivatograph. All measurements were performed in an inert atmosphere under constant purging of nitrogen with a 20 mL/min flow rate. Thermogravimetric data were recorded in the temperature range of 25−600 °C, with a heating speed of 10 °C/min. A thermal analysis in dynamic conditions (TG, DTG, and DTA) was also performed and Star software was used for data investigation. Infrared spectra were recorded at room temperature on an ATR−FTIR spectrometer (attenuated total reflectance−Fourier transform infrared), model IdentifyIR from SmithDetection. The samples were measured as powders, with no previous dilution in KBr. The scans were collected with a resolution of 4 cm−1 over the spectral range 600 − 4000 cm−1. The MT loading within CHT microparticles and the concentration of MT in the release media were assessed by means of HPLC analysis using a Shimadzu instrument equipped with a Prominence SIL-20AC autoinjector, a Prominence LC-20AD quaternary pump, a five channels Prominence DGU-20A5 degasser, a Prominence CTO-20AC column oven, and a Prominence SPD-M20A DAD detector (scanning range 200−360 nm, slit 1.2, acquisition frequency of 1.5265 Hz). 2.4. HPLC Analysis and Method Validation. The chromatographic method applied for assessment of microencapsulation efficiency and for in vitro release tests was developed and completely validated in terms of system suitability, method linearity, precision, and accuracy, establishing limits of detection and limits of quantification. Details on HPLC method development and validation are given in Supporting Information.

PY =

practical amount (microparticle) × 100 theoretical amount (CHT + MT)

(1)

Drug Content Evaluation. The amount of MT encapsulated in CHT microparticles was assessed by HPLC using the fully validated method described above. For this, 10 mg of each sample, accurately weighted, was dispersed in 7 mL of methanol and centrifuged for 10 min at 3900 rpm. The supernatant was quantitatively transferred into a volumetric flask of 10 mL. Then, 0.5 mL internal standard solution (1.0 mg/mL PRP) was added and completed to the mark with methanol. Microencapsulation Efficiency (ME). ME was calculated as the ratio of MT content in CHT microparticles (determined by HPLC) to the theoretical MT content and was expressed in percentages. For each sample, three sets of determinations were performed and the final results were calculated as a average of these three determinations, calculating also the standard deviation. 2.6. In Vitro Dissolution Release Study. In vitro dissolution tests were performed on the three studied MT− CHT microparticle formulations, the marketed product (Egilok, conventional tablets containing 50 mg of metoprolol tartrate) (EKG), and on unencapsulated metroprolol tartrate in order to verify and compare the release profile of MT. All tests were conducted in two different dissolution media, simulated gastric fluid (SGF) (pH 1.2) and simulated intestinal fluid (SIF) (pH 6.8), using a SR 8 Plus Series (AB & L Jasco) apparatus 2 (paddles). The experimental protocol was set as follows: 50 mg of MT, one tablet of Egilok, and microparticle samples (ranging from 180 mg to 370 mg, depending on the drug to polymer ratio and the microencapsulation efficacy) were introduced in the vessels containing 500 mL of dissolution medium with the following conditions: the bath temperature was 37 °C ± 0.5 °C; the rotation speed was 50 rpm; the sampling interval was set at 5 min during the first 30 min of the test and to 30 min for the next 4.30 h (2 mL aliquots were withdrawn and to subjected to HPLC analysis in order to determine the amount of MT released; after every sampling, these aliquots were replaced with the same medium volume at 37 °C). All the dissolution tests were made in triplicate, with the mean values reported in graphics (relative standard deviation, RSD < 5%). In order to emphasize the prolonged release of MT from the CHT−microparticulate formulations, the results of the in vitro dissolution test were compared with those obtained on commercially available product containing MT (i.e., Egilok 50 mg/tablet) by means of f 2 similarity factor, defined by the formula: ⎧⎡ ⎪ 1 f2 = 50log⎨⎢1 + ⎪⎢⎣ n ⎩

n

∑ (R t − Tt ) t=1

⎤−0.5

2⎥

⎦⎥

⎫ ⎪ × 100⎬ ⎪ ⎭

(2)

where n is the number of sampling time points, Σ is the sum of all sampling time points, Rt is the released MT, expressed as percentage, at time point t of the reference product, Tt is the released MT percentage at time point t of the test. 2.7. Analysis of in Vitro Drug Release Kinetics and Mechanism. In order to predict and correlate the in vitro drug release behavior from formulated chitosan microparticles containing MT, it is necessary to fit into a suitable mathematical model. The experimental data obtained after in vitro MT 17434



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Figure 2. SEM images of placebo−CHT (Fb) and MT−CHT microparticles (F1, F2, F3).

dissolution tests d on the new formulations were investigated by mathematical modeling using four mathematical models: zero-order and first-order kinetics, Higuchi, and Korsmeyer− Peppas models.41−43 The R2 values of these models were determined for evaluation of accuracy. The simulation analysis, plotting, and data fitting have been performed using Matlab 7.1 software.

in clusters, showing nonhomogeneous particles sizes. According to recorded SEM micrographs, the F2 microparticulate drug system containing a MT:CHT ratio of 1:2 shows the most homogeneous particle size distribution, although part of these microparticles are raspberry-like rather than spherical. The formation of microparticles of this form was previously observed for chitosan-based microspheres containing pharmaceutically active substances prepared by the spray-drying method.40 In order to verify this finding that the most homogeneous particle size distribution is obtained on F2 sample, we have subjected all samples to particle size analysis by laser diffraction. The results are shown in Figure 3. It is obvious that the initial MT:CHT ratio affects the mean size of the final microparticulate drug system. According to the results obtained by this technique, the mean size of the microparticles in the F2 formulation is 2.83 μm, very close to the mean size of the microparticles in the placebo Fb sample formulation (2.74 μm),

3. RESULTS AND DISCUSSIONS 3.1. Morphological and Particle Size Distribution Investigations. Representative SEM micrographs of placebo−CHT (Fb) and MT-encapsulated CHT microparticles (F1, F2, F3) are displayed in Figure 2. As clearly observed, in all cases, microparticles show spherical shape with flower-like surface and sizes below 5 μm, previously reported for chitosan microspheres prepared by spray-drying method.40,44 However, the sample F1, containing the higher amount of drug (MT:CHT = 1:1), is more irregular in shape and is gathered 17435



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Figure 3. Particle size distribution of CHT and MT−CHT microparticles, determined by laser diffraction.

Figure 4. DTG thermograms of MT, CHT, and prepared microparticles.

from the particle size point of view, F2 formulation is the best suited. 3.2. Thermal Behavior Investigations. The TG/DTG technique was used to investigate the thermal properties of MT substance, CHT biopolymer, placebo−CHT, and MTencapsulated CHT microparticles. The results are summarized in Figure 4 and Table 1, revealing that MT substance follows one thermal decomposition step, from 175 to 380 °C, while CHT biopolymer shows three steps of weight loss in the temperature range of 85, 299, and 565 °C. The first weight loss,

whereas the mean size of the microparticles in the F1 and F3 formulations is 5.11 and 3.13 μm, respectively. Moreover, the curve allure obtained on sample F2 fits best the Gaussian model, showing narrow particle size distribution, compared with those of sample F1 and F3, which show wider particle size distributions. It is well-known that when formulating microparticulated drug carriers, homogeneity of particles size, expressed in terms of narrow particles size distribution, is highly desirable. Thus, 17436



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Figure 5. ATR−FTIR spectra of MT substance, CHT, and MT−CHT microparticles.

at 85 °C, corresponds to the loss of adsorbed water. The second loss at a temperature around 299 °C was due to the onset of chitosan decay. The final stride of weight loss was due to complete decomposition of chitosan. The two-step weight loss in the temperature range between 55 and 200 °C in placebo−CHT microparticles (Fb) is related to the evaporation of adsorbed water and showed higher weight loss (17.69%) than simple CHT biopolymer (8.77%), which may be due to the higher water retention capacity of microparticulated chitosan. Because of their smaller particle sizes, chitosan microparticles have significantly increased surface area and implicitly greater numbers of hydroxyl groups available to bind water molecules, resulting in higher water retention capacity. Regarding the MT−CHT microparticulate formulations, the TG/DTG curve follow thermal trends of the components, with three decomposition steps; the first two follow the same trend as in placebo−CHT microparticles and occur in the temperature range between 55 and 200 °C, showing evaporation of adsorbed water. The weigh losses increase from 14.68% up to 19.57% as the amount of MT encapsulated in chitosan microparticles decreases (MT:CHT ratio goes from 1:1 in F1 to 1:4 in F3). A third decomposition step is obvious on all three MT−CHT microparticulated formulas. The temperature where the maximum weight loss occurs in this third step slightly changes from 268 °C in F1 to 294 °C in F3, because of a higher amount of chitosan. Moreover, it can be clearly observed that the intensity of this peak decreases as the amount of MT in the formula decreases, suggesting different weight losses, from 64.77% in F1 to 56.02% in F3. This behavior leads to the conclusion that different amounts of MT are indeed microencapsulated, while CHT plays an important additional role in the thermal stabilization of MT during the microencapsulation process. It can be observed that, generally, the practical residual weight at 600 °C is in good agreement with the theoretical residual weight at the same temperature.

3.3. Structural Investigations. In order to check the integrity of microencapsulated MT, FTIR spectra of MT substance, placebo−CHT microparticles, and MT−CHT microparticles were collected and compared. The IR spectrum of chitosan, displayed in Figure 5e, shows characteristic bands at 3275 (νOH), 2850 (νCH), 1620 (νNH2), 1565 (δNH), 1390 (δCH), and 1050−1030 cm−1 (νCH). This ATR−FTIR spectrum is in agreement with previously reported spectra of chitosan.45,46 In the IR spectrum of MT substance (Figure 5a), the band located at high wave numbers (≈3450 and ≈3000− 2800 cm−1) can be assigned to the absorption for the aliphatic hydroxyl group (νO−H) and to the stretching vibrations of either the NH or the CH species. Also, other spectral components are evident in the 1100−1600 cm−1 range, most likely due to (i) the stretching vibration of CC bond of the aromatic ring (≈1600 cm−1), (ii) the νOCO stretching (asymmetric and symmetric) modes of all carboxylate species (≈1580 and ≈1400 cm−1, respectively) and (iii) the asymmetric δCH3 deformation (≈1450 cm−1). The other two components located at ≈1570 and ≈1515 cm−1 are ascribable, on the basis of their spectral behavior, to the (asymmetric and symmetric) bending modes of all NH-containing species. Finally, the band at ≈820 cm−1 is usually assigned to aromatic CH bending. These results conform to previously reported IR spectra registered on metoprolol tartrate samples.47 The spectra of MT−CHT microparticles, displayed in Figure 5b,c,d appear as a combination of the characteristic spectrum of metoprolol tartrate and chitosan. Bands characteristic to chitosan (marked with *) occur at 895, 1020, 1065, 1150, 1400, and 1550 cm−1 in all spectra recorded on the three formulations. Likewise, three important bands, characteristic to MT, occur at 820, 1245, 1510, and 1585 cm−1 in all three formulations and are marked with an arrow in Figure 5. The intensity of these bands increases with the content of metoprolol in the formulated microparticles. Furthermore, due to the presence of chitosan, these bands become wider as 17437



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Table 2. Theoretical and Calculated Values Obtained for Three Sets of Determinations formula

MT:CHT gravimetric ratio

theoretical drug content (%) ± SD

practical drug content (%) ± SD

microencapsulation efficacy (%) ± SD

Fb F1 F2 F3

1:1 1:2 1:3

50 33.33 25

45.06 ± 0.17 29.06 ± 0.03 21.21 ± 0.07

90.13 ± 0.34 87.19 ± 0.09 84.85 ± 0.33

the amount of the chitosan increases in the microcapsules. Only for the F1 sample, a weak band occurs at 1109 cm−1. This band is characteristic to MT, as can be clearly seen in Figure 5a, where this band is very intense in the IR spectrum of pure MT substance. In addition, spectra recorded on CHT and MT− CHT microparticles show a broad band at ≈3275 cm−1, which is the normal range of adsorption for the aliphatic hydroxyl group from CHT. The intensity of this band is identical for the CHT and microparticle formulations, indicating that no Hbonding is formed between the drug substance and chitosan. Moreover, the presence in the spectra of the three formulations of the two bands around 1050−1030 cm−1 assigned to stretching vibration of CH from chitosan stands for the same idea. In conclusion, the spectra registered with F1, F2, and F3 samples show individual bands characteristic to both the drug substance and chitosan. 3.4. Determination of Drug Content, Microencapsulation Efficacy and Production Yield. Drug content, microencapsulation efficacy, and production yield are important parameters for evaluating the quality of developed microparticulated systems and the applicability of the formation process. The experimental results are summarized in Table 2, and they revealed an average production yield of 72%. In this study, practical drug content and microencapsulation efficacy are assessed by measuring with HPLC the concentration of MT in solution after dissolving process. Taking into account data from the microencapsulation efficiency calculations, it is obvious that losses affect MT, as well as CHT, and probably occur after the microparticle formation, in the drying chamber, where due to the adhesive properties of CHT, the alreadyformed microparticles adhere to the chamber wall. This conclusion is sustained by the result regarding the production yield of the Fb sample (that is the placebo−CHT sample). 3.5. In Vitro Dissolution Tests. Figure 6 shows the dissolution profiles for MT−CHT microparticulated drug delivery systems compared to those of the marketed product (Egilok 50 mg/tablet) and pure MT substance in simulated gastric fluid (SGF; pH = 1.2) (Figure 6A) and in simulated intestinal fluid (SIF; pH = 6.8) (Figure 6B). The results show the significant influence of the pH value of the dissolution media on the release behavior. Thus, in the SGF dissolution test, performed at pH 1.2, where CHT is soluble, the release of MT from CHT microparticles follows the same trend with commercially available Egilok product, which is considered a product with conventional release rate. Approximatively 40% of MT is released even in the first 15 min (Figure 6A), revealing a burst release effect. The solubility of CHT in this medium was easily observed, because after 5 h of testing, no microparticle agglomerates could be observed in the stirring flask. This is contrary to the observed phenomenon in SIF medium, where even after 5 h, small particle agglomerates are still visible. For all formulations, a decrease of the release rate was observed after 60 min. However, while 92% of MT was released after 60 min from Egilok formulation, a slightly sustained

production yield (%) ± SD 70.33 74.15 71.30 72.16

± ± ± ±

0.77 0.91 1.01 0.72

Figure 6. Dissolution profile for pure MT, MT−CHT microparticulated formulations, and the commercially available Egilok product in (A) SGF (pH = 1.2) and (B) SIF (pH = 6.8).

release was observed when MT−CHT microparticulate formulations were used, with more pronounced sustained release for F1 and F2 formulations (from 70% after 60 min to 85% after 300 min). Among MT−CHT microparticulated formulations, the F3 formula (that contains the highest amount of CHT) releases the highest amount of MT. On the other hand, the microparticle behavior was totally different when the test was performed in SIF media, at pH of 6.8. As Figure 6B shows, the significant decrease of the release rate is reached after 120 min and this phenomenon is assigned to the CHT insolubility in the pH 6.8 medium. The hydration of microparticles is in SIF media, compared to SGF and the encapsulated MT release from the microspheres takes place based on a diffusion process. Confirmation of this hypothesis is presented in section 3.6 (Analysis of in Vitro Drug Release Kinetics and Mechanism). Thus, the release behavior is different than that of Egilok, and the dissolution curves do not follow the same trend for MT− CHT microparticles as for marketed product. Once again, no significant differences are marked between the release behavior of F1 and F2 formulations. Moreover, the lowest amount of 17438



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MT is released by the F3 formula (81% after 300 min), in contrast to its behavior in pH = 1.2 release environment, revealing sustained-release of encapsulated MT. During the in vitro dissolution test performed in this SIF medium, the prepared microparticulate formulation had a different behavior that could be observed visually, as well. Thus, the microparticles of the F1 formulation formed aggregates/ agglomerates that moved to the flask bottom after 150 min, while the F2 and F3 type microparticles (with increased content of CHT) formed aggregates that were visible at the surface after 300 min. In conclusion, the retardation action of CHT toward the release of encapsulated MT is more obvious in this SIF medium. By presenting these experimental information, we intended to highlight the different behavior of MT−CHT microparticles in the two release media, taking into account the solubility of CHT, the MT:CHT ratio. We concluded that as the content of CHT in the formulation increases the insolubility of the microparticles is accentuated, forming visible aggregates during the entire in vitro dissolution test (300 min). In order to emphasize the prolonged release of MT from the CHT−microparticulate formulations, the results of the in vitro dissolution test were analyzed and compared with those obtained on commercially available product containing MT (i.e., Egilok 50 mg/tablet) by means of f2 similarity factor. Two dissolution profiles are considered similar if f2 value is higher than 50.48,49 As expected, the f2 obtained values show different dissolution patterns for MT release from this commercial product and the prepared MT−CHT microparticulate formulations, in both SGF and SIF media (Table 3). The only observation that should be noted is that the f2 values obtained in SIF medium (pH 6.8) are lower than the f2 values obtained in SGF medium (pH 1.2).

Table 4. Results of Curve Fitting of the In Vitro MT Release Profile from Formulated Chitosan Microparticles Korsmeyer− Peppas model

dissolution media SGF (pH 1.2)

SIF (pH 6.8)

reference formula (Rf) (Egilok)

test formula (Tt)

f2 value

1.2

F1 F2 F3 F1 F2 F3

39.821 42.767 49.273 34.385 35.750 31.155

6.8

first order model (R2)

Higuchi model (R2)

(R2)

n

F1

0.6536

0.9688

0.8429

0.9712

0.22

F2 F3 F1

0.6676 0.6476 0.7146

0.9669 0.9801 0.9821

0.8526 0.8417 0.8933

0.9743 0.9554 0.9650

0.22 0.23 0.27

F2 F3

0.7189 0.6857

0.9809 0.9925

0.8953 0.8684

0.9645 0.9360

0.27 0.28

analysis of the formulas to distinguish between release mechanisms: Fickian release (diffusion controlled release), non-Fickian (anomalous transport), and case-II transport (relaxation-controlled release). According to this model, values of n below 0.43 indicate a Fickian release. When n is approximately 0.5 indicates the pure Fickian diffusion controlled mechanism from spherical forms. Values of n between 0.5 and 1 indicate anomalous transport kinetics.50,51 The data reveal, for all three chitosan-based microparticulate formulations (F1, F2, F3) on the two pH values under study, a first-order release kinetic, because the values of R2 are higher than 0.95. The diffusion release mechanism is confirmed. The values of the exponential release coefficient n in Korsmeyer− Peppas model are ranged in the interval 0.22−0.28, defining a Fickian diffusion process, as expected. It was the purpose of this research work to prepare chitosanbased microparticles for sustained linear release of metoprolol. Therefore, it is highly desirable that the release profile fit best with the first order kinetic model, as is the case herein.

Table 3. Similarity Factor Values f2 of MT Microparticles Based on CHT pH value

formula

zero order model (R2)

4. CONCLUSIONS The goal of the present research study was to develop microparticulated therapeutic delivery systems based on chitosan for improved patient compliance on oral administration of metoprolol tartrate, a selective β1-receptor blocker used in treatment of several diseases of the cardiovascular system, especially hypertension. This goal was successfully achieved by microencapsulation of metoprolol tartrate into biocompatible chitosan matrix by means of spray-drying method. Three different formulations were prepared with different drug-to-polymer ratios: F1, F2, and F3 with a MT:CHT ratio of 1:1, 1:2, and 1:4 respectively. Before in vitro dissolution testing, the newly developed microparticulate therapeutic systems were fully investigated using SEM, laser diffraction, FTIR, and TGA in order to understand their morphology, structure, and thermal behavior. The results of the thermogravimetric analysis reveal the stability and the compatibility of MT and CHT for preparing microparticles by the spray-drying technique, in the studied experimental conditions, while FTIR measurements showed that drug molecules are physically adsorbed within microparticles, thus, its release being easily possible in the proper release environment. The microencapsulation method efficiency, expressed as percentages after the loading of the resulted microparticles, as well as the production yield, are satisfactory for all three

3.6. Analysis of In Vitro Drug Release Kinetics and Mechanism. The experimental data obtained after in vitro MT dissolution tests d on the new formulations were investigated by mathematical modeling using four mathematical models: zero-order and first-order kinetics, Higuchi, and Korsmeyer− Peppas models.41−43 The Higuchi mechanism describes the cumulative percentage of release versus square root of time dependent process based on Fickian diffusion. It is usually applied to describe drug dissolution from transdermal patches and tableted matrices with water-soluble drugs. The Krosmeyer−Peppas mechanism is a simple, semiempirical model in which diffusion is the main drug release mechanism, relating the drug release to the elapsed time exponentially. It is used to describe the drug release from microcapsules and microspheres. The results of the curve fitting into various mathematical models are summarized in Table 4. The Korsmeyer−Peppas model was employed in the in vitro drug release behavior 17439



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formulations. Thus, it is justified to conclude that this method is efficient for the preparation of microparticulate therapeutic systems containing metoprolol tartrate as the active substance and chitosan as the encapsulation material. After performing in vitro dissolution tests, it was concluded that the drug-to-polymer ratio is an important element in controlling the release features of microparticulate systems based on CHT. On the other hand, the pH of the dissolution fluid plays an important role in the release of the drug substance from the microspheres. Thus, it was observed that at pH 6.8, the MT release rate from the microparticles varies inversely with the amount of CHT in the formula. This conclusion points to an important characteristic of the MT microparticles based on CHT with prolonged release, intended for oral use, taking into account the pH of the metoprolol physiological absorption area (the superior area of the thin intestine duodenum−jejunum). The calculated values of f2 similarity factor show a prolonged profile for MT release from the microparticulate system compared to the commercially available product Egilok currently used in practical therapeutics. Additionally, the analysis of the release kinetic mechanism concluded that in SGF media as well as in SIF media, the MT from the chitosan-based microparticles is released by means of a Fickian diffusion process. These findings, corroborated with the high amount of drug encapsulated per gram of microparticles stand for less frequent administration of metoprolol tartrate when formulated as chitosan-based microparticles, without going on underdosing.

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ASSOCIATED CONTENT

S Supporting Information *

This information is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Dr. Alina Maria Tomoiaga. Phone: +40-74-922-5995. E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Author Contributions ¶

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ABBREVIATIONS MT = metoprolol tartrate CHT = chitosan SEM = scanning electronic microscopy TGA/DSC = thermogravimetric analysis/differential scanning calorimetry FTIR = Fourier transform infrared spectroscopy HPLC = high performance liquid chromatography SGF = simulated gastric fluid SIF = simulated intestinal fluid

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REFERENCES

(1) U.S.Pharmacopeial Medicines Compendium. https://mc.usp. org/monographs/metoprolol (accessed Dec 9, 2012). 17440



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