microencapsulation of metoprolol tartrate into chitosan ...stoleriu/iecr.pdf · improved oral...

Microencapsulation of Metoprolol Tartrate into Chitosan forImproved Oral Administration and Patient ComplianceLacramioara Ochiuz,†,¶ Gratiela Popa,† Iulian Stoleriu,‡ Alina Maria Tomoiaga,*,§,¶ 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 carriersystems for the sustained release of orally administered drugs. In the current study, we followed a microencapsulation techniquebased 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 behaviorby optical microscopy, infrared spectroscopy, and thermogravimetric analysis. Microencapsulation efficiency and drug contentwere assessed by a validated HPLC method. In vitro dissolution tests performed in simulated gastric fluid (SGF) (pH 1.2) andsimulated intestinal fluid (SIF) (pH 6.8) revealed that the drug-to-polymer ratio is an important element in controlling therelease features of microparticulate systems based on CHT. Also, the pH of the dissolution fluid plays an important role in therelease of the drug substance from the microspheres. Additionally, the analysis of the release kinetic mechanism concluded that inSGF media as well as in SIF media, the MT from the chitosan-based microparticles is released by means of a Fickian diffusionprocess.

1. INTRODUCTION

In the past decade, more and more people suffer from variousforms of cardiovascular diseases, affecting the human longevity.Thus, tremendous efforts are made by chemists, pharmacists,and medical doctors worldwide for finding new deliveryapproaches, new modes of action, and new pharmaceuticallyactive substances to treat these life-threatening illnesses.Metoprolol tartrate (MT) with the chemical formula

(C15H25NO3)2C4H6O6 (Figure 1A) is a β1-selective andrenergicblocker 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 water-soluble and permeable drug.4 Because the biological half-life ofMT ranges to 3−4 h,1 multiple doses are needed to maintain aconstant plasma concentration for a good therapeutic response.It has also been reported that MT absorption in the duodenumand jejunum is directly proportional to the dose availability.5,6

The conventional oral administration dosage form fails tomaintain the drug plasma concentration over the extendedperiod of time, resulting in frequent administration of the drugwith a higher dose, causing unwanted toxic effects. Beside this,if the entire amount of drug is released at once, dose dumpingoccurs and a low therapeutic response is obtained. Therefore,the success of a drug delivery formulation depends on theability to build a biocompatible carrier, capable of high drug

Received: August 10, 2013Revised: October 31, 2013Accepted: November 5, 2013Published: November 5, 2013

Figure 1. (A) Chemical structure of metoprolol and (B) chemicalstructural representation of chitin and chitosan depicting thecopolymer character of the biopolymers.

Article

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

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loadings and controlled release, thus improving patientcompliance and convenience.Biopolymer-based microparticles are good examples of

modern therapeutic systems that offer many advantages suchas decreased adverse secondary effects, controlled/sustainedrelease of the active agent, resulting in reduced administrationfrequencies and optimization of the therapeutic effect.7−10

Thus, the formulation of MT as sustained release micro-particulate drug delivery systems seems one of the most feasibleideas to overcome some of the problems concerning itspharmacological availability. Various polymers have beenevaluated to develop therapeutic microsystems capable ofdelivering drugs in a controlled manner over a prolongedperiod. Among them, chitosan (CHT), a natural linearpolysaccharide obtained from the deacetylation of chitin11

(Figure 1B), has many advantages that are highly important forthe pharmaceutical development field, namely its biodegrad-ability and biocompatibility,12−15 its ability to control therelease of drug substance,12,16−19 its solubility in aqueous acidicsolutions, thus avoiding the use of hazardous organic solventswhile obtaining particles,20 its excellent surface chemistry basedon a high number of free amino groups that are readily availablefor cross-linking,21−24 and its cationic nature that allows ioniccross-linking with multivalent anions.25 Over the years, CHThas been used in the preparation of mucoadhesive formula-tions26,27 in order to improve the dissolution rate of poorlysoluble drugs,28 for drug targeting,19,29,30 for pH-stimulateddrug delivery,31 for enhancement of poorly absorbable drug andpeptide absorption,31−34 and as microcarriers for nanoparticledelivery.16 There are few previous reports on the preparation ofmetoprolol-encapsulated microparticles for various pharma-ceutical applications. For example, Shabaraya reported thepreparation of metoprolol tartrate-loaded microspheres byphase separation−emulsification technique.35 Palanisamy re-ported the preparation of metoprolol succinate chitosan-basedmicrospheres by a cross-linking method.36 The encapsulationefficiency in this case was only 65−70%, and the size of themicrospheres was of 330 − 527 nm. In 2013, Adi andcollaborators reported the preparation of metoprolol tartratechitosan-based microspheres by ionic precipitation andchemical cross-linking method.37 The preparation method

was proven to be very efficient, and the resultant microsphereswere used for intranasal delivery of MT. Spray drying is widelyused method for the preparation of microparticulate drugdelivery systems. In the past few years, this method has beenintensively investigated mainly for aqueous polymeric for-mulations as an alternative to the conventional methods thatgenerally use organic solvents, which involves a high risk ofproduct contamination, toxicity, and explosion hazards, aswell.38 It has several advantages including high reliability,reproducibility, and control of particle size.39 More, chitosanmicroparticles prepared by the spray-drying method arecharacterized by high sphericity and specific surface area,parameters that are very important for the research field dealingwith drug development.40

Therefore we have chosen to prepare chitosan-basedmicroparticles encapsulating metoprolol tartrate by means ofthe spray-drying method using different ratios of MT:CHT.The newly formulated CHT−MT therapeutic microsystemswere investigated using SEM, FTIR, and TGA/DSC techniquesin order to monitor the modifications occurred at micro-encapsulation of various amounts of MT into chitosan.Production yield, microencapsulation efficiency, and drugcontent were also assessed by means of gravimetric andHPLC measurements. Fully validated HPLC method was used(see Supporting Information for details). In vitro release studieswere performed in order to establish the dissolution profiles ofnewly formulated microsystems.

2. MATERIALS AND METHODS

2.1. Materials. Metoprolol tartrate, propranolol hydro-chloridum, and practical-grade chitosan with a degree ofdeacetylation >85% were supplied by Sigma-Aldrich, Germany.Chromatographic-grade methylic alcohol and triethanolaminewere purchased from Merck. Metoprolol tartrate CRS wasacquired from EDQM, France. Ammonium acetate, glacialacetic acid, and phosphoric acid were kindly supplied byChemical Company, Romania. Metoprolol tartrate tablets(Egilok 50 mg/tablet, Egis Pharmaceuticals, Hungary) werepurchased from a local pharmacy. All chemicals were ofanalytical grade and used as received.

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

sample stage Tonset Tpeak Tendset Wloss (%) Mean ± SD Wres (%) Mean ± SD Wtheo (%)

MT I 200 284 378 91.98 ± 0.84 8.01 ± 0.82CHT I 61 85 114 8.77 ± 0.18 29.37 ± 0.38

II 272 299 328 46.21 ± 0.71III 494 565 15.65 ± 0.32

Fb I 55 82 108 11.85 ± 0.25 31.14 ± 0.21II 152 174 200 5.84 ± 0.14III 270 289 399 51.17 ± 0.19

F1 I 59 75 100 7.22 ± 0.32 20.37 ± 0.66 18.74II 140 155 210 7.64 ± 0.80III 210 268 451 64.77 ± 0.94

F2 I 52 77 98 8.32 ± 1.02 24.27 ± 8.84 22.19II 140 155 210 7.64 ± 0.80III 216 290 412 61.40 ± 0.87

F3 I 60 78 104 11.52 ± 0.72 24.31 ± 0.69 24.35II 140 152 222 8.05 ± 0.53III 222 294 470 56.12 ± 0.79

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

Industrial & Engineering Chemistry Research Article

dx.doi.org/10.1021/ie402625h | Ind. Eng. Chem. Res. 2013, 52, 17432−1744117433

2.2. Preparation of Chitosan-Based MicroparticlesContaining Metoprolol Tartrate. Three different formula-tions of CHT−MT microparticles are prepared with variousdrug-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 vigorouscontinuous stirring, resulting in solutions containing variabledrug-to-polymer ratios. Further, the resulting solutions aresprayed through the nozzle of a Mini Spray Dryer B29 (Buchi,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 therotameter); nozzle cleaner, 3. A placebo microparticle formulawas prepared under the same condition by spraying a CHTsolution 1% (w/v). Through the whole procedure, temperatureand humidity were controlled and maintained at 23 ± 1 °C and55 ± 2% relative humidity. Each formulation was carried out intriplicate.2.3. Characterization Methods and Equipment. The

morphological characteristics of chitosan and MT-encapsulatedchitosan microparticles were examined using a TESCAN VEGAII SBH scanning electron microscope (SEM). Each micro-particulated sample was coated with a 15 nm layer of gold bymeans of cathode pulverization. Imaging parameters (kV,magnification, etc.) can be visualized on the registeredmicrographs. The mean microparticles diameter and the sizedistribution were assessed by laser diffraction using aSHIMADZU−SALD 7001 particle size analyzer. All measure-ments were carried out in triplicate on suspensions of particlesin acetone after thorough sonication. Thermal stability of MT,CHT, and prepared microparticles was investigated with aMettler Toledo TGA-SDTA 851e derivatograph. All measure-ments were performed in an inert atmosphere under constantpurging of nitrogen with a 20 mL/min flow rate. Thermogravi-metric data were recorded in the temperature range of 25−600°C, with a heating speed of 10 °C/min. A thermal analysis indynamic conditions (TG, DTG, and DTA) was also performedand Star software was used for data investigation. Infraredspectra were recorded at room temperature on an ATR−FTIRspectrometer (attenuated total reflectance−Fourier transforminfrared), model IdentifyIR from SmithDetection. The sampleswere measured as powders, with no previous dilution in KBr.The scans were collected with a resolution of 4 cm−1 over thespectral range 600 − 4000 cm−1.The MT loading within CHT microparticles and the

concentration of MT in the release media were assessed bymeans of HPLC analysis using a Shimadzu instrumentequipped with a Prominence SIL-20AC autoinjector, aProminence LC-20AD quaternary pump, a five channelsProminence DGU-20A5 degasser, a Prominence CTO-20ACcolumn oven, and a Prominence SPD-M20A DAD detector(scanning range 200−360 nm, slit 1.2, acquisition frequency of1.5265 Hz).2.4. HPLC Analysis and Method Validation. The

chromatographic method applied for assessment of micro-encapsulation efficiency and for in vitro release tests wasdeveloped and completely validated in terms of systemsuitability, method linearity, precision, and accuracy, establish-ing limits of detection and limits of quantification. Details onHPLC method development and validation are given inSupporting Information.

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

=+

×PYpractical amount (microparticle)

theoretical amount (CHT MT)100

(1)

Drug Content Evaluation. The amount of MT encapsulatedin CHT microparticles was assessed by HPLC using the fullyvalidated method described above. For this, 10 mg of eachsample, accurately weighted, was dispersed in 7 mL of methanoland centrifuged for 10 min at 3900 rpm. The supernatant wasquantitatively transferred into a volumetric flask of 10 mL.Then, 0.5 mL internal standard solution (1.0 mg/mL PRP) wasadded and completed to the mark with methanol.

Microencapsulation Efficiency (ME). ME was calculated asthe ratio of MT content in CHT microparticles (determined byHPLC) to the theoretical MT content and was expressed inpercentages. For each sample, three sets of determinations wereperformed and the final results were calculated as a average ofthese three determinations, calculating also the standarddeviation.

2.6. In Vitro Dissolution Release Study. In vitrodissolution tests were performed on the three studied MT−CHT microparticle formulations, the marketed product(Egilok, conventional tablets containing 50 mg of metoprololtartrate) (EKG), and on unencapsulated metroprolol tartrate inorder to verify and compare the release profile of MT. All testswere conducted in two different dissolution media, simulatedgastric 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 asfollows: 50 mg of MT, one tablet of Egilok, and microparticlesamples (ranging from 180 mg to 370 mg, depending on thedrug to polymer ratio and the microencapsulation efficacy)were introduced in the vessels containing 500 mL of dissolutionmedium with the following conditions: the bath temperaturewas 37 °C ± 0.5 °C; the rotation speed was 50 rpm; thesampling interval was set at 5 min during the first 30 min of thetest and to 30 min for the next 4.30 h (2 mL aliquots werewithdrawn and to subjected to HPLC analysis in order todetermine the amount of MT released; after every sampling,these aliquots were replaced with the same medium volume at37 °C). All the dissolution tests were made in triplicate, withthe mean values reported in graphics (relative standarddeviation, RSD < 5%). In order to emphasize the prolongedrelease of MT from the CHT−microparticulate formulations,the results of the in vitro dissolution test were compared withthose obtained on commercially available product containingMT (i.e., Egilok 50 mg/tablet) by means of f 2 similarity factor,defined by the formula:

∑= + − ×=

−⎧⎨⎪⎩⎪

⎡⎣⎢⎢

⎤⎦⎥⎥

⎫⎬⎪⎭⎪

fn

R T50log 11

( ) 100t

n

t t21

20.5

(2)

where n is the number of sampling time points, Σ is the sum ofall sampling time points, Rt is the released MT, expressed aspercentage, at time point t of the reference product, Tt is thereleased MT percentage at time point t of the test.

2.7. Analysis of in Vitro Drug Release Kinetics andMechanism. In order to predict and correlate the in vitro drugrelease behavior from formulated chitosan microparticlescontaining MT, it is necessary to fit into a suitable mathematicalmodel. The experimental data obtained after in vitro MT



dissolution tests d on the new formulations were investigatedby 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 weredetermined for evaluation of accuracy. The simulation analysis,plotting, and data fitting have been performed using Matlab 7.1software.

3. RESULTS AND DISCUSSIONS

3.1. Morphological and Particle Size DistributionInvestigations. Representative SEM micrographs of place-bo−CHT (Fb) and MT-encapsulated CHT microparticles (F1,F2, F3) are displayed in Figure 2. As clearly observed, in allcases, microparticles show spherical shape with flower-likesurface and sizes below 5 μm, previously reported for chitosanmicrospheres 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

in clusters, showing nonhomogeneous particles sizes. Accordingto recorded SEM micrographs, the F2 microparticulate drugsystem containing a MT:CHT ratio of 1:2 shows the mosthomogeneous particle size distribution, although part of thesemicroparticles are raspberry-like rather than spherical. Theformation of microparticles of this form was previouslyobserved for chitosan-based microspheres containing pharma-ceutically active substances prepared by the spray-dryingmethod.40

In order to verify this finding that the most homogeneousparticle size distribution is obtained on F2 sample, we havesubjected all samples to particle size analysis by laser diffraction.The results are shown in Figure 3. It is obvious that the initialMT:CHT ratio affects the mean size of the final micro-particulate drug system. According to the results obtained bythis technique, the mean size of the microparticles in the F2formulation is 2.83 μm, very close to the mean size of themicroparticles in the placebo Fb sample formulation (2.74 μm),

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



whereas the mean size of the microparticles in the F1 and F3formulations is 5.11 and 3.13 μm, respectively. Moreover, the

curve allure obtained on sample F2 fits best the Gaussianmodel, showing narrow particle size distribution, compared

with those of sample F1 and F3, which show wider particle sizedistributions.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,

from the particle size point of view, F2 formulation is the bestsuited.

3.2. Thermal Behavior Investigations. The TG/DTGtechnique was used to investigate the thermal properties of MTsubstance, CHT biopolymer, placebo−CHT, and MT-encapsulated CHT microparticles. The results are summarizedin Figure 4 and Table 1, revealing that MT substance followsone thermal decomposition step, from 175 to 380 °C, whileCHT biopolymer shows three steps of weight loss in thetemperature range of 85, 299, and 565 °C. The first weight loss,

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.



at 85 °C, corresponds to the loss of adsorbed water. Thesecond loss at a temperature around 299 °C was due to theonset of chitosan decay. The final stride of weight loss was dueto complete decomposition of chitosan. The two-step weightloss in the temperature range between 55 and 200 °C inplacebo−CHT microparticles (Fb) is related to the evaporationof adsorbed water and showed higher weight loss (17.69%)than simple CHT biopolymer (8.77%), which may be due tothe higher water retention capacity of microparticulatedchitosan. Because of their smaller particle sizes, chitosanmicroparticles have significantly increased surface area andimplicitly greater numbers of hydroxyl groups available to bindwater molecules, resulting in higher water retention capacity.Regarding the MT−CHT microparticulate formulations, theTG/DTG curve follow thermal trends of the components, withthree decomposition steps; the first two follow the same trendas in placebo−CHT microparticles and occur in the temper-ature range between 55 and 200 °C, showing evaporation ofadsorbed water. The weigh losses increase from 14.68% up to19.57% as the amount of MT encapsulated in chitosanmicroparticles decreases (MT:CHT ratio goes from 1:1 in F1to 1:4 in F3). A third decomposition step is obvious on all threeMT−CHT microparticulated formulas. The temperature wherethe maximum weight loss occurs in this third step slightlychanges from 268 °C in F1 to 294 °C in F3, because of a higheramount of chitosan. Moreover, it can be clearly observed thatthe intensity of this peak decreases as the amount of MT in theformula decreases, suggesting different weight losses, from64.77% in F1 to 56.02% in F3. This behavior leads to theconclusion that different amounts of MT are indeed micro-encapsulated, while CHT plays an important additional role inthe thermal stabilization of MT during the microencapsulationprocess.It can be observed that, generally, the practical residual

weight at 600 °C is in good agreement with the theoreticalresidual weight at the same temperature.

3.3. Structural Investigations. In order to check theintegrity of microencapsulated MT, FTIR spectra of MTsubstance, placebo−CHT microparticles, and MT−CHTmicroparticles were collected and compared. The IR spectrumof chitosan, displayed in Figure 5e, shows characteristic bandsat 3275 (νOH), 2850 (νCH), 1620 (νNH2

), 1565 (δNH),1390 (δCH), and 1050−1030 cm−1 (νCH). This ATR−FTIRspectrum is in agreement with previously reported spectra ofchitosan.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 aliphatichydroxyl group (νO−H) and to the stretching vibrations of eitherthe NH or the CH species. Also, other spectral componentsare evident in the 1100−1600 cm−1 range, most likely due to(i) the stretching vibration of CC bond of the aromatic ring(≈1600 cm−1), (ii) the νOCO stretching (asymmetric andsymmetric) modes of all carboxylate species (≈1580 and≈1400 cm−1, respectively) and (iii) the asymmetric δCH3

deformation (≈1450 cm−1). The other two componentslocated at ≈1570 and ≈1515 cm−1 are ascribable, on thebasis of their spectral behavior, to the (asymmetric andsymmetric) bending modes of all NH-containing species.Finally, the band at ≈820 cm−1 is usually assigned to aromaticCH bending. These results conform to previously reportedIR spectra registered on metoprolol tartrate samples.47 Thespectra of MT−CHT microparticles, displayed in Figure 5b,c,dappear as a combination of the characteristic spectrum ofmetoprolol tartrate and chitosan. Bands characteristic tochitosan (marked with *) occur at 895, 1020, 1065, 1150,1400, and 1550 cm−1 in all spectra recorded on the threeformulations. Likewise, three important bands, characteristic toMT, occur at 820, 1245, 1510, and 1585 cm−1 in all threeformulations and are marked with an arrow in Figure 5. Theintensity of these bands increases with the content ofmetoprolol in the formulated microparticles. Furthermore,due to the presence of chitosan, these bands become wider as

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



the amount of the chitosan increases in the microcapsules. Onlyfor the F1 sample, a weak band occurs at 1109 cm−1. This bandis characteristic to MT, as can be clearly seen in Figure 5a,where this band is very intense in the IR spectrum of pure MTsubstance. In addition, spectra recorded on CHT and MT−CHT microparticles show a broad band at ≈3275 cm−1, whichis the normal range of adsorption for the aliphatic hydroxylgroup from CHT. The intensity of this band is identical for theCHT and microparticle formulations, indicating that no H-bonding is formed between the drug substance and chitosan.Moreover, the presence in the spectra of the three formulationsof the two bands around 1050−1030 cm−1 assigned tostretching vibration of CH from chitosan stands for thesame idea. In conclusion, the spectra registered with F1, F2,and F3 samples show individual bands characteristic to both thedrug substance and chitosan.3.4. Determination of Drug Content, Microencapsu-

lation Efficacy and Production Yield. Drug content,microencapsulation efficacy, and production yield are importantparameters for evaluating the quality of developed micro-particulated systems and the applicability of the formationprocess. The experimental results are summarized in Table 2,and they revealed an average production yield of 72%. In thisstudy, practical drug content and microencapsulation efficacyare assessed by measuring with HPLC the concentration of MTin solution after dissolving process. Taking into account datafrom the microencapsulation efficiency calculations, it isobvious that losses affect MT, as well as CHT, and probablyoccur after the microparticle formation, in the drying chamber,where due to the adhesive properties of CHT, the already-formed microparticles adhere to the chamber wall. Thisconclusion is sustained by the result regarding the productionyield 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 drugdelivery systems compared to those of the marketed product(Egilok 50 mg/tablet) and pure MT substance in simulatedgastric fluid (SGF; pH = 1.2) (Figure 6A) and in simulatedintestinal fluid (SIF; pH = 6.8) (Figure 6B). The results showthe significant influence of the pH value of the dissolutionmedia on the release behavior. Thus, in the SGF dissolutiontest, performed at pH 1.2, where CHT is soluble, the release ofMT from CHT microparticles follows the same trend withcommercially available Egilok product, which is considered aproduct with conventional release rate. Approximatively 40% ofMT is released even in the first 15 min (Figure 6A), revealing aburst release effect. The solubility of CHT in this medium waseasily observed, because after 5 h of testing, no microparticleagglomerates could be observed in the stirring flask. This iscontrary to the observed phenomenon in SIF medium, whereeven 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 releasedafter 60 min from Egilok formulation, a slightly sustained

release was observed when MT−CHT microparticulateformulations were used, with more pronounced sustainedrelease for F1 and F2 formulations (from 70% after 60 min to85% after 300 min). Among MT−CHT microparticulatedformulations, the F3 formula (that contains the highest amountof 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 of6.8. As Figure 6B shows, the significant decrease of the releaserate is reached after 120 min and this phenomenon is assignedto the CHT insolubility in the pH 6.8 medium. The hydrationof microparticles is in SIF media, compared to SGF and theencapsulated MT release from the microspheres takes placebased on a diffusion process. Confirmation of this hypothesis ispresented in section 3.6 (Analysis of in Vitro Drug ReleaseKinetics 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, nosignificant differences are marked between the release behaviorof F1 and F2 formulations. Moreover, the lowest amount of

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

formulaMT:CHT gravimetric

ratiotheoretical drug content (%) ±

SDpractical drug content (%) ±

SDmicroencapsulation efficacy (%) ±

SDproduction yield (%) ±

SD

Fb 70.33 ± 0.77F1 1:1 50 45.06 ± 0.17 90.13 ± 0.34 74.15 ± 0.91F2 1:2 33.33 29.06 ± 0.03 87.19 ± 0.09 71.30 ± 1.01F3 1:3 25 21.21 ± 0.07 84.85 ± 0.33 72.16 ± 0.72

Figure 6. Dissolution profile for pure MT, MT−CHT micro-particulated formulations, and the commercially available Egilokproduct in (A) SGF (pH = 1.2) and (B) SIF (pH = 6.8).



MT is released by the F3 formula (81% after 300 min), incontrast 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 adifferent 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 increasedcontent of CHT) formed aggregates that were visible at thesurface after 300 min. In conclusion, the retardation action ofCHT toward the release of encapsulated MT is more obviousin this SIF medium. By presenting these experimentalinformation, we intended to highlight the different behaviorof MT−CHT microparticles in the two release media, takinginto account the solubility of CHT, the MT:CHT ratio. Weconcluded that as the content of CHT in the formulationincreases the insolubility of the microparticles is accentuated,forming visible aggregates during the entire in vitro dissolutiontest (300 min).In order to emphasize the prolonged release of MT from the

CHT−microparticulate formulations, the results of the in vitrodissolution test were analyzed and compared with thoseobtained on commercially available product containing MT(i.e., Egilok 50 mg/tablet) by means of f2 similarity factor. Twodissolution profiles are considered similar if f2 value is higherthan 50.48,49

As expected, the f2 obtained values show different dissolutionpatterns for MT release from this commercial product and theprepared MT−CHT microparticulate formulations, in bothSGF and SIF media (Table 3). The only observation thatshould 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).

3.6. Analysis of In Vitro Drug Release Kinetics andMechanism. The experimental data obtained after in vitro MTdissolution tests d on the new formulations were investigatedby mathematical modeling using four mathematical models:zero-order and first-order kinetics, Higuchi, and Korsmeyer−Peppas models.41−43 The Higuchi mechanism describes thecumulative percentage of release versus square root of timedependent process based on Fickian diffusion. It is usuallyapplied to describe drug dissolution from transdermal patchesand tableted matrices with water-soluble drugs. The Kros-meyer−Peppas mechanism is a simple, semiempirical model inwhich diffusion is the main drug release mechanism, relatingthe drug release to the elapsed time exponentially. It is used todescribe the drug release from microcapsules and microspheres.The results of the curve fitting into various mathematical

models are summarized in Table 4. The Korsmeyer−Peppasmodel was employed in the in vitro drug release behavior

analysis of the formulas to distinguish between releasemechanisms: Fickian release (diffusion controlled release),non-Fickian (anomalous transport), and case-II transport(relaxation-controlled release). According to this model, valuesof n below 0.43 indicate a Fickian release. When n isapproximately 0.5 indicates the pure Fickian diffusioncontrolled mechanism from spherical forms. Values of nbetween 0.5 and 1 indicate anomalous transport kinetics.50,51

The data reveal, for all three chitosan-based microparticulateformulations (F1, F2, F3) on the two pH values under study, afirst-order release kinetic, because the values of R2 are higherthan 0.95. The diffusion release mechanism is confirmed. Thevalues of the exponential release coefficient n in Korsmeyer−Peppas model are ranged in the interval 0.22−0.28, defining aFickian diffusion process, as expected.It was the purpose of this research work to prepare chitosan-

based microparticles for sustained linear release of metoprolol.Therefore, it is highly desirable that the release profile fit bestwith the first order kinetic model, as is the case herein.

4. CONCLUSIONSThe goal of the present research study was to developmicroparticulated therapeutic delivery systems based onchitosan for improved patient compliance on oral admin-istration of metoprolol tartrate, a selective β1-receptor blockerused in treatment of several diseases of the cardiovascularsystem, especially hypertension. This goal was successfullyachieved by microencapsulation of metoprolol tartrate intobiocompatible chitosan matrix by means of spray-dryingmethod. Three different formulations were prepared withdifferent drug-to-polymer ratios: F1, F2, and F3 with aMT:CHT ratio of 1:1, 1:2, and 1:4 respectively. Before invitro dissolution testing, the newly developed microparticulatetherapeutic systems were fully investigated using SEM, laserdiffraction, FTIR, and TGA in order to understand theirmorphology, structure, and thermal behavior. The results of thethermogravimetric analysis reveal the stability and thecompatibility of MT and CHT for preparing microparticlesby the spray-drying technique, in the studied experimentalconditions, while FTIR measurements showed that drugmolecules are physically adsorbed within microparticles, thus,its release being easily possible in the proper releaseenvironment.The microencapsulation method efficiency, expressed as

percentages after the loading of the resulted microparticles, aswell as the production yield, are satisfactory for all three

Table 3. Similarity Factor Values f2 of MT MicroparticlesBased on CHT

pH value test formula (Tt) f2 value

reference formula (Rf) (Egilok) 1.2 F1 39.821F2 42.767F3 49.273

6.8 F1 34.385F2 35.750F3 31.155

Table 4. Results of Curve Fitting of the In Vitro MT ReleaseProfile from Formulated Chitosan Microparticles

Korsmeyer−Peppas model

dissolutionmedia formula

zeroordermodel(R2)

firstordermodel(R2)

Higuchimodel(R2) (R2) n

SGF (pH1.2)

F1 0.6536 0.9688 0.8429 0.9712 0.22

F2 0.6676 0.9669 0.8526 0.9743 0.22F3 0.6476 0.9801 0.8417 0.9554 0.23

SIF (pH6.8)

F1 0.7146 0.9821 0.8933 0.9650 0.27

F2 0.7189 0.9809 0.8953 0.9645 0.27F3 0.6857 0.9925 0.8684 0.9360 0.28



formulations. Thus, it is justified to conclude that this methodis efficient for the preparation of microparticulate therapeuticsystems containing metoprolol tartrate as the active substanceand chitosan as the encapsulation material.After performing in vitro dissolution tests, it was concluded

that the drug-to-polymer ratio is an important element incontrolling the release features of microparticulate systemsbased on CHT. On the other hand, the pH of the dissolutionfluid plays an important role in the release of the drugsubstance from the microspheres. Thus, it was observed that atpH 6.8, the MT release rate from the microparticles variesinversely with the amount of CHT in the formula. Thisconclusion points to an important characteristic of the MTmicroparticles based on CHT with prolonged release, intendedfor oral use, taking into account the pH of the metoprololphysiological absorption area (the superior area of the thinintestine duodenum−jejunum). The calculated values of f2similarity factor show a prolonged profile for MT releasefrom the microparticulate system compared to the commer-cially available product Egilok currently used in practicaltherapeutics. Additionally, the analysis of the release kineticmechanism concluded that in SGF media as well as in SIFmedia, the MT from the chitosan-based microparticles isreleased by means of a Fickian diffusion process. Thesefindings, corroborated with the high amount of drugencapsulated per gram of microparticles stand for less frequentadministration of metoprolol tartrate when formulated aschitosan-based microparticles, without going on underdosing.

■ ASSOCIATED CONTENT*S Supporting InformationThis information is available free of charge via the Internet athttp://pubs.acs.org/.

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

Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.Author Contributions¶These authors contributed equally.

NotesThe authors declare no competing financial interest.

■ ABBREVIATIONSMT = metoprolol tartrateCHT = chitosanSEM = scanning electronic microscopyTGA/DSC = thermogravimetric analysis/differential scan-ning calorimetryFTIR = Fourier transform infrared spectroscopyHPLC = high performance liquid chromatographySGF = simulated gastric fluidSIF = simulated intestinal fluid

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