2014

http://informahealthcare.com/mncISSN: 0265-2048 (print), 1464-5246 (electronic)

J Microencapsul, 2014; 31(1): 1622! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2013.799242

ORIGINAL ARTICLE

Polymer encapsulation of amoxicillin microparticles by SAS process

A. Montes, E. Baldauf, M. D. Gordillo, C. M. Pereyra and E. J. Martnez de la Ossa

Department of Chemical Engineering and Food Technology, University of Cadiz, Puerto Real (Cadiz), Spain

Abstract

Encapsulation of amoxicillin (AMC) with ethyl cellulose (EC) by a supercritical antisolventprocess (SAS) was investigated. AMC microparticles obtained previously by an SAS processwere used as host particles and EC, a biodegradable polymer used for the controlled release ofdrugs, was chosen as the coating material. In this work, a suspension of AMC microparticles in asolution of ethyl cellulose in dichloromethane (DCM) was sprayed through a nozzle intosupercritical CO2. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS)and HPLC analyses were carried out. The effects of AMC:EC ratio, the initial polymerconcentration of the solution, temperature and pressure on the encapsulation process wereinvestigated. Although all the experiments led to powder precipitation, the AMC encapsulationwas achieved in only half of the cases, particularly when the lower drug:polymer ratios wereassayed. In general, it was observed that the percentages of AMC present in the precipitateswere higher on increasing the AMC:EC ratio. In these cases composites rather than encapsulateswere obtained. The in vitro release profiles of the resulting materials were evaluated in order toascertain whether composites can be used as encapsulated systems for drug delivery systems.

Keywords

Coating, ethyl cellulose, microparticles,supercritical antisolvent

History

Received 13 November 2012Revised 13 February 2013Accepted 28 March 2013Published online 23 May 2013

Introduction

Microparticle encapsulation has been a field of interest for someconsiderable time. The multiple applications of encapsulatedmaterials in the pharmaceutical, electronic, food, cosmetic, textileand biomedical industries make this kind of process veryattractive. In particular, in the pharmaceutical field, micro- andnano-encapsulates provide the benefits of protection from rapiddegradation, increased capability to pass materials throughphysiological membranes and allow targeting of the organ, cellor tissue where the drug must act. Furthermore, this approach canmask the taste and/or odor of a material and enable control ofactive substance delivery of pharmaceutical compounds (Mi-Kyeong et al., 2010).

Conventional techniques for the encapsulation of microparti-cles are usually associated with adverse environmental impact dueto the large amounts of organic solvents, surfactants and otheradditives used, leading to volatile organic compound emissionsand other waste streams (Wang et al., 2005; Esmaeili et al., 2012).In addition, the combination of high temperatures and chemicalsrequired in the majority of these processes can lead to degradationof the encapsulates.

An alternative to the conventional encapsulation process is theapplication of supercritical fluids, an approach that has previouslyproven to be successful. In particular, a large number of studiesconcerning the encapsulation of microparticles with polymersusing the supercritical antisolvent process (SAS) have been

reported (Tu et al., 2002; Liu et al., 2005; Kalogiannis et al.,2006; Kalani et al., 2011; Zu et al., 2011).

The SAS technique is applicable for encapsulation due to thelow solubility of pharmaceutical compounds in supercriticalfluids once they are dissolved in the organic phase. Thus,supercritical fluids dissolve the organic solvents and cause theprecipitation of the pharmaceutical compound in question.

Ethyl cellulose (EC), a polymer widely used for the controlledrelease of pharmaceuticals, is the selected carrier for AMCencapsulation because it fulfills all the requirements of thepharmaceutical field, including biocompatibility, non-toxicityand suitable resistance to preserve the properties and the activityof the active substance. AMC was used as a model antibioticbecause it is one of the most widely prescribed drugs of its type.Kalogiannis studied the encapsulation of AMC with poly(L-lacticacid) (PLLA) using SEDS (solution enhanced dispersion bysupercritical fluid) process: At 200 bar and 323.15 K the loadingpercentages and efficiencies were higher, but with AMC outsidesurface (Kalogiannis et al., 2006). In previous studies, coprecipita-tion of amoxicillin with EC was carried out (Montes et al., 2011)and the resulting composites were produced by the simultaneousprecipitation of the drug and the polymer, leading to a dispersion ofparticles of the drug into a matrix of polymer. In this case the drugwas not into the core and the polymer was not the coating materialsince there is no core-shell morphology. Spherical microparticleswere obtained in most of the composites except when the SASexperiment was carried out at the highest temperature, in whichcase agglomerates of particles formed by irregular blocks wereobserved. Other authors have carried successfully out severalcomposite precipitations using the SAS process (Elvassore et al.,2001; Moneghini et al., 2001; Kang et al., 2008).

In the work described here, it was aimed to obtain encapsulatesin which the coating material was precipitated as a shell over a

Address for correspondence: A. Montes, Department of ChemicalEngineering and Food Technology, Faculty of Sciences, University ofCadiz, International Excellence Agrifood Campus (CeiA3), 11510 PuertoReal (Cadiz), Spain. Tel: +34-956-016-458. Fax: +34-956-016-411.E-mail: antonio.montes@uca.es

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core material particle (Cocero et al., 2009). In this case the AMCparticles would be surrounded by an ethyl cellulose layer. For thispurpose, spherical AMC microparticles were used that had beensuccessfully precipitated from N-methylpyrrolidone (NMP) by anSAS process (Montes et al., 2010). These microparticles weresuspended in a polymer solution and then sprayed into thechamber by a peristaltic pump, in contrast to the coprecipitationprocess where a solution of both solutes is sprayed into the vessel.

Materials and methods

Amoxicillin (AMC) (purity 97%), ethyl cellulose (EC) (4849.5% ethoxy content) and dichloromethane (DCM) (purity99.5%) were purchased from Sigma-Aldrich Chemical (Spain).CO2 with a minimum purity of 99.8% was supplied by Linde(Spain). SEM pictures of the amoxicillin and ethyl cellulose areshown in Figures 1 and 2, respectively. Amoxicillin and ethylcellulose microparticles precipitated previously by an SASprocess are shown in Figure 3. The amoxicillin microparticlesshown in Figure 3 were used to prepare the suspensions assayed.

The experiments were carried out in pilot plant developed byThar Technologies (model SAS 200). A schematic diagram of thisequipment is shown in Figure 4. This plant was explained in detailin a previous work (Tenorio et al., 2007). The SAS 200 systemcomprises the following main components: two high-pressurepumps, one for the CO2 (P1) and the other for the solution (P2),which incorporate a low-dead-volume head and check valves toprovide efficient pumping of CO2 and a range of solvents; astainless steel precipitator vessel (V1) of 2 L volume, consisting oftwo parts, the main body and the frit, all surrounded by an electricalheating jacket (V1-HJ1); an automated high precision back-pressure regulator (ABPR1), attached to a motor controller witha position indicator; and a jacketed (CS1-HJ1) stainless steelcyclone separator (CS1) with 0.5 L volume, to separate the solventand CO2 once the pressure is released by the manual back-pressureregulator (MBPR1). The following auxiliary elements were alsonecessary: a low pressure heat exchanger (HE1), cooling lines, anda cooling bath (CWB1) to keep the CO2 inlet pump cold and to chillthe pump heads; an electrical high-pressure heat exchanger (HE2)to preheat rapidly the CO2 in the precipitator vessel to the requiredtemperature; thermocouples placed inside (V1-TS2) and outside(V1-TS1) the precipitator vessel, inside the cyclone separator(CS1-TS1), and on the electric high pressure heat exchanger toobtain continuous temperature measurements; and a FlexCORcoriolis mass flowmeter (FM1) to measure the CO2 mass flow rateand other parameters such as total mass, density, temperature,volumetric flow rate and total volume.

In order to study the ability of EC to encapsulate AMC, severalsuspensions of AMC microparticles in a solution of EC in DCMwere used. All the operating conditions are summarized in Table 1.Pressure, temperature, drug:polymer ratio and polymer concentra-tion effects have been evaluated. These AMC microparticlesuspensions (20 to 200 mg of AMC) in a 20 mL DCMEC solutionwere sprayed through a nozzle using a P305 peristaltic pump (asopposed to the SAS200 sample pump) to avoid blocking. Thesupercritical CO2 acts as an antisolvent for the DCM. A rapid mutualdiffusion between the supercritical CO2 and the organic solventcauses supersaturation of the polymer solution, leading to nucleationand precipitation of the polymer to encapsulate the AMC particles.In the precipitation carried out on a suspension of particles, theparticles behave as nuclei for the precipitation of the polymer and apolymer matrix of encapsulated particles is produced by agglom-eration (Wang et al., 2004; Cocero et al., 2009).

In this work, DCM was selected as the organic solvent toprecipitate EC using the SAS technique because it was previouslyshown that the precipitated particles are not agglomerated

(Duarte et al., 2006). The low solubility of ethyl cellulose incarbon dioxide and their relatively high solubility in DCMprovided suitable conditions to employ the SAS process forcontrolled particle formation. As a consequence, the encapsula-tion process was carried out by suspending AMC microparticlesin polymer solutions with different AMC:polymer ratios, i.e. 1:1,1:2, 1:5 and 1:10. The conditions for AMC encapsulation aresummarized in Table 1. Different initial concentrations of thepolymer solution (5, 10 and 15 mg/mL) were considered in orderto establish the relationship between the particle sizes ofencapsulates and the initial concentration of the solution.Pressures in the range 80150 bar and temperatures in the range3565 C were investigated. The liquid flow rate (QL) was2 mL/min in order to ensure that the solution was atomized and asmall nozzle diameter (n) of 100mm was used to guarantee theturbulence and higher supersaturations at the border of the jet inorder to obtain small particles (Tenorio et al., 2009).

Encapsulate powder precipitated on the wall was observedusing a QUANTA 200 scanning electron microscope (SEM). Thesamples had previously been placed on carbon tape and thencovered with a coating of gold using a sputter coater. The appliedcurrent was 15 mA and the coating time was 150 s.

LP mass of AMCmass of AMCmass of EC

precipitated

100

LE LPLP0

Figure 2. SEM image of unprocessed amoxicillin.

Figure 1. SEM image of unprocessed ethyl cellulose.

DOI: 10.3109/02652048.2013.799242 Polymer encapsulation of amoxicillin 17

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Amoxicillin loading in the encapsulates was determined byhigh-performance liquid chromatography (HPLC). The HPLCequipment consisted of a chromatograph (Agilent Technologies1100 Series) with a UVvisible detector. A C18 Hypersil ODS(5mm particle size) column (250 mm 4.6 mm) (Supelco) wasused. The samples were dissolved with the aid of stirring and

sonication during 5 minutes in a mixture of methanol:distilledwater (90:10) to ensure complete dissolution of the drug. AMCwas eluted using a mixture of 80% phosphate buffer at pH 2, 16%acetonitrile and 4% methanol (Kalogiannis et al., 2006) as themobile phase with a flow rate of 0.3 mL/min and detection at230 nm. The amoxicillin loading percentage (LP), the theoretical

Figure 4. Schematic diagram of the pilot plant.

Table 1. Encapsulation experiments with SAS process.

RunAMC:EC

ratio (wt/wt)

Ethylcelluloseconcentration

(mg/mL)Pressure

(bar)Temperature

(C)XPS*

(%Nitrogen)

Loadingpercentages

(%)Loading

efficiency (%)Encapsulation

success

1 1:1 10 80 35 3.67 0.18 39.32 78.00 2 1:2 10 80 35 1.06 0.22 13.98 41.94 3 1:5 10 80 35 0.00 0.00 8.47 48.19 4 1:10 10 80 35 0.00 0.00 3.72 44.44 5 1:2 5 80 35 1.96 0.12 13.28 39.39 6 1:2 15 80 35 0.86 0.08 14.02 42.42 7 1:10 10 110 35 0.00 0.00 4.25 47.22 8 1:10 10 150 35 0.00 0.00 3.39 37.66 9 1:10 10 150 50 0.00 0.00 3.41 37.88 10 1:10 10 150 65 0.00 0.00 3.25 36.11 Note: *XPSX-ray photoelectron spectroscopy.

Figure 3. SEM image of microparticlesprecipitated by SAS process of (a) amoxicil-lin obtained at 55 C (T), 180 bar (P), 66 g/min (QCO2), 5 mL/min (QL), 180 min (tw),100mm (n) and (b) ethyl cellulose at 35 C(T), 80 bar (P), 11 g/min (QCO2), 2 mL/min(QL), 90 min (tw) and 100 mm (n).

18 A. Montes et al. J Microencapsul, 2014; 31(1): 1622

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loading of amoxicillin in the organic solution (LP0) and loadingefficiency (LE) were calculated as follows:

Drug release profiles were obtained for the pure drug and forthe precipitated microparticles. Simulated gastric fluid (SGF)without pepsin and simulated intestinal fluid (SIF) without trypsinwere prepared to carry out the release experiments. SGF wasmade by dissolving sodium chloride (2 g L1) and hydrochloricacid (0.2 N) and adjusting to pH 1.2 0.1, and SIF was made bydissolving monobasic potassium phosphate (6.8 g L1) in asolution of 0.2 N sodium hydroxide and adjusting to pH6.8 0.1. In these experiments 40 mg of AMC were added to20 mL of simulated fluid and the solutions were kept at 37 C withcontinuous stirring at 170 r.p.m. The absorbance at 288 nm wasmeasured on a Shimadzu mini 1240 UV-visible spectrophotom-eter and this was used for quantitative analysis of AMC on thebasis of a calibration curve. Samples were collected at regularintervals within 24 h.

X-ray photoelectron spectroscopy (Kratos Axis Ultra DLD)was used to determine the possible location of AMC in theprecipitates, with chemical analysis of the surface of thesemicrospheres also carried out because SEM images are notsuitable to observe the distribution of both compounds: the AMCcould be located on the surface of the microspheres and/or withinthe core. It is possible to differentiate between composites, whichare formed by dispersions of particles within the core material in amatrix of coating material, and encapsulates, which are producedwhen the coating material is precipitated as a thin shell over apreviously existing core material particle (Cocero et al., 2009).So, XPS was used to determine the success of the encapsulationprocess (Morales et al., 2007). In this procedure the powder

samples were mounted on a double-sided adhesive conductingpolymer tape and analyzed without any further treatment. Theelements that differentiate AMC from EC are sulfur (S) andnitrogen (N), so the absence of these elements in the XPS analysiswould indicate that the drug was contained within the core of theencapsulates. However, the detection of these elements wouldindicate that AMC is present at the surface and that there is AMCthat is not encapsulated. The XPS data are collected in Table 1.

Results and discussion

The successful precipitation of amoxicillin and ethyl cellulose onthe wall of the precipitator vessel was achieved in all experiments.SEM images of the products are shown in Figures 5 and 6. In mostcases, the formation of spherical or quasi-spherical agglomeratedmicroparticles was obtained. In any case, the resulting particleswere significantly smaller than the unprocessed compounds(Figures 1 and 2) but, according to the SEM images, larger thanAMC and EC precipitated separately by an SAS process(Figure 3). The SAS process also led to marked improvementsin the morphologies of particles. The morphology of the particleschanged from irregular blocks to spherical microparticles.

Drug polymer ratio

In the first set of experiments (runs 14) the effect of AMC:ECratio on size and morphology was investigated and AMC loadingpercentages (LP) and efficiencies (LE) of the resulting systemswere calculated. As one would expect, the LP and LE increasedon increasing the AMC:EC ratio, with the highest LP obtainedwhen the drug and polymer were in the same ratio. In general, the

Figure 5. SEM images of microparticles ofamoxicillin and ethylcellulose obtained at35 C (T), 80 bar (P), 11 g/min (QCO2),2 mL/min (QL), 90 min (tw) and 100 mm(n).

DOI: 10.3109/02652048.2013.799242 Polymer encapsulation of amoxicillin 19

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efficiency of the process was consistently around 4048%(Table 1). However, it was observed that the success of theencapsulation process was greater when the drug:polymer ratiowas lower (runs 3 and 4). When the ratio was higher there wasinsufficient ethyl cellulose to cover completely the amoxicillinparticles. Thus, there is a balance between drug content andsuccess of the encapsulation process and this aspect must be takeninto account. On the other hand, a significant difference inparticle size and morphology of the resulting systems was notfound according to the SEM images, as can be seen in Figure 5.

Polymer concentration

Experiments were carried out with different initial EC concen-trations in the solution pumped into the vessel (runs 2, 5 and 6).The EC concentration used in this work is not a critical parameterfor the success of the encapsulation, as can be seen from theresults in Table 1. However, the particle size of encapsulates washigher when the EC initial concentration was higher, as shown inFigure 5.

The relationship between the initial concentration of thesolution and particle size has two possible influences. On onehand, a higher concentration allows higher supersaturation to beachieved, thus leading to a smaller particle size. On the otherhand, an increase in the condensation rate at higher concentrationsincreases the particle size. The operating conditions used in thiswork led to the second effect prevailing; so, the higher the initialconcentration of the solution, the higher the condensation rate andtherefore the larger the particle size produced (Martin and Cocero,2004). This same trend for the initial concentration effect has beenobserved in previous studies (Montes et al., 2010, 2012).

Pressure and temperature effects

Pressure and temperature assayed did not have an appreciableeffect on success of encapsulation. On the other hand, as can be

seen in Figure 6, the particle size of systems obtained decreasedslightly as the pressure increased, as can be observed in the SEMimages. This result can be explained by considering that anincrease in pressure at constant temperature enhances the solventpower of supercritical CO2 towards DCM, thus the liquid solventmolecules are more strongly captured by the CO2. In contrast, thepossibility of interaction between DCM and AMC is reduced(Reverchon and Della Porta, 1999; Snavely et al., 2002). On theother hand, pressure seems to be able to affect the processefficiency being the optimum pressure 110 bar and after that theefficiency decreases as can be seen in Table 1.

The effect of temperature on the process is illustrated by runs810. As can be seen in Figure 7, the particle size of theencapsulates increased as the operating temperature wasincreased. CO2 and solvent mass transfer rates are expected tobe increased only slightly by small increases in temperature. Theincreases in mass transfer rates, which would tend to increasesupersaturation ratios and decrease particle sizes, may be offset byparticle growth rates that increase with temperature (Randolphet al., 1993). Increasing the temperature has no advantages on theprocess efficiency as can be seen in Table 1.

Drug location

It was not possible to use the SEM images to determine whetherthe AMC was within the core of the precipitates covered by ECand therefore we were unable to gauge the success of theencapsulation process. The AMC could be located on the surfaceof the microspheres and/or within the core. In a previous study thepresence of AMC on the surface was detected when AMC and ECwere coprecipitated by an SAS process to give composites(Montes et al., 2011). As a result, XPS was used to determine thesuccess of the encapsulation process through chemical analysisof the particles on the precipitate surface as it was explained inthe previous section. The XPS data are collected in Table 1.

Figure 7. SEM images of microparticles of amoxicillin and ethylcellulose obtained at polymer concentration of 10 mg/mL with 1:10 amoxicillin:ethylcellulose ratio at different operating temperature.

Figure 6. SEM images of microparticles of amoxicillin and ethylcellulose obtained at polymer concentration of 10 mg/mL with 1:10amoxicillin:ethylcellulose ratio at different operating pressure.

20 A. Montes et al. J Microencapsul, 2014; 31(1): 1622

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AMC was present on the particle surface in experiments withhigher AMC:EC ratios (runs 1, 2, 5 and 6). At the same time, theLP values calculated by HPLC analyses confirmed that AMC ispresent in the products from all of the experiments, as can be seenfrom the results in Table 1. This finding could be due to thepresence of excess AMC microparticles with the EC unable tocover all of the AMC in the precipitation process. As a result,composites with AMC on the surface and encapsulates withoutAMC on the surface were obtained.

Drug release

The drug release behavior was evaluated in simulated fluidsprepared in the laboratory: simulated gastric fluid (SGF), pH1.2 0.1, and simulated intestinal fluid (SIF), pH 6.8 0.1, wereobtained as described previously in the Materials and methodssection. Samples and pure drugs were taken periodically andmeasured at 288 nm in these fluids. The profiles show the

percentage of antibiotic released with time. A comparison of theseresults is presented in Figures 8 and 9. It was found that allformulations led to slower release than the pure drug.

The delayed release from the precipitated particles incomparison to the pure drug is more marked in SIF than SGF.In a previous study (Montes et al., 2011) a relationship wasestablished between the presence of N, i.e. AMC on theprecipitate surface, and the release profile. The release of thedrug from precipitates in which N was present on the surface wasfaster than in cases where N was not present: i.e. AMC on thesurface is more accessible for rapid delivery. A similar trend canbe seen in this work for the SIF profile. Thus, in runs 1, 2, 5 and 6in SIF there was AMC on the surface and in these cases there is aburst release during the first hour, during which around 5070% of the total drug is released.

Drug release from microcapsules should theoretically beslower as the amount of polymer is increased due to the increasein the path length through which the drug has to diffuse. The total

t(h)0 2 4 6 8

Dru

g Fr

actio

n Re

leas

e (%

)

20

40

60

80

100

run 1-SGFrun 2-SGFrun 3-SGFrun 4-SGFrun 5-SGFrun 6-SGFrun 7-SGFrun 8-SGFrun 9-SGFrun 10-SGFamoxicillin

Figure 9. Release profile of the amoxicillin from obtained systems in Simulated Gastric Fluid.

t(h)0 2 4 6 8

Dru

g Fr

actio

n Re

leas

e (%

)

0

20

40

60

80

100

120

run 1-SIFrun 2-SIFrun 3-SIFrun 4-SIFrun 5-SIFrun 6-SIFrun 7-SIFrun 8-SIFrun 9-SIFrun 10-SIFamoxicillin

Figure 8. Release profile of the amoxicillin from obtained systems in Simulated Intestinal Fluid.

DOI: 10.3109/02652048.2013.799242 Polymer encapsulation of amoxicillin 21

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cumulative quantity of drug released at the end of the 12 hdissolution test was below 100% for all dosage forms. This may inpart be due to the relatively slow erosion of the matrix under thetest conditions, with a resultant slow release of entrapped drugfrom the matrices under investigation.

The influence of the operating conditions on the releaseprofiles was analyzed, although it is difficult to establishrelationships between drug release and the pressures andtemperatures investigated. However, in run 1 a slower releasetook place and this reached only 510% of the AMC in the firsthour. It can be seen from the results for runs 58 (assayed in SIF)that it is possible to tune the operating conditions to developdifferent release behavior.

Conclusions

Encapsulation of amoxicillin in ethyl cellulose from a suspensionof amoxicillin microparticles in a solution of ethyl cellulose indichloromethane was studied. The supercritical antisolvent pro-cess provides a feasible approach for the formation of encapsu-lates and composites of these two compounds. Encapsulates wereobtained with a lower amoxicillin:ethyl cellulose ratio andcomposites with a higher ratio. It was observed that thepercentages of amoxicillin are maintained in the precipitateswith regard to the initial suspended drug and there is nosignificant difference in particle size and morphology onchanging the drug:polymer ratio. However, SEM images showthat the particle size of encapsulates was higher on increasing theinitial ethyl cellulose concentration. The effects of pressure andtemperature on particle size were also evaluated. The sizedecreased on increasing pressure but increased as the operatingtemperature was increased. The release of drug from precipitatesin which N was present on the surface was faster than in caseswhere N was absent. However, in both cases the release wasslower than the dissolution profile of the pure drug.

Declaration of interest

The authors report no declarations of interest. We gratefully acknowledgethe Spanish Ministry of Science and Technology (Project CTQ2010-19368) for financial support.

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