Curcumin Encapsulation in Submicrometer Spray-Dried Chitosan/Tween 20 ParticlesMartin G. O’Toole, Richard M. Henderson, Patricia A. Soucy, Brigitte H. Fasciotto, Patrick J. Hoblitzell,Robert S. Keynton, William D. Ehringer, and Andrea S. Gobin*

Department of Bioengineering, University of Louisville, Louisville, Kentucky 40292, United States

*S Supporting Information

ABSTRACT: Optimal curcumin delivery for medicinalapplications requires a drug delivery system that bothsolubilizes curcumin and prevents degradation. To achievethis, curcumin has been encapsulated in submicrometerchitosan/Tween 20 particles via a benchtop spray-dryingprocess. Spray-drying parameters have been optimized using aTaguchi statistical approach to minimize particle size and tofavor spheroid particles with smooth surfaces, as evaluatedwith scanning electron microscopy (SEM) imaging. Nearlyspherical particles with 285 ± 30 nm diameter and 1.21 axialratio were achieved. Inclusion of curcumin in the spray-drying solution results in complete encapsulation of curcumin within thechitosan/Tween 20 particles. Release studies confirm that curcumin can be released completely from the particles over a 2 hperiod.

■ INTRODUCTIONOptimized drug delivery systems for curcumin, a majorcomponent of turmeric extracted from the rhizome of Curcumalonga, would offer new treatment options for a variety ofdiseases.1 However, despite displaying potent antioxidant,2,3

anti-inflammatory,4,5 antiseptic,6 and analgesic7 activity, theefficacy and widespread clinical use of curcumin is hampered bypoor bioavailability. Although high doses of curcumin havebeen shown to be pharmacologically safe, oral administration ofcurcumin is rapidly metabolized and excreted.8 Solubilization ofcurcumin in aqueous solution typically requires addition oforganic solvents (e.g., methanol, dimethyl sulfoxide (DMSO))or alkaline conditions. Unfortunately, curcumin quicklydecomposes under these conditions, with a pH-dependenthalf-life on the order of seconds to minutes.9−11 Theinsolubility and instability of curcumin thus provide a primeopportunity for use of drug delivery agents to enhancecurcumin’s clinical relevance. To this effect, spray-driedchitosan particles have been chosen as a potential drug deliveryvehicle to both solubilize and protect curcumin for medicinalapplications.Chitosan, an amine-rich polysaccharide derived from chitin,

is well-known for its ability to form biodegradable andbiocompatible particulate systems for drug delivery.12−18

Chitosan has been shown to serve as a protective agent againstphotolytic and hydrolytic degradation of curcumin.19 Chitosan-based drug carriers for curcumin have also been shown toprotect curcumin from degradation, increase drug uptake, andmaintain therapeutic benefits.20 The carrier used in the previousstudy, however, required several purification steps duringproduction and had a tendency to aggregate in solution.

Several methods are available for producing nanoparticles fromchitosan, including ionic gelation with tripolyphosphate,microemulsion, emulsification solvent diffusion, polyelectrolytecomplexation, and spray-drying.12,21 Spray-drying offers theadvantage of producing a dry drug-loaded particle from apolymer/drug solution in a single step. Several reports havedemonstrated the feasibility of spray-drying chitosan for drugdelivery formulations.21−34 However, typical particle sizes forthese methods have ranged from 1 to 50 μm. For intravenousdrug delivery applications, particle sizes should fall in thesubmicrometer regime in order to traverse capillary networkswithout causing embollism.35 Curcumin has also beensuccessfully formulated into drug delivery vehicles throughspray-drying with polymers such as ethyl cellulose, starch/gelatin, malodextran, polyvinylpyrrolidone, and solid lipidparticles.33,36−41 However, there are little if any availableaccounts of encapsulating curcumin in chitosan particles viaspray-drying.A recent advance for producing submicrometer-sized

particles via spray-drying on a laboratory scale is thecommercial availability of the Buchi B-90 Nanospray spraydryer.42,43 During the spray-drying process with the B-90, apolymer solution is sonicated and forced through a fine (4−7μm) mesh into a heated air stream. Particles are formed fromthe aerosolized polymer during flight down a heated columnduring which the solvent rapidly evaporates. Addition of smallmolecules to the spray-drying mixture allows for encapsulation

Received: April 11, 2012Revised: June 26, 2012Published: June 27, 2012

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within the polymeric particle framework. The dry particles arethen collected onto a charged precipitation drum at the end ofthe airshaft. Several user-defined variables impact the resultantsize and morphology of the spray-dried particles, includingsolvent composition, polymer concentration, surfactant con-centration, spray-drying temperature, and rate of airflowthrough the spray dryer. Proper optimization of the spray-drying parameters can achieve submicrometer particle sizes.In this study, we examined the ability of curcumin to be

encapsulated into submicrometer-sized chitosan particles foruse in drug delivery. Tween 20 has been incorporated into theformulation as a solubilizing agent for curcumin.44,45 Spray-drying conditions have been optimized for small particle sizeand spherical morphology using Taguchi statistical analysis ofmeasurements from scanning electron microscopy (SEM)images. Naturally fluorescent curcumin and fluorescentlylabeled Tween 20 have been utilized to image the particleswith incorporated surfactant using confocal microscopy.Curcumin stability after release from particles was determinedvia spectroscopic analysis. Our findings suggest that theseparticles will provide a platform to overcome current problemsassociated with curcumin delivery. The bioactivity of curcumin,once released from the particles, will be the subject of a futurereport.

■ MATERIALS AND METHODSChemical Supplies. Chitosan 90/200 was purchased from Heppe

Medical Chitosan, GmbH (Halle, Germany). Curcumin (85% pure;demethoxycurcumin and bis-demethoxycurcumin as impurities), aceticacid, 1-octanol, ascorbic acid, 3,5-di-tert-butyl-4-hydroxytoluene(BHT), carbonyldiimidazole (CDI), dichloromethane, diethyl ether,DMSO, polyethylene glycol sorbitan monolaurate (Tween 20), andpolyethylene glycol sorbitan monooleate (Tween 80) were purchasedfrom Sigma Aldrich (St. Louis, MO, USA). Magnesium sulfate waspurchased from Fisher Scientific (Pittsburgh, PA USA). Texas RedHydrazide and SlowFade antifade reagent were purchased fromInvitrogen Corporation (Carlsbad, CA, USA).Curcumin Saturation Studies. Curcumin (50 mg) was added to

100 mL 1% acetic acid containing 0−0.05 w/v% Tween 20 with orwithout 0.05 w/v% chitosan. The solution was stirred for 1 h in thedark followed by centrifugation (3489g, 5 min). The supernatant wasdecanted and the UV/visible spectrum collected on a BeckmannCoulter DB-525 spectrometer (Brea, CA). The absorbance at 425 nmwas used to quantify curcumin based on calibrations made with knownamounts of curcumin dissolved in 1% acetic acid with 0.1 w/v%Tween 20.Synthesis of Texas Red-Labeled Tween 20. CDI-activated

Tween 20 was prepared according to published protocols.46,47 Theprocedure for preparation of Texas Red-labeled Tween 20 from CDI-activated Tween 20 is a modification of the procedure published byYoshimura, et al.48 Care was taken during all manipulations tominimize light exposure. CDI-activated Tween 20 (6 mg, 4.8 μmol)was dissolved in 25 mL phosphate-buffered saline (PBS) buffer with 3mg (4.8 μmol) of Texas Red Hydrazide (Invitrogen). The reaction wasstirred for 2 h at room temperature. The dark red product wasextracted with 3 × 50 mL dichloromethane, dried over MgSO4, gravityfiltered, and the solvent was removed by rotary evaporation leaving abright red oily product.Determination of Detergents for Spray-Drying Formulation.

Chitosan-based particles were produced using a Buchi B-90 Nanosprayspray dryer (New Castle, DE). Solutions 0.05 w/v% chitosan with orwithout 0.025 w/v% detergent (Tween 20 or Tween 80) were stirredovernight and filtered through a 0.45 μm syringe filter before spray-drying. Solutions were spray-dried through a 4 μm spray mesh usingan inlet temperature of 120 °C and air flow rate of 150 L/min. Dryparticles were collected from the collecting drum using a particlescraper and stored in a desiccator.

Chitosan-Based Particle Production via Spray-Drying.Chitosan-based particles were produced using a Buchi B-90 Nanosprayspray dryer (New Castle, DE). Solutions of various concentrations ofchitosan and detergent were stirred overnight and filtered through a0.45 μm syringe filter before spray-drying. Solutions of polymer withand without detergent or curcumin were spray-dried through a 4 μmspray mesh using various combinations of the following parameters:Inlet temperature = 80, 100, or 120 °C; air flow rate = 100, 120, or 150L/min; chitosan concentration = 0.025, 0.05, or 0.1 w/v%; Tween 20concentration = 0, 0.025, or 0.05 w/v%. Specific combinations ofparameters were determined using a Taguchi array. Dry particles werecollected from the collecting drum using a particle scraper and storedin a desiccator.

Particle Characterization. Spray-dried particles were character-ized using a Zeiss Supra 35 (Carl Zeiss Microscopy, LLC, Thornwood,NY) field emission scanning electron microscope. Samples wereprepared by placing dry particles onto sample pedestals coated withsilver paint followed by coating with gold/palladium alloy for 30 susing a plasma coater. Images were captured using EHT = 2 kV.Particle diameter and axial ratio measurements were determined usingImageJ software on randomly selected particles (150 particles/image)from three separate SEM images for each sample (n = 3 samples foreach spray-drying condition; 1350 particles for each condition).Particle diameter was based on the longest particle dimension. Theaxial ratio is defined as the ratio of the largest particle dimension to thesmallest. Particle morphology is determined from the axial ratiomeasurement, with spherical defined as an axial ratio of 1. Statisticalcomparison of size was performed using Student t tests with p ≤ 0.05considered significantly different.

Statistical Analysis of Particle Size and Morphology.Optimization of spray-drying parameters was completed using theTaguchi-based statistical method, which is designed for optimizing alarge number of parameters using the fewest number of trials.42,49,50

Parameters to be optimized were chitosan concentration, detergentconcentration, spray dryer inlet temperature, and airflow rate. Eachparameter was evaluated at three different levels (Table S1). An L9orthogonal array (four parameters, three parameter levels, nineexperiments) was used to determine the instrumental parameters.Design criteria for particle size and morphology were to produce thesmallest particles that were as close to spherical as possible. Parameteroptimization was quantified by maximizing the experimental signal-to-noise (S/N) ratio using the formula:

∑= − ·=

y n(S/N) 10 log [ ( / )]in

i

i10

12

where y is the value returned for the parameter being optimized, and nis the number of experimental trials. Particle diameters for eachcondition were compared.

Confocal Imaging. Spray-dried particles were imaged on a NikonA1 laser scanning confocal microscope (Melville, NY). Samples wereprepared by placing a small amount of dried particles (with or withoutcurcumin and with or without Texas Red-labeled Tween 20) on amicroscope slide followed by a few drops of SlowFade antifadereagent. A glass coverslip was then affixed over the sample. Curcuminfluorescence was monitored on the green channel (Ex/Em = 488/525nm), and Texas Red was monitored on the red channel (Ex/Em =561/595 nm)

Curcumin Release Studies. Curcumin or curcumin encapsulatedin chitosan/Tween 20 particles (2 mg/mL) was placed in 1% aceticacid or 1× PBS buffer with 1% ascorbic acid as preservative at 37 °C inthe dark.51 Curcumin release from the particles was evaluated bypartitioning a small aliquot of the release mixture with 1-octanolcontaining 0.1% BHT as preservative at various time points. Theaqueous/organic mixture was centrifuged for 5 min at 218g to aid inseparation of layers. The organic layer was removed, and curcumincontent was evaluated by measuring the absorbance at 425 nm.

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■ RESULTS AND DISCUSSIONDetergent Choice and Solubilizing Curcumin. Due to

the poor solubility of curcumin in 1% acetic acid solution,fabrication of curcumin chitosan particles via spray-drying wasnot successful. To improve curcumin’s solubility, Tween 20 andTween 80 were screened as potential solubilizing agents basedon known biocompatibility and previous successful use inspray-drying experiments.42 Chitosan (0.05 w/v%) wasprepared for spray-drying by filtering through a 0.45 μm filterafter stirring in 0.025 w/v% Tween 20 or Tween 80 in an aceticacid solution overnight. Samples of chitosan were spray-driedthrough a 4 μm mesh with and without the presence of Tween20 or Tween 80 using a chitosan concentration of 0.05 w/v%,0.025 w/v% detergent, 120 °C inlet temperature, and 150 L/min flow rate.42 SEM analysis of the resulting particles showedthat average particle size for samples with Tween 20, Tween 80,and samples without detergent were not statistically different(360 ± 164, 325 ± 145, and 346 ± 37 nm, respectively; Figure1a). The surfaces of the particles spray-dried without detergent

or with Tween 20 appeared smooth, whereas the samplesspray-dried with Tween 80 displayed “pitted” surfaces. Tween20 was chosen for inclusion in future spray-drying experiments,as it did not detrimentally affect the particle size and producedparticles with comparatively smooth surfaces (Figure 1).The ability of Tween 20 to solubilize curcumin in 1% acetic

acid was evaluated by measuring the curcumin concentration incurcumin-saturated acetic acid solutions with varying amountsof Tween 20. The acidic nature of these solutions allowed forprolonged monitoring of curcumin with minimal degradation.Solubility results were compared with identical solutions thatalso contained 0.05 w/v% chitosan (Figure 2). The curcuminsaturation point increases linearly with Tween 20 concentrationthroughout the range used in these experiments, with an upperlimit of 294 μM curcumin with 0.05 w/v% Tween 20. Thisrepresents a 12.7-fold increase in curcumin solubility. At eachTween 20 concentration, the presence of chitosan in thesolution increases the curcumin saturation point by an average

of 19.6%. The ability of chitosan to help solubilize curcumin hasbeen attributed to hydrogen bonding interactions betweenchitosan −NH2 and curcumin phenol −OH groups.19

Spray-Drying Optimization. Four spray-drying parame-ters were chosen for optimization (Table S1). All samples weresprayed through a 4 μm spray head mesh, as the 5.5 and 7 μmmeshes provided with the spray dryer produced particles thatwere deemed too large for circulatory system drug delivery(data not shown). Chitosan concentration levels were restrictedto 0.1 w/v% or less to avoid clogging the spray head. Table S2lists the Taguchi orthogonal array utilized for this experiment aswell as the resulting S/N ratios calculated from the resultingparticle size and axial ratio data. The smallest particle sizes (285± 30 nm) were produced in trial 9, using a chitosanconcentration of 0.025 w/v%, no Tween 20, 120 °C inlettemperature, and 120 L/min flow rate. The most sphericalparticles (axial ratio = 1.12) were produced using theparameters for trial 2: chitosan concentration of 0.05 w/v%,Tween 20 concentration of 0.5 w/v%, 120 °C inlet temper-ature, and 100 L/min.By averaging all of the trial results for each level of a given

parameter (e.g., all of the trials using a chitosan concentrationof 0.05 w/v%), it is possible to determine which parameter hadthe greatest impact on particle size and morphology. Theimpact of each parameter is ranked according to the degree ofvariance of the experimental result obtained from varying thatparameter (Tables S3 and S4). Both particle size andmorphology were impacted most by choice of chitosanconcentration, with the smallest and most spheroid particlesbeing produced with the lowest chitosan concentration. Particlesize was also greatly impacted by airflow rate, although not in areliably predictive manner. Representative SEM images of theparticles showing the effect of chitosan concentration onparticle size are displayed in Figure 3. Note that with the highconcentrations of chitosan, a bimodal distribution of particlesize occurs. As samples spray-dried with low chitosanconcentrations had unimodal size distributions, the standarddeviation of these trials were markedly lower (±30 nm versus±569 nm for the bimodal high concentration trials).Although in preliminary trials particles produced with and

without Tween 20 had smooth surfaces, during theoptimization trials samples tended to yield more particleswith a wrinkled appearance (Figure 3c). This effect was moresubstantial with higher chitosan concentrations. When using 0.1

Figure 1. SEM images of chitosan particles spray-dried in the presenceof (a) Tween 20 (T20), (b) Tween 80 (T80), and (c) no surfactant(NS). Size analysis results are displayed in bar graph form (d). Spray-drying conditions: chitosan concentration = 0.05 w/v%, surfactantconcentration = 0.025 w/v%, inlet temperature = 120 °C, airflow =150 L/min.

Figure 2. Solubility of curcumin in 1% acetic acid with increasingconcentration of Tween 20 displayed with no chitosan added (graybars) and with 0.05 w/v% chitosan (white bars). Curcuminconcentration monitored at 420 nm in saturated solutions.

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w/v% chitosan and no detergent, 20−50% of the particlesdisplayed a wrinkled appearance. When the chitosan concen-tration was lowered to 0.05 w/v%, only 5−10% of the particleshad wrinkled surfaces. Several incompletely formed particleswere found during SEM analysis. These particles containedlarge holes in the polymer shell that allow confirmation that theparticles are hollow, as is typical for spray-dried particles(Figure 3d).While the spray-drying optimization results can be used to

produce either the smallest or most spherical particles, theseattributes alone may not be the most important to considerwhen choosing an optimum set of conditions for encapsulationof curcumin. For example, the parameters that produced thesmallest particles were from trial 9: 0.025 w/v% chitosan, 0Tween 20, 120 °C inlet temperature and 120 L/min airflow.These parameters involved no detergent, which in ourpreliminary studies was deemed necessary for solubilizingcurcumin in the spray-drying solution, and therefore makes apoor set of conditions for curcumin encapsulation. Additionally,the use of only 0.025 w/v% chitosan resulted in much longerspray-drying times required for producing an adequate amountof material. The most spherical particles were produced in trial2, using 0.05 w/v% chitosan, 0.05 w/v% Tween 20, 120 °C, and100 L/min air flow. This trial included equal amounts, weightfor weight, of chitosan and Tween 20. Comparing results oftrial 2 with trials using less Tween 20 reveals that decreasedTween 20 has little detriment to particle shape. Additionally,this trial yielded only the fifth smallest batch of particles.Therefore, in the interest of minimizing both particle size andthe amount of “auxiliary” components in the spray-dryingmixture, this trial was rejected as a candidate for curcuminencapsulation.The ideal set of conditions for encapsulating curcumin via

spray-drying would therefore produce small, nearly sphericalparticles using a minimum amount of Tween 20 to solubilizecurcumin and a chitosan concentration large enough to reducespray-drying times. Perusing the data presented in Table S2(Supporting Information) shows that the conditions and resultsfrom trial 1 meet all of these criteria. The conditions of trial 1(0.05 w/v% chitosan, 0.025 w/v% Tween 20, 150 °C, and 120

L/min air flow) produced the second smallest batch of particleswith an axial ratio that was within 5% of that obtained for trial 2(the condition producing the most spherical particles).

Curcumin Encapsulation. Curcumin was spray-driedusing the parameters from chitosan trial 1. UV−visiblespectroscopy was used to probe for curcumin decompositiondue to the spray-drying process (Figure 4). Curcumin

decomposition is easily monitored by a decrease in intensityof the peak at 425 nm in concert with a broadening of theabsorption band and appearance of two new peaks at 365 and335 nm due to degradation of curcumin to trans-6-(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5-hexenal.52 Despite the hightemperature of the spray head (150 °C) and sonication, theUV−visible spectrum of curcumin after spray-drying is identicalin content to that of the same solution before spray-drying. An8.5 ± 0.6% (n = 3) reduction in peak intensity at 425 nmrepresents minimal loss due to the spray-drying process. Todetermine the effect of curcumin loading on particle size,particles were spray-dried using the previously determinedoptimized conditions with the inclusion of various curcuminconcentrations below the saturation point (Table S5). SEMmeasurements reveal that encapsulation of curcumin results in anearly linear increase in chitosan particle diameter. Attempts toextract curcumin from the dry particles with repeated ethylacetate washings with vigorous shaking resulted in nodetectable traces of curcumin in the ethyl acetate, implyingthat all curcumin in the sample is trapped within the chitosanparticles. From this it is determined that curcuminencapsulation efficiency for this process is nearly 100%.53 It istherefore assumed that the increase in particle size in theseexperiments is due to curcumin loading.

Particle Composition. The fate of Tween 20 present in thespray-drying mixture was determined through spray-dryingparticles with Tween 20 covalently modified with Texas Redfluorescent dye.48 Confocal microscopy shows that the Tween20 dye is incorporated into the chitosan particle structure andtends to be concentrated on the outer periphery of theparticles, suggesting that the detergent is accumulated aroundthe chitosan shell, most likely due to hydrogen bondinginteractions (Figure 5a).54 The naturally occurring fluorescenceof curcumin can be used to visualize its compartmentalizationwithin the particles as well (Figure 5b). Unlike the Texas Red−Tween 20, the curcumin appears to be evenly distributedthroughout the particle. An overlay of both fluorescence images

Figure 3. SEM images of chitosan particles spray-dried with (a) 0.025w/v% chitosan and 0 w/v% Tween 20, (b) 0.05 w/v% chitosan and0.025 w/v% Tween 20, (c) 0.1 w/v% chitosan and 0.025 w/v% Tween20, and (d) 0.05 w/v% chitosan and 0.05 w/v% Tween 20. Figure 4. UV/visible spectrum of curcumin before (solid trace) and

after (dashed trace) spray-drying in 1% acetic acid with 0.025 w/v%Tween 20 and 0.05 w/v% chitosan. Spray-dried at 120 °C with 150 L/min air flow.

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displays the curcumin and Tween 20 spatial distribution withinthe particles (Figure 5c,d, respectively).Curcumin Release Studies. Curcumin release studies were

conducted in both 1% acetic acid and 1× PBS buffer to ensuredissolution of the chitosan/Tween 20 particles. The releasemedium was fortified with 1% ascorbic acid and the organiclayer with 0.1% BHT as preservatives.51 Release studies in thepresence of preservatives in either solvent show a burst releaseprofile for curcumin, with nearly 40% of curcumin released inthe first 5 min of the experiment (Figure 6). All detectablecurcumin release was complete within 2 h (Figure 6).

■ CONCLUSIONS

The feasibility of encapsulating curcumin in submicrometerchitosan particles via spray-drying has been demonstrated. The

spray-drying process has been optimized for encapsulation ofcurcumin in the smallest, most spherical particles possible withthe smoothest morphology. Release studies confirm a burstrelease profile for curcumin. Future studies will involve chitosanderivatives that display better solubility under physiologicalconditions for applications in systemic drug delivery. Inaddition, the use of cross-linking agents will be explored foraltering curcumin release kinetics under physiologic conditions.The incorporation of preservative molecules in addition tocurcumin within the chitosan capsules will also be explored.

■ ASSOCIATED CONTENT

*S Supporting InformationTables S1−S5 are included in the Supporting Information forthis manuscript. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Tel: 502 852 8321. Fax: 502 852 6806. E-mail: andrea.gobin@louisville.edu.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

Support for this work was in part provided by NASA GrantNNX10AJ36G. The authors wish to acknowledge Joe Williamsand Dhruvinkumar Patel for their assistance with SEM imaging.

Figure 5. Confocal microscopy images (single xy plane of a z-stack) of curcumin-loaded chitosan/Tween 20 particles: (a) Tween 20−Texas Redshown in red, (b) curcumin shown in green, (c) overlay of Texas Red and FITC channels, and (d) 10× zoom of overlay of Texas Red and FITCchannels of curcumin-loaded chitosan/Texas Red-labeled Tween 20 particles. Scale bar = 10 μm.

Figure 6. Release profile of curcumin from chitosan/Tween 20particles in 1% acetic acid (⧫) and PBS buffer (○) (n = 3).

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