Design and Development of Novel Mitochondrial TargetedNanocarriers, DQAsomes for Curcumin InhalationSpela Zupancic,† Petra Kocbek,† M. Gulrez Zariwala,‡ Derek Renshaw,‡ Mine Orlu Gul,§ Zeeneh Elsaid,§

Kevin M. G. Taylor,§ and Satyanarayana Somavarapu*,§

†Faculty of Pharmacy, University of Ljubljana, Askerceva Cesta 7, 1000 Ljubljana, Slovenia‡Faculty of Science & Technology, University of Westminster, 115 New Cavendish Street, London W1W 6UW, United Kingdom§Department of Pharmaceutics, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom

*S Supporting Information

ABSTRACT: Curcumin has potent antioxidant and anti-inflammatory properties but poor absorption following oraladministration owing to its low aqueous solubility. Develop-ment of novel formulations to improve its in vivo efficacy istherefore challenging. In this study, formulation of curcumin-loaded DQAsomes (vesicles formed from the amphiphile,dequalinium) for pulmonary delivery is presented for the firsttime. The vesicles demonstrated mean hydrodynamic diametersbetween 170 and 200 nm, with a ζ potential of approximately+50 mV, high drug loading (up to 61%) and encapsulationefficiency (90%), resulting in enhanced curcumin aqueous solubility. Curcumin encapsulation in DQAsomes in the amorphousstate was confirmed by X-ray diffraction and differential scanning calorimetry analysis. The existence of hydrogen bonds andcation−π interaction between curcumin and vesicle building blocks, namely dequalinium molecules, were shown in lyophilizedDQAsomes using FT-IR analysis. Encapsulation of curcumin in DQAsomes enhanced the antioxidant activity of curcumincompared to free curcumin. DQAsome dispersion was successfully nebulized with the majority of the delivered dose deposited inthe second stage of the twin-stage impinger. The vesicles showed potential for mitochondrial targeting. Curcumin-loadedDQAsomes thus represent a promising inhalation formulation with improved stability characteristics and mitochondrial targetingability, indicating a novel approach for efficient curcumin delivery for effective treatment of acute lung injury and the rationale forfuture in vivo studies.

KEYWORDS: acute lung injury, antioxidant, curcumin, DQAsomes, nebulization

■ INTRODUCTION

Acute lung injury is defined as a secondary illness that occurs inresponse to various primary etiologies such as severepneumonia, sepsis, multiple traumas, and massive bloodtransfusion.1 The pathogenesis of acute lung injury involvesinflammatory cells, cytokines, and chemokines, as well asactivators and inhibitors of apoptosis which give rise to thegeneration of free radicals and reactive oxygen species. Thisresults in injury of lung endothelium and epithelium, leading toan increase in lung vascular and epithelial permeability, causingpulmonary edema due to the passage of protein-rich fluid intothe alveolar air spaces.2 Promising initial investigations focusedon pharmaco-therapies such as administration of exogenoussurfactant and inhalation of nitric oxide, glucocorticoids, andlysofylline. However, comprehensive studies have failed todemonstrate favorable clinical outcomes.3 Novel potentialtherapeutic strategies include mesenchymal stem cell therapy4

and application of anti-inflammatory and antioxidant moleculesthat act on diverse signaling pathways.5

Curcumin, a polyphenolic compound derived from thetuberaceous plant Curcuma longa, has been shown to exhibit a

wide range of therapeutic properties, such as antimicrobial andchemo-preventive properties in several types of cancer andhepato-protective, antiaggregatory, antioxidant, and anti-inflammatory effects. In an induced lung injury model in rats,curcumin treatment significantly inhibited inflammatoryresponse and prevented inhibition of the antioxidant enzymessuperoxide dismutase and glutathione peroxidase.6 In a cecalligature puncture induced model of acute lung injury in rats,curcumin treatment was demonstrated to exert protectiveeffects and significantly increase the survival rate of animals by40−50%.7The clinical applications of curcumin are severely limited due

to its low oral bioavailability, which is a consequence of its lowaqueous solubility, poor cellular uptake, high rate of metabolismin the intestine, and rapid elimination from the body.8

Pulmonary delivery offers a promising approach that may

Received: January 3, 2014Revised: April 8, 2014Accepted: May 22, 2014

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overcome some of the limitations mentioned and enhance thetherapeutic potential of curcumin, particularly for local lungconditions. The reasons are the high local concentration ofdrug that can be achieved via pulmonary application andreduced exposure to enzymatic activity as compared to deliveryvia the oral route. In addition, a large alveolar surface area isalso available for absorption when delivered via the pulmonaryroute.9 Sandersen et al. showed positive results in an in vivostudy of pulmonary delivery of a soluble curcumin derivative,which had an inhibitory effect in neutrophil inducedinflammation in the lower airways of horses affected byrecurrent airway obstruction.10 Swellable nano- and micro-particulate systems with encapsulated curcumin exhibited goodcontrolled release and showed promising aerosolizationcharacteristics in the in vitro model.11

Dequalinium (DQA) has been used for more than 50 yearsas an antimicrobial agent, and its oral health care application isFDA approved. In an aqueous medium, DQA single-chain bola-amphiphile molecules self-associate and form vesicles namedDQAsomes.12 The cationic nature and mitochondria-targetingproperties of DQAsomes have been exploited for delivery ofexogenous DNA into mitochondria, which was shown to besuccessful but with low transfection efficiency (1−5%).13DQAsomes resulted in a 5-fold increase in the apoptoticactivity of paclitaxel in colo205 cancer cell lines.14 These studiesclearly show the ability of DQAsomes to overcome cellularmembranes and deliver the payload intracellularly.In this study, a novel curcumin-loaded nanoformulation was

developed to enable its delivery via in vitro pulmonaryadministration, as an alternative to the oral route. Curcuminwas incorporated in DQAsomes with the aim of enhancing itsaqueous solubility and maintaining its antioxidant potential.The DQAsome formulation was nebulized to explore itspotential for pulmonary delivery for local treatment of acutelung injury. Finally, preliminary studies of the mitochondrialtargeting ability of the DQAsomes were performed so as toascertain the potential of these carriers for future in vivo studies.

■ MATERIALS AND METHODS

Materials. Unless otherwise stated, all chemicals wereanalytical grade. Dequalinium chloride hydrate (DQA; 95%),trifluoroacetic acid (99%), RPMI-1640, L-glutamine, fetalbovine serum (FBS), Dulbecco’s phosphate buffered saline(PBS), and pyrene (98%) were purchased from Sigma-Aldrich,U.K. Curcumin (total curcuminoid content 95%) from turmericrhizome was obtained from Alfa Cesar, USA. Acetonitrile,methanol, and water were HPLC grade and were supplied byFisher Scientific, U.K. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium com-pound (MTS) and phenazine methosulfate (PMS) whereobtained from Promega, CA, USA. Caco-2 cells were purchasedfrom the European Collection of Cell Cultures (Catalogue no.

09042001, ECACC, U.K.). Dulbecco’s Modified Eagle Medium(DMEM), Minimum Essential Media (MEM), 4-morpholinee-thanesulfonic acid 2-(N-morpholino)ethanesulfonic acid hy-drate buffer (MES) and fetal calf serum (FCS), and 100×antibiotic-antimycotic were produced from Invitrogen, U.K.MitoTracker Red CMXRos and TO-PRO-3 were obtainedfrom Life Technologies Ltd., Paisley, U.K. Culture plates andflasks were from Nunc, Denmark.

Preparation of Curcumin-Loaded DQAsomes. DQA-somes were prepared using a thin-film hydration method withminor modifications.12 Briefly, DQA and curcumin at certainmolar ratios (Table I) were dissolved in 20 mL of methanol.The concentration of DQA was kept constant at 5 mg/mL, andthe concentration of curcumin varied to achieve the requiredDQA:curcumin ratios. The solvent was then evaporated using arotary evaporator (Hei-VAP Advantage Rotary Evaporator,Heidolph, Germany) at 150 rpm, 80 °C and under vacuum(KNF Laboport, KNF Neuberger, Germany) for 10 min toobtain a thin film. The resultant thin film was hydrated with 20mL of water and mixed thoroughly at 25 or 80 °C for 2 minand sonicated using a VWR Ultrasonic cleaner bath USC300T(VWR International Limited, U.K.) for 20 min. The solutionobtained was filtered twice through a sterile 0.45 μm filter(Millex-MP, Millipore, Carrigtwohill, Ireland) to removeunentrapped curcumin. Samples were prepared in triplicateand lyophilized using a Virtis AdVantage 2.0 BenchTop freeze-dryer (SP Industries, U.K.) for storage and further analysis.

Size and Surface Charge of DQAsomes. Followingdilution in deionized water, the size distribution of theDQAsomes was obtained as ZAve hydrodynamic diameter andpolydispersity index (PDI) by photon correlation spectroscopy.The surface charge was measured by laser Doppler micro-electrophoresis. These studies were performed using theZetasizerNano ZS (Malvern Instruments, U.K.). All experi-ments were performed in triplicate, and results are presented asmean ± SD.

HPLC Analysis of Curcumin. Curcumin concentration wasdetermined by reversed-phase high performance liquidchromatography (HPLC) using the Discovery HS F5 HPLCcolumn (L × I.D. 15 cm × 4.6 mm, 5 μm particle size, 120 Åpore diameter, Supelco, USA) at 25 °C and UV detection at428 nm. The mobile phase was a mixture of water andacetonitrile 55:45 (v/v), supplemented with 0.1% (v/v)trifluoroacetic acid. The flow rate was 1 mL/min and runtime 15 min. The representative chromatogram of curcumin(Supporting Information Figure SI) dissolved in methanol isavailable online at http://pubs.acs.org/. The method wasvalidated according to ICH guidelines Validation of analyticalprocedures: text and methodology15 and FDA reviewer guidanceValidation of chromatographic methods.16

Quantification of DQA. The concentration of DQA incurcumin-free DQAsomes was spectrophotometrically deter-

Table I. Hydrodynamic Diameter (d), Polydispersity Index (PDI), Surface Charge, Drug Loading (DL) and EncapsulationEfficiency (EE) of Curcumin-Loaded DQAsomes Prepared at 25 and 80 °C (mean ± S.D., n = 3)

sample nDQA:ncur d (nm) PDI surface charge (mV) DL (%) EE (%)

DQAsomes25(1:0.5) 1:1 176.1 ± 19.3 0.25 ± 0.02 +46.8 ± 1.5 9.2 ± 1.5 35.5 ± 5.7DQAsomes25(1:2) 1:2 205.8 ± 8.0 0.27 ± 0.07 +48.6 ± 4.3 31.9 ± 5.5 54.8 ± 19.1DQAsomes(1:0.5) 1:0.5 160.7 ± 2.9 0.29 ± 0.07 +52.3 ± 1.7 22.5 ± 1.0 86.7 ± 3.9DQAsomes(1:1) 1:1 173.2 ± 21.1 0.23 ± 0.03 +53.7 ± 1.9 38.1 ± 1.9 92.6 ± 4.6DQAsomes(1:2) 1:2 203.2 ± 15.9 0.25 ± 0.03 +49.9 ± 6.5 53.4 ± 2.4 91.6 ± 4.1DQAsomes(1:3) 1:3 203.5 ± 9.9 0.24 ± 0.05 +44.0 ± 2.0 61.0 ± 2.0 90.2 ± 3.0

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mined at 328 nm after dissolution of DQAsomes in methanol(Jenway 7315 UV/visible spectrophotometer, Camlab, Stafford-shire, U.K.).Determination of Drug Loading and Encapsulation

Efficiency. DQAsomes were dissolved with methanol toachieve the theoretical concentration of curcumin in thesolution 14 ± 2 μg/mL. Five microliters of the sample wasinjected into the HPLC system and analyzed. The amount ofcurcumin encapsulated in DQAsomes, i.e. drug loading (DL),was calculated using eq 1, and encapsulation efficiency (EE)was calculated using eq 2.

=DL(%) m(cur)/m(DQAsomes) (1)

= ‐EE(%) m(cur)/m(cur T) (2)

m(cur) = determined amount of curcumin in DQAsomesm(DQAsomes) = amount of curcumin loaded DQAsomesm(cur-T) = theoretical amount of curcumin in DQAsomeXRD Analysis. X-ray diffraction patterns were obtained for

pure curcumin, DQA, their physical mixture (molar ratioDQA:curcumin of 1:2), lyophilized DQAsomes(1:0.5), andDQAsomes(1:2) using an X-ray diffractometer (Rigaku MiniFlex600, Miniflex, Japan). The samples were analyzed at roomtemperature in the angle range 5−35° with a step size of 0.01°and scanning rate 2°/min.DSC Analysis. Thermal analysis was performed using the

DSC Q2000 module (TA Instruments, USA). A 2−5 mgsample was weighed in a Tzero aluminum pan (TAInstruments, U.K.) and covered with a lid having a 50 μmpinhole. Curcumin, the physical mixture of DQA and curcuminin the molar ratio 1:2, and lyophilized formulations ofDQAsomes(1:0.5) and DQAsomes(1:2) were analyzed using aheating rate of 10 °C/min in the temperature range 0 to 110°C. When the sample reached 110 °C, it was kept at isothermalconditions for 5 min prior to cooling to 0 °C and reheating upto 200 °C. DQA was analyzed using the same conditions withthe exception of the second heating cycle, which was continuedto 350 °C. The measurements were performed in an inertnitrogen atmosphere with a flow rate of 50 mL/min. DSCheating curves were analyzed using Universal Analysis 2000software (TA Instruments). Thermal transitions reported hereare based on the second heating cycle.FT-IR Analysis. The chemical structure of DQA, curcumin,

their physical mixtures (molar ratios DQA:curcumin of 1:0.5,1:1, and 1:2), and lyophilized formulations of DQAsomes(1:0.5),DQAsomes(1:1), and DQAsomes(1:2) were analyzed using aPerkinElmer Spectrum 100 FT-IR spectrometer (PerkinElmer,USA) from 650 to 4000 cm−1 at a resolution of 4 cm−1. Eachsample was measured with 20 scans. Data were analyzed usingthe PerkinElmer Spectrum Express software (PerkinElmer,USA).Morphology of DQAsomes. Morphological analysis of

plain DQAsomes and DQAsomes(1:0.5), just after preparationand after 40 days, was performed by transmission electronmicroscopy (TEM) using an FEI CM 120 BioTwin trans-mission electron microscope (Philips Electron Optics BV,Netherlands) using acceleration voltage 120.0 kV. Approx-imately 40 μL of the DQAsome dispersion was placed on aFormvar/carbon coated copper grid and negatively stained with1% uranyl acetate. Digital images were taken at 65,000, 93,000,and 135,000 times magnification.Critical Micelle Concentration of DQAsomes. Critical

micelle concentration is the concentration at which the DQA

micelles form. This was determined using the pyrenefluorescence assay, using a fluorescence spectrometer (Perki-nElmer precisely LS55 luminescence spectrometer, Wellesley,USA). The vibration band intensities of the fluorescent probe,pyrene, change in response to the polarity of the surroundingenvironment. Above the critical micelle concentration, adecrease is observed in the ratio between the first and thirdvibrational peaks (I1 and I3, respectively) of the emissionspectra obtained. This corresponds to the entrapment of pyrenewithin the interior hydrophobic core of the micelles.17

The critical micelle concentration of DQA micelles wasdetermined via a serial dilution of the formulation with theaddition of pyrene at a concentration of 6 × 10−7 M. Thefluorescence spectra were then read at an emission wavelengthof 332 nm and an excitation of 335 nm. The critical micelleconcentration value was taken as the point of intersectionbetween the two tangents drawn from the graph of the intensityratio (I1/I3) against the log DQA concentration.18

Physical Stability of DQAsomes at Room Temper-ature. All formulations of curcumin-loaded DQAsomesprepared by the thin film method at 80 °C were stored atroom temperature, protected from light for 40 days. Followingthis period, the size distribution, surface charge, drug loading,and encapsulation efficiency were measured, as describedpreviously. Drug loading and encapsulation efficiency analysiswere performed after the samples were filtered once through asterile 0.45 μm filter to remove any aggregates or precipitateddrug present in the samples.

Antioxidant Properties of Curcumin. The antioxidantactivity of curcumin-loaded DQAsomes was determined usingthe ferric ion reducing antioxidant power (FRAP) assay, withsome modification.19 Acetate buffer (pH 3.6), tripyridyltriazine, and iron(III) chloride were mixed to prepare theFRAP reagent mixture. The concentrations of curcumin inDQAsome formulations (DQAsomes(1:0.5), DQAsomes(1:1), andDQAsomes(1:2)) were spectrophotometrically determined justafter preparation and after 60 days using a microtiter platereader (VersaMax, Molecular Devices, USA) at 428 nm. Toanalyze antioxidant activity, all DQAsome samples andcurcumin stock solution in methanol (2.7 mM) were dilutedwith either water or methanol to the final concentration ofcurcumin 500 μM. FRAP assay was carried out by addition ofsamples (DQAsomes or curcumin solution) (30 μL) to FRAPreagent (900 μL), and the reaction mixture was incubated for30 min at 25 °C. 300 μL of the samples obtained were thentransferred into a 96-well microtiter plate and absorbancemeasured at 593 nm. FRAP reagent mixture without anyadditives was used as a blank. The antioxidant activity ofDQAsome samples was compared to the antioxidant activity ofcurcumin in methanol solution.

Determination of the Aerosol Properties of Curcu-min-Loaded DQAsomes Delivered from a Jet Nebulizer.The twin-stage impinger (TSI, Copley Scientific Limited, U.K.)was comprised of two stages with a cutoff aerodynamicdiameter between the stages of 6.4 μm at a flow rate of 60 L/min (Figure 1).20 To collect the nebulized aerosols, 7 and 30mL of water were placed in the upper and the lower stages,respectively. DQAsomes(1:0.5) (2 mL) were added to a Pari LCSprint nebulizer attached to a TurboBoy N compressor (PariMedical Ltd., GmbH, Starnberg, Germany) directed toward thethroat of the TSI. The pump was switched on 10 s before thenebulizer was run for 60 s, and for an additional 5 s after thepump was switched off. Liquid and washings from the nebulizer

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and lower and upper stages were collected for particle size andsurface charge measurements and for determination ofcurcumin. The total mass balance of curcumin in the TSIand nebulizer was determined according to the proceduredescribed in the European Pharmacopoeia.20

Evaluation of Formulation Safety Using the MTSAssay. The human epithelial cell line A549 was expanded inRPMI-1640 medium comprised of 10% FBS, 1% L-glutamine,and 1× antibiotic-antimycotic at 37 °C in a humidifiedincubator containing 5% CO2. Cells were seeded onto 96-well plates at 2000 cells per well, with cell viability beingquantitatively assessed using MTS with PMS. Cells wereexposed to DQAsomes(1:2) and curcumin in DMSO (0.6%DMSO in media) for 72 h, after which 20 μL of MTS (5 mg/mL in PBS) was added to the wells and incubated for a further2 h. MTS then yields a water-soluble formazan product and theoptical density of the wells was measured using a multidetectionmicroplate reader (Synergy HT, Bio-Tek Instruments, VT,USA), at an absorbance maximum of 490 nm. Cell viability wasexpressed as a percentage relative to control cells, with cellsexposed to media alone acting as the positive control and thoseexposed to 1% Triton-x as the negative control. Data was givenas mean ± standard deviation.Ability of DQAsomes to Target Mitochondria. Caco-2

cells were obtained at passage 20, and passages 45 to 55 wereused in experiments. Cells were seeded onto 6-well plates at aninitial seeding density of 3 × 104 cells/cm2 and cultured at 37°C in an atmosphere with 5% CO2 in air. The medium (FCSsupplemented DMEM) was replaced every 2 days. Theexperiments were carried out 14 days post-seeding, whenCaco-2 cells differentiated to a fully matured gastrointestinaltract phenotype.On the 14th day post-seeding, cell culture medium was

aspirated and cell monolayer washed twice with sterile DPBS.DQAsome(1:0.07) sample (1:0.07 molar ratio of DQA:curcumin)was added to achieve a final concentration of 50 μM curcuminin the incubation media. DQAsome sample was prepared bydilution of DQAsome(1:0.07) formulation with a serum-freeMEM, pH 5.8 adjusted with 10 mM MES buffer. Caco-2 cellswere then incubated for 2 h to allow the DQAsomes to beinternalized. Following incubation, medium was removed andcells were washed with DPBS and incubated for 30 min with a200 nM solution of red fluorescent mitochondrial dye

MitoTracker Red CMXRos in MEM pH 5.8 adjusted with 10mM MES buffer. The cell monolayer was then washed twicewith DPBS and fixed with 3.9% paraformaldehyde solution.Cell nuclei were stained with TO-PRO-3 (1 μM) for 1 h atroom temperature. The samples were examined under aconfocal microscope (Leica TCS SP2, Leica microsystems,Milton Keynes, U.K.) to assess mitochondrial targetingproperties of curcumin-loaded DQAsomes. Images wereanalyzed using the Leica LCS Lite software suite (Leicamicrosystems, Milton Keynes, U.K.).

Statistical Analysis. The data were expressed as mean ±standard deviation (S.D.). The results were statistically analyzedusing paired Student’s t test or one way analysis of variance(ANOVA) followed by Turkey’s posthoc test (PRISM softwarepackage, Version 4, Graphpad Software Inc., San Diego, USA).

■ RESULTSPreparation and Physical Characterization of DQA-

somes. Preliminary experiments using a solvent dialysismethod indicated this was unsuitable for preparation ofcurcumin-loaded DQAsomes (data not shown). Curcumin,however, was successfully encapsulated in DQAsomes using thethin-film hydration method (Table I). The yields for drug-freeDQAsomes were 77.4 ± 4.2% and 98.2 ± 3.4%, for thoseprepared at 25 and 80 °C, respectively.DQAsomes prepared at 25 °C were larger at certain molar

ratios and had lower drug loading compared to those preparedat 80 °C. The surface charge, expressed as ζ potential, wasapproximately +50 mV, independent of DQA to curcuminmolar ratio and the preparation temperature. The meanhydrodynamic diameter of DQAsomes prepared at highertemperature was between 170 and 200 nm, and the sizeincreased with the amount of encapsulated curcumin.DQAsomes(1:0.5) were significantly smaller than DQAsomes(1:2)or DQAsomes(1:3) (p < 0.05). Drug loading increased withmolar ratio from 9.2% to 61.0%, and PDI was between 0.23 and0.29. Encapsulation efficiency was approximately 90%,independent of DQA to curcumin molar ratio. Surface charge,PDI, and encapsulation efficiency did not significantly differ (p> 0.05) between the DQAsome formulations.

Characterization of Solid Samples. XRD Analysis. XRDpatterns of curcumin, DQA, their physical mixture, andlyophilized formulations of DQAsomes(1:0.5) and DQA-somes(1:2) are presented in Figure 2. The spectrum of DQAshows the main peaks at 9.4, 22.5, 23.3, 23.5, and 25.6°,indicating a high level of crystallinity (Figure 2a). The spectrum

Figure 1. TSI used in our study: (a) jet nebulizer, (b) throat, (c)upper stage, and (d) lower stage. The TSI was equipped also with (e)a compressor and (f) a vacuum pump.

Figure 2. XRD patterns of (a) DQA, (b) curcumin, (c) a physicalmixture of DQA and curcumin in the molar ratio of 1:2, (d)lyophilized DQAsomes(1:0.5) and (e) lyophilized DQAsomes(1:2).

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of curcumin shows the main peaks at 8.8, 12.0, 14.3, 17.2, 18.0,20.9, 23.2, 24.4, 25.4, 27.2, and 28.8° (Figure 2b), alsoindicating a high level of crystallinity. The physical mixtureexhibits characteristic peaks of both components with theintensity proportional to their fraction in the mixture (Figure2c). The spectra of both lyophilized DQAsome formulationsdid not reveal any characteristic peaks, indicating theamorphous state of both components (Figure 2d and e).DSC Analysis. The DSC curves shown in Figure 3 show the

thermal characteristics of curcumin, DQA, their physical

mixture, and lyophilized DQAsomes(1:0.5) and DQAsomes(1:2).The DSC curve of pure DQA exhibits a single endothermicpeak at 337.96 °C, due to its melting and degradation (Figure3a). The characteristic sharp endothermic melting peak ofcurcumin is seen at 176.11 °C (Figure 3b), while the physicalmixture of DQA and curcumin shows a broader melting peak atslightly lower temperature (170.12 °C) (Figure 3c). Lyophi-lized samples of DQAsomes(1:0.5) and DQAsomes(1:2) exhibitglass transitions at 101.91 and 96.68 °C (Figure 3d and e).FT-IR Analysis. FT-IR was used to explore the interactions

between curcumin and DQA in DQAsomes (Figure 4). TheFT-IR spectrum of DQA (Figure 4a) exhibits two absorptionpeaks for primary amine groups at 3343 and 3268 cm−1 forasymmetric and symmetric N−H stretches. The strong broadpeak at 3085 cm−1 and the sharp peak at 1609 cm−1 are due tovibration of aromatic CC bonds. The peaks at 2929 and2847 cm−1 result from stretching and deformation of methylgroups. The FT-IR spectrum of curcumin (Figure 4b) shows asharp peak at 3516 cm−1 and a broad peak at 3292 cm−1 whichindicate the presence of −OH groups. Vibrations of CC andCO bonds are combined in a strong peak at 1629 cm−1. Thestrong band at 1604 cm−1 is caused by symmetric aromatic ringstretching vibrations. The stretching of the carbonyl group isvisible as a peak at 1508 cm−1, the enol group at 1279 cm−1,and the C−O−C fragment at 1024 cm−1. Benzoate trans-CHvibrations are visible at 962 cm−1, and cis −CH vibrations ofthe aromatic ring at 713 cm−1.The FT-IR spectrum of the physical mixture exhibits peaks

corresponding to both components of the mixture (Figure 4c,d, and e). The signals corresponding to curcumin were lessintense, at lower molar ratio of DQA:curcumin. For lyophilizedcurcumin-loaded DQAsomes, a decrease in resolution ofinfrared spectra was observed compared to the physical mixtureof the same composition (Figure 4f, g, and h). The positions ofthe peaks in the FT-IR spectra of the physical mixture orlyophilized DQAsomes are at the same wavenumber with

deviations of ±1 cm−1 independent of molar ratio of DQA andcurcumin in DQAsomes. The positions of the characteristicpeaks, which are shifted in the case of lyophilized DQAsomeformulations, are shown in Table II.The FT-IR spectrum of lyophilized DQAsomes exhibited

fewer intense and sharp peaks between 1659 and 1604 cm−1

compared to the FT-IR spectrum of the physical mixture. Thepeak corresponding to the phenol C−O bond of curcumin at1427 cm−1 and the peaks in the range 1235−1181 cm−1

disappeared or were less pronounced in lyophilized DQAsomeformulations. Small peaks at 1316, 977, and 886 cm−1 are notvisible in the FT-IR spectra of DQAsomes.

Morphology of DQAsomes. TEM was used to character-ize the morphology of plain DQAsomes prepared at 80 °C andcurcumin-loaded DQAsomes(1:0.5) just after preparation andafter prolonged storage (Figure 5). Plain DQAsomes werespherical, with a mean size of 100−220 nm (Figure 5a). PureDQA formed, beside DQAsomes, also smaller particles of about30 nm (Figure 5a, marked with arrow). DQAsomes(1:0.5)exhibited different morphology, namely irregularly sphericalwith wrinkles and with an average size of 200 nm (Figure 5b).This remained unchanged after prolonged storage (Figure 5c).

Critical Micelle Concentration of DQAsomes. Thecritical micelle concentration of DQAsomes was calculated tobe 1.14 × 10−6 M according to the pyrene fluorescence assay.This value was at least 1000-fold lower than those ofconventional detergents and 10-fold lower than those ofother micellar preparations,21 and this suggests that theintegrity of the micelles would be maintained post-dilution.

Physical Stability Following 40 day Storage at RoomTemperature. The physical characteristics of DQAsomes80

after prolonged storage are shown in Table III. Compared tothe characteristics of particles following preparation (Table I), asignificant increase in mean particles was observed forDQAsomes(1:0.5) and an increase in surface charge forDQAsomes(1:3). Drug loading and encapsulation efficiencydecreased depending on curcumin to DQA ratio. At the highestinvestigated curcumin to DQA ratio, most of the drugprecipitated during storage.

Antioxidant Properties of Curcumin. The antioxidantactivity of curcumin-loaded DQAsomes was evaluated using theFRAP test and was compared to the antioxidant activity ofcurcumin in a solution of methanol used as a standard (Figure6). The antioxidant activity of pure curcumin in methanol didnot significantly differ compared to that of DQAsome samplesin methanol (ANOVA, p > 0.05). But the antioxidant activity ofpure curcumin in water was significantly lower compared to theantioxidant activity of curcumin-loaded DQAsomes in water.The antioxidant activities of all curcumin-loaded DQAsomeformulations was comparable (ANOVA, p > 0.05). DQAsomesdissolved in methanol exerted significantly higher (9.4 ± 4.0%)antioxidant activities compared to the antioxidant activity of thesame formulation in water (paired t test, p < 0.001). Theantioxidant activity of curcumin dissolved in water was 45%lower compared to that of curcumin dissolved in methanol and41% lower compared to those of the samples of DQAsomes inwater. No significant difference in antioxidant activity wasobserved between samples, after preparation and after prolongstorage in either methanol or water (data not shown). PlainDQAsomes did not show any antioxidant activity.

Aerosol Properties of Curcumin-Loaded DQAsomesDelivered from a Jet Nebulizer. DQAsome(1:0.5) formulationwas nebulized using a jet nebulizer (Pari LC Sprint, Pari

Figure 3. DSC curves of (a) DQA, (b) curcumin, (c) a physicalmixture of DQA and curcumin in the molar ratio of 1:2, (d)lyophilized DQAsomes(1:0.5) and (e) lyophilized DQAsomes(1:2).

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Medical Ltd., GmbH, Starnberg, Germany) in a TSI for 1 min.The total mass balance for curcumin is presented in Table IVand was within the pharmacopeial limit, being 75−125% of theaverage delivered dose.20 Figure 7 represents only the totalcurcumin dose delivered to the throat, upper stage, and lowerstage excluding the curcumin remaining in the air-jet nebulizer.More than 85% of the delivered curcumin was deposited in the

lower stage of the TSI, indicating the ability of an air-jetnebulizer to produce DQAsome formulation aerosols, with adroplet size small enough to reach the deepest part of theapparatus. Size, PDI, and surface charge of DQAsomes(1:0.5)before nebulization, of DQAsomes(1:0.5) that remained in thedevice, and of DQAsomes(1:0.5) deposited in TSI are shown inTable V. No significant difference was found in the mean size

Figure 4. FT-IR spectra in the ranges (A) 3600−2800 cm−1 and (B) 1700−650 cm−1 for (a) DQA, (b) curcumin, physical mixtures of DQA andcurcumin in the molar ratios (c) 1:0.5, (d) 1:1, and (e) 1:2, (f) lyophilized DQAsomes(1:0.5), (g) lyophilized DQAsomes(1:1) and (h) lyophilizedDQAsomes(1:2). Decrease in resolution and disappearance of characteristic peaks is emphasized with rectangles, and main shifts are marked witharrows.

Table II. Characteristic Peak Positions in FT-IR Spectra of Various Samples

curcumin(cm−1)

DQA(cm−1)

physical mixturea

(cm−1)lyophilized DQAsomesb

(cm−1) peak assignment according to Kolev et al.34

3508 3508 3331 OH stretching of phenol group3056 3056 3100 aromatic CC

1626 1625 1621 CO, CC aromatic stretching1505 1506 1509 CO stretching, C−C−C, C−CO in plane bending1151 1155 1152 1122 C−O−C stretching, in plane bending of aromatic and skeletal CCH1025 1025 1030 out of plane bending of CH3, in plane bending of aromatic CCH855 854 856 846 out of plane bending of aromatic and skeletal CCH807 808 808 818 out of plane bending of aromatic CCH

aAverage of analyzed physical mixtures DQA:curcumin in molar ratios 1:0.5, 1:1, and 1:2. bAverage of analyzed lyophilized DQAsomes(1:0.5),DQAsomes(1:1), and DQAsomes(1:2).

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or charge of DQAsomes(1:0.5) in the device before and afternebulization (p > 0.05). However, PDI was significantly higher(p < 0.05) after nebulization. The average size and PDI ofDQAsomes(1:0.5) in the upper and lower stage was significantlylarger after nebulization compared to the formulation beforenebulization. The surface charge of DQAsomes(1:0.5) depositedin the upper and lower stage was significantly lower than thatbefore nebulization.Evaluation of Formulation Safety Using the MTS

Assay. A549 cell viability following 72 h exposure to curcumin-loaded DQAsomes and curcumin dissolved in DMSO wasassessed according to the MTS assay, shown in Figure 8. Aswith curcumin dissolved in DMSO, curcumin-loaded DQA-somes demonstrated cell viability greater than 80% when usedat concentrations below 3 μM. This preliminary data providespromising information that supports the application ofcurcumin-loaded DQAsomes for pulmonary delivery applica-tions.Ability of DQAsomes to Target Mitochondria.

Confocal microscopy imaging revealed high levels of curcuminaccumulation in Caco-2 cells (Figure 9), indicating high cellularuptake of DQAsomes. Cell mitochondria and nuclei werestained with specific markers, and the merged image clearlydemonstrates mitochondrial-specific accumulation of curcumin.Thus, curcumin-loaded DQAsomes exhibited efficient cellularuptake and possessed the ability to specifically target thecellular mitochondria.

■ DISCUSSIONPreparation and Physical Characterization of DQA-

somes. Two methods were evaluated for the preparation ofcurcumin-loaded DQAsomes, namely the solvent dialysis andthin-film methods. However, curcumin was only successfullyincorporated in DQAsomes using the thin film method. Thehigher temperature (80 °C) used in the DQAsome preparationled to a significantly improved curcumin encapsulationefficiency and drug loading compared to preparation at 25 °C

Figure 5. TEM images of (a) plain DQAsomes80 and smaller particlesof DQA (marked with an arrow) and DQAsomes(1:0.5) (b) just afterpreparation and (c) after storage for 40 days.

Table III. Hydrodynamic Diameter (d), Polydispersity Index (PDI), Surface Charge, Drug Loading (DL) and EncapsulationEfficiency (EE) of Curcumin-Loaded DQAsomes after 40 days of Storage (mean ± S.D., n = 3)

sample nDQA:ncur d (nm) PDI surface charge (mV) DL (%) EE (%)

DQAsomes(1:0.5) 1:0.5 186.7 ± 11.4a 0.28 ± 0.05 +55.2 ± 3.1 18.4 ± 1.5a 71.1 ± 5.6a

DQAsomes(1:1) 1:1 170.7 ± 7.3 0.29 ± 0.07 +60.9 ± 1.5 22.0 ± 0.9b 53.4 ± 2.3b

DQAsomes(1:2) 1:2 200.7 ± 37.0 0.22 ± 0.03 +53.6 ± 0.9 24.3 ± 2.1b 41.6 ± 3.6b

DQAsomes(1:3) 1:3 225.1 ± 12.2 0.34 ± 0.05 +59.7 ± 2.2a 7.1 ± 1.4b 10.4 ± 2.1b

ap < 0.05. bp < 0.001.

Figure 6. Antioxidant activity of pure curcumin and curcumin-loadedDQAsomes. Samples were diluted with methanol or water to achieve500 μM final concentration of curcumin (mean ± S.D., n = 3).

Table IV. Distribution of Curcumin Delivered asDQAsomes(1:0.5) between the Nebulizer and Various Parts ofthe TSI (mean ± S.D., n = 3)

% of curcumin dose

Nebulizer 80.1 ± 4.9Throat 0.7 ± 0.1Upper stage 1.8 ± 0.2Lower stage 14.0 ± 0.6Total 96.5 ± 5.5

Figure 7. Distribution of delivered dose of curcumin (DQA-somes(1:0.5)) in the TSI (mean ± S.D., n = 3).

Table V. Hydrodynamic Diameter (d), Polydispersity Index(PDI) and Surface Charge of DQAsomes(1:0.5) before andafter Nebulization, and Deposited in the TSI

d (nm) PDISurface charge

(mV)

Prenebulization 170.4 ± 4.8 0.25 ± 0.03 +59.0 ± 2.1Device (nebulizer) 187.9 ± 27.6 0.35 ± 0.07a +59.3 ± 1.3Upper stage 276.0 ± 21.0a 0.40 ± 0.09a +28.7 ± 0.9a

Lower stage 366.0 ± 14.2a 0.27 ± 0.05a +49.6 ± 1.5a

ap < 0.05.

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(Table I). The increase in solubility of DQA at highertemperatures may be an important factor contributing to theincreased drug loading, as was shown in the case of the yieldsfor drug-free DQAsomes. The mean size of DQAsomesincreased with drug loading, due to incorporation of the drugin DQA vesicles. DQAsomes showed a very high capacity forcurcumin incorporation, with theoretically one molecule ofDQA in DQAsomes entrapping three molecules of curcuminwith ∼90% efficiency (DQAsomes(1:3)). No previous study hasdemonstrated drug loading as high as 61% (DQAsomes(1:3))and curcumin solubility of 9.3 mg/mL, as achieved in thisstudy. Tonnesen et al. reported the solubility of curcumin inwater to be only 1.1 × 10−5 mg/mL.22 Yang et al. (2012)formulated micelles wherein curcumin was conjugated topoly(lactic acid) via tris(hydroxymethyl)aminomethane, achiev-ing an encapsulation efficiency of 18.5 ± 1.3%,23 while a drugloading of 12.95 ± 0.15% curcumin was achieved usingmonomethoxy poly(ethylene glycol)-poly(ε-caprolactone) mi-celles.24

The surface charge of all curcumin-loaded DQAsomeformulations was ∼ +50 mV, whereas ∼ +34 mV has beenreported for plain DQAsomes by Vaidya et al.25 The dataindicate that incorporation of curcumin results in increasedsurface charge and, therefore, improved physical stability. Thehydrodynamic diameter of DQAsomes(1:0.5) determined byphoton correlation spectroscopy (160.7 ± 2.9 nm) wasunchanged after storage for 40 days.

TEM imaging revealed the presence of two differentstructures in plain DQAsome samples: larger spheres, withoutany visible substructure, and much smaller particles (Figure 5a).The spherical morphology of plain DQAsomes has beenreported previously by Weissig et al.12 The smaller particles canbe, according to Attwood et al. (1980), DQA micelles, whichform spontaneously in an aqueous environment.26 On the otherhand, Weissig et al. (1998) observed formation of DQAaggregates with a diameter between about 70 and 700 nm,which were too large to be micelles.12 However, in ourexperiment the critical micelle concentration of DQAsomes wasdetermined to be 1.14 × 10−6 M. Curcumin-loaded DQAsomesexhibited a unique new shape (Figure 5b), which is similarneither to plain DQAsomes nor to the previously reportedshape of paclitaxel-loaded DQAsomes, which measured 673 ±19 nm and were rode-like in shape.25,27 The curcumin-loadedparticles formulated in this study were approximately spherical;however, their surface seemed folded, unlike the smooth surfacetypically seen for plain DQAsomes (Figure 5). A consequenceof the folded structure was an increase in surface area;therefore, larger numbers of DQA cations could have beenexposed on the particle surface. The ζ potential wasconsequently increased up to ∼ +50 mV compared to plainDQAsomes with ζ potential +34 mV.XRD studies revealed that curcumin in DQAsomes was in

the amorphous form, in agreement with previous reports forcurcumin inclusion into cyclodextrins.28 Thermal analysis wasperformed using two heating cycles. The first heating up to 110°C caused water removal from samples, and the second heatingproduced the DSC curve of dry sample. The DSC curve ofcurcumin shows a melting peak at 176 °C (Figure 3b), aspreviously reported.29 The melting peak of curcumin inphysical mixtures with DQA was slightly broader and shiftedto lower temperature (170 °C), since DQA behaves as animpurity in curcumin (Figure 3c). DSC studies confirmed theresults of XRD, which demonstrated the amorphous state ofcurcumin in lyophilized DQAsomes. The incorporation ofcurcumin in nanovesicles suppressed the crystallization ofcurcumin resulting in an amorphous state.30 The lyophilizationprocess on the other hand often generates amorphoussubstances, as for instance in the study of curcumin byHegge et al.31 Therefore, the change in the curcumin state wasdue to its incorporation in DQAsomes; however, following thelyophilization process, the amorphous state was preserved.The localization of curcumin in dry DQAsome formulations

was investigated using FT-IR. The peaks of curcumin spectraagreed with previous reports.28 The shifts of characteristicpeaks observed in FT-IR spectra can represent changes in

Figure 8. A549 cell viability, post 72 h exposure with curcumin-loadedDQAsomes (■) and curcumin dissolved in DMSO (●) as per theMTS assay. Results given as average (n = 3) ± standard deviation.

Figure 9. Mitochondrial targeting with DQAsomes in Caco-2 cells: (a) internalized curcumin-loaded DQAsomes (green), (b) Mitotracker-stainedcell mitochondria (red), (c) stained cell nuclei (blue), (d) merged image, showing significant overlap of curcumin-loaded DQAsome and cellmitochondria fluorescence (yellow).

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conformation of a substance, as it interacts with othercompounds present in the sample.32 FT-IR spectra oflyophilized DQAsome formulations showed some differencescompared to spectra of pure curcumin or physical mixtures ofcurcumin and DQA (Figure 4). Absorption peaks at 3292 and3516 cm−1 observed in spectra of curcumin and the physicalmixture were for lyophilized curcumin-loaded DQAsomesshifted and merged in one broader peak at 3331 cm−1 (TableII). Li et al. (2013) reported that the peak broadensconsiderably if curcumin is in an amorphous state. Such apeak has a maximum at around 3400 cm−1 and a shoulder at3500 cm−1, due to a difference in the molecular environment ofthe hydroxyl groups in amorphous relative to crystallinecurcumin.33 Kolev et al. (2005) investigated curcumin inacetonitrile solution and confirmed hydrogen bonding betweenthe hydroxyl group of curcumin and the nitrogen atom ofacetonitrile close to 3360 cm−1.34 Therefore, the presence ofhydrogen bonding between the hydroxyl group in curcuminand the amino group in DQA can also be expected. Thedecrease in resolution and the disappearance of and decrease inthe intensity of some peaks in the FT-IR spectra of all threelyophilized DQAsome formulations compared to the physicalmixtures of curcumin and DQA indicated incorporation ofcurcumin in DQAsomes. The presence of a π−π interactionbetween the aromatic rings of DQA and curcumin can be seenfrom a shift of the aromatic CC bond peak from 3056 to3100 cm−1 (Figure 4). There were several other shifts ofaromatic and skeletal C−C−C, C−H and CC peaksobserved as a result of CH−π interactions and cation−πinteractions, which indicate a different conformation of thelipophilic parts of curcumin and DQA in lyophilizedDQAsomes compared to their conformation in the physicalmixture.25,32

The high drug loading achieved is likely to be a consequenceof strong interactions and good compatibility between bothDQAsome building blocks, namely DQA and curcumin.Structurally, curcumin has two aryl rings with several functionalgroups, including β-diketone, methoxy, and hydroxyl groups,while DQA has two cationic lipophilic groups linked via analkyl chain spanning between them. Therefore, there arepossible electrostatic interactions, hydrophobic forces, andhydrogen bonding between DQA and curcumin. Since thecalculated log P of curcumin is 2.56 (determined usingChemBioDraw Ultra 13.0; Cambridgesoft Corporation, Cam-bridge, MA, USA), it is poorly water-soluble and is thereforeincorporated in the vesicle bilayer, as was also shown by Hunget al.35 It is known that lipophilic drugs (log P > 5) areincorporated within the bilayers, while drugs with log P below1.7 usually reside within the aqueous core of bilayered vesicles.The incorporation of drugs with log P values between 1.7 and5, such as curcumin, is more difficult;36 therefore, their lossfrom the vesicles can occur. Physical stability data support thisstatement, since drug precipitation was observed afterprolonged storage, resulting in decreased encapsulationefficiency and drug loading of DQAsomes. Weissig et al.(1998) have presented DQAsomes as single layer vesicles.37

Curcumin can be anchored in a lipid layer, but it has a tendencyto expose its hydroxyl groups on either site of the surface of thelipid layer and its incorporation can cause thinning of the lipidlayer.35,38 The hydroxyl group of curcumin may form hydrogenbonds either with water molecules or with the amino group ofDQA. Wang et al. (2009) showed that positively charged DQAmicelles or vesicles exerted strong interactions with curcumin

phenoxide ion, which resulted in a shift in peak of curcuminabsorbance.39 In the case of DQAsomes(1:0.5) and DQA-somes(1:2) in water, we did not observe any shift compared tocurcumin in water; therefore, it can be concluded that themajority of the curcumin was in the nonionized state (data notshown). Furthermore, the absorbance spectra were similar tothose for pure curcumin in water. This phenomenon indicatesthat curcumin may have interacted strongly with watermolecules in the Stern layer of the vesicle, i.e. the layerbetween the core/water interface and the hydrodynamic shearsurface.40 The π−cation interactions between curcumin arylrings and DQA cation confirmed by FT-IR studies may have animportant influence on the drug loading. To conclude, theincorporation of curcumin in DQAsomes was achieved bypassive loading since no additional forces were employed toenhance drug loading. Curcumin was incorporated in theDQAsome lipid layer and successfully stabilized the vesiclestructure with hydrophobic interactions and hydrogen bondsbetween hxdroxyl groups at the surface of vesicles and watermolecules or the amino group of DQA. The presence of the π−cation interactions additionally contributed to the achievementof very high drug loading.

Antioxidant Properties of Curcumin. Antioxidantactivity was determined by FRAP assay, which is a redox-linked colorimetric assay using antioxidant to reduce ferrictripyridyl triazine to ferrous form at low pH and has beenvalidated for 39 different antioxidants in various solvents.41 Itwas previously used for determination of curcumin reducingpower.42 A key advantage of the experimental approach used inour study is a direct evaluation of the antioxidant activity ofcurcumin incorporated in DQAsomes. The structure ofDQAsomes was preserved upon dilution with water, contraryto the method where DQAsomes are diluted with methanoland disintegrated, resulting in a methanol solution of DQA andcurcumin. Therefore, FRAP assay represents a significantadvantage over the more commonly used DPPH assay sinceit enables the evaluation of the antioxidant activity of drugincorporated in the nanodelivery system without the need todisintegrate the carrier. Several previous studies have usedantioxidant assays to evaluate the antioxidant activity ofcurcumin incorporated in nanoparticles.30,42a However, a directcomparison of their findings with our results is not possible dueto wide variations in the antioxidant tests used (e.g., FRAP,DPPH, ABTS), the procedures employed for preparation of thecurcumin standard solution, incubations times, and the mode inwhich the results were presented, i.e. absorbance of the testsolution, FRAP values, Trolox equivalents, or IC50 value forcurcumin.30,41−43

The antioxidant activities of DQAsomes and pure curcumindissolved in methanol were not significantly different in ourstudy, indicating that preparation of curcumin-loaded DQA-somes did not adversely affect the antioxidant activity ofcurcumin. Plain DQAsomes did not show any antioxidantactivity, and consequently, the antioxidant activity of curcumin-loaded DQAsomes dissolved in methanol was not increasedcompared to that of pure curcumin. Furthermore, theantioxidant activity of DQAsomes after prolonged storage wascomparable to that immediately after preparation. Theantioxidant activity of the aqueous DQAsome dispersion was90% of the antioxidant activity of pure curcumin or DQAsomesdissolved in methanol, indicating highly preserved antioxidantpotential of curcumin in DQAsomes, before its release from thenanodelivery system. Since the curcumin-loaded DQAsomes

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exerted antioxidant activity, this indicates that curcumin wasincorporated in the vesicles in a manner that enableslocalization of its functional groups which are important forantioxidant activity on the surface, being accessible to ferricions. The result is in line with data previously published forliposome bilayers.44 The small decrease in antioxidant activityof curcumin in DQAsomes may be attributed to theinteractions of DQA cation with the electronegative group ofcurcumin, as previously shown by Ke et al. (2011) in the studyof cationic surfactants.45 Comparison between the antioxidantactivity of curcumin in DQAsomes and in aqueous solutionindicated significantly improved antioxidant potential. Theresult is in agreement with a study reporting improvedantioxidant potential following curcumin incorporation in ananoparticulate system;30,46 however, the possible disturbancesin the structure of a nanoparticulate system after addition ofDPPH in methanol were not taken into account.Aerosol Properties of Curcumin-Loaded DQAsomes

Delivered from a Jet Nebulizer. Based on the demonstratedpotential of a curcumin dry powder inhalation formulation,11

we have developed a novel curcumin-loaded inhalationformulation for mitochondrial targeting. Curcumin-loadedDQAsomes were nebulized with an air-jet nebulizer, since itwas often used in previous studies for investigation of liposomenebulization in the early stages of research.47 The advantages ofthis device are its wide applicability, since almost every liquiddrug formulation can be nebulized, large dose volumes can beadministered, and it enables formation of sufficiently small sizedaerosol appropriate for inhalation.47b DQAsomes(1:0.5) were themost physically and chemically stable of the investigatedformulations, and therefore, they were chosen for nebulizationexperiments. The results of these, using a jet nebulizer, havedemonstrated that curcumin-loaded DQAsomes were predom-inantly delivered (>85% of delivered dose) in the lower stage ofthe TSI (Figure 7). This demonstrates the potential ofDQAsome formulations for deep lung deposition, essentialfor effective local treatment of acute lung injury. In the upperstage of the device, some precipitation was detected, indicatingleakage of the drug from DQAsomes in the nebulizationprocess. Several studies showed that vesicular systems are notstable in the nebulization process, which causes the leakage ofthe drug from the vesicles due to the breakage of liposomes andliposome aggregates, when they pass through the nebuli-zer.47b,48 Nebulization resulted in an increase in the meandiameter and polydispersity of DQAsomes deposited in theTSI. Other studies have reported a decrease in the size ofliposomes due to nebulization.47b,c The mechanism is recyclingof large liposomes in the nebulizer until their size is smallenough to be included in the secondary aerosol emitted by thenebulizer.47b However, results of these studies are in line withour data, reporting an increase in the size of nanodeliverysystems, such as liposomes, biodegradable nanoparticles, andalso polystyrene microparticle after nebulization.49 The authorssuggested various reasons for this phenomenon. Abu-Dahab etal. (2001) proposed that an increase in the average size afternebulization may be due to the collapse of some liposomes andtheir aggregation or cleavage.47a The jet nebulizer causes a highfrequency of particle collisions, resulting in aggregation due tothe hydrophobic interactions between them as reported byDailey et al.49a The aggregation is therefore closely related tothe concentration of DQAsomes in the aerosol. Chattopadhyayet al. studied the effect of nebulization on liposome dispersionswith different concentrations (0.1 and 1.0 mg/mL).50 The size

of liposomes in the sample with smaller concentrationremained unchanged, whereas the size of liposomes in a ten-times more concentrated dispersion significantly increased afternebulization. The reason was probably the high concentrationof liposomes in the aerosol droplets, which may be pushedtogether due to droplet collision in the baffle. The increasedinterparticle interaction during this process can triggeraggregation in the absence of strong hydration and repulsiveforces.50 This mechanism can also be applied in the case of ourcurcumin-loaded DQAsomes, since the concentration of anebulized dispersion of curcumin-loaded DQAsomes wasapproximately 4 mg/mL.

Curcumin Delivered by DQAsomes Can Reach CellMitochondria. The subcellular target sites of treatment withcurcumin are mitochondria, since they represent the site ofmajor intracellular free radical formation. Therefore, the abilityof DQAsomes to enter the cells and deliver curcumin inmitochondria was evaluated in a preliminary in vitro test usingthe Caco-2 cell model. The aim was to provide exploratoryinformation about the ability of DQAsomes to target cellularmitochondria using an epithelial cell line taken as arepresentative model. The assumption about the similarity inthe transport characteristics between Caco-2 cells and airwaycell cultures, e.g., Calu-3 and 16HBE14o-, has already beenproposed by other researchers.51 Furthermore, Ma et al. (2003)reported similar results on the uptake of chitosan nanoparticlesin Caco-2 and adenocarcinomic human alveolar basal epithelialcells (A549).52 Our results indicate high cellular uptake ofcurcumin-loaded DQAsomes and demonstrate their ability tospecifically target the cellular mitochondrial membrane (Figure9). Similar confocal microscopy images, indicating mitochon-drial targeting, were obtained by Marrache and Dhar, whoinvestigated the influence of size and charge of DQAsomes onmitochondrial uptake.53 They showed that a high positivesurface charge (+34.5 mV; as compared with the surface chargeof our DQAsomes; ∼ +50 mV) improves their mitochondrialuptake due to electrostatic attraction with the negativemembrane potential of mitochondria.53 The targeting proper-ties of DQAsomes are of key importance, since curcumin-loaded DQAsomes are intended for treatment of acute lunginjury, which is caused by hypoxia of lung cells, leading torelease of reactive oxygen species from the inner mitochondrialmembrane to the intermembrane space. The reactive oxygenspecies cause the activation of transcription factors, includinghypoxia-inducible factor, activation of hypoxic pulmonaryvasoconstriction, activation of AMP-dependent protein kinase,and internalization of the membrane Na,K-ATPase from thebasolateral membrane of alveolar epithelial cells.54 Admin-istration of a mitochondria-targeted formulation can deliver thedrug to the site where free radicals are formed, thereforeimproving the treatment efficiency and decreasing side effects.Curcumin, as an antioxidant encapsulated in a nanodeliverysystem with expressed mitochondria targeting properties, candecrease the production of reactive oxygen species as previouslypublished for other mitochondria targeting antioxidants, such asMitoVit-E and MitoQ10,55 and consequently reduce lunginjury. In summary, the confirmed targeting of curcumin inmitochondria by DQAsomes is a promising characteristic of anovel curcumin nanoformulation for the treatment of acutelung injury.

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■ CONCLUSIONIn this study, formulation of curcumin-loaded DQAsomes forpulmonary delivery is presented for the first time. The highdrug loading, enhanced curcumin aqueous solubility, successfulDQAsome nebulization with the majority of the delivered dosedeposited in the second stage of the in vitro lung model,preserved antioxidant activity and potential for mitochondrialtargeting demonstrate that curcumin-loaded DQAsomes are apromising formulation approach for the achievement ofimproved curcumin bioavailability. The design and develop-ment of these novel nanocarriers and their detailed analysis invitro in this study gives way to future in vivo work. In summary,the targeting of curcumin in mitochondria by application of aninhalation formulation offers a novel approach for efficientcurcumin delivery and potential effective treatment of acutelung injury.

■ ASSOCIATED CONTENT*S Supporting InformationChromatogram of curcumin dissolved in methanol. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel: ++44 (0) 207 753 5987. Fax: ++44 (0) 207 753 5942. E-mail: s.somavarapu@ucl.ac.uk.NotesThe authors declare no competing financial interest.

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Molecular Pharmaceutics Article

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Molecular Pharmaceutics Article

dx.doi.org/10.1021/mp500003q | Mol. Pharmaceutics XXXX, XXX, XXX−XXXL