Design and Development of Novel Mitochondrial Targeted

Article pubs.acs.org/molecularpharmaceutics

Design and Development of Novel Mitochondrial Targeted Nanocarriers, DQAsomes for Curcumin Inhalation Špela Zupančič,† 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, Aškerčeva 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 antiinflammatory properties but poor absorption following oral administration owing to its low aqueous solubility. Development of novel formulations to improve its in vivo efficacy is therefore challenging. In this study, formulation of curcuminloaded DQAsomes (vesicles formed from the amphiphile, dequalinium) for pulmonary delivery is presented for the first time. The vesicles demonstrated mean hydrodynamic diameters between 170 and 200 nm, with a ζ potential of approximately +50 mV, high drug loading (up to 61%) and encapsulation efficiency (90%), resulting in enhanced curcumin aqueous solubility. Curcumin encapsulation in DQAsomes in the amorphous state was confirmed by X-ray diffraction and differential scanning calorimetry analysis. The existence of hydrogen bonds and cation−π interaction between curcumin and vesicle building blocks, namely dequalinium molecules, were shown in lyophilized DQAsomes using FT-IR analysis. Encapsulation of curcumin in DQAsomes enhanced the antioxidant activity of curcumin compared to free curcumin. DQAsome dispersion was successfully nebulized with the majority of the delivered dose deposited in the second stage of the twin-stage impinger. The vesicles showed potential for mitochondrial targeting. Curcumin-loaded DQAsomes thus represent a promising inhalation formulation with improved stability characteristics and mitochondrial targeting ability, indicating a novel approach for efficient curcumin delivery for effective treatment of acute lung injury and the rationale for future in vivo studies. KEYWORDS: acute lung injury, antioxidant, curcumin, DQAsomes, nebulization

■

INTRODUCTION Acute lung injury is defined as a secondary illness that occurs in response to various primary etiologies such as severe pneumonia, sepsis, multiple traumas, and massive blood transfusion.1 The pathogenesis of acute lung injury involves inflammatory cells, cytokines, and chemokines, as well as activators and inhibitors of apoptosis which give rise to the generation of free radicals and reactive oxygen species. This results in injury of lung endothelium and epithelium, leading to an increase in lung vascular and epithelial permeability, causing pulmonary edema due to the passage of protein-rich fluid into the alveolar air spaces.2 Promising initial investigations focused on pharmaco-therapies such as administration of exogenous surfactant and inhalation of nitric oxide, glucocorticoids, and lysofylline. However, comprehensive studies have failed to demonstrate favorable clinical outcomes.3 Novel potential therapeutic strategies include mesenchymal stem cell therapy4 and application of anti-inflammatory and antioxidant molecules that act on diverse signaling pathways.5 Curcumin, a polyphenolic compound derived from the tuberaceous plant Curcuma longa, has been shown to exhibit a © XXXX American Chemical Society

wide range of therapeutic properties, such as antimicrobial and chemo-preventive properties in several types of cancer and hepato-protective, antiaggregatory, antioxidant, and antiinflammatory effects. In an induced lung injury model in rats, curcumin treatment significantly inhibited inflammatory response and prevented inhibition of the antioxidant enzymes superoxide dismutase and glutathione peroxidase.6 In a cecal ligature puncture induced model of acute lung injury in rats, curcumin treatment was demonstrated to exert protective effects and significantly increase the survival rate of animals by 40−50%.7 The clinical applications of curcumin are severely limited due to its low oral bioavailability, which is a consequence of its low aqueous solubility, poor cellular uptake, high rate of metabolism in the intestine, and rapid elimination from the body.8 Pulmonary delivery offers a promising approach that may Received: January 3, 2014 Revised: April 8, 2014 Accepted: May 22, 2014

A

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

Molecular Pharmaceutics

Article

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

nDQA:ncur

DQAsomes25(1:0.5) DQAsomes25(1:2) DQAsomes(1:0.5) DQAsomes(1:1) DQAsomes(1:2) DQAsomes(1:3)

1:1 1:2 1:0.5 1:1 1:2 1:3

d (nm) 176.1 205.8 160.7 173.2 203.2 203.5

± ± ± ± ± ±

19.3 8.0 2.9 21.1 15.9 9.9

PDI 0.25 0.27 0.29 0.23 0.25 0.24

± ± ± ± ± ±

surface charge (mV)

0.02 0.07 0.07 0.03 0.03 0.05

+46.8 +48.6 +52.3 +53.7 +49.9 +44.0

± ± ± ± ± ±

1.5 4.3 1.7 1.9 6.5 2.0

DL (%) 9.2 31.9 22.5 38.1 53.4 61.0

± ± ± ± ± ±

1.5 5.5 1.0 1.9 2.4 2.0

EE (%) 35.5 54.8 86.7 92.6 91.6 90.2

± ± ± ± ± ±

5.7 19.1 3.9 4.6 4.1 3.0

09042001, ECACC, U.K.). Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Media (MEM), 4-morpholineethanesulfonic acid 2-(N-morpholino)ethanesulfonic acid hydrate 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 obtained from Life Technologies Ltd., Paisley, U.K. Culture plates and flasks were from Nunc, Denmark. Preparation of Curcumin-Loaded DQAsomes. DQAsomes were prepared using a thin-film hydration method with minor modifications.12 Briefly, DQA and curcumin at certain molar ratios (Table I) were dissolved in 20 mL of methanol. The concentration of DQA was kept constant at 5 mg/mL, and the concentration of curcumin varied to achieve the required DQA:curcumin ratios. The solvent was then evaporated using a rotary evaporator (Hei-VAP Advantage Rotary Evaporator, Heidolph, Germany) at 150 rpm, 80 °C and under vacuum (KNF Laboport, KNF Neuberger, Germany) for 10 min to obtain a thin film. The resultant thin film was hydrated with 20 mL of water and mixed thoroughly at 25 or 80 °C for 2 min and sonicated using a VWR Ultrasonic cleaner bath USC300T (VWR International Limited, U.K.) for 20 min. The solution obtained was filtered twice through a sterile 0.45 μm filter (Millex-MP, Millipore, Carrigtwohill, Ireland) to remove unentrapped curcumin. Samples were prepared in triplicate and lyophilized using a Virtis AdVantage 2.0 BenchTop freezedryer (SP Industries, U.K.) for storage and further analysis. Size and Surface Charge of DQAsomes. Following dilution in deionized water, the size distribution of the DQAsomes was obtained as ZAve hydrodynamic diameter and polydispersity index (PDI) by photon correlation spectroscopy. The surface charge was measured by laser Doppler microelectrophoresis. These studies were performed using the ZetasizerNano ZS (Malvern Instruments, U.K.). All experiments were performed in triplicate, and results are presented as mean ± SD. HPLC Analysis of Curcumin. Curcumin concentration was determined by reversed-phase high performance liquid chromatography (HPLC) using the Discovery HS F5 HPLC column (L × I.D. 15 cm × 4.6 mm, 5 μm particle size, 120 Å pore diameter, Supelco, USA) at 25 °C and UV detection at 428 nm. The mobile phase was a mixture of water and acetonitrile 55:45 (v/v), supplemented with 0.1% (v/v) trifluoroacetic acid. The flow rate was 1 mL/min and run time 15 min. The representative chromatogram of curcumin (Supporting Information Figure SI) dissolved in methanol is available online at http://pubs.acs.org/. The method was validated according to ICH guidelines Validation of analytical procedures: text and methodology15 and FDA reviewer guidance Validation of chromatographic methods.16 Quantification of DQA. The concentration of DQA in curcumin-free DQAsomes was spectrophotometrically deter-

overcome some of the limitations mentioned and enhance the therapeutic potential of curcumin, particularly for local lung conditions. The reasons are the high local concentration of drug that can be achieved via pulmonary application and reduced exposure to enzymatic activity as compared to delivery via the oral route. In addition, a large alveolar surface area is also available for absorption when delivered via the pulmonary route.9 Sandersen et al. showed positive results in an in vivo study of pulmonary delivery of a soluble curcumin derivative, which had an inhibitory effect in neutrophil induced inflammation in the lower airways of horses affected by recurrent airway obstruction.10 Swellable nano- and microparticulate systems with encapsulated curcumin exhibited good controlled release and showed promising aerosolization characteristics in the in vitro model.11 Dequalinium (DQA) has been used for more than 50 years as an antimicrobial agent, and its oral health care application is FDA approved. In an aqueous medium, DQA single-chain bolaamphiphile molecules self-associate and form vesicles named DQAsomes.12 The cationic nature and mitochondria-targeting properties of DQAsomes have been exploited for delivery of exogenous DNA into mitochondria, which was shown to be successful but with low transfection efficiency (1−5%).13 DQAsomes resulted in a 5-fold increase in the apoptotic activity of paclitaxel in colo205 cancer cell lines.14 These studies clearly show the ability of DQAsomes to overcome cellular membranes and deliver the payload intracellularly. In this study, a novel curcumin-loaded nanoformulation was developed to enable its delivery via in vitro pulmonary administration, as an alternative to the oral route. Curcumin was incorporated in DQAsomes with the aim of enhancing its aqueous solubility and maintaining its antioxidant potential. The DQAsome formulation was nebulized to explore its potential for pulmonary delivery for local treatment of acute lung injury. Finally, preliminary studies of the mitochondrial targeting ability of the DQAsomes were performed so as to ascertain the potential of these carriers for future in vivo studies.

■

MATERIALS AND METHODS Materials. Unless otherwise stated, all chemicals were analytical grade. Dequalinium chloride hydrate (DQA; 95%), trifluoroacetic acid (99%), RPMI-1640, L-glutamine, fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (PBS), and pyrene (98%) were purchased from Sigma-Aldrich, U.K. Curcumin (total curcuminoid content 95%) from turmeric rhizome was obtained from Alfa Cesar, USA. Acetonitrile, methanol, and water were HPLC grade and were supplied by Fisher Scientific, U.K. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium compound (MTS) and phenazine methosulfate (PMS) where obtained from Promega, CA, USA. Caco-2 cells were purchased from the European Collection of Cell Cultures (Catalogue no. B



Article

micelles form. This was determined using the pyrene fluorescence assay, using a fluorescence spectrometer (PerkinElmer precisely LS55 luminescence spectrometer, Wellesley, USA). The vibration band intensities of the fluorescent probe, pyrene, change in response to the polarity of the surrounding environment. Above the critical micelle concentration, a decrease is observed in the ratio between the first and third vibrational peaks (I1 and I3, respectively) of the emission spectra obtained. This corresponds to the entrapment of pyrene within the interior hydrophobic core of the micelles.17 The critical micelle concentration of DQA micelles was determined via a serial dilution of the formulation with the addition of pyrene at a concentration of 6 × 10−7 M. The fluorescence spectra were then read at an emission wavelength of 332 nm and an excitation of 335 nm. The critical micelle concentration value was taken as the point of intersection between the two tangents drawn from the graph of the intensity ratio (I1/I3) against the log DQA concentration.18 Physical Stability of DQAsomes at Room Temperature. All formulations of curcumin-loaded DQAsomes prepared by the thin film method at 80 °C were stored at room temperature, protected from light for 40 days. Following this period, the size distribution, surface charge, drug loading, and encapsulation efficiency were measured, as described previously. Drug loading and encapsulation efficiency analysis were performed after the samples were filtered once through a sterile 0.45 μm filter to remove any aggregates or precipitated drug present in the samples. Antioxidant Properties of Curcumin. The antioxidant activity of curcumin-loaded DQAsomes was determined using the ferric ion reducing antioxidant power (FRAP) assay, with some modification.19 Acetate buffer (pH 3.6), tripyridyl triazine, and iron(III) chloride were mixed to prepare the FRAP reagent mixture. The concentrations of curcumin in DQAsome formulations (DQAsomes(1:0.5), DQAsomes(1:1), and DQAsomes(1:2)) were spectrophotometrically determined just after preparation and after 60 days using a microtiter plate reader (VersaMax, Molecular Devices, USA) at 428 nm. To analyze antioxidant activity, all DQAsome samples and curcumin stock solution in methanol (2.7 mM) were diluted with either water or methanol to the final concentration of curcumin 500 μM. FRAP assay was carried out by addition of samples (DQAsomes or curcumin solution) (30 μL) to FRAP reagent (900 μL), and the reaction mixture was incubated for 30 min at 25 °C. 300 μL of the samples obtained were then transferred into a 96-well microtiter plate and absorbance measured at 593 nm. FRAP reagent mixture without any additives was used as a blank. The antioxidant activity of DQAsome samples was compared to the antioxidant activity of curcumin in methanol solution. Determination of the Aerosol Properties of Curcumin-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 aerodynamic diameter 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 30 mL of water were placed in the upper and the lower stages, respectively. DQAsomes(1:0.5) (2 mL) were added to a Pari LC Sprint nebulizer attached to a TurboBoy N compressor (Pari Medical Ltd., GmbH, Starnberg, Germany) directed toward the throat of the TSI. The pump was switched on 10 s before the nebulizer was run for 60 s, and for an additional 5 s after the pump was switched off. Liquid and washings from the nebulizer

mined at 328 nm after dissolution of DQAsomes in methanol (Jenway 7315 UV/visible spectrophotometer, Camlab, Staffordshire, U.K.). Determination of Drug Loading and Encapsulation Efficiency. DQAsomes were dissolved with methanol to achieve the theoretical concentration of curcumin in the solution 14 ± 2 μg/mL. Five microliters of the sample was injected into the HPLC system and analyzed. The amount of curcumin 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 DQAsomes m(DQAsomes) = amount of curcumin loaded DQAsomes m(cur-T) = theoretical amount of curcumin in DQAsome XRD Analysis. X-ray diffraction patterns were obtained for pure curcumin, DQA, their physical mixture (molar ratio DQA:curcumin of 1:2), lyophilized DQAsomes(1:0.5), and DQAsomes(1:2) using an X-ray diffractometer (Rigaku MiniFlex 600, Miniflex, Japan). The samples were analyzed at room temperature 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 mg sample was weighed in a Tzero aluminum pan (TA Instruments, U.K.) and covered with a lid having a 50 μm pinhole. Curcumin, the physical mixture of DQA and curcumin in the molar ratio 1:2, and lyophilized formulations of DQAsomes(1:0.5) and DQAsomes(1:2) were analyzed using a heating rate of 10 °C/min in the temperature range 0 to 110 °C. When the sample reached 110 °C, it was kept at isothermal conditions for 5 min prior to cooling to 0 °C and reheating up to 200 °C. DQA was analyzed using the same conditions with the exception of the second heating cycle, which was continued to 350 °C. The measurements were performed in an inert nitrogen atmosphere with a flow rate of 50 mL/min. DSC heating curves were analyzed using Universal Analysis 2000 software (TA Instruments). Thermal transitions reported here are 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 a PerkinElmer Spectrum 100 FT-IR spectrometer (PerkinElmer, USA) from 650 to 4000 cm−1 at a resolution of 4 cm−1. Each sample was measured with 20 scans. Data were analyzed using the PerkinElmer Spectrum Express software (PerkinElmer, USA). Morphology of DQAsomes. Morphological analysis of plain DQAsomes and DQAsomes(1:0.5), just after preparation and after 40 days, was performed by transmission electron microscopy (TEM) using an FEI CM 120 BioTwin transmission electron microscope (Philips Electron Optics BV, Netherlands) using acceleration voltage 120.0 kV. Approximately 40 μL of the DQAsome dispersion was placed on a Formvar/carbon coated copper grid and negatively stained with 1% 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 C



Article

MitoTracker Red CMXRos in MEM pH 5.8 adjusted with 10 mM MES buffer. The cell monolayer was then washed twice with DPBS and fixed with 3.9% paraformaldehyde solution. Cell nuclei were stained with TO-PRO-3 (1 μM) for 1 h at room temperature. The samples were examined under a confocal microscope (Leica TCS SP2, Leica microsystems, Milton Keynes, U.K.) to assess mitochondrial targeting properties of curcumin-loaded DQAsomes. Images were analyzed using the Leica LCS Lite software suite (Leica microsystems, Milton Keynes, U.K.). Statistical Analysis. The data were expressed as mean ± standard deviation (S.D.). The results were statistically analyzed using paired Student’s t test or one way analysis of variance (ANOVA) followed by Turkey’s posthoc test (PRISM software package, Version 4, Graphpad Software Inc., San Diego, USA).

■

RESULTS Preparation and Physical Characterization of DQAsomes. Preliminary experiments using a solvent dialysis method indicated this was unsuitable for preparation of curcumin-loaded DQAsomes (data not shown). Curcumin, however, was successfully encapsulated in DQAsomes using the thin-film hydration method (Table I). The yields for drug-free DQAsomes were 77.4 ± 4.2% and 98.2 ± 3.4%, for those prepared 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 prepared at 80 °C. The surface charge, expressed as ζ potential, was approximately +50 mV, independent of DQA to curcumin molar ratio and the preparation temperature. The mean hydrodynamic diameter of DQAsomes prepared at higher temperature was between 170 and 200 nm, and the size increased 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 with molar ratio from 9.2% to 61.0%, and PDI was between 0.23 and 0.29. 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. XRD patterns of curcumin, DQA, their physical mixture, and lyophilized formulations of DQAsomes(1:0.5) and DQAsomes(1:2) are presented in Figure 2. The spectrum of DQA shows 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.

and lower and upper stages were collected for particle size and surface charge measurements and for determination of curcumin. The total mass balance of curcumin in the TSI and nebulizer was determined according to the procedure described in the European Pharmacopoeia.20 Evaluation of Formulation Safety Using the MTS Assay. The human epithelial cell line A549 was expanded in RPMI-1640 medium comprised of 10% FBS, 1% L-glutamine, and 1× antibiotic-antimycotic at 37 °C in a humidified incubator containing 5% CO2. Cells were seeded onto 96well plates at 2000 cells per well, with cell viability being quantitatively assessed using MTS with PMS. Cells were exposed 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 further 2 h. MTS then yields a water-soluble formazan product and the optical density of the wells was measured using a multidetection microplate reader (Synergy HT, Bio-Tek Instruments, VT, USA), at an absorbance maximum of 490 nm. Cell viability was expressed as a percentage relative to control cells, with cells exposed to media alone acting as the positive control and those exposed to 1% Triton-x as the negative control. Data was given as mean ± standard deviation. Ability of DQAsomes to Target Mitochondria. Caco-2 cells were obtained at passage 20, and passages 45 to 55 were used in experiments. Cells were seeded onto 6-well plates at an initial seeding density of 3 × 104 cells/cm2 and cultured at 37 °C in an atmosphere with 5% CO2 in air. The medium (FCS supplemented DMEM) was replaced every 2 days. The experiments were carried out 14 days post-seeding, when Caco-2 cells differentiated to a fully matured gastrointestinal tract 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 curcumin in the incubation media. DQAsome sample was prepared by dilution of DQAsome(1:0.07) formulation with a serum-free MEM, pH 5.8 adjusted with 10 mM MES buffer. Caco-2 cells were then incubated for 2 h to allow the DQAsomes to be internalized. Following incubation, medium was removed and cells were washed with DPBS and incubated for 30 min with a 200 nM solution of red fluorescent mitochondrial dye

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



Article

deviations of ±1 cm−1 independent of molar ratio of DQA and curcumin in DQAsomes. The positions of the characteristic peaks, which are shifted in the case of lyophilized DQAsome formulations, 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. The peak corresponding to the phenol C−O bond of curcumin at 1427 cm−1 and the peaks in the range 1235−1181 cm−1 disappeared or were less pronounced in lyophilized DQAsome formulations. Small peaks at 1316, 977, and 886 cm−1 are not visible in the FT-IR spectra of DQAsomes. Morphology of DQAsomes. TEM was used to characterize the morphology of plain DQAsomes prepared at 80 °C and curcumin-loaded DQAsomes(1:0.5) just after preparation and after prolonged storage (Figure 5). Plain DQAsomes were spherical, with a mean size of 100−220 nm (Figure 5a). Pure DQA formed, beside DQAsomes, also smaller particles of about 30 nm (Figure 5a, marked with arrow). DQAsomes(1:0.5) exhibited different morphology, namely irregularly spherical with wrinkles and with an average size of 200 nm (Figure 5b). This remained unchanged after prolonged storage (Figure 5c). Critical Micelle Concentration of DQAsomes. The critical micelle concentration of DQAsomes was calculated to be 1.14 × 10−6 M according to the pyrene fluorescence assay. This value was at least 1000-fold lower than those of conventional detergents and 10-fold lower than those of other micellar preparations,21 and this suggests that the integrity of the micelles would be maintained post-dilution. Physical Stability Following 40 day Storage at Room Temperature. The physical characteristics of DQAsomes80 after prolonged storage are shown in Table III. Compared to the characteristics of particles following preparation (Table I), a significant increase in mean particles was observed for DQAsomes(1:0.5) and an increase in surface charge for DQAsomes(1:3). Drug loading and encapsulation efficiency decreased depending on curcumin to DQA ratio. At the highest investigated curcumin to DQA ratio, most of the drug precipitated during storage. Antioxidant Properties of Curcumin. The antioxidant activity of curcumin-loaded DQAsomes was evaluated using the FRAP test and was compared to the antioxidant activity of curcumin in a solution of methanol used as a standard (Figure 6). The antioxidant activity of pure curcumin in methanol did not significantly differ compared to that of DQAsome samples in methanol (ANOVA, p > 0.05). But the antioxidant activity of pure curcumin in water was significantly lower compared to the antioxidant activity of curcumin-loaded DQAsomes in water. The antioxidant activities of all curcumin-loaded DQAsome formulations was comparable (ANOVA, p > 0.05). DQAsomes dissolved in methanol exerted significantly higher (9.4 ± 4.0%) antioxidant activities compared to the antioxidant activity of the same formulation in water (paired t test, p < 0.001). The antioxidant activity of curcumin dissolved in water was 45% lower compared to that of curcumin dissolved in methanol and 41% lower compared to those of the samples of DQAsomes in water. No significant difference in antioxidant activity was observed between samples, after preparation and after prolong storage in either methanol or water (data not shown). Plain DQAsomes did not show any antioxidant activity. Aerosol Properties of Curcumin-Loaded DQAsomes Delivered from a Jet Nebulizer. DQAsome(1:0.5) formulation was nebulized using a jet nebulizer (Pari LC Sprint, Pari

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), also indicating a high level of crystallinity. The physical mixture exhibits characteristic peaks of both components with the intensity proportional to their fraction in the mixture (Figure 2c). The spectra of both lyophilized DQAsome formulations did not reveal any characteristic peaks, indicating the amorphous 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

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

mixture, and lyophilized DQAsomes(1:0.5) and DQAsomes(1:2). The DSC curve of pure DQA exhibits a single endothermic peak at 337.96 °C, due to its melting and degradation (Figure 3a). The characteristic sharp endothermic melting peak of curcumin is seen at 176.11 °C (Figure 3b), while the physical mixture of DQA and curcumin shows a broader melting peak at slightly lower temperature (170.12 °C) (Figure 3c). Lyophilized samples of DQAsomes(1:0.5) and DQAsomes(1:2) exhibit glass 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). The FT-IR spectrum of DQA (Figure 4a) exhibits two absorption peaks for primary amine groups at 3343 and 3268 cm−1 for asymmetric and symmetric N−H stretches. The strong broad peak at 3085 cm−1 and the sharp peak at 1609 cm−1 are due to vibration of aromatic CC bonds. The peaks at 2929 and 2847 cm−1 result from stretching and deformation of methyl groups. The FT-IR spectrum of curcumin (Figure 4b) shows a sharp peak at 3516 cm−1 and a broad peak at 3292 cm−1 which indicate the presence of −OH groups. Vibrations of CC and CO bonds are combined in a strong peak at 1629 cm−1. The strong band at 1604 cm−1 is caused by symmetric aromatic ring stretching vibrations. The stretching of the carbonyl group is visible 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-CH vibrations are visible at 962 cm−1, and cis −CH vibrations of the 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 less intense, at lower molar ratio of DQA:curcumin. For lyophilized curcumin-loaded DQAsomes, a decrease in resolution of infrared spectra was observed compared to the physical mixture of the same composition (Figure 4f, g, and h). The positions of the peaks in the FT-IR spectra of the physical mixture or lyophilized DQAsomes are at the same wavenumber with E



Article

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 and curcumin 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) lyophilized DQAsomes(1:2). Decrease in resolution and disappearance of characteristic peaks is emphasized with rectangles, and main shifts are marked with arrows.

Table II. Characteristic Peak Positions in FT-IR Spectra of Various Samples curcumin (cm−1)

DQA (cm−1)

3508 3056 1626 1505 1151 1025 855 807

1155 854 808

physical mixturea (cm−1)

lyophilized DQAsomesb (cm−1)

peak assignment according to Kolev et al.34

3508 3056 1625 1506 1152 1025 856 808

3331 3100 1621 1509 1122 1030 846 818

OH stretching of phenol group aromatic CC CO, CC aromatic stretching CO stretching, C−C−C, C−CO in plane bending C−O−C stretching, in plane bending of aromatic and skeletal CCH out of plane bending of CH3, in plane bending of aromatic CCH out of plane bending of aromatic and skeletal CCH out of plane bending of aromatic CCH

a

Average 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).

Medical Ltd., GmbH, Starnberg, Germany) in a TSI for 1 min. The total mass balance for curcumin is presented in Table IV and was within the pharmacopeial limit, being 75−125% of the average delivered dose.20 Figure 7 represents only the total curcumin dose delivered to the throat, upper stage, and lower stage 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-jet nebulizer to produce DQAsome formulation aerosols, with a droplet size small enough to reach the deepest part of the apparatus. Size, PDI, and surface charge of DQAsomes(1:0.5) before nebulization, of DQAsomes(1:0.5) that remained in the device, and of DQAsomes(1:0.5) deposited in TSI are shown in Table V. No significant difference was found in the mean size F



Article

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

Figure 6. Antioxidant activity of pure curcumin and curcumin-loaded DQAsomes. Samples were diluted with methanol or water to achieve 500 μM final concentration of curcumin (mean ± S.D., n = 3).

or charge of DQAsomes(1:0.5) in the device before and after nebulization (p > 0.05). However, PDI was significantly higher (p < 0.05) after nebulization. The average size and PDI of DQAsomes(1:0.5) in the upper and lower stage was significantly larger after nebulization compared to the formulation before nebulization. The surface charge of DQAsomes(1:0.5) deposited in the upper and lower stage was significantly lower than that before nebulization. Evaluation of Formulation Safety Using the MTS Assay. A549 cell viability following 72 h exposure to curcuminloaded DQAsomes and curcumin dissolved in DMSO was assessed according to the MTS assay, shown in Figure 8. As with curcumin dissolved in DMSO, curcumin-loaded DQAsomes demonstrated cell viability greater than 80% when used at concentrations below 3 μM. This preliminary data provides promising information that supports the application of curcumin-loaded DQAsomes for pulmonary delivery applications. Ability of DQAsomes to Target Mitochondria. Confocal microscopy imaging revealed high levels of curcumin accumulation in Caco-2 cells (Figure 9), indicating high cellular uptake of DQAsomes. Cell mitochondria and nuclei were stained with specific markers, and the merged image clearly demonstrates mitochondrial-specific accumulation of curcumin. Thus, curcumin-loaded DQAsomes exhibited efficient cellular uptake and possessed the ability to specifically target the cellular mitochondria.

Table IV. Distribution of Curcumin Delivered as DQAsomes(1:0.5) between the Nebulizer and Various Parts of the TSI (mean ± S.D., n = 3) % of curcumin dose Nebulizer Throat Upper stage Lower stage Total

80.1 0.7 1.8 14.0 96.5

± ± ± ± ±

4.9 0.1 0.2 0.6 5.5

Figure 7. Distribution of delivered dose of curcumin (DQAsomes(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 and after Nebulization, and Deposited in the TSI

■

DISCUSSION Preparation and Physical Characterization of DQAsomes. Two methods were evaluated for the preparation of curcumin-loaded DQAsomes, namely the solvent dialysis and thin-film methods. However, curcumin was only successfully incorporated in DQAsomes using the thin film method. The higher temperature (80 °C) used in the DQAsome preparation led to a significantly improved curcumin encapsulation efficiency and drug loading compared to preparation at 25 °C

d (nm) Prenebulization Device (nebulizer) Upper stage Lower stage a

170.4 187.9 276.0 366.0

± ± ± ±

4.8 27.6 21.0a 14.2a

PDI

Surface charge (mV)

± ± ± ±

+59.0 +59.3 +28.7 +49.6

0.25 0.35 0.40 0.27

0.03 0.07a 0.09a 0.05a

± ± ± ±

2.1 1.3 0.9a 1.5a

p < 0.05.

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

a

sample

nDQA:ncur

DQAsomes(1:0.5) DQAsomes(1:1) DQAsomes(1:2) DQAsomes(1:3)

1:0.5 1:1 1:2 1:3

d (nm) 186.7 170.7 200.7 225.1

± ± ± ±

11.4a 7.3 37.0 12.2

PDI 0.28 0.29 0.22 0.34

± ± ± ±

0.05 0.07 0.03 0.05

surface charge (mV) +55.2 +60.9 +53.6 +59.7

± ± ± ±

3.1 1.5 0.9 2.2a

DL (%) 18.4 22.0 24.3 7.1

± ± ± ±

1.5a 0.9b 2.1b 1.4b

EE (%) 71.1 53.4 41.6 10.4

± ± ± ±

5.6a 2.3b 3.6b 2.1b

p < 0.05. bp < 0.001. G



Article

TEM imaging revealed the presence of two different structures in plain DQAsome samples: larger spheres, without any visible substructure, and much smaller particles (Figure 5a). The spherical morphology of plain DQAsomes has been reported previously by Weissig et al.12 The smaller particles can be, according to Attwood et al. (1980), DQA micelles, which form spontaneously in an aqueous environment.26 On the other hand, Weissig et al. (1998) observed formation of DQA aggregates with a diameter between about 70 and 700 nm, which were too large to be micelles.12 However, in our experiment the critical micelle concentration of DQAsomes was determined to be 1.14 × 10−6 M. Curcumin-loaded DQAsomes exhibited a unique new shape (Figure 5b), which is similar neither to plain DQAsomes nor to the previously reported shape of paclitaxel-loaded DQAsomes, which measured 673 ± 19 nm and were rode-like in shape.25,27 The curcumin-loaded particles formulated in this study were approximately spherical; however, their surface seemed folded, unlike the smooth surface typically seen for plain DQAsomes (Figure 5). A consequence of the folded structure was an increase in surface area; therefore, larger numbers of DQA cations could have been exposed on the particle surface. The ζ potential was consequently increased up to ∼ +50 mV compared to plain DQAsomes with ζ potential +34 mV. XRD studies revealed that curcumin in DQAsomes was in the amorphous form, in agreement with previous reports for curcumin inclusion into cyclodextrins.28 Thermal analysis was performed using two heating cycles. The first heating up to 110 °C caused water removal from samples, and the second heating produced the DSC curve of dry sample. The DSC curve of curcumin shows a melting peak at 176 °C (Figure 3b), as previously reported.29 The melting peak of curcumin in physical mixtures with DQA was slightly broader and shifted to lower temperature (170 °C), since DQA behaves as an impurity in curcumin (Figure 3c). DSC studies confirmed the results of XRD, which demonstrated the amorphous state of curcumin in lyophilized DQAsomes. The incorporation of curcumin in nanovesicles suppressed the crystallization of curcumin resulting in an amorphous state.30 The lyophilization process on the other hand often generates amorphous substances, as for instance in the study of curcumin by Hegge et al.31 Therefore, the change in the curcumin state was due to its incorporation in DQAsomes; however, following the lyophilization process, the amorphous state was preserved. The localization of curcumin in dry DQAsome formulations was investigated using FT-IR. The peaks of curcumin spectra agreed with previous reports.28 The shifts of characteristic peaks observed in FT-IR spectra can represent changes in

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

(Table I). The increase in solubility of DQA at higher temperatures may be an important factor contributing to the increased drug loading, as was shown in the case of the yields for drug-free DQAsomes. The mean size of DQAsomes increased with drug loading, due to incorporation of the drug in DQA vesicles. DQAsomes showed a very high capacity for curcumin incorporation, with theoretically one molecule of DQA in DQAsomes entrapping three molecules of curcumin with ∼90% efficiency (DQAsomes(1:3)). No previous study has demonstrated drug loading as high as 61% (DQAsomes(1:3)) and curcumin solubility of 9.3 mg/mL, as achieved in this study. Tonnesen et al. reported the solubility of curcumin in water to be only 1.1 × 10−5 mg/mL.22 Yang et al. (2012) formulated micelles wherein curcumin was conjugated to poly(lactic acid) via tris(hydroxymethyl)aminomethane, achieving an encapsulation efficiency of 18.5 ± 1.3%,23 while a drug loading of 12.95 ± 0.15% curcumin was achieved using monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) micelles.24 The surface charge of all curcumin-loaded DQAsome formulations was ∼ +50 mV, whereas ∼ +34 mV has been reported for plain DQAsomes by Vaidya et al.25 The data indicate that incorporation of curcumin results in increased surface charge and, therefore, improved physical stability. The hydrodynamic diameter of DQAsomes(1:0.5) determined by photon correlation spectroscopy (160.7 ± 2.9 nm) was unchanged after storage for 40 days.

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



Article

phenoxide ion, which resulted in a shift in peak of curcumin absorbance.39 In the case of DQAsomes(1:0.5) and DQAsomes(1:2) in water, we did not observe any shift compared to curcumin in water; therefore, it can be concluded that the majority of the curcumin was in the nonionized state (data not shown). Furthermore, the absorbance spectra were similar to those for pure curcumin in water. This phenomenon indicates that curcumin may have interacted strongly with water molecules in the Stern layer of the vesicle, i.e. the layer between the core/water interface and the hydrodynamic shear surface.40 The π−cation interactions between curcumin aryl rings and DQA cation confirmed by FT-IR studies may have an important influence on the drug loading. To conclude, the incorporation of curcumin in DQAsomes was achieved by passive loading since no additional forces were employed to enhance drug loading. Curcumin was incorporated in the DQAsome lipid layer and successfully stabilized the vesicle structure with hydrophobic interactions and hydrogen bonds between hxdroxyl groups at the surface of vesicles and water molecules or the amino group of DQA. The presence of the π− cation interactions additionally contributed to the achievement of very high drug loading. Antioxidant Properties of Curcumin. Antioxidant activity was determined by FRAP assay, which is a redoxlinked colorimetric assay using antioxidant to reduce ferric tripyridyl triazine to ferrous form at low pH and has been validated for 39 different antioxidants in various solvents.41 It was previously used for determination of curcumin reducing power.42 A key advantage of the experimental approach used in our study is a direct evaluation of the antioxidant activity of curcumin incorporated in DQAsomes. The structure of DQAsomes was preserved upon dilution with water, contrary to the method where DQAsomes are diluted with methanol and disintegrated, resulting in a methanol solution of DQA and curcumin. Therefore, FRAP assay represents a significant advantage over the more commonly used DPPH assay since it enables the evaluation of the antioxidant activity of drug incorporated in the nanodelivery system without the need to disintegrate the carrier. Several previous studies have used antioxidant assays to evaluate the antioxidant activity of curcumin incorporated in nanoparticles.30,42a However, a direct comparison of their findings with our results is not possible due to wide variations in the antioxidant tests used (e.g., FRAP, DPPH, ABTS), the procedures employed for preparation of the curcumin standard solution, incubations times, and the mode in which the results were presented, i.e. absorbance of the test solution, FRAP values, Trolox equivalents, or IC50 value for curcumin.30,41−43 The antioxidant activities of DQAsomes and pure curcumin dissolved in methanol were not significantly different in our study, indicating that preparation of curcumin-loaded DQAsomes did not adversely affect the antioxidant activity of curcumin. Plain DQAsomes did not show any antioxidant activity, and consequently, the antioxidant activity of curcuminloaded DQAsomes dissolved in methanol was not increased compared to that of pure curcumin. Furthermore, the antioxidant activity of DQAsomes after prolonged storage was comparable to that immediately after preparation. The antioxidant activity of the aqueous DQAsome dispersion was 90% of the antioxidant activity of pure curcumin or DQAsomes dissolved in methanol, indicating highly preserved antioxidant potential of curcumin in DQAsomes, before its release from the nanodelivery system. Since the curcumin-loaded DQAsomes

conformation of a substance, as it interacts with other compounds present in the sample.32 FT-IR spectra of lyophilized DQAsome formulations showed some differences compared to spectra of pure curcumin or physical mixtures of curcumin and DQA (Figure 4). Absorption peaks at 3292 and 3516 cm−1 observed in spectra of curcumin and the physical mixture were for lyophilized curcumin-loaded DQAsomes shifted and merged in one broader peak at 3331 cm−1 (Table II). Li et al. (2013) reported that the peak broadens considerably if curcumin is in an amorphous state. Such a peak has a maximum at around 3400 cm−1 and a shoulder at 3500 cm−1, due to a difference in the molecular environment of the hydroxyl groups in amorphous relative to crystalline curcumin.33 Kolev et al. (2005) investigated curcumin in acetonitrile solution and confirmed hydrogen bonding between the hydroxyl group of curcumin and the nitrogen atom of acetonitrile close to 3360 cm−1.34 Therefore, the presence of hydrogen bonding between the hydroxyl group in curcumin and the amino group in DQA can also be expected. The decrease in resolution and the disappearance of and decrease in the intensity of some peaks in the FT-IR spectra of all three lyophilized DQAsome formulations compared to the physical mixtures of curcumin and DQA indicated incorporation of curcumin in DQAsomes. The presence of a π−π interaction between the aromatic rings of DQA and curcumin can be seen from a shift of the aromatic CC bond peak from 3056 to 3100 cm−1 (Figure 4). There were several other shifts of aromatic and skeletal C−C−C, C−H and CC peaks observed as a result of CH−π interactions and cation−π interactions, which indicate a different conformation of the lipophilic parts of curcumin and DQA in lyophilized DQAsomes compared to their conformation in the physical mixture.25,32 The high drug loading achieved is likely to be a consequence of strong interactions and good compatibility between both DQAsome building blocks, namely DQA and curcumin. Structurally, curcumin has two aryl rings with several functional groups, including β-diketone, methoxy, and hydroxyl groups, while DQA has two cationic lipophilic groups linked via an alkyl chain spanning between them. Therefore, there are possible electrostatic interactions, hydrophobic forces, and hydrogen bonding between DQA and curcumin. Since the calculated log P of curcumin is 2.56 (determined using ChemBioDraw Ultra 13.0; Cambridgesoft Corporation, Cambridge, MA, USA), it is poorly water-soluble and is therefore incorporated in the vesicle bilayer, as was also shown by Hung et al.35 It is known that lipophilic drugs (log P > 5) are incorporated within the bilayers, while drugs with log P below 1.7 usually reside within the aqueous core of bilayered vesicles. The incorporation of drugs with log P values between 1.7 and 5, such as curcumin, is more difficult;36 therefore, their loss from the vesicles can occur. Physical stability data support this statement, since drug precipitation was observed after prolonged storage, resulting in decreased encapsulation efficiency 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 tendency to expose its hydroxyl groups on either site of the surface of the lipid layer and its incorporation can cause thinning of the lipid layer.35,38 The hydroxyl group of curcumin may form hydrogen bonds either with water molecules or with the amino group of DQA. Wang et al. (2009) showed that positively charged DQA micelles or vesicles exerted strong interactions with curcumin I



Article

of liposomes in the sample with smaller concentration remained unchanged, whereas the size of liposomes in a tentimes more concentrated dispersion significantly increased after nebulization. The reason was probably the high concentration of liposomes in the aerosol droplets, which may be pushed together due to droplet collision in the baffle. The increased interparticle interaction during this process can trigger aggregation in the absence of strong hydration and repulsive forces.50 This mechanism can also be applied in the case of our curcumin-loaded DQAsomes, since the concentration of a nebulized dispersion of curcumin-loaded DQAsomes was approximately 4 mg/mL. Curcumin Delivered by DQAsomes Can Reach Cell Mitochondria. The subcellular target sites of treatment with curcumin are mitochondria, since they represent the site of major intracellular free radical formation. Therefore, the ability of DQAsomes to enter the cells and deliver curcumin in mitochondria was evaluated in a preliminary in vitro test using the Caco-2 cell model. The aim was to provide exploratory information about the ability of DQAsomes to target cellular mitochondria using an epithelial cell line taken as a representative model. The assumption about the similarity in the transport characteristics between Caco-2 cells and airway cell cultures, e.g., Calu-3 and 16HBE14o-, has already been proposed by other researchers.51 Furthermore, Ma et al. (2003) reported similar results on the uptake of chitosan nanoparticles in Caco-2 and adenocarcinomic human alveolar basal epithelial cells (A549).52 Our results indicate high cellular uptake of curcumin-loaded DQAsomes and demonstrate their ability to specifically target the cellular mitochondrial membrane (Figure 9). Similar confocal microscopy images, indicating mitochondrial targeting, were obtained by Marrache and Dhar, who investigated the influence of size and charge of DQAsomes on mitochondrial uptake.53 They showed that a high positive surface charge (+34.5 mV; as compared with the surface charge of our DQAsomes; ∼ +50 mV) improves their mitochondrial uptake due to electrostatic attraction with the negative membrane potential of mitochondria.53 The targeting properties of DQAsomes are of key importance, since curcuminloaded DQAsomes are intended for treatment of acute lung injury, which is caused by hypoxia of lung cells, leading to release of reactive oxygen species from the inner mitochondrial membrane to the intermembrane space. The reactive oxygen species cause the activation of transcription factors, including hypoxia-inducible factor, activation of hypoxic pulmonary vasoconstriction, activation of AMP-dependent protein kinase, and internalization of the membrane Na,K-ATPase from the basolateral membrane of alveolar epithelial cells.54 Administration of a mitochondria-targeted formulation can deliver the drug to the site where free radicals are formed, therefore improving the treatment efficiency and decreasing side effects. Curcumin, as an antioxidant encapsulated in a nanodelivery system with expressed mitochondria targeting properties, can decrease the production of reactive oxygen species as previously published for other mitochondria targeting antioxidants, such as MitoVit-E and MitoQ10,55 and consequently reduce lung injury. In summary, the confirmed targeting of curcumin in mitochondria by DQAsomes is a promising characteristic of a novel curcumin nanoformulation for the treatment of acute lung injury.

exerted antioxidant activity, this indicates that curcumin was incorporated in the vesicles in a manner that enables localization of its functional groups which are important for antioxidant activity on the surface, being accessible to ferric ions. The result is in line with data previously published for liposome bilayers.44 The small decrease in antioxidant activity of curcumin in DQAsomes may be attributed to the interactions of DQA cation with the electronegative group of curcumin, as previously shown by Ke et al. (2011) in the study of cationic surfactants.45 Comparison between the antioxidant activity of curcumin in DQAsomes and in aqueous solution indicated significantly improved antioxidant potential. The result is in agreement with a study reporting improved antioxidant potential following curcumin incorporation in a nanoparticulate system;30,46 however, the possible disturbances in the structure of a nanoparticulate system after addition of DPPH in methanol were not taken into account. Aerosol Properties of Curcumin-Loaded DQAsomes Delivered from a Jet Nebulizer. Based on the demonstrated potential of a curcumin dry powder inhalation formulation,11 we have developed a novel curcumin-loaded inhalation formulation for mitochondrial targeting. Curcumin-loaded DQAsomes were nebulized with an air-jet nebulizer, since it was often used in previous studies for investigation of liposome nebulization in the early stages of research.47 The advantages of this device are its wide applicability, since almost every liquid drug formulation can be nebulized, large dose volumes can be administered, and it enables formation of sufficiently small sized aerosol appropriate for inhalation.47b DQAsomes(1:0.5) were the most physically and chemically stable of the investigated formulations, and therefore, they were chosen for nebulization experiments. The results of these, using a jet nebulizer, have demonstrated that curcumin-loaded DQAsomes were predominantly delivered (>85% of delivered dose) in the lower stage of the TSI (Figure 7). This demonstrates the potential of DQAsome formulations for deep lung deposition, essential for effective local treatment of acute lung injury. In the upper stage of the device, some precipitation was detected, indicating leakage of the drug from DQAsomes in the nebulization process. Several studies showed that vesicular systems are not stable in the nebulization process, which causes the leakage of the drug from the vesicles due to the breakage of liposomes and liposome aggregates, when they pass through the nebulizer.47b,48 Nebulization resulted in an increase in the mean diameter and polydispersity of DQAsomes deposited in the TSI. Other studies have reported a decrease in the size of liposomes due to nebulization.47b,c The mechanism is recycling of large liposomes in the nebulizer until their size is small enough to be included in the secondary aerosol emitted by the nebulizer.47b However, results of these studies are in line with our data, reporting an increase in the size of nanodelivery systems, such as liposomes, biodegradable nanoparticles, and also polystyrene microparticle after nebulization.49 The authors suggested various reasons for this phenomenon. Abu-Dahab et al. (2001) proposed that an increase in the average size after nebulization may be due to the collapse of some liposomes and their aggregation or cleavage.47a The jet nebulizer causes a high frequency of particle collisions, resulting in aggregation due to the hydrophobic interactions between them as reported by Dailey et al.49a The aggregation is therefore closely related to the concentration of DQAsomes in the aerosol. Chattopadhyay et al. studied the effect of nebulization on liposome dispersions with different concentrations (0.1 and 1.0 mg/mL).50 The size J



■

Article

Karasalihoglu, S. Preventive effects of curcumin on different aspiration material-induced lung injury in rats. Pediatr. Surg. Int. 2009, 25 (1), 83−92. (c) Gunaydin, M.; Guzel, A.; Guzel, A.; Alacam, H.; Salis, O.; Murat, N.; Gacar, A.; Guvenc, T. The effect of curcumin on lung injuries in a rat model induced by aspirating gastrointestinal decontamination agents. J. Pediatr. Surg. 2012, 47 (9), 1669−76. (7) Xiao, X.; Yang, M.; Sun, D.; Sun, S. Curcumin protects against sepsis-induced acute lung injury in rats. J. Surg. Res. 2012, 176 (1), e31−9. (8) Nagpal, M.; Sood, S. Role of curcumin in systemic and oral health: An overview. J. Nat. Sci. Biol. Med. 2013, 4 (1), 3−7. (9) Beck-Broichsitter, M.; Merkel, O. M.; Kissel, T. Controlled pulmonary drug and gene delivery using polymeric nano-carriers. J. Controlled Release 2012, 161 (2), 214−24. (10) Sandersen, C.; Olejnik, D.; Franck, T.; Neven, P.; Serteyn, D.; Art, T. Inhalation with NDS27 attenuates pulmonary neutrophilic inflammation in recurrent airway obstruction. Vet. Rec. 2011, 169 (4), 100. (11) El-Sherbiny, I. M.; Smyth, H. D. Controlled release pulmonary administration of curcumin using swellable biocompatible microparticles. Mol. Pharmaceutics 2012, 9 (2), 269−80. (12) Weissig, V.; Lasch, J.; Erdos, G.; Meyer, H. W.; Rowe, T. C.; Hughes, J. DQAsomes: a novel potential drug and gene delivery system made from Dequalinium. Pharm. Res. 1998, 15 (2), 334−7. (13) Lyrawati, D.; Trounson, A.; Cram, D. Expression of GFP in the mitochondrial compartment using DQAsome-mediated delivery of an artificial mini-mitochondrial genome. Pharm. Res. 2011, 28 (11), 2848−62. (14) D’Souza, G. G.; Cheng, S. M.; Boddapati, S. V.; Horobin, R. W.; Weissig, V. Nanocarrier-assisted sub-cellular targeting to the site of mitochondria improves the pro-apoptotic activity of paclitaxel. J. Drug Target 2008, 16 (7), 578−85. (15) International Conference on Harmonization (ICH), Q2(R1): Validation of analytical procedures: text and methodology. Retrieved November 6, 2005. (16) Reviewer Guidance, Validation of Chromatographic Methods. Center for Drug Evaluation and Research, US Food and Drug Administration: 1994. (17) Zhang, X.; Jackson, J. K.; Burt, H. M. Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique. J. Biochem. Biophys. Methods 1996, 31 (3−4), 145−50. (18) Kalyanasundaram, K.; Thomas, J. K. Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems. J. Am. Chem. Soc. 1977, 99 (7), 2039−2044. (19) Sessa, M.; Tsao, R.; Liu, R.; Ferrari, G.; Donsi, F. Evaluation of the stability and antioxidant activity of nanoencapsulated resveratrol during in vitro digestion. J. Agric. Food Chem. 2011, 59 (23), 12352− 60. (20) 2.9.18. Preparations for Inhalation: Aerodynamic Assessment of Fine Particles. In: European pharmacopoeia 7.0; Council of Europe, European Directorate for the Quality of Medicines & HealthCare: Strasbourg, 2010: pp 274−75. (21) Lukyanov, A. N.; Gao, Z.; Mazzola, L.; Torchilin, V. P. Polyethylene glycol-diacyllipid micelles demonstrate increased acculumation in subcutaneous tumors in mice. Pharm. Res. 2002, 19 (10), 1424−9. (22) Tonnesen, H. H.; Masson, M.; Loftsson, T. Studies of curcumin and curcuminoids. XXVII. Cyclodextrin complexation: solubility, chemical and photochemical stability. Int. J. Pharm. 2002, 244 (1− 2), 127−35. (23) Yang, R.; Zhang, S.; Kong, D.; Gao, X.; Zhao, Y.; Wang, Z. Biodegradable polymer-curcumin conjugate micelles enhance the loading and delivery of low-potency curcumin. Pharm. Res. 2012, 29 (12), 3512−25. (24) Gou, M.; Men, K.; Shi, H.; Xiang, M.; Zhang, J.; Song, J.; Long, J.; Wan, Y.; Luo, F.; Zhao, X.; Qian, Z. Curcumin-loaded

CONCLUSION In this study, formulation of curcumin-loaded DQAsomes for pulmonary delivery is presented for the first time. The high drug loading, enhanced curcumin aqueous solubility, successful DQAsome nebulization with the majority of the delivered dose deposited in the second stage of the in vitro lung model, preserved antioxidant activity and potential for mitochondrial targeting demonstrate that curcumin-loaded DQAsomes are a promising formulation approach for the achievement of improved curcumin bioavailability. The design and development of these novel nanocarriers and their detailed analysis in vitro in this study gives way to future in vivo work. In summary, the targeting of curcumin in mitochondria by application of an inhalation formulation offers a novel approach for efficient curcumin delivery and potential effective treatment of acute lung injury.

■

ASSOCIATED CONTENT

S Supporting Information *

Chromatogram of curcumin dissolved in methanol. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: ++44 (0) 207 753 5987. Fax: ++44 (0) 207 753 5942. Email: [email protected]. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Bernard, G. R.; Artigas, A.; Brigham, K. L.; Carlet, J.; Falke, K.; Hudson, L.; Lamy, M.; Legall, J. R.; Morris, A.; Spragg, R. The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am. J. Respir. Crit. Care. Med. 1994, 149 (3 Pt 1), 818−24. (2) (a) Galani, V.; Tatsaki, E.; Bai, M.; Kitsoulis, P.; Lekka, M.; Nakos, G.; Kanavaros, P. The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): an up-to-date cellspecific review. Pathol. Res. Pract. 2010, 206 (3), 145−50. (b) Matthay, M. A.; Zemans, R. L. The acute respiratory distress syndrome: pathogenesis and treatment. Annu. Rev. Pathol. 2011, 6, 147−63. (3) Johnson, E. R.; Matthay, M. A. Acute lung injury: epidemiology, pathogenesis, and treatment. J. Aerosol. Med. Pulm. Drug. Delivery 2010, 23 (4), 243−52. (4) Maron-Gutierrez, T.; Silva, J. D.; Asensi, K. D.; Bakker-Abreu, I.; Shan, Y.; Diaz, B. L.; Goldenberg, R. C.; Mei, S. H.; Stewart, D. J.; Morales, M. M.; Rocco, P. R.; Dos Santos, C. C. Effects of mesenchymal stem cell therapy on the time course of pulmonary remodeling depend on the etiology of lung injury in mice. Crit. Care Med. 2013, 41 (11), e319−33. (5) (a) Lim, S. B.; Rubinstein, I.; Sadikot, R. T.; Artwohl, J. E.; Onyuksel, H. A novel peptide nanomedicine against acute lung injury: GLP-1 in phospholipid micelles. Pharm. Res. 2011, 28 (3), 662−72. (b) Melo, A. C.; Valenca, S. S.; Gitirana, L. B.; Santos, J. C.; Ribeiro, M. L.; Machado, M. N.; Magalhaes, C. B.; Zin, W. A.; Porto, L. C. Redox markers and inflammation are differentially affected by atorvastatin, pravastatin or simvastatin administered before endotoxin-induced acute lung injury. Int. Immunopharmacol. 2013, 17 (1), 57− 64. (6) (a) Xu, F.; Lin, S. H.; Yang, Y. Z.; Guo, R.; Cao, J.; Liu, Q. The effect of curcumin on sepsis-induced acute lung injury in a rat model through the inhibition of the TGF-beta1/SMAD3 pathway. Int. Immunopharmacol. 2013, 16 (1), 1−6. (b) Guzel, A.; Kanter, M.; Aksu, B.; Basaran, U. N.; Yalcin, O.; Guzel, A.; Uzun, H.; Konukoglu, D.; K



Article

K.; Parthasarathy, K.; Gokaraju, G. R.; Gokaraju, R. R.; Bhupathiraju, K.; Mandapati, V. N. S. R. R.; Somashekara, N. Curcuminoids and its metabolites for the application in allergic ocular/nasal conditions. Patent US 20120010297 A1, 2012. (44) Kristl, J.; Volk, B.; Gasperlin, M.; Sentjurc, M.; Jurkovic, P. Effect of colloidal carriers on ascorbyl palmitate stability. Eur. J. Pharm. Sci. 2003, 19 (4), 181−9. (45) Ke, D.; Wang, X.; Yang, Q.; Niu, Y.; Chai, S.; Chen, Z.; An, X.; Shen, W. Spectrometric study on the interaction of dodecyltrimethylammonium bromide with curcumin. Langmuir 2011, 27 (23), 14112− 7. (46) Kakran, M.; Sahoo, N. G.; Tan, I. L.; Li, L. Preparation of nanoparticles of poorly water-soluble antioxidant curcumin by antisolvent precipitation methods. J. Nanopart. Res. 2012, 14 (3), 1− 11. (47) (a) Abu-Dahab, R.; Schafer, U. F.; Lehr, C. M. Lectinfunctionalized liposomes for pulmonary drug delivery: effect of nebulization on stability and bioadhesion. Eur. J. Pharm. Sci. 2001, 14 (1), 37−46. (b) Bridges, P. A.; Taylor, K. M. G. Nebulisers for the generation of liposomal aerosols. Int. J. Pharm. 1998, 173 (1−2), 117− 125. (c) Zaru, M.; Mourtas, S.; Klepetsanis, P.; Fadda, A. M.; Antimisiaris, S. G. Liposomes for drug delivery to the lungs by nebulization. Eur. J. Pharm. Biopharm. 2007, 67 (3), 655−66. (48) Desai, T. R.; Hancock, R. E.; Finlay, W. H. A facile method of delivery of liposomes by nebulization. J. Controlled Release 2002, 84 (1−2), 69−78. (49) (a) Dailey, L. A.; Schmehl, T.; Gessler, T.; Wittmar, M.; Grimminger, F.; Seeger, W.; Kissel, T. Nebulization of biodegradable nanoparticles: impact of nebulizer technology and nanoparticle characteristics on aerosol features. J. Controlled Release 2003, 86 (1), 131−44. (b) Chattopadhyay, S.; Ehrman, S. H.; Bellare, J.; Venkataraman, C. Morphology and bilayer integrity of small liposomes during aerosol generation by air-jet nebulisation. J. Nanopart. Res. 2012, 14 (4), 1−15. (50) Chattopadhyay, S.; Ehrman, S.; Bellare, J.; Venkataraman, C. Morphology and bilayer integrity of small liposomes during aerosol generation by air-jet nebulisation. J. Nanopart. Res. 2012, 14 (4), 1−15. (51) (a) Johansson, F.; Hjertberg, E.; Eirefelt, S.; Tronde, A.; Hultkvist Bengtsson, U. Mechanisms for absorption enhancement of inhaled insulin by sodium taurocholate. Eur. J. Pharm. Sci. 2002, 17 (1−2), 63−71. (b) Tronde, A.; Krondahl, E.; von Euler-Chelpin, H.; Brunmark, P.; Bengtsson, U. H.; Ekstrom, G.; Lennernas, H. High airway-to-blood transport of an opioid tetrapeptide in the isolated rat lung after aerosol delivery. Peptides 2002, 23 (3), 469−78. (52) (a) Ma, Z.; Lim, L.-Y. Uptake of Chitosan and Associated Insulin in Caco-2 Cell Monolayers: A Comparison Between Chitosan Molecules and Chitosan Nanoparticles. Pharm. Res. 2003, 20 (11), 1812−1819. (b) Huang, M.; Ma, Z.; Khor, E.; Lim, L. Y. Uptake of FITC-chitosan nanoparticles by A549 cells. Pharm. Res. 2002, 19 (10), 1488−94. (53) Marrache, S.; Dhar, S. Engineering of blended nanoparticle platform for delivery of mitochondria-acting therapeutics. Proc. Natl. Acad. Sci. U S A 2012, 109 (40), 16288−93. (54) Schumacker, P. T. Lung cell hypoxia: role of mitochondrial reactive oxygen species signaling in triggering responses. Proc. Am. Thorac. Soc. 2011, 8 (6), 477−84. (55) Li, X.; Fang, P.; Mai, J.; Choi, E. T.; Wang, H.; Yang, X. F. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J. Hematol. Oncol. 2013, 6, 19.

biodegradable polymeric micelles for colon cancer therapy in vitro and in vivo. Nanoscale 2011, 3 (4), 1558−67. (25) Vaidya, B.; Vyas, S. P. Transferrin coupled vesicular system for intracellular drug delivery for the treatment of cancer: development and characterization. J. Drug Target. 2012, 20 (4), 372−80. (26) Attwood, D.; Natarajan, R. Micellar properties and surface activity of some bolaform drugs in aqueous solution. J. Pharm. Pharmacol. 1980, 32 (7), 460−2. (27) Vaidya, B.; Paliwal, R.; Rai, S.; Khatri, K.; Goyal, A. K.; Mishra, N.; Vyas, S. P. Cell-selective mitochondrial targeting: A new approach for cancer therapy. Cancer Therapy 2009, 7, 141−148. (28) Mohan, P. R. K.; Sreelakshmi, G.; Muraleedharan, C. V.; Joseph, R. Water soluble complexes of curcumin with cyclodextrins: Characterization by FT-Raman spectroscopy. Vib. Spectrosc. 2012, 62 (0), 77−84. (29) Mohanty, C.; Sahoo, S. K. The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials 2010, 31 (25), 6597−611. (30) Yen, F. L.; Wu, T. H.; Tzeng, C. W.; Lin, L. T.; Lin, C. C. Curcumin nanoparticles improve the physicochemical properties of curcumin and effectively enhance its antioxidant and antihepatoma activities. J. Agric. Food Chem. 2010, 58 (12), 7376−82. (31) Hegge, A. B.; Vukicevic, M.; Bruzell, E.; Kristensen, S.; Tonnesen, H. H. Solid dispersions for preparation of phototoxic supersaturated solutions for antimicrobial photodynamic therapy (aPDT): Studies on curcumin and curcuminoides L. Eur. J. Pharm. Biopharm. 2013, 83 (1), 95−105. (32) Bourassa, P.; Kanakis, C. D.; Tarantilis, P.; Pollissiou, M. G.; Tajmir-Riahi, H. A. Resveratrol, genistein, and curcumin bind bovine serum albumin. J. Phys. Chem. B 2010, 114 (9), 3348−54. (33) Li, B.; Konecke, S.; Wegiel, L. A.; Taylor, L. S.; Edgar, K. J. Both solubility and chemical stability of curcumin are enhanced by solid dispersion in cellulose derivative matrices. Carbohydr. Polym. 2013, 98 (1), 1108−16. (34) Kolev, T. M.; Velcheva, E. A.; Stamboliyska, B. A.; Spiteller, M. DFT and experimental studies of the structure and vibrational spectra of curcumin. Int. J. Quantum Chem. 2005, 102 (6), 1069−1079. (35) Hung, W. C.; Chen, F. Y.; Lee, C. C.; Sun, Y.; Lee, M. T.; Huang, H. W. Membrane-thinning effect of curcumin. Biophys. J. 2008, 94 (11), 4331−8. (36) Perrie, Y. Pharmaceutical nanotechnology and nanomedicines. In Aulton’s pharmaceutics: the design and manufacture of medicines, 4th ed.; Aulton, M. E., Taylor, K., Eds.; Churchill Livingstone/Elsevier: Edinburgh, 2013; pp 777−96. (37) Weissig, V. From serendipity to mitochondria-targeted nanocarriers. Pharm. Res. 2011, 28 (11), 2657−68. (38) Barry, J.; Fritz, M.; Brender, J. R.; Smith, P. E.; Lee, D. K.; Ramamoorthy, A. Determining the effects of lipophilic drugs on membrane structure by solid-state NMR spectroscopy: the case of the antioxidant curcumin. J. Am. Chem. Soc. 2009, 131 (12), 4490−8. (39) Wang, Z.; Leung, M. H.; Kee, T. W.; English, D. S. The role of charge in the surfactant-assisted stabilization of the natural product curcumin. Langmuir 2010, 26 (8), 5520−6. (40) Adhikary, R.; Carlson, P. J.; Kee, T. W.; Petrich, J. W. Excitedstate intramolecular hydrogen atom transfer of curcumin in surfactant micelles. J. Phys. Chem. B 2010, 114 (8), 2997−3004. (41) Halvorsen, B. L.; Blomhoff, R. Validation of a quantitative assay for the total content of lipophilic and hydrophilic antioxidants in foods. Food Chem. 2011, 127 (2), 761−8. (42) (a) Sonkaew, P.; Sane, A.; Suppakul, P. Antioxidant activities of curcumin and ascorbyl dipalmitate nanoparticles and their activities after incorporation into cellulose-based packaging films. J. Agric. Food. Chem. 2012, 60 (21), 5388−99. (b) Weber, W. M.; Hunsaker, L. A.; Abcouwer, S. F.; Deck, L. M.; Vander Jagt, D. L. Anti-oxidant activities of curcumin and related enones. Bioorg. Med. Chem. 2005, 13 (11), 3811−20. (43) (a) Benzie, I. F.; Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure of ″antioxidant power″: the FRAP assay. Anal. Biochem. 1996, 239 (1), 70−6. (b) Chaniyilparampu, R. N.; Nair, A. L


Design and Development of Novel Mitochondrial Targeted

Recommend Documents