Abstract
With a view to improving the solubility and delivery characteristics of poorly water-soluble drugs, we prepared β-cyclodextrin-curcumin (βCD-C) inclusion complexes (hydrophilic curcumin) and entrapped both native curcumin (hydrophobic) and the complexes separately into liposomes; these were then assessed for in vitro cytotoxicity in lung and colon cancer cell lines. Optimization of curcumin entrapment within βCD was achieved, with the resultant βCD-C complexes prepared by methanol reflux. Inclusion complexes were confirmed using UV spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction. The water solubility of βCD-C complexes improved markedly (c.f. native curcumin) and successful entrapment of complexes into liposomes, prepared using a thin-film hydration approach, was also achieved. All the liposomal formulations were characterized for curcumin and βCD-C complex entrapment efficiency, particle size, polydispersity and stability at 2–8°C. Curcumin, βCD-C complex and their optimized liposomal formulations were evaluated for anticancer activity in lung (A-459) and colon (SW-620) cancer cell lines. All curcumin-containing formulations tested were effective in inhibiting cell proliferation, as determined via an MTT assay. The median effective dose (EC50) for all curcumin formulations was found to be in the low µM range for both lung and colon cancer cell lines tested. Our results confirm that βCD inclusion complexes of poorly water soluble drugs, such as curcumin can be entrapped within biocompatible vesicles such as liposomes, and this does not preclude their anticancer activity.
Keywords::
Introduction
Curcumin, a low-molecular-weight polyphenol derived from turmeric (Curcuma longa L.), has not only been long used as a spice, but it has had a firm place for centuries in Ayurvedic and Chinese medicine formularies. A wide range of beneficial pharmacological effects of curcumin have been demonstrated, including antioxidant, antibacterial, hypocholesterolemic, anticoagulant, anti-inflammatory, hepatoprotective and antitumor (reviewed by Zhou et al. Citation2011). However, its particularly low water solubility, poor absorption upon oral administration and extensive pre-systemic metabolism leads to unfavourable pharmacokinetics (Ireson et al. Citation2002; Sharma et al. Citation2005). With a view to improving the delivery of curcumin in its native hydrophobic form, various encapsulation-based formulations, such as polymer nanoparticles (Bisht et al. Citation2007; Shaikh et al. Citation2009), surfactants (Tonnesen et al. 2002; Wang et al. Citation2010), phospholipids (Maiti et al. Citation2007), and polyethylene glycol conjugates (Safavy et al. Citation2007) have been reported. Liposome-encapsulated curcumin renders the drug amenable to intravenous administration therefore improving its bioavailability (Takahashi et al. Citation2009), as has been reported in antitumor animal models (Li et al. Citation2005).
To circumvent issues associated with the delivery of poorly water soluble drugs the use of cyclodextrin (CD)-drug inclusion complexes has also been a proven and very successful strategy (Loftsson and Brewster, Citation2010). Various forms of CDs are commercially available and their basic structure comprises a cyclic chain of carbohydrates oriented so the inner cavity is hydrophobic in character, while the outer surface is flanked by hydrophilic hydroxyl moieties. This allows hydrophobic molecules to be entrapped within the inner cavity of CDs while their outer surface permits aqueous miscibility. β-cyclodextrin (βCD) is particularly widely used to enhance drug delivery due to its low toxicity (Loftsson and Brewster Citation2010). Using βCD, various pharmacologically active hydrophobic agents including curcumin (Yadav et al. Citation2009; Yallapu et al. Citation2010), prednisolone (Fatouros et al. Citation2001), doxorubicin (Al-Omar et al. Citation1999), paclitaxel (Bouquet et al. Citation2007), and various non-steroidal anti-inflammatory drugs (Castelli et al. Citation1992) have been formulated, resulting in improved physicochemical properties and delivery efficiency.
Inclusion of curcumin within CD has been shown to improve in vitro cytotoxicity in a prostate cancer cell line (Yallapu et al. Citation2010) and in vivo anti-inflammatory activity in rats (Yadav et al. Citation2009). However, circulating CD-drug complexes are still subject to rapid elimination by organs of the reticuloendothelial system, namely, the spleen and lymph tissue (Sakai et al. Citation2004). To circumvent this, further packaging CD-drug complexes into nanovesicular liposomes, may be expected to evade the reticuloendothelial system, and if appropriately functionalised (e.g. with PEGylation) can achieve prolonged circulation times with preferential deposition in target tumour tissue (Maruyama, Citation2011; Moghimi and Szebeni, Citation2003). The encapsulation of other CD-drug complexes within liposomes has drawn much attention by researchers (Arima et al. Citation2006; Fatouros et al. Citation2001; Kaur et al. Citation2010; McCormack and Gregoriadis, Citation1994). Here we report the preparation of βCD-curcumin inclusion complexes and their entrapment within liposomes followed by subsequent assessment of in vitro cytotoxicity using model lung and colon cancer cell lines.
Materials and methods
Preparation of β-cyclodextrin-curcumin complex
βCD-C inclusion complexes were prepared following a modified methanol reflux method (Tang et al. Citation2002). Curcumin and βCD were dissolved in methanol (500 µl) and water (4 ml) respectively in ratios 1:0.5, 1:1, 1:2, 1:5 (w/w) at 60°C for 5 min. The methanolic curcumin solution was added drop-wise into aqueous βCD solution with continuous and intensive stirring. The mixture was refluxed with vigorous agitation at 70°C for 4 h. The methanol was removed from the mixture by stirring for 1 h at 70°C without reflux, followed by rotary evaporation. The aqueous mixture was cooled to room temperature and stirred for 1 h before freezing and lyophilizing to produce an amorphous powder of βCD-C complexes. A physical mixture of curcumin and βCD was prepared by mixing both in appropriate ratios (as detailed above for the inclusion complexes) and continuous stirring for 4 h at room temperature. All of the crude products were analysed for curcumin entrapment. Any unassociated-curcumin was removed by dissolving the crude mixture in cold water (4°C) followed by gravity filtration. Fresh cold water was repeatedly passed over the filter paper until clear water fractions were eluted. The water fractions were pooled and lyophilized. Residual curcumin remaining on the filter paper was dissolved using ethanol as solvent, which was then evaporated in vacuo. All samples were prepared in triplicate.
Characterization of βCD-C complexes
Dry or lyophilized powders of βCD-C complexes, curcumin, βCD, and physical mixtures of curcumin and βCD were visually examined and compared for their colour and texture. They were each also assessed using UV, Fourier transformed infrared spectroscopy (FT-IR) and X-ray diffraction. For UV, samples were dissolved in ethanol and diluted with water to make the stock solution 6 × 10−4 mol/l, which was further diluted to 6 × 10−6 mol/l. The absorbance was measured from 300 to 600 nm using a Shimadzu UV-265 spectrophotometer. For FT-IR, using a Nicolet 6700 FT-IR spectrophotometer, 40 scans from 4000 to 400 cm−1 were performed at a resolution of 4 cm−1 measuring the absorbance of KBr discs containing 1–2 mg of each sample. X-ray diffraction patterns were determined using a Rigaku Miniflex X-ray diffractometer (Rigaku Americas, Texas, USA) equipped with an X-ray source with graphite monochromator, X-ray tube and wide angle goniometer. The powder samples were placed on the sample holder after mixing with a few drops of ethanol followed by oven drying at 50°C for 3 min. Each sample was scanned (2θ) from 15° to 40° at a speed of 2° per min.
Curcumin estimation in βCD-C complexes
Two independent methods were used to estimate the efficiency of curcumin loading into βCD. Firstly, a UV-spectroscopic method was used (Yallapu et al., Citation2010). Here, βCD-C (1 mg equivalent curcumin) was dissolved in 50 ml dimethyl sulphoxide (DMSO) and vigorously agitated for 24 h at room temperature to aid curcumin dissociation from βCD. The sample was then centrifuged at 14000 rpm for 30 min to separate βCD from curcumin. The supernatant containing DMSO (and extracted curcumin) was removed, diluted and measured using UV-spectroscopic analysis at 425 nm. A standard curve prepared using curcumin in DMSO was used to quantify curcumin in the supernatant.
Secondly, curcumin was estimated using RP-HPLC. The samples were dissolved in acetonitrile (HPLC grade) at a concentration of 1 mg/ml and passed through a 0.45 μm filter (Millipore Corp., USA). The Agilent HPLC instrument was equipped with degasser, binary solvent delivery pump, auto sampler and photodiode array detector. Chromatographic separation was performed using an Agilent C-18 column (2.1 μm pore size, 3.5 mm × 150 mm) and the column maintained at 25°C. The mobile phase was composed of a mixture of phosphoric acid (0.05%) and acetonitrile in the ratio 75:25 (v/v) at 1 ml/min for 20 min. A 50 µl sample volume was injected and curcumin eluted at 10.6 min; curcumin concentration was calculated using a highly reproducible standard curve using peak area.
Determination of curcumin solubility
Solubility protocols were modified from a previously reported method (Yadav et al. Citation2009). Samples of βCD-C complex (curcumin:βCD 1:2 and 1:5), a physical mixture of curcumin with βCD (1:2 and 1:5), and curcumin alone, where curcumin concentration was 5 mg/ml for each sample, were dispersed in distilled water (Millipore) and stored in screw-capped vials. All vials were shaken continuously for 2 h at room temperature to obtain a supersaturated solution. Each solution was passed through a 0.45 μm nylon filter and diluted with water. An aliquot of each sample (100 μl) was further diluted with water and its UV absorbance measured at 425 nm. Standard curves for curcumin and βCD-C complexes were used to determine the solubility of curcumin in the samples.
Preparation of curcumin and βCD-C complexes entrapped liposomes
PEGylated liposomes composed of EggPC/Cholesterol/DSPE-PEG (85:10:5, w/w) were prepared for both curcumin and βCD-C complexes by a modified thin-film hydration method (Yang et al. Citation2007). Lipid alone or lipid/curcumin were dissolved in 10 ml of chloroform and transferred to a suitable round bottomed flask (RBF). The solvent was steadily removed in vacuo while maintaining the water bath at 40°C. Thin films were carefully prepared using varying rotary speeds, time cycles and vacuum pressures. This was followed by residual solvent removal by placing the RBF in a vacuum desiccator for 30 min. After nitrogen flushing, 1 ml PBS or βCD-C complex dissolved in PBS was added to the dried lipid film followed by vigorous shaking at 40°C. The sealed RBF containing a dispersion was then stored for 1.5 h in the dark to allow for complete film hydration. The formulations were then serially extruded through polycarbonate membranes (0.4 µm pore size × 8 times) under nitrogen gas. Lipid and curcumin ratios of 5:1, 10:1 and 15:1 (w/w) were used to prepare liposomes containing curcumin, and ratios of 20:0.5, 20:1.0 and 20:1.5 (w/w) were used to prepare liposomes containing βCD-C complexes. Un-entrapped curcumin and βCD-C complexes were separated from liposomes by centrifugation. Diluted liposomal formulations were centrifuged at 10000 rpm for 10 min and the supernatant removed. The supernatant for each sample was further centrifuged at 40000 rpm for 30 min. Finally, pellets were collected and resuspended with PBS. The supernatant was diluted and its absorbance at 425 nm measured. Absorbance values were then used to determine the amount of un-entrapped curcumin and βCD-C complexes present, using standard curcumin and βCD-C complex concentration curves. Finally entrapment efficiency was calculated by subtracting the un-entrapped amount from the initial amount added to each formulation. All liposomes were prepared in triplicate.
Particle size and polydispersity index
The average diameter and polydispersity index (scale, 0–1) of the liposomes were determined by photon correlation spectroscopy, using a Zetasizer Nano ZS particle sizing instrument (Malvern Instruments, Malvern, UK) at a temperature of 25 ± 0.1°C with diluted samples. From the analysis, the Z-average value was used, which is an approximation of the diameter of the prepared liposomes.
Investigation of liposome stability
Stability of the formulations was assessed with their particle size and polydispersity variation when stored at 2–8°C. Particle size and polydispersity were measured after their preparation and 12 h, 24 h, 3, 7, 10, 14, 21 and 28 days of storage. The experiment was performed triplicate.
In vitro cytotoxicity
To evaluate the anticancer potential of curcumin within βCD-C complexes and liposomes, cell proliferation assays were performed using A-549 lung and SW-620 colon cancer cell lines. The selected cells were grown at 37°C in 5% CO2 and seeded (at 10,000 cells/well) in 96-well plates. The cells were allowed to attach for 24 h before replacing the media and cells were then ready for the test formulations. After adding the samples (curcumin-entrapped liposomes, βCD-C complex-entrapped liposomes, curcumin, βCD-C complexes, PBS, 0.25% DMSO and βCD) which were prepared in PBS except curcumin (dissolved in 0.25% DMSO), cells were incubated for 48 h and finally an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay was performed (Scudiero et al. Citation1988). Equivalent amounts of PBS, DMSO and βCD were also used as controls. The anti-proliferative activity was calculated as a percentage of cell growth with respect to the activity found with empty liposomes (control). Finally, EC50 values of each formulation were estimated by interpolating from the graph. The experiment was performed in triplicate. Differences in EC50 between the formulations containing curcumin were compared using one-way ANOVA with a Bonferroni post hoc test and a significance level of p < 0.05.
Results
Confirmation of complex formation
In powder form, βCD-C complexes prepared by methanol reflux appeared as an amorphous substance that was a lighter yellow colour than the distinctly orange-yellow appearance of curcumin. The physical mixture of curcumin and βCD retained more of curcumin’s orange colour and appeared darker than the inclusion complex. All of the βCD-C complexes prepared absorbed less light than native curcumin or the physical mixture of curcumin and βCD (). The FT-IR spectra also confirmed βCD-C complex formation. Curcumin showed some prominent peaks at 3595 cm−1 for hydroxyl (phenolic) vibration, 1600 cm−1 for benzene ring skeleton stretching, 1280 cm−1 for Ar-O stretching and 1510 cm−1 for C=O and C=C vibrations (). The spectra of the physical mixture retained most of the characteristic peaks of curcumin and βCD, whereas βCD-C complexes were similar to the βCD spectrum and did not exhibit any of the characteristic peaks of curcumin. The X-ray diffraction of curcumin showed many characteristic peaks with high intensity. The physical mixture of curcumin and βCD retained some of the peaks from curcumin within a pattern that was very similar to βCD alone. However the βCD-C complexes lost all the characteristic peaks of curcumin and appeared very similar to βCD (). Therefore, all methods indicated successful inclusion of curcumin into βCD.
Figure 1. UV absorption spectra for curcumin, βCD, a physical mixture of curcumin and βCD (1:5), and βCD-C complexes (curcumin:βCD 1:1, 1:2 and 1:5). Bars (smaller than the symbols) indicate SEM of three replicates.
Figure 2. FT-IR spectra of curcumin, βCD, a physical mixture of curcumin and βCD (1:5), and βCD-C complex (curcumin:βCD 1:5).
Figure 3. General X-ray diffraction patterns of curcumin, βCD, a physical mixture of curcumin and βCD (1:5), and βCD-C complex (curcumin:βCD 1:5).
Efficiency of complexation
Greater than 80% entrapment of curcumin within βCD was achieved when ratios of 1:1, 1:2 and 1:5 (curcumin: βCD, w/w) were used, whether determined using UV spectroscopy or RP-HPLC (). Entrapment efficiency was lower, at around 35%, for the 1:0.5 ratio. The curcumin and βCD physical mixture showed low levels (< 40%) of curcumin entrapment (). Based on these results it is apparent that use of a ratio of 1:2 or 1:5 is associated with an entrapment efficiency of approximately 90%.
Figure 4. Curcumin entrapment efficiency into βCD-C determined using UV-spectroscopy (A) and RP-HPLC (B). Bars indicate SEM of three replicates.
Water solubility of curcumin and βCD-C complexes
All βCD-C complexes appeared to readily dissolve in water producing a deep red coloured solution; βCD-C complexes prepared at a ratio of 1:5 (curcumin: βCD) showed the greatest solubility, which was approximately two-fold higher than complexes prepared at 1:2 ratio (). Curcumin alone remained as a suspension in water with negligible solubility (0.4 µg/ml). Physical mixtures of curcumin and βCD showed low solubility, and appeared as a light yellow coloured suspension.
Figure 5. Water solubility of curcumin alone, in physical mixtures of curcumin and βCD (1:2 and 1:5), and in βCD-C complexes (curcumin:βCD 1:2 and 1:5) at room temperature (22°C). Bars indicate SEM of three replicates.
Curcumin and βCD-C complexes entrapment into liposomes
The lipid film hydration method was used to prepare empty liposomes, and liposomes containing curcumin and βCD-C complexes. Entrapment of βCD-C complexes into liposomes increased with the amount of lipid present, from 67% entrapment with 5:1 ratio (lipid: βCD-C complex) to 91% for 20:1.5 (). Entrapment efficiency for native curcumin was also found to increase as the lipid concentration was raised, with 72% for a 5:1 ratio improving to 90% for a 10:1 ratio ().
Table 1. Entrapment efficiency (mean ± standard deviation, n = 3) of curcumin and βCD-C complexes into liposomes with varying amounts of lipid and βCD-C complexes.
Liposome stability
Liposomes containing curcumin or βCD-C were approximately 420 nm in size at preparation, and this increased gradually upon storage at 2–8°C for 28 days to around 600 nm (). Control liposomes, which were prepared in the same way but contained neither form of curcumin, increased from a starting size of 350 nm to 500 nm during storage. Polydispersity, as a measure of particle size variation, was similar for all the formulations, slowly increasing from 0.4 to ≈ 0.65 upon storage ().
Figure 6. Changes in particle size (A) and polydispersity (B) of empty liposomes (control) or liposomes containing βCD-C complexes or curcumin during storage at 2–8°C. Bars (smaller than the symbols) indicate SEM of three replicates.
In vitro cytotoxicity
The median effective dose (EC50) of the formulations on colon cancer cells were calculated to be 0.96 µM for curcumin-entrapped liposomes, 1.9 µM for curcumin, 2.95 µM for βCD-C complexes and 3.25 µM for liposomes containing βCD-C (). The EC50 of the formulations on lung cancer cells followed the same pattern, being 0.90 µM for curcumin-entrapped liposomes, 1.5 µM for curcumin, 2.4 µM for βCD-C and 2.9 µM for liposomes containing βCD-C (). All of the controls (empty liposome, PBS, βCD and 0.25% DMSO) had no effect. The EC50 values for each of the four curcumin formulations were significantly (p < 0.05) different to each other in both colon and lung cancer cells tests, with liposomal formulations containing native curcumin the most effective in preventing cell proliferation. However, all curcumin-containing formulations appeared to be effective in inhibiting cell proliferation, as EC50 was maintained in the low µM range in each case.
Figure 7. Cytotoxicity of curcumin formulations in SW-620 colon cancer cells (A) and A-549 lung cancer cells (B). All other controls (empty liposome, βCD and 0.25% DMSO) produced no effect or same as PBS. Bars indicate SEM of three replicates.
Discussion
Curcumin, a hydrophobic compound, possesses enormous potential as a therapeutic in numerous areas of treatment including cancer. It has been investigated as model drug in a wide range of studies for its anticancer potential (Sharma et al. Citation2005; Zhou et al. Citation2011). However, its hydrophobic character, poor bioavailability and pharmacokinetics limit its use as a therapeutic agent (Ireson et al. Citation2002; Sharma et al. Citation2005). In order to improve the delivery characteristics we investigated the anticancer potential of liposomes containing native and βCD-enclosed curcumin in two different kinds of cancer cells, lung and colon.
We used a widely accepted pharmaceutical excipient, βCD (Loftsson and Brewster Citation2010) to form βCD-C complexes using a methanol reflux method (Tang et al. Citation2002) and measured the effectiveness of the method. Like previous studies (Tang et al. Citation2002; Yadav et al. Citation2009; Yallapu et al. Citation2010), characterization using FT-IR, X-ray diffraction, UV-spectroscopy and colour with texture change indicated successful complex formation between βCD and curcumin. Determination of curcumin entrapment into βCD was carried out using UV spectroscopy, and further confirmed using RP-HPLC, with comparable data obtained for both techniques (). In agreement with previous studies (Baglole et al. Citation2005; Tang et al. Citation2002; Yallapu et al. Citation2010) maximum entrapment (90%) of curcumin into βCD was achieved with the 1:2 (w/w) of curcumin and βCD ratios. A physical mix of curcumin and βCD prepared by non-thermal treatment showed complex formation to some extent (20%) while the βCD-C complexes prepared by thermal treatment (reflux at 70°C) indicated complex formation to a much greater extent (90%). The mild treatment temperature, which is just above the boiling point of methanol (69°C) is likely to have played a role in enhanced entrapment of curcumin into βCD. These findings suggest that this methanol reflux method can be used to produce inclusion complexes with other hydrophobic drugs.
βCD is generally used to enhance the water solubility of hydrophobic compounds (Loftsson and Brewster Citation2010; Yallapu et al. Citation2010). Our synthesized βCD-C complexes showed remarkable water solubility (c.f. native curcumin), readily producing a concentrated and deep-red coloured transparent solution in water, whereas native curcumin remained suspended in water. The enhanced water solubility of curcumin in βCD-C complexes are in good agreement with those recently reported (Yadav et al. Citation2009; Yallapu et al. Citation2010). In terms of the optimal quantity of βCD required to complex with curcumin, a ratio of 1:5 showed around a two-fold greater water solubility than a ratio of 1:2 even though both had similar entrapment efficiencies of ≈ 90%.
Liposomes are known to enhance the therapeutic efficacy and decrease non-specific toxicity of encapsulated anticancer drugs (Maruyama, Citation2011). Liposomes are US-FDA approved and widely used as a biocompatible delivery system due to their ability to selectively deliver therapeutics including anticancer drugs. Furthermore, liposomal delivery of cyclodextrin-enclosed hydrophobic drugs has the potential to combine all the delivery advantages that both cyclodextrin and liposomes can offer individually (Arima et al. Citation2006; Fatouros et al. Citation2001; Kaur et al. Citation2010; McCormack and Gregoriadis, Citation1994). Therefore, we used liposomes as our model delivery system to improve the delivery characteristics of both curcumin and its complex with βCD. We prepared liposomes with a widely used film hydration method that can entrap both hydrophobic as well as βCD-enclosed hydrophobic compounds (MaestrelLi et al. Citation2005). High drug entrapment levels were achieved, with around 90% entrapment of curcumin or βCD-C complexes into the PEGylated liposomes prepared with EggPC, cholesterol and DSPE-PEG. However, these liposomes gradually increased in particle size and polydispersity when stored at fridge temperature (2–8°C) for 4 weeks, indicating that further work would be required to improve liposome stability or mode of storage.
All of the formulations that contained curcumin exhibited anticancer activity in vitro in colon and lung cancer cell lines. All the formulations including liposomal curcumin and liposomal βCD-C complexes had relatively low EC50 values in both cell lines tested. Liposomes containing curcumin have previously been reported to increase curcumin delivery in other types of cancer cells (Li et al. Citation2005), while curcumin complexed with βCD maintains cytotoxicity (Yallapu et al. Citation2010), and other hydrophobic compounds complexed with CD and packaged into liposomes also retain their activity (Agashe et al. Citation2011; Arima et al. Citation2006; Kaur et al. Citation2010). In our study, we have shown that liposomes containing βCD-C complexes also retain their anticancer activity. Importantly, a biocompatible liposomal delivery system can be used to package and deliver hydrophilic curcumin (βCD-C complex) to cancer cells with good efficacy.
Conclusions
Delivering hydrophobic agents to target tissue remains a significant hurdle and preparing curcumin as βCD inclusion complexes within liposomes represents a viable model to address this, while also overcoming concerns associated with unwanted hydrophobic drug-liposomal lipid interactions that may interfere with vesicular bi-layer formation (McCormack and Gregoriadis, Citation1994). Using our approach, poorly water soluble therapeutics such as curcumin can be rendered wholly hydrophilic, and is likely to be retained within the aqueous compartment(s) of liposomes thereby not impeding nor hampering lipid self-rearrangement during liposome formation. This process now paves the way for the entrapment of numerous hydrophobic drugs into biocompatible lipid vesicles with high entrapment efficiency.
Acknowledgements
First author is grateful to the School of Pharmacy, The University of Queensland for a PhD scholarship.
Declaration of interest
This research was funded by the School of Pharmacy, The University of Queensland, Australia.
References
- Agashe H, Sahoo K, Lagisetty P, Awasthi V. 2011. Cyclodextrin-mediated entrapment of curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA in liposomes for treatment of xenograft lung tumor in rats. Colloids Surf B Biointerfaces 84:329–337.
- Al-Omar A, Abdou S, De Robertis L, Marsura A, Finance C. 1999. Complexation study and anticellular activity enhancement by doxorubicin-cyclodextrin complexes on a multidrug-resistant adenocarcinoma cell line. Bioorg Med Chem Lett 9:1115–1120.
- Arima H, Hagiwara Y, Hirayama F, Uekama K. 2006. Enhancement of antitumor effect of doxorubicin by its complexation with γ-cyclodextrin in pegylated liposomes. J Drug Target 14:225–232.
- Baglole KN, Boland PG, Wagner BD. 2005. Fluorescence enhancement of curcumin upon inclusion into parent and modified cyclodextrins. J. Photochem. Photobiol. A 173:230−237.
- Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, Maitra A. 2007. Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin”): a novel strategy for human cancer therapy. J Nanobiotechnology 5:3.
- Bouquet W, Ceelen W, Fritzinger B, Pattyn P, Peeters M, Remon JP, Vervaet C. 2007. Paclitaxel/β-cyclodextrin complexes for hyperthermic peritoneal perfusion: formulation and stability. Eur. J. Pharm. Biopharm. 66:391−397.
- Castelli F, Puglisi G, Giammona G, Ventura CA. 1992. Effect of the complexation of some nonsteroidal antiinflammatory drugs with β-cyclodextrin on the interaction with phosphatidylcholine liposomes. Int. J. Pharmaceut. 88:1−8.
- Fatouros DG, Hatzidimitriou K, Antimisiaris SG. 2001. Liposomes encapsulating prednisolone and prednisolone-cyclodextrin complexes: comparison of membrane integrity and drug release. Eur. J. Pharm. Sci. 13:287−296.
- Ireson CR, Jones DJ, Orr S, Coughtrie MW, Boocock DJ, Williams ML, Farmer PB, Steward WP, Gescher AJ. 2002. Metabolism of the cancer chemopreventive agent curcumin in human and rat intestine. Cancer Epidemiol Biomarkers Prev 11:105–111.
- Kaur N, Puri R, Jain SK. 2010. Drug-cyclodextrin-vesicles dual carrier approach for skin targeting of anti-acne agent. AAPS PharmSciTech 11:528–537.
- Li L, Braiteh FS, Kurzrock R. 2005. Liposome-encapsulated curcumin: in vitro and in vivo effects on proliferation, apoptosis, signaling, and angiogenesis. Cancer 104:1322–1331.
- Loftsson T, Brewster ME. 2010. Pharmaceutical applications of cyclodextrins: basic science and product development. J Pharm Pharmacol 62:1607–1621.
- Maestrelli F, Gonzalez-Rodriguez ML, Rabasco AM, Mura P. 2005. Preparation and characterisation of liposomes encapsulating ketoprofen–cyclodextrin complexes for transdermal drug delivery. Int. J. Pharmaceut. 298:55−67.
- Maiti K, Mukherjee K, Gantait A, Saha BP, Mukherjee PK. 2007. Curcumin- phospholipid complex: preparation, therapeutic evaluation and pharmacokinetic study in rats. Int. J. Pharmaceut. 330:155−163.
- Maruyama K. 2011. Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects. Adv Drug Deliv Rev 63:161–169.
- McCormack B, Gregoriadis G. 1994. Drugs-in-cyclodextrins-in liposomes: a novel concept in drug delivery. Int. J. Pharmaceut. 112:249−258.
- Moghimi SM, Szebeni J. 2003. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog Lipid Res 42:463–478.
- Safavy A, Raisch KP, Mantena S, Sanford LL, Sham SW, Krishna NR, Bonner JA. 2007. Design and development of water-soluble curcumin conjugates as potential anticancer agents. J Med Chem 50:6284–6288.
- Sakai H, Masada Y, Horinouchi H, Ikeda E, Sou K, Takeoka S, Suematsu M, Takaori M, Kobayashi K, Tsuchida E. 2004. Physiological capacity of the reticuloendothelial system for the degradation of hemoglobin vesicles (artificial oxygen carriers) after massive intravenous doses by daily repeated infusions for 14 days. J Pharmacol Exp Ther 311:874–884.
- Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR. 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48:4827–4833.
- Shaikh J, Ankola DD, Beniwal V, Singh D, Kumar MN. 2009. Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. Eur J Pharm Sci 37:223–230.
- Sharma RA, Gescher AJ, Steward WP. 2005. Curcumin: the story so far. Eur J Cancer 41:1955–1968.
- Takahashi M, Uechi S, Takara K, Asikin Y, Wada K. 2009. Evaluation of an oral carrier system in rats: bioavailability and antioxidant properties of liposome-encapsulated curcumin. J Agric Food Chem 57:9141–9146.
- Tang B, Ma L, Wang HY, Zhang GY. 2002. Study on the supramolecular interaction of curcumin and β-cyclodextrin by spectrophotometry and its analytical application. J Agric Food Chem 50:1355–1361.
- Tønnesen HH, Másson M, Loftsson T. 2002. Studies of curcumin and curcuminoids. XXVII. Cyclodextrin complexation: solubility, chemical and photochemical stability. Int J Pharm 244:127–135.
- Wang Z, Leung MH, Kee TW, English DS. 2010. The role of charge in the surfactant-assisted stabilization of the natural product curcumin. Langmuir 26:5520–5526.
- Yadav VR, Suresh S, Devi K, Yadav S. 2009. Effect of cyclodextrin complexation of curcumin on its solubility and antiangiogenic and anti-inflammatory activity in rat colitis model. AAPS PharmSciTech 10:752–762.
- Yallapu MM, Jaggi M, Chauhan SC. 2010. β-Cyclodextrin-curcumin self-assembly enhances curcumin delivery in prostate cancer cells. Colloids Surf B Biointerfaces 79:113–125.
- Yang T, Choi MK, Cui FD, Kim JS, Chung SJ, Shim CK, Kim DD. 2007. Preparation and evaluation of paclitaxel-loaded PEGylated immunoliposome. J Control Release 120:169–177.
- Zhou H, Beevers CS, Huang S. 2011. The targets of curcumin. Curr Drug Targets 12:332–347.