Curcumin Encapsulated Alginate / Pluronic Block Copolymer Micelles as a Promising Therapeutic Agent

Article Information Received 15 May 2014 Received in revised form 28 June 2014 Accepted 29 June 2014 Abstract Curcumin in turmeric is responsible for most of the therapeutic effects exhibited by this natural product. Solubility of this dietary compound is very low in water at neutral pH and room temperature, which decreases its bioavailability. This in turn proves to be a major setback in making use of curcumin as a therapeutic agent against many diseases, including cancer. Alginate/Pluronic® tri-block copolymer P123 micelles has the potential for the formulation of curcumin. Inner core of the micelles effectively trap-free curcumin as evidenced by analytical data. The drug-encapsulated micelles were spherical in shape with diameters below 100 nm and found to be spherical particles with an average size of 50–100 nm, being suitable for drug delivery applications. Release profile of curcumin was investigated in aqueous PBS solution at pH 7.4 (37 ± 0.2 oC). Fitting of drug release data in the Korsmeyer–Peppas model suggested sustained release with diffusion mechanism (0.5 < n). The in vitro cytotoxicity of the drug formulation was investigated with L 929 cancer cells. Polymer encapsulated curcumin showed comparable anticancer activity with free Cur.


Figure 1 Structure of Curcumin
Cancer refers to a state where cells grow and divide uncontrollably.
Cancer cells follow a number of mutagenic processes, which imparts unlimited proliferation potential and self-sufficiency in growth signals 5 .Instances where combination therapy in cancer treatment causes major side effects to the patients, therapeutic modalities with no or minimal side effects to normal organs have been investigated.
As a potential drug candidate, Cur has been receiving considerable attention because of its putative cancer prevention and anti-cancer activities, which are mediated through multiple signalling pathways [6][7][8] .
In spite of the promising anticancer activities, low solubility of Cur in water restricts its use in intravenous administration and is associated with poor absorption in the intestine upon oral administration.Rapid degradation by enzymes residing the intestinal tract causes low oral bioavailability of curcumin 9 .In order to improve the aqueous solubility and stability of Cur, polymeric micellar encapsulation is extensively investigated.Traditional methods to improve the bioavailability of poorly soluble drugs include encapsulating them in nanosized carriers such as liposomes 10 , emulsions, polymer micelles, niosomes 11 , lipid particles etc. Poly ethylene glycol (PEG) based block copolymers have the distinct advantage as compared to other delivery systems due to its ability to encapsulate large amounts of drug, having stealth nature and well defined size with narrow polydispersity 12 .Pluronic® block copolymers (also termed poloxamers) were used as effective carriers for hydrophobic drugs with long-circulating characteristics through avoidance of trapping by the reticular endothelial system (RES).Pluronic® block copolymers are approved by FDA for pharmaceutical and medical applications.

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They find use as emulsification, solubilization, dispersion, thickening, coating, and wetting agents.
Major aim of this study is to develop a new polymeric micellar formulation for Cur, intended to be intravenously administered.
Pluronic® P123 (PEO20-PPO70-PEO20) was chosen for micellar encapsulation due to its commercial availability, biocompatibility and safety 13,14 .Paclitaxel (PTX)-loaded P123 micelles were studied by Han et al 15 .The results showed that P123 micelles may efficiently load, protect and retain PTX in the biological environment.Moreover, they could increase blood circulation time and reduce the distribution in liver.

Preparation of Cur loaded Pluronic® micelles
Pluronic® P123, Cur, Sodium alginate from brown algae (viscosity of 2% solution at 25 ºC: ∼250 cps) were purchased from Aldrich (Bangalore, India).All the salts and solvents used in the study were purchased from Merck (Mumbai, India).All other reagents and buffer solution components were analytical grade preparations.Distilled and deionized water was used in all experiments.Cur loaded Pluronic® micelles were prepared by thin-film hydration method [16][17][18] .
Stock solutions of Cur (1 mg/mL) and Pluronic® copolymer (10 mg/mL) were prepared in dichloromethane.Required amount of stock solutions was transferred to separate round-bottomed flasks to obtain different drug to polymer ratio.The solvent was evaporated by rotary evaporation at 50 ºC for about 1 hour to obtain drug-containing polymer films.The residual organic solvent remaining in the films was removed by keeping them in the vacuum overnight at room temperature.After that, the films were rehydrated in water and extensively vortexed to prepare drug-encapsulated micelles.Then, the resultant thin film was hydrated with 5 mL water at 40°C, then stirred at 500 rpm for 1 hour to obtain a clear micelle solution, which was filtered through 0.22 μm filter (Millipore) to remove the nonencapsulated drug, followed by lyophilization.Quantitative estimation of the released curcumin in phosphate-buffer saline (PBS) (pH 7.4) was performed using HPLC.

Determination of drug content in micelles
The drug concentration was determined by RP-HPLC method (LC 2010 C HT Shimadzu, Kyoto, Japan).A 65:35 (v/v) degassed mixture of acetonitrile and water was used as the mobile phase.The reverse phase column (Kintex 5u C18, 250 mm × 4.6 mm, Phenomenex, California, USA).The column temperature was maintained at room temperature.The flow rate was set at 1.0 mL/min and the samples were monitored at 227 nm 19 .Sample solution was injected at a volume of 20 μL.Micelles were dissolved in acetonitrile and vortexed to get a clear solution.The concentration of Cur in samples was determined by HPLC.Drug-loading (DL %) and encapsulation efficacy (EE %) were calculated as per Equations ( 1) and ( 2), respectively 20 .
Lyophilized formulation was obtained by freeze-drying Cur encapsulated Pluronic® micelles in a freeze dryer.Drug-loaded micelle samples were rapidly frozen by liquid N2 and attached to the freeze dryer.

In vitro release of Cur from micelles
In vitro Cur release profiles from encapsulated formulation were obtained as follows.Alginate (Alg) 2 wt% solution was prepared.
Alginate/P123/Cur loaded films were produced by casting/solvent evaporation technique in the different drug polymer ratio (shown in table 1).The drug loaded films were suspended in glass vessels containing 50 mL of 0.01 M PBS (pH 7.4).At appropriate time intervals aliquots of the solutions were withdrawn and the amount of Cur released were evaluated by UV spectrophotometer at 420 nm.
The release was quantified as follows:

Release kinetics
The Korsmeyer-Peppas model 21  and attached to the freeze dryer.

In vitro cytotoxic activity
In vitro cytotoxic activity of Cur-P123 micelles were tested using  between % cumulative release and time (Fig. 3).Table 1 show the values of the kinetic exponent (n) and regression coefficient (r 2 ) for all the formulations.It was noted that curcumin is released by the diffusion mechanism 22,23 .The percentage loading efficiency (% LE) and percentage drug loading (% DL) was calculated from equations ( 1) and ( 2) respectively.The best DL% and ER% were 1.39 ± 0.04% and 95.09 ± 2.91% respectively for the composition of 1:8 (w/w) Cur:

Drug loading and release studies
P123.This optimized matrix was further used for other characterization studies.) with the probable release pattern is given

Release kinetics
The release constants and release exponents (n) were determined by fitting the release data into the respective equations along with regression coefficients (r 2 ).Analysis of regression coefficients for different kinetic models revealed the mechanism of Cur release from Cur-P123 formulation.Tabular values (Table 1) of r 2 showed that the power law was best fitted with release kinetic data of Cur.

Biological studies
Cytotoxicity profile was established using MTT Assay.Since cancer cells are sensitive towards Cur, we chose to use L 929, a normal mouse fibroblast like connective tissue for cytotoxicity analysis with encapsulated Cur and free Cur.The results clearly, indicated that within the low concentrations range, these mammalian cells are more sensitive to encapsulate Cur than to non-encapsulated Cur (Fig. 8).It can be concluded that encapsulated Cur do not possess any significant cytotoxic effect in normal cells.Cells treated with the highest concentration of polymer encapsulated drug molecules showed a viability of more than 60%.Encapsulated matrix is highly biocompatible and do not possess any significant toxicity in vitro.Focus of the reported study is to formulate an aqueous soluble matrix for delivery of Cur which is extremely hydrophobic and the most difficult candidate for controlled release from the polymeric matrix.Liposomes are reported as a promising carrier for curcumin which is known to improve the bioavailability 31 .However, an enhancement in aqueous solubility evidenced by the quantitative data shows that block copolymers represent an efficient scaffold for the curcumin encapsulation.We present curcumin entrapment in P123 matrix by thin film hydration method.This represents a complete novel carrier system for hydrophobic curcumin molecule 32 was used to analyze the drug release mechanism from various formulations differing in the drug polymer ratio.This model could be represented as Power law model: Mt/M∞ = kt n … … …(4)where Mt/M∞ is the fraction of drug released at time "t" and "k" represent Korsemeyer-Peppas constant.In the power law model, "n" is the release exponent, indicative of the mechanism of drug release.The values of "k" and "n" were determined by fitting the release data into the respective equation.Lyophilized formulation was obtained by freeze-drying of Cur encapsulated Pluronic® micelles in a freeze dryer.Drug-loaded micelle samples were rapidly frozen by liquid N2 L929 cells by MTT assay.Three different concentrations of each sample were tested in L929 cell line (mouse fibroblast like connective tissue).The cells were grown in DMEM (Dulbecco's Modified Eagle Medium) and 10% FBS (Fetal bovine serum).Briefly, cells were seeded in 96-well plates at the density of 4000 viable cells per well and incubated 24 h to allow cell attachment.Cells were then treated with a series of doses of blank micelles, Cur-P123-micelles, respectively, at 37 °C.After 96 hour incubation, 20 μL of MTT (5 mg/mL) was added to each well of the plate.After incubating for additional 4 hour, MTT was aspirated off and 200 μL/well of DMSO (Dimethyl sulfoxide) was added to dissolve the formazan crystals.The plates were vigorously shaken before taking measurement of relative colour intensity.The absorbance at 570 nm of each well was measured by a micro plate reader.The results were expressed as mean values ± standard deviation of three measurements.2.6 Analytical characterization and MorphologyFluorescence spectra were recorded with F-2500 Fluorescence spectrometer (Hitachi, Japan).UV spectroscopic analysis was carried out on Shimadzu (UV-2450) UV-Visible double beam spectrophotometer with matched pair of stoppered fused silica cells of 1 cm optical path length.Infrared spectra were recorded on Fourier transform infrared (FTIR) spectrometer SHIMADZU, using KBr pellets, in the region of 400-4000 cm −1 .Thermo gravimetric analysis (TGA) was carried out using TGA-Pyres-6TGA (Perkin Elmer, UK) by heating from 40 ο C to 730 ο C at a heating rate of 10 ο C min −1 under N2 flow.Differential scanning calorimetry (DSC) experiments were carried out in a DSC Q100 V7.3 Build 249 (DSC standard cell) at a heating rate of 20 ο C min −1 in the range of 40 ο C to 300 ο C under steady flow of N2.Drug loading characteristics of P123 encapsulated Cur was further established by 1 H NMR spectra recorded on a Varian 400 MHz spectrometer (Varian, Palo Alto, CA, USA) using deuterated dimethyl sulfoxide (DMSO-d6) as solvent at room temperature.Morphology of Cur-P123 was elucidated under scanning electron microscope (SEM; LEO 1430 VP, Leo Electron Microscopy Ltd., Cambridge, United Kingdom).

Fig. 2
Fig. 2 depicts the release profile of Cur between % cumulative release versus time with varying Cur and P123 ratio.It has been found that approximately 40%, 60% and 70% of curcumin were released within 20, 40 and 60 min of incubation from the 1:8 matrix (w/w), while approximately 60%, 80% and 90% from 1:2 matrix, 10%, 15% and 20% curcumin were released from the 1:80 matrix.The probable mechanism of drug release from all formulations was explained by plotting the Korsmeyer-Peppas model (equation 4)

Figure 2 Figure 3 Figure 4
Figure 2 In vitro Cur release (%) from Cur-P123-Alg in PBS (pH 7.4) at 37 °C.The error bars represent mean and standard deviations of the experiments of raw Cur and Curencapsulated P123 shows that P123-Cur follows the weight-loss pattern of P123 (data not shown).The weight-loss in raw Cur occurs around 200°C, while that of the encapsulated Cur occurs around 300°C.The weight loss in Cur follows a gradual decrease while that of Cur-encapsulated P123 occur more rapidly.The raw Cur and the Cur loaded P123 left some residual matter after the end of thermal decomposition, while the P123 is burnt completely without leaving behind any residual matter (Fig.5 (a)).The Cur entrapment into the inner core of the P123 micelles was confirmed by DSC.As shown in Fig. 5 (b), the DSC curve indicates an amorphous or disorder crystalline phase of a molecular dispersion or a solid solution state in the polymer matrix.

Figure 7 FTIR 1 H
Figure 7 FTIR spectra of Cur; Pluronic P123; and Cur-P123 (a). 1 H NMR spectrum of P123-Cur micelles (b).The small signals from Cur are indicated by * Successful incorporation of Cur in P123 and its release in DMSO-d6 is established by 1 H NMR spectroscopy 30 .Figure7 (b) shows the 1 H NMR spectra of Cur encapsulated P123 polymer matrix. 1 H NMR spectral characterization of paclitaxel encapsulated P123/F127 Pluronic® polymer was already reported. 21In DMSO-d6, the aromatic proton signals corresponding to Cur in P123 polymer matrix is seen with lower intensity than compared with the Pluronic® counterpart.This clearly indicates that Cur got entrapped in the hydrophobic PPO core of the co polymer.

Figure 8
Figure 8 Effect of different curcumin formulations on the viability of L 929.Values represent the mean ± SD of triplicate experiments (n = 3) after 24 h of exposure to Cur 3.8 Morphology Drug loaded micellar solution obtained was clear and yellowish as seen in Fig. 4. Suspension of crude Cur dispersed in water at the same drug concentration was employed as the control.The size and morphology of the polymer-encapsulated Cur was confirmed by SEM imaging.SEM image (Fig.9) of the Cur encapsulated P123 shows that the particles have an average size of 50-100 nm, and lie in the optimal size range (below 200 nm) suitable for drug delivery applications.There is a significant decrease in the size of Cur compared to free Cur.This is probably because the hydrophilic polymers prevent the aggregation of hydrophobic Cur.

Figure 9 :
Figure 9: SEM image of curcumin-loaded ALG-P123 film showing distinct encapsulated particles with solid dense structure .

1 H
NMR spectrum confirmed the entrapment of the drug within the polymeric core along with Fluorescence, UV-Visible and FT-IR spectroscopic techniques.The in vitro cytotoxic assay results indicated that Cur can be released from the conjugate without losing cytotoxicity thus confirming the biocompatibility of P123 carrier.Cur is released from the matrix by diffusion mechanism (0.5 < n).The biocompatibility of the polymeric matrix can serve as an ideal formulation for tissue engineering and controlled drug delivery applications.In order to study the release profile of curcumin, Alginate was incorporated in the P123 matrix.Although further conclusions can only be drawn only after a thorough in vivo study, the biocompatible nature of Cur, the long history of its therapeutic use, low cost of the material and the easiness in encapsulating with the suggested polymer enhances its role as a promising drug.