From micron to nano-curcumin by sophorolipid co-processing: highly enhanced bioavailability, fluorescence, and anti-cancer efficacy

Abstract

Co-sonication of curcumin and acidic sophorolipid in aqueous solution is shown to lead to a dramatic enhancement of curcumin bioavailability through size reduction and encapsulation. The interaction between the two is studied and discussed based on optical absorption, photoluminescence, dynamic light scattering (DLS), zeta potential, FE-SEM, TEM, Infrared spectroscopy and X-ray diffraction measurements. The cytotoxicity effects of curcumin on breast cancer cell lines, MCF-7 and MDA-MB-231, are shown to be significantly enhanced by the formation of its complex with sophorolipid. The relative cytotoxicity of curcumin with its SL(A) complex is more due to the presence of the glucose moiety. The results further suggest that sophorolipid based formulations, which solubilize and nano-encapsulate curcumin after lipid digestion, show great potential for curcumin cell entry.

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Introduction

Curcumin is known to have antioxidant, anti-inflammatory, chemo-preventive and chemo-therapeutic properties.¹ The bioavailability² of curcumin is determined by the rate at which it enters the plasma and reaches the target sites and the corresponding concentration. The oral bioavailability³ of curcumin is low because a major portion of the compound remains unabsorbed due to a fairly low intestinal absorption capacity. Even a minor amount which is actually absorbed is rapidly metabolized in the liver and thrown out of the body by the gall bladder.^4–6

Various studies have verified that even a very high oral dose of curcumin (up to 1 g kg⁻¹ of the body weight) is almost completely eliminated by the human metabolic system.

Curcumin (diferuloylmethane), a bright orange yellow pigment, is the main active ingredient of turmeric; an ancient spice known for its medicinal uses.⁷ Curcumin exhibits tautomerism in its molecular structure and thus exists in the enol form in non-polar solvents, because of intra-molecular hydrogen bond formation. In polar solvents, however, it is observed in the diketo form.^3,8 The keto form of curcumin acts as a proton donor in acidic and neutral media. At pH values above 8.0, the enol form dominates, acting as an electron donor.

The phenolic, b-diketone, and methoxy groups of curcumin contribute to its free-radical scavenging property. This property imparts the anti-cancer nature to this compound.^9,10 However, as mentioned previously, these attributes are not reflected well in clinical studies because of the low oral bioavailability of curcumin.¹¹ Therefore several soft materials systems including liposomes,^12,13 dendrimers,¹⁴ microspheres,¹⁵ micelles,¹⁶ and lipid nanoparticles^17–20 have been explored to design specific drug-delivery vehicles for curcumin. These nano-assembly forming procedures designed to improve the bio-availability of curcumin are all inherently expensive and hence there is a strong urge to obtain cost-effective replacements for this system.

Biosurfactants ²¹ derived from microorganisms are an interesting category of bio-organic systems with potential applications in biomedical science. They can be produced from renewable feedstock or waste material^22,23 by natural fermentation. These amphiphilic compounds are known to easily form self assemblies at different pH conditions in aqueous environment. Sophorolipids are an eco-friendly and biocompatible class of amphiphilic biosurfactants which easily form emulsions in aqueous solution to reduce the surface tension and interfacial energies.²⁴ They possess unique structures that can be engineered to suit specific application domains.²⁵ Sophorolipid exists in two forms: acidic and lactonic. Acidic sophorolipids (SL(A)), are known to form micelles, which interact depending on the pH of the system.²⁶ In SL(A), a sophorose unit is attached to an oleic acid moiety through an ether bond on the C17 carbon atom.²⁷ This particular characteristic leaves the COOH group available and responsive to changes in pH of the solution giving rise to the possibility of a series of self assembled structures.

In this study, we have developed a novel formulation, namely a complex of acidic sophorolipid (ESI Fig. 1†) and curcumin (SL(A) + Cur), to improve the water solubility, stability and bioavailability of curcumin in order to enhance its effectiveness in the context of anti-cancer activity. SL(A) + Cur complexes were prepared by sonication driven supramolecular self-assembly (Fig. 1), and were characterized by using UV-Vis and photoluminescence (PL) spectroscopy, dynamic light scattering (DLS), zeta potential, Fourier-transform infrared (FTIR)], X-ray diffraction and scanning as well as transmission electron microscopy (SEM and TEM, respectively). The as-synthesized curcumin formulation showed significantly improved bioavailability in cancer cells compared to the curcumin in ethanol. The optimized curcumin formulation also exhibited more cytotoxicity in cancer cells. This study thus suggests a new cost-effective nanoscale self-assembly approach for improved curcumin delivery and therapeutic efficacy in cancer.

Fig. 1 Structure of SL(A) + Cur self-assembly. SL(A) is seen to completely encapsulate curcumin through hydrophobic part because of its hydrophobic nature. The hydrophillic part of SL(A) makes this assembly soluble in aqueous environment and prevents it from degradation.

Result and discussion

Optical properties

The optical properties of sophorolipid acidic SL(A), curcumin (Cur) and sophorolipid–curcumin (SL(A) + Cur) complex show distinct significant differences. SL(A) appears transparent, curcumin solution looks turbid, whereas the SL(A) + Cur solution appears transparent yellow indicating curcumin solubilization. The photo-physical properties of curcumin are very sensitive to the medium. Curcumin has strong absorption around 420 nm in organic solvent but it decreases in aqueous solution due to degradation of curcumin in water by a reaction at the keto–enol group. Interestingly though, the sophorolipid shell imparts a hydrophobic surface to the curcumin core in the SL(A) + Cur complex and the outer hydrophilic portion of the sophorolipid assists in the solubility of the complex in water (Fig. 2A). This assembly in aqueous solution greatly assists in stabilization of the complex giving rise to the enhanced absorption at 420 nm in the aqueous medium.^8,11

Fig. 2 (A) UV-visible spectra of SL(A), curcumin and SL(A) + Cur solutions. SL(A) solution shows absorbance at λ = 234 nm and curcumin solution at λ = 344 and 420 nm while SL(A) + Cur showing increase absorption at one peak at 420 nm; (B) photoluminescence study of SL(A), curcumin and SL(A) + Cur solutions. SL(A) solution shows no PL, curcumin solution shows PL at 550 nm while SL(A) + Cur showing very strong Photoluminescence at 500 nm; (C) photoluminescence quenching and right shift of the PL of SL(A) + Cur self assembly on gradually addition of ethanol solvent.

Photoluminescence (PL) of the SL(A), curcumin and SL(A) + Cur samples was recorded for comparison by excitation at the same wavelength of 420 nm in an aqueous solution. SL(A) sample exhibits no PL, while curcumin exhibits weak excitonic emission at 550 nm due to low solubility.²⁸ However, on addition of SL(A) in curcumin aqueous solution a strong emission is seen at 500 nm reflecting tremendous enhancement of the fluorescence intensity (Fig. 2B). The fluorescence maximum shifts from a broad unremarkable 550 nm band to a remarkable blue shifted band at 500 nm.^11,29 To confirm the improvement in PL, we performed some additional experiments. When we add ethanol gradually in this mixture, photoluminescence quenching is observed. This is probably due to disturbance of nonpolar region around curcumin nanoparticles created by SL(A) self-assembly. Furthermore, a red shift is observed (from 500 nm to 550 nm) with the addition of ethanol to the SL(A) + Cur aqueous solution, clearly indicating a gradual degradation of the self assembly yielding the original structure itself (Fig. 2C).

As described earlier, a large blue shift results when curcumin is bound to the SL(A) micelles. This can occur due to the fact that curcumin in SL(A) micelles could create a lipophylic condition via binding to the hydrophobic regions. Besides the shift in the fluorescence maximum, there was a remarkable improvement in the fluorescence intensity of curcumin upon formation of the self-assembly with SL(A) (Fig. 3). As seen from Fig. 3B a very feeble fluorescence is seen in the case of curcumin particulates, while the well-dispersed and well distributed SL(A) + Cur nanoparticulates exhibit enhanced fluorescence (Fig. 3D).

Fig. 3 (A and C) Optical microscopy images of curcumin and SL(A) + Cur self-assembly; (B and D) fluorescence images of curcumin and SL(A) + Cur self assembly (scale bar 100 μm).

Particles size distribution and zeta potential

In this experiment, all solutions were analyzed in the DLS apparatus at a constant shutter opening diameter. DLS measurements for curcumin, SL(A) and SL(A) + Cur (10 mg of each dissolved in 10 ml H₂O) exhibit hydrodynamic radii of about 818 nm, 6.8 nm and 15.5 nm, respectively (Fig. 4A, C and E). This was also confirmed with the results obtained by SEM and TEM analysis. These data clearly show that the size of the SL(A) + Cur particles is ∼6–7 nm indicating that there is a decreased agglomeration of curcumin in aqueous solution due to its capping by SL(A) and there is a definite increase in the size of SL(A) + Cur complex as compared to only SL(A) because of the additional encapsulation of curcumin nanoparticles. The percentage of curcumin encapsulated in nano-complex was calculated by UV-Vis spectrophotometer (ESI Fig. 2†).

Fig. 4 (A) DLS and zeta potential results for (A) curcumin showing hydrodynamic radius of 818.6 nm and (B) corresponding curcumin zeta potential; (C) SL(A) showing hydrodynamic radius of 6.8 nm and (D) corresponding SL(A) zeta potential; (E) SL(A) + Cur showing hydrodynamic radius 15.5 nm and (F) corresponding SL(A) + Cur zeta potential.

To understand the stability of SL(A) + Cur, zeta potential measurements were done on all the three samples. The zeta potential values for the three sample were SL(A) = −17.30 mV, curcumin = −15.14 mV, SL(A) + Cur = −24.38 mV (Fig. 4B, D and F respectively).

The increase in the zeta potential of SL(A) + Cur compared to individual SL(A) and curcumin can be taken as an indication of an increased stability of the self-assembled complex. The stability of the nano-complex in aqueous environment over a period of four month was confirmed by UV/Vis spectra, PL spectra and NMR spectra analysis (ESI Fig. 3, 4 and 5†)

Microscopy studies

SL(A), curcumin and SL(A) + curcumin samples were further examined by scanning electron microscopy (Fig. 5A–C). SL(A) exhibits a ribbon type morphology (Fig. 5A), curcumin appears as large chunks forming undefined shape (Fig. 5B), whereas SL(A) + Cur shows a fibrous morphology (Fig. 5C) which differs distinctly from that of SL(A). Remembering that these morphologies evolve in the solution drying process and do not represent the situation in the solution, it is clear that morphological organization of SL around curcumin with hydrophilic groups protruding outside would change their organization and therefore the morphology during the drying process as compared to SL itself which does not have such preferential molecular organization in the solution. The TEM pictures at different levels of magnification (Fig. 5D–F) apparent in SEM (Fig. 5C) due to the corresponding limited resolution show tiny fairly uniformly dispersed curcumin nanoparticles (<about 20 nm, some agglomerated on TEM grid) which were not apparent in SEM (Fig. 5C) due to the corresponding limited resolution.

Fig. 5 (A) SEM of SL(A) showing ribbon structure; (B) SEM of curcumin showing chunks of undefined structure; and (C) SEM of SL(A) + curcumin exhibiting morphological change vis a vis SL (A); (D–F) high resolution TEM images of SL(A) + Cur (scale bar sizes for A, B, C, D, E, F are 0.5, 10, 5, 0.5, 0.2 and 0.1 μm respectively).

FTIR analysis

Fig. 6 shows the FTIR spectra for SL(A), curcumin and SL(A) + Cur after sonication treatment for 30 minutes. The SL(A) shows a broad band at 3350 cm⁻¹ corresponding to the O–H stretch frequency in the glucose moiety. The asymmetrical and symmetrical stretch modes of methylene(CH₂) groups occur at 2928 and 2854 cm⁻¹, respectively. Sophorolipid also has two strong absorption bands arising from C–O– and C–O stretching; the C–O absorption band at 1744 cm⁻¹ may include contributions from the acid group. Moreover, sugar C–O– stretch of C–O–H groups is found at 1048 cm⁻¹ and the band at 1452 cm⁻¹ corresponds to the C–O–H in-plane bending of carboxylic acid (–COOH) in the product.

Fig. 6 FTIR analysis of SL(A), curcumin and SL(A) + Cur.

All these details are in conformity with the literature reports.²⁹ The FTIR spectrum of curcumin shows one sharp peak at 3508 cm⁻¹ indicating the presence of OH. The strong peak at 1626 cm⁻¹ has a predominantly mixed (C=C) and (C=O) character. Another strong band at 1601 cm⁻¹ is attributed to the symmetric stretching vibrations of the aromatic ring (C=C ring). The 1508 cm⁻¹ peak is assigned to the (C=O), while enol C–O peak was obtained at 1272 cm⁻¹. The other assignments are as follows: C–O–C peak at 1023 cm⁻¹, benzoate trans-CH vibration at 959 cm⁻¹ and cis-CH vibration of aromatic ring at 713 cm⁻¹.³⁰ The FTIR spectrum of SL(A) + Cur shows all the peaks related to SL(A) and curcumin peaks are seen to have been suppressed due to nano-encapsulation.

X-ray diffraction (XRD) analysis

To examine the crystallinity of micelle-encapsulated SL(A) + Cur, XRD analysis was performed. XRD analysis of samples (Fig. 7) was done over broad angle range (2θ = 10–80 degrees). The powder X-ray diffractograms of SL(A), curcumin and SL(A) + Cur dried powders are shown in Fig. 7. The characteristic peaks of curcumin appeared at 2θ values at 7.96, 8.90, 12.26, 14.54, 17.24°, etc. implying the presence of curcumin in crystalline form.³¹ It is found that the characteristic diffraction peaks of the curcumin are absent in the spectrum of the SL(A) + Cur nano-encapsulation, which suggests that curcumin being nano-encapsulated its peaks are too broadened and the XRD pattern in dominated by the structure of sophorolipid component. Interestingly, the diffraction patterns of the material obtained after dissolution in chloroform showed a pattern similar to that of pure curcumin, indicating that the complex is broken down and the encapsulated curcumin is released.

Fig. 7 XRD spectra of SL(A) + Cur self assembly. Black line explain the curcumin XRD pattern, red SL(A), green SL(A) + Cur while blue SL(A) + Cur after dissolve in chloroform solvent. Blue line showed unaffected nature of curcumin encapsulated in SL(A) self assembly.

Cytotoxicity assay

To evaluate whether SL(A) enhanced the bioavailability of curcumin in the cancer cells, cytotoxic potential of aqueous SL(A) + Cur was tested in breast cancer cell lines, MCF-7 and MDA-MB-231 and compared with that of aqueous SL(A), curcumin in water, and curcumin dissolved in ethanol, the latter being used as a positive control. Curcumin was administered to the cells at non-toxic doses of ethanol. After treatment with SL(A), both the breast cancer cell lines exhibited 80–100% viability upto the concentration of 160 μg ml⁻¹ showing that it was almost non-toxic to the cells. It can be observed that in SL(A) + Cur nano-complex, curcumin exhibited anticancer activity at extremely low doses starting from 6.66 μg ml⁻¹ compared to 40 μg ml⁻¹ concentration of curcumin dissolved in ethanol in MCF-7 cells (Fig. 8A). As seen in the case of MCF-7 cells, curcumin dissolved in ethanol is more toxic than nano-complexes at moderate and low concentrations. In the low to moderate dose range (5–20 μg ml⁻¹), curcumin dissolved in ethanol has more concentration compared to the concentration of curcumin present in the SL(A) + Cur nano-complexes (0.83–3.33 μg ml⁻¹). Thus the nano-complexes do not show effective killing within this concentration. However, at higher dose range (40–160 μg ml⁻¹), the concentration of curcumin in SL(A) + Cur is also increasing (6.66 to 26.66 μg ml⁻¹) and thus we observe the increase in cytotoxicity. Similarly, in MDAMB-231 cells (Fig. 8B), SL(A) + Cur exhibits cytotoxicity at 3.33 μg ml⁻¹ compared to 20 μg ml⁻¹ concentration of curcumin dissolved in ethanol. We have also added the data of curcumin (in water) that shows no cytotoxicity since curcumin does not go in water. These results clearly show that the bioavailability of curcumin is increased in the SL(A) + curcumin complex as compared to the of curcumin dissolved in ethanol.

Fig. 8 Cytotoxicity analysis of SL(A), curcumin and SL(A) + curcumin by MTT assay in (A) MCF-7 and (B) MDA-MB-231 and (C) HEK 293 cells. All the data are presented as mean ± SD of five independent experiments at p < 0.0001, indicating statistically significant differences compared to the control untreated group.

Interestingly, MCF-7 shows increased susceptibility to anticancer drugs at lower doses compared to MDAMB-231, the reason being the difference in estrogen and progesterone (ER/PR) receptor status in the cell lines. MCF-7 is ER/PR positive while MDAMB-231 is ER/PR negative, thereby being slightly insensitive to lower concentrations of anticancer drugs. Moreover, SL(A) makes the curcumin present in the NPs more bio-available at lower doses as compared to higher doses of curcumin dissolved in ethanol which explains the increased cytotoxicity of nano-complex.³² This improved cytotoxicity of SL(A) + Cur complexes compared to curcumin in ethanol in cancer cell lines may be due to tautomeric molecular form of curcumin after SL(A) encapsulation.

We have compared the cytotoxicity of curcumin in water, curcumin in ethanol, SL(A) and SL(A) + curcumin in non-cancerous cell line HEK 293 (Fig. 8C). Interestingly, except for curcumin in ethanol (at higher doses), all others are non-toxic to the cells.^34–40

Experimental

Synthesis procedure

SL(A) dissolved in 25 ml distilled water (1 mg ml⁻¹) was taken in a 50 ml beaker, and was kept for bath sonication for 30 minutes. 5 ml curcumin solution (1 mg ml⁻¹) in distilled water was mixed drop wise during sonication at the rate of 0.5 ml min⁻¹. The final volume was dried by rotavapor and then 5 ml water was added for complete dispersion. The solution was filtered through a 0.22 μm filter paper to make sure that only the well-dispersed compound will go across the membrane.

UV-Vis and photoluminescence studies

UV-Vis absorption spectra were recorded on Varian CARY 100 Bio UV-Vis spectrophotometer, with 10 mm quartz cell at 25 ± 0.1 °C. For recording the spectra, 3 ml solutions of SL(A), curcumin and SL(A) + Cur solution were prepared with concentration of 100 μg ml⁻¹. The solutions were mixed gently and subsequently the spectra were recorded.

DLS measurements

The dynamic light scattering (DLS) measurements were carried out on Brookhaven Instrument model 90 Plus Particle Size Analyzer.

Zeta potential

The surface charges of the SL(A), curcumin and SL(A) + Cur were determined using a zeta potential analyzer (Brookhaven Instruments Corporation, NY). The average zeta potentials of the nano self-assembly dispersions were determined without any dilution.

FTIR analysis

FTIR spectra were recorded with KBr pellets in transmission mode using a Nicolet Magna IR-750 spectrophotometer at 4 cm⁻¹ resolution with 64 scans between 4000 and 400 cm⁻¹. Two milligram of dried powder was mixed with 198 milligram KBr and analyzed.

NMR study

¹H was recorded on Bruker Avance DPX 200 and DPX 400 instruments operating at 200 MHz (¹H).

Scanning Electron Microscopy (SEM)

Field emission scanning electron microscopy images were acquired on FEI QUANTA 200 microscope, equipped with a tungsten filament gun, operating at WD 10.6 mm and 20 kV. A 10 μl aliquots of all three sample solution were placed on silicon wafer and these were fixed on copper stubs with the help of carbon tape. The samples were dried at room temperature overnight and images were recorded without gold coating.²⁵

Cell lines and reagents

The human breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231 used in the study were obtained from National Centre for Cell Science (NCCS), Pune, India.³³ The cells were grown in DMEM containing 2 mM L-glutamine supplemented with 10% fetal bovine serum and 100 U ml⁻¹ of penicillin–streptomycin. The cells were incubated in a humidified 5% CO₂ incubator at 37 °C. Tissue culture plastic ware was purchased from BD Biosciences, CA, USA. curcumin, Dulbecco's Modified Eagles Medium (DMEM), Fetal Bovine Serum (FBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylthiazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). Penicillin–streptomycin and L-glutamine were obtained from Gibco BRL, CA, USA.

MTT assay

The cell viability was determined by MTT dye uptake as described previously.³³ Briefly, the cells were seeded at a density of 1 × 10⁵ cells per ml in 96-well plates. An untreated group was also kept as a negative control. The cells were treated with different concentrations (0–160 μg ml⁻¹) of SL(A), curcumin (dissolved in ethanol) and SL(A) + Cur. MTT solution (concentration 5 mg ml⁻¹) was added to each well and the cells were cultured for another 4 h at 37 °C in 5% CO₂ incubator. The formazan crystals formed were then dissolved by addition of 90 μl of SDS–DMF (20% SDS in 50% DMF). After a duration of 15 min, the amount of colored formazan derivative was determined by measuring the optical density (OD) using the ELISA microplate reader (Biorad, Hercules, CA) at 570 nm (OD 570–630 nm). The percentage viability was calculated as:

% Viability = [OD of treated cells/OD of control cells] × 100

Statistical analysis

All the experiments were performed in triplicates and repeated twice and the data are presented as mean ± SD.³³ Statistical analysis was conducted with the Graph Pad 4 prism program using one-way ANOVA. The p-values used for comparisons were <0.05. IC₅₀ values were calculated using Kyplot software.

Conclusions

Sophorolipid, an environmentally friendly, biocompatible and important class of biosurfactants is complexed with curcumin to increase its solubility, stability, fluorescence and bioavailability. Particle size distribution, TEM and zeta potential analyses suggest that the increase in cellular uptake could be attributed to the nano-encapsulation of curcumin, its solubilization and stability in aqueous solution. It has been clearly established that the complex formation of curcumin with SL(A) significantly reduced its therapeutic index, reflecting its increased bioavailability. This study emphasizes that solubilization by nano-encapsulation is an effective aspect of designing drug delivery systems.

Acknowledgements

This work is financially supported by the Council of Scientific and Industrial Research (CSIR), New Delhi India.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07300b

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