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

This journal is © The Royal Society of C sophorolipid co-processing: highly enhanced bioavailability, fluorescence, and anti-cancer efficacy Pradeep Kumar Singh, Kirtee Wani, Ruchika Kaul-Ghanekar, Asmita Prabhune* and Satishchandra Ogale* Correction for ‘From micron to nano-curcumin by sophorolipid co-processing: highly enhanced bioavailability, fluorescence, and anti-cancer efficacy’ by Pradeep Kumar Singh et al., RSC Adv., 2014, 4, 60334–60341.


Introduction
Curcumin is known to have antioxidant, anti-inflammatory, chemo-preventive and chemo-therapeutic properties. 1 The bioavailability 2 of curcumin is determined by the rate and concentration at which it enters the plasma and reaches the target sites.The oral bioavailability 3 of curcumin is low because a major portion of the compound remains unabsorbed due to a fairly low intestinal absorption capacity.5][6] Several 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. 7Curcumin exhibits tautomerism in its molecular structure and thus exists in the enol form in nonpolar solvents, because of intra-molecular hydrogen bond formation.In polar solvents, however, it is observed in the diketo form. 8The keto form of Curcumin acts as a proton donor in acidic and neutral media, whereas at pH values above 8.0, the enol form dominates and acts as an electron donor.
The existence of the phenolic, b-diketone, as well as the methoxy groups of Curcumin contribute to its free-radical scavenging property.0] However, as mentioned previously, these results have not been reflected well in clinical studies mainly due to the low oral bioavailability of Curcumin.Therefore several soft materials systems including liposomes, [11][12][13] microspheres, 14 dendrimres, 15 micelles, 16 hydrogels and solid lipid nanoscale particles [17][18][19][20] have been explored to design specific drug-delivery systems for Curcumin.These nano-assembly forming procedures designed to improve the bioavailability 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 bioorganic systems with potential applications in biomedical science.They can be produced from renewable feedstock or waste material [22][23]  aqueous solution to reduce the surface tension and interfacial energies. 24They possess unique structures that can be engineered to suit specific application domains. [25]phorolipid exists in two forms: acidic and lactonic. SL(A), is composed of a sophorose unit attached to an oleic acid moiety through an ether bond on the C17 carbon atom of the fatty acid chain.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.

Synthesis of acidic sophorolipid (SL(A))
Crude sophorolipid mixture was synthesized by Candida bombicola processed by alkaline hydrolysis to convert it as acidic sophorolipid under heat treatment.In brief 20 gm.crude sophorolipid mixture was added in 50 mL 5M NaOH solution and reflux under stirred condition.Temperature of the solution was rises to 90 ºC at constant rate 2ºC/min, and then leaves the solution for 10 minutes and finally it was cool to room temperature.Color of the solution was yellow/pale yellow from the start point to the end.Temperature control is a crucial parameter because once it reaches beyond 90 ºC, would reduce the final product due to the formation of oxidized species.Acidic sophorolipid was collected at pH 4 by adding 40 mL 18.5 wt% HCL solutions.

Synthesis procedure for the SL(A)+Cur assembly
SL(A) dissolved in 25 ml distilled water (1 mg/ml) was taken in a 50 ml beaker, and was kept for bath sonication for 30 minutes. 5 ml Curcumin solution (1mg/ml) in distilled water was mixed drop wise during sonication at the rate of 0.5 ml/min.The final volume was dried by rota-vapor 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.

Characterization
In this study, we have developed a novel formulation, namely a complex of acidic sophorolipid 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 (

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.The solutions were mixed gently and subsequently the spectra were recorded.

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.

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. 10 μL aliquots of all three sample solution were placed on silicon wafer Pradeep Kumar Singh 86 Savitribai Phule Pune University 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.

Sample preparation for NMR
Chloroform was added in SL(A)+Cur aqueous solution to extract curcumin from nano-complex.The sample was rotator evaporator and vacuum dried for 60 min to remove chloroform.Curcumin crystals, as a control, were dissolved in chloroform and vacuum-dried as above the encapsulated curcumin.The obtained curcumin powders were dissolved in deuterated chloroform for 1H NMR study.

Cell lines and reagents
The human breast adenocarcinoma cell lines, MCF-

MTT assay
The cell viability was determined by MTT dye uptake as described previously. 32iefly, the cells were seeded at a density of 1 × 10 5 cells/ml density in 96-well plates.
An untreated group was kept as a negative control.The cells were treated with different concentrations (0-160 µg/ml) of SL(A), Curcumin (dissolved in ethanol) and SL(A)+Cur.MTT solution (5 mg/ml) was added to each well and the cells were

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 photophysical properties of curcumin are very sensitive to the medium.Curcumin has extensive absorption around 420 nm in organic solvent.However, its absorbance 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 (Figure 4.2 A).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] toluminescence (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 (Figure 4.2 B).The fluorescence maximum shifts from a broad unremarkable 550 nm band to a remarkable blue shifted band at 500 nm. 11,29To confirm the improvement in PL, we performed some additional experiments.

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;
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 (Figure 4.2 C).
As described earlier, a large blue shift was observed when Curcumin was bound to the SL(A) micelles signifying that Curcumin in SL(A) micelles creates nonpolar

Particles Size Distribution and Zeta Potential
In this experiment, all solutions were analyzed at a constant shutter opening diameter in the DLS apparatus.DLS measurements for Curcumin, SL(A) and SL(A)+Cur (10 mg of each dissolved in 10 ml H 2 O) exhibit hydrodynamic radii of about 818 nm, 6.8.nm and 15.5 nm, respectively (Figure 4.4 A, C, 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

X-ray diffraction (XRD) analysis
To examine the crystallinity of micelle-encapsulated SL(A)+Cur, XRD analysis was performed.XRD analysis of samples was done over broad angle range (2Ɵ = 10-80 degrees).The powder X-ray diffractograms of SL(A), Curcumin and SL(A)+Cur dried powders are shown in Figure 4.7

Nuclear magnetic resonance analysis
We have confirmed the stability of curcumin after four month storage in refrigerator condition through NMR study of as synthesized and after four storage sample study.

Figure 4 . 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, 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 costeffective nanoscale self-assembly approach for improved curcumin delivery and therapeutic efficacy in cancer.

cultured for another 4 h%
at 37°C in 5% CO 2 incubator.The formazan crystals formed were dissolved by addition of 90 μl of SDS-DMF (20% SDS in 50% DMF).After 15 min, the amount of colored Formazan derivative was determined by measuring optical density (OD) using the ELISA micro plate reader (Biorad, Hercules, CA) at 570 nm (OD570-630 nm).The percentage viability was calculated as: Viability = [OD of treated cells/OD of control cells] × 100Statistical AnalysisAll the experiments were performed in triplicates and repeated twice and the data are presented as mean ± SD.Statistical analysis was conducted with the Graph Pad 4 prism program using one-way ANOVA.The p-values used for comparisons were <0.05.IC 50 values were calculated using Kyplot software.
environment, possibly by binding to the hydrophobic regions of SL(A) micelles.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) (Figure 4.3).As seen from Figure 4.3 B a very feeble fluorescence is seen in the case of Curcumin particulates, while the well-dispersed and therefore well distributed SL(A)+Cur nanoparticulates exhibit enhanced fluorescence ( Figure 4.3D).
increase in the size of SL(A)+Cur complex as compared to only SL(A) because of the additional encapsulation of Curcumin nanoparticles.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) = -17.30mV, Curcumin = -15.14mV, SL(A)+Cur= -24.38 mV (Fig.4 B, D, Frespectively).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.

Figure 4 .
Figure 4.6 shows the FTIR spectra for SL(A), Curcumin and SL(A)+Cur after sonication treatment for 30 minutes.The SL(A) reveals a broad band at 3350 cm −1

Figure 4 . 7
Figure 4.7 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

Figure 4 . 8 (
Figure 4.8 (A) NMR spectra of curcumin in SL(A)+ Cur complex and (B) curcumin in SL(A)+ Cur complex after four month stability.Sample preparation for the NMR study perform by addition of chloroform in