Soluble 1:1 Stoichiometry curcumin binary complex for potential apoptosis in human colorectal adenocarcinoma cells (SW480 and Caco-2 cells)

 

Muthu Mohamed J*¹, Fazil Ahmad², Narra Kishore¹, Abeer M. Al-Subaie³

¹Department of Pharmaceutical Technology, BIT Campus, Anna University, Tiruchirappalli 620024,

Tamil Nadu, India.

²Department of Anesthesia Technology, College of Applied Medical Sciences in Jubail, Imam Abdulrahman Bin Faisal University, P.O. Box 4030, Jubail, Saudi Arabia

³Department of Clinical laboratory Sciences, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam, Saudi Arabia.

 

ABSTRACT:

This study investigates the solid dispersion (SD) of curcumin (CMN) enhance the solubility, which can be employed for the treatment of colorectal cancer (CRC) greater to pure CMN. Solid dispersion (SD) prepared by a hot melt method using CMN with several carriers of poloxamers (P-407 and P-188), gelucire 50/13 (GLR) and mannitol (MNT). Prior, phase solubility studies were performed with drug and carriers. The SD characterized by in vitro drug release and novel dyeing test. Additionally, the cytotoxicity and apoptosis studies resolved to utilize the colorectal adenocarcinoma cell lines. The result showed that CMN-P-407 complex produced significant properties towards solubility (318±14.46 fold) and dissolution (91±0.43% at 30 min). The IC₅₀ value for complex found to be 74 and 52µM/mL, while that for pure CMN ranged from 146 and 116µM/mL on the SW480 and Caco-2 cells respectively. Apoptosis study revealed that the cells are undergoing cell death by apoptosis and the small number of necrosis. The profound efficiency of soluble 1:1 stoichiometry curcumin binary complex (CMN-P-407 SD) indicated its potential application for CRC treatment by showing a higher capability of inhibiting cell growth compared to that of pure CMN.

 

 

 

INTRODUCTION:

Primarily, colorectal cancer (CRC) is the major type of cancer in both women and men. In men, CRC is the third most common type of cancer and in women the second most typical type of cancer, and the third highest cause for death worldwide especially, in developed countries (60% of all CRC cases). The risk factors of CRC include colon polyps, long-standing ulcerative colitis, and genetic family history, and it is readily diagnosed in people aged 65–74¹.

 

Several studies have found that curcumin (CMN) shows a wide variety of pharmacological activities and has been considered as one of the most chemo-preventive agents that can induce apoptosis in numerous cellular systems. It is also reported to have the potential impact against an assortment of cancer cell growth². As a result of many toxicological studies, the drug is proved to be non-toxic even at high concentrations.

 

The enhancement of the solubility of insoluble drugs is an important one among the most challenging task in present-day research. The diverse physicochemical methodologies that have been practice to enhance the solubility addition of surfactants by nanosizing and micronization, reduce the particle size, improve of wettability of powders, utilization of prodrugs and its salts, the liposome approach³, and solid dispersion (SD).

 

This research is aimed for (i) the utilization of different carriers in the preparation of SD, (ii) the prepared complex evaluated for aqueous solubility and in vitro dissolution, and (iv) upgrading CMN in hostile to cancer action by altering its mild cytotoxic profile against the SW480 and Caco-2 cell lines of CRC.

 

2. EXPERIMENTAL:

2.1. Materials:

Following chemicals were purchased:  curcumin (CMN; purity > 99%; SRL Pvt. Ltd, Maharashtra, India), poloxamers (P-407 and P-188; BASF Corporation, Mumbai, India) and mannitol (MNT; S.D. Fine Chem. Pvt. Ltd, India). Gelucire 50/13 was gift samples from Gattefosse Pvt. Ltd, Mumbai, India. The colorectal adenocarcinoma cell lines of SW480 and Caco-2 were obtained from National Center for Cell Science (NCCS), Pune, India. Other reagents and chemicals utilized were based on the grade of analytics.

 

2.2. Phase Solubility Studies:

The study of phase solubility is done by the approach explained by Higuchi and Connors⁴. Briefly, CMN is done by adding an excess amount of CMN in 25 mL aqueous solutions of different concentrations (1 to 15 %) to the carriers (P-407, P-188, GLR, and MNT). The Eppendorf tubes containing the arrangement is set in a water bath at a constant temperature of 25 and 37± 0.5 °C for 24 h until the point that balance is accomplished, being shaken often at 30-minute interims. Hence, the content filtered by the Millipore membrane filter (0.45µm), is diluted appropriately, and absorbance is measured at 425nm with UV-spectrophotometer (Agilent Cary 60 UV-Vis Spectrophotometer, USA.). The complexation constant (K_1:1) is ascertained (Eqn. 1) by using slope and intercept evaluation of the phase solubility curve, where the intercept is the intrinsic solubility of the drug.

 

                     Slope

K_1:1 = ––––––––––––––––––………………………..(1)

           Intercept (1-Slope)

 

Also, the change in enthalpy (ΔH) on complexation determined from Van’t Hoff equation, (Eqn. 2)

 

ln(K₂/K₁) = ΔH (T₂-T₁)/(RT₂T₁)….……………...… (2)

 

Where K₂, K₁ and T₂ and T₁ have referred to the stability constants and corresponding temperatures in Kelvin of 37 and 25 °C respectively. The change in Gibbs pure energy (ΔG) and entropy (ΔS) upon complexation /solubilization were computed from the equations (Eqn. 3 and 4) respectively.

 

ΔG = -RT lnK…………………………….................. (3)

where R is the gas constant (R = 8.314J/mol K)

          ΔH-ΔG

ΔS = –––––––…………..………………...............… (4)

             ΔT

 

2.3. Preparation of SD and PM:

The solid dispersions (SD) of varying compositions (1:3 to 1:7) were prepared by a melting method by adding CMN to the molten carrier at 70°C with continuous stirring of 700 RPM for 15 min until a homogeneous dispersion obtained. The resultant melt was allowed to solidify, cooled at room temperature (28°C), pulverized, sieved (# 120; 150-125µm) and stored at 25°C in a desiccator⁵. The physical control blends of the same compositions prepared by utilizing a mortar and pestle as indicated by the guidelines of geometrical mixing, following by screening.

 

2.4. Aqueous Solubility study:

Milli Q water by means of excess amount of test samples (CMN, PM and SD) in a volumetric flask were placed in a water bath at a constant temperature of 25 and 37±0.5°C for 24 h and shaken in between at 30 min intervals⁶. In this way, the substance was filtered through a Millipore membrane filter (0.45µm), diluted appropriately, and the UV absorbance was measured.

 

2.5. In vitro drug release:

The dissolution study was done at 37±0.5°C in 900mL of double distilled water at 50rpm by USP dissolution apparatus II (DS 8000, Lab India, India). The sample placed into the jar and time set as zero. At every 5min time interval up to 30 min, 5mL of samples were withdrawn and filtered by Whatman filter paper (pore size 11µm) then, the dissolved amount of CMN was determined spectrophotometrically. A correction factor represented for the cumulative dilution initiated by replacement of the sample with a unique medium to keep up the sink condition⁷.

 

2.6. Dyeing experiment:

A simple novel test facilitates how efficiently compound (usually colored) get dissolved in an aqueous medium [8]. Briefly, 10mg of CMN and its SDs (equivalent wt.) was added to 15mL of double distilled water then sonicated for 5 min then filtered, formerly pictures of the solutions had taken.  The white linens clothes of similar size (8 × 4.5 cm²) were soaked into the above solution, it has already diluted to 50mL for 1.5 h.

 

2.7. In vitro cytotoxicity study:

The cytotoxicity of samples against SW480 and Caco-2 cell were estimated utilizing an MTT assay. Briefly, 96-well culture plates used for seeding the cells at a cell density of 5 x 10³ cells/mL (200µL/well). DMSO solution used as a solvent control, after 24 h of incubation in different concentration of complexes, the MTT solution (5mg/mL, 20µL/well) was added to the media, followed by incubation at 37°C for 4 h. The obtained purple formazan product dissolved by 100μL of DMSO solution to each well⁹. The absorbance estimations of each well were estimated at 570nm utilizing a plate reader (Bio-Rad, iMark, USA). The IC₅₀ value was estimated, as the complex concentration is necessary to reduce the absorbance to a half the amount of that control.

 

2.8. Apoptosis study by AO/EB staining:

Acridine orange (AO) and ethidium bromide (EB) staining method used to investigate apoptotic morphology with some modifications. Briefly, the cells were treated with the IC₅₀ concentration of compounds for 24 h were collected and washed with chilly PBS. The cell shots were resuspended and diluted with PBS to a concentration of 5 x 10⁵ cells/mL and mixed with 25µL of staining solution (3.8µM of AO and 2.5µM of EB in PBS) on a clean slide. Quickly analyzed under a fluorescent microscope (Carl Zeiss, Axioscope 2plus, Germany) with UV filter (450-490nm). Three 100 cells for each sample were counted for live, apoptotic or necrotic by staining the nucleus structure, membrane integrity and percentage calculated¹⁰. Morphological variations were also observed and photographed (400x magnification).

 

2.9. Apoptosis study by Hoechst staining:

The Sw480 and Caco-2 cells were cultured in separate 6-well plates and treated with IC₅₀ concentrations of samples. Control and treated cells were collected after 24 h of incubation and stained (Hoechst 33258 stain; mg/mL; aqueous) at room temperature for 5 min¹¹. The fluorescent microscope fitted with a 377- 355 nm filter, randomly 300 cells observed with 400x magnification.

 

3. RESULTS:

3.1 Phase Solubility Studies

A standard linear curve obtained as a result of the concentration ranging from 5.97 × 10^-5 to 5.7 × 10^-4 mM and 8.14 × 10^-5 to 7.8 × 10^-4 mM for CMN at 25 and 37⁰C respectively (Table 1). The result is revealing A_L type phase-solubility profile¹².

 

The apparent K_1:1 computed from the slope and intrinsic intercept values of solubility curves acquired by plotting concentration (% w/v) of dissolved CMN against concentration (% w/v) of the carrier (Fig. 1a and b). The stability constant (Ka) attained from the complex ranked in the order of 25 and 37°C as P-407 (631.9 and 524.9M^-1) > P-188 (436.48 and 388.28 M^-1) > GLR (100.14 and 112.05 M^-1) > MNT (10.88 and 11.90 M^-1). Entropy (ΔS), Gibbs pure energy (∆G) and enthalpy (ΔH) were additionally ascertained from PS chart.

 

Figure 1: Phase solubility diagram of CMN in water (a) at 25 and (b) 37 °C in the presence of P-407, P-F188, GLR, and MNT.

 

 

Figure 2: In-vitro release of CMN from (a) PM and (b) SD (mean ± SD, n = 3)

3.2 Aqueous solubility:

The effect of carrier concentration on the solubility of pure CMN in Milli Q water at 37°C found to be 0.004 mg/mL for 24 h. The solubility of CMN-SD (1:3 to 1:7) was enhanced by ~270-322 and ~169-195 fold with P-407 and P-188 respectively (Table 2), due to strong surface-active property surface of drug molecules well adsorbed by the carrier¹³.

 

3.3 In vitro dissolution Studies:

The mean dissolution curves of CMN and SD presented in Fig. 2. It is evident that rate of dissolution of pure CMN was very slow (1.62%) at the end of 30 min because of its native (high hydrophobicity - floats on the medium) characteristics, could prevent to contact with the bulk of the solution. The rate of dissolution of CMN-SD twofold framework with respective carriers demonstrated high burst release (20-80%) in the initial 5-6min showing perfect complex developed with the carrier.

 

 

Figure 3: Photograph of solution of (a) (i) CMN (ii) CMN- P-407 SD (iii) CMN- P-F188 SD and (iv) CMN- GLR SD (b) Photograph of cotton clothes dyed in the solution of (i) CMN (ii) CMN- P-407 SD (iii) CMN- P-F188 SD and (iv) CMN- GLR SD

After the burst release, the constant rate release profile observed with all SDs (Fig. 2b). To explore further solubility and dissolution, P-188 which has a lower molecular weight than P-407, (P-188 of average MW 8500 compared with the average MW of 12600 for P-407) used. The CMN-GLR SD (1:6) showed a considerable release rate of about 56.15% at the end of 30 min. At MNT-SD complex obtained were not satisfactory in comparison to other SDs¹⁴.  

 

3.4 Dyeing effect: 

The CMN and CMN- SDs of P-407, P-188, and GLR showed in Fig. 3, when 10mg CMN added to 15mL of water, CMN float on the water due to the lipophilicity nature of the drug. However, the solution was a bright and characteristic color of poloxamers complex with CMN. Hence, GLR showed yellow color (Fig. 3a).  Fig. 4b shows that the cloth dyed in the solution was colorless (CMN), characteristic deep yellow (P-407), yellow (P-188) and pale yellow color (GLR) respectively¹⁵.

 

3.5 MTT assay:

The cytotoxic results of the MTT-reduction assay of pure CMN and CMN-P-407(SD) on SW480 (Fig. 4a) and Caco-2 (Fig. 4b) cells lines are shown the IC₅₀ value for SD was estimated around 74µM/mL and 52µM/mL, while that of pure CMN ranged from 132µM/mL and 116µM/mL respectively.  

 

3.6 Analysis of cell death:

The cytotoxic effect caused by CMN-P-407(SD) occurred in Sw480, and Caco-2 treated are shown as control or viable cells appeared as bright green color and having uniform chromatin with an intact cell membrane that they did not undergo any apoptotic changes. The stained cells characterized by SD caused more effective cell death than pure CMN.

 

 

Figure 4: In vitro cytotoxic effect of CMN and CMN-SD on (a) SW480 and (b) Caco-2 cells lines.

(Mean ± SD, n = 3)

Increased apoptotic cells and necrotic type of cell death also appeared in both samples (Fig. 5e).

 

After treatment with IC₅₀ concentrations of the complex for 24 h, the Sw480 and Caco-2 cells were observed for cytological changes¹⁶. The manual count of normal and abnormal, i.e., apoptotic cells in percentage from Hoechst 33258 staining in Sw480 and Caco-2 cells image illustrated in Fig. 5II; c and d.

 

4. DISCUSSION:

The PS study acknowledged as a valuable data on the effect of the various carrier responsible for the solubility of CMN. The solubility of the CMN expanded straightly as a component of carrier fixation, i.e., increasing temperature and concentration of carrier, the solubility of CMN increased probably due to the changes in the interaction forces, such as hydrophobic forces and Vander Waals and between CMN and carriers. The slope of the PS diagram obtained (>1) in all carriers indicated the 1:1 complex stoichiometry¹⁷. From all the carriers, poloxamers and GLR showed ideal complexation constant in the ranges of 100 to 1000 M^-1, in contrary, MNT showed the weak interaction with CMN [18]. The figured estimations of ΔG were revealed negative in all carriers, thus confirmed spontaneity of binding and decreased with increase in the carrier’s molecular weight. The calculated values of ΔH were found negative (exothermic) in P-407 and P-188 complexes except for the solubility system with GLR and MNT (Table 1).

 

Similarly, the ΔS value in CMN-GLR and CMN-MNT system found to be marginally high (6 and 4 J/mol K) showing that the reaction type is endothermic¹⁹. The very strong binding constant of poloxamer due to a polyoxyethylene-polypropylene block copolymer nonionic surfactant with an HLB value of 18-23. Polypropylene oxide (PPO) for the most part frame a focal hydrophobic center wherein methyl group associated employing Vander Waals force with CMN undergoing solubilization²⁰. However, solubility due to polyoxyethylene oxide (PEO) blocked by hydrogen bonding interaction of either oxygen with water molecules. Endothermic (+ΔH) process of binding with high (+ΔS) value (6.3 J/mol/deg.) of GLR enhanced wettability while surrounded with a hydrophilic matrix, by reduction of interfacial tension among drug and water. The solubility system of the CMN increased with an increase in concentrations indicating the solvent properties of GLR for the drug²¹.

 

An exception was noticed from MNT where high (+ΔS) value (4.4 J/mol/deg.) obtained due to ligand molecules are ionized, and water molecules are less ordered. The binding process is endothermic (+ΔH); ΔH favors ΔG and ΔS, and spontaneity ensured by negative ΔG refers to a least complex formation between MNT and CMN. These obtained results established that the soluble complex formed between hydrophilic carriers with lipophilic drugs.

 

The aqueous solubility of GLR binary system showed passionate enhancement of solubility around 121-144 fold higher than pure CMN could be due to more hydrogen bonding of water molecules to electron-rich GLR chain containing oxygen atoms (Table 2). In contrary, MNT complex showed odder enhancement of solubility 16-23 fold due to less interaction with drug molecules²².

 

The in vitro release demonstrate after the burst release, the constant rate release profile observed with all SDs. These may occur due to the metastable supersaturation of CMN in the wet carrier matrix during dissolution.

 

Table 1: Thermodynamic parameters of CMN with various carriers at 25 and 37 °C (mean ± SD, n = 3)

 

Table 2: Aqueous solubility data of CMN-SD

Aqueous solubility (mg/mL)
D:C ratio*	1:3	1:4	1:5	1:6	1:7
Pure CMN at 37 °C                                                  0.004 ± 0.0003
P-407	1.0812 ± 0.022	1.110 ± 0.0162	1.21 ± 0.0239	1.25 ± 0.0225	1.29 ± 0.0264
P-F188	0.6791 ± 0.028	0.7017 ± 0.016	0.7423 ± 0.024	0.7601 ± 0.022	0.7798 ± 0.026
GLR	0.4872 ± 0.028	0.4919 ± 0.016	0.5288 ± 0.024	0.5525 ± 0.025	0.5791 ± 0.024
MNT	0.0671 ± 0.028	0.0698 ± 0.025	0.0795 ± 0.022	0.0812 ± 0.032	0.0912± 0.034

^* Drug: carrier ratio

 

Aside from non-ionic attributes, viscosity suggested a perfect part in the dissolution of the drug by delaying the contact of the drug with the dissolution medium. At CMN-SD with MNT obtained were not satisfactory in comparison to other carriers because the solubility due to a polar effect of carbohydrates and the formation of a hydrogen bond may improve solubilization²³. The result showed the limited dissolution rate of MNT might be incomplete amorphization of drug particles compared with pure drug powder.

 

Among all carriers, P-407 showing better physicochemical properties, subsequently these complex developed for cytotoxic contribution. The results from MTT assay specifies that the complex revealed better cytotoxic activity than pure CMN²⁴. These findings could be due to the effect of the variations in the cellular uptake profile leading to the better action of SD as suggested by Manju and coworker²⁵. The results of this assay indicate that the complex of SD can efficiently deliver the drug to SW480 and Caco-2 cells using active targeting via endocytic process shared with improved cytotoxicity and aqueous solubility of CMN-SD. The cell viability with both SD and pure CMN decreases with increasing the concentration of cytotoxicity, whereas SD was significantly better than that of pure CMN.

 

Figure 5: Apoptotic morphology of AO/EB and Hochest staining visualized under fluorescent microscope with (I and II; a) SW480 and (I and II; b) Caco-2 cells; (i) control; (ii) pure CMN; and (iii) CMN-SD and (c and d) percentage of normal, apoptotic and necrotic cells at 24 treatment. Data are expressed as (mean ± SD (%); n=3)

 

Morphological image changes during cell death are important criteria in apoptosis that can be measured by AO/EB staining. Collectively, if the cells were undergoing the specific form of cell death by AO/EB staining, cytological changes have been observed.  As indicated by the fluorescence emission and geologies of the chromatin, cells can be characterized into viable cells; these are very uniform and sorted out structure with green fluorescing cores (Fig. 5; Ia and b; i). Early apoptotic cells, stayed intact membranes yet had quite recently initiated fragmentation of DNA with green fluorescing cores, yet chromatin buildup is perinuclear and visible splendid green pieces or patches (Fig. 5; Ia and b; ii and iii). Late apoptotic cells were divided or condensed chromatin with fluorescing nuclei (orange to red) (Fig. 5I; a and b; ii and iii). Necrotic cells are substantial, or swollen structure had fluorescing nuclei (orange to red) consistently through a nonappearance of chromatin fragmentation (Fig. 5; Ia and b; ii and iii)²⁶ These results suggest that complex treatment caused cell death through apoptosis and necrosis. Altogether, a higher level of cell death was observed contrasted with necrotic cell death (Fig. 5c and d) of Sw480 and Caco-2 than pure CMN treatment. Hoechst staining revealed the deviations in cytology of the cell, with particular reference to cytoplasm and nucleus core at the primary level to identify the apoptosis (Fig. 5; IIa and b; ii and iii). The CMN-SD observation was showing that the early apoptotic highlights, such as cell shrinkage, chromatin buildup, and discontinuity had seen in treated cells and small quantities of necrotic cells²⁷ were observed.

 

5. CONCLUSION:

This study evolution the trend to examine the solubility and dissolution properties of the curcumin complex with various carriers and evaluated the anticancer potency of soluble curcumin on the colorectal adenocarcinoma cell lines of SW480 and Caco-2. The cytotoxic results of the MTT-reduction assay of pure CMN and CMN-SD-P-407 (1:5) on colorectal adenocarcinoma cells and IC₅₀ value was very much lower than pure CMN. The profound efficiency of 1:1 stoichiometry soluble curcumin indicated its potential application for CRC treatment. The research provided an existing and novel method for the implementation of a valuable cancer therapy. We hope the present investigation will inspire further performing along these lines.

 

6. ACKNOWLEDGMENTS:

We thank Prof. K. Ruckmani (HOD), Department of Pharmaceutical Technology, Anna University, Tiruchirappalli and Prof. S. Shanmuganathan, Department of Pharmacy, Sri Ramachandra University, Chennai as doctoral committee members to one of the authors, Muthu Mohamed J research work.

 

7. CONFLICT OF INTEREST:

The authors declare no conflicts of interests.

 

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Received on 13.12.2019           Modified on 14.02.2020

Accepted on 01.04.2020         © RJPT All right reserved