INTRODUCTION:

In the present era the costs for parenteral dosage forms are increasing day by day hence oral drug delivery is the most prominent and economical option for the cancer treatment. Compare to other drug delivery system, oral route has maximum surface area for loading different type of drugs. Pharmaceutical solid dosage forms include tablets, spheroids, capsules, granules, etc. and among all tablets are mostly used solid dosage forms. Accounting for about 70% of all approved pharmaceutical solid dosage forms used worldwide¹.

Cancer is the unintended growth of the cells in an uncontrolled manner in our body. Colon cancer is one among the 100 types of cancer and the main reason behind its prevalence is life style changes. The chemotherapy declines in the efficacy as the drug fails to reach to its target site. Targeting of drugs to specific colonic region can improve the therapeutic efficacy of the treatment. Colon targeting of drug can be achieved by using various formulation aspects such as controlled drug release by transit time, different pH condition and intestinal micro flora². The colon is a perfect site for absorption of drugs for both local as well as systemic drug delivery. Due to different conditions of gastrointestinal tract such as pH, enzymes, microbial flora, transit time, formulation of drug should have special targeting mechanism to reach and absorb via colon. Compare to stomach colon has a longer transit time. The site of colon can have a number of serious disease conditions such as ulcerative colitis, colorectal cancer and other inflammatory conditions. So if the drug targeted to colon, it could be treated more efficiently³. In the Gastrointestinal tract pH gradually increases when moves down from stomach to the terminal ileum, where the pH of stomach is1.5to 3 and when it comes to the terminal ileum it becomes pH 7-8.4. Upon the combination of different polymers to develop single coating system dosage forms the pH dependent drug release can be obtained. Optimization of factors such as the composition of coating material and thickness, are helpful in targeting the colon through the pH dependent delivery⁵.

From the ancient time itself the use of the curcumin in traditional medicine is being noted significantly in India and China⁷. Curcumin is extracted from the rhizomes of the turmeric (Curcuma longa), chemically a polyphenolic compound and yellow in colour. It provides a wide range of pharmaceutical activities such as antioxidant, anti-inflammatory, anticancer and so on. It also has an advantage as compared to the synthetic agents as it produces low toxic effect to the body. Previous investigations on curcumin in different human cancer cell lines have identified that the curcumin in free form stimulates the obstruction of cell cycle or apoptosis and then inhibits the nuclear factor kappa B (NF-κB) activation⁸. Results also supported that curcumin is used to treat responses in malignancy where it causes weakening of pre-malignant lesions over skin, cervix, bladder, Gastrointestinal tract and the soft palate⁹. The main disadvantage for the formulation of the curcumin to be administered orally is its poor water solubility and decrease in its absorption in the blood that limits it from the systemic bioavailability. The bioavailability problem associated with curcumin can be overcome by incorporating in the SNEDDS (Self nano emulsifying drug delivery system). After the incorporation of drug in SNEDDS it can be loaded on spheroids by using extrusion and spheronization equipment¹⁰. Thus, an effort has taken to deliver bioactive curcumin to the colon for treating the colon cancer, with the use of pectin and hydroxyl propylmethylcellulose (HPMC) as release modifiers. In the physiological ambience of stomach and small intestine Pectin and HPMC remains undiminished and gets degraded in colon due to bacterial inhabitants of the human colon¹¹. Pectin gets solubilised in water; because of its hydrophilic character it loses its shield when it crosses through the stomach and small intestine. To overcome from this problem HPMC, a hydrophilic rate-controlling polymer is used. The drug release rate can be modified by change in viscosity of the polymer grade, adjust the polymer concentration, and by adding different types of excipients to HPMC matrix. The core reason for picking up spheroids as formulation is because of its fast gastric emptying time. Therefore, it was hypothesized that with the positive input of HPMC-Pectin would be a good nominee for aiming the bioactive curcumin for colonic delivery through spheroid formulation¹².

MATERIALS AND METHODS:

Materials:

The following chemicals were purchased: Curcumin (Hi Media Ltd, India), Pectin (Hi Media Ltd., India), sodium starch glycolate (Sigma Ltd., India), microcrystalline cellulose (MCC PH 101) (Signet Corporation Ltd., Mumbai), hydroxyl propyl methylcellulose (Fourts India Ltd., India), sodium carboxymethyl cellulose (sigma Ltd., India), Capryol 90, peanut oil Labrafac PG, Labrafac Lipophile WL 1349 (Gatteffosse India Pvt .Ltd., Mumbai), Cremophore EL (Sigma Aldrich Corporation, Bangalore). Ethanol (Merck chemicals India Pvt. Ltd. Mumbai)

Solubility of the Oil:

Standard calibration curve of the drug (l_max) was found out by using the UV-VIS spectrophotometer. By using the different concentration of the standard solution linearity range was obtained¹³.Solubility of the curcumin in oil was found out by using Shake flask method by adding excess amount of drug in 1ml of the vehicle (Capryol90, Labrafac Lipophile WL 1349, Labrafac PG, Peanut oil) in a volumetric flask and is mixed by using a vortex mixture. Then the vials were kept at a temperature range of 25±10°C in an isothermal shaker for 72h to reach steadiness. The equilibrated sample was extracted using organic solvent (ethanol) by centrifugation at 3000rpm for 20min. The supernatant was passed through a 0.45μm membrane filter. Using UV spectroscopy the concentration of drug was established at 425nm respectively¹⁴.The selection of criteria for surfactants and cosurfactants were based on the ability to form a homogeneous emulsion between the oil, surfactant and cosurfactants mixed together.

Purity of the Drug:

Purity of the Curcumin was found out by using DSC (Differential Scanning Calorimetry). DSC curves were recorded in a TA Instrument using in temperature range of 20-250º C and heating rate of 20 º C/min with a stream of nitrogen. Sealed empty pan was used as a reference sample. The analysis was performed, and the graph was evaluated.

Compatibility Studies:

Compatibility studies for the drug and the physical mixture was found out by using the FT-IR. Here the oil, Surfactant, Co-surfactant, sodium starch glycolate, microcrystalline cellulose, sodium carboxymethyl cellulose and formulation was studied. A physical mixture of drug, Lipid, surfactants, Co-surfactant, sodium starch glycolate, microcrystalline cellulose, sodium carboxymethyl cellulose (either alone or in combination) was prepared and mixed with anhydrous potassium bromide (KBr) in 1:4 ratio. About 100mg of this mixture was ground into fine powder using mortar and pestle followed by compression to form a transparent KBr pellet by using a hydraulic press at 15tons pressure. Each KBr pellet was scanned at 4mm/s at a resolution of 2cm over a wave number region from 4000 to 400cm^-1 in a FTIR spectrophotometer (Shimadzu, Japan). The IR spectrum of the physical mixture (1:4 ratios) was evaluated with that of pure drug, lipid, surfactant, Co-surfactant, sodium starch glycolate, microcrystalline cellulose, sodium carboxymethyl cellulose and IR peak matching was carried out. It was used to identify any disappearance or appearance of peaks. FTIR was also carried out for optimized batch of drug loaded spheroid formulation.

Formulation of nanoemulsion (NE) by spontaneous emulsification method:

Based on the solubility study, Capryol90 was selected for NE formulation. NE was formulated by using simple spontaneous emulsification technique¹⁵. The NE formulations were prepared by blending oil, surfactant, and co-surfactant, in the right proportion, with mild agitation.

Selection of NE formulation:

From Visual observations during phase titration method using SCoS (Surfactant and Co-surfactant) 1:1 to 1:3 ratios, the correct ratio of SCoS was selected for incorporation of drug and subjected for Particle size distribution, Polydispersity index (PDI) and Zeta potential. Determination was done by using the Malvern Zetasizer Nano, Series ZEN1002 instrument and dynamic light scattering (DLS) technique was used as the method.

Formulation of curcumin spheroids with selected oil, surfactant, co-surfactant ratio and other excipients:

Preparation of the Self-emulsifying mixture¹⁶:

Drug, Oil and SCoS were mixed together at room temperature, this was followed by mixing and blend until both are mixed thoroughly.

Extrusion/Spheronization:

Nano emulsion (NE) concentrate containing curcumin was mixed with the optimal ratio of microcrystalline cellulose (MCC PH 101) and sodium starch glycolate and to form a viscoelastic mass it was moistened with binder solution (sodium carboxymethyl cellulose).Then the obtained load was extruded from the roller extruder (Plate 1), then was subjected to spheronization (Plate 2) at 1500rpm speed and rounded off to obtain the spherical section. The obtained spheroids were dried for 1h in a hot air oven at 50°C and were kept in a sealed container, at a dark area.

Optimization of spheronization speed and time: Spheronization speed and time is optimized with different speed (rpm) and time interval (min), the effect of increase in speed, shape of spheroids were examined as to obtain a narrow range of spheroid¹⁷.

Coating of Spheroids:

The spheroids prepared were coated by using a fabricated coating pan¹⁸ using P1-pectin (1%), Pectin: HPMC P2- (0.5:1), P3-(1:0.5), P4- (1:1), P5-(1:2) and P6-(1:3)¹⁹. In which the coating solutions were made using the above-mentioned ratio and it is made to rotate at 20rpm. Coating was continued until spheroids achieved its complete polymer weight. After the coating, the spheroids were kept in an oven for 24h at 40°C. Then the pH dependent polymeric coated spheroids were kept for drug release studies in different pH condition.

Characterisation of Curcumin spheroids:

Particle size:

The particle size of the formulated spheroids was determined with the aid of mechanically sieve shaker. 200mg of sample was placed on top of the sieve and mechanically shaken then the sieves were removed; spheroids retained on each sieve were weighed²⁰. The percentage weights of spheroids were evaluated by using the equation 1. The percentage yield of the spheroids was obtained by equation 2 ²¹.

Particle seize= (weight size)/100………………….. (1 a)

Weight size =

Mean size of sieve opening × % weight retained on smaller sieve………………………………………. (1 b)

Percentage Yield = (Total weight of the spheroids)

----------------------------------------×100

(Total weight of excipient and drug) (2)

Spheroids bulk density was determined by using the equation 3.

Bulk density=M/Vb…………………………………..(3)

Where M= Mass of the spheroids:

Vb= Bulk volume

After determining the tapped density and the bulk density of the spheroids, the compressibility was determined by using the Carr’s index formula. Carr’s index was obtained by using the equation 4²².

(Tapped density-Bulk density)

Carr’s index= --------------------------------------…….. (4)

(Tapped density)

Determination of angle of repose: Method used for finding out the angle of repose of the spheroids was fixed funnel method. Until the formation of the conical pile the spheroids were poured through a funnel. The radius (r) and the height (h) of the pile were noted. The angle of repose (θ) was found out by using the equation 5.

θ = tan^-1 h/r……………………………………………(5)

Friability: Roche Friabilator was used to find out the friability. 10g of the spheroids were weight and tested at 25rpm for 4min. The resultant spheroids were weighed sieved, the spheroids on the sieve were weighted and its percentage friability was estimated by using equation 6.

(Initial weight-final weight)

% Friability = ----------------------------------------- × 100

(Initial weight)

Drug content determination:

10mg of the spheroids were weighed and dissolved in ethanol and sonicated for 1h, it is filtered to form a clear solution. Then the drug content was determined by using UV-Vis spectrophotometer at wavelength of 425nm.

Drug loading and encapsulation efficiency:

Specific amount of the drug was weighed, crushed and suspended in 100ml of 6.8pH phosphate buffer and was agitated at room temperature for 24h. After filtering the sample it was analysed spectrophotometrically at a wavelength of 425nm. The % drug entrapment and % drug loading were evaluated by using the equation 7&8 respectively.

(Practical drug content)

% Drug entrapment = ---------------------------------×100

(Theoretical drug content)

(Amount of drug in sample pellets)

% Drug loading = -------------------------------------------- × 100

(Weight of the pellets

In vitro dissolution studies:

In vitro drug release studies were conducted by modifying the method used by Sarasija and Hota, by the incorporation of a different pH medium on the basis of the transit time of the drug from stomach to colon with the use of the paddle apparatus by USP-XXIII-dilution method. Thus various pH environment (pH 1.2 for 2hr, pH 3.0 for 1hr, pH 7.2 for 1hr, and pH 6.8 for up to 8hr) based on the in vivo conditions were maintained. Around 500 ml of buffer pH 1.2 medium was kept in a basket, then spheroids were added and the paddle was kept for rotation at 50 rpm. The 1 ml of samples was removed on fixed time intervals and was restored with equivalent amount of new buffer solution. 1 hour later, a particular amount of 0.1 M tri sodium phosphate was further added, to alter the pH of the test medium to pH 3.0, without discontinuing the dissolution procedure. The quantity of drug released was measured using the UV-visible spectrophotometer at wavelength of 425 nm²³.

Scanning electron microscopic (SEM) studies:

The shape and surface morphology of spheroids were analyzed by using scanning electron microscope (SEM) Model Joel- LV-5600, USA, photographs were taken at the necessary magnification at room temperature.

Cell-line study:

Effect of Curcumin loaded spheroid formulation on cell growth was determined on human colon carcinoma, HT-29 cell line (National Centre for Cells studies (NCCS), PUNE).The inhibitory activity of sample over the HT-29 cell line is done by (MTT) colorimetric assay which involves 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide²⁴. Cell lines were kept in DMEM (Dulbecco's Modified Eagle's Medium) under suitable conditions and 10% fetal bovine serum (FBS)). The HT-29 cells were kept at condition of about 37°C and in an atmosphere containing 5% CO₂/95% relative humidity (NAPCO series 5400 CO₂ incubator). Once the cells became confluent they were subjected to trypsinization and then inoculated into 96-well culture plates at the density of about 2 × 10³cells per well and incubated at the same condition previously mentioned. 24 later, the old medium was carefully aspirated and the cells were incubated in a logarithmic growth phase with various concentrations ranging from 0 to 600 𝜇g/mL of free Curcumin, equivalent curcumin loaded spheroids. The old medium was altered with newly prepared medium after 24 h of incubation, each well should be added with MTT dye in the concentration of 0.5mg/mL, 20 𝜇L and this is allowed for further incubation at 37⁰C for 4 hrs. This favour the living cells to change into purple formazan crystal from MTT dye. Once the incubation was over the medium was aspirated and dimethyl sulphoxide (DMSO) (200𝜇L) was added. Using a micro plate reader optical density was measured at 450nm. High concentration of dye color leads to high optical density readings which shows high tendency of viable cells to metabolize MTT salts. Using the equation 8 the fractional absorption of the sample was calculated²⁵.

RESULTS AND DISCUSSION:

Preformulation studies:

From the calibration curve of Curcumin the “Slope (K)” and “Intercept (C)” value was found to be 0.007x and 0.014 respectively and linearity R² value was found to be 0.996. Solubility studies of the drug in different oil were performed and the maximum solubility of the drug in oil was selected for further aspects. From Table 1, it was apparent that Capryol 90 showed maximum solubility of Curcumin 103.8±0.18 mg/mL. Thus Capryol 90 was chosen for the formulation of SNEDDS. The solubility study of the drug in deferent oils was given in Table 1.

Table 1 Solubility studies of drug in different oils

Oil	Curcumin (mg/ml)
Labrafac PG	3.557± 0.410
Capryol 90	103.802± 2.401
Labrafac Lipophile WL 1349	3.567±0.351
Peanut oil	0.163±0.030

After the screening of the surfactant and co-surfactants, Cremophor EL and Ethanol were selected for the formulation. SCoS mixture of Cremophor EL and Ethanol, Capryol 90 (oil) was found to produce clear and uniform o/w emulsion.

The DSC thermograms of Curcumin showed single sharp peak at 183.06°C, pectin and HPMC at 92.61°C and 89.02°C respectively. The formulation thermogram exhibited the endothermic peak of drug and polymer at 176.81°C and 91.01°C indicating their crystalline nature and are compatible with each other. Curcumin displayed a strong endothermic peak at 183.06°C, due to crystalline nature of curcumin, whereas the formulation displayed no peak in this region indicating Curcumin changes from crystalline to amorphous form. The melting points of different excipients along with drug are shown in Table 2. The purity of curcumin was measured using DSC and found to be 99.25%. This method has an advantage as it does not require a pure standard.

Table 2 Melting point studies

S. No	Substance	Melting Point
1.	Curcumin	183.06^oC
2.	Microcrystalline cellulose	97.73^oC
3.	Sodium Starch glycolate	106.82^oC
4.	Pectin	92.61^oC
5.	Hydroxypropyl methyl cellulose	89.02^oC
6.	Sodium carboxy methyl cellulose	144.34^oC
7.	Capryol 90	122^oC

Compatibility studies:

The prominent peaks in Curcumin spectra is as follows 1)3014.84 cm^-1 to CH stretching vibration 2) 3000-3500 cm^-1 to OH stretching vibration 3)1627.97 cm^-1 to-C=O bending vibration 4)1114.87 cm^-1 to -C-O-C stretching vibration. As evident from the results, the –OH, -C-H, -C=O, -C-O-C- all group are intact and there were no interactions between Curcumin and selected excipients and as all the peaks were present in the formulation spectra.

Formulation of nanoemulsion (NE) by spontaneous emulsification method:

In this method following observation was recorded. Capryol 90:

In SCoS ratio 1:1 when surfactant and co-surfactants are in equal ratio the ratio of Oil and SCoS i.e. from formulation (F5 to F9) translucent formation of NE was observed. In the SCoS ratio 1:2 the ratio of Oil and SCoS i.e. from formulation (F8 to F9) translucent formation of NE was observed and final ratio of SCoS 1:3 the ratio of Oil and SCoS from formulation (F1 to F9) only milky and emulsion gel was observed. Therefore 1:1 ratio of SCoS having formulation (F5) was selected for incorporation in the final formulation of spheroids. The visual observations of during aqueous titration were given in table 3.

Table 3 Visual observations during aqueous phase titration using SCoS 1:1 to 1:3

SCoS	Oil: SCoS
SCoS	F1	F2	F3	F4	F5	F6	F7	F8	F9
1:1	1:1	1:2	1:3	1:4	1:5	1:6	1:7	1:8	1:9
	E	E	E	E	NE	NE	NE	NE	NE
1:2	1:1	1:2	1:3	1:4	1:5	1:6	1:7	1:8	1:9
	M	E	E	EG	E	M	E	NE	NE
1:3	1:1	1:2	1:3	1:4	1:5	1:6	1:7	1:8	1:9
	E	E	M	EG	EG	E	EG	E	M

NE= Nanoemulsion, E= Emulsion

EG=Emulsion gel, M= Milky

Selection of NE formulation:

From visual observations during aqueous phase titration using SCoS 1:1 to 1:3, the correct ratio of SCoS was found to be SCoS (1:1) ratio having the Oil: SCoS from formulation (F5 to F9), were selected for formulation and it was subjected for further studies.

Particle size distribution, Zeta potential and Polydispersity index (PDI):

As the intensity of oil in formulation rises, particle size enhances and when the strength of SCoS increases, the particle size reduces. Different ratios of SCoS (1:1 to 1:3), the ratio of SCoS (1:1) having formulation F5 (1:5) was observed to form the NE and which was found to have a particle size distribution of 25.9nm with a PDI 0.169, and zeta potential -2.00mV with 100% transmission was selected for incorporating into spheroid formulation, since in other formulations mean droplet size and PDI were found to be larger than other SCoS ratio and wider range of particle size distribution which is not desirable. The optimized SCoS ratio (1:1) having formulation F5 (1:5) was selected based on least particle size and PDI. The other parameters like % transmission and conductivity were found to be satisfactory for all the formulations. The results were displayed in the table 4. Since the formulations are translucent, % transmission was almost greater. The PDI was also less, zeta potential was found to be -2.00, which shows that the SCoS (1:1) ratio having formulation F5 (1:5) was stable.

Formulation of curcumin spheroids with selected oil, surfactant, co-surfactant ratio and other excipients:

The compositions of self-emulsifying formulation consisted of drug, oil and SCoS. The proper ratio of oil and SCoS were mixed with the drug. The formulations showed no signs of phase separation at the ratio of 1:1 of SCoS and oil and SCoS ratio of 1:5 (F5) was selected for the incorporation into the spheroids.

Table 4 Characterization of NE

Sample	Particle size distribution (nm)	PDI	Zeta Potential (mV)	% Trans mission	Conductivity (µS/cm)
1	25.9	0.169	-2.00	100	166.8
2	21.2	0.263	-3.2	98.7	160
3	23.1	0.250	-3.34	97.9	155
4	19.4	0.226	-4.71	94.9	154

Extrusion/Spheronization:

The optimized formulation F5 was further selected for the preparation of spheroids. During the process the speed and time were optimized to get uniform spherical sized spheroids.

Optimization of spheronization speed and time:

Spheronization speed and time was optimized by using different speed (rpm) and time interval (min). With the increase in speed the shape of spheroid were examined, to obtain a narrow size range of spheroid. Time also has an effect on the shape of spheroid, with increasing time a drastic change in the shape of the spheroid is observed. Narrow size of spheroid is needed for good flow and with minimum size range.

Table 5 Optimization of curcumin spheroids spheronization speed

Spheronization speed (rpm)	Spheroid description
400	Dumb bell shape
600	Dumb bell shape
1200	Dumb bell shape
1500	Spheroids with narrow size range

Optimisation of the curcumin spheroids and spheronization speed is given in table 5. At low spheronization speed, were obtained rod and dumbbell shape spheroids and when the speed was increased to 1500 rpm, spherical shape spheroids were obtained. Characteristics of the product being used and particle size required were the factors on which the speed depends. The spheronization time were also optimized. At low spheronization time i.e. < 10 min, extrudes were not completely transformed to spheroids and the obtained yield were also low. From the table 6, it was found that at spheronization time of 15 min was sufficient to generate spheroids with highest yield.

Table 6 Optimization of Curcumin spheroids spheronization time

Spheronization time (min)	Spheroid description
5	Spheroids not formed
10	Spheroids not formed
15	Spheroids formed

Coating of spheroids:

The prepared spheroids were coated with the help of a fabricated coating pan. The % weight gain was observed in the table 7. It was clearly understood that as the polymer content increased the weight gain were also found to be increased. Gradually the HPMC ratio was increased while the Pectin is kept constant for delay the release.

Table 7 Different batches with % polymeric weight gain

Sl.No	BATCH	% POLYMERIC WEIGHT GAIN
1.	P1- pectin (1%)	02.70
2.	P2- (0.5:1)	04.00
3.	P3- (1:0.5)	05.00
4.	P4-(1:1)	05.40
5.	P5- (1:2)	07.89
6.	P6- (1:3)	10.25

Characterization of curcumin spheroids:

The micrometric properties of spheroids were shown in Table 8, like particle size, percentage yield, bulk density, angle of repose, compressibility, drug content and friability were also found to be within limit. Therefore, from the above micrometric data it was obvious that formulation batch prepared possess a comparable particle size. In particle size analysis, it was identified that spheroids have the particle size of 482.9μm. The percentage yield was found to be 66.61%. The bulk densities of the spheroids were established with the range of 0.60-0.70gm/cm³. The lesser values of compressibility (14%) and the angle of repose less than 25° shows a good flow property of spheroids, it depicts that the spheroids are with smooth flow property ensuring a homogenous filling capacity. The friability of spheroids was evaluated to be < 1% w/w. The drug content of curcumin in spheroids was found by using a visible spectrophotometer at 425nm.

Drug loading and Entrapment efficiency:

Drug loading is essential with concern to release characteristics. An acceleration of the drug released can be achieved with the increase in drug loading. The % drug entrapment and drug loading was found to be 93.10% and 0.77%, which was within the range which was given in table 9.

Scanning electron microscopic (SEM) studies:

The shape and surface morphology of the Pectin–HPMC (1:3) coated spheroids can be described by SEM (Figure 1) the spheroids were spherical and uniform in shape. The SEM micrographs of spheroids show a rough and folded surface morphology with porous in nature. The cross-section micrograph shows the coated layer around the spheroids which is the rate limiting factor for delayed release.

Figure 3: Bar- diagram showing IC₅₀of formulation and free curcumin

Cell-line study:

The cytotoxicity study of Curcumin loaded spheroid formulation on cell growth was determined on human colon carcinoma, HT-29 cell line. The inhibitory activity of sample over the HT-29 cell line is done by (MTT) colorimetric assay which involves 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the Viability of cells was determined. Table 11 and Fig. 3 shows the results of cell viability assay. The IC₅₀value of the formulation was found to be 21.1𝜇g/mL, while that of free curcumin was 36𝜇g/mL. When compared with the treated cells there is no relevant cytotoxicity in cells treated to blank formulation. There is no significant change was found even the test performed in the same manner up to 72 hrs.

The particle size, PDI, zeta potential, % Transmission and conductivity of the selected formulations was found to be 25.9nm, 0.169, -2.00mV, 100, and 166.8μS/cm respectively by formulation F5. The formulation F5 was selected for incorporation of the curcumin. SCoS gets adsorbed at the interface, reducing the energy required for NE formation thus improving the thermodynamic stability of the NE formulation. The curcumin loaded NE formulation was incorporated into spheroids and performed the micrometric characterization, in vitro study and cell-line studies. The Preformulation studies like DSC, FTIR were performed to know the compatibility between drug and excipients used in the formulation. In DSC analysis, curcumin illustrated a strong endothermic peak at temperature at 183.06°C, due to crystalline nature of curcumin, while the formulation didn’t show any peak in this area indicating curcumin changes from crystalline to amorphous. From the DSC study it was concluded that, the pectin and HPMC were found to be compatible in entrapping the selected drug curcumin. From the FTIR reports the –OH, -C-H, -C=O, -C-O-C-groups indicates no interactions between Curcumin and selected excipients. Hence, pure drug of curcumin and other excipients are compatible with each other. The spheroid formulation was formed and was optimized by spheronization speed and time. The spheronization speed at 1500rpm more energy is passed on to the particles, leads to production of more force during collision. Smaller particles require higher speed. It was found that spheronization time of 15min was sufficient to get spheroids with maximum yield. The increase in time also affects the shape of spheroid, at different time interval with increasing time, changes in the shape of spheroids were observed. Narrow size ranges of spheroids were needed for good flow and with minimum size range. The shape and surface morphology of spheroids were analyzed by using scanning electron microscope (SEM) Model Joel- LV-5600, USA, photographs were taken and observed to verify spherical nature of the spheroids.

Table 11 % Cell viability of formulation and free curcumin

Sl. No	Weight of spheroid (mg)	% Cell viability
Sl. No	Weight of spheroid (mg)	Formulation	Free curcumin
1.	1.56	68	70
2.	3.125	51	62
3.	6.25	45	55
4.	12.5	30	48
5.	25	25	35
6.	50	22	30
7.	100	21	28

By performing the solubility studies of drug in different oils, the curcumin loaded spheroid formulation was optimized. From the solubility studies, it was clear that capryol90 was the oil in which drug showed maximum solubility. The composition of NE containing oil and SCoS (Cremophore EL, Ethanol) was optimized by visual observation of formation to form translucent NE for the incorporation of drug. Four different formulations are subjected for particle size distribution, PDI and Zeta potential. The ratio 1:1 (Oil: SCoS) having formulation F5 (1:5) was selected for the formulation.

The micrometric properties of spheroids, like particle size, percentage yield, bulk density, angle of repose, compressibility, drug content and friability are found to be within limit. The % drug entrapment and drug loading was found to be 93.10% and 0.77%.In- vitro dissolution study by using the batch prepared by 1:3 ratio (Pectin: HPMC) showed a minimum release of 0.18% at pH 1.2 and maximum release of 88.91% at pH 6.8.In SEM study the shape and surface morphology of the pectin–HPMC (1:3) coated pellets show a rough and folded with porous in nature. Coated layer around the pellets is the rate limiting factor for delayed release. Cell-line study was done on HT-29 cell-line in which IC₅₀value of the formulation was found to be 21.1𝜇g/mL, while that of free curcumin had 36𝜇g/mL, which conclude the formulation is effective than the free drug.

CONCLUSION:

The spheroid formulation containing curcumin loaded SNEDDS was prepared and evaluated. From the results it can be concluded that the ratio 1:1 (Oil: SCoS) formulation (F5) exhibited particle size, PDI, zeta potential, % Transmission and conductivity of 25.9nm, 0.169, -2.00mV, 100, and 166.8μS/cm respectively. From the DSC study it was reported that, the pectin and HPMC were found to be compatible in entrapping with the selected drug curcumin. FTIR showed Pure drug of Curcumin and other excipients are compatible with each other, the groups (–OH, -C-H, -C=O, -C-O-C-) are intact and there were no interactions between Curcumin and selected excipients and as all the peaks were present in the formulation spectra. The optimized speed of spheronization 1500rpm and time 15min was sufficient to produce spheroids with maximum yield.

In vitro dissolution study of batch made via 1:3 ratio (Pectin: HPMC) revealed a minimum release of 0.18% at pH 1.2 and maximum release of 88.91% at pH 6.8. HT-29 cell-line in which IC₅₀value of the formulation was found to be 21.1𝜇g/mL, while that of free curcumin had 36𝜇g/mL, which conclude the formulation was effective than the free drug.

Therefore, curcumin loaded/entrapped in SNEDDS and formulated to spheroids can be used successfully in the treatment of colorectal cancer to get maximum therapeutic effect.

CONFLICT OF INTEREST:

Conflict of interest declared none.

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Received on 31.01.2019 Modified on 28.02.2019

Research J. Pharm. and Tech. 2019; 12(7):3338-3346.

DOI: 10.5958/0974-360X.2019.00563.8

% Product yield	% Drug content	Particle size (µm)	Bulk density (gm/cm³)	Compressibility	Angle of repose	Friability
66.61	93.10	482.9	0.60-0.70	14	21^o	0.55

pH	Time in hours	Pectin: HPMC (0.5:1)	Pectin (1%)	Pectin: HPMC (1:0.5)	Pectin: HPMC (1:1)	Pectin: HPMC (1:2)	Pectin: HPMC (1:3)	Pectin: HPMC (1:3)
-	0	0	0	0	0	0	0	0
1.2	1	16.06	10.72	5.60	4.07	3.19	0.18	-
1.2	2	19.57	15.90	9.52	5.27	4.77	0.31	-
3	3	40.39	42.45	18.74	9.10	7.56	0.33	-
7.2	4	57.82	62.57	60.39	38.42	36.46	19.70	-
6.8	5	62.42	88.37	64.57	41.33	40.24	22.41	27.70
	6	80.00	98.96	88.27	43.81	42.84	32.11	34.60
	7	97.61		98.65	60.23	50.64	36.75	55.25
	8				62.83	52.60	42.40	60.23
	9				64.57	55.90	44.84	65.32
	10				84.32	60.50	48.32	70.45
	11				88.28	74.07	52.31	74.52
	12				97.34	78.63	57.60	88.91

Formulation	% Drug entrapment efficiency	% Drug loading
Curcumin spheroid	93.10	0.77