1. INTRODUCTION:

Development of multiparticulate systems had gained much fame over the single unit systems for oral drug delivery applications as per reports of last few decades¹. It has proved to be a potential system due to numerous reasons viz., predictable gastric emptying, reduced risk of toxicity, reduced dose dumping, reduced local irritation, reduced inter-intra subject variability, increased bioavailability, and improved stability. Multiparticulate system mostly used for oral routes includes microspheres, beads, granules, nanoparticles, microparticles, etc., that ensure forunique release profiles, uniform drug dispersion and absorption into the gastrointestinal (GI) tract. The system developed for colonic targeting were able to pass through the upper GI tract easily, while reaches colon region at predictable time and retained at ascending for longer period of time^2,3,4. Colorectal cancer is the third mainly common cancer in the world, with nearly 1.8 million novel cases diagnosed in 2018, and has deprived prognosis when metastasized to lymph nodes or distant organs⁵. In Europe, it is the second mainly common cancer and in the United States, the third most common form of cancer and second-leading cause of deaths^{6, 7}. In fact, colorectal cancer is responsible for a high rate of morbidity and mortality according to global cancer statistics⁸. Colorectal cancer manifests as cancerous growths in the colon, rectum and appendix. Colorectal cancer is the second most ordinary cancer killer overall and third most common cause of cancer-related death in the United States in both males and females. Oral colon-specific drug delivery system is more advantageous over conventional cancer chemotherapy as it is unproductive in delivering drugs to the colon due to absorption or degradation of the active ingredient in the upper gastrointestinal tract. CDDS as an effective and safe therapy for colon cancer provides therapeutic concentrations of anticancer agent at the site of action and spare the normal tissues, with reduced dose and reduced duration of therapy. The effective focused on delivery of medication to the colon by means of the gastrointestinal tract requires the security of a medication from debasement and discharge in the stomach and small digestive system and afterward guarantees unexpected or controlled discharge in the proximal colon⁹.In pH controlled discharge frameworks, the distinctive pH of human GIT is abused by covering the measurement structure with pH subordinate polymers which stays accordingly in the upper GIT and debase in the digestive organ where the pH is high i.e. Subsidiaries of acrylic acid and cellulose are the for the most part utilized pH-subordinate polymers. On the activity of polymers and their solvency at different pH condition, delivery frameworks have been intended to pass on medications at the specific target site. Most regularly utilized pH-subordinate polymers are methacrylicacid copolymer (i.e., Eudragit L100 and S100), which disintegrate at pH 6.0 and 7.0 separately^10,11. These polymers don't break down in stomach and intestinal pH because of hydrogen holding between the hydroxyl gatherings of the carboxylic moiety and the carbonyl oxygen of ester bunches in the polymer particles. Be that as it may, they breaks up in the colon as a result of the ionization of their carboxyl practical gatherings and discharges the medication in the colon¹².It is possible to modify the polymer characteristics by using the combination of Eudragit S100 and L100 in varying ratio^13,14,15. The addition of Eudragit L100 to S100 in varying ratios altered the pH at which the polymer solubilized to produce formulations with high accuracy. Methotrexate (MTX) is used as anti-cancer drug, acting as a dihydrofolate reductase inhibitor. It is used in the colo-rectal cancer¹⁶. High-portion MTX is settled for the treatment of strong tumors and leukemias ^17,18 while low-portion regimens are generally utilized in the treatment of immune system diseases^19,20,21 and as of late, as immunosuppressive agent in organ transplantation²².MTX was introduced to clinics over six decades ago and is one of the most widely used and studied anticancer agents. Its administration however, has the potential of severe side effects, including neurologic toxicity, renal failure due to tubular obstruction by crystal deposits of MTX and its primary metabolite, 7-hydroxy-methotrexate (7-OH-MTX), myelosuppresion, and mucositis. The effectiveness of HDMTX therapy has been greatly enhanced by the observation that patients at high risk of serious toxicity may be detected by monitoring serum MTX concentrations. Therefore, the routine monitoring of drug serum concentrations is important in guiding leucovorin rescue and is considered to be imperative for both patient safety and evaluation of therapeutic concentrations of MTX. The objective of the present investigation was to formulate and characterize the microspheres of MTX using polymers Eudragit S 100 and sodium lauryl sulfate for colon targeting.

2. MATERIALS AND METHODS:

2.1 Materials

MTX was acquired from Khandelwal Laboratory Pvt. Ltd. Mumbai. Eudragit S 100 was acquired from Evonik Degussa India Pvt. Ltd. Mumbai. DCM (Dichloro methane) and SLS (Sodium lauryl sulfate) were acquired from HiMedia Laboratory Pvt. Mumbai, Maharashtra (India).All other reagents and chemicals used were of analytical grade. Triple distilled water was generated in house.

2.2 Preformulation Studies

The preformulation studies of drug was carried out by physical examination i.e., colour, texture, odour etc. The solubility of the drug was determined by taking small quantity of drug (aprox. 10 mg) in the 10 ml volumetric flasks separately and added the 10 ml of the solvent (water, ethanol, methanol, 0.1N HCL, 0.1N NaOH, chloroform and 7.4 pH buffer) Shake vigorously and kept for some time. Note the solubility of the drug in various solvents (at room temperature).Melting point was determined by placing small quantity of powder into a fusion tube. That tube was placed in the melting point determining apparatus (Chemline) containing castor oil. The temperature of the castor oil was gradual increased automatically and read the temperature at which powder started to melt and the temperature when all the powder gets melted²³.Quantitative estimation of drug was performed by determination of λ _maxof methotrexate. Accurately weighed 10 mg of drug was dissolved in 10 ml of phosphate buffer pH 7.4 solution in 10 ml of volumetric flask. The resulted solution 1000µg/ml and from this solution 1 ml pipette out and transfer into 10 ml volumetric flask and volume make up with phosphate buffer pH 7.2 solution. Prepare suitable dilution to make it to a concentration range of 10-30µg/ml. The spectrum of this solution was run in 200-400 nm range in U.V. spectrophotometer (Shimadzu UV1800, Japan). A graph of concentration Vs absorbance was plotted.

2.3 FTIR spectroscopy

The concentration of the sample in KBr should be in the range of 0.2% to 1 %. The pellet is a lot thicker than a liquid film, consequently a decrease concentration in the sample is required (Beer's Law). For the die set that you'll be the usage of, about 80 mg of the mixture is wanted. Too excessive of an attention causes typically difficulties to obtain clean pellets. This pellet keeps into the sample cell and scanned between 4000-400 c.m^-1 and IR spectra are obtained²⁴.

2.4 Process variables

There are many procedure factors, which could influence the arrangement and properties of the microspheres, were recognized and considered. The technique for planning was in like manner advanced. These are the procedure factors of microspheres arrangement were chosen for optimization of plan

· Concentration of polymer.

· Stirring rate.

Total 9 formulations were designed on the basis of these variables. The formulation code and respectable variables used in the preparation of microspheres are given in Table 1. The effects of these variables were observed on particle size, % yield, % drug entrapment and % drug release. The procedure adopted in the optimization of the variables was follows,

· Concentration of polymer: To optimize the formulation, varying concentration of drug polymer i.e. 1:1, 1:2 and 1:3 were taken by keeping drug and emulsifying agent constant.

· Stirring rate: Stirring rate for the preparation was optimized by keeping the microsphere at different stirring speed i.e. 900, 1200 and 1500 rpm, while keeping all the parameters constant as described in the procedure for the preparation of microspheres.

2.5 Fabrication of Eudragit microspheres containing drug

Eudragit containing microspheres were fabricated by emulsion-solvent evaporation technique^25,
26. MTX and Eudragit were used in ratios 1:1, 1:2 and 1:3 to obtain significant different characteristics. The required amount of the polymer was dissolved in 10ml of a mixture of dichloromethane and ethanol (1:1 v/v). The calculated amount of MTX powder was dissolved in the polymeric solution. The prepared dispersion was slowly poured into 100 ml of 0.2 % w/v SLS aqueous solution and was emulsified by vigorous stirring at (900, 1200, and 1500rpm) at room temperature using a three-blade mechanical stirrer. The dispersed drug and polymer were immediately transformed into fine droplets, which were subsequently solidified into rigid microspheres due to solvent evaporation. Stirring was continued for 3-4 hrs until all solvent was evaporated. Finally, the microspheres were filtered, washed with distilled water, and dried at a fixed temperature of 37 ⁰C for 24 h to yield free flowing discrete microspheres. The microspheres were dried and stored in air tight containers until further analysis. Formulations with different drug to polymer ratios were prepared as shown in table 1.

Table 1: Composition of various formulation of microsphere

S. No	Formulation code	Drug: Polymer ratio	Stirring speed (rpm)	Concentration of emulsifying agent (%w/v)
1	SS 1a	1:1	900	0.2
2	SS 2a	1:2	900	0.2
3	SS 3a	1:3	900	0.2
4	SS 1b	1:1	1200	0.2
5	SS 2b	1:2	1200	0.2
6	SS 3b	1:3	1200	0.2
7	SS 1c	1:1	1500	0.2
8	SS 2c	1:2	1500	0.2
9	SS 3c	1:3	1500	0.2

2.6 Percentage yield:

The prepared microspheres with a size range of 1μm to 1000μm were collected and weighed from different formulations. The determined weight was separated by the aggregate sum of all non-unpredictable parts which were utilized for the planning of the microspheres.

Actual weight of product

% Yield = -------------------------------------- x 100

Total weight of drug and polymer

2.7 Percentage drug entrapment:

Percent drug entrapment determination is the most important parameter to study the efficiency of the process. Percent drug entrapment of all the batches prepared was determined by using spectrophotometer to study the effect of various variables.²⁷ An accurately weighed 100 mg microspheres containing MTX were washed with specific amount of methylene chloride. At that point microspheres were dissolved in 20 ml of ethanol. The arrangement was sifted with a whatman paper (# 40) and 1 ml of this arrangement was around weakened to 10 ml utilizing ethanol and examined spectrophotometrically at 258 nm utilizing UV-spectrophotometer (Shimadzu UV1800, Japan).

Calculated drug content

% Drug Entrapment = ^{---------------------------------------------------------------------------------------------------------------------------------------------} × 100

Theoretical drug content

2.8 Measurement of mean particle size:

The particle size determination of prepared multiparticulate system was performed by optical microscopic method. The size of microsphere was measured. The mean of 100 microspheres was noted as particle size. All the readings of particle size were the mean of three trials ± S.D. The eyepiece micrometer was previously calibrated with a standard stage micrometer. The prepared microsphere was taken on the clean glass slide and the size of the particles was determined by utilizing eyepiece micrometer.

2.9 Differential scanning calorimetry:

The possible interaction between MTX and Eudragit S 100 during the processing of microspheres is assessed by carrying out the thermal analysis of pure drug along with excipients. The stability of a formulation depends upon the compatibility of the drug with excipients. It is of significance to detect any possible physical (or) chemical interaction, since it can affect the bioavailability and stability. DSC is quick and solid techniques to screen medicates excipients similarity and give most extreme data about the conceivable cooperation. Thermal analysis dose not replace stability test, but is valuable tool at the preformulation stage. DSC in combination with short time stress tests is recommended for easy evaluation and interpretation of DSC curves. The ratio of drug excipients used in the study is subjected to the discretion of the formulator. However, Van Dooren recommends ratio of 1:5 for diluents, 3:1 for binders or disintegrates, 5:1 for lubricants and 10:1 for colorant etc. In DSC an interaction is concluded by elimination of endothermic peak(s), appearance of new peak(s), change in peak shape and its onset, peak temperature/melting point and relative peak area or enthalpy. DSC examination was conducted for the optimized formulation, pure drug, SLS and the polymer using DCS instrument (DSC-4000, Perkin Elmer). Samples (2-5 mg) were weighed and hermetically sealed in flat bottomed aluminum pans. These samples were heated over a temperature range of 50-400⁰C in an atmosphere of nitrogen (50 ml/min) at a constant rate of 10⁰C/min, with alumina being the reference standard.²⁸

2.10 X-Ray diffraction:

In order to determine the physical state of drug i.e. amorphous or crystalline nature in formulation, XRD was done. The sharp peak determines the crystalline nature of the drug. To characterize the physical state of MTX, Polymers and formulations, X-ray diffraction analysis was performed in an X-ray diffractometer (Rigaku X-ray diffractometer). The characteristic X-ray diffraction spectra of pure drug and formulation were presented in Fig: 6. The powder drug were recorded using Ni-filter, anode material Cu, K-alpha radiation (1.54060 and 1.5443 Å), scan type continuous, a voltage of 30Kv, a current of 15 mA, scan speed 40 min^-1 over the 0^o to 90^odiffraction angle (2θ) range and the count range 2000cps. The stability of a formulation depends upon the compatibility of the drug with excipients. It is of significance to detect any possible physical (or) chemical interaction, since it can affect the bioavailability and stability. XRD is a fast and reliable method to screen drug-excipients compatibility and provide maximum information about the possible interaction.

2.11 Shape and surface morphology:

From the formulated batches of microspheres, formulations (SS 3b) which showed an appropriate balance between the percentage drug releases was examined for surface morphology and shape using scanning electron microscope (Jeol, Japan)_.²⁹Sample was fixed on carbon tape and fine gold sputtering was applied in a high vacuum evaporator. The acceleration voltage was set at 10KV during scanning. Microphotographs were taken on different magnification (1000 and 2500X) was used for surface morphology.

2.12 In-Vitro Drug Release:

The prepared microspheres were evaluated for in vitro drug release by using USP II Basket type dissolution test apparatus. A weighed quantity of formulation (equivalent to 30mg) was filled in capsule and kept in basket of dissolution apparatus with dissolution media (900 ml) at 37±0.2°C. Samples were withdrawn at different time interval and compensated with same amount of fresh dissolution medium.³⁰ Volume of sample withdrawn was made up to 5ml by media. The samples withdrawn were assayed spectrophotometrically at 258 nm for percent of release of MTX using UV visible spectrophotometer. The release of MTX was calculated with the help of standard curve of MTX. The scheme of using the simulated fluids at different timing was as follows:

1^st hour:Simulated gastric fluid (SGF) of pH 1.2.

3^rd hour:Simulated intestinal fluid (SIF) of pH 6.8.

6^th hour and onward:SIF pH 7.4

2.13 Drug release kinetic data analysis:

Several kinetic models have been proposed to describe the release characteristics of a drug from matrix. The following five equations were zero-order, first-order, Higuchi’s, Hixon and Korsemeyer-Peppas equation used to determine the mechanism of drug release_.³¹

Equation 1, the zero-order model equation (Plotted as cumulative percentage of drug released vs time); Equation 2, the first-order model equation (Plotted as log cumulative percent Drug remaining Vs time); Equation 3, Higuchi’s square-root equation (Plotted as cumulative percentage of drug released vs square root of time); Equation 4, Hixon equation (Plotted as percentage cube root of drug remaining vs time); and Equation 5, the Korsemeyer-Peppas equation (Plotted as Log cumulative percentage of drug released vs Log time).

2.14 Stability studies for optimized formulation:

Accurately weighed 100 mg microsphere prepared with the optimized formulation parameters was stored at three different conditions: Room temperature (RT), and accelerated temperature (40°C). MTX content of the samples was analyzed at predetermined time intervals (Initial day, 1 month, 2 month, 3 month and 6 month)³².

3. RESULTS AND DISCUSSIONS:

The melting point of MTX (pure drug) was found to be 182-189⁰C. MTX was insoluble in ethanol, methanol, water and ether, practically soluble in 0.1 N HCl, and soluble in 0.1 N NaOH and phosphate buffer pH 7.4. Identification of MTX was done by FTIR spectroscopy with respect to marker compound. It was identified from the result of IR spectrum as per specification fig. 1. The calibration curve of MTX was found to be linear in the concentration range of 2-20 µg/ml at 258 nm fig. 2. Partition coefficient and; moisture content of MTX was found to be 0.005 K and 0.0711 respectively.

Fig. 1 FTIR Spectrum of pure MTX drug

Fig. 2 Wavelength maxima of MTX in phosphate buffer pH 7.4

During preparation of microsphere of MTX, change in particle size of microsphere on varying the concentration of Eudragit in drug-polymer ratio has been observed. The size of microsphere was ranges from 25.12 to 42.32 µm. The formulation SS 1c displayed lowest size of microsphere 25.12 µm, while the formulations SS 3a revealed largest size of microspheres. Table 2 depicted that the formulation containing lower concentration of Eudragit produces smallest size of microsphere. In contrast the formulation comprising higher concentration of Eudragit, it increases the size of microspheres due to aggregation of particles. It clearly indicates that the size of microsphere was found to be dependent on concentration of Eudragit.

The average particle size of Eudragit microspheres decreased as agitation speed increased from 900 to 1500 rpm. This was expected because high turbulence caused frothing, results in decreased in mean particle size of microspheres. These results are in agreement with the result of sahoo et al, (2005) demonstrated that an increased in stirring rate shows a decreased in the mean particle size of eugragit microspheres because of high turbulence.

The percentage yield and entrapment efficiency of the microspheres are listed in Table 2. Table 2 exhibited that on increasing the polymer in formulation it improves the percentage yield, and its corresponding encapsulation efficiency also improved. The percentage yield and entrapment efficiency ranges from 68.89 to 80.33% and 65.00 to 74.00%, respectively.

The incorporation efficiency of MTX was found to be good at all loadings. The high entrapment efficiency of the drug is believed to be due to its poor non aqueous solubility. The extent of loading appears to influence the particle size distribution of microspheres.

Table 2: Particle size analysis of formulations

Formulation code	Average particle size (µm)	Yield (%)	Drug entrapment (%)
SS 1a	36.90 ± 3.97	68.89 ± 1.2	65.00 ± 2.9
SS 2a	38.06 ± 3.67	73.48 ± 0.9	67.08 ± 3.1
SS 3a	42.32 ± 3.22	76.66 ± 1.8	69.78 ± 1.6
SS 1b	29.03 ± 2.90	73.30 ± 0.4	68.60 ± 2.9
SS 2b	32.01 ± 4.63	75.01 ± 1.3	69.71 ± 2.3
SS 3b	39.87 ± 3.06	79.48 ± 1.6	72.09 ± 2.6
SS 1c	25.12 ± 3.28	75.52 ± 2.5	70.07 ± 4.1
SS 2c	30.12 ± 2.67	78.06 ± 3.1	71.88 ± 3.4
SS 3c	33.98 ± 2.34	80.33 ± 0.7	74.00 ± 2.3

Values are mean ± S.D.

The surface morphology of the drug loaded microsphere was investigated by scanning electron microscopy. Studies using SEM provided a better understanding of the morphological characteristics of the microspheres. The microsphere prepared by higher Eudragit concentration the microsphere with smooth surface was obtained (Fig 3). When the inner water phase is evaporated the crust is destroyed, the outer surface collapses and as a result, small pores are formed (Fig 4). The entrapped substance is drained, affecting the loading efficiency. Furthermore, it will concentrate towards the microparticle surface contributing to the initial burst release. Surface hollows could be attributed to the subsequent shrinkage of the microspheres after solidification.

Figure 3 Scanning electron photomicrograph of Eudragit microsphere (magnification 1000x).

Figure 4 Scanning electron photomicrograph of Eudragit microsphere (magnification 2500x).

The stability of a formulation depends upon the compatibility of the drug with excipients. It is of significance to detect any possible physical (or) chemical interaction, there is no peak of SLS in DSC study which was absence of in the formulation. So there is no interference of formulation additive in the estimation of MTX by DSC study (Fig 5). XRD determines the crystalline nature of MTX, amorphous nature of polymer and formulation Figure 6.

Fig. 5 DSC data of SS 3b microsphere formulation

Fig. 6 XRD data of (a) Methotrexate (MTX), (b) Eudragit S 100 and (c) microsphere formulation SS 3b

Study of in vitro drug-release profile of MTX from different formulation batches in pH 1.2, pH 6.8 and followed by pH 7.4. The microspheres of Eudragit S 100 also released the drug in pH 1.2 and pH 6.8, which may be due to the presence of pores on the microspheres surface. Released of drug from microspheres in pH 1.2 and pH 6.8 may be due to diffusion process but released at pH 7.4 could be followed both diffusion and erosion mechanism. From Fig. 8 in-vitro dissolution studies revealed that 92.65% of drug release from SS 1a at 14 hrs. The 50% of the drug was released from the formulations SS 1a within 8 hrs, while SS 2a, SS 3a, SS 1b, SS 2b, SS 3b, SS 1c, SS 2c and SS 3c release 50% drug after 8 hrs. All the formulation release maximum drugs at 14 hrs, but SS 3b showed 85.24% drug release at 14 hrs. It was considered as sustained release of drug from microspheres. From above finding it has been noticed that on increasing the concentration of polymer it decreased the drug release from microspheres. The high concentration of polymer makes the microsphere stiff. This hardness of microsphere decreases the rate of drug release from microspheres.

Fig 7: In-vitro drug release profile of MTX microsphere

The formulations of MTX microsphere were subjected to five model fitting analysis namely, zero order, first order, Higuchi, Hixon and Korsmeyer-peppas model (Table 3).Table 3 indicate that all the formulations follow the Higuchi order kinetics as the co-efficient of regression (R²) was more near to unity as compared to the regression value of zero order, first order and Hixon model. It indicates that the release of drug from matrix as a square root of time dependent process based on non Fickian diffusion. Among all the formulations it was observed that R² value of formulation SS 3b was more near to one than other formulations. On the basis of this parameter, SS 3b was selected for further study.

Further, to understand the drug release mechanism, the data were fitted to Peppas exponential equation Mt/Mi = Ktⁿ, where Mt/Mi is the fractional drug release into the dissolution medium, K is a constant which incorporates the properties of the macromolecular polymeric system and drug and n is the diffusional exponent, which characterizes the drug transport mechanism. When n = 0.5, it indicates quasi-Fickian diffusion mechanism. For n > 0.5, an anomalous non-Fickian diffusion and the special case of n = 1 that has gained importance due to its potential application in the development of swelling controlled drug delivery systems with zero-order kinetics indicate pseudo-case-II transport mechanism. In the present study also it was observed (Table 4) that the entire formulated microsphere followed non-Fickian diffusion mechanism, which indicates the drug release through diffusion and relaxation.

4. CONCLUSION:

The stable colon targeted SLS and Eudragit S-100 methotrexate microspheres with for the treatment of colon target were successfully developed in this research work. The microspheres were prepared successfully which was confirmed by characterization analysis of formulation. The value of particle size, %yield and entrapment efficiency of formulated microspheres were acceptable. All the formulations showed drug release for 14 hrs. The R²value of SS 3b was more near to one than other formulations. Hence it was considered as best formulations. Thus, the designed formulation can act as potential drug delivery system to colon by resisting drug release in stomach and in upper small intestine but maximum amount of drug release in colon.

5. CONFLICT OF INTEREST:

The authors declare there is no conflict of interest.

6. ACKNOWLEDGEMENT:

One of the authors is thankful to UGC-BSR, New Delhi for providing such a platform for research work.

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Received on 18.03.2019 Modified on 10.04.2019

Research J. Pharm. and Tech. 2019; 12(5):2067-2074.

DOI: 10.5958/0974-360X.2019.00342.1

Formulation code	Zero order	First order	Higuchi	Hixon	Korsmeyer and Peppas Model
Formulation code	R²	R²	R²	R²	n	R²
SS 1a	0.916	0.898	0.965	0.941	0.514	0.963
SS 2a	0.961	0.906	0.964	0.942	0.662	0.958
SS 3a	0.964	0.909	0.970	0.943	0.610	0.963
SS 1b	0.964	0.896	0.964	0.941	0.548	0.962
SS 2b	0.963	0.897	0.967	0.939	0.682	0.963
SS 3b	0.980	0.905	0.981	0.940	0.552	0.984
SS 1c	0.975	0.899	0.966	0.941	0.593	0.958
SS 2c	0.962	0.893	0.971	0.935	0.601	0.968
SS 3c	0.958	0.885	0.978	0.927	0.605	0.979

Formulation code	Value of n	R²Value	Mode of transport
SS 1a	0.514	0.963	Non Fickian Anamolous
SS 2a	0.662	0.958	Non Fickian Anamolous
SS 3a	0.610	0.963	Non Fickian Anamolous
SS 1b	0.548	0.962	Non Fickian Anamolous
SS 2b	0.682	0.963	Non Fickian Anamolous
SS 3b	0.552	0.984	Non Fickian Anamolous
SS 1c	0.593	0.958	Non Fickian Anamolous
SS 2c	0.601	0.968	Non Fickian Anamolous
SS 3c	0.605	0.979	Non Fickian Anamolous

Parameter	Initial day	1 Month	2 Month	3 Month	6 Month
Particle size (µm)	39.86±0.85	39.43±0.63	39.27±0.74	39.17±0.47	38.59±0.63
Percentage yield	80.73±0.36	80.52±0.46	80.21±0.57	79.73±0.64	79.48±0.54
DEE (%)	74.92±0.41	74.63±0.28	74.14±0.32	73.84±0.19	73.36±0.35
Surface morphology	-	-	-	-	-

Parameter	Initial day	1 Month	2 Month	3 Month	6 Month
Particle size (µm)	40.25±0.49	40.12±0.52	40.63±0.24	39.54±0.76	39.32±0.34
Percentage yield	80.27±0.37	80.05±0.41	79.68±0.94	79.43±0.53	79.21±0.47
DEE (%)	74.52±0.18	74.13±0.73	73.61±0.34	73.28±0.81	72.86±0.29
Surface morphology	-	-	-	-	-