Keywords
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| -cyclodextrin ,Curcumin, Ethylcellulose , Modified emulsion solvent diffusion method, Microsponge |
Introduction
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| Microsponges are polymeric delivery systems composed of porous microspheres. They are tiny sponge like spherical particles that consists of interconnecting voids within a non-collapsible structure with a large porous surface [1] with particle size ranging from 5-150μm [2].Curcumin (CUR) is the source of turmeric having potential application in tumour treatment. CUR is polyphenolic compound with low intrinsic toxicity. The drug not only suppresses the onset of tumours as well as their and metastasis. Although curcumin showed high safety and efficacy profile, poor aqueous solubility and low systemic bioavailability limits its therapeutic utility [3-6]. Cyclodextrin –based nanosponge have been used to increase the solubility of curcumin and to control its release [3]. CD is regarded as safe by the FDA for use as a food additive [7]. Thus CD can be used safely in the formulation. Moreover for an effective anticancer therapy, the drug molecule must be present at the site of the tumour for a longer time period to exert its therapeutic action. Investigations are being carried on to improve the solubility of curcumin as well as to prolong its release at the site of its action. One possible way to increase its therapeutic utility is to encapsulate the curcumin within the internal cavity of nanocarrier, which improves the solubility as well, increases the drug release rate [6]. Microsponges as controlled release drug delivery systems are used recently for oral purpose [2]. |
| The purpose of the present work is to develop EC microsponge loaded with CUR-CD inclusion complex prepared by modified emulsion solvent diffusion method. Inclusion complex of CUR with CD were prepared and then incorporating this complex in the ethyl cellulose microsponge matrix by the use of modified emulsion solvent diffusion method and thereby evaluated the formulations by using various invitro physicochemical characterization and report the best formulation. |
Materials and Methods
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Materials
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| All chemicals and reagents were of analytical grade and used as received without further purification. -cyclodextrin (CD) was generously gifted by Roquette Signet Chemicals Corp. Pvt. Ltd.(India). Curcumin was purchased from Cayman Chemical Company, India; Ethyl cellulose was purchased from CDH Chemicals, Delhi, India; Polyvinyl alcohol was purchased from CDH, New Delhi, India; Dichloromethane (DCM) was purchased from CDH, New Delhi, India; Sodium lauryl Sulfate was procured from CDH, New Delhi, India; Mili Q water (Milipore) was used throughout the studies was obtained from Elix Milipore water purifier. |
Methods
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Preparation of ethyl cellulose microsponge loaded with curcumin-cyclodextrin inclusion complex
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| The method used for preparation of microsponge is modified emulsion solvent diffusion technique [7].Initially a inclusion complex of CUR was prepared with CD by co-grinding technique [8, 9] in a china dish, where mixing of CUR was done in a ratio of 1:1(w/w ratio) with CD in a glass mortar for 30 minutes, and stored in a dessicator. Then the weighed quantity of ethyl cellulose was dissolved in DCM to form the polymeric organic phase. To this organic phase the inclusion complex was added. To this mixture of the organic phase-inclusion complex aqueous phase containing 1% w/v PVA solution (100ml) was added rapidly with constant stirring at speed ranging from 400-1200 rpm for 2 hours to form an o/w emulsion. The mixture was stirred at variable r.pm ranging from 400-1200 rpm for 2 hours for complete evaporation of the DCM (8ml) from the reaction flask. The formed microsponges were filtered and dried at room temperature. The compositions of the microsponge are given in Table 1. |
Phase solubility studies of CUR
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| The phase solubility study was carried as per the method described by Connors et al [10].An excess of CUR (20mg) was added to 50ml volumetric flasks containing 50 ml phosphate buffer of pH 5 and pH 7.4, with successively increasing quantities of (0, 2, 4, 6, 8, 10,12mM) of -CD [11]. Flasks were sealed and brought to solubility equilibrium at room temperature after shaking for 72 hours in a water bath shaker (Remi water bath shaker). After equilibrium, the content of each flask was filtered through a Millipore membrane (0.45μm) and appropriately diluted with methanol and determined spectrophotometrically the amount of dissolved CUR, at 430.5nm (using Shimadzu UV-1800).The phase solubility diagram was plotted as total dissolved CUR against total CD concentration as given in Table 2. |
Physicochemical Characterizations
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Determination of encapsulation efficiency
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| 30mg of microsponge were taken and crushed in a glass mortar-pestle. The powdered microsponge was mixed with methanol to make up the volume upto 50ml and placed the whole system in a sonicator for 30 min to get the maximum extraction of CUR in the solvent [12]. The sample so obtained were filtered to obtain clear solution and assayed for the drug content spectrophotometrically at 430.5 nm. |
Particle size
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| The particle size were determined by using ocular microscopy by using compund microscope of Olympus. Each 100 number of microsponge were observed under the microscope for three times and thus calculated the particle size. For the confirmation of particle size, the formulation was again observed in a SEM. |
Morphology
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| The morphology and surface characterization of the microsponge were observed using SEM analysis using SEM analyzer (Jeol Japan, JSM- 6390LV), after coating the micropsonges with gold coating. |
Determination of Angle of repose, % Carr’s consolidation index and Hausner’s ratio
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| Angle of repose, % Carr’s consolidation index and Hausner’s ratio were determined to determine the flow properties of formulations [13]. |
Analytical studies of the microsponge
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Fourier Transformed Infrared (FTIR) spectroscopy
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| Pure CUR, physical mixture of pure CUR with CD and EC, formulation F4, pure EC and pure CD were subjected to FTIR spectroscopic studies by potassium bromide disc method using Shimadzu Corporation FT-IR 8400S , in the region of 4000-1 - 400cm-1. |
Differential Scanning calorimetry (DSC)
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| Pure CUR, physical mixture of pure CUR with CD and EC, formulation F4, pure EC and pure CD were subjected to DSC studies using Differential Scanning calorimetry DSC-50, TA-60WS of Shimadzu Corporation, Japan, with 3mg of the samples in the standard aluminium pans. The samples were heated at a constant rate of 100C /min under nitrogen atmosphere. The measurements were done in the range from 300C to 2500C. |
Invitro dissolution studies
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| The invitro dissolution studies were carried out using USP basket type dissolution apparatus (Electrolab TDT-08L) in 900 ml of 0.5% sodium lauryl sulphate solution [14, 15].100mg of the microsponge were taken and filled in a muslin cloth bag and placed in the basket of the dissolution apparatus and started the study at 50rpm [16].The study was continued for a period of 10 hours. 5ml of the sample was withdrawn after every 0.5, 1, 2, 3,4,5,6,7,8,9 and 10 hours and analyzed spectrophotometrically at 430.5nm and calculated the cumulative drug release. The drug release study was done in triplicate. |
Kinetic modelling of drug release pattern
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| The data obtained from invitro release studies were fitted to various kinetic equations to find out the mechanisms of the drug release. The kinetic models used were Zero order equation, first order Equation, Higuchi equation, Hixson Crowell Equation, Peppas-Korsmeyer Equation [17]. |
Results
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Effect of method on the formation of microsponge
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| CUR microsponge prepared by modified emulsion solvent diffusion technique was found to be free flowing and spherical in shape. The preparation method was found to be advantageous for entrapment of water insoluble drugs. The production of microsponge varied with different ratios of polymer. The results are shown in Table 1. |
Phase solubility studies of CUR
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| The phase solubility studies of CUR was done in two different phosphate buffer medium of pH 5 and pH 7.4.It was found that with the increase in pH , the solubility of CUR was found to increase [18].Moreover as from earlier studies , it is confirmed, that with the increase in pH the solubility of the drug increases. As from the Figure 1, the apparent stability Ks for pH 5 is found out to be 6314.327M-1 and for pH 7.4 is 2816.3 M-1. |
Encapsulation efficiency of formulations
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| The microsponge prepared by modified emulsion solvent diffusion technique showed more encapsulation. Encapsulation efficiency of the formulations is shown in Table 1. |
Morphological studies of formulations
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| The particle size of the optimized formulation F4 as observed under the optical microscope is 52.83±6.112 (mean±S.D, n=3) as given in Table 2.The mean particle size of the formulation F4 was further confirmed by SEM photograph as shown in Figure 2. Also there is an evident relationship between the speed and the particle size as shown in Figure 3.Moreover the pores formed on the surface of the formulation are shown in Figure 4. |
Physiochemical properties
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| From data of angle of repose, % carr’s consolidation index, and hausner’s ratio as given in Table 3, it is clear that the formulations ofmicrosponge are sufficiently free flowing and require no other excipients for their smooth movement which is in confirmation with the previous datas [19]. |
Invitro Release study
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| The release profile obtained for the microsponge formulations are presented in Figure 5. A biphasic release pattern of the CUR was observed. |
Fourier transform infrared spectroscopy
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| Figure 6 shows the comparison of FTIR spectra of pure curcumin , physical mixture of curcumin with CD, formulation F4.Pure drug shows the characteristic peak at around 3508.63 cm-1 due to the phenolic O-H stretching vibration, 3347.57cm-1 due to bonded N-H group, sharp absorption bands at 1602.90 cm-1 due to the stretching vibrations of benzene ring, 1511.28cm- 1 due to the C=O and C=C vibration,1429.30cm-1 due to olefinic C-H bending vibration, 1283.67cm- 1 due to aromatic C-O stretching vibrations of curcumin were noticed. |
Differential scanning calorimetry studies
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| Figure 7 shows the comparison between the pure drug, formulation F4, and physical mixture of pure drug with beta-cyclodextrin with ethylcellulose. The thermogram of the pure drug curcumin shows a sharp melting point at 179 0C. There is no sharp peak observed in the F4. |
Kinetics of drug release
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| After plotting the release data of formulation F4, in various kinetic models, it is found out that the release of curcumin followed Korsmeyer- peppas model, showing anomalous transport mechanism. The data of the kinetic modelling are given in Table 4. |
Discussion
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Effect of method on the formation of microsponge
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| The product yield was found to be higher by the modified emulsion solvent diffusion technique. The high yield may be due to the entire mass of the polymer available for coacervation. As the large volume of aqueous phase was added to the small organic phase, the organic phase immediately coacervates getting less time for distribution and thus the yield is more. When the volume was decreased, very good microsponges were formed which is confirmed from the earlier studies [1]. |
Encapsulation efficiency of Curcumin microsponges
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| The high encapsulation efficiency may be due to that when the aqueous phase (which is large in quantity) is added to the organic phase which is very less in quantity, the organic phase immediately starts phase coacervation and the drug is more encapsulated in the polymer matrix. |
Morphological studies
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| The speed had a great impact on the particle size of microsponge formulation. With the increase in speed the particle size decreased. The data are presented in Table 2 and Figure 2. The present study shows a very good correlation between particle size and speed of rotation. With the increase in speed, there is a decrease in the particle size as shown in Figure 3. The topography of the formulations were observed by using SEM and from the photographs as given in Figure 4, it is evident that the pores formed on the microsponge are of the size of 409.166 nm (0.409166μm) ±1.687. |
Invitro Release study
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| The observed initial release may be due to the curcumin which is present in the matrix form.Subsequently, sustained release of the drug was observed due to the presence of curcumin in the inclusion complex [3]. Based on all the above studies of particle size, entrapment efficiency and other micromeritic properties, the formulation F4 is considered as the best formulation. But after the dissolution studies, it is found out that formulation F4 , F5 and F6 gives release of 80%, 93% and 81% respectively in 10 hours of study. Thus formulation F4 is considered as the best formulation because it not only maintains the sustained effect in comparison to the rest of the formulations but also have the least particle size and highest entrapment efficiency. Thus further analytical studies were done for F4 to observe any possible interactions. |
Fourier transform infrared spectroscopy
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| In the FTIR spectra of physical Mixture of CUR with CD and EC, almost all the characteristics peaks of CUR are present. In the FTIR spectra of F4, some characteristic peak of the pure drug of wavelength 3347.57cm-1 have disappeared, which indicates that some complexation of drug has occurred with CD. These studies are in confirmation with the previous datas [3]. |
Differential scanning calorimetry studies
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| The thermogram of the pure drug CUR as shown in Figure 7 shows a sharp melting point at 1790C. But this characteristic peak was not observed in the formulation F4, and also in the physical mixture the peak was a bit shifted towards the lower the lower temperature, which shows that the drug CUR is well complexed with CD. |
Conclusion
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| The study conducted above indicates that the CUR microsponge prepared by modified emulsion solvent diffusion technique shows much smaller particle size and is effective in controlled release of CUR, which may be helpful in targeting drugs to organs. Moreover the formulations can be tested invitro and invivo for evidences of targeted anticancer therapies. |
Acknnowledgements
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| The author deeply acknowledges the contributions from Central instrumentation facility (CIF), BIT, Mesra, for carrying out the analytical procedures successfully. |
Tables at a glance
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Table 3 |
Table 4 |
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Figures at a glance
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Figure 7 |
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