INVESTIGATION OF ANTICANCER DRUG CURCUMIN RELEASE FROM CHITOSAN/CELLULOSE MICROCRYSTALS (CMC)
Materials and methods
Chitosan with a degree of deacetylation (DD) of 93% and vis-cosity average molecular weight of 1463 kDa was obtained fromResearch Lab, Mumbai, India and was used with no further treat-ment. Cellulose micro crystals (CMC), liquid ammonia, various saltsto produce required relative humidity (RH) were obtained fromHi Media Chemicals, Mumbai, India and were analytical grade.Turmeric was purchased from the local market. The double distilled water was used throughout the investigations.2.2. Extraction of CC from turmeric Curcumin (Cur) was obtained from turmeric following the method proposed elsewhere 16. In brief, 15 g of fine turmericpowder was suspended in 150 ml of acetone under moderate stir-ring for 72 h at 30?C. The mixture was filtered and the filtrate was poured into a Petri plate and the solvent was evaporated under vacuum to obtain semi-dry oily mass. The oily mass was weighed and dissolved in 50 ml of dimethyl sulfoxide (DMSO) to give a reddish-brown curcumin solution.
Preparation of Cur-loaded Ch/CMC films by VIPI approach
In order to prepare the Cur-loaded films, 2% chitosan solution was prepared in 2% solution of acetic acid, followed by addition of pre-calculated quantity of CMC under vigorous stirring for 1 hto ensure complete missing. Now, a pre-calculated volume of cur-cumin dispersion was also mixed thoroughly into the above film forming solution and the solution was transferred into Petri plates, followed by their immediate exposure to NH3gas (for detailed procedure see our ref.15). In all four films were prepared, each one containing same amount of curcumin, i.e. 450 _g per g of film. These films were designated as Ch/CMC (0)450, Ch/CMC(10)450,Ch/CMC(20)450, and Ch/CMC(30)450, where the number in subscriptis the amount of curcumin in g present in 1 g of the film.
Characterization of films
The Cur-loaded films were characterized by SEM, XRD, TGA and mechanical analysis. The X-ray diffraction (XRD) method was used to measure the crystalline nature of Ch/CMC(20) and Cur-loaded Ch/CMC(20)450films. These measurements were carried out on a Rikagu Diffractometer (Cu radiation = 0.1546 nm) running at 40 kVand 40 mA. The diffractogram was recorded in the 2_ range of 5–60?at the rate of 2?min?1. The surface morphology of the plain and Curloaded films was investigated using a JEOL 6400F microscope oper-ated with an accelerating voltage of 2 kV and a working distance of4.4 mm. 50 _l sediment suspension (0.01 wt%) was dropped ontoclean silicon wafers followed by air-drying for 24 h and then sput-tered with an approximately 6 nm layer of gold/palladium. The mechanical properties of the films were determined according to the procedure reported elsewhere 17.
Curcumin release studies
The pre-weighed Cur loaded film was placed in 25 ml of phys-iological fluid (PF) at 37?C. The amounts of curcumin released at different time intervals were determined spectrophotometri-cally (Shimadzu, Genesis 10-S) at 526 nm. After each measurement, the film was transferred in to 25 ml of fresh physiological fluid. Calibration curve, prepared for the curcumin solutions of known concentrations in the appropriate range, was used to determine the amount of curcumin in the release media.
A representative sample Ch/CMC(20)450was used to determine the total protein adsorption using the procedure given in a report from the International Standard Organization (ISO)
Antibacterial test by the ‘zone inhibition method’
The antibacterial tests were carried out with curcumin loadedfilm sample Ch/CMC (20)450by ‘zone of inhibition’ method. In brief, the culture medium was prepared by mixing nutrient agar (2.8 g)and agar powder (1 g) in 100 ml of distilled water in a conical flaskand autoclaved for 30 min. The medium was transferred into steril-ized Petri plates in a laminar air flow. After solidification of media, Escherichia coli culture was streaked on its surface. Now, the film sample Ch/CMC (20)450was placed in center of the Petri plate and the Petri plate was incubated for 2 days at 37?C in the incubator
We also carried out the antifungal activity of the film sample Ch/CMC (20)450 against Candida albicans, and Candida parapsilosis. As even to fourteen days old culture was obtained from Fungal Dis-ease Diagnostic Center, Jabalpur (India). For disc diffusion test, films were cut into disc shape with a diameter of 5 mm, then sterilized by autoclaving for 30 min at 120?C, and finally placed on different cultured agar plates. The plates were incubated for 1 day at 37?Cin an incubation chamber.
Results and discussion
Characterization of films
The surface morphology of the film samples Ch/CMC (20)and Ch/CMC(20)450was determined using scanning electronmicroscopy. The results are shown in Fig. 1. The SEM images of filmsamples Ch/CMC(20) and Ch/CMC(20)450with 100× magnificationsare shown in Fig. 1(a) and (b), respectively.It is observed that surface texture of film Ch/CMC(20) is quiterough due to the presence of well dispersed cellulose micro crys-tals throughout the matrix. Overall, a uniform distribution of CMC is CMCis visible (although with a few agglomerations). However, thesurface texture of Cur loaded film sample Ch/CMC (20)450exhibitsdifferent texture. It may be noticed that the whole surface have arelatively smoother texture and the curcumin molecules seems tohave superimposed on the cellulose crystals and the grooves visi-ble in the plain film are not so pronounced now. In other words,the pronounced appearance of the cellulose crystals in the filmsample Ch/CMC(20) is depressed greatly due to presence of cur-cumin which must have formed a layer on the cellulose crystalsthroughout the film matrix. The SEM images with 200 times mag-nifications are also shown in Fig. 1(c) and (d), respectively. Theseimages further support our observations.In order to investigate the crystalline nature of the films, XRDanalysis of film samples Ch/CMC (20) and Ch/CMC (20)450was carried out. The diffractograms of films Ch/CMC (20) and Ch/CMC(20)450are shown in Fig. 2(a) and (b), respectively.
In the diffractogram (a), two characteristic peaks of chitosanare observed at 10?and 23?22. It is also seen that three mod-erate peaks, corresponding to (1 0 1), (1 0 1) planes of cellulose-Iand (1 1 0) plane of cellulose-II, also appear at 14.6?, 16.3?and 20?,respectively 23. The most intense peak, occurring at 22.5?, corre-sponds to the (0 0 2) lattice plane of cellulose-I. In fact, the twopeaks, namely one at 22.5?for cellulose-I and the other one at23?for chitosan superimpose and cannot be distinguished sepa-rately. The diffraction pattern of sample Ch/CMC (20)450, is shownin Fig. 2(b). It contains a number of peaks, though not prominentlyvisible, in the 2_ range of 10–30?. These peaks indicate the pres-ence of highly crystalline curcumin in the film matrix 24. TheFig XRD of pure curcumin is also shown in the inset for comparison.It is noteworthy here that we did not get such prominent peaks inthe diffractogram of Ch/CMC (20)450, probably be due to the very low concentration of curcumin in the film.
Fig. 1. SEM image of (a) plain Ch/CMC(0) and (b) Ch/CMC(20)450film samples at 100× magnifications., (c) plain Ch/CMC(0) and (d) Ch/CMC(20)450film samples at 200×magnifications.