Gelatin-Coated Zeolite Y For Controlled Release of Anticancer Drug (Zerumbone)

Norashikin Salleh, Umi Sarah Jais*, Siti Halimah Sarijo Metals, Ceramics, and Composite Group, Faculty of Applied Sciences

Universiti Teknologi Mara, Shah Alam Selangor, Malaysia

*umisarah@salam.uitm.edu.my

Abstract—An oral slow release drug delivery system of zerumbone-zeolite Y-gelatin composites were prepared by two coating techniques namely dipping already shaped drug loaded zeolite y beads in gelatin and by blending gelatin with drug loaded zeolite Y powder followed by pelletizing by drop wise addition into sunflower oil. Zeolite Y acted as the support material in the nanocomposite preparation. In both coating techniques, pellets obtained were spherical in shape. Zerumbone (ZER) a natural anticancer drug was extracted from local Zingiber Zerumbet and loaded into zeolite Y via wet impregnation method. The composites were tested via UV-VIS for in- vitro drug delivery study and were characterized using TLC, SEM and XRD methods. From the data obtained, dip coated composite samples showed best sustained release of zerumbone for 24 h compared to blended zerumbone-zeolite Y-gelatin pellet. The inert zeolite Y acted as a carrier and support for drug, and the pores encapsulated, protected and controlled the released of the drug.

Keywords; Controlled drug release; Gelatin; zeolite y; Zerumbone

I. INTRODUCTION Conventional oral drug administration typically does not provide rate-controlled release of drug. In many cases, conventional drug delivery such as oral tablet consumption and injection provide rapid increase in the drug concentration thus repeatedly achieving toxic level followed by relatively short period of the therapeutic level before it finally drops off for re-administration of the drug [1].Thus, controlled release of drug is necessary to be more effective. Controlled release refers to the capability of a drug delivery system to release drug over an extended period of time at a controlled rate. It is becoming popular to treat diseases such as cancer, diabetes and tumors [2]. It allows the pharmological effectiveness of the drug to be maximized, thus minimizing potential side effects and allowing less of the expensive drug compound to be used. At above a certain level of drug concentration, side effects may occur while below certain concentration, the drug will have no beneficial effect [3].

In recent years much research in slow drug delivery has been focused on degradable polymer [1] and since then, synthetic and natural polymers have been accepted for the

production of biodegradable drug delivery system. Along with several natural polymers, gelatin has some beneficial material properties. Its proteinaceous nature is available for chemical modifications [4]. Gelatin is also known for its excellent biodegradability and biocompatibility [5]. Moreover, gelatin is “Generally Recognized as Safe” (GRAS) substance in the area of food additives by the U.S. Food and Drug Administration (FDA). The use of gelatin in controlled release devices for bioactive molecules like protein or plasmid DNA was lately reviewed [6]. Active agent is usually dispersed in polymer like gelatin by means of compression, dissolution or melting, and agent release is dependent on the polymer and the loading level of the agent. However, because of the un-uniformity in degradation, the biodegradable polymeric drug delivery vehicles are still questionable [7].

Today, nanoparticle-mediated drug release and drug targeting are intensively studied [8]. There are various engineered constructs, assemblies, and particulate systems, whose combining aspect is in the nanometer scale size range to be used in the controlled release drug delivery. The therapeutic agents can be encapsulated, covalently attached, or adsorbed on to such nanocarriers. Several carriers, however have poor capability to incorporate active compounds due to the of 5–10 nm size of the composites [9]. Therefore, porous materials with well-ordered structures are attractive candidates for storage and release of organic guest molecules [10]. In this study, Zeolite Y with a three-dimensional structure of 7.4 Aº diameter window openings connecting 13Aº diameter supercages has been chosen as the host carrier. Zeolite Y is small size has relatively high surface area and high porosity so high drug amount can be encapsulated. Therefore, a well-ordered structure of inert and porous Zeolite Y is an attractive candidate for storage and release of organic molecules [11].

The objective of this study is to prepare and evaluate a formulation of biodegradable gelatin-Zeolite Y composite and determine the mechanism for oral chemotherapy sustained-release delivery system by using zerumbone as anticancer drug. Studies proved that zerumbone is one of the most gifted chemo preventive agents against colon, skin and breast cancer [12-13].

2012 IEEE Symposium on Business, Engineering and Industrial Applications

978-1-4577-1634-8/12/$26.00 ©2012 IEEE 124

II. MATERIALS AND METHODS Zingiber Zerumbet Smith, Lempoyang, Gelatin purchased from Halagel (M) Sdn Bhd. Zeolite (Sigma-Aldrich)

A. Zerumbone extraction 1) Extraction and Isolation of Zerumbone from Zingiber

Zerumbet Smith

The isolation of Zerumbone from Zingiber Zerumbet was adopted from Abdul A.B.H. et al (2008). 4 kg of Zingiber zerumbet was obtained from a wet market in Kuala Lumpur, cut into slices (1-2 mm) and dried in the oven at 37 °C for 2 weeks. The dried Zingiber zerumbet slices were ground and soaked in absolute methanol for 3 days and strained. The methanol extract was evaporated at 40 °C using a rotary evaporator to obtain the crude extract. The crude zerumbone was separated by separating funnel method using absolute hexane The hexane layer was collected in a round bottom flask and evaporated. 5g of silica gel was added and evaporation was continued to dryness. The powdered form was mixed with 10 ml of hexane and transferred onto a glass column (3.5 x 30.0 cm) packed with silica gel. The silica gel column was eluted with 100% hexane, followed by three hexane and ethyl acetate mixtures in the ratio of 9:1, 8.5:1.5 and finally 8:2. The ratio of 8:2 was used repeatedly until the fraction containing zerumbone was isolated from the column. The zerumbone portion was allowed to dry to form crystals and purified by recrystallization with hexane.

B. Preparation of Samples 1) Zerumbone loaded Zeolite Y

10 mg of zerumbone was measured and diluted in 100 mL of ethanol. 900 mL of deionized water was added to prepare stock solution with concentration of 400 μM. The stock solution was further diluted to give a final concentration of 100 M. Zerumbone loaded zeolite Y was prepared by suspending zeolite Y in 100 μM of zerumbone at 6:10 wt/v ratio for 20 min, dried for 24h and kept for further use.

2) Zerumbone loaded Gelatin

10g of gelatin was dissolved in 50 mL of deionized water at 70ºC to obtain homogenous solution. 5g of glycerin was added to act as plasticizer. The loaded zeolite prepared as in B (I) was then added to gelatin solution under constant stirring until homogenous mixture formed and was then palletized by releasing the mixture drop wise from a syringe (60ºC) into 200 mL of cooled sunflower oil (10 ºC) with constant stirring.

3) Zerumbone -Zeolite Y -Gelatin composites

Gelatin solution was prepared as in B (2). The zerumbone loaded zeolite suspension prepared in B (I) then added to gelatin solution under constant stirring until homogenous mixture formed.

Zerumbone loaded gelatin-zeolite Y composite was pelletized by releasing the mixture drop wise from a syringe (60 ºC) into a cooled sunflower oil (10 ºC) with constant stirring.

In another experiment, the pellets were formed manually by shaping the zerumbone/zeolite Y mixture of similar composition as B(1) and B(2) into small spheres of 50 mg each and let dry at room temperature for 2h. The dried pellets were then dipped into gelatin solution and dropped into cooled sunflower oil (10 ºC) with constant stirring.

The resulting composites of B(2) and B(3) were decanted, freed of sunflower oil by repeated washings with 200 mL isopropyl alcohol and finally air dried over a period of 24 hours. The dried pellets were stored in a desiccator at ambient temperature until further evaluation. The compositions and preparation methods of each sample are summarized in Table 1 and Fig.1, Fig 2 and Fig 3.

TABLE I. COMPOSITION OF ZERUMBONE-ZEOLITE Y- GELATIN COMPOSITES

Sample Identity

Sample Description

Zerumbone

Zeolite Y (g)

Gelatin (g)

Glycerin (g)

S1 Pure ZER 10 mg - - -

S2 ZER-Gelatin 1 mL - 10 5

S3 ZER-Zeolite Y

1 mL 0.6 - -

S4 Blended ZER/Zeo/Gel

1mL 0.6 10 5

S5 Dip Coated ZER/Zeo/Gel

1mL 0.6 10 -

Figure1. Preparation of zerumbone loaded zeolite Y

Dilute zerumbone in ethanol and deionized water

Load zerumbone solution in zeolite Y

Manually shaped zerumbone-zeolite Y into small spheres

Dry (24h) at room temperature

Store at ambient temperature

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Figure2. Preparation of zerumbone loaded gelatin

Figure3. Preparation of zerumbone-zeoliteY-gelatin composite

C. Characterization 1) Thin Layer Chromatography (TLC)

Thin-Layer Chromatography (TLC) was used to determine the purity of the extracted ZER. A small quantity of ZER solution collected from column chromatography was dropped as a small spot on a TLC plate, which consisted of a thin layer of silica gel (SiO2) on a glass plate purchased from Merck. The plate was then positioned in a chamber which had been saturated with the mobile phase (hexane). The TLC of synthesized ZER was compared with the pure zerumbone compound obtained from Sigma- Aldrich.

2) X-Ray Diffraction (XRD)

The XRD analysis was carried out on a model Panalytical X’Pert PRO to determine the XRD patterns of standard gelatin powder, ZER loaded gelatin, blended ZER-Zeo-Gel, coated ZER-Zeo-Gel, ZER loaded Zeolite Y and standard Zeolite Y. All samples were triturated into fine powder before taking the scan from 0 - 2 .

3) Scanning Electron Microscopy (SEM)

The microstructures of the pellets were determined using SEM model Carl Zeiss SMT, SUPRA’40VP. All the samples were coated with gold before analysis.

D. Drug Release Analysis The procedure of drug release experiment was similar to

that reported by Pralhad and Rajendrakumar [14]. Drug released experiments were performed at 37 ºC using an ultrasonic bath sonicator at speed of 100 rpm. 250ml of phosphate buffer solution (pH 7.4) was used as the dissolution media to simulate gastrointestinal tract (GIT) conditions, 0.5g sample pellet/s was placed in 250 mL of the buffer solution and 5 ml aliquot was pipetted each time to determine the zerumbone released at 1 hr time interval over a period of 24 hr. The dissolution media was refilled with a fresh buffer solution after each removal. The amount of zerumbone released was analyzed using a UV spectrophotometer model PERKIN ELMER UV/VIS Lambda 20 at wavelength of 280 nm.

III. RESULTS AND DISCUSSIONS

A. Characterization of samples 1) Thin Layer Chromatography

TLC analysis of the sample solution containing zerumbone showed only one single spot on TLC plate which signifies the purity of the compound isolated. It also indicate the compound extracted and isolated from crude plant extract consisted mainly of one single compound. The TLC plates for both extracted and purchased zerumbone show single spot on the plate and confirmed the purity of the extracted zerumbone.

Gelatin-zerumbone-zeolite Y mixture

Add glycerin Manually shaped into small pellets

Stir until homogenous Dry at room

temperature

Palletize by releasing drop wise from a

syringe (60ºC) into sunflower oil (10ºC) with constant stirring

Dip pellets in gelatin

Drop into sunflower oil (10ºC)

Wash with isopropyl alcohol

Dry (24h) at room temperature

Store at ambient temperature

(i) (ii)

Dissolve gelatin in water at 70ºC

Add glycerin and zerumbone solution

Stir until homogenous

Palletize by releasing drop wise from a syringe (60ºC) into sunflower oil (10ºC) with constant

stirring

Wash with isopropyl alcohol

Dry (24h) at room temperature

Store at ambient temperature

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2) X-Ray Diffraction Analysis

The XRD patterns of standard gelatin, zeolite Y and composites samples are shown in Figure 4. The diffraction patterns show broad peaks for samples 4(a) and 4(b) indicating that the samples are amorphous while 4(d), 4(e) and 4(f) exhibit XRD profiles characteristic of zeolite Y. XRD pattern for 4(c), however characteristic peaks of zeolite Y are less intense due to the less amount of zeolite Y incorporated in the composite sample.

Figure 4: X-Ray diffraction for composite samples

3) Scanning Electron Microscope (SEM)

The scanning electron micrographs of formulated samples are shown in Fig 5, Fig 6, Fig 7 and Fig 8. Fig 5(a) and 5(b) confirmed that zeolite Y acquired porous structure. Fig 6(a) showed surface topography of ZER loaded gelatin and sphere ZER-gelatin pellet in 6(b) . After addition of zeolite Y in Fig 7(a) sample, the surface of composite seem coarse, indicating an improved toughness and Fig 7(b) showes the sphere shaped pellet of directly blended zerumbone, zeolite Y and gelatin. Fig 8 (a) shows sphere shaped of manually blended composites and 8(b) shows the composites cross section. Dense surface of zerumbone loaded zeolite Y located at center core surrounded by a layer of gelatin membrane with average thickness of 163 μm

Figure 5. SEM image for (a) zeolite Y, (b) zeolite Y pores

Figure 6: Images of Zerumbone-gelatin (a) SEM (b) camera of spherical pellets

Figure 7: Images of Zerumbone- ZeoliteY-gelatin (a) SEM and (b)

camera image of spherical pellets

a b

a b

a b

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Figure 8. Image of (a) camera images of Zerumbone-zeoliteY-gelatin and (b) SEM image of cross section of dip coated Zerumbone-zeoliteY-gelatin sample

B. In Vitro Drug Release Studies The release profiles of zerumbone released over a period of 24 h are shown in Figure 9. Dip - coated composite sample (S5) shows sustained release of zerumbone for 24h which indicates a controlled and prolonged drug released. Pure zerumbone crystal, (S1) as expected shows maximum drug released of 96.7% after 2h. For the ,zerumbone loaded in gelatin, S2 the release profile shows 47.2% of zerumbone released for first 1h and the sample completely dissolved after 2h. For, zerumbone loaded zeolite (S3), 69% of drug was released after 2h and completely released during the third hour. Blended composite, S4 shows 27.3% of zerumbone was released and the process was complete in the sixth hour. Zerumbone crystal (S1) shows the fastest release compared to the other samples because the drug did not have any protective coating to slow down the process when immersed in the phosphate buffer. Similar observation was seen for S2. Although encapsulated in gelatin, most of the zerumbone completely burst out in the second hour. This is probably because gelatin is hygroscopic, it tends to swell when pH buffer penetrated the pellets, and dissolved the gelatin allowing zerumbone to diffuse out from the gelatinous layer. At maximum swelling, the membrane deteriorated and finally burst releasing all the drug. In the case of S3, where zerumbone was loaded into zeolite, the zeolite did not appeare to slow down the process either. The release was fast in the first hour possibly because the pore size of zeolite Y is small (7.4Aº pore diameter) providing large surface area. The zerumbone entered the pores as very small particles which eould easily be dissolved by phosphate buffer resulting in fast drug release although slightly slower than S2. Gelatin blended zerumbone - zeolite, S4 composite lasted up to 6h before completely releasing the drug. Due the addition of zeolite, however the release for S4 sample lasted longer. This could be due to the porosity of zeolite Y. Zeolite Y did not encapsulate only zerumbone, but gelatin as well. Due to the availability of more free voids of zeolite, lesser number of drug molecules could diffuse out of zeolite Y because it was also in addition encapsulated in gelatin. S5 pellets were larger in size (10 mm) compared to S2,S3 and S4 pellets (2 mm) and SEM image shows zerumbone loaded zeolite completely coated with a layer

of gelatin ( Figure 8(b) ). The gelatin layer swelled when pH buffer penetrated, thus retarding the drug transporting out by delaying the buffer reaching the zeolite containing zerumbone core.

Figure 9: In Vitro release studies of zerumbone

IV. CONCLUSIONS

Composites of zerumbone, zeolite Y and gelatin were prepared by different gelatin coating methods and characterized by X-ray diffractometry, scanning electron microscopy and UV-VIS spectroscopy. This composites were loaded with a fixed amount of natural anticancer drug, zerumbone, to study the drug release behavior. All samples were spherical in shape. The drug was released in a controlled sustained manner for sample that was manually shaped and coated with gelatin compared to others. Zeolite Y contributed to slower release of zerumbone due to principally of zeolite Y voids. The drug release mechanism presumably involved water penetration, swelling, diffusion of the drug (polymer hydrofusion), and/or erosion of the gelatin layer. It also depend on the type of polymer used, pore characteristics of host carrier and pellet size.

Acknowledgements: This work has been supported by Universiti Teknologi MARA and the Ministry of Higher Education, Malaysia (Fundamental Research Grant no. 600-RMI/ST/FRGS 5/3/Fst (7/2010)

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