Calcium alginate-carboxymethyl cellulose beads for colon-targeted drug delivery

Calcium alginate-carboxymethyl cellulose beads for colon-targeted drug delivery

Accepted Manuscript Title: Calcium Alginate - Carboxymethyl Cellulose Beads for Colon Targeted Drug Delivery Author: Tarun Agarwal S.N. Gautham Hari N...

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Accepted Manuscript Title: Calcium Alginate - Carboxymethyl Cellulose Beads for Colon Targeted Drug Delivery Author: Tarun Agarwal S.N. Gautham Hari Narayana Kunal Pal Krishna Pramanik Supratim Giri Indranil Banerjee PII: DOI: Reference:

S0141-8130(15)00071-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.12.052 BIOMAC 4875

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

18-10-2014 12-12-2014 15-12-2014

Please cite this article as: T. Agarwal, S.N.G.H. Narayana, K. Pal, K. Pramanik, S. Giri, I. Banerjee, Calcium Alginate - Carboxymethyl Cellulose Beads for Colon Targeted Drug Delivery, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2014.12.052 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Calcium Alginate - Carboxymethyl Cellulose Beads for

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Colon Targeted Drug Delivery

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Tarun Agarwal, Gautham Hari Narayana S.N., Kunal Pal, Krishna Pramanik, Supratim Giri#,

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Indranil Banerjee*

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Department of Biotechnology and Medical Engineering, Department of Chemistry,

National Institute of Technology Rourkela, Odisha, Pin: 769008, India

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Tarun Agarwal Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela Odisha, Pin: 769008. India Email: [email protected] Phone: +919933968910

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Gautham Hari Narayana .S.N Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela Odisha, Pin: 769008. India Email: [email protected] Phone: +917750826049

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Dr. Kunal Pal Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela Odisha, Pin: 769008. India Email: [email protected] Phone: 0661-2462289 Dr. Krishna Pramanik Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela Odisha, Pin: 769008. India Email: [email protected] 1 Page 1 of 37

Phone: 0661-2462283

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*Author for correspondence: Dr. Indranil Banerjee Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela Odisha, Pin: 769008. India E-mail: [email protected] Phone: +91-9438507035

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Dr. Supratim Giri Department of Chemistry, National Institute of Technology Rourkela Odisha, Pin: 769008. India Email: [email protected] Phone: 0661-2462666

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HIGHLIGHTS

 CA-CMC bead formulation showed pH dependent swelling and colon

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mucoadhesivity

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 CA-CMC bead showed preferential degradation in presence of colonic microflora

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 CA-CMC bead formulation ensures sustained but complete delivery of drug at colon

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 The drug loaded CA-CMC bead had efficacy against HT29 colon cancer cell line

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ABSTRACT

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The present study delineates preparation, characterization and application of calcium alginate

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(CA) -carboxymethyl cellulose (CMC) beads for colon specific oral drug delivery. Here, we

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exploited

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biodegradability of the formulations for colon specific drug delivery. The CA-CMC beads were

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prepared by ionic gelation method and its physico-chemical characterization was done by SEM,

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XRD, EDAX, DSC and texture analyzer. The swelling and mucoadhesivity of the beads was

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found higher at the simulated colonic environment. Variation was more prominent in

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compositions with lower CMC concentrations. CA-CMC formulations degraded slowly in

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simulated colonic fluid, however, degradation rate increased drastically in the presence of

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colonic microflora. In vitro release study of anticancer drug 5-Fluorouracil (5-FU) showed a

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release (>90%) in presence of colonic enzymes. A critical analysis of drug release profile along

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with FRAP (Fluorescence Recovery after Photobleaching) study revealed that the presence of

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CMC in the formulation retarded the release rate of 5-FU. 5-FU loaded formulations were tested

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swelling,

mucoadhesivity

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colonic

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against colon adenocarcinoma cells (HT-29). Cytotoxicity data, nuclear condensation -

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fragmentation and apoptosis analysis (by flow cytometry) together confirmed the therapeutic

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potential of the CA-CMC formulations. In conclusion, CA-CMC beads can be used for colon

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specific drug delivery.

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Keywords:

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Calcium Alginate, Carboxymethyl Cellulose, Bead, Colon Specific Delivery, pH sensitivity,

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Mucoadhesivity

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1. INTRODUCTION

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The colon targeted oral drug delivery is desirable in order to treat a variety of colon diseases

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such as, ulcerative colitis, Crohn’s disease, amebiosis, colonic cancer, etc. [1-6]. In recent years,

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there have been a number of developments for the improvement of target specificity of colon

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targeted delivery systems [7]. The primary approaches pertaining to the colon specific delivery

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include: (i) covalent linkage of a drug with polymers as a prodrug, (ii) coating of the delivery

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system with the pH sensitive polymers (e.g. Eudragit polymers), bioadhesive polymers (e.g.

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Polycarbophil based polymers) or biodegradable polymers and (iii) microbially triggered release

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of the drug. In addition, some of the novel drug delivery approaches have also been introduced

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such as: (i) pressure controlled drug delivery, (ii) CODESTM (combined approach of pH

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dependent and microbially triggered drug delivery), (iii) osmotic pressure controlled drug

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delivery through a semipermeable membrane and (iv) multiparticulate systems like microspheres

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and nanoparticles [1]. However, in an recent review, Talaei and Atyabi et. al. highlighted that

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although the novel drug delivery systems have shown good potential, yet further improvements

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are needed before their full translation into clinical use [8].

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Calcium alginate (CA) and carboxymethyl cellulose (CMC) are two biopolymers that can be

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used for developing oral drug delivery systems. Alginate (salts of Alginic acid) is a linear

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polysaccharide composed of alternating blocks of β (1→4) linked d-mannuronic acid and α

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(1→4) linked l-guluronic acid residues [9-14] whereas CMC consists of linear chains containing

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β (1→4)-linked glucopyranose residues [15]. These biopolymers have been reported to show a

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pH dependent swelling behavior [13, 16-18]. Both the polymers are anionic in nature due to the

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presence of negatively charged carboxyl groups at pH > 5. These negative charges allow the

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polymer to shrink in the acidic pH and to swell when they are exposed to neutral or basic pH.

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This properties make these polymers suitable for applications in the design of oral drug delivery

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systems. Apart from the pH sensitivity, SA and CMC have also been known to possess an

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excellent mucoadhesive property [19].

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Keeping the aforesaid perspective in mind, here we have explored the potential of calcium

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alginate (CA) - carboxymethyl cellulose (CMC) bead as a colon specific drug delivery system.

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We hypothesize that an appropriate composition of CA-CMC will ensure: (1) least amount of

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drug release at non specific sites (stomach and small intestine) during its transit through the GI

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tract; (2) higher adhesion to the colonic mucosa in comparison to other parts of the GI tract; and

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(3) controlled degradation of the formulations by the colonic microflora. These factors are

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expected to promote sustained release of the drug in the colon. The rationale behind such

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ventures is: (i) to ensure the appropriate therapeutic dose at colon for effective treatment, (ii) to

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avoid dose and activity loss of the therapeutics during the GI transit and (iii) to minimize the

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adverse side effects of the therapeutics caused from absorption at non-specific tissue locations. 5-

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Fluorouracil (5-FU) was taken as the reference drug in this study.

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2. MATERIALS AND METHODS

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2.1 Materials

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Sodium alginate (SA) (molecular weight: 7.72x104g/mol, degree of polymerization: 476, M/G

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ratio 1.08) was bought from SDFCL, Mumbai, India. Calcium Chloride (CaCl2, fused) was

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purchased from MERCK, Mumbai, India. Glutaraldehyde (25% aqueous solution) was procured

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from LOBA Chemie, Mumbai, India. Carboxymethyl cellulose sodium (CMC) salt (molecular

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weight: 6.62x105g/mol, degree of polymerization: 3062, degree of substitution: 0.68), MaCoy’s

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5A media, Dulbecco’s Phosphate Buffer Saline (DPBS), Trypsin-EDTA solution, Fetal Bovine

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Serum, Antibiotic-Antimycotic solution, MTT assay kit and Nutrient Broth were purchased from

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Himedia, Mumbai, India. HT29 adenocarcinoma cell line was procured from NCCS, Pune. 5-FU

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and FITC-Dextran (Molecular weight: 10kDa) were obtained from Sigma-Aldrich, Mumbai.

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2.2 Methods

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2.2.1. Preparation of CA-CMC Beads

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Preparation of the CA-CMC beads was done by ionic gelation method as described by

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Girhepunje et. al. [20]. Both SA and CMC were dissolved in deionized water at a specific

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concentration (Table 1). Thereafter, the prepared polymeric solution was extruded as droplets

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using a syringe (28G) and poured into 2% calcium chloride (w/v) solution under constant stirring

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at 80rpm and 37oC and cured for 10min. Then, 1.1ml of glutaraldehyde reagent [Glutaraldehyde

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(25%, 0.5ml) + Ethanol (0.5ml) + HCl (0.1N, 100l)] was added to 50ml of calcium chloride

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solution and bead curing was done for another 10min. The beads were washed with deionized

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water, neutralized with glycine and dried overnight at 40oC. To determine the average size of the

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swollen and dried beads, images were taken using the camera (Canon A2400 IS) and the images

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were analyzed by NIH ImageJ software.

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2.2.2. Physico-chemical characterization of the beads:

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Morphological characterization of the dried beads was carried out using scanning electron

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microscopy (JOEL India JSM-6480Lv) at 15kV after platinum sputter coating. The calcium

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content of the beads was analyzed by energy dispersive X-Ray spectroscopy. Variation in

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percentage crystallinity of CA-CMC beads were recorded using X-Ray diffraction (Philips XRD-

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PW1700 diffractometer). Scanning was done in the range of 5-60°2 with a step size of

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0.02°/second using monochromatic CuKα radiation of wavelength (λ=1.514Å). Differential

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scanning calorimetry analysis was carried out by heating 20mg of CA-CMC beads from 35°C to

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250°C, at a rate of 5oC/min using DSC-200-F3 MAIA instrument (Netzsch, Germany). Bulk

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compressive strength of the CA-CMC beads was analyzed by TA.XT2i Texture analyzer (Stable

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Micro Systems Ltd, Surrey, UK). The analysis was done using 30mm probe, 1mm/sec test speed

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and auto (force) mode (5g, 5mm).

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2.2.3 Swelling Analysis

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Swelling of the beads in simulated GI fluids was studied following the protocol described by

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Pasparakis et. al. [21]. For this, accurately weighed, dried CA-CMC beads were immersed in

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phosphate buffer saline (PBS; pH 7.4 & 6.8) and in 0.1N HCl (pH 1.2) at 37oC. At defined time

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intervals, the beads were withdrawn from the solution and increase in the weight of the beads

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was measured as a function of time. Swelling ratio (SR) was expressed as:

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where, W1 and W2 represent the dry and wet weight of the beads respectively.

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SR = (W2 – W1) / W1

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2.2.4. Mucoadhesivity

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Mucoadhesivity testing was carried out following in vitro wash-off protocol as reported by Lehr

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et. al. [22]. In brief, fresh tissue portions from the goat stomach and colon were obtained from a

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commercial slaughter house and cleaned with cold normal saline. The tissues (1.5cm x 1.5cm)

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were fixed on a glass slide using adhesive glue keeping the mucosal surface upward. 50mg of the

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beads were placed on the mucosa and a 5g load was applied on to it for 15min to ensure uniform

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adhesion of the bead on the mucosa. Thereafter, bead loaded stomach and colon mucosa were

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placed in 0.1 N HCl (pH adjusted to 1.2, specific to stomach) and PBS (pH 6.8, specific to colon)

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respectively, on to the groves of USP24 tablet disintegration apparatus. The disintegration

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apparatus was then operated in a way that ensured up and down movement of tissue specimen in

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one liter of buffer at 37°C. The experiment was run for 24h and the time corresponding to the

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complete wash off of the beads was noted.

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2.2.5. Colonic microflora specific biodegradation

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Colonic microflora was obtained from human stool culture of a healthy volunteer. The culture

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was inoculated in nutrient broth at 37 ºC and 50mg of the dried CA-CMC beads were added to

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the culture. A similar set of experiment was also performed in phosphate buffer saline (pH 6.8)

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and nutrient broth (pH 6.8, without bacteria). The study was monitored for one month and time

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required for the complete degradation was recorded. Also, the interaction of the colonic

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microflora with the bead material was examined through field emission scanning electron

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microscope (FESEM) (Nova NanoSEM 450) after gold sputter coating (Quorum Technologies,

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Q150R ES) at 3kV.

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2.2.6. In vitro drug release study

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Loading of 5-FU in the CA- CMC bead was done by swelling [23]. In brief, 10mg of the dried

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beads were incubated in to 50 µl of aqueous drug solution (10mg/ml) at pH 7.4 for 9h. Volume

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of the drug solution and duration of loading was fixed on the basis of swelling data. After 9h,

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beads were taken out from the drug solution washed gently with PBS and then subjected for drug

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release study. To analyze the extent of loading, each set was dried separately, then crushed using

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mortar pestle and total drug was extracted using 10ml of PBS (pH 7.4). Extraction was done in a

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step wise manner using 2 ml of extraction buffer at a time. Concentration of drug in the extract

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was measured using UV-Vis spectrophotometry at 266nm. In vitro drug release from the beads

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was carried out in simulated gastrointestinal environment as described by Ahmad et. al. [24]. In

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brief, 5-FU was loaded into the polymeric solution at a concentration of 5% (w/w) of the total

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polymer concentration. Thereafter, accurately weighed 100mg of the drug loaded dried beads

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were initially incubated in 50ml 0.1N HCl (simulated gastric fluid, pH adjusted to 1.2) for 2h,

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then incubated in 50ml of PBS (simulated small intestinal fluid, pH 7.4) for 3h, and finally

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transferred to 50ml of PBS (simulated colonic fluid, pH 6.8) and kept for another 115h [14, 20].

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In addition, a similar analysis was also performed wherein the enzyme cocktail released by the

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colonic microflora was added to PBS (pH 6.8) in 1:10 ratio. The enzyme cocktail was prepared

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from the human stool culture of a healthy volunteer. For this, the stool culture was centrifuged at

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5000rpm for 10min. The supernatant, thus obtained was filtered using 0.22m filter and then

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used for the analysis. The beads in the release medium were kept under shaking conditions in a

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shaker incubator (WADEGATI Labequip Pvt. Ltd.) at 60rpm and 37°C. At definite time

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intervals, 3ml of the sample was taken out of the flasks and was replaced by 3ml of fresh PBS

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and analyzed spectrophotometrically at 266nm. Furthermore, mobility of large molecules

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entrapped in the CA-CMC beads at simulated intestinal fluid (PBS, pH 6.8) with or without

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enzyme cocktail was analyzed by Fluorescence Recovery After Photobleaching (FRAP) using

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confocal scanning laser microscope (Olympus IX 81 confocal microscope using Fluoview1000).

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For this purpose, FITC Dextran (Mol wt. 10kDa) entrapped beads were subjected to bleaching in

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a specific region of interest (ROI) using 95% intensity of a multi-argon laser (40mW, 488nm).

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Fluorescence recovery in the region of interest was recorded at 0.2% intensity using the same

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laser source.

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2.2.7. In vitro evaluation of therapeutic potential of drug loaded beads

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Efficacy of the drug loaded bead was tested against HT-29 colon adenocarcinoma cell line by

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MTT assay, Flow cytometry and immunocytochemistry. In brief, the cell line was maintained in

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MaCoy’s 5A supplemented with 10% FBS in a humidified (95%), CO2 (5%) incubator at 37oC.

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Cells were harvested using 0.25% Trypsin-EDTA solution. Thereafter, the cells were seeded in a

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12 well plate at a viable cell concentration of 1 x 105cells/ml. Dried beads (blank and drug

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loaded) of each formulation were UV sterilized and 10 beads were placed in each well. The

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culture plate was incubated for the next 48h. Cytotoxicity of the formulation was first analyzed

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using MTT Assay. For flow cytometry based apoptosis analysis, 1x105 cells were incubated for

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48h in a six well plate in presence of drug loaded beads (10mg). After 48h of incubation,

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percentage of apoptosis was analyzed by Flow Cytometer (BD Accuri) using PE Annexin V

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apoptosis detection kit (BD pharmigen). Furthermore, HT-29 cells exposed to the drug ( leachant

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from the 5-FU loaded beads) was checked for nuclear condensation and fragmentation using

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DAPI staining (1:300 dilution) by Confocal microscopy (Olympus IX 81 confocal microscope

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using Fluoview1000) [25].

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2.2.8. Statistical Analysis

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All the data were reported as mean ± S.D (Standard deviation). For evaluating statistical

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significance of the data, one way ANOVA was performed.

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3. RESULTS AND DISCUSSION

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3.1. Bead Preparation

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Calcium alginate-carboxymethyl cellulose (CA-CMC) beads were prepared by the ionic gelation

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method. In this process, anionic carboxylic groups present in alginate and carboxymethyl

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cellulose interacted with bivalent calcium ion to form the gel. It was observed that an increase in

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the CMC concentration resulted in an overall decrease in the percentage yield. The percentage

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yield of T1G beads was found 95.93±2.3% and that of T3G and T5G was 92.04±1.9% and

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88.56±2.7% respectively. In an earlier study, Arica et. al. reported similar trend while working

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with alginate beads [26]. In practice, with an increase in the biopolymer concentration (here,

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CMC), the viscosity of the solution increases which contributes towards higher retention of

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liquid volume at the tip of the nozzles and thus increased bead size [14]. The average size of

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dried T1G, T3G and T5G beads was found to be 738.51±19.41µm, 875.81±25.37µm and

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918.92±37.45µm, respectively.

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Preliminary stability study showed that beads crosslinked by calcium chloride were degraded

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completely over a period of 12h in PBS (pH 7.4) [27]. This happened because of the release of

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Ca+2 from the beads to the solution which leads to the disruption of the polymeric network. The

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stability of the beads was increased by crosslinking with 0.25% glutaraldehyde. In this case,

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glutaraldehyde crosslinked the polymers via acetal bond formation [28-29]. The swollen bead

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formulations appeared white, translucent and spherical in shape. The average diameter of

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swollen T1G, T3G, and T5G were found 1938.16±37.15, 2156.45±57.56 and 2270.11±73.78 μm

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respectively (Fig. 1G).

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3.2. Physico-chemical characterization:

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Analysis of surface morphology of the dried beads by scanning electron microscopy revealed

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that pure calcium alginate beads were spherical with a smooth surface topography. Beads

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appeared solid (devoid of any core) and without any micropores on their surface. A critical

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examination showed that an increase in the CMC concentrations resulted in an increase in the

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bead size and formation of wrinkles on the bead surface (Fig. 1A-H). Similar morphological

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changes were reported earlier by Kim et. al. [30]. It is important to mention that there were

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cracks on the surface of the beads. However, such cracks were not seen under light microscope

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and might have developed during the sample processing for scanning electron microscopy. The

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elemental analysis of the beads demonstrated that with an increase in the CMC concentration, the

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calcium content increased significantly from 12.89±0.37% (%w) in T1G to 31.74±0.93% (%w)

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in T5G (p < 0.05) (Fig. 1I). Calcium is involved in the crosslinking of both alginate and CMC

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[31].

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[Insert Fig. 1]

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The XRD analysis of the pure sodium alginate demonstrated characteristic peaks at 13.6o 2 and

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22.1o 2, while the diffractogram of CMC consisted of a broad characteristic peak at 20o 2.

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Aforementioned three characteristics peaks corresponding to SA and CMC were all present in

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the XRD profile of CA-CMC beads. However, the peaks were broadened suggesting an increase

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in the amorphous nature of the formulations (Fig. 2).

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[Insert Fig. 2]

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The DSC thermogram of the CA-CMC beads showed a broad endothermic peak near 100oC

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which may be due to the evaporation of water molecules. The peak was found to be more intense

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in T1G followed by T3G and T5G respectively, which suggest the presence of higher proportion

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of water in T1G. It is also important to mention that a secondary endothermic peak was also

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observed which may be associated with the bound water molecules. The secondary peak in T1G,

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T3G and T5G were found to be present at ~186oC, ~186oC and ~193oC respectively. The

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presence of secondary peak at elevated temperature indicates thermal degradation of the polymer

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matrix (Fig. 3A). The bulk compressive modulus of T3G and T5G was found 0.4605±0.004MPa

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and 0.3726±0.517MPa respectively, which are significantly lower in comparison to that of T1G

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(1.331±0.181MPa). This could possibly due to inherent property (compressive strength) of the

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polymeric formulation or the variation in the packing of the CA-CMC beads. The packing of the

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beads is dependent on their size. As already mentioned, T1G have smaller and regular bead size,

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owing to its proper packing and higher bulk compressive strength (Fig. 3B).

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3.3 Swelling Analysis

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Calcium alginate and carboxymethyl cellulose, being polyelectrolytes, exhibit pH dependent

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swelling. Such pH responsive swelling property is attributed to the presence of negatively

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charged carboxyl groups present in the polymer backbone. In acidic pH, carboxylic acid groups,

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remain undissociated and therefore no net charge is developed in the polymeric network. Once

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exposed to neutral or alkaline medium, carboxylic acid group converts to negatively charged

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carboxylate ions resulting in an electrostatic repulsion amongst the different polymer chains.

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This in turn, compels the polymer network to swell. Such pH dependent swelling often

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modulates release of a drug molecule from a carrier system in oral drug delivery.

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The swelling study of the beads was carried out in three different pH conditions: 1.2, 7.4 and 6.8

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corresponding to the pH of the stomach, small intestine and colon respectively. The highest

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swelling for all formulations took place at pH 7.4 while lowest swelling happened at pH 1.2.

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Difference in swelling at pH 6.8 and 7.4 was insignificant for all the formulations. Interestingly,

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at higher pH, i.e. at pH 6.8 and 7.4, the swelling was reduced with an increase in the CMC

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concentration while a reverse trend was observed at pH 1.2. When compared to T1G (without

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CMC), there was a 1.81 and 3.26 fold decrease in the swelling index in T3G (with 0.5% CMC)

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and T5G (with 1.0% CMC), respectively, at pH 7.4 (Fig. 3C). On the other hand, the swelling

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ratio of the T3G and T5G beads in pH 1.2 increased by 1.3 and 1.6 folds in comparision to T1G,

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respectively. Swelling of T1G, T3G and T5G at pH 6.8 followed a similar pattern as in case of

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pH 7.4 with swelling index of 30.05±1.277, 17.278±0.955 and 8.914±0.97 respectively. With an

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increase in the CMC concentration, relative charge density of the beads tends to increase. At pH

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1.2, the negative charges present on the surface tend to get shielded; however, the charges

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present in the core of beads may contribute to a slight increase in the swelling index due to

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inadequate shielding [30]. A critical analysis of the swelling profile of all the formulation at pH

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7.4 and 6.8 revealed that the extent of swelling for all the three formulations at initial phase (upto

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1h) was significantly different irrespective of the formulation studied.

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[Insert Fig. 3]

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3.4. Mucoadhesivity of the beads at different parts of the GI tract

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Mucoadhesivity is a property of the polymeric formulation which allows it to adhere onto the

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mucus membrane [33]. Mucoadhesivity needs special consideration while designing an oral drug

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delivery system as it ensures prolonged retention of the formulation at a specific location in the

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GI tract and thus helps in achieving a sustained drug release for a longer period [33-34]. It is

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important to mention that the entire gastrointestinal tract in humans is lined by a mucus

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membrane but the characteristics of this mucosal lining tend to vary from one region of the GI

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tract to the other [35]. The major component of this mucus membrane is a branched glycoprotein

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named mucin which shows a pH dependent variation in its configuration [36]. Such pH

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dependence alters its affinity for mucoadhesive materials. This implies that the formulation

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having the maximum mucoadhesivity for colon mucosa is desirable for an effective colon

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targeted delivery. Keeping this fact in mind, the mucoadhesiveness of the beads was analyzed for

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colon and stomach mucosa at pH 6.8 and 1.2, respectively (Table 2). Data showed that at pH 6.8,

362

a major fraction of the beads remained adhered to the colon mucosa surface after 24h while

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15 Page 15 of 37

significant decrease of mucoadhesiveness was observed in case of stomach mucosa at pH 1.2. At

364

acidic pH, all three formulations showed weak mucoadhesivity and beads were washed off from

365

the mucosal surface within 2-3h. In the acidic pH, the intrinsic negative charges of the beads get

366

shielded off, which may play a critical role for the adhesiveness of the beads with the mucus

367

membrane. Also, at neutral pH, the charges of the beads tend to get expose, allowing it to

368

interact with the mucus membrane through strong electrostatic interactions and thus showing a

369

greater mucoadhesivity.

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370

[Insert Table 2]

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3.5. Colonic microflora specific degradation of the beads

374

The colonic region of the gastrointestinal tract is a known habitat of over 400 distinct bacterial

375

species, including Bacteroides, Bifidobacterium, Eubacterium, Peptococcus, Lactobacillus,

376

Clostridium and Escherichia coli [1, 6, 9, 24, 35, 37]. These bacterial species produces a number

377

of reductive and hydrolytic enzymes such as β-glucuronidase, β-xylosidase, β-galactosidase, α-

378

arabinosidase, nitroreductase, azoreductase, deaminase and urea hydroxylase. This enzymatic

379

cocktail produced by the colonic microflora help in the drug release by degrading the

380

biopolymeric matrix of the delivery system [6, 9, 24]. It is usually observed that before reaching

381

the colon region of the GI tract, a significant amount of the entrapped drug is retained inside the

382

beads. So, it is very essential that the drug delivery systems must be degraded completely by the

383

microbial population residing in the colon and release the remaining drug entrapped within them.

384

In this regard, we tried to analyze in vitro, the potential of the intrinsic colon bacteria to degrade

385

beads of the CA-CMC formulation. In vitro degradation studies carried out in the presence of

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16 Page 16 of 37

native colon microflora obtained from the human stool culture, revealed that the prepared beads

387

were completely degradable in such environment (Fig. 4A). Although the time required for

388

complete degradation of the beads varied largely from one formulation to another. It was

389

observed that the pure alginate beads took approximately 42h to get degraded while T5G alginate

390

beads containing 1% CMC get degraded in around 98h. An increase in the CMC concentration in

391

the beads tends to slow down the rate of degradation. A comparative kinetic study of bead

392

degradation revealed that the beads of all the formulations failed to get degraded in the absence

393

of bacteria in phosphate buffer saline (pH 7.4) and nutrient broth (pH 7.4) within a considerable

394

time frame and took a time span of four weeks. In PBS and media, T1G took approximately 12-

395

13 days for degradation while T5G took approximately 600- 640 h (4 weeks). The analysis

396

demonstrated that the addition of CMC had a profound effect on the degradation of the beads

397

irrespective of the environmental conditions. In PBS and nutrient broth, addition of CMC in the

398

formulation significantly reduced the degradation rate of the formulated beads (p < 6.5E-6).

399

Furthermore, with the addition of the microbial culture to the degradation media to simulate

400

colonic condition, the degradation rate of all the formulations increased significantly (p < 5.00E-

401

6), however degradation profile followed the same trend as in the case of PBS and nutrient broth.

402

Here, it is important to mention that the transit time for the colon is reported to be 8 – 72h [38].

403

Our previous data already suggests that beads of all formulations are highly mucoadhesive to

404

colon mucosa at pH 6.8. The high mucoadhesivity of the beads may increase their retention time,

405

which may provide a chance for the complete degradation of these beads and thus resulting in the

406

complete drug release. A microscopic analysis pertaining to the interaction between beads and

407

microbes (Fig. 4B-D) revealed a preferential bacterial adhesion on a pure alginate composition

408

with formation of bacterial biofilm on T1G bead surface. However, the bacterial adhesion was

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17 Page 17 of 37

409

found to decrease with the addition of CMC. This explains about a higher degradation rate of

410

T1G in comparison to T3G and T5G in the presence of the bacterial microflora.

411

[Insert Fig. 4]

ip t

412 413

3.6. In vitro Drug Release Study

415

The colon specific drug delivery systems must release a negligible or low amount of the loaded

416

drug in the stomach or small intestine. The residence time of any solid dose in human stomach

417

and small intestine during GI transit is 2-3h and 3-4h respectively [38]. Since, it is difficult to

418

control the transit time, therefore efforts have been made to tailor the release of the drugs from

419

the carrier system during transit. Thus, it becomes essential to analyse the percentage of the drug

420

released from the formulations before they reach the colon.

421

Drug loading is an important parameter in this regard because it determines the theoretical limit

422

of maximum drug release. Here drug loading was done by swelling method. Analysis of drug

423

loading showed that percentage loading of 5-FU in dried alginate bead (T1G) was 66% of the

424

initial drug take. The same for T3G and T5G were 72% and 82% respectively. The loading with

425

respect to the dry weight of the bead was 30mg/g for T1G, 33mg/g for T3G and 41mg/g for T5G.

426

It is important to mention that extent of drug loading in the beads did not follow the swelling

427

profile rather a reverse trend was observed. There could be a drug-CMC interaction that caused

428

higher retention of the drug molecules by the polymer. Analysis of the drug loaded beads by

429

XRD revealed that there was a variation in crystallite structure of CMC in presence of 5-FU

430

(data not shown) which indicates about such possibility.

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18 Page 18 of 37

The drug release analysis showed that the T3G and T5G beads released 6% and 4.7% of the

432

drug, respectively, in comparision to 8.6% drug release in T1G after 2h of analysis at pH 1.2. A

433

sudden release of the drug was observed in case of beads of all formulations when they were

434

transferred from simulated gastric fluid (pH 1.2) to simulated intestinal fluid (pH 7.4), although

435

this burst release significantly reduced with an increase in the CMC concentration (p < 0.05) [39-

436

40]. A cumulative release of 34% and 27% was observed in T3G and T5G respectively, after 5h

437

of analysis at pH 7.4. T1G beads showed significant difference with respect to T3G (p < 0.005)

438

and T5G (p < 0.005) under same conditions with a cumulative release of 41%. Further, when the

439

beads were transferred to the simulated colonic fluid (pH 6.8), a cumulative release of 75.5%,

440

60.6% and 51.5% was observed in case of T1G, T3G and T5G, respectively, at the end of

441

analysis. This data showed that the typical drug release in the colon compartment is 34.2%

442

(T1G), 26.5% (T3G) and 24.36% (T5G). It is important to mention that a higher drug release

443

occurred in the presence of enzymes with respect to control (without enzymes). A critical

444

analysis of the drug release in simulated colonic fluid (pH 6.8) demonstrated that the drug

445

released from the beads of all three formulations in presence/absence of enzymes was not

446

significant during the initial 24h of the analysis (p > 0.05). Thereafter, considerable differences

447

were observed in comparison to the control set (without enzymes). A cumulative drug release of

448

94.7%, 75.9% and 60.8% occurred from T1G, T3G and T5G beads, respectively, at the end of

449

the analysis (Fig. 5A). In the absence of enzymes, the amount of the drug released in the colon,

450

followed a ratio of 1.0: 0.78: 0.71. This ratio changes significantly in the presence of colonic

451

enzyme cocktail and becomes 1.0: 0.74: 0.62. It is important to mention that all though the bead

452

formulations released the drug mostly in simulated colonic environment, however, a small

453

portion of the loaded drug also get released at other part of GI tract (simulated stomach and small

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19 Page 19 of 37

intestine environment). This could further be prevented by finer tuning of the polymeric

455

formulations or using an enteric coating on the beads with polymers such as Eudragit®. The

456

enteric coating polymers offer various advantages including pH-dependent drug release,

457

protection of actives sensitive to gastric fluid, protection of gastric mucosa from aggressive

458

actives. In addition, these polymers offers GI and colon targeting depending on their grades, for

459

example; Eudragit® S100-55, Eudragit® L100 and Eudragit® S100 dissolves above pH 5.5, 6 and

460

7 respectively, thus offering selectivity in targeted site for delivery. However, the coating must

461

not interfere with the mucoadhesive property of the CA-CMC beads formulations. Our previous

462

biodegradation study has suggested that in all CA-CMC beads formulations, T1G showed the

463

highest rate of biodegradation followed by T3G and T5G respectively. However, our

464

mucoadhesivity study suggests that beads of all formulations exhibited higher adhesion with

465

colonic mucosa at pH 6.8. This clearly indicates the higher retention of the CA-CMC beads in

466

the colon which will allow the degradation of the beads by colonic microflora, aiding in a faster

467

and complete drug release. Such variations in the rate of degradation along with higher

468

mucoadhesivity will allow the usage of CA-CMC formulations for colon specific drug delivery

469

applications. Further, the FRAP analysis of CA-CMC beads in PBS (pH 6.8) demonstrated that

470

the recovery of the FITC-Dextran at the bleached ROI attained 84%, 79% and 77% of its initial

471

intensity in T1G, T3G and T5G, respectively (Fig. 5B). Such variations in the FRAP profile are

472

due to their compositions which aids in the variation in the local viscosity of the formulations

473

and microarchitecture of the beads. These factors contribute to the variations in the internal

474

solute transport. However, the rate of recovery of fluorophore increased when the beads were

475

exposed to the microbial enzyme cocktail. A complete recovery (approx 100%) was obtained in

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20 Page 20 of 37

476

case of T1G when exposed to the enzyme cocktail while in case of T3G and T5G; nearly 90%

477

and 81%, respectively, of the recovery could be achieved.

478

[Insert Fig. 5]

ip t

479 480

3.7. In vitro evaluation of therapeutic potential of anticancer drug loaded beads

482

5-FU loaded beads were evaluated for their potential as a drug carrier system against HT-29

483

adenocarcinoma cell lines. The viability of the cells was estimated by MTT assay [41]. The

484

results demonstrated that the cell viability decreased significantly after 48h of exposure of cells

485

to the drug loaded beads of all formulations. The percentage cell death in case of T1G was found

486

to be 1.29 folds higher than T3G and 1.42 folds higher than T5G (Fig. 6A). The percent cell

487

death observed in all the three formulations was significant with respect to the control (without

488

drug) and amongst themselves (p < 0.005). It is also important to mention that the blank beads of

489

all the formulations were found to be biocompatible (data not shown). Furthermore, in response

490

to the bead treatment, apoptosis in HT-29 cells was analyzed by flow cytometry (Fig. 6B) and by

491

checking nuclear condensation and fragmentation (Fig. 6C). Cell cycle analysis showed that the

492

percentage apoptosis was highest for T1G (22.5%; early apoptotic 6.97, late apoptotic 15.5),

493

followed by T3G (15.7%; early apoptotic 5.61%, late apoptotic 10.08%) and T5G (15.4% early

494

apoptotic 5.88%, late apoptotic 9.55% respectively). The same for control was 7% (early

495

apoptotic 1.72%, late apoptotic 5.32%). Further, immunocytochemistry data showed that the

496

cells, which were exposed to leachant from 5-FU drug loaded beads, underwent nuclear

497

condensation and fragmentation. In comparison, the leachants from the blank beads did not show

498

any such apoptotic response. This data clearly suggest that the CA-CMC beads can work as an

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21 Page 21 of 37

499

effective colon targeted drug delivery system and the efficacy of the formulation can be modified

500

be by changing the composition of the bead.

501

[Insert Fig. 6]

ip t

502 503

4. CONCLUSION

505

Conventional strategies of colon targeted drug delivery rely upon the exploitation of either pH

506

sensitive property of the polymer or biodegradability (especially microbial degradation of the

507

carrier matrix). Recently, combined approach of pH dependent and microbially triggered drug

508

delivery (CODESTM) system is employed for the aforesaid purpose with reasonable success.

509

However, none of the existing system has exploited the combined effect of pH sensitivity,

510

colonic microbial degradation and colon mucosa specific preferential mucoadhesivity. In this

511

regard, the present study is novel in the field of colon targeted delivery system. In this study, we

512

have successfully proven that calcium alginate-carboxymethyl cellulose beads are potential

513

candidates for colon specific oral delivery of therapeutics. We showed that these beads can

514

effectively protect the therapeutics from the harsh-lytic environment of the stomach during its

515

transit through the GI tract and can preferentially deliver the drug at colon under the influence of

516

colonic pH and microflora. It was evident from the study that such formulations are working well

517

in in vitro colon cancer model. The study gives a clear indication that such formulation can be

518

used for delivery of other therapeutics. The system reported here is a prototype only and

519

improvement in colon specific delivery of therapeutics from the formulation is highly possible

520

by changing the composition. However, the study needs to be substantiated in in vivo system for

521

further progress.

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522 22 Page 22 of 37

5. ACKNOWLEDGEMENT

524

The authors would like to thank Dr. T. K. Maiti and Dr. Bibhas Roy Department of

525

Biotechnology, Indian Institute of Technology, Kharagpur for providing confocal imaging

526

facility.

ip t

523

527

6. REFERENCES

cr

528

us

529

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531

approaches, Oman Med. J., 25 (2010) 79-87.

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crosslinked with Ca2+ and Ba2+ ions, React. Funct. Polym., 59 (2004) 129-140.

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potential of calcium alginate beads for use in embolisation, J. Mater. Sci. - Mater. Med., 21

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[11] D. Tahtat, M. Mahlous, S. Benamer, A.N. Khodja, H. Oussedik-Oumehdi, F. Laraba-

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[13] S.C. Kumar, A.K. Kalekar, Performance Evaluation of Mucoadhesive Potential of Sodium

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Alginate on Microspheres Containing an Anti-Diabetic Drug: Glipizide, Int. J. Pharm. Sci. Drug

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[14] K. Manjanna, K.T. Pramod, B. Shivakumar, Calcium alginate cross-linked polymeric

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[15] J.-F. Su, Z. Huang, X.-Y. Yuan, X.-Y. Wang, M. Li, Structure and properties of

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carboxymethyl cellulose/soy protein isolate blend edible films crosslinked by Maillard reactions,

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[16] M.A. Abd El-Ghaffar, M.S. Hashem, M.K. El-Awady, A.M. Rabie, pH-sensitive sodium

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alginate hydrogels for riboflavin controlled release, Carbohydr. Polym., 89 (2012) 667-675.

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[17] J.-P. Zhang, Q. Wang, X.-L. Xie, X. Li, A.-Q. Wang, Preparation and swelling properties of

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pH-sensitive sodium alginate/layered double hydroxides hybrid beads for controlled release of

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diclofenac sodium, J. Biomed. Mater. Res., Part B, 92B (2010) 205-214.

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[18] A. Sannino, C. Demitri, M. Madaghiele, Biodegradable cellulose-based hydrogels: design

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and applications, Materials, 2 (2009) 353-373.

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[19] V. Grabovac, D. Guggi, A. Bernkop-Schnürch, Comparison of the mucoadhesive properties

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of various polymers, Adv. Drug Deliv. Rev., 57 (2005) 1713-1723.

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[20] K. Girhepunje, V. Krishnapiillai, R. Pal, H. Gevariya, N. Thirumoorthy, Celecoxib loaded

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microbeads: A targeted drug delivery for colorectal cancer, Int. J. Curr. Pharm. Res., 2 (2010)

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46-55.

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[21] G. Pasparakis, N. Bouropoulos, Swelling studies and in vitro release of verapamil from

579

calcium alginate and calcium alginate–chitosan beads, Int. J. Pharm., 323 (2006) 34-42.

580

[22] C.-M. Lehr, J.A. Bouwstra, J.J. Tukker, H.E. Junginger, Intestinal transit of bioadhesive

581

microspheres in an in situ loop in the rat—A comparative study with copolymers and blends

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based on poly(acrylic acid), J. Control Release, 13 (1990) 51-62.

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[23] P. Sriamornsak, J. Nunthanid, K. Cheewatanakornkool, S. Manchun, Effect of drug loading

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method on drug content and drug release from calcium pectinate gel beads, AAPS

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PharmSciTech, 11 (2010) 1315-1319.

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[24] M.Z. Ahmad, S. Akhter, M. Anwar, F.J. Ahmad, Assam Bora rice starch based

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biocompatible mucoadhesive microsphere for targeted delivery of 5-fluorouracil in colorectal

588

cancer, Mol. Pharm., 9 (2012) 2986-2994.

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membrane blebbing and nuclear condensation, Mol. Biol. Cell, 12 (2001) 1569-1582.

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[26] B. Arica, S. Calis, P. Atilla, N. Durlu, N. Cakar, H. Kas, A. Hincal, In vitro and in vivo

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studies of ibuprofen-loaded biodegradable alginate beads, J. Microencapsulation, 22 (2005) 153-

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165.

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[27] A.K. Anal, D. Bhopatkar, S. Tokura, H. Tamura, W.F. Stevens, Chitosan-alginate multilayer

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beads for gastric passage and controlled intestinal release of protein, Drug Dev. Ind. Pharm., 29

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[28] S. Distantina, R. Rochmadi, M. Fahrurrozi, W. Wiratni, Preparation and Characterization of

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Glutaraldehyde-Crosslinked Kappa Carrageenan Hydrogel, Engineering Journal, 17 (2013) 57-

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66.

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[29] A.W. Chan, R.A. Whitney, R.J. Neufeld, Semisynthesis of a controlled stimuli-responsive

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alginate hydrogel, Biomacromolecules, 10 (2009) 609-616.

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[30] M.S. Kim, S.J. Park, B.K. Gu, C.-H. Kim, Ionically crosslinked alginate–carboxymethyl

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cellulose beads for the delivery of protein therapeutics, Appl. Surf. Sci., 262 (2012) 28-33.

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[31] M. Saeedi, J. Akbari, R. Enayatifard, K. Morteza-Semnani, M. Tahernia, H. Valizadeh, In

605

situ cross-linking of polyanionic polymers to sustain the drug release from theophylline tablets,

606

Iran. J. Pharm. R., 8 (2010) 241-249.

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[32] J. Wang, P. Somasundaran, Adsorption and conformation of carboxymethyl cellulose at

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solid–liquid interfaces using spectroscopic, AFM and allied techniques, J. Colloid Interface Sci.,

609

291 (2005) 75-83.

610

[33] T.R.R.S. Rahamatullah Shaikh, M.J. Garland, A.D. Woolfson, R.F. Donnelly,

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Mucoadhesive drug delivery systems, J. Pharm. Bioallied Sci., 3 (2011) 89-100.

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[34] A. Ahuja, R.K. Khar, J. Ali, Mucoadhesive drug delivery systems, Drug Dev. Ind. Pharm.,

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23 (1997) 489-515.

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[35] Y.K. Yadav, A.B. Gupta, R. Kumar, J.S. Yadav, B. Kumar, Mucoadhesive Polymers:

615

Means of Improving the Mucoadhesive Properties of Drug Delivery System, , J. Chem. Pharm.

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Res., 2 (2010) 418-432.

617

[36] X. Cao, R. Bansil, K.R. Bhaskar, B.S. Turner, J.T. LaMont, N. Niu, N.H. Afdhal, pH-

618

dependent conformational change of gastric mucin leads to sol-gel transition, Biophys. J., 76

619

(1999) 1250-1258.

620

[37] I. Sekirov, S.L. Russell, L.C.M. Antunes, B.B. Finlay, Gut microbiota in health and disease,

621

Physiol. Rev., 90 (2010) 859-904.

622

[38] T.T. Kararli, Comparison of the gastrointestinal anatomy, physiology, and biochemistry of

623

humans and commonly used laboratory animals, Biopharm. Drug Dispos. , 16 (1995) 351-380.

624

[39] K. Kesavan, G. Nath, J.K. Pandit, Sodium alginate based mucoadhesive system for

625

gatifloxacin and its in vitro antibacterial activity, Sci. Pharm., 78 (2010) 941-957.

626

[40] K. Nishida, Y. Ando, M. Enomoto, Interaction of 5-fluorouracil with sodium

627

carboxymethylcellulose, Colloid Polym. Sci., 260 (1982) 511-513.

628

[41] P. Twentyman, M. Luscombe, A study of some variables in a tetrazolium dye (MTT) based

629

assay for cell growth and chemosensitivity, Br. J. Cancer, 56 (1987) 279-285.

cr

us

an

M

d

te

Ac ce p

630

ip t

612

631 632 633 634

27 Page 27 of 37

635 636 637

ip t

638 639

cr

640

us

641 642

Table 1: Composition of the CA-CMC beads

an

643

Polymer concentrations

645 646 647

T1G

2

T3G

3

T5G

M

Concentration

Sodium

Carboxymethyl

Alginate

Cellulose

d

1

1.8

Glutaraldehyde

(%)

-

0.25

1.8

0.5

0.25

1.8

1

0.25

Ac ce p

644

Samples *

te

S.No.

(Weight %)

* G stands for glutaraldehyde crosslinked beads

648 649 650 651

28 Page 28 of 37

652 653 654

ip t

655 656

cr

657

us

658 659

663 664 665 666

2

T3G

3

T5G

1.9

>24

2.5

>24

2.6

>24

M

T1G

pH 6.8

te

662

1

pH 1.2

* Mucoadhesivity is expressed in terms of retention time of beads on mucosal surface.

Ac ce p

661

Mucoadhesivity (Hours)*

Sample

d

S.No.

an

Table 2: Differential mucoadhesion of CA-CMC beads

660

667 668 669 670 671 29 Page 29 of 37

672 673 674

ip t

675 676

cr

677

us

678 679

FIGURE CAPTION LIST

an

680 681

 Fig. 1: Scanning electron micrograph of dried CA-CMC bead. (A, B) T1G; (C, D) T3G; (E,

683

F) T5G. Average diameter of the swollen (G) and dried (H) beads. (I) EDAX based analysis

684

of calcium content (%w) in the CA-CMC beads. Sodium alginate (SA) and Carboxymethyl

685

cellulose (CMC) were taken as reference.

687 688 689

d

te

 Fig. 2: XRD profile of dried CA-CMC beads. Sodium alginate (SA) and Carboxymethyl

Ac ce p

686

M

682

cellulose (CMC) were taken as reference.  Fig. 3: (A) DSC thermogram of the CA-CMC beads, (B) Bulk compressive strength of CACMC beads, (C) Swelling of CA-CMC beads in different GIT simulated fluids.

690

 Fig. 4: A) Degradation of CA-CMC beads at colonic pH. Degradation was studied in

691

presence and absence of colonic microflora and expressed in terms of time (hours) required

692

for complete degradation of 100 mg beads of different formulations. Data was expressed as

693

Mean ± SD (*, ** p<0.005). B) Differential adhesions of colonic microflora on CA-CMC

694

beads at colonic pH. A) T1G, B) T3G, 5) T5G

30 Page 30 of 37

 Fig. 5: (A) Analysis of release of 5 Fluorouracil from CA-CMC matrix in presence and

696

absence of colonic enzymes in simulated fluids corresponds to different compartment of the

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GI tract. The residence time of the bead at each compartment was fixed as per the actual in

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vivo value. Data are expressed as Mean SD. All data are statistically significant with respect

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to groups (p<0.005). (B) Analysis of solute mobility inside CA-CMC matrix through

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fluorescence recovery after photobleaching. Percentage recovery corresponds to the free

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mobility of solute molecules (FITC-Dextran, 10 kDa). E stands for the experimental

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conditions in which colonic enzymes are present and active.

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 Fig. 6: Study of the efficacy of 5-FU loaded CA-CMC beads on HT-29 cells. Cell viability

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was measured by MTT after 48hours of drug exposure. B) Flow cytometry analysis of

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apoptosis in HT-29 cells using PE –annexin V. The cells were exposed to the drug loaded

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formulation for 48h.

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fragmentation using DAPI (Blue) I) Control, II) T1G, III) T3G, IV) T5G. Arrow designates

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the nuclear condensation and fragmentation.

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C) Immunocytochemistry based study of nuclear condensation and

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