INTRODUCTION:

The development of mucoadhesive drug delivery system has received increased attention generating a large range of applications in pharmaceutical research. In biomedical field various formulations are based in mucoadhesion based delivery system, aiming the controlled release of drugs. These systems further present the versatility of allowing the incorporation of suitable amounts of drugs and improving the bioavailability of degradable drugs and the permeation of hydrophilic substances across epithelial layers. Mucoadhesive polymers are synthetic or natural macromolecules capable of attaching to mucosal surfaces. Mucoadhesive polymers may fulfil the desirable features of a prolonged residence time at the site of drug absorption owing to increased contact with the absorbing mucosa, resulting in a steep concentration gradient to favour drug absorption, and localization in specified regions to improve the bioavailability of drugs.

The basic components of mucus are mucin glycoproteins which form an unstirred gel layer over the epithelial cells of the mucosa. For optimum mucoadhesion, there has to be an intimate contact between the adhesive and the substrate and interpenetration of the polymer chains with the mucin glycoprotein network. Chitosan interacts with mucin by multiple modes, namely due to molecular attractive forces formed by electrostatic interaction between positively charged chitosan and negatively charged mucosal surfaces (sialic acid). Thus the electrostatic attraction between the positively charged mucoadhesive chitosan and negatively charged mucus glycoprotein plays an important role in the adsorption of chitosan on mucin.

The exceptional mucoadhesive properties of chitosan, in comparison to other polymers like polycarbophil, used as a reference substance, were found for the first time employing chitosan films in pig intestinal mucosa investigated the mucoadhesive properties of several polymers by measurement of time of mucoadhesion on porcine small intestinal mucosa and of total work of adhesion by tensile studies. It was showed that the mucoadhesive profile of the precipitated and lyophilized form of chitosan was pH dependent. At acid medium (pH 3.0) chitosan was immediately disintegrated. The best results of chitosan occurred for lyophilized form, at higher pH values (pH 6.5 and 7.0).

Chitosan, a natural linear bio-polyaminosaccharide, is usually obtained by alkaline deacetylation of chitin. Chitosan solutions, gels, films, sponges, micro and nanoparticles have demonstrated to promote absorption of small polar molecules and peptide/protein drugs through ocular, nasal, pulmonary, oral and intestinal mucosa by using animal models and human volunteers. Factors such as chemical modification can significantly influence the mucoadhesivity of polymeric micropaticles^5-6.

The studies were carried out on modifying the chitosan to their Schiff bases. These Schiff bases were compared with the non modified chitosan for their mucoadhesion assessment. This was done by using ‘Tensiometry’ method. The forces required for detachment of respective compounds from mucin was reported for precise mucoadhesivity of each compound.

EXPERIMENTAL^7-8:

Chemistry

General method for the synthesis of Schiff base of chitosan:

Chitosan (1gm) was firstly dissolved in 0.2M acetic acid (pH 4, 60ml). The solution was diluted with Methanol (80ml) before the addition of designated aromatic aldehydes. The reaction mixture was stirred at room temperature for 2 hrs. Then, the pH of the reaction mixture was adjusted to 7 with 15% (w/v) NaOH. The product was isolated by dialysis using water for 4 days and recrystalised from alcohol.

Table 1: Physical data of Schiff base of chitosan derivatives

Compd Name	Name Of Substituted Aldehyde (R)	Molecular Formula	Molecular Weight
3a	Ortho-chloro benzaldehyde	CH₅O₂ (C₂₀H₂₂ClN₂O₈)_n	49.04+(453.85)_n
3b	p-dimethylamino benzaldehyde	CH₅O₂ (C₂₂H₂₈N₃O₈)_n	49.04+(462.47)_n
3c	2,4-dimethoxy benzaldehyde	CH₅O₂ (C₂₂H₂₇N₂O₁₀)_n	49.04+(479.45)_n
3d	2-hydroxy naphthaldehyde	CH₅O₂ (C₂₄H₂₅N₂O₉)_n	49.04+(485.46)_n

Infrared (IR) and Proton Nuclear Magnetic Resonance (¹H-NMR) were used to confirm the structures of all the synthesized compounds.IR spectra were recorded on a Jasco FTIR-410 spectrophotometer using KBr pellets. ¹H-NMR spectra were recorded on Varian mercury YH-300 using CF₃COOD as solvent at 300 MHz. TMS was used as an internal reference standard for relative proton chemical shift.

Evaluation of the Mucoadhesive Strength

The mucoadhesive potential of each derivative was determined by measuring the force required to detach the derivative from stomach mucosal tissue using a modified method². In brief, stomach tissues were carefully removed from the stomach of rat. Tissues were immediately used after their separation. At the time of testing, a section of stomach tissue was secured (keeping the mucosal side out) to the upper probe using a cyanoacrylate adhesive. The upper probe was attached to precalibrated force displacement transducer SS12LA, (BIOPAC Systems) connected to the Biopac MP-30 data acquisition system (BIOPAC Systems). The surface area of each exposed mucosal membrane was 0.680cm². At room temperature, fixed amount of samples of each derivative were placed on the lower probe. The probes were equilibrated and maintained at 34°C. Probe with stomach tissue was lowered until the tissue contacted the surface of the sample. Immediately, a force of 0.1 N was applied for 2 minutes to ensure intimate contact between the tissues and the samples. The probe was then moved upwards at a constant speed of 0.15 mm/s. The bioadhesive force, expressed as the detachment stress in dyne/cm², was determined from the minimal weights that detached the tissues from the surface of each derivative using the following equation ^{3, 4}.

Detachment Stress (Dyne/ cm²) = m g a

Where m is the weight added to the balance in grams; g is the acceleration due to gravity taken as 980 cm/s²; and A is the area of tissue exposed. Measurements were repeated thrice for each of the derivative, but before each measurement a fresh smooth gel surface was created.

Effect of Initial Contact Time on Mucoadhesive Strength

Effect of varying contact time (1, 2, 3, 5, and 10 minutes) was investigated for some of the gel preparations to optimize initial contact time. In brief, derivatives were allowed to be in contact with mucosa for carrying contact times (1, 2, 3, 5, and 10 minutes), and the bioadhesive force was determined as discussed above. Contact time which has resulted in maximum bioadhesive strength was assessed as optimum contact time required for proper adhesion.

RESULTS AND DISCUSSION:

Synthesis of Schiff base of Chitosan derivative:

The derivatives of chitosan (CS), Schiff bases of chlorobenzyl chitosan [3a], N-(4-N, N-dimethylaminobenzyl) Chitosan [3b]; N-(2, 4-dimethoxybenzyl) Chitosan [3c]; N-(2-hydroxynaphthyl) Chitosan [3d] with various aromatic aldehydes were successfully synthesized. The reaction was carried out by using homogeneous reaction between chitosan and aromatic aldehyde in methanolic acetic acid. The series of 4 Schiff bases of chitosan derivatives with different electron withdrawing and electron donating substituents were obtained. The representation of synthesis of Schiff base of chitosan derivatives is presented in scheme.1. The compounds were identified by IR and ¹H-NMR ⁹.

Table 2: IR and NMR spectral values for probable derivatives of Schiff bases

No.	IR		NMR
No.	OH stretching	Imine C=N	Tetrahydron	Imine	Aromatic	Acetyl
3a	3447	1646	4.161	9.983	7.995	3.739
3b	3380	1633	4.101	10.289	8.524	2.247
3c	3419	1607	4.170	10.870	7.270	2.380
3d	3425	1636	4.182	11.501	8.701	2.544

Table 3: Test for Mucoadhesion of Schiff base of chitosan. (By Tensiometry)

Sample	Weight required for detachment(mg)	Stress (N/cm²)	Stress (dynes/cm²)
CS	1168.6	0.0180	1800
3a	2971.3	0.0457	4579
3b	2605.8	0.0401	4012
3c	2473.0	0.0380	3808
3d	2100.5	0.0323	3236

Earlier work with Chitosan polymers has clearly indicated that it is the availability of hydrogen bonding groups that determines bioadhesion; Chitosan has major functionalities for hydrogen bonding with sugar residues in oligosaccharide chains in the mucus membrane, resulting in formation of a strengthened network between polymer and mucus membrane. Thus, Chitosan having high density of available hydrogen bonding groups would be able to interact more strongly with mucin glycoproteins. In addition, Chitosan derivatives may also adopt more favorable macromolecular conformation with increased accessibility of its functional groups for hydrogen bonding. It is speculated that the higher mucoadhesive strength of the delivery system may lead to the prolonged retention and increased absorption across mucosal tissues ^1-3_.

NMR data:

1. 3a

2. 3b:

3. 3c:

4. 3d:

Figure 1: NMR spectra for 3a, 3b, 3c, and 3d

CONCLUSION:

Three kinds of derivatives have been synthesized as representatives of the Schiff base derivative class viz; Schiff base of Chloro benzyl chitosan, Dimethyl amino benzyl Chitosan, 2, 4-dimethoxybenzyl Chitosan and 2-hydroxy naphthyl Chitosan. They have shown promising and greater mucoadhesive strength than unmodified Chitosan.

IR data

ACKNOWLEDGEMENT:

We are really thankful to Prof. D. D. Chougule, Dr. C. S. Magdum, for their kind support and time to time encouragement. I also want to thank Dr. J. I. D’souza for his kind support. We also pleased to thank department of pharmaceutical chemistry of the college.

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Received on 12.07.2011 Modified on 09.09.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1099-1103