Preliminary spectroscopic characterization of PEGylated mucin , a novel polymeric drug delivery system

1 Drug Delivery Research Unit, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka 410001, Enugu State, Nigeria. 2 Department of Pharmaceutical Technology and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka 410001, Enugu State, Nigeria. 3 Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.


INTRODUCTION
Drug delivery can be of importance for both new chemical entities as well as established drugs.The basic goal of any drug delivery system is to achieve steady state of blood concentration or tissue level that is therapeutically effective and non-toxic for an extended period of time (Muller et al., 2000;Gillies and Frechet, 2004).Many biolo-gically active molecules are limited in their therapeutic values by properties such as poor solubility, limited bioavailability, and rapid elimination (Muller et al., 2000).In addition, while the beneficial effects of many drugs arise from their interaction with specific tissues, their exposure to other cell types frequently leads to undesirable side effects and toxicity (Gillies and Frechet, 2004).In recent decades, there has been increased awareness of the need to develop drug delivery systems to improve the properties of therapeutic compounds, increase their effectiveness, and reduce their harmful side effects.Oral administration of therapeutic agents represents by far the easiest, safest and most convenient route of drug delivery, especially in the case of chronic therapies (Kreuter, 1991).Unfortunately, the oral delivery route is beset with problems such as gastrointestinal (GI) destruction of labile molecules and low levels of macromolecular absorption (Francis et al., 2004).The development of oral forms of many drugs however remains a challenge either on account of their stability or their absorption from the GIT thus leading to sub-therapeutic bioavailability (Kreuter, 1991).To reduce the impact of digestive enzymes and ensure the absorption of bioactive agents in an unaltered form, molecules may be incorporated into polymeric matrices, which could shield the encapsulated drug from the external harsh conditions, and may favour uptake by intestinal cells (Delie and Blanco-Prieto, 2005).Many polymers have been used in the formulation of polymeric matrices (Ravi-Kumar, 2000;Pamujula et al., 2004;Lagarce et al., 2005;Bernkop-Schnurch et al., 2004;Attama and Nwabunze, 2007).The engineering of new polymer biomaterials with special physicochemical properties appears to be an answer to the afore-mentioned problems.To obtain new polymer biomaterials with better physicochemical qualities that will meet the carrier needs of challenging drug molecules, blending of polymers with desirable properties becomes imperative.Thus, mucin and PEG could be blended to improve the physico-chemical properties of the individual polymers.
Mucins are high molecular weight (0.2 -10 million Dalton) glycoproteins containing both highly glycosylated domains and naked domains.The glycosylated domains are enriched in serine and threonine residues which serve as anchoring points for oligosaccharide chains.These Olinked oligosaccharide side chains are complex both in terms of composition and length.The naked domains are typically found in the N-terminal and C-terminal parts of the protein and are enriched in cysteine residues (Carlstedt et al., 1983).The cysteine residues can form intermolecular bonds, and in the native state mucins are often found as oligomers composed of several end-to-end linked mucin subunits (Perez and Proust, 1987).A common feature of mucins, apart from a high molecular weight and a high carbohydrate content (68 -81%), is the abundance of negatively charged groups.The negative charges arise mainly from sialic acid residues and in some cases from sulphated sugars.These acidic groups account for the low isoelectric point (2-3) of mucins (Durrer et al., 1995).The basic amino acids of the protein component are serine and threonine.The glycosylated regions of mucins interact favourably with water and force the molecule to an extended random coil conformation, and the high molecular weight enables individual mucin molecules to overlap and entangle at relatively low concentrations (Lee et al., 2005).These features are ideal with respect to the formation of hydrogels.Studies have shown that reconstucted mucous gels from mucins have similar rheological properties as native mucous gels at physiological concentrations (Raynal et al., 2003).The ability of mucin to form the structural backbone of the mucous gel is one of its most important functions (Raynal et al., 2003).
There is an on-going effort aimed at improving the potential use of mucin in controlled delivery of certain drugs.One of the strategies that have been successfully employed is PEGylation (Momoh et al., 2010;Momoh et al., 2011;Momoh et al., 2010).Previous studies on PEGylation (the process of attaching PEG to any polymer, or the molecular attachment to polyethylene glycols with different molecular weights to active drug molecules or surface treatment of drug-bearing particles with PEGs) have shown that the interaction between PEG and mucin is either molecular interaction in a solvent which resulted in the formation of new bond or attachment of the PEG to a functional group in the protein molecule to form a copolymer (Schnurrer and Lehr, 1996).This technology is one of the most promising and extensively studied strategies for improving the pharmacokinetics of drugs (Abuchowski et al., 1977).Clinically proven PEGylation technology can improve the performance and dosing convenience of peptides, proteins, some water soluble drugs, antibodies, oligonucleotides and many small molecules by optimizing pharmacokinetics, increasing bioavailability and decreasing immunogenicity and dosing frequency.PEGylation has been shown to increase therapeutic efficacy by enabling increased drug concentration, improved biodistribution, and longer dwelling time at the site of action (Kodera et al., 1998).This would result in the achievement of therapeutic drug concentrations with less frequent dosing which would be a significant benefit to patients who are taking injected drugs and long time medication for example in diabetes management.Therefore, among the different polymer-based drug delivery systems, PEGylated polymeric bioadhesive microparticles represent a promising delivery vehicle especially intended for short-acting active pharmaceutical ingredients (APIs) employed in chronic therapies in order to improve their therapeutic effects.
Recently, PEGyalted mucin matrices were characterized with respect to thermal properties.The results of the thermal properties of PEGylated mucin which was measured by differential scanning calorimetry (DSC) indicated that an interaction occurred between PEG and mucin (Momoh et al., 2010).This serves as a basis for further characterization of the interaction between PEG and mucin in PEGylated mucin matrices for drug delivery application.Fourier transform infrared (FT-IR) spectroscopic analysis can be used to determine the functional groups of the molecules (Sahoo et al., 2012;Sahoo et al., 2012;Sahoo et al., 2011).Therefore, to obtain more information in detail about chemical interaction between PEG and mucin, the FT-IR analysis was carried out.In this study, admixtures of PEG 4000 and mucin obtained from African land snail (Archachatina marginata) were prepared by combining the two at different ratios.Some physicochemical properties as well as compatibility of the PEGylated mucin matrices were assessed.The objective of this study was to evaluate, spectroscopically, the compatibility of non-mucinated polyethylene glycol (PEG) 4000 and non-PEGylated mucin in a PEGylated mucin matrices for drug delivery application.

MATERIALS AND METHODS
The following materials were purchased from their local suppliers and used without further purification: polyethylene glycol-4000 (Ph.Eur.Carl Roth GmbH + Co.KG Karlsruhhe Germany), citric acid, sodium hydroxide (Merck, Germany), methyl red, ethanol, nitric acid, silver nitrate, tetraoxosulphate (vi) acid, sodium chloride, acetone, concentrated hydrochloric acid (BDH, England).The giant African land snails (A.marginata) used were procured from a local market in Nsukka, Enugu State.All other reagents were of analytical grade and were used as such.

Extraction of snail mucin (slime)
The method of Adikwu and Nnamani (2005) was employed for the extraction with modifications.The giant African land snails (A.maginata) were washed with clean water and the shells were knocked open at the apex and a spirally coiled rod was inserted to remove the fleshy body.The mucus layer (slime mucus) was gently scraped off from the fleshy parts, pooled together in a container and precipitated with chilled acetone.These precipitates were then dried with a flush of cold air (-4°C) to obtain greyish-brown flakes of the snail mucin, which were powdered and used for the study.

Physicochemical properties of snail mucin
Some physicochemical properties of the snail mucin were also assessed using standard procedures (Adikwu and Ikejiuba, 2005).

Biuret test
Two drops of water and 1 ml of dilute sodium hydroxide were added to 2 % dispersion of mucin in water.Two drops of 1 % copper sulphate solution were added with the solution shaken thoroughly after each drop and observed.A purple or pink colour shows the presence of proteins.

Xanthoproteic reaction
Two drops of concentrated nitric acid were carefully added to 2% dispersion of mucin in water.A white precipitate was formed, which turned yellow on heating.The contents of the test tube were cooled, three drops of dilute sodium hydroxide solution added and the precipitate observed.A yellow colour which changes to orange indicates the presence of proteins.

Test for fats and oils
A drop of the acetone extract of the mucin was placed on a filter paper.The solvent was allowed to evaporate and the filter paper observed carefully for any transluscence.

Test for sugars
Three drops of freshly prepared Fehling's solutions I and II were added to 1% w/v aqueous dispersion of snail mucin which was then heated in a boiling water bath for 5 min and observed.

Iodine test
Two drops of 1% iodine solution were added to 1 ml of 1% w/v of mucin and then observed for blue-black colouration.

Molisch's test
Two drops of ᾳ-naphthol solution was added to 2 ml of the snail mucin dispersion and the two mixed thoroughly.Then 1 ml of concentrated sulphuric acid was gently poured down the side of the tube and observed.

Tollen's reagent test
Tollen's reagent prepared as 1 ml of 5% silver nitrate solution was treated with a few drops of 5 % sodium hydroxide solution.A volume of aqueous ammonia just enough to redissolve the precipitate was added to 3 drops of the snail mucin dispersion and the mixture warmed in a boiling water bath for few minutes.The colour of the precipitate formed was observed.

Solubility profile of snail mucin
The solubility of snail mucin in several solvents was determined by dispersing 100 mg of the snail mucin in definite volumes of each solvent-acetone, ethanol, water, sodium hydroxide, hydrochloric acid and ammonium hydroxide respectively at different temperatures (25, 30, 40°C).

Preparation of PEGylated-mucin matrices
Polymer hybrids (matrices) were generated from mucin and poly(ethylene glycol) (PEG M w 4000) by controlled coacervation in aqueous medium (Momoh et al., 2010;Momoh et al., 2011;Momoh et al., 2010).The matrices were prepared in the following molecular ratios (M:P): 1:1, 2:1, 3:1, 2:0, 0:2.Briefly, known quantities of mucin and PEG 4000 were weighed into separate 100 ml beakers and 20 ml of distilled water was added into the samples and allowed to stand for 72 h for interaction of the solvent and the polymers.The contents of the two beakers were then mixed and allowed to stand for another 72 h for molecular interaction.The hybrids were precipitated with chilled acetone, dried, pulverized and kept in an airtight container until used.

Spectroscopic characterization of the matrices
FTIR spectroscopy study was conducted using a Shimadzu FTIR 8300 Spectrophotometer (Shimadzu, Tokyo, Japan) and the spectrum was recorded in the wavelength region of 4000 to 400 cm - `1 with threshold of 1.303, sensivity of 50 and resolution of 2 cm -`1 range.The procedure consisted of dispersing a 5 mg sample in KBr and compressing into discs by applying a pressure of 5 tons for 5 min in a hydraulic press.The pellet was placed in the light path and the spectrum obtained (Sahoo et al., 2011(Sahoo et al., , 2012(Sahoo et al., , 2012;;Builders et al., 2008).
Although PEGyalted mucin matrices (prepared with PEG 2000 and snail mucin) (Momoh et al., 2010;Momoh et al., 2011;Momoh et al., 2010) had been characterized with respect to thermal properties by differential scanning calorimetry (DSC) (Momoh et al., 2010), this serves as a basis for further characterization of the interaction between PEG and mucin in PEGylated mucin matrices for drug delivery application.FT-IR spectroscopic analysis can be used to determine the functional groups of the molecules (Sahoo et al., 2012;Sahoo et al., 2012;Sahoo et al., 2011).Therefore, to obtain more information in detail about chemical interaction between PEG and mucin, the FT-IR analysis was carried out on PEGylated mucin matrices prepared with PEG 4000 and snail mucin.The presence of different peaks in the FT-IR spectra of the PEGylated mucin matrices compared with the non-PEGylated mucin (2:0) matrix and non-mucinated PEG 4000 (0:2) matrix indicated the formation of new polymers, which could be employed in drug delivery.Results obtained in this study is in agreement with earlier reports on the drug delivery potential of PEGylated mucin (Momoh et al., 2010).The novelty embodied in this research is that this is the first spectroscopic study to be carried out on PEGylated mucin using PEG 4000 and snail mucin.

Conclusion
The engineering of new polymer biomaterials could be employed to address some problems associated with delivery of some APIs and biomolecules.To obtain new polymer biomaterials that will meet the carrier needs of challenging drug molecules, blending of polymers with desirable properties becomes imperative.In this study, admixtures of PEG 4000 and mucin obtained from African land snail (A.marginata) were prepared by combining the two at different ratios.Preliminary spectroscopic characterization was performed on the PEGylated mucin matrices using fourier transform infra-red spectroscopic characterization.Results obtained indicated that a new polymeric carrier was made from admixtures of mucin and PEG 4000 by PEGylation technology.Further solid state spectroscopic charcterization (WAXD and SAXD) studies on PEGylated mucin are currently ongoing in our laboratory.

Table 2 .
Solubility profile of snail mucin.