Carbamoylethyl pullulan: QbD based synthesis, characterization and corneal wound healing potential

https://doi.org/10.1016/j.ijbiomac.2018.07.107Get rights and content

Highlights

  • Carbamoylethyl pullulan synthesis process was optimized using statistical approach.

  • Improved rheological properties of carbamoylethyl pullulan were attained.

  • Carbamoylethyl pullulan possessed enhanced wound healing potential.

  • Impact of corneal adhesiveness and O2 neutralizing ability on ocular wound healing.

Abstract

Corneal injuries are the major problem arises these days due to excessive use of mobile phone, TV, environment pollution, etc. Thus, a need is to evaluate materials having good corneal wound healing potential. To achieve this goal, pullulan was derivatized using carbamoylethylation and 24 factorial design has been found a good approach for the optimization of synthesis process of carbamoylethyl pullulan (CMEP). The design expert software applied for this purpose suggested significance of the generated model. ATR-FTIR, NMR and XRD spectral attributes validated the structure of CMEP. Interestingly, derivatization of pullulan to CMEP has increased its ΔH from 171.552 J/g to 203.5 J/g and decreased the endothermic transition from 323.44 °C to 248.67 °C. Further, rheological behavior suggested enhanced viscosity after carbamoylethylation indicating increase in H-bonding that further participate in the enhancement of corneal adhesive strength. The CMEP possessed high corneal wound healing property that was associated with its high corneal adhesive strength and oxygen neutralizing capacity. Thus, the findings suggested overwhelming influence of CMEP as a corneal wound healing agent.

Introduction

Wound healing is a process to repair the damaged tissues and regains their functional properties under suitable conditions [1]. The ideal material for wound healing should be biocompatible with the damaged tissue and maintain a healing environment [2]. The wound healing process mainly involves cell migration and regeneration. Along with other organs, eyes are the most important and highly sensitive part of human body. The cornea of eye is the most vital refractive medium in the anterior part of the eye and has high susceptibility to environment, foreign body invasion and physical or chemical damage. The corneal wounds are responsible for blindness worldwide as most of the ocular refractive power is controlled by cornea. Various natural biopolymers have been studied for peripheral eye disease i.e. collagen [3], gelatin [4], fibrin [5], alginate [6], chitosan [7], etc. However, the epithelium of cornea and conjunctiva has tight sealed intercellular junctions, which limits the entry or retention of any external material along with healing remedies on the surface of eye and lowers the efficient treatment of corneal diseases [8]. The major loss of healing solution requires more frequent instillations for proper healing which may lead to various adverse effects. Hence, to achieve efficient cornea wound healing potential, the healing solution should have prolonged adhesive strength in addition to good oxidative stress neutralizing capacity required for accelerating the wound healing [9].

Pullulan is one of the potential biopolymers commercially produced by yeast-like fungus Aureobasidium pullulans [[10], [11], [12]]. It is produced extracellularly by A. pullulans and can be extracted easily from the cell-free extract by using organic solvent precipitation [13]. Pullulan is off-white to white colored powder without any toxicity, mutagenicity or carcinogenicity. It is an edible non-ionic polymer with easy solubility properties. The basic structure of pullulan consists of maltotriose as repeating units, which constitutes α-(1 → 4) bonded glucopyranose rings interlinked by α-(1 → 6) linkages, i.e. [α-d-Glcp-(1 → 4)-α-d-Glcp-(1 → 4)-α-d-Glcp-(1 → 6)]n [14]. Each maltotriose unit has nine hydroxyl groups on the glucopyranose rings. These hydroxyl groups can be substituted to synthesize different derivatives via various chemical reactions [15]. Pullulan has been derivatized into a number of derivative forms which have significant biomedical applications [16]. Moreover, pullulan and its derivatives, its conjugation with other polymers and complex forms possess great applications in tissue engineering [17], drug delivery and gene delivery [18]. However, pullulan is a low viscosity polysaccharide [10] which has low cornea adhesive strength. This may be the reason that the literature reports on cornea wound healing potential of pullulan or its derivatives is not available. Thus the main focus of present investigation is to utilize pullulan (either in native form or after best possible derivatization) for the benefit of corneal injuries.

Carbamoylethylation of polymers is a strategy to improve physico-chemical performance. So far, the carbamoylethylation of Cassia tora gum [19], guar gum [20], Cassia occidentalis seed gum [21], and Cassia angustifolia seed gum [22] have been reported. The carbamoylethylation involves addition of amide group which leads to an increase in nitrogen content of polymer. Carbamoylethylation have significant effect on the rheological properties of the gums. All gums derivatives have non-Newtonian pseudoplastic behavior regardless of nitrogen content, but different polymer derivatives have varied viscous properties. The carbamoylethyl solution of Cassia tora gum and Cassia occidentalis seed gum showed increase in viscosity with increase in nitrogen content of the product [19,21]. On the other hand, apparent viscosity of carbamoylethyl guar gum is inversely related with the nitrogen content of polymer at a constant shear rate [20]. The carbamoylethyl solution of Cassia angustifolia seed gum has higher stability than native gum [22]. Hence, carbamoylethylation of polysaccharide improves the rheological properties. The enhancement in viscosity after derivatization of polysaccharide is associated with changes in H-bonding attributes [23]. The H-bonding characteristic is known to be responsible for changes in mucoadhesive strength. Therefore it was expected that carbamoylethylation of pullulan could enhance the cornea adhesive strength thereby can be used for wound healing purpose.

Although, pullulan has been explored for its applications in pharmaceutical field, but there is no report on direct usage of pullulan or its derivatives as cornea wound healer. Thus, the present work was aimed to synthesize a novel pullulan derivative carbamoylethyl pullulan to explore its wound healing potential. The synthesis process of carbamoylethyl pullulan (CMEP) was optimized using design expert software 11.0.3.0 software (State-Ease Inc., Minneapolis, MN, USA) employing 24 factorial design. The structural elucidation of CMEP was performed by employing different spectral attributes and CMEP was also evaluated for its rheological properties. Further, CMEP was investigated for its early recovery from corneal injuries and further investigated to explore the mechanism behind wound healing process.

Section snippets

Materials

Sodium hydroxide, acrylamide, glacial acetic acid and methanol were of laboratory grade (SD Fine-Chem. Ltd., Mumbai, India). Dialysis membrane-150 (molecular weight cut off 12,000 to 14,000; pore size 2.4 nm; capacity 5.07 mL/cm) was purchased from Himedia Chemicals Mumbai, India.

Production and recovery of pullulan

Pullulan was produced from the yeast-like fungal strain of Aureobasidium pullulans according to the method reported by Singh and Saini [13]. In brief, A. pullulans (MTCC 6994) was grown for 7 days under agitation

Experimental design

Carbamoylethylation is generally carried out by reacting polysaccharide (dissolved in water) with acrylamide in presence of sodium hydroxide. Thus, the carbamoylethylation reaction lead to the attachment of amide moieties linked with ethylene to hydroxyl group on the extremities of backbone of polysaccharide chain [36]. Carbamoylethylation of pullulan (PU) was assured by determining the percentage nitrogen content of the derivatized product [27]. On reacting pullulan with acrylamide in the

Conclusions

The 24 factorial design was found to be good approach for optimizing the synthesis process of carbamoylethyl pullulan (CMEP). The process that met critical quality attributes for the synthesis of CMEP involves addition of 250 mmol acrylamide in 40 mmol alkaline pullulan solution with reaction time of 4 h at 70 °C. The carbamoylethylation added carbamoyl moieties to native pullulan structure that enhances the rheological performance of CMEP along with its porous surface. However, non-Newtonian

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