Preparation and characterisation of gelatin–gum arabic aldehyde nanogels via inverse miniemulsion technique

Abstract

Gelatin–gum arabic aldehyde nanogels designed by a nanoreactor concept using inverse miniemulsion technique were reported. Stable separate miniemulsions were prepared from gelatin (Gel) and gum arabic aldehyde (GAA). These emulsions were intermixed under sonication to obtain cross-linked nanogels. During fusion, cross-linking occurred between aldehyde groups of GAA and amino groups of gelatin. The concentration of the surfactant and weight fraction of water in the inverse miniemulsion was optimised so as to yield nanogels with controlled particle size. Properties of the nanogels were studied by FT-IR spectroscopy, particle size analysis and XRD. Surface morphology of the nanogels was established by Scanning Electron Microscopy (SEM). SEM and particle size analysis confirmed that nanogels possess spherical morphology with an average diameter of 151 ± 6 nm. Hemolysis property of the nanogels was examined and the results indicated that the nanogels were hemocompatible. The in vitro cytotoxicity of the nanogels towards MCF-7 cells was evaluated by MTT assay and the nanogels showed nontoxic behaviour towards the cells. All these studies confirm that these nanogels are potential candidates in applications such as drug and gene delivery.

Introduction

The increasing interest on nanoscale materials with biocompatibility, biodegradability and nontoxicity has accelerated research on development of new nanomaterials and new synthetic routes. Nanogels are one such class of materials which bagged great attention from different areas of biology, chemistry, physics and medicine because of their unique properties offered by its nano size [1]. Nanogels are nanometer sized counterparts of hydrogels, possessing special properties such as huge surface area, colloidal stability and elevated drug loading ability offered by its small size in addition to the properties of hydrogels [2], [3]. Because of their potential as versatile carriers for different therapeutic and diagnostic agents, nanogels find applications in biomedical fields, such as gene delivery, drug delivery and bioimaging [3], [4], [5]. Nanogels have been prepared using synthetic as well as natural polymers. Although synthetic polymers like poly(ethylene glycol) [6], poly(lactic acid) [7] and poly(ɛ-caprolactone) [8] proved their ability as suitable materials for development of nanogels, natural polymers like chitosan [9], dextran [10] and hyaluronic acid [11] received more importance owing to their nontoxicity, biocompatibility and biodegradability. Jayakumar and co-workers developed pH responsive chitin and chitosan nanogels and examined the viability for drug delivery to cancer cells, gene therapy, biosensing and bioimaging [12], [13], [14], [15] applications. Pullulan, a highly hydrophilic polysaccharide was hydrophobically modified with cholesterol/spiropyran and self assembled nanogels were prepared from the resultant amphiphilic polysaccharide [16], [17].

For the present work, natural polymers, namely, gelatin and gum arabic (GA) were selected. Gelatin is a well exploited natural protein with fascinating biomedical properties such as biocompatibility, biodegradability, non-immunogenicity and safety. It is extensively used in food industry, in pharmaceutical industry as drug carrier, and in biomedical field as tissue engineering matrix [18], [19]. Koul et al. [20] prepared nanogels of interpenetrating polymer network of gelatin and polyacrylic acid by one pot inverse miniemulsion technique and this nanogels could be used as drug delivery vehicles for cancer targeting. Paclitaxel-loaded gelatin nanoparticles exhibited rapid release of the drug and showed significant activity towards human cancer bladder cells [21]. Tseng et al. [22] developed gelatin nanoparticles by desolvation method and its surface was modified with NeutrAvidin^FITC-biotinylated epidermal growth factor (GP-Av-bEGF) for targeting to lung cancer cells. Gelatin nanoparticles were also utilised for loading and delivery of protein and peptide drugs such as insulin [23], bovine serum albumin (BSA) [24], alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), tissue-type plasminogen activator (t-PA) [25] and angiogenic basic fibroblast growth factor (bFGF) [26]. Other applications of gelatin nanoparticles include ocular drug delivery, nutraceutical delivery, pulmonary drug delivery, enzyme immobilization etc. [27].

For overcoming the poor mechanical properties and to improve strength and aqueous stability, gelatin based hydrogel systems were cross-linked with different cross-linking agents including glutaraldehyde, diisiocyanates, genipin, carbodiimide [28], [29], [30], [31] and oxidised polysaccharides [32], [33]. Cross-linking of gelatin with oxidised polysaccharides is a safer method to improve the properties compared to the cross-linking with toxic agents like glutaraldehyde, diisocyanates and carbodiimide. In the present work, a plant polysaccharide, namely gum arabic was selected to prepare the cross-linked nanogels. Gum arabic is obtained from the exudates of acacia tree. It has a complex branched structure with rhamnose, galactose and glucuronic acid residues. The back bone and side chains consist of 1,3 linked β-d-galactopyranosyl units with side chain joined to the main chain by 1,6 linkages [34]. High water solubility, biocompatibility and low cost are the main attractions for selecting this polysaccharide for this work. GA is widely used in the food industry as stabilizing, emulsifying and thickening agent. Even though GA was used for the preparation of microparticles and nanoparticles, biomedical applications of this potential polysaccharide were not explored in greater detail. Avadi et al. [35] developed gum arabic and chitosan based nanoparticle system for oral delivery of insulin. Release properties of gum arabic microparticles were investigated with vettiver essential oil and camphor oil as models [36], [37]. Nishi et al. prepared oxidised GA and conjugated it with different drugs and evaluated its application in drug delivery [38], [39], [40]. The preparation, characterisation and in vitro biomedical applications of gum arabic aldehyde cross-linked gelatin hydrogels [41], [42] have been reported by us recently. However, no research effort has been made to prepare GA based nanogels. In this work, nanogels from oxidised gum arabic (GA) and gelatin was prepared by fusion of two separate inverse miniemulsions.

Less energy intensive technique is the preferred choice of researchers to produce nanomaterials. One of the highly explored, such method is water-in-oil (w/o) mimiemulsion and it was used to prepare semiconductors [43], polymeric nanoparticles [44], drug nanocrystals [45] and magnetic particles [46]. The ease with which miniemulsion can be prepared makes it a favourable method for developing nanoparticles. For the preparation of Gel–GAA nanogels, fission and fusion of separate miniemulsions of GAA and gelatin was adopted. These two miniemulsions contain individual reactants in aqueous phase and during fusion, inter micellar exchange and cross-linking of individual reactants occur leading to the formation of nanogels. Processing parameters such as concentration of surfactant and aqueous fraction in the inverse miniemulsion were optimised to obtain Gel–GAA nanoparticles with controlled size. Physicochemical properties of the nanogels were analysed by particle size analysis, SEM, FT-IR, and XRD. Hemocompatibility studies and MTT assay were performed to assess the blood and cytocompatibility of the nanogels.