Development of biopolymer nanocomposite for silver nanoparticles and Ciprofloxacin controlled release
Introduction
Intestinal infections become sometimes a difficult pathology to treat because of different bacterial survival strategies such as multidrug resistant and/or biofilms formation [1]. For example in the cystic fibrosis disease, the bacteria colonize the intestine making biofilms by mucoid polymers protecting them against antibiotics. Moreover, the nutrient absorption of the patients is strongly compromised and followed by a slow death if is not properly treated. Also, the treatment of these pathologies generally requires the use of several doses of high antibiotic concentrations, which are associated to undesirable side effects including drug accumulation in non-targeted organs, hypersensitivity, immune-suppression, and allergic reactions [2].
In the recent years, silver nanoparticles (Ag-NPs) have emerged as a powerful tool to treat microbial infections including multi-resistant pathogens. Ag-NPs inactivate a panel of drug-resistant and drug-susceptible bacteria (Gram positive and Gram negative), exert their antibacterial activity through a bactericidal rather than bacteriostatic mechanism, and inhibiting the bacterial growth rate from the time of first contact with the bacteria [3]. Hence, Ag-NPs have been applied in diverse medical fields such as topical ointments and creams containing silver to prevent infection of burns and open wounds or in the development of medical devices and implants prepared with silver-impregnated polymers [3]. In addition to their direct bactericidal activity, Ag-NPs are also known to be capable of interact and disrupt biofilm formation [4], [5]. However, high concentrations of Ag-NPs were reported as toxic in living organisms which are remarking the need of a carefully adjusted dose as key information on potential adverse side effects toward establishing a treatment on humans [6]. Besides, human cells were found to have a high resistance to the toxic effects of silver nanoparticles compared to other organisms. Even, silver nanoparticles present a lower toxicity compared with silver ions and nanoparticle encapsulation also lowered its toxicity [7]. In particular, oral delivery of Ag-NPs was recently reported and found that were not observable clinically relevant toxicity markers in an exposed time of 14 days in humans [8]. However, chemically synthesized Ag-NPs are requiring protection by coating to prevent phenomena like aggregation, oxidation and destabilization, parameters that determine their toxicity [9], [10].
Metal nanoparticles-embedded hydrogels, generally described as polymer matrix-nanocomposites, have been postulated as an emergent new generation of antimicrobials [3], [11]. Three dimensional, matrices based on various natural hydrophilic polymers like starch, gelatin, cellulose, chitosan, sodium alginate, and their derivatives such as carboxymethyl cellulose or alkyl amine guar gum, are known to extend effective stabilization of the nanoparticles for a range of applications in catalysis, biomedicine, optics and pharmaceuticals [12], [13]. Additionally, in recent work the relevance of Ag-NPs co-administration with an antibiotic to produce a synergic antibacterial activity was reported [14].
Among antibiotics, Ciprofloxacin (Cip) is the fifth largest generic antibiotic produced in the world keeping 24% of the therapeutic prescription market (about US$ 2.3 million dollars/year). Cip belong to the fluoroquinolone family, a wide class of antibiotics with broad antibacterial spectrum currently used in many infections [15]. However, ciprofloxacin administration is currently associated with many undesirable and diverse toxic side effects that involve many organs and behavior [16]. Many of the reported problems could be associated to a poor Cip solubility and biodisponibility under physiological media [17]. In addition, Cip stacking phenomena can surpass drug solubility and becoming highly toxic to humans [18].
Guar Gum (GG) is a biopolymer synthesized by the seed legume Cyamopsis tetragonolobus commonly found in the Indian subcontinent. GG is a galactomannan composed by a linear chain of β 1,4-D-mannopyranoses in where D-galactopyranoses residues are α-1,6 linked at every second mannosyl residue forming short side-branches (2:1 ratio), and average molecular weight of 220 kDa. Guar gum has approximately 8-times the water-thickening potency of corn starch, and can be used in multi-phase formulations as an emulsifier preventing oil droplets from coalescing, and/or as a stabilizer in order to prevent molecular aggregation and particles from settling, and it can form gels in presence of calcium ions [19]. However, GG has a low solubility in aqueous solutions (about 1.0 mg/ml). A common strategy to increase the solubility of GG is by introducing polar groups in the main structure of polymer. Alkylation of GG with ethylamine increases polymer solubility and it has been proposed as a matrix for drug controlled release and as a stabilizer of silver nanoparticles [12], [20].
Alginates (ALG) are linear anionic polysaccharides widely used in food and pharmaceutical industries. ALG are made of β-manuronic acid (M units) and α-guluronic acid (G units) linked by 1-4 bounds. Their properties of being non-toxic, showing no immunologic response, high biocompatibility and gelation in presence of divalent ions by making “egg box” structure made them very suitable gels for many therapeutic applications [21]. ALG gels can be enzymatically hydrolyzed by alginate lyase (AL). AL is able to hydrolyze alginate polysaccharides (tetramers or higher) in an endo-type manner at the β-1,4-glycosidic linkage via β-elimination reaction to produce mono-, di-, and trisaccharide products [22]. Its importance in the release of cargo molecules with high molecular weight or a size higher than the matrix porosity becomes an interesting tool to overcome the diffusional limitations of encapsulated silver nanoparticles from a biopolymeric gel structure based on alginate.
Pectins are water-soluble polysaccharides present in the plant cell wall. The use of pectins as matrix in oral drug delivery devices were proposed [23]. Pectins are composed of linear polysaccharides of partially metoxylated poly α-(1,4)-D-galacturonic acids. The esterification degree (DE) of pectins has strong influence on biopolymer properties. Pectins are grouped into Low Methoxylated (LM) with DE below of 40%, Medium Methoxylated (MM) with DE range between 40 and 60%, and High Methoxylated pectins (HM) with DE higher than 60%. LM and MM pectins can be gelled by multivalent cations, meanwhile HM only by acid pH [24].
Matrices composed of alginate, pectin and their coacervates modulating the Ciprofloxacin release profile and providing protection of labile molecules against harsh environments were previously reported by our laboratory [25], [26].
The aim of the present work is the co-encapsulation of silver nanoparticles stabilized into Alkyl Amine Guar Gum (Ag-NPs(AAGG)) and Ciprofloxacin into biopolymeric gel beads to provide a dual-component drug delivery system for the treatment of intestinal infections and prevention of chronic colonization of pathogens. The release profile of both antimicrobials was analyzed at different pHs and the diffusional mechanism was established. Also, stability of the Ag-NPs incorporated into the matrix was evaluated against simulated physiological environments (gastric and intestinal) to validate their potential application in oral delivery. Characterization of the matrices was carried out by statistical analysis of scanning electron microscopy (SEM) pictures. Finally, microbiocidal properties against Gram positive and Gram negative strains as model recurrent opportunistic pathogens were tested and correlated by TEM images.
Section snippets
Materials
Ciprofloxacin (Cip, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-ox-o-7-(1-piperazinyl)-3-quinoline carboxylic acid) was purchased from Sigma-Aldrich (Buenos Aires, Argentina). Low viscosity sodium Alginate (ALG) (average Mη 1.0 × 105 Da) was obtained from Biochem SA (Buenos Aires, Argentina). Low Methoxylated (LMP, ED 33%, average Mη 1.56 × 105 Da) and High Methoxylated (HMP, ED 74%, average Mη 1.60 × 105 Da) pectins were kindly provided by C.P. Kelco (Buenos Aires, Argentina). All other reagents used were of
Determination of Ag-NPs(AAGG) interactions with Cip
In order to establish a comprehensive analysis of the experimental conditions for the nanocomposite formulation, interactions between the cargo components were under study. By co-precipitation method, an interaction between Cip and AAGG of about 50% was found. Also, the interaction was independent of Ag-NPs content, even when NPs concentration was increased 20 times (p ≤ 0.05) (Fig. 1S, Supporting Material). This result is indicating the specific interaction of the Cip with the modified guar gum,
Conclusions
Novel biopolymer nanocomposite microbeads were developed as a matrix for controlled release of silver nanoparticles and Ciprofloxacin. The co-encapsulation of both antimicrobials is relevant in the field since multi-resistant opportunist pathogens are reported more often and novel strategies of delivering drugs based on the use of green synthesis and non-toxic materials are required. The present study reports a facile preparation of a NiMDS composed of Alginate/HM Pectin made by ionic gelation
Acknowledgements
The present work was supported by grants from Fundación Argentina de Nanotecnología, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; PIP 0498, Argentina), Universidad Nacional de La Plata of Argentina (11/X545) and Agencia Nacional de Promoción Científica y Técnica (PICT 2011-2116, Argentina). The doctoral fellowship to GAI from the UNLP was gratefully acknowledged.
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