Colloids and Surfaces A: Physicochemical and Engineering Aspects
Factorial design as tool in chitosan nanoparticles development by ionic gelation technique
Graphical abstract
Introduction
Chitosan is a polysaccharide composed of β(1→4) linked units of N-acetyl-d-glucosamine and d-glucosamine forming a long and linear chain [1]. This polymer can be naturally found in the cell wall of certain groups of fungi, particularly zygomycetes [2] and also can be obtained from partial deacetylation, by alkaline or enzymatic hydrolysis, of chitin which is naturally found in insects, arachnids, and crustaceans.
Since Bodmeier et al. [3] reported that small particles can be obtained by dripping a solution of CTS onto a solution of TPP, many researchers have explored this property in pharmaceutical studies [4], [5], [6], [7], [8], [9].
Due to the advantageous biological properties of CTS, such as wound healing effect [9], antimicrobial activity [10], low toxicity, biocompatibility, biodegradability [11], cationic properties and bioadhesive characteristics [12], nanoCTS have been extensively applied.
NanoCTS have been widely used in pharmaceutical applications mainly as drug carrier [4], [13] and for DNA delivery [14], [15]. The nanoparticles can easily penetrate through capillary and epithelial tissue and this allows an efficient delivery of therapeutic agents, such as drugs, to target sites in the body. According to Gan et al. [16], smaller size nanoparticles (∼100 nm) demonstrated more than 3-fold greater arterial uptake compared to larger nanoparticles (∼275 nm).
CTS has antibacterial activity that is associated with the positive charge of the amino groups, which can bind to the bacterial cell surface and interfere with normal functions of the membrane, inhibiting their growth [17]. When CTS is in nanoparticles form, exhibits higher antibacterial activity than CTS powder because the polycationic nanoCTS has higher surface area and charge density and can interact to a greater degree with the negatively charged surface of the bacterial cell [18]. Therefore, the antibacterial activity of nanoCTS has been explored by many researchers [19], [20], [21].
Different methods for development of nanoCTS can be found in literature as: ionic gelation [7], [12], [22], emulsion [23], synthesis with carboxymethyl cellulose [24], formulations using glutaraldehyde [25], synthesis with alginate [26], coacervation [27], reverse micellar [28] and polymerization with poly(hydroxyethyl methacrylate) [29].
Ionic gelation technique presents the following advantages over other methods: the nanoparticles are obtained spontaneously under mild control conditions without involving high temperatures, organic solvents, or sonication [30] and the TPP is a multivalent polyanion, with low toxicity and cost, unlike other cross-linkers, it presents no severe constraints of handling and storage. After adding TPP solution, nanoparticles form immediately through inter and intramolecular linkages created between TPP phosphates and CTS amino groups [4]. Generally, in this method nanoparticles are prepared by addition of TPP solution (pH 7–9) into an acidic solution (pH 4–6) of chitosan [31].
The particle size and surface zeta potential can be manipulated by variation of the development conditions such as CTS:TPP ratio, CTS concentration and pH solution [16], [32], [33]. Since there are different kinds of CTS, with distinct deacetylation degrees and molar weights, it is very important to study, with an experimental design, which is the best condition in order to obtain nanoparticles with size and superficial charge appropriate.
Optimization of experiments, such as those used in nanoparticles development, can lead to useful savings of scientific resources. Factorial experimental designs are commonly used to optimize experiments and discover which factors influence the outcome of the experiment and what levels of these factors lead to a test with the better response [34].
There are a few works in the literature [35], [36] that demonstrate how to obtain nanoCTS by ionic gelation method with desired characteristics using a factorial design, however, these works present nanoCTS with diameter greater than 100 nm.
This study aims to investigate the influence of acetic acid concentration, pH and ratio of CTS:TPP in the size and zeta potential of the nanoparticles obtained through ionic gelation. The nanoparticles formulation was optimized using a 23 factorial design (FFD), with 8 experiments (in duplicate), to analyze the effects of the three selected factors.
Section snippets
Materials
CTS with deacetylation degree of 81.9% and molar mass of 111.01 kDa was supplied by Purifarma (Brazil), sodium tripolyphosphate and potassium bromide from Sigma–Aldrich (USA), sodium hydroxide and glacial acetic acid were obtained from Synth (Brazil).
Analysis of different ratios of CTS:TPP
NanoCTS were produced according ionic gelation technique described by Calvo, Remuñán-López, Vila-Jato and Alonso [30]. CTS solution, with concentration of 2.5 mg mL−1, was prepared by dissolving the polymer with sonication in 1% (w/v) acetic acid
Analysis of different ratios of CTS:TPP
The use of chitosan for nanoparticles formation was available, as preliminary study. In this way, different formulations composed of CTS and TPP were evaluated. The formulations were easily prepared based on the ionic gelation of positively charged amino groups of CTS with TPP anions.
Observing in Fig. 1, there is a tendency to increase the zeta potential of the nanoparticles with the increment of the CTS concentration, which is attributed to the increase of NH3+ protonated groups of CTS, as
Conclusion
The analysis of different ratios of CTS:TPP showed that the 3:1 proportion is the best for nanoCTS production. The development of nanoCTS was analyzed by using a 23 factorial design, which considered simultaneously three main factors: pH, CTS:TPP ratio and acetic acid concentration. The parameter that had a significant influence in the size and zeta potential of the nanoparticles was the ratio between CTS and TPP. The best conditions to form the particles were pH 4.4, CTS:TPP of 3:0.8 and 0.1 M
Acknowledgements
The authors thank CAPES for their financial support and scholarship and the Central Laboratory of Electron Microscopy (LCME) at Federal University of Santa Catarina (UFSC) for EDX and TEM analysis.
References (46)
The cellular basis of chitin synthesis in fungi and insects: common principles and differences
Eur. J. Cell Biol.
(2011)- et al.
Chitosan from Mucor rouxii: production and physico-chemical characterization
Process Biochem.
(2005) - et al.
Preparation of aspirin and probucol in combination loaded chitosan nanoparticles and in vitro release study
Carbohydr. Polym.
(2009) - et al.
Chitosan/beta-lactoglobulin core–shell nanoparticles as nutraceutical carriers
Biomaterials
(2005) - et al.
Saponin-loaded chitosan nanoparticles and their cytotoxicity to cancer cell lines in vitro
Carbohydr. Polym.
(2011) - et al.
In vivo evaluation of curcumin nanoformulation loaded methoxy poly(ethylene glycol)-graft-chitosan composite film for wound healing application
Carbohydr. Polym.
(2012) - et al.
Synthesis and characterization of chitosan and silver loaded chitosan nanoparticles for bioactive polyester
Carbohydr. Polym.
(2011) - et al.
Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N,O-carboxymethyl chitosan nanoparticles
Carbohydr. Polym.
(2009) - et al.
Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique
Colloids Surf. B
(2012) - et al.
Preparation and evaluation of warfarin-β-cyclodextrin loaded chitosan nanoparticles for transdermal delivery
Carbohydr. Polym.
(2012)