Design of Antimicrobial Release Systems Based on Chitosan and Copper Nanoparticles for Localized Periodontal Therapy

Research Article

J Dent & Oral Disord. 2016; 2(7): 1035.

Design of Antimicrobial Release Systems Based on Chitosan and Copper Nanoparticles for Localized Periodontal Therapy

González JP¹, Covarrubias C¹*, Cádiz M¹, Corral C², Cuadra F¹, Fuentevilla I³ and Bittner M3,4

¹Laboratory of Nanobiomaterials, Institute for Research in Dental Sciences, Faculty of Dentistry, University of Chile, Chile

²Restorative Dentistry Department, Faculty of Dentistry, University of Chile, Chile

³Laboratorio de Microbiología Oral y Biotecnología, Faculty of Biological Sciences, University Andrés Bello, Chile

4Laboratorio de Microbiología Oral y Biotecnología, Faculty of Dentistry, Universidad Andrés Bello, Santiago, Chile

*Corresponding author: Covarrubias C, Laboratory of Nanobiomaterials, Institute for Research in Dental Sciences, Faculty of Dentistry, University of Chile, Santiago, Chile

Received: August 10, 2016; Accepted: September 08, 2016; Published: September 09, 2016


Background: The aim of this study was to design an antimicrobial release system for periodontal therapy based on chitosan and copper nanoparticles and assess its in vitro antibacterial activity against Aggregatibacter actinomycetemcomitans.

Methods: Copper nanoparticles were synthesized into a chitosan, starch and ascorbic acid bio-friendly system. Copper nanoparticles/chitosan gel nanocomposites were used to produce solid sponges and gel spheres with 100 μg/mL copper content. The nanocomposite materials were characterized by scanning electron microscopy and attenuated total reflectance with Fourier transform infrared spectroscopy. The antimicrobial activity was tested against A. actinomycetemcomitans by halo inhibition assay on semisolid agar medium. Copper release from the nanocomposites was measured up to 10 days of incubation in artificial saliva at 37°C by analyzing the Cu concentration with a Copper Ion Selective Electrode.

Results: The formation of sponges and gel spheres of chitosan loaded with nanometric copper particles was confirmed. These materials inhibited the growth of A. Actinomycetemcomitans. Sphere nanocomposites presented higher stability in saliva and exhibited a controlled and sustained release of bactericidal copper concentrations.

Conclusions: Copper nanoparticles/chitosan nanocomposites effectively inhibit growth of A. Actinomycetemcomitans and appear as promissory systems for the development of localized periodontal therapies.

Keywords: Copper nanoparticles; Periodontal therapy; Aggregatibacter actinomycetemcomitans; Chitosan


Cunp: Copper Nanoparticles; UV-Vis: Ultraviolet-Visible Spectrophotometry; SEM: Scanning Electron Microscopy; ATRFTIR: Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy; BS: Backscattering; EDX: Energy-Dispersive X-Ray Spectroscopy; ANOVA: Analysis of Variance; SD: Standard Deviation


Periodontitis is one of the most common inflammatory diseases of humans, characterized by progressive destruction of the toothsupporting tissues; which can lead to the loss of teeth [1]. Although, the clinical distinction between chronic and aggressive periodontitis is not clear cut, one of the features of localized aggressive periodontitis is the relatively low level of gingival inflammation compared with chronic forms of periodontitis [2]. Elevated proportions of Aggregatibacter actinomycetemcomitans, and in some cases, of Porphyromonas gingivalis as well are commonly found in patients with aggressive periodontitis [3]. Aggregatibacter actinomycetemcomitans is a Gramnegative, nonmotile, facultative anaerobic cocobacillus bacterium that produces a variety of virulence factors, such as lipopolysaccharide, leukotoxin and Cytolethal Distending Toxin (CDT) and has been found be particularly more resistant to antibiotic treatment [4].

The current periodontal therapy is based on the mechanical removal of bacterial deposits from the root tooth surface [5] and on the use of systemic antibiotics with numerous side effects and development of bacterial resistance [6-8]. Several studies and recent reviews present metallic nanoparticles like silver and copper as a new generation of antimicrobial agents for biomedical applications [9- 13]. Both agents have proven efficacy as antimicrobials over a wide range of pathogens [14], however bactericide properties of copper can be achieved at much less cost than silver [15]. Although zerovalent Copper Nanoparticles (CuNP) exhibit a wide spectrum of antimicrobial activity against different species of microorganisms, including fungi and Gram-positive and Gram-negative bacteria, there are not reports about their activity against dental pathogens. The potential use of CuNP in localized antimicrobial therapies would require regulating the rate of activity in the site of action within an acceptable therapeutic time and concentration [16]. In the case of periodontal disease treatment, it is also desirable that the antimicrobial material have physical properties to be handled and molded into any desired shape and placed to the infected site [17]. Hydrogels such as chitosan have been used in transporting drugs with excellent results. Chitosan is a cationic amino polysaccharide obtained from chitin [18,19], and presents properties such as biodegradability, biocompatibility, non-toxicity, antimicrobial effects, hemostasis, bioadhesivity and it promotes drug absorption [20]. These properties and the ability of chitosan to form flexible physical presentations such as hydrogels, sponges, and microspheres, make it a viable option as a carrying matrix for CuNPs [18,19].

The synthesis of CuNPs is commonly carried out by using relatively toxic reducing agents [21,22], which are no compatible with medical applications. It has been previously optimized the synthesis of CuNP by using starch and ascorbic acid as more biocompatible chemical agents [23]. The appropriate incorporation of these bio-friendly nanoparticles into chitosan matrices could generate new antimicrobial systems for localized periodontal therapy. Chitosan gel doped with silver nanoparticles has demonstrated to have significantly higher antimicrobial activity against Escherichia coli than its components at their respective concentrations [24]. However, the preparation and antimicrobial activity of chitosan composite materials with CuNP have been not reported. Sponges and macrospheres of chitosan doped with CuNP could show better release properties compared to the fluid gel forms as they can behave as stronger and more resistance vehicles and adapt to different physical locations [18,19]

In this work the preparation of a new antimicrobial release systems based on CuNP-chitosan composites in the form of macrospheres and sponges with potential properties for localized periodontal therapy is presented. The antimicrobial activity of the composite is demonstrated against Aggregatibacter actinomycetemcomitans, pathogen often associated with aggressive periodontitis.

Materials and Methods

Preparation of CuNP/chitosan nanocomposites

Preparation of sponge nanocomposites: Sponge composites were produced from a CuNP/chitosan gel. For this purpose, 2.13 g of chitosan and 1.06 g of starch were dissolved in 73.2 mL of 10% ascorbic acid to obtain a viscous gel. Then, 838.4 μL of copper acetate 0.2 M (Cu(CH3COO)2) were added and heated in a microwave for 1 minute in two series of 30 seconds each. Thus, CuNP were in situ synthesized in the chitosan gel. After that, 26 mL of a 0.3% sodium polymetaphosphate /4% sodium hydroxide solution were added to obtain 100 mL of a 100 ppm CuNP/Chitosan crosslinked gel. Sponges were prepared individually placing 5 mL of CuNP/ chitosan crosslinked-gel into 24 well plates. The samples were frozen at -80º C for 24 hours and then freeze dried for 2-3 days to obtain sponge nanocomposites. Neat chitosan sponges were also prepared as control by using the same procedure and replacing the volume of CuNP suspension by distilled water.

Preparation of bead nanocomposites: Bead-shaped composites were prepared by adding 26 ml of distilled water to 73 mL of the CuNP/chitosan gel nanocomposite. Then, the gel was vertically dripped into a 100 mL of a 0.3% /sodium polymetaphosphate 4% NaOH solution by using a 10 mL syringe with a needle of 0.45 mm in diameter. Thus, nanocomposite beads of approximately 1.5 mm in diameter were produced and separated through a stainless steel sieve. Neat chitosan beads were also prepared as control following the same procedure and by using pure chitosan gel.

Synthesis of CuNP suspension

CuNP suspensions were used as control. The synthesis of CuNP was carried out following the procedure reported in a previous work [23]. Briefly, 0.242 g of starch was dissolved in 96.8 mL of ascorbic acid 10% for 1 min in a microwave (600 W). Then, 3.14 mL of copper acetate 0.2 M solution was added and heated in the microwave for 1 minute in two series of 30 seconds each. This procedure was used to obtain 100 ml of 400 ppm CuNP suspension.

Material characterization

The CuNP was identified through the surface plasmon resonance (a physical property of metallic nanoparticles) determined by Ultraviolet-Visible Spectrophotometry (UV-Vis). The size and morphology of CuNP were examined by scanning electron microscopy (SEM) with a Jeol JSM 5410 microscope. Chemical structure of the nanocomposite materials was characterized by Attenuated Total Reflectance with Fourier Transform Infrared Spectroscopy (ATR - FTIR) on an Agilent Cary 630 ATR-FTIR spectrometer. The microstructure of the sponge nanocomposite was characterized by SEM in Backscattering (BS) mode associated with Energy-Dispersive X-Ray Spectroscopy (EDX) for analysis of the chemical composition.

Antimicrobial activity

The antimicrobial activity of the CuNP/chitosan composites was tested against Aggregatibacter actinomycetemcomitans (serotype b). The strain was grown in BHI broth or agar (Brain Heart Infusion, Oxoid, Wesel, Germany) and incubated in a 5% CO2 atmosphere at 37°C for 48 h. From a grown plate, bacteria were transferred to a fresh BHI liquid medium, to a density equivalent to 0.5 McFarland. Then, 1 mL of the bacterial suspension was added to 7 mL of semi-solid BHI agar supported on 20 mL of solid medium. The antibacterial activity of the materials was assessed through the inhibition halo test. For this purpose, each sample (sponges, spheres, suspensions and controls) was individually placed into an approximately 2.5 cm³cavity created in the agar medium. After 48 h of incubation at 37°C in a 5% CO2 atmosphere, the diameters of the halos of bacterial growth inhibition were measured and photographed.

Cu release measurement

The release of Cu ions from the different composite materials was measured up to 10 days of incubation in artificial saliva pH 6.5 at 37°C [25]. Approximately 1 cm³ in volume of each material was immersed in 5 mL of artificial saliva. The Cu ion concentration in the aqueous phase was analyzed at different time intervals with a Copper Ion Selective Electrode (Hanna HI4115). The amount of copper released was calculated as copper mass per material volume (μg/cm³).

Statistical analysis

Statistical analysis of the mean values of the inhibition halos of each material was performed by one-way analysis of variance (ANOVA) followed by multiple comparison Tukey’s test. Statistical significance was set at p < 0.05. Each standard deviation (SD) serves as the estimate for the standard uncertainty associated with a particular measurement.


The UV-vis absorption spectrum of the synthesized CuNP suspension displays a peak centered at 593 nm Figure 1, which correspond to the characteristic surface plasmon resonance of copper particles with nanometric dimensions. The size of the CuNPs, as estimated through SEM, was of approximately 90 nm.