Factors affecting the antibacterial activity of chitosan‐silver nanocomposite

Abstract

This study provides the optimum preparation parameters of chitosan‐silver nanoparticles composite (CSNC) with promising antibacterial activity against the most common bacterial infections found on burn wounds. CSNC was synthesised by simple green chemical reduction method with different preparation factors. Chitosan was used to reduce silver nitrate and stabilise silver nanoparticles in the medium. For this reason, spectroscopic and microscopic techniques as, ultraviolet‐visible Fourier transform infrared spectroscopy and transmission electron microscopy were used in the study of the molecular and morphological properties of the resultant composites. Furthermore, the composite was assessed in terms of Ag‐ions release by AAS and its efficacy as antibacterial material. As a result, CSNC showed stronger antibacterial effect than its individual components (chitosan and silver nitrate solutions) towards Gram‐positive (Staphylococcus aureus) and Gram‐negative (Pseudomonas aeruginosa and Escherichia coli) bacteria. CSNC prepared in this study showed highest inhibition percentage of bacterial growth up to 96% at concentration of 220 μg/ml.

1 Introduction

Chitosan is a polymeric 1–4 linked 2‐amino 2‐deoxy‐ß‐D glucose [1]. Chitosan is a transformed polysaccharide prepared by deacetylation of natural chitin, which is one of the important natural polymers constituting the shells of crustaceans and the cell wall of many fungi [2]. Chitin and chitosan production is currently based on discard waste crab and shrimp shells by the canning industries in Oregon, Washington, Virginia and Japan and by various finishing fleets in the Antarctic [3]. The importance of chitosan as a natural polymer is increasing due to its unique properties such as biodegradability, biocompatibility, bioactivity and attractive physical, and mechanical properties [4]. The antimicrobial activity of chitosan has been well documented and its antibacterial activity has been widely explored as well [5, 6, 7]. Novel α‐chitin/nanosilver composite scaffolds for wound healing applications have been recently developed by Madhumadi et al. [8]. Chitosan is reported as reducing and capping agent in many references. This action may be attributed to the expected electrostatic interaction between OH/˗NH₂ groups of chitosan and electropositive metal cations [9].

Anciently, silver has been extensively studied and used to fight against infections and prevent spoilage [10]. Currently, the antibacterial and multi‐functional properties of Ag nanoparticles have attracted many researchers. That is due to large surface area/volume ratio, high catalytic capabilities and ability to generate reactive oxygen species (ROS) [11, 12, 13, 14] beside the growing microbial resistance against antibiotics and the development of resistant strains [15, 16]. Antibacterial activity of Ag nanoparticles was suggested in reducing infections when treating burns as well as in preventing bacterial colonisation of prostheses and catheters [17, 18, 19, 20]. Ag nanoparticles are reported as non‐toxic and non‐tolerant disinfectant [10, 21, 22, 23]. Argyria (skin discoloration) and argyremia (elevated silver concentration in blood) may be reduced safely using Ag nanoparticles [24, 25]. Ag nanoparticles exhibited considerable safety due to their minimal penetration into skin [26].xxx

The antibacterial and catalytic activities of the Ag‐nanoparticles are dependent on their size, structure, shape and size distribution as well as chemical‐physical environment [27]. The biocompatibility of the Ag‐nanoparticles can be improved efficiently and reliably by reducing their particles size [28].

It's believed that Ag‐ions released in aqueous solution of silvers and nanosilver interact with three main components of the bacterial cell; the peptidoglycan cell wall [29], the plasma membrane [28]; cytoplasmic DNA [30, 31] and proteins [25, 29, 30]. In general, Ag ions are known to interact with a number of electron donor functional groups like thiols, phosphates, hydroxyls, imidazoles, indoles and amines [32, 33].

The antimicrobial dressing materials based on silver impregnated or surface coated with silver membranes are reported to remove 99% pathogens [34]. The United States Food and drug administration (USFDA) and the European Food Safety Authority (EFSA) have approved many silver‐based commercial products for wound dressing and antibacterial applications [35]. The most common pathogens on infected wound burn producing biofilms such as Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae and Enterobacter sp. [36, 37]. Synthesis of Ag nanoparticles by chemical reducing agents often involves absorption of harsh chemicals on the surfaces of nanoparticles raising their toxicity. Also, Ag nanoparticles have a tendency to agglomerate, so the unique properties associated with the nanosize are lost [38]. In our previous work, extensive spectroscopic study on several parameters regarding the synthesis protocols of CSNC was investigated [39]. This study is a complementary work to investigate the efficiency of the optimised CSNC as antimicrobial material. It would be able to overcome the antibiotic resistance of microorganisms for skin burn wound treatment.

In this work, chitosan is used in preparation of CSNC as a reducing, stabilising and capping agent. Different preparation factors (AgNO₃ : Chitosan ratios, reduction temperature and time) are used in order to investigate their corresponding structural properties and release behaviour. The antibacterial activity of CSNC are studied against the most common bacterial strains, found on infected burn wounds, such as Staphylococcus aureus, P. aeruginosa and E. coli.

2 Materials and methods

2.1 Materials

High molecular weight Chitosan powder with practical grade > 75% decetylation, molecular weight range from 31,000 to 37,000 and the viscosity range from 800 to 2000mPas (Sigma‐Aldrich, St. Louis, MO) is used for preparation of chitosan‐Ag nanoparticles composite. Silver nitrate salt and acetic acid were of analytical grade and supplied by SRL Company, India. Solutions were prepared with de‐ionised water.

2.2 Preparations

CSNC was prepared using chemical reduction method [3]. Stock of chitosan solution was prepared by dissolving 6.92 g of chitosan in 1 l of (1%) acetic acid solution. Stock of Ag nitrate solution was prepared by dissolving 8.84 g of Ag nitrate salt in 1 l of de‐ionised water. Each solution was stirred overnight at 25°C till homogenous solution was reached. A series of mixtures of the two stocks was prepared using different AgNO₃ : Chitosan ratios; 1:3, 1:2, 1:1, 2:1 and 3:1(v/v) producing AgNO₃ final concentration 2.21, 2.95, 4.42, 5.89 and 6.63 mg/ml, respectively. The mixture was stirred overnight at 25 ± 1°C then it was kept at 70°C, 80°C or 90°C for 12 h or 16 h according to the desired conditions for the reduction process.

Thin films (0.01‐0.05 mm) of the obtained CSNC were prepared by casting in petri dish and dried at room temp (21 ± 1°C). These films were used in order to assess the Ag‐ions release behaviour.

2.3 Characterisation

CSNC was characterised by UV‐visible spectrophotometer Model V‐570 operating in the absorption mode in the range of 200–1000 nm. FTIR spectra were recorded on a Fourier‐transform infrared instrument (JASCO FTIR‐400 Japan) in the spectral range of 4000–400 cm⁻¹. Transmission electron microscope (TEM) images were taken by using JEOL JEM 2010 transmission electron microscope. The revolution 4pi‐Analysis‐v1.6.0b195 program was applied to the transmission electron microscopy (TEM) micrographs to measure the nanosize of the particles. Ag‐ions released were determined by Flame Atomic Absorption Spectrometer, A.A.S., (Varian Australia, SPECTRAA 220).

2.4 Release test

To mimic the Ag‐ions release in the physiological environment of the superficial wound burn, the CSNC films (2 × 2 cm²) were incubated in distilled water (50 ml) of pH 6.9‐7 at 37°C. After 15 min, the film pieces were removed and placed into new fresh 50 ml distilled water (pH 6.9‐7). The above step was repeated by incubation at 37°C for 0.25, 0.50, 1, 2, 3, 6, 9, 12, 24 and 38 h. The Ag‐ions concentrations of the samples were measured by A.A.S. The measurements were carried out in triplicates.

2.5 Bacterial stains

Three bacterial species were used; Gram‐positive {Staphylococcus aureus (ATCC 25923)} and Gram‐negative {P. aeruginosa (ATCC 27853) and E. coli (ATCC 35218)}. These bacterial strains were maintained on nutrient broth and were kindly provided by Department of Zoonotic Diseases, National Research Center, Egypt.

2.6 Antibacterial assay

The bacterial suspension was prepared and adjusted by comparison against 0.5 Mc‐Farland turbidity standard (5 × 10⁷ cells/ml) tubes. It was further diluted to obtain a final of 5 × 10⁶ cells/ml. These bacterial strains were enriched on nutrient broth as well as on selective broth for bacterial propagation [40]. The broth was inoculated by the 0.20 μl/10 ml broth Staphylococcus aureus, P. aeruginosa and E. coli, and then added 40 μl of CSNC solution [41]. CSNC solution was added to broth inoculated with isolates of different bacterial species. The tubes were incubated at 37°C for 24 h. The turbidity test and Percentage of bacterial growth inhibition were measured by S110 spectrophotometer (WPA LINTON Cambridge UK). The bacterial growth was indicated by optical density (OD) at wavelength 595 nm. The assay was repeated three times for each bacterial strain and the mean values of inhibition were calculated [42, 43, 44].

2.7 Statistical analysis

The results obtained in the present work are represented as mean ± standard error, and were analysed using analysis of variance (ANOVA). The significance of difference between mean values at P  < 0.05 was calculated using the Duncan Multiple Range Test [45].

3 Results and discussion

3.1 UV‐visible spectroscopy

The ultraviolet‐visible (UV‐Vis) spectra of the CSNC (Fig. 1 a) prepared using different AgNO₃ : Chitosan ratios provide strong evidence for the formation of Surface Plasmon Resonance (SPR) peak of Ag nanoparticles. The position and singularity of the SPR band of Ag nanoparticles in the range 420–450 nm suggest that the resultant Ag nanoparticles have spherical shape [14, 46, 47, 48]. The relationship between the concentration of AgNO₃ in the composite samples and the absorbance are plotted in Fig. 1 b with Gaussian fitting curve. The absorbance of the SPR peak increases with increasing the concentration of AgNO₃ up to 4.42 mg/ml (AgNO₃ : Chitosan 1:1) above this concentration the absorbance decreases, rapidly as seen in Fig. 1 b.

Fig. 1.

UV‐VIS spectroscopic study of CSNC prepared with different AgNO₃ : Chitosan ratios. (a) UV‐VIS absorption spectra of CSNC as function of AgNO₃ : Chitosan ratios, (b) The Relationship between the different concentrations of AgNO₃ in CSNC samples and the SPR peak maximum

3.2 FTIR spectroscopy

Detailed investigation of the deconvoluted FTIR spectra (an example spectrum is shown in Fig. 2 a) of all CSNC samples under investigation showed remarkable shift of the OH (3362 cm⁻¹) and NH (3291 cm⁻¹) stretching vibration bands of the chitosan to the lower frequency side to 3327 cm^˗1 and 3268 cm^˗1, respectively. This shift assumes that NH stretching vibration was affected by the attachment of silver [48, 49, 50]. It was notable that the treatment of chitosan with AgNO₃ solution resulted in an appearance of new band at 1760 cm^˗1 which can be assigned to carbonyl stretching vibration indicating that the reduction of Ag‐ions is coupled to the oxidation of the OH group in the chitosan molecules [40, 48, 50]. Also, amid I (1647 cm⁻¹) and amid II (1561 cm^˗1) bands were shifted to the lower frequency side, increasingly with increasing the AgNO₃ : Chitosan ratio as shown in Fig. 2 b. This figure suggests that as the AgNO₃ concentration increases in the salt: chitosan ratio as the attachment of silver to the nitrogen atoms increases leading to red frequency shift due to the heavier molecular weight produced. These are consisted with Wei et al., 2009 and Vimala et al., 2010 [38, 48].

Fig. 2.

FTIR spectroscopic study of CSNC prepared with different AgNO₃ : Chitosan ratios. (a) FTIR spectrum of deconvoluted CNSC with AgNO₃ : Chitosan ratio 1:2, (b) Frequency shift of the amide I and amide II absorption bands in the deconvoluted FTIR spectra of CSNC as function of AgNO₃ : Chitosan ratio

3.3 Transmission electron microscopy

The TEM images of CSNC prepared from AgNO₃ : Chitosan ratios namely 1:3, 1:2, 1:1 and 3:1 at 80°C for 16 h are shown in Fig. 3 and their particles size distribution histogram with Gaussian fitting curve are inset figures. The TEM micrographs show that in case of low ratios (1:3 and 1:2) the Ag nanoparticles are very small (4.6 nm and 7.3 nm), smooth, spherical, loose and freely distributed Figs. 3 a and b, respectively. In case of high AgNO₃ : Chitosan ratios (1:1 and 3:1), the Ag nanoparticles are larger (22.88 and 29.01 nm) smooth, spherical but tend to form clusters Figs. 3 c and d, respectively. We suggested that the increase of chitosan ratio in the composite produce considerable increase in separation dispersion, owing to the protection of sufficient chitosan fragment to Ag nanoparticles. The effect of the reduction temperature on the shape and size of the CSNC produced is illustrated in Fig. 4. It reveals that small spherical particles (p.s. range 2–16 nm with an average ∼7.3 nm) were formed at 80°C. On the other side, 90°C reduction temperature produced much larger particles (p.s. range 57–70 nm with an average 62 nm) but still spherical shaped as well. This is can be explained by the fact that the temperature enhances the rate of reduction. At 70°C and 80°C, the temperature was suitable to initiate and preserve reduction rate to form nucleation. Thus small nanoparticles were produced. Consequently, increasing the temperature up to 90°C the reduction rate was enhanced enough for more particle growth, producing larger nanoparticles [51]. Tran et al. [52] reported the shape and particle distribution are affected by reduction temperature. Fig. 5 shows TEM micrographs of CSNC samples prepared from 1:2 ratio at 80°C for 12 and 16 h, respectively. CSNC prepared at 12 h reduction time produced spherical and hexagonal particles (p.s. range 14–162 nm with an average 88 nm) while those prepared at 16 h produced homogenous spherical nanoparticles (p.s. range 2–16 nm with an average 7.3 nm). This figure indicates that the reduction time played an important role in the shape of the nanoparticles produced.

Fig. 3.

TEM image of Ag nanoparticles prepared at 80°c for 16 h using different AgNO₃ : Chitosan ratios (a) 1:3, (b) 1:2, (c) 1:1, (d) 3:1.

The inset figures show the particle size distribution of each TEM image with Gaussian fitting curve

Fig. 4.

TEM image of CSNC prepared using 1:2 AgNO₃ : Chitosan ratio reduced for 16 h at different reducing temperatures (a) 70°C, (b) 80°C, (c) 90°C

Fig. 5.

TEM image of CSNC prepared using 1:2 AgNO₃ : Chitosan ratio prepared at 80°C for different reducing times (a) 12 h, (b) 16 h

3.4 Atomic absorption spectroscopy

The effect of AgNO₃ : Chitosan ratio, reduction temperature, and reduction time on the Ag‐ions releasing behaviour from the CSNC films, using the atomic absorption spectroscopy (AAS), are illustrated in Figs. 6 a –c, respectively. Fig. 6 a indicates that all samples show initial burst within the 1st hour followed by gradual increase up to 12 h then reaching a plateau up to 36 h. The rate of this behaviour is increasing with the increase of AgNO₃ concentration in the composites. Although the minimum Ag‐ions releasing was obtained from the composite films prepared from 1:2 AgNO₃ : chitosan ratio, this ratio was selected to investigate the effect of reduction temperature and time because of their homogenous distribution as revealed from TEM micrograph. Composite films (Fig. 6 b) prepared at 80°C showed the highest rate of Ag‐ions releasing followed by those prepared at 70°C and 90°C, respectively. This may be attributed to that the amount of Ag nanoparticles formed at 70°C (SPR max 0.15) was less than that produced at 80°C (SPR max 0.30), while CSNC prepared at 90°C produced much more amount (SPR max 0.87) of clustered Ag nanoparticles. Fig. 6 c demonstrates that 16 h reduction time of the CSNC prepared provides maximum rate of Ag‐ions releasing compared to those prepared at 12 h.

Fig. 6.

Silver ion releasing behaviour of the composite films for different

(a) AgNO₃ : Chitosan ratios at 80°C and 16 h, (b) reduction temperatures using the AgNO₃ : chitosan ratio 1:2 at 16 h, (c) reduction times ratio 1:2 at 80°C

3.5 Antibacterial activity

The antibacterial activity of chitosan, AgNO₃ and CSNC solutions toward Gram‐positive (S. aureus) and Gram‐negative bacteria (P. aeruginosa and E. coli) are shown in Fig. 7. The bacterial growth is expressed by the increase of O.D. (Y‐axis) whereas the antibacterial activity is expressed by the decrease of the O.D. Generally, CSNC solutions reveal higher antibacterial effect than their individual components against both G+ve and G−ve bacteria. Results of S. aureus Gram‐positive bacteria show gradual decrease in antibacterial activity as AgNO₃ : Chitosan ratio increases [Fig. 7 a].

Fig. 7.

The bacterial growth expressed by O.D. of untreated, Chitosan‐treated, AgNO₃ ‐treated and CSNC‐treated bacteria prepared by

(a) Different AgNO₃ : Chitosan ratios at reduction temperature 80°C and reduction time 16 h, (b) Different reduction temperatures using AgNO₃ : Chitosan ratio 1:2 and reduction time 16 h, (c) Different reduction time using AgNO₃ : Chitosan ratios 1:2 at reduction temperature 80°C (N.B. The arrow indicates the direction of increase)

CSNC formed at AgNO₃ : Chitosan ratio 1:3 has the highest antibacterial effect on P. aeruginosa while this effect decreases dramatically as the AgNO₃ increase in the ratio. All composite solutions of other ratios also cause decrease in the antibacterial activity but to more or less to constant value compared to the 1:3 ratio. E‐coli represents the least response to the antibacterial effect of all CSNC under investigation, in comparison to the response of S. aureous and P. aeruginosa.

Ag nanoparticles of CSNC composites prepared from low AgNO₃ : chitosan ratios namely; 1:3 and 1:2 revealed maximum antibacterial activity against G+ve and G−ve correlated with the smallest particle size of homogeneous spherical distribution combined with the appropriate concentration, 4.5 nm ± 0.2 (max SPR 0.3) and 7.3 nm ± 0.4 (max SPR 0.36), respectively regardless to the amount of Ag‐ions released. This comes in agreement with literature [1, 38] and can be attributed to the great surface area offered by the much small particles’ size, reaching the nuclear content of bacteria easily [48].

Wei et al. (2009) [48] stated that chitosan‐based Ag nanoparticles have a dual mechanism of action for antibacterial activity, namely, the bactericidal effect of Ag nanoparticles and cationic effect of chitosan. Three most common mechanisms proposed are: (i) gradual release of free Ag‐ions followed by disruption of ATP production and DNA replication, (ii) Ag nanoparticles direct damage to cell membranes, and (iii) Ag nanoparticles and Ag‐ions generation of ROS. Several studies insist that the mode of antimicrobial action of Ag nanoparticles is similar to that of Ag‐ions [53], which contradict to our results. Many authors demonstrate that the small particles mainly in the range of 1–10 nm could enter the cell based on indirect microscopic evidences [14].

Results of Fig. 7 b indicate that CSNC prepared at reduction temperature 90°C has the highest antibacterial activity against Gram‐positive and Gram‐negative bacteria though it has higher particle size 62 nm ± 3.12 and lower Ag‐ions releasing concentration (shown in Fig. 6 b). This finding points to the vital role of the nanoparticles concentration besides to their size in killing the bacteria. Fig. 7 c shows that the composites prepared at 12 h reduction time have higher antibacterial activity, in general. TEM investigation (micrographs) of CSNC prepared at 12 h reveal mixture of spherical and hexagonal Ag nanoparticles (Fig. 5 a), which may take part in the process of bacterial inhibition by different mechanisms according to the nanoparticles shape and size. Fig. 7 c reveals also that the shape of the nanoparticles, produced as a result of the reduction time, has an effective role in the antibacterial activity in addition to their size and concentration. The presence of hexagonal shaped nanoparticles at 12 h (of size range 103–193 nm with average 148 nm) and spherical ones (p.s. 14–162 nm with average 88 nm) in amount relatively 30% less than that of the spherical shape only (at 16 h) produced higher antibacterial activity against P. aeroginosa (36.67%) and E. coli (23.68%).

Series of five concentrations (10 folds dilution) of a selected composites prepared from 1:3 AgNO₃ : Chitosan ratio at 80°C for 16 h, 1:2 AgNO₃ : chitosan ratio at 80°C for 16 h and 1:2 AgNO₃ :chitosan ratio at 90°C for 16 h were examined for their inhibition percentage to G+ve and G−ve bacteria. Results showed that all composites under investigation at the highest concentration 220 µg/ml produced maximum inhibition % in the range 89–96% for all bacteria. The minimum inhibition % range (56–74%) was achieved by the smallest three concentrations 2.2‐0.02 µg/ml for all bacteria.

4 Conclusion

Results of this study indicated that the concentration of AgNO₃ in the solution, reduction temperature and reduction time play a key role in the Ag nanoparticles dispersion, shape, size and size distribution in the preparation of silver nanocomposite. CSNC showed efficient continuous release of Ag+ up to 36 h, that is may be correlated with its stronger antimicrobial effect than that of the individual chitosan or AgNO₃ solutions towards Gram‐positive (S. aureus) and Gram‐negative (P. aeruginosa and E. coil) bacteria. All preparation factors under investigation were optimized to AgNO₃ : Chitosan ratio 1:3, reduction temperature 80°C and reduction time 16 h to obtain a potentially effective antibacterial nanocomposite capable of inhibiting the bacterial growth up to 96% at concentration 220 µg/ml.