Comparative study of preparation, characterization and anticandidal activities of a chitosan silver nanocomposite (CAgNC) compared with low molecular weight chitosan (LMW-chitosan)

Abstract

A chitosan-silver nanocomposite (CAgNC) was synthesized in a green manner using low molecular weight chitosan (LMW-chitosan) and silver nitrate without applying external chemical reducing agents. The newly synthesized CAgNC was characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), field emission electron microscopy (FE-SEM and FE-TEM), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), particle size and zeta potential analysis. The average sizes of LMW-chitosan and CAgNC were 1776 ± 23 nm and 240.1 ± 23.6 nm, respectively. The zeta potential of CAgNC was observed as +41.1 mV. The AgNPs that are deposited on the chitosan matrix had average size ranges between 5 and 50 nm. The Ag content of the CAgNC was determined as 0.696 ± 0.054% (w/w). The minimum inhibitory concentration (MIC) values of LMW-chitosan and CAgNC against Candida albicans were determined as 100 and 50 μg mL⁻¹, whereas the minimum fungicidal concentration (MFC) values were recorded as 400 and 150 μg mL⁻¹, respectively. Propidium iodide (PI) uptake results suggested that CAgNC affected the permeability of the cell membrane of C. albicans. Moreover, CAgNC induced the level of reactive oxygen species (ROS) at a higher level when compared to the LMW-chitosan, in a concentration-dependent manner. This report illustrates the eco-friendly approach for the reduction of silver ions using LMW-chitosan as a reducing agent to make a biologically active composite (CAgNC) and as a potential antifungal agent against C. albicans.

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1. Introduction

Chitosan is a natural cationic polysaccharide that consists of co-polymers of glucosamine (β 1-4-linked 2-amino-2-deoxy-d-glucose) and N-acetyl glucosamine (2-acetamido-2-deoxy-d-glucose). Chitosan is derived from partial deacetylation of chitin obtained from crustaceans or the mycelium of fungi.¹ Bacteriostatic² and fungistatic effects³ due to reactive amino groups and metal ion chelating activity associated with linear polyamine (poly-D-gulcosamine) structures are the main functional properties of chitosan. Recently, applications of chitosan have extended to various fields such as medicine, food, chemical engineering, pharmaceuticals, nutrition, environmental protection and agriculture.⁴ In particular, the antifungal and antibacterial activities of chitosan have been investigated against wide range of pathogenic strains.⁵ The size and zeta potential of chitosan particles are critical properties when considering its bioactivities.⁶

Nanosilver (silver nanoparticles, AgNPs, or Ag⁰_nano) is considered as zero valent silver (Ag⁰) having a less than 100 nm of particle diameter. AgNPs are commonly synthesized through chemical reduction methods in which silver salts, such as AgNO₃,⁷ or silver perchlorate/AgClO₄,⁸ can be reduced by reducing agent like glucose,⁷ or sodium borohydride/NaBH₄.⁹ In function, AgNPs displays stronger, longer-term, and broader spectrum of antimicrobial activities when compare with other metallic nano particles.¹⁰ Meanwhile coating agents or stabilizers, such as polysaccharides,¹¹ poly vinyl alcohol/PVA,¹² poly ethylene glycol/PEG,^13,14 or citrate,¹⁵ are generally used to prevent aggregation of AgNPs. To achieve better biomedical performances AgNPs, many researches have tested polymer based composite materials combined with AgNPs.¹⁶

Moreover, polymer embedded AgNPs have been shown superior characteristics such as longer stability, better dispersion and low toxicity levels. Chitosan-silver nano composite (CAgNC) is one of the composite materials which can be synthesized via electrochemical,¹⁷ chemical,¹⁸ green synthesis,^19,20 and biosynthesis methods.²¹ It possesses antimicrobial activity,^22,23 bio sensing potential,²² and dye oxidation properties.²⁴ In recent years, severe fungal infections have caused increasing morbidity and mortality among immunocompromised patients who need intensive treatments.²⁵ C. albicans is the most widespread species among other Candida species such as C. tropicalis, C. glabrata, C. kruisei.²⁶ Therefore, it is an urgent need for development of new and non-toxic antifungal agents against, C. albicans.

Our main objective of the present study was to compare the physiochemical properties and anticandidal properties of CAgNC with its precursor LMW-chitosan. For that we firstly prepared the CAgNC using LMW-chitosan and determined the physiochemical properties such as particle size, zeta potential, UV-vis absorption, FE-SEM, FE-TEM and XRD. In order to make functional comparison, antifungal activity against C. albicans was assessed under various parameters such as MIC, MFC, cell viability, change of cell membrane structure, capacity of ROS production and PI uptake. Based on the results and interpretation of possible mode of action, we conclude that newly synthesized CAgNC has superior antifungal activities than LMW-chitosan.

2. Experimental section

2.1 Synthesis and characterization of CAgNC from LMW-chitosan

CAgNC was green synthesized by reduction method²⁷ using LMW (50–150 kDa)-chitosan with a deacetylation degree of ∼85% (Sigma-Aldrich, USA). In brief, the CAgNC was synthesized by adding 4 mL of freshly prepared 0.01 M AgNO₃ solution (Sigma Aldrich, USA) followed by addition of 400 μL of 0.5 M NaOH solution (Biosesang, Korea) to 100 mL of 0.2% (w/v) LMW-chitosan solution with constant stirring at 95 °C. The formation of AgNPs was indicated by the appearance of a yellow color about 1 min after the addition of the NaOH solution. After 15 min, the resulting suspension was filtered and washed several times using distilled water and then dried at 60 °C for 6 h. In order to confirm the formation of AgNPs, UV-vis spectroscopy was carried out using double beam UV-vis spectrophotometer (Mecasys, Korea), over a range of 300–800 nm. The percentage of Ag in CAgNC was determined using an ICP-AES (Perkin-Elmer Optima, USA). FT-IR spectra was recorded in the wavelength region 4000 to 600 cm⁻¹ using Bio-Rad 175C FTS spectrophotometer in Attenuated Total Reflectance (ATR) mode. The surface morphology was examined by FE-SEM analysis (Hitachi S-4800, Japan) operating at an accelerating voltage of 3.0 kV. The particle size and shape of the CAgNC was analyzed using FE-TEM, (Model Tecnai G2 F30 S-Twin, FEI, USA) operating at 300 keV. The phase analysis was done by observing the SAED pattern to confirm the crystal structure of CAgNC. X-ray diffraction (XRD) is a versatile, non-destructive analytical method for the identification and quantitative determination of various crystalline phases. Powder XRD analysis was conducted via Philips PW 1710 diffractometer with Cu Kα radiation (λ = 1.5406 Å) and graphite monochromator, operated at 45 kV; 30 mA and 25 °C. Particle size distribution and zeta potential of CAgNC and LMW-chitosan were determined by Zetasizer S-90 Malvern instruments (Malvern, UK) using diluted and dispersed solution of CAgNC in 0.25% (v/v) acetic acid.

2.2 Analysis of anticandidal activities of CAgNC and LMW-chitosan

MIC and MFC of CAgNC and LMW-chitosan against C. albicans were determined via turbid metric assay as described previously.²⁸ The different concentrations of CAgNC and LMW-chitosan (25, 50, 75, 100, 150 and 200 μg mL⁻¹) were added to 4 mL of potato dextrose broth (PDB, Difco-USA) with C. albicans at 0.05 OD: 600 nm (10⁵ CFU mL⁻¹) and incubated at 30 °C while shaking at 150 rpm for 24 h. The corresponding control test was carried out without CAgNC and LMW-chitosan, whereas the positive control was conducted with 10 μg mL⁻¹ nystatin. All experiments were carried out in triplicates.

2.3 Determination of ROS production and cell viability

To determine the ROS production and the cell viability in C. albicans culture (0.05 OD, 600 nm) with different concentrations of CAgNC and LMW-chitosan (0 to 100 μg mL⁻¹) was kept in a shaking incubator for 6 h at 30 °C. ROS generated cells were stained with 30 μg mL⁻¹ 5-(and-6)-carboxy-2′,7′ dichloro dihydro fluorescein diacetate (H₂DCFDA) followed by 30 min incubation and harvesting by centrifugation at 13 000 rpm for 2 min. Cells were washed and dissolved using ×1 PBS to quantify ROS generation using the FACScaliber flow cytometer (Becton Dickinson, USA). Cell viability was determined by MTT assay. Briefly, after 24 h incubation period, the samples were treated with 70 μg μL⁻¹ of MTT solution (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and incubated for additional 30 min. Harvested cells were re-suspended in DMSO (200 μg per μL per well) and cell viability was detected at OD 570 using a micro plate reader (Thermo, USA) attached to a computer.

2.4 Effect on CAgNC and LMW-chitosan on plasma membrane of C. albicans by PI assay

Cell membrane integrity of CAgNC and LMW-chitosan treated C. albicans was assessed by monitoring the uptake of the fluorescent probe, PI (Sigma Aldrich, USA). For the determination of the PI uptake, cell suspensions of the control, MIC and MFC levels treated samples were centrifuged (3500 rpm, 2 min) and the pellets were re-suspended in PBS. The treated cells were incubated with PI (5 μg mL⁻¹) at 30 °C for 15 min in dark. Over staining were washed twice with PBS. Finally, one drop of each suspensions was placed on the cover slip and observed using a Zess LSM 510 meta confocal laser scanning microscope (CLSM) scan head integrated with the Axiovert 200 M inverted microscope (Carl Zeiss, Jena, Germany). C. albicans cells were observed through a 40 × 1.3 oil objective and PI was excited with the 543 laser line and the emission was recorded through a 585 long-pass filter.

2.5 Statistical analysis

All the data related to the cell viability was illustrated as means ± SD for triplicate reactions. Statistical analysis was performed using unpaired, two-tailed t-test to calculate the P-value using GraphPad program (GraphPad Software, Inc.). The significant difference was defined at P < 0.05.

3. Result and discussion

3.1 Synthesis and characterization of CAgNC

Present study describes the use of LMW-chitosan with AgNPs to develop biologically active and superior anticandidal agent against C. albicans. The first part of this work is synthesis of CAgNC using LMW-chitosan and physiochemical characterization. The progress of the AgNPs synthesis was tracked by using UV-vis spectroscopy. The UV-visible absorption spectra of LMW-chitosan and CAgNC are shown in Fig. 1. The spectra exhibited an absorption band around 415 nm for CAgNC. However, there is no specific absorption spectrum observed in LMW-chitosan. The surface plasmon resonance (SPR) band of spherical AgNPs was observed around 420 nm and it clearly evidences for the formation of AgNPs as previously reported.²⁹ To convert the Ag⁺ into metallic Ag, an electron supplier or a reducing agent should be added. When NaOH is added to an AgNO₃ aqueous solution, the pH of the solution can be increased and Ag₂O is precipitated as solid mass.³⁰ However, in this study, a solid gray precipitate of Ag₂O was not formed because Ag⁺ stabilized by the basic chitosan suspension. Then Ag⁺/chitosan complex has allowed Ag⁺ to collect electrons from the basic suspension and to be reduced to an Ag atom. Twu et al.,²⁰ has suggested the greater probability of supplying electron by degradation products of low-molecular weight chitosan (e.g. glucosamide) and functioning as a reducing agent.

Fig. 1 Product of CAgNC and UV-visible spectroscopy analysis. (A) Formation of AgNPs on chitosan matrix which develops yellow color. (B) UV-vis spectrum of AgNPs presenting an absorption peak at 410 nm due to surface plasma resonance (LMW-chitosan as precauser).

We studied the chemical interaction between LMW-chitosan and Ag in the CAgNC matrix by FT-IR spectral analysis. Results shows the FT-IR spectrum of CAgNC indicating the band at 3366 cm⁻¹ which confirm the stretching vibrations of –OH and –NH groups (Fig. S1†). Moreover, additional bands were displayed at 2871 cm⁻¹, 1645 cm⁻¹, 1375 cm⁻¹, 1060 cm⁻¹ which are ascribed to the asymmetric stretching vibrations of –CH group, amide group (C–O stretching along-N–H deformation), COO⁻ group carboxylic acid salt, and stretching vibrations of C–O–C in the glucose unit, respectively. The LMW-chitosan shows all the corresponding bands of CAgNC. However, the spectrum of the CAgNC was shifted towards lower wave numbers (amine group was shifted from 1658 cm⁻¹ to 1645 cm⁻¹) when compare with the spectrum of LMW-chitosan. This suggests the attachment of Ag into N atoms (amino groups), which reduces the vibration intensity of the N–H bond due to the greater molecular weight of CAgNC due to the incorporation of Ag atoms in to LMW-chitosan as described previously.³¹

The surface morphology of the synthesized CAgNC and LMW-chitosan was analyzed using FE-SEM and images are presented in Fig. 2A and B. The FE-TEM image (Fig. 2C) implies the presence of spherical AgNPs in the chitosan suspension. Also, it clearly indicated that AgNPs were deposited on chitosan matrix and the average particle size of AgNPs lies between 5 and 50 nm. The three diffraction patterns observed in the selected area of electron diffraction (SAED) pattern are shown in Fig. 2D, and it can be indexed to a face centered cubic lattice. The first strongest ring is the combination of both (111) and (200) planes, whereas the second ring corresponds to the crystallographic plane of (220). The third ring represents the (311) plane of Ag. The SAED pattern was completely aligned with the XRD pattern.

Fig. 2 The FE-SEM and FE-TEM image of CAgNC. (A) FE-SEM image of CAgNC (B) FE-SEM image of LMW-chitosan (C) FE-TEM image of CAgNC (D) SEAD pattern of CAgNC.

XRD is a versatile and non-destructive analytical method for the identification and quantitative determination of various crystalline phases. The structural properties of CAgNC were analyzed using XRD technique. XRD analysis results revealed that pattern of CAgNC was clearly differed from that of LMW-chitosan (Fig. 3). The peak for LMW-chitosan was appeared at 2θ value of the broad peak around 5–25° (Fig. S2†). The XRD pattern of powdered CAgNC showed Bragg reflections with 2θ values of 38.12, 44.22, 64.36 and 77.32 for a set of lattice planes which could be indexed to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic geometry of Ag and the existence of broad peak between 5–25° which can be attributed to the presence of LMW-chitosan in the CAgNC. The results showed that the synthesized CAgNC contains AgNPs in crystalline structure, since the position and the relative intensity of all the diffraction peaks of the samples were consistent with the crystalline pattern of Ag.³² The lattice parameters were determined to be a = 4.0580 that matches with the Joint Committee on Powder Diffraction Standards (JCPDS) file no. 87-0720. There were no additional peaks in the spectra, indicating the purity of CAgNC sample and no detectable impurities present.

Fig. 3 The XRD graph of the CAgNC.

The particle size distribution of LMW-chitosan and CAgNC was determined using Zetasizer Nano-ZS90. The analysis was performed in triplicates for each sample and presented as mean ± standard deviation (SD) in Table 1. Agreeing to the result of this analysis, the average size of LMW-chitosan and CAgNC were 1776 ± 23 nm and 240.1 ± 23.6 nm, respectively (Fig. S3A and B†). Zeta potential of synthesized CAgNC measured at pH = 4.6 was found as +41.1 mV (Fig. S3C†). The value of zeta potential enables determination of colloid stability and particle aggregation.³³ Therefore, the positive value of the zeta potential of CAgNC could have evidenced the presence of positively charged polymeric layer on AgNPs surface.

Table 1 Comparison of particle size, zeta potential, MIC and MFC of LMW-chitosan and CAgNC

Compound name	Particle size (nm) (mean ± SD)	Zeta potential (mV) (mean ± SD)	MIC (μg mL^−1)	MFC (μg mL^−1)
LMW-chitosan	1776 ± 23.00	—	100	400
CAgNC	240.1 ± 23.26	+41.6 ± 4.64	50	150

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3.2 Anticandidal of LMW-chitosan and CAgNC

After characterization of CAgNC, we investigated the antifungal activity against C. albicans. The synthesized CAgNC showed superior antifungal activity against C. albicans compared to LMW-chitosan. It was found that MIC and MFC of LMW-chitosan as 100 400 μg mL⁻¹, respectively (Table 1).

Interestingly CAgNC showed significantly lower MIC of 50 μg mL⁻¹ (2 times lower than LMW-chitosan) and MFC of 150 μg mL⁻¹ (2.7 times lower than LMW-chitosan), respectively. Ing et al.,³⁴ showed that LMW-chitosan solution has higher MIC₉₀ value (3 mg mL⁻¹) compare with chitosan nano particle (0.25 mg mL⁻¹) against C. albicans. Panacek et al.,²⁵ showed that MIC of stabilized AgNPs varied from 0.052 to 0.84 mg L⁻¹ with Candida sp. The size of particles plays an important role in determination of antimicrobial activity of nanoparticles as they enter the cell walls of microbes through carrier proteins or ion channel and smaller nanoparticles result in a better uptake into microbial cells.⁶ Zeta potential has been suggested as a key factor that is contributing to antifungal effect of chitosan through the interaction with negatively charged microbial surface.³⁵ The synthesized CAgNC has shown higher anticandidal activity because of its low particle size and higher zeta potential value when compare with LMW-chitosan. The mode of action of CAgNC against C. albicans is not fully understood and therefore further investigations are required to establish in future.

Recent study³⁶ suggested that the accumulation of ROS induces and regulates the apoptotic pathway in yeast. Thus, to examine the relationship between the accumulation of ROS and the induction of apoptosis, an experiment was conducted to find out the effect of different concentration of LMW-chitosan and CAgNC on the ROS production and cell viability in C. albicans. ROS level was slightly increased until 75 μg mL⁻¹ and beyond 100 μg mL⁻¹ (for LMW, MIC is 100 μg mL⁻¹) in LMW-chitosan treated (12.5, 25, 50 and 75 μg mL⁻¹) C. albicans (Fig. 4). Furthermore, C. albicans samples which were treated with 12.5 μg mL⁻¹ and 25 μg mL⁻¹ concentrations of CAgNC have demonstrated sequentially increased ROS levels. Also, at 50 μg mL⁻¹ (MIC of 50 μg mL⁻¹ for CAgNC) a steep increase was observed while it slightly increased again at 100 μg mL⁻¹. Moreover similar ROS values were obtained both control and acetic acid treated samples as well as for positive control (10 mM H₂O₂) treated samples. Further, ROS result showed slight increased value for CAgNC treated sample comparing to the LMW-chitosan. The reason for such observation could be that AgNPs have the capacity to inhibit the C. albicans by increasing the oxidative stress.

Fig. 4 Effect of LMW-chitosan and CAgNC on ROS production in C. albicans cells. (A) LMW-chitosan (B) CAgNC. 0.25% (v/v). AC: acetic acid (negative control), 10 mM H₂O₂ (positive control).

The overall results suggest that LMW-chitosan and CAgNC trigger the oxidative stress by generating ROS which causes various damages to macromolecules such as DNA, RNA, and proteins as well as other cellular components.^36,37 The production of ROS may be interfered with the essentials of electronic transport chain which may cause the reduction of cellular energy production.³⁸ Additionally, the excessive production of ROS may damage plasma membrane and intra cellular organelles which may leads to cell death.³⁹ The cell viability of C. albicans was decreased significantly (P < 0.001) with increasing the concentration of LMW-chitosan and CAgNC (Fig. 5). Furthermore, highest and lowest cell viability in LMW-chitosan treatment was observed in control and H₂O₂ treated groups, respectively. Whereas highest and lowest cell viability was observed in control and 100 μg mL⁻¹ of CAgNC treated group, respectively. All the CAgNC treated groups were shown lower cell viability than LMW-chitosan. However, significant difference (P < 0.05) in cell viability was observed in CAgNC and LMW-chitosan treatments from 12.5–100 μg mL⁻¹ concentration. Thereby, lowest cell viability (28%) was observed in 100 μg mL⁻¹ CAgNC treatment. Whereas, cell viability for positive control was 34% at the 10 mM H₂O₂ and negative control 100% at 0.25% AC.

Fig. 5 Comparison of the effect of LMW-chitosan and CAgNC on cell viability of C. albicans. Cell viability was assessed by MTT assay (n = 3) after treatment with different concentration of LMW-chitosan and CAgNC (12.5–100 μg mL⁻¹). Significant differences in C. albicans cell viability were obtained with respect to untreated control (P ≤ 0.05). The treatments with * mark represent the significant cell viability (%) between LMW-chitosan and CAgNC. Bars with no asterisk were not significantly difference in cell viability. Acetic acid 0.25% (v/v) as negative control and, 10 mM H₂O₂ as positive control.

The PI uptake result is associated with the occurrence of substantial damage to the membrane, indicating alteration of cell membrane potential, which finally causes cell death. PI could enter the cell and bind to DNA, showing red fluorescence.⁴⁰ PI uptake by C. albicans cells show concentration dependent mortality in both treated groups where control and MIC treatment have the least number of PI stained C. albicans cells which indicates the least number of cell death (Fig. S4†). However, almost all C. albicans cells in both treatments at the MFC level have shown higher red florescence (Fig. 6). When cationic chitosan bind to the negatively charge cell surface it may cause to increase hyperpolarization of the plasma membrane.⁴¹ Also, some amount of AgNPs on the surfaces could be ionized and produce cationic silver (Ag⁺) traces. This Ag⁺ traces flowing in to the cell with the cationic influx generated due to hyperpolarized cell membrane.⁴²

Fig. 6 Effect of LMW-chitosan and CAgNC on cell membrane permeability by PI staning. Merged image of C. albicans (by confocal laser scanning microscopy) showing the dead C. albicans cells at MFC treatment level. (A) LMW-chitosan (400 μg mL⁻¹) (B) CAgNC (150 μg mL⁻¹).

4. Conclusions

In summary, we synthesized the CAgNC using LMW-chitosan without external chemical reducing agent and compared their physiochemical properties and anticandidal action. First we prepared the improved version of chitosan nano composite format with unique characteristics such as smaller particle size (240.1 nm) higher zeta potential (+41.1 mV) and lower amount of AgNPs (0.69%). Moreover, CAgNC had superior anticandidal activities (MIC 50 μg mL⁻¹, MFC 100 μg mL⁻¹), than the precause LMW-chitosan suggesting that it has great potential to be developed as antifungal agent against wide array of Candida species.

Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A2A1A11054585), research fund of Chungnam National University and part of the project titled ‘Development of Fish Vaccines and Human Resource Training’, funded by the Ministry of Oceans and Fisheries, Republic of Korea.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03917k

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