Antibacterial activity and in vitro cytotoxicity evaluation of alginate-AgNP fibers

Abstract

Biopolymer nanocomposites containing metal nanoparticles have attracted much attention due to their excellent properties and broad applications. In this work, alginate fibers embedded with silver nanoparticles (AgNPs) were prepared. The as-obtained alginate-AgNP fibers exhibited antibacterial activity against both Gram microorganisms of model microbes Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). A growth kinetic study with S. aureus and E. coli displayed the inhibition of bacterial growth at the logarithmic phase. The cytotoxic effect of the fibers in human cervical cancer (HeLa) cells was assessed by cell counting kit-8 (CCK-8) assay and flow cytometry. The as-prepared alginate-AgNP fibers, particularly with high amount and long treatment time, showed high cell-killing efficiency. These findings emphasize that such alginate-AgNP fibers with multifaceted biological activities are a promising material for applications in the textile or biomedical fields.

Keywords

alginate fiber silver nanoparticles antibacterial activity cytotoxicity

Silver, both in metallic and ionic form, exhibits strong antimicrobial activities toward many different bacteria, fungi, viruses, and several antibiotic resistant strains and its use as an antibacterial agent is well known.¹ Compared with the bulk form, silver nanoparticles (AgNPs) have an extremely large specific surface area to volume ratio, thus increasing their contact with bacteria or fungi and vastly improving their bactericidal activities.^2,3 Unfortunately, colloidal AgNPs tend to easily aggregate and oxidize, resulting in a significant reduction of the antibacterial efficiency. Therefore, immobilization of AgNPs onto adequate substrates is advantageous because it can effectively inhibit oxidation and aggregation of the immobilized AgNPs.^4,5 There are numerous reports on incorporation of AgNPs into polymers such as poly(vinyl alcohol),^6,7 polyimide,^8,9 cellulose,¹⁰ chitosan,^11,12 etc. The resulting polymer nanocomposites containing AgNPs combine the outstanding characteristics of polymer with the remarkable properties of metal nanoparticles, which make the composites suitable for different unique applications.^13,14 For example, Song et al. reported AgNPs embedded into cationic polymer nanofibers and their antibacterial activity.¹⁵ El-Rafie et al. reported the bio-synthesis and applications of AgNPs onto cotton fabrics.¹⁶ Although the literature reports various studies related to silver nanocomposites with antimicrobial applications in the textile and biomedical fields, few studies concerning addition of AgNPs to alginate fibers have been published.

Alginate, isolated from marine algae, is a copolymer of β-d-mannuronic acid (M) and α-l-guluronic acid (G). Alginate fiber, as bio-based fiber, can be prepared by wet spinning sodium alginate into a coagulating bath containing CaCl₂ aqueous solution. Wet spinning is one of the main spinning methods for chemical fibers. Wet spinning involves the following processes: (1) preparation of spinning solution; (2) solution extrusion from the spinneret hole, forming a trickle; (3) solidification of the liquid trickle into the nascent fibers; (4) roll or post-processing of the primary fiber. The preparation and application of alginate fibers are springing up in the field of high value textile and medical materials due to their superior properties, including excellent biocompatibility, non-toxicity, and potential bioactivity. However, alginate fiber in its native form does not possess an antimicrobial property and as a natural polysaccharide may act as a nutrient, thus becoming a suitable medium for bacterial growth.¹⁷ Consequently, an alginate fiber with incorporated antimicrobial activity is desirable.

As a part of our ongoing investigation on the functionality and application of alginate fibers, an extensive screening study was carried out involving several methods to combine the AgNPs with alginate fiber to endow alginate fiber with special performance. In a recent paper, we reported the preparation of alginate-AgNP composite fibers by a blending method. In brief, AgNPs were first prepared with sodium alginate, then the as-prepared AgNP colloid solution was used directly to prepare sodium alginate-AgNP spinning dope and the resultant spinning dope was spun to prepare alginate-AgNP composite fibers. In the present work, AgNPs were incorporated in alginate fibers via an in situ reduction method. Alginate fibers are favorable supporting material to stabilize AgNPs. The prepared alginate-AgNP fibers were characterized and their antibacterial activities were explored. Moreover, the in vitro cytotoxic effect of the fibers was evaluated in human cervical cancer (HeLa) cell lines.

Experimental details

Preparation of alginate-AgNP fibers

Pure alginate fibers were first prepared by the wet spinning technique. Briefly, sodium alginate (Jiejing Seaweed Co. Ltd., Shandong Province, China) was dissolved in water under stirring to obtain a homogenous, well-flowing viscous solution for spinning. Then, the spinning solution was extruded through a stainless steel spinneret (30-hole, 0.08 mm diameter) by a metering pump into a coagulation bath (5 wt % CaCl₂). The resulting alginate fibers were stretched, washed with water, and dried at room temperature. The prepared alginate fibers (0.4 g) were soaked in 24 mL AgNO₃ aqueous solutions (12–24 mmol/L) for 1 h at room temperature. After that, 1 mL glucose solution (1 wt %) was added as reducing agent and the mixture was heated at 60℃ for 1 h in an oscillating water bath. Finally, the fibers were taken out, rinsed, and dried at room temperature. The fibers prepared are named hereafter as alginate-AgNP fibers.

Characterization of the fibers

Field emission-scanning electron microscope (FE-SEM) images were obtained using a JSM-7500F microscope equipped with an energy dispersive X-ray spectroscopy (EDS) detector at an operating voltage of 5 kV. Transmission electron microscope (TEM) images were recorded by a JEM-1200EX microscope. The fiber was first embedded into an epoxy resin, then cut to ultrathin sections (60 nm thickness) with an ultramicrotome (PowerTome XL, RMC, USA). The sections were analyzed using TEM to reveal the distribution of nanoparticles. X-ray diffraction (XRD) measurements were performed with a powder X-ray diffractometer (D/MAX-RB) using Cu K_α radiation (λ = 0.15418 nm) over a 2θ range 5°–80° with a step size of 0.05°.

Antibacterial activity

The antimicrobial behavior of the fibers was evaluated using the disk diffusion method. Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 35218) were used as model test strains for Gram-positive and Gram-negative bacteria, respectively. Nutrient agar medium was prepared by using peptone (5.0 g), beef extract (3.0 g), agar (15.0 g), and sodium chloride (5.0 g) in 1000 mL distilled water; the pH was adjusted to 7.0. The agar medium was sterilized in an autoclave at 121℃ for 20 min. Sterilized nutrient agar was transferred into sterilized petri dishes. After solidification of the agar, bacterial culture was inoculated on the surface of the sterile agar plate and swabbed with sterile cotton swab. Then the test specimens were gently pressed to the agar surface with a sterilized spatula. All the plates were incubated for 18–24 h at 37℃ and examined whether a zone of inhibition was produced around the samples.

The inhibitory effect of fibers on the different phases of bacterial growth was assessed using E. coli and S. aureus culture by constructing the growth curve. The bacterial growth curve includes four phases of growth, namely lag, logarithmic, stationary, and decline phases. In this study, microbial growth curves were determined by turbidimetry. The fibers were added to the liquid culture of bacteria in a 250 mL culture flask and kept in an incubator shaker at 37℃, 200 r/min. The absorbance of the bacterial culture was measured at 550 nm at 1 h intervals by a Shimadzu UV3150 UV-vis spectrophotometer. The growth curve of bacteria was constructed by plotting absorbance versus time. E. coli and S. aureus cultured in media without fibers were used as controls.

For 2 h, 4 h, and 7 h E. coli bacterial culture, after centrifugation (5000 r/min, l0 min), the E. coli was washed with phosphate buffer solution twice, then fixed with 2.5% glutaraldehyde, and TEM images observed after staining.

Cell viability assay

Cell viability was measured using CCK-8 assay (Cell Counting Kit-8, Beyotime, China) according to the manufacturer’s instruction. The HeLa cells were cultivated in 96-well plates (10⁴ cells per well) and treated with a series of alginate-AgNP fibers for 6, 12, 24, or 48 h. CCK-8 reagent was then added to each well and cells were incubated for another 4 h; then the absorbance was recorded using a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm. The cell viability fraction (%) was calculated as the following formula: where A₀ is the absorbance of the cells untreated with fibers as the negative control, A₁ is the absorbance of the cells treated with fibers as experimental measurements, and A₂ is the absorbance of the media plus fibers as the condition controls.

Apoptosis assay

The treated cells were stained according to the Annexin V/propidium iodide (PI) double staining method. Briefly, 1 × 10⁵ cells were stained with incubation buffer consisting of 2 mL Annexin V and 2 mL PI for 20 min at room temperature in the dark. The apoptotic/necrotic cells were analyzed with flow cytometry (FCM).

Results and discussion

SEM, TEM, and XRD analysis

FE-SEM analysis was carried out to understand the surface morphology and fine structures of the fibers. As shown in Figure 1(a), the pure alginate fibers prepared by the wet spinning technique showed a homogenous structure without any contaminating particles on their surfaces. Compared to the pure alginate fibers, the morphology of alginate-AgNP fibers has no obvious change (Figure 1(b) and (c)). Nevertheless, AgNPs coated on the surface of the fibers can be clearly observed by higher magnification (the inset figures of Figure 1(b) and (c)). The presence of AgNPs on the fibers was further confirmed by the EDS spectrum. As shown in Figure 1(d), the peak at around 3 keV is clearly seen and assigned to the silver signal.^18,19 In addition, the silver content of the alginate-AgNP fibers was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, Perkin-Elmer Optima 8000, USA) according to the previous work.²⁰ The content of Ag increased from 1.03 wt % to 2.26 wt % when AgNO₃ concentration increased from 12 mmol/L to 24 mmol/L, but the distribution of AgNPs on the fiber surface was still relatively homogeneous.

Figure 1.

FE-SEM micrographs of (a) pure alginate fibers, (b) alginate-AgNP fibers synthesized at 60℃, 12 mM AgNO₃, and (c) alginate-AgNPs fiber synthesized at 60℃, 24 mM AgNO₃. (d) EDS spectrum of alginate-AgNP fibers synthesized at 60℃, 12 mM AgNO₃.

Typical TEM images of alginate-AgNP fibers in cross-section are presented in Figure 2(a) and (b). From the TEM images, it can be seen clearly that spherical AgNPs (the small black dots) are not only located on the surface but also inside of the fibers. When alginate fibers were immersed in AgNO₃ solution, Ag⁺ could diffuse easily into alginate fibers and reacted with the fibers via ion exchange with calcium ions or sodium ion in the fibers due to the negatively charged alginate facilitating the attraction of the positively charged silver ions by electrostatic attraction. Then, Ag⁺ ions were reduced in situ and the formed AgNPs closely deposited in the alginate fibers. Moreover, no aggregation was observed, indicating that the in situ formed AgNPs could be effectively stabilized by the alginate fibers. Particle size distribution histograms of AgNPs in the fibers (Figure 2(c) and (d)) illustrate that the particle sizes seem to be in the range 10–25 nm. The particle size marginally increases with the increase of AgNO₃ concentration.

Figure 2.

(a, b) TEM images of alginate-AgNP fibers in cross-section and (c, d) particle size distribution histograms of AgNPs in the fibers. (a, c) Alginate-AgNP fibers synthesized at 60℃, 12 mM AgNO₃; (b, d) alginate-AgNP fibers synthesized at 60℃, 24 mM AgNO₃.

The XRD technique was used to determine the crystal structure of AgNPs. XRD patterns of pure alginate fibers and alginate-AgNP fibers are shown in Supplementary figure S1 online. Pure alginate fiber do not reveal any diffraction peak of silver, while, alginate-AgNP fibers reveal distinct diffraction peaks at approximately 38.3°, 44.3°, 64.6°, and 77.4°, which corresponds to (111), (200), (220), and (311) crystalline planes, respectively, of face centered cubic silver. Moreover, the interplanar spacing (d_hkl) values (2.348, 2.037, and 1.440 Å) and the lattice constant (4.065 Å) calculated from the XRD spectrum are consistent with the standard silver values (JCPDS No. 04-0783). This result further confirmed the presence of AgNPs in the fibers.²¹

Mechanical properties of the prepared fibers were measured on an XQ-1 fiber tensile tester (Shanghai New Fiber Instrument Co. Ltd., China) with an extension speed of 5 mm/min under equilibrium conditions at 25℃ and 65% relative humidity. The statistical results came from 50 measurements for each sample. Initial modulus, breaking tenacity and elongation of pure alginate fibers and alginate-AgNP fibers were obtained. The results are shown in Supplementary table S1 online. Initial modulus decreased from 45.2cN/dtex for pure alginate fiber to 38.5 and 37.2 cN/dtex for alginate-AgNPs composite fibers synthesized with 12 and 24 mM AgNO₃, respectively. Breaking tenacity and elongation showed no noticeable change between the fibers. From the obtained data, it can be concluded that the prepared fibers can be used for the fabrication of textile or medical materials by classical textile techniques (woven or non-woven textiles).

Antimicrobial activity

A recent study indicated that impregnation, instead of coating on the fiber surface, with AgNPs improved the antimicrobial activity of fibers.^22,23 This could prolong the antimicrobial effect and lower the possibility of normal human tissue damage due to the slow and continual release of AgNPs, which are slowly changed to silver ions under our physiological system. The released silver ions prefer to interact with sulfur-containing proteins present in the cell membrane, cytoplasm, and inner membrane of mitochondria, as well as with phosphorus-containing compounds like DNA, or preferably attack the respiratory chain, finally resulting in bacteria division and death.^24,25 Fiber samples were placed on bacteria-inoculated agar plates and were visualized for antibacterial activity. The pure alginate fiber was used as control. The results shown in Figure 3(a) indicated that alginate-AgNP fibers have significant inhibition effect toward both S. aureus and E. coli. The diameters of the inhibition zones are summarized in Table 1 to demonstrate the antibacterial effects of the fibers. Pure alginate fibers did not show any antibacterial activity, indicating that antibacterial activity is mainly caused by the AgNPs.²⁶

Figure 3.

(a) Zone of inhibition of alginate-AgNP fibers against (i) S. aureus, (ii) E. coli: (0) is pure alginate fibers as control, (1), (2) are alginate-AgNP fibers synthesized at 60℃, 12 mM AgNO₃, (3), (4) are alginate-AgNP fibers synthesized at 60℃, 24 mM AgNO_3. (b) Effects of fibers on bacterial growth of S. aureus (left) and E. coli (right): (i) control, (ii) pure alginate fibers, (iii) alginate-AgNP fibers.

Table 1.

Diameters (cm) of inhibition zones of pure alginate fibers and alginate-AgNP fibers against S. aureus and E. coli

	Pure alginate fibers	Alginate-AgNP fibers synthesized with 12 mM AgNO₃	Alginate-AgNP fibers synthesized with 24 mM AgNO₃
S. aureus	–	2.30 ± 0.10	2.55 ± 0.15
E. coli	–	1.52 ± 0.05	1.70 ± 0.10

Zone of inhibition is expressed as the diameter in centimeters. Values represent mean ± standard deviations of inhibition zones from three individual experiments.

The antibacterial effect of the fibers on various phases of bacterial growth was analyzed by growth kinetic studies in S. aureus and E. coli, and the growth curves are shown in Figure 3(b). For the control and the pure alginate fibers, the growth pattern of bacteria showed a lag phase at the initial 5 h. Then, the bacterial growth exhibited the logarithmic phase, which is the active phase when the bacterial cells show exponential growth. The pure alginate fibers did not significantly affect the growth profiles of the bacteria. However, alginate-AgNP fibers disturbed the growth pattern of bacteria by interfering in the bacterial growth at the logarithmic phase and caused a reduction in the number of viable cells. In addition, the lag phases of S. aureus and E. coli prolonged to more than 12 h, indicating the long-term inhibition capability of alginate-AgNP fibers. A similar observation was reported earlier with AgNP-coated silk fiber in E. coli and S. aureus.²⁷ The data from inhibition zone assays and growth curve revealed that the as-prepared alginate-AgNP fibers possess good antibacterial property.

The effect of alginate-AgNP fibers on the microstructure of E. coli was further observed by TEM; the results are shown in Figure 4. Apparently, the cell wall structure of the untreated E. coli was intact, and the cytoplasm was uniform; however, the cell wall of E. coli was damaged, appeared empty, and the surface of bacteria became rough after treated 2h with the fibers (Figure 4(b)). Serious damage further appeared on the E. coli cell wall after being treated for 4 h and the cytoplasm appeared to have a large number of holes (Figure 4(c)). Furthermore, the cell wall and cell membrane of the E. coli were blurred, indicating that many bacteria cells had been cracked.

Figure 4.

The structure of E. coli observed by TEM. (a) Native E. coli cells; (b–d) E. coli treated with alginate-AgNP fibers for 2 h, 4 h, and 7 h, respectively.

CCK-8 assay

Although negative perceptions concerning the toxicity of AgNPs sometimes hinder their application, toxicity itself can be useful for cancer therapies. AgNPs have proven promising antitumor effects.²⁸ The cytotoxicity of alginate-AgNP fibers to HeLa cells was assessed by CCK-8 assay. CCK-8 is a cell viability assay reagent with higher sensitivity and better reproducibility than traditional MTT assay.²⁹ The cells were treated with various concentrations of fibers ranging from 0.5 mg/mL to 4 mg/mL for 6, 12, 24, and 48 h and the viabilities of cell proliferation are shown in Figure 5. Data obtained from the CCK-8 assay indicated that the cell viability decreased with increasing concentrations of the fibers with prolonged incubation time. No obvious cytotoxicity was found for alginate-AgNP fibers synthesized with 12 mM AgNO₃ at 0.5 mg/mL. However, when the concentration reached 2 mg/mL, the percentage of viability of HeLa cells was found to be 54.36 ± 2.07 and 29.53 ± 4.58 for 12 h and 24 h respectively, indicating significant cytotoxicity (Figure 5(a)). Alginate-AgNP fibers synthesized with 24 mM AgNO₃ showed cytotoxicity to HeLa cells at low concentration 0.5 mg/mL, the percentage of viability was 72.93 ± 2.94 and 42.47 ± 3.65 for 12 h and 24 h, respectively. This promoted cell-killing effect could be ascribed to the higher silver content in the fibers.

Figure 5.

CCK-8 assay of the HeLa cells treated with alginate-AgNP fibers: (a) alginate-AgNP fibers synthesized at 60℃, 12 mM AgNO₃; (b) alginate-AgNP fibers synthesized at 60℃, 24 mM AgNO₃.

Typical optical microscopic observations of the treated HeLa cells are shown in Figure 6. The healthy HeLa cells (see Figure 6(a)) displayed an elongated shape since they attached to the plate. Compared to the healthy HeLa cells, the cells treated with pure alginate fibers (Figure 6(b)) showed minor morphological changes, indicating that pure alginate fibers have good biocompatibility. However, when HeLa cells were treated with alginate-AgNP fibers, obvious morphological changes could be observed (see Figure 6(c) and (d)). In particular, many cells were observed with small and spherical morphology due to the cell pull-off from the plate, which means these cells were dead already.³⁰

Figure 6.

Microscopy images of HeLa cells: (a) control group, (b) treated with pure alginate fibers, (c, d) cultured with alginate-AgNP fibers synthesized with 12 mM AgNO₃ and 24 mM AgNO₃, respectively.

Apoptosis assay

The FCM assay technique was employed to further confirm the cytotoxic potential of alginate-AgNP fibers in HeLa cells and to assess the percentage of cells in the early and late stages of apoptosis. In the early and intermediate apoptotic cells, phosphatidylserine (PS) is transferred from the inner part of the plasma membrane to the outer layer. Annexin V, which is a Ca²⁺-dependent phospholipid binding protein, was used to detect the apoptotic cells due to its high affinity for PS. PI, a nucleic acid dye that can penetrate into the late apoptotic cells as well as dead cells because of the increase of cell membrane permeability and binds with the DNA of the cells, will make the cell nucleus red.²⁸ As shown in Figure 7, when the cells are viable, the black spots will be located in lower-left quadrants (Q1-LL), while the spots in upper-left quadrants (Q1-UL) indicate nuclear debris. On the other hand, the spots in lower-right quadrants (Q1-LR) indicate the cells are in the early- to mid-stages of apoptosis, and furthermore, the spots located in upper-right quadrants (Q1-UR) present the cells are in the late-stages of apoptosis or neurosis. From the result we can see that almost of the control group maintained bioactivity due to very few spots appearing in the right quadrant for the control, while when the HeLa cells coexisted with the pure alginate fibers, the spot in the right quadrant increased slightly, indicating that the minor effect of cell apoptosis emerged. More importantly, the apoptotic effect became much more obvious when the HeLa cells were treated with the as-prepared alginate-AgNP fibers. After culture with pure alginate fibers (1 mg/mL) for 12 h, the apoptosis ratio was about 5.1% (early apoptosis) and 12.9% (late apoptosis), slightly higher than that of the control group (early apoptosis 3.3%, late apoptosis7.5%). When the cells were incubated with as-prepared alginate-AgNP fibers, the corresponding ratios increased to 19.3% and 12.4%(early apoptosis), 31.0% and 25.9% (late apoptosis), respectively, which are much higher than that cultured with pure alginate fibers. In an earlier study AgNPs inducing DNA damage in HeLa and A549 cells has also been observed by TUNEL assay.³¹ Our findings from FCM assay along with the CCK-8 analysis absolutely confirm the cytotoxic effects of the as-prepared fibers in cancer cells through induction of apoptosis. The results suggest that the prepared alginate-AgNP fibers possess potential application in cancer chemoprevention and chemotherapy.³²

Figure 7.

Representative fluorometric assays of apoptosis measured by Annexin V/PI staining following treatment with fibers in HeLa cells. Events in each of the four quadrants are as follows: lower-left: viable cells; lower-right: cells in the early to mid-stage of apoptosis; upper-right: cells in the late stages of apoptosis or neurosis; upper-left: mostly nuclear debris. (a) Control, (b) pure alginate fiber, (c) alginate-AgNP fibers synthesized at 60℃, 12 mM AgNO₃, (d) alginate-AgNP fibers synthesized at 60℃, 24 mM AgNO₃.

Conclusions

In summary, alginate fibers embedded with silver nanoparticles (AgNPs) were prepared via in situ reduction of Ag⁺-alginate fibers. Spherical AgNPs with diameter 10–25 nm can be effectively dispersed and stabilized in alginate fibers. The synthesized alginate-AgNP fibers showed excellent antibacterial activity against both Gram classes of bacteria. They could hinder the bacterial growth at the logarithmic phase. In addition, alginate-AgNP fibers had dose-dependent cytotoxic effects against HeLa cell through induction of apoptosis. The high performance may strongly encourage the practical applications as a promising material in textile and biomedical field.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Natural Science Foundation of China (grant numbers 51503110, 51303089), the Taishan Scholar Program of Shandong Province, the Special Fund for Self-directed Innovation of Shandong Province of China (grant number 2013CXB80201), and the Science and Technology Program of Qingdao (grant number 14-2-3-61-nsh).

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