Preparation of silver nanoparticle functionalized amphiphilic bio-carbon particles and its bacteria-killing effect and blood compatibility

Abstract

Catheter-related bloodstream infection (CRBSI) has become the bacterial infection of special concern considerably causing the prolonged hospitalization and the delayed clinical treatment. In this paper, silver nanoparticles (AgNPs) were stabilized in sodium polyethylene sulfonate (PVS) solution (PVS-Ag). Amphiphilic bio-carbon particles (ACPs) were synthesized from S. cerevisiae cells and employed as the matrix. Chitosan (CS) modification on ACPs (ACPs-CS) facilitated the PVS-Ag grafting with stability and efficiency. Results of morphologic analysis indicated that CS and PVS-Ag modification distinctly changed the morphological-structural properties of the ACPs surface. Surface potentials of ACPs, ACPs-CS and the obtained functionalized bio-carbon material (ACPs-CS-PVS-Ag) were −38.0 mV, +36.1 mV and −53.1 mV, respectively, revealing that the anionic groups enriched on the PVS-Ag surface had largely consumed the amino groups around the surface of ACPs-CS. Results from X-ray photoelectron spectroscopy (XPS) and thermogravimetric analysis (TGA) further demonstrated that AgNPs had successfully attached to the ACPs-CS surface. The anionic amphiphiles (ACPs-CS-PVS-Ag) exerted high blood-compatibility and bio-safety, especially the broad-spectrum antibacterial property at the microgram scale. In conclusion, the ACPs-CS-PVS-Ag assembly has potential value in the development of biomedical devices and/or implants for the safe-and-effective elimination of bacterial infections.

Keywords:

• Catheter-related bloodstream infection (CRBSI)
• amphipathic bio-carbon particles (ACPs)
• chitosan (CS)
• anionic silver nanoparticles (AgNPs)
• antibacterial

1. Introduction

Catheter-related bloodstream infection (CRBSI) is one of the most common bacterial infection diseases caused by multiple nosocomial pathogens during the medical attention [1]. It mainly involves the bacteremia resulting from the microbial infection during the clinical treatment or 48h after removal of medical catheters for central vein catheterization, intravenous infusion and blood infusion, with pathological characteristics of fever and shivering. The preliminary diagnosis includes the redness and swelling, the induration and/or the exudate at the catheter position [2,3]. Significantly, the incidence of CRBSI has climbed upwards with the widespread application of medical catheters in recent years. A whole range of complications have been closely associated with diagnostic mistakes and improper treatment, including bacteremia, septicemia, pseudoaneurysm and arteriorrhexis, which further leads to the high morbidity and mortality, the extended hospital stays and the increment of medical expense, and seriously impacts the prognosis and life quality of patients [4,5]. Hitherto, several antimicrobial treatments have been practiced for avoiding CRBSI, such as the empirical antimicrobial therapy [6], the targeted antimicrobial therapy [5], Curos™ Disinfection Caps [2]. Besides, the antimicrobial catheter impregnated with disinfectants has remarkably reduced the incidence of CRBSI, which has become the major intervention against CRBSI at present [7]. But few researches are available for the mechanisms of bacterial infection and blood coagulation involved with the catheter-related diseases. It’s of essence to improve the preparation of certain bio-functional materials with antibacterial effects specific to the CRBSI prevention.

Chitosan (CS), the only cationic natural polysaccharide deacetylated from chitin, possesses good moisture-retentive ability and high adsorptive capability [8–10]. The insolubility of CS in water and most organic solvents, however, has restricted its application in many fields, which could be ameliorated by CS derivates obtained from the chemical modification of the functional groups (-OH and -NH₂, etc.). It’s been reported that several CS derivates have the improved biocompatibility, bioactivity and biodegradability and retain the intrinsic pharmacologic effects, which are applicable for inducing the erythrocyte aggregation, promoting the platelet activation, and activating the complement system [11–14]. With the rapid development of nanotechnology, CS derivates of nanoparticles, hydrogels, microspheres, and micelles have been developed with the antimicrobial potential greatly enhanced [8].

Currently, most of antimicrobial materials have been fabricated via supplementing the antibiotic active substance at quantum satis into certain matrices to exert the bacteriostasis activity and bactericidal effect [15,16]. Among those, silver-based antibacterial agents have attracted extensive attention of the bio-medical field, which are prone to be adsorbed on the major microbial biomolecules (i.e. DNA, membrane proteins, enzymes and/or intracellular cofactor) to decompose the active sites [17,18], thus achieving the bactericidal effectiveness remarkably [19]. Studies have found that silver nanoparticles (AgNPs) are appropriate for the CRBSI treatment due to the high stability and safety, upstanding antimicrobial property [20] and excellent biocompatibility [21,22], and nontoxicity even at low concentrations [23,24].

The silver-based antimicrobial agents are very active chemically with potent germicidal effects, which, however, would seriously damage normal cells of a human body while inhibiting or killing pathogens simultaneously. This study aimed to introduce a simple and efficient processing method of nano-silver antibacterial material derived from the composite chitosanizated bio-carbon material (Figure 1). The fabricated nano-silver bio-carbon material was expected to perform the bacteriostatic effect and reduce the toxicity to normal cells stably and remarkably.

Figure 1. Construction of the functionalized bio-carbon material.

2. Materials and methods

2.1. Cells

S. cerevisiae cells were purchased from Omkang Technology (Zhengzhou, China).

2.2. Synthesis of amphiphilic bio-carbon particles (ACPs)

50.0 g S. cerevisiae cells was homogeneously dispersed in 200 mL deionized water. After placing at room temperature for 10 min, the bacterial suspension was filtered to remove the surface impurities and then the upper dispersion was collected for centrifugation at 8000 rpm for 3 min with the supernatant discarded. The centrifugated deposit was supplemented with a given concentration of acetone and stirred thoroughly in TS-100 decolorizing shaker (Kylin-Bell, Haimen, China) for 20 min, which was uniformly re-centrifuged and re-dispersed by acetone. Repeat the procedure twice to remove cellular contents, and then wash the sediment with deionized water for three times to remove the residual acetone solvent. Subsequently, the pre-prepared 2% glutaral-phosphate buffer solution was added to homogeneously disperse the precipitate, which finally transferred into the reaction kettle for hydrothermal reaction (180 °C, 12 h). With the mixed-system reaction being completed, the floating impurities around the superstratum were removed at room temperature, and the sediment was progressively rinsed with deionized water until none of black impurities left. The uniformly dispersed mixture was centrifuged and the precipitate was collected and vacuum-dried to obtain the yellow fine powder, i.e. ACPs.

2.3. Synthesis of ACPs-CS

150 mg of ACPs synthesized in Section 2.2 was ultrasonically dispersed in 40 mL phosphate buffer solution (PBS, pH = 5.3). Two carboxyl activators, i.e. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 100 mg) and N-hydroxy succinimide (NHS, 100 mg), were consecutively dissolved in 10 mL PBS (pH = 5.3). Intermix the above two mixtures on the shaker at 180 rpm for 2 h under room temperature to fully activate the carboxylation.

200 mg of chitosan (CS; degree of deacetylation: 80.0% ∼ 95.0%) was completely dissolved in CH₃COOH (1 wt.%) aqueous solution and then mixed with the carboxyl-activated ACPs under the incubation overnight at 180 rpm on the shaker under room temperature. Then the deposit was further retained by centrifugation, washed twice by CH₃COOH (1 wt.%) aqueous solution and re-centrifuged to remove the residual CS. The precipitate was further collected, washed twice by deionized water and re-centrifuged to remove the residual CH₃COOH. Finally, the substance was rinsed by anhydrous ethanol and centrifuged with the remaining sediment vacuum-dried to obtain the light-yellow fine powder, i.e. ACPs-CS.

2.4. Synthesis of PVS-Ag

In the opaque background, 16.9 mg AgNO₃ was dissolved completely with 46 mL deionized water under the ultrasonic technique and magnetic stirring at 300 rpm. 10 μL sodium polyethylene sulfonate (PVS; 20 wt.%) aqueous solution was fully diluted in 2 mL deionized water on the vortex oscillator, which was then dropwise added into the above AgNO₃ solution with a disposable pipette and stirred for 20 min.

Subsequently, 3.78 mg NaBH₄ dissolved with 2 mL deionized water was supplemented into the above reaction system at the rate of one drop per 10 s under the magnetic stirring for 5 h, and continue stirring the solution magnetically for another 5 h after the rapid color change (from colorless to yellowish brown). The colloidal solution of AgNPs coated with PVS was obtained and recorded as PVS-Ag (2 mmol·L⁻¹).

2.5. Synthesis of ACPs-CS-PVS-Ag

60 mg of ACPs-CS prepared in Section 2.3 and 36 mL of the PVS-Ag colloidal solution prepared in Section 2.4 were intermixed and shaken on the vortex oscillator at room temperature for 8 h under the dark condition. After the completion of reaction, the mixed system was centrifuged at 8000 rpm for 3 min with the supernatant discarded. Repeat the operation of washout by deionized water and centrifugation to remove the unloaded PVS-Ag particles until the cleansing solution became colorless. Then the residual was dried at 60 °C for 6 h to obtain the functional amphiphilic bio-carbon material loaded with AgNPs recorded as ACPs-CS-PVS-Ag.

2.6. Analysis of scanning electron microscope (SEM)

Three materials, i.e. ACPs, ACPs-CS, and ACPs-CS-PVS-Ag, were uniformly dispersed in the anhydrous ethanol with the sample concentration controlled within the appropriate range, 20 μL of which was respectively pipetted and dispensed on the silicon wafer to perform the spray-gold treatment and SEM examination at a voltage of 20 kV and photographed. Besides, morphological characteristics of AgNPs in PVS-Ag colloidal solution was observed after natural drying

2.7. Measurements of particle size and Zeta-potential

All the three powdered samples (ACPs, ACPs-CS, and ACPs-CS-PVS-Ag) were mixed with deionized water to form the dispersion system (1 mg·mL⁻¹). Then the particle size and Zeta-potential of PVS-Ag, ACPs, ACPs-CS, and ACPs-CS-PVS-Ag solution was individually detected by Malvern laser particle size and Zeta potential analyzer.

2.8. Amphiphilicity

ACPs, ACPs-CS and ACPs-CS-PVS-Ag dispersions (1 mg·mL⁻¹) were prepared by H₂O, DMF, CHCl₃, and the mixture of H₂O and CHCl₃ as solvents with the material amphiphilicity observed and photographed, respectively.

2.9. Analysis of X-ray photoelectron spectroscopy (XPS)

The three powdered samples prepared in Section 2.2, Section 2.3 and Section 2.5 were pressed into thin sheets, fixed and vacuumed. Then the elemental compositions of ACPs, ACPs-CS and ACPs-CS-PVS-Ag were analyzed by X-ray photoelectron spectroscopy (XPS).

2.10. Thermogravimetric Analysis (TGA)

The thermogravimetric curves of powdered samples (3 mg–5 mg) were determined on the DTG-60 thermal analyzer (Shimadzu, Japan) under the nitrogen environment (50 mL·min⁻¹) with the heating rate of 10 °C·min⁻¹ and the maximum temperature of 600 °C.

2.11. Determination of the binding amount of AgNPs in ACPs-CS-PVS-Ag

By using the UV-VIS full-wavelength scanning, the maximum absorption peak of PVS-Ag solution was determined as 397 nm. Then the PVS-Ag solution with different concentrations (0.01 mmol·L⁻¹,0.02 mmol·L⁻¹,0.03 mmol·L⁻¹,0.04 mmol·L⁻¹,0.05 mmol·L⁻¹, and 0.06 mmol·L⁻¹) was further analyzed to obtain the UV full-wavelength absorption data within the range of 320 nm ∼ 700 nm and plot the standard curve of UV characteristic absorption peak. The content of PVS-Ag loaded on the surface of ACPs-CS-PVS-Ag was calculated by the difference of the absorbance value measured at 397 nm of the supernatant obtained by centrifugation before and after the interaction between PVS-Ag and ACPs-CS.

2.12. Antibacterial activity of ACPs-CS-PVS-Ag

A certain amount of ACPs, ACPs-CS and ACPs-CS-PVS-Ag was individually transferred into the 4 mL EP tube with 75% ethanol added, which were fully shaken for 15 min and centrifuged to remove the supernatant. The sediment was washed by PBS (pH = 7.4) for 4 times and then dried, into which PBS (pH = 7.4) was added to prepare the stock solution (0.5 mg·mL⁻¹). Four strains, including S. aureus, E. coli, C. albicans and Methicillin-resistant S. aureus (MRSA), were counted and serially diluted into the bacteria solution (3 × 10⁵ CFU·mL⁻¹). The minimum inhibitory concentration (MIC) of ACPs-CS-PVS-Ag was conducted with the different concentrations of ACPs-CS-PVS-Ag (0 μg·mL⁻¹, 0.1 μg·mL⁻¹, 0.5 μg·mL⁻¹, 2.0 μg·mL⁻¹ and 3.0 μg·mL⁻¹) added into the 3 mL intermixture, where the number of bacterial cells was maintained as 3 × 10⁴ CFU·mL⁻¹. Meanwhile, 45 μL of the stock solutions (ACPs, ACPs-CS and ACPs-CS-PVS-Ag) were individually mixed with 300 μL of S. aureus suspension and 2.655 mL of PBS to prepare the suspensions, where the concentration of material sample and bacterial cells were 3.0 μg·mL⁻¹ and 3 × 10⁴ CFU·mL⁻¹. The inhibition zone test was performed to observe the bactericidal effects. All the EP tubes were sealed and shaken under 37 °C for 6 h ∼ 8 h. Under aseptic conditions, 100 μL of the above suspensions were evenly spread on the surface of the sterilized agar medium, which were then cultured under constant temperature (37 °C) and humidity for 18 h ∼ 24 h with the colony growth observed.

2.13. Hemolysis of ACPs-CS-PVS-Ag

1 mL of human blood was diluted 10 times with PBS and then centrifuged (10000 rpm, 10 min) to obtain the red blood cells (RBCs) that was washed with PBS and dispersed into 5 × PBS buffer. 0.2 mL of the diluted RBCs solution and 0.8 mL of the ACPs-CS-PVS-Ag solution with different concentrations (i.e. 0 μg·mL⁻¹, 200 μg·mL⁻¹,400 μg·mL⁻¹, 600 μg·mL⁻¹, 800 μg·mL⁻¹, and 1000 μg·mL⁻¹) were homogenously processed by vortex oscillation, settled at room temperature for 3 h, and centrifuged (10000 rpm, 3 min). Then the supernatant were collected with the absorbance at 570 nm measured, of which the absorbance at 655 nm was taken as control. The hemolysis ratio (%) was calculated as followed: $$\mathit{\text{Hemolysis Ratio}}=\frac{{\mathit{\text{OD}}}_{\mathit{\text{Sample}}}-{\mathit{\text{OD}}}_{\mathit{\text{Negative control}}}}{{\mathit{\text{OD}}}_{\mathit{\text{Positive control}}}-{\mathit{\text{OD}}}_{\mathit{\text{Negative control}}}}\times 100\mathrm{\%}$$ where, OD_Sample, OD_{Negative control} or OD_{Positive control} stands for the absorbance of the mixture of the ACPs-CS-PVS-Ag solution with different concentrations, the deionized water or PBS, and the diluted RBCs solution placed at room temperature for 3h.

2.14. Cytotoxicity of ACPs-CS-PVS-Ag

Normal human liver cells HL-7702 were digested and centrifuged. 100 μL of the cell suspension was inoculated into the 96-well plate (5000 cells per well) and cultured for 24 h. Then 100 μL of the ACPs-CS-PVS-Ag solution with different concentrations (i.e. 0 μg·mL⁻¹, 0.1 μg·mL⁻¹, 1 μg·mL⁻¹, 10 μg·mL⁻¹, 100 μg·mL⁻¹, and 1000 μg·mL⁻¹) was added to each well, respectively. After 24 h, 20 µL methyl thiazolyl tetrazolium (MTT) solution was added to each well, mixed and cultured at 37 °C for 4 h. Subsequently, the plate was washed with PBS with 100 µL dimethyl sulfoxide (DMSO) added into each well, the absorbance of which at 490 nm was detected by microplate reader.

3. Results and analysis

3.1. PVS-Ag modification changed the surface morphology and structure of ACPs-CS

Surface characteristics of ACPs, ACPs-CS and ACPs-CS-PVS-Ag were individually observed by SEM. The results showed that ACPs was the ellipsoidal particle with slightly rough surface. ACPs-CS was also the ellipsoid with smoother and more homogeneous surface in comparison with ACPs, indicating the successful grating of CS on the ACPs surface. Certain micro-particles were detected on the ACPs-CS-PVS-Ag surface, revealing that PVS-Ag modification had re-configured the ACPs-CS surface (Figure 2).

Figure 2. PVS-Ag modification changed the surface morphology and structure of ACPs-CS. SEM images of ACPs (A), ACPs-CS (B) and ACPs-CS-PVS-Ag (C).

3.2. CS and PVS-Ag modifications changed the particle size and potential of ACPs

Potential changes before and after the ACPs-CS-PVS-Ag synthesis was evaluated. As indicated in Figure 3A, the particle size of PVS-Ag was uniform and stable with the distribution range of 23 nm. SEM characterization further demonstrated that AgNPs distributed within the PVS-Ag solution maintained stable and homogeneous. Moreover, the PVS-Ag potential was stabilized at −28.3 mV (Figure 3B). Figure 3C showed that the potential of ACPs, ACPs-CS and ACPs-CS-PVS-Ag was −38.0 mV, + 36.1 mV and −53.1 mV, respectively. Negative-charged groups (e.g. carboxyl and hydroxyl groups) were enriched on the surface of ACPs prepared by the hydrothermal method, causing the negative Zeta-potential measured, which conformed to the expected characteristics and requirements of the synthetic materials. CS belongs to the polymer rich in positive-charged groups (e.g. amino group). Hence the surface potential of ACPs altered with CS modification due to the amino group largely exposed on the material surface. In addition, ACPs-CS-PVS-Ag was negatively charged. The PVS-Ag surface was possessed of numerous anionic groups (e.g. the sulfonic acid group), which were acylated with the positive groups (e.g. the amino group) on the ACPs-CS surface during the process of covalent binding between the two materials, causing the consumption of amino groups and the exposure of negatively-charged groups around the material surface. The above results indicated that the anionic ACPs-CS-PVS-Ag had been successfully prepared by grafting PVS-Ag onto the ACPs-CS surface.

Figure 3. CS and PVS-Ag modifications changed the particle size and potential of ACPs. (A) Particle size distribution and SEM analysis of PVS-Ag. (B) Zeta potential of PVS-Ag. (C) Zeta potential distribution of ACPs (black), ACPs-CS (red) and ACPs-CS-PVS-Ag (blue).

3.3. CS and PVS-Ag modifications exerted no effects on the amphiphilicity of ACPs

Effects of CS and PVS-Ag modifications on the amphiphilicity of ACPs were further investigated. As shown in Figure 4, ACPs-CS and ACPs-CS-PVS-Ag maintained excellent homo-disperse stability in three different polar solvents, i.e. H₂O, DMF and CHCl₃, indicating that ACPs-CS and ACPs-CS-PVS-Ag materials were amphiphilic due to the ACPs matrix. Furthermore, the two materials could disperse in the mixed polar solvent of H₂O and CHCl₃ and stabilize at the oil-water interface. Therefore, the results demonstrated that both the covalent grafting of CS to ACPs surface and the PVS-Ag coating on ACPs-CS surface didn’t alter the amphiphilicity of ACPs.

Figure 4. CS and PVS-Ag modifications exerted no effects on the amphiphilicity of ACPs. Dispersion of ACPs-CS (A) and ACPs-CS-PVS-Ag (B) in different polar solvents.

3.4. AgNPs were successfully adhered to the surface of ACPs-CS

The elementary composition of three materials was analyzed by XPS to evaluate the AgNPs adhesion to the ACPs-CS surface. As indicated in Figure 5A, all the three materials obviously presented the absorption peaks of C1s, O1s, and N1s. Furthermore, it’s distinct that ACPs-CS-PVS-Ag exhibited the absorption peaks of Ag3d and S2p in comparison with the other two materials. Ag3d absorption peak illustrated that AgNPs had been successfully grafted, and S2p absorption peak was detected due to sulfur element contained in sulfonate of the anionic stabilizer PVS, further proving the AgNPs adhesion on the surface of ACPs-CS particles.

Figure 5. Silver nanoparticles successfully adhered to the surface of ACPs-CS particles. (A) XPS results of ACPs (a), ACPs-CS (b) and ACPs-CS-PVS-Ag (c). (B) Thermogravimetric curves of ACPs (black), ACPs-CS (red) and ACPs-CS-PVS-Ag (blue). (C) UV-VIS full-wavelength analysis of PVS-Ag with different concentrations. (D) Standard curve for the PVS-Ag content determined by UV-VIS full-wavelength scanning.

Figure 5B showed changes in the thermogravimetric loss of the three materials with the temperature increment ranging from 70 °C to 640 °C, which began to plunge from 260 °C, decelerated appeared around 490 °C and finally stabilized at 600 °C. Compared with ACPs, ACPs-CS exhibited 9.88% weight gain due to the large amount of benzene ring contained in CS, which were indestructible for ACPs covalently grafting with CS before the temperature reaches 750 °C. The weight increment further indicated the successful modification of CS on the ACPs surface. The thermal weight loss of ACPs-CS-PVS-Ag decreased by 3.27% when compared with ACPs-CS. As with PVS-Ag, the AgNPs were thermostable, while the anionic stabilizer PVS tended to be pyrolytically decomposed, causing the thermal weight loss of ACPs-CS-PVS-Ag increased in the temperature range from 260 °C to 450 °C and then decreased since 450 °C in comparison with ACPs-CS.

Subsequently, the PVS-Ag content of ACPs-CS-PVS-Ag was analyzed. From Figure 5C, the maximum absorption peak of PVS-Ag was observed at 397 nm. The PVS-Ag solution was brownish yellow, clear, and transparent, which could be stably preserved at 4 °C in the dark. Finally, the absorbance value of the supernatant obtained by centrifugation after the reaction between PVS-Ag and ACPs-CS was measured as 0.236, which could be substituted into the standard curve equation [$y=0.073x-0.001$; $R^2=0.9986$] (Figure 5D) to calculate that the PVS-Ag content on the surface of ACPs-CS-PVS-Ag was 0.72 µmol·mg⁻¹.

3.5. ACPs-CS-PVS-Ag exhibited good antibacterial effects

In this study, S. aureus, E. coli, C. albicans and MRSA, were selected to evaluate the antibacterial properties of ACPs-CS-PVS-Ag and the survival ratio was measured. Figure 6A showed that antimicrobial effects of ACPs-CS-PVS-Ag were significantly enhanced with the concentration increment. Growth of 56.3% S. aureus was suppressed by ACPs-CS-PVS-Ag at 0.1 µg·mL⁻¹, and the antibiotic efficiency reached to 99% for the ACPs-CS-PVS-Ag concentration of 3.0 µg·mL⁻¹. It showed that the ACPs-CS-PVS-Ag material prepared in this study enabled the inhibitory activity against Gram-positive bacteria (represented by S. aureus), Gram-negative bacteria (represented by E. coli), fungi (represented by C. albicans) and drug-resistant strains (represented by MRSA), which could be further interpreted by the colony growth of blank control (PBS) and the other two materials (i.e. ACPs and ACPs-CS) on S. aureus, E. coli, C. albicans and MRSA as photographed in Figure 6B-E, respectively. Result of the inhibition zone further validated the release-type bactericidal mechanism of ACPs-CS-PVS-Ag (Figure 6F).

Figure 6. ACPs-CS-PVS-Ag had good antibacterial performance. (A) Survival ratios of S. aureus, E. coli, C. albicans and MRSA (3 × 10⁴ CFU·mL⁻¹) incubated in ACPs-CS-PVS-Ag under different concentrations. (B) Antibacterial activity of the blank bacteria (a), ACPs (b), ACPs-CS (c) and ACPs-CS-PVS-Ag (d) under S. aureus exposure. (C) Antibacterial activity of the blank bacteria (a), ACPs (b), ACPs-CS (c) and ACPs-CS-PVS-Ag (d) under E. coli exposure. (D) Antibacterial activity of the blank bacteria (a), ACPs (b), ACPs-CS (c) and ACPs-CS-PVS-Ag (d) under C. albicans exposure. (E) Antibacterial activity of the blank bacteria (a), ACPs (b), ACPs-CS (c) and ACPs-CS-PVS-Ag (d) under MRSA exposure. (F) Results of the inhibition zone for the blank bacteria (a), ACPs (b), ACPs-CS (c) and ACPs-CS-PVS-Ag (d) under S. aureus exposure.

3.6. ACPs-CS-PVS-Ag had good blood compatibility and biological safety

The hemolysis reaction was conducted to illustrate whether ACPs-CS-PVS-Ag could cause the erythrocyte fragmentation. From Figure 7A and B, the hemolytic reaction was simply notable in the deionized water group (the positive control), which was undetected in the normal saline group (the negative control). Besides, the hemolytic reaction remained unobserved for erythrocytes maintained in PBS (pH = 7.4) containing ACPs-CS-PVS-Ag of different concentrations for 3 h. These results indicated that ACPs-CS-PVS-Ag had good hemocompatibility.

Figure 7. ACPs-CS-PVS-Ag had excellent hemocompatibility. (A) Hemolysis Assay of ACPs-CS-PVS-Ag. (B) Photograph of RBCs incubated with ACPs-CS-PVS-Ag under different concentrations for 3 h. (C) Cell viability of HL-7702 maintained in ACPs-CS-PVS-Ag under different concentrations for 24h.

Furthermore, the cytotoxicity of ACPs-CS-PVS-Ag to human normal heptical cell line (HL-7702) was investigated. As shown in Figure 7C, little cytotoxicity appeared for the ACPs-CS-PVS-Ag concentration less than 100 μg·mL⁻¹ when compared with the control group for the cell viability rate above 95%. But when the ACPs-CS-PVS-Ag concentration reached 1000 μg·mL⁻¹, the cell survival rate decreased to 78.79%, presenting low cytotoxicity. Considering the excellent antibacterial performance of ACPs-CS-PVS-Ag as previously investigated, the effective antibiosis concentration of which was far below 100 μg·mL⁻¹. Hence the ACPs-CS-PVS-Ag material would maintain the bio-safety under concentrations for application.

4. Discussion

In the field of pharmaceutical research, AgNPs with various properties are mostly loaded onto the surface of graphene [25], calcium phosphate [26] and titanium dioxide [27] etc., the application of which, however, has been seriously restricted due to the undesirable properties, including brittleness, low mechanical performance and poor oil-water dispersion. Hence, it’s of great importance to construct certain biomaterials with excellent biocompatibility, high mechanical strength, good oil-water dispersion and large load capacity.

Hitherto, there remains no report about the immobilization of CS and AgNPs on the ACPs surface. In this study, the anionic stabilizer PVS was utilized to prepare AgNPs (PVS-Ag), further improving the stability and greatly enhancing the antibacterial activity. As the biological carrier prepared from S. cerevisiae cells by the hydrothermal synthesis method, the ACPs matrix were dispersible well in most of polar and nonpolar solvents due to the carboxyl and aromatic groups contained on the surface. Furthermore, the nano-scale ACPs was characterized by the large specific surface area and strong adsorptive capacity, making it applicable for modification. Covalent grafting of CS onto the ACPs surface was performed in our study via the carboxylation reaction catalyzed by EDC and NHS. The obtained ACPs-CS was enriched by amino groups to reinforce the anionic AgNPs loading and improve the biocompatibility. We selected the biocompatible CS as the intermediate medium to concatenate ACPs and PVS-Ag, where the anionic PVS-Ag adhered onto the ACPs-CS surface via the electrostatic interaction. Synthesis of the functional biomaterial ACPs-CS-PVS-Ag was further validated by the determination of Zeta-potential and particle size, the thermogravimetric analysis, the amphiphilicity, blood compatibility and bio-safety detection. Results revealed that multiple modifications didn’t affect the amphiphilic properties of ACPs and the release of AgNPs, and the non-toxicity, high blood compatibility, and excellent antibiotic effects further expand the application field of ACPs-CS-PVS-Ag.

It’s been reported that the potential mechanisms involved in the nano-silver antibacterial materials mainly summarized the three followings: (1) Ag-NPs can accumulate on the surface of bacterial cytoderm and cytomembrane to decompose the lipopolysaccharide molecules and increase the membrane permeability, causing the cytoplasm leakage [28]. (2) The nano-silver biomaterials can destroy the functional groups of active enzymes and block the protein synthesis by releasing silver ion to destruct the cytomembrane and diffuse into the cytoplasm, further inhibiting the cellular metabolism and proliferation. Besides, the silver ion can also impact the structure of nucleic acid and interrupt the DNA replication, inhibiting the metabolic capacity at the gene level [29]. (3) AgNPs and silver ion function as the catalytic active center under certain wavelengths to induce the production of reactive oxygen species (ROS; e.g. the hydroxyl radical) suppressing the bacterial proliferation, interfering nucleic acid replication and enzyme activity and eventually leading to bacterial death [30]. In consideration of the significant advantages of AgNPs, including the high bio-safety, strong antibacterial ability, sustainable bacteriostasis and low toxicity, nano-silver antibacterial materials have been widely applied in the field of medical treatment. We found that the functionalized bio-carbon material had good hemocompatibility, almost presenting nontoxicity towards HL-7002 at effective concentrations. In addition, the MIC of ACPs-CS-PVS-Ag prepared in this study maintained at the microgram level (∼3.0 μg·mL⁻¹), with excellent antibacterial activity against S. aureus, E. coli, C. albicans and MRSA, indicating the notable antibiotic performance against Gram-positive bacteria, Gram-negative bacteria, and fungi. It’s been expected to reinforce the practical application of ACPs-CS-PVS-Ag in preparing the functional catheters and provide the applicable foundation for the CRBSI treatment.

5. Conclusions

Our study introduced a simple and efficient method for preparing the functionalized bio-carbon material ACPs-CS-PVS-Ag via obtaining the ACPs from S. cerevisiae cells and selecting CS as the intermediate medium to adhere AgNPs stabilized in PVS. The constructed ACPs-CS-PVS-Ag avoids the defects of conventional antibiotic biomaterials, such as he brittleness, low mechanical performance, and poor oil-water dispersion. Besides the potent antimicrobial effect, the good blood compatibility and biological safety of ACPs-CS-PVS-Ag prepared in this study further improve the development of medical catheter in the CRBSI remission.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datas are available from the corresponding author on reasonable request.

Additional information

Funding

This study was supported by the Henan Provincial Science and Technology Research Program (No. 242102230118).