# Multivalent and synergistic chitosan oligosaccharide-Ag nanocomposites for therapy of bacterial infection

Jun 19, 2020

### Orthogonal array design for optimizing the synthesis conditions of AgNPs-COS

AgNPs-COS were synthesized via reduction of AgNO3 by CA at 80 °C in the presence of COS in water. The amine groups of COS can complex with silver cations and then conjugated on the growing AgNPs surface following reduction process. The antibacterial activity of AgNPs-COS was related to the amount of AgNO3, COS, and CA in synthesis process. The amount of AgNO3 and CA was found to be related to the size of AgNPs-COS and then can affect the antibacterial activity of AgNPs-COS. COS as capping groups to the AgNPs surface can stabilize AgNPs and then affect the surface plasmon resonance absorbance of AgNPs. OAD can quickly generate useful information on key variable by arranging different factors within a single experiment. The results of OAD can then be analyzed by analysis of variance. Optimization of synthesis condition via an OAD would reduce the amount of experiments and costs17,18. Therefore, OAD was used to optimize experimental conditions of AgNPs-COS. In this study, the synthesis conditions include three factors: the amount of AgNO3, COS, and CA. Therefore, an OAD L9 (34) was used to evaluate effects of these factors. Each factor was evaluated in three levels. Experiments were carried out with 1.0% (w/w) AgNO3 solution at 300, 500, or 700 µL, 1.0% (w/w) COS solution at 200, 500, or 800 µL, and 1.0% (w/w) CA solution at 300, 600, or 900 µL, respectively. The OAD experiments were performed according to Table 1. Subsequently, the MIC values against S. aureus and E. coli were determined to evaluate the antibacterial activity of the nine synthesized AgNPs-COS and the results were shown in Tab. 1. Clearly, AgNPs-COS synthesized by the optimum synthesis conditions (No.5 in Table 1) can obtain the lowest MIC value and act synergistic antibacterial functions against gram-positive (S. aureas) and gram-negative bacteria (E. coli). Therefore, the optimum conditions were 500 µL of 1.0% (w/w) AgNO3 solution, 500 µL of 1.0% (w/w) COS solution, and 900 µL of 1.0% (w/w) CA solution, respectively. Under this condition, synthesized AgNPs-COS achieved the desired antimicrobial activities toward both gram-positive and gram-negative bacteria.

### Characterization of AgNPs-COS

UV-vis absorption spectrum was used to confirm the formation of AgNPs-COS. The spectra of AgNPs and AgNPs-COS were shown in Fig. 1. Compared with the absorption spectrum of AgNPs, the maximum absorption wavelength of AgNPs-COS showed a blue shift from 400 to 391 nm. Moreover, the peak intensity of AgNPs-COS was higher than that of AgNPs. COS as stabilizers was beneficial to the nucleation and growth of nanoparticles and avoided the formation of these large nanoparticles. In addition, the surface charge analysis was performed. The zeta potential of AgNPs was −20.3 mV. Although AgNPs with a large number of negative charges can maintain stability in aqueous solution, the interaction between AgNPs and the bacteria is impeded by electrostatic repulsion. However, the zeta potential of AgNPs-COS was 11.3 mV, which could enhance their adsorption to negatively charged bacterial membranes by electrostatic interaction. This result indicated that COS molecules successfully binding to the AgNPs surface.

The morphology of AgNPs-COS was observed by TEM (Fig. 1, inset A). It can be found that the obtained nanoparticles were spherical with good dispersibility and COS coated on AgNPs surface in the core-shell module. The average diameter of AgNPs-COS was 36.7 nm (Fig. 1, inset B). Because of conjugation of COS onto AgNPs surface, the edge of AgNPs-COS emerged light corona (Fig. 1, inset A) compared with AgNPs (see Supplementary Fig. S1 online).

### Study of synergistic antimicrobial activity

The FIC index was employed to detect any synergistic antimicrobial effect between AgNPs and COS by a two-dimensional microdilution assay. The FIC was calculated as follows:

$${rm{FIC}}=frac{{rm{MIC}},{rm{of}},{rm{compound}},{rm{A}},{rm{in}},{rm{combination}}}{{rm{MIC}},{rm{of}},{rm{compound}},{rm{A}},{rm{alone}}}+frac{{rm{MIC}},{rm{of}},{rm{compound}},{rm{B}},{rm{in}},{rm{combination}}}{{rm{MIC}},{rm{of}},{rm{compound}},{rm{B}},{rm{alone}}}$$

If the FIC index was less than 0.5, the interaction was defined as synergistic effect19. The results showed that the FIC indices were 0.34 and 0.29 against S. aureus and E. coli, respectively, indicating that COS binding to the AgNPs can obtain synergistic antimicrobial effect.

The synergistic effects between AgNPs and COS was also verified by zone of the inhibition test. For this purpose, COS, AgNPs, and AgNPs-COS at a final concentration of 100, 64, and 64 μg/mL, respectively, was prepared. From Fig. 2, the diameters of zone of inhibition of AgNPs-COS were significantly larger than those of COS and AgNPs, indicating that AgNPs-COS had better antimicrobial performance compared with COS and AgNPs. The outstanding synergistic activity of COS and AgNPs was also confirmed against both gram-negative and gram-positive bacteria. Moreover, COS was used as stabilizers to protect AgNPs against agglomeration for retaining their diffusivity and enhancing antimicrobial property.

### Effect of Mg2+ ions on the antibacterial activity of AgNPs-COS

To explore the binding sites of AgNPs-COS on the bacterial surface, the effects of Mg2+ ion on the bacterial growth inhibition were examined. From Fig. 3, OD600 value of S. aureus and E. coli were obviously increased when Mg2+ ions were added to the bacterial suspensions in the presence of AgNPs-COS with different concentration. It was indicated that the antimicrobial activity of the nanoparticles decreased. It has been reported that the lipopolysaccharide (on the surface of the gram-negative bacteria, e.g. E. coli) linked with Mg2+ ions via electrostatic interaction to form a stable structure on the surface of the bacterial membrane20,21. However, amino group of COS can chelate Mg2+ ions by metal-to-ligand π-bonding. Then the lipopolysaccharide was isolated and dispersed to the medium, result in the damage of the outer membrane of the bacteria. When Mg2+ ions were added to the bacterial suspension, the COS of the nanoparticle surface would chelate Mg2+ ions in the medium, thereby avoiding replacement of these ions from their binding sites in lipopolysaccharide. Similarly, in gram-positive bacteria (e.g. S. aureus), the teichoic acids of the bacterial cell wall can attract Mg2+ ions to provide rigidity to the cell wall22. When COS of the nanoparticle surface chelated Mg2+ ions from these original sites on the bacterial surface, teichoic acid would be dispersed to the medium and the bacterial cell wall would be damaged. Therefore, AgNPs-COS can interact with the bacteria by binding to Mg2+ of the bacterial surface.

### The permeability of outer membrane

The interaction of AgNPs-COS with outer membrane of E. coli cells was studied using the hydrophobic fluorescent probe NPN, which has strong fluorescence in hydrophobic environments and weak fluorescence in aqueous environments23,24. When AgNPs-COS disorganized outer membrane of bacterial cell, NPN could partition into the phopholipid layer of the outer membrane, which can increase NPN fluorescence intensity. As shown in Fig. 4, NPN fluorescent intensity in E. coli suspensions was increased with the increase in the nanoparticles concentration and interaction time. The fluorescent intensity reached a plateau after approximately 20 min, which indicated that AgNPs-COS can permeabilizate the cell membrane and destroy the integrality of bacterial cell.

### Morphological change of the bacteria by SEM observation

The morphological change of S. aureus and E. coli before and after incubation with AgNPs-COS were investigated via SEM observations. Untreated S. aureus and E. coli had a smooth surface with the integrity of membrane structure (Fig. 5A,C). In contrast, the morphology of the treated bacteria is altered significantly after incubation with 4.0 µg/mL AgNPs-COS for 10 min (Fig. 5B,D). The cell walls of S. aureus had formed a large number of vesicles (Fig. 5B). Furthermore, the leakage of large cytoplasmic components can be observed on the cytomembrane of E. coli (Fig. 5D). Similar SEM images of various bacteria had been also observed in that of the treated bacteria25,26,27. The thickness of the peptidoglycan layer of gram-positive and gram-negative bacteria was difference. When the bacteria interacted with AgNPs-COS for 10 min, S. aureus with the thick peptidoglycan layer formed the vesicles (small leakage of cytoplasmic components) and E. coli with the thin peptidoglycan layer represented large leakage of cytoplasmic components. It indicates that AgNPs-COS can disrupt the bacterial membrane, leading to leakage of cytoplasm. Then the damaged membrane can destroy the structural integrity and the membrane ability, resulting in bacterial death.

The antimicrobial mechanisms of AgNPs-COS are possibly as follows. AgNPs-COS interact with the bacteria by binding to Mg2+ ions of the bacterial surface. Then, the nanoparticles disrupt bacterial membrane via increasing the permeability of the outer membrane, resulting in leakage of cytoplasm. These are possibly reasons causing bacterial cell death.

### Cytotoxicity assay

The cytotoxicity of AgNPs-COS was investigated in Raw246.7 cells by MTT assay. As shown in Fig. 6, no obvious cytotoxicity was observed at a concentration up to 128 μg/mL, which had exceeded 160-fold and 250-fold MIC against S. aureus and E. coli, respectively (Table 1, No.5). Moreover, the toxicity of AgNPs can be improved by binding to the COS with good biocompatibility. Therefore, the nanoparticles had the potential for in vivo use.

### In vivo study

To explore the healing status of the wound infection with S. aureus, 8.0 µg/mL AgNPs-COS, 75% ethanol or 0.9% NaCl solution were administered to the wounds of every group for daily therapy, respectively. The physical measurement of wound area before the treatment and during the treatment were evaluated by wound photographs. From Fig. 7, the wounds of AgNPs-COS group and ethanol group were healed after 12 days. However, the wounds treated with NaCl and without treatment were not healed. Histological evaluation of rat dermal wound was performed at the 12th days after treatment and representative optical micrographs by H&E staining were showed in Fig. 8. The epithelialization underlying wound connective tissues were observed by the therapy of AgNPs-COS or ethanol. Meanwhile, a few inflammatory cells emerged from the 75% ethanol-treated wounds. However, massive inflammatory cells appear from the 0.9% NaCl solution-treated and untreated wounds. From immunohistochemical staining of rat epidermal tissues, NaCl group and blank control group had more collagen I (brown parts or dots) than ethanol group and AgNPs-COS group. Moreover, as shown in Fig. 9, relative expression levels of collagen I were significantly increased in the rat skin tissues of NaCl group and blank control group, indicated inflammation still exists. Meanwhile, the results of relative expression levels of keratin, fibronectin and laminin illustrated that AgNPs-COS can promote wound healing of bacterial infection without inflammation28,29,30,31.