Honghai Biotechnology Co. Ltd. (Qingdao, China) provided pharmaceutical grade CMCS (amino substituted ratio, 95.1%). N-trimethyl chitosan chloride was obtained from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Food grade sodium alginate was obtained from Bright Moon Seaweed Group Co., Ltd. (Qingdao, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was obtained from Tokyo Chemical Industry (Japan).

High-sugar and high-lipid food was obtained from Tianqin Biotechnology Co., Ltd. (Changsha, China). Blood glucose test strips were obtained from Acon Laboratories, Inc. (Hangzhou, China). Streptozocin (STZ) was obtained from Sigma-Aldrich LLC (Germany). Imipenem and cilastatin sodium for injection were obtained from Merck & Co., Inc. (Germany). Recombinant Human Epidermal Growth Factor Hydro Gel was obtained from Pavay Gene Pharmaceutical Co., Ltd. (Guilin, China). A silver ion detection kit (WAK-Ag) was obtained from Kyoritsu Chemical-Check Lab., Corp. (Japan).

Kelong Chemical Reagent Factory (Chengdu, China) provided all other compounds.

Commercial CMCS was used in this experiment. 0.1% CMCS aqueous solution was prepared. It had been detected by ICS-3000 ion chromatography (Dionex, USA), the chloride ion concentration of this solution was 14 mg/L (≈ 0.4 mM). AgNO₃ aqueous solution (0.6 M, 200 μL) and the CMCS stock solution (100 mL) was mixed. Then, the reaction solution was irradiated under sunlight for 8 h with continual stirring.

CMCS solution (10 mL; 1% w/v) and the unpurified solution (10 mL; Ag⁺:Cl⁻ = 3:1) were mixed. The mixture was then poured into a 24-well plate (1 mL in each well), frozen overnight at –50°C, and then freeze-dried overnight in an Edwards freeze dryer.

Human skin fibroblast cells (CRL-2522 ATCC, USA) were used in this study. The cell line was cultured in ATCC-recommended media with 10% FBS and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% CO₂. The cells were plated in a 96-well plate at 5 × 10³ cells per well in 100 mL medium, grown for 24 h, and then exposed to different concentrations of diluted silver nanoparticle solutions from the concentrated reaction solution for 48 h. Cell viability was measured using the CCK-8 assay, as described previously [20].

Aqueous polymer solutions (N-trimethyl chitosan chloride or sodium alginate; 0.1% (w/v)) were prepared. The mixture stirred overnight to obtain stock solutions for use in different silver nanoparticle preparations.

Aqueous polymer solutions (N-trimethyl chitosan chloride or sodium alginate; 0.5%, w/v) were prepared and stirred overnight. A mixture of 60 μL polymer aqueous solution, 20 μL DPPH ethanol solution (0.05%, w/v), and 220 μL DMSO was monitored using a Varioskan™ LUX multimode microplate reader (Thermo Scientific, Finland).

Nanoparticles were purified by centrifugation and resuspension for three times before characterization. The silver nanoparticle sizes were measured using a Nano ZS90 Zetasizer (Malvern, UK). JEM-2100f TEM (JEOL, Japan) was used for TEM (transmission electron microscopy) and SAED (selected area electron diffraction) experiments. Rigaku X-ray diffractometer (D/MAX-2500PC, Japan) was used for XRD (X-ray diffraction) experiments (Cu Kα radiation, 40 kV, 150 mA). The films of silver nanoparticle were used for the two tests mentioned above.

After three weeks of treatment, the rat wounds in all the experimental groups were significantly healed, except in the negative control group (Fig. 1, first group). Compared with growth factors, antibiotics and silver nanoparticles (Fig. 1, last two groups) exhibit better healing effects, probably because the wounds in the rats with diabetes are susceptible to infection; therefore, an anti-infective treatment is crucial.

The histological analysis of the healing process in negative control group demonstrated tissue necrosis of the wound (Fig. 2, first group). This phenomenon was not found in treatment group. Both fibrin and inflammatory cells were found in the growth factors group (Fig. 2, second group). These suggest that wound healing was accompanied by an inflammatory response in this group. By contrast, the inflammation in the last two groups was much less intense (Fig. 2, last two group). Significant granulation tissue was also found in the both of two groups.

Furthermore, no remnant silver ions were detected in the major organs of these rats (Fig. 3) after the application of silver nanoparticles (Fig. 2).

In our previous study, we proposed that silver nanoparticles prepared using CMCS can be used for downstream applications (e.g., bacteriostatic agents or dressings) without further purification. Accordingly, we intend to test the cellular toxicity of the unpurified reaction solutions containing the prepared silver nanoparticles. For this experiment, human skin fibroblast cells were used.

Based on cell viability, no toxicity to human skin fibroblast cells was observed in the diluted reaction solutions until the silver content was increased to 0.4 mM. However, sudden proliferation occurred when the human skin fibroblast cells were exposed to the silver nanoparticles (Fig. 4), suggesting a plausible explanation for the healing effect observed in the wounds of rats with diabetes.

To determine the growth process of silver nanoparticles in CMCS, the reaction solutions were analyzed at different time intervals via TEM and XRD, as well as using a Zetasizer. An obvious breakage process of the particles is observed during the growth of nanocrystalline silver in CMCS (Fig. 5, 30 min and 1 h).

Cl^• are primarily responsible for the transformation of Ag⁺ to Ag⁰ [21]. Therefore, the above experimental phenomena may be attributed to the increasing concentration of Cl^• in the crystalline core.

The XRD spectrum of the silver nanoparticle solution reaction indicates that prolonging the reaction time reduces the intensity of the AgNO₃ diffraction peak until it disappears completely (Fig. 6a). Almost the entirety of the Ag⁺ is reduced to Ag⁰, despite the molar ratio of Ag⁺ and Cl^- being 3:1. Since the molar ratio of Ag and Cl is 1:1 in AgCl, it can be assumed that Cl^- was recycled after Ag⁺ was reduced to Ag⁰ in this reaction. Accordingly, the mechanism of the silver nanoparticle preparation method using CMCS is presumed to arise from the transformation of Ag⁺ to Ag⁰ (Fig. 6b) [20].

In this study, we also attempted to determine whether this method is suitable for synthesizing silver nanoparticles using other similar polymers and halide ions. N-trimethyl chitosan chloride and sodium alginate were used in this particular experiment.

From the UV-vis spectra, the characteristic UV-vis absorbance peak of silver nanoparticles was found only in the reaction mixture containing N-trimethyl chitosan chloride (Fig. 7a), probably because of the free radical activities of the two polymers. There was a significant difference in this index between the two polymers (Fig. 7b). Notably, the silver nanoparticles were successfully prepared using N-trimethyl chitosan chloride; however, an increased amount of precipitation was observed.

Interestingly, silver nanoparticles were synthesized successfully, and absolutely no precipitation was observed when both polymers were used together (Fig. 8). This may be due to the free radical activity compromising the mixed reaction system.