The virus dilution that reduces the optical density of the sample compared to the optical density of the cell control by 50% was determined, which is the titer and is expressed in TCID50/ml. The virucidal effect was determined by the reduction in virus titer as the difference between the log10TCID50/ml values of the virus control and the test sample.
Before the formation of polyelectrolyte complexes, the concentration of functional groups of oppositely charged biopolyelectrolytes was determined by the conductometric titration method. It was found that the concentration of functional groups of CLMW, CMMW, CHMW and Na-CMC is 4.00, 3.52, 3.63 and 2.57 mmol/g, respectively. Then, the charge of cationic and anionic polyelectrolytes in aqueous solutions was studied by adding 0.1 M solutions of HCl or NaOH to them (Fig. 1).
It was found that for chitosan, regardless of its molecular weight, with an increase in the pH of the solution from 2 to 10, the zeta potential of the degree of charge of the polyelectrolyte decreases. For an anionic polyelectrolyte, with an increase in the pH of the solution from 2 to 10, the degree of charge of the sodium salt of carboxymethylcellulose increases. For the formation of polyelectrolyte complexes, the optimal pH value of the solutions was chosen, which is 5 (Fig. 1).
Analysis of wide-angle X-ray diffraction patterns of the initial cationic polyelectrolytes: chitosan of low, medium and high molecular weight showed that they are all characterized by an amorphous-crystalline structure. This is indicated by the presence of two diffraction peaks at 2θ ~ 9.2° and 19.9° on the curves (Fig. 2). It was established that the structure of chitosan of low, medium and high molecular weight is practically identical.
The relative crystallinity level (X) of chitosan of low, medium and high molecular weight was estimated according to the equation:
where Q is the area of diffraction maxima that characterize the crystalline structure of the polymer; Q + Q is the area of the entire diffractogram in the range of scattering angles (2θ ÷ 2θ) in which the amorphous-crystalline structure of the polymer is manifested, showed that relative crystallinity level does not depend on the molecular weight of chitosan and is about 58%.
In turn, the assessment of the effective size of crystallites L of chitosan of different molecular weights was carried out by the Scherrer method:
where K is a constant related to the shape of crystallites (if their shape is unknown, K = 0.9), and β is the angular half-width (width at half height) of the diffraction maximum, showed that the average value for CLMW is L ≈ 2.9 nm, for CMMW -- L ≈ 5.3 nm, for CHMW -- L ≈ 4.4 nm (for calculations, diffraction maxima at 2θ = 9.2 and 19.9° were used).
Anionic polyelectrolyte -- Na-CMC has an amorphous structure of both the main macrochains and the side branches (methylcarboxyl and methylcarboxylate). This is evidenced by the presence of two asymmetric diffuse diffraction maxima (amorphous halos) of varying intensity on the X-ray diffractogram of this polyelectrolyte (Fig. 3, curve 1), with the angular position of the primary intensity maximum at 19.6° and the secondary at 9.9°.
Considering the number of atoms (electrons) in the glucosidic ring and the methylcarboxylate side branch, the more intense amorphous halo at 2θₘₐₓ ≈ 19.6° corresponds to short-range ordering associated with the spatial (volumetric) translation of main macromolecular chain fragments within the Na-CMC sample. The less intense halo at 2θₘₐₓ ≈ 9.9° characterises short-range ordering in the spatial arrangement of methylcarboxyl and methylcarboxylate side chain fragments.
The average value of the period d of the short-range ordering of Na-CMC macromolecular chain fragments in their spatial arrangement (within the volume of the anionic polyelectrolyte), according to the Bragg equation:
where λ is the wavelength of characteristic X-ray radiation (λ = 1.54 Å for CuK-radiation), for the main macromolecular chains is 4.5 Å, and for their side branches - 8.9 Å.
When mixing anionic and cationic polyelectrolytes -- Na-CMC and chitosan, polyelectrolyte complexes with a new amorphous structure are formed (Fig. 3, curves 1-3). This is indicated by the amorphous halo at 2θ ~ 24.0°, which characterizes the structure caused by the electrostatic interaction of oppositely charged polyelectrolytes (Fig. 3, curves 2).
It should be noted that the angular position of the amorphous halo of samples of polyelectrolyte complexes based on carboxymethylcellulose and chitosan of different molecular weights is significantly shifted to the region of larger angles compared to the amorphous halo of the anionic polyelectrolyte -- Na-CMC (Fig. 3a-c).
This indicates a decrease in the average Bragg distance between the macromolecular chains of oppositely charged polyelectrolytes -- carboxymethylcellulose and chitosan of low, medium or high molecular weight in polyelectrolyte complexes.
Analysis of the structure of the studied samples showed that in the diffraction patterns of polyelectrolyte complexes based on Na-CMC and chitosan with different molecular weights, the angular position of the amorphous halo, which characterizes the structure of the PEC, does not change. At the same time, the average Bragg distance between the macromolecular chains of the anionic and cationic polyelectrolytes remains constant when using chitosan of low, medium or high molecular weight in the composition of the PEC (Fig. 3a-c, curves 2).
After the reduction of silver ions in polymer systems based on CMC and low molecular weight chitosan using green tea extract, the diffractogram of the resulting sample shows two intense diffraction peaks at 2θ ~ 38.2° and 44.2°, which correspond to the crystallographic planes of the face-centered cubic lattice of silver, characterized by indices (111) and (200), respectively, and confirm the presence of metallic silver in the polymer system (Fig. 4, curve 1). There are also three low-intensity peaks at 2θ ~ 27.7°, 32.2° and 46.2°, which correspond to the planes (110), (111) and (211), respectively. These correspond to a face-centered cubic structure and are consistent with previous studies on biogenic AgO particles.
When reducing Ag ions in polyelectrolyte-metal complexes based on CMC and chitosan of medium and high molecular weight, the intensity of the peaks at 2θ ~ 27.7°, 32.2° and 46.2°, which characterize the AgO structure, increases in the diffraction patterns of these samples. This indicates an increase in the amount of silver oxide in the polymer systems (curves 2, 3).
When forming nanocomposites by reducing Ag ions in polymer systems based on CMC and chitosan of various molecular weights using ginger extract, the diffractogram of the CMC -- Ag/AgO-CLMW sample contains low-intensity peaks at 2θ ~ 38.0° and 44.0°, which characterize the structure of metallic silver, and peaks at 2θ ~ 27.7°, 32.2°, and 46.2°, which confirm the presence of AgO particles (Fig. 5, curve 1). When using medium and high molecular weight chitosan to form PECs with anionic polyelectrolyte -- CMC, the intensity of the peaks responsible for the AgO structure significantly increases in the diffractograms of these samples. At the same time, the diffraction peaks that characterize the structure of metallic silver practically do not change compared to the CMC sample -- Ag/AgO-CLMW (curves 2, 3 and 1). This indicates that CMMW and CHMW contribute to better reduction of silver ions with the subsequent formation of a larger number of AgO particles.
When using propolis extract to form silver-containing nanocomposites, the diffractograms of CMC samples with low and medium molecular weight chitosan contain only peaks corresponding to the structure of metallic silver, located at 2θ ~ 38.2° and 44.2° (Fig. 6, curves 1, 2). However, when forming a sample with high molecular weight chitosan, its diffractogram contains peaks that are responsible for both the structure of Ag and AgO (curve 3).
According to the manufacturer of the extracts used in this study, the acid number is 6.1, 9.8 and 11 mg KOH per 1 g of ginger, green tea and propolis extracts, respectively. The highest value for propolis extract may indicate a higher content of organic acids in this extract. According to the literature, green tea extracts are characterized by a high content of polyphenols, while propolis extracts contain a large amount of phenolic compounds and flavonoids. In ginger extracts, the content of these components is usually much lower, which leads to a lower reducing capacity during the synthesis of silver nanoparticles.
Phenolic compounds play a dual role in the green synthesis of metal nanoparticles, acting as both reducing and stabilizing agents. The presence of functional groups such as hydroxyl and carboxyl allows them to effectively reduce Ag⁺ ions while stabilizing the resulting nanoparticles through surface adsorption. In addition, phenolic compounds are well known for their intrinsic bioactivity, including antioxidant, antimicrobial, anti-inflammatory, and anticancer properties. When interacting with the surface of nanoparticles, they enhance the biological functionality of nanocomposites through synergistic effects.
The most likely mechanism for the formation of Ag/AgO during the interaction of CMC-Chit-Ag⁺ complexes with extract components is the initial formation of AgOH through a reaction between silver ions and the hydroxyl groups of phenolic compounds. Then, AgOH, which is unstable, converts to AgO and is reduced by various reducing agents contained in the extracts to Ag. For ginger extract, the reduction reaction practically does not occur due to the extract's low reducing ability. Complete reduction was observed for low molecular weight chitosan and green tea or propolis extracts. However, incomplete reduction was observed for high molecular weight chitosan. This may be due to the formation of denser complex structures that reduce access to silver atoms for reducing agents. A thorough review of the extant literature reveals that, in contrast to the chemical reduction reactions that used NaBH, ascorbic acid, sodium citrate, or other reducing agents, a mixture of Ag/AgO or pure AgO is typically formed when plant extracts are utilized.
Figure 7 presents transmission electron microscopy micrographs of silver-containing nanocomposites based on CMC and low molecular weight chitosan. Analysis of the micrographs showed that when using green tea extract as a reducing agent for Ag ions in polymer systems, Ag/AgO nanoparticles with an average size of 11 nm are formed (Fig. 7a), when using ginger and propolis extracts, larger particles with an average size of 20 and 44 nm are formed, respectively (Fig. 7b, c).
As can be seen from the micrographs, when using various extracts for the reduction of silver ions, nanoparticles are formed that are statistically distributed in polymer matrices. The smallest particles and the smallest dispersion in the size of nanoparticles are inherent in the CMC sample -- Ag/AgO-CLMW, where the reducing agent was green tea extract (Fig. 7a). In samples synthesized using ginger extract, nanoparticles are more prone to aggregation, which may indicate a lower activity of this reducing agent (Fig. 7b). When using propolis extract to form samples, particles of the largest size are formed (Fig. 7c).
Literature data indicate that the specific surface area of silver nanoparticles synthesized using extracts can vary significantly depending on the phytochemical composition of the plant extract. For example, a specific surface area of 28.2 m/g was found for silver nanoparticles synthesized using Moringa oleifera leaf extract. Similarly, silver nanoparticles were synthesized using Coriandrum sativum (parsley) leaf extract, and the surface area of the silver nanoparticles was 33.72 m/g. In another study, silver nanoparticles synthesized using Brassica oleracea var. botrytis (cauliflower) extract had a specific surface area of 19.22 m/g. Such data indicate the key role of plant-derived phytochemicals in influencing the morphology of nanoparticles and surface characteristics.
The polymeric systems CMC-Ag/Ag₂O-CLMW exhibited significant antibacterial activity (Fig. 8). The inhibition zone diameters for S. aureus were as follows: 22.63 ± 1.20 mm (reducing agent: green tea extract, sample 1); 24.00 ± 1.40 mm (reducing agent: ginger extract, sample 2); 22.83 ± 1.10 mm (reducing agent: propolis extract, sample 3). The silver-containing CMC-Ag/Ag₂O-CLMW samples showed slightly lower antibacterial activity against E. coli. The inhibition zone diameters were recorded as follows: 18.36 ± 0.60 mm (reducing agent: green tea extract, sample 1); 15.63 ± 0.30 mm (reducing agent: ginger extract, sample 2); 16.51 ± 0.50 mm (reducing agent: propolis extract, sample 3) (Table 1).
The studied CMC- CLMW samples obtained with the addition of green tea and ginger extracts, which did not contain silver nanoparticles (Fig. 8, Table 1, samples 4 and 5), showed no antibacterial activity against the S. aureus and E. coli. However, the CMC-CLMW (propolis extract) sample exhibited slight activity against S. aureus (Fig. 8). The inhibition zone diameter was 11.58 ± 0.40 mm (Table 1, sample 6). The localized antimicrobial effect against S. aureus may be associated with the natural antimicrobial properties of propolis. The absence of activity against E. coli may indicate the lower effectiveness of the propolis extract against Gram-negative bacteria.
A similar effect was found for biomaterials synthesized by combining sodium alginate and chitosan with three different beekeeping substances -- honey, propolis and royal jelly, as well as with a mixture of these three compounds. Antibacterial studies have shown that among the synthesized samples, films enriched with propolis tincture or a mixture of beekeeping products demonstrated high antibacterial activity. In other studies, it was found that the combination of several antibacterial agents in one composite allows to obtain an effective antibacterial film due to synergy. Composite films were synthesized using compounds such as hydroxyethyl cellulose, ZnO, mesoporous silica and cinnamon essential oil (spice extract).
Chitosan-based films with ZnO and Ag nanoparticles were produced by casting. The nanoparticles were loaded with citronella essential oil (CEO) to enhance the antimicrobial activity of the nanocomposite films. The synergistic effect of the synthesized antimicrobial films was confirmed by comparing the results of the antimicrobial action of the composite with simple chitosan/ZnO/CEO or chitosan/Ag/CEO films.
A moderate virucidal effect against influenza A (H1N1) virus was demonstrated by the CMC-Ag/AgO-CLMW (ginger extract), CMC-Ag-CLMW (propolis extract) and CMC-CLMW (propolis extract): they reduce the infectious titer by (1.87 ± 0.09), (1.99 ± 0.10) and (2.01 ± 0.10)logTCID/ml, respectively. The samples CMC-CLMW (green tea extract), CMC-CLMW (ginger extract), and CMC-CLMW did not affect IAV (Fig. 9a).
The sample CMC-Ag/AgO-CLMW (green tea extract) showed the best effect in inhibiting the influenza virus. It demonstrated a decrease in infectious titer by (8.91 ± 0.45)logTCID/ml, which is a complete inhibition of virus reproduction in MDCK cells (Fig. 9a).
The studied silver-containing samples CMC-Ag/AgO-CLMW (ginger extract) and CMC-Ag- CLMW (propolis extract) reduced the infectious titer of herpes simplex virus by (3.79 ± 0.27) and (3.02 ± 0.23)logTCID/ml, respectively. The CMC-Ag/AgO-CLMW (green tea extract) reduced the titer of HSV-1 significantly less: by (1.23 ± 0.06)logTCID/ml.
Contact of HSV-1 with CMC-CLMW (green tea extract), CMC-CLMW (ginger extract) and CMC-CLMW (propolis extract) did not lead to a change in its infectious titer compared to the control (Fig. 9b).
The studied polymer silver-containing nanocomposites based on CMC and CLMW, formed by reducing Ag ions with green tea, ginger, and propolis extracts, as well as the CMC-CLMW samples filled with extracts did not exhibit a toxic effect on the MDCK cell culture (Fig. 10a). All undiluted samples reduced the cell viability by no more than 28.5%.
The studied nanocomposites in the tested dilutions inhibited the functional activity of Vero cells from 1.4 to 14.7%, which indicates the absence of their cytopathic properties (Fig. 10b). Thus, the samples did not exhibit a significant toxic effect on the studied cell cultures.