The effect of molecular weight of chitosan on the size of chitosan-Cu2+ complex-stabilized sulfur nanoparticles

Chitosan (CS) samples with average molecular weight (Mw) of 80.4, 61.8, and 45.2 kDa were prepared by the heterogeneous degradation of CS using 1% H2O2 solution under ambient conditions. The degree of deacetylation (DD) of the resultant CS was almost unchanged compared with that of the original CS. The sulfur nanoparticles (SNPs) were prepared by acidification of sodium thiosulfate (Na2S2O3) in a CS-Cu2+ complex solution. The influence of Mw CS on the size of SNPs was investigated. The average size of the SNPs/CS-Cu2+ complexes measured through Transmission electron microscopy (TEM) images was 25.1, 32.3, and 48.3 nm for using CS with Mw of 80.4, 61.8, and 45.2 kDa, respectively. The obtained SNPs/CS-Cu2+ complexes were also characterized by Ultraviolet-visible (UV-Vis), Fourier-transform infrared (FTIR), and x-ray diffraction (XRD) measurements. The synthetic method is favorable for large-scale production. Furthermore, the SNPs/CS-Cu2+ complex can be used as an agent for controlling plant disease in agriculture.

Sulfur is a non-metallic element with antibacterial [9], anticancer [10], and antioxidant [11] properties.It has been used in agriculture as a pesticide [12] and a fungicide [13].Additionally, sulfur compounds are the nutrients for plants, used in fertilizers to improve the nutritional quality of phosphate and nitrogen fertilizers [14].Currently, several methods have been applied to prepare sulfur nanoparticles (SNPs) such as the acidification of sodium thiosulfate (Na 2 S 2 O 3 ) or sodium polysulfide, and stabilization in polyethylene glycol-400 [15], poly-N-vinylpyrrolidone [16], or chitosan (CS) [10]; microemulsion method [17]; and electrochemical method [18].Green chemical method to synthesize SNPs by plant extracts has also been studied [19,20].
CS is a linear polysaccharide composed of D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (unit containing an acetyl group) linked at the β−1,4 position [21].It is derived from chitin, which is found in the shells of some crustaceans (shrimps, crabs, and squids), or fungi, and insects [22,23].CS is a non-toxic, biocompatible, biodegradable polymer that exhibits special biological activities, such as antioxidant, anticancer [24], antibacterial [25][26][27], antifungal [28,29], immunity stimulation, and has the potential for use as a plantdisease control agent [25].However, commercial CS often has a high molecular weight (Mw), which makes it difficult to dissolve at high concentrations in an acidic medium.Therefore, its applicability is limited [30].Low Mw CS is more flexible than high Mw CS and is known as a potent biotic elicitor that increases plant resistance to diseases [31,32].Therefore, studying the degradation of CS to low Mw has received special attention.The methods used to reduce the Mw of CS by breaking the β−1,4 glucoside bond include microwave irradiation [33], Co-60 gamma irradiation [34], combining Co-60 gamma irradiation with H 2 O 2 [35], ultrasound [36], UV-irradiation [37], and chemical degradation agent [38][39][40], etc.Among them, the method of heterogeneous degradation of CS with H 2 O 2 was considered effective due to its environmental safety, energy saving, and low cost [41].
Based on the biological properties of CS, studies on modifying CS to enhance its properties have also gained much interest.Typically, the complexation of CS with metal ions is considered a common method to modify CS.Since CS contains -NH 2 and -OH functional groups, it can form complexes with several metal ions such as Cu 2+ , Zn 2+ , Ti 4+ , Ag + , Fe 3+ [42][43][44][45][46], or iodine [47].There have been many studies demonstrating that the antimicrobial activity of the CS-Cu 2+ complex was higher than that of CS.Gu et al (2022) reported that the CS-Cu 2+ complex had more antibacterial activity against Escherichia coli and Staphylococcus aureus than CS [48].In addition, reducing the Mw of CS in the CS-Cu 2+ complex also increased its antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Candida parapsilosis [49].The complexation of CS with metal ions also increased the positive charge, thus enhancing its ability to interact with the negatively charged cell membrane of microorganisms [49].
So far, studies on the dependence of the size of SNPs stabilized in the CS-Cu 2+ complex on different Mw of CS have not been published.Consequently, in this study, we reported a method to reduce the original CS Mw from 132.5 kDa to 80.4-45.2kDa by oxidative hydrolysis with 1% H 2 O 2 solution and using them to complex with Cu 2+ as a stabilizer for SNPs.The characteristic properties of degraded CS and SNPs/CS-Cu 2+ complex were determined by Ultraviolet-visible (UV-Vis), x-ray diffraction (XRD), and Fourier-transform infrared (FTIR) measurements.In addition, the effect of Mw CS used to form CS-Cu 2+ complex on the size of SNPs was also investigated.

Experimental methods
Preparation of degraded CS: Three degraded CS samples were prepared with three replicates.For each sample, 10g CS was soaked in a 100 ml H 2 O 2 1% solution for 30 min, left at room temperature, and then stirred after 24 h.After 24, 48, and 72 h of reaction, the CS/H 2 O 2 mixture was filtered and washed several times with water and vacuum dried to a constant weight to obtain degraded CS powder.Refer to the method of Nguyen et al (2023) [41], based on the amount of degraded CS powder, the CS powder recovery efficiency (E) was calculated according to the formula (1): Where m is a mount of degraded CS powder (g) and m o is the amount of initial CS powder (g).Preparation of SNPs/CS-Cu 2+ complexes: 4g degraded CS was dissolved in 80 ml lactic acid 5% (w/v), the mixture was soaked for 8 h and then filtered through a steel mesh filter with a pore size of 100 μm to remove residue.Subsequently, 2.7g Cu(NO 3 ) 2 .3H 2 O was added to obtain the CS-Cu 2+ complex solution with the [-NH 2 ]:[Cu 2+ ] molar ratio of 2:1, stirred for 60 min.Afterward, a 20 ml solution of 500 mM Na 2 S 2 O 3 was added drop by drop into the 80 ml solution of CS-Cu 2+ complex while stirring on a magnetic stirrer until the Na 2 S 2 O 3 solution was completely added and continue stirring for 30 min for the Na 2 S 2 O 3 hydrolysis reaction to occur completely.The resulting in a 100 ml SNPs/CS-Cu 2+ complex containing 100 mM S, 4% CS, and 0.7% Cu (w/v), pH after the reaction was determined to be ∼5.The reaction to form SNPs occurred according to the following equation: Preparation of SNPs/CS-Cu 2+ powder samples for XRD and FTIR measurements: Following the method of Cuppett et al (2006) [50] and Xie et al (2003) [51] with modifications.The SNPs/CS-Cu 2+ complex solution was precipitated with 97% C 2 H 5 OH with the volume ratio of 3 C 2 H 5 OH:1 solution sample (v/v), then 2 ml of 2% NaOH was added to adjust the pH of the mixture to ∼7.The mixture was stirred and allowed to settle for 2 h, then the precipitate was filtered through filter paper, washed three times with 97% C 2 H 5 OH, and then dried at 60°C to a constant weight to obtain a powder sample of SNPs/CS-Cu 2+ complex.The amount of Cu in the filtrate was determined by inductively coupled plasma mass spectroscopy (ICP-MS) on a PerkinElmer's NexION ® 2000, American.The complexation efficiency of CS with Cu2+ (H) was calculated according to formula (4): Where m o (g) is the initial amount of Cu and m (g) is the amount of Cu in filtrate.
The degree of deacetylation (DD) of CS was determined through Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy on a 500 MHz Advance III HD nuclear magnetic resonance spectrometer (Bruker Biospin, Switzerland), using D 2 O + CD 3 COOH as the solvent.DD of CS was calculated from the 1 H NMR spectrum according to formula (5) [52]: Where I CH3 is the sum of the signal integral of the protons of the acetyl group, I H2-H6 is the integration the signal of the proton from peak H 2 to H 6 .
The Mw of CS samples was measured by Gel permeation chromatography (GPC) spectroscopy on a GPC LC-20AD (Shimazu, Japan), using a 0.25 M CH 3 COOH + 0.25 M CH 3 COONa mixture as eluent and pullulan with Mw from 1.3 to 110 kDa as standard.This device uses an RID 20A detector and Shodex SB803 column HQ [41].
The particle size was calculated from Transmission electron microscopy (TEM) images measured on a JEM 1010 (JEOL, Japan) and presented as average size ± standard deviation (SD).The particle size and particle size distribution were determined using ImageJ 1.54g and MS Excel 2016 software.
The crystal structure was measured on an XRD D8 Advance x-ray diffractometer (Bruker, Germany), using Cu kα (λ = 1.5406Å) radiation at 40 kV and 40 mA, 2θ diffraction angle from 5°to 80° [55].The average size of crystalline (D) was calculated from the XRD pattern according to the Debye -Scherrer formula (6) [56]: Where k is a dimensionless shape factor (k = 0.9), λ (Å) is the x-ray wavelength (λ = 1.5406), β (rad) is the full width at half the maximum intensity (FWHM), and θ (°) is the Bragg angle.The bonds and functional groups formed in the materials were performed on an FTIR 840S spectrometer (Shimadzu, Japan) in the wavenumber range of 4000-400 cm -1 .The samples were prepared in 0.25 mm thickness KBr pellets and stabilized under controlled relative humidity before acquiring the spectrum [57].

Heterogeneous degradation of CS powder by H 2 O 2
The results in table 1 showed that the CS powder recovery efficiency in the samples reached ∼100%, proving that the degrading process did not create water-soluble CS.The Mw of CS determined through GPC spectroscopy (figure 1(a)) was about 80.4, 61.8, and 45.2 kDa after 24, 48, and 72 h, respectively.These results showed that the Mw of CS decreased with reaction time.Additionally, the PI values of CS increased from 1.90 to 2.18 after 72 h of reaction; it might be due to the heterogeneous degrading process, where the oxidizing agent H 2 O 2 preferentially reacted with CS molecules on the solid and liquid phase interface, thus, obtaining CS with uneven Mw [41].Furthermore, the DD of CS samples calculated according to formula (5) through the 1 H NMR spectra  (figure 1(b)) did not change remarkably.This result was also consistent with previously published studies when using H 2 O 2 with a concentration < 2% to degrade CS with little change in the structure of CS [35,58].
The UV-Vis spectra of CS samples are presented in figure 2 showed that there was an absorption peak at 220 nm assigned to the n-σ * transition for the amido group [59] The UV-Vis spectra of degraded CS appeared a peak at 256 nm (figure 2 The XRD patterns of CS samples with different Mw are shown in figure 3.All CS samples showed two typical peaks of CS at 2θ ∼10°and ∼20° [61][62][63] corresponding to the crystal planes (001) and (110), characteristic of the monoclinic crystal lattice [64].Notably, for the heterogeneous degraded CS samples, these peaks decreased in intensity compared to the CS0 samples.The degradation of CS into smaller molecules reduced intramolecular hydrogen bonds, thereby reducing crystallinity compared to CS0 [65].This result is also consistent with the research of some authors when degrading CS using different methods [66,67].
The FTIR spectra of CS samples in figure 4 indicated that all CS samples exhibited characteristic peaks for vibrations of the bonds in CS as follows: The peak at 3437 cm -1 was due to the hydroxyl stretching vibrations [68,69] which shifted to a lower wavenumber for heterogeneous degraded CS samples due to the reduction of intramolecular hydrogen [70].The peaks at 2920-2861 cm -1 were typical for the C-H symmetric and asymmetric stretching vibrations [71,72].Two peaks at 1598 cm -1 and 1650 cm -1 characterized the N-H bending, vibration while the peak at 1420 cm -1 was typical for the stretching vibration of the -CH 2 group [73,74].The FTIR spectra of degraded CS samples did not appear the characteristic peak for the carboxylic at 1735 cm -1 [66], which proved that the degraded CS samples did not create acidic group and have a structure almost similar to CS0. 2+ complexes TEM images and the particle size distribution histogram of SNPs/CS-Cu 2+ complex samples are presented in figure 5, showing that the SNPs had an angular shape.It could be seen that the lower the Mw of CS, the larger the size of SNPs; specifically, the size of SNPs was 25.1, 32.3, and 48.3 nm when stabilized in CS80-Cu 2+ , CS62-Cu 2+ , CS45-Cu 2+ complex solutions, respectively.The above results demonstrated that the Mw of CS had a strong influence on the size of the SNPs.This result was also consistent with the study of Aranaz et al (2018) when stabilizing Ag nanoparticles in CS solution with different Mw.Particularly, CS with Mw > 30 kDa had the effect of creating smaller and more stable Ag NPs than CS with Mw from 5 to 30 kDa [75].Similarly, Phu et al (2010) also demonstrated that CS with higher Mw had a better protective effect on Ag NPs than CS with smaller Mw [76].It is due to steric and electrostatic effects [76,77].Long-chain polymers with more functional groups create an effect that prevents nanoparticles from coming close to each other and agglomeration [76].

SNPs/CS-Cu
The UV-Vis spectra of SNPs/CS-Cu 2+ complexes in figure 6 showed that the characteristic peaks at 256, 260, and 265 nm assigned to CS were almost not changed.The characteristic peak for SNPs in figure 6(a) at 276 nm which shifted to a higher wavelength was 280 nm (figure 6(b)) and 285 nm (figure 6(c)), it might be due to the increase in the size of SNPs as the Mw of CS decreased.In addition, all SNPs/CS-Cu 2+ complexes also appeared a new peak at 680 nm characterizing the d -d electron transfer between the amino group with Cu 2+ ions when forming the complex [54].
After precipitation of the SNPs/CS-Cu 2+ complex with ethanol, the filtrate no longer has the characteristic blue color of Cu 2+ ions (figure 7).The complex formation efficiency of the SNPs/CS80-Cu 2+ , SNPs/CS62-Cu 2+ , and SNPs/CS45-Cu 2+ complexes calculated according to formula (4) was 99.88%, 99.86%, and 99.88%, respectively.As is known, CS is precipitated in water at pH > pKa ∼6.5 [78] or ethanol [51].According to Kim et al (2010), ethanol is a solvent that has the ability to dissolve Cu(NO) 3 .3H 2 O salt much less than water and methanol [79], so using ethanol to precipitate the SNPs/CS-Cu 2+ complex is a reasonable method.Cuppett et al (2006) reported that the maximum solubility of Cu 2+ was 4 mg L −1 at pH 6.5, and the maximum solubility was 1.3 mg L −1 at pH 7.4 [50].The results of analyzing the Cu content in the filtrate solution after precipitation of SNPs/CS-Cu 2+ complex with ethanol at pH ∼6.8 demonstrated that the Cu content in the obtained powder was not lost.
The size of SNPs of SNPs/CS-Cu 2+ complexes calculated by formula (6) through XRD patterns are presented in table 2. The results in table 2 showed that the particle size of SNPs calculated through the XRD pattern was larger than through the TEM image and followed the law of increasing with the decrease in Mw of CS.Pabisch et al (2012) when studying to determine the size of zirconia and silica nanoparticles using many different techniques showed that there were differences in particle size between measurement methods [83].Similarly, Kashkarov et al (2016) also reported a difference in particle size when determined by XRD and TEM imaging for diamond nanoparticles in explosive products [84].
The FTIR of SNPs/CS-Cu 2+ complexes in figure 9 showed that the most characteristic peaks of CS were retained.Although, the peak at 1420 cm -1 decreased in intensity, and two peaks at 1590 cm -1 and 1650 cm -1 were overlapped and shifted to a lower wavenumber of 1550 cm -1 , owing to the formation of a complex between CS and   Cu 2+ ions [42].When the pH is 5.3-5.8, the complexation between the -NH 2 group of CS, and Cu 2+ ion mainly formed [Cu(-NH 2 )] 2+ complex [42,85].This complex had a higher positive charge than NH 3 + (the protonated anime group of CS).Consequently, the CS-Cu 2+ complex created a higher electrostatic interaction with the SNPs than CS, so the SNPs had better stability.Furthermore, the characteristic peaks for the S element also appeared at 451, 644, 898, and 913 cm -1 [81].In addition, two new peaks also appeared at 625 cm -1 and 584 cm -1 in spectra of SNPs/CS-Cu 2+ complex samples, corresponding to the stretching vibration of Cu-N [86].The above results clearly proved the formation of SNPs in the CS-Cu 2+ complex solution by the Na 2 S 2 O 3 hydrolysis method.

Conclusion
In this study, the SNPs/CS-Cu 2+ complexes were successfully synthesized by the acidification of Na 2 S 2 O 3 in the CS-Cu 2+ complex solution.Low Mw CSs (80.4,61.8, and 45.2 kDa) were prepared by the heterogeneous degradation in 1% H 2 O 2 solution at ambient conditions and used to form the complex with Cu 2+ ions.The obtained SNPs had an angular shape, and monoclinic crystal structure with an average size in the range of 25.1-48.3nm depending inversely on the Mw of CS.The obtained SNPs/CS-Cu 2+ complex can be potentially applied as an disease and nematode control agent for plants due to the antimicrobial activity of individual substances.

Figure 7 .
Figure 7. Photographs of the SNPs/CS-Cu 2+ complex solutions before (a) and after (b) precipitation by ethanol.

Table 1 .
The CS powder recovery efficiency (E), Mw, polydispersity index (PI), and DD of CS samples according to the reaction time.

Table 2 .
The particle size of nano sulfur calculated through XRD patterns.