Development of a Novel Antibacterial Hydrogel Scaffold Based on Guar Gum/Poly (methylvinylether-alt-maleic Acid) Containing Cinnamaldehyde-Loaded Chitosan Nanoparticles

In this study, we produced a novel chemical cross-linked Guar gum/Poly(methylvinylether-alt-maleicacid) (GG/PMVE-MA) hydrogels with various blending weight ratio of GG, and PMVE-MAn (GG/P20, GG/P40, and GG/P70). These produced hydrogels were analyzed by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), thermogravimetric analysis (TGA), swelling degree, and mechanical characteristics. The results demonstrated that with increasing PMVE-MAn content, thermal stability, swelling degree, and mechanical characteristics of hydrogels were improved. As a result, the GG/P70 hydrogel was selected as an optimal hydrogel. Moreover, MTT analysis indicated that these hydrogels were non-toxic and any reduction or stop of cells growth wasn't observed over time. Additionally, encapsulation of cinnamaldehyde (CA)-loaded chitosan nanoparticles (CSNPs) into optimal hydrogel formulation significantly (P < 0.05) increased scavenging of 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical about 60%. In addition, the inhibition capability of GG/P70/CA-loaded CSNPs hydrogel against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), demonstrating that the hydrogel had high antibacterial and antioxidant activities. The general results showed that this composite hydrogel (GG/P70/CA-loaded CSNPs) could be useful for various applications such as drug delivery, tissue engineering and food industry.


Introduction
Newly, biopolymer-based hydrogels have been extensively identified as biomaterials due to their high biocompatibility, safety, bioactivity, and biodegradability [1][2][3][4]. Hydrogels are unique category of biomaterials with three-dimensional polymeric structures which can absorb high content of water or biological fluids, and display swelling behavior instead of dissolving in aqueous environment [5,6]. Hydrogels can be applied in many applications for example; drug delivery, tissue engineering, wound dressing and food packaging [7]. Guar gum (GG) is one of the suitable biopolymer for synthesis of hydrogels due to the low cost, hydrophilic nature and non-ionic polysaccharide which containing of galactose and mannose units [8][9][10][11]. However, the main limitation of GG for hydrogel fabrication is its high hydrophilic properties, and also reduction in viscosity [12,13]. These limitations of GG can be overcome by combining it with a synthetic/ semi synthetic polymer, and chemical modification to produce an effective hydrogel [14]. Thereby, choosing the suitable synthetic polymer to overcome the limitations of GG is important.
Poly [(methyl vinyl ether)-alt-(maleic anhydride)] (PMVE-MAn) is a hydrophilic and biocompatible polymer which known as Gantrez®AN. The hydrolyzed form of poly [methylvinylether-alt-maleic acid] (PMVE-MA) with the carboxylic acid functional groups can be used as support for drug delivery and cell growth. Hydrogels can be obtained by esterification grafting crosslinking reaction between maleic anhydride groups of PMVE-MAn and hydroxyl or amine groups of natural polymers [15]. Therefore, this synthetic polymer can act as a cross linker and create chemical cross-linking with GG.
Cinnamaldehyde (CA) is one of important constituent of cinnamon (Cinnamomum zeylanicum) which is existent in its essential oil. Because of high safety, and antibacterial effects against Gram positive and Gram negative bacteria, CA has many applications in several fields [16]. However, CA is unstable, volatile, and has a low water solubility which limits its application [17,18]. In order to overcome these limitations, CA can be encapsulated in natural safe nanoparticles [19]. Chitosan (CS) is widely applied as nontoxic material to produce nanoparticles as drug delivery system. CS is a natural, non-toxicity, biodegradable, biocompatible, and de-acetylated polysaccharide derived of chitin. Additionally, as a cationic polymer, it can be suitable for encapsulating active ingredient [20].
In this current study, the GG/PMVE-MA composite hydrogels were successfully produced. The various combination ratios of GG and PMVE-MAn were evaluated to assess their effects on the physicochemical properties of hydrogels. Chemical structure, morphology, thermal stability, mechanical properties, swelling ratio, and cell viability of hydrogel samples were determined. After that, CA was successfully encapsulated in the CS nanoparticles (CSNPs) and loaded in optimal GG/PMVE-MA composite hydrogels. Additionally, the particle size of CSNPs, antioxidant activity, antibacterial activity of optimal hydrogel containing CA-loaded CSNPs were assessed. This paper presents a scientific report of developed GG/PMVE-MA/ CA-loaded CSNPs composite hydrogels for use in different industrial applications.

Synthesis of CSNPs and CA-Loaded CSNPs
CA-loaded CSNPs were prepared using ionic gelation assay with minor change [21]. In briefly, CA (0.3 mL, 1 v/v%) was added slowly into preheated CS solution (0.4% w/v) under magnetic stirring. After that, TPP solution (0.3% w/v) was added gradually into solution under magnetic stirring (at 800 rpm) for 20 min at room temperature to obtain CA-loaded CSNPs. Tween 80% was used as a surfactant. The nanodispersion was then centrifuged at 10,000×g for 1 h and washed by distilled water (four times) to remove the unloaded CA.

Particle Size, Zeta Potential and Encapsulation Efficiency (EE) Studies
The particle size and zeta potential of CSNPs were calculated with a dynamic light scattering (DLS) (Zetasizer Nano-ZS90, Malvern Instruments Ltd., Worcestershire, UK) under vacuum condition [22,23]. All evaluations were performed at room temperature. The EE% of CA-loaded CSNPs was calculated by UV-Vis spectrophotometer (Spectrum SP-UV500DB) at 285 nm.

Preparation of Composite Hydrogels and CA-Loaded CSNPs Incorporated with Hydrogels
GG (0.2% w/v)/PMVE-MA (2% w/v) hydrogel samples were prepared as follows; aqueous solutions comprising various weight ratios of GG, and PMVE-MAn were put in 24-well plates. Solutions were freeze-dried for 72 h. The final hydrogel samples were taken from the 24-well plates. The freeze-dried hydrogel samples were put into an oven at 75 °C for 3 days and 8 h for each day. The final obtained hydrogels were cited in a high content of water for 24 h. Finally, these hydrogel samples were frozen in liquid nitrogen and after that freezedried for 48 h.
To prepare of hydrogels containing CA-loaded CSNPs, the certain amount (10% wt versus total polymers weight) of CA-loaded CSNPs was dispersed in GG solution (0.2% w/v) under continuous mechanical stirring for 24 h and sonicated for 20 min. After that, this solution was mixed with different amounts of PMVE-MAn solution (2% w/v) to obtain the formulations coded as GG/P20, GG/P40, and GG/P70 (Table 1).

Characterization
FTIR spectra were recorded for GG, PMVE-MA, and final hydrogels with Thermo Avatar 370 spectrometer (Ten-sor27, Bruker Co., Ettlingen, Germany) in transmittance mode amongst 400-4000 cm −1 . The morphology of hydrogels was analyzed by a scanning electron microscopy (SEM, Hitachi High-Tech HITACHI, Tokyo, Japan). SEM images were analyzed and evaluated for surface pore size and the appearance of an interconnecting pore structure, and average pore diameter. To investigate the thermal stability of produced hydrogels, thermogravimetric analysis (TGA) was performed with a LINSEIS SPA PT 1600 device (Germany) at a range of 20-600 °C, at a heating rate of 10 °C/min and under N 2 .

Swelling and Degradation Behaviour
To measure the swelling degree of hydrogel samples, gravimetric analysis was performed [24]. In short, at first the dry weight of the hydrogels was measured (W d ), after that the hydrogels were immersed in 30 mL of PBS solution at room temperature. After that, the swollen hydrogels were weighed after 2, 4, 7, 10, 12, 24, 36, 72, and 96 h (W s ). The swelling degree (SD) was measured according with the equation: In the presence of lysozyme, the mass change of the dried samples could evaluate the degradability test of the hydrogels after the incubation in degradation solutions (shaking at 37 °C, at about 50 rpm) [25]. The solution was changed every day by a fresh solution. Control group was incubated in PBS, while other groups were rinsed in a buffer containing enzyme (100 U per mL gel). At the specified times, the samples were removed, washed with distilled water and then freeze dried to obtain a dried sample. The mass weight loss was calculated using the following equation: W i and W f indicated the initial and final dry weight of the hydrogels samples, respectively.

Mechanical Characteristics
Using a universal testing system (INSTRON series 3366), the mechanical properties of the samples were investigated. The initial elastic modulus was calculated from the slope of the initial linear segment of stress-train curves, Tensile strength was taken as the maximum force divided by the minimum cross-sectional area of the specimen to find a precise tensile strength. The strain value corresponding to the maximum stress was calculated as a strain at break.

Release Behavior of CA
The in-vitro release of CA from the CSNPs and hydrogels was determined using the dispersion of freeze-dried NP powders and hydrogels in prepared PBS solution (0.01 M, at pH 7.4) and incubated for 48 h at 37 °C. After incubation period, samples were centrifuged for 15 min at 8960×g (Universal 32R, Hettich, Tuttlingen, Germany). Then, 100 μL of supernatant was mixed with 1.9 mL of anhydrous ethanol and its UV absorbance was measured at 289 nm. The concentration of the released CA was measured from a calibration curve of CA in ethanol [26].

Cell Viability Study
The MTT technique was accomplished to explore the cell viability of hydrogel samples [27][28][29]. In summary, the hydrogel samples were cut into 12 mm 2 and immersed in 75% ethanol for 60 min to be sterilized. After that, 1 mL of cell suspension including 5 × 10 4 cells was seeded onto sterilized hydrogel samples and incubated in RPMI-1640 including 10% FBS at 37 °C with 5% CO 2 . After 1, 3, 5 and 7 days, 200 μL DMSO was added to dissolve formazan crystals formed inside cells. Then, absorbance was determined using spectrophotometer (UV-2550, Shimadzu, Japan) at 570 nm.

Antioxidant Capacity
The antioxidant capacity of the optimal hydrogel and optimal hydrogel containing CA-loaded CSNPs was calculated with the DPPH radical scavenging assay during 9 h of incubation [30]. In summary, 0.1 mM of DPPH solution in ethanol was prepared. Next, 1 mL of DPPH solution was added to 3 mL of solution containing hydrogel and hydrogel/CAloaded CSNPs. Finally, after 30 min, the absorbance was measured at 517 nm assessed with a UV-Vis spectrophotometer (Spectrum SP-UV500DB) after 3, 6, and 9 h. The DPPH radical scavenging was calculated with the equation: 1 3 where Abs DPPH was the absorbance of DPPH ethanolic solution and Abs extract was the absorbance of the hydrogels.

Antibacterial Activity
Antibacterial activities of the optimal hydrogel and optimal hydrogel containing CA-loaded CSNPs against S. aureus and E. coli bacteria were measured using disk diffusion methods [8]. In short, 3-5 mm disk of hydrogel sample was exposed on Mueller-Hinton agar plate that cultured by 1.5 × 10 8 CFU/mL concentration of each bacterial suspension. After 24 h, inhibition of bacterial growth area about the hydrogel sample disk was determined and expressed.

Statistical Analysis
Statistical differences (P < 0.05) were analyzed using oneway ANOVA followed by Tukey's test for multiple comparisons by GraphPad Prism 5. Obtained results were expressed as mean ± standard deviation (SD). The level of significance was considered P < 0.05.

Synthesis of Cross-Linked GG/ PMVE-MA Hydrogels
In a preliminary experiment, the non-cross-linked samples were unstable in water due to the dissolution of polymer chains. To synthesize the cross-linked hydrogels, the aqueous solutions comprising various concentrations and weight ratios of GG and PMVE-MAn were mixed. In this regard, two condensation reactions could take place: (i) the anhydrization between the carboxylic groups of PMVE-MAn (ii) the esterification between the carboxylic groups of PMVE-MAn and the hydroxyl groups of GG [31]. By the formation of inter-macromolecular ester bonds, the cross-linked hydrogels were prepared by thermal treatment of the samples.
DPPH radical scavenging(%) = Abs DPPH − Abs extract Abs DPPH × 100 After that, the hydrogels were washed in a large amount of water to remove the soluble fraction of the polymer. During washing, the anhydrides bonds were hydrolyzed, re-forming the free carboxylic groups. The schematic image of the synthesized hydrogels is shown in Scheme 1.

The Assessment of Particle Size, Zeta Potential and TEM Image of CA-Loaded CSNPs
The TEM image confirmed non-aggregation of CA-loaded CSNPs with an average diameter of approximately 220 nm, which is similar to the report of PCS analysis (Fig. 1). According to this image, the NPs were well dispersed, smooth and spherical in shape. The formation and size distribution of CA-loaded CSNPs was investigated using DLS analysis. The size of CA-loaded CSNPs was 256 ± 42 nm. The CA-loaded CSNPs nanodispersion revealed a poly dispersity index (PDI) of 0.277 and zeta potential of 35 ± 8 mV.
The EE% was calculated using a standard curve of CA. The EE (%) of CA-loaded CSNPs was observed to be 69.25 ± 1.2%.

FTIR Characterization
The FTIR spectra of GG, PMVE-MAn, and GG/PMVE-MA hydrogels were demonstrated in Fig. 2. The FTIR spectrum of GG showed an extensive peak at 3539 cm −1 attributed to the OH stretching and the peak at ∼ 2920 cm −1 related to the CH stretching. Also, the peak at 1641 cm −1 attributed to C-OH vibrations [8]. The FTIR spectrum of PMVE-MAn indicates the attendance of a peak at 1107 cm −1 owing to C-O-C group. The peak at 1701 cm −1 attributed to the carbonyl groups of acidic groups of PMVE-MA [32,33]. In the FTIR spectrum of GG/PMVE-MA hydrogels, the peak at 1701 cm −1 which related to the carbonyl groups of PMVE-MAn was shifted to 1732 cm −1 , which indicates the occurrence of a reaction between PMVE-MAn and GG. Additionally, the peak at 1641 cm −1 which related to C-OH vibrations of GG, was shifted to 1648 cm −1 , that approved the occurrence of ester and anhydride bonds in the GG/PMVE-MA hydrogels.

Morphological Studies
The morphology of GG/PMVE-MA hydrogel samples was investigated with SEM analysis. The SEM results of freezedried hydrogels with various combination ratios of GG, and PMVE-MAn was demonstrated in Fig. 3. As is clear in Fig. 3, the structure of the all hydrogel samples was porous. However, the changes of the PMVE-MAn content seem to affect the morphology of the hydrogels. The hydrogels containing a large content of PMVE-MAn (GG/P70 sample) demonstrated a spongy, and dense structure with smaller pores. This result can be related to the increasing of crosslinking density resulted from the occurrence of esterification and anhydrization reaction in the GG/PMVE-MA hydrogels. The similar results can be seen in previous studies [34][35][36]. Porosity also plays an important role in the function of the implant as mass transport of nutrients and must be possible to facilitate seeding and subsequent cell proliferation. Important parameters are pore size and size distribution, pore interconnection size, total pore surface area, and morphology [37][38][39]. The pore size must be within a critical range of 20-200 µm, depending upon application, so that the cells are housed in a compartment with sufficient space to grow and attach well to the material. Porous networks extending through an implant increases the surface area, allowing a greater number of cells to attach to the matrix. This enhances the regenerative properties of the implant by allowing the tissue ingrowth directly onto the interior of the matrix. A minimum pore size of 150 µm has been suggested in the literature for bone [40] and 200-250 µm for soft tissue [41]. Therefore, the objective of this work was to produce degradable scaffolds, consisting of pores with an average diameter between 100 and 300 µm. As revealed in Fig. 3, scaffolds produced with different content of PMVE-MAn as crosslinker agent were found to have a significant variation in mean pore size between the transverse and cross sections, suggesting that, for GG/P70 sample with higher content of PMVE-MAn, the more pores are formed rather than GG/P40 and GG/P20 samples. is related to the loss of free and bound water adsorbed in the structure of hydrogels. The second stage of weight loss occurred at the range of 150-380 °C which is associated to the chemical decomposition of the GG [42]. Weight loss in the second stage for hydrogel sample with high content of GG (GG/P20) occurred with more rate. By increasing the content of PMVE-MAn in the structure of GG/PMVE-MA hydrogels the thermal stability was improved owing to the chemical crosslinking reaction between the -OH groups of the GG and the carbonyl groups of the PMVE-MAn [32,34].

Swelling and Degradation Behavior
One of the most crucial parameters in the application of hydrogels in biomedical applications is their ability to absorb fluids and exchange nutrients and waste. Hydrogels owing to their cross-linked structure are 3-dimensional polymeric scaffolds that are capable of absorbing large volumes of water and swelling without dissolving. Meanwhile, the hydrogel swelling nature could affect the mechanical features and hydrolysis rate; therefore, the hydrogels hydrophilicity may be as an essential property for the tissue engineering uses. The equilibrium swelling degree and the elastic modulus of hydrogels depend on the cross-link and charge densities of the polymer [43]; the amount of water absorbed by the gel depends on the porousness of the structure, the type of materials used and the crosslink density [44]. Crosslinking density also influences the swelling capacity of a hydrogel. If a higher concentration of cross-linking agent produces the higher cross-link density, it results in additional network formation taking place. Subsequently the network structure of hydrogel is disturbed and a compact structure is formed which repels penetration of the larger amounts of water. As reported in previous literatures, by increasing the swelling degree, the mechanical strength of hydrogels decreased and suffer from weak mechanical properties [45][46][47]. The swelling degree of the hydrogel samples with various blending weight ratios of GG, and PMVE-MAn was determined in PBS (pH 7.4) solution at 37 °C after 2, 4, 7, 10, 12, 24, 36, 72, and 96 h. Figure 5 presented the swelling profile of hydrogel samples. As can be seen from this figure, the swelling ratio of hydrogel samples decreased over time with increasing the PMVE-MAn content. So, GG/P70 and GG/P40 showed the lower swelling degree in comparing with GG/P20. As reported by Wu et al. hydrogels present a typical swelling-weakening phenomenon because of the dilution of the network, which always suffers from a sharp decline in mechanical strength after swelling and largely limits the application, particularly when a given mechanical strength is required, such as biological glues or artificial tissues [47]. Therefore, GG/P20 could not to be a suitable platform for biological application.
In-vitro degradation study was applied to estimate the degradation behavior of hydrogels and the results are presented in Fig. 6. As shown in this figure, the hydrogels with low content of PMVE-MAn (GG/P20) were more rapidly degraded than the other hydrogel formulations (GG/P40 and GG/P70). The increase in the degradation of the hydrogel structure with increasing immersion time was detected after 12 days. After 12 days of incubation, the hydrogel with the low content of PMVE-MAn showed a mass loss significantly higher than the other samples which may be due to the decreased crosslinking density of the network and consequently more penetration of water molecules in enzymatic condition. At this time point, the GG/P40 and GG/P70 hydrogels showed the same degradation trend that were presenting a similar degradation profile. As mentioned above, the increased swelling ratio of hydrogel displays its increased rate of degradation. Therefore, the GG/P70 and GG/P40 can be used as a suitable acellular matrix to retain the cell for a long time.

Mechanical Characterization
The mechanical characteristics of hydrogels are one of the important parameters for evaluating their durability and resistance to external forces [48]. The mechanical characteristics of the hydrogels were investigated by tensile strength (TS), elongation at break (EAB), and elastic modulus [49]. Since pure GG-based hydrogels have weak and undesirable mechanical properties, improving, and reinforcing these properties by adding synthetic polymer and or cross-linking can be very effective [50]. In this paper, the mechanical characteristics of hydrogels were slightly improved by increasing the content of PMVE-MAn. As shown in Fig. 7, the order of the mechanical characteristics of GG/PMVE-MA with different blending weight ratio of GG, and PMVE-MAn was found as: GG/P70 > GG/P40 > GG/P20. The obtained results showed that with more PMVE-MAn content, the mechanical characteristics of the GG/PMVE-MA hydrogels were improved. It was due to the reduced cross-linking density in the samples with lower PMVE-MA content (GG/P20), as mentioned by Bahadoran et al. [51]. However, no significant difference in elongation at break was observed between GG/ P70 and GG/P40 (P > 0.05). Furthermore, Elastic moduli of 90 ± 4.2 MPa, 83 ± 1.75%, and 90.2 ± 1.4 kPa, were achieved for the hydrogels with the samples coded as: GG/P70, GG/ P40, GG/P20, respectively. This result suggested that elastic moduli of the GG/P70 and GG/P40 were significantly higher than GG/P20, resulting in a stiffer hydrogel (P < 0.05). Since the cells exposed to stiffer substrates experience a higher elastic modulus in their plasma membrane with a betterorganized actin cytoskeleton, so the cells cultured on stiffer substrates proliferate faster and migrate slower compared to those cultured on soft substrates [52]. Besides, the elastic performance of hydrogels is achieved via the control over properties such as Young's modulus (E) [53], tensile strength [54], failure strain [55] and compressive strength [56]. Therefore, the TS, EAB, and elastic modulus of GG/ PMVE-MA hydrogel with ratio of GG/P20 may not to be a suitable substrate in comparing to GG/P70 and GG/P40 for biological application [51]. These results were related to the degree of chemical crosslinking between the -OH groups of GG and carbonyl groups of PMVE-MAn which had hardened the hydrogel structure. This result is consistent with the results of other research in this area [57][58][59][60].

Release Behavior
CA is known to exhibit antimicrobial activity against a wide range of bacterial species. Therefore, controlled release of CA from the CA-loaded CSNPs and hydrogel containing CA-loaded CSNPs were studied. Release profiles showed during 48 h in Fig. 8. The CA release from hydrogels is achieved by degradation of the polymer matrix of hydrogels, which leads to its release. Therefore, as shown in Fig. 8, CA-loaded CSNPs delivers more CA than polymer hydrogels which has released about 50% in 48 h. As is clear in Fig. 8, the release of CA from the optimal hydrogel (GG/ P70) was relatively slow over 48 h. Moreover, the release rate of hydrogels containing 10% CA-loaded CSNPs was calculated to be around 39%.

Cell Viability
The biocompatibility of the hydrogel samples for cell growth was investigated using MTT assay. Figure 9, illustrated the cell viability of NIH3T3 fibroblast cells in the attendance of the GG/PMVE-MA hydrogels with different blending weight ratios of GG, and PMVE-MAn (GG/P20, GG/P40, and GG/ P70). As realized from this figure, all hydrogel samples were appropriate for cell growth and survival and the cell growth wasn't stop or even decrease. This obtained result could be related to the safety of GG, and PMVE-MAn which have the capability to interactions between growth factors and other Extra Cellular Matrix (ECM) proteins which can enhanced cellular activities [61,62].

Antioxidant Capacity
To induce antioxidant and antimicrobial capability for the produced GG/PMVE-MA hydrogels, CA-loaded CSNPs was encapsulated in the optimal hydrogel formulation (GG/P70). In this regard, the hydrogel extract exposed to DPPH radicals and the resulting adsorption was determined at 517 nm after different reaction times (3, 6, and 9 h) to assess the antioxidant capacity. The results of DPPH radical scavenging activity assay were displayed in the Fig. 10. DPPH method indicated that the DPPH radical scavenging rate of blank hydrogel was only 6.21 ± 1.23%, and also wasn't observed significant changes in the DPPH radical scavenging rate with increasing reaction time, indicating that its antioxidant capacity was low. Whereas, the encapsulation of CA-loaded CSNPs into the optimal hydrogel formulations (GG/P70) induced the antioxidant activity and increased the DPPH radical scavenging rate. This result is consistent with the results of other research in this area [63][64][65][66][67][68]. Also, the DPPH radical scavenging rate of hydrogel containing CAloaded CSNPs increased with increasing reaction time that can be associated to the increased CA release, resulting in increased reaction rate and DPPH radical scavenging.

Antibacterial Activity
To evaluate the antibacterial effect of optimal hydrogel (GG/ P70) containing CA-loaded CSNPs, the agar disc diffusion assay was used to found the amount of clear zone obtained from a circular hydrogel sample disk. The obtained results indicated that the control sample (GG/P70) did not inhibit the growth of the pathogenic bacteria. As shown in Fig. 11, E. coli (Gram negative bacteria), and S. aureus (Gram positive bacteria) were sensitive to CA essential oil. In this content, the diameter of the clear zone is 14.3 ± 1.12 mm for S. aureus. There are similar results in many previous studies confirmed our results [18,63,65,69,70]. Since, the Gramnegative bacteria have a more complex wall structure, so, the Gram-negative bacteria outer mats prevents the penetration of CS-loaded CSNPs into the bacterial cell.

Conclusion
The chemical cross-linked GG/PMVE-MA hydrogels with various combination weight ratios (GG/P20, GG/P40, and GG/P70) were successfully produced. The biological and physicochemical properties of produced hydrogels were investigated using different analysis. The hydrogel with more content of PMVE-MAn (GG/P70) demonstrated the high cross-linking density and spongy structure with smaller and more pores. Also, by enhancing the content of PMVE-MAn, mechanical properties and swelling ratio significantly Overall results showed that this composite hydrogel (GG/P70/CAloaded CSNPs) can be useful for various applications such as drug delivery, tissue engineering and food industry.