Gel fraction analysis was performed to determine the degree of crosslinking of hydrogels produced by irradiation. The effects of different radiation doses and CS/PVA ratios on the gel fractions of hydrogels are shown in Fig. 1. With an increase in the radiation dose, the gel fraction increased until it reached a maximum at 25 kGy, after which it started to decrease owing to the predominance of chain scission over crosslinking (Tan et al. 2021). At 10 kGy and 30 kGy, 75/25 CS/PVA could not form a gel; meanwhile, at 25 kGy, approximately 20% gel fraction was attained. The gel fraction of the 50/50 CS/PVA hydrogel exhibited significant change greater than 4 times as compared to those of the 75/25 CS/PVA hydrogel at 25 kGy. The fraction of the insoluble part increased with an increase in the PVA content in the composite hydrogel due to the increase in the number of PVA radicals to form 3D networks. Thus, crosslinking predominated degradation. Nevertheless, the gel fraction substantially decreased with an increase in the CS content, because a higher amount of CS as compared to that of PVA hindered radical recombination. Consequently, no crosslinking occurred. In addition, water in the polymer solution played a crucial role in realizing maximum crosslinking, resulting in a higher number of free radicals, thereby improving the generation of macroradicals. Furthermore, H atoms and –OH radicals can produce additional macroradicals by eliminating H from PVA molecules (Jeon et al. 2018). During irradiation, radical crosslinking and chain scission are two main reactions that simultaneously occur.
The ability of a hydrogel to retain water/biological fluid is a critical factor in determining the suitability of the hydrogel for biomaterial applications. The hydrogel experiences a change in swelling rate in a selected pH environment for an excellent tailored drug release.
In DI water, crosslink density is primarily responsible for the swelling abilities of hydrogels. The SRs of hydrogels at different radiation doses and CS/PVA content as a function of time are depicted in Fig. 2. Hydrogels prepared at 10 kGy swelled at different rates when compared with those of the hydrogels fabricated at higher radiation doses (25 and 30 kGy). At 10, 25, and 30 kGy, the equilibrium swelling degrees of the 50/50 CS/PVA hydrogel were 8.4-, 4.7-, and 5.2-fold compared to the dried state after 24 h, respectively. In contrast to the case at 10 kGy, the higher crosslinking of polymer chains at 25 kGy formed a stronger network with higher resistance to expansion, thereby reducing the swelling degree. However, owing to predictable polymer chain scission and a resultant decrease in the crosslinking density, the swelling degree of the hydrogel generated at 30 kGy slightly increased when compared with that of the hydrogel produced at 25 kGy. The amount of absorbed water in the gel network considerably increased before reaching a plateau. Moreover, the SR decreased with an increase in the PVA content, because with an increase in the PVA concentration of hydrogels, the crosslink density increased, thereby decreasing the SR.
Swelling indices in different pH solutions
The swelling behavior of hydrogels is a complicated phenomenon that involves three consecutive steps: diffusion of the solvent into the network, chain relaxation inside the hydrated gels, and network expansion. Functional groups lead to electrostatic repulsion inside the network, thus expanding the gel and ultimately resulting in equilibrium. The presence of more free amino groups in the network leads to stronger electrostatic repulsion between polymer chains and a faster swelling rate (Li 2009). According to the findings of this study, the pH sensitivity of the hydrogel originates from the different swelling degrees of the hydrogel in buffer solutions with different pH values. At pH 7, 10, and 13, the 50/50 CS/PVA hydrogel prepared at 25 kGy had a lower SR than those at pH 1 and 4. The swelling degree of the hydrogel at pH 1 increased by twofold to approximately 7 (g/g) compared to that at pH 13. Furthermore, the ionic strength of the solution may influence the swelling degree. High Mw CS provides a better possibility for crosslinking either chemically or physically which forms a 3D network structure by increasing the entanglement to reduce repulsive force which tends to increase intramolecular interaction at pH 5.5–6 of CS/PVA blended hydrogel. The amino groups of high Mw CS can be protonated (NH3+) in acidic fluids at low pH, and the electrostatic repulsions induced by these ionic groups can increase the hydrophilicity of the hydrogel, thereby expanding the hydrogel networks. In contrast, swelling decreases under neutral (pH 7) and alkaline (pH 10 and 13) conditions due to the deprotonation of these amino groups. Owing to the increasing demand for controlled drug delivery with high accuracy, the pH-sensitive swelling behaviors of hydrogels may be advantageous for regulating drug release (Almáši et al. 2020; Ding et al. 2021; Fan et al. 2019; Yang et al. 2008). During swelling, water molecules invade the hydrogel surface and diffuse inside. Fick’s law is employed to explain the process by which the water molecules diffuse through swollen materials, such as hydrogels. Fick’s laws are depicted in Fig. 3, and the values of k and n are presented in Table 1. For all CS/PVA samples, the estimated n values ranged between 0.2211 and 0.3556, which were all less than 0.5 for the hydrogels investigated herein, which was consistent with the previously reported results (Wang et al. 2008). With an increase in pH, the mobilities of polymer chains reduced; simultaneously, the affinity between the water molecules and the polymer substantially decreased, resulting in a sluggish rate of diffusion of water molecules into the gel; therefore, the n values decreased (Wang et al. 2008). The n values implied that all the CS/PVA samples exhibit Fickian water transport. CS/PVA samples with minimal swelling demonstrate Fickian (less relaxation-controlled) behaviors, because hydrogel ionization is dominant and the H bond regulates solvent transport. The ionization of functional groups affects the mechanism of water diffusion, influencing both the relative magnitude of diffusion and the swelling degree (Ghobashy et al. 2021).
Chemical structures of crosslinked CS hydrogels
Distinctive functional groups and newly formed bonds between neat CS, pure PVA, and CS/PVA hybrid hydrogels were assessed via FTIR absorption spectroscopy and solid-state 13C NMR spectroscopy. FTIR spectra of unirradiated and gamma-irradiated CS/PVA hydrogels are shown in Fig. 4.
FTIR spectrum of pure PVA (Fig. 4a) shows a broad absorption band at 3301 cm−1, corresponding to the stretching and bending vibrations of the –OH group. The peaks at 2923, 1433, and 1090 cm−1 are ascribed to the C–H stretching of alkyl groups, C–H bending, and C–O group stretching, respectively (Abureesh et al. 2016). The peak of neat CS at 3330 cm−1 is attributed to the vibrational stretching of the N–H and O–H intermolecular and intramolecular H bonds. Stretching vibration absorption peaks of C–H on the CS chain were observed at approximately 2879 cm−1. Moreover, the absorption peaks of CS were noticed at 1645 (amide I), 1567 (amide II), and 1387 cm−1 (amide III), which primarily originated from the stretching vibration of C–O, the vibration of the N–H bond, and the stretching vibration of the C–N bond, respectively (Kong & Yu 2007). Peaks related to the antisymmetric stretching of the C–O–C bridge and C–O vibration of the ring, which are characteristic peaks of the saccharide backbone, were observed at 1141 and 1021 cm−1, respectively (Bisen et al. 2017). FTIR spectrum of the hybrid hydrogel exhibits all the characteristic peaks of both CS and PVA hydrogels. The peaks of the non-irradiated samples shifted from 3304 to 3291, 3279, and 3287 cm−1 compared to those of the irradiated samples because of the formation of an intermolecular H bond between CS and PVA. This H bond functions as a connector between the two polymers. In addition, the obtained data indicated that no substantial degradation of CS occurred in the case of the 50/50 hydrogel samples irradiated at different radiation doses (Fig. 4b) as the intensity of the peak at 1567 cm−1 had slightly increased (Mozafari et al. 2012; Bisen et al. 2017; Casimiro et al. 2021; Casimiro et al. 2021).
Chemical structures of hydrogels were verified by solid-state 13C NMR spectroscopy. Table 2 presents the integrals of the NMR resonances of the particular functional groups discovered in pure CS, before and after irradiation of neat PVA, irradiated CS/PVA hydrogel. The corresponding assignments are as follows: 0–49 ppm: alkyl C; 49–62 ppm: N–CH; 62–94 ppm: O-alkyl C; 94–110 ppm: O–C–O anomeric C; and 160–188 ppm: COO and N–C–O (Duarte et al. 2020). 13C MAS NMR spectra clearly show the peak of methylene C (–CH2–) at 44.80 ppm and methine C (– CH–) resonances at 64.53, 70.48, and 75.32 ppm. The methylene carbon is responsible for a well-separated peak at 44 ppm in the both non-irradiated and irradiated PVA spectrum. Carbon connected to OH groups may be allocated to the peaks at 64, 70, and 76 ppm (Jayasekara et al. 2004). Peaks I and II have been ascribed to the isotactic structure with two intramolecular H bonds and the heterotactic structure with one intramolecular H bond, respectively, whereas Peak III has been assigned to the syndiotactic structure with no intervening intramolecular H bonds. Comparing neat PVA before and after irradiation spectra to those of the equivalent dry gels demonstrates that the gelation process fragments the network of the intramolecular hydrogen bonds based on previously reported NMR studies on PVA (Padavan et al. 2011; Lai et al. 2002). In blended hydrogel (Fig. 4c), the combination of PVA and CS peaks was obtained. In addition, the C3 and C5 peaks at 75.32 ppm were two overlapping peaks, which arose from the intramolecular H bond (Wang et al. 2019). C peaks were observed in the spectrum of pure CS (C = O: 174 ppm; C1: 104 ppm; C4/C3: 85–82 ppm; C5: 75 ppm; C2: 60; C6: 58 ppm; and CH3: 23 ppm). Herein, the C1 peak showed minor low-field shifts after the irradiation of the hybrid hydrogel as compared to the case of pure CS and intramolecular/intermolecular H bonds formed around C2, C3, C5, and C6 during crosslinking, resulting in slight low-field shifts of the corresponding peaks, which were in agreement with the findings reported in the literature (Yang et al. 2021; Heux et al. 2000).
To elaborate the crosslinking mechanism of CS/PVA hydrogel by gamma irradiation, the proposed crosslinking process of CS/PVA hydrogel under gamma irradiation is shown in Scheme 1. Water in aqueous solutions absorbs the majority of the gamma-radiation energy. Radiolytic products of water are mainly formed by indirect action on water molecules yielding radicals ·OH, e−aq, and ·H. (Makuuchi and Cheng 2012). The main reactive species is •OH, which readily removes H from polymer chains and causes the production of CS and PVA radicals and water. In the final phase, covalent bonds between the polymer chains are generated by recombining two macro radicals which are PVA–CS, PVA–PVA, and CS–CS radicals.
TGA was conducted to better understand the thermal characteristics of the hydrogels. Figure 5a shows the weight loss (TG) and derivate (DTG) curves of pure PVA, neat CS, and blended CS/PVA hydrogels with different contents of CS and PVA and at various radiation doses. According to the TGA data, CS deteriorated in two stages. The first signs of degradation appeared at 60 °C, resulting in a 10% weight loss due to the loss of water molecules. Thermal and oxidative degradations of CS were responsible for the subsequent 49% weight loss between 280 and 350 °C. It was caused by the breakdown of the primary components of CS, most notably the heat degradation of the pyranose ring and the fracture of the b-glycosidic bonds connecting the glucosamine and N-acetylglucosamine moieties (Pawlak and Mucha 2003; Martel-Estrada et al. 2014). Moreover, the main decomposition (DTG curve) occurred between 300 and 414 °C and was attributed to the dehydroxylation of PVA, suggesting the initiation of polymeric chain decomposition, and the subsequent decomposition occurred between 414 and 475 °C. This phase involved the continuation of the polyene structure via the generation of C and hydrocarbons (Release 2017). The decomposition temperatures (Td) of CS, PVA, and blended hydrogels were 223, 267, and 240–266 °C, respectively (Fig. 5a). Thus, PVA had the highest Td and was most thermally stable owing to intramolecular and intermolecular H-bonding between its chains. Furthermore, hybrid hydrogels with 50–75% PVA were more thermally stable than those with 25% PVA. This improvement in thermal stability originated from the high degree of crosslinking induced by gamma radiation, which resulted in the formation of a network structure. When the radiation dose was increased from 10 to 30 kGy, the Td of the 50/50 CS/PVA hydrogel slightly increased, revealing higher crosslink densities at higher radiation doses (Fig. 5b).
Morphologies of the crosslinked CS hydrogels
For migration, drugs absorb and release in a 3D network; thus, the morphology and interconnectivity between pores are crucial factors in this regard. Topologies of the hydrogel networks with various CS/PVA ratios were examined using SEM. The average diameter of 50/50 CS/PVA hydrogels at 10, 25, and 30 kGy was determined from SEM images employing a magnification of 1000x. Twenty locations were randomly chosen and measured for each sample using ImageJ software. The hydrogel framework obtained at 10 kGy had fewer holes and larger pore sizes of approximately 8.83 µm, indicating limited crosslinking points between polymers. The greater crosslinked network structure was formed when the radiation dose was increased to 25 and 30 kGy. The average pore sizes of the samples decreased to 3.59 µm when the radiation dosage was increased from 10 to 25 kGy and slightly expanded to 4.63 µm at 30 kGy (Fig. 6). With a further increase in the radiation dose, a minor alteration in the porous structure was noticed. With an increase in the PVA concentration, numerous connected chains were generated, reducing the average pore size. Moreover, the morphology of neat PVA, which exhibited an extremely inferior porous pattern, was different from those of the hybrid hydrogels. The porous interpenetrating mesh structure provided excellent permeability, which improved drug transport through the hydrogels. Due to their highly interconnected porous structures forward the moderate pore size, the blended hydrogels may be used for the loading and release of medicines owing to their better swelling characteristics as compared to those of other hydrogels (Vo et al. 2020).
In vitro amoxicillin release studies
The goal of localized release systems is to concentrate medicine concentration in the targeted organ to reduce side effects caused by unfocused release points (Ilgin et al. 2019), Fig. 7a. In this condition, the amount of amoxicillin uptake of CS/PVA gels was 11.133 ± 0.231 µg/mg polymer. At 37 °C, UV spectrophotometry was employed to monitor amoxicillin in vitro release from hydrogel networks over 1440 min following pH changes in PBS medium and DI water. Figure 7b depicts the drug release findings. It is noted that the fast release of amoxicillin in all of the samples at different pH values took place in the first 300 min. The percentage release of amoxicillin from hydrogel at 1440 min was estimated at 85%, 50% at pH 2.1 and 7.4 in PBS media; at 34% at pH 5.5 in DI water. Under acidic conditions, the amount of drug released from the hydrogel increased owing to electrostatic repulsion induced by the protonation of amino groups as this repulsion offered a larger surface area for drug release. Furthermore, the porous structure played a crucial role in drug release as it enhanced the drug permeation ability (Aycan and Alemdar 2018; Constantin et al. 2017; Mulchandani et al. 2017). According to the results, the release of amoxicillin in the physiological environment of PBS is greater compared to in DI water. The higher percentage of released drugs from CS/PVA hydrogel might be due to amoxicillin solubility which is impacted by pH and ionic strength (Palma et al. 2016). Higher ionic strengths were expected to weaken the molecular structure of the polymer by increasing repulsive electrostatic interactions between charged polymer molecules (Vigata et al. 2020).
The use of mathematical modeling to achieve this goal is extremely advantageous, since it allows for the estimation of release kinetics before the creation of free release systems. Typically, the model is created by measuring several critical physical characteristics, such as the drug diffusion coefficient and experimental release data (Ilgin et al. 2019). Model-linked techniques, including zero-order, first-order, Hixson–Crowell, Higuchi, and Korsmeyer–Peppas models were utilized to examine the optimal drug release kinetic mechanism explaining the solution profile, and are summarized in Table 3.
In a zero-order model, drug elimination is constant regardless of concentration, whereas, in a first-order model, drug elimination rises proportionately as concentration increases (Rungrod et al. 2021). The plot according to the zero-order and first-order equations showed not best fitted with r2 values obtained around 0.76 and 0.90 for amoxicillin release in PBS at both 2.1 and 7.4. Meanwhile, zero-order kinetics describes the process of constant drug release from a drug delivery system that corresponds to drug release in DI medium with an r2 value of up to 0.97. It is noted that the drug release mechanism in DI water is governed by the relaxing of polymeric chains and has a constant release rate regardless of the concentration of the drug.
Higuchi drug release is a diffusion method based on Fick’s law, which proposes that matrix swelling and evaporation are minor or insignificant and have a square-root time dependency (Kumari & Meena 2021). The correlation coefficients obtained for the Hixon–Crowell model (0.79–0.91) were lower than those found for the Higuchi model, indicating that this model could not suit the release mechanism. The diffusion-controlled release was eventually discovered to be the primary mechanism of drug kinetics compared to a change in the surface area and diameter of particles.
To explore the drug release mechanism from hydrogel or exist in more than one sort of release phenomenon, the Korsmeyer–Peppas model is helpful. In this model, a range of parameters composing polymer swelling, erosion, matrix porosity, and drug diffusion rates in swelling systems was investigated (Ilgin et al. 2019). The reported literature reveals that if n < 0.45, solvent penetration into the hydrogels follows the Fickian process. Furthermore, if n is between 0.45 and 0.89, the drug release is controlled by diffusion and polymer network relaxation. This is referred to as a non-Fickian process. However, several n larger than 0.89 represents drug release as a function of polymer gel system expansion or relaxation. In this work, amoxicillin releases followed non-Fickian with n values from 0.61 to 0.72 for different pH environments and the r2 value applied for all conditions was greater than 0.95, as shown in Table 3. As a result, drug release occurs in response to both diffusion and swellable porous matrix.
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