Introduction

Thermosensitive hydrogels [1] are prepared based on a "sol–gel" transition principle. It is a three-dimensional network polymer with high adsorption capacity [2], porous structure [3], excellent biocompatibility [4, 5], bioadhesion, adequate mechanical strength and mechanical properties [6,7,8]. Poly(n-isopropylacrylamide) (PNIPAM) [9, 10] is a heat-responsive material with reversible swelling and distension, which conforms to the "sol–gel" transition law of hydrogel. Its low critical solution temperature (LCST) is 32 °C, which is close to the temperature of human skin. Below the LCST, the intermolecular hydrogen bonding of PNIPAM leads to a negative entropy change, so that PNIPAM mixes with the solvent to become a flowing sol. Above the LCST, the PNIPAM molecular chains are wound and compressed to expel the solvent, resulting in a structurally compact gel. Due to this unique behavior, it has received extensive attention as a novel biomedical hydrogel in embolic agents, drug delivery, human implantable materials, and biological cartilage tissue [11, 12]. Iatridi et al. [13] synthesized a thermo-responsive copolymer of N-isopropylacrylamide-grafted sodium alginate P(NIPAM-co-NtBAM)-NH2), enriched with hydrophobic N-tert-butylamide monomer, and studied its thermal response and shear response properties through rheology. The graft copolymer formed a 3D network through thermo-induced hydrophobic association of the thermo-responsive P(NIPAM-co-NtBAM) side chains in water. It is shown that the thermo-induced thickening effect was mainly due to the slowing down of the P(NIPAM90-co-NtBAM10) associative side chains exchange dynamics. Moreover, the combination of shear- and thermo-responsiveness provided excellent hydrogel injectability with instantaneous gelation at physiological temperature. Kalyani [14] is focused on the preparation of novel thermally sensitive nano ZnO imprinted poly(N-isopropylacrylamide)/polyethylene glycol-dextran (PNIPAM/PEG-D/ZnO) nanocomposite hybrid hydrogels through in situ technique for the in vitro release of ciprofloxacin (CFX) antibiotic drugs. The results indicated that the PNIPAM/PEG-D/ZnO) nanocomposite hybrid hydrogels exhibited pH-sensitivity and could be applied efficiently as biodegradable carriers for in vitro release of CFX model drug.

With the widespread use of hydrogels, injectable hydrogels for the human body require higher safety and non-toxicity. The most important is the choice of cross-linking agent to be green. Biodegradable crosslinkers are compounds that have a structure that can be hydrolyzed or hydrolyzed enzymatically under physiological conditions. It is mainly divided into two categories: small molecules and polymers. In the traditional hydrogel synthesis process, small molecule cross-linking agents [15], such as glutaraldehyde, are often added. Although these crosslinkers have a good cross-linking effect on the formation of hydrogels, they are also recognized as toxic crosslinkers [16,17,18]. Therefore, the research and development of non-toxic new polymer cross-linking agents is more necessary.

In this paper, a series of new maleic acid-chitosan (MCS) crossliners were prepared by modifying chitosan with maleic acid. Besides, the thermosensitive hydrogel P(NIPAM-co-MA)/MCS was prepared by copolymerization of N-isopropylacrylamide (NIPAM) monomer with maleic acid (MA) as a hydrophilic modifier. One of the carboxyl groups of maleic acid was used in amidation with the amino group of chitosan to increase the hydrophilicity of chitosan. At the same time, carbon–carbon double bonds were introduced into the structure of chitosan, so that it could be co-polymerized with monomers containing double bonds in situ through chemical cross-linking. The preparation method is green, non-toxic, simple and efficient, which provides ideas and methods for the development and preparation of new cross-linking agents and the study of injectable thermosensitive hydrogels.

Experimental

Materials

N-Isopropyl acrylamide (NIPAM containing stabilizer MEHQ) was obtained from Maclin (Shanghai, China). Maleic acid (MA, AR), chitosan (CS, 100–200 mPa s), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC 98.5%) and N-hydroxysuccinimide (NHS 98%) were obtained from Aladdin (Shanghai, China). Absolute ethanol (AR), ammonium persulfate (APS), sodium hydroxide (NaOH, AR) and hydrochloric acid (HCl, AR) were obtained from Kemio (Tianjing, China).

Fourier transform infrared spectroscopy (FTIR, Bruker VERTEX70), nuclear magnetic resonance (NMR, Bruker AVANCELL III 600 MHz), scanning electron microscopy (SEM, HITACHI SU8010) and synchronous thermal analyzer (TG-DSC, TA), contact angle measuring instrument (SDC-100) were performed in this study.

Preparation of thermosensitive hydrogel P(NIPAM-co-MA)/MCS

Preparation of the crosslinker MCS

Preparation of chitosan solution

A 50 mL of water, 1 g of chitosan, and 0.696 g of maleic acid were added to a beaker. Chitosan solution with mass concentration of 2% was prepared by stirring at 35 °C for 2 h. This solution is denoted as solution A.

Activation of maleic acid 1.710 g of EDC, 1.035 g of NHS, and 25 mL of water were added to a round-bottom flask. One gram of maleic acid was activated by carboxyl groups for 2 h under ice bath conditions. This solution is denoted as solution B.

Preparation of the cross-linking agent Solution A was slowly dropped into solution B at low temperature. A yellow product was obtained after a period of reaction. Finally, the reaction liquid was dropped into excess absolute ethanol to precipitate the solid material. The white solid product MCS was obtained by pumping, washing, re-pumping and freeze-drying. The mechanism of maleic acid activation and crosslinker preparation is shown in Fig. 1.

Fig. 1
figure 1

a Mechanism of maleic acid activation, and b mechanism of crosslinker preparation

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The orthogonal test of three factors and three levels L9 (33) was used to explore the influence of three factors, namely reaction temperature, reaction time and a molar ratio of feed (CS: MA), on the degree of acylation at their respective three levels (0, 10, 20 °C), (12, 24, 36 h) and (1:1, 1:1.5, 1:2), as shown in Table 1, in an attempt to find the best preparation scheme of crosslinking agent.

Table 1 Factors and levels
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Preparation of thermosensitive hydrogel P(NIPAM-co-MA)/MCS

The P(NIPAM-co-MA)/MCS hydrogels were prepared by free radical polymerization according to the following procedure. First, the temperature was set at 55 °C and the magnetic stirring speed at 600 r/min. At that time, 2 g of isopropyl acrylamide, 0.5 g of maleic acid, 20 mL of deionized water, and 0.2 g of cross-linker were added to the three-mouth flask and stirred for half an hour under a vacuum. Then the initiator of 2% ammonium persulfate was added, and the reaction lasted for 12 h to obtain the thermosensitive hydrogel product. The mechanism of hydrogel preparation is shown in Fig. 2. To explore the effect of maleic acid content on the LCST of hydrogel, the mass ratio of NIPAM to MA was changed to 10:1, 8:1, 6:1 and 4:1, respectively, and P(NIPAM-co-MA(1–4))/MCS was prepared. Without maleic acid, the pure NIPAM polymer is P(NIPAM-MA0)/MCS, and the specific composition of the hydrogel raw materials is shown in Table 2.

Fig. 2
figure 2

Mechanism of hydrogel preparation

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Table 2 Ratio of raw material for hydrogel preparation
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Characterization

The Fourier transform infrared (FTIR) spectra of MCS were recorded in transmittance mode for the wavelength range of 500–4000 cm−1 with a resolution of 4 cm−1, using Bruker VERTEX70 FTIR Spectrometer.

The nuclear magnetic resonance (NMR) spectra of MCS were recorded in transmittance mode for the chemical shift range of 1–10, using Bruker AVANCELL III 600 MHz NMR spectrometer.

The acylation degree was measured by titration: 0.2 g of the crosslinker product was placed in a 500 mL conical flask and 30 mL of 0.1 mol/L NaOH standard solution was added. The excess NaOH was titrated with 0.1 mol/L HCl standard solution using phenolphthalein as an indicator. The degree of acylation can be calculated by the following formula:

$$\mathrm{DC}=\frac{\left({V}_{\mathrm{NaOH}}\times {c}_{\mathrm{NaOH}}-{V}_{\mathrm{HCl}}\times {c}_{\mathrm{HCl}}\right)\times M\times 100}{m}$$

where VNaOH and CNaOH are the volume and concentration of NaOH standard solution, VHCl and CHCl are the volume and concentration of hydrochloric acid consumed, M is the molecular weight of the substituent group, and m is the mass of the sample to be measured.

The pretreatment was performed on the samples used for testing SEM[1]. P (NIPAM-co-MA)/MCS hydrogel was swelled by water absorption, and then freeze-dried. Before SEM analysis, the surface of the P(NIPAM-co-MA)/MCS hydrogels was Au–Pd coated.

The LCSTs of the P(NIPAM-co-MA)/MCS hydrogels were determined using a differential scanning calorimeter DSC [1]. Before DSC analysis, the P(NIPAM-co-MA)/MCS hydrogels were swollen in distilled water for 1 h at room temperature and then placed into the refrigerator for 2 h to achieve uniform diffusion of water molecules throughout the hydrogel network. The DSC measurements were carried out using hermetic aluminum pans under a nitrogen purge gas flow of 30 mL min−1. The P(NIPAM-co-MA)/MCS samples in the swollen state (10 ± 0.5 mg) were equilibrated at 20 °C and heated up to 50 °C at 1 °C min−1.

The sample was placed in a constant temperature blast oven at 35 ℃ and continuously dried to constant weight. The dried sample was placed in 100 mL of distilled water and subjected to swelling kinetics testing at room temperature. At the beginning of the experiment, samples were taken out at a fixed time. After absorbing surface moisture with filter paper, the mass of each sample was accurately weighed. As the experiment progresses, the experiment was stopped until there is almost no change in the mass of the sample between the two measurements. Finally, the average of the three measurements was taken. In addition, the sample with swelling equilibrium was tested for weight under different temperature conditions.

The water contact angle of P(NIPAM-MA)/MCS was recorded using an SDC-100 contact angle tester.

Results and discussion

FTIR analysis

Figure 3 shows the FTIR spectra of MA, CS and MCS. According to the infrared spectrum of MA, the C=O absorption peak at 1700 cm−1 is mainly due to the bimolecular association in the maleic acid molecule, which makes it move to the low wavenumber direction. According to the IR spectra of CS, the bending vibration absorption peak of -NH2 was found at 1600 cm−1 [19]. According to the IR spectra of MCS, the C=O stretching vibration absorption peak of the carboxyl group was at 1710 cm−1. The absorption peak at 1630 cm−1 is the superposition of C=C and amide C=O stretching vibration absorption peaks. The in-plane bending vibration absorption peak of the N–H in the second band was 1570 cm−1, and the C–N absorption peak of the third band was 1270 cm−1. It can be seen that MCS contains amide bonds and carbon–carbon double bonds, which proves that the carboxyl group of MA and the amino group of CS react, proving that the cross-linking agent is successfully prepared.

Fig. 3
figure 3

FTIR spectra of: (1) MA, (2) CS, and (3) MCS

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NMR analysis

The NMR spectra of CS and MCS are shown in Fig. 4. The peak at 11.30 ppm in spectrum (1) is the deuterated trifluoroacetic acid peak. The peak at 4.89 ppm in the spectrum (2) is the deuterated heavy water peak. The peak between 2.5 and 3.6 ppm was reduced to the proton hydrogen peak on the chitosan ring, and it was found by integration that the peak areas in this range were almost the same. By comparing the NMR spectra of CS and MCS, a new characteristic peak of –CH=CH2 appeared at 6.52 ppm, indicating the introduction of a double bond of maleic acid into the chitosan structure. NMR spectra showed that the characteristic structures of MA and CS were present in the product, and the integrated infrared test results showed that the MCS were successfully prepared.

Fig. 4
figure 4

NMR spectra of: (1) CS and (2) MCS

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Acylation degree analysis

The results of MCS acylation degree of the crosslinker prepared by the orthogonal test are shown in Table 3. K1, K2, and K3 are the sum of the data at each level of each factor. For each factor, the largest value of K corresponds to the best level. R is the range value of the factor, that is, the maximum value minus the minimum value of K under each factor. The larger the R, the greater the effect of the change in the level of this factor on the trial. According to the range analysis, the influence degree of the three factors A, B and C on the acylation degree was A > C > B. Among them, temperature is the optimal factor, showing that the reaction temperature is the main factor affecting the degree of acylation of the product.

Table 3 Acylation test result of the orthogonal test
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As can be seen from the table, the acylation test results of the nine groups of experiments prepared were 62.1%, 65.2%, 67.3%, 63.0%, 71.5%, 62.8%, 62.3%, 58.9% and 56.3%, respectively. Among them, the highest acylation degree was 71.5% in experiment 5. The optimal preparation conditions for this experiment were A2B2C3, that is, the temperature was 10 °C, the reaction time was 24 h, and the feeding ratio CS:MA = 1:2.

In this experiment, the crosslinker product is well soluble and is a white powder after drying and grinding. In the process of product processing, it was found that the crosslinker prepared at 20 °C (experiments 7, 8, 9) had a yellow color and poor water solubility. The reason may be that the activation of maleic acid is affected, and the reaction ratio of the chitosan amino group is reduced, making the acylation at higher temperatures less effective. When the temperature was reduced (experiments 1, 2, and 3), the degree of acylation was between 60 and 70%, which was still less than 71.5% of experiment 5. The reason is that although the low temperature is beneficial to the activation of maleic acid by the activator, the continuous low temperature during the reaction after the activation will affect the activation energy of the reaction, and the effective collision rate between the reactant molecules is not high, so the acylation effect is not optimal. When the temperature was 10 °C (experiments 4, 5, and 6), the secondary factor affecting the degree of acylation was the feeding ratio. When the amount of maleic acid is increased, the molar ratio of the carboxyl group is excessive, which can react better with the amino group of macromolecular chitosan and achieve the effect of increasing the degree of acylation.

SEM analysis

The SEM micrographs of P(NIPAM-co-MA)/MCS hydrogels are shown in Fig. 5. As can be seen from Fig. 5, the hydrogel has regularly arranged pores and a characteristic spongy porous structure. This result is mainly due to the presence of a large number of carboxyl groups (derived from MA). The more hydrophilic hydrogels absorbed more water and produced larger space inside them. Wei et al. reported that fewer closed pores (with small size) were observed in conventional PNIPAM hydrogel [20]. In another report, Liu et al. reported that conventional PNIPAM hydrogel showed the typical microporous morphology with dense walls [21]. In this study, due to the introduction of MA in the molecular chain, the content of -COOH significantly increases, which can form hydrogen bonds with water molecules. The water absorption ability of the hydrogel is improved, which further leads to the expansion of the polymer network and the expansion of the pore size.

Fig. 5
figure 5

SEM micrograph of the P(NIPAM-co-MA)/MCS hydrogel

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LCST analysis

The LCST of the hydrogel was determined by endothermic peak analysis by DSC [1]. Figure 6 shows the temperature transition point test results of thermosensitive hydrogels with different acid contents. It can be seen from the figure that the temperature transition point of the hydrogel is continuously increased with the increase of MA content compared with pure PNIPAM. The LCSTs were 33.50, 36.29, 36.66, 37.88 and 38.31 °C, respectively. The analysis showed that the increased LCST value may be the result of an enhanced hydrophilicity of the hydrogel. The increase in maleic acid content (–COOH) allowed an increase in the hydrophilic group in the hydrogel, interrupting the arrangement of the hydrophobic group (N-isopropyl group) of the heat-sensitive NIPAM. In other words, the hydrophobic/hydrophilic nature of the copolymer determines the heat of phase transition and the LCST value of the NIPAM copolymer [1]. Due to the occurrence of copolymerization, the amount of N-isopropyl group in the monomer molecule is reduced. Therefore, the increase of LCST would become proportional to the content of hydrophilic copolymer (MA). On the other hand, as a macromolecular cross-linking agent, MCS itself can increase the density inside the hydrogel, which can improve the thermosensitive transition point of the hydrogel.

Fig. 6
figure 6

Temperature transition point of thermosensitive hydrogels with different acid contents

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In this study, the ratio of the weight of hydrogel at different times to the weight of blank hydrogel was used to explore its swelling rate by soaking the hydrogel in deionized water. Moreover, the degree of deswelling is explored by weighing the swelling-balanced hydrogel at different temperatures. Figure 7 shows the research results of hydrogel swelling kinetic curves in deionized water at 20 °C and swelling ratio values in deionized water as a function of temperature from 20 to 50 °C. It can be seen from Fig. 7a that with the increase of maleic acid content, the swelling degree of the hydrogel increases first and then decreases, which are 26.9, 39.9, 123, 140 and 180, respectively. Among them, the equilibrium swelling of P(NIPAM-MA4)/MCS is the largest, which can be explained by the hydrophilicity of MA introduced into the hydrogel [22]. When the MA content increases, the hydrogels become more hydrophilic and absorb more water, leading to an increased equilibrium swelling ratio. In addition to high hydrophilicity, the free space inside the hydrogels may be an important factor influencing the swelling rate and equilibrium swelling ratio. Figure 7b shows the thermal sensitivity of the hydrogel. It can be seen that in the temperature range of 20–30 °C, the hydrogel has a high swelling property. When the temperature increases from 30 to 35 °C, the swelling ratio decreases significantly, which is mainly due to the temperature sensitivity of the hydrogel. When the temperature is above 40 °C, the swelling property of all hydrogels decreases to the minimum. This result is due to the LCST of all hydrogels below 40 °C. Generally, the thermo-responsive hydrogel shrink above the LCST and swell below the LCST. When the temperature is above 40 °C, the swelling property of all hydrogels decreases to the minimum. This result is due to the LCST of all hydrogels below 40 °C. At high temperatures, the molecular chain of hydrogel shrinks to the smallest volume.

Fig. 7
figure 7

Photographs of a swelling kinetic curves in deionized water at 20 °C, and b swelling ratio values in deionized water as a function of temperature from 20 to 50 °C

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Hydrophilic effect analysis

The contact angle is a simple and effective method to evaluate the hydrophilicity and hydrophobicity of a material surface. The maleic acid variable was used as a single factor to explore its effect on the hydrophilicity of the hydrogel. The results of the contact angle test are shown in Fig. 8. The test results of (1)–(5) correspond to each sample in Table 2. Figure 8 shows that the hydrophilic angles of the hydrogel materials are 66.879°, 63.494°, 58.385°, 45.782° and 36.282°, respectively. It can be seen that with the increase of maleic acid content, the water contact competition of the hydrogel material gradually becomes smaller, that is, the hydrophilicity is becoming better. This result is due to the increasing content of carboxyl groups in MA. When the water droplets touch the surface of the material, the water molecules combine with the carboxyl groups to form hydrogen bonds due to their interaction forces. Thus, the wetting effect becomes better. Among them, when NIPAM:MA = 4:1, the hydrophilicity is the best, but combined with the temperature sensitivity analysis, the temperature transformation point under this condition is 38.31 °C, which is very high to be conducive to the hydrogel formation. Considering the temperature of the hydrogel in human practical application, 38.31 °C has exceeded the normal temperature range. Comprehensive analysis showed that when NIPAM:MA = 8:1, the hydrophilicity and temperature sensitivity of the hydrogel are better. The P(NIPAM-MA2)/MCS hydrogel material has a better comprehensive effect.

Fig. 8
figure 8

Contact angle test results of the hydrogel

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Conclusion

An acylated chitosan crosslinker was prepared by low-temperature activation of maleic acid by EDC/NHS, followed by grafting modification of chitosan. From FTIR and NMR analysis, the carbon–carbon double bond of maleic acid was successfully introduced into the chitosan structure. A novel macromolecular crosslinking agent-acylated chitosan was successfully prepared, and the acylation degree of the crosslinking agent reached 71.5%.

N-Isopropyl acrylamide and maleic acid were successfully cross-linked to form hydrogel under the action of MCS crosslinker. SEM characterization results showed that the hydrogel of a porous structure. DSC characterization results showed that the hydrogel had better temperature response performance, and the temperature transition point increased to a certain extent compared with pure poly(N-isopropyl acrylamide). NIPAM has both hydrophilic and hydrophobic groups, so the hydrogel has good temperature sensitivity. In addition, the carboxyl group of maleic acid was successfully introduced into the hydrogel system, which also played a role in regulating the hydrophilicity of the hydrogel. The LCST of hydrogel is improved to a certain extent, which can reach the physiological temperature.

The novel crosslinking agent prepared in this paper is expected to replace the traditional small molecule hydrogel crosslinking agent. In addition, the thermosensitive hydrogel is injectable and temperature sensitive at the same time. The area required for implantation can be used at syringe pressure, and after injection, gelation occurs after reaching the temperature transition point, which can avoid its diffusion in the surrounding tissues. These hydrogels are expected to have excellent performance in drug delivery or absorption applications. In future experiments, we will analyze the mechanical properties and cytotoxicity of the carrier. This study can provide a certain research basis and relevant theoretical basis for clinical practice.