Investigation on pH-switchable (itaconic acid/ethylene glycol/acrylic acid) based polymeric biocompatible hydrogel

Abstract

In this paper, a new variety of pH-sensitive polymeric hydrogels (IAE) have been developed and evaluated as biocompatible hydrogels using synergetic combinations of itaconic acid (IA), acrylic acid (AA), and ethylene glycol (EG) in water medium by free radical polymerization. The prepared hydrogels were characterised by FT-IR and SEM, which revealed the presence of functional groups and the morphological aspects of the IAE based hydrogels. The detailed swelling behaviour of various stoichiometric amounts of IAE based hydrogels has been studied at varying pH (1.2, 6.0, 7.4 and 10.0) with respect to time from 0 to 360 minutes. The comparative swelling equilibrium, biodegradation and biocompatibility studies of hydrogels have also been investigated. The antibacterial activities of the IAE based hydrogels were examined using pathogenic microorganisms viz. E. coli, S. aureus and B. cereus. Furthermore, the IAE hydrogels were also subjected to antifungal activity against Aspergillus niger & Candida albicans using the agar well diffusion method at various concentrations. The improved biocompatible properties of these gels imply that the IAE hydrogels have good potential for future biomedical applications in controlled drug release.

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1. Introduction

Hydrogels are an insoluble, three dimensional crosslinked macromolecular network consisting of hydrophilic, homo-, hetero-, or co-polymeric units which have the ability to absorb enormous amounts of water.¹ Since they possess a significant amount of water content, their degree of flexibility and texture are highly comparable with natural tissues. The hydrophilic and hydrophobic harmonizing character of a gel can provide properties for appropriate applications.² Hydrogels are versatile biomaterials for various applications which include biomedical applications, drug delivery, contact lenses, dialysis membranes, tissue engineering, wound healings, heart-valves, compressed tablets, site specific drug-carriers, water purification, membranes for molecular separation etc.; however, the poor biocompatibility of hydrogels restrains their utility in many cases, especially in drug delivery applications.³ Itaconic acid (IA) and acrylic acid (AA) are widely used monomers for the preparation of pH-sensitive hydrogels because they are biocompatible in nature and can be copolymerized easily.⁴ Itaconic acid has a high potential for substitution with acrylic acid due to the ionized hydrogen bonds. Hence, itaconic acid can easily facilitate itself to form hydrogen bonds with other carboxylic side groups.⁵ Unlike monocarboxylic acrylic acid, itaconic acid possesses two carboxylic (COOH) groups with different pK_a values. Hence, a smaller amount of itaconic acid can enhance pH-sensitivity and increase the swelling nature. Itaconic acid based hydrogels are hydrophilic and biocompatible due to their availability from natural resources.^6,7 In general pH-sensitive hydrogels contain pendant acidic or basic groups (carboxylic acids, sulphonic acids, primary amines, or ammonium salts). This pH-responsive swelling behavior is due to the ionization of functional groups in the gels, with response to changes in the pH. The pH-sensitive hydrogels can be used to release drugs towards different pH environments due to the hydrogel’s varied swelling ability, namely the controllable release of the drug to the target position. The pH-sensitive hydrogel changes it’s swelling behavior, according to the natural pH environment of the gastrointestinal tract in the mammalian body, which varies from acidic in the stomach to neutral in the intestinal track, hence pH sensitive hydrogels have valuable applications in the biomedical field.⁸

In recent years, biodegradable hydrogels have been applied in the field of drug delivery, to release low molecular weight drugs as well as bioactive agents such as proteins, fatty acids and other nutrients.^9,10 Biodegradable hydrogels are either prepared by a synthetic or natural manner. The efficiency of drug release hydrogels can be controlled by regulating the degradation rate. Biodegradable hydrogels can release drugs at a specific site in the body. In other words, drug pointing can be attained using biodegradable hydrogels. Biodegradable hydrogels can also own other properties, such as bioadhesive, pH-sensitive, temperature-sensitive, or other environment-sensitive properties. These possessions can be used to design self-regulated drug delivery systems. The drug release rate of biodegradable hydrogels can be controlled by a number of influences, such as the biodegradation kinetics of the polymeric hydrogel,^11–13 the physical and chemical properties of the hydrogel and drug etc.^14,15

Previously, many researchers have reported the synthesis of hydrogels by using toxic organic solvents.¹⁶ Green chemistry is aimed at obtaining greater alternatives for toxic solvents, minimizing the synthesis procedure and simplifying purification techniques. In recent years, solvent-free reactions have drawn considerable attention and popularity.¹⁷ Hence, the present investigation aimed to develop pH-responsive hydrogels under organic solvent-free reaction conditions and a simple methodology was adopted with a shorter reaction duration. In this current study, IAE based biocompatible, non-toxic, anti-bacterial and anti-fungal hydrogel based biomaterials were prepared. A biomaterial can be a biological or synthetic substance which can be introduced into body tissue as part of an implanted medical device or used to replace an organ, bodily function, etc. A good biomaterial shows more than 50% of the cytotoxic index (IC > 50%). In general, hydrogels are most important for drug carriers to attend to a patient’s physiological need at a stipulated time or a proper site. Hence, a new variety of biocompatible hydrogels has been developed with unique pH-sensitivity. The performance of the hydrogels that can be controlled by the architecture has a pronounced effect on swelling, morphology, porosity and the diffusion process. The cytotoxicity of the hydrogels was evaluated using a 3T3 fibroblast cell line (MTT) assay. The cell viability at the time of measurement was more than 90%, which indicated that the product of degradation and any by-products of the synthesis were non-toxic to the mammalian cell culture. Further, powerful antibacterial activity against Staphylococcus aureus (Gram +ve), Escherichia coli (Gram −ve) and Bacillus cereus (Gram +ve) was shown by the synthesized hydrogels. The detailed investigation progressed to reveal that the synthesized itaconic acid based hydrogels are pH-sensitive to the external environment. Subsequently, cell proliferation, anti-bacterial and anti-fungal activity and in vitro degradation behaviour were also studied in this paper.

2. Experimental

2.1. Materials

Itaconic acid (IA) was procured from Sigma Aldrich Company (Bangalore, India). Ethylene glycol (EG) and acrylic acid (AA) were obtained from Merck. AA was vacuum distilled at 54 °C/25 mm Hg to remove the inhibitor hydroquinone. Potassium persulphate (K₂S₂O₈) was obtained from Merck. N,N′-Methylene bis-acrylamide was purchased from Sigma Aldrich. Demineralized water was used for both polymerizations and the preparation of the buffer solution. Double distilled water was used throughout the experiments.

2.2. Preparation of polymeric hydrogel IAE

The pH-sensitive hydrogel was prepared based on Guhanathan et al. 2015, with minor modifications.¹⁸ At first, an equimolar mixture of itaconic acid and ethylene glycol was dissolved in a polymerization tube at 60 °C for 30 minutes with continuous stirring under an inert nitrogen atmosphere. The obtained product was labelled as pre-polyester. Subsequently, the initiator K₂S₂O₈ (0.05 g) and N,N′-methylene bis-acrylamide (0.05 g) cross linker were also introduced along with a stoichiometric amount of acrylic acid (AA). The free radical polymerization was progressed for 1 h 30 min at 60 °C with constant stirring under nitrogen atmosphere in the polymerization tube. The completion of the polymerization reaction was ensured when a straw yellowish insoluble product was obtained (Scheme 1). The IAE hydrogels have been prepared using constant acrylic acid while changing the stoichiometry of selected monomers of itaconic acid and ethylene glycol. The prepared hydrogels were immersed in distilled water for about seven days to remove traces of unreacted monomers with a subsequent change of DM water regularly. The notation and description of the IAE based hydrogels are listed in Table 1.

Scheme 1 The mechanism for the formation of the IAE hydrogels.

Table 1 Stoichiometric amounts and physical appearance of polymeric hydrogels based on IA, EG and AA (unit: mol) and distilled water (unit: ml)

S. No.	Sample	Composition (mol)				Observations
		IA	AA	EG	Distilled water
1	I1A1E4	0.01	0.025	0.04	10.0	Straw yellowish gel, insoluble in water
2	I2A1E3	0.02	0.025	0.03	10.0	Straw yellowish gel, insoluble in water
3	I3A1E2	0.03	0.025	0.02	10.0	Straw yellowish gel, insoluble in water

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2.3. FT-IR studies

Infrared spectras were recorded using an Alpha Bruker Fourier-transform infrared spectrometer (FTIR), with a resolution of 2 cm⁻¹. The hydrogel samples were prepared by adding polymeric hydrogel (1 g l⁻¹) on a pressed KBr pellet. The measurements were carried out at room temperature. Spectra were recorded between 4000 and 400 cm⁻¹ at 2 cm⁻¹ resolution.

2.4. pH-Sensitive swelling studies

Swelling is an important property of polymeric hydrogels. The dried hydrogel discs were immersed in 50 ml of carbonate buffer solution (CBS) of desired pH values ranging from 1.2 to 10.0 at ambient temperature. The swelling tendency of the hydrogels was analysed periodically. Swollen hydrogels were taken out from the buffer solution, blotted with clean tissue paper and the hydrogel samples were weighed at necessary intervals. Subsequently they were transferred in the same flask. The required pH of the buffer medium was adjusted through 0.01 M dil HCl and 0.01 M dil NaOH, checked by the pH meter (Citizen 3000). All of the swelling experiments were performed in triplicate. The degree of swelling (S%) was calculated through the following eqn (1)where W_d is the initial weight of the dried hydrogel, and W_t is the weight of the swollen sample at regular time interval t. W_eq is the weight of the swollen sample at equilibrium. The equilibrium degree of swelling (S_eq%) was calculated by eqn (2)

2.5. Scanning electron microscopy (SEM)

Microstructure characterization of the IAE based hydrogels was performed by JSM-6701S scanning electron microscopes. All of the samples were freeze dried and kept in vacuum until the silver sputtering treatment prior to SEM analysis.

2.6. In vitro antibacterial activity assay

The IAE hydrogels were inoculated in tubes with sterile saline solution (3 ml) for 24 h at 37 °C. The selected microorganisms were Staphylococcus aureus (MTCC430), Bacillus cereus (MTCC3311) and Escherichia coli (MTCC739). The test was performed in Nutrient Agar (NA) plates seeded with an 8 h broth culture of different bacteria. In each of these plates, wells were cut out using a sterile cork borer. The samples were carefully added with different concentrations (500, 1000, 1500 and 2000 μg) into the wells by using a sterilized dropping pipette. The wells were allowed to diffuse at room temperature for 2 h. Later the plates were then incubated in a closed container at 37 °C for 18–24 h. Gentamicin (10 μg) was chosen as a positive control and distilled water was used as a negative control. The antimicrobial activity of the IAE hydrogels was evaluated by measuring the diameter of the inhibition zone.

2.7. Anti-fungal assay

The antifungal activity of the IAE hydrogels was determined by using the well diffusion method. Petri plates were prepared with 20 ml of sterile MHA (Hi-media, Mumbai). The test culture was swabbed on top of the solidified media and allowed to dry for 10 min. Wells were made on the media using a well borer. Different concentrations of the sample (0.5, 0.75 & 1 mg per well) were loaded on the wells. Ketakonazole (10 μg per well) was used as a positive control. These plates were incubated for 48 h at 28 °C under closed conditions. The zone of inhibition was measured in millimeters (mm).

2.8. In vitro cytotoxicity studies

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for high throughput screening (HTS). The MTT tetrazolium assay technology has been widely adopted and remains popular in academic labs as evidenced by thousands of published articles. The MTT substrate is prepared in a physiologically balanced solution, added to cells in culture, usually at a final concentration of 0.2–0.5 mg ml⁻¹, and incubated for 1 to 4 hours. The quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. A reference wavelength of 630 nm is sometimes used, but is not necessary for most assay conditions. Cytotoxicity was determined using Graph pad prism-5 software.

2.9. Soil buried biodegradability

Biodegradable hydrogel provides a special advantage by degrading in an aqueous environment, thereby avoiding the need for removal after usage. The biodegradability of the IAE hydrogels was determined under a natural environment. 0.5 g of the IAE based hydrogel was buried 15 cm deep in soil. The place was enriched with varieties of plants. The relative humidity was maintained at about 60%. The IAE hydrogels were allowed to stand at atmospheric conditions for 90 days. The degree of degradation was monitored at 15 day intervals. In order to observe biodegradation, the IAE hydrogels were removed from the soil and washed with water to remove environmental impurities. After washing, the hydrogels were placed in a vacuum for 8 h at 37 °C to attain a constant weight. The rate of degraded weight% was calculated by the weight loss method using following formula (3).where M_t1 is the pre weight of dried hydrogel and M_t2 is the post weight of degraded hydrogel at each time interval.

3. Results and discussion

3.1. FT-IR spectroscopic analysis of IAE hydrogels

FT-IR was used to confirm the composition of the IAE based hydrogels. The FT-IR spectra of the I₃A₁E₂ hydrogel are shown in Fig. 1a. The broad peak at 3564.1 cm⁻¹ corresponds to the hydrogen bonded O–H stretching vibration.^19,20 The peak observed around 1696.7 cm⁻¹ belongs to the C=O stretching vibration.²¹ The clear peak at 2916.5 cm⁻¹ was attributed to aliphatic CH₂ stretching frequencies.^22,23 Many peaks appeared in the range of 1500 to 700 cm⁻¹ due to the presence of CH, CH₂, C–C, C–O and OH groups.^24,25 The short band at 1618.2 cm⁻¹ might be due to the presence of carboxylate symmetric and asymmetric stretching.^26,27 In general, the hydroxyl group (O–H) enhances the hydrophilicity, biocompatibility and mechanical properties of the hydrogels.^28–30 The FT-IR spectra of the I₁A₁E₄ based hydrogel are shown in Fig. 1b. A broad absorption band appeared at 3564.1 cm⁻¹ due to the hydrogen bonded O–H group. The intensive absorption peak at 1696 cm⁻¹ was assigned to the stretching vibration of C=O. A clear band around 2925.7 cm⁻¹ corresponded to the presence of CH₂ stretching frequencies. A doublet sharp peak at 1206.2 cm⁻¹ denoted the presence of C–O stretching. C–O–H in plane and out of plane bending was noticed at 1409 and 900.5 cm⁻¹, respectively. Similar to our concern, Guadalupe Rodr et al.³¹ have also observed. Fig. 1c shows the FT-IR spectra of the I₂A₁E₃ hydrogel. A clear peak was observed at 1686.5 cm⁻¹ due to the presence of the C=O stretching frequency. The stretching band in the region of 2917.3 cm⁻¹ might be due to CH₂ absorption. A broad peak originated at 3536.7 cm⁻¹ due to hydrogen bonded O–H absorption. Similarly, a C–O stretching peak was observed at 1215 cm⁻¹.^32,33 The fingerprint region of EG was observed at 1600–800 cm⁻¹ for all obtained IAE hydrogels.³⁴ The stoichiometric amount of itaconic acid and ethylene glycol in IAE based hydrogels exhibits noticeable differences in the IR spectrum. The FT-IR spectroscopic analysis indicates the conversion of all monomeric units into the polymeric hydrogel network. The necessary functional groups were not lost during the hydrogel formation.

Fig. 1 Comparative FT-IR spectra of IA, I₃A₁E₂ (a), I₁A₁E₄ (b) and I₂A₁E₃ (c).

3.2. pH-Sensitive swelling of IAE based hydrogels

Fig. 2 shows the stoichiometric effects of the monomer on the swelling nature of the IAE based hydrogels. Fig. 2a shows that S% of the I₁A₁E₄ hydrogel at 1 h was 150, 130, 450 and 560%. The swelling % at 6 h was 230, 650, 590 and 740%. As a result, the swelling increases with increasing pH. The maximum swelling was attained at pH 7.4 and 10.0 due to the ionization of the carboxylic group (COO⁻) of IA because the pK_a values of IA are pK_a1 = 3.85 and pK_a2 = 5.43.³⁵ Fig. 2b shows the S% of the I₂A₁E₃ hydrogel. S% at 1 h was 100, 120, 190 and 250%, and after 6 h S% was 120, 520, 690 and 490%. The swelling percentage of the I₂A₁E₃ hydrogel reached a maximum at pH 7.4. This is due to the IA and AA content producing higher electrostatic repulsion of the negatively charged ions, which enhanced the swelling behaviour.³⁶ Fig. 2c shows that the S% of the I₃A₁E₂ hydrogel at 1 h was 250, 240, 400 and 330% and the increased swelling after 6 h was 510, 1450, 1470 and 1400%. The swelling nature of the I₃A₁E₂ hydrogel increased with the increasing pH of the external medium.^37–42 The result of the highly acidic pH was the S% falling due to the formation of the hydrogel bonded complex. This complex boosts the polymeric matrix as hydrophilic in nature. The various compositions of the prepared IAE based hydrogel showed the extent of swelling at neutral and basic pH due to the formation of carboxylate interionic repulsion and the osmotic pressure of the external pH medium. The pH tuneable nature of the IAE based hydrogels may be used for drug release applications.^43,44

Fig. 2 The swelling percentage of I₁A₁E₄ (a), I₂A₁E₃ (b), I₃A₁E₂ (c) based hydrogels at various pH with respect to time. The (d) comparative swelling equilibrium percentage of I₁A₁E₄, I₂A₁E₃, I₃A₁E₂ based hydrogels with varying pH of 1.2, 6.0, 7.4 and 10.0.

3.3. Equilibrium swelling studies of IAE hydrogels

The pH-sensitive swelling of hydrogels is an essential factor for pharmaceutical applications.⁴⁵ Equilibrium swelling studies of IAE based hydrogels were carried out at different pH values from 1.2 to 10.0 in ambient temperature for 24 h. The enhancement of swelling was monitored gravimetrically. Fig. 2d shows the experimental swelling equilibrium results of the I₁A₁E₄, I₂A₁E₃ and I₃A₁E₂ hydrogels. The prepared hydrogels saw sharp increases in swelling equilibrium at basic pH and nominal increases at acidic pH. The swelling equilibrium of the IAE hydrogels attained a maximum at a pH of 6.0 and 7.4. This is due to the H bonds breaking from the monomers of IA and AA, which accelerates complete ionization of the IAE based hydrogels, resulting in electrostatic repulsion leading to a hydrogel network extension. The role of IA and AA is to increase the number of –COO⁻ ions along with free H⁺ ions within the polymeric matrix. This causes electrostatic repulsion between alike carboxylate groups and osmotic swelling. The S_eq% of the IAE based hydrogels is lower at a pH of 1.2 because the H-bonding compresses the hydrogel network leading to a rejection of water molecules from the polymeric hydrogel networks.⁴⁶ In general, the presence of the CH₃ group in the polymeric backbone shrinks the surface area of the non-polar (CH₂) part in the hydrogel by increasing its free volume and flexibility. Hence, the hydrophilicity increases in the polymer network thereby enhancing the swelling equilibrium.⁴⁷

3.4. SEM morphologies of IAE hydrogels

Scanning electron microscopy was performed to identify the surface morphology (Fig. 3) of the IAE hydrogels. The micro photographs of the IAE based hydrogels confirm that radical polymerization and the crosslinking reaction were carried out uniformly throughout the preparation. Fig. 3a and b show the cauliflower-like surface morphology of the I₁A₁E₄ hydrogel. It has globular loop holes which support the permeation of external stimuli. The I₂A₁E₃ hydrogel in Fig. 3c and d exhibits a fractured surface morphology. This revealed irregular pores with rigid structures within the hydrogel matrix. Fig. 3e and f show minute voids present on the surface of the I₃A₁E₂ hydrogel. The results of the morphological studies of the IAE based hydrogels support the nature of swelling and porosity. The flake like morphology of the IAE based hydrogels might be used to undergo degradation easily. S_eq% of the prepared hydrogels at various pH is arranged in the ascending order of I₂A₁E₃ < I₁A₁E₄ < I₃A₁E₂. The porous morphology can facilitate use in biomedical applications.

Fig. 3 SEM images of the I₁A1E₄ (a and b), I₂A₁E₃ (c and d) and I₃A₁E₂ (e and f) hydrogels.

3.5. Antibacterial activity of IAE hydrogels

Hydrogels have been broadly used for biomedical applications such as wound healing, contact lenses, artificial skin, in drug delivery systems etc.⁴⁸ Therefore, taking precaution of their contamination with microorganisms is very essential. Antibacterial activity is a very worthy property for biological applications and has been reported for many polymers.⁴⁹ To identify the qualitative determination of antibacterial assessment the obtained IAE based hydrogels underwent antibacterial analysis. The results of antibacterial activity against Staphylococcus aureus (Gram +ve), Escherichia coli (Gram −ve) and Bacillus cereus (Gram +ve) are listed below. The inhibition zones of the I₃A₁E₂ (a–c) and I₁A₁E₄ (d–f) hydrogels are shown in Fig. 4. Gentamicin was chosen for its broad spectrum of antibiotics. The I₃A₁E₂ polymeric hydrogel showed inhibition against S. aureus at 0 mm, 25.56 mm, 34.46 mm, and 36.98 mm. The inhibition zones of the I₃A₁E₂ hydrogel against E. coli were 0 mm, 27.34 mm, 33.82 mm and 38.84 mm. The zones of inhibition for the I₃A₁E₂ hydrogel against B. cereus were 0 mm, 29.56 mm, 32.17 mm and 38.27 mm. From the results, the I₃A₁E₂ hydrogel exhibited better inhibition activity towards E. coli and B. cereus. The I₁A₁E₄ hydrogel showed inhibition zones against S. aureus at 0 mm, 27.66 mm, 33.34 mm and 39.0 mm. The inhibition against E. coli was 21.52 mm, 23.41 mm, 25.96 mm and 29.11 mm. For B. cereus it was 0 mm, 29.56 mm, 32.17 mm and 38.27 mm respectively. These results suggest that the I₃A₁E₂ hydrogel showed better zones of inhibition against S. aureus, E. coli and B. cereus than the I₁A₁E₄ hydrogel. The obtained results concluded that the I₃A₁E₂ hydrogel shows better anti-bacterial activity against Gram positive bacteria due to the itaconic acid proportion in the IAE based hydrogel which increases the diameter of the antimicrobial inhibition zone (mm). The itaconic acid moieties can interact with a particular constituency of the cytoplasmic membrane of bacteria, causing structural and degradation changes to occur which lead to the death of anti-bacterial cells.^50,51 S. L. Tomic et al., 2009 prepared itaconic acid based hydrogel which showed an antimicrobial inhibition zone of ∼70% for antibiotic loaded hydrogel.⁵² The prepared IAE hydrogels produced a ∼40% zone of inhibition against various anti-bacterial pathogens even for antibiotic unloaded samples. This revealed that the enhancement of itaconic acid and acrylic acid content in IAE based hydrogels extends the chain length of the polymeric backbone and obviously supports the inhibition nature against anti-bacterial pathogens.⁵³ According to the obtained results the IAE based hydrogels are more effective towards inhibiting microbial growth due to the presence of acidic moieties.^54,55 The zone of inhibition against anti-bacterial pathogens is significantly characterized for wound dressings, particularly in shielding the wound from further infection.

Fig. 4 Anti-bacterial activity of the I₃A₁E₂ (a–c) and I₁A₁E₄ (d–f) hydrogels.

3.6. Anti-fungal activity of IAE hydrogels

The anti-fungal activity of a material is a valuable property for biomedical applications. Aspergillus niger is one of the most common species from the genus Aspergillus. It may cause some disease called black mould on certain fruits and vegetables and is a common contaminant of food etc. For human beings it produces fungal ear infections and serious lung disease. Candida albicans originated from the Candida genus and produces many common types of disease like candidiasis, fungemia, morbidity, etc. The IAE based hydrogels were subjected to anti-fungal pathogens and the results are shown in Fig. 5 and Table 2. The I₃A₁E₂ hydrogel showed the maximum inhibition against C. albicans and A. niger. The I₁A₁E₄ hydrogel also showed a maximum inhibition against C. albicans but no significant inhibition zone for A. niger. The results conclude that both IAE based hydrogels have significant inhibition activity against C. albicans. The zone of inhibition of I₃A₁E₂ against anti-fungal pathogens is higher than the I₁A₁E₄ hydrogel. This might be due to the increasing stoichiometric amount of itaconic acid.⁵⁶ In general monomers containing ionic groups like itaconic acid (IA) are highly hydrophilic in nature and have been recently studied by researchers in detail to improve the swelling capacity, antimicrobial activity and biocompatibility. In addition, those which are obtained from natural renewable sources could be considered as a good barrier against microbes. The mechanism for the antimicrobial activity of the IAE hydrogels is shown in Fig. 6. The increased IA content in all of the hydrogels was found to exhibit higher antimicrobial activity. The enhanced biological activity might be due to the increasing contribution of ionic moieties in the hydrogel network by IA.^57,58

Fig. 5 Antifungal activity of the I₃A₁E₂ (a and b) and I₁A₁E₄ (c and d) hydrogels.

Table 2 Antifungal activity of the IAE hydrogels by using the well diffusion method

S. No.	Sample	Concentration (μg)	Composition (mol)
			A. niger	C. albicans
1	I1A1E4	450	—	—
		900	—	11
		135	—	13
		180	—	14
	Control	100	19	20
2	I3A1E2	450	—	11
		900	—	13
		135	15	14
		180	17	16
	Control	100	17	20

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Fig. 6 The general mechanism for antimicrobial activity of the IAE hydrogels.

3.7. In vitro cytotoxicity studies

Biocompatibility of polymeric biomaterial is an important property to assist in biomedical applications.⁵⁹ The in vitro cytotoxicity assay of the IAE hydrogels was observed with an MTT assay using 3T3 fibroblast cell lines with respect to various concentrations. The cell viability% (triplicate values) and microscopic images of the I₁A₁E₄ and I₃A₁E₂ hydrogels are represented in Table 3 and Fig. 7. The cell proliferation of the I₃A₁E₂ and I₁A₁E₄ hydrogels against the control was 100%. The I₁A₁E₄ hydrogel showed ∼83% cell proliferation for 250 μg. In the meantime the I₃A₁E₂ hydrogel exhibited ∼91%. The rate of replication of both of the hydrogels was greater than 80%. So the IAE hydrogels are a suitable mechanism for cell viability. The results of the cell culture method showed that the I₃A₁E₂ hydrogel had superb biocompatibility compared to the I₁A₁E₄ hydrogel. The biocompatibility of the IAE based hydrogels increased according to the entrapment of itaconic acid and this is a key role in biomedical applications.^60–62 The concentration of hydrogel which causes a 50% destruction of cell viability is known as the cytotoxic index (IC 50%).⁶³ According to GB/T 16886.5-2003 (ISO 10993-5:1999), samples with a cell viability larger than 75% can be considered as showing no cytotoxicity.⁶⁴ Hence the synthesized hydrogel can be considered non-toxic in nature and thus compatible with living tissues.

Table 3 The cell viability percentage of the IAE based hydrogels

S. No.	Concentration (μg ml^−1)	% of cell viability (triplicate values)
		I1A1E4			I3A1E2
1	10	94	95	94	98	99	99
2	25	92	92	92	97	97	97
3	50	89	88	88	95	96	95
4	100	88	86	87	92	94	93
5	250	83	83	83	90	91	91
6	Control	100	100	100	100	100	100

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Fig. 7 Microscopic images of the 3T3 fibroblast cell lines treated with the I₁A₁E₄ and I₃A₁E₂ hydrogels with respect to concentration.

3.8. Soil buried biodegradability

A polymer broken into smaller molecules or fragments by microorganisms is called a biodegradable polymer. Biodegradability is a very eminent property of hydrogels in biomedical applications due to their activity towards biological interaction. To determine the biodegradation of the IAE polymeric hydrogels, a soil buried test was carried out. The results are shown in Fig. 8. Initially, the weight of the IAE based hydrogels was increased due to the water imbibing capacity of the hydrogels from atmospheric soil; subsequently a weight loss occurred which strongly supports the biodegradation process. According to the results, the I₁A₁E₄ hydrogel showed ∼60% weight loss in 90 days. In the meantime, the I₃A₁E₂ exhibited ∼82% weight loss. In general, soil possess many microorganisms like bacteria, fungi, protozoa, actinomycetes etc. The microorganisms wish to evacuate their cellular enzymes on the surface of the cleavable polymeric moieties of the IAE based hydrogels. Hence, the hydrogels undergo surface erosion which leads to formation of biodegradable intermediates. At last, the final product by aerobic degradation might be CO₂, H₂O, and CH₄ etc. The degradation rate of the IAE based hydrogels mainly depends upon the presence of hydrolysable ester group and its substituents (R₁–R₂), for instance, electron withdrawing groups enhance hydrolysis and electron donating groups inhibit hydrolysis. The degradation process concludes that the IAE based hydrogels have ‘n’ number of ester groups which facilitates biodegradation easily due to the substitution of weakly electron donating (–R alkyl) groups.^65–67

Fig. 8 The degradable weight loss percentage of the I₁A₁E₄ and I₃A₁E₂ hydrogels.

4. Conclusions

IAE based hydrogels were prepared by a simple, organic solvent-less, meek purification, with less time duration. The results of FT-IR confirmed the formation and structure of the IAE polymeric hydrogels. The swelling equilibrium of the IAE based hydrogels showed a maximum at a basic pH range due to the complete dissociation of the COOH moieties present in itaconic acid and acrylic acid. The swelling investigations confirmed that the IAE based hydrogels were a precise candidate for drug release carriers at stimulated intestinal fluid pH conditions. The SEM morphology strongly supported the porous morphology of the IAE hydrogels. The antimicrobial activity of the IAE based hydrogels increases with an increasing ratio of itaconic acid. The itaconic acid based hydrogel has obvious antimicrobial inhibition zones even for the antibiotic unloaded sample. The IAE based hydrogels exhibited very good cell proliferation above 80%, which concludes that they were not harmful for usual metabolic activities. The presence of the easily hydrolysable ester functional group in the IAE based hydrogels underwent biodegradation easily, hence the IAE based hydrogels are biodegradable. Therefore, the itaconic acid based hydrogels have appreciable and controlled drug release mechanisms, a swelling nature, and biodegradation that facilitates these IAE hydrogels as super imposable with biological body fluids.

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