Synthesized cryogels, eMIP, which contains GA was observed in darker yellow tones, while NIP, which is the control group, was observed in light yellow tones and a color closer to white (Fig. 1).

Optical photographs of eMIP (left) and NIP (right) cover materials

GA was used as the epitope of TA. Before loading TA to the eMIPs the GA should be removed from the cover materials properly to obtain GA unit spesific recognition cavities. GA spesific cavities obtained, by applying an elution agent (0.5 M NaCl in PBS pH 7.4) to the eMIP. After four cycles of elution, approximately 96% of GA unit molecules were removed from the cavities. The obtained GA spesific cavities then used for the selective binding of TA (Fig. 2).

Reprinted with permission from (American Chemical Society, 2018).

Illustration of the process of GA-epitope imprinting on eMIP and its removal from the structure.

Swollen samples (18 mm in diameter) were used to conduct swelling test studies. Swelling rate, porosity and polymerization yield were determined by swelling tests. It was observed that the inclusion of GA in the structure reduced the gelation efficiency of the cryogel, but it did not have a negative effect on the macroporosity and swelling ratios (Table 1).

SEM images of eMIP and NIP showed that both of them have homogeneously distributed interconnected macropores (Zenger and Peşint 2022) (Fig. 3).

SEM images of NIP (a, c, e) and eMIP (b, d, f) (250×, 500×, 1000×, respectively)

Structural analysis of cryogels was evaluated via FTIR-ATR for eMIP and NIP. The spectra of GA, VIM and GA-VIM precomplex (1:8) are given below, respectively (Fig. 4a).

When the FTIR spectrum of GA is examined, the intense peak at 3491 cm⁻¹ is indicative of a robust O–H stretching vibration, pointing towards the presence of hydroxyl groups in the GA molecule. At 3266 cm⁻¹, a strong O–H stretching peak emerges, specifically attributed to the carboxylic acid group. This observation reinforces the identification of the carboxylic acid functional group within the GA. The region between 2600 and 2800 cm⁻¹ displays medium-intensity peaks corresponding to C–H stretching vibrations. These peaks affirm the presence of carbon-hydrogen bonds in the compound, contributing to the overall structure of GA. The peaks at 1539 and 1604 cm⁻¹ are associated with C=C stretching vibrations, characteristic of aromatic rings. These peaks serve as a distinct marker for the presence of aromatic rings within the molecular structure of GA. In the range of 1000–1300 cm⁻¹, strong peaks are observed, indicating C–O stretching vibrations. These peaks are particularly relevant to the functional groups involving oxygen atoms and contribute essential information regarding the molecular configuration of GA. Finally, the strong peaks within the 700–900 cm⁻¹ range are attributed to C–H bending vibrations. These peaks emphasize the presence of carbon-hydrogen bonds and their specific bending motions within the GA molecule. In the spectrum of VIM, The peak at 3109 cm⁻¹ signifies the C-H stretching of the aromatic ring, confirming the presence of the structural feature of an aromatic ring within the molecule. Medium intensity peaks around 1646 cm⁻¹ predominantly represent the C=C double bonds and N–H bending associated with the aromatic ring. This indicates the distinct impact of double bonds and amine groups on the FTIR spectrum. The peaks at 1509 and 1492 cm⁻¹ correspond to C–N stretching, demonstrating the presence of C–N bonds within the molecule. These peaks specifically highlight the stretching vibrations of these bonds. The peaks in the range of 1300–1400 cm⁻¹ indicate strong C-N stretching vibrations within the aromatic ring. These peaks are unique to VIM and leave a prominent signature in the FTIR spectrum. When the FTIR spectrum of GA-VIM pre-complex is examined; The large peak observed at 3326 cm⁻¹ is attributed to O–H stretching vibrations, believed to be associated with GA. This broadening suggests hydrogen bonding, emphasizing the interaction between GA-VIM. The peak at 1648 cm⁻¹ signifies C=C stretching vibrations and N–H bending, characteristics associated with aromatic rings. This observation supports the successful inclusion of the aromatic structures from both GA and VIM in the complex. The peaks at 1503 and 1233 cm⁻¹ are thought to be associated with VIM, specifically corresponding to C–N stretching vibrations within aromatic rings. This provides further evidence of the incorporation of VIM into the complex, emphasizing the interaction between the two molecules. Additionally, the peak at 1084 cm⁻¹ is attributed to C–H stretching vibrations, indicating the presence of carbon-hydrogen bonds. This observation further supports the successful formation of the preliminary complex. In conclusion, the FTIR analysis underscores the successful complexation of GA and VIM, as evidenced by the distinctive peaks associated with the individual components. The observed vibrational modes and peak patterns strongly indicate the formation of hydrogen bonds and interactions between the aromatic structures.

Below are the characteristic FTIR spectra for eMIP and NIP (Fig. 4b). Since the structures of eMIP and NIP are quite similar, their spectra also exhibit significant similarities. Despite the masking effect of the abundant content of Gelatin, HEMA and EGDMA in the structure, the presence of VIM and GA molecules has been successfully demonstrated. First of all, the wide O–H stretching band at 3376 cm⁻¹ seen in both spectra is the hydrogen containing gelatin and HEMA. The C-H stretching peaks at 2948 cm⁻¹ originate from the methylene groups of EGDMA and the methylene and methenyl groups of HEMA. The C=O stretching peaks at 1711 cm⁻¹ represent the carboxyl or ester groups of ethylene glycol dimethacrylate and HEMA. Additionally, the C–H deformation peaks at 1450 cm⁻¹ indicate the presence of methylene or methenyl groups, while the peaks at 1388 cm⁻¹ and 1245 cm⁻¹ reflect C–H deformations along with C–N or C–O stretching vibrations. Unlike in NIP, the band observed in eMIP at 1322 cm⁻¹ corresponds to amine group stretching bands, suggesting the presence of amine groups within the imprinted cavities. This observation may serve as evidence that eMIP forms more hydrogen bonds than NIP. The existence of this peak can also be attributed to the inclusion of the VIM molecule in the structure. However, the frequency shift at 1711 cm⁻¹, caused by C=O vibrations, to a higher frequency in eMIP, along with the difference in peak intensity compared to NIP, supports the presence of the pre-complex molecule in the structure. These variations observed in the spectra strongly indicate the incorporation of the GA-VIM pre-complex into the structure of eMIP.

FTIR Spectrum of a GA, VIM and GA: VIM (1:8 /n: n) pre-complex, b eMIP and NIP

GA epitope removed cavities were loaded with TA and the maximum TA loading capacity determined by interacting different concentrations of TA solutions (between 1.5 and 10 mg/mL) to the cover materials. The maximum TA adsorption for eMIP and NIP polymers was calculated as 210.27 mg and 24.74 mg per gram cover material, respectively (Fig. 5).

TA loading amounts of eMIP and NIP. mdry: 0.1155 g, V: 5 mL, time: 120 min, T: 25 ℃, pH: 7.4

The Langmuir and Freundlich adsorption isotherm models were used to investigate the interaction behavior of stnthesized cover materials with TA. Figure 6 shows the Langmuir and Freundlich adsorption isotherms of TA adsorption on eMIP. The Langmuir adsorption isotherm describes monolayer adsorption in which it is assumed that all binding sites are homogeneous, energitally equal and adsorbed species don’t interact with each other within themselves (Foo and Hameed 2010). The Langmuir model is defined by the following equation:

$$}_}} /}_}} = }/}_}}} + }/}_}}} \times } \times }_}}$$

(9)

When the Eq. 9 is linearized, the following equation is obtained:

$$}_}} /}_}} = }/}\left( }_}}} .}} \right)} + }\left( }_}} /}_}}} } \right)$$

(10)

The Ce versus Ce/Qe plotted to obtain the intersection of the line formed gives 1/Qmax*b, and its slope gives 1/Qmax. Here Qe is TA binding capacity (mg/g), Ce is equilibrium TA concentration (mg/mL), b is Langmuir constant (mL/mg), Qmax is maximum TA adsorption capacity (mg/g).

The maximum TA adsorption capacity (Qmax) 588.23 mg/g, and the Langmuir constant (b) calculated as 0.08 mL/mg from the slope and cut of the graph in Fig. 6(Table 2).

The Freundlich isotherm describes multilayer adsroption, it assumes that the binding sites are not homogeneous, energitacaly not equal, adsorbed species can interact with each other within themselves. The equation for the Freundlich adsorption isotherm is as follows:

$$}_}} = }_}} \times }_}} ^}/}}}$$

(11)

When the Eq. 11 is linearized, the following equation is obtained:

$$}_}} = }_}} + }/} \times }_}}$$

(12)

here Qe is TA binding capacity (mg/g); Ce is equilibrium TA concentration (mg/mL); Qf is the Freundlich adsorption capacity of the adsorbent (mg/g); 1/n is the freundlich constant. The lnCe against lnQe plotted to obtain 1/n and lnQf values from the slope and the intersect of the lines. Figure 6.b shows the Freundlich adsorption isotherm of TA adsorption of eMIP. The 1/n was found 0.6767 and the Qf was calculated as 53 mg/g (Table 2).

Adsorption isotherm plots of eMIP a Langmuir, b Freundlich

Among the important parameters affecting the drug release rate is the amount of drug initially loaded into the polymeric system. In this study, where TA release from cryogel cover materials was examined, TA was loaded by adsorption at 5 different concentrations (1.5 mg/mL, 3 mg/mL, 5 mg/mL, 7 mg/mL, 10 mg/mL). In order to determine the effect of loading TA amount on release kinetics; release pH (7.4) and temperature (25 ℃) were kept constant. Figure 7 shows that the release rate increases as the initial TA loading amount increases. Cumulative release analysis studies of TA loaded cryogels showed that the maximum release rate is reached in the first 2 h (Fig. 7a-b).

Effect of TA concentration on release depending on time (T: 25 °C, pH:7.4, DI) a Cumulative release (mg/g), b Percentage release (%)

Time-dependent cumulative release percentage (%) rates of each cryogel evaluated in Fig. 7b. Cryogels released almost all of the TA in their structures nearly in 2 h. All release behavior almost the same for each cryogel cover materials (Fig. 8).

Optical images of antimicrobial eMIPs and NIPs against; a S. aureus and b E. coli

Time-dependent cumulative release rates of cryogels were evaluated with the parameters of Korsmeyer-Peppas kinetic release models (Table 3). The relative effect of macromolecular structure on the drug release mechanism can be easily determined by modifying the experimental data to Eq. 6. However, this equation is applicable only for the initial 60% of the total released drug.

Equation 6 defines the type of diffusion process that depends on the value of n and describes the overall transport behavior of the solvent in the polymer. Plotting the data from this equation on a logarithmic scale and using linear regression to calculate the slope allowed for the determination of the diffusional exponent, or n.through a diffusion process of a non-Fickian type.

Table 3 shows the release coefficients ​​n and k of eMIP cryogel cover materials containing different amounts of TA, and also the regression coefficients. As given in Table 3, n values are smaller than 0.5.

eMIP cryogel cover materials loaded with three different concentrations of TA showed antimicrobial activity against S. aureus and E. coli. Inhibition zone diameters of cover materials were determined to be larger at highest concentration of TA which is 5 mg/mL according to 1.5 and 3 mg/mL. The highest antimicrobial activity with 15 mm inhibition zone was observed against S. aureus. On the other hand, inhibition zone of 12 mm was observed against E. coli.

The viability of HaCaT cells cultured on synthesized cryogels

MTT assay results revealed that all synthesized materials had no cytotoxic effect on HaCaT cells and were suitable for cell growth (Fig. 9). It was determined that as the incubation time increased, cell proliferation in eMIP cryogels increased slightly and viability was slightly higher than in the 2D control group (100.98% and 101.76% respectively). Cell viability for the NIP cryogel was very close to the 2D control at both incubation times (99.38% and 100.4% for 24 and 48 h respectively). Indeed, cell viability for both materials showed no significant difference compared to the 2D control in either incubation period. This indicated that neither the material nor the TA loaded into it has a toxic effect on the cells and does not inhibit their proliferation.