Modification of Collagen Derivatives with Water-Soluble Polymers for the Development of Cross-Linked Hydrogels for Controlled Release

Novel cross-linked hydrogels were synthesized as potential materials for the development of smart biofertilizers. For this purpose, hydrogels were prepared using collagen hydrolysate recovered from tannery waste. The water-soluble polymer poly(sodium 4-styrenesulfonate-co-glycidyl methacrylate) (P(SSNa-co-GMAx)) was among others used for the cross-linking reaction that combined hydrophilic nature with epoxide groups. The synthetic procedure was thoroughly investigated in order to ensure high percentage of epoxide groups in combination with water-soluble behavior. The copolymer did not show cytotoxicity against normal lung, skin fibroblasts, or nasal polyps fibroblasts. Through the present work, we also present the ability to control the properties of cross-linked hydrogels by altering copolymer’s composition and cross-linking parameters (curing temperature and time). Hydrogels were then studied in terms of water-uptake capacity for a period up to six days. The techniques Proton Nuclear Magnetic Resonance (1H NMR), Thermogravimetric Analysis (TGA), Size Exclusion Chromatography (SEC), and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) were applied for the characterization of the synthesized copolymers and the cross-linked hydrogels. Three samples of biofertilizers based on collagen hydrolysate functionalized with P(SSNa-co-GMAx) and starch and having nutrients encapsulated (N, P, K) were prepared and characterized by physical–chemical analysis and Energy Dispersive X-ray analysis-Scanning Electron Microscope (EDAX-SEM) in terms of microstructure. Preliminary tests for application as fertilizers were performed including the release degree of oxidable organic compounds.


Introduction
The industries all over the world are the major source of pollution with a significant impact on the environment. Industrial wastes may cause a severe pollution in river systems, lakes, soil, and sea and also atmospheric pollution by fume release. The improper waste management and their disposal to recipients will lead to harmful effects on the biotic and abiotic ecosystems disturbing the ecological Table 1. Characterization results for the P(SSNa-co-GMAx) copolymers.

Modification of Collagen Hydrolysate with P(SSNa-co-GMAx)
The collagen hydrolysate was mixed in different proportions with the copolymer P(SSNa-co-GMAx) at room temperature under stirring conditions. After short time, a viscous mass was precipitated. The precipitated mass was separated from the supernatant solution and remained for different time intervals at room temperature (R.T.) or thermally treated at 80 • C or 120 • C. Finally, both the soluble (water soluble) and insoluble (residue) fractions were dried and then their weights were calculated. The insoluble part was varied between 70 and 100 wt%. The results for selected preparations are shown in Tables 3-6. Table 3. Residues and water-uptake capacities for blends of collagen hydrolysate/P(SSNa-co-GMAx) copolymers in 80/20 composition after thermal treatment at 80 • C for 20 h.

Modification of Collagen Hydrolysate with Encapsulated Nutrients and Polymeric Materials
In this case, the untanned hide waste was treated by hydrolysis of protein waste in acidic medium, resulting in a protein hydrogel, which in combination with other polymers, such as P(SSNa-co-GMAx) and starch, resulted in various hydrogels with a collagen structure.
As an example, 6800 g of pelt waste are weighed, washed, and delimed with 4-4.5% ammonium sulfate, followed by dilution in 5-6.5 L industrial water containing 2-3.5% concentrated H 2 SO 4 , 2.5-3.5% dipotassium phosphate (K 2 HPO 4 ·3H 2 O), and 0.5-1% industrial salt (NaCl). This mixture is poured into a 50 L autoclave with double jacket and shaker, at 80-98 • C for 2. 5-4.5 h. This product is gelatinous, elastic, and semi-transparent, with pH of 5.8-6.8, containing collagen hydrogel with encapsulated nutrients (K, P), and will be considered as a reference organic fertilizer for agriculture applications, denoted as CH-Ref [26]. In this stage, 5% copolymer P(SSNa-co-GMA20) with IN20c code is added to obtain a similar fertilizer functionalized with this synthetic copolymer, and will be denoted as CH-IN. If instead of IN20c in the autoclave is added 36-60% corn starch and a methylene bis-acrylamide as chemical bonding agent (0.5 g dissolved in 50 mL water) to form a chemically bounded starch copolymer, a starch graft copolymer is obtained, able to absorb liquid fluids without dissolution. Hydrolysis is continued for 1.5-2 h, at a temperature of 78-85 • C resulting in a superabsorbent hydrogel in the form of a white paste, with a pH about 6.7 and marked as CH-Starch.

Proton Nuclear Magnetic Resonance ( 1 H NMR)
The samples for 1 H NMR characterization were prepared by dissolving the P(SSNa-co-GMAx) copolymers in D 2 O. 1 H NMR spectra were obtained at 400 MHz at 300 K on a Bruker AVANCE DPX 400 spectrometer (Bruker BioSpin GmbH, Magnet Division, Karlsruhe, Germany). The 1 H NMR spectra were used to determine the chemical composition of the copolymers.

Thermal Analysis
Thermogravimetric analysis (TGA) was carried out in alumina crucibles in a Labsys™TG (Caluire, France) apparatus of Setaram from 25 to 800 • C under nitrogen and at a heating rate of 10 • C/min.

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR)
The Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectra of the copolymers, the collagen hydrolysate, and the collagen-P(SSNa-co-GMA) cross-linked hydrogels were recorded using Bruker Optics' Alpha-P Diamond ATR Spectrometer of Bruker Optics GmbH (Ettlingen, Germany).

Energy Dispersive X-ray analysis-Scanning Electron Microscope (EDAX-SEM)
Scanning electron microscopy of cross-linked hydrogels (SEM, JEOL 6300, Tokyo, Japan, instrument equipped with an X-ray Energy Dispersive Spectrometer, EDS) was performed to investigate their morphologies. The samples were obtained through freeze-drying and sputtered with gold to produce electric conductivity before SEM examination.
The morphology of collagen hydrolysate with encapsulated nutrients and polymeric materials was conducted through Scanning Electron Microscopy on SEM, Hitachi, S-3400 N type II Model, Secondary Electrons, High Vacuum, 10-3 Pa, 3 nm at 30 kV. The Energy Dispersive X-ray Spectroscopy (EDS/EDX) analysis was performed using Thermo Ultra Dry, Noran System 7, NSS Model (2,000,000 counts/s).

Cell Lines and Cell Culture Conditions
Lung and skin fibroblasts were isolated from lung and skin tissues, respectively, of healthy donors. Nasal polyps fibroblasts were isolated from polyps' tissues obtained from patients undergoing sinus surgery for treatment of polyposis. The isolation and culture of fibroblasts were performed as previously described for eye pterygium fibroblasts [27]. All cells were cultured as monolayers in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% penicillin and streptomycin, in a humidified 5% CO 2 atmosphere.

Cell Viability
i Toxicity Test of the Copolymer P(SSNa-co-GMA20) For cell viability, the tetrazolium/formazan assay was used, as previously described [28]. Fibroblasts in DMEM-10% FCS were seeded onto 96 well plates. When the cell monolayers reached confluent, the conditioned medium was replaced with solutions of copolymer at different concentrations (0.125-100 µg/mL) in the same medium, and the cells were cultured for different time intervals. Then, the conditioned medium was removed and a solution of MTT (1 mg/mL) in the same medium, was added. After incubation at 37 • C for 4 h in a humidified 5% CO 2 atmosphere, the solution was thoroughly aspirated, the crystals of formazan were dissolved in DMSO, and the absorbance at 540 nm was measured, using a microplate reader (TECAN infinite M200, Grödig, Austria).
ii Toxicity Test of the Collagen-P(SSNa-co-GMAx) Cross-linked Network Hydrogels The biocompatibility of prepared hydrogels was characterized as previously described [29,30]. In brief, small disks were produced from the cross-linked network hydrogels collagen-P(SSNa-co-GMAx), where x = 30 mol% GMA (Hydrogel 1) and x = 20 mol% GMA (Hydrogel 2), placed into 96-well plates, and then sterilized under UV irradiation. Then, nasal polyps fibroblasts of passage 7 in DMEM-10% FCS (2 × 104 cells per well) were seeded on hydrogels' surface and cultured at 37 • C, under a 5% CO 2 humidified atmosphere. Hydrogel disks were also placed in wells without cells and subjected to the same treatment as the corresponding wells containing gels and cells together, in order to be used as blank. After 24 h and 72 h of culture, the conditioned medium was removed and replaced by the same medium containing 1 mg/mL MTT (100 µL/well). The next steps were performed as described above. The net absorbance at 540 nm of wells, containing gels and cells together, was obtained after a subtraction of absorbance of wells containing only the gels.

Water-Uptake Measurements
In order to study the water-uptake capacity of the materials, 1 g of dry sample was soaked in 250 mL of deionized water for 6 days to be swelled completely. The weight of the hydrogel was measured after removing the excess of water (decantation). According to the weight of the swollen (M 2 ) and dried (M 1 ) samples, the water-uptake capacity (Q) was calculated according to the equation below:

Release Degree of Oxidable Compounds in Water
The release degree of oxidable compounds (organic and inorganic) in distilled water of biofertilizers CH-Ref, CH-IN, and CH-Starch reflects their stepwise decomposition in aquatic medium in dynamic conditions [29]. A glass column of 2.8 cm inner diameter and 20 cm height was used, which was loaded with three layers of sand and gravel to ensure a uniform flow in the column among stagnant areas. Each layer had a height of 5 cm, arranged from the bottom to upwards, while the first gravel had the diameter between 6 and 9 mm, the second gravel between 4 and 6 mm, and the third between 0.4 and 1.8 mm. An amount of 5 g of each biofertilizer was placed on the sand layer. For each biofertilizer, two series of determinations was made. A constant flow of distilled water was ensured by connecting the column with a reservoir; the flow rate was 225 mL/h. Fractions of 100 mL were collected and the oxidable compound degree was determined using chemical oxygen demand CODMn method.
The initial weight of oxidable compounds was determined using a sample of 0.1 g of each fertilizer dissolved in 100 mL hot distilled water. The experiments occurred at 25 • C.
The quantitative evaluation of release degree (R) of oxidable compounds was performed by applying the relation: where R is the amount of oxidable compounds released (%), W i is the amount of oxidable compounds released at t moment (g) and W t is the total amount of oxidable compounds released (g).

Synthesis and Characterization of P(SSNa-co-GMAx) Copolymers
The synthesis of the P(SSNa-co-GMAx) copolymers was conducted through free radical polymerization, as described previously, with x = 2, 5, 20, 30, and 40 mol% GMA. The chemical composition of the synthesized copolymers was estimated through 1 H NMR spectroscopy. Figure 1 shows the 1 H NMR spectra of the P(SSNa-co-GMAx) copolymers in D 2 O with 30 and 40 mol% GMA content. The peaks of GMA are clearly seen as shown by Figure 1A,B. The presence of SSNa from the aromatic (e, e') protons at 6.0-7.8 ppm is clearly noticed at both spectra. Concerning GMA, the methylene protons of the epoxy ring of GMA correspond to the peak at 3.8 and 4.2 ppm (h', h type protons), the methine proton was observed at 3.2 ppm (f type protons), and the two protons for the methylene of the ring were observed at 2.7 and 2.9 ppm (g, g' type protons) [24,30]. The protons (b, c, d) of the copolymer's backbone are shown at 1.1-2.0 ppm and the methyl protons of GMA (a) at 0.5 ppm.   Due to the fact that in some cases the peaks of GMA in the copolymers with GMA content lower than 30% are not clearly seen in 1 H NMR spectra, thermogravimetric analysis was also applied in order to determine copolymers' chemical composition. Figure 2 shows the thermal analysis of the copolymers IN20a, IN20b, IN30, and IN40, with 20, 30, and 40 mol% GMA content, respectively, as well as the homopolymers PSSNa and poly(glycidyl methacrylate) (PGMA). More specifically, since the PSSNa homopolymer is thermally stable up to 400 • C while PGMA shows thermal decomposition above 250 • C and its thermal decomposition is completed above 450 • C, the weight loss of the copolymers at 500 • C was considered to be related to GMA content. Based on the above consideration, GMA content was calculated taking into account the weight loss of the copolymers at 500 • C. The monomer feed composition and the characterization results from 1 H NMR spectroscopy and TGA analysis of the P(SSNa-co-GMAx) synthesized copolymers are shown in Table 1.
The synthesized copolymers were examined in terms of chemical structure via ATR-FTIR spectroscopy and the spectra are presented in Figure 3. The spectra of poly(sodium 4-styrenesulfonate) (PSSNa) and poly(glycidyl methacrylate)(PGMA) homopolymers are also reported for comparison reasons.
The presence of PSSNa units in the copolymers was confirmed by the characteristic peaks shown with an asterisk. More specifically, the absorption band at 1010 and 1128 cm −1 assigned to the in-plane bending and the in-plane skeleton vibration of benzene ring, respectively. Moreover, the peaks at 1040 and 1182 cm −1 are related to the symmetric and antisymmetric vibration absorption peaks of SO 3 − group, respectively. The peak at 676 cm −1 may be related to aromatic C-H out-of-plane bending (deformation) vibration [31,32]. The existence of the GMA unit in P(SSNa-co-GMAx) copolymers is verified by the characteristic band at 850 and 909 cm −1 assigned to the epoxy ring vibrations and the peak at 1255 cm −1 to the C−H of the epoxy group. The peak at 750 cm −1 corresponds to the C-H bending vibration of the epoxide ring. Moreover, in the region of 1720 cm −1 is the characteristic stretching of an ester group, related to the carbonyl group of the glycidyl methacrylate segment (C=O) [33,34]. As it may be seen from the ATR-FTIR spectra of Figure 3, the peak at 909 cm −1 of the epoxy group is obvious in all the copolymers.    The molecular weight distributions of the P(SSNa-co-GMAx) copolymers were determined through size exclusion chromatography in aqueous 0.1 M LiNO3 solution, using poly(ethylene oxide) standards. The number-average molecular weights, Mn, weight-average molecular weights, Mw, and polydispersity indices, PDI, are presented in Table 2. Part of the results is also graphically shown in Figure 4. As we can see, high molecular weights (Mn values up to 83,000) were obtained and the polydispersity indices were quite high. In some cases, a bimodal distribution in the molecular weights and high DPn values were observed. The main driving force of this behavior is the Trommsdorff-Norrish effect, which induces an increase in the viscosity of the polymerizing medium leading to delayed reaction terminations. This in turn causes a rapid increase in the overall reaction rate, leading to a potential uncontrolled reaction and altering the characteristics of the produced polymer (auto  The molecular weight distributions of the P(SSNa-co-GMAx) copolymers were determined through size exclusion chromatography in aqueous 0.1 M LiNO 3 solution, using poly(ethylene oxide) standards. The number-average molecular weights, Mn, weight-average molecular weights, Mw, and polydispersity indices, PDI, are presented in Table 2. Part of the results is also graphically shown in Figure 4. As we can see, high molecular weights (Mn values up to 83,000) were obtained and the polydispersity indices were quite high. In some cases, a bimodal distribution in the molecular weights and high DPn values were observed. The main driving force of this behavior is the Trommsdorff-Norrish effect, which induces an increase in the viscosity of the polymerizing medium leading to delayed reaction terminations. This in turn causes a rapid increase in the overall reaction rate, leading to a potential uncontrolled reaction and altering the characteristics of the produced polymer (auto acceleration) [35]. The copolymer P(SSNa-co-GMA20) was then tested in terms of toxicity against normal lung and skin fibroblasts as well as against nasal polyps fibroblasts, and the cells viability was determined ( Figure 5). These cells were selected because the corresponding organs and tissues are most likely to come into contact with the copolymer. As shown in Figure 5, the copolymer, independently of origination of cells, did not exhibit cytotoxic effects in all concentrations used at both exposure periods of 24 h and 48 h. Only at higher concentrations of copolymer (10-100 µg/mL) and for 48 h exposure period, a lower decrease of cells' viability ranging from 15.3 to 31.8% for lung and 11.0 to 22.6% for skin fibroblasts, respectively, was observed, which, however, was significantly different compared to control in the case of lung fibroblasts ( Figure 5, middle panel). The nasal polyps fibroblasts, the viability of which was not affected even at 100 µg/mL concentration of copolymer and 48 h exposure period, were further tested against the copolymer for longer exposure times 76 h and 92 h ( Figure 5, lower panel). As shown in Figure 5 (lower panel), the copolymer, in both exposure times, caused statistically significant decrease of cells viability of about 30% only at the concentration of 100 µg/mL, supporting the suggestion that it does not exhibit cytotoxicity.
In addition, the nasal polyps fibroblasts were cultured in the absence or presence of copolymer at concentrations 5 and 100 µg/mL for 24, 48, and 76 h, and the morphology of cells were examined by phase contrast microscopy on an Olympus CKX41 microscope carrying a MicroPublisher 3.3RTV camera ( Figure 6). The microscopy revealed that the copolymer, at both concentrations used and in all exposure times, did not alter the cell morphology. The copolymer P(SSNa-co-GMA20) was then tested in terms of toxicity against normal lung and skin fibroblasts as well as against nasal polyps fibroblasts, and the cells viability was determined ( Figure 5). These cells were selected because the corresponding organs and tissues are most likely to come into contact with the copolymer. As shown in Figure 5, the copolymer, independently of origination of cells, did not exhibit cytotoxic effects in all concentrations used at both exposure periods of 24 h and 48 h. Only at higher concentrations of copolymer (10-100 µg/mL) and for 48 h exposure period, a lower decrease of cells' viability ranging from 15.3 to 31.8% for lung and 11.0 to 22.6% for skin fibroblasts, respectively, was observed, which, however, was significantly different compared to control in the case of lung fibroblasts ( Figure 5, middle panel). The nasal polyps fibroblasts, the viability of which was not affected even at 100 µg/mL concentration of copolymer and 48 h exposure period, were further tested against the copolymer for longer exposure times 76 h and 92 h ( Figure 5, lower panel). As shown in Figure 5 (lower panel), the copolymer, in both exposure times, caused statistically significant decrease of cells viability of about 30% only at the concentration of 100 µg/mL, supporting the suggestion that it does not exhibit cytotoxicity.
In addition, the nasal polyps fibroblasts were cultured in the absence or presence of copolymer at concentrations 5 and 100 µg/mL for 24, 48, and 76 h, and the morphology of cells were examined by phase contrast microscopy on an Olympus CKX41 microscope carrying a MicroPublisher 3.3RTV camera ( Figure 6). The microscopy revealed that the copolymer, at both concentrations used and in all exposure times, did not alter the cell morphology. Materials 2019, 12, x FOR PEER REVIEW 11 of 23 Figure 5. Effects of P(SSNa-co-GMA20) copolymer on lung, nasal polyps, and skin fibroblasts' viability for different exposure periods. A range of concentrations from 0.125 to 100 µg/mL of copolymer was assayed. The results are expressed as mean ± SD of three separate experiments in triplicate. Statistically significant differences were evaluated using the unpaired Student's t-test. (*) indicates statistically significant decrease (p < 0.05) compared to control.

Preparation of the Collagen-P(SSNa-co-GMAx) Cross-Linked Network Hydrogels
In order to develop materials that will efficiently incorporate the desired fertilizers by hydrogel formation, the collagen matrix was modified with functional polymeric electrolytes. Particularly, negatively charged polyelectrolytes were used as additives in order to interact with collagen hydrolysate, which in acidic conditions has a positively charged amino group and a carboxyl group. The cross-linking reaction of collagen hydrolysate and P(SSNa-co-GMAx) copolymers is schematically shown in Scheme 1. Based on our previous experience, we used the copolymer P(SSNa-co-GMAx), which has a dual functionality: the epoxy groups may create a chemical bond with the carboxyl groups of collagen hydrolysate and the SO3 − groups may electrostatically interact with the positively charged amino groups of collagen hydrolysate. For the formation of a cross-linking network between collagen hydrolysate and the P(SSNa-co-GMAx) copolymers, synthesized in the present work, various parameters were examined for the optimization of the reaction process. Scheme 1. Cross-linking reaction of collagen hydrolysate and P(SSNa-co-GMAx) copolymers. Figure 6. Phase contrast microscopy of nasal polyps fibroblasts cultured in the absence or presence of P(SSNa-co-GMA20) copolymer at concentrations 5 and 100 µg/mL for different exposure periods. All micrographs were taken at an original magnification of ×10.

Preparation of the Collagen-P(SSNa-co-GMAx) Cross-Linked Network Hydrogels
In order to develop materials that will efficiently incorporate the desired fertilizers by hydrogel formation, the collagen matrix was modified with functional polymeric electrolytes. Particularly, negatively charged polyelectrolytes were used as additives in order to interact with collagen hydrolysate, which in acidic conditions has a positively charged amino group and a carboxyl group. The cross-linking reaction of collagen hydrolysate and P(SSNa-co-GMAx) copolymers is schematically shown in Scheme 1. Based on our previous experience, we used the copolymer P(SSNa-co-GMAx), which has a dual functionality: the epoxy groups may create a chemical bond with the carboxyl groups of collagen hydrolysate and the SO 3 − groups may electrostatically interact with the positively charged amino groups of collagen hydrolysate. For the formation of a cross-linking network between collagen hydrolysate and the P(SSNa-co-GMAx) copolymers, synthesized in the present work, various parameters were examined for the optimization of the reaction process.
Materials 2019, 12, x FOR PEER REVIEW 12 of 23 Figure 6. Phase contrast microscopy of nasal polyps fibroblasts cultured in the absence or presence of P(SSNa-co-GMA20) copolymer at concentrations 5 and 100 µg/mL for different exposure periods. All micrographs were taken at an original magnification of ×10.

Preparation of the Collagen-P(SSNa-co-GMAx) Cross-Linked Network Hydrogels
In order to develop materials that will efficiently incorporate the desired fertilizers by hydrogel formation, the collagen matrix was modified with functional polymeric electrolytes. Particularly, negatively charged polyelectrolytes were used as additives in order to interact with collagen hydrolysate, which in acidic conditions has a positively charged amino group and a carboxyl group. The cross-linking reaction of collagen hydrolysate and P(SSNa-co-GMAx) copolymers is schematically shown in Scheme 1. Based on our previous experience, we used the copolymer P(SSNa-co-GMAx), which has a dual functionality: the epoxy groups may create a chemical bond with the carboxyl groups of collagen hydrolysate and the SO3 − groups may electrostatically interact with the positively charged amino groups of collagen hydrolysate. For the formation of a cross-linking network between collagen hydrolysate and the P(SSNa-co-GMAx) copolymers, synthesized in the present work, various parameters were examined for the optimization of the reaction process. Scheme 1. Cross-linking reaction of collagen hydrolysate and P(SSNa-co-GMAx) copolymers. Scheme 1. Cross-linking reaction of collagen hydrolysate and P(SSNa-co-GMAx) copolymers.
Firstly, several experiments were conducted in order to test the difference between well-known water-soluble polymer electrolytes with the copolymers P(SSNa-co-GMAx), where x = 2, 5, 20, 30, and 40 mol% GMA. Thus, mixtures of the collagen hydrolysate with the homopolymers PSSNa, PAA, PEO and the copolymer poly(4-styrenesulfonic acid-co-maleic acid), and sodium salt P(SSNa-co-MA) were prepared in 80/20 (%) dry weight. These mixtures were stirred at R.T. for 1 h. Only in the case of PSSNa, a substantial quantity of a viscous mass was precipitated, separated from the supernatant solution and thermally treated at 80 • C for 20 h. The residue was determined around 60 wt%, which was eventually completely soluble in water.
The next step was to compare the polymer electrolyte PSSNa with the P(SSNa-co-GMAx) copolymers, where x = 2, 5, 20, 30, and 40 mol% GMA. Blends with collagen hydrolysate and the P(SSNa-co-GMAx) were prepared in the same conditions, as mentioned above. The thermal treatment was conducted at 80 • C for various time intervals. Thermal treatment at 120 • C was also tested for the reaction of collagen hydrolysate with the copolymer P(SSNa-co-GMA20).
Residues in the range of 70-100 wt% were obtained in all cases. Solubility tests where then applied to all residues and in the cases where swelling was observed and water-uptake measurements were performed. Representative results for the blends collagen hydrolysate/P(SSNa-co-GMAx) copolymers in 80/20 composition, after thermal treatment at 80 • C for 20 h, are shown in Table 3.
In the case of the copolymers with x = 2% and 5%, no swelling appeared since the immersion of the residue in excess water led to turbid solutions. On the other hand, P(SSNa-co-GMAx) with 20, 30, and 40 mol% GMA led to high residues insoluble in water. As it may be seen from Table 3, an higher water-uptake capacity was obtained when the copolymer P(SSNa-co-GMA20) was used. In the case of 30 and 40 mol% GMA, lower water-uptake capacities were observed showing that the GMA content in the copolymer may affect the swelling capacity of the cross-linked hydrogels.
Additionally, the quantity of the copolymer P(SSNa-co-GMA20) was investigated by varying the blend composition. In fact, blends of 99/1, 93/7, and 88/12 were compared with the blend 80/20 that was tested at first. From the results presented in Table 4, blend 99/1 led to a cloudy mass, whereas the rest compositions had a lower water retention compared to the composition 80/20. Experiments were also conducted with copolymers with 30 and 40 mol% GMA.
In Figure 7, the ATR-FTIR spectra of the dry collagen hydrolysate, the copolymer P(SSNa-co-GMA20), and the cross-linked hydrogel obtained from the 80/20 are presented. As it may be seen, the broad and intense peak at 3284 cm −1 is characteristic of the O-H and -NH 2 groups of collagen hydrolysate. The existence of the transmittance band at 3069 cm −1 was related to the benzene ring. The bands appeared at 2948 and 2877 cm −1 are attributed to the -CH 2 and -CH 3 groups, respectively. In addition, the bands at 1629 and 1524 cm −1 correspond to the stretching vibration of C=O and bending vibration of N-H in the amide group (-CO-NH-), respectively [36]. The peaks at 1444 and 1405 cm −1 were assigned to the symmetric stretching vibrations of carboxylate (COO − ) groups [4]. Moreover, the peak observed at 1324 cm −1 is due to the stretching vibration of -CH 2 and bending vibration of C-H [37]. Another peak is located at 1236 cm −1 from the combination peaks between N-H deformation and C-N stretching vibrations [37,38]. Obviously, the bands obtained at 1119, 1079, and 1030 cm −1 represent stretching vibrations of C-O and C-O-C of ether groups. For the confirmation of the cross-linking reaction, we examined the peak of the epoxide group at 909 cm −1 . As it may be seen from the spectrum of the cross-linked hydrogel, the peak was significantly decreased showing the successful reaction between collagen hydrolysate and epoxide groups of the copolymer P(SSNa-co-GMA20). [37]. Another peak is located at 1236 cm −1 from the combination peaks between N-H deformation and C-N stretching vibrations [37,38]. Obviously, the bands obtained at 1119, 1079, and 1030 cm −1 represent stretching vibrations of C-O and C-O-C of ether groups. For the confirmation of the cross-linking reaction, we examined the peak of the epoxide group at 909 cm −1 . As it may be seen from the spectrum of the cross-linked hydrogel, the peak was significantly decreased showing the successful reaction between collagen hydrolysate and epoxide groups of the copolymer P(SSNa-co-GMA20). The curing temperature and time were also important parameters that needed to be investigated. For the cross-linking reaction between collagen and P(SSNa-co-GMA20) copolymers, the precipitated mass was left at the desired temperature (R.T., 80 °C or 120 °C) for a period of 15 min up to 20 h, depending on the curing temperature. According to a previous work that had been conducted by our group, curing at high temperatures would allow the epoxide ring opening of GMA [25]. When the curing of the precipitated mass was conducted at R.T., the cross-linked hydrogel was eventually completely soluble in water. Moreover, as mentioned above, the heat treatment was carried out for a period of 15 min up to 20 h, when the curing temperature was 80 °C, whereas at 120 °C, the tested curing period was 15 min to 2 h. The results for collagen hydrolysate/P(SSNa-co-GMA20) blends in composition 80/20 cured at 80 °C and 120 °C are presented in Tables 5 and 6.
Finally, the water-uptake capacity of the cross-linked hydrogels was investigated. Examination of water absorbency behavior is essential for many applications in agriculture. As described in the experimental part, the hydrogels were immersed in excess water and left for 6 days. In Figure 8, a visual observation of the hydrogels cured at 120 °C at 2 and 6 days of the swelling period can be seen. As it may be seen, an increased swelling behavior was observed for all the hydrogels at the 6th day, even if a delayed swelling appeared for the ones cured at low time intervals (15 to 45 min). Almost the same attitude was observed for the hydrogels cured at 80 °C where in all cases the hydrogels showed The curing temperature and time were also important parameters that needed to be investigated. For the cross-linking reaction between collagen and P(SSNa-co-GMA20) copolymers, the precipitated mass was left at the desired temperature (R.T., 80 • C or 120 • C) for a period of 15 min up to 20 h, depending on the curing temperature. According to a previous work that had been conducted by our group, curing at high temperatures would allow the epoxide ring opening of GMA [25]. When the curing of the precipitated mass was conducted at R.T., the cross-linked hydrogel was eventually completely soluble in water. Moreover, as mentioned above, the heat treatment was carried out for a period of 15 min up to 20 h, when the curing temperature was 80 • C, whereas at 120 • C, the tested curing period was 15 min to 2 h. The results for collagen hydrolysate/P(SSNa-co-GMA20) blends in composition 80/20 cured at 80 • C and 120 • C are presented in Tables 5 and 6.
Finally, the water-uptake capacity of the cross-linked hydrogels was investigated. Examination of water absorbency behavior is essential for many applications in agriculture. As described in the experimental part, the hydrogels were immersed in excess water and left for 6 days. In Figure 8, a visual observation of the hydrogels cured at 120 • C at 2 and 6 days of the swelling period can be seen. As it may be seen, an increased swelling behavior was observed for all the hydrogels at the 6th day, even if a delayed swelling appeared for the ones cured at low time intervals (15 to 45 min). Almost the same attitude was observed for the hydrogels cured at 80 • C where in all cases the hydrogels showed high swellings after the 6th day (figure not shown). The water-uptake capacity of the cross-linked hydrogels cured at 80 and 120 • C at various time intervals are graphically presented at Figure 9A,B, where a third-order polynomial was applied for the curve fitting.
From the results of Table 5 and Figure 9A, we may notice the highest water-uptake capacity at curing time 90 min. Meanwhile, the results of Table 6 and Figure 9B showed a high value of water-uptake capacity at 15 min, which successively decreased with time. This behavior may be attributed to the fact that the cross-linking reaction accelerates at higher temperatures, and, thus in the case of 120 • C, higher water-uptake capacities were observed at lower curing times. The highest water-uptake capacity values observed in both curing temperatures indicate the formation of a cross-linked hydrogel network. The decrease of Q values after this point may be owed to a continuing cross-linking reaction leading to a more closed network that does not allow adequate swelling of the polymer hydrogels. Thus, the key point in the present work is not only to obtain high cross-linking ratios but also to achieve the ideal fraction between cross-linking and water-uptake capacity of the obtained hydrogels. In this point, it is very important to be able to control these factors according to the desired application.  The morphological characterization of the cross-linked hydrogels for the blends of collagen hydrolysate/P(SSNa-co-GMA30) was conducted through Scanning Electron Microscopy (SEM). After the thermal treatment at 80 °C and swelling for 6 days, the hydrogels were obtained through freezedrying for SEM characterization. Figure 10 shows the micrographs of the cross-linked hydrogels. From magnifications of 1000× ( Figure 10A), 2000× ( Figure 10B), and 2700× ( Figure 10C). The surface of the samples revealed a 3-D structure with a porous morphology with pore size ranging from 1-6 µm.  The morphological characterization of the cross-linked hydrogels for the blends of collagen hydrolysate/P(SSNa-co-GMA30) was conducted through Scanning Electron Microscopy (SEM). After the thermal treatment at 80 °C and swelling for 6 days, the hydrogels were obtained through freezedrying for SEM characterization. Figure 10 shows the micrographs of the cross-linked hydrogels. From magnifications of 1000× ( Figure 10A), 2000× ( Figure 10B), and 2700× ( Figure 10C). The surface of the samples revealed a 3-D structure with a porous morphology with pore size ranging from 1-6 µm. Experiments conducted by Saruchi et al. [39] by using hydrogel interpenetrating polymer network (hydrogel-IPN) of natural gum polysaccharide with acrylamide and methacrylic acid showed swelling ratios between 165% and 259%. Moreover, Dang et al. [3] measured swelling rates for various blends of collagen protein powder with corn starch in water for 24 h reaching the maximum value of 89%. Li et al. [19] measured the swelling behavior of wheat straw cellulose hydrogels in various conditions, which showed values between 70 and 180 g/g.
Comparing the water-uptake capacities of the cross-linked hydrogels synthesized in the present work with the above-mentioned results published in literature, we may notice a very interesting behavior of the current materials since they show higher water-uptake values in the range of 800-1800% at short time intervals. The results are very encouraging in order to proceed to a further investigation of these or other systems.
The morphological characterization of the cross-linked hydrogels for the blends of collagen hydrolysate/P(SSNa-co-GMA30) was conducted through Scanning Electron Microscopy (SEM). After the thermal treatment at 80 • C and swelling for 6 days, the hydrogels were obtained through freeze-drying for SEM characterization. Figure 10 shows the micrographs of the cross-linked hydrogels. From magnifications of 1000× ( Figure 10A), 2000× (Figure 10B), and 2700× ( Figure 10C). The surface of the samples revealed a 3-D structure with a porous morphology with pore size ranging from 1-6 µm. The hydrogel's cytotoxicity was also tested against the nasal polyps fibroblasts ( Figure 11). As shown in Figure 11, the fibroblasts viability was not affected after 72 h of being in contact with hydrogels. On contrary, in the presence of materials, a significant increase of cells proliferation was observed. It should also be noticed that the degradation by-products that could be produced during the 72 h incubation did not affect cell viability. Figure 11. Evaluation of cell viability after 24 and 72 h culture of cells in contact with hydrogels. Control: cells in the absence of hydrogels. Each result is the mean ± SD of at least three independent experiments. Statistically significant differences were evaluated using the unpaired Student's t-test. (*) indicates statistically significant increase (p < 0.05) compared to control.

Characterization of Collagen Hydrogel Fertilizers
Chemical modification of collagenous materials may occur with a micro-or macromolecular reagent, capable of interacting with one or more types of functional groups attached to the polypeptide chain. The encapsulation of nutrients into the collagenous materials and the modification with functional polymers led to the development of hydrogels that may be applied as potential fertilizers. Based on this methodology, the copolymer P(SSNa-co-GMA20) was used in order to ensure functionality with the collagenous materials containing nutrients. In parallel, starch was used as a biopolymer in combination with the cross-linker methylene bis-acrylamide for the modification of the The hydrogel's cytotoxicity was also tested against the nasal polyps fibroblasts ( Figure 11). As shown in Figure 11, the fibroblasts viability was not affected after 72 h of being in contact with hydrogels. On contrary, in the presence of materials, a significant increase of cells proliferation was observed. It should also be noticed that the degradation by-products that could be produced during the 72 h incubation did not affect cell viability. The hydrogel's cytotoxicity was also tested against the nasal polyps fibroblasts ( Figure 11). As shown in Figure 11, the fibroblasts viability was not affected after 72 h of being in contact with hydrogels. On contrary, in the presence of materials, a significant increase of cells proliferation was observed. It should also be noticed that the degradation by-products that could be produced during the 72 h incubation did not affect cell viability. Figure 11. Evaluation of cell viability after 24 and 72 h culture of cells in contact with hydrogels. Control: cells in the absence of hydrogels. Each result is the mean ± SD of at least three independent experiments. Statistically significant differences were evaluated using the unpaired Student's t-test. (*) indicates statistically significant increase (p < 0.05) compared to control.

Characterization of Collagen Hydrogel Fertilizers
Chemical modification of collagenous materials may occur with a micro-or macromolecular reagent, capable of interacting with one or more types of functional groups attached to the polypeptide chain. The encapsulation of nutrients into the collagenous materials and the modification with functional polymers led to the development of hydrogels that may be applied as potential fertilizers. Based on this methodology, the copolymer P(SSNa-co-GMA20) was used in order to ensure functionality with the collagenous materials containing nutrients. In parallel, starch was used as a biopolymer in combination with the cross-linker methylene bis-acrylamide for the modification of the Figure 11. Evaluation of cell viability after 24 and 72 h culture of cells in contact with hydrogels. Control: cells in the absence of hydrogels. Each result is the mean ± SD of at least three independent experiments. Statistically significant differences were evaluated using the unpaired Student's t-test. (*) indicates statistically significant increase (p < 0.05) compared to control.

Characterization of Collagen Hydrogel Fertilizers
Chemical modification of collagenous materials may occur with a micro-or macromolecular reagent, capable of interacting with one or more types of functional groups attached to the polypeptide chain. The encapsulation of nutrients into the collagenous materials and the modification with functional polymers led to the development of hydrogels that may be applied as potential fertilizers. Based on this methodology, the copolymer P(SSNa-co-GMA20) was used in order to ensure functionality with the collagenous materials containing nutrients. In parallel, starch was used as a biopolymer in combination with the cross-linker methylene bis-acrylamide for the modification of the nutrient encapsulated collagen. Various parameters, such as temperature and polymer concentration, were examined in order to obtain the optimum conditions for the functionalization of enriched collagen hydrogels with functional polymers in order to obtain biofertilizers with controlled release.
Chemical analysis of the three fertilizers (Table 7) reveals similar features between reference sample and the sample functionalized with the synthetic copolymer IN20c, while the collagen hydrolysate functionalized with starch shows minimum content in dry substance, ash, and total nitrogen. The fertilizing capacity is conferred mainly by the content of the nutrients encapsulated, quantified in Table 8. Their presence is confirmed by EDAX analysis (Figure 12A-C), where the signal specific to potassium is dominant in all three samples, while the presence of other useful elements is detected, such as phosphorous, sulfur, magnesium, calcium, sodium, etc. A different note among the samples is manifested by the carbon content, which is the highest for collagen hydrolysate functionalized with starch, as expected. Moreover, examination of SEM images reveals similar microstructures for CH-Ref and CH-IN samples ( Figure 12A,B) that could be explained by quite a low content of synthetic copolymer (5% IN20c) imposed by biodegradability requirements of fertilization, which does not create major changes in the collagen microstructure. At the same time, the high content of highly biodegradable biopolymer starch results in important changes in the morphology of these samples as compared with the reference sample ( Figure 12C).
In Figure 13, the variation of release degree for oxidable compounds in time for these biofertilizers is illustrated.
The experimental data concerning the release degree of oxidable compounds in free water of these biofertilizers increases in time to maximum after about 26 h: 99.9%, 87%, and over 90%, for CH-Ref, CH-IN, and CH-Starch, respectively ( Figure 13). Reference collagen hydrolysate exhibits a higher capacity to release of oxidable compounds as compared to the other two samples, thus revealing that functionalization of collagen results in slowing their release capacity.  In Figure 13, the variation of release degree for oxidable compounds in time for these biofertilizers is illustrated.  The experimental data concerning the release degree of oxidable compounds in free water of these biofertilizers increases in time to maximum after about 26 h: 99.9%, 87%, and over 90%, for CH-Ref, CH-IN, and CH-Starch, respectively ( Figure 13). Reference collagen hydrolysate exhibits a higher capacity to release of oxidable compounds as compared to the other two samples, thus revealing that functionalization of collagen results in slowing their release capacity.
As referring to the release mechanism, one may suppose that in the first stage, the organic and inorganic compounds with high solubility are removed, like peptides with short chains, soluble polymers (starch), aminoacids, glucides (mono-and di-zaharides), nitrate salts, acids, etc. In the second stage, the rate of release of oxidable compounds is decreasing for all fertilizers and can be assessed to organic compounds with low solubility, such as proteins, collagen, and degradable polymers [29].

Conclusions
The combination of collagen hydrolysate as a matrix with the water-soluble polymers provides the development of cross-linked network hydrogels with the prospect to obtain "smart" organic biofertilizers. In the present work, copolymers with various GMA content were synthesized and their characterization in terms of stoichiometry, thermal behavior, molecular weight, and chemical structure was performed. It was also assessed that the prepared copolymer, as well as the respective hydrogels, was not cytotoxic against fibroblasts of different origin, normal or pathologic, and it did not alter the cells' morphology. In a further step, various blends of collagen hydrolysate with different functionalized water-soluble polymers were made in order to prepare cross-linked network hydrogels. ATR-FTIR analysis confirmed the cross-linking reaction between collagen hydrolysate and epoxide groups of the copolymer. Depending on the cross-linking ratio, we were able to control the wateruptake capacity of the cross-linked hydrogels.
Moreover, preliminary tests on three samples of collagen hydrolysate with encapsulated nutrients (N, P, K), functionalized with the synthetic copolymer and starch as biopolymer, led to the development of hydrogels that may be applied as potential fertilizers. The variation of release degree for oxidable compounds in time for these biofertilizers showed that the enriched collagen functionalized with the polymers led to a slower release capacity of oxidable compounds than the unfunctionalized enriched collagen.
It is worth to note that the current work presents a concept with high environmental impact, since the recovery of collagen hydrolysate from tannery waste contributes to a circular economy model As referring to the release mechanism, one may suppose that in the first stage, the organic and inorganic compounds with high solubility are removed, like peptides with short chains, soluble polymers (starch), aminoacids, glucides (mono-and di-zaharides), nitrate salts, acids, etc. In the second stage, the rate of release of oxidable compounds is decreasing for all fertilizers and can be assessed to organic compounds with low solubility, such as proteins, collagen, and degradable polymers [29].

Conclusions
The combination of collagen hydrolysate as a matrix with the water-soluble polymers provides the development of cross-linked network hydrogels with the prospect to obtain "smart" organic biofertilizers. In the present work, copolymers with various GMA content were synthesized and their characterization in terms of stoichiometry, thermal behavior, molecular weight, and chemical structure was performed. It was also assessed that the prepared copolymer, as well as the respective hydrogels, was not cytotoxic against fibroblasts of different origin, normal or pathologic, and it did not alter the cells' morphology. In a further step, various blends of collagen hydrolysate with different functionalized water-soluble polymers were made in order to prepare cross-linked network hydrogels. ATR-FTIR analysis confirmed the cross-linking reaction between collagen hydrolysate and epoxide groups of the copolymer. Depending on the cross-linking ratio, we were able to control the water-uptake capacity of the cross-linked hydrogels.
Moreover, preliminary tests on three samples of collagen hydrolysate with encapsulated nutrients (N, P, K), functionalized with the synthetic copolymer and starch as biopolymer, led to the development of hydrogels that may be applied as potential fertilizers. The variation of release degree for oxidable compounds in time for these biofertilizers showed that the enriched collagen functionalized with the polymers led to a slower release capacity of oxidable compounds than the un-functionalized enriched collagen.
It is worth to note that the current work presents a concept with high environmental impact, since the recovery of collagen hydrolysate from tannery waste contributes to a circular economy model involving socio-economic advantages. Moreover, the P(SSNa-co-GMAx) is a cost-effective copolymer since the monomers may be provided in bulk quantities with low cost.
These findings provide new insights and foster a better understanding of the behavior of the cross-linked network hydrogels based on the combination of collagen hydrolysate and hydrophilic polymers with functional groups, which may open the way for new practical applications of these systems in agriculture as a controlled-release biostimulant for plant growth.
Author Contributions: J.K.K., conceptualization and experimental design; G.C.L. and I.T., writing-Original draft preparation; J.K.K., A.M., and G.Z., review and editing; A.M. and G.Z. provided the collagen hydrolysate; G.C.L. and I.T. performed the experiments concerning the synthesis of copolymers and cross-linked network hydrogels development; S.S. performed the experiments with nutrients encapsulated in the hydrogels and release degree of oxidable organic compounds; A.J.A. performed the experiments concerning the copolymer's toxicological assessment.