Chitosan Sponges for Efficient Accumulation and Controlled Release of C-Phycocyanin

The paper proposed a new porous material for wound healing based on chitosan and C-phycocyanin (C-PC). In this work, C-PC was extracted from the cyanobacteria Arthrospira platensis biomass and purified through ammonium sulfate precipitation. The obtained C-PC with a purity index (PI) of 3.36 ± 0.24 was loaded into a chitosan sponge from aqueous solutions of various concentrations (250, 500, and 1000 mg/L). According to the FTIR study, chitosan did not form new bonds with C-PC, but acted as a carrier. The encapsulation efficiency value exceeded 90%, and the maximum loading capacity was 172.67 ± 0.47 mg/g. The release of C-PC from the polymer matrix into the saline medium was estimated, and it was found 50% of C-PC was released in the first hour and the maximum concentration was reached in 5–7 h after the sponge immersion. The PI of the released C-PC was 3.79 and 4.43 depending on the concentration of the initial solution.


Introduction
Phototrophic microorganisms are a promising material for the environmentally sustainable production of a wide range of high value-added compounds (proteins, carbohydrates, lipids, and pigments) through the efficient use of artificial and actual sunlight. Among commercially used microalgae species, the cyanobacterium p. Arthrospira (commercially known as Spirulina) has been cultivated on a large scale for over 40 years as the basis of nutritional supplements and animal feed due to its high nutrient content and easy digestibility [1]. In addition, the presence of primary amino groups is the basis for most of the biological effects of chitosan, namely, antimicrobial, anti-inflammatory, and fungicidal action, as well as hemostatic and wound healing ability.
The wound-healing effect of chitosan oligomers is associated with their ability to stimulate the production of a number of enzymes-lysozyme, chitinase, and N-acetyl-b-D-glucosaminidase-the hydrolytic effect of which promotes macrophage activation and collagen synthesis [19]. Wound healing based on the activation of macrophages by chitosan is associated with the presence of receptors for Glc-NAc-glycoproteins in the latter.
The use of a chitosan-based sponge impregnated with fibroblast growth factors (FGF) has been described in [20]. The inclusion of growth factors in sponges made of chitosan ensures a uniform distribution of the active substance and minimizes local irritation during drug administration [21].
A positive effect of various active substances loaded into the matrix of chitosan is shown in the process of healing diabetic wounds [22,23].
Upon contact with physiological fluids, the chitosan sponge forms a hydrogel, thus ensuring the delivery of the active substance even to deep wounds, unlike gels or ointments. As the sponge is destroyed under the action of enzymes, a steady release of the active substance is carried out. In addition, removal of the sponge after the wound has healed is most often not required.
The ability of chitosan to stabilize phycocyanin, preventing its thermal degradation has also been reported [24,25].
The aim of this work is to prepare chitosan sponges and study in vitro accumulation of C-PC isolated from the biomass of the cyanobacterium Arthrospira platensis by them, as well as the C-PC's release properties and its characteristics after release.

Strain and Cultivation Conditions
C-phycocyanin was obtained by exhaustive extraction from the biomass of cyanobacterium Arthrospira platensis B-12619 (Russian National Collection of Industrial Microorganisms). A. platensis was grown in modified Zarrouk medium [26] consisting of (g/L): In addition, the presence of primary amino groups is the basis for most of the biological effects of chitosan, namely, antimicrobial, anti-inflammatory, and fungicidal action, as well as hemostatic and wound healing ability.
The wound-healing effect of chitosan oligomers is associated with their ability to stimulate the production of a number of enzymes-lysozyme, chitinase, and N-acetyl-b-D-glucosaminidase-the hydrolytic effect of which promotes macrophage activation and collagen synthesis [18]. Wound healing based on the activation of macrophages by chitosan is associated with the presence of receptors for GlcNAc-glycoproteins in the latter.
The use of a chitosan-based sponge impregnated with fibroblast growth factors (FGF) has been described in [19]. The inclusion of growth factors in sponges made of chitosan ensures a uniform distribution of the active substance and minimizes local irritation during drug administration [20].
A positive effect of various active substances loaded into the matrix of chitosan is shown in the process of healing diabetic wounds [21,22].
Upon contact with physiological fluids, the chitosan sponge forms a hydrogel, thus ensuring the delivery of the active substance even to deep wounds, unlike gels or ointments. As the sponge is destroyed under the action of enzymes, a steady release of the active substance is carried out. In addition, removal of the sponge after the wound has healed is most often not required.
The ability of chitosan to stabilize phycocyanin, preventing its thermal degradation has also been reported [23,24].
The aim of this work is to prepare chitosan sponges and study in vitro accumulation of C-PC isolated from the biomass of the cyanobacterium Arthrospira platensis by them, as well as the C-PC's release properties and its characteristics after release.

C-Phycocyanin Extraction and Purification
For exhaustive C-PC extraction, the A. platensis wet biomass was subjected to three cycles of freezing/thawing, dissolved in 0.2 M sodium phosphate buffer (pH 7.00 ± 0.02), and left incubating at 4 • C in the dark overnight [28]. Next, the samples were centrifuged (17,300× g, 30 min, 4 • C) to separate the supernatant, which was further purified and used in all experiments.
The C-PC crude extracts were purified by ammonium sulfate precipitation. At each purification step, the C-PC concentration and purity were controlled. Ammonium sulfate was gradually added to the crude extracts to achieve 20, 40, and 60% saturation with continuous stirring. The resulting solutions were kept overnight at 4 • C and centrifuged (17,300× g, 30 min, 4 • C). The obtained precipitate was dissolved in 0.2M sodium phosphate buffer (pH 7.00 ± 0.02). At the final stage (60% ammonium sulfate saturation) the C-PC precipitate was dissolved in deionized water and dialyzed against 100 volumes of deionized water overnight in the dark at 4 • C. The mixture was freeze-dried.

C-Phycocyanin Quantification and Purity Determination
The C-PC concentration was calculated according to Equation (1) [29], taking the absorption spectra with a Genesys 10S UV-visible spectrophotometer (Thermo Scientific, USA).
where A λ is the absorbance of the extract at λ nm. The C-PC purity index (PI) was determined according to Equation (2) [30].
where A λ is the absorbance of the solution at λ nm. Equivalent amounts of the protein fractions were analyzed by protein gel electrophoresis SDS-PAGE (13%).

High-Performance Liquid Chromatography
HPLC was performed on an Agilent Technologies 1200 chromatograph equipped with a DAD detector on a Kromasil C 5 (250 × 4.6 mm) column. The composition of the mobile phase was acetonitrile containing 0.1% trifluoroacetic acid (solution A) and 0.1% aqueous solution of trifluoroacetic acid (solution B). The flow rate was 1.2 mL/min. The solution ratio solution A/solution B was increased from 25% to 100% within 15 min. The volume of the injected sample was 50 µL. The column effluent was monitored using a photodiode array detector at 620 nm.

Antioxidant Activity Measurements
The DPPH free radical scavenging activity of the C-PC samples was evaluated according to [28]. The C-PC sample or antioxidant standard (Trolox) ethanol solution was added to the same volume of a 0.1 mM DPPH (Sigma-Aldrich, Burlington, NJ, USA) ethanol solution and incubated at 25 • C in the dark for 30 min. The absorbance was measured at 517 nm using ethanol as a blank. The percentage inhibition of free radicals was determined according to Equation (3).
where A sample is the absorbance of the treated sample, and A control is the absorbance of 0.05 mM DPPH ethanol solution.
The antioxidant activity of the C-PC samples was expressed in terms of the effective concentration of IC 50 , i.e., the concentration required to inhibit 50% of the DPPH free radicals, which was calculated using the linear regression formula.
The Trolox equivalent antioxidant capacity (TEAC) was calculated according to Equation (4) using the DPPH free radicals inhibition curves for Trolox and the C-PC samples. The result was expressed in terms of micromoles of trolox equivalent (TE) per gram of the C-PC (µM TE/g):

Chitosan Sponge Preparation
Sponges were prepared from Primex ChitoClear ® HQG 1600 (Iceland). Then, 200-300 µL of a 25% sodium hydroxide solution was added dropwise to a 2% (wt.) solution of chitosan in 2% (wt.) acetic acid with vigorous stirring to reach pH 5.7, and then 1% (wt.) of glutaraldehyde (Russia) was added (calculated on the dry weight of chitosan). The resulting solution was poured into a plate and frozen, after which it was freeze-dried on Martin Crist ALPHA 1-4LSC (Germany).

Incorporation of C-PC into a Chitosan Sponge, Encapsulation Efficiency (Entry Efficiency), Load Capacity, and In Vitro Release Study
C-PC was introduced into the chitosan sponge matrix by immersing the sponge (29.21 ± 2.24 mg) in 5 mL of an aqueous solution of C-PC at three test concentrations of 250, 500, and 1000 µg/mL (hereinafter, CPC-250, CPC-500, and CPC-1000) for 24 h at room temperature in the dark.
Next, the sponges were freeze-dried and stored at 4 • C until testing. The relative concentration of C-PC (C r , %) is the remaining concentration of C-PC (C t ) as a percentage of the initial concentration (C 0 ) according to the equation: The amount of C-PC transferred to the sponge was determined by the formula: where C 0 , V 0 and C t , V t are concentrations and volumes of C-PC solution at the initial and final time, respectively. The entry efficiency (EE) was calculated by the formula: where m 0 and m un are the initial mass and mass of unloaded C-PC, respectively. Load capacity (LC): where m L (mg) and m S (g) are the mass of the loaded C-PC and the mass of the sponge, respectively.
To study the release of loaded C-PC, C-PC loaded sponge samples were placed in 10 mL of saline (pH 7.4) and kept at 37 • C and 100 rpm for 24 h.

Microscopy Imaging
Microscopy images were obtained by using a fluorescence microscope BX61 (Olympus, Tokyo, Japan) with a DP71 digital microscope camera (Olympus, Japan).

Fourier Transform Infrared Spectroscopy Measurements (FTIR)
IR spectra were recorded using a Nicolet iS5 IR Fourier spectrometer (Thermo Fisher Scientific, USA) with an iD5 ATR frustrated total internal reflection attachment (crystaldiamond). The spectral range was 4000-550 cm −1 , the spectral resolution was 4 cm −1 , the number of scans was 32. The spectra were recorded and processed using the standard software of the instrument (Omnic 8.2).

Scanning Electron Microscopy of Sponges (SEM)
SEM images of the chitosan-based sponge were obtained using a scanning electron microscope Thermo Scientific Phenom XL with a backscattered electron detector at an accelerating voltage of 5 kV and a pressure of 60 Pa without a conductive coating.

Statistical Analysis
Numerical results are presented as means ± SD of at least three independent replicates. One-way analysis of variance (ANOVA) followed by Tukey's test were used to verify significant differences considering a confidence level of 95% (p < 0.05).

Results and Discussion
At the moment, the market price of C-PC with a purity of more than 3.5 exceeds 150EUR per 1 mg [31], and commercial products containing C-PC of similar purity will have a high cost. Therefore, in our studies, we used C-PC with a PI of 3.36 ± 0.24, obtained using a simple and cheap process for extracting C-PC from the wet biomass of the cyanobacterium A. platensis. Figure 2 shows optical and fluorescent microscopy of the cyanobacterium A. platensis, as well as the absorption and fluorescence spectra of the C-PC isolated from it, which was used for the experiments with sponges. Microscopy images were obtained by using a fluorescence microscope BX61 (Olympus, Tokyo, Japan) with a DP71 digital microscope camera (Olympus, Japan).

Fourier Transform Infrared Spectroscopy Measurements (FTIR)
IR spectra were recorded using a Nicolet iS5 IR Fourier spectrometer (Thermo Fisher Scientific, USA) with an iD5 ATR frustrated total internal reflection attachment (crystaldiamond). The spectral range was 4000-550 cm -1 , the spectral resolution was 4 cm -1 , the number of scans was 32. The spectra were recorded and processed using the standard software of the instrument (Omnic 8.2).

Scanning Electron Microscopy of Sponges (SEM)
SEM images of the chitosan-based sponge were obtained using a scanning electron microscope Thermo Scientific Phenom XL with a backscattered electron detector at an accelerating voltage of 5 kV and a pressure of 60 Pa without a conductive coating.

Statistical Analysis
Numerical results are presented as means ± SD of at least three independent replicates. One-way analysis of variance (ANOVA) followed by Tukey's test were used to verify significant differences considering a confidence level of 95% (p < 0.05).

Results and Discussion
At the moment, the market price of C-PC with a purity of more than 3.5 exceeds 150EUR per 1 mg [32], and commercial products containing C-PC of similar purity will have a high cost. Therefore, in our studies, we used C-PC with a PI of 3.36 ± 0.24, obtained using a simple and cheap process for extracting C-PC from the wet biomass of the cyanobacterium A. platensis. Figure 2 shows optical and fluorescent microscopy of the cyanobacterium A. platensis, as well as the absorption and fluorescence spectra of the C-PC isolated from it, which was used for the experiments with sponges.

Extraction and Purification of Phycocyanin
C-PC was extracted from the wet biomass and isolated by 3 cycles of freeze/thawing in phosphate buffer (pH 7.0). As a result, a crude extract was obtained, the absorption spectrum of which had two main maxima in the region of 280 nm (protein part) and 620 nm (chromophore group), as well as a small shoulder at 650 nm (Figure 3), indicating the presence of trace amounts of allophycocyanin (APC), the PI was 1.53. Purification of the

Extraction and Purification of Phycocyanin
C-PC was extracted from the wet biomass and isolated by 3 cycles of freeze/thawing in phosphate buffer (pH 7.0). As a result, a crude extract was obtained, the absorption spectrum of which had two main maxima in the region of 280 nm (protein part) and 620 nm (chromophore group), as well as a small shoulder at 650 nm (Figure 3), indicating the presence of trace amounts of allophycocyanin (APC), the PI was 1.53. Purification of the BioTech 2023, 12, 55 7 of 14 C-PC was carried out by stepwise precipitation with ammonium sulfate. For this, 20% ammonium sulfate was initially added to the crude extract, the precipitate was discarded, and 40% ammonium sulfate was added to the supernatant solution. At this stage, the separation of the C-PC that precipitated and the APC that remained in the solution occurred, as evidenced by the absence of a shoulder at 650 nm in the absorption spectrum. Next, the saturation with ammonium sulfate was increased to 60%, which led to the complete precipitation of the C-PC. The precipitated pigment was dissolved in milliQ, dialyzed overnight against water, and then lyophilized. The resulting powder had a PI of 3.36 ± 0.24. Prior to testing, C-PC powder was stored at 4 • C.
During the purification of the crude C-PC, the change in the PI, along with the absorption spectra, was also controlled by gel electrophoresis. Figure 3b clearly shows two bands at about 17 kDa and 19 kDa, corresponding to the αand β-subunits of C-PC. In addition, these pictures show that with each subsequent stage of purification, the PI of the C-PC increased, the intensity of the absorption maximum at 280 nm decreased (Figure 3a), while the number of bands corresponding to impurity proteins (molecular weight different from the mass of αand β-subunits) also decreased ( Figure 3b).
BioTech 2023, 4, x FOR PEER REVIEW 7 of 14 C-PC was carried out by stepwise precipitation with ammonium sulfate. For this, 20% ammonium sulfate was initially added to the crude extract, the precipitate was discarded, and 40% ammonium sulfate was added to the supernatant solution. At this stage, the separation of the C-PC that precipitated and the APC that remained in the solution occurred, as evidenced by the absence of a shoulder at 650 nm in the absorption spectrum. Next, the saturation with ammonium sulfate was increased to 60%, which led to the complete precipitation of the C-PC. The precipitated pigment was dissolved in milliQ, dialyzed overnight against water, and then lyophilized. The resulting powder had a PI of 3.36 ± 0.24. Prior to testing, C-PC powder was stored at 4 °C. During the purification of the crude C-PC, the change in the PI, along with the absorption spectra, was also controlled by gel electrophoresis. Figure 3b clearly shows two bands at about 17 kDa and 19 kDa, corresponding to the α-and β-subunits of C-PC. In addition, these pictures show that with each subsequent stage of purification, the PI of the C-PC increased, the intensity of the absorption maximum at 280 nm decreased (Figure 3a), while the number of bands corresponding to impurity proteins (molecular weight different from the mass of α-and β-subunits) also decreased (Figure 3b).

Characteristics of Pure C-PC
Also, pure C-PC was further characterized by reverse phase HPLC (Figure 4), FTIR, and its AOA was also determined.

Characteristics of Pure C-PC
Also, pure C-PC was further characterized by reverse phase HPLC (Figure 4), FTIR, and its AOA was also determined.
BioTech 2023, 4, x FOR PEER REVIEW 7 of 14 C-PC was carried out by stepwise precipitation with ammonium sulfate. For this, 20% ammonium sulfate was initially added to the crude extract, the precipitate was discarded, and 40% ammonium sulfate was added to the supernatant solution. At this stage, the separation of the C-PC that precipitated and the APC that remained in the solution occurred, as evidenced by the absence of a shoulder at 650 nm in the absorption spectrum. Next, the saturation with ammonium sulfate was increased to 60%, which led to the complete precipitation of the C-PC. The precipitated pigment was dissolved in milliQ, dialyzed overnight against water, and then lyophilized. The resulting powder had a PI of 3.36 ± 0.24. Prior to testing, C-PC powder was stored at 4 °C. During the purification of the crude C-PC, the change in the PI, along with the absorption spectra, was also controlled by gel electrophoresis. Figure 3b clearly shows two bands at about 17 kDa and 19 kDa, corresponding to the α-and β-subunits of C-PC. In addition, these pictures show that with each subsequent stage of purification, the PI of the C-PC increased, the intensity of the absorption maximum at 280 nm decreased (Figure 3a), while the number of bands corresponding to impurity proteins (molecular weight different from the mass of α-and β-subunits) also decreased (Figure 3b).

Characteristics of Pure C-PC
Also, pure C-PC was further characterized by reverse phase HPLC (Figure 4), FTIR, and its AOA was also determined.  Detection at 620 nm revealed two major peaks (7.66 and 8.02 min) which were identified as the αand β-subunits of C-PC. The β-subunit contains twice as much phycocyanobilin chromophore as the α-subunit, so the peak attributed to the β-subunit is more intense. The results obtained are consistent with the data from the literature [32][33][34].
The pure C-PC was also examined by FTIR ( Figure 5). The IR spectrum of the C-PC contains specific bands of amide I (C=O stretching vibrations) and amide II (N-H bending + C-N stretching) at 1650 cm −1 and 1536 cm −1 , respectively; and the intensity of the amide I band is higher than amide II. The position and shape of the amide I band are used to analyze the secondary structure of the protein. The sharp peak of the amide I band points to the α-helix as the main element of its secondary structure [11]. It should be noted that the IR spectrum of the lyophilized pure C-PC additionally confirmed the absence of impurities of inorganic sulfates and phosphates (absence of intense bands in the region of 1040 and 1015 cm −1 ). Detection at 620 nm revealed two major peaks (7.66 and 8.02 min) which were identified as the α-and β-subunits of C-PC. The β-subunit contains twice as much phycocyanobilin chromophore as the α-subunit, so the peak attributed to the β-subunit is more intense. The results obtained are consistent with the data from the literature [33][34][35].
The pure C-PC was also examined by FTIR ( Figure 5). The IR spectrum of the C-PC contains specific bands of amide I (C=O stretching vibrations) and amide II (N-H bending + C-N stretching) at 1650 cm −1 and 1536 cm −1 , respectively; and the intensity of the amide I band is higher than amide II. The position and shape of the amide I band are used to analyze the secondary structure of the protein. The sharp peak of the amide I band points to the α-helix as the main element of its secondary structure [12]. It should be noted that the IR spectrum of the lyophilized pure C-PC additionally confirmed the absence of impurities of inorganic sulfates and phosphates (absence of intense bands in the region of 1040 and 1015 cm −1 ). Antioxidant activity was determined by the radical scavenging activity of DPPH (2,2-diphenylpicrylhydrazyl). For the pure C-PC, the IC50 was 212.73 µg/mL, 174.47 TEAC.

Chitosan Sponges
Based on chitosan Primex ChitoClear ® HQG 1600, sponges were obtained by lyophilization of an acidic aqueous solution. The standard method involves freeze-drying an aqueous chitosan solution in acetic acid. However, in this work, the approach was slightly modified. An important factor affecting the stability of phycocyanin is the acidity of the medium. According to the data from the literature [36], C-phycocyanin retains its stability at medium with pH in the range of 5.5-6.0. At higher or lower values, degradation of C-phycocyanin occurs with a change in its spectral properties and color.
A chitosan sponge placed in an aqueous solution causes a significant decrease in the pH of the medium by washing out the acetic acid remaining in the sponge after lyophilization. In order to combine the sponge with the phycocyanin solution, before lyophilization, the pH of the aqueous acid solution of chitosan was adjusted to a value of 5.7 by adding a small amount (200-300 µL) of a 25% aqueous solution of sodium hydroxide. This pH value approximately corresponds to the complete neutralization of free protons not bound to the amino groups of chitosan (Figure 6). At the same time, the polymer does not precipitate due to intensive mixing. The use of a concentrated alkali solution makes it possible to avoid dilution of the chitosan solution and, as a result, a decrease in the mechanical characteristics of the sponges. Antioxidant activity was determined by the radical scavenging activity of DPPH (2,2-diphenylpicrylhydrazyl). For the pure C-PC, the IC 50 was 212.73 µg/mL, 174.47 TEAC.

Chitosan Sponges
Based on chitosan Primex ChitoClear ® HQG 1600, sponges were obtained by lyophilization of an acidic aqueous solution. The standard method involves freeze-drying an aqueous chitosan solution in acetic acid. However, in this work, the approach was slightly modified. An important factor affecting the stability of phycocyanin is the acidity of the medium. According to the data from the literature [35], C-phycocyanin retains its stability at medium with pH in the range of 5.5-6.0. At higher or lower values, degradation of C-phycocyanin occurs with a change in its spectral properties and color.
A chitosan sponge placed in an aqueous solution causes a significant decrease in the pH of the medium by washing out the acetic acid remaining in the sponge after lyophilization. In order to combine the sponge with the phycocyanin solution, before lyophilization, the pH of the aqueous acid solution of chitosan was adjusted to a value of 5.7 by adding a small amount (200-300 µL) of a 25% aqueous solution of sodium hydroxide. This pH value approximately corresponds to the complete neutralization of free protons not bound to the amino groups of chitosan (Figure 6). At the same time, the polymer does not precipitate due to intensive mixing. The use of a concentrated alkali solution makes it possible to avoid dilution of the chitosan solution and, as a result, a decrease in the mechanical characteristics of the sponges.
When the first equivalence point is reached, the free acid is neutralized. The first equivalence point roughly corresponds to the solution pH of 5.6. Further titration is based on the interaction of the titrant with the protonated amino groups of chitosan. When the first equivalence point is reached, the free acid is neutralized. The first equivalence point roughly corresponds to the solution pH of 5.6. Further titration is based on the interaction of the titrant with the protonated amino groups of chitosan. Since the pore wall thickness of chitosan sponges is quite small, the pore wall thickness does not exceed 100 µm (Figure 7d), and most of the protonated amino groups of chitosan are probably located on the wall surface. At pH 5.7, phycocyanin, whose isoelectric point corresponds to pH 4.6, is predominantly negatively charged, which contributes to more efficient adsorption of the protein complex on the surface of the chitosan sponge due to the forces of electrostatic interaction.

Sponge Chitosan-Phycocyanin
With the introduction of C-PC into the matrix of the chitosan sponge, three concentrations were tested: 250, 500, and 1000 µg/mL (hereinafter, CPC-250, CPC-500, and CPC-1000). The choice of tested concentrations was based on the analysis of the data from the literature [6][7][8][9][37][38][39][40][41][42]. As can be seen in Figure 8, at the lowest concentration tested (CPC-250), half of the initial amount of C-PC passed into the sponge in about 2.5 h, while the other two concentrations took longer, 4 and 5 h, respectively, for CPC-500 and CPC-1000. After 8 h from the beginning of the experiment, 72.35 ± 2.39, 64.64 ± 1.26 and 57.05 ± 0.73% of the initial amount of C-PC passed into the sponges for CPC-250, CPC-500, and CPC-1000, respectively. Over the next 16 h, the amount of C-PC that did Since the pore wall thickness of chitosan sponges is quite small, the pore wall thickness does not exceed 100 µm (Figure 7d), and most of the protonated amino groups of chitosan are probably located on the wall surface. At pH 5.7, phycocyanin, whose isoelectric point corresponds to pH 4.6, is predominantly negatively charged, which contributes to more efficient adsorption of the protein complex on the surface of the chitosan sponge due to the forces of electrostatic interaction. When the first equivalence point is reached, the free acid is neutralized. The first equivalence point roughly corresponds to the solution pH of 5.6. Further titration is based on the interaction of the titrant with the protonated amino groups of chitosan. Since the pore wall thickness of chitosan sponges is quite small, the pore wall thickness does not exceed 100 µm (Figure 7d), and most of the protonated amino groups of chitosan are probably located on the wall surface. At pH 5.7, phycocyanin, whose isoelectric point corresponds to pH 4.6, is predominantly negatively charged, which contributes to more efficient adsorption of the protein complex on the surface of the chitosan sponge due to the forces of electrostatic interaction.

Sponge Chitosan-Phycocyanin
With the introduction of C-PC into the matrix of the chitosan sponge, three concentrations were tested: 250, 500, and 1000 µg/mL (hereinafter, CPC-250, CPC-500, and CPC-1000). The choice of tested concentrations was based on the analysis of the data from the literature [6][7][8][9][37][38][39][40][41][42]. As can be seen in Figure 8, at the lowest concentration tested (CPC-250), half of the initial amount of C-PC passed into the sponge in about 2.5 h, while the other two concentrations took longer, 4 and 5 h, respectively, for CPC-500 and CPC-1000. After 8 h from the beginning of the experiment, 72.35 ± 2.39, 64.64 ± 1.26 and 57.05 ± 0.73% of the initial amount of C-PC passed into the sponges for CPC-250, CPC-500, and CPC-1000, respectively. Over the next 16 h, the amount of C-PC that did

Sponge Chitosan-Phycocyanin
With the introduction of C-PC into the matrix of the chitosan sponge, three concentrations were tested: 250, 500, and 1000 µg/mL (hereinafter, CPC-250, CPC-500, and CPC-1000). The choice of tested concentrations was based on the analysis of the data from the literature [6][7][8][9][36][37][38][39][40][41]. As can be seen in Figure 8, at the lowest concentration tested (CPC-250), half of the initial amount of C-PC passed into the sponge in about 2.5 h, while the other two concentrations took longer, 4 and 5 h, respectively, for CPC-500 and CPC-1000. After 8 h from the beginning of the experiment, 72.35 ± 2.39, 64.64 ± 1.26 and 57.05 ± 0.73% of the initial amount of C-PC passed into the sponges for CPC-250, CPC-500, and CPC-1000, respectively. Over the next 16 h, the amount of C-PC that did not load into the sponge (remaining in the solution) was 18.90, 20.20, and 24.25%, respectively.
Based on the results obtained, the entry efficiency (EE) and load capacity (LC) were calculated (Table 1).   As can be seen from the table, the entry efficiency (EE), regardless of the initial concentration of C-PC, was more than 90%, which exceeded the available literature data on loading C-PC into polymer carriers: 67% and 72% with STMP/STPP C-PC encapsulation cross-linked starches from different botanical sources [30] and phycocyanin-alginate beads [43], respectively.
After the introduction of C-PC into the sponge and subsequent drying, the sponges acquired a blue tint, the intensity of which depended on the concentration of C-PC tested. Optical and fluorescence microscopy of thin sections of the sponges was carried out for the sponges loaded with C-PC (Figure 9), and the FTIR spectrum was also taken ( Figure  5).  As can be seen from the table, the entry efficiency (EE), regardless of the initial concentration of C-PC, was more than 90%, which exceeded the available literature data on loading C-PC into polymer carriers: 67% and 72% with STMP/STPP C-PC encapsulation cross-linked starches from different botanical sources [29] and phycocyanin-alginate beads [42], respectively.
After the introduction of C-PC into the sponge and subsequent drying, the sponges acquired a blue tint, the intensity of which depended on the concentration of C-PC tested. Optical and fluorescence microscopy of thin sections of the sponges was carried out for the sponges loaded with C-PC (Figure 9), and the FTIR spectrum was also taken ( Figure 5).   As can be seen from the table, the entry efficiency (EE), regardless of the initial concentration of C-PC, was more than 90%, which exceeded the available literature data on loading C-PC into polymer carriers: 67% and 72% with STMP/STPP C-PC encapsulation cross-linked starches from different botanical sources [30] and phycocyanin-alginate beads [43], respectively.
After the introduction of C-PC into the sponge and subsequent drying, the sponges acquired a blue tint, the intensity of which depended on the concentration of C-PC tested. Optical and fluorescence microscopy of thin sections of the sponges was carried out for the sponges loaded with C-PC (Figure 9), and the FTIR spectrum was also taken ( Figure  5).  Figure 5 shows the spectra of both the initial C-PC, the chitosan sponge, and the sponge loaded with C-PC. Chitosan contains a large number of amino groups in its composition, as a result, amide I and II bands are also present in its spectrum, and the intensity of the amide I band is significantly lower than amide II. As a consequence, an increase in the intensity of the peak at the amide I band in the spectrum of the loaded sponge compared to the spectrum of the original sponge indicates the inclusion of C-PC in the polymer matrix, and the absence of new peaks indicates the absence of the formation of new bonds.

C-PC Release from the Sponge
In this study, the release of C-PC from the sponges into saline (pH 7.4) was studied. The in vitro release profiles of C-PC from chitosan sponges are shown in Figure 10.
At the lowest of the tested concentrations (CPC-250), the maximum concentration of C-PC released from the sponge into the solution was reached in 5 h after the sponge was immersed in saline and remained practically unchanged until the end of the experiment, while more than 50% of the initial amount remained in the sponge. Figure 9. Appearance of the chitosan sponges (a,f,k) and thin section optical (b,d,g,i,l,n) and fluorescent (c,e,h,j,m,o) microscopy with magnification x40 (b,c,g,h,l,m) and x100 (d,e,h,i,n,o): CPC-250 (a-e), CPC-500 (f,g), CPC-1000 (k-o). Figure 5 shows the spectra of both the initial C-PC, the chitosan sponge, and the sponge loaded with C-PC. Chitosan contains a large number of amino groups in its composition, as a result, amide I and II bands are also present in its spectrum, and the intensity of the amide I band is significantly lower than amide II. As a consequence, an increase in the intensity of the peak at the amide I band in the spectrum of the loaded sponge compared to the spectrum of the original sponge indicates the inclusion of C-PC in the polymer matrix, and the absence of new peaks indicates the absence of the formation of new bonds.

C-PC Release from the Sponge
In this study, the release of C-PC from the sponges into saline (pH 7.4) was studied. The in vitro release profiles of C-PC from chitosan sponges are shown in Figure 10.
At the lowest of the tested concentrations (CPC-250), the maximum concentration of C-PC released from the sponge into the solution was reached in 5 h after the sponge was immersed in saline and remained practically unchanged until the end of the experiment, while more than 50% of the initial amount remained in the sponge. For the other two concentrations, the release rate was faster: 50% of C-PC from the sponges obtained with CPC-500 was released after 1 h, and it took less than 1 h for CPC-1000. The maximum amount of C-PC came out at 7 h, and the residual content of C-PC in sponges after 24 h was about 20% and 15%, respectively.
For C-PC, released from the sponge, the PI and AOA were determined ( Table 2). According to the results, for all variants of the experiment, the PI of the C-PC released from the sponge was higher (therapeutic and analytical purity) than that of the For the other two concentrations, the release rate was faster: 50% of C-PC from the sponges obtained with CPC-500 was released after 1 h, and it took less than 1 h for CPC-1000. The maximum amount of C-PC came out at 7 h, and the residual content of C-PC in sponges after 24 h was about 20% and 15%, respectively.
For C-PC, released from the sponge, the PI and AOA were determined ( Table 2). According to the results, for all variants of the experiment, the PI of the C-PC released from the sponge was higher (therapeutic and analytical purity) than that of the initial C-PC. In addition, the AOA was also higher than the initial value (by 32, 45, and 82%, respectively, for CPC-250, CPC-500, and CPC-1000), and there is a clear dependence of the increase in the AOA with increasing the PI.

Conclusions
In the work, sponges based on chitosan containing C-PC were obtained. The encapsulation efficiency value exceeded 90%, and the maximum loading capacity was 172.67 ± 0.47 mg/g. FTIR analysis showed that no new bonds are formed during the introduction of C-PC into the sponge. An in vitro study showed that about 50% of C-PC was released in the first hour, and the released C-PC reached a maximum concentration at 5 h, which remained at a constant level during the next 19 h of control. The released C-PC was characterized by a PI consistent with pharmaceutical (3.79) and analytical (4.43) uses and an increased AOA.
Thus, as a result of the studies carried out, a C-PC-containing sponge was obtained, which can be used as an integumentary material for wound healing.