Biomimetic Materials Based on Poly-3-Hydroxybutyrate and Chlorophyll Derivatives

Biomimetic materials are substances that replicate natural structures. There are several methods for producing such materials with highly developed surfaces from a wide range of polymers, but electrospinning is of particular interest due to its simplicity of implementation and the ability to modify the produced fibrous materials resembling structures found in living organisms. This study explores new biomimetic materials based on polyhydroxyalkanoates, specifically poly-3-hydroxybutyrate, modified with chlorophyll derivatives. The research investigates the impact of chlorophyll derivatives on the morphology, supramolecular structure, and key properties of nonwoven materials. The obtained results are of interest for the development of new biomimetic environmentally friendly materials for biomedicine with special properties.

Keywords:

Subject: Chemistry and Materials Science - Biomaterials

1. Introduction

Tetrapyrroles and their derivates are the basis of essential physiological functions in most living organisms [1]. In this connection, tetrapyrroles and their derivatives are of a great interest for the creation of new materials and biomedical tools [2,3].

Scientists have achieved great success in the synthesis and modification of tetrapyrroles due to the directional design of photosensitive molecules with varying the nature of the metal, peripheral substituents or the length of the conjugated double bond system [4]. It is important to emphasize the successes in the organic synthesis of chlorophyll and its derivatives [5]. A great attention is drawn to chlorophyll derivatives due to a set of its unique properties.

Chlorophyll’s functions include accumulation, transport of energy and driving charge separation reactions in reaction centers [6]. It possesses antioxidant, antigenotoxic and antimutagenic properties [7,8,9]. Its derivatives are bioactive, easily modified and their synthesis cost is low [5,10,11]. Industrial forms of chlorophyll are well known, and they are successfully used in food industry, in cosmetics and medicine [9,12].

A particular success should be considered the effective use of chlorophyll derivatives in photodynamic therapy [5]. Their effectiveness is due to combination of light, photosensitizer and cytotoxic agent [13]. In case of chlorophyll derivatives, the cytotoxic agent is the molecular oxygen which is produced only upon in situ irradiation [14]. So, it is possible to control the work of such remedies for therapeutic purposes effectively.

Nanoparticles based on chlorophyll derivatives for photodynamic therapy are well known due to their good photosensitizing properties caused by the energy transfer from the nanoparticle to the photosensitizer [15].

In general, success in combining porphyrins and polymers as the carriers is observed in a large number of scientific papers. Systems with polymers such as chitosan [16]; polyethylene glycol [17]; poly(lactide-co-glycolide) [18,19] are well studied. But special attention should be paid to such a polymer as poly-3-hydroxybutyrate (PHB) [20,21]. PHB has many advantages over other polymers, among which the key ones are: high biocompatibility, stability of properties and controlled biodegradation in a wide temperature range [22,23].

In this work, it was decided to create biomimetic materials based on PHB and various chlorophyll derivatives for a detailed study of their suitability for the production of polymer-porphyrin systems for biomedicine. As a method of obtaining biomimetic systems electrospinning (ES) was chosen. ES allows to achieve maximum similarity of created materials to the structures produced by nature due to the formation of a highly developed hierarchical structure of fibrous material [24]. Biomimetic materials based on PHB-porphyrin electrospun composites are already known: polyhydroxybutyrate/5,10,15,20-tetrakis(4-hydroxy-phenyl)-21H,23H-porphine [25]; polystyrene/polyhydroxybutyrate/graphene/tetraphenylporphyrin [26], polyhydroxybutyrate/Hemin [27], polyhydroxybutyrate/tetraphenylporphyrin [28].

Thus, the aim of the work is to study biomimetic systems of PHB-chlorophyll derivatives for biomedicine.

2. Materials and Methods

2.1. Materials

For this study, was chosen biopolymer poly-3-hydroxybutyrate (PHB), obtained through microbiological synthesis (16F series, produced by BIOMER, Frankfurt, Germany). This polymer exhibited a crystallinity of 60%, a molecular weight of 206 kDa, a density of 1.248 g/cm³, and a melt flow index of 10 g/10 min (180°C, 5 kg). Powdered derivatives of chlorophyll: mC₂N+, mC₂N, mC₂NH₂, mC₃OH and mC₄ (Figure 1) were utilized as functional additives. The synthesis of the amide derivatives of chlorin e₆ from pheophorbide a methyl ester was described in detail earlier in articles [29] for compound mC₃OH, [30] for compound mC₄, [31] for compound mC₂NH2. 13¹-N-(2-N’, N’-diethylaminoethyl) amide-15²,17³-dimethyl ester of chlorin e₆ (mC2N(Et)2) was synthesized and then quaternized by methyl iodide (mC₂N(Et)₂Me⁺ I^-) according to the reported procedures in [32]. Both substances were dissolved in chloroform (Biolot, St. Petersburg, Russia) for the experiments.

2.2. Methods

2.2.1. Obtaining of nonwoven fibrous materials

Fibrous nonwoven materials were obtained using the electrospinning (ES) method utilizing a single-capillary setup (Figure 2). Homogeneous solutions were prepared, including a 7% concentration solution of PHB and chlorophyll derivatives dissolved in chloroform with an additive concentration of 0.03%. Each solution was used in a volume of 25 mL.

The forming process proceeded as follows: under the influence of electrostatic force, fibers were drawn from the solution in the cell and deposited on a substrate located on the lower electrode. During the forming process, the solvent evaporated, and the fiber solidified. The obtained nonwoven materials were subjected to drying to remove solvent residues and excess moisture for 48 h at 24 °C.

2.2.2. Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) images of electrospun PHB-chlorophyll derivatives composites were obtained using the Tescan VEGA SBU II (Brno, Czech Republic) on samples coated with a platinum layer for 10 min. Structural changes, including variations in fiber diameter, defects and morphology, were analyzed using the Olympus BX43 microscope (Olympus, Tokyo, Japan). The primary morphological properties of the fibers were measured through micrography, facilitated by Olympus Stream Basic software.

2.2.3. Differential Scanning Calorimetry (DSC)

Differential Scanning Calorimetry (DSC) is an experimental method used to study the thermal properties of materials. In this study, the DSC 214 Polyma (Netzsch, Selb, Germany) was employed. The methodology involved two heating cycles (from 20 °C to 220 °C) and two cooling cycles (from 220 °C to 20 °C) with a temperature change rate of 10 K/min, samples were analyzed in an argon atmosphere. DSC measures the heat flow absorbed or released by a substance as its temperature changes, so this method allows for the determination of heat capacity, thermal transitions, crystalline and amorphous properties of the material.

2.2.4. Fourier-Transform Infrared Spectroscopy (FTIR)

The spectra of the samples were obtained using the Lumos BRUKER infrared spectrometer (Karlsruhe, Germany) employing the multiple attenuated total internal reflection method on a diamond crystal. The spectral resolution was set at 2 cm⁻¹. The analysis was conducted in the wavelength range from 400 to 4000 cm⁻¹. This method enabled a detailed examination of the material’s interaction with infrared radiation, providing valuable insights into its chemical structure and composition.

2.2.5. Wettability

The surface wettability of PHB/Chlorophyll derivatives samples was measured using the contact angle (CA) method, which quantifies surface moisture. Water droplets (2 µL) were dispensed onto the material’s surface using an automatic dispenser, and the contact angle formed between the droplet and the surface was measured. We utilized an optical microscope, M9 №. 63649, with an FMA050 lens (AmScope, Moscow, Russia), and analyzed the data using Altami Studio 3.4 software.

2.2.6. Air Permeability

The air permeability of membranes, represented by the Gurley number, was measured following the standard protocol [33]. The applied pressure was 1.22 MPa, the air volume was 100 mL, and the test area was 6.5 cm². The relative experimental error was ± 5%.

2.2.7. Antimicrobial Properties

Antimicrobial activity of the samples was studied using the strains of Escherichia coli O157:H7. The lysis zone was considered as a marker of antimicrobial activity. The Petri dishes were inoculated with a standardized inoculum of test microorganisms. The samples were placed into the Petri dishes. The strain was grown in BSA/agar at 37 °C and allowed to stay with the samples for 24 h.

2.2.8. Mechanical Properties

The mechanical properties of the material, such as strength (the maximum load the material can withstand) and elongation at break (how much the material can stretch before breaking) were measured using the testing machine DVT GP UG 5. The stretching speed was 25 mm per minute. Elongation at break was calculated as the difference between the final and initial length of the sample, expressed as a percentage of the initial length. The results were averaged based on no fewer than 5-7 measurements, with a measurement error of ±0.2%.

3. Results

3.1. Morphology of PHB/chlorophyll derivatives electrospun materials

Electrospun materials can be characterized as fibrous materials with highly developed surface area. Such materials have a high degree of similarity to living structures, that is, they are biomimetics. The key to such a highly developed complex hierarchy of structure is morphology. The morphology of the fibers largely depends on the conditions of ES. The key parameters of ES should include: voltage, properties and flow rate of the spinning solution, the distance between the electrodes, the pressure exerted on the solution [34]. Table 1 shows the most important parameters of fiber molding based on solutions of PHB/chlorophyll derivatives. The capillary configuration has a great influence on the formation of fibers of the set diameter, it remained constant and was 0.1 mm. Other important parameters were selected experimentally. Thus, the introduction of the additive had an effect on the viscosity of the molding solution, increasing it by an average of 20%.

The formation process of the PHB/mC₂N solution was hindered; the fibers did not adhere properly to the substrate due to possible high humidity, leading to incorrect distribution and accumulation of fibers on the substrate.

In the current work, 5 chlorophyll derivatives (Figure 1) are considered, differing in the structure of the R₁ radical. The types of R₁ are shown in Figure 1. A number of studies have shown that additives containing polar groups even in very small concentrations (proportion of percents) have a significant effect on molding properties [35,36]. The chlorophyll compounds considered in this work are distinguished by the presence of such polar groups. They contribute to improving the electrical conductivity of the solution, and, as a result, the ability to form fibers at a higher voltage with a higher efficiency. A higher voltage contributes to the formation of thinner fibers; however, it may be accompanied by an increase in the flow velocity of the solution jet, resulting in fibers with a thicker diameter [37]. In this regard, it is important to ensure optimal viscosity of the solution, as well as the pressure of the air column on the spinning solution.

It is important to note that despite a very small percentage of the additive (0.03%), a significant effect on the material was mentioned. In all five cases, the formation of a highly developed material structure was observed. However, differences should be noted. Figure 3 shows SEM images of PHB based electrospun materials with different chlorophyll derivatives.

Nonwovens based on PHB are distinguished by the presence of specific defects – thickening (Figure 3a). Such defects are often found for pure PHB fibrous materials. In a number of works scientists use various methods of reducing thickenings: preparing PHB based blends with other polymers, for instance, PHB/PEG in works by Thanh et al. [38] or PHB/PCL in works by Borisova et al. [39]; using plasticizers like polyanilines in works by Ahmed et al. [40] or oligomeric lactic acid in works by Arrieta et al. [41]; using nanoparticles in works by Romero et al. [42] and others.

In the case of materials based on PHB/chlorophylls, it should be noted that PHB/mC₄ (Figure 3b) and PHB/mC₂N+ (Figure 3e) allowed to minimize and almost completely reduce the number of such defects respectively. In these cases, the number of thickenings is minimal, there are no gluing’s completely, the fibers are well cured. While other chlorophylls did not show a satisfactory result, moreover, they even worsened the quality of the material. So PHB/mC₃OH (Figure 3c) promotes the formation of highly porous fibers, which is usually associated either with a high rate of solvent evaporation [43] or with excessive friability of the amorphous phase due to the intermolecular interaction of the polymer and the additive [44]. While PHB/mC₂N (Figure 3f) shows a significant increase in defects in the form of glues and smudges that make the material unusable, reducing its mechanical properties to a minimum.

Table 2 shows the most significant parameters of the morphology of the fibrous material. It is clearly seen that in all cases the additives contributed to a decrease in average fiber diameters and only mC₂N had the opposite effect. In addition to the defects that made the material brittle and unusable, the diameter of the fibers increased by 1.4 times in case of PHB/mC₂N. For the rest of the additives, there was some variation in fiber distribution (Figure 3), but, in general, the average diameter of the fibers decreased by about 1.5 times. The smallest diameter was observed for the mC₃OH sample with the highest porosity. All additives led to a decrease in bulk density of the material by about 1.5 times, however, there was no obvious correlation between the average diameter of the fibers and the density of their laying, as can be seen from the results (Table 2).

Changes in the porosity of the material and the proportion of open pores were estimated using the Gurley method. It is clearly seen that the introduction of the additive significantly increased the proportion of open pores capable of passing a larger volume of air per unit of time (Table 2). This characteristic directly demonstrates the importance of the introduction of additives for the planning of high-pored materials. Thus, the highest values were shown by materials containing mC₂NH₂ and mC₃OH, where the smallest average diameters were detected. To the surprise, materials with the least number of defects PHB/mC₄ and mC₂N+ showed a less high increase in the proportion of open pores, which may be due to a greater degree of surface development and a smaller proportion of open space between the pores due to high fiber entanglement.

3.2. Chemical structure PHB/chlorophyll derivatives electrospun materials

Figure 4 shows FTIR spectra of PHB/chlorophyll derivatives electrospun materials. It is clearly seen that the obtained spectra are identical to the spectrum of pure PHB. The observed effect is due to a very low concentration of additives (0.03%) in the material and a low probability of its localization on the fiber surface.

The most notable for the identification and evaluation of PHB are the peaks: C=O carbonyl at 1720 cm^−1, -C-O-C- ester linkage at 1150–1300 cm⁻¹, -C-H bonds in methyl radical at 2900–3100 cm⁻¹, -CH3 at 1380 cm⁻¹ [45]. The region 400-1300 cm⁻¹ is responsible for a complicated series of absorptions due to all manners of bending vibrations of PHB [46].

In works by Luo et al. [47], Kovalcik et al. [48], Xu et al. [49] was proposed to evaluate the state of the crystalline phase of PHB with ratio of the peak intensities of 1225 cm⁻¹ and 1453 cm⁻¹, which allows us to characterize changes in the size of the crystalline fraction of the PHB. The ratios were obtained: 3.63 for PHB; 3.64 for PHB/mC₄; 3.22 for PHB/mC₃OH; 3.59 for PHB/mC₂NH₂; 3.88 for PHB/mC₂N+; 3.13 for PHB/mC₂N. These ratios are very close values and indicate weak changes in the structure of the crystalline phase of PHB. Although this method should be subjected to some criticism, since the signal is taken from the surface and does not give a complete picture of the structural organization in the mass of fibers, and not only on their surface, which is more influenced by environmental factors during electroforming.

3.3. Thermophysical characteristics of PHB/chlorophyll derivatives electrospun materials

Chlorophylls and their derivatives are thermally stable in the range from 20 to 200 ^°С, and thermal decomposition begins no earlier than at 300 ^°С [50]. According to this, we can evaluate the effect of additives on the formation of the supramolecular structure of the PHB, focusing on the thermophysical properties of the polymer. Thus, the first heating allows us to assess the state of the polymer in the form of fibers, the supramolecular structure of which is largely determined by the peculiarities of the electroforming process [51]. The second heating allows us to see the thermophysical behavior of the native state of PHB formed under the influence of chlorophyll derivatives. DSC curves are shown on Figure 5.

The enthalpy and melting point of PHB/chlorophyll derivatives electrospun materials are shown in Table 3. Thus, the melting temperature changes slightly, and therefore the size of the crystallites remains quite close, because the larger crystallites melt at higher temperatures, and smaller ones at lower temperatures [52]. It is important to note that the introduction of all additives reduced the enthalpy of melting by 8-17% at the first melting and by 12-19% at the second melting. These data indicate the plasticizing effect of additives. Moreover, it is important to note that the type of the selected radical, as can be seen from the results, influenced the structure of the PHB in its own way. This is especially evident in the form of peaks during the second heating. it can be seen that when all additives are introduced, a low-temperature shoulder appears in the range of 155-165 degrees. This shoulder indicates the formation of defective crystallites or a fine crystalline fraction during the formation of fibers. And consequently, at the stage of curing of the polymer melt, additives can prevent the slow crystallization of a more regular fraction of PHB, acting as crystallization aggregates.

Additives probably occupy a position in the amorphous phase of the polymer, influence the crystallization process, contribute to the formation of two fractions of PHB crystallites: large and small after the first heating. However, with slow curing during electrospinning, crystallization centers can act in all cases, except for mC₂N, which has a pronounced shoulder already at the first heating. This effect is probably manifested in the formation of defects in the form of glues and smudges on the material (Figure 2f). And the material that differed in the smallest number of defects and the smallest bulk density (Figure 2e) has the most pronounced peak consumption, which indicates the significant role of the additive in rapid crystallization under experimental conditions.

3.4. Wettability of PHB/chlorophyll derivatives electrospun materials

The chemical structure of PHB contains hydrophobic methyl group and hydrophilic ester groups, but in general PHB is hydrophobic polymer [53]. PHB is a hydrophobic material. The control of its hydrophilicity is an important task, the solution of which can greatly expand the areas of biomedical introduction of this polymer [54]. Figure 6 shows the changes in wettability of PHB based electrospun materials with different chlorophyll derivatives.

As can be seen from the Figure 6, chlorophyll additives affect the wettability of the material in different ways, leading to a change in its surface characteristics. In the works of Russie et al. it was possible to obtain hydrophobic materials based on chlorophyll derivatives [55]. The greatest increase in hydrophilicity can be seen for mC₃OH, to a less extent for mC₂N, which may be due to the appearance of open pores on the surface of fibers that contribute to wettability. The mC₂NH₂ additive has even less effect. An increase in the hydrophilicity of the material can occur due to the presence of a polar amino group, which is able to form hydrogen bonds with water molecules. In other cases, we see a slight increase in hydrophobicity, which is inherent in chlorophyll derivatives.

3.5. Antimicrobial properties of PHB/chlorophyll derivatives electrospun materials

Many tetrapyrroles have notable antimicrobial characteristics [56,57,58]. Chlorophyll derivatives are considered good antimicrobial agents in the case of photodynamic therapy. Due to singlet oxygen the antimicrobial effect of chlorophyll derivatives has been shown in relation to Staphylococcus aureus, Escherichia coli, Candida albicans, and Artemia salina in works by Gerola et al., Indrawati et al., Suvorov et al. [59,60,61]. However, of interest is the possibility of tetrapyrroles to exhibit antimicrobial properties in the absence of excitation, but for the time being in the material. To answer this question, samples of PHB/chlorophyll derivatives electrospun materials were placed in medium with E. Coli. The results are shown in Figure 7.

So, it is observed that the additives themselves do not have antimicrobial properties, but they suppress the activity of E. coli directly under the material layer. The lysis zone does not go beyond the boundaries of the material, moreover, it practically does not differ from the one obtained for pure PHB. This is probably due to the low concentration of the additive and the absence of photo excitation.

3.6 Mechanical properties of PHB/chlorophyll derivatives electrospun materials.

It is important to note that materials based on PHB are characterized by low strength, high brittleness. Often biomimetic medical materials are not required to withstand high loads, so this is not a problem. In many respects, high brittleness is a consequence of the semi-crystalline nature of the polymer, but also, due to the low glass transition temperature, the amorphous phase is in a glazed state and is quite fragile. Glass transition temperature of PHB is 4-7 ^oC [62].

Although a number of scientists note that various additives are able to affect the glass transition temperature of PHB, which indicates changes in the state of the amorphous phase and, as a consequence, the mechanical properties [63,64].

So with the introduction of additives, we see the appearance of a peak in the range 47-50 ^oC (Figure 5 1 heating). These peaks may indicate a shift in the glass transition temperature, which means that the additive is localized in the amorphous phase. Thus, at room temperature, the amorphous phase of PHB with chlorophyll derivatives is in an elastic rubber state. However, this did not allow to increase or improve the mechanical properties of nonwovens based on PHB. Mechanical properties (tensile strength and elongation at break) are shown in Table 4.

Unfortunately, only 2 samples – PHB/mC₄ and PHB/mC₂N+ were suitable for investigation on the test machine. Only these chlorophyll derivatives allowed to obtain a material with the least number of structural defects since these two additives resulted in a decrease of structural defects. Other additives (PHB/mC₃OH; PHB/mC₂NH₂; PHB/mC₂N) were ripped apart without any signals.

Examples of Stress-strain curves of PHB/mC₄ and PHB/mC₂N+ electrospun materials are shown in Figure 8. It can be seen that the mechanical properties are influenced by the strength of individual fibers, which are still brittle, although well cured (Figure 8 B).

From the data obtained, it can be seen that the strength of the material has changed slightly, remaining very low. Elongation decreased despite the fact that the number of defects decreased, and the state of the amorphous phase changed. Nevertheless, the materials are characterized by high porosity, have a controlled surface, which can offset their low mechanical properties sufficient for use in biomedicine.

4. Conclusions

In the study, new materials based on the biopolymer poly-3-hydroxybutyrate (PHB) modified with chlorophyll derivatives were proposed. These electrospun materials exhibit high environmental friendliness and possess biomimetic characteristics. All five examined chlorophyll derivatives enabled the production of nonwoven materials through the electrospinning method. However, mC₂N had a negative impact on the mechanical properties, making this additive unsuitable. All additives led to a reduction in the crystallinity of PHB while preserving the highly developed material structure. Additives mC₄ and mC₂N+ resulted in a decrease in structural defects, with mC₄ producing the thickest fibers and mC₂N+ achieving the lowest bulk density of the material. These additives can be employed to control the hydrophilicity and hydrophobicity of the produced material. Further research is planned to explore the biomedical properties of the new materials with the most successful combinations of properties relevant to solving healthcare and medical challenges.

Author Contributions

Conceptualization, P.T. and M.T.; methodology, R.R.; software, R.R.; validation, P.T.; formal analysis, K.G.; investigation, K.G, L.M., E.K., R.R.; resources, E.L. and M.T.; data curation, P.T.; writing—original draft preparation, K.G.; writing—review and editing, P.T.; visualization, R.R.; supervision, M.T.; project administration, P.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Acknowledgments

The study was carried out using scientific equipment of the Center of Shared Usage «New Materials and Technologies» of Emanuel Institute of Biochemical Physics and the Common Use Centre of Plekhanov Russian University of Economics.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Structural formulas of chlorophyll derivatives: (a) mC₂N+; (b) mC₂N; (c) mC₂NH₂; (d) mC₃OH; (e) mC₄.

Figure 2. Single-capillary ES setup.

Figure 3. SEM images of electrospun materials based on PHB with different chlorophyll derivatives: (a) PHB; (b) PHB/mC₄; (c) PHB/mC₃OH; (d) PHB/mC₂NH₂; (e) PHB/mC₂N+; (f) PHB/mC₂N.

Figure 4. FTIR spectra of electrospun materials based on PHB with different chlorophyll derivatives: (a) PHB; (b) PHB/mC₄; (c) PHB/mC₃OH; (d) PHB/mC₂NH₂; (e) PHB/mC₂N+; (f) PHB/mC₂N.

Figure 5. DSC curves of electrospun materials based on PHB with different chlorophyll derivatives.

Figure 6. Wettability (contact angle) of electrospun materials based on PHB with different chlorophyll derivatives.

Figure 7. The microbiological test of electrospun materials based on PHB with different chlorophyll derivatives against E. coli (red arrows show lysis zone).

Figure 8. Mechanical analysis of PHB/chlorophyll with different chlorophyll derivatives: (A) PHB/mC₄; (B) PHB/mC₂N+.

Table 1. Features of the ES process of PHB/chlorophyll derivatives electrospun materials.

Sample	Diameter of capillary, mm	Distance between the electrodes, mm	Gas pressure on the solution, kg(f)/cm²	Voltage, Kv	Viscosity, Pa s
PHB	0.1	210	10	18	1.0
PHB/mC₂NH₂		220	10	17-18	1.2
PHB/mC₂N		220	10	17-18	1.2
PHB/mC₂N+		220	10	17-18	1.2
PHB/mС₄		220	10	17-18	1.2
PHB/mC₃OH		220	10	17-18	1.2

Table 2. Morphology of PHB/chlorophyll derivatives electrospun materials.

Sample	Average diameter, µm	Bulk density, g/cm3	Air permeability, mL	Time by Guerly method, s
PHB	3.5	0.30	0.38	50.0
PHB/mC₂NH₂	2.24	0.18	3.75	26.7
PHB/mC₂N	4.86	-	-	-
PHB/mC₂N+	2.27	0.13	2.78	36.0
PHB/mС₄	2.63	0.16	2.35	42.6
PHB/mC₃OH	2.08	0.16	4.35	23.0

Table 3. Thermal Properties of PHB- chlorophyll derivatives, where χ - crystallinity degree Δ±2.5%, ∆H -melting enthalpy Δ±2.5%, Tm – melting temperature Δ±2%.

Sample	First heating run		Second heating run
Sample	T_m, ⁰C	ΔH, J/g	T_m, ⁰C	ΔH, J/g
PHB	175	93.1	170	90.8
PHB/mC₂NH₂	176	83.3	174	78.7
PHB/mC₂N	176	85.8	173	81.2
PHB/mC₂N+	177	80.6	173	79.8
PHB/mС₄	176	82.0	174	78.9
PHB/mC₃OH	175	77.4	171	74.9

Table 4. Mechanical properties of PHB/chlorophyll derivatives electrospun materials.

Sample	Tensile strength, MPa Δ ±0.02 MPa	Elongation at break, % Δ ±0.2 %
PHB	1.7	3.6
PHB/mC₂NH₂	-	-
PHB/mC₂N	-	-
PHB/mC₂N+	1.5	1.3
PHB/mС₄	1.5	0.6
PHB/mC₃OH	-	-

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