Comparative Structure-Property Characterization of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)s Films under Hydrolytic and Enzymatic Degradation: Finding a Transition Point in 3-Hydroxyvalerate Content

The hydrolytic and enzymatic degradation of polymer films of poly(3-hydroxybutyrate) (PHB) of different molecular mass and its copolymers with 3-hydroxyvalerate (PHBV) of different 3-hydroxyvalerate (3-HV) content and molecular mass, 3-hydroxy-4-methylvalerate (PHB4MV), and polyethylene glycol (PHBV-PEG) produced by the Azotobacter chroococcum 7B by controlled biosynthesis technique were studied under in vitro model conditions. The changes in the physicochemical properties of the polymers during their in vitro degradation in the pancreatic lipase solution and in phosphate-buffered saline for a long time (183 days) were investigated using different analytical techniques. A mathematical model was used to analyze the kinetics of hydrolytic degradation of poly(3-hydroxyaklannoate)s by not autocatalytic and autocatalytic hydrolysis mechanisms. It was also shown that the degree of crystallinity of some polymers changes differently during degradation in vitro. The total mass of the films decreased slightly up to 8–9% (for the high-molecular weight PHBV with the 3-HV content 17.6% and 9%), in contrast to the copolymer molecular mass, the decrease of which reached 80%. The contact angle for all copolymers after the enzymatic degradation decreased by an average value of 23% compared to 17% after the hydrolytic degradation. Young’s modulus increased up to 2-fold. It was shown that the effect of autocatalysis was observed during enzymatic degradation, while autocatalysis was not available during hydrolytic degradation. During hydrolytic and enzymatic degradation in vitro, it was found that PHBV, containing 5.7–5.9 mol.% 3-HV and having about 50% crystallinity degree, presents critical content, beyond which the structural and mechanical properties of the copolymer have essentially changed. The obtained results could be applicable to biomedical polymer systems and food packaging materials.

. Biosynthesis of copolymers of sodium phosphate buffer (PBS) with the A. chroococcum 7B producer strain in the culture medium with sucrose as the main carbon source and salts of carboxylic acids as additional sources of carbon and precursors for biosynthesis of the copolymers of poly (3- 1H-NMR spectra of copolymer samples with the maximum inclusion of HV in the PHBV 17 mol. % (with the application of 20 mM valerate) and a minimum inclusion of HV PHBV 2.5% (option with 20 mM propionate), as well as a homopolymer of PHB (sucrose only), were showed in Figure S1. The signal of the methyl group HV was detected at the chemical shift value of 0.89 ppm ( Figure S1a,b), whereas in the spectrum of the PHB homopolymer (Figure S1c), this signal was absent. Thus, PHBV copolymer with different molar percentage incorporation in the HV polymer chain can be obtained by adding to the culture medium valeric acid, propionic acid, and hexanoic acid.
A new PHB4MV copolymer was synthesize by adding the 4-methylvaleric acid to the culture medium as an additional carbon source and the precursor of 3H4MV monomer in the copolymer chain. The incorporation of 3H4МV residues into the synthesized polymer PHB4МV was also confirmed by 1 H-NMR. On the 1 H-NMR spectrum, the 4-methyl group (e) and the -CH group (g) of the 3H4МV monomer appear as peaks at 0.90 ppm and 1.91, respectively ( Figure S2), while the PHB homopolymer and the PHBV copolymer in this region have no signals. For the synthesis of new copolymers with polyethylene glycol-300, polyethylene glycol (PEG) was used as additives in the culture medium. The addition of these components at a concentration of 150 mM with sucrose also leads to the inclusion of ethylene glycol monomers in the synthesized PHB polymer. The inclusion of monomers was confirmed by 1 H-NMR spectroscopy of the newlysynthesized PHB-PEG copolymer [3,4]. The melting and crystallization temperatures of the polymers were measured using differential scanning calorimetry. The melting points of both homopolymers and copolymers had a forked peak. Figure S4. Thermogram of the PHBV copolymer 17.6% 1190 obtained by the DSC method. The thermogram shows two heating curves (red and purple lines) and two cooling curves of the sample (white blue and deep blue lines).
The bifurcated peak in homopolymers is explained by the presence of crystallites of varying degrees of perfection, which may be due to both the thermal history of the sample and the wide molecular weight distribution. In copolymers, the possible appearance of a double peak is explained by the presence of copolymer residues having a lower melting point.
To analyze the synthesized poly(3-hydroxybutyrate), gel permeation chromatography was used. The solvent was chloroform with the addition of 3% vol. methanol. The elution rate was 1 mL/min. The detector is a refractometer. The concentration of the sample is 5 mg/mL. Sample volume −100 μL. Columns-Pl gel 50, 10 3 , 10 5 A. Calibration was carried out using polystyrene reference samples with a narrow distribution (less than 1.1). According to the table it can be seen that the polymers have a small polydispersity. The data obtained by gel permeation chromatography corresponded to those obtained by the method of viscometry. Therefore, for further study of molecular weight, the method of viscometry was used.
Young's modulus of the films was measured by nanoindentation. The main point of the method is that an indenter with a diamond pyramid at the end is immersed in the sample with a certain force. Further, the load curves (indenter deepening) and unloading curves (indenter output) are taken. The resulting curves are processed and Young's modulus is calculated. The characteristic curve is shown Figure S5.

S2. Lipase Concentration Selection
In the literature devoted to the problem of PHB biodegradation, own bacterial enzymes are widely used as an enzyme decomposing a polymer [6][7][8]. However, the decomposition of the polymer will be affected not only by the enzyme but also by the buffer solution in which this enzyme is dissolved [1]. Therefore, the choice of a buffer solution in which polymer degradation will take place is an important task. To compare the biodegradation rate of the polymer, two buffer solutions were selected: 0.01 M sodium phosphate buffer (PBS) and a buffer simulating blood plasma (SBF), as well as various concentrations (0; 0.1; 0.25; 1 mg/mL) of pork pancreatic lipase in these solutions. All solutions had a pH of 7.4. PHB films with a molecular weight of 105 kDa were placed in these solutions. The polymer films were stored at a temperature of 37 °C for a month. Figure S7 shows diagrams of changes in the weight of PHB films after 1 week and a month of biodegradation under the action of pancreatic lipase. According to the decrease in the wight of PHB films during degradation in both buffer solutions, it can be said that these solutions do not interfere with polymer degradation; however, a lipase solution in sodium phosphate buffer has a greater effect on polymer degradation. After a month, a solution with a lipase concentration of 0.25 mg/mL had the strongest effect on the polymer film, whose wight decreased by 7% from the original values. At lipase concentrations of 0.1 mg/mL and 1 mg/mL, the weight loss of the films averaged 4.7% per month in sodium phosphate buffer and 4% in a buffer simulating blood plasma. This partly confirms the results of [10], in which blood plasma had a negligible effect on polyhydroxybutyrate samples. The effect of lipase concentration can be explained by the fact that at 1 mg/mL the process of substrate inhibition occurs, similar to that described for PHB-depolymerase. At a high concentration of the enzyme, the degradation rate decreases due to the blocking of access of the catalytic domain of the enzyme to polymer molecules [11]. At a concentration of 0.1 mg/mL, not the entire polymer surface is occupied by enzyme molecules; therefore, the degradation rate does not reach its peak. Based on the results obtained, a concentration of 0.25 mg/mL is optimal for experiments on the degradation of PHB, and this concentration of lipase corresponds to the concentration of pancreatic lipase in the human body [12]. To describe the morphological changes induced by enzymatic degradation, the films were studied by AFM. The lamellar structure is easily distinguished by phase imaging, so this method was applied. Three types of morphological changes were observed: the emergence of new lamellar structures, fragmentation of lamellar structures, and the disappearance of lamellar structures The first effect of decomposition-the appearance of new lamellar structures ( Figure S8). The arrow shows an extended structure, which can be interpreted as a single lamella ( Figure S8A). After treatment with a lipase solution, it becomes more visible, and another lamella appears to the right of it ( Figure S8B). These changes in the morphology of the film explained as the stack of lamellas is coated with a thin layer of an amorphous polymer. Lipase, which had adsorbed on the surface of the film, begins to decompose amorphous polymer, exposing the crystalline structures under them. The second change in the morphology -the fragmentation of lamellar structures. Figure S9A shows an image of PHB film before enzymatic hydrolysis. The selected area contains a system of parallel strips, interpreted as a stack of lamellae. After treatment with lipase ( Figure S9B), lamellae are fragmented. Before enzymatic degradation, lamellae length in the stack was 300 ± 20 nm; after the degradation, the length of the fragments had decreased to 80 ± 10 nm. The reason for that is probably the defects in the crystal structure. Lipase attacks such defects, which result in the disintegration of lamellae into smaller pieces. The third morphological change is the disappearance of lamellar structures. Figure S10 shows the PHB film images before and after lipase treatment. A system of parallel strips structure interpreted by us as a stack of lamellae was found on a PHB film before treatment with lipase. After hydrolysis in the lipase solution, this structure has changed dramatically: most of the lamellae have disappeared, and the remaining were highly fragmented ( Figure S10). The reason for the disappearance of lamellae is the same, as in the situation with fragmentation of lamellae-there are defects in lamellae, but in this case, their number is much greater, allowing the lipase to almost completely destroy lamellae stack during the experiment [13].