The Effect of Betulin Diphosphate in Wound Dressings of Bacterial Cellulose-ZnO NPs on Platelet Aggregation and the Activity of Oxidoreductases Regulated by NAD(P)+/NAD(P)H-Balance in Burns on Rats

The inhibition of platelet aggregation, and the activity of oxidoreductases and microhemocirculation in a burn wound on the treatment of burns with wound dressings based on bacterial nanocellulose (BC)-zinc oxide nanoparticles (ZnO NPs)-betulin diphosphate (BDP) were studied. The control of the treatment by BC-ZnO NPs-BDP on burned rats by the noninvasive DLF method showed an increase in perfusion and the respiratory component in wavelet spectra, characterizing an improvement in oxygen saturation in the wound. The study on the volunteers’ blood found the inhibition of ADP-induced platelet aggregation by 30–90%. Disaggregation depends on the dose under the action of the ionized form of BDP and ZnO NPs-BDP in a phosphate buffer; it was reversible and had two waves. It was shown on rats that the specific activity of LDHreverse and LDHdirect (control-intact animals) on day 21 of treatment increased by 11–38% and 23%, respectively. The LDHreverse/LDHdirect ratio increased at BC-ZnO NPs-BDP treatment, which characterizes efficient NAD+ regeneration. AlDH activity increased significantly in the first 10 days by 70–170%, reflecting the effectiveness of the enzyme and NAD+ in utilizing toxic aldehydes at this stage of burn disease. The activities of GR and G6PDH using NADP(H) were increased with BC-ZnO NPs-BDP treatment.


Introduction
Oxidative stress and a decrease in antioxidant enzymatic protection, coupled with a change in the ratio of the level of pairs of pyridine nucleotides NAD+/NADH and NADP+/NADPH, are links in the triggering mechanism of energy and redox metabolism disorders that are associated with the causes of many diseases [1][2][3][4]. To treat such serious diseases as burn disease, it is also important to understand cellular redox homeostasis and energy metabolism induced by hypoxia (hypoxia-inducible factor) [2].
NADH/NAD+ and NAD+/NADH redox imbalance are the initiators of reductive stress and oxidative stress in different diseases dealing with hypoxia. Previously, the NAD+/NADH redox state was proposed as a potential biomarker and therapeutical target, for example, in neuroprotection in glaucoma [5]. The NADH/NAD+ redox imbalance can deal with diabetic kidney disease [6]. It has been shown that circulating markers of NADHreductive stress correlate with mitochondrial disease severity [7]. A study of burn injuries betulin diphosphate. We studied: (i) microcirculation in a burn wound using a noninvasive method of laser Doppler flowmetry; (ii) the ability of betulin diphosphate to inhibit the platelets aggregation induced by ADP using in vitro experiments on human blood; (iii) the activity of oxidoreductases coupled with the redox balance of the pair of coenzymes NAD+/NADH and the level of NADPH in experiments on rats.

Results
Groups of rats with burns without treatment (Day 0 and Day 10) were used as negative controls. The study on Day 21 in the negative control groups was not carried out, because the results were unreproducible due to the death of some rats. The group treated by Oleogel ZnO NPs-BDP and the Intact group were used as positive controls. The general appearance and condition of the animals during the treatment with wound dressings BC-ZnO NPs-BDP, BC-BDP, and BC-ZnO NPs significantly improved in comparison with the group without treatment. The burnt wound image of rats after the treatment by BC-ZnO NPs-BDP was better than those of BC-BDP and BC-ZnO NPs ( Figure S1). The wound area reduced on days 10 and 21 by 10.33% and 42.31%, respectively, using BC-ZnO NPs-BDP wound dressings (Table S1).
The effect of BDP having two ionizable phosphate groups on the healing of burnt wounds may be due to the following events: respiration in the wound; ADP production after severe burn leading to platelet aggregation; and NADH/NAD+ ratio, determining oxidoreductases activity.

Study of Microhemocirculation in a Burn Wound
Microhemocirculation in the wound before and after the burn on Day 10 in the treatment of BC-ZnO NPs-BDP and BC-ZnO NPs was studied by the noninvasive laser Doppler flowmetry (LDF). We studied the state of the flow of erythrocytes in the blood (perfusion) and tissue oxygenation in the wound, which is important for cellular respiration [43,44].
The LDF spectra after the wavelet transform were analyzed by the approach described in [44], using the frequency regions of the Doppler reflected signal, characterizing the endothelial (E), myogenic (M), neurogenic (N), respiratory (R), and cardiological (C) components of blood circulation in a burn wound ( Figure 1).
Microhemocirculation spectra of the Intact group during the experiment (21 days) were not changed, because the conditions of keeping the animals (23 ± 2 • C, air humidity 50 ± 10%, ventilation mode 30 min/h, 12/12 h lighting rhythm) were constant. Changes in the microcirculation index in animals without trauma were within the physiological norm (13.26 ± 1.21 perf. un.).
The most changes in the wavelet-DLF spectra (amplitude) during the treatment by BC-ZnO NPs and BC-ZnO NPs-BDP occurred in the frequency range of 0.5-4.5 Hz, reflecting the respiratory component R of microhemocirculation as a factor of oxygen saturation. Endothelial E, neurogenic N, and myogenic M components of the wavelet-DLF spectra changed slightly in comparison with the Intact group ( Figure 1). Microhemocirculation indexes were assessed using R/E, R/N, and R/C ratios of the intensity of the wavelet spectrum amplitude ( Table 1). The ratio in the respiratory R region to the amplitude intensity in the neurogenic N and endothelial E regions (R/E and R/N) significantly increased on Day 10 of treatment by all studied dosage forms.   The big differences in the R/E, R/N, and R/C ratios in the wavelet-LDF-spectra after the treatment with BC-ZnO-BDP and Oleogel ZnO-BDP are probably due to the influence of the lipophilicity of dosage forms on oxygen saturation in the wound. Unlike the lipophilic oil medium in Oleogel, hydrophilic bacterial cellulose can retain large amounts of water, provide wound hydration and absorption of exudate, and remove unwanted products from the wound.
The perfusion as microhemocirculation indexes (MIs) caused by the flow of erythrocytes in the blood had values close to those of the MIs of healthy animals on the third day, which changed up to Day 21 insignificantly. Big changes in the wavelet LDF spectra on 10-21 days of treatment with BC-ZnO-BDP can be due to an improvement in oxygen saturation into the burn zone under the influence of BDP.
In general, the most intense microcirculation in a burn wound during treatment with BC-ZnO-BDP wound dressings can be the key to improving oxygenation in the area of a burn wound and, accordingly, accelerating its healing.

Study of Platelet Aggregation under Action of BDP
We studied the effect of BDP on platelet aggregation in vitro in human platelet-rich plasma (PRP) activated by the agonist ADP using the turbidimetric method by the Born method [45]. Figures 2 and 3 show the inhibition of platelet aggregation over time under the action of ADP and the studied substances. The black lines on Figures 2 and 3 mean the control aggregation process under ADP only, without adding studied substances (BDP, ZnO NPs, their mixtures, and acetylsalicylic acid). In this case, the platelet aggregation process was the same under the constant experiment conditions. Colored lines on Figures 2 and 3 show platelet aggregation under the action of the studied substances introduced after 2 min after the addition of ADP.
Acetylsalicylic acid (ASA), which exhibits both antiplatelet and anticoagulant properties, was chosen as a positive control.
The data in Figure 2 and Table 2 demonstrate the complex process of inhibition of ADP-induced platelet aggregation by 6.65 mM of BDP, ZnO NPs-BDP 6.65 mM, and acetylsalicylic acid (ASA).
In all experiments, the agonist ADP was added to platelet-rich plasma (PRP) after 10 s. In the control in the absence of inhibitors under the ADP action, platelet aggregation occurred (Figure 2a-c, black line). Aggregation inhibitors were added to PRP with ADP after two minutes. We observed the formation of a turbid solution within the next minute due to reversible platelet disaggregation. At 3.5 min (about 210 s), the second wave of platelet disaggregation began. The percent inhibition of aggregation by the addition of various agents in the presence of an ADP agonist is shown in Table 2. In all experiments, the agonist ADP was added to platelet-rich plasma (PRP) after 10 s. In the control in the absence of inhibitors under the ADP action, platelet aggregation occurred (Figure 2a-c, black line). Aggregation inhibitors were added to PRP with ADP after two minutes. We observed the formation of a turbid solution within the next minute due to reversible platelet disaggregation. At 3.5 min (about 210 s), the second wave of platelet disaggregation began. The percent inhibition of aggregation by the addition of various agents in the presence of an ADP agonist is shown in Table 2. The obtained data show that BDP exhibited antiaggregatory properties similar to acetylsalicylic acid. Zinc ions (ZnO NPs-BDP) reduced the inhibition of platelet aggregation twice at 2-3 min: 28% compared to 58% under the action of BDP only. Figure 3 and Table 3 show that BDP inhibited platelet aggregation in a dose-dependent manner.  The obtained data show that BDP exhibited antiaggregatory properties similar to acetylsalicylic acid. Zinc ions (ZnO NPs-BDP) reduced the inhibition of platelet aggregation twice at 2-3 min: 28% compared to 58% under the action of BDP only. Figure 3 and Table 3 show that BDP inhibited platelet aggregation in a dose-dependent manner. Several disaggregation waves were observed at a low BDP concentration (0.44 mM), but complete disaggregation was not achieved. At an average concentration of BDP (2.22 mM), complete aggregation was observed at the fifth minute of the experiment, with an intermediate first stage of disaggregation by almost 90%. At a high concentration of BDP (4.43 mM), stable platelet disaggregation was achieved at 5 min (complete inhibition of aggregation), while a reversible first stage of disaggregation was observed at 3 min.
Pure ZnO NPs without immobilized BDP did not have a disaggregation effect, but ZnO NPs-BDP showed the dose-dependent anti-aggregation activity.
The effect of triterpenoid concentration on platelet aggregation was studied using betulinic acid by authors [42].   Several disaggregation waves were observed at a low BDP concentration (0.44 mM), but complete disaggregation was not achieved. At an average concentration of BDP (2.22 mM), complete aggregation was observed at the fifth minute of the experiment, with an intermediate first stage of disaggregation by almost 90%. At a high concentration of BDP (4.43 mM), stable platelet disaggregation was achieved at 5 min (complete inhibition of aggregation), while a reversible first stage of disaggregation was observed at 3 min.
Pure ZnO NPs without immobilized BDP did not have a disaggregation effect, but ZnO NPs-BDP showed the dose-dependent anti-aggregation activity.
The effect of triterpenoid concentration on platelet aggregation was studied using betulinic acid by authors [42].

LDH and AlDH-Specific Activities Controlled by Pairs of Coenzymes NAD+/NADH in Burns during Treatment with Wound Dressings Based on Bacterial Cellulose
The LDH reversible oxidation/reduction of lactate-pyruvate can be easily pushed to either lactate→pyruvate or pyruvate→lactate without the requirement for any special process or mechanism. Written in the direction of pyruvate (Pyr − ) reduction to lactate (La − ), the reaction is as follows: Pyr − + NADH + H + La − + NAD+. At a pH near 7.0, the proton can be omitted. In the direction: We can write the mass action ratio (MAR) of the reaction as: We suggested using the specific activity ratio (SAR) of LDH direct LDH reverse or LDH reverse LDH direct determined using [NAD+] and [NADH] concentrations and calculated by the Michaelis-Menten equation as an empirical biochemical parameter-reflected redox imbalance in this reaction.
We studied groups of intact animals, untreated animals (negative control) treated by oleogel (positive control), and groups treated by wound dressings (Table 4). Studies have shown that the specific activity of LDH in direct and reverse reactions decreases after a burn without treatment on Day 3 and Day 7 but increases on Day 10 up to 96.43% in the direct and up to 72.72% in the reverse reaction.
On Day 3 and Day 7, the increase in LDH activity in direct and reverse reactions after treatment by Oleogel was 15-30%, compared with the untreated burn (Table 4). On the 10th and 21st days, the LDH activity in the reverse reaction returned to normal (96%), while the LDH activity in the direct reaction increased by 11% ( Table 4).
Evaluation of the specific activity of LDH reverse and LDH direct in the treatment of wound dressings based on bacterial cellulose BC-ZnO NPs-BDP showed a better result than Oleogel (the positive control) did. On Day 21, under treatment by BC-ZnO NPs-BDP, LDH activity in the direct and reverse reactions increased by 38% and 23%, respectively, compared with intact animals. The LDH activity under the action of BC-BDP wound dressings was slightly lower than that of BC-ZnO NPs-BDP (Table 4).
In this work, we evaluated the change in the specific activity of LDH in the direct and reverse reactions, by the spectral method (λ max = 340 nm for NADH) in erythrocytes (Table 4). [NAADH] ratio using the data in Table 4. The LDH reverse LDH direct ratio decreased in the group of burned animals without treatment, and this ratio was lower than for intact animals, which indirectly characterizes the low NAD+ regeneration in the reaction of pyruvate with NADH. This means a decrease in the concentration of NAD+ during the burn disease compared to the initial burn wound state.
Cytosolic lactate-pyruvate and NAD+/NADH ratios determined by routine assays of lactate and pyruvate in resting skeletal muscle yield a ratio (lactate/pyruvate) of about 10 [46].
LDH is a very-high-activity cytosolic enzyme with a strong advance in response to small changes in a substrate or product concentrations, and finally, small changes in the SAR.
We also studied the specific activity of the aldehyde dehydrogenase (AlDH), which is involved in many reactions in the human body. One of the most important AlDH reactions is utilizing the lipid peroxidation product malondialdehyde by the pair NAD+/NADH's participation.
In the rats' burn wound model, we showed that the AlDH-specific activity under treatment by the studied drugs increased compared to untreated burned animals during the initial treatment period (10 days) by 70-170% (Table 6).  Table 7 shows data on the specific activity of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) by the action of the studied drugs (Oleogel ZnO NPs-BDP, BC-BDP, and BC-ZnO NPs-BDP). On the 21st day, an increase in the level of G6PDH to 91% compared to the control was observed. An important function of this enzyme is the formation of cellular NADPH from NADP+, which is necessary to maintain the level of reduced glutathione in the cell, and the synthesis of fatty acids and isoprenoids.

Discussion
The increase in the respiratory component's amplitude in wavelet-LDF spectra and in microhemocirculation indexes (Table 1, Figure 1) confirmed an intensification of oxygen saturation in a burn wound under the action of BC-ZnO NPs-BDP and a decrease in hypoxia. In turn, the enhancement of microcirculation in burn wounds acts not only on the blood flow rate in arterioles, capillaries, venules, and other vessels, but is also able to influence the blood coagulation system under hypoxia. An improvement in oxygen saturation due to a regulatory mechanism-respiratory control-is also associated with the increased oxidation of substrates (fatty acids, glycolysis products, and participants in the Krebs cycle). The oxidation of substrates is mainly regulated by allosteric mechanisms and is most consistently stimulated by an excess of ADP [47]. In addition, oxidative phosphorylation also depends on the presence of O 2 , NADH, ADP, and the phosphate residue Pi in the substrate and controls the rate of this process. For example, severe hypoxia or vascular occlusion restricts oxygen availability and can accelerate cell death. Such oxygen starvation reflexively increases the concentration of ADP and NADH while the concentration of ATP decreases.
The introduction of ADP into platelet-rich plasma (PRP) in the presence of the studied antiplatelet agents (BDP, ZnO NPs-BDP, and ASA) causes not only platelet aggregation but also causes the process of platelet disaggregation (Table 2 and Figure 2, Table 3 and Figure 3).
These results can be explained by the contribution of charge-charge interactions between the ADP agonist and BDP immobilized in BC and ZnO NPs. To substantiate the charge-charge interactions, we carried out electrokinetic studies to measure the zeta potential and hydrodynamic diameter of wound dressings component ZnO NPs, modified with compounds with phosphate groups. We used a BDP solution in a phosphate buffer solution at pH 7.44, in which phosphate groups are ionized and exist partially in the form of sodium salts. For comparison, we carried out studies in a solution containing phytic acid and sodium phytate.
The zeta potential of ZnO NPs (sizes are equal to 12-20 nm according to PXRD data) in distilled water is +15.9 ± 1.20 mV. From the data in Table 8, it follows that ZnO NPs in aqueous solutions of BDP (water or phosphate buffer solution with pH 7.20-7.44) has a negative charge and a large hydrodynamic diameter, which makes it possible to stabilize the ZnO NPs-BDP colloidal system without additional stabilizers. The negative charge characterizes the ZnO particle with adsorbed BDP in ionized form with a phosphate ion group (Figure 4).  The negative charge characterizes the ZnO particle with adsorbed BDP in ionized form with a phosphate ion group (Figure 4). The reversibility of disaggregation-aggregation under the action of ADP in the BDP and ZnO NPs-BDP system, as well as phytic acid and sodium phytate, is probably due to the appearance of AMP and changes in the phosphate pool in the system, including the ionization (degree of ionization) of phosphate groups. Thus, the authors of [48] showed that the sodium salt of phytic acid, which has an extremely high charge density of 12 anionic charges per inositol moiety, demonstrated strong anticoagulant activity using in vitro and in vivo experiments (on rats and healthy volunteers). In contrast, phytic acid increased the size of aggregates under the action of agonists [49]. The authors of [50] suggested that inhibition of agonist-induced platelet aggregation promoted the treatment of oncological diseases by the example of colon cancer. We can assume that the inhibition of aggregation under the action of BDP and ZnO NPs-BDP, in which phosphate groups are ionized, will promote the healing of burn wounds.
The interruption of blood microcirculation in a burn wound, leading to a lack of oxygen (hypoxia), causes a change in the ratio of LDH activity in the direct and reverse reactions (Table 4), and, accordingly, leads to an imbalance in the pairs of coenzymes NAD+/NADH. L-lactate dehydrogenase converts lactate in a direct reaction to pyruvate, which is then used in the Krebs cycle. NAD+ is regenerated by converting pyruvate to lactate (reverse reaction) ( Figure 5). The reversibility of disaggregation-aggregation under the action of ADP in the BDP and ZnO NPs-BDP system, as well as phytic acid and sodium phytate, is probably due to the appearance of AMP and changes in the phosphate pool in the system, including the ionization (degree of ionization) of phosphate groups. Thus, the authors of [48] showed that the sodium salt of phytic acid, which has an extremely high charge density of 12 anionic charges per inositol moiety, demonstrated strong anticoagulant activity using in vitro and in vivo experiments (on rats and healthy volunteers). In contrast, phytic acid increased the size of aggregates under the action of agonists [49]. The authors of [50] suggested that inhibition of agonist-induced platelet aggregation promoted the treatment of oncological diseases by the example of colon cancer. We can assume that the inhibition of aggregation under the action of BDP and ZnO NPs-BDP, in which phosphate groups are ionized, will promote the healing of burn wounds.
The interruption of blood microcirculation in a burn wound, leading to a lack of oxygen (hypoxia), causes a change in the ratio of LDH activity in the direct and reverse reactions (Table 4), and, accordingly, leads to an imbalance in the pairs of coenzymes NAD+/NADH. L-lactate dehydrogenase converts lactate in a direct reaction to pyruvate, which is then used in the Krebs cycle. NAD+ is regenerated by converting pyruvate to lactate (reverse reaction) ( Figure 5). In most tissues, the total concentration of [NAD+] + [NADH] is ⁓10 −5 M, while the ratio NAD+(oxidation)/NADH(reduction) is large, which favors the transfer of the hydride ion from the substrate to NAD+ with the formation of NADH.
From the data of [2], it follows that with burns, the NADH concentration changes insignificantly (within 7 days after the burn), "−"0.012-0.014 nmol/mg wet weight, which is a percentage of 8.4 (12 h) to 11.9 (7 days) from the total concentration of [NAD+]+ [NADH]. For intact animals, the NADH value was 18.2%.
Our data on animals with burns without treatment are consistent with work [2] on the measurement of the concentration of NAD+ during the burn disease in an in vitro experiment on mice. In the study [2], the concentration of NAD+ on Day 3 decreased almost twice with the burn disease without treatment. On Day 7, the NAD+ concentration decreased almost four times compared with the initial state of the burn: from 0.374 to 0.167 nmol/mg wet weight (Day 3) and up to 0.089 nmol/mg wet weight (Day 7).
When treated with Oleogel BC-BDP, BC-ZnO, and NPs-BDP, the ratio of LDH activity ( ) on Day 3 increased compared to the burn to 8.70, which indirectly characterizes the increase in the concentration of NAD+, which is required for obtaining pyruvate and its further participation in the Krebs cycle. With further treatment, this value decreased slightly and became close to the norm (7.60). Therefore, the empirical assessment of the ratio of LDH activity ( ) can be used as a predictor of the course of burn disease and the effectiveness of burn treatment. The level of cellular NAD(H)/NADP(H) is essential for maintaining redox homeostasis. Deficiency in these redox couples can lead to oxidative or reductive stress, depending on the redox ratio of each. Both oxidative stress and reductive stress are detrimental to normal cell functions. This dual role complicates the use of global antioxidants as rational and effective therapeutic approaches to redox stress disorders. An imbalance of these two redox couples directly affects energy metabolism. This imbalance alters NAD(H)/NADP(H)-dependent enzymes and, thus, affects their functions in regulating cellular metabolism [4].
The increase in GR activity under the treatment by BC-ZnO NPs-BDP in comparison with the untreated burnt group leads to an increase in the fraction of antioxidants glutathione (GSH) and NADPH in cells.
NADPH and glutathione serve as dual-function participants in maintaining cellular redox homeostasis. GSH is a cosubstrate (antioxidant) for hydrogen peroxide (H2O2) re- In most tissues, the total concentration of [NAD+] + [NADH] is~10 −5 M, while the ratio NAD+(oxidation)/NADH(reduction) is large, which favors the transfer of the hydride ion from the substrate to NAD+ with the formation of NADH.
From the data of [2], it follows that with burns, the NADH concentration changes insignificantly (within 7 days after the burn), "−"0.012-0.014 nmol/mg wet weight, which is a percentage of 8.4 (12 h) to 11.9 (7 days) from the total concentration of [NAD+]+[NADH]. For intact animals, the NADH value was 18.2%.
Our data on animals with burns without treatment are consistent with work [2] on the measurement of the concentration of NAD+ during the burn disease in an in vitro experiment on mice. In the study [2], the concentration of NAD+ on Day 3 decreased almost twice with the burn disease without treatment. On Day 7, the NAD+ concentration decreased almost four times compared with the initial state of the burn: from 0.374 to 0.167 nmol/mg wet weight (Day 3) and up to 0.089 nmol/mg wet weight (Day 7).
When treated with Oleogel BC-BDP, BC-ZnO, and NPs-BDP, the ratio of LDH activity ( LDH reverse LDH direct ) on Day 3 increased compared to the burn to 8.70, which indirectly characterizes the increase in the concentration of NAD+, which is required for obtaining pyruvate and its further participation in the Krebs cycle. With further treatment, this value decreased slightly and became close to the norm (7.60).
Therefore, the empirical assessment of the ratio of LDH activity ( LDH reverse LDH direct ) can be used as a predictor of the course of burn disease and the effectiveness of burn treatment.
The level of cellular NAD(H)/NADP(H) is essential for maintaining redox homeostasis. Deficiency in these redox couples can lead to oxidative or reductive stress, depending on the redox ratio of each. Both oxidative stress and reductive stress are detrimental to normal cell functions. This dual role complicates the use of global antioxidants as rational and effective therapeutic approaches to redox stress disorders. An imbalance of these two redox couples directly affects energy metabolism. This imbalance alters NAD(H)/NADP(H)-dependent enzymes and, thus, affects their functions in regulating cellular metabolism [4].
The increase in GR activity under the treatment by BC-ZnO NPs-BDP in comparison with the untreated burnt group leads to an increase in the fraction of antioxidants glutathione (GSH) and NADPH in cells.
NADPH and glutathione serve as dual-function participants in maintaining cellular redox homeostasis. GSH is a cosubstrate (antioxidant) for hydrogen peroxide (H 2 O 2 ) removal by glutathione peroxidases (GPxs). NADPH functions as an indispensable cofactor for glutathione reductase (GR) that is essential for GPx-mediated peroxide removal. GR catalyzes the recycling of GSH from its oxidized form (GSSG). Excess accumulation of GSH and/or NADPH leads to reductive stress, and may directly contribute to the production of "O 2−" and H 2 O 2 .
It can be proposed that ionizable phosphate groups of BDP regulate the NADPH functions of GR.
Another principal source of cytosolic NADPH is G6PDH. It affects cellular NADPH levels and consequently the cellular redox state and biological functions.
Data of Table 6 on the significant increase in G6PDH activity under the treatment by BC-ZnO NPs, BC-BDP, and BC-ZnO NPs-BDP indirectly characterize the increase in cytosolic NADPH concentration.
It is difficult to assess the effect of wound dressings components (BDP or ZnO NPs) on improving biochemical indexes such as the activity of the enzymes GR and G6PDH.
One of reasons for GR and G6PDH activation can be the ability of BDP to contribute to the phosphate pool in the biosystem.

Preparation of BC
BC was produced using previously described methods [29,51]. In a typical preparation, BC was obtained in a static culture medium by Komagataeibacter sucrofermentans H-110, which was isolated from Kombucha tea and identified by sequencing the amplified product of 16S rRNA. For the production of BC, a Hestrin and Schramm (HS) medium that contained glucose (20 g·L −1 ), peptone (5 g·L −1 ), yeast extract (5 g·L −1 ), citric acid (1.15 g·L −1 ), and disodium hydrogen phosphate (2.7 g·L −1 ) at a pH of 6.0 was used. The culture medium was autoclaved and inoculated with 10% (v/v) inoculum. After incubation at 28 • C for 5 days, the BC was collected, washed, and the bacterial cells were removed. The 13 C nuclear magnetic resonance (NMR) spectrum of dried BC used in this study is a typical spectrum of the BC crystal structure [29].

Zinc Oxide Nanoparticles
Two methods of ZnO NPs preparation were used in this work.

Preparation of BC-ZnO NPs-BDP Composites and Their Properties
The BC film was placed in aqueous alcohol (1:1) solutions containing BDP·11.5H 2 O (1%) at pH 7.44 for swelling for 30 min. After swelling, a suspension of an aqueous-alcoholic solution of zinc oxide nanoparticles modified by BDP was sprayed onto the surface of an elastic polymer mesh at the weight ratio of ZnO NPs to initial BC film equal to 5:100. After removing the excess solvent by vacuum drying, the films were closed on both sides with parchment and sealed in cellophane envelopes.
When treating rats with BC-ZnO NPs, BC-BDP, and BC-ZnO NPs-BDP composite coatings, the composite coatings were additionally moistened with 0.9% sodium chloride solution in the presence of 0.01% lidocaine and 0.01% benzalkonium chloride.

FTIR Analysis
FTIR spectra in the 400-4000 cm −1 range were measured by an IR Prestige-21 FTIR spectrometer (Shimadzu, Kyoto, Japan) equipped with a KBr beam splitter. A pellet from a well-dried KBr was prepared according to standard cold pressing. The resolution was 0.5 cm −1 , and the number of scans was 45.

RP-HPLC Analysis
Reversed phase high-performance liquid chromatography analysis was carried out on an LC-20Avp (Shimadzu, Kyoto, Japan) with UV-detection, and the column was a Discovery C18 (25 cm × 4.6 mm, 5 µm, Supelco).

Specific Surface Areas Analysis
Specific surface areas (SSAs) of the ZnO NPs were analyzed using an analyzer of specific surface area and adsorption porosity ASAP 2020 (Micromeritics, Norcross, GA, USA).

Chemical Composition of BC
The chemical composition of BC was analyzed by determining C/O/N using a Flash EA 1112 CN analyzer (NEOLAB Ltd., VELLETRI, ROMA, Italy). Analysis for major and trace elements was performed with an atomic absorption spectrophotometer AA-7000 (Shimadzu, Kyoto, Japan) after wet mineralization of cellulose samples with a mixture of perchloric and nitric acids.

Surface Charge and Dynamic Light Scattering Measurements
The surface charge and average hydrodynamic diameter of the ZnO NPs were measured using NanoBrook Omni (Brookhaven Instruments, NY, USA). Suspensions with a solid loading of 0.00625-0.02500% were prepared in the presence (5 × 10 −4 M) of BDP in different mediums and were allowed to equilibrate for 24 h to reach the steady state. The zeta potential was determined by phase analysis electrophoretic light scattering (PALS). The Smoluchowski model was used to convert the electrophoretic mobility values to the zeta potential values.
The hydrodynamic diameter of ZnO nanoparticles was determined by dynamic light scattering (DLS) in the mode of multimodal analysis of the correlation function. The measurements were carried out at 25 ± 0.1 • C at an angle of 90 • in the range from 0.1 to 5000 nm in polystyrene cuvettes (1 cm). The accumulation time of the correlation function was 180 s (n = 10).

Biological Activity
Male Wistar rats (150-200 g) were involved in the study. The animals were purchased from the Animal Breeding Facilities "Andreevka" Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies" of the Federal Medical and Biological Agency (Andreevka, Moscow region, Russia). The animals were handled humanely, kept in plastic suspended cages, and placed in a well-ventilated and hygienic rat house under suitable conditions of room temperature (27 ± 2 • C) and humidity. They were given food and water ad libitum and subjected to a natural photoperiod cycle of 12 h light and 12 h dark. The animals were allowed two weeks of acclimatization before the commencement of all animal model experiments in the study.
All blood taking and withdrawal of the animals for the experiment were performed under anesthesia, all efforts being made to minimize suffering.
The animal study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Ethics Committee of Privolzhsky Research Medical University, Russian Federation (protocol No. 1 from 18 January 2021).

Modeling of Thermal Burns in Animals
The deep second-degree thermal burns of the rat's back was made using electromagnetic radiation from an infrared soldering station YaXunXY865D following the requirements of Good Laboratory Practice for the experimental modeling of thermal burns in laboratory animals. Standard thermal burns had an area of 14.0 ± 0.5 cm 2 , and the average weight of the rats was 285.0 ± 5.0 g.

Microcirculation Research
Microcirculation was assessed quantitatively using the LAKK-02 (LASMA, Moscow, Russia). This device transmits continuous-wave laser light (30 mW, 890 nm) and white light (20 W, 500-900 nm) to skin tissue near the wound, where it is scattered and collected on the skin surface with fibers of the probe. The movement of erythrocytes causes a Doppler shift, which, in turn, is detected by the laser light and analyzed by the LAKK-02, which is then computed and displayed as the blood flow velocity. The detected laser signal correlates with the number of moving erythrocytes in tissue and blood flow velocity for calculation of the microcirculation parameters, using such arbitrary (relative) units (arb. un.) as perfusion units (perf. un.). The rate of microcirculation (the microcirculation level), the regulatory activity of its components, and the degree of shunt path participation with an allowance for the frequency range intervals of the blood flow oscillations in the rats' microvessels were investigated [56,57].

Biological Analysis In Vitro
In vitro biological analysis was performed using blood stabilized with sodium citrate. Erythrocytes were washed twice with 0.9% NaCl by centrifugation for 10 min at 1600× g. Glutathione reductase activity (EC 1.8.1.7) was studied by spectrophotometry based on the oxidized glutathione reduction [58]. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was determined in hemolysate of erythrocytes by spectrophotometry based on glucose-6-phosphate oxidation to the phosphoglucolactone with the formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) [59]. The energy metabolism in erythrocytes was studied using the catalytic activity of LDH (LDH, EC 1.1.1.27), directly (LDH direct , substrate-50 mM of sodium lactate) and in reverse (LDH reverse , substrate-23 mM of sodium pyruvate) reactions [60]. The activity of aldehyde dehydrogenase (EC 1.2.1.3) was estimated spectrophotometrically in accordance with previous methods [61]. The specific activity of the enzymes was calculated from the protein concentration analyzed by the modified Lowry method [62].

Platelet-Rich Plasma (PRP) and Platelet-Poor Plasma (PPP) Preparation
This study was performed in human platelets from 10 healthy volunteers aged 18 to 50 years, which were free of medication for 7 days. Volunteers signed the informed consent before participating in this study. This study was approved by the Local Ethics Committee of Privolzhsky Research Medical University, Russian Federation (protocol No. 1 from 18 January 2021). Blood samples (30 mL) from overnight fasting healthy volunteers were collected by venipuncture and put into plastic tubes containing 3.2% sodium citrate (blood:buffer = 9:1 v/v). The platelet-rich plasma (PRP) was prepared by centrifugation of blood samples at 21 • C, 200× g (1300 rpm) for 7 min, and the top layer was collected as PRP. Isolation of the platelet-poor plasma (PPP) was performed by further centrifugation of the rest of the blood samples at 21 • C, 2000× g (4000 rpm) for 15 min. PPP was used as a reference to define the theoretical point of 100% light transmission. Aggregation testing was performed at least 15 min after PRP was prepared to recover from refractoriness and used within 4 h of sample collection.

Platelet Aggregation Measurement
The platelet aggregation test was performed in accordance with [63], which was modified from the Born turbidimetric aggregometry method [45] using an aggregometer ALAT-2 laser aggregation analyzer (BIOLA, Moscow, Russia). PRP (300 µL) was set as 0% light transmission. PPP (300 µL) was set as 100% light transmission. We recorded changes in the light transmittance (at 785 nm wavelength) for 5 min after adding 15 µL of ADP solution. The submaximal concentration of ADP (the final concentration was equal to 3.75 µM) was used to induce platelet aggregation. Two minutes after the start of the experiment, 100 µL of BDP solution or ZnO NPs-BDP suspension was added to the studied solution. Acetylsalicylic acid (0.1 mM) was used as a positive control. PRP and all used solutions were pre-incubated for 5 min at 37 • C. Each sample was analyzed in triplicate. The light transmission dependence on time corresponding to the dependence of the percentage of platelet aggregation on time was presented.

Statistical Analysis
Statistical data processing was performed by Statistica 6.0 software (StatSoft Inc., Tulsa, OK, USA). The normality of the distribution of results was shown using the Shapiro-Wilk test. The significance of differences between groups was assessed using Student's t-test and one-way analysis of variance (ANOVA). The differences were considered statistically significant at p < 0.05.

Conclusions
It has been shown that BDP and ZnO NPs-BDP can inhibit aggregation due to ionized phosphate groups in BDP. This anti-aggregation effect promotes the healing of burn wounds. To inhibit ADP-induced platelet aggregation in plasma under the action of betulin diphosphate, it is important to ionize its two phosphate groups, which are potential donors of phosphoryl groups and can affect the total phosphate pool. This ability to inhibit platelet aggregation is retained upon introducing BDP into the bacterial cellulose matrix or upon immobilization in ZnO NPs. The antiaggregatory effect can be enhanced by bacterial cellulose in polyanionic form. We can assume that polyanionic compounds with a high charge, with ionized phosphate or phosphoryl groups, will be advantageous as antiplatelet agents.
The work demonstrates the high enzymatic activity of oxidoreductases (LDH, AlDH, GR, and G6PDH) under conditions of hypoxia caused by a burn in the treatment of wound dressings based on bacterial cellulose and zinc oxide nanoparticles with betulin diphosphate. The ratio of specific LDH activity in reverse and direct reactions LDH reverse LDH direct can be used as an empirical assessment of NAD+/NADH imbalance in the development of burn disease and a predictor of the effectiveness of burn treatment.
Supplementary Materials: The following are available online, Figure S1: Images of intact (a) and burnt rats without treatment (Day 0, b and Day 10, c), and under the treatment of BC-BDP (d, g), BC-ZnO NPs (e, h), and BC-ZnO NPs-BDP (f, i), Table S1: The decrease in wound area in % of an initial burn wound state during the treatment, Figure S2  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.