The development of advanced biomaterials and constructs for accelerated recovery of damaged tissues is a key direction in regenerative medicine. Biocompatible scaffolds based on natural biopolymers are widely used for these tasks. Organ decellularization enables obtaining a cell-free extracellular matrix (ECM) with preserved composition and biological activity. The objectives of the present work were combining these two approaches for the development of a composite scaffold based on silk fibroin and ECM microparticles and assessing its structure, biological properties, and regenerative potential. ECM microparticles were obtained by grinding the decellularized matrix of Wistar rat liver in liquid nitrogen. Scaffolds in the form of films were prepared by the casting method. The sinuous and rough topography of the scaffold surface was assessed by the scanning probe nanotomography (SPNT) technique. The inclusion of ECM microparticles in the composition did not affect the elasticity and tensile strength of the scaffolds. The obtained scaffold was non-toxic to cells, maintained high levels of adhesion and proliferation of mouse 3T3 fibroblast and Hep-G2 cells, and showed high regenerative potential, which was studied in the experimental model of full-thickness rat skin wound healing. The wound healing was accelerated by 1.74 times in comparison with the control. The primary tasks of tissue engineering and regenerative medicine are manufacturing scaffolds, imitating the microstructure of the native extracellular matrix of tissues and organs, and recreating a favorable environment for cell adhesion and proliferation. At the same time, an important task is to develop and create constructions with not only the necessary biochemical, immunological, and mechanical properties for cell adhesion, cell-to-cell communication, and their production of the extracellular matrix in vivo, but also a defined three-dimensional micro- and nanoscale morphology. Commercial medical implants made of synthetic materials have some advantages , but the methods for obtaining such implants do not allow repeating the necessary architecture, micro- and nanostructural features, and functional activity of native tissues. This problem can be solved by the use in regenerative medicine of biological grafts that include components of native tissue.Decellularized tissues and organs may serve as sources of cell-free ECM, which is used for such regenerative medicine tasks as 1) fabrication of specific microcarriers for cells, (2) fabrication of hydrogels from lyophilized decellularized tissue treated with pepsin, followed by dissolving in phosphate-buffered saline, and (3) fabrication of coatings from a solution of the components of the extracellular matrix obtained from lyophilized decellularized tissue [ fabricatMicrocarriers can be used as supporting material in transplantation in dry form or in the form of suspensions for injection as a minimally invasive method of therapy. For example, microparticles from decellularized skin tissue were used to regenerate the skin .The traditionally used hepatocyte transplantation has practical limitations, mainly owing to the difficulties associated with obtaining of a sufficient number of functioning hepatocytes necessary for therapeutic efficacy . In addiIn reconstructive surgery, it is often necessary to restore adipose tissue in the event of postoperative, congenital, or post-traumatic loss, which can lead to scar tissue formation or loss of function. The literature describes the use of decellularized human greater omentum from cadaveric donors , as wellThe obtained decellularized adipose tissue has been used as a material for various products: hydrogel scaffolds based on decellularized tissue  and compAccording to the literature, decellularized myocardial tissue has also been used for the manufacture of hydrogels. After grinding the extracellular matrix, the decellularized tissue was treated with pepsin, and the self-organizing hydrogel with preserved bioactivity was obtained. This was demonstrated in vitro using a culture of mouse fibroblasts HL-1, where the extracellular matrix maintained high metabolic activity and cell proliferation. In an ischemia simulation experiment, microparticles of decellularized heart tissue had a specific cell protector effect on cardiomyocytes .Pancreas transplantation is the only reliable long-term therapy for patients with insulin-dependent diabetes mellitus. Since the incidence of diabetes mellitus among adults and children is growing every year, and at the same time there are a number of problems with transplantation, such as a lack of donor organs, the high cost of transplantation and subsequent maintenance immunosuppressive therapy, the necessity to develop new approaches to create tissue-engineered constructions for the treatment of pancreatic diseases is more relevant than ever. Decellularization of the human pancreas was performed by perfusion with a solution of Triton X-100 and ammonium hydroxide or by treatment with deoxycholate. Furthermore, the obtained extracellular matrix was lyophilized and subjected to cleavage in a solution of pepsin in hydrochloric acid, as a result of which a hydrogel of the pancreatic extracellular matrix was obtained. The obtained hydrogel was recellularized with various types of pancreatic cells and showed biocompatibility with the insulinoma cell line, beta-like stem cells, and endothelial cells, which can subsequently contribute to adequate graft vascularization. The absence of immunogenicity of the obtained hydrogel was also demonstrated in vivo in mice .The decellularization method was also applied to the human placenta, owing to the high content of extracellular matrix components and endogenous growth factors . Cells wMany experimental data on the possibility of using decellularized tissue in various fields of regenerative medicine, as well as on the study of the unique biological properties of the extracellular matrix, determine the relevance of the development of alternative methods of decellularization for the creation of grafts for liver regeneration. The goal of the present work is the development of a novel kind of biocomposite material combining silk fibroin, as a base biopolymer, and ECM microparticles. We report on the development of composite scaffolds based on silk fibroin with microparticles of decellularized rat liver tissue, investigation of its structural and biological properties, and in vivo assessment of its regenerative potential in a rat skin wound healing model.We used male Wistar rats (250–350 g) both for liver explantation with consequent decellularization and for investigation of the scaffold regenerative potential in a model of full-thickness skin wound healing. All experiments with animals were approved by the Local Ethical Committee of the V.I. Shumakov National Medical Research Center of Transplantology and Artificial Organs. Experiments were conducted in accordance with Directive 210/63/EU of the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes .Hepatic portal vein cannulation was performed with the use of a Vasofix Certo 18 G catheter . After that, the liver was explanted from the rat organism, and a perfusion pump  was used to remove the blood from the organ; the liver was perfused with 200 mL of phosphate-buffered saline (PBS) . Decellularization was conducted by sequential 3-stage perfusion of the explanted liver with solutions of 0.1% sodium dodecyl sulfate  in PBS (500 mL) with increasing concentrations of Triton X-100 : 1%, 2% and 3%. The organ was perfused with these detergent solutions over 72 h. Then, to remove the detergents contained in the solutions from the liver, it was perfused with PBS. A perfusion rate of 150 mL/h was used in all stages. The obtained decellularized liver was stored at 4 °C until use .g. The precipitate was milled in liquid nitrogen using a pre-cooled pestle and mortar for 15 min. The obtained particles were transferred to a clean pre-cooled tube, and the volume was adjusted to 35 mL with distilled water with stirring.Decellularized rat liver tissue was ground with surgical scissors, transferred to a tube, and the volume was adjusted to 15 mL with a solution of 15% glycerol in PBS (pH = 7.4). Then tissue was incubated for 20 min and centrifuged for 10 min at 8500× g twice. Then the supernatant was centrifuged for 10 min at 720× g. The obtained supernatant was centrifuged for 10 min at 12,100× g 4 times. After that, the supernatant was taken completely, and the obtained precipitate was diluted in double-distilled water in a volume 10 times smaller than the volume of the selected suspension of particles after grinding in liquid nitrogen.After subsidence of the obtained suspension of particles during the day, the upper 1/3 of the suspension of settled particles was taken, which was centrifuged for 10 min at 1355× Bombyx mori silkworm. In the first stage, the threads were cut into 5 cm fragments and purified from sericin. For this, 1 g of threads was boiled in a 500 mL of 2.52 M sodium bicarbonate aqueous solution in a water bath for 40 min and washed with 3.6 L of water. Then, the silk was boiled in 500 mL of water in a water bath for 30 min and washed with 3.6 L of distilled water. The last procedure was repeated 3 times. The purified silk fibroin was dried at room temperature.The composite scaffolds in the form of films were obtained using the casting method . Silk fig. The supernatant was dialyzed against double-distilled water and centrifuged for 7 min at 12,100× g. The fibroin solution concentration was measured with a Thermo Genesis 10 UV spectrophotometer  at a 280 nm wavelength (molar extinction coefficient of fibroin 1.07). To obtain a film with a diameter of 1 cm2, 100 μL of the working silk fibroin solution with a concentration of 20 mg/mL was deposited on the surface of the polished Teflon and dried for 2 days at room temperature. The films were incubated in 96% ethanol for 15 min and were separated by scalpel [An aqueous solution of fibroin (1 mL) was prepared by boiling a solution of 389 mg of calcium chloride, 388 μL 96% ethanol, 544 μL double-distilled water, and 130 mg of silk fibroin for 5 h in a water bath ,27. Then scalpel .For the fabrication of the composite scaffolds based on silk fibroin in the form of films, a suspension of microparticles of decellularized rat liver tissue was added to a working solution of silk fibroin. Before adding, the suspension was carefully dispersed and added to the solution for fabricating a scaffold to a final concentration of microparticles of 8 mg/mL.Mechanical characteristics of fabricated scaffolds were studied using a Zwick/Roell BZ 2.5/TNIS testing machine . There were two groups of samples: group 1—scaffolds in the form of films based on silk fibroin, group 2—composite scaffolds in the form of films based on silk fibroin with microparticles of decellularized rat liver tissue. Samples with dimensions of 1.5 × 5.0 cm were prepared for testing. Mechanical measurements were performed at a speed of 50 mm/min with preload of 0.05 N. As a result, two basic characteristics were determined for each sample: elasticity or elongation as a percentage of the initial length of the sample and tensile strength (MPa). Statistical processing of the data obtained was conducted using the TestXpert software version III .Samples for SEM were fixed in a solution of 2.5% glutaraldehyde  in PBS for 2 h in the dark at 4 °C. Fixed samples were washed 5 times for 5 min in PBS and dehydrated by incubation in ethanol solutions with increasing concentrations (10%–30%–50%–70%–80%–96%) for 30 min in each solution. Then, samples were transferred into acetone. Before the measurements, critical point transition drying of the samples was conducted with a K850 Critical point dryer . A 10 nm thick layer of gold was deposited on the dried samples at an ion current of 20 mA in argon atmosphere (pressure: 1 mbar) with use of a Q150R ES  vacuum spraying device.SEM analysis of the prepared samples was conducted using a Tescan Vega 3  scanning electron microscope. Images were processed by Tescan VegaTC software version 4.2.17.0  .The three-dimensional structure of fabricated scaffolds was studied by the scanning probe nanotomography (SPNT) technique. This method utilizes a specialized integrated SPM/ultramicrotome apparatus combining a scanning probe microscope and an ultramicrotome . It enables acquiring SPM data from the sample blockface surface immediately after ultrathin sectioning by an ultramicrotome diamond knife .Samples for SPNT analysis were fixed, dehydrated, and embedded in epoxy embedding medium  using a protocol described in . Consecu2, the culturing medium was changed and scaffold samples were brought to the wells: one 5 × 5 mm film sample per well. Scaffold samples were also incubated for 7 days at 37 °C, 5% CO2 conditions, while the culture plastic was used as a control. Then, each well was supplied with 60 μL of a 5 mg/mL methylthiazolyldiphenyl-tetrazolium bromide  solution, which was incubated for 4 h at 37 °C, 5% CO2, followed by precipitation of dark blue formazan crystals. After removal of scaffold samples, the plate was centrifuged at 885 g for 5 min. The formazan precipitates were dissolved in 300 μL of dimethyl sulfoxide  for 20 min, and the solution optical density at a wavelength of 540 nm was measured with a Picon spectrophotometer  for each well. The experiment was conducted on 5 samples of each silk fibroin scaffold and composite scaffold group, and the experiment was conducted in duplicate.Mouse 3T3 fibroblasts were utilized for cytotoxicity analysis performed using the MTT test. Cells were cultured in 300 μL of Dulbecco’s Modified Eagle’s Medium (DMEM) with low-glucose concentration  containing 10% fetal bovine serum , 40 μg/mL gentamicin , and 4 mM glutamine . After 3-day culturing in a 96-well plate in an incubator at 37 °C, 5% CO2. 3T3 line fibroblasts were incubated in DMEM Low glucose medium, while Hep-G2 cells were incubated in DMEM High glucose  and Ham’s F-12  mixed culture media; all media contained 10% fetal bovine serum, 10 mg/mL gentamicin solution, and 0.324 mg/mL glutamine.Cell proliferation on fabricated scaffolds was studied using two standard cell lines: the mouse 3T3 fibroblasts  and the Hep-G2 human hepatocarcinoma cells . Both cell lines were obtained from Dr. M.E.Krasheninnikov . Scaffolds of both groups were placed in the wells of the 96-well plate, while culture plastic was used as a control. The cell suspension in the medium was transferred into 96-well plates at the rate of 1000 cells per well. Cells were incubated for 7 days at 37 °C and 5% CO2. Then, the samples were washed twice with PBS.The samples were stained with DAPI  fluorescence dye, and 3 µg aqueous DAPI solution was added at a rate of 150 µL per well and incubated for 5 min at 37 °C, 5% COCell adhesion and proliferative activity were investigated by fluorescence optical microscopy using a Carl Zeiss Axio Vert.A1 microscope  with an objective lens LD Plan-Neofluar 20x/0.4 Corr M27  and a filter cube adjusted for DAPI dye, having an excitation range of 360–370 nm, an emission range of 420–470 nm. Images of cells in the 425 × 350 µm field of view were obtained with an Axiocam 305 color camera  and processed by the Zen 2.3 Blue Edition program . Quantity of cells for each sample was obtained by direct counting of DAPI-stained nuclei in the obtained images. The experiment was conducted on 5 samples of each experimental group, and the experiment was conducted in duplicate.An in vivo experiment was performed on a model of the rat skin wound healing. The animals were divided into three groups: group 1—control group, where the wounds were not covered with any material or construction that promoted skin healing; group 2—experimental group, where the wounds were covered with scaffolds in the form of films based on silk fibroin; group 3—experimental group, where the wounds were covered with composite scaffolds in the form of films based on silk fibroin with microparticles of decellularized rat liver tissue. There were 5 rats in each group. Ether was used for inhalation anesthesia at the rate of 50 mg/kg for all surgery manipulations with animals, which were in spontaneous respiration. Full-thickness wounds of circular shape were cut with surgical scissors on the back in the area of the rat shoulder after hair removal. Average diameter of wounds was 14 mm. An aqueous solution (0.05%) of chlorhexidine bigluconate  was used for wound treatment. Sterile gauze dressing was utilized for covering the wounds of control group rats. Photographs of wounds were acquired on the 3rd, 9th, 14th, 18th, and 23rd day of the experiment for the experimental group, and on the same days plus the 28th, 35th, 40th day for the control group, with a Nikon D5100 18-105 VR Kit camera with a Nikon AF-S DX 18–105 mm f/3.5–5.6 G ED VR lens . The progress of skin wound healing was assessed quantitatively using a wound-healing area parameter, calculated according to the formula:Obtained quantitative data enabled us to plot healing diagrams of skin wounds in all three groups. After complete healing of a full-thickness skin wound was confirmed visually, samples of skin tissue with dimensions of 20 × 20 mm were collected for consequent histological analysis.Samples of skin tissue were fixed using a mixture of formalin, ethanol, and acetic acid in a volume ratio of 4:1:0.3, incubated into paraffin for 48 h at room temperature, and embedded in paraffin. Then, embedded samples were sectioned with a Thermo Scientific HM 325 Rotary Microtome  and sections of 14 μm thickness were collected. All sections were stained with Masson’s trichrome stain , embedded in Canada balsam, and studied with a Carl Zeiss Axio Vert.A1 optical microscope  with an Axiocam 305 color camera . Images were processed using Zen 2.3 Blue Edition software .The data were processed by ANOVA. The statistical significance of differences in the results was evaluated by the Mann–Whitney U test. The level of statistical significance α was considered equal to 0.05.Liver tissue microparticles were obtained by grinding the decellularized matrix in liquid nitrogen. The fraction of microparticles of the rat liver extracellular matrix with an average size of 1–5 μm was 20% by weight of the total amount of tissue .Scaffolds in the form of films based on silk fibroin with microparticles of decellularized rat liver tissue included in the composition were made by the casting method . Two groThe surface structure of the obtained composite scaffolds was analyzed by scanning electron microscopy. The surface topography of the scaffold, with included microparticles, was rougher and non-uniform compared with the surface topography of a non-composite scaffold . The micMicroparticles of decellularized tissue were observed on the surface of the composite scaffolds, in addition to microparticles located in the volume of the film. At the same time, microparticles of decellularized rat liver tissue were spread over the surface of the scaffold and created a rough relief of the film surface.Based on the obtained SPM images, a quantitative analysis of the roughness of the studied scaffolds was conducted. Data on the roughness of the scaffold surfaces are presented in The data presented in the table indicated that the inclusion of the extracellular matrix of the liver tissue in the composition of the microparticles increased the surface roughness of the construction five-fold.The analysis of the micro- and nanostructure of the obtained composite construction was conducted by the SPNT method. For quantitative analysis of the construction, three-dimensional reconstructions were created by combining a series of SPM-sections of scaffolds , depictiThe three-dimensional reconstructions were obtained to calculate the ratio of the volume of microparticles to the volume of the scaffold. For a scaffold in the form of a film based on silk fibroin, with microparticles of decellularized rat liver tissue included in the composition, this ratio was 1.4% ± 0.4.A study of the effect of inclusion of microparticles of decellularized tissue into the composition of scaffolds in the form of films based on silk fibroin on the mechanical properties of the constructions was conducted. The samples were subjected to deformation on a Zwick/Roell BZ 2.5/TNIS tensile testing machine, and the values of the material’s tensile strength (MPa) and elasticity (% elongation) were evaluated . AccordiAnalysis of the cytotoxicity of the obtained scaffolds was conducted using the 3T3 mouse fibroblast cell line. During the experiment, samples of two types of scaffolds were placed next to a monolayer of mouse fibroblasts and incubated in a culture medium for 7 days. The obtained scaffolds were not toxic to the 3T3 mouse fibroblast cells. .Adhesion and proliferation of 3T3 mouse fibroblasts and Hep-G2 human hepatocarcinoma cells were studied on samples of the obtained scaffolds in the form of films based on silk fibroin. Culture plastic was used as a control. Fluorescent microscope images of the number of cells and their distribution on the scaffolds during the experiment are shown in Quantitative results of proliferative activity analysis of 3T3 and Hep-G2 culture cells are depicted in The fabricated composite scaffolds with microparticles of decellularized rat liver tissue included in the composition were successfully used as wound dressings for the full-thickness rat skin wound-healing model. Images of wounds during the experiment are shown in To quantify the wound-healing process, wound diameters were measured on the 0th, 3rd, 9th, 14th, 18th, 23rd, 28th, 35th, and 40th days of the experiment , and heaAll scaffolds were hydrophilic, so they were easily positioned on the wound and did not require additional fixation with suture material. According to the obtained results, an increase in the rate of wound healing in the experimental group was observed after 9 days. Complete wound healing occurred in group 3 on day 23, and in group 2 on day 28, while in the control group complete wound closure was observed at day 40 .After complete wound healing on the aforementioned days of the experiment for each group, sections of skin tissue samples from the wound areas were collected from all three groups, stained with Masson’s trichrome, and studied by optical microscope. Thus, we compared completely regenerated skin tissue in the control group on day 40 with samples of regenerated skin tissues from the experimental groups from considerably earlier periods to prove the observed acceleration of full skin regeneration in the experimental groups by histology means. A histological evaluation of skin samples in the area of complete wound healing for each group (A = 100%) revealed the restoration of three layers of skin in all the experimental animals—epidermis, dermis, and hypodermis—which indicated successful wound healing . MoreoveA main approach of tissue engineering and regenerative medicine in the restoration of organ and tissue pathological injuries is the fabrication of construction that mimics the structure of tissues and organs. The relevant selection of the materials is a crucial parameter for the manufacturing of such constructions. It is essential to use materials that combine the properties to provide both high regeneration efficiency and the possibility of implantation into the body.Constructions that are made of synthetic materials, such as polycaprolactone, polyglycolide, and polyamide, generally have good mechanical properties, allowing them to adjust their shape, structure, and physical parameters, but their degradation products can be toxic to cells.The utilization of natural materials, which are able to mimic the microenvironment of native tissue as closely as possible, is associated with a number of difficulties. These constructions are obtained from native tissues, which implies high costs, ethical issues, and low mechanical properties of obtained grafts. This leads to the emergence of prerequisites for the development of new constructions for regenerative medicine.Bombyx mori [One promising material for the creation of biocompatible constructions for tissue engineering and regenerative medicine is silk fibroin from the cocoons of the silkworm byx mori . The strbyx mori . It was Within our framework, scaffolds in the form of films based on silk fibroin with microparticles of decellularized rat liver tissue were fabricated by the casting method. Microparticles were obtained by grinding decellularized liver tissue in liquid nitrogen. All scaffolds were colorless, transparent, and had a thickness of 20 to 60 µm.The surface structure of the obtained scaffolds was studied with SEM. The surface of the composite scaffolds had a rough structure compared to the unmodified scaffold. Moreover, the surface topography of the composite scaffold was uneven over the entire surface area with distinct local clusters of microparticles.The micro- and nanostructure of the surface of the obtained scaffolds, as well as the structure of the microparticles in the scaffold composition, were studied with scanning probe microscopy. It was shown that microparticles of decellularized tissue were present both in the thickness of composite scaffolds and on their surface. Thus, it can be concluded that the chosen method of manufacturing composite scaffolds was effective, while some microparticles were not shielded by silk fibroin, which can affect the interaction of cells with the construction before the scaffold degradation begins. In the quantitative analysis of SPM images, it was shown that the inclusion of liver tissue in the microparticles of the extracellular matrix in the scaffold composition increased the surface roughness of the construction by 5 times compared with non-composite scaffolds. Substrate roughness is known to be an important parameter that determines the level of cell adhesion, proliferation, and differentiation ,41.The study of the micro- and nanostructure of the obtained composite scaffolds was conducted with the SPNT method. It was shown that rat liver microparticles were flattened on the scaffold surface and had a large area of contact with it. The unique capabilities of the SPNT method made it possible to identify the morphology of the contact between the microparticle and the scaffold core, which was impossible using other microscopy methods, including SEM ,42.Bombyx mori silk fibroin aqueous solution had tensile strength and elasticity comparable to products actively used in regenerative medicine [Important properties of materials and constructions for regenerative medicine are the mechanical characteristics, which allow performing surgical procedures during implantation of the obtained constructions. Constructions based on medicine . At the Furthermore, a study of the biocompatibility and an assessment of the regenerative potential in vivo of the obtained scaffolds in the form of films were conducted. Before conducting these experiments, an analysis of the cytotoxicity of the scaffolds was conducted, which did not reveal a cytotoxic effect on the cells of the obtained scaffolds based on silk fibroin.The biocompatibility of the constructions was assessed using two cell cultures, 3T3 mouse fibroblasts and Hep-G2 human hepatocarcinoma cells. During the experiment, it was shown that in all scaffolds based on silk fibroin support cell adhesion, there were no significant differences between different groups of samples and control. On composite scaffolds, the proliferative activity of cells on the 7th day of observation was higher than on unmodified scaffolds, which may be because of the optimal roughness of composite constructions. The largest number of cells of both cultures on the 7th day of the experiment was registered on scaffolds in the form of a film based on silk fibroin, with microparticles of decellularized rat liver tissue included in the composition. This may have depended on the shape and position of decellularized tissue microparticles on the scaffold surface; they were spread over the surface of the construction, which created an optimal relief and substrate roughness for cell adhesion and proliferation. This may also have been connected with the tissue-specific properties of the microparticles of decellularized tissue  or due tThe regenerative potential and the possibility of application of composite scaffolds based on silk fibroin were studied in a full-thickness rat skin wound-healing model.All obtained scaffolds were successfully implanted. Scaffolds based on silk fibroin were hygroscopic, fixed on the wound without suture material, retaining moisture throughout the entire procedure . ScaffolSkin healing was quantified by measuring the diameter of wounds during the experiment. Scaffolds based on silk fibroin of both groups accelerated wound healing compared with controls.The shape of the curve of the dynamics of wound healing with composite scaffolds as wound dressing did not differ from the shape of the wound healing curve of the control. The utilization of scaffolds based on silk fibroin with microparticles of decellularized tissue accelerated wound healing by 17 days (1.74 times) compared with the control, and by 5 days (1.22 times) compared with the unmodified scaffold.An investigation of the quality of the tissue restoration in the full-thickness skin wounds was performed by histological analysis of sections of skin tissue samples obtained after complete healing of the wounds. The study indicated that all three layers of skin tissue were presented, similar to the native skin, indicating successful regeneration. The sections from the experimental group using an SF scaffold demonstrated a more pronounced stratum corneum of the epidermis and a loose structure of the dermis. Importantly, no macrophage presence or inflammation foci were observed, which also proved the quality of skin healing. The presence of hair follicles in groups of composite scaffolds was shown, which also indicated a complete functional restoration of the skin.It makes sense to compare observed acceleration of skin regeneration with the results obtained previously for other scaffolds studied in the similar full-thickness rat skin wound-healing model, namely electrospun microfibrous scaffolds based on silk fibroin  and lyopSilk fibroin-based microfibrous scaffolds demonstrated a regenerative effect analogous to SF-MP scaffolds studied in the present work , while LThus, it can be concluded that the obtained scaffolds based on silk fibroin with microparticles of decellularized rat liver tissue have high regenerative potential in vitro and in vivo and may be considered as a basis for development of new biomedical products for regenerative medicine.


The fracture behavior of the Cu/Sn-3.0Ag-0.5Sn (SAC305)/Cu solder joint was investigated by conducting tensile tests with in situ X-ray micro-computed tomography (μ-CT) observation, and finite element (FE) simulation. The tensile fracture process of solder joints with a real internal defect structure was simulated and compared with the experimental results in terms of defect distribution and fracture path. Additionally, the stress distribution around the defects during the tensile process was calculated. The experimental results reveal that the pores near the intermetallic compound (IMC) layers and the flaky cracks inside the solder significantly affected the crack path. The aggregation degree of the spherical pores and the angle between the crack surface and the loading direction controlled the initiation position and propagation path of the cracks. The fracture morphology indicates that the fracture of the IMC layer was brittle, while the solder fracture exhibited ductile tearing. There are significant differences in the fracture morphology under tensile and shear loading. The failure of a solder joint is a problem that must be urgently addressed in the manufacturing industry of electronic and microelectronic products. Defects such as pores and microcracks in solder joints are a main cause for the low reliability of modern electronic equipment ,2. The dThe SAC305 alloy is the most important solder material, and its mechanical properties have attracted widespread attention. To date, studies have conducted several types of tests, including uniaxial tension tests , creep tExisting studies have conducted extensive numerical evaluations, but have mainly predicted the mechanical behavior of solder joints using a finite element (FE) model without internal defects ,20 or byCurrently, many related studies have been carried out in the fields of electronics and microelectronics. For example, X-ray μ-CT has been used to quantitatively characterize the number and volumes of pores ,28, the In view of the present research situation, this paper would set up an in situ tensile system under The solder used in the experiment was the commercial SAC305 alloy, its nominal composition was displayed in Finally, to satisfy the size requirements of the tensile setup and X-ray image, the uniaxial tensile specimen was designed in a dog-bone shape. During the manufacturing process, the solder alloy and the Cu bars were fixed by the self-designed clamp of the Al alloy, as shown in A 3D X-ray μ-CT system  with a resolution of 1.00 μm was used to obtain the internal microstructure of the solder joints. The inspection area was indicated by the blue rectangle in To observe the evolution of the internal defects during the tensile process, a CT5000 Deben  tensile setup was placed in the X-ray μ-CT scanning environment cavity, which was installed on the CT rotatable test table, as shown in 3 were retained. The obtained FE mesh model is shown in A FE model with real internal defects was established to simulate the tensile fracture process of solder joints. First, the CT scanning data of the solder joint were imported into the Avizo software to generate the internal structure model. Considering that larger defects and defects near the boundary greatly influence the fracture of the material, in the FE modeling process, the defects with a volume larger than L was the length of the element and The material property of the SAC305 alloy was set to isotropic hardening. The material parameters were mainly obtained from the experimental tensile curve , respectively, were extracted as shown in The volume and shape evolution of the internal defects were quantitatively characterized. Generally, the shape characteristics of the defects were characterized by the sphericity parameters, that is, the ratio between the spherical surface, which was the same as the volume of the defect, and the defective surface . Figure Defects were inevitable for solder joints . As showThe position and shape of defects in the solder joints had a significant influence on the stress distribution. As shown in As shown in 5Sn6 phase, which proved that the fracture surfaces of regions Q2 and Q3 were IMC layers. Overall, the IMC layers were brittle, and had many pores around them, which was the main reason why the IMC layers became the weak link between the SAC305 solder and the Cu bar. Another reason was that a large concentration of stress existed on the lower and upper IMC layers, owing to the aggregation of the pores. Additionally, interface debonding between the Cu6Sn5 particles and the β-Sn matrix was observed on the fracture surface, as shown in 6Sn5 phase was approximately three times equal to that of the β-Sn matrix. Therefore, the deformation between the Cu6Sn5 phase and the Additionally, in the partially enlarged image in (1)The tensile strength of the Cu/SAC305/Cu solder joint is 51.52 MPa, which is close to the fracture strength of the solder materials, and its fracture strain is 19.87% due to the great toughness of the solder material.(2)In terms of the crack propagation path, the simulation results are in good agreement with the experimental results. The reliability of the simulation results obtained by this study was verified by the in situ X-ray μ-CT observation of the 3D crack path.(3)The fracture morphology of the solder joints can be divided into four regions, namely, regions Q1, Q2, and Q3, which comprise the tensile fracture zone, and region S4, which is the shear fracture zone. As region Q2 is a brittle IMC layer and contains aggregated pores, its crack propagation rate was significantly higher compared with region Q1.(4)6Sn5 phase and the β-Sn matrix debonded, owing to the inconsistent deformation.In the tensile fracture zone of Q2 and Q3, on one side of the solder, the tearing ridges of the aggregated pores were linear, owing to the large deformation, while those on the other side were arc-shaped. Additionally, the interface between the CuThis study used in situ X-ray μ-CT to quantitatively and visually characterize the evolution of the internal defects of Cu/SAC305/Cu solder joints under uniaxial tensile loading. The influence of the location, distribution, and aggregation of the defects on the fracture behavior of the solder joints was characterized by CT images. The FE model was established using the CT reconstruction data of the solder joints at the initial state. Based on the experimental and simulation results, the main conclusions drawn from this study are as follows:6Sn5, Ag3Sn phase in IMC layers, could not be established in the FE model, thus the accuracy of the simulation results needs to be further improved.Although some results had been obtained in the fracture research of the Cu/SAC305/Cu solder joint, there were still some shortcomings, which should also be the prospect of future work. In microelectronics, the service of solder joints was mainly caused by the synergy of electrical current and mechanical load, so the influence of electrical current on fracture behaviors could not be ignored. On the other hand, due to the limited resolution of μ-CT, some microstructures, such as the Cu


Ppargc1a decreased and Drp1 and Parkin gene expression increased, suggesting the disturbances of mitochondrial biogenesis, fission, and mitophagy, respectively. Uridine treatment of diabetic mice restored the mRNA level of Ppargc1a and enhanced Pink1 gene expression, which may indicate an increase in the intensity of mitochondrial biogenesis and mitophagy, and as a consequence, mitochondrial turnover. Uridine also reduced oxidative phosphorylation dysfunction and suppressed lipid peroxidation, but it had no significant effect on the impaired calcium retention capacity and potassium transport in the heart mitochondria of diabetic mice. Altogether, these findings suggest that, along with its hypoglycemic effect, uridine has a protective action against diabetes-mediated functional and structural damage to cardiac mitochondria and disruption of mitochondrial quality-control systems in the diabetic heart.Long-term hyperglycemia in diabetes mellitus is associated with complex damage to cardiomyocytes and the development of mitochondrial dysfunction in the myocardium. Uridine, a pyrimidine nucleoside, plays an important role in cellular metabolism and is used to improve cardiac function. Herein, the antidiabetic potential of uridine  and its effect on mitochondrial homeostasis in the heart tissue were examined in a high-fat diet–streptozotocin-induced model of diabetes in C57BL/6 mice. We found that chronic administration of uridine to diabetic mice normalized plasma glucose and triglyceride levels and the heart weight/body weight ratio and increased the rate of glucose utilization during the intraperitoneal glucose tolerance test. Analysis of TEM revealed that uridine prevented diabetes-induced ultrastructural abnormalities in mitochondria and sarcomeres in ventricular cardiomyocytes. In diabetic heart tissue, the mRNA level of  Diabetes mellitus (DM) is a group of metabolic disorders of different etiologies that are associated with insufficient secretion of insulin from the β-cells of the pancreatic islets and variable degrees of peripheral insulin resistance, resulting in hyperglycemia . The seaUridine is a pyrimidine nucleoside that plays an important role in maintaining cellular function and energy metabolism . UridineUridine and its derivatives have been widely used to reduce cytotoxicity, suppress drug-induced hepatic steatosis, and improve neurophysiological functions ,13,14,15At the same time, data on the effects of uridine on the progression of diabetes mellitus and insulin-resistant states are rather contradictory. On the one hand, oral administration of uridine with food was shown to improve insulin sensitivity in healthy mice and animals kept on a high-fat diet ,21,22. LGiven the beneficial effects of uridine on cell-energy metabolism and redox status, we suggested that the nucleoside could serve as a supplemental therapy to ameliorate metabolic disorders associated with mitochondrial alterations. Mitochondrial dysfunction is known to play an important role in both the development and progression of DM, particularly due to excessive production of ROS, a loss of mitochondrial membrane potential, impaired oxidative phosphorylation, and increased susceptibility of the organelles to the permeability transition pore (PTP) opening, which eventually could lead to mitochondrial damage. In parallel, DM is accompanied by a malfunction of the mitochondrial quality-control system and a disruption of mitochondrial dynamics and mitochondrial biogenesis, resulting in the accumulation of defects in the mitochondrial network and the dysregulation of cellular energy supply ,27,28. ITaking into account the above, the aim of the present work was to evaluate the antidiabetic potential of uridine  and to study the effect of the nucleoside on the development of mitochondrial dysfunction in the heart tissue in the C57BL/6 mouse model of a high-fat diet–streptozotocin-induced DM.The design of the experiments and the scheme for induction of DM are outlined in Drp1 (dynamin-related protein 1) and Parkin, suggesting the stimulation of the processes of mitochondrial fission and mitophagy, respectively. In parallel, the mRNA level of Ppargc1a  was decreased, which may indicate the suppression of mitochondrial biogenesis in the DM group.In the next part of the work, we determined changes in the level of expression of genes encoding proteins involved in mitochondrial fusion/fission, mitochondrial biogenesis, and mitophagy in the experimental groups . One canPpargc1a gene, but did not affect the expression of other genes studied. It should be noted that in the DM + U group, the mRNA level of Pink1 (PTEN-induced putative kinase 1) increased, which may additionally indicate the activation of mitophagy. The mRNA level of the Mfn2 (mitofusin 2) gene, which is involved in the mitochondrial fusion process, did not differ in any experimental group.Administration of uridine to diabetic mice normalized the expression of the DNP, respectively) (DNP respiration only tended to restore). This suggests that uridine may contribute to the normalization of cardiomyocyte energy supply.Ultrastructural abnormalities and dysregulation of the quality-control system of mitochondria can promote dramatic changes in mitochondrial functions, the main one of which is the ability to produce ATP through oxidative phosphorylation. The DM group demonstrated a decrease in the rates of ADP- and 2,4-dinitrophenol (DNP)-stimulated respiration of the heart mitochondria (state 3 and 3Uctively) . In paraThe oxidative damage to the heart mitochondria was next examined by the index of lipid peroxidation products  using the thiobarbituric acid (TBA) assay . One canATP) [+ ions from mitochondria, which was mediated by the inversion of the mitoKATP [+ release from the heart mitochondria. The administration of uridine to diabetic animals did not lead to a statistically significant increase in the rate of K+ release from the mitochondria (only a trend towards an increase in this parameter was observed).Uridine is known to be a precursor of uridine 5′-diphosphate (UDP), an activator of the mitochondrial ATP-dependent potassium channel (mitoKATP) . In thismitoKATP . As can 2+-dependent PTP opening [2+ retention capacity (CRC) index was reduced in the DM group. Uridine treatment caused a 1.25-fold increase in this parameter, but this effect was statistically insignificant.As shown earlier, DM can be accompanied by a decrease in the resistance of heart mitochondria to the induction of the Ca opening ,33,34,35Accumulating evidence suggests that the pyrimidine nucleoside uridine is essential for maintaining a number of cellular functions and energy metabolism . The dynIn this work, we demonstrate the protective effect of chronic administration of uridine against the development of mitochondrial dysfunction in the heart of C57BL/6 mice with DM induced by a high-fat diet combined with low-dose streptozotocin injections. According to the literature data, short-term administration of uridine to mice on a high-fat diet can significantly improve glucose tolerance . Herein,The development of diabetic cardiomyopathy is one of the severe consequences of DM, which is defined as the occurrence of abnormal myocardial structure and function, with the exclusion of hypertension, coronary artery disease, and other preexisting cardiovascular conditions ,5,6. DiaSome studies revealed that the protective effect of uridine against ischemia-induced damage to the heart, as well as in some other cellular pathologies associated with oxidative stress, may be related to the normalization of the structure and function of mitochondria . It was The administration of uridine (30 mg/kg/day) to diabetic mice for three weeks leads to an improvement in the morphology of the organelles in ventricular cardiomyocytes. In the DM + U group, the ultrastructure of the mitochondrial matrix and cristae, the size of mitochondria, and their number significantly differed from those in the DM group and were similar to those the CTR group. It is important to note that treatment with uridine leads not only to the preservation of mitochondrial ultrastructure but also to the normalization of other structures of cardiomyocytes. In particular, the DM + U group showed an increase in sarcomere length compared to the DM group.Ppargc1a gene responsible for the synthesis of the PGC1α protein, the main regulator of mitochondrial biogenesis. Administration of uridine to diabetic mice resulted in a significant increase in the level of Ppargc1a mRNA. This may indicate the normalization of mitochondrial biogenesis in cardiomyocytes of the DM + U group. We did not observe a significant change in Drp1 and Parkin mRNA levels after uridine treatment, which are responsible for the synthesis of Drp1 and Parkin proteins involved in the processes of mitochondrial fission and mitophagy. Nevertheless, the DM + U group showed an increase in the level of Pink1 mRNA . This suggests that uridine stimulates mitophagy in diabetic animals. The increased exclusion of defective mitochondria along with stimulation of mitochondrial biogenesis by uridine may lead to the occurrence of a sufficient population of healthy mitochondria in diabetic cardiomyocytes. It should be noted that although the changes observed in the mRNA levels of the proteins studied are indirect evidence, they are fully consistent with the literature data that diabetes mellitus is accompanied by an increase in mitochondrial fission and a decrease in mitochondrial biogenesis and mitophagy [Normalization of the ultrastructure and number of cardiomyocyte mitochondria in the case of uridine administration to diabetic animals may be associated with the restoration of systems responsible for mitochondrial homeostasis, namely, mitochondrial biogenesis, dynamics, and mitophagy. Indeed, we found that the DM group showed a decrease in the expression of the itophagy .Indeed, as shown in + in the mitochondria of the heart. It is believed that the transport of K+ through the inner mitochondrial membrane is mediated by the mitoKATP channel, which can be activated by UDP and whose metabolic precursor is uridine [+ in the DM + U group compared to the DM group (only an upward trend was observed). One can suggest that this concentration of uridine is not sufficient to restore this parameter in diabetic mice.Mitochondrial dysfunction in DM is mediated not only by a violation of oxidative phosphorylation and excessive production of ROS but also by the dysregulation of ion homeostasis . As show uridine ,44,45. W2+ concentration, and a number of other stimuli. The main structural components of the PTP are cyclophilin D, adenylate translocator, and ATP synthase [2+-dependent PTP opening. Thus, under our experimental conditions, uridine was unable to inhibit the formation of the PTP in diabetic mitochondria. Taken together, our findings suggest that, along with its glucose-lowering effect, uridine has a protective action against diabetes-mediated functional and structural damage to cardiac mitochondria and disruption of mitochondrial quality-control systems in the diabetic heart.The development of DM is accompanied by a decrease in the resistance of mitochondria of the cardiac and skeletal muscles to the PTP opening ,46,47,48synthase ,50. The synthase ,46,47. Tn = 10); (2) control + uridine (CTR + U) (n = 10); (3) DM (n = 10); and (4) mice with DM treated with uridine (DM + U) (n = 10). The induction of diabetes in experimental mice was carried out using a combination of a high-fat diet with multiple injections of low doses of streptozotocin according to the protocol described in our previous work [Male mice of the C57BL/6NCrl line weighing 22–24 g were used. The animals were purchased from the Animal Breeding Facility, Branch of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, . The mice were randomly divided into four experimental groups: (1) vehicle-treated control (CTR)  for the transmission electron microscopy (TEM) imaging were taken from decapitated animals and fixed for 2 h in a 2.5% glutaraldehyde solution in 0.1 M PBS (pH = 7.4) . The preRplp2 mRNA, and a comparative CT method was used to quantify the results [Total RNA was isolated from 100 mg of deep-frozen tissue samples from the quadriceps using an ExtractRNA kit  in accordance with the protocols of the manufacturer. Real-time PCR was performed on a QuantStudio 1  using the qPCRmix-HS SYBR reaction mixture . The selection and analysis of gene-specific primers were performed using Primer-BLAST  [2PO4, 2.5 mM potassium malate, 2.5 mM potassium glutamate, and 10 mM HEPES/KOH (pH 7.4). Ca2+ transport in mitochondria was estimated with an Arsenazo III indicator at 675–685 nm using a Tecan Spark 10M plate reader  [2PO4, 2.5 mM malate, 2.5 mM glutamate, 10 μM EGTA, 50 μM Arsenazo III, and 10 mM HEPES-KOH (pH 7.4.). The total amount of the added Ca2+ ions that induced their spontaneous release from mitochondria due to the induction of the PTP opening was interpreted as the calcium retention capacity (CRC) index of the organelles. Transport of K+ in mitochondria was determined by the rate of DNP-induced release of K+ from the organelles [+ efflux was measured with an ion-selective electrode . The incubation medium contained 180 mM sucrose, 70 mM mannitol, 5 mM NaH2PO4, 1 μg/mL oligomycin, and 10 mM Tris/HCl, pH 7.4. Intensity of lipid peroxidation in mitochondrial membranes was assessed spectrophotometrically by quantification of thiobarbituric acid-reactive substances represented by malondialdehyde and some other minor aldehyde species, as described in [Mitochondria were isolated from fresh mouse heart tissue by differential centrifugation . Final sAustria) . The reazerland) . The reaganelles . The DNPribed in .p-values < 0.05 were considered statistically significant.The data were analyzed using the GraphPad Prism 7.0 software  and are presented as means ± SD of 4–10 biological replicates (excluding electron microscopy data). We used the Shapiro–Wilk normality test to verify that the data had Gaussian distribution. If the data fit the normal distribution, the statistical significance of the differences between the experimental groups was analyzed with one-way analysis of variance (ANOVA), followed by the Tukey multiple comparison post hoc test. For not normally distributed data, one-way ANOVA using the Kruskal-Wallis test were used. Ppargc1a and enhanced Pink1 gene expression, which may indicate an increase in the intensity of mitochondrial biogenesis and mitophagy, and as a consequence mitochondrial turnover; and (4) uridine restored the mitochondrial oxidative phosphorylation capacity and prevented oxidative damage to the organelles, but did not affect the systems responsible for mitochondrial ion transport in the hearts of mice with DM. As mentioned above, diabetic mice demonstrated a significant decrease in uridine levels in blood plasma, bile, heart, and brown adipose tissue [In the present work, we investigated the effect of long-term administration of uridine (30 mg/kg/day for 3 weeks) on the course of DM and its concomitant mitochondrial dysfunction in heart tissue in the high-fat diet–low-dose streptozotocin-induced diabetic mouse model. The following results were obtained: (1) uridine treatment increased the rate of glucose utilization during the IPGTT in the DM group; (2) uridine normalized the ultrastructure and the number of mitochondria in ventricular cardiomyocytes of diabetic mice; (3) uridine administration to diabetic mice restored the mRNA level of e tissue ; therefo


Snca, Drd2, Rab5a, Anxa2, and Nsf) in the brain tissues and peripheral blood of mice with MPTP -induced models of PD. We detected changes in the expressions of Drd2, Anxa2, and Nsf at the earliest modeling stages. Additionally, we have identified conspicuous changes in the expression level of Anxa2 in the striatum and substantia nigra of mice with MPTP-induced models of PD in its early stages. These data clearly suggest the involvement of protein products in these genes in the earliest stages of the pathogenesis of PD.Processes of intracellular and extracellular transport play one of the most important roles in the functioning of cells. Changes to transport mechanisms in a neuron can lead to the disruption of many cellular processes and even to cell death. It was shown that disruption of the processes of vesicular, axonal, and synaptic transport can lead to a number of diseases of the central nervous system, including Parkinson’s disease (PD). Here, we studied changes in the expression of genes whose protein products are involved in the transport processes ( Among the most important processes in cell function are intracellular and extracellular transport ,2,3. TheOf particular importance is the cell transport in neurons. This is because of the structural features of these cell types, as in addition to the soma, they have very long processes (axons and dendrites) ,5,6,7. TNerve impulse transmission at the synapse is a complex physiological process that proceeds in several stages and includes multiple participants, such as synaptic vesicles, receptors, neurotransmitters, and transporters . VesiculChanges to transport mechanisms in a neuron can lead to the disruption of many cellular processes and even to cell death. Changes or disturbances in synaptic transmission can lead to a slowdown or interruption in the transmission of nerve impulses and to the disorganization of the brain and the organism as a whole ,17.Disruption of the processes of vesicular, axonal, and synaptic transport can lead to a number of diseases of the central nervous system ,26,27,28Previously, we performed a whole-transcriptome analysis of mouse brain tissues with MPTP -induced models of the early stages of PD ,41. It wHere, we present an account of the data obtained as a result of whole-transcriptome analysis and a study of the changes in expression of selected genes in MPTP-induced models of the early stages of PD.We used models of the presymptomatic stage of PD with decapitation of mice 6 h after last injection of MPTP (6 h-PSS), the presymptomatic stage of PD with decapitation of mice 24 h after last injection of MPTP (24 h-PSS), and the advanced presymptomatic (AdvPSS) and early symptomatic (ESS) stages of PD. Models were created as previously described ,43. DetaAll efforts were made to minimize the potential suffering and discomfort of animals and care was taken according to the 3R rule .RNA isolation from tissue samples from each animal as well as analysis of mRNA levels using reverse transcription and real-time PCR  was carried out in accordance with the protocols described previously .Snca, Drd2, Rab5a, Anxa2, and Nsf genes and the reference genes Bcat2 and Psmd7 [Primer and Probe design was produced using Beacon designer 7.0 software  and the nucleotide sequences of the nd Psmd7 ,46,47,48The protocol of statistical and bioinformatic analysis is described in detail in the work carried out in our laboratory earlier .Previously, the whole-transcriptome analysis of the murine striatum and substantia nigra in models of the presymptomatic stage of PD with the decapitation of mice 6 h after the last injection of MPTP (6 h-PSS), the presymptomatic stage of PD with the decapitation of mice 24 h after the last injection of MPTP (24 h-PSS), and the advanced presymptomatic (AdvPSS) and early symptomatic (ESS) stages of PD was carried out in our laboratory. As a result, 170 differentially expressed genes that belonged to the “transport” clusters were identified .® software 12.0  [Snca, Drd2, Rab5a, Anxa2, and Nsf, was built, corresponding to all keywords as closely as possible  ,50. A nepossible .Snca and Drd2 genes in the brain tissues and peripheral blood using the AdvPSS and ESS models of PD [Further, an analysis of the expression levels of selected genes was performed at the early stages of the development of PD in the brain tissues and peripheral blood of mice using MPTP-induced models of the early stages of the disease . Earlierls of PD . This wols of PD  and currDrd2 in the peripheral blood of mice in the 6 h-PSS and 24 h-PSS models of PD were under the detection level of the methods used.The data presented in Fewer genes changed their expression levels in the tissues of animals with the 6 h-PSS and 24 h-PSS models than in those using the models of the more advanced stages of PD (12 versus 19).Anxa2 and Drd2 increased in the substantia nigra while Snca, Rab5a, and Anxa2 transcript levels changed in the peripheral blood.At the earliest stage modeled (6 h-PSS), there were changes in gene expression only in the substantia nigra and peripheral blood, whereas there were increases in the relative mRNA levels for all genes; the relative expression levels of Anxa2 in all studied tissues at 24 h after MPTP administration. Moreover, a large increase in mRNA level—almost 7.5-fold—was observed in the striatum. There was a significant decrease in the relative mRNA level of Drd2 in the substantia nigra and an increase in the striatum by more than 1.5-fold. A change in expression at the mRNA level was also detected for Nsf in the striatum and peripheral blood.There was a change in the relative mRNA level of Anxa2 by 3.5-fold and a 2.5-fold increase for Rab5a in the substantia nigra. Decreased expression levels of Rab5a mRNA were also detected in the striatum and peripheral blood.There were significant changes in gene expression in the AdvPSS model of PD. There was an abrupt decrease in the relative mRNA level of Nsf and Rab5a in the substantia nigra. In addition, there was an abrupt 9-fold decrease for Nsf and an almost 2-fold increase for Anxa2 in the striatum.For the ESS model of PD, we found increases in the relative mRNA levels of Drd2 and Anxa2, with 7 of 12 analyzed samples for each.It is worth noting that the maximum number of significant data points on relative mRNA levels was obtained for SNCA, VPS35, LRRK2, and ATP13A2 serve to maintain normal neuronal transport functions [Proteins encoded by the human genes of the monogenic forms of PD, unctions ,54,55,56Snca, Drd2, Rab5a, Nsf, and Anxa2 in the brain tissues and peripheral blood of mice containing MPTP-induced models of PD.The whole-transcriptome analysis of mouse brain tissues ,41 and sIn the present study, we used MPTP-induced models of the presymptomatic stages of PD, 6 h-PSS and 24 h-PSS, and of the advanced presymptomatic (AdvPSS) and early symptomatic (ESS) stages of PD (with decapitation 2 weeks after MPTP administration) ,43. The 2O2), which is not only cytotoxic but also causes the formation of oxygen radicals [Other types of models have a number of their own features. In addition to MPTP, one of the most common toxins is 6-hydroxydopamine (6-OHDA). The introduction of this toxin promotes the degeneration of catecholamine neurons, including DAergic neurons, since 6-OHDA has a high affinity for DA transporters. The neurotoxic effect of 6-OHDA is mediated by a two-stage mechanism: at the first stage, the toxin accumulates in catecholamic neurons; at the second stage, a change in cellular homeostasis and neuron death occurs. Because of its similarity to endogenous catecholamines, 6-OHDA is taken up by the DAT and NAT transporters. The oxidation of 6-OHDA by monoamine oxidase (MAO-A) promotes the formation of hydrogen peroxide  suggested that changes in the Snca mRNA levels did not occur at the early stages of neurodegeneration. Here, we observed an increase in the relative level of Drd2 mRNA in the striatum and a decrease in the substantia nigra 24 h after the last injection of MPTP. Thus, this gene might be involved in pathogenesis at the very early stages of PD.In our previous study, we obtained data on the expression of S models  . In this case, it is likely that the cells were trying to transport RNA to the neuronal terminal, which is reflected in the striatum in the 24 h-PSS model of PD. This indicates that neurons are trying to compensate for the lack of resources caused by the development of neurodegeneration.To maintain proper synaptic functioning, a neuron needs to concentrate all its resources precisely in the near-synaptic region, including the enhanced expression of proteins involved in vesicular transport. Our data support this assumption . We obseSnca and Drd2 mRNAs was revealed in the substantia nigra and peripheral blood [The study of gene expression in the blood opens up broad prospects in the search for genes that change expression at different stages of the disease, which will make it possible to better understand the mechanisms of PD pathogenesis. Blood cells, in particular lymphocytes, have some characteristics typical of a dopaminergic neuron: they contain both enzymes for dopamine synthesis, a dopamine membrane transporter, and a number of dopamine receptors are expressed. It has been assumed that the changes found in the peripheral blood of patients with PD might reflect the processes occurring in the DAergic neurons of the substantia nigra ,100,101 al blood . Our worSnca, Drd2, Rab5a, Anxa2, and Nsf) in the brain tissues and peripheral blood of mice with MPTP-induced models of PD. We detected changes in the expressions of Drd2, Anxa2, and Nsf at the earliest modeling stages. These findings suggest that changes in transport already occur in the early stages of neurodegeneration.Here, we studied changes in the expression of genes whose protein products are involved in transport processes (Anxa2 in the striatum and substantia nigra of mice with MPTP-induced models of the early stages of PD. These data clearly suggest the involvement of this gene in the compensatory mechanisms in the early stages of the pathogenesis of PD.Moreover, we are the first to report conspicuous changes in the expression levels of


The possibility of controlling the sizes, shapes and aerial density of the nanoparticles by the variation of the deposition and irradiation parameters was systematically investigated. Scanning electron microscopy studies of the samples deposited and irradiated in different conditions allowed for analysis of the morphological features of the nanoparticles and the distribution of their sizes and allowed for determination of the optimal parameters for the formation of the plasmonic-active structures. Additionally, the plasmonic properties of the resulting nanoparticles were characterized by means of linear spectroscopy and surface-enhanced Raman spectroscopy. Hereby, in this work, we demonstrate the possibility of the fabrication of silver nanoparticles with a widely varied range of average sizes and aerial density by means of a post-deposition ion irradiation technique to form nanostructured surfaces which can be applied in sensing technologies and surface-enhanced Raman spectroscopy (SERS).The possibility of controlled scalable nanostructuring of surfaces by the formation of the plasmonic nanoparticles is very important for the development of sensors, solar cells, etc. In this work, the formation of the ensembles of silver nanoparticles on silicon and glass substrates by the magnetron deposition technique and the subsequent low-energy Ar The controllable nanostructuring of surfaces by the formation of the nanoparticles of noble metals such as gold, silver and copper, which support the excitation of localized surface plasmon resonance (LSPR), are of increasing interest for a wide range of applications, such as non-linear optics, electronics, catalysis, sensing, enhanced hot-electron and photocurrent generation ,2,3,4. CTherefore, the methods of the formation of nanostructured surfaces via several-step processes are being extensively analyzed and developed . A numbe+ ions.The aim of the study was to prove that low-energy ion irradiation of Ag films makes it possible to manufacture Ag NPs with controllable properties which can be applied as SERS-active substrates. Additionally, the ion-induced processes were investigated. As point-of-care diagnostics via SERS for biomedical, biochemical and food analysis are widely developed nowadays ,33, it w+ flux adjustment and its effect on the resulting silver nanostructures were studied in [+ beam created by the Hall-effect ion source may be applied to irradiate the Ag nanostructures, allowing for simple and reliable variation of the dose of Ar+ with selected energy. In this work, we discussed the impact of the magnetron deposition time and ion-beam irradiation dose on the morphological properties of the silver nanoparticles, which allowed us to summarize the influence of ion-induced sputtering, surface diffusion and defect-assisted nucleation on the properties of ion-beam-treated Ag nanostructures. We showed that the discussed method ensures the control of the size and surface coverage of Ag NPs, and we anticipate the obtained results to be very promising for their further plasmonic sensing applications.Previously, Ag film treatment by plasma exposure was reported in ,34,35. Audied in , impedin−3 Pa, followed by synthesis in an argon atmosphere at the pressure of 1 × 10−1 Pa. The diameter of the used 99.999% Ag target was 75 mm, while the distance between the substrate and the target was 200 mm. The angle between the flux of the Ag particles and the substrate surface was kept at 45°. During the deposition, the samples were fixed on the substrate holder, located on the carousel of the 20 mm diameter with five positions for substrate holders. Four of them were shuttered from the flux of the particles during the deposition. The active substrate holder was exposed for the particle beam. Prior to the deposition, the surface of the target was decontaminated by 60 s magnetron sputtering at the discharge power of ~30 W, during which the flux of the sputtered particles was aimed at the substrate holder which had no loaded samples. The cover glass Deltalab D101818 and silicon wafer were used as substrates. The structures deposited on glass substrates were used for transmittance measurements, which allowed us to investigate the absorbance spectra. Before applying the coating, the substrate surfaces were etched by the ion beam of 1 keV energy for 5 min. A thin coating was deposited by radio-frequency magnetron sputtering of a silver target. The discharge power was approximately 30 W. Three sets of samples, indicated as “I”, “II” and “III” throughout the manuscript, were deposited within 22, 45 and 67 s, respectively. The thicknesses were 10, 20 and 30 nm, respectively. The thickness of the films was measured by the detector based on a quartz oscillator . The sensor worked in the mass assessment mode. The value of the mass of the deposited material detected after the deposition of each sample was used to estimate the thickness of the films. After the deposition, the argon ion irradiation of 150 eV energy and 20 mA/cm2 current density was applied to the deposited films. The angle between the beam and the substrate surface was 45°. The samples were irradiated at the doses of 2.2 × 1016 ion/cm2, 4.5 × 1016 ion/cm2 and 6.7 × 1016 ion/cm2. The ion beam was produced by the ion source with cold hollow cathode Klan 53-M .The coating process was carried out in a high-vacuum chamber with a preliminary pumping up to 1.3 × 10Samples visualization was implemented by scanning electron microscopy , transmission electron microscopy  and by an optical microscope  with a digital camera DS-Fi3. UV–visible absorption (UV–Vis) spectra of the samples were measured by the UV–Vis–NIR spectrophotometer Agilent Technologies Cary 5000 .Transmission electron microscopy  was used to characterize the nanoparticles. The used electron microscopy grids consisted of copper frames with 40 μm cells and thin (~5 nm) polymer film deposited on the upper side of the frame. To transfer the structure to these grids, the samples were mechanically pressed to the polymer-coated side of the grids and removed after 5–10 s of grid-sample interaction.2, and scanning step was ~0.7 μm (15 × 15 points in map).The chemical state of the samples was controlled by X-ray photoelectron spectroscopy (XPS). The measurements were performed by the Theta Probe  system with a monochromatic Al Ka (1486.6 eV) X-ray source. The photoelectron spectra were acquired in the fixed analyzer transmission mode with the pass energy of 50 eV. The cleaning of the surface by Ar ions was not carried out, since ion etching could cause a change in the chemical state of Ag NPs. The SERS performance of the silver nanoparticles was investigated by the Raman microscope Horiba LabRAM HR Evolution . The measurements were performed with a 632.8 nm laser wavelength, 600 lines/mm diffraction grating, 0.5 sec. integration time, ×100 objective (N.A. = 0.90), and with an incident power ~0.3 mW. The spot size was ~0.43 μm, scanned area was 10 × 10 μm−7 M solution of crystal violet (CV) in distilled water and dried under ambient conditions.Before SERS measuring, the samples were covered with the 10The low-energy ion irradiation of the Ag films leads to their structural rearrangement. The film modification can be controlled by a dose variation, inducing processes such as sputtering and resputtering of the material and the initiation of the surface diffusion of adatoms and clusters .+ ions. The Ar+ energy of 150 eV was chosen according to the studies, indicating that energies exceeding 200 eV can lead to ion-beam mixing, ion incorporation and silver amorphization [The thickness of the deposited silver film is another parameter that determines the structure of the resulting material by affecting the degree of the interaction between ion beam and substrate. Substrate exposure to a low-energetic ion beam initiates multi-step structural rearrangement of irradiated material which involves the accumulation of the defects and subsequent relaxation resulting in the formation of point defects on the surface . The defhization ,42,43,4416 ion/cm2 dose deposited on a Si substrate, was performed  of 0.85 eV, corresponding to the resolution of the XPS set-up. It confirms that the formed NPs consist of silver in a metallic state [2 substrate. The concentration of the detected carbon is low, and its presence apparently corresponds to the minor surface carbon contamination due to the interaction of the samples with the atmosphere.In order to clarify that the used method allows the formation of Ag NPs without significant impurities, XPS analysis of sample II, which was irradiated with a 4.5 × 10erformed a,b. The ic state . On the ic state ,47 were 16 ion/cm2 dose deposited on the Si substrate. This result confirms that ion irradiation leads to the formation of individual NPs from continuous films. As the SERS intensity of continuous films is lower than the one of individual nanoparticles [The TEM images  show thearticles ,48, the articles ,50. HoweIn http://gwyddion.net/, accessed on 25 August 2022). The NP size estimated via this software is a doubled radius of the disc of the projected area similar to the one of the NP . To investigate the evolution of the samples, SEM images were processed with Gwyddion software  to 5.5–6.3 nm for the samples irradiated with the (4.5–6.7) × 1016 ion/cm2 dose are shown by the violet line in Size distributions of the NPs obtained after ion irradiation of sample I  and their approximations with Gaussian lines are shown in 16 ion/cm2 dose is 8.4 nm. The increased ion treatment dose resulted in the decrease in aerial density of the particles and their enlargement. As shown by the green line in 16 ion/cm2-irradiated NP, the average size was 9.6 nm, while for the 6.7 × 1016 ion/cm2 treatment, it reached the value of 10.5 nm.The distributions of the particles observed for irradiated sample II  c and the16 ion/cm2. Such irradiation parameters are favorable for the formation of mostly laced agglomerates, whereas a subsequent dose increase to 6.7 × 1016 ion/cm2 leads to the separation of the particles. We should note that morphologically, set III features irregular shapes, so the depicted “size” can poorly reflect the real width, extension, or any linear dimension of the branched particle. The Gaussian fittings , the resulting agglomerates were, apparently, not entirely separated from the continuous Ag layer by ion irradiation. Therefore, the morphological features of this sample are hereinafter referred to as “convexes”. The ion irradiation of sample III resulted in the surface development and the formation of these agglomerates consisting of convexes interconnected with narrow “bridges”. The size distributions e in thisfittings e of the The differences in the distribution of the Ag NPs formed from the films with various thicknesses can be explained by various manifestations of the irradiation-induced processes, such as the sputtering and the resputtering of the material; the enhancement of the mobility of the resputtered adatoms and nuclei; and the defect-induced nucleation of the resputtered material. As the initial thickness of sample I (~10 nm) is low, the morphological features of set I may be related to the prominent ion-substrate interaction. The high aerial density of the sub-7 nm particles in this case is explained by the set of interconnected processes: first, during the ion-beam treatment, the surface of the substrate was exposed and irradiated, resulting in the formation of the defects on it. Second, the defects formed on the substrate surface act as favorable nucleation centers for the surface-diffusing silver adatoms and clusters formed during ion irradiation . In the For set II, the ion-induced defect formation on the substrate surface for thicker films is suppressed. In this case, the number of nucleation centers on the substrate is decreased, and ion irradiation predominantly affects nanoparticles by enhancing their surface mobility, thereby leading to the enlargement of the particles and a decrease in their numbers. The overall aerial density of the particles for samples I and II irradiated with various doses is shown in For sample III, the results showing the enlargement of the particles and branching of the structure are in correspondence with  findingsThe variation in the average feature size with the irradiation dose a confirmcR − oR)/oR, where cR and oR are the radii of the circumscribed and inscribed circles [To investigate the morphological changes of the sets, the shape parameter of the particles was assessed as an  × 1016 ion/cm2 range. The elongation and the branching of the particles are interconnected in this case, as both of the processes are caused by the formation of the narrow “bridges” between the individual particles resulting in the formation of the “wormhole” network rather than the enlargement of the nanoparticles. The initial increase in the shape parameter of set III with the dose enlargement is notably different from the decreasing tendency observed for samples I and II. As set III is apparently formed by the convexes on the continuous film rather than by the NPs, the ion-induced surface diffusion of the resputtered material is more prominent for this set. Intensive diffusion leads to the condensation of the “bridges”, while the “erosion” of the material results in the decrease of its overall thickness rather than in the shrinking of the NPs. Apparently, both of the decreasing trends observed in The averaged shape parameter of sample III irradiated with various doses , as the structure in the vicinity of the enhancing agent will remain shadowed from the ion beam.To discuss the possibility of the formation of the plasmonic-active media on the surface of various samples, the variability of the Ag NPs formation on different substrates should be addressed. We haven’t observed any influence of the surface morphology on the Ag NPs distribution, as the ion irradiation of ~200 eV at similar ion currents is known to create ~50–100 nm-sized ripples on the surface , leading2.Firstly, the processes of the formation of ion-induced defects and surface diffusion may occur differently on various substrates. The materials with ionic bonding are more likely to undergo modification under ion irradiation than the materials with covalent bonding, as the preferential sputtering leads to the removal of the anions from the subsurface layers, resulting in the pronounced formation of point defects . Therefo2) [2) [Secondly, the wettability between the Ag NP and the substrate affects the shape of the condensing particles . General2)  is signi2) [2) ,61. As a2) [2) .Something this is noteworthy is that ion assistance may introduce some additional effects into the nucleation process. Ion-induced surface development may change the contact surface, thus reducing the critical nuclei size , while p2 it is 0.32 eV [Thirdly, the substrate type may also affect the mobility of the adatoms. As the variation of the substrate material changes the activation energies of diffusion and desorption ,66, the  0.32 eV . TherefoTo sum up, the difference in the NP particle size distribution on different substrates may in general be caused by the variation of the morphology of substrates, the variability of the processes of condensation, surface diffusion and wetting. However, we do not expect a significant difference in the processes of condensation, surface diffusion of resputtered material as well in the wetting. As the wettability of Si and glass to Ag is poor, we expect a spheroidal or ellipsoidal shape of Ag NPs in the studied case.16 ion/cm2 for subsequent SERS-related studies discussed in The important practical aspect of the presented analysis of the Ag NPs’ distributions is the demonstration of various exposure of the coated substrates to ion irradiation. The size distribution in the NPs of set II shows that the ion-induced defect formation is less prominent than the one in set I. It allows us to suggest that for set II, the processes related to the interaction between the substrate and ion beam are suppressed by the thicker Ag layer. This is confirmed by the particle increase and the distribution widening tendency with irradiation dose increase, which may be adequately explained by the ion and silver interaction summarized in 16 dose are shown in The selected sample was also characterized by linear and Raman spectroscopy. The experimental extinction spectrum of Ag nanoparticles on the glass substrate recorded for sample II and the 4.5 × 10−7 M concentration are shown in To estimate the applicability of the fabricated samples for SERS, the studies of Raman spectroscopy were carried out. The spectra of the crystal violet solution of the 105 for the ensembles of NPs. Notably, the used Raman excitation wavelength of 632 nm is relatively far from the observed SPR peak of 418 nm, which demonstrates the versatility of the properties of the samples. We suggest that the use of the 488 nm excitation wavelength can enhance several times the EF of the studied nanoparticles [3 typical for the Ag nanoparticles unable to form the hotspots [The optical images and SERS mappings  is suppressed, compared with set I . The listed factors allowed us to choose the irradiated 20 nm-thick Ag film for the SERS studies. The formed Ag NPs manufactured from the film of 20 nm in thickness with the 4.5 × 10


The objective of the study was to develop a new method for the determination of the total content of vitamin C and dehydroascorbic acid in food, based on the technique of differential pulse voltammetry with the use of a boron-doped diamond electrode modified with mercury film. A comparison was made between the results obtained with the developed method and a proposed reference method based on high-performance liquid chromatography with spectrophotometric detection. The reduction of dehydroascorbic acid was performed with the use of tris(2-carboxyethyl)phosphine. The interference caused by the presence of tris(2-carboxyethyl)phosphine during the voltammetric determination of ascorbic acid was effectively eliminated through a reaction with N-ethylmaleimide. The conducted validation of the voltammetric method indicated that correct results of analysis of the total content of vitamin C and ascorbic acid were obtained. Analysis of the content of dehydroascorbic acid was imprecise due to the application of the differential method. The results of the analyses and the determined validation parameters of the developed method are characterised by a high degree of conformance with the results obtained with the chromatographic reference method, which indicates the equivalence of the two methods. The name vitamin C covers ascorbic acid (AA) and dehydroascorbic acid (DHAA), as both forms display high biological activity. Dehydroascorbic acid displays the properties of vitamin C, as it is easily reduced in the organism to ascorbic acid. One of the most important biological functions of ascorbic acid, resulting from its reducing properties, is its capacity for stopping the radical chain reaction . In addiIn food analysis, the correct method for the determination of vitamin C should measure the combined content of ascorbic acid and dehydroascorbic acid to determine the total content of vitamin C. There are a large number of methods permitting direct analysis of ascorbic acid based on its physicochemical properties, as opposed to dehydroascorbic acid ,9,10. FoHigh-performance liquid chromatography (HPLC) is undoubtedly the most extensively used technique for determining vitamin C in food products. The advantages of this method include high specificity, sensitivity, and relative ease of performing the analysis. Ascorbic acid is electrochemically active, which allows for the development of numerous analytical procedures, making use of polarographic and voltammetric techniques. Procedures involving the use of the dropping mercury electrode are less frequently employed, giving ground to the voltammetric technique, which makes use of electrodes made of various materials. In many cases, the surface of the electrodes is modified to improve their analytical properties ,14,15,16Tris(2-carboxyethyl)phosphine (TCEP) is a compound with an application similar to that of dithiothreitol, which can be considered as an alternative. As opposed to dithiothreitol, it reacts within a broad range of pH values and does not undergo oxidation by atmospheric oxygen, and does not have the unpleasant smell characteristic of thiols . Trisphosphine (TCEP) which, at present, is the most frequently used reagent capable of DHAA reduction in extraction environments with pH < 2. A comparison was made between the validation parameters of the developed voltammetric method and a reference method based on the use of high-performance liquid chromatography with diode-array detection (HPLC-DAD).At the initial stage of the research, a voltammetric analysis of ascorbic acid in food samples  was carried out on a non-modified boron-doped diamond electrode, using the parameters described in the publication by Sadok et al. . InterfeAt the next stage of the study, it was tested whether tris(2-carboxyethyl)phosphine does not have its voltammetric peak within the range of potentials used in the analysis of ascorbic acid, or does not cause distortion of the AA peak. The voltammogram of tris(2-carboxyethyl)phosphine solution with a concentration of 10 mM presented in Research results presented in the literature indicate a possibility of effective reduction of dehydroascorbic acid with the use of tris(2-carboxyethyl)phosphine in the environment of metaphosphoric acid with pH 1.9, as well as hydrochloric acid with pH ≈ 1 . To veria (4.751 nA) and the residual standard deviation sxy (11.328 nA):The limit of detection (LOD) was determined based on the parameters of the standard curve obtained on the basis of analysis of standard solutions prepared within the concentration range from 4.95 to 44.55 µg/mL. The limit of detection was calculated on the basis of the value of the standard deviation of the free term sThe inclusion of the residual standard deviation resulted in a higher value of the limit of detection as, in this case, one should also take into account the variation of the slope of the calibration curve. The mean value was adopted as the value of the limit of detection.The correctness of the determination of the limit of detection was verified . The lowThe limit of quantification (LOQ) was determined on the basis of the formula:HPLC-DAD = 0.82 µg/mL, LOQHPLC-DAD = 2.46 µg/mL [The values of the limits of detection and quantification of the voltammetric method and the chromatographic reference method—LOD46 µg/mL —are simiThe results of the analysis of food products with the differential pulse voltammetric (DPV) method with the use of tris(2-carboxyethyl)phosphine are presented in Analysis of the total content of vitamin C in a sample of the certified reference material BCR 431 and comparison of the obtained average value with the certified value allowed the determination of the correctness of the results obtained. t-test indicates an absence of statistically significant differences in the results obtained in the case of all samples, except for the analysis of the total content of vitamin C in tomato and of the content of dehydroascorbic acid in cauliflower.t-test indicate an absence of statistically significant differences between the obtained mean value of recovery and the value of 100%.The results of the analysis of the recovery of ascorbic acid in the voltammetric method of vitamin C determination with the use of tris(2-carboxyethyl)phosphine are presented in The following materials were used: juices from local producers , fruits and vegetables , fruit cream in powder, infant milk powder, and multivitamin syrup. All samples were purchased from local supermarkets in Lublin, Poland. The certified reference material BCR-431 (Brussels sprout) was purchased from the Institute of Reference Materials and Measurements (IRMM), European Commission Joint Research Centre .2+ = 1 g/L) were purchased from the company Sigma-Aldrich , metaphosphoric acid was purchased from Merck , and sodium acetate and glacial acetic acid were purchased from POCH S.A . All reagents were of analytical grade purity.Ascorbic acid, dehydroascorbic acid, N-ethylmaleimide, tris(2-carboxyethyl)phosphine hydrochloride, and mercury standard stock solution (Hgsm). Following that, 2% solution of metaphosphoric acid was added and the sample was weighed again (s+am). The dilution coefficient (F) was calculated and subsequently used to quantify the analyte in the sample.Each sample was weighed to the nearest 1 mg  and the other was used for the determination of the total content of vitamin C (TC) after the reduction of DHAA.The sample was homogenised for 1 min using an Ultra Turrax  homogeniser and then centrifuged for 5 min at 12,000× During all stages of analysis, the temperature in the laboratory was between 23 and 25 °C. All analyses were repeated three times, and the results are expressed as mg of analyte/100 g fresh mass in the case of fruits and vegetables and mg of analyte/100 g of product for the remaining samples.The amount of 0.2 mL 100 mM of tris(2-carboxyethyl)phosphine was added to 1.8 mL of the extract and then the sample was shaken for 30 min on a mechanical shaker. In the case of preparation of a sample for the voltammetric analysis, 2 mL of 80 mM solution of N-ethylmaleimide was added and shaken for 30 min.The analysis of ascorbic acid was performed using the reversed-phase high-performance liquid chromatography in conformance with the method described by Mazurek and Jamroz . A diode2. The electrode was then transferred into the plating solution containing 20 mg/L Hg(II) and the Hg film was formed by holding the working electrode potential at −1.30 V for 180 s. To minimise the effect of substances present in the matrix of the analysed samples on the electrochemical properties of the electrode, a fresh coat of mercury was applied before the analysis of every sample.Before measurement, the boron-doped diamond electrode  of 3 mm in diameter was thoroughly polished using a 0.3 μm alumina slurry on a Buehler polishing pad, rinsed with ethanol and water in an ultrasonic bath, and dried with NVitamin C determinations were performed by the method of differential pulse voltammetry, using a voltammetric trace analyser Metrohm 797 VA . A three-electrode system was used—a boron-doped diamond electrode modified by the mercury film as the working electrode and a platinum electrode as the auxiliary electrode. The potential of the working electrode was measured in relation to an Ag/AgCl/KCl reference electrode. A quantitative assay was performed with the method of triple standard addition using an automatic dosing unit Dosino 700 . Qualitative determination of ascorbic acid was performed based on the comparison of the potential of its peak  with the potential of peaks obtained after adding the standard solution three times to the sample. Ten mL of acetate buffer with pH 4.6 and 0.5 mL of an extract of the analysed sample were added directly to the voltammetric vessel. Before voltammetric measurement, the solution was flushed with argon for 5 min to remove oxygen. The following conditions were applied during the differential pulse voltammetric analysis: initial potential −50 mV, final potential 200 mV, voltage step time 0.6 s, voltage step 6 mV, pulse duration 40 ms, pulse amplitude 50 mV, the rate of potential change 10 mV/s.s Equation (2)), then quantitative reduction of dehydroascorbic acid was carried out using tris(2-carboxyethyl)phosphine, and total vitamin C content (TC) was calculated based on Equation (3). The content of dehydroascorbic acid (DHAAs) was calculated by subtracting the initial ascorbic acid content from the total amount of vitamin C (subtraction method).AAC—concentration of ascorbic acid in extract (µg/mL)TAC—concentration of ascorbic acid after the reduction step (µg/mL)The first step consisted of determining ascorbic acid content in the sample phosphine as a reducing reagent. The determined validation parameters of the voltammetric method indicate that the method provides correct results of analysis of the total content of vitamin C and ascorbic acid. Analysis of the content of dehydroascorbic acid is imprecise due to the employment of the subtraction method . The val