Background and Objectives: One of the most difficult aspects of hip revision is to remove the stem from the femoral canal with or without cement while maintaining the maximal amount of bone stock to obtain the best possible press-fit of the revision prosthesis. The transfemoral approach ensures direct access to the medullary canal so that the content removal is completed under direct control, while protecting the bone. This type of approach is particularly efficient for special conditions, such as deformation of the proximal femoral region, broken stems, or the presence of cement over a long distance distal to the prosthesis. The aim of this study was to evaluate the main advantages of transtrochanteric approach in hip revisions. Materials and Methods: Our series included 36 revisions performed using the transfemoral approach. We have analyzed the following postoperative radiological aspects: the length of the fixation zone distal to the osteotomized bone fragment (OBF), the gap between the OBF and the diaphysis, stem subsidence over time, and OBF consolidation. Results: The results were very good, both in terms of the rate of intraoperative complications and postoperative evolution. The fixation zone length was 4.2 cm on average (range: 2 to 5.8 cm). The distal gap between the OBF and the diaphysis was 1.2 cm on average (range: 0 to 2.3 cm). Stem subsidence was noted in four cases (11.1%). In all cases, stem subsidence occurred between three and six months and was 6 to 8 mm without affecting hip stability. OBF consolidation was radiologically confirmed for all cases at one year follow-up. Clinical assessment based on the Harris Hip Score showed an overall improvement from 43.2 preoperatively to 79.7 at 12 months and 83 at two years, respectively. The most important rate of progress was between 6 months and 12 months. Conclusions: The transtrochanteric approach has been shown to be very efficient for hip revisions. Understanding the hip biomechanics, applying a less aggressive surgical technique, and using efficient fixation methods such as cables significantly improved the results. In recent decades, hip arthroplasty has been one of the most successful orthopedic procedures and is practiced to an increasing degree. The revisions have multiplied significantly, as well as the degree of complexity, particularly their difficulty. An essential aspect of revision is the removal of the femoral component, with or without cement, which depends on the type of prosthesis and requires preservation of the bone stock. In some cases, this is difficult to achieve with an endofemoral approach, leading to either fractures or the sacrifice of a significant amount of bone, which has an unfavorable effect on the fixation of the revision stem. In septic conditions, the complete removal of any potentially contaminated material, such as cement and distal bone plug, increases the chances of infection healing. Other conditions, such as periprosthetic fracture, broken stems, and varus remodeling of the femur, are good indications for a transfemoral approach. This approach was popularized in the 1960s by Sir John Charnley, who used it for both primary arthroplasties and hip revisions, developing Ollier’s ideas from the previous century (1881). In 1999, Wagner proposed an extended lateral femoral osteotomy, using a transfemoral approach and osteotomizing half of the proximal femur. This method provided excellent results in cases of prosthetic revision ,3. The mThe aim of this study was to evaluate the main advantages of a transtrochanteric approach in hip revisions.This is a prospective study reviewing 36 stem revisions that used the transfemoral approach and were performed between 2017 and 2019. The minimum follow-up was 24 months. Twenty-seven cases were total revision, and nine cases were stem revisions only. Twenty-two cases were cemented stems, and 14 were cementless. The initial diagnoses for hip replacement procedures were the following: hip arthritis (23 cases), rheumatoid arthritis (five cases), aseptic necrosis of the femoral head (five cases), and femoral neck fracture (three cases). Twenty-two patients were women, and 14 were men. The average age was 70.4 years (range 54–82 years). The BMI range was between 25.5 and 33.2. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Emergency Hospital of Saint Pantelimon in Bucharest.The indications for stem revision were the following: (a) 17 cases were stem loosening (47.2%), with varus remodeling of the proximal femur in four cases and a long cement plug distally to the stem tip in five cases; (b) six cases were periprosthetic joint infections, with a 22-month interval after the index procedure on average; one case was an early infection after revision (16.6%). For these cases, both the initial procedure  and revision were performed using a transfemoral approach; (c) four cases were Vancouver B2 periprosthetic fractures (11.2%); (d) seven cases were well-fixed stems associated with acetabular loosening, in which the revision was performed at both levels (19.4%); (e) two cases were broken stems (5.6%).The cases were classified according to the Paprosky system for classifying femoral bone defects: type 1—two cases (5.5%); type 2—11 cases (30.5%); type 3A—15 cases (41.7%); type 3B—six cases (16.6%); type 4—two cases (5.5%). ® titanium alloy. The proximal  component was cylindrical or conical with dorsal and ventral ribs; the cylindrical component showed less prominent ribs in the sagittal plane, a slim design that prevented proximal jamming. The type was chosen depending on the aspect of the femoral metaphysis . Thirty-two distal components were short (140 and 200 mm), with press-fit fixation at the level of the femoral isthmus, and four were long (260 mm), distally locked stems.The prosthesis used was a modular curved conical cementless revision stem, Revitan . The distal  component was 2° tapered with longitudinal ribs, and a cover of ProtasulThe length of the osteotomized bone fragment (OBF) is essential; it must be long enough to allow the easy removal of the stem with or without cement but without affecting the primary stability of the revision stem. Accurate preoperative planning is very important for this reason. On the right magnification A-P and profile hip X-rays, we measured the length of the stem and the length of the distal cement plug including the length of the plastic restrictor, taking the tip of the greater trochanter as a reference point. For cementless stems, it is not necessary for the OBF to reach the level of the stem tip, only up to approximately 2 cm, whenever it is preoperatively intact . MaintaiThe posterior approach was used in all the cases. The vastus lateralis and its insertion on the OBF must be protected as much as possible while the vascular branches coming from the muscle are decisive in ensuring osteotomy consolidation . At the Removing the prosthesis and the cement from the medullary canal is usually easy; however, difficulties can be associated with removing a cementless long stem prosthesis, which is integrated distally to the OBF ,9. In adAdapting the shape of the trochanteric region of the OBF to the lateral profile of the prosthesis had to be performed very carefully using thorough debridement. The OBF was fixed using two or three Dall–Miles cables, adequately tensioned, depending on the length. No difference was found between two- and three-cable fixation in terms of the peak force, stiffness of the construction, or angular or axial displacement of the osteotomy ,11. ReceFixation of periprosthetic fractures was performed in all four cases in our series using Dall–Miles plates with distal screws and proximal cables after exchanging the primary prosthesis with a long revision stem which bypassed the fracture. This internal fixation method for periprosthetic fractures has been shown to be efficient in many biomechanical studies .The surgical time was 210 min on average with a range between 150 and 320 min. The average perioperative blood loss (intraoperative and first 24 h) was 450 mL (200–1250 mL), mainly occurring intraoperatively. The amount of blood loss decreased significantly with the use of IV tranexamic acid on a regular basis.The intraoperative complications were the following: five fractures of the OBF (13.8%) that did not require an additional fixation apart from three Dall–Miles cables, usually used for OBF fixation; three fractures of the trochanter (8.3%) that required fixation with wire (two cases); a trochanter plate (one case); and one undisplaced fracture of the femoral shaft, distal to the OBF (2.8%) fixed with another Dall–Miles cable. The most common intraoperative complication, OBF fracture, occurred in most cases due to mishandling the fragment during surgery and, much less frequently, during osteotomy. The general percentage of femur fractures was similar to the incidence reported in previous studies in the literature ,17.The X-ray postoperative protocol followed four aspects: length of the fixation (anchorage) zone, distal to the OBF; the gap between the osteotomy and the diaphysis; stem subsidence over time; and OBF consolidation.Theoretically, primary stability by press-fit in the diaphysis must be achieved on a 5 cm length. When a good press-fit is hard to obtain, especially in Paprosky type 4 bone defects, the fixation of the stem must be supplemented by distal locking with two or three screws. Among the 36 cases of our study, we used distal locking in four cases (two for type 3B and two for type 4). For the other 32 cases, the fixation zone was 4.2 cm on average (ranging between 2 and 5.8 cm).The distal gap between the OBF and the diaphysis was 1.2 cm on average (range: 0 to 2.3 cm). The distance had not increased in any of the cases at subsequent radiological follows-up, which demonstrated the solidity of the fixation of the OBF with cables. At one year, this space disappeared in all cases with an initial length of less than 1.5 cm (20 cases). For the gaps larger than 1.5 cm, complete filling was noted in 10 out of 16 cases. In the other six cases, partial filling was obtained but without any functional effect.Stem subsidence was noted in four cases (11.1%). In all cases, stem subsidence occurred between three and six months. The defect type was Paprosky 2 B in two cases, 3A in one case, and 3B in one case. For cases with a 2B defect, the fixation area was 4.8, respectively, 5.2 cm in length. However, the stem diameter was too small, which caused a subsidence of 8, respectively, 6 mm. For both the 3A and 3B defect type cases, the subsidence was 8 mm. Subsidence did not lead to the dislocation of the prosthesis in either of the cases. For the remaining cases, subsidence was not noted, independently to the length of the fixation zone or the type of bone defect, even for the case with an undisplaced diaphyseal fracture fixed with a cable.OBF consolidation was radiologically confirmed for all cases at one-year follow-up, despite delayed consolidation noted at six months after surgery for nine cases.The clinical evolution was determined using the Harris hip score. All the statistical analysis was performed in Python version 3.7.12 using the libraries NumPy (version 1.19.5) and Matplotlib (version 3.2.2).As expected, regardless of the type of bone defect, the Harris score showed continuous improvement in successive examinations at 3, 6, 12, and 24 months. Younger patients had the best preoperative scores, regardless of the type of defect, respectively, the cases with a lower degree of bone defects. The same cases showed a faster improvement of the Harris score. The lowest preoperative score and the lowest rate of improvement in the Harris score was observed in elderly patients and patients with major bone defects (3 and 4 Paprosky). In these cases, there were no differences between the cases in which the fixation was press-fit and cases where distal locking was used .p < 0.005).The statistical comparison of the Harrison Hip Score between the preoperative period and all follow-up periods showed statistically significant differences , confirmed during the second-stage procedure ,24, and The presence of remnant cement fragments can deflect the tools used for preparing the medullary canal  or even of the revision stems, leading to perforations or fractures of the diaphyseal cortex.Another issue related to the presence of cement inside the medullary canal is the integration of the cementless revision stem by limiting the contact surface with the bone as well as under-sizing the stem. Thoroughly removing the cement also improves the bone ingrowth of the revision stem. Bone ingrowth is also promoted by the creation of the OBF itself; it has the same healing mechanism as a fracture since it is actually a controlled fracture. The reduction of this controlled fracture of the stem and the close apposition of the bone fragments induces much wider contact between the bone and the prosthesis than in an endofemoral approach, where three points of contact are used .In 30% of stem loosening cases, varus remodeling of the metaphyseal–diaphyseal area of the femur has been observed . In thesTypically, the femoral OBF can be reduced so that the gap between the OBF and the diaphysis is minimal. However, this kind of reduction cannot always be achieved, maintaining a significant distance which fills with bone in the healing process. From this practical aspect, the proximal or distal displacement of the OBF to correct the tension in the abductors is quite easy to achieve, and the results are very good .The displacement of the femoral OBF provides excellent visualization of the acetabulum and the posterior column, which is beneficial for reconstructing it. Good visualization is also useful in cases where the removal of the acetabular component is difficult either due to intrapelvic displacement or when it is not possible to disengage the prosthetic components, such as in hemiarthroplasties. In these situations, attempting to dislocate the prosthesis can cause fractures of either the acetabulum or the femur. The transtrochanteric approach ensures easy removal of the acetabular component without creating additional tension in the bone ,28,29.An important indication of the transtrochanteric approach is periprosthetic fractures. Vancouver type B2 and B3 fractures, in which the fracture is near or just distal to the loose stem (B2) or loosely associated with a poor bone stock (B3), are suitable for this kind of approach. Protection of the bone is the most important aspect in stem removal, especially for B3 type fractures when the bone is very thin and fragile . In our The non-union rate of the osteotomy is very low (0–4.4%) in the last period of time ,35. DataThe transtrochanteric approach has been shown to be very efficient for hip revisions. Understanding the hip biomechanics, applying a less aggressive surgical technique, and using efficient fixation methods such as cables significantly improved the results. Despite seeming to be a more aggressive procedure, the transfemoral approach is a relatively easy procedure that makes it possible to remove the prosthesis quickly and clean the medullary canal. The newly formed bone forms within several months after surgery.The main advantage of this method is the ease of stem removal, both of a cemented prosthesis but also a well-integrated cementless prosthesis especially. Cement removal is also much easier than in an endofemoral approach, thus reducing both the operating time and possible complications such as perforation or fracture of the femur; the complete removal of the cement is mandatory for septic revisions. Periprosthetic fractures, especially Vancouver types B2 and B3, can be effectively treated using this type of approach. The visualization of the acetabulum and the correct tension of the abductors by repositioning the trochanter are other advantages of this method.The excellent results obtained in terms of the reduction in the operative time, lower rate of complications, and postoperative functional recovery make this method the method of choice for us in most hip revisions.


Inorder to fully characterize the obtained molecules, the comprehensive computational chemistry studywas performed. The influence of the intermolecular interactions for their absorption in solution wasanalyzed. The theoretical data entirely support the experimental outcomes.The present study is devoted to the combined experimental and theoretical descriptionof the photophysical properties and photodegradation of the new boron-dipyrromethene (BODIPY)derivatives obtained recently for biomedical applications, such as bacteria photoinactivation. Absorption and emission spectra for a widegroup of solvents of different properties for the analyzed BODIPY derivatives were investigatedin order to verify their suitability for photopharmacological applications. Additionally, the photostabilityof the analyzed systems were thoroughly determined. The exposition to the UV light wasfound first to cause the decrease in the most intensive absorption band and the appearance of thehypsochromically shifted band of similar intensity. On the basis of the chromatographic and computationalstudy, this effect was assigned to the detachment of the iodine atoms from the BODIPY core.After longer exposition to UV light, photodegradation occurred, leading to the disappearance of theintensive absorption bands and the emergence of small intensity signals in the strongly blue-shiftedrange of the spectrum. Since the most intensive bands in original dyes are ascribed to the molecularcore bearing the BF Among td, etc.) ,3 or novPhotodynamic therapy has gained large interest, due to its weak side effects, as an alternative to damaging surgical, chemo- and radiotherapeutic treatment ,5,6. It The commonly applied PDT photosensitizers include porphyrins, chlorins and phthalocyanines, among others; however, their side effects, such as low bioavailability, susceptibility for aggregation, which satisfies the desired photophysical properties, or decomposition under light or oxygen presence induce further needs for improvements ,5. An efPresently, there is a wide variety of fluorescent and luminescent materials available, such as organic compounds, molecular complexes, biomacromolecules (such as proteins), and nanoparticles . An exammeso-position together with the presence of singlet oxygen quenchers has been shown to diminish the photodegradation rate.One of the important features, which need to be exhibited by efficient photosensitizers, is high photostability under UV and Vis radiation, together with easy photoactivation within the tissue. Therefore, it is crucial to determine the behavior of the photosensitizer under light, together with its careful absorption and emission characteristics—the determination of the photodegradation products is a required step in the conventional drug pre-formulation process. Despite the big interest in highly-fluorescent photostable BODIPY derivatives for biomedical applications, still only a few studies on the mechanisms of their photodegradation are available . Most ofAccording to our knowledge, the photodegradation phenomena of BODIPY dyes has been scarcely investigated so far with computational chemistry tools ,30. In m1 is unsubstituted, while system 2 contains iodine heavy atoms, incorporated in the structure to increase the intersystem crossing to the triplet state. This substitution is expected to quench the fluorescence quantum yield of the system and simultaneously increase the generation of singlet oxygen reactive species. Due to the synergistic action of both the BODIPY core, exhibiting green light absorption [The two systems investigated in the present study were synthesized previously for possible biomedical applications for bacteria inactivation . They disorption ,42,43, asorption  with the1 and 2 were purified, using flash chromatography. It was carried out using a chromatographic system with a mass detector. Furthermore, the identity of the obtained products was confirmed, using NMR, UV–Vis spectroscopy and mass spectrometry. Organic solvents of high purity were obtained from Sigma-Aldrich and Avantor Performance Materials, Poland.Two investigated BODIPY systems are presented in 1 and 2 and a reference ΦF=ΦF1 and 2 was investigated at an ambient temperature, using MeOH as the solvent, in accordance with the previously described procedure [2, 55 W/m2 and 18.7 W/m2 for UVA, UVB and UVC, respectively. Radiation intensity was measured with a radiometer HD 9021  at the sample position, i.e., at 10 cm distance from the radiation source. The products of the photochemical reaction were identified, using the Shimadzu Prominence HPLC system . The instrument includes a binary solvent delivery system (LC-20 AD), an autosampler (SIL-20A), and a column thermostat (CTO-10AS VP), absorbance spectrophotometric diode array UV–Vis detector (SPD-M20A) and a data acquisition station. Separation was performed in gradient elution from 70% to 100% of methanol in water over 10 min on the Kinetex Phenyl-Hexyl  2.1 × 100 mm chromatographic column with particle size 1.7 μm. The flow rate of the mobile phase was 0.25 mL/min, the injection volume was 10 μL, and the separation temperature was 35 °C. The investigated sample of 1 was separated with the HP-LC method, and for the photodegradation products, the UV spectrum was simultaneously registered. During the investigation, samples were collected after every 15 min of irradiation, starting with 5 min, then 20 min, and so on. The composition of the irradiated mixture was analyzed with the use of a DAD detector recording the change in the absorption of products leaving the chromatographic column. The wavelength range selected for the detector was 200–600 nm.Photochemical stability under the UV–Vis irradiation of rocedure . A high-meso-position and denoted as 3 and 4 for the pristine and iodinated system, respectively, were also included into the present considerations in order to improve the method calibration and simplify the computational part relying on the conformational flexibility of the analyzed dyes. For all of the four investigated systems, the absorption spectrum in vacuum and in the selected solvents was calculated, using the vertical approach in linear response formalism. The solvent model was applied in the polarizable continuum version as implemented in Gaussian16 [Conformational analysis for the investigated BODIPY derivatives, presented in ussian16 . Due to ussian16 ,49,50. Sendence. ,52,53,54For the detailed description of both the intramolecular and intermolecular non-covalent interactions (NCI), the NCIPlot program was applied ,56 for tAccording to the experimental measurements for the BODIPY derivatives after UV irradiation, five photodegradation mechanisms are proposed on the basis of the earlier literature reports. The computational chemistry tools are applied in order to verify the possible decomposition ways by comparison of the experimental shape of the spectra before and after irradiation with the computational absorption spectra for the hypothetical photodegradation products.1 and 2, presented in 1 and 2, due to the evident stacking interaction between the BODIPY core and the phthalimide substituent, independently, on the iodine presence. The visualization of these effects is presented in meso-position is aligned almost perpendicularly to the BODIPY core. This is expected to significantly affect the extent of the Among the stable conformers of 1 and 2. The increase in the basis set to the def2-TZVP one causes the noticeable decrease in the relative energy to 5.42 and 5.83 kcal/mol for 1 and 2, respectively. The substitution of 1 by the iodine in the 2 and 6 positions, leading to the 2 structure, does not significantly modify the relative energy between the most stable bent and elongated (unfolded) form. These energies were compared to the phthalimide-BODIPY core interaction energy, calculated for the optimized complexes in the supermolecular manner with the B3LYP-D3/def2-SVP approach . For the unsubstituted core 3 , tetrahydrofuran (THF), dichloromethane (DCM), methanol (MeOH), acetonitrile (MeCN) and dimethyl sulfoxide (DMSO) in order to confirm the stability of the investigated systems in various environments. Due to the similar shape of the spectrum in all the solvents, only the spectra recorded in acetonitrile are shown in The electronic absorption spectra of BODIPY derivatives 1. The spectrum of the iodinated derivative 2 exhibited an intensive band in the region of about 500–550 nm and a weak, flat band between 350 and 430 nm. The long-wavelength bands, around 450–550 nm, can be attributed to the n orbitals of oxygen atom in the ether moiety. The molecular orbitals involved in the above transitions  and stronger for the 413 nm transition in 2 , arising from the shift of the electron density from the phenyl ring to the BODIPY core upon excitation. Moreover, one should notice the mild contribution of the iodine atoms to these singlet-singlet transitions in 2, which is of importance in light of the photodegradation mechanisms discussed in the further part of this manuscript.Two absorption bands were found for each compound. The sharp, intensive bands located in the range 470–520 nm and the much weaker, broad and flat bands in the range of 320–400 nm were observed for 1 exhibits a maximum between 496 and 502 nm, depending on the solvent used. On the other hand, a broader, bathochromically shifted band with the maximum wavelength in the range of 528–536 nm is observed for 2. The red-shift of the absorption band of 2 in comparison to 1 of about 30 nm can be explained by the heavy-atom effect of iodine substituted to the BODIPY core. The solvatochromic effect of the investigated dyes is tiny in size and the largest difference between the maximum of absorption in toluene and acetonitrile does not exceed 7 and 6 nm for 1 and 2, respectively. This small bathochromic shift between the maximum absorption in different solvents is also in line with the general behavior of BODIPYs [1 and 2, recorded in different solvents, results mainly from solvation phenomena.The insertion of the halogen atom at 2 and 6 positions in the core introduces the gentle absorption spectrum modification. The long-wavelength, sharp band of the absorption spectrum of  BODIPYs ,59,60. H1 and 2 remains red-shifted by about 6 to 7 nm with respect to the unfolded, elongated conformer and the selected conformation affects neither the qualitative discussion of the obtained data nor the comparison between the theory and experiment. The frontier orbitals involved in the most intensive transitions are presented in The theoretical TD-DFT calculations for BODIPY dyes are known to suffer from the deficiencies in the description of the multiconfigurational systems and provide systematically underestimated absorption wavelengths ,38,40,611 with three molecules of toluene, methanol and acetonitrile are presented in 1 and −12.39 to −9.50 kcal/mol for 2, depending on the arrangement of the aromatic rings). Aromatic rings of toluene eagerly stack either with the BODIPY core or with the phthalimide substituent. The phenyl ring in the meso-position of the BODIPY molecule is, on the other hand, protected by the methyl groups in 1 and 7 positions; due to sterical reasons, it does not create an effective stacked complex. On the other hand, the smaller size of the acetonitrile and methanol molecules allow to penetrate easily, even in more crowded areas, and align parallel to the phenyl ring. However, the hydroxyl group of the alcohol prefers to form strong hydrogen bonds to the ether oxygen rather than to encounter dispersion or C-H...1 is of the order of 6–9 kcal/mol, depending on their position, and substitution with the iodine atom does not influence these interactions significantly.In order to shed more light on the nature of the solute–solvent interaction, particularly in the case of the toluene solutions, theoretical calculations for the BODIPY, explicitly solvated with three solvent molecules, were performed for toluene as well as for methanol and acetonitrile within the B3LYP-D3/def2-SVP approach. The optimized complexes of 1 and 2 were recorded in the same series of solvents as the absorption spectra and are presented in 1 exhibits the intensive emission in all of the solvents. The emission maximum wavelength for 1 in DMSO is equal to 510.5 nm, Stokes shifts amounts to 0.051 eV, and the high values of n-hexane and toluene, the emission quantum yield is much lower and equal to 0.19 and 0.22, respectively. On the other hand, the iodinated BODIPY derivative 2 exhibits weak emission properties with a maximum wavelength of about 543 nm in all solvents. The quantum yield of fluorescence is very low and smaller than 0.02 in all the solvents. The substantial decrease in 2 with respect to the original system 1 is in agreement with previous reports and can be ascribed to the heavy atom effect, which provides an effective intersystem crossing process from the singlet excited state to the triplet state . Already after 5 min of irradiation, the peak of the substance P9 occurs in the chromatogram with the maximum absorption at 512 nm. After the irradiation time t = 20 min, another peak appears in the chromatogram, with 1. Thus, 1 is one of the products of photodegradation that occur under the influence of the irradiation of 2 during fragmentation, according to the photodegradation mechanism I (PM-I), presented in 1. Additionally, it is important to note that the P7  and P9 products absorb only in the UV range.A different course of the photodegradation process was observed for 2, the kinetics of the photochemical reaction in the first 50 min of irradiation is determined. Since the change in concentration of the investigated substances is directly proportional to the area of the chromatographic peak A . Furthermore, the formation of singlet oxygen induces photo-oxidation processes that can lead to a wide variety of products that are not recorded by the selected chromatography method. Photo-oxidation may also explain the lower amount of products with an absorption maximum at 496 nm, observed for 2 than for 1. If such products are obtained, their concentration may not be high enough for their detection.For c peak A , the logc peak A . Based oz et al.  for 2 inOne can see that besides the strong confirmation of the iodine detachment in the first stage of the photodegradation PM-I,  by the e1 and 2 bent and unfolded conformations with the PBE03/def2-TZVP approach in methanol and are presented in The Wiberg bond indexes, which allow to estimate the weakest bonds, were calculated for 2 was assumed to lose the iodine atoms either in a concerted or rather stepwise manner, as presented in 1 and 2. One can clearly see that the computational results indicate the hypsochromic shift of the maximum absorption wavelength by about 34 nm when going from the iodinated 2 to unsubstituted 1 molecule for the elongated (unfolded) conformers. The corresponding shift for the lowest energy bent conformations amounts to 32 nm in methanol. Additionally, the second band calculated for 1 at 378 nm significantly decreases for the unsubstituted unfolded molecule 2. The dominating orbitals contributing to these excitations are localized at the BODIPY core with only minor influence arising from iodine atoms. The presence of the metoxy group arising from the probable radical reactions involving the solvent molecules in the BODIPY core only moderately affects the calculated spectrum; therefore, one can expect that this is not the radical solute–solvent interaction crucial for the observed absorption changes upon irradiation. Taking into account the agreement of the initially recorded modification of the experimental spectrum, namely, the signal shift after the irradiation times up to 60 min of the order of 30 nm  are included in the considerations.First of all, the iodinated derivative  compare , one canmeso-substituent reveal relatively weak bonding of this fragment (1.0308 for unfolded 1 structure); therefore, one of the considered photodegradation mechanisms, depicted in meso-substituent and its BODIPY core counterpart (denoted here as 3) is presented in panel (b) of meso-substituent bearing the phenyl ring and the phthalimide moiety bound by the aliphatic chain and ether oxygen does not exhibit absorption in the visible region and only weakly depends on the conformation of the chain. Therefore, its presumptive presence in the photodegradation products mixture is of no importance for the recorded absorption spectra after irradiation  or between the ether oxygen and phthalimide group (O21–C22 Wiberg bond index 0.9127) leads to the degradation products with the absorption spectra shifted negligibly with respect to the original 1 molecule, with no observable intensity modification. Therefore, one can assume again that it is not this photodegradation path that is responsible for the experimentally recorded changes of the absorption spectra upon the long irradiation presented in The photodegradation mechanism III, investigated in the present study , includeAnother possibility of the influence of UV irradiation is the destruction of the phthalimide moiety itself, presented in meso-position, produces the photoproduct of the weak absorption at 425, 394 and 341 nm, while the further total devastation of the system by the B4–N11 bond breaking  to the generation of the single substituted succinimide, brings the only weak absorption band to 314 nm. All of the remaining bands in the spectrum vanish for this small system. All of these theoretical calculations together with the experimental observations of the irradiated system spectra lead to the rationalization of the initial assumption that the modification of the spectrum upon UV–Vis irradiation is a stepwise process, including the detachment of the iodine atoms in the first stage, probable meso-substituent detachment  and finally, the photo-oxidation of the BODIPY core in the last stage.The remaining possible, and, at the same time, the strongest, structural modification deeply affecting the molecule character and thus the absorption of the BODIPY dyes is connected to the boron heterocyclic ring breaking . This pathway was suggested by Mula et al.  with themeso-position differ by the absence/presence of the iodine substituents in 2 and 6 positions for 1 and 2, respectively. The meso-position occupied by the aromatic system and connected with the BODIPY core by the long linker allows the adaptation of different structures, benefiting either from the intramolecular dispersion interaction for bent molecules, or from the maximized intermolecular 1 and 2, the photodegradation process in both systems differs to a high extent and discrepancies are observed in the shape of the spectra upon irradiation, the timescale of the fragmentation and the kinetics of the reactions. The DFT verification of the absorption spectra of the hypothetical irreversibly destructed photodegradation products seems to inevitably confirm that the total diminishing of the strong absorption bands upon the irradiation arises from the destruction of the BODIPY core by the photo-oxidation process. This observation remains in agreement with the larger singlet oxygen quantum yield in the case of 2, which enables easier oxidation of the BODIPY core than in 1.The present study devoted to the photostability of the two BODIPY derivatives, synthesized previously for bacteria photoinactivation, combines the theoretical and chromatographic approaches. The two systems containing the phthalimide moiety in the


Molybdenum blue dispersions were synthesized by reducing an acidic molybdate solution with glucose, hydroquinone and ascorbic acid. The influence of the H/Mo molar ratio on the rate of formation of molybdenum particles was established. For each reducing agent, were determined the rate constant and the order of the particle formation and were established the conditions for the formation of aggregative stable dispersion with the maximum concentration of particles. The dispersed phase is represented by toroidal molybdenum oxide nanoclusters, which was confirmed by the results of UV/Vis, FTIR, XPS spectroscopy and DLS. By their behavior, dispersions of molybdenum blue are close to micellar solutions of surfactants, classical representatives of associated colloids ,17,18,19It should be noted that in the case of dispersions of molybdenum oxide nanoclusters, lyophilization of the surface is provided by the formation of a hydrated shelf on the nanocluster surface. However, this factor is not sufficient to ensure the aggregative stability of this type of dispersed systems. That is, unlike associated colloids, molybdenum blue dispersions still require additional stabilization. Due to the features of the synthesis of polyoxometalate complexes, a reducing agent, a necessary component in the process of self-assembly of clusters, can act as a stabilizer. This role can also be performed by the oxidation products of some of the organic reducing agents. Moreover, if organic compounds are chosen as a reducing agent, then it is possible to immediately solve not only the problem of the formation of particles (clusters), but their further stabilization also ,25,26,27+6 compounds, high solubility and further chemical stability of oxidized products in an aqueous solution. The following organic compounds fulfill these requirements: glucose, hydroquinone and ascorbic acid. In our previous works, it was shown that these reducing agents make it possible to synthesize stable dispersions of molybdenum blue [Besides it, the reducing agent has to match some other conditions, among which it is the redox potential sufficient for the partial reduction of Monum blue ,30,31,32However, the difference in the structure of these organic compounds should affect the process of self-organization of molybdenum blue nanoparticles and the further behavior of the dispersed system in time. Taking into account the previously obtained data, we assumed that hydroquinone acts only as a redox agent, glucose and ascorbic acid, in addition to the reducing function, play the role of a stabilizer.A study of the processes leading to the formation of particles in stable colloidal systems would lead to a more complete understanding of the synthesis of molybdenum blue as colloidal systems. The scientific novelty of this work is in the results of the comparative analysis of the kinetic study of the particle formation and the behavior in time of dispersions of molybdenum blue, synthesized using organic reducing agents of various nature. Such an analysis will be carried out for the first time and will make it possible to establish the factors of aggregation stability of systems unique for colloidal chemistry—dispersions of molybdenum blue.The aim of this work was to investigate the properties of molybdenum blue dispersions and to estimate the role of each organic reducing agent in the process of self-assembly of nanoparticles. +5 and Mo+6 is required. For the obtaining of Mo+5 complexes it is necessary to carry out a partial reduction of the molybdate ions in solutions [It is known that the formation of clusters of molybdenum blue occurs as a result of self-organization (self-assembly) of molybdenum complexes . For theolutions . In thisolutions . It is iThus, to obtain dispersions of molybdenum clusters (herein after referred to as molybdenum blue), it is necessary to determine the optimal molar ratios of the reagents: reducing agent/Mo (R/Mo), acid/Mo (H/Mo) and, also, to establish the pH value at which stable molybdenum blue hydrosols are formed. In this work, glucose, hydroquinone and ascorbic acid were used as the reducing agent. Earlier research ,18,19 fo154−x family [The interaction of ammonium heptamolybdate with reducing agents in the above ratios leads to the formation of stable dispersions of molybdenum blue. The fact of their formation is confirmed by the appearance of an intense blue color and a change in the UV/Vis spectrum. In x family ,36.max = 745 nm) is directly proportional to the change in the concentration of nanoclusters in the dispersion. It should be noted that UV/Vis spectroscopy can also be used as a quantitative method for analyzing the dispersions of toroidal molybdenum oxide nanoclusters ,33. The max = 745 nm, which corresponds to the absorption maximum of toroidal molybdenum oxide nanoclusters [A kinetic experiment was carried out to establish and compare the rates of particle formation in the presence of various organic reducing agents. Absorbance was measured at the absorption maximum λclusters . SamplesThe initial linear region of the kinetic plot was selected to estimate the formation rate. The results of the rate calculation are shown in As can be seen from the presented data, the rate and order of particle formation using ascorbic acid and hydroquinone are similar. In the case of glucose, the formation of particles occurs seven to eight times slower, the magnitude of the order of formation of nanoparticles changes.Thus, the formation of nanoparticles of molybdenum blue in the presence of ascorbic acid and hydroquinone occurs much faster than in the presence of glucose, which may be due to the high reducing ability of hydroquinone and ascorbic acid. However, despite the fact that the rates of particle formation in the case of hydroquinone and ascorbic acid are similar, the highest concentration of particles, all other things being equal, is formed in the presence of ascorbic acid.The low constant rate of self-assembly in the case of glucose can also be associated with a high concentration of glucose in the dispersion, which leads to an increase in the viscosity of the dispersion medium and, accordingly, to a decrease in the rate of self-assembly of molybdenum blue nanoparticles.It is known that the self-assembly of molybdenum-oxide clusters proceeds during time . To estaOver time, for systems prepared with glucose and hydroquinone, an increase in the absorbance and hence the number of particles of molybdenum blue is observed. The rate of their formation depends on the molar ratio of (H/Mo). There is a range of values in which the rate of formation is higher. For glucose this region is 0.5–0.8 and maximum of particles of molybdenum blue is observed at (H/Mo) = 0.5. At values of (H/Mo) less than 0.5 molybdenum blue formation does not occur. For hydroquinone this region is 1.0–5.0 and maximum of particles is observed at (H/Mo) = 1.5.A different situation is observed in systems synthesized with ascorbic acid. Particle formation occurs on the first day in the entire range of the studied (H/Mo) ratios, that is probably due to the higher reducing capacity of ascorbic acid. The largest number of clusters is formed when the ratio (H/Mo) = 1. However, over time the absorbance decreases to a certain value, whereas the character of the spectrum does not change (the absorption maximum is 745 nm). Such behavior can be explained with a long time to establish equilibrium between the formed nanoclusters and the initial building blocks.Despite the reducing agent used a constant value of absorbance of the synthesized dispersions is observed 21 days after synthesis. In contrast to small inorganic ions, which usually reach equilibrium state quickly giant molecules of polyoxometalates need time to reach an equilibrium state. The results obtained are in good agreement with the data available in the literature ,27.h = 1.5 nm) did not change during the study period for all investigated samples (30 days).The particle-size distributions in molybdenum blues dispersions determined by DLS method are shown in In the UV/Vis and FTIR spectroscopy is used to characterize molybdenum blue particles and its structure . Figure 138, Mo150, Mo154 and Mo176). Comparing the spectrum for the isolated particles with the spectrum of the initial dispersion, we concluded that after the separation of the particles, their structure does not change.The electronic absorption spectrum for all investigated samples contains a wide absorption band with maximum at 750 nm. The observed band, according to the literature , is charThe typical IR spectrum for the molybdenum blue nanoparticles synthesized using glucose is shown in −1. These results are in good agreement with published data, which repeatedly noted a high content of OH groups on the surface of toroidal particles, as well as the presence of aqua ligands and intracluster water molecules  [−1 correspond to deformation vibrations of water δH2O.Molybdenum blue particles have many hydrogen bonds ν (OH...H), as evidenced by the presence of a wide band in the region of 3100–3500 cmcluster) . The ban−1 contains bands related to vibrations of the polyoxomolybdate framework, excepting band 1406 cm−1 corresponding to the δNH4+. In the range of 950–990 cm−1, closely spaced bands are observed, which are related to the Mo=O bond. A band of 957 cm−1 is also presented in this region, corresponding to stretching vibrations of the Mo–O–Mo bond [−1 refer to deformation vibrations of the δ (Mo–Oμ) bond, where oxygen is bonded to two or three molybdenum atoms.The region below 1000 cm–Mo bond . The ban154 toroidal clusters [The number of absorption bands observed on the spectra of the synthesized samples is in good agreement with the literature data and corresponds to the structure of the Moclusters ,25.V in the analyzed samples, XPS spectroscopy was used , Mo+5 (d5/2 and d3/2 electrons) and Mo+6 (MoO3) (electrons with binding energies 234.6 and 237.7 eV).As can be seen from the data presented, the spectrum of Mo3d electrons for samples of molybdenum blue synthesized using glucose and hydroquinone is represented by three doublets: Mo+6 d5/2 and Mo+6 d3/2 and Mo+5 d5/2 and Mo+5 d3/2. Mo+6 in the form of MoO3 is absent or its amount is insignificant.Molybdenum in molybdenum oxide clusters synthesized in the presence of ascorbic acid is presented in two forms: Mo+5 in the composition of molybdenum oxide clusters. The content of reduced molybdenum Mo+5 in molybdenum blues synthesized using glucose and hydroquinone is about 12%, with the use of ascorbic acid 29%. The results obtained agree with the literature data on the degree of reduction of molybdenum in toroidal clusters.Thus, XPS spectroscopy confirms the presence of reduced molybdenum Mo2C [Methods for the synthesis of aggregative stable molybdenum blue hydrosols using organic reducing agents  have been developed. The synthesis conditions (R/Mo); (H/Mo) were established, which make it possible to obtain stable dispersions with reproducible properties, suitable for the further preparation of catalysts based on Mo2C ,38,39,40From the results presented it follows that to obtain the maximum concentration of particles of molybdenum blue, the most effective reducing agent is ascorbic acid, the use of which allows the synthesis of dispersions even at a molar ratio of (R/Mo) up to 1.0. Therefore, the process of particle formation ends on the first day after synthesis. The stability of molybdenum blue dispersions maintains for no less than 1 month. Significant differences in the behavior of these systems are due to the more complicated mechanism of redox process with ascorbic acid . So, staWhen using hydroquinone, a larger amount of reducing agent is required (R/Mo) = 4, the process of particle formation is completed 20 days after synthesis. Samples synthesized under optimal conditions are stable for 10 days. Hydroquinone acts only as a reducing agent and cannot provide the stability of dispersions.The largest amount of reducing agent (R/Mo) = 7 is required to obtain molybdenum blue using glucose, which correlates with the lowest reducing ability of this compound under the selected synthesis conditions. The formation of particles occurs over a longer time (22 days).Dispersions of molybdenum blue synthesized using glucose are the most stable. The results obtained also indicate that glucose and its oxidation products have a stabilizing effect on the dispersion of molybdenum blue, which is not observed when using hydroquinone.2C [2C .4)6Mo7O24∙4H2O, reagent grade), crystalline glucose , hydroquinone , ascorbic acid  and hydrochloric acid . All reagents were delivered by CT Lantan .Molybdenum blue dispersions were synthesized at room temperature using the reagents ammonium heptamolybdate ((NHDispersions of molybdenum blue were synthesized via the reduction of molybdate solution by organic reducing agent. The synthesis was carried out at the constant molybdenum concentration 0.07 M at different molar ratios hydrochloric acid/molybdenum (H/Mo) and reducing agent/molybdenum (R/Mo), depending on the type of organic reducing agent. The system with glucose was synthesized at the constant molar ratio (R/Mo) = 7.0 and in the range (H/Mo) from 0.3 to 1.0. The systems with hydroquinone were synthesized at the constant molar ratio(R/Mo) = 4.0 and in the range (H/Mo) from 0.4 to 5.0. The systems with ascorbic acid were synthesized at the constant molar ratio (R/Mo) = 1.0 and in the range (H/Mo) from 0.3 to 3.0. The kinetic study of the nanoparticle self-assembly during the synthesis was conducted using UV/Vis spectroscopy. Samples of molybdenum blue dispersions, synthesized using glucose (R/Mo) = 7.0; (H/Mo) = 0.5; hydroquinone (R/Mo) = 4.0; (H/Mo) = 3.0 and ascorbic acid (R/Mo) = 1.0; (H/Mo) = 0.8, were prepared at the concentration of molybdate ion in the range 0.01–0.1 M.max = 745 nm) was taken for 100 s.The reagents  were mixed in the quartz cell (volume—3 mL) directly before measurement. Time scan at the absorption maximum  with a combined electrode. UV/Vis spectra were recorded by Leki SS2110 UV scanning spectrophotometer  using quartz cells.The hydrodynamic radii of the particles in the molybdenum blue dispersions were determined via dynamic light scattering by Photocor Compact-Z analyzer . The signal acquisition time was 30 min at a laser power of 20 mW and a wavelength of 658 nm.−1. The XPS spectra were recorded on ESCA + X-ray photoelectron spectrometer . The samples were exposed to X-rays (AlKα with excitation energy 1486.6 eV) under ultra-high vacuum conditions. The pass energy of the analyzer was 100 eV for registration of survey and 50 eV for measuring high-resolution spectra. The XPS peaks position were standardized by the C1s peak of hydrocarbon impurities from the atmosphere, the binding energy eV was taken 285.0 eV. The spectrometer is calibrated by the Au4f7/2 and Cu2p3/2 . Decomposition XPS spectra into components after background subtraction was conducted according to the Shirley method. The peak position was determined with an accuracy of ±0.1 eV. For spectrum interpretation the software UniFit was used.FTIR spectra were measured by using Nicolet 380 IR Fourier spectrometer  in compressed KBr pellets in the range from 350 to 4000 cmAt first time a comparative analysis of the properties  of unique dispersed systems—molybdenum blues, synthesized using organic reducing agents of various nature, was done.The influence of the (H/Mo) molar ratio on the rate of formation of molybdenum particles was established. For each reducing agent, the conditions (molar ratios of (H/Mo), pH value) for the formation of aggregative stable dispersion of nanoclusters with the maximum concentration of particles are determined. The kinetic experiment for dispersions obtained under optimal conditions made it possible to estimate the values of the rate constant and the order of formation of nanoparticles in the case of various organic reducing agents. It was shown that the rates of particle formation in the case of hydroquinone and ascorbic acid are close, while when glucose is used, the rate of formation of nanoclusters decreases by seven to eight times.Based on the UV/Vis, FTIR, XPS spectroscopy and DLS data, it was shown that the particles of the dispersed phase are represented by nanocluster of a toroidal shape.It was established, that hydroquinone acts exclusively as a reducing agent, ascorbic acid is a strong reducing agent, but in a certain range of molar ratios it can have a stabilizing effect, and glucose and its oxidation products are stabilizers.


The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes , as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels. For many years, ischemic stroke has been one of the main causes of mortality and disability among the populations of many developed countries. The search for new therapeutic strategies for stroke treatment remains an urgent problem. A significant contribution to the solution can be made by a detailed study of the mechanisms of action of drugs that have a neuroprotective effect against cerebral ischemia. Peptides of the melanocortin family are actively involved in the functioning of the central nervous system because they act as neuroprotective agents . The neuroprotective effects of melanocortins in acute and chronic neurodegenerative conditions are manifested through the antagonism of excitotoxic, inflammatory, and apoptotic reactions, which are the main damaging mechanisms associated with ischemia, as well as through neurogenic effects ,3,4,5. MIn particular, α-MSH, the amino acid sequence of which coincides completely with the site of amino acids 1–13 of ACTH, exhibits noticeable anti-inflammatory, neurogenic, and neuroprotective effects in models of experimental ischemia ,7. The sWithout hormonal and toxic side effects, the peptide increases the rate and extent of recovery of neurological functions , reducesThe study of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide acts on the brain transcriptome: it enhances the transcription of neurotrophins and their receptors ,14, signThe aim of the present study was to evaluate the effect of Semax on the expression of several key proteins involved in inflammation and cell death processes, as well as neuroprotection and recovery in stroke. In this work, we used a rat model of transient occlusion of the middle cerebral artery (tMCAO) to investigate the effect of Semax on the expression level of the protein products of several genes that are the target of the α-MSH neuroprotective action in the subcortical structures of the brain that contained the focus of ischemic injury, as well as in the adjacent frontoparietal cortex at 24 h after occlusion. As the role of these proteins in the pathogenesis of stroke is well known, this may contribute to the understanding of the molecular mechanisms of action of this peptide. These proteins include matrix metalloproteinase 9 (MMP-9), which is activated during inflammation and may serve as a prognostic indicator of stroke severity ; c-Jun NWe found that, under the influence of Semax, the expression profiles of several of the studied proteins in the subcortical structures and the cortex of ischemic rats differed markedly. However, the general effect of the drug in both tissues was to compensate for the level of expression of most of the proteins that were impaired by ischemia.In standard coronal sections from experimental animals that were stained with hematoxylin and eosin (H&E), an ischemic lesion of the brain tissue in the form of “enlightenment” was found in the lateral zones of the subcortical region of the right brain hemisphere of rats at 24 h after tMCAO. The developmental regions of the subcortical lesion zone were observed in the caudoputamen region, from +1.7 to −3.3 mm from the bregma. At the level of the hippocampus, the infarction zone exhibited an elongated shape in the dorsoventral direction and captured most of the caudoputamen lateral region a,b, withConcomitantly, the area of the developing ischemic damage covered the caudoputamen to the outer capsule. Microscopic examination revealed a drop in the lumen of the capillaries and their emptying, pronounced perivascular edema, sparsity and bleaching of the neuropil caused by edema and vesiculation, and the appearance of numerous hyperchromic neurons with pericellular edema.According to pathomorphological criteria, a fraction of the neurons located within the outer boundary zone of the lesion were normal; many neurons exhibited hypoxic damage ; and death (nuclear pyknosis with increased cytoplasmic eosinophilia (red neurons), destruction of nuclei, and cytoplasmic lysis) c. VacuolThere were no microscopic signs of ischemic damage to the nervous tissue in the cortex of the right hemisphere. The cytoarchitecture of the cerebral cortex along the layers of neurons was without features e. In theUsing the Western blot method, we evaluated the changes in the total content of the active forms of JNK, namely phospho-(Thr183/Tyr185)-SAPK/JNK 54 kDa and 46 kDa (pJNK) and active phospho-(Ser133)-CREB (pCREB), as well as in the level of the MMP-9 and c-Fos proteins in the subcortical structures and cortex of rats at 24 h after the onset of occlusion in the groups of animals with “ischemia–reperfusion” (IR) relative to the animals of the group with “sham operation” (SH), as well as under the effect of Semax in ischemia (IS) relative to the IR group. The β-actin protein was used as the internal control.The total content of pJNK was increased by more than two-fold in the subcortex a and cerThe level of pCREB was decreased by more than 1.4-fold in the subcortex during ischemia, whereas Semax increased its levels by more than 1.5-fold c. The leAccording to the Western blot analysis, in the subcortical structures of the brain of animals in the IR group, the total content of the MMP-9 protein was increased by approximately 1.7-fold relative to the SH group a. A decrIn the cerebral cortex of the animals in the IR group, the levels of the MMP-9 c and c-FMmp9 and Fos genes in the frontoparietal cortex in the IR relative to the SH group, as well as in the IS relative to the IR group. The mRNA level of the Gapdh and Rpl3 genes was used as the internal control. We found that, under the conditions of ischemia–reperfusion, the levels of the Mmp9 and Fos mRNAs were increased by more than two-fold in the frontoparietal cortex of the rats adjacent to the damaged area  signaling pathways. In contrast, the administration of Semax under ischemic conditions downregulated pJNK, which led to a change in the activity of four signaling pathways . The activity of all of these signaling pathways was modulated by ischemia–reperfusion in the damaged area compared with the corresponding brain regions in the SH group .As we did not record the changes in the level of the MMP-9 and c-Fos proteins in subcortical structures during ischemia and Semax treatment, we were not able to illustrate the spectrum of signaling pathways associated with these proteins. However, we found that c-Fos was involved in the functioning of four signaling pathways , whereas MMP-9 was involved in estrogen signaling pathway functioning. Moreover, these pathways might be modulated by the activity of CREB or JNK under the action of Semax after the establishment of tMCAO conditions.The peptide drug Semax has a pronounced neuroprotective effect in the treatment of cerebrovascular insufficiency and acute cerebral stroke ,24,25,26In this work, to further investigate the mechanisms underlying the neuroprotective action of Semax under the conditions of the tMCAO model, the expression of the pJNK, MMP-9, c-Fos, and pCREB proteins, which are actively involved in the pathogenesis of ischemic stroke, was analyzed by immunodetection in the subcortical structures and the frontoparietal cortex of the rat ipsilateral hemisphere.pJNK, MMP-9, and c-Fos are important factors in the progression of ischemic stroke. JNK belongs to the family of MAPK that respond to stress, including stress caused by reactive oxygen species, excitotoxicity, and inflammatory cytokines. Research found that stress stimuli cause the rapid and long-term activation of the JNKs involved in ischemic cell death and neuroinflammation, while their inhibition following experimental ischemia causes a pronounced neuroprotective effect ,28. PhosThe activation of the MMP-9 protein, which disrupts the stability of the blood–brain barrier, promotes edema and stroke progression . It has IL-6, TNF-α, CCL2, matrix metalloproteinase genes, and genes involved in apoptosis. However, by activating late effector genes involved in post-stroke regeneration , c-Fos is involved in the prevention of neuronal death in ischemic conditions [The transcription factor c-Fos plays a significant role in post-ischemic inflammation . As partnditions . There inditions .Mmp9 gene both at the mRNA and protein levels  regulates the transcriptional programs of synaptic plasticity, neurogenesis, and neuronal survival in the brain . CREB alMany drugs, such as cerebrolysin and resveratrol, which have a neuroprotective effect in stroke, also increase the level of active CREB in ischemic brain tissues ,41. MoreThus, the changes in the expression of the pJNK, pCREB, MMP-9, and c-Fos proteins in the brain of rats subjected to ischemia–reperfusion detected under the action of Semax confirm the protective effect of the peptide, which had been previously revealed at the transcriptomic level. The observation that the effect of Semax on the pCREB content was manifested only in the subcortex, whereas that of the MMP-9 and c-Fos proteins was observed in the cortex and that of pJNK was detected in the cortex and subcortex, apparently reflects both significant differences and similarities in the molecular and cellular mechanisms underlying the pathogenesis of damage, and neuroprotection in the zones of infarction and areas of the brain outside ischemia.It is well known that ischemic injury results in the formation of an infarct nucleus surrounded by an ischemic penumbra. According to the results of our histological examination at 24 h after tMCAO, the infarction nucleus and the penumbra zone were localized in the subcortical region of the brain, while no gross pathological changes were detected in the frontoparietal cortex. In turn, the role of pJNK and pCREB under the action of Semax may be associated with their participation in the modulation of neurotransmission and inflammation signaling pathway activity in subcortical structures . We haveThus, pJNK and pCREB are likely to be involved in the functioning of signaling pathways in the area of ischemic injury, to a large extent, independently of each other. We showed that pJNK was upregulated in this region of the brain during ischemia, followed by its downregulation after the administration of Semax; in contrast, pCREB was downregulated during ischemia and upregulated under the effect of Semax. Thus, the activity of pJNK and pCREB may be an important element in the mechanism underlying the neuroprotective action of Semax, including explaining the multiple compensatory effects of the peptide in the zone of damage after ischemia–reperfusion.A limitation of our study is the analysis of the expression of only four proteins; the genome-wide proteomic analysis may reveal some additional proteins involved in the response to Semax administration during ischemia. A detailed study of the expression of mRNA and proteins will definitely confirm the features of the functioning of genes under the influence of the peptide during ischemia in various brain structures.The role of the cortical cells lying outside the area of visible damage in the molecular and cellular mechanisms of ischemic damage and the possible protective effect of drugs remains an open question. It is known that, together with excitotoxicity, oxidative stress, and inflammation, a significant role is played by the spreading depression (SD) in the pathophysiology of ischemic stroke . This isMmp9 and Fos genes at the mRNA and protein levels detected here in the ipsilateral region of the rat frontoparietal cortex under IR conditions are largely attributable to the SD effect. Semax may weaken the secondary damage caused by the destructive effects of SD by reducing the expression of the Mmp9 and Fos genes in the cortex and has a neuroprotective effect in this area of the brain.The SD is an important factor in secondary injury in stroke and leads to profound transcriptomic, metabolic, and hemodynamic effects ,45,46. IWhite 2-month-old male rats of the Wistar line  were obtained from the Experimental Radiology sector in A. Tsyb Medical Radiological Research Center, Obninsk, Russian Federation. The animals were maintained on a 12-h light/dark cycle at a temperature of 22–24 °C, with free access to food and water.The tMCAO model was induced by endovascular occlusion of the right middle cerebral artery using a monofilament  for 90 min and was performed under the control of magnetic resonance imaging (MRI), as described previously . Ninety-Animals that had been subjected to 1.5-h occlusion and subsequent reperfusion were treated with intraperitoneal injections of Semax at a dose of 100 μg/kg (IS) or saline (0.9% NaCl) (IR) immediately after occlusion and at 1.5- and 5-h after the onset of reperfusion. Sham-operated controls (SH) were treated with saline at the same time points. The rats were decapitated 24 h after tMCAO. The experiment design is schematically shown in In our conditions, according to an MRI investigation of the ischemic animals, the damaged area was localized in the right subcortex  or in the subcortex plus the cortex. To obtain the most representative results, animals with a subcortical localization of the damaged area were used exclusively, for the immunodetection of proteins in the rat brain and analysis of gene expression using real-time reverse transcription polymerase chain reaction (RT–PCR) in the cortex. Tissues were placed in RNAlater solution  for 24 h at 0 °C and then stored at –70 °C. The total RNA from the subcortex, was isolated using TRI Reagent  and acid guanidinium thiocyanate–phenol–chloroform extraction . The iso18 primers were used to analyze the mRNA.cDNA synthesis was conducted in 20 μL of reaction mixture containing 2 mg of RNA using the reagents of a RevertAid First Strand cDNA Synthesis Kit  in accordance with the manufacturer’s instructions. Oligo (dT)The 25 μL polymerase chain reaction (PCR) mixture contained 2 μL of 0.2× reverse transcriptase reaction sample, forward and reverse primers (5 pmol each), 5 μL of 5× reaction mixture  including PCR buffer, Taq DNA polymerase, deoxyribonucleoside triphosphates (dNTP), and the intercalating dye SYBR Green I. Primers specific to the genes studied were selected using OLIGO Primer Analysis Software version 6.31  and were synthesized by the Evrogen Joint Stock Company . The ampGapdh and Rpl3, were used to normalize the cDNA samples [Two reference genes,  samples . Calcula samples  and Rela samples . The manCt(ref)/EfCt(tar), where Ef is the PCR efficiency, Ct(tar) is the average threshold cycle (Ct) of the target gene, Ct(ref) is the average Ct of the reference gene, and EfCt(ref) is the geometric average EfCt of the reference genes. The PCR efficiencies were assessed using the amplification of a series of standard dilutions of cDNAs and computed using REST software [p < 0.05. Additional calculations were performed using Microsoft Excel .The values were calculated as Efsoftware . The effThe total protein content was isolated from brain tissues simultaneously with a total RNA extraction using the TRI Reagent  according to the manufacturer’s protocol. Protein pellets were precipitated with acetone from the organic phase and dissolved in 2% sodium dodecylsulfate (SDS). Protein concentrations were determined using a Lowry-based protein assay . Samples of 12–45 µg were separated using SDS–PAGE according to the method of Laemmli  and tranThe membranes were blocked and incubated overnight with primary antibodies against phospho-(Thr183/Tyr185)-SAPK/JNK , phospho-(Ser133)-CREB , MMP9 , c-Fos (MyBioSource), or β-actin . Subsequently, the membranes were incubated with a specific peroxidase-conjugated secondary antibody  for 2 h at 4 °C. The signals were visualized using the chemiluminescent peroxidase substrate Lumigen ECL Ultra  and imaged using a ChemiDoc MP Visualization System  or Hyperfilm ECL . The protein signals were quantified with scanning densitometry using the ImageQuant 5.2 software . The protein abundance was normalized to that of β-actin.p < 0.05. The most representative blots are shown in The protein levels in the rat groups  were expressed as the mean ± standard error of the mean (S.E.M.). Student’s t test was used to evaluate the differences between groups. The significance was set at The preparation of materials for studies with a histotopographic notch of the brain and frontal orientation of tissue blocks for their subsequent microtomy in the caudorostral direction was as reported previously .Sections of tissue samples of rat brains collected at 24 h after occlusion (n = 4) with a thickness of 5–6 µm obtained through 0.5–1 mm on a microtome  were stained with H&E  after dewaxing. Histological specimens were examined under a microscope (Leica DM 1000), and micrographs were acquired using a digital camera (Leica ICC50 HD). Morphological analysis was performed with an allowance for normal and pathological central nervous system variants ,57,58. SThe participation of the active forms of JNK and CREB in the signaling pathways that were previously identified by our group ,22 was aOur data revealed the effect of Semax on the activity of several proteins that play an essential role in the processes of damage and recovery of brain cells under the pathological effects of ischemia. Concomitantly, the results obtained here allow us to note that, at 24 h after the occlusion of the middle cerebral artery, Semax exerted various effects in the subcortical structures of the brain that contained the focus of ischemic damage, as well as in the adjacent area of the frontoparietal cortex. In the subcortex, Semax inhibited pJNK and activated pCREB without affecting the level of MMP-9 and c-Fos; however, in the cortex (which contained the area adjacent to the ischemic core), Semax downregulated pJNK, MMP-9, and c-Fos, and did not change the levels of pCREB. Nevertheless, the general effect of the drug on both brain tissues occurred via its ability to compensate for the expression profiles of these proteins, which were disturbed by ischemia. It should be noted that CREB activation and JNK inhibition are involved in the anti-inflammatory and neuroprotective action of melanocortins in models of ischemic and inflammatory processes in the brain ,60. The modulation of the activity of the pJNK, pCREB, MMP-9, and c-Fos proteins in various brain regions of animals revealed here may contribute significantly to the mechanisms underlying the neuroprotective action of Semax in the brain under tMCAO conditions. Our study of the neuroprotective properties of Semax was limited to the analysis of the expression of several proteins. Further in-depth studies of the effect of the peptide on the molecular pathways of stroke at the proteome level will definitely clarify the mechanism underlying the neuroprotection afforded by Semax.


Engineered electrospun membranes have emerged as promising materials in guided tissue regeneration, as they provide an appropriate framework for the formation of new functional periodontal tissues. The development of multifunctional local drug delivery systems with sustained release of drugs for prolonged infection control can be used in periodontal surgical interventions to simultaneously prohibit epithelium downgrowth and ensure proper healing and regeneration of damaged periodontal tissues. The aim of the present study was the fabrication of novel composite membranes from PLGA/moxifloxacin-loaded mesoporous nanocarriers through electrospinning and the evaluation of their drug release profiles. The addition of moxifloxacin-loaded mesoporous nanocarriers in PLGA yielded a sustained and prolonged drug release, while maintaining satisfactory mechanical strength. The freshly fabricated membranes were found to be biocompatible at masses less than 1 mg after exposure to healthy erythrocytes. Increase in the amount of polymer led to more uniform fibers with large diameters and pores. The study of the parameters of the electrospinning process indicated that increase in the applied voltage value and rotation speed of the collector led to more uniform fibers with higher diameter and larger pores, suitable for tissue regeneration applications, such as periodontal tissue regeneration. Periodontitis is a multifactorial, multifaceted chronic inflammatory condition that affects the periodontium (the soft and hard tissues that surround the tooth) causing irreversible loss of tooth retention. The failure to diagnose and treat the disease can lead to severe damage to the alveolar bone and tooth support causing functional alterations that could lead to premature tooth loss. The consequences of untreated periodontal disease have a wide range of effects on a person’s quality of life. Thus, the goal of periodontal restoration is to maintain and/or regenerate the architecture of the dental support system in order to maintain its function ,2. A widWithin this framework, guided tissue regeneration (GTR) was introduced as an effective strategy for the treatment of periodontal infections . This apBiodegradable polymers with high processing flexibility are the most sought-after candidates for tissue engineering applications. Synthetic biodegradable polymers with a well-defined structure and without immune reactions widely used in tissue engineering include polyesters, polyhydrates, polyurethanes, etc. . The mosThe application of the systemic administration of growth factors and/or drugs for the enhancement of bone regeneration is accompanied by significant side effects and limited bioavailability ,16. AlthS. aureus, which is the major pathogen associated with osteomyelitis. MOX is superior to ciprofloxacin, levofloxacin, and ofloxacin against Staphylococcus aureus, and could therefore be applied locally for bacterial eradication  × 100% (1). Statistical analysis was performed using a paired sample w/v PLGA with the addition of 0 , obtained under similar synthesis conditions, were selected for further study.SEM images of groups 30A and 20A at ×4000 magnification (data not shown) showed better dispersion of the added nanoparticles within the fibers in the sample 30A2 , in combThe measurements of all fibrous scaffolds revealed that increase in the voltage led to an increase in the size of the fibers and the size of the pores. In addition, increasing the speed of the rotating collector did not seem to significantly affect the average fiber size, but it did seem to result in a small increase in pore size. However, there was a simultaneous increase in fiber diameter with increasing pore size.−1 were attributed to the vibrations of carbonyl and C-O-C stretching peaks of the PLGA group. The peaks around 1455 and 1423 cm−1 were attributed to the C-H stretching in methyl groups. These particular peaks were assigned to the C-CH3 stretching vibrations [−1 was attributed to the vibration of the C-O-C bond, the peak around 1049 cm−1 was also attributed to the C-CH3 vibration, while the peak at 1270 cm−1 was attributed to the PLGA ester groups [−1, which was not observed in the spectrum of sample 30N2 (neat PLGA electrospun membrane). This peak was attributed to the bending vibration of the Si-O-Si bond due to the presence of silica-based mesoporous nanocarriers. In addition, the presence of mesoporous nanocarriers was also confirmed by the widening of the broad peak between 900 and 1542 cm−1, because the wide peak of typical silicate glasses (900–1200 cm−1 was attributed to the asymmetric vibration of the Si-O-Si bond) overlaps with those of the synthetic polymer PLGA in this region [Typical spectra of PLGA polymer-based and composite membranes were found from ATR analysis for the selected samples 30N2, 30A2, and 30B . The banbrations . The pear groups . The spes region .Uniaxial tensile tests were performed on the electrospun membranes. Compared with the neat PLGA membranes (30N2), for the PLGA/MSN membranes (30A2 and 30B) the breaking strength decreased with the addition of MSNs. This may be due to poor interfacial adhesion between MSNs and PLGA or poor distribution of the nanoparticles in the polymeric matrix.The degradation rate of neat PLGA and composite membranes was performed for 0, 7, 14, and 28 days, as shown in The drug loading of MOX for the electrospun nanofibers was also studied. −1, 2521 cm−1 and 1355 cm−1 were attributed to the vibrations of ((CH)-CH2), ν(NH2+) and δb(-CH2), respectively [−1 was attributed to the bending vibration of the N-H bond (due to presence of quinolones), the peaks at 801 and 990 cm−1 were attributed to the bending vibration of the C-H bond and the peak around 1711 cm−1 corresponded to the vibration of the deformed δb(COO-) bond [w/v) and the low percentage of encapsulation of the drug in them (~31%). In addition, this was also confirmed by the determination of the loading of the MSN-incorporated fibrous membranes (30A2DL) (27.5 ± 0.2%), which was lower than the fibrous membranes without MSNs (30N2DL) (37.2 ± 0.1%) sample.ectively . In addiO-) bond ,68,69,70The SEM microphotographs and the mean size distribution of the fibers and pores of the fabricated membranes after the encapsulation of the drug are presented in PLGA copolymer undergoes degradation by hydrolysis or biodegradation through cleavage of its backbone ester linkages into oligomers and, finally, monomers. The degradation process for these polymers mainly takes place over the entire polymer matrix, leading to a uniform mode of erosion, called a ‘bulk’ pathway . In thisIt is well known that the crystallinity of the pharmaceutical ingredient affects the dissolution rate and bioavailability. For this reason, XRD studies were performed to examine the crystallinity of MOX before and after its adsorption onto MSNs and the electrospun membrane. Hemocompatibility assay of electrospun membranes after direct exposure to healthy human erythrocytes was performed after 60 min and 24 h of incubation. 2/g) and pore volume (1.618 cm3/g) and typical mesoporous structure, making them ideal drug carriers for drug loading and release studies. These MSNs have shown crystalline hydroxyapatite formation after immersion in simulated body fluid [In this study, nanocomposite membranes were fabricated by the incorporation of Ca/Mg/Sr-containing MCM-41 mesoporous nanoparticles in electrospun copolymer poly(lactic-glycolic acid) (PLGA) fibers for the delivery of the antibacterial drug moxifloxacin, with optimum morphology, drug release, degradation rate and hemocompatibility. According to our previous study , the seldy fluid  and theidy fluid . Electrody fluid . Cellulady fluid ,79,80,81According to previous studies, it is possible to achieve a correspondence between the size of the pores and the size of the fiber diameter . More spw/v MSNs) in combination with neat membranes 30N2 and 30B (addition of 3% w/v MSNs) were obtained under similar synthesis conditions and were selected for further study. The study of the mechanical properties of the membranes revealed that incorporation of MSNs in PLGA fibers led to a reduction in the breaking strength. Similarly, according to Jia et al. [SEM images of groups 30A and 20A indicated a better dispersion of the intermediate nanopores within the fibers in the sample 30A2, in combination with the increased porosity and the large diameter of the fibers. The samples 30A2  were produced by the electrospinning technique. Study of the polymer concentration and the synthesis conditions during the fabrication of the fibrous membranes revealed their effect on the size and morphology of the fibers. In particular, lower polymer concentration resulted in the formation of less uniform and indistinguishable fibers of smaller size. Study of the parameters during fiber formation showed that increase in the voltage led to increase in fiber diameter and the size of the pores. At the same time, increase in the collector speed fabricated membranes of slightly higher porosity. The use of MSNs as MOX nano-carriers in fibrous PLGA membranes seemed to improve the release of the drug by creating a more controlled and prolonged release profile and enhancing their hemolytic behavior, while the encapsulation of moxifloxacin presented a protective effect on the erythrocytes. In addition, it appears that increase in the polymer concentration yielded fibers with better dispersion of the MSNs, combining increased porosity and large fiber diameter. However, the addition of the MSNs limited the tensile and elongation strength. In addition, the rate of degradation and hydrophilicity increased with increasing MSNs amount. However, the encapsulation of the drug in membranes without the addition of MSNs was higher. Moreover, the addition of MSNs after encapsulation of the MOX did not change the morphology of the fibers, while the neat polymeric fibers, after the addition of the drug, led to a reduction in fiber cross-links. The proposed composite electrospun membranes could be an effective alternative strategy for the treatment of periodontal infections due to their controllable release of moxifloxacin ensuring antibacterial activity against an extensive variety of periodontal pathogens with a flexible degradation rate. Such barrier membranes could overcome the constraints related to hydrophobicity and restricted bioactivity and induce osteogenetic and angiogenic capacity.