Retrieval of such a large molecular structure is not possible with other variants of LIED or ultrafast electron diffraction. Combining electron diffraction with machine learning presents new opportunities to image complex and larger molecules in static and time-resolved studies.Ultrafast diffraction imaging is a powerful tool to retrieve the geometric structure of gas-phase molecules with combined picometre spatial and attosecond temporal resolution. However, structural retrieval becomes progressively difficult with increasing structural complexity, given that a global extremum must be found in a multi-dimensional solution space. Worse, pre-calculating many thousands of molecular configurations for all orientations becomes simply intractable. As a remedy, here, we propose a machine learning algorithm with a convolutional neural network which can be trained with a limited set of molecular configurations. We demonstrate structural retrieval of a complex and large molecule, Fenchone (C 10H16O), from laser-induced electron diffraction data without the need for fitting or ab initio calculations.Diffraction imaging offers high spatiotemporal resolution, but fitting complex molecular structure data is expensive. Here, the authors use a machine learning algorithm with a convolutional neural network to retrieve a complex and large molecule, fenchone (C Simple convergence strategies are readily implemented for small molecules, but such methods quickly become intractable for complex systems. For example, relativistic 3.7 MeV ultrafast electron diffraction (UED) time resolved the ring-opening reaction of 1,3-cyclohexadiene, but the method could not identify the complex transient structure beyond 3 Å, which is expected to arise from three possible isomers2. X-ray diffraction imaging with free-electron laser pulses requires a careful balance between sufficient beam brightness and avoiding structural damage by the strong X-ray pulses5. Such issues severely limit the number of studies and necessitate ab initio calculations8. Laser-induced electron diffraction (LIED)22 is a powerful laser-based UED method that images even singular molecular structures with combined sub-atomic picometer and femtosecond-to-attosecond spatiotemporal resolution. Here the challenge of structural retrieval arises from the strong-field nature of self-imaging the structure24 by recolliding a laser-driven attosecond wavepacket after photo ionization. In LIED, an electron wave packet is: (i) tunnel ionized from the parent molecule in the presence of strong laser field; (ii) accelerated and driven back by the oscillating electric field of the laser; and (iii) rescattered against the parent ion’s atomic cores. The geometrical information of the target nuclei is encoded in the detected momentum distribution of rescattered electrons. LIED often uses the quantitative rescattering (QRS) theory14 to retrieve the structure of simple molecular systems. The QRS theory enables the extraction of field-free elastic electron scattering cross-sections from electron rescattering measurements performed under the presence of a strong laser field. The molecular structure is embedded onto the momentum distribution of the highly energetic rescattering electrons. But for large and complex molecular structures, the QRS retrieval quickly becomes intractable due to the difficulty in identifying a unique solution in the multi-dimensional solution space. A somewhat remedy is found by reducing the dimensionality of the problem with FT-LIED25. Nevertheless, a multi-peak fitting procedure is needed to identify bond distances. Such an approach becomes ambiguous when the radial distribution function does not exhibit clear and separable structures. We note that similar problems arise in essentially all implementations of structural imaging and many retrieval algorithms severely limit the size of the molecular system under investigation.The retrieval of complex molecular structures with electron or X-ray diffraction is challenging due to the multi-dimensional solution space in which a global extremum has to be found2H2), an asymmetric top 2D system, carbon disulfide (CS2) and a complex large 3D system, (+)-fenchone (C10H16O). For the specific problem of LIED, we employ ML with a convolutional neural network (CNN). Comparing with the commonly used artificial neural networks such as a fully connected neural network26 or recurrent neural network (RNN)27, the CNN is well suited for problems in image recognition to identify subtle features from an image at different levels of complexity similar to a human brain29. ML algorithms are usually trained either with supervised or unsupervised learning. Under the supervised learning scenario, both the classification and regression methods are commonly chosen. The classification method is generally used to predict a specific category of data, such as in facial recognition29 and crystal determination30. Here, we implement the regression method to quantitatively extract the structural parameters that identify the molecular structure. The molecular structure is found via the relationship between the molecular configuration and the molecular interference signal through the corresponding two-dimensional differential cross-sections (2D-DCSs) from its database. Using 2D-DCSs as an input, our ML algorithm takes full advantage of the complete molecular interference signal rather than only considering a one-dimensional (1D) portion of the interference signal as is typically used in other methods17. Moreover, the ML CNN is capable to interpolate between samples in the database of precalculated structures to provide a meaningful configuration to measured data. This feature of ML CNN is especially important to identify large and complex molecular structures, since it is simply impossible to calculate all possible molecular structures with sufficient structural resolution and due to the many degrees of freedom. We show a sufficiently reduced database suffices, which only considers (i) changes in a few important groups of atoms and (ii) a molecule-wide global change, allowing the algorithm to learn the relationship between the molecular structures and corresponding interference signals in our input database. Thus, our ML model, together with the CNN algorithm and regression method, provides a new way forward to identify the structure of large complex molecules and transient structures with similar geometric configurations in time-resolved pump-probe measurements.To overcome these limitations, we employ a machine learning (ML) algorithm for LIED (ML-LIED) to accurately extract the three-dimensional (3D) molecular structure of larger and more complex molecules. The ML-LIED method avoids the use of chi-square fitting algorithms, multi-peak identification procedures, and ab initio calculations. The method draws from the interpolation and learning capabilities of ML which significantly reduces the required molecular configurations to train the system for a much larger solution space. We demonstrate our method’s capability by extracting a single accurate molecular structure with sub-atomic picometer spatial resolution on the symmetric top linear system acetylene -fenchone, which failed to retrieve with FT-LIED and QRS-LIED. Lastly, we discuss the advantages, limitations, and many applications of the ML framework in retrieving large complex gas-phase molecular structures in the context of structural retrieval and time-resolved imaging of chemical reactions.This paper starts with a description of the ML scheme for LIED, followed by details on our ML model’s training with a CNN and its subsequent validation. We then predict the molecular structure of C32. The 2D-DCSs are calculated as a function of the electron’s return energy  and rescattering angle . The measured 2D-DCS signal, σtot, is comprised of two components: (i) the incoherent sum of atomic scatterings, σatom, and (ii) a modulating coherent molecular scattering signal, σcoherent, which is approximately one order lower than σatom, i.e. σtot = σatom + σcoherent. Here, the σatom signal contributes as a background signal to our total scattering signal and it depends on the number and types of atoms but it is independent of the molecular structure. The two-center σcoherent signal is dependent on the internuclear distance between two atoms. Next, we subtract the slowly varying background from the 2D-DCS maps of the ML database34 and from the measured data to enhance the DCS. We note that the exact functional form for the subtraction is irrelevant as it is applied to database entries and data. For simplicity, one can use the equilibrium structure for a known molecular system. This procedure leads to clearly visible fringe patterns in the resulting difference 2D-DCS maps. We show them for the two small molecules in Fig. 2H2 and CS2. Once the model is validated, we use the experimental 2D-DCS map as the input for our ML model to extract the molecular structure that most likely corresponds to the measured LIED signal . The evolution of the cost value (blue dots) after five iterations is shown. The change in gradient of the cost function is also given by the red arrows, with the arrow length illustrating the step size taken at each iteration, called the learning rate (α). The weights are randomly initialized at the beginning. After five iterations, we observe a decrease in the cost function, which indicates that the model is optimized and that the predicted values are close to the real values. In reality, our CNN model calculates a cost function based on thousands of parameters  instead of only two weights, as discussed above. At each iteration, all the parameters are simultaneously updated and optimized to minimize the cost function and the difference between predicted and real value. This capability is unique to the ML CNN and does not exist for simple regression methods or evolutionary algorithms.Figure 34. The normalization process ensures that the experimental and theoretical DCS values are on the same order of magnitude. We also avoid the need for a fitting factor as is typically used in QRS-LIED  of RCC = 1.23 ± 0.11 Å and RCH = 1.08 ± 0.03 Å (RCS = 1.87 ± 0.14 Å and θSCS = 104.7 ± 6.4°). These values are in excellent agreement with the values retrieved by the QRS model; see the comparison in Tab. 1. After training our ML model on five separate datasets, the predicted structures vary slightly. This is because the neural network during the training process uses a random number generator to select the input–target pairs of data. Thus, each neuron’s corresponding initial weights and biases are also randomly chosen, leading to slightly different starting conditions in the CNN training of the ML model. Using random initial conditions for the neurons ensures that systematic errors are minimized. In addition, we generated predicted structures from the ML model, trained on five separate occasions, to ensure the reliability of the predicted values. We find that the uncertainty in our ML-predicted structural parameters arise from two contributions: the predicted model error and the experimental statistical error. The predicted model error is obtained by calculating the MAE between the absolute and calculated value of the structural parameter using the test set as described earlier. The experimental error arises from the standard error in our experimental DCS, following a Poissonian statistical distribution , measured with LIED. For such a large and complex 3D molecule, the ML has the decisive advantage to interpolate and learn between the course grids of precalculated structures and to take into account a manifold of degrees of freedom in the solution space. Thus, we can use a sufficiently reduced database that only considers (i) four groups of atoms of the molecule  3D Cartesian coordinates of seven atoms of (+)-fenchone (green circles). We find that the ML-LIED-measured (+)-fenchone structure shows only slight deviations from the equilibrium ground-state neutral molecular structure (red triangles) which are involuntarily caused by the presence of the LIED laser field. The degree of uncertainty of the predicted 3D positions (green circles) are shown on top of the predicted 3D molecular structure in Fig. With the successful retrieval of the smaller 1D and 2D molecules, we put our ML framework to the test to extract the structure of gas-phase (+)-fenchone -fenchone on a standard desktop computer . A 20-atom system with n = 5 steps will require an unrealistic 1.4 × 109 h of calculation time. Thus, it is not feasible at the present time to extract complex molecular structures via calculating all possible configurations for all degrees of freedom. To overcome the unfavorable scaling of the problem, we make use of the fact that a ML framework can simultaneously identify a large multitude of features by pattern matching on an interpolated dataset, despite having initially trained the ML framework on a coarse ensemble of structures. We showed that this permits ML-LIED to reveal the 3D location of each atom in the molecule, providing significantly more detailed structural information than any other method that relies on the identification of non-overlapping peaks in the scattering radial distribution function. Such identification is near impossible for larger and complex molecular structures due to the large number of peaks in the radial distribution that overlap due to the multitude of unresolvable two-atom combinations. The reduced computational demands for complex molecules may prove decisive to address transient structures and transition states of complex molecules. ML-LIED has the advantage that once the ML model is validated, the molecular structures can be identified for each experimental 2D-DCS map, avoiding the high computational cost of repeated chi-square fittings as in time-resolved QRS-LIED. Lastly, using 2D-DCSs as an input instead of 1D-DCSs utilizes the complete measured molecular interference signal thus maximizing confidence in the identification of the measured molecular structure. We also note that the ML framework draws from unambiguous pattern matching conditions. It is conceivable, though very unlikely, that different non-equilibrium structures may be identified as the same structure. The ML framework is amendable to be adapted and trained on the existence of a combined total interference signal from two or more molecular structures. This may allow to predict the multiple molecular structures contributing to the total measured interference signal. It should be noted that the same problem of contributions from multiple molecular structures to the measured signal can also arise in other LIED and UED methods, and it is not unique to just ML-LIED. This possibility for ML-LIED may overcome standing problems in time-resolved UED studies of isomerization or ring-opening reactions, e.g., where the molecular structure changes will lead to subtle changes in the measured 2D-DCS. Thus, the combination of LIED with a ML framework and CNN provides a powerful new opportunity to determine the structure of large molecules.This work establishes a ML-based framework to overcome present limitations in structural retrieval from diffraction measurements. The problem with present methods is the need to compare a measured diffraction pattern with a precalculated structure and the extremely poor scaling of pattern matching methods with the quickly increasing number of degrees of freedom of larger complex molecular structures. This is compounded by the need precalculate a very large set of molecular configurations in different orientations and with high resolution. Further, identification of such a precalculated set reduces to finding a global extremum in a multi-dimensional solution space which is a difficult inverse problem to solve. These issues are tractable for small molecular systems and for systems where dimensionality of the problem can be reduced significantly. This is however not possible for large and complex molecular structures and the total calculation time scales as 2H216, CS220 and (+)-fenchone, which were measured with a reaction microscope35 to demonstrate our ML algorithm. We calculated the 2D-DCS of 40,000–120,000 possible molecular structures in our database using the IAM.In this work, we used experimental data for C® Tesla® V100). To predict a molecular structure with an experimental 2D-DCS input requires less than 1 min of calculation time.We generate thousands of structures and calculated their 2D DCS as an input dataset. Our CNN contained 3 convolutional layers and 30 fully connected layers, with the first convolutional layer containing 32 filters with kernel size 5 × 5, the second layer has 32 filters with kernel size 3 × 3, and the third has 32 filters with kernel size 3 × 3. A batch size of 120 was used, and batch normalization is used to avoid overfitting. Our model was trained and validated with an iteration number of greater than 50 to generate a model that does not overfit or underfit the input data. The training of the ML model costs 1–2 h to run on the Google cloud GPU (NVIDIASupplementary information.


HypC has an additional function in transferring the Fe(CN)2CO group to the apo-precursor of the Hyd catalytic subunit. Bacteria that synthesize more than one Hyd enzyme often have additional HypC-type chaperones specific for each precursor. The specificity determinants of this large chaperone family are not understood. Escherichia coli synthesizes two HypC paralogs, HypC and HybG. HypC delivers the Fe(CN)2CO group to pre-HycE, the precursor of the H2-evolving Hyd-3 enzyme, while HybG transfers the group to the pre-HybC of the H2-oxidizing Hyd-2 enzyme. We could show that a conserved histidine residue around the amino acid position 50 in both HypC and HybG, when exchanged for an alanine, resulted in a severe reduction in the activity of its cognate Hyd enzyme. This reduction in enzyme activity proved to be due to the impaired ability of the chaperones to interact with HypD. Surprisingly, and only in the case of the HybGH52A variant, its co-synthesis with HypD improved its interaction with pre-HycE, resulting in the maturation of Hyd-3. This study demonstrates that the conserved histidine residue helps enhance the interaction of the chaperone with HypD, but additionally, and in E. coli only for HybG, acts as a determinant to prevent the inadvertent maturation of the wrong large-subunit precursor. This study identifies a new level of control exerted by a bacterium synthesizing multiple [NiFe]-Hyd to ensure the correct enzyme is matured only under the appropriate physiological conditions.The biosynthesis of the NiFe(CN) Escherichia coli synthesizes three comparatively abundant [NiFe]-hydrogenases (Hyd) (2 oxidation (2-evolving formate hydrogenlyase (FHL) complex  activation by the enzyme 2CO group to the Hyd large-subunit precursors  . Of thesxidation , while t complex . All thre enzyme . The bioe enzyme . While tecursors . They coO ligand ; the celCO group , 2004. Oecursors . The rolpre-HyaB . The aim2CO from the HypD to pre-HybC pre-HybC . These fpre-HybC . Becausehyb structural gene operon, which encodes Hyd-2 , there is a highly conserved histidine residue at amino acid position 51   , which iCO group . The limvolution , while aynthesis . The exaproteins , which mproteins .E. coli as a model system and made a new variant by exchanging this residue for alanine in both proteins (position 52 in HybG). Using combined in vitro and in vivo experimental approaches, we show that this conserved histidine residue is indeed important to allow HypC and HybG to interact effectively with HypD. Unexpectedly, however, our studies also revealed that the HybGH52A variant gains the ability to interact with pre-HycE, thus identifying this histidine residue as a key determinant in ensuring that HybG normally does not mature Hyd-3 in vivo.In an attempt to resolve the function of this residue in the chaperone we have used HypC and HybG of E. coli strains used in this study included MC4100 , and itC ΔhybG) . All of pDCStrep , respectybGstrep  and pT-hybGstrep , respect–1, respectively. Cells were harvested anaerobically when cultures had reached an OD600 nm of between 0.8 and 1.2 by centrifugation at 5,000 g for 15 min at 4°C. Cell pellets were either used immediately or stored at –20°C until use.In preparation for routine microbiology and molecular biology experiments, such as cloning, strains were grown on LB-agar plates or in LB-broth at 37°C . AnaerobE. coli strain SHH228 (ΔhypC ΔhybG) was transformed with the indicated plasmids using standard procedures  of 0.4 was reached. To increase the amount of protein for purification purposes, the expression of the plasmid pASK-IBA3-borne hypC and hybG genes was induced by the addition of 0.2 μg ml–1 anhydrotetracycline (AHT), whereas for pT7-7- and pACYC-Duet1-based plasmids, gene expression was induced by the addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Incubation of the cultures was continued at 30°C for 3 h, after which the cells were harvested by centrifugation at 5000 × g for 15 min at 4°C. The filling of bottles and tubes for the centrifugation of cultures and cell suspensions was performed under anoxic conditions in an anaerobic chamber . Cell pellets, derived by centrifugation, were either used immediately or stored at – 20°C until use.For anaerobic protein overproduction experiments, ocedures . Cultivaocedures . Dependi2 production. One involved measuring cumulative H2 production after anaerobic cultivation of strains, while the other determined continuous H2 production in cell suspensions. Cumulative H2 content was determined by growing strains in 15 ml Hungate tubes  containing 8 ml of culture medium. The cultures were incubated for 20 h at 30°C and the H2 concentration was measured by removing 200 μl aliquots from the headspace and analyzing the gas-phase using gas chromatography with a GC2010 Plus Gas Chromatograph  as described  to reverse the polarizing voltage to –0.7 V, essentially as described (2 produced was determined using pure H2 gas as described (Continuous Hescribed . Cells wescribed . The ass–1 and 0.2 mM phenylmethylsulfonyl fluoride per 1 g wet weight. Cells were disrupted by sonication (20 W for 2 min with 0.5 s pulses). Cell debris and unbroken cells were removed by centrifugation for 20 min at 21,000 × g at 4°C. The supernatant (crude extract) was carefully decanted into a fresh tube and was used immediately. Protein concentration was determined as described before  as described , exactly as described . Wet cell paste was suspended in 2 ml buffer W  per 1 g cell paste. Phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 0.8 mM and DNAse I to a final concentration of 10 μg/ml to the cell suspension. Cells were disrupted by sonication  on ice. Cell debris and unbroken cells were removed by centrifugation at 21,000 × escribed . His-tagPurified proteins were buffer-exchanged into anaerobic 50 mM Tris, pH 8, using 5 ml PD-10 columns containing G-25 matrix (Cytiva). The resulting protein samples were concentrated using Amicon centrifugal concentration filters (cut-off of 5 kDa for HypC and HybG proteins and 50 kDa for pre-HycE samples). Purified protein samples were stored at –80°C.WT, HybGH52A, HypCWT, or HypCH51A with His-tagged pre-HycE 150 μg of each protein (5:1 mol excess of chaperone) was mixed and incubated at 30°C under anoxic conditions for 2 h. After incubation, the mixture was loaded onto either a 0.5 ml Strep-tactin sepharoseXT column or a 0.5 ml cobalt-charged TALON Superflow agarose column for the enrichment of the interaction partners. Columns were pre-equilibrated with buffer W . After loading of sample, the columns were washed with 10 column volumes of buffer W to remove unbound proteins. Bound proteins were subsequently eluted with buffer W containing 50 mM biotin (Strep-tactin sepharoseXT columns), or with buffer W containing 300 mM imidazole . Fractions of 0.5 ml were collected and aliquots from these were analyzed by electrophoresis on 12.5% (w/v) or 1% (w/v) denaturing polyacrylamide sodium dodecylsulfate (SDS)-PAGE (To examine the interaction between Strep-tagged HybGDS)-PAGE . After tDS)-PAGE . After bDS)-PAGE . The det2CO group of the [NiFe]-cofactor into pre-HyaB and pre-HybC, the respective precursors of Hyd-1 and Hyd-2 . It is important to note that in all experiments described in the current study, HypC and HybG both carried a C-terminal Strep-tag II. The presence of this StrepII-tag does not interfere with the functionality of either protein with respect to Hyd precursor maturation nd Hyd-2 . As the structed . Derivatturation . Consequ2-oxidizing Hyd enzyme activity  had no detectable activity and acted as the negative control strep, H51A is still functional in the maturation of Hyd-3, but is less efficient at completing maturation unless HypD and the HypC proteins are co-over-produced.As the  enzymes , we wishypCstrep . When thhybG gene, encoding HybG, is located within the hyb operon  failed to result in restoration of wild-type total Hyd activity to the mutant and activity was barely detectable .The b operon  and doestectable . It shoun 10–20% . This doe MC4100 , which ae MC4100 . Note thhybG gene resulting in exchange of histidine for alanine and delivering HybGH52A, failed to yield measurable Hyd activity (hypD was co-expressed with this mutated hybG gene on pT-hybG(H52A)-hypDEF, the total Hyd activity measured was similar to that measured for hypC-hypD on pT-hypDCStrep in SHH228 (H52A protein when it was co-over-produced with HypD.The anticipated transformation of SHH288 with plasmid phybG(H52A)strep, which has a mutation in codon 52 in the activity . Surprisn SHH228 . This re2 during glucose fermentation  accumulated no detectable H2 gas (H52A) accumulated H2 to a level that was slightly more than that of the parental MC4100 strain, indicating that Hyd-3 was active. The same experiment performed with a plasmid bearing hypD and the parental hybG genes (pT-hybG-hypDEF) showed essentially no H2 accumulation, as did SHH228 transformed with plasmids carrying only the parental hybG or only the mutated hybG genes lacking the additional hypD gene strep had an H2-evolving activity that was only 10% of that of the wild-type strain MC4100  N and O, which is a side-reaction of these enzymes , and in the absence of any HypC, Hyd-3 activity could be visualized (hypC gene was expressed on its own from plasmid phypCstrep (H52A can mature the large subunit precursor (pre-HycE) of Hyd-3.Bands corresponding to Hyd-1, Hyd-2, and Hyd-3 can be readily distinguished after native PAGE followed by staining the gel specifically for Hyd enzyme activity . Moreove enzymes , but is  enzymes , we firs enzymes  and stat enzymes  for evidobserved . In contsualized . AlthougypCstrep . This rehybG when introduced into SHH228 and the very low total Hyd activity measured in H52A variant, showed no activity bands corresponding to either Hyd-1 or Hyd-2 when the cognate gene was expressed on its own from plasmid phybG(H52A)strep strep . Notablynd Hyd-2 . These rhypC and hypD . The resulting complexes were separated by SDS-PAGE and visualized using Coomassie Blue staining (H52A:HypD and ∼85% lower for HypCH51A:HypD).HypC-HypD and HybG-HypD complexes can be readily isolated from anaerobically cultivated cells  and it hstaining . While tH52A, as well as a His-tagged derivative of pre-HycE, the precursor of the Hyd-3 large subunit. These were then used in interactions experiments  to test whether HybGH52A could form a complex with pre-HycE 2CO group to three separate large-subunit precursors. Although both chaperones interact (with different effectiveness) with pre-HyaB, the Hyd-1 large-subunit precursor, only HypC delivers the iron group to pre-HycE, while pre-HybC receives the group exclusively from HybG 2CO group] and the pre-HybC large-subunit precursor is not strongly compromised by the exchange, and the observed reduction in Hyd-2 activity might be due simply to the poorer interaction of HybGH52A with HypD. Nevertheless, future quantitative assessment of binding affinities for these various interaction partners will be needed to verify this proposal.In rom HybG . Here, wCO group . This al E. coli . Based o E. coli , this hirecursor , 2012. TH52A and pre-HycE, which resulted in an unprecedented HybG-dependent maturation of Hyd-3. This result suggests that the histidine residue functions to prevent HybG from interacting with pre-HycE. When histidine is substituted by the much smaller alanine residue, this inhibition is relieved, allowing interaction and maturation to take place. A similar residue exchange in HypC (H51A) did not have any effect on Hyd-2 enzyme activity, only causing decreased H2 production, because pre-HycE is HypC’s principal interaction partner  as well as pre-HybC, and it appears to be more effective at this than HypC 2CO group occurs to facilitate the maturation of all three Hyd large subunit precursors.Thus, two features of the HybG chaperone seem to be important in ensuring it only matures the Hsee also . The fachybG and hypD genes still failed to result in inadvertent maturation of pre-HycE, but not when codon 52 of hybG coded for an alanine residue. The selective maintenance of H52 in HybG thus assures effective HybG-HypD scaffold complex formation, while at the same time guaranteeing that pre-HycE cannot be matured by HybG. This study thus identifies a new layer of control during Hyd maturation. Controlling which chaperone (HypC or HybG) preferentially interacts with HypD determines which Hyd precursor is matured in accordance with the demands set by the physiological conditions. This ultimately results in the balanced production of H2-evolving and H2-oxidizing enzyme activities.Secondly, the H52 residue, as well as being required for optimal interaction with HypD, acts to prevent HybG from interacting with pre-HycE. Non-native co-overexpression of the native The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AH and RGS conceived and designed the study, analyzed the data, and drafted the manuscript. AH performed all of the experiments. Both authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Various solutions have been suggested and among them, the increase of active sites through surface modification is one of the most studied methods. A porous nanostructure with a large specific surface area is an attractive solution to increasing active sites, and in the electrospinning process, mesoporous nanofibers can be obtained by controlling the composition of the precursor solution. This study successfully carried out surface modification of TiO2 nanofibers by mixing polyvinylpyrrolidone with different molecular weights and using diisopropyl azodicarboxylate (DIPA). The morphology and crystallographic properties of the TiO2 samples were analyzed using a field emission electron microscope and X-ray diffraction method. The specific surface area and pore properties of the nanofiber samples were compared using the Brunauer-Emmett-Teller method. The TiO2 nanofibers fabricated by the precursor with K-30 polyvinyl pyrrolidone and diisopropyl azodicarboxylate were more porous than the TiO2 nanofibers without them. The modified nanofibers with K-30 and DIPA had a photocatalytic efficiency of 150% compared to TiO2 nanofibers. Their X-ray diffraction patterns revealed anatase peaks. The average crystallite size of the modified nanofibers was calculated to be 6.27–9.27 nm, and the specific surface area was 23.5–27.4 m2/g, which was more than 150% larger than the 17.2 m2/g of ordinary TiO2 nanofibers.The superior chemical and electrical properties of TiO Environmental pollution caused by industrial waste disrupts sustainable development and causes multiple problems. The use of fossil fuels, such as coal, oil, and natural gas, increases the amount of carbon dioxide and fine dust in the atmosphere, and causes air pollution ,4,5,6,7;operties ,23,24. Hle pairs ,27,28,29ructures ,35,36,37ructures ,43,44,45reported ,48,49,502 nanofibers (TNF) and ordinary nanofibers, a methylene blue (MB) photodegradation test was carried out using a UV lamp, and the absorbance of the MB solution was measured using UV–Vis. The test results confirmed that the modified nanofibers achieved improved photocatalytic efficiency over ordinary nanofibers [In this study, we modified mesoporous nanofibers using polyvinylpyrrolidone with different molecular weights and diisopropyl azodicarboxylate (DIPA) for a precursor solution. Previous studies using titanium butoxide, polyvinyl pyrrolidone (PVP), and polyvinyl alcohol have been reported for the mesoporous electrospun nanofibers fabrication; however, studies using a titanium tetraisopropoxide (TTIP) and PVP mixture of different molecular weights are difficult to find. The surface and pore properties of the nanofibers were improved via control of the calcination temperature ramping speed. To compare the modifed TiOnofibers ,52.PVP , , and DIPA  were purchased from Alfa Aesar Korea Co., Ltd. . TTIP  and ACAC  were purchased from Junsei Co., Ltd. . EtOH  was purchased from Daejung .As shown in The prepared precursor solutions were electrospun using a nozzle capillary with a diameter of 0.34 mm (23 gauge) at each 1.0 mL/h flow rate. The tip-to-collector distance was kept at 15 cm, and the applied voltage was set to 15 kV. The process was carried out at room temperature and humidity below 50%, and aluminum foil was used as the collector.A schematic diagram of the electrospinning process is shown in As-spun nanofibers were dried in an oven heated to 60 °C for 2 h and then, calcined by heating in a box furnace to 500 °C.2 nanofibers were obtained using a Fourier transform infrared spectrometer , with a KBr pellet in the range 400 and 4000 cm−1. The crystal structure and crystallinity of the TiO2 sample were analyzed using the diffraction pattern obtained using an X-ray diffractometer . The specific surface area and pore characteristics were analyzed using a Brunauer-Emmett-Teller surface area analyzer , and the adsorption/desorption of N2 gas was carried out at 77.3 K.Thermogravimetric analysis  was carried out to decide the calcination temperature of as-spun nanofibers. The surface and average diameter of each sample were measured using a field emission scanning electron microscope . FT-IR spectra of the calcined TiO2 nanofiber sample and 20 mL of deionized water were added to a quartz beaker, and stirred for 30 min. After that, 200 mL of MB aqueous solution was added to the TiO2 dispersed water and stirred for 2 h in a dark room, with temperature control for mixing and stabilizing. After that, the distance between the UV lamp  and the quartz beaker was fixed at 10 cm, and the beaker was stirred at 240 rpm during the photodegradation reaction. The reaction was carried out for 3 h, and the mixed solution was sampled every 30 min using a syringe. The TiO2 photocatalyst in the sampled solution was filtered and removed using a syringe filter ; in addition, the filtered solution was stored in a cuvette and refrigerated to block the incident light.The MB degradation test was carried out in a dark room to compare the photocatalytic efficiency of each sample. An aqueous solution of 5 mg/L was prepared using MB as a pollutant; in addition, 0.2 g of the TiO2 gas, and each TNF sample weighed 10 mg. The change of the TGA curve was observed in three steps, and the evaporation of the solvent and adsorbed water evaporation was not significant due to the drying process. The first section at ~150 °C was considered as mass reduction via the evaporation of moisture and residual solvents. Starting at 210 °C, the second section can be considered the glass transition and combustion phase. At this time, the decrease rate of TNF 1 was greater than that of TNF 0, which is considered due to the lower burning point of DIPA and low molecular weight PVP. When the section is above 390 °C, the thermal decomposition of the carbonized compound and the residual polymer starts. After 450 °C, the final weights of TNF 0 and TNF 1 were 29.6 wt% and 32.1 wt%, respectively; in addition, it was confirmed that there was no significant difference and the thermal behavior was ended. The calcination temperature was determined to be 500 °C to prevent rutile growth starting at 600 °C and higher, while providing a sufficient driving force for crystallization [A TGA analysis was carried out to determine the calcination temperature of TNF, including a blowing agent. The thermal behavior of TNF0 and TNF1 was compared to confirm that the same calcination temperature of ordinary TNF could be applied, and the results are shown in lization ,54.2 nanofiber (TNF) samples were analyzed using FE-SEM as shown in 2 and various gases. DIPA contained inside the nanofibers is gasified at high temperatures, and mesopores are formed where the gas was released. Since the speed at which the blowing agent is gasified and released depends on the heating rate, the pore properties can be controlled using this [The surface structure and microstructure of the TiOing this ,56. In pMore than 300 diameter values were measured using FE-SEM images, and the average diameter graph is shown in 2 nanofibers at different calcination heating rates. The analysis of chemical bonding and various compositions was performed by FT-IR in 4000 cm−1 and 400 cm−1, as shown in −1. The peak at 3432 cm−1 represents the O–H bonding stretching vibration associated with absorbed water. The peak at 2970 cm−1 is attributed to C–H stretching in the polymer (CH2). The peak is also due to the stretching of C=O found in PVP and has a wave number of 1650 cm−1. The vibrational peak at 516 cm−1 was found to be related to the characteristic Ti–O–Ti bonds.FT-IR measurements were recorded using a tungsten halogen NIR lamp. XRD analysis was carried out to verify the crystal structure of the TNF samples and the results are shown in 2 gas was carried out at 77.3 K and 2. The specific surface area and pore properties calculated using BET theory and the Barrett-Joyner-Halenda (BJH) absorption/desorption method are summarized in 2 nanofibers had a mesoporous surface with a large specific surface area. The specific surface area of each sample was calculated as 17.2 m2/g, 23.8 m2/g, 27.4 m2/g, and 23.5 m2/g for TNF 0, 1, 2, and 3, respectively. Pore volume means the volume of all the open pores formed on the surface of a material. The BJH desorption average pore width represents the average throat size of pores, and the micropore area represents the pore area per unit mass of the sample. Theoretically, an increase in the specific surface area leads to an increase in the active site and has a proportional relationship with the photocatalytic activity. However, in reality, the photocatalytic activity is rather reduced due to the surface tension of the solution, and the ease of adsorption and desorption of the generated gas, depending on the shape or diameter of the pores. These results show that the specific surface area of nanofibers can be increased by controlling the molecular weight of PVP and adding DIPA. Compared to TNF 0, the mesoporous nanofibers exhibited a specific surface area increased by up to 159%. The average pore widths of TNF 0, 1, 2, and 3 calculated by the BJH method were 13.67 nm, 15.64 nm, 8.7 nm, and 16.2 nm, respectively. TNF 2 was the highest in specific surface area, whereas TNF 3 had better characteristics, such as pore volume, size, and micropore area.The specific surface area and pore properties were confirmed using a BET method. Adsorption/desorption of N2 nanofiber samples). In order to compare the photocatalytic activity, we performed a methylene blue (MB) photodegradation test by TNFs (TiO0) and ln (C0/C) by taking the absorbance at 665 nm to C value, where 665 nm is the lambda max of methylene blue. The C/C0 and ln(C/C0) values as time passed are shown in We obtained normalized values (C/C2 nanofibers, we prepared modified surfaces using electrospinning and compared the photodegradation efficiency. The precursor solution composition for electrospinning was controlled using the PVP molecular weight and DIPA to obtain the modified nanofibers. An anatase single crystal phase, high specific surface area, and improved photocatalytic activity were achieved in the modified nanofibers. Fabricated nanofibers using DIPA and K-30 showed mesoporous adsorption/desorption curves, and the specific surface area increased from 17.2 m2/g to 27.4 m2/g compared to the ordinary nanofibers.To improve the photocatalytic activity of TiO2 for the field of photocatalyst materials, the mesoporous surface of TiO2 obtained using a simple method such as the one proposed here can be used with other techniques to improve the activity of photocatalysts, such as doping and heterointerface formation.The photodegradation rate of methylene blue was increased up to 150%, which validates the improvement of the photocatalytic activity of modified nanofibers. Different crystallinity and surface properties were obtained according to the heating rate for the calcination and composition of precursor solutions; in addition, it was necessary to optimize these factors to improve the photocatalytic activity. To contribute to the potential of TiO


N-arginine and alginate, giving rise to a colloidal polyelectrolytic complex of pH-responsive properties. The inclusion of the polypeptide in the bioparticle structure largely increases the binding sites of complexation during the bioparticles production, leading to its effective incorporation. After lyophilization, detailed evaluation of colloidal structure of redispersed bioparticles evidenced nano or microparticles with size, polydispersity and zeta potential dependent on pH and ionic strength, and the dependence was not withdrawn with the polypeptide inclusion. Significant increase of pore edge tension in giant vesicles evidenced effective interaction of the polypeptide-bioparticle with lipid model membrane. Antibacterial activity against Aeromonas dhakensis was effective at 0.1% and equal for the isolated polypeptide and the same complexed in bioparticle, which opens perspectives to the composite material as an applicable antibacterial system.The development of biomaterials to enable application of antimicrobial peptides represents a strategy of high and current interest. In this study, a bioparticle was produced by the complexation between an antimicrobial polypeptide and the biocompatible and biodegradable polysaccharides chitosan- Bacterial infections and antibiotic resistance are among the main concerns in human and veterinary medicine. In the development of new strategies in the fight against bacterial threats, the potential of diverse drug delivery systems, such as micro and nanoparticles, liposomes, hydrogels, nanofibers, as carriers of antibacterial drugs and antimicrobial bioactives, has been evidenced ,5,6,7,8.Polypeptides are generally cationic and amphiphilic, capable of antibacterial activity by interacting with the pathogen’s negatively charged components or cell surface receptors . Most caEscherichia coli and Staphylococcus aureus [The association of bioactives with micro or nanoparticles has proven advantages for allowing further specific applications, specially concerning pharmacological improvement concomitant with elimination or lowering of side effects, besides reduction of prolonged treatment regimens and costs. In this context, antimicrobial peptides were complexed with nanoparticles and the improved biological activity has been demonstrated ,13,14,15s aureus .The chitosan biopolymer has a macromolecular structure, with bioactive and versatile physicochemical qualities of high interest to research and industrial biotechnology ,22. AddiN-arginine was complexed with the negatively charged alginate in the production of nanoparticles of pH-responsive structure, profitable for drug delivery via oral administration [Previously, chitosan was modified by chemically linking arginine to part of the biopolymer monomers . Argininstration ,27,28.N-arginine and alginate, providing a bioparticle intended for antibacterial applications. The bioparticle was obtained with the inclusion of JM72, which is a polypeptide produced from the isolated antibacterial peptide longipinene, extracted from the hemolymph of Acustisoma longipes, with molecular structure rich in arginine (R) and tryptophan (W) and with primary sequence: Ac-SGWLPGREWVWRWRGRVF-NH2 [In the present study, a polypeptide was complexed with chitosan-GRVF-NH2 . The JM7Aeromonas dhakensis, a pathogenic bacteria found in fish and water streams and an increasingly recognized human pathogen [The bioparticle as a result of complexation between the biopolymers and the polypeptide was thoroughly studied since its formation in controlled conditions by evaluating the thermodynamics of molecular interactions. As a polyelectrolytes bioparticle, which may be highly susceptible to the ionic effects of the application media ,31, leadpathogen ,33,34.Macrocystis pyrifera; Sigma-Aldrich, St. Louis, MO, USA) has 61% mannuronic acid and 39% guluronic acid, with average molecular weight Mw = 200 kDa (1143 uronic acid units) and low viscosity (5 to 40 cP). L-arginine (98%) was purchased from Sigma-Aldrich. JM72 polypeptide (extracted from Acustisoma longipes hemolymph) has a mean molecular weight of 2372 Da and was obtained and characterized as previously described [N-arginine was obtained and characterized in our previous studies [The chitosan used  has 95% deacetylation degree, average molecular weight Mw = 130 kDa (corresponding to an average of 797 monomers per molecule) and low viscosity (<20 cP). Sodium alginate  and then submitted for 30 min in an ultrasonic bath . Subsequently, the solutions were filtered with 0.8 μm filters (Millipore) to eliminate impurities. Finally, the alginate solution was titrated dropwise (2 mL/min) into the chitosan-N-arginine solution under magnetic stirring  and after the mixture was kept under stirring overnight, thus obtaining the colloidal bioparticles through electrostatic interactions between the two oppose charged macromolecules [N-arginine solution, before the addition of alginate, leaving it under stirring overnight . After preparation, the colloidal particles were frozen with dry ice and lyophilized for 48 h, obtaining them as a fine dry powder. The material was kept in a refrigerator at 4 °C.Two 50 mL biopolymer solutions were prepared by shaking in acetate buffer  the following concentrations: 0.1 mg/mL alginate solution and 1 mg/mL chitosan-sn-glycero-3-phosphocholine; 99%, Avanti Polar Lipids) dissolved in chloroform (1 mg/mL) and using the electroformation procedure [For the biological model membrane, giant unilamellar vesicles (GUVs) were produced with POPC  for the application of electrical pulses . From the pulse application and subsequent opened pores, individual pore dynamics was analyzed. The theoretical work from Brochard-Wyart [R as a four-stage process: (1) growth, (2) stabilization, where it reaches its maximum radius, (3) leakage stage, which is the longest, and finally (4) quick closing. The third stage, which is linear, can be used for pore edge tension determination. By recording the time dependence of the pore closure, pore edge tension R is the GUV radius after pore closure, r is the pore radius over time t, η is the viscosity of the aqueous solution and C is a constant dependent on each experiment. Upon pulse application, pore dynamics was evaluated automatically with PoET [η we used 1.133 × 10−3 Pa.s. The mean values of edge tension for independent groups were compared via monofactorial analysis of variance followed by Bonferroni post-test.For the observation and electroporation experiments, 100 µL of GUVs suspension was placed in an electroporation chamber , consisting in two parallel platinum electrodes 500 µm apart. Subsequently, 870 µL of glucose solution (1.9 mM) with 130 µL of NaCl solution (0.5 mM) and suspension of JM72-bioparticles (15 µM JM72) dispersed in the same glucose solution at amounts of 10, 20 and 30 µL for independent experiments were carefully included in the cell with micropipette. GUVs were observed under an inverted microscope  equipped with a 20×, NA 0.4 objective and a sCMOS PCO.edge 4.2 digital camera. The employed acquisition rate was between 400 and 600 frames per second (fps). The chamber was connected to a multiporator  were characterized in buffers with pH 2.5 (citrate), 4.5 (acetate), 6.5 (phosphate) and 7.8 (phosphate), all in two concentrations of 8 mM and 40 mM. Quasi-elastic light scattering and zeta potential measurements were performed with a Nano-ZS90 Malvern ZetaSizer equipment  operating with a 4 mW HeNe laser at a wavelength of 632.8 nm in a temperature-controlled chamber at 25 °C and detection at an angle of 173°. 0.1 mg/mL of bioparticle was dispersed in each buffer. Then, measurements were carried out in the equipment folded capillary cell with data acquisition and analysis using Malvern software. The intensity-weighted size distribution was obtained by fitting data with a discrete Laplace inversion routine. Size determination was made using the Stokes–Einstein relation and the polydispersity was acquired by cumulant analysis of the obtained correlation functions applying the amplitude of the correlation function and the relaxation frequency. The second-order cumulant was used to compute the polydispersity of each sample. Each result corresponds the analysis of three consecutive measurements.Zeta potential based on laser Doppler velocimetry was concomitantly measured with data acquired performing at least 100 runs per sample. The electrophoretic mobility was converted to zeta potential in mV using the Helmholtz Smoluchowski relationship.N-arginine solution in 80 mM acetate buffer, pH 4.50, without JM72 polypeptide or containing 2.5, 7.5 and 15 µM of the polypeptide, respectively, for individual experiments. On the other hand, alginate solution (5 mM) in the same acetate buffer was filled in the injection syringe to dose 1 aliquot of 2 µL, followed by 27 aliquots of 10 µL of this solution into the cell, at 400 s intervals, mixing at 307 rpm at 25 °C. The data acquisition and analyses were carried out with Origin software provided by MicroCal and the data fitting was performed applying the single set of identical binding sites model, as previously described [ITC measurements were performed in a MicroCal Inc.  microcalorimeter. The equipment cell, which has a volume of 1.442 mL, was filled with 0.5 mM chitosan-escribed .Aeromonas dhakensis bacteria was isolated from a moribund juvenile pacu fish (P. mesopotamicus) obtained from the fish farm of the National Center for Continental Fish Research and Conservation (CEPTA/ICMBio) in the municipality of Pirassununga, State of Sao Paulo, Brazil and was identified by phenotypic and molecular identification, as described in details in Carriero et al. [TM) and incubated for 24 h at 30 °C. The inhibitory effects of the free polypeptide JM72 and the same carried in the bioparticles was accessed by means of turbidity measurements. Optical densities (OD) of bacterial cultures were measured at 590 nm and adjusted to 1.5. For antibacterial analysis, aliquots of JM72 or JM72-bioparticle suspensions (15 µM JM72) in acetate buffer (pH 5.5) and autoclaved at 121 °C for 20 min were added to the bacterial broth and the bacteria proliferation assays were conducted by measuring the turbidity of Aeromonas dhakensis incubated in the samples from 0 to 0.8% (w/v) in 96-well microtiter plates. The kinetics of bacterial growth was measured at 30 °C during 24 h and keeping constant slight shaking, in a multiskan microplate reader . Triplicate of each condition was performed and data expressed as mean ± standard error of mean.The o et al. . The colN-arginine (CHarg) and JM72 polypeptide.To evaluate the physicochemical interaction between the polyelectrolytes during the bioparticle formation process, the ITC technique was used, which provided the heat released in the titration of ionized alginate (Alg) in protonated chitosan-K, the number of binding sites, N, and the enthalpy variation, ΔH, of the complexation of macromolecules during particle formation. With K, we calculate the Gibbs energy applying ΔG = −RTln(55.55 K), where ΔG is defined for standard state of molar fraction, T is the temperature and 55.55 introduces the water concentration. Applying TΔS = ΔH − ΔG the entropy gain is also estimated. Thus, a complete thermodynamic characterization of the particle production was provided  in the structure, in buffers with a concentration of 8 mM , at pHs Considering the concentration of 40 mM buffer solutions , differeFor JM72-bioparticles, structural and charge contribution of the polypeptide must be considered, which has a high number of amino groups and also arginine, and is therefore similar to CHarg at these points. Comparing Regarding zeta potential, the difference between the values characterized at pH 2.5 for the two conditions was remarkable. At a low ionic concentration, the mean value of 31.1 mV is significantly higher than the mean value of 2.4 mV for the high concentration of buffer. Adding to this, the results of having aggregates in the first case and of having a single structure with an average diameter of 88 nm in the second—the smallest characterized in the study—evidence the importance of ionic concentration of the medium as an influential parameter in the structure of the bioparticle.It is also worth pointing out that the bioparticle without JM72 also presented a single and reduced hydrodynamic diameter, with an average value of 101 nm, only at pH 2.5 in the high buffer concentration condition . These rFurther comparing the zeta potential results of bioparticles with and without JM72 in the two conditions , a smallIn general, comparing Giant liposomes, also known as giant unilamellar vesicles (GUVs), were applied in the present study as a model membrane in order to evaluate the interaction of the bioparticles with a simplified cell membrane made only with lipids. The GUVs advantage is that the micrometer dimension allows direct microscopic observation of the membrane dynamic properties through optical microscopy under controlled parameters. Hence, the changes caused by the variation of physical-chemical conditions of the medium and the interaction with different molecules or structures can be studied, since the behavior of the vesicle membrane can be monitored.In electroporation of GUVs, the same were in a special electric field chamber, containing saline solution and also dispersed in aqueous solutions containing different concentrations of polypeptide bioparticles. The electric pulses lead to deformation of the vesicles and opening of pore on the membrane. The edge tension of the pores was measured for each individual monitored vesicle. p < 0.05) in the presence of small concentrations of bioparticles is evidenced, compared to the result in absence of bioparticles.In determining the pore edge tension, the stage of pore closing is monitored by extracting the time dependence of the pore size, as previously described ,47. The It is apparent that in the presence of bioparticles, more energy is required to rearrange POPC lipids along the pore walls, leading to edge tension increase, similar to the effect provided by increasing concentrations of cholesterol in GUVs membranes . In our Aeromonas dhakensis isolated from diseased pacu fish (Piaractus mesopotamicus) [The antibacterial activity of polypeptide JM72 alone and complexed to the bioparticle (15 µM JM72 sample) was in vitro evaluated against tamicus) . Figure In both independent experiments, the control (0%) and the lower concentration of polypeptide 0.025%) showed the same results with a log phase at 6 and 8 h, respectively. At 0.05%, a slight difference was noticeable with a log phase at 11 h for the pure JM72, but an increase to 14 h for JM72-bioparticles. This result may suggest a slight improvement of the antibacterial action of the relatively low concentration of polypeptide when the same is transported by the bioparticle, corroborating that the effective interaction of the bioparticles with cell membranes may improve the effective action of carried bioactives, as previously described for similar praziquantel and invermectin drug release nanoparticles % showed ,27,28.Aeromonas dhakensis colonies was detected during 24 h exposure. Thereby, it is evidenced that the antibacterial action of JM72 is effective and that the complexation of the polypeptide with the bioparticles is not inhibiting the antibacterial effect. The unveiled results further evidence that association of the polypeptide with chitosan-N-arginine and alginate biopolymers can be advantageous as an antimicrobial delivery system.In the presence of 0.1% (and above) of free or bioparticle-carried JM72, no visible growth of Aeromonas dhakensis represents a bacteria with multifactorial pathogenicity [The genicity  and a nugenicity ,50,51. Sgenicity , cause bgenicity  and gastgenicity . As a bagenicity , the devAeromonas dhakensis of the polypeptide complexed with biopolymers highlights perspectives of the composite bioparticle as an efficient antibacterial material for biomedical applications.The complexation between biopolymers and JM72 polypeptide led to large negative variation in enthalpy, Gibbs free energy and entropy, providing the production of biopolymers-polypeptide bioparticles with thermodynamic favored conditions. In re-dispersing the freeze-dried particles in aqueous conditions of different ionic strength and various pHs, the bioparticle structural dependence on the solution conditions was roughly maintained with the presence of JM72, hence its inclusion in the bioparticle preserves the bioparticle colloidal characteristics. Moreover, the physical change of increase of pore edge tension on the lipid bilayer of giant vesicles evidenced effective interaction of JM72-bioparticles with the simplified model membrane. Finally, the in vitro antibacterial activity on


Electrospun techniques are promising and flexible technologies to fabricate ultrafine fiber/nanofiber materials from diverse materials with unique characteristics under optimum conditions. These fabricated fibers/nanofibers via electrospinning can be easily assembled into several shapes of three-dimensional (3D) structures and can be combined with other nanomaterials. Therefore, electrospun nanofibers, with their structural and functional advantages, have gained considerable attention from scientific communities as suitable candidates in biomedical fields, such as the regeneration of tissues and organs, where they can mimic the network structure of collagen fiber in its natural extracellular matrix(es). Due to these special features, electrospinning has been revolutionized as a successful technique to fabricate such nanomaterials from polymer media. Therefore, this review reports on recent progress in electrospun nanofibers and their applications in various biomedical fields, such as bone cell proliferation, nerve regeneration, and vascular tissue, and skin tissue, engineering. The functionalization of the fabricated electrospun nanofibers with different materials furnishes them with promising properties to enhance their employment in various fields of biomedical applications. Finally, we highlight the challenges and outlooks to improve and enhance the application of electrospun nanofibers in these applications. During the last century, the utilization of polymers has rapidly increased, leading to the development of techniques used to produce polymer fibers to satisfy the high-performance requirements of different industries and modern applications . NanofibTissue engineering is focused on fabricating scaffolds that can temporarily substitute for the native extracellular matrix (ECM), to guide and regenerate specific tissue functions. Tissue engineering technologies encompass rapidly advancing techniques, which combine features and advantages of biochemicals/biomaterials sciences and transplantation of cells to generate bioartificial tissues or organs to cure damage to skin , cartilaNanoparticles suffer from aggregations in liquid during use forming a slurry, and these powders are difficult to remove after treatment operation, which reduces their applicable viability. In this respect, nanofibers introduce a valuable substrate for the nano-powder, whether the nanoparticles are embedded into  or decorAlthough there are a great many published reviews that represent the applications of electrospun nanofibers in particular fields, such as wastewater treatment ,29,30,31Electrospinning is a simple, cost-effective, and highly versatile technique used to fabricate a layer of nanofibers with diameters of 3.0–>5.0 nm, in the presence of a high-voltage electric field used on injected polymer solutions to stretch the droplets ,53,54,55Consequently, this technology has attracted great attention in fabricating different polymer ultrafine fibers . RecentlRecently, the fabrication of biomimetic scaffolds, using electrospun nanofibers, has attracted great attention regarding its use in tissue engineering applications. A densely packed 2D electrospun nanofibrous scaffold is considered a limitation impeding utilization in regeneration of tissues and organs . These cFabricated electrospun nanofibers have promising characteristics to use in biomedical applications, where they have the ability to be assembled in several shapes of 2D and 3D structures, with considerable surface/volume ratios, porosity, and adaptable pore sizes, and, furthermore, they can mimic fibers in a natural extracellular matrix.According to the chemical composition of the fabricated nanofibers, they can be classified into four major types: 1—Inorganic nanofibers, 2—Carbon nanofibers, 3—Polymer-based nanofibers, 4—Composite nanofibers . All of During the last decade, electrospun nanofibers have attracted tremendous attention from researchers in materials science, nanotechnology, environmental science, and biomedical applications. Nanofibers’ superior characteristics and functions have attracted great attention and widespread application in different emerging fields. Their morphologies, chemical characteristics, and spatial distributions determine the potential application and functional performance ,57. TherDue to the efficiency and promising properties of nanofibers in the treatment and curing of medical complications, like bone cell proliferation, vascular scaffolds, and skin tissue engineering, recent developments in nanofibers have made great progress in biomedical fields ,57. ConsBone cell proliferation processes are complicated and have many defects where self-regeneration is restricted. Therefore, scaffolds are widely utilized in replacement and regeneration processes for damaged bone tissues. During the last decade, various types of bionanomaterials have been used to design porous scaffolds that mimic the structure of the original ECM ,59,60. TTo culture the bone marrow endothelial progenitor cells (BEPCs), two varied hybrid scaffolds, composed of collagen/polycaprolactone (PCL) (70:30%) and gelatin (Gel)/PCL (70:30%), were prepared by electrospinning . BEPCs wRecently, gelatin nanofibers have acquired considerable attention due to their promising characteristics as eco-friendly materials and they have considerable influences on cell adhesion, proliferation, and differentiation of diverse tissues . Glucosed,l-lactide, gelatin, and RKKP glass-ceramics were used to form a tricomponent electrospun scaffold. The bioactivity RKKP glass-ceramic system arises from La3+ and Ta5+ ions, which act as growth supporting agents for bone. RKKP glass-ceramics have encapsulated the microfibers of electrospun scaffolds, and the results confirm its homogenous distribution in the fibrous composite.Moreover, the in vivo cranial bone regeneration investigation found that cross-linked gel/zein scaffolds showed high positive effects on the regeneration of cranial bone with enhanced new bone volume and connective tissue formation. On the other hand, the presence of zein in the gelatin scaffolds did not favor the regeneration mechanism. Furthermore, cross-linking of gelatin scaffold decreases bone resorption, which hinders osteoclasts’ differentiation . Also, p3+ and Ta5+, in the RKKP incorporated polymeric nanocomposite scaffolds could improve cell growth and allow differentiation , were prepared by an electrospinning strategy for bone tissue engineering. In detail, PANI-co-PDA was prepared through a one-step chemical oxidization technique. After that, d,l-lactaide monomer was blended onto the PDA part via a ring-opening polymerization (ROP) to form PANI-co-(PDA-g-PLA) terpolymer. Finally, a solution of the prepared terpolymer was electrospun to form a conductive nanofiber scaffold. The experimental results showed enhancement in physicochemical properties, such as mechanical properties, conductivity, electroactivity, hydrophobicity, and morphology, as well as biological features involving biocompatibility, biodegradability, and improvement in the cells’ attachment and proliferation , many methods have investigated mimicking natural vascular 3D architecture to guide SMC action . In thisogenesis , Figure Also, a stromal cell-derived factor-1-alpha (SDF-1α)-aligned-(SF)/3D porous bladder cellular matrix graft (3D-BAMG) hybrid scaffold for long-section ventral urethral construction and repair in vivo was developed, as represented in Considering the complexities of the native artery wall structure and the challenges facing present treatment strategies, many works have been investigated to develop biomimetic tri-layers tissue-engineered vascular grafts . A bio-iOn the other hand, nanofibrous polyurethane scaffolds immobilized with resveratrol drug were prepared and evaluated towards viability of endothelial and smooth muscle cells. The results indicated that the loading of resveratrol significantly enhanced tensile strength and Young’s modulus of the scaffold and they became closer to native vessels. The release profile resveratrol presented smooth release from the nanofibrous scaffolds. In addition, resveratrol-loaded nanofibers possessed increase in anti-thrombogenicity with respect to polyurethane alone, enlarging time of human blood clotting and decreasing hemolysis. Moreover, the resveratrol-immobilized nanofibers resulted in suitable antithrombotic function and composition of a monolayer on the scaffold surface of endothelial cells and decreased the growth of smooth muscle cells .Generally, electrospinning technology succeeded in developing various types of nanofibrous scaffolds and creating tubular scaffolds which are utilized in vascular tissue engineering. Published works have investigated the performance of artificial vascular grafts over the last years and the considerable progress achieved in large-diameter ones. Many challenges still remain for small diameter vascular tissues, due to various reasons, such as thrombogenesis or intimal hyperplasia. Also, there are challenges to develop an engineered scaffold graft that increases bioactivities and can effectively and quickly reconstruct urethral defects and rebalance the epithelialization of the urinary tract and other smooth muscles . TherefoSkin is the outermost and the most important barrier organ in the body and plays a vital role in protection of internal tissues from external damage ,97,98. TPolymeric nanofibers can be incorporated with nanofillers, such as nanoparticles and nanotubes, to improve their superior properties and enhance applications in skin tissue engineering. Electrospun nanofibers of glucose (G)-reduced graphene oxide (rGO) (0–1.0 wt%) blended with PVA to form PVA/GrGO scaffolds, referred as (PG) scaffolds, and crosslinked chemically with acidic glutaraldehyde (GA) in acetone medium to mimic the (ECM) to apply in skin tissue engineering. Further increase in concentrations of G-rGO in PG scaffolds cause a decrease in tensile strengths and elongations, increasing the thermal properties. The biological properties of PG scaffolds were examined using in vitro hemolysis, using CCD-986Sk (a human skin fibroblast cell line). Results indicated G-rGO incorporation in PVA nanofibers induced a small shift from hydrophilic to hydrophobic. Moreover, the PVA/G-rGO scaffolds did not possess hemolysis of red blood cells, even at a G-rGO immobilizing of 1.0 wt%, and PG-1.0 scaffold (with a GRGO loading of 1.0 wt%) there was good compatibility with fibroblasts and highly increased metabolic activity after seed for twenty-one days, as compared with PG-0 controls .Another nanofiber scaffold was synthesized from PU and cellulose acetate via electrospinning. RGO/Ag nanocomposite and/or curcumin was incorporated with the materials attributed to the antibacterial activity of rGO/Ag-nanocomposite. The scaffolds could prevent both of Gram-negative and Gram-positive bacteria via direct contact with them. In vivo histopathological experiments demonstrated that the scaffolds of the blended rGO/Ag nanocomposites and curcumin showed excellent wound healing and could promote the healing rate of artificial wounds, which provides considerable biomedical potential for nanomaterials in wound healing . DevelopEscherichia coli and Staphylococcus aureus. A schematic illustrates the fabrication of PVA/Chi/SS-TC electrospun nanofibers was represented in On the other hand, many works illustrate nanofiber materials containing sericin being utilized to reconstruct epidermal–dermal tissues . TherefoN-α-amido-glutamic acid) 1,7 heptane tetracarboxylate and functionalized with a mix of the tyrosine-rich amylogenin peptide sequence MPLPPHPGHPGYINF followed by polygalacturnonic acid and HP derived from salmon or red-snapper fish scales. The scaffolds-based salmon scale derived HP exhibit larger mechanical strength and Young’s Modulus than snapper scale-derived scaffolds, as shown in New peptide-based composite scaffold was fabricated by incorporating two varied fish scale-derived hydroxyapatite with modified peptide nanofibers to apply in periodontal tissue regeneration. The nanofibers were synthesized by self-immobilization of the newly constructed peptide bolaamphiphile Bis  nanofiber scaffold. The fabricated nanofibers, PLA/CA, exhibit a homogenous surface morphology with elongation at break more than pure PLA). The addition of CA increases the hydrophilicity of polylactic acid nanofibers, as well as supporting cell attachment of periodontal ligament cells (PDLCs) and bone marrow stromal cells (BMSCs). Moreover, the calcium alginate enhances the cell mineralization gene’s expression level and the composition of mineralization knots (BMSCs). On the other hand, both neat polylactic acid nanofibers and polylactic acid/calcium alginate nanofibers could enhance the expression level of inflammatory mediators and (TLR4) of human periodontal ligament cells (hPDLCs) .v/v mixture of concentrated sulfuric (H2SO4) and nitric acid (HNO3) for various periods. The characterization results exhibit the presence of –COOH and –OH groups due to the oxidation process. Furthermore, a composite polycaprolactone (PCL), as a matrix, and the CNF, as the reinforcement, was fabricated. The storage module of the acid treated CNFs was enhanced compared with untreated CNFs. Furthermore, the scaffolds of PCL/oxidized CNFs improve the cell proliferation [Inorganic/organic compositions incorporated into porous biomaterials could be convenient for skin tissue engineering. Therefore, much research has been done to study the suitability of carbon nanofibers (CNFs) in utilization as a reinforcement agent in the enhancement of bone scaffolds . Therefoferation .Staphylococcus aureus was carried out in vitro and explored the inhibition zone to be two folds of the control group treated with a model aminoglycoside antibiotic, tobramycin [In some particular wounds, such as nonhealing diabetic ulcers, bioactive glass (BG) materials have been recognized to bond well with hard and soft tissues. The reactions of such bioactive materials with biological fluids can enhance the vascularization of endothelial cells (ECs) and increase osteogenesis and angiogenesis . In thisbramycin .E. coli compared with the ciprofloxacin activity, while it monitored 43.0% for S. aureus. Furthermore, increasing the deposition time of nanoparticles for AgNPs@Se-CHA/PCL improves both cell growth and bacterial inhibition [Hydroxyapatite (HA) is an important biomaterial and has unique properties in versatile biomedical applications such as skin tissue engineering and recently in wound healing. Therefore, many works have studied the antibacterial behavior of HA structure and the influence of silver (Ag) ionic dopant into HA structure and their antibacterial behaviors . Therefohibition .Recently, various types of biomaterials have been fabricated and investigated to apply in skin tissue engineering applications, especially for serious skin defects. Many works investigate nanofibrous scaffolds of chitosan (Chi), and its compounds as a wound dressing, due to its multiple characterizations. Therefore, further modifications for chitosan nanofibrous scaffolds have been carried out to enhance its broad-spectrum antimicrobial activity ,107,108.Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). Meanwhile, they support the proliferation of pig iliac endothelial cells (PIECs). Rats’ wounds treated by GO-coated Chi/PLLA nanofibrous scaffolds were more excellently cured than other groups on pathological section [Due to the unique surface properties of graphene oxide (GO), it is widely used as a new antimicrobial agent to prevent bacterial infections . Graphen section . Further section . Also, d section . Since m section .Also, electrospun curcumin (Cur)-mixed Chi/PVA/carbopol/PCL nanofibrous composite for concurrent delivery of the buccal fat pad-derived mesenchymal stem cells (BFP-MSCs) and Cur to a full-thickness wound on the mouse model. Results showed that the prepared composite scaffolds have an affinity for cell culturing and promote their growth and proliferation, as shown in E. coli and S. aureus were 98.87% and 98.26%, respectively, while the SIS provided no antibacterial ability. Finally, in vivo experiments presented that the Chi/ES-SIS composite could support tissue repair upon implantation without any inflammatory reaction [A biomimetic small intestinal submucosa (SIS) -based biocomposite (Chi/ES-SIS) for abdominal wall healing, chitosan (Chi), and elastin (ES) electrospun nanofibers were utilized to develop biodegradability, antibacterial activity, and angiogenesis. The results showed that the dialysis rate of Chi/ES-SIS composites was lower by about 24.5% than that of SIS. Moreover, the Chi/ES-SIS composites exhibited high microstructure stability and micromechanical characteristics compared with SIS. The antibacterial activity of Chi/ES-SIS composites towards reaction .Gelatin is a hydrophilic, adhesive, and biocompatible polymer widely utilized in wound healing systems and as an absorbent pad during surgery, where it is a bio-protein that contains high amounts of proline, glycine, and hydroxyproline ,114,115.l-lactide-co-d,l-lactide (PLDLLA) and PVA, including triclosan (Tri), as antibacterial drugs were developed. The results showed that the hybrid material exhibited improved hydrophilicity with water contact angle (WCA) of 53° over the blend material with WCA of 73° which was further confirmed by a swelling test. The antibacterial performance presented excellent results for hybrid-Tri with inhibition zones of 35 mm and 48 mm for E. coli and S. aureus, respectively. Also, the hybrid membrane exhibited significant mechanical behavior. Furthermore, the SNL 76/7 fibroblast cell line culture indicated that the hybrid-Tri nanofibrous sample possessed more excellent proliferation performance than the blend-Tri sample attributed to minimal cytotoxicity and maximal cell viability by MTT and acridine orange/ethidium bromide staining [Also, cinnamon (cin) was immobilized into poly(ε-caprolactone)/gelatin (PCL/Gel) nanofibrous matrices to produce a suitable material to enhance wound healing. Fabricated materials with cinnamon and free of cinnamon were employed to cure full-thickness excisional wounds in Wistar rats. The findings provided that the cinnamon content directly induced porosity, mechanical features, swelling, water contact angle, rate of transmission of water vapor and cell proliferation. Moreover, the data obtained from the in vivo test demonstrated that wound closure was (98%) after 14 days curing with PCL/Gel 5%, which was the best among other groups . Anotherstaining .On the other hand, fabricated electrospun nanofibers that contain gelatin were investigated in wound healing applications as composite scaffolds, and as efficient artificial biomimetic skin substitute nanofiber membranes in vitro and in vivo studies ,117,118.Moreover, biomimetic skin substitute properties of PLLA/Gel nanofiber membranes were evaluated in facilitating chronic cutaneous wound healing. The material physical and biological characteristics were evaluated. Clinical applications further confirmed safety and effectiveness. The proliferation of fibroblasts and monocytes was highly enhanced at the seventh day seeded on the substitute with obviously inhibited bacterial growth. In the clinical case experiment, the wound healing closed with 93.3%, and the recovery rate was 6.7% . Also, bw/v for PMS: PLA 60:40A homogenous nanofibers of size 235 ± 38 nm resulted. The in vitro and in vivo biocompatibility tests provide high cytocompatibility, proliferation, and tissue responses of PMS: PLA nanofibrous scaffolds with suitable interactions between cell and scaffold. Moreover, inflammatory responses of PMS: PLA nanofibrous scaffolds were negligible [Bio-based PLA polymers are becoming the most important biomedical polymers due to their promising properties, such as considerable cytocompatibility, biodegradability, processability, and mechanical characteristics . Thus, tgligible .Clinical research illustrated that keratin can stimulate keratinocyte cells existing in the wounds and activate these cells to quickly enter a hyper-proliferative phase . TherefoRecently, elastomers have been extensively used to investigate cell physiology in fields such as mechanobiology, but their undesirable properties, such as intrinsic high hydrophobicity, render its surface discordant with prolonged cell adhesion and proliferation. So, many works have been done to improve the mechanical and chemical stability bonding between an elastomeric bulk substrate and electrospun nanofibers, limiting the presence of fibers to a thin interfacial layer . To achil-lacticco-ε-caprolactone (PLCL) to form nanofibrous scaffolds via electrospinning. Spidroins consisted of an N-terminal module (NT) from major ampullate spidroins, a C-terminal module (CT) from minor ampullate spidroins and 1-4 repeat modules (W) from aciniform spidroins. The increase in W modules content will lead to increase in both the tensile strength and elongation of blend scaffolds. In vitro experiments, using human umbilical vein endothelial cells (HUVEC) seeded on NTW4CT/PLCL (25/75) scaffolds possessed higher activity of proliferation and adhesion than on pure PLCL scaffolds [During the last decade, many biomaterials have attracted great attention. One of these promising biomaterials is silk fibroin (SF) which is a natural protein, has abundant supply, unique biological characteristics and can be reformed into various structures . Biomimecaffolds .Another vital adhesive protein that has attracted considerable attention and been utilized as material surface modification is 3,4-dihydroxyphenylalanine (DOPA) and its derivatives which are extracted from marine mussels . DOPA waTo circumvent many limitations facing the applications of some electrospun nanofibers, such as poor mechanical properties, an online suture approach to prepare electrospun polycaprolactone (PCL) fibrous yarns incubated with both collagen (COL) and bFGF growth factor, to prepare bFGF-COL@PCL sutures was devised. The in vivo experiments revealed that, as compared to commercialized vicryl suture, bFGFCOL@ PCL sutures highly support wound healing at various stages by decreasing granulation tissue building time, collagen formation, and re-epithelialization. The improved wound healing activity of bFGF-COL@PCL sutures is referred to two synergistic factors: (i) the excellent-oriented nanofibrous texture minimizes tissue hindrance to reduce their trauma and (ii) the introduction of both collagen and bFGF improve the basement membrane (BM) reconstruction, cell proliferation, and angiogenesis . Also, eRecently, due to the unique physicochemical properties of graphene and its derivatives, it has been widely used as an additive in the field of biomaterials to improve mechanical performance, hydrophilicity, and interaction between cell and scaffold . Highly 2 aqueous solution. Mechanical experiments indicated that the addition of CNC improved the tensile modulus by 65%.Due to the versatile properties of (PVA), such as its nontoxicity, water-soluble synthetic-polymers, considerable biocompatibility and eventual biodegradability in the body, it has great attention as a hydrophilic polymer in biomedical applications ,131,132.Moreover, the crosslinked samples raised elongation at break and improved the sample wettability. In addition, Cell NIH/3T3 viability was highly enhanced (90%) with the addition of CNC to PCL-CaAlg nanofibers . AnotherN-acetylcysteine to compose of controlled release and chemically crosslinked COL/NAC (N-acetylcysteine) hybrid with PA nanofibers to improve the mechanical function of COL and prepare this multi-layered (PA-COL/NAC) scaffold. The results showed that the prepared scaffolds had an excellent porous structure and swelling properties. Moreover, the PA-COL/NAC scaffold could simply release NAC over fourteen days. After implantation of the cell, the PA-COL/NAC scaffold exhibited enhanced cell proliferation and migration over the other groups. In vivo, PA-COL/NAC scaffolds could support wound dressing better than all other groups [H. perforatum oil blende zein film containing MMT and the bottom layer was composed of 3D electrospun zein/MMT nanofibers to stimulate wound dressing with controlled release of H. perforatum oil. Results indicated that the produced monolayer films exhibited suitable mechanical and gas barrier features and surface wettability for wound dressing. H. perforatum oil was released from the prepared membranes up to 48 h. Bilayer metrics presented antimicrobial behavior against E. coli, S. aureus, and C. albicans without any toxic effect on NIH3T3 mouse fibroblasts and HS2 keratinocyte cell lines. In vitro scratch analysis results demonstrated that H. perforatum oil had a wound healing activity by encouraging fibroblast migration. The proliferation tests presented an increase in fibroblast proliferation on H. perforatum oil assembled bilayer membranes [The mechanical characteristics of the scaffolds are critical factors. Much work has been done to develop alginate nanofibers. A two-step approach for coaxial electrospinning and post electrospinning is an efficient experiment for preparing superfine nanofibers with highly swellable hydrogels. Alginate and PCL were co-electrospun through fibrous meshes with a coaxial nozzle; alginate was encapsulated in PCL and then cross-linked in calcium chloride solution. Polyε-caprolactone shell was dissolved from the meshes by washing with organic-phase. The cross-linked hydro=nanofiber possesses high stiffness and Derjaguin–Müller–Toporov modulus. Moreover, NIH 3T3 cells seed on the material presented deeply infiltrated to the bottom of the mesh . Also, ar groups . Recentlembranes . Also, aembranes .2O2) released from Inflammation or infectious wounds may prevent wound healing processes. Also, such reactive oxygen-species (ROS) have a considerable pivotal effect in healing processes, cascading at multiple stages [2O2-responsive smart dressing, prepared by mixing Eu CPs into (PAN) nanofibers meshes, where europium (Eu3+) Coordination Polymers (Eu-CPs) showed properties to determine the level of H2O2 and allow wound healing. Therefore, this theranostic wound dressing can assay H2O2 level of the wound microenvironment via a facile visible color change. In addition, in vitro cytocompatibility tests showed that human umbilical vein endothelial cells (HUVEC) and fibroblast cells (L929) could well adhere and proliferate onto the PAN-Eu-CPs nanofibers materials. Moreover, in vivo animal experiments provided that PAN-Eu-CPs nanofibers materials could reduce wound healing period by allowing neovascularization performed by immunohistochemical analysis [High levels of hydrogen peroxide (He stages . Therefoanalysis .Recently, developments in the fabrication of multifunctional electrospun nanofiber nanomaterials have significantly contributed to the enhancement of wound healing scaffold nanomaterials with notable potential for skin regeneration in vitro as well as in vivo. On the other hand, one of the biggestt challenges facing the wound healing during the last years is development of a novel smart wound dressings which have the ability to interact with the wounds and effectively facilitate wound healing with consiedrable sensing characteristics and multiple smart properties using smart nanomaterials as well as stimuli-responsive and self-healing nanomaterials .Finally, In this review, we discussed recent progress and potential applications of electrospinning nonofibers in biomedical applications. Also, this review represents enormous promising works which have recently been done to develop and improve the utilization and application of electrospun nanofibers.Nowadays, electrospun nanofibers with superior performance have illustrated considerable and promising results for biomedical applications. Their unique characteristics makes them convenient to apply in various fields. However, fabrication of nanofibers with distinct morphological and superior properties and considerable yield is still challenging. Such fabricated nanofibers can be functionalized through various techniques to widespread application in different strategic fields. Hence, considerable developments have been achieved even though there are still various challenges. Much effort has been put into improving desired performance of fabricated electrospun nanofiber and functionalized nanofiber application in specific areas. However, further work still needs to be done to obtain more promising results and eventual practical electrospun nanofibrous materials by advanced electrospinning techniques and scaling up the fabrication of nanofibers from the laboratory to commercial scales, especially for biomedical applications. The development of nanomaterials/biomaterials from biodegradable, biobased electrospun nanofibers is imperative to improve the applications of electrospun nanofibers in diverse fields, such as biomedical applications.Also, electrospun nanofiber materials can play a vital role to overcome many challenges facing tissue engineering technologies in regeneration of tissues and organs where the scaffolds fabricated by electrospun nanofibers materials can mimic 3D with other physiological properties of tissues and organs in vitro to then transplant in vivo. Also, they are promising materials to regenerate injured nerve through tissue engineering technologies. Another serious challenge facing tissue engineering technologies to mimic and fabricate nerve grafts is losing the structural integrity, or biological functionality, of some natural polymer nanofibers used in a media simulating that in the human body. Therefore, further intensive research is required to explore and fabricate new materials to overcome such challenges. Using nanomaterials has many disadvantages, such as aggregation, uncontrollable release, costs, and potential cellular toxicity. Therefore, further research is needed to avoid such disadvantages.On the other hand, current utilization of electrospun nanofibers in biomedical applications, such as bone cell proliferation, nerve regeneration, vascular tissue, and skin tissue engineering has been demonstrated. Many of these results were obtained for small rodent models which may be accompanied by restrictions regarding their immunological responses compared with humans. Therefore, these studies must be confirmed with suitable, and larger, animal models.