The catecholamine neurotransmitter dopamine has the potential to act as an endogenous neurotoxin when its vesicular sequestration is dysregulated. Despite postmortem analyses from patients with Parkinson’s disease that demonstrate decreased vesicular sequestration of dopamine with a corresponding increase in dopamine metabolism, dopamine’s contribution to nigrostriatal dopaminergic degeneration in Parkinson’s disease has been debated. Here, we present a new in vivo model demonstrating the induction of Parkinson’s disease-associated pathogenic mechanisms of degeneration resulting from acquired dysregulation of dopamine sequestration in nigrostriatal dopaminergic neurons in adult rats. Utilizing adeno-associated virus (serotype 2), viral-mediated small-hairpin RNA interference of endogenous vesicular monoamine transporter 2 (VMAT2) expression resulted in a loss of VMAT2 protein expression in transduced dopaminergic cell bodies in the substantia nigra with a corresponding loss of VMAT2 protein within the striatal terminals. The loss of VMAT2 resulted in an accumulation of cytosolic dopamine and subsequent increased dopamine metabolism, deficits in dopamine-mediated behaviors, and degeneration of nigrostriatal dopaminergic neurons that was rescued through reintroduction of exogenous VMAT2, demonstrating that the toxicity was specific to the loss of VMAT2. Analysis of parkinsonian pathogenic mechanisms of degeneration identified oxidative damage, activation of Parkinson’s disease-associated kinase LRRK2, and the formation of aberrant α-synuclein. This model demonstrates that a progressive acquired loss of VMAT2 expression in adulthood is sufficient to induce Parkinson’s disease-associated pathogenic mechanisms of degeneration and provides a new model to further investigate the consequences of cytosolic dopamine. While multiple neuronal populations are affected in Parkinson’s disease, the nigrostriatal dopaminergic neurons appear to have increased vulnerability to degeneration and the loss of these neurons results in the cardinal motor symptoms that lead to a diagnosis of Parkinson’s disease. It is hypothesized that dopamine, the neurotransmitter these neurons use to regulate motor output, contributes to the vulnerability to degeneration; this is thought to be due to dopamine’s potential to act as an endogenous neurotoxin through two processes that generate reactive metabolites and reactive oxygen species: oxidation and enzymatic catabolism4.Parkinson’s disease is a progressive neurodegenerative disease characterized by motor deficits resulting from the degeneration of dopaminergic neurons in the nigrostriatal pathway. Postmortem analyses of brains from Parkinson’s patients show a loss of neuromelanin containing neuronal cell bodies in the substantia nigra and a progressive loss of dopaminergic terminals in the striatum. Within remaining neurons, there is evidence of neuronal dysfunction in the form of protein inclusions called Lewy bodies—a primary component of which is aggregated α-synuclein. Parkinson’s disease cases resulting solely from genetic factors are rare, and the majority of cases are believed to be the result of complex interactions between predisposition genes, a lifetime accumulation of environmental exposures, and deficits due to normal aging processes2O2 or superoxide as a byproduct5. The dopamine quinone will bind to free and protein-bound nucleophilic cysteine, as well as highly nucleophilic selenocysteine found in selenoproteins, such as the mitochondrially-localized antioxidant selenoprotein glutathione peroxidase 4 (GPx4)6. The dopamine quinone has been shown to impair several neuronal processes with mitochondrial dysfunction being a common downstream consequence10. If dopamine does not undergo oxidation, it is susceptible to enzymatic catabolism, which generates the metabolite DOPAL while producing H2O2 as a byproduct11. Similarly to the dopamine quinone, upon accumulating in the cytosol, DOPAL will react with proteins and generate additional reactive oxygen species as a byproduct12. To minimize the accumulation of cytosolic dopamine to preserve neuronal health, there exist physical interactions between dopamine synthesis, packaging, and reuptake machinery14. Following synthesis in the cytosol or presynaptic terminal reuptake, dopamine is rapidly sequestered into synaptic vesicles by the vesicular monoamine transporter 2 (VMAT2) thereby isolating and stabilizing dopamine. However, there is significant evidence of VMAT2 deficiency in Parkinson’s disease, which implicates dysregulation of dopamine handling in the development and/or progression of the disease17.Dopamine oxidation occurs either by autooxidation or enzymatic oxidation and generates the highly reactive and electrophilic dopamine quinone, producing either H15. Furthermore, VMAT2 isolated from the striatal terminals from postmortem tissue of Parkinson’s patients shows an impaired rate of dopamine uptake, suggesting that in addition to a loss of VMAT2, the remaining VMAT2 is less efficient at sequestering dopamine16. Accordingly, analysis from postmortem tissue of Parkinson’s disease patients demonstrates increased cytosolic dopamine metabolism as evidenced by an increase in the amount of the dopamine metabolites DOPAC, HVA, and DOPAL relative to the amount of dopamine18. In addition, VMAT2 mRNA is decreased in circulating platelets from Parkinson’s disease patients, which suggests a systemic deficiency in VMAT2 involved in the pathogenesis of Parkinson’s disease17. Testing the hypothesis that a deficiency in VMAT2 contributes to the development of Parkinson’s disease, a genetic mouse model investigated the consequence of systemic VMAT2 deficiency and revealed the replication of motor and nonmotor Parkinson’s symptoms as well as age-dependent dopaminergic neurodegeneration20. Building on fundamental in vivo and in vitro experiments investigating the neurotoxic consequences of cytosolic dopamine, here we present a new in vivo model of acquired dopaminergic dysregulation through viral-mediated interference of VMAT2 expression in adult rats.Analysis from postmortem tissue from Parkinson’s disease patients revealed decreased expression of VMAT2, which suggests a deficiency in synaptic vesicle dopaminergic sequestrationt-test, n = 37 cells in shControl treatment and 29 cells in shVMAT2 treatment, p < 0.0001)   was targeted by ribonucleic acid interference using a short-hairpin RNA (shRNA) in a dopaminergic neuronal cell line (RCSN-3) derived from the midbrain of an adult rat as the initial step to test the feasibility of knocking-down VMAT2 expression in adult animals. Transfections with shVMAT2 were performed with coadministration of GFP to identify transfected cells, and efficiency of shVMAT2-mediated knockdown was quantified compared to cells transfected with a scramble shRNA (shControl). Analysis of VMAT2 protein immunoreactivity at 48 h post-transfection demonstrated a 41.4% decrease compared to shControl transfected cells    under the CMV promoter. AAV2-shVMAT2 virus was administered in vivo through unilateral injections in male adult Lewis rats targeting the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Analysis performed at 6 weeks post-transduction demonstrated robust GFP immunoreactivity in tyrosine hydroxylase (TH) immunoreactive neurons in the SNpc indicating efficient viral transduction of dopaminergic (TH-positive) neurons Fig. . VMAT2 i05) Fig. . Howevers.) Fig. . In addis.) Fig.  with mais.) Fig. .Fig. 1sht-test, n = 5 animals, p = 0.0012)   Fig. . There w12) Fig. . This co15) Fig. .Fig. 2sht-test, n = 4 animals, p = 0.0433)    Fig.  left; ho38) Fig.  middle. 09) Fig.  right.t-test, n = 5 animals, p = 0.0177)   demonstrated no loss in TH-positive neurons, however a significant loss of TH-positive neurons was observed at 8 and 12 weeks post-transduction . Striatal dopamine levels showed a further decrease of −50.37% and −78.52% at 8 and 12 weeks, respectively   Fig.  left. St46) Fig.  middle. 22. The forepaw contralateral to AAV2-shVMAT2 (affected paw) had a 78.0% increase in distance to trigger compared to the forepaw associated with the AAV2-shControl injected hemisphere, which performed at 2.5 cm    Fig. . As an a01) Fig. . These d24. Decreased VMAT2 expression resulted in a 26.7% increase in 4HNE within transduced TH-positive neurons as compared to nontransduced TH-positive neurons in the SNpc , whereas neurons transduced with AAV2-shControl showed no increase in the amount of 4HNE  , whereas neurons transduced with AAV2-shControl showed no increase in the amount of 3NT   and 4-hydroxynonenal (4HNE) are markers of oxidative damage and have classically been used as an index of oxidative stress within neurons25. Recently we have shown that proximity ligation assay (PLA) can be used to quantify the amount of autophosphorylated LRRK2, which is an indirect measure of LRRK2 activity25. The immunoreactivity of autophosphorylated LRRK2 was increased within transduced TH-positive neurons by 28.4% compared to nontransduced neurons in the SNpc      compared to the nontransduced hemisphere  Fig. . As an a13) Fig. . Consistt-test, n = 3 animals, n.s.)   or pSer129-syn compared to nontransduced TH-positive neurons in the SNpc    Fig. , phospho14) Fig. . In cont49) Fig. . Althougn = 4–6 experimental replicates, *p < 0.05, **p < 0.01, ***p < 0.001) . The construct, first tested in vitro, expresses human VMAT2 with a myc-DDK tag. Co-transfection of RCSN-3 cells with shVMAT2 and wild-type VMAT2 (wtVMAT2) did not rescue VMAT2 immunoreactivity. However, co-transfection with shVMAT2 and kdrVMAT2 restored VMAT2 immunoreactivity    Fig. . Fluores46) Fig. . These dA summary of animal cohorts analyzed in this study is shown in Supplementary Table Although the contribution of dopamine to the pathogenesis of Parkinson’s disease has been controversial, there is a wealth of in vitro and in vivo literature demonstrating toxic consequences to dysregulated cytosolic dopamine and dopamine oxidation. Here, we demonstrate that acquired dopamine dysregulation in adulthood is sufficient to induce Parkinsonian degeneration in rats. Through viral-mediated RNA interference of VMAT2 expression, a decrease in VMAT2 immunoreactivity is observed in dopaminergic cell bodies in the substantia nigra pars compacta and within striatal terminals. As an index of cytosolic dopamine following VMAT2 knockdown, analysis of striatal tissue demonstrated a loss of dopamine with a corresponding increase in dopamine turnover, as measured by the ratio of DOPAC to dopamine, as well as an increase in dopamine quinone formation, as measured by protein cysteinyl-dopamine adducts.Stereological analysis performed by identifying overlap of TH, microtubule-associated protein 2 (MAP2), and DAPI demonstrated a loss of dopaminergic neurons with a corresponding focal loss of TH in the dorsolateral striatum that is characteristic of dopaminergic degeneration. There was an extensive loss of MAP2 immunoreactivity across the substantia nigra, which is indicative of dopaminergic degeneration rather than only a loss of TH-phenotype. Furthermore, this degeneration was shown to be time dependent as analysis performed at earlier timepoints demonstrated dysregulation in dopamine packaging before the onset of degeneration. Replicating what has been previously demonstrated in rodent models of Parkinson’s disease, the nigrostriatal dopaminergic degeneration resulted in deficits in two dopamine-mediated behaviors commonly used to measure motor deficits. Furthermore, pathogenic mechanisms associated with Parkinsonian degeneration were evaluated within the remaining transduced TH-positive neurons and demonstrated an increase in LRRK2 activity as well as the formation of aberrant α-synuclein. Importantly, the neurodegeneration following knockdown of endogenous VMAT2 expression was able to be prevented by reintroducing exogenous VMAT2 expression. Our data suggest the induction of Parkinson’s-associated pathogenic mechanisms of degeneration as a result of direct interactions with cytosolic dopamine and its metabolites, and from the production of reactive oxygen species due to cytosolic dopamine as summarized in Fig. 27. Additionally, VMAT2 hypomorph mice that express 5% of the normal amount of VMAT2 showed Parkinsonian dopaminergic degeneration in aged mice (18 months), as well as the replication of other features Parkinson’s disease28. In an animal model dysregulating dopamine homeostasis by targeting the plasmalemmal dopamine transporter (DAT), overexpression of DAT resulted in increased in dopamine metabolism associated with a loss of TH-positive neurons in the SNpc, motor deficits, and increased susceptibility to dopaminergic toxins29. Furthermore, Chen et al. (2008) demonstrated that introducing the DAT into non-dopaminergic striatal neurons that lack VMAT2, and therefore the machinery required for proper sequestration, resulted in oxidative damage and neurodegeneration, providing further evidence that dopamine without proper sequestration can induce non-dopaminergic neurotoxicity30. Given the nature of the mechanism of knockdown employed in our model, VMAT2 is lost gradually over time as shRNA blocks the translation of new VMAT2 and old VMAT2 is degraded. Therefore, in comparison to these previously established models with lifelong alterations in dopamine handling, the model we characterize here demonstrates the consequences of an acquired loss of VMAT2 expression in adulthood.Previous research has investigated the consequences of genetically mediated dysregulation of dopamine handling. While VMAT2 knockout mice die shortly after birth, experiments performed in heterozygote VMAT2 knockout mice show increased susceptibility to dopaminergic toxins31. However, a study published by Xiong et al. in 2016 identified two polymorphisms in SLC18A2 within a Parkinson’s disease cohort32. Furthermore, rare mutations in SLC18A2 result in infantile Parkinsonism35. In contrast, a number of studies have identified variants outside the coding region of SLC18A2 that increase expression of VMAT2 resulting in a gain-of-function and decreased likelihood of developing Parkinson’s disease38. These studies suggest that increased sequestration of dopamine can be protective against the development of Parkinson’s disease. Accordingly, when tested in vivo, mouse and drosophila models overexpressing VMAT2 demonstrate protection against dopaminergic neurotoxins and Parkinson’s disease-associated environmental toxins, further supporting the hypothesis that proper dopaminergic handling is required to maintain neuronal health42.Until recently, analysis of the gene (SLC18A2) for VMAT2 within Parkinson’s disease cohorts was unable to conclude that variants in SLC18A2 result in increased likelihood of Parkinson’s disease18. Previous work has also shown that the sequestration of dopamine is impaired downstream of Parkinson’s disease-associated environmental factors, and many studies have identified dysregulation of dopamine sequestration and release downstream of other Parkinson’s-associated genetic factors including misfolded α-synuclein, VPS35 variants, and LRRK2 variants49.It is possible that independently of genetically derived alterations in VMAT2 expression, other factors may result in altered VMAT2 expression, thereby contributing to the propagation of the disease process through further oxidative stress and interactions with Parkinson’s disease-associated mechanisms of degeneration. Centrally, analysis from postmortem tissue identified decreased striatal VMAT2 immunoreactivity as well as decreased VMAT2 activity from isolated synaptic vesicles, whereas peripheral analysis revealed decreased VMAT2 mRNA in circulating platelets from Parkinson’s disease patients suggesting a possible systemic deficiency in Parkinson’s disease pathogenesis57. Previous experiments have suggested dopamine mediates α-synuclein-induced toxicity both in cell culture and animal modeling experiments, and other recent studies have implicated calcium as a susceptibility factor mediating dopaminergic neuron vulnerability to α-synuclein59. Furthermore, Sang et al. (2007) demonstrated the bidirectional influence of VMAT on parkin-mediated parkinsonian phenotypes in drosophila. This study showed exacerbated parkinsonian toxicity in drosophila deficient in VMAT and with Parkinson’s-associated parkin mutations, whereas overexpression of VMAT attenuated mutant parkin-associated toxicity. These data suggest an interaction between cytosolic dopamine and parkin-mediated pathogenic mechanisms in Parkinson’s disease60. The cumulation of these studies demonstrate that impairment in dopamine sequestration is a convergent mechanism downstream of a variety of processes implicated in Parkinson’s disease, and that dopamine can contribute to the propagation of these same pathogenic mechanisms.In addition, the dopamine quinone has also been shown to interact with Parkinson’s disease-associated chaperone protein DJ-1, and dopamine and its metabolic products have also been shown to interact with α-synuclein, which can cause downstream deficits in chaperone mediated autophagy, decreased glucocerebrosidase activity, damage to synaptic vesicles, and mitochondrial dysfunction19. However, we detected an increase in both the amount of phosphorylated α-synuclein and the association between α-synuclein and TOM20, which we have shown blocks mitochondrial import9. Both of these observations suggest the presence of aberrant forms of α-synuclein, possibly including dopamine-modified α-synuclein9. It is likely that although the total amount of monomeric α-synuclein did not change in our model, given the abundance of evidence demonstrating an interaction between dopamine and α-synuclein, VMAT2 knockdown resulted in aberrant α-synuclein that contributed to the neurodegenerative process. This is further supported by the evidence of increased phosphorylated α-synuclein as the phosphorylation of α-synuclein at Ser129 has been extensively characterized for its pathogenicity61.Although an increase in α-synuclein following VMAT2 knockdown has been previously reported, we did not detect a significant increase in total α-synuclein25. This increase corresponds with an increase in the amount of phosphorylated Rab10—a substrate for LRRK2. Taken together, these data suggest that increased LRRK2 activity is a common pathogenic mechanism in Parkinson’s disease. In the current study, we observed an increase in proximity ligation assay signal for LRRK2 and as an additional measure of LRRK2 activity, an increase in phosphorylated Rab10 following VMAT2 knockdown. The induction of LRRK2 activity downstream of cytosolic dopamine has previously been unreported in vivo. We have previously shown that LRRK2 activity can be induced by reactive oxygen species, and we hypothesized that oxidative stress produced as a consequence of increased cytosolic dopamine may induce LRRK2 activity25. Accordingly, our data demonstrated that following VMAT2 knockdown, there are increased oxidative modifications, which are an indirect marker of reactive oxygen species, and increased LRRK2 activity. We are the first to demonstrate the induction of LRRK2 activity downstream of dysregulated dopamine homeostasis in vivo.Recently, we have demonstrated an increase in activated LRRK2 as measured by proximity ligation assay in idiopathic Parkinson’s disease62. However, these experiments employed bilateral knockdown of VMAT2 resulting in a motor phenotype characterized by a decrease in total movement as early as 8 weeks following knockout, as well as a significant decrease in food and water intake with weight loss at 16 weeks. Interestingly, the decrease in movement, feeding, and drinking corresponded with a decrease in survival. It is possible that the bilateral loss of VMAT2 caused animals to succumb due to dehydration or starvation before neurodegeneration was observed. We hypothesize that a unilateral loss in VMAT2 expression in these mice may protect the animals against a loss of total movement, allowing them to survive for analysis at later timepoints, which may reveal dopaminergic neurodegeneration.It is important to mention that work published by Isingirini et al. did not detect degeneration following the conditional loss of VMAT2 in adult mice64. GFP-positive and TH-negative neurons were excluded from analysis in order to ensure the amount of proteins of interest quantified was only collected from confirmed transduced dopaminergic neurons. To that end, analysis within the transduced dopaminergic neurons in the substantia nigra pars compacta demonstrated no difference in TH immunoreactivity. By excluding the population of neurons expressing GFP but lacking TH, we may be reporting a conservative value of protein immunoreactivity changes as some of the GFP-positive and TH-negative neurons may have been dopaminergic neurons undergoing degeneration. However, additional analysis on this population of neurons may reveal more insight into the mechanisms active during degeneration.The experiments presented here provide the foundation for several follow-up studies that will further elucidate the toxic consequences of dysregulated dopamine homeostasis. As demonstrated in Fig. In addition, to better understand the induction of pathogenic mechanisms, a thorough time-course can be investigated. Here, we have presented data from a number of timepoints post-transduction including 4, 6, 8, 12, and 20 weeks. Our initial analysis has demonstrated that the degeneration of dopaminergic neurons following a loss of VMAT2 indicates a trend toward progressive degeneration over time; however, additional studies are necessary to more clearly elucidate the temporal induction of pathogenic mechanisms. Additionally, we did not observe a consistent change in behavior at 6 weeks post-transduction likely due to compensatory mechanisms protecting extracellular dopamine levels in striatum with the loss of dopamine terminals. It is known that neurological deficits associated with dopamine loss do not appear until striatal dopamine levels are decreased by 80–85% of control levels. Given the biochemical analyses of striatal dopamine levels in this model, it is likely that behavioral deficits would have been observed as early as 12 weeks. Thus, in addition to the development of pathogenic mechanisms, the development of motor symptoms can be deeply characterized in future studies.65.While we were able to identify indices of Parkinson’s-associated mechanisms of degeneration such as aberrant α-synuclein and induction of LRRK2 activity, additional experiments outside the scope of this manuscript are necessary to determine whether these mechanisms are causal or correlative to the degenerative process. Future experiments will investigate the role of these mechanisms in neurodegeneration by employing interventions targeting the individual processes and looking for neuroprotection. For example, a LRRK2 inhibitor could be administered to determine whether LRRK2 activation mediates the degenerative process downstream of dysregulated dopamine sequestration. Additionally, decreasing α-synuclein expression utilizing a viral approach previously published by our group would be useful in determining whether decreasing the amount of α-synuclein is be protective against dopamine-mediated degeneration66. Investigating how the dopaminergic neurons in the ventral tegmental area respond differently to cytosolic dopamine may provide insight both into why these neurons are resistant to degeneration and which pathogenic mechanisms are necessary mediators of dopamine-induced toxicity.As an additional direction to understand the vulnerability to degeneration of the dopaminergic neurons in the substantia nigra, AAV2-shVMAT2 can be transduced within the ostensibly less-vulnerable dopaminergic neurons in the ventral tegmental area to evaluate whether Parkinson’s-associated mechanisms of degeneration are similarly induced and whether neurodegeneration occurs. Previous work has shown resistance to dopaminergic degeneration within the ventral tegmental area of mice deficient in VMAT2, which mimics data from human studies demonstrating resistance to degeneration in the ventral tegmental area of Parkinson’s disease patientsAs summarized above, our model of acquired dysregulation of dopamine homeostasis can be used to further understand the mechanisms by which dopamine to acts an endogenous neurotoxin and investigate how these processes relate to Parkinson’s disease pathogenesis. There are several benefits to the viral-mediated knockdown of VMAT2 employed in this study. The virus can be injected directly into the brain of adult animals targeting specific areas rather than having global knockdown from birth. A unilateral injection of the virus also has the advantage of an internal control on the contralateral hemisphere. The extent of knockdown can be modulated through diluting the virus to introduce fewer viral particles or adjusting timepoint for experiments, and with neurodegeneration observed as early as 6 weeks post-transduction, this allows for more feasible testing of therapeutic interventions on a faster timescale. Furthermore, the acquired loss of VMAT2 prevents the development of compensatory mechanisms characteristic of genetic models, and models an acute loss of VMAT2 activity in adulthood. Collectively, this model of dysregulated dopamine sequestration has demonstrated that cytosolic dopamine is sufficient to induce Parkinson’s disease-associated pathogenic mechanisms of neurodegeneration. The implications of which further suggests a therapeutic potential for VMAT2 in the treatment of Parkinson’s disease.2. Predesigned small-hairpin ribonucleic acid (shRNA) constructs against rat vesicular monoamine transporter 2 (VMAT2) were obtained from Origene and the shRNA with best knockdown efficiency was chosen for viral production . A scramble control shRNA construct was provided from Invitrogen and used for in vitro experiments. Cells were transfected with a construct for green fluorescent protein (GFP) and shRNA constructs using Lipofectamine LTX and Plus Reagent and collected at 48 h post-transfection for RNA and protein analysis. Immunocytochemistry was used to visualize protein immunoreactivity and protein quantification was performed on images obtained on an Olympus IXB1 confocal in Fluoview software. Regions of interest (ROIs) were drawn around GFP-positive cells. RNA was isolated utilizing the RNeasy Mini Kit . For q-RT-PCR analysis, cDNA was generated with the iScript cDNA Synthesis Kit . Primers were obtained from Real Time Primers . A list of primers with sequences can be found in Supplementary Table Cell culture experiments were performed in RCSN-3 cells: an immortalized dopaminergic neuronal cell line derived from the substantia nigra of an adult rat. RCSN-3 cells were obtained through an agreement with the University of South Florida and the University of Chile and maintained in media  in an incubator at 37 °C with 100% humidity and an atmosphere of 10% COThe oligonucleotide encoding the small-hairpin ribonucleic acid for VMAT2 is targeted against the following nucleotide sequence: [TCA][ACA][GTT][ATG][TTT][GCC][TTC][TCC][AGC].21. Adeno-associated virus (serotype 2) vector particles containing viral genomes derived from the pAAV vector were produced by the Penn Vector Core. The resulting shVMAT2 vector (AAV2-shVMAT2) expresses the shRNA construct under the human U6 promoter and eGFP under the CMV promoter with a titer of 3.56 × 1013 genome copies/mL. A previously established non-targeting shRNA control vector based on the same vector genome was used as a control21. The knockdown resistant VMAT2 construct expresses human VMAT2 with four silent mutations and a myc-DDK tag. This construct was cloned into pZac2.1 plasmid and the AAV2-kdrVMAT2 virus was produced by the Penn Vector Core with a titer of 2.00 × 1012 genome copies/mL. A control virus overexpressing GFP (AAV2-GFP) was ordered from Penn Vector Core with a titer of 2.00 × 1012 genome copies/mL. The AAV2-shVMAT2 virus was diluted to equal titer (2.00 × 1012 genome copies/mL) in sterile PBS for rescue experiments. For rescue experiments, fresh viral aliquots and dilutions as necessary were prepared on the day of the surgery. Viruses were diluted to the same viral titer in sterile PBS and equal proportions of each virus were mixed thoroughly. Animals received 2 μL of mixed viral preparations (AAV2-GFP + AAV2-shVMAT2 or AAV2-shVMAT2 + AAV2-kdrVMAT2) equivalent to 1 μL each of the individual viruses injected as described below. In the study presented here, the knockdown resistant virus was administered at the same time as the knockdown virus, which models the protective replacement of exogenous VMAT2 expression as endogenous VMAT2 is lost. This study design was chosen as an initial proof of principle experiment to demonstrate the specificity of neurodegeneration as a result of VMAT2 knockdown and to circumvent possible confounding variables or issues surrounding multiple viral injections into the brain.The nucleotide sequence in the knockdown resistant VMAT2 viral vector is: [TCT][ACG][ATC][ATG][TTC][GCC][TTC][TCC][AGC]. The oligonucleotide encoding the shRNA construct with optimal VMAT2 knockdown  was annealed into the BamHI/EcoRI sites of pAAV-D(+)-U6-siRNA-CMV-GFPAdult (≥3 months) male and female Lewis rats were obtained from Envigo . Rats were singly housed in temperature-controlled conditions under a 12:12 light-dark cycle with ad libitum access to food and water. All experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.Stereotaxic surgery was performed to deliver virus to the substantia nigra . Animals were deeply anesthetized under isoflurane (2–4%) for the entirety of the surgery. Two microliter of virus was infused over a 10-min period (0.2 uL/min). Animals were closely monitored on a heated surface during recovery of surgery. Animals received analgesia in the form of ketoprofen (3.0 mg/kg s.c.) with the first dose presurgery and 2×/day for 3 days following surgery. Animals were sacrificed by transcardial perfusion with phosphate buffered saline followed by 4% paraformaldehyde. The brains were harvested and sectioning was performed on a freezing-stage microtome (ThermoFisher HM 450 sliding microtome) at 35 µm. Sections were maintained in cryoprotectant at −20 °C until analysis.22. For the postural instability test, the distance the animal is moved to trigger a corrective step was measured for each forepaw. The task was performed in triplicate and an average in performance was calculated per day. For the cylinder test, the animal was placed in a clear cylinder and allowed to freely rear for a 5-min period. The cylinder test was repeated three times across a 2-week period for each animal to aggregate total number of paw touches.Deficits in dopamine-mediated behaviors were evaluated by two previously established tests of dopamine-mediated deficits in Parkinson’s disease models—the postural instability test and the cylinder testImmunohistochemistry was performed on free-floating sections before mounting on glass slides for imaging. Primary antibodies used for immunohistochemical analysis are listed in Table Proximity ligation assay was analyzed utilizing PLA probes  and visualized with orange  and far-red  kits.For quantification of immunofluorescence, confocal images of both immunohistochemistry and proximity ligation assay were taken on an Olympus IXB1 confocal. Intensity measurements were obtained in Fluoview software by circling ROIs around TH-positive neurons in the nontransduced hemisphere and TH- and GFP-positive neurons in the transduced hemisphere while blinded to the protein of interest. Intensity values of the protein of interest were compared between transduced and nontransduced TH-positive neurons. Number of objects analysis was performed in Nikon Elements software by circling ROIs around TH-positive neurons in the nontransduced hemisphere and TH- and GFP-positive neurons in the transduced hemisphere while blinded to the protein of interest. Within ROIs, a threshold was applied to the protein of interest in order to achieve unbiased counts of objects. Images to visualize DAB immunoreactivity were taken on an Olympus BX61VS microscope and analyzed for pixel saturation in CellSens software.67. Briefly, tissue was immunolabeled for TH, MAP2, and DAPI and imaged at ×20 on a Nikon90i fluorescent microscope in the Center for Biologic Imaging at the University of Pittsburgh. Images were analyzed in Nikon Elements software counting the number of dopaminergic neurons determined by overlap between DAPI, MAP2, and TH within a region of interest defining the substantia nigra pars compacta as previously described67. Subsequent experiments were performed with a modified method of stereology utilizing overlap between TH and fluorescent Nissl  within the substantia nigra pars compacta as previously described69. These images were obtained on an Olympus BX61VS slide scanning microscope, and analysis was performed utilizing Nikon Elements software.Stereology was initially performed on serial sections from the midbrain following a protocol adapted from Tapias et al. 201470. Striatal tissue was homogenized in 0.1 M perchloric acid. Homogenates were centrifuged  to separate the acid precipitated protein from the acid-soluble components and stored at −80 until analysis. The acid-soluble component was filtered  and analyzed by HPLC-ED . The protein pellet was washed by resuspension and recentrifugation in 0.1 M perchloric acid containing 0.2 mM sodium bisulfite and then subjected to acid hydrolysis in 6 N HCl as we previously described70. Following drying and resuspension, the catechol moieties were extracted with alumina and the cysteinyl catechol were analyzed using HPLC-ED ). The mobile phase contained 0.05 M sodium phosphate, 0.01 M citric acid, 0.01 M sodium acetate, 0.32 mM sodium octyl sulfate, and 0.1 mM EDTA, 10% (vol/vol) methanol, and was titrated to pH 2.5 with HCl. Compounds were identified by their elution positions and quantified by comparison to catechol standards.Animals were sacrificed at 8 weeks post-transduction via transcardial perfusion with phosphate buffered saline (PBS). Following PBS perfusion, the striatal tissue was dissected, weighed (12–30 mg), and rapidly frozen on dry ice. Tissues were stored at −80 °C until used for the analyses of free and protein-bound cysteinyl catechols and parent catechol levels. Tissue was analyzed as previously describedt-tests were used to evaluate within animal protein immunoreactivity, neurochemical measurements, neuronal counts, and behavior. Unpaired t-tests were used to compare viral treatment groups. One-way ANOVA with Tukey’s post-hoc test was performed to evaluate mRNA.Data are presented as individual data points corresponding to an individual animal and “n” equal to the number of animals unless otherwise specified. Graphs demonstrate mean and standard error of the mean. Paired Experimental approval was obtained through the Institutional Animal Care and Use Committee at the University of Pittsburgh.Further information on research design is available in the Supplementary Figures and TablesReporting Summary FLAT


Artificial extracellular matrices (aECMs) are an extension of biomaterials that were developed as in-vitro model environments for tissue cells that mimic the native in vivo target tissues’ structure. This bibliometric analysis evaluated the research productivity regarding aECM based on tissue engineering technology. The Web of Science citation index was examined for articles published from 1990 through 2019 using three distinct aECM-related topic sets. Data were also visualized using network analyses (VOSviewer). Terms related to in-vitro, scaffolds, collagen, hydrogels, and differentiation were reoccurring in the aECM-related literature over time. Publications with terms related to a clinical direction  have steadily increased, as have the number of countries and institutions involved in the artificial extracellular matrix. As progress with 3D scaffolds continues to advance, it will become the most promising technology to provide a therapeutic option to repair or replace damaged tissue. The field of tissue engineering has an extensive range of potential applications in tissue repair and regeneration . The rolArtificial extracellular matrices (aECMs) are an extension of biomaterials that were developed to provide a model environment for tissue cells in-vitro that mimic the native surroundings of the target tissue in vivo ,18,19. TThe significant attraction in the study of artificial ECMs is largely attributed to their potential applications in the field of regenerative medicine ,30,31. TMany reviews highlight the current strategies and future prospects of aECMs in tissue engineering; however, there is a lack of bibliometric reviews on the research output of publications. We aimed to evaluate and document how this field is expanding. We assessed the literature on aECMs based on tissue engineering over three decades by performing a bibliometric analysis that highlights (1) the most common terms occurring in related papers and (2) research volume output by authors, institutions, countries, and journals and their impact.(1)Topic set 1 = (tissue engineer and regeneration);(2)Topic set 2 = ;(3)Topic set 3 =  and .Data were obtained from the Web of Science online database  in January 2020. Web of Science is a universal scientific citation index used to retrieve scholarly articles and academic literature ,42. ThisThe Web of Science Core Collection Citation Indexes: Science Citation Index Expanded, Social Sciences Citation Index from 1990 through 2019 was applied to all three topic sets. The “custom year range” timespan of 1990 through 2019 was subdivided into three time periods  for each set to analyze temporal trends. Book chapters, proceedings papers, and retracted publications were excluded from the results of all three topic sets. Web of Science citation data extracted for each topic set and selected time period were downloaded as full records and cited references and saved in a tab-delimited file format. The data that were downloaded included publication by institution, publication by country, publication by funding agency, and publication by author.The extracted citations were imported into VOSviewer version 1.6.14   for the The output of research related to topic set 1 of our terms search, in the form of publications, increased continuously from 1990 through 2019, resulting in a total of 17,082 publications. The first paper was published in 1990 and publications reached double-digit numbers per year in 1994. Since 2012, over 1000 papers have been published per year. A. The ouThe term maps generated from VOSviewer from topic set 1 are displayed in The term map generated from VOSviewer using topic set 3 are displayed in Between 1990 through 1999 papers about topic set 1 originated mainly in the USA followed by England . During Similar to topic set 1, papers about topic set 2 originated mainly in the USA, followed by England in the 1990s . The USABetween 1990 through 1999, papers about topic set 3 originated mainly in the USA, followed by Germany . Japan, Throughout this time period, Reis R.L. ranked first with 195 publications, followed by Ramakrishna S. with 141 publications . Wang Y.Throughout this time period, the most productive organization in terms of publication output was Harvard University with 446 total publications . The UniBibliometric analysis is a tool used to quantitatively evaluate the output of scholarly journal publications and measure the significance of studies in the scientific community ,45,46. TThe field of TE-related research is continuously growing, as presented in the rising number of publications and the expansion of research areas involved. TEs’ in-vitro research is gradually being transitioned to being applied to in vivo studies with a more clinical focus. This trend is reflected in the last decade as more terms gradually appear to be related to clinical topics like biocompatibility, grafts, transplantation, wound healing, nerve regeneration and bone regeneration. This is evident in the term maps of the most recent publications that highlight groups of tissue regeneration terms that overlap in several publications . The curBiomaterials are important components of tissue engineering studies as shown in the term maps generated for topic set 1. The appearance of terms such as biodegradable polymer, hydrogels, scaffolds, collagen, alginate and fibrin were shown throughout the time period, with scaffolds appearing consistently . During Regarding the global trend for topic set 3, there was a large output of publications from the USA. Japan and Germany also contributed a substantial amount of publications related to topic set 3 throughout the time period. The extensive number of publications from these countries were reflected in the large number of articles that cited these publications. For topic set 2, the USA and China were leading in both publication output and citing articles. However, countries like South Korea and Germany were not consistently the top producers of publications, but they did contribute a considerable number of citing articles. For topic set 1, the USA was again the lead producer of publication output throughout the entire period. In the 1990s, China was not a top publication producer, but that shifted in the 2000s and the 2010s as their publications begin to increase. Both countries also continued to lead in citing articles throughout each decade. Although the USA produced the most research for each topic set, their relative contribution to the percentage of the total global output decreased as the other counties began to maintain a significant contribution to their publication output each decade.Many of the top authors results for each of the topic sets identified individuals that came from the same lab or collaborating labs. A considerable amount of the top authors also coincided with the top country results. For topic set 3, the primary rank for publication output continued to shift throughout each decade. Yannas and Minuth remained in the top five producing authors until the last decade, when all new authors took the lead. Authors of the top citing articles varied greatly from the authors with the greatest publication output. However, there could be a possibility that some of the top producing authors collaborated on publications with the authors from the top citing articles. This trend continued into publications related to topic set 2, aside from Kaplan, Reis and Mikos, who appeared in the top rank for both the publication output and the citing articles at least once in each decade. For topic set 1, quite a few of the top producing authors concur with the authors from the top citing articles throughout the time period. The institutional results for topic set 3 correspond with the results from the top producing countries based on the geographical location of the institution. For topic set 3 in the 1990s and the 2000s, the USA was the leading producer and citer of publications. This aligns with the results of American institutions such as MIT, Harvard University and the University of California System leading in publication output and citing articles. In the 2010s, Dresden University of Technology in Germany was the leading institution for publications and the Chinese Academy of Sciences in China was the leading citer of publications. This also coincides with the data from the countries during that time period. For topic set 2, during the entire time period the USA and the China continued to the lead the top charts for both publications and citing articles. For topic set 1, the American institutions dominated the publication output and the citing articles for the first two decades. In the 2010s, Chinese organizations such as Shanghai Jiao Tong University were the top producers and the top citer of publications was the Chinese Academy of Sciences .In conclusion, this analysis demonstrated that the artificial extracellular matrix literature based on tissue engineering technology is growing exceptionally fast in research related to biomaterials and regenerative medicine. It is expected that we will continue to see an increasing research output in the near future as 3D scaffolds continue to advance their design to resemble the natural ECM as closely as possible. As progress with 3D scaffolds continue to advance it will become the most promising technology to provide a therapeutic option to repair damaged tissue.


Herein, recent developments of metal–organic frameworks (MOFs) structured into nanofibers by electrospinning are summarized, including the fabrication, post‐treatment via pyrolysis, properties, and use of the resulting MOF nanofiber architectures. The fabrication and post‐treatment of the MOF nanofiber architectures are described systematically by two routes: i) the direct electrospinning of MOF‐polymer nanofiber composites, and ii) the surface decoration of nanofiber structures with MOFs. The unique properties and performance of the different types of MOF nanofibers and their derivatives are explained in respect to their use in energy and environmental applications, including air filtration, water treatment, gas storage and separation, electrochemical energy conversion and storage, and heterogeneous catalysis. Finally, challenges with the fabrication of MOF nanofibers, limitations for their use, and trends for future developments are presented. Metal–organic framework based nanofibers with hierarchical porous structure fabricated via electrospinning are reviewed for energy and environmental applications. The mechanism of electrospinning is based on the ejection and elongation of a viscous polymer melt or solution under a high‐voltage electric field which is then solidified in the electrified fluid jet on a collector.Electrospinning has recently been reported as an elegant approach for shaping various MOFs into hybrid materials with multiscale porosity and additional functionalities. In the first pioneering work from 2011 by Ostermann et al.,FigureThe synthesis, chemical modification, and potential applications of MOFs have been widely reviewed previously.2Two major routes have been developed for the structuring of MOF‐polymer nanofiber architectures based on electrospinning, which are “direct electrospinning” and “surface decoration.” MOF nanofibers obtained from both routes can be subsequently converted by post‐treatments into derivatives of MOF nanofibers. We give selected examples for the two routes in Sections In direct electrospinning, a mixture of a slurry that contains MOF particles and a polymer solution is directly electrospun into a composite nanofiber.2.12.1.12 support tubes with MOF nanofiber layers for use in a gas separation membrane. Figure2 support. Figure MOF‐polymer nanofibers were fabricated initially by a simple three‐step procedure: MOF powder particle synthesis, preparation of a MOF‐polymer slurry, and direct electrospinning of the slurry.2, MOF‐199, Mg‐MOF‐74 and their combination with the polymers PAN and PS. Nanofiber diameter, porosity, and pore size of the MOF‐polymer nanofibers could be controlled by optimizing the electrospinning conditions, such as applied electric voltage and flow rate, resulting in MOF nanofibers with different microstructures, as shown in Figure Zhang et al.Figure2Cl2 was selected as solvent to disperse the ZIF‐8 into the PLA solution. The high volatility of the CH2Cl2 ensures fast evaporation during the flight of the electrospinning jet and the addition of ZIF‐8, which results in the formation of a large number of mesopores with large pore area fraction in the resulting PLA/ZIF‐8 nanofibers. The diameter and porous structure of the PLA/ZIF‐8 nanofibers could be efficiently adjusted by varying the loading fraction of the MOF particles.In MOF‐polymer composite nanofibers, the polymer phase acts as a binder supplying the structure with flexibility. However, the polymer might cover the surface and micropores of the MOF could negatively affect the performance in some specific applications, for example, the adsorption of gases on the MOFs. To overcome this problem, Dai et al.2 isotherms for as‐synthesized Mat:PEO:ZIF‐8 nanofibers with different ratio before (“unwashed”) and after PEO removal (“washed”) are shown in Figure 2 adsorption could be achieved by creation of pore channels in the polymer by washing  and by the increase of the ZIF‐8 to polymer ratio. This process is similar to solvent extraction in injection molded ceramic parts before the thermal debinding step. Solvent extraction is a generic method which can be used to create porosity in a variety of MOF/polymer nanofiber composites with implications for the use in the field of filtration, adsorption, sensing, or catalysis.Armstrong et al.2.2The second route to produce hierarchical porous MOF‐polymer composite nanofibers is called “surface decoration” and describes the growth of MOF nanoparticles on the surface of nanofibers that have been produced by electrospinning. This route can overcome some of the drawbacks of the direct electrospinning route discussed in Section 2.2.1i)In “seed‐assisted growth” , MOF crystallite seeds are incorporated in a polymer nanofiber during electrospinning and the MOF growth is started by immersion of the seeded nanofibers in a MOF precursor solution.ii)In another approach, one of the MOF precursors  is added to the polymer nanofiber via the electrospinning process and the MOF crystallization is initialized by immersion of the precursor‐polymer nanofiber in a solution of the other precursor.iii)In direct solvo or hydrothermal growth, a MOF is directly grown on the surface of a polymer nanofiber by immersion in a MOF precursor solution . The polymer nanofiber usually needs to be activated for in situ growth by a nanofiber surface pretreatment to achieve sufficient attachment. The application of the MOF precursors can be done in one step  or sequentially.During in situ growth, MOF precursors  are dissolved in a suitable solvent and subsequently MOF nanoparticles are crystallized on the surface of a polymer nanofiber to obtain a MOF‐polymer nanofiber composite.2(bpdc)2(bpee) to demonstrate the effectiveness of MOF seeding in nanofibers to initiate subsequent intimate growth of MOF particles inside and on the surface of a MOF‐seeded polymer nanofiber structure by secondary growth to achieve a continuous, uniform, and defect‐free MOF membrane. The process is illustrated in FigureAll in situ growth approaches include multistep processing. Selected examples are discussed in the following section, including the use of specific fabrication techniques. In seed‐assisted growth, MOF seeds are embedded in the polymer nanofiber matrix to initiate further growth of the MOF particles. Wu et al.2/PAN nanofiber. The nanofiber was subsequently immersed in a solution of the organic linker 2‐methylimidazole (HmIM) in ethanol to grow a homogeneous layer of ZIF‐67 nanocrystals on the PAN nanofiber surfaces  on PAN nanofibers for toxic chemical removal. This approach resulted in higher MOF loading and better adherence due to more homogeneous in situ growth of the MOF particles from the precursor solution on the PAN/ATA nanofiber surface compared to the growth on bare PAN nanofibers  to prepare the HKUST‐1. LBL assembly is a feasible technique to produce nanolayers with controlled thickness on the surface of nanofibers but the multistep operation is time‐consuming.MOFs can be synthesized directly on the surface of nanofibers (according to approach iii), but in most cases, the interface bonding between the MOF and the polymer is a challenge due to the different chemical properties. The activation of polymer nanofiber surfaces is a simple approach to anchor MOF particles to nanofiber surfaces during in situ growth from a MOF precursor solution. Laurila et al.2 on PAN nanofibers in acetone (instead of DMF) because PAN is stable in acetone. Ji et al.Often, in situ growth of MOFs on polymer nanofibers is restricted by the stability of the polymeric nanofiber in the solvent which is required for the growth of the specific MOFs. Some MOFs are, for example, solvothermally prepared using DMF as solvent, which would dissolve polymer nanofibers made of PVP, PAN, or PVA. In addition, the crystallization of MOFs often requires relatively high reaction temperatures. Therefore, different approaches have been reported to stabilize polymer nanofiber structures for growth of MOFs. A simple approach (if applicable) is the replacement of a common solvent by a more appropriate solvent that is compatible with the polymeric nanofiber backbone and still allows the crystallization of the desired MOF structure. For example, Lu et al.2, ZnO, TiO2) have been developed to obtain more stable nanofiber substrate structures. Liu et al.2 nanofibers under harsh solvothermal reaction condition in different solvents and at temperatures as high as 200 °C. Furthermore, these nanofiber structures revealed desirable mechanical properties such as malleability with a high breaking elongation. Liu et al.2 hybrid nanofiber for the in situ growth of different types of MOFs , ZIF‐8, and MIL‐88B(Fe)) due to a large number of available active surface hydroxyl and carboxyl groups for anchoring the MOF precursors.For more severe conditions during in situ growth, cross‐linking of the polymers can be applied to stabilize the structure of polymers. This is a common approach which has been commonly used in polymer chemistry.2 layer was deposited on freestanding polyamide‐6 nanofibers to enhance the subsequent heterogeneous nucleation of Zr‐MOFs on the TiO2 surface  image of PA‐6@TiO2@UiO‐66‐NH2 shows Zr‐MOF nanoparticles that are fully covering the nanofibers, indicating strong attachment to the substrates , respectively  which is incorporated in the surface of the polymer nanofiber. The precursor is converted into the MOF structure during in situ growth in a second step. A kinetic matching between the structure of the precursor and the targeted MOF structure is enabling the phase transformation. Liang et al.s Figure b. Compary Figure b. Intere2 g−1 could be achieved by this process.Holopainen et al.2.3Limited electrical conductivity and stability of MOFs have restricted their use in many energy and storage applications. MOFs as templates have been post‐treated to fabricate different kind of derivatives with high surface area, ordered pore structures, and uniform pore sizes for the use in energy and storage applications. Nevertheless, conventionally fabricated MOF derivatives still suffer from insufficient electrochemical performance related to a lack of electrical connectivity between the particles and limited mass and electron transfer. Therefore, considerable research efforts have been recently devoted to the development of the derivatives of MOF nanofibers to overcome these limitations. A few, selected examples of such type of nanostructures are discussed in this section and in Section 2 atmosphere for the use in supercapacitors  for use as anode materials in lithium‐ion batteries (LIB) 2. Upon pyrolysis of the nanofiber under high temperature and subsequent removal of residual metals by acid (HCl), N‐doped carbon hollow tubules (CHTs) were obtained, covered with a large fraction of MOF‐derived graphitic carbon with large interlayer spacing (0.38–0.44 nm). The resulting N‐doped porous CHT paper yielded in an outstanding cycling life (10 000 cycles), with high‐rate capabilities of Na+ intercalation and deintercalation and no decline in capacity. Li et al.Chen et al.e Figure e. For th33.1Figure2 with high selectivity. Compared with pure PAN filters, the UiO‐66‐NH2/PAN (0.019 g g−1) and MOF‐199/PAN (0.014 g g−1) nanofiber filters displayed a largely improved SO2 adsorption performance  and toxic gases, has become an increasing public health hazard globally. To achieve efficient removal of PMs and toxic gases, the design of air filters with high selectivity and low cost has drawn considerable attention.e Figure b. The ree Figure c.2O3 mat (77% and 72%) and Al2O3 mat (67% and 63%). The long‐term filtration test shows a sufficient capture capability to block tobacco smoke during 24 h  was below 10 ppb in the permeate, meeting the requirement for drinking water. Further investigations showed that the strong interactions between the Pb (II) and the Fe‐MOF contributed to the effective removal of the heavy metal, including competitive ion exchange (CIE), electrostatic interactions with the MOF crystals or polymer, and binding to open metal sites of the MOFs. In addition to the high removal efficiency, excellent durability of the MOF‐PAN nanofibers makes these membranes suitable for practical water treatment applications. For a similar application, Shooto et al.Figure−1 at a pH of 3.0. The removal of the U(VI) ions is due to the surface complexation of the 2‐Hmim linker in ZIF‐8 with U(VI), as indicated in Figure 22+‐ions over Ln3+‐ions.Worldwide water pollution and scarcity of clean drinking water have led to a strong need for the development of energy‐savings and cost‐efficient water purification technologies.Li et al.o and a high water contact angle in oil of 155° could be reached, which is favorable for improving fast separation dynamics. Accordingly, the hierarchically structured PAN‐ZIF‐8 nanofibers could effectively separate surfactant‐stabilized oil‐in‐water and water‐in‐oil emulsions with higher separation efficiency and flux compared to conventional membranes in pressure‐driven separation process.Oil spill from different sources, for example, from tankers or oil drilling, is regarded as one of the most severe water pollution problems.Dai et al.3.32 capture, natural gas sweetening, oxygen purification, hydrocarbon separation, or noble gas separation. Storage of gases, such as H2 or CH4, by adsorption on microporous materials in pressure vessels is developed as an alternative technology to replace fossil fuels in vehicles. Porous MOFs have been investigated for gas storage and separation because of extremely high accessible internal surface area, tunable surface chemistry, and pore sizes in the range of the kinetic diameter of different gas molecules.The industrial separation of gas mixtures is of great importance for CO2 g−1 compared to 1219 m2 g−1 for the unstructured, pure ZIF‐8 powder. H2 and CO2 adsorption capacities of 8.1 and 13.3 m3 g−1 at 106 kPa have been measured for PAN@ZIF‐8 nanofibers. Ren et al.2 storage with a Zr‐MOF (UiO‐66) and a Cr‐MOF (MIL‐101) embedded in a PAN polymer matrix by utilizing a vacuum degassing step to create the required open porosity in the PAN polymer matrix. Interestingly, structuring the MOF into MOF‐PAN nanofibers with a MOF mass fraction as low as 20 wt% could still achieve about 60% of the H2 uptake capacity of the pure MOF nanocrystals at 900 mbar , indicating that a molecular sieving effect was achieved by using a ZIF‐8/PVP nanofiber membrane with intergrown ZIF‐8 crystals as a separation layer. Due to the large surface area, tailored surface chemistry, and well‐defined pore channels of MOFs, MOF nanofibers are very interesting materials for use in gas separation and storage.Wu et al.d Figure b. Fan et3.43O4 hollow nanoparticles and carbon nanotubes for LIB, based on the heat treatment of ZIF‐67‐PAN nanofibers as template. The formation of the CNT/Co3O4 microtubes is visualized in Figure3O4 hollow nanoparticles and CNT subunits not only enables a short diffusion distance for fast diffusion of Li+ ions but also provides sufficient contact between the active material and the electrolyte for the rapid charge‐transfer. The ZIF‐67 derivative can alleviate the volume variation during the electrochemical reactions, and the CNTs can facilitate the fast electron transfer and avoid the aggregation of Co3O4 nanoparticles during cycling. As a result, the obtained CNT/Co3O4 microtubes show a high capacity of 782 and 577 mAh g−1 after 200 cycles at 1 and 4 A g−1, respectively  and lithium‐ion conductivity. The incorporation of the MOF particles alleviates the decomposition of the electrolyte, enhances the electrode reaction kinetics, and reduces the interface resistance between the electrolyte and the electrodes. As a result, the MOF‐PVA nanofiber separator showed enhanced rate capability and cycling durability for use in conventional LIBs.Zhang et al.Lee et al.2O molecules as the vehicles to transport protons, which limits the fuel cell operation temperature to below 100 °C. Wu et al.2(C2O4)(C2N4H3)2(H2O)0.5 (ZCCH) shows significant proton conductivity at high temperature and anhydrous conditions because protonic charge carriers  can be incorporated in the pores. The proton‐conductive Zn‐MOF was mixed with the polymer SPPESK and electrospun into a highly oriented nanofiber membrane, to construct a continuous proton‐conducting path  should be operated at high temperature >100 °C) to eliminate CO poisoning and to promote the electrochemical reactions. However, perfluorinated sulfonic acid (PFSA) membranes, such as Nafion, use H0 °C to eFigure−2  within 10 min. The catalytic sites are protected by the rigid framework of ZIF‐67 embedded in a flexible nanofiber structure. This architecture ensures a stable catalytic activity and the catalyst can easily be separated (recycled) after the reaction is completed. Furthermore, the work demonstrated that ZIF‐67/PAN nanofibers are an efficient catalyst for the oxidative catalytic degradation of other pollutants for the recovery of waste water, proving the decomposition of 68.3% of tetracycline (TC), 100% of bisphenol A (BPA), and 98.3% of rhodamine B (RhB) by ZIF‐67/PAN.Heterogeneous catalysis, in which the catalyst occupies a different phase than the reactants and products, enables large‐scale production, and selective product formation. MOFs are regarded as single‐site catalysts because the coordinatively unsaturated metal ions, substituents at the organic linkers or guest species located inside the framework can play the role of the active site.3+ metal center and 1,3,5‐tris(4‐carboxyphenyl)benzene (BTB) as the organic ligand  nanofibers. Subsequently, UiO‐66‐NH2 was grown on the surface of the metal oxide coated nanofibers. The hydrolysis of a simulant material for CWAs, 4‐nitrophenyl phosphate (DMNP), was used as an example to evaluate the catalytic performance of MOFs nanofibers for this application  and high conversion rate (>90%) within 1 h. As compared with that of PP nanofiber, the TiO2 coating enabled a more effective and uniform MOF nanofiber adhesion, resulting in a rapid catalytic degradation rate. These results show that Zr‐MOFs have the capability of catalytically hydrolyze phosphonate ester bonds. In addition, Zhao et al.2@UiO‐67 nanofibers for the catalytic degradation of the nerve agent soman   and high conversion (80%) within 10 min, due to the optimal microstructure of UiO‐67 that allowed easy diffusion of reactants into the pores and fast reaction at the catalytic active sites of the frameworks. This work demonstrated, for the first time, an effective approach to decompose a real CWA compound using MOF nanofibers.Lee et al.n Figure h. As com) Figure i,j. The Figure2 nanofibers for the Knoevenagel condensation ‐NH2 was structured into nanofibers. As conclusion, in heterogeneous catalysis MOF nanofiber structures can significantly reduce the loss of catalytic sites (MOFs), improve durability, and enhance mass transfer due to efficient fixation of the catalytic sites, high surface area, and a hierarchical porous catalyst support structure.In order to improve the stability of catalysts and to avoid leaching problems, Leus et al.n Figure c. The Knh Figure d. The MIs Figure e whereasd Figure f, which d Figure g. The ex4Intensive research has been reported for the structuring of MOFs into hierarchical porous nanofibers. Two routes are developed: i) direct electrospinning of a MOF‐polymer dispersion or ii) surface decoration of MOF on the surface of polymer nanofibers. The developed hierarchical nanofiber architectures show interesting properties, which can be utilized for various energy and environmental applications. In many areas, including gas separation, air filtration, water treatment, and heterogeneous catalysis, the research will most likely focus on the development of MOF‐polymer nanofibers because the functionality of the original MOF structures is required for the application. In other application, mostly those related to electrochemical energy conversion, for example, electrodes in batteries or fuel cells, sufficiently high electronic conductivity is required. It is expected that the research in these areas would be mostly devoted to the derivatives of MOF nanofibers, such as MOF nanofiber derived 1D porous or hollow carbon‐based nanofibers.Considering the complexity of structuring MOF nanofibrous architectures and the rapid progress in energy and environmental applications, a couple of challenges could be addressed in the future: i) Investigation of a range of polymers that can be processed into nanofiber systems and can withstand MOF synthesis conditions under harsh conditions  during MOF decoration. (ii) Exploring the implementation of postfabrication processes for MOFs nanofiber structures, which are already used for processing of conventional nonwoven fibers in textile industry or related industries. Examples are vacuum forming of fiber blankets, calendaring, or needle punching.Commercial products of MOF nanofibers have so far not been reported in energy and environmental applications. A combination of relatively high costs for the expensive raw materials (MOFs) and for complex fabrication processes (relative low production rates in electrospinning and MOF growth) could be factors for inhibiting the commercialization of MOF nanofiber products. MOF nanofiber products are expected to enter markets first in those areas in which they can provide significant performance improvements and create high value. Examples are the efficient capture/removal of emerging pollutants from air or water, replacement of expensive noble metals in electrocatalysis, high value biomedical and healthcare products, or drug or enzyme delivery.In conclusion, electrospinning is a new path for structuring of functional MOFs into hierarchical nanofiber structures, which allows the creation of a range of fascinating new properties. We hope that this review can guide and inspire researchers to do further work on MOF nanofibers, which should eventually lead to functional MOF nanofibers used in energy and environmental applications.The authors declare no conflict of interest.


A receptors, and GABAA receptors can regulate dopamine release, we hypothesized that allopregnanolone would reduce phasic fluctuations in mesolimbic dopamine release that are important in learning and reward processing. We used fast-scan cyclic voltammetry in anesthetized female and male rats to measure dopamine release in the nucleus accumbens evoked by electrical stimulation of the ventral tegmental area, before and after administration of allopregnanolone. Allopregnanolone  reduced evoked dopamine release in both male and female rats, compared to β-cyclodextrin vehicle. In males, all doses of allopregnanolone decreased dopamine transmission, with stronger effects at 15 and 25 mg/kg allopregnanolone. In females, 15 and 25 mg/kg allopregnanolone reduced dopamine release, while 7.5 mg/kg allopregnanolone was no different from vehicle. Since allopregnanolone is derived from progesterone, we hypothesized that high endogenous progesterone levels would result in lower sensitivity to allopregnanolone. Consistent with this, females in proestrus (high progesterone levels) were less responsive to allopregnanolone than females in other estrous cycle stages. Furthermore, 30 mg/kg progesterone reduced evoked dopamine release in males, similar to allopregnanolone. Our findings confirm that allopregnanolone reduces evoked dopamine release in both male and female rats. Moreover, sex and the estrous cycle modulated this effect of allopregnanolone. These results extend our knowledge about the pharmacological effects of neurosteroids on dopamine transmission, which may contribute to their therapeutic effects.Mesolimbic dopamine transmission is dysregulated in multiple psychiatric disorders, including addiction. Previous studies found that the endogenous GABAergic steroid -3-hydroxy-5-pregnan-20-one  modulates dopamine levels in the nucleus accumbens and prefrontal cortex. As allopregnanolone is a potent positive allosteric modulator of GABA Some steceptors  may proveceptors , but theeceptors . Previoueceptors . Moreoveeceptors . Furthereceptors  and modueceptors . Howevereceptors .A receptors, and based on the above evidence that drugs that enhance GABAA receptor activity reduce dopamine release, we hypothesized that it would inhibit phasic dopamine release. To test this, we used fast-scan cyclic voltammetry, a technique that offers the temporal, spatial, and chemical resolution required to assess fast dopamine release events  were assessed for estrous cycle stage by cell morphology after vaginal lavage with 20 µl of saline, immediately after the experiment. Data collection occurred between 1200 and 1700 h each experimental day, for all the male and female animals. Vaginal lavage occurred at the end of the experiment – typically between 1400–1500 h, but occasionally as late as 1700 h. All procedures were approved by the Institutional Animal Care and Use Committee of University of North Carolina at Chapel Hill.Adult male  and female  Sprague–Dawley rats  were used in the present study. Animals were housed in a temperature-controlled environment (21 ± 1°C) with 12-h light/dark cycles and Allopregnanolone, purchased from the late Dr. R. H. Purdy , was added to 45% hydroxypropyl-β-cyclodextrin  in water, vortexed, mixed in an ultrasonic water bath for 15–20 min, then stored while continuously stirring at 4°C, and kept for a maximum of two days after preparation. Progesterone  was added to the 45% hydroxypropyl-β-cyclodextrin solution, vortexed, then stored while continuously stirring at 4°C. The doses of allopregnanolone and progesterone used in the study were selected as doses with neuroactive (anti-seizure) effects but minimal sedative effects in rats .in vivo  and secured in a stereotaxic frame on a heated pad. Urethane was chosen due to modest effects on multiple neurotransmitter-gated ion channels  and the in vivo . Anterioin vivo . A bipolin vivo .Voltammetric parameters, electrical stimulation parameters, and data acquisition were controlled by a computer using LabVIEW instrumentation software  as previously described . In briein vivo electrodes used in the lab that were calibrated in a flow-cell with 1 µM DA in TRIS buffer  in females and 8.5 (range: 8.1–8.9 mm) in males. The average final DV placement of the carbon-fiber electrode in the NAc was 6.5 mm (range: 6.2–7.1 mm) in females and 6.8 (range: 6.2–7.6 mm) in males. The experimental recording began when evoked dopamine release reached a minimum of 2.5 nA and when the signal-to-noize ratio was higher than 25. Thereafter, dopamine release was evoked at 5-min intervals, and an average of nine electrically-evoked signals were collected before starting the experiment in order to confirm stability of the signal. Next, to ensure that injections did not alter the dopamine signal and that the signal was stable, animals received two saline injections (IP), 15 min apart. Finally, animals received allopregnanolone, progesterone, or vehicle (IP), followed by 60 min of electrochemical recording. The average calibration factor used to estimate dopamine concentration from current was 1 nA = 0.103 ± 0.017 µM, extracted from a library of 107 S buffer .max (maximum dopamine concentration) and T½  as measures of dopamine release and clearance, respectively  ANOVA, followed by Holm-Sidak post-hoc comparisons, as appropriate. For these analyses, we used raw T½ values and normalized [DA]max values. Specifically, the raw [DA]max data did not pass the Shapiro-Wilk normality test, and to account for individual variability due to electrode placement or length, [DA]max data were normalized within each rat to the evoked dopamine levels during the saline injections ; the transformed data passed the normality test.To broadly compare evoked dopamine release and clearance at baseline by sex or by estrous cycle stage, we used Mann-Whitney U tests or Kruskal-Wallis one-way ANOVA on ranks due to the non-normal distribution of the data. For these comparisons, we used raw  [DA]η2 of at least 0.0099, 0.0588, and 0.1379, respectively, based on values of ƒ as described by Cohen , resulting in seven time points . For the animals that received allopregnanolone (Allo) or vehicle, the pre-injection bins were indicated as BL , Sal  1, and Sal 2, and the post-injection as Allo 1, Allo 2, Allo 3, and Allo 4. For those that received progesterone (Prog), the bins were BL, Sal 1 and Sal 2, followed by Prog 1 to Prog 4. Effect sizes were considered small, medium, or large if they corresponded to partial by Cohen . Analysemax or T½; these data are reported in detail in the max  at the final time bin  representing the maximal effect of allopregnanolone. Allopregnanolone reduced evoked dopamine release at multiple doses in both males and females  and sex , with no significant interaction . Post-hoc comparisons following on the main effect of dose found that, collapsed across sex, both the 15 and 25 mg/kg doses were significantly different from vehicle (p < 0.05). The main effect of sex indicated that, collapsed across dose, males exhibited larger reductions in [DA]max after injections than females. Due to this sex difference, we next analyzed the dose-dependent effects of allopregnanolone over time separately within males and females.Allopregnanolone (or vehicle) was administered at 7.5, 15, and 25 mg/kg to separate groups of male and female rats. We first compared baseline dopamine signals between males and females and found no difference in [DA] females . The 2-wmax as compared to the vehicle group, although the timing of this effect differed , as well as main effects of dose  and time . Post-hoc analysis revealed that both 15 and 25 mg/kg allopregnanolone decreased [DA]max within 15 min after injection when compared to the vehicle group (Allo 1: both p < 0.05), and this effect was maintained throughout the collection time . In addition, 7.5 mg/kg allopregnanolone significantly reduced [DA]max compared to the vehicle group during the Allo 2 bin (p < 0.05). We also made within-group comparisons of each bin to the Sal 2, the bin immediately before the allopregnanolone or vehicle injection. None of the timepoints differed in the vehicle group, while all post-injection timepoints differed from baseline in the 15 and 25 mg/kg groups , and the final three bins differed from baseline in the 7.5 mg/kg group . We also compared T½ across groups, and found that allopregnanolone did not change dopamine clearance; these data are reported in detail in the In male rats, all doses of allopregnanolone reduced [DA]differed . The two6,30 = 11.1, p < 0.001), similar to the effect of 7.5 mg/kg dose.As we observed that 7.5 mg/kg allopregnanolone significantly reduced evoked [DA]max, we conducted a follow-up experiment with a lower dose–5 mg/kg – in a separate group of male rats (n = 6). As described in the max revealed a significant interaction between dose and time , as well as a main effect of time  and a marginal effect of dose . Post-hoc comparison confirmed that 15 and 25 mg/kg allopregnanolone decreased [DA]max in the Allo 2 bin when compared to the vehicle group (both p < 0.05), while the 7.5 mg group did not differ from vehicle at any time. Likewise, [DA]max in the vehicle and 7.5 mg allopregnanolone groups did not differ from the Sal 2 (the bin immediately preceding injection) at any time, while all post-injections timepoints were significantly reduced from baseline in the 15 and 25 mg/kg groups . Thus, while the 7.5 mg/kg dose effectively reduced dopamine release in males both within and between groups, it was ineffective in females. We also compared T½ across groups, and found that allopregnanolone did not change dopamine clearance; these data are reported in detail in the In female rats, only the 15 and 25 mg/kg doses effectively reduced evoked dopamine release . The two2 = 4.29, p < 0.12). We also found no significant difference in T½ across cycle stage; these data are reported in detail in the 6,42 = 3.99, p < 0.01), as well as significant main effects of time  and cycle stage . Post-hoc comparisons showed that rats in proestrus were less sensitive to the allopregnanolone effect on dopamine transmission than rats in non-proestrus cycle stages at all post-injection times .It is possible that the effect of allopregnanolone to decrease dopamine release in the NAc was affected by hormonal fluctuations during the estrous cycle. In rats, the estrous cycle includes proestrus, estrus, metestrus, and diestrus stages, and proestrus is the phase when the plasma levels of progesterone  are highest . Moreovemax . When compared to Sal 2, [DA]max in all post-injection time points was significantly reduced . Thus, administration of the precursor to allopregnanolone reduced evoked dopamine release similarly to allopregnanolone itself.As a proof of principle, to assess the effect of allopregnanolone’s precursor on dopamine transmission, we administered 30 mg/kg progesterone to male rats and measured evoked dopamine release in the NAc . Male raA receptors, and as other GABAergic compounds reduce evoked dopamine release  reduced evoked dopamine release compared to vehicle in males, while only the two higher doses reduced evoked dopamine release compared to vehicle controls in females. Other studies confirm similar sex differences in allopregnanolone action; for example, females that received allopregnanolone were less sedated and recovered more rapidly from the neurosteroid effect than males at the same dose . Similarsame age . We can  females . AlternaA receptors in multiple brain regions also fluctuates across the ovarian cycle  research award, and Bowles Center for Alcohol Studies at the University of North Carolina. MHM was supported on “UNC PREP in the Biomedical Sciences” (NIH R25 GM089569). AD and GM were supported on Postdoctoral Program Abroad Fellowships (CAPES)—Coordination of Improvement of Higher Education Personnel (Brazil).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.


With the increased interest in information on gut microbes, people are realizing the benefits of probiotics to health, and new technologies to improve the viability of probiotics are still explored. However, most probiotics have poor resistance to adverse environments. In order to improve the viability of lactic acid bacteria, polylactic acid (PLA) nanofibers were prepared by coaxial electrospinning. The electrospinning voltage was 16 kV, and the distance between spinneret and collector was 15 cm. The feed rates of the shell and core solutions were 1.0 and 0.25 mL/h, respectively. The lactic acid bacteria were encapsulated in the coaxial electrospun nanofibers with PLA and fructooligosaccharides (FOS) as the shell materials. Scanning electron microscopy, transmission electron microscopy, and laser scanning confocal microscopy showed that lactic acid bacteria were encapsulated in the coaxial electrospun nanofibers successfully. The water contact angle test indicated that coaxial electrospun nanofiber films had good hydrophobicity. An in vitro simulated digestion test exhibited that the survival rate of lactic acid bacteria encapsulated in coaxial electrospun nanofiber films was more than 72%. This study proved that the viability of probiotics can be improved through encapsulation within coaxial electrospun PLA nanofibers and provided a novel approach for encapsulating bioactive substances. Escherichia coli, Staphylococcus epidermidis, and T4 bacteriophage have also been encapsulated in electrospun nanofibers [Lactobacillus rhamnosus in poly  and sodium alginate-based electrospun nanofibers [Probiotics are defined by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO) as living microorganisms that, when administered in adequate amounts, confer a health benefit on the “host” . Probiotnofibers ,17. Bifinofibers  and Lactnofibers . Althougnofibers ,21,22. Inofibers .2 and H2O, which can be excreted out of the body through the kidneys. The US Food and Drug Administration (FDA) has long approved PLA use for absorbable surgical sutures, injection capsules, orthopedic fixation materials, microspheres for drug release, and tissue-engineered stents [Polylactic acid (PLA) is usually synthesized through lactic acid fermentation. Lactic acid is a type of organic acid that is widely found in animals and plants; therefore, PLA has good biocompatibility . PLA cand stents . At presd stents ,27.In this study, the probiotics represented by lactic acid bacteria were encapsulated in PLA nanofibers by coaxial electrospinning, and fructooligosaccharides (FOS) were added as a prebiotic to improve the viability of probiotics in the digestive system. The physicochemical properties of the electrospun nanofibers were analyzed by means of scanning electron microscopy (SEM) micrographs, Fourier transform infrared spectroscopy (FT-IR), water contact angle, and differential scanning calorimetry (DSC). The survival rate of probiotics was studied via in vitro simulated digestion experiments.N,N-Dimethylformamide (DMF) were obtained from TianJin Fine Chemicals Co., Ltd. . Disodium hydrogen phosphate (Na2HPO4) and potassium dihydrogen phosphate (KH2PO4) were purchased from Aladdin Reagent Co., Ltd. . Fructooligosaccharides (FOS), Rhodamine 123, and de Man−Rogosa−Sharpe (MRS) medium were obtained from Beijing Solarbio Science & Technology Co., Ltd. . Lactic acid bacteria (Lactobacillus plantarum 1.0665) were donated by the College of Food Science, Northeast Agricultural University .PLA (4032D) was purchased from NatureWorks LLC. . Dichloromethane (DCM) and w/v), and the concentration of FOS was 2.5% (w/v). The core solution was prepared by inoculating 1% (v/v) L. plantarum 1.0665 into the MRS medium [8 CFU/mL.The shell solution was prepared by dissolving PLA in a mixture of DCM and DMF with a volume ratio of 7:3, and then FOS was added. The shell solution was magnetically stirred at room temperature (20–25 °C) for 6 h until it was dissolved completely. The concentration of PLA was 8%  with an s61 rotor at 20 °C. The depth of the immersion solution was based on the groove above the probe . The datThe electrospinning nanofiber films were cut out and adhered to the sample stage with a conductive tape. After sputtering Au and carbon coating with an ion sputtering apparatus, the microscopic morphology of the nanofibers were observed using a scanning electron microscope ; the acceleration voltage was 5 KV. The diameter of the nanofibers was measured using Image-Pro Plus software. The average diameter of nanofibers was calculated from the obtained data and plotted as a diameter distribution histogram.−1, wavenumber range of 4000–400 cm−1, and 64 scans [The electrospun nanofiber films and the materials were analyzed using a FT-IR spectrometer  with an Attenuated Total Reflection (ATR) device. The electrospinning nanofiber films were cut into 3 cm × 3 cm sheets and placed on the ATR device, and the materials were measured using a KBr disk with spectral resolution of 4 cm64 scans .The thermal behavior of the electrospun nanofiber films and materials was analyzed using a differential scanning calorimeter . Accurately weighed samples of 6–10 mg were placed in sealed aluminum crucibles. An empty crucible under the same conditions was used as a reference. The samples were heated from 20 to 230 °C with a heating rate of 10 °C/min under a nitrogen atmosphere.The water contact angles of the electrospun nanofiber films were measured using a water contact angle meter . The electrospun nanofiber films were cut into 10 cm × 10 cm sheets and fixed to glass slides. A droplet of water was added to the surface of the electrospinning nanofiber films. The averages of five measurements recorded at different areas of each sample were taken as the water contact angles. The water contact angles were measured from 0° to 180° with an accuracy of ± 0.1°. The Young–Laplace equation was used to calculate the water contact angles.The coaxial electrospun nanofibers were collected directly onto the copper grids. The morphology and core/shell structure of the coaxial electrospinning nanofibers were observed by using a transmission electron microscope . The images were captured using an Olympus imaging system .The lactic acid bacteria were stained by Gram stain and observed using an inverted light microscope. The presence and distribution of lactic acid bacteria stained with Rhodamine 123 were observed using an inverted laser scanning confocal microscope  with an excitation wavelength of 488 nm to check whether the lactic acid bacteria were properly encapsulated within the coaxial electrospinning nanofibers ,32.2PO4 6.8 g/L, pH = 7.0) and incubated at 37 °C for 2 h. The digestive fluid was taken out, and the concentration of the lactic acid bacteria was measured by the standard plate count. Undigested coaxial electrospun nanofiber films under the same conditions were used as a control, the concentration of which was considered as 100%.The in vitro digestion studies were carried out and improved following the methodology proposed by Marysol Aceituno-Medina, et al. . CoaxialExperiments were performed in triplicate. Statistical analysis was performed using SPSS Statistics Software . The obtained data are presented as the mean ± standard deviation.1/2/cm3/2, which is equivalent to that of non-polar materials. Chloroform, dichloromethane, tetrahydrofuran and others could be used as good solvents for PLA. However, DCM has a relatively lower toxicity than the aforementioned solvents. The conductivity of DCM can be increased by mixing DMF with high charge induction capability to stabilize the jet flow. The solubility parameter of the mixed solvent system (δ2) is 17.9~19.85 J1/2/cm3/2, 0.35 ≤| δ1 − δ2 |≤ 3.9. Therefore, the mixed solvent could be used as a good solvent for PLA electrospinning.Three conditions should be satisfied for a solution to produce a stable jet in an electrostatic field. Firstly, there should be the ability to generate inductive charge, which causes the charged jet to form a wave back and to form the spinning body in the electrostatic field. Secondly, the solvent should have a good solubility for the solute to ensure that solute molecules in the solvent are evenly dispersed and at the receiving end to form a spinneret. Finally, the solvent system can be composed of multiple solvents that are volatile ,35. AfteThe viscosity of the electrospun solution directly affects the morphology and properties of the nanofibers obtained by electrospinning. The higher the viscosity of the solution, the more easily the polymer molecular chain will become entangled and the more unstable the jet flow will be, which makes the electrospinning process more difficult and it is not easy to produce nanofibers with a uniform diameter distribution. However, when the viscosity of solution is low, the solution cannot form a jet in the electrostatic field, but only form microdroplets. Therefore, the key of electrospinning is to prepare a solution with appropriate viscosity . The visThe microstructure of the shell and coaxial electrospinning nanofibers were analyzed by scanning electron microscopy. As shown in −1, while the spectra of FOS had characteristic absorption peak (O–H stretching) at 3385 cm−1. Similarly, the absorption peaks (O–H stretching) of shell and coaxial electrospinning nanofiber films occur at 3410/3420 cm−1, indicating that FOS was successfully electrospun into the shell nanofibers. The carboxyl group in PLA caused the O–H stretching peak of the shell and coaxial electrospinning nanofiber films to exhibit a blue-shift. The spectra of PLA possessed peaks at 2967/2857 cm−1 (CH3 inverse/symmetric), 1733 cm−1 (C=O), 1154 cm−1 (C–O–C), and 1045 cm−1 (C–C). The spectra of FOS showed peaks at 2939/2902 cm−1 (CH3inverse/symmetric), 1146 cm−1 (C–C), and 1058 cm−1 (C–O–C). Most of the peaks of PLA and FOS with different strengths were found in the spectra of shell and coaxial electrospun nanofiber films, indicating that both the substances exist in the shell electrospun nanofibers.The FT-IR spectra of PLA, FOS, shell and coaxial electrospun nanofiber films are shown in The viscoelastic and viscoplastic properties of polymers are related to their melting and glass transition temperatures, respectively. PLA is a semi-crystalline polymer containing both amorphous and crystalline domains; therefore, it shows both glass transition and melting temperatures . The resHydrophobicity is an important parameter of microcapsule encapsulation materials, which is usually characterized by the water contact angle. In general, a material is called hydrophilic when the intrinsic water contact angle is smaller than 90°, and a material is called hydrophobic when the intrinsic water contact angle is greater than 90° . As showInverted optical microscope images of lactic acid bacteria and laser scanning confocal microscope images of lactic acid bacteria and coaxial electrospun nanofibers were captured. 4 CFU/mg. Under the condition of simulating digestion of intestinal solution for 2 h, the viable count of lactic acid bacteria was 8 × 103 CFU/mg, which remained above 72%. According to the FT-IR results, FOS was contained in shell electrospun nanofibers, which could selectively promote the further growth of lactic acid bacteria and improve their activity. Yoha et al. [p < 0.5). The above discussion confirms that electrospinning is a successful microencapsulation technique. However, there are a few limitations that will hinder the progress of its applications. For instance, in the food industry, the biocompatibility of materials is necessary. In order to utlize electrospun products prepared in laboratory for practical applications, further research is required to have an in-depth understanding of the materials and address the limitations [The food needs to be digested in the stomach across four stages for about 2 h. The free lactic acid bacteria without encapsulation decreased rapidly after the simulated digestion for 0–2 h, and the number of viable bacteria decreased to zero . The elea et al.  used spra et al.  prepareditations .L. plantarum were prepared. The average diameter of the coaxial electrospun nanofibers was 676 ± 162 nm. As observed in the LSCM image, the lactic acid bacteria exhibited a linear arrangement, indicating that the bacteria were successfully encapsulated in the coaxial electrospun nanofibers. In addition, the DSC and FT-IR analysis of the coaxial electrospun nanofiber films showed the characteristics of PLA, FOS, and nanofiber films, which provided further evidence for the successful preparation of coaxial electrospun nanofibers. The water contact angle of coaxial electrospun nanofiber films was 113.5 ± 0.1°, thus indicating their hydrophobic nature. Moreover, the in vitro simulated digestion of lactic acid bacteria showed that during digestion, the number of viable bacteria remained above 72%. Therefore, with PLA and fructooligosaccharides (FOS) as shell materials and by encapsulating lactic acid bacteria using the coaxial electrospinning technique, the nanofiber films were found to exhibit good hydrophobicity and biocompatibility, and the encapsulated lactic acid bacteria showed good activity. This study provides a novel approach to the encapsulation of bioactive substances.In this study, coaxial PLA electrospun nanofibers containing