Although both males and females become addicted to cocaine, females transition to addiction faster and experience greater difficulties remaining abstinent. We demonstrate an oestrous cycle-dependent mechanism controlling increased cocaine reward in females. During oestrus, ventral tegmental area (VTA) dopamine neuron activity is enhanced and drives post translational modifications at the dopamine transporter (DAT) to increase the ability of cocaine to inhibit its function, an effect mediated by estradiol. Female mice conditioned to associate cocaine with contextual cues during oestrus have enhanced mesolimbic responses to these cues in the absence of drug. Using chemogenetic approaches, we increase VTA activity to mechanistically link oestrous cycle-dependent enhancement of VTA firing to enhanced cocaine affinity at DAT and subsequent reward processing. These data have implications for sexual dimorphism in addiction vulnerability and define a mechanism by which cellular activity results in protein alterations that contribute to dysfunctional learning and reward processing. et al. report oestrous cycle-dependent fluctuations in firing of VTA dopamine neurons that drive alterations in DAT function expressed in terminals in the NAc. These differences underlie enhanced cocaine reward processing during oestrus.Sex differences in reward processing are at present poorly understood. Calipari and Juarez  Drug addiction is a debilitating neuropsychiatric disorder characterized by high levels of drug intake, drug seeking and repeated cycles of abstinence and relapse3459In addition to females displaying evidence for higher risk for addiction than males, there are also several reports of menstrual cycle-dependent fluctuations in the subjective effects of cocaine and drug craving in females1111Circulating ovarian hormones, including oestrogens, act throughout the entire nervous system via non-genomic and genomic mechanisms. During development, X-chromosome genes play a role in developmental programming of the dopamine system via genetic and hormonal factors that can lead to sex differences in both basal function and the hormonal control over these processes141517in vivo calcium imaging via fibre photometry to study the ability of male and naturally cycling, intact female mice in either dioestrus (low circulating hormones) or oestrus (high circulating hormones) to associate the rewarding effects of cocaine with the contextual cues that predict cocaine administration. By monitoring cycle stages of female mice, we determined how oestrous cycle-dependent fluctuations in reward system activity underlie reward processing and how this can influence addiction vulnerability. Here we outline a mechanism by which enhanced function of the dopamine system converges with increased pharmacological effects of cocaine to enhance these associations.Studies in animal models have implicated ovarian hormone-dependent changes in drug reward functioning−1 intraperitoneally (i.p.) cocaine and confined to one side of a three-chamber CPP apparatus. In alternating sessions, the other chamber was paired with saline (We first conducted conditioned place preference (CPP) for cocaine, which provides an indirect measure of drug reward, in male and intact female C57BL/6J mice. Animals were injected with 10 mg kgh saline . VaginalBoth male and female mice spent more time on the side that was previously paired with cocaine; however, female mice exhibited higher levels of place preference compared with males, indicating enhanced reward to this moderate cocaine dose . InteresBoth drug reward and cue-reward associations  are dependent on dopaminergic transmission from the ventral tegmental area (VTA) to the nucleus accumbens (NAc)in vivo electrophysiology in drug-naive male mice, dioestrus female mice and oestrus female mice, we found that female mice overall exhibit increased basal putative VTA dopamine neuron activity when compared with male mice , the rate-limiting enzyme for dopamine synthesis, were not changed, nor was the phosphorylation of TH at any of the sites that alter its catalytic activity . Howeverin vivo during associative learning for drug rewards, we injected male and female C57BL/6J mice with AAV5-CamKII-GCaMP6f into the VTA and recorded Ca2+ transients through two separate optic fibres implanted in the VTA and the NAc during cocaine and saline conditioning  and magnitude (% ΔF/F) in all groups  is increased in oestrus females  within each animal on blood collected immediately before FSCV recordings. We found that circulating estradiol levels were significantly higher in females in oestrus as compared with both dioestrus females and males . FurtherKm (affinity) of dopamine for DAT was not significantly different before cocaine bath application, suggesting that the effects of cocaine on DAT are distinct from any direct effects of estradiol on the transporter  was significantly higher in oestrus-conditioned female mice , combined with viral expression of Cre-dependent AAV5-DIO-GCaMP6f in the VTA, to record dopamine neuron-specific Ca2+ transients through two separate optic fibres implanted in the VTA and the NAc during cocaine and saline conditioning  was significantly higher in oestrus-conditioned female mice when recording specifically from VTA dopamine cell bodies  we were able to use clozapine-N-oxide (CNO) to enhance VTA dopamine neuron firing selectively in males and dioestrus females  and elevate dopamine levels is increased in oestrus females . TH-BAC-Cre mice from GENSTAT were bred and maintained by the Center for Comparative Medicine and Science staff at Icahn School of Medicine at Mount Sinai Mice until transfer for experimental use. All mice were housed three to five per cage. Female mice were subjected to daily vaginal smears and vaginal cytology was conducted to determine oestrous cycle phase. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Icahn School of Medicine at Mount Sinai, which approved and supervised all animal protocols. Experimenters were blind to experimental group and order of testing was counterbalanced during behavioural experiments.Male and female 6- to 8-week-old C57BL/6J and −1 in 5% dimethyl sulfoxide in normal saline (Sigma-Aldrich). CNO was administered in the following dose of 1 mg kg−1, i.p. An equal (matched) volume of vehicle  was used as the control. Cocaine HCl was provided by the NIDA drug supply programme and administered at a 10 mg kg−1 doses i.p. For slice voltammetry experiments, Cocaine (NIDA Drug Supply), Quinpirole (Sigma-Aldrich) and Estradiol (Sigma-Aldrich) were perfused over slices in artificial cerebrospinal fluid (aCSF) solution.CNO (Sigma-Aldrich) was dissolved at a concentration of 2.5 mg mlTH-BAC-Cre mice. For DREADD experiments in dopamine populations of the VTA, TH-BAC-Cre animals were injected with AAV2-hSyn-DIO-hM3Dq-mCherry that was purchased from the viral core at the University of North Carolina, Chapel Hill.For general GCaMP expression in VTA neuronal populations, GCaMP6f was cloned into the adeno-associated virus (AAV) vector, AAVdj-CaMKIIα-GCaMP6f-WPRE. For dopamine-specific GCaMP expression in TH(+) populations of the VTA, cre-inducible (DIO) GCaMP6f was cloned into the AAV vector, AAVdj-DIO-EF1α-GCaMP6f-WPRE, and injected into the VTA of −1)/xylazine (10 mg kg−1) anaesthesia, mice were positioned in a stereotaxic frame (Kopf Instruments) and the VTA was targeted . Ophthalmic ointment was applied to the eyes to prevent drying. A midline incision was made down the scalp and a craniotomy was made using a dental drill. A 10 μl Nanofil Hamilton syringe  with a 34-gauge beveled metal needle was used to infuse 0.5 μl virus at a rate of 100 nl min−1. Following infusion, the needle was kept at the injection site for 10 min and then slowly withdrawn. Chronically implantable optic fibres constructed with 400 μm core 0.48 numerical aperture (NA) optic fibre and unilaterally implanted into the VTA  and NAc , and were cemented to the skull using dental acrylic (Grip Cement Dentsply). For robust viral expression, viral infusions and implantation occurred a minimum of 4–6 weeks before recordings.Under ketamine . For calcium imaging during conditioning or during choice tests, a 3 m-long fibre-optic patch-cord (Doric Lenses) was connected to the chronically implanted optical fiber with a zirconia sheath and was suspended above the behavioural testing environment, to allow animals to move freely during stimulation. Noldus software running Ethovision 10.0 recorded behavioural outputs.TH-BAC-Cre mice. The virus was allowed to express for 5 weeks and then the animals went through CPP as described above. CNO  was administered 2 h before cocaine pairing sessions. Time spent in each area of the conditioning chamber was recorded as before and animals were considered to have developed a preference when the difference in time spent between the drug paired and chamber paired with saline was significantly greater than before the pairings.To determine whether basal increases in VTA firing were sufficient to enhance reward processing, AAV-DIO-hM3Dq was injected into the VTA of In vivo extracellular, single-unit recordings to determine basal activity of VTA dopamine neurons of anaesthetized intact mice were conducted following published methods−1, i.p.) and head fixed to a stereotaxic frame. The skull was exposed to identify bregma and coordinates were measured to determine VTA location where a skull window was created . Extracellular single-unit signals were amplified using a DP-311 Differential Amplifier and filtered (0.3–1 kHz band pass). Voltage data were acquired using an Axon Digidata Data Acquisition System, sampled using 16-bit resolution at 32 kHz and stored using pCLAMP for further offline analysis.R as previously describedPutative dopamine cells in the VTA were identified according to standard electrophysiological criteria: a stark, triphasic waveform with set filters; action potential duration from start to negative trough ≥1.1 ms; slow firing rate (<10 spikes per second); and bursting behaviour with onset beginning when two spikes occurred within <80 ms and offset set when no activity occurred for ≥160 msEx vivo FSCV was used to characterize D2/D3 autoreceptor function, DAT activity, dopamine release and cocaine potency in the NAc. Voltammetry experiments were conducted 1–2 weeks following the completion of the CPP experiments. Recordings were made directly at the end of the fiberoptic probe, to ensure that the recordings were from the same location in the NAc as calcium-imaging experiments. A vibrating tissue slicer was used to prepare 250 μm-thick coronal brain sections containing the NAc, which were immersed in oxygenated aCSF containing (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2), NaHCO3 (25), glucose (11), L-ascorbic acid (0.4) and pH was adjusted to 7.4. The slice was transferred to the testing chambers containing aCSF at 32 °C with a 1 ml min−1 flow rate. A carbon fibre microelectrode  and bipolar stimulating electrode were placed into the NAc where GCaMP-GFP terminal expression was high and near the optic fibre track to get the best representation of release near the recorded regions. Dopamine release was evoked by a single electrical pulse  applied to the tissue every 5 min. The extracellular dopamine level was recorded by applying a triangular waveform . Once the peak of evoked dopamine release was stabilized (three collections with <10% variability), the amount of evoked dopamine release and a maximal rate of uptake (Vmax) were assessed. Subsequently, tonic and phasic stimulations were applied to slices, to determine the responsivity of VTA terminals in the NAc to tonic and phasic firing parameters. Tonic stimulations consisted of one pulse stimulations. Phasic stimulations consisted of five pulses at either 5, 10 or 20 Hertz. These stimulation parameters were selected based on the physiological firing properties of VTA dopamine neurons in vivo.To assess autoreceptor function, quinpirole, a D2/D3 receptor agonist, was applied cumulatively to the brain slice, to determine a concentration–response curve (0.01–1 μM) to assess quinpirole effects on dopamine release. In a slice preparation, presynaptic autoreceptors are dissociated from postsynaptic D2 receptors. As quinpirole activates autoreceptors, evoked dopamine release is reduced. The resulting concentration–response curve is then fit to determine the IC50 of quinpirole in each condition. Increases in autoreceptor sensitivity increase the IC50 of qunipirole on dopamine release.−1) was applied and resulting current versus time plots were analysed at each concentration, to determine the ability of cocaine to inhibit DAT and elevate synaptic dopamine levels.To determine cocaine potency, cocaine was applied cumulatively to brain slices. Once the extracellular dopamine response was stable, cocaine (0.03–30 μmol l−1) within each group.To determine the local effects of estradiol on NAc brain slices, estradiol was bath applied at 1 or 3 μM. Recordings were taken for 60 min at each dose, to ensure that estradiol had sufficient time to induce changes. Once dopamine measurements were stable, a concentration response curve for cocaine was run  to a known concentration of dopamine (3 μM) using a flow-injection system. This was used to convert an electrical current to a dopamine concentration.Vmax and cocaine potency. The quinpirole concentration–response curves were assessed by determining the peak height of dopamine release at each dose and expressed as a percentage of the pre-drug basal dopamine release.Demon voltammetry and analysis software was used for all analysis of FSCV dataKm (App. Km), a measure of the ability of a drug to inhibit dopamine clearance, was used to evaluate changes in cocaine potency. App. Km was determined from the decreased affinity of dopamine (Km) for the DAT. As the ability of DAT to clear dopamine is decreased (following application of cocaine), the extent to which this occurs can be calculated. The decreased affinity of dopamine (Km) for DAT is then used to determine an App. Km of the drug for the transporter. For cocaine concentration–response curves, App. Km, a measure of apparent affinity for the DAT, was used to determine changes in ability of the cocaine to inhibit dopamine uptake. As App. Km increases, uptake inhibition increases and changes in drug potency are seen as shifts in the curve.To evaluate dopamine kinetics and drug potency, evoked levels of dopamine were modelled using Michaelis–Menten kinetics. Apparent Ki) were determined by plotting the linear concentration-effect profiles and determining the slope of the linear regression. The Ki was calculated by the equation Km/slope. Ki values are reported in μM and are a measure of the drug concentration that is necessary to produce 50% uptake inhibition. Ki is a measure of the actual drug affinity for the DAT in concentration of drug, whereas Km is a dopamine-related parameter that estimates uptake inhibition.Inhibition constants  and the plate was incubated for 30 min in the dark. After substrate incubation, the reaction was halted with the addition of an acid-based stop solution (100 μl per well). The plate was read using a Spectra max plus plate reader equipped with a 450 nm filter (Molecular Devices).ELISA kits for estradiol and progesterone  were used to measure serum samples from oestrus and dioestrus females and males. Blood samples were taken immediately before FSCV recordings. The appropriate estradiol or progesterone standards of the manufacturer, and negative and positive controls were run on each plate. A 10 μl aliquot of diluted samples was pipetted manually into the plate wells in duplicate. The ELISA assays were then run according to the instructions of the manufacturers. The samples were incubated in microplate wells coated with fixed amounts of oriented high-affinity purified polyclonal antibody and the mixture was incubated at room temperature for 60 min in the dark. After incubation, the plate was washed six times with deionized HTH-BAC-Cre mice. The virus was allowed to express for 5 weeks. Vaginal cytology was done starting 2 weeks before the voltammetry experiments, to determine the cycle stage of the animals. For all groups DREADDs were expressed in VTA dopamine neurons and activated with CNO (1 mg kg−1). Vaginal cytology was done before FSCV recordings, to confirm cycle stage on day of recording.To determine whether basal increases in VTA firing were sufficient to alter basal dopamine system function, AAV-DIO-hM3Dq-mCherry was injected into the VTA of male and female Fibre photometry uses the same optic fibre to both excite and record from the genetically encoded calcium indicator GCaMP6f in real time and thus can allow for temporally precise measurements of neuronal activity that can then be time-locked to a specific behavioural output2627Analysis of the signal was accomplished with custom-written MatLab softwareF/F was then calculated for each behavioral session as: /. We calculated a single baseline fluorescence value, either as the median of the entire trace (which robustly estimated the baseline fluorescence) or by manually defining the baseline during visually identified periods of rest. We calculated the normalized change in fluorescence (ΔF/F) by subtracting the baseline fluorescence from the fibre fluorescence at each time point and dividing that value by the baseline fluorescence. Peak reward ΔF/F was calculated as the maximum ΔF/F value in the paired chamber in a 10 s period around the entry. It is important to note that this window is only applied around trials where the animal is facing the chamber and subsequently enters the chamber.To do this, both signals were first filtered at 40 Hz. A least-squares linear fit was applied to the 405 nm control signal, to align it to the 490 nm signal. The ΔBehavioural variables, such as paired and unpaired chamber entries, were marked in the signalling traces via the real-time processor as electrical pulses from Noldus Ethovision software. This allowed for precise determinations of the temporal profile of signals in relation to paired and unpaired chamber entries. For each mouse, the time at which the animal entered the drug-paired chamber was determined and a 5 s window around that point was analysed. We subsequently determined the maximum or minimum fluorescent signal that occurred within that window over both consecutive entries and across behavioural tasks/sessions.Peak analysis to determine frequency and amplitude of the signals was carried out by determining the median average deviation of the corrected/normalized data set and peaks were identified as events that exceeded the median average deviation by 2.91 deviations.Bilateral 14-gauge NAc punches were collected on ice after rapid decapitation and then immediately placed on dry ice and stored at −80 °C until use. Protein concentrations of whole-cell lysates were determined using a DC protein assay (Bio-Rad). Within each fraction, equal amounts of protein were mixed with reducing sample buffer containing SDS and dithiothreitol, and samples heated to 95 °C for 5 min. Samples were separated by molecular weight using SDS–PAGE with Criterion 4–15% Tris-HCl, 1.0 mm precast gels (Bio-Rad) and proteins transferred onto Immobilon-P 0.45 μm pore side membranes (Millipore) at 100 V for 1 h at 4 °C in SDS-free transfer buffer containing 15% methanol. Membranes were blocked in Tris-buffered saline containing 5% BSA and 0.1% Tween-20 for 1 h at room temperature and membranes incubated with a polyclonal primary antibody to DAT , pDAT , TH , pS40 TH , Erk1/2  or pERK1/2  in blocking solution overnight at 4 °C. Membranes then were incubated with a peroxidase-conjugated secondary antibody  in blocking solution at room temperature for 2 h, washed and developed using chemiluminescent substrate (Thermo Scientific). The amount of protein expressed in each lane was quantified by densitometry using Image J . Actin levels were used to normalize measurements. For DAT, two bands (at 75 and 55 kDA) are obtained due to different glycosylation states of the mature protein. Each band was quantified separately and the ratio of the 75 kDA band to the 55 kDA band was compared. Images in Ki), VTA contextual cue responses, baseline VTA activity as measured via fibre photometry, cycle-dependent changes in uptake rates and cycle-dependent changes in Km were analysed by a one-way analysis of variance with Tukey's post-hoc test. Data of VTA dopamine neuron in vivo firing rate, VTA dopamine neuron burst length and NAc contextual cue-responses were analysed using a Kruskal–Wallis χ2-test because of non-normal distributions followed by Wilcoxon test. Data for phasic stimulations responses across groups, quinpirole concentration response curves, cocaine-induced inhibition of VTA and NAc activity using fibre photometry, cocaine concentration–response curves, cocaine-induced dopamine release and effects of estradiol and CNO during FSCV recordings were analysed by a two-way analysis of variance with Bonferroni post-hoc test. Initial male versus female comparisons for CPP and basal VTA electrophysiology were compared using a Student's t-test. Correlational analyses were used to compare CPP difference score with cocaine effects in the VTA and NAc, contextual-cue response with difference score and maximal rate of uptake with cocaine affinity (Ki). P-levels <0.05 were considered to be statistically significant for all data.Graph Pad Prism  or R were used to statistically analyse data sets and create graphs. Normality of data was assessed and, when data were not normally distributed, non-parametric analyses were performed. Data for CPP across oestrous cycle, VTA dopamine neuron percent spikes within a burst, voltammetrically recorded dopamine release, cocaine effects during fibre photometry recordings, cocaine affinity measurements .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures


All membrane‐bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron‐transfer iron sulfur cluster closest  to the [NiFe] H2‐binding active site. Replacement of this amino acid with alanine induces O2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O2‐induced over‐oxidation of the [Fe4S3Cys2] proximal cluster possessed by all O2‐tolerant MBH. We have created an Escherichia coli Hyd‐1 His‐to‐Ala variant and report O2‐free electrochemical measurements at high potential that indicate the histidine‐mediated [Fe4S3Cys2] cluster‐opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His‐to‐Ala replacement in Escherichia coli Hyd‐2, a [NiFe]‐MBH that contains a [Fe4S4] center.[NiFe] hydrogenases are electrocatalysts that oxidize H Proton transfer is then initiated from Glu73  is a common choice as a replacement residue when probing the biochemical function of the amino acids in a protein structure because it possesses sufficient steric bulk to stabilize the resultant variant structure but does not introduce any new acid/base or redox reactivity.However, in our work on the 2‐sensitive [NiFe] hydrogenases such as E. coli Hyd‐2, the proximal cluster is a standard ferredoxin‐like [Fe4S4]2+/1+ center.2‐sensitive [NiFe] hydrogenases also show a large‐subunit His residue close to the proximal cluster,E. coli Hyd‐2 .In OE. coli Hyd‐1 and His‐214 was replaced with Ala in E. coli Hyd‐2 is described in SI. The purification of these variants and the “native” His‐containing unmodified versions of the enzymes was enabled by the incorporation of small subunit, C‐terminus hexa‐His tags . The purity of the isolated hydrogenases was checked with SDS‐PAGE gels, as shown in Figure S2. All purified enzymes contained a band at ∼65 kDa and a band at ∼40 kDa, denoting the large and small subunits, respectively, of the hydrogenase dimer. Additional protein bands were also visible, particularly in the Hyd‐1 variant and both Hyd‐2 samples. The band at ∼25 kDa has been identified in other hydrogenase samples and has been characterized as cAMP Receptor Protein, which has been named as one of the most common “contaminant” E. coli proteins seen in Ni‐affinity chromatography.2‐exposed dialysis or centrifugation steps. In electrochemical experiments, the only detectable redox activity is assignable to hydrogenase catalysis indicating that either the contaminating proteins are non‐redox active or that they fail to adsorb to the electrode, with film formation essentially acting as a final purification step.The procedure for the successful generation of variants in which His‐229 was replaced with Ala in S. enterica Hyd‐5,It is notable that the final protein yields of the variants were approximately half of those of the respective native enzymes . It is therefore suggested that whatever the role in tuning chemical reactivity may be, in [NiFe] hydrogenases the structural integrity of the dimeric enzyme unit benefits from the presence of a large subunit His located at the large‐small protein dimer interface. Lower protein yields for a His‐to‐Ala variant were not noted in the purification of the 2. At pH 6.0 both native Hyd‐2 and the Hyd2‐H214A variant exhibit the characteristic aerobic reactivity of O2‐sensitive hydrogenases;2 into the gas mixture their catalytic activity tends towards zero . When O2 is removed from the gas stream, there is no significant difference in the extent of reactivation of native Hyd‐2 and Hyd2‐H214A, suggesting that the His does not act in a protective manner against formation of the slow‐reactivating, O2‐inihibited Ni−A state which O2‐sensitive [NiFe] hydrogenases are known to form.Figure S. enterica Hyd‐5, exchanging the conserved His for Ala in E. coli Hyd‐1 decreases O2 tolerance at pH 6.0.2 sensitivity of Hyd‐1 than Hyd‐2 at pH 6.0 supports this model. The impact of pH on the O2‐tolerance of Hyd‐1 is discussed later.As summarized in Table 2. Chronoamperometry experiments conducted at pH 4.5  further confirm that the amino acid exchange has not impacted the catalytic bias, as do cyclic voltammograms under different partial pressures of H2 . Analogous catalytic bias potential‐step and voltammetric experiments comparing native Hyd‐1 and the Hyd1‐H229A variant also indicate that the His residue close to the proximal cluster plays a minimal role in controlling the ratio of oxidation to reduction current in Hyd‐1  Conversely, the value of kA for the Hyd1‐H229A variant is consistently lower than that of native Hyd‐1. This correlates with the difference in Eswitch values.Figure l Scheme . The avekA due to the one electron Ni3+/2+ reductive activation of the active site proceeding via electron transfer from the proximal cluster oxidizing from the +4 to the +5 state. Alternatively, if the active form of the enzyme is assumed to require the proximal cluster in the +3 or +4 state, then slow reductive activation of the cluster may be interpreted as the reason for the variants decreased kA. It is suggested that to deconvolute the mechanism more precisely a more extended family of variants and wider suite of techniques is required. The amino acid residues which Dance predicts act as a communication relay between the active site and His‐229, particularly Thr‐80, which is thought to H‐bond to His‐229, should be exchanged for Ala or possibly valine in the presence and absence of the His‐to‐Ala exchange. Electrochemistry, EPR and ideally X‐ray crystallography data is then required to underpin more extensive calculations. Ideally, we would like to use the more sophisticated technique of Fourier transform large amplitude alternating current voltammetry to interrogate such variants, since this may enable measurement of the iron sulfur cluster redox transitions.According to the Dance model, removal of the His would impede the oxidative opening of the proximal cluster. Disrupting this process may decrease kI is insensitive to changes in the proximal cluster redox chemistry suggests that in the native and variant enzyme the rate‐determining step of oxidative inactivation remains unchanged. This can be interpreted by considering the work by Jones et al.2‐sensitive [NiFe]‐hydrogenase the chemical binding of hydroxide was the rate‐limiting process during anaerobic oxidative inactivation, rather than the secondary electrochemical step.The fact that the inactivation rate 2 tolerance experiments  should be addressed to the authors.SupplementaryClick here for additional data file.


Antimicrobial resistance (AMR) is a growing public health problem across the world. As the negative consequences of AMR become apparent at local, national and international levels, more attention is being focussed on the variety of mechanisms by which AMR is potentiated. We explore how interactions between pet owners and veterinarians represent a key arena in which AMR-related behaviours can be shaped.n = 23) and vets (n = 16) across the UK in 2017. A thematic analysis approach was taken, with inductively gathered data analysed deductively using a behavioural framework to identified key behaviours emerging from participant accounts which were amenable to change.In depth semi-structured interviews were carried out with pet owners (Interactions between vets and pet owners were characterised by misunderstandings and misconceptions around antibiotics by pet owners, and a lack of clarity about the positions and intentions of the other party. Vets and pet owners had differing perceptions of where pressure to prescribe antibiotics inappropriately originated. Vets perceived it was mostly pet owners who pushed for inappropriate antibiotics, whereas pet owners reported they felt it was vets that overprescribed. Low levels of understanding of AMR in general were apparent amongst pet owners and understandings with regard to AMR in pets specifically were almost non-existent in the sample.Improved use of antibiotics could be assisted by educating the pet owning public and by guideline development for companion animal vets, concurrent development of mandatory legislation, increased consultation time to facilitate better communication, development of vet training on antimicrobial therapy and stewardship led interactions with pet owners, and increased levels of knowledge of pet-related AMR amongst pet owners. Interdisciplinary consensus identifies AMR as an increasingly severe global concern , 2. AntiAnimal health care is implicated in the problem of AMR. Animals have been shown to have potential to act as reservoirs for resistant organisms, especially when infections are not comprehensively and successfully treated. Moreover, veterinary science acknowledges that a unique and critical aspect of AMR in pets is their close physical contact with humans , 10. ThiTo combat these drivers of AMR, the effective use of antibiotics in animal health depends on pet-owners and their collaborations with prescribing vets. Pet owners mediate the treatment of their animals; they control antibiotic usage and other actions that can directly determine potential for AMR in their animals . In the This paper addresses the pet-owner – vet prescribing interaction through qualitative interviews focussed on experiences and knowledge of antibiotics and AMR, to i) shed light on pet-owners and vets experiences, beliefs and intentions with regard to their interactions on antibiotic prescription, and ii) to identify approaches for behaviour change interventions targeting pet owner-vet interactions as a primary site for mutual understanding between parties to develop more responsible antibiotic use in pets.What do vets believe are the most important aspects of interaction between pet owners and themselves that can lead to increases in AMR?What do the pet owning public believe are the most important aspects of interactions between themselves and vets that can lead to increases in AMR?How do these aspects help or hinder the development and maintenance of antimicrobial stewardship?n = 22) and small animal vets (n = 16) across the UK in 2017.We took a qualitative approach, using semi-structured interviews with two types of study group: pet owners .Pet owners were recruited through: adverts on social media (Facebook and Twitter); convenience sampling at a veterinary practice; snowballing via recruited participants; and through personal contacts from the research team. Inclusion criteria for pet owners were: having a dog, cat or rabbit; having received antibiotics for their pet in the last year; and not having recently bereaved of a pet. Vets were recruited through: nationwide veterinary practices, networks and connections from Health Protection Scotland (HPS) and the wider Control of Antimicrobial Resistance Scotland (CARS) programme Animal Health and AMR group. The vet inclusion criterion was that they had to be in small animal practice.Topic guides for interviews were developed with expert advice from the study advisory group. Both topic guides were used to balance participant led data with our focus upon understandings and perceptions of AMR and the drivers of AMR.Twenty one pet owner participants were interviewed of which 14 were female and seven were male. For pet owners, eight face to face interviews and 13 phone interviews were carried out. The duration of the interviews ranged from 28 min to one hour and 30 min. Pet owners ranged in age from 32 to 77 years old.n = 10), Second opinion (n = 2), vet hospital (n = 1), emergency out of hours (n = 1) and vet school (n = 1). These interviews lasted between 25 min and 40 min.Sixteen vets took part in interviews. Nine of the participants were male and seven were female. Eleven of the vets were based in Scotland, four in England and one in Northern Ireland. Recruited participants were identified as holding various positions within the industry including: front-line ; and interacting with vets. This paper examines the behavioural domain of interactions from the perspectives of both groups of participants. A continuous process of cross checking was carried out between researchers to ensure analytical rigour.The following sections consider important AMR-related aspects of the broad behavioural domain of interactions between vets and pet owners, firstly from the perspective of vets, then from the perspective of pet owners.Vets gave their perspectives on their interactions with pet owners, and identified the behaviours, and barriers and facilitators they thought contributed to inappropriate antibiotic decision-making. These relate primarily to perceived pressures from pet owners and possible discussions around antimicrobial resistance.There's a pressure when an owner comes in and pays 35 or 40 pounds , for a consultation, because their animal is unwell, that they want to go home with a medication that in inverted commas “fixes” their animal, and even though the bill will be higher with that medication, so you sort of have that feeling that if you have an unwell animal and if you send it home with nothing then you'll get a raised eyebrow, or for example they will say to you, “Well, so and so , the senior partner gave my older dog antibiotics just last week for similar symptoms” so, you know whatever, but there is a sort of general expectation I think of owners, that you will treat their animal when they pay for a consultation. (Vet 10)Vets reported that their interactions with pet owners were complex when it came to their decision making concerning antibiotics. There was a common tension between an awareness of the need for AMR stewardship and the perceived satisfaction of the pet owner. The satisfaction of the pet owner, from a vet perspective, was dependent on a myriad of factors which extended beyond the appropriate use of an antibiotic relating to clinical need, including: payment for consultation and the expectation of active treatment; ease of administering the treatment; previous experiences with receiving antibiotics for their pets; and pet owners’ emotional connections with their animals. Vets commonly reported prescribing ‘just in case’ in response to their perceptions of client anxiety about pet health and welfare. From the vet’s point of view, clients appeared to expect the provision of a clearly defined treatment (i.e. an antibiotic prescription), as the following example indicates:As this extract suggests, expectations from clients to prescribe antibiotics were often inferred rather than explicitly stated. Vets saw that pet owners appeared to perceive prescription as a binary of presence or absence of treatment and therefore action versus inaction. Prescription appeared to be received as a measurable, visible action taken for the health of the pet, whereas a lack of prescription meant nothing was being done to help the animal.Tacit pressure was also framed by vets in terms of pet owners’ expectations for transaction, that is, payment exchanged for a tangible outcome in the form of medicine for the pet. This expectation was exacerbated by the fact that veterinarian practices are profit making enterprises, and seeking to keep customers loyal, sometimes against appropriate clinical action. This pressure was felt by vets because customers are able to go to other practices where they may get what they want:Yeah, I mean these people are our customers and they are, what keeps the business going, so if we annoy them and there is another vet practice they can go to where they may just be handed out antibiotics, and [the vet] might take the opinion “well, here’s a client, if I give him a course of antibiotics, he’ll just take his dog to me from now on and I’ve gained a client” (Vet 1)So for example, if you’ve got a cat with cystitis and a lot of [vets] will give them antibiotics when actually a lot of them don't need antibiotics. So I think a lot of is through fear as well. You want to make the animal better, therefore you think oh sod it, I'll just give them a jag. (Vet 13)These market pressures can therefore be construed as potential drivers of inappropriate antibiotic prescribing among other mechanisms. Other vet accounts show that the AMR related consequences of over-prescription are not high on their agenda when acquiescing to pet owner pressure to provide treatment. Vets cited professional tendencies to prescribing, i.e., giving animals antibiotics as a quick and easy way to deal with an infection, even if that was not necessarily the best course of action:As this quotation indicates, the risks involved in inappropriate use of an antibiotic were outweighed by the risk losing a client. Potential, ambiguous and non-present consequences of AMR for vets were not as salient as the present lived experiences of pressure from pet owners, and business pressures around client satisfaction.They'll be a little bit annoyed that I haven't really given them anything, even though there's nothing that I could really give them that would improve their cat's position. And a lot of the time, they will sort of be like, well, what about some antibiotics, what about this, what about that, and I'll have to be, like, well, no, your cat doesn't need it and it's not going to help. But they sort of feel like I'm not really listening to their issue or that I'm not trying to help their cat because I'm not giving them this medicine that they perceive will be helpful to them, basically. (Vet 3)Vets were asked to what extent they felt they could use the concept of AMR to negotiate with pet owners in the interactions described above, to improve overall antimicrobial stewardship. Vets identified that pet owner understanding and willingness to discuss antibiotics and AMR were important determinants of their eventual prescribing decisions. The majority of vets reported that their clients tended to have some knowledge of the importance of correct antibiotic use; particularly that partial use of a course of antibiotics, or using them unnecessarily, had some kind of negative effects concerning future treatment outcomes. However vets reported a general lack of knowledge of the wider and longer term, inter specifies effects of over- or mis-use of antibiotics, and an almost total lack of knowledge of about the phenomena of AMR. This was also borne out in data from pet owners. Vet accounts also reported that clients were often unaware of disease processes. The limits of pet owners’ knowledge about appropriate antibiotic use could lead them to believe that an antibiotic would be appropriate treatment when it would not be suitable, for example, for a viral infection:You can tell very quickly if someone’s going to be receptive to that [appropriateness of antibiotics] conversation or not, and if they’re not, then obviously sometimes you just have to accept that that’s what the client wants, and if we don’t do that then the animal is going to suffer anyway, so we just say, well, there’s no point even discussing this. (Vet 15)Most vets in the sample perceived that whilst this lack of knowledge was an issue, awareness was improving, making it easier to carry out AMR negotiations without jeopardising pet owner satisfaction. However, some vets reported that there were still those who would not engage in discussion about appropriate use of antibiotics:This extract shows that vets are required to make judgements which balance outcomes for clients and pets, revealing prescribing decisions to be complexly embedded in management of the pet’s illness, the owner’s expectations and the reality of a competitive business environment. Antimicrobial stewardship does not easily factor into these complex deliberations.I think a lot of it boils down to communication, and it does get easier as you get older. As a younger vet - I'm kind of middle aged now, somewhere in the middle - as a younger vet, it's difficult, you often feel pressured from clients, you feel pressured to conform to what the older vets would do, if they would do something in a certain way. But now that I'm an established vet in my own right with my own ideas and opinions, as long as I get the time to actually communicate that with the client and talk effectively, I can usually talk them round to my point of view and they will see that's reasonable enough, yeah. And I think it depends where you work. If you're in a very busy practice or if you're a younger vet feeling pressure from clients or from senior staff, then it is going to be difficult, you're just going to grab the antibiotics, that's the easy option. (Vet 2)Vets also reported that their professional experience played an important role in empowering them to be able to assert a more appropriate course of action, skills which took time to develop:This account reflects the social norms and dynamics which influence prescribing decisions and underlines that the development of communication skills was an important factor in being able to negate pressures from misinformed pet owners and, it seemed, other vets. Identifying and sharing these skills could be a useful tactic for the advancement of stewardship.Pet owners also gave their perspectives on their interactions with vets, and identified the behaviours, and barriers and facilitators they thought contributed to antibiotic decision-making and antibiotic use for their pets. These relate primarily to pet owner expectations around antibiotic prescribing and discussions related to antimicrobial resistance.I would say ultimately it’s my decision but the vet is the expert… I mean if they tell me it’s needed, I’ll not, I’m not going to, I might try and ask sensible questions… but I’m not going to dispute that. .In contrast to vet perspectives that pet owner satisfaction was the main source of inappropriate prescribing, pet owners, on the whole, denied that they would be dissatisfied if their pets were not prescribed antibiotics. The majority of our respondents indicated that they would follow the vet’s instructions because they perceived vets to have expertise in an area where they had little to no knowledge.But I have been to the vet before and got antibiotics and questioned it and asked why they were taking them, when, where… they said right we’ll put them on antibiotics and I said I’m not really sure that they need them and do we really need to go ahead with them and they said to me, yes we really, really, need to go ahead, then I would go ahead with it. But there has been instances where I’ve said, I don’t, I’m not sure that we need them, are you absolutely sure? And they said OK, no. We’ll wait a while. .However, some participants reported that they thought it was vets who promoted antibiotics unnecessarily:Andrea’s account of questioning the prescription was rare in our interviews as most reported that they had not had any disagreements with vets over their expectations that antibiotics would be prescribed. Some pet owners reported assuming that antibiotics were required and had those assumptions negated by the expertise of the vet. However, in contrast to vet perceived risks of not keeping the client satisfied, there were no cases where pet owners had major disagreements which resulted in moving to other vets. These accounts indicate that pet owners may differ markedly from vets regarding their experiences of interactions and that there was a shared tendency among pet-owners and vets to externalise the drivers of inappropriate prescribing. This understandable self-protecting bias among both vets and owners is an important consideration for the development of effective stewardship, as we discuss below.As with vets, being able to understand and discuss AMR was seen by pet owners as an important factor for their own enactment of stewardship principles.Maura, dog and cat owner).“I think we’re just reaping the [consequences] of maybe people having antibiotics unnecessarily.” I'm sure there was something on the BBC news about pigs, I'm sure there was pig farming because I was just tuning in and out of it. I wondered why they had pigs and then they had people in a hospital bed and I was getting them confused. I know it was something about pigs being prescribed antibiotics and that is somehow having a knock on effect for us [laughter] but the steps on that chain I'm not sure about. I suppose with any of these future problems, problems are easily described as being something that’ll affect us in the distant future, there’s an issue with knowing how serious a risk is this, how much needs to be done Another challenge for pet-owners was the long term and collective nature of AMR which was not easily related to their own immediate predicament with their pet in the present:As in this example, for most pet owners, AMR was perceived as distant, future problem, with many ambiguous and unknown dimensions. Some participants contrasted the seemingly distant threat of AMR with the immediacy of their pets’ health, which was more directly experienced as part of their everyday lives, and therefore of a bigger influence on decision-making processes about antibiotics. AMR was of such negligible concern, due to its abstract, complex and temporally distant nature, that it did not influence decisions about the immediate concern of pet health and wellbeing. In contrast, the suffering of the animal that many considered to be “part of the family” was a visceral, potently experienced phenomenon that pushed individuals to make financial and other sacrifices to ensure their pet got better.Figure how vets and pet-owners experience antibiotic decision-making, and identifying their subjective views on the factors which influence effective stewardship. This novel and inductive approach makes a valuable contribution by identifying the antecedents which underpin antibiotic prescribing behaviour (in vets) and usage (in pet owners). As such, it identifies several key ingredients for future intervention development.This paper is the first of its kind to examine First, it is important to acknowledge the interaction between pet owners and vets in relation to behaviours which might drive AMR. Vets appeared focussed upon the prescribing of antibiotics. They attributed over-prescribing to the complexity of the following drivers; perceived client satisfaction, commercial pressures to retain clients, the symbolic value of giving treatment, the influence of senior vets within practice, under-developed communication and persuasion skills, pet owners partial knowledge of appropriate antibiotic use and their very poor knowledge of antimicrobial resistance and interspecies transmission. Second, we must consider the nature of the interaction between pet owners and vets which may in itself facilitate AMR. For example, in the current study, pet owners had very poor knowledge and understanding of AMR and inter-species transmission but were aware of the negative consequences of inappropriate antibiotic use. They considered vets as being fundamentally responsible for prescribing decisions . Pet owners did not report having high expectations of receiving antibiotic prescription. Finally, several key points are illuminated in terms of antimicrobial stewardship. Dissonance between the pet owner and vet appears to exist between their perceptions of where responsibility should rest in relation to the decision to prescribe antibiotics. Low levels of knowledge and understanding of AMR and its drivers amongst pet owners are important to note in this regard as they have the potential to constrain antimicrobial stewardship.The apparent dissonance in perceptions of responsibility for antibiotic decision making for pets should be factored into effective stewardship interventions. Alternatively, vets and pet-owners only notice differing prescribing decisions which conflict with their a priori expectations . This possible explanation for dissonance implies that future work should focus on sensitising vets and owners to these situations. Dissonance may also result from a breakdown in communication, that is, misinterpretation of behavioural cues, mistaken inferences and so on, undertaken in the challenging context of time limited consultations with a pet-owner and their pet. The facilitation of more effective discussions between pet owner and vets around antibiotics might therefore be useful. Interventions should include professional development for vets, particularly those new to practice, to specifically facilitate more effective discussion around antibiotics. This may enable a more accurate identification and mitigation of pet owner pressures to prescribe. Potentially, standardised guidelines, vignettes and protocols could help pet owners and vets manage their interactions more effectively. Concurrent to the development of guidelines, there is potential to develop mandatory legislation, however this is contingent on political will and identifying suitable bodies to legislate and enforce. Specific training and guidelines with step-by-step protocols for engaging in conversations about antibiotic prescription have the potential to assist professionals and their clients ensure better decisions on antibiotics , 20. As Raising awareness and knowledge of AMR in pet owners themselves could be key to facilitating more productive interactions and discussions with vets around appropriate antibiotic use for their pets. Increased emphasis on appropriate, responsible stewardship could be especially effective in countering potential vet pressures to overprescribe, as pet owners might be more likely to resist with better knowledge resources. If negative consequences of over prescription were framed in terms of side effects for the animal (lessened future efficacy of antibiotics) it might be particularly effective. In this regard, increasing the self-efficacy of pet owners by teaching skills to participate in consultations might be useful. For example, vets claimed pet owners were unsatisfied if they did not receive antibiotics as they did not get “their money’s worth”. If pet owners’ awareness of the vets’ diagnostic processes was improved and therefore understanding of the informed decisions vets make was increased, potentially they would be more likely to perceive vet’s expertise as a reasonable outcome for the money they have spent.There are some possible barriers to enhancing antibiotic decision-making, however. Research in other areas has shown that awareness alone is not sufficient to change individual behaviours , particuIt is important to consider the key strengths and limitations of the current study. First, this paper presents a novel piece of work, original in its inductive, qualitative approach and innovative in its dual-focus (considering both the vet and pet-owners perspectives). This study contributes a rich, insightful understanding of AMR enabling behaviours from the perspectives of the participants themselves. It promotes increased awareness of the key drivers for antibiotic use/prescription, including underlying cognitive processes such as decision making and dissonance.Various limitations have been identified for this study, primarily sources of bias within samples. The pet-owner sample were predominantly from middle-class, well-educated backgrounds (due to relatively poor success of other recruitment approaches). Vets who were recruited were likely to be already interested in AMR, and therefore potentially biased towards stewardship. Vets may have also been reserved in their disclosure due to a desire to project professionalism. The sample size reflects accepted standards for exploratory, theory-building qualitative health research. However, a larger sample could have alleviated some of the issues. The next step in exploring this topic would be a quantitative design informed by this research exploring behaviours of pet owners and vets regarding antibiotics use.This paper provides original evidence of the power of interactions between vets and pet owners to influence appropriate antibiotic prescribing and use in companion animals. In the absence of open communication, both vets and pet owners are inclined to misinterpret the intentions of the other, potentially leading to unnecessary prescription and inappropriate use of antibiotics.Targeted multi-modal strategies which increase knowledge and awareness of antimicrobial resistance and appropriate antibiotic use in the pet owning public are important. These would provide a strong foundation on which effective interactions between vets and pet owners can be built, thus enhancing antibiotic stewardship behaviours. In addition, adequate time within consultations is required to enable vets to provide relevant information and to educate per owners around alternatives to antibiotic use.By tackling the barriers of potential misunderstandings, lack of awareness of AMR, and lack of opportunity or capacity to discuss AMR during consultations, whilst enhancing the enablers of skilled communication which focuses on best clinical judgement as a precursor to client satisfaction, more effective antimicrobial stewardship can be achieved.


Introduction.Vibrio vulnificus (V. vulnificus) causes a severe infection that develops in the compromised host. Its pathophysiology is classified into three types: (1) primary septicaemia, (2) gastrointestinal illness pattern and (3) wound infection pattern. Of these, primary septicaemia is critical. V. vulnificus can be classified into three biotypes and two genotypes and its pathogenicity is type-dependent.Case presentation. A 47-year-old man presented to a local hospital with chief complaints of fever, bilateral lower limb pain and diarrhoea. He had no history of foreign travel or known medical problems. He was in septic shock and developed fulminant purpura within 24 h of the onset. High-dose vasopressor and antibiotic administration failed to alter his status and he died 3 days after the onset of symptoms. V. vulnificus was isolated from blood, skin and nasal discharge cultures. Biotype and gene analysis of the microbe isolated identified it as Biotype 3, mainly reported in Israel in wound infections, and Genotype E, implicating an environmental isolate. These typing analyses indicated that the microbe isolated could be classified as a type with low pathogenicity.Conclusion. This case highlighted that Biotype 3 and Genotype E can also cause primary septicaemia. Although the majority of reports on Biotype 3 have been from the Middle East, this experience with the present case provided evidence that the habitat of Biotype 3 V. vulnificus has been extending to East Asia as well. Vibrio vulnificus is a Gram-negative, facultatively anaerobic rod distributed in warm, brackish water areas, and is known to cause severe infectious disease in the compromised host such as those with liver disorders or diabetes mellitus. Infection routes of V. vulnificus are oral and wound infection. Its pathophysiology is classified into three types: (1) primary septicaemia, (2) gastrointestinal illness pattern and (3) wound infection pattern. Primary septicaemia is caused by oral route infection with ingestion of raw seafood and results in a systemic infection with necrotizing fasciitis within 48 h [V. vulnificus infection manifest as primary septicaemia, with a mortality rate of up to 70 %, and more than one-half of cases die within 3 days [V. vulnificus classified as low pathogenicity type by biotype and genotype analysis.hin 48 h . In JapaA 47-year-old man with a history of hypertension and cholecystolithiasis was brought to the emergency department of a local hospital because of fever over 38 °C, bilateral lower limb pain and diarrhoea. He was a carpenter and was a habitual alcohol drinker (120 g per day) and smoker. One day prior to the presentation, he had a slight fever and anorexia. He was unconscious on arrival with Glasgow Coma Scale of E3V4M6 and blood pressure of 85/45 mmHg, and a heart rate of 110 beats per minute. He was intubated, and vasopressors were administered intravenously. Under a diagnosis of septic shock, 1 g doripenem was administered. Twelve hours after the presentation, erythema spread to the trunk and purpura developed on the lower limbs. Three hours later, purpura appeared on the face and trunk as well and he was transferred to our hospital because of persistent circulatory failure. Although the details of his dietary habits were unknown, he resided in an inland area and had no contact with seawater or history of foreign travel.−1 min−1. His body temperature was high, up to 40.5 °C. No abnormalities were noted on thoracic auscultation or abdominal palpation. On the external surface of his body, there were no visible scars, but severe oedema and systemic purpura were seen  were uploaded to the RAST  server for annotation and functional characterization based on comparison with the V. vulnificus CMCP6 genome. Also, using Genetyx ver.11 (GENETYX), the primer sequence shown by Rosche et al. [V. vulnificus CMCP6  and environmental type (E-type) according to gene sequences. In PCR analysis using prepared primers based on these genotypes, the C-type sequence was found in 90 % of strains of clinical isolates, and the E-type sequence in 93 % of strains of environmental isolates.iotyping . Conventiotyping . Type 3 iotyping . Reportso Israel . Rosche  [et al.  reportedV. vulnificus with Biotype 3 and Genotype E. This type of V. vulnificus had been believed to be a less severe pathological strain. Furthermore, this is the first case report in East Asia about Biotype 3 V. vulnificus infection, which has hitherto been reported only in the Middle East.We presented a case of fulminant infection by V. vulnificus of Biotype 3 or Genotype E thus far considered as being of low pathogenicity can cause primary septicaemia and suggested that the habitat of Biotype 3 strains has been extending to East Asia.In conclusion, this case highlighted that even


Electrospinning has emerged as an effective method of producing nanoscale fibers for use in multiple fields of study. One area of significant interest is nanofiber utilization for tissue engineering because the nanofibrous mats can mimic the native extracellular matrix of biological tissues. A logical next step is the inclusion of certain molecules and compounds to accelerate or increase the efficacy of tissue regeneration. Two methods are under scrutiny for their capability to encapsulate therapeutic compounds within electrospun nanofibers: emulsion and coaxial electrospinning. Both have advantages and disadvantages, which need to be taken into careful consideration when deciding to use them in a specific application. Several examples are provided here to highlight the vast potential of multilayered nanofibers as well as the emergence of new techniques to produce three-dimensional scaffolds of nanofibers for use in the field of tissue engineering. The technique languished for many years until the early 1990s when interest was renewed largely as a result of the need for a process which would enable the fabrication of fibers on the nanometer scale. In this regard, Reneker, Yarin, and other researchers began to examine more closely the fibers that were formed by this process and investigate the physics governing their formation.20–31The technique known as electrospinning was discovered and described over a century ago18 is formed as the drop distorts and a small strand of solution, or jet, is ejected from its tip, generating a flow-modified Taylor cone. The excess electrical charges within the jet repel one another, the result of which leads to rapid elongation of the expelled strand of solution. The jet of solution travels to a collector, which is usually in the form of a flat, electrically conductive plate. As the strand of solution travels to the collector, the solvent evaporates to leave solid fibers.Electrospinning is possible because of an imbalance between two physical properties common to the process, surface tension, and electric field strength. A solution or melt with sufficiently high viscosity passes through an orifice, usually at the end of a needle or metal cone with a small hole at its tip, to generate a small droplet . When su21 and they have led to the creation of electrospinning designs that have tips and collectors either submerged in liquid32 or sealed in chambers with tightly controlled atmospheric conditions.33 Melt-based electrospinning is manipulated by some of the same factors as solvent-based electrospinning , but it can also be modified by altering other aspects, such as the temperature of the melted polymer.Many parameters may be manipulated to optimize the electrospinning process. A few factors often altered when solvent-based methods are employed include applied voltage, distance between the electrospinning tip and collector, solvent volatility, electrical conductivity of the solution, and solution viscosity. These parameters have significant effects on the morphological characteristics of the formed nanofibers. Environmental factors such as temperature and humidity may also have a marked effect on the morphology of the fibers formed,34–36 filtration devices,37 protective clothing and textiles,38 sensors,39 tissue scaffolds,41 and drug delivery systems.43 All of these areas benefit from the high surface area-to-mass ratio of the nanofiber mats. As an example, it is interesting to consider drug delivery in this context. A significant problem encountered in drug delivery is maintaining a therapeutic dosage to a patient over a specified period of time. Controlling the dosage rate is especially important in the case of drugs with a low therapeutic index, such as doxorubicin (dox)42 or digoxin,44 as they must be administered more often and in smaller amounts to minimize the risk of toxic side effects and remain within the window of concentration where they will be effective. However, as the frequency of required doses increases, patient compliance generally decreases. An effective way in which to ensure patients will adhere to the dosage schedule is to place the therapeutic agent within a device or chemical compound that will control the rate at which the agent is released and decrease the frequency of required doses. Insulin pumps, nicotine patches, and time-release tablets are a few examples of products currently in the consumer marketplace which utilize a form of controlled drug delivery. Hydrogels,45–47 dendrimers,48–51 liposomes,52–55 and microelectromechanical systems (MEMS)56–59 represent a small fraction of technologies under investigation for their use in drug delivery.A variety of nonwoven nanofiber mats may be generated by electrospinning. These have been utilized in applications for wound dressings,36 Second, the electrospun mats do not need to be subjected to the same rigorous testing protocols as a product used for implantation into the human body. Third, the morphological characteristics of the nanofibrous mats can be adjusted to allow air to pass through the mats while controlling or preventing the flow of moisture or dirt.38 Perhaps the most interesting feature of the nanofiber mats is the ability to couple them with antibacterial or regenerative agents to increase the rate of healing.One of the advantages of utilizing electrospun nonwoven nanofiber mats for drug delivery derives from the capability to employ them as wound dressings or tissue scaffolds for tissue engineering. The therapeutic agent can be loaded into the electrospun fibers and released at a steady rate over a period of time. Early biological applications of electrospun fibers concentrated on the field of surface wound repair for a few reasons. First, the materials used can be nonbiodegradable and consist of polymers which are already designed to form nanofiber mats.35Small molecules or particles may become trapped within nanofibers as the solutions are electrospun. For instance, titanium dioxide nanoparticles have been added to nanofibers of polyurethane, which demonstrated increased antibacterial activity and significant water vapor permeability, but exhibited relatively low cytotoxicity to cultured L929 (mouse fibroblast) cells. Water vapor permeability was examined because the desired application of these nanofibers was the treatment of burn wounds, which require moisture to prevent dehydration and promote tissue regeneration.36 The primary issue encountered in utilizing nonbiodegradable materials is that they remain in place if they are implanted into the human body. To circumvent this problem, biodegradable polyesters such as polyglycolic acid (PGA), poly-l-lactic acid (PLLA), and polycaprolactone (PCL) were examined with the intent of creating biodegradable nanofibers that may be implanted into the human body and dissolved slowly over time with few, if any, deleterious effects.Skin ulcers associated with Leishmaniasis were treated effectively through the use of electrospun mats which were designed to release nitric oxide upon the addition of a small amount of water.42 PGA,60 PCL,43 polylactic acid (PLA),60 and various copolymers of these compounds have been utilized to great effect to form tissue scaffolds of electrospun nanofibrous mats. Natural polymers, such as collagen and elastin, have also been successfully electrospun to provide nanofiber mats that closely resemble those components of the extracellular matrix of some biological systems.61 Chitosan,62–65 alginate,66 and hyaluronan68 are more natural materials, which have gained interest in light of their availability, affordability, and cellular compatibility. Whether synthetic or natural materials are used for electrospinning, living cells appear to attach and proliferate more readily on the nanofiber mats than when grown in monolayer culture.69–71d,l-lactic acid [PDLLA]) and those grown on nanofibers (PDLLA fibers). The results for day 3 show a lower number of cells initially attached to the nanofibers. However, by day 14, the cell density on the nanofibrous materials has increased significantly compared with the cell density present on the spin-coated PDLLA. Synthetic, bioresorbable materials, such as polyethylene oxide (PEO),Significant advances have been realized in recent years with regard to the utilization of electrospun nanofiber mats composed of biodegradable materials for tissue engineering. The relative simplicity of the electrospinning technique, coupled with the morphological characteristics of the fibrous mats, make it an attractive method for forming tissue scaffolds. By controlling the size, density, composition, and orientation of the fibers, it is possible to produce materials which approximate closely the fibrous structures that are present in the extracellular matrix of almost any tissue.For tissue engineering, the nanofiber mats containing a therapeutic agent can be utilized as part of a tissue scaffold. In such an application, the electrospun fibers should serve to mimic the three-dimensional environment of the extracellular matrix of the target tissue as well as release small amounts of the desired agent in a controlled fashion. The composition of the fibers may be altered to optimize their drug-loading efficiency, activity, and biocompatibility.72 The viability of mouse neuronal stem cells was not significantly affected by the alignment of the fibers. However, the alignment of the fibers served to influence the cells to grow in a more directed fashion similar to those found within native nerve tissue. Also, the nerve cells on aligned fibers demonstrated a significant increase in neurite length.With respect to a specific example, PLLA was selected as a suitable material for fabricating mats of random and aligned nanofibers in at least one case of neural tissue engineering.61 Vascular grafts could be tailored to specific situations with only minor modifications to the nanofiber collector. Electrospun collagen/elastin mats could also be utilized to repair the outer walls of the stomach or intestinal tract.Attempts to utilize natural polymers, such as collagen and elastin, have been met with success in some instances. The use of collagen/elastin mats for smooth muscle tissue engineering demonstrates promise because the fibers exhibit similar mechanical properties as the smooth muscle tissue located within blood vessel walls.73 The nanofibrous mats produced were referred to as a “guided bone regeneration membrane” and it was postulated that the nanoparticles would facilitate the proliferation and differentiation of osteoblasts.73 Early results indicated that the scaffolds did not exhibit any cytotoxic effects on human osteoblasts or rat fibroblasts.73 In addition, the osteoblasts grown on nanofibers containing calcium carbonate were examined under scanning electron microscopy and demonstrated the initial signs of mineralization with the appearance of small crystalline granules. A notable potential consequence of inducing and maintaining osteoblast differentiation was mentioned in the publication. The study found that the osteoblasts which had differentiated slowed considerably with respect to proliferation.73 A similar situation was mentioned by Owen et al. in a study of rat osteoblasts grown in vitro.74Wutticharoenmongkol et al. generated nanofibrous mats of PCL that contained calcium carbonate or hydroxyapatite nanoparticles for bone tissue regeneration.75 This technique has proven successful in encapsulating nerve growth factor (NGF),60 proteinase K,76 lysozyme,77 cytochrome C,78 and bovine serum albumin.79 Emulsion electrospinning is also being investigated because it accommodates the use of water as the solvent instead of more hazardous chemicals.80 In many cases, surfactants or other amphiphilic compounds are used to generate the emulsified droplets in water-based systems.80Although the electrospinning technique is relatively simple, difficulties may occur with the method in aspects of loading therapeutic agents into the fibers. Problems may be encountered, for example, with proteins because of their size and requirements relative to their three-dimensional structure. In this regard, small conformational changes could occur as a result of the electrospinning process  and they may render a protein inactive or possibly even toxic. To circumvent such a situation, proteins may require the use of more complex techniques, such as emulsion or coaxial electrospinning, to be loaded efficiently and effectively into nanofibers to be electrospun. In the case of both emulsion and coaxial electrospinning, the nanofibers generated consist of an outer sheath and inner core of differing composition. Emulsion electrospinning relies on chemical means of separation through the creation of an emulsion within a single solution and the subsequent organization of the emulsified droplets into two distinct phases as the solvent evaporates from the electrospun fibers.81 SF is similar to collagen in that it is a biopolymer obtained from natural sources and contains cell recognition peptides such as RGD (arginine-glycine-aspartic acid), which facilitate cell attachment and proliferation.83 The main focus in generating SF/PCL fibers was to capitalize on the mechanical strength of the PCL while exploiting the advantages of SF as a tissue engineering material. Testing in vitro using primary fibroblast cells from human skin (FEK4) demonstrated an increase in initial cell attachment as well as a slight increase in cell proliferation rate.Before work with encapsulating therapeutic agents, suitable solution and electrospinning parameters should be determined. For example, core–sheath-structured nanofibers of silk fibroin (SF) and PCL have been constructed utilizing emulsion electrospinning.84 Hyaluronan, or hyaluronic acid (HA), is a glycosaminoglycan found in the extracellular matrix of many tissues and it can promote cellular adhesion and proliferation.67 The purpose of its inclusion into the SF/PCL emulsion was to provide some measure of control over the adsorption of proteins to the surface of the nanofibers while still enhancing infiltration of cells into the scaffold. Water swelling and contact angle measurements indicated an increase in hydrophilicity of the nanofibrous scaffolds as a result of HA addition. Also, the rate of FEK4 cell infiltration and proliferation was significantly higher after 5 days when compared with scaffolds composed only of SF and PCL.84After positive results were obtained from the SF/PCL combination, properties of the nanofibers were optimized to a greater degree by the addition of hyaluronan to the electrospinning solution.85 Other solvent systems were developed to combat this issue and can be used to great effect with the emulsion electrospinning methodology.86One potential challenge for researchers intending to capitalize on the success of this study and others like it results from the use of a fluorinated solvent, in this case, hexafluoroisopropanol (HFIP). HFIP is a corrosive, volatile solvent, which may not be suitable for biological molecules or desired therapeutic agents.42 The thickness of the inner and outer layers of the nanofibers can be altered by changing solution parameters as well. When Xu et al. encapsulated dox within PLA/PEO nanofibers, they discovered that increasing the concentration of dox within the core of the fibers shrinks the diameter of the core as a result of the favorable association between dox molecules.42 The association and smaller diameter core also had the effect of significantly decreasing the release rate of the drug from 60% to only 20% released within the first 5 h. More importantly, studies such as this one demonstrate the feasibility of producing nanofibers containing aqueous and organic phases. This dual-phase nature is critical not only in the case of small molecule therapeutics, but even more so for proteins and growth factors.As outlined in the 78 The work showed that a change of 1–3% PLLA concentration significantly slowed the initial release of cytochrome C from the core of the fibers, essentially eliminating the burst-release phenomenon.78 This investigation highlights the concept that manipulating the release rate of the therapeutic agent can be achieved through altering the sheath of the nanofibers. The medium into which the nanofiber mats are placed can exhibit a marked effect on release characteristics as well. This research group demonstrated that the release rate of cytochrome C could also be altered through the addition of hydrophilic polymers such as polyethyleneimine (PEI) or poly-l-lysine (PLL).78 By addition of one of these polymers to the emulsified electrospinning solution, the hydrophobicity of the nanofiber mats was decreased and the release rate of the protein was increased, results which also reintroduced the initial burst-release phenomenon. The capability to manipulate and control the release process shown by this study is critical in many cases for effective administration of therapeutic agents. Such control is especially important with respect to growth factor-centered treatments as it may be beneficial to release certain factors in high concentrations initially and slowly decrease the dosage rate over time.A study by Maretschek et al. demonstrated the encapsulation of a relatively small heme protein, cytochrome C, within nanofibers of PLLA.34 Specifically, this group focused on the release of basic fibroblast growth factor (bFGF) sequestered within polyethylene glycol/poly-DL-lactide (PELA) nanofibers. in vitro. The initial 12 h after incubation  of the drug-loaded nanofiber mats demonstrated the burst-release phenomenon, freeing ∼14% of the total bFGF from the nanofibers. Over the next 15 days, another 60% of the growth factor was liberated from the nanofibers during the “fast” phase of release. Following another 10 days, the release rate tapered off significantly and resulted in 15% of the protein being freed from the fibers during the “slow” release phase.A more thorough examination of the release profiles for encapsulated proteins was undertaken by Yang et al.in vitro for compatibility with living cells using mouse embryo fibroblasts. For testing in vivo, the growth factor-loaded nanofiber mats were applied to dorsal wounds in diabetic rats.34 After 1 week, the wounds covered with the bFGF-loaded PELA nanofibers had already begun to heal, whereas the PELA nanofiber mats without the growth factor and containing free growth factor had not. Within 3 weeks, the wounds covered with growth factor-loaded nanofibers had completely healed. The other testing groups required an additional week to heal, and the control group still had not healed fully at the end of 4 weeks. Such marked success in wound healing was attributed by Yang et al. to three factors: encapsulation of bFGF prevented its early degradation in the proteolytic environment of the wound, the nanofibers kept the factor localized to the wound, and sustained release of bFGF was maintained over time.34 In this case, the triphasic release profile of bFGF may have been beneficial to wound healing. The initial burst of the growth factor may facilitate rapid proliferation of fibroblasts to start the healing process. The second and third phases of release follow with decreasing release rates, prolonging the presence of the growth factor within the wound while slowing the proliferation of cells as the wound shrinks and eventually closes.The bFGF-loaded nanofiber mats were tested 72 Certain processing parameters and solution properties such as inner and outer solution flow rates, viscosities, and electrical conductivities are often taken into account when attempting to apply the coaxial technique. The coaxial electrospinning process can be utilized to create drug delivery systems of varying complexity. For instance, the core and sheath compositions of the fibers could be chosen for strength and cell attachment properties, respectively. The approach has been demonstrated by electrospinning nanofibers with a thermoplastic polyurethane core and a collagen sheath.87 Fibers such as these provide desirable mechanical strength as a result of the polyurethane core and facilitate increased cell attachment and proliferation because of the presence of the collagen sheath.Coaxial electrospinning differs significantly from emulsion-centered methods in that it generates core–sheath fibers by physical separation through the utilization of two electrospinning tips and two solutions.87 The polyurethane core contributed significant mechanical strength to the nanofiber mats, which would not have been realized using a pure collagen scaffold. Stress–strain curves  (PGS) is a degradable synthetic material which exhibits mechanical properties similar to elastin.90 Nanofibers of PGS are difficult to produce under basic electrospinning conditions because they require curing after the fibers are produced to generate the crosslinks which allow the material to display three-dimensional morphologies similar to elastin and collagen. During the curing process, the nanofibers need to remain separated from one another to prevent them from melting and merging into larger fibers or thin films. Yi and LaVan were successful in generating nanofiber mats of PGS by utilizing PLLA as a sheath to sequester the uncured PGS and ensure it would crosslink while remaining as nanofibers.90 Once the PGS was crosslinked, the PLLA was removed using dichloromethane to leave a nanofibrous mat of pure PGS. They proposed using the PGS nanofiber scaffolds for tissue engineering in microvasculature and showed the nanofibers had no significant effects on viability of human dermal microvascular endothelial cells.90The coaxial technique has been employed to produce single-phase, solid nanofibers from materials which would be extremely troublesome to electrospin under normal conditions.91 and vascular endothelial growth factor (VEGF) within Dextran/PLGA92 as well. In both cases, the encapsulated growth factors maintained significant bioactivity and were not altered by the coaxial electrospinning process. In the instance of NGF encapsulated within PLGA, Wang et al. demonstrated a controlled, linear-release profile following an initial burst-release of 29.5%.91 Also, the nanofibrous mats were electrospun in an aligned fashion and rolled into small tubes for testing as a potential nerve guidance conduit (NGC). Results from studies in vivo indicated the NGF-loaded nanofibers were as effective as autograft nerve tissue for regenerating tissue in a rat sciatic nerve model.91in vivo. No significant adverse health effects were noted throughout the course of the experiment. It appeared that the NGF was released locally around the nerve defect and did not have a systemic effect.91Research groups have demonstrated methods for encapsulating successfully NGF in poly(lactic-co-glycolic acid) (PLGA)92 It was noted that the cells began to infiltrate the pores of the VEGF-loaded nanofibrous mats as soon as day 3 after seeding and commenced secreting extracellular matrix, which began to be incorporated with the nanofibers to provide a suitable environment for additional cell growth.92In a separate study, dextran/PLGA nanofiber mats loaded with VEGF were tested for cell proliferation and growth factor viability using human umbilical vein endothelial cells.93 Coaxial nanofibers with a core containing VEGF and a shell composed of polyurethane were produced and demonstrated more efficient and even loading of the factor into the nanofibers when compared to simple electrospinning. Also, sustained release of the factor occurred over the course of 30 days with initial burst release occurring over the first 6 days in culture. Cell-seeded constructs were implanted into hemophilic mice. The skeletal muscle-engineered constructs demonstrated marked improvement compared with the control groups. Histological examination showed an increase in vascularity and presence of significantly more and larger lymphatic vessels.93Another significant advantage of coaxial electrospinning for tissue scaffolds and controlled drug delivery is seen in an experiment conducted using myoblasts isolated from the skeletal muscle of mice.77 Emulsion electrospinning also has the advantage of simplicity, but it lacks well defined control over the placement of the therapeutic agent within either the core or shell of the structure. Enabling precise control over the location of the drug within the core or shell of the nanofibers is one of the advantages of using coaxial electrospinning instead of emulsion.94 Reproducibility and high-throughput are two other advantages in designing drug delivery devices using coaxial electrospinning. The major disadvantage with coaxial electrospinning lies in the complexity of its setup. A coaxial needle and multiple syringe pumps are required to control the precise formation of the core–sheath structures. The syringe pumps are relatively simple to arrange for effective delivery of polymer solutions. The design of the coaxial needle is critical to the ultimate arrangement of the core–sheath fibers. The simplest and most often utilized needle arrangement is a single core centered within the outer sheath.87 More complex strategies place multiple needles within the center to produce nanofibers having more than a single core.95–97 The fabrication of intricate needle designs such as these serves to add costs to the initial setup of the coaxial electrospinning apparatus and is investigated in situations where a simpler technique is not viable.Emulsion, compared to coaxial, electrospinning is the more prevalent technique because it provides protein solvation within a mild solvent and separation from the harsh solvent required to dissolve the desired polymer.97 For example, the inner layer of the electrospun fibers could be composed of PCL, loaded with osteogenic protein-1 (OP-1), whereas the outer layer could consist of collagen, loaded with bFGF. The reason for utilizing OP-1 and bFGF in this case is that OP-1 has been shown to promote differentiation of chondroblasts and osteoblasts98 and bFGF has a demonstrated capability to increase cell proliferation.100 Conceptually then, bFGF loaded into the outer sheath of the nanofibers would be released into the tissue environment before OP-1 encapsulated within the core of the fibers. Thus, this type of scaffold could promote delivery of factors or drugs in two stages, the first for enhancing cell proliferation and the second for ensuring that the cells differentiate into the desired cell type. A potential pitfall, which could hinder this approach, might result from the use of PCL as the material for the inner layer and the application of an organic solvent required for PCL dissolution. The organic solvent selected could alter the activity of OP-1, potentially reducing or eliminating the advantageous effects of its incorporation into the nanofibers.Utilization of the coaxial electrospinning technique could allow creation of more complex drug delivery systems as well.78 As the nanofiber degradation will not be completely uniform, an overlap in the release profiles between OP-1 and bFGF may be present. It should be possible to adjust this window of overlap to maintain an optimal ratio of the two factors. Such an approach would be beneficial in many applications which require multiple drugs to be released in a sequential manner.Release profiles of OP-1 and bFGF should be governed by the thickness of the inner core and outer sheath as well as the chemical composition of each electrospun fiber layer.101 The approach required two extra layers of fibers to delay the release of the second drug and produced fibers of differing morphologies in all four layers. As the fibers in every layer were composed of poly-l-lactide-co-ɛ-caprolactone (PLCL), macroscopic separation of the mesh layers did not appear to be an issue. However, the multilayered mats were being examined principally for applications in sequential chemotherapy, not in tissue engineering.101 Therefore, the meshes were not created to optimize attachment and proliferation of cells. Also, using multilayered fibers could prove to be problematic in tissue engineering applications because the cells may infiltrate all the layers and begin degrading them simultaneously, resulting in premature release of the second drug. This situation could be prevented, in concept, by generating nanofibrous mats having sufficient fiber density and small pore size to prevent rapid infiltration of cells. Small pore electrospun mats that hinder cell infiltration might be advantageous in wound healing applications, for example, in which growth factors and related molecules could penetrate a fiber mesh relatively easily, whereas cells, bacteria, and other such larger elements could not. Considerations of fiber size and porosity are important related to diffusion of various molecules and infiltration of cells into electrospun mats for tissue engineering applications. Discussion of these factors has been presented in multiple studies.102–105Research has already demonstrated the capability to fabricate multidrug nanofiber mats using sequential electrospinning to generate a multilayered mesh .101 The 106 This possibility is of particular interest for many therapeutic formulations in the pharmaceutical industry. Long-term factor or drug storage can be a significant obstacle to production of compounds, which are not required on a regular basis or require transport to locations lacking the necessary infrastructure to preserve them. Proper preparation is still important when storing or transporting therapeutic agents in this fashion. For instance, PCL, PLA, and PGA undergo hydrolytic degradation under normal environmental conditions. It would, therefore, be prudent to store drug-loaded nanofibers containing such compounds in an environment with very low humidity. While significant advances are being made in developing water-soluble108 and chemically stable solutions,109–111 the pharmaceutical industry will benefit greatly from the introduction of simple preservation methods to prolong the useful life and bioavailability of unstable, yet effective, formulations.In any case, the aforementioned applications of coaxial electrospinning in tissue engineering and drug delivery increase the complexity of the basic technique. Controlling the release profiles and thickness of the inner and outer electrospun fiber layers may be challenging, for instance. Any therapeutic agents loaded into the electrospinning solutions must also remain active after being subjected to the various organic solvents and the electric field in the electrospinning operation. As a beneficial side effect, the sequestration of a drug within electrospun nanofibers could prolong the amount of time a drug could be stored.Obstacles preventing the use of electrospinning for mass production of drug delivery devices obviously still exist. Establishing general parameters for successful encapsulation and release of therapeutic agents is a difficult task because of the myriad of polymers, solvents, experimental conditions, and drugs which may be utilized. Basic guidelines regarding solvent properties, solution viscosities, and experimental parameters have been set forth by multiple researchers for basic and coaxial electrospinning. It is critical to determine the specific application in which the drug-loaded nanofibers will be utilized. In the instances where the therapeutic agent is not affected by the solvent used to prepare a polymer for electrospinning, basic electrospinning may be sufficient to produce the desired result. Utilization of coaxial or emulsion electrospinning to generate core-sheath-structured nanofibers may be required to construct a suitable drug delivery device if the drug or growth factor is not suitable for basic electrospinning.112–115 This result may complicate matters further with respect to selection of appropriate solvents and electrospinning conditions. Particle size and composition may be critical to preservation of particle structure and release characteristics. In addition, the presence of particles within the fibers can affect the physical properties of the electrospun mats.116–118 Modifications to the coaxial electrospinning approaches facilitated creation of multiaxial designs and trilayer nanofibers that exhibit three distinct layers in individual fibers. Drugs may be loaded into any or all of the layers to produce systems with highly specific applications.119–121The examples provided in the preceding pages represent but a small fraction of the research being conducted in the field of drug delivery using coaxial and emulsion electrospinning. In addition, multiple drug delivery technologies were combined in a number of studies to various rates of success. Sequestration of drug molecules within a carrier before production of nanofiber structures can increase the efficacy of delivery and control release profiles to great effect.® E.122 A solvent-based methodology was compared with melt-based electrospinning, and one finding was that the structure of carvedilol was retained in its amorphous form using both methods. The primary advantage of employing the melt-based technique developed from the lack of a solvent, thus simplifying the process and saving the cost and difficulty of dealing with various solvents. Also, the high surface area of the nanofibers permitted more rapid release of carvedilol into solution when compared with the bulk, crystalline form of the drug. An extensive review of melt electrospinning was published recently by Brown et al., exploring many general and specific applications of melt electrospinning techniques.123All of the examples of drug-loaded nanofibers presented until now in this review rely on solvent-based methodologies to suspend therapeutic agents within solutions that are then transformed into nanofibers by the electrospinning process. Melt electrospinning is another potential technique that could be employed to include drug formulations within nanofibers. In fact, Nagy et al. demonstrated such a process when this research group loaded carvedilol into nanofibers composed of EUDRAGIT124 Li et al. generated coaxial nanofibers using a melt-based system as well.125 The core of the nanofibers contained thermochromic compounds, which resulted in a color change to the nanofibers in response to increases or decreases in temperature. These reports could offer insight into developing coaxial, melt electrospinning systems that include growth factors and other therapeutic agents within core–shell nanofibers.To date, there have not been many documented attempts to include therapeutics within nanofibers fabricated using melt electrospinning. The process offers a unique concept and approach for accomplishing such an advance as it removes the requirement of a solvent, but it introduces new challenges as well. Elevated temperatures necessary for the melt electrospinning process could result, for example, in complete denaturation or deactivation of certain proteins and growth factors. For compounds that retain their activity after exposure to high temperature, melt electrospinning could be a viable option for developing nanofiber-based drug-delivery systems. Additionally, the literature is limited with respect to reports that describe the incorporation of solution-based, coaxial electrospinning methodology into melt-based electrospinning technology. McCann et al. detailed production of phase-change nanofibers consisting of a composite sheath and long-chain hydrocarbon cores that demonstrated the capability of absorbing significant quantities of thermal energy.123 However, the simplest examples of these experiments are seen in a number of reports detailing methods of forming small tubes composed of electrospun nanofibers with the intent of utilizing them to tissue engineer blood vessels.126–129 In the 1970s, Annis et al. detailed the creation of vascular prostheses of electrospun polyurethane nanofibers that were implanted into minipigs for up to a year.19 In addition to significant tissue regeneration, they noted that the mechanical properties of the synthetic vascular graft prevented formation of aneurysms. Recent attempts to create synthetic blood vessels highlight multilayering techniques as well as incorporation of natural polymers into the nanofibrous structure.126–128Examples have been noted in the previous paragraphs that only touch on the use of drug/growth factor-loaded nanofibers as simple, flat sheets of material. Flat materials provide some data on cellular interaction with nanofibrous sheets, but they lack the capability of producing complex, three-dimensional structures. Attempts to produce electrospun materials with greater three-dimensional complexities have met with some success in recent years. Controlled deposition of nanofibers affords the possibility of generating patterned structures in two dimensions, which could be layered to form more complex three-dimensional structures.129 While the overall structure of the valve was three-dimensional, it was still composed of a two-dimensional sheet of fibers. Shim et al. showed it is possible to modify sheets of nanofibers post-production to expand them into three dimensions.130 Specifically, this group used a small metal comb to pull the nanofibers apart to decrease the overall nanofiber density and increase porosity of the PLLA scaffolds. Blakeney et al. approached the dimensionality issue by modifying the nanofiber collector, altering the structure of a PCL nanofiber scaffold as the nanofibers were deposited.131 The scaffolds in this instance were structured similar to a cotton ball and were called Focused, Low density, and Uncompressed nanoFibrous (FLUF) mesh. Jha et al. produced three-dimensional, aligned nanofiber scaffolds for nerve regeneration using a two-pole air gap technique, another modification to the nanofiber collection method.132 Cai et al. manipulated the conductivity of the polymer solution to create three-dimensional nanofiber structures.133 Hybrid scaffolds were also produced, incorporating electrospun nanofibers as a thin coating over the surfaces of pre-existing materials.134Expansion of nanofibers to more complex three-dimensional structures is problematic. In addition to blood vessels, Gaudio et al. demonstrated fabrication of a trileaflet heart valve composed of PCL nanofibers.An interesting extension of these approaches would incorporate drug-loaded nanofibers into more intricate, three-dimensional structures. Such systems would increase the complexity and potentially complicate determination of therapeutic agent loading and release in some instances, but the benefits may outweigh these issues for certain cases.1 However, that statement was a bit premature as new applications for electrospun nanofibers are being discovered constantly in many research institutions around the globe.Ultimately, the intended application and its requisite components drive the development of electrospinning methods to enclose, protect, and/or control release rates for a particular drug delivery system or other purpose. This review has noted many such approaches. Over a century has passed since Boys wrote, “Fibres spun by the electrical method are so brittle that they do not seem to be of any practical use.”