Glyphosate, formulated as glyphosate-based herbicides (GBHs) including the best-known formulation Roundup, is the world's most widely used herbicide. During the last years, the growing and widespread use of GBHs has raised a great concern about the impact of environmental contamination on animal and human health including potential effect on reproductive systems. Using an in vitro model of pig oocyte maturation, we examined the biological impact of both glyphosate and Roundup on female gamete evaluating nuclear maturation, cytoplasmic maturation and developmental competence of oocytes, steroidogenic activity of cumulus cells as well as intracellular levels of glutathione (GSH) and ROS of oocytes. Our results indicate that although exposure to glyphosate and Roundup during in vitro maturation does not affect nuclear maturation and embryo cleavage, it does impair oocyte developmental competence in terms of blastocyst rate and cellularity. Moreover, Roundup at the same glyphosate-equivalent concentrations was shown to be more toxic than pure glyphosate, altering steroidogenesis and increasing oocyte ROS levels, thus confirming that Roundup adjuvants enhance glyphosate toxic effects and/or are biologically active in their side-effect and therefore should be considered and tested as active ingredients. N-(phosphonomethyl)glycine, is a non-selective herbicide widely used worldwide to control weeds1. Gly is commonly applied as part of glyphosate-based herbicides (GBHs), which include the popular commercial formulation Roundup (R), in which adjuvants enhance the herbicidal properties.Glyphosate (Gly), or 3. Only a small amount of Gly is metabolized by mammals, while the majority is excreted unmodified by urine in which Gly residues have been detected in both humans4 and animals as rats5, cows7, rabbits7, dogs and cats8.During the last years, the growing and widespread use of GBHs has raised a great concern about the impact of environmental contamination on animal and human health. Human and animal Gly exposure may occur through various routes such as food and drinking water, skin contact or by inhalation14. However, findings of both in vitro and in vivo studies are conflicting and several authors concluded that Gly is safe at levels below regulatory permissible limits22.The possible risk associated with Gly exposure to human and animal health is a matter of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including potential adverse effects on nervous, digestive, endocrine and reproductive systems24, thus determining an endocrine disarray in cell lines (e.g. 26). Furthermore, R exposure in rats has been demonstrated to interfere with both steroidogenic enzymes and reproductive health28. It has been suggested that the modification in reproductive hormone concentrations induced by GBHs could be due to changes in the number and activity of Leydig cells and modification of steroidogenic acute regulatory protein (StAR) or aromatase levels and activity30. According to another study, R seems to exert an inhibitory effect at the hypothalamic-pituitary level and to disrupt cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and corticosterone synthesis in the adrenal gland31. Moreover, Gly and R have been demonstrated to impair bovine and swine granulosa cells growth and steroid production34. In female rat and mice, Gly and GBHs have been reported to cause hormonal imbalances, oxidative stress and alterations in folliculogenesis including an increase of atretic follicles36. Recently Yahfoufi et al.37 reported that Gly exposure of mature mouse oocyte (MII) induces spindle fibre destruction, disturbance in chromosomal alignment, depletion of intracellular zinc bioavailability and ROS accumulation. Similar effects were found in mouse embryos exposed to Gly during embryo culture37. Induction of oxidative stress and apoptosis were also observed in bovine embryos cultured in presence of R38.Nevertheless, GBHs have been clearly demonstrated to exert their effects through a chemical endocrine disruption; in fact, they have been shown to impair the androgen/estrogen balance39. Zang et al.40 observed that Gly interferes with in vitro mouse oocyte maturation impairing nuclear maturation, generating oxidative stress and inducing DNA damage and early apoptosis.However, very few data are available in literature on possible effects of Gly and GBHs on mammal oocyte maturation, which prepare oocyte for fertilization events and affect early embryonic development41.On these bases, the objective of this study was to characterize the impact of Gly and R on female gamete using an “in vitro” model of pig oocyte maturation (IVM) evaluating nuclear maturation, cytoplasmic maturation and developmental competence of oocytes, steroidogenic activity of cumulus cells as well as intracellular levels of glutathione (GSH) and ROS of oocytes. We tested concentrations ranging from either 360 µg/mL Gly or 0.1% Roundup (containing 360 µg/mL Gly) to 70-fold lower on the basis of previous in vitro studies on reproductive tissues and gametesWhen COCs were matured in presence of Gly at 0, 5, 10, 100, 200 and 360 µg/mL or R at the same Gly-equivalent doses, no significant variations in the proportion of oocytes completing nuclear maturation showing a MII nuclear morphology were recorded  did not affect the cleavage rate, it caused a significant p < 0.01) reduction of the percentage of oocytes that developed to blastocyst stage at the higher concentration (360 µg/mL). Moreover, a significant decrease in the mean number of blastomeres per blastocyst was observed starting from Gly 200 µg/mL (p < 0.05)  it induced a significantly lower blastocyst rate  and mean number of blastomeres per blastocyst (p < 0.01 and p < 0.001 for R 200 and R 360 respectively) compared to control  at 44 h of culture compared to 22 h irrespective of Gly and R concentrations.E2 and P4 production was not affected by Gly exposure in none of the 2 days of culture   decreased the proportion of polar body extrusion of mouse oocytes due to misaligned chromosomes and abnormal spindle morphology; the authors suggested that Gly toxicity on mouse oocytes could be mediated by the increase of intracellular ROS levels.As a first step of this study we investigated the effect of Gly and R exposure during IVM on nuclear maturation of pig oocytes. None of the concentrations tested  modified the percentage of oocytes reaching MII stage compared to control. These results are in contrast with the only study performed up to now on the effect of Gly exposure during IVM on nuclear maturation of mammalian oocytes40 and our results could be due to a species-dependent sensitivity to Gly and/or it can be ascribed to different cultural conditions: Zang et al.40 matured mouse oocytes in M2 medium, while, in our model, pig IVM was performed in NCSU 37 medium supplemented with cysteine and β-mercaptoethanol, molecules known to reduce pig oocyte ROS levels, and 10% porcine follicular fluid (pFF), endowed with high radical scavenging activity elicited from SOD isoenzymes48; these supplementations, increasing the antioxidant activity of the system, may have masked the potential Gly toxic effects on nuclear maturation. In fact, it has been recorded that cell damage encompassed by ROS are difficult to detect in pig oocytes cultured in a medium supplemented with 10% pFF, even in the presence of ROS generated by the hypoxanthine-xanthine oxidase system47. Anyway, it must be kept in mind that in vivo oocytes are normally protected from the harmful effects of ROS by anti-oxidant enzymes which are present in the follicular fluid49 and therefore it can be assumed that our culture system is capable of closely mimicking the in vivo environment during oocyte maturation. In our pig model, a significant increase of the intracellular levels of ROS with R at the concentration of 360 µg/mL, but not with pure Gly, was recorded; this increase, however, was not as dramatic as that recorded in mouse oocyte after Gly exposure40.The discrepancy between Zang et al.50. The maintained sperm nuclear decondensing ability of the exposed oocytes agree well with the absence of any effect of either Gly or R on intracellular GSH levels which was recorded in this study. Even if an inverse relationship between oocytes intracellular ROS and GSH levels has been observed by many authors48, in this study, R 360 significantly increased the intracellular levels of ROS; it is likely that this rise was not so strong to significantly reduce oocyte GSH levels and, in turn, to impair oocyte decondensing activity.In order to evaluate the potential toxic effect of Gly and R exposure on cytoplasmic maturation, as a first step, fertilization parameters and oocytes ability to decondense sperm head and sustain male pronucleus formation after in vitro fertilization were evaluated. As observed for nuclear maturation, no detrimental effect of all the Gly and R doses tested on these parameters was recorded. Adequate oocyte GSH levels are needed in order to reduce protamine disulfide bonds that represent the first step in the induction of sperm nuclear decondensation and hence male pronucleus formation after in vitro fertilizationThe developmental competence of exposed oocytes after IVF was used as a further parameter of proper cytoplasmic maturation. Even the highest doses tested of both Gly and R had no effect on embryo cleavage. By contrast, R, and to a lesser extent Gly, induced a dose dependent reduction of both blastocyst rate and blastomere number per blastocyst. Therefore, oocyte exposure to R and Gly during IVM impaired the acquisition of a proper cytoplasmic maturation leading to a reduction of developmental competence, even if pesticides were not more present during embryo culture. This toxic effect was more evident with R compared to Gly, thus suggesting a synergistic effect provoked by the adjuvants present in the commercial formulation.52, likely due to cumulus cell differentiation-luteinization and R was markedly effective in inhibiting this P4 increase as P4 produced after 48 h of culture was significantly lower in R treated groups, starting from 100 µg/mL, as compared to control. Similarly, treatment of bovine granulosa cells with Gly (10 and 300 µg/mL) had no effect on P4 and E2 production33 while R (10 µg/mL) dramatically decreased steroid levels (P4 and E2). Other researches carried out by Gigante et al.34 on swine granulosa cell showed that Gly  induced a significant inhibitory effect on granulosa cell E2 secretion, viability and proliferation; in contrast, P4 secretion was stimulated at all tested concentrations. It must be considered that the researches carried out by Perego et al.33 and Gigante et al.34 were performed on plated granulosa cell with the addition of testosterone or androstenedione as estradiol precursors while our model, consisting of cumulus cell-oocyte complexes, is completely different. In fact, cumulus cells and mural granulosa cells are phenotypically/functionally different: cumulus cells play an essential role in the normal growth and development of the oocyte, while mural granulosa cells primarily exert an endocrine function and support follicle growth53. Moreover, evidence exists that oocyte plays an active role in determining the fate of follicle somatic cells; results obtained by Coskun et al.54 demonstrated that porcine oocytes secrete molecule(s) that inhibits steroid production by cumulus and granulosa cells. Therefore, based on these information we cannot strictly compare our results on steroidogenesis with those obtained by Perego et al.33 and Gigante et al.34.Concerning the effects on cumulus cell steroidogenesis, while Gly did not induce any alteration of E2 and P4 levels, R decreased P4 (but not E2) production. In our system, P4 secretion by COCs dramatically increased during the second half of culture, as already observed56. Therefore, the more serious negative effects of R compared to the pure molecule on oocyte developmental competence can possibly be due, at least in part, to the observed perturbation induced by R on P4 production by cumulus cells. Anyway, the mechanism through which R exerts its effects on cumulus cell steroidogenesis needs further investigations.P4 produced by cumulus complexes has been reported to positively influence porcine oocyte cytoplasmic maturation and to improve developmental competence to the blastocyst stage following IVF57.The decrease in P4 production by cumulus cells recorded in presence of R could have contributed to the increase in intracellular ROS levels induced by R 360. Previous studies, in fact, demonstrated that progesterone possesses antioxidant properties that are blocked by RU486, a P4 receptor antagonist58 or exert an intrinsic toxicity inducing membrane disruption59, apoptosis60, inhibition of mitochondrial respiration61 and DNA damage62.All these results support the hypothesis that surfactants/adjuvants present in GBHs are responsible for the increased toxicity of GlyTo the best of our knowledge, the present study is the first work describing the effects of Gly and R on pig oocytes maturation.Our results indicate that the exposure to Gly and its commercial formulation R during IVM, even if it does not affect nuclear maturation and embryo cleavage, impairs oocyte developmental competence in term of blastocyst rate and cellularity. Moreover, R at the same Gly-equivalent concentrations resulted to be more toxic than pure Gly, altering steroidogenesis and increasing oocyte ROS levels, thus confirming that R adjuvants enhance Gly toxic effects and/or are biologically active in their side-effect and therefore should be considered and tested as active ingredients.65; mean levels of 0.26 µg/mL (range < 0.020–17.2 µg/mL) in occupationally exposed workers have been recently reported66. These concentrations are far lower than those observed to be toxic in this study. Blood glyphosate levels recorded in human acute intoxications were 61 µg/mL (range 0.6–150 µg/mL) and 838 µg/mL respectively in mild–moderate and severe intoxication cases67, concentrations of the order of magnitude of those that were toxic in our study.Glyphosate concentrations detected in human urine has been reported to be at ng/mL levels with higher level in specifically exposed individualsObviously, the effects induced by Gly and formulants should be lower in vivo than in culture, and in vitro methods cannot provide the information that can be derived from in vivo tests. Nevertheless, in vitro maturation of pig oocytes, that can be obtained in large number from ovaries collected at the slaughterhouse, can be used as a reliable model to screen toxic agent for female gamete allowing the reduction of the number of laboratory animals used in vivo accordingly to 3R principles.The consequences of the massive use of GBHs remain a matter of concern on public health. We found that Gly and R exposure during IVM detrimentally affect the subsequent developmental ability of embryos, providing further evidence of their potential toxic effect on female reproductive system that is worth of a deeper investigation.N-(Phosphonomethyl)glycine  as well as the other chemicals, unless otherwise specified, were purchased from Sigma-Aldrich  except Roundup Bioflow  containing 360 g/L of glyphosate acid in the form of 480 g/L isopropylamine salts of glyphosate (41.5%), water (42.5%) and surfactant .68 supplemented with 5 μg/mL insulin, 1 mM glutamine, 0.57 mM cysteine, 10 ng/mL epidermal growth factor (EGF), 50 μM β-mercaptoethanol and 10% porcine follicular fluid (IVM medium), groups of 50 COCs were transferred to a Nunc 4-well multidish containing 500 μL of the same medium per well and cultured at 39 °C in a humidified atmosphere of 5% CO2 in air. For the first 22 h of in vitro maturation the medium was supplemented with 1.0 mM db-cAMP and 0.12 IU/mL Pluset . For the last 22 h COCs were transferred to fresh maturation medium69.Ovaries were collected from pre‐pubertal gilts at a local slaughterhouse and transported (in 0.9% wt/vol NaCl solution) to the laboratory within 2 h. Cumulus‐oocyte complexes (COCs) were aspirated from antral follicles, 3–6 mm in diameter, with a 18‐gauge needle fixed to a 10‐mL disposable syringe. Intact COCs were selected under a stereomicroscope and only COCs with more than three layers of compact cumulus cells and with uniform cytoplasm were transferred into a petri dish  prefilled with 2 mL of modified PBS supplemented with 0.4% BSA. After three washes in NCSU 37In order to assess the effect of Gly and R on nuclear maturation, pig COCs were exposed during in vitro maturation period (44 h) to 0, 5, 10, 100, 200 and 360 µg/mL Gly or R at the same Gly-equivalent doses.70.At the end of the maturation period the oocytes were denuded by gentle repeated pipetting and then mounted on microscope slides, fixed in acetic acid/ ethanol (1:3) for 24 h and then stained with Lacmoid. The oocytes were observed under a phase contrast microscope in order to evaluate the meiotic stage achieved . Oocytes with a nuclear morphology corresponding to metaphase-II stage (MII) were considered matureCytoplasmic maturation was assessed by evaluating:a) insemination parameters and ability of oocytes to sustain male pronucleus formation after in vitro fertilization. or R at the same Gly-equivalent doses, the oocytes were fertilized with frozen boar semen purchased from a commercial company . Straws were thawed in a waterbath at 37 °C under agitation for 30 s and immediately diluted, at the same temperature, in Beltsville Thawing Solution (BTS) at a dilution rate 1:3.71 supplemented with 12% fetal calf serum  and 0.7 mg/mL caffeine (IVF medium). Forty-five to fifty oocytes freed from cumulus cells were washed twice in IVF medium and transferred to 500 µL of the same medium containing 0.25 × 106 sperm/mL. After 1 h of gamete coincubation, oocytes were transferred to fresh IVF medium previously equilibrated under 5% CO2 and cultured for 17 h until fixation as above described .After 1 h semen was washed twice with BTS and finally resuspended with Brackett and Oliphant’s mediumParameters evaluated were: penetration rate , monospermy rate  and the ability of oocytes to sustain male pronucleus formation.Degenerated and immature oocytes were not counted.b) developmental competence of embryos after 7 days of in vitro culture. FCS to reach a final FCS concentration of 10% (v/v). At Day 7 post-fertilization, percent of blastocysts and number of blastocyst nuclei were determined by fixing and staining embryos as above described for oocytes . Embryos with at least 20 blastomeres and a clearly visible blastocoel were considered as blastocysts.Based on the results obtained from in vitro fertilization, a set of experiments was carried out to evaluate the effect of Gly and R exposure during IVM on embryonic development of oocytes after IVF. At the end of maturation period in presence of different concentrations of Gly  or R at the same Gly-equivalent doses, oocytes were coincubated with frozen-thawed spermatozoa for 1 h as described above, washed twice in IVF medium and incubated 3 h in the same medium. Then presumptive zygotes were washed twice in NCSU-23g for 5 min and the supernatants were stored at − 20 °C until assayed for progesterone (P4) and estradiol-17β (E2) by validated radioimmunoassays52. At the end of the maturation period, cumulus cells were counted using a Thoma’s hemocytometer, after being freed from matured oocytes by gentle repeated pipetting. For P4, the intra- and interassay coefficients of variation were 6.8% and 10.1%, respectively; assay sensitivity was 4.4 pg/tube. The intra- and interassay coefficients of variation for E2 were 5.4% and 10.5%, respectively; assay sensitivity was 1.7 pg/tube. Steroid concentrations are expressed as ng/106 cells.IVM media of both the first and the second day of culture of COCs in presence of Gly  or R at the same Gly-equivalent doses, were collected, centrifuged at 900×36. From each treatment group, oocytes were incubated in the dark for 30 min at 39 °C in PBS/0.1% (wt/vol) PVA supplemented with 10 μM H2DCFDA or 10 μM CellTracker Blue. Following incubation, the oocytes were washed in PBS/0.1% (wt/vol) PVA, placed into 10‑μL droplets, and fluorescence was evaluated under a Nikon Eclipse E 600 epifluorescence microscope . The fluorescence images were analysed with Image J software (public domain). Relative oocyte fluorescence was measured by normalizing the oocyte fluorescence with the background and with each oocyte area. Five independent experiments were performed .Intracellular GSH and ROS levels of oocytes at the end of maturation period in presence of Gly  or R at the same Gly-equivalent doses, were determined using 4‑chloromethyl‑6.8‑difluoro‑7‑hydroxycoumarin  or 2′,7′‑dichlorodihydrofluorescein diacetate  respectively as previously described72. Values are expressed as mean ± standard deviation (SD) and level of significance was at p < 0.05.Statistical analyses were performed using R (version 3.4.0)Data on nuclear maturation, IVF trials, blastocyst formation and cumulus cell steroidogenesis were analysed using a general linear model with binomial distribution and a Tukey post-hoc test was subsequently run to determine differences between treatments.Data on blastomere number were analysed using a Poisson distribution and a Tukey post-hoc test was subsequently run to determine differences between treatments.Data on GSH and ROS intracellular levels, after being tested for normality and homogeneity of variances through Shapiro–Wilk test, were analysed using Non-parametric Kruskal–Wallis Test and Wilcoxon test was subsequently used to assess differences between treatments.


Calendula officinalis L., and recently proved as a promising anti-Alzheimer. The effect of the critical process parameters (CPP) viz. PLGA amount, Wlecithin/WPLGA ratio, and Tween 80 concentration on critical quality attributes (CQA); entrapment, size and size distribution, was statistically analyzed via design of experiments, and optimized using the desirability function. The optimized CPP were maintained while substituting Tween 80 with other PEG-SAA. All hybrid particles exhibited spherical shape with perceptible lipid shells. The biocompatibility of the prepared NPs was confirmed by hemolysis test. The pharmacokinetic assessments, post-intravenous administration to rats, revealed a significant higher RU bioavailability for NPs relative to drug solution. Biodistribution studies proved non-significant differences in RU accumulation within brain, but altered phagocytic uptake among various LPHNPs. The present study endorses the successful development of LPHNPs using PEG-SAA, and confirms the prospective applicability of TPGS and Solutol in enhancing brain delivery.The blood–brain barrier is considered the leading physiological obstacle hindering the transport of neurotherapeutics to brain cells. The application of nanotechnology coupled with surfactant coating is one of the efficacious tactics overcoming this barrier. The aim of this study was to develop lipid polymer hybrid nanoparticles (LPHNPs), composed of a polymeric core and a phospholipid shell entangled, for the first time, with PEG-based surfactants (SAA) viz. TPGS or Solutol HS 15 in comparison with the gold standard Tween 80, aiming to enhance brain delivery and escape opsonization. LPHNPs were successfully prepared using modified single-step nanoprecipitation technique, loaded with the flavonoid rutin (RU), extracted from the flowers of  The effectiveness of these actives is generally compromised due to failure reaching the site of action sufficiently. The blood–brain barrier (BBB) is considered the most dominant physiological barrier impeding the passage of neuropharmaceuticals to the brain cells. It is a highly fortified membrane system, composed of specialized capillary endothelial cells, that protects the brain from extraneous organisms and harmful chemicals, and supplies the brain with the nutrients required  is considered the gold standard effectively crossing BBB. The fact is due to the preferential adsorption of apolipoprotein E (Apo E), present in blood, on NP surfaces coated with Tween 80, rendering particles resembling the low density lipoproteins (LDL), hence interacting with LDL receptors on BBB and enhancing their cellular uptake via receptor-mediated transcytosis mechanism  and Solutol® HS 15 (polyethylene glycol-15-hydroxy stearate) -mediated drug transport to the brain has been governed by particle coating with surfactants. The surfactant Tweenco-glycolide (PLGA), respectively. They exhibit a number of advantages compared with liposomes; the extended stability upon storage, the robust structural integrity and the controlled release pattern for encapsulated drugs , was developed combining the characteristics of both systems. These platforms are composed of a polymeric core surrounded by a self-assembly phospholipid shell intertwined with a PEG-containing substrate, typically a PEG-lipid warranting the protracted circulation time  is the most common neurodegenerative disorder causing dementia among the elderly population in the world. It is usually manifested by a bundle of disruptions in memory, thinking, understanding, learning abilities, linguistics, calculation and coordination , the glycoside of the flavonol quercetin, is found in many plants, for instance, citrus fruits , apples, berries, peaches and green tea , a novel promising candidate reported to exert a boosting anti-AD effect. RU . The results were statistically analyzed and then optimized through the desirability function parameter. A comparative study was then developed to compare different coating materials in terms of characterization, biocompatibility, pharmacokinetic and biodistribution studies.In the present work, a novel attempt was studied targeting the anti-AD polyphenol drug (RU) to the brain via the innovative nanoconstructs ‘LPHNPs’ coupled with surfactant coating. As a new approach, the classical PEG-lipid component in LPHNPs was substituted, for the first time, with PEG-based surface active agents (SAA) aiming to afford equivalent stealth character to the hybrid particles. Among these SAA, Tween 80, TPGS and Solutol HS 15 are prototypes. These SAA also combined the reported persuasive roles in enhanced brain delivery. In this context, RU was first extracted from the flowers of 2.2.1.2.1.1.C. officinalis L., family Asteraceae were collected from the Botanical Garden of El-Orman, Giza, Egypt. The plant was authenticated by Mrs. Trease Labib, Plant Taxonomy Consultant at the Ministry of Agriculture. A voucher specimen has been deposited at Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Abbassiah, Cairo, Egypt (PHG-P-CO-1). All used solvents used for extraction were of high analytical grade.The edible flowers of 2.1.2.dl-lactide-co-glycolide (PLGA)  was gently provided from Corbion Purac Biomaterials . The lecithin, Soybean phosphatidylcholine  was kindly supplied by Lipoid GmbH . TPGS was generously delivered by Isochem . Tween® 80 and Solutol® HS 15 were obtained from Sigma-Aldrich, Chemical Co. . Methanol (HPLC grade) was purchased from Fluka . Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride (KCl), acetone, dimethyl sulfoxide (DMSO) and ortho-phosphoric acid were obtained from Adwia, El-Nasr Pharmaceutical Co. (Egypt). Spectra/Por® dialysis membrane, 12,000–14,000 MWCO was purchased from Spectrum Medical Industries . Nanosep® centrifuge tubes, fitted with an ultra-filter of 100 kDa MWCO, were provided from Pall Life Sciences . Deionized water provided from Milli-Q Gradient A10 System was employed all over the research study. All other chemicals and reagents were of analytical grade.Poly-2.2.2.2.1.C. officinalis L.) flowers (2 kg) were dried in shade, and extracted with neat methanol several times till complete exhaustion. The methanol in the combined extracts was distilled off at 50 °C in a rotary evaporator. The remaining concentrated extract was then subjected to preparative TLC using the BAW solvent system butanol: acetic acid: water . A major band showing a dark purple color upon using UV lamp at 365 nm and quenched the UV light at 254 nm, was scratched, dissolved in methanol and the solvent was evaporated in vacuo at 50 °C, to obtain a yellow powder that was subjected to UV and NMR spectroscopic analysis. 1H and 13C-NMR spectroscopic data were measured at 400 and 100 MHz, respectively, in DMSO on a Bruker Ascend-400 spectrometer . The chemical shifts (recorded in ppm) were measured using tetramethyl silane (TMS) as internal standard. The UV spectroscopic measurement was performed on UV-Spectrophotometer .The marigold  was dispersed in 10 ml deionized water heated to 65 °C. The resulting PLGA/SPC/RU organic solution was then added into the preheated SAA solution dropwise under gentle stirring. The mixed solution was then agitated vigorously for 5 min. followed by gentle stirring for 2 h at room temperature to ensure complete evaporation of the organic solvent. The drug-loaded PLGA NPs were also prepared, for comparison purpose, using the same method described above except lecithin was excluded from the preparation.2.2.3.X1), Wlecithin/WPLGA ratio (X2) and Tween 80 concentration (X3), each at two levels; 50 and 100 mg, 1/1 and 3/1, 0.5 and 1%, respectively. The critical quality attributes (CQA) considered were Y1; the percent of drug entrapment efficiency (EE), Y2; the particle size (PS) and Y3; the polydispersity index (PDI) of the formed NPs. The typical statistical design of the experiment is displayed in A response surface methodology (RSM) using two-level full factorial design was implemented aiming to optimize the critical process parameters (CPP) of LPHNPs. The experiment studied the effect of three CPP, namely PLGA amount . A sample of 50 µl of NP dispersion was diluted with 450 µl deionized water, placed in the upper part of a Nanosep2.2.5.2.Particle size and polydispersity index measurements were accomplished using dynamic light scattering (DLS) technique via Zetasizer Nano ZS instrument . The PS and PDI of three independent samples were measured in disposable cuvettes at a temperature of 25 ± 0.5 °C after 50-fold dilution with deionized water.2.2.5.3.Laser Doppler anemometry (LDA) technique was applied for zeta potential (ZP) measurements using Zetasizer Nano ZS instrument . Three independent samples were suitably diluted with 10 mM KCl and then placed in zeta cells for measurement at a temperature of 25 ± 0.5 °C.2.2.5.4.via HR-TEM  with an acceleration voltage at 200 kV. All samples for TEM imaging were initially prepared by allowing a single drop of NP suspension to dry at room temperature on a carbon-coated copper meshwork after being stained with 1% phosphotungstic acid.The imaging of the selected RU-loaded LPHNPs, as well as PLGA NPs, was performed 2.2.5.5.The thermal properties of different samples of RU, PLGA, SPC and selected loaded NP formulations were studied using a differential scanning calorimeter . The NP formulations were left to dry overnight in a desiccator. Powdered samples were sealed in aluminum pans with lids and heated from 25 to 300 °C at a rate of 10°C/min. under nitrogen flow at a rate of 25 ml/min.In vitro release experiments of RU from loaded LPHNPs were performed in PBS (pH 7.4) for 12 h. An aliquot of NPs, equivalent to 1 mg RU, was introduced in a dialysis bag and then placed in a closed container containing 50 ml PBS, acquiring the sink conditions, adjusted at 37 °C under gentle magnetic stirring. At predetermined time intervals, 1 ml of the medium was withdrawn and then replaced with an equal volume of fresh medium. The amount of RU released was calculated by HPLC at λmax 360 nm. The release of free drug was also performed similarly. The in vitro release experiments were done in triplicate for each formulation.λmax 414 nm. The hemolysis index was then calculated using the following equation:t, ODnc, and ODpc are the optical densities of the test sample, the negative control and the positive control, respectively.Erythrocytes separated from rat blood was used to evaluate the potential hemolytic activity of the prepared SAA-coated LHPNPs. Serial dilutions of each nanosuspension type were prepared to assess the biocompatibility of each SAA at different concentration levels in the blood. The hemolytic assay was conducted according to the method adopted by Mourtas et al.  with fewThe negative and positive controls were prepared by incubating the erythrocytes suspension with normal saline or deionized water, respectively.2.2.8.3-U100 insulin syringe equipped with 28 G needle at RU dose level of 5 mg/kg  were divided into 4 groups each of six animals. The study protocol was reviewed and approved by the Experiments and Advanced Pharmaceutical Research Unit (EAPRU), Faculty of Pharmacy, Ain Shams University on the use of the animals. The treatment was performed as follows; group I received RU solution prepared in 10% DMSO while groups II, III and IV received RU-loaded Tween 80-LPHNPs, TPGS-LPHNPs, Solutol-LPHNPs, respectively. All formulae were injected into the tail vein of the rats using a 1 cm/kg Zhu, . SamplesCmax) and the time to reach Cmax (Tmax) were detected from plasma drug charts. The areas under the curve from time zero to last sampling time (AUC0−t), the area under the curve from time zero to infinity (AUC0−∞), and those under the first moment curve (AUMC) were determined. Mean residence time (MRT) was computed by dividing AUMC by AUC. The relative bioavailability (Fr), defined as the ratio of AUC0−∞ of either TPGS or Solutol-LPHNPs to that of Tween 80-LPHNPs at the same doses administered, was also calculated.The plasma RU concentrations versus time data for different groups were analyzed by non-compartmental estimations using PKsolver, the add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Maximum plasma concentration  were randomly divided into three groups administered the three loaded formulae under study  intravenously through the tail vein via a 28 G-needle insulin syringe at a dose of 5 mg/kg. At different time intervals after drug injection; 0.25, 0.5, 1, 2, 4 and 6 h, three mice from each group were sacrificed by decapitation. Different organs; brain, liver, spleen, and kidney were dissected from each mouse, washed with normal saline, blotted with filter paper to remove excess fluid, weighed and then stored at −20 °C until analysis.On the day of analysis, organ samples are allowed to thaw and then subjected to homogenization after the addition of a calculated volume of normal saline. The tissue homogenates were centrifuged at 4000 rpm for 15 min, and the supernatants were collected for drug extraction. The RU concentrations in tissue samples were analyzed by LC/MS, similarly to plasma samples, and the mean RU amounts per g organ were then calculated and plotted versus time, and all pharmacokinetic parameters were computed as described earlier.2.2.10.in vitro samples according to Kuntić et al.  was used and maintained at temperature =40 °C. The mobile phase constituted of a binary mixture of methanol–water 1:1 (v/v), pH 2.8 (adjusted with phosphoric acid) adjusted at flow rate of 1 mL/min. The wavelength of UV detector was set at λmax 360 nm. The method was first validated according to the International Conference on Harmonization guidelines . The data was analyzed using ChemStation B.04.01 software .An isocratic RP-HPLC was adopted to quantify RU in all ć et al.  using Ag2.2.11.m/z 608.9 precursor ion to the m/z 300.0 for RU and m/z 295.6 precursor ion to the m/z 268.9 for IS. Quadrupoles Q1 and Q3 were set on unit resolution and the analytical data were processed using Analyst software (Version 1.4.2).A sensitive, selective and accurate LC–MS/MS method was developed and validated for the determination of RU concentrations in plasma. Stock solution of hydrochlorothiazide internal standard (IS) was prepared by dissolving 10 mg in methanol and serially diluted with mobile phase to give a final working concentration of 200 ng/ml. A shimadzu Prominence  series LC system equipped with degasser (DGU-20A3), solvent delivery unit (LC-20AB) along with auto-sampler (SIL-20 AC) was used to inject 20 µl aliquots of the processed samples on a Luna C  (50 × 4.6) mm, 5 μm particle size. The isocratic mobile phase (pH 4.5) consisted of acetonitrile and (0.02 M) ammonium acetate buffer  and 0.1% formic acid which was delivered at a flow rate of 0.50 ml/min into the mass spectrometer’s electrospray ionization chamber. All analysis was carried out at room temperature. Quantitation was achieved by MS/MS detection in negative ion mode for both RU and IS, using a MDS Sciex  API-3200 mass spectrometer, equipped with a Turbo ionspray interface at 400 °C. The ion spray voltage was set at −4500 V. The nebulizer gas was set at 30 psi, curtain gas at 15 psi, auxiliary gas at 55 psi and collision gas at 9 psi. The compound parameters, namely, declustering potential, collision energy, entrance potential and collision exit potential were −1 V, −52 V, −10 V, and 19 V for RU and −45 V, −22 V, −10 V, and −12 V for hydrochlorothiazide (IS), respectively. The ions were detected in the multiple reaction monitoring mode, monitoring the transition of the 2.2.12.in vitro experiment was performed in triplicates; the average data and their standard deviations (SD) were then calculated. Results of the in vivo studies were expressed as mean ± standard error of the mean (SE). For statistical comparisons, a paired t-test was used; p < 0.01 was considered significant statistically.Each ® version 7.0.0 software  applying design of experiment (DoE) by means of RSM and two-level full factorial design. The following polynomial equation model was employed fitting the experimental data and predicting the new trials (out of the design): Y = β0 + β1X1 + β2X2 + β3X3 + β12X1X2 + β13X1X3 + β23X2X3 + € where Y represents the predicted response, X1, X2, and X3 are the independent variables, β0 is the intercept, β1, β2, and β3 are the main effect coefficients, while β12, β13, and β23 are the two-way interaction coefficients, and €corresponds to the model residual. The quality of fit the experimental data by the polynomial model equation was expressed by the coefficient of determination (R2) and its adjusted R2. The analysis of variance (ANOVA) was adopted. The significance of each coefficient term and the lack of fit of the suggested model were evaluated via p-value and F-value with 95% confidence level. Main effects, contour and 3D-response surfaces plots were all designed.The statistical relationships between the CPP and CQA in the experimental design were adopted using Design Expert2.2.13.After statistical analysis of the experimental design data, a numerical optimization was statistically adopted by applying the desirability function (D) which transforms the values of a response into  where 0 stands for a non-acceptable value of the response and 1 for ideal target values of this response  of 0.5 on TLC using the solvent system butanol:acetic acid:water  which was in accordance with the authentic compound Rf value.Phytochemical investigation of λmax (nm) MeOH: 257, 266 (sh), 301 (sh), 362. 1 H-NMR  δ ppm: 1.00 , 3.10-3.72 , 5.11 , 5.35 , 6.20 , 6.41 , 6.85 , 7.55 , 7.59 , 9.20 , 9.68 , 10.85 , 12.59 . 13 C-NMR  δ ppm: 18.19 (C-6′′′), 68.70 (C-5′′′), 67.45 (C-6′′), 70.45 (C-2′′′), 70.83 (C-3′′′), 71.01 (C-4′′′), 72.30 (C-4′′), 74.52 (C-2′′), 76.35 (C-5′′), 77.30 (C-3′′), 94.05 (C-8), 99.14 (C-6), 101.19 (C-1′′), 101.60 (C-1′′′), 104.42 (C-10), 115.68 (C-2′), 116.72 (C-5′), 121.63 (C-6′), 122.05 (C-1′), 133.74 (C-3), 145.19 (C-3′), 148.85 (C-4′), 156.87 (C-2), 157.07 (C-5), 161.66 (C-9), 164.51 (C-7), 177.80 (C-4), as illustrated in Figure S2.Yellow amorphous powder (100 mg), UV 3.2.Wlecithin/WPLGA ratios, as previously reported in such cases , not dispersed in water as usual, together with the polymer and drug. It could be assumed that the precipitation of PLGA into NPs simultaneously occurred with self-assembling of lecithin around them owing to hydrophobic interactions. The lipid hydrophobic tails were anchored to the polymeric hydrophobic core while the hydrophilic head was extended to the external aqueous medium stabilizing the hybrid NPs formed. The steric stabilization of LPHNPs was conventionally afforded by the PEG chains of a lipid-PEG element. Nevertheless, this was compensated in this study by; (1) higher Wlecithin/WPLGA ratios and Tween 80 concentrations was studied on drug EE, PS, and PDI. The formulation results, depicted in The effect of different PLGA amounts, 3.3.3-full factorial design was applied and the product design space was defined leading to eight sets of experiments with three replicates. The statistical significance of three CPP, i.e. polymer amount, Wlecithin/WPLGA ratio and Tween 80 concentration, each at two levels, on different LPHNPs CQA; EE, PS, and PDI; were considered using RSM.A DoE was conducted to evaluate and quantify the effect of independent factors on the selected CQA. A 2Table S1.The Box–Cox Plot for power transforms was initially diagnosed. This is typically used as a guideline for selecting the correct power law transformation (lambda) set at the minimum point of the curve generated by the natural log of the sum of squares of the residuals  for both EE and PS while the linear equation was recommended for PDI. The coefficient of determination (R2) was calculated to be higher than 0.9 for all responses under study (Table S1) indicating the satisfactory adjustment of the suggested models to the experimental data as well as the high predictability of these models. The non-significant ‘Lack of Fit’ (p > 0.05) for all models is considered a good sign implying its insignificance relative to the pure error. The ‘Predicted R2’ were in reasonable agreement with the ‘Adjusted R2’ for all models under study. The ‘Adequate Precision’ measures the signal to noise ratio. A ratio greater than 4 is desirable indicating an adequate signal, hence all models can be used to navigate the design space.The statistical models were developed by regression analysis using the experimental data for the drug EE, PS, and PDI, as displayed in Table S2, it was found that all main effects model terms  were found significant in terms of EE and PS (p < 0.05). In the case of PDI, only X1 was considered significant. The interaction model terms were found significant in EE and PS models only. Note that the non-significant model terms were omitted, so that the model reduction may improve its applicability.By means of observation of ANOVA analysis, based on Table S2) show that all factors studied had significant effects on RU EE (p < .01), being the effect of Wlecithin/WPLGA ratio the highest. A clear positive influential EE response was associated with PLGA amount and Wlecithin/WPLGA ratio and, whereas the SAA concentration impacted a negative effect, as presented in Table S1 equations. As illustrated in contour and 3D-response surface plots (Wlecithin/WPLGA ratio parameter. The highest RU EE was attained at the highest levels (+1) of both factors; 100 mg PLGA and 3/1 Wlecithin/WPLGA ratio.The ANOVA results  with the most prominent effect goes for PLGA amount. Increasing polymer amount led to an enlargement in PS while the opposite followed in case of Wlecithin/WPLGA ratio as well as SAA concentration, as shown in X1 and X2 where at high level (+1) of Wlecithin/WPLGA ratio (3/1), the both lowest and highest PS were manifested yet when combined with the low (−1) and high (+1) levels of PLGA amount, respectively. Also, the lowest PS was reached when the lower level (−1) of PLGA amount was accompanied with the higher level (+1) of Tween 80 concentration, as revealed in The effect of polymer amount in the prepared hybrid NPs was found similar to that in case of nonhybrid (polymeric) NPs. Increasing polymer content increased drug EE, PS and heterogeneity of the NPs formed. This could be ascribed to the rapid precipitation of the polymer on the surface of the internal droplets hindering drug diffusion, hence EE improved. The increased viscosity of the dispersed phase increases the diffusion resistance of drug molecules from the organic to the aqueous phase leading to the formation of non-uniform larger particles of longer diffusional pathways for the drug, thereby reducing its loss and increasing its entrapment ) were constructed relating X1 and X2 at 0.5 and 1% Tween 80 concentration, respectively. These plots show the optimal goal parameters for attaining the QTPP required. As displayed in Figure S3(A and B), the highest D attained was 0.69 and 0.64, at 0.5% and 1% SAA concentration, respectively. The target areas in plots are manifested in yellowish green color. Therefore and based on D values, the optimal CPP; PLGA amount, Wlecithin/WPLGA ratio and Tween 80 concentration, were adjusted to 75 mg, 3/1 and 0.5%, respectively.After statistical analysis, a numerical optimization was conducted aiming to select the most optimum CPP for the preparation of LPHNPs. The optimal quality target product profile (QTPP) was set in terms of EE, PS, and PDI, as tabulated in 3.5.Figure S3(C–E), the predicted CQA of the optimized formula consisting of 75 mg PLGA, 3/1 Wlecithin/WPLGA ratio and 0.5% Tween 80 were 69.13%, 241.89 nm and 0.3 for EE, PS, and PDI, respectively. The optimized formulation was then prepared using the optimized CPP and all actual CQA were measured, recorded in Table S4 and compared with the predicted values. By computing the prediction error percent, it could be concluded the validity and predictability of the model equations generated from DoE owing to the small %bias which did not exceed 20%  the highest EE and the lowest PS among all prepared NPs. The fact could be attributed to the apparent increase in viscosity of the aqueous phase in which SAA was dissolved, owing to the higher Mw of TPGS (1513 g/mol) compared with other SAA (Tween 80 Mw = 1310 g/mol and Solutol HS15 Mw= 963.24 g/mol), which would reduce the drug diffusion and loss in the surrounding medium, and hence improve drug EE. In the same context, the efficiency of TPGS as emulsifier has been previously reported during the preparation of polymeric NPs  of the prepared NPs were also measured and tabulated in 3.7.p > .05) in PS and surface charge of the dialyzed suspensions (data not shown) in comparison with the non-dialyzed ones, as recorded in in vitro hemolysis assay.A dialysis-based purification step was applied aiming to recover NPs as well as eliminate excess SAA and unentrapped drug from the prepared nanosuspensions. Only 30 min. was found sufficient to get rid of all free drug, after which the release of entrapped RU began. Though this short dialysis duration was found not satisfactory to wipe out completely the excess SAA present, this was confirmed by the non-significant alteration (3.8.3.8.1.Tg)  Percent Hemolysis induced by incubation of various types of RU-loaded LPH NPs with rat blood at different SAA concentrations. Each data represents the mean ± SD (n = 3). The black dotted line in (d) denotes the permissible threshold hemolysis percent. NS; Nonsignificant difference at p > .05.(A) DSC thermograms of RU, PLGA, SPC and loaded LPH NPs. (B) HR-TEM micrographs of RU-loaded Tween 80-LPH NPs (a), TPGS-LPH NPs (b), Solutol-LPH NPs (c) and polymeric NPs prepared with 50 mg PLGA and 1% Tween 80 (d). (C) 3.8.2.co-exist adjacent to hybrid particles, as pointed out by red arrows in TEM was used to visualize the morphology of RU-loaded LPHNPs coated with different PEG-based SAA. As illustrated in in vitro release study of RU from different formulations was performed using dialysis bag technique in PBS pH 7.4 adjusted at 37 °C under sink conditions. The comparative drug release profiles of different types of loaded LPHNPs were illustrated in .,The p < .05) compared with the corresponding nanocarriers either coated with TPGS or Solutol at all SAA concentration levels. The low toxicities of TPGS and Solutol were previously reported by Pooja et al. (p > .05) in hemolysis was detected in case of Tween 80 between concentrations 0.25 and 0.5 mg/ml, 0.5 and 1 mg/ml while for TPGS and Solutol the insignificance was recognized at concentration range 0.1–0.5 mg/ml. Based on numerous studies, the in vitro percent hemolysis is assessed as ‘no concern’ when it ranges from 5% to 25%  by about 160-fold, 98-fold, and 159-fold was perceived for Tween, TPGS, and Solutol-based particles, respectively, relative to the drug solution  and Fr values almost equal 1 , expressed by non-significant differences in AUCs  especially in liver and spleen, expressed by lower AUCs. This excitingly reveals the efficiency of this PEG-SAA in sheathing NPs away from macrophages recognition, compared with other SAAs. Unexpectedly, TPGS-LPHNPs exhibited higher drug accumulation in RES organs; liver > kidney > spleen, reflected by significant higher AUCs (p < .05) compared with Tween-sheathed particles, translated by high Fr values, computed as 2.26, 2.69 and 1.14 in liver, kidney and spleen, respectively. The predominant cause of NPs loss from the systemic circulation is its phagocytic uptake by RES. These results demonstrate that the surface modification with TPGS did not avoid the opsonization process and engulfment of particles by macrophages. Similar finding was previously reported by Durán-Lobato et al.  for all prepared hybrid NPs relative to the drug solution. However, TPGS–LPHNPs exhibited a much lower drug bioavailability compared with the other NPs. The biodistributions of different LPHNPs were also studied. Nonsignificant differences in brain drug uptake (p > .05) were detected among the different types of LPHNPs However, NP surface treatment with TPGS specifically exhibited a noticeable higher phagocytic uptake in RES organs. The results of the present study highlighted the potential applicability of Solutol and TPGS in brain targeting being equivalent to the gold standard Tween 80. This study opens the door for further investigations in the field of neuro-pharmaceuticals aiming to confirm the competency of these PEG-SAA in brain delivery, phagocytic uptake evasion, and long circulation in blood.We have successfully developed the novel LPHNPs composed of a PLGA polymeric core and soybean lecithin intertwined, for the first time, with a PEG-SAA instead of PEG-lipid, forming the lipid shell for the effective delivery of RU to the brain. Single-step modified nanoprecipitation method was adopted to prepare LPHNPs using Tween 80. A 2Click here for additional data file.


People living with Long Term Neurological Conditions (LTNCs) value peer support and social activities. Psychological support and wellbeing enables them to manage their condition. Social prescribing is a formal process of referring patients to a link worker to co-design a plan to improve their health and wellbeing. Intervention involves supporting participation in activities based within the individual’s local community. This study aimed to explore the barriers and enablers to accessing social prescribing for people living with LTNCs (plwLTNCs).A total of four focus groups were carried out with 17 participants, including different neurological conditions such as multiple sclerosis, Fragile X Syndrome, epilepsy, and traumatic brain injury. Two participants were family carers and supported people living with epilepsy and motor neurone disease. Findings were analysed using thematic analysis.Five themes were identified: (1) Lack of knowledge; (2) Service provision difficulties; (3) Benefits of social prescribing activities; (4) Physical barriers and (5) Psychological barriers. There was a lack of knowledge about social prescribing and what it actually was. Participants anticipated service provision difficulties relating to funding, link workers need for knowledge of LTNC’s and for activities to be varied and individualised. The potential benefits of social prescribing activities were recognised across the groups especially its potential to tackle loneliness and to offer plwLTNC’s purpose. Participants highlighted a number of physical barriers such as transport and accessibility; and psychological barriers such as anxiety and stigma.Social prescribing aims to address the health inequalities of those living with long-term conditions, however currently it is likely to exclude plwLTNCs. Recommendations for practice and future research are made.The online version contains supplementary material available at 10.1186/s12913-021-07213-6. There are an estimated 14.7 million neurological cases in England, equating to at least 1 in 6 people living with one or more neurological condition . Neurolon = 3459) of the neurology patients they surveyed reported living with at least one other co-morbid condition  prior to study commencement.A copy of the topic guide can be found in supplementary materials  is an occupational therapist and working in the field of long-term neurological conditions, with an MRes degree and some experience in qualitative data. She received further qualitative data support from CG, educated at PhD level in Psychology, who has in-depth experience in conducting qualitative research with vulnerable population groups. Both of their backgrounds made them aware of the population groups as a whole, whilst they were solely guided by the data and not by their background.A total of four focus groups (minimum 4 participants) were carried out with a total of 17 participants, 12 female and 5 male. Participants represented the views of people living with a variety of neurological conditions, this included multiple sclerosis (2), Fragile X Syndrome (1), epilepsy (4), traumatic brain injury (1), essential tremor (2), ataxia (2) and subarachnoid haemorrhage (3). Two participants were family carers and supported people living with epilepsy (1) and motor neurone disease (1).Across the four focus groups five themes were identified: (1) Lack of knowledge; (2) Service provision difficulties; (3) Benefits of social prescribing activities; (4) Physical barriers and (5) Psychological barriers. Within these themes subthemes were identified and are outlined.The majority of participants had not heard of the term social prescribing. Those who had had been made aware of it by the charity where the focus groups took place or had seen information in the media. Reference was made to the term social prescribing and its link to a medical model of care and the potential difficulties the term would introduce when searching for information.“I don’t think I’d necessarily heard the term but I know what that means. I’d seen stuff about people gardening and people being outdoors so I presumed it was all interlinked in to that side of things.”Focus Group 1 Participant“I have heard it in the media and the papers and actually I think there was something on BBC news actually all about it. I might not be right but I think it is all about coming up with something that you can do to aid your condition or your recovery after what might … all of us have some sort of neurological condition so it is about that.”Focus Group 4 Participant“ … because it was just like a two-minute thing my consultant recommended coming here because there was no more that he could do, because he was so busy it was just like go there for help with practical support, I hadn’t even thought of it but I was told here (referring to the charity).”Focus Group 2 Participant“I think also it is getting GPs on board as well because if they do, GPs are obviously snowed under aren’t they?”Focus Group 4 ParticipantNone of the participants had knowingly accessed a social prescribing service. For the few who had accessed activities or groups, the trigger was a passing comment made by a health professional or was the outcome of their own motivation to participate. Participants highlighted the need for education of health care professionals about social prescribing so that they could consistently signpost or refer people living with LTNCs.Participants felt link workers would need to have knowledge and understanding of LTNCs. There was recognition amongst the groups that this would be a challenge given the number of neurological conditions and the vast array of difficulties faced by people living with LTNCs. Participants made reference to link workers needing good communication and social skills. Participants felt some people living with their conditions would need link workers to use a variety of communication approaches such as e-mail, text message and sykpe as well as face to face to engage with people. For those with memory problems the use of text reminders was highlighted as an important enabler. They felt given the challenge link workers would face, they would need to feel valued and have access to training and support.“I think something like this (referring to the charity), or like a booklet for people potentially going in as link workers would help them. Like the different diagnosis, what to expect … ”Focus Group 2 Participant“You need people who are like, no matter who the person is or whether they are autistic or epileptic or I don’t know, no matter what the problem is they’ve got to be able to actually communicate with that person which would probably be difficult to find”Focus Group 3 ParticipantThe need for knowledge from link workers extended to services and activity providers. In order to achieve this understanding, emphasis was placed on the need to involve people living with LTNCs in the development and delivery of services. Participants recognised the cost of providing services and that activities needed to be adequately funded.“I mean the big thing is funding isn’t it, I mean, the NHS is struggling and we, you know social prescribing requires money to have a link worker and valuing the fact that this link worker is important. You know you can go to your GP and go and see the specialist nurse and whatever but they don’t fully understand what your condition is and there are so many different conditions so funding is a big thing”Focus Group 4 Participant“It’s all more workers thought isn’t it? You know it’s not an easy fix you know. One to one is all more work and it’s all more expense if it’s being paid for by the NHS.”Focus Group 2 ParticipantRecognition of individual differences despite the same diagnosis was important with participants emphasising that one size does not fit all and that activities would need to be varied. Ensuring services got to know the person living with a LTNC and focused on their abilities rather than their disabilities was a priority. Participants considered the impact finances had on the ability to participate in activities as many were unemployed or living on a reduced income. Some highlighted problems negotiating the benefits system.“Cause like you were saying before, if your benefit that you’re entitled to is stopped your income is going to go down, so of course money to access anywhere or there being places that are local for you to go to. It’s more expensive to get to if they’re not local anymore”Focus Group 2 Participant“I think with social prescribing for neurological conditions there has to be realisation that not one size fits all, because of the variety of the conditions you can’t just say you’ve got MS, you’ve got epilepsy whatever maybe I’ll send you off to a gardening group. So there has got to be a real thought process behind what’s being prescribed for people.”Focus Group 1 Participant“Maybe it will put a lot of emphasis on the things that you can’t do anymore, and even if you can’t do things anymore, they don’t put the emphasis on all of the stuff that you actually still can do and focus on that instead.”Focus Group 3 Participant“I think sometimes with neurological conditions the person needs to be there to support them a little bit longer than maybe someone who doesn’t have a neurological condition”Focus Group 4 ParticipantParticipants felt access to support to attend groups would be important and they felt consideration was needed as to how long this support was provided. Participants felt people living with LTNCs may require a more prolonged period of support to be able to continue participation independently. Due to the high levels of anxiety and reduced confidence experienced by plwLTNCs, they believed it was unlikely a single accompanied visit would be sufficient.Participants discussed the difficulties faced by people living with LTNCs. Social isolation was seen as a significant problem in all focus groups. Participants expressed feelings of loss, difficulties maintaining relationships, managing families concerns and the impact of not working.“The isolation is the worst part because you go from, well depending on what issue you have, I have gone from what I had which was a very pressurised work environment, kind of working at the top of what I could do in a very busy, I suppose in and I know this sounds stupid, but being quite important in terms of what I did to everything has gone.”Focus Group 1 Participant“I think getting a diagnosis is isolating in itself because you can still have your friends and your family and everyone that you used to have still around you but it’s something that has only happened to you. So things to help people feel less isolated”Focus Group 3 ParticipantSocial prescribing was considered an opportunity to reduce social isolation for people living with LTNCs. Activities provided the opportunity to socialise and connect with others. Attending activities could provide a reason to leave the house and participation in activities provided a sense of purpose. New activities offered the opportunity to learn a new skill and for some this was felt to act as a form of rehabilitation promoting further recovery. Others felt activities provided important mental stimulation. For those participants living with ataxia physical exercise and sport was seen as a valuable activity for supporting mental and physical health as well as providing opportunity to socialise. Participants living brain injury felt participation in activities could aid recovery, whilst others with degenerative conditions focused more on the sense of purpose activities provided.“I do a stitch club because my brain has not been working properly and I have been like well if my brain needs to work out new ways to work let’s do something that your brain has never done before, so I stitch and actually from stitching that got me involved in other bits and bobs so it’s just been super cool”Focus Group 1 Participant“I think volunteering gives you a real sense of purpose. It’s really important. Obviously it’s easier to access for people with neurological conditions, looking for a job might be difficult, but getting in to volunteering can be easier”Focus Group 2 Participant“I don’t want to sit at home and not doing anything, I don’t think anyone wants that”Focus Group 4 ParticipantParticipants made particular reference to the benefits of volunteering and peer support. Opportunities to volunteer were key to providing people with feelings of purpose and could provide an alternative to paid employment. Meeting with people with the same or similar conditions was important for many of the participants. Attendees could choose to talk about their conditions and gain support, alternatively they could talk about anything other than their condition, but knew they were with people who understood.“That’s actually probably the best thing about me being in an environment with people who have got head injuries is that nobody talks to me about it.”Focus Group 1 ParticipantParticipants raised a number of concerns regarding accessing social prescribing activities. The availability of activities and the accessibility of buildings was recognised to be a physical barrier to participation. For those participants who were wheelchair users lack of access to an appropriately adapted toilet was often a barrier to participation, some participants explained that they would either not drink or minimise the length of time they were out. Timing of activities was also discussed in relation to how medications or symptoms of a neurological condition can impact on a person’s ability to attend activities at certain times particularly early in the morning. Fatigue was a symptom experienced by the majority of participants and is recognised as a symptom of many neurological conditions.“Yes, and even the time of day perhaps as well. I know speaking perhaps for XXX is that she is always better, livelier because of medications in the morning, and we’ve spoken to other people and by the afternoon because of the medications they are on they’re weary and need a rest and tend not to go out in the evenings as well for various reasons, so mornings tend to be, certainly for us and others that we know, are better.”Focus Group 1 ParticipantSome participants highlighted the need to consider the activities and the potential need to adapt an activity to enable participation. This was especially pertinent to those who were wheelchair users or had reduced hand function. Risk assessment was also important for those living with epilepsy and having the option to complete activities whilst seated.“I have tonic clonic seizures so I could go from kind of like standing up to automatically being on the floor and therefore I am not able to kind of work in a garden, whereas I could quite happily be watching kind of a film … .or discuss books or that sort of thing, it’s a safer environment.”Focus Group 1 ParticipantDiagnosis with a neurological condition can result in temporary or permanent restrictions on driving. All the groups talked about the impact of being unable to drive and issues relating to accessing public or private hire transport.“I’m sort of the isolated sort of way because I’m unable to drive due to my condition so when my mum who is also sitting next to me, is out then I can’t drive so I’m sort of isolated. I’m just sort of sat there and there is no outreach like kind of groups for me unless I’m able to get taxis to places”Focus Group 1 Participant“You go out and you might have a seizure on the train or on the train platform or on a bus, and people won’t help you or in a taxi and you might get chucked out the taxi just anywhere or the taxi driver’s going to act real funny about it. So you end up sort of thinking ‘oh, you know, it’s just sensible if I stay at home’ … and then you do stay at home and then you never leave … and then you just get comfortable staying in”Focus Group 3 ParticipantAll groups highlighted a number of psychological barriers. Reduced confidence and anxiety was regarded as a barrier to people with LTNCs visiting new places or joining groups and activities. Concerns related to physical symptoms, communication and duration of increasing isolation was discussed. Families’ worries about the person living with a LTNC and needing to manage their concerns was explored as another potential barrier.“I’d love to get out and about a bit more but the fear of going out and the fear of tripping that’s what makes me think about, I know I shouldn’t because I’m over reacting or you may think so but me, I’m not. I panic, I am a panicker.”Focus Group 3 Participant“I think one of the biggest issues is the actual carer or family that are trying to maybe cocoon the person with the neurological condition and making assumptions like ‘well I don’t think they can do that’ because they are trying to be overprotective. So I think if the link worker tried to work with the family to ensure the family are and the carers are comfortable because once they feel that comfort then they will work with the link worker”Focus Group 3 ParticipantStigma and lack of acceptance by others was seen as a significant barrier for people living with LTNCs. The general public’s awareness of LTNCs was felt to be poor in particular their understanding of hidden or invisible disabilities. Those participants living with epilepsy felt many groups immediately put up barriers when hearing their diagnosis and were worried something might happen to trigger a seizure and the consequences of this for the group. Some participants felt their mental capacity was questioned when they revealed their diagnosis and people would change their behaviour towards them often in a negative way. There appeared to be a strong desire to be accepted and treated like everyone else.“Well the main worry is if you haven’t gone so long without a seizure and you’re frightened of people’s reactions... Cause I’ve been stopped by the police for being drunk and I don’t even drink … because they don’t understand the condition, you know there’s people with different disabilities they don’t understand. Because I always say role reversal, you’ve got to have been through it to understand it”Focus Group 3 Participant“I think with anybody who has had a neurological condition physically you look fine, it is what’s going on in the inside and people don’t, don’t know and that is due to lack of knowledge, lack of education.”Focus Group 4 Participant“A lot of people are like ‘oh you can’t do this and you can’t do that’ and you know, you can’t do these things and you’ve got to take these meds and you can’t stay out late at night and you can’t do all these myriad of things where really I probably can or could if I had some support.”Focus Group 3 ParticipantThese are amongst the first findings to explore the potential benefits and perceived barriers to accessing social prescribing for people living with LTNCs. Participants highlighted that there was very little knowledge of social prescribing amongst people living with LTNCs. Those who had accessed social prescribing activities had done so by chance and as a result of their own proactiveness. The lack of signposting by health professionals was apparent and there was an agreed need for health professionals to be educated about social prescribing. Bickerdike et al. (2017) reported that social prescribing was unfamiliar to many GPs and in order to engage participants they required a good clear explanation . This seThe benefits of participation in activities was recognised by all the focus groups. Participation was seen to provide meaning and purpose. A strong emphasis was placed on the social benefits of participation. Similar findings have been demonstrated in social prescribing studies. An evaluation of a social prescribing service in Northern England found that accessing a range of activities provided a sense of independence and gave individuals a sense of purpose . A recenStigma was found to impact on social identity leading to loss of confidence and anxiety. Research into stigma particularly the impact of living with epilepsy has shown that stigma can result in a perceived reduction in social value and poor quality of life . InterveSupport to return to work or to find volunteering opportunities was a desirable area for link worker support for many of the participants. People with LTNCs who fail to return to work after injury or onset, or who are encouraged to relinquish work prematurely may be financially disadvantaged, have a poorer quality of life and suffer adverse health outcomes such as anxiety and depression –63. For Participants discussed the physical barriers to accessing social prescribing activities. Transport was seen as a significant barrier to participation. Many studies have highlighted that transportation is often a barrier to participation in community based activities , 68–70. Participants emphasised the need for knowledgeable link workers and services given the complexity of LTNCs, the challenges faced by many living with a neurological condition and the importance of individuality. A recent systematic review  examinedThe provision of group and individual programmes that promote health, social interaction and self-efficacy have been at the forefront of occupational therapy intervention historically . AccordiOccupational therapists should be recruited to work alongside link workers to reach those living with LTNCs in order to tackle many of the barriers highlighted in this study. With the push to introduce New Roles in primary care networks this could be achieved through direct referral to occupational therapists based within primary care who would then work collaboratively with a link worker . This moThe National Institute for Health and Care Excellence (NICE) public health guideline ‘Mental Wellbeing in over 65’s’  focuses This study was subject to some limitations. It is recognised that the study included a small number of plwLTNC and participants and may not be representative of all LTNCs. As a result of convenience sampling, our study included people with multiple sclerosis, Fragile X Syndrome, epilepsy, traumatic brain injury, essential tremor, ataxia and subarachnoid haemorrhage. The family carers supported people living with epilepsy and motor neurone disease. The investigators took the decision to keep demographic data to a minimum in order to help participants to feel their responses would be completely anonymised especially given the small sample size. This article will be accessible to all and that will include the charity where the study took place. Details regarding age, ethnicity and locality were not collected and quotes do not indicate who is responding or their diagnosis as this would identify who the respondent was especially single representatives of a condition. However, this is one of the very first studies exploring social prescribing with plwLTNCs, indicating steps for future research to build on, such as expanding the participant pool to incorporate a greater variation of LTNCs. The majority of participants were regular attendees at the charity where the focus groups were run and this may have influenced their responses leading to potential response bias. This might have included avoiding making critical comments about the activities run by the charity or adapting responses due to being familiar with other members of the focus group. The decision to have mixed groups of family carers and plwLTNCs is acknowledged to be another limitation of the study as the presence of each might have resulted in people holding back to avoid offending or upsetting another participant due to their status. The study may not represent those plwLTNC who experience significant difficulty leaving the house and are in greater need of social prescribing than those people represented in this study. Future research should aim to reach out to those who are unable to leave their homes to establish if they experience the same or different barriers to social prescribing activities. Research should also examine the impact the pandemic has had on the wellbeing of plwLTNCS. This should include potential positive changes such as online groups and activities which aimed to enable social interaction and inclusion during the pandemic , but alsThis study emphasises the need to consider the whole system and how social prescribing can be framed to meet the needs of plwLTNC. Future research should explore how best to enable participation by overcoming physical and psychological barriers and by identifying interventions that reduce the impact of psychological barriers such as stigma. A particular focus needs to be set on the impact of the COVID-19 pandemic on engaging with social prescribing, as this is likely to have thrown up further barriers in accessing general social support, as emerging evidence highlights. The pandemic has also provided positive learning opportunities and the potential to utilise technology in the delivery of social prescribing going forward. Local infrastructure needs to evolve in order to reduce the physical barriers faced by plwLTNC in particular transport. Without major changes, plwLTNC will endure further inequalities relating to the delivery and accessibility of social prescribing. This paper has made recommendations to consider the role occupational therapists could take in the development and delivery of social prescribing given their expertise.Additional file 1. Focus group topic guide.


Volumetric muscle loss (VML) is associated with irreversibly impaired muscle function due to traumatic injury. Experimental approaches to treat VML include the delivery of basic fibroblast growth factor (bFGF) or rehabilitative exercise. The objective of this study was to compare the effects of spatially nanopatterned collagen scaffold implants with either bFGF delivery or in conjunction with voluntary exercise. Aligned nanofibrillar collagen scaffold bundles were adsorbed with bFGF, and the bioactivity of bFGF-laden scaffolds was examined by skeletal myoblast or endothelial cell proliferation. The therapeutic efficacy of scaffold implants with either bFGF release or exercise was examined in a murine VML model. Our results show an initial burst release of bFGF from the scaffolds, followed by a slower release over 21 days. The released bFGF induced myoblast and endothelial cell proliferation in vitro. After 3 weeks of implantation in a mouse VML model, twitch force generation was significantly higher in mice treated with bFGF-laden scaffolds compared to bFGF-laden scaffolds with exercise. However, myofiber density was not significantly improved with bFGF scaffolds or voluntary exercise. In contrast, the scaffold implant with exercise induced more re-innervation than all other groups. These results highlight the differential effects of bFGF and exercise on muscle regeneration. Volumetric muscle loss (VML) is characterized by irreversible damage in skeletal muscle structure and function due to the loss of a significant portion of skeletal muscle. Traumatic injuries leading to VML are associated with impaired endogenous muscle regeneration, long-term disability, and cosmetic deformities ,2. CurreSkeletal muscle is generally composed of bundles of parallel-aligned myofibers, interspersed with blood vessels within close proximity  that proThe promise of regenerative medicine is the full regeneration of damaged tissues, either by promoting repair from endogenous stem cells or by the transplantation of cells to enhance regeneration . To dateRecent studies suggest that rehabilitative exercise may be beneficial for the treatment of VML ,25. In pSince rehabilitative exercise and bFGF elaboration are two potential strategies for the treatment of VML, the objective of this study was to compare the effects of implanted spatially nanopatterned bFGF-releasing scaffolds to that of implanted scaffolds in conjunction with voluntary caged wheel exercise. By evaluating muscle physiology and tissue histology in a mouse model of VML, we show that bFGF and voluntary exercise have differential therapeutic benefits for the treatment of VML.−1 in 0.02 N in acetic acid (pH 3.5) and then extruded from a 22 G blunt tip needle onto glass slides that were submerged in phosphate-buffered saline  at 37 °C, leading fibrillogenesis along the direction of extrusion. To fabricate parallel-aligned 3D scaffold bundles, 16 scaffolds strips were intrinsically sticky and adhered in parallel to one another before immobilizing the scaffold bundle onto surface-reactive Nexterion H  slides. The scaffolds were washed with 1× PBS, dried overnight in a laminar flow hood, and then disinfected with 70% ethanol.Aligned nanofibrillar collagen scaffolds were fabricated as described in our previous work ,16,28. I−1, Peprotech, Cranbury, NJ, USA) overnight at 37 °C and 5% CO2. Control scaffolds were incubated in 0.1% bovine serum albumin . After overnight incubation, scaffold bundles were removed from the Nexterion H Slides and then trimmed to 4 mm × 3 mm × 2 mm scaffolds for implantation or in vitro studies. The surface topography of the scaffolds was assessed by routine scanning electron microscopy, based on previous methods [In order to prepare growth factor-laden scaffolds, the scaffolds were submerged within a solution of recombinant human bFGF .To determine the kinetics of bFGF release from the scaffolds over time, the bFGF-laden scaffolds or control scaffolds were incubated in 0.1% BSA. At time points over 21 days, the supernatant was sampled and then replaced with fresh BSA. The release of bFGF was quantified by an enzyme-linked immunosorbent assay (ELISA) using a Human bFGF Quantikine ELISA Kit  according to the manufacturer’s instructions. Readings were taken at 10 min following the addition of the stop solution at a wavelength of 450 nm with correction at 540 nm . The Ki67 + cells were expressed as a percentage of the total cells (n = 3).To demonstrate the bioactivity of bFGF-releasing scaffolds, the mitogenic effect of bFGF was quantified by the proliferation of mouse myoblasts  and human endothelial cells . Each cell type was cultured on bFGF-laden nanofibrillar scaffolds or control nanofibrillar scaffolds in growth media . After 4 days, the cells were fixed in 4% paraformaldehyde for immunofluorescence staining of the S-phase cell cycle marker, Ki67. Immunofluorescence staining was carried out based on previously published methods ,30,31. IC57BL/6 mice  underwent a bilateral induction of VML. The animals were anesthetized with isoflurane (2–3%) at an oxygen flow rate of 1 L/min. A longitudinal incision was made in the skin directly above the tibialis anterior (TA) to expose the muscle. The TA was dissected from the extensor digitorum longus (EDL). A spatula was placed in between the EDL and TA muscles, and then a 2-mm diameter biopsy punch was applied to produce two adjacent holes in the central region of the TA, creating a 4 mm × 4 mm × 2 mm full-thickness defect that was approximately 20% of the weight of the TA. Immediately after tissue ablation, scaffold bundles that contained either bFGF or vehicle control (BSA) were placed into the defect, and the sides of the ablation were approximated using an 8–0 suture (S&T). The skin was then closed with an 8–0 nylon suture. Animals were placed in a cage on top of a warming pad to recover. The animals received sustained-release buprenorphine  for analgesia along with antibiotics .After induction of VML, the mice were allowed to rest for 7 days before further randomizing to receive non-exercise or exercise regimens, leading to four treatment groups with the following denotations: (1) scaffold (control), (2) bFGF-laden scaffold (bFGF scaffold), (3) scaffold with voluntary caged wheel exercise; or (4) bFGF-laden scaffold with caged wheel exercise. The mice in exercise groups were placed into running wheel cages  from days 7 through 21 post-surgery for voluntary exercise. To track running habits, each caged wheel was attached to an electronic counter that recorded the time and distance traveled every 15 s , as was conducted previously ,28. The n = 6). The running distances of the mice were recorded for 2 weeks to evaluate the post-injury timepoint in which running habits reach steady-state levels.To identify an optimal time after induction of VML to administer rehabilitative exercise, a separate animal study was performed in which C57BL/6 mice were allowed to run on caged wheels starting from the day of VML induction (n = 7–8).On day 21, after induction of VML, the mice underwent TA force measurement testing. An incision was made longitudinally in the skin of the lower leg to expose the TA. The distal tendon of the TA was severed, and the muscle was partially dissected so that the surrounding muscles did not contribute to the measurements. The dissected end of the tendon was attached to a 5–0 suture and secured to the transducer . A needle was placed through the leg above the patella to secure the knee. Two electrodes were placed touching but not penetrating the TA to stimulate the muscle directly for isometric contraction. The Instantstim function from Dynamic Muscle Control (DMC) LabBook software  was used to stimulate twitch contractions in the TA muscle using a pulse of 0.2 ms and a train frequency of 0.5 Hz. While the muscle was being stimulated at 2 s intervals, the current was slowly increased until the TA achieved maximal stimulation. After the current was optimized, the length of the muscle was slowly increased until the optimal resting length was obtained. A single twitch force was then performed with a pulse width of 0.2 ms per leg for analysis (The TA muscles were explanted for routine cryosectioning and hematoxylin and eosin (H&E) staining. Immunofluorescence staining of tissue samples was carried out by fixation in 4% paraformaldehyde, followed by permeabilization in 0.5% Triton-X-100 ,33. For 2, n = 5). For the quantification of neuromuscular junction formation, the total number of α-bungarotoxin-expressing junctions were quantified within a 500-µm radial distance from the scaffold and expressed as neuromuscular junction density .To quantify muscle regeneration at the periphery of the scaffold implants, the total number of laminin + myofibers with centrally located nuclei within 500 µm radial distance from the implanted scaffold’s periphery was quantified. The data were expressed as regenerating muscle density  with post hoc Tukey’s adjustment  was performed. Data are shown as a mean ± standard deviation (SD). Significance was accepted at p < 0.05 (*). All graphs were created in Microsoft Excel. Sample size reflects per operated leg unless otherwise noted.Statistical analysis was performed using an unpaired Aligned nanofibrillar scaffolds were fabricated by a facile shear-based extrusion technique, in which monomeric collagen was extruded from a syringe needle into a pH neutral saline, leading to rapid fibrillogenesis. The resultant collagen scaffolds were long and thin, with a scaffold width of approximately 300 µm. To create a scaffold bundle, 16 strips were immobilized adjacent to one another onto a glass substrate A. Owing p < 0.05). Additionally, skeletal myoblasts showed nearly a five-fold increase in proliferation on bFGF-laden scaffolds, compared to scaffolds without bFGF (p < 0.05). These studies demonstrated that the bFGF released from the scaffolds were bioactive in inducing cellular proliferation.To confirm the bioactivity of bFGF upon release from the scaffolds, we performed in vitro experiments to characterize the mitogenic response of skeletal muscle myoblasts or endothelial cells to bFGF stimulation. Skeletal myoblasts or endothelial cells were seeded onto the bFGF-laden scaffolds for 4 days before fixing the samples for immunofluorescence staining of the S-phase cell cycle marker, Ki67 A. QuantiUpon validating the bioactivity of the bFGF-laden scaffolds in vitro, we next tested the therapeutic efficacy of bFGF-laden scaffolds in a murine model of VML. The bFGF-laden scaffolds were manually detached from the glass substrates to form a delaminated bundle of 16 scaffolds. The mice underwent bilateral full-thickness ablation of the TA muscle, followed by acute implantation of the bFGF-laden scaffold or the control scaffold into the site of muscle ablation A,B. The The selection of a 7-day delay for voluntary caged wheel running was based on a study in which animals were allowed to undergo caged wheel running immediately after VML induction. Based on their daily running habits, we observed an unusually high degree of running distance on day 1, presumably due to the effect of post-surgery analgesics that masked the pain associated with running . Startinp < 0.05). To assess for differences in running habits of animals with implants of the control scaffold or bFGF-laden scaffolds, the daily running distance was recorded for 21 days after induction of VML. The voluntary running habits showed a cumulative increase in running distance over the course of 21 days for animals treated with control scaffolds or bFGF-laden scaffolds, but no significant differences between these two exercise groups  was significantly higher than in animals treated with the combination of the bFGF-laden scaffold with exercise (69 ± 52 mN), whereas exercise alone appeared to not have led to significant improvements in twitch force  staining. From the H&E staining, the partially degraded collagen scaffold could be observed within the ablated muscle region, based on its characteristic ribbon-like fibrillar appearance . Regardl2), compared to the scaffold treatment group . Interestingly, the combined treatment of bFGF-laden scaffold with exercise did not show any significant difference compared to the control group  bFGF and voluntary caged wheel exercise have differential effects on the treatment of VML; and (2) treatment of scaffolds releasing bFGF significantly improved muscle physiology after 3 weeks, whereas caged wheel exercise promoted neuromuscular junction formation. VML is a traumatic injury in which improved treatment strategies are in high demand. The delivery of pro-regenerative factors such as bFGF is a promising off-the-shelf therapy that can be applied in even austere settings. Since bFGF has been demonstrated to be safe in clinical trials for other indications such as wound healing and peripheral arterial disease ,35, it iTo account for the large volume of muscle defect, an aligned nanofibrillar scaffold was implanted to the scaffold for the spatially controlled release of bFGF as well as for promoting cellular infiltration into or near the scaffold. Collagen was selected because of the ease of fabricating aligned nanofibrillar scaffold bundles using a highly facile extrusion process ,20,29. NIn recognition that rehabilitative strategies can augment the benefits of tissue regeneration, we compared the effect of voluntary caged wheel running to that of localized bFGF release from nanofibrillar collagen scaffold. Similar to our previous findings using a partial thickness TA muscle ablation model, voluntary caged wheel running stimulation and the implantation of aligned nanofibrillar scaffolds promoted neuromuscular junction formation . Our finOur results also show that the interaction between bFGF and voluntary caged wheel exercise is complex. For example, the implantation of bFGF-releasing scaffolds significantly increased twitch force generation A compareAlthough bFGF-releasing scaffolds could significantly improve muscle physiology, histological analysis of regenerating myofiber density did not reveal a statistically significant increase in regenerating myofiber density. Since the twitch force measurements quantitatively reflect the global function of the entire muscle, whereas histological methods semi-quantitatively reflect muscle regeneration in smaller regions of interest, it is plausible that the findings between both assays might contrast. However, the global nature of muscle force quantification possibly suggests a more reliable metric than tissue histological quantification.In summary, we demonstrated that bFGF-laden aligned nanofibrillar collagen scaffolds for the treatment of VML in full-thickness muscle ablation model led to significantly improved muscle physiology, whereas collagen scaffold implants with voluntary caged wheel exercise promoted greater neuromuscular junction formation. Intriguingly, the combination of bFGF-laden scaffolds with rehabilitative exercise showed no therapeutic effect on the treatment of VML. Further studies to explore the optimal timing, dosing, and duration of exercise and bFGF treatments for therapeutic efficacy are warranted. This study highlights the differential therapeutic benefits of these two therapies separately, but not when prescribed in combination.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) emerged in China in December 2019. Healthcare workers (HCWs) are one of the high-risk groups of infection and knowledge of the seroprevalence of SARS-CoV-2 antibodies among this class is very important, not only to understand the spread of COVID-19 among health institutions but also to assess the success of public health interventions. The objective of this prospective study was to determine the seroprevalence of COVID-19 immunoglobulin G (IgG) antibodies after vaccine administration and assess the symptomatology associated with the number of IgG antibodies. A total of 75 HCWs from an intensive care unit were studied three and six months after the second administration of the COVID-19 vaccine. They were divided into three groups: IgG antibodies between 4,160 and 6,350 (group one), greater than 6,350 (group two), and less than 4,160 (group three). After the first administration of the vaccine, 80% had symptoms in both groups one and two, whereas only 13.8% had symptoms in group three. After the second dose of the vaccine, all elements of group one and 80% of group two developed symptoms, but only 40% of group three manifested symptoms. With the exception of one, all professionals showed a decrease in the number of IgG antibodies from three to six months. Our findings show that professionals with a higher number of IgG antibodies had more symptoms and that these rapidly declined over the three-to-six-month period. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swept the globe at a shocking rate, with a reported 265,970,247 cases and 5,267,137 deaths [HCWs were informed about the conduction of the COVID-19 antibody serological study and approval was acquired from the hospital's ethics committee  to analyze the prevalence of SARS-CoV-2 antibodies among HCWs of the ICU after Pfizer-BioNTech vaccine administration. The HCWs included doctors (n = 5), nursing staff (n = 57), operational assistants (n = 11), and administrative staff (n = 2).The study population was divided into three groups: immunoglobulin G (IgG) antibodies between 4,160 and 6,350 (group one), greater than 6,350 (group two), and less than 4,160 (group three). Clinical and demographic data such as sex, age, symptoms after the first and second administration of the vaccine, contact with positive patients, comorbidities, and the difference in antibodies between three and six months were analyzed.The SARS-CoV-2 IgG II Quant assay is a chemiluminescent microparticle immunoassay used for the qualitative and quantitative determination of IgG antibodies to the receptor binding domain of the S1 subunit of the spike protein of SARS-CoV-2 in human serum and plasma. If the IgG antibodies were between 4,160 and 6,350, there was a 95% probability of presence of neutralizing antibodies, and if they were superior to 6,350, there was a 99% probability.Data analysis was performed using the chi-square test for categorical variables and the one-way analysis of variance (ANOVA) test to compare means between groups. The Wilcoxon test was used to analyze differences between antibodies. Data analysis was performed using SPSS for Windows version 25.0 .A total of 75 HCWs were included in this study. Group one had an average age of 33 years and all were female. As for group two, the average age was 40 years, where 60% were female and 40% were male. Group three had an average age of 39 years and 61.5% were female and 38.5% were male. In group one, most HCWs did not have comorbidities (60%); however, in groups two and three, most had associated pathologies . The most frequent were hypertension, hypothyroidism, and asthma.There was a prevalence of 6.66% of SARS-CoV-2 antibodies in groups one and two and 86.7% in group three. After the first administration of the vaccine, 80% of HCWs had symptoms in both groups one and two and only 13.8% had symptoms in group three. After the second dose of the vaccine, all elements of group one and 80% of group two had symptomatology, but only 40% of group three developed symptoms.Table Symptoms included fever, asthenia, myalgia, arthralgia, cephalgia, vomiting, diarrhea, and dizziness. The frequency of symptoms in the three groups after the first dose of the COVID-19 vaccine is shown in Table After the second administration of the vaccine, the frequency of symptoms was higher. The frequency of symptoms in the three groups after the second dose of the COVID-19 vaccine is depicted in Table Most professionals in group two had regular contact with infected patients (80%) as well as group three (63.1%); however, the majority of group one had no contact with these patients (60%).At three months, mean and median IgG antibodies were 2668.6 and 2373.1, respectively, where the minimum was 3.4 and the maximum was 8336.8. At six months, we calculated a mean of 1373.5 and a median of 1256.9 IgG antibodies, where the minimum was 4.6 and the maximum was 5192.7.The mean and median of IgG antibodies at three and six months after the second dose of the COVID-19 vaccine are depicted in Figure With the exception of one, all HCWs showed a decrease in the number of antibodies in the three-to-six-month period after the administration of the second dose.Humoral and cell-mediated response against SARS-CoV-2 is one of the major aspects for understanding viral clearance and vaccination effectiveness. One study confirmed that HCWs reach adequate humoral and cellular response after a single dose of an mRNA vaccine . Since SIn the present prospective study, we found a substantial decline in IgG levels in the three-to-six-month period after the second dose of the COVID-19 vaccine, except in one HCW. Our findings are similar to those of Nag et al. . Mishra Studies observed that spike IgG concentrations are durable, with modest declines after six to eight months. Kinetics of humoral response over a longer time period should be evaluated to obtain information on the long-term immunity in vaccinated subjects .Vaccination has been shown to provide significantly better protection against severe diseases. Boosting with vaccines is an effective strategy to combat the waning of immunity and the current variants of concern .In group two, we found HCWs who presented with the highest number of IgG antibodies . This group was the one with a higher average age and female ratio, with a significant percentage of comorbidities and symptomatology after the first and second administration of the COVID-19 vaccine and all had consistent contact with infected patients. In group one, we found the professionals with antibodies between 4,160 and 6,350. They were all female, younger, mostly with no comorbidities, and had no contact with COVID-19 patients. Like group two, they predominantly had symptoms after the first and second vaccine administration. Group three comprised HCWs with the lowest number of antibodies, inferior to 4,160. This group was mostly female with associated previous pathologies and with contact with infected patients. However, they had fewer symptoms after vaccine administration.Limitations of our clinical study include the small sample size.Our study concluded that professionals with a higher number of antibodies presented with more symptoms after vaccine administration. It also came to light that in the three-to-six-month period after vaccine administration, there was a significant decrease in the number of antibodies.However, it is important to note that this is an area of investigation where data are still quite scarce and further studies are needed on this subject, especially about the third dose of the vaccine.