Blood nerve barrier permeability enables nerve targeting of circulating nanoparticles in experimental autoimmune neuritis

Blood nerve barrier permeability enables nerve targeting of circulating nanoparticles in experimental autoimmune neuritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Blood nerve barrier permeability enables nerve targeting of circulating nanoparticles in experimental autoimmune neuritis Kelly Langert, Chanpreet Kaur, Ellaina Villarreal, Maleen Cabe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4631228/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Guillain-Barré syndrome (GBS) is a devastating autoimmune disease of the peripheral nervous system (PNS) for which treatment options are strictly palliative. Several studies have shown attenuation of the well-characterized preclinical experimental autoimmune neuritis (EAN) model with systemically administered therapeutic compounds via a range of anti-inflammatory or immunomodulatory mechanisms. Despite this, clinical advancement of these findings is limited by dosing that is not translatable to humans or is associated with off-target and toxic effects. This is due, in part, to the blood-nerve barrier (BNB), which restricts access of the circulation to peripheral nerves. Here, we assessed the degree to which BNB permeability and immune cell infiltration over the course of EAN enable passive accumulation of circulating nanoparticles. We found that at stages of EAN defined by distinct clinical scores and pathology (onset, intermediate, peak), intravenously administered small molecules and nanoparticles ranging from 50–150 nm can permeate into the endoneurium from the endoneurial vasculature in a size- and disease stage-dependent manner. This permeation occurs uniformly in both sciatic nerves and in proximal and distal regions of the nerves. We propose that this passive targeting serves as a platform by which potential therapies for GBS can be reevaluated and investigated preclinically in nanoparticle delivery systems. Biomedical Engineering Neurobiology of Disease Peripheral nerve nanoparticle blood-nerve barrier experimental autoimmune neuritis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Guillain-Barre syndrome (GBS) is a devastating autoimmune disease of the peripheral nervous system (PNS) for which there is no targeted therapy [ 1 , 2 ]. Experimental autoimmune neuritis (EAN) is a well-established preclinical model of the acute inflammatory demyelinating polyneuropathy (AIDP) subtype of GBS that has advanced our understanding of mechanism by which pathology develops and progresses [ 3 – 5 ]. Several previous studies have shown attenuation of EAN with systemic administration of potential therapeutic compounds that span a range of anti-inflammatory or immunomodulatory strategies [ 6 – 11 ]. While shedding light on the mechanisms of disease, clinical advancement of these findings is limited by dosing that is not translatable to humans or is associated with off-target and toxic effects. The blood nerve barrier (BNB) represents a key obstacle to delivering therapeutics to peripheral nerves [ 12 ]. This barrier consists of layers of connective tissue as well as specialized microvascular endothelial cells linked by tight junctions, together creating an isolated endoneurial microenvironment. Several studies have investigated the role of the BNB in restricting access of circulating molecules to the endoneurium under physiological conditions [ 13 ]. During acute neuroinflammation, such as that associated with AIDP, the normally restrictive BNB exhibits increased vascular permeability and enables immune cell infiltration [ 14 , 15 ]. While these pathological changes contribute to disease, they may also offer a unique window of opportunity to access the otherwise restricted peripheral nerve microenvironment for therapeutic delivery. It is established that fenestrated vasculature, like those featured in sites of localized inflammation, can promote passive accumulation of circulating nanoparticles (NPs) with favorable size and surface properties [ 16 – 18 ]. This phenomenon is dependent on prolonged circulation and avoidance of clearance, which are best achieved by NPs in the range of 50–200 nm [ 19 ]. Despite promising preclinical results for enhanced permeation and retention (EPR) to promote accumulation of NPs in tumors [ 20 ], successful translation of EPR for chemotherapeutic delivery has been low [ 21 , 22 ]. Established contributing factors include properties and kinetics of tumor development in preclinical models poorly reflect human pathology and tumor microenvironments contain both avascular regions and tortuous vessels, resulting in heterogenous NP distribution [ 21 , 22 ]. Given the established validity of the EAN model and linearity of peripheral nerve vasculature [ 15 , 23 ], the potential for EPR to facilitate delivery to peripheral nerves in AIDP is an exciting area for preclinical investigation [ 18 ]. In this study, we assessed whether the BNB permeability and immune cell infiltration over the course of EAN enable passive accumulation of circulating nanoparticles. We identified stages of EAN, onset, intermediate, and peak, defined by distinct clinical scores and pathology. We found that intravenously administered small molecules (69 kDa) and NPs ranging from 50–150 nm can permeate into the endoneurium from the endoneurial vasculature in a size- and disease stage-dependent manner. We propose that this passive targeting serves as an opportunity by which potential therapies for GBS can be reevaluated and investigated preclinically in NP delivery systems. Materials and Methods Materials Carboxylated modified Fluospheres™, specifically 40 nm red (F8793, λ ex /λ em = 580/605) and 100 nm yellow-green (F8803, λ ex /λ em = 505/515), 20x borate buffer, glycine, and ICAM-1 antibody (MA5407) were purchased from Thermofisher Scientific (Waltham, MA, USA). Evans blue dye (E2129), N-(3-Dimethylaminopropyl)-N’- ethyl carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), Amicon Ultra centrifugal filters (10 and 100 kDa), and incomplete Freund's adjuvant were purchased from Sigma-Aldrich (St Louis, MO, USA). Methoxy poly(ethylene) glycol (mPEG)-amine (2 kDa) was obtained from Creative PEGWorks (Chapel Hill, NC). Phosphate buffered saline (10x PBS) and mouse anti rat CD68 primary antibody was obtained from Bio-Rad (Hercules, CA, USA). P2 peptide was obtained from Peptide Synthesis Core Facility of the Simpson Querrey Institute, Northwestern University. Heat-inactivated Mycobacteria tuberculosis (strain H37Ra) was purchased from Becton, Dickinson, and Company (Sparks, MD 21152, USA). Nanoparticle PEG conjugation (PEGylation) Carboxylated FluoSpheres ™ were PEGylated with mPEG-amine using carbodiimide chemistry and previously published methods (17). First, 500 µL of FluoSpheres (2% (yellow green) and 5% (red) suspension from the manufacturer) were washed once using Amicon centrifugal filters (10 kDa MWCO) at 14,000 x g for 15 min. Next, mPEG-amine was added at 5x molar excess followed by NHS (6.5 mg), EDC (15.4 mg) and borate buffer (6 ml, pH 8) and allowed to stir for 3 hours. The reaction was quenched with glycine (75 mg, 100mM) for 30 min. PEGylated FluoSpheres, herein referred to as NPs, were washed and collected using Amicon centrifugal filters (100 kDa MWCO) and resuspended to original concentration in filtered 1x PBS for storage at 4°C. NP characterization Hydrodynamic diameter and zeta potential of NPs were quantitatively assessed with dynamic light scattering using Zetasizer Nano ZS90 (Malvern Panalytical). A suspension of NPs (0.1 mg/ml) was prepared in deionized water and transferred to a disposable polystyrene cuvette or a disposable capillary cell. Suspension was equilibrated for 3 minutes in the cuvette and measured at 90° angle. Each sample was measured 4 times. EAN induction Experiments were conducted using protocols approved by the Edward Hines, Jr., VA Hospital Institutional Animal Care and Use Committee in accordance with the principles of laboratory animal care. Adolescent male Lewis rats were housed in pairs, allowed free access to standard rat chow and water, and maintained on a 10h/14h light/dark cycle. Naïve rats (Envigo, Indianapolis, IN) were induced with EAN using P2 peptide as we have previously described [ 24 ]. Briefly, anesthetized rats (ketamine (100 mg/kg)-xylazine (5 mg/kg)) were injected with a freshly prepared emulsion containing (1:1 v/v) purified P2 myelin neuritogenic peptide (residues 53–78, 100 µg) suspended in sterile saline and Freund’s adjuvant supplemented with a final concentration of 5 mg/ml heat-inactivated Mycobacterium tuberculosis (strain H37RA) into one hind footpad. Control rats received a footpad injection of sterile saline. Weights and clinical progression of disease were assessed daily beginning on Day 7. Rats receive a score of 1 (loss of tail tone), 2 (disrupted gait), 3 (mild hemiparesis), 4 (severe paraparesis), or 5 (paraplegia), including increments of 0.5 [ 24 , 25 ]. Intravenous injection of tracers On the designated day, ketamine-anesthetized control and EAN rats received a single intravenous (tail vein) infusion of Evans blue dye (2% w/v suspended in PBS) or a cocktail of NPs (8 mg total/rat, suspended in diH 2 O). Time points for administration of Evans blue or NPs included EAN onset (Day 11), intermediate EAN (Day 13) and peak EAN (Day 15). Tissue collection Thirty minutes after infusion of tracers, rats were euthanized, and sciatic nerves were collected. Nerves were fixed for 4 hours in 4% paraformaldehyde followed by incubation in 30% sucrose/PBS overnight at 4°C. The next day, nerves were incubated in 50% optimal cutting temperature (OCT) medium/50% sucrose/PBS for 2 hours followed by embedding in OCT and snap freezing in liquid nitrogen. Embedded tissue was stored at -80°C until further analysis. Immunohistochemistry and Evans blue accumulation Serial 10µm cross and longitudinal sections were prepared from nerves using a Leica CM 1850 Cryostat, and the content and distribution of CD68 + immune infiltrates was determined by immunohistochemistry as we have previously described [ 24 ]. Washed sections were blocked for 30 min at room temperature in PBS containing 5% (v/v) normal goat serum and 0.05% (v/v) Triton X-100 (blocking buffer). Cross sections were incubated with a 1:250 dilution of mouse anti-CD68 (macrophage marker, clone ED1, purified IgG primary antibody) followed by a 1:1000 dilution of Alexa Fluor 488 anti-mouse secondary antibody and coverslipped with prolong diamond antifade mountant containing DAPI. Infiltrating CD68 + macrophages were visualized on a Nuhsbaum inverted confocal microscope and semi-quantified in a blinded manner with ImageJ software. Longitudinal sections were incubated with a 1:100 dilution of mouse anti-CD54 (ICAM-1, clone W3/25, purified IgG) primary antibody followed by 1:1000 dilution of Alexa Fluor 488 anti-mouse secondary antibody and similarly coverslipped. CD54 + endothelial cells were visualized as above, and ICAM-1 expression was qualitatively assessed. Evans blue accumulation was assessed in the same sections at 10x magnification at laser setting of 561 nm (photomultiplier tube (PMT) = 610 nm − 677 nm, 27.754% laser intensity). Z-stacked images of nerves were obtained and converted to one image using maximum projection on Leica LAS-X software. Five random regions of interest (ROI) were chosen in the cross-section images (8 nerve sections per animal, 4 animals per cohort) and fluorescence intensity was quantified using ImageJ software. NP accumulation Serial 10µm cross sections were prepared from nerves using a Leica CM 1850 Cryostat and mounted onto Superfrost Plus microscope slides. Slides were incubated at room temperature for 30 min, washed once with PBS, and air dried. Coverslips were gently placed using Prolong Diamond Antifade mountant (Thermofisher Scientific). NP permeation was visualized using confocal microscopy. Images were captured at laser settings of 405nm, 488nm, 561nm (PMT: 432–482 nm, 522–585 nm, and 610–677 nm) and laser intensity (17.07%, 21.35%, 27.74% respectively) was standardized for all nerve sections. Z-stacked images (40x magnification) of nerves were obtained and converted to one image using maximum projection on Leica LAS-X software. Standardized areas of ROI around endoneurial blood vessels were chosen in the cross-section images (up to 10 nerve sections per animal, 4–5 animals per cohort) and fluorescence intensity was quantified using ImageJ software. Statistics All statistical analyses were conducted using GraphPad Prism 10.2.1 (397). Data are expressed as the mean ± SEM of N observations, and p < 0.05 was considered statistically significant. Statistical significance across multiple experimental groups (EAN score and weight, leukocytic infiltrates, Evans blue permeation, NP accumulation comparison at onset, intermediate, and peak of EAN) was conducted using one-way ANOVA followed by Fishers LSD and Tukey’s post-hoc test. Statistical significance of paired groups was conducted using multiple unpaired t-tests. Statistical significance and R value of clinical score and NP accumulation was conducted using simple linear regression correlation analysis. Results Experimental autoimmune neuritis clinical and pathological presentation. We and others have previously shown that EAN induced with complete Freund’s adjuvant (CFA) and P2 peptide manifests as a robust, monophasic course of ascending weakness [ 24 , 26 ]. Here, the observed disease course develops and progresses similarly, with onset on day 11 post induction presenting as loss of tail tone and the peak of disease on day 15 post induction ( Fig. 1 a ) . Rats lose weight over the course of EAN, with a significant reduction compared to their peak weight beginning on Day 13 ( Fig. 1 b ) . In this study, we focus on three distinct stages of disease: EAN onset (Day 11), intermediate EAN (Day 13), and peak EAN (Day 15). The observed clinical scores ( Fig. 1 a ) at each of these stages are distinct and significantly different from baseline and from each other (p < 0.05). It is well established that immune cells, including CD68 + macrophages, infiltrate nerves at the peak of EAN [ 5 , 11 , 24 ]. Here, to better understand the ability of immune cells to gain access to the endoneurium at different stages of disease, we quantified CD68 + macrophages in cross sections of sciatic nerves collected from animals at onset, intermediate, and peak EAN. Nerves from naïve animals contain a limited number of macrophages (7.9 ± 4.0 cells per mm 2 , Fig. 2 a ) . At intermediate EAN, nerves contain a significant increase in macrophages (337.8 ± 93.4 cells per mm 2 ). This number doubles over the next two days (p = 0.036), and nerves collected at EAN peak contain 678 ± 94.49 CD68 + cells per mm 2 . These quantitative findings are depicted by representative cross sections shown in Fig. 2 b. In addition to the immune cells that migrate into nerves during the course of EAN, the endothelial cells that form the blood nerve barrier (BNB) exhibit increased cell adhesion molecule expression and chemokine release [ 14 , 27 ]. We qualitatively assessed intercellular adhesion molecule (ICAM)-1 in longitudinal sections of sciatic nerves over the course of EAN. ICAM-1 was not detected in nerves from naïve animals, but staining was apparent at EAN onset and persisted through the peak of disease ( Fig. 2 c ) . Permeation of a small molecule tracer over the course of EAN. We utilized Evans blue dye as a small molecule tracer to assess changes in vascular permeability over the course of EAN. Evans Blue binds to serum albumin at high affinity upon intravenous administration resulting in an approximate molecular weight of 69 kDa [ 28 , 29 ]. We observed a significant increase (p < 0.05) in dye permeation at EAN onset (Day 11) compared with naïve control rats ( Fig. 3 a ) . In control rats, the dye was restricted to the outer periphery of nerve cross sections ( Fig. 3 b ) . At EAN peak (Day 15), Evan’s blue dye permeation was at its maximum, a greater than 4-fold increase over control ( Figs. 3 a and 3 b ) . Nanoparticle PEGylation and characterization. We investigated the accumulation of two sizes of polystyrene NPs corresponding with separate spectral wavelengths (λ ex /λ em = 580/605 and λ ex /λ em = 505/515). NPs were obtained as carboxylated FluoSpheres with nominal diameters of 40 nm and 100 nm, respectively (Table 1 ). For prolonged systemic circulation and reduction of protein opsonization (19), we covalently attached mPEG-amine to carboxyl functional groups via carbodiimide chemistry ( Fig. 4 a ) . PEGylation was confirmed by an increase in NP hydrodynamic diameter and a shift in surface charge or zeta potential. NP size increased from 53.15 nm to 64.5 ± 2.7 nm (red) and from 124.3 nm to 136.5 ± 1.3 nm (green, Fig. 4 b ) . This change in size with PEGylation was identical for both FluoSphere formulations and is in line with values in the literature (17,30). Zeta potential of carboxylated NPs (-18 mV) shifted to -1.5 mV for both FluoSpheres (Fig. 4 c ) . Accumulation of nanoparticle tracers over the course of EAN. To investigate the potential of vascular permeability to facilitate delivery of NPs to peripheral nerves over the course of EAN, we administered PEGylated NPs of varying diameters and wavelength (64 nm, λ ex /λ em = 580/605 and 136 nm, λ ex /λ em = 505/515) and quantified distribution in sciatic nerve cross sections using confocal microscopy ( Figs. 5 a and 5 b ) . Standardized regions of interest (ROIs) were selected around endoneurial vessels for quantitative assessment of NP accumulation relative to a respective control. Both NP sizes exhibited disease-stage dependent accumulation in nerves. Accumulation of 64 nm NPs increased significantly between onset and intermediate EAN (p < 0.05), but the increase from intermediate to peak was not significant (p = 0.2), Fig. 5 a ) . In contrast, 136nm NP accumulation was not significantly increased at EAN onset (p = 0.66), but accumulation increased significantly between intermediate and peak EAN (p < 0.05, Fig. 5 b). Representative sciatic nerve cross sections illustrate both a lack of apparent endoneurial vessel accumulation in naïve animals as well as a time dependent increases in fluorescence intensity of NPs ( Fig. 5 c ) . We further compared accumulation of each tracer (small molecule and NP) at each stage of EAN ( Fig. 6 ) . At the disease onset, the increases in Evans blue associated fluorescence (234 ± 30.9%) were greater than those of the 64 nm and 136 nm NP tracers (153.1 ± 18.31%, 119.6 ± 5.57%), respectively (Fig. 6 a). At intermediate and peak EAN, no significant difference was observed between the accumulation of each of the three tracers ( Fig. 6 b and 6 c ) . Analysis of NP accumulation in different regions of the nerve over the course of EAN. Given that we induce EAN with a single injection of the antigen/adjuvant emulsion into the left hind footpad, and the injected foot exhibits local inflammation and edema over the course of disease, we sought to assess any lateral differences in distribution throughout the nerves. For both NP sizes, no significant difference was found between the accumulation in the left or right sciatic nerve over the disease course ( Fig. 7 a and 7 b ) . Further, given potential regional variation in blood flow through the microcirculation, we assessed NP accumulation in proximal and distal regions of the sciatic nerves. No significant difference was found between either of these regions over the course of the disease ( Fig. 7 c and 7 d ) . Correlation between NP accumulation and EAN clinical score. We analyzed our data by day post-EAN induction at defined disease stages: onset, intermediate, and peak of EAN. It follows that clinical scores also increase over the course of EAN. To confirm our hypothesis that increased clinical scores are associated with increased fluorescence, and to extrapolate our findings to other EAN models that may exhibit onset or peak on different days, we analyzed the correlation between clinical score and NP accumulation. We found a strong positive correlation between clinical score and NP accumulation for both 64 nm ( Fig. 8 a ) and 136 nm ( Fig. 8 b ) NPs, suggesting that more severe clinical symptoms are associated with increased BNB breakdown and enhanced vascular permeability. Discussion The BNB represents a key obstacle in delivering therapeutic compounds to peripheral nerves under physiological conditions. Given advances in nanomedicine that have enabled passive delivery strategies to locally inflamed sites [ 18 ], including the inflamed or injured brain [ 16 , 17 ], we hypothesized that EAN-associated pathology provides an opportunity to access the peripheral nerves for targeted delivery. Our work builds upon early EAN studies demonstrating nerve permeation of horseradish peroxidase [ 3 , 31 ] and Evans blue dye [ 32 ]. While these studies were instrumental in identifying BNB permeability as a key pathological feature, the imaging data were not quantified and analyzed statistically over the course of disease. Further, while small molecule tracers can inform solute permeability, the approximate hydrodynamic diameters (~ 4 nm for Evans blue associated with serum albumin [ 33 ]) do not represent the dimensions of potential NP drug delivery systems [ 19 ]. Here, we assessed immune cell infiltration, adhesion molecule expression, and BNB permeability to small molecules and two distinct sizes of NP tracers at defined stages of disease progression. Our data demonstrate endothelial ICAM-1 expression and small molecule permeability at EAN onset. At intermediate stages of EAN development, 64 nm NPs reach maximum accumulation in nerves, and at EAN peak 136 nm NPs reach maximum accumulation. These findings indicate that inflamed nerves can be accessed from the systemic circulation for therapeutic delivery. Many previous studies by different groups have demonstrated protective or therapeutic attenuation of EAN with systemically administered compounds spanning a range of anti-inflammatory or immunomodulatory mechanisms. Each of these studies has contributed to our understanding of EAN development and progression. However, the investigated therapies themselves are not feasible treatment options due to supraphysiological required doses ranging from 40–300 mg/kg [ 8 , 10 , 34 , 35 ], in some cases twice a day dosing [ 8 , 9 ]. Further, many compounds exhibit off target effects and toxicity [ 7 , 10 , 11 , 27 ]. While some examples, such as sphingosine-1-phosphate inhibitors [ 27 ] or peroxisome proliferator-activated receptor gamma (PPARγ) antagonists [ 36 ], act systemically on immune organs and circulating immune cells, several examples act on sites within affected nerves. Given our findings that circulating NPs accumulate in affected nerves, PEGylated nanoformulations may represent a delivery strategy that allows for effective doses to be achieved at the desired sites. Therapeutic targets within nerves may include axonal transcription factors, or channels or receptors at the nodes of Ranvier. Pitarokoili et al demonstrated that dimethyl fumarate (DMF) reduces EAN severity by upregulating axonal levels of the transcription factor Nrf-2, leading to subsequent reduced demyelination and increased axonal survival [ 8 ]. DMF would benefit from a NP delivery system to increase bioavailability and nerve levels, given its poor water solubility and 45 mg/kg twice a day dosing. Thrombin receptors localized at nodes of Ranvier may also serve as potential therapeutic targets [ 37 ]. Thrombin activity increases during EAN, and activation of the axonal thrombin receptor protease activated receptor (PAR)-1 contributes to conduction blocks and destruction at the nodes of Ranvier. Administration of either a nonselective thrombin inhibitor (4.4 mg/kg/day) or a highly specific thrombin inhibitor (69.8 mg/kg/day) attenuated EAN. Given the role of thrombin inhibitors as anticoagulants, excessive bleeding was observed in animals post-mortem. Targeted delivery may alleviate this risk in GBS/EAN. Finally, the sodium channel blocker flecainide attenuated EAN when administered at 30 mg/kg twice daily starting after disease onset. The authors propose that by blocking sodium currents, flecainide prevents detrimental accumulation of sodium at sites of inflammation and prevents subsequent increases in axonal calcium ions. Sodium channel blockers are associated with arrhythmias and cardiac side effects [ 38 ], which precludes systemic administration for GBS/EAN, particularly given the dosing for which beneficial effects on EAN were observed. Nerve endothelial cells, macrophages, or motor proteins may also represent therapeutic targets that would benefit from NP delivery systems. Sarkey et al found that lovastatin therapeutically attenuated EAN (25 mg/kg/day) by limiting immune cell trafficking into nerves [ 11 ]. Our subsequent work demonstrated that statins act directly on the BNB [ 24 ] and that the therapeutic mechanism may involve inhibition of chemokine release from BNB endothelial cells [ 39 , 40 ]. The non-nitrogen containing bisphosphonate, clodronate, was shown to eliminate nerve macrophages and attenuate EAN when administered therapeutically after symptom onset [ 6 ]. The kinesin-5 inhibitor monastrol reduced EAN by enhancing neurite outgrowth within axons [ 7 ]. Importantly, this therapy was administered at peak of disease, which reflects the time when most patients are diagnosed with EAN. Despite clear size- and disease stage-dependent patterns in NP associated fluorescence, and a lack of lateral or longitudinal variation, NP accumulation appears to be centered upon endoneurial blood vessels (see Fig. 5 ). We focused on defined regions of interest that centered on endoneurial blood vessels to quantify our data. This observation has implications for the types of small molecules that would benefit from a NP delivery system. In the case of statins [ 11 , 24 , 40 ], the therapeutic target (endothelial cells) is clearly accessible via passive accumulation of NPs. Clodronate may also act closely to the BNB to eliminate macrophages. In the case of axonal and neuronal targets, targets may not be fully accessible by passively accumulating NPs. Active targeting strategies may enable further penetration of NPs into the endoneurium [ 41 – 43 ], and lipophilic payloads may diffuse more deeply into the endoneurium once released from polymeric NPs [ 44 , 45 ]. We induce EAN with a single injection of the antigen/adjuvant emulsion into the left hind footpad, and the injected foot exhibits local inflammation and edema over the course of disease. Further, nerves exhibit regional variation in blood flow through the microcirculation as well as inflammation associated edema [ 46 , 47 ]. We assessed lateral and longitudinal distribution throughout the nerves to determine any regional variation in passive accumulation, and we did not observe any significant differences in NP accumulation between left and right or proximal and distal regions of the sciatic nerves. We did not assess permeation into nerve roots, given that the intermediate regions of the sciatic nerve are most restrictive to circulating molecules during homeostasis [ 13 , 48 ]. However, others have demonstrated that nerve roots become fenestrated earlier than the sciatic nerve during EAN [ 32 ], and we anticipate that nerve roots would promote NP accumulation no less than observed in sciatic nerves. Collectively, our findings confirm that inflammation in EAN is not localized to one region and is multifocal and widespread along the length of nerves. This supports the need for systemic, rather than local, therapeutic approaches. For extrapolation of our findings to the investigations of systemically administered compounds discussed above, we must consider our defined disease stages in the context of other preclinical EAN studies. The clinical course of EAN in rat models induced with P2 peptide does not vary significantly between groups, with onset around Day 10 ± 1 and peak at Day 16 ± 1 [ 5 , 24 , 36 ]. Elahi et al [ 49 ] induced “mild” and “severe” phenotypes by varying the concentration of CFA (1–3 mg/ml) and number of injection sites (hind foot pad ± base of tail). In the severe phenotype, EAN onset was on Day 8 ± 1. While we analyzed mean fluorescence intensity at different days post-induction, we observed a strong correlation between clinical score and fluorescence intensity (Fig. 8 ), suggesting that our findings can be applied to other EAN models, even if onset and peak are not at Day 11 and Day 15. For future translational therapeutic studies, it is critical to also consider how our findings align with the course of GBS/AIDP in human patients. Most GBS patients reach peak neuropathy within four weeks of onset [ 50 ], and diagnosis based on electrodiagnostic data is not possible until closer to peak disease [ 15 ]. Therefore, while early diagnosis occurs in some cases [ 15 ], EAN onset does not directly correspond with GBS diagnosis. In EAN rats, we demonstrated that 64 nm NPs can access the endoneurium before clinical disease has peaked, at an intermediate stage between EAN onset and peak. Given that the time spent in each stage for patients is on the order of weeks, not days, we anticipate that both patients presenting with early symptoms as well as patients receiving a diagnosis at peak of GBS would be candidates for nanomedicine approaches. Importantly, one EAN study demonstrated benefits of their systemically administered compound (monastrol) when it was administered after peak disease [ 7 ]. Further, while intravenous administration is difficult to incorporate into outpatient medication paradigms [ 51 , 52 ], it will blend seamlessly with ongoing standard of care for GBS, including plasmapheresis and IVIg [ 1 , 2 ]. Additional study of BNB permeability in GBS patients, as well as continued investigation of NP fate throughout the body in EAN, is needed. Conclusions In this study, we characterized BNB breakdown over the course of EAN. We demonstrated a disease stage dependent increase in immune infiltrates, adhesion molecule expression, and permeation of a small molecule dye. We demonstrated that NPs with a hydrodynamic diameter of 64 nm can cross the BNB and accumulate in nerves at intermediate EAN, and NPs with a hydrodynamic diameter of 136 nm cross the BNB and accumulate in nerves at peak EAN. These findings establish that EAN associated pathology enables passive nerve targeting of circulating NPs and set the foundation for further development of NP drug delivery strategies in diseases like inflammatory peripheral neuropathies that necessitate targeted, systemic administration. Declarations Acknowledgements: This work was supported, in part, by funding from the Department of Veterans Affairs (RX002305) and start-up funding from Loyola University Chicago. We thank Zoe Adelstein for technical assistance. Peptide Synthesis was performed at the Peptide Synthesis Core Facility of the Simpson Querrey Institute for BioNanotechnology at Northwestern University. References Van Doorn PA (2009) What’s new in Guillain-Barre syndrome in 2007–2008? J Peripher Nerv Syst 74:72–74 Willison HJ, Jacobs BC, van Doorn PA (2016) Guillain-Barré syndrome. Lancet 388(10045):717–727 Spies JM, Westland KW, Bonner JG, Pollard JD (1995) Intraneural activated t cells cause focal breakdown of the blood-nerve barrier. Brain 118(4):857–868 Lu MO, Zhu J (2011) The role of cytokines in Guillain-Barré syndrome. J Neurol 258(4):533–548 Tomikawa E, Mutsuga M, Hara K, Kaneko C, Togashi Y, Miyamoto Y (2019) Time Course of Axon and Myelin Degeneration in Peripheral Nerves in Experimental Autoimmune Neuritis Rats. Toxicol Pathol 47(4):542–552 Katzav A, Bina H, Aronovich R, Chapman J (2013) Treatment for experimental autoimmune neuritis with clodronate (Bonefos). Immunol Res 56(2–3):334–340 Kohle F, Ackfeld R, Hommen F, Klein I, Svačina MKR, Schneider C et al (2023) Kinesin-5 inhibition improves neural regeneration in experimental autoimmune neuritis. J Neuroinflammation [Internet]. ;20(1):1–13. https://doi.org/10.1186/s12974-023-02822-w Pitarokoili K, Ambrosius B, Meyer D, Schrewe L, Gold R (2015) Dimethyl fumarate ameliorates lewis rat experimental autoimmune neuritis and mediates axonal protection. PLoS ONE 10(11):1–17 Bechtold DA, Yue X, Evans RM, Davies M, Gregson NA, Smith KJ (2005) Axonal protection in experimental autoimmune neuritis by the sodium channel blocking agent flecainide. Brain 128(1):18–28 Yi C, Zhang Z, Wang W, Zug C, Schluesener HJ, Zhang Z (2011) Doxycycline attenuates peripheral inflammation in rat experimental autoimmune neuritis. Neurochem Res 36(11):1984–1990 Sarkey JP, Richards MP, Stubbs EB (2007) Lovastatin attenuates nerve injury in an animal model of Guillain-Barré syndrome. J Neurochem 100(5):1265–1277 Langert KA, Brey EM (2018) Strategies for targeted delivery to the peripheral nerve. Front Neurosci 12(NOV):1–10 Abram SE, Yi J, Fuchs A, Hogan QH (2006) Permeability of injured and intact peripheral nerves and dorsal root ganglia. Anesthesiology 105(1):146–153 Putzu GA, Figarella-Branger D, Bouvier-Labit C, Liprandi A, Bianco N, Pellissier JF (2000) Immunohistochemical localization of cytokines, C5b-9 and ICAM-1 in peripheral nerve of Guillain-Barre Syndrome. J Neurol Sci 174(1):16–21 Berciano J, Sedano MJ, Pelayo-Negro AL, García A, Orizaola P, Gallardo E et al (2017) Proximal nerve lesions in early Guillain–Barré syndrome: implications for pathogenesis and disease classification. J Neurol 264(2):221–236 Bennett J, Basivireddy J, Kollar A, Reickmann P, Jefferies W, McQuaid S (2010) Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE. J Immunol 229(1–2):180–191 Bharadwaj VN, Lifshitz J, Adelson PD, Kodibagkar VD, Stabenfeldt SE (2016) Temporal assessment of nanoparticle accumulation after experimental brain injury: Effect of particle size. Sci Rep [Internet]. ;6(April):1–12. http://dx.doi.org/10.1038/srep29988 Durymanov M, Kamaletdinova T, Lehmann SE, Reineke J (2017) Exploiting passive nanomedicine accumulation at sites of enhanced vascular permeability for non-cancerous applications. J Control Release [Internet]. ;261(June):10–22. https://doi.org/10.1016/j.jconrel.2017.06.013 Alexis F, Pridgen E, Molnar LK, Farokhzad OC (2008) Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 5(4):505–515 Rafiei P, Haddadi A (2017) Docetaxel-loaded PLGA and PLGA-PEG nanoparticles for intravenous application: Pharmacokinetics and biodistribution profile. Int J Nanomed 12:935–947 Bae YH, Park K (2011) Targeted drug delivery to tumors: Myths, reality and possibility. J Control Release [Internet]. ;153(3):198–205. http://dx.doi.org/10.1016/j.jconrel.2011.06.001 Subhan MA, Yalamarty SSK, Filipczak N, Parveen F, Torchilin VP (2021) Recent advances in tumor targeting via epr effect for cancer treatment. J Pers Med. ;11(6) Berciano J (2021) Axonal degeneration in Guillain–Barré syndrome: a reappraisal. J Neurol 268(10):3728–3743 Langert KA, Goshu B, Stubbs EB (2017) Attenuation of experimental autoimmune neuritis with locally administered lovastatin-encapsulating poly(lactic-co-glycolic) acid nanoparticles. J Neurochem Calik MW, Shankarappa SA, Langert KA, Stubbs EB (2015) Forced exercise preconditioning attenuates experimental autoimmune neuritis by altering Th1 lymphocyte composition and egress. ASN Neuro [Internet]. ;7(4). https://doi.org/10.1177/1759091415595726 Calik MW, Shankarappa SA, Langert KA, Stubbs EB (2015) Forced exercise preconditioning attenuates experimental autoimmune neuritis by altering Th1 lymphocyte composition and egress. ASN Neuro. ;7(4) Ambrosius B, Pitarokoili K, Schrewe L, Pedreiturria X, Motte J, Gold R (2017) Fingolimod attenuates experimental autoimmune neuritis and contributes to Schwann cell-mediated axonal protection. J Neuroinflammation 14(1):1–9 Yao L, Xue X, Yu P, Ni Y, Chen F (2018) Evans Blue Dye: A Revisit of Its Applications in Biomedicine. Contrast Media Mol Imaging 2018:18–24 Scalisi J, Balau B, Deneyer L, Bouchat J, Gilloteaux J, Nicaise C (2021) Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome. Neurosci Lett [Internet]. ;746(August 2020):135665. https://doi.org/10.1016/j.neulet.2021.135665 Householder KT, Dharmaraj S, Sandberg DI, Wechsler-Reya RJ, Sirianni RW (2019) Fate of nanoparticles in the central nervous system after intrathecal injection in healthy mice. Sci Rep 9(1):1–11 Powell HC, Braheny S, Myers RR, Rodriguez M, Lampert P (1983) Early changes in experimental allergic neuritis. Lab Investig 48(3):332–338 Hahn A, Feasby T, Gilbert J (1985) Blood nerve barrier studies in experimental allergic neuritis. Acta Neuropathol 68:101–109 Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A (2006) Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst 98(5):335–344 Zhang Z, Zhang ZY, Fauser U, Schluesener HJ (2008) Valproic acid attenuates inflammation in experimental autoimmune neuritis. Cell Mol Life Sci 65(24):4055–4065 Han F, Luo B, Shi R, Han C, Zhang Z, Xiong J et al (2014) Curcumin ameliorates rat experimental autoimmune neuritis. J Neurosci Res 92(6):743–750 Ramkalawan H, Wang YZ, Hurbungs A, Yang YF, Tian FF, Zhou W, Bin et al (2012) Pioglitazone, PPARgamma agonist, attenuates experimental autoimmune neuritis. Inflammation 35(4):1338–1347 Shavit-Stein E, Aronovich R, Sylantiev C, Gera O, Gofrit SG, Chapman J et al (2019) Blocking thrombin significantly ameliorates experimental autoimmune neuritis. Front Neurol 10(JAN):3–12 Munger MA, Olğar Y, Koleske ML, Struckman HL, Mandrioli J, Lou Q et al (2020) Tetrodotoxin-sensitive neuronal-type na + channels: A novel and druggable target for prevention of atrial fibrillation. J Am Heart Assoc. ;9(11) Langert KA, Pervan CL, Stubbs EB (2014) Novel role of cdc42 and RalA GTpases in TNF-α mediated secretion of CCL2. Small GTPases. ;5 Langert KA, Von Zee CL, Stubbs EB (2013) Cdc42 GTPases facilitate TNF-α-mediated secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells. J Peripher Nerv Syst 18(3):199–208 Chittasupho C, Xie S-X, Baoum A, Yakovleva T, Siahaan T, Berkland CJ (2009) ICAM-1 targeting of doxorubicin-loaded PLGA nanoparticles to lung epithelial cells. Eur J Pharm Sci 37(2):141–150 Yang F, Cabe MH, Ogle SD, Sanchez V, Langert KA (2021) Optimization of critical parameters for coating of polymeric nanoparticles with plasma membrane vesicles by sonication. Sci Rep [Internet]. ;11(1):1–13. https://doi.org/10.1038/s41598-021-03422-5 Li R, He Y, Zhu Y, Jiang L, Zhang S, Qin J et al (2019) Route to Rheumatoid Arthritis by Macrophage-Derived Microvesicle-Coated Nanoparticles. Nano Lett 19(1):124–134 Cook RL, Householder KT, Chung EP, Prakapenka AV, Diperna DM, Sirianni RW (2015) A critical evaluation of drug delivery from ligand modified nanoparticles: Confounding small molecule distribution and efficacy in the central nervous system. J Control Release 220:89–97 Medina DX, Householder KT, Ceton R, Kovalik T, Heffernan JM, Shankar RV et al (2017) Optical barcoding of PLGA for multispectral analysis of nanoparticle fate in vivo. J Control Release [Internet]. ;253:172–82. http://dx.doi.org/10.1016/j.jconrel.2017.02.033 Prahm C, Heinzel J, Kolbenschlag J (2022) Blood Supply and Microcirculation of the Peripheral Nerve. In: Philips JB (ed) Peripheral Nerve Tissue Engineering and Regeneration. Springer Nature Switzerland, pp 35–79 Kozu H, Tamura E, Parry G (1992) Endoneurial blood supply to peripheral nerves is not uniform. J Neurol Sci 111:204–208 Liu H, Chen Y, Huang L, Sun X, Fu T, Wu S et al (2018) Drug distribution into peripheral nerve. J Pharmacol Exp Ther 365(2):336–345 Elahi E, Ali ME, Zimmermann J, Getts DR, Müller M, Lamprecht A (2022) Immune Modifying Effect of Drug Free Biodegradable Nanoparticles on Disease Course of Experimental Autoimmune Neuritis. Pharmaceutics. ;14(11) Ubogu EE (2015) Inflammatory neuropathies: pathology, molecular markers and targets for specific therapeutic intervention. Acta Neuropathol 130(4):445–468 Lucas M, Hugh-Jones K, Welby A, Misbah S, Spaeth P, Chapel H (2010) Immunomodulatory therapy to achieve maximum efficacy: Doses, monitoring, compliance, and self-infusion at home. J Clin Immunol 30(SUPPL 1):84–89 Mejia-Chew C, Heuring B, Salmons J, Weilmuenster L, Beggs J, Kleinschmidt G et al (2024) IVsight as an Infusion Monitor for Patients Receiving Intravenous Therapy: An Exploratory, Unblinded, Single-Center Trial. Curr Ther Res - Clin Exp [Internet]. ;100:100747. https://doi.org/10.1016/j.curtheres.2024.100747 Tables Table 1 Nominal, measured, and PEGylated nanoparticle diameters. Nominal diameter (nm) Measured diameter (nm) PEGylated diameter (nm) Red 40 53.15 ± 0.5 64.5 ± 2.7 Green 100 124.3 ± 0.9 136.5 ± 0.8 Additional Declarations The authors declare potential competing interests as follows: No authors have interests, affiliations, or associations that might be perceived to influence the results and/or discussion reported in this preprint submission. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4631228","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":318412083,"identity":"cd1e4618-36b2-456a-bc13-b51ccc0d3cc9","order_by":0,"name":"Kelly Langert","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIie3LMQrCMBiG4ZRAuwS6/j9Rz1AJxKGXiVMPUJAORQqB9gpew8ULBOrSMwguzhVEOhUJ1rlxE8w7fcP3EOLz/WAQTSN+D+pApg+g/pokxpWgprfro7jshWHrnhTptpojnIYbsehykIYJIF02T1aUSI61skSSoDYuJHpyHBUIbcnoQDhlEu+VgoRaUjkQ1GzHSavwYMIcVJuJWQLn5oRDqeK40ce+L9PlLLFR9lnK5W4LBtenz+fz/WcvOiAx7vbzky8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3017-1380","institution":"Loyola University Chicago","correspondingAuthor":true,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Langert","suffix":""},{"id":318412084,"identity":"b8215b70-bc2b-4126-8f69-61581da8442b","order_by":1,"name":"Chanpreet Kaur","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Chanpreet","middleName":"","lastName":"Kaur","suffix":""},{"id":318412085,"identity":"e1bded34-c3d9-49fb-a291-6db3011bf7f2","order_by":2,"name":"Ellaina Villarreal","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Ellaina","middleName":"","lastName":"Villarreal","suffix":""},{"id":318412086,"identity":"531caac0-6cec-4913-969e-5f257ed842e0","order_by":3,"name":"Maleen Cabe","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Maleen","middleName":"","lastName":"Cabe","suffix":""}],"badges":[],"createdAt":"2024-06-24 15:33:12","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4631228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4631228/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59189715,"identity":"b9f76308-a07e-416d-aa1b-7e37fd299c4a","added_by":"auto","created_at":"2024-06-27 12:55:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114741,"visible":true,"origin":"","legend":"\u003cp\u003eEAN clinical presentation\u003cstrong\u003e. \u003c/strong\u003eEAN was induced with P2 peptide (residues 53-78) and complete Freund’s adjuvant on Day 0. \u003cstrong\u003e(a)\u003c/strong\u003e Daily clinical scores of naïve control and EAN rats. Key disease stages include Day 11 (onset), Day 13 (intermediate), and Day 15 (peak). \u003cstrong\u003e(b)\u003c/strong\u003e Daily weights of naïve control and EAN rats, as a percentage of peak weight. Data shown are the mean ± SEM, n=8, *p\u0026lt;0.05, Ordinary one-way ANOVA with Tukey’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/e12a0c7099002b932cb39f2a.png"},{"id":59190447,"identity":"83dc975d-23d4-4e48-a36d-b19927d25c04","added_by":"auto","created_at":"2024-06-27 13:03:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":496889,"visible":true,"origin":"","legend":"\u003cp\u003eCellular changes in nerves over the course of EAN\u003cstrong\u003e.\u003c/strong\u003e Sciatic nerves were harvested at key disease stages and immune and endothelial cells were assessed with immunohistochemistry. \u003cstrong\u003e(a)\u003c/strong\u003e Quantification of CD68\u003csup\u003e+\u003c/sup\u003e macrophages in transverse sections. Data shown are the mean ± SEM, n=3-4 rats, *p\u0026lt;0.01, one-way ANOVA followed by Fisher’s LSD multiple comparison. \u003cstrong\u003e(b)\u003c/strong\u003e Representative images of data quantified in (A). Green, CD68; blue, DAPI; scale bar, 40 µm. \u003cstrong\u003e(c)\u003c/strong\u003e Representative longitudinal sections stained for the presence of intercellular adhesion molecule (ICAM)-1 expressing endothelial cells. Green, CD54; blue, DAPI; scale bar, 40 µm.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/1952c9124303fed266b29ecf.png"},{"id":59190448,"identity":"386d085e-c7c9-4848-99d9-0ba10c885575","added_by":"auto","created_at":"2024-06-27 13:03:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":519019,"visible":true,"origin":"","legend":"\u003cp\u003ePermeation of a small molecule tracer over the course of EAN\u003cstrong\u003e. \u003c/strong\u003eRats\u003cstrong\u003e \u003c/strong\u003ereceived a tail vein injection of Evans blue dye (EVB, 69 kDa) at key disease stages, and nerves were collected 30 minutes later. \u003cstrong\u003e(a)\u003c/strong\u003e Quantitative analysis of EVB permeation (λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e = 610/680 nm) in transverse nerve sections at indicated days. Data shown are the mean ± SEM, n=4 rats/group, *p\u0026lt;0.05, Ordinary one-way ANOVA followed by Fisher’s LSD multiple comparison.\u003cstrong\u003e (b) \u003c/strong\u003eRepresentative images of data quantified in (A). Scale bar, 100 µm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/31f7a87e10f35f363e6fe404.png"},{"id":59189719,"identity":"b52596b7-16b7-4613-9bf1-422be60d0b09","added_by":"auto","created_at":"2024-06-27 12:55:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":150892,"visible":true,"origin":"","legend":"\u003cp\u003eModification and characterization of polystyrene nanoparticles\u003cstrong\u003e. (a)\u003c/strong\u003e Schematic demonstrating the covalent attachment of poly(ethylene) glycol (PEG)-amine to carboxylated NPs using carbodiimide chemistry (EDC/NHS). \u003cstrong\u003e(b)\u003c/strong\u003e Size (nm) and \u003cstrong\u003e(c) \u003c/strong\u003ezeta potential (mV) of carboxylated NPs and PEGylated NPs. Values shown are mean ± SEM, n=5 separate batches, *p\u0026lt;0.05 vs. carboxylated counterpart, multiple unpaired t-tests.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/cc930d558392d42af5c78ba3.png"},{"id":59189723,"identity":"62e5c59c-3b73-4242-8cb1-b84cdc6c5d1d","added_by":"auto","created_at":"2024-06-27 12:55:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":740012,"visible":true,"origin":"","legend":"\u003cp\u003eAccumulation of nanoparticle tracers over the course of EAN\u003cstrong\u003e.\u003c/strong\u003e Rats received a tail vein injection of a cocktail of \u003cstrong\u003e(a)\u003c/strong\u003e 64 nm and \u003cstrong\u003e(b)\u003c/strong\u003e 136 nm PEGylated NPs at key disease stages, and nerves were collected 30 minutes later. Fluorescence intensity in regions of interest around endoneurial vessels in transverse sections was assessed. Data shown are the mean ± SEM, n=4-5 rat/group, *p\u0026lt;0.05, Ordinary one-way ANOVA followed by Fisher’s LSD multiple comparison. \u003cstrong\u003e(c)\u003c/strong\u003e Representative images of data quantified in A, B. Scale bar, 30 µm. \u003cstrong\u003e(d) \u003c/strong\u003eRepresentative 10x images of data quantified in A, B. Scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/c67e4f461d813347c788d242.png"},{"id":59189716,"identity":"901f333b-bfea-4d6c-a417-99a6448facf9","added_by":"auto","created_at":"2024-06-27 12:55:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90638,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of size-dependent permeation over the course of EAN. Fluorescence of three different sized tracers in transverse sections of sciatic nerves collected were expressed as an increase over each tracers’ respective control (Day 0) and directly compared at \u003cstrong\u003e(a)\u003c/strong\u003e onset, \u003cstrong\u003e(b)\u003c/strong\u003e intermediate, and \u003cstrong\u003e(c)\u003c/strong\u003epeak of EAN. Data shown are the mean ± SEM, n=4-5 rat/group, *p\u0026lt;0.05, Ordinary one-way ANOVA followed by Fisher’s LSD multiple comparison.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/a51e4a2096cc0467dd7f9065.png"},{"id":59189720,"identity":"802961ad-9c5b-43f3-8db1-ee890549b9ac","added_by":"auto","created_at":"2024-06-27 12:55:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":250430,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial analysis of nanoparticle tracers over the course of EAN. Quantitative analysis of \u003cstrong\u003e(a, b)\u003c/strong\u003e lateral and \u003cstrong\u003e(c, d)\u003c/strong\u003e longitudinal distribution of 64 nm and 136 nm NP tracers, as indicated. Shown in A and B is a comparison between the nerve closest to the hind paw injection of CFA + antigen (ipsilateral, solid bars) and the contralateral nerve (patterned bars). Shown in C and D is a comparison between proximal (solid bars) and distal (patterned bars) regions of the nerves. All data shown are the mean ± SEM, n=4-5 rats/group, multiple unpaired t tests.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/e2b589fef37ea32a05477b13.png"},{"id":59189718,"identity":"b2939649-a8fc-4d46-8a6b-30791ff05f72","added_by":"auto","created_at":"2024-06-27 12:55:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":114036,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelative analysis of clinical score and NP accumulation\u003cstrong\u003e.\u003c/strong\u003e Dot plot showing positive correlation between clinical score and\u003cstrong\u003e (a) \u003c/strong\u003e64 nm NP-associated fluorescence \u003cstrong\u003e(b) \u003c/strong\u003e136 nm NP-associated fluorescence. Values plotted are for n =16-17 rats.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/27990bbcc8ea1cc69ab4010a.png"},{"id":59191157,"identity":"35c12e88-2507-4739-96bb-0a364d64c802","added_by":"auto","created_at":"2024-06-27 13:11:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2924695,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4631228/v1/c68bc16a-2014-481f-83b8-2b1fd37582bf.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: No authors have interests, affiliations, or associations that might be perceived to influence the results and/or discussion reported in this preprint submission.","formattedTitle":"\u003cp\u003eBlood nerve barrier permeability enables nerve targeting of circulating nanoparticles in experimental autoimmune neuritis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGuillain-Barre syndrome (GBS) is a devastating autoimmune disease of the peripheral nervous system (PNS) for which there is no targeted therapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Experimental autoimmune neuritis (EAN) is a well-established preclinical model of the acute inflammatory demyelinating polyneuropathy (AIDP) subtype of GBS that has advanced our understanding of mechanism by which pathology develops and progresses [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Several previous studies have shown attenuation of EAN with systemic administration of potential therapeutic compounds that span a range of anti-inflammatory or immunomodulatory strategies [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While shedding light on the mechanisms of disease, clinical advancement of these findings is limited by dosing that is not translatable to humans or is associated with off-target and toxic effects.\u003c/p\u003e \u003cp\u003eThe blood nerve barrier (BNB) represents a key obstacle to delivering therapeutics to peripheral nerves [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This barrier consists of layers of connective tissue as well as specialized microvascular endothelial cells linked by tight junctions, together creating an isolated endoneurial microenvironment. Several studies have investigated the role of the BNB in restricting access of circulating molecules to the endoneurium under physiological conditions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During acute neuroinflammation, such as that associated with AIDP, the normally restrictive BNB exhibits increased vascular permeability and enables immune cell infiltration [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While these pathological changes contribute to disease, they may also offer a unique window of opportunity to access the otherwise restricted peripheral nerve microenvironment for therapeutic delivery.\u003c/p\u003e \u003cp\u003eIt is established that fenestrated vasculature, like those featured in sites of localized inflammation, can promote passive accumulation of circulating nanoparticles (NPs) with favorable size and surface properties [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This phenomenon is dependent on prolonged circulation and avoidance of clearance, which are best achieved by NPs in the range of 50\u0026ndash;200 nm [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Despite promising preclinical results for enhanced permeation and retention (EPR) to promote accumulation of NPs in tumors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], successful translation of EPR for chemotherapeutic delivery has been low [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Established contributing factors include properties and kinetics of tumor development in preclinical models poorly reflect human pathology and tumor microenvironments contain both avascular regions and tortuous vessels, resulting in heterogenous NP distribution [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Given the established validity of the EAN model and linearity of peripheral nerve vasculature [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the potential for EPR to facilitate delivery to peripheral nerves in AIDP is an exciting area for preclinical investigation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we assessed whether the BNB permeability and immune cell infiltration over the course of EAN enable passive accumulation of circulating nanoparticles. We identified stages of EAN, onset, intermediate, and peak, defined by distinct clinical scores and pathology. We found that intravenously administered small molecules (69 kDa) and NPs ranging from 50\u0026ndash;150 nm can permeate into the endoneurium from the endoneurial vasculature in a size- and disease stage-dependent manner. We propose that this passive targeting serves as an opportunity by which potential therapies for GBS can be reevaluated and investigated preclinically in NP delivery systems.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eCarboxylated modified Fluospheres\u0026trade;, specifically 40 nm red (F8793, λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;580/605) and 100 nm yellow-green (F8803, λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;505/515), 20x borate buffer, glycine, and ICAM-1 antibody (MA5407) were purchased from Thermofisher Scientific (Waltham, MA, USA). Evans blue dye (E2129), N-(3-Dimethylaminopropyl)-N\u0026rsquo;- ethyl carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), Amicon Ultra centrifugal filters (10 and 100 kDa), and incomplete Freund's adjuvant were purchased from Sigma-Aldrich (St Louis, MO, USA). Methoxy poly(ethylene) glycol (mPEG)-amine (2 kDa) was obtained from Creative PEGWorks (Chapel Hill, NC). Phosphate buffered saline (10x PBS) and mouse anti rat CD68 primary antibody was obtained from Bio-Rad (Hercules, CA, USA). P2 peptide was obtained from Peptide Synthesis Core Facility of the Simpson Querrey Institute, Northwestern University. Heat-inactivated Mycobacteria tuberculosis (strain H37Ra) was purchased from Becton, Dickinson, and Company (Sparks, MD 21152, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle PEG conjugation (PEGylation)\u003c/h2\u003e \u003cp\u003eCarboxylated FluoSpheres\u003cb\u003e\u0026trade;\u003c/b\u003e were PEGylated with mPEG-amine using carbodiimide chemistry and previously published methods (17). First, 500 \u0026micro;L of FluoSpheres (2% (yellow green) and 5% (red) suspension from the manufacturer) were washed once using Amicon centrifugal filters (10 kDa MWCO) at 14,000 x g for 15 min. Next, mPEG-amine was added at 5x molar excess followed by NHS (6.5 mg), EDC (15.4 mg) and borate buffer (6 ml, pH 8) and allowed to stir for 3 hours. The reaction was quenched with glycine (75 mg, 100mM) for 30 min. PEGylated FluoSpheres, herein referred to as NPs, were washed and collected using Amicon centrifugal filters (100 kDa MWCO) and resuspended to original concentration in filtered 1x PBS for storage at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNP characterization\u003c/h2\u003e \u003cp\u003eHydrodynamic diameter and zeta potential of NPs were quantitatively assessed with dynamic light scattering using Zetasizer Nano ZS90 (Malvern Panalytical). A suspension of NPs (0.1 mg/ml) was prepared in deionized water and transferred to a disposable polystyrene cuvette or a disposable capillary cell. Suspension was equilibrated for 3 minutes in the cuvette and measured at 90\u0026deg; angle. Each sample was measured 4 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEAN induction\u003c/h2\u003e \u003cp\u003e Experiments were conducted using protocols approved by the Edward Hines, Jr., VA Hospital Institutional Animal Care and Use Committee in accordance with the principles of laboratory animal care. Adolescent male Lewis rats were housed in pairs, allowed free access to standard rat chow and water, and maintained on a 10h/14h light/dark cycle. Na\u0026iuml;ve rats (Envigo, Indianapolis, IN) were induced with EAN using P2 peptide as we have previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, anesthetized rats (ketamine (100 mg/kg)-xylazine (5 mg/kg)) were injected with a freshly prepared emulsion containing (1:1 v/v) purified P2 myelin neuritogenic peptide (residues 53\u0026ndash;78, 100 \u0026micro;g) suspended in sterile saline and Freund\u0026rsquo;s adjuvant supplemented with a final concentration of 5 mg/ml heat-inactivated Mycobacterium tuberculosis (strain H37RA) into one hind footpad. Control rats received a footpad injection of sterile saline. Weights and clinical progression of disease were assessed daily beginning on Day 7. Rats receive a score of 1 (loss of tail tone), 2 (disrupted gait), 3 (mild hemiparesis), 4 (severe paraparesis), or 5 (paraplegia), including increments of 0.5 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIntravenous injection of tracers\u003c/h2\u003e \u003cp\u003eOn the designated day, ketamine-anesthetized control and EAN rats received a single intravenous (tail vein) infusion of Evans blue dye (2% w/v suspended in PBS) or a cocktail of NPs (8 mg total/rat, suspended in diH\u003csub\u003e2\u003c/sub\u003eO). Time points for administration of Evans blue or NPs included EAN onset (Day 11), intermediate EAN (Day 13) and peak EAN (Day 15).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTissue collection\u003c/h2\u003e \u003cp\u003eThirty minutes after infusion of tracers, rats were euthanized, and sciatic nerves were collected. Nerves were fixed for 4 hours in 4% paraformaldehyde followed by incubation in 30% sucrose/PBS overnight at 4\u0026deg;C. The next day, nerves were incubated in 50% optimal cutting temperature (OCT) medium/50% sucrose/PBS for 2 hours followed by embedding in OCT and snap freezing in liquid nitrogen. Embedded tissue was stored at -80\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and Evans blue accumulation\u003c/h2\u003e \u003cp\u003eSerial 10\u0026micro;m cross and longitudinal sections were prepared from nerves using a Leica CM 1850 Cryostat, and the content and distribution of CD68\u003csup\u003e+\u003c/sup\u003e immune infiltrates was determined by immunohistochemistry as we have previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Washed sections were blocked for 30 min at room temperature in PBS containing 5% (v/v) normal goat serum and 0.05% (v/v) Triton X-100 (blocking buffer). Cross sections were incubated with a 1:250 dilution of mouse anti-CD68 (macrophage marker, clone ED1, purified IgG primary antibody) followed by a 1:1000 dilution of Alexa Fluor 488 anti-mouse secondary antibody and coverslipped with prolong diamond antifade mountant containing DAPI. Infiltrating CD68\u003csup\u003e+\u003c/sup\u003e macrophages were visualized on a Nuhsbaum inverted confocal microscope and semi-quantified in a blinded manner with ImageJ software. Longitudinal sections were incubated with a 1:100 dilution of mouse anti-CD54 (ICAM-1, clone W3/25, purified IgG) primary antibody followed by 1:1000 dilution of Alexa Fluor 488 anti-mouse secondary antibody and similarly coverslipped. CD54\u003csup\u003e+\u003c/sup\u003e endothelial cells were visualized as above, and ICAM-1 expression was qualitatively assessed.\u003c/p\u003e \u003cp\u003eEvans blue accumulation was assessed in the same sections at 10x magnification at laser setting of 561 nm (photomultiplier tube (PMT)\u0026thinsp;=\u0026thinsp;610 nm \u0026minus;\u0026thinsp;677 nm, 27.754% laser intensity). Z-stacked images of nerves were obtained and converted to one image using maximum projection on Leica LAS-X software. Five random regions of interest (ROI) were chosen in the cross-section images (8 nerve sections per animal, 4 animals per cohort) and fluorescence intensity was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eNP accumulation\u003c/h2\u003e \u003cp\u003eSerial 10\u0026micro;m cross sections were prepared from nerves using a Leica CM 1850 Cryostat and mounted onto Superfrost Plus microscope slides. Slides were incubated at room temperature for 30 min, washed once with PBS, and air dried. Coverslips were gently placed using Prolong Diamond Antifade mountant (Thermofisher Scientific). NP permeation was visualized using confocal microscopy. Images were captured at laser settings of 405nm, 488nm, 561nm (PMT: 432\u0026ndash;482 nm, 522\u0026ndash;585 nm, and 610\u0026ndash;677 nm) and laser intensity (17.07%, 21.35%, 27.74% respectively) was standardized for all nerve sections. Z-stacked images (40x magnification) of nerves were obtained and converted to one image using maximum projection on Leica LAS-X software. Standardized areas of ROI around endoneurial blood vessels were chosen in the cross-section images (up to 10 nerve sections per animal, 4\u0026ndash;5 animals per cohort) and fluorescence intensity was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using GraphPad Prism 10.2.1 (397). Data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of N observations, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical significance across multiple experimental groups (EAN score and weight, leukocytic infiltrates, Evans blue permeation, NP accumulation comparison at onset, intermediate, and peak of EAN) was conducted using one-way ANOVA followed by Fishers LSD and Tukey\u0026rsquo;s post-hoc test. Statistical significance of paired groups was conducted using multiple unpaired t-tests. Statistical significance and R value of clinical score and NP accumulation was conducted using simple linear regression correlation analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExperimental autoimmune neuritis clinical and pathological presentation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe and others have previously shown that EAN induced with complete Freund\u0026rsquo;s adjuvant (CFA) and P2 peptide manifests as a robust, monophasic course of ascending weakness [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Here, the observed disease course develops and progresses similarly, with onset on day 11 post induction presenting as loss of tail tone and the peak of disease on day 15 post induction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Rats lose weight over the course of EAN, with a significant reduction compared to their peak weight beginning on Day 13 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. In this study, we focus on three distinct stages of disease: EAN onset (Day 11), intermediate EAN (Day 13), and peak EAN (Day 15). The observed clinical scores \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e at each of these stages are distinct and significantly different from baseline and from each other (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eIt is well established that immune cells, including CD68\u003csup\u003e+\u003c/sup\u003e macrophages, infiltrate nerves at the peak of EAN [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Here, to better understand the ability of immune cells to gain access to the endoneurium at different stages of disease, we quantified CD68\u003csup\u003e+\u003c/sup\u003e macrophages in cross sections of sciatic nerves collected from animals at onset, intermediate, and peak EAN. Nerves from na\u0026iuml;ve animals contain a limited number of macrophages (7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 cells per mm\u003csup\u003e2\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. At intermediate EAN, nerves contain a significant increase in macrophages (337.8\u0026thinsp;\u0026plusmn;\u0026thinsp;93.4 cells per mm\u003csup\u003e2\u003c/sup\u003e). This number doubles over the next two days (p\u0026thinsp;=\u0026thinsp;0.036), and nerves collected at EAN peak contain 678\u0026thinsp;\u0026plusmn;\u0026thinsp;94.49 CD68\u003csup\u003e+\u003c/sup\u003e cells per mm\u003csup\u003e2\u003c/sup\u003e. These quantitative findings are depicted by representative cross sections shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb.\u003c/p\u003e \u003cp\u003eIn addition to the immune cells that migrate into nerves during the course of EAN, the endothelial cells that form the blood nerve barrier (BNB) exhibit increased cell adhesion molecule expression and chemokine release [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We qualitatively assessed intercellular adhesion molecule (ICAM)-1 in longitudinal sections of sciatic nerves over the course of EAN. ICAM-1 was not detected in nerves from na\u0026iuml;ve animals, but staining was apparent at EAN onset and persisted through the peak of disease \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePermeation of a small molecule tracer over the course of EAN.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe utilized Evans blue dye as a small molecule tracer to assess changes in vascular permeability over the course of EAN. Evans Blue binds to serum albumin at high affinity upon intravenous administration resulting in an approximate molecular weight of 69 kDa [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We observed a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in dye permeation at EAN onset (Day 11) compared with na\u0026iuml;ve control rats \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. In control rats, the dye was restricted to the outer periphery of nerve cross sections \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. At EAN peak (Day 15), Evan\u0026rsquo;s blue dye permeation was at its maximum, a greater than 4-fold increase over control \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNanoparticle PEGylation and characterization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe investigated the accumulation of two sizes of polystyrene NPs corresponding with separate spectral wavelengths (λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;580/605 and λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;505/515). NPs were obtained as carboxylated FluoSpheres with nominal diameters of 40 nm and 100 nm, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For prolonged systemic circulation and reduction of protein opsonization (19), we covalently attached mPEG-amine to carboxyl functional groups via carbodiimide chemistry \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. PEGylation was confirmed by an increase in NP hydrodynamic diameter and a shift in surface charge or zeta potential. NP size increased from 53.15 nm to\u003c/p\u003e \u003cp\u003e64.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 nm (red) and from 124.3 nm to 136.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 nm (green, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. This change in size with PEGylation was identical for both FluoSphere formulations and is in line with values in the literature (17,30). Zeta potential of carboxylated NPs (-18 mV) shifted to -1.5 mV for both FluoSpheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAccumulation of nanoparticle tracers over the course of EAN.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential of vascular permeability to facilitate delivery of NPs to peripheral nerves over the course of EAN, we administered PEGylated NPs of varying diameters and wavelength (64 nm, λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;580/605 and 136 nm, λ\u003csub\u003eex\u003c/sub\u003e/λ\u003csub\u003eem\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;505/515) and quantified distribution in sciatic nerve cross sections using confocal microscopy \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Standardized regions of interest (ROIs) were selected around endoneurial vessels for quantitative assessment of NP accumulation relative to a respective control. Both NP sizes exhibited disease-stage dependent accumulation in nerves. Accumulation of 64 nm NPs increased significantly between onset and intermediate EAN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but the increase from intermediate to peak was not significant (p\u0026thinsp;=\u0026thinsp;0.2), Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. In contrast, 136nm NP accumulation was not significantly increased at EAN onset (p\u0026thinsp;=\u0026thinsp;0.66), but accumulation increased significantly between intermediate and peak EAN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Representative sciatic nerve cross sections illustrate both a lack of apparent endoneurial vessel accumulation in na\u0026iuml;ve animals as well as a time dependent increases in fluorescence intensity of NPs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe further compared accumulation of each tracer (small molecule and NP) at each stage of EAN \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. At the disease onset, the increases in Evans blue associated fluorescence (234\u0026thinsp;\u0026plusmn;\u0026thinsp;30.9%) were greater than those of the 64 nm and 136 nm NP tracers (153.1\u0026thinsp;\u0026plusmn;\u0026thinsp;18.31%, 119.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.57%), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). At intermediate and peak EAN, no significant difference was observed between the accumulation of each of the three tracers \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of NP accumulation in different regions of the nerve over the course of EAN.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that we induce EAN with a single injection of the antigen/adjuvant emulsion into the left hind footpad, and the injected foot exhibits local inflammation and edema over the course of disease, we sought to assess any lateral differences in distribution throughout the nerves. For both NP sizes, no significant difference was found between the accumulation in the left or right sciatic nerve over the disease course \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Further, given potential regional variation in blood flow through the microcirculation, we assessed NP accumulation in proximal and distal regions of the sciatic nerves. No significant difference was found between either of these regions over the course of the disease \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation between NP accumulation and EAN clinical score.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe analyzed our data by day post-EAN induction at defined disease stages: onset, intermediate, and peak of EAN. It follows that clinical scores also increase over the course of EAN. To confirm our hypothesis that increased clinical scores are associated with increased fluorescence, and to extrapolate our findings to other EAN models that may exhibit onset or peak on different days, we analyzed the correlation between clinical score and NP accumulation. We found a strong positive correlation between clinical score and NP accumulation for both 64 nm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e and 136 nm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e NPs, suggesting that more severe clinical symptoms are associated with increased BNB breakdown and enhanced vascular permeability.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe BNB represents a key obstacle in delivering therapeutic compounds to peripheral nerves under physiological conditions. Given advances in nanomedicine that have enabled passive delivery strategies to locally inflamed sites [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], including the inflamed or injured brain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we hypothesized that EAN-associated pathology provides an opportunity to access the peripheral nerves for targeted delivery. Our work builds upon early EAN studies demonstrating nerve permeation of horseradish peroxidase [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and Evans blue dye [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. While these studies were instrumental in identifying BNB permeability as a key pathological feature, the imaging data were not quantified and analyzed statistically over the course of disease. Further, while small molecule tracers can inform solute permeability, the approximate hydrodynamic diameters (~\u0026thinsp;4 nm for Evans blue associated with serum albumin [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]) do not represent the dimensions of potential NP drug delivery systems [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Here, we assessed immune cell infiltration, adhesion molecule expression, and BNB permeability to small molecules and two distinct sizes of NP tracers at defined stages of disease progression. Our data demonstrate endothelial ICAM-1 expression and small molecule permeability at EAN onset. At intermediate stages of EAN development, 64 nm NPs reach maximum accumulation in nerves, and at EAN peak 136 nm NPs reach maximum accumulation. These findings indicate that inflamed nerves can be accessed from the systemic circulation for therapeutic delivery.\u003c/p\u003e \u003cp\u003eMany previous studies by different groups have demonstrated protective or therapeutic attenuation of EAN with systemically administered compounds spanning a range of anti-inflammatory or immunomodulatory mechanisms. Each of these studies has contributed to our understanding of EAN development and progression. However, the investigated therapies themselves are not feasible treatment options due to supraphysiological required doses ranging from 40\u0026ndash;300 mg/kg [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], in some cases twice a day dosing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Further, many compounds exhibit off target effects and toxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. While some examples, such as sphingosine-1-phosphate inhibitors [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] or peroxisome proliferator-activated receptor gamma (PPARγ) antagonists [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], act systemically on immune organs and circulating immune cells, several examples act on sites within affected nerves. Given our findings that circulating NPs accumulate in affected nerves, PEGylated nanoformulations may represent a delivery strategy that allows for effective doses to be achieved at the desired sites.\u003c/p\u003e \u003cp\u003eTherapeutic targets within nerves may include axonal transcription factors, or channels or receptors at the nodes of Ranvier. Pitarokoili \u003cem\u003eet al\u003c/em\u003e demonstrated that dimethyl fumarate (DMF) reduces EAN severity by upregulating axonal levels of the transcription factor Nrf-2, leading to subsequent reduced demyelination and increased axonal survival [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. DMF would benefit from a NP delivery system to increase bioavailability and nerve levels, given its poor water solubility and 45 mg/kg twice a day dosing. Thrombin receptors localized at nodes of Ranvier may also serve as potential therapeutic targets [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thrombin activity increases during EAN, and activation of the axonal thrombin receptor protease activated receptor (PAR)-1 contributes to conduction blocks and destruction at the nodes of Ranvier. Administration of either a nonselective thrombin inhibitor (4.4 mg/kg/day) or a highly specific thrombin inhibitor (69.8 mg/kg/day) attenuated EAN. Given the role of thrombin inhibitors as anticoagulants, excessive bleeding was observed in animals post-mortem. Targeted delivery may alleviate this risk in GBS/EAN. Finally, the sodium channel blocker flecainide attenuated EAN when administered at 30 mg/kg twice daily starting after disease onset. The authors propose that by blocking sodium currents, flecainide prevents detrimental accumulation of sodium at sites of inflammation and prevents subsequent increases in axonal calcium ions. Sodium channel blockers are associated with arrhythmias and cardiac side effects [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which precludes systemic administration for GBS/EAN, particularly given the dosing for which beneficial effects on EAN were observed.\u003c/p\u003e \u003cp\u003eNerve endothelial cells, macrophages, or motor proteins may also represent therapeutic targets that would benefit from NP delivery systems. Sarkey \u003cem\u003eet al\u003c/em\u003e found that lovastatin therapeutically attenuated EAN (25 mg/kg/day) by limiting immune cell trafficking into nerves [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our subsequent work demonstrated that statins act directly on the BNB [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and that the therapeutic mechanism may involve inhibition of chemokine release from BNB endothelial cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The non-nitrogen containing bisphosphonate, clodronate, was shown to eliminate nerve macrophages and attenuate EAN when administered therapeutically after symptom onset [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The kinesin-5 inhibitor monastrol reduced EAN by enhancing neurite outgrowth within axons [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Importantly, this therapy was administered at peak of disease, which reflects the time when most patients are diagnosed with EAN.\u003c/p\u003e \u003cp\u003eDespite clear size- and disease stage-dependent patterns in NP associated fluorescence, and a lack of lateral or longitudinal variation, NP accumulation appears to be centered upon endoneurial blood vessels (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We focused on defined regions of interest that centered on endoneurial blood vessels to quantify our data. This observation has implications for the types of small molecules that would benefit from a NP delivery system. In the case of statins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], the therapeutic target (endothelial cells) is clearly accessible via passive accumulation of NPs. Clodronate may also act closely to the BNB to eliminate macrophages. In the case of axonal and neuronal targets, targets may not be fully accessible by passively accumulating NPs. Active targeting strategies may enable further penetration of NPs into the endoneurium [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and lipophilic payloads may diffuse more deeply into the endoneurium once released from polymeric NPs [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe induce EAN with a single injection of the antigen/adjuvant emulsion into the left hind footpad, and the injected foot exhibits local inflammation and edema over the course of disease. Further, nerves exhibit regional variation in blood flow through the microcirculation as well as inflammation associated edema [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. We assessed lateral and longitudinal distribution throughout the nerves to determine any regional variation in passive accumulation, and we did not observe any significant differences in NP accumulation between left and right or proximal and distal regions of the sciatic nerves. We did not assess permeation into nerve roots, given that the intermediate regions of the sciatic nerve are most restrictive to circulating molecules during homeostasis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, others have demonstrated that nerve roots become fenestrated earlier than the sciatic nerve during EAN [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and we anticipate that nerve roots would promote NP accumulation no less than observed in sciatic nerves. Collectively, our findings confirm that inflammation in EAN is not localized to one region and is multifocal and widespread along the length of nerves. This supports the need for systemic, rather than local, therapeutic approaches.\u003c/p\u003e \u003cp\u003eFor extrapolation of our findings to the investigations of systemically administered compounds discussed above, we must consider our defined disease stages in the context of other preclinical EAN studies. The clinical course of EAN in rat models induced with P2 peptide does not vary significantly between groups, with onset around Day 10\u0026thinsp;\u0026plusmn;\u0026thinsp;1 and peak at Day 16\u0026thinsp;\u0026plusmn;\u0026thinsp;1 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Elahi \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] induced \u0026ldquo;mild\u0026rdquo; and \u0026ldquo;severe\u0026rdquo; phenotypes by varying the concentration of CFA (1\u0026ndash;3 mg/ml) and number of injection sites (hind foot pad\u0026thinsp;\u0026plusmn;\u0026thinsp;base of tail). In the severe phenotype, EAN onset was on Day 8\u0026thinsp;\u0026plusmn;\u0026thinsp;1. While we analyzed mean fluorescence intensity at different days post-induction, we observed a strong correlation between clinical score and fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), suggesting that our findings can be applied to other EAN models, even if onset and peak are not at Day 11 and Day 15.\u003c/p\u003e \u003cp\u003eFor future translational therapeutic studies, it is critical to also consider how our findings align with the course of GBS/AIDP in human patients. Most GBS patients reach peak neuropathy within four weeks of onset [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], and diagnosis based on electrodiagnostic data is not possible until closer to peak disease [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, while early diagnosis occurs in some cases [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], EAN onset does not directly correspond with GBS diagnosis. In EAN rats, we demonstrated that 64 nm NPs can access the endoneurium \u003cem\u003ebefore\u003c/em\u003e clinical disease has peaked, at an intermediate stage between EAN onset and peak. Given that the time spent in each stage for patients is on the order of weeks, not days, we anticipate that both patients presenting with early symptoms as well as patients receiving a diagnosis at peak of GBS would be candidates for nanomedicine approaches. Importantly, one EAN study demonstrated benefits of their systemically administered compound (monastrol) when it was administered after peak disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Further, while intravenous administration is difficult to incorporate into outpatient medication paradigms [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], it will blend seamlessly with ongoing standard of care for GBS, including plasmapheresis and IVIg [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Additional study of BNB permeability in GBS patients, as well as continued investigation of NP fate throughout the body in EAN, is needed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we characterized BNB breakdown over the course of EAN. We demonstrated a disease stage dependent increase in immune infiltrates, adhesion molecule expression, and permeation of a small molecule dye. We demonstrated that NPs with a hydrodynamic diameter of 64 nm can cross the BNB and accumulate in nerves at intermediate EAN, and NPs with a hydrodynamic diameter of 136 nm cross the BNB and accumulate in nerves at peak EAN. These findings establish that EAN associated pathology enables passive nerve targeting of circulating NPs and set the foundation for further development of NP drug delivery strategies in diseases like inflammatory peripheral neuropathies that necessitate targeted, systemic administration.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis work was supported, in part, by funding from the Department of Veterans Affairs (RX002305) and start-up funding from Loyola University Chicago. We thank Zoe Adelstein for technical assistance. Peptide Synthesis was performed at the Peptide Synthesis Core Facility of the Simpson Querrey Institute for BioNanotechnology at Northwestern University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVan Doorn PA (2009) What\u0026rsquo;s new in Guillain-Barre syndrome in 2007\u0026ndash;2008? J Peripher Nerv Syst 74:72\u0026ndash;74\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillison HJ, Jacobs BC, van Doorn PA (2016) Guillain-Barr\u0026eacute; syndrome. Lancet 388(10045):717\u0026ndash;727\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpies JM, Westland KW, Bonner JG, Pollard JD (1995) Intraneural activated t cells cause focal breakdown of the blood-nerve barrier. Brain 118(4):857\u0026ndash;868\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu MO, Zhu J (2011) The role of cytokines in Guillain-Barr\u0026eacute; syndrome. J Neurol 258(4):533\u0026ndash;548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomikawa E, Mutsuga M, Hara K, Kaneko C, Togashi Y, Miyamoto Y (2019) Time Course of Axon and Myelin Degeneration in Peripheral Nerves in Experimental Autoimmune Neuritis Rats. Toxicol Pathol 47(4):542\u0026ndash;552\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatzav A, Bina H, Aronovich R, Chapman J (2013) Treatment for experimental autoimmune neuritis with clodronate (Bonefos). Immunol Res 56(2\u0026ndash;3):334\u0026ndash;340\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohle F, Ackfeld R, Hommen F, Klein I, Svačina MKR, Schneider C et al (2023) Kinesin-5 inhibition improves neural regeneration in experimental autoimmune neuritis. J Neuroinflammation [Internet]. ;20(1):1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12974-023-02822-w\u003c/span\u003e\u003cspan address=\"10.1186/s12974-023-02822-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePitarokoili K, Ambrosius B, Meyer D, Schrewe L, Gold R (2015) Dimethyl fumarate ameliorates lewis rat experimental autoimmune neuritis and mediates axonal protection. PLoS ONE 10(11):1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBechtold DA, Yue X, Evans RM, Davies M, Gregson NA, Smith KJ (2005) Axonal protection in experimental autoimmune neuritis by the sodium channel blocking agent flecainide. Brain 128(1):18\u0026ndash;28\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi C, Zhang Z, Wang W, Zug C, Schluesener HJ, Zhang Z (2011) Doxycycline attenuates peripheral inflammation in rat experimental autoimmune neuritis. Neurochem Res 36(11):1984\u0026ndash;1990\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkey JP, Richards MP, Stubbs EB (2007) Lovastatin attenuates nerve injury in an animal model of Guillain-Barr\u0026eacute; syndrome. J Neurochem 100(5):1265\u0026ndash;1277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangert KA, Brey EM (2018) Strategies for targeted delivery to the peripheral nerve. Front Neurosci 12(NOV):1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbram SE, Yi J, Fuchs A, Hogan QH (2006) Permeability of injured and intact peripheral nerves and dorsal root ganglia. Anesthesiology 105(1):146\u0026ndash;153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePutzu GA, Figarella-Branger D, Bouvier-Labit C, Liprandi A, Bianco N, Pellissier JF (2000) Immunohistochemical localization of cytokines, C5b-9 and ICAM-1 in peripheral nerve of Guillain-Barre Syndrome. J Neurol Sci 174(1):16\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerciano J, Sedano MJ, Pelayo-Negro AL, Garc\u0026iacute;a A, Orizaola P, Gallardo E et al (2017) Proximal nerve lesions in early Guillain\u0026ndash;Barr\u0026eacute; syndrome: implications for pathogenesis and disease classification. J Neurol 264(2):221\u0026ndash;236\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennett J, Basivireddy J, Kollar A, Reickmann P, Jefferies W, McQuaid S (2010) Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE. J Immunol 229(1\u0026ndash;2):180\u0026ndash;191\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBharadwaj VN, Lifshitz J, Adelson PD, Kodibagkar VD, Stabenfeldt SE (2016) Temporal assessment of nanoparticle accumulation after experimental brain injury: Effect of particle size. Sci Rep [Internet]. ;6(April):1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1038/srep29988\u003c/span\u003e\u003cspan address=\"10.1038/srep29988\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurymanov M, Kamaletdinova T, Lehmann SE, Reineke J (2017) Exploiting passive nanomedicine accumulation at sites of enhanced vascular permeability for non-cancerous applications. J Control Release [Internet]. ;261(June):10\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jconrel.2017.06.013\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2017.06.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexis F, Pridgen E, Molnar LK, Farokhzad OC (2008) Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 5(4):505\u0026ndash;515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafiei P, Haddadi A (2017) Docetaxel-loaded PLGA and PLGA-PEG nanoparticles for intravenous application: Pharmacokinetics and biodistribution profile. Int J Nanomed 12:935\u0026ndash;947\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBae YH, Park K (2011) Targeted drug delivery to tumors: Myths, reality and possibility. J Control Release [Internet]. ;153(3):198\u0026ndash;205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.jconrel.2011.06.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2011.06.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubhan MA, Yalamarty SSK, Filipczak N, Parveen F, Torchilin VP (2021) Recent advances in tumor targeting via epr effect for cancer treatment. J Pers Med. ;11(6)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerciano J (2021) Axonal degeneration in Guillain\u0026ndash;Barr\u0026eacute; syndrome: a reappraisal. J Neurol 268(10):3728\u0026ndash;3743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangert KA, Goshu B, Stubbs EB (2017) Attenuation of experimental autoimmune neuritis with locally administered lovastatin-encapsulating poly(lactic-co-glycolic) acid nanoparticles. J Neurochem\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalik MW, Shankarappa SA, Langert KA, Stubbs EB (2015) Forced exercise preconditioning attenuates experimental autoimmune neuritis by altering Th1 lymphocyte composition and egress. ASN Neuro [Internet]. ;7(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/1759091415595726\u003c/span\u003e\u003cspan address=\"10.1177/1759091415595726\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalik MW, Shankarappa SA, Langert KA, Stubbs EB (2015) Forced exercise preconditioning attenuates experimental autoimmune neuritis by altering Th1 lymphocyte composition and egress. ASN Neuro. ;7(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbrosius B, Pitarokoili K, Schrewe L, Pedreiturria X, Motte J, Gold R (2017) Fingolimod attenuates experimental autoimmune neuritis and contributes to Schwann cell-mediated axonal protection. J Neuroinflammation 14(1):1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao L, Xue X, Yu P, Ni Y, Chen F (2018) Evans Blue Dye: A Revisit of Its Applications in Biomedicine. Contrast Media Mol Imaging 2018:18\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScalisi J, Balau B, Deneyer L, Bouchat J, Gilloteaux J, Nicaise C (2021) Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome. Neurosci Lett [Internet]. ;746(August 2020):135665. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neulet.2021.135665\u003c/span\u003e\u003cspan address=\"10.1016/j.neulet.2021.135665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHouseholder KT, Dharmaraj S, Sandberg DI, Wechsler-Reya RJ, Sirianni RW (2019) Fate of nanoparticles in the central nervous system after intrathecal injection in healthy mice. Sci Rep 9(1):1\u0026ndash;11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell HC, Braheny S, Myers RR, Rodriguez M, Lampert P (1983) Early changes in experimental allergic neuritis. Lab Investig 48(3):332\u0026ndash;338\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHahn A, Feasby T, Gilbert J (1985) Blood nerve barrier studies in experimental allergic neuritis. Acta Neuropathol 68:101\u0026ndash;109\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A (2006) Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst 98(5):335\u0026ndash;344\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Zhang ZY, Fauser U, Schluesener HJ (2008) Valproic acid attenuates inflammation in experimental autoimmune neuritis. Cell Mol Life Sci 65(24):4055\u0026ndash;4065\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan F, Luo B, Shi R, Han C, Zhang Z, Xiong J et al (2014) Curcumin ameliorates rat experimental autoimmune neuritis. J Neurosci Res 92(6):743\u0026ndash;750\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamkalawan H, Wang YZ, Hurbungs A, Yang YF, Tian FF, Zhou W, Bin et al (2012) Pioglitazone, PPARgamma agonist, attenuates experimental autoimmune neuritis. Inflammation 35(4):1338\u0026ndash;1347\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShavit-Stein E, Aronovich R, Sylantiev C, Gera O, Gofrit SG, Chapman J et al (2019) Blocking thrombin significantly ameliorates experimental autoimmune neuritis. Front Neurol 10(JAN):3\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunger MA, Olğar Y, Koleske ML, Struckman HL, Mandrioli J, Lou Q et al (2020) Tetrodotoxin-sensitive neuronal-type na\u0026thinsp;+\u0026thinsp;channels: A novel and druggable target for prevention of atrial fibrillation. J Am Heart Assoc. ;9(11)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangert KA, Pervan CL, Stubbs EB (2014) Novel role of cdc42 and RalA GTpases in TNF-α mediated secretion of CCL2. Small GTPases. ;5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangert KA, Von Zee CL, Stubbs EB (2013) Cdc42 GTPases facilitate TNF-α-mediated secretion of CCL2 from peripheral nerve microvascular endoneurial endothelial cells. J Peripher Nerv Syst 18(3):199\u0026ndash;208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChittasupho C, Xie S-X, Baoum A, Yakovleva T, Siahaan T, Berkland CJ (2009) ICAM-1 targeting of doxorubicin-loaded PLGA nanoparticles to lung epithelial cells. Eur J Pharm Sci 37(2):141\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang F, Cabe MH, Ogle SD, Sanchez V, Langert KA (2021) Optimization of critical parameters for coating of polymeric nanoparticles with plasma membrane vesicles by sonication. Sci Rep [Internet]. ;11(1):1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-03422-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-03422-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R, He Y, Zhu Y, Jiang L, Zhang S, Qin J et al (2019) Route to Rheumatoid Arthritis by Macrophage-Derived Microvesicle-Coated Nanoparticles. Nano Lett 19(1):124\u0026ndash;134\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCook RL, Householder KT, Chung EP, Prakapenka AV, Diperna DM, Sirianni RW (2015) A critical evaluation of drug delivery from ligand modified nanoparticles: Confounding small molecule distribution and efficacy in the central nervous system. J Control Release 220:89\u0026ndash;97\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedina DX, Householder KT, Ceton R, Kovalik T, Heffernan JM, Shankar RV et al (2017) Optical barcoding of PLGA for multispectral analysis of nanoparticle fate in vivo. J Control Release [Internet]. ;253:172\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1016/j.jconrel.2017.02.033\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel.2017.02.033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrahm C, Heinzel J, Kolbenschlag J (2022) Blood Supply and Microcirculation of the Peripheral Nerve. In: Philips JB (ed) Peripheral Nerve Tissue Engineering and Regeneration. Springer Nature Switzerland, pp 35\u0026ndash;79\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozu H, Tamura E, Parry G (1992) Endoneurial blood supply to peripheral nerves is not uniform. J Neurol Sci 111:204\u0026ndash;208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Chen Y, Huang L, Sun X, Fu T, Wu S et al (2018) Drug distribution into peripheral nerve. J Pharmacol Exp Ther 365(2):336\u0026ndash;345\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElahi E, Ali ME, Zimmermann J, Getts DR, M\u0026uuml;ller M, Lamprecht A (2022) Immune Modifying Effect of Drug Free Biodegradable Nanoparticles on Disease Course of Experimental Autoimmune Neuritis. Pharmaceutics. ;14(11)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUbogu EE (2015) Inflammatory neuropathies: pathology, molecular markers and targets for specific therapeutic intervention. Acta Neuropathol 130(4):445\u0026ndash;468\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLucas M, Hugh-Jones K, Welby A, Misbah S, Spaeth P, Chapel H (2010) Immunomodulatory therapy to achieve maximum efficacy: Doses, monitoring, compliance, and self-infusion at home. J Clin Immunol 30(SUPPL 1):84\u0026ndash;89\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMejia-Chew C, Heuring B, Salmons J, Weilmuenster L, Beggs J, Kleinschmidt G et al (2024) IVsight as an Infusion Monitor for Patients Receiving Intravenous Therapy: An Exploratory, Unblinded, Single-Center Trial. Curr Ther Res - Clin Exp [Internet]. ;100:100747. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.curtheres.2024.100747\u003c/span\u003e\u003cspan address=\"10.1016/j.curtheres.2024.100747\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNominal, measured, and PEGylated nanoparticle diameters.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNominal diameter (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured diameter (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePEGylated diameter (nm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e53.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e64.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e124.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e136.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"c30db6cd-84ef-45b1-b797-8bd52e601cb0","identifier":"10.13039/100000738","name":"U.S. Department of Veterans Affairs","awardNumber":"RX002305","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Loyola University Chicago","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Peripheral nerve, nanoparticle, blood-nerve barrier, experimental autoimmune neuritis","lastPublishedDoi":"10.21203/rs.3.rs-4631228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4631228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGuillain-Barr\u0026eacute; syndrome (GBS) is a devastating autoimmune disease of the peripheral nervous system (PNS) for which treatment options are strictly palliative. Several studies have shown attenuation of the well-characterized preclinical experimental autoimmune neuritis (EAN) model with systemically administered therapeutic compounds via a range of anti-inflammatory or immunomodulatory mechanisms. Despite this, clinical advancement of these findings is limited by dosing that is not translatable to humans or is associated with off-target and toxic effects. This is due, in part, to the blood-nerve barrier (BNB), which restricts access of the circulation to peripheral nerves. Here, we assessed the degree to which BNB permeability and immune cell infiltration over the course of EAN enable passive accumulation of circulating nanoparticles. We found that at stages of EAN defined by distinct clinical scores and pathology (onset, intermediate, peak), intravenously administered small molecules and nanoparticles ranging from 50\u0026ndash;150 nm can permeate into the endoneurium from the endoneurial vasculature in a size- and disease stage-dependent manner. This permeation occurs uniformly in both sciatic nerves and in proximal and distal regions of the nerves. We propose that this passive targeting serves as a platform by which potential therapies for GBS can be reevaluated and investigated preclinically in nanoparticle delivery systems.\u003c/p\u003e","manuscriptTitle":"Blood nerve barrier permeability enables nerve targeting of circulating nanoparticles in experimental autoimmune neuritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 12:55:03","doi":"10.21203/rs.3.rs-4631228/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea052d60-3f49-44f9-8110-3ad85921d068","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33667367,"name":"Biomedical Engineering"},{"id":33667368,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2024-06-27T12:55:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-27 12:55:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4631228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4631228","identity":"rs-4631228","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}