Longitudinal dynamics of microvascular recovery after acquired cortical injury.

Acquired brain injuries due to trauma damage the cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury, but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14 and 21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (> 200 µm), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.

Viral mimetic triggers cerebral arteriopathy in juvenile brain via neutrophil elastase and NETosis.

Stroke is among the top ten causes of death in children but has received disproportionally little attention. Cerebral arteriopathies account for up to 80% of childhood arterial ischemic stroke (CAIS) cases and are strongly predictive of CAIS recurrence and poorer outcomes. The underlying mechanisms of sensitization of neurovasculature by viral infection are undefined. In the first age-appropriate model for childhood arteriopathy-by administration of viral mimetic TLR3-agonist Polyinosinic:polycytidylic acid (Poly-IC) in juvenile mice-we identified a key role of the TLR3-neutrophil axis in disrupting the structural-functional integrity of the blood-brain barrier (BBB) and distorting the developing neurovascular architecture and vascular networks. First, using an array of in-vivo/post-vivo vascular imaging, genetic, enzymatic and pharmacological approaches, we report marked Poly-IC-mediated extravascular leakage of albumin (66kDa) and of a small molecule DiI (∼934Da) and disrupted tight junctions. Poly-IC also enhanced the neuroinflammatory milieu, promoted neutrophil recruitment, profoundly upregulated neutrophil elastase (NE), and induced neutrophil extracellular trap formation (NETosis). Finally, we show that functional BBB disturbances, NETosis and neuroinflammation are markedly attenuated by pharmacological inhibition of NE (Sivelestat). Altogether, these data reveal NE/NETosis as a novel therapeutic target for viral-induced cerebral arteriopathies in children.

Acute Treatment With Gleevec Does Not Promote Early Vascular Recovery Following Intracerebral Hemorrhage in Adult Male Rats.

Intracerebral hemorrhage (ICH) remains one of the most debilitating types of stroke and is characterized by a sudden bleeding from a ruptured blood vessel. ICH often results in high mortality and in survivors, permanent disability. Most studies have focused on neuroprotective strategies designed to minimize secondary consequences and prevent further pathology. Lacking is an understanding of how ICH acutely affects cerebrovascular components and their response to therapeutic interventions. We hypothesized that ICH alters cortical vessel complexity in the parenchyma adjacent to site of the initial vascular disruption and that vascular abnormalities would be mitigated by administration of the PDGFR inhibitor, Imatinib mesylate (Gleevec). Briefly, ICH was induced in male adult rats by injection of collagenase into basal ganglia, followed by Gleevec administration (60 mg/kg) 1 h after injury. Rats were then perfused using vessel painting methodology (Salehi et al., 2018b) to stain whole brain vascular networks at 1 day post-ICH. Axial and coronal wide field fluorescence microscopy was performed. Analyses for vascular features were undertaken and fractal analysis for vascular complexity. Data were collected from four groups of rats: Sham + Vehicle; Sham + Gleevec; ICH + Vehicle; ICH + Gleevec. Microscopy revealed that cortical vessels in both ipsi- and contralateral hemispheres exhibited significantly reduced density and branching by 22 and 34%, respectively. Fractal measures confirmed reduced complexity as well. Gleevec treatment further reduced vascular parameters, including reductions in vessel density in tissues adjacent to the ICH. The reductions in brain wide vascular networks after Gleevec in the current study after ICH is contrasted by previous reports of improved behavioral outcomes and decreased lCH lesion volumes Reductions in the vascular network after Gleevec may be involved in long-term repair mechanisms by pruning injured vessels to ultimately promote new vessel growth.

Characterization and preclinical evaluation of the cGMP grade DNA based vaccine, AV-1959D to enter the first-in-human clinical trials.

The DNA vaccine, AV-1959D, targeting N-terminal epitope of Aß peptide, has been proven immunogenic in mice, rabbits, and non-human primates, while its therapeutic efficacy has been shown in mouse models of Alzheimer's disease (AD). Here we report for the first time on IND-enabling biodistribution and safety/toxicology studies of cGMP-grade AV-1959D vaccine in the Tg2576 mouse model of AD. We also tested acute neuropathology safety profiles of AV-1959D in another AD disease model, Tg-SwDI mice with established vascular and parenchymal Aß pathology in a pre-clinical translational study. Biodistribution studies two days after the injection demonstrated high copy numbers of AV-1959D plasmid after single immunization of Tg2576 mice at the injection sites but not in the tissues of distant organs. Plasmids persisted at the injection sites of some mice 60 days after vaccination. In Tg2576 mice with established amyloid pathology, we did not observe short- or long-term toxicities after multiple immunizations with three doses of AV-1959D. Assessment of the repeated dose acute safety of AV-1959D in cerebral amyloid angiopathy (CAA) prone Tg-SwDI mice did not reveal any immunotherapy-induced vasogenic edema detected by magnetic resonance imaging (MRI) or increased microhemorrhages. Multiple immunizations of Tg-SwDI mice with AV-1959D did not induce T and B cell infiltration, glial activation, vascular deposition of Aß, or neuronal degeneration (necrosis and apoptosis) greater than that in the control group determined by immunohistochemistry of brain tissues. Taken together, the safety data from two different mouse models of AD substantiate a favorable safety profile of the cGMP grade AV-1959D vaccine supporting its progression to first-in-human clinical trials.

Acute intranasal osteopontin treatment in male rats following TBI increases the number of activated microglia but does not alter lesion characteristics.

Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood-brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.

CX3CR1-CCR2-dependent monocyte-microglial signaling modulates neurovascular leakage and acute injury in a mouse model of childhood stroke.

Stroke is among the top 10 causes of death in children. The developmental stage of the brain is central to stroke pathophysiology. The incidence of childhood arterial ischemic stroke (CAIS) is lower than of perinatal arterial ischemic stroke but the rate of recurrence is strikingly high. Vascular inflammation is seen as major contributor to CAIS but the mechanisms that govern structural-functional basis of vascular abnormalities remain poorly understood. To identify the contribution of immune-neurovascular interactions to CAIS, we established stroke model in postnatal day 21 (P21) mice. We demonstrate acute functional deficits and histological injury and chronic MRI-identifiable injury, brain atrophy and marked derangements in the vascular network. In contrast to negligible albumin leakage and neutrophil infiltration following acute perinatal stroke, CAIS leads to significantly increased albumin leakage and neutrophil infiltration in injured regions of wild type mice and mice with functional CX3CR1-CCR2 receptors. In mice with dysfunctional CX3CR1-CCR2 signaling, extravascular albumin leakage is significantly attenuated, infiltration of injurious Ccr2+-monocytes essentially aborted, accumulation of Ly6G+ neutrophils reduced and acute injury attenuated. Unique identifiers of microglia and monocytes revealed phenotypic changes in each cell subtype of the monocyte lineage after CAIS. Taken together, CX3CR1-CCR2-dependent microglia-monocyte signaling contributes to cerebrovascular leakage, inflammation and CAIS injury.

Repeated Pediatric Concussions Evoke Long-Term Oligodendrocyte and White Matter Microstructural Dysregulation Distant from the Injury.

Concussion or mild traumatic brain injury (mTBI) is often accompanied by long-term behavioral and neuropsychological deficits. Emerging data suggest that these deficits can be exacerbated following repeated injuries. However, despite the overwhelming prevalence of mTBI in children due to falls and sports-related activities, the effects of mTBI on white matter (WM) structure and its development in children have not been extensively examined. Moreover, the effect of repeated mTBI (rmTBI) on developing WM has not yet been studied, despite the possibility of exacerbated outcomes with repeat injuries. To address this knowledge gap, we investigated the long-term effects of single (s)mTBI and rmTBI on the WM in the pediatric brain, focusing on the anterior commissure (AC), a WM structure distant to the injury site, using diffusion tensor imaging (DTI) and immunohistochemistry (IHC). We hypothesized that smTBI and rmTBI to the developing mouse brain would lead to abnormalities in microstructural integrity and impaired oligodendrocyte (OL) development. We used a postnatal day 14 Ascl1-CreER: ccGFP mouse closed head injury (CHI) model with a bilateral repeated injury. We demonstrate that smTBI and rmTBI differentially lead to myelin-related diffusion changes in the WM and to abnormal OL development in the AC, which are accompanied by behavioral deficits 2 months after the initial injury. Our results suggest that mTBIs elicit long-term behavioral alterations and OL-associated WM dysregulation in the developing brain. These findings warrant additional research into the development of WM and OL as key components of pediatric TBI pathology and potential therapeutic targets.

A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents.

We introduce a novel protocol to stain, visualize, and analyze blood vessels from the rat and mouse cerebrum. This technique utilizes the fluorescent dye, DiI, to label the lumen of the vasculature followed by perfusion fixation. Following brain extraction, the labeled vasculature is then imaged using wide-field fluorescence microscopy for axial and coronal images and can be followed by regional confocal microscopy. Axial and coronal images can be analyzed using classical angiographic methods for vessel density, length, and other features. We also have developed a novel fractal analysis to assess vascular complexity. Our protocol has been optimized for adult rat, adult mouse, and neonatal mouse studies. The protocol is efficient, can be rapidly completed, stains cerebral vessels with a bright fluorescence, and provides valuable quantitative data. This method has a broad range of applications, and we demonstrate its use to study the vasculature in assorted models of acquired brain injury.

Corpus Callosum Vasculature Predicts White Matter Microstructure Abnormalities after Pediatric Mild Traumatic Brain Injury.

Emerging data suggest that pediatric traumatic brain injury (TBI) is associated with impaired developmental plasticity and poorer neuropsychological outcomes than adults with similar head injuries. Unlike adult mild TBI (mTBI), the effects of mTBI on white matter (WM) microstructure and vascular supply are not well understood in the pediatric population. The cerebral vasculature plays an important role providing necessary nutrients and removing waste. To address this critical element, we examined the microstructure of the corpus callosum (CC) following pediatric mTBI using diffusion tensor magnetic resonance imaging (DTI), and investigated myelin, oligodendrocytes, and vasculature of WM with immunohistochemistry (IHC). We hypothesized that pediatric mTBI leads to abnormal WM microstructure and impacts the vasculature within the CC, and that these alterations to WM vasculature contribute to the long-term altered microstructure. We induced in mice a closed-head injury (CHI) mTBI at post-natal day (P) 14; then at 4, 14, and 60 days post-injury (DPI) mice were sacrificed for analysis. We observed persistent changes in apparent diffusion coefficient (ADC) within the ipsilateral CC following mTBI, indicating microstructural changes, but surprisingly changes in myelin and oligodendrocyte densities were minimal. However, vascular features of the ipsilateral CC such as vessel density, length, and number of junctions were persistently altered following mTBI. Correlative analysis showed a strong inverse relationship between ADC and vessel density at 60 DPI, suggesting increased vessel density following mTBI may restrict WM diffusion characteristics. Our findings suggest that WM vasculature contributes to the long-term microstructural changes within the ipsilateral CC following mTBI.

Male and Female Mice Exhibit Divergent Responses of the Cortical Vasculature to Traumatic Brain Injury.

We previously reported that traumatic brain injuries (TBI) alter the cerebrovasculature near the injury site in rats, followed by revascularization over a 2-week period. Here, we tested our hypothesis that male and female adult mice have differential cerebrovascular responses following a moderate controlled cortical impact (CCI). Using in vivo magnetic resonance imaging (MRI), a new technique called vessel painting, and immunohistochemistry, we found no differences between males and females in lesion volume, neurodegeneration, blood-brain barrier (BBB) alteration, and microglia activation. However, females exhibited more astrocytic hypertrophy and heme-oxygenase-1 (HO-1) induction at 1 day post-injury (dpi), whereas males presented with increased endothelial activation and expression of ß-catenin, shown to be involved in angiogenesis. At 7 dpi, we observed an increase in the number of vessels and an enhancement in vessel complexity in the injured cortex of males compared with females. Cerebrovasculature recovers differently after CCI, suggesting biological sex should be considered when designing new therapeutic agents.

Up-regulation of Wnt/ß-catenin expression is accompanied with vascular repair after traumatic brain injury.

Recent data suggest that repairing the cerebral vasculature after traumatic brain injury (TBI) may help to improve functional recovery. The Wnt/ß-catenin signaling pathway promotes blood vessel formation during vascular development, but its role in vascular repair after TBI remains elusive. In this study, we examined how the cerebral vasculature responds to TBI and the role of Wnt/ß-catenin signaling in vascular repair. We induced a moderate controlled cortical impact in adult mice and performed vessel painting to visualize the vascular alterations in the brain. Brain tissue around the injury site was assessed for ß-catenin and vascular markers. A Wnt transgenic mouse line was utilized to evaluate Wnt gene expression. We report that TBI results in vascular loss followed by increases in vascular structure at seven days post injury (dpi). Immature, non-perfusing vessels were evident in the tissue around the injury site. ß-catenin protein expression was significantly reduced in the injury site at 7 dpi. However, there was an increase in ß-catenin expression in perilesional vessels at 1 and 7 dpi. Similarly, we found increased number of Wnt-GFP-positive vessels after TBI. Our findings suggest that Wnt/ß-catenin expression contributes to the vascular repair process after TBI.

Traumatic brain injury results in acute rarefication of the vascular network.

The role of the cerebrovascular network and its acute response to TBI is poorly defined and emerging evidence suggests that cerebrovascular reactivity is altered. We explored how cortical vessels are physically altered following TBI using a newly developed technique, vessel painting. We tested our hypothesis that a focal moderate TBI results in global decrements to structural aspects of the vasculature. Rats (naïve, sham-operated, TBI) underwent a moderate controlled cortical impact. Animals underwent vessel painting perfusion to label the entire cortex at 1 day post TBI followed by whole brain axial and coronal images using a wide-field fluorescence microscope. Cortical vessel network characteristics were analyzed for classical angiographic features (junctions, lengths) wherein we observed significant global (both hemispheres) reductions in vessel junctions and vessel lengths of 33% and 22%, respectively. Biological complexity can be quantified using fractal geometric features where we observed that fractal measures were also reduced significantly by 33%, 16% and 13% for kurtosis, peak value frequency and skewness, respectively. Acutely after TBI there is a reduction in vascular network and vascular complexity that are exacerbated at the lesion site and provide structural evidence for the bilateral hemodynamic alterations that have been reported in patients after TBI.

Mild Concussion, but Not Moderate Traumatic Brain Injury, Is Associated with Long-Term Depression-Like Phenotype in Mice.

Mild traumatic brain injuries can lead to long-lasting cognitive and motor deficits, increasing the risk of future behavioral, neurological, and affective disorders. Our study focused on long-term behavioral deficits after repeated injury in which mice received either a single mild CHI (mCHI), a repeated mild CHI (rmCHI) consisting of one impact to each hemisphere separated by 3 days, or a moderate controlled cortical impact injury (CCI). Shams received only anesthesia. Behavioral tests were administered at 1, 3, 5, 7, and 90 days post-injury (dpi). CCI animals showed significant motor and sensory deficits in the early (1-7 dpi) and long-term (90 dpi) stages of testing. Interestingly, sensory and subtle motor deficits in rmCHI animals were found at 90 dpi. Most importantly, depression-like behaviors and social passiveness were observed in rmCHI animals at 90 dpi. These data suggest that mild concussive injuries lead to motor and sensory deficits and affective disorders that are not observed after moderate TBI.

Susceptibility-Weighted Imaging Identifies Iron-Oxide-Labeled Human Neural Stem Cells: Automated Computational Detection.

Neonatal hypoxic-ischemic brain injury (HII) can lead to devastating neurological outcomes such as cerebral palsy, epilepsy, and mental retardation. Human neural stem cell (hNSC) therapy provides new hope for the treatment of neonatal HII. These multipotent cells can aid in HII recovery by activating multiple reparative mechanisms including secretion of neurotrophic factors that enhance brain repair and plasticity. For clinical use of implanted hNSCs, methods are required to identify, quantify, track, and visualize migration and replication in an automated and reproducible fashion. In the current study, we used a model of unilateral HII in 10-day-old rat pups that were implanted with 250,000 Feridex-labeled hNSCs into the contralateral ventricle 3 days after HII. In addition to standard noninvasively acquired serial magnetic resonance imaging (MRI) sequences (11.7 and 4.7 T) that included diffusion-weighted imaging and T2-weighted imaging, we also acquired susceptibility-weighted imaging (SWI) 1-90 days after hNSC implantation. SWI is an advanced MRI method that enhances the visualization of iron-oxide-labeled hNSCs within affected regions of the injured neonatal brain. hNSC contrast was further enhanced by creating minimal intensity projections from the raw SWI magnitude images combined with phase information. Automated computational analysis using hierarchical region splitting (HRS) was applied for semiautomatic detection of hNSCs from SWI images. We found hNSCs in the ipsilateral HII lesion within the striatum and cortex adjacent to the lesion that corresponded to histological staining for iron. Quantitative phase values (radians) obtained from SWI revealed temporally evolving increased phase which reflects a decreased iron oxide content that is possibly related to cell division and the replicative capacity of the implanted hNSCs. SWI images also revealed hNSC migration from the contralateral injection site towards the ipsilateral HII lesion. Our results demonstrate that MRI-based SWI can monitor iron-labeled hNSCs in a clinically relevant manner and that automated computational methods such as HRS can rapidly identify iron-oxide-labeled hNSCs.

Neuroprotective effect of hyperbaric oxygen therapy in a juvenile rat model of repetitive mild traumatic brain injury.

Repetitive mild traumatic brain injury (rmTBI) is an important medical concern for adolescent athletes that can lead to long-term disabilities. Multiple mild injuries may exacerbate tissue damage resulting in cumulative brain injury and poor functional recovery. In the present study, we investigated the increased brain vulnerability to rmTBI and the effect of hyperbaric oxygen treatment using a juvenile rat model of rmTBI. Two episodes of mild cortical controlled impact (3 days apart) were induced in juvenile rats. Hyperbaric oxygen (HBO) was applied 1 hour/day × 3 days at 2 atmosphere absolute consecutively, starting at 1 day after initial mild traumatic brain injury (mTBI). Neuropathology was assessed by multi-modal magnetic resonance imaging (MRI) and tissue immunohistochemistry. After repetitive mTBI, there were increases in T2-weighted imaging-defined cortical lesions and susceptibility weighted imaging-defined cortical microhemorrhages, correlated with brain tissue gliosis at the site of impact. HBO treatment significantly decreased the MRI-identified abnormalities and tissue histopathology. Our findings suggest that HBO treatment improves the cumulative tissue damage in juvenile brain following rmTBI. Such therapy regimens could be considered in adolescent athletes at the risk of repeated concussions exposures.

Claudin-4: functional studies beyond the tight junction.

Claudin-4 is an unusual member of the claudin family; in addition to its role in epithelial tight junction barrier function, it is a receptor for the Clostridium perfringens enterotoxin. We have also found that claudin-4 is regulated in mucosal epithelium M cells, both in increased expression of the protein and in redistribution into endocytosis vesicles. Our ongoing studies are studying the potential for developing ligands specific to claudin-4 for targeted delivery of cargo such as proteins and poly(DL-lactide-co-glycolide) nanoparticles to mucosal M cells. Methods for the study of claudin-4 movement within epithelial cells, and delivery of nanoparticles through targeted binding of claudin-4 are described.

CD137 is required for M cell functional maturation but not lineage commitment.

Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's patch and nasopharyngeal associated lymphoid tissue to transport particles to underlying lymphocytes. M cell development is associated with B lymphocytes in a basolateral pocket, but the interactions between these cells are poorly understood. In a cell culture model of M cell differentiation, we found lymphotoxin/tumor necrosis factor alpha induction of CD137 (TNFRSF9) protein on intestinal epithelial cell lines, raising the possibility that CD137 on M cells in vivo might interact with CD137L expressed by B cells. Accordingly, while CD137-deficient mice produced UEA-1+ M cell progenitors in nasopharyngeal associated lymphoid tissue and Peyer's patch epithelium, they showed an abnormal morphology, including the absence of basolateral B cell pockets. More important, CD137-deficient nasopharyngeal associated lymphoid tissue M cells were defective in microparticle transcytosis. Bone marrow irradiation chimeras confirmed that while induction of UEA-1+ putative M cell precursors was not CD137-dependent, full M cell transcytosis function required expression of CD137 by radioresistant stromal cells as well as by bone marrow-derived cells. These results are consistent with a two-step model of M cell differentiation, with initial CD137-independent commitment to the M cell lineage followed by a CD137-CD137L interaction of M cells with CD137-activated B lymphocytes or dendritic cells for functional maturation.

Intranasal M cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength.

In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery.

Claudin 4-targeted protein incorporated into PLGA nanoparticles can mediate M cell targeted delivery.

Polymer-based microparticles are in clinical use mainly for their ability to provide controlled release of peptides and compounds, but they are also being explored for their potential to deliver vaccines and drugs as suspensions directly into mucosal sites. It is generally assumed that uptake is mediated by epithelial M cells, but this is often not directly measured. To study the potential for optimizing M cell uptake of polymer microparticles in vivo, we produced sub-micron size PLGA particles incorporating a recombinant protein. This recombinant protein was produced with or without a c-terminal peptide previously shown to have high affinity binding to Claudin 4, a protein associated with M cell endocytosis. While the PLGA nanoparticles incorporate the protein throughout the matrix, much of the protein was also displayed on the surface, allowing us to take advantage of the binding activity of the targeting peptide. Accordingly, we found that instillation of these nanoparticles into the nasal passages or stomach of mice was found to significantly enhance their uptake by upper airway and intestinal M cells. Our results suggest that a reasonably simple nanoparticle manufacture method can provide insight into developing an effective needle-free delivery system.