Intraluminal Behavior of Various Transporter Substrates in the Rat Gastrointestinal Tract.

PURPOSE: The aim of this study was to evaluate the intraluminal behavior of various transporter substrates in different regions of the gastrointestinal (GI) tract. METHODS: Drug solutions containing non-absorbable FITC-dextran 4000 (FD-4), were orally administered to rats. Residual water was sampled from the GI regions to measure the luminal drug concentration. RESULTS: Cephalexin (CEX), a substrate of the proton-coupled oligopeptide transporter, was absorbed rapidly, and no drug was detected in the lower small intestine. Saquinavir (SQV) was primarily absorbed in the upper region. However, unlike CEX, SQV was detected even in the lower segment probably due to the efflux of SQV via P-glycoprotein (P-gp). The concentration of methotrexate (MTX) showed a similar pattern to that of non-absorbable FD-4. The low absorption of MTX was probably due to efflux via several efflux transporters, and the limited expression of proton-coupled folate transporter, an absorptive transporter for MTX, in the upper region. CONCLUSION: This study revealed that the luminal concentration pattern of each drug differed considerably depending on the site because of the different absorption properties and luminal volumes. Although further investigation using a specific transporter inhibitor or transporter-knockout animals are necessary to clarify the actual contribution of each transporter to the drug absorption, this information will be valuable in evaluating transporter-mediated drug absorption in in vitro transport studies for ensuring optimal drug concentrations.

Impact of Dietary Intake of Medium-Chain Triacylglycerides on the Intestinal Absorption of Poorly Permeable Compounds.

The present study sought to demonstrate the effect of dietary intake of medium-chain triacylglycerides (MCTs) on the intestinal absorption of a poorly permeable compound of intermediate molecular weight (FITC-dextran 4000 [FD-4]). As a model of MCTs, C8-C12 fatty acid triacylglyceride (COCONAD ML) was mainly used, and the dose strength of each triglyceride was set with consideration of the dietary ingestion dose (12.5 mg/rat). When FD-4 with MCTs dispersed in fasted state simulated intestinal fluid containing surfactants was administered into the rat jejunum, the intestinal absorption of FD-4 was significantly higher than when administered with a similar solution with or without corn oil (long-chain triglycerides). The effects of pretreatment by MCT lipolysis, inhibition of endogenous lipases, and different dose timings of MCTs and FD-4 on the intestinal absorption of FD-4 indicated that medium-chain fatty acids, such as caprylic acid and capric acid, released from MCTs by lipolysis in the small intestine significantly enhanced the intestinal absorption of FD-4, but the effect was transient. In addition, a similar effect was observed when MCTs were dispersed in soymilk, although large interindividual variation was detected. These findings suggested that dietary intake of MCTs might affect the intestinal absorption of poorly permeable compounds.

Weak Electric Current Treatment to Artificially Enhance Vascular Permeability in Embryonated Chicken Eggs.

Technologies that overcome the barrier presented by vascular endothelial cells are needed to facilitate targeted delivery of drugs into tissue parenchyma by intravenous administration. We previously reported that weak electric current treatment (ET: 0.3-0.5 mA/cm2) applied onto skin tissue in a transdermal drug delivery technique termed iontophoresis induces cleavage of intercellular junctions that results in permeation of macromolecules such as small interfering RNA and cytosine-phosphate-guanine (CpG) oligonucleotide through the intercellular space. Based on these findings, we hypothesized that application of ET to blood vessels could promote cleavage of intercellular junctions that artificially induces increase in vascular permeability to enhance extravasation of drugs from the vessels into target tissue parenchyma. Here we investigated the effect of ET (0.34 mA/cm2) on vascular permeability using embryonated chicken eggs, which have blood vessels in the chorioallantoic membrane (CAM), as an animal model. ET onto the CAM of the eggs significantly increased extravasation of intravenously injected calcein (M.W. 622.6), a low molecular weight compound model, and the macromolecule fluorescein isothiocyanate (FITC)-dextran (M.W. 10000). ET-mediated promotion of penetration of FITC-dextran through vascular endothelial cells was also observed in transwell permeability assay using monolayer of human umbilical vein endothelial cells without induction of obvious cellular damage. Confocal microscopy detected remarkable fluorescence derived from injected FITC-dextran in blood vessel walls. These results in embryonated chicken eggs suggest that ET onto blood vessels could artificially enhance vascular permeability to facilitate extravasation of macromolecules from blood vessels.

FXR modulates the gut-vascular barrier by regulating the entry sites for bacterial translocation in experimental cirrhosis.

BACKGROUND & AIMS: Pathological bacterial translocation (PBT) in cirrhosis is the hallmark of spontaneous bacterial infections, increasing mortality several-fold. Increased intestinal permeability is known to contribute to PBT in cirrhosis, although the role of the mucus layer has not been addressed in detail. A clear route of translocation for luminal intestinal bacteria is yet to be defined, but we hypothesize that the recently described gut-vascular barrier (GVB) is impaired in experimental portal hypertension, leading to increased accessibility of the vascular compartment for translocating bacteria. MATERIALS: Cirrhosis was induced in mouse models using bile-duct ligation (BDL) and CCl4. Pre-hepatic portal-hypertension was induced by partial portal vein ligation (PPVL). Intestinal permeability was compared in these mice after GFP-Escherichia coli or different sized FITC-dextrans were injected into the intestine. RESULTS: Healthy and pre-hepatic portal-hypertensive (PPVL) mice lack translocation of FITC-dextran and GFP-E. coli from the small intestine to the liver, whereas BDL and CCl4-induced cirrhotic mice demonstrate pathological translocation, which is not altered by prior thoracic-duct ligation. The mucus layer is reduced in thickness, with loss of goblet cells and Muc2-staining and expression in cirrhotic but not PPVL mice. These changes are associated with bacterial overgrowth in the inner mucus layer and pathological translocation of GFP-E. coli through the ileal epithelium. GVB is profoundly altered in BDL and CCl4-mice with Ileal extravasation of large-sized 150 kDa-FITC-dextran, but only slightly altered in PPVL mice. This pathological endothelial permeability and accessibility in cirrhotic mice is associated with augmented expression of PV1 in intestinal vessels. OCA but not fexaramine stabilizes the GVB, whereas both FXR-agonists ameliorate gut to liver translocation of GFP-E. coli. CONCLUSIONS: Cirrhosis, but not portal hypertension per se, grossly impairs the endothelial and muco-epithelial barriers, promoting PBT to the portal-venous circulation. Both barriers appear to be FXR-modulated, with FXR-agonists reducing PBT via the portal-venous route. LAY SUMMARY: For intestinal bacteria to enter the systemic circulation, they must cross the mucus and epithelial layer, as well as the gut-vascular barrier. Cirrhosis disrupts all 3 of these barriers, giving bacteria access to the portal-venous circulation and thus, the gut-liver axis. Diminished luminal bile acid availability, cirrhosis and the associated reduction in farnesoid x receptor (FXR) signaling seem, at least partly, to mediate these changes, as FXR-agonists reduce bacterial translocation via the portal-venous route to the liver in cirrhosis.

Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance.

BACKGROUND: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1ß (IL-1ß) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1ß production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1ß source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. METHODS: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1ß-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1ß production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1ß expression in the SFO perivascular macrophages. RESULTS: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1ß in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1ß expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1ß, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. CONCLUSIONS: The current data indicate that perivascular macrophages enable the SFO to produce IL-1ß in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.

HIV-1 Tat and opioids act independently to limit antiretroviral brain concentrations and reduce blood-brain barrier integrity.

Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. Tat and morphine effects on the leakage of fluorescently labeled dextrans (10-, 40-, and 70-kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat- mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. Morphine exposure, and, to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within the striatum and significantly less dolutegravir within the hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.

Age differences in photodynamic therapy-mediated opening of the blood-brain barrier through the optical clearing skull window in mice.

BACKGROUND: Photodynamic therapy (PDT), a minimally invasive therapeutic tool, has been an important option for post-surgical treatment of malignant gliomas (MGs) in both adult and young patients. Recent studies have shown that PDT can also open the blood-brain barrier (BBB). However, there are no optimized parameters of PDT for patients at different ages. To determine whether there are age differences in PDT effects on the BBB, we studied PDT-related BBB opening through the optical clearing skull window in healthy 4- and 8-week-old mice. METHODS: In this work, we realized BBB opening by combining PDT with the optical clearing skull window by using different radiant exposures (635 nm, 10-20-30-40 J/cm2 ) and 5-aminole-vulinic acid (5-ALA, 20 mg/kg). Then, we evaluated BBB permeability by: (i) spectrofluorimetric measuring of Evans Blue dye (EBd) leakage; (ii) confocal imaging of 70 kDa FITC-dextran extravasation and the BBB integrity; and (iii) histological analysis of brain tissues. RESULTS: Using the skull optical clearing method, we demonstrated PDT-induced BBB opening to EBd and FITC-dextran in a radiant exposure manner. The histological analysis revealed the different severities of vasogenic edema corresponding to radiant exposures. Besides, the PDT-related increase in the BBB permeability to high weight molecules (EBd and FITC-dextran) and solutes (vasogenic edema) was more pronounced in 4-week-old mice than in 8-week-old mice. CONCLUSIONS: The more pronounced PDT-induced BBB disruption in juvenile mice compared with adult mice suggests age differences in PDT-related BBB opening. This might be an important informative platform for a new application of PDT as a method for brain drug delivery, especially for post-surgical treatment of MGs. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

A Novel Method for Rapid Bedside Measurement of GFR.

Background Direct quantitative measurement of GFR (mGFR) remains a specialized task primarily performed in research settings. Multiple formulas for estimating GFR have been developed that use the readily available endogenous biomarkers creatinine and/or cystatin C. However, eGFR formulas have limitations, and an accurate mGFR is necessary in some clinical situations and for certain patient populations. We conducted a prospective, open-label study to evaluate a novel rapid technique for determining plasma volume and mGFR.Methods We developed a new exogenous biomarker, visible fluorescent injectate (VFI), consisting of a large 150-kD rhodamine derivative and small 5-kD fluorescein carboxymethylated dextrans. After a single intravenous injection of VFI, plasma volume and mGFR can be determined on the basis of the plasma pharmacokinetics of the rhodamine derivative and fluorescein carboxymethylated dextrans, respectively. In this study involving 32 adults with normal kidney function (n=16), CKD stage 3 (n=8), or CKD stage 4 (n=8), we compared VFI-based mGFR values with values obtained by measuring iohexol plasma disappearance. VFI-based mGFR required three 0.5-ml blood draws over 3 hours; iohexol-based mGFR required five samples taken over 6 hours. Eight healthy participants received repeat VFI injections at 24 hours.Results VFI-based mGFR values showed close linear correlation with the iohexol-based mGFR values in all participants. Injections were well tolerated, including when given on consecutive days. No serious adverse events were reported. VFI-based mGFR was highly reproducible.Conclusions The VFI-based approach allows for the rapid determination of mGFR at the bedside while maintaining patient safety and measurement accuracy and reproducibility.

Characterization of tumor vascular permeability using natural dextrans and CEST MRI.

PURPOSE: To investigate the use of natural dextrans as nano-sized chemical exchange saturation transfer (CEST) MRI probes for characterizing size-dependent tumor vascular permeability. METHODS: Dextrans of different molecular weight (10, 70, 150, and 2000 kD) were characterized for their CEST contrast. Mice (N = 5) bearing CT26 subcutaneous colon tumors were injected intravenously with 10 kD (D10, 6 nm) and 70 kD (D70, 12 nm) dextran at a dose of 375 mg/kg. The CEST-MRI signal in the tumors was assessed before and approximately 40 min after each injection using a dynamic CEST imaging scheme. RESULTS: All dextrans of different molecular weights have a strong CEST signal with an apparent maximum of approximately 0.9 ppm. The detectability and effects of pH and saturation conditions (B1 and Tsat ) were investigated. When applied to CT26 tumors, the injection of D10 could produce a significant "dexCEST" enhancement in the majority of the tumor area, whereas the injection of D70 only resulted in an increase in the tumor periphery. Quantitative analysis revealed the differential permeability of CT26 tumors to different size particles, which was validated by fluorescence imaging and immunohistochemistry. CONCLUSIONS: As a first application, we used 10- and 70-kD dextrans to visualize the spatially variable, size-dependent permeability in the tumor, indicating that nano-sized dextrans can be used for characterizing tumor vascular permeability with dexCEST MRI and, potentially, for developing dextran-based theranostic drug delivery systems. Magn Reson Med 79:1001-1009, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Cytosolic Uptake of Large Monofunctionalized Dextrans.

Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. Here, we report on a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos.

Bioorthogonal Masking of Circulating Antibody-TCO Groups Using Tetrazine-Functionalized Dextran Polymers.

Pretargeting strategies have gained popularity for the in vivo imaging and therapy of cancer by combining antibodies with small molecule radioligands. In vivo recombination of both moieties can be achieved using the bioorthogonal inverse electron demand Diels-Alder (IEDDA) chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). An issue that arises with pretargeting strategies is that while part of the antibody dose accumulates at antigen-expressing tumor tissue, there is a significant portion of the injected antibody that remains in circulation, causing a reduction in target-to-background ratios. Herein, we report the development of a novel TCO scavenger, the masking agent DP-Tz. DP-Tz is based on Tz-modified dextran polymers (DP, MW = 0.5-2 MDa). Large dextran polymers were reported to exhibit low penetration of tumor vasculature and appeared nontoxic, nonimmunogenic, and easily modifiable. Our newly developed masking agent deactivates the remaining TCO-moieties on the circulating mAbs yet does not impact the tumor uptake of the Tz-radioligand. In pretargeting studies utilizing a 68Ga-labeled tetrazine radioligand ([68Ga]Ga-NOTA-PEG11-tetrazine), DP-Tz constructs (Tz/DP ratios of 62-254) significantly increased TTB ratios from 0.8 ± 0.3 (control cohorts) to up to 5.8 ± 2.3 at 2 h postinjection. Tumor tissue delineation in PET imaging experiments employing DP-Tz is significantly increased compared to control. Uptake values of other significant organs, such as heart, lungs, pancreas, and stomach, were decreased on average by 2-fold when using DP-Tz. Overall, pretargeting experiments utilizing DP-Tz showed significantly improved tumor delineation, enhanced PET image quality, and reduced uptake in vital organs. We believe that this new masking agent is a powerful new addition to the IEDDA-based pretargeting tool box and, due to its properties, an excellent candidate for clinical translation.

Role of the Basement Membrane as an Intestinal Barrier to Absorption of Macromolecules and Nanoparticles.

Full understanding of the barrier property of mucosal tissues is imperative for development of successful mucosal drug delivery strategies, particularly for biologics and nanomedicines. The contribution of the mucosal basement membrane (BM) to this barrier is currently not fully appreciated. This work examined the role of the BM as a barrier to intestinal absorption of model macromolecules (5 and 10 kDa dextrans) and 100 nm polystyrene nanoparticles. Dextrans and nanoparticles were applied either directly to BM-coated inserts or to an intestinal model, namely, differentiated intestinal epithelial monolayers (Caco-2) cultured on BM-modified inserts. The work shows that the BM per se does not impact the diffusion of dextran macromolecules but severely hinders the movement of nanoparticles. However, importantly, Caco-2 monolayers cultured on BM-coated inserts, which show a remarkably different morphology, display a significantly larger barrier to the translocation of one dextran, as well as nanoparticle systems compared to cells cultured on unmodified inserts. Therefore, this work shows that, in addition to presenting a direct physical barrier to the movement of nanoparticles, the BM also exerts an indirect barrier effect, likely due to its influence on epithelial cell physiology. This work is important as it highlights the currently unmet need to consider and further study the barrier properties of the BM in mucosal delivery of biologics and nanomedicines.

Glucose Responsive Rheological Change and Drug Release from a Novel Worm-like Micelle Gel Formed in Cetyltrimethylammonium Bromide/Phenylboronic Acid/Water System.

A novel glucose (Glc)-responsive gel formed by worm-like micelles (WLMs) has the potential to provide a self-regulating insulin delivery system. We have prepared a WLM gel system using 75 mM cetyltrimethylammonium bromide, 75 mM phenylboronic acid, and water. At pH 9.4, this gel-like system was highly viscous and supported its own weight, and dynamic viscoelasticity measurement indicated that it contained long and entangled WLMs. The visual observation of gels prepared to include >6 mM Glc revealed that these adopted a sol-like appearance, whereas those prepared to include a control compound (2-10 mM diethylene glycol) retained their gel-like appearance. The storage modulus ( G') of this system decreased as the Glc concentration increased (2-10 mM), indicating a gradual shortening of the WLMs. In vitro release was evaluated using a test compound (fluorescein isothiocyanate dextran) in a microsized flow system. By 120 min, the release of this compound from the WLM gel was around 27-fold greater in the presence of 100 mM Glc than without Glc or with 100 mM diethylene glycol. This demonstrated the successful preparation of a WLM gel that showed an altered drug release rate, depending on Glc concentration.

Reinforcing Mucus Barrier Properties with Low Molar Mass Chitosans.

The mucus gel covers the wet epithelia that forms the inner lining of the body. It constitutes our first line of defense protecting the body from infections and other deleterious molecules. Failure of the mucus barrier can lead to the inflammation of the mucosa such as in inflammatory bowel diseases. Unfortunately, there are no effective strategies that reinforce the mucus barrier properties to recover or enhance its ability to protect the epithelium. Herein, we describe a mucus engineering approach that addresses this issue where we physically cross-link the mucus gel with low molar mass chitosan variants to reinforce its barrier functions. We tested the effect of these chitosans on mucus using in-lab purified porcine gastric mucins, which mimic the native properties of mucus, and on mucus-secreting HT29-MTX epithelial cell cultures. We found that the lowest molar mass chitosan variant (degree of polymerization of 8) diffuses deep into the mucus gels while physically cross-linking the mucin polymers, whereas the higher molar mass chitosan variants (degree of polymerization of 52 and 100) interact only superficially. The complexation resulted in a tighter mucin polymer mesh that slowed the diffusion of dextran polymers and of the cholera toxin B subunit protein through the mucus gels. These results uncover a new use for low molar mass mucoadhesive polymers such as chitosans as noncytotoxic mucosal barrier enhancers that could be valuable in the prevention and treatment of mucosal diseases.

Extended Pharmacokinetic Model of the Rabbit Eye for Intravitreal and Intracameral Injections of Macromolecules: Quantitative Analysis of Anterior and Posterior Elimination Pathways.

PURPOSE: To extend the physiological features of the anatomically accurate model of the rabbit eye for intravitreal (IVT) and intracameral (IC) injections of macromolecules. METHODS: The computational fluid dynamic model of the rabbit eye by Missel (2012) was extended by enhancing the mixing in the anterior chamber with thermal gradient, heat transfer and gravity, and studying its effect on IC injections of hyaluronic acids. In IVT injections of FITC-dextrans (MW 10-157 kDa) the diffusion though retina was defined based on published in vitro data. Systematic changes in retinal permeability and convective transport were made, and the percentages of anterior and posterior elimination pathways were quantified. Simulations were compared with published in vivo data. RESULTS: With the enhanced mixing the elimination half-lives of hyaluronic acids after IC injection were 62-100 min that are similar to in vivo data and close to the theoretical value for the well-stirred anterior chamber (57 min). In IVT injections of FITC-dextrans a good match between simulations and in vivo data was obtained when the percentage of anterior elimination pathway was over 80%. CONCLUSIONS: The simulations with the extended model closely resemble in vivo pharmacokinetics, and the model is a valuable tool for data interpretation and predictions.

Tissue-Clearing Techniques Enable Three-Dimensional Visualization of Aerosolized Model Compound and Lung Structure at the Alveolar Scale.

In this study, we examined the usefulness of a tissue-clearing technique for the evaluation of the lung distribution of aerosolized drugs. An aerosol formulation of TexasRed dextran (70 kDa), a model compound of drug carrier for aerosolized drugs, was administered intrapulmonarily to mice using a MicroSprayer, and then DyLight 488-conjugated tomato lectin was administered intravenously to visualize general lung structure via the fluorescent labeling of alveolar and bronchial epithelial cells. Tissue clearing followed by laser scanning confocal microscopy enabled the three-dimensional visualization of intrapulmonary TexasRed dextran and the evaluation of its distribution at the alveolar scale without the preparation of thin tissue sections. These findings suggest that tissue-clearing techniques are useful for the evaluation of intrapulmonary distribution and development of pulmonary drug delivery systems.

The Effect of Absorption-Enhancement and the Mechanism of the PAMAM Dendrimer on Poorly Absorbable Drugs.

The polyamidoamine (PAMAM) dendrimer is a highly efficient absorption promoter. In the present study, we studied the absorption-enhancing effects and the mechanism of PAMAM dendrimers with generation 0 to generation 3 (G0⁻G3) and concentrations (0.1⁻1.0%) on the pulmonary absorption of macromolecules. The absorption-enhancing mechanisms were elucidated by microarray, western blotting analysis, and PCR. Fluorescein isothiocyanate-labeled dextrans (FDs) with various molecular weights were used as model drugs of poorly absorbable drugs. The absorption-enhancing effects of PAMAM dendrimers on the pulmonary absorption of FDs were in a generation- and concentration-dependent manner. The G3 PAMAM dendrimer with high effectiveness was considered to the best absorption enhancer for improving the pulmonary absorption of FDs. G3 PAMAM dendrimers at three different concentrations were non-toxic to Calu-3 cells. Based on the consideration between efficacy and cost, the 0.1% G3 PAMAM dendrimer was selected for subsequent studies. The results showed that treatment with a 0.1% G3 PAMAM dendrimer could increase the secretion of organic cation transporters (OCTs), OCT1, OCT2, and OCT3, which might be related to the absorption-enhancing mechanisms of the pulmonary absorption of FDs. These findings suggested that PAMAM dendrimers might be potentially safe absorption enhancers for improving absorption of FDs by increasing the secretion of OCT1, OCT2, and OCT3.

Imaging and quantification of iron-oxide nanoparticles (IONP) using MP-RAGE and UTE based sequences.

PURPOSE: To investigate two-dimensional (2D) and three-dimensional (3D) ultrashort echo time (UTE) and 3D magnetization-prepared rapid gradient-echo (MP-RAGE) sequences for the imaging of iron-oxide nanoparticles (IONP). METHODS: The phantoms were composed of tubes filled with different IONP concentrations ranging from 2 to 45 mM. The tubes were fixed in an agarose gel phantom (0.9% by weight). Morphological imaging was performed with 3D MP-RAGE, 2D UTE, 2D adiabatic inversion recovery-prepared UTE (2D IR-UTE), 3D UTE with Cones trajectory (3D Cones), and 3D IR-Cones sequences. Quantitative assessment of IONP concentration was performed using R2*(1/T2*) and R1 (1/T1 ) measurements using a 3 Tesla (T) scanner. RESULTS: The 3D MP-RAGE sequence provides high-contrast images of IONP with concentration up to 7.5 mM. Higher IONP concentration up to 37.5 mM can be detected with the UTE sequences, with the highest IONP contrast provided by the 3D IR-Cones sequence. A linear relationship was observed between R2* and IONP concentration up to ∼45 mM, and between R1 and IONP concentration up to ∼30 mM. CONCLUSION: The clinical 3D MP-RAGE sequence can be used to assess lower IONP concentration up to 7.5 mM. The UTE sequences can be used to assess higher IONP concentration up to 45 mM. Magn Reson Med 78:226-232, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Assessment of mucosal integrity by quantifying neutrophil granulocyte influx in murine models of acute intestinal injury.

Intact epithelial body surfaces represent physical barriers which protect the organism from invading pathogens and loss of nutrients. Barrier malfunction is closely linked to disorders such as inflammatory bowel disease and graft-versus-host disease. In fact, several pharmacological or radiobiological therapeutic strategies have side effects that affect epithelial surfaces. In this context, assays that accurately assess epithelial barrier integrity in patients and animal models are crucial to create a better understanding of the mechanisms leading to disease or limiting therapeutic approaches due to barrier disruption. Here, we tested the ability of the widely used FITC-dextran intestinal permeability analysis to evaluate loss of intestinal barrier integrity in different murine models of gut mucosal damage and established influx of neutrophil granulocytes into the intestinal lamina propria (LP) as an alternative approach. We demonstrate that the sensitivity and specificity of FITC-dextran intestinal permeability analysis is relatively low: Although it did represent severe forms of mucosal damage due to intensive conditioning therapy (high doses of either total body irradiation (TBI) or chemotherapy) or after conditioning and allogeneic stem cell transplantation, it did not recognize less severe forms of damage as after lower doses of TBI or chemotherapy alone. In addition, discrimination of untreated from irradiated mice by differences in FITC-dextran translocation was not exact. In contrast, influx of neutrophil granulocytes into the intestinal LP, which reflects immune activation due to translocation of microbes and microbial products during intestinal barrier breech, quantitatively correlated with the severity of intestinal barrier damage. It accurately represented both severe and less severe forms of intestinal damage as after high or lower dose TBI or chemotherapy and correctly discriminated treated from untreated animals. Taken together, we demonstrate the limitations of FITC-dextran intestinal permeability analysis and identify intestinal neutrophil influx as a powerful additional tool to measure breakdown of intestinal barrier function.

Functionalized superparamagnetic iron oxide nanoparticles provide highly efficient iron-labeling in macrophages for magnetic resonance-based detection in vivo.

BACKGROUND AIMS: Tracking cells during regenerative cytotherapy is crucial for monitoring their safety and efficacy. Macrophages are an emerging cell-based regenerative therapy for liver disease and can be readily labeled for medical imaging. A reliable, clinically applicable cell-tracking agent would be a powerful tool to study cell biodistribution. METHODS: Using a recently described chemical design, we set out to functionalize, optimize and characterize a new set of superparamagnetic iron oxide nanoparticles (SPIONs) to efficiently label macrophages for magnetic resonance imaging-based cell tracking in vivo. RESULTS: A series of cell health and iron uptake assays determined that positively charged SPIONs (+16.8 mV) could safely label macrophages more efficiently than the formerly approved ferumoxide (-6.7 mV; Endorem) and at least 10 times more efficiently than the clinically approved SPION ferumoxytol (-24.2 mV; Rienso). An optimal labeling time of 4 h at 25 µg/mL was demonstrated to label macrophages of mouse and human origin without any adverse effects on cell viability whilst providing substantial iron uptake (>5 pg Fe/cell) that was retained for 7 days in vitro. SPION labeling caused no significant reduction in phagocytic activity and a shift toward a reversible M1-like phenotype in bone marrow-derived macrophages (BMDMs). Finally, we show that SPION-labeled BMDMs delivered via the hepatic portal vein to mice are localized in the hepatic parenchyma resulting in a 50% drop in T2* in the liver. Engraftment of exogenous cells was confirmed via immunohistochemistry up to 3 weeks posttransplantation. DISCUSSION: A positively charged dextran-coated SPION is a promising tool to noninvasively track hepatic macrophage localization for therapeutic monitoring.