Molecular Weight FITC-dextran Research Articles

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

Responsive Hyaluronic Acid-Ethylacrylamide Microgels Fabricated Using Microfluidics Technique.

Volume changes of responsive microgels can probe interactions between polyelectrolytes and species of opposite charges such as peptides and proteins. We have investigated a microfluidics method to synthesize highly responsive, covalently crosslinked, hyaluronic acid microgels for such purposes. Sodium hyaluronate (HA), pre-modified with ethylacrylamide functionalities, was crosslinked in aqueous droplets created with a microfluidic technique. We varied the microgel properties by changing the degree of modification and concentration of HA in the reaction mixture. The degree of modification was determined by 1H NMR. Light microscopy was used to investigate the responsiveness of the microgels to osmotic stress in aqueous saline solutions by simultaneously monitoring individual microgel species in hydrodynamic traps. The permeability of the microgels to FITC-dextrans of molecular weights between 4 and 250 kDa was investigated using confocal laser scanning microscopy. The results show that the microgels were spherical with diameters between 100 and 500 µm and the responsivity tunable by changing the degree of modification and the HA concentration. Microgels were fully permeable to all investigated FITC-dextran probes. The partitioning to the microgel from an aqueous solution decreased with the increasing molecular weight of the probe, which is in qualitative agreement with theories of homogeneous gel networks.

Endothelin-axis antagonism enhances tumor perfusion in pancreatic cancer

Delivery of therapeutic agents in pancreatic cancer (PC) is impaired due to its hypovascular and desmoplastic tumor microenvironment. The Endothelin (ET)-axis is the major regulator of vasomotor tone under physiological conditions and is highly upregulated in multiple cancers. We investigated the effect of dual endothelin receptor antagonist bosentan on perfusion and macromolecular transport in a PC cell-fibroblast co-implantation tumor model using Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI). Following bosentan treatment, the contrast enhancement ratio and wash-in rates in tumors were two- and nine times higher, respectively, compared to the controls, whereas the time to peak was significantly shorter (7.29 ± 1.29 min v/s 22.08 ± 5.88 min; p = 0.04). Importantly, these effects were tumor selective as the magnitudes of change for these parameters were much lower in muscles. Bosentan treatment also reduced desmoplasia and improved intratumoral distribution of high molecular weight FITC-dextran. Overall, these findings support that targeting the ET-axis can serve as a potential strategy to selectively enhance tumor perfusion and improve the delivery of therapeutic agents in pancreatic tumors.

Fabrication and development of controlled release PLGA microneedles for macromolecular delivery using FITC-Dextran as model molecule

The use of biodegradable polymers has been prominent in the field of drug delivery due to its degradability and biocompatible properties. These polymers when used to prepare microneedles, impart high mechanical strength, sustained drug release profile, and long term biocompatibility. In this study, PLGA (poly (D, L-lactic co-glycolic acid)) and PVA (Polyvinyl Alcohol) were used in combination to optimize the microneedle fabrication for high molecular weight FITC Dextran (4 kDa MW) to be used as a model drug. FITC-Dextran loaded PLGA microneedles were fabricated using a mold casting technique. MNs were characterized for the textural properties, FITC-Dextran release profile, and in-vitro permeation profile. The fate of microneedles in the skin (ex vivo) was characterized using fluorescence and confocal imaging. The FITC -Dextran MNs (10 × 10 array) had a dimension of 415 ± 2.65 μm height, 207 ± 1.63 μm width of the base and a needle pitch of 463 ± 1.45 μm. The texture analysis data showed puncture force for FITC-Dextran loaded PLGA microneedles (28.46 ± 0.23 g) and PLGA microneedles (16.92 ± 0.04 g). The Cumulative permeation of FITC-Dextran after 48 h was 2.34 ± 0.40 μg/cm2 and it showed a 31 fold higher permeation profile than its respective control. Ex vivo imaging studies were used to characterize the fluorescence intensity of FITC-Dextran in the skin. This study demonstrates that incorporating higher molecular weight molecules into PLGA microneedles proved to be an effective strategy to sustain the release of macromolecules across stratum corneum/epidermis for transdermal delivery.

Quantifying blood-brain-barrier leakage using a combination of evans blue and high molecular weight FITC-Dextran

BackgroundEvans blue (EB) is the most widely used tracer to assess BBB leakage. However, a well-established method to obtain visualized and quantitative results of EB extravasation is presently unavailable. New MethodWe reported a novel method to quantify BBB leakage by combining EB and high molecular weight FITC-Dextran (2000 kDa). EB was used for a long circulation duration (60 min) to detect BBB leakage. FITC-Dextran was used for a short circulation duration (10 min) to outline vascular contours. Confocal microscope imaging was used to obtain visualized images of BBB leakage. The result of dividing integrated optical density of EB by vascular areas outlined by FITC-Dextran was treated as the quantification of BBB leakage. ResultsThis method proved workable in quantifying BBB leakage of specific regions in lipopolysaccharide-induced BBB disruption mice and apoE−/− mice. Sections processed with this method enabled further immunofluorescence staining. Through combining the results of EB extravasation and immunofluorescence staining, the colocalization of specific proteins and BBB disruption was achieved. Comparison with Existing MethodsColorimetric and spectrophotometric methods provide us with quantitative results of EB extravasation but fail to locate the specific regions. Fluorescence microscopy imaging can locate specific regions of EB extravasation but a well-established quantitative method is presently unavailable. Our method combines advantages of above two classic methods, providing us with visualized and quantitative information of BBB leakage based on EB extravasation in specific cerebral regions. ConclusionsThe proposed method proved powerful in quantifying BBB leakage of specific regions, which may benefit studies regarding BBB disruption.

Abstract 2963: Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development

Abstract Background: Inactivation of the TGF-β signaling occurs in 50-75% of all colorectal cancer cases. Down-regulation of Smad4, the central mediator of TGF-β signaling, occurs in >50% of stage III patients. How Smad4 contributes to normal intestinal homeostasis and functions as a tumor suppressor are incompletely understood. We reported that β-catenin mRNA expression is inhibited by BMP-mediated Smad4 signaling, and loss of Smad4 signaling results in biologically significant amplification of the Wnt/β-catenin signal. Here, we report on the consequences of Smad4 loss for intestinal homeostasis. Methods: We generated mice genetically engineered to undergo inducible, tissue-specific deletion of Smad4 within intestinal epithelial cells. K19-CreERT x Smad4 flx/flx and Lrig1-CreERT x Smad4 flx/flx mice were treated with vehicle or tamoxifen to induce recombination at the Smad4 locus in the intestinal stem cell compartment. Intestines from sacrificed mice were cleansed for enteroid isolation, Ussing chamber studies, RNA-seq analysis and preservation by formalin fixation and paraffin embedding (FFPE). We validated Smad4 status via immunohistochemistry and stained FFPE sections for different cell populations within the crypt. To evaluate inflammatory cytokines, we performed Luminex cytokine array analysis on colonic mucosa from control and tamoxifen treated animals. To determine the role for Smad4 in intestinal response to injury, we also treated control and tamoxifen treated animals with dextran sodium sulfate (DSS). We utilized RNA-Seq analysis to determine an intestinal Smad4 loss gene signature. Results: Smad4 deficient crypts exhibited an expansion of the Ki67 positive cells in the proliferative zone and fewer Carbonic anhydrase II staining cells suggesting fewer mature enterocytes. We found that loss of Smad4 in the intestinal crypt is accompanied by an accumulation of pericryptal CD3+ lymphocytes and F4/80+ macrophages and is associated with increased intestinal permeability as measured by low molecular weight FITC Dextran using the Ussing chamber. Cytokine analysis revealed Macrophage Inflammatory Protein 3a (MIP3a) to be up-regulated in Smad4 knockout mice. Guided by our RNA-Seq analysis, we observe increased levels of pErk in Smad4 deplete tissue. We also observed that mice with intestinal epithelial Smad4 depletion developed advanced mucinous adenocarcinoma after recovery from an inflammatory stimulus induced by DSS. Conclusions: These results suggest that Smad4 loss disrupts intestinal homeostasis by enabling persistently high levels of Erk signaling to interfere with cell maturation, thereby contributing to increased inflammation. We postulate that the increased inflammation observed in Smad4 knockout mice leads to a tumorigenic environment. Citation Format: Tanner J. Freeman, Jillian Pope, Josh Smith, Daniel Sharbel, Kay Washington, Xi Chen, Rupesh Chaturvedi, Keith Wilson, Noah Shroyer, Punita Dhawan, Anna Means, Natasha G. Deane, R. Daniel Beauchamp. Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2014-2963

Platelet endothelial cell adhesion molecule‐1 and capillary density in the retina of diabetic rats

The objective of this study was to evaluate potential changes in platelet endothelial cell adhesion molecule‐1 (PECAM‐1) and capillary density occurring early in the diabetic retina. In these experiments, streptozotocin (STZ; or vehicle alone, for controls) was injected into rats (30 mg/kg for 3 days) to induce a model of type I diabetes. Eight weeks later, a fluorescent plasma tracer (high molecular weight FITC‐dextran) was injected i.v. into the anesthetized rats, and the eyes were excised just prior to euthanasia. One eye was used in a flatmount of the retinal tissue, with capillary density quantified using images captured through a microscope camera. The other eye was used for western blot analysis. Levels of PECAM‐1 were found to decrease by 44% (p<0.05), but in contrast, no changes were observed in the number of perfused capillaries (that is, perfused with FITC‐dextran). Further studies will be needed to examine the possibility, and implications, of diabetic retinal capillaries expressing lower levels of PECAM‐1. Funded by NIH EY017599 (NRH).

High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)–Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations

This note is to report how histological preparation techniques influence the extravasation pattern of the different molecular sizes of fluorescein isothiocyanate (FITC)–dextrans, typically used as markers for blood-brain barrier leakage. By using appropriate preparation methods, false negative results can be minimized. Wistar rats underwent a 2-h middle cerebral artery occlusion and magnetic resonance imaging. After the last imaging scan, Evans blue and FITC–dextrans of 4, 40, and 70 kDa molecular weight were injected. Different histological preparation methods were used. Sites of blood-brain barrier leakage were analyzed by fluorescence microscopy. Extravasation of Evans blue and high molecular FITC–dextrans (40 and 70 kDa) in the infarcted region could be detected with all preparation methods used. If exposed directly to saline, the signal intensity of these FITC–dextrans decreased. Extravasation of the 4-kDa low molecular weight FITC–dextran could only be detected using freshly frozen tissue sections. Preparations involving paraformaldehyde and sucrose resulted in the 4-kDa FITC–dextran dissolving in these reactants and being washed out, giving the false negative result of no extravasation. FITC–dextrans represent a valuable tool to characterize altered blood-brain barrier permeability in animal models. Diffusion and washout of low molecular weight FITC–dextran can be avoided by direct immobilization through immediate freezing of the tissue. This pitfall needs to be known to avoid the false impression that there was no extravasation of low molecular weight FITC–dextrans.

Methamphetamine induces macropinocytosis in differentiated SH-SY5Y human neuroblastoma cells

Acute and chronic abuses of psychostimulant drugs such as methamphetamine (METH) have been known to cause cell death. In particular, neurotoxicity caused by such drugs is one of the most serious adverse events in humans. Although various effects on neuronal cells caused by METH have been studied, the cellular and molecular mechanisms of METH-induced neurotoxicity remain to be elucidated. To investigate the mechanism of METH-induced cytotoxicity, we studied cytological as well as biochemical changes in retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells. Marked cell death was observed with more than 7 mM METH, although caspase-dependent apoptotic cell death was not observed with any concentration of METH treatment. The most prominent cytomorphological effect by METH was the formation of large cytoplasmic vacuoles which were not colocalized with either GFP-LC3 or HSP47-GFP, autophagosome and ER markers respectively. In contrast, many of these vacuoles incorporated large molecular weight FITC-dextran and were confirmed as macropinosomes. Our results indicate that METH-induced cytomorphological effects on RA-differentiated SH-SY5Y human neuroblastoma cells involve macropinocytosis and the hyperstimulation of this process may be involved in METH-caused cytotoxicity.

Targeting therapy of pigment epithelium derived factor mediated by ultrasound activated immunoliposome for choroidal neovascularization

目的 研究超声联合载色素上皮源性因子(PEDF)免疫纳米脂质体对脉络膜新生血管(CNV)的靶向治疗作用.方法 以CNV内皮细胞上血管内皮生长因子受体-2(VEGFR-2)为靶向位点,将其配体寡肽连接到纳米脂质体上,再反向装载PEDF,制备能与CNV内皮细胞特异性结合的免疫纳米脂质体.对48只BN大鼠右眼行半导体倍频激光(波长为532nm)光凝术建立CNV模型.光凝后7d,实验组鼠尾静脉分别注射游离PEDF、载PEDF脂质体、载PEDF免疫脂质体,联合眼局部超声波辐照20s,并设立玻璃体腔注射PEDF组和对照组.光凝后14d,脉络膜巩膜冰冻切片观察免疫脂质体对CNV的靶向性.FITC-右旋糖酐标记脉络膜巩膜铺片行新生血管定量分析.结果 载PEDF免疫脂质体可特异性靶向结合CNV.CNV与FITC标记的PEDF免疫纳米脂质体结合而呈现绿色荧光,正常脉络膜血管不显现荧光.玻璃体腔注射PEDF组及鼠尾静脉注射游离PEDF、载PEDF脂质体、载PEDF免疫脂质体组CNV面积均较对照组减少,差异有统计学意义(P＜0.05).超声联合PEDF免疫纳米脂质体组对CNV的抑制作用强于玻璃体腔注射PEDF组(P＜0.05).结论 动物实验证实PEDF免疫纳米脂质体可特异性结合CNV细胞,超声可增强PEDF免疫纳米脂质体对CNV的抑制作用,该方法有望为眼新生血管的治疗提供新途径。

Determination of human lens capsule permeability and its feasibility as a replacement for Bruch's membrane

We have investigated human anterior lens capsule as a potential replacement for Bruch's membrane as a treatment for age-related macular degeneration. Any substrate to replace Bruch's membrane should possess certain characteristics to maintain proper function of the overlying retina. One of the important properties of Bruch's membrane is allowing the flow of nutrients and waste between the retinal pigment epithelium and the choriocapillaris. Here, we measured the permeability of the lens capsule by studying the diffusion of various molecular weight FITC–dextran molecules. Expressions for extraction of diffusion coefficients from concentration vs. time data from a blind-well chamber apparatus were derived for both a single and double membrane experiments. The diffusion coefficients in the lens capsule were found to be in the range of 10 −6 to 10 −10 cm 2/s. We demonstrated a power law relationship, with the diffusion coefficient possessing a −0.6 order dependence on molecular weight. The molecular weight exclusion limit was determined to be 150±40 kDa. We have compared this value with reported values of Bruch's membrane molecular weight exclusion limit and find that the lens capsule has the potential to act as a substitute Bruch's membrane.

Highly Hydrophilic Fused Aggregates (Microsponges) from a C12 Spermine Bolaamphiphile

Bolaamphiphiles consist of two hydrophilic headgroups joined by a hydrophobic linker. They are known to self-assemble into vesicles, micelles, tubules and fibers. Here we report on the self-assembly of a 1,12-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G1) in aqueous media into discrete nonbilayer nanoparticles and unusual fused highly hydrated sponge like structures. 12G1, 1,10-[ N,N-bis(3-aminopropyl)-1,4-butane diamine] decane (10G1), 1,8-[N,N-bis(3-aminopropyl)-1,4-butane diamine] octane (8G1), and 1-[N,N-bis(3-aminopropyl)-1,4-butane diamine] dodecane (12G0) were synthesized and their self-assembly studied. 12G0 was relatively insoluble in water and multilamellar vesicles were produced with this amphiphile and cholesterol. While no aggregation in aqueous media was recorded for either 10G1 or 8G1 up to a concentration of 20 mM, aggregation of 12G1 molecules was seen to take place at an aqueous concentration of 1.75 mM as determined using various fluorescent and colorimetric probes and surface tension measurements. Electron microscopy images revealed that 12G1 formed monolayer vesicles of 20 nm in size at a concentration of 2.6 mM and even larger fused aggregates when the concentration was increased to 35 mM. Fusion resulted from the bridging of vesicles by the bolaamphiphile molecules. 12G1 aggregates encapsulated 23X more low molecular weight (4kD) FITC-dextran than high (250kD) molecular weight FITC-dextran, an indication of the limited volume of the hydrophilic domains. 12G1 also encapsulated 137X more 4kD FITC-dextran than was encapsulated by conventional sorbitan monostearate vesicles. We conclude that highly hydrated aggregates consisting of fused monolayer vesicles with small discrete hydrophilic domains in a sponge like structure may be formed by the self-assembly of bolaamphiphiles.

Quantitative Image Analysis of Laser-induced Choroidal Neovascularization in Rat

Rodent models of laser-induced choroidal neovascularization (CNV) are now extensively used to identify angiogenic proteins, determine the role of specific genes with knockout mice, and evaluate the efficacy and safety of anti-angiogenic therapies. CNV is typically evaluated by fluorescein angiography or vascular endothelial cell labeling in histologic sections. The current study examined an alternative method using high molecular weight FITC-dextran (MW 2×106) for high resolution angiography in RPE-choroid-sclera flat mounts. At 24hr after lasering, the lesions appeared as a circular weakly fluorescent area of approximately equal diameter to the laser spot. No FITC-dextran labeled blood vessels were visible in the lesion at day 1. Three days after lasering, 47% of the lesions showed FITC-dextran labeling indicative of CNV. The incidence (71%) and extent of CNV increased by day 6, and by day 10 all lesions were vascularized, and the maximal area was attained. No significant change followed day 10, and the neovascular area remained constant through day 31. The highest rate of blood vessel growth (between 3 and 10 days after laser) correlates with the peak expression of VEGF, bFGF, and their receptors shown in previous studies. Morphologic analysis of flat mounts and histologic sections showed that the neovascular plexus in most lesions originates from deeper choroidal vessels in the center of the lesion, grows towards the neural retina, then branches circumferentially to anastamose with uninjured choriocapillaris. The microvessels in these lesions are broad and flat, similar to normal choriocapillaris. In a separate study, rats were treated daily with the angiostatic corticosteroid dexamethasone (20–500μ g kg−1day−1), and CNV was examined at day 10 in FITC-dextran labeled flat mounts and histologic sections. Dexamethasone dose-dependently inhibited CNV, and its highest dose inhibited approximately 95% of CNV labeled by FITC-dextran and resulted in lesions with no detectable Factor VIII immunostaining. High resolution angiography with FITC-dextran is reproducible and quantifiable, and it may accelerate the discovery of therapeutic agents that modulate choroidal neovascularization.

A bioresorbable, polylactide reservoir for diffusional and osmotically controlled drug delivery.

The purpose of this study was to design and characterize a zero-order bioresorbable reservoir delivery system (BRDS) for diffusional or osmotically controlled delivery of model drugs including macromolecules. The BRDS was manufactured by casting hollow cylindrical poly (lactic acid) (PLA): polyethylene glycol (PEG) membranes (10 x 1.6 mm) on a stainless steel mold. Physical properties of the PLA:PEG membranes were characterized by solid-state thermal analysis. After filling with drug (5 fluorouracil [5FU] or fluorescein isothiocyanate [FITC]-dextran:mannitol, 5:95 wt/wt mixture) and sealing with viscous PLA solution, cumulative in vitro dissolution studies were performed and drug release monitored by ultraviolet (UV) or florescence spectroscopy. Statistical analysis was performed using Minitab (Version 12). Differential scanning calorimetry thermograms of PLA:PEG membranes dried at 25 degrees C lacked the crystallization exotherms, dual endothermal melting peaks, and endothermal glass transition observed in PLA membranes dried at -25 degrees C. In vitro release studies demonstrated zero-order release of 5FU for up to 6 weeks from BRDS manufactured with 50% wt/wt PEG (drying temperature, 25 degrees C). The release of FITC dextrans of molecular weights 4400, 42 000, 148 000, and 464 000 followed zero-order kinetics that were independent of the dextran molecular weight. When monitored under different concentrations of urea in the dissolution medium, the release rate of FITC dextran 42 000 showed a linear correlation with the calculated osmotic gradient(DeltaPi). This study concludes that PEG inclusion at 25 degrees C enables manufacture of uniform, cylindrical PLA membranes of controlled permeability. The absence of molecular weight effects and a linear dependence of FITC-dextran release rate on DeltaPi confirm that the BRDS can be modified to release model macromolecules by an osmotically controlled mechanism.

Transdermal delivery of macromolecules using skin electroporation.

(1) To evaluate the feasibility of transdermal delivery of macromolecules by skin electroporation. (2) To assess the influence of the molecular weight of the permeant on transport and examine whether there exists a "cut-off" value of molecular weight. (3) To localize the transport pathways of the macromolecules in the skin. FITC-dextran (FD) of increasing molecular weight (4.4, 12 and 38 kDa) were used as model macromolecules to study the extent of transport across hairless rats skin in vitro and to localize their distribution in the skin by confocal scanning laser microscopy. Electroporation enhanced the transport of the macromolecules as compared to passive diffusion. The transdermal delivery by skin electroporation of FITC and FD 4.4 was equivalent whereas transport of higher molecular weight FD was lower but significant. FITC and FD 38 were observed in the epidermis both around and in the keratinocytes. Transdermal and topical delivery of macromolecules of at least 40 kDa can be achieved by skin electroporation.