Conjugated Wheat Germ Agglutinin Research Articles

Podocyte glycocalyx plays an essential role in the function of GFB and the development of albumin leakage

The role of the cell surface layer, glycocalyx is emerging in various areas of cell biology including signaling, metabolism, and immune response. Although a decrease of podocyte glycocalyx depth in glomerular pathologies has been reported before, the specific role of specialized glycocalyx between the podocyte foot processes and the glomerular basement membrane (GBM) in the function and dysfunction of the glomerular filtration barrier (GFB) is elusive. Here we aimed to develop podocyte or endothelial glycocalyx targeting strategies to gain mechanistic understanding of the specific roles of the glycocalyx in the function of GFB and in the development of albumin leakage. Our overall hypothesis is that the specialized podocyte glycocalyx forms an important additional layer of the GFB and changes in the structure and composition of podocyte glycocalyx leads to GFB dysfunction and the development of albumin leakage. New genetic mouse models were generated by the knock-in of pCAG-Lox-STOP-Lox-EGFP-Sdc1 to generate animals with Cre-dependent expression of a syndecan-1 (Sdc1) fusion protein with N-terminal, extracellular GFP. Sdc1-GFPflox animals were crossed with either Podocin-Cre or Cdh5-Cre mice for the specific genetic labeling of the glycocalyx in podocytes or endothelial cells, respectively. Intravital multiphoton microscopy (MPM) was used to measure changes in the depth of glycocalyx and corresponding changes in GFB function in vivo over time. Endoglycosidase enzymes, such as hyaluronidase (50U/animal), heparinase (0.7 U/mouse), and chondroitinase (1 U/mouse) were used to acutely modify the composition of glycocalyx. Alexa-680 conjugated wheat germ agglutinin (WGA) lectin was used to label the glycosaminoglycan (GAG) component of the glycocalyx; Alexa-594 conjugated albumin was used to visualize GFB permeability. Classic phenotyping of Pod and Cdh5 Sdc1-GFP animals showed no significant difference in terms of systolic blood pressure, glomerular filtration rate, body and kidney weight between the Sdc1-GFP and wild type littermates. The co-localization of the genetic GFP and anti-podocin immunofluorescence co-staining confirmed the podocyte-specific expression of Sdc1-GFP. In vivo MPM imaging of Pod-Sdc1-GFP mouse kidneys revealed intense linear labeling of the podocyte cell surface including the apical and basolateral surfaces of the cell body as well as the surfaces of primary, secondary, and foot processes. Interestingly, the thickness and fluorescence intensity of Sdc1-GFP labeled glycocalyx was greater on the basal surface of the podocyte at the GBM as compared to the apical surface implying the role of glycocalyx in the function of GFB. In addition, labeling of the basolateral surface of the podocyte cell body and podocyte foot processes enabled the visualization of sub-podocyte space. Acute injection of endoglycosidase enzymes significantly (3-fold) reduced Sdc1-GFP fluorescence intensity and the thickness of the linear labeling suggesting the acute shedding of podocyte glycocalyx. Importantly, increased albumin permeability of the GFB was observed simultaneously with the shedding of podocyte glycocalyx, indicating the disruption of GFB charge and size-selective functions. In conclusion, the specialized podocyte glycocalyx at the GBM may function as an important new layer of the GFB and may play an important role in the disruption of GFB functions in disease. ASN Carl W. Gottschalk Research Scholar Grant University of Southern California Dean's Pilot Project Award DK123564. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Using 5-fluorouracil-encored plga nanoparticles for the treatment of colorectal cancer: the in-vitro characterization and cytotoxicity studies

Objective(s): Colorectal cancer (CRC) is a prevalent cancer worldwide. The present study aimed to synthesize and investigate the potential of wheat germ agglutinin (WGA) conjugated with polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) incorporating 5-fluorouracil (5-FU). Materials and Methods: The NPs were investigated in terms of various characteristics, such as the particle size, surface charge, surface morphology, entrapment efficiency rate, and in-vitro drug release profile in simulated gastric and intestinal fluids. The optimized NPs were conjugated with WGA and characterized for the WGA conjugation efficiency, mucoadhesion, and cytotoxicity studies. Results: The zeta potential of the WGA-conjugated NPs decreased (-17.9±1.4 mV) possibly due to the conjugation of the NPs with WGA, which reduced the zeta potential. The WGA-conjugated NPs exhibited sustained drug release effects (p<0.05) compared to the marketed formulation containing 5-FU after 24 hours. In addition, the optimized NPs followed the Higuchi kinetics, showing diffusion-controlled drug release mechanisms. Finally, the WGA-conjugated PLGA NPs could significantly inhibit the growth of colon cancer cells (HT-29 and COLO-205) compared to the non-conjugated NPs and pure drug solution (P<0.05). Conclusion: According to the results, the WGA-conjugated NPs could be potential carrier systems compared to the non-conjugated NPs for the effective management of CRC.

Engineering the surface of prostate tumor cells and hyaluronan/chitosan multilayer films to modulate cell-substrate adhesion properties

This paper explores different film assembly conditions of the polyelectrolyte solutions of hyaluronan (HA) and chitosan (CHI), as well as both substrate and cell surface modifications, to investigate PC3 cells adhesion properties. UV–Visible, AFM-IR and Zeta potential techniques indicate that the solution ionic strength is a relevant parameter to modulate the free carboxylic groups of HA on the film surface. In addition, capacitive coupling measurements suggest that assembly conditions that favor surface charge mobility inhibit cell adhesion due to polymer rearrangements that support non-specific electrostatic interactions of positively charged CHI residues and the negatively charged cell moieties, rather than specific CD44-hyaluronan interactions. Moreover, the PC3 cells treatment with hyaluronidase and anti-CD44 antibody also highlighted the importance of CD44 binding site availability on the tumor cell adhesion properties. Finally, the conjugation of wheat germ agglutinin on the film surface proved to be a suitable strategy to boost the PC3 cell adhesion properties. Our results reveal the remarkable capacity of HA/CHI films to modulate cell-substrate properties, which pave the road for the development of surfaces suitable for several applications based on biosensing.

TiO2 Nanoparticles and Commensal Bacteria Alter Mucus Layer Thickness and Composition in a Gastrointestinal Tract Model.

Nanoparticles (NPs) are used in food packaging and processing and have become an integral part of many commonly ingested products. There are few studies that have focused on the interaction between ingested NPs, gut function, the mucus layer, and the gut microbiota. In this work, an in vitro model of gastrointestinal (GI) tract is used to determine whether, and how, the mucus layer is affected by the presence of Gram-positive, commensal Lactobacillus rhamnosus; Gram-negative, opportunistic Escherichia coli; and/or exposure to physiologically relevant doses of pristine or digested TiO2 NPs. Caco-2/HT29-MTX-E12 cell monolayers are exposed to physiological concentrations of bacteria (expressing fluorescent proteins) and/or TiO2 nanoparticles for a period of 4 h. To determine mucus thickness and composition, cell monolayers are stained with alcian blue, periodic acid schiff, or an Alexa Fluor 488 conjugate of wheat germ agglutinin. It is found that the presence of both bacteria and nanoparticles alter the thickness and composition of the mucus layer. Changes in the distribution or pattern of mucins can be indicative of pathological conditions, and this model provides a platform for understanding how bacteria and/or NPs may interact with and alter the mucus layer.

Modification of curcumin-loaded liposome with edible compounds to enhance ability of crossing blood brain barrier

This research explored the potential of food grade sialic acid (SA), polymerized SA (PSA), and their oxidized form to conjugate with wheat germ agglutinin (WGA) on liposome to deliver curcumin to brain. Results showed that curcumin was entrapped in liposome with high encapsulation efficiency (90.77%) and the vector had a size of about 115 nm in diameter. The thermogravimetric analysis (TGA) confirmed the successful presence of SA and PSA in the lipid bilayers of liposomes. The TGA curves below 100 °C suggested that WGA acts as a protective layer for SA and PSA. In general, the liposomes modified by the un-oxidized SA or PSA could conjugate more WGA than that oxidized counterparts, and polymerization of SA did not increase WGA conjugation on the liposomes. More than 40% of WGA on the surface of modified liposomes remained after digestive enzymes challenge. The amide peaks at 1241 cm-1 and 533-581 cm-1 in the Fourier transform infrared (FTIR) spectrum of the modified liposomes indicated the covalent bonding between carboxyl group of SA and amine group of WGA. These characteristic peaks still existed after digestive enzymes challenge, demonstrating that the conjugation of WGA and SA on the modified liposome can resist the digestive enzymes to a certain extent. SEM images showed that the modified liposomes after digestive enzymes treatment were still spherical with clear rim, and the enzyme-treated vectors had a higher permeation rate crossing blood-brain barrier (BBB) endothelial cell monolayer than the un-modified liposome without enzyme treatment. This study demonstrates how the surface modification can enhance the function of liposome as a phytochemical carrier for brain disease.

Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes.

Small-molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes, because they bind to the cell's glycocalyx. Here, we describe the derivatization of WGA with a pH-sensitive rhodamine fluorophore (pHRho; pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform and did not appreciably alter the voltage gating of glycosylated ion channels, and the extracellular changes in pH correlated with proton channel activity. Using single-plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small-molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and nonelectrogenic events on the extracellular side of the plasma membrane.

Detection of white head symptoms of panicle blast caused by Pyricularia oryzae using cut-flower dye

BackgroundBreeding of rice with panicle resistance to rice blast disease caused by Pyricularia oryzae is a challenge towards sustainable rice production. Methods for accurate estimation of disease severity can support breeding. White head symptoms are a commonly used index of panicle blast in the field. As the development mechanism of this symptom remains unclear, we used cut-flower dye (CFD) solution to visualize the infected panicle tissues.ResultsCFD delineated the edge of white head symptoms in rice panicles artificially infected with P. oryzae. Hyphae within the tissues were confirmed through staining with a fluorescent wheat germ agglutinin conjugate. Hyphal density was obviously diminished at the dye edge. Growing hyphae preferred to move along the vascular bundles; infected tissues lost the ability to transport water, leading to white head formation. By marking the edge of the white heads, this simple dyeing technique precisely reveals the extent of infection. Further, digital imaging allowed dried samples to be stored and reassessed later.ConclusionsThe CFD detection technique served as a powerful tool for estimating disease severity by color, as it clearly revealed lesions in both the panicles and leaves. Combined with reliable methods for artificial inoculation and observation of infecting hyphae, this technique will advance the research and breeding of panicle blast-resistant rice.

Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-infarction cardiomyocyte remodeling by maintaining transverse-tubule (TT) structures, thus improving calcium dynamics and contractility.MethodsMyocardial infarction (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5μg/kg/d) or vehicle in drinking water for 16 wks (n = 10–11/group). After in vivo echocardiographic and hemodynamic analyses, left ventricular myocytes were isolated by collagenase digestion and simultaneous calcium and contractile transients in single cardiomyocytes were recorded using IonOptix imaging. Live cardiomyocytes were stained with AlexaFluor-488 conjugated wheat germ agglutinin (WGA-488) or di-8-ANEPPS, and multiple z-stack images per cell were captured by confocal microscopy for analysis of TT organization. RTqPCR and immunoblot approaches determined expression of TT proteins.ResultsEchocardiography and in vivo hemodynamic measurements showed significant improvements in systolic and diastolic function in T3- vs vehicle-treated MI rats. Isolated cardiomyocyte analysis showed significant dysfunction in measurements of myocyte relengthening in MI hearts, and improvements with T3 treatment: max relengthening velocity (Vmax, um/s), 2.984 ± 1.410 vs 1.593 ± 0.325, p < 0.05 and time to Vmax (sec), 0.233 ± 0.037 vs 0.314 ± 0.019, p < 0.001; MI + T3 vs MI + Veh, respectively. Time to peak contraction was shortened by T3 treatment (0.161 ± 0.021 vs 0.197 ± 0.011 s., p < 0.01; MI + T3 vs MI + Veh, respectively). Analysis of TT periodicity of WGA- or ANEPPS-stained cardiomyocytes indicated significant TT disorganization in MI myocytes and improvement with T3 treatment (transverse-oriented tubules (TE%): 9.07 ± 0.39 sham, 6.94 ± 0.67 MI + Veh and 8.99 ± 0.38 MI + T3; sham vs MI + Veh, p < 0.001; MI + Veh vs MI + T3, p < 0.01). Quantitative RT-PCR showed that reduced expression of BIN1 (Bridging integrator-1), Jph2 (junctophilin-2), RyR2 (ryanodine receptor) and Cav1.2 (L-type calcium channel) in the failing myocardium were increased by T3 and immunoblot analysis further supporting a potential T3 effect on the TT-associated proteins, BIN1 and Jph2.In conclusion, low dose T3 treatment initiated immediately after myocardial infarction attenuated adverse TT remodeling, improved calcium dynamics and contractility, thus supporting the potential therapeutic utility of T3 treatment in heart failure.

Enhanced anti-colon cancer efficacy of 5-fluorouracil by epigallocatechin-3- gallate co-loaded in wheat germ agglutinin-conjugated nanoparticles

Colon adenocarcinoma is the third most common cause of cancer-related deaths worldwide owing to its aggressive nature. Here, we developed a novel oral drug delivery system (DDS) that comprised active targeted nanoparticles made from gelatin and chitosan (non-toxic polymers). The nanoparticles were fabricated using a complex coacervation method, which was accompanied by conjugation of wheat germ agglutinin (WGA) onto their surface by glutaraldehyde cross-linking. Specifically, we integrated 5-fluorouracil (5-FU), the first-line treatment agent against colon cancer, and (−)-epigallocatechin-3-gallate (EGCG), which inhibits tumor growth via anti-angiogenesis and apoptosis-inducing effects, into the nanoparticles, named WGA-EF-NP. The 5-FU and EGCG co-loaded nanoparticles showed sustained drug release, enhanced cellular uptake, and longer circulation time. WGA-EF-NP exhibited superior anti-tumor activity and pro-apoptotic efficacy compared to the drugs and nanoparticles without WGA decoration owing to better bioavailability and longer circulation time in vivo. Thus, WGA-EF-NP shows promise as a DDS for enhanced efficacy against colon cancer.

Abstract 444: ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted

Abstract GRP78 (a Mr 78kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr (i.e., N-linked glycosylation). This important biochemical event is essential for glycoprotein folding and function. When targeted in ER-/PR-/HER2+ breast tumor in athymic nude mice with the N-glycosylation inhibitor tunicamycin, the cancer growth was reduced ~55% in three weeks. Paraffin sections of the tumor exhibited reduced N-glycan level, and reduced angiogenesis but increased GRP78 expression in microvasculature as well as in the tumor tissue (J. Biol. Chem. 286, 29127–29138, 2011). This supported the presence of ER stress. We have now evaluated the effect of tunicamycin on the proliferation of metastatic ER-/PR-/HER2- (MDA-MB-231) and non-metastatic ER+ (MCF-7) human breast cancer cells. Tunicamycin inhibited proliferation of both cells types in a time and dose-dependent manner. Interestingly, GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 µg/ml) treated MDA-MB-231 and in MCF-7 cells and supported by both Western blotting and qPCR. Since GRP78 is an ER stress marker, so we have followed its intracellular localization by immunofluorescence microscopy after treating the cancer cells with tunicamycin as well as after subjecting them to various stress conditions. We have used unfixed cells and stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) or with anti-GRP antibody. There were expression of N-glycans but not GRP78. GRP78 however became detectable after permeabilization of cells with a brief exposure to ice-cold methanol. GRP78 was not detected in the conditioned media of the cancer cell but a high level of MMP-1 did. We have therefore, concluded that GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. This is obviously, contradicted the current dogma that GRP78 expression on the tumor cell surface interferes with the cancer therapeutic(s) and make them as tumor promoters and not tumor suppressors. Our study, thus, suggests strongly that anti-angiogenic and anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast tumor and all other sold tumors. Supported in part by the university funds and NIH/NIMHD G12MD007583 (KB). Citation Format: Jesus E. Serrano, Andrea Rivera-Ruiz, Krishna Baksi, Dipak K. Banerjee. ER Stress marker GRP78 is not expressed on ER-/PR-/Her2- human breast cancer cell surface or secreted [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 444.

Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.

GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) is located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Nonmetastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/mL) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A) or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

The effect of rebamipide ophthalmic suspension on ocular surface mucins in soft contact lens wearers.

To evaluate the changes in ocular surface mucins with 2%rebamipide ophthalmic suspension treatment in soft contact lens (SCL) wearers. Rebamipide suspension is a mucin secretagogue approved for the treatment of dry eye syndrome in Japan. In this study, the fluorescence intensity of wheat germ agglutinin conjugate of fluorescein (F-WGA) was used as a marker of membrane-associated mucins, and sialic acid concentration in tear fluids as a marker of secreted mucins. Thirty-two eyes of 16 SCL wearers with discomfort were treated with rebamipide suspension at a dose of one drop in each eye four times daily for two weeks. The parameters of clinical efficacy were tear break-up time, fluorescein staining scores for the cornea and conjunctiva, and Schirmer test values. Fluorescence intensities in the central cornea were measured by fluorophotometry after the application of 5% F-WGA solution. Tears collected by Schirmer test strips were analyzed by high-performance liquid chromatography, and the concentrations of sialic acid, total protein, and the four major tear proteins, namely secretory IgA, lactoferrin, lipocalin-1, and lysozyme were measured. Significant increases in F-WGA fluorescence intensities (p < 0.005) were seen in the corneal surfaces. Sialic acid concentrations increased over time; however, the differences were not statistically significant. Except for a slight increase in kerato-conjunctival staining scores (p < 0.05) and secretory IgA (p < 0.05), no other significant differences were seen among clinical parameters or tear proteins. Topical application of rebamipide suspension significantly increased F-WGA intensity, a marker of membrane-associated mucins in SCL wearers.

Targeted systemic mesenchymal stem cell delivery using hyaluronate - wheat germ agglutinin conjugate.

A variety of receptors for hyaluronate (HA), a natural linear polysaccharide, were found in the body, which have been exploited as target sites for HA-based drug delivery systems. In this work, mesenchymal stem cells (MSCs) were surface-modified with HA - wheat germ agglutinin (WGA) conjugate for targeted systemic delivery of MSCs to the liver. WGA was conjugated to HA by coupling reaction between aldehyde-modified HA and amine group of WGA. The conjugation of WGA to HA was corroborated by gel permeation chromatography (GPC) and the successful surface modification of MSCs with HA-WGA conjugate was confirmed by confocal microscopy. The synthesized HA-WGA conjugate could be incorporated onto the cellular membrane by agglutinating the cell-associated carbohydrates. Fluorescent imaging for invivo biodistribution visualized the targeted delivery of the HA-WGA/MSC complex to the liver after intravenous injection. This new strategy for targeted delivery of MSCs using HA-WGA conjugate might be successfully exploited for various regenerative medicines including cell therapy.

Diquafosol for Soft Contact Lens Dryness: Clinical Evaluation and Tear Analysis.

To evaluate the efficacy of topical application of 3% diquafosol tetrasodium solution for the treatment of soft contact lens (SCL) wearers with dryness. In addition to clinical tests and subjective symptoms, we assessed the fluorescence intensity of wheat germ agglutinin conjugate of fluorescein (F-WGA) used as a marker of membrane-associated mucins and sialic acid concentration in tear fluids as a marker of secreted mucins. Twelve SCL wearers with dryness symptoms were treated with diquafosol for 4 weeks. Clinical tests included the tear film break-up time (BUT), corneal and conjunctival fluorescein staining scores, and Schirmer values. Subjective symptoms were evaluated by the Dry Eye-Related Quality-of-Life Score (DEQS). Fluorescence intensities in the central cornea were measured by fluorophotometry at 5 minutes after a 5% F-WGA solution was applied to the eye. The tears collected by the Schirmer test strips were analyzed by high-performance liquid chromatography (HPLC), and the concentrations of sialic acid; total protein; and the four major tear proteins secretory IgA, lactoferrin, lipocalin-1, and lysozyme proteins were measured. Comparing the results before and after diquafosol treatment, BUT (p < 0.01), kerato-conjunctival staining score (p < 0.05), corneal staining score (p < 0.05), and DEQS score (p < 0.01) showed statistically significant improvements. The F-WGA fluorescence intensities (p < 0.0001) significantly increased after treatment, whereas the concentrations of sialic acid and tear proteins remained unchanged. Topical application of diquafosol solution to the SCL wearers with dryness improved biomarker of membrane-associated mucins, BUT, staining of cornea and conjunctiva, and subjective symptoms.

Inhibition of bacterial adhesion and biofilm formation by antimicrobial functionalized textured biomaterial surfaces

Event Abstract Back to Event Inhibition of bacterial adhesion and biofilm formation by antimicrobial functionalized textured biomaterial surfaces Lichong Xu1, Yaqi Wo2, Mark E. Mayerhoffb2, Zhicheng Tian3, Harry Allcock3 and Christopher A. Siedlecki1, 4 1 Penn State College of Medicine, Department of Surgery, United States 2 University of Michigan, Department of Chemistry, United States 3 The Pennsylvania State University, Department of Chemistry, United States 4 Penn State College of Medicine, Department of Bioengineering, United States Introduction: Infection due to bacterial adhesion and biofilm formation represetns a major drawback to the use of blood-contacting devices. Previously we have developed textured polyurethane biomaterials for reducing bacterial adhesion and inhibiting biofilm formation. In this work, we applied the texturing pattern to functionalized polymers bearing antimicrobial properties, and show a combination of antimicrobial functionalization and physical approaches can produce additive effects on biological responses and enhance the hemocompatibility of biomaterials. Material and Methods: Carbosil polyurethane (PU, 2080A) and phosphazene polymers (trifluoroethoxy (TFE), crosslinakble trifluoroethoxy (XLTFE), and octafluoropentoxy (OFP)) were textured with ordered arrays of pillars using a soft lithography two-stage replication molding technique[1]. The Carbosil polyurethane material was doped with S-nitroso-N-acetylpenicillamine (SNAP) with different contents of SNAP in PU (5, 10, and 15 wt%) for nitric oxide (NO) release[2]. Antimicrobial properties of materials were evaluated by the optical density (OD600) of microbial cultures incubated with materials and strain S. epidermidis RP62A. Bacterial adhesion and biofilm formation on material surfaces were carried out in a multiwell plate or rotating disk system (RDS)[1] with same strain. Bacterial adhesion was counted under a fluorescent optical microscope. To observe biofilm formation, samples were in incubated in multiwell plates under static condition for 2 days or RDS system under shear for 8 days. Samples were stained with FTIC conjugated wheat germ agglutinin, and biofilm observed by fluorescence. Results and Discussion: Characterization of biomaterial surfaces: AFM images showed material surfaces textured with ordered arrays of pillars, as expected, except for TFE and XLTFE materials that were difficult to texture.Water contact angle measurements show surface hydrophobicity increased after texturing due to entrapped air. Antimicrobial properties of materials:The optical densities of culture media indicate the growth of bacteria in the presence of a variety of polymers (Fig. 1). Carbosil PU 0% SNAP and PUU films show the similar optical density as that of control (no polymers). Significant inhibition of bacterial growth was observed in the cultures for Carbosil 10% and 15% SNAP polymers, indicating NO release inhibits the growth of bacteria. A slight decrease of OD600 was observed for all phosphazene polymers. Fig. 1 Optical density of culture media indicating antimicrobial properties of polymers. Bacterial adhesion on functionalized textured polymer surfaces: Higher bacterial adhesion was observed on smooth surfaces without functionalization (Fig. 2). Surface texturing with pattern decreased bacterial adhesion on all surfaces, except for TFE and XLTFE polymers, indicating the importance of surface texturing in inhibiting adhesion. Furthermore, a significant decrease in adhesion was observed on textured PU surfaces containing 10% or 15% SNAP as well as on OFP textured surfaces, suggesting antimicrobial functionalization and texturing produce a synergistic effect on inhibiting bacterial adhesion, as the OFP polymer alone inhibited bacterial growth only slightly. Fig. 2 Bacterial adhesion on polymer surface in PBS for 1 hr at 37°C under static condition. Biofilm formation: Under static condition, biofilms were observed on Carbosil PU surfaces containing 0% SNAP and 5% SNAP after 2 days, while no biofilms were observed on surfaces containing 10% and 15% SNAP. Under shear stresses, no biofilms were observed on smooth or textured PU surfaces containing 15% SNAP after 8 days. Long term exposure experiments for biofilm formation on functionalized textured surfaces are on-going. Fig. 3 Biofilm on textured 500/500 nm patterned surfaces containing (a) 0% SNAP, (b) 15% SNAP under static condition for 2 days, and (c) 15% SNAP under shear for 8 days. Conclusion: A combination of antimicrobial functionalization of polymers and surface texturing provided an improved approach to inhibiting bacterial adhesion and biofilm formation, and thus to potentially preventing biomaterial related infections.

Multi-functional Liposomes Enhancing Target and Antibacterial Immunity for Antimicrobial and Anti-Biofilm Against Methicillin-Resistant Staphylococcus aureus.

The aim of this study was to prepare wheat germ agglutinin (WGA)-modified liposomes encapsulating clarithromycin and to evaluate their in vitro and in vivo efficacy against Methicillin-resistant Staphylococcus aureus (MRSA). Physicochemical parameters, minimum inhibitory concentrations, in vitro killing kinetic, cellular uptake, biofilm formation inhibition and pre-formed biofilm destruction, biodistribution, in vivo antibacterial efficacy against MRSA, and phagocytosis into macrophages for liposomes loading clarithromycin were determined. The minimum inhibitory concentration and the time-kill curve for WGA-modified liposomal clarithromycin were better than those of free and nonmodified liposomal clarithromycin. Flow cytometry analysis displayed that liposomes could deliver more Coumarin 6, a fluorescent probe, into bacteria because of the conjugation of WGA. Besides, WGA-modified liposomal clarithromycin inhibited formation of S. aureus (ATCC 29213) and MRSA biofiom, and prompted the biofilm disassembly at lower concentrations below MIC. Effective accumulation of liposomes was displayed in the enterocoelia of the mice because of WGA. The number of MRSA colony-forming units in the kidney and spleen in mice treated with WGA-modified liposomal clarithromycin was significantly lower than that treated with free and nonmodified clarithromycin (p < 0.05). Intracellular localization of MRSA occurred in a significantly higher proportion of macrophage exposed to WGA-modified liposomes compared to those exposed to nonmodified liposomes. Liposome modified by WGA is a promising formulation for bacteria targeted delivery and immunity defensive system through macrophage improving uptake of bacteria, biodistribution, in vitro and in vivo antibacterial efficacy against MRSA.

Characterization, biorecognitive activity and stability of WGA grafted lipid nanostructures for the controlled delivery of Rifampicin

Targeted nanomedicines improve the delivery of drugs by increasing the drug concentration at target site, protecting the premature degradation and releasing the encapsulated drug in controlled manner. To make rifampicin (RFN) delivery more effective, we designed and characterized wheat germ agglutinin (WGA) conjugated, RFN loaded solid-lipid nanoparticles (WRSN). Nanoparticles were prepared by solvent emulsification/evaporation and conjugated with fluorescein isothiocyanate-labeled WGA. Important characteristics, such as particle size, zeta potential, encapsulation efficiency, conjugation efficiency and in vitro drug release behavior, were investigated. WGA conjugation to the nanoparticles was confirmed by Fourier Transform Infrared (FTIR) analysis. Conjugation efficiency was determined by fluorescent spectroscopy and Bradford assay. RFN was released from nanoparticles via the diffusion-controlled, non-fickian and supercase II mechanism. A haemaglutination test confirmed that WGA retained its bio-recognition activity and sugar-binding specificity after it was coupled with the nanoparticles. In vitro experiments demonstrated that WRSN interacted more than non-conjugated nanoparticles with porcine mucin. WRSN were stable in the presence of electrolytes up to 1M concentration. Therefore, WGA-conjugated solid lipid nanoparticles could be a promising tool for the controlled delivery of RFN or other anti-tubercular drugs.

Abstract 315: Inflamed HDL Loses Its Anti-inflammatory Properties on Adipocytes: Role of Interaction of Serum Amyloid A with the Extracellular Matrix

Obesity induces adipocyte hypertrophy and accumulation of macrophages in adipose tissue, which associates with insulin resistance and cardiovascular disease risk. HDL and apoA-I have anti-inflammatory properties in addition to facilitating reverse cholesterol transport. We recently showed that HDL from healthy humans and lean mice inhibits palmitate-induced monocyte chemotactic factor expression by decreasing lipid raft (LR) content in adipocytes. We also have shown that adipocytes produced extracellular matrix (ECM) in vivo and in vitro when they became hypertrophic. Moreover, inflamed HDL loses its anti-inflammatory effect and ability to stimulate cholesterol efflux from adipocytes. From gain of function and loss of function experiments, it appears that serum amyloid A (SAA) is partially responsible for these effects. However, the mechanism by which SAA impairs HDL’s anti-inflammatory function is unknown. To generate HDL under conditions of sterile inflammation, HDL was purified from plasma of AgNO 3 -injected C57BL/6 mice. Also, to generate SAA-enriched HDL, HDL was purified after HDL from healthy humans was exposed to 293 HEK cells with lentiviral overexpression of SAA2. Control, inflamed and SAA-enriched HDL were labelled using DiI dye and the ECM of adipocytes was stained by Alexa 488 conjugated wheat germ agglutinin. After exposure of control HDL to adipocytes, there was no co-localization of DiI and Alexa 488 staining. However, we found that inflamed and SAA-enriched HDL were co-localized with the ECM of adipocytes, suggesting that inflamed and SAA-enriched HDL are being trapped at the ECM and therefore are blocked from accessing the plasma membrane. Enzymatic digestion of the ECM restored inflamed and SAA-enriched HDL’s abilities to facilitate cholesterol efflux from adipocytes and to inhibit palmitate-induced chemotactic factor expression. Taken together, our results suggest that the presence of SAA on HDL makes the lipoprotein lose its anti-inflammatory effect on adipocytes by blocking accessibility of HDL to the plasma membrane. The interaction between SAA in HDL and the ECM of adipocytes should be considered as a new therapeutic target for improvement of HDL’s anti-inflammatory properties.

Diquafosol sodium ophthalmic solution for the treatment of dry eye in soft contact lens wearers

AbstractPurpose Ocular dryness, experienced by up to 75% of soft contact lens (SCL) wearers, is the main reason for its discontinuation. The aim of the study is to evaluate the efficacy of topical application of diquafosol sodium for the treatment of dry eye in SCL wearers. In addition to clinical tests, we used a wheat germ agglutinin conjugate of fluorescein (F‐WGA) and fluorophotometry to evaluate the glycocalyx in the ocular surface in vivo.Methods Twelve SCL wearers with dry eye symptoms (31.3 ± 6.8 year‐old) were treated with 3% diquafosol eyedrops for 4 weeks. The clinical tests included tear break‐up time (BUT), Schirmer test, fluorescein staining score, and subjective symptoms evaluated by the Dry Eye Related Quality of Life Score (DEQS). Five minutes after a 5% F‐WGA solution was applied to the eye, fluorescent intensities in the central cornea were measured by fluorophotometry.Results When comparing before and after diquafosol treatment for 4 weeks, BUT (3.6±2.2 sec to 5.0±2.1 sec, p=0.003), kerato‐conjunctival staining score (2.8±1.8 to 2.1±1.4, p=0.045) and DEQS score (19.2±12.7 to 10.6±12.5, p=0.0032) showed statistically significant improvements (Wilcoxon signed‐rank test). F‐WGA fluorescent intensities in the central cornea (571.8±227.8 to 794.6±219.4, p&lt;0.0001) significantly increased, and had a significant correlation with BUT.Conclusion Fluorophotometric measurement of WGA staining appears to reflect the glycocalyx in the corneal epithelium, and therefore can be a good indicator of the ocular surface wettability. Topical application of diquafosol sodium improved subjective symptoms of SCL wearers with dry eye, possibly by enhancing tear film stability and reducing damage of ocular surface epithelia.

Poly(lactic acid) nanoparticles coated with combined WGA and water-soluble chitosan for mucosal delivery of β-galactosidase

A combinatorial design, physical adsorption of water-soluble chitosan (WSC) to particle surface and covalent conjugation of wheat germ agglutinin (WGA) to WSC, was applied to surface modification of poly(lactic acid) nanoparticles (NPs) for targeted delivery of β-galactosidase to the intestinal mucosa. All the surface-engineered NPs in the size range of 500–600 nm were prepared by a w/o/w solvent diffusion/evaporation technique. β-Galactosidase encapsulated in these NPs was well protected from external proteolysis and exerted high hydrolytic activity on the permeable lactose. The presence of WSC coating, whether alone or with WGA, highly improved the suspension stability of NPs and tailored the particle surface positively charged. In comparison to NPs modified with WGA or WSC alone, the synergistic action of WGA and WSC greatly enhanced the NP–mucin interactions in vitro. The highest amount of NPs was found in the small intestine at 24 h after oral administration in rats. Notably, calculated half-life of WGA–WSC–NPs in the small intestine was 6.72 h, resulting in 2.1- and 4.3-fold increase when compared to WGA–polyvinylalcohol (PVA)–NPs and WSC–NPs, much longer than that of control PVA–NPs (6.9-fold). These results suggest that NPs with the combined WGA and WSC coating represent promising candidates for efficient mucosal drug delivery as well as biomimetic treatment of lactose intolerance.