Uptake Of Heparin Research Articles

Modified Diatomaceous Earth in Heparin Recovery from Porcine Intestinal Mucosa.

Heparin, a highly sulfated glycosaminoglycan, is a naturally occurring anticoagulant that plays a vital role in various physiological processes. The remarkable structural complexity of heparin, consisting of repeating disaccharide units, makes it a crucial molecule for the development of commercial drugs in the pharmaceutical industry. Over the past few decades, significant progress has been made in the development of cost-effective adsorbents specifically designed for the adsorption of heparin from porcine intestinal mucosa. This advancement has been driven by the need for efficient and scalable methods to extract heparin from natural sources. In this study, we investigated the use of cationic ammonium-functionalized diatomaceous earth, featuring enhanced porosity, larger surface area, and higher thermal stability, to maximize the isolated heparin recovery. Our results showed that the higher cationic density and less bulky quaternary modified diatomaceous earth (QDADE) could adsorb up to 16.3 mg·g-1 (31%) of heparin from the real mucosa samples. Additionally, we explored the conditions of the adsorbent surface for recovery of the heparin molecule and optimized various factors, such as temperature and pH, to optimize the heparin uptake. This is the introductory account of the implementation of modified diatomaceous earth with quaternary amines for heparin capture.

Efficient Heparin Recovery from Porcine Intestinal Mucosa Using Zeolite Imidazolate Framework-8.

Heparin is one of the most valuable active pharmaceutical ingredients, and it is generally isolated from porcine intestinal mucosa. Traditionally, different types of commercial resins are employed as an adsorbent for heparin uptake; however, using new, less expensive adsorbents has attracted more interest in the past few years to enhance the heparin recovery. Zeolite imidazolate framework-8 (ZIF-8), as a metal–organic framework (MOF) with a high surface area, porosity, and good stability at high temperatures, was selected to examine the heparin recovery. In this research, we demonstrate that ZIF-8 can recover up to ~70% (37 mg g−1) of heparin from porcine intestinal mucosa. A mechanistic study through kinetic and thermodynamic models on the adsorption revealed appropriate surface conditions for the adsorption of heparin molecules. The effect of different variables such as pH and temperature on heparin adsorption was also studied to optimize the recovery. This study is the first to investigate the usage of MOFs for heparin uptake.

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.

High-Throughput, Green, Low-Cost, and Efficient Recovery of Heparin from a Biological Mixture Using Bio-Originated Magnetic Nanofibers

Heparin is an anticoagulant commercially derived from porcine intestinal mucosa that is challenging to selectively isolate for further purification. In this study, we propose the fabrication of amine-functionalized magnetic nanobiofibers (NBF@Fe-NH2), 1–2 μm long and 50–200 nm wide with mostly tube-shaped structures, originating from grape seeds as green and efficient bioadsorbents for the efficient recovery of heparin. To rapidly screen for heparin adsorption, we used a novel high-throughput technique based on UV–vis spectroscopy and 96-well microplates. The heparin uptake performance of the fabricated adsorbents vs Amberlite FPA98 Cl, a conventional absorbent utilized in industry for heparin recovery, was examined in a mixture containing heparin isolated from porcine intestinal mucosa. The NBF@Fe-NH2 showed a higher heparin uptake perf compared to the Amberlite FPA98 Cl. Furthermore, we also performed a sheep plasma clotting assay and found that the heparin sample separated by NBF@Fe-NH2 featured a high...

Anticalcification potential of heparin on hydroxyapatite seeds using a constant composition in vitro model

Calcification is among the principal causes of biological heart valve substitute failure. Glycosaminoglcans (GAGs) are negatively charged molecules, possessing anticoagulation and anti-inflammatory activity that make them a potential solution against calcification. In the present work, the anticalcification potential of heparin was investigated under constant supersaturation conditions with respect to hydroxyapatite (Ca5(PO4)3OH; HAP). Heparin concentration in the supersaturated solutions was in the range between 0.25 and 3 ppm, at pH 7.40 and 37 °C. Heparin showed inhibitory activity, which was attributed to adsorption at the active crystal growth centres. Heparin concentration as low as ca. 50 μM, reduced the rate of HAP crystal growth by more than 80%, while further increase (up to 200 μM) failed to completely inhibit the process beyond 90%. Heparin uptake studies at equilibrium conditions and analysis of the kinetics data in the presence of heparin, strongly suggest that the inhibition is due to the adsorption of heparin onto the HAP crystals, which resulted in the poisoning of the active growth sites.

Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs.

Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions.

Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin

Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.

Cellular uptake and activity of heparin functionalised cerium oxide nanoparticles in monocytes

Cerium oxide nanoparticles (nanoceria) are effective in scavenging intracellular reactive oxygen species (ROS). In this study nanoceria synthesized by flame spray pyrolysis (dXRD = 12 nm) were functionalised with heparin via an organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria were functionalised with approximately 130 heparin molecules per nanoparticle as determined by thermo gravimetric analysis. Heparin functionalised nanoceria were more effectively internalised by the human monocyte cell line, U937, and U937 cells that had been activated with phorbol 12 myristate 13-acetate (PMA) than bare nanoceria. The heparin functionalised nanoceria were also more effective in scavenging ROS than nanoceria in both activated and unactivated U937 cells. Heparin coupled nanoceria were found to be biologically active due to their ability to bind fibroblast growth factor 2 and signal through FGF receptor 1. Additionally, the heparin-coupled nanoceria, once internalised by the cells, were found to be degraded by 48 h. Together these data demonstrated that heparin enhanced the biological properties of nanoceria in terms of cellular uptake and ROS scavenging, while the nanoceria themselves were more effective at delivering heparin intracellularly than exposing cells to heparin in solution.

Sulfation Patterns Determine Cellular Internalization of Heparin-Like Polysaccharides

Heparin is a highly sulfated polysaccharide that serves biologically relevant roles as an anticoagulant and anticancer agent. While it is well-known that modification of heparin's sulfation pattern can drastically influence its ability to bind growth factors and other extracellular molecules, very little is known about the cellular uptake of heparin and the role sulfation patterns serve in affecting its internalization. In this study, we chemically synthesized several fluorescently labeled heparins consisting of a variety of sulfation patterns. These polysaccharides were thoroughly characterized using anion exchange chromatography and size exclusion chromatography. Subsequently, we utilized flow cytometry and confocal imaging to show that sulfation patterns differentially affect the amount of heparin uptake in multiple cell types. This study provides the first comprehensive analysis of the effect of sulfation pattern on the cellular internalization of heparin or heparan sulfate like polysaccharides. The results of this study expand current knowledge regarding heparin internalization and provide insights into developing more effective heparin-based drug conjugates for applications in intracellular drug delivery.

Intracellular Delivery of Heparin Complexed with Chitosan-g-Poly(Ethylene Glycol) for Inducing Apoptosis

Chitosan-g-PEG/heparin polyelectrolyte complex micelles were prepared for inducing apoptotic death of cancer cells. The cytotoxicity of polyelectrolyte complex micelles was evaluated by examining the growth inhibition of mouse melanoma B16F10 cells. Cellular uptake and apoptosis-inducing effect were investigated by confocal laser scanning microscopy and flow cytometric analysis, respectively. The prepared polyelectrolyte complex micelles had a spherical shape with an average diameter of 162.8 +/- 18.9 nm. They were highly stable and well dispersed even in the presence of serum due to the presence of hydrophilic PEG shell layer surrounding the micelles. Moreover, they exhibited significantly higher cytotoxic activity against B16F10 cells compared to heparin or chitosan-g-PEG at the same concentration. The polyelectrolyte complex micelles were internalized by cancer cells to a greater extent than free heparin alone, indicating that the dramatic cell death was attributed to the increased cellular uptake of heparin. The internalized heparin was shown to induce apoptotic death of the cancer cells via a caspase-dependent pathway. Nanosized and stable chitosan-g-PEG/heparin polyelectrolyte complex micelles were produced by a self-assembly process. The polyelectrolyte complex micelles facilitated the intracellular delivery of heparin, triggered the caspase activation, and consequently promoted apoptotic death of cancer cells.

Synthesis, characterization, and intracellular delivery of reducible heparin nanogels for apoptotic cell death

Reducible heparin nanogels cross-linked with disulfide linkages were developed for efficient cellular uptake of therapeutic heparin to induce apoptotic cell death. The heparin nanogels were synthesized by forming nanocomplexes between thiolated heparin and poly(ethylene glycol) in a selected organic solvent, and subsequently producing intermolecular disulfide bonds between thiolated heparin molecules by ultrasonication. The resultant heparin nanogels had a stable structure with an average diameter of 248.7 ± 26.8 nm in aqueous solution. However, they rapidly disintegrated and released free heparin molecules under reductive environments, such as intracellular cytosol, through the cleavage of disulfide cross-links within their network structure. Confocal laser scanning microscopy and flow cytometric analysis revealed that these heparin nanogels significantly inhibited proliferation of mouse melanoma cells by inducing caspase-mediated apoptotic cell death. The present study suggested that the reducible heparin nanogels exhibiting a remarkable apoptotic activity could be potentially applied for cancer cell targeted delivery when combined with various therapeutic and diagnostic agents.

Sustained release and activation of the growth factor basic fibroblast growth factor from loaded scaffolds in heart valve tissue engineering

Objectives. Loading of biological matrices offers an opportunity to induce specific cell behaviour. We previously reported the use of growth factors to promote cell invasion and proliferation in tissue valve engineering.We investigated biological matrices preloaded with heparin as an ionically attractive template for the binding, activation and sustained release of basic fibroblast growth factor (bFGF).Methods. Heparin loading concentrations were evaluated and different incubation times were tested. Heparin and heparin-bound bFGF uptake and release were evaluated by 123I radio-labelling. Biological activity of bFGF was evaluated in vitro.Results. Maximum heparin uptake was observed for 2000 μg/ml at 2 h and stabilized thereafter. bFGF-loaded matrices showed an initial burst release of 15% within 4 h and thereafter sustained release reaching 21% at 24 h. Released bFGF was bioactive.Conclusions. This model would be useful in tissue engineering using porcine aortic matrices and could be applied using other growth factors or combinations.

Improvement of the intestinal membrane permeability of low molecular weight heparin by complexation with stem bromelain

The aim of this study was to investigate the influence of the proteolytic enzyme bromelain on the permeation of heparin across the gastrointestinal epithelial barrier. Stability of the complex and effect of heparin on the enzymatic activity of bromelain was analysed photometrically by measuring bromelain enzymatic activity in complex with the heparin. In vitro permeation studies were performed with Caco-2 cell monolayer and rat small intestinal mucosa in Ussing-type chambers, respectively. Results revealed that enzymatic activity of bromelain remained uninfluenced by the immobilization of heparin on it. Transport studies across Caco-2 cell monolayer and rat small intestine showed that the permeation of heparin could be significantly increased in presence of bromelain. In the study with Caco-2 cells, the most effective molar ratio of bromelain to heparin was 2:1, leading to 6.7-fold improvement in uptake, whereas the molar ratio 1:1 showed the highest permeation enhancing effect in the study on intestinal mucosa. This study provides evidence that heparin and bromelain form stable complexes leading to a significantly improved uptake of heparin.

Heparin-Induced Cancer Cell Death

Abstract Heparin uptake into cancer cells can be promoted by conjugation to poly (β-amino ester)s. Internalized heparin is cytotoxic, causing cancer cell death by interfering with transcription factor activity and inducing apoptosis, but only certain poly(β-amino ester)s promote this activity.

Development and in vivo evaluation of an oral delivery system for low molecular weight heparin based on thiolated polycarbophil.

It was the purpose of this study to develop a new oral drug delivery system for low molecular weight heparin (LMWH) providing an improved bioavailability and a prolonged therapeutic effect. The permeation enhancing polycarbophil-cysteine conjugate (PCP-Cys) used in this study displayed 111.4 +/- 6.4 microM thiol groups per gram polymer. Permeation studies on freshly excised intestinal mucosa were performed in Ussing chambers demonstrating a 2-fold improved uptake of heparin as a result of the addition of 0.5% (w/v) PCP-Cys and the permeation mediator glutathione (GSH). Tablets containing PCP-Cys, GSH, and 279 IU of LMWH showed a sustained drug release over 4 h. To guarantee the swelling of the polymeric carrier matrix in the small intestine tablets were enteric coated. They were orally given to rats. For tablets being based on the thiomer/GSH system an absolute bioavailability of 19.9 +/- 9.3% (means +/- SD; n = 5) vs. intravenous injection could be achieved. whereas tablets comprising unmodified PCP did not lead to a significant (p < 0.01) heparin concentration in plasma. The permeation enhancing effect and subsequently a therapeutic heparin level was maintained for 24 h after a single dose. Because of the strong and prolonged lasting permeation enhancing effect of the thiomer/GSH system, the oral bioavailability of LMWH could be significantly improved. This new delivery system represents therefore a promising tool for the oral administration of heparin.

Novel Poly(urethane-aminoamides): an in vitro study of the interaction with heparin

In order to obtain heparin-binding polyurethanes, tertiary amino-groups have been introduced in the polymer backbone by attributing a key-role to the chain extender, i.e. substituting butanediol, commonly used in polyurethane synthesis, with a tailor-made diamino-diamide-diol. In this work a poly(ether-urethane-aminoamide) (PEU/PIME/al) was obtained with poly(oxytetramethylene) glycol 2000, 1,6-hexamethylene-diisocyanate and the new chain extender, in the molar ratio 1 :2 : 1. The heparin binding capacity of PEU/PIME/al was evaluated with 125I labelled heparin, using for comparison the analogous polymer obtained with a diamide-diol (i.e. the poly(ether-urethane-amide) PEU/PIBLO/al), and two commercially available biomedical polyurethanes (Pellethane 2363 and Corethane). pH and ionic strength dependence of the heparin uptake were investigated by treating all the polyurethanes with solutions of 125I heparin into buffers from pH 4 to 9 or NaCl molarity from 0.0 to 1.0. The stability of the interaction with bound heparin was investigated by sequential washing treatments (PBS, 1 N NaOH, 2% SDS solution), then analysing the residual radioactivity on the materials. Results indicated that the heparin binding of PEU/PIME/al is significantly higher and more stable than that of the other polyurethanes, with a time-dependent kinetic. The interaction with heparin appears to be prevalently ionic, with the contribution of other electrostatic and hydrophobic interactions. Activated partial thromboplastin time (APTT), performed on human plasma with polyurethane-coated, heparinized test tubes, indicated that bound heparin maintains its biological activity after the adsorption.

Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: Comparison between heparin-sensitive and heparin-resistant cultures

We studied the binding and entry of fluorescein (FITC)-labeled heparin derivatives into rat aortic smooth muscle cells (SMC) by confocal microscopy. FITC-labeled heparin fractions or FITC-labeled SR 80037A, a potent antiproliferative heparin derivative (Bârzu et al., Eur. J. Pharmacol., 219:225-233 1992), were prepared and their antiproliferative activity was confirmed. By incubating SMC with FITC-labeled heparins, a specific cell-associated fluorescence was found. Cellular fluorescence was mostly located around the nucleus and at the level of cell contacts or cell adhesion. The fluorescence was displaced neither by chasing with excess of unlabeled heparins nor by washing with 1 M NaCl, which proved that labeled heparins had been internalized by SMC. Kinetics of internalization of FITC-heparins suggested receptor-mediated endocytosis of heparins by SMC. Double labeling of SMC with biotinylated Concanavalin A and FITC-SR 80037A also indicated that heparin derivative enters the endocytic pathway. The process was accelerated when serum was present in the incubation medium. Treatment of cells with chloroquine (50 microM) induced accumulation of FITC-SR 80037A in the late endosomes, around the nucleus. No fluorescence labeling could be evidenced inside the nucleus. Neither electron microscopy nor cell fractionation experiments performed with SMC previously incubated with [3H]-heparin were able to ascertain nuclear uptake of heparin, as proposed by other workers (Busch et al., Cell Biol., 116:31-42; 1992; Sing et al., Drug Dev. Res., 29:129-136 1993). The cell-associated fluorescence was very weak in SMC resistant to the antiproliferative activity of heparin, selected by long-term heparin treatment (HT-SMC) as previously shown [Bârzu et al., J. Cell. Physiol., 160:239-248, 1994]. The HT-SMC differed from control SMC with regard to expression of extracellular matrix proteins. These cells exhibited very low expression of fibronectin and prevalent expression of laminin and synthesized less cell-associated glycosaminoglycans. From our results, the following conclusions can be drawn: (1) the antiproliferative heparins are bound and internalized by SMC without being taken up into the nucleus; (2) there is a correlation between the binding and/or the internalization process and the sensitivity of SMC to the antiproliferative activity of heparins; and (3) selection of heparin-resistant SMC by long treatment with heparin results in particular growth pattern of SMC (absence of focal overgrowth), associated with changes in the expression of the extracellular matrix components (fibronectin, laminin, and cell-bound glycosaminoglycans).

The Mechanism of Nitroglycerine Induced Reduction in Plasma Heparin Levels Does Not Appear to Involve Altered Heparin Uptake Or Release by Endothelial Cells

The Mechanism of Nitroglycerine Induced Reduction in Plasma Heparin Levels Does Not Appear to Involve Altered Heparin Uptake Or Release by Endothelial Cells

Therapeutic levels of nitroglycerine do not affect the uptake and release of heparin by endothelial cells in vitro

Intravenous heparin and nitroglycerin are frequently given in combination to patients with acute coronary syndromes such as unstable angina and post myocardial infarction angina. Heparin is prescribed since it has been shown that intracoronary thrombus formation is important in the pathophysiology of these acute conditions. However, it has been demonstrated that intravenous nitroglycerin can interfere with the anticoagulant effect of heparin. The exact mechanism of the interaction is unknown but it has been suggested that there is a direct effect on plasma heparin characterised by a reduction in circulating plasma heparin levels. Heparin binds to the surface of endothelial cells in a process that is time dependent, reversible and exhibits saturation kinetics. A possible mechanism of the observed effects on the plasma heparin levels produced by nitroglycerin may be the altered handling of heparin by endothelial cells. We have investigated this further by assessing the effects of therapeutic doses of nitroglycerin on heparin uptake and release by endothelial cells, using 35S labelled heparin and human umbilical vein endothelial cell cultures.

Performance of a New Vascular Xeno Prosthesis

To date no prosthetic vascular prosthesis performs as well as the autologous saphenous vein in diameters of 6 mm and less. The purpose of this study was to compare the in vitro and in vivo performance of a new glutaraldehyde stabilized, narrow diameter (4 mm), vascular prosthesis fashioned from bovine ureters (Flonova®) with the saphenous vein and polytetrafluoroethylene (PTFE) prostheses. The methods used for the comparative in vitro analysis of the bovine ureter and saphenous vein were modifications of tests recommended by the American National Standards for Vascular Prostheses. Blood compatibility was evaluated by measuring platelet consumption in an artificial circulation, and heparin uptake was quantified using tritiated heparin. The results indicate that the bovine ureter grafts have an adequate degree of mural integrity and hemocompatible properties comparable to saphenous vein. The in vivo patency in a canine bilateral femoral interposition model was 62.5% (10/16) for the bovine ureters and 43.75% (7/16) for PTFE. Bovine ureter grafts appear to have an excellent potential for use in narrow diameter peripheral and coronary arterial bypass procedures and warrant further investigation.