Heparin Affinity Research Articles

Highly pure measles virus generated by combination of salt-active nuclease treatment and heparin affinity chromatography

Highly purified virus preparations are essential for accurate activity and potency determination. This requires simple and efficient purification methods, especially in the early stages of research and development. While heparin affinity chromatography has been already successfully used for the purification of several enveloped viruses and virus-like particles, we extended its use to purification of very sensitive measles virus. The performance of heparin and heparin-like affinity chromatography was evaluated for the purification of recombinant measles virus, a large and labile enveloped virus used as vaccine or cancer therapy. Since DNA, particularly in the form of chromatin is a critical impurity in enveloped virus preparations, the effect of integration of an endonuclease (Benzonase® or M-SAN) treatment prior to chromatography was also investigated.Both, Capto™ DeVirS (heparin-like) and Capto™ Heparin were able to capture measles viruses directly from clarified cell culture supernatant. Despite capturing 100% of infectious measles virus, low recovery (8%) was observed for Capto™ DeVirS. For Capto™ Heparin recoveries up to 85% were observed. The combination of M-SAN with Capto™ Heparin enabled the production of highly purified measles virus with a yield of 62% and a final purity of 10.2 ng dsDNA per dose (1×105), outperforming the processes without endonuclease treatment with a yield of 18%, and a purity of 66.7 ng dsDNA/dose or using Benzonase® with a yield of 38% and a purity of 21.2 ng dsDNA/dose. As the developed method is simple and scalable it could also be integrated in a downstream process train for measles virus manufacturing.

Determination of Bovine Lactoferrin in Powdered Infant Formula and Adult Nutritionals by Heparin Affinity Extraction and Reverse-Phase High-Performance Liquid Chromatography/Ultraviolet Detection (HPLC/UV): Single-Laboratory Validation, First Action 2021.10.

Infant formulas, and pediatric and adult nutritional products, are being fortified with bovine lactoferrin (bLF) due to its beneficial impacts on immune development and gut health. Lactoferrin supplementation into these products requires an analytical method to accurately quantify the concentrations of bLF to meet global regulatory and quality standards. To develop and validate a lactoferrin method capable of meeting the AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) 2020.005. Powder formula samples are extracted using warm dibasic phosphate buffer, pH 8, then centrifuged at 4°C to remove insoluble proteins, fat, and other solids. The soluble fraction is further purified on a HiTrap heparin solid-phase extraction (SPE) column to isolate bLF from interferences. Samples are filtered, then analyzed by LC-UV using a protein BEH C4 analytical column and quantitated using an external calibrant. The LOQ (2 mg/100 g), repeatability (RSD: 2.0-4.8%), recovery (92.1-97.7%), and analytical range (4-193 mg/100 g) all meet the method requirements as stated in SMPR 2020.005 for lactoferrin. The reported single-laboratory validation (SLV) results demonstrate the ability of this lactoferrin method to meet or exceed the method performance requirements to measure soluble, intact, non-denatured bLF in infant and adult nutritional powder formulas. The use of a heparin affinity column to isolate lactoferrin from bovine milk products combined with a selective analytical chromatographic column provides suitable analyte specificity without requiring proprietary equipment or reagents.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

A Magnetic Beads-Based Sandwich Chemiluminescence Enzyme Immunoassay for the Rapid and Automatic Detection of Lactoferrin in Milk.

Lactoferrin (LF), an iron-binding glycoprotein with immunological properties and a high nutritional value, has emerged as a prominent research focus in the field of food nutrition. Lactoferrin is widely distributed in raw milk and milk that has undergone low-temperature heat treatment during pasteurization, making its rapid and accurate detection crucial for ensuring the quality control of dairy products. An enzyme-linked immunosorbent assay-based analytical protocol has often been referred to for the detection of LF in real samples. Signal amplification was accomplished using the streptavidin-biotin system. Here, an automated magnetic beads-based sandwich chemiluminescence enzyme immunoassay (MBs-sCLEIA) system was developed for the quantification of lactoferrin in pasteurized milk. The MBs-sCLEIA system consists of an automated chemiluminescence-based analyzer and a lactoferrin MBs-sCLEIA assay kit. Notably, our proposed method eliminates the need for pretreatment procedures and enables the direct addition of milk samples, allowing for the automatic quantitative detection of lactoferrin within a rapid 17 min timeframe for up to eight samples simultaneously. The MBs-sCLEIA was linear over the range of 7.24-800 ng/mL and displayed a limit of detection (LOD) of 2.85 ng/mL. As its good recovery and CV values indicate, the method exhibited high precision and accuracy. Furthermore, it was verified that it was selective towards five additional common milk proteins. A good correlation was observed between the results from the MBs-sCLEIA and heparin affinity column-HPLC (r2 = 0.99042), which proves to be a useful and practicable way of conducting an accurate analysis of lactoferrin in dairy products.

Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy.

Introduction: Human basic fibroblast growth factor (hbFGF) is a highly valuable multifunctional protein that plays a crucial role in various biological processes. In this study, we aim to accomplish the scaling-up production of mature hbFGF (146aa) by implementing a high cell-density fermentation and purification process on a 500-L scale, thereby satisfying the escalating demands for both experimental research and clinical applications. Methods: The hbFGF DNA fragment was cloned into a mpET-3c vector containing a kanamycin resistance gene and then inserted into Escherichia coli BL21 (DE3) plysS strain. To optimize the yield of hbFGF protein, various fermentation parameters were systematically optimized using BOX-Behnken design and further validated in large-scale fermentation (500-L). Additionally, a three-step purification protocol involving CM-Sepharose, heparin affinity, and SP-Sepharose column chromatography was developed to separate and purify the hbFGF protein. Isoelectric focusing electrophoresis, MALDI-TOF/MS analysis, amino acid sequencing, CD spectroscopy, and Western blotting were performed to authenticate its identity. The biological efficacy of purified hbFGF was evaluated using an MTT assay as well as in a diabetic deep second-degree scald model. Results: The engineered strain was successfully constructed, exhibiting high expression of hbFGF and excellent stability. Under the optimized fermentation conditions, an impressive bacterial yield of 46.8 ± 0.3g/L culture with an expression level of hbFGF reaching 28.2% ± 0.2% was achieved in 500-L scale fermentation. Subsequently, during pilot-scale purification, the final yield of purified hbFGF protein was 114.6 ± 5.9mg/L culture with RP-HPLC, SEC-HPLC, and SDS-PAGE purity exceeding 98%. The properties of purified hbFGF including its molecular weight, isoelectric point (pI), amino sequence, and secondary structure were found to be consistent with theoretical values. Furthermore, the purified hbFGF exhibited potent mitogenic activity with a specific value of 1.05 ± 0.94 × 106AU/mg and significantly enhanced wound healing in a deep second-degree scald wound diabetic rat model. Conclusion: This study successfully established a stable and efficient large-scale production process of hbFGF, providing a solid foundation for future industrial production.

Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106cells/mL, a plasmid concentration of 2µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2mM of sodium butyrate added 18h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32nm with 91.4% of protein similarity to exosomes.

Chemically heparinized PEEK via a green method to immobilize bone morphogenetic protein-2 (BMP-2) for enhanced osteogenic activity.

Osseointegration remains one of the major challenges in the success of bone-related implants. Recently, polyetheretherketone (PEEK) has emerged as an alternative material in orthopedic and dental applications due to its bone-mimicking mechanical properties. However, its bioinertness resulting in poor osseointegration has limited its potential application. So, the surface modification of PEEK with bone morphogenetic protein-2 (BMP-2) can be a potential approach for improving osseointegration. In this study, we proposed the chemical modification of heparin onto PEEK through an environmentally benign method to exploit the BMP-2 binding affinity of heparin. The heparin was successfully functionalized on the PEEK surface via a combination of ozone and UV treatment without using organic solvents or chemicals. Furthermore, BMP-2 was efficiently immobilized on PEEK and exhibited a sustained release of BMP-2 compared to the pristine PEEK with enhancement of bioactivity in terms of proliferation as well as osteogenic differentiation of MG-63. The significant synergistic effect of BMP-2 and heparin grafting on osteogenic differentiation of MG-63 was observed. Overall, we demonstrated a relatively safe method where no harsh chemical reagent or organic solvent was involved in the process of heparin grafting onto PEEK. The BMP-2 loaded, heparin-grafted PEEK could serve as a potential platform for osseointegration improvement of PEEK-based bone implants.

Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

Quantification of lactoferrin in dietary supplements using a Heparin affinity clean-up column by HPLC PDA detector

The use of dietary supplements containing lactoferrin is a growing trend. As a result, there is a need for a simple and reliable analytical method to determine the content of lactoferrin in these supplements to ensure food quality. In this study, an HPLC method was developed and validated for determining lactoferrin in dietary supplements using a HiTrap Heparin clean-up column in combination with HPLC. The study involved selecting optimal conditions for extracting lactoferrin from the sample matrix, followed by chromatography conditions using a C4 column (150 mm × 4.6 mm, 3.5 µm). The limit of detection (LOD) and limit of quantification (LOQ) were determined, with the achieved values being 1.2 mg/100g and 4.0 mg/100g, respectively. The method has shown good recovery (99.5–103.4%) and precision (RSD 1.76–3.44%), meeting the method performance requirements outlined in AOAC SMPR 2020.005. The validated method was applied to determine the contents of lactoferrin in dietary supplement samples.

Construction of a highly sensitive detection platform for heparin based on a “turn-off” cationic fluorescent dye

A highly sensitive detection platform for heparin was constructed via the utilization of a commercially available cationic fluorescent dye (cresyl violet acetate, CV) as a fluorescence probe. The electrostatic binding between CV and heparin quenched the fluorescence in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic (HEPES) buffer solution (10 mM, pH 7.1). CV was highly selective towards heparin over other potential inferring substances. The detection limit of heparin detection was 5.19 ng/mL, and the linear working range was 0 ∼ 1 μg/mL in HEPES solution. In 1 % serum, the detection platform based on the fluorescence “turn-off” behavior of CV was also successfully constructed with a detection limit of 5.86 ng/mL in the linear range of 0 ∼ 0.8 μg/mL. Moreover, the CV-heparin complex was considered a potential sensor platform for the detection of protamine because of its stronger affinity for heparin and protamine.

Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea

Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.

Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation

Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1–183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60–80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.

Downstream process design for Gag HIV-1 based virus-like particles.

Virus-like particles-based vaccines have been gaining interest in recent years. The manufacturing of these particles includes their production by cell culture followed by their purification to meet the requirements of its final use. The presence of host cell extracellular vesicles represents a challenge for better virus-like particles purification, because both share similar characteristics which hinders their separation. The present study aims to compare some of the most used downstream processing technologies for capture and purification of virus-like particles. Four steps of the purification process were studied, including a clarification step by depth filtration and filtration, an intermediate step by tangential flow filtration or multimodal chromatography, a capture step by ion exchange, heparin affinity and hydrophobic interaction chromatography and finally, a polishing step by size exclusion chromatography. In each step, the yields were evaluated by percentage of recovery of the particles of interest, purity, and elimination of main contaminants. Finally, a complete purification train was implemented using the best results obtained in each step. A final concentration of 1.40 × 1010 virus-like particles (VLPs)/mL with a purity of 64% after the polishing step was achieved, with host cell DNA and protein levels complaining with regulatory standards, and an overall recovery of 38%. This work has resulted in the development of a purification process for HIV-1 Gag-eGFP virus-like particles suitable for scale-up.

Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers

BackgroundProteins of the TGFβ family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFβ signaling.ResultsIn this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers.ConclusionsUltimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.

Characterization of bioactive substances involved in the induction of bone augmentation using demineralized bone sheets

PurposeTo investigate the bone augmentation ability of demineralized bone sheets mixed with allogeneic bone with protein fractions containing bioactive substances and the interaction between coexisting bioactive substances and proteins.MethodsFour types of demineralized bone sheets mixed with allogeneic bone in the presence or absence of bone proteins were created. Transplantation experiments using each demineralized bone sheet were performed in rats, and their ability to induce bone augmentation was analysed by microcomputed tomography images. Bioactive substances in bone proteins were isolated by heparin affinity chromatography and detected by the measurement of alkaline phosphatase activity in human periodontal ligament cells and dual luciferase assays. Noncollagenous proteins (NCPs) coexisting with the bioactive substances were identified by mass spectrometry, and their interaction with bioactive substances was investigated by in vitro binding experiments.ResultsDemineralized bone sheets containing bone proteins possessed the ability to induce bone augmentation. Bone proteins were isolated into five fractions by heparin affinity chromatography, and transforming growth factor-beta (TGF-β) was detected in the third fraction (Hep-c). Dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE), and biglycan (BGN) also coexisted in Hep-c, and the binding of these proteins to TGF-β increased TGF-β activity by approximately 14.7% to 32.7%.ConclusionsDemineralized bone sheets are capable of inducing bone augmentation, and this ability is mainly due to TGF-β in the bone protein mixed with the sheets. The activity of TGF-β is maintained when binding to bone NCPs such as DMP1, MEPE, and BGN in the sheets.

Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation.

The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies.

The Role of Disordered Regions in Orchestrating the Properties of Multidomain Proteins: The SARS-CoV-2 Nucleocapsid Protein and Its Interaction with Enoxaparin.

Novel and efficient strategies need to be developed to interfere with the SARS-CoV-2 virus. One of the most promising pharmaceutical targets is the nucleocapsid protein (N), responsible for genomic RNA packaging. N is composed of two folded domains and three intrinsically disordered regions (IDRs). The globular RNA binding domain (NTD) and the tethered IDRs are rich in positively charged residues. The study of the interaction of N with polyanions can thus help to elucidate one of the key driving forces responsible for its function, i.e., electrostatics. Heparin, one of the most negatively charged natural polyanions, has been used to contrast serious cases of COVID-19 infection, and we decided to study its interaction with N at the molecular level. We focused on the NTR construct, which comprises the NTD and two flanking IDRs, and on the NTD construct in isolation. We characterized this interaction using different nuclear magnetic resonance approaches and isothermal titration calorimetry. With these tools, we were able to identify an extended surface of NTD involved in the interaction. Moreover, we assessed the importance of the IDRs in increasing the affinity for heparin, highlighting how different tracts of these flexible regions modulate the interaction.

Biochemical characterization of a disease-causing human osteoprotegerin variant

Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.

Covalent Immobilization of REDV Peptide on Heparin Coating Promotes Selective Adhesion of Endothelial Cells

Forming an endothelial cell (EC) monolayer on the surface of blood-contacting devices is an ideal way to improve their blood compatibility, this process is known as endothelialization. Heparin is a proteoglycan with a strong negative charge, which is a commonly used antithrombotic agent. Heparin coatings also has been successfully used to improve the hemocompatibility of various biomaterials. In this study, a short peptide Arg-Glu-Asp-Val (REDV) with specific affinity for endothelial cells was covalently conjugated with maleimide-functionalized heparin. The conjugate retains the negative charge of heparin and can be directly immobilized on the surface of positively charged poly-L-lysine. This specific reaction to the carboxyl group of heparin could potentially reduce the shedding and steric hindrance effects of REDV peptide and enhancing the affinity and selectivity of heparin for EC.

Front Cover: Efficient Imidazolium‐Biomolecule Interaction‐Assisted Amplified Quenching for Ultrasensitive Detection of Heparin (Chem. Asian J. 18/2022)

A micelle-based efficient sensing approach, exhibiting stronger amplified fluorescence quenching by introducing imidazolium capable of enhancing affinity for heparin (HP), is described. The pyrene aggregates in the micelle provide an efficient sensing platform for amplified quenching, and the C2 proton of imidazolium on the micelle surface attracts HP very effectively, as if black holes exert pressure on their environment. A micelle-based fluorescent sensor with these features detects HP more sensitively than conventional fluorescent sensors with aliphatic ammonium. More information can be found in the Research Article by Seoung Ho Lee et al.