323 research outputs found
Protein repelling coatings based on stimuli-responsive aqueous microgels decorated with oligo ethylene glycols
In the present work novel, biocompatible, thermo-responsive microgels were synthesized by surfactant-free precipitation copolymerization of N-vinylcaprolactam (VCL) with 2-methoxyethyl acrylate (MEA). MEA was chosen as comonomer because its polymer is known for his excellent blood compatibility and low protein adsorption[1,2] and therefore is already in use as coating material for artificial organs.[3]
Using optimized precipitation polymerization procedure we could incorporate up to 32 mol% of MEA into the PVCL-based microgels without loss of colloidal stability. The microgel composition was quantified by Raman-Spectroscopy. Both, a high resolution transverse relaxation NMR study and calorimetric measurements delivered that MEA is located mainly in the microgel shell due to its lower polymerization rate in comparison to VCL. With increasing comonomer content the swelling degree ΔRh(20°C-50°C) of the microgels decreased. This behavior can be explained by the temperature-responsivity of both monomers. While pure PVCL microgels exhibit a LCST = 31 °C,[4] linear PMEA has a very low LCST = 0-5 C°C.[5,6] It follows that with increasing MEA-content the VPTT of the microgels is shifted to lower values. Additionally, the microgel shell is already collapsed at room temperature which prevents the swelling of the PVCL-rich core. Consequently the PVCL/MEA microgels become more rigid and less temperature-sensitive with increasing MEA-content. While the particles with low MEA-content exhibit a core-shell like structure which indicates that the core is denser than the shell, the particles with high MEA-content are rigid and compact that can be approved by AFM measurements. Furthermore, the protein repellent properties of microgel films were tested. High protein repellence could be obtained for PVCL/MEA microgels, as expected (Figure 1). At last different functionalities like carboxylic acid groups or epoxy groups were integrated into the PVCL/MEA microgels by copolymerization with acrylic acid (AA) or glycidyl methacrylate (GMA). While the carboxylic acid groups lead to additional pH-dependence of the microgels that can be used for pH-triggered uptake and release mechanisms, the epoxy groups are known to react specifically with primary amines and thiols. Therefore proteins, drugs and dyes can be bound to the microgels covalently.
Please click Additional Files below to see the full abstract
Enzyme-mediated surface functionalisation of stimuli-responsive microgels
Aqueous microgels based on N-Vinylcaprolactam (VCL) gained much attention in the biomedical research due to their temperature-sensitive behavior and their high biocompatibility.[1] However, the post-modification of PVCL-microgels with proteins is still challenging. The so-called sortase-mediated ligation is one possible method for the conjugation of biomolecules to polymers.[2]
Sortases are bacterial enzymes with transpeptidase activity, responsible for the attachment of proteins to the cell wall of gram-positive bacteria. Sortases of class A (SrtA) recognize a LPXTG-sorting motif (X being any amino acid) at the C-terminus of the targeted protein, cleave it between the threonine and the glycine and ligate it to a second protein via an oligoglycine nucleophile.[3]
In this work, we present the use of sortase-mediated ligation for the conjugation of different proteins to PVCL-microgels with the aim to incorporate special functionalities. For this purpose, microgels based on PVCL containing 5 mol% glycidyl methacrylate (GMA) as comonomer in the particles shell were synthesized and modified with the specific recognition peptide sequence LPETG for SrtA. The coupling of the LPETG sequence was analyzed via UV-Vis spectroscopy and Raman spectroscopy. To perform Sortase-mediated ligation, oligoglycin-tagged enhanced Green Fluorescent Protein (GGG‑eGFP) was used as a model protein. The conjugation of the eGFP to the microgel was investigated qualitatively via confocal microscopy and quantitatively via fluorescence intensity measurements. It was shown that the fluorescence intensity increased linearly with increasing eGFP-concentration and exponentially with increasing reaction time up to seven hours. Additionally, we were able to show that also oligoglycin-tagged CueO-laccase can be conjugated to the PVCL-microgel using sortase-mediated ligation. To this end, the protein activity of the microgels was measured using ABTS as laccase substrate and the amount of conjugated protein was analyzed via BCA assay. These results indicate that sortase-mediated ligation is a very promising and powerful tool for the modification of microgels with biomacromolecules for applications in drug delivery, biointerface coatings and sensors.
Aqueous microgels based on N-Vinylcaprolactam (VCL) gained much attention in the biomedical research due to their temperature-sensitive behavior and their high biocompatibility.[1] However, the post-modification of PVCL-microgels with proteins is still challenging. The so-called sortase-mediated ligation is one possible method for the conjugation of biomolecules to polymers.[2]
Sortases are bacterial enzymes with transpeptidase activity, responsible for the attachment of proteins to the cell wall of gram-positive bacteria. Sortases of class A (SrtA) recognize a LPXTG-sorting motif (X being any amino acid) at the C-terminus of the targeted protein, cleave it between the threonine and the glycine and ligate it to a second protein via an oligoglycine nucleophile.[3]
In this work, we present the use of sortase-mediated ligation for the conjugation of different proteins to PVCL-microgels with the aim to incorporate special functionalities. For this purpose, microgels based on PVCL containing 5 mol% glycidyl methacrylate (GMA) as comonomer in the particles shell were synthesized and modified with the specific recognition peptide sequence LPETG for SrtA. The coupling of the LPETG sequence was analyzed via UV-Vis spectroscopy and Raman spectroscopy. To perform Sortase-mediated ligation, oligoglycin-tagged enhanced Green Fluorescent Protein (GGG‑eGFP) was used as a model protein. The conjugation of the eGFP to the microgel was investigated qualitatively via confocal microscopy and quantitatively via fluorescence intensity measurements. It was shown that the fluorescence intensity increased linearly with increasing eGFP-concentration and exponentially with increasing reaction time up to seven hours. Additionally, we were able to show that also oligoglycin-tagged CueO-laccase can be conjugated to the PVCL-microgel using sortase-mediated ligation. To this end, the protein activity of the microgels was measured using ABTS as laccase substrate and the amount of conjugated protein was analyzed via BCA assay. These results indicate that sortase-mediated ligation is a very promising and powerful tool for the modification of microgels with biomacromolecules for applications in drug delivery, biointerface coatings and sensors.
Please click Additional Files below to see the full abstract
Recommended from our members
Dual Stimuli-Responsive Self-Assembly Behavior of a Tailor-Made ABC-Type Amphiphilic Tri-Block Copolymer
This investigation describes the synthesis of a dual stimuli-responsive, amphiphilic ABC tri-block copolymer (BCP) based on the functional monomers via RAFT polymerization. In this case, ABC-type BCP was prepared based on N-isopropylacrylamide, n-butyl acrylate, and 4-vinylpyridine in DMF solvent using cyanomethyl dodecyl trithiocarbonate as the RAFT agent and azobisisobutyronitrile as a thermal initiator in a subsequent macro-RAFT approach, respectively. The BCPs were characterized by SEC, 1H-NMR, FTIR spectroscopy, and DSC analyses. Temperature and pH-dependent properties of the smart BCP micelles in aqueous medium were investigated using dynamic light scattering. Transmission electron microscopic images were taken at cryogenic and dry conditions to study the morphology of molecular assemblies of block copolymers in an aqueous medium. The phase and topographical images were captured by atomic force microscopy to understand the assembly of block copolymers in solvents of different polarities. The morphology of BCP micelles was transformed from flower-like to spherical in the presence of solvents with different polarities (H2O or CHCl3). © 2020 The Authors. Journal of Polymer Science published by Wiley Periodicals, Inc
Recommended from our members
Biadhesive Peptides for Assembling Stainless Steel and Compound Loaded Micro-Containers
Biadhesive peptides (peptesives) are an attractive tool for assembling two chemically different materials—for example, stainless steel and polycaprolactone (PCL). Stainless steel is used in medical stents and PCL is used as a biodegradable polymer for fabrication of tissue growth scaffolds and drug delivering micro-containers. Biadhesive peptides are composed of two domains (e.g., dermaseptin S1 and LCI) with different material-binding properties that are separated through a stiff peptide-spacer. The peptesive dermaseptin S1-domain Z-LCI immobilizes antibiotic-loaded PCL micro-containers on stainless steel surfaces. Immobilization is visualized by microscopy and field emission scanning electron microscopy analysis and released antibiotic from the micro-containers is confirmed through growth inhibition of Escherichia coli cells
Повышение качества поверхностей глубоких отверстий при сверлении ружейными сверлами в единичном и мелкосерийном производствах
В работе определены технологические возможности ружейных сверл в единичном и мелкосерийном производствах. Разработаны рекомендации по применению ружейных сверл на многооперационных станках.In this work defines the technological capabilities of the gun drills in single and smal-scale type of production.
Recommendations for the use of gun drills on multi-operational machines have been developed
Recommended from our members
Mechanoresponsive diselenide-crosslinked microgels with programmed ultrasound-triggered degradation and radical scavenging ability for protein protection
In the context of controlled delivery and release, proteins constitute a delicate class of cargo requiring advanced delivery platforms and protection. We here show that mechanoresponsive diselenide-crosslinked microgels undergo controlled ultrasound-triggered degradation in aqueous solution for the release of proteins. Simultaneously, the proteins are protected from chemical and conformational damage by the microgels, which disintegrate to water-soluble polymer chains upon sonication. The degradation process is controlled by the amount of diselenide crosslinks, the temperature, and the sonication amplitude. We demonstrate that the ultrasound-mediated cleavage of diselenide bonds in these microgels facilitates the release and activates latent functionality preventing the oxidation and denaturation of the encapsulated proteins (cytochrome C and myoglobin) opening new application possibilities in the targeted delivery of biomacromolecules
- …