The choice of biopolymer is crucial to trigger angiogenesis with vascular endothelial growth factor releasing coatings

Bio-based coatings and release systems for pro-angiogenic growth factors are of interest to overcome insufficient vascularization and bio-integration of implants. This study compares different biopolymer-based coatings on polyethylene terephthalate (PET) membranes in terms of coating homogeneity and stability, coating thickness in the swollen state, endothelial cell adhesion, vascular endothelial growth factor (VEGF) release and pro-angiogenic properties. Coatings consisted of carbodiimide cross-linked gelatin type A (GelA), type B (GelB) or albumin (Alb), and heparin (Hep), or they consisted of radically cross-linked gelatin methacryloyl-acetyl (GM5A5) and heparin methacrylate (HepM5). We prepared films with thicknesses of 8–10 µm and found that all coatings were homogeneous after washing. All gelatin-based coatings enhanced the adhesion of primary human endothelial cells compared to the uncoated membrane. The VEGF release was tunable with the loading concentration and dependent on the isoelectric points and hydrophilicities of the biopolymers used for coating: GelA-Hep showed the highest releases, while releases were indistinguishable for GelB-Hep and Alb-Hep, and lowest for GM5A5-HepM5. Interestingly, not only the amount of VEGF released from the coatings determined whether angiogenesis was induced, but a combination of VEGF release, metabolic activity and adhesion of endothelial cells. VEGF releasing GelA-Hep and GelB-Hep coatings induced angiogenesis in a chorioallantoic membrane assay, so that these coatings should be considered for further in vivo testing.DFGProjekt DEA

Stimulus‐Responsive Regulation of Enzyme Activity for One‐Step and Multi‐Step Syntheses

Multi-step biocatalytic reactions have gained increasing importance in recent years because the combination of different enzymes enables the synthesis of a broad variety of industrially relevant products. However, the more enzymes combined, the more crucial it is to avoid cross-reactivity in these cascade reactions and thus achieve high product yields and high purities. The selective control of enzyme activity, i.e., remote on-/off-switching of enzymes, might be a suitable tool to avoid the formation of unwanted by-products in multi-enzyme reactions. This review compiles a range of methods that are known to modulate enzyme activity in a stimulus-responsive manner. It focuses predominantly on in vitro systems and is subdivided into reversible and irreversible enzyme activity control. Furthermore, a discussion section provides indications as to which factors should be considered when designing and choosing activity control systems for biocatalysis. Finally, an outlook is given regarding the future prospects of the field

Photoinduced cleavage and hydrolysis of o-nitrobenzyl linker and covalent linker immobilization in gelatin methacryloyl hydrogels

Light‐induced release systems can be triggered remotely and are of interest for many controlled release applications due to the possibility for spatio‐temporal release control. In this study a biotin‐functionalized photocleavable macromer is incorporated with an o‐nitrobenzyl moiety into gelatin methacryloyl(‐acetyl) hydrogels via radical cross‐linking. Stronger immobilization of streptavidin‐coupled horseradish peroxidase occurs in linker‐functionalized hydrogels compared to pure gelatin methacryloyl(‐acetyl) hydrogels, and a controlled release of the streptavidin conjugate upon UV‐irradiation is possible. Liquid chromatography coupled to mass spectrometry (LC‐MS) analysis of aqueous linker solutions allows the identification of the main cleavage products and the cleavage kinetics. Thus, it is shown that a significant hydrolysis of the linker occurs at 37 °C. Nevertheless the system reported here is a promising controlled release scaffold for proteins and application in tissue engineering, if background releases of the immobilized drug are tolerable

Expanding the range of available isoelectric points of highly methacryloylated gelatin

Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix in tissue engineering. However, so far, isoelectric points (IEPs) of highly methacryloylated GM derivatives are limited to IEPs below 7 due to the consumption of amino groups in the modification reaction. In this contribution, the synthesis of a new GM derivative, gelatin methacryloyl-aminoethyl (GME), with an IEP above 7 similar to the gelatin type A (GA) starting material is reported, together with a high degree of methacryloylation. GME is obtained by reacting GM with ethylenediamine (EDA). The impact of the EDA functionalization on the properties of GM and GME derivatives is characterized thoroughly via 1H-NMR, 1H-13C-HSQC-NMR, IEP, solution viscosity, gel point, and physico-chemical properties of resulting hydrogels. The second functionalization step results in amino group concentrations similar to GA and with that in an IEP of 9.9. The data suggest that amino functionalization with EDA prevents physical cross-linking in the same way as described before for acetyl functionalization. Therefore, GME hydrogels are less stiff than GM hydrogels from GMs with a comparable amount of methacrylic functions. With GME, a gelatin derivative is available that is positively charged at neutral pH without being limited to low methacryl modifications

Controlled Release of Vascular Endothelial Growth Factor from Heparin-Functionalized Gelatin Type A and Albumin Hydrogels

Bio-based release systems for pro-angiogenic growth factors are of interest, to overcome insufficient vascularization and bio-integration of implants. In this study, we investigated heparin-functionalized hydrogels based on gelatin type A or albumin as storage and release systems for vascular endothelial growth factor (VEGF). The hydrogels were crosslinked using carbodiimide chemistry in presence of heparin. Heparin-functionalization of the hydrogels was monitored by critical electrolyte concentration (CEC) staining. The hydrogels were characterized in terms of swelling in buffer solution and VEGF-containing solutions, and their loading with and release of VEGF was monitored. The equilibrium degree of swelling (EDS) was lower for albumin-based gels compared to gelatin-based gels. EDS was adjustable with the used carbodiimide concentration for both biopolymers. Furthermore, VEGF-loading and release were dependent on the carbodiimide concentration and loading conditions for both biopolymers. Loading of albumin-based gels was higher compared to gelatin-based gels, and its burst release was lower. Finally, elevated cumulative VEGF release after 21 days was determined for albumin-based hydrogels compared to gelatin A-based hydrogels. We consider the characteristic net charges of the proteins and degradation of albumin during release time as reasons for the observed effects. Both heparin-functionalized biomaterial systems, chemically crosslinked gelatin type A or albumin, had tunable physicochemical properties, and can be considered for controlled delivery of the pro-angiogenic growth factor VEGF

Beyond the modification degree: impact of raw material on physicochemical properties of gelatin type A and type B methacryloyls

Gelatin methacryloyl (acetyl) (GM(A)) is increasingly investigated for various applications in life sciences and medicine, for example, drug release or tissue engineering. Gelatin type A and type B are utilized for GAM(A) and GBM(A) preparation, but the impact of gelatin raw material on modification reaction and resulting polymer properties is rather unknown so far. Therefore, the degrees of modification (DMA) and physicochemical properties of five GAM(A) and GBM(A) derivatives are compared: The degrees of methacryloylation (0.32–0.98 mmol g−1) are indistinguishable for GAM(A) and GBM(A) as are the sol‐gel temperatures. Isoelectric points, solution viscosities, and hydrodynamic radii which are distinct for GA and GB, converge with increasing DMA. Interestingly, differences are measured for the storage moduli and equilibrium degrees of swelling of respective GA and GB derivative‐based hydrogels, in spite of their comparable DMA. This underlines the importance of GM(A) characterization beyond the modification degree

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

In vitro enzyme cascades possess great benefits, such as their synthetic capabilities for complex molecules, no need for intermediate isolation, and the shift of unfavorable equilibria towards the products. Their performance, however, can be impaired by, for example, destabilizing or inhibitory interactions between the cascade components or incongruous reaction conditions. The optimization of such systems is therefore often inevitable but not an easy task. Many parameters such as the design of the synthesis route, the choice of enzymes, reaction conditions, or process design can alter the performance of an in vitro enzymatic cascade. Many strategies to tackle this complex task exist, ranging from experimental to in silico approaches and combinations of both. This review collates examples of various optimization strategies and their success. The feasibility of optimization goals, the influence of certain parameters and the usage of algorithm-based optimizations are discussed

Modulation of transaminase activity by encapsulation in temperature‐sensitive poly(N‐acryloyl glycinamide) hydrogels

Smart hydrogels hold much potential for biocatalysis, not only for the immobilization of enzymes, but also for the control of enzyme activity. We investigated upper critical solution temperature-type poly N-acryloyl glycinamide (pNAGA) hydrogels as a smart matrix for the amine transaminase from Bacillus megaterium (BmTA). Physical entrapment of BmTA in pNAGA hydrogels results in high immobilization efficiency (>89 %) and high activity (97 %). The temperature-sensitiveness of pNAGA is preserved upon immobilization of BmTA and shows a gradual deswelling upon temperature reduction. While enzyme activity is mainly controlled by temperature, deactivation tended to be higher for immobilized BmTA (≈62–68 %) than for free BmTA (≈44 %), suggesting a deactivating effect due to deswelling of the pNAGA gel. Although the deactivation in response to hydrogel deswelling is not yet suitable for controlling enzyme activity sufficiently, it is nevertheless a good starting point for further optimization

Interactions of methacryloylated gelatin and heparin modulate physico-chemical properties of hydrogels and release of vascular endothelial growth factor

Gelatin hydrogels are used as tissue engineering scaffolds and systems for controlled release due to their inherent biodegradability and biocompatibility. In this study gelatin methacryloyl(-acetyl) (GM/A) with various degrees of methacryloylation (DM) and methacryl-modified heparin (HepM) were cross-linked radically via thermal-redox initiation. Investigation of gel yields (79.4%-85.8%) and equilibrium degrees of swelling (EDS; 564.8%-750.3%) by an experimental design approach suggested interaction effects between the applied HepM mass fraction and the DM of gelatin. HepM reduced the cross-linking effectivity (gel yield) only when added to GM with low DM (83% without HepM, 79% with HepM) but not when added to GM with high DM. For EDS combined impacts of the physical and chemical nature of the applied biopolymers are indicated: the elevated hydrophilicity and low cross-linking potential of HepM enhanced EDS in GM gels with low DM (O 1.1-fold increase), and lowered the storage moduli of all GM formulations (O 1.2-fold decrease). Vascular endothelial growth factor (VEGF) loading before cross-linking of gels resulted in major loss of functional growth factor (O 0.5% release), while loading after cross-linking was successful and significant release was detected over 28 days (6.4%-10.4% release). Release kinetics were mainly controlled by the VEGF concentration used for loading, and thus VEGF release and physico-chemical properties of the hydrogels can be tuned independently from each other in a broad range

Bio-based heparin-functionalized hydrogels for controlled VEGF-release II: Radical-initiated cross-linking

Purpose Presently insufficient oxygen and/or nutrient supply in tissue engineered grafts resulting in improper integration or death of encapsulated cells remain current limitations for clinical translation. [1] One strategy to overcome this is the controlled release of proangiogenic growth factors to induce vascularization and thereby bio-integration of the scaffold. Here we describe the preparation and characterization of hydrogels with tunable physico-chemical properties based on radical cross-linkable gelatin and heparin with application perspective as a tissue engineering scaffold allowing for storage and controlled release of proangiogenic growth factors. Methods Cross-linkable gelatin and heparin derivatives were prepared by reaction of the biopolymers with methacrylic anhydride. [2] Their degrees of methacrylation were quantified by nuclear magnetic resonance spectroscopy. Chemical cross-linking of hydrogels based on gelatin methacrylamide (9 % w/w) and heparin methacrylate (1 % w/w) was achieved by thermal-induced radical cross-linking in the presence of a water-soluble redox initiator system (APS Ammoniumperoxodisulfate / TEMED N,N,N′,N′-Tetramethylethylenediamine). Hydrogels were characterized concerning gel yield and equilibrium degree of swelling by gravimetric analysis. Viscoelastic properties were tuned by degree of modification of gelatin and characterized using rheological measurements. Release of VEGF165 (vascular endothelial growth factor) from the hydrogels was quantified by ELISA (enzyme-linked immunosorbent assay). Furthermore cytocompatibility of hydrogels was investigated with human primary endothelial cells. Results We will present results on biopolymer derivatization and physico-chemical properties of hydrogels of different compositions. Three gelatin derivatives with methacrylation-degrees between 0.78 mmol/g and 1.45 mmol/g and methacrylated heparin with 0.17 mmol methacrylate groups per gram were prepared. A suitable initiator concentration range for cross-linking of hydrogels consisting of the different derivatives was found by varying the TEMED / APS ratio between 0.0 and 0.5 and the APS concentration between 0.025 M and 0.100 M. We show results on the impact of gel composition, cross-linking time, initiator concentration, and composition on gel yield, equilibrium degree of swelling, and viscoelastic properties. The gels had lower swelling capacities and higher mechanical stiffness’s with higher methacrylation degrees of gelatin, whereas gel yield of all preparations were comparable. Furthermore we observed a significant effect of hydrogel drying on its swelling behavior. The swelling capacity was on average halved for all dried hydrogels compared with hydrogels swollen directly after preparation. We will also show effects of gel composition on release of VEGF. Finally, we aim to show results on the reaction of HDMVEC (human dermal microvascular endothelial cells) towards the hydrogel system. Conclusion Chemically cross-linked hydrogels composed of gelatin methacrylamide and heparin methacrylate have tunable physico-chemical properties. Hence this system might be suitable as a tissue engineering scaffold for storage and controlled release of growth factors