Evaluation of Hydrogels Based on Oxidized Hyaluronic Acid for Bioprinting.

In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers.

An Albumin-Oligonucleotide Assembly for Potential Combinatorial Drug Delivery and Half-Life Extension Applications.

The long blood circulatory property of human serum albumin, due to engagement with the cellular recycling neonatal Fc receptor (FcRn), is an attractive drug half-life extension enabling technology. This work describes a novel site-specific albumin double-stranded (ds) DNA assembly approach, in which the 3' or 5' end maleimide-derivatized oligodeoxynucleotides are conjugated to albumin cysteine at position 34 (cys34) and annealed with complementary strands to allow single site-specific protein modification with functionalized ds oligodeoxynucleotides. Electrophoretic gel shift assays demonstrated successful annealing of complementary strands bearing Atto488, 6-carboxyfluorescein (6-FAM), or a factor IXa aptamer to the albumin-oligodeoxynucleotide conjugate. A fluorometric factor IXa activity assay showed retained aptamer inhibitory activity upon assembly with the albumin and completely blocked factor IXa at a concentration of 100 nM for 2 hr. The assembled construct exhibited stability in serum-containing buffer and FcRn engagement that could be increased using an albumin variant engineered for higher FcRn affinity. This work presents a novel albumin-oligodeoxynucleotide assembly technology platform that offers potential combinatorial drug delivery and half-life extension applications.

Albumin-based drug delivery using cysteine 34 chemical conjugates - important considerations and requirements.

The long blood circulation time of albumin has been clinically utilized as a half-life extension technology for improved drug performance. The availability of one free thiol for site-selective chemical conjugation offers an alternative approach to current genetic fusion and association-based products. This special report highlights important factors for successful conjugation that allows the reader to design and evaluate next-generation albumin conjugates. Albumin type, available conjugation chemistries, linker length, animal models and influence of conjugation on albumin pharmacokinetics and drug activity are discussed.

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding.

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Control of Nanoparticle Release Kinetics from 3D Printed Hydrogel Scaffolds.

The convergence of biofabrication with nanotechnology is largely unexplored but enables geometrical control of cell-biomaterial arrangement combined with controlled drug delivery and release. As a step towards integration of these two fields of research, this study demonstrates that modulation of electrostatic nanoparticle-polymer and nanoparticle-nanoparticle interactions can be used for tuning nanoparticle release kinetics from 3D printed hydrogel scaffolds. This generic strategy can be used for spatiotemporal control of the release kinetics of nanoparticulate drug vectors in biofabricated constructs.

Thiol-ene Clickable Poly(glycidol) Hydrogels for Biofabrication.

In this study we introduce linear poly(glycidol) (PG), a structural analog of poly(ethylene glycol) bearing side chains at each repeating unit, as polymer basis for bioink development. We prepare allyl- and thiol-functional linear PG that can rapidly be polymerized to a three-dimensionally cross-linked hydrogel network via UV mediated thiol-ene click reaction. Influence of polymer concentration and UV irradiation on mechanical properties and swelling behavior was examined. Thiol-functional PG was synthesized in two structural variations, one containing ester groups that are susceptible to hydrolytic cleavage, and the other one ester-free and stable against hydrolysis. This allowed the preparation of degradable and non-degradable hydrogels. Cytocompatibility of the hydrogel was demonstrated by encapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Rheological properties of the hydrogels were adjusted for dispense plotting by addition of high molecular weight hyaluronic acid. The optimized formulation enabled highly reproducible plotting of constructs composed of 20 layers with an overall height of 3.90 mm.

Albumin-based drug delivery: harnessing nature to cure disease.

The effectiveness of a drug is dependent on accumulation at the site of action at therapeutic levels, however, challenges such as rapid renal clearance, degradation or non-specific accumulation requires drug delivery enabling technologies. Albumin is a natural transport protein with multiple ligand binding sites, cellular receptor engagement, and a long circulatory half-life due to interaction with the recycling neonatal Fc receptor. Exploitation of these properties promotes albumin as an attractive candidate for half-life extension and targeted intracellular delivery of drugs attached by covalent conjugation, genetic fusions, association or ligand-mediated association. This review will give an overview of albumin-based products with focus on the natural biological properties and molecular interactions that can be harnessed for the design of a next-generation drug delivery platform.

Side-chain cysteine-functionalized poly(2-oxazoline)s for multiple peptide conjugation by native chemical ligation.

We prepared statistical copolymers composed of 2-methyl-2-oxazoline (MeOx) in combination with 2-butenyl-2-oxazoline (BuOx) or 2-decenyl-2-oxazoline (DecOx) as a basis for polymer analogous introduction of 1,2-aminothiol moieties at the side chain. MeOx provides hydrophilicity as well as cyto- and hemocompatibility, whereas the alkene groups of BuOx and DecOx serve for functionalization with a thiofunctional thiazolidine by UV-mediated thiol-ene reaction. After deprotection the cysteine content in functionalized poly(2-oxazoline) (POx) is quantified by NMR and a modified trinitrobenzenesulfonic acid assay. The luminescent cell viability assay shows no negative influence of cysteine-functionalized POx (cys-POx) concerning cell viability and cell number. cys-POx was used for multiple chemically orthogonal couplings with thioester-terminated peptides through native chemical ligation (NCL), which was performed and confirmed by NMR and MALDI-ToF measurements.

Cysteine-functional polymers via thiol-ene conjugation.

A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials.

Controlled ring-opening polymerization of substituted episulfides for side-chain functional polysulfide-based amphiphiles.

We used initiation solutions of DBU and different thiols for the controlled ring-opening homo- and copolymerization of ethoxy ethyl thio glycidyl ether (EETGE) and allyl thio glycidyl ether (ATGE) to side-chain multifunctional polysulfides. Optimized preparation conditions allow the syntheses of monomodal homopolysulfides and monomodal polysulfide-block-mPEG copolymers. Furthermore, copolymers of EETGE and mPEG are firstly synthesized, characterized, and finally deprotected to yield intact poly(thio glycidol)-block-poly(ethylene glycol) copolymers. These amphiphiles are suitable to form particles in aqueous solutions as confirmed by DLS and cryo-SEM measurements.

A new polymorph of dimesitylborinic acid.

A new polymorph of dimesitylborinic acid (or hydroxy-dimesitylborane), C(18)H(23)BO, showcasing different crystal packing and symmetry, complements the previously reported polymorph [Weese, Bartlett, Murray, Olmstead & Power (1987 ▶). Inorg. Chem.26, 2409-2413; Entwistle, Batsanov & Marder (2007 ▶). Acta Cryst. E63, o2639-o2641]. The structure of the title compound contains only one mol-ecule in the asymmetric unit, and no O-H⋯O inter-actions are observed. However, mol-ecules are linked by weak inter-molecuar O-H⋯π(arene) inter-actions to form centrosymmetric dimers.