All sorted: The enzyme Sortase A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.

Nano-/micrometer-scaled films and capsules made of low-fouling materials such as poly(ethylene glycol) (PEG) are of interest for drug delivery and tissue engineering applications. Herein, the assembly and degradation of low-fouling, alkyne-functionalized PEG (PEG(Alk) ) multilayer films and capsules, which are prepared by combining layer-by-layer (LbL) assembly and click chemistry, are reported. A nonlinear, temperature-responsive PEG(Alk) is synthesized, and is then used to form hydrogen-bonded multilayers with poly(methacrylic acid) (PMA) at pH 5. The thermoresponsive behavior of PEG(Alk) is exploited to tailor film buildup by adjusting the assembly conditions. Using alkyne-azide click chemistry, PEG(Alk)/PMA multilayers are crosslinked with a bisazide linker that contains a disulfide bond, rendering these films and capsules redox-responsive. At pH 7, by disrupting the hydrogen bonding between the polymers, PEG(Alk) LbL films and PEG(Alk) -based capsules are obtained. These films exhibit specific deconstruction properties under simulated intracellular reducing conditions, but remain stable at physiological pH, suggesting potential applications in controlled drug release. The low-fouling properties of the PEG films are confirmed by incubation with human serum and a blood clot. Additionally, these capsules showed negligible toxicity to human cells.

Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.

Das Enzym Sortase A zur Funktionalisierung PEGylierter Kapseln mit einem aktivationsspezifischen einkettigen Antikörper (scFv) gegen Plättchen ermöglicht die schnelle, kovalente und ortsspezifische Funktionalisierung von Transportvehikeln unter milden Bedingungen. Mit scFv bedeckte PEGylierte Kapseln binden hoch selektiv an Thrombi, was auf einen möglichen Einsatz in der Thrombosetherapie hindeutet.

The engineering of layer-by-layer (LbL) hybrid click capsules that are responsive to biological stimuli is reported. The capsules comprise a pH-sheddable, non cross-linked outer coating that protects enzyme-cleavable inner layers. Upon cellular uptake, the outer coating is released and the capsules are enzymatically degraded. In vitro cell degradation results in rapid capsule degradation (10 min) upon cellular internalization.