Orthogonal Covalent Entrapment of Cargo into Biodegradable Polymeric Micelles via Native Chemical Ligation

Abstract

Polymeric micelles (PMs) are promising platforms for enhanced tissue targeting of entrapped therapeutic agents. Strategies to circumvent premature release of entrapped drugs include cross-linking of the micellar core as well as covalent attachment of the drug cargo. The chemistry employed to obtain cross-linked micelles needs to be mild to also allow entrapment of fragile molecules, such as certain peptides, proteins, oligonucleotides, and fluorescent dyes. Native chemical ligation (NCL) is a mild bio-orthogonal reaction between a N-terminal cysteine residue and a thioester that proceeds under physiological conditions. Here, we designed a trifunctional cross-linker containing two cysteine residues for the micelle core-cross-linking reaction and an azide residue for ring-strained alkyne conjugation of fluorescent dyes. We applied this approach to thermosensitive methoxypolyethylene glycol-b-N-(2-hydroxypropyl)methacrylamide-lactate (mPEG-b-HPMAmLac n ) based block copolymers of a core-cross-linked polymeric micelle (CCPM) system by attaching thioester residues (using ethyl thioglycolate-succinic anhydride, ETSA) for NCL cross-linking with the trifunctional cross-linker under physiological conditions. By use of mild copper-free click chemistry, we coupled fluorescent dyes, Sulfo.Cy5 and BODIPY, to the core via the azide residue present on the cross-linker by triazole ring formation. In addition, we employed a recently developed cycloheptyne strain promoted click reagent (TMTHSI, CliCr) in comparison to the frequently employed cyclooctyne derivative (DBCO), both achieving successful dye entrapment. The size of the resulting CCPMs could be tuned between 50 and 100 nm by varying the molecular weight of the thermosensitive block and ETSA content. In vitro cell experiments showed successful internalization of the dye entrapped CCPMs, which did not affect cell viability up to a polymer concentration of 2 mg/mL in PC3 cells. These fluorescent dye entrapped CCPMs can be applied in diagnostic imaging and the chemistry developed in this study serves as a steppingstone toward covalently entrapped fragile drug compounds with tunable release in CCPMs.

Mechanistic Study on the Degradation of Hydrolysable Core-Crosslinked Polymeric Micelles

“…The polydispersity index (PDI) increased during degradation, which is also in line with previously published findings, being associated with the degradation of particles in batch DLS measurements. 10,34 Similar results were obtained by accelerated degradation of the CCPMs at pH 9.5, 25 °C (Figure S4.1). The accelerated degradation is due to the increased rate of lactate ester hydrolysis, which is driven by first-order kinetics with respect to OH − concentration above a pH of 5.…”

“…10,33 Recently, we reported the use of NCL (a reaction between a cysteine and a thioester forming an amide bond) as an orthogonal crosslinking reaction in mPEG-b-PHPMAmLac n block copolymers for the formation of CCPM using a bifunctional crosslinking agent. 34 The advantage of this NCL crosslinking strategy is that no free radical reactions are needed that can potentially damage sensitive cargo. Briefly, by increasing the temperature of an aqueous solution of thermosensitive mPEG-b-PHPMAmLac n block copolymers modified with thioesters, micelles were formed, which were subsequently stabilized by a crosslinker with two cysteine handles, allowing amide bond formation via NCL.…”

High Rates of Quinone‐Alkyne Cycloaddition Reactions are Dictated by Entropic Factors

“…Firstly, we compared the recently published THS 2 – a highly strained 7‐membered cyclic alkyne with unprecedented reaction rate constants in SPAAC reactions with azides [22,23] – with BCN−OH 3 , an 8‐membered cyclic alkyne that is currently one of the benchmark reagents for metal‐free click chemistry applications [24] . [Of note: DBCO, by far the most frequently employed cyclooctyne for SPAAC reactions, was found not to display significant reactivity in SPOCQ and was therefore omitted from the matrix [1] …”

Bottom–up on-surface synthesis based on click-functionalized peptide bundles

“…In addition, after the preparation of a rigid rod, its exposed end still has unreacted clickable groups that can be used for additional functional modifications. To illustrate this advantage, PEG, which is a polymer having excellent hydrophilicity, biocompatibility, low/non-toxicity, and broad use in imaging, 33,34 self-assembly [35][36][37] and anticancer 38,39 fields, was selected as the functional group to modify the exposed end of a rigid rod. Using CUAAC click chemistry, linear PEG (PEGalkynyl 10 kDa) with one-terminal click-functional was grafted alone on an azide-modified mica sheet, showing a height of approximately 9 nm (Fig.…”