Organized three-dimensional (3D) nanomaterial architectures are promising candidates for applications in optoelectronics, catalysis, or theranostics owing to their anisotropy and advanced structural features that allow tailoring their physical and chemical properties. The synthesis of such complex but well-organized nanomaterials is difficult because the interplay of interfacial strain and facet-specific reactivity must be considered. Especially the magnetic anisotropy with controlled size and morphology plays a decisive role for applications like magnetic resonance imaging (MRI) and advanced data storage. We present a solution phase seed mediated synthesis of colloidal, well dispersible iron oxide superparticles with flower- and hedgehog-like morphology starting from dispersible spherical maghemite (SPH) and nanoplate hematite (HEX) templates. In the superparticles the templates are epitaxially decorated with nanodomains and nanorods as shown by (high-resolution) transmission electron microscopy (TEM), orientation mapping, and electron diffraction (ED). While the templates determine the morphology of the superparticles, the solution chemistry determines the phase identity. Oxidation of Fe(CO)5 during superparticle formation reaction leads to maghemite nanodomains and nanorods decorating the templates, unveiled by a combination of X-ray diffraction (XRD) and Mössbauer spectroscopy (MS). After hydrophilic surface functionalization the superparticles are well dispersible. The cytotoxicity of templates and superparticles is low. The magnetic resonance imaging R2-relaxivity of the flower-like superparticles could be increased by a factor 2.5 compared to its spherical nanoparticle template due to direct interfacial connection resulting from the unique nanoarchitecture.
Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer−protein hybrids to the broad field of available functional RAFT-derived polymers.
The reversible addition−fragmentation chain-transfer (RAFT) polymerization provides access to a broad variety of biocompatible and functional macromolecules for diverse polymer−drug conjugates. Due to thiocarbonylthio groups at the ends of each growing polymer chain, they can straightforwardly be converted into disufilde-containing self-immolative motives for reversible drug conjugation by traceless linkers. This may be relevant for RAFT-polymerized poly(N,N-dimethylacrylamide) (pDMA), which has been demonstrated to provide similar properties as poly(ethylene glycol) (PEG) in terms of improving the drug's poor pharmacokinetic profile or enhancing its bioavailability. For that purpose, we established a highly efficient one-pot reaction procedure for introducing various functionalities including both primary and secondary amines and primary alcohols and demonstrated their reversible conjugation and traceless release from pDMA's polymer chain end. Next, a first polymer−drug conjugate with a Toll-like receptor agonist exhibited significantly increased activity in vitro compared to conventional irreversibly covalently fixed variants. Finally, α-ω-bifunctional dye or drug conjugates could be generated by a cholesterol-modified RAFT chain-transfer agent. It facilitated the polymer−drug conjugate's internalization at the cellular level monitored by flow cytometry and confocal imaging. This approach provides the basis for a variety of potentially impactful polymer−drug conjugates by combining versatile small molecular drugs with a plethora of available RAFT polymers through reductive-responsive self-immolative linkers.
Herein, N-heterocyclic olefins (NHOs) are utilized as catalysts for the ring-opening polymerization (ROP) of functional aliphatic carbonates. This emerging class of catalysts provides high reactivity and rapid conversion. Aiming for the polymerization of monomers with high side chain functionality, six-membered carbonates derived from 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) served as model compounds. Tuning the reactivity of NHO from predominant side chain transesterification at room temperature toward ring-opening at lowered temperatures (−40 °C) enables controlled ROP. These refined conditions give narrowly distributed polymers of the hydrophobic carbonate 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MTC-OBn) (Đ < 1.30) at (pseudo)first-order kinetic polymerization progression. End group definition of these polymers demonstrated by mass spectrometry underlines the absence of side reactions. For the active ester monomer 5-methyl-5-pentafluorophenyloxycarbonyl-1,3-dioxane-2-one (MTC-PFP) with elevated side chain reactivity, a cocatalysis system consisting of NHO and the Lewis acid magnesium iodide is required to retune the reactivity from side chains toward controlled ROP. Excellent definition of the products (Đ < 1.30) and mass spectrometry data demonstrate the feasibility of this cocatalyst approach, since MTC-PFP has thus far only been polymerized successfully using acidic catalysts with moderate control. The broad feasibility of our findings was further demonstrated by the synthesis of block copolymers for bioapplications and their successful nanoparticular assembly. High tolerability of NHO in vitro with concentrations ranging up to 400 μM (equivalent to 0.056 mg/mL) further emphasize the suitability as a catalyst for the synthesis of bioapplicable materials. The polycarbonate block copolymer mPEG44-b-poly(MTC-OBn) enables physical entrapment of hydrophobic dyes in sub-20 nm micelles, whereas the active ester block copolymer mPEG44-b-poly(MTC-PFP) is postfunctionalizable by covalent dye attachment. Both block copolymers thereby serve as platforms for physical or covalent modification of nanocarriers for drug delivery.
Applications of antibody‐drug conjugates are rapidly growing, however, arduous fabrication of antibodies and impairment of highly potent drugs by covalent fixation to the protein is urging for alternatives to these conventional strategies. Here, a procedure on genetically engineered single domain antibodies, so‐called nanobodies, is demonstrated for their site‐specific reversible bioconjugation using self‐immolative linkers (SILs). Straight‐forward fluorescent labelling at their C‐terminal cysteine can be reversed under reductive conditions due to its disulfide‐containing SIL. Flow cytometry and microscopy images demonstrate cellular uptake and confirm the integrity of the nanobodies’ biological affinity notwithstanding being modified. Following this strategy, a potent small molecular immunomodulator can be installed and its stimulatory effect on a cellular level is boosted in vitro compared to non‐degradable alternatives. Furthermore, this protocol is extended to further therapeutically relevant representatives of nanobodies, underlining the versatility of this reversible reductive‐responsive bioconjugation for a broad field of applications.
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