Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEGderived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are aminereactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.

Interactive materials that can respond to atrigger by changing their morphology,b ut that can also gradually degrade into af ully soluble state,a re attractive building blocks for the next generation of biomaterials.H erein, we design such transiently responsive polymers that exhibit UCST behaviour while gradually losing this property in response to ahydrolysis reaction in the polymer side chains.The polymers operate within ap hysiologically relevant window in terms of temperature,p H, and ionic strength. Whereas such behaviour has been reported earlier for LCST systems,i ti sa tp resent unexplored for UCST polymers.Furthermore,wedemonstrate that, in contrast to LCST polymers,i na queous medium the UCST polymer forms ac oacervate phase belowt he UCST, which can entrap ah ydrophilic model protein, as well as ah ydrophobic dye.B ecause of their non-toxicity,w ea lso provideinvivo proof of concept of the use of this coacervate as aprotein depot, in view of sustained-release applications.Stimuli-responsive polymers,a lso called "smart polymers", respond to chemical or physical changes by ac hange in solution behaviour. [1][2][3] This unique property has fuelled interest in these materials for an umber of applications, including biomaterials design, separation, catalysis,a nd sensors. [4][5][6][7][8][9] Amongst the most intensively studied stimuliresponsive polymers are those that exhibit lower critical solution temperature (LCST) behaviour. [10,11] Such polymers

Poly(2-oxazoline)s and, more recently, also poly(2-oxazine)s represent an emerging class of polymers with a broad range of applications. Surprisingly, to date, the statistical copolymerization of these two cyclic imino ether monomers has not yet been reported. Herein, we demonstrate that the statistical copolymerization of 2-oxazines with 2oxazolines can lead to the formation of amphiphilic gradient copolymers in a single step. These gradient copolymers combine the high structural modularity of poly(2-oxazoline)s with the excellent biological properties of poly(2-oxazine)s, especially poly(2-methyl-2-oxazine). The copolymerization was found to proceed in a nonexpected way with the relative incorporation rates of the monomers being opposite to the reactivity observed for the corresponding homopolymerizations. In fact, the statistical copolymerizations lead to faster incorporation of the 2-oxazine followed by a gradual transition toward the 2oxazoline. The self-assembly properties of the prepared amphiphilic poly[(2-methyl-2-oxazine)-grad-(2-butyl-2-oxazoline)] (PMeOzi-grad-PBuOx) as well as the thermoresponsive poly[(2-methyl-2-oxazine)-grad-(2-propyl-2-oxazoline)] (PMeOzigrad-PPrOx) confirmed their potential as stimuli-responsive nonionic surfactants for various applications. Finally, the noncytotoxic character and cellular uptake of PMeOzi-grad-PBuOx copolymers was confirmed in vitro in SKOV3 cells.

Conjugation of nanoparticles (NP) to the surface of living cells is of interest in the context of exploiting the tissue homing properties of ex vivo engineered T cells for tumor‐targeted delivery of drugs loaded into NP. Cell surface conjugation requires either a covalent or non‐covalent reaction. Non‐covalent conjugation with ligand‐decorated NP (LNP) is challenging and involves a dynamic equilibrium between the bound and unbound state. Covalent NP conjugation results in a permanently bound state of NP, but the current routes for cell surface conjugation face slow reaction kinetics and random conjugation to proteins in the glycocalyx. To address the unmet need for alternative bioorthogonal strategies that allow for efficient covalent cell surface conjugation, we developed a 2‐step click conjugation sequence in which cells are first metabolically labeled with azides followed by reaction with sulfo‐6‐methyl‐tetrazine‐dibenzyl cyclooctyne (Tz‐DBCO) by SPAAC, and subsequent IEDDA with trans‐cyclooctene (TCO) functionalized NP. In contrast to using only metabolic azide labeling and subsequent conjugation of DBCO‐NP, our 2‐step method yields a highly specific cell surface conjugation of LNP, with very low non‐specific background binding.