Zn2+ plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn2+ homeostasis and the putative signaling role of Zn2+, various fluorescent sensors have been developed that allow monitoring of Zn2+ concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn2+ sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer and co-workers. Both have been successfully used to measure cytosolic free Zn2+, but distinctly different concentrations have been reported when using these sensors to measure Zn2+ concentrations in the ER and mitochondria. Here, we report the development of a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was created on the surface of the donor and acceptor fluorescent domains. This eZinCh-2 sensor binds Zn2+ with a high affinity that is similar to that of eCALWY-4 (Kd = 1 nM at pH 7.1), while displaying a substantially larger change in emission ratio. eZinCh-2 not only provides an attractive alternative for measuring Zn2+ in the cytosol but was also successfully used for measuring Zn2+ in the ER, mitochondria, and secretory vesicles. Moreover, organelle-targeted eZinCh-2 can also be used in combination with the previously reported redCALWY sensors to allow multicolor imaging of intracellular Zn2+ simultaneously in the cytosol and the ER or mitochondria.

Supramolecular polymers are an emerging family of nanosized structures with potential use in materials chemistry and medicine. Surprisingly, application of supramolecular polymers in the field of drug delivery has received only limited attention. Here, we explore the potential of PEGylated 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers for intracellular delivery. Exploiting the unique modular approach of supramolecular chemistry, we can coassemble neutral and cationic BTAs and control the overall properties of the polymer by simple monomer mixing. Moreover, this platform offers a versatile approach toward functionalization. The core can be efficiently loaded with a hydrophobic guest molecule, while the exterior can be electrostatically complexed with siRNA. It is demonstrated that both compounds can be delivered in living cells, and that they can be combined to enable a dual delivery strategy. These results show the advantages of employing a modular system and pave the way for application of supramolecular polymers in intracellular delivery.

Extracellular vesicles (EVs) are small vesicles secreted by cells and have gained increasing interest as both drug delivery vehicles or as cell‐free therapeutics for regenerative medicine. To achieve optimal therapeutic effects, strategies are being developed to prolong EV exposure to target organs. One promising approach to achieve this is through EV‐loaded injectable hydrogels. In this study, the use of a hydrogel based on ureido‐pyrimidinone (UPy) units coupled to poly(ethylene glycol) chains (UPy‐hydrogel) is examined as potential delivery platform for EVs. The UPy‐hydrogel undergoes a solution‐to‐gel transition upon switching from a high to neutral pH, allowing immediate gelation upon administration into physiological systems. Here, sustained EV release from the UPy‐hydrogel measured over a period of 4 d is shown. Importantly, EVs retain their functional capacity after release. Upon local administration of fluorescently labeled EVs incorporated in a UPy‐hydrogel in vivo, EVs are still detected in the UPy‐hydrogel after 3 d, whereas in the absence of a hydrogel, EVs are internalized by fat and skin tissue near the injection site. Together, these data demonstrate that UPy‐hydrogels provide sustained EV release over time and enhance local EV retention in vivo, which could contribute to improved therapeutic efficacy upon local delivery and translation toward new applications.

Information about the in vivo location, shape, degradation, or erosion rate of injected in situ gelating hydrogels can be obtained with magnetic resonance imaging (MRI). Herein, an injectable supramolecular ureidopyrimidinone-based hydrogel (UPy-PEG) is functionalized with a modified Gadolinium(III)-DOTA complex (UPy-Gd) for contrast enhanced MRI. The contrast agent is designed to supramolecularly interact with the hydrogel network to enable high-quality imaging of this hydrogel. The applicability of the approach is demonstrated with successful visualization of the Gd-labeled UPy-PEG hydrogel after targeted intramyocardial catheter injection in a pig heart.

Ureidopyrimidinone-based polymers in solution provide a new platform for intracellular drug delivery.