The effect of charge and albumin on cellular uptake of supramolecular polymer nanostructures.

Intracellular delivery of functional biomolecules by using supramolecular polymer nanostructures has gained significant interest. Here, various charged supramolecular ureido-pyrimidinone (UPy)-aggregates were designed and formulated via a simple "mix-and-match" method. The cellular internalization of these UPy-aggregates in the presence or absence of serum proteins by phagocytic and non-phagocytic cells, i.e., THP-1 derived macrophages and immortalized human kidney cells (HK-2 cells), was systematically investigated. In the presence of serum proteins the UPy-aggregates were taken up by both types of cells irrespective of the charge properties of the UPy-aggregates, and the UPy-aggregates co-localized with mitochondria of the cells. In the absence of serum proteins only cationic UPy-aggregates could be effectively internalized by THP-1 derived macrophages, and the internalized UPy-aggregates either co-localized with mitochondria or displayed as vesicular structures. While the cationic UPy-aggregates were hardly internalized by HK-2 cells and could only bind to the membrane of HK-2 cells. With adding and increasing the amount of serum albumin in the cell culture medium, the cationic UPy-aggregates were gradually taken up by HK-2 cells without anchoring on the cell membranes. It is proposed that the serum albumin regulates the cellular internalization of UPy-aggregates. These results provide fundamental insights for the fabrication of supramolecular polymer nanostructures for intracellular delivery of therapeutics.

Injectable Supramolecular Ureidopyrimidinone Hydrogels Provide Sustained Release of Extracellular Vesicle Therapeutics.

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.

Controlled Release of RNAi Molecules by Tunable Supramolecular Hydrogel Carriers.

Local, sustained release and presentation of RNAi therapeutics can be achieved with hydrogel delivery systems. Here we show the development of a supramolecular hydrogel into a local RNAi delivery system. By careful material design, two simple but effective strategies are introduced to obtain controlled release of two classes of RNAi therapeutics, that is, microRNA and antimiR. It was shown that the release of microRNA could be regulated using cholesterol-modification for interaction with the supramolecular hydrogel. Non-modified antimiR release could be controlled via supramolecular introduction of positively charged additive molecules into the supramolecular hydrogel. In this way, either the cholesterol-modification on the drug or the charge introduction into the hydrogel provides handles for controlled RNAi therapy.

Cholesterol Modification of an Anticancer Drug for Efficient Incorporation into a Supramolecular Hydrogel System.

Treatment of cancer in the peritoneal cavity may be improved with macroscale drug delivery systems that offer control over intraperitoneal concentration of chemotherapeutic agents. Currently, suitable drug carriers to facilitate a sustained release of small hydrophilic drugs such as mitomycin C are lacking. For this purpose, a pH-responsive supramolecular hydrogel based on ureido-pyrimidinone (UPy) chemistry is utilized here. In order to provide a sustained release profile, a lipophilicity-increasing cholesterol conjugation strategy is proposed that enhances affinity between the modified drug (mitomycin-PEG24 -cholesterol, MPC) and the hydrophobic compartments in the UPy gel. Additional advantages of cholesterol conjugation include improved chemical stability and potency of mitomycin C. In vitro the tunability of the system to obtain optimal effective concentrations over time is demonstrated with a combinatorial treatment of mitomycin C and MPC in one UPy hydrogel delivery system.

MRI Visualization of Injectable Ureidopyrimidinone Hydrogelators by Supramolecular Contrast Agent Labeling.

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.

Multicomponent Supramolecular Polymers as a Modular Platform for Intracellular Delivery.

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.

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging.

Zn(2+) plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn(2+) homeostasis and the putative signaling role of Zn(2+), various fluorescent sensors have been developed that allow monitoring of Zn(2+) concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn(2+) 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 Zn(2+), but distinctly different concentrations have been reported when using these sensors to measure Zn(2+) 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 Zn(2+) 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 Zn(2+) in the cytosol but was also successfully used for measuring Zn(2+) 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 Zn(2+) simultaneously in the cytosol and the ER or mitochondria.