Supramolecular Assembly in Live Cells Mapped by Real-Time Phasor-Fluorescence Lifetime Imaging.

The complex dynamics and transience of assembly pathways in living systems complicate the understanding of these molecular to nanoscale processes. Current technologies are unable to track the molecular events leading to the onset of assembly, where real-time information is imperative to correlate their rich biology. Using a chemically designed pro-assembling molecule, we map its transformation into nanofibers and their fusion with endosomes to form hollow fiber clusters. Tracked by phasor-fluorescence lifetime imaging (phasor-FLIM) in epithelial cells (L929, A549, MDA-MB 231) and correlative light-electron microscopy and tomography (CLEM), spatiotemporal splicing of the assembly events shows time-correlated metabolic dysfunction. The biological impact begins with assembly-induced endosomal disruption that reduces glucose transport into the cells, which, in turn, stymies mitochondrial respiration.

Macrocyclic Dual-Locked "Turn-On" Drug for Selective and Traceless Release in Cancer Cells.

Drug safety and efficacy due to premature release into the bloodstream and poor biodistribution remains a problem despite seminal advances in this area. To circumvent these limitations, we report drug cyclization based on dynamic covalent linkages to devise a dual lock for the small-molecule anticancer drug, camptothecin (CPT). Drug activity is "locked" within the cyclic structure by the redox responsive disulfide and pH-responsive boronic acid-salicylhydroxamate and turns on only in the presence of acidic pH, reactive oxygen species and glutathione through traceless release. Notably, the dual-responsive CPT is more active (100-fold) than the non-cleavable (permanently closed) analogue. We further include a bioorthogonal handle in the backbone for functionalization to generate cyclic-locked, cell-targeting peptide- and protein-CPTs, for targeted delivery of the drug and traceless release in triple negative metastatic breast cancer cells to inhibit cell growth at low nanomolar concentrations.

A Versatile and Efficient Method to Isolate DNA-Polymer Conjugates.

We present a facile and adaptable method to purify and isolate DNA-polymer conjugates from different uncharged homo, random, or block copolymer families. Anion exchange chromatography is used to separate the reaction solution and retrieve the excess unreacted polymer and oligonucleotide. The stationary phase has a high efficiency (25 nmol of DNA per run), facilitating the purification of large batches without compromising the peak shape and resolution. To demonstrate the versatility of this method, different types of polymers, including acrylates, methacrylates, and acrylamides containing hydrophilic and hydrophobic blocks, were purified with high yields. Additionally, DNA-polymer conjugates with various DNA block lengths were also successfully purified, further highlighting the broad applicability of this method.

Engineering the BASHY Dye Platform toward Architectures with Responsive Fluorescence.

A set of nine boronic-acid-derived salicylidenehydrazone (BASHY) complexes has been synthesized in good to very good chemical yields in a versatile three-component reaction. In an extension to previous reports on this dye platform, the focus was put on the electronic modification of the "vertical" positions of the salicylidenehydrazone backbone. This enabled the observation of fluorescence quenching by photoinduced electron transfer (PeT), which can be reverted by the addition of acid in organic solvent (OFF-ON fluorescence switching). The resulting emission is observed in the green-to-orange spectral region (maxima at 520-590 nm). In contrast, under physiological pH conditions in water, the PeT process is inherently decativated, thereby enabling the observation of fluorescence in the red-to-NIR region (maxima at 650-680 nm) with appreciable quantum yields and lifetimes. The latter characteristic supported the application of the dyes in fluorescence lifetime imaging (FLIM) of live A549 cells.

Introducing carbohydrate patterning in mannose-decorated supramolecular assemblies and hydrogels.

Benzene-1,3,5-tricarboxamide (BTA) glyco-monomers containing one, two or three mannose units are synthesized and formulated into differently patterned supramolecular glycopolymers through homo-assembly or co-assembly with non-functionalized BTAs. Unfortunately, no cellular activity could be detected. Excitingly, these glyco-BTA monomers could be formulated into hydrogels, paving the way for (immune) cell culture.

Polymer Chemistry in Living Cells.

The polymerization of biomolecules is a central operation in biology that connects molecular signals with proliferative and information-rich events in cells. As molecules arrange precisely across 3-D space, they create new functional capabilities such as catalysis and transport highways and exhibit new phase separation phenomena that fuel nonequilibrium dynamics in cells. Hence, the observed polymer chemistry manifests itself as a molecular basis leading to cellular phenotypes, expressed as a multitude of hierarchical structures found in cell biology. Although many milestone discoveries had accompanied the rise of the synthetic polymer era, fundamental studies were realized within a closed, pristine environment and that their behavior in a complex multicomponent system remains challenging and thus unexplored. From this perspective, there is a rich trove of undiscovered knowledge that awaits the polymer science community that can revolutionize understanding in the interactive nanoscale world of the living cell.In this Account, we discuss the strategies that have enabled synthetic polymer chemistry to be conducted within the cells (membrane inclusive) and to establish monomer design principles that offer spatiotemporal control of the polymerization. As reaction considerations such as monomer concentration, polymer growth dynamics, and reactivities are intertwined with the subcellular environment and transport processes, we first provide a chemical narrative of each major cellular compartment. The conditions within each compartment will therefore set the boundaries on the type of polymer chemistry that can be conducted. Both covalent and supramolecular polymerization concepts are explored separately in the context of scaffold design, polymerization mechanism, and activation. To facilitate transport into a localized subcellular space, we show that monomers can be reversibly modified by targeting groups or stimulus-responsive motifs that react within the specific compartment. Upon polymerization, we discuss the characterization of the resultant polymeric structures and how these phase-separated structures would impact biological processes such as cell cycle, metabolism, and apoptosis. As we begin to integrate cellular biochemistry with in situ polymer science, we identify landmark challenges and technological hurdles that, when overcome, would lead to invaluable discoveries in macromolecular therapeutics and biology.

In Situ Assembly of Platinum(II)-Metallopeptide Nanostructures Disrupts Energy Homeostasis and Cellular Metabolism.

Nanostructure-based functions are omnipresent in nature and essential for the diversity of life. Unlike small molecules, which are often inhibitors of enzymes or biomimetics with established methods of elucidation, we show that functions of nanoscale structures in cells are complex and can implicate system-level effects such as the regulation of energy and redox homeostasis. Herein, we design a platinum(II)-containing tripeptide that assembles into intracellular fibrillar nanostructures upon molecular rearrangement in the presence of endogenous H2O2. The formed nanostructures blocked metabolic functions, including aerobic glycolysis and oxidative phosphorylation, thereby shutting down ATP production. As a consequence, ATP-dependent actin formation and glucose metabolite-dependent histone deacetylase activity are downregulated. We demonstrate that assembly-driven nanomaterials offer a rich avenue to achieve broad-spectrum bioactivities that could provide new opportunities in drug discovery.

Nanoscale patterning of polymers on DNA origami.

The combination of DNA-origami and synthetic polymers paves the way to a new class of structurally precise biohybrid nanomaterials for diverse applications. Herein, we introduce the grafting to method with high conversions (70-90%) under ambient conditions to generate DNA-polymer conjugates, which can hybridized precisely to DNA-origami architectures. We generated homo and block copolymers from three different polymer families (acrylates, methacrylates and acrylamides), coupled them to single stranded DNA (ssDNA) and pattern different DNA-origami architectures to demonstrate the formation of precise surface nanopatterns.

Multiple Wavelength Photopolymerization of Stable Poly(Catecholamines)-DNA Origami Nanostructures.

The synthesis of multicomponent polymer hybrids with nanometer precision is chemically challenging in the bottom-up synthesis of complex nanostructures. Here, we leverage the fidelity of the DNA origami technique to install a multiple wavelength responsive photopolymerization system with nanometer resolution. By precisely immobilizing various photosensitizers on the origami template, which are only activated at their respective maximum wavelength, we can control sequential polymerization processes. In particular, the triggered photosensitizers generate reactive oxygen species that in turn initiate the polymerization of the catecholamines dopamine and norepinephrine. We imprint polymeric layers at designated positions on DNA origami, which modifies the polyanionic nature of the DNA objects, thus promoting their uptake into living cells while preserving their integrity. Our herein proposed method provides a rapid platform to access complex 3D nanostructures by customizing material and biological interfaces.

Assembly of pH-Responsive Antibody-Drug-Inspired Conjugates.

With the advent of chemical strategies that allow the design of smart bioconjugates, peptide- and protein-drug conjugates are emerging as highly efficient therapeutics to overcome limitations of conventional treatment, as exemplified by antibody-drug conjugates (ADCs). While targeting peptides serve similar roles as antibodies to recognize overexpressed receptors on diseased cell surfaces, peptide-drug conjugates suffer from poor stability and bioavailability due to their low molecular weights. Through a combination of a supramolecular protein-based assembly platform and a pH-responsive linker, the authors devise herein the convenient assembly of a trivalent protein-drug conjugate. The conjugate should ideally possess distinct features of ADCs such as 1) recognition sites that recognize cell receptor and are arranged on 2) distinct locations on a high molecular weight protein scaffold, 3) a stimuli-responsive linker, as well as 4) an attached payload such as a drug molecule. These AD-like conjugates target cancer cells that overexpress somatostatin receptors, can enable controlled release in the microenvironment of cancer cells through a new pH-responsive biotin linker, and exhibit stability in biological media.

Controlling Polymer Morphologies by Intramolecular and Intermolecular Dynamic Covalent Iron(III)/Catechol Complexation-From Polypeptide Single Chain Nanoparticles to Hydrogels.

Responsive biomaterials, tunable from the molecular to the macroscopic scale, are attractive for various applications in nanotechnology. Herein, a long polypeptide chain derived from the abundant serum protein human serum albumin is cross-linked by dynamic-coordinative iron(III)/catechol bonds. By tuning the binding stoichiometry and the pH, reversible intramolecular folding into polypeptide nanoparticles with controllable sizes is achieved. Moreover, upon varying the stoichiometry, intermolecular cross-links become predominant yielding smart and tunable macroscopic protein hydrogels. By adjusting the intra- and intermolecular interactions, biocompatible and biodegradable materials are formed with varying morphologies and dimensions covering several lengths scales featuring rapid gelation without toxic reagents, fast and autonomous self-healing, tunable mechanical properties, and high adaptability to local environmental conditions. Such material characteristics can be particularly attractive for tissue engineering approaches to recreate soft tissues matrices with highly customizable features in a fast and simple fashion.

Reversing Aß Fibrillation and Inhibiting Aß Primary Neuronal Cell Toxicity Using Amphiphilic Polyphenylene Dendrons.

Uncontrolled amyloid-beta (Aß) fibrillation leads to the deposition of neurotoxic amyloid plaques and is associated with Alzheimer's disease. Inhibiting Aß monomer fibrillation and dissociation of the formed fibers is regarded as a promising therapeutic strategy. Here, amphiphilic polyphenylene dendrons (APDs) are demonstrated to interrupt Aß assembly and reduce Aß-cell interactions. Containing alternating negatively charged sulfonic acid and hydrophobic n-propyl peripheral groups, APDs bind to the secondary structure of the Aß aggregates, inhibiting fibrillation and disassemble the already formed Aß fibrils. APDs reveal vesicular cellular uptake in endosomes as well as cell compatibility for endothelial and neuronal cells, and significantly reduce Aß-induced neuron cytotoxicity in vitro. Moreover, they are transported into the brain and successfully cross the blood-brain barrier after systemic application in mice, indicating their high potential to inhibit Aß fibrillation in vivo, which can be beneficial for developing therapeutic strategy for Alzheimer's disease.

Designing bioresponsive nanomaterials for intracellular self-assembly.

Supramolecular assemblies are essential components of living organisms. Cellular scaffolds, such as the cytoskeleton or the cell membrane, are formed via secondary interactions between proteins or lipids and direct biological processes such as metabolism, proliferation and transport. Inspired by nature's evolution of function through structure formation, a range of synthetic nanomaterials has been developed in the past decade, with the goal of creating non-natural supramolecular assemblies inside living mammalian cells. Given the intricacy of biological pathways and the compartmentalization of the cell, different strategies can be employed to control the assembly formation within the highly crowded, dynamic cellular environment. In this Review, we highlight emerging molecular design concepts aimed at creating precursors that respond to endogenous stimuli to build nanostructures within the cell. We describe the underlying reaction mechanisms that can provide spatial and temporal control over the subcellular formation of synthetic nanostructures. Showcasing recent advances in the development of bioresponsive nanomaterials for intracellular self-assembly, we also discuss their impact on cellular function and the challenges associated with establishing structure-bioactivity relationships, as well as their relevance for the discovery of novel drugs and imaging agents, to address the shortfall of current solutions to pressing health issues.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

Polymer cyclization for the emergence of hierarchical nanostructures.

The creation of synthetic polymer nanoobjects with well-defined hierarchical structures is important for a wide range of applications such as nanomaterial synthesis, catalysis, and therapeutics. Inspired by the programmability and precise three-dimensional architectures of biomolecules, here we demonstrate the strategy of fabricating controlled hierarchical structures through self-assembly of folded synthetic polymers. Linear poly(2-hydroxyethyl methacrylate) of different lengths are folded into cyclic polymers and their self-assembly into hierarchical structures is elucidated by various experimental techniques and molecular dynamics simulations. Based on their structural similarity, macrocyclic brush polymers with amphiphilic block side chains are synthesized, which can self-assemble into wormlike and higher-ordered structures. Our work points out the vital role of polymer folding in macromolecular self-assembly and establishes a versatile approach for constructing biomimetic hierarchical assemblies.

Disaccharide Residues are Required for Native Antifreeze Glycoprotein Activity.

Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP's unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.

Functional DNA-Polymer Conjugates.

DNA nanotechnology has seen large developments over the last 30 years through the combination of solid phase synthesis and the discovery of DNA nanostructures. Solid phase synthesis has facilitated the availability of short DNA sequences and the expansion of the DNA toolbox to increase the chemical functionalities afforded on DNA, which in turn enabled the conception and synthesis of sophisticated and complex 2D and 3D nanostructures. In parallel, polymer science has developed several polymerization approaches to build di- and triblock copolymers bearing hydrophilic, hydrophobic, and amphiphilic properties. By bringing together these two emerging technologies, complementary properties of both materials have been explored; for example, the synthesis of amphiphilic DNA-polymer conjugates has enabled the production of several nanostructures, such as spherical and rod-like micelles. Through both the DNA and polymer parts, stimuli-responsiveness can be instilled. Nanostructures have consequently been developed with responsive structural changes to physical properties, such as pH and temperature, as well as short DNA through competitive complementary binding. These responsive changes have enabled the application of DNA-polymer conjugates in biomedical applications including drug delivery. This review discusses the progress of DNA-polymer conjugates, exploring the synthetic routes and state-of-the-art applications afforded through the combination of nucleic acids and synthetic polymers.

DNA Origami Meets Polymers: A Powerful Tool for the Design of Defined Nanostructures.

The combination of DNA origami nanostructures and polymers provides a new possibility to access defined structures in the 100 nm range. In general, DNA origami serves as a versatile template for the highly specific arrangement of polymer chains. Polymer-DNA hybrid nanostructures can either be created by growing the polymer from the DNA template or by attaching preformed polymers to the DNA scaffold. These conjugations can be of a covalent nature or be based on base-pair hybridization between respectively modified polymers and DNA origami. Furthermore, the negatively charged DNA backbone permits interaction with positively charged polyelectrolytes to form stable complexes. The combination of polymers with tuneable characteristics and DNA origami allows the creation of a new class of hybrid materials, which could offer exciting applications for controlled energy transfer, nanoscale organic circuits, or the templated synthesis of nanopatterned polymeric structures.

Controlled Supramolecular Assembly Inside Living Cells by Sequential Multistaged Chemical Reactions.

Synthetic assembly within living cells represents an innovative way to explore purely chemical tools that can direct and control cellular behavior. We use a simple and modular platform that is broadly accessible and yet incorporates highly intricate molecular recognition, immolative, and rearrangement chemistry. Short bimodular peptide sequences undergo a programmed sequence of events that can be tailored within the living intracellular environment. Each sequential stage of the pathways beginning with the cellular uptake, intracellular transport, and localization imposes distinct structural changes that result in the assembly of fibrillar architectures inside cells. The observation of apoptosis, which is characterized by the binding of Annexin V, demonstrates that programmed cell death can be promoted by the peptide assembly. Higher complexity of the assemblies was also achieved by coassembly of two different sequences, resulting in intrinsically fluorescent architectures. As such, we demonstrate that the in situ construction of architectures within cells will broaden the community's perspective toward how structure formation can impact a living system.

Amphiphilic dendrimers control protein binding and corona formation on liposome nanocarriers.

Amphiphilic polyphenylene dendrimers (PPDs) with distinct lipophilic and positively or negatively charged surface groups were adsorbed onto liposomes and their impact on protein adsorption in blood plasma was studied. The PPD corona reduced binding of specific opsonins and increased the adsorption of proteins controlling cellular uptake based on their surface patches.