Effect of Particle Heterogeneity in Catalytic Copper-Containing Single-Chain Polymeric Nanoparticles Revealed by Single-Particle Kinetics.

Single-chain polymeric nanoparticles (SCPNs) have been extensively explored as a synthetic alternative to enzymes for catalytic applications. However, the inherent structural heterogeneity of SCPNs, arising from the dispersity of the polymer backbone and stochastic incorporation of different monomers as well as catalytic moieties, is expected to lead to variations in catalytic activity between individual particles. To understand the effect of structural heterogeneities on the catalytic performance of SCPNs, techniques are required that permit researchers to directly monitor SCPN activity at the single-polymer level. In this study, we introduce the use of single-molecule fluorescence microscopy to study the kinetics of Cu(I)-containing SCPNs towards depropargylation reactions. We developed Cu(I)-containing SCPNs that exhibit fast kinetics towards depropargylation and Cu-catalyzed azide-alkyne click reactions, making them suitable for single-particle kinetic studies. SCPNs were then immobilized on the surface of glass coverslips and the catalytic reactions were monitored at a single-particle level using total internal reflection fluorescence (TIRF) microscopy. Our studies revealed the interparticle turnover dispersity for Cu(I)-catalyzed depropargylations. In the future, our approach can be extended to different polymer designs which can give insights into the intrinsic heterogeneity of SCPN catalysis and can further aid in the rational development of SCPN-based catalysts.

Enhanced Efficiency of Pd(0)-Based Single Chain Polymeric Nanoparticles for in Vitro Prodrug Activation by Modulating the Polymer's Microstructure.

Bioorthogonal catalysis employing transition metal catalysts is a promising strategy for the in situ synthesis of imaging and therapeutic agents in biological environments. The transition metal Pd has been widely used as a bioorthogonal catalyst, but bare Pd poses challenges in water solubility and catalyst stability in cellular environments. In this work, Pd(0) loaded amphiphilic polymeric nanoparticles are applied to shield Pd in the presence of living cells for the in situ generation of a fluorescent dye and anticancer drugs. Pd(0) loaded polymeric nanoparticles prepared by the reduction of the corresponding Pd(II)-polymeric nanoparticles are highly active in the deprotection of pro-rhodamine dye and anticancer prodrugs, giving significant fluorescence enhancement and toxigenic effects, respectively, in HepG2 cells. In addition, we show that the microstructure of the polymeric nanoparticles for scaffolding Pd plays a critical role in tuning the catalytic efficiency, with the use of the ligand triphenylphosphine as a key factor for improving the catalyst stability in biological environments.

Imaging Diffusion and Stability of Single-Chain Polymeric Nanoparticles in a Multi-Gel Tumor-on-a-Chip Microfluidic Device.

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.

Bisurea-Based Supramolecular Polymers for Tunable Biomaterials.

Water-soluble supramolecular polymers show great potential to develop dynamic biomaterials with tailored properties. Here, we elucidate the morphology, stability and dynamicity of supramolecular polymers derived from bisurea-based monomers. An accessible synthetic approach from 2,4-toluene diisocyanate (TDI) as the starting material is developed. TDI has two isocyanates that differ in intrinsic reactivity, which allows to obtain functional, desymmetrized monomers in a one-step procedure. We explore how the hydrophobic/hydrophilic ratio affects the properties of the formed supramolecular polymers by increasing the number of methylene units from 10 to 12 keeping the hydrophilic hexa(ethylene glycol) constant. All bisurea-based monomers form long, fibrous structures with 3-5 monomers in the cross-section in water, indicating a proper hydrophobic\hydrophilic balance. The stability of the supramolecular polymers increases with an increasing amount of methylene units, whereas the dynamic nature of the monomers decreases. The introduction of one Cy3 dye affords modified supramolecular monomers, which co-assemble with the unmodified monomers into fibrous structures. All systems show excellent water-compatibility and no toxicity for different cell-lines. Importantly, in cell culture media, the fibrous structures remain present, highlighting the stability of these supramolecular polymers in physiological conditions. The results obtained here motivate further investigation of these bisurea-based building blocks as dynamic biomaterial.

Amphiphilic polymeric nanoparticles enable homogenous rhodium-catalysed NH insertion reactions in living cells.

Rh-catalysed NH carbene insertion reactions were exported to living cells with help of amphiphilic polymeric nanoparticles. Hereto, hydrophobic dirhodium carboxylate catalysts were efficiently encapsulated in amphiphilic polymeric nanoparticles comprising dodecyl and Jeffamine as side grafts. The developed catalytic nanoparticles promoted NH carbene insertions between α-keto diazocarbenes and 2,3-diaminonaphthalene, followed by intramolecular cyclisation to form fluorescent or biologically active benzoquinoxalines. These reactions were studied in reaction media of varying complexity. The best-performing catalyst was exported to HeLa cells, where fluorescent and cytotoxic benzoquinoxalines were synthesized in situ at low catalyst loading within a short time. Most of the developed bioorthogonal transition metal catalysts reported to date are easily deactivated by the reactive biomolecules in living cells, limiting their applications. The high catalytic efficiency of the Rh-based polymeric nanoparticles reported here opens the door to expanding the repertoire of bioorthogonal reactions and is therefore promising for biomedical applications.

Protein-Inspired Control over Synthetic Polymer Folding for Structured Functional Nanoparticles in Water.

The folding of proteins into functional nanoparticles with defined 3D structures has inspired chemists to create simple synthetic systems mimicking protein properties. The folding of polymers into nanoparticles in water proceeds via different strategies, resulting in the global compaction of the polymer chain. Herein, we review the different methods available to control the conformation of synthetic polymers and collapse/fold them into structured, functional nanoparticles, such as hydrophobic collapse, supramolecular self-assembly, and covalent cross-linking. A comparison is made between the design principles of protein folding to synthetic polymer folding and the formation of structured nanocompartments in water, highlighting similarities and differences in design and function. We also focus on the importance of structure for functional stability and diverse applications in complex media and cellular environments.

Simultaneously controlling conformational and operational stability of single-chain polymeric nanoparticles in complex media.

Single-chain polymeric nanoparticles (SCPNs) comprising a solvatochromic pyrazoline adduct show conformational and operational stability in complex media and in cellular compartments; the connectivity of the adduct is crucial in modulating interactions with the surrounding media.

Spectrally PAINTing a Single Chain Polymeric Nanoparticle at Super-Resolution.

Folding a polymer chain into a well-defined single-chain polymeric nanoparticle (SCPN) is a fascinating approach to obtaining structured and functional nanoparticles. Like all polymeric materials, SCPNs are heterogeneous in their nature due to the polydispersity of their synthesis: the stochastic synthesis of polymer backbone length and stochastic functionalization with hydrophobic and hydrophilic pendant groups make structural diversity inevitable. Therefore, in a single batch of SCPNs, nanoparticles with different physicochemical properties are present, posing a great challenge to their characterization at a single-particle level. The development of techniques that can elucidate differences between SCPNs at a single-particle level is imperative to capture their potential applications in different fields such as catalysis and drug delivery. Here, a Nile Red based spectral point accumulation for imaging in nanoscale topography (NR-sPAINT) super-resolution fluorescence technique was implemented for the study of SCPNs at a single-particle level. This innovative method allowed us to (i) map the small-molecule binding rates on individual SCPNs and (ii) map the polarity of individual SCPNs for the first time. The SCPN designs used here have the same polymeric backbone but differ in the number of hydrophobic groups. The experimental results show notable interparticle differences in the binding rates within the same polymer design. Moreover, a marked polarity shift between the different designs is observed. Interestingly, interparticle polarity heterogeneity was unveiled, as well as an intraparticle diversity, information which has thus far remained hidden by ensemble techniques. The results indicate that the addition of hydrophobic pendant groups is vital to determine binding properties and induces single-particle polarity diversity. Overall, NR-sPAINT represents a powerful approach to quantifying the single-particle polarity of SCPNs and paves the way to relate the structural heterogeneity to functionality at the single-particle level. This provides an important step toward the aim of rationally designing SCPNs for the desired application.

En Route to Stabilized Compact Conformations of Single-Chain Polymeric Nanoparticles in Complex Media.

Precise control over the folding pathways of polypeptides using a combination of noncovalent and covalent interactions has evolved into a wide range of functional proteins with a perfectly defined 3D conformation. Inspired hereby, we develop a series of amphiphilic copolymers designed to form compact, stable, and structured single-chain polymeric nanoparticles (SCPNs) of defined size, even in competitive conditions. The SCPNs are formed through a combination of noncovalent interactions (hydrophobic and hydrogen-bonding interactions) and covalent intramolecular cross-linking using a light-induced [2 + 2] cycloaddition. By comparing different self-assembly pathways of the nanoparticles, we show that, like for proteins in nature, the order of events matters. When covalent cross-links are formed prior to the folding via hydrophobic and supramolecular interactions, larger particles with less structured interiors are formed. In contrast, when the copolymers first fold via hydrophobic and hydrogen-bonding interactions into compact conformations, followed by covalent cross-links, good control over the size of the SCPNs and microstructure of the hydrophobic interior is achieved. Such a structured SCPN can stabilize the solvatochromic dye benzene-1,3,5-tricarboxamide-Nile Red via molecular recognition for short periods of time in complex media, while showing slow exchange dynamics with the surrounding complex media at longer time scales. The SCPNs show good biocompatibility with cells and can carry cargo into the lysosomal compartments of the cells. Our study highlights the importance of control over the folding pathway in the design of stable SCPNs, which is an important step forward in their application as noncovalent drug or catalyst carriers in biological settings.

Controlling the Processability and Stability of Supramolecular Polymers Using the Interplay of Intra- and Intermolecular Interactions.

Polymer networks crosslinked via non-covalent interactions afford interesting materials for a wide range of applications due to their self-healing capability, recyclability, and tunable material properties. However, when strong non-covalent binding motifs in combination with high crosslink density are used, processing of the materials becomes troublesome because of high viscosities and the formation of insoluble gels. Here, we present an approach to control the processability of grafted polymers containing strong non-covalent interactions by balancing the interplay of intra- and intermolecular hydrogen bonding. A library of copolymers with different degrees of polymerization and content of protected ureido-pyrimidinone-urea (UPy-urea) grafts was prepared. Photo-deprotection in a good solvent like tetrahydrofuran (THF) at low concentrations (≤1 mg mL-1) created intramolecularly assembled nanoparticles. Remarkably, the intrinsic viscosity of these nanoparticle solutions was an order of magnitude lower compared to solutions of the intermolecularly assembled analogues, highlighting the crucial role of intra- versus intermolecular interactions. Due to the strong hydrogen bonds between UPy dimers, the intramolecularly assembled structures were kinetically trapped. As a result, the polymer nanoparticles were readily processed into a bulk material, without causing major changes in the morphology as verified by atomic force microscopy. Subsequent intermolecular crosslinking of the nanoparticle film, by heating to temperatures where the hydrogen-bond exchange becomes fast, resulted in a crosslinked network. The reversibility of the hereby obtained polymer networks was shown by retrieving the intramolecularly assembled nanoparticles via redissolution and sonication of the intermolecularly crosslinked film in THF with a small amount of acid. Our results highlight that the stability and processability of highly supramolecularly crosslinked polymers can be controlled both in solution and in bulk by using the interplay of intra- and intermolecular non-covalent interactions in grafted polymers.

Dilution-induced gel-sol-gel-sol transitions by competitive supramolecular pathways in water.

Fascinating properties are displayed by synthetic multicomponent supramolecular systems that comprise a manifold of competitive interactions, thereby mimicking natural processes. We present the integration of two reentrant phase transitions based on an unexpected dilution-induced assembly process using supramolecular polymers and surfactants. The co-assembly of the water-soluble benzene-1,3,5-tricarboxamide (BTA-EG4) and a surfactant at a specific ratio yielded small-sized aggregates. These interactions were modeled using the competition between self-sorting and co-assembly of both components. The small-sized aggregates were transformed into supramolecular polymer networks by a twofold dilution in water without changing their ratio. Kinetic experiments show the in situ growth of micrometer-long fibers in the dilution process. We were able to create systems that undergo fully reversible hydrogel-solution-hydrogel-solution transitions upon dilution by introducing another orthogonal interaction.

Direct C-H Trifluoromethylation of (Hetero)Arenes in Water Enabled by Organic Photoredox-Active Amphiphilic Nanoparticles.

Photoredox-catalyzed chemical conversions are predominantly operated in organic media to ensure good compatibility between substrates and catalysts. Yet, when conducted in aqueous media, they are an attractive, mild, and green way to introduce functional groups into organic molecules. We here show that trifluoromethyl groups can be readily installed into a broad range of organic compounds by using water as the reaction medium and light as the energy source. To bypass solubility obstacles, we developed robust water-soluble polymeric nanoparticles that accommodate reagents and photocatalysts within their hydrophobic interior under high local concentrations. By taking advantage of the high excited state reduction potential of N-phenylphenothiazine (PTH) through UV light illumination, the direct C-H trifluoromethylation of a wide array of small organic molecules is achieved selectively with high substrate conversion. Key to our approach is slowing down the production of CF3 radicals during the chemical process by reducing the catalyst loading as well as the light intensity, thereby improving effectiveness and selectivity of this aqueous photocatalytic method. Furthermore, the catalyst system shows excellent recyclability and can be fueled by sunlight. The method we propose here is versatile, widely applicable, energy efficient, and attractive for late-stage introduction of trifluoromethyl groups into biologically active molecules.

Structure and Dynamics of Supramolecular Polymers: Wait and See.

The introduction of stereogenic centers in supramolecular building blocks is used to unveil subtle changes in supramolecular structure and dynamics over time. Three stereogenic centers based on deuterium atoms were introduced in the side chains of a benzene-1,3,5-tricarboxamide (BTA) resulting in a supramolecular polymer in water that at first glance has a structure and dynamics identical to its achiral counterpart. Using three different techniques, the properties of the double helical polymers are compared after 1 day and 4 weeks. An increase in helical preference is observed over time as well as a decrease in the helical pitch and monomer exchange dynamics. It is proposed that the polymer of the chiral monomer needs time to arrive at its maximal preference in helical bias. These results indicate that the order and tight packing increase over time, while the dynamics of this supramolecular polymer decrease over time, an effect that is typically overlooked but unveiled by the isotopic chirality.

Tuning the donor-acceptor interactions in phase-segregated block molecules.

The assembly of donor-acceptor molecules via charge transfer (CT) interactions gives rise to highly ordered nanomaterials with appealing electronic properties. Here, we present the synthesis and bulk co-assembly of pyrene (Pyr) and naphthalenediimide (NDI) functionalized oligodimethylsiloxanes (oDMS) of discrete length. We tune the donor-acceptor interactions by connecting the pyrene and NDI to the same oligomer, forming a heterotelechelic block molecule (NDI-oDMSPyr), and to two separate oligomers, giving Pyr and NDI homotelechelic block molecules (Pyr-oDMS and NDI-oDMS). Liquid crystalline materials are obtained for binary mixtures of Pyr-oDMS and NDI-oDMS, while crystallization of the CT dimers occurred for the heterotelechelic NDI-oDMS-Pyr block molecule. The synergy between crystallization and phase-segregation coupled with the discrete length of the oDMS units allows for perfect order and sharp interfaces between the insulating siloxane and CT layers composed of crystalline CT dimers. We were able to tune the lamellar domain spacing and donor-acceptor CT interactions by applying pressures up to 6 GPa on the material, making the system promising for soft-material nanotechnologies. These results demonstrate the importance of the molecular design to tune the CT interactions and stability of a CT material.

Elucidating the Supramolecular Copolymerization of N- and C-Centered Benzene-1,3,5-Tricarboxamides: The Role of Parallel and Antiparallel Packing of Amide Groups in the Copolymer Microstructure.

An in-depth study of the supramolecular copolymerization behavior of N- and C-centered benzene-1,3,5-tricarboxamides (N- and C-BTAs) has been conducted in methylcyclohexane and in the solid state. The connectivity of the amide groups in the BTAs differs, and mixing N- and C-BTAs results in supramolecular copolymers with a blocky microstructure in solution. The blocky microstructure results from the formation of weaker and less organized, antiparallel hydrogen bonds between N- and C-BTAs. In methylcyclohexane, the helical threefold hydrogen-bonding network present in C- and N-BTAs is retained in the mixtures. In the solid state, in contrast, the hydrogen bonds of pure BTAs as well as their mixtures organize in a sheet-like pattern, and in the mixtures long-range order is lost. Drop-casting to kinetically trap the solution microstructures shows that C-BTAs retain the helical hydrogen bonds, but N-BTAs immediately adopt the sheet-like pattern, a direct consequence of the lower stabilization energy of the helical hydrogen bonds. In the copolymers, the stability of the helical aggregates depends on the copolymer composition, and helical aggregates are only preserved when a high amount of C-BTAs is present. The method outlined here is generally applicable to elucidate the copolymerization behavior of supramolecular monomers both in solution as well as in the solid state.

Elucidating the Stability of Single-Chain Polymeric Nanoparticles in Biological Media and Living Cells.

The controlled folding of synthetic polymer chains into single-chain polymeric nanoparticles (SCPNs) of defined size and shape in water is a viable way to create compartmentalized, nanometer-sized structures for a range of biological applications. Understanding the relationship between the polymer's microstructure and the stability of folded structures is crucial to achieving desired applications. Here, we introduce the solvatochromic dye Nile red into SCPNs and apply a combination of spectroscopic and microscopic techniques to relate polymer microstructure to nanoparticle stability in complex biological media and cellular environments. Our experimental data show that the polymer's microstructure has little effect on the stability of SCPNs in biological media and cytoplasm of living cells, but only SCPNs comprising supramolecular benzene-1,3,5-tricarboxamide (BTA) motifs showed good stability in lysosomes. The results indicate that the polymer's microstructure is vital to ensure nanoparticle stability in highly competitive environments: both hydrophobic collapse and a structured interior are required. Our study provides an accessible way of probing the stability of SCPNs in cellular environments and paves the way for designing highly stable SCPNs for efficient bio-orthogonal catalysis and sensing applications.

Choline-Functionalized Supramolecular Copolymers: Toward Antimicrobial Activity against Streptococcus pneumoniae.

Dynamic binding events are key to arrive at functionality in nature, and these events are often governed by electrostatic or hydrophobic interactions. Synthetic supramolecular polymers are promising candidates to obtain biomaterials that mimic this dynamicity. Here, we created four new functional monomers based on the benzene-1,3,5-tricarboxamide (BTA) motif. Choline or atropine groups were introduced to obtain functional monomers capable of competing with the cell wall of Streptococcus pneumoniae for binding of essential choline-binding proteins (CBPs). Atropine-functionalized monomers BTA-Atr and BTA-Atr3 were too hydrophobic to form homogeneous assemblies, while choline-functionalized monomers BTA-Chol and BTA-Chol3 were unable to form fibers due to charge repulsion. However, copolymerization of BTA-Chol3 with non-functionalized BTA-(OH)3 yielded dynamic fibers, similar to BTA-(OH)3. These copolymers showed an increased affinity toward CBPs compared to free choline due to multivalent effects. BTA-based supramolecular copolymers are therefore a versatile platform to design bioactive and dynamic supramolecular polymers with novel biotechnological properties.

Temperature-dependent modulation by biaryl-based monomers of the chain length and morphology of biphenyl-based supramolecular polymers.

Supramolecular copolymerizations offer attractive options to introduce structural and functional diversity in supramolecular polymer materials. Yet, general principles and structure-property relationships for rational comonomer design remain lacking. Here, we report on the supramolecular (co)aggregation of a phenylpyridine and bipyridine derivative of a recently reported biphenyl tetracarboxamide-based monomer. We show that both arylpyridines are poor monomers for supramolecular homopolymerizations. However, the two arylpyridines efficiently influence supramolecular polymers of a biphenyl-based polymer. The phenylpyridine derivatives primarily sequestrate biphenyl monomers, while the bipyridine intercalates into the polymers at high temperatures. Thereby, these two poorly homopolymerizing monomers allow for a fine control over the length of the biphenyl-based supramolecular polymers. As such, our results highlight the potential to control the structure and morphology of supramolecular polymers by tailoring the electronic properties of additives.

Introducing Hyaluronic Acid into Supramolecular Polymers and Hydrogels.

The use of supramolecular polymers to construct functional biomaterials is gaining more attention due to the tunable dynamic behavior and fibrous structures of supramolecular polymers, which resemble those found in natural systems, such as the extracellular matrix. Nevertheless, to obtain a biomaterial capable of mimicking native systems, complex biomolecules should be incorporated, as they allow one to achieve essential biological processes. In this study, supramolecular polymers based on water-soluble benzene-1,3,5-tricarboxamides (BTAs) were assembled in the presence of hyaluronic acid (HA) both in solution and hydrogel states. The coassembly of BTAs bearing tetra(ethylene glycol) at the periphery (BTA-OEG4) and HA at different ratios showed strong interactions between the two components that led to the formation of short fibers and heterogeneous hydrogels. BTAs were further covalently linked to HA (HA-BTA), resulting in a polymer that was unable to assemble into fibers or form hydrogels due to the high hydrophilicity of HA. However, coassembly of HA-BTA with BTA-OEG4 resulted in the formation of long fibers, similar to those formed by BTA-OEG4 alone, and hydrogels were produced with tunable stiffness ranging from 250 to 700 Pa, which is 10-fold higher than that of hydrogels assembled with only BTA-OEG4. Further coassembly of BTA-OEG4 fibers with other polysaccharides showed that except for dextran, all polysaccharides studied interacted with BTA-OEG4 fibers. The possibility of incorporating polysaccharides into BTA-based materials paves the way for the creation of dynamic complex biomaterials.

Photoredox-Catalyzed Reduction of Halogenated Arenes in Water by Amphiphilic Polymeric Nanoparticles.

The use of organic photoredox catalysts provides new ways to perform metal-free reactions controlled by light. While these reactions are usually performed in organic media, the application of these catalysts at ambient temperatures in aqueous media is of considerable interest. We here compare the activity of two established organic photoredox catalysts, one based on 10-phenylphenothiazine (PTH) and one based on an acridinium dye (ACR), in the light-activated dehalogenation of aromatic halides in pure water. Both PTH and ACR were covalently attached to amphiphilic polymers that are designed to form polymeric nanoparticles with hydrodynamic diameter DH ranging between 5 and 11 nm in aqueous solution. Due to the hydrophobic side groups that furnish the interior of these nanoparticles after hydrophobic collapse, water-insoluble reagents can gather within the nanoparticles at high local catalyst and substrate concentrations. We evaluated six different amphiphilic polymeric nanoparticles to assess the effect of polymer length, catalyst loading and nature of the catalyst (PTH or ACR) in the dechlorination of a range of aromatic chlorides. In addition, we investigate the selectivity of both catalysts for reducing different types of aryl-halogen bonds present in one molecule, as well as the activity of the catalysts for C-C cross-coupling reactions. We find that all polymer-based catalysts show high activity for the reduction of electron-poor aromatic compounds. For electron-rich compounds, the ACR-based catalyst is more effective than PTH. In the selective dehalogenation reactions, the order of bond stability is C-Cl > C-Br > C-I irrespective of the catalyst applied. All in all, both water-compatible systems show good activity in water, with ACR-based catalysts being slightly more efficient for more resilient substrates.