Tailoring of Physical Properties in Macroporous Poly(isocyanopeptide) Cryogels.

Over the years, synthetic hydrogels have proven remarkably useful as cell culture matrixes to elucidate the role of the extracellular matrix (ECM) on cell behavior. Yet, their lack of interconnected macropores undermines the widespread use of hydrogels in biomedical applications. To overcome this limitation, cryogels, a class of macroporous hydrogels, are rapidly emerging. Here, we introduce a new, highly elastic, and tunable synthetic cryogel, based on poly(isocyanopeptides) (PIC). Introduction of methacrylate groups on PIC facilitated cryopolymerization through free-radical polymerization and afforded cryogels with an interconnected macroporous structure. We investigated which cryogelation parameters can be used to tune the architectural and mechanical properties of the PIC cryogels by systematically altering cryopolymerization temperature, polymer concentration, and polymer molecular weight. We show that for decreasing cryopolymerization temperatures, there is a correlation between cryogel pore size and stiffness. More importantly, we demonstrate that by simply varying the polymer concentration, we can selectively tune the compressive strength of PIC cryogels without affecting their architecture. This unique feature is highly useful for biomedical applications, as it facilitates decoupling of stiffness from other variables such as pore size. As such, PIC cryogels provide an interesting new biomaterial for scientists to unravel the role of the ECM in cellular functions.

Probing the Lewis Acidity of Boronic Acids through Interactions with Arene Substituents.

Boronic acids are Lewis acids that exist in equilibrium with boronate forms in aqueous solution. Here we experimentally and computationally investigated the Lewis acidity of 2,6-diarylphenylboronic acids; specially designed phenylboronic acids that possess two flanking aromatic rings with tunable aromatic character. Hammett analysis of 2,6-diarylphenylboronic acids reveals that their Lewis acidity remains unchanged upon the introduction of EWG/EDG at the distant para position of the flanking aromatic rings. Structural and computational studies demonstrate that polar-π interactions and solvation effects contribute to the stabilization of boronic acids and boronate forms by aromatic rings. Our physical-organic chemistry work highlights that boronic acids and boronates can be stabilized by aromatic systems, leading to an important molecular knowledge for rational design and development of boronic acid-based catalysts and inhibitors of biomedically important proteins.

Through-Space Stabilization of an Imidazolium Cation by Aromatic Rings.

Imidazole-based compounds are widely found in natural products, synthetic molecules, and biomolecules. Noncovalent interactions between the imidazole ring and other functional groups play an important role in determining the function of diverse molecules. However, there is a limited understanding of the underlying noncovalent interactions between imidazoles and aromatic systems. In this work, we report physical-organic chemistry studies on 2-(2,6-diarylphenyl)-1H-imidazoles and their protonated forms to investigate the noncovalent interactions between the central imidazole ring and two flanking aromatic rings possessing substituents at the para/meta position. Hammett analysis revealed that pKa values and proton affinities correlate well with Hammett σ values of para-substituents at the flanking rings. Additional quantitative Kohn-Sham molecular orbital and energy decomposition analyses reveal that through-space π-π interactions and NH-π interactions contribute to the intramolecular stabilization of the imidazolium cation. The results are important because they clearly demonstrate that the imidazolium cation forms energetically favorable noncovalent interactions with aromatic rings via the through-space effect, a knowledge that can be used in rational drug and catalyst design.

Probing Noncovalent Interactions in [3,3]Metaparacyclophanes.

Arene-arene interactions are fundamentally important in molecular recognition. To precisely probe arene-arene interactions in cyclophanes, we designed and synthesized (2,6-phenol)paracyclophanes and (2,6-aniline)paracyclophanes that possess two aromatic rings in close proximity. Fine-tuning the aromatic character of one aromatic ring by fluorine substituents enables investigations on the intramolecular interactions between the electron-rich phenol and aniline with tetra-H- and tetra-F-substituted benzene. pKa measurements revealed that the tetra-F-template increases the acidity of the phenol (ΔpKa = 0.55). X-ray crystallography and computational analyses demonstrated that all [3,3]metaparacyclophanes adopt cofacial parallel conformations, implying the presence of π-π stacking interactions. Advanced quantum chemical analyses furthermore revealed that both electrostatic interactions and orbital interactions provide the key contribution to the structure and stability of [3,3]metaparacyclophanes.

Do Sulfonamides Interact with Aromatic Rings?

Aromatic rings form energetically favorable interactions with many polar groups in chemical and biological systems. Recent molecular studies have shown that sulfonamides can chelate metal ions and form hydrogen bonds, however, it is presently not established whether the polar sulfonamide functionality also interacts with aromatic rings. Here, synthetic, spectroscopic, structural, and quantum chemical analyses on 2,6-diarylbenzenesulfonamides are reported, in which two flanking aromatic rings are positioned close to the central sulfonamide moiety. Fine-tuning the aromatic character by substituents on the flanking rings leads to linear trends in acidity and proton affinity of sulfonamides. This physical-organic chemistry study demonstrates that aromatic rings have a capacity to stabilize sulfonamides via through-space NH-π interactions. These results have implications in rational drug design targeting electron-rich aromatic rings in proteins.

Through-Space Polar-π Interactions in 2,6-Diarylthiophenols.

Molecular recognition between polar groups and aromatic molecules is fundamentally important to rational drug design. Although it has been well established that many polar functionalities interact with electron-rich aromatic residues through energetically favorable polar-π interactions, there is a limited understanding of the association between thiols and aromatic systems. Herein we report physical-organic chemistry studies on 2,6-diarylthiophenols that possess the central thiophenol ring and two flanking aromatic rings with tunable electronic properties caused by substituents at distant para position. Hammett analysis revealed that pKa values and proton affinities correlate well with Hammett sigma values of substituents. Additional energy decomposition analysis supported the conclusion that both through-space SH-π interactions and S- -π interactions contribute to intramolecular stabilization of 2,6-diarylthiophenols.

Through-Space Polar-π Interactions in 2,6-Diarylthiophenols.

The front cover artwork is provided by Marijn Maas from the group of Prof. Jasmin Mecinovic (University of Southern Denmark). The image shows the stabilization of thiols by aromatic rings, as a result of energetically favorable SH-π interactions in a designed small molecule and in proteins. Read the full text of the Article at 10.1002/cphc.202000132.

Synthetic Semiflexible and Bioactive Brushes.

Polymer brushes are extensively used for the preparation of bioactive surfaces. They form a platform to attach functional (bio)molecules and control the physicochemical properties of the surface. These brushes are nearly exclusively prepared from flexible polymers, even though much stiffer brushes from semiflexible polymers are frequently found in nature, which exert bioactive functions that are out of reach for flexible brushes. Synthetic semiflexible polymers, however, are very rare. Here, we use polyisocyanopeptides (PICs) to prepare high-density semiflexible brushes on different substrate geometries. For bioconjugation, we developed routes with two orthogonal click reactions, based on the strain-promoted azide-alkyne cycloaddition reaction and the (photoactivated) tetrazole-ene cycloaddition reaction. We found that for high brush densities, multiple bonds between the polymer and the substrate are necessary, which was achieved in a block copolymer strategy. Whether the desired biomolecules are conjugated to the PIC polymer before or after brush formation depends on the dimensions and required densities of the biomolecules and the curvature of the substrate. In either case, we provide mild, aqueous, and highly modular reaction strategies, which make PICs a versatile addition to the toolbox for generating semiflexible bioactive polymer brush surfaces.

Probing Through-Space Polar-π Interactions in 2,6-Diarylphenols.

Although it is well established that the acidity of phenol can be fine-tuned with substituents on its aromatic ring via through-bond effects, the role of through-space effects on the acidity of phenols is presently poorly understood. Here, we present integrated experimental and computational studies on substituted 2,6-diarylphenols that demonstrate the essential contribution from through-space OH-π interactions and O--π interactions in the observed trends in proton affinities and acidities of 2,6-diarylphenols.

Stress-stiffening-mediated stem-cell commitment switch in soft responsive hydrogels.

Bulk matrix stiffness has emerged as a key mechanical cue in stem cell differentiation. Here, we show that the commitment and differentiation of human mesenchymal stem cells encapsulated in physiologically soft (∼0.2-0.4 kPa), fully synthetic polyisocyanopeptide-based three-dimensional (3D) matrices that mimic the stiffness of adult stem cell niches and show biopolymer-like stress stiffening, can be readily switched from adipogenesis to osteogenesis by changing only the onset of stress stiffening. This mechanical behaviour can be tuned by simply altering the material's polymer length whilst maintaining stiffness and ligand density. Our findings introduce stress stiffening as an important parameter that governs stem cell fate in a 3D microenvironment, and reveal a correlation between the onset of stiffening and the expression of the microtubule-associated protein DCAMKL1, thus implicating DCAMKL1 in a stress-stiffening-mediated, mechanotransduction pathway that involves microtubule dynamics in stem cell osteogenesis.

Affinity-Based Purification of Polyisocyanopeptide Bioconjugates.

Water-soluble polyisocyanopeptides (PICs) are a new class of synthetic polymers that mimic natural protein-based filaments. Their unique semiflexible properties combined with a length of several hundred nanometers have recently enabled a number of biomedical applications ranging from tissue engineering to cancer immunotherapy. One crucial step toward the further development of PICs for these applications is the efficient and controlled synthesis and purification of PIC-biomolecule conjugates. Considering the large size of PICs and the biomolecules to be conjugated, conjugation reactions do usually not proceed to completion due to steric effects. As a consequence, purification of the reaction mixture is necessary to separate the obtained bioconjugates from unreacted biomolecules. As a direct result of the semiflexible nature of PICs, standard polymer and protein purification methods based on molecular weight have not been successful. Here, we introduce a new affinity-based purification method utilizing biotin as an affinity tag. PICs decorated with a controlled and tunable density of biotin molecules (biotinPICs) were efficiently bound to and eluted from a monoavidin resin in buffered aqueous solution. Using these biotinPICs, two different protein conjugates were synthesized, one carrying the enzyme alkaline phosphatase (PhoA) and the other T-cell activating anti-CD3 antibodies. The resulting biotinPIC-protein conjugates were successfully obtained in high purity (>90%) and without any loss of protein activity. The high purity greatly simplifies the analysis of biotinPIC bioconjugates, such as the determination of the average number of biomolecules conjugated per biotinPIC chain. Most importantly, it allows for the direct and straightforward application of the obtained bioconjugates in the desired applications. The new method developed may further be adapted for the purification of other advanced bioconjugates that are difficult to obtain in high purity with the available standard methods.

Biomimetic Stress Sensitive Hydrogel Controlled by DNA Nanoswitches.

One of the most intriguing and important aspects of biological supramolecular materials is its ability to adapt macroscopic properties in response to environmental cues for controlling cellular processes. Recently, bulk matrix stiffness, in particular, stress sensitivity, has been established as a key mechanical cue in cellular function and development. However, stress-stiffening capacity and the ability to control and exploit this key characteristic is relatively new to the field of biomimetic materials. In this work, DNA-responsive hydrogels, composed of semiflexible PIC polymers equipped with DNA cross-linkers, were engineered to create mimics of natural biopolymer networks that capture these essential elastic properties and can be controlled by external stimuli. We show that the elastic properties are governed by the molecular structure of the cross-linker, which can be readily varied providing access to a broad range of highly tunable soft hydrogels with diverse stress-stiffening regimes. By using cross-linkers based on DNA nanoswitches, responsive to pH or ligands, internal control elements of mechanical properties are implemented that allow for dynamic control of elastic properties with high specificity. The work broadens the current knowledge necessary for the development of user defined biomimetic materials with stress stiffening capacity.

Antifibrotic properties of hyaluronic acid crosslinked polyisocyanide hydrogels.

Fibrosis is characterized by the formation of fibrous connective tissue in response to primary injury. As a result, an affected organ may lose part of its functionality due to chronic, organ-specific tissue damage. Since fibrosis is a leading cause of death worldwide, targeting fibrotic diseases with antifibrotic hydrogels can be a lifesaving therapeutic strategy. This study developed a novel hybrid antifibrotic hydrogel by combining the synthetic polyisocyanide (PIC) with hyaluronic acid (HA). Gels of PIC are highly tailorable, thermosensitive, and strongly biomimetic in architecture and mechanical properties, whereas HA is known to promote non-fibrotic fetal wound healing and inhibits inflammatory signaling. The developed HA-PIC hybrids were biocompatible with physical properties comparable to those of the PIC gels. The antifibrotic nature of the gels was assessed by 3D cultures of human foreskin fibroblasts in the presence (or absence as control) of TGFß1 that promotes differentiation into myofibroblasts, a critical step in fibrosis. Proliferation and macroscopic contraction assays and studies on the formation of stress fibers and characteristic fibrosis markers all indicate a strong antifibrotic nature of HA-PIC hydrogel. We showed that these effects originate from both the lightly crosslinked architecture and the presence of HA itself. The hybrid displaying both these effects shows the strongest antifibrotic nature and is a promising candidate for use as in vivo treatment for skin fibrosis.

Immunofilaments Are Well Tolerated after Local or Systemic Administration in Mice.

The invention of nanosized biomaterials has paved the way for novel therapeutics that can manipulate cells on a nanoscale. Nanosized immunofilaments (IFs) are synthetic filamentous polymers consisting out of polyisocyanopeptides, which have been recently established as a powerful platform to activate specific immune cells in vivo such that they raise an antitumor immune response. However, toxicological effects or immunogenicity toward the IFs have not yet been investigated. In this study, we evaluated potential toxic or immunogenic effects in C57BL/6 mice upon intravenous or subcutaneous injection of nonfunctionalized IFs or immunostimulatory IFs over 30 days. We here present a detailed analysis of the gross pathology, hematological parameters, blood biochemistry, histology, and antibody-response against the IF backbone. Our results demonstrate that IFs do not induce severe acute or chronic toxicity in mice. After 30 days, we only found elevated IgG-titers in intravenously injected but not subcutaneously injected mice. In summary, we demonstrate that IFs can be administered into a living organism without adverse side effects, thereby establishing the safety of IFs as a therapeutic intervention.

Probing Polar-π Interactions Between Tetrazoles and Aromatic Rings.

The heterocyclic tetrazole, a well-established bioisosteric replacement of carboxylic acid, plays an important role in medicinal chemistry. To deepen the functional understanding of tetrazoles in chemical sciences, it is essential to investigate the noncovalent interactions between the tetrazole ring and aromatic rings. Here, we report synthetic, spectroscopic, structural and quantum chemical analyses on specially designed 2-arylphenyl-1H-tetrazoles to study the underlying noncovalent interactions between the tetrazole ring and the neighboring aromatic ring possessing substituents at para/meta position. pKa values and proton affinities of 2-arylphenyl-1H-tetrazoles correlate well with Hammett sigma values of para-substituents at the flanking aromatic ring. Molecular orbital and energy decomposition analyses reveal that through-space NH-π interactions and π-π interactions contribute to the trend of pKa values and proton affinities of 2-arylphenyl-1H-tetrazoles. The electrostatic interaction between tetrazole/tetrazolide interacting with the aromatic rings appears responsible for the observed acidity trends. These results will be helpful for the rational design of tetrazole-based drugs and materials.

RETRACTED: P-377 CD38 targeting in multiple myeloma.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. This abstract was submitted to the International Myeloma Society and the meeting organizers by the first author without informed consent of all co-authors.

Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis.

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Multifunctional, Multivalent PIC Polymer Scaffolds for Targeting Antigen-Specific, Autoreactive B Cells.

Multivalent scaffolds that carry multiple molecules with immunophenotyping or immunomodulatory properties are invaluable tools for studying and modulating specific functions of human immune responses. So far, streptavidin-biotin-based tetramers have been widely used for B-cell immunophenotyping purposes. However, the utility of these tetramers is limited by their tetravalency, the inherent immunogenicity of streptavidin (a bacterial protein that can potentially be recognized by B cells), and the limited feasibility to functionalize these reagents. This has rendered tetramers suboptimal for studying rare, in particular, antigen-specific B-cell populations in the context of clinical applications. Here, we used polyisocyanopeptides (PICs), multivalent polymeric scaffolds functionalized with around 50 peptide antigens, to detect autoreactive B cells in the peripheral blood of patients with rheumatoid arthritis. To explore the potential immunomodulatory functionalities, we functionalized PICs with autoantigenic peptides and a trisaccharide CD22 ligand to inhibit autoreactive B-cell activation through interference with the B-cell receptor activation pathway, as evidenced by reduced phospho-Syk expression upon PIC binding. Given the possibilities to functionalize PICs, our data demonstrate that the modular and versatile character of PIC scaffolds makes them promising candidates for future clinical applications in B-cell-mediated diseases.

Artificial Antigen-Presenting Cell Topology Dictates T Cell Activation.

Nanosized artificial antigen-presenting cells (aAPCs), synthetic immune cell mimics that aim to activate T cells ex or in vivo, offer an effective alternative to cellular immunotherapies. However, comprehensive studies that delineate the effect of nano-aAPC topology, including nanoparticle morphology and ligand density, are lacking. Here, we systematically studied the topological effects of polymersome-based aAPCs on T cell activation. We employed an aAPC library created from biodegradable poly(ethylene glycol)-block-poly(d,l-lactide) (PEG-PDLLA) polymersomes with spherical or tubular shape and variable sizes, which were functionalized with αCD3 and αCD28 antibodies at controlled densities. Our results indicate that high ligand density leads to enhancement in T cell activation, which can be further augmented by employing polymersomes with larger size. At low ligand density, the effect of both polymersome shape and size was more pronounced, showing that large elongated polymersomes better activate T cells compared to their spherical or smaller counterparts. This study demonstrates the capacity of polymersomes as aAPCs and highlights the role of topology for their rational design.

Semiflexible Immunobrushes Induce Enhanced T Cell Activation and Expansion.

A variety of bioactive materials developed to expand T cells for adoptive transfer into cancer patients are currently evaluated in the clinic. In most cases, T cell activating biomolecules are attached to rigid surfaces or matrices and form a static interface between materials and the signaling receptors on the T cells. We hypothesized that a T cell activating polymer brush interface might better mimic the cell surface of a natural antigen-presenting cell, facilitating receptor movement and concomitant advantageous mechanical forces to provide enhanced T cell activating capacities. Here, as a proof of concept, we synthesized semiflexible polyisocyanopeptide (PIC) polymer-based immunobrushes equipped with T cell activating agonistic anti-CD3 (αCD3) and αCD28 antibodies placed on magnetic microbeads. We demonstrated enhanced efficiency of ex vivo expansion of activated primary human T cells even at very low numbers of stimulating antibodies compared to rigid beads. Importantly, the immunobrush architecture appeared crucial for this improved T cell activating capacity. Immunobrushes outperform current benchmarks by producing higher numbers of T cells exhibiting a combination of beneficial phenotypic characteristics, such as reduced exhaustion marker expression, high cytokine production, and robust expression of cytotoxic hallmarks. This study indicates that semiflexible immunobrushes have great potential in making T cell-based immunotherapies more effective.