Glycooligomer-Functionalized Catalytic Nanocompartments Co-Loaded with Enzymes Support Parallel Reactions and Promote Cell Internalization.

A major shortcoming associated with the application of enzymes in drug synergism originates from the lack of site-specific, multifunctional nanomedicine. This study introduces catalytic nanocompartments (CNCs) made of a mixture of PDMS-b-PMOXA diblock copolymers, decorated with glycooligomer tethers comprising eight mannose-containing repeating units and coencapsulating two enzymes, providing multifunctionality by their in situ parallel reactions. Beta-glucuronidase (GUS) serves for local reactivation of the drug hymecromone, while glucose oxidase (GOx) induces cell starvation through glucose depletion and generation of the cytotoxic H2O2. The insertion of the pore-forming peptide, melittin, facilitates diffusion of substrates and products through the membranes. Increased cell-specific internalization of the CNCs results in a substantial decrease in HepG2 cell viability after 24 h, attributed to simultaneous production of hymecromone and H2O2. Such parallel enzymatic reactions taking place in nanocompartments pave the way to achieve efficient combinatorial cancer therapy by enabling localized drug production along with reactive oxygen species (ROS) elevation.

Poly(2-oxazoline)/saRNA Polyplexes for Targeted and Nonviral Gene Delivery.

RNA delivery has been demonstrated to be a potent method of vaccine delivery, as demonstrated by the recent success of the COVID-19 vaccines. Polymers have been shown to be effective vehicles for RNA delivery, with poly(ethylene imine) (PEI) being the current gold standard for delivery. Nonetheless, PEI has toxicity concerns, and so finding alternatives is desirable. Poly(2-oxazoline)s are a promising alternative to PEI, as they are generally biocompatible and offer a high degree of control over the polymer structure. Here, we have synthesized an ionizable primary amine 2-oxazoline and combined it with a double bond containing oxazoline to synthesize a small library of charged statistical and block copolymers. The pendant double bonds were reacted further to decorate the polymers with glucose via a thiol-ene click reaction. All polymers were shown to have excellent cell viability, and the synthesized block polymers showed promising complexation efficiencies for the saRNA, demonstrating a clear structure-property relationship. The polymer transfection potential was tested in various cell lines, and a polymer composition with an amine/glucose ratio of 9:27 has demonstrated the best transfection potential across all cell lines tested. Overall, the results suggest that block polymers with a cationic segment and high levels of glycosylation have the best complexation efficiency and RNA expression levels.

Mechanism of Action of Oxazoline-Based Antimicrobial Polymers Against Staphylococcus aureus: In Vivo Antimicrobial Activity Evaluation.

Antimicrobial-resistant pathogens have reached alarming levels, becoming one of the most pressing global health issues. Hence, new treatments are necessary for the fight against antimicrobial resistance. Synthetic nanoengineered antimicrobial polymers (SNAPs) have emerged as a promising alternative to antimicrobial peptides, overcoming some of their limitations while keeping their key features. Herein, a library of amphiphilic oxazoline-based SNAPs using cationic ring-opening polymerization (CROP) is designed. Amphipathic compounds with 70% cationic content exhibit the highest activity against clinically relevant Staphylococcus aureus isolates, maintaining good biocompatibility in vitro and in vivo. The mechanism of action of the lead compounds against S. aureus is assessed using various microscopy techniques, indicating cell membrane disruption, while the cell wall remains unaffected. Furthermore, a potential interaction of the compounds with bacterial DNA is shown, with possible implications on bacterial division. Finally, one of the compounds exhibits high efficacy in vivo in an insect infection model.

Step-Growth Glycopolymers with a Defined Tacticity for Selective Carbohydrate-Lectin Recognition.

Glycopolymers are potent candidates for biomedical applications by exploiting multivalent carbohydrate-lectin interactions. Owing to their specific recognition capabilities, glycosylated polymers can be utilized for targeted drug delivery to certain cell types bearing the corresponding lectin receptors. A fundamental challenge in glycopolymer research, however, is the specificity of recognition to receptors binding to the same sugar unit (e.g., mannose). Variation of polymer backbone chirality has emerged as an effective method to distinguish between lectins on a molecular level. Herein, we present a facile route toward producing glycopolymers with a defined tacticity based on a step-growth polymerization technique using click chemistry. A set of polymers have been fabricated and further functionalized with mannose moieties to enable lectin binding to receptors relevant to the immune system (mannose-binding lectin, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin, and dendritic and thymic epithelial cell-205). Surface plasmon resonance spectrometry was employed to determine the kinetic parameters of the step-growth glycopolymers. The results highlight the importance of structural complexity in advancing glycopolymer synthesis, yet multivalency remains a main driving force in lectin recognition.

Microphase separation assisted reduction in the percolation threshold of MWCNT/block polymer composites.

Block copolymers continue to attract a great deal of interest since they allow the formation of microphase-separated domains, useful for nanopatterning/templating. Herein, we present the drastic effect of microphase separation of a diblock copolymer on the electrical properties of polymer nanocomposites. Microphase-separated poly(styrene-b-2-ethylhexyl acrylate) (P(St-b-EHA)) block copolymers having different block lengths were synthesized and utilized as templates for multi-walled carbon nanotubes (MWCNTs). The percolation threshold of the films decreased from 0.46 to 0.19 vol% with decreasing styrene phase fraction. More importantly, we observed a non-linear and unique reduction in percolation threshold with transforming the phase into lamellar structures.

Polymers without Petrochemicals: Sustainable Routes to Conventional Monomers.

Access to a wide range of plastic materials has been rationalized by the increased demand from growing populations and the development of high-throughput production systems. Plastic materials at low costs with reliable properties have been utilized in many everyday products. Multibillion-dollar companies are established around these plastic materials, and each polymer takes years to optimize, secure intellectual property, comply with the regulatory bodies such as the Registration, Evaluation, Authorisation and Restriction of Chemicals and the Environmental Protection Agency and develop consumer confidence. Therefore, developing a fully sustainable new plastic material with even a slightly different chemical structure is a costly and long process. Hence, the production of the common plastic materials with exactly the same chemical structures that does not require any new registration processes better reflects the reality of how to address the critical future of sustainable plastics. In this review, we have highlighted the very recent examples on the synthesis of common monomers using chemicals from sustainable feedstocks that can be used as a like-for-like substitute to prepare conventional petrochemical-free thermoplastics.

Glycopolymer-Functionalized MOF-808 Nanoparticles as a Cancer-Targeted Dual Drug Delivery System for Carboplatin and Floxuridine.

Codelivery of chemotherapeutics via nanomaterials has attracted much attention over the last decades due to improved drug delivery to tumor tissues, decreased systemic effects, and increased therapeutic efficacies. High porosities, large pore volumes and surface areas, and tunable structures have positioned metal-organic frameworks (MOFs) as promising drug delivery systems (DDSs). In particular, nanoscale Zr-linked MOFs such as MOF-808 offer notable advantages for biomedical applications such as high porosity, good stability, and biocompatibility. In this study, we report efficient dual drug delivery of floxuridine (FUDR) and carboplatin (CARB) loaded in MOF-808 nanoparticles to cancer cells. The nanoparticles were further functionalized by a poly(acrylic acid-mannose acrylamide) (PAAMAM) glycopolymer coating to obtain a highly selective DDS in cancer cells and enhance the therapeutic efficacy of chemotherapy. While MOF-808 was found to enhance the individual therapeutic effects of FUDR and CARB toward cancerous cells, combining FUDR and CARB was seen to cause a synergistic effect, further enhancing the cytotoxicity of the free drugs. Enhancement of CARB loading and therefore cytotoxicity of the CARB-loaded MOFs could be induced through a modified activation protocol, while coating of MOF-808 with the PAAMAM glycopolymer increased the uptake of the nanoparticles in cancer cells used in the study and offered a particularly significant selective drug delivery with high cytotoxicity in HepG2 human hepatocellular carcinoma cells. These results show how the enhancement of cytotoxicity is possible through both nanovector delivery and synergistic treatment, and that MOF-808 is a viable candidate for future drug delivery studies.

Hierarchy of Complex Glycomacromolecules: From Controlled Topologies to Biomedical Applications.

Carbohydrates bearing a distinct complexity use a special code (Glycocode) to communicate with carbohydrate-binding proteins at a high precision to manipulate biological activities in complex biological environments. The level of complexity in carbohydrate-containing macromolecules controls the amount and specificity of information that can be stored in biomacromolecules. Therefore, a better understanding of the glycocode is crucial to open new areas of biomedical applications by controlling or manipulating the interaction between immune cells and pathogens in terms of trafficking and signaling, which would become a powerful tool to prevent infectious diseases. Even though a certain level of progress has been achieved over the past decade, synthetic glycomacromolecules are still lagging far behind naturally existing glycans in terms of complexity and precision because of insufficient and inefficient synthetic techniques. Currently, specific targeting at a cellular level using synthetic glycomacromolecules is still challenging. It is obvious that multidisciplinary collaborations are essential between different specialized disciplines to enhance the carbohydrate receptor-targeting paradigm for new biomedical applications. In this Perspective, recent developments in the synthesis of sophisticated glycomacromolecules are highlighted, and their biological and biomedical applications are also discussed in detail.

Synthetic Glycomacromolecules of Defined Valency, Absolute Configuration, and Topology Distinguish between Human Lectins.

Carbohydrate-binding proteins (lectins) play vital roles in cell recognition and signaling, including pathogen binding and innate immunity. Thus, targeting lectins, especially those on the surface of immune cells, could advance immunology and drug discovery. Lectins are typically oligomeric; therefore, many of the most potent ligands are multivalent. An effective strategy for lectin targeting is to display multiple copies of a single glycan epitope on a polymer backbone; however, a drawback to such multivalent ligands is they cannot distinguish between lectins that share monosaccharide binding selectivity (e.g., mannose-binding lectins) as they often lack molecular precision. Here, we describe the development of an iterative exponential growth (IEG) synthetic strategy that enables facile access to synthetic glycomacromolecules with precisely defined and tunable sizes up to 22.5 kDa, compositions, topologies, and absolute configurations. Twelve discrete mannosylated "glyco-IEGmers" are synthesized and screened for binding to a panel of mannoside-binding immune lectins (DC-SIGN, DC-SIGNR, MBL, SP-D, langerin, dectin-2, mincle, and DEC-205). In many cases, the glyco-IEGmers had distinct length, stereochemistry, and topology-dependent lectin-binding preferences. To understand these differences, we used molecular dynamics and density functional theory simulations of octameric glyco-IEGmers, which revealed dramatic effects of glyco-IEGmer stereochemistry and topology on solution structure and reveal an interplay between conformational diversity and chiral recognition in selective lectin binding. Ligand function also could be controlled by chemical substitution: by tuning the side chains of glyco-IEGmers that bind DC-SIGN, we could alter their cellular trafficking through alteration of their aggregation state. These results highlight the power of precision synthetic oligomer/polymer synthesis for selective biological targeting, motivating the development of next-generation glycomacromolecules tailored for specific immunological or other therapeutic applications.

Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition-fragmentation chain transfer polymerisation and post-modification utilising copper alkyne-azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Synthetic Routes to Single Chain Polymer Nanoparticles (SCNPs): Current Status and Perspectives.

Recent advances in polymer science make it possible to create single chain polymer nanoparticles (SCNPs), which can mimic the folding of natural macromolecules, such as protein and nucleic acid, in terms of their native and functional state. Even though considerable progress has been done during the last years, the synthesis of relatively controlled SCNPs with a good folding accuracy is still challenging due to lack of appropriate chemical synthesis techniques. Different types of SCNPs are developed with enhanced properties and used for various applications, e.g., delivery systems, imaging agents, and nanomedicine. As it is believed that SCNPs are so crucial to elucidate single chain technology, in this review, recent developments in SCNPs are discussed comprehensively according to their synthetic approaches to keep readers updated on this important research field. First, selective point folding methods are classified and highlighted, and then repeat unit folding routes are discussed with exciting examples.

Self-Assembled Multi- and Single-Chain Glyconanoparticles and Their Lectin Recognition.

In this work, we describe the physicochemical characterization of amphiphilic glycopolymers synthesized via copper(0)-mediated reversible-deactivation radical polymerization (Cu-RDRP). Depending on the chemical composition of the polymer, these glycopolymers are able to form multi-chain or single-chain polymeric nanoparticles. The folding of these polymers is first of all driven by the amphiphilicity of the glycopolymers and furthermore by the supramolecular formation of helical supramolecular stacks of benzene-1,3,5-tricarboxamides (BTAs) stabilized by threefold hydrogen bonding. The obtained polymeric nanoparticles were subsequently evaluated for their lectin-binding affinity toward a series of mannose- and galactose-binding lectins via surface plasmon resonance. We found that addition of 2-ethylhexyl acrylate to the polymer composition results in compact particles, which translates to a reduction in binding affinity, whereas with the addition of BTAs, the relation between the nature of the particle and the binding ability system becomes more unpredictable.

Multi-Arm Star-Shaped Glycopolymers with Precisely Controlled Core Size and Arm Length.

Star-shaped glycopolymers provide very high binding activities toward lectins. However, a straightforward synthesis method for the preparation of multi-arm glycopolymers in a one-pot approach has been challenging. Herein, we report a rapid synthesis of well-defined multi-arm glycopolymers via Cu(0)-mediated reversible deactivation radical polymerization in aqueous media. d-Mannose acrylamide has been homo- and copolymerized with NIPAM to provide linear arms and then core cross-linked with a bisacrylamide monomer. Thus, the arm length and core size of multi-arm glycopolymers were tuned. Moreover, the stability of multi-arm glycopolymers was investigated, and degradation reactions under acidic or basic conditions were observed. The binding activities of the obtained multi-arm glycopolymers with mannose-specific human lectins, DC-SIGN and MBL, were investigated via surface plasmon resonance spectroscopy. Finally, the encapsulation ability of multi-arm glycopolymers was examined using DHA and Saquinavir below and above the lower critical solution temperature (LCST) of P(NIPAM).

Effect of Arm Number and Length of Star-Shaped Glycopolymers on Binding to Dendritic and Langerhans Cell Lectins.

Many cell types in Nature are covered by glycans with a sugar shell on their surface. Synthetic glycopolymer-based materials can mimic these glycans in terms of their variety of biological processes, such as cell growth regulation, adhesion, inflammation by bacteria and viruses, and immune responses. However, the complexity of glycans is still very challenging to be mimicked completely to obtain specific and selective binding ability. Therefore, in this study we aimed to understand how the complexity in the sense of the effect of number of arms and lengths in star-shaped glycopolymers affect the binding activity with different lectins. The Cu-mediated reversible deactivation radical polymerization (Cu-RDRP) technique was employed for the synthesis of mannose containing star-shaped glycopolymers with varying arm number and length. Two sets of star-shaped glycopolymers with on average 1, 3, 7, 8, and 15 arms were successfully synthesized and characterized via 1H NMR, GPC, and DLS. The first set of glycopolymers (Set S1) encompasses 5 star-shaped glycopolymers with a different amount of arms per macromolecule but with equal arm length, whereas in the second set of 5 glycopolymers (Set S2), the amount of sugars per macromolecule was kept constant to obtain glycopolymers with similar glycovalency but in different configuration. Both glycopolymer sets were subsequently evaluated for their lectin-binding affinity toward a series of both newly and previously studied C-type mannose specific lectins present on dendritic and Langerhans cells. Briefly, while Set S1 glycopolymers with the same arm length and different molecular weight showed considerably different biological activities, Set S2 glycopolymers with different arm lengths and the same molecular weight displayed very similar binding abilities, which can indicate that multivalency can be more important than structure complexity to improve the binding behavior of glycopolymers.

Bottlebrush Glycopolymers from 2-Oxazolines and Acrylamides for Targeting Dendritic Cell-Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin and Mannose-Binding Lectin.

Lectins are omnipresent carbohydrate binding proteins that are involved in a multitude of biological processes. Unearthing their binding properties is a powerful tool toward the understanding and modification of their functions in biological applications. Herein, we present the synthesis of glycopolymers with a brush architecture via a "grafting from" methodology. The use of a versatile 2-oxazoline inimer was demonstrated to open avenues for a wide range of 2-oxazoline/acrylamide bottle brush polymers utilizing aqueous Cu-mediated reversible deactivation radical polymerization (Cu-RDRP). The polymers in the obtained library were assessed for their thermal properties in aqueous solution and their binding toward the C-type animal lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and mannose-binding lectin (MBL) via surface plasmon resonance spectrometry. The encapsulation properties of a hydrophobic drug-mimicking compound demonstrated the potential use of glyco brush copolymers in biological applications.

Mannosylated Poly(ethylene imine) Copolymers Enhance saRNA Uptake and Expression in Human Skin Explants.

Messenger RNA (mRNA) is a promising platform for both vaccines and therapeutics, and self-amplifying RNA (saRNA) is particularly advantageous, as it enables higher protein expression and dose minimization. Here, we present a delivery platform for targeted delivery of saRNA using mannosylated poly(ethylene imine) (PEI) enabled by the host-guest interaction between cyclodextrin and adamantane. We show that the host-guest complexation does not interfere with the electrostatic interaction with saRNA and observed that increasing the degree of mannosylation inhibited transfection efficiency in vitro, but enhanced the number of cells expressing GFP by 8-fold in human skin explants. Besides, increasing the ratio of glycopolymer to saRNA also enhanced the percentage of transfected cells ex vivo. We identified that these mannosylated PEIs specifically increased protein expression in the epithelial cells resident in human skin in a mannose-dependent manner. This platform is promising for further study of glycosylation of PEI and targeted saRNA delivery.

Peptide Cross-Linked Poly(2-oxazoline) as a Sensor Material for the Detection of Proteases with a Quartz Crystal Microbalance.

Inflammatory conditions are frequently accompanied by increased levels of active proteases, and there is rising interest in methods for their detection to monitor inflammation in a point of care setting. In this work, new sensor materials for disposable single-step protease biosensors based on poly(2-oxazoline) hydrogels cross-linked with a protease-specific cleavable peptide are described. The performance of the sensor material was assessed targeting the detection of matrix metalloproteinase-9 (MMP-9), a protease that has been shown to be an indicator of inflammation in multiple sclerosis and other inflammatory conditions. Films of the hydrogel were formed on gold-coated quartz crystals using thiol-ene click chemistry, and the cross-link density was optimized. The degradation rate of the hydrogel was monitored using a quartz crystal microbalance (QCM) and showed a strong dependence on the MMP-9 concentration. A concentration range of 0-160 nM of MMP-9 was investigated, and a lower limit of detection of 10 nM MMP-9 was determined.

Self-Assembling Hydrogels Based on a Complementary Host-Guest Peptide Amphiphile Pair.

Supramolecular polymer-based biomaterials play a significant role in current biomedical research. In particular, peptide amphiphiles (PAs) represent a promising material platform for biomedical applications given their modular assembly, tunability, and capacity to render materials with structural and molecular precision. However, the possibility to provide dynamic cues within PA-based materials would increase the capacity to modulate their mechanical and physical properties and, consequently, enhance their functionality and broader use. In this study, we report on the synthesis of a cationic PA pair bearing complementary adamantane and ß-cyclodextrin host-guest cues and their capacity to be further incorporated into self-assembled nanostructures. We demonstrate the possibility of these recognition motifs to selectively bind, enabling noncovalent cross-linking between PA nanofibers and endowing the resulting supramolecular hydrogels with enhanced mechanical properties, including stiffness and resistance to degradation, while retaining in vitro biocompatibility. The incorporation of the host-guest PA pairs in the resulting hydrogels allowed not only for macroscopic mechanical control from the molecular scale, but also for the possibility to engineer further spatiotemporal dynamic properties, opening opportunities for broader potential applications of PA-based materials.

Peptide Cross-Linked Poly (Ethylene Glycol) Hydrogel Films as Biosensor Coatings for the Detection of Collagenase.

Peptide cross-linked poly(ethylene glycol) hydrogel has been widely used for drug delivery and tissue engineering. However, the use of this material as a biosensor for the detection of collagenase has not been explored. Proteases play a key role in the pathology of diseases such as rheumatoid arthritis and osteoarthritis. The detection of this class of enzyme using the degradable hydrogel film format is promising as a point-of-care device for disease monitoring. In this study, a protease biosensor was developed based on the degradation of a peptide cross-linked poly(ethylene glycol) hydrogel film and demonstrated for the detection of collagenase. The hydrogel was deposited on gold-coated quartz crystals, and their degradation in the presence of collagenase was monitored using a quartz crystal microbalance (QCM). The biosensor was shown to respond to concentrations between 2 and 2000 nM in less than 10 min with a lower detection limit of 2 nM.

Single-Chain Glycopolymer Folding via Host-Guest Interactions and Its Unprecedented Effect on DC-SIGN Binding.

Reversible self-folding actions of natural biomacromolecules play crucial roles for specific and unique biological functions in Nature. Hence, controlled folding of single polymer chains has attracted significant attention in recent years. Herein, reversible single-chain folded glycopolymer structures in α-shape with different density of sugar moieties in the knot were created. The influence of folding as well as the sugar density in the knot was investigated on the binding capability with lectins, such as ConA, DC-SIGN, and DC-SIGNR. The synthesis of triblock glycocopolymers bearing ß-CD and adamantane for the host-guest interaction and also mannose residues for the lectin interaction was achieved using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique. The reversible single-chain folding of glycopolymers was achieved under a high dilution of an aqueous solution and the self-assembled folding was monitored by 2D nuclear overhauser enhancement spectroscopy (NOESY) NMR and dynamic light scattering. The lectin binding profiles consistently provided an unprecedented effect of single chain folding as the single-chain folded structures enhanced greatly the binding ability in comparison to the unfolded linear structures.