Multi-targeting oligopyridiniums: Rational design for biofilm dispersion and bacterial persister eradication.

The development of effective antibacterial drugs to combat bacterial infections, particularly the biofilm-related infections, remains a challenge. There are two important features of bacterial biofilms, which are well-known critical factors causing biofilms hard-to-treat in clinical, including the dense and impermeable extracellular polymeric substances (EPS) and the metabolically repressed dormant and persistent bacterial population embedded. These characteristics largely increase the difficulty for regular antibiotic treatment due to insufficient penetration into EPS. In addition, the dormant bacteria are insensitive to the growth-inhibiting mechanism of traditional antibiotics. Herein, we explore the potential of a series of new oligopyridinium-based oligomers bearing a multi-biomacromolecule targeting function as the potent bacterial biofilm eradication agent. These oligomers were rationally designed to be "charge-on-backbone" that can offer a special alternating amphiphilicity. This novel and unique feature endows high affinity to bacterial membrane lipids, DNAs as well as proteins. Such a broad multi-targeting nature of molecules not only enables its penetration into EPS, but also plays vital roles in the bactericidal mechanism of action that is highly effective against dormant and persistent bacteria. Our in vitro, ex vivo, and in vivo studies demonstrated that OPc3, one of the most effective derivatives, was able to offer excellent antibacterial potency against a variety of bacteria and effectively eliminate biofilms in zebrafish models and mouse wound biofilm infection models.

A Membrane-Embedded Macromolecular Catalyst with Substrate Selectivity in Live Cells.

Substrate selectivity is one of the most attractive features of natural enzymes from their "bind-to-catalyze" working flow and is thus a goal for the development of synthetic enzyme mimics that mediate abiotic transformations. However, despite the recent success in the preparation of substrate-selective enzyme mimics based on single-chain nanoparticles, examples extending such selectivity into living systems have been absent. In this article, we report the in cellulo substrate selectivity of an enzyme-mimicking macromolecular catalyst based on a cationic dense-shell nanoparticle (DSNP) scaffold. With a systematic study on DSNP's structure-activity relationship, we demonstrate that the DSNP has excellent membrane affinity that is governed by several contributing factors, namely, charge density, type of charge, and particle size, and the best-performing phosphonium-rich DSNP can be used as a membrane-embedded catalyst (MEC) for efficient on-membrane synthesis. Importantly, the DSNP catalyst retains its selectivity toward lipophilic and anionic substrates when working as an MEC for on-membrane ligation. The usefulness of such substrate selectivity and on-membrane catalysis strategy was exemplified with several molecules of interest with low cell permeability and anionic nature, which were successfully transported into eukaryotic cells by after their formation directly on the cell membrane.

Chemical Biology Approach to Reveal the Importance of Precise Subcellular Targeting for Intracellular Staphylococcus aureus Eradication.

Intracellular bacterial pathogens, such as Staphylococcus aureus, that may hide in intracellular vacuoles represent the most significant manifestation of bacterial persistence. They are critically associated with chronic infections and antibiotic resistance, as conventional antibiotics are ineffective against such intracellular persisters due to permeability issues and mechanistic reasons. Direct subcellular targeting of S. aureus vacuoles suggests an explicit opportunity for the eradication of these persisters, but a comprehensive understanding of the chemical biology nature and significance of precise S. aureus vacuole targeting remains limited. Here, we report an oligoguanidine-based peptidomimetic that effectively targets and eradicates intracellular S. aureus persisters in the phagolysosome lumen, and this oligomer was utilized to reveal the mechanistic insights linking precise targeting to intracellular antimicrobial efficacy. The oligomer has high cellular uptake via a receptor-mediated endocytosis pathway and colocalizes with S. aureus persisters in phagolysosomes as a result of endosome-lysosome interconversion and lysosome-phagosome fusion. Moreover, the observation of a bacterium's altered susceptibility to the oligomer following a modification in its intracellular localization offers direct evidence of the critical importance of precise intracellular targeting. In addition, eradication of intracellular S. aureus persisters was achieved by the oligomer's membrane/DNA dual-targeting mechanism of action; therefore, its effectiveness is not hampered by the hibernation state of the persisters. Such precise subcellular targeting of S. aureus vacuoles also increases the agent's biocompatibility by minimizing its interaction with other organelles, endowing excellent in vivo bacterial targeting and therapeutic efficacy in animal models.

Combination of Backbone Rigidity and Richness in Aryl Structures Enables Direct Membrane Translocation of Polymer Scaffolds for Efficient Gene Delivery.

The development of cell-penetrating polymers with endocytosis-independent cell uptake pathways has emerged as a prominent strategy to enhance the transfection efficiency. Inspired by the rigid α-helical structure that endows polypeptides with cell-penetrating ability, we propose that a rigid backbone can facilitate the corresponding polymer vector's performance in gene delivery by bypassing the difficult endosomal escape process. Meanwhile, the installation of aromatic domains, as a way to promote gene transfection efficiency, is employed through the construction of a poly(benzyl ether) (PBE)-based scaffold in this work. We demonstrate that the direct membrane translocation capability of the synthesized PBE contributes to its enhanced transfection performance and excellent biocompatibility profile, rendering the imidazolium-functionalized PBE scaffold with higher activity and biocompatibility. Molecular details of the PBE-lipid interaction are also revealed in molecular dynamics simulations, indicating the important roles of individual structural elements on the polymeric scaffold in the membrane penetration process.

Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1.

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

Dendronized Arm Snowflake Polymer as a Highly Branched Scaffold for Cellular Imaging and Delivery.

Incorporation of branched structures is a major pathway to build macromolecules with desired three-dimensional (3D) structures, which are of high importance in the rational design of functional polymeric scaffolds. Dendrimers and hyperbranched polymers have been extensively studied for this purpose, but proper gain-of-function for these structures usually requires large enough molecular weights and a highly branched interior so that a spherical 3D core-shell architecture can be obtained, yet it is generally challenging to achieve precise control over the structure, high molecular weight, and high degree of branching (DoB) simultaneously. In this article, we present a set of snowflake-shaped star polymers with functional cores and dendronized arms, which ensure a high DoB and an overall globular conformation, thus facilitating the introduction of functional moieties onto the easily achieved scaffold without the need for high-generation dendrons. Using a polyglycerol dendron (PGD) as a proof of concept, we propose that this dendronized arm snowflake polymer (DASP) structure can serve as a better performing alternative to high-generation PGDs. DASPs with molecular weights of 750, 1220, 2120, and 3740 kDa were prepared with >85% yields in all cases, and we show that these DASPs have high encapsulating efficiency of Nile Red due to their high DoB and high biocompatibility due to their hydroxyl-rich nature after ketal removal, as well as high cell permeability that is molecular-weight-dependent. Introduced fluorophores such as fluorescein and difluoroboron 1,3-diphenylaminophenyl ß-diketonate with suitable excitation wavelengths may turn the DASPs into stable, endosome-staining fluorophores with ultra-large Stokes shifts, narrowed emission bands, and suitability for long-term cellular tracing. Moreover, the scaffold can encapsulate antibiotic molecules and deliver them into phagolysosomes for efficient elimination of intracellular Staphylococcus aureus, which is insensitive toward many antibiotics but is a key target for the clinical success of methicillin-resistant Staphylococcus aureus infection treatment. Elimination of Staphylococcus aureus could be improved to >99.9% for chloramphenicol at 32 µg/mL with 450 µg/mL DASP.

Designed transition metal catalysts for intracellular organic synthesis.

The development of synthetic, metal-based catalysts to perform intracellular bioorthogonal reactions represents a relatively new and important area of research that combines transition metal catalysis and chemical biology. The ability to perform reactions in cellulo, especially those transformations without a natural counterpart, offers a versatile tool for medicinal chemists and chemical biologists. With proper modification of the metal catalysts, it is even possible to direct a reaction to certain intracellular sites. This review highlights advances in this new area, from early work on intracellular functional group conversions to recent advances in intracellular synthesis of drugs, including cytotoxic agents. Both the fundamental and applied aspects of this approach to intracellular synthesis are reviewed.

Enzyme-like Click Catalysis by a Copper-Containing Single-Chain Nanoparticle.

A major challenge in performing reactions in biological systems is the requirement for low substrate concentrations, often in the micromolar range. We report that copper cross-linked single-chain nanoparticles (SCNPs) are able to significantly increase the efficiency of copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reactions at low substrate concentration in aqueous buffer by promoting substrate binding. Using a fluorogenic click reaction and dye uptake experiments, a structure-activity study is performed with SCNPs of different size and copper content and substrates of varying charge and hydrophobicity. The high catalytic efficiency and selectivity are attributed to a mechanism that involves an enzyme-like substrate binding process. Saturation-transfer difference (STD) NMR spectroscopy, 2D-NOESY NMR, kinetic analyses with varying substrate concentrations, and computational simulations are consistent with a Michaelis-Menten, two-substrate, random-sequential enzyme-like kinetic profile. This general approach may prove useful for developing more-sustainable catalysts and agents for biomedicine and chemical biology.

Bottom-Up Strategy To Prepare Nanoparticles with a Single DNA Strand.

We describe the preparation of cross-linked, polymeric organic nanoparticles (ONPs) with a single, covalently linked DNA strand. The structure and functionalities of the ONPs are controlled by the synthesis of their parent linear block copolymers that provide monovalency, fluorescence and narrow size distribution. The ONP can also guide the deposition of chloroaurate ions allowing gold nanoparticles (AuNPs) to be prepared using the ONPs as templates. The DNA strand on AuNPs is shown to preserve its functions.

A Highly Efficient Single-Chain Metal-Organic Nanoparticle Catalyst for Alkyne-Azide "Click" Reactions in Water and in Cells.

We show that copper-containing metal-organic nanoparticles (MONPs) are readily synthesized via Cu(II)-mediated intramolecular cross-linking of aspartate-containing polyolefins in water. In situ reduction with sodium ascorbate yields Cu(I)-containing MONPs that serve as highly efficient supramolecular catalysts for alkyne-azide "click chemistry" reactions, yielding the desired 1,4-adducts at low parts per million catalyst levels. The nanoparticles have low toxicity and low metal loadings, making them convenient, green catalysts for alkyne-azide "click" reactions in water. The Cu-MONPs enter cells and perform efficient, biocompatible click chemistry, thus acting as intracellular nanoscale molecular synthesizers.

Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting.

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.

Reversibly cross-linked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity.

Polycations often suffer from the irreconcilable inconsistency between transfection efficiency and toxicity. Polymers with high molecular weight (MW) and cationic charge feature potent gene delivery capabilities, while in the meantime suffer from strong chemotoxicity, restricted intracellular DNA release, and low stability in vivo. To address these critical challenges, we herein developed pH-responsive, reversibly cross-linked, polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA) for the effective and targeted gene delivery to cancer cells. Low-MW PEI was cross-linked with the ketal-containing linker, and the obtained high-MW analogue afforded potent gene delivery capabilities during transfection, while rapidly degraded into low-MW segments upon acid treatment in the endosomes, which promoted intracellular DNA release and reduced material toxicity. HA coating of the polyplexes shielded the surface positive charges to enhance their stability under physiological condition and simultaneously reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene therapy.

Orientation-controlled membrane anchoring of bioorthogonal catalysts on live cells via liposome fusion-based transport.

An obstacle to conducting diverse bioorthogonal reactions in living systems is the sensitivity of artificial metal catalysts. It has been reported that artificial metallocatalysts can be assembled in "cleaner" environments in cells for stabilized performance, which is powerful but is limited by the prerequisite of using specific cells. We report here a strategy to establish membrane-anchored catalysts with precise spatial control via liposome fusion-based transport (MAC-LiFT), loading bioorthogonal catalytic complexes onto either or both sides of the membrane leaflets. We show that the inner face of the cytoplasmic membrane serves as a reliable shelter for metal centers, protecting the complexes from deactivation thus substantially lowering the amount of catalyst needed for effective intracellular catalysis. This MAC-LiFT approach makes it possible to establish catalyst-protective systems with exclusively exogenous agents in a wide array of mammalian cells, allowing convenient and wider use of diverse bioorthogonal reactions in live cellular systems.

A nanoscale MOF-based heterogeneous catalytic system for the polymerization of N-carboxyanhydrides enables direct routes toward both polypeptides and related hybrid materials.

Synthetic polypeptides have emerged as versatile tools in both materials science and biomedical engineering due to their tunable properties and biodegradability. While the advancements of N-carboxyanhydride (NCA) ring-opening polymerization (ROP) techniques have aimed to expedite polymerization and reduce environment sensitivity, the broader implications of such methods remain underexplored, and the integration of ROP products with other materials remains a challenge. Here, we show an approach inspired by the success of many heterogeneous catalysts, using nanoscale metal-organic frameworks (MOFs) as co-catalysts for NCA-ROP accelerated also by peptide helices in proximity. This heterogeneous approach offers multiple advantages, including fast kinetics, low environment sensitivity, catalyst recyclability, and seamless integration with hybrid materials preparation. The catalytic system not only streamlines the preparation of polypeptides and polypeptide-coated MOF complexes (MOF@polypeptide hybrids) but also preserves and enhances their homogeneity, processibility, and overall functionalities inherited from the constituting MOFs and polypeptides.

Novel Organic Superbase Dopants for Ultraefficient N-Doping of Organic Semiconductors.

Doping is a powerful technique for engineering the electrical properties of organic semiconductors (OSCs), yet efficient n-doping of OSCs remains a central challenge. Herein, the discovery of two organic superbase dopants, namely P2-t-Bu and P4-t-Bu as ultra-efficient n-dopants for OSCs is reported. Typical n-type semiconductors such as N2200 and PC61 BM are shown to experience a significant increase of conductivity upon doping by the two dopants. In particular, the optimized electrical conductivity of P2-t-Bu-doped PC61 BM reaches a record-high value of 2.64 S cm-1 . The polaron generation efficiency of P2-t-Bu-doped in PC61 BM is found to be over 35%, which is 2-3 times higher than that of benchmark n-dopant N-DMBI. In addition, a deprotonation-initiated, nucleophilic-attack-based n-doping mechanism is proposed for the organic superbases, which involves the deprotonation of OSC molecules, the nucleophilic attack of the resulting carbanions on the OSC's π-bonds, and the subsequent n-doping through single electron transfer process between the anionized and neutral OSCs. This work highlights organic superbases as promising n-dopants for OSCs and opens up opportunities to explore and develop highly efficient n-dopants.

Amphiphilic Nano-Swords for Direct Penetration and Eradication of Pathogenic Bacterial Biofilms.

Bacterial biofilms are major causes of persistent and recurrent infections and implant failures. Biofilms are formable by most clinically important pathogens worldwide, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, causing recalcitrance to standard antibiotic therapy or anti-biofilm strategies due to amphiphilic impermeable extracellular polymeric substances (EPS) and the presence of resistant and persistent bacteria within the biofilm matrix. Herein, we report our design of an oligoamidine-based amphiphilic "nano-sword" with high structural compacity and rigidity. Its rigid, amphiphilic structure ensures effective penetration into EPS, and the membrane-DNA dual-targeting mechanism exerts strong bactericidal effect on the dormant bacterial persisters within biofilms. The potency of this oligoamidine is shown in two distinct modes of application: it may be used as a coating agent for polycaprolactone to fully inhibit surface biofilm growth in an implant-site mimicking micro-environment; meanwhile, it cures model mice of biofilm infections in various ex vivo and in vivo studies.

Improving the Hemocompatibility of Antimicrobial Peptidomimetics through Amphiphilicity Masking Using a Secondary Amphiphilic Polymer.

Antimicrobial peptidomimetics (AMPMs) have received widespread attention as potentially powerful weapons against antibiotic resistance. However, AMPMs' membrane disruption mechanism not only brings resistance-resistant nature, but also nonspecific binding and disruption toward eukaryotic cell membranes, and consequently, their hemolytic activity is the primary concern on clinical applications. Here, the preparation and screening of an AMPM library is reported, through which a best-performing hit, PT-b1, can be obtained. To further improve PT-b1's hemocompatibility, a strategy is devised to mask the amphiphilicity of the AMPM using a charge-free, FDA-approved amphiphilic polymer, Pluronic F-127 (PF127). A PF127 solution containing PT-b1 can form a temperature-sensitive, absorbable hydrogel at higher concentration, but dissolve and complex with PT-b1 through hydrophobic interactions at lower concentration or lower temperature. The complexation from PF127 can mask the amphiphilicity of PT-b1 and render it extremely hemocompatible, yet the reversibility in such nanocomplexation and the existence of a secondary mechanism of action ensure that the AMPM's potency remains unchanged. The in vivo effectiveness of this antimicrobial hydrogel system is demonstrated using a mice wound infection model established with Methicillin-resistant Staphylococcus aureus, and observations indicate the hydrogel can promote wound healing and suppress bacteria-caused inflammation even when resistant pathogens are involved.

Revealing the Electrophilic-Attack Doping Mechanism for Efficient and Universal p-Doping of Organic Semiconductors.

Doping is of great importance to tailor the electrical properties of semiconductors. However, the present doping methodologies for organic semiconductors (OSCs) are either inefficient or can only apply to some OSCs conditionally, seriously limiting their general applications. Herein, a novel p-doping mechanism is revealed by investigating the interactions between the dopant trityl tetrakis(pentafluorophenyl) borate (TrTPFB) and poly(3-hexylthiophene) (P3HT). It is found that electrophilic attack of the trityl cations on thiophenes results in the formation of tritylated thiophenium ions, which subsequently induce electron transfer from neighboring P3HT chains to realize p-doping. This unique p-doping mechanism enables TrTPFB to p-dope various OSCs including those with high ionization energy (IE ≈ 5.8 eV). Moreover, this doping mechanism endows TrTPFB with strong doping capability, leading to doping efficiency of over 80% in P3HT. The discovery and elucidation of this novel doping mechanism not only points out that strong electrophiles are a class of efficient p-dopants for OSCs, but also provides new opportunities toward highly efficient doping of various OSCs.

Molecularly pure miktoarm spherical nucleic acids: preparation and usage as a scaffold for abiotic intracellular catalysis.

We present a fullerene-based strategy that allows the synthesis of molecularly pure miktoarm spherical nucleic acids (SNAs) with diverse structures, which, with post-functionalization, could serve as efficient scaffolds for intracellular catalysis. The SNA structure promotes cell permeability, nucleic acid stability, and catalytic efficiency, making the platform ideal for in cellulo reactions. Consequently, the tris(triazole)-bearing miktoarm SNA was able to effectively mediate intracellular copper-catalyzed alkyne-azide cycloaddition at nanomolar level of copper, and facilitate the same reaction in live zebrafish.

An alternatingly amphiphilic, resistance-resistant antimicrobial oligoguanidine with dual mechanisms of action.

The increasing number of infections caused by multi-drug resistance (MDR) bacteria is an omen of a new global challenge. As one of the countermeasures under development, antimicrobial peptides (AMPs) and AMP mimics have emerged as a new family of antimicrobial agents with high potential, due to their low resistance generation rate and effectiveness against MDR bacterial strains resulted from their membrane-disrupting mechanism of action. However, most reported AMPs and AMP mimics have facially amphiphilic structures, which may lead to undesired self-aggregation and non-specific binding, as well as increased cytotoxicity toward mammalian cells, all of which put significant limits on their applications. Here, we report an oligomer with the size of short AMPs, with both hydrophobic carbon chain and cationic groups placed on its backbone, giving an alternatingly amphiphilic structure that brings better selectivity between mammalian and bacterial cell membranes. In addition, the oligomer shows affinity toward DNA, thus it can utilize bacterial DNA located in the vulnerable nucleoid as the second drug target. Benefiting from these designs, the oligomer shows higher therapeutic index and synergistic effect with other antibiotics, while its low resistance generation rate and effectiveness on multi-drug resistant bacterial strains can be maintained. We demonstrate that this alternatingly amphiphilic, DNA-binding oligomer is not only resistance-resistant, but is also able to selectively eliminate bacteria at the presence of mammalian cells. Importantly, the oligomer exhibits good in vivo activity: it cleans all bacteria on Caenorhabditis elegans without causing apparent toxicity, and significantly improves the survival rate of mice with severely infected wounds in a mice excision wound model study.