Reversing Anticancer Drug Resistance by Synergistic Combination of Chemotherapeutics and Membranolytic Antitumor ß-Peptide Polymer.

Multidrug resistance is the main obstacle to cancer chemotherapy. Overexpression of drug efflux pumps causes excessive drug efflux from cancer cells, ultimately leading to drug resistance. Hereby, we raise an effective strategy to overcome multidrug resistance using a synergistic combination of membranolytic antitumor ß-peptide polymer and chemotherapy drugs. This membrane-active ß-peptide polymer promotes the transmembrane transport of chemotherapeutic drugs by increasing membrane permeability and enhances the activity of chemotherapy drugs against multidrug-resistant cancer cells. As a proof-of-concept demonstration, the synergistic combination of ß-peptide polymer and doxorubicin (DOX) is substantially more effective than DOX alone against drug-resistant cancer both in vitro and in vivo. Notably, the synergistic combination maintains a potent anticancer activity after continuous use. Collectively, this combination therapy using membrane lytic ß-peptide polymer appears to be an effective strategy to reverse anticancer drug resistance.

An Assay for Immunogenic Detection of Anti-PEG Antibody.

With the increasing use of polyethylene glycol (PEG) based proteins and drug delivery systems, anti-PEG antibodies have commonly been detected among the population, causing the accelerated blood clearance and hypersensitivity reactions, poses potential risks to the clinical efficacy and safety of PEGylated drugs. Therefore, vigilant monitoring of anti-PEG antibodies is crucial for both research and clinical guidance regarding PEGylated drugs. The enzyme-linked immunosorbent assay (ELISA) is a common method for detecting anti-PEG antibodies. However, diverse coating methods, blocking solutions and washing solutions have been employed across different studies, and unsuitable use of Tween 20 as the surfactant even caused biased results. In this study, we established the optimal substrate coating conditions, and investigated the influence of various surfactants and blocking solutions on the detection accuracy. The findings revealed that incorporating 1 % bovine serum albumin into the serum dilution in the absence of surfactants will result the credible outcomes of anti-PEG antibody detection.

Peptide-Mimicking Poly(2-oxazoline)s Possessing Potent Antifungal Activity and BBB Penetrating Property to Treat Invasive Infections and Meningitis.

Invasive fungal infections, including meningitis, cause a high mortality rate due to few available antifungal drugs and frequently associated side effects and quick emergence of drug-resistant fungi. The restrictive permeability of the blood-brain barrier (BBB) further limits the efficacy of antifungal agents substantially in treating meningitis. Hereby, we design and synthesize guanidinium-functionalized poly(2-oxazoline)s by mimicking cell-penetrating peptides. The optimal polymer, PGMeOx10 bearing a methylene spacer arm, displays potent activities against the drug-resistant fungi and biofilm, negligible toxicity, and insusceptibility to antimicrobial resistance. Moreover, PGMeOx10 can break BBB retractions to exert promising antifungal functions in the brain. PGMeOx10 demonstrates potent in vivo antifungal therapeutic efficacy in mouse models including skin infection, systemic infections, and meningitis. PGMeOx10 effectively rescues infected mice and reduces fungal burden and inflammation in the brain. These results and the excellent biosafety of poly(2-oxazoline)s indicate the effectiveness and potential of our strategy to design promising antifungal agents in treating systemic infections and meningitis.

Universal and One-Step Modification to Render Diverse Materials Bioactivation.

Bioactive materials that can support cell adhesion and tissue regeneration are greatly in demand in clinical applications. Surface modification with bioactive molecules is an efficient strategy to convert conventional bioinert materials into bioactive materials. However, there is an urgent need to find a universal and one-step modification strategy to realize the above transformation for bioactivation. In this work, we report a universal and one-step modification strategy to easily modify and render diverse materials bioactivation by dipping materials into the solution of dibutylamine-DOPA-lysine-DOPA (DbaYKY) tripeptide-terminated cell-adhesive molecules, ß-peptide polymer, or RGD peptide for only 5 min. This strategy provides materials with a stable surface modification layer and does not cause an undesired surface color change like the widely used polydopamine coating. This one-step strategy can endow material surfaces with cell adhesion properties without concerns on nonspecific conjugation of proteins and macromolecules. This universal and one-step surface bioactivation strategy implies a wide range of applications in implantable biomaterials.

Advance in the Polymerization Strategy for the Synthesis of ß-Peptides and ß-Peptoids.

Peptide mimics, possessing excellent biocompatibility and protease stability, have attracted broad attention and research in the biomedical field. ß-Peptides and ß-peptoids, as two types of vital peptide mimics, have demonstrated great potential in the field of foldamers, antimicrobials and protein binding, etc. Currently, the main synthetic strategies for ß-peptides and ß-peptoids include solid-phase synthesis and polymerization. Among them, polymerization in one-pot can minimize the repeated separation and purification used in solid-phase synthesis, and has the advantages of high efficiency and low cost, and can synthesize ß-peptides and ß-peptoids with high molecular weight. This review summarizes the polymerization methods for ß-peptides and ß-peptoids. Moreover, future developments of the polymerization method for the synthesis of ß-peptides and ß-peptoids will be discussed.

Secondary Amine Pendant ß-Peptide Polymers Displaying Potent Antibacterial Activity and Promising Therapeutic Potential in Treating MRSA-Induced Wound Infections and Keratitis.

Interest in developing antibacterial polymers as synthetic mimics of host defense peptides (HPDs) has accelerated in recent years to combat antibiotic-resistant bacterial infections. Positively charged moieties are critical in defining the antibacterial activity and eukaryotic toxicity of HDP mimics. Most examples have utilized primary amines or guanidines as the source of positively charged moieties, inspired by the lysine and arginine residues in HDPs. Here, we explore the impact of amine group variation (primary, secondary, or tertiary amine) on the antibacterial performance of HDP-mimicking ß-peptide polymers. Our studies show that a secondary ammonium is superior to either a primary ammonium or a tertiary ammonium as the cationic moiety in antibacterial ß-peptide polymers. The optimal polymer, a homopolymer bearing secondary amino groups, displays potent antibacterial activity and the highest selectivity (low hemolysis and cytotoxicity). The optimal polymer displays potent activity against antibiotic-resistant bacteria and high therapeutic efficacy in treating MRSA-induced wound infections and keratitis as well as low acute dermal toxicity and low corneal epithelial cytotoxicity. This work suggests that secondary amines may be broadly useful in the design of antibacterial polymers.

Heterochiral ß-Peptide Polymers Combating Multidrug-Resistant Cancers Effectively without Inducing Drug Resistance.

Multidrug resistance to chemotherapeutic drugs is one of the major causes for the failure of cancer treatment. Therefore, there is an urgent need to develop anticancer agents that can combat multidrug-resistant cancers effectively and mitigate drug resistance. Here, we report a rational design of anticancer heterochiral ß-peptide polymers as synthetic mimics of host defense peptides to combat multidrug-resistant cancers. The optimal polymer shows potent and broad-spectrum anticancer activities against multidrug-resistant cancer cells and is insusceptible to anticancer drug resistance owing to its membrane-damaging mechanism. The in vivo study indicates that the optimal polymer efficiently inhibits the growth and distant transfer of solid tumors and the metastasis and seeding of circulating tumor cells. Moreover, the polymer shows excellent biocompatibility during anticancer treatment on animals. In addition, the ß-peptide polymers address those prominent shortcomings of anticancer peptides and have superior stability against proteolysis, easy synthesis in large scale, and low cost. Collectively, the structural diversity and superior anticancer performance of ß-peptide polymers imply an effective strategy in designing and finding anticancer agents to combat multidrug-resistant cancers effectively while mitigating drug resistance.

Two Reference-Quality Sea Snake Genomes Reveal Their Divergent Evolution of Adaptive Traits and Venom Systems.

True sea snakes (Hydrophiini) are among the last and most successful clades of vertebrates that show secondary marine adaptation, exhibiting diverse phenotypic traits and lethal venom systems. To better understand their evolution, we generated the first chromosome-level genomes of two representative Hydrophiini snakes, Hydrophis cyanocinctus and H. curtus. Through comparative genomics we identified a great expansion of the underwater olfaction-related V2R gene family, consisting of more than 1,000 copies in both snakes. A series of chromosome rearrangements and genomic structural variations were recognized, including large inversions longer than 30 megabase (Mb) on sex chromosomes which potentially affect key functional genes associated with differentiated phenotypes between the two species. By integrating multiomics we found a significant loss of the major weapon for elapid predation, three-finger toxin genes, which displayed a dosage effect in H. curtus. These genetic changes may imply mechanisms that drove the divergent evolution of adaptive traits including prey preferences between the two closely related snakes. Our reference-quality sea snake genomes also enrich the repositories for addressing important issues on the evolution of marine tetrapods, and provide a resource for discovering marine-derived biological products.

Quaternized Polysaccharide-Based Cationic Micelles as a Macromolecular Approach to Eradicate Multidrug-Resistant Bacterial Infections while Mitigating Antimicrobial Resistance.

Microbial infections and microbial resistance lead to a high demand for new antimicrobial agents. Quaternized polysaccharides are cationic antimicrobial candidates; however, the limitation of homogeneous synthesis solvents that affect the molecular structure and biological activities, as well as their drug resistance remains unclear. Therefore, the authors homogeneously synthesize a series of quaternized chitin (QC) and quaternized chitosan (QCS) derivatives via a green and effective KOH/urea system and investigate their structure-activity relationship and biological activity in vivo and in vitro. Their study reveals that a proper match of degree of quaternization (DQ) and degree of deacetylation (DD') of QC or QCS is key to balance antimicrobial property and cytotoxicity. They identify QCS-2 as the optimized antimicrobial agent with a DQ of 0.46 and DD' of 82%, which exhibits effective broad-spectrum antimicrobial properties, good hemocompatibility, excellent cytocompatibility, and effective inhibition of bacterial biofilm formation and eradication of mature bacterial biofilms. Moreover, QCS-2 exhibits a low propensity for development of drug resistance and significant anti-infective effects on MRSA in vivo comparable to that of vancomycin, avoiding excessive inflammation and promoting the formation of new blood vessels, hair follicles, and collagen deposition to thus expedite wound healing.

Advance and Designing Strategies in Polymeric Antifungal Agents Inspired by Membrane-Active Peptides.

The high-mortality invasive fungal infections seriously threaten the lives of immunocompromised people. Host defense peptides and cell-penetrating peptides are representative membrane-active peptides with different functions. Among them, host defense peptides mimicking is a valid strategy in the design of synthetic antifungal agents. Despite the brilliance in the field of intracellular delivery, the potential of cell-penetrating peptides and their mimics for designing antifungal agents has been overlooked. In this concept article, we describe the structural design of synthetic antifungal polymers as mimics of host defense peptides, and highlight the effectiveness and potential of cell-penetrating peptide-inspired strategy in designing potent and selective antifungal polymeric agents. In addition, an outlook for further expanding the design horizons of antifungal polymers is also presented.

Interplay of Fusion, Leakage, and Electrostatic Lipid Clustering: Membrane Perturbations by a Hydrophobic Antimicrobial Polycation.

Membrane active compounds are able to induce various types of membrane perturbations. Natural or biomimetic candidates for antimicrobial treatment or drug delivery scenarios are mostly designed and tested for their ability to induce membrane permeabilization, also termed leakage. Furthermore, the interaction of these usually cationic amphiphiles with negatively charged vesicles often causes colloidal instability leading to vesicle aggregation or/and vesicle fusion. We show the interplay of these modes of membrane perturbation in mixed phosphatidyl glycerol (PG)/phosphatidyl ethanolamine (PE) by the statistical copolymer MM:CO comprising, both, charged and hydrophobic subunits. MM:CO is a representative of partially hydrophobic, highly active, but less selective antimicrobial polycations. Cryo-electron microscopy indicates vesicle fusion rather than vesicle aggregation upon the addition of MM:CO to negatively charged PG/PE (1:1) vesicles. In a combination of fluorescence-based leakage and fusion assays, there is support for membrane permeabilization and pronounced vesicle fusion activity as distinct effects. To this end, membrane fusion and aggregation were prevented by including lipids with polyethylene glycol attached to their head groups (PEG-lipids). The leakage activity of MM:CO is very similar in the absence and presence of PEG-lipids. Vesicle aggregation and fusion however are largely suppressed. This strongly suggests that MM:CO induces leakage by asymmetric packing stress because of hydrophobically driven interactions which could lead to leakage. As a further membrane perturbation effect, MM:CO causes lipid clustering in model vesicles. We address potential artifacts and misinterpretations of experiments characterizing leakage and fusion. Additional to the leakage activity, the pronounced fusogenic activity of the polymer and potentially of many other similar compounds likely has implications for antimicrobial activity and beyond.

Advance in Hybrid Peptides Synthesis.

Hybrid peptides with heterogeneous backbone are a class of peptide mimics with adjustable proteolytic stability obtained from incorporating unnatural amino acid residues into peptide backbone. α/ß-peptides and peptide/peptoid hybrids are two types of hybrid peptides that are widely studied for diverse applications, and several synthetic methods have been developed. In this mini review, the advance in hybrid peptide synthesis is summarized, including solution-phase method, solid-phase method, and novel polymerization method. Conventional solution-phase method and solid-phase method generally result in oligomers with defined sequences, while polymerization methods have advantages in preparing peptide hybrid polymers with high molecular weight with simple operation and low cost. In addition, the future development of polymerization method to realize the control of the peptide hybrid polymer sequence is discussed.

Design, Synthesis and Antifungal Evaluation of Novel Pyrylium Salt In Vitro and In Vivo.

Nowadays, discovering new skeleton antifungal drugs is the direct way to address clinical fungal infections. Pyrylium salt SM21 was screened from a library containing 50,240 small molecules. Several studies about the antifungal activity and mechanism of SM21 have been reported, but the structure-activity relationship of pyrylium salts was not clear. To explore the chemical space of antifungal pyrylium salt SM21, a series of pyrylium salt derivatives were designed and synthesized. Their antifungal activity and structure-activity relationships (SAR) were investigated. Compared with SM21, most of the synthesized compounds exhibited equivalent or improved antifungal activities against Candida albicans in vitro. The synthesized compounds, such as XY10, XY13, XY14, XY16 and XY17 exhibited comparable antifungal activities against C. albicans with MIC values ranging from 0.47 to 1.0 µM. Fortunately, a compound numbered XY12 showed stronger antifungal activities and lower cytotoxicity was obtained. The MIC of compound XY12 against C. albicans was 0.24 µM, and the cytotoxicity decreased 20-fold as compared to SM21. In addition, XY12 was effective against fluconazole-resistant C. albicans and other pathogenic Candida species. More importantly, XY12 could significantly increase the survival rate of mice with a systemic C. albicans infection, which suggested the good antifungal activities of XY12 in vitro and in vivo. Our results indicated that structural modification of pyrylium salts could lead to the discovery of new antifungal drugs.

Short Guanidinium-Functionalized Poly(2-oxazoline)s Displaying Potent Therapeutic Efficacy on Drug-Resistant Fungal Infections.

New antifungals are urgently needed to combat invasive fungal infections, due to limited types of available antifungal drugs and frequently encountered side effects, as well as the quick emergence of drug-resistance. We previously developed amine-pendent poly(2-oxazoline)s (POXs) as synthetic mimics of host defense peptides (HDPs) to have antibacterial properties, but with poor antifungal activity. Hereby, we report the finding of short guanidinium-pendent POXs, inspired by cell-penetrating peptides, as synthetic mimics of HDPs to display potent antifungal activity, superior mammalian cells versus fungi selectivity, and strong therapeutic efficacy in treating local and systemic fungal infections. Moreover, the unique antifungal mechanism of fungal cell membrane penetration and organelle disruption explains the insusceptibility of POXs to antifungal resistance. The easy synthesis and structural diversity of POXs imply their potential as a class of promising antifungal agents.

Recombinant expression, purification and characterization of human soluble tumor necrosis factor receptor 2.

TNFR2 is aberrantly expressed on various cancer cells and highly immunosuppressive regulatory T cells (Tregs) accumulated in tumor microenvironment. As an oncoprotein and a stimulator of the immune checkpoint Tregs that promote cancer cell survival and tumor growth, TNFR2 is considered to be a prospective target for cancer immunotherapy with the blockers developed to simultaneously inhibit abundant TNFR2+ tumor-associated Tregs and directly kill TNFR2-expressing tumors. The soluble ectodomain of TNFR2 has also been successfully applied in clinical treatment for TNF-related autoimmune diseases. Research practices on these therapeutic strategies need recombinant protein of human soluble TNFR2 (hsTNFR2); however, mass production of such biologics using eukaryotic cells is generally high-cost in culture materials and growth conditions. This study aimed to establish an efficient methodology to prepare bioactive hsTNFR2 through a prokaryotic expression system. Recombinant vector pMCSG7-hsTNFR2 was constructed and the His-tagged fusion protein expressed in E. coli was enriched in inclusion bodies. Recombinant hsTNFR2 was denatured, refolded, and then purified by affinity chromatography, tag removal, ion-exchange chromatography and gel filtration chromatography. A protein yield of 8.4 mg per liter of bacterial culture liquid with a purity of over 97% was obtained. Purified hsTNFR2 exhibited strong affinity to human TNF-α with a KD of 10.5 nM, and inhibited TNF-α-induced cytotoxicity in L929 cells with an EC50 of 0.57 µg/ml. The biological activity assessed in vitro indicated that this soluble protein can be promisingly used in drug discovery for immunotherapy of TNFR2+ cancers and treatment of autoimmune diseases featured by TNF-α overload.

Hidden complexity in membrane permeabilization behavior of antimicrobial polycations.

A promising alternative to classical antibiotics are antimicrobial peptides and their synthetic mimics (smAMPs) that supposedly act directly on membranes. For a more successful design of smAMPs, we need to know how the type of interaction with the membrane determines the type of membrane perturbation. How this, in turn, transfers into selectivity and microbial killing activity is largely unknown. Here, we characterize the action of two smAMPs: MM:CO (a copolymer of hydrophobic cyclooctyl subunits and charged ß-monomethyl-α-aminomethyl subunits) and the highly charged poly-NM (a homopolymer of α-aminomethyl subunits). By thorough characterization of vesicle leakage experiments, we elucidate complex membrane perturbation behavior in zwitterionic or negatively charged vesicles. Vesicle leakage data does not entirely agree with the growth inhibition of microbes. Our ensemble of advanced membrane permeabilization approaches clarifies these discrepancies. Long cumulative leakage kinetics show that the two smAMPs act either by transient leakage or by rare stochastic leakage events that occur at charge neutralization in the sample. We determine the strengths of individual leakage events induced by the smAMPs in membranes of various compositions. These strengths indicate changes in leakage mechanism over time and concentration range. Thus, our sophisticated analysis of vesicle leakage experiments reveals a fine-tuned flexibility in membrane permeabilization mechanisms. These details are indispensable in judging and designing membrane-active compounds.

Superfast and Water-Insensitive Polymerization on α-Amino Acid N-Carboxyanhydrides to Prepare Polypeptides Using Tetraalkylammonium Carboxylate as the Initiator.

We design the tetraalkylammonium carboxylate-initiated superfast polymerization on α-amino acid N-carboxyanhydrides (NCA) for efficient synthesis of polypeptides. Carboxylates, as a new class of initiator for NCA polymerization, can initiate the superfast NCA polymerization without the need of extra catalysts and the polymerization can be operated in open vessels at ambient condition without the use of glove box. Tetraalkylammonium carboxylate-initiated polymerization on NCA easily affords block copolymers with at least 15 blocks. Moreover, this method avoids tedious purification steps and enables direct polymerization on crude NCAs in aqueous environments to prepare polypeptides and one-pot synthesis of polypeptide nanoparticles. These advantages and the mild polymerization condition of tetraalkylammonium carboxylate-initiated NCA polymerization imply its great potential in functional exploration and application of polypeptides.

Silk-Inspired ß-Peptide Materials Resist Fouling and the Foreign-Body Response.

The functions of implants like medical devices are often compromised by the host's foreign-body response (FBR). Herein, we report the development of low-FBR materials inspired by serine-rich sericin from silk. Poly-ß-homoserine (ß-HS) materials consist of the hydrophilic unnatural amino acid ß-homoserine. Self-assembled monolayers (SAMs) of ß-HS resist adsorption by diverse proteins, as well as adhesion by cells, platelets, and diverse microbes. Experiments lasting up to 3 months revealed that, while implantation with control PEG hydrogels induced obvious inflammatory responses, collagen encapsulation, and macrophage accumulation, these responses were minimal with ß-HS hydrogels. Strikingly, the ß-HS hydrogels induce angiogenesis in implant-adjacent tissues. Molecular dynamics simulations indicated that the low FBR performance of ß-HS results from what we term "dual hydrogen bonding hydration", wherein both the backbone amide groups and the sidechain hydroxyl groups of ß-HS undergo hydration.

Poly(2-Oxazoline)-Based Functional Peptide Mimics: Eradicating MRSA Infections and Persisters while Alleviating Antimicrobial Resistance.

Peptides have important biological functions. However, their susceptibility to proteolysis limits their applications. We demonstrated here for the first time, that poly(2-oxazoline) (POX) can work as a functional mimic of peptides. POX-based glycine pseudopeptides, a host defense peptide mimic, had potent activities against methicillin-resistant S. aureus, which causes formidable infections. The POX mimic showed potent activity against persisters that are highly resistant to antibiotics. S. aureus did not develop resistance to POX owning to the reactive oxygen species related antimicrobial mechanism. POX-treated S. aureus is sensitive to common antibiotics, demonstrating no observable antimicrobial pressure or cross-resistance in using antimicrobial POX. This study highlights POX as a new type of functional mimic of peptides and opens new avenues in designing and exploring peptide mimetics for biological functions and applications.

Water-Insensitive Synthesis of Poly-ß-Peptides with Defined Architecture.

Biocompatible and proteolysis-resistant poly-ß-peptides have broad applications and are dominantly synthesized via the harsh and water-sensitive ring-opening polymerization of ß-lactams in a glovebox or using a Schlenk line, catalyzed by the strong base LiN(SiMe3 )2 . We have developed a controllable and water-insensitive ring-opening polymerization of ß-amino acid N-thiocarboxyanhydrides (ß-NTAs) that can be operated in open vessels to prepare poly-ß-peptides in high yields, with diverse functional groups, variable chain length, narrow dispersity and defined architecture. These merits imply wide applications of ß-NTA polymerization and resulting poly-ß-peptides, which is validated by the finding of a HDP-mimicking poly-ß-peptide with potent antimicrobial activities. The living ß-NTA polymerization enables the controllable synthesis of random, block copolymers and easy tuning of both terminal groups of polypeptides, which facilitated the unravelling of the antibacterial mechanism using the fluorophore-labelled poly-ß-peptide.