Single-Cell Oral Delivery Platform for Enhanced Acid Resistance and Intestinal Adhesion.

Oral delivery of cells, such as probiotics and vaccines, has proved to be inefficient since cells are generally damaged in an acidic stomach prior to arrival at the intestine to exert their health benefits. In addition, short retention in the intestine is another obstacle which affects inefficiency. To overcome these obstacles, a cell-in-shell structure was designed with pH-responsive and mucoadhesive properties. The pH-responsive shell consisting of three cationic layers of chitosan and three anionic layers of trans-cinnamic acid (t-CA) was made via layer-by-layer (LbL) assembly. t-CA layers are hydrophobic and impermeable to protons in acid, thus enhancing cell gastric resistance in the stomach, while chitosan layers endow strong interaction between the cell surface and the mucosal wall which facilitates cell mucoadhesion in the intestine. Two model cells, probiotic L. rhamnosus GG and dead Streptococcus iniae, which serve as inactivated whole-cell vaccine were chosen to test the design. Increased survival and retention during oral administration were observed for coated cells as compared with naked cells. Partial removal of the coating (20-60% removal) after acid treatment indicates that the coated vaccine can expose its surface immunogenic protein after passage through the stomach, thus facilitating vaccine immune stimulation in the intestine. As a smart oral delivery platform, this design can be extended to various macromolecules, thus providing a promising strategy to formulate oral macromolecules in the prevention and treatment of diseases at a cellular level.

Nontoxic, Biodegradable Hyperbranched Poly(ß-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(ß-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Hydrogel dressings with intrinsic antibiofilm and antioxidative dual functionalities accelerate infected diabetic wound healing.

Chronic wounds are often infected with biofilm bacteria and characterized by high oxidative stress. Current dressings that promote chronic wound healing either require additional processes such as photothermal irradiation or leave behind gross amounts of undesirable residues. We report a dual-functionality hydrogel dressing with intrinsic antibiofilm and antioxidative properties that are synergistic and low-leaching. The hydrogel is a crosslinked network with tethered antibacterial cationic polyimidazolium and antioxidative N-acetylcysteine. In a murine diabetic wound model, the hydrogel accelerates the closure of wounds infected with methicillin-resistant Staphylococcus aureus or carbapenem-resistant Pseudomonas aeruginosa biofilm. Furthermore, a three-dimensional ex vivo human skin equivalent model shows that N-acetylcysteine promotes the keratinocyte differentiation and accelerates the re-epithelialization process. Our hydrogel dressing can be made into different formats for the healing of both flat and deep infected chronic wounds without contamination of the wound or needing other modalities such as photothermal irradiation.

A hydrophilic polyimidazolium antibiotic targeting the membranes of Gram-negative bacteria.

OBJECTIVES: The rise of MDR Gram-negative bacteria (GNB), especially those resistant to last-resort drugs such as carbapenems and colistin, is a global health risk and calls for increased efforts to discover new antimicrobial compounds. We previously reported that polyimidazolium (PIM) compounds exhibited significant antimicrobial activity and minimal mammalian cytotoxicity. However, their mechanism of action is relatively unknown. We examined the efficacy and mechanism of action of a hydrophilic PIM (PIM5) against colistin- and meropenem-resistant clinical isolates. METHODS: MIC and time-kill testing was performed for drug-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. N-phenyl-1-naphthylamine and propidium iodide dyes were employed to determine membrane permeabilization. Spontaneous resistant mutants and single deletion mutants were generated to understand potential resistance mechanisms to the drug. RESULTS: PIM5 had the same effectiveness against colistin- and meropenem-resistant strains as susceptible strains of GNB. PIM5 exhibited a rapid bactericidal effect independent of bacterial growth phase and was especially effective in water. The polymer disrupts both the outer and cytoplasmic membranes. PIM5 binds and intercalates into bacterial genomic DNA upon entry of cells. GNB do not develop high resistance to PIM5. However, the susceptibility and uptake of the polymer is moderately affected by mutations in the two-component histidine kinase sensor BaeS. PIM5 has negligible cytotoxicity on human cells at bacterial-killing concentrations, comparable to the commercial antibiotics polymyxin B and colistin. CONCLUSIONS: PIM5 is a potent broad-spectrum antibiotic targeting GNB resistant to last-resort antibiotics.

Cationic Carbon Monoxide-Releasing Polymers as Antimicrobial and Antibiofilm Agents by the Synergetic Activity.

Recent studies indicate that carbon monoxide-releasing molecules (CORMs), a class of organometallic compounds, exert antibacterial activities through the delivery of carbon monoxide (CO) molecules. We developed a new-class CO-delivery system by conjugating classical low-molecular-weight CORMs (i.e., [Ru(CO)3Cl2]2 and Mn(CO)5Br) onto a positively charged carrier, polyimidazolium (PIM), giving cationic CO-releasing polymers Ru@PIM and Mn@PIM, respectively. Compared with low-molecular-weight CORMs, our polymeric CO vehicles showed improved water solubility, reduced cytotoxicity, significantly extended CO-releasing duration, and enhanced antimicrobial ability against both planktonic and biofilm microorganisms. Ru@PIM and Mn@PIM inhibited the growth of a broad spectrum of free Gram-positive and Gram-negative bacteria as well as fungus with the lowest minimum inhibitory concentration (MIC) at 8 µg/mL. They were effective in preventing pathogenic Pseudomonas aeruginosa biofilm formation with biofilm reduction by more than 92% at 16 µg/mL and 99% at 32 µg/mL. They also demonstrated potent dispersal efficacy on recalcitrant well-established biofilms through a synergetic activity with a biofilm log10 reduction of 2.5-3.2 ≥ 64 µg/mL and nearly 2.0 at the concentration of as low as 16 µg/mL. This CO-releasing system may retain long-time antimicrobial ability after the complete release of CO molecules owing to the cationic structure. The novel CO-releasing polymers have great potential as antimicrobial and antibiofilm agents in biomedical applications.

Membrane Potential-Dependent Uptake of Cationic Oligoimidazolium Mediates Bacterial DNA Damage and Death.

The treatment of bacterial infections is becoming increasingly challenging with the emergence of antimicrobial resistance. Thus, the development of antimicrobials with novel mechanisms of action is much needed. Previously, we designed several cationic main-chain imidazolium compounds and identified the polyimidazolium PIM1 as a potent antibacterial against a wide panel of multidrug-resistant nosocomial pathogens, and it had relatively low toxicity against mammalian epithelial cells. However, little is known about the mechanism of action of PIM1. Using an oligomeric version of PIM1 with precisely six repeating units (OIM1-6) to control for consistency, we showed that OIM1-6 relies on an intact membrane potential for entry into the bacterial cytoplasm, as resistant mutants to OIM1-6 have mutations in their electron transport chains. These mutants demonstrate reduced uptake of the compound, which can be circumvented through the addition of a sub-MIC dose of colistin. Once taken up intracellularly, OIM1-6 exerts double-stranded DNA breaks. Its potency and ability to kill represents a promising class of drugs that can be combined with membrane-penetrating drugs to potentiate activity and hedge against the rise of resistant mutants. In summary, we discovered that cationic antimicrobial OIM1-6 exhibits an antimicrobial property that is dissimilar to the conventional cationic antimicrobial compounds. Its killing mechanism does not involve membrane disruption but instead depends on the membrane potential for uptake into bacterial cells so that it can exert its antibacterial effect intracellularly.

Correction: Polymers as advanced antibacterial and antibiofilm agents for direct and combination therapies.

[This corrects the article DOI: 10.1039/D1SC05835E.].

Chemical Basis of Combination Therapy to Combat Antibiotic Resistance.

The antimicrobial resistance crisis is a global health issue requiring discovery and development of novel therapeutics. However, conventional screening of natural products or synthetic chemical libraries is uncertain. Combination therapy using approved antibiotics with inhibitors targeting innate resistance mechanisms provides an alternative strategy to develop potent therapeutics. This review discusses the chemical structures of effective ß-lactamase inhibitors, outer membrane permeabilizers, and efflux pump inhibitors that act as adjuvant molecules of classical antibiotics. Rational design of the chemical structures of adjuvants will provide methods to impart or restore efficacy to classical antibiotics for inherently antibiotic-resistant bacteria. As many bacteria have multiple resistance pathways, adjuvant molecules simultaneously targeting multiple pathways are promising approaches to combat multidrug-resistant bacterial infections.

Designer co-beta-peptide copolymer selectively targets resistant and biofilm Gram-negative bacteria.

New antimicrobials are urgently needed to combat Gram-negative bacteria, particularly multi-drug resistant (MDR) and phenotypically resistant biofilm species. At present, only sequence-defined alpha-peptides (e.g. polymyxin B) can selectively target Gram-negative bacterial lipopolysaccharides. We show that a copolymer, without a defined sequence, shows good potency against MDR Gram-negative bacteria including its biofilm form. The tapered blocky co-beta-peptide with controlled N-terminal hydrophobicity (#4) has strong interaction with the Gram-negative bacterial lipopolysaccharides via its backbone through electrostatic and hydrogen bonding interactions but not the Gram-positive bacterial and mammalian cell membranes so that this copolymer is non-toxic to these two latter cell types. The new #4 co-beta-peptide selectively kills Gram-negative bacteria with low cytotoxicity both in vitro and in a mouse biofilm wound infection model. This strategy provides a new concept for the design of Gram-negative selective antimicrobial peptidomimetics against MDR and biofilm species.

Polyimidazolium Protects against an Invasive Clinical Isolate of Salmonella Typhimurium.

Frequent outbreaks of Salmonella Typhimurium infection, in both animal and human populations and with the potential for zoonotic transmission, pose a significant threat to the public health sector. The rapid emergence and spread of more invasive multidrug-resistant clinical isolates of Salmonella further highlight the need for the development of new drugs with effective broad-spectrum bactericidal activities. The synthesis and evaluation of main-chain cationic polyimidazolium 1 (PIM1) against several Gram-positive and Gram-negative bacteria have previously demonstrated the efficacy profile of PIM1. The present study focuses on the antibacterial and anti-biofilm activities of PIM1 against Salmonella in both in vitro and in ovo settings. In vitro, PIM1 exhibited bactericidal activity against three strains of Salmonella at a low dosage of 8 µg/mL. The anti-biofilm activity of PIM1 was evident by its elimination of planktonic cells within preformed biofilms in a dose-dependent manner. During the host cell infection process, PIM1 reduces the extracellular bacterial load, which reduces adhesion and invasion to limit the establishment of infection. Once intracellular, Salmonella strains were tolerant and protected from PIM1 treatment. In a chicken egg infection model, PIM1 exhibited therapeutic activity for both Salmonella strains, using stationary-phase and exponential-phase inocula. Moreover, PIM1 showed a remarkable efficacy against the stationary-phase inocula of drug-resistant Salmonella by eliminating the bacterial burden in >50% of the infected chicken egg embryos. Collectively, our results highlight the potential for PIM1 as a replacement therapy for existing antibiotic applications on the poultry farm, given the efficiency and low toxicity profile demonstrated in our agriculturally relevant chicken embryo model.

Smart Multifunctional Polymer Systems as Alternatives or Supplements of Antibiotics To Overcome Bacterial Resistance.

In recent years, infectious diseases have again become a critical threat to global public health largely due to the challenges posed by antimicrobial resistance. Conventional antibiotics have played a crucial role in combating bacterial infections; however, their efficacy is significantly impaired by widespread drug resistance. Natural antimicrobial peptides (AMPs) and their polymeric mimics demonstrate great potential for killing bacteria with low propensity of resistance as they target the microbial membrane rather than a specific molecular target, but they are also toxic to the host eukaryotic cells. To minimize antibiotics systemic spread and the required dose that promote resistance and to advocate practical realization of the promising activity of AMPs and polymers, smart systems to target bacteria are highly sought after. This review presents bacterial recognition by various specific targeting molecules and the delivery systems of active components in supramolecules. Bacteria-induced activations of antimicrobial-based nanoformulations are also included. Recent advances in the bacteria targeting and delivery of synthetic antimicrobial agents may assist in developing new classes of highly selective antimicrobial systems which can improve bactericidal efficacy and greatly minimize the spread of bacterial resistance.

Caging Cationic Polymer Brush-Coated Plasmonic Nanostructures for Traceable Selective Antimicrobial Activities.

Cationic polymers are under intense research to achieve prominent antimicrobial activity. However, the cellular and in vivo toxicity caused by nonspecific electrostatic interaction has become a major challenge for their practical applications. Here, the development of a "caging" strategy based on the use of a block copolymer consisting of a stealth block and an anionic block that undergoes degradation in presence of enzymes secreted by selective bacterial pathogens of interest is reported. The results have shown that antimicrobial cationic polymer brushes-coated gold nanorods (AuNRs) can be caged by the block polymer of poly(ethylene glycol) and anionic, lipase-degradable block of ε-caprolactone and methacrylic acid copolymer to afford neutrally charged surfaces. The caged AuNRs are activated by lipase released by bacteria of interest to endow an excellent bactericidal effect but show minimal binding and toxicity against mammalian cells and nonspecific bacteria that do not produce lipase. In this design, AuNRs play multifunctional roles as the scaffolds for polymer brushes, photothermal transducers, and imaging probes for traceable delivery of the activation and delivery of bactericidal cationic polymer brushes. The caging strategy opens new opportunities for the safe delivery of antimicrobial materials for the treatment of bacterial infections.

Polymers as advanced antibacterial and antibiofilm agents for direct and combination therapies.

The growing prevalence of antimicrobial drug resistance in pathogenic bacteria is a critical threat to global health. Conventional antibiotics still play a crucial role in treating bacterial infections, but the emergence and spread of antibiotic-resistant micro-organisms are rapidly eroding their usefulness. Cationic polymers, which target bacterial membranes, are thought to be the last frontier in antibacterial development. This class of molecules possesses several advantages including a low propensity for emergence of resistance and rapid bactericidal effect. This review surveys the structure-activity of advanced antimicrobial cationic polymers, including poly(α-amino acids), ß-peptides, polycarbonates, star polymers and main-chain cationic polymers, with low toxicity and high selectivity to potentially become useful for real applications. Their uses as potentiating adjuvants to overcome bacterial membrane-related resistance mechanisms and as antibiofilm agents are also covered. The review is intended to provide valuable information for design and development of cationic polymers as antimicrobial and antibiofilm agents for translational applications.

Enzyme- and Relative Humidity-Responsive Antimicrobial Fibers for Active Food Packaging.

Active food packaging materials that are sustainable, biodegradable, and capable of precise delivery of antimicrobial active ingredients (AIs) are in high demand. Here, we report the development of novel enzyme- and relative humidity (RH)-responsive antimicrobial fibers with an average diameter of 225 ± 50 nm, which can be deposited as a functional layer for packaging materials. Cellulose nanocrystals (CNCs), zein (protein), and starch were electrospun to form multistimuli-responsive fibers that incorporated a cocktail of both free nature-derived antimicrobials such as thyme oil, citric acid, and nisin and cyclodextrin-inclusion complexes (CD-ICs) of thyme oil, sorbic acid, and nisin. The multistimuli-responsive fibers were designed to release the free AIs and CD-ICs of AIs in response to enzyme and RH triggers, respectively. Enzyme-responsive release of free AIs is achieved due to the degradation of selected polymers, forming the backbone of the fibers. For instance, protease enzyme can degrade zein polymer, further accelerating the release of AIs from the fibers. Similarly, RH-responsive release is obtained due to the unique chemical nature of CD-ICs, enabling the release of AIs from the cavity at high RH. The successful synthesis of CD-ICs of AIs and incorporation of antimicrobials in the structure of the multistimuli-responsive fibers were confirmed by X-ray diffraction and Fourier transform infrared spectrometry. Fibers were capable of releasing free AIs when triggered by microorganism-exudated enzymes in a dose-dependent manner and releasing CD-IC form of AIs in response to high relative humidity (95% RH). With 24 h of exposure, stimuli-responsive fibers significantly reduced the populations of foodborne pathogenic bacterial surrogates Escherichia coli (by ∼5 log unit) and Listeria innocua (by ∼5 log unit), as well as fungi Aspergillus fumigatus (by >1 log unit). More importantly, the fibers released more AIs at 95% RH than at 50% RH, which resulted in a higher population reduction of E. coli at 95% RH. Such biodegradable, nontoxic, and multistimuli-responsive antimicrobial fibers have great potential for broad applications as active and smart packaging systems.

Nanosensor Detection of Synthetic Auxins In Planta using Corona Phase Molecular Recognition.

Synthetic auxins such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) have been extensively used in plant tissue cultures and as herbicides because they are chemically more stable and potent than most endogenous auxins. A tool for rapid in planta detection of these compounds will enhance our knowledge about hormone distribution and signaling and facilitate more efficient usage of synthetic auxins in agriculture. In this work, we show the development of real-time and nondestructive in planta NAA and 2,4-D nanosensors based on the concept of corona phase molecular recognition (CoPhMoRe), to replace the current state-of-the-art sensing methods that are destructive and laborious. By designing a library of cationic polymers wrapped around single-walled carbon nanotubes with general affinity for chemical moieties displayed on auxins and its derivatives, we developed selective sensors for these synthetic auxins, with a particularly large quenching response to NAA (46%) and a turn-on response to 2,4-D (51%). The NAA and 2,4-D nanosensors are demonstrated in planta across several plant species including spinach, Arabidopsis thaliana (A. thaliana), Brassica rapa subsp. chinensis (pak choi), and Oryza sativa (rice) grown in various media, including soil, hydroponic, and plant tissue culture media. After 5 h of 2,4-D supplementation to the hydroponic medium, 2,4-D is seen to accumulate in susceptible dicotyledon pak choi leaves, while no uptake is observed in tolerant monocotyledon rice leaves. As such, the 2,4-D nanosensor had demonstrated its capability for rapid testing of herbicide susceptibility and could help elucidate the mechanisms of 2,4-D transport and the basis for herbicide resistance in crops. The success of the CoPhMoRe technique for measuring these challenging plant hormones holds tremendous potential to advance the plant biology study.

Antibiofilm Activity of Gallium(III) Complexed Anionic Polymers in Combination with Antibiotics.

Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening pathogen associated with multiantibiotic resistance, which is largely caused by its strong ability to form biofilms. Recent research has revealed that gallium (III) shows an activity against the biofilm of P. aeruginosa by interfering with Fe metabolism. The antibacterial activity of the combination of Ga3+ ion and antibiotic rifampicin (RMP) against P. aeruginosa PAO1 is investigated. An anionic polymer poly{{2-[(2-methylprop-2-enoyl)oxy]ethyl}phosphonic acid} (PDMPOH) is exploited to form complexes (GaPD) with Ga3+ . The GaPD complexes act as a carrier of Ga3+ and release Ga3+ via enzymatic degradation by bacterial lipases. GaPD is found to damage the outer membrane, leading to enhanced cellular uptake of RMP and Ga3+ due to increased outer membrane permeability, which inhibits the RNA polymerase and interferes with Fe metabolism. The antibiofilm activity and biocompatibility of the GaPD system offer a promising treatment option for P. aeruginosa biofilm-related infections.

Cyanine-Dyad Molecular Probe for the Simultaneous Profiling of the Evolution of Multiple Radical Species During Bacterial Infections.

Real-time monitoring of the evolution of bacterial infection-associated multiple radical species is critical to accurately profile the pathogenesis and host-defense mechanisms. Here, we present a unique dual wavelength near-infrared (NIR) cyanine-dyad molecular probe (HCy5-Cy7) for simultaneous monitoring of reactive oxygen and nitrogen species (RONS) variations both in vitro and in vivo. HCy5-Cy7 specifically turns on its fluorescence at 660 nm via superoxide or hydroxyl radical (O2.- , . OH)-mediated oxidation of reduced HCy5 moiety to Cy5, while peroxynitrite or hypochlorous species (ONOO- , ClO- )-induced Cy7 structural degradation causes the emission turn-off at 800 nm. Such multispectral but reverse signal responses allow multiplex manifestation of in situ oxidative and nitrosative stress events during the pathogenic and defensive processes in both bacteria-infected macrophage cells and living mice. Most importantly, this study may also provide new perspectives for understanding the bacterial pathogenesis and advancing the precision medicine against infectious diseases.

DNA-derived nanostructures selectively capture gram-positive bacteria.

We report the first demonstration of the efficient bacteria targeting properties of DNA-based polymeric micelles with high-density DNA corona. Nanoscale polymer micelles derived from DNA-b-polystyrene (DNA-b-PS) efficiently selected most tested Gram-positive strains over Gram-negative strains; single-strand DNAs were 20-fold less selective. We demonstrate that these targeting properties were derived from the interaction between densely packed DNA strands of the micelle corona and the peptidoglycan layers of Gram-positive bacteria. DNA-b-PS micelles incorporating magnetic nanoparticles (MNPs) can efficiently capture and concentrate Gram-positive bacteria suggesting the simple applications of these DNA block copolymer micelles for concentrating bacteria. Adenine (A), thymine (T), cytosine (C), and guanine (G)-rich nanostructures were fabricated, respectively, for investigating the effect of sequence on Gram-selective bacteria targeting. T-rich micelles showed the most efficient targeting properties. The targeting properties of these DNA nanostructures toward Gram-positive bacteria may have applications as a targeted therapeutic delivery system.

Mixed-charge pseudo-zwitterionic copolymer brush as broad spectrum antibiofilm coating.

Zwitterionic polymers are classical antifouling polymers but they require specialized monomers that have cationic and anionic charges integrated into a single monomer. Herein, we show that pseudo-zwitterionic copolymers synthesized from a mixture of 2 monomers each having a single opposite polarity has excellent antibiofilm efficacy. We have discovered a new mixed-charge copolymer brush (#1-A) synthesized from 2 oppositely charged monomers, the anionic SPM (3-Sulfopropyl methacrylate) and the cationic AMPTMA ((3-Acrylamidopropyl) trimethylammonium chloride), that achieves broad spectrum in vitro antibiofilm effect of greater than 99% reductions against all six Gram-positive and Gram-negative bacteria tested. In the murine subcutaneous wound catheter infection models, the #1-A has good long-term anti-biofilm efficacy against MRSA and Pseudomonas aeruginosa of 3.41 and 3.19 orders respectively, outperforming previous mixed-charge copolymer coatings. We discovered a new method to choose the cationic/anionic pair combination to form the best antibiofilm copolymer brush coating by exploiting the solution polymerization kinetics disparity between the cationic and anionic monomers. We also showed that #1-A is softer and has higher hydration than the classical zwitterionic polymer. This study shows the possibility of achieving potent antibiofilm efficacy by combining readily available opposite singly charged monomers.

Smart nanomicelles with bacterial infection-responsive disassembly for selective antimicrobial applications.

New generation antimicrobial agents are expected to exhibit non-metabolic killing mechanisms, high killing potency and biocompatibility. We synthesized a cationic chitosan derivative and an anionic chitosan derivative - specifically an α-poly(l)lysine side-grafted chitosan (CS-PLL) and an anionic citraconyl anhydride (CA) modified polylysine side graft for chitosan (CS-PLL-CA). The ß-carboxylic amide of CS-PLL-CA is pH-labile and self-cleavable under pH 6 or below. When we mixed the cationic (CS-PLL) and anionic (CS-PLL-CA) peptidosaccharide copolymers, they self-assembled, due to electrostatic charge interactions, into nanomicelles (NMs) with the oppositely charged peptides in the core and the chitosan polysaccharide arms on the shell. The NMs exhibited high hemo- and cytocompatibility (nontoxic) at physiological pH of 7.4, due to the chitosan protection on the shell and charge neutralization on the core. Upon reaching the bacterial infection site, the chitosan shell interacted and accumulated around the bacteria. The bacterial infection sites in the body usually show localized acidity as a result of the combined actions of bacterial metabolism and host immune response, and the pH can decrease to as low as 5.5. At this low pH, the ß-carboxylic amide bond of the anionic polypeptide gradually hydrolyzed to expose the initial cationic amine moieties, causing the NMs to 'decompose' into individual CS-PLL and 'spill' the cationic molecules which then disrupted and killed the bacteria. This 'smart' bacteria-recognizing chitosan-decorated nanosystem opens the pathway to explore other anionic and cationic and biocompatible polymers for 'stealth' delivery of antimicrobial polypeptide, and 'on-demand' recovery of the cationic parts to kill bacteria at infection sites.