Lipopolysaccharide Neutralization by Cationic-Amphiphilic Polymers through Pseudoaggregate Formation.

Synthetic polymers incorporating the cationic charge and hydrophobicity to mimic the function of antimicrobial peptides (AMPs) have been developed. These cationic-amphiphilic polymers bind to bacterial membranes that generally contain negatively charged phospholipids and cause membrane disintegration resulting in cell death; however, cationic-amphiphilic antibacterial polymers with endotoxin neutralization properties, to the best of our knowledge, have not been reported. Bacterial endotoxins such as lipopolysaccharide (LPS) cause sepsis that is responsible for a great amount of mortality worldwide. These cationic-amphiphilic polymers can also bind to negatively charged and hydrophobic LPS and cause detoxification. Hence, we envisaged that cationic-amphiphilic polymers can have both antibacterial as well as LPS binding properties. Here we report synthetic amphiphilic polymers with both antibacterial as well as endotoxin neutralizing properties. Levels of proinflammatory cytokines in human monocytes caused by LPS stimulation were inhibited by >80% when coincubated with these polymers. These reductions were found to be dependent on concentration and, more importantly, on the side-chain chemical structure due to variations in the hydrophobicity profiles of these polymers. These cationic-amphiphilic polymers bind and cause LPS neutralization and detoxification. Investigations of polymer interaction with LPS using fluorescence spectroscopy and dynamic light scattering (DLS) showed that these polymers bind but neither dissociate nor promote LPS aggregation. We show that polymer binding to LPS leads to sort of a pseudoaggregate formation resulting in LPS neutralization/detoxification. These findings provide an unusual mechanism of LPS neutralization using novel synthetic cationic-amphiphilic polymers.

Side Chain Degradable Cationic-Amphiphilic Polymers with Tunable Hydrophobicity Show in Vivo Activity.

Cationic-amphiphilic antibacterial polymers with optimal amphiphilicity generally target the bacterial membranes instead of mammalian membranes. To date, this balance has been achieved by varying the cationic charge or side chain hydrophobicity in a variety of cationic-amphiphilic polymers. Optimal hydrophobicity of cationic-amphiphilic polymers has been considered as the governing factor for potent antibacterial activity yet minimal mammalian cell toxicity. However, the concomitant role of hydrogen bonding and hydrophobicity with constant cationic charge in the interactions of antibacterial polymers with bacterial membranes is not understood. Also, degradable polymers that result in nontoxic degradation byproducts offer promise as safe antibacterial agents. Here we show that amide- and ester (degradable)-bearing cationic-amphiphilic polymers with tunable side chain hydrophobicity can modulate antibacterial activity and cytotoxicity. Our results suggest that an amide polymer can be a potent antibacterial agent with lower hydrophobicity whereas the corresponding ester polymer needs a relatively higher hydrophobicity to be as effective as its amide counterpart. Our studies reveal that at higher hydrophobicities both amide and ester polymers have similar profiles of membrane-active antibacterial activity and mammalian cell toxicity. On the contrary, at lower hydrophobicities, amide and ester polymers are less cytotoxic, but the former have potent antibacterial and membrane activity compared to the latter. Incorporation of amide and ester moieties made these polymers side chain degradable, with amide polymers being more stable than the ester polymers. Further, the polymers are less toxic, and their degradation byproducts are nontoxic to mice. More importantly, the optimized amide polymer reduces the bacterial burden of burn wound infections in mice models. Our design introduces a new strategy of interplay between the hydrophobic and hydrogen bonding interactions keeping constant cationic charge density for developing potent membrane-active antibacterial polymers with minimal toxicity to mammalian cells.

Surviving sepsis in the era of antibiotic resistance: are there any alternative approaches to antibiotic therapy?

Sepsis, a serious cause of morbidity in humans, has no proper single medication dedicated to it. In this review, we look at the current treatment modalities, the different approaches attempted towards treating it and alternative approaches that could be implemented to counter this neglected disease condition. The use of antibiotics towards treatment of sepsis, use of combinations and strategies derived from natural antimicrobial peptides have been dealt in detail. The social and technical difficulties associated with treating sepsis and the possible ways of combating them have also been discussed.

Cleavable cationic antibacterial amphiphiles: synthesis, mechanism of action, and cytotoxicities.

The development of novel antimicrobial agents having high selectivity toward bacterial cells over mammalian cells is urgently required to curb the widespread emergence of infectious diseases caused by pathogenic bacteria. Toward this end, we have developed a set of cationic dimeric amphiphiles (bearing cleavable amide linkages between the headgroup and the hydrocarbon tail with different methylene spacers) that showed high antibacterial activity against human pathogenic bacteria (Escherichia coli and Staphylococcus aureus) and low cytotoxicity. The Minimum Inhibitory Concentrations (MIC) were found to be very low for the dimeric amphiphiles and were lower or comparable to the monomeric counterpart. In the case of dimeric amphiphiles, MIC was found to decrease with the increase in the spacer chain length (n = 2 to 6) and again to increase at higher spacer length (n > 6). It was found that the compound with six methylene spacers was the most active among all of the amphiphiles (MICs = 10-13 µM). By fluorescence spectroscopy, fluorescence microscopy, and field-emission scanning electron microscopy (FESEM), it was revealed that these cationic amphiphiles interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thus killing the bacteria. All of the cationic amphiphiles showed low hemolytic activity (HC(50)) and high selectivity against both gram-positive and gram-negative bacteria. The most active amphiphile (n = 6) had a 10-13-fold higher HC(50) than did the MIC. Also, this amphiphile did not show any cytotoxicity against mammalian cells (HeLa cells) even at a concentration above the MIC (20 µM). The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.30-0.11 mM) and were 10-27 times smaller than the corresponding monomeric analogue (CMC = 2.9 mM). Chemical hydrolysis and thermogravimetric analysis (TGA) proved that these amphiphiles are quite stable under both acidic and thermal conditions. Collectively, these properties make the newly synthesized amphiphiles potentially superior disinfectants and antiseptics for various biomedical and biotechnological applications.

Temporal release of a three-component protein subunit vaccine from polymer multilayers.

Sustained antigen and adjuvant availability have been shown to improve antiviral immune responses following vaccination. Transcutaneous delivery of vaccines using microneedles has also shown promise and may be particularly relevant for mosquito-borne viruses. We aim to combine these traits to create a three-component Protein Subunit vaccine on Microneedle Arrays (PSMNs) for transcutaneous delivery using layer-by-layer (LbL) assembly. Polymer multilayer thin films were generated to co-deliver a model combination of three chemically distinct vaccine components, a dengue virus Envelope protein Domain III (EDIII) subunit antigen and two adjuvants, a double-stranded RNA (Poly (inosinic:cytidylic acid) (PolyI:C)) and an amphiphilic hexapeptide, Pam3CSK4. Following application of PSMNs to the skin, implanted thin films facilitated sustained and temporal release of individual vaccine components from polymer multilayers. By modulating LbL composition and architecture, component release profiles in the skin could be independently tuned to allow release of adjuvants and antigen from days up to two weeks. Uptake of antigen and adjuvant from implanted vaccine films by antigen-presenting cells was demonstrated using in vivo mouse and ex vivo human skin models. Overall, we believe that such modular vaccine strategies offer design principles for enhancing the immunogenicity of protein subunit vaccines.

Microneedle-based intradermal delivery of stabilized dengue virus.

Current live-attenuated dengue vaccines require strict cold chain storage. Methods to preserve dengue virus (DENV) viability, which enable vaccines to be transported and administered at ambient temperatures, will be decisive towards the implementation of affordable global vaccination schemes with broad immunization coverage in resource-limited areas. We have developed a microneedle (MN)-based vaccine platform for the stabilization and intradermal delivery of live DENV from minimally invasive skin patches. Dengue virus-stabilized microneedle arrays (VSMN) were fabricated using saccharide-based formulation of virus and could be stored dry at ambient temperature up to 3 weeks with maintained virus viability. Following intradermal vaccination, VSMN-delivered DENV was shown to elicit strong neutralizing antibody responses and protection from viral challenge, comparable to that of the conventional liquid vaccine administered subcutaneously. This work supports the potential for MN-based dengue vaccine technology and the progression towards cold chain-independence. Dengue virus can be stabilized using saccharide-based formulations and coated on microneedle array vaccine patches for storage in dry state with preserved viability at ambient temperature (VSMN; virus-stabilized microneedle arrays).

Synthetic Charge-Invertible Polymer for Rapid and Complete Implantation of Layer-by-Layer Microneedle Drug Films for Enhanced Transdermal Vaccination.

The utility of layer-by-layer (LbL) coated microneedle (MN) skin patches for transdermal drug delivery has proven to be a promising approach, with advantages over hypodermal injection due to painless and easy self-administration. However, the long epidermal application time required for drug implantation by existing LbL MN strategies (15-90 min) can lead to potential medication noncompliance. Here, we developed a MN platform to shorten the application time in MN therapies based on a synthetic pH-induced charge-invertible polymer poly(2-(diisopropylamino) ethyl methacrylate- b-methacrylic acid) (PDM), requiring only 1 min skin insertion time to implant LbL films in vivo. Following MN-mediated delivery of 0.5 µg model antigen chicken ovalbumin (OVA) in the skin of mice, this system achieved sustained release over 3 days and led to an elevated immune response as demonstrated by significantly higher humoral immunity compared with OVA administration via conventional routes (subcutaneously and intramuscularly). Moreover, in an ex vivo experiment on human skin, we achieved efficient immune activation through MN-delivered LbL films, demonstrated by a rapid uptake of vaccine adjuvants by the antigen presenting cells. These features, rapid administration and the ability to elicit a robust immune response, can potentially enable a broad application of microneedle-based vaccination technologies.

l-Lysine based lipidated biphenyls as agents with anti-biofilm and anti-inflammatory properties that also inhibit intracellular bacteria.

l-Lysines were conjugated to lipidated biphenyls using simple synthetic chemistry to obtain selective membrane-active antibacterial agents that inhibit cell-wall biosynthesis. The most selective compound bore promising activity against biofilm-related infections and intracellular bacteria, and also suppressed the stimulation of TNF-α induced by lipoteichoic acid. Belligerent to resistance development, it was active in a murine model of MRSA infection.

Membrane-active macromolecules kill antibiotic-tolerant bacteria and potentiate antibiotics towards Gram-negative bacteria.

Chronic bacterial biofilms place a massive burden on healthcare due to the presence of antibiotic-tolerant dormant bacteria. Some of the conventional antibiotics such as erythromycin, vancomycin, linezolid, rifampicin etc. are inherently ineffective against Gram-negative bacteria, particularly in their biofilms. Here, we report membrane-active macromolecules that kill slow dividing stationary-phase and antibiotic tolerant cells of Gram-negative bacteria. More importantly, these molecules potentiate antibiotics (erythromycin and rifampicin) to biofilms of Gram-negative bacteria. These molecules eliminate planktonic bacteria that are liberated after dispersion of biofilms (dispersed cells). The membrane-active mechanism of these molecules forms the key for potentiating the established antibiotics. Further, we demonstrate that the combination of macromolecules and antibiotics significantly reduces bacterial burden in mouse burn and surgical wound infection models caused by Acinetobacter baumannii and Carbapenemase producing Klebsiella pneumoniae (KPC) clinical isolate respectively. Colistin, a well-known antibiotic targeting the lipopolysaccharide (LPS) of Gram-negative bacteria fails to kill antibiotic tolerant cells and dispersed cells (from biofilms) and bacteria develop resistance to it. On the contrary, these macromolecules prevent or delay the development of bacterial resistance to known antibiotics. Our findings emphasize the potential of targeting the bacterial membrane in antibiotic potentiation for disruption of biofilms and suggest a promising strategy towards developing therapies for topical treatment of Gram-negative infections.

Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.

Bacterial biofilms represent the root-cause of chronic or persistent infections in humans. Gram-negative bacterial infections due to nosocomial and opportunistic pathogens such as Acinetobacter baumannii are more difficult to treat because of their inherent and rapidly acquiring resistance to antibiotics. Due to biofilm formation, A. baumannii has been noted for its apparent ability to survive on artificial surfaces for an extended period of time, therefore allowing it to persist in the hospital environment. Here we report, maleic anhydride based novel cationic polymers appended with amide side chains that disrupt surface established multi-drug resistant A. baumannii biofilms. More importantly, these polymers significantly (p < 0.0001) decrease the bacterial burden in mice with chronic A. baumannii burn wound infection. The polymers also show potent antibacterial efficacy against methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococci (VRE) and multi-drug resistant clinical isolates of A. baumannii with minimal toxicity to mammalian cells. We observe that optimal hydrophobicity dependent on the side chain chemical structure of these polymers dictate the selective toxicity to bacteria. Polymers interact with the bacterial cell membranes by causing membrane depolarization, permeabilization and energy depletion. Bacteria develop rapid resistance to erythromycin and colistin whereas no detectable development of resistance occurs against these polymers even after several passages. These results suggest the potential use of these polymeric biomaterials in disinfecting biomedical device surfaces after the infection has become established and also for the topical treatment of chronic bacterial infections.

A Biodegradable Polycationic Paint that Kills Bacteria in Vitro and in Vivo.

Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and ß-lactam-resistant Klebsiella pneumoniae. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive S. aureus and Gram-negative E. coli, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15-20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under in vivo conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections.

Aryl-alkyl-lysines: Membrane-Active Small Molecules Active against Murine Model of Burn Infection.

Infections caused by drug-resistant Gram-negative pathogens continue to be significant contributors to human morbidity. The recent advent of New Delhi metallo-ß-lactamase-1 (blaNDM-1) producing pathogens, against which few drugs remain active, has aggravated the problem even further. This paper shows that aryl-alkyl-lysines, membrane-active small molecules, are effective in treating infections caused by Gram-negative pathogens. One of the compounds of the study was effective in killing planktonic cells as well as dispersing biofilms of Gram-negative pathogens. The compound was extremely effective in disrupting preformed biofilms and did not select resistant bacteria in multiple passages. The compound retained activity in different physiological conditions and did not induce any toxic effect in female Balb/c mice until concentrations of 17.5 mg/kg. In a murine model of Acinetobacter baumannii burn infection, the compound was able to bring the bacterial burden down significantly upon topical application for 7 days.

Correction: Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics.

Gram-negative 'superbugs' such as New Delhi metallo-beta-lactamase-1 (blaNDM-1) producing pathogens have become world's major public health threats. Development of molecular strategies that can rehabilitate the 'old antibiotics' and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs)that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards blaNDM-1 Klebsiella pneumonia and blaNDM-1 Escherichia coli clinical isolates.Organismic studies showed that bacteria had an increased and faster uptake of tetracyclinein the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover,bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.

Aryl-Alkyl-Lysines: Agents That Kill Planktonic Cells, Persister Cells, Biofilms of MRSA and Protect Mice from Skin-Infection.

Development of synthetic strategies to combat Staphylococcal infections, especially those caused by methicillin resistant Staphyloccus aureus (MRSA), needs immediate attention. In this manuscript we report the ability of aryl-alkyl-lysines, simple membrane active small molecules, to treat infections caused by planktonic cells, persister cells and biofilms of MRSA. A representative compound, NCK-10, did not induce development of resistance in planktonic cells in multiple passages and retained activity in varying environments of pH and salinity. At low concentrations the compound was able to depolarize and permeabilize the membranes of S. aureus persister cells rapidly. Treatment with the compound not only eradicated pre-formed MRSA biofilms, but also brought down viable counts in bacterial biofilms. In a murine model of MRSA skin infection, the compound was more effective than fusidic acid in bringing down the bacterial burden. Overall, this class of molecules bears potential as antibacterial agents against skin-infections.

Membrane-active macromolecules resensitize NDM-1 gram-negative clinical isolates to tetracycline antibiotics.

Gram-negative 'superbugs' such as New Delhi metallo-beta-lactamase-1 (blaNDM-1) producing pathogens have become world's major public health threats. Development of molecular strategies that can rehabilitate the 'old antibiotics' and halt the antibiotic resistance is a promising approach to target them. We report membrane-active macromolecules (MAMs) that restore the antibacterial efficacy (enhancement by >80-1250 fold) of tetracycline antibiotics towards blaNDM-1 Klebsiella pneumonia and blaNDM-1 Escherichia coli clinical isolates. Organismic studies showed that bacteria had an increased and faster uptake of tetracycline in the presence of MAMs which is attributed to the mechanism of re-sensitization. Moreover, bacteria did not develop resistance to MAMs and MAMs stalled the development of bacterial resistance to tetracycline. MAMs displayed membrane-active properties such as dissipation of membrane potential and membrane-permeabilization that enabled higher uptake of tetracycline in bacteria. In-vivo toxicity studies displayed good safety profiles and preliminary in-vivo antibacterial efficacy studies showed that mice treated with MAMs in combination with antibiotics had significantly decreased bacterial burden compared to the untreated mice. This report of re-instating the efficacy of the antibiotics towards blaNDM-1 pathogens using membrane-active molecules advocates their potential for synergistic co-delivery of antibiotics to combat Gram-negative superbugs.

Broad spectrum antibacterial and antifungal polymeric paint materials: synthesis, structure-activity relationship, and membrane-active mode of action.

Microbial attachment and subsequent colonization onto surfaces lead to the spread of deadly community-acquired and hospital-acquired (nosocomial) infections. Noncovalent immobilization of water insoluble and organo-soluble cationic polymers onto a surface is a facile approach to prevent microbial contamination. In the present study, we described the synthesis of water insoluble and organo-soluble polymeric materials and demonstrated their structure-activity relationship against various human pathogenic bacteria including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and beta lactam-resistant Klebsiella pneumoniae as well as pathogenic fungi such as Candida spp. and Cryptococcus spp. The polymer coated surfaces completely inactivated both bacteria and fungi upon contact (5 log reduction with respect to control). Linear polymers were more active and found to have a higher killing rate than the branched polymers. The polymer coated surfaces also exhibited significant activity in various complex mammalian fluids such as serum, plasma, and blood and showed negligible hemolysis at an amount much higher than minimum inhibitory amounts (MIAs). These polymers were found to have excellent compatibility with other medically relevant polymers (polylactic acid, PLA) and commercial paint. The cationic hydrophobic polymer coatings disrupted the lipid membrane of both bacteria and fungi and thus showed a membrane-active mode of action. Further, bacteria did not develop resistance against these membrane-active polymers in sharp contrast to conventional antibiotics and lipopeptides, thus the polymers hold great promise to be used as coating materials for developing permanent antimicrobial paint.

Small molecular antibacterial peptoid mimics: the simpler the better!

The emergence of multidrug resistant bacteria compounded by the depleting arsenal of antibiotics has accelerated efforts toward development of antibiotics with novel mechanisms of action. In this report, we present a series of small molecular antibacterial peptoid mimics which exhibit high in vitro potency against a variety of Gram-positive and Gram-negative bacteria, including drug-resistant species such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. The highlight of these compounds is their superior activity against the major nosocomial pathogen Pseudomonas aeruginosa. Nontoxic toward mammalian cells, these rapidly bactericidal compounds primarily act by permeabilization and depolarization of bacterial membrane. Synthetically simple and selectively antibacterial, these compounds can be developed into a newer class of therapeutic agents against multidrug resistant bacterial species.

Polymers with tunable side-chain amphiphilicity as non-hemolytic antibacterial agents.

Quaternized polymers mimicking the antimicrobial peptides were created by tuning the side-chain amphiphilicity using a first-time approach of post-functionalization. They displayed excellent efficacy against pathogenic bacteria even in human plasma and membrane disruptive mode of action. The optimized polymers and degraded products were non-hemolytic.