Jiaul Hoque

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.

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.

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.

Aggregation Properties of Amide Bearing Cleavable Gemini Surfactants by Small Angle Neutron Scattering and Conductivity Studies

The micellar aggregation of different amide bearing cleavable gemini surfactants with varying methylene spacer chain length (m = 2, 4, 6, 8, and 12) along with the corresponding monomeric surfactant in aqueous media has been investigated by conductometric and small angle neutron scattering (SANS) studies. The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.08-0.19 mM) and were 23-55 times lesser than the corresponding monomeric analogue (CMC = 4.4 mM). With increase in the spacer chain length, CMC was found to decrease whereas the degree of ionization was found to increase. SANS data have been analyzed by considering the screened coulombic interactions between the micelles to compute the interparticle structure factor S(Q). The extent of micellar growth and the variation of shapes of the micelles formed by these new surfactants in aqueous solution have been found to depend strongly on the spacer chain length. It was observed that the extent of micellar growth and variation of micellar shapes are more pronounced for surfactants with short spacer chain length (m ≤ 4), whereas the surfactants with a long spacer chain length (m ≥ 6) showed slight variation of these properties in aqueous solution. The effects of the variation of the concentration and temperature on the SANS spectra (and hence on the microstructure) of the gemini surfactant (m = 4) were also examined. With an increase in concentration the aggregation number (N) and size of the micelles (the ratio of semimajor axis (a) to semiminor axis (b = c)) increased whereas opposite phenomena was observed with an increase in temperature.

Membrane Active Small Molecules Show Selective Broad Spectrum Antibacterial Activity with No Detectable Resistance and Eradicate Biofilms

Treating bacterial biofilms with conventional antibiotics is limited due to ineffectiveness of the drugs and higher propensity to develop bacterial resistance. Development of new classes of antibacterial therapeutics with alternative mechanisms of action has become imperative. Herein, we report the design, synthesis, and biological evaluations of novel membrane-active small molecules featuring two positive charges, four nonpeptidic amide groups, and variable hydrophobic/hydrophilic (amphiphilic) character. The biocides synthesized via a facile methodology not only displayed good antibacterial activity against wild-type bacteria but also showed high activity against various drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and β-lactam-resistant Klebsiella pneumoniae. Further, these biocides not only inhibited the formation of biofilms but also disrupted the established S. aureus and E. coli biofilms. The membrane-active biocides hindered the propensity to develop bacterial resistance. Moreover, the biocides showed negligible toxicity against mammalian cells and thus bear potential to be used as therapeutic agents.

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.

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.

Direct Synthesis of Dextran-Based Antibacterial Hydrogels for Extended Release of Biocides and Eradication of Topical Biofilms

Cationic small molecular biocides have been developed as promising antibiofilm agents because of their tunability in chemical structures and their ability to disrupt established biofilms. However, the impact of biocides in antibiofilm treatment is largely limited due to the lack of an effective delivery system that can ensure sustained release of biocides at the target site. Herein we report a biocide-encapsulated antibacterial and antibiofilm hydrogel that acts as an efficient delivery vehicle for the biocide and eradicates matured bacterial biofilm. The hydrogels are prepared using dextran methacrylate (Dex-MA), a biocompatible and photopolymerizable polymer, and a nontoxic cationic biocide with two cationic charges, two nonpeptidic amide bonds, and optimized amphiphilicity, which is capable of eradicating established bacterial biofilms. The gels, prepared via direct loading of the biocide and with highly controllable amounts, display 100% activity against both drug-sensitive and drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Importantly, the gels are shown to release the biocide and kill bacteria for an extended period of time (until day 5). When being treated with the established bacterial biofilms, the released biocide from the gel is shown to completely eradicate establishedS. aureus, Escherichia coli, and MRSA biofilms, the most common biofilm forming bacteria that cause severe infections (e.g., skin infections, urinary tract infections, etc.) in humans. Moreover, the gels were shown to annihilate preformed MRSA biofilm with >99.99% bacterial reduction under in vitro and in vivo conditions in a superficial MRSA infection model in mice. Notably, when tested, excellent skin compatibility is observed for these materials in various animal models such as a rat model of acute dermal toxicity, guinea pig model of skin sensitization, and rabbit model of skin irritation. The biocompatible antibacterial and antibiofilm hydrogels developed herein thus might be useful in treating bacterial biofilm associated infections, especially topical infections.

Dual Function Injectable Hydrogel for Controlled Release of Antibiotic and Local Antibacterial Therapy

We present vancomycin-loaded dual-function injectable hydrogel that delivers antibiotic locally suitable for treatment of infections in avascular or necrotic tissues. The syringe-deliverable gels were developed using polydextran aldehyde and an inherently antibacterial polymer N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride along with vancomycin. The antibiotic was primarily encapsulated via reversible imine bonds formed between vancomycin and polydextran aldehyde in the hydrogel which allowed sustained release of vancomycin over an extended period of time in a pH-dependent manner. Being inherently antibacterial, the gels displayed excellent efficacy against bacteria due to dual mode of action (killing bacteria upon contact as well as by releasing antibiotics into surroundings). Upon subcutaneous implantation, the gel was shown to kill methicillin-resistant Staphylococcus aureus (>99.999%) when bacteria were introduced directly into the gel as well as at distal site from the gel in a mice model. These materials thus represent as novel noninvasive drug-delivery device suitable for local antibiotic therapy.