Broad-Spectrum Antimicrobial/Antifouling Soft Material Coatings Using Poly(ethylenimine) as a Tailorable Scaffold.

Microbial colonization and biofilm formation is the leading cause of contact lens-related keratitis. Treatment of the condition remains a challenge because of the need for prolonged therapeutic course and high doses of antimicrobial agents especially for biofilm eradication. The development of strategies to prepare nonfouling contact lens surfaces is a more practical way to ensure users' safety and relieve the excessive public healthcare burden. In this study, we report a series of polymers that were modified to introduce functionality designed to facilitate coating adhesion, antimicrobial and antifouling properties. Cyclic carbonate monomers having different functional groups including adhesive catechol, antifouling poly(ethylene glycol) (PEG), and hydrophobic urea/ethyl were conjugated onto branched poly(ethylenimine) (bPEI, 25 kDa) at various degrees in a facile and well-controlled manner using a simple one step, atom economical approach. Immersion of contact lenses into an aqueous solution of the catechol-functionalized polymers at room temperature resulted in robust and stable coating on the lens surfaces, which survived the harsh condition of autoclaving and remained on the surface for a typical device application lifetime (7 days). The deposition of the polymer was unambiguously confirmed by static contact angle measurement and X-ray photoelectron spectroscopy (XPS). Polymer coating did not change light transmission significantly. Combinatorial optimization demonstrated that lenses coated with bPEI functionalized with catechol, PEG (5 kDa) and urea groups at 1:12:3:23 molar ratio for 18 h provided the highest antifouling effect against four types of keratitis-causing pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Fusarium solani, after 7 days of incubation. The polymer coating also inhibited protein adsorption onto the contact lens surfaces after exposure to bovine serum albumin solution for up to 24 h, owing to the flexible and large PEG constituent. Notably, all the polymer coatings used in this study were biocompatible, achieving ≥90% cell viability following direct contact with human corneal epithelial cells for 24 h. Hence, these polymer coatings are envisaged to be promising for the prevention of contact lens-related keratitis.

Organic acid-catalyzed polyurethane formation via a dual-activated mechanism: unexpected preference of N-activation over O-activation of isocyanates.

A systematic study of acid organocatalysts for the polyaddition of poly(ethylene glycol) to hexamethylene diisocyanate in solution has been performed. Among organic acids evaluated, sulfonic acids were found the most effective for urethane formations even when compared with conventional tin-based catalysts (dibutyltin dilaurate) or 1,8-diazabicyclo[5.4.0]undec-7-ene. In comparison, phosphonic and carboxylic acids showed considerably lower catalytic activities. Furthermore, sulfonic acids gave polyurethanes with higher molecular weights than was observed using traditional catalyst systems. Molecular modeling was conducted to provide mechanistic insight and supported a dual activation mechanism, whereby ternary adducts form in the presence of acid and engender both electrophilic isocyanate activation and nucleophilic alcohol activation through hydrogen bonding. Such a mechanism suggests catalytic activity is a function of not only acid strength but also inherent conjugate base electron density.

Antimicrobial polycarbonates: investigating the impact of balancing charge and hydrophobicity using a same-centered polymer approach.

Biodegradable antimicrobial polymers are a promising solution for combating drug resistant microbes. When designing these materials, the balance between charge and hydrophobicity significantly affects the antimicrobial activity and selectivity toward microbes over mammalian cells. Furthermore, where the charge and hydrophobicity is located on the molecules has also proven to be significant. A series of antimicrobial homopolymer polycarbonates were synthesized, where the hydrophobic/hydrophilic balance was controlled by varying the spacer between the charged quaternary ammonium moiety and the polymer backbone (a "same-centered" structure where the hydrophobic moiety is directly attached to the charged moiety). These homopolymers were active against all microbes tested but depending on the spacer length some hemolytic activity was observed. To reduce the polymer hemolytic activity we systematically varied the polymer composition by copolymerizing the different monomers used in the "same center" homopolymers. By maintaining charge on each repeat unit but copolymerizing monomers having varied hydrophobic side chain lengths, polymers with high activity and selectivity were achieved. In addition, these macromolecules act via a membrane disruption mechanism, making them less likely to induce resistance.

Broad-spectrum antimicrobial and biofilm-disrupting hydrogels: stereocomplex-driven supramolecular assemblies.

Fighting the resistance: biodegradable and injectable/moldable hydrogels with hierarchical nanostructures were made with broad-spectrum antimicrobial activities and biofilm-disruption capability. They demonstrate no cytotoxicity in vitro, and show excellent skin biocompatibility in animals. These hydrogels have great potential for clinical use in prevention and treatment of various multidrug-resistant infections.

Enhanced stability of polymeric micelles based on postfunctionalized poly(ethylene glycol)-b-poly(γ-propargyl L-glutamate): the substituent effect.

One of the major obstacles that delay the clinical translation of polymeric micelle drug delivery systems is whether these self-assembled micelles can retain their integrity in blood following intravenous (IV) injection. The objective of this study was to evaluate the impact of core functionalization on the thermodynamic and kinetic stability of polymeric micelles. The combination of ring-opening polymerization of N-carboxyanhydride (NCA) with highly efficient "click" coupling has enabled easy and quick access to a family of poly(ethylene glycol)-block-poly(γ-R-glutamate)s with exactly the same block lengths, for which the substituent "R" is tuned. The structures of these copolymers were carefully characterized by (1)H NMR, FT-IR, and GPC. When pyrene is used as the fluorescence probe, the critical micelle concentrations (CMCs) of these polymers were found to be in the range of 10(-7)-10(-6) M, which indicates good thermodynamic stability for the self-assembled micelles. The incorporation of polar side groups in the micelle core leads to high CMC values; however, micelles prepared from these copolymers are kinetically more stable in the presence of serum and upon SDS disturbance. It was also observed that these polymers could effectively encapsulate paclitaxel (PTX) as a model anticancer drug, and the micelles possessing better kinetic stability showed better suppression of the initial "burst" release and exhibited more sustained release of PTX. These PTX-loaded micelles exerted comparable cytotoxicity against HeLa cells as the clinically approved Cremophor PTX formulation, while the block copolymers showed much lower toxicity compared to the cremophor-ethanol mixture. The present work demonstrated that the PEG-b-PPLG can be a uniform block copolymer platform toward development of polymeric micelle delivery systems for different drugs through the facile modification of the PPLG block.

Effects of side group functionality and molecular weight on the activity of synthetic antimicrobial polypeptides.

The rapid emergence of antibiotic-resistant bacteria along with increasing difficulty in biofilm treatment has caused an immediate need for the development of new classes of antimicrobial therapeutics. We have developed a library of antimicrobial polypeptides, prepared by the ring-opening polymerization of γ-propargyl-L-glutamate N-carboxyanhydride and the alkyne-azide cycloaddition click reaction, which mimic the favorable characteristics of naturally occurring antimicrobial peptides (AmPs). AmPs are known not to cause drug resistance as well as prevent bacteria attachment on surfaces. The ease and scale of synthesis of the antimicrobial polypeptides developed here are significantly improved over the traditional Merrifield synthetic peptide approaches needed for naturally occurring antimicrobial peptides and avoids the unique challenges of biosynthetic pathways. The polypeptides range in length from 30 to 140 repeat units and can have varied side group functionality, including primary, secondary, tertiary, and quaternary amines with hydrocarbon side chains ranging from 1 to 12 carbons long. Overall, we find these polypeptides to exhibit broad-spectrum activity against both Gram positive and Gram negative bacteria, namely, S. aureus and E. coli , while having very low hemolytic activity. Many of the polypeptides can also be used as surface coatings to prevent bacterial attachment. The polypeptide library developed in this work addresses the need for effective biocompatible therapeutics for drug delivery and medical device coatings.

Broad Spectrum Macromolecular Antimicrobials with Biofilm Disruption Capability and In Vivo Efficacy.

In this study, antimicrobial polymers are synthesized by the organocatalytic ring-opening polymerization of an eight-membered heterocyclic carbonate monomer that is subsequently quaternized with methyl iodide. These polymers demonstrate activity against clinically relevant Gram-positive Staphylococcus epidermidis and Staphylococcus aureus, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and fungus Candida albicans with fast killing kinetics. Importantly, the polymer efficiently inhibits biofilm growth and lyses existing biofilm, leading to a reduction in biomass and cell viability. In addition, the macromolecular antimicrobial is less likely to induce resistance as it acts via a membrane-lytic mechanism. The polymer is not cytotoxic toward mammalian cells with LD50 of 99.0 ± 11.6 mg kg-1 in mice through i.v. injection. In an S. aureus blood stream infection mouse model, the polymer removes bacteria from the blood more rapidly than the antibiotic Augmentin. At the effective dose, the polymer treatment does not damage liver and kidney tissues or functions. In addition, blood electrolyte balance remains unchanged after the treatment. The low cost of starting materials, ease of synthesis, nontoxicity, broad spectrum activity with fast killing kinetics, and in vivo antimicrobial activity make these macromolecular antimicrobials ideal candidates for prevention of sepsis and treatment of infections.

Enhancement of cationic antimicrobial materials via cholesterol incorporation.

Cationic antimicrobial materials are an attractive option for treating drug-resistant bacteria. Their membrane lytic mechanism can provide broad spectrum antimicrobial activity while largely negating natural resistance development. Selectivity is achieved using non-specific electrostatic interactions since microbial membranes display significantly more peripheral negative charge than due eukaryotic bilayers. Following membrane association, structural changes occur causing bilayer destabilization and cell lysis. Herein, antimicrobial effects of enhanced membrane assimilation are examined. Cholesterol, a functionalizable small molecule that assimilates abundantly within cell membranes, is chosen to increase membrane penetration ability to improve antimicrobial activity. Furthermore, cholesterol has an ability to template interesting nanostructures due to its propensity for rotative face-on-face stacking. The installation of cationic polycarbonates with systematically varied chain lengths from three separate cholesteryl initiators is accomplished using organocatalytic ring-opening polymerization. Introduction of cholesteryl oligomers into aqueous media creates "coin" shaped self-assemblies possessing high exterior cationic charge density. Continued evaluation of these assemblies demonstrates broad spectrum activity against S. epidermidis, S. aureus, E. coli, P. aeraginosa, and C. albicans. Additional results show that, despite repeated sub-lethal dosing, E. coli does not evolve drug-resistance and maintains the wild-type minimum inhibitory concentration of 31.3 mg L(-1) .

Tetra-n-butylammonium Fluoride as an Efficient Transesterification Catalyst for Functionalizing Cyclic Carbonates and Aliphatic Polycarbonates.

We have developed a general method for the functionalization of cyclic carbonate monomers having a pentafluorophenyl ester substituent at the 5-position (MTC-OC6F5), as well as the postpolymerization modification of the subsequent polymer, poly(MTC-OC6F5), with alcohols. The transesterifications are achieved under mild conditions using catalytic tetra-n-butylammonium fluoride (TBAF) as the nucleophilic acyl transfer agent. As an organic-soluble form of fluoride, TBAF loadings as low as 5 mol % were sufficient in bringing about high conversions at room temperature. The mild reaction conditions preserved the integrity of the sensitive carbonate moieties even without the use of Schlenk techniques. In addition to commercial TBAF solutions, we also found solid-supported forms of TBAF to be effective for transesterification, thus enabling facile postreaction workup and purification. More importantly, with only minor adjustments to the reaction conditions, we show that TBAF also promotes the postpolymerization modification of poly(MTC-OC6F5), whereby fluoride-mediated transesterification with various alcohols proceeded quantitatively across the pendant pentafluorophenyl esters. Synthesizing a series of pendant ester-functionalized polycarbonates from a common precursor polymer was previously unattainable with existing methods, an issue that is now resolved by the current work.

Mitigated cytotoxicity and tremendously enhanced gene transfection efficiency of PEI through facile one-step carbamate modification.

Extremely efficacious gene transfection vector: The rapid and facile modification of PEI with commercially available TMC produces an extremely efficacious gene delivery vector with minimal cytotoxicity. Functionalization of PEI is easily controlled by PEI:cyclic carbonate feed ratios and allows for the addition of functionality. Modified PEIs hold great potential as gene delivery systems due to easy synthesis, scalability, low cost, low toxicity, and outstanding transfection capacity.

Polycarbonate-Based Brush Polymers with Detachable Disulfide-Linked Side Chains.

We have successfully designed and synthesized polycarbonate-based brush polymers with detachable, disulfide-linked side chains. A polycarbonate backbone with disulfide-linked, hydroxyl-terminated pendant side chains was first prepared. Poly(trimethylene carbonate) or poly(l-lactide) brushes were then grafted from the terminal hydroxyl groups using an acid- or base-catalyzed ring-opening polymerization. Inspired by how cells use glutathione to mediated reduction of disulfides in cytoplasmic proteins, we also demonstrate that the side chains are easily detached under mild reductive conditions (e.g., with 1,4-dithiothreitol). l-Lactide and trimethylene carbonate were selected as model building blocks for the polymer grafts because of their commercial availability and routine use in polymeric drug delivery systems.

Polymerizing Base Sensitive Cyclic Carbonates Using Acid Catalysis.

Organic acids were explored as a means to expand the library of cyclic carbonate monomers capable of undergoing controlled ring-opening polymerization. Various nitrogenous bases have proven incredibly adept at polymerizing cyclic carbonates; however, their use has largely precluded monomers with an acidic proton. Molecular modeling of acid catalysis provided new mechanistic insight, wherein a bifunctional activation pathway was calculated. Depending on acid structure, modeling experiments showed both monomer carbonyls and propagating hydroxyl groups undergo hydrogen bonding activation. The dual activation mechanism suggests acid strength, as well as conjugate base effects, play vital roles in catalyzing cyclic carbonate polymerizations. Moreover, the use of acid catalysis was shown to be compatible with amide-containing monomers while promoting controlled polymerizations.

Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 µm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and ~100% substitution of primary amine groups leads to significantly lower gene transfection efficiency. These findings provide insights to modification of PEI for development of effective and non-cytotoxic non-viral vectors.

Supramolecular high-aspect ratio assemblies with strong antifungal activity.

Efficient and pathogen-specific antifungal agents are required to mitigate drug resistance problems. Here we present cationic small molecules that exhibit excellent microbial selectivity with minimal host toxicity. Unlike typical cationic polymers possessing molecular weight distributions, these compounds have an absolute molecular weight aiding in isolation and characterization. However, their specific molecular recognition motif (terephthalamide-bisurea) facilitates spontaneous supramolecular self-assembly manifesting in several polymer-like properties. Computational modelling of the terephthalamide-bisurea structures predicts zig-zag or bent arrangements where distal benzyl urea groups stabilize the high-aspect ratio aqueous supramolecular assemblies. These nanostructures are confirmed by transmission electron microscopy and atomic force microscopy. Antifungal activity against drug-sensitive and drug-resistant strains with in vitro and in vivo biocompatibility is observed. Additionally, despite repeated sub-lethal exposures, drug resistance is not induced. Comparison with clinically used amphotericin B shows similar antifungal behaviour without any significant toxicity in a C. albicans biofilm-induced mouse keratitis model.

Dual responsiveness of a tunable thermo-sensitive polypeptide.

The temperature and pH dependent solubility of poly(γ-propargyl L-glutamate) (PPLG) functionalized through copper catalyzed 1,3 cycloaddition reaction between an alkyne and azide can be tuned with precision over a broad range of conditions by varying the ratio of substitution of short oligo ethylene glycol and diisopropylamine side groups.

Antibacterial and antifouling catheter coatings using surface grafted PEG-b-cationic polycarbonate diblock copolymers.

Intravascular catheter-associated infections (CAIs), which are normally induced by microbial adhesion and subsequent biofilm formation, are a major cause of morbidity and mortality. Therefore, strategies to prevent CAIs are in great demand. In this study, a series of diblock copolymers of PEG and cationic polycarbonate with various compositions were synthesized by metal-free organocatalytic ring-opening polymerization, and coated onto silicone rubber (a commonly used catheter material) at different concentrations via a reactive polydopamine coating. Static contact angle and X-ray photoelectron spectroscopy measurements proved the successful coating, and quartz crystal microbalance results showed that the coating thickness increased as polymer concentration increased. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates - leading causes of intravascular CAIs - were employed to evaluate the antibacterial and antifouling activities of the polymer coatings. Polymer coatings with a hydrophobic component effectively killed planktonic MSSA and MRSA in solution and prevented their fouling on silicone rubber surface. Live/dead cell staining experiments revealed that polymer coatings with the optimal polymer composition possessed significantly higher antifouling activity than PEG coating. In addition, scanning electron microscopic studies showed that the polymer coating inhibited S. aureus biofilm formation over a period of 7 days. Furthermore, the polymer coating caused no significant hemolysis, and there was no blood protein adsorption or platelet adhesion observed. Therefore, PEG-b-cationic polycarbonates with optimal compositions are effective antifouling and antibacterial coatings for the prevention of intravascular CAIs.

Catalyst Chelation Effects in Organocatalyzed Ring-Opening Polymerization of Lactide.

(-)-Sparteine is a proven organocatalyst for the ring-opening polymerization (ROP) of l-lactide, which affords polymers of controlled molecular weight and narrow polydispersity. The recent worldwide shortage of (-)-sparteine has necessitated the identification of simple and cost-effective replacement ROP catalysts. A series of commercially available molecules was first identified through molecular modeling and then subsequently investigated for polymerizing l-lactide. The modeling proved very useful at predicting spatial relationships and nitrogen geometries that greatly aided in the rapid identification of various alkyl amines as alternative organocatalysts.

Broad-spectrum antimicrobial supramolecular assemblies with distinctive size and shape.

With the increased prevalence of antibiotic-resistant infections, there is an urgent need for innovative antimicrobial treatments. One such area being actively explored is the use of self-assembling cationic polymers. This relatively new class of materials was inspired by biologically pervasive cationic host defense peptides. The antimicrobial action of both the synthetic polymers and naturally occurring peptides is believed to be complemented by their three-dimensional structure. In an effort to evaluate shape effects on antimicrobial materials, triblock polymers were polymerized from an assembly directing terephthalamide-bisurea core. Simple changes to this core, such as the addition of a methylene spacer, served to direct self-assembly into distinct morphologies-spheres and rods. Computational modeling also demonstrated how subtle core changes could directly alter urea stacking motifs manifesting in unique multidirectional hydrogen-bond networks despite the vast majority of material consisting of poly(lactide) (interior block) and cationic polycarbonates (exterior block). Upon testing the spherical and rod-like morphologies for antimicrobial properties, it was found that both possessed broad-spectrum activity (Gram-negative and Gram-positive bacteria as well as fungi) with minimal hemolysis, although only the rod-like assemblies were effective against Candida albicans.