Shaoqiong Liu

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.

Thermally sensitive micelles self-assembled from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) for controlled delivery of paclitaxel

Thermally sensitive micelles self-assembled from poly(N-isopropylacrylamide-co- N,N-dimethylacrylamide)-b-poly(d,l-lactide-co-glycolide)[P(NIPAAm-co-DMAAm)-b-PLGA] are fabricated and used as a carrier for the controlled delivery of paclitaxel. Paclitaxel is efficiently loaded into the micelles by a membrane dialysis method. The lower critical solution temperature (LCST) of the micelles is 39.0 degrees C in PBS. Encapsulation efficiency and loading level of paclitaxel are affected by the initial loading level of paclitaxel, fabrication temperature and polymer composition. The blank and paclitaxel-loaded micelles are characterized by particle size analysis (DLS), morphology (TEM and AFM) and paclitaxel distribution (NMR, DSC and WAXRD). The micelles are spherical in shape, having an average size less than 130 nm. Paclitaxel is molecularly distributed within the core of micelles. Sustained release of paclitaxel is achieved, which is much faster at a temperature above the LCST than at the normal body temperature (37 degrees C). Cytotoxicity of free paclitaxel and paclitaxel-loaded micelles against a human breast carcinoma cell line (MDA-MB-435S) is studied at different temperatures. The cytotoxicity of the paclitaxol-loaded micelles is greater as compared to free paclitaxel. Enhanced cytotoxicity is achieved by the paclitaxol-loaded micelles when the environmental temperature increases slightly above the LCST. Paclitaxel-loaded P(NIPAAm-co-DMAAm)-b-PLGA micelles may provide a good formulation for cancer therapy.

Anti-mycobacterial activities of synthetic cationic α-helical peptides and their synergism with rifampicin.

The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic α-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti-mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK)2M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB.

Nanostructured PEG-based hydrogels with tunable physical properties for gene delivery to human mesenchymal stem cells.

Effective delivery of DNA to direct cell behavior in a well defined three dimensional scaffold offers a superior approach in tissue engineering. In this study, we synthesized biodegradable nanostructured hydrogels with tunable physical properties for cell and gene delivery. The hydrogels were formed via Michael addition chemistry by reacting a four-arm acrylate-terminated PEG with a four-arm thiol-functionalized PEG. Nanosized micelles self-assembled from the amphiphilic PEG-b-polycarbonate diblock copolymer, having reactive end-groups, were chemically incorporated into the hydrogel networks at various contents. The use of Michael addition chemistry allows for in situ hydrogel formation under the physiological conditions. Mechanical property analysis of the hydrogels revealed a correlation between the content of micelles and the storage modulus of the hydrogels. Internal morphology of hydrogels was observed using a field emission scanning electron microscope, which showed that the number and/or size of the pores in the hydrogel increased with increasing micelle content due to reduced crosslinking degree. There exists an optimal micelle content for cell proliferation and gene transfection. MTT assays demonstrated the highest cell viability in the hydrogel with 20% micelles. The gene expression level in hMSCs in the hydrogel with 20% micelles was also significantly higher than that in the hydrogel without micelles. The enhanced cell viability and gene expression in the hydrogel with the optimized micelle content are likely attributed to the physical properties that provide a better environment for cell-matrix interactions. Therefore, incorporating micelles into the hydrogel is a good strategy to control cellular behavior in 3-D through changes in physical properties of the microenvironment.

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.

Branched and 4‐Arm Starlike α‐Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity

2 The innate immune system of multicellular organisms fights against microbial infections by secreting antimicrobial peptides (AMPs). In the midst of emerging multidrug-resistant bacteria, these peptides have been acknowledged as the blueprint for the new class of antibiotics.[1,2] They exhibit an antimicrobial effect by interacting with the outer envelope of microbes, leading to eventual lysis and cell death. The most common characteristic of these peptides is that they contain both cationic and hydrophobic amino acids. The cationic charge is reported to increase the long-range interactions with negatively charged microbial surfaces, while the hydrophobic part is required to attain anchorage on the lipid bilayer of microbial membranes to form pores and induce membrane lysis.[3,4] Since then, synthetic materials, like nonnatural peptides[5–7] and polymers,[8–11] have been designed to mimic the amphiphilicity and antimicrobial activity of these natural peptides. However, selectivity of these materials towards microbes over mammalian cells remains a challenge, which hinders their applications in clinical settings.[12,13] It is desirable that these materials interact more readily with microbes than with mammalian cells to reduce their toxicity. Recently, we reported an approach to designing new AMPs from the basic protein-folding theories.[5] AMPs with a general peptide sequence of (XXYY)n (X: hydrophobic amino acid; Y: cationic amino acid) folded into α-helices upon interaction with bacterial membrane mimics. The length of peptides (with n repeat units) dictated the readiness to form the helical structures. Aside from helical propensity, an increase in the number of repeat units also increases the cationic and hydrophobic contents of the AMP, without disturbing the cationic–hydrophobic balance. This might also contribute to the increased antimicrobial activity. However, the selectivity towards microbes was grossly reduced with increasing numbers of repeat units.[5] In another study for oligomeric and polymeric antimicrobial materials, the selectivity towards microbial cells was reported to be associated with the rigidity of the material backbone. More rigid

pH and redox dual-responsive biodegradable polymeric micelles with high drug loading for effective anticancer drug delivery

Diblock copolymers of poly(ethylene glycol) (PEG) and biodegradable polycarbonate functionalized with GSH-sensitive disulfide bonds and pH-responsive carboxylic acid groups were synthesized via organocatalytic ring-opening polymerization of functional cyclic carbonates with PEG having different molecular weights as macroinitiators. These narrowly-dispersed polymers had predictable molecular weights, and were used to load doxorubicin (DOX) into micelles primarily through ionic interactions. The DOX-loaded micelles exhibited the requisite small particle size (<100 nm), narrow size distribution and high drug loading capacity. When exposed to endolysosomal pH of 5.0, drug release was accelerated by at least two-fold. The introduction of GSH further expedited DOX release. Effective DOX release enhanced cytotoxicity against cancer cells. More importantly, the DOX-loaded micelles with the optimized composition showed excellent antitumor efficacy in nude mice bearing BT-474 xenografts without inducing toxicity. These pH and redox dual-responsive micelles have the potential as delivery carriers to maximize the therapeutic effect of anticancer drugs.

Broad-Spectrum Antimicrobial Star Polycarbonates Functionalized with Mannose for Targeting Bacteria Residing inside Immune Cells.

In this study, a series of star-shaped polycarbonates are synthesized by metal-free organocatalytic ring-opening polymerization of benzyl chloride (BnCl) and mannose-functionalized cyclic carbonate monomers (MTC-BnCl and MTC-ipman) with heptakis-(2,3-di-O-acetyl)-β-cyclodextrin (DA-β-CD) as macroinitiator. The distributions and compositions of pendent benzyl chloride and protected mannose group (ipman) units are facilely modulated by varying the polymerization sequence and feed ratio of the monomers, allowing precise control over the molecular composition, and the resulting polymers have narrow molecular weight distribution. After deprotection of ipman groups and quaternization with various N,N-dimethylalkylamines, these star polymers with optimized compositions of cationic and mannose groups in block and random forms exhibit strong bactericidal activity and low hemolysis. Furthermore, the optimal mannose-functionalized polymer demonstrates mannose receptor-mediated intracellular bactericidal activity against BCG mycobacteria without inducing cytotoxicity on mammalian cells at the effective dose. Taken together, the materials designed in this study have potential use as antimicrobial agents against diseases such as tuberculosis, which is caused by intracellular bacteria.

Biodegradable functional polycarbonate micelles for controlled release of amphotericin B

Amphotericin B (AmB), a poorly soluble and toxic antifungal drug, was encapsulated into polymeric micelles self-assembled from phenylboronic acid-functionalized polycarbonate/PEG (PEG-PBC) and urea-functionalized polycarbonate/PEG (PEG-PUC) diblock copolymers via hydrogen-bonding, boronate ester bond, and/or ionic interactions between the boronic acid group in the micellar core and amine group in AmB. Three micellar formulations were prepared: AmB/B micelles using PEG-PBC, AmB/U micelles using PEG-PUC and AmB/B+U mixed micelles using 1:1molar ratio of PEG-PBC and PEG-PUC. The average particle sizes of the micelles were in the range of 54.4-84.8nm with narrow size distribution and zeta potentials close to neutral. UV-Vis absorption analysis indicated that AmB/B micelles significantly reduced AmB aggregation status due to the interactions between AmB and the micellar core, while Fungizone® and AmB/U micelles had no effect. AmB/B+U mixed micelles exerted an intermediate effect. Both AmB/B micelles and AmB/B+U mixed micelles showed sustained drug release, with 48.6±2.1% and 59.2±1.8% AmB released respectively after 24hunder sink conditions, while AmB/U micelles displayed a burst release profile. All AmB-loaded micelles showed comparable antifungal activity to free AmB or Fungizone®, while AmB/B micelles and AmB/B+U mixed micelles were much less hemolytic than other formulations. Histological examination showed that AmB/B and AmB/B+U micelles led to a significantly lower number of apoptotic cells in the kidneys compared to Fungizone®, suggesting reduced nephrotoxicity of the micellar formulations in vivo. These phenylboronic acid-functionalized polymeric micelle systems are promising drug carriers for AmB to reduce non-specific toxicities without compromise in antifungal activity. STATEMENT OF SIGNIFICANCE There is a pressing need for a novel and cost-effective delivery system to reduce the toxicity induced by the antifungal agent, amphotericin B (AmB). In this study, phenylboronic acid-functionalized polycarbonate/PEG diblock copolymers were used to fabricate micelles for improved AmB-micelle interaction via the manipulation of hydrogen-bonding, boronate ester bond, ionic and hydrophobic interactions. Compared to free AmB and Fungizone®, the resultant micellar systems displayed improved stability while reducing non-specific toxicities without a compromise in antifungal activity. These findings demonstrate the potential of biodegradable functional polycarbonate micellar systems as promising carriers of AmB for the treatment of systemic fungal infections.

Simple and cost-effective polycondensation routes to antimicrobial consumer products

In this study, cost-effective macromolecular antimicrobials for applications in consumer care products were targeted. Our strategy for inexpensive yet highly efficacious macromolecular antimicrobials employs organocatalytic step-growth polymerization of commercially available monomers and catalysts. Importantly, bulk polymerization conditions were sought to mitigate the cost, reduce solvent waste, and eliminate polymer purification and isolation steps. Moreover, diffusion-controlled, bulk polymerization conditions limited the polymer number-average molecular weights (Mn) to ∼5000–10000 g mol−1, as the activity and selectivity was independent of molecular weight. The modest molecular weights enable the polymers to be soluble/processable for subsequent quaternization. A number of polymer-forming reactions were investigated including ester, amide, urea, and guanidinium formation. Of these polymers, polyamides quaternized with methyl iodide or benzyl bromide exhibited excellent water-solubility and potent antimicrobial activity against a panel of clinically relevant microbes including multidrug-resistant P. aeruginosa. These polymers contain amide bonds, which remain intact in aqueous solution (even in a weakly alkaline environment), thereby increasing their suitability for personal care products due to the long shelf-life. The introduction of Jeffamine to the polymers as a means to further reduce cost does not change antimicrobial potency, but significantly increases compatibility to mammalian cells, further justifying its potential use in personal care products. The advantages of this approach addresses not only the cost-related challenges of polymerization scale-up, but also the synthetic versatility necessary to explore a variety of chemical functional groups and tune the polymer amphiphilicity for targeted antimicrobial performance, as well as cytotoxicity.