Quantified Membrane Permeabilization Indicates the Lipid Selectivity of Membrane-Active Antimicrobials.

Most antimicrobial peptides (AMPs) and their synthetic mimics (SMAMPs) are thought to act by permeabilizing cell membranes. For antimicrobial therapy, selectivity for pathogens over mammalian cells is a key requirement. Understanding membrane selectivity is thus essential for designing AMPs and SMAMPs to complement classical antibiotics in the future. This study focuses on membrane permeabilization induced by SMAMPs and their selectivity for membranes with different lipid compositions. We measure release and fluorescence lifetime of a self-quenching dye in lipid vesicles. Apart from the dose-response, we quantify the strength of individual leakage events, and, employing cumulative kinetics, categorize permeabilization behavior. We propose that differing selectivities in a series of SMAMPs arise from a combination of the effect of the antimicrobial agent and the susceptibility of the membrane (with a given lipid composition) for certain types of leakage behavior. The unselective and hemolytic SMAMP is found to act mainly by the asymmetry stress mechanism, mediated by hydrophobic insertion of SMAMPs into lipid layers. The more selective SMAMPs induced leakage events occurring stochastically over several hours. Lipid intrinsic properties might additionally amplify the efficiency of leakage events. Leakage behavior changes with both the design of the SMAMP and the lipid composition of the membrane. Understanding how leakage behavior contributes to the selectivity and activity of antimicrobial agents will aid the design and screening of antimicrobials. An understanding of the underlying processes facilitates the comparison of membrane permeabilization across in vitro and in vivo assays.

Structure-Property Relationships of Amine-rich and Membrane-Disruptive Poly(oxonorbornene)-Coated Gold Nanoparticles.

The article describes the interactions between poly (oxonorbornenes) (PONs)-coated gold nanoparticles (AuNPs) with phospholipid vesicles and shows that the strength of these interactions strongly depends on the molecular structure of PONs, specifically their amine/alkyl side chain ratio. PONs, which are a recently introduced class of cationic polyelectrolytes, can be systematically varied to control the amine/alkyl ratio and to explore how the chemical character of cationic polyelectrolytes affects their interactions and the interactions of their nanoparticle conjugates with model membranes. Our study shows that increasing the amine/alkyl ratio by copolymerization of diamine and 1:1 amine/butyl oxonorbornene monomers impacts the availability of PONs amine/ammonium functional groups to interact with phospholipid membranes, the PONs surface coverage on AuNPs, and the membrane disruption activity of free PONs and PONs-AuNPs. The study makes use of transmission electron microscopy, UV-vis spectroscopy, dynamic light scattering, thermogravimetric analysis, fluorescamine assay, ζ-potential measurements, and X-ray photoelectron spectroscopy measurements to characterize the PONs-AuNPs' size, size distribution, aggregation state, surface charge, and PONs surface coverage. The study also makes use of real-time fluorescence measurements of fluorescent liposomes before and during exposure to free PONs and PONs-AuNPs to determine the membrane disruption activity of free PONs and PONs-AuNPs. As commonly observed with cationic polyelectrolytes, both free PONs and PONs-AuNPs display significant membrane disruption activity. Under conditions where the amine/alkyl ratio in PONs maximizes PONs surface coverage, the membrane disruption activity of PONs-AuNPs is about 10-fold higher than the membrane disruption activity of the same free PONs. This is attributed to the increased local concentration of ammonium ions when PONs-AuNPs interact with the liposome membranes. In contrast, the hydrophobicity of amine-rich PONs, which are made for example from diamine oxonorbornene monomers, is significantly reduced. This leads to a significant reduction of PON surface coverage on AuNPs and in turn to a significant decrease in membrane disruption.

A New Alkamide with an Endoperoxide Structure from Acmella ciliata (Asteraceae) and Its in Vitro Antiplasmodial Activity.

From the aerial parts of Acmella ciliata (H.B.K.) Cassini (basionym Spilanthes ciliata Kunth; Asteraceae), three alkamides were isolated and identified by mass- and NMR spectroscopic methods as (2E,6E,8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol, (1)), N-(2-phenethyl)-2E-en-6,8-nonadiynamide (2) and (2E,7Z)-6,9-endoperoxy-N-isobutyl-2,7-decadienamide (3). While 1 and 2 are known alkamides, compound 3 has not been described until now. It was found that the unusual cyclic peroxide 3 exists as a racemate of both enantiomers of each alkamide; the 6,9-cis- as well as the 6,9-trans-configured diastereomers, the former represents the major, the latter the minor constituent of the mixture. In vitro tests for activity against the human pathogenic parasites Trypanosoma brucei rhodesiense and Plasmodium falciparum revealed that 1 and 3 possess activity against the NF54 strain of the latter (IC50 values of 4.5 and 5.1 µM, respectively) while 2 was almost inactive. Compound 3 was also tested against multiresistant P. falciparum K1 and was found to be even more active against this parasite strain (IC50 = 2.1 µM) with considerable selectivity (IC50 against L6 rat skeletal myoblasts = 168 µM).

Bioinspired All-Polyester Diblock Copolymers Made from Poly(pentadecalactone) and Poly(2-(2-hydroxyethoxy)benzoate): Synthesis and Polymer Film Properties.

The bioinspired diblock copolymers poly(pentadecalactone)-block-poly(2-(2-hydroxyethoxy)-benzoate) (PPDL-block-P2HEB) were synthesized from pentadecalactone and dihydro-5H-1,4-benzodioxepin-5-one (2,3-DHB). No transesterification between the blocks was observed. In a sequential approach, PPDL obtained by ring-opening polymerization (ROP) was used to initiate 2,3-DHB. Here, the molar mass Mn of the P2HEB block was limited. In a modular approach, end-functionalized PPDL and P2HEB were obtained separately by ROP with functional initiators, and connected by 1,3-dipolar Huisgen reaction ("click-chemistry"). Block copolymer compositions from 85:15 mass percent to 28:72 mass percent (PPDL:P2HEB) were synthesized, with Mn of from about 30,000-50,000 g mol-1. The structure of the block copolymer was confirmed by proton NMR, FTIR spectroscopy, and gel permeation chromatography. Morphological studies by atomic force microscopy (AFM) further confirmed the block copolymer structure, while quantitative nanomechanical AFM measurements revealed that the DMT moduli of the block copolymers ranged between 17.2 ± 1.8 MPa and 62.3 ± 5.7 MPa, i.e. between the values of the parent P2HEB and PPDL homopolymers (7.6 ± 1.4 MPa and 801 ± 42 MPa, respectively). Differential scanning calorimetry showed that the thermal properties of the homopolymers were retained by each of the copolymer blocks (melting temperature 90 °C, glass transition temperature 36 °C).

Bioinspired All-Polyester Diblock Copolymers Made from Poly(pentadecalactone) and Poly(3-hydroxycinnamate): Synthesis and Polymer Film Properties.

A bioinspired diblock copolymer was synthesized from pentadecalactone and 3-hydroxy cinnamic acid. Poly(pentadecalactone) (PPDL) with a molar mass of up to 43,000 g mol-1 was obtained by ring-opening polymerization initiated propargyl alcohol. Poly(3-hydroxy cinnamate) (P3HCA) was obtained by polycondensation and end-functionalized with 3-azido propanol. The two functionalized homopolymers were connected via 1,3-dipolar Huisgen addition to yield the block copolymer PPDL-triazole-P3HCA. The structure the block copolymer was confirmed by proton NMR, FTIR spectroscopy and GPC. By analyzing the morphology of polymer films made from the homopolymers, from a 1:1 homopolymer blend, and from the PPDL-triazole-P3HCA block copolymer, clearly distinct micro- and nanostructures were revealed. Quantitative nanomechanical measurements revealed that the block copolymer PPDL-triazole-P3HCA had a DMT modulus of 22.3 ± 2.7 MPa, which was lower than that of the PPDL homopolymer (801 ± 42 MPa), yet significantly higher than that of the P3HCA homopolymer (1.77 ± 0.63 MPa). Thermal analytics showed that the melting point of PPDL-triazole-P3HCA was similar to PPDL (89-90 °C), while it had a glass transition was similar to P3HCA (123-124 °C). Thus, the semicrystalline, potentially degradable all-polyester block copolymer PPDL-triazole-P3HCA combines the thermal properties of either homopolymer, and has an intermediate elastic modulus.

Poly(oxanorbornene)-Coated CdTe Quantum Dots as Antibacterial Agents.

In this study, synthetic mimics of antimicrobial peptides based on poly(oxanorbornene) molecules (or PONs) were used to coat CdTe quantum dots (QDs). These PONs-CdTe QDs were investigated for their activity against Escherichia coli, a bacterium with antibiotic resistant strains. At the same time, the antibacterial activity of the PONs-CdTe QDs was compared to the antibacterial activity of free PONs and free CdTe QDs. The observed antibacterial activity of the PONs-CdTe QDs was additive and concentration dependent. The conjugates had a significantly lower minimum inhibitory concentration (MIC) than the free PONs and QDs, particularly for PONs-CdTe QDs which contained PONs of high amine density. The maximum activity of PONs-CdTe QDs was not realized by conjugating PONs with the highest intrinsic antibacterial activity (i.e., the lowest MIC in solution as free PONs), indicating that the mechanism of action for free PONs and PONs-CdTe QDs is different. Equally important, conjugating PONs to CdTe QDs decreased their hemolytic activity against red blood cells compared to free PONs, lending to higher therapeutic indices against E. coli. This could potentially enable the use of higher, and therefore more effective, PONs-QDs concentrations when addressing bacterial contamination, without concerns of adverse impacts on mammalian cells and organisms.

On the Limits of Benzophenone as Cross-Linker for Surface-Attached Polymer Hydrogels.

The synthesis of different photo-reactive poly(alkenyl norbornenes) and poly(oxonorbornenes) containing benzophenone (BP) via ring-opening metatheses polymerization (ROMP) is described. These polymers are UV irradiated to form well-defined surface-attached polymer networks and hydrogels. The relative propensity of the polymers to cross-link is evaluated by studying their gel content and its dependency on BP content, irradiation wavelength (254 or 365 nm) and energy dose applied (up to 11 J·cm-²). Analysis of the UV spectra of the polymer networks demonstrates that the poly(oxonorbornenes) show the expected BP-induced crosslinking behavior at 365 nm, although high irradiation energy doses and BP content are needed. However, these polymers undergo chain scission at 254 nm. The poly(alkenyl norbornenes), on the other hand, do not cross-link at 365 nm, whereas moderate to good cross-linking is observed at 254 nm. UV spectra demonstrate that the cross-linking at 254 nm is due to BP cross-linking combined with a [2 + 2] cylcoaddition of the alkenyl double bonds. This indicates limitations of benzophenone as a universally applicable cross-linking for polymer networks and hydrogels.