Mechanism of Action and Design of Potent Antibacterial Block Copolymer Nanoparticles.

Self-assembled polymer nanoparticles are promising antibacterials, with nonspherical morphologies of particular interest as recent work has demonstrated enhanced antibacterial activity relative to their spherical counterparts. However, the reasons for this enhancement are currently unclear. We have performed a multifaceted analysis of the antibacterial mechanism of action of 1D nanofibers relative to nanospheres by the use of flow cytometry, high-resolution microscopy, and evaluations of the antibacterial activity of pristine and tetracycline-loaded nanoparticles. Low-length dispersity, fluorescent diblock copolymer nanofibers with a crystalline poly(fluorenetrimethylenecarbonate) (PFTMC) core (length = 104 and 472 nm, height = 7 nm, width = 10-13 nm) and a partially protonated poly(dimethylaminoethyl methacrylate) (PDMAEMA) corona (length = 12 nm) were prepared via seeded growth living crystallization-driven self-assembly. Their behavior was compared to that of analogous nanospheres containing an amorphous PFTMC core (diameter of 12 nm). While all nanoparticles were uptaken into Escherichia coli W3110, crystalline-core nanofibers were observed to cause significant bacterial damage. Drug loading studies indicated that while all nanoparticle antibacterial activity was enhanced in combination with tetracycline, the enhancement was especially prominent when small nanoparticles (ca. 15-25 nm) were employed. Therefore, the identified differences in the mechanism of action and the demonstrated consequences for nanoparticle size and morphology control may be exploited for the future design of potent antibacterial agents for overcoming antibacterial resistance. This study also reinforces the requirement of morphological control over polymer nanoparticles for biomedical applications, as differences in activity are observed depending on their size, shape, and core-crystallinity.

Length-Controlled Nanofiber Micelleplexes as Efficient Nucleic Acid Delivery Vehicles.

Micelleplexes show great promise as effective polymeric delivery systems for nucleic acids. Although studies have shown that spherical micelleplexes can exhibit superior cellular transfection to polyplexes, to date there has been no report on the effects of micelleplex morphology on cellular transfection. In this work, we prepared precision, length-tunable poly(fluorenetrimethylenecarbonate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PFTMC16-b-PDMAEMA131) nanofiber micelleplexes and compared their properties and transfection activity to those of the equivalent nanosphere micelleplexes and polyplexes. We studied the DNA complexation process in detail via a range of techniques including cryo-transmission electron microscopy, atomic force microscopy, dynamic light scattering, and ζ-potential measurements, thereby examining how nanofiber micelleplexes form, as well the key differences that exist compared to nanosphere micelleplexes and polyplexes in terms of DNA loading and colloidal stability. The effects of particle morphology and nanofiber length on the transfection and cell viability of U-87 MG glioblastoma cells with a luciferase plasmid were explored, revealing that short nanofiber micelleplexes (length < ca. 100 nm) were the most effective delivery vehicle examined, outperforming nanosphere micelleplexes, polyplexes, and longer nanofiber micelleplexes as well as the Lipofectamine 2000 control. This study highlights the potential importance of 1D micelleplex morphologies for achieving optimal transfection activity and provides a fundamental platform for the future development of more effective polymeric nucleic acid delivery vehicles.

Structure-activity relationship studies on divalent naphthalene diimide G quadruplex ligands with anticancer and antiparasitic activity.

Naphthalene diimide (NDI) is a central scaffold that has been commonly used in the design of G-quadruplex (G4) ligands. Previous work revealed notable anticancer activity of a disubstituted N-methylpiperazine propyl NDI G4 ligand. Here, we explored structure-activity relationship studies around ligand bis-N,N-2,7-(3-(4-methylpiperazin-1-yl)propyl)-1,4,5,8-naphthalenetetracarboxylic diimide, maintaining the central NDI core whilst modifying the spacer and the nature of the cationic groups. We prepared new disubstituted NDI derivatives of the original compound and examined their in vitro antiproliferative and antiparasitic activity. Several N-methylpiperazine propyl NDIs showed sub-micromolar activity against Trypanosoma brucei and Leishmania major parasites with up to 30 fold selectivity versus MRC-5 cells. The best compound was a dimorpholino NDI with an IC50 of 0.17 µM against T.brucei and 40 fold selectivity versus MRC-5 cells. However, no clear correlation between G4 binding of the new NDI derivatives and antiproliferative or antiparasitic activity was observed, indicating that other mechanisms of action may be responsible for the observed biological activity.

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity*.

A facile imide coupling strategy for the one-step preparation of G-quadruplex ligands with varied core chemistries is described. The G-quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV-Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G-quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3, which was based on a perylene scaffold and exhibited 40-fold selectivity for a telomeric G-quadruplex over duplex DNA. Surprisingly, a tetra-substituted flexible core, compound 11, also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78-fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.

Extending the Scope of "Living" Crystallization-Driven Self-Assembly: Well-Defined 1D Micelles and Block Comicelles from Crystallizable Polycarbonate Block Copolymers.

Fiber-like block copolymer (BCP) micelles offer considerable potential for a variety of applications; however, uniform samples of controlled length and with spatially tailored chemistry have not been accessible. Recently, a seeded growth method, termed "living" crystallization-driven self-assembly (CDSA), has been developed to allow the formation of 1D micelles and block comicelles of precisely controlled dimensions from BCPs with a crystallizable segment. An expansion of the range of core-forming blocks that participate in living CDSA is necessary for this technique to be compatible with a broad range of applications. Few examples currently exist of well-defined, water-dispersible BCP micelles prepared using this approach, especially from biocompatible and biodegradable polymers. Herein, we demonstrate that BCPs containing a crystallizable polycarbonate, poly(spiro[fluorene-9,5'-[1,3]-dioxan]-2'-one) (PFTMC), can readily undergo living CDSA processes. PFTMC- b-poly(ethylene glycol) (PEG) BCPs with PFTMC:PEG block ratios of 1:11 and 1:25 were shown to undergo living CDSA to form near monodisperse fiber-like micelles of precisely controlled lengths of up to ∼1.6 µm. Detailed structural characterization of these micelles by TEM, AFM, SAXS, and WAXS revealed that they comprise a crystalline, chain-folded PFTMC core with a rectangular cross-section that is surrounded by a solvent swollen PEG corona. PFTMC- b-PEG fiber-like micelles were shown to be dispersible in water to give colloidally stable solutions. This allowed an assessment of the toxicity of these structures toward WI-38 and HeLa cells. From these experiments, we observed no discernible cytotoxicity from a sample of 119 nm fiber-like micelles to either healthy (WI-38) or cancerous (HeLa) cell types. The living CDSA process was extended to PFTMC- b-poly(2-vinylpyridine) (P2VP), and addition of this BCP to PFTMC- b-PEG seed micelles led to the formation of well-defined segmented fibers with spatially localized coronal chemistries.

Divalent Naphthalene Diimide Ligands Display High Selectivity for the Human Telomeric G-quadruplex in K+ Buffer.

Selective G-quadruplex ligands offer great promise for the development of anti-cancer therapies. A novel series of divalent cationic naphthalene diimide ligands that selectively bind to the hybrid form of the human telomeric G-quadruplex in K+ buffer are described herein. We demonstrate that an imidazolium-bearing mannoside-conjugate is the most selective ligand to date for this quadruplex against several other quadruplex and duplex structures. We also show that a similarly selective methylpiperazine-bearing ligand was more toxic to HeLa cancer cells than doxorubicin, whilst exhibiting three times less toxicity towards fetal lung fibroblasts WI-38.

A flexible solution to anion transport: powerful anionophores based on a cyclohexane scaffold.

Transmembrane anion carriers (anionophores) have potential for biological activity, including the treatment of channelopathies such as cystic fibrosis. A new family of anionophores has been synthesized, in which three thiourea groups are mounted on a cyclohexane-based scaffold. Though conceptually related to earlier polycyclic systems, these molecules are simpler and far more accessible. Preorganization is somewhat reduced compared to earlier systems, and anion affinities are correspondingly lower. However, transport activities set new records. This surprising performance suggests a role for controlled flexibility in the design of transmembrane anion carriers.

Optimization of precision nanofiber micelleplexes for DNA delivery.

As nucleic acid (NA) technologies continue to revolutionize medicine, new delivery vehicles are needed to effectively transport NA cargoes into cells. Uniform and length-tunable nanofiber micelleplexes have recently shown promise as versatile polymeric delivery vehicles for plasmid DNA, however the effects of several key parameters on micelleplex transfection and stability remain unknown. In this work, we compare poly(fluorenetrimethylenecarbonate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PFTMC-b-PDMAEMA) nanofiber micelleplexes to nanosphere micelleplexes and PDMAEMA polyplexes, examining the effects of complexation buffer, the temporal and serum stability of nanofiber micelleplexes, as well as the effects of cell density, cell type, and polymer DPn upon transfection efficiency and cell viability. These studies are vital for understanding the formation and biological activity of micelleplexes in more detail and should inform the future design of more advanced polymeric NA delivery systems.

Cellular uptake and targeting of low dispersity, dual emissive, segmented block copolymer nanofibers.

Polymer-based nanoparticles show substantial promise in the treatment and diagnosis of cancer and other diseases. Herein we report an exploration of the cellular uptake of tailored, low dispersity segmented 1D nanoparticles which were prepared from an amphiphilic block copolymer, poly(dihexylfluorene)-b-poly(ethyleneglycol) (PDHF13-b-PEG227), with a crystallizable PDHF core-forming block and a 'stealth' PEG corona-forming block with different end-group functionalities. Segmented C-B-A-B-C pentablock 1D nanofibers with varied spatially-defined coronal chemistries and a selected length (95 nm) were prepared using the living crystallization-driven self-assembly (CDSA) seeded-growth method. As the blue fluorescence of PDHF is often subject to environment-related quenching, a far-red BODIPY (BD) fluorophore was attached to the PEG end-group of the coronal B segments to provide additional tracking capability. Folic acid (FA) was also incorporated as a targeting group in the terminal C segments. These dual-emissive pentablock nanofibers exhibited uptake into >97% of folate receptor positive HeLa cells by flow cytometry. In the absence of FA, no significant uptake was detected and nanofibers with either FA or BD coronal groups showed no significant toxicity. Correlative light and electron microscopy (CLEM) studies revealed receptor-mediated endocytosis as an uptake pathway, with subsequent localization to the perinuclear region. A significant proportion of the nanofibers also appeared to interact with the cell membrane in an end-on fashion, which was coupled with fluorescence quenching of the PDHF core. These results provide new insights into the cellular uptake of polymer-based nanofibers and suggest their potential use in targeted therapies and diagnostics.

Is UV-Induced Electron-Driven Proton Transfer Active in a Chemically Modified A·T DNA Base Pair?

Transient electronic and vibrational absorption spectroscopies have been used to investigate whether UV-induced electron-driven proton transfer (EDPT) mechanisms are active in a chemically modified adenine-thymine (A·T) DNA base pair. To enhance the fraction of biologically relevant Watson-Crick (WC) hydrogen-bonding motifs and eliminate undesired Hoogsteen structures, a chemically modified derivative of A was synthesized, 8-(tert-butyl)-9-ethyladenine (8tBA). Equimolar solutions of 8tBA and silyl-protected T nucleosides in chloroform yield a mixture of WC pairs, reverse WC pairs, and residual monomers. Unlike previous transient absorption studies of WC guanine-cytosine (G·C) pairs, no clear spectroscopic or kinetic evidence was identified for the participation of EDPT in the excited-state relaxation dynamics of 8tBA·T pairs, although ultrafast (sub-100 fs) EDPT cannot be discounted. Monomer-like dynamics are proposed to dominate in 8tBA·T.