Facile Modification of Medical-Grade Silicone for Antimicrobial Effectiveness and Biocompatibility: A Potential Therapeutic Strategy against Bacterial Biofilms.

A one-step modification of biomedical silicone tubing with N,N-dimethyltetradecylamine, C14, results in a composition designated WinGard-1 (WG-1, 1.1 wt % C14). A surface-active silicon-amine phase (SAP) is proposed to account for increased wettability and increased surface charge. To understand the mechanism of antimicrobial effectiveness, several procedures were employed to detect whether C14 leaching occurred. An immersion-growth (IG) test was developed that required knowing the bacterial Minimum Inhibitory Concentrations (MICs) and Minimum Biocidal Concentrations (MBCs). The C14 MIC and MBC for Gm- uropathogenic E. coli (UPEC), commonly associated with catheter-associated urinary tract infections (CAUTI), were 10 and 20 µg/mL, respectively. After prior immersion of WG-1 silicone segments in a growth medium from 1 to 28 d, the IG test for the medium showed normal growth for UPEC over 24 h, indicating that the concentration of C14 must be less than the MIC, 10 µg/mL. GC-MS and studies of the medium inside and outside a dialysis bag containing WG-1 silicone segments supported de minimis leaching. Consequently, a 5 log UPEC reduction (99.999% kill) in 24 h using the shake flask test (ASTM E2149) cannot be due to leaching and is ascribed to contact kill. Interestingly, although the MBC was greater than 100 µg/mL for Pseudomonas aeruginosa, WG-1 silicone affected an 80% reduction via a 24 h shake flask test. For other bacteria and Candida albicans, greater than 99.9% shake flask kill may be understood by proposing increased wettability and concentration of charge illustrated in the TOC. De minimis leaching places WG-1 silicone at an advantage over conventional anti-infectives that rely on leaching of an antibiotic or heavy metals such as silver. The facile process for preparation of WG-1 silicone combined with biocidal effectiveness comprises progress toward the goals of device designation from the FDA for WG-1 and clearance.

PEAMOtecan, a novel chronotherapeutic polymeric drug for brain cancer.

Glioblastoma multiforme (GBM) is an aggressive and difficult to treat form of brain cancer. In this work, we report on a novel chronotherapeutic polymeric drug, PEAMOtecan, for GBM therapy. PEAMOtecan was synthesized by conjugating camptothecin, a topoisomerase I inhibitor, to our proprietary, 'clickable' and modular polyoxetane polymer platform consisting of acetylene-functionalized 3-ethyl-3-(hydroxymethyl)oxetane (EAMO) repeat units (Patent No.: US 9,421,276) via the linker 3,3'-dithiodipropionic acid (DDPA) with a disulfide bond (SS) extended by short-chain polyethylene glycol (PEG). We show that PEAMOtecan is a highly modular polymer nanoformulation that protects covalently bound CPT until slowly being released over extended periods of time dependent on the cleavage of the disulfide and ester linkages. PEAMOtecan kills glioma cells by mitotic catastrophe with p53 mutant/knockdown cells being more sensitive than matched wild type cells potentially providing cancer-specific targeting. To establish proof-of-principle therapeutic effects, we tested PEAMOtecan as monotherapy for efficacy in a mouse orthotopic glioma model. PEAMOtecan was administered by one-time, convection-enhanced delivery (CED) intra-tumorally to achieve superior distribution and extended drug release over time. In addition, the near-infrared (NIR) dye Cy5.5 was coupled to the polymer providing live-animal imaging capability to track tissue distribution and clearance of the injected polymer over time. We show that PEAMOtecan significantly improves the survival of mice harboring intra-cranial tumors (p = .0074 compared to untreated group). Altogether, these results support further development and testing of our nanoconjugate platform.

Surface Characterization, Antimicrobial Effectiveness, and Human Cell Response for a Biomedical Grade Polyurethane Blended with a Mixed Soft Block PTMO-Quat/PEG Copolyoxetane Polyurethane.

Infection is a serious medical complication associated with health care environments. Despite advances, the 5-10% incidence of infections for hospital patients is well documented. Sources of pathogenic organisms include medical devices such as catheters and endotracheal tubes. Offering guidance for curbing the spread of such infections, a model antimicrobial coating is described herein that kills bacteria on contact but is compatible with human cells. To achieve these characteristics, a novel blend of a conventional biomedical grade polyurethane (Tecoflex) with mixed soft block polyurethane is described. The functional polyurethane (UP-C12-50-T) has a copolyoxetane soft block P-C12-50 with quaternary ammonium (C12) and PEG-like side chains and a conventional poly(tetramethylene oxide) (PTMO, T) soft block. DSC and DMA data point to limited miscibility of UP-C12-50-T with Tecoflex. The blend of Tecoflex with 10 wt % UP-C12-50-T designated UP-C12-50-T-10 radically changed surface properties. Evidence for surface concentration of the P-C12-50 soft block was obtained by atomic force microscopy (AFM), dynamic contact angles (DCAs), zeta potentials (ζ), and X-ray photoelectron spectroscopy (XPS). The antimicrobial effectiveness of the blend coatings was established by the ASTM E2149 "shake flask" test for challenges of E. coli and a methicillin resistant strain of S. epidermidis. Cytocompatibility was demonstrated with an in vitro test designed for direct contact (ISO 10993-5). Growth of human mesenchymal stem cells (MSCs) beside and under UP-C12-50-T-10 indicated remarkable biocompatibility for a composition that is also strongly antimicrobial. Overall, the results point to a model coating with a level of P-C12-50 that combines high antimicrobial effectiveness and low toxicity to human cells.

Development of mannose functionalized dendrimeric nanoparticles for targeted delivery to macrophages: use of this platform to modulate atherosclerosis.

Dysfunctional macrophages underlie the development of several diseases including atherosclerosis where accumulation of cholesteryl esters and persistent inflammation are 2 of the critical macrophage processes that regulate the progression as well as stability of atherosclerotic plaques. Ligand-dependent activation of liver-x-receptor (LXR) not only enhances mobilization of stored cholesteryl ester but also exerts anti-inflammatory effects mediated via trans-repression of proinflammatory transcription factor nuclear factor kappa B. However, increased hepatic lipogenesis by systemic administration of LXR ligands (LXR-L) has precluded their therapeutic use. The objective of the present study was to devise a strategy to selectively deliver LXR-L to atherosclerotic plaque-associated macrophages while limiting hepatic uptake. Mannose-functionalized dendrimeric nanoparticles (mDNP) were synthesized to facilitate active uptake via the mannose receptor expressed exclusively by macrophages using polyamidoamine dendrimer. Terminal amine groups were used to conjugate mannose and LXR-L T091317 via polyethylene glycol spacers. mDNP-LXR-L was effectively taken up by macrophages (and not by hepatocytes), increased expression of LXR target genes (ABCA1/ABCG1), and enhanced cholesterol efflux. When administered intravenously to LDLR-/- mice with established plaques, significant accumulation of fluorescently labeled mDNP-LXR-L was seen in atherosclerotic plaque-associated macrophages. Four weekly injections of mDNP-LXR-L led to significant reduction in atherosclerotic plaque progression, plaque necrosis, and plaque inflammation as assessed by expression of nuclear factor kappa B target gene matrix metalloproteinase 9; no increase in hepatic lipogenic genes or plasma lipids was observed. These studies validate the development of a macrophage-specific delivery platform for the delivery of anti-atherosclerotic agents directly to the plaque-associated macrophages to attenuate plaque burden.

A Polycation Antimicrobial Peptide Mimic without Resistance Buildup against Propionibacterium Acnes.

A preliminary study is reported for a polycation antimicrobial peptide (AMP) mimic against Propionibacterium acnes, which is associated with acne vulgaris, a common skin condition. Antibiotics are commonly used against P. acnes but buildup of resistance is well-known. Worse, antibiotic regimens build up resistance for more sensitive bacteria such as Staphylococcus epidermidis. The polycation AMP mimic C12-50, 1, is chosen for the present study as it has been previously shown to have high antimicrobial effectiveness. This study reports that C12-50 is active against P. acnes (strain ATCC 6919) with a minimum inhibitory concentration (MIC) of 6.3 µg mL-1 . To monitor resistance build-up ten passages are conducted with C12-50 against P. acnes. The MIC remains constant with no resistance buildup. Parallel studies with erythromycin confirm previously reported resistance buildup. The results point to a promising pathway to applications for polycation AMP mimics against P. acnes.

Nanoconjugated NAP as a Potent and Periphery Selective Mu Opioid Receptor Modulator To Treat Opioid-Induced Constipation.

Opioids are the mainstay for cancer and noncancer pain management. However, their use is often associated with multiple adverse effects. Among them, the most common and persistent one is probably opioid-induced constipation (OIC). Periphery selective opioid antagonists may alleviate the symptoms of OIC without compromising the analgesic effects of opioids. Recently our laboratories have identified one novel lead compound, 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-[(4'-pyridyl)acetamido]morphinan (NAP), as a peripherally selective mu opioid receptor ligand carrying subnanomolar affinity to the mu opioid receptor and over 100-folds of selectivity over both the delta and kappa opioid receptors, with reasonable oral availability and half-life, and potential to treat OIC. Nanoparticle-based drug delivery systems are now widely considered due to their technological advantages such as good stability, high carrier capacity, low therapeutic side effects, etc. Herein we report nanoparticle supported NAP as a potential candidate for OIC treatment with improved peripheral selectivity over the original lead compound NAP.

Real-Time Observation of Antimicrobial Polycation Effects on Escherichia coli: Adapting the Carpet Model for Membrane Disruption to Quaternary Copolyoxetanes.

Real-time atomic force microscopy (AFM) was used for analyzing effects of the antimicrobial polycation copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12-50 showed a 4 log kill at 15 µg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 µg/mL C12-50 are therefore reasonably attributed to cell death. At the highest concentration (60 µg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water-soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12-50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level. In future work this approach will provide important insights into structure-property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.

Click synthesis of a polyamidoamine dendrimer-based camptothecin prodrug.

In the present work we report on the click synthesis of a new camptothecin (CPT) prodrug based on anionic polyamidoamine (PAMAM) dendrimer intended for cancer therapy. We applied 'click' chemistry to improve polymer-drug coupling reaction efficiency. Specifically, CPT was functionalized with a spacer, 1-azido-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (APO), via EDC/DMAP coupling reaction. In parallel, propargylamine (PPA) and methoxypoly(ethylene glycol) amine were conjugated to PAMAM dendrimer G4.5 in sequence using an effective coupling agent 4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM). CPT-APO was then coupled to PEGylated PAMAM dendrimer G4.5-PPA via a click reaction using copper bromide/2,2'-bipyridine/ dimethyl sulfoxide (catalyst/ligand/solvent). Human glioma cells were exposed to the CPT-conjugate to determine toxicity and cell cycle effects using WST-1 assay and flow cytometry. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 µM, a 185-fold increase relative to free CPT, presumably as a result of slow release. As expected, conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. Altogether, highly efficient click chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery.

Click hybridization of immune cells and polyamidoamine dendrimers.

Immobilizing highly branched polyamidoamine (PAMAM) dendrimers to the cell surface represents an innovative method of enhancing cell surface loading capacity to deliver therapeutic and imaging agents. In this work, hybridized immune cells, that is, macrophage RAW264.7 (RAW), with PAMAM dendrimer G4.0 (DEN) on the basis of bioorthogonal chemistry are clicked. Efficient and selective cell surface immobilization of dendrimers is confirmed by confocal microscopy. Viability and motility of RAW-DEN hybrids remain the same as untreated RAW cells according to WST-1 assay and wound closure assay. Furthermore, Western blot analysis reveals that there are no significant alterations in the expression levels of signaling molecules AKT, p38, and NFκB (p65) and their corresponding activated (phosphorylated) forms in RAW cells treated with azido sugar and dendrimer, indicating that the hybridization process neither induced cell stress response nor altered normal signaling pathways. Taken together, this work shows the feasibility of applying bioorthogonal chemistry to create cell-nanoparticle hybrids and demonstrates the noninvasiveness of this cell surface engineering approach.

Poly (ethylene glycol)-armed hyperbranched polyoxetanes for anticancer drug delivery.

A facile method for synthesis of polyethylene glycol (PEG)-armed hyperbranched polyoxetanes is presented as well as characterization and use in drug delivery. A series of hyperbranched polyoxetanes with multiple PEG arms were synthesized via a one-pot cationic ring-opening polymerization of 3-ethyl-3-hydroxymethyloxetane (EHMO) and its PEGylated derivative (EPMO), in which the feed mass ratio of EHMO to EPMO was 98:2, 96:4, 74:26, or 17:83. Characterization methods included NMR, DLS, FT-IR, DSC, and SEM. Toxicity of the synthesized polymers to human dermal fibroblasts was evaluated using the MTT assay. Formulation into particles was carried out to encapsulate the anticancer drug camptothecin using the single oil-in-water (o/w) solvent evaporation method. The resulting drug encapsulated particles were evaluated for antitumor activity using HN12 cells.

Synthesis of water-soluble camptothecin-polyoxetane conjugates via click chemistry.

Water-soluble camptothecin (CPT)-polyoxetane conjugates were synthesized using a clickable polymeric platform P(EAMO) that was made by polymerization of acetylene-functionalized 3-ethyl-3-(hydroxymethyl)oxetane (i.e., EAMO). CPT was first modified with a linker 6-azidohexanoic acid via an ester linkage to yield CPT-azide. CPT-azide was then click coupled to P(EAMO) in dichloromethane using bromotris(triphenylphosphine)copper(I)/N,N-diisopropylethylamine. For water solubility and cytocompatibility improvement, methoxypolyethylene glycol azide (mPEG-azide) was synthesized from mPEG 750 g mol(-1) and click grafted using copper(II) sulfate and sodium ascorbate to P(EAMO)-g-CPT. (1)H NMR spectroscopy confirmed synthesis of all intermediates and the final product P(EAMO)-g-CPT/PEG. CPT was found to retain its therapeutically active lactone form. The resulting P(EAMO)-g-CPT/PEG conjugates were water-soluble and produced dose-dependent cytotoxicity to human glioma cells and increased γ-H2AX foci formation, indicating extensive cell cycle-dependent DNA damage. Altogether, we have synthesized CPT-polymer conjugates able to induce controlled toxicity to human cancer cells.

Competition between Ultrafast Energy Flow and Electron Transfer in a Ru(II)-Loaded Polyfluorene Light-Harvesting Polymer.

This Letter describes the synthesis and photophysical characterization of a Ru(II) assembly consisting of metal polypyridyl complexes linked together by a polyfluorene scaffold. Unlike many scaffolds incorporating saturated linkages, the conjugated polymer in this system acts as a functional light-harvesting component. Conformational disorder breaks the conjugation in the polymer backbone, resulting in a chain composed of many chromophore units, whose relative energies depend on the segment lengths. Photoexcitation of the polyfluorene by a femtosecond laser pulse results in the excitation of polyfluorene, which then undergoes direct energy transfer to the pendant Ru(II) complexes, producing Ru(II)* excited states within 500 fs after photoexcitation. Femtosecond transient absorption data show the presence of electron transfer from PF* to Ru(II) to form charge-separated (CS) products within 1-2 ps. The decay of the oxidized and reduced products, PF(+•) and Ru(I), through back electron transfer are followed using picosecond transient absorption methods.