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Nitric oxide releasing poly(vinylidene fluoride-co-hexafluoropropylene) films using a fluorinated nitric oxide donor to greatly decrease chemical leaching.

ABSTRACT: Nitric oxide (NO) releasing polymers have been widely applied as biomaterials for a variety of biomedical implants and devices. However, the chemical leaching of NO donors and their byproduct species is almost always observed during the application of polymers doped with NO donors, unless the donor is covalently linked to the polymer. Herein, we report the first NO releasing poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) fluorinated copolymer prepared by incorporating a fluorinated S-nitrosothiol as the NO donor. Under physiological conditions, the resulting polymeric films can release NO for 16?days. Importantly, due to both fluorine-fluorine and electrostatic charge interactions between the fluorinated NO donor and the PVDF-HFP copolymer, the total chemical leaching of the fluorinated NO donor and its disulfide product after 9?day was only 0.6% (mol%) of the initial amount of NO donor loaded into the film. These new NO release PVDF-HFP films exhibit antimicrobial and anti-biofilm activities against both Gram positive S. aureus and Gram negative P. aeruginosa strains. The NO-releasing PVDF-HFP polymer can also be coated on Teflon tubing to release NO under physiological conditions for extended time periods. This NO-releasing PVDF-HFP copolymer with greatly reduced chemical leaching could help enhance the biocompatibility and antimicrobial activity of various biomedical devices. STATEMENT OF SIGNIFICANCE: Fluoropolymers have been widely used in creating various biomedical implants and devices. However, nitric oxide (NO) release fluoropolymers have not been well studied to date. Additionally, in the application of biomaterials doped with NO donors, a significant amount of NO donors and their byproducts almost always leach into aqueous environment. We now report an NO releasing poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) fluoropolymer by incorporating a new fluorinated S-nitrosothiol. The NO release can last for 16?days under physiological conditions. The total chemical leaching was determined to be only 0.6?mol% of the initial S-nitrosothiol loaded. As expected, significant antimicrobial/anti-biofilm activities of the NO release PVDF-HFP film were observed against Gram positive S. aureus and Gram negative P. aeruginosa bacterial strains.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC6513704 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Project description:Wild-type Campylobacter jejuni NCTC11168 was grown in 2 homemade, parallel chemostats. Cells were grown in 125 ml volumes of Mueller-Hinton Broth where the growth rate was controlled by the dilution rate (0.1 h-1). A gas mix of 80% nitrogen, 10% carbon dioxide and 10% oxygen was pumped into the head-space of the vessels at a rate of 0.25 l/min to obtain a microaerobic environment and the culture was maintained at 42 ÂºC via a water jacket. Cells were grown as above until steady-state had been reached. At steady-state one of the cultures was exposed to 10 uM NOC-5 & -7 (final concentration) for a period of 10 min. Samples of both the control and stressed cultures were harvested into phenol/ethanol to stabilize the RNA and total RNA was purified using Qiagenâ??s RNeasy Mini kit (as recommended by the suppliers) prior to use in microarray analysis. Equal quantities of RNA from control and GSNO-supplemented cultures were labelled using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 4). The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. C. jejuni NCTC11168 was grown in two parallel custom-built chemostats based on Sigma Proculture Dynalift spinner flasks with a working volume of 125 ml, and the dilution rate (which at steady state is equal to the specific growth rate) of 0.1 h-1. A gas mix of 10% O2, 10% CO2 and 80% N2 was passed into the head space of the culture vessel at a rate of 0.25 l min-1 to obtain a microaerobic atmosphere, and silicone water jackets maintained the temperature at 42 ÂºC. The flasks were placed on KMO 2 basic IKA-Werke stirrers (arbitrary setting, 260; Scientific Laboratory Supplies), which gaveeffective transfer of O2 from the gaseous atmosphere to the culture by virtue of a stable vortex. A fresh overflow sample was used to check the pH at the time of harvest (which was consistently between 6.6 and 7) and to ensure that there was no contamination by plating a 20 Î¼l sample onto both nutrient agar plates incubated at 37 Â°C at atmospheric oxygen, and Mueller-Hinton plates incubated at 42 Â°C in the microaerophilic work station. When staedy-state was reached, as verified by collecting at least 5 culture volumes, NOC-5 & -7 (final concentration 10 uM) was added to one culture. After a further 10 min the 125 ml volume was separated into 3 equal volumes and each was mixed immediately on ice with 3.56 ml 100% ethanol and 185 ml phenol to stabilize the RNA. The cells were subsequently harvested by centrifugation. Total RNA was purified by using a Qiagen RNeasy Mini kit as recommended by the supplier. cDNA synthesis was carried out using 12 Âµg of starting material, which was primed with 9 Âµg of pd(N)6 random hexamers (Amersham Biosciences). Reaction mixtures (20 Âµl) containing 0.5 mM ATP, 0.5 mM TTP, 0.5 mM GTP, 0.2 mM CTP, and 1 mM Cy3- or 1 mM Cy5-dCTP were incubated for 2 h at 42 Â°C with 200 U of Superscript II RNase H reverse transcriptase (Invitrogen). Following synthesis, cDNA was purified by using a PCR purification kit (Qiagen) to remove the unincorporated deoxynucloside triphosphates, fluorescent dye, and primers. Equal volumes of cDNA were combined and evaporated to near dryness with an SPD121P Speed Vac (Thermon Savant). For hybridisation to the microarray slides, cDNA was resuspended in an appropriate volume of salt-based hybridization buffer (provided by Ocimum Biosolutions). Prior to addition to the slides, cDNA samples were heated to 95 Â°C for 3 min, and then placed on ice for no more than 3 min. The slides were placed in MWG Biotech hybridization chambers and incubated for 16 to 24 h in a shaking water bath at 110 rpm and 42 Â°C. Following incubation , the slides were washed sequentially for 5 min in 2x SSC-1 % sodium dodecyl sulphate, 1x SSC, 0.5x SSC and 0.1x SSC at 37 Â°C with gentle agitation (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The slides were dried by centrifugation at 254 xg for 5 min and subsequently scanned with an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained by using commercially available software from Biodiscovery, Inc. (Imagine version 4.0, and Genesight, version 3.5). The mean values from each channel were log2 transformed and normalized by using the subtract-by-mean method to remove intensity-dependent effects in the log2 (ratio) values. The Cy3/Cy5 fluorescence ratios were calculated from the normalized values. Biological experiments (i.e. chemostat growth with and without 0.25 mM GSNO addition) were carried out three times and a dye swap was performed for two of the three experiments which provided two technical repeats, one each for two of the three biological repeats. Data from the independent experiments were combined. Genes that were differentially expressed â?¥ twofold and displayed and P value of â?¤ 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

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Nitric oxide releasing poly(vinylidene fluoride-co-hexafluoropropylene) films using a fluorinated nitric oxide donor to greatly decrease chemical leaching.

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