Small Molecule Interferences in Resazurin and MTT-Based Metabolic Assays in the Absence of Cells.

In vitro assays (such as resazurin and MTT) provide an opportunity to determine the cytotoxicity of novel therapeutics before moving forward with expensive and resource-intensive in vivo studies. A concern with using these assays, however, is the production of false responses in the presence of particular chemical functionalities. To better understand this phenomenon, 19 small molecules at 6 concentrations (1 µM-100 mM) were tested in the presence of resazurin and MTT reagents to highlight potential interfering species. Through the use of absorbance measurements (using well-plate assays and UV-vis spectroscopy) with parallel MS analysis, we have shown that significant conversion of the assay reagents readily occurs in the presence of many tested interfering species without the need for any cellular activity. The most attributable sources of interference seem to arise from the presence of thiol and carboxylic acid moieties. Interestingly, the detectable interferences were more prevalent and larger in the presence of MTT (19 species with some deviations >3000%) compared to resazurin (16 species with largest deviation of ∼150%). Additionally, those deviations in the presence of resazurin were only substantial at high concentrations, while MTT showed deviations across the tested concentrations. This comprehensive study gives insight into chemical functional groups (thiols, amines, amides, carboxylic acids) that may interfere with resazurin and MTT assays in the absence of metabolic activity and indicates that proper control studies must be performed to obtain accurate data from these in vitro assays.

Surface-Anchored Metal-Organic Framework-Cotton Material for Tunable Antibacterial Copper Delivery.

In the present study, a new copper metal-organic framework (MOF)-cotton material was strategically fabricated to exploit its antibacterial properties for postsynthetic modification (PSM) to introduce a free amine to tune the physicochemical properties of the material. A modified methodology for carboxymethylation of natural cotton was utilized to enhance the number of nucleation sites for the MOF growth. Subsequently, MOF Cu3(NH2BTC)2 was synthesized into a homogenous surface-supported film via a layer-by-layer dip-coating process. The resultant materials contained uniformly distributed 1 µm × 1 µm octahedral MOF crystals around each carboxymethylated fiber. Importantly, the accessible free amine of the MOF ligand allowed for the PSM of the MOF-cotton surface with valeric anhydride, yielding 23.5 ± 2.2% modified. The Cu2+ ion-releasing performance of the materials was probed under biological conditions per submersion in complex media at 37 °C. Indeed, PSM induces a change in the copper flux of the material over the first 6 h. The materials continue to slowly release Cu2+ ions beyond 24 h tested at a flux of 0.22 ± 0.003 µmol·cm-2·h-1 with the unmodified MOF-cotton and at 0.25 ± 0.004 µmol·cm-2·h-1 with the modified MOF-cotton. The antibacterial activity of the material was explored using Escherichia coli by testing the planktonic and attached bacteria under a variety of conditions. MOF-cotton materials elicit antibacterial effects, yielding a 4-log reduction or greater, after 24 h of exposure. Additionally, the MOF-cotton materials inhibit the attachment of bacteria, under both dry and wet conditions. A material of this type would be ideal for clothing, bandages, and other textile applications. As such, this work serves as a precedence toward developing uniform, tunable MOF-composite textile materials that can kill bacteria and prevent the attachment of bacteria to the surface.

Biodegradable crosslinked polyesters derived from thiomalic acid and S-nitrosothiol analogues for nitric oxide release.

Crosslinked polyesters with Young's moduli similar to that of certain soft biological tissues were prepared via bulk polycondensation of thiomalic acid and 1,8-octanediol alone, and with citric or maleic acid. The copolymers were converted to nitric oxide (NO)-releasing S-nitrosothiol (RSNO) analogues by reaction with tert-butyl nitrite. Additional conjugation steps were avoided by inclusion of the thiolated monomer during the polycondensation to permit thiol conversion to RSNOs. NO release at physiological pH and temperature (pH 7.4, 37 °C) was determined by chemiluminescence-based NO detection. The average total NO content for poly(thiomalic-co-maleic acid-co-1,8-octanediol), poly(thiomalic-co-citric acid-co-1,8-octanediol), and poly(thiomalic acid-co-1,8-octanediol) was 130 ± 39 µmol g-1, 200 ± 35 µmol g-1, and 130 ± 11 µmol g-1, respectively. The antibacterial properties of the S-nitrosated analogues were confirmed against Escherichia coli and Staphylococcus aureus. The hydrolytic degradation products were analyzed by time-of-flight mass spectrometry after a 10-week study to investigate their composition. Tensile mechanical tests were performed on the non-nitrosated polymers as well as their S-nitrosated derivatives and suggested that the materials have appropriate Young's moduli and elongation values for biomedical applications.

Critical nitric oxide concentration for Pseudomonas aeruginosa biofilm reduction on polyurethane substrates.

Bacterial colonies that reside on a surface, known as biofilms, are intrinsically impenetrable to traditional antibiotics, ultimately driving research toward an alternative therapeutic approach. Nitric oxide (NO) has gained attention for its biologically beneficial properties, particularly centered around its antibacterial capabilities. NO donors that can release the molecule under physiological conditions (such as S-nitrosothiols) can be utilized in clinical settings to combat bacterial biofilm infections. Herein the authors describe determining a critical concentration of NO necessary to cause >90% reduction of a Pseudomonas aeruginosa biofilm grown on medical grade polyurethane films. The biofilm was grown under optimal culture conditions [in nutrient broth media (NBM) at 37 °C] for 24 h before the addition of the NO donor S-nitrosoglutathione (GSNO) in NBM for an additional 24 h. The cellular viability of the biofilm after the challenge period was tested using varying concentrations of NO to determine the critical amount necessary to cause at least a 90% reduction in bacterial biofilm viability. The critical GSNO concentration was found to be 10 mM, which corresponds to 2.73 mM NO. Time kill experiments were performed on the 24 h biofilm using the critical amount of NO at 4, 8, 12, and 16 h and it was determined that the 90% biofilm viability reduction occurred at 12 h and was sustained for the entire 24 h challenge period. This critical concentration was subsequently tested for total NO release via a nitric oxide analyzer. The total amount of NO released over the 12 h challenge period was found to be 5.97 ± 0.66 × 10(-6) mol NO, which corresponds to 1.49 ± 0.17 µmol NO/ml NBM. This is the first identification of the critical NO concentration needed to elicit this biological response on a medically relevant polymer.

Plasma-modified nitric oxide-releasing polymer films exhibit time-delayed 8-log reduction in growth of bacteria.

Tygon(®) and other poly(vinyl chloride)-derived polymers are frequently used for tubing in blood transfusions, hemodialysis, and other extracorporeal circuit applications. These materials, however, tend to promote bacterial proliferation which contributes to the high risk of infection associated with device use. Antibacterial agents, such as nitric oxide donors, can be incorporated into these materials to eliminate bacteria before they can proliferate. The release of the antimicrobial agent from the device, however, is challenging to control and sustain on timescales relevant to blood transport procedures. Surface modification techniques can be employed to address challenges with controlled drug release. Here, surface modification using H2O (v) plasma is explored as a potential method to improve the biocompatibility of biomedical polymers, namely, to tune the nitric oxide-releasing capabilities from Tygon films. Film properties are evaluated pre- and post-treatment by contact angle goniometry, x-ray photoelectron spectroscopy, and optical profilometry. H2O (v) plasma treatment significantly enhances the wettability of the nitric-oxide releasing films, doubles film oxygen content, and maintains surface roughness. Using the kill rate method, the authors determine both treated and untreated films cause an 8 log reduction in the population of both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Notably, however, H2O (v) plasma treatment delays the kill rate of treated films by 24 h, yet antibacterial efficacy is not diminished. Results of nitric oxide release, measured via chemiluminescent detection, are also reported and correlated to the observed kill rate behavior. Overall, the observed delay in biocidal agent release caused by our treatment indicates that plasma surface modification is an important route toward achieving controlled drug release from polymeric biomedical devices.

Nitric oxide release from a biodegradable cysteine-based polyphosphazene.

Nitric oxide (NO) is a unique bioactive molecule that performs multiple physiological functions and has been found to exhibit antithrombotic, antimicrobial, and wound-healing effects as an exogenous therapeutic agent. NO release from polymeric materials intended for use in biomedical applications has been established to reduce their thrombogenicity and decrease the likelihood of infection and inflammation that frequently produce medical complications. As a result, numerous NO-releasing polymers have been developed in an effort to utilize the beneficial properties of NO to improve the performance of implantable materials. The majority of synthetic NO-releasing biodegradable polymers that have been reported to date are polyesters, and there is significant interest in the development of new NO-releasing materials with improved or distinctive physicochemical characteristics. Polyphosphazenes are polymers with inorganic phosphorus-nitrogen backbones, and hydrolytically-sensitive derivatives with organic substituents have been prepared that degrade under physiological conditions. For this reason, biodegradable poly(organophosphazenes) are interesting candidate materials for applications such as tissue engineering, where the addition of NO release capability may be therapeutically useful. Herein, we report the first development and characterization of an NO-releasing poly(organophosphazene) from poly(ethyl S-methylthiocysteinyl-co-ethyl cysteinyl phosphazene) (POP-EtCys-SH). The thiolated polymer was synthesized from the reaction of poly(dichlorophosphazene) with ethyl S-methylthiocysteinate, followed by partial cleavage of the disulfide linkages to form free thiol groups. The conversion of thiol to the NO-releasing S-nitrosothiol functional group with tert-butyl nitrite resulted in a polymer (POP-EtCys-NO) with an average NO content of 0.55 ± 0.04 mmol g-1 that was found to release a total of 0.35 ± 0.02 mmol NO g-1 over 24 h under physiological conditions (37 °C, pH 7.4 phosphate buffered saline). Extracts obtained from both the thiolated and S-nitrosated polymers were not found to significantly impair the viability of human dermal fibroblasts or induce morphological changes, indicating that this cysteine-based polyphosphazene may possess potential utility as an NO-releasing biomaterial.

Reprint of: Nitric oxide-releasing polysaccharide derivative exhibits 8-log reduction against Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus.

Health-care associated infections (HAIs) and the increasing number of antibiotic-resistant bacteria strains remain significant public health threats worldwide. Although the number of HAIs has decreased by using improved sterilization protocols, the cost related to HAIs is still quantified in billions of dollars. Furthermore, the development of multi-drug resistant strains is increasing exponentially, demonstrating that current treatments are inefficient. Thus, the quest for new methods to eradicate bacterial infection is increasingly important in antimicrobial, drug delivery and biomaterials research. Herein, the bactericidal activity of a water-soluble NO-releasing polysaccharide derivative was evaluated in nutrient broth media against three bacteria strains that are commonly responsible for HAIs. Data confirmed that this NO-releasing polysaccharide derivative induced an 8-log reduction in bacterial growth after 24h for Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus. Additionally, the absence of bacteria after 72 h of exposure to NO illustrates the inability of the bacteria to recover and the prevention of biofilm formation. The presented 8-log reduction in bacterial survival after 24h is among the highest reduction reported for NO delivery systems to date, and reaches the desired standard for industrially-relevant reduction. More specifically, this system represents the only water-soluble antimicrobial to reach such a significant bacterial reduction in nutrient rich media, wherein experimental conditions more closely mimic the in vivo environment than those in previous reports. Furthermore, the absence of bacterial activity after 72 h and the versatility of using a water-soluble compound suggest that this NO-releasing polysaccharide derivative is a promising route for treating HAIs.

Nitric oxide-releasing polysaccharide derivative exhibits 8-log reduction against Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus.

Health-care associated infections (HAIs) and the increasing number of antibiotic-resistant bacteria strains remain significant public health threats worldwide. Although the number of HAIs has decreased by using improved sterilization protocols, the cost related to HAIs is still quantified in billions of dollars. Furthermore, the development of multi-drug resistant strains is increasing exponentially, demonstrating that current treatments are inefficient. Thus, the quest for new methods to eradicate bacterial infection is increasingly important in antimicrobial, drug delivery and biomaterials research. Herein, the bactericidal activity of a water-soluble NO-releasing polysaccharide derivative was evaluated in nutrient broth media against three bacteria strains that are commonly responsible for HAIs. Data confirmed that this NO-releasing polysaccharide derivative induced an 8-log reduction in bacterial growth after 24h for Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus. Additionally, the absence of bacteria after 72h of exposure to NO illustrates the inability of the bacteria to recover and the prevention of biofilm formation. The presented 8-log reduction in bacterial survival after 24h is among the highest reduction reported for NO delivery systems to date, and reaches the desired standard for industrially-relevant reduction. More specifically, this system represents the only water-soluble antimicrobial to reach such a significant bacterial reduction in nutrient rich media, wherein experimental conditions more closely mimic the in vivo environment than those in previous reports. Furthermore, the absence of bacterial activity after 72h and the versatility of using a water-soluble compound suggest that this NO-releasing polysaccharide derivative is a promising route for treating HAIs.

Nitric oxide-releasing S-nitrosated derivatives of chitin and chitosan for biomedical applications.

Nitric oxide (NO)-releasing derivatives of chitin and chitosan were prepared through incorporation of the symmetrical dithiols 1,2-ethanedithiol, 1,3-propanedithiol, and 1,6-hexanedithiol, followed by S-nitrosation with tert-butyl nitrite. The NO loading of the materials and their real-time NO release profiles under physiological conditions (pH 7.4 phosphate buffered saline, 37 °C) were recorded over 24 hours, and in vitro cytotoxicity studies were performed using human dermal fibroblasts (HDF) to assess the suitability of the materials for biomedical applications. Of the six thiolated parent materials, five exhibited cell viability higher than 70% (MTS assay), an outcome that was corroborated by LIVE/DEAD assay. In all cases, HDF morphology was unaffected by the presence of extracts obtained from the thiolated materials.