Improving antimicrobial release kinetics from hydrophobic polymer implants via ion pairing to combat biomedical-device related infection

Purpose The aim of this study is to improve the drug release properties of antimicrobial agents from hydrophobic biomaterials using using an ion pairing strategy. In so doing antimicrobial agents may be eluted and maintained over a sufficient time period thereby preventing bacterial colonisation and subsequent biofilm formation on medical devices. Methods The model antimicrobial agent was chlorhexidine and the selected fatty acid counter ions were capric acid, myristic acid and stearic acid. The polymethyl methacrylate films were loaded with 2% of fatty acid:antimicrobial agent at the following molar ratios; 0.5:1M, 1:1M and 2:1M and thermally polymerized using azobisisobutyronitrile initiator. Drug release experiments were subsequently performed over a 3-month period and the mass of drug released under sink conditions (pH 7.0, 37oC) quantified using a validated HPLC-UV method. Results In all platforms, a burst of chlorhexidine release was observed over the initial 24-hour period. Similar release kinetics were observed between the formulations during the initial 28 days. However, as time progressed, the chlorhexidine baseline plateaued after 56 days whereas formulations containing the counterions appeared to continuously elute linearly with time. As can be observed in figure 1, the rank order of total chlorhexidine release in the presence of 0.5M fatty acid was myristic acid (40%) > capric acid (35%) > stearic acid (30%)> chlorhexidine baseline (15%). Conclusion The incorporation of fatty acids within the formulation significantly improved chlorhexidine solubility within both the monomer and the polymer and enhanced the drug release kinetics over the period of study. This is attributed to the greater diffusivity of chlorhexidine through PMMA in the presence of fatty acids. In th absence of fatty acids, chlorhexidine release was facilitated by dissolution of surface associated drug particles. This study has illustrated the ability of fatty acids to modulate chlorhexidine release from a model biomaterial through enhanced diffusivity. This strategy may prove advantageous for improved medical devices with enhanced resistance to infection.

The antimicrobial potential of ionic liquids: A source of chemical diversity for infection and biofilm control

Although described almost a century ago, interest in ionic liquids has flourished in the last two decades, with significant advances in the understanding of their chemical, physical and biological property sets driving their widespread application across multiple and diverse research areas. Significant progress has been made through the contributions of numerous research groups detailing novel libraries of ionic liquids, often ‘task-specific’ designer solvents for application in areas as diverse as separation technology, catalysis and bioremediation. Basic antimicrobial screening has often been included as a surrogate indication of the environmental impact of these compounds widely regarded as ‘green’ solvents. Obviating the biological properties, specifically toxicity, of these compounds has obstructed their potential application as sophisticated designer biocides. A recent tangent in ionic liquids research now aims to harness tuneable biological properties of these compounds in the design of novel potent antimicrobials, recognising their unparalleled flexibility for chemical diversity in a severely depleted antimicrobial arsenal. This review concentrates primarily on the antimicrobial potential of ionic liquids and aims to consolidate contemporary microbiological background information, assessment protocols and future considerations necessary to advance the field in light of the urgent need for antimicrobial innovation.

Extracellular DNA impedes the transport of vancomycin in Staphylococcus epidermidis biofilms pre-exposed to sub-inhibitory concentrations of vancomycin

Staphylococcus epidermidis biofilm formation is responsible for the persistence of orthopedic implant infections. Previous studies have shown that exposure of S. epidermidis biofilms to sub-MICs of antibiotics induced an increased level of biofilm persistence. BODIPY FL-vancomycin (a fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC-vancomycin-treated S. epidermidis biofilms was impeded compared to that of control, untreated biofilms. Further experiments showed an increase in the extracellular DNA (eDNA) concentration in biofilms preexposed to sub-MIC vancomycin, suggesting a potential role for eDNA in the hindrance of vancomycin activity. Exogenously added, S. epidermidis DNA increased the planktonic vancomycin MIC and protected biofilm cells from lethal vancomycin concentrations. Finally, isothermal titration calorimetry (ITC) revealed that the binding constant of DNA and vancomycin was 100-fold higher than the previously reported binding constant of vancomycin and its intended cellular D-Ala-D-Ala peptide target. This study provides an explanation of the eDNA-based mechanism of antibiotic tolerance in sub-MIC-vancomycin-treated S. epidermidis biofilms, which might be an important factor for the persistence of biofilm infections.

Dengue Envelope Domain III-Peptide Binding Analysis via Tryptophan Fluorescence Quenching Assay

In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300–400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt ina concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not dueto a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain whenincubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenientspectrophotometric binding assay for the analysis of EIII–peptide interactions in a drug screening application.

Novel monoclonal antibody and peptide binders for Mycobacterium avium subsp. paratuberculosis and their application for magnetic separation

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Identification of Peptide Inhibitors of Enveloped Viruses Using Support Vector Machine

The peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. There are three types of envelope proteins each exhibiting distinct structure folds. Although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. The common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. Here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. The model displayed 92% prediction accuracy with the Matthew’s correlation coefficient of 0.84, obviously superior to those using physicochemical properties and amino acid decomposition as input. The predictive support vector machine model for self- derived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

Intermedin and calcitonin gene-related peptide fail to shine in acute coronary syndrome

Aims: To measure levels of intermedin and calcitonin gene-related peptide (CGRP) in acute coronary syndrome (ACS) and to determine if they are elevated. 
Methods and results: 81 patients admitted with suspected ACS were enrolled into the study. 50 were confirmed ACS by ACC (2000) guidelines and 31 were in a control group as non-cardiac chest pain. Intermedin was nonsignificantly elevated 6.14 pg/ml vs 4.84 pg/ml b8 h in the ACS group; sensitivity 68%, specificity 63% on presenting sample. Intermedinwas significantly elevated in those patientswho had an initially negative troponin T (b0.03 ng/ml) on presentation, 6.67 pg/ml vs 4.84 pg/ml, p = 0.03. CGRP was significantly elevated in ACS patients, 8–b16 h after pain onset, 8.67 pg/ml vs 7.08 pg/ml, p= 0.036. However, it didn't aid diagnosis in initially negative troponin patients; sensitivity 61%, specificity 60% on presenting sample. Both intermedin and CGRP were elevated in STEMI patients on a first sample, but only intermedin was significantly elevated; 7.03 pg/ml vs 4.84 pg/ml, p =0.02 and 8.87 pg/ml vs 7.03 pg/ml p = 0.093, respectively. High sensitivity troponin T was significant elevated in the ACS group at b8 h (414.9 vs 17.22, p= 0.006) and at 8–b16 h (3325.27 vs 21.54, p = 0.02). 
Conclusions: Both intermedin and CGRP are detectable in human patients. Levels showa trend to elevation in ACS, with CGRP being significantly raised N8 h after pain onset. The degree of elevation will have limited clinical applicability.

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae

Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.

Methylene Blue-Loaded Dissolving Microneedles: Potential Use in Photodynamic Antimicrobial Chemotherapy of Infected Wounds

Photodynamic therapy involves delivery of a photosensitising drug that is activated by light of a specific wavelength, resulting in generation of highly reactive radicals. This activated species can cause destruction of targeted cells. Application of this process for treatment of microbial infections has been termed "photodynamic antimicrobial chemotherapy" (PACT). In the treatment of chronic wounds, the delivery of photosensitising agents is often impeded by the presence of a thick hyperkeratotic/necrotic tissue layer, reducing their therapeutic efficacy. Microneedles (MNs) are an emerging drug delivery technology that have been demonstrated to successfully penetrate the outer layers of the skin, whilst minimising damage to skin barrier function. Delivering photosensitising drugs using this platform has been demonstrated to have several advantages over conventional photodynamic therapy, such as, painless application, reduced erythema, enhanced cosmetic results and improved intradermal delivery. The aim of this study was to physically characterise dissolving MNs loaded with the photosensitising agent, methylene blue and assess their photodynamic antimicrobial activity. Dissolving MNs were fabricated from aqueous blends of Gantrez(®) AN-139 co-polymer containing varying loadings of methylene blue. A height reduction of 29.8% was observed for MNs prepared from blends containing 0.5% w/w methylene blue following application of a total force of 70.56 N/array. A previously validated insertion test was used to assess the effect of drug loading on MN insertion into a wound model. Staphylococcus aureus, Escherichia coli and Candida albicans biofilms were incubated with various methylene blue concentrations within the range delivered by MNs in vitro (0.1-2.5 mg/mL) and either irradiated at 635 nm using a Paterson Lamp or subjected to a dark period. Microbial susceptibility to PACT was determined by assessing the total viable count. Kill rates of >96%, were achieved for S. aureus and >99% for E. coli and C. albicans with the combination of PACT and methylene blue concentrations between 0.1 and 2.5 mg/mL. A reduction in the colony count was also observed when incorporating the photosensitiser without irradiation, this reduction was more notable in S. aureus and E. coli strains than in C. albicans.

Glucagon-like peptide-1 based therapies as a novel approach for chronic heart failure

Glucagon-like peptide-1 (GLP-1) is an endogenous peptide hormone whose metabolic effects have been exploited for glycaemic control in diabetes, but which also exerts important cardiovascular actions. We have recently reported that the GLP-1 mimetic, exendin-4, exerts clear benefits post-myocardial infarction via specific effects on extracellular matrix remodelling which is dysregulated in the diabetic heart (Robinson E et al, Basic Res Cardiol 2015; 110: 20), and have now shown similar cardioprotective actions in experimental diabetes, which are mediated via direct effects on infiltrating macrophages (Tate M et al, Basic Res Cardiol 2015; in press). Taken together with the apparent complexity of GLP-1 signalling and disappointing results of recent cardiovascular trials, our work strongly suggests that selective targeting of GLP-1 may be required in order to realise therapeutic benefit for both diabetic and non-diabetic heart failure patients. This is particularly important given the epidemic increase in the incidence of diabetes which is associated with a markedly enhanced susceptibility to cardiovascular stress.

Glucagon-like peptide-1 based therapies as a novel approach for chronic heart failure

Glucagon-like peptide-1 (GLP-1) is an endogenous peptide hormone whose metabolic effects have been exploited for glycaemic control in diabetes, but which also exerts important cardiovascular actions. We have recently reported that the GLP-1 mimetic, exendin-4, exerts clear benefits post-myocardial infarction via specific effects on extracellular matrix remodelling which is dysregulated in the diabetic heart (Robinson E et al, Basic Res Cardiol 2015; 110: 20), and have now shown similar cardioprotective actions in experimental diabetes, which are mediated via direct effects on infiltrating macrophages (Tate M et al, Basic Res Cardiol 2016; 111: 1). Taken together with the apparent complexity of GLP-1 signalling and disappointing results of recent cardiovascular trials, our work strongly suggests that selective targeting of GLP-1 may be required in order to realise therapeutic benefit for both diabetic and non-diabetic heart failure patients. This is particularly important given the epidemic increase in the incidence of diabetes which is associated with a markedly enhanced susceptibility to cardiovascular stress.