PurposeTo characterise a biorelevant simulated lung fluid (SLF) based on the composition of human respiratory tract lining fluid. SLF was compared to other media which have been utilized as lung fluid simulants in terms of fluid structure, biocompatibility and performance in inhalation biopharmaceutical assays.MethodsThe structure of SLF was investigated using cryo-transmission electron microscopy, photon correlation spectroscopy and Langmuir isotherms. Biocompatibility with A549 alveolar epithelial cells was determined by MTT assay, morphometric observations and transcriptomic analysis. Biopharmaceutical applicability was evaluated by measuring the solubility and dissolution of beclomethasone dipropionate (BDP) and fluticasone propionate (FP), in SLF.ResultsSLF exhibited a colloidal structure, possessing vesicles similar in nature to those found in lung fluid extracts. No adverse effect on A549 cells was apparent after exposure to the SLF for 24 h, although some metabolic changes were identified consistent with the change of culture medium to a more lung-like composition. The solubility and dissolution of BDP and FP in SLF were enhanced compared to Gamble’s solution.ConclusionThe SLF reported herein constitutes a biorelevant synthetic simulant which is suitable to study biopharmaceutical properties of inhalation medicines such as those being proposed for an inhaled biopharmaceutics classification system.

self-assembled monolayers; SLD, scattering length density; SMW, silicon matched water; SSI, surgical site infections; SRS, standard reaction solution; TMPA, 3-(trimethoxysilyl)propyl acrylate; TSA, tryptone soya agar. ABSTRACTHT61 is a quinoline-derived antimicrobial, which exhibits bactericidal potency against both multiplying and quiescent methicillin resistant and sensitive Staphylococcus aureus, and has been proposed as an adjunct for other antimicrobials in order to extend their usefulness in the face of increasing antimicrobial resistance. In this study we have examined HT61's effect on the permeability of Staphylococcus aureus membranes and whether this putative activity can be attributed to an interaction with lipid bilayers. Using membrane potential and ATP release assays, we have shown that HT61 disrupts the membrane enough to results in depolarisation of the membrane and release of intercellular constituents at concentrations above and below the minimum inhibitory concentration of the drug. Utilising both monolayer subphase injection and neutron reflectometry we have shown that increasing the anionic lipid content of the membrane leads to a more marked effect of the drug. In bilayers containing 25 mol% phosphatidylglycerol, neutron reflectometry data suggests that exposure to HT61 increases the level of solvent in the hydrophobic region of the membrane, which is indicative of gross structural damage. Increasing the proportion of PG elicits a concomitant level of membrane damage resulting in almost total destruction when 75 mol% phosphatidylglycerol is present.We therefore propose that HT61's primary action is directed towards the cytoplasmic membrane of Gram positive bacteria.The quinoline-derived cationic antimicrobial HT61 [1] was initially developed to improve the success of nasal decolonisation interventions aimed at decreasing the risk of post-operative surgical site infections (SSI) posed by carriage of methicillin resistant Staphylococcus aureus (MRSA) [2,3] . However, more recently it has been proposed as a resistance breaker to be used in conjunction with other more established antimicrobials [4] . Although the protein synthesis inhibitor mupirocin is currently widely used for anti-MRSA nasal decolonisation, it is not active against non-multiplying persister bacteria which constitute a reservoir for recolonisation [5] . In addition to this, an increased prevalence of mupirocin resistant MRSA [6] has led to the development of more effective alternatives, including the experimental antimicrobials LTX-109 [7] and XF-73 [8] in addition to HT61 [1] .The mode of action of HT61 has not hitherto been thoroughly investigated, however initial cell-based assays showed that HT61 is capable of depolarising the cytoplasmic membrane ofGram positives with further evidence from electron microscopy suggesting that HT61 causes lysis of either the membrane or cell wall [1,4] . The putative membrane-targeting of HT61 may provide some explanation for its potency against non-multiplying MRSA [9] , and suggests that...

π-Conjugated polymer nanoparticles (CPNs) are under investigation as photoluminescent agents for diagnostics and bioimaging. To determine whether the choice of surfactant can improve CPN properties and prevent protein adsorption, five nonionic polyethylene glycol alkyl ether surfactants were used to produce CPNs from three representative π-conjugated polymers. The surfactant structure did not influence size or yield, which was dependent on the nature of the conjugated polymer. Hydrophobic interaction chromatography, contact angle, quartz crystal microbalance, and neutron reflectivity studies were used to assess the affinity of the surfactant to the conjugated polymer surface and indicated that all surfactants were displaced by the addition of a model serum protein. In summary, CPN preparation methods which rely on surface coating of a conjugated polymer core with amphiphilic surfactants may produce systems with good yields and colloidal stability in vitro, but may be susceptible to significant surface alterations in physiological fluids.

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Gram-negative bacteria possess numerous defenses against antibiotics, due to the intrinsic permeability barrier of their outer membrane, explaining the recalcitrance of some common and life-threatening infections. We report the formulation of a new drug, PPA148, which shows promising activity against all Gram-negative bacteria included in the ESKAPEE pathogens. PPA148 was solubilized by inclusion complexation with cyclodextrin followed by encapsulation in liposomes. The complex and liposomal formulation presented increased activity against E. coli compared to the pure drug when assessed with the Kirby Bauer assay. The novel formulation containing 1 μg PPA148 reached similar efficacy levels equivalent to those of 30 μg pure rifampicin. A range of biophysical techniques was used to explore the mechanism of drug uptake. Langmuir trough (LT) and neutron reflectivity (NR) techniques were employed to monitor the interaction between the drug and the formulation with model membranes. We found evidence for fluidosome fusion with the model Gram-negative outer membrane and for cyclodextrins acting as inner membrane permeation enhancers without presenting intrinsic antimicrobial activity. An antibiotic-in-cyclodextrin-in-liposomes (ACL) formulation was developed, which targets both the bacterial OM and IM, and offers promise as a means to breach the Gram-negative cell envelope.