A Langmuir approach on monolayer interactions to investigate surface active peptides

The Langmuir Blodgett apparatus provides a versatile system for studying the interfacial properties of peptides and peptide-membrane interactions under controlled conditions. Using amphiphilic α-helical peptides to highlight studies undertaken, here we discuss the use of this system to provide information on the surface activity of peptides and describe the insights these studies give into biological functio

A theoretical analysis of secondary structural characteristics of anticancer peptides

Here, cluster analysis showed that a database of 158 peptides formed 21 clusters based on net positive charge, hydrophobicity and amphiphilicity. In general these clusters showed similar median toxicities (p = 0.176) against eukaryotic cell lines and no single combination of these properties was found optimal for efficacy. The database contained 14 peptides, which showed selectivity for tumour cell lines only (ACPCT), 123 peptides with general toxicity to eukaryotic cells (ACPGT) and 21 inactive peptides (ACPI). Hydrophobic arc size analysis showed that there was no significant difference across the datasets. Even though there was no correlation there was no correlation observed, peptides with wide hydrophobic arcs (> 270°) appeared less toxic. Extended hydrophobic moment plot analysis predicted that over 50% of ACPCT and ACPGT peptides would be surface active, which led to the suggestion that amphiphilicity is a key driver of the membrane interactions for these peptides but probably plays a role in their efficacy rather than their selectivity. This analysis also predicted that only 14% of ACPCT peptides compared to 45% of ACPGT peptides were candidates for tilted peptide formation. This implies that those peptides with non-specific activity may have a tendency towards the utilisation of membrane disruptive structures such as tilt peptides which led to the suggestion that the absence of this structure may support cancer cell selectivity. However, these analyses predicted that ACPI peptides, which possess no anticancer activity, would also form surface active and tilted a-helices, clearly showing that other factors are involved in determining the efficacy and selectivity of ACPs

Challenging the Clostridium botulinum toxin type A (BoNT/A) with a selection of microorganisms by culture methods and extended storage of used vials to assess the loss of sterility

In 2002, botulinum toxin type A (BoNT/A) was approved by the US Food and Drug Administration (FDA) for cosmetic use. However, there may be procedural differences between the ways in which a clinician handles, applies and stores the product compared to the suggested guidelines of the manufacturer for handling and storage. To this end vials (N = 12) of BoNT/A were tested for the incidence of microbial contamination followed by challenging the product with a selection of microorganisms by culture methods and by using a calcein release assay to contaminate multi-dose vials at the single concentration used for facial aesthetics. A culture, droplet method was used to count microorganisms challenged with the therapeutic product and to compare viability levels in appropriate controls as well as measuring their lytic properties via an existing cell-free system involving calcein release. Counts of test organisms within the droplets, with the product and the controls without the product were undertaken using Image J software. The result from the incidence of in-vial contamination was inconclusive. Bacterial levels between controls and product challenged groups demonstrated no differences in the growth of viable microorganisms following immediate contact (p = ≥ 0.05). The cell-free calcein release assay demonstrated differences at all time points for low levels of lysis in each case with bacterial lipid extract and were statistically significant (p = 0.011). Although these data appear to correlate with the minimum inhibitory concentration, the additives and vial integrity are also likely to contribute to the maintenance of BoNT/A sterility

Development of a novel, multifunctional, membrane-interactive pyridinium salt with potent anticancer activity

The synthesis and biological evaluation of a novel pyridinium salt is reported. Initial membrane interaction with isolated phospholipid monolayers was obtained with the pyridinium salt, and two neutral analogues for comparison, and the anticancer effects of the best compound established using a cytotoxicity screening assay against glioma cells using both an established cell line and three short-term cell cultures – one of which has been largely resistant to all chemotherapeutic drugs tested to date. The results indicate that the pyridinium salt exhibits potent anticancer activity (EC50s = 9.8-312.5 μM) on all cell types, including the resistant one, for a continuous treatment of 72 hours. Microscopic examination of the treated cells using a trypan blue exclusion assay showed membrane lysis had occurred. Therefore, this letter highlights the potential for a new class of pyridinium salt to be developed as a much needed alternative treatment for glioma chemotherapy

Toward Rational Design of Selective Molecularly Imprinted Polymers (MIPs) for Proteins: Computational and Experimental Studies of Acrylamide Based Polymers for Myoglobin

Molecularly imprinted polymers (MIPs) have potential as alternatives to antibodies in the diagnosis and treatment of disease. However, atomistic level knowledge of the prepolymerization process is limited that would facilitate rational design of more efficient MIPs. Accordingly, we have investigated using computation and experiment the protein-monomer binding interactions that may influence the desired specificity. Myoglobin was used as the target protein and five different acrylamide-based monomers considered. Protein binding sites were predicted using SiteMap and binding free energies of monomers at each site calculated using MM-GBSA. Statistical thermodynamic analysis and study of atomistic interactions facilitated rationalization of monomer performance in MIP rebinding studies (% rebind; imprinting factors). CD spectroscopy was used to determine monomer effects on myoglobin secondary structure, with all monomers except the smallest monomer (acrylamide) causing significant changes. A complex interplay between different protein-monomer binding effects and MIP efficacy was observed. Validation of hypotheses for key binding features was achieved by rational selection of two different co-monomer MIP combinations that produced experimental results in agreement with predictions. The co-monomer studies revealed that uniform, non-competitive binding of monomers around a target protein is favourable. This study represents a step towards future rational in silico design of MIPs for proteins

Rapid Sub-nanomolar Protein Determination in Serum using Electropolymerized Molecularly Imprinted Polymers (E-MIPs)

Rapid detection of biologicals is important for a range of applications such as medical screening and diagnostics. Antibodies are typically employed for biosensing with high sensitivity and selectivity but can take months to prepare. Here, we investigate electropolymerized molecularly imprinted polymers (E-MIPs), which are produced in minutes as alternative-antibody rapid biosensors for the selective recognition of model proteins bovine haemoglobin (BHb) and bovine serum albumin (BSA). We evaluated two disposable screen-printed electrodes (SPE) designated AT-Au and BT-Au based on their different annealing temperatures. E-MIPs for BHb demonstrated an imprinting factor of 146:1 at 1nM and 12:1 at 0.1nM, showing high effectiveness of E-MIPs compared to their control non-imprinted polymers. The BHb imprinted E-MIP, when tested against BSA as a non-target protein, gave a selectivity factor of 6:1 for BHb. Sensor sensitivity directly depended on the nature of the SPE, with AT-Au SPE demonstrating limits of detection in the sub-micromolar range typically achieved for MIPs, while BT-Au SPE exhibited sensitivity in the sub-nanomolar range for target protein. We attribute this to differences in electrode surface area between AT-Au and BT-Au SPEs. The E-MIPs were also tested in calf serum as a model biological medium. The BT-Au SPE MIPs detected the presence of target protein in < 10 min with an LOD of 50 pM and LOQ of 100 pM, suggesting their suitability for protein determination in serum with minimal sample preparation. Using electrochemical impedance spectroscopy, we determine equilibrium dissociation constants (KD) for E-MIPs using the Hill-Langmuir adsorption model. KD of BHb E-MIP was determined to be 0.86 ± 0.11nM

Evaluation of acrylamide-based molecularly imprinted polymer thin-sheets for specific protein capture - a myoglobin model

We evaluate a series of thin-sheet hydrogel molecularly imprinted polymers (MIPs), using a family of acrylamide-based monomers, selective for the target protein myoglobin (Mb). The simple production of the thin-sheet MIP offers an alternative biorecognition surface that is robust, stable and uniform, and has the potential to be adapted for biosensor applications. The MIP containing the functional monomer N-hydroxymethylacrylamide (NHMAm), produced optimal specific rebinding of the target protein (Mb) with 84.9 % (± 0.7) rebinding and imprinting and selectivity factors of 1.41 and 1.55, respectively. The least optimal performing MIP contained the functional monomer N,Ndimethylacrylamide (DMAm) with 67.5 % (± 0.7) rebinding and imprinting and selectivity factors of 1.11 and 1.32, respectively. Hydrogen bonding effects, within a protein-MIP complex, were investigated using computational methods and Fourier transform infrared (FTIR) spectroscopy. The quantum mechanical calculations predictions of a red shift of the monomer carbonyl peak is borneout within FTIR spectra, with three of the MIPs, acrylamide, N-(hydroxymethyl) acrylamide, and N-(hydroxyethyl) acrylamide, showing peak downshifts of 4, 11, and 8 cm-1, respectively

Porphyromonas gingivalis LPS and Actinomyces naeslundii conditioned medium enhance the release of a low molecular weight, transcriptionally active, fragment of glycogen synthase-3 kinase in IMR-32 cell line

Background: Glycogen synthase-3 kinase (GSK3) is one of the major contributors of tau hyperphosphorylation linked to neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD). Objectives: To determine a mechanism of GSK-3β activation by two periodontal bacteria consistently confirmed in AD autopsied brains. Methods: Porphyromonas. gingivalis FDC381 and Actinomyces naeslundii ATCC10301 (designated An) conditioned media were collected. IMR-32 cells were challenged for 48h with the conditioned media alongside P. gingivalis (ATCC33277) ultrapurified lipopolysaccharide (LPS) designated Pg.LPS under established cell culture conditions either, alone or combined. Gene expression, and protein analyses for GSK-3β were carried out. Results: qPCR demonstrated that GSK-3β gene was over-expressed in IMR-32 cells treated with Pg.LPS with a 2.09 fold change (p=0.0005) whilst An treated cells demonstrated 1.41 fold change (p=0.004). Western blotting of the cells challenged with Pg.LPS (p=0.01) and An conditioned medium (p=0.001) demonstrated the 37 kDa band for each treatment with variable intensity across the medium control. Immunohistochemistry with the GSK-3β of the IMR-32 cells challenged with Pg.LPS and An alone demonstrated cytoplasmic and nuclear localisation. Conclusions: Exposure to various bacterial factors up-regulated the gene expression of GSK-3β. Western blotting for GSK-3β confimed the presence of the cleaved fragment by Pg.LPS (37 kDa band p=0.01) and An conditioned medium (37 kDa band p=0.001). Immunostaining demonstrated both cytoplasmic and nuclear localisation of GSK-3β. Therefore, Pg.LPS and an unknown factor from the An conditioned medium mediated GSK-3β activation via its transcriptionally active, cleaved, fragment. These virulence factors in the body appear to be detrimental to brain health

Aerosolizable Lipid-Nanovesicles Encapsulating Voriconazole Effectively Permeate Pulmonary Barriers and Target Lung Cells

The entire world has recently been witnessing an unprecedented upsurge in microbial lung infections. The major challenge encountered in treating the same is to ensure the optimum drug availability at the infected site. Aerosolization of antimicrobials, in this regard, has shown immense potential owing to their localized and targeted effect. Efforts, therefore, have been undertaken to systematically develop lung-phosphatidylcholine-based lipid nanovesicles of voriconazole for potential management of the superinfections like aspergillosis. LNVs, prepared by thin-film hydration method, exhibited a globule size of 145.4 ± 19.5 nm, polydispersity index of 0.154 ± 0.104 and entrapment efficiency of 71.4 ± 2.2% with improved in vitro antifungal activity. Aerodynamic studies revealed a microdroplet size of ≤5 μm, thereby unraveling its promise to target the physical barrier of lungs effectively. The surface-active potential of LNVs, demonstrated through Langmuir-Blodgett troughs, indicated their ability to overcome the biochemical pulmonary surfactant monolayer barrier, while the safety and uptake studies on airway-epithelial cells signified their immense potential to permeate the cellular barrier of lungs. The pharmacokinetic studies showed marked improvement in the retention profile of voriconazole in lungs following LNVs nebulization compared to pristine voriconazole. Overall, LNVs proved to be safe and effective delivery systems, delineating their distinct potential to efficiently target the respiratory fungal infections

In Vitro Characterization of Inhalable Cationic Hybrid Nanoparticles as Potential Vaccine Carriers

In this study, PGA-co-PDL nanoparticles (NPs) encapsulating model antigen, bovine serum albumin (BSA), were prepared via double emulsion solvent evaporation. In addition, chitosan hydrochloride (CHL) was incorporated into the external phase of the emulsion solvent method, which resulted in surface adsorption onto the NPs to form hybrid cationic CHL NPs. The BSA encapsulated CHL NPs were encompassed into nanocomposite microcarriers (NCMPs) composed of l-leucine to produce CHL NPs/NCMPs via spray drying. The CHL NPs/NCMPs were investigated for in vitro aerosolization, release study, cell viability and uptake, and stability of protein structure. Hybrid cationic CHL NPs (CHL: 10 mg/mL) of particle size (480.2 ± 32.2 nm), charge (+14.2 ± 0.72 mV), and BSA loading (7.28 ± 1.3 µg/mg) were produced. The adsorption pattern was determined to follow the Freundlich model. Aerosolization of CHL NPs/NCMPs indicated fine particle fraction (FPF: 46.79 ± 11.21%) and mass median aerodynamic diameter (MMAD: 1.49 ± 0.29 µm). The BSA α-helical structure was maintained, after release from the CHL NPs/NCMPs, as indicated by circular dichroism. Furthermore, dendritic cells (DCs) and A549 cells showed good viability (≥70% at 2.5 mg/mL after 4–24 h exposure, respectively). Confocal microscopy and flow cytometry data showed hybrid cationic CHL NPs were successfully taken up by DCs within 1 h of incubation. The upregulation of CD40, CD86, and MHC-II cell surface markers indicated that the DCs were successfully activated by the hybrid cationic CHL NPs. These results suggest that the CHL NPs/NCMPs technology platform could potentially be used for the delivery of proteins to the lungs for immunostimulatory applications such as vaccines