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Knowledge of odor thresholds is very important for the perfume industry. Due to the difficulty associated with measuring odor thresholds, empirical models capable of estimating these values can be an invaluable contribution to the field. This work developed a framework based on scientific machine learning strategies. A transfer learning-based strategy was devised, where information from a graph convolutional network predicting semantic odor descriptors was used as input data for the feedforward neural network responsible for estimating odor thresholds for chemical substances based on their molecular structures. The predictive performance of this model was compared to a benchmark odor threshold prediction model based on molecular structures that did not utilize transfer learning. Furthermore, the prediction was compared to a correlation previously proposed in the literature and a dummy regressor. Results demonstrated that the transfer learning-based strategy displayed a better predictive performance, suggesting this technique can be useful for predicting odor thresholds., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Cationic and hydrophilic coatings based on casting and drying water dispersions of two different nanoparticles (NPs) onto glass are here described and evaluated for antimicrobial activity. Discoid cationic bilayer fragments (BF) surrounded by carboxy-methylcellulose (CMC) and poly (diallyl dimethyl ammonium) chloride (PDDA) NPs and spherical gramicidin D (Gr) NPs dispersed in water solution were cast onto glass coverslips and dried, forming a coating quantitatively evaluated against Pseudomonas aeruginosa , Staphylococcus aureus and Candida albicans . From plating and colony forming units (CFU) counting, all strains interacting for 1 h with the coatings lost viability from 10 5 to 10 6 , to zero CFU, at two sets of Gr and PDDA doses: 4.6 and 25 μg, respectively, or, 0.94 and 5 μg, respectively. Combinations produced broad spectrum, antimicrobial coatings; PDDA electrostatically attached to the microbes damaging cell walls, allowing Gr NPs interaction with the cell membrane. This concerted action promoted optimal activity at low Gr and PDDA doses. Further washing and drying of the deposited dried coatings showed that they were washed out so that antimicrobial activity was no longer present on the glass surface. Significant applications in biomedical materials can be foreseen for these transient coatings.

The current methods applied to material screening for adsorption-based heat pumps are based on a fixed set of temperatures or their independent variation, providing a limited, insufficient, and unpractical evaluation of different adsorbents. This work proposes a novel strategy for the simultaneous optimization and material screening in the design of adsorption heat pumps by implementing a meta-heuristic approach, particle swarm optimization (PSO). The proposed framework can effectively evaluate variable and broad operation temperature intervals to search for viable zones of operation for multiple adsorbents at once. The criteria for selecting the adequate material were the maximum performance and the minimum heat supply cost, which were considered the objective functions of the PSO algorithm. First, the performance was assessed individually, followed by a single-objective approximation of the multi-objective problem. Next, a multi-objective approach was also adopted. With the results generated during the optimization, it was possible to find which adsorbents and temperature sets were the most suitable according to the main objective of the operation. The Fisher-Snedecor test was applied to expand the results obtained during PSO application and a feasible operating region built around the optima, enabling the arrangement of close-to-optima data into practical design and control tools. This approach allowed for a fast and intuitive evaluation of multiple design and operation variables., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Flavor is an essential component in the development of numerous products in the market. The increasing consumption of processed and fast food and healthy packaged food has upraised the investment in new flavoring agents and consequently in molecules with flavoring properties. In this context, this work brings up a scientific machine learning (SciML) approach to address this product engineering need. SciML in computational chemistry has opened paths in the compound's property prediction without requiring synthesis. This work proposes a novel framework of deep generative models within this context to design new flavor molecules. Through the analysis and study of the molecules obtained from the generative model training, it was possible to conclude that even though the generative model designs the molecules through random sampling of actions, it can find molecules that are already used in the food industry, not necessarily as a flavoring agent, or in other industrial sectors. Hence, this corroborates the potential of the proposed methodology for the prospecting of molecules to be applied in the flavor industry., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Flavor is the focal point in the flavor industry, which follows social tendencies and behaviors. The research and development of new flavoring agents and molecules are essential in this field. However, the development of natural flavors plays a critical role in modern society. Considering this, the present work proposes a novel framework based on scientific machine learning to undertake an emerging problem in flavor engineering and industry. It proposes a combining system composed of generative and reinforcement learning models. Therefore, this work brings an innovative methodology to design new flavor molecules. The molecules were evaluated regarding synthetic accessibility, the number of atoms, and the likeness to a natural or pseudo-natural product. This work brings as contributions the implementation of a web scraper code to sample a flavors database and the integration of two scientific machine learning techniques in a complex system as a framework. The implementation of the complex system instead of the generative model by itself obtained 10% more molecules within the optimal results. The designed molecules obtained as an output of the reinforcement learning model's generation were assessed regarding their existence or not in the market and whether they are already used in the flavor industry or not. Thus, we corroborated the potentiality of the framework presented for the search of molecules to be used in the development of flavor-based products.

Model-based optimization of simulated moving bed reactors (SMBRs) requires efficient solvers and significant computational power. Over the past years, surrogate models have been considered for such computationally demanding optimization problems. In this sense, artificial neural networks-ANNs-have found applications for modeling the simulated moving bed (SMB) unit but not yet been reported for the reactive SMB (SMBR). Despite ANNs' high accuracy, it is essential to assess its capacity to represent the optimization landscape well. However, a consistent method for optimality assessment using surrogate models is still an open issue in the literature. As such, two main contributions can be highlighted: the SMBR optimization based on deep recurrent neural networks (DRNNs) and the characterization of the feasible operation region. This is done by recycling the data points from a metaheuristic technique-optimality assessment. The results demonstrate that the DRNN-based optimization can address such complex optimization while meeting optimality., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The separation of the proteins Bovine Serum Albumin (BSA) and Myoglobin (Mb) was achieved by Size-Exclusion Simulated Moving Bed (SE-SMB) and performed experimentally in the FlexSMB® unit, an SMB unit designed and built in the Laboratory of Separation and Reaction Engineering. Before accomplishing the separation experiments in the mentioned unit, separation regions were computed by simulation based on a phenomenological mathematical model to determine appropriate operating conditions. The developed model was validated in advance, against fixed-bed dynamic adsorption experimental results, for pure component and binary mixtures. Then the SMB experiments were carried out, and purities of the Mb on the extract and BSA on the raffinate streams were 98% and 96%, respectively. The achieved recoveries were 80% of Mb on the extract and 94% of BSA on the raffinate. Lastly, productivities of 6.4 g protein ⋅l ads -1 ⋅day -1 for the extract and 28.8 g protein ⋅l ads -1 ⋅day -1 for the raffinate were obtained., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The SMB unit developed by the Laboratory of Separation and Reaction Engineering (FlexSMB-LSRE ® ) was used to perform tartronic acid (TTA) and glyceric acid (GCA) separation and to validate the mathematical model in order to determine the optimum operating parameters of an industrial unit. The purity of the raffinate and extract streams in the experiments performed were 80% and 100%, respectively. The TTA and GCA productivities were 79 and 115 kg per liter of adsorbent per day, respectively and only 0.50 cubic meters of desorbent were required per kilogram of products. Under the optimum operating conditions, which were determined through an extensive simulation study based on the mathematical model developed to predict the performance of a real SMB unit, it was possible to achieve a productivity of 86 kg of TTA and 176 kg of GCA per cubic meter of adsorbent per day (considering the typical commercial purity value of 97% for both compounds) with an eluent consumption of 0.30 cubic meters per kilogram of products., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This work was motivated by the need of stimuli responsive drug carriers, which can be activated by low cost non-invasive stimuli such as external magnetic field (EMF). Thus, novel antimicrobial materials based on xanthan gum (XG), magnetic nanoparticles (MNP), bovine serum albumin (BSA) and amoxicillin (Amox) were designed in order to promote the release of Amox under magnetic stimuli. Firstly, surfaces with different functionalities were prepared by sequential deposition of thin layers on Si wafers and characterized by means of ellipsometry and atomic force microscopy. Amox adsorbed preferentially onto XG or BSA films. In solution, favorable interactions between Amox and BSA were evidenced by substantial changes in the BSA secondary structure, as revealed by circular dichroism. Patches of XG and XG/MNP/BSA were immersed in 2 g L -1 Amox, yielding 10 ± 3 and 17 ± 4 μg/cm 3 Amox loading, respectively. The inclusion of 0.2 wt% Fe 3 O 4 in the patches and their exposure to EMF enabled in vitro release of Amox, at pH 5.5 and 0.02 mol L -1 NaCl, following the quasi-Fickian behavior. Amox diffused from XG/MNP/BSA patches in agar medium containing Staphylococcus aureus and Escherichia coli, inhibiting their growth. The inhibition of E. coli growth was particularly efficient under EMF., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nanostructured particles of polystyrene sulfate (PSS) covered by a cationic lipid bilayer of dioctadecyldimethylammonium bromide (DODAB) incorporated gramicidin D (Gr) yielding optimal and broadened bactericidal activity against both Escherichia coli and Staphylococcus aureus . The adsorption of DODAB/Gr bilayer onto PSS nanoparticles (NPs) increased the zeta-average diameter by 8-10 nm, changed the zeta-potential of the NPs from negative to positive, and yielded a narrow size distributions for the PSS/DODAB/Gr NPs, which displayed broad and maximal microbicidal activity at very small concentrations of the antimicrobials, namely, 0.057 and 0.0057 mM DODAB and Gr, respectively. The results emphasized the advantages of highly-organized, nanostructured, and cationic particles to achieve hybrid combinations of antimicrobials with broad spectrum activity at considerably reduced DODAB and Gr concentrations., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The optimization of bilayer coverage on particles is important for a variety of biomedical applications, such as drug, vaccine, and genetic material delivery. This work aims at optimizing the deposition of cationic bilayers on silica over a range of experimental conditions for the intervening medium and two different assemblies for the cationic lipid, namely, lipid films or pre-formed lipid bilayer fragments. The lipid adsorption on silica in situ over a range of added lipid concentrations was determined from elemental analysis of carbon, hydrogen, and nitrogen and related to the colloidal stability, sizing, zeta potential, and polydispersity of the silica/lipid nanoparticles. Superior bilayer deposition took place from lipid films, whereas adsorption from pre-formed bilayer fragments yielded limiting adsorption below the levels expected for bilayer adsorption., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.

The control of Simulated Moving Bed (SMB) units is challenging due to their complex dynamic behaviour and the difficulty of measuring their main properties. Furthermore, for the SMB units, the transfer function identification when the unit is operating at its optimal point is not easy to be done through the usual way. This work presents the development of a novel strategy to identify transfer functions of TMB/SMB and its application on classical linear model predictive controllers (MPC). However, for the process in study, due its unique dynamics, only the identification of the linear model is not enough to solve its control problem. Therefore, it is proposed a modification in the MPC prediction, that consists in a strategy based on a switching system where the most adequate transfer function is employed in the controller to overcome the problems related with the process dynamic behaviour. The results show that the used methodology enables the easy identification of transfer functions at the process optimal operating point and that the MPC can control the process in both the servo and regulator problem cases. It is also showed that the transfer function identified can be applied in the control of a SMB unit with four columns, under its optimal conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hydroxypropyl methylcellulose (HPMC) and xyloglucan (XG) crosslinked with citric acid over a range of HPMC/XG weight ratios formed sustainable blend films characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, tensile tests, circular dichroism and determination of inhibitory activity against Staphylococcus aureus and Escherichia coli. Both in solution and in the crosslinked films, HPMC chains lost the original ordered conformation upon interacting with XG, giving rise to an entropic gain. The highest values of tensile strength (25MPa) and Young's modulus (689MPa) occurred for the 50:50 HPMC/XG blend films. In vitro loading of gentamicin sulfate (GS) in the films amounted to 0.18±0.05 -0.37±0.05g of GS per g polymer. At pH 7.4 and 37°C, the GS release kinetics from the films fitted with the Korsmeyer-Peppas model revealed a non-Fickian release mechanism with diffusional coefficient n∼0.7. The cross-linked films of HPMC, XG and their blends loaded with GS showed outstanding antibacterial activity against Staphylococcus aureus and Escherichia coli, disclosing their potential for novel biomedical applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: The aim of the study was to investigate prognostic factors in feline mammary gland neoplasms, correlating them with overall survival (OS)., Methods: Fifty-six primary malignant mammary gland neoplasms and 16 metastatic lymph nodes from 37 female cats were analyzed. Clinical staging, histologic type and grade, and immunohistochemistry for Ki-67, progesterone and estrogen receptor, human epidermal growth factor receptor type 2 (HER-2), cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were evaluated. Follow-up was performed in order to correlate prognostic factors with OS., Results: Lymph node metastasis was found in 35% of cases. Clinical stage III, tubulopapillary carcinomas and histologic grade II cases prevailed in the study. Most neoplasms were positive for hormonal receptors, negative for HER-2 overexpression and presented VEGF overexpression. Immunoreactivity for Ki-67 (P = 0.046) and COX-2 (P = 0.007) was higher in metastases than in primary tumors. COX-2 (P = 0.089), HER-2 (P = 0.012) and histologic grade (P = 0.080) were correlated with OS., Conclusions and Relevance: The data suggest that inhibition of ovarian hormones and COX-2 may represent a therapeutic option for malignant feline mammary gland neoplasms. When evaluating disease progression, COX-2 scores and Ki-67 index should be analyzed in primary tumors and metastases. Histologic grade, HER-2 status and COX-2 scores were found to have a direct influence on OS. Prognostic factors allow for a better understanding of disease outcome in a condition that is characterized by a poor prognosis. The present work highlights the need for further studies on endocrine therapy and COX-2 inhibitors, which could influence OS., (© The Author(s) 2015.)

In the last years several studies were carried out in order to separate gas mixtures by SMB technology; however, this technology has never been implemented on an industrial scale. In the present work, a gas phase SMB bench unit was built and tested for the separation of propane and propylene mixtures, using 13X zeolite extrudates as adsorbent and isobutane as desorbent. Three experiments were performed to separate propane/propylene by gas phase SMB in the bench scale unit with a 4-2-2 configuration, i.e., open loop circuit by suppressing section IV (desorbent regeneration followed by a recycle). Consequently, all the experiments were conducted using an external supply of pure isobutane as desorbent. Parameters such as switching time, extract and raffinate stream flow rates were changed to improve the efficiency of the process. Experimental results have shown that it is feasible to separate propylene from propane by gas phase SMB at a bench scale and that this process is a potential candidate to replace the conventional technologies for the propane/propylene separation. The performance parameters obtained are very promising for future development of this technology, since propylene was obtained in the extract stream with a purity of 99.93%, a recovery of 99.51%, and a productivity of [Formula: see text] . Propane was obtained in the raffinate stream with a purity of 98.10%, a recovery of 99.73% and a productivity of [Formula: see text] . The success of the above mentioned bench scale tests is a big step for the future implementation of this technology in a larger scale., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Peroxidases are enzymes catalyzing redox reactions that cleave peroxides. Their active redox centers have heme, cysteine thiols, selenium, manganese, and other chemical moieties. Peroxidases and their mimetic systems have several technological and biomedical applications such as environment protection, energy production, bioremediation, sensors and immunoassays design, and drug delivery devices. The combination of peroxidases or systems with peroxidase-like activity with nanostructures such as nanoparticles, nanotubes, thin films, liposomes, micelles, nanoflowers, nanorods and others is often an efficient strategy to improve catalytic activity, targeting, and reusability.

Damage control refers to those actions made towards minimizing damage or loss. Depending on the context, these can range from emergency procedures dealing with the sinking of a ship or to a surgery dealing with severe trauma or even to an imaginary company in Marvel comics, which repairs damaged property arising from conflicts between super heroes and villains. In the context of host microbe interactions, tissue damage control refers to an adaptive response that limits the extent of tissue damage associated with infection. Tissue damage control can limit the severity of infectious diseases without interfering with pathogen burden, conferring disease tolerance to infection. This contrasts with immune-driven resistance mechanisms, which although essential to protect the host from infection, can impose tissue damage to host parenchyma tissues. This damaging effect is countered by stress responses that confer tissue damage control and disease tolerance to infection. Here we discuss how the stress response regulated by the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) acts in such a manner., (© 2015 Authors.)

Dioctadecyldimethylammonium bromide (DODAB) is an antimicrobial lipid that can be dispersed as large closed bilayers (LV) or bilayer disks (BF). Gramicidin (Gr) is an antimicrobial peptide assembling as channels in membranes and increasing their permeability towards cations. In mammalian cells, DODAB and Gr have the drawbacks of Gram-positive resistance and high toxicity, respectively. In this study, DODAB bilayers incorporating Gr showed good antimicrobial activity and low toxicity. Techniques employed were spectroscopy, photon correlation spectroscopy for sizing and evaluation of the surface potential at the shear plane, turbidimetric detection of dissipation of osmotic gradients in LV/Gr, determination of bacterial cell lysis, and counting of colony-forming units. There was quantitative incorporation of Gr and development of functional channels in LV. Gr increased the bilayer charge density in LV but did not affect the BF charge density, with localization of Gr at the BF borders. DODAB/Gr formulations substantially reduce Gr toxicity against eukaryotic cells and advantageously broaden the antimicrobial activity spectrum, effectively killing Escherichia coli and Staphylococcus aureus bacteria with occurrence of cell lysis.

Poly(ethylene glycol) (PEG)-decorated polystyrene (PS) nanoparticles with mean hydrodynamic diameter (D) and zeta-potential (ζ) of (286 ± 15) nm and (-50 ± 5) mV, respectively, were modified by the adsorption of Congo red (CR). The PS/PEG/CR particles presented D and ζ values of (290 ± 19) nm and (-36 ± 5) mV, respectively. The adsorption of lipase onto PS/PEG or PS/PEG/CR particles at (24 ± 1) °C and pH 7 changed the mean D value to (380 ± 20) and (405 ± 11) nm, respectively, and ζ value to (-32 ± 4) mV and (-25 ± 2) mV, respectively. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase, lipase immobilized onto PS/PEG and PS/PEG/CR particles. Lipase on PS/PEG/CR presented the largest Michaelis-Menten constant (KM), but also the highest Vmax and kcat values. Moreover, it could be recycled seven times, losing a maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. Although lipases immobilized onto PS/PEG particles presented the smallest KM values, the reactions were comparatively the slowest and recycling was not possible. Hydrolysis reactions performed in the temperature range of 25 °C to 60 °C with free lipases and lipases immobilized onto PS/PEG/CR particles presented an optimal temperature at 40 °C. At 60 °C free lipases and lipases immobilized onto PS/PEG/CR presented ~80% and ~50% of the activity measured at 40 °C, indicating good thermal stability. Bioconjugation effects between CR and lipase were evidenced by circular dichroism spectroscopy and spectrophotometry. CR molecules mediate the open state conformation of the lipase lid and favor the substrate approaching.

Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

The purpose of this Article is to characterize polymeric particles of poly(methylmethacrylate) (PMMA) synthesized in the presence of one of two different quaternary ammonium surfactants (QACs): cetyltrimethylammonium bromide (CTAB) or dioctadecyldimethylammonium bromide (DODAB). The methods used are dynamic light scattering for sizing, polydispersity and zeta potential analysis, scanning electron microscopy (SEM) for morphology visualization, and plating plus colony-forming unities (CFU) counting for the determination of antimicrobial activity. The results point out the high QAC concentration required to obtain cationic and bioactive antimicrobial particles with good colloidal stability and a permanent load of the polymeric network with QACs. Over a range of micromolar QAC concentrations, there is remarkable antimicrobial activity of PMMA/CTAB or PMMA/DODAB particles, which is much higher than those determined for the QACs by themselves. Loading the biocompatible polyacrylate particles with QACs is a facile, fast, low-cost approach to obtaining highly efficient antimicrobial nanoparticles.

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern-Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5'-TTGACGTTCG-3') assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mM DODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mM DODAB, 0.1 mg/mL OVA and 0.006 mM CpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The interaction between horseradish peroxidase (HRP) and dioctadecyldimethylammonium bromide (DODAB) bilayers supported on polystyrene microspheres (PSS) or on flat silicon wafers was evaluated from the following techniques: (1) dynamic light-scattering for determining size distributions, zeta-potentials and polydispersities for dispersions; (2) spectrophotometric determination of HRP concentration in supernatants of centrifuged mixtures; (3) in situ ellipsometry for mean thickness of deposited layers on wafers; (4) kinetics of product appearance for oxidation of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid by H(2) O(2) in presence of free or immobilized enzyme. HRP incorporation (3.0 mg/m(2)) did not alter mean diameter and zeta-potential of PSS/DODAB particles but reduced enzyme activity by 50%, though activity persisted after several rinsing steps. In situ ellipsometry could not detect any HRP layer on top of the DODAB bilayer. HRP insertion in the bilayer core explained all results for both systems. Useful biotechnological applications are anticipated for such assemblies., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Hybrid films from poly (methylmethacrylate) (PMMA) and dioctadecyldimethylammonium bromide (DODAB), cetyltrimethylammonium bromide (CTAB), or tetrapropylammonium bromide (TPAB) were characterized by determination of wettability, ellipsometry, atomic force microscopy, active compounds diffusion to water, X-ray photoelectron spectroscopy (XPS) with determination of atomic composition on the films surface, and biocidal activity against Pseudomonas aeruginosa or Staphylococcus aureus. QAC mobility in the films increased from DODAB to CTAB to TPAB. Diffusion and optimal hydrophobic-hydrophilic balance imparted the highest bioactivity to CTAB. DODAB sustained immobilization at the film surface killed bacteria upon contact. TPAB ability to diffuse was useless because of its unfavorable hydrophobic-hydrophilic balance for bioactivity., (© 2011 American Chemical Society)

Background: Cationic bilayers based on the inexpensive synthetic lipid dioctadecyldimethylammonium bromide (DODAB) have been useful as carriers for drug delivery, immunoadjuvants for vaccines and active antimicrobial agents., Methods: Rifampicin (RIF) or isoniazid (ISO) interacted with DODAB bilayer fragments (BF) or large vesicles (LV). Dispersions were evaluated by dynamic light-scattering for zeta-average diameter (Dz) and zeta-potential (ζ) analysis; dialysis for determination of drug entrapment efficiency; plating and CFU counting for determination of cell viability of Mycobacterium smegmatis or tuberculosis, minimal bactericidal concentration (MBC) and synergism index for DODAB/drug combinations., Results: DODAB alone killed micobacteria over a range of micromolar concentrations. RIF aggregates in water solution were solubilised by DODAB BF. RIF was incorporated in DODAB bilayers at high percentiles in contrast to the leaky behavior of ISO. Combination DODAB/RIF yielded MBCs of 2/2 and 4/0.007 μg/mL against Mycobacterium smegmatis or Mycobacterium tuberculosis, respectively. Synergism indexes equal to 0.5 or 1.0, indicated synergism against the former and independent action, against the latter species., Conclusions: In vitro, DODAB acted effectively both as micobactericidal agent and carrier for rifampicin. The novel assemblies at reduced doses may become valuable against tuberculosis.

The interaction between cationic bilayer fragments and a model oligonucleotide was investigated by differential scanning calorimetry, turbidimetry, determination of excimer to monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidyl-choline in bilayer fragment dispersions and dynamic light scattering for sizing and zeta-potential analysis. Salt (Na₂HPO₄), mononucleotide (2'-deoxyadenosine-5'-monophosphate) or poly (dA) oligonucleotide (3'-AAA AAA AAA A-5') affected structure and stability of dioctadecyldimethylammonium bromide bilayer fragments. Oligonucleotide and salt increased bilayer packing due to bilayer fragment fusion. Mononucleotide did not reduce colloid stability or did not cause bilayer fragment fusion. Charge neutralization of bilayer fragments by poly (dA) at 1:10 poly (dA):dioctadecyldimethylammonium bromide molar ratio caused extensive aggregation, maximal size and zero of zeta-potential for the assemblies. Above charge neutralization, assemblies recovered colloid stability due to charge overcompensation. For bilayer fragments/poly (dA), the nonmonotonic behavior of colloid stability as a function of poly (dA) concentration was unique for the oligonucleotide and was not observed for Na₂HPO₄ or 2'-deoxyadenosine-5'-monophosphate. For the first time, such interactions between cationic bilayer fragments and mono- or oligonucleotide were described in the literature. Bilayer fragments/oligonucleotide assemblies may find interesting applications in drug delivery., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CMC, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta = 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of P. aeruginosa viability (10(7) CFU/mL) at 1 or 2 microg/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 microg/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed as one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration.

Background: Silica particles cationized by dioctadecyldimethylammonium bromide (DODAB) bilayer were previously described. This work shows the efficiency of these particulates for antigen adsorption and presentation to the immune system and proves the concept that silica-based cationic bilayers exhibit better performance than alum regarding colloid stability and cellular immune responses for vaccine design., Results: Firstly, the silica/DODAB assembly was characterized at 1 mM NaCl, pH 6.3 or 5 mM Tris.HCl, pH 7.4 and 0.1 mg/ml silica over a range of DODAB concentrations (0.001-1 mM) by means of dynamic light scattering for particle sizing and zeta-potential analysis. 0.05 mM DODAB is enough to produce cationic bilayer-covered particles with good colloid stability. Secondly, conditions for maximal adsorption of bovine serum albumin (BSA) or a recombinant, heat-shock protein from Mycobacterium leprae (18 kDa-hsp) onto DODAB-covered or onto bare silica were determined. At maximal antigen adsorption, cellular immune responses in vivo from delayed-type hypersensitivity reactions determined by foot-pad swelling tests (DTH) and cytokines analysis evidenced the superior performance of the silica/DODAB adjuvant as compared to alum or antigens alone whereas humoral response from IgG in serum was equal to the one elicited by alum as adjuvant., Conclusion: Cationized silica is a biocompatible, inexpensive, easily prepared and possibly general immunoadjuvant for antigen presentation which displays higher colloid stability than alum, better performance regarding cellular immune responses and employs very low, micromolar doses of cationic and toxic synthetic lipid.

The interaction between giant bacteriophage DNA and cationic biomimetic particles was characterized from sizing by dynamic light-scattering, zeta-potential analysis, turbidimetry, determination of colloid stability, visualization from atomic force microscopy (AFM), and determination of cytotoxicity against E. coli from colony forming unities counting. First, polystyrene sulfate (PSS) particles with different sizes were covered by a dioctadecyldimethylammonium bromide (DODAB) bilayer yielding the so-called cationic biomimetic particles (PSS/DODAB). These cationic particles are highly organized, present a narrow size distribution and were obtained over a range of particle sizes. Thereafter, upon adding lambda, T5 or T2-DNA to PSS/DODAB particles, supramolecular assemblies PSS/DODAB/DNA were obtained and characterized over a range of DNA concentrations and particle sizes (80-700 nm). Over the low DNA concentration range, PSS/DODAB/ DNA assemblies were cationic, colloidally stable with moderate polydispersity and highly cytotoxic against E. coli. From DNA concentration corresponding to charge neutralization, neutral or anionic supramolecular assemblies PSS/DODAB/DNA exhibited low colloid stability, high polydispersity and moderate cytotoxicity. Some nucleosome mimetic assemblies were observed by AFM at charge neutralization (zeta-potential equal to zero).

Intermolecular associations between a cationic lipid and two model polymers were evaluated from preparation and characterization of hybrid thin films cast on silicon wafers. The novel materials were prepared by spin-coating of a chloroformic solution of lipid and polymer on silicon wafer. Polymers tested for miscibility with the cationic lipid dioctadecyldimethylammonium bromide (DODAB) were polystyrene (PS) and poly(methyl methacrylate) (PMMA). The films thus obtained were characterized by ellipsometry, wettability, optical and atomic force microscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and activity against Escherichia coli. Whereas intermolecular ion-dipole interactions were available for the PMMA-DODAB interacting pair producing smooth PMMA-DODAB films, the absence of such interactions for PS-DODAB films caused lipid segregation, poor film stability (detachment from the silicon wafer) and large rugosity. In addition, the well-established but still remarkable antimicrobial DODAB properties were transferred to the novel hybrid PMMA/DODAB coating, which is demonstrated to be highly effective against E. coli.

Background: Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans., Results: Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings., Conclusion: Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.

Dioctadecyldimethylammonium bromide (DODAB)/dipalmitoylphosphatidylcholine (DPPC) large and cationic vesicles obtained by vortexing a lipid film in aqueous solution and above the mean phase transition temperature (T(m)) are characterized by means of determination of phase behaviour, size distribution, zeta-potential analysis and colloid stability. The effect of increasing % DODAB over the 0-100% range was a nonmonotonic phase behaviour. At 50% DODAB, the mean phase transition temperature and the colloid stability were at maximum. There is an intimate relationship between stability of the bilayer structure and colloid stability. In 1, 50 and 150mM NaCl, the colloid stability for pure DPPC or pure DODAB vesicles was very low as observed by sedimentation or flocculation, respectively. In contrast, at 50% DODAB, remarkable colloid stability was achieved in 1, 50 or 150mM NaCl for the DODAB/DPPC composite vesicles. Vesicle size decreased but the zeta-potential remained constant with % DODAB, due to a decrease of counterion binding with vesicle size. This might be important for several biotechnological applications currently being attempted with cationic bilayer systems.

The adsorption behavior of horseradish peroxidase (HRP) onto hybrid particles of poly(methylmethacrylate) (PMMA) and carboxymethylcellulose (CMC) was investigated by means of spectrophotometry. Dispersions of PMMA/CMC particles were characterized by light scattering, zeta potential measurements and scanning electron microscopy before and after HRP adsorption. HRP adsorbed irreversibly onto PMMA/CMC particles; the adsorption isotherm showed an initial step and an adsorption plateau. The enzymatic activity of free HRP and immobilized HRP (plateau region) was monitored by means of spectrophotometry as a function of storing time. Upon adsorbing HRP there is little (up to 20%) or no reduction of enzymatic activity in comparison to that observed for free HRP in solution. After storing free HRP and HRP-covered PMMA/CMC particles for 18 days the level of enzymatic activity is kept. HRP-covered PMMA/CMC particles dispersions, which were dried and re-dispersed, retained 50% of their catalytic properties. These interesting findings were discussed in the light of a beneficial effect of a hydrated microenvironment for maintenance of enzyme conformation and activity.

The interaction between composite dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) bilayer vesicles in the gel state and silica is investigated over the 0-20% DODAB range from determination of adsorption curves, silica sedimentation, particle sizing and zeta-potentials. At 1 mg/mL silica, 0% DODAB, pH 6.3, over the 0-150 mM NaCl range of ionic strengths, high affinity adsorption curves were barely affected by ionic strength and all of them exhibited limiting adsorption values above the level expected for single bilayer deposition. At 1 mg/mL silica, 2% DODAB, pH 6.3 and 1 mM NaCl, high affinity adsorption curves fortuitously presented limiting adsorption indicative of one bilayer deposition on each silica particle. At %DODAB<2% or %DODAB>2%, limiting adsorption was above and below the level expected for bilayer deposition, respectively. Increasing %DODAB in the vesicle composition negatively modulated the limiting adsorption on silica despite the increasing surface charge on vesicles and electrostatic attraction between vesicles and particles. The results point out the difficulty of closed vesicle disruption (required for bilayer deposition from vesicles) when the bilayer is tightly packed in the rigid gel state and might be of interest for analytical applications of immobilized vesicles on silica.

Chitosan (CH) decorated polystyrene (PS) particles were synthesized within complexes of CH, a polycation under acid conditions, and tiny amounts of sodium dodecylsulfate (SDS). Particle characterization was performed by means of dynamic light scattering, zeta potential measurements, and transmission electron microscopy. All dispersions were stable in the ionic strength of 2.0 mol L-1 NaCl during 2 months. The outstanding colloidal stability was attributed to the presence of a hydrated CH layer around the particles. CH decorated PS particles were attached to atomic force microscopy cantilevers and probed against Si wafers in water and in NaCl 0.01 mol/L. The mean thickness of CH layer amounted to 35 +/- 11 and 16 +/- 6 nm, when the medium was water and NaCl 0.01 mol/L, respectively. Adsorption isotherm of hexokinase (HK) onto PS/CH particles studied by means of spectrophotometry showed three regions: an initial step; adsorption plateau and multilayer formation. Enzymatic activity of free HK and immobilized HK was monitored by means of spectrophotometry as a function of storing time and reuse. After 3 days, storing HK free in solution dramatically lost its catalytic properties. On the contrary, HK-covered PS/CH particles retained enzymatic activity over 1 month. Moreover, HK-covered PS/CH particles could be reused in the determination of glucose two times consecutively, without losing activity. These interesting findings were discussed in light of the role of water in enzyme conformation.

The study on the adsorption of horseradish peroxidase (HRP) onto silicon wafers was carried out by means of in situ ellipsometry, atomic force microscopy (AFM) and contact angle measurements. A smooth HRP layer adsorbed onto Si wafers. The enzymatic activity of free or adsorbed HRP was determined by the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and by the emulsion polymerization of ethylene glycol dimethacrylate (EGDMA). Upon adsorbing, HRP molecules might have undergone some conformational changes, which caused a small reduction of enzymatic activity in comparison to that observed for HRP solution. However, it was possible to reuse the same HRP-covered Si wafer as catalyst in the polymerization of EGDMA three times.

Therapeutic activity of an effective and less nephrotoxic amphotericin B (AMB) formulation with dioctadecyldimethylammonium bromide (DODAB) bilayer fragments (named DODAB/AMB) inspired this toxicity survey in mice. At low drug to lipid molar ratios, hepatotoxicity, spleen damage and blood changes in comparison to DOC/AMB (sodium desoxycholate/amphotericin B, Fungizone) are evaluated ultimately showing toxic effects associated to DODAB only. Swiss Webster female mice were given DODAB, DODAB/AMB or DOC/AMB intraperitonially (ip) for 10 consecutive days (0.4 mg/kg/day AMB; 80 mg/kg/day DODAB) and repeated dose-toxicity was evaluated at the end of the treatment period (on day 11) and after a recovery period of 6 months from biochemical and hematological parameters plus histopathological examination of spleen and liver. Both at day 11 and 180, DODAB in the formulation administered ip causes irreversible changes in spleen such as fibrosis and leukocytes infiltration as a consequence of the administration route. Whereas focal necrosis is induced by DODAB in liver at day 180, DOC/AMB causes more severe multifocal necrosis both at day 11 and day 180. In the kidneys, the novel formulation preserves integrity of tubules and glomeruli in contrast to the serious damage caused by DOC/AMB as shown previously. The majority of the toxic effects observed for the novel formulation were due to the DODAB carrier used at 10mg/mL, i.e., at a rather high concentration and further studies should minimize DODAB dose.

Objectives: To determine the mechanism of antimicrobial action for cationic lipid dioctadecyldimethylammonium bromide (DODAB) and hexadecyltrimethylammonium bromide (CTAB) against Candida albicans., Methods: Determination of DODAB or CTAB adsorption isotherms; cell viability; cell electrophoretic mobility (EM); and leakage of small phosphorylated compounds, proteins or DNA from fungus or haemoglobin from erythrocytes., Results: High affinity isotherms for CTAB and DODAB adsorption onto fungus cells (10(8) cfu/mL) yield limiting adsorption at 7.8 and 3.7 x 10(9) molecules per cell, respectively. Negatively charged C. albicans cells (10(6) cfu/mL) remain viable whereas positively charged ones die. At 0.3 mM CTAB or 0.01 mM DODAB, EM is zero and fungus viability is 50%. Cells start to die at submicellar CTAB concentrations and fungus lysis does not play a significant role in the mechanism of antifungal action. Over 0.1-10 mM CTAB or DODAB, there is no leakage of tested compounds from C. albicans cells despite the low cell viability. In contrast to the fungus, under isotonic conditions, cationic amphiphiles induce haemolysis over a range of low DODAB (>0.01 mM) and CTAB (>0.001 mM) concentrations., Conclusions: The critical phenomenon determining antifungal effect of cationic surfactants and lipids is not cell lysis but rather the change of cell surface charge from negative to positive.

Objectives: Coverage of antifungal drug particles (miconazole and amphotericin B) with cationic lipid and evaluation of a synergistic action between lipid and drug., Methods: Miconazole and amphotericin B were mixed with cationic bilayer fragments (BF) of dioctadecyldimethylammonium bromide (DODAB) at extreme drug to lipid molar proportions (P). Light scattering for particle sizing and zeta-potential analysis evaluated effects of cationic lipid on drug particle size and charge. Colony counts evaluated viability of Candida spp. and Cryptococcus neoformans over a range of P., Results: BF loading capacity for monomeric amphotericin B is 0.1 mM amphotericin B at 2 mM DODAB (P = 1:20). Above this low P, amphotericin B aggregates in the dispersion. At high P, addition of chaotropic K2HPO4 (0.2-2 mM) converts miconazole or amphotericin B aggregates into negatively charged particles with affinity for cationic lipid, which then surrounds each drug particle with a cationic layer. The combined in vitro action of lipid-covered drug particles against Candida and C. neoformans depends on P and interaction time. DODAB by itself kills C. neoformans and Candida at 2 and 2 to > 250 mg/L minimal fungicidal concentration (MFC). In combination, over the first hour, fungicidal activity is due to DODAB with lipid capsules retarding drug action. At 48 h interaction time and 10(4) cfu/mL, MFC (mg/L) against Candida albicans is reduced from 4 to 1 amphotericin B (at 2 DODAB), and from 8 to 1 miconazole (at 1 DODAB)., Conclusions: DODAB may be a suitable candidate for use in combination with miconazole for antifungal therapy.

The effect of monovalent salt nature and concentration over a range of low ionic strengths (0-10 mM LiCl, NaCl, KCl, or CsCl) and at two different pH values (6.3 and 10.0) on adsorption of dioctadecyldimethylammonium bromide (DODAB) bilayer fragments (BF) onto flat SiO(2) surfaces was systematically evaluated by means of in situ ellipsometry. High-affinity adsorption isotherms fitted by the Langmuir model indicated that adsorption maxima were consistent with bilayer deposition only around 10 mM monovalent salt at both pH values. In pure water, the mean thickness of the DODAB adsorbed layer was close to zero with bilayer deposition taking place only around 10 mM ionic strength. In the presence of 10 mM CsCl or LiCl, the highest and the lowest affinity constants for DODAB adsorption onto SiO(2) were, respectively, obtained consistently with the expected facility of cation exchange at the surface required for DODAB adsorption. The cation more tightly bound to the solid surface should be Li(+), which would present the largest resistance to displacement by the DODAB cation, whereas the less tightly bound cation should be Cs(+) due to its largest ionic radius and lowest charge density. In other words, DODAB adsorption proceeds in accordance with charge density on the solid surface, which depends on the nature and concentration of bound counterions as well as DODAB cation ability to displace them. AFM images show a very smooth DODAB film adsorbed onto the surface in situ with a large frequency of BF auto-association from their edges. The present results for flat surfaces entirely agree with previous data from our group for DODAB adsorption onto silica particles.

Biomimetic particles supporting lipid bilayers are becoming increasingly important to isolate and reconstitute protein function. Cholera toxin (CT) from Vibrio cholerae, an 87-kDa AB5 hexameric protein, and its receptor, the monosialoganglioside GM1, a cell membrane glycolipid, self-assembled on phosphatidylcholine (PC) bilayer-covered silica particles at 1 CT/5 GM1 molar ratio in perfect agreement with literature. This receptor-ligand recognition represented a proof-of-concept that receptors in general can be isolated and their function reconstituted using biomimetic particles, i.e., bilayer-covered silica. After incubation of colloidal silica with small unilamellar PC vesicles in saline solution, pH 7.4, PC adsorption isotherms on silica from inorganic phosphorus analysis showed a high PC affinity for silica with maximal PC adsorption at bilayer deposition. At 0.3 mM PC, fluorescence of pyrene-labeled GM(1) showed that GM(1) incorporation in biomimetic particles increased as a function of particles concentration. At 1 mg/mL silica, receptor incorporation increased to a maximum of 40% at 0.2-0.3 mM PC and then decreased as a function of PC concentration. At 5 microM GM(1), 0.3 mM PC, and 1 mg/mL silica, CT binding increased as a function of CT concentration with a plateau at 2 mg bound CT/m2 silica, which corresponded to the 5 GM(1)/1 CT molar proportion and showed successful reconstitution of receptor-ligand interaction.

Our previous work has shown that dioctadecyldimethylammonium bromide (DODAB) bilayer fragments (BF) presented antimicrobial activity, solubilized fungicides, e.g., amphotericin B and miconazole (MCZ), stabilized hydrophobic drug particles and were effective in vivo. Here, the interaction between MCZ and DODAB BF is evaluated from determination of BF loading capacity and effects of drug-to-lipid molar proportion (MP) on particle size, zeta potential and gel-to-liquid-crystalline phase transition T(m). DODAB BF solubilized MCZ over a range of MP. BF loading capacity was 0.5 mM MCZ at 5 mM DODAB. Above this limit, the drug aggregated in the dispersion. At pH 6.3, BF zeta potentials decreased with MP, suggesting insertion of deprotonated drug into the bilayer. MCZ optical spectra in BF were similar to those in best organic solvent, confirming drug solubilization. At MP 1:10, BF T(m) remained unchanged, suggesting drug capture at BF hydrophobic edges. At MP 1:10, T(m) decreased, showing MCZ insertion into DODAB bilayer. However, drug was expelled from the bilayer core upon lowering temperature. Minimal fungicidal concentrations against C. albicans were synergically reduced by 10 times for drug/BF.

The effect of ionic strength and pH on phosphatidylcholine (PC) adsorption from vesicles on silica nanoparticles was investigated over a range of NaCl concentrations (0.1-150 mM) at pH 6.3 and 7.4 from determination of adsorption isotherms, colloid stability, particle sizing, and zeta-potentials. At and above 10 mM ionic strength, pH 6.3, high-affinity adsorption isotherms with limiting adsorption indicative of one-bilayer deposition on each silica particle were obtained. At 10 mM ionic strength, adsorption isotherms indicated lower affinity between PC and silica at pH 7.4 than at pH 6.3, suggesting a role of hydrogen bonding between silanol on silica and phosphate on PC in promoting bilayer deposition at low pH. Under conditions where high affinity and bilayer deposition were achieved, silica sedimentation documented from photographs was absent, suggesting particle stabilization induced by bilayer coverage. However, at physiological (150 mM NaCl) or close to physiological ionic strength (140 mM NaCl), the large colloid stability similarly achieved at pH 6.3 or 7.4 suggested the major role of van der Waals attraction between the PC bilayer vesicle and silica particle in determining bilayer deposition. The effect of increasing ionic strength was increasing van der Waals attraction, which caused PC vesicle disruption with bilayer deposition and bilayer-induced silica stabilization.

The interaction of cytochrome c (cyt c) with mitochondrial mimetic vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and heart cardiolipin (PCPECL) was investigated over the 7.4-6.2 pH range by means of turbidimetry and photon correlation spectroscopy. In the presence of cyt c, the decrease of pH induced an increase in vesicle turbidity and mean diameter resulting from vesicle fusion as determined by a rapid decrease in the excimer/monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidylcholine (PyPC). N-acetylated cyt c and protamine, a positively charged protein, increased vesicle turbidity in a pH-independent manner, whereas albumin did not affect PCPECL vesicle turbidity. pH-dependent turbidity kinetics revealed a role for cyt c-ionizable groups with a pK(a)((app)) of approximately 7.0. The carbethoxylation of these groups by diethylpyrocarbonate prevented cyt c-induced vesicle fusion, although cyt c association to vesicles remained unaffected. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that Lys-22, Lys-27, His-33, and Lys-87 cyt c residues were the main targets for carbethoxylation performed at low pH values (<7.5). In fact, these amino acid residues belong to clusters of positively charged amino acids that lower the pK(a). Thus, at low pH, protonation of these invariant and highly conserved amino acid residues produced a second positively charged region opposite to the Lys-72 and Lys-73 region in the cyt c structure. These two opposing sites allowed two vesicles to be brought together by the same cyt c molecule for fusion. Therefore, a novel pH-dependent site associating cyt c to mitochondrial mimetic membranes was established in this study.