In situ interaction between the hormone 17α-ethynylestradiol and the liquid-ordered phase composed of the lipid rafts sphingomyelin and cholesterol.

Hormone treatments are frequently associated with cardiovascular diseases and cancers in women. Additionally, the detrimental effects of their presence as contaminants in water remain a concern. The transport of hormones through cell membranes is essential for their biological action, but investigating cell permeability is challenging owing to the experimental difficulty in dealing with whole cells. In this paper, we study the interaction of the synthetic hormone 17α-ethynylestradiol (EE2) with membrane models containing the key raft components sphingomyelin (SM) and cholesterol (Chol). The models consisted of Langmuir monolayers and giant unilamellar vesicles (GUVs) that represent bilayers. EE2 induced expansion of SM monolayers upon interacting with the non-hydrated amide group of SM head, but it had practically no effect on SM GUVs because these group are not available for interaction in bilayers. In contrast, EE2 interacted with hydrated phosphate group (PO2-) and amide group of SM/Chol mixture monolayer, which could explain the loss in phase contrast of liquid-ordered GUVs suggesting pore formation. A comparison with reported EE2 effects on GUVs in the fluid phase, for which no loss in phase contrast was observed, indicates that the liquid-ordered phase consisting of lipid rafts is relevant to be associated with the changes on cell permeability caused by the hormones.

A sandwich-type electrochemical immunosensor based on Au-rGO composite for CA15-3 tumor marker detection.

A sensitive detection of carbohydrate antigen 15-3 (CA15-3) levels may allow for early diagnosis and monitoring the treatment of breast cancer, but this can only be made in routine clinical practice if low-cost immunosensors are available. In this work, we developed a sandwich-type electrochemical immunosensor capable of rapid detection of CA15-3 with an ultra-low limit of detection (LOD) of 0.08 fg mL-1 within a wide linear concentration range from 0.1 fg mL-1 to 1 µg mL-1. The immunosensor had a matrix of a layer-by-layer film of Au nanoparticles and reduced graphene oxide (Au-rGO) co-electrodeposited on screen-printed carbon electrodes (SPCE). The high sensitivity was achieved by using secondary antibodies (Ab2) labeled with horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) as signal amplifiers, and hydroquinone (HQ) was used as an electron mediator. The immunosensor was selective for CA15-3 in human serum and artificial saliva samples, robust, and stable to permit storage at 4 °C for more than 30 days. With its high performance, the immunosensor may be incorporated into future point-of-care (POC) devices to determine CA15-3 in distinct biological fluids, including in blood and saliva samples.

Chain Cleavage of Bioinspired Bacterial Membranes Photoinduced by Eosin Decyl Ester.

Photodynamic therapy (PDT) is promising for bacterial inactivation since cellular internalization of photosensitizers (PS) is not crucial for the treatment effectiveness. Photoinduced damage in the lipid envelope may already induce microbial inactivation, which requires PS capable of easily penetrating into the membrane. Herein, we report on the insertion of the PS eosin decyl ester (EosDec) into Langmuir films of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), and cardiolipin (CLP) used as mimetic systems of bacterial membranes. Surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) indicated that the hydrophobic nature of EosDec favored deeper penetration in all the phospholipid monolayers. The incorporation of EosDec led to monolayer expansion, especially in the anionic DOPG and CLP owing to repulsive electrostatic interactions, and induced disorder in the lipid chains. Light irradiation of DOPE, DOPG, and CLP monolayers containing EosDec increased the rate of material loss to the subphase, which is attributed to cleavage of lipid chains triggered by contact-dependent reactions between excited states of EosDec and lipid unsaturations. The latter is key for membrane permeabilization and efficiency in microbial inactivation.

Role of Toluidine Blue-O Binding Mechanism for Photooxidation in Bioinspired Bacterial Membranes.

The alarming increase in bacterial resistance to antibiotics has demanded new strategies for microbial inactivation, which include photodynamic therapy whose activity relies on the photoreaction damage to the microorganism membrane. Herein, the binding mechanisms of the photosensitizer toluidine blue-O (TBO) on simplified models of bacterial membrane with Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) were correlated to the effects of the photoinduced lipid oxidation. Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were also used as a reference of mammalian membranes. The surface pressure isotherms combined with polarization-modulated infrared reflection absorption spectroscopy revealed that TBO expands DOPC, DOPE, and DOPG monolayers owing to electrostatic interactions with the negatively charged groups in the phospholipids, with a stronger adsorption on DOPG, which has a net surface charge. Light irradiation made the TBO-containing DOPC and DOPE monolayers less unstable as a result of the singlet oxygen (1O2) reaction with the chain unsaturation and hydroperoxide formation. In contrast, the decreased stability of the irradiated TBO-containing DOPG monolayer suggests the cleavage of carbon chains. The anionic nature of DOPG allowed a deeper penetration of TBO into the chain region, favoring contact-dependent reactions between the excited triplet state of TBO and lipid unsaturations or/and hydroperoxide groups, which is the key for the cleavage reactions and further membrane permeabilization.

Evaluation of EGCG Loading Capacity in DMPC Membranes.

Catechins are molecules with potential use in different pathologies such as diabetes and cancer, but their pharmaceutical applications are often hindered by their instability in the bloodstream. This issue can be circumvented using liposomes as their nanocarriers for in vivo delivery. In this work, we studied the molecular details of (-)-epigallocatechin-3-gallate (EGCG) interacting with 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) monolayer/bilayer systems to understand the catechin loading ability and liposome stability, using experimental and computational techniques. The molecular dynamics simulations show the EGCG molecules deep inside the lipid bilayer, positioned below the lipid ester groups, generating a concentration-dependent lipid condensation. This effect was also inferred from the surface pressure isotherms of DMPC monolayers. In the polarization-modulated infrared reflection absorption spectra assays, the predominant effect at higher concentrations of EGCG (e.g., 20 mol %) was an increase in lipid tail disorder. The steady-state fluorescence data confirmed this disordered state, indicating that the catechin-induced liposome aggregation outweighs the condensation effects. Therefore, by adding more than 10 mol % EGCG to the liposomes, a destabilization of the vesicles occurs with the ensuing release of entrapped catechins. The loading capacity for DMPC seems to be limited by its disordered lipid arrangements, typical of a fluid phase. To further increase the clinical usefulness of liposomes, lipid bilayers with more stable and organized assemblies should be employed to avoid aggregation at large concentrations of catechin.

Femtosecond Laser Patterning of the Biopolymer Chitosan for Biofilm Formation.

Controlling microbial growth is crucial for many biomedical, pharmaceutical and food industry applications. In this paper, we used a femtosecond laser to microstructure the surface of chitosan, a biocompatible polymer that has been explored for applications ranging from antimicrobial action to drug delivery. The influence of energy density on the features produced on chitosan was investigated by optical and atomic force microscopies. An increase in the hydrophilic character of the chitosan surface was attained upon laser micromachining. Patterned chitosan films were used to observe Staphylococcus aureus (ATCC 25923) biofilm formation, revealing an increase in the biofilm formation in the structured regions. Our results indicate that fs-laser micromachining is an attractive option to pattern biocompatible surfaces, and to investigate basic aspects of the relationship between surface topography and bacterial adhesion.

Lipid interaction triggering Septin2 to assembly into ß-sheet structures investigated by Langmuir monolayers and PM-IRRAS.

The molecular mechanisms responsible for protein structural changes in the central nervous system leading to Alzheimer's disease are unknown, but there is evidence that a family of proteins known as septins may be involved. Septins are a conserved group of GTP-binding proteins which participate in various cellular processes, including polarity determination and membrane dynamics. SEPT1, SEPT4, and SEPT2 have been found in deposits known as neurofibrillary tangles and glial fibrils in Alzheimer's disease. In this study, we provide molecular-level information for the interaction of SEPT2 with Langmuir monolayers at the air/water interface, which are used as simplified membrane models. The high surface activity of SEPT2 causes it to adsorb onto distinct types of lipid Langmuir monolayers, namely dipalmitoylphosphatidylcholine and PtdIns(4,5)P2. However, the interaction with PtdIns(4,5)P2 is much stronger, not only leading to a higher adsorption, but also to SEPT2 remaining inserted within the membrane at high surface pressures. Most importantly, in situ polarization-modulated infrared reflection absorption spectroscopy results indicated that the native secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when dipalmitoylphosphatidylcholine is at the air/water interface. Taken together, the results presented here suggest that the interaction between SEPT2 and the cell membrane may play an important role in the assembly of SEPT2 into amyloid-like fibers.

Immunosensor for HIV-1 diagnostics based on immobilization of the antigenic peptide p24-3 into liposomes.

In this work we developed an immunosensor for HIV-1 diagnostics that exploits the biorecognition between the antibody anti-p24 and the antigenic peptide p24-3 (AMATLRAEQASQEVKNWMTETL- LVQNA) derived from the HIV-1 p24 protein. p24-3 was encapsulated in phospholipid liposomes and immobilized in layer-by-layer (LbL) films produced with polyethyleneimine (PEI). The incorporation of p24-3 into liposomes was investigated using circular dichroism (CD) spectroscopy, from which an increase in the alpha helix conformation could be noted. The maximum fluorescence emission for p24-3 occurred at 340 nm in solution, compatible with the tryptophan residue being exposed to the solvent, and at 335 and 322 nm when in liposomes and PEI/p24-3-liposome LbL films, respectively. This blue shift is consistent with the tryptophan being in a partially buried environment. With the preserved structure in the LbL films, p24-3 could recognize the anti-p24 antibody in impedance spectroscopy measurements. Therefore, LbL films containing p24-3 may be suitable for detecting HIV-1 in a low-cost, easy-to-use immunosensing assay.

The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems.

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure-area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC(236)), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC(236) the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity.

Characterization of PAH/DPPG layer-by-layer films by VUV spectroscopy.

The spectroscopic characterization of layer-by-layer (LbL) films containing liposomes is essential not only for determining the precise film architecture but also to guide the design of drug delivery systems. In this study we provide the first report of vacuum ultraviolet spectroscopy (VUV) characterization of LbL films made with liposomes from 1.2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG) alternated with poly(allylamine hydrochloride) (PAH). Measurements in the 6.0-9.5eV range allowed us to identify the electronic transitions responsible for the spectra, which were assigned to carboxyl, hydroxyl and phosphate groups in DPPG while the PAH spectra were governed by electronic transitions in the amino groups. The surface mass density of the LbL films could be determined, from which the formation of a DPPG bilayer was inferred. This rupture of the liposomes into bilayers was confirmed with atomic force microscopy measurements. In subsidiary experiments we ensured that the UV irradiation in vacuum had negligible damage in the DPPG liposomes during the course of the VUV measurements. In addition to demonstrating the usefulness of VUV spectroscopy, the results presented here may be exploited in biological applications of liposome-containing films.

Bending of layer-by-layer films driven by an external magnetic field.

We report on optimized architectures containing layer-by-layer (LbL) films of natural rubber latex (NRL), carboxymethyl-chitosan (CMC) and magnetite (Fe3O4) nanoparticles (MNPs) deposited on flexible substrates, which could be easily bent by an external magnetic field. The mechanical response depended on the number of deposited layers and was explained semi-quantitatively with a fully atomistic model, where the LbL film was represented as superposing layers of hexagonal graphene-like atomic arrangements deposited on a stiffer substrate. The bending with no direct current or voltage being applied to a supramolecular structure containing biocompatible and antimicrobial materials represents a proof-of-principle experiment that is promising for tissue engineering applications in biomedicine.

Incorporation of acridine orange and methylene blue in Langmuir monolayers mimicking releasing nanostructures.

The efficiency of methylene blue (MB) and acridine orange (AO) for photodynamic therapy (PDT) is increased if encapsulated in liposomes. In this paper we determine the molecular-level interactions between MB or AO and mixed monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) and cholesterol (CHOL) using surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). To increase liposome stability, the effects from adding the surfactants Span® 80 and sodium cholate were also studied. Both MB and AO induce an expansion in the mixed monolayer, but this expansion is less significant in the presence of either Span® 80 or sodium cholate. The action of AO and MB occurred via coupling with phosphate groups of DPPC or DPPG. However, the levels of chain ordering and hydration of carbonyl and phosphate in headgroups depended on the photosensitizer and on the presence of Span® 80 or sodium cholate. From the PM-IRRAS spectra, we inferred that incorporation of MB and AO increased hydration of the monolayer headgroup, except for the case of the monolayer containing sodium cholate. This variability in behaviour offers an opportunity to tune the incorporation of AO and MB into liposomes which could be exploited in the release necessary for PDT.

Tailored SERS substrates obtained with cathodic arc plasma ion implantation of gold nanoparticles into a polymer matrix.

This manuscript reports on the fabrication of plasmonic substrates using cathodic arc plasma ion implantation, in addition to their performance as SERS substrates. The technique allows for the incorporation of a wide layer of metallic nanoparticles into a polymer matrix, such as PMMA. The ability to pattern different structures using the PMMA matrix is one of the main advantages of the fabrication method. This opens up new possibilities for obtaining tailored substrates with enhanced performance for SERS and other surface-enhanced spectroscopies, as well as for exploring the basic physics of patterned metal nanostructures. The architecture of the SERS-active substrate was varied using three adsorption strategies for incorporating a laser dye (rhodamine): alongside the nanoparticles into the polymer matrix, during the polymer cure and within nanoholes lithographed on the polymer. As a proof-of-concept, we obtained the SERS spectra of rhodamine for the three types of substrates. The hypothesis of incorporation of rhodamine molecules into the polymer matrix during the cathodic arc plasma ion implantation was supported by FDTD (Finite-Difference Time-Domain) simulations. In the case of arrays of nanoholes, rhodamine molecules could be adsorbed directly on the gold surface, then yielding a well-resolved SERS spectrum for a small amount of analyte owing to the short-range interactions and the large longitudinal field component inside the nanoholes. The results shown here demonstrate that the approach based on ion implantation can be adapted to produce reproducible tailored substrates for SERS and other surface-enhanced spectroscopies.

Chitosan effects on monolayers of zwitterionic, anionic and a natural lipid extract from E. coli at physiological pH.

Langmuir monolayers are used to simulate the biological membrane environment, acting as a mimetic system of the outer or the inner membrane leaflet. Herein, we analyze the interaction of membrane models with a partially N-acetylated chitosan (Ch35%) possessing a quasi-ideal random pattern of acetylation, full water solubility up to pH ≈ 8.5 and unusually high weight average molecular weight. Lipid monolayers containing dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethalonamine (DPPE), dipalmitoyl phosphatidyl glycerol (DPPG) or E. coli total lipid extract were spread onto subphases buffered at pH 4.5 or 7.4. The incorporation of Ch35% chitosan caused monolayer expansion and a general trend of decreasing monolayer rigidity with Ch35% concentration. Due to its relatively high content of N-acetylglucosamine (GlcNAc) units, Ch35% interactions with negatively charged monolayers and with E. coli extract were weaker than those involving zwitterionic monolayers or lipid rafts. While the smaller interaction with negatively charged lipids was unexpected, this finding can be attributed to the degree of acetylation (35%) which imparts a small number of charged groups for Ch35% to interact. Chitosan properties are therefore determinant for interactions with model cell membranes, which explains the variability in chitosan bactericide activity in the literature. This is the first study on the effects from chitosans on realistic models of bacterial membranes under physiological pH.

Using machine learning and an electronic tongue for discriminating saliva samples from oral cavity cancer patients and healthy individuals.

The diagnosis of cancer and other diseases using data from non-specific sensors - such as the electronic tongues (e-tongues) - is challenging owing to the lack of selectivity, in addition to the variability of biological samples. In this study, we demonstrate that impedance data obtained with an e-tongue in saliva samples can be used to diagnose cancer in the mouth. Data taken with a single-response microfluidic e-tongue applied to the saliva of 27 individuals were treated with multidimensional projection techniques and non-supervised and supervised machine learning algorithms. The distinction between healthy individuals and patients with cancer on the floor of mouth or oral cavity could only be made with supervised learning. Accuracy above 80% was obtained for the binary classification (YES or NO for cancer) using a Support Vector Machine (SVM) with radial basis function kernel and Random Forest. In the classification considering the type of cancer, the accuracy dropped to ca. 70%. The accuracy tended to increase when clinical information such as alcohol consumption was used in conjunction with the e-tongue data. With the random forest algorithm, the rules to explain the diagnosis could be identified using the concept of Multidimensional Calibration Space. Since the training of the machine learning algorithms is believed to be more efficient when the data of a larger number of patients are employed, the approach presented here is promising for computer-assisted diagnosis.

Wearable sensors made with solution-blow spinning poly(lactic acid) for non-enzymatic pesticide detection in agriculture and food safety.

On-site monitoring the presence of pesticides on crops and food samples is essential for precision and post-harvest agriculture, which demands nondestructive analytical methods for rapid, low-cost detection that is not achievable with gold standard methods. The synergy between eco-friendly substrates and printed devices may lead to wearable sensors for decentralized analysis of pesticides in precision agriculture. In this paper we report on a wearable non-enzymatic electrochemical sensor capable of detecting carbamate and bipyridinium pesticides on the surface of agricultural and food samples. The low-cost devices (

Immobilization of ibuprofen-containing nanospheres in layer-by-layer films.

Liposomes have been applied to many fields as nanocarriers, especially in drug delivery as active molecules may be entrapped either in their aqueous interior or onto the hydrophobic surface. In this paper we describe the fabrication of layer-by-layer (LbL) films made with liposomes incorporating the anti-inflammatory ibuprofen. The liposomes were made with dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol (DPPG) and palmitoyl oleoyl phosphatidyl glycerol (POPG). LbL films were assembled via alternate adsorption of the polyamidoamine dendrimer (PAMAM), generation 4, and liposomes containing ibuprofen. According to dynamic light scattering measurements, the incorporation of ibuprofen caused DPPC and DPPG liposomes to become more stable, with a decrease in diameter from 140 to 74 nm and 132 to 63 nm, respectively. In contrast, liposomes from POPG became less stable, with an increase in size from 110 to 160 nm after ibuprofen incorporation. These results were confirmed by atomic force microscopy images of LbL films, which showed a large tendency to rupture for POPG liposomes. Film growth was monitored using nanogravimetry and UV-Vis spectroscopy, indicating that growth stops after 10 bilayers. The release of ibuprofen obtained with fluorescence measurements was slower for the liposomes, with decay times of 9.2 and 8.5 h for DPPG and POPG liposomes, respectively, than for the free drug with a decay time of 5.2 h. Ibuprofen could also be released from the LbL films made with DPPG and POPG liposomes, which is promising for further uses in patches.

Cholesterol modulates the interaction between paclitaxel and Langmuir monolayers simulating cell membranes.

The composition of Langmuir monolayers used as cell membrane models is an essential factor for the interaction with biologically-relevant molecules, including pharmaceutical drugs. In this paper, we report the modulation of effects from the antineoplastic drug paclitaxel by the relative concentration of cholesterol in the Langmuir monolayers of ternary mixtures of dipalmitoylphosphatidylcholine, sphingomyelin, and cholesterol. Since the dependence on cholesterol concentration for these monolayers simulating lipid rafts is non-monotonic, we analyzed the surface pressure and compressibility modulus data with the multidimensional projection technique referred to as interactive document mapping (IDMAP). The maximum expansion induced by paclitaxel in surface pressure isotherms was observed for 27% cholesterol, while the compressibility modulus decreased most strongly for the monolayer with 48% cholesterol. Therefore, the physiological action of paclitaxel may vary depending on whether it is associated with penetration in the membrane or with changes in the membrane elasticity.

Bacterial Photoinactivation Using PLGA Electrospun Scaffolds.

The use of ultraviolet (UV) and blue irradiation to sterilize surfaces is well established, but commercial applications would be enhanced if the light source is replaced with ambient light. In this paper, it is shown that nanofibers can be explored as an alternative methodology to UV and blue irradiation for bacterial inactivation. It is demonstrated that this is indeed possible using spun nanofibers of poly[lactic-co-(glycolic acid)] (PLGA). This work shows that PLGA spun scaffolds can promote photoinactivation of Staphylococcus aureus and Escherichia coli bacteria with ambient light or with laser irradiation at 630 nm. With the optimized scaffold composition of PLGA85:15 nanofibers, the minimum intensity required to kill the bacteria is much lower than in antimicrobial blue light applications. The enhanced effect introduced by PLGA scaffolds is due to their nanofiber structures since PLGA spun nanofibers were able to inactivate both S. aureus and E. coli bacteria, but cast films had no effect. These findings pave the way for an entirely different method to sterilize surfaces, which is less costly and environmentally friendly than current procedures. In addition, the scaffolds could also be used in cancer treatment with fewer side effects since photosensitizers are not required.

Interaction of a C-terminal peptide of Bos taurus diacylglycerol acyltransferase 1 with model membranes.

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and dedicated step in the synthesis of triacylglycerol, which is believed to involve the lipids oleoyl coenzyme A (OCoA) and dioleoyl-sn-glycerol (DOG) as substrates. In this work we investigated the interaction of a specific peptide, referred to as SIT2, on the C-terminal of DGAT1 (HKWCIRHFYKP) with model membranes made with OCoA and DOG in Langmuir monolayers and liposomes. According to the circular dichroism and fluorescence data, conformational changes on SIT2 were seen only on liposomes containing OCoA and DOG. In Langmuir monolayers, SIT2 causes the isotherms of neat OCoA and DOG monolayers to be expanded, but has negligible effect on mixed monolayers of OCoA and DOG. This synergistic interaction between SIT2 and DOG+OCoA may be rationalized in terms of a molecular model in which SIT2 may serve as a linkage between the two lipids. Our results therefore provide molecular-level evidence for the interaction between this domain and the substrates OCoA and DOG for the synthesis of triacylglycerol.