Lipid Bilayer Of Liposomes Research Articles

Selective uptake and modulation of nanometal surface energy transfer from quantum dot to Au nanoparticle across lipid bilayer of liposomes

Understanding the initial adsorption and subsequent translocation of nanoparticles (NPs) across the phospholipid bilayer of liposome find importance in drug delivery and in vivo bio-imaging applications. Herein, we have investigated the time-dependent selective uptake of 2.0 ± 0.3 nm-sized CdTe quantum dots (QDs) into small unilamellar vesicles (SUV) of dipalmitoylphoshatidylcholine (DPPC) in its gel phase and subsequent alteration of nanometal surface energy transfer (NSET) in the presence of 18.0 ± 0.2 nm-sized citrate-capped gold NPs (Au NPs). The interactions between CdTe QD and Au NP across the lipid bilayer of DPPC liposome has been explored by monitoring the NSET process using fluorescence spectroscopy. The time-dependent selective partitioning of QDs into the liposomal phase has been demonstrated using confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). It has been observed that instant mixing of QDs and Au NPs with liposomes does not alter the extent of NSET between QD-Au NP pair relative to that in the absence of liposome. However, equilibrating the binary mixture of liposome and QDs for 24 h at room temperature leads to the formation of liposome encapsulated QD (LipQD) which does not take part in NSET with aqueous phase Au NPs. In contrast, equilibrating the ternary mixture of QDs, liposomes, and Au NPs for 24 h results in the formation of Au NP adsorbed QD-encapsulated liposomes (AuLipQD) which exhibit moderate amount of NSET from encapsulated QDs to the adsorbed Au NPs at the surface of liposomes. It has been observed that the efficiency of NSET decreases from 62% in bulk aqueous medium to 24% in AuLipQD complex. Our present findings may be useful to understand the fundamental interaction of other metal and semiconductor NPs with liposomes and cell membrane for various drug-delivery and bio-imaging applications.

Photosensitizer-Doped and Plasma Membrane-Responsive Liposomes for Nuclear Drug Delivery and Multidrug Resistance Reversal.

Clinically approved doxorubicin (Dox)-loaded liposomes (e.g., Doxil) guarantee good biosafety, but their insufficient nuclear delivery of Dox (<0.4%) after cellular uptake significantly hampers their final anticancer efficacy. Here, we report that simply doping protoporphyrin IX (PpIX, a commonly used hydrophobic photosensitizer) into the lipid bilayers of Dox-loaded liposomes (the resultant product is termed PpIX/Dox liposomes) is a feasible way to promote the nuclear delivery of Dox. This facile strategy relies on a unique property of PpIX-it presents considerably higher affinity for the real plasma membrane over its liposomal carrier, which drives the doped PpIX molecules to detach from the liposomes when encountering cancer cells. We demonstrate that this process can trigger the efficient release of the loaded Dox molecules and allow them to enter the nuclei of MCF-7 breast cancer cells without being trapped by lysosomes. Regarding the drug-resistant MCF-7/ADR cells, the aberrant activation of the efflux pumps in the plasma membranes expels the internalized Dox. However, we strikingly find that the robust drug resistance can be reversed upon mild laser irradiation because the photodynamic effect of PpIX disrupts the drug efflux system (e.g., P-glycoprotein) and facilitates the nuclear entry of Dox. As a proof-of-concept, this PpIX doping strategy is also applicable for enhancing the effectiveness of cisplatin-loaded liposomes against both A549 and A549/DDP lung cancer cells. In vivo experimental results prove that a single injection of PpIX/Dox liposomes completely impedes the growth of MCF-7 tumors in nude mice within 2 weeks and, in combination with laser irradiation, can synergistically ablate MCF-7/ADR tumors. Biosafety assessments reveal no significant systemic toxicity caused by PpIX/Dox liposomes. This work exemplifies a facile method to modulate the subcellular fate of liposomal drugs and may inspire the optimization of nanopharmaceuticals in the near future.

Physicochemical properties of L-alpha dipalmitoyl phosphatidylcholine large unilamellar vesicles: Effect of hydrophobic block (PLA/PCL) of amphipathic diblock copolymers

In the present work, we show how amphipathic diblock copolymers affect the physicochemical properties of the lipid bilayer of DPPC liposome. Diblock copolymers proposed for this study are focused in the difference between PLA and PCL hydrophobic block, because PLA and PCL differ in their glass transition temperature, where a higher ratio of PLA, lowers the flexibility of the diblock copolymer. On the contrary, a greater proportion of PCL makes the diblock copolymer more flexible. This flexibility difference between hydrophobic block would affect the physicochemical properties of lipid bilayer of DPPC. The difference of rigidity or flexibility of hydrophobic block and their interaction with DPPC large unilamellar vesicles (LUVs) was evaluated at low and high copolymers concentration. The copolymer concentrations used were chosen based on their respective cmc. We measure (a) Thermotropic behavior from GP of Laurdan and fluorescence anisotropy of DPH; (b) Relation between wavelength excitation and generalized polarization of Laurdan; (c) Time-resolved fluorescence anisotropy of DPH; (d) Water outflow through the lipid bilayer and (e) calcein release from DPPC LUVs. Furthermore, large unilamellar vesicles in the absence and in the presence of different copolymers were characterized by size and zeta-potential. The results show that the diblock copolymer at high PLA/PCL ratio, that is, greater rigidity of hydrophobic block produces an increase of the phase transition temperature (Tm). For DPPC LUVs, Tm increase 3.5 °C at low and about 4.5 °C at high copolymers concentration, sensed by Laurdan and DPH fluorescent probes, although the DPPC/copolymers molar ratio for Cop4 is higher than Cop3, Cop2 and Cop1. In addition, we observed a decrease in the polarity of microenvironments in the bilayer and an increase in the order of the acyl chains in the bilayer to a high proportion of PLA. Furthermore, the presence of diblock copolymer with high proportion of PLA, decreases water outflow from DPPC liposome and water efflux is slower; leading to a decrease in calcein release from DPPC liposomes. Our results clearly show that the greater the stiffness of the hydrophobic block, greater degree of packaging of the lipid bilayer, greater the order of the acyl chains, and greater retention of water and calcein inside the liposome. Therefore, the presence of AB-type diblock copolymers with a more rigid hydrophobic block, stabilizes the lipid bilayer and would allow a more controlled release of water, and encapsulated molecules inside of the DPPC liposome.

Transforming a toxic drug into an efficacious nanomedicine using a lipoprodrug strategy for the treatment of patient-derived melanoma xenografts

Despite the progress made with the recent clinical use of the anticancer compound cabazitaxel, the efficacy in patients remains unsatisfactory, largely due to the high in vivo toxicity of the agent. Therefore, strategies that achieve favorable outcomes and good safety profiles will greatly expand the repertoire of this potent agent. Here, we propose a combinatorial strategy to reform the cabazitaxel agent and the use of sequential supramolecular nanoassembly with liposomal compositions to assemble a prodrug-formulated liposome, termed lipoprodrug, for safe and effective drug delivery. Reconstructing cabazitaxel with a polyunsaturated fatty acid (i.e., docosahexaenoic acid) via a hydrolyzable ester bond confers the generated prodrug with the ability to be readily integrated into the lipid bilayer of liposomes for systemic administration. The resulting lipoprodrug scaffold showed significantly sustained drug release profiles and improved pharmacokinetics in rats as well as a reduction in systemic toxicity in vivo. Notably, the lipoprodrug outperformed free cabazitaxel in terms of in vivo therapeutic efficacy in multiple separate tumor xenograft-bearing mouse models, one of which was a patient-derived xenograft model. Surprisingly, the lipoprodrug was able to reduce tumor invasiveness and reprogram the tumor immunosuppressive microenvironment by proinflammatory macrophage polarization. Our findings validate this lipoprodrug approach as a simple yet effective strategy for transforming the highly toxic cabazitaxel agent into an efficacious nanomedicine with excellent in vivo tolerability. This approach could also be applied to rescue other drugs or drug candidates that have failed in clinical trials due to poor pharmacokinetic properties or unacceptable toxicity in patients.

Modification of curcumin-loaded liposome with edible compounds to enhance ability of crossing blood brain barrier

This research explored the potential of food grade sialic acid (SA), polymerized SA (PSA), and their oxidized form to conjugate with wheat germ agglutinin (WGA) on liposome to deliver curcumin to brain. Results showed that curcumin was entrapped in liposome with high encapsulation efficiency (90.77%) and the vector had a size of about 115 nm in diameter. The thermogravimetric analysis (TGA) confirmed the successful presence of SA and PSA in the lipid bilayers of liposomes. The TGA curves below 100 °C suggested that WGA acts as a protective layer for SA and PSA. In general, the liposomes modified by the un-oxidized SA or PSA could conjugate more WGA than that oxidized counterparts, and polymerization of SA did not increase WGA conjugation on the liposomes. More than 40% of WGA on the surface of modified liposomes remained after digestive enzymes challenge. The amide peaks at 1241 cm-1 and 533-581 cm-1 in the Fourier transform infrared (FTIR) spectrum of the modified liposomes indicated the covalent bonding between carboxyl group of SA and amine group of WGA. These characteristic peaks still existed after digestive enzymes challenge, demonstrating that the conjugation of WGA and SA on the modified liposome can resist the digestive enzymes to a certain extent. SEM images showed that the modified liposomes after digestive enzymes treatment were still spherical with clear rim, and the enzyme-treated vectors had a higher permeation rate crossing blood-brain barrier (BBB) endothelial cell monolayer than the un-modified liposome without enzyme treatment. This study demonstrates how the surface modification can enhance the function of liposome as a phytochemical carrier for brain disease.

The simultaneous quantification of phytosterols and tocopherols in liposomal formulations using validated atmospheric pressure chemical ionization- liquid chromatography –tandem mass spectrometry

A novel liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and β-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05−10 μg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25−10 μg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25−10 μg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC–MS/MS method addresses three major analytical shortcomings, namely i)longer run time, ii)complex gradient elution and iii)poor baseline separation of phytosterols and tocopherols.

Interaction of amphiphilic coumarin with DPPC/DPPS lipid bilayer: effects of concentration and alkyl tail length.

In this work, interactions between amphiphilic amino methyl coumarin and dipalmitoyl-sn-glycero-3-phosphocholine/dipalmitoyl-sn-glycero-3-phosphoserine (DPPC/DPPS) lipid bilayer were investigated. A combination of experimental techniques (zeta potential, fluorescence spectroscopy, and differential scanning calorimetry) along with molecular dynamics simulations was employed to examine the influence of alkyl tail length and concentration of the amphiphilic coumarin on the lipid bilayer. Alkyl tails comprising 5(C5), 9(C9), and 12(C12) carbon atoms were conjugated to amino methyl coumarin via a single-step process. The binding and insertion mechanisms of the amphiphilic coumarins were studied in increasing concentrations for short-tailed (C5) and long-tailed (C12) coumarins. The simulation results show that C5 coumarin molecules penetrate the lipid bilayer, but owing to the short alkyl tail, they interact primarily with the lipid head groups resulting in lipid bilayer thinning; however, at high concentrations, the C5 coumarins undergo continuous insertion-ejection from the outer leaflet of the lipid bilayer. In contrast, C12 coumarins interact favorably with the hydrophobic lipid tails and lack the ejection-reinsertion behavior. Instead, the C12 coumarin molecules undergo flip-flops between the outer and inner leaflets of the lipid bilayer. At high concentrations, the high-frequency flip-flops lead to lipid destabilization, causing the lipid bilayer to rupture. The simulation results are in excellent agreement with the toxicity of amphiphilic coumarin activity in cancer cells. The efficacy of amphiphilic coumarins in liposomal lipid bilayers demonstrates the promise of these molecules as a tool in the treatment of cancer.

Development and Characterization of the Solvent-Assisted Active Loading Technology (SALT) for Liposomal Loading of Poorly Water-Soluble Compounds.

A large proportion of pharmaceutical compounds exhibit poor water solubility, impacting their delivery. These compounds can be passively encapsulated in the lipid bilayer of liposomes to improve their water solubility, but the loading capacity and stability are poor, leading to burst drug leakage. The solvent-assisted active loading technology (SALT) was developed to promote active loading of poorly soluble drugs in the liposomal core to improve the encapsulation efficiency and formulation stability. By adding a small volume (~5 vol%) of a water miscible solvent to the liposomal loading mixture, we achieved complete, rapid loading of a range of poorly soluble compounds and attained a high drug-to-lipid ratio with stable drug retention. This led to improvements in the circulation half-life, tolerability, and efficacy profiles. In this mini-review, we summarize our results from three studies demonstrating that SALT is a robust and versatile platform to improve active loading of poorly water-soluble compounds. We have validated SALT as a tool for improving drug solubility, liposomal loading efficiency and retention, stability, palatability, and pharmacokinetics (PK), while retaining the ability of the compounds to exert pharmacological effects.

Evaluation of the effect of liposomes loaded with chlorogenic acid in treatment of 2,4,6-trinitrobenzenesulfonic acid-induced murine colitis.

Crohn's Disease (CD), one of the types of inflammatory bowel disease, poses a significant challenge to modern healthcare. This condition severely impacts patients' quality of life, and its incidence is continuously rising. Despite constant research, current treatment options are limited and largely unsuccessful and result in serious side effects, therefore new therapy alternatives are needed. Liposomal formulation provides a new hope for disease management. In our study, we characterized the anti-inflammatory activity of mesalazine (5-ASA) and chlorogenic acid (CGA) encapsulated in liposomal formulation in the animal model of CD. Liposomes were obtained by thin film hydration method and characterized in terms of suspension stability and particle size and distribution. Colitis was induced in mice by intracolonic (i.c.) administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The effect of treatment with liposomal suspensions of 5-ASA and CGA was evaluated macroscopically and by measuring myeloperoxidase (MPO) activity. We observed that liposome-encapsulated 5-ASA (5 mg/kg), but not CGA (20 mg/kg) attenuated colitis as evidenced by a decreased macroscopic and microscopic scores. It may be hypothesized that the composition of liposomal lipid bilayer as well as the switch in macrophage populations leading to unfavorable accumulation of anti-inflammatory agents in the cells may underly the efficiency of obtained liposomes and need to be taken into consideration in further studies on drug delivery.

Novel Class of Ultrasound-Triggerable Drug Delivery Systems for the Improved Treatment of Tumors.

The controlled release of anticancer drugs at the tumor site is a central challenge in treating cancer. To achieve this goal, our strategy was based on tumor-specific targeting and ultrasound-triggered release of an anticancer agent from liposomal nanocarriers. To enhance the ultrasound-triggered drug release, we incorporated a lipophilic sonosensitizer, chlorin e6 (Ce6) ester, into the lipid bilayer of liposomes. Additionally, asparagine-glycine-arginine (NGR) that binds to CD13, which is overexpressed in tumor cells, was introduced into these liposomes. Under the navigation effects of the NGR, the novel ultrasound-triggerable NGR-modified liposomal nanocarrier (NGR/UT-L) accumulates in tumor sites. Once irradiated by ultrasound in tumor tissues, the sonodynamic effect produced by Ce6 could create more efficient disruptions of the lipid bilayer of the liposomal nanocarriers. After encapsulating doxorubicin (DOX) as the model drug, the ultrasound triggered lipid bilayer breakdown can spring the immediate release of DOX, making it possible for ultrasound-responsive chemotherapy with great selectivity. By combining tumor-specific targeting and stimuli-responsive controlled release into one system, NGR/UT-L demonstrated a perfect antitumor effect. Moreover, this report provides an example of controlled-release by means of a novel class of ultrasound triggering systems.

Reversible Aggregation of Chlorophyll Derivative Induced by Phase Transition of Lipid.

Controlling the supramolecular organization of pigment molecules will provide innovative materials that exhibit variable optical properties. In nature, photosynthetic systems employ chlorophyllous supramolecules in which each pigment molecule is suitably organized in proteins, and their properties are adequately optimized by changing the structures of the surrounding amino acid residues. Here, we report a strategy for varying the aggregation behavior of a chlorophyll derivative by using a phase-transition phenomenon of lipid bilayers. Methyl pyropheophorbide a (MPP) was employed as a chlorophyllous pigment in our artificial system, and synthetic phosphatidylcholines with saturated acyl chain(s) were also used. The MPP molecules successfully accumulated within the lipid bilayer of liposomes without changing the vesicular structure. When the lipid bilayer was in a gel form (under the phase-transition temperature, Tm), the embedded MPP aggregated to yield a dimeric form showing red-shifted absorption bands and circular dichroism signals. When the solutions of MPP-containing liposomes were heated to higher temperatures than their Tm, MPP disaggregated to monomeric form as the absorption spectrum changed into its original fashion in dichloromethane. The reversible thermochromic (dis)aggregation of the MPP molecules had good cyclability. Additional careful examination of the phase transition in the MPP-lipid co-assemblies clarified that the critical temperatures of the MPP (dis)aggregation were in good agreement with the phase-transition temperatures of the pigment-containing bilayers. The reversible MPP aggregation in the lipid bilayers occurred in a wide range of temperatures (around 10-55 °C) by changing the length of the diacyl side chains of phospholipids. The reversible thermochromism of the chlorophyllous system was established by varying the nature of the surrounding lipid bilayer. This study can provide a useful strategy for making variable tetrapyrrolic aggregate systems induced by mild extrinsic stimuli.

Implication of Cholesterol in Regulating the Pore‐formation Mechanism of Vibrio Cholerae Cytolysin, a β‐barrel Pore‐forming toxin

Vibrio cholerae cytolysin (VCC) is a β‐barrel pore‐forming toxin with potent cell‐killing cytolytic/cytotoxic activity. To exert it's cytolytic effect, VCC binds to the target cell membranes and forms transmembrane oligomeric β‐barrel pores. Earlier studies have indicated that cholesterol might play a critical role in regulating the mode of action of VCC. However, exact mechanistic basis of the process still remains unknown. In the present study, we have explored and examined the roles of cholesterol in the different steps of VCC mode of action. Employing the membrane lipid bilayer of lipid vesicles or liposomes containing varying amount of cholesterol, we have shown that cholesterol governs the efficient interaction of the toxin with the membrane lipid bilayer that in turn facilitates subsequent oligomeric pore‐formation process. We have also studied the physiological relevance of membrane cholesterol in the context of the biomembranes of the eukaryotic cells. Our results have shown that the depletion of membrane cholesterol from erythrocytes compromises the pore‐forming activity of VCC. In the biomembrane of erythrocytes, where accessory interactions are available, binding of VCC is not affected to any significant extent. However, upon depletion of membrane cholesterol in the erythrocytes membranes, membrane‐bound fraction of VCC remains trapped in the form of abortive oligomeric assembly, and does not form the functional pores. Our study has also revealed that VCC shows a marked tendency to associate with the cholesterol‐rich membrane micro‐domains on erythrocytes that, in turn, appears to facilitate cholesterol‐dependent pore‐formation mechanism of the toxin. In sum, our results suggest that the presence of cholesterol in the target membranes play critical role for the pore‐formation mechanism of VCC.Support or Funding InformationWe acknowledge the support through funding from the Department of Biotechnology (DBT), Govt. of India [DBT grant no. BT/HRD/NBA/37/01/2014 (x)] and also the support through funding under the Centre of Excellence (COE) in Frontier Areas of Science and Technology (FAST) program of the Ministry of Human Resource Development, Govt. of India, in the area of protein science, design, and engineering (to K.C.). We also thank the Indian Institute of Science Education and Research Mohali for the support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A226: Cell-free membrane reconstitution system for cis and trans interaction of T-cell co-receptors and ligands

Abstract Immunotherapy, which harnesses the immune system to combat cancers, is a paradigm-shifting approach in oncology. One important branch of immunotherapy is to use drugs to perturb molecular interactions at the T-cell–antigen presenting cell (APC) interface. In particular, antibodies that block the T-cell coinhibitory receptor PD-1 or its ligand PD-L1 have produced unprecedented clinical activities against a subset of human cancers in a fraction of patients. Extending immunotherapy to a larger patient population requires a better understanding of the interaction network at the T-cell-APC interface. Typically, it is believed that ligands from APCs bind to receptors on T-cells (in trans) to trigger intracellular signaling. A largely overlooked fact is that many receptors often co-exist with ligands on T-cells and APCs, including PD-1/PD-L1 and CD28/B7.1. Using novel reconstitution approaches, we recently discovered that co-expressed PD- 1 and PD-L1 bind to each other in cis and this cis interaction competes with their trans interaction to inhibit PD-1 signaling. We hypothesize that cis interaction occurs with many other ligand-receptor pairs and may regulate other aspects of tumor immunity by perturbing their trans interactions. We are currently using our robust reconstitution systems to ask whether other immunoreceptors and ligands interact in cis and how the putative cis interaction affect their trans interactions. Specifically, to detect cis interaction, we co-attach the ligand and receptor to liposomes, and probe their cis interaction using a fluorescence resonance energy transfer (FRET) readout. To detect trans interaction, we have developed a liposome-lipid bilayer conjugation assay, in which binding between ligand- labeled liposomes and receptor-labeled supported lipid bilayer (SLB) is used as an index for trans interaction. To determine whether cis interaction inhibits trans interaction, we ask in the liposome-bilayer system if addition of receptor on liposomes decreases liposome-SLB conjugation. Our preliminary data revealed that CD28 only binds B7.1 in trans, but not in cis. In contrast, PD-1 binds with PD-L1 both in cis and trans, and the two modes of interaction compete with each other. Further experiments in cells confirmed our finding in vitro, demonstrating the utility of our cell-free reconstitution systems in detecting cis and trans interaction of T-cell co-receptors. Insights from this study will help us clarify the ligand-receptor interaction network at the immunological synapse. Citation Format: Yunlong Zhao, Enfu Hui. Cell-free membrane reconstitution system for cis and trans interaction of T-cell co-receptors and ligands [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A226.

Investigating Electrochemical Stability and Reliability of Gold Electrode‐electrolyte Systems to Develop Bioelectronic Nose Using Insect Olfactory Receptor

AbstractThis article aims to demonstrate an electrochemically stable and reliable gold electrode‐electrolyte system to develop an insect odorant receptor (Drosophila melanogaster Or35a) based bioelectronic nose. Cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS) of bare gold electrodes, after modification with the self‐assembled monolayer (SAM) of 6‐mercaptohexanoic acid (MHA) and after immobilization with Or35a integrated into the lipid bilayers of liposomes were conducted in the presence of four different redox probes. Potassium ferri/ferrocyanide [Fe(CN)6]3−/[Fe (CN)6]4− and hydroquinone (H2Q) redox probes revealed variable and irreversible signals at the time scale of our measurements, with atomic force microscopy (AFM) images and x‐ray photoelectron spectroscopy (XPS) results suggesting gold surface etching due to the presence of CN− ions in case of [Fe(CN)6]3−/[Fe (CN)6]4−. Although the hexaammineruthenium complex showed stable electrochemical behaviour at all stages of biosensor development, changes in CV and EIS readings after each surface modifications were insignificant. PBS buffer as a non‐Faradaic medium, was found to provide reliable systems for electrochemical probing of modified gold electrodes with Or35a/liposomes in aqueous media. Using this system, we have shown that this novel biosensor can detect its known odorant E2‐hexenal selectively compared to methyl salicylate down to femtomolar concentration.

Development and characteristics of novel sonosensitive liposomes for vincristine bitartrate

The aim of drug delivery is to increase therapeutic efficacy. Externally triggered drug delivery systems enable site-specific and time-controlled drug release. To achieve this goal, our strategy was based on ultrasound-triggered release of an anticancer agent from sonosensitive liposomes (SL). To realize the ultrasound-triggered drug release, a lipophilic sonosensitizer, hematoporphyrin monomethyl ether (HMME) was incorporated into the lipid bilayer of liposomes. Once irradiated by the ultrasound in tumor tissues, the sonodynamic effect generated by HMME could lead to an efficient disruption of the lipid bilayer in the SL. After encapsulating vincristine bitartrate (VIN) as the model drug, the ultrasound-triggered lipid bilayer breakdown can trigger the instant release of VIN, enabling ultrasound-controlled chemotherapy with great specificity. In the in vitro and in vivo studies, by integrating tumor-specific targeting and stimuli-responsive controlled release into one system, VIN-loaded SL showed excellent antitumor efficacy. The SL could potentially produce viable clinical strategies for improved targeting efficiency of VIN for the treatment of related cancer. More importantly, this report provides an example of controlled release by means of a novel class of ultrasound triggering system.

An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants

Herein, we present that insect odorant receptors reconstituted into the lipid bilayers of liposomes can be successfully immobilized onto a gold surface and selectively and sensitively detect odorant molecules. The odorant receptors (OrXs) Or10a, Or22a, and Or71a from the common fruit fly, Drosophila melanogaster, were recombinantly expressed, purified and integrated into nano-liposomes (100–200 nm). These liposomes were covalently attached to the self-assembled monolayers (SAMs) of a 6-mercaptohexanoic acid (MHA)-modified gold surface. X-ray Photo Electron Spectroscopy (XPS) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements confirmed the successful modification of the gold surface and immobilization of liposomes. Atomic Force Microscopy (AFM) revealed that the liposomes were covalently attached to the surface without any disruption of vesicles. The liposomes tethered to the gold sensor surface were then treated with a range of known ligands of various concentrations. We demonstrated by Electrochemical Impedance Spectroscopy (EIS) that an OrX/liposome EIS sensor can sensitively and selectively detect its known ligand to femtomolar concentrations by detecting a change in electrical signal upon binding. Our study is the first step towards using purified insect odorant receptors alone in biosensors to enable the development of novel ultrasensitive volatile sensors for medical diagnostic, air quality, food safety and border security applications.

Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing.

Increasing antibiotic resistance in pathogenic microorganisms has led to renewed interest in bacteriophage therapy in both humans and animals. A “Trojan Horse” approach utilizing liposome encapsulated phages may facilitate access to phagocytic cells infected with intracellular pathogens residing therein, e.g., to treat infections caused by Mycobacterium tuberculosis, Listeria, Salmonella, and Staphylococcus sp. Additionally, liposome encapsulated phages may adhere to and diffuse within mucosa harboring resistant bacteria which are challenges in treating respiratory and gastrointestinal infections. Orally delivered phages tend to have short residence times in the gastrointestinal tract due to clinical symptoms such as diarrhea; this may be addressed through mucoadhesion of liposomes. In the present study we have evaluated the use of a microfluidic based technique for the encapsulation of bacteriophages in liposomes having mean sizes between 100 and 300 nm. Encapsulation of two model phages was undertaken, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). The yield of encapsulated T3 phages was 109 PFU/ml and for phage K was much lower at 105 PFU/ml. The encapsulation yield for E. coli T3 phages was affected by aggregation of T3 phages. S. aureus phage K was found to interact with the liposome lipid bilayer resulting in large numbers of phages bound to the outside of the formed liposomes instead of being trapped inside them. We were able to inactivate the liposome bound S. aureus K phages whilst retaining the activity of the encapsulated phages in order to estimate the yield of microfluidic encapsulation of large tailed phages. Previous published studies on phage encapsulation in liposomes may have overestimated the yield of encapsulated tailed phages. This overestimation may affect the efficacy of phage dose delivered at the site of infection. Externally bound phages would be inactivated in the stomach acid resulting in low doses of phages delivered at the site of infection further downstream in the gastrointestinal tract.

Preparation of liposomes encapsulated Epirubicin-gold nanoparticles for Tumor specific delivery and release

Liposome/metal nanoparticle conjugates are found to be a promising carrier tool for effectively delivering wide variety of drugs in a targeted site and without arguing toxic side effects of existing anticancer drugs. In this scenario, the present work was performed to choose Epirubicin-HCl (EPI) as model anticancer drug to formulate citrate stabilized EPI-gold nanoparticles (EPI-GNPs). Further, the biocompatible EPI-GNPs incorporated with lipid bilayer of liposomes formulated to exhibit effective drug delivery system. The biosynthesized EPI-GNPs and its incorporation in a lipid bilayer of liposomes was confirmed by UV-visible spectrophotometer, drug encapsulation, loading capacity, pH stability and drug releasing capacity were studied. Simultaneously, drug and carrier interaction, physical nature, stability, morphological and particle size characteristics were also studied by FTIR, XRD, SEM, TEM and Zeta size analyzer. The obtained characterization data confirmed the properties of EPI-GNPs encapsulated liposomes. The in vitro cytotoxic screening of EPI-GNPs encapsulated liposomes arrest the proliferation of MCF-7 breast cancer cell lines, at 0.1 μg/ml of liposomes displayed 52% of cell death at 24 h. The EPI-GNPs encapsulated liposomes induced time and dose dependent apoptosis compared with untreated control cancer cells.

Oligomerisation of Synaptobrevin-2 Studied by Native Mass Spectrometry and Chemical Cross-Linking

Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies. Here, we employ three Synaptobrevin-2 variants—the full-length protein Syb(1-116), the soluble, cytosolic variant Syb(1-96) as well as a shorter version Syb(49-96) containing structured segments but omitting a trigger site for SNARE complex formation—to study oligomerisation in the absence of interaction partners or when incorporated into the lipid bilayer of liposomes. Combining native mass spectrometry with chemical cross-linking, we find that the truncated versions show increased oligomerisation. Our findings from both techniques agree well and confirm the presence of oligomers in solution while membrane-bound Synaptobrevin-2 is mostly monomeric. Using ion mobility mass spectrometry, we could further show that lower charge states of Syb(49-96) oligomers, which most likely represent solution structures, follow an isotropic growth curve suggesting that they are intrinsically disordered. From a technical point of view, we show that the combination of native ion mobility mass spectrometry with chemical cross-linking is well-suited for the analysis of protein homo-oligomers.Graphical ᅟ

Structural characterization of multilamellar liposomes coencapsulating curcumin and vitamin D3

This study aimed the production of multilamellar liposomes coencapsulating two hydrophobic bioactives, curcumin and vitamin D3, which present antioxidant and nutraceutical properties. Liposomes were produced by proliposomes hydration and stabilized with a mixture of xanthan and guar gums. Proliposomes were produced by coating of micronized sucrose method. Physicochemical properties of the dispersions were evaluated over 42 days of refrigerated storage. Microstructural characterization were performed by differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS) and small deformation rheological analyses. Liposomes showed spherical and near-spherical shapes and presented hydrodynamic diameters in the 680–1580 nm range. Curcumin and cholecalciferol were loaded into the same vesicles, with preservation efficiencies ranging from 89.6 to 93.3% and from 88.3 to 91.3%, respectively, after 42 days of storage. SAXS data showed that the lipid bilayer of liposomes produced in the presence of curcumin, vitamin D3, or a mixture of both, are very similar. Also, higher amounts of vitamin D3 did not affect negatively the stability of phospholipid vesicles. Overall, it was demonstrated that the coencapsulation of curcumin and vitamin D3 by liposomes is feasible and high contents of bioactives were retained throughout the storage time.