Phospholipid Suspension Research Articles

Rapid production of liposomes using high pressure carbon dioxide and direct ultrasonication

Herein, we introduce a protocol for preparing liposomes using high-pressure CO2, water and direct ultrasonication (HPC-D) allowing rapid formation of liposomes in a single-step. In the HPC-D method, phospholipid suspensions were treated at temperatures from 25 °C to 70 °C and pressures from 4 MPa to 6.8 MPa with direct ultrasonication. The liposomes produced from HPC-D had an average particle size of 159 ± 2 nm to 136 ± 8 nm at liposome recovery yields up to 95.3 ± 4.6 %. A mechanism for the HPC-D method is proposed, in which the micro-phase separation between water-CO2 interface increases surface area and phospholipids act as surfactants and reassemble into small liposome particles. Drug loading (DL) and encapsulation efficiency (EE) of liposomes obtained with the HPC-D method for Cyclosporin A gave DL values of 37.4 ± 3.4 % and EE of 79.7 ± 2.5 %, confirming efficient entrapment.

Abdominal Contrast Enhanced Ultrasound, A Practical Review

Abstract Abdominal contrast enhanced ultrasound (CEUS) is a relatively new investigation method that combines conventional ultrasound with a small amount of intravenous contrast through a peripheral vein (usually the cubital vein). The contrast is made by very small gas microbubbles floating in a phospholipid suspension. The size of microbubbles are between 1-10 micrometers, being to large to go out of the vessels, but sufficiently small to stay into the capillaries, and this is the reason for they can be used as vascular tracers. So, it is possible to obtain images with microvessels inside of different organs in different vascular phases (arterial, portal, venous), images that are similar with the one obtained in CECT and CEMRI. The intravenous contrast used in CEUS eliminates trough the lungs, during expiration. These is the reason why the most important contraindication for using iv contrast is severe respiratory failure. Also, it is important that intravenous contrast is not allergenic, the percentage of allergic reaction being extremly small (0,001%). The most important advantages of CEUS are: • It can be safely used for the pacients that are allergic to intravenous contrast used in CECT and CEMRI; • It can be used in pacients with chronic kidney disease; Abdominal contrast enhanced ultrasound is used in day-to-day practice mainly for evaluation of hepatic lesions, but also for pancreatic, kidney, testis pathology, as well as for inflammatory bowel diseases.

A Phase 1a/1b Trial of ST-001, a Fenretinide Phospholipid Suspension for 4-Hour Intravenous Infusion in Relapsed/Refractory T-Cell Non-Hodgkin's Lymphoma

A phase 1a/1b trial is planned for specific types of T-cell non-Hodgkin's lymphoma (T-NHL) using a new formulation of fenretinide for intravenous (IV) administration (ST-001, IND# 135475). Despite documented anticancer properties, clinical development of fenretinide has been hampered by poor bioavailability of the oral preparations and poor water solubility for IV infusion although some progress has been made. A phase 1 clinical trial of a 20% soy oil-in-water emulsion containing 2mg/mL of fenretinide, administered via 24-hour daily infusions x5 q3wks, demonstrated activity in mycosis fungoides /Sézary syndrome (MF/SS) and in angioimmunoblastic T-cell lymphoma (AITL) (Mohrbacher et al 2017). However, all doses exhibiting disease activity were associated with grade 4 hypertriglyceridemia. ST-001 from SciTech Development, LLC is a second generation, triglyceride-free, phospholipid-based nano-dispersion formulation of higher potency fenretinide (12.5mg/mL) suitable for IV infusion. The SciTech Delivery Vehicle (SDV) is composed of 4 clinically acceptable phospholipids (DPPC, DOPC, DMPC, and DMPG) which, in the presence of fenretinide, form lamellar bilayer structures <200nm in diameter. Pharmacokinetic (PK) studies showed that IV administration of ST-001 in the Sprague-Dawley rat model achieved fenretinide blood levels required for in vitro cytotoxicity to human cancer cell lines (including T- and B-NHL). The planned US multicenter phase 1a trial utilizes an Accelerated Titration (Simon) 4B design, with 100% dose escalation in single-patient cohorts until first instance of grade 3 or higher toxicity is encountered, at which point the trial reverts to a 'standard', 3 patient-cohort approach and 40% dose escalation. To maximize each patient's chance to be treated at a potentially active dose, intrapatient dose escalation is allowed for patients who remain on study and have no evidence of toxicity once the safety of the next dose level is established in a drug-naïve patient. The primary study objective is determining the maximum tolerated dose (MTD) of ST-001 in adult patients with T-NHL. Secondary objectives are to describe the toxicity profile of ST-001, observe and record anti-tumor activity of ST-001 in patients with T-NHL, describe the PK of ST-001 when given at the specified dose/schedule in this study, and evaluate potential mechanisms of action of ST-001 using pharmacodynamic (PD) biomarkers. Eligible patients will be 18 years or older with histologically confirmed diagnosis of MF/SS (stages IB-IV according to TNMB system), or Ann Arbor stage II-IV non-cutaneous peripheral T-cell NHL (PTCL), AITL, or follicular T-NHL (FTCL) as defined in the 2016 WHO classification of lymphoid malignancies. Patients must have received at least 2 prior systemic therapies, have good performance status, and acceptable organ function. The drug will be administered IV over 4 hours daily for 5 consecutive days as outpatient starting at a dose of 1.25mg/m2/day. Treatment will be repeated every 3 weeks for up to 8 cycles. Once MTD is determined, an expanded cohort (phase 1b) will be conducted at MTD for diseases that demonstrate efficacy signals in phase 1a; however, patients must have at least one measurable disease site to be eligible for 1b. Up to 44 patients are projected to be enrolled (24 for phase 1a, and 20 for 1b). All patients who receive at least one dose of ST-001 will be evaluated for safety and toxicity according to the Common Terminology Criteria for Adverse Events (CTCAE) v4.03. Patients with at least one measurable site of disease and have received at least one full cycle (5-day infusion) of ST-001 are eligible for response evaluation. Descriptive statistics will be used for PK analysis to characterize the range, median and mean of calculated values of clearance, volume of distribution, and half-lives of distribution and elimination phases across all evaluable patients. PD analysis will use each patient's baseline value as the control and normalize PD values as % change from baseline. Effect of ST-001 on specific immune functions including cytokines and immune effector cell function will be evaluated at baseline and after therapy. It is anticipated that ST-001 will demonstrate activity in T-NHL in the absence of excipients that contribute to triglyceride elevations. Disclosures Kuzel: SciTech Development, LLC: Membership on an entity's Board of Directors or advisory committees. Parchment:SciTech Development, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Investigating the Role of Lipid Transfer in Microbubble-Mediated Drug Delivery.

Sonoporation, the permeabilization of cell membranes following exposure to microbubbles and ultrasound, has considerable potential for therapeutic delivery. To date, engineering of microbubbles for these applications has focused primarily upon optimizing microbubble size and stability, or attachment of targeting species and/or drug molecules. In this work, it is demonstrated that the microbubble coating can also be tailored to directly influence cell permeabilization. Specifically, lipid exchange mechanisms between phospholipid microbubbles and cells can be exploited to significantly increase sonoporation efficiency in vitro. A theoretical analysis of the energy required for pore formation was carried out. From this, it was hypothesized that sonoporation could be promoted by the transfer of lipid molecules with appropriate carbon chain length and/or shape (cylindrical or conical). Spectral imaging with a hydration-sensitive membrane probe (C-Laurdan) was used to measure changes in the membrane lipid order of A-549 cancer cells following exposure to suspensions of different phospholipids. Two candidate lipids were identified, a short-chain-length phospholipid (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) and a medium-chain-length lysolipid (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC)). Microbubbles were prepared with matched concentrations, size distributions, and acoustic responses. Confocal microscopy was used to measure cell uptake of a model drug (propidium iodide) with and without ultrasound exposure (1 MHz, 250 kPa peak negative pressure, 1 kHz pulse repetition frequency, 10% duty cycle, 15 s exposure). Despite significantly decreasing the cell membrane lipid order, DLPC did not increase sonoporation. Microbubbles containing 16:0 lyso-PC, however, produced a ∼5-fold increase in sonoporation compared to control microbubbles. Importantly, the lyso-PC molecules were incorporated into the microbubble coating and did not affect cell permeability prior to ultrasound exposure. These findings indicate that microbubbles can be engineered to exploit lipid exchange between microbubble shells and cell membranes to enhance drug delivery, a new optimization route that may lead to enhanced therapeutic efficacy of ultrasound-mediated treatments.

Raman spectroscopy for quantification of water-to-lipid ratio in phospholipid suspensions

In this paper, a way of quantification of the water-to-lipid ratio in aqueous suspensions of phospholipid vesicles by Raman spectroscopy is suggested. We measured the ratio of Raman intensities of the OH stretching band and the symmetric CH2-stretching mode for series of water suspensions of different phosphatidylcholines with varied water-to-phospholipid ratio. It is shown that there is a simple linear relation between the ratio of Raman intensities and the water-to-lipid ratio if the number of CH2 groups in the hydrocarbon chains is taken into account. The proportionality coefficient of this relation is determined for the gel and fluid phases of phospholipids.

Preparation of liposomes using supercritical carbon dioxide technology: Effects of phospholipids and sterols

Liposomes were prepared utilizing a supercritical carbon dioxide (SC-CO2) process. A phospholipid suspension was first equilibrated with CO2 at 300bar and then depressurized at a constant pressure and rate. The effects of phospholipid concentration, phospholipid type, sterol concentration and sterol type on particle size, uniformity, zeta potential and morphology were investigated. With increasing soy lecithin concentration (5–30mM) at 50°C, the smallest particle size of liposomes (146.1±0.8nm) was obtained at 30 mM with the polydispersity index (PdI) of 0.398±0.008. Increased phospholipid concentration was favorable for the formation of smaller size vesicles with higher uniformity. Longer chain length of fatty acids in pure phospholipids resulted in a larger particle size with more spherical shape while a phospholipid with unsaturated fatty acyl chains resulted in increased asymmetry. With elevated β-sitosterol concentration (10%–50%), particle size and PdI increased to 245.5±7.14nm and 0.514±0.018, respectively, with decreased absolute zeta potential. 6-Ketocholestanol showed the smallest diameter and PdI of liposomes with the most spherical shape among all sterol types tested. Soy lecithin exhibited the highest stability of vesicular systems due to its highest absolute zeta potential (−58.3±2.17mV) among all the phospholipid types. The SC-CO2 method demonstrated superior characteristics of liposomes over traditional thin film hydration method for a smaller size and PdI as well as enhanced intactness without leakage. It might offer a promising way to reduce usage of sterol in liposome formulations while eliminating organic solvent usage.

Preparation of liposomes using a modified supercritical process via depressurization of liquid phase

Liposomes were prepared utilizing a modified supercritical process. In this method, phospholipid suspension in water was equilibrated with high pressure CO2 and the CO2-expanded liquid phase was depressurized via two protocols. In protocol (I), depressurization rate was kept constant while in protocol (II), both the depressurization rate and the pressure inside the vessel were constant. Pressure, depressurization rate and temperature were the processing parameters evaluated in terms of their effect on the properties of the liposomes obtained. Particle size of liposomes prepared by the modified supercritical method was as small as 265±4nm and 214±3nm for protocols (I) and (II), respectively. Elevated pressure contributed to a smaller size while higher depressurization rates resulted in enhanced uniformity. Amount of unhydrated CO2 dissolved in the phospholipid bilayer seems to be important for a small particle size while release rate from between the bilayers may affect size distribution. Liposomes prepared by the modified supercritical method were stable for up to 8 and 6 weeks for protocols (I) and (II), respectively, with a high zeta potential. Protocol (II) yielded smaller and more uniform particles while protocol (I) gave higher storage stability. The modified supercritical method is more effective in liposome preparation than conventional thin film hydration method for a reduced size and enhanced stability.

Solid self-emulsifying phospholipid suspension (SSEPS) with diatom as a drug carrier

We report the application of diatom as a solid carrier for water insoluble drugs applied in oral drug delivery system based on the self-emulsifying drug delivery system (SEDDS) caprylocaproyl macrogol-8 glycerides/lecithin/propylene glycol/caprylic/capric triglyceride. Diatoms are fossilized skeletons of photosynthetic algae with complex 3-dimensional (3D), porous structure consisting of amorphous silica, obtained by purification of diatomaceous earth. Different solid samples of carbamazepine (CBZ) suspension in SEDDS, called solid self-emulsifying phospholipid suspension (SSEPS), were prepared using two methods: adsorption of CBZ dispersion in SEDDS by gentle mixing with diatoms in mortar with pestle (Method A) or dispersion of diatoms in ethanol solution of CBZ and SEDDS components, followed by ethanol evaporation (Method B). Release rate of CBZ from SSEPS was significantly higher in comparison to pure drug, physical mixture of diatoms and CBZ as well as solid dispersion of pure CBZ and diatoms obtained by ethanol evaporation. The dissolution of CBZ from SSEPS sample prepared using method B was faster than from the sample prepared by the method A. Higher dissolution for sample prepared by the method B can be attributed to the partial adsorption (deeper localization) of liquid material inside the pores of diatoms. Upon storage of the samples under accelerated conditions (40°C and 70% RH) for 10weeks no significant changes in CBZ crystallinity and dissolution was in case of SSEPS, contrary to solid dispersion with increased crystallinity, indicating that diatoms with adsorbed liquid CBZ-loaded SEPS can maintain initial CBZ characteristics.

Morphological changes of bacterial model membrane vesicles

Unilamellar vesicles frequently serve as model membrane systems to study membrane‐peptide and membrane‐protein interactions. They are formed either from natural lipid extracts that contain complex lipid mixtures, or from synthetic one‐, two‐, or multi‐component lipid mixtures mimicking the lipid composition of biological membranes. Unilamellar vesicles can be prepared by extrusion of aqueous multilamellar phospholipid suspensions and they can have limited stability due to aggregation and fusion processes. We used cryo‐transmission electron microscopy (cryoTEM) to study the morphology of vesicles of phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), and cardiolipins (CLs) that represent the major lipid components of cytoplasmic membranes of Gram‐negative bacteria. Our results show that single‐component vesicles of PE and PG stored at RT are stable in shape and size for at least 1 wk. In contrast, two‐component vesicles of PE/PG and PE/CL transform into lamellar sheets already several hours after preparation, which is probably due to a compensation of different spontaneous curvatures of PG lipids compared to PE and CL. With this investigation we intend to alert researchers working with model membranes to scrutinize the shape and stability of vesicles before using them in protein‐vesicle interaction studies.Practical applications: Liposomes represent widely‐used model membrane systems that are applied to mimic natural membranes. Usually, these liposomes are composed of one or two lipid components representing the major lipid constituents of the respective natural membrane. Their long‐term shape, aggregation, and fusion stability is an important prerequisite for the study of peptide‐liposome and protein‐liposome interactions, where the binding of peptides or proteins can have serious impacts on the liposomal shape and size. Furthermore, shape and size stability is of outstanding importance for the application of liposomes as drug‐delivery system. We showed that single‐component liposomes composed of the major lipid components of cytoplasmic membranes of Gram‐negative bacteria (PE and PG) are stable in shape and size for at least 1 wk, whereas the corresponding two‐component liposomes become unstable within hours and transform into discs and sheets. This knowledge will be indispensable for future studies on interactions of liposomes with peptides and proteins.Cryo‐transmission electron microscopy is the method of choice to study the size, morphology, and shape stability of liposomes being composed of the phospholipids PE, PG, and CL that represent the major lipid components of cytoplasmic membranes of Gram‐negative bacteria.

Mass-Action Model Analysis of the Apparent Molar Volume and Heat Capacity of Pluronics in Water and Liposome Suspensions at 25 °C

Pluronics are block copolymers composed of a central block of polypropylene oxide and two side chains of polyethylene oxide. They are used in water to generate aggregates and gels or added to phospholipid suspensions to prepare microparticles for drug delivery applications. The structure of these systems has been widely investigated. However, little is known about the mechanisms leading to these structures. This investigation compares the apparent molar volumes and heat capacities of Pluronics F38, F108, F127, P85, P104, and P103 at 25 °C in water and in the presence of lecithin liposomes. The changes in molar volumes, heat capacities, and enthalpies generated by a mass-action model are in good agreement with the loss of hydrophobic hydration of the polypropylene oxide central block of the Pluronics. However, the molecularity of the endothermic transitions is much smaller than the aggregation numbers reported in the literature for the same systems. It is suggested that Pluronics go through dehydration of their central block to form unimolecular or small entities having a hydrophobic polypropylene oxide core. In water, these entities would assemble athermally to form larger aggregates. In the presence of liposomes, they would be transferred into the hydrophobic lecithin bilayers of the liposomes. Light transmission experiments suggest that the liposome suspensions are significantly altered only when the added Pluronics are in the dehydrated state.

Tuning the aggregation behaviour of single-chain bolaphospholipids in aqueous suspension: from nanoparticles to nanofibres to lamellar phases

The aggregation behaviour in aqueous suspensions of symmetrical bipolar phospholipids (bolalipids) composed of one long alkyl chain and two polar headgroups were studied as a function of their chemical structure, i.e. the length of their chain, the modification of the chain by introduction of hetero-atoms, triple bonds, or phenyl rings, and the size of the headgroups. Three types of aggregate structures are formed by these bolalipids, namely helical nanofibres, micelle-like aggregates and lamellar sheets. The type of aggregate formed depends not only on the chemical structure, particularly the ratio of the cross-sectional area of the headgroup and the chain, but also on the presence of attractive interactions via hydrogen bonds or repulsive electrostatic interactions between the headgroups.

Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. PA phosphatase contains five tryptophan residues and exhibited inherit fluorescence that increased upon interaction with phosphatidylcholine liposomes. The interaction was enhanced by inclusion of other phospholipids and especially the substrate PA. Interaction was dependent on both the concentration of phosphatidylcholine-PA liposomes as well as the surface concentration of PA in liposomes. Mg(2+) ions, which were required for catalysis, did not affect PA phosphatase interaction with phosphatidylcholine-PA liposomes. PA phosphatase was a substrate for protein kinase A, protein kinase C, and casein kinase II, and these phosphorylations decreased PA phosphatase interaction with phosphatidylcholine-PA liposome membranes.

Structural and biophysical characterization of an antimicrobial peptide chimera comprised of lactoferricin and lactoferrampin

Lactoferricin and lactoferrampin are two antimicrobial peptides found in the N-terminal lobe of bovine lactoferrin with broad spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacteria as well as Candida albicans. A heterodimer comprised of lactoferrampin joined to a fragment of lactoferricin was recently reported in which these two peptides were joined at their C-termini through the two amino groups of a single Lys residue (Bolscher et al., 2009, Biochimie 91(1):123–132). This hybrid peptide, termed LFchimera, has significantly higher antimicrobial activity compared to the individual peptides or an equimolar mixture of the two. In this work, the underlying mechanism behind the increased antibacterial activity of LFchimera was investigated. Differential scanning calorimetry studies demonstrated that all the peptides influenced the thermotropic phase behaviour of anionic phospholipid suspensions. Calcein leakage and vesicle fusion experiments with anionic liposomes revealed that LFchimera had enhanced membrane perturbing properties compared to the individual peptides. Peptide structures were evaluated using circular dichroism and NMR spectroscopy to gain insight into the structural features of LFchimera that contribute to the increased antimicrobial activity. The NMR solution structure, determined in a miscible co-solvent mixture of chloroform, methanol and water, revealed that the Lys linkage increased the helical content in LFchimera compared to the individual peptides, but it did not fix the relative orientations of lactoferricin and lactoferrampin with respect to each other. The structure of LFchimera provides insight into the conformation of this peptide in a membranous environment and improves our understanding of its antimicrobial mechanism of action.

Phosphoinositide Binding Differentially Regulates NHE1 Na+/H+ Exchanger-dependent Proximal Tubule Cell Survival

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

Enhanced bioavailability and retinal accumulation of lutein from self-emulsifying phospholipid suspension (SEPS)

Ability of any formulation to keep the drug in solubilized form in vivo is essential for bioavailability (BA) enhancement rather than the solubility of drug in the formulation vehicle/matrix itself. Besides, utilization of an excess amount of surfactants/co-surfactants to solubilize the drug in the lipid formulation poses potential pharmaceutical as well as health problems. To address this problem, self-emulsifying phospholipid suspension (SEPS) consisting of high amount of phospholipid (an endogenous lipid with efficient in vivo emulsification capability) and relatively low amount of surfactant/co-surfactant has been proposed to enhance the bioavailability (BA) of lutein. In this study, the ability of SEPS formulation to enhance the BA of lutein was assessed from three SEPS formulations with various amounts of phospholipid (SEPS-0, SEPS-I, and SEPS-II with 0 mg, 250 mg, and 500 mg of Phosal ® 53 MCT, respectively) in beagle dogs following a single oral administration of lutein equivalent to 100 mg, and were compared with commercial formulation (CF). In addition, the retinal accumulation of lutein in Sprague Dawley (SD) rats’ eyes from SEPS-II formulation (lutein dose of 100 mg/kg/day) was investigated following single daily oral administration for a period of 14 days. CF and placebo (vegetable oil without lutein) were also administered for the same period of time and were compared with the SEPS-II formulation. In the relative BA study in beagle dogs, no significant differences were observed between the pharmacokinetic (PK) parameters of formulation SEPS-O and CF. However, the C max in comparison to CF was 3.70 folds and 11.76 folds higher for SEPS-I and SEPS-II, respectively. Relative BA compared to CF was 178.88% and 473.13% for SEPS-I and SEPS-II, respectively. The retinal lutein accumulation was 0.91 ± 0.31 ng/g, 3.45 ± 1.63 ng/g, and 14.72 ± 2.02 ng/g for placebo, CF, and SEPS-II, respectively. This enhancement was about 16.1 folds and 4.27 folds compared to placebo and CF, respectively. The relative BA study in dogs and retinal accumulation study in rats demonstrated the excellent ability of SEPS to enhance the BA of lutein. For this reason, SEPS containing lutein could be a promising lipid based delivery system for the prevention of ocular diseases.

Controlling size and size distribution of electrohydrodynamically prepared microbubbles

AbstractThere are several methods for producing commercial quantities of coated microbubbles with diameters <10 μm and a low polydispersivity index for use as ultrasound contrast agents, drug and gene delivery vehicles. Co-axial electrohydrodynamic microbubbling is a recently developed method which can generate microbubbles at a high rate. In this method, two capillary needles are concentrically aligned and subjected to a potential difference applied between the needles and a ground electrode with a stream of gas pushed through the inner needle and a stream of liquid through the outer so that a two-phase jet is formed at the outlet. The jet breaks up into microbubbles which are collected in a suitable liquid. In this work, co-axial electrohydrodynamic microbubbling was used to prepare microbubbles from a phospholipid suspension containing 2 wt-% Tween 80, a surfactant. Microbubbling was carried out under different electrical fields and air/liquid flowrates in order to prepare microbubbles with a low polyd...

Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein

alpha-Synuclein (alpha-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related alpha-synulceinopathies. However, whether phosphorylation promotes or inhibits alpha-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of alpha-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type alpha-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated alpha-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of alpha-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of alpha-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern alpha-syn aggregation and toxicity in PD and related alpha-synulceinopathies.

Generation of microbubbles for diagnostic and therapeutic applications using a novel device

Developments in both diagnostic and therapeutic applications of microbubbles have greatly increased the need for more advanced preparation technologies which provide a well-defined, narrow microbubble size-distribution. In this paper, we demonstrate the use of a new device, consisting of a T-junction whose outlet capillary is fitted with an electrohydrodynamic spraying arrangement, to prepare phospholipid-coated air microbubbles, making significant advances in controlling and decreasing the size and size-distribution, and increasing the stability/lifetime of the bubbles prepared. The microbubbles were characterised via optical microscopy to determine the relationship between the size-distribution obtained and the process variables, specifically the flow rates of the phospholipid suspension and air (Ql and Qg), and the applied voltage (V). The formation of microbubbles in the device was also studied using high-speed photography. For the range of parameters investigated, the bubble diameter was found to scale with the product of the flow rate ratio (Ql/Qg) and the applied voltage, with a consistent bubble diameter of 5.1 ± 2 μm being obtained at Ql/Qg = 1.7 and V = 18 kV. The bubbles prepared using this method were found to be stable for at least 2 h at ambient temperature and pressure.

Novel methods for preparing phospholipid coated microbubbles

Two new methods for preparing phospholipid coated microbubble suspensions are elucidated. Firstly, co-axial electrohydrodynamic atomisation was utilized to generate 3-7 microm diameter microbubbles. Secondly, a specially designed and constructed T-junction device was used to prepare monodisperse microbubbles. Characteristics of microbubbles prepared by these two methods are compared with those obtained by sonication of the phospholipid suspension.

Inhibition of Gastric Cancer Cell Adhesion in Nude Mice by Inraperitoneal Phospholipids

Adhesion of tumor cells to mesothelial cells or extracellular matrix components is a pivotal step in developing peritoneal dissemination after gastric cancer. As phospholipids were found to reduce adhesion formation, especially at sites of peritoneal lesions, we assessed the inhibition of attachment of NUGC-4 gastric cancer cells by local treatment with phospholipids to the peritoneum in nude mice. Gastric cancer cells (1xl0(6)) suspended in either normal saline (controls) or phospholipid suspension 75 mg/kg body weight (PL75) or 150 mg/kg (PL150) were injected intraperitoneally into 90 female BALB/c nu/nu mice. The treatment groups were subdivided into animals with defined peritoneal lesions and animals without lesions. After 30 days the extent of peritoneal carcinosis and the Peritoneal Cancer Index were evaluated. Statistical analysis was performed with two factorial ANOVAs. The level of significance was adjusted according to Bonferrorni (alpha = 0.00278). During a 90-day observation period the survival rate was determined using the log rank test. After 30 days the intraperitoneal tumor volume was reduced by PL150 up to 0.6 ml (SEM 0.16) and 0.48 ml (SEM 0.09) in mice with peritoneal lesions compared to 0.9 ml (SEM 0.2) and 0.9 ml (SEM 0.1) in the control group (P = 0.04). The mean area of tumor adhesion amounted to 145 mm(2) (SEM 17) (P = 0.08) and 164 mm(2) (SEM 32.8) (P = 0.049) with peritoneal lesions after treatment with PL150 [controls: 216 mm(2) (SEM 28.5) and 245 mm(2) (SEM 29.3)]. The peritoneal cancer index was 16.4 (SEM 1.7) in the control group and 9 (SEM 1.68) with PL150 (P = 0.0002). In the subgroup with peritoneal lesions, the respective values were as follows: controls: 20.8 (SEM 0.85); PL 150:14.3 (SEM 1.07) (P = 0.0001). We found a prolonged survival rate after treatment with PL150. However, this effect was not significantly different to that seen in the control group. Treatment with PL75 had no significant influence. Phospholipids may be an efficacious and economic tool for reducing peritoneal tumor cell adhesion and consequently the development of peritoneal carcinosis after resection of gastric cancer.