Acoustic Droplet Vaporization of Perfluorohexane Emulsions Induced by Heterogeneous Nucleation at an Ultrasonic Frequency of 1.1 MHz.

Droplets made of liquid perfluorocarbon undergo a phase transition and transform into microbubbles when triggered by ultrasound of intensity beyond a critical threshold; this mechanism is called acoustic droplet vaporization (ADV). It has been shown that if the intensity of the signal coming from high ultrasonic harmonics are sufficiently high, superharmonic focusing is the mechanism leading to ADV for large droplets (>3 µm) and high frequencies (>1.5 MHz). In such a scenario, ADV is initiated due to a nucleus occurring at a specific location inside the droplet volume. But the question on what induces ADV in the case of nanometer-sized droplets and/or at low ultrasonic frequencies (<1.5 MHz) still remains. We investigated ADV of perfluorohexane (PFH) nano- and microdroplets at a frequency of 1.1 MHz and at conditions where there is no superharmonic focusing. Three types of droplets produced by microfluidics were studied: plain PFH droplets, PFH droplets containing many nanometer-sized water droplets, and droplets made of a PFH corona encapsulating a single micron-sized water droplet. The probability to observe a vaporization event was measured as a function of acoustic pressure. As our experiments were performed on droplet suspensions containing a population of monodisperse droplets, we developed a statistical model to extrapolate, from our experimental curves, the ADV pressure thresholds in the case where only one droplet would be insonified. We observed that the value of ADV pressure threshold decreases as the radius of a plain PFH droplet increases. This value was further reduced when a PFH droplet encapsulates a micron-sized water droplet, while the encapsulation of many nanometer-sized water droplets did not modify the threshold. These results cannot be explained by a model of homogeneous nucleation. However, we developed a heterogeneous nucleation model, where the nucleus appears at the surface in contact with PFH, that successfully predicts our experimental ADV results.

In vitro evaluation of polymeric nanoparticles with a fluorine core for drug delivery triggered by focused ultrasound.

Polymeric nanoparticles are being intensively investigated as drug carriers. Their efficiency could be enhanced if the drug release can be triggered using an external stimulus such as ultrasound. This approach is possible using current commercial apparatus that combine focused ultrasound with MRI to perform ultrasonic surgery. In this approach, nanoparticles made of a perfluoro-octyl bromide core and a thick polymeric (PLGA-PEG) shell may represent suitable drug carriers. Indeed, their perfluorocarbon core are detectable by 19F MRI, while their polymeric shell can encapsulate drugs. However, their applicability in ultrasound-triggered drug delivery remains to be proven. To do so, we used Nile red as a model drug and we measured its release from the polymeric shell by spectrofluorometry. In the absence of ultrasound, only a small amount of Nile red release was measured (<5%). Insonations were performed in a controlled environment using a 1.1 MHz transducer emitting tone bursts for a few minutes, whereas a focused broadband hydrophone was used to detect the occurrence of cavitation. In the absence of detectable inertial cavitation, less than 5% of Nile red was released. In the presence of detectable inertial cavitation, Nile red release was ranging from 10% to 100%, depending of the duty cycle, acoustic pressure, and tank temperature (25 or 37 °C). Highest releases were obtained only for duty cycles of 25% at 37 °C and 50% at 25 °C and for a peak-to-peak acoustic pressure above 12.7 MPa. Electron microscopy and light scattering measurements showed a slight modification in the nanoparticle morphology only at high release contents. The occurrence of strong inertial cavitation is thus a prerequisite to induce drug release for these nanoparticles. Since strong inertial cavitation can lead to many unwanted biological effects, these nanoparticles may not be suitable for a therapeutic application using ultrasound-triggered drug delivery.

Ultrasound-triggered delivery of paclitaxel encapsulated in an emulsion at low acoustic pressures.

We investigated the in vitro ultrasound-triggered delivery of paclitaxel, a well known anti-cancerous drug, encapsulated in an emulsion and in the presence of CT26 tumor cells. The emulsion was made of nanodroplets, whose volume comprised 95% perfluoro-octyl bromide and 5% tributyl O-acetylcitrate, in which paclitaxel was solubilized. These nanodroplets, prepared using a high-pressure microfluidizer, were stabilized by a tailor-made and recently patented biocompatible fluorinated surfactant. The delivery investigations were performed at 37 °C using a high intensity focused ultrasound transducer at a frequency of 1.1 MHz. The ultrasonic pulse was made of 275 sinusoidal periods and the pulse repetition frequency was 200 Hz with a duty cycle of 5%. The measured viabilities of CT26 cells showed that paclitaxel delivery was achievable for peak-to-peak pressures of 0.4 and 3.5 MPa, without having to vaporize the perfluorocarbon part of the droplet or to induce inertial cavitation.

Characterization of a Biomimetic Mesophase Composed of Nonionic Surfactants and an Aqueous Solvent.

We have investigated the physical and biomimetic properties of a sponge (L3) phase composed of pentaethylene glycol monododecyl ether (C12E5), a nonionic surfactant, an aqueous solvent, and a cosurfactant. The following cosurfactants, commonly used for solubilizing membrane proteins, were incorporated: n-octyl-ß-d-glucopyranoside (ß-OG), n-dodecyl-ß-d-maltopyranoside (DDM), 4-cyclohexyl-1-butyl-ß-d-maltoside (CYMAL-4), and 5-cyclohexyl-1-pentyl-ß-d-maltoside (CYMAL-5). Partial phase diagrams of these systems were created. The L3 phase was characterized using crossed polarizers, diffusion of a fluorescent probe by fluorescence recovery after pattern photobleaching (FRAPP), and freeze fracture electron microscopy (FFEM). By varying the hydration of the phase, we were able to tune the distance between adjacent bilayers. The characteristic distance (db) of the phase was obtained from small angle scattering (SAXS/SANS) as well as from FFEM, which yielded complementary db values. These db values were neither affected by the nature of the cosurfactant nor by the addition of membrane proteins. These findings illustrate that a biomimetic surfactant sponge phase can be created in the presence of several common membrane protein-solubilizing detergents, thus making it a versatile medium for membrane protein studies.

Perfluorocarbon nanodroplets stabilized by fluorinated surfactants: characterization and potentiality as theranostic agents.

We aim to produce emulsions that can act as contrast agents and drug carriers for cancer imaging and therapy. To increase tumor detection and decrease drug side effects, it is desirable to take advantage of the enhanced permeability and retention effect that allows nanoparticles to accumulate in tumor tissues. To do so, the emulsion droplets need to be small enough and stable over time in addition to enhancing image contrast and carrying a drug payload. In the present study, we have investigated the properties and potentiality as theranostic agents of perfluorocarbon emulsions stabilized by a biocompatible fluorinated surfactant called FTAC. To obtain better control of our system, the synthesis of those surfactants was studied and their physico-chemical properties were explored in different configurations such as micelles, in the perfluorocarbon droplet shell and at water/air and water/perfluorocarbon interfaces. The originality of this work lies in the determination of numerous characteristics of emulsions and fluorinated surfactants including surface tension, interfacial tension, critical micelle concentration, adiabatic compressibility, density, size distribution (aging studies), and ultrasonic echogenicity. These characterization studies were conducted using different types of FTAC and several perfluorocarbons (perfluoropentane, perfluorohexane, and perfluorooctyl bromide). We have also shown that a hydrophobic drug could be encapsulated in the FTAC-stabilized perfluorocarbon droplets thanks to triacetin addition. Finally, the perfluorocarbon emulsions were detectable in vitro by a clinical 3 T MRI scanner, equipped with a double frequency 19F/1H transmit-receive coil.

Unfolding and refolding of bovine serum albumin at acid pH: ultrasound and structural studies.

Serum albumin is the most abundant protein in the circulatory system. The ability of albumins to undergo a reversible conformational transition, observed with changes in pH, is conserved in distantly related species, suggesting for it a major physiological role possibly related to the transport of small molecules including drugs. We have followed changes of bovine serum albumin (BSA) in volume by densimetry and in adiabatic compressibility during its conformational transition from pH 7-2, using ultrasound measurements. In parallel, circular dichroism was measured. The volume and adiabatic compressibility decrease from pH 4 to 2. The change in ellipticity shows a decrease over the same pH range from 70% to 40% of its alpha-helix content. Sorbitol, at concentrations from 0 to 2 M, led to the progressive restoration of BSA volume and compressibility values, as well as a substantial recovery of its original alpha-helix content. This finding implies that the compressibility variation observed reflects the conformational changes during the transition. The mutual interactions of the mechanical properties and structural features of BSA reported here are important in biotechnology for research in material sciences and for the design and the development of new, tailor-made drug carriers.

Ultrasonic studies of alcohol-induced transconformation in beta-lactoglobulin: the intermediate state.

In mixed alcohol-water solvents, bovine beta-lactoglobulin undergoes a cooperative transition from beta-sheet to a high alpha-helix content conformer. We report here the characterization of beta-lactoglobulin by compressibility and spectroscopy measurements during this transconformation. Both the volume and compressibility increase as a function of alcohol concentration, up to maximal values which depend on the chemical nature of the three alcohols used: hexafluoroisopropanol, trifluoroethanol, and isopropanol. The order of effectiveness of alcohols in inducing the compressibility transition is identical to that previously reported for circular dichroism and thus independent of the observation technique. The highly cooperative sigmoidal curves found by compressibility determination match closely those obtained by circular dichroism at 222 nm, indicating a correlation between the two phenomena measured by the two different techniques. The presence of an equilibrium intermediate form was shown by the interaction of beta-lactoglobulin with 8-anilino-1-naphthalene sulfonic acid, a probe widely used to detect molten-globule states of proteins. It was correlated with the plateau region of the volume curves and with the inflexion points of the sigmoidal compressibility curves. Ultrasound characterization of proteins can be carried out in optically transparent or nontransparent media.

Quantal transmitter release by glioma cells: quantification of intramembrane particle changes.

Glial cells in situ are able to release neurotransmitters such as glutamate or acetylcholine (ACh). Glioma C6BU-1 cells were used to determine whether the mechanisms of ACh release by a glial cell line are similar or not to quantal release from neurones. Individual C6BU-1 cells, pre-filled with ACh, were moved into contact with a Xenopus myocyte that was used as a real-time ACh detector. Upon electrical stimulation, C6BU-1 cells generated evoked ACh impulses which were Ca(2+)-dependent and quantal (quantal steps of ca. 100 pA). Changes in plasma membrane ultrastructure were investigated by using a freeze-fracture technique designed for obtaining large and flat replicas from monolayer cell cultures. A transient increase in the density of medium and large size intramembrane particles--and a corresponding decrease of small particles--occurred in the plasma membrane of C6BU-1 cells stimulated for ACh release. Changes in interaction forces between adjacent medium and large particles were investigated by computing the radial distribution function and the interaction potential. In resting cells, the radial distribution function revealed a significant increase in the probability to find two particles separated by an interval of 24 nm; the interaction potential suggested repulsive forces for intervals shorter than 24 nm and attractive forces between 24 and 26 nm. In stimulated cells, this interaction was displaced to 21 nm and made weaker, despite of the fact that the overall particle density increased. The nature of this transient change in intramembrane particles is discussed, particularly with regard to the mediatophore proteolipid which is abundant in the membranes C6-BU-1 like in those of cholinergic neurones. In conclusion, evoked ACh release from pre-filled C6-BU-1 glioma cells is quantal and Ca(2+)-dependent. It is accompanied by a transient changes in the size distribution and the organisation of intramembrane particles in the plasma membrane. Thus, for the release characteristics, glioma cells do not differ fundamentally from neurones.

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.

Unbinding-binding transition induced by molecular snaps in model membranes.

We have used a lamellar phase made of a nonionic surfactant, dodecane and water, as a model membrane to investigate its interactions with macromolecular inclusions bringing together two membranes, i.e., acting as macromolecular snaps. In systems devoid of inclusions, the interlamellar distance depends on the total volume fraction of membranes Phi. We show that, in presence of a transmembrane protein, or of several de novo designed peptides of different length and composition, the lamellar phase undergoes a binding transition. Under such conditions, the interlamellar distance is no longer proportional to Phi(-1), but rather to the surface concentration of snaps within the membrane. It also appears that, in the presence of the hydrophobic segment of peptide snaps, the length of the inclusions must be at least equal to the hydrophobic length of the membrane to be active. Experimental results have been precisely fitted to a model of thermally stabilized membranes, decorated with snaps. However, in the presence of inclusions, the parameter describing the interactions between membranes, has to take into account the length of the inclusion to preserve good predictive capabilities.