Reentrant Stabilization of Grafted Nanoparticles in Polymer Solutions.

Polymer chains grafted onto nanoparticles may facilitate the dispersion of such particles in a polymer solution. We explore the optimal strategy for stabilizing polymer-grafted nanoparticles using self-consistent field theory and experiments. The best results are obtained for relatively low grafting densities and for chain lengths of the brush polymer NB larger than that of the freely floating polymers Nf. When Nf > NB, one finds a compatibilization gap and re-entrant stabilization: At both very low and very high polymer concentrations particles disperse in the polymer solution, while at intermediate concentrations the particles lose their colloidal stability. At low grafting densities the underlying surface is in contact with the solvent. Particles covered by a bidisperse brush can combine a low grafting outer region with full coverage of the surface by a densely grafted inner layer. Using classical colloid-chemical stabilization criteria the region in the phase diagram for which the particles are expected to mix with a concentrated polymer solution opens up. Now, also upon an increase in the length of the freely dispersed polymers, a re-entrant colloid-chemical stabilization is found for particles on the nanometer length scale: At both short and long polymer chains in solution the particles will not aggregate, whereas at intermediate lengths the colloidal stability is marginal. This multi re-entrant behavior is found from numerical self-consistent field calculations, and these predictions are consistent with corresponding experiments.

Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: a case for bolus injections.

Localized gene delivery has many potential clinical applications. However, the nucleic acids (e.g. pDNA and siRNA) are incapable of passively crossing the endothelium, cell membranes and other biological barriers which must be crossed to reach their intracellular targets. A possible solution is the use of ultrasound to burst circulating microbubbles inducing transient permeabilization of surrounding tissues which mediates nucleic acid extravasation and cellular uptake. In this study we report on an optimization of the ultrasound gene delivery technique. Naked pDNA (200 µg) encoding luciferase and SonoVue® microbubbles were co-injected intravenously in mice. The hindlimb skeletal muscles were exposed to ultrasound from a non-focused transducer (1 MHz, 1.25 MPa, PRI 30s) and injection protocols and total amounts as well as ultrasound parameters were systemically varied. Gene expression was quantified relative to a control using a bioluminescence camera system at day 7 after sonication. Bioluminescence ratios in sonicated/control muscles of up to 101× were obtained. In conclusion, we were able to specifically deliver genetic material to the selected skeletal muscles and overall, the use of bolus injections and high microbubble numbers resulted in increased gene expression reflected by stronger bioluminescence signals. Based on our data, bolus injections seem to be required in order to achieve transient highly concentrated levels of nucleic acids and microbubbles at the tissue of interest which upon ultrasound exposure should lead to increased levels of gene delivery. Thus, ultrasound mediated gene delivery is a promising technique for the clinical translation of localized drug delivery.

Ultrafast vapourization dynamics of laser-activated polymeric microcapsules.

Precision control of vapourization, both in space and time, has many potential applications; however, the physical mechanisms underlying controlled boiling are not well understood. The reason is the combined microscopic length scales and ultrashort timescales associated with the initiation and subsequent dynamical behaviour of the vapour bubbles formed. Here we study the nanoseconds vapour bubble dynamics of laser-heated single oil-filled microcapsules using coupled optical and acoustic detection. Pulsed laser excitation leads to vapour formation and collapse, and a simple physical model captures the observed radial dynamics and resulting acoustic pressures. Continuous wave laser excitation leads to a sequence of vapourization/condensation cycles, the result of absorbing microcapsule fragments moving in and out of the laser beam. A model incorporating thermal diffusion from the capsule shell into the oil core and surrounding water reveals the mechanisms behind the onset of vapourization. Excellent agreement is observed between the modelled dynamics and experiment.

Real-time imaging and kinetics measurements of focused ultrasound-induced extravasation in skeletal muscle using SPECT/CT.

Drugs need to overcome several biological barriers such as the endothelium and cellular membranes in order to reach their target. Promising new therapeutics, many of which are charged and macromolecular, are not able to passively extravasate, let alone cross cell membranes, and stay mainly in the blood pool upon intravenous injection until clearance. Using focused ultrasound (fUS) in combination with circulating microbubbles (MBs) leads to temporary localized tissue permeabilization allowing extravasation of (macro) molecules from the vascular system. Thus, fUS is a promising approach for localized drug delivery. However, little is known about the permeabilization kinetics in skeletal muscle. In this study, we used single photon emission computed tomography (SPECT) to characterize the kinetics of extravasation of ¹¹¹In-labeled bovine serum albumin (BSA), a model macromolecular drug, in muscle treated with fUS and MBs. The same fUS protocol was applied to 6 groups of mice with different times, ∆t, between fUS application and BSA injection (∆t=-10, 2.5, 10, 30, 60, 90 min) followed by SPECT imaging. For ∆t ≤30min we observed an exponential accumulation of activity in an area of the treated muscle which extended to a volume larger than the fUS pattern with highest accumulation for short waiting times ∆t. The extent of extravasation decreased exponentially with increasing ∆t, with a calculated half-life of ca. 21 min, defining the time window of extravasation. The same treatment without MBs did not induce extravasation of BSA thus supporting MBs and drug co-injection strategies. These results provide essential information for the development of fUS based strategies for localized drug delivery.

Rupture threshold characterization of polymer-shelled ultrasound contrast agents subjected to static overpressure.

Polymer-shelled micro-bubbles are employed as ultrasound contrast agents (UCAs) and vesicles for targeted drug delivery. UCA-based delivery of the therapeutic payload relies on ultrasound-induced shell rupture. The fragility of two polymer-shelled UCAs manufactured by Point Biomedical or Philips Research was investigated by characterizing their response to static overpressure. The nominal diameters of Point and Philips UCAs were 3 µm and 2 µm, respectively. The UCAs were subjected to static overpressure in a glycerol-filled test chamber with a microscope-reticule lid. UCAs were reconstituted in 0.1 mL of water and added over the glycerol surface in contact with the reticule. A video-microscope imaged UCAs as glycerol was injected (5 mL∕h) to vary the pressure from 2 to 180 kPa over 1 h. Neither UCA population responded to overpressure until the rupture threshold was exceeded, which resulted in abrupt destruction. The rupture data for both UCAs indicated three subclasses that exhibited different rupture behavior, although their mean diameters were not statistically different. The rupture pressures provided a measure of UCA fragility; the Philips UCAs were more resilient than Point UCAs. Results were compared to theoretical models of spherical shells under compression. Observed variations in rupture pressures are attributed to shell imperfections. These results may provide means to optimize polymeric UCAs for drug delivery and elucidate associated mechanisms.

Monodisperse polymeric particles prepared by ink-jet printing: double emulsions, hydrogels and polymer mixtures.

Submerged ink-jetting produces a monodisperse emulsion that can be converted into monodisperse particles. As the initial droplet size is known and the final particle size can be easily measured, such a method can be used to quantify the shrinkage and the swelling of polymer particles made from double emulsions, polymer mixtures and hydrogel forming polymers. It is found that at the same starting concentration and initial emulsion droplet size poly-lactide-co-glycolide particles made from an ink-jetted emulsion have the same size as particles ink-jetted from a solution, however with a more porous structure. The total pore volume, however, is negligible compared to the polymer volume of the particle. If polymers containing a poly-ethylene glycol block are included, particles with internal porosity are formed, even if no double emulsion process is applied. Still the final particle size is the same. Only if typical hydrogel forming polymers are used, in which water is distributed more homogeneously, significantly higher particle diameters are found; for a four-arm PEG-poly-caprolacton a degree of swelling of 3.3 is found.

Effect of molecular weight, crystallinity, and hydrophobicity on the acoustic activation of polymer-shelled ultrasound contrast agents.

Polymer-shelled microbubbles are applied as ultrasound contrast agents. To investigate the effect of the polymer on microbubble preparation and acoustic properties, polylactides with systematic variations in molecular weight, crystallinity, and end-group hydrophobicity were used. Polymer-shelled cyclodecane filled capsules were prepared by emulsification, and the cyclodecane was removed by lyophilization to obtain hollow capsules. Complete removal of cyclodecane from the microcapsules was only achieved for short chain (about M(w) 6000) crystalline polymers. The pressure threshold for acoustic destruction of the microbubbles was found to increase with molecular weight. Noncrystalline polymers showed a higher threshold for destruction than crystalline polymers. Hydrophobically modified short chain crystalline polymers showed the steepest increase in acoustic destruction after the threshold as a function of the applied pressure, which is a favorable characteristic for ultrasound mediated drug delivery. Microcapsules made with such polymers had an inhomogeneous surface including pores through which cyclodecane was lyophilized efficiently.

Ultrasound triggered image-guided drug delivery.

The integration of therapeutic interventions with diagnostic imaging has been recognized as one of the next technological developments that will have a major impact on medical treatments. Important advances in this field are based on a combination of progress in guiding and monitoring ultrasound energy, novel drug classes becoming available, the development of smart delivery vehicles, and more in depth understanding of the mechanisms of the cellular and molecular basis of diseases. Recent research demonstrates that both pressure sensitive and temperature sensitive delivery systems hold promise for local treatment. The use of ultrasound for the delivery of drugs has been demonstrated in particular the field of cardiology and oncology for a variety of therapeutics ranging from small drug molecules to biologics and nucleic acids.

Oil-filled polymer microcapsules for ultrasound-mediated delivery of lipophilic drugs.

The use of ultrasound contrast agents as local drug delivery systems continues to grow. Current limitations are the amount of drug that can be incorporated as well as the efficiency of drug release upon insonification. This study focuses on the synthesis and characterisation of novel polymeric microcapsules for ultrasound-triggered delivery of lipophilic drugs. Microcapsules with a shell of fluorinated end-capped poly(L-lactic acid) were made through pre-mix membrane emulsification and contained, apart from a gaseous phase, different amounts of hexadecane oil as a drug-carrier reservoir. Mean number weighted diameters were between 1.22 microm and 1.31 microm. High-speed imaging at approximately 10 million fames per second showed that for low acoustic pressures (1 MHz, 0.24 MPa) microcapsules compressed but remained intact. At higher diagnostic pressures of 0.51 MPa, microcapsules cracked, thereby releasing the encapsulated gas and model lipophilic drug. Using conventional ultrasound B-mode imaging at a frequency of 2.5 MHz, a marked enhancement of scatter intensity over a tissue-mimicking phantom was observed for all differently loaded microcapsules. The partially oil-filled microcapsules with high drug loads and well-defined acoustic activation thresholds have great potential for ultrasound-triggered local delivery of lipophilic drugs under ultrasound image-guidance.

Adhesion and ultrasound-induced delivery from monodisperse microbubbles in a parallel plate flow cell.

Microbubbles, ultrasound contrast agents currently in development as ultrasonically activated drug delivery vehicles, were studied using a novel flow cell design. The flow cell combined ultrasound compatibility, a planar optical configuration, and a Cartesian orientation of buoyant, shear, and acoustic forces. The set-up enabled measurements of buoyant rise and adhesive sensitivity to shear forces for individual biotinylated, monodisperse, polymer-shelled microbubbles near a NeutrAvidin-coated polystyrene substrate. Analysis of the velocity history demonstrated that adhesion depended on the buoyant rise to the surface before attachment to the substrate: only when the distance parallel to the substrate in the flow direction was between 10 and 20 microm from the stopping position could specific molecular recognition events occur. Low intensity ultrasound caused strong two-dimensional mobility leading to reversible clustering of microbubbles, even though they interacted strongly with the substrate through biotin-NeutrAvidin bonds. At higher acoustic pressure, local gas release took place. With sufficient acoustic intensity, the agents demonstrate potential as large payload carriers for biomolecularly targeted therapeutic delivery. However, difficulties may limit the range of targeting applications: large sizes may render microbubbles susceptible to detachment at the shearing forces present in many regions of the vasculature and secondary radiation forces may reduce targeting effectiveness.