Microfluidic manufacture of rt-PA -loaded echogenic liposomes.

Echogenic liposomes (ELIP), loaded with recombinant tissue-type plasminogen activator (rt-PA) and microbubbles that act as cavitation nuclei, are under development for ultrasound-mediated thrombolysis. Conventional manufacturing techniques produce a polydisperse rt-PA-loaded ELIP population with only a small percentage of particles containing microbubbles. Further, a polydisperse population of rt-PA-loaded ELIP has a broadband frequency response with complex bubble dynamics when exposed to pulsed ultrasound. In this work, a microfluidic flow-focusing device was used to generate monodisperse rt-PA-loaded ELIP (µtELIP) loaded with a perfluorocarbon gas. The rt-PA associated with the µtELIP was encapsulated within the lipid shell as well as intercalated within the lipid shell. The µtELIP had a mean diameter of 5 µm, a resonance frequency of 2.2 MHz, and were found to be stable for at least 30 min in 0.5 % bovine serum albumin. Additionally, 35 % of µtELIP particles were estimated to contain microbubbles, an order of magnitude higher than that reported previously for batch-produced rt-PA-loaded ELIP. These findings emphasize the advantages offered by microfluidic techniques for improving the encapsulation efficiency of both rt-PA and perflurocarbon microbubbles within echogenic liposomes.

Plasmin-loaded echogenic liposomes for ultrasound-mediated thrombolysis.

Plasmin, a direct fibrinolytic, shows a significantly superior hemostatic safety profile compared to recombinant tissue plasminogen activator (rtPA), the only FDA-approved thrombolytic for the treatment of acute ischemic stroke. The improved safety of plasmin is attributed to the rapid inhibition of free plasmin by endogenous plasmin inhibitors present in very high concentrations (1 µM). However, this rapid inhibition prevents the intravenous (IV) administration of plasmin. In emergency situations, catheter-based local administration is not practical. There is a need for an alternative technique for IV administration of plasmin. A possible solution is the encapsulation of plasmin in echogenic liposomes (ELIP) for protection from inhibitors until ultrasound (US)-triggered release at the clot site. ELIP are bilayer phospholipid vesicles with encapsulated gas microbubbles. US induces oscillation and collapse of the gas bubbles, which facilitates ELIP rupture and delivery of the encapsulated contents. Plasmin-loaded ELIP (PELIP) were manufactured and characterized for size, gas and drug encapsulations, and in vitro thrombolytic efficacy using a human whole blood clot model. Clots were exposed to PELIP with and without exposure to US (center frequency 120 kHz, pulse repetition frequency 1667 Hz, peak-to-peak pressure of 0.35 MPa, 50 % duty cycle). Thrombolytic efficacy was calculated by measuring the change in clot width over a 30-min treatment period using an edge detection MATLAB program. The mean clot lysis obtained with PELIP in the presence of US exposure was 31 % higher than that obtained without US exposure and 15 % higher than that obtained with rtPA treatment (p < 0.05).The enhanced clot lysis is attributed to the US-mediated release of plasmin from the liposomes.

In vivo testing of a non-invasive prototype device for the continuous monitoring of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a stroke subtype with the highest mortality rate. Hematoma expansion and re-bleeding post-ICH are common and exacerbate the initial cerebral insult. There is a need for continuous monitoring of the neurologic status of patients with an ICH injury. NEW METHOD: A prototype device for non-invasive continuous monitoring of an ICH was developed and tested in vivo using a porcine ICH model. The device consists of receiving and transmitting antennae in the 400-1000 MHz frequency range, placed directly in line with the site of the ICH. The device exploits the differences in the dielectric properties and geometry of tissue media of a healthy brain and a brain with an ICH injury. The power received by the receiving antenna is measured and the percent change in power received immediately after infusion of blood and 30 min after the infusion, allowing for the blood to clot, is calculated. RESULTS: An increase in the received power in the presence of an ICH is observed at 400 MHz, consistent with previous in vitro studies. Frequency sweep experiments show a maximum percent change in received power in the 750-1000 MHz frequency range. COMPARISON WITH EXISTING METHODS: Currently, CT, MRI and catheter angiography (CA) are the main clinical neuroimaging modalities. However, these techniques require specialized equipment and personnel, substantial time, and patient-transportation to a radiology suite to obtain results. Moreover, CA is invasive and uses intra-venous dye or vascular catheters to accomplish the imaging. CONCLUSIONS: The device has the potential to significantly improve neurologic care in the critically ill brain-injured patient.

A prototype device for non-invasive continuous monitoring of intracerebral hemorrhage.

A prototype for a non-invasive, real-time, monitoring device was developed to detect changes in the brain secondary to disease or injury such as intracerebral hemorrhage (ICH). The eventual goal is a non-invasive, real time sensor that can alert the clinician to alterations in the comatose patient's brain resulting from hemorrhage, seizure or stroke. In this work, a 400 MHz electromagnetic (EM) signal was transmitted with an antenna (T), incident on a 'brain gel' in vitro ICH model, and received by a receiving (R) antenna. Changes in the received signal were found to be induced by the presence of blood. The received power (P(R)) was found to be a linear function of the cross sectional area of blood, as measured normal to the incident wave. In addition, the sensor was able to detect as little as 1 mL of blood in this 1000 mL in vitro model.

Comparison of electrical conductivities of various brain phantom gels: Developing a 'Brain Gel Model'

The use of conducting gels to mimic brain and other tissues is of increasing interest in the development of new medical devices. Currently, there are few such models that can be utilized at physiologic temperatures. In this work, the conductivities of agar, agarose and gelatin gels were manipulated by varying NaCl concentration from 0-1 mg/ml. The AC conductivity was measured at room and physiological temperatures (37°C) in the 100-500 Hz frequency range. Conductivity (σ) was nearly independent of frequency but increased linearly with NaCl concentration and was higher at physiological temperatures in these gels. A formula for predicting conductivity as a function of NaCl concentration was derived for each gel type. The overall goal is to develop a 'brain gel model', for studying low frequency electrical properties of the brain and other tissues at physiological temperatures.

Short-term high-dose effect of lovastatin on thrombolysis by rt-PA in a human whole-blood in vitro clot model.

High-dose hydroxymethylglutaryl coenzyme. A reductase inhibitor (statin) administration reduces neuronal injury and improves outcomes in experimental models of acute ischemic stroke, and has been shown to be safe in a phase 1 dose-escalation study using lovastatin at doses higher than currently approved for daily use. Statins also affect the hemostatic system by upregulating t-PA expression and decreasing plasminogen activator inhibitor (PAI-1) expression, platelet adhesion and thrombus formation in animal models. Since a thrombolytic agent, recombinant tissue plasminogen activator (rt-PA), is currently the only FDA-approved therapy for use in ischemic stroke patients, it is important to ascertain whether high statin doses impact the efficacy of rt-PA. The main goal of this study was to evaluate the effect of a high dose of lovastatin and its active form, lovastatin hydroxy acid, on rt-PA thrombolysis in an in vitro model. Percentage clot lysis was measured in the presence and absence of rt-PA in three different treatment groups: lovastatin, lovastatin hydroxy acid, and ethanol. The effect of ethanol on clot lysis was studied since ethanol was used to disperse the highly hydrophobic lovastatin. The decrease in clot width over time was measured using microscopic imaging of an in vitro human whole blood clot model; an approximately 400 µm diameter clot was formed on suture silk, suspended in human fresh frozen plasma (hFFP) and exposed to treatment. In the absence of rt-PA, clot lysis did not show statistically significant differences in the percentage clot lysis between different treatment groups (p=0.103). In the presence of rt-PA, clot lysis was greater than in the absence of rt-PA for all groups, but there were no statistically significant differences between treatment groups (p=0.385). In this in vitro study, high doses of lovastatin neither impaired nor enhanced the lytic efficacy of rt-PA.

Polypeptide grafted hyaluronan: A self-assembling comb-branched polymer constructed from biological components.

Rheological evidence is provided demonstrating that covalent grafting of monodisperse isotactic poly(L-leucine) branches onto linear hyaluronan (HA) polysaccharide chains yields comb-branched HA chains that self-assemble into long-lived physical networks in aqueous solutions driven by hydrophobic interactions between poly(L-leucine) chains. This is in stark contrast to native (unmodified) HA solutions which exhibit no tendency to form long-lived physical networks.

Comparison of surfactants used to prepare aqueous perfluoropentane emulsions for pharmaceutical applications.

Perfluoropentane (PFP), a very hydrophobic, nontoxic, noncarcinogenic fluoroalkane, has generated much interest in biomedical applications, including occlusion therapy and controlled drug delivery. For most of these applications, the dispersion within aqueous media of a large quantity of PFP droplets of the proper size is critically important. Surprisingly, the interfacial tension of PFP against water in the presence of surfactants used to stabilize the emulsion has rarely, if ever, been measured. In this study, we report the interfacial tension of PFP in the presence of surfactants used in previous studies to produce emulsions for biomedical applications: polyethylene oxide-co-polylactic acid (PEO-PLA) and polyethylene oxide-co-poly-epsilon-caprolactone (PEO-PCL). Because both of these surfactants are uncharged diblock copolymers that rely on the mechanism of steric stabilization, we also investigate for comparison's sake the use of the small-molecule cationic surfactant cetyl trimethyl ammonium bromide (CTAB) and the much larger protein surfactant bovine serum albumin (BSA). The results presented here complement previous reports of the PFP droplet size distribution and will be useful for determining to what extent the interfacial tension value can be used to control the mean PFP droplet size.

Poly(L-lactide) (PLLA)/multiwalled carbon nanotube (MWCNT) composite: characterization and biocompatibility evaluation.

Much effort has been directed at the fabrication of carbon nanotubes (CNTs)/polymer composites and the characterization of their physical properties. Among them, composites comprising CNTs and the biocompatible polymers are of special interest due to their potential for specific biomedical applications. we report the preparation of the MWCNT/poly(L-lactide) composite and the corresponding spectroscopic (Raman) and the microscopic (SEM, TEM) characterization. The electronic transport, thermal properties, and biocompatibility of this composite have also been investigated. The Raman spectroscopic analysis suggests the interaction between PLLA and MWCNT occurs mainly through the hydrophobic C-CH3 functional groups. The DC conductivity of the composite increases as the MWCNT loading is increased. Such behavior can be described by a percolation mechanism in which a percolation threshold at about 14 wt % MWCNT loading is observed with the maximum end conductivity of 0.1 S x cm(-1). The DSC study of the PLLA/MWCNT composite reveals that the MWCNTs in the composite have the effect of inducing crystallization and plasticizing the polymer matrix. The results from the cell culture test suggest that the presence of MWCNT in the composite inhibits the growth of the fibroblast cells.