Tyrone M. Porter

Thermosensitive liposomes for localized delivery and triggered release of chemotherapy

Liposomes are a promising class of nanomedicine with the potential to provide site-specific chemotherapy, thus improving the quality of cancer patient care. First-generation liposomes have emerged as one of the first nanomedicines used clinically for localized delivery of chemotherapy. Second-generation liposomes, i.e. stimuli-responsive liposomes, have the potential to not only provide site-specific chemotherapy, but also triggered drug release and thus greater spatial and temporal control of therapy. Temperature-sensitive liposomes are an especially attractive option, as tumors can be heated in a controlled and predictable manner with external energy sources. Traditional thermosensitive liposomes are composed of lipids that undergo a gel-to-liquid phase transition at several degrees above physiological temperature. More recently, temperature-sensitization of liposomes has been demonstrated with the use of lysolipids and synthetic temperature-sensitive polymers. The design, drug release behavior, and clinical potential of various temperature-sensitive liposomes, as well as the various heating modalities used to trigger release, are discussed in this review.

Effect of high-intensity focused ultrasound on whole blood with and without microbubble contrast agent

Using human whole blood samples with and without contrast agent (CA), we evaluated the effect of exposures to focused, continuous wave (CW) 1.1-MHz ultrasound for durations of 10 ms to 1 s at spatial average intensities of 560 to 2360 W/cm2. Cavitation was monitored with a passive cavitation detector and hemolysis was determined with spectroscopy. In whole blood alone, no significant cavitation, heating or hemolysis was detected at any exposure condition. Conversely, cavitation and hemolysis, but not heating, were detected in whole blood with CA. A CA concentration as low as 0.28 microL CA per mL whole blood at an intensity of 2360 W/cm2 for 1 s resulted in measurable cavitation and a 6-fold increase in hemolysis compared to shams. Cavitation and hemolysis increased proportional to the concentration of CA and duration of exposure. In samples containing 4.2 microL CA per mL whole blood exposed for 1 s, a threshold was seen at 1750 W/cm2 where cavitation and hemolysis increased 10-fold compared to exposures at lower intensities. HIFU exposure of whole blood containing CA leads to significant hemolysis in vitro and may lead to clinically significant hemolysis in vivo.

An in vitro study of a phase-shift nanoemulsion: a potential nucleation agent for bubble-enhanced HIFU tumor ablation.

Phase-shift nanoemulsions have the potential to nucleate bubbles and enhance high-intensity focused ultrasound (HIFU) cancer therapy. This emulsion consists of albumin-coated dodecafluoropentane (DDFP) droplets with a mean diameter of approximately 260 nm at 37°C. It is known that superheated perfluorocarbon droplets can be vaporized with microsecond long ultrasound pulses if the acoustic pressure exceeds a specific threshold. In addition, it is well documented that particles smaller than 400 nm can extravasate through leaky tumor vessels and accumulate in the tumor interstitial space. Thus, nanoemulsions may passively target solid tumors, thus localizing cavitation nuclei for bubble-enhanced HIFU-mediated heating. In this study, we investigate the acoustic droplet vaporization of a DDFP nanoemulsion in tissue-mimicking gels and demonstrate the ability to nucleate inertial cavitation (IC) and enhance HIFU-mediated heating. The nanoemulsion was dispersed throughout albumin-acrylamide gel phantoms and sonicated with microsecond-length HIFU pulses (f = 2 MHz). The pressure threshold needed to vaporize the nanoemulsion was measured as a function of degree of superheat, pulse length and nanoemulsion concentration. It was determined that the vaporization threshold was inversely proportional with degree of superheat and independent of pulse length and concentration within the range of values tested. It was also shown that the bubbles formed from vaporized nanoemulsions reduced the IC threshold in the gel phantoms. Finally, it was demonstrated that cavitation from vaporized nanoemulsions accelerated HIFU-mediated heating. The results from this study demonstrate that phase-shift nanoemulsions can be combined with HIFU to provide a high degree of spatial and temporal control of bubble-enhanced heating.

Thermosensitive liposomes modified with poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers for triggered release of Doxorubicin

A novel polymer-modified thermosensitive liposome (pTSL) was developed for the delivery of Doxorubicin (DOX) for cancer therapy. Copolymers containing temperature-responsive N-isopropylacrylamide (NIPAAm) and pH-responsive propylacrylic acid (PAA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, yielding copolymers with dual pH/temperature-dependent phase transition properties. When attached to liposomes, these copolymers were membrane-disruptive in a pH/temperature-dependent manner. pTSL demonstrated enhanced release profile and significantly lower thermal dose threshold when compared to traditional thermosensitive formulations and were stable in serum with minimal drug leakage over time. These liposomes thus have the potential to dramatically reduce the risk of damage to healthy tissues that is normally associated with liposomal cancer therapy.

Acoustic characterization of echogenic liposomes: Frequency-dependent attenuation and backscatter

Ultrasound contrast agents (UCAs) are used clinically to aid detection and diagnosis of abnormal blood flow or perfusion. Characterization of UCAs can aid in the optimization of ultrasound parameters for enhanced image contrast. In this study echogenic liposomes (ELIPs) were characterized acoustically by measuring the frequency-dependent attenuation and backscatter coefficients at frequencies between 3 and 30 MHz using a broadband pulse-echo technique. The experimental methods were initially validated by comparing the attenuation and backscatter coefficients measured from 50-μm and 100-μm polystyrene microspheres with theoretical values. The size distribution of the ELIPs was measured and found to be polydisperse, ranging in size from 40 nm to 6 μm in diameter, with the highest number observed at 65 nm. The ELIP attenuation coefficients ranged from 3.7  ±  1.0 to 8.0  ±  3.3 dB/cm between 3 and 25 MHz. The backscatter coefficients were 0.011  ±  0.006 (cm str)(-1) between 6 and 9 MHz and 0.023 ± 0.006 (cm str)(-1) between 13 and 30 MHz. The measured scattering-to-attenuation ratio ranged from 8% to 22% between 6 and 25 MHz. Thus ELIPs can provide enhanced contrast over a broad range of frequencies and the scattering properties are suitable for various ultrasound imaging applications including diagnostic and intravascular ultrasound.

Localized delivery of doxorubicin in vivo from polymer-modified thermosensitive liposomes with MR-guided focused ultrasound-mediated heating

Thermosensitive liposomes have emerged as a viable strategy for localized delivery and triggered release of chemotherapy. MR-guided focused ultrasound (MRgFUS) has the capability of heating tumors in a controlled manner, and when combined with thermosensitive liposomes can potentially reduce tumor burden in vivo. However, the impact of this drug delivery strategy has rarely been investigated. We have developed a unique liposome formulation modified with p(NIPAAm-co-PAA), a polymer that confers sensitivity to both temperature and pH. These polymer-modified thermosensitive liposomes (PTSL) demonstrated sensitivity to focused ultrasound, and required lower thermal doses and were more cytotoxic than traditional formulations in vitro. A set of acoustic parameters characterizing optimal release from PTSL in vitro was applied in the design of a combined MRgFUS/PTSL delivery platform. This platform more effectively reduced tumor burden in vivo when compared to free drug and traditional formulations. Histological analysis indicated greater tumor penetration, more extensive ECM remodeling, and greater cell destruction in tumors administered PTSL, correlating with improved response to the therapy.

Acoustic techniques for assessing the Optison destruction threshold.

Objective. The purpose of this study was to identify the pressure threshold for the destruction of Optison (octafluoropropane contrast agent; Amersham Health, Princeton, NJ) using a laboratory‐assembled 3.5‐MHz pulsed ultrasound system and a clinical diagnostic ultrasound scanner. Methods. A 3.5‐MHz focused transducer and a linear array with a center frequency of 6.9 MHz were positioned confocally and at 90° to each other in a tank of deionized water. Suspensions of Optison (5–8 × 104 microbubbles/mL) were insonated with 2‐cycle pulses from the 3.5‐MHz transducer (peak rarefactional pressure, or Pr, from 0.0, or inactive, to 0.6 MPa) while being interrogated with fundamental B‐mode imaging pulses (mechanical index, or MI, = 0.04). Scattering received by the 3.5‐MHz transducer or the linear array was quantified as mean backscattered intensity or mean digital intensity, respectively, and fit with exponential decay functions (Ae−kt + N, where A + N was the amplitude at time 0; N, background echogenicity; and k, decay constant). By analyzing the decay constants statistically, a pressure threshold for Optison destruction due to acoustically driven diffusion was identified. Results. The decay constants determined from quantified 3.5‐MHz radio frequency data and B‐mode images were in good agreement. The peak rarefactional pressure threshold for Optison destruction due to acoustically driven diffusion at 3.5 MHz was 0.15 MPa (MI = 0.08). Furthermore, the rate of Optison destruction increased with increasing 3.5‐MHz exposure pressure output. Conclusions. Optison destruction was quantified with a laboratory‐assembled 3.5‐MHz ultrasound system and a clinical diagnostic ultrasound scanner. The pressure threshold for acoustically driven diffusion was identified, and 3 distinct mechanisms of ultrasound contrast agent destruction were observed with acoustic techniques.

Triggered drug release from superhydrophobic meshes using high-intensity focused ultrasound.

Application of high-intensity focused ultrasound to drug-loaded superhydrophobic meshes affords triggered drug release by displacing an entrapped air layer. The air layer within the superhydrophobic meshes is characterized using direct visualization and B-mode imaging. Drug-loaded superhydrophobic meshes are cytotoxic in an in vitro assay after ultrasound treatment.

Accumulation of Phase-Shift Nanoemulsions to Enhance MR-Guided Ultrasound-Mediated Tumor Ablation In Vivo

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is being explored as a non-invasive technology to treat solid tumors. However, the clinical use of HIFU for tumor ablation applications is currently limited by the long treatment times required. Phase-shift nanoemulsions (PSNE), consisting of liquid perfluorocarbon droplets that can be vaporized into microbubbles, are being developed to accelerate HIFU-mediated heating. The purpose of this study was to examine accumulation of PSNE in intramuscular rabbit tumors in vivo. MR images were acquired before and after intravenous injection of gadolinium-containing PSNE. MR signal enhancement was observed in rabbit tumors up to six hours after injection, indicating that PSNE accumulated in the tumors. In addition, PSNE vaporization was detected in the tumor with B-mode ultrasound imaging, and MR thermometry measurements indicated that PSNE accelerated the rate of HIFU-mediated heating. These results suggest that PSNE could dramatically improve the efficiency and clinical feasibility of MRgHIFU.

Synthesis and Characterization of Hybrid Polymer/Lipid Expansile Nanoparticles: Imparting Surface Functionality for Targeting and Stability

The size, drug loading, drug release kinetics, localization, biodistribution, and stability of a given polymeric nanoparticle (NP) system depend on the composition of the NP core as well as its surface properties. In this study, novel, pH-responsive, and lipid-coated NPs, which expand in size from a diameter of approximately 100 to 1000 nm in the presence of a mildly acidic pH environment, are synthesized and characterized. Specifically, a combined miniemulsion and free-radical polymerization method is used to prepare the NPs in the presence of PEGylated lipids. These PEGylated-lipid expansile NPs (PEG-L-eNPs) combine the swelling behavior of the polymeric core of expansile NPs with the improved colloidal stability and surface functionality of PEGylated liposomes. The surface functionality of PEG-L-eNPs allows for the incorporation of folic acid (FA) and folate receptor-targeting. The resulting hybrid polymer/lipid nanocarriers, FA-PEG-L-eNPs, exhibit greater in vitro uptake and potency when loaded with paclitaxel compared to nontargeted PEG-L-eNPs.