Chris Oerlemans

Evidence for a new mechanism behind HIFU-triggered release from liposomes.

A promising approach for local drug delivery is high-intensity focused ultrasound (HIFU)-triggered release of drugs from stimuli-responsive nanoparticles such as liposomes. The aim of this study was to investigate whether another release mechanism is involved with HIFU-triggered release from liposomes beside cavitation and temperature. Furthermore, it was studied whether this new release mechanism allows the release of lipophilic compounds. Therefore, both a lipophilic (Nile red) and a hydrophilic (fluorescein) compound were loaded into thermosensitive (TSL) or non-thermosensitive liposomes (NTSL) and the liposomes were subjected both to continuous wave (CW)- and pulsed wave (PW)-HIFU. The mean liposome size varied from 97 to 139 nm with a polydispersity index (PDI)≤0.06 for the different formulations. The Tm of the phospholipid bilayer of the TSL was around 42°C. Approximately 80% of fluorescein was released within 15 min from TSL at temperatures≥42°C. In contrast, no fluorescein release from NTSL and NR release from both TSL and NTSL was observed at temperatures up to 60 °C. CW-HIFU exposure of TSL resulted in rapid temperature elevation up to 52°C and subsequently almost quantitative fluorescein release. Fluorescein release from NTSL was also substantial (~64% after 16 min at 20 W). Surprisingly, CW-HIFU exposure (20W for 16 min) resulted in the release of NR from TSL (~66% of the loaded amount), and this was even higher from NTSL (~78%). PW-HIFU exposure did not result in temperatures above the Tm of TSL. However, nearly 85% of fluorescein was released from TSL after 32 min at 20W of PW-HIFU exposure, whereas the release from NTSL was around 27%. Interestingly, NR release from NTSL was~30% after 2 min PW-HIFU exposure and increased to~70% after 32 min. Furthermore, addition of microbubbles to the liposomes prior to PW-HIFU exposure did not result in more release, which suggests that cavitation can be excluded as the main mechanism responsible for the triggered release of both a hydrophilic and a lipophilic model compound from liposomes. Dynamic light scattering analysis showed that the mean size and PDI of the liposomes did not significantly change after CW- and PW-HIFU exposure. Taken together, it is therefore concluded that neither temperature elevation nor inertial cavitation is essential for the release of both hydrophilic and lipophilic compounds from liposomes. It is assumed that the release originates from radiation force-induced acoustic streaming, causing the liposomes to collide at the walls of the exposure chamber leading to shear forces which in turn results in reversible liposome destabilization and release of both hydrophilic and lipophilic compounds.

Triggered Release of Doxorubicin from Temperature-Sensitive Poly(N-(2-hydroxypropyl)-methacrylamide mono/dilactate) Grafted Liposomes

The objective of this study was to design temperature-sensitive liposomes with tunable release characteristics that release their content at an elevated temperature generated by high intensity focused ultrasound (HIFU) exposure. To this end, thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide mono/dilactate of different molecular weights and composition with a cholesterol anchor (chol-pHPMAlac) were synthesized and grafted onto liposomes loaded with doxorubicin (DOX). The liposomes were incubated at different temperatures and their release kinetics were studied. A good correlation between the release-onset temperature of the liposomes and the cloud point (CP) of chol-pHPMAlac was found. However, release took place at significantly higher temperatures than the CP of chol-pHPMAlac, likely at the CP, the dehydration and thus hydrophobicity is insufficient to penetrate and permeabilize the liposomal membrane. Liposomes grafted with chol-pHPMAlac with a CP of 11.5 °C released 89% DOX within 5 min at 42 °C while for the liposomes grafted with a polymer with CP of 25.0 °C, a temperature of 52 °C was needed to obtain the same extent of DOX release. At a fixed copolymer composition, an increase in molecular weight from 6.5 to 14.5 kDa decreased the temperature at which DOX was released with a release-onset temperature from 52 to 42 °C. Liposomes grafted with 5% chol-pHPMAlac exhibited a rapid release to a temperature increase, while at a grafting density of 2 and 10%, the liposomes were less sensitive to an increase in temperature. Sequential release of DOX was obtained by mixing liposomes grafted with chol-pHPMAlac having different CPs. Chol-pHPMAlac grafted liposomes released DOX nearly quantitatively after pulsed wave HIFU. In conclusion, the release of DOX from liposomes grafted with thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide mono/dilactate can be tuned to the characteristics and the grafting density of chol-pHPMAlac, making these liposomes attractive for local drug delivery using hyperthermia.

Alginate–lanthanide microspheres for MRI-guided embolotherapy

In cancer therapy, a promising treatment option to accomplish a high tumor-to-normal-tissue ratio is endovascular intervention with microsized particles, such as embolotherapy. In this study, alginate microspheres (ams) were prepared with the JetCutter technique, which is based on cutting a sodium alginate solution jet stream into small droplets of uniform size which are then cross-linked with different lanthanides or iron-III, resulting in microspheres of a predefined size which can be visualized by magnetic resonance imaging (MRI). The microspheres were investigated for their size and morphology (light microscopy and scanning electron microscopy analysis), cation content and MRI properties. The lanthanide-ams formulations, with a uniform size of 250 μm and a cation content between 0.72-0.94%, showed promising results for MR imaging. This was further demonstrated for Ho(3+)-cross-linked alginate microspheres (Ho(3+)-ams), the most potent microsphere formulation with respect to MR visualization, allowing single sphere detection and detailed microsphere distribution examination. Intravascular infusion of Ho(3+)-ams by catherization of ex vivo rabbit and porcine liver tissue and assessment of the procedure with MRI clearly showed accumulation and subsequently embolization of the targeted vessels, allowing accurate monitoring of the microsphere biodistribution throughout the tissue. Therefore, the different alginate-lanthanide microsphere formulations developed in this study show great potential for utilization as image-guided embolotherapy agents.

New insights into the HIFU-triggered release from polymeric micelles.

Continuous wave (CW), low frequency, high intensity focused ultrasound (HIFU) is a promising modality to trigger release of active compounds from polymeric micelles. The aim of the present study was to investigate whether high frequency CW as well as pulsed wave (PW) HIFU can induce the release of a hydrophobic agent from non-cross-linked (NCL) and core cross-linked (CCL) poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAm-Lac(n))) micelles. It was shown that high frequency CW as well as PW HIFU was able to trigger the release (up to 85%) of a hydrophobic compound (i.e., nile red, NR) from NCL and CCL micelles. No changes in size distribution of the micelles after CW and PW HIFU exposure were observed and no degradation of polymer chain had occurred. We therefore hypothesize that the polymeric micelles are temporally destabilized upon HIFU exposure due to radiation force induced shear forces, leading to NR release on demand.

Holmium-lipiodol-alginate microspheres for fluoroscopy-guided embolotherapy and multimodality imaging.

Embolotherapy is a minimally invasive transcatheter technique aiming at reduction or complete obstruction of the blood flow by infusion of micro-sized particles in order to induce tumor regression. A major drawback of the current commercially available and clinically used microspheres is that they cannot be detected in vivo with medical imaging techniques, impeding intra- and post-procedural feedback. It can be expected that real-time monitoring of microsphere infusion and post-procedural imaging will result in better predictability and higher efficacy of the treatment. In this study, a novel microsphere formulation has been developed that can be visualized with fluoroscopy, X-ray computed tomography (CT) and magnetic resonance imaging (MRI). The microspheres were prepared with the JetCutter technique and consist of alginate (matrix-forming polymer), holmium (cross-linking and MRI contrast agent), lipiodol (radiopaque contrast agent) and Pluronic F-68 (surfactant). The mean size (±SEM) of the hydrated holmium-lipiodol-alginate microspheres (Ho-lip-ams) was 570±12 μm with a holmium content of 0.38±0.01% (w/w). Stability studies showed that the microspheres remained intact during incubation for two weeks in fetal calf serum (FCS) at 37 °C. The inclusion of lipiodol in the microspheres rendered excellent visualization capabilities for fluoroscopy and CT, whereas the holmium ions, which keep the alginate network together, also allow MR imaging. In this study it was shown that single sphere detection was possible by fluoroscopy, CT and MRI. The Ho-lip-ams were visualized in real-time, during infusion in a porcine kidney using fluoroscopy, and post-procedural, the deposition of the microspheres was examined with fluoroscopy, (cone beam rotational) CT and MRI. The different imaging modalities showed similar deposition patterns of the microspheres within the organ. The combination of intra-procedural visualization, multimodality imaging for patient follow-up and the possibility of quantification offers a new and promising method for more safe, efficient and successful embolization treatment.

A novel approach to identify non-palpable breast lesions combining fluorescent liposomes and magnetic resonance-guided high intensity focused ultrasound-triggered release.

The combination of fluorescein-containing liposomes (FCL) and magnetic resonance-guided high intensity focused ultrasound (MR-HIFU)-triggered release is a promising approach for lesion demarcation and more efficient removal of non-palpable breast lesions. Exposure of FCL to ablation temperatures (60 °C) using MR-HIFU would result in palpable, stained tumors, which are more easy to identify during surgical resection. In this study, proof-of-concept concerning fluorescent FCL for MR-HIFU-triggered release and tumor demarcation of non-palpable breast lesions is presented. Ex vivo experiments in human blood and porcine muscle tissue showed increased label release from the liposomes, clear fluorescence enhancement and diffusion of the released compound after heating to 60 °C. Next, fluorescein release of FCL was observed after MR-HIFU-mediated mild hyperthermia (42 °C) and ablation temperature (60 °C) for a short period (30s), which is in line with the clinically relevant MR-HIFU treatment parameters. These results indicate the potential of the FCL as a tool to improve tumor demarcation in patients by MR-HIFU-triggered release. Therefore, this method may offer a new tool for efficient surgical resection of non-palpable breast tumor lesions by enabling proper discrimination between tumor tissue and adjacent healthy tissue.

MRI monitoring of nanocarrier accumulation and release using Gadolinium-SPIO co-labelled thermosensitive liposomes

Encapsulation of anticancer drugs in triggerable nanocarriers can beneficially modify pharmacokinetics and biodistribution of chemotherapeutic drugs, and consequently increase tumor drug concentration and efficacy, while reducing side effects. Thermosensitive liposomes release their contents triggered by hyperthermia, which can be, for example, precisely delivered using an MR Imaging-guided focused ultrasound procedure. In such a scenario, it is attractive to demonstrate the accumulation of liposomes before applying hyperthermia, as well as to document the release of liposome content using MRI. To address this need, thermosensitive liposomes were developed and characterized, which were doubly loaded by iron oxide nanoparticles and Gd-chelate, as opposed to loading with a single contrast agent. When intact, the transverse relaxivity of the liposomes is high allowing detection of carriers in tissue. After heating the longitudinal relaxivity steeply increases indicating release of the small molecular contents. By choosing the appropriate MR sequences, availability and release can be evaluated without interference of one contrast agent with the other. Copyright

Rapid dynamic R1 /R2 */temperature assessment: a method with potential for monitoring drug delivery.

Local drug delivery by hyperthermia‐induced drug release from thermosensitive liposomes (TSLs) may reduce the systemic toxicity of chemotherapy, whilst maintaining or increasing its efficacy. Relaxivity contrast agents can be co‐encapsulated with the drug to allow the visualization of the presence of liposomes, by means of R2*, as well as the co‐release of the contrast agent and the drug, by means of R1, on heating. Here, the mathematical method used to extract both R2* and R1 from a fast dynamic multi‐echo spoiled gradient echo (ME‐SPGR) is presented and analyzed. Finally, this method is used to monitor such release events. R2* was obtained from a fit to the ME‐SPGR data. Absolute R1 was calculated from the signal magnitude changes corrected for the apparent proton density changes and a baseline Look–Locker R1 map. The method was used to monitor nearly homogeneous water bath heating and local focused ultrasound heating of muscle tissue, and to visualize the release of a gadolinium chelate from TSLs in vitro. R2*, R1 and temperature maps were measured with a 5‐s temporal resolution. Both R2*and R1 measured were found to change with temperature. The dynamic R1 measurements after heating agreed with the Look–Locker R1 values if changes in equilibrium magnetization with temperature were considered. Release of gadolinium from TSLs was detected by an R1 increase near the phase transition temperature, as well as a shallow R2* increase. Simultaneous temperature, R2* and R1 mapping is feasible in real time and has the potential for use in image‐guided drug delivery studies. Copyright