Mercy Afadzi

Mechanisms of the ultrasound-mediated intracellular delivery of liposomes and dextrans

The mechanism involved in the ultrasound-enhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.

Effect of Ultrasound Parameters on the Release of Liposomal Calcein

The ultrasound exposure parameters that maximize drug release from dierucoyl-phosphatidylcholine (DEPC)-based liposomes were studied using two transducers operating at 300 kHz and 1 MHz. Fluorescent calcein was used as a model drug, and the release from liposomes in solution was measured using a spectrophotometer. The release of calcein was more efficient at 300 kHz than at 1 MHz, with thresholds of peak negative pressures of 0.9 MPa and 1.9 MPa, respectively. Above this threshold, the release increased with increasing peak negative pressure, mechanical index (MI), and duty cycle. The amount of drug released followed first-order kinetics and increased with exposure time to a maximal release. To increase the release further, the MI had to be increased. The results demonstrate that the MI and the overall exposure time are the major parameters that determine the drugs release. The drugs release is probably due to mechanical (cavitation) rather than thermal effects, and that was also confirmed by the detection of hydroxide radicals.

Contact-mediated intracellular delivery of hydrophobic drugs from polymeric nanoparticles

Encapsulation of drugs in nanoparticles can enhance the accumulation of drugs in tumours, reduce toxicity toward healthy tissue, and improve pharmacokinetics compared to administration of free drug. To achieve efficient delivery and release of drugs at the target site, mechanisms of interaction between the nanoparticles and cells and the mechanism of delivery of the encapsulated drug are crucial to understand. Our aim was to determine the mechanisms for cellular uptake of a fluorescent hydrophobic model drug from poly(butylcyanoacrylate) nanoparticles. Prostate adenocarcinoma cells were incubated with Nile Red-loaded nanoparticles or free Nile Red. Uptake and intracellular distribution were evaluated by flow cytometry and confocal laser scanning microscopy. The nanoparticles mediated a higher intracellular level and more rapid uptake of encapsulated Nile Red compared to model drug administered alone. The main mechanism for delivery was not by endocytosis of nanoparticles but by nanoparticle-cell contact-mediated transfer directly to the cytosol and, to a smaller extent, release of payload from nanoparticles into the medium followed by diffusion into cells. The payload thus avoids entering the endocytic pathway, evading lysosomal degradation and instead gains direct access to intracellular targets. The nanoparticles are promising tools for efficient intracellular delivery of hydrophobic anticancer drugs; therefore, they are clinically relevant for improved cancer therapy.

ULTRASOUND IMPROVES THE UPTAKE AND DISTRIBUTION OF LIPOSOMAL DOXORUBICIN IN PROSTATE CANCER XENOGRAFTS

Combining liposomally encapsulated cytotoxic drugs with ultrasound exposure has improved the therapeutic response to cancer in animal models; however, little is known about the underlying mechanisms. This study focused on investigating the effect of ultrasound exposures (1 MHz and 300 kHz) on the delivery and distribution of liposomal doxorubicin in mice with prostate cancer xenografts. The mice were exposed to ultrasound 24 h after liposome administration to study the effect on release of doxorubicin and its penetration through the extracellular matrix. Optical imaging methods were used to examine the effects at both microscopic subcellular and macroscopic tissue levels. Confocal laser scanning microscopy revealed that ultrasound-exposed tumors had increased levels of released doxorubicin compared with unexposed control tumors and that the distribution of liposomes and doxorubicin through the tumor tissue was improved. Whole-animal optical imaging revealed that liposomes were taken up by both abdominal organs and tumors.

Ultrasound stimulated release of liposomal calcein

Ultrasound exposure parameters that maximize drug release from liposomes were studied using two ultrasound transducers (300 kHz and 1 MHz). Variations in acoustic peak negative pressure (260–2037 kPa), temporary average intensity (0.05–6.08 W/cm2), mechanical index (MI) (0.4–3.0), insonation time (0.5–20 minutes), pulse repetition frequency (PRF) (100–1000 Hz) and pulse length (0.05–0.4 ms) were studied. Drug release was more efficient at 300 kHz compared to 1 MHz. A certain threshold in peak negative pressure had to be overcome to obtain drug release, and the pressure needed was lower at 300 kHz (0.72 MPa) than at 1 MHz (1. 39 MPa) which corresponds to MI values of 1.30 and 1.39 respectively. Above the threshold the release increased with increasing temporal average intensity, peak negative pressure, MI and duty cycle (i.e PRF and pulse length). The release was found to increase with exposure time, where the profile followed a first-order kinetics. The first-order rate constant for the release increased linearly with MI. This indicates that the release of the drug from liposomes was caused by mechanical rather than thermal effects. The results demonstrate that ultrasound has a potential in enhancing drug release from liposomes and can potentially improve cancer therapy.

Abstract 5618: Ultrasound-mediated delivery of a novel nanoparticle-microbubble platform.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ultrasound-mediated delivery of a novel nanoparticle-microbubble platform. A major obstacle in delivery of nanoparticles (NPs) to tumor cells is the low uptake and heterogeneous distribution of the NPs in tumor tissue. Ultrasound (US) may improve the delivery of encapsulated drug in various ways depending on the frequency and intensity applied, by inducing heating, radiation force or cavitation. We have developed a novel multimodal, multifunctional drug delivery system consisting of microbubbles stabilized by polymeric NPs to be used in US-mediated delivery of NPs. Miniemulsion polymerization was used to prepare NPs of the biocompatible and biodegradable polymer poly(butyl-2-cyanoacrylate) (PBCA). The NPs were coated with PEG to improve the circulation time and biodistribution. Microbubbles stabilized by these NPs were prepared by mixing the NP dispersion with proteins and air using an ultra-turrax. The aim of the present work was to study the cellular uptake of the NP in vitro and the microdistribution of the NP in tumor tissue in vivo. Human prostate cancer cells were incubated with fluorescently labeled (Nile red and DiR) NPs and the cellular uptake measured by flow cytometry and confocal laser scanning microscopy (CLSM). Prostate cancer xenografts were grown subcutaneously on the leg of athymic mice, and NP alone or NPs stabilizing microbubbles were injected intravenously. The particles circulated for 5 min or 24 hr, before the tumors were exposed to US, thus the effect of US both on extravasation and penetration through the extracellular matrix could be studied. The tumors were exposed to a focused US beam at low (300 kHz or 1 MHz) or high (5 MHz) frequency, applying various intensities. The blood vessels were visualized by injection of FITC- tomato lectin 5 min before euthanizing the mice. The distribution of NPs was studied by CLSM, imaging frozen tumor sections along a radial track from the periphery of the tumor sections. The biodistribution of NPs comparing the uptake in normal and tumor tissue, was studied by whole animal optical imaging. The cellular uptake of the NPs in vitro depended on the length and type of PEG used. In vivo, ultrasound enhanced the uptake and improved the distribution of the NPs in the extracellular matrix. In untreated tumors only small amounts of NPs were observed and they were located close to the blood vessels. In the US-exposed tumors, the uptake was enhanced and the NP had penetrated further away from the blood vessels compared with unexposed tumors. US administered 5 min after NP-injection was more efficient than US given after 24 h. This demonstrates that the effect of US on extravasation is more important than the effect on penetration of NPs through the extracellular matrix. A prerequisite for successful cancer therapy is that the cytotoxic drugs reach all the cancer cells. The present results demonstrate that US improves the delivery of NPs, and mainly by increasing the permeability of the capillary wall. Citation Format: Catharina De Lange Davies, Siv Eggen, Stein-Martin Fagerland, Mercy Afadzi, Audun Dybvik Bohn, Hakon Furu, Rune Hansen, Bjorn Angelsen, Per Stenstad, Yrr Morch. Ultrasound-mediated delivery of a novel nanoparticle-microbubble platform. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5618. doi:10.1158/1538-7445.AM2013-5618

Ultrasonic drug release: Impact on liposomal doxorubicin in collagen gels

Ultrasound enhances the release of drugs from liposomes, and in solution this is shown to be caused by cavitation. However, the mechanism in tissue is unclear, and there is a need to bridge the gap between results obtained in solution and in tissue. Thus, we studied the release of liposomal doxorubicin in 5% w/v type I rattail collagen gels when subjected to 300 MHz ultrasound. Confocal laser scanning imaging was used to characterize the gels before and after the treatment, and for determining the release of doxorubicin. The results indicate that drug release in collagen gels is similar to that in liquid, due to mechanical effects (cavitation) rather than thermal ones. Release was shown to be highly dependent on the presence of gas bubbles by degassing and adding controlled amounts of gas contrast bubbles, and the drug release correlated with the presence of OH radicals. Compared to experiments in solution, the threshold at which such effects happen is similar, at a negative pressure of 0.9 MPa, but the maximum release is halved for identical treatment.

Multifunctional nanoparticles for drug delivery and imaging: Effect of ultrasound on cellular uptake and tumor tissue distribution

This study is focused on cellular uptake (in vitro) and microdistribution (in vivo) using a novel multifunctional drug delivery system consisting of microbubbles (MBs) stabilized by polymeric nanoparticles (NPs). Prostate cancer cells in suspension were incubated with NP-loaded MBs and then exposed to focused ultrasound (US, 300 kHz). Cellular uptake was measured by flow cytometry. To study microdistribution of the NPs in prostate tumors, particles were injected intravenously into mice bearing subcutaneous prostate xenografts. The tumors were exposed to focus US 5 min or 24 h after the injection using 300 kHz or 5 MHz US. Tumors were frozen and sections were analyzed by confocal laser scanning microscope (CLSM). Fluorescent labeled lectin was used to stain the blood vessels. The in vitro study shows enhancement of cellular uptake in the presence of US compared to untreated cells. Cellular uptake of NPs increased with increase in mechanical index. Analysis of confocal images of tumor slices showed an enhanced uptake and improved penetration of NPs after US exposure. These effects might be due to cavitation and radiation forces. The results show the potential of the novel multifunctional drug delivery system to improve cancer therapy.

Ultrasound mediated delivery of liposomal doxorubicin in prostate tumor tissue

Combining ultrasound exposure and liposomal encapsulated anti-cancer drugs has beneficial synergistic effects in cancer therapy, although little is known about the underlying mechanisms. This study has focused on investigating the effect of different ultrasound exposures (1 MHz and 300 kHz) on delivery and distribution of liposomal doxorubicin in Balb/c nude mice bearing prostate cancer xenografts. Ultrasound exposure was done 24 h after administration of liposomes to study the effect on liposomes present in extracellular matrix. Optical imaging methods were used to evaluate the effects of the ultrasound exposures both on subcellular microscopic level and macroscopically of organs. Confocal laser scanning microscopy revealed that ultrasound-exposed tumors had increased amounts of released doxorubicin in tumor tissue compared to unexposed control tumors. Image analysis demonstrated an increased distance from blood vessels to areas with liposomes and released doxorubicin in tumors exposed to 1 MHz ultrasound. Whole animal optical imaging showed uptake of liposomes both in normal tissue as well as tumor tissue.

Intracellular delivery of nanoparticles using ultrasound

The effects of ultrasound on cellular uptake of FITC-dextrans governed by sonoporation or endocytosis were studied. Hela cells in suspension with FITC-dextran (MW 4-2000 kDa) were exposed to ultrasound using different acoustic parameters (0.0 to 0.58 MPa peak negative pressures, 33 μs pulse length, 3 kHz pulse repetition frequency and 120s insonication time) in the presence or absence of microbubbles. After ultrasound exposure, the cellular uptake of FITC-dextran and cell viability were measured using flow cytometry. Confocal microscopy was used to localize the uptake of nanoparticles in cells. The role of endocyctosis was investigated using endocytic inhibitors; genistein, wortmannin and chlorpromazine inhibiting respectively caveolae-mediated endocytosis, macropinocytosis and clathrin-mediated endocytosis. Ultrasound in the presence of microbubbles enhanced the cellular uptake of dextran more than ultrasound alone. At a constant duty cycle (10%) and insonication time (120s), the percentage of cells internalizing dextran increased to 65% with increase in acoustic peak negative pressure (145 to 576 kPa, i.e., MI of 0.26 to 1.05). There was no statistical difference (p ≥ 0.3) between the uptakes of different sizes of dextran (MW 4-2000 kDa) in the presence of microbubbles. Inhibition of the endocytic pathways resulted in significant decrease in the cellular uptake (29% for genistein, 37% for wortmannin and 45% for chlorpromazine). The result indicates that ultrasound in the presence of microbubbles enhances cellular uptake of nanoparticles whereas ultrasound alone has hardly any effect. The improved uptake might be due to both poration and endocytosis. The mechanism of uptake of dextran is size independent (up to 2 MDa), thus either the size of the pores is larger than the largest dextran molecule used, or endocytosis is size independent. The results show that ultrasound enhances cellular uptake of therapeutic molecules and has the potential to improve cancer therapy.