Per Stenstad

Monodisperse magnetic polymer particles: new biochemical and biomedical applications

The method of activated swelling of polymer particles developed by the authors allows the preparation of monodisperse spherical beads of predictable size from 1 to 100 microns in diameter. The polymer particles may be prepared from a number of different monomeric materials and with various morphologies including macroporous structures. The porous beads form the basis for magnetizable monodisperse polymer particles which have magnetic iron oxides distributed as small grains all through the volume of the beads. The magnetic particles are being used extensively for selective cell separation and for immunomagnetic separation within microbiology and molecular biology. A review of recent work within these fields is given. New methods for positive cell separation are announced.

Fischer–Tropsch diesel emulsions stabilised by microfibrillated cellulose and nonionic surfactants

Water-in-diesel emulsion fuels have been prepared with a combination of sorbitan monolaurate and glycerol monooleate as emulsifier and with microfibrillated cellulose (MFC) of different hydrophilic/hydrophobic character as stabilizer. The MFC was treated with either octadecylamine or poly(styrene-co-maleic anhydride), resulting in very hydrophobic fibrils. The most stable emulsion was achieved with a combination of hydrophilic (untreated) and hydrophobic MFC and only minute amounts of the stabilizer gave a pronounced effect. Even with the optimized formulation the lifetime of the emulsion was shorter than previously reported when a conventional polymeric stabilizer was used, however. The water drop sizes in the emulsions were determined by three methods: optical images, light scattering, and NMR diffusometry. All three methods gave water drops sizes of ca 2 μm. The NMR diffusometry indicated that besides the micrometer-sized emulsion drops a significant fraction of the water is present in small droplets of micelle size. The chemical exchange of water between these two populations of pools is believed to be the reason for the relatively low stability of the system.

Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)-coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air-liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP-MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs.

Biomedical Applications of Monodisperse Magnetic Polymer Particles

The method developed by Ugelstad and coworkers1-3 for preparation of monosized polymer particles allows preparation of particles of any size from 1 to 100 µm with standard deviation in diameter ~ 1%. Also the method allows preparation of particles from a large number of monomers. Particles with highly crosslinked polymer of high mechanical strength, porous macroreticular particles and core and shell particles are prepared. The application of the porous macroreticular particles in fast protein liquid chromatography (FPLC)4 as marketed by Pharmacia is already well established and has in many cases led to very significant improvements in analysis and separation of complex protein mixtures.

Preparation and Application of Monosized Magnetic Particles in Selective Cell Separation

Some of the basic principles of formation of monosized macroporous particles by the method of “activated swelling” and the preparation of superparamagnetic particles based upon these particles are discussed. A short review of the applications of monodisperse, magnetic polymer particles in cell separation, with emphasis on recent work, is given. Some new work concerning non-specific adhesion of antibodies and cells on different beads, and the prevention of this unwanted phenomenon by help of casein are described. A method for the removal of excess magnetic beads by gradient centrifugation is presented. Positive cell separation, with easy liberation of free cells after magnetic isolation of rosetted cells, is obtained by use of particles with covalently coupled aminophenyl boronic acid at the surface.

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

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.