Delivery of nanoparticles in tumour tissue and cells

Catharina Davies in the laboratory. Photo: Irene Aspli

Nanotechnology has started a new era in engineering multifunctional nanoparticles for improved cancer diagnosis and therapy, incorporating both contrast agents for imaging and therapeutics into so called theranostics NPs.

Encapsulating the drugs into nanoparticles improves the pharmacokinetics and reduces the systemic exposure due to the leaky capillaries in tumours. However, the distribution of nanoparticles in tumour tissue is heterogeneous. We are studying how ultrasound can improve the delivery of nanoparticles to cancer cells, and penetrate the blood-brain barrier thereby making it possible to treat disorder in the central nervous system.
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Research topics

Multifunctional nanoparticles in diagnosis and therapy

Lipid based nanoparticles, such as liposomes and emulsions, are widely employed as in vivo carrier and delivery systems. These systems are well suited to incorporate, transport and deliver high payloads of pharmaceutical and/or contrast agents in vivo.

Multifunctional nanoparticles in diagnosis and therapy

Lipid based nanoparticles, such as liposomes and emulsions, are widely employed as in vivo carrier and delivery systems. These systems are well suited to incorporate, transport and deliver high payloads of pharmaceutical and/or contrast agents in vivo.

Up to date, only a very limited number of nanoparticulate pharmaceutical or diagnostic agents have entered the clinic. An important reason for this is that the in vivo behavior of nanoparticles needs further elucidation before the therapeutic effect or diagnostic outcome can be sufficiently predicted, understood and fine tuned to specific pathologies. In this project newly developed lipid based nanoparticles are studied using both in vivo confocal laser scanning microscopy (CLSM) and magnetic resonance imaging (MRI).

Cartoon of the nanoemulsions, with a legend on the left. Developed by Willem Mulder et al, Mount Sinai School of Medicine, New York, and published in Biomaterials (2009) 6947-6954

The nanoparticle platform used in this project is a newly developed oil-in-water nanoemulsion (see figure). Contrast or pharmaceutical agents can be incorporated in both the hydrophobic core and the lipid layer. The particles can be prepared in sizes ranging from 30 to 100 nm and in order to direct them to specific cell types, a variety of targteting ligands can be conjugated.

Multimodal imaging combining MRI and CLSM is performed using Fe3O4-crystals (MRI contrast agent) dispersed in the oil core and a fluorescently labeled lipid added to the lipid mixture. As a targeting ligand, tripeptide arginine-glycine-aspartic (RGD) is used, which specifically binds to angiogenic endothelium. Angiogenesis is both a promising target for tumor diagnostics as well as anti-cancer therapies.

After intravenous injection of these particles in tumor bearing mice, CLSM is used to characterize the tissue and particle distribution and dynamics on a (sub)cellular resolution. This information will be used to explain the clinically relevant enhancement in MR images recorded after injection and thus to assess the diagnostic value of the nanoparticles.

Contact: Sjoerd Hak


Tue, 25 Nov 2014 18:33:25 +0100

Degradation and remodelling of the extracellular matrix to improve tumour uptake of nanomedicine

The interstitium or extracellular matrix consists of a protein network embedded in a hydrophilic gel of glycosaminoglycans and proteoglycans. Collagen is the most important structural network in the extracellular matrix.

Degradation and remodelling of the extracellular matrix to improve tumour uptake of nanomedicine

The interstitium or extracellular matrix consists of a protein network embedded in a hydrophilic gel of glycosaminoglycans and proteoglycans. Collagen is the most important structural network in the extracellular matrix.

It is not clear whether the network of fibrillar collagen or the glycosaminoglycan gel plays the most important role in limiting transport of macromolecules through the extracellular matrix.

Makromolecule alone
Makromolecule alone

Makromolecule and hyaluronidase
Makromolecule and hyaluronidase

Periphery Centre

We have been studying how enzymatic degradation of the extracellular matrix using collagenase to degrade collagen, or hyaluronidase to degrade the glycosaminoglycan gel improves the distribution and uptake of therapeutic agents.

Collagenase seems to be more important in improving the uptake of larger molecules (immunoglobulins), whereas hyaluronidase increases the uptake of small cytotoxic agens such as doxorubicin. Ionizing radiation is also found to improve the distribution and uptake of liposomal drugs, and chemoirradiation induces synergistic treatment effects. The ionizing radiation induces apoptosis and cell shrinkage, thereby destabilizing the extracellular matrix.

The improved uptake of the various therapeutic agents is shown to partly be due to the induction of a transcapillary pressure gradient and reducing the interstitial fluid pressure, and partly due to enhanced diffusion.

Contact: Catharina Davies


Tue, 25 Nov 2014 18:41:54 +0100

Gene therapy: Delivery of DNA-chitoan nanoparticles

The success of gene therapy depends on efficient delivery of DNA to diseased cells and involves several steps, including penetration through the extracellular matrix to target cells, intracellular uptake, trafficking to the nucleus, and efficient transcription of the transgene.

Gene therapy: Delivery of DNA-chitoan nanoparticles

The success of gene therapy depends on efficient delivery of DNA to diseased cells and involves several steps, including penetration through the extracellular matrix to target cells, intracellular uptake, trafficking to the nucleus, and efficient transcription of the transgene.

Fig 1. Intracellular localization of DNA (green)-chitosan (red). Nucleus (blue), lysosomes (yellow).The size and charge of the DNA molecule impede several of these transport processes. However, gene delivery efficiency can be improved by using a cationic vector that condenses DNA and protects it from degradation by nucleases in the intra- and extracellular environment (fig. 1).

Although viral gene delivery vectors have so far yielded higher transfection efficacy, safety concerns have drawn attention to non-viral vectors. Among these potential non-viral vectors is chitosan, a cationic polysaccharide derived from chitin which is found in e.g. the exoskeleton of crabs and shrimps, and presents a low cytotoxicity. Chitosans of different molecular weights, structures, and charges can be produced, and chitosan has been shown to efficiently compact DNA into nanoparticles and protects it from degradation by nucleases.

The efficiency of chitosan-based gene delivery seems to depend on the strength of the interaction between chitosan and DNA, which is determined by the molecular structure of chitosan. Aiming to improve gene therapy, we study the influence of chitosan molecular weight, structure, and charge on DNA packing, delivery, and transfection. On that purpose we use a variety of techniques such as dynamic light scattering and atomic force microscopy to characterize the nanoparticles; gel electrophoresis, and fluorescence correlation spectroscopy to study DNA-chitosan interactions; flow cytometry to measure cellular uptake; and confocal laser scanning microscopy to study intracellular trafficking and colocalization of the nanoparticles with the endosomal-lysosomal compartments, cytosol and nucleus (2-5).

Contact: Catharina Davies


Tue, 25 Nov 2014 18:43:42 +0100

Ultrasound in drug delivery and early diagnosis of cancer

Topics

Ultrasound in early diagnosis of cancer, Ultrasound and drug delivery and Ultrasound and drug delivery to the brain.

Ultrasound in drug delivery and early diagnosis of cancer

Topics

Ultrasound in early diagnosis of cancer, Ultrasound and drug delivery and Ultrasound and drug delivery to the brain.

Ultrasound in early diagnosis of cancer

Contrast-enhanced ultrasound is a promising method to improve early detection of tumours, and for monitoring therapeutic response.

Ultrasound contrast agents are encapsulated micro-bubbles containing gas with a diameter of a few micrometer. The size of the micro-bubbles constrains them to the vasculature, not being able to cross the capillary wall. By labelling the micro-bubbles with a ligand or antibody binding specifically to endothelial cells receptors in tumour vasculature, contrast-enhanced ultrasound is used to detect angiogenesis in tumour and thereby depicting tumours more efficiently.

A prerequisite for tumour growth is the development of new blood vessels in the tumour, a process called angiogenesis. The capillary wall in tumours consist of endothelial cells, and activated tumour endothelial cells engaged in angiogenesis express an elevated level of several surface receptors such as vascular endothelial growth factor receptor 2 (VEGFR2), P-selectin, leukocyte adhesion molecule 1 (ICAM-1), and dvß3 integrin. Micro-bubbles may thus specifically target tumour vasculature.

Together with SINTEF Materials and Chemistry which develop new gas bubbles, we are studying the potential of early detection of neoangiogenesis in tumours thereby improving diagnosis.

Ultrasound and drug delivery

Ultrasound has the potential of improving drug delivery by various means: Ultrasound may increase the release of cytotoxic drugs from liposomes or other capsules, improve the transcapillary transport, improve the transport through the extracellular matrix, and facilitate the cellular uptake.

The mechanisms may be due to either thermal or non-thermal effects, and cavitation and radiation force are important non-thermal mechanisms. Cavitation is the oscillation of gas bubbles upon exposure to pressure waves, and is divided into two types: stable or collapse. Stable cavitation is the repeatable oscillation of bubbles without leading to bubble collapse, and it occurs at relatively lower acoustic intensities. At higher intensities collapse cavitation occurs and the bubbles oscillate sufficiently that the inertia of the inward moving water causes the bubbles to collapse violently, producing shock waves, jet streams, high temperatures, and free radicals.

The radiation force is a result of the acoustic pressure gradient that is created by the energy lost by the ultrasound wave as it propagates through the tissue. To obtain radiation force the energy flux density must be high as the radiation force is proportional with the intensity of the incoming ultrasound beam, and increases with the frequency.

Ultrasound and drug release from liposomes

The increased release of drugs from capsules or liposomes may be due to either thermal effects "melting" the membrane of the capsules or cavitation producing pores in the capsules or destroying them. The mechanism depends on the ultrasound exposure and the properties of the capsules. Increasing the permeability of the capillary wall by the formation of pores and by the radiation force may cause improved transcapillary transport. Enhanced transport through the extracellular matrix which is the main barrier for delivery of capsules and drugs, may be caused by several ultrasound-related effects such as:  increased diffusion due to heating of the tissue, and to mechanical effects caused by cavitation such as the production of shock waves, acoustic streaming and jet streams, as well as by radiation force pushing the nanoparticles further away from the blood vessel wall. The permeability across the cell membrane is found to be enhanced by ultrasound which forms transient pores in the plasma membrane.

All these effects depend on ultrasound exposure parameters such as frequency, intensity/acoustic pressure, length and number of ultrasound pulses, and duration of ultrasound exposure. In collaboration with professor Bjørn Angelsen and Ultrasound research group at Department of circulation and medical imaging NTNU we are characterizing the ultrasound exposure to determine the optimal treatment for drug release and delivery. The project is also a collaboration with the company Epitarget as which develops acoustic active liposomes containing drugs.

Ultrasound and drug delivery to the brain

To treat brain, or central nervous system (CNS), diseases like Parkinson's disease, Alzheimer's disease and brain cancer (glioblastoma multiforme), one has to penetrate the blood-brain barrier (BBB).

The BBB protects the brain and the spinal cord from unwanted toxic substances and have two main barrier functions. Firstly, the epithelial cells that are the lining of all blood vessels are extra tightly packed in the BBB and this hinders molecules from passing into the CNS between the cells. Secondly, if a molecule manages to diffuse into an epithelial cell, it is most likely transported out into circulation again due to high abundance of Glycoprotein efflux pumps. The BBB is so effective that only about 2 % of all small molecules (<600 dalton) are allowed through and none of the larger ones.

Since the BBB is so restrictive, a novel way of transporting drugs across the BBB is needed. Instead of modifying functioning drugs to accommodate BBB-passage (which is very time consuming and not always feasible) it would be better to have a generic vessel that can pass the BBB and then release its cargo once in the CNS. For this we are using the microbubble/focused ultrasound technique. The cavitation from microbubbles has been shown to open the BBB and hence the nanoparticles that compose the microbubble can break off and "ride" on the jet streams into the CNS. Once inside the nanoparticle will degrade and release the drug.

In order to further increase the efficacy of treatment we also use Au@Fe nanoparticles that have silencing RNA attach to it. The silencing RNA can turn off the efflux-pumps and hence the drug will stay inside the CNS for a longer period of time.

This interdisciplinary project is a collaboration with the ultrasound group mentioned above, SINTEF Material and Chemistry making novel polymeric nanoparticle-microbubble to be used to penetrate the BBB, and Wilhelm Glomm Dept of Chemical engineering, NTNU making the Au@Fe nanoparticles.

Contact: Catharina Davies


Tue, 25 Nov 2014 18:46:01 +0100