Drug delivery across blood-brain barrier: use of multifunctional nanoparticles, ultrasound, and silencing of efflux transporters
Drug delivery to the brain is strongly restricted by the blood brain barrier (BBB). The presence of tight junctions, efflux pumps, and a decreased number of endocytotic vesicles in endothelial cells maintaining the BBB, prevent most medicines to penetrate the BBB and limits the entry into brain tissue. Ultrasound (US) in the presence of microbubbles (MBs) has been shown to disrupt tight junctions, increase sonoporation and endocytosis in vivo. The aims of this project were (1) to participate to the development of multifunctional nanoparticles (NPs) to stabilize MBs, encapsulate drugs, and improve penetration through the BBB, (2) to combine these NPs with iron-gold nanoparticles Fe@Au grafted with Si-RNA to silence P-glycoprotein P-gp efflux pumps, (3) to develop an in vitro BBB model to reduce the number of animals used for optimizing US parameters, NPs and Fe@Au NPs. The composition of the multifunctional NPs developed in collaboration with SINTEF was optimized to control their degradation rate and their circulation time. Flow cytometry, and imaging techniques were used to show that degradation rates and endocytosis of poly(butyl cyanoacrylate) PBCA and poly(octyl cyanoacrylate) POCA NPs depends on cell type (Sulheim et al., J. of Nanobiotechnology, 2016). Poly(ethylene glycol) (PEG) is used to prevent NPs aggregation and prolong circulation time in the blood. The influence of PEG length and density on the circulation time is currently studied in vivo. The ability of NPs coated with different length and density of PEG to penetrate in vitro models of the extracellular matrix was studied using correlation microscopy. Additionally, it was found that NPs' load such as fluorescent dyes influences NPs' uptake, and that uptake also depends on cell type (Snipstad et al., submitted manuscript). Most importantly, in vivo studies performed on rats have recently shown that it is possible to transiently open the BBB using PBCA NPs and US, and penetration of PBCA NPs into brain tissue was demonstrated (Åslund et al., J. of Controlled Release, 2015). Silencing of efflux pumps using Fe@Au NPs have shown limited results due to the toxicity of Fe@Au NPs. These NPs are currently optimized by the Department of Chemistry at NTNU. Alternatively, silencing of P-gp efflux pumps is being studied on an in vitro BBB model consisting of immortalized rat brain endothelial cells RBE4 using mesoporous silica particles. An in vitro BBB model consisting of primary porcine brain endothelial cells (PBEC), which provides one of the closest models to human BBB, has been developed with the collaboration of Wolfgang Sattler's group at the University of Graz, Austria. Measurement of the electrical resistance of the cells as well as immunostaining of Claudin-3, one of the protein forming tight junctions, and flow cytometry measurements show that our PBEC cells are able to form good tight junctions and produce P-gp. With the help of SINTEF Medical Technology and the Department of Circulation and Medical Imaging, we used this in vitro BBB model based on PBEC to study the influence of acoustic pressure on tight junctions and endocytosis. SonoVue, a commercially available and well characterized contrast agent was used in combination with US. Our results seem to indicate that lower acoustic pressure producing stable oscillation of the MBs and inducing shear stress might increase drug transport in a more efficient way than higher acoustic pressures leading to MBs collapse and jetting. However, in vitro model based on PBEC seem to be very sensitive to medium change and culture conditions, which might limit its use to study the effects of acoustic pressure and pulse length on endothelial cells (manuscript in preparation). Finally, preliminary experiments confirm that PBEC monolayers can be used to investigate the uptake of polymeric NPs. In particular, PBEC based in vitro models will be helpful to optimize polymeric NPs loaded with anti-cancer drug such as doxorubicin.
The project supported by HMN is part of a larger project supported by the Research Council of Norway, Nano2021. While the HMN project has worked on the in vitro models for BBB, the Nano2021 also includes preclinical work using rats. We have found that a novel nanoparticle-microbubble platform combined with focused ultrasound opens safely and temporarily the BBB, and the NPs are accumulated in brain tissue (Åslund et al., J. of Controlled Release, 2015). This opens new possibilities to treat diseases in the central nervous system such as cancer, Alzheimer, or Parkinson diseases. Further funding from NFR will help us to start a new phase of the project where polymeric NPs will be used in combination with US to treat gliomas and to detect plaques in Alzheimer disease AD. This work will be done in collaboration with Lars Nilsson, University of Oslo, and Rolf Bjerkvig, from the University of Bergen, who have developed respectively genetically modified mice developing Alzheimer plaques and glioma model from patients. AD is a neurodegenerative disorder associated with huge socio-economic costs and accounts for 60% to 70% of cases of dementia. The number of people affected by AD is estimated to be doubled by the year of 2050, and more than 100 million people worldwide will be affected by this disease. Gliomas are extremely difficult to treat, with around 30% of the patients surviving one year and only 14% surviving five years. Therefore, new and more effective treatments would have significant health impact . However there are few drugs on the marked partly due to problems for successful delivery. Our project, by showing that it is possible to open the BBB, demonstrates to the pharmaceutical industry new possibilities in treating diseases in the central nervous system in general.
Investigating the effects of ultrasound and microbubbles on the opening of the blood brain barrier in-vitro
The brain is protected from pathogens and toxic molecules, but also from medicines, at its interface with the blood. Ultrasounds and microbubbles can temporarily open the blood brain barrier BBB, which might facilitate non-invasive brain treatments and greatly improve drug delivery.
Molecules circulating in the blood might access the brain via two routes. The very small interstices between the cells regulate the passage of water, oxygen, and ions only. Larger and more lipophilic substances must be trafficked through the endothelial cells before accessing the brain. Medicines are usually large molecules, and only 2% are able to reach the brain. By using microbubbles and ultrasounds, one expects to be able to "poke" the endothelial cells, thereby creating new routes for medicines to access the brain. Microbubbles subjected to ultrasounds can expend and shrink repetitively, or collapse and produce a jet, depending on ultrasound parameters. Cells close to a microbubble will be "massaged" by the bubble's oscillation, or poked during microbubble's jetting, which induces sonoporation. Both stable oscillation and sonoporation might enhance drug delivery through the cell. Importantly, ultrasound can be focused on a very small area of the brain and do not require opening of the skull to be applied. Hence it is possible to treat the diseased area only and to spare healthy tissues. In addition, two types of nanoparticles are used in order to improve drug delivery: (1) Nanoparticles developped by SINTEF Material and chemistry are able to stabilize microbubbles and can be loaded with drugs. After ultrasound exposure and microbubbles' collapse, the nanoparticles are free to enter the capillaries via sonoporation, and the drugs they contain might be released in the brain. (2) Silencing RNA grafted nanoparticles are used to lower the selectivity of the cells towards xenobiotics. Previous experiments involving nanoparticles with a magnetic core, designed by the Department of Chemistry at NTNU, have shown some toxicity. Recent experiments using a mesoporous silica core and silencing RNA seem to show more promising results. This project uses an in-vitro model of the BBB to investigate the effects of ultrasound and microbubbles on macromolecular transport in endothelial cells. We use an in-vitro BBB model based on primary cells isolated from pig brains' microcapillaries. The cells are grown as a monolayer on permeable polyethylene terephthalate membranes and have a very low permeability, which reflects quite well brain microcapillaries' permeability. A tank has been custom-made by NTNU Finmekanisk verksted to accommodate the ultrasound transducer and the membrane hosting the cells. The effect of ultrasound and microbubbles on the cells is tested by measuring (1) the resistance of the cells monolayer to ions transport, and (2), the transport of fluorescent macromolecules across the monolayer after exposition to the ultrasound. The monolayer is also observed by fluorescent microscopy. The in-vitro BBB model has been established with the help of Wolfgang Sattler's group at the University of Graz, Austria. Ultrasound experiments are performed in collaboration with SINTEF Medical Technology and with the Department of Circulation and Medical Imaging. This work is part of a larger project using our nanoparticle-microbubbles in combination with ultrasound to open the BBB in rats and aiming at treating gliomas and other diseases in the central nervous system. The in-vivo experiments performed on rats are encouraging and have shown that the nanoparticle-microbubbles temporary open the BBB in combination with ultrasound. The clinical relevance of this project in insured through the collaboration with researcher at St. Olavs hospital.
Improving treatment of diseases in the brain through the use of ultrasound, microbubbles and nanoparticles
The interface between the blood and the brain, the blood brain barrier BBB, protects the brain against viruses, bacteria and toxins, but also against medicines. By combining ultrasound, microbubbles and nanoparticles this project aims to disrupt the BBB and increase the delivery of drugs to specific areas of the brain such as tumors for instance.
In the brain, the cells forming the blood vessels are packed so closely that only small molecules such as water can pass between them. Larger molecules such as sugar access the brain by going through the cells. However, passages through the cells are highly regulated. Indeed cells forming the BBB use proteins present on their membrane as "border patrol agents" and reject systematically into the blood molecules able to bind these proteins, which includes many cancer drugs. As a result, less than 2% of all the medicines produced are able to reach the brain, and diseases such as cancer, alzheimer or parkinson are very difficult to treat. The purpose of this project is to increase the amount of drugs accessing the brain by combining different technologies. It involves two steps: first it aims to prevent the cells forming the BBB to produce the proteins rejecting drugs back to the blood. This part of the project involves collaboration with the Department of Chemistry at NTNU, where magnetic nanoparticles are designed and covered with silencing RNA. The magnetic core of the nanoparticles allows them to be visualised during their transport into the body, while the silencing RNA temporarily prevents the cells to produce the proteins rejecting drugs.
In a second step, ultrasounds are used in combination with microbubbles and nanoparticles loaded with drugs. In the bloodstream, when microbubbles coated with nanoparticles are subjected to ultrasounds, their size shrinks and expands very quickly. This can temporarily damage neighbouring cells membranes. Ultrasounds can also induce microbubbles collapse, which leads to the release of the nanoparticles coating their surface. Nanoparticles loaded with drugs are then free to enter the cells. Such nanoparticles and microbubbles are designed at SINTEF Material and chemistry. Using ultrasounds requires the collaboration of SINTEF Medical Technology and of the Department of Circulation and Medical Imaging.
The development of this project requires the use of in vitro and in vivo models to optimize the different nanoparticles used as well as ultrasounds parameters. In vitro models are based on an immortalised cell line from rats and on primary cells from pigs brain blood vessels. These cells are grown on transparent plastic membranes and present similar properties to the cells forming the BBB in live brains. After exposition to the different nanoparticles and ultrasounds, the cells are observed using different techniques such as microscopy and flow cytometry. This allows a first optimization of the nanoparticles composition prior to animal testing, and therefore reduces the number of animal used in the project. However, the ultimate goal is to treat diseases located in human brains. Therefore optimizing ultrasound parameters and nanoparticles composition requires working on live brains protected by a skull. In vivo experiments are currently performed on rats. Collaboration with researcher at St. Olavs hospital is also essential to ensure the clinical relevance of this project.
Moreover, this research is part of a larger and ambitious project which goal is to combine the use of ultrasounds, microbubbles and nanoparticles to improve cancer treatment in the whole body. Previous experiments on mice have shown that the combine use of ultrasound, microbubbles and nanoparticles does have the potential to increase the amount of drug accumulating in tumour tissues, which makes this research very promiseful.
Labeling nanoparticles – dye leakage and altered cellular uptake
submitted to Cytometry
An in vitro model to optimize contrast agents
Manuscript in preparation
PEGylation of PBCA nanoparticles, a quantitative and qualitative study
Manuscript in preparation