1. Ultrasound-assisted transport of nanoparticles across the blood- brain barrier Habib Baghirov, Department of Physics, NTNU

2. Blood-brain barrier (BBB) Physical and biochemical wall between blood and the brain. Backbone: brain endothelial cells. Properties: tight junctions, lack of fenestrations, small number of pinocytotic vesicles. Astrocytes and pericytes support BBB development and integrity. BBB takes care of brain homeostasis and decides what the brain gets from blood circulation. It carries essential nutrients to the other side but is generally prejudiced against hydrophilic and/or large molecules. What tight junctions are to paracellular route, efflux transporters are to transcellular passage.

3. Nanocarrier-assisted drug delivery across the blood-brain barrier BBB filters out the vast majority of drugs, letting through only small lipophilic molecules mostly used in the treatment of affective disorders, epilepsy, depression and such. Treatment of Alzheimer’s, Huntington’s, brain cancer, stroke, brain injury, multiple sclerosis, amyotrophic lateral sclerosis and many other brain disorders requires efficient delivery, not only the actual discovery of drugs. Nanocarriers: possible answer to this challenge. Why? 1. Ability to carry many drug molecules. 2. Functionalization to confer specific properties or avoid early degradation. 3. Targeting for more efficient delivery and reduced non-specific toxicity. 4. Combination of imaging with theranostics. 5. One box to ship them all: once a nanocarrier is made to cross the BBB, it can, with some adaptation, be used to deliver a large variety of drugs.

4. Study objectives Use ultrasound exposure get polymeric nanoparticles across the blood-brain barrier. Evidence shows that the delivery of nanoparticles across the BBB, even with targeting ligands, is a matter of chance, with no uniform delivery method developed so far. Ultrasound has been shown to open the BBB transiently and non-destructively, and can secure the passage of nanoparticles with their cargo. Nanoparticles can incorporate gas bubbles, providing a distinct advantage in ultrasound treatment. Use nanoparticles get small interfering RNAs into endothelial cells and silence efflux pums, such as P- glycoprotein. Efflux pumps prevent the transport of those relatively small and lipophilic drugs that would otherwise cross the BBB. Silencing them will eliminate this issue. Ideally, doing it with iron-containing nanoparticles will also allow tracing the nanoparticles with magnetic resonance imaging.

5. In vitro models of the blood-brain barrier Cells: 1: RBE4 rat brain endothelial cell line. 2: MDCK II canine kidney cells Cells are cultured on permeable membranes that allow the formation of tight monolayers. RBE4 are of brain endothelial origin, but MDCK II cells form tighter monolayers Methods: Transepithelial resistance (TER) and passage of a zero permeability marker to verify monolayer integrity. Fluorometry to study the transport of nanoparticles across the monolayers Flow cytometry and confocal microscopy to verify uptake of nanoparticles. Immunocytochemistry to verify the formation of tight junctions and their their status under different experimental conditions. From Nat Protoc. 2009;4(5):662-73., modified

6. Overview of microbubble-cell interations induced by ultrasound exposure Stable and inertial cavitation. (A) Schematic representation of an acoustic pressure wave. (B) and (C) show, respectively, stable and inertial cavitation of microbubbles Biological effects of microbubbles. (A) - pore formation. (B) – formation of reactive oxygen species. (C) Deformation of cell membrane caused by shear stress induced by microstreams.

7. Monolayer formation in RBE4 cells – CellMask stain Center Slice Red – CellMask DeepRed staining

8. Zonula occludens 1 (ZO-1) staining Center Slice Green – ZO-1 rabbit primary antibody / Alexa Fluor 488 goat anti-rabbit secondary antibody Monolayer formation in RBE4 cells – Zo-1 stain

9. Uptake of PBCA and POCA nanoparticles by RBE4 cells

10. Maximum intensity projection Blue – nuclei (Hoechst) Green – F-actin (stained with phalloidin) Red – nanoparticles Uptake of PBCA nanoparticles in RBE4

11. PBCA Uptake by RBE4 – late endosomes Live cell imaging, upward of 3 hours.

12. PBCA Uptake by RBE4 after 4 hours – early endosomes

13. PBCA Uptake by RBE4 after 4 hours – late endosomes

15. PBCA Uptake by RBE4 after 4 hours – lysosomes

16. PBCA Uptake by RBE4 after 4 hours - overview Early endosomes Late endosomes Lysosomes

17. PBCA and POCA Uptake Kinetics in PC3 and RBE4 Uptake kinetics in PC3 and RBE4 over 24 hours Inhibition of NP uptake by PC3 and RBE4 by clathrin and caveolin-mediated endocytosis inhibitors

18. PBCA and POCA nanoparticle cytotoxicity

21. P-gp silencing by MSN nanoparticles

22. Method description RBE 4 cells were incubated with NPs containing Abcb1a rat siRNA and negative control siRNA. Medium changed after 3, 6 or 24 hours. On day 3, cells were incubated with Rhodamine 6G for 1 hour. After that, medium was changed and the cells allowed to efflux Rhodamine 6G for 2 hours. After 2 hours, cells were washed several times and prepared for flow cytometry. Additional controls: cells incubated with Rhodamine 6G and blank cells.

23. P-gp silencing in RBE4 cells with MSN NPs: 3 hours

24. P-gp silencing in RBE4 cells with MSN NPs: 6 hours

25. P-gp silencing in RBE4 cells with MSN NPs: 24 h

27. Ultrasound-induced transport of MSN nanoparticles across the BBB or lack thereof

28. Setup description PBS-filled chamber containing a 1 MHz ultrasound transducer and a holder for semi-permeable inserts. Inserts are placed in the chamber upside down. Microbubbles (HEPS or Sonovue) are injected to cover the cell layer. After ultrasound exposure (2 minutes, 0.4 MI thought to induce inertial cavitation, or 0.1 MI thought to induce stable cavitation) the membranes are transferred to cell culture plates and a transport assay is performed as usual.

29. Meta-analysis results

30. Outlook and prospects Polymeric poly(butyl) and poly(octyl)cyanoacrylate nanoparticles have excellent uptake profile in rat brain endothelial cells – something to build upon. However, there are technical issues with their transport across an in vitro model. Mesoporous silica nanoparticles have been used in in vitro ultrasound- assisted transport studies instead, with currently uncertain results. As of now, FeAu nanoparticle loaded with P-glycoprotein siRNA have failed to successfuly silence P-gp in RBE4 cells. Mesoporous silica nanoparticles have succeded, but to a limited extent only. Further in vitro and, potentially, in vivo experiments will be done with better MSN batches. In vivo studies using ultrasound and polymeric nanoparticle will be carried out with a melanoma brain metastasis model and ultrasound exposure.

31. The End Thank you for your attention! I am grateful to my supervisor, Catharina de Lange Davies, and co-supervisors Wilhelm Robert Glomm and Rune Hansen, as well as to Yrr Mórch, Sulalit Bandyopadhyay, Einar Sulheim, Andreas Åslund and other colleagues from the NTNU Department of Physics and elsewhere for their input used in this study and in the making of this presentation.