Silver Lining: Explore the Lee lab use of rabies virus to target tumors

Everything has a silver lining. The rabies virus, infamous for killing beloved Old Yeller, is deadly because of its dangerously efficient method of attacking the central nervous system and the brain. As scary as the rabies virus is, it is admirable because it is one of the few that is able to penetrate the fortified blood brain barrier. The Lee lab saw this deadly mechanism as a silver lining. They used nanogold particles to mimic the shape of the rabies virus in order to bypass the blood brain barrier and target tumors.

The blood brain barrier is one of the biggest obstacles for neurological treatments. The same mechanism meant to protect the precious brain serves as one of the biggest obstacles for medical treatments. Around “98% of small molecules as well as colloidal-sized particles are restricted from entry into the brain”. This is one of the biggest obstacles physicians face when trying to treat brain tumors. When faced with such a daunting challenge, the Lee Lab turned to naturally occurring breaches of the BBB. That is when they came across the silver lining of rabies. The rabies virus has many proteins that surround its outer body but one specific protein, the rabies virus glycoprotein (RVG), allows the virus to use neural pathways, travel to the CNS, and cross the blood brain barrier. Thus, they decided to create a nanoparticle that would be coated with RVG which would allow it to pass through the BBB and target brain tumors.

Now that they knew what the outside of the nano particle should be, they had to figure out what the inside material should be. The material needed to be inorganic, flexible, and unique photothermal properties. An inorganic substance was needed so that it would be inert and would not react with the other biomaterials in the body. In regards to structure, there are many factors that influence the uptake and biodistribution of nanoparticles inside the body like charge, shape, size, etc. However, “particle shape is considered to be one of the most powerful tools for enhancing nanoparticle internalization”. Thus, flexibility was important because different shapes and configurations of the nanoparticles needed to be tested. The material of choice also had to have photothermal properties because the plan was to have the nanoparticle pass the BBB and then use a laser to activate the nanoparticles only in the region of the brain tumor. This was crucial because, in theory, when the nanoparticles are released into the bloodstream, they are going to be found throughout the body and the brain. Thus, the nanoparticles either had to be made so that they targeted just the tumor or they had to be made so that they could be anywhere but would only activate when in the tumor region. The latter would be much easier because it is often hard to differentiate between a tumor cell and a regular cell using proteins. Thus, having unique photothermal properties would allow the nanoparticle to overheat when target with a laser and burn the tumor tissue around it.

The material that best fitted the job description was gold. Gold is a flexible inorganic metal that is inert and nontoxic. Gold also has unique photothermal properties. It has a very high rate of absorption: ~105 times conventional dyes. In addition to having high absorption which allows for better imaging, gold can increase from a range of 10 oC to 1000 oC. This property is important because it needs to be able to heat up enough to kill the tumor cells surrounding it. Luckily, some studies show that tumor cells are more susceptible to damage due to heat than regular cells.It is also generates a strong the localized surface plasmon resonance (LSPR) when in presence of near infrared light (NIR) which is a bunch of fancy words for allows for better imaging when certain wavelengths of light interact with it. This allows for better data analysis and tracking of what the nanogold rods are doing in the brain. Even better, gold has a strong LSPR in the “wavelength range of 700–950 nm (maximally 1200 nm), which is the best spectral region for imaging and therapy due to the so-called “water window” of all aqueous tissues”. It is important to note that this ideal wavelength is only possible when the gold is molded into rods and not spheres. This ends up being ideal because they are trying to mimic rabies virus which is “bullet-like shape with one rounded end and one planar end”. It is also very helpful that the absorption range of gold can be adjusted by changing the aspect ratio of the gold rods which will allows room for experimentation to find the shape that can heat up the most and be efficiently internalized. After trying different aspect ratios, it was experimentally determined that the optimal ratio for the rods was 2.34 (~120 nm in length and ~50 nm in width) which is very similar to that of the rabies virus. This shape combined with the RVG protein coating allowed the gold nanorods to mimic the rabies virus. Experimentally, it was shown that gold nanorods that had the RVG protein coating and the specific aspect ratio were more present in spinal and brain cells than regular gold nanorods.

Now that the ideal structure was determined, the theory was put to the test. Male mice were injected with N2a cells - tumor cells. The N2a cells were injected outside the brain and inside the brain. This means that some were injected onto the right dorsal side of the mouse and some were injected into the striatum of the brain. This was done in order to test for the effectiveness of the RVG treated gold nanorods on general tumor cells and then specifically on tumor cells in the brain. Next, some mice were injected with plain gold nanorods while others were injected with RVG gold nanorods. Finally, the tumor cells were irradiated with an 808 nm laser. It is important to note that the laser itself did not cause any damage to the skin or induce any tumor suppression. The results were very promising.

The results showed that RVG treated gold nanorods were able to greatly decrease the size of tumors that were present on the side flank of the mice (not in the brain) when compared to the using regular gold nanorods or saline. In fact, a couple of the mice had tumors that completely went away and minimal skin damage, due to the treatment, was healed within thirteen days. After seeing preliminary success outside the brain, the real test was conducted. It was the moment of truth. We knew that the RVG gold nanorods can greatly decrease tumors however, would they be able to overcome the abominable BBB and have an impact on the tumor cells? The Lee lab was proud to publish that the RVG gold nanorods were in fact able to cross the BBB and were able to greatly suppress tumors especially when compared to regular nanorods and saline models.