Nanozyme: eliminating the leading cause of death worldwide.
Atherosclerosis is responsible for about 50% of deaths globally including ischemic strokes and heart disease. We are leveraging nanotechnology and gene editing to destroy the root cause of these deaths— plaques.
This article was written by Hung, Sualeha, Neha and me, Dasha.
The headline might sound like clickbait, yet it’s not. We have the solution.
Cardiovascular diseases are the leading cause of death globally.
According to many credible organizations, including World Health Organisation: cardiovascular diseases are the leading cause of death globally.
They are claiming nearly 17.9 million lives annually. Household names such as heart attacks and ischemic strokes are included within the umbrella of cardiovascular diseases (CVD’s), which combined are the cause of over 85% of CVD-related deaths. Cardiovascular diseases such as coronary artery disease(ischemic heart disease) amount to over 31 % of deaths globally.
Atherosclerosis is the root cause behind cardiovascular diseases(and many others).
Underlying the majority of these notorious diseases is the silent but deadly condition known as atherosclerosis.
It’s estimated that atherosclerosis is the root cause of about 50% of all deaths in westernized society.
Atherosclerosis is a disease that causes plaques of fatty material to build up on the inner walls of the arteries. Depending on where they are in the body, they cause different diseases and medical conditions.
- Plaques prevent normal blood flow as they narrow the arteries.
This can cause blood pressure to be lower in some places and higher in others(as plaque is an obstacle after the plaque blood will travel slower). Moreover, plaques prevent enough blood from reaching organs and tissues.
- Plaques rapture causing thrombosis — completely blocking off the blood flow in the artery.
Once enough plaque deposits with the artery walls, they can become vulnerable to rupture. When they do rupture, their core content exits into the bloodstream to cause blood clotting. As a result, blot clots(thrombus) will grow at the point of rupture and enlarge to fill the entirety of the artery.
Parts of the blood clot can travel with the blood flow, and once they reach the narrow place in the artery — they will block the blood flow there — in a different place from where the plaque was initially.
This ultimately deprives major organs in the body, like the brain, of oxygen-rich blood and causes disease in many organs and irreversible neuron death in the brain.
Depending on where the plaques are in the body, they cause different damage.
From heart to brain, to kidneys and legs.
Plaques in the heart or arteries leading to heart cause ischemic heart disease(or CAD). Those plaques rapturing can cause a medical condition like a heart attack or heart failure. Each year, 9 million people die of CAD.
Plaques in the heart can play a role in the development of arrhythmia, and other diseases.
Plaques in arteries leading to the brain(carotid artery disease) or in the brain can cause a TIA(mini-stroke) — TIA is not dangerous and the symptoms disappear in 24 hours. Those plaques also cause ischemic stroke which is far more risky with 3 million deaths every year.
What determines how bad the stroke is?
- where in the brain it happens + how much tissue is actually damaged.
When a clot blocks an artery the tissue starts to die off in minutes as the blood is not able to reach it. Severely reduced blood flow is called ischemia. A very straightforward illustration of ischemic stroke:
The blood clot can happen in an artery that supplies a small insignificant area of the brain. In this case, the patient is likely to recover without much damage.
However, blockages do occur in the most vital parts of the brain — like the brain stem which is responsible for functions like breathing, swallowing, eye movement, facial movement and sensation, hearing, heart rate, blood pressure. Here, death or severe disabilities are the most likely outcomes.
The time that a brain area spent with no blood circulation directly affects how much damage has been done. In some cases, parts of the tissue die, and the brain area can keep on functioning(not as well as before). In other cases, whole areas die off. Often surrounding brain areas can help manage the function of a damaged area. The significance of the areas affects the patient's life/death battle, as well as further well-being.
Brain and heart atherosclerosis is the one talked about. In reality, there are much more conditions provoked by plaques.
Plaques in the arteries leading to kidneys reduce the amount of blood that kidneys get. Normally, they filter all the blood in your body more than 30 times a day but plaques can interfere with the process. In a long run, it can cause kidney failure.
Peripheral artery disease — plaques in arms and legs that prevent normal blood circulation. It can cause claudication.
PAD can make your limbs less sensitive — you won’t feel the heat and cold as well as before which raises the risk of burns and frostbite.
In severe cases, it can cause tissue death — gangrene.
Atherosclerosis can damage different parts of the body and cause disability, or death.
Stealthy atherosclerosis — it’s hard to diagnose.
It is important to note that almost everyone has plaques in their blood vessels. The size of the plaque and the location define the risk. Atherosclerosis can develop in early childhood and not cause much damage for decades. It can also develop fast and lead to severe consequences.
The problem is, it’s almost impossible to notice and diagnose atherosclerosis on time. The symptoms are very vague and can be taken for something else, or simply ignored.
⛔️You should specifically go through diagnostics for atherosclerosis — for this, you need to suspect that you might have it. Most people don’t until they’re in intense care for a heart attack or ischemic stroke.
Medications for atherosclerosis are ignoring the real problem when masking the symptoms and risk factors.
After having a medical condition occur, if a patient does survive then they are put on life-long medications. Those pills are supposed to:
- lower the chance of plaque bursting by keeping blood pressure low,
- decrease the ability of the blood to clot(blood thinners/anticoagulants) so if the plaque does burst, the blood clot(thrombus) doesn’t form,
- lower cholesterol to reduce the probability of a plaque happening in the first place(and exciting ones growing).
When those medications do reduce the risk of platelet clumping or imbalances in the bloodstream, they do not directly address plaque build up in the arteries — nor prevent it, nor get rid of exciting plaques. Therefore, they can’t guarantee high efficiency and safety for the patient’s life.
Current medications attempt to act as risk reducers for CVDs instead of solving the root cause for them — plaques.
They reduce the risk for thrombosis(vessel blockage) developing on top of atherosclerosis. They don’t treat atherosclerosis.
Moreover, the study shows that 62% of people forget to take their medication. With cardiovascular disease, it’s crucial to take your pills on time.
Patients who suffer from cardiovascular disease incur medical costs of upwards of $19000 per year in treatments. Furthermore, a common surgical procedure such as coronary bypass surgery, on average costs $40,000 per surgery. Surgery is performed in cases with severe blockages in the arteries to remove plaques.
*The costs differ around the world.
52 million years of healthy life is lost each year due to ischemic stroke-related death and disability
50% of healthy life lost due to ischemic stroke-related death and disability affects people under the age of 70 years.
Resource: World Stroke Organisation
The science behind our solution
How plaques form
Plaque buildup happens in the arteries in the innermost layer. For context, the artery is made up of 3 main layers:
- Tunica Externa — the outermost layer; consists of connective tissue, such as collagen, to protect the artery from high blood pressures.
- Tunica Media — the middle layer; made of elastic tissue and smooth muscle cells, which functions to expand or contract to control blood flow.
- Tunica Intima — the innermost layer; contains a single layer (monolayer) of endothelial cells and connective tissue with one-way valves to force blood flow in only one direction.
Encapsulated within these 3 layers is the lumen, the opening in which blood flows.
Key drivers for plaque formation
The main drivers of plaque formation in atherosclerosis include:
- Chronic inflammation of the blood vessels
- High blood pressure (hypertension)
- High levels of lipid fats, especially cholesterol, in the bloodstream (hyperlipidemia)
Along with these key factors, behaviors such as smoking and poor dieting can further contribute to the damage of the inner artery walls(endothelium). As a consequence, the damaged endothelium will experience dysfunctional adhesive molecular behavior that leads to plaque buildup.
In a healthy endothelium, there is little cellular adhesion to the singular layer of endothelial cells on the inner wall and blood flows regularly. Furthermore, the blood vessel wall is less permeable and does not release any inflammation factors. However, a damaged endothelium behaves in the opposite manner. The vessel wall becomes more permeable and allows for more molecular penetration. It also experiences an increase in the expression of its adhesion molecules, which allows for more circulating immune cells to stick to its surface.
The increased permeability allows for deposits of low-density lipoproteins (LDLs), which are primary carriers of cholesterol in the bloodstream, to form within the inner vessel walls. Monocytes that penetrate the endothelium due to increased adhesion specialize (differentiate) to become macrophages and produce reactive oxygen species (ROS) signaling molecules and chemokine signaling proteins to attract other immune cells to the site of infection or inflammation. After LDLs enter the endothelium, they lose electrons (oxidation) to become oxidized low-density lipoproteins, or oxLDLs, due to the increased production of reactive oxygen species.
The macrophages now within the endothelium will then attempt to clean up the site by ingesting the oxLDLs. Unfortunately, oxidized LDLs are more resistant to breakdown by macrophages than their non-oxidized counterparts, which causes the macrophages ingesting them to be overly burdened with lipids and become foam cells due to a lower rate of outflux of cholesterol. Once macrophage cells die due to programmed cell death (PrCD, otherwise known as apoptosis), their cellular components and lipids remain within the endothelium. This then aggregates to eventually form a necrotic core of dead cellular debris with increased apoptosis (programmed cell death) of macrophage foam cells in more advanced plaques.
In the usual case, the process of cellular cleanup (efferocytosis) allows for dead cells and other components to be disassembled by macrophages in a matter of minutes. But during atherosclerosis, the process of efferocytosis is impaired and reduced by a factor of 20. When macrophages engulf cellular debris and cholesterol oxLDLs for breakdown (phagocytosis), the influx of cholesterol more than doubles its current cholesterol levels since cholesterol also plays an essential role in the integrity of cellular membranes (in dead and live cells). Since many mammalian cells struggle with the process of cholesterol breakdown, the body has a process called reverse cholesterol transport to combat dramatic influxes in cholesterol. The reverse cholesterol transport mechanism is a multi-step process that allows the body to use high-density lipoproteins (HDLs) to transport cholesterol LDLs back to the liver for redistribution or disposal. The impairment of this assistive process greatly limits macrophages from effectively digesting its cellular membrane debris and cholesterol, which leads to foam cell formation and the inability to clean up other dead foam cells.
Due to the impairment of efferocytosis, the anti-inflammatory factors released by macrophages during successful cleanup of cellular debris do not happen. As the necrotic core within the plaque lesion continues to develop, inflammatory factors (such as cytokines) continue to get released. In response, the immune system continues sending more and more monocyte immune cells to the lesion, a mechanism that leads to the formation of more foam cells. Meanwhile, the endothelial cells will continue to grow and proliferate over the plaque to form an extracellular matrix fibrous cap. The consequences of continued development of this process includes the hardening and narrowing of the artery walls since plaque structures limit vascular smooth muscle movement.
As long as the fibrous cap over the lesion does not break, there is no immediate risk other than more restricted blood flow. However, the aggregated cell debris that builds up under the plaque contains chemicals that can attack the fibrous cap and reduce its thickness, leaving the lesion vulnerable to rupture. When the plaque lesion does rupture, a thrombus blood clot will occur at the rupture site as the body attempts to patch up the wound. Because of this thrombus, the artery becomes even more blocked, which then leads to either greatly reduced or completely restricted blood flow, which is the cause of ischemic strokes.
When first looking at the pathway of this disease, it is no doubt overwhelming and challenging to find a place to start attacking the problem. Our initial hypothesis began as an idea that we could create nanobots and deploy them into the body to clean up the plaque. However, as great of an amazing future as that can be, that is just too easy. In fact, researchers at Drexel University have created a molecular corkscrew robot that can be controlled by external magnetic fields and complex microfluidics algorithms to drill through clogged arteries. Not to discredit the fact that accomplishing something like that is incredible, but accomplishing it in a fast, scalable, and affordable way within 2 to 5 years was not in the books. With the vast majority of the victims of cardiovascular disease being in developing countries, the deployment time and costs of hardware systems and nanobots will be tremendous and slow. This forces us to go back to the drawing board.
As we attempted to devise a new solution to this behemoth of a problem, we had to establish a set of values and standards to follow. A set of high values and standards.
Safety — derived from our value in having INTEGRITY and transparency.
Nanotechnology is great. But so is its ability to intoxicate the human body if not properly designed to be biocompatible and safe. We want to make the world healthier and safer without risking the lives of those that we wish to help.
Our goal is to make a product that we would feel safe to put in our own bodies. It should be safe and effective — people should feel safe to use it. As much as we can share about our company, we will share with the public to build trust.
First Principles — derived from our value of challenging the status quo with COURAGE.
In the process of addressing the health problems related to atherosclerosis, most of the world has only tackled symptoms of the problem or created reactive solutions. As great as using nanobots to unclog arteries is, this is a reactive solution that waits until things go wrong to jump in.
We aim to create a proactive solution that solves the root problem, not the consequences. It’s bold to go against the system which already makes a ton of money on sh#t solutions. We value courage.
With our established compass, we then began the process of establishing boundaries for our solution by asking essential design questions:
- How can we make the solution safe?
- How might we make it scalable and affordable?
- How can we be proactive instead of reactive?
- Where in the disease pathway will we tackle the problem?
- How can we deliver the solution to the plaque sites in the body?
- How can we trigger the drug to work at the target site?
In an attempt to answer those design questions, we developed an initial set of corresponding hypotheses:
- Safety: our solution’s components will need to be biocompatible.
- Scalability & affordability: our solution will likely need to inhabit the form of an injection in a syringe so we can take advantage of global vaccine supply lines.
- Proactive: our solution needs to address plaque build-up before it results in blood clotting and causes conditions on top of it.
- Disease pathway: our solution will need to leverage the body’s own cleanup mechanisms since we do not want to have fancy nanobots to do the work.
- Drug delivery: our solution needs to find unique biomarkers that it can target
- Drug deployment: our solution needs to trigger based on the unique biomarkers of the plaque’s environment.
After binge reading dozens and dozens of research articles, we have finally converged on a solution as we put the puzzle pieces together. Now mind you, this was only a week so we still have a lot to learn :)
We will use mesoporous biocompatible silicon nanospheres equipped with miRNA-20a/b regulation factors that will control expression of the ABCA1 gene responsible for reverse cholesterol transport.
Our solution will be injected directly into the bloodstream for immune cells like monocytes to engulf them in the process of phagocytosis. Since immune cells often travel to inflamed sites within the body, they will take this drug with them to plaque lesion sites, which are chronically inflamed. To make sure that our solution triggers only in this environment, it will trigger only in environments that are both overly lipid-rich and have high numbers of reactive oxygen species (ROS), mimicking a similar micelle-based mechanism using a copolymer poly.
Why approach the problem with the reverse cholesterol transport mechanism?
We initially hypothesized that we could recruit the body’s cleanup cells to attack the plaque and get rid of it. Understanding the process of atherosclerosis development gave us further insight into the problem. What we did not know before was that the body was already trying to clean up the plaque — albeit very poorly. With the variety of pathways that we could leverage, we decided on reverse cholesterol transport since it is the safest and most effective. If we had tried anything else like tagging even more plaque structures and apoptotic and dead cells, we would still have the problem of weak reverse cholesterol transport, based on a study that suggested necrotic cells do not trigger reverse cholesterol transport like regular apoptotic cells. Even if we had gone after pathways such as finding a way to mark plaque with “eat-me” ligands that will draw macrophages to them for engulfment, that drug in itself presents a risk if mistriggered or if it deploys in the wrong location since it can trigger the attacking of healthy cells and tissue by immune cells.
Since cholesterol efflux from macrophage is an essential component that frees up space in the cell for continued cleanup, the inhibition of this process is incredibly detrimental. The continual build-up of cholesterol in macrophages without any enhancement of the reverse cholesterol transport cellular machinery simply leads to more and more foam cells building up, which inhibits the cleanup process. As a matter of fact, a match-pair study found that there was a “28% reduced odds of incident coronary events per SD increase in [cholesterol efflux capacity]” where the presence of “carotid plaque is a robust predictor of ischemic stroke events.” Simply put, the higher rate of cholesterol efflux from cells contributes to the reduction of risk of coronary-related incidents as cholesterol gets transported back to the liver for regulation.
Why miRNA-20a/b and ABCA1?
The ABCA1 gene is responsible for making transport proteins that move molecules across the cell membrane. This ABCA1 transport protein is found in many regions of the body, but it is especially prevalent in the liver and macrophages. In atherosclerosis, however, a study linked the effects of deficient levels of ABCA1 proteins and others to impaired cholesterol efflux, increased release of inflammatory factors, and increased cell death apoptosis rates when macrophages burdened with cholesterol or oxLDLs. Furthermore, this implies that to bring back healthy rates of cholesterol efflux for transport, we need to upregulate this gene to produce more transport proteins to keep up with the high rates of cholesterol influx.
A study connected the mechanisms of inhibiting miRNA-20 with increased expression of the ABCA1 gene. This means that when miRNA-20 was inhibited, there was an increase in cholesterol efflux from the cell, which consequently reduced cell cholesterol content to inhibit the formation of foam cells. Furthermore, this miRNA-20 is also responsible for regulating high-density lipoproteins for transporting cholesterol back to the liver and the development of atherosclerosis. In all, controlling this micro RNA can be the key to solving atherosclerosis.
Why mesoporous silicon nanospheres?
Many studies such as this one study, have so far found mesoporous nanoparticles to be safe as “it was demonstrated that none of the Fe3O4@MSN caused toxicity to the liver, kidney, and spleen tissues at the administered doses. Moreover, no immunotoxic effect was observed at the animal level.” This is especially promising as one of the greatest drawbacks of using nanomedicine is its accumulation in the body and the toxic effects that could follow.
Mesoporous nanoparticles have a porous solid structure, which means it has a lot of surface area for drug-carrying capacity. There are also several other benefits to using these nanoparticles, as reported in this study. The surfaces of these particles can be modified to react and trigger their environment. This high degree of programmability and stability has made mesoporous nanoparticles one of the most useful types of potential drug delivery vessels. Research for biodegradable mesoporous nanoparticles and shows promise to further reduce the risk of accumulated toxicity.
Why utilize phagocytosis?
The delivery of our drug will be a key factor in its scalability and cost. If we require external machinery to produce magnetic fields to deliver the particles to the desired sites, then we will run into the challenges of scaling up our solution since it requires external infrastructure. With that in mind, we hypothesized that we could perhaps use macrophages and find a way to have them deliver us to the plaque sites. Due to the fact that atherosclerosis involves increased artery permeability, swelling, and foam cell formation, these are key factors that can facilitate macrophage attraction to the site. One way that we can use macrophages to deliver our drug is through the process of engulfment by macrophages, which is called phagocytosis.
Surviving engulfment is critical because if the drug delivery vessel breaks down prematurely, then that could have potentially catastrophic consequences for the body. Fortunately, we found that mesoporous silica nanoparticles have been shown to be able to survive engulfment until they reach the delivery site in cancer experiments. With these promising results from cancer research, we can also hope to transfer and apply that mechanism for atherosclerosis treatments.
Why trigger based on ROS & lipid richness?
Once the macrophages reach their destination, we need to consider designing a trigger mechanism that is unique to the cholesterol-rich plaque sites. With this in mind, we are hypothesizing that we can use the present reactive oxygen species (ROS) combined with the lipid-rich environment to create a release mechanism. As a matter of fact, we did find a study that utilized the (ROS) mechanism for drug release, but we have to find a combination of the two together.