Designing the Future of Medicine: an Artificial Red Blood Cell

Trying out an a new article style — let me know what you think by emailing me at aahaanmaini@gmail.com

My Proposal: Artificial Red Blood Cell

More specifically, polyethylene glycol-polylactic acid copolymer nanocapsule encapsulating polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase, Apabetalone, the chemical inhibitor of CD47-SIRPα, Cyclodextrin-statin, Aliskiren and Midodrine. The nanocapsule’s surface is configured with polyethylene glycol, oxLDL, tissue plasminogen activator, various antibodies, bradykinin receptors and renin receptors.

TLDR

As requested by several readers, here’s a quick outline of the purpose of each of the aspects mentioned above.

PEG-PLA Membrane: Avoids detection by immune system and increases circulation time in the body.

PolyHb-SOD-CAT-CA: More efficient transport of gasses, while displaying antioxidant properties.

Apabetalone: Drug involved in treatment of atherosclerosis — increases HDL for increasing reverse cholesterol transport

Chemical Inhibitor of CD47-SIRPa: Drug involved in treatment of atherosclerosis — deactivates signal that evades phagocyte detection

Cyclodextrin-Statin: Drug involved in treatment of atherosclerosis — tackles each stage of the process

Aliskiren: Drug used in the treatment of hypertension post-diagnosis

Midodrine: Drug used in the treatment of hypotension post-diagnosis

Aptamer: Synthetic antibodies used to flag and remove harmful pathogens from the bloodstream

oxLDL: Protein used to target plaques in the bloodstream by targeting LOX-1 receptors

Tissue Plasminogen Activator: Protein used to dissolve blood clots in the bloodstream

Renin receptor: Receptor used to diagnosis hypertension through signalling vasoconstriction

Bradykinin receptor: Receptor used to diagnosis hypotension through signalling vasodilation

Principles

Primarily, this was designed as an enhanced artificial red blood cell, meaning it would perform all the regular functions of the red blood cell, but also include capabilities for the proactive diagnosis and treatment of various diseases and viral/bacterial infections.

The nanocapsule was intended for use in healthy individuals to optimize the functions of red blood cells, as well as post-diagnosis in individuals who suffer from specific diseases. Though there is a multitude of possibilities for the latter option, it starts to enter the field of targeted drug delivery, hence this proposal has a higher emphasis on the former.

In healthy individuals, the nanoparticle will efficiently perform the required functions and serve as a “superhuman” version of a traditional red blood cell but it is equipped with mechanisms to diagnose important diseases from the bloodstream itself. This will then trigger the treatment of the disease, often through the release of a certain drug, while still at an early stage, eliminating the need for medical intervention.

Polylactic acid nanocapsules can also be configured for pre-mediated release of drugs and depending on the specific disorder, doctors can encapsulate certain drugs into the nanocapsule to be released after a certain amount of time. I want to reiterate that while this is a possibility, this is not the primary focus as there are other nanoparticles intended for use in targeted drug delivery.

I’ll start by explaining how the nanocapsule performs the traditional functions of a red blood cell (RBC), and then move into the customizations for the proactive diagnosis and treatment.

Quick Information

Important information that doesn’t require a full explanation

Size of the nanocapsule: 80–120 nm, included membrane size is 5–15nm

Life cycle inside the body: <29 hours — explained in much more detail down in the PEG: Evading the RES section

Energy generation: N/A — passive nanostructure

Administration: Intravenously — required to be located in the bloodstream

Concentration: Requires lab testing which has not been done because this is a proprietary proposal

PolyHb-SOD-CAT-CA — Oxygen and Carbon Dioxide Transport + Antioxidant Mechanism

As I am sure you’re aware, the primary function of red blood cells is the delivery of oxygen from the lungs to all the cells around the body, and the reverse function of delivering carbon dioxide.

Source

Traditional RBCs utilize hemoglobin for this delivery. Near the lungs, oxygen binds with hemoglobin to form oxyhemoglobin. Each hemoglobin molecule contains 4 atoms of iron, which can therefore bind 4 molecules of oxygen.

Let’s say the RBC reaches a tissue cell in the body, carbon dioxide diffuses into the RBC. Two possibilities may occur, but I am going to be focusing on the one relating to carbon dioxide binding. Carbon dioxide reacts with the oxyhemoglobin, which displaces the oxygen and produces Carbaminohemoglobn (hemoglobin bound with carbon dioxide) plus hydrogen ions. The oxygen diffuses into the cell and the hydrogen ion enters a different process which also results in oxygen being displaced and diffusing into the cell.

Visualization of the interactions of O₂ and CO₂ with a traditional tissue cell. Source

Instead of using regular hemoglobin, polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (PolyHb-SOD-CAT-CA) has been tested to be more efficient at transporting oxygen and carbon dioxide. Apart from that, PolyHb-SOD-CAT-CA has been shown to express antioxidant properties, similar to the innate antioxidant mechanism of traditional RBCs.

Polyhemoglobin (PolyHb) is formed by the crosslinking of hemoglobin, automatically making it more efficient than traditional singular hemoglobin particles. The incorporation of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) can remove harmful oxygen radicals plus stabilize the crosslinked hemoglobin.

In terms of safety, all of the included molecules are naturally found in the body. On top of that, PolyHb-SOD-CAT-CA has been tested in rats and showed no toxicity and no side effects after up to 30 days.

Polyethylene Glycol — Evading the RES

The reticuloendothelial system (RES) also known as the mononuclear phagocyte system, is a subsection of the immune system to remove dead or abnormal cells, tissues and foreign substances, especially nanoparticles. According to Gref et al., Unprotected nanoparticles, of all different compositions, can be removed by the RES within seconds.

The process by which the removal occurs is called opsonization. Opsonin is a circulating protein that absorbs onto the surface of the nanoparticle, flagging it for phagocytes to engulf and eventually remove it.

Process of Opsonization. Opsonin (blue), Nanoparticle (Red) and Phagocyte (Yellow). Source

Avoiding detection by the RES has arguably been the most difficult challenge for the implementation of nanoparticles in the body.

Polyethylene glycol (PEG) is widely considered the best approach for the development of “stealth” coatings to evade detection. The addition of PEG polymer chains to the surface of nanoparticles in a process known as PEGylation has been proven to increase circulation time.

PEG exhibits several other desirable properties of high hydrophilicity and biocompatibility. PEG has even been FDA-approved for use in humans, which is ultimately what drove me to utilize this compared to its alternatives.

Example of a PEG-coated gold nanoparticle. Source

The primary mechanism, as described by Jeon et al., is that the PEG polymer chain develops a flexible surface barrier layer that prevents adhesion of opsonin to the nanoparticle surface. The secondary mechanism is that hydrophobic particles are more easily opsonized, as supported by Carrstensen et al. and Müller et al.

Fam et al. described several coating parameters that are vital for developing long-circulating PEG-coated nanoparticles. Firstly, the molecular weight of the grafted PEG chains, in other words, the thickness of the layer. PEG molecular weight of at least 2kDa has been generally accepted, however, Gref et al. concluded that protein absorption significantly decreased when the molecular weight was increased from 2 kDa to 5 kDa, but no further reduction. Secondly, the smaller the size the more optimal, as concluded by Fang et al. and Cui et al.

Exclusively using PEG for a coating on nanoparticles upgraded the circulation half time from 0.55h (unprotected) up to 19.5 hours, as reported by Perry et al.

For this proposal, a polyethylene glycol-polylactic acid copolymer membrane was developed, which can increase the circulation half-life up to 42.5 hours.

Circulation half-life is a metric used when the concentration of an external substance reaches in the bloodstream reaches 50%. This is used to determine the circulation time of an average nanoparticle since 50% represents the average time of removal. For example, if the circulation half-life of a nanoparticle is 10 hours, that means it takes 10 hours to reach 50% of the initial concentration of nanoparticles in the bloodstream, which makes sense because 10 hours is the average time — certain nanoparticles may get removed at 1 hour or 19 hours.

Polyethylene Glycol — Polylactic Acid Copolymer Membrane

Polylactic acid (PLA) polymers are the copolymers used in the development of the nanocapsule, due to their high biocompatibility, low toxicity and approval by the FDA for medical implantation.

PLA is a biodegrade polymer that dissolves into lactic acid, which then breaks down into carbon dioxide and water.

In two studies conducted by Liu et Chang available here and here, PLA showed no effects in the livers, spleens and kidneys of rats in up to 3 weeks.

Chang, 2012 developed a PEG-PLA membrane artificial red blood cell with a circulation half-life of 29 hours in rats, which is 42.5 hours when extrapolated in humans.

For the development of the membrane, a 17:1 ratio of molecular glutaraldehyde (cross-linker) to hemoglobin for the formation of PolyHb, used a 1.5:1 ratio of PLA to PEG and crosslinked the nanoparticles after production to increase stability, all of which resulted in the increased circulation time.

Membrane encapsulation is one of the three primary methods for internal enzymes to interact with external substances while avoiding protein sensitization, anaphylactic reaction, or antibody production.

Proactive Diagnosis and Treatment of Atherosclerosis

Finally, time to get down to the meat of the article. Similar to any proactive medicine approach, a continuous diagnosis mechanism is required. In our case, a dual mechanism of using oxLDL to target LOX-1 receptors and incorporating interleukin-10, both on the surface of the nanoparticle, is used for the diagnosis.

Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) is the primary receptor for oxLDL, present on macrophages and endothelial cells and smooth muscle cells. Plaques are combinations of damaged endothelial cells, cholesterol-filled macrophages and more cholesterol. LOX-1 presence is enhanced in areas of inflammation inside the skin (macrophages plus endothelial cells), therefore they are a perfect target for the diagnosis of atherosclerosis plaques. As mentioned, the method of targeting LOX-1 is by incorporating oxLDL, which will lead the nanoparticle to the site of the plaque, if it occurs. This mechanism has been proved successful by Dayuan et al. who concluded that LOX-1 can be used as a target for atherosclerosis plaque in vivo.

Once an atherosclerosis plaque has been confirmed, the nanoparticle approaches the site via the mechanisms explained. If there is a complete blockage of the artery, there will likely be a clot of red blood cells preventing access to the plaque. Blood clots are formed when fibrin is polymerized and it along with platelets forms a plug to restrict blood flow. The nanoparticle will then release the tissue plasminogen activator, a protein responsible for the conversion of plasminogen to plasmin, a natural substance that dissolves fibrin in blood clots.

Now the blood clot is mostly dissolved and the ~100nm size of the nanocapsule enables its access to the plaque buildup. In case a blockage of red blood cells isn’t there (due to a variety of reasons), the tissue plasminogen activator doesn’t interact with anything until it reaches the plaque. Once a plaque gets to a certain stage, a fibrous “cap” is formed to prevent the blood from directly interacting with the cholesterol and foam cells. In that case, the tissue plasminogen activator may interact with it and speed up the process of dissolving the first layer of the plaque.

Source

One out of the three mechanisms of drug release that are activated by the oxLDL-LOX-1 reaction is the release of Apabetalone. Apabetalone triggers increased expression of ApoA-1, which leads to increased efficacy of HDL and increased efficiency of reverse cholesterol transport. Let’s slow down and take it step by step.

Similar to most other malfunctions in the human body, it has a way to treat it, but it doesn’t do a good enough job. Reverse cholesterol transport is the body’s mechanism of removing excess cholesterol from any area. The process works through a particle named HDL, which engulfs cholesterol particles from foam cells in the plaque and transports them to the liver for excretion. ApoA1 is the primary protein in HDL, making up over 70% of its volume and generating its anti-atherosclerotic properties.

Oversimplified diagram of reverse cholesterol transport. Cholesterol (blue), HDL (green). Source

Tying it back to Apabetalone, the drug works by increasing the production of ApoA1, which increases the amount of HDL, which can therefore transport more liver from plaques to the liver. Bailey et al. tested the effects of Apabetalone (RVX-208) on ApoA1 and HDL in vivo and concluded that it was an effective treatment. It also follows our strict safety policy as the drug is currently in stage III clinical trials.

The second released drug mechanism is all about the CD47-SIRPa complex. CD47 is an antibody that when configured with SIRPa, forms a protein signal that evades phagocyte detection. It is commonly found on cancer cells and in this case, is used by [where cd47 is found]. Inhibiting the expression of this protein allows phagocytes to elicit an immune response against the plaque. The chemical inhibitor of CD47-SIRPa is the second drug that is released by the oxLDL-LOX-1 plaque diagnosis.

The final drug release removes plaque from the blood vessel by tackling each of the root causes, Cyclokinded-Statin.

Regulating Blood Pressure

Unhealthy blood pressure consists of hypertension (too high) or hypotension (too low).

Hypertension is caused by vasoconstriction of the vessels, which decreases the diameter and increases pressure to unhealthy levels. Hypotension is caused by vasodilation of the blood vessels, which increases the diameter and decreases the pressure.

Proactive diagnosis of this is a very difficult process since the modulation of the vessel is an autonomous function that isn’t driven by a few key biomarkers.

Blood vessels don’t just constrict randomly, but rather based on a chemical signal, which can be harnessed as the biomarker for diagnosis. In the case of vasoconstriction, the protein Renin. Extrapolating this to hypertension, if a large amount of Renin is detected, we can assume an increase in blood pressure. Renin receptors are configured onto the surface of the artificial red blood cell, and only when all of them are activated the drug release is activated.

The specific number of receptors is determined by the doctor, according to the patient’s blood pressure. If someone has higher blood pressure, fewer receptors are required because even less vasoconstriction will result in unhealthy levels. Whereas if someone has regular blood pressure, more receptors are required to be activated because if only a portion of them is activated it may still be in the healthy range.

Aliskiren is an FDA-approved drug to lower blood pressure, so instead of overcomplicating the solution, a simple controlled release of this drug when all the Renin receptors are activated will regulate hypertension.

Now the opposite.

Applying the same principle as hypertension, the protein Bradykinin signals the vasodilation of the vessel. Bradykinin receptors are configured that only release the drug if they are all activated and the specific amount depends on the patient’s blood pressure.

Midodrine is the FDA-approved drug to increase blood pressure, therefore regulating hypotension.

Diagnosis to Treatment of Infections

Ah, the simplest of all the functions of this artificial cell: infection treatment through aptamers (artificial antibodies).

Bacteria and viruses have specific antigens that once inside the body, the immune system develops antibodies against, to prevent the same infection from harming your body twice.

Source

These antibodies bind to antigens on the surface of the invasive pathogen, signalling phagocytes to engulf and remove it. Thus, configuring antibodies against common infections can prevent the patient from contracting them.

That is exactly what is being done, but enhanced. Aptamers are strands of DNA or RNA that are configured to bind exclusively to a molecule, peptide or proteins like antibodies, that can then elicit an immune response to remove the target. In other words, they act as synthetic antibodies that can bind to specific identifiers of infection-and-disease-causing cells.

Aptamers were used over traditional antibodies because they exhibit higher binding affinity, they can bind to any target and the scalability of their production.

Solving Blood Incompatibility

Blood types are one of the major issues with traditional blood transfusions, however, they can be eliminated when red blood cells are engineered.

A blood types are red blood cells with the A antigen and B antibodies. In contrast, B blood types have red blood cells with the B antigen and A antibodies.

AB blood types are red blood cells with both A and B antigens and no antibodies. O blood types are red blood cells with no antigens but both A and B antibodies.

Blood types are incompatible when the host cell has an antibody for the foreign antigen — this causes an immune response and rejection of the blood. The same blood type can exchange freely with each other.

Therefore, A and B types cannot exchange blood, AB type can revive from anyone (no antibodies), labelling them as universal receivers. O type can give to anyone (no antigens) but can only receive from other O blood, labelling them universal donors.

Another important aspect is the Rh factor, which can be either positive, to represent having the Rh antigen or negative, to represent not having the antigen.

To remove all the issues of blood compatibility, we can engineer the cells to have no antigens and without the, mimicking the O blood type and acting as a universal donor. The solution is that simple.

Nanocapsule Production

Red blood cells are by far the most abundant cell in the body, accounting for over 80% of all cells, therefore the production of artificial red blood cells needs to be scalable and efficient.

The artificial cell is produced following the general method of production of nanocapsules: emulsion solvent evaporation.

Diagram of each step of emulsion. Source

The process outline:

  1. The drugs and polymers are dissolved in a solvent
  2. The polymers evenly disperse and form copolymers — undergo emulsification
  3. The copolymers surround the dissolved drugs
  4. The solvent evaporates and the mixture forms into a nanocapsule
Another representation of the process of emulsion solvent evaporation. Source

In this case, the 5 drugs along with the Polyethylene glycol polymers and polylactic acid polymers are dissolved in ethyl acetate, and then the rest of the procedure is carried out.

Final Thoughts

I loved working on this project and designing the artificial cell because it is leveraging nanotech to tackle such an important problem to treat diseases in the present, but also pioneer a future of proactive diagnosis and treatment in the body. While there are several logistical roadblocks to making this proposal a large-scale reality, I definitely want to continue to work to develop a prototype.

Thanks for Reading!

Feel free to connect with me on LinkedIn if you enjoyed this article, or email me at aahaanmaini@gmail.com if you have any questions/want to discuss more! If you want to stay up to date on all of the cool things I am doing, subscribe to my monthly newsletter!

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Aahaan Maini

Aahaan Maini

16-year-old ML dev currently building Circulate to tackle the blood shortage in India