Engineering A Polymeric Nanoparticle for Precise and Targeted Drug Delivery
One day I was walking back home with one of my family members from our local health clinic, we had just picked up a prescription to treat their diabetes called Metformin, a drug commonly used to treat diabetes in people when diet and exercise have unfortunately not been enough to control blood sugar levels.
We were really hungry, so when we got back home, we immediately ate, this was perfect since the medicine is required to be taken with food which I thought was extremely interesting and at the same time although I did not understand this at all!
A few weeks had passed and all was well, however, one night they began to experience extreme diarrhoea, loss of their appetite, and extreme pain in their liver and abdominal areas.
The issue was the medicine. You see, prescription drugs work to tackle areas in your body, in the case of metformin it reduces the amount of sugar your liver excretes into the body and allows your body to react better to insulin. However, prescription drugs often have the ability to also harm parts of the body completely separate from the issue, which is where side effects come in as the medicine not only targets the area of the problem but other parts as well. Although these drugs are given written instructions on what to do to decrease the severity of side effects (in the case of metformin the variable of food works to solve this), they still take place in smaller amounts and begin to accumulate.
So, when we went back to the doctor one day, she told us this was a normal side effect and that there is not really anything you or we can do about it, just try to eat a little less of the medicine, take one tablet instead of two.
Hearing this I was quite baffled and all the more intrigued, on one hand eating the medicine is causing further problems in one’s body and on the other hand to slow this down helps but means putting your body at the risk of high blood sugar levels!
We have been relying on prescription drugs for many years now, and although they save countless lives each year, their efficacy is becoming iffier and iffier.
There is a solution though through the use of nanotechnology!
Nanotechnology
Before delving into how we can better target weaker parts of our body, we need to first learn about the vast universe of nanotechnology.
Nanotechnology: the study of our ability to essentially make things happen at an extremely small scale.
How small you ask? Well, smaller than how we measure specimens in a microscope. That’s right, smaller than a micrometre, and to put it into better perspective a nanometre is 1 billionth of a metre, and it is 100 000 times smaller than a strand of human hair. 🤯
Nanotechnology comes with changes in the physical and chemical properties of materials by simply changing their size and shape. How this works is extremely interesting as you can break up gold known for its shiny yellow colour and see it become purple and or red in colour at such small measurements!
This is because at this scale all normal on the surface is washed away. The conductivity, hardness, reactivity, etc. are all changed when the surface area to volume ratio changes!
It is because of this that nanotechnology has many uses and applications in vast areas such as healthcare and energy.
Going back to our problem of precision medicine, nanotechnology is rising in this area with the ability to solve this issue with drumroll please… 🥁 drug-eluting nanoparticles!
Introducing Drug-Eluting Nanoparticles (DEN’s): Our Body’s Tiny Surgeons
Drug-Eluting NPs, are engineered technologies that can be synthesized from more biological methods such as with amino acids, as well as with materials including gold, and palladium, or a mixture of both! Drug delivery systems with DENs allow for a controlled and maintainable release of drugs and therapeutics to specific areas in the body, resulting in less severe and frequent side effects, as well as the quantity of dosage, putting less strain on areas of your body such as your liver.
DEN’s can be characterized into three categories.
The first category or class of a DEN is: inorganic. These consist of silica NP’s, quantum dots, iron oxide NP’s, and gold NP’s, which have magnetic, electrical, and optical characteristics that can effectively be used for thermal medicines, imaging applications in vitro, which essentially means that this imaging is for procedures conducted outside of an organism’s common biological environment.
The second type is lipid-based NP’s. This category consists of liposomes, liquid NP’s, and emulsion NP’s. Lipid-based NP’s have a liquid centre that is surrounded by two layers of fat cells put into two thin “sheets” this helps to protect what is inside, so that the inside can organically be released into the body as the lipid or fat layer is ionizable, meaning it can dissolve in the appropriate conditions within the body!
Last but not least, we have polymeric nanoparticles. The subsets of this class are polymersomes, dendrimers, and polymer micelles. Polymeric NP’s in simple terms can be thought of as a combination between lipid-based and inorganic NP’s as they are made with both natural and artificial substances. This allows those polymeric, to get the best of both world’s, allowing them to be so effective in drug delivery, which is why today we are going to be learning how they work, what these NP’s do exactly, and how they are engineered! Let’s get into it!
This is an example of a polymeric nanoparticle — but only just one type. Polymeric nanoparticles (PNP’s) can come in many different structures with unique characteristics as they can again be made from artificial and natural materials as well as monomers and preformed polymers, and so we have a variety to choose from!
From PNPs, the most commonly used can be broken down into two groups: nanocapsules, and nanospheres. These two groups are then broken down further to produce dendrimers, polymersomes, and polymer micelles.
Each has their own benefits and unique characteristics, however, today we are going to specifically be looking at dendrimers a subclass of nanocapsules. A nanocapsule can range in size from 10 nm (nanometres) to 100 nm. Generally, they can be made up of a liquid or solid core, and are surrounded by a membrane made up of natural or artificial polymers.
Part 1: Introduction of a Dendrimer Nanocapsule
Dendrimers are described as hyperbranched macromolecules (HMs) and this means that they have a perfectly controlled and defined structure. Dendrimers are also constructed with careful steps called a layer-by-layer construction, making them very different from other branched polymers.
Dendrimers a subclass of nanocapsules can most be recognized for their branch-like features it is for this reason that they have a complex three-dimensional structure, that resembles that of the dendrites on neurons in our brains!
It is because of this unique shape made using a hyperbranched polymer that the shape itself, the surface potential for eluting drugs, and weight can easily be manipulated making them extremely efficient for precision medicine.
With dendrimers, their exterior, as well as their interior, allows for effective use. The exterior of a dendrimer can be used for allowing substances to sit on the surface and interact with the surface, in addition to this, drugs can effectively be loaded into the interior and they also have the ability to deliver and hold a wide variety of drugs!
Part 2: Structure & Function of a Dendrimer Nanocapsule
A Dendrimer has 3 most important and basic layers to its structure. The method we will be going through is the nanoconstruct approach, where the dendrimer is covalently built.
- The CORE
- The Dendrons (Subunits)
- The Surface Ligands (Endgroups)
The CORE
The core of the dendrimer can be a group of bonded atoms or one central atom. This core is multifunctional as this is where the drug is actually loaded, and also allows for the growth of the dendrimer simulated through the dendrons, but we’ll get more into this later!
For now, it’s imperative we learn the actual structure of the core and its function. Since the dendrimer is a type of nanocapsule it consists of an inner and outer shell. The inner shell consists of the core, and the internal drug cavities, while the outer shell includes the endgroups and dendrons.
To start off with the core reagent, this part is extremely reactive as it needs to facilitate other reactions that will form and make the foundation for the other layers of the dendrimer. We can use the example of the radical NH2. NH2 is an amino radical, and it was once Ammonia (NH3), however, due to chemical interactions Ammonia can lose one hydrogen atom, causing the compound to lose 1 electron, making it a radical. We know that atoms like to be stable, they become stable by gaining or losing electrons for a full outer shell, and so when an atom is not stable it will do anything in its power to gain electrons or lose!
This is where our core reagent comes in NH2 or the Aminyl Radical with its high reactivity comes into contact with CO2H or Carboxylic Acid. When a radical such as NH2 comes into contact with CO2H, something very particular happens because CO2H is a functional carboxy group, it consists of groups of carbonyl (C=O) which means the carbon is double-bonded to an oxygen atom, with a hydroxyl group (H-O) bonded to the carbonyl. Now, when the aminyl radical is near a carbonyl it will form an amide. An amide singlehandedly controls the entire foundation as it is essentially a strong bond between a carbonyl and a nitrogen group, otherwise known as a peptide bond.
Being the entire foundation of the core is not easy, however, this amide bond is one of the most common, and important chemical bonds, this gives them a really large advantage to come together with other molecules for effective chemical, molecular, and biological sensing and targeting. This conjugation is carried out through amid linkage. When amid linkage takes place the amino acid (NH2) links with the carboxyl group! The linkage is initiated through a condensation reaction, so the carbonyl and anime will link with each other and release water, so our core is wet! With these changes what also ends up happening is the formation of a polar hydrophilic amino acid.
Our body is an aqueous environment, and so a dendrimer works perfectly in this sense as when a polar amino acid, which is simply an amino acid with an OH or NH2 group is in an aqueous environment it is able to conjugate with other groups to make hydrogen bonds. These types of amino acids have lipophilic properties and have the tendency to stick to the outside of proteins as sidechains. So, these lipophilic amino acids attached to our proteins form a sort of circle. This also makes the core love to accumulate lipids and does not want water molecules to come in, but lipids, and it is also important to remember our core does consist of reactions that release H2O.
We have seen quite a prominence of hydrogen in this, and this is because hydrogen bonds are the strongest bonds you can have on a molecular level. Since a hydrogen atom only has one electron, it has much less negative influence, this makes the bonds stronger, as hydrogen is a weak atom, allowing for a stronger atom to bond to it easily.
So, now we have the core settled, but there is another integral part that the core facilitates. It is because of the amino acids layer we discussed that a drug can be added because it is protected and held in until the time comes to release it by a circle of lipid-like structures! These are known as internal cavities.
It’s important to remember that dendrimers as they are formed through chemical reactions and smaller than the width and length of a strand of hair are essentially self-constructing especially in the nanoconstruct approach, this is why the loading of the drug is not directly facilitated, but rather through the hydrophilic and lipophilic properties of certain lipids.
We want these NPs to go through cells, however, there is one problem! Cells have a semi-permeable membrane meaning, they only allow certain things to go in, and even if they allow certain substances to go in, every substance varies on how much time it takes to go through the membrane. This is why drugs for nanoparticles are designed as lipid-based.
Lipids are used as they improve absorption, meaning in a cell they can diffuse the fastest to attack the infected areas.
Now, we know our drug is lipid-based, and our core is protected by amino acids that have lipophilic properties, which means they like lipids and want to bring them closer to them! If this was not already so efficient our core is also wet, which may seem that is can damage the drug, but the types of lipids, particularly for NPs, are called membrane lipids. These lipids hence the name are the groups of lipids that make the protective lipid bilayer of our cells. You may be wondering that usually, things are either hydrophilic or lipophilic, but with lipids this is possible because lipids are amphiphilic, meaning that they have one region that is soluble and attracted in lipid-like areas, and another region that is soluble and attracted to water. These lipids can then come in and bind to each part perfectly!
Finally, our drug is in, and the inner shell is complete! It’s now important we move on to the second layer: the sub-branching units. Since, if this dendrimer was really forming, this would be happening right now! If you didn’t get that no worries, all the more reason to keep on reading! So, let us get into…
The DENDRONS
Dendrons the second basic layer in the dendrimer can be thought of as essentially having the same structure as branches on a tree, or dendrites on the neurons in our brain! There is one catch though, these dendrons are almost completely identical allowing for effective drug delivery as each branch is easily in control and facilitated by the initiator core!
This is where the concrete construction of the dendrimer begins, in an effort to not get into the synthesis too much I will say that through chemical reactions these branching layers are added over and over again, with more on each new layer called a generation. The result: dendrimers can actually be beautiful works of art, just look at the one below!
But aside from the beauty, these dendrons play an important role in the drug delivery of the dendrimer.
The dendrons have a focal point where a single reactive function exists, and terminal groups where the dendron “chain” essentially ends, or else our dendrons would grow to extremely large lengths.
In the image we can see the focal point, an area of the dendrons located extremely close to the core, this area is extremely reactive and allows the branching units to form, the G1, G2, and G3, each stand for a new layer (Generation 1, 2, and 3) and you can see that with each new layer the size doubles! What is actually doubling is the number of amino acids and thus more proteins!
As the dendrons branch into new larger layers, they make globular proteins, that have hydrophilic and lipophilic regions. What is key about globular proteins is that they fold in such a way that the hydrophilic region of the proteins is faced outwards, and the hydrophobic inwards, this keeps the core protected from exterior water, but attracts lipids that are likened to water.
The beginning of a protein starts when an N-terminus is formed, this is the start of the protein chain and it describes a loose anime group that is, a group that contains nitrogen and is a radical of Ammonia (NH3), which we saw in our core as (NH2), this again happens because one hydrogen atom has been replaced with a different group such as Akyl. Akyl can be groups of unsaturated hydrocarbons such as methane, and ethane, and the concept of this is intentionally a little vague to make room for multiple different possibilities of substitutes.
The terminal is the part of the amino acid sequence called a C-Terminus, where a loose carboxyl group will come and essentially terminate the bond so that it stops growing. This is because again, the peptide chain of the amino acid continues to grow as each amino acid has a carboxyl and an amine group, so the amino acids link to each other and form this chain by taking the amine group of one amino acid and it binds to the carboxyl group of another, and releases water, so the end is then simply a carboxyl group that is not bound to an amine group, this then stops the cascading reactions in the dendrons. This is also because there are only so many ways in which these atoms can keep growing, as eventually there will be no more substances to interact with unless, of course, interference takes place.
These reactions are imperative to the functionality of the dendrimer, although these NPs are already on an extremely small scale, the NP needs a large enough surface area on the nanoscale to effectively entrap and deliver the drug within the body to cover more area means to work 10x better!
Of course, though, the core and dendrons would be irrelevant if what is on the absolute surface did not correctly correlate to all of these amazing processes. So, let’s get into the last but not least surface layer of a dendrimer!
The SURFACE LIGANDS
The surface ligands are also known as the peripheral groups or endgroups in the dendrimer are all responsible for the recognition, detection, and delivery.
The highlighted green parts are actually peripheral molecules, but these molecules are not on the particles during synthase but are formed in the body once the NP is inserted.
Peripheral molecules are proteins that are formed from the amino acids of the dendrons, when they interact with the lipid bilayer of a cell membrane.
The peripheral proteins then become bounded to the cell temporarily, however, do not enter it. You may be asking then how does an NP actually detect if it is targeting an infected cell? Well, it is similar to how we might dip a donut into powdered sugar. The donut will collect some of the powdered sugar, and so now the powdered sugar is a part of the donut. In a similar way, the peripheral molecules are able to interact with an infected cell in essence and their powdered sugar are proteins.
Since proteins are the basis of cells most stay, but those that leave take waste and unnecessary fluids from the cell, so these proteins’ amino acids will then interact with those in the dendrimer, but since, they are coming from a diseased cell they are flawed. The peripherals instead of recognizing them will begin to deteriorate as they will try to form bonds. Each generation of dendrons then begins to as well, causing the drug from the centre to leave, and releasing the drug into the infected cell, through the hydrophobic tails, as it is likened to lipids and not water, causing the drug to make its way into the cell!
Part 3: How Dendrimer Nanocapsules are Synthesized
Method Engineering
In the synthase of dendrimers, there are two methods by which the dendrimer is formed.
- Convergent — In the convergent method of synthesis, the dendrons are made first, it is the dendrons then that grow inward and form the core.
- Divergent — In the divergent method the synthesis is initiated by the core, and the dendrimer grows outwards. This is the method we discussed in!
Physical Engineering
Now that we’ve talked method, let’s get into the actual physical engineering of these drug-eluting polymeric nanocapsule dendrimers! The method we are going to specifically be looking into is with a microfluidizer!
Step 1*
We need to review the materials required to make them. As a reminder, we discussed dendrimers, and although the machine does not specifically develop dendrimers, it does go through the overarching groups dendrimers are in polymeric nanoparticles, which as we can see in our dendrimers consist of the following materials:
- A Solvent
- A Polymer
- H2O
- A Sulfactant
This is because our dendrimers as we saw with our hydrophilic and hydrophobic properties consist of two vital stages: an oil (lipid) and a water phase. We saw this with our dendrimers as we had active processes with both lipids and water!
The water phase consists of the surfactant which can and is most often PVA (Polyvinyl Alcohol). The oil phase then consists of the solvent and the polymer. The solvent is water-immiscible meaning it will not mix with water, as for example oil and water do not mix. However, the solvent in this case we can use solvents such as dichloromethane or ethyl acetate. Polymers can consist of most commonly PLGA ( polylactic co-glycolic acid). Intentionally within the oil phase other materials are added as well but not specified as this is what can allow a dendrimer to form a polymersome or a polymer micelle! These materials can be contrasting agents: substances that are used to enhance the area of target cells, by altering the way radiation and sound waves make their way through the body. They can also be other types of activating substances, this depends on again the type you are attempting to make, but since we focused on dendrimers, contrasting agents are what we are looking for!
It is important to note that water phases can be described at 95% of the particle, and oil the rest, as we’ve seen with the structure and function this is what makes our particles have biocompatible characteristics and help for solubility.
As an example, if we were using PLGA and Dichloromethane in our oil phase, we would be putting the PLGA into the dichloromethane with precise measurements. And for our water phase, we would put PVA into DI (deionized water). Then the mixing of these two would result in one oil phase being in nineteen parts of water, and the formulation of our NPs.
Step 2*
To start the process, PVA is put into a beaker and is dissolved into deionized water, the water during this time is heated to approximately 80 degrees celsius. Once the PVA becomes dissolved the water in the beaker will return to a transparent state. This is being facilitated on a hot plate with a magnetic stirring bar that works by attraction to the metal hot plate, allowing the mixture to be mixed without interference. The mixture is then allowed to rest so that it can cool back down to room temperature (20 degrees celsius).
Step 3*
Next, in the microfluidizer, the black compartment with the large spring is called a cooling tray. Here ice is actually placed in so that the dichloromethane does not evaporate. From here, the machine is turned on and prepared for use.
Step 4*
The oil phase is prepared by taking the PLGA and dissolving it into the dichloromethane. It is then taken and a small measured bit of it is added into the water phase, with again fewer parts per water.
Step 5*
The mixture is put into the LM 10 reservoir, which is the silver tube attached at the top, that resembles a siphon gun. Then the little silver straw at the bottom left corner of the machine is what pumps out the prepared mixture. After this step, the mixture is added into large jars, where they are left open so that the solvent can evaporate.
Here is an example of an end product!
We’ve now learned all about dendrimer nanocapsules, their structure and function, and how we can synthesize them! So, let’s go make them! You can check out my video here, where I used Blender to make a 3D animation of the application of a dendrimer nanocapsule!
Thank you so much for tuning in!
