Abstract
When I was first asked to give this talk, I was thinking what exactly I should present: some new data, old data. I looked at my history and decided that I had been in the field of drug delivery for so many years that I could give some historical aspects. So whatever I show you here is my own perspective. I know many of you are in industry. If you disagree with me, I will be happy to talk with you about the issues. First, I will give a historical aspect of the transformation of biotechnologies even before drug delivery became a very major player in our field. Then I will talk about how drug delivery systems were viewed by people in the past. Then I will talk about some research done in my lab with adhesions and improved delivery based on nanocapsules. This will involve cancer therapy and oral delivery of proteins, which depends on particle uptake in the GI. And then I will end with the topic of tissue engineering and its relation to encapsulation of cytokines, vasculargenic factors, and recruitments of stem cells.
Many systems experience huge difficulties transitioning from research and development to product-based therapies. The pharmaceutical industry in 1945–1955 had difficulty bringing products to market for human use. The controlled-release science that I know most about had this struggle in the 1970s and 1980s, and as a matter of fact, during that time, we were always accused at conferences of developing a great delivery system, but there was no product on the market. We were accused of just developing some kind of engineering tools that will never materialize.
Alza achieved the change in 1980–1981. The development of a product in any field is not something that occurs overnight. An example is in the pharmaceutical industry between the 1930s and 1940s, after Bayer discovered Sulfanilamide in 1935. It was very difficult to patent this molecule. As a result, many companies synthesized this molecule, and because of the war, we don’t really know what the profit from this product was. Another example is penicillin, which was actually never patented. It was discovered in 1928 and was first used in clinical studies in 1941. Because of a joint U.S. and British effort, it was not brought to full use as a result of lack of patenting. But because of lack of patenting, profit was low, and profit is usually what major industries really want to see. These products have been used but had a hard time getting into the market. Cortisone is another example. It was used as a pep pill for pilots in the wartime. When the war was over, it was discovered to be effective in treating rheumatoid arthritis. This was one of the drugs that were extremely useful, and the companies were producing it, but the amount was so huge that they almost went into bankruptcy because the starting materials were very hard to synthesize.
So the point is that all of these companies are really facing a lot of struggle, and so did drug delivery. Thalidomide is another example of a drug that was never approved here in the United States, because there was one person in the FDA that refused to accept it. It was used in Europe and caused developmental damage. This is why major companies completely changed the models/structure and the way companies approved drugs. Today, pharmaceutical companies are the major health and money creators in our society. So, we actually learn a lot from the history.
So, what is a drug delivery system? The field started when we first developed collars for dogs to repel insects. What we wanted to do was use polymers and incorporate them inside drugs, any kind of drugs—low molecular weight, high molecular weight—and try to release them in a controlled condition.
In the case of the collar for the dog, the molecule size of the active material is very small, so when you incorporate it in a polymer like Siloxane, it can diffuse out and act as a delivery system. It’s more difficult when you start to think about proteins, and in the beginning, we didn’t have the tools to release proteins out of polymers.
So, people would accuse us, “Is it a science at all?” It was probably the only field of the interdisciplinary-sciences nature that involved everyone—medicine, chemists, pharmacists, and engineers. It was so interdisciplinary and so applied science that most of the elite universities really considered it a real “sin.” When I finished my postdoc at MIT, I got a position at a major institution, but the chairman called me in and said, “Edith, what are you planning to do?” and I said, “I’m going to continue to do drug delivery, it’s a new field,” and he said, “I’m not going to give you any tenure in my department. I know that you have an offer, but you won’t get any tenure, because if you take from the ‘drug delivery’ the word ‘drug,’ you don’t have any science left.” Well, I was shocked at that time, and we are talking 1988 when people really looked down at the field of drug delivery systems. They looked at it as being composed of a group of engineers creating solutions to nonexisting problems. However, the tools of creating those “nonexisting problems” first of all helped us advance the field, and today, there is a new field based on drug delivery, which is tissue engineering. And they are using all the tools that were developed in the field of drug delivery to create new scaffold.
So we learn from different generations; even though we have struggled along the way, we learned a lot to develop the next field. So, microspheres in general, or drug delivery systems—they don’t have to be microspheres—usually have a reservoir-type delivery system where you have core and a well-defined polymer around it. The core can be gas, if you want to use it for imaging, or solids for any kind of therapeutic or liquid for encapsulation. This is what we call reservoir-type delivery system. We can also have metrics-type delivery systems in which we have a continuous polymer and the drug is dispersed throughout it, just so we can understand what we are talking about here. Now, there are tons of ways in which you can make those delivery systems, but I am not going to talk about it today, because I just cannot, but there are many ways.
What will determine what method you are using is the type of molecule you want to encapsulate. This is what will determine what kind of encapsulation method you will use and what type of polymer. Alza, in 1968, was the company that saved our pride, because we didn’t have any product. This drug did not sell so well. But they were the first company that showed that they could develop a drug delivery system. They used to be a purely engineering company and they could engineer anything you wanted. So they also studied the transdermal nitroglycerine in 1981 and the transdermal scopolamine, which was actually very successful. If you look at the history of Alza, in late 1978, they had less than $1 million in the bank. And in the beginning, it was tough. The company survived because it was bought by Ciba-Geigy. They have developed some of the most exciting delivery systems that are being used even today.
The schematic here shows osmotic-type delivery systems. And I have to mention that type of delivery system, because any major company, before considering developing a major drug delivery system, will take osmotic pump and try to evaluate if the concept is useful for the drug of concern. It is a container that has one orifice. The red area is a semipermeable membrane to water: water can penetrate inside. The blue area is a salt, which creates osmotic pressure, and the green area is the drug. When water penetrates in, the drug is being pushed out in a zero-order release.
So, if I summarized the field
In 1975, Judith Folkman and Bob Langer came out with a very innovative approach at that time. They actually showed that you can release proteins from polymers. Everyone met the concept with a lot of objection. Polymer chemists knew that proteins could not diffuse through polymers because they are so large. And before that, people never even tried to do that. So, what happened? What makes this research so exciting is that they prepared a sustained of BSA and incorporated it inside hydrophobic polymer. They immersed it in water and found that a protein was coming out. Why is that? If you take cross-sections of the polymer before release occurs, you can see continuous polymer, and the protein is somehow incorporated inside it. If you immerse it in water, what you see after taking cross-section is small channels that are created inside the delivery system. And by the end of the release, one sees large pores inside this polymer matrix. So the proceeds are diffusion in pores. So if you have your hydrophobic polymer (green) and fill it with hydrophilic drug (in red here) so the first molecule to come up is the protein that is close to the aqueous solution, it will come out and create a hole, and then water will penetrate, dissolve the rest of the protein, and so on and so forth. At the beginning, we were all sure that just taking the protein and plugging it into a polymer solves the problem.
So the first effort was really toward trying to calculate the controlled-release rate, but then, to our big disappointment, we found that you have to find ways to stabilize the protein and that the polymer is not enough to stabilize the protein. So this is again a new field that basically emerged after the 1980s that we had to deal with. So in many companies now, like Alkemes and Takeda, you really have to find a way to stabilize your protein inside a polymeric solution. So, the engineers discovered how to control the rate of release. But protein chemists had to work on stabilization of the protein inside the polymer. And then we can move to a different type of polymers, polymers that are bioerodable.
Most of you are probably familiar with polylactic acid. The polymers that we use for the injectable system basically can hydrolyze in our bodies and degrade to a very small, nontoxic molecule such as lactic acid. We can control the degradation rate in such a way that you can have a delivery system that degrades in 3 weeks, 4 weeks, or a couple of months with this polymer. The polylactic acid is known to erode by what we call bulk erosion, and what it means is that if you take the polymer and put it in aqueous solution, you are going to have, at the beginning, almost no degradation. Water will penetrate the system, and only after a while, slight degradation will occur. Then a major degradation will take place, and the entire system will collapse. How will the release from such system look? If we assume that this is the microsphere loaded with drug inside, at time zero, there is no release after immersion in aqueous solution. All the molecules that are close to the surface will come out by diffusion. And then, after a certain time, the polymer does not degrade. The release is slowed down, and after a while, the polymer completely disintegrates, the rest of the drug comes out, and you usually have a second burst of release. So this is the typical release out of bioerodable polylactic glycolide. However, companies that can apply this shape actually introduce different excipients, which cause the release to be almost zero.
The LHRH that was introduced by Takeda in 1984 is basically a system in which you have a needle—this is the original system, microspheres—and a liquid that you mix together and inject into a patient. The new system looks more elegant. You have one needle, the liquid is here, the microspheres are here, and when you push the liquid through the microspheres, you can inject them. They have products now that last for 1 month, 3 months, and I think even more. This is a general picture of what they have here. Small micro size is 120 microns, and they contain LHRH with a concentration of about 10%. This is one of the most successful products, as I said, and one of the most important that came in the field of drug delivery.
In the 1980s there was a need to develop polymers that have different behavior than bulk erosion. Polyanhydrides had been developed in Japan many years earlier, but Professor Langer actually used them as drug delivery systems. They are unique in that you can make the backbone extremely hydrophobic so that the polymer will repel the water, but the bond that binds them is extremely hydrophilic and labile as a result, and there are high chances that degradation will occur only at the surface of the device. It will usually go down almost as a zero-order release. I say almost because it depends on the structure, the morphology of the polymer here. So, if this is a dense structure, you can get zero-order release and surface erosion. However, if it’s a porous structure, you will get bulk erosion. Gilford used this polymer as a brain implant. This is one of the products that came from the collaboration of MIT and Johns Hopkins. The reason why I am mentioning this polymer is because I was there when the process really developed, and it was interesting to see how we were working on synthesizing the polymer. Within the life of my postdoc, it became a real product used in patients. So this is one of the examples I am very excited to discuss.
My research at Brown University has focused, since 1991, on developing bioadhesive polymers, or adhesive delivery systems that stick to the GI. We also developed a new nano-encapsulation technique. We call it phase inversion nano-encapsulation. It is a process in which, by taking the polymer solution and a drug and inverting it into a nonsolvent, spontaneous microspheres are achieved. The sizes can be controlled extremely well only by changing the polymer concentration. This method was indeed invented for proteins, because the method does not require shearing of the protein before encapsulation. However, we also found it to be very helpful for encapsulation of genes and encapsulation of low-molecular-weight materials like paclataxol. There are many methods to make nanoparticles, I am not saying this is the only one. This is just the one I am using at Brown. In the first topic, I wanted to share with you work done with Najad Engelmes at Buffalo University. So, we encapsulated Interleukin 12 (IL12). We studied, after encapsulation, the activity, and the activity seemed to be reserved.
We did release studies over 30 days, and we had very nice release studies. Why is it so impressive? With nanoparticles, it’s very hard to get release for 30 days out of such small particles. You really have to have good control over the size of the particle and good control of the activity. And with this, what Najad did was to inject IL 12 into mice and see if this can actually reduce the growth of tumor. The interleukin 12 dramatically changed the tumor size over time. So from this point, what he is planning to do is to take, now let’s say, a model of lung cancer and inject the microspheres, and they are small, they are nanoparticles. Well, nanoparticles, in my language, are smaller than 1 micron. But as a matter of fact, I have a combination of nanoparticles up to 2 microns. So, when I say nanoparticles, I really mean the range of 1 to 2 microns. The small microspheres, 1 to 2 microns, are injected into this tumor, and because they are so small, they spread very nicely. They actually check this phenomenon. Local inflammation is created and developed systemically, the main tumor is removed, and then this immune response is supposed to treat all the metastasis. So this work is done with Engelmes, where my lab is actually making the microspheres. He studied the same thing in mice. What he did here was he induced a subcutaneous tumor (primary tumor) and then let the tumor grow and metastasize to the lung. He then removed the primary tumor and then studied how long the mice survived. And what he found was that if you don’t inject the interleukin 12, all of the mice are dead within 8 weeks, while those injected with IL 12 last for much longer. We are now all trying to get into humans with this system. We have had difficulties raising the money to do that.
Everyone prefers to take pills orally, but there are some drugs that you cannot deliver orally, like insulin. The problems with delivering some drugs orally are the size of the degradation, low permeability, and definitely low bioavailability. So, our approach is that we are going to try to encapsulate different drugs (for example, insulin) in small microspheres, 0.5 to 1 microns in size. The challenge here with nanoparticles is that first of all, you encapsulate the drug to make sure that your drug itself is small enough. If the drug is not small enough, you cannot encapsulate it in nanoparticles. In addition, we want to combine bioadhesion because bioadhesion will give us intimate contact with absorptive cells, increase residence time, increase absorption rate, and increase bioavailability. The polymer that we synthesized, or developed, supplies the bioadhesion. We have developed a method to measure bioadhesion, and we have different particles that we interact with intestinal tissue. What you see here is tensile-type measurement, and you have to trust me, you can find some polymers that are extremely adhesive. Basically, it takes a huge amount of force to detach from the tissue. And those are the polymers that we would like to use for our system here. The first study that we did was to take polystyrene microspheres. Now, polystyrenes are definitely not used for therapeutic applications but for studying the phenomenon of biodistribution.
With polystyrenes you can get at different sizes and administer to an isolate loop in the GI tract, the jejunum or the ileum. The reason why we study the jejunum is there is more surface area. We wanted to see if there is a chance to get absorption in that area. And we are going to deliver those to the isolated loop and try to see where the particles are found in cell level using TEM. In this study, we took 290 polystyrene microspheres. The wide structure is seen here, and you see only a few beads maybe penetrating through the membrane. However, if you studied more, you see a huge amount of microspheres in the cell and you see that many microspheres are located in the separation between the 2 cells. Here is another example with microspheres the size of 200 nanometers close to the tight junction of the cells. Here, we have 2 nanometers after 30 minutes. The biggest slides are at 5 minutes. After 30 minutes, you can actually see a much higher amount of microspheres inside the cells in different areas of the cells. Here is a higher magnification of 200 nanometers, and here you can see a huge amount of particles inside cells.
The studies are extremely important for us because many scientists try to deliver particles orally, but it’s very hard to detect them with TEM. Because most of us are actually working with PLGA, they degrade very quickly. So this is why we decided to take these microspheres and evaluate them this way. This is a particle size of 500 nanometers, and you can see that here, trying to get into microvillus. But you do see some of them do get inside. It is tedious work. This is another one, 500 nanometers, and again, it shows different microspheres at different stages of processing in the tissue. This is another one. You can see very nicely in the zone here. We are not sure what the mechanism is. We are studying it now. This is another slide of 1-micron microspheres, and even those are being taken up. And here is a slide where we took polyhydride, and even here, this was published in Nature and you can see a huge amount of microspheres in this particular case-encaspsulated marker. And basically, this is what we would like to use, this type of polymer, as a delivery system. So, just to summarize: We can see particles smaller than 0.5 nanometers. Traffic of particles seems to be between cells; particles can move through the cytoplasm. Larger particles were observed by light microscopy, and we carried a lot of confocal studies too, I didn’t show you, but I do have a lot of confocal studies and have published about it.
So the next idea was to identify how they are coming in. And we believe that there are four mechanisms in which the microspheres can get here. The approach we take is to quantify, first of all, the uptake in the ileum—how much is taken up by all these mechanisms. The way we are going to do that is we are going to extract the polystyrene because its nonerodable, quantify in different orders of the animal where those microspheres are, and maybe find a relationship between uptake and biodistribution. Here is the experiment: Again, we are doing isolated loop, and we introduce the microspheres in specific regions. After 1 or 5 hours, we sacrifice the animal and we process the tissue. We take the tissue, different organs, liver, lung, and spleen, whatever we can. And because polystyrene is hydrophobic, we can extract it by organic solvents and directly inject it GPC. It was clear that with 0.5 microns, we could get absorption up to 46% and 35% in the ileum. This was huge. When we went to 1 micron, the absorption was a little smaller but still quite distinct. And when you go to 2 to 5 microns, you get about 16% absorption. So we really would like to optimize this uptake, and while we know the small particles are the most effective ones, they are the ones that have the least ability to encapsulate, let’s say, insulin, for example. So we would like to see what the upper range is where we can still get good uptake and also efficient encapsulation.
We also did other studies where we administered different sizes, and we actually looked at how is the biodistribution in the body. You will see that there is high uptake, but in some cases, you can direct the microspheres to the liver. This is important for insulin because you really want to have them delivered to the liver. So you can see the smaller one will go to the liver. But can you control it from the oral route? To summarize, we believe that endocytosis is the dominant mechanism in uptake here.
Next, I am going to show my studies with insulin. The insulin studies are interesting because when we did the studies 6 years ago, we did not have the knowledge of the optimal particle size. So the particle size that you see here is 2 to 4 microns. So when you encapsulate the insulin and give it to diabetic rats, if you don’t treat them at all, what you see is that the glucose level goes up. If you give them the microspheres orally, you basically see that you can get basal lateral control of the insulin, and we believe that eventually, this could be one of the ways to treat the patient, because these microspheres were directly delivered to the stomach, so there is the dilution in the stomach and the dilution in the GI tract. The other study that I did was directly to the small intestine. We are thinking of how to develop a delivery system that will rupture in the intestine and deliver particles there.
The bioavailability of this system is only 6%. And it’s again 6% because the particle size is not optimal. We will repeat these studies now, and based on the knowledge that we learned with the mechanism, hopefully, we would get better results. The next topic is more tissue engineering. What we wanted to do here is develop a small vascular graft, and we can design a sophisticated delivery system. You can see here this is a cross-section, and these are little microspheres. But we could not really control what we were going to attract to this side. So we decided to go and do something very simple. We wanted to encapsulate VGF and GMCSF again in microspheres, and this tech seemed to be very useful for protein encapsulation. All activity is retained. And then you put those little microspheres in a tea bag, and you plant it subcutaneously in the back of a rat. When we did the studies, we found huge vascularization of the area, and then we started to analyze the cells coming to it. It was very indicative that we did have some progenitor cells, but then the question was, is it really coming from the bone marrow? To do that, we used the Rosa mouse. We destroyed all the bone marrow cells of a rat and then injected bone marrow with a marker beta galactoside so it carried a blue color. Then, we repeated the entire study with this blue marker. If you have cells coming from the bone marrow, they must be blue inside the mesh. When you put together the VGF and GMCSF, you see a huge amount of staining inside the mesh. In most drug delivery, we are trying to cure some kind of therapy; here, the microspheres are actually attracting the cells to the system. And we intend to do that in the vascular graft later on. Now, we also show here that you really need the delivery system to get the results. If you have just the drugs themselves, it doesn’t work.
So today, drug delivery systems can be found in many areas, from developing chips, osmotic pumps, liposomes, nanoparticles, and different therapies to delivery. It’s a very broad field, and what we have learned from history is that we don’t learn from history. And this is true; however, we do build a strong foundation on which we drive and excel.
Controlled release and drug delivery system is a major player in our research institute and industry today. Controlled release is the foundation of tissue engineering today.