Encapsulation: A Novel Approach in Curing Atherosclerosis

Tunaidi Ansari, Jun Hong Introduction Atherosclerosis is the leading cause of coronary heart disease and stroke and is caused by an accumulation of plaque in the arteries. Nearly 4.6 million people in the United States suffer from this disease with a fatality rate of almost 15,000 deaths per year [2] [12]. Atherosclerosis describes the reduction of blood flow within the arteries through a gradual process, in which deposits of fatty substances, cholesterol, cellular waste products, calcium, and other substances build up on their inner linings [1]. Narrowing due to this plaque buildup not only substantially reduces blood flow, but engenders damaging consequences as well. When plaque becomes frail and ruptures, blood clots form causing additional blockage to blood flow or migrating to other parts of the body [6]. Depending on where stenosis, or unnatural narrowing, of the arteries occurs or where these residues travel, there will be different negative scenarios. Blockage of blood flow to the heart will result in heart attacks; blockage of blood flow to the brain will result in strokes; and blockage of blood flow to the arms or legs will result in difficulties in walking and mobility [7] [8] [10]. The most detrimental and prevailing substances found in plaque are oxidized low density lipoproteins (LDL), commonly termed as bad cholesterol, which initiates and promotes the stenosis in a complicated process. Oxidized LDL damages the arterial lining, causing an immune response by white blood cells (WBC) in an attempt to absorb the LDL. The WBCs inability to effectively remove the threat causes the LDL-WBC complex to grow and rupture, and subsequently signaling more WBCs to conglomerate. This vicious cycle repeats until the artery undergoes stenosis due to the increasing layers of cells that grow over the affected area [11]. Currently, there are various methods of atherosclerosis treatments. These include both medication and surgical procedures. Medication usually involves daily ingestion of one of the following or a mixture of the following: drugs that lower LDL; drugs that increase high density lipoprotein (HDL) concentration, the good cholesterol; anti-platelet drugs that reduce the likelihood of platelets clotting the arteries; anticoagulants that help in thinning blood, preventing blood clots; or drugs that control the blood pressure in order to slow the accumulation of plaque. One of the more invasive treatments includes angioplasty, in which a long thin tube, called a catheter, is inserted into the affected artery accommodating both a wire and a deflated balloon, which will be inflated to widen the artery so that a mesh tube, or stent, can be permanently placed there to keep the artery open. Other treatments are endarterectomy, in which plaque is surgically removed from the narrowed arteries; thrombolytic therapy, in which a clot-dissolving drug is inserted into the affected artery; and bypass surgery, in which an artificial vessel is created in order for blood to flow around the narrowed artery [5].

The purpose of our research is to develop a novel approach at curing atherosclerosis for an enhanced long term effect that surpasses the benefits of currently administered methods and treats the source of the problem, oxidized LDL. Methods Initially, we will genetically modify arterial epithelial cells so that they will secrete lipoprotein lipase, an enzyme with the ability to degrade LDL in clogged arteries. This will be accomplished via the recombinant cell method [4]. In fabricating the capsule, we will employ the etching technique, in which a strong acid is used to remove the exposed metal. Holes will be created in the thin polysilicone membrane by etching a thermally grown oxide layer with chlorine plasma [4] (Fig. 1-a). Then, thermal oxidation of the subsequent membrane in dry oxygen will induce the growth of pore sacrificial oxide on the exposed area (Fig. 1-b). The thickness of this layer will determine the size of the pores through which nutrients, metabolites, and enzymes pass and, thus, the diffusion rate of those molecules [4]. The size of these pores, however, will be miniscule enough to prevent the encapsulated cells from contact with immune components of the patient. We will deposit a second silicone layer over this membrane and remove the section above the entrance of the pores (Fig. 1-c, d). The bath in KOH will release the processed membrane from the bulk silicone, and the subsequent treatment with HF will remove the pore sacrificial oxide layer [4] (Fig. 1-d). We will insert a polymethylmethacrylate (PMMA) spacer, where the recombinant cell suspension will be loaded, in between the two membranes so that the portion of the PMMA spacer outside of the overlaid area can help in anchoring the encapsulated device on the arterial wall (Fig. 2). That part of the PMMA will be covered with epithelial cell-targeting ligands so that the cells of the tissue in contact with the device can attach to the polymer and grow over that area, providing a mechanical support for the device to stay fixed at the appropriate location. The outside portion of the PMMA will also contain adhesive molecules for the epithelial cells and their extracellular matrix, promoting the initial device fixation after surgery. The dimensions of our device will be 2 mm x 3 mm x 2 mm, including the PMMA insert thickness. The lipoprotein lipase will diffuse through the membrane in a controlled manner and will be able to act on the accumulated LDL. We will implant the cell-containing device (Fig. 3) into the arteries using a catheter, which will be inserted along an artery of the arm or leg until it reaches the intended location. This will be monitored via fluoroscopy. The surgeon would insert an intravenous line into veins or arteries, where dye or contrast substance, which provides a more detailed inner body structure, would be injected [9]. A continuous x-ray is then passed through the patient, in which a special x-ray scanner detects the x-rays running through the body and transmits the corresponding signals to a monitor [9]. This real-time imaging with x-ray video will be able to detect the position of the catheter carrying the encapsulated device. Once the position is determined, the mechanical micro-robotic arm at the tip of the catheter will place the capsules onto the sides of the artery.

Figure 1. Above are the schematic pictures illustrating the etching procedure: (a) etching of holes in structural silicon layer; (b)growth of sacrificial oxide by dry oxidation; (c) deposition of second structural silicon layer; and (d) planarization of second silicon layer and removal of sacrificial oxide and protective nitride layers [4].

Figure 2. Above are the schematic views of the device with the PMMA between two polysilicone membranes. The grey material is the polysilicone material, while the white one is the PMMA [4].

Figure 3. Above is the schematic design of our cell-encapsulating device [4]. Experiments First, we will create an in vitro flow system (Fig. 4) and place an encapsulation device on a thin glass side, upon which the normal epithelial cells similar to the ones found in the artery tissue will be cultured (Fig. 5). Then, we will measure the concentration and rate of enzyme secretion by taking a sample solution from the system. The flow system will have a circulating cell culture medium with appropriate nutrients and metabolites such as L-glutamine, serum, and sodium pyruvate essential for the cells survival. To control this rate of enzyme secretion, we can adjust the pore size of the polysilicone membrane or the concentration of the cell suspension to be loaded inside the device. Using this in vitro system with LDL inside, we can also measure the rate of degradation of LDL with different concentration of enzyme-secreting cells in the device and different densities of LDL by performing several assays, determining the LDL concentration in a sample solution taken from the system. Thus, we will be able to select the optimum concentration and the secretion rate of lipase for the desired degradation of LDL, while ensuring that the cells surrounding the device are still viable. For stability testing, we will place the cell-containing device in the in vitro flow system and measure the change in size of the device or any signs of deterioration on its surface at different times. The encapsulated recombinant cells viability will also be tested by opening the device that has been in the flow system for some time. For toxicity testing, we will use the same in vitro flow system method with arterial epithelial cells and observe the cell viability over certain periods of time. As implied previously, we can improve the cell viability by adjusting the concentration or the secretion rate of lipase from the device. It is also possible to perform our proposed experiments inside a mouses artery to observe the stability and toxicity effect of the device by inspecting the device inside the mouse and examining the tissue surrounding the device some time after implantation.

Figure 4. Above is the schematic layout of the in vitro system: (a) motor circulating the fluid inside the system; (b) assembled plate. Arrows indicate the flow direction of media.

Figure 5. Above is the schematic design of the simulated encapsulation device: (a) top metal holder; (b) top thin glass layer; (c) rubber spacer; (d) bottom thin glass layer with cultured cells (represented by red dots); (e) bottom metal holder. There will be openings where fluid can flow in and out of the plate at both lateral ends of the assembled plate. Discussion Our proposed design is theoretically feasible and testable. It incorporates many advantages into its system and improves upon the disadvantages of previous methods of atherosclerosis cures. Since our goal is to focus on the long term benefits of a one-time surgical procedure, our encapsulation technique outperforms currently known medical treatments, and any downfall that can be associated with it is duly compensated with its effectiveness.

We do not consider drugs to be an efficient solution to atherosclerosis because they must be repeatedly taken in order to produce their desired effect. Neither do we deem temporary surgical applications, such as endarterectomy and thrombolytic therapy, useful in the same regard. Thus, we aim to compare the encapsulation technique with angioplasty, the solution proven to be most effective on the long term. A thorough analysis of angioplasty leads us to believe several preconceptions: atherosclerosis may reoccur, where plaque may continue to build up over the stents and rupture even larger clumps when the time comes; reoccurrence of stenosis requires invasive surgery to be performed again, requiring the cleanup of plaque and the replacement of stents; and angioplasty does not solve the root of the problem, which involves LDL breakdown. On the contrary, the encapsulation technique is a one-time procedure, where secreted enzymes continually degrade LDL after the enzymes diffuse with a highly precise mechanism across the silicon membrane. The effect is long term and permanent until atherosclerosis is fully cured and the root of the problem is destroyed. In addition, our proposed system offers high bioavailability and sustainability. Silicone is chemically stable and inert and has been previously used as a biomaterial in implants. The nutrient-nourished encapsulated environment has a high longevity with research proving that cells can secrete certain proteins and enzymes for several months [3] [4]. Taking patient compliance into consideration, it is far more acceptable to undergo surgical procedures just once rather than risking a second or third attempt. Thus, it is in the patients best interest to favor our design over angioplasty. Though this product will be difficult to manufacture, our treatment protocol embodies a wealth of advantages that substantially outweigh any foreseeable disadvantages. The approval of this innovation will not only provide an alternative cure for atherosclerosis, but may also be used in future medical applications. A precise control on enzyme release via encapsulation will certainly prove to be useful for various diseases and conditions. Whether the enzymes will be used to degrade unwanted pathogens or to catalyze the synthesis of desired molecules, our novel design has the potential to influence a large spectrum of applications. References [1] American Heart Association. Atherosclerosis. http://www.americanheart.org/presenter.jhtml?identifier=4440 [2] eMedicine. Atherosclerosis. http://emedicine.medscape.com/article/150916-overview [3] G. Voskericiana, M. S. Shivea, R. S. Shawgoc, H. von Recumd, J. M. Andersona, M. J. Cimac, R. Langere. Biocompatibility and biofouling of MEMS drug delivery devices. Biomaterials. 24 (2003) 1959-1967. [4] L. Leoni, T. A. Desai. Micromachined biocapsules for cell-based sensing and delivery. Advanced Drug Delivery Reviews. 56 (2004) 211-229.

[5] MayoClinic. Atherosclerosis. http://www.mayoclinic.com/health/arteriosclerosisatherosclerosis/DS00525/DSECTION=treatments-and-drugs [6] Medline Plus. Atherosclerosis. http://www.nlm.nih.gov/medlineplus/ency/article/000171.htm [7] Merck. Atherosclerosis. http://www.merck.com/mmhe/sec03/ch032/ch032a.html [8] National Heart Lung and Blood Institute. What is Atherosclerosis? http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html [9] University of Maryland. Radiology. http://www.umm.edu/radiology/fluroscopy.htm [10] US Against Athero. About Atherosclerosis. http://www.usagainstathero.com/aboutatherosclerosis/index.aspx?source=332&WT.mc_id=332&WT.srch=1 [11] W. Youichiro, S. Akira, et al. Lipid Accumulation in Smooth Muscle Cells Under LDL Loading Is Independent of LDL Receptor Pathway and Enhanced by Hypoxic Conditions. Arteriosclerosis, Thrombosis, and Vascular Biology. 22 (2002) 1712-1719. [12] Wrong Diagnosis. Statistics about Atherosclerosis. http://www.wrongdiagnosis.com/a/atherosclerosis/stats.htm