Cardiovascular disease is one of the leading causes of deaths occurring globally. Various surgical therapies for treating such diseases are the catheter based procedures, bypass surgery, balloon angioplasty, atherectomy etc to reduce the narrowing caused by blood vessels plagues. However, these techniques have some disadvantages like uneven rough expansion and recoil of the channel causing it to become smaller after being expanded by balloon. Intra-vascular stents have evolved to overcome these short comings. Because of the stent's effectiveness, they are now used in the vast majority of surgical treatments for alleviating diminished blood flow to organs by preventing artery reclosing. They help to treat angina pectoris, myocardial infarction, atherosclerosis, blockage due to kidney stones and tumours. However, as they are composed of metals, stents have a potential disadvantage of causing hypersensitivity, thrombosis and restenosis. Hence various approaches are being explored to make them tissue compatible and avoid such adverse effects. The market for stents is expanding as safer devices are being developed. Some of these approaches are discussed in this article.
What is a stent?
A stent is a cage like tube with slots or reservoirs that is inserted into a natural duct of the body to prevent or counteract a disease-induced localized flow constriction. Their size ranges from 2-15 mm in diameter and 8-38 mm in length, depending upon the vessel size. Stents can be placed in central and peripheral arteries and veins, bile ducts, esophagus, colon, trachea or large bronchi, ureters, and urethra. Vascular stents are implanted into blood vessels with the help of a balloon catheter. They are of three types: First generation bare metal stents, second generation drug eluting stents and third generation cell seeded or living stents.
First generation bare metal stents
Bare metal stents are made up of metals like stainless steel (316), tantalum, titanium, titanium alloys (like nitinol) and transition metals (like zirconium); metal alloys like cobalt-chrome alloy, platinum-iridium alloy and metal oxides. The desirable properties of metals are biocompatibility, corrosion resistance, radial strength, elasticity and shape memory. In order to provide a biologically inert barrier between the stent surface, circulating blood and endothelial wall, a variety of stent coatings can be applied like coating of gold, heparin, carbon (diamond like), silicon carbide, etc. However, such stents have complications like hypersensitivity, scar formation, subacute thrombosis, and restenosis.
Second generation drug eluting stents
This is the first approach to counteract the adverse effects of bare metal stents. Drug eluting stents are placed into narrowed, diseased arteries and slowly release a drug to a specific site to block cell proliferation and thrombus formation. The released drug bypasses the first pass metabolism and hence no adverse effect is seen. Various types of drugs like antimitotic, anti-inflammatory, anti-coagulant, immunosupressive agents, angiogenic substance, etc are used. A biocompatible polymer coating is applied on the stent surface which acts as drug reservoir and slowly releases the drug in a controlled manner over months. The coating thickness can range from 2-8 microns. The composition of polymeric carrier influences the release profiles and elution of the drug. There are two types of polymeric carriers:
● Bioabsorbable or biodegradable polymer - poly (caprolactone), poly (L-lactate) (PLLA), poly (lactide-co-glycolide) (PLGA), fibrin, elastomers, collagen, etc.
● Biostable or non-biodegradable polymer - poly (n-butylacrylate), polyurethanes, polyvinyl acetate, etc.
The various drug release mechanisms from the stent surface are:
● Degradation of the carrier by enzymatic hydrolysis (breaking of chemical bonds), exposure to water (that causes swelling of matrix and subsequent bursting) or bulk erosion (due to haemodynamic stress) causing drug release.
● Some carriers are permeable and release the drug by simple diffusion against concentration gradient.
● A stent can be bilayered a drug loaded and a drug free protective layer on top of it to provide unidirectional drug delivery and prevent washout of drug by body fluids.
Drug eluting stents are manufactured by preparing a polymer-drug mixture in suitable solvents and applied to the surfaces of the stent by any technique such as air suspension, dip coating, spray coating, brush coating and plasma polymerization. The solvent is allowed to evaporate, leaving a polymer film containing the drug. The drug can also be encapsulated in the mixture as a micro bead or micro particle.
There are marketed products of drug eluting stents like Sirolimus Cypher stent by Cordis Corporation and Johnson & Johnson and Paclitaxel Infinnium stent by Sahajanand Medical Technologies Pvt Ltd.
Third generation cell seeded or living stent
This is another approach in stent technology wherein stents are coated with a layer of stem cells or any other cells of the body. They have angiogenic effects and antiapoptotic effects and prevent the defense mechanism of the body from kicking in against the stent. Types of stem cells mainly seeded on stents are mesenchymal stem cells, which differentiate into fibroblasts, myocytes and adipocytes forming connective tissue and endothelial progenitor cells (EPCs), which differentiate into endothelial cells forming blood vessel lining.
The types of living stents are: Seeded stents, Tissue wrapped stents and Genetically modified stents.
Seeded stents
This involves coating the surface of stent with a biofunctional coating composition which comprises of two types of peptides:
● Surface-binding domain (SBD) - It binds to the surface of stent made of metal or polymer. They include peptides of 7-60 amino acids.
● Endothelial-binding domain (EBD) - It binds to endothelial cells or EPC’s.
A chemical group can be added to the N-terminal or C-terminal of amino acid of a synthetic peptide to reduce susceptibility by proteinase digestion in the biological fluids and prolong the half-life of peptides. N-terminal groups are lower alkanoyl, acetyl, acyl or sulphonyl groups. C-terminal groups are an ester or amide group.
The two domains are coupled together in following ways:
● Side chain to side chain bond with N-terminal and C-terminal.
● Synthetic or recombinant expression in which a peptide of an SBD can be coupled directly to a peptide of an EBD by synthesizing or expressing both peptides as a single peptide.
● Use of linkers which acts as a molecular bridge to couple at least two different molecules. Linkers are short peptides between 3 and 15 amino acids like polylysines, polycysteines, polyglutamic acid or polymers like polyethylene glycol, collagen, etc. A linker may vary in length and composition for optimizing properties like preservation of biological function, stability and resistance to certain chemical or temperature parameters.
Coating stabilization methods:
● By using an elastomeric polymer along with biofunctional coating which adds stability to the stent when it is subjected to mechanical forces.
● Matrix which supports endothelialization can be added to the coated surface. Components of such a matrix can include collagen, vitrogen, laminin, fibronectin, glycans and growth factors supporting endothelial cell growth.
Techniques of seeding
In vitro seeding: It involves attaching the endothelial cells prior to implantation of the device. A solution or suspension of biocompatible coating composition is applied on stents by spraying or immersing and allowed to remain in contact for 15-60 minutes at 25-35 degree Celsius. The EPCs are attached to the coated stents by similar methods and incubated. The stents are sterilized by autoclaving at 121 degree Celsius for 20 minutes. Seeded EPCs forms confluent monolayers onto the stent surface. After few days of implantation, EPCs migrates from the stent struts, proliferates and endothelialize the luminal surfaces of the vascular medial tissue. Besides that, even growth factors like Vascular Endothelial Growth Factor (VEGF), Basic Fibroblast Growth Factor (BFGF), stem cell growth factor, can be seeded on the openings of the stent. Stem cells are then delivered to the blood vessels containing the stents by intravenous or intracoronary injections. The presence of growth factor on the stent allows adhesion and proliferation of stem cells on stents.
In vivo or auto seeding: When the stent is implanted, the endothelial cells or progenitors adhere to the surface of the stent coated with biofunctional coating composition. An example of such stent is anti CD34 coated stents.
CD34 molecule is a cluster of differentiation molecule. It is a cell surface glycoprotein and functions as a cell-cell adhesion factor. Cells expressing CD34 are normally found in the umbilical cord, bone marrow, EPC’s and endothelial cells of blood vessels. A bare metal surface of stent is coated with immobilized CD34 antibodies forming an immuno-affinity surface. These antibodies attract the antigens on endothelial progenitor cells circulating in blood stream and cause them to attach to stent at a much faster rate. Once captured, EPCs flatten out and mature into endothelial cells; forming a functional nascent endothelial layer over the stent. The Genous Bio-engineered R stent, by Orbus Neich Company, was the first stent of its kind featuring this technology. . The process of EPC capture is shown in figure 1.
Figure 1: Capture of endothelial progenitor cells on the stents
Method of manufacture
The stent surface is initially primed to prepare a functionalized surface and applied with a thin biocompatible molecular coating layer. This coating increases the number of antigen binding site and create a favourable environment for the endothelial progenitor cells. An aqueous solution of antibodies is applied on stent. It is then dried and sterilized by gamma radiation so that the antibodies are covalently bonded to the surface.
Tissue wrapped stent
Stents can also be made by a tissue engineering method. This method involves culturing a population of cells in a tissue culture container in the presence of one or more tissue control rods. A tissue control rod is used to secure a growing tissue sheet to the culture container, to prevent spontaneous detachment and facilitate sheet handling. Conditions are maintained to allow the formation of a robust tissue sheet containing living cells and extracellular matrix. The formed tissue is removed from the culture container and rolled one or more times to form a tubular stent like structure or can also be wrapped around the stent. The stents are sterilized by gamma radiation. The extracellular matrix offer a complex extracellular environment to the cells (similar to a physiological tissue environment), and forms a three dimensional organization for the tissue. The tissue control rods are secured using magnets placed on the outside of a culture container and then left in place till a tissue sheet envelops them and reaches a desired thickness (50-200 µm). They can be peeled off a culture substrate with regular tweezers.
Genetically modified stents
These stents are coated with genes that code for proteins which inhibit proliferation or inflammation of arterial walls. They can also be coated with transformed cells (preferably EPCs) containing such genes. The genes encode therapeutic proteins like:
● Proteins which prevents cell proliferation-endostatin, angiostatin, inhibitor of VEGF, inhibitor of BFGF.
● Antisense oligonucleotides
● Anti-restenotic or anti-thrombogenic agent - nitric oxide synthatase, tissue plasminogen activator, heparin, anti-inflammatory agents.
Strategy of designing
Transfection refers to the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer. It involves opening transient pores in cell plasma membrane to allow the uptake of genetic material in the form of super coiled plasmid DNA. The schematic representation of transfection is depicted in figure 2.
Figure 2: Diagrammatic representation of transfection
A polymer coating (1-2 microns thick) that adheres to the stent incorporates the DNA or transformed cells. It facilitates DNA delivery and transfection of cells within the injured vessel wall. The intimate contact between the DNA containing the therapeutic transgene within the stent coating and vessel wall cells leads to efficient incorporation of the DNA into these cells. Once the cell is transfected with the gene, it codes for the therapeutic protein.
The coating also acts as a support for the binding of collagen to the stent. The collagen matrix facilitates two important features:
● It provides efficient contact between DNA and cells on that part of the vessel in which the stent is deployed.
● It will provide a “DNA/collagen barrier” to cells migrating from tunica media or tunica adventitia on their way to form the expanding neointima. These cells will transiently reside in the barrier and get transfected.
Transfection method
● DEAE-dextran-mediated transfection: use of cationic polymers such as DEAE-dextran or polyethylenimine as stent coatings. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.
● Liposome mediated transfection (lipofection): involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. A liposome is a spherical vesicle composed of phospolipid and cholesterol bilayer membrane around a core material.
Conclusion
Although much research has been done in various labs, more studies related to the properties of these implants need to be executed for the stents to be successful in the clinical trials, for their final approvals in the markets. It is also envisaged that ultimately the development of stents that are biocompatible in that they avoid immune stimulation will lead to an expansion in the clinical roles of this technology. Researches made on cell seeded stents coated with endothelial cells or its progenitors are a very challenging stage in the development of stents. Tissue engineering techniques can also be utilized. Stents are also genetically modified to deliver genes to the luminal cells responsible for inhibiting translation of inflammatory proteins. These developments are still under research and clinical phases. Research is directed not just towards providing an effective therapy, but to also provide a cost effective therapy. In conclusion, these latest technologies in stent development will eliminate the surgical and drug related adverse effects for cardiovascular disorders and provide a better and safer therapy to such patients.
(The authors are with Department of Pharmaceutical Sciences and Technology, ICT, Mumbai)