Prodigiosin Encapsulated Poly Lactide-CO-Glycolide (PLGA)-Coated Stent for Coronary Cardiovascular Interventions

Prodigiosin Encapsulated Poly Lactide-CO-Glycolide (PLGA)-Coated Stent for Coronary Cardiovascular Interventions

ABSTRACT

This research focuses on the design of a robust but flexible prodigiosin eluting stent coating for possible coronary cardiovascular implant. When coated with the drug embedded polymer matrix, the stent would be expected to dilate the vessel around the fatty blockages while the drug eluting polymer membrane delivers anti-proliferative drugs over a period of time to prevent the restenosis that otherwise would occur. The goal of this work is to incorporate anti-cancerous drug prodigiosin in the PLGA polymer matrix and then ascertain its release kinetics. In this research, Poly vinyl Pyrrolidone was used as a binder and cross-linker to create adhesion between the metallic stent strut and the drug encapsulated polymer matrix as well as between the polymer and the drug. This work also explores diffusion and degradation phenomena to explain the transport, dissemination, dispersion and absorption of drugs at the interface between the stent and the vessel wall. The expected results will then be discussed for potential applications via the incorporation of these prodigiosin-eluting stents for the treatment of coronary cardiovascular diseases.

Keywords: stent, degradable stent coating, controlled release, coronary disease, poly(lactide-co-glycolide), Poly (vinyl pyrolidone), prodigiosin, diffusion and degradation mechanism.

TABLE OF CONTENTS

ABSTRACT …………………………………………………………………………………………………………………… ii
DEDICATION ………………………………………………………………………………………………………………. iii
Table of Contents ……………………………………………………………………………………………………………. v
List of Figures ……………………………………………………………………………………………………………… viii
CHAPTER ONE …………………………………………………………………………………………………………….. 1
1.0 BACKGROUND AND INTRODUCTION ……………………………………………………………… 1
1.1 STATEMENT OF THE PROBLEM ……………………………………………………………………. 2
1.2 CORONARY ARTERY DISEASE (ATHEROSCLEROSIS) …………………………………. 3
1.2.1 Mechanism of Atherosclerosis ………………………………………………………………………. 4
1.2.2 Treatment Trends ………………………………………………………………………………………… 9
1.3 UNRESOLVED ISSUES ………………………………………………………………………………….. 10
1.4 SCOPE OF WORK ………………………………………………………………………………………….. 14
References ……………………………………………………………………………………………………………………. 14
CHAPTER TWO ………………………………………………………………………………………………………….. 17
2.0 LITERATURE REVIEW …………………………………………………………………………………….. 17
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2.1 DRUG ELUTING STENTS (DESs) …………………………………………………………………… 17
2.1.1 First Generation Drug Eluting Stents ……………………………………………………………. 18
2.4 COATING OF STENTS …………………………………………………………………………………… 26
2.4.2 Spraying of Stent……………………………………………………………………………………….. 28
2.4.3 Ultrasonic Nozzles for Stent Coating …………………………………………………………… 30
2.4.4 Electronanospray……………………………………………………………………………………….. 32
2.5 CONTROL DELIVERY OF DRUG…………………………………………………………………… 34
2.5.1 Drug Release Mechanisms ………………………………………………………………………….. 36
2.5.2 Factors Influencing Drug Release ………………………………………………………………… 38
2.5.3 Drug Delivery from stents ………………………………………………………………………….. 38
2.6 DRUG-POLYMER ADHESION ……………………………………………………………………. 45
2.6.1 Polymer-Stent Adhesion …………………………………………………………………………….. 47
References ………………………………………………………………………………………………………………… 48
CHAPTER THREE ………………………………………………………………………………………………………. 56
3.0 METHODOLOGY ………………………………………………………………………………………………….. 56
3.1 MATERIALS AND METHOD ……………………………………………………………………………… 56
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COATING OF STENTS …………………………………………………………………………………………….. 57
3.2 PREPARATION OF PBS SOLUTION ………………………………………………………………. 58
3.3 PURIFICATION OF PRODIGIOSIN ………………………………………………………………… 59
3.4 COATING METHODS…………………………………………………………………………………….. 59
References ………………………………………………………………………………………………………………… 60
CHAPTER FOUR …………………………………………………………………………………………………………. 61
4.0 DISCUSSION OF RESULTS………………………………………………………………………………. 61
4.1 PROSCOPE ANALYSIS ………………………………………………………………………………….. 61
4.2 PHASES OF DRUG RELEASE ………………………………………………………………………… 64
4.4 Gravimetric Analysis………………………………………………………………………………………… 71
4.3 UV-VIS SPECTROPHOTOMETRY …………………………………………………………………. 72
References ……………………………………………………………………………………………. 73
CHAPTER FIVE ………………………………………………………………………………………………………….. 75
5.0 CONCLUSION AND FUTURE WORK ……………………………………………………………….. 75
5.1 CONCLUSION ……………………………………………………………………………………………….. 75
5.2 FUTURE WORK …………………………………………………………………………………………….. 76
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5.2.1 NEW STENT COATING TECHNOLOGY ………………………………………………….. 77

CHAPTER ONE

1.0 BACKGROUND AND INTRODUCTION

Cardiovascular disease is currently the leading cause of death in Africa and the world at large, accounting for at least 30% of all death. One of the major causes of cardiovascular disease is arteriosclerosis. It is as a consequence of the build-up of fatty cells and tissues within blood vessels. In most cases, this built-up is not detected until complete blockage which prevents the flow of blood and subsequently leads to heart attack, stroke or death. The objective is to develop prodigiosin embedded polymer-coated cardiovascular stents that will successfully deliver drugs and bioactive agents to blood vessels and treat coronary cardiovascular diseases while at the same time preventing restenosis-the narrowing of the blood vessel after angioplastic procedure. The polymer earmarked for these biodegradable coating is poly lactic-co-glycolic acid (PLGA) because of it biocompatibility and high rate of biodegradation. Over the past decade, drug eluting stents have been used to treat arteriosclerosis. Essentially, the stent dilate the vessel around the fatty blockages while the drug eluting coatings deliver anti-cancer drugs to prevent the restenosis that otherwise would occur. However, the stents remain permanently in the blood vessels after the duration of drug elution. Furthermore, vessel irritation, endothelial dysfunction, vessel hypersensitivity and chronic inflammation at the site of implantation are critical parameters that have attracted serious attention. There is therefore a need for multi-component, multifunctional materials for novel cardiovascular stents whereby, one important function should be degradability of the polymer so that after degradation, a functional vessel wall is regenerated [1].

There is also an equal need for reasonable stent coatings that can degrade gradually and be absorbed slowly by the body without creating the afore-mentioned adverse side effects.

1.1 STATEMENT OF THE PROBLEM

Cardiovascular disease (CVD) is a result of the disorder of the heart and blood vessels. Atherosclerosis, the main cause of coronary artery disease (CAD), is an inflammatory disease in which immune mechanisms interact with metabolic risk factors to initiate, propagate and activate lesions in the arterial wall [2]. About two decades ago, it was widely expected that the treatment of hypercholesterolemia and hypertension would eliminate CADs by the end of the 20th century but this has not been the case. CVDs include coronary heart disease (heart attacks), cerebrovascular disease (stroke), high blood pressure (hypertension), peripheral artery disease, rheumatic heart disease, congenital heart disease, and heart failures [3]. The first two are the foremost causes of death in Africa and the world over. In 2008, about 17.3 million deaths were as a result of cardiovascular diseases. These represented 30% of the global death toll in 2008. Out of this figure, 7.3 million were due to coronary artery disease (Atherosclerosis), 6.2 million were due to stroke and 5.8 million were jointly caused by hypertension, high blood pressure, diabetes, and heart failure. Moreover, Coronary Artery Disease (CAD) is the principal cause of death in both males and females in the low-income and high-income countries. There is, therefore, an urgent need for improved approaches for the treatment of CAD in both developed developing and underdeveloped countries. People in low and middle-income countries are usually more exposed to risk factors of CVDs and do not often benefit from prevention programs as do people in high-income countries. As a result, over 80% of all deaths due to CVDs occur in low and middle-income countries. This huge statistical disproportion in low and middle income countries is also due to the lack of effective and affordable health care services including early detection and treatment of the disease. This leads to the early death of people from the disease in these regions, often in their most productive years. It has been estimated that the number of people who die of CVDs will increase to 23.3 million by 2030 and is likely to remain the largest killer of people [4].

1.2 CORONARY ARTERY DISEASE (ATHEROSCLEROSIS)

Coronary artery disease simply refers to the buildup of fatty cells in the coronary artery. As a consequence, the regular flow of blood is retarded due to the narrowing of the arterial vessel. This accumulated reduction in blood flow eventually lead to ischemia and myocardial infraction-conditions that result from the damage of the heart tissues. Any arteries in the body, including arteries in the heart, brain, arms, legs, and pelvis, can be clotted by plaques. As a result, different diseases may develop based on which arteries are affected. Factors such as high blood pressure, high cholesterol levels, and low fatty acid metabolism precede the onset of coronary artery disease. Essentially, the most famous approach available for treating atherosclerosis is the insertion of a stent in the arterial wall which then prop opens the vessel to allow the flow of blood. Current drug-eluting stents are achieved by combining different materials: metals for mechanical strength, polymer coating for hemocompability, and drug to be release for prevention of restenosis, the recurrence of the narrowing of the blood vessel (e.g., by ingrowth of smooth muscle cells into the arterial wall leading to restricted blood flow) [1].

1.2.1 Mechanism of Atherosclerosis

Coronary arteries are hollow tube-like blood vessels through which oxygen-rich blood flows to the heart muscles. The muscular walls of the coronary arteries are normally smooth and elastic. Lining the walls are layers of cells called the endothelium. The endothelium provides a physical barrier (protective layer) between the blood stream and the coronary artery walls, while regulating the function of the artery by releasing chemical signals in response to various stimuli.

Figure 1.1 Structural arrangement of the coronary artery

A fully developed artery should consist of three morphologically distinct layers. The intima, the innermost layer, is bounded by a monolayer of endothelial cells on the luminal side and a sheet of elastic fibers, the internal elastic lamina, on the peripheral side. The normal intima is a very thin region (size exaggerated in this figure) and consists of extracellular connective tissue matrix, primarily proteoglycans and collagen. The media, the middle layer, consists of smooth muscle cells (SMCs). The adventitia, the outer layer, consists of connective tissues with interspersed fibroblasts and SMCs [5]. The pathogenesis of coronary artery disease (Atherosclerosis) is associated with the buildup or accumulation of cholesterol (low-density lipoprotein), fatty cells, calcium and other blood nutrients on the walls of coronary artery. Atherosclerosis starts when the endothelium becomes damaged, allowing low-density lipoprotein cholesterol to accumulate in the artery wall. The body instructs macrophage white blood cells to clean up the cholesterol. In the event of doing so, they sometimes get stuck at the affected site. Over time this results in plaque built-up, consisting of bad cholesterol (LDL cholesterol) and macrophage white blood cells [6]. The early stage of Atherosclerosis; Fatty streak, which is common in infants and young children, is a pure inflammatory lesion consisting of monocytes-derived macrophages and T- lymphocytes [7] (Figure 1.2 (a) [6]. As one advances in age, the fat keeps building up and damages the protective layer of the wall; the endothelium. This is termed endothelial dysfunction. Consequently, some of the fat, calcium and other nutrients within the blood escape in to the smooth muscles (Figure 1.2(b)) and affects the dilation and contraction of the smooth muscles which controls the flow of blood within the coronary artery. Over time, the inside of the arteries develop plaques of different sizes (c). Several of the plaque deposits are soft on the inside with a hard fibrous “cap” covering the outside. If the hard surface cracks or tears, the soft, fatty inside is exposed (d). Platelets (disc-shaped particles in the blood that patronize clotting) come to the area, and blood clots form around the plaque (e-f). The endothelium can also become irritated and fail to function properly, causing the muscular artery to squeeze at inappropriate times. This may cause the artery to narrow even more. Sometimes, the blood clot breaks apart, and blood supply is restored. In other cases, the blood clot (coronary thrombus (g)) may swiftly block the blood supply to the heart muscle (coronary occlusion (h)), causing one of three serious conditions called acute coronary syndromes [8].

(a) Normal artery (b) Fatty streak

(c) Stable plaque growth (d) Unstable plaque growth

(e) Rupture of plaque (f) Thrombosis

(g) Thrombosis grows (h) Occlusion

Figure 1.2 Progression of Atherosclerotic plaque

1.2.2 Treatment Trends

A number of treatment options are available for the treatment of coronary Heart disease. They include but are not limited to angioplasty, bypass surgery, and medication. Why not a coronary bypass surgery or just angioplasty? A coronary bypass surgery is usually done by creating an alternative pathway through which the heart muscles can be fed with blood nutrients and other essentials. However, a lot more time will be required for wound healing than with the case of stent insertion. Moreover, there could be a recurrence of the narrowing of the vessel after a few months. A mechanically robust, copolymer stent that releases anti-cancerous drug will therefore, more often than not, prevent thrombosis and restenosis after stent insertion. The first of such procedure for treating the disease was performed by Dr. Andreas Gruentzig on 14th September 1977 in Zurich, Switzerland on a 38 year old patient [9, 11]. The procedure was called Percutaneous Transluminal Coronary Angioplasty (PTCA).

Transluminal Coronary Angioplasty (PTCA) otherwise known as balloon procedure [5]. Furthermore, the establishment of a comprehensive knowledge and understanding that atherosclerosis is an inflammatory disease, offers novel opportunities for prevention and treatment of CAD. The use of efficient immunosuppressant or anti-inflammatory agents would serve to provide attractive treatments for acute coronary syndromes [2]. Cyclosporine, sirolimus and paclitaxel are just a few of the immunosuppressive drugs that inhabit the activation of T cells. At relatively high concentrations, they also prevent the proliferation of smooth-muscles cells by inhabiting intimal lesion. Previous generations of drug eluting stents have employed the use of sirolimus and paclitaxel polymers coatings for the prevention of restenosis after angioplasty [2].

1.3 UNRESOLVED ISSUES

There has been a considerable shift from the use of bare metal stent to the implantation of first and second generation stent primarily because of the early restenosis associated with its implantation. Additionally, first and second generation stents have been quite effective in the treatment of early restenosis but poses a high risk of late restenosis and thrombosis. These have been attributed to the Presence of durable polymer that induces inflammation and local drug toxicity [15]. Also, preclinical analysis confirmed that these durable polymers were associated with delimitation and webbed polymer surface subsequently leading to stent expansion followed by plastic deformation. Hence, the focus is now on the development of novel DESs with biodegradable drug carriers from which drug can be dispensed in a modulated manner.

Shown in figure 1.3 are cross-sections and platforms of a bare-metal stent (Section A) and a drug-eluting stent (Section B). Stent implantation causes an arterial injury that activates vascular smooth-muscle cells and drives their migration and proliferation, with extracellular-matrix formation resulting in the production of neointimal tissue. Extreme neointimal hyperplasia leads to restenosis within the treated segment, with ischemia requiring repeat revascularization [9]. DESs provide site-specific, controlled release of anti-proliferative agents targeting the suppression of neointimal hyperplasia. Anti-proliferative drugs used in in first and second generation DESs and their mechanisms of action are shown in Section C.

Figure 1.3 Mechanism of Stent Thrombosis [9, 10] and nejm.org, Retrieved, October 2014

These agents bind to the intracellular receptor FKBP12, impeding the mammalian target of rapamycin (mTOR), which results in up-regulation of cyclin-dependent kinase inhibitor p27Kip1. This blocks the proliferation of smooth-muscle cells in the gap 1 (G1) phase of the cell cycle [9]. On the other hand, paclitaxel, used in first generation DESs, binds to the β-tubulin subunit of microtubules, inhibiting the disassembly of microtubules and thereby arresting cell replication in the G0–G1 and mitotic phases of the cycle of smooth-muscle cells [9].

Figure 1.4 Cell-cycle and mechanism of action of sirolimus, zotarolimus, everolimus & paclitaxel.

In spite of all these precautions, stent thrombosis remains a critical issue to consider. Stent thrombosis, a rare but problematical phenomenon arising from the treatment of coronary heart disease, has been attributed to procedural factors and inadequate platelet inhibition during post implantation as well as to chronic inflammation and delayed arterial healing during late follow-up [9, 10]. Numerous definitions for stent thrombosis have been proposed by a number of researchers thus, creating lots of confusion. However, the Academic Research Consortium provides a standardized time-regulated definition (i.e., early, ≤1 month; late, >1 month to ≤1 year; or very late, >1 year) and the definite, probable or possible degree of certainty in diagnosis [9, 12].

Studies conducted by [13, 14] revealed that sirolimus and paclitaxel-eluting stents induced higher risk of very late stent thrombosis in comparison to bare metal stents.

Since the development and subsequent implantation of coronary stents in humans, concerns about their long term safety have frequently been raise. The need to exploit the safety of the bare metal stent combine with the efficacy of a durable biodegradable polymer in the formulation of a novel DES has ever been of paramount concern. The localized elution of the anti-proliferative drug from the proposed novel DES is expected to drastically inhabit restenosis as well as reduced thrombosis and target lesion revascularization rates.

1.4 SCOPE OF WORK

This work highlights the use of prodigiosin loaded PLGA polymer for cardiovascular stent coating. The mechanism of drug release from the polymer matrix, aimed at preventing restenosis, thrombosis and inflammation will be comprehensively explained. To this effect, the drug loading efficiency which corresponds to optimum therapeutic effect will be established. Diffusion and degradation release processes and their influences on the release of drug from the stent strut will be extensively elucidated. By measuring the change in drug concentration with respect to time, one can develop a model to fully explain the drug release profile. The Implication of the results thereof will be discussed for the design of drug eluting biodegradable, polymer-coated stents for cardiovascular interventions.

References

[1] Jung F., Wischke C., Lendlein A., Degradable, Multifunctional Cardiovascular Implants: Challenges and Hurdles, MRS Bulletin 607-610 (2010)
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[2] G. K. Hansson; Inflammatory Atherosclerosis and Coronary Artery Disease, New England Journal of Medicine, 2005 10.1056/NEJM 200101113440217

[3] World Health Organization Statistics Atlas for Cardiovascular Disease, 2011

[4] Mathers C.D, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med, 2006, 3(11):e442.

[5] PTCA /stent procedure/ www.stormontvail.org

[6] Russell Ross: Atherosclerosis Inflammatory Disease, the New England Journal of Medicine, 005, 340

[7] David Martin et al.: Drug eluting stents for coronary artery disease (review), Med Eng Phys,
2010

[8] Sydell and Arnold Miller: Coronary Artery Disease Treatment Guide, Heart & Vascular Institute, Cleveland Clinic

[9] Stefanini, G. G., and Holmes, D. R., Drug-Eluting Coronary-Artery Stents, New England Journal of Medicine, 2013;368:254-65, copyright @ 2013, Massachusetts Medical Society

[10] Holmes D.R., Kereiakes, D.J., Garg, S., et al. Stent thrombosis. J Am Coll Cardiol 2010;56:1357-65.
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[11] Holmes Dr. et al : Restenosis After Percutaneous Transluminal Coronary Angioplasty (PTCA), Am J Cardiol 1984:53 (Suppl C), 77-81

[12] Cutlip D.E., Windecker S., Mehran R., et al. Clinical end points in coronary stent trials: a case for standardized definitions. Circulation 2007;115:2344-51.

[13] Stone GW, Moses JW, Ellis SG, et al. Safety and efficacy of sirolimus- and paclitaxel-eluting Coronary Stents N Engl J Med 2007;356:998-1008.

[14] Räber L, Magro M, Stefanini GG, et al. Very late coronary stent thrombosis of a newer-generation everolimus-eluting stent compared with early-generation drug-eluting stents: a prospective cohort study. Circulation 2012;125:1110-21.

[15] Stettler C, Wandel S, Allemann S, et al. Outcomes associated with drug-eluting and bare-metal stents: a collaborative network meta-analysis. Lancet 2007;370: 937-48.