The capability to deliver high effective dosages to specific sites in the Human body has become the holy grail of drug delivery research. Drugs with proven effectiveness under in vitro investigation often reach a major roadblock under in vivo testing due to a lack of an effective delivery strategy.
MAGNETIZABLE IMPLANTS FOR TARGETED DRUG DELIVERY
OBJECTIVE OF STUDY
The capability to deliver high effective dosages to specific sites in the Human body has become the holy grail of drug delivery research. Drugs with proven effectiveness under in vitro investigation often reach a major roadblock under in vivo testing due to a lack of an effective delivery strategy. This project proposes a method for targeted drug delivery by applying high magnetic field gradients within the body to an injected perparamagnetic colloidal fluid carrying a drug, with the aid of modest uniform magnetic field. The design involves patterning of endovascular implants, such as coronary stents, with soft magnetic coatings capable of applying high local magnetic field gradients within the body.
Examination of the feasibility of the design has been focused around the treatment of coronary restenosis following angioplasty. Drug-eluting stents, which have debuted in hospitals over the past two years, have thus far reduced restenosis rates to below 10%. Our local drug delivery system is a viable alternative or enhancement to drug-eluting stents, offering increased clinician control of dose size, the ability to treat a site repeatedly, and a wide array of applications for treatment of other pathologies. The theoretical models, parallel plate and pipe flow analysis, and cell culture models presented give insight into the use of micron and sub-micron scale magnetic particles for site-specific delivery of pharmaceuticals and magnetically labeled cells
1: INTRODUCTION
As the pharmaceutical industry continues to develop new and effective medications, the need to develop efficient, minimally invasive delivery techniques is paramount. A successful targeted drug delivery system will allow clinical usage of drugs not currently accessible to physicians, as well as a more efficient means for delivering those already available.
Drug targeting is the delivery of drugs to receptors or organ or any other specific part of the body to which one wishes to deliver the drug exclusively. Various nonmagnetic microcarries (nanoparticles, microspheres and microparticles etc.) are successfully utilized for drug targeting but they show poor site specificity and are rapidly cleared off by RES (reticuloendothelial system) under normal circumstances. Magnetism play an important role in these case, magnetic particles composed of magnetite which are well tolerated by the body, magnetic fields are believed to be harmless to biological systems and adaptable to any part of the body1. Up to 60% of an injected dose can be deposited and released in a controlled manner in selected nonreticuloendothelial organs. So magnetic microcarriers were developed to overcome two major problems encountered in drug targeting namely RES clearance and target site specificity2. Magnetism has application in numerous fields like diagnostics, drug targeting, molecular biology, cell isolation, cell purification, hyperthermia, and radioimmunoassay. This article discusses the potential applications of magnet in drug targeting, magnet containing particles & mechanism of targeted drug delivery by magnetism.
Popular cancer drugs have been found to have applications in many realms of clinical medicine74. The best approach for treating tumors and other localized medical defects is to administer drugs only at the site of complication. By delivering the drug locally, the toxicity of the drug to the rest of the body can be reduced while maintaining the desired therapeutic benefit at the site of interest. Many exciting drugs developed by the pharmaceutical industry have shown remarkable success during in vitro testing and animal trials, but have yielded undesirable results in clinical trials due to systemic toxicity of the drug to other parts of the body. Thus, the ability to deliver large concentrations of drugs only at the site of complication is of major importance for both the pharmaceutical industry and for clinicians.
In most cases, however, the drug delivery vehicles have not been advanced to a state where it is possible to deliver locally high concentrations of drugs with minimally invasive techniques. This is especially true when repeat dosing is required. The magnetic drug delivery system proposed herein overcomes many of these difficulties, and provides a method for concentrating drugs at selected sites in the body with minimal stress on the patient.
1.1: DEFINITION OF MAGNETIC IMPLANT
Magnetic implants are based on the same principles as high gradient magnetic separation (HGMS). When a ferromagnetic element (e.g., an implant) is placed in a magnetic field, it becomes magnetically energized creating a very strong but localized magnetic field that is far more capable of concentrating magnetic particles at the site of the implant compared to the magnetic field alone. Invasive magnetic implants can be made of needles, wires, stents, catheter tips, and even very magnetic (non-drug carrying) particles. Wires can also be placed just outside the body near the target zone to improve the collection efficiency of the MDCPs. Finally, magnetic implants can be placed in the body at the target site by transdermal injection through the skin using a specially designed syringe, or through the use of catheters.
1.2: HISTORY OF MAGNETIC IMPLANTS
The association between magnets, health and well-being is currently enjoying a revival, but the approach is not new. Magnetic therapy possesses an ancient heritage and has occupied a central role in Chinese medicine for over 2000 years. Magnetic therapy is mentioned in some of the earliest writings in Egypt, India and Greece. Until recently the scientific explanation of magnetic action was not available. Magnetic therapy utilizes the natural energy of magnetism that is important to human existence and overall health. A magnetic field provides a (natural) way to assist the body’s normal healing processes as it passes through all tissues and cells. Studies show that magnets can be an effective therapy for the relief of pain by blocking pain sensations. Applying magnetic fields to an injured area improves blood flow and oxygen to enhance the body’s natural healing process. The improved blood flow and fluid exchange to the injured tissue helps reduce pain and inflammation.
Magnetic implants have been used for several years in dentistry and reconstructive surgery, but their inclusion in the body modification world has been quite a recent one.
Having magnets implanted under the skin allows the wearer to attach magnetic items to the outside of the skin, and also enables the wearer to sense electromagnetic fields.
Samppa implanted a few magnets in himself and close friends in the late 1990's, but they weren't very strong and were only capable of picking up small items.
Jesse Jarrell and Steve Haworth decided to develop magnetic implants which were both smaller, and stronger, than those used by Samppa. The magnets used were neodymium iron boron alloy, with a thin gold plating, encapsulated in silicone. While most of the people who received these implants were satisfied with the post procedural result, many have gone on to develop problems with the implants which have necessitated their removal. The main problems have been caused by the rupturing of the silicone covering, bringing the metal into contact with bodily tissues, which in most cases caused it to rapidly break down.
2:VARIOUS APPROCHES OF MAGNETIC DRUG DELIVERY
1. Magnetic Nanoparticles
2.Magnetic Microspheres
3.Magnetic Implants
4.Magnetic Liposomes
5.Magnetic Emulsion
6.Magnetically Resealed Erythrocytes
1.Magnetic nanoparticles:
Magnetic nanoparticles are particles in nano size range containing polymers, drug along with ferromagnetic particles (magnetite). In recent years the separation of cells, viruses, and bio-molecules using magnetic microparticles has gained increasing popularity. Hence, new technologies using magnetic microparticles or nanoparticles are emerging. With magnetic separation, it is possible to achieve very high efficiency of separation in complex media. Other applications of magnetic particles include immunoassays, drug targeting, drug transporting, and biosensing 21.
2.Magnetic microspheres :
Magnetic microspheres are supramolecular particles that are small enough to circulate through capillaries without producing embolic occlusion (<4 μm) but are sufficiently susceptible (ferromagnetic) to be captured in microvessels and dragged in to the adjacent tissues by magnetic fields of 0.5-0.8 tesla (T)2. Magnetic microspheres were prepared by mainly two methods namely phase separation emulsion polymerization (PSEP) and continuous solvent evaporation (CSE). The amount and rate of drug delivery via magnetic responsive microspheres can be regulated by varying size of microspheres, drug content , magnetite content , hydration state and drug release characteristic of carrier 4. the amount of drug and magnetite content of microspheres needs to be delicately balanced in order to design an efficient therapeutic system. magnetic microsphere are characterized for different attributes such as particle size analysis including size distribution ,surface topography, and texture etc. using scanning electron microscopy (SEM), drug entrapment efficiency, percent magnetite content, and in vitro magnetic responsiveness and drug release.
3.Magnetic Implants:
Magnetic implants are based on the same principles as high gradient magnetic separation (HGMS). When a ferromagnetic element (e.g., an implant) is placed in a magnetic field, it becomes magnetically energized creating a very strong but localized magnetic field that is far more capable of concentrating magnetic particles at the site of the implant compared to the magnetic field alone. Invasive magnetic implants can be made of needles, wires, stents, catheter tips, and even very magnetic (non-drug carrying) particles. Wires can also be placed just outside the body near the target zone to improve the collection efficiency of the MDCPs. Finally, magnetic implants can be placed in the body at the target site by transdermal injection through the skin using a specially designed syringe, or through the use of catheters.
4.Magnetic Liposomes:
Liposomes are simple microscopic vesicles in which lipid bilayer structures are present with an aqueous volume entirely enclosed by a membrane, composed of lipid molecule. There are a number of components present in liposomes, with phospholipids and cholesterol being the main ingredients but in case of magneto liposomes magnetite is one of the component of the liposomes 8. Generally these are magnetic carrier which can be prepared by entrapment of Ferro fluid within core of liposomes 9, 10 . Magnetoliposome can also be produced by covalent attachment of ligands to the surface of the vehicles or by incorporation of target lipids in the matrix of structural phospholipids 11. Alternatively magnetoliposomes are prepared using the phospholipid vesicle as a nanoreactor for the in situ precipitation of magnetic nanoparticles 12. Vesicles are also prepared containing didodecyl methyl ammonium bromide; contain an ionic magnetic fluid 13. These magnetoliposomes were effectively used for site specific targeting, cell sorting & as magnetic resonance contrast enhancing agent. Thermo sensitive magnetioliposomes can release the entrapped drug after selective heating caused by the electromagnetic fields 14. Magnetofluorescent liposomes were used for increasing sensitivity of immunofluorescence.
5. Magnetic Emulsion:
Besides magnetic modulated systems, like microcapsules/microspheres Magnetic emulsion was also tried as drug carrier for chemotherapeutic agents. The emulsion is magnetically responsive oil in water type of emulsion bearing a chemotherapeutic agent which could be selectively localized by applying an external magnetic field to specific target site29.Akimoto and Morimoto prepared magnetic emulsion by utilizing ethyl oleate based magnetic fluid as the dispersed phase, casein solution as the continuous phase and anticancer agent, methyl CCNU trapped in the oily dispersed phase as active chemotherapeutic agent. Magnetic emulsion appears to have potential in conferring site specificity to certain chemotherapeutic agent
6.Magnetically Resealed Erythrocytes:
Resealed erythrocytes have various advantages as drug carriers such as it is biodegradable, biocompatible, large quantity of variety of material can be encapsulated within small volume of cell and can be utilized for organ targeting etc. Due to these advantages of resealed erythrocytes, magnetic resealed erythrocytes came in to existence which contains ferrofluides (magnetite) along with loaded drugs within the cell. Magnetically responsive ibuprofen-loaded erythrocytes were prepared and characterized in vitro by Vyas and Jain 28. The erythrocytes loaded with ibuprofen and magnetite (ferrofluids) using the preswell technique. The loaded cell effectively responded to an external magnetic field. Various process variables including drug concentration, magnetite concentration, sonication of ferrofluids that could affect the loading of drugs and magnetite were studied. The loaded erythrocytes were characterized for in vitro drug efflux, hemoglobin release, morphology osmotic fragility, in vitro magnetic responsiveness and percent cell recovery.
3: PRINCIPLE OF MAGNETIC IMPLANTS
One of the key problems associated with drug administration is the difficulty to target specific areas or sites in the body, like cancerous tumors or arterial blockage. Typically in these cases, exceedingly large doses of a drug are needed to ensure that some of the drug reaches a specific site, a fact which unavoidably imposes substantial toxic side effects at non-targeted organs. One way to achieve drug targeting in the body is to incorporate magnetic particles into drug carriers and then to retain them at the site using an externally applied magnetic field.
This process is referred to as magnetic drug targeting (MDT). However, the main limitation of MDT is that under a given set of conditions an externally applied magnetic field alone may not be able to retain a sufficient number of magnetic drug carrier particles (MDCPs) to justify its use. Such a limitation may not exist if high gradient magnetic implants (HGMIs) are used to assist MDT.
Magnetic implants are based on the same principles as high gradient magnetic separation (HGMS). When a ferromagnetic element (e.g., an implant) is placed in a magnetic field, it becomes magnetically energized creating a very strong but localized magnetic field that is far more capable of concentrating magnetic particles at the site of the implant compared to the magnetic field alone. Invasive magnetic implants can be made of needles, wires, stents, catheter tips, and even very magnetic (non-drug carrying) particles. Wires can also be placed just outside the body near the target zone to improve the collection efficiency of the MDCPs. Finally, magnetic implants can be placed in the body at the target site by transdermal injection through the skin using a specially designed syringe, or through the use of catheters.The figure 3a.
The Implant: The implant consists of a telescopic shaft which is extended by a power screw jacking the two halves apart. The power screw is driven by a stainless steel gearbox (Ø = 21.5mm and l = 18.5mm) mounted in the knee producing an overall speed reduction of 13061:1 and an output torque of 4Nm. The input shaft of the gearbox is connected to a NdFeB toroidal magnet encapsulated in titanium. Figure 3b
The Drive Unit:
Once the prosthesis has been implanted, it is extended by placing the limb with the implant in the drive unit. A rotating magnetic field of speed 3000RPM is produced by passing electric current through a set of coils. The field produced has the strength to generate the predefined torque, coupling with the implant magnet. As the magnetic field is turned on, it captures the implant magnet causing it to rotate. At full speed the implant grows at a rate of 0.23mm per minute.
The magnetic force can be maximized by using a relatively strong uniform field to magnetize the magnetic nanoparticle-loaded cells, and this effect also is superimposed on high-level magnetic gradients within the steel stent wire network induced by the presence of a uniform field. Specifically, a uniform magnetic field is supplied by electromagnetic
coils positioned external to the body, and the short-range, high-gradient magnetic fields are produced by the magnetizable wires of a stent for the purpose of maximizing the regional magnetic force on endothelial cells loaded with magnetic nanoparticles (Fig. 3c). In practice, this technique enables a far greater fraction of the delivered materials to be captured than if a single source magnetic field were used.
4: MATERIAL SELECTION FOR MAGNETIC IMPLANTS
To simulate a stent-like surface as a plane instead of a circular cross-section, the first material selected for electroplating was a woven, 316L stainless steel wire mesh (140μm wire diameter, 400μm apertures), which can be seen in Figure 4a. This particular material was selected due to its large strut spacing, and extremely low saturation magnetization. As a result, a large difference in response to applied magnetic fields, and subsequently in capture ability, can be compared between a virtual non-magnetic 316L mesh and a CoNi electroplated 316L mesh.
The second stent-like material selected was a molded 304 grade stainless steel (150μm wire diameter, 450μm apertures). This material has a more level, consistent surface, as opposed to a woven geometry. In addition, it has stronger inherent magnetic properties than 316L steel. An image of this material can be found in Figure 4b
With the aid of the Drexel Machine shop, stent-like tubes of this 304 steel were rolled by heating the mesh, and sealing the roll using silver solder. Tubes were rolled to a 5mm external diameter, 2cm in length. An image of a rolled tube can be seen in Figure 4c
The final material selected were industrial application 302 stainless steel compression springs (2cm long spring, 3mm diameter, 355μm wire diameter). These springs are more stent-like in geometry and flexibility, but their wire diameter is 3-5 times thicker than struts in a typical stent. These springs are highly magnetic, and provide an upper bound for examining different alloys and their inherent abilities to capture particles under the application of external magnetic fields. An image of a 302 steel compression spring can
be seen in Figure 4d
5: METHOD OF DEPOSITION OF MAGNETIC PARTICLES ON IMPLANTS AND THEIR EVALUATION
Electroplating is a process of depositing a coating (commonly) of silver, gold, cobalt, or nickel on an inferior metal, by means of electricity.
Electroplating Procedure
Before each electroplating session, a fresh bath was prepared. 100 mL of bath was prepared of the following makeup: 0.45 M NiCl2, 0.65 M CoCL2, 30 g/dm-3 H3BO3 and a trace of Saccharin (Sigma, MO).
The bath solution was placed in a 1L glass beaker, heated to 55ºC. A thermometer was stabilized for constant temperature measurement. An air bubbler, connected to the house air system, was fixed at the bottom of the beaker. A Princeton Applied Research 363 Potentiostat was used as the current controller for electroplating. To the anode an industrial-grade sheet of cobalt (EMI, CA) 2 x 2 inches in size was connected, and connected to the cathode was the piece of implant-simulating material.
In order to maintain reproducibility, it was entirely necessary to account for how much magnetic material was deposited on a given sample. While this can be calculated loosely by ion concentrations in the bath, the dimensions of the sample, and the applied current, some of these factors may be inconsistent. Plating height was determined by measuring the mass of the sample before and after plating, using a digital balance with a resolution of 10-5 grams. By using an air-bubbler, keeping the sample close to the anode, and rotating the sample 180 degrees at the half time mark of each plating session, it is most
reasonable to assume near uniform plating. So assuming uniform plating, with
knowledge of the geometry and dimensions of each sample, electroplating height was calculated.
The method for reproducing the same height in each session, was correlating the exact sample size, ion concentrations, and plating time. This was accomplished with ease due to the quality of the electroplating setup, and strict monitoring of these governing factors.
Evaluation of Magnetic Properties of Model Implants
Magnetic properties of model implants were measured using a Princeton Measurements MicroMag Alternating Gradient Magnetometer (AGM) Images of the AGM can be found in Figure 5b
The MicroMag AGM is a highly sensitive instrument for detecting changes in the magnetic properties of materials. 5mm circular punch-outs of each mesh were made using an industrial holepunch, as well as individual coils from the 302 compression spring. In order to normalize saturation magnetization to mass, each sample was weighed and its mass documented. A piece of mesh or spring was then mounted on the end of a cantilevered rod that incorporates a piezoelectric sensor (the perpendicular probe was selected). A dc field then magnetizes the sample while simultaneously subjecting it to a small alternating field gradient. This gradient exerts an alternating force on the sample, which is proportional to the magnitude of the field gradient and to the magnetic moment of the sample. The resulting deflection of the rod is detected by the piezoelectric element. Computer software then generates hysteresis curves and saturation magnetization data. Five samples of each material were measured by AGM, and the normalized results averaged to obtain Ms per gram data.
6: IN VITRO EVALUATION
In all magnetic drug delivery systems, the magnet serves two purposes; first, to magnetize the drug, and secondly to provide magnetic field gradients to capture the drug. Although large magnets held near or implanted in the body provide strong magnetic fields to magnetize the drug, these large magnets inherently produce weak magnetic field gradients. On the other hand, micron-sized magnets provide very strong magnetic field gradients, however their fields are short-range and they cannot by themselves efficiently magnetize the drug.
By using micron-sized magnetic implants in combination with long-range magnetic fields, it is possible to both magnetize the drug and create strong magnetic field gradients for more efficient capture and localization of the drug.
It is performed by following methods
6.1 Parallel plate flow Technique
6.2 Pipe flow characterization and quantitative evaluation of magnetic particle capture
6.1 parallel plate flow Technique
A parallel plate flow chamber (PPFC) is selected as the best system for applying a laminar flow of particles to a magnetically patterned chip or mesh contained within the chamber60. The PPFC to be used consists of three main layers: a 4” glass wafer with a mounted sample of magnetic mesh, a layer of Dow Corning Brand Sylgard poly(dimethyl siloxane) (PDMS) with a cut-out channel, and a 6mm thick abrasion-resistant cast acrylic coverpiece. The coverpiece contains two reservoirs (flow in / flow out) that connect to the pump and the waste receptacle, respectively. In addition there is one vacuum connection, which feeds a milled out loop around the circumference of the piece. This top piece mainly serves to assure correct parallel plate flow, and to seal the top of the channel firmly to the PDMS layer.
The PDMS layer itself is glued atop of a glass wafer using a thinly applied micro-layer of uncured PDMS, and is held to the top coverpiece by vacuum. This method for attaching the PDMS layer has been tested for stability, endurance, leakage, as well as its ability to withstand sterilization procedures and has been successful in all tests. At the end of the channel, an approximately 2 x 2 cm magnetically electroplated stainless steel mesh is also attached by an ultra-thin layer of PDMS at the end of the actual channel space. A description of the preparation and design of the mesh is included in above section. Diagrams of overhead and side views of the flow chamber can be seen in the Figure
A Fisherbrand variable flow chemical pump (Fisher, IL) is used to draw in solution containing the magnetic particles and outputs the particles into the mouth of the PPFC.
Flow chamber output is released into a waste bottle. Photographs of the chamber and flow chamber experimental setup can be seen in Figure 6.1
Figure 6.1b : Flow chamber
Although in the model the variable parameter is often the vessel diameter, since focusing here on a flow chamber scenario the characterization will be somewhat different. The design of our flow chamber on an average sized
coronary artery of diameter 3mm. The parameters of the channel in the flow chamber are Height: 2r, Width: 18r, Pattern Width 14r (2r clearance on each side), and Mesh Length: 2cm, where r is the radius of the average coronary artery: 1.5mm.
The goal in designing the pattern dimensions was to simulate an ‘unrolled’ stent (of diameter 3mm and length 2cm) and to continue with the limiting-case scenario analysis, channel height was set at 2r instead of r in order to have Vmax in the center of the channel. Since each mesh/PDMS-gasket combination is single use, numerous other configurations are possible, providing increased functionality to the design.
In addition, the pattern is aligned towards the end of the channel, to ensure many channel diameters worth of distance from the entering reservoir to minimize entrance effects at the pattern itself, and to hence obtain steady parallel plate flow. It is important to note that while the actual channel itself is 4cm in length, the full cutout length in the PDMS is 6.5cm, allowing for space at either end of the channel at which the reservoir feeds or empties the channel.
The reservoir dimensions are the same at the entrance and exit, and are sized at approximately 5 times the cross-sectional area of the flow channel to ensure appropriate flow parameters and speed in the channel itself. As calculated from resting blood flow rates in the coronary artery of 1.25mL/sec, an approximately 15.0cm/s flow velocity for the channel was used in the experiments (which can be increased or decreased without modification of the flow chamber itself, and adjusted for different channel heights).
a woven stainless steel mesh was selected as the primary model implant for parallel plate flow experiments. The 304 stainless steel mesh was also tested in the flow chamber.
Three different particle types were used in experiments. Particles used in the first two series of flow experiments were commercially available superparamagnetic polystyrene beads (Spherotech, IL). These beads, composed of 20% _-Fe2O3 magnetite by weight and labeled with nile red fluorescent pigment, had a nominal diameter of 2μm with approximately 10% variance in size. Particles come in 2mL water solutions concentrated at 1% w/v or 2.274 x 109 particles/mL.
Another sample of beads used from Spherotech, were also 20% _-Fe2O3 magnetite by weight and labeled with nile red fluorescent pigment, had a nominal diameter of 350nm with approximately 10% variance in size. Particles come in 2mL water solutions concentrated at 1% w/v or 4.8 x 1011 particles/mL.
another set used to prepare fluorescent-labeled (BIODPY 564/570) biodegradable polylactic acidbased particles loaded with magnetite. This population of nanoparticles had an average diameter of 370nm with sizes spanning the range of 364 -380 nm. These nanoparticles were found to have similar magnetic material properties (c » 2.5 ) as the commercially available beads from Spherotech.
Magnetic capture can be done on the following systems
1.Magnetic Capture to 316L Stainless Steel Mesh (Figure 6.1d)
2.Magnetic Capture of 2μm and 350nm Magnetic Particles (Figure6.1e)
3.Magnetic Capture across a Silicone Barrier (Figure 6.1f)
6.2 PIPE FLOW CHARACTERIZATION AND
QUANTITATIVE ANALYSIS OF MAGNETIC PARTICLE CAPTURE
The selection of the appropriate implant models and execution of proper electroplating technique allowed for a suitable “target” for magnetic capture. The optimization of a reusable and multi-functional parallel plate flow chamber provided an appropriate system for determining an ideal working range for both particle size and concentration.
Before this magnetic drug delivery system can be tested in vivo, pipe flow experiments are needed to obtain a stronger starting point for dose ranging. Large and average size vessels are accounted for, as are differing geometries and materials of the implant models used.
Two separate materials were selected for pipe flow experiments: 304 stainless steel large diameter molded stents, and 302 stainless steel compression springs. Both of these materials have a moderately high saturation magnetization before any soft magnetic material is plated on them, allowing analysis of magnetic alloys to capture particles with the aid of a external, uniform magnetic field.
With the aid of the Drexel Machine shop, stent-like tubes of the 304 grade steel were rolled by heating the mesh, and sealing the roll using silver solder. Tubes were rolled to a 5mm external diameter, 2cm in length. An image of a rolled tube can be seen in Figure 6.2a.
Two separate plating heights of CoNi were electroplated onto these stents: 3.1 and 5.2 μm. The geometry and malleability of these stents are quite different from properties seen in commercially manufactured stents. But as this technology is being considered for numerous applications where implants may not be used for keeping a vessel open, but rather having drug delivery as its primary function, it’s useful to examine a model with a significantly different geometry, as well as a larger vessel diameter.
The final materials selected were industrial application 302
stainless steel compression springs (2cm long spring, 3mm outer diameter, 355μm wire diameter). These springs are more stent-like in geometry and
flexibility, but their wire diameter is 3-5 times thicker than struts in a typical stent. These springs are highly magnetic, and provide an upper bound for examining different alloys and their inherent abilities to capture particles under the application of external magnetic fields. An image of a 302 steel compression spring can be seen in Figure (b) Two separate plating heights of CoNi were deposited onto these springs: 2.5 and 5.5μm.
These compression springs have a much smaller surface area than the molded stents, and may actually be an ideal starting design for manufacturing an endovascular implant that has the sole function of capture magnetic drug.
Spherotech magnetic particles were selected for all pipe flow experiments. The sample of beads used from Spherotech, were 20% _-Fe2O3 magnetite by weight and labeled with nile red fluorescent pigment, had a nominal diameter of 350nm with approximately 10% variance in size (Spherotech, IL). Particles come in 2mL water solutions concentrated at 1% w/v or 4.8 x 1011 particles/mL.
Pipe Flow Setup
Two separate views of the pipe flow experimental setup can be found in Figure 6.2b
The setup consists of a 50mL beaker that supplies the flow pump with solution via a 20mm long polyvinyl chloride (PVC) tube. The pump passes the particle dose through a 60mm long PVC tube, in which the stent or spring was gently inserted within. The spring is mounted between two Tesla coils controlled by a bipolar operational power supply/amplifier (Kepco, NY) was used to generate the external magnetic field.
A variable flow peristaltic pump (Fisher, IL) was used for these flow experiments. A much lower volumetric flow rate was required to maintain a 15cm/s coronary flow velocity, dependent on the diameter and length of the tubing, and averaged out at approximately 1.25ml/s. The flow is not continuous, but rather pulsatile, unlike the flow chamber experiments which required the use of a variable flow chemical pump for its ability to deliver fluid at rates of up to 2L/min. Experiment performed as the following Molded 304 Stent Pipe Flow Experiments
1.Compression Spring Pipe Flow Experiments
2.Alternating Gradient Magnetometer Analysis of Flow Capture Efficiency
7: BIOCOMPATIBLE EVALUATION
If the magnetic drug delivery system described within this thesis were to be completely successful in capturing magnetic particles to implanted stents, and even in the therapeutic transfer of drugs from carriers to tissue, proper risk analysis of numerous factors must still be considered in the development of the system. For instance, if it was to be accepted that a 1-10μm thick coating was to be deposited over the entire surface of a stent (disregarding here the question of potential mechanical consequences of such a layer), the issue of chemical reactivity of the CoNi alloy within the body comes immediately into play. Numerous studies have indicated the toxicity of free Cobalt within the body87 . The coating must either be heat treated to anneal the plated layer, or passivated by means of a metal, ceramic, or polymer coating. The selection of material is a more of a manufacturing issue, but a flexible non-degradable polymer, or a nanolayer of gold have arisen as acceptable choices.
The effects of magnetic fields (both static and high gradient) on biological materials have been analyzed extensively in the literature, with a variety of theories regarding safety from the macro-scale to single cells61, as well as the abilities of large (6T+) magnetic fields to 96 sediment erythrocytes81. Additional studies have found magnetite and magnetitebased composites to be extremely well tolerated by the body87. For the modestly sized fields and magnetic features presented within the proposed design, consultation of this literature increased confidence in low risks regarding these issues. the examination of any effects on the growth, morphology, or behavior of endothelial cells due to the magnetic field gradients of the wires of an electroplated 316L Stainless Steel mesh. The magnetic field gradients of the CoNi coating rapidly decay after the removal of an applied magnetic field. Therefore, endothelial cell cultures were investigated with and without constant application of an external field to saturate the magnetization of the coating. Interestingly enough, one original desire was to examine if cells would survive the delivery of large numbers of magnetic particles to cultures, but were actually found to compartmentalize the particles into the cell. As a result, investigations studying the ability for endothelial cells with internalized magnetic particles to maintain normal growth, morphology, and behavior, as well as to be captured magnetically to mesh surfaces were also performed.
Procedure
Culture of Endothelial Cells Bovine aortic endothelial cells (BAECs) were selected as an appropriate culture model.
These cells were previously isolated by standard
technique at the University of Pennsylvania in 2002. All experiments were be performed at low-passages (<10). Cells were routinely cultured in low glucose DMEM (Sigma, MO) supplemented with 10% Qualified Heat inactivated Fetal Bovine Serum (Sigma, MO) and 1% 2.5mM L-glutamine (Sigma, MO). After expansion of the culture using 100μg/mL streptomycin (Sigma, MO), and 100U/mL penicillin (Sigma, MO) per 500mL batch of medium, it was determined that BAECs could be cultured with ease without the use of antibiotics or antimycotics, so they were removed from future batches of culture medium.
Cells were routinely cultured in 75cm2 flasks in a Fisherbrand cell culture incubator at 37°C and 5.0 % CO2 (Fisher, IL). In preliminary experiments determining the preparation of mesh for culturing, the BAECs were shown to grow poorly on silicone and glass surfaces, and when these surfaces were treated with coated with a 1% Rat Tail Type I Collagen (Sigma, MO), cells grew robustly, maintaining a growth rate and visible morphology alike to BAECs cultured in T75 flasks.
Culture of BAECs on Magnetic Mesh
5mm punch-outs of magnetic and non-magnetic mesh were prepared, and treated with PDMS as described above. Prior to use, each of the 6 cover slips (25mm diameter x 0.25mm thick, 3 with magnetic mesh, 3 with non-magnetic mesh) were washed with Ivory soap and water, and rinsed thoroughly. Following cleaning, each mesh was placed in a glass petri dish within a laminar flow hood, and soaked in 70% ethanol for 30 minutes. After 30 minutes, all ethanol was aspirated, and the cover slips were allowed to dry for 20 minutes.
Two separate 6-well plates were obtained (one for magnetic mesh, the other for nonmagnetic mesh), and 3 slips of each experimental group were placed in each. A preparation of 1% by volume Rat Tail Type I Collagen (Sigma, MO) in Phosphate Buffered Saline (Sigma, MO) was used to add 150μL to each well. After 30 minutes, the collagen solution was aspirated, the slips rinsed twice with an equal volume of PBS, and allowed to dry for 10 minutes within the laminar flow hood.
BAECs, routinely cultured as described above, were then seeded to each cover slip at 1:2 split ratio (adjusted to cm2 growth area). 3cm long by 0.5cm tall by 0.5cm wide pieces of neodymium permanent magnets were placed under each well, for both non-magnetic and magnetic mesh, and separated by plastic spacers. These pieces applied an approximately 500 Gauss magnetic field at the center of the mesh as measured by a handheld Gaussmeter (Lakeshore Cryotronics, OH). These magnets were kept in place, under the wells, and placed in the cell culture incubator for 30 minutes while cells were allowed to attach to the surface of the collagen-coated PDMS. 30 minutes was selected as a modest
period for which a magnetic drug delivery injection may be performed, saturating the magnetic moment of the material for that period. At the conclusion of 30 minutes, the magnets were removed and the samples were left in culture, and imaged by phase contrast at 24 hours. vascular endothelial cells succeed in growth within microns of distance from magnetically plated mesh under the influence of a field, but can be labeled with magnetic
particles, delivered by magnetic force to the surface of the mesh and survive. By placing a permanent magnet underneath the culture continuously during particle seeding to a culture, these particles may have an increased tendency to form chains or large aggregates too large for delivery to the inside of a cell,
and may therefore remain on top of the culture. These large aggregates may even be pulled through intracellular space to the bottom of the culture, beneath the cells, but if the field is left in place throughout, one would expect minimal internalization for micron and even large sub-micron particles.
8: APPLICATION OF MAGNETIC IMPLANTS
Stents are commonly used in a variety of biomedical applications. For example, stents are routinely implanted in patients to keep blood vessels open in the coronary arteries, to keep the esophagus from closing due to strictures of cancer, to keep the ureters open for maintenance of kidney drainage, and to keep the bile duct open in patients with pancreatic cancer. Such stents are usually inserted percutaneously under radiological guidance. Stents comprise a tube made of metal or polymer, in a wide range of physiologically appropriate diameters and lengths. Currently the most commonly used materials for stents are 316L Stainless Steel or Nickel-Titanium.
General stent design varies in the number of intersections and interstrut area, the in-strut configuration, and the metal-to-artery ratio. The two different expansion principles for stents are balloon-expansion and self-expansion, and the design types can be categorized into five types: ring, tubular, multi-design, coil, and mesh. Stents have been routinely used over the last 15 years in percutaneous transluminal coronary angioplasty (PTCA), a procedure for the treatment of severe, symptomatic coronary stenosis74.
Coronary Restenosis
In-stent restenosis (the re-closing of the vessel) remains a major limitation in coronary stenting. Restenosis is generally considered a local vascular manifestation of the biological response to injury. The injury as a result of catheter insertion consists of denudation of the intima (endothelium) and stretching of the media (smooth muscle). The wound-healing reaction consists of an inflammatory phase, a granulation phase, and a remodeling phase. The inflammation is characterized by growth factor and platelet
activation, the granulation by smooth muscle cell and fibroblast migration and proliferation into the injured area, and the remodeling phase by proteoglycan and collagen synthesis, replacing early fibronectin as the major component of extracellular matrix74.
Coronary stents comprise mechanical scaffolding that almost completely eliminates recoil and remodeling. However, neo-intimal growth or proliferation is still a problem. Neo-intimal proliferation occurs principally at the site of the primary lesion within the first 6 months after implantation, a major checkpoint for patient health post-surgery101. Neo-intima forms during the first week after PTCA and the progress is well under way after 4 weeks, with continued progression over the following months79. This neo-intima is an accumulation of smooth muscle cells within a proteoglycan matrix that narrows the previously enlarged lumen. Its formation is triggered by a series of molecular events including leukocyte infiltration, platelet activation, smooth muscle cell expansion, extracellular matrix elaboration, and reendothelialization101.
Current Treatment Options
There are several treatment options in practice or under investigation for treating restenosis. Local drug delivery provides limited systemic release, thereby reducing the risk of systemic toxicity. Techniques for local drug delivery to arterial tissue that have been described include, but are not limited to, direct coating of the stent with drug, coating of the stent with a drug-containing biodegradable polymer, and hydrogel/drug coating. Biodegradable stents have also been described74.
Problems with these technologies, include damage to the polymer layer during implantation (causing portions to sever, break off and cause clot formation), the inability to deliver effective concentrations, one-time dosage limitations, and, in the case of biodegradable stents, mechanical compromise. An additional concern with the polymercoated drug-eluting stents is limitation of the endothelialization necessary to cover the stent and prevent the bare metal from coming in long contact with the blood, thereby leading to clot formation.
PROPOSED DESIGN OF MAGNETICALLY TARGETED DRUG DELIVERY
Embedded Particles in Vessel Walls
One possible approach to this problem is to use magnetic material embedded in blood vessel walls as the source of strong localized magnetic field gradients at defined locations in the body. The proposed design involves seeding magnetic particles onto blood vessel walls at designated sites through specific receptor-ligand recognition69. Although this approach also uses external magnetic fields, these fields are uniform and the intent is to magnetize the anchored particles to produce the field gradients rather than to use the external field gradient itself to direct the particle.
Typical techniques for drug delivery involve saturation of site receptors with an appropriate ligand chemically attached to a desired drug. In contrast with these techniques, our method relies on saturating receptors with inert superparamagnetic particle anchors coated with specific ligands for the site of interest (containing no drug). High concentrations of anchors can be applied to saturate receptors without fear of harmful side-effects. One can also imagine scenarios in which magnetic anchors can be implanted in blood vessel walls through catheter based insertion methods. Once the anchors or implants are in place, uniform magnetic fields may be used to attract an injected drug-infused superparamagnetic colloidal fluid to the anchored particles, thereby allowing high local concentrations of otherwise systemically toxic drugs to be captured at the site of interest.
Magnetic Implants for Local Drug Delivery
The primary focus of this thesis is an implant-based drug delivery system, which operates by placement of a magnetic implant (a stent or cylindrical endovascular implant) at designated sites in the cardiovascular system and then attracting to the designated sites injected doses of magnetically susceptible drugs. This is accomplished with the aid of a modest and uniform, external magnetic field. Magnetic microspheres or nanospheres, which can be designed to carry virtually any type of drug or medical agent, are attracted to regions of the strongest magnetic field gradients. Consequently, our research is focused on designing biocompatible implants that produce strong magnetic field gradients near the surface of the implant so that sufficient doses of drug can be captured.
To maximize the magnetic field gradients, the implant is embedded in blood vessel walls in order to be in proximal contact with the blood flow. One key advantage to this drug delivery system is that the implant can be inserted by minimally invasive techniques such as catheterization, as opposed to surgical techniques used to implant other drug delivery devices. To accommodate more serious cases where open chest or otherwise more invasive procedures are necessary, surgical placement of magnetized implants solely for the purpose of targeted delivery of therapeutics may also be viable. A conceptual drawing of the implant-based magnetic drug delivery system can be found in Figure
The therapeutic agents that can be delivered to the implant by this method include pharmaceutical drugs, radioactive polymers, an cells. For example, in the treatment of coronary atherosclerosis, the ability to deliver growth-inhibiting drugs to the site of stent implantation can greatly reduce restenosis (the in-growth of arterial tissue following angioplasty). In addition, numerous low molecular weight polymer sphere formulations have been examined for their ability to deliver poorly water soluble drugs (such as paclitaxel, in the case of restenosis) by burst release, over a range of weight distributions for particle content65. The proposed design is perfectly in league with current technology in polymer science.
This drug delivery system could also offer significant benefits in the treatment of hepatic, renal, pancreatic, prostate and other cancers where the ability to provide multiple doses is of enormous importance. It may be possible to adapt currently used devices, such as the FDA approved coronary stent, with a pattern of magnetic material to attract magnetic drug to its surface. This approach is convenient for use in large vessels (i.e. vessels larger than 3 mm diameter) that are easily accessible by catheterization.
UNIQUENESS OF APPROACH
Magnetic targeting of drugs to specific sites in the body enjoys certain advantages over other drug delivery methods. Magnetic colloids can be injected into the bloodstream and guided to the targeted area with external magnetic fields, which is far less invasive than surgical methods for targeted drug delivery. Magnetic particles in fluids interact strongly with each other, which facilitates the delivery of high concentrations of drug to targeted
areas. Magnetic particles composed of magnetite are well tolerated by the human body.
In addition, magnetic fields are well suited for biological applications as they are not screened by biological fluids and do not interfere with most biological processes.
In most applications, however, the magnetic field gradients used to guide the magnetic drug are generated externally. The disadvantage to this approach is that externally generated magnetic fields apply relatively small and insufficiently local forces on micron-sized magnetic particles. Furthermore, the production of high gradient external magnetic fields causes the drug to be pulled towards the surface of the body and away from the site of interest. In cases where the treatment site is deep in the interior of the body, this technique may not be a practical solution.
For these reasons, there has been great interest in devising systems that produce strong and highly localized field gradients in the interior of the body. The use of internal magnetic field gradients has been employed to create embolisms and controlled blockages at the site of vascular defects, such as aneurysms and arterio-venous malformations (AVMs). These applications, which attempt to block the flow through a blood vessel, are solutions for occluding cardiovascular defects rather than for drug delivery.
The efficiency of chemotherapy treatment may be enhanced to a great extent by magnetically assisted delivery of cytotoxic agent to the specific site. There are a large number of magnetic carrier systems which demonstrates increasing drug concentration efficiency at the tumor site35.
In transdermal drug delivery it is useful to over come transport barrier: dermal, epidermal and endothelial layers
9: CONCLUSIONS AND ADVANTAGES
This has provided a proof of concept to the proposed magnetically targeted drug delivery system. This system operates by first embedding magnetic implants, such as a stent, at designated sites in the cardiovascular system, and then attracting injected doses of magnetically susceptible drugs to those implant sites with the aid of a modest external magnetic field. Previous attempts to use magnetic particles in these applications have relied on high gradient magnetic fields produced by magnets external to the body to direct magnetic particles to specific locations. This limits the range of their applications. The main disadvantage of this approach is that externally generated magnetic fields apply relatively small and insufficiently local forces on micron and nano-scale magnetic particles. As a result, there has been great interest in devising systems that produce strong and highly localized field gradients in the interior of the body.
Rational magnetic implant design began with the selection of stent-simulating materials of different geometries, mesh sizes, and metallic content, suitable for in vitro flow experimentation. Stainless steel materials ranging from 316 to 302 grade were chosen, in grid-like mesh geometries, as well as in the form of a compression spring. A soft magnetic alloy of Cobalt-Nickel was selected as a practical material for increasing the saturation magnetization of the materials, while retaining a very low state of magnetization in the absence of an externally applied magnetic field. An electroplating setup was developed utilizing a cobalt anode, borate bath containing scaled concentrations of cobalt and nickel, and controlled by a potentiostat. By combining the use of very weakly magnetic materials (316L SS) and highly magnetic materials (302 SS) with varied plating heights of soft magnetic alloy, flow experiments were able to examine
the scalability of magnetic capture over a range of saturation magnetizations.
Two flow systems were used to test the proposed design for magnetic particle capture to the wires of model stent materials. The first system employed was a project specific design of a parallel plate flow chamber (PPFC) adaptable to various channel heights and capable of sustaining high volumetric flow rates needed to obtain physiologically significant flow velocities. The second system for flow analysis of the proposed design utilizes three-dimensional model implants placed within a pipe flow system.
The final area of examination for completing a thesis on a magnetically targeted drug delivery system was indeed biocompatibility.
ADVANTAGES AND RISKS
As implants are foreign bodies, and we know the body will always recognize them as such, there remains a long-term threat for future inflammation, thrombus formation, and other complications. In our current fast food culture with patients as young as their 30s receiving coronary stents, if such patients make the appropriate lifestyle adjustments, they may otherwise live to be 90 years old. What happens to these stents as the patient ages? It became obvious to me that if one could safely adapt stents magnetically for the delivery of magnetic drug, the ability to dose the site repeatedly over time would provide a clear clinical advantage. Not only was repeatability advantageous, but if a high degree of control over magnetic dosing could be built into the design, clinicians could have more flexibility for treating on a patient to patient basis. The long shelf-life of controlled release spheres and non-drug-coated stents, compared to the expensively sterilized and briefly storable drug-eluting stents were other obvious benefits.
Proposed method for local drug delivery does not have to be disruptive, but
rather complementary; meaning that it can be paired with drug-eluting stent technology to provide an enhancement for additional doses along the lifetime of the implant. Drug eluting stents are limited by some of their problems: complications related to implantation, cracking of the polymer layer, limited dose size, shelf life, and the fact that they can only provide a single dose. For the other uses of stents (biliary, renal, brain), pacemakers, and orthopedic implants, a dose at implantation may not be necessary, making the addition of magnetic drug delivery functionality beneficial for prevention of complications in the future as needed. It also considered the use of endovascular and extravascular implants for treatment of localized tumors. Implants in these cases
would have the sole or primary function of facilitating local magnetic drug delivery, and could be implanted by catheter, or in cases where open chest surgery is already required to excise tumors, implanted extravascularly over vessels or organs. This could provide a future option for local chemotherapy at the same site should carcinomas be found to be re-growing during remission.
Obviously the benefits of this system must not come at the cost of increased risk in other arenas, such as chemical tolerance of a magnetic coating or final compositions of polymer and magnetite crystals. The approach I have taken with this thesis utilized previously FDA approved magnetic particle composites, as well as both soft magnetic coatings and magnetic alloys in order to explore the range of manufacturing capabilities that maintain the fundamental essence of the technology: controllable local delivery of magnetic drug to the wires of a stent. As biocompatibility surely represent a roadblock in clinical testing, having flexibility in the design will make the system that much more attractive to industry. Development and in vivo testing of final designs should heavily extend efforts to construct a magnetic drug delivery system that will allow for safe and effective MRI procedures for patients receiving the implants.
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