Super paramagnetic iron oxide nanoparticle coupled tissue soldering

Field of the invention

The present invention relates to the field of sutureless thermal mediated tissue fusion by using superparamagnetic iron oxide nanoparticles (SPIONs) dispersed in or immobilized to a solder interface, for the development of minimally invasive surgical procedures.

Background of the invention

Minimizing tissue injury has been a key interest ever since surgery has been performed. Development of micro-optics has permitted a constant improvement towards minimally invasive and endoscopic approaches (Quayle S. S. et al., Urology 66:489-493, 2005). For endovascular procedures the point of interest is remote to the point of entry. Parallel to these developments, the underlying treatment of the pathology needs to be addressed specifically, hence miniaturized and elaborated, such as by using sutureless tissue fusion techniques. Research in these directions was emphasized over the last three decades, focusing on either a micromechanical, gluing or soldering technique (Constantinescu M.A. et al., Lasers Med Sci 22:10-14, 2007). From these three techniques, soldering appears to have the best profile for clinical application. Adding proteins or polymers to the solder has further improved the connective tensile strength of the tissue fusion, but reproducibility remained problematic (Lauto A. et al., Lasers Surg Med 35:140-145, 2004). Described soldering techniques almost unanimously consisted of optical systems, with highly variable heat deposition resulting from the irregularity of laser beam tissue penetration. Furthermore inhomogeneity in solder deposition complicates the regularity of the resulting connective tensile strength. In contrast, electromagnetic induced thermal induction is less susceptible to these parameters and thus seems advantageous for tissue soldering, especially when the electromagnetic source can be applied close to the site of action.

Techniques for tissue fusion should result in an immediate connection, responding to the condition required for the respective application. This can be achieved only by using a mediator such as a mechanical connector, biochemical glue or a soldering technique. For all these techniques the balance between strength of the tissue connection and tissue destruction resulting from long term effects, such as fibrosis or intimal hyperplasia in the case of vascular anastomosis need to be determined. Furthermore new techniques for tissue fusion need to compete with the standard of care such as microsurgery using

microsutures or newly non-occlusive anastomosis techniques in order to reduce treatment and pathology related morbidity. Therefore all new developments need to consider the integration of minimally invasive techniques such as endoscopic or endovascular assisted methods. Furthermore the transmitting medium needs to be biocompatible or bioresorbable.

Superparamagnetic iron oxide nanoparticles (SPIONs) are obtainable by many different methods, including co-precipitation, thermal decomposition and/or reduction, micelle synthesis, hydrothermal synthesis, and laser pyrolysis techniques (Lu A.H. et al., Angew Chem lnt Ed 46:1222-1244, 2007). Heating physics of the SPIONs is essentially different from previous descriptions of radiofrequency tissue fusion (US 2004/0210282; US 2003/0236518; Flock ST. and Marchitto K.S., Otolaryngol Clin North Am 38:295-305, 2005). SPIONs are non-conductive and thus are not electromagnetically inducible.

Summary of the invention

The invention relates to superparamagnetic iron oxide nanoparticles for use in sutureless thermal mediated tissue fusion. The invention furthermore relates to a pharmaceutical preparation comprising superparamagnetic iron oxide nanoparticles, preferably with a diameter of below 20 nm, and such nanoparticles coated with an organic compound and/or imbedded in a biocompatible material, for example a soldering agent.

The invention likewise relates to the use of superparamagnetic iron oxide nanoparticles for the manufacture of a medicament for use in tissue fusion with an alternating electromagnetic field, and to a method of tissue fusion wherein superparamagnetic iron oxide nanoparticles dispersed in a biocompatible interface material are heated by application of an alternating electromagnetic field.

Brief description of the figures

Figure 1: Schematic stimulating setup. A high frequency power generator (PG) creates an electromagnetic field, inside a thin water cooled cooper coil (CC). A fluoro-optical temperature sensor system is used for temperature feedback (TM) which allows controlling the negative feedback loop by computer steering (CS). Electromagnetic tissue fusion site (B).

Figure 2: Haematoxylin and eosin (HE) staining of side to side SPION dispersed albumin soldering of two abdominal rabbit aortas, showing the tight contact of the two layers. Collagen structures are minimally altered.

Figure 3: Electron micrograph of superparamagnetic iron oxide nanoparticles as they were used for tissue soldering in Figure 2.

Figure 4: Schematic drawing of nanoparticles coated with polyvinyl-alcohol and proteinous macromolecules bonded to it, for specialized soldering or fixation to a scaffold structure.

Figure 5: MRI signal decay curve for liver as the region of interest (ROI) of 7 rats (sham, 1d, 3d, 2w, 4w). The slopes of the curves do not significantly differ for rats with implanted film and sham treated rats as well as for different implantation times. X-axis represents TE relaxation time and y-axis represents T2-intensity.

Figure 6: T2w sagittal and axial image showing susceptibility at the soldered site (arrows) 4 days after subcutaneous tissue soldering with SPIONs in the neck of the rat.

Detailed description of the invention

The present invention is directed to the use of superparamagnetic iron oxide nanoparticles (SPIONs) dispersed in biocompatible material to produce tissue fusion by application of an alternating electromagnetic field and thus heat generation. The invention as described herein offers a solution to the problem of tissue fusion and responds to the needs of the skilled person in the art.

Superparamagnetic iron oxide nanoparticles are known and can be obtained by known methods. As used herein, superparamagnetic iron oxide nanoparticles also includes corresponding superparamagnetic nanoparticles obtained from iron-platinum (FePt). Of particular use in the method of the invention are SPIONs of a diameter of below 20 nm, for example between 5 nm and 20 nm, e.g. around 15 nm. These nanoparticles may be applied as a dispersion in a biocompatible material, whereby such a dispersion may consist of completely dispersed nanoparticles or may also contain agglomerates comprising a few nanoparticles, e.g. agglomerates of 2 to 50, or preferably 2 to 20, nanoparticles.

A biocompatible material is a material that may be applied to the animal body, in particular to the human body, and is non-toxic, causes no or only limited irritation at the site of application, does not cause a rejection by the animal or human body, and may be temporarily or permanently integrated into structures of the animal or human body.

Examples of biocompatible materials are low molecular weight compounds, e.g. water, solutions of inorganic salts in water, for example physiological saline, aqueous solutions of amino acids and/or sugars, aqueous solutions comprising excipients such as are used for the preparation of injectable pharmaceutical preparations, proteins, peptides and glycopeptides of animal or human origin, and biocompatible polymers.

Such biocompatible polymers are polystyrenes, for example poly(styrene-co-chloromethyl- sytrene), poly(styrene-co-chloromethylstyrene-co-methyl-4-vinylbenzyl) ether, poly (styrene-co-chloromethylsytrene), their derivatives and copolymers; polyphosphoester, for example poly[1 ,4-bis(hydroxyethyl)terephthalate-co-ethyloxyphosphate], poly((lactide-co- ethyleneglycol)-co-ethyloxyphosphate), poly(1 ,4-bis(hydroxyethyl)terephthalate-co- ethyloxyphosphate), their derivatives and copolymers; polyphosphazenes, for example poly(bis(4-carboxyphenoxy)phosphazene), poly(bis(1-(ethoxycarbonyl)methylamino) phosphazene), poly(bis(1-(ethoxycarbonyl)-2-phenylethyl)phosphazene), their derivatives and copolymers; aliphatic polyesters, for example polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers, for example poly(lactide-co-caprolactone); polyhydroxybutyric acid (PHB), polyhydroxyvaleric acid (PHV), and their copolymers, for example poly(hydroxybutyric acid-co-hydroxyvaleric acid), their derivatives and copolymers; polybutylene succinates, their derivatives and copolymers.

Further biocompatible polymers considered are those containing a hydrolyzable backbone, for example poly(amide-enamines), their derivatives and copolymers; poly(anhydrides), for example poly(adipic anhydride), their derivatives and copolymers; natural polymers and polysaccharides, such as cellulose, chitosan, maltodextrin, starch, agar, alginic acids and their copolymers and derivatives; polypeptides, for example gelatin, their derivatives and copolymers, poly(ethylene glycol) (PEG) based polymers, their derivatives and copolymers; polypropylene, its derivatives and copolymers, for example poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); polyanhydrides, for example (4-carboxyphenoxy)propane, their derivatives and copolymers; polymers with carbon backbones, for example polyvinyl alcohol) and poly(vinylacetate), their derivatives and copolymers; dendrimers, for example, PAMAM

dendrimers, cyclotriphosphazene-PMMH-6 dendrimer, PAMAM-OS-trimethoxysilyl dendrimer, their copolymers and derivatives; hydrogels and crosslinked polymers, for example poly(acrylic acid-co-acrylamide), poly(isobutylene-co-maleic acid), poly (isobutylene-co-maleic acid), poly(N-isopropylacrylamide), poly (isobutylene-co-maleic acid), poly(acrylic acid), and their derivatives; hydrophilic polymers, for example lignosulfonic acid, polyacrylamide, polyacrylic acid, poly(acrylonitrile), poly(allylamine), poly(isobutylene), poly(4-vinylpyridine), polyvinylpyrrolidone, their derivatives and copolymers; hydrophobic polymers, for example cucurbit[x]uril, polyacrylonitrile poly (1-decene-sulfone), poly(2-ethylacrylic acid), poly(i-hexadecene-sulfone), poly(ethylene terephthalate), poly(hexafluoropropylene oxide), poly(i -hexene-sulfone), poly(methyl vinyl ether), poly(i -octene-sulfone), poly(propylene glycol), poly(propylene glycol) diglycidyl ether, poly(propylene glycol) methacrylate, poly (propylene glycol) monobutyl ether, poly(propylene glycol) 4-nonylphenyl ether, poly(i -tetradecene-sulfone), poly(tetra- hydrofuran), poly(vinylbenzyl chloride), polyvinyl chloride, poly(4-vinylphenol), poly (4-vinylpyridine-co-styrene), their derivatives and copolymers; polyaminoacids, for example polylysine, their derivatives and copolymers; polycarbonates, polysulfones, polymethacrylate, and salts of these mentioned polymers.

Preferred biocompatible materials are proteins, peptides and glycopeptides of animal or human origin, and biocompatible polymers.

SPIONs are dispersed in a solder for improvement of tissue fusion. A "solder" as understood in the context of the invention is a biocompatible material, for example as described hereinbefore, which assists in gluing tissue when heated. The solder may contain small or large molecules suitable for tissue soldering. Such molecules are biocompatible proteins, crosslinkers or polymerisates. Preferred substances are serum proteins and other proteins present in the animal or human body not having any immunogenic properties, and thus being clinically applicable, for example albumin, fibrin, fibrinogen, myoglobin, and collagen of different types. Particularly preferred is albumin.

SPIONs may be dispersed in a biocompatible scaffold. The scaffold is either of solid, flexible, and/or semi-liquid-state. The scaffold may have different porosity. Suitable biocompatible material forming such a scaffold are listed hereinbefore.

Common clinically established polymers preferred as scaffolds comprise polyglycolide, polylactides, polycaprolactone; di- and tri-block polymers, such as polymers incorporating

polyethyleneoxides or polycaprolactone, and other resorbable polymers in different chain arrangements which confer both degradation and mechanical property customization; other polymeric biomaterials, such as polyorthoesters, polyanhydrides, polyhydroxy- alkanoates, polypyrroles, poly(ether ester amides), elastic shape-memory polymers; hydrogels; and biomimetic materials, supramolecular polymers formed by self-assembly, and matrices presenting distinctive or a variety of biochemical cues (Polym lnt 56:145- 157, 2007). The scaffolds include furthermore foam material that can be stabilized by SPIONs or can contain SPIONs.

The morphology, magnitude, and interconnection of the scaffolds' porosity are critical factors in assessing their viability as tissue engineering devices (Adv Polym Sci 200:209- 231 , 2006). The porosity can be introduced into the scaffolds by methods such as melt extruding methods, solvent casting and particulate leaching methods, phase separation techniques, freeze drying, carbon dioxide foaming, chemical crosslinking of the polymers by crosslinking techniques including as well thermal and photocrosslinking, solid free form fabrication (Adv Biochem Engin/Biotechnol 102:187-238, 2006), wet spinning, electrospinning, or self-assembly techniques. Likewise sintered materials containing superparamagnetic nanoparticles can be used. The solder material can be either incorporated directly in the scaffold material or added subsequently into the pores. A further useful method consists of dissolving a polymer without and with nanoparticles in a solvent, and adding particles of a leachable porogen, such as a salt or glucose. The mixture is left to dry in air or in a vacuum until the solvent has evaporated completely. The porogen is then dissolved in a suitable solvent leaving behind a network of interconnected pores. Porosities can be controlled by the size of the porogen (10 μm to 2 mm) and the weight % of the inserted porogen (10 - 98 wt%). Phase separation methods consist of inducing a solid-liquid or liquid-liquid phase separation of the polymer solution. The polymer is dissolved in a solvent and quenched at a certain temperature ranging from 0 ⁰ C to -196 ⁰ C. The solutions are finally freeze-dried for several days in vacuum. With this method microstructured materials can be created. Structures can be controlled by varying certain processing parameters such as the quenching temperature, the freeze-drying temperature, and the polymer concentration (Adv Polym Sci 200: 209-231 , 2006).

The SPION dispersed scaffold may contain further bioactive molecules enabling a long term stimulative or inhibitory effect on surrounding viable tissue. Bioactive molecules may be of enzymatic, hormone, cytoinductive or cytoinhibitory effect. Examples of preferred bioactive molecules are vascular endothelial growth factor (VEGF), nerve growth factor

(NGF) ₁ brain derived neurothropic factor (BDNF), glial derived neurotropic factor (GDNF), erythropoietin (EPO) ₁ fibroblast growth factor (FGF), transforming growth factor (TGF), insulin-like growth factor (IGF) ₁ and subgroups of the mentioned compounds.

Further stimulative or inhibitory molecules considered are activins, inhibins and receptors, angiogenic factors, chemokines, ephrin and ephrin receptors, epidermal growth factors (EGF) ₁ hematopoietic cytokines, hepatocyte growth factor, interferons (IFN), interleukins (IL), neurotrophic factors, oncostatin M (OSM) ₁ platelet-derived growth factor (PDGF) and corresponding receptors, pleiotrophin, tumor necrosis factor (TNF), and subgroups of the corresponding families.

Further substrates or organic or inorganic additives may be added to the solder in order to cause a conformational change resulting in unique properties in conjunction with the nanoparticle tissue fusion by electromagnetic stimulation. Such additives may permit a physical conformation change of the solder during the fusion procedure from liquid to solid or from solid to liquid state, or help keep the solder liquid or solid, respectively. Additives may, for example, be activated by heat. Particular additives considered are acrylates, tensides, inorganic salts, surfactants, lipids and sphingolipids.

The present invention is directed to the fusion of biological tissue surfaces independent of their nature. Particularly preferred biological tissue surfaces are skin, or the surface of musculoskeletal, visceral, vascular, or neuro-glial tissue. The tissue is fused using the intermediacy of biocompatible material containing nanoparticles of the invention. The strength of fusion may be anywhere from weak to strong, and may be for temporary binding or permanent binding whereby natural tissue replaces the fusion over time.

Superparamagnetic iron oxide nanoparticles (SPIONs) are bioresorbable and are known to be eliminated by macrophages of the mononuclear phagocytosis system.

The present invention is directed to pharmaceutical preparations comprising superparamagnetic iron oxide nanoparticles (SPIONs), in particular such nanoparticles having a diameter of below 20 nm, for example between 5 and 20 nm, such as around 15 nm. The nanoparticle may be used as such or coated with an organic compound, for example coated with a polymer layer. This coating material may be further connected to proteins or biocompatible polymers. The pharmaceutical preparation may further comprise a

biocompatible material as defined hereinbefore, in particular a soldering agent, and other excipients conventionally used in injectable solutions.

Such excipients considered are, for example, preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity- increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80 (polyoxy- ethylene(20)sorbitan mono-oleate).

The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers.

The present invention uses an electromagnetic wave generator setup including a thermal feedback steering system, for stimulation of superparamagnetic iron oxide nanoparticles (Figure 1).

In electrically conductive metal parts the main heating effect in an electromagnetic alternating field is resistive heating caused by magnetically induced currents called eddy currents. The heating of the SPIONs as described in this invention is due to Neel relaxation, which occurs in superparamagnetic particles exposed to an alternating magnetic field. The extent of the relaxation mechanism contributing to the heating is dependent on the size of the particle, the frequency, and temperature. Heating of the nanoparticles occurs by Neel relaxation, which itself depends on the volume of the particles and the anisotropic constant. Neel relaxation is caused by the reorientation of the magnetization vector inside the (fixed) magnetic particle against an energy barrier. Each magnetization and demagnetization of the superparamagnetic particles leads to the dissipation of heat, which can be increased by increasing the applied frequency. The specific absorption rate (SAR) quantifies the rate of energy deposition in tissue in hyperthermia, which is a measure of the amount of energy converted by the magnetic particles from the magnetic field into heat per unit time and mass. Usually SAR is described by power losses due to Brownian and Neel relaxation mechanisms:

(m//ωr _c πT

P =

2τ _cir k _n TV{ l + ω ² τl _tt )

The SAR depends on the applied magnetic field strength (H), the frequency (ω), the effective relaxation time which combines the Brownian and Neel relaxation (τ _eff ), the temperature (T) and the volume of the particles (V). k _B is the Boltzman constant and m the magnetic moment of the particles. Physical limits of the above equation are given by the superparamagnetic limit, which is for iron oxide between room temperature and 10O ⁰ C at around 20 nm particle diameter. This means that the particle has to be smaller than 20 nm. The limit for the maximal frequency is given by the relaxation time which is determined by the volume of the particles and the magnetic exchange energy. For a typical heating application the diameter of the particles has to be between 10 and 20 nm, preferably between 15 to 20 nm, and the frequency between 1 and 30 MHz, preferably between 20 and 30 MHz. At these high frequencies, the second relaxation mechanism, the Brownian relaxation (particles rotate according the applied field) does not contribute to the heating.

SPIONs may act in agglomerates or as individual particles. In general, with increasing frequency the heat generation increases. This effect is most important around the optimal particle size of around 15 nm.

The electromagnetic field transmitter device design and power specifications are dependent on the site of the field application in the body (at the surface or deep in the body), site access (wide or narrow), and the necessary SPION dispersed solder interface heating specifications. For example fusion of vascular tissue may necessitate a different solder type and thus SPION dispersion than neuronal, dermal or visceral tissue. A deep seated structure in the depth of a craniotomy gives less freedom of movement than a structure located at the body surface. Thus the design of the transmitter device can vary form slim and long to short and wide. The device may be miniaturized and serve in minimally invasive surgery and in endoscopy. The device comprises, for example, a fluoro-optical temperature sensor. Other temperature feedback techniques may be used, e.g. a thermocouple, laseroptical or a thermocamera system.

Locally applied electromagnetic stimulation results in heating of the SPIONs and the biocompatible material comprising the SPIONs in dispersed form. This leads to fusion of the tissue in contact.

The transmitter induction properties should demonstrate a clinically suitable time lapse to reach the preferred target temperature of between 4O ⁰ C and 150 ⁰ C, in particular 8O ⁰ C ± 5,

for surgical reasons such as limited immobilization of the soldering site and the changes in the soldering environment. Furthermore the temperature profile and especially the temperature feedback mechanism should result in minimal fluctuations in the temperature profile. Temperature fluctuations observed are minimal with the SPIONs dispersed in solders. Furthermore SPIONs are homogenously dispersed over the tissue interface, permitting a good temperature feedback control.

Electromagnetic heating of SPIONs is dependent on the size of the particles and the medium they are dispersed in. Conductivity (S/m) is defined as the inverse resistance and describes how much the electromagnetic wave is attenuated as it transits the material. Conductivity reduces the electromagnetic radiation penetration into or through material, as the free electrons and ions in the material are moved by the incoming wave's field. Conductivity is dependent on the applied frequency and the tissue exposed to the irradiation. For example, the conductivity is expected to be between 0.1 and 1 S/m at the particular exemplified frequency of 1.8 MHz. The conductivity loss over a tissue segment of a thickness between 0.5 to 1 mm is thus negligible in a local applicable induction system.

The IEC guidelines recommend not exposing human whole body to more than 4 W/kg in a high frequency field, and the electromagnetic field produced by local transmitters such as cellular phones (900-1800MHz) is controversially discussed. Although the electromagnetic induction power unit used in the exemplification of this invention is designed for fusion of metal parts, the frequency at which the induction is performed (1 -30 MHz) as well as the dimension of the coil and thus the field dimension are not of safety concern for a whole body exposure as well as local close application to biological tissue, as long as not metal parts, or metal dust is involved.

The SPIONs obtained by the co-precipitation method generally yield individual particles with an average diameter around 10 nm, for example 9 nm. Depending on the measurement technique, size distributions from 5-15 nm (number weighted) are obtained. These individual particles may be coated with synthetic polymers, fatty acids, proteins, carbohydrates, or inorganic materials to yield either individually coated single particles, or agglomerates of the latter. This is influenced by the intrinsic properties (e.g. hydrophilicity, hydrophobicity) of the coating material used. While, for example, polymer chains adsorb on the surface of the nanoparticles, covalent coupling to the oxidic surface is achieved

using silanes such as aminopropyltriethoxysilane (APS), tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), or derivates thereof.

Coating substances considered are, for example, the biocompatible polymers listed hereinbefore, and biocompatible proteins, crosslinkers or molecules enhancing the tissue fusion. These coating substances may be attached directly or through an intermediary functional silane as described hereinbefore. Preferred coating substances are serum proteins and other proteins present in the animal or human body not having any immunogenic properties, and thus being clinically applicable, for example albumin, fibrin, fibrinogen, myoglobin, and collagen of different types. The particle surfaces can be further functionalized according to standard bioconjugate coupling techniques. Molecules to be coupled to the particles are either directly covalently attached to the SPIONs or via standard cross-linkers. The chemistry of reactive groups is well known and depends on two interrelated chemical reactions: the reactive functional groups present on the various cross-linking or derivatizing reagents and the functional groups present on the target macromolecules to be modified. An example of a polymer coated nanoparticle linked to further peptides is shown schematically in Figure 4.

The compounds of the invention may not only be used for fusion of biological tissue, but also for fusion of biological tissue to implants. For that purpose, the SPION dispersed solder composition optionally comprises additives depending on the implant material to which fusion is required. For example, the material used in a vascular graft is typically manufactured from polytetrafluoroethylene (PTFE). The SPION dispersed fusion composition will then be adapted to preferentially adhere to PTFE. As a result, the biological tissue is fused to the implanted material, and the implant is thereby sealed and stabilized. A particular application is the fusion of a PTFE vessel graft to a blood vessel for bypass surgery. Other implantable materials may be on the basis of polygly-co-lactide (PGA-PGLA), or any one of the biocompatible polymers listed hereinbefore.

Experimental part

Superparamagnetic iron oxide nanoparticles (SPIONs)

Superparamagnetic iron oxide nanoparticles are prepared by alkaline co-precipitation of ferric and ferrous chlorides in aqueous solution as described by Chastellain M. et al., J Colloid Interface Sci 278:353-360, 2004. The obtained black precipitate is washed several times with ultra-pure water and the remaining solid refluxed in nitric acid (10 ^'2 M) in the

presence of iron-(lll)-nitrate. The obtained brown suspension is dialyzed against 0.01 M nitric acid for two days, and stored at 4 ⁰ C.

SPIONs embedded in BSA SPIONs are concentrated from the original dispersion (10 mg/ml Fe) by different methods. a) Solvent evaporation: The concentration of iron is augmented in a graduated cylinder at 9O ⁰ C up to 300 mg/ml Fe. b) Precipitation: The original SPIONs are precipitated with 1 M NaCI. The precipitated particles are centrifuged at 5000 g to separate the particles from the salt solution. The sediment is redispersed in 0.01 M nitric acid. The final concentration of SPIONs is 300 mg/ml. These dispersions are diluted to a final concentration of 5%, 10%, and 20% iron containing 20% (w/w) BSA, which is added as a powder to the dispersed particles.

SPIONs in scaffolds Polymer film preparation: 15 ml of SPIONs (7.7 mg/ml Fe) are mixed with 7.5 ml of 1 M ammonia (Fluka), 150 μl oleic acid (Fluka, cat. no. 75093) and 105 ml chloroform (Fluka). The resulting mixture is centrifuged at 3000 g for 10 minutes to transfer the SPIONs into the organic chloroform phase. The water phase is discarded and the particle dispersion concentrated to a final iron concentration of 115 mg/ml (4 ml) under the fume hood.

Nonporous films of polycaprolactone containing 5, 10, 20, 30, and 40% (w/w) iron are prepared by using a solvent-casting technique. For the preparation of a scaffold containing 40% (w/w) iron, 56 mg of polycaprolactone is dissolved in 1 ml of chloroform. Subsequently, 466 μl of the SPION dispersion described above is added and mixed. For scaffolds with lower iron content this value is adapted. This solution is spread to cover the bottom surface of a 32 mm Petri dish that is cleaned first with chloroform, followed by ethanol, and ultra-filtered deionized water. After air drying (24 h), the films are hydrated for 1 hour and then carefully peeled off the bottom of the Petri dish. Then, the resulting polymer films are air dried and stored at room temperature.

Porous films of polycaprolactone containing 5, 10, 20, 30, and 40% (w/w) iron are prepared by using a solvent-casting technique and particulate leaching technique. For the preparation of a scaffold containing 40% (w/w) iron, 56 mg of polycaprolactone is dissolved in 1 ml of chloroform. Subsequently, 466 μl of the SPION dispersion described above is added and mixed. Then sodium chloride particles of a size between 100 and 125 μm at concentrations of 70 to 90 (w/w) are added, corresponding to 217.7 and 839.3 mg,

respectively. For scaffolds with lower iron content these values are adapted. This solution is spread to cover the bottom surface of a 32 mm Petri dish that is cleaned first with chloroform, followed by ethanol, and ultra-filtered deionized water. After air drying for 24 h, the films are hydrated for 1 hour and then carefully peeled off the bottom of the Petri dish. Then the resulting polymer films are air dried and stored at room temperature.

Polycaprolactone may also be replaced by poly (DL-lactide-co-caprolactone) 40:60, poly (DL-lactide) and other copolymers.

Induction heating system setup

For the soldering experiments a system consisting of a high frequency generator (Power Controller 32/1800, Ceia, Italy) operated at a frequency of 1.8 MHz and a magnetic flux density of 40 mT is used. The generator is connected to a control unit (Power controller v2, Ceia, Italy), which sets the energy output of the system at a predefined optimum for the special coil. The alternating current is transferred on a water cooled coil consisting of a thin copper tube which induces the electromagnetic field. This coil has an outer diameter of 5.5 mm, the tube itself has a diameter of 1.5 mm. Cooling is achieved by continuous water flow through the tube. The output power is controlled by reduction of the pulse length of the electromagnetic field for lower power levels or by changing the strengths of the electromagnetic field by changing the current.

A second and custom made induction device consisting of a frequency generator, an amplifier and a resonant circuit harbouring a ferrite core has been realized. The resonant circuit is justified for maximal energy absorption in the tissue. The operating frequency is adaptable from 30 kHz to 470 MHz, and the amplifier power is 2000 W.

For the temperature feedback loop a fluoro-optical temperature sensor (FOT Lab-Kit, Luxtron, CA, USA) is used, allowing the recording of a contact temperature profile at a pickup frequency of 4 Hz. A negative feedback control consisting of a control unit (Power controller v2, Ceia, Italy) between the temperature measurement system and the high frequency generator enables a fast and constant levelling of the temperature (Figure 1).

Metallic structures as energy mediator

A platinum ring (MR) (inner diameter: 2.8 mm, wire diameter: 0.25 mm) (ELANA BV, Utrecht, The Netherlands) and a stainless steel grid (MG) (BOPP, Zurich, Switzerland) with a wire diameter: 0.065 mm, mesh opening: 0.3 mm are used as solid mediators for

conversion of the electromagnetic to thermal energy and are compared with dispersed agglomerates of SPION with particles size of 15 nm in diameter.

Solder Solder consists of 36% (w/w) of bovine serum albumin (BSA) dissolved in pure sterile water for the experiments in conjunction with the solid metal parts (MR and MG). BSA concentration is kept at 20% (w/w) in combination with SPIONs.

Teflon receptacle A teflon receptacle is used for the analysis of the temperature profiles for MR and

SPIONs. The distance from the induction coil to MR and MG is between 1.2 mm and 2.4 mm and for the SPIONs 2.4 mm defined as the distance from the centre of the measured volume (30 μl) in the receptacle to the coil.

Soldering temperature parameters

The total induction time is dependent on the time needed to reach the target temperature of 8O ⁰ C ± 5 ⁰ C and the holding time. A tissue soldering holding time of 60 seconds is considered suitable for tissue fusion.

Vascular tissue and tensile strength measurement

Pieces of rabbit abdominal aorta (15 x 10 mm) are used for the in vitro soldering experiments (Soltermann, Thδrigen, Switzerland). The two adventitial surfaces are soldered together with an overlapping area of 1 cm ² responsible for the connection. The tissue is placed in a custom made tissue holder made out of Perspex, enabling a constant distance of the tissue and the induction coil.

The strength of the so induced tissue connection is quantified using a tensile strength measurement setup. Both pieces of soldered tissue are fixed with surgical clamps, one of them attached to a force gauge measuring the maximum tensile strength in mN during the rupture procedure.

Induction of the MR in teflon receptacle

The heating of the MR in 30 μl of albumin solder in the teflon receptacle is tested. Temperature heating curves are analyzed in dependence of power output and distance of the induction coil to the metal parts. A maximum temperature of 100 ⁰ C is reached. Induction time is 30 s.

Solder temperatures increased in dependence of power and distance during the 30 s induction time. The optimal distance for induction of the MR for tissue fusion is found to be in the range of 2 to 4 mm. The optimal range for power is found to be between 20 and 50%, always dependent on distance (distance 4 mm: 50% power, distance 2 mm: 20% power).

Induction of SPION in teflon receptacle

30 μl of SPION dispersed in 20% (w/w) albumin solder are induced in a teflon receptacle and analyzed with respect to their heating ability. 5, 10 and 20% (w/w) of SPIONs are analyzed. Distance from the coil to the nanoparticles is kept at the closest distance possible to the middle of the fluid volume of 2.4 mm, and the power output of the electromagnetic high frequency generator is set to 90% for an overall activation time of 30 s. Increasing the SPION concentration from 5%, 10% to 20% results in an increase in solder temperature.

Tissue soldering: Induction of MR and MG with vessels

Pieces of vascular tissue are adapted as described above. Between the two tissue pieces a MR as well as 30 μl of BSA solder without and with an oxidized cellulose gauze (Tabotamp, Johnson and Johnson, Switzerland) is placed. This complex is exposed to the electromagnetic field for soldering. The grid is immersed in 30 μl of solder before placing it between the two tissue pieces. Temperature is regulated with the above described feedback mechanism at 80 ⁰ C. Induction time is 60 s after reaching 80"C. In order to maintain this temperature over 60 seconds, the power of the electromagnetic high frequency generator is regulated by the temperature control feedback to a mean power output of 20%.

Induction of SPION with vessels

Pieces of vascular tissue are adapted as described above. 30 μl of SPION (20% w/w) dispersed in BSA are used. The induction setup needs to be adapted in order to reach sufficient induction parameters to reach the acceptable temperatures. Temperature is regulated with the above described feedback mechanism at 8O ⁰ C. Induction time is 60 s after reaching 80 °C. The power of the electromagnetic high frequency generator is set at 90%, according to the findings from the teflon receptacle studies.

Tissue soldering compared with suturing

The most constant temperature profile was recorded for the SPION tissue soldering. Tensile strength of MR, MR-cellulose, MG and SPION were 2922 ± 623, 2668 ± 176, 3692 ± 190 and 3078 ± 852 mN (n=5), respectively. Findings are reported in mean ± standard deviaton (SD). For the analysis of the tensile strength between MR, MG and SPION the ANOVA test is used. Significance is defined at a p<0.05. Further, for comparison five pairs of tissue samples, identical to those used for the soldering experiments were sutured in conventional manner. A (8-0) Prolene thread (Ethicon J&J, Spreitenbach, Switzerland) was used to accomplish interrupted suture, seven stitches were performed along the tissue edge of 1 cm. Thereafter, tensile strength of these samples were measured, too, yielding a rupture force of 2580 ± 950 nM.

Histology

Soldered tissue is fixated in formaldehyde and later embedded into methylacrylate resin over a 30 day period for slow hardening in order to perform the tissue slices. The tissue blocks are then cut with a diamond band saw with a slice thickness of 150 μm (Exakt Apparatebau, Nordersted-Hamburg, Germany). Thereafter the slices are polished down to a thickness of 50 μm. The slices are stained using haematoxylin and eosin (HE). The histological HE staining of the electromagnetically induced vessel tissue fusion using SPION demonstrates the close contact of the abdominal aorta vessel tissue pieces (Figure 2).

In vivo tests

Subcutaneous implantation of SPION-BSA-PVA solder into 10 living rats (wistar rats) is performed in order to observe eventual pathologic effects on the living body, such as those observed in the iron storage disease called haemochromatosis.This hereditary disease is characterized by improper dietary iron metabolism (making it an iron overload disorder), which causes the accumulation of iron in a number of body such as liver, kidney and pancreas. Since nanoparticles are theoretically small enough to pass the blood-brain barrier, the brain is also investigated to achieve certainty that it is not affected. MRI is performed on the rats, and iron presence is checked in the regions of interest as a mean to measure iron content variations. To crosscheck the measurements, the rats are euthanized right after MRI and histology is performed on the organs of interest. This enables a control of the measured T2 ^* relaxation times (MRI). The resulting tissue iron content correlates with microscopical analyses of particular organs by special iron stainings.

Further information is collected about the behaviour of the film in living tissue. In order to find out how the surrounding tissue reacts to the implant, histology is performed on the implantation region.

Solder film composition was 25 % (w/w) SPION, 42 % (w/w) BSA and 8 % (w/w) water. Sample dimensions were 5 mm x 5 mm x 0.5 mm. Films were implanted subcutaneously into the neck of 8 rats; 4 of the films were then electromagnetically soldered, while 4 of them were kept in their raw state. MRI and euthanization was performed after 1 , 4, 15 and 28 days. MRI was done on one sham rat, and histology was made on two sham rats as well as on all other rats. Figure 5 shows that there are no significant changes in the presence of the SPION-BSA- PVA implant in the liver, spleen, kidney, brain and pancreas by MRI. However, a broad signal loss is achieved in the rats neck (Figure 6). Histology results prove that there is no inflammation occurring in the region of interest. Lack of presence of SPION in the region of interest is shown by staining with Prussian blue. The results prove that SPION film is relatively inert and SPIONs do not concentrate in investigated organs over a timerange of 4 weeks, and thus do not affect the function of those particular organs. In histology of the liver, a slight increase of Kupffer cells counts (macrophages of the liver) are observed, which indicates that some SPIONs are degraded.