EP4096624A1

EP4096624A1 - Paste comprising magnetic alkoxysilane-coated metal containing nanoparticles

Info

Publication number: EP4096624A1
Application number: EP21703186.3A
Authority: EP
Inventors: Stefan GERBES; Andreas Jordan
Original assignee: Magforce AG
Current assignee: Magforce AG
Priority date: 2020-01-31
Filing date: 2021-01-29
Publication date: 2022-12-07
Also published as: IL295186A; KR20220133893A; WO2021152136A1

Abstract

The present invention provides a paste comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles obtainable by a method comprising the step of thermally treating an aqueous suspension comprising agglomerates of alkoxysilane-coated magnetic metal containing nanoparticles; a medical device or a pharmaceutical composition comprising said paste; the paste for use in treating or preventing proliferative diseases; and a method of producing the paste. The paste is characterized by the presence of yield stress and by shear thinning behavior, allowing the paste to be drawn up into e.g. a syringe, whereby upon injection into a tumor, the paste remains at the site of injection.

Description

Paste comprising magnetic alkoxysilane-coated metal containing nanoparticles

Magnetofluids comprising magnetic alkoxysilane-coated nanoparticles are known in the art. For example, aqueous suspensions comprising magnetic alkoxysilane-coated metal containing nanoparticles, methods of producing them and their use in the treatment of proliferative diseases have been described in WO 2013/020701.

Magnetofluids comprising magnetic alkoxysilane-coated nanoparticles are injected into tumors for treating the tumors by hyperthermia upon applying an alternating magnetic field. Therein, the nanoparticles agglomerate and form a depot, a kind of implant. Since more than one depot can be generated by this process, the entirety of all depots in their shape, position and distribution determines the temperature distribution that is achieved when the particles are subsequently activated in the tumor by an alternating magnetic field. Optimally, the tumor is covered exactly by the therapeutically necessary minimum temperature and the areas of high temperature in which the tissue is directly killed lie exclusively within the tumor.

When injected into tumor, it may take some time before the magnetofluid solidifies due to the agglomeration of the magnetic alkoxysilane-coated nanoparticles. This process takes place in a nanoparticle depot from the outside to the inside, whereby the solid surface can tear, so that nanoparticles can flow off through cracks following internal surfaces, gaps or other weak points in the tissue into other areas outside the target area. Thus, the desired particle distribution in the tumor tissue cannot be achieved and, consequently, the desired therapeutic temperatures cannot be reached in the tumor tissue or healthy tissue may be damaged. This is a problem especially with small tumors.

Accordingly, there is a need to provide alternative magnetic particle compositions which avoid the flowing off of magnetic nanoparticles into areas outside the target area after injection into a target site.

The present invention relates to a magnetic particle composition which overcomes the disadvantage of deferred solidification after injection into the target site and the further disadvantage of spreading and scattering of the nanoparticles or agglomerates of nanoparticles into neighboring tissue.

The inventors of the present invention have surprisingly found that a particular pasty composition, i.e. a composition in the form of a paste, can be provided by thermal treatment when taking an aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles as a starting material. A pasty composition or paste is characterized by the presence of yield stress and by shear thinning behavior; allowing the paste on the one hand to be drawn up into e.g. a syringe, e.g. via a needle, or into a cannula. Upon injection into a tumor, but on the other hand to remain at the site of injection, as it is injected into the tumor as a composition which, after injection, immediately adopts a non-movable state, as compared to a fully liquid suspension which did not undergo any thermal treatment. This immobilization prevents the flowing off of nanoparticles and agglomerates into non-target area.

The present invention provides a paste comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles which can be injected in the daily routine precisely into tissue and remains at the injection site, without spreading or substantially spreading to surrounding tissue. This allows a more accurate hyperthermia treatment particularly of small tumors and of tumorous tissue in the vicinity of channels, where the suspension of magnetic nanoparticles would otherwise leave the treatment area. Furthermore, the present invention provides a manufacturing process for such paste.

In the following, the present invention is disclosed in detail. The features of the present invention are described in individual paragraphs. This, however, does not mean that a feature described in a paragraph stands isolated from a feature or features described in other paragraphs. Rather, a feature described in a paragraph can be combined with a feature or features described in other paragraphs.

In a first aspect, the present invention provides a paste comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles obtainable by a method, wherein the method comprises the step of thermally treating an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles.

In a preferred embodiment of the invention, the paste is characterized by plastic behavior. ln a further preferred embodiment of the invention, the paste is characterized by the presence of yield stress. The yield stress is preferably between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa.

In an equally preferred embodiment, the paste has a shear thinning behavior.

In a preferred embodiment of the invention, the paste is characterized by a viscosity, wherein the viscosity of the paste is between 0.03 and 7 Pa s, more preferably between 0.03 and 5 Pa s, still more preferably between 0.08 and 3.5 Pa s, still more preferably between 0.1 and 3.5 Pa s, still more preferably between 0.1 and 3.0 Pa s, still more preferably between 0.5 and 3.0 Pa s, still more preferably between 0.7 and 3.0 Pa s, still more preferably between 1 and 3.0 Pa, measured at a shear rate of 50/s.

In a preferred embodiment of the invention, the step of thermally treating is carried out at a temperature between 25 and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60 °C.

In a preferred embodiment of the invention, the step of thermally treating is carried out for a time period of between 0.1 to 96 hours (hrs), preferably for a time period of between 0.5 to 72 hours (hrs), more preferably for a time period of between 1 to 48 hours (hrs).

In an another preferred embodiment of the invention, the aqueous suspension comprises a concentration of at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M, as determined by its metal content.

In yet another preferred embodiment of the invention, the method further comprises the step of incubating the aqueous suspension of magnetic metal containing nanoparticles with an alkoxysilane before thermally treating the aqueous suspension.

In an embodiment of the invention, the step of incubating is carried out in the absence of an added organic solvent.

In an embodiment of the invention, the alkoxysilane is a trialkoxysilane, preferably selected from the group consisting of 3-(2-aminoethylamino)-propyl-trimethoxysilane, 3- aminopropyltriethoxysilane, trimethoxysilylpropyl-diethylenetriamine and N-(6- aminohexyl)-3-aminopropyltrimethoxysilane, especially 3-(2-aminoethylamino)-propyl- trimethoxysilane, and/or 0.3 to 0.6 x 10³ mol, preferably 0.4 to 0.5 x 10³ mol, more preferably 0.43 to 0.45 x 10³ mol, alkoxysilane per 0.9 mol metal is added in the incubating step.

In an embodiment of the invention, the magnetic metal containing nanoparticles comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, preferably iron salts, more preferably wherein the iron salt is an iron oxide, preferably magnetite and/or maghemite.

In an embodiment of the invention, the iron oxide nanoparticles are provided

(a) by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide, or

(b) by thermal decomposition of an iron salt or an iron complex compound.

In an embodiment of the invention, the method further comprises the step of ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles before thermally treating the aqueous suspension.

In an embodiment of the invention, the specific absorption rate (SAR) of the nanoparticles is larger or equal than 2 W/g Me, preferably larger or equal than 3 W/g Me, more preferably 4 to 12 W/g Me, as determined at a magnetic field strength of 3.5 kA/m and a frequency of 100 kHz.

In a further aspect, the present invention relates to a medical device comprising the paste of the present invention.

In another aspect, the invention relates to a pharmaceutical composition comprising the paste of the present invention.

In yet another aspect, the paste of the invention is for use in a method for treating or preventing proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection in a patient. In a further aspect, the pharmaceutical composition comprising the paste of the invention is for use in a method for treating or preventing proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection in a patient.

Equally, the present invention relates to a method of preventing or treating a proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection in a subject in need thereof, comprising administering the paste or the pharmaceutical composition of the invention to the subject in need thereof.

In yet another aspect, the present invention provides a method for producing the paste of the invention, wherein the method comprising any of the steps as detailed above.

In particular, the method comprises the step of thermally treating an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles. This thermal treating step may be carried out at a temperature between 25 and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60°C, and/or for a duration of time of between 0.1 to 96 h, preferably 0.5 to 72 h, more preferably 1 to 48 h. Preferably, the aqueous suspension comprises a concentration of at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M, as determined by its metal content. The method may further comprise the step of incubating the aqueous suspension comprising magnetic metal containing nanoparticles with an alkoxysilane before the step of thermally treating the aqueous suspension. The incubating step may be carried out in the absence of an added organic solvent. The alkoxysilane may be a trialkoxysilane, preferably selected from the group consisting of 3- (2-aminoethylamino)-propyl-trimethoxysilane, 3-aminopropyltriethoxysilane, trimethoxysilylpropyl-diethylenetriamine and N-(6-aminohexyl)-3- aminopropyltrimethoxysilane, especially 3-(2-aminoethylamino)-propyl-trimethoxysilane. 0.3 to 0.6 x 10³ mol, preferably 0.4 to 0.5 x 10³ mol, more preferably 0.43 to 0.45 x 10³ mol, alkoxysilane per 0.9 mol metal may be added in the incubating step. The magnetic metal containing nanoparticles may comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, preferably iron salts, more preferably wherein the iron salt is an iron oxide, preferably magnetite and/or maghemite, whereby the iron oxide nanoparticles may be provided by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide, or by thermal decomposition of an iron salt or an iron complex compound. Definitions

The term “comprise/es/ing”, as used herein, is meant to “include or encompass” the disclosed features and further features which are not specifically mentioned. The term “comprise/es/ing” is also meant in the sense of “consist/s/ing of” the indicated features, thus not including further features except the indicated features.

The term ..viscosity" refers to the quotient of shear stress t and shear rate

The first is a measure for the applied force F on the liquid in direction of flow, divided by the area A in direction of the flow (F/A). The latter is a measure for the velocity-gradient dv/dy in the liquid perpendicular to the flow of the liquid (see Fig. 1).

“Newtonian behavior” means that for such a liquid, h is a constant depending only on the temperature. That is, a graph of stress t versus shear rate g yields a straight line through the origin with h as its slope (see Figure 2a).

Further, “non-Newtonian behavior” means, that h is also a function of shear stress t and shear rate A good way to describe such a behavior is the equation of Herschel and Bulkley (1926):

T = T₀ + K†ⁿ

T₀= yield stress

K = consistency (slope at zero shear rate) n = flow index (curvature of the graph; referring to shear thinning (n < 1) or shear thickening (n > 1))

“Yield stress” refers to the necessary shear stress, above which a paste is movable, e.g. behaves like a liquid and/or adopts a liquid state. In a graph of shear stress t versus shear rate g, the yield stress is the value of the intersection of the graph and the y-axis (see Figure 2a). Yield stress is a property of the paste of the present invention, adopted by thermal treatment, allowing the paste to adopt a movable state by application of a shear stress above the yield stress, e.g. to behave like a liquid and/or to become a liquid, and to adopt a non-movable state by application of a shear stress below the yield stress or by not applying a shear stress. In this case the paste may e.g. behave like a solid and/or is a solid. The paste of the present invention shows non-Newtonian behavior, thus, the yield stress is above zero.

“Movable”, as used herein, refers to the adoption of a state of a paste, in which adopted state the paste is able to move or flow. This state is adopted by the influence of shear stress which converts the non-movable paste into a state, in which the paste can move or flow.

“Non-movable”, as used herein, refers to a state of the paste, in which the paste is not able to move or flow by itself. In the non-movable state, the paste may behave like a solid or is a solid. However, in the non-movable state, the paste can be deformed by applying shear stress in an elastic way only -in other words: is reversible-, as long as the applied shear stress is lower than the yield stress. Applying a shear stress higher than the yield stress transfers the paste from the non-movable to the movable state. “Solid”, as used herein, means that the paste is in a firm and stable shape.

“Shear thinning behavior” refers to a composition, where the viscosity decreases with increasing shear stress or shear rate. This results in lower necessary force if, e.g. the composition is forced through a small opening such as present in a syringe, needle or cannula.

“Plastic behavior” refers to a composition which is non-movable, e.g. behaves like a solid or adopts a solid state, below a certain value of shear stress (yield stress), but is movable, e.g. behaves like a liquid or adopts a liquid state (see Figure 2a).

“Paste” means a composition showing plastic behavior. A paste can have a non-movable state, (below its yield stress) or a paste can have a movable state (above its yield stress).

“Pasty” means the state adopted by a paste.

By “agitation”, as used herein, is meant any strong impact that is applied to the paste of the present invention. This includes strong shaking, preferably over a long time such as 6 to 24 hours, heavy vibration such as vortexing or application of a high kinetic impulse. Agitation changes the consistency of the paste having been thermally treated from a non movable composition such as a solid back to the original liquid consistency of the aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles, from which the paste is formed by thermal treatment. By “shearing” is meant any flowing movement of the material, e.g. paste, whereby a force in the direction of flow is applied on the material, e.g. paste. This includes the drawing up into a syringe or flowing through a thin cannula, spreading of the paste or the application of stress on the paste. The changes made by this type of influence are reversible, i.e. if the shear stress falls under the yield stress, the movement of the paste stops and the paste adopts its non-movable state, e.g. behaves like a solid or is a solid, without that thermal treatment is applied. If the shear stress rises over the yield stress, the paste gets into movement. Thus, “shearing” and “agitation” are different insofar, as changes by “shearing” are reversible, the paste remains a paste, by themselves, whereas changes by “agitation” -paste -> liquid- are reversible only upon thermal treatment.

“Specific Absorption Rate” (SAR) is a measure for the rate at which energy is absorbed by the nanoparticles upon exposure to the alternating magnetic field. It is dependent on the magnetic field strength and the frequency of the alternation of the polarization of the magnetic field. The SAR is preferably determined according to the method developed by Jordan et al. (1993) at a frequency of 100 kHz and a field strength of up to 18 kA/m, preferably at 4 kA/m and refers to the mass of used metal, e.g. iron (unit W/g metal).

"Zeta potential" refers to measured electrical potential of a colloidal nanoparticle in aqueous environment, measured with an instrument such as a Malvern ZetaSizer 3000 HSA at pH 5.2 and a conductivity of 2.0 mS/cm (each determined at 25°C). The zeta potential describes the potential at the boundary between bulk solution and the region of hydrodynamic shear or diffuse layer.

“Hydrodynamic diameter” describes the size of the agglomerates as found in the suspension or in the paste. It is determined by light scattering. In this context, the average size is determined in water according to example 3. With this light scattering measurement, the size of agglomerates of nanoparticles is determined - in contrast to the size of the ball-shaped or cubic electron-dense single nanoparticles (“primary particles”) which are forming such agglomerates.

“Z-average” with respect to the size of agglomerates means the readout of the light scattering size determination as carried out in example 3. Z-average values above the provided ranges lead to sedimentation of the nanoparticles and are therefore generally not suitable for the foreseen applications of these nanoparticles. “Hydrodynamic volume” means the average space needed for an average agglomerate. It can be calculated from the hydrodynamic diameter by the formula:

V_H = 1/6 nd wherein V_H is the hydrodynamic volume and d_H is the hydrodynamic diameter.

Surprisingly, it was found that the thermal treatment leads to an increase in hydrodynamic diameter hence in hydrodynamic volume. For example, within 48 at 60°C, the hydrodynamic volume has risen nearly by the factor of 2, as shown in Example 3.

The hydrodynamic size, the z-average- as well as the viscosity values seem to have a maximum, until which the measured values grow with time in an asymptotic manner, as shown in Example 6.

The hydrodynamic volume still includes water. Thus, by increasing the volume of the agglomerates, the volume of the suspension does not change. Thus, the hydrodynamic volume of the agglomerates is not exclusive. Nevertheless, it restricts the space for movement of the agglomerates. If the agglomerates are seen as spheres, there is a theoretical maximum for filling a space with spheres. This is about 74 % in case of ideal spheres of same size in a hexagonal or cubic dense packing in a perfect crystal lattice (Hollemann, 1985). In reality, the agglomerates will interact with each other even at much lower space filling, starting with thickening, shear thinning and at higher space filling plastic behavior (Mueller, 2010). Thus, the maximum for the growth of the agglomerates seems to be due to maximum space filling.

In the context of the present invention, the term “about” means a deviation from the given number or value of ± 10 %, preferably of ± 5 % and especially of ± 1 %.

The term “magnetic” incorporates magnetic, paramagnetic, ferromagnetic, anti ferromagnetic, ferrimagnetic, antiferrimagnetic and superparamagnetic. Preferably, the nanoparticles are paramagnetic, more preferably ferromagnetic, ferrimagnetic, antiferrimagnetic or superparamagnetic, particularly preferably, the nanoparticles are superparamagnetic.

The term “nanoparticles” shall mean nanoparticles in the nanometer range, meaning nanoparticles from 1 to 100 nm with respect to its metal core, as can be determined by electron microscopy. Preferably, the nanoparticles have a size of 5 to 25 nm, more preferably 7 to 20 nm and still more preferably 9 to 15 nm.

“Metal nanoparticle” refers to a magnetic nanoparticle, which contains metal or metal ions. The term “alkoxysilane coating” refers to a coating resulting from the polycondensation of alkoxysilanes, a process which is also referred to as “aminosilane coating”. The term “polycondensation” as used herein generally means any condensation reaction of a monomer with two functional groups which leads to the formation of a polymer and water.

“Agglomerating” means that several individual nanoparticles form agglomerates or clusters of nanoparticles. “Agglomerates” refer to agglomerated nanoparticles or clusters of nanoparticles.

As shown in the examples, it has been found in the context of the present invention that upon thermal treatment, the consistency of an aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles is changed from liquid into a pasty. Thus, the aqueous suspension becomes pasty.

The paste is characterized by the presence of a yield stress and by a shear thinning behavior, meaning that the paste can adopt a non-movable, such as a solid, state and a movable, such as a liquid, state. If a shear stress higher than said yield stress is applied to the paste, the paste adopts a movable state, e.g. the paste may show the characteristics of a liquid, e.g. it may become a viscous liquid, whereby the viscosity decreases with increasing shear stress or shear rate. Preferably, the paste of the present invention is characterized by a yield stress of between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa. Thus, the paste of the present invention shows a “non-Newtonian behavior”, preferably a “plastic behavior”, as defined above.

Upon agitation, as defined herein, the paste is converted a liquid suspension with nearly the same rheological properties as the suspension from which it is formed by thermal treatment, without plastic behavior. The viscosity and the yield stress can be newly set to obtain desired values by thermal treatment. This means, the conversion of the liquid suspension to the paste is reversible.

Thus, the special properties - the yield stress and/or the elevated viscosity - gained by the thermal treatment are reversible upon agitation. Due to such reversibility, yield stress and/or viscosity can be adjusted to any levels which are desired. For example, yield stress can be adjusted to between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa and/or viscosity can be adjusted to values between 0.03 and 7 Pa s, more preferably between 0.03 and 5 Pa s, still more preferably between 0.08 and 3.5 Pa s, still more preferably between 0.1 and 3.5 Pa s, still more preferably between 0.1 and 3.0 Pa s, still more preferably between 0.5 and 3.0 Pa s, still more preferably between 0.7 and 3.0 Pa s, still more preferably between 1 and 3.0 Pa s, as determined by rotational rheometry at 20°C, measured at a shear rate of 50/s.

Upon shearing, as defined herein, the paste leaves its non-movable state, however, only to an extent that reversibility from the adopted movable state to the non-movable state is achieved by itself, i.e. if the shear stress is below the yield stress and without thermal treatment. Thus, yield stress and shear thinning behavior have also the effect that the paste is processable, as, due to the adoption of a movable state by applying shear stress, the paste can be for example drawn up into a cannula or into a syringe, e.g. through a needle, as shown in example 7. When the shear stress is diminished or stopped, the movable state of the paste is reversed into the non-movable state, e.g. solid state. This means that the paste, when drawn up into a cannula or syringe, adopts its movable state as long as moving through narrow openings or through a cannula, so that it can easily be handled. This further means that upon injection into a desired site, the paste gets non movable, due to the drop of shear stress between hard cannula and soft tissue after leaving the narrow cannula. This process of adoption of the previous state regarding movability and/or viscosity is much quicker than the agglomeration/coagulation process which occurs in a non-thermally treated suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles at contact with a physiological saline solution, as e.g. present in a body, leading to solidification.

This behavior is shown in Figure 4. In both beakers, a 6 M aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles was dropped into a physiological salt solution (0.9 % by weight of NaCI). In the left beaker, the solidification of the thermally treated (1 h, 60°C) suspension into hard and sticky droplets of compacted state is shown, whereas in the right beaker, a spread and scattered mass of the agglomerates of the aqueous suspension, not subjected to thermal treatment, is visible. In both cases, the suspension solidifies, as the agglomerates fall through the salt solution. While in the left beaker, the paste from the thermally treated suspension regains its solid character directly after leaving the cannula and therefore keeps the form of the droplets, the droplets of the non- thermally treated suspension entering the salt solution as a liquid are spread and scattered. In the latter case, the formation of a solid mass is too slow to maintain the droplet form during the way to the bottom of the beaker. Consequently, the paste of the present invention is highly suitable for forming a solid depot or implant upon injection into a body, as the paste regains its non-movable, e.g. solid character, immediately after leaving the injection cannula or needle. Thus, flowing off nanoparticles and formation of undesired off-target depots are prevented.

In contrast thereto, a suspension comprising magnetic agglomerates of alkoxysilane- coated metal containing nanoparticles which is not thermally treated has a delayed solidification, resulting in spreading and/or scattering of the agglomerated nanoparticles and escape from the injection site into the surrounding tissue.

Thus, the paste of the present invention is associated with a more precisely controllable injection into tumorous tissue leading to a much better control over the heat distribution in the tumor and a lower occurrence of depots outside the treatment area lowering the efficacy of a hyperthermia treatment, as compared to a suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles which is not thermally treated. Thereby, the higher is the viscosity of the paste and its yield stress, the less nanoparticles will escape from the injection site.

In the context of the present invention, the term “thermally treating” or “thermal treatment” means subjecting the aqueous suspension comprising agglomerates of alkoxysilane- coated magnetic metal containing nanoparticles to a temperature of between 25°C and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60°C. Thus, the temperature may be 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or 121 °C.

By heating the aqueous suspension, the viscosity is increased. Thereby, the level of increase of viscosity depends on the temperature. Higher temperatures result in higher viscosities. The temperatures may be between 25°C and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60°C, and may be 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or 121 °C. The increase of the viscosity also depends on the duration of the thermal treating step. The longer the thermal treating is, the higher the viscosity becomes. Preferably, the thermal treating step is carried out for a time period of between 0.1 to 96 hours (hrs), preferably for a time period of 0.5 to 72 hours (hrs), more preferably for a time period of 1 to 48 hours (hrs). Notably, a maximum viscosity can be reached during thermal treating, whereby still longer thermal treating may result in a decrease of viscosity, as can be taken from Table 5.

The viscosity of the paste (and also of the aqueous suspension) also depends on the molarity or concentration, as determined by the metal content. Thereby, higher molarities result in higher viscosities. The molarity may be at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M. The desired metal concentration can be adjusted, e.g. by evaporating water from the aqueous suspension in a rotation evaporator. Samples can be analyzed regarding solids content and metal- concentration using the method disclosed below (see, for example, Example 2). Higher molarity of metal content results in a higher specific absorption rate (SAR) based on volume. With respect to iron, 2 M equals 112 mg/ml. The concentration of metal can be determined by photometry of certain metal complexes, e.g. iron can be determined after transformation into an iron(ll) phenanthroline complex, as described in Example 2.

By thermally treating the aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles, the viscosity is increased. Preferably, also the z-average value and the hydrodynamic volume per particle are increased.

Preferably, the viscosity of the paste is between 0.03 and 7 Pa s, more preferably between 0.03 and 5 Pa s, still more preferably between 0.08 and 3.5 Pa s, still more preferably between 0.1 and 3.5 Pa s, still more preferably between 0.1 and 3.0 Pa s, still more preferably between 0.5 and 3.0 Pa s, still more preferably between 0.7 and 3.0 Pa s, still more preferably between 1 and 3.0 Pa s, as determined by rotational rheometry at 20°C, measured at a shear rate of 50/s. Rotational rheometry according to the present invention is, for example, exemplified in Example 6. Notably, a maximum viscosity may be reached during thermal treating, as can be taken from Table 3 and as depicted in Figure 3a.

By thermal treatment, the size of the agglomerates of the aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles is increased, as exemplarily presented in Table 1. Preferably, the agglomerates in the paste have an average size of 40 to 460 nm, more preferably of 60 to 360 nm, still more preferably of 80 to 310 nm, still more preferably of 100 to 200 nm, still more preferably of 140 to 160 nm, as determined by light scattering. Notably, a maximum size of agglomerates may be reached during thermal treating. The size of agglomerated nanoparticles can be measured, as for example described in Example 3.

The size of the metal cores of the nanoparticles, the shape of the nanoparticles and the SAR values of the nanoparticles in the paste are the same as in the aqueous suspension, from which the paste is formed by thermal treatment.

The paste of the present invention can be injected (e.g. via syringe or cannula) in the daily routine into tissues such as tumors, remains within the tissue at the site of injection and therefore can be used for precisely placed hyperthermia and/or thermoablation. It has surprisingly been found that thermal treatment of an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles leads to the increase of viscosity to become a paste which can, due to the presence of yield stress and its shear-thinning properties, be easily moved, e.g. drawn up in a syringe, allowing injection into tissues where the suspension immediately thickens due to its plastic behavior to achieve the solidification state of the original paste. Thus, the agglomerates stay in close proximity within the injection site without escaping to neighboring environment. This makes the paste especially suitable for achieving of an arrangement of depots or implants with high precision in respect to location and size, e.g. for hyperthermia and/or thermoablation.

In the present invention, an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles may be produced by any suitable method. Methods of producing such aqueous suspensions are known to the skilled person and have been described, for example, in WO 2013/020701.

In a preferred embodiment of the production method, an aqueous suspension comprising metal containing magnetic nanoparticles is incubated with an alkoxysilane to produce agglomerates of the magnetic alkoxysilane-coated metal containing nanoparticles.

The term “incubating” or “incubation” means any experimental setup, experimental condition(s) or reaction mixture(s) which allow for the polycondensation of alkoxysilanes and thereby for the aminosilane coating of nanoparticles. The incubation of an aqueous suspension comprising magnetic metal containing nanoparticles with alkoxysilanes is preferably carried out essentially in the absence of an organic solvent, more preferably in the absence of an added organic solvent. “Essentially in the absence” in the context of organic solvents means that small traces of organic solvents may be present, preferably the amount of organic solvents is smaller than 10 % by volume, more preferably smaller than 5 % by volume, still more preferably smaller than 1 % by volume, especially smaller than 0.5 % by volume. For example, minor amounts of methanol may be produced during the reaction and, therefore, to some extent may remain in the product. In a preferred embodiment, the coating is carried out in absence of an organic solvent, especially the coating is carried out in the absence of an added organic solvent. The preferred solvent for the coating reaction is water. Without being bound to any scientific theory, the inventors assume that these reaction conditions lead to a defined, however incomplete condensation reaction of the alkoxysilanes which translates into the agglomeration properties of the nanoparticles. An organic solvent may be a liquid organic compound, i.e. a carbohydrate, with the power to dissolve solids, gases, or liquids. Examples of organic solvents include, but are not limited to, ethylene glycol, acetone, toluene and equivalents.

Thus, the aqueous suspension comprising the agglomerates of magnetic alkoxysilane- coated metal containing nanoparticles is essentially free of organic solvents. “Essentially free of organic solvents” in this context means that the small traces of organic solvents may be present, e.g. the amount of organic solvents is smaller than 5% by volume, preferably 1 % by volume, more preferably smaller than 0.5 % by volume, especially smaller than 0.1 % by volume. In an especially preferred embodiment, no organic solvent can be detected in the nanoparticle preparation by customary methods.

The production method of the agglomerates is preferably carried out in the absence of ethylene glycol. Ethylene glycol interferes with the coating reaction. Furthermore, it is at least very difficult if not impossible to remove it completely from the nanoparticle preparation, as usually relatively large amounts of ethylene glycol remain attached to the coating of the nanoparticles and due to its high boiling point of 197 °C. This applies also to the preparations prepared according to Lesniak et al. (1997). According to the European Pharmacopeia only 600 ppm of ethylene glycol are allowed in the final medical product, which makes nanoparticle preparations with higher amounts of ethylene glycol inacceptable for commercial clinical use. In one embodiment, the metal nanoparticles comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, whereas iron salts are preferred. Iron comprising nanoparticles are preferred due to their low toxicity compared to other magnetic metals such as cobalt or nickel. In a preferred embodiment the iron complex compounds, the iron carbonyl compounds or iron salts are essentially free of other metals and other contaminants in order to avoid toxicities. It is well known in the art that chemicals may contain traces of contaminants. Therefore, “essentially free” in this context means preferably that less than 1% by weight, preferably less than 0.1% by weight of other contaminants is comprised within the iron complex compounds, iron carbonyl compounds or iron salts. Especially preferred are iron salts essentially free of other contaminants.

In an especially preferred embodiment, the iron salt is an iron oxide, preferably magnetite and/or maghemite.

Such iron nanoparticles made of iron oxide can be manufactured by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide. “Iron nanoparticles” as used herein are nanoparticles containing Fe atoms or Fe ions. Accordingly, in a preferred embodiment, the iron oxide nanoparticles are provided by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide. Here, the ratio between iron(lll) chloride and iron(ll) chloride is preferably of about 2:1.

In the context of the present invention, the terms “iron nanoparticles” and “iron oxide nanoparticles” are equivalently used.

Suitable precipitation reactions and conditions have been described by Massart (1981) and reviewed by Mohapatra and Anand (2010). Preferred conditions for the precipitation reaction are (i) a ratio of Fe(lll)chloride and Fe(ll)chloride of about 2:1; (ii) pouring the Fe(lll)chloride and Fe(ll)chloride solution into a sodium hydroxide solution with a concentration of about 2.13 M; (iii) precipitation temperature of about 25°C; and (iv) time for the precipitation reaction of about 52 min. Optionally, the sodium hydroxide solution is poured into the iron chloride solution (instead of vice e versa) during a period of about 39 min at about 15°C. A method for producing coated iron oxide nanoparticles by means of precipitating iron salts in solution is, for example, exemplified in Example 1.1.

Alternatively, the iron oxide nanoparticles can be provided by thermal decomposition of an iron salt or an iron complex compound. The term “iron complex compound” as used herein generally means any complex containing iron, preferably any compound comprising complexed iron. Suitable methods have been described by Waldoefner and Stief (2011). Briefly, an iron-containing compound and an organic solvent are kept for 10 min at a temperature between 50°C and 50°C below the reaction temperature. Next, the solution is heated to 200 to 400°C to yield nanoparticles. The nanoparticles are oxidized with oxygen, peroxide or a tertiary amineoxide, and treated with nitric acid and ironnitriate resulting in maghemite nanoparticles. Another suitable method for the preparation of iron oxide nanoparticles by thermal decomposition has been described by Guardia et al. (Guardia et al. 2010 a; Guardia et al. 2010 b; Guardia et al. 2012). Briefly, iron (III) acetylacetonate is mixed with decanoic acid in dibenzyl ether. The solution is constantly heated up to 200°C. After 2 h at 200°C the solution is heated up to reflux and kept at this temperature for 1 h and finally cooled down to room temperature, washed and collected by centrifugation. Both methods are preferred due the high SAR of the resulting nanoparticles. A method for producing iron oxide nanoparticles by means of thermal decomposition is, for example, described in Example 1.2.

Accordingly, in an alternatively preferred embodiment, the iron oxide nanoparticles are provided by thermal decomposition of an iron salt or an iron complex compound.

Iron salts and iron complexes which are applicable in the method of producing iron oxide nanoparticles are well known to the person skilled in the art and include, but are not limited to, iron(lll) chloride, iron(ll) chloride, iron (III) acetylacetonate, iron carbonyls and equivalents.

The metal nanoparticles may be treated with H2O2 prior to the incubation with alkoxysilane. This optional step is preferred as the iron is fully oxidized to Fe₂0₃ (maghemite) under defined conditions and, as a consequence, subsequent reaction steps can be conducted in the absence of a protective gas (e.g. argon). Otherwise in the absence of H2O2, it is preferred to work under protective gas such as argon in order to control reaction conditions.

The alkoxysilan is preferably a trialkoxysilane. It is more preferably selected from the group consisting of 3-(2-aminoethylamino)-propyl-trimethoxysilane (DIAMO), 3- aminopropyltriethoxysilane (APTES), trimethoxysilylpropyl-diethylenetriamine (TRIAMO) and N-(6-aminohexyl)-3-aminopropyltrimethoxysilane. In an especially preferred embodiment, the alkoxysilane is 3-(2-aminoethylamino)-propyl-trimethoxysilane. In a further preferred embodiment, the coating reaction is carried out by adding 0.3 to 0.6 x 10³ mol, preferably 0.4 to 0.5 x 10³ mol and especially 0.43 to 0.45 x 10³ mol trialkoxysilane per 0.9 mol of the metal.

Preferably, the incubation with alkoxysilane is performed at a pH of between 2 and 6 (which means that also a pH of 2 or 6 is included into this range), more preferably of between 2.5 and 5.5, still more preferably of 4.5 ± 1. During the incubation/, the pH may be adjusted to said values, if required. Acetic acid can be used to adjust the pH accordingly.

Preferably, the metal magnetic nanoparticles are disintegrated prior to the incubation with alkoxysilane. The nanoparticles are disintegrated preferably by ultrasound treatment in order to generate a suspension of ball-shaped or cubic electron-dense nanoparticles which can then be subjected to the coating reaction. Ultrasound treatment may be done in an ultrasonic bath at 45 kHz 30 min to 2 h, especially for about 1 h. This disintegration method preferably is carried out at acidic conditions, preferably between pH 2.5 and 3.0. Disintegration of nanoparticles according to the present invention is, for example, described in Example 1.1.

Another suitable method for disintegrating nanoparticles is laser-based deagglomeration / laser fragmentation technique (Schnoor et al. 2010).

The method of producing agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles may further comprise the step of disintegrating the agglomerates in the aqueous suspension after starting the incubation with alkoxysilane, which can be carried out as described above. Preferably, disintegration of the nanoparticles starts with or after the coating step. Alternatively, the disintegration step may start prior to the coating step and may be further carried out simultaneously with and/or after the coating step. Still more preferably, disintegration is started prior to the coating step and is continued during and after the coating step. Preferably, disintegration is carried out for a total of about 24 h or more.

Upon possible disintegration and coating, a suspension can be generated that can stably be stored at room temperature. It is assumed that most individual nanoparticles are completely coated with the alkoxysilane, easily adhere to neighboring nanoparticles and form agglomerates. Still, the suspension is an aqueous fluid suspension which is fluent enough to easily pass through syringes and to be injectable into tissue such as tumor tissue.

Preferably, an additional step for removing incompletely coated and/or very large agglomerates (e.g. agglomerates of more than 2,000 nanoparticles) from the suspension is carried out. Suitable methods for this step are centrifugation (e.g. for 10 min at 2,000 rpm) and filtration (e.g. through a pleated filter with a pore size of 12-25 pm). Especially preferred, both centrifugation and filtration are carried out. It has been observed that predominantly and completely alkoxysilane-coated nanoparticles do not sediment from the suspension e.g. if centrifuged for 10 min at 2,000 rpm. Accordingly, the supernatant of the centrifugation and/or the flow-through of the filtration is/are a suspension which does/do not show sedimentation over one day, preferably one week, especially one month, and therefore can be stored over a long time.

On the other hand, incompletely coated nanoparticles can be removed to a large extent from the suspension e.g. by such centrifugation. Such removal of incompletely coated nanoparticles is preferred, as incompletely coated nanoparticles have a reduced SAR which therefore reduce the volume SAR of a suspension.

The disintegration step(s) and optionally the removal step is/are preferably carried out, until the agglomerates of the metal nanoparticles have an average size (z-average) of 30 to 450 nm, preferably of 50 to 350 nm and more preferably of 70 to 300 nm, as determined by light scattering. In this context, the average size is determined in water according to example 3. With this light scattering measurement, the size of agglomerates of nanoparticles is determined - in contrast to the size of the ball-shaped or cubic electron-dense single nanoparticles which are forming such agglomerates. “Z-average” with respect to the size of agglomerates means the readout of the light scattering size determination as carried out in example 3. Z-average values above the provided ranges lead to sedimentation of the nanoparticles and are therefore generally not suitable for the foreseen applications of these nanoparticles. Even if the dispersion may be reconstituted prior to instillation of a tumor, larger agglomerates may lead to serious problems, as the dispersion may partially separate into buffer and agglomerates while passing through the needle leading to an uneven distribution of the nanoparticles within the tissue.

Preferably, the agglomerates in the aqueous suspension have an average size of 30 to 450 nm, more preferably of 50 to 350 nm and still more preferably of 70 to 300 nm, as determined by light scattering. The size of agglomerated nanoparticles can be measured, as for example described in Example 3.

The metal nanoparticles are preferably nanoparticles having a metal core with a size of 5 to 25 nm, preferably with a size of 7 to 20 nm, more preferably with a size of 9 to 15 nm, as determined by electron microscopy. The agglomerates of the suspension are preferably composed of dozens to hundreds of such individual nanoparticles, whereas any or only very few are small agglomerates of less than ten nanoparticles, as may be determined in transmission electron microscopy (TEM) e.g. according to the method described by Jordan et al. (1996, on page 712, 3.2.2), preferably less than 3 agglomerates of 10 or less nanoparticles in a representative TEM picture displaying 700 nm by 700 nm and at least 1000 nanoparticles.

In turn, in a representative TEM picture displaying 700 nm by 700 nm and at least 1000 nanoparticles, less than 10 individual nanoparticles, preferably less than 5 individual nanoparticles, especially one or none individual nanoparticle can be detected. A nanoparticle in this context is one basically ball-shaped or cubic electron-dense nanoparticle visible in transmission electron micrographs. A single nanoparticle is a nanoparticle which is not attached to at least one other nanoparticle.

Preferably, the shape of the single nanoparticles is ball-shaped or cubic. Size and shape of the nanoparticles can be tailored by adjusting pH, ionic strength, temperature, nature of the salts (perchlorates, chlorides, sulfates, and nitrates), or the Fe(ll)/Fe(l II) concentration ratio (reviewed by Mohapatra and Anand 2010).

The suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles may have a zeta potential of 25 to 80 mV, preferably of 35 to 70 mV, especially of 45 to 60 mV. The zeta potential is determined as described in Example 4 at pH 5.2 and at a conductivity of 2.0 mS/cm (each determined at 25°C). The zeta potential is dependent on the successful coating of the nanoparticles as it depends on the amino groups of the alkoxysilanes. Lower zeta potentials indicate an insufficient coating of the nanoparticles The correct zeta potential within the provided ranges contributes to the properties of the nanoparticles upon injection into tissue, i.e. that injected nanoparticles remain at or near the injection site within, for example, the tumor, and do not spread to surrounding tissue, which would limit the applicable magnetic field and thereby the success of the treatment. Furthermore, the zeta potential in the provided ranges ensures optimal colloidal stability and therefore extends the shelf life of the nanoparticle composition.

Preferably, the agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles are suspended in a water-based physiologically acceptable buffer. Physiologically acceptable buffers are known in the art and include for example acetate, citrate, carbonate or phosphate at a pH (at 25 °C) between 5 and 8, preferably between 5 and 6, and especially between 5.1 and 5.8 and a conductivity (at 25 °C) of 1.5 to 2.5 mS/cm, preferably 1.7 to 2.3 mS/cm. The osmolality of a suitable suspension is 0.01 to 0.09 Osmol/kg, preferably 0.02 to 0.07 Osmol/kg.

In a preferred embodiment, the specific absorption rate (SAR) of the nanoparticles is larger or equal than 2 W/g of the respective metal (e.g. iron), more preferably larger or equal than 3 W/g of the respective metal and still more preferably 4 to 50 W/g of the respective metal, as determined at a magnetic field strength of 4 kA/m and a frequency of 100 kHz according to the method as described by Jordan et al. (1993). The SAR value is determined as described in Example 5. Generally, high SAR values are preferred, as consequently higher temperatures can be achieved during exposure to an alternating magnetic field. If the SAR value of the nanoparticles is too low, i.e. lower than the provided numbers, it is likely that upon exposure to an alternating magnetic field achieved temperatures throughout the tumor are too low to reach a therapeutic effect. The thermal treatment disclosed by this invention does not change the SAR- value of the suspension as shown in example 5. In contrast, other measures to adjust consistency like adjectives, gelling agents etc. might decrease the SAR-value.

In a further preferred embodiment, the method further comprises the step of ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles before thermally treating the aqueous suspension. Preferably, this step of ultrasound treatment is performed after nanoparticle preparation and optional coating and before thermal treatment.

Usually, the aqueous suspension after its preparation and before thermal treatment and instillation is stored at temperatures lower than room temperatures. But if not, the step of ultrasound treatment may be performed after storage and before thermally treating the aqueous suspension in order to ensure that the suspension after storage is still an homogeneously dispersed aqueous fluid suspension, which then by thermal treatment forms a homogeneous paste. A homogeneous paste passes more easily through syringes and is injectable into tissue such as tumor tissue. For example, before the aqueous solution is filled into a syringe for further instillation, it may be objected to ultrasound treatment and then thermally treated in the syringe.

Ultrasound treatment may be done in an ultrasonic bath at any common frequency. Using a higher frequency may be required for shorter treatment times and using a lower frequency may be sufficient for longer treatment times. For example, ultrasound treatment may be performed at 45 kHz for 30 min to 4 h, preferably for 1 h to 3 h, more preferably for 1.5 h to 2.5 h, most preferably for about 2 h. Preferably, ultrasound treatment is performed at room temperature. This ultrasound treatment of nanoparticles according to the present invention is, for example, described in Example 8.

The present invention relates to a medical device comprising the paste of the present invention. As the magnetic nanoparticles exert their therapeutic effect upon exposure to an alternating magnetic field through generation of heat as a physical mode of action and do not directly interact with the metabolism of the patient, these nanoparticles are classified in multiple jurisdictions as medical devices. Still, they can be used as powerful tools for the treatment or prophylaxis of tumor diseases and other diseases through hyperthermia and/or thermoablation, where cells are malfunctioning in a certain region of the body.

Examples for such other diseases which can be treated with the paste of the present invention are rheumatism, arthritis, arthrosis and bacterial infections. Tumor diseases which can be treated with the paste of the present invention are preferably solid tumors, especially local or locally advanced tumors or systemic tumor diseases which cause local problems such as inoperable metastasis. Examples are brain tumors, e.g. glioblastoma and astrocytoma, brain metastasis, prostate cancer, prancreatic cancer, hepatocellular carcinoma, head and neck cancer, bladder cancer, gastric cancer, renal cell carcinoma, ovarian carcinoma, cervical carcinoma, sarcoma, basal cell carcinoma and melanoma.

The present invention relates to a pharmaceutical composition comprising the paste of the present invention. The aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles can be formulated with active pharmaceuticals such as anti-cancer agents, e.g. chemotherapeutic agents (which can be grouped into alkylating agents, antineoplastic antibiotics, anti-metabolites, natural source derivatives), hormones/growth factors or hormone/growth factor analogues or inhibitors, signal transduction inhibitors and immune therapeutics. Suitable pharmaceuticals are listed for example in Waldoefner and Stief (2011, paragraphs [0096] to [0102]). Accordingly, it is within the invention that the nanoparticles are combined with such active pharmaceuticals. The present invention relates to the paste of the present invention or the medical device or the pharmaceutical composition for use in a method of treating or preventing the diseases, as referred to above, including proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection, in a patient.

The present invention relates to a method of treating or preventing the diseases, as referred to above, including proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection, comprising the step of administering the paste of the present invention to a human or animal patient.

Preferably, the paste of the present invention for use in a method of treating or preventing the diseases, as referred to above, or the method of treating or preventing the diseases further includes exposing the patient to an alternating magnetic field. Usually, the alternating magnetic field is applied hours or days after injecting the paste into the target region, e.g. tumor, of the patient (Johannsen et al. 2007; Thiesen and Jordan 2008; Maier- Hauff et al. 2011).

The paste of the present invention can further be used in a method for increasing the activity of an anti-cancer agent comprising the steps of administering to a patient in need thereof a pharmaceutical composition comprising the paste of the present invention and administering at least one anti-cancer agent together with at least one pharmaceutically acceptable excipient, carrier and/or solvent. The two administrations may be simultaneous, e.g. as separate formulations or the aqueous suspension is formulated with active pharmaceuticals, or one after the other (first nanoparticles, second anti-cancer agent or vice versa), however, in such a way that nanoparticles and anti-cancer agent are present at the same time within the patient’s body, in order to be able to act together and enhance each other’s therapeutic effect. Whereas, according to the present invention, the nanoparticle agglomerates remain within the tissue for months or years within the target area and can generate heat upon exposure to an alternating magnetic field, an administered anti-cancer agent typically acts for hours or days. “Act together” in this context therefore means, that still sufficient pharmacologically active levels of the anti cancer agent are present in the tissue. Accordingly, the present invention relates to the paste of the present invention for use in a method for the prophylaxis and/or treatment of tumor diseases, wherein the paste is administered together with an anti-cancer agent in such a way, that nanoparticles and anti-cancer agent are present at the same time within the patient’s body.

The agglomerates of nanoparticles may be complexed with or covalently coupled to an active pharmaceutical agent or to a targeting agent such as antibodies, antibody fragments or ligands, as known in the art. For example, the coupling of active pharmaceuticals and/or ligands to nanoparticles is described in Jordan et al. (2008), Gao et al. (2011), Waldoefner and Stief (2011) and Ivkov et al. (2005).

Other suitable medical device forms of the paste of the present invention for application to the body are solid, semi-solid or gel-like medical devices, sponges or films for use as an implant or combinations with a carrier e.g. a porous matrix which may be bioresorbable Generally, pharmaceutical compositions or medical devices of the present invention can easily be combined with conventional therapies used for the respective treatment or prophylaxis of the disease, such as chemotherapy or radiation. They can be used either to increase the effectiveness of the individual treatment and/or reduce side effects of the conventional therapy by lower their dose if combined with the pharmaceutical compositions or medical devices of the present invention.

In light of the foregoing general discussion, the specific figures and examples presented below are illustrative only and are not intended to limit the scope of the invention.

Figures

Figure 1 : Schematic drawing showing the principles of viscosity. Force F is applied on the liquid in direction of flow; A is the area in direction of the flow; v is velocity; y the axis of the distance between the fluid layers parallel to the surface A, r is the distance between the surface of the streaming liquid and the mid of the stream.

Figure 2: Six schematic drawings depicting the graphs f or t (shear stress) vs. g (shear rate) (left column) and h (viscosity) vs. g (shear rate) (right column), T₀= yield stress.

Figure 3a: Impact of thermal treatment on viscosity of 6 M suspensions comprising magnetic alkoxysilane-coated metal containing nanoparticles. Figure 3a shows viscosity versus duration of heating at 60°C. The viscosity increases with increasing duration of heating. After 48 hours, the maximal viscosity is nearly reached. Figure 3b: Impact of thermal treatment on yield stress of a 6 M suspension comprising alkoxysilane-coated magnetic metal containing nanoparticles. Figure 3b shows viscosity versus the duration of heating at 60°C. The yield stress increases with increasing duration of heating. After 48 hours, the maximal yield stress is nearly reached.

Figure 4: Photograph showing the impact of thermal treatment of a 6 M suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles on the behavior in a physiological sodium chloride solution. The left beaker shows what happens to the paste of the present invention, exemplified by the thermally treated 6 M suspension of magnetic alkoxysilane-coated metal containing nanoparticles, heated at 60°C for 1 h. The paste is dropped from a syringe into the solution. Droplets are on the bottom of the beaker. The right beaker contains a non-thermally treated 6 M suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles from the same production batch dropped from a syringe into the solution. Rough distributed heaps of solid matter are scattered on the bottom of the beaker.

Examples

1 Manufacturing of coated magnetic iron oxide nanoparticles

1.1 By means of precipitating iron oxide from iron salt solutions

Precipitation and Washing: NaOH is weighted out into a flask, is solved in purified water to a concentration of 2.13 M and is subsequently chilled to 25°C. Fe(lll)chloride and Fe(ll)chloride (ratio 2:1) are filled into a glass bottle and solved in purified water to get a 0.48 M Fe(lll)chloride / 0.24 M Fe(ll)chloride solution. The iron chloride solution is poured into the NaOH solution and is mixed during a period of about 53 min, while the temperature is constantly held at 25 °C. The generated nanoparticles are left to sediment and the supernatant is removed. The nanoparticles are washed with degassed water until the supernatant reaches a conductivity of < 5mS/cm. The nanoparticles used in the following are prepared by this method. Optionally, the NaOH solution may be poured into the iron chloride solution (instead of vice e versa) during a period of about 39 min at 15°C.

Coating and Disintegration The nanoparticle suspension from above is adjusted with diluted HCI until pH is between 2.5 and 3.0. Afterwards the flask is positioned in an ultrasonic bath and treated with ultrasound at 45 kHz for 1 h while stirring. Now over a time of 90 min 3-(2- aminoethylamino)propyl)trimethoxysilane (Fluka, 48 ml per 1,2 I nanoparticle suspension) is added dropwise, while the pH is kept below a threshold of 5.5 by adding drops of acidic acid, but the pH shall not get lower than 5.0. After this step, the pH is adjusted to 4.65 with diluted HCI and the suspension is further treated with ultrasound for 23 hours.

Optionally, the nanoparticles may be treated with H2O2 for two days prior to the coating in order to achieve a finer dispersion of the nanoparticles and a better colloidal stability. Further H2O2 may be used in order to completely oxidize Fe under controlled conditions to Fe₂0₃. As a result, subsequent reactions can be performed in the absence of a protections gas (e.g. argon). This optional step has not been performed to the nanoparticles used herein.

Dialysis: The suspension is purified with a blood dialysis cartridge (Fresenius F8 HPS) against degassed ultrapure water until a conductivity of 400 pS/cm is reached.

Centrifugation and Concentration: One half of the resulting suspension is filled in a centrifuge bucket and centrifuged for 10 min at 2,000 rpm. Next the supernatant is filtered through a pleated filter (12-25 pm) into a glass bottle, which has previously been rinsed for 5 min with Argon. This procedure is repeated identically with the second of the suspension. Afterwards, the nanoparticle suspension is concentrated with a rotation evaporator to the desired Fe concentration (e.g. 112 mg/ml Fe equals 2 M Fe or 335 mg/ml Fe equal 6 M Fe). Nanoparticle samples can be analyzed regarding solids content and Fe-concentration.

1.2 By means of thermal decomposition of iron complex

Nanoparticles may also be produced similar to the methods described in Waldoefner and Stief (2011). Briefly, iron(lll) chloride sodium acetate, diaminohexane and ethyleneglycole were combined in a three necked flask and stirred until a homogeneous solution was obtained. Then the mixture was heated strongly until near boiling and was refluxed for five hours. After washing and collecting the particles via centrifugation the dried particles were mixed with trimethyleneoxide in ethylene glycol and heated to 130°C and kept for 2 h. Then the mixture was heated under reflux for 1 h. For the following oxidation step the washed particles were resuspended in nitric acid and treated with iron nitride. Then, after washing and collecting after the particles by centrifugation, the particles were coated with a tetraalkoxysilane in order to form a thick SiC>2-shell. Resulting particles were collected by centrifugation and resuspended in water. The final coating, disintegration and purification (dialysis, centrifugation and concentration) can be done in the same way as disclosed above. Nanoparticles may also be produced similar to the methods described by Guardia et al. (2010 a; 2010 b; 2012).

A solution of iron(lll) acetylacetonate and decanoic acid in dibenzyl ether were rapidly heated up to 200°C under stirring. Then the mixture was stirred for 2 h at this temperature and heated within 15 min to 298°C. This temperature was held for another hour. Finally, the suspension was allowed to cool down to room temperature. Then, acetone was added to the mixture and the precipitate was air-dried. The particles were resuspended in water. The final coating, disintegration and purification can be done in the same way as disclosed above for the particles as used herein.

1.3 Conditioning

The suspensions, individually filled in 15 ml injection vials with stopper and metal cap, were heated in a water bath at a fixed temperature for the specified time. Designation of sample, its production batch as well as time and temperature for the thermal treatment are given in the respective tables.

2. Iron Concentration/Solids Content Determination

Determination of the iron concentration within a suspension is based on the photometric measurement of the extinction of an iron(ll) phenanthroline complex. The complex is generated by extraction of the nanoparticles with hydrochloric acid until the extraction is complete as determined by visual inspection. All iron contained is reduced to iron (II) using hydroxylamine-hydrochloride and transformed into the phenanthroline complex in acetic acid/acetate buffer. Extinction of the complex is determined at 513 nm using a Shimadzu UV-1700 Pharmaspec against an iron(ll) ethylendiammonium sulfate standard (Merck, Darmstadt). The solid content of a suspension is determined by weighing e.g. 1 ml of the suspension prior to and after evaporation of the solvent (e.g. water).

3. Particle / Agglomerate Size Measurement

To measure the average size of the nanoparticles a light scattering procedure is used to determine the hydrodynamic size of the nanoparticle preparation (e.g. Malvern ZetaSizer 3000 HSA or Malvern Zetasizer Nano ZS). The primary parameter is the z-average value, which is weighted by the scattering intensity. Therefore, in case of a polydisperse distribution, larger nanoparticles are weighted stronger than smaller ones. Furthermore, this method determines the average size of the nanoparticle agglomerates, and not the size of the single or primary nanoparticles. Principle: If the nanoparticles or molecules are illuminated with a laser, the intensity of the scattered light fluctuates at a rate that is dependent upon the size of the nanoparticle/ agglomerates as smaller nanoparticles are “kicked” further by the solvent molecules and move more rapidly. Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the nanoparticle size using the Stokes-Einstein relationship. Procedure: A small part of the test substance is diluted dependent on its concentration (1:1000 up to 1:3000). A sample of the diluted suspension is placed in the measurement device and treated according to recommendations of the Malvern ZetaSizer Nano ZS. Table 1: Measured z-average values for a 6 molar suspension heated at 60°C

4. Zeta Potential Measurement

To measure the Zeta potential of the nanoparticles, a sample is vortexed for 30 sec. 75 ml of a 1:1000 dilution of the solution with a concentration of about 0.11 mg/ml for Fe (or other metal) in ultrapure water is prepared and treated for 15 min with ultrasound. 20 ml of the solution are injected in the measuring cell of the Malvern ZetaSizer 3000 HSA (or Malvern Zetasizer Nano ZS) and measured according to the recommendations of the manufacture. The pH of the solution is determined with a separate pH meter.

5. SAR Measurement

The SAR of samples from Example 1 was determined according to the method developed by Jordan et al. (1993) at a frequency of 100 kHz and different field strengths of up to 15 kA/m. Results for a thermally treated (1h 60°C) and a non-thermally treated sample are shown in Table 2. Both samples are derived from the same production batch (02-2017- 010). Table 2: Specific absorption rate 6. Rotational Rheometry/Viscosity Measurement

The viscosity of the nanoparticle samples was determined by Malvern material characterization services using rotational rheometry at 20°C, wherein the shear viscosity (in Pa s) was determined in dependence of the shear rate (from 7 to 1500 /s).

The samples were measured in a rotational rheometer (Bohlin GEMIN 200, Malvern) with cone-plate geometry (2°, 60mm 0), whereby the samples were kept at 20°C each time.

1.) Conditioning of a 6-molar suspension at 60°C

Table 3: Measured rheological data for a 6 molar suspension heated at 60°C

Table 3 shows that the viscosity of the suspension increases with increasing duration of heating. After 48 hours, the maximal viscosity does not seem to have been reached. A graph of the viscosity values is shown in Figure 3a and the yield stress values are shown in Figure 3b.

2.) Conditioning of a 6-molar suspension at 40°C or 50°C

Table 4: Measured data for a 6 molar suspension heated at 40°C or 50°C

Table 3 shows that the viscosity of the suspension increases with increasing heating temperature. After 48 hours, the maximal viscosity does not seem to have been reached.

3.) Conditioning of a 2-molar suspension at 60°C

Table 5: Measured data for a 2 molar suspension heated at 60°C Table 5 shows that the viscosity of the suspension increases with increasing duration of heating. After 48 hours, the maximal viscosity seems to have been reached. The viscosity of the suspension is lower as compared to a 6 molar suspension (see Table 3). The behavior of the suspension seems to be similar to the 6-molar suspension.

7. Agglomeration in physiological saline

Two vials of a 6 M suspension of magnetic alkoxysilane-coated metal containing nanoparticles from the same production batch (batch-number: 02-2017-010) were used. One was thermally treated for one hour at 60°C (designation: SG-F06-08-02), the other one (designation: SG-F06-08-01) was not treated. A physiological saline solution was prepared by dissolving of about 0.9 g NaCI in 1 I of deionized water. Two 150 ml beakers were filled with 50 ml of that physiological saline solution.

Both vials were opened by removing of stopper and metal cap. The content of the non- treated sample was liquid, the content of the thermally treated sample looks like a solid, even turning it upside down does not change this state.

From both vials (thermally threated and non-treated) about 1 ml were taken up into a 1 ml syringe (B. Braun, Omnifix 0.01-1 ml / Luer Solo) each through a 19 G-cannula (B. Braun, Sterican 1.10 x 50 mm BI/LB, 19 G x 2”). For taking up the thermally treated sample, a little more force was needed to pull the paste into the syringe.

Each of the samples was dropped from the syringe via the 19 G-cannula into one of the beakers with the physiological saline. The distance between the end of the cannula and surface of the saline solution was approximately 3 to 5 cm.

The result is shown in Figure 4. The left beaker shows what happens to the paste of the present invention, the thermally treated 6 M suspension of magnetic alkoxysilane-coated metal containing nanoparticles, heated at 60°C for 1h. It is readily visible that the form of the drops leaving the syringe is maintained during their way sinking through the physiological saline solution. Obviously, the material regained immediately its pasty consistency before entering the salt solution. Thus, it was not scattered before completion of agglomeration. The right beaker contains a non-thermally treated 6 M suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles from the same production batch dropped from a syringe into the solution. It is readily visible that the drops leaving the syringe are scattered by the impact into the salt-solution, before they can solidify by agglomeration. 8. Ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles

In this study, the influence of ultrasound treatment on the aqueous suspension of magnetic metal containing nanoparticles was investigated.

The prostate used in this study was collected from a patient on Day 1 of the study and received on Day 2. The study was performed on the day the prostate was received.

The aqueous suspension of magnetic metal containing nanoparticles used in this study was filled in vials and had been stored therein for about 3 years and 10 months.

Two vials of such aqueous suspension of magnetic metal containing nanoparticles were used and divided into Group I and Group II.

The vial of Group I was put into an ultrasonic water bath at room temperature for 30 minutes. Subsequently, 5ml of the aqueous solution were filled into each of the two syringes and incubated for 2 hours at 60°C.

The vial of Group 2 was not treated in an ultrasonic water bath. Subsequently, also in this case 5ml of the aqueous solution were filled into each of the two syringes and incubated for 2 hours at 60°C.

Instillation (i.e. introduction of the paste into the prostate tissue) was performed using IV extension tubing (fill volume of 2.1ml) and 18G 10" Chiba needles. Pumps were run at 0.7ml/hr for a total infusion volume of 0.25ml.

The right side of all prostates was a control using Group 2 aqueous solution (60°C for 2 hours).

Procedure:

Tubing and needle were primed in each channel before insertion. The pump was started as soon as the needle was placed. The channels were primed one by one and only right before the needle was placed. The pump was run to dispense volume of 0.25ml from each channel. It was waited for 10 min after instillation was complete in each channel before the needles were removed.

Results from Computed Tomography (CT) measurements: Discussion/Conclusion:

Pretreating the aqueous suspension of magnetic metal containing nanoparticles in the sonicator had a significant effect on how the aqueous suspension of magnetic metal containing nanoparticles behaves in tubing and needle. There was much less resistance when priming the tubing. However, pre-treating the aqueous suspension of magnetic metal containing nanoparticles via ultrasound treatment helped to make the viscosity more consistent and allowed for easier flow in the tubing while still maintaining higher concentrations in the tissue and less loss due to efflux through the urethra.

Literature

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Claims

1. A paste comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles obtainable by a method, wherein the method comprises the step of thermally treating an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles.

2. The paste of claim 1, wherein the paste is characterized by plastic behavior.

3. The paste of claim 1 or 2, wherein the paste is characterized by the presence of yield stress, wherein the yield stress is preferably between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa.

4. The paste of any one of claims 1 to 3, wherein the paste has a shear thinning behavior.

5. The paste of any one of claims 1 to 4, wherein the past is characterized by a viscosity of between 0.03 and 7 Pa s, more preferably of between 0.03 and 5 Pa s, still more preferably of between 0.08 and 3.5 Pa s, still more preferably of between 0.1 and 3.5 Pa s, still more preferably of between 0.1 and 3.0 Pa s, still more preferably of between 0.5 and 3.0 Pa s, still more preferably of between 0.7 and 3.0 Pa s, still more preferably of between 1 and 3.0 Pa, measured at a shear rate of 50/s.

6. The paste of any one of claims 1 to 5, wherein the step of thermally treating is carried out at a temperature of between 25 and 130°C, preferably of between 30 and 120°C, more preferably of between 40 and 100°C, still more preferably of between 40 and 80°C, still more preferably of between 40 and 60 °C.

7. The paste of any one of claims 1 to 6, wherein the step of thermally treating is carried out for a time period of between 0.1 to 96 hours, preferably for a time period of 0.5 to 72 hours, more preferably for a time period of 1 to 48 hours.

8. The paste of any one of claims 1 to 7, wherein the aqueous suspension comprises a concentration of at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M, as determined by its metal content.

9. The paste of any one of claims 1 to 8, wherein the method further comprises the step of incubating the aqueous suspension of magnetic metal containing nanoparticles with alkoxysilane before thermally treating the aqueous suspension.

10. The paste of any one of claims 1 to 9, wherein the step of incubating is carried out in the absence of an added organic solvent.

11. The paste of any one of claims 1 to 10, wherein the alkoxysilane is a trialkoxysilane, preferably selected from the group consisting of 3-(2-aminoethylamino)-propyl- trimethoxysilane, 3-aminopropyltriethoxysilane, trimethoxysilylpropyl- diethylenetriamine and N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, especially 3-(2-aminoethylamino)-propyl-trimethoxysilane, and/or

0.3 to 0.6 x 10³ mol, preferably 0.4 to 0.5 x 10³ mol, more preferably 0.43 to 0.45 x 10³ mol, alkoxysilane per 0.9 mol metal is added in the incubating step.

12. The paste of any one of claims 1 to 11 , wherein the magnetic metal containing nanoparticles comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, preferably iron salts, more preferably wherein the iron salt is an iron oxide, preferably magnetite and/or maghemite.

13. The paste of any one of claims 1 to 12, wherein the iron oxide nanoparticles are provided

(b) by thermal decomposition of an iron salt or an iron complex compound.

14. The paste of any one of claims 1 to 13, wherein the method further comprises the step of ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles before thermally treating the aqueous suspension.

15. The paste of any one of claims 1 to 14, wherein the specific absorption rate (SAR) of the nanoparticles is larger or equal than 2 W/g Me, preferably larger or equal than 3 W/g Me, more preferably 4 to 12 W/g Me, as determined at a magnetic field strength of 3.5 kA/m and a frequency of 100 kHz.

16. A medical device comprising the paste of any one of claims 1 to 15.

17. A pharmaceutical composition comprising the paste of any one of claims 1 to 15.

18. The paste of any one of claims 1 to 15 or the pharmaceutical composition of claim 17 for use in a method for preventing or treating a proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or a bacterial infection in a subject in need thereof.

19. A method for producing the paste of any one of claims 1 to 15, wherein the method comprises the step of thermally treating an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles.

20. The method of claim 19, wherein the method is further characterized by any of the steps as defined in any one of claims 6 to 14.