WO2005097672A2

WO2005097672A2 - Method for the production of carbon nanotubes that are ferromagnetically filled in part and carry biomolecules, and use thereof in diagnosis and therapy

Info

Publication number: WO2005097672A2
Application number: PCT/EP2005/003790
Authority: WO
Inventors: Jens Ingolf MÖNCH; Axel Meye; Albrecht Leonhardt; Gerd Hammermann; Manfred P. Wirth; Bernd BÜCHNER; Kai KRÄMER
Original assignee: Leibniz-Institut Für Festkörper- Und Werkstoffforschung Dresden; Technische Universität Dresden; Hamstein Consult Gmbh
Priority date: 2004-04-07
Filing date: 2005-04-07
Publication date: 2005-10-20
Also published as: WO2005097672A3; EP1747018A2; DE102004017705A1

Abstract

The invention relates to a method for producing single-wall or multiple-wall carbon nanotubes (CNTs) that are ferromagnetically filled in part with iron, cobalt, nickel, an alloy thereof, or an alloy of said elements and platinum while carrying biomolecules which are enclosed within the tube and/or are associated with the outer surface of the tube. In a preferred embodiment of the invention, the disclosed nanotubes can contain two or several active substances in the tube which are released one after another when used in the body. The invention further relates to the use of the inventive carbon nanotubes that are ferromagnetically filled in part for diagnostic and therapeutic purposes, especially for the diagnosis and treatment of tumors.

Description

Process for the production of partially ferromagnetic carbon nanotubes carrying biomolecules and their use in diagnosis and therapy

The invention relates to a process for the production of partially ferromagnetically filled single or multi-walled carbon nanotubes (carbon nanotubes, CNTs) which are filled with iron, cobalt, nickel or an alloy thereof or an alloy of these elements with platinum and carry biomolecules where - with the biomolecules enclosed in the tube and / or with the outer

Surface of the tube are associated. In a preferred embodiment of the invention, the nanotubes according to the invention can contain two or more different active substances in the tube, which are released one after the other when used in the body. The invention also relates to the use of the partially ferromagnetically filled materials according to the invention

Carbon nanotubes in diagnosis and therapy, especially tumor diagnosis and therapy.

There is still a need for diagnostic and therapeutic agents that can be used in human or veterinary medicine.

Nanoparticles for use in diagnostics and therapy are already known from the literature. So z. B. in the patent EP 0625055 (DE 69328550 T2) fullerene-like compounds with magnetic molecules, ions, etc. and used in particular for diagnostic imaging methods. The aim here is to realize particles that are as small as possible and thus superparamagnetic, which is useful for the contrast agents sought in diagnostic imaging methods. Due to the superparamagic state, however, an energy conversion and thus, in addition to the diagnosis, a therapeutic application, eg. B. in hyperthermia, only possible to a limited extent.

Magnetic nanoparticles for use in diagnosis and therapy are also known from the literature (compare, for example, QA Pankhurst et al., J. Phys. D .: Appl. Phys. 36 (2003), R161-R181). But they show an irregular

Geometry and have a considerable proportion of nanocrystalline or amorphous phase in their structure. This adversely affects the magnetic properties. For a high energy turnover, e.g. B. For use in hyperthermia, the largest possible area under the magnetization curve is sought. The known magnetic nanoparticles are not optimal for this application and also do not allow simultaneous loading with several therapeutic agents and / or diagnostic agents.

Diagnostic and therapeutic agents also strive for a very tight tolerance in the geometric dimensions and the diagnostic / therapeutic volume of the active phase. These requirements cannot be met with the geometrically irregular nanoparticles.

The object of the invention was therefore to provide magnetic compounds which can be used universally as diagnostic and therapeutic agents and which can advantageously simultaneously serve both as diagnostic agents and as therapeutic agents for the same disease and, if appropriate, can carry several therapeutic agents or can be implemented as a depot form. The object of the invention was, in particular, to specify a method for producing such connections with their geometric dimensions and their magnetic properties can be produced in a stable and defined manner.

The object of the invention is achieved according to the independent claims. The subclaims represent preferred embodiments.

It has been found that with the method according to the invention, partially ferromagnetic, with iron, cobalt, nickel or an alloy thereof or an alloy of iron, cobalt, nickel with platinum, filled carbon nanotubes can be produced in a targeted and reproducible manner and in the desired manner biochemically can be functionalized. In a preferred embodiment of the invention, the metal filling is monocrystalline or polycrystalline to a high degree by volume and has a length-width ratio greater than 1. The metal filling in the nanotubes is preferably only partially formed in order to to have the remaining volume available for diagnostically and / or therapeutically active compounds. A 10 percent ferromagnetic filling, for example, is already sufficient to use the carbon nanotubes according to the invention in imaging processes, e.g. B. the MRI to use. According to the invention, the ferromagnetic filling in the carbon nanotube has a higher coercive force than the same ferromagnetic material in the bulk material.

The carbon nanotubes according to the invention can be multi-walled but also single-walled. The single-wall carbon nanotubes are preferred for the use according to the invention and were produced for the first time using the method according to the invention.

The partially ferromagnetically filled carbon nanotubes according to the invention are produced by providing a substrate over the entire surface with a layer on which the ferromagnetically filled carbon nanotubes do not grow, for example a gold layer, the thickness of which is selected depending on the desired length of the nanotubes. subsequently a photoresist layer is applied to this layer, subsequently a dot pattern in the form of holes, preferably cylindrical holes, of the same size is introduced by means of lithographic methods, the diameter of which is selected as a function of the desired outer diameter of the resulting nanotubes, now after exposure and resist development by a directional etching process at an angle of approx. 90 °, the layer on which the ferromagnetic carbon nanotubes do not grow is thinned at the points not covered with photoresist so that holes i n the depth are formed, which correspond ^• ren the desired length of Nanoröh-, then the bottom of the holes is coated with a catalytically active metal layer, hereinafter referred to primarily in the holes by means of chemical vapor deposition (by pyrolysis of metallor- ganic compounds and / or hydrocarbons) carbon nanotubes partially filled with the ferromagnetic materials are deposited, then superficial protrusions are removed by a directional etching process, then those partially with the ferromagnetic Materials filled nanotubes optionally a thermal treatment ^■ lung are subjected, and then the nanotubes a) on the open side with diagnostically and / or therapeutically active compounds, sealed with a biocompatible and degradable in the body or disclose polymer or gel which detached from the substrate and optionally biofunctionalized on the surface by means of a carrier compound or b) sealed with a biocompatible polymer or gel, detached from the substrate and biofunctionalized on the surface with diagnostically and / or therapeutically active compounds and optionally additionally by means of a carrier compound ,

Metal locenes of the elements iron, cobalt, nickel or their mixtures, carbonyls of these metals or their mixtures or other organometallic compounds of these elements are used individually or as mixtures, optionally in a mixture with readily thermally decomposable platinum compounds, as starting materials for chemical vapor deposition.

The nanotubes that can be produced with the method according to the invention have outer diameters between 10 and 100 nm and preferably a maximum

Length of 500 nm.

It is known in principle that ferromagnetic carbon nanotubes can be produced by chemical vapor deposition (Chemical Vapon Deposition, CVD) (A. Leonhardt et al. In "Diamond and Related Materials 3-7: 790-793 (2002)") However, the production of partially ferromagnetically filled nanotubes is not described and is not trivial. On the one hand, the nanotubes of the invention should contain sufficient ferromagnetic filling, for example, to also be used in hyperthermia, be associable.

In chemical vapor deposition, the growth of the nanotubes as partially filled tubes can be carried out according to the invention using a Two-zone furnaces can be realized, the sublimation of the metal-containing compound used being implemented in the first low-temperature zone and the thermal decomposition of the sublimed compound and the hydrocarbon used being carried out in the high-temperature zone. The temperature ranges are advantageously realized spatially separated.

When using several precursors with clearly differing sublimation temperatures, it can also be advantageous to control the temperature for the decomposition of the individual starting materials separately and thus to switch from a two-zone furnace to a multi-zone furnace. The composition of the individual ferromagnetic components in the carbon nanotubes can then also be controlled in a simple manner by the temperature for the decomposition of the individual components.

It is advantageous to carry out the sublimation of the metal-containing starting compounds in the temperature range from 100 ° C. to 175 ° C. The actual growth of the metal-filled carbon nanotubes advantageously takes place in a temperature range from 850 ° C to 1,050 ° C. For this purpose, the sublimed metallocenes (Fe, Co, Ni), carbonyls of these metals or organometallic compounds see individually or as a mixture over a metal-coated

Substrate (preferably Fe) transported.

A hydrocarbon can optionally be added to the gas. The location of the nanotube growth is determined by the location of the catalytic metal particles on the precoated substrate. The length of the filled tubes is determined by the duration of the CVD process, the sublimation temperature and thus the concentration of the precursors, the reaction temperature and the flow conditions in the reactor. For example, The growth of an empty carbon tube can first be achieved by introducing only the hydrocarbon (eg acetylene) and thereby the tubes into the

Holes which are coated with the catalyst (eg Fe) grow unfilled and then the tubes continue to grow filled by changing the gas phase (introduction of an organometallic compound and termination of the hydrocarbon supply). This change in the gas phase (e.g. metallocene / acetylene Change) can be done several times, so that alternately filled and unfilled

Areas are created in the nanotubes.

In order to produce single-walled nanotubes, which are preferred according to the invention, two- or multi-component alloys are used as the catalytic metal coating on the bottoms of the etched holes in the process described

(preferably Fe / Mo) applied in order to stabilize sufficiently small active catalyst particles, preferably Fe, which are embedded in the alloy. Single-walled nanotubes can also be produced by strongly shifting the ratio of metal component to carbon component to the side of the metal component in the gas phase in such a way that the carbon component is completely converted in the gas phase.

It has further been shown that the nanotubes produced can be thermally treated before the biofunctionalization so that the carbon shell is stable enough for biofunctionalization or for the connection of carrier molecules which are intended to bring the nanotubes to the site of action. This would require approx. 3,000 ° C to 4,000 ° C. In a preferred embodiment of the invention, however, this thermal treatment is carried out as a plasma chemical treatment in an argon or argon / hydrogen atmosphere (at approx. 800 ° C. to 900 ° C.), as a result of which any structural defects in the carbon shell can be effectively healed and thereby a high one Degree of crystalline perfection is achieved.

In a further preferred embodiment of the invention, the ferromagnetic filling, which may also be carbide or paramagnetic

Components can contain, subsequently (but before the biofunctionalization) thermally treated and converted in such a way that the filling consists exclusively of the ferromagnetic modifications (usually the α-modifications) of the elements iron, cobalt or nickel. As a result, 20% less filling and less energy is required for use in diagnostic or / and therapeutic methods with the nanotubes according to the invention. The subsequent thermal conversion can take place, for example, by annealing, in the case of an iron filling at approx. 650 ° C., just below the Curie temperature. The annealing temperature for nickel fillings is 350 ° C Cobalt filling at 1000 ° C. For alloy fillings (e.g. Ni / Co) one must

Annealing temperature can be selected, which, depending on the composition of the alloy, lies between the Curie temperatures of the individual components (here between 350 and 1000 ° C).

For the purposes of the invention, thermal treatment therefore means a plasma-chemical treatment for healing structural defects in the carbon shell and / or a thermal treatment of the filling in order to achieve an exclusively ferromagnetic filling.

It is further preferred according to the invention to use the directional etching method

Removing the superficially protruding portions at an angle of 2 to 45 ° to the surface of the substrate, preferably from 5 to 15 °. The directional etching with ions is preferably carried out by means of IBE (Ion Beam Etching), RIBE (Reactive Ion Beam Etching) or CAIBE (Chemical Assisted Ion Beam Etching).

The biocompatible and biodegradable polymer with which the nanotubes produced can be closed is, for example, polyethylene glycol, a copolymer of lactic acid and glycolic acid, a copolymer of lactic acid, glycolic acid and PEG or other structurally related polymers. The release of an active ingredient from the nanotubes depends on the type of polymer or gel used. Hydrogels which contain water as a solvent have proven to be particularly favorable. These hydrogels are harmless to health and have favorable thermosensitive swelling behavior. For example, hydroxypropyl

(HPC) has a UKLT (lower critical solution temperature) of 44 ° C +/- 2 ° C and is therefore particularly suitable for the desired effect. A thermo-reversible connection can be achieved by further crosslinking with divinyl sulfone, but this entails high toxicity. This problem can be solved by crosslinking on an isocyanate basis, since a urethane bond is formed in the course of the reaction. According to the invention, such polymers / gels are used to close the nanotubes, the swelling and shrinking behavior of which can be influenced in a defined manner by varying the degree of crosslinking. Depending on the temperature, a reversibly adjustable degree of swelling between 100% and> 1,500% can be achieved. With this type of release is above the

UKLT greatly reduces the volume of the gel, so that the agent is released by diffusion processes (variant F1).

When using gels that increase the volume extremely above a critical temperature, the nanotube is irreversibly destroyed by the volume increase (variant F2) and the agent is thereby released. Such gels are e.g. Block copolymers based on PNIPAAm (poly (N-isopropylacrylamide)). The block copolymers should be produced with a modified poly (ethylene oxide) PEO macroinitiator by ATRP (Atom Transfer Radical Polymerization). By a suitable choice of the polymer composition and the degree of crosslinking, the temperature of the volume increase can be in sufficiently wide temperature ranges, e.g. can be set to 47 ° C and the opening of the nanotubes can be achieved by increasing the volume of the polymer. Other possible materials for variants F1 or F2 are known. The solubility properties of the gels used can vary according to the expected environmental conditions, e.g. Tissue, blood or other body fluids can be adjusted and thus the opening of the nanotubes in the body can be controlled.

Filling the unilaterally opened nanotube with a diagnostically and / or therapeutically active compound can e.g. by separating this compound from the gas phase. These can also be fullerenes containing metal ions, for example.

In a particularly preferred embodiment of the invention, the nanotubes produced and partially filled with ferromagnetic materials, which are still open at one end, are alternately filled with therapeutically / diagnostically active compounds and biocompatible polymers, the biocompatible polymers being selected such that each is different from the other Conditions, preferably at a different temperature in the body is degradable.

As a result, intrinsic forms of custody account can be realized. In a preferred embodiment of the invention, partially ferromagnetically filled carbon nanotubes of basic type 1 (FIG. 6) and basic type 2 (FIG. 7) are produced.

To produce the basic type 1, the nanotubes are first filled and then unfilled to such a length that in process step a), after filling with a first active ingredient and sealing with a first polymer / gel (polymer 1), another active ingredient or the same Active ingredient can be introduced again and with a second, for example degradable in the body at a lower temperature, polymer / gel (polymer 2) is closed. In the therapeutic application, for example

42 ° C removes the polymer 2 and thus releases the active ingredient, which can react at the cellular level. At a later time, the temperature is raised to 47 ° C. and the active ingredient in the second chamber is released. If it is the same active ingredient (e.g. cis-platinum), the active ingredient concentration can be kept constant over a longer period of time.

In basic type 2 (see Fig. 7), the nanotubes are manufactured as described above in such a way that the ferromagnetic filling is included in the center and the same or different active substances are enclosed on both sides by polymers / gels that are degradable in the body at different temperatures. Since nanotubes are closed on one side during the manufacturing process, they have to be opened on the closed side. This is preferably achieved with an argon / oxygen low-pressure plasma.

The closing of the nanotubes filled with active ingredient is preferably by means of

Argon plasma polymerization of monomers that are temperature sensitive.

The bio-functionalization of the nanotubes according to the invention on the outer surface is carried out using known methods. These are described in detail in EP 0625055, for example.

The biofunctionalization can be carried out, for example, via a covalent substitution on the carbon shell or, as described above, a closable polymer cap. Functional group means a chemical group that can bind to a specific component and thus associates the nanocontainer with a specific component. Examples include monoclonal or polyclonal antibodies, Fab fragments of polyclonal and Fab2 fragments of monoclonal antibodies, fused and unfused single and multi-chain recognition motifs.

In a particularly preferred embodiment, the biofunctionalization of the outer surface of the carbon nanotubes is carried out in such a way that the nanotubes are partially provided with a metal on the outside, which enables the targeted connection of the diagnostically and / or therapeutically active compounds at this point.

In principle, it is possible to provide the ferromagnetically filled carbon nanotubes with additional “docking points” for biomolecules. This is achieved, for example, by generating the cylindrical ones

Holes in the gold layer according to embodiment 1 are not first deposited as a metal which acts catalytically for the growth of the nanotubes, but rather a metal which is preferred for binding the biomolecules, e.g. Niobium and only in the subsequent coating step the catalytically active metal. After removal of the photoresist, this is also done here

Embodiment 1 the growth of the ferromagnetic partially filled carbon nanotubes. In the display process, growth takes place starting from the metal layer and implementing these metal parts. In the later separation process by etching the gold layer, the niobium is not removed from the ferromagnetic and possibly agent-filled nanotubes and is therefore available for binding the biomolecules. In this production variant, the partially coated portion is located opposite the nanotube opening that is closed by the biocompatible polymer.

The biofunctionalized carbon

After production, nanotubes are subjected to the necessary cleaning and rinsing processes, the nanotubes being held to the vessel wall by permanent magnets. After that, they are ready for use according to pharmaceutical formulation for diagnosis and / or therapy. The manufacturing process cedures, preparation, functionalization and galenical formulation are always carried out under sterile conditions as are customary for the production of pharmaceutical agents.

A major advantage of using substrates in the manufacturing process of nanotubes is that the diameter of the biofunctionalized

Carbon nanotubes are decisively determined or co-determined by the diameter of the structure etching, the length of the biofunctionalized carbon nanotubes by the depth of this etching in the gold layer. This is the only way - in contrast to other manufacturing methods - that close tolerances in the end product can be maintained.

If an agent is not filled, the use of substrates can in principle be dispensed with and the ferromagnetically filled carbon nanotubes can be represented as "powder" in the CVD reactor. The biofunctionalization takes place in an analogous manner after the additional chemical cleaning steps then required.

For the purposes of the invention, diagnostic units are understood to be biomolecules, elements with atomic numbers above 50, chromophores or fluorophores. According to the invention, therapeutically active units are molecules which represent chemically or genetically engineered pharmaceutical active substances, e.g. B. Chemotherapy drugs. For the purposes of the invention, biomolecules are preferably peptides, proteins, nucleic acids, antibodies, their fragments, enzymes, hormones or polysaccharides.

The diagnostically effective biomolecules are affinity molecules for specific recognition and binding to one or more target molecules. These can be, for example, peptides or proteins (e.g. antibodies, receptors, antigens, lectins, avidine oligopeptides, lipoproteins, glycoproteins, peptide hormones), nucleic acids, carbohydrates or ligands with a low molecular weight.

The nanotubes according to the invention can be used on the outer surface exclusively or in addition to the diagnostic or therapeutic inputs. Carrier molecules, such as cationic lipids or antibodies, carry the

Arrange transport to the target cell or the preferred site of action.

The biomolecules and / or carrier molecules associated on the outer surface of the tube can be conjugated or bound directly or via a linker.

In addition to the production method according to the invention, the invention also relates to the carbon nanotubes produced in this way.

The carbon nanotubes according to the invention can advantageously be used for therapy. The biomolecules on the surface of the nanotube allow, for example, the nanotubes to be transported to a specific biological unit (cell, tissue or organ) in the body. For example, a nanotube coated with antibodies is transported in the body to the corresponding antigen. At the same time, the carbon nanotubes according to the invention can contain an active ingredient, e.g. an antitumor agent (doxorubicin (trade name Adriamycin), paclitaxel and its derivatives (taxol, taxanes, etc.), vincristine, carboplatin, cisplatin, epirubicin, fluorouracil, ifosfamide, methotrexate, mitomycin, vinblastine, irinotecan, or containing mitoxantruide, in the desired target region, e.g. the tumor can be transported. The carbon nanotubes according to the invention can also function as depot forms, which release the drug with a defined delay, the depot function being realized by various sections of the tube containing the active substance, the boundaries of which are gradually broken down and the active substance is thus released at different times and in doses. The gradual dismantling can e.g. depending on the temperature.

For therapeutic purposes, it is also possible with the carbon nanotubes according to the invention to enclose a nucleic acid-based vector which carries the therapeutic gene or parts thereof, and to bring this into contact with the tissue at the desired site of action in order to increase the efficiency of the To increase gene transfection and expression. It is also possible to bring the viral vector that carries the therapeutic gene onto the surface of the carbon nanotubes. Due to external magnetic fields This carrier can then be kept at the destination to keep the virus in for extended periods

Bring contact with the appropriate tissue.

An important area of application of the nanotubes according to the invention is hyperthermia, in which the nanotubes according to the invention are excited by a magnetic field or thermally to absorb heat, which they then transmit to the focus of the disease, e.g. the tumor.

It is also possible according to the invention to inject nanotubes with a length between approximately 10-20 μm into malignant tissue, so that local concentration and the release of active substance or thermal excitation can take place there. If, according to the invention, the nanotubes are additionally provided with diagnostic units, tumor diagnosis (primary tumor, recurrence and / or metastasis) can be carried out pretherapeutically. In addition, the nanotubes can also be biofunctionalized in such a way that they are increasingly taken up by tumor cells (cf. Example 2).

An alternative therapeutic approach uses a magnetically-based in vivo movement of nanotubes of minimal dimensions, the length of which does not exceed 100 nm: with miniaturized super magnets that are implemented in a targeted manner, individually or multiple, temporarily peritumoral, biofunctionalized nanotubes can be used systemically or in close proximity to the focus of the tumor into a body fluid, concentrated at the focus of the tumor. The efficiency of this transfer can be directly influenced by various parameters (e.g. strength of the magnetic field and degree of filling of the nanotubes). After the local therapy, the miniaturized super magnets are explanted again.

In a further embodiment, nanotubes are irradiated ex vivo, whereby the iron, which is the partial filling of the nanotubes, is converted to radioactive iron. These radionuclides can then be used as a therapeutic agent, packaged in the form of a nanotube. In addition to the diagnostic and therapeutic methods described, the ferromagnetic carbon nanotubes according to the invention are also very suitable for diagnosis even without enclosed or associated biomolecules in the outer surface, since they can be excited magnetically and / or thermally by the ferromagnetic properties. The biomolecules associated on the carbon surface have the advantage that additional binding to and transport to the target molecule can take place.

The invention also relates to the use of the carbon nanotubes produced according to the invention as a diagnostic and / or therapeutic agent, in particular for tumor diagnosis and therapy. The diagnostic and / or therapeutic agent is provided by formulating the physiologically compatible carbon nanotubes produced according to the invention with pharmaceutically compatible adjuvants. Auxiliaries in the sense of the invention are formulation auxiliaries, such as e.g. Carriers, antioxidants, stabilizers or buffers.

In a preferred embodiment of the invention, the pharmaceutical compositions are intended for i.v. application. The appropriate infusion or injection solutions are preferably provided with physiological saline. The diagnostic or therapeutic agents can also i.m., i.p. or administered orally. In the case of solid tumors treated with the CNTs of the invention, e.g. Prostate carcinoma and bladder carcinoma, the CNTs are peri- or intratumoral, i.e. injected into the adjacent tumor tissue or directly into the tumor.

The invention thus also relates to a method for diagnosing and / or treating a tumor disease, in particular bladder and prostate cancer, in which the subject has an effective amount, preferably 10 mg to. 500 mg per target lesion depending on the size of the tumor which is administered according to the invention carbon nanotubes.

The ferromagnetically filled and biofunctionalized carbon nanotubes produced by the method according to the invention have a number of significant advantages over the prior art. They are especially special in their geometrical dimensions and their magnetic properties can be produced in a defined manner and can also be specifically filled through the possibility of controlled opening and closing. Through the process control in the representation of the carbon nanotubes according to the invention, it is also possible to influence the number of carbon shells around the core and thus to adapt the chemical resistance to the desired conditions of use. Single-wall ferromagnetic carbon nanotubes are being produced for the first time. The CNTs produced according to the invention are extremely stable. In contrast to any other manufacturing method previously described for nanoparticles, the nanotubes can be partially provided with another metal on the outside, which enables the targeted connection of biomolecules at these locations.

The invention is explained in more detail below using exemplary embodiments.

example 1

Manufacture of nanotubes filled with ferromagnetic active ingredients and bio-functionalized on the surface

The production of the partially ferromagnetic and partial according to the invention

Agent-filled nanotubes can advantageously be realized by growth and manipulation of the nanomaterials on substrates. Figures 1 to 4 show the basic sketch for the production. Fig. 1 shows a substrate with a 5 nm thick Fe layer on Au and on photoresist. Fig. 2 shows the substrate with partially filled nanotube after the CVD

Growth. The direction of etching in argon ion beam etching to remove the protruding portions is indicated by arrows.

Fig. 3 shows the substrate after shortening, filling and sealing the nanotubes. Fig. 4 shows the end product after etching the Au layer and processing the functionalized nanotubes.

A thermally oxidized silicon wafer with an oxide layer thickness of 1 μm serves as the substrate. Using suitable coating methods - e.g. thermal Evaporation - a 200 nm thick gold layer is applied to the entire surface of this substrate. Then a 1 μm thick photoresist layer is applied. In the following process step, a regular dot pattern is realized using lithographic methods, for example electron beam lithography. The individual structures have a diameter of 80nm at a distance of 400nm. The later outer diameter of the carbon nanotubes can be significantly determined by the size of these structures. After the exposure and the necessary resist development, the substrates are rinsed in ultrapure water and dried. Argon ion beam etching at an angle of 90 ° relative to the sample surface partially thinned the 200nm thick gold layer - at the locations that are not covered with photoresist - so that holes about 150nm deep were created in the Au layer. The later length of the biofunctionalized nanotubes is determined by this etching depth. This etching process is carried out under the following conditions: etching gas: argon, pressure: 6x10 ^"4 mbar, acceleration voltage: 400V, microwave power: 170 W.

In the next step, the etched substrates are subjected to a further coating with the photoresist still present. An approximately 5 nm thick iron layer is deposited by electron beam evaporation and the coated substrate is subsequently treated in acetone (15 min). The photoresist is detached and the Fe layer portions deposited on the photoresist are lifted off. The catalytic Fe components are now only in the deeper etched Au areas. Before the actual production of the nanotubes begins, it is advantageous to clean the pre-structured substrate in a hydrogen plasma.

The pre-structured substrate is then heated to 900 ° C. in the reaction zone of a chemical vapor deposition (CVD) reactor. The CVD reactor used allows the temperature in several zones to be set independently of one another and the growth process to be carried out under inert gas or vacuum conditions. Ferocenes (0.3 g in a crucible) are used as the starting material for the production of the partially ferromagnetically filled carbon nanotubes. This precursor is sublimed at a temperature of 180 ° C outside the actual reaction zone. As a carrier gas, which sublimates the precursor into the actual reaction zone transported, argon is used. The metallocene used here is both

Source of ferromagnetic material as well as source of carbon for the shell. Under the realized conditions, completely Fe-filled carbon tubes are deposited within the first 3 minutes, preferably within 1 minute, about 50 nm long. To achieve the end result of only partially filled nanotubes, the temperature of the metal precursors is now very quickly reduced from 180 ° C to a temperature <100 ° C and a gaseous hydrocarbon (acetylene) is introduced into the reactor. This ensures that after a short time only the hydrocarbon is in the gas phase and the nanotubes only continue to grow unfilled, since no further filler material from the gas phase is offered.

As a result of small temperature fluctuations, different flow conditions and / or fluctuations in the precursor concentration, the carbon nanotubes produced can have a variation in length. In order to produce partially filled carbon nanotubes of the same length, they are subjected to a directional etching process in the subsequent technology step. The portions above the unetched Au surface are removed by argon ion beam etching at an angle of 8 ° relative to the sample surface. This etching process is carried out under the following conditions: etching gas: argon, pressure: 6x10 ^"4 mbar, acceleration voltage: 400V, microwave power: 170 W.

The partially ferromagnetically filled carbon nanotubes are thus open on the side facing away from the substrate and can be treated with a first agent, e.g. a therapeutic agent can be partially filled. The length of the agent filling is e.g. 20nm. For the defined termination of the agent, the still unfilled part of the carbon nanotube is filled with a biocompatible polymer that is degradable in the body at a certain temperature. This biocompatible polymer is e.g. Degradable at 42.5 ° C. If necessary, additional sections with alternating proportions of agent and biocompatible polymer can be realized. Here is the melting point of the biocompatible

To reduce polymers by 1 ° C, so that the release of the therapeutic agent can be controlled by changing the temperature in the later therapeutic application. The change in temperature can can be easily controlled via the energy converted in the ferromagnetic filling.

The biocompatible polymers must therefore be selected so that the polymer that closes the tube is first broken down at the lowest temperature and then the further polymers are gradually broken down as the temperature increases gradually, so that one active ingredient is released after the other. If the same active ingredient is found in the individual sections of the tube, a delayed release (depot form, sustained release) can be achieved in this way. After the desired fillings have been realized, the functionalized carbon nanotubes are separated from the substrate. For this, the gold on the substrate is treated in a solution (consisting of 0.06 mol / l KJ, 0.09 mol / l J ₂ and 0.005 mol / l alcohol) for 10 min. Similar to a "lift-off" process, the gold located under the functionalized nanotubes is removed, thereby removing the hold from the nanotubes. The nanotubes are now in the etching solution. Since the partially filled nanotubes have ferromagnetic properties, they can easily be held on the vessel wall by a permanent magnet. In this state, rinsing with water to remove the etchant residues is possible. The use of permanent or electromagnets simultaneously separates the ferromagnetically filled from the insufficiently ferromagnetically filled nanotubes. The insufficiently filled tubes are removed with the water. Of course, the etchant residues can also be removed by flushing using ultrasound. It is advantageous to add a volume fraction of at least 1% alcohol to the water. Hereby the realization of the water / nanotube

Suspension much easier.

In a subsequent step, the outer surface of the tubes is biofunctionalized in solution. Analogous to the possibilities described in the literature for the functionalization of carbon nanotubes (Zheng et al. (2003), Nature Materials 2: 338-342), individual carbon

Nanotubes, for example, are bound with single-stranded (ssDNA) and therapeutically active DNA molecules via "π-stacking". The binding of an ssDNA molecule is achieved via various mechanisms and can take place partially or over the full length of this structure, whereby Examples include a helical packaging with left and right turns and a simple surface adsorption. Such a dispersion functionalizes the carbon nanotube per se, since the hybrids are negatively charged due to the phosphate group of the ssDNA molecule. This dispersion can also be used, for example short double-stranded DNA molecules (dsDNA), siRNA constructs or RNA molecules.

In these cases, the carbon nanotube serves as a combined carrier-shuttle functional unit for the transport of therapeutically active substances to a site of action in the body of a living being. Such a structural unit can also be encased and moved using an additional carrier, for example a cationic lipid.

Example 2

Chemical treatment of advanced prostate cancer (PCa) As part of the treatment of advanced tumor disease, such as diffuse metastatic prostate cancer, nanotubes are sometimes injected into the malignant tissue peritumorally or intratumorally after conventional diagnostic localization of the metastatic foci. When using "long" nanotubes with a length of approx. 10-20 μm, which guarantees a local concentration in the target tissue (no free diffusion possible), there is a thermal excitation or release of those attached or attached to the nanotube For this purpose, for example, the nanotubes can be labeled with choline, which leads to a relatively increased uptake by the tumor cells (in comparison to the surrounding, non-malignant cells) The metastatic foci can be represented in this way from a size of a few millimeters, and can be treated immediately afterwards. Example 3

Local Treatment of Bladder Carcinoma (BCa) With the carbon nanotubes produced according to Example 1, an instillation therapy of the bladder of a patient suffering from bladder carcinoma is carried out. Using a highly precise magnetic field applied to the outside of the bladder wall, the ferromagnetically filled carbon nanotubes according to Example 1 are concentrated at the resection site of a superficial bladder tumor area. In a subsequent step, the active substance is released in the resection bed of the tumor under thermal excitation. Fig. 5 shows the clinically local therapy against superficial tumors of the human urinary bladder (bladder carcinoma, BCa) using the carbon nanotubes produced according to Example 1, which are coated with antisense DNA constructs. The tumor is removed using electroresection. The resection margins (sediment margins after removal of the tumor mass) become corresponding

Storage of the bladder overlays one or two times with a concentrated, defined solution and incubated. This form of treatment can be repeated or modified as part of post-resections (secondary transurethral bladder tumor resection, TUR-B, or in the case of cystoscopic follow-up). It can also be combined with conservative local chemotherapy (mitomycin-C, Bacillus-Calmette-Guerin) These active ingredients can both be implemented in the nanotube and released in a targeted manner, and can also be used in the form of an external complexation with the nanotubes.

Example 4

Nuclear thermal tumor treatment

The carbon nanotubes produced in accordance with Example 1 are excited after application and targeting to a bladder carcinoma (BCa) by external radiation to absorb heat. Their localization requires a local heating of the tumor tissue and thus a therapy-efficient and selective destruction of tumor cells. In addition, the carbon nanotubes according to the invention can be used to diagnose (locate) this tumor if they carry appropriate diagnostic units. This enables a timely and metered combination of tumor diagnosis and tumor treatment.

Example 5

Intracellular detection of CNT clusters

Adherently growing human bladder carcinoma cells (EJ28 cell line) were incubated for 2 hours with a mixture of partially ferromagnetic-filled CNTs without active ingredient and a cationic lipid, then the cells were washed with PBS and detached from the culture vessel by means of trypsin-EDTA digestion, centrifuged and according to standard protocols embedded in resin. The transmission electron microscopic examination (top) of 55 nm thin sections shows an internalization of the CNT into the cytoplasm of the cells. On the basis of the EDX analysis carried out in the transmission electron microscope on the nanotubes taken into the tumor cell and cross-sectioned by the preparation, it is clearly demonstrated that the localized structures are filled with iron. In addition to the characteristic lines Fe-L and Fe-K, only carbon is detected as a further main component. Fig. 10 shows a TEM image and an EDX spectrum of an Fe-filled one

Carbon nanotube in an EJ28 tumor cell (before hyperthermia treatment).

Example 6

Flow cytometry (FACS analysis in FSC-SSC dot blot: FACScan. Becton Dickinson) on PC-3 prostate cancer cells after incubation with CNTs (see Fig. 11).

A CNT solution (without cells, left) can be characterized using FACS (presentation of the overall result for 2x10E4 cells examined), whereby the detectable particles have a wide range of granularity, which indicates a significant adhesion or clustering of the CNT. If untreated cells (middle) are compared with the same cells, which are incubated for 2 h with a CNT solution and treated as in Example 5 (trypsinization and centrifugation) (right side), you can see a clear one

Increase in relative granularity in the majority of cells analyzed. This is independent evidence that CNT is associated with cells in vitro. However, this FSC-SSC analysis cannot differentiate between extracellular adhesion of the CNTs to the cell membrane or a localization in the cell membrane and a cytoplasmic localization. The

Evidence of the uptake of ferromagnetically filled carbon nanotubes into the tumor cells was provided by TEM investigations.

Example 7

Magnetization curves of a tumor cell-CNT mixture The EJ28 cells were incubated for 2 h in petri dishes with CNT-lipid mixtures according to Example 5, then washed twice with PBS and stored as a cell pellet after trypsin digestion until the measurement.

Figure 8 shows magnetization curves of Fe-filled carbon nanotubes, transferred into tumor cells, whereby these cells were recovered from treated tissue. The magnetization curves show a typical ferromagnetic behavior of the Fe-filled carbon nanotubes taken up in the cells. A pronounced anisotropy cannot be demonstrated by the measurements. This is not to be expected due to the lack of preferential orientation in cells. The level of saturation magnetization is clearly dependent on the transferred amount of α-iron in the filled carbon nanotubes. A correlation of the detected elementary magnetic units (emu), which is directly related to the mass of the α-iron absorbed into the cells, proves that enough iron was absorbed into the cells for successful hyperthermia. The meanings are: μ ₀ H = applied magnetic induction m = magnetic moment Example 8

Application example for CNTs according to the invention with two different active ingredients inside

With a standardized measuring cell that allows both magnetic and resulting heat manipulation from the outside and local control of the heat development (principle of the measuring chamber using the example of a CNT

Treatment of tumor cells, see Fig. 9) inside the chamber, the therapeutic effect of a CNT treatment can be demonstrated and optimized. These measurements can be carried out both on tissue level (freshly obtained tissue thick sections and placed in a physiological solution) as well as on explanted organs and are shown below using an example:

A PCa xenograft tumor of human origin (PC3 cells) was explanted as a solid tumor from the nude mouse. From this, rough tissue sections with a thickness of 5 mm were obtained and transferred to the solution of the measuring chamber. After the tissue section had been fixed in the measuring chamber, an intratumoral injection of a CNT solution (100 μg CNT in total, at 6 different injection sites, distributed over the tissue section area) was carried out as described in basic type 1, i.e. sealed with two different polymers and filled with carboplatin and with a nucleic acid construct of non-human origin.

The marked injection sites (marked and coupled with heat sensors by contact-free heating of the CNTs to a temperature of 44 ° C (average temperature in a 3 mm radius around the injection site or sensor location) were then heated by applying an external magnetic field. When this temperature was reached There was a change in conformity of polymer / gel 1 with subsequent release / exposure of carboplatin The local antitumor effect of the therapeutic agent was determined by comparative studies with a placebo (filling with a non-toxic polymer) using a histological (TUNEL assay for late apoptosis detection) and Pathomorphological detection of apoptosis / necrosis in the tissue section in the period 24-96 h after release of the therapeutic agent or by FACS analysis (Annexin-V-PI analysis to detect early and late apoptosis and necrosis). Furthermore, some cuts were half

Hours after the release of carboplatin and non-contact heating heated to 48 ° C, the melting point of the second polymer (hereinafter referred to as CNT-based heated tissue sample). The detection of the release of nucleic acid fragments, which exemplarily represented the filling with the second therapeutic agent, was carried out by specific PCR detection of the non-human fragment (luciferase gene fragment), two different tissue samples serving as controls, which on the one hand filled with the same amount of CNT, but had not been heated (negative control 1) and, on the other hand, tissue samples without CNT injection, but with magnetic field treatment like CNT-injected sample (negative control 2). It could be clearly and exclusively for the double, CNT-based heated tissue sample that after the carboplatin release also at a locally detected temperature of 48 ° C, luciferase DNA fragments were released into the injected tissue.

Example 9

Another application example of the CNTs according to Example 8

An anesthetized nude mouse, which had carried a PCa xenograft tumor (PC3 cells, as in Example 8) of 1 cm maximum diameter subcutaneously, is fixed in the measuring chamber according to Fig. 9. After injection of CNT according to Example 8 (total dose 200 μg) via the tail vein, an external magnetic field is applied after 2 h. A frequency of 40 kHz was realized in a magnetic field of 15 kA / m. After an optimized magnetic field incubation of the entire animal body in preliminary tests, whereby several temperature probes had been implemented in the tumor border area, via which a local temperature measurement was carried out, the target temperature of 44 ° C was detected in peritumoral areas, with the mouse subsequently and without further magnetic field exposure 2 was kept under anesthesia for additional hours. Then followed in the periods 24/48/72

Tissue removal. TEM examinations of the outer tumor edges of the tumor confirmed the accumulation of CNT in and between tumor cells. Under histological control (H&E staining), therapy-induced antiproliferative effects (apoptosis induction) were also detected in the outer tissue areas. A second mouse was like above the. A second mouse was treated identically as described above, only after reaching target temperature 1 (44 ° C) was treatment continued in the magnetic field until target temperature 2 (48 ° C) was reached. The nucleic acid fragments released in this way could be made visible via a non-radioactive label in the tissue association. These results confirmed the feasibility of local and CNT-induced hyperthermia in combination with chemotherapy and their therapeutic efficiency.

Claims

claims

1. A process for the production of partially ferromagnetic with one of the elements iron, cobalt, nickel, an alloy thereof or an alloy of these elements with platinum-filled carbon nanotubes which carry diagnostically and / or therapeutically active compounds, characterized in that a The entire surface of the substrate is provided with a layer on which the ferromagnetically filled carbon nanotubes do not grow, the thickness of which is selected as a function of the desired length of the nanotubes, then a photoresist layer is applied to this layer, subsequently a dot pattern using lithographic methods in the form of holes of the same size, the diameter of which is selected as a function of the desired outer diameter of the resulting nanotubes, now after exposure and resist development by a directional etching process at an angle of approximately 90 °, the layer on which the ferr do not grow the magnetically filled carbon nanotubes, thin the areas not covered with photoresist so that holes are created in the depth corresponding to the desired length of the nanotubes, then the bottom of the holes is coated with a catalytically active metal layer, below carbon nanotubes partially filled with the ferromagnetic materials are deposited in the holes by chemical vapor deposition (catalytic pyrolysis of organometallic compounds and / or hydrocarbons), then superficial protrusions are removed by a directional etching process, then the nanotubes partially filled with the ferromagnetic materials, if necessary are subjected to a thermal treatment and then the nanotubes a) are filled via the open side with diagnostically and / or therapeutically active compounds, with a biocompatible and in the body a Buildable polymer or gel closed, detached from the substrate and optionally biofunctionalized on the surface by means of a carrier compound or b) sealed with a biocompatible polymer or gel, detached from the substrate and biofunctionalized on the surface with diagnostically and / or therapeutically active compounds and optionally additionally by means of a carrier compound.

2. The method according to claim 1, characterized in that a plasma chemical treatment in argon or argon / hydrogen atmosphere is carried out at about 800-900 ° C as the thermal treatment.

3. The method according to claim 1, characterized in that as a thermal treatment, an annealing of the filled carbon nanotubes is carried out shortly below the Curie temperature of the corresponding filler material such that a complete conversion of all phases of the filler material into the ferromagnetic modification is achieved.

4. The method according to claim 1, characterized in that the starting materials for the chemical vapor deposition are metallocenes of the elements iron, cobalt, nickel or their mixtures, carbonyls of these metals or their mixtures, optionally in a mixture with thermally easily decomposable platinum compounds.

5. The method according to claim 1, characterized in that for the production of single-walled nanotubes in chemical vapor deposition as catalyst particles on the substrate multi-component alloys, preferably iron / molybdenum, are used, in which the active catalyst, preferably iron, is embedded and thereby its size is stabilized.

6. The method according to claim 1, characterized in that in the chemical vapor deposition the growth of the empty carbon nanotubes is achieved by introducing the hydrocarbon alone, when a mixture of hydrocarbon and organometallic compound is introduced, the growth is partially filled and when exclusively introducing organometallic compounds the filled carbon tubes.

7. The method according to claim 1, characterized in that the directional etching process for removing the superficial protruding portions is carried out at an angle of 2 to 45 ° to the surface of the substrate, preferably from 5 to 15 °.

8. The method according to claim 7, characterized in that the directional etching with ions by means of IBE (Ion Beam Etching), RIBE (Reactive Ion Beam Etching) or CAIBE (Chemical Assisted Ion Beam Etching) is carried out.

9. The method according to claim 1, characterized in that the filling of the nanotubes with diagnostic and / or therapeutically active compounds is carried out by deposition from the gas phase or by liquid processes.

10. The method according to claim 1, characterized in that the sealing of the nanotubes with a biocompatible polymer is carried out by means of argon plasma polymerization.

1 1. The method according to claim 1, characterized in that a polyethylene glycol, a copolymer of lactic acid and glycolic acid, a copolymer of lactic acid, glycolic acid and PEG or other structurally related and biocompatible polymers are used as the biocompatible polymer.

12. The method according to claim 1, characterized in that partially filled with ferromagnetic materials nanotubes are produced, which contain the ferromagnetic filling in the center, the closed side of the tubes is opened in an argon / oxygen low-pressure plasma, the tubes on both sides with the same or with different active ingredients are filled and the two ends are closed with the same or different polymers or gels, the polymers / gels being selected so that each is degradable in the body at a different temperature.

13. The method according to claim 1, characterized in that the biofunctionalization of the outer surface of the carbon nanotubes is carried out in such a way that the nanotubes are partially provided on the outer surface with a metal which enables the targeted connection of the diagnostically and / or therapeutically active compounds enabled at these points.

14. The method according to claim 1, characterized in that the nanotubes are produced in such a length that in step a) after filling with a first active ingredient and sealing with a first polymer or gel, a second active ingredient or again the first active ingredient can be sealed with a second polymer or gel that degrades at a lower temperature in the body.

15. The method according to claim 1, characterized in that biomolecules, elements with atomic numbers above 50, chromophores or fluorophores are used as diagnostically or therapeutically active compounds with which the carbon nanotubes are filled and / or biofunctionalized on the surface.

16. The method according to claim 15, characterized in that peptides, proteins, nucleic acids, antibodies, their fragments, enzymes, hormones or polysaccharides are used as biomolecules.

17. The method according to claim 15, characterized in that chemically or genetically engineered active substances, in particular antitumor agents, are used as therapeutically active compounds.

18. Partially ferromagnetically filled carbon nanotubes which carry diagnostically and / or therapeutically active compounds, produced by the method of claims 1 to 17.

19. Carbon nanotubes according to claim 18 for use as a diagnostic and / or therapeutic agent.

20. Use of carbon nanotubes according to claim 18 for the production of a diagnostic and / or therapeutic agent, in particular for the diagnosis and / or therapy of tumor diseases.

21. Use of carbon nanotubes according to claim 20 for the diagnosis and / or therapy of bladder or prostate cancer.

22. A method for diagnosing and / or treating a tumor disease, in particular bladder or prostate cancer, characterized in that the test subject is administered an effective amount of the carbon nanotubes according to claim 18.