GB2415374A - Targeted delivery of biologically active substances using iron oxide/gold core-shell nanoparticles - Google Patents

Abstract

Iron oxide/gold core-shell nanoparticulate systems are prepared in which a biologically active substance is either bound to the surface of the gold shell of the nanoparticle or is contained within the gold shell of the nanoparticle. Such systems are used for the targeted delivery of biologically active substances, hyperthermia treatment, imaging or combinations thereof. The core diameter of the said nanoparticles is between 5-150nm and thickness of the gold shell is typically between 1-50nm. Depending on the size of the core, the particles are single domain magnetic or superparamagnetic. The biologically active substances used may be attached to the shell through chemical bonding, for example by using thiol or amine groups. Optical properties of the nanoparticles (i.e., the wavelength at which the nanoparticles may absorb) can be controlled by varying shell thickness and core size.

Description

24 1 5374

NANOPARTICLES AND USES THERE OF

FIELD OF THE INVENTION

The present invention involves the gram-scale synthesis of magnetic coreshell nanoparticles, where the core is iron oxide or more specifically maghemite (-Fe2O3) and the shell is gold, more particularly it concerns surface functionalized magnetic core-gold shell nanoparticles.

Furthermore, the invention concerns the synthesis of said particles where the shell is coated with biological or organic compounds. The present invention is aimed at using these particles as agents for magnetic targeted drug delivery, hyperthermia treatment, imaging or combinations there of.

BACKGROUND OF THE INVENTION

Magnetic nanoparticles can be prepared by a variety of methods. However, all synthetic methods are either based on the decomposition of organic iron-complexes in solution or on the formation of ferric oxy-hydroxide and subsequent dehydration. Both processes have been reported numerous times.' The advantage of synthetic methods based on the decomposition of iron-complexes is that quasi-monodisperse nanoparticles are formed, while the advantages of the other method are easy handling, less hazard and absence of inert gas during reaction.

Furthermore, this process lends itself to industrial gram scale operations.

The magnetism of small ferromagnetic particles is dominated by two key features: there is a size limit below which the specimen can no longer gain favorable energy configuration by breaking up into domains; hence it remains with one domain, the single domain magnetic (SDM) nanoparticles.

The thermal energy can, with small enough size, decouple the magnetization from the particle itself to give rise to the phenomenon of superparamagnetism, the superparamagnetic (SPM) nanoparti cl es.

These two key features are represented by two key sizes (or the length scale), the single domain size and the superparamagnetic size, both of which are well documented in literature.2 1 e :-::: ..: :- :: .

The key difference between them lies in the fact that SDMs show affinity towards self assembly to form strings of magnetic particles resulting in net magnetic moment,3 whereas SPMs need an external magnetic field to align themselves. An advantage in use of SDMs is that they show very large coercivity (the ability to retain their magnetic properties against any demagnetization force).4 Literature search revealed that one of the largest range of single-domain size belongs to maghemite (-Fe2O3), from 20 to 166 nm, with maximum saturation magnetization at 66 nm..5 Furthermore, since iron has its highest oxidation state (Fe+3) in y-Fe2O3, the particles are

stable.

A major challenge in the area of the parental administration of biologically active materials is the development of a controlled target specific delivery device that is small enough for intravenous application with long circulating half-life. Biologically active materials administered in such controlled fashion into tissue or blood are expected to exhibit decreased toxic side effects compared to traditional drug administration, and may reduce degradation of sensitive compounds in the plasma. Gold nanoparticles have been investigated for use as a carrier of different biologically active materials and recently, gold nanoparticles with an average diameter of 25 nm, have been proposed as an efficient delivery system of anticancer drugs.6 However, while delivery of biologically active compounds by gold is more efficient and less toxic than traditional administration of drugs, the delivery is not necessarilly target- specific. Magnetic nanoparticles (dressed with biologically active compounds), on the other hand, can be guided to a specific site in the human body by means of an external magnetic field (magnetic targeted drug delivery). Furthermore, in situ position monitoring is possible by MRI. A related application of magnetic particles is in the field of hyperthermia.

Hyperthermia is heat treatment where the temperature of the tissue (for example a tumor) is raised artificially with the aim of receiving therapeutic benefits. For example, the viability of cancer cells is reduced and their sensitivity to chemotherapy and radiation increases when the malignant cells are heated to about 45 C. Magnetic nano- particles embedded in biocompatible materials can be injected into the affected area and alternating magnetic fields are used to heat the magnetic particles embedded in the affected area. Magnetic y-Fe2O3 with 2 e e e e e . _.

diameters between 5 - 500 rim have been considered for all these applications, and are commercially availably A combination of magnetic yFe2O3 and gold on the other hand, more specifically in the form of a coreshell nanoparticle, has not been considered for targeted drug delivery, hyperthermia treatment and MRI imaging or a combination there of. However, such particles may suit the purpose best for the ease of targetting and in situ position monitoring (by MRI) the magnetic core will allow. Furthermore, since gold coated iron oxide is strongly light absorbing, with tunable optical resonances, laser induced hyperthermia (or thermal ablative therapy)8 becomes feasible, in addition to or instead of the use of an alternating magnetic field. By tuning the core-shell nanoparticles to strongly absorb light in the near infrared, where the optical transmission of tissue is optimal, a therapeutic dose of heat can be delivered by exposure to near infrared laserlight. Since the particles are also magnetic, they can be delivered to a specific target in the body by an external magnet, followed by laser irradiation to provide the necessary dose of heat.

SUMMARY OF THE PRESENT INVENTION

The present invention concerns the gram-scale synthesis of such coreshell particles to be used for targeted drug delivery, hyperthermia treatment and imaging, or combinations there of. The core diameter of these particles is typically between 5 - 150 rim and the thickness of the gold shell is typically between 1 - 50 nm. Depending on the size of the core, the particles are single domain magnetic or superparamagnetic. A preferred core diameter can for instance be 15 - 120 nm, more preferably it is 20 - 80 rim and most preferably between 35 to 50 nm.

The shell of the particles can be dressed with a variety of biologically active compounds through chemical bonding with functional groups such as thiol or amine. The optical properties of the particles (and therefore the wavelength at which the particles absorb) can be controlled by varying shell thickness and core size.

Magnetic core-shell nanoparticles such as iron-gold, cobalt-gold and magnetite (Fe30,) -gold have been reported,9 where the gold shell serves as a protection against the toxicity or instability of the core material. These particles are being considered for MRI applications. In the present invention, the purpose of the gold layer is not protection but instead serves a dual 3. . e e . .. e. .,

purpose, i.e. as an anchoring material for biologically active compounds and as absorbing medium for incoming light in hypertherrnia therapy. Figure 1 briefly presents a schematic description of magnetic targeted drug delivery inside human body to a target tumor or cancer site.

The present invention involves a delivery system for targeted delivery of a component to treat a subject, characterized in that the delivery system comprises iron magnetic core-shell nanoparticles wherein the core is iron oxide, in particular maghemite (-Fe203) and the shell is gold and the component is attached on or comprised in the shell. Such component may for instance be a drug to treat the subject or it may be heat that is induced when the core shell of the nanoparticles absorbs light in the near infrared. The drugs can in particular be coated on the gold core shell through chemical bonding with functional groups such as thiol or amine.

Ideally such delivery system is configured for locoregional delivery for instance by guiding the nanoparticles by a magnetic field to the target site for instance to the targeted tissue. : .

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended text. The above and further advantages of this invention may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which: Figure 1 is a schematic description of targeted magnetic drug delivery to be used in human body. The drug carrier coated magnetic core-shell particles of this invention may be used as the magnetic carriers in such a system.

Figure 2 outlines the reaction involved in polyol process to prepare magnetic iron oxide core.

Figure 3 describes layered structure of the particles described in this invention; Functionality means any organic compound with a free thiol or amine group which allows specific binding to gold shell, the shell can be made of either gold or any other non-toxic noble metal.

Figure 4 describes 1) representative SEM picture of maghemite particles, 2) size distribution of these particles, 3) representative SEM picture of gold coated maghemite particle, 4) EDAX analysis on representative gold coated maghemite particles.

Figure 5 shows a representative absorption spectrum of gold coated maghemite particles in solution. Two representative plasmon resonances are visible, one at 590 nm and and other at 730 nm. The position of the plasmon resonance at 730 rim can be shifted to higher or lower wavelengths by adjusting shell sickness. This clearly shows that these particles are suitable to be used in laser ablative hyperthermal treatment.

DETAILED DESCRIPTION:

The invention is based on a method for preparing gold coated magnetic nanoparticles using polyol process. The method is based on the hydrolysis of and iron(III) salt followed by a dehydration step in any poly-alcohol (or polyol) medium (for example ethylene glycol, propylene glycol and various polyethylene glycols), to form maghemite nanoparticles. In a next step, a gold salt is reduced in the presence of the maghemite particles and in the same polyol medium to provide the gold-shell. The method yields uniformly sized particles composed of a magnetic core enveloped by a gold shell. The sickness of the gold-shell can be varied by adjusting the amount of gold salt in the reaction. Since such gold-coated particles have strong plasmon resonances in the visible region of the spectrum that are strongly correlated with the thickness of the shell, one can tune the absorption wavelength of the core - shell particles to be used effectively in laser ablative hyperthermia treatment. The gold shell 5. . . . . : . . . can be further coated with a variety of organic and biomolecules such as bovine serum albumin. Bovine serum albumin and human serum albumin are widely used as drug carriers.

Thus drugs can be attached to the magnetic core - shell particles to be used in magnetic targeted drug delivery. Figure 3 describes the target form of the coated core shell particle.

The polyol process involves hydrolysis followed by inorganic polymerization (dehydration reaction) of transition metal salts in a polyol medium. The polyol acts as a solvent for the inorganic precursor salt as well as the organic capping agent allowing one to carry out the hydrolysis and stabilizing the nanoparticles formed. The reaction involved is outlined in Figure 2. When FeCl3, 6H2O is used as the metal salt, as preferred in this invention, the resultant iron oxide is maghemite (-Fe2O3). These particles could be redispersed into hexane, chloroform, toluene and xylene in presence of 3 % (v/v) octylamine resulting in organic ferrofluid. In presence of tetramethylammoniumhydroxide these particles could be dispersed in water at very high concentration resulting in highly viscous aqueous ferro fluid.

Varying the ratio of stabilizing agent to metal salts, it is possible to achieve particles of different dimensions ranging from 5 to 150 nm. This process also allows to prepare cores consisting of magnetite (Fe3O4), greigite (Fe3S4) and cobalt ferrite (CoFe2O4) by chasing appropriate metal salts. Particles prepared by this process is generally of spheroid shape.

Depending on particular application the magnetic core can be either super paramagnetic or single domain (i.e. with fixed magnetic moment). Superparamagnetic particles are preferred in applications where particles having a fixed magnetic moment are not desired, such as, in in -situ MRI; whereas the single domain particles are preferred in applications like magnetic recording media and magnetic targeted drug delivery that requires mechanical transduction (single domain particles used to impart torque).

In a second step this invention uses polyol process to reduce gold from gold salt such as hydrogen tetrachloro aurate to form a complete shell of gold to envelope the magnetic core.

Use of tetramethylammonium hydroxide as stabilizing agent enables the same to be dispersed in water. The shell can be made of either gold or any other non toxic noble metal. The shell can be of any thickness (i.e. length from outside surface of core to outside surface of shell) and with the preferred method of forming the shell used in this invention, the shell may have a thickness ranging from 1 to 50 nm.

.' ,, In Figure 3, the shell is shown to be completely covering the core and thus sequestering core from outside environment. In the case of this invention, the shell protects the core's catalytic ability and bigincompatibility. The surface of the shell can be stabilized and / or functionalized using organic or biomolecules that contain an amine or thiol groups or functional groups that can be converted to amine or thiol.

The shell allows for easy functionalisation of the particles with a variety of compounds, since gold binds strongly with a thiol or amine function.

Hence it becomes possible to functionalise the core-shell particles with various organic compounds and to introduce a specific functionality to the particles. Such compounds can be for instance compounds of the structure R-S-H, which have a terminal thiol group or free thiol group.

In one embodiment of present invention the core-shell is functionalised by nucleotides ( a RNA or a DNA molecule) comprising a terminal thiol group. These are for instance obtainable by transcription in vitro of RNAs that carry free thiol groups, using ribonucleoside triphosphate analogs containing a substituent with a terminal thiol group on their heterocyclic ring. A nucleoside triphosphate (NTP) analog containing a substituent with a terminal thiol group on its heterocyclic ring (NTP-SH) is incorporated into RNA in a standard runoff transcription reaction, driven by RNA polymerase and using an appropriately cut plasmid as a template for transcription. A method for producing thiol-modified nucleic acids has been described in W09820020.

In another embodiment of present invention the core-shell is functionalised by thiol- containing metal chelators. Methods of making such reagents, and methods of using the radiopharmaceuticals produced therefrom are also provided in EP0804252. Bryson et al., 1990, Inorg. Chem. 29: 2948-2951 describe chelators containing two amide groups, a thiol group and a substituted pyridine group. These chelators are particularly suitable for the preparation of radiopharmaceuticals for diagnostic and therapeutic purposes.

In another embodiment of present invention the core-shell is functionalised by thiol- containing (poly)peptides that contain one or more free thiol groups. Particularly suitable are antibodies, F(ab)l, F(ab)2, Fab' and Fab fragments, single-chain sub-fragments such as sFvs, divalent constructs such as dsFvs. Peptides that originally do not comprise a free thiol group 7. 3.. ..

can be modified to add a free thiol group by methods known to those skilled in the art. For example, the peptide can be thiolated with reagents such as 2-iminothiolane, or intrinsic disulfide bonds such as cysteine residues can be reduced. A combination of both modifications also can be performed, such as the acylation of Iysine residues with N succinimidyl-(2-pyridylthio)-propionate (SPDP) followed by the controlled reduction of the appended disulfide bond.

In one example the gold coated magnetic particles are coated with bovine serum albumin.

The serum albumins are used as standard drug carriers in human body. Since the bovine serum albumin coats the core-shell particle completely, the final particles shall be bio- compatible. Human serum albumin can also be used instead of bovine serum albumin.

The core-shell can also be functionalised by compounds of the structure RNH2. For this purpose other functional groups which may be first converted into amines such groups include nitro, halogen, hydroxyl, aside, -S02R, -OR, -SR, and -N=NAr where R is alkyl and Ar is aromatic, see, J. March, Advanced Organic Chemistry, 4th edn., Wiley, Chichester, 1991.

Some of the compounds suitable for functionalising the core-shell may be for instance free amine benzophenanthridine alkaloids (which are known to have antibacterial and antifungal properties (WO9219242)), peptides with a free amine group at the N-terminus or protein chains with a free amine (for instance N terminus) group, saccharides with a free amine group (such as GlcN, GalN, or GlcN), small molecules with a free amine group (such as the biomarine product (3-trimethoxysilylpropyl) diethylene- triamine) and free amine group chelators (such as deferoxamine)

Examples

Example 1

Preparation of Maghemite (y-Fe2O3) nanoparticles by Polyol Process In the procedure 4.0 g of FeCl3, 6H2O dissolved in 2.5 mL of MilliQ water and 10 mL of ethylene glycol is injected onto a refluxing mixture of 50 mL of ethylene glycol and 25 mL of 8.. ...

octylamine at 190 C while stirring. This mixture is refluxed for 6 furs. During this period initial brown precipitate slowly turns into black powder. The reaction mixture is cooled to room temperature and poured onto 250 mL of absolute ethanol. The precipitate was collected with the help of a magnet. Non-magnetic precipitate and supernatant reactant mixtures were decanted off. Magnetic residue thus collected was further washed with 100 mL (x 3) acetone and dried on air. The dried powder weighs 1.5 g resulting in 82 /0 yield (weight of octyl amine attached to the particles is not considered). The particles thus prepared can be stored as a suspension in 99 mL of ethanol and 1 mL of octylamine. The diameter of the particles can be varied by adjusting reaction conditions as shown in Table 1.

Table 1: reaction conditions used to vary the diameter of maghemite nanoparticles Amount of FeCI3, 6H2O octylamine water reflux time size (diameter) 4g 30 ml 5ml 12hrs 12+3nm 4g 25 ml 2.5 ml 6 hrs 21 + 4 nm 4g 15ml Oml 5hrs 32+5nm 4g 10 ml O ml 5 hrs 44 + 4 rim

Example 2

Preparation of Maghemite (-Fe203) Au core-shell nanoparticles by Polyol Process In a typical preparation 10 mg of maghemite particles (12 nm diameter) is dispersed in 1 mL of octylamine by sonication for 15 minutes. This is diluted to 5 mL with ethylene glycol.

This suspension is heated to 150 C. 30 mg of HAuC14, 3H20 dissolved in 1 mL of ethylene glycol was added to the solution while stirring the mixture efficiently. 100 pL of aqueous tetramethylammonium hydroxide (50 % solution) is added to the reaction mixture dropwise.

Dark orange color of the solution slowly changes to deep blue over a period of 30 minutes.

After 45 minutes the reaction mixture is cooled and poured onto acetone. The precipitate settled was collected over a magnet and washed four times with acetone (25 mL x 4). It was dried on air and redispersed in 25 mL of Milli Q water by sonication. By adjusting the amount og gold in this procedure, the shell sickness can be varied as shown in Table 2.

Table 2: Shell sickness vs amount of gold used in example 2 : ... 9... . . ...

:::: .: :e:: ë . . Amount HAuC14.3H2O Core diameter Shell sickness 30mg 12nm 2.5 nm 80mg 12nm 8.5 nm 600 mg 12 rim 23.5 nm

Example 3

Preparation of Bovine Serum Albumin coated Maghemite (-Fe2O3) Au coreshell Nanoparticles.

To the aqueous dispersion of gold coated maghemite nanoparticles prepared in the previous section, 150 pL of tetramethylammonium hydroxide was added to adjust the pH to 7.4. 150 mg of bovine serum albumine (BSA, pH 7. 2 fraction) was added to the dispersion and agitated at 30 C overnight. This mixture was poured onto 250 mL of absolute ethanol to precipitate the coated colloidal particles. Precipitate was collected over magnet and washed with ethanol. The particles thus prepared are purified by redispersing in water and precipitating back using ethanol. Finally, these BSA coated maghemite - gold core - shell nanoparticles can be stored in water.

- . . . . References to this application 1. a) T. Hyeon et. al., J. Am. Chem. Soc., 2001, 123, 12798-12801.

b) M. Rajamathi et. al., Curr. Opin. Solid State & Mat. Sci., 2002, 6, 337-345 2. Alex Hubert, Rudolf Schafer Magnetic Domains: The Analysis of Magnetic Microstructures, Springer Verlag; (October 1998).

3. R. E. Rosenweig, Ferrohydrodynamics, Cambridge University Press, Cambridge, 1985.

4. J. D. Kraus, Electromagnetics, McGraw Hill International Book Company, 3r Edition, USA, 1984.

5. C. M. Sorensen, chapter on Magnetism, in Nanoscale materials in Chemistry, Ed. K. J. Klabunde, John Wiley and Sons, Inc. New York, 2001.

6. Patent by CytImmune Sciences, Inc. (US 2001/0055581 At; US 2004/0115208 Al) 7. Many different types of magnetic particles (nanoparticles or micron sized particles) are commercially available from several different manufacturers including: Bangs Laboratories (Fishers, Ind.), Promega (Madison, Wis.), Dynal Inc. (Lake Success, N. Y.), Advanced Magnetics Inc. (Surrey, UK), CPG Inc. (Lincoln Park, N.J.), Cortex Biochem (San Leandro, Calif.), European Institute of Science (Lund, Sweden), Ferrofluids Corp. (Nashua, N.H.), FeRx Inc. (San Diego, Calif.), Immunicon Corp. (Huntingdon Valley, Pa), Magnetically Delivered Therapeutics Inc. (San Diego, Calif.), Miltenyi Biotech GmbH (USA), Microcaps GmbH (Rostock, Germany), PolyMicrospheres Inc. (Indianapolis, Ind.), Polysciences Inc. (Warrington, PA), Scigen Ltd. (Kent, UK), Seradyn Inc. (Indianapolis, Ind. ), Spherotech Inc. (Libertyville, Ill), Stereotaxis Inc. (St. Luis, Missouri), Indicia Biotechnology (Oullins, France), Estapor Microspheres, Merck, Liquids Research (North Wales, UK), and LyonBioAdvisor (Lyon, France). Most of these particles are made using conventional techniques, such as, grinding and milling, emulsion polymerization, block copolymerization and microemulsion.

8. L.R. Hirsch et al, PNAS, 2003,100, 13549-13554.

9. a) M. Mikhaylova et. al., Langmuir, 2004, 20, 2471-2477.

b) M. Toprak et. al., Mat. Res. Soc. Symp. Proc., 2002, 704, w6.29.1- w6. 29.6.

c) J. Lin et.al.,J. Solid State Chem., 2001, 159, 26-31.

11. . . . .- . .

Claims (1)

1. NANOPARTICLES AND USES THERE OF

   1) A delivery system for targeted delivery of a component to treat a subject, characterized in that the delivery system comprises iron magnetic core-shell nanoparticles wherein the core is iron oxide, in particular maghemite (y-Fe203) and the shell is gold and the component is attached on or comprised in the shell 2) The delivery system of claim 1, wherein the component is drug.

   3) The system of claim 2, wherein the gold shell is coated with the drug.

   4) The delivery system of claim 3, wherein the drug is a biological component.

   5) The delivery system of claim 3, wherein the drug is an organic component.

   6) The system of the claims 3, 4 or 5, wherein the drug is coupled to the gold shell through chemical bonding with functional groups such as thiol or amine.

   7) The delivery system of claim 1, wherein the component is heat which is inducible by radiation of the iron magnetic core-shell nanoparticles.

   8) The system of the claims 1 to 7, configured for locoregional delivery of the component.

   9) The system of the claims 8, configured for locoregional delivery by a magnetic field.

   10) The system of the claims 9, further configured for parenteral administration and target specific delivery of the iron magnetic coreshell nanoparticles in a subject.

   1. . . . . . ... . .