MAGNETIC PARTICLES

Materials and methods for making small magnetic particles, e.g., clusters of metal atoms, which can be employed as a substrate for immobilizing a plurality of ligands. Also disclosed are uses of these magnetic nanoparticles as therapeutic and diagnostic reagents, and in the study of ligand-mediated interactions.

DETAILED DESCRIPTION

Pharmaceutical Compositions

The nanoparticles described herein or their derivatives can be formulated in pharmaceutical compositions, and administered to patients in a variety of forms. Thus, the nanoparticles may be used as a medicament for tumour targeting and hyperthermic therapies, for in vivo cell and tissue labelling, or as contrast enhancement media in magnetic resonance imaging.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant or an inert diluent. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0.1 wt % of the compound.

Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol), and rectal systemic routes. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.

In addition to one or more of the compounds, optionally in combination with other active ingredient, the compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, isotonicizing agent, preservative or anti-oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. orally or parenterally.

Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0, more preferably between about 4.5 and 8.5 and still more preferably between about 5.0 and 8.0. The pH of a composition can be maintained by the use of a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine, typically employed in the range from about 1 mM to 50 mM. The pH of compositions can otherwise be adjusted by using physiologically acceptable acids or bases.

Preservatives are generally included in pharmaceutical compositions to retard microbial growth, extending the shelf life of the compositions and allowing multiple use packaging. Examples of preservatives include phenol, meta-cresol, benzyl alcohol, para-hydroxybenzoic acid and its esters, methyl paraben, propyl paraben, benzalkonium chloride and benzethonium chloride. Preservatives are typically employed in the range of about 0.1 to 1.0% (w/v).

Preferably, the pharmaceutically compositions are given to an individual in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful activity providing benefit to the individual. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition, 1995, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), and Handbook of Pharmaceutical Excipients, 2nd edition, 1994. By way of example, and the compositions are preferably administered to patients in dosages of between about 0.01 and 100 mg of active compound per kg of body weight, and more preferably between about 0.5 and 10 mg/kg of body weight.

Antibodies

The nanoparticles may be used as carriers for raising antibody responses against the ligands linked to the core particles. These antibodies can be modified using techniques which are standard in the art. Antibodies similar to those exemplified for the first time here can also be produced using the teaching herein in conjunction with known methods. These methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the nanoparticle(s). Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a nanoparticle, an antibody specific for the ligand and/or nanoparticle may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the nanoparticles, or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

The term “monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibodies, i.e. the individual antibodies comprising the population are identical apart from possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies can be produced by the method first described by Kohler and Milstein, Nature, 256:495, 1975 or may be made by recombinant methods, see Cabilly et al, U.S. Pat. No. 4,816,567, or Mage and Lamoyi in Monoclonal Antibody Production Techniques and Applications, pages 79-97, Marcel Dekker Inc, New York, 1987.

In the hybridoma method, a mouse or other appropriate host animal is immunised with the antigen by subcutaneous, intraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the nanoparticles used for immunisation. Alternatively, lymphocytes may be immunised in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell, see Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).

Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody producing cells, and are sensitive to a medium such as HAT medium.

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the nanoparticles/ligands. Preferably, the binding specificity is determined by enzyme-linked immunoabsorbance assay (ELISA). The monoclonal antibodies of the invention are those that specifically bind to the nanoparticles/ligands.

In a preferred embodiment of the invention, the monoclonal antibody will have an affinity which is greater than micromolar or greater affinity (i.e. an affinity greater than 10−6mol) as determined, for example, by Scatchard analysis, see Munson & Pollard, Anal. Biochem., 107:220, 1980.

After hybridoma cells are identified that produce neutralising antibodies of the desired specificity and affinity, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include Dulbecco's Modified Eagle's Medium or RPM1-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumours in an animal.

Nucleic acid encoding the monoclonal antibodies of the invention is readily isolated and sequenced using procedures well known in the art, e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies. The hybridoma cells of the invention are a preferred source of nucleic acid encoding the antibodies or fragments thereof. Once isolated, the nucleic acid is ligated into expression or cloning vectors, which are then transfected into host cells, which can be cultured so that the monoclonal antibodies are produced in the recombinant host cell culture.

Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression. The invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.

Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody” should-be construed as covering any binding substance having a binding domain with the required specificity. Thus, the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope, here a carbohydrate ligand as defined herein.

Examples of antibody fragments, capable of binding an antigen or other binding partner, are the Fab fragment consisting of the VL, VH, Cl and CH1 domains; the Fd fragment consisting of the VH and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab′)2fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.

A hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies, humanised antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A, GB 2 188 638 A or EP 0 239 400 A. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A.

EXPERIMENTAL SECTION

A method of synthesising magnetic glyconanoparticles covalently bound to ligands was devised. By way of example, thiol derivatised neoglycoconjugates 1 and 2 of two significant oligosaccharides, the non-antigenic disaccharide maltose (Glcα(1→4)Glcβ1-OR) and the antigenic lactose (Galβ(1→4)Galβ1-OR), were prepared to functionalise in situ magnetic nanoparticles (FIG. 3, scheme 1). The synthesis of the disulfides 1 and 2 was carried out by glycosidation of the conveniently protected maltose and lactose derivatives with 11-acetylthio-undecanol and 11-acetylthio-3,6,9-trioxa-undecanol, respectively.[12] Both linkers have been used to test the influence of their hydrophobic or hydrophilic nature in the properties of the whole material. Compounds 1 and 2 were isolated as disulfide forms, and used in this form for the preparation of gold-iron protected glyconanoparticles. The water-soluble glyconanoparticles 1-AuFe (malto-AuFe) and 2-AuFe (lacto-AuFe) were obtained in methanol/water mixtures using one-pot synthesis. FeCl3and HAuCl4in a ratio 1:4 were reduced with NaBH4in the presence of disulphides 1 or 2. The protection of the metal core with the neoglycoconjugate monolayers results in highly stable and bio-functional nanoclusters. They have been purified by means of centrifugal filtering and characterised by1H-NMR, UV-vis, ICP, TEM, EDX and SQUID.

Iron analysis of the particle, carried out by means of inductively coupled plasma-atomic emission spectrometry (ICP), indicated 0.27% and 2.81% iron content for 1-AuFe and for 2-AuFe, respectively. These data correspond to an average Au:Fe ratio of 5:0.1 and 5:1 respectively.FIG. 1shows Zero-Field Cooling and Field Cooling magnetisation curves obtained for the lacto-AuFe (A) and malto-AuFe (B) nanoparticles by means of Superconducting Quantum Interference Device (SQUID) between 5 k and 300 k in a field of 500 Oe. From the magnetic measurements it is inferred that both a superparamagnetic and ferromagnetic behaviour are present between 5 k and 300 k. SQUID measurements confirm the superparamagnetic character of the glyconanoparticles which have a blocking temperature (TB) below 5K (FIG. 1), which would be expected for a magnetic nanoparticle of 2 nm diameter. The superparamagnetic component is clearly observed from a) the partial fitting of the experimental thermal dependence of magnetisation to the Curie-Weiss law; b) the partial dependence of the hysteresis loop on the ration between the applied field and the temperature (H/T); and c) the difference between ZFC and FC curves.

FIG. 2shown transmission electron micrographs (left) and core-size distribution histograms (right) for the lacto-AuFe (A) and malto-AuFe (B) nanoparticles. Each black dot corresponds to a single particle. The dots are regularly separated by the ligands (neoglycoconjugate) attached to the core and they form ordered monolayers. The TEM was recorded at a 200 kV electron beam energy on a Philips CM200 microscope.

In the case of the 2-AuFe sample (lacto-AuFe), the glyconanoparticles are dispersed, spherical and homogeneous. The mean diameter of the gold/iron cluster was evaluated to be 2 nm. A few isolated particles with a size of about 10 nm have been found in some regions of the grid, but these particles have not been included in the histogram. In the case of the sample 1-AuFe (malto-AuFe), the glyconanoparticle presents a bimodal particle size distribution, as indicated by the corresponding histogram (FIG. 2B). Particles with a mean diameter of the gold/iron cluster about 2.5 nm and less than 1.5 nm have been found. Worthy of note is the spontaneous formation of aligned chains in extended regions of the grid, indicating an additional magnetostatic force (FIG. 2B). This behaviour could be attributed to dipole-dipole magnetic forces or quantum tunneling among the nanoparticles. The aligned arrangement was not observed in the micrographs obtained for the 2-AuFe nanodots, although a high ordered monolayer is observed.

Preparation

A solution of FeCl3(2 mg; 0.013 mmol; 0.25 equiv) in water (0.5 mL) was added to a solution of disulfide 1 (80 mg; 0.075 mmol; 3 equiv.) in MeOH (11.5 mL) followed by the addition of a solution of HauCl4(17 mg; 0.05 mmol; 1 equiv) in water (2 ml). NaBH41 M (52 mg; 1.38 mmol; 27.5 equiv) was then added in small portions with rapid stirring. The black suspension formed was stirred for an additional 2 h and the solvent removed under vacuum. The glyconanoparticles are insoluble in MeOH but soluble in water.

A solution of FeCl3(1 mg; 0.0065 mmol; 0.25 equiv) in water (0.25 mL) was added to a solution of disulfide 2 (70 mg; 0.07 mmol; 5.5 equiv.) in MeOH (12 mL) followed by the addition of a solution of HAuCl4(8 mg; 0.025 mmol; 1 equiv) in water (1 mL). NaBH41 M (26 mg; 0.69 mmol; 27.5 equiv) was then added in small portions with rapid stirring. The black suspension formed was stirred for an additional 2 h and the solvent removed under-vacuum. The glyconanoparticles are insoluble in MeOH but soluble in water.

Purification was performed by centrifugal filtration. The crude product was dissolved in water (˜15 mL) NANOpure and the solution was loaded into a centrifugal filter device (CENTRIPLUS YM30, MICROCON, MWCO=30000), and subjected to centrifugation (3000×g, 40 min). The dark glyconanoparticle residue was washed with MeOH and water and the process repeated several times until the starting material could no longer be detected by thin layer chromatography (TLC). The residue was dissolved in water and centrifuged several times to eliminate insoluble materials. The clear solution was lyophilised and the products obtained were free of salts and starting material (absence of signals from disulfide and Na+ions in1H and23Na NMR spectroscopy).

TEM examination of the samples was carried out at 200 KV (Philips CM200 microscope). A single drop (20 μL) of the aqueous solutions of the Au/Fe glyconanoparticles were placed onto a copper grid coated with a carbon film. The grid was left to dry in air for several hours at room temperature. Particle size distributions of the Au/Fe clusters were evaluated from several micrographs using an automatic image analyser. EDX analysis was performed with a Philips DX4 equipment attached to the microscope. ICP analysis was performed by Agriquem S. L. following PEC-009 protocol. UV spectra were obtained by a UV/vis Perkin Elmer Lambda 12 spectrophotometer.1H-NMR spectra were acquired on Bruker DRX-500 spectrometers and chemical shifts are given in ppm (δ) relative to D2O.

Magnetic Au Nanoparticles

Water soluble gold glyconanoparticles (GNPs) stabilized with self-assembled monolayers (SAMs) of different carbohydrate molecules were prepared by the chemical reduction of a metal salt precursor in aqueous solution in the presence of an excess of thiol derivatised neoglycoconjugates. The preparation sample procedure used as a starting point the Penadés et al [11] [19] that produces gold GNPs in which the metal cluster has been at same time protected and functionalised with the organic molecule. The formation of Au—S covalent bonds isolate the metal cluster preventing its growth (core diameter≈2 nm) and confer on the nanoclusters exceptional stability in solution.

In this example, we report on the experimental observation of magnetic hysteresis up to room temperature in gold glyconanoparticles with average diameters of 1.4 and 1.5 nm. By increasing the ratio of thiol:gold in the Penadés procedure, GNPs sample with diameter of less than 1.5 nm can be obtained. This is illustrated by the preparation and the magnetic properties of Au-GNPs obtained using the maltose neoglycoconjugate 1 as thiol linker species (FIG. 4).

Preparation of Gold Glyconanoparticles malto-Au:

An aqueous solution of tetrachloroauric acid (HAuCl4, 0.018 mmol) and an excess of disulfide neoglycoconjugate 1 (0.2 mmol) was reduced with sodium borohydride (NaBH4, 22 equiv) at room temperature. A brown suspension was immediately formed. The suspension was shaken for about two hours, then the solvent was removed and the glyconanoparticles (GNPs) were purified by washing with water and centrifugal filtering (CENTRIPLUS, Mr 30000, 1 h, 3000×g). The residue in the filter was dissolved in water and lyophilized. The GNPs were characterised by transmission electron microscopy (TEM), and1HNMR and UV-visible spectroscopy, induced coupling plasma (ICP) and elemental analysis. TEM: average diameter and; number of Au atoms, 1.5 nm and 79, respectively. UV-VIS (H2O): λ=520 nm. ICP: 28% Au. Elemental analysis calculated for (C23H43O11S)nAun(n=79): C, 38.18; H, 5.98; S, 4.40; Au, 27.18. Found: C, 39.5; H, 6.07; Au, 28.0.

FIG. 4shows in a) the synthetic scheme for the malto-Au GNPs and the corresponding TEM micrographs for the malto-Au GNPs and the corresponding particle size distribution histograms for the samples; and in b) the1HNMR spectra in D2O and in DMSO-d6are also shown. The malto-Au GNPs present, in all the cases, narrow particle size distribution with an average size of 1.5 nm or less. High resolution electron micrograph (HRTEM) indicating the fcc structure of the thiol protected malto-Au GNPs is show inFIG. 5.

Superconducting Quantum Interference Devise (SQUID) magnetometry indicated ferromagnetic behaviour even up to room temperature. Hysteresis loop measured at 5K exhibits a coercive field of 120 Oe. The blocking temperature, obtained from the thermal dependence of coercivity, was found to be 395 K that corresponds to an effective anisotropy constant of 10 meV/atom which is similar to that observed in a single Co atom onto platinum (III) surface [20]. The magnetisation did not conform to the Curie-Weiss law, but showed a much slower T-dependence. An atomic magnetic moment of around 0.003 μBper Au atom was derived from low T magnetic measurements.

FIG. 6show the hysteresis loops measured at 5K for gold thiol capped malto-Au GNPs. It is evident fromFIG. 5the magnetization process of thiol protected glyconanoparticles exhibit similar behaviour as typical ferromagnetic materials describing a hysteresis loops even at room temperature. In addition, it was observed that the samples are not saturated at any temperature. Remanence values around half of the magnetisation value at 1 T are measured, which implies that atoms as well as GNPs hold a permanent magnetic moment and that the gold GNPs system consists of an assembly of magnetic moments randomly distributed in orientation.

One can argue whether the observed behaviour is due to the presence of ferromagnetic impurities. Inductive Coupled Plasma (ICP) analysis indicated that the amount of Fe impurities (0.007% wt.) in the malto-Au is very low to account for the obtained magnetization values. In spite of that analysis, samples of malto-Au Fe GNPs containing 0.2% wt of iron have been prepared to characterized the influence of Fe on the magnetic behavior.FIG. 6shows the hysteresis loops measured at 5 K for both set of GNPs. It is clear that the presence of increased amounts of iron (ferromagnetic impurities) in the malto-AuFe nanoparticles reduces the ferromagnetic behaviour at this temperature, whereas the hysteresis loop still remains for malto-Au samples. As the GNPs are dispersed, inter-particle interactions can only be of magnetostatic nature. The average distance between gold core is determined by the length of two consecutive molecules of the maltose neoglycoconjugate 1 (6 nm). As the permanent magnetic moment of each particle is very low, the magnetic field acting on a GNP by a single neighbour GNP is lower than 10 Oe. Therefore, the influence of the stray fields can be neglected.

Since bulk Au is diamagnetic, the ferromagnetic behaviour may be due to the combination of both size and bonding effects [21]. The discrete electronic energy structure [22], the presence of stacking faults [23], as well as the extremely high percentage (≧80%) of surface atoms [24], covalently bonded to S, may be the possible causes of the onset of ferromagnetism.

In conclusion, it has been shown (FIG. 6) that very small thiol protected gold glyconanoparticles exhibit a localized permanent magnetism in contrast to the metallic diamagnetism characteristic of other non-thiol protected gold nanoparticles or bulk gold. This observation point out that the thiol-gold bonding induces in gold glyconanoparticles permanent magnetic moments probably associated with the extra d-holes localized near to the Au bonds. This suggest the technological application of the nanoparticles of the present invention for magnetic recording. Furthermore, the water solubility and the biological label of these GNPs amplify enormously their application in the biological field.

Gold glyconanoparticle (GNPs) may be complexed to Gd(III) and other lanthanides to give new contrast agent. The neoglycoconjugate ligands present in the GNPs (60 to 100 molecules) are the chelating moiety.

Preparation of lactoEG4-Au(Gd) Glyconanoparticles:

To a solution of the corresponding gold glyconanoparticle (20.0 mg) in water (1 mL) a solution of GdCl3. (0.5 M, 1.08 mL) was added. The mixture was stirred in the absence of light during 20 h. The solution was filtered by centrifugation (MICROCON YM30, 13000 rpm, 8 min). The residue was washed (8×0.5 mL, methanol/water, 1/3). The nanoparticles were dissolved in water and lyophilized to give 17.5 mg of dark violet nanoparticles. TEM: average diameter 2.5 nm. EDX: Gd 6.8%; Au 33:2% atomic.

Determination of Relaxivities:

1H NMR relaxation times T1and T2(37° C., pH 7.2) of the water protons in aqueous solution were measured at 1.5 Tesla in a Brucker Minispec NMR spectrometer. T1values were determined by the inversion-recovery method and the T2values were determined by the Carr-Purcell-Maiboom-Gill sequence. Solutions of the lacto-Au(Gd) nanoparticles at five different concentration (0.01, 0.1, 1, 10, 100 μg/mL) were prepared in Hepes buffer with 150 mM of NaCl. The relaxivities were calculated from the differences in longitudinal and transversal relaxation rates (1/T1(2)) of the water protons in the presence and absence of the glyconanoparticles, and the concentration of Gd(III) expressed in mM.FIGS. 7 and 8show the results.

In conclusion, in the examples shown herein, the inventors have developed a simple methodology to prepare water-soluble, superparamagnetic nanoparticles covalently linked to antigenic oligosaccharides. The methodology can be extended to the preparation of hybrid nanoparticles incorporating carbohydrates and other molecules. Carbohydrate-receptor interactions can direct the magnetic glyconanoparticles to target cells and tissues allowing their selective labelling. This demonstrates that this type of polyvalent magnetic glyconanoparticles complements the scarcely available bioactive magnetic nanoparticles.[9] [10] [17] Accordingly, the easy preparation and purification, their small core size and their stability and solubility in physiologically conditions of nanoparticles of the present invention convert these tools in potential candidates for diagnostic, tumour targeting [15], hyperthermia [16], and magnetic resonance imaging [17] applications.

REFERENCES