WO2005094884A2 - Boron containing nanoparticles targeted to t-cells - Google Patents

Abstract

Boron compound nanoparticles for use as a medicament, especially for ingestion in T-cells and administration prior to boron neutron capture therapy may be provided with reactive surface groups by milling and/or may be functionalised by reaction of said surface groups to form covalent linkages to organic molecules, including TAT or other cell penetration peptides. Alternatively, organic functionalising compounds may be adsorbed to boron carbide nanoparticles. Both the particles and the TAT peptide may be labelled with respective fluorophores.

Description

Boron Containing Nanoparticles

The invention relates to the use of improved agents for use in boron neutron capture therapy and compositions, T-cell preparations, and methods relevant to such therapy, including functionalised boron rich nanoparticles. WO01/70938 proposed the use of boron compound nanoparticles enriched in ¹⁰B for use in medical treatment by neutron capture radiotherapy. It was disclosed that large quantities of boron presented as boron compound nanoparticles could be endocytosed by T-cells that would, upon administration, home to a site of disease. Alternatively, boron compounds could be bound to T-cells. Once the boron had been carried to the target site, neutron bombardment could be used to produce radioactive decay of the boron with consequent local cytotoxic effects. Materials disclosed for this purpose were boron compounds incorporated into micro-capsules, micro-beads, or liposomes, and bound to T-cells by antibody linkage. Boronated porphyrins, nucleosides, nucleotides and other boron compounds, including boron compound nanoparticles, were disclosed to be ingestible by T-cells. It was disclosed that colloidal boron nanoparticles could be stabilised by coating with dextran and could be derivatised with any suitable membrane translocation signal, such as TAT (trans-activating transcriptional activator) peptide. It was also disclosed that boron could be coated over other particles such as iron oxide. Conjugation of boron enriched streptavidin to biotinylated T-cells was disclosed. W093/15768 discloses boron doped fullerenes for use in therapy. US4331647 discloses antibody fragments containing a radioisotope label and an addend containing significant numbers of boron atoms . These may be present in a boron-rich coupling agent such as the diazonium ion derived from l-(4- aminophenyl) -1, 2-dicarba-closo-dodecarborane . These again are for use in neutron capture radiotherapy. US4665897 discloses antibody tagging of boron oxide powder or boronic acid esters . Incorporation of boron compounds for therapeutic purposes into liposomes is disclosed in US5328678. Disclosed compounds include Na₂Bι₀Hι₀, Na₂Bι₂HnSH and a₂BιoOι_e. Na₂Bι₂HnSH is also used for boronating dextran for conjugation to antibodies in US5846741. There is a continuing need however for further convenient methods for enabling substantial numbers of boron atoms to be incorporated into medicaments for use especially in boron neutron capture therapy. The term ^Nanoparticles' is used herein of particles of less than 500 nm, more preferably 200 nm, still more preferably 100 nm largest dimension. This excludes nanotubes and like structures having one dimension that is in the range of 500 nm to 100 μm. Preferred nanoparticles are of no more than 50 nm, more preferably no more than 20 nm, still more preferably no more than 10 nm in their largest dimension. ^ΛCell penetrating peptide' is used to refer to any peptide capable of carrying a ^payload' conjugated thereto through a cell membrane by the mechanism of protein transduction. Many cell penetrating peptides are known. An extensive discussion of cell penetrating peptides (also referred to as ^λcell permeable peptides') is found in Zhao and eisselder, ^ΛIntracellular Cargo Delivery Using Tat Peptide and Derivatives', Medicinal Research Reviews, Vol. 24, No. 1, 1-12, 2004. Several proteins such as HIV-1 Tat (alternatively written TAT' : trans-acting transcriptional activator) , Drosophila Antennapedia homeoprotein, and HSV-1 VP22 have been shown to traverse cell membranes by a process called protein transduction and to reach the cell nucleus while retaining their biological activity. The mechanism underlying this process is still being studied. However, it is mediated by the presence of short domains in the proteins and peptides derived from these domains can be internalised in most types of cells and can convey a payload, such as a conjugated or fused biomolecule or a solid particle, into the cell. These peptides seem to operate by virtue of a predominantly cationic character, preferably contributed by the guanidinium side chains of arginine . They are typically not sequence specific in their action. Peptide sequences derived from HIV-1 Tat that act as cell penetrating peptides include GRKKRRQRRRGYKC and GRKKRRQRRRPP. Other cell penetrating peptide sequences include -(Arg)g- and other poly -Arg- sequences, transportan, Penetratin™ (a 16 amino acid peptide from the third helix of the homeodomain of the Antennapedia protein of Drosophila melanogaster) and analogues thereof. Transportan is a 27- residue peptide (G TLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chi eric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan (a wasp venom toxin peptide) in the C-terminus and a connecting lysine. Cell penetrating peptides useful in the invention described herein include known and yet to be discovered cell penetrating peptides, whether naturally occurring or otherwise . Cell penetrating peptide analogues' includes peptides comprising or consisting of D-amino acids and having cell penetrating properties. These may be D-amino acid analogues of the particular peptides described above. They may be peptoids in which D and L amino acids are both incorporated randomly. Analogues included here may instead or additionally involve modification of the peptide backbone. For instance, this may be replaced by an oligocarbamate structure . 'Boron neutron capture therapy' is a well known radiotherapeutic procedure in which a site of pathology, typically a cancer, is infiltrated with a boron compound containing the isotope ¹⁰B . Desirably, the boron content is highly enriched with ¹⁰B . The treatment zone is then exposed to bombardment with thermal (<0.4 eV) or epithermal (0.4 eV- 10 keV neutrons) which cause fission of ¹⁰B to produce energetic ⁷Li and ⁴He particles that cause fatal damage to cells within about one cell diameter of the B atom. Nanoparticles of boron carbide are available for use in for instance abrasives. We have now found that such particles, especially when further reduced in size, have particular suitability for use as boron neutron capture agents and have developed methods for improving their suitability for such purposes . With regard to the foregoing, the present invention now provides boron compound nanoparticles comprising boron carbide for use as a medicament. They are especially useful for use in boron neutron capture therapy. The invention includes therefore the use of boron carbide nanoparticles for the preparation of a medicament for use in a method of boron neutron capture therapy. An advantage of boron carbide nanoparticles for use in therapy is their low aqueous solubility and lack of any tendency to leach boron into solution in water or physiological fluids. It is desirable for several reasons to facilitate the conjugation to boron carbide nanoparticles of other atoms, molecules or functional groups. These may include traceable labels such as radiolabels, fluorescent labels, or magnetic resonance labels. They may include therapeutic agents such as cytostatic or cytotoxic agents. They may include cell penetrating peptides or analogues thereof as discussed above. To this end, the invention provides boron rich nanoparticles comprising elemental boron or boron carbide, bearing amine surface groups, which may be primary amine groups. These may be used to bond to linker compounds that can be used to space other molecules or atoms from the particle surface. Fluorescent labels may be used to assist in monitoring uptake of the boron particles by biological cells. FITC (fluorescein isothiocyanate) is well known as a label for peptides to which it is attachable at primary amine, e.g. lysine residues, typically an added terminal lysine. Tat peptides and other cell penetrating peptides are available in FITC labelled form and can be conjugated to boron containing particles in the same way as unlabelled cell penetrating peptides. Alternatively, FITC may be used to label amine bearing nanoparticles directly. Many other fluorescent labels are known, especially for labelling proteins or peptides, and generally all such labels may be employed in this invention. A suitable example is lissamine. In one aspect, the invention includes boron rich nanoparticles comprising a first detectable label covalently bonded to the surface thereof and a second detectable label independently covalently bonded to the surface thereof via a linker comprising a cell penetrating peptide or cell penetrating peptide analogue. Preferably, the two detectable labels, which are different, are fluorescent labels having different fluorescence emission wavelengths, or different fluorescence stimulation wavelengths, or differing fluorescence lifetimes. This enables the presence of both labels at the same location to be demonstrated readily by observation of the different fluorescence properties. As one label is linked to the particle via the cell penetrating peptide or analogue and the other is linked differently to the particle, the presence of both labels provides evidence that both the peptide and the particle are present. Thus, if labelled particles are presented to cells in order for them to be taken up, there may be a possibility that the peptide could be cleaved off and taken up by the cells without the boron rich nanoparticle . If this happened, both labels would not then be seen. In this specification, the term ^λboron rich nanoparticle' refers to a particle of nano-dimensions containing at least 10%, preferably at least 20%, more preferably at least 70% boron by weight. Such materials include iron boride, zirconium diboride, boron carbide, boron nitride and elemental boron itself. Suitable reactive surface groups which may be used to form covalent bonds to compounds to be bound to the surface of boron rich particles include amino groups, hydroxyl groups and thiol groups. These may be provided by milling in the presence of appropriate reactive gases. Generally, any chemical grouping by which the nanoparticles can be reacted with atoms or molecules such as those described above may be used as reactive surface groups. In order to facilitate the conjugation to the nanoparticles of a useful number of other molecules such as those discussed above, there are preferably at least 10 such active groups per particle, more preferably at least 100 active groups per particle, more preferably still at least 1000 active groups per particle. Active groups may be attached to the surface atoms of boron carbide nanoparticles covalently at carbon or at boron. Preferably, a significant proportion and preferably a substantial proportion of the boron atoms lying at the surface of said particles bear active groups such as amine groups. Preferably, at least 1%, more preferably at least

5%, still more preferably at least 10% and most preferably at least 50% of the surface boron atoms bear active groups, especially amino groups . Such amino groups or other surface groups may be used as reaction sites by which the nanoparticles described above can be caused to bear a cell penetrating peptide or cell penetrating peptide analogue. The invention includes boron or boron carbide nanoparticles bearing a cell penetrating peptide or cell penetrating peptide analogue. The cell penetrating peptide or analogue may preferably be a TAT peptide, antennapedia, transportan, or HSV-1 VP22 peptide or an analogue of such a peptide. Preferably there are at least 10 cell penetrating peptide molecules per particle, more preferably at least 100 cell penetrating peptide molecules per particle, still more preferably at least 1000 cell penetrating peptide molecules per particle. The invention includes boron carbide nanoparticles having an organic compound on the surface thereof. Said organic compound may be covalently bonded to said nanoparticles, e.g. via -N- or -0- linkages, which may be directed to boron in the particles and so may be B-N- or B-0- linkages or may be directed to carbon and so may be C-N- or C-O- linkages. Alternatively, said organic compound may be adsorbed onto the surface of said nanoparticles. To assist this, preferably said organic compound has multiple positively charged sites. It may for instance be a polyalkyleneimine , such as polyethyleneimine, or may be polylysine, poly (diallyldimethyl) ammonium chloride, or poly- (allylamine) hydrochloride or any one of many other similarly charged polymers. Preferably, such nanoparticles bear alternating layers of positively and negatively charged polymeric material, e.g. at least 3 layers (+/-/+) . Negatively charged layers can be formed from polymers having repeated anionic groups such as carboxylic acid groups or more preferably sulphonic acid groups such as polystyrenesulphonic acid. The organic molecule on the surface of the nanoparticles preferably has a chain length of at least 4 atoms, or at least 6 atoms, e.g. of at least 10 atoms. These chains may be carbon atom chains, but may also include hetero atoms, e.g. in a peptide or other polyamide chain or a polyester or other polymer chain.

Said organic compound may have a reactive functional group, suitably spaced by at least 6, more preferably at least 10 atoms from a covalent attachment to the surface of the particle. The functional group such as amine or hydroxyl may be used for conjugating other molecules to the particle and may for instance be a primary or secondary amine, hydroxyl, carboxylic acid, carboxylic acid anhydride, and derivatives thereof or other reactive species . Preferably, in any of the nanoparticles described above the boron is enriched in ¹⁰B, e.g. to comprise at least 50%, more preferably at least 80%, still more preferably at least 90% ¹⁰B . The higher the enrichment, the less boron may need to be got to a treatment site for effective boron neutron capture therapy to be carried out. For the delivery of boron containing nanoparticles to a treatment site, one especially preferred vehicle is a biological cell having the ability to migrate to the treatment site and to accumulate there selectively. Cells that may fulfil this purpose will vary according to the nature of the pathology and may include T-cells, macrophages, tumour infiltrating lymphocytes (TIL) , natural killer cells (NK) , LAK-cells, or dendritic cells. The invention accordingly includes biological cells, e.g. T-cells containing ingested nanoparticles of the invention of the kind described above. Preferably, such T- cells have a predetermined antigen specificity. In some embodiments such specificity is for a cancer antigen. Preferably, such a cell contains at least 10 pg/cell of boron, more preferably at least 20 pg/cell. Higher loadings such as up to 40 pg/cell may be used, and this is not considered to be an ultimate upper limit. These are appropriate loadings for T-cells which have a diameter of 5- 10 μm, where 20 pg/cell is typically about 40,000 ppm. Cells of a different size may appropriately contain similar levels on a ppm by weight basis. For use as described above, the invention includes a process for producing boron rich nanoparticles bearing reactive surface groups such as amine or hydroxyl or thio groups comprising milling (grinding) boron rich particles to reduce their particle size under an elemental or compound nitrogen containing atmosphere such as air or ammonia or with alkali metal (e.g. sodium) amide for forming amino groups or in an oxygen containing atmosphere for producing hydroxyl groups or under hydrogen sulphide or with sulphur for forming thio groups. It is a highly surprising finding that boron rich particles such as boron carbide or boron nanoparticles can be surface aminated simply by grinding in the presence of nitrogen gas . The exact mechanism involved has not yet been elucidated. It is thought that the fracture of the surface of such extremely small particles in the grinding process reveals new surface atoms under conditions of heat (generated by the frictional forces involved) and pressure and that bonding requirements of the newly revealed surface atoms may be momentarily unsatisfied by adjacent atoms, leading to reaction with for instance available nitrogen. It is presently uncertain whether reaction takes place to aminate surface boron atoms only or also surface carbon atoms (or even surface carbon atoms only) , when boron carbide is used. It is thought that even if the reactants and the apparatus are subjected to normal drying measures, there is sufficient water available to supply the hydrogen atoms needed to form amine groups. Instead of using gaseous nitrogen as the source of nitrogen for such groups one may use nitrogen compounds. Similarly, other active chemical groups may be produced by conducting grinding in the presence of suitable source reactants such as sulphur or sulphur compounds for -SH groups . In a further aspect, the invention includes a process for forming boron rich nanoparticles bearing alkyl or alkylene chains comprising forming amine groups on the surface of boron rich nanoparticles and reacting said amine groups with an alkylating agent or an acylating agent. In a further aspect, the invention includes a process for forming boron rich nanoparticles bearing alkyl or alkylene chains comprising reacting hydroxy groups on the surface of boron rich nanoparticles with a silane derivative such as an alkylchlorosilane, an alkoxysilane, an aminosilane, an alkoxychlorosilane, or a bromosilane or with a carboxylic acid or reactive derivative thereof, such as an acyl halide or anhydride. If the organic moiety of these reagents has a suitable functionality, e.g. a halo (e.g. chloro or bromo) , amine, azide, or hydroxyl functionality, that may be used as an attachment ^• point for forming a covalent linkage to a detectable label and/or a cell penetration peptide or analogue. For instance, boron carbide nanoparticles having hydroxyl surface groups may be reacted with 11-bromoundecyltrichlorosilane (BUTS) or an alternative ω-haloalkyltrichlorosilane or morre generally other functional group substituted-trichlorosilane . The bromo functionality may be replaced with azide by reaction of the functionalised nanoparticles with sodium azide and the azide moiety may be subjected to cyclisation using an appropriate alkyne. The alkyne may be selected to have substitution providing suitable functionality for conjugation to the desired label or cell penetration peptide or analogue. In a still further aspect, the invention includes a process for forming boron rich nanoparticles bearing a cell penetrating peptide or cell penetrating peptide analogue, comprising forming boron rich nanoparticles having covalently bonded thereto an organic compound providing a spacer-linker moiety, and bonding to said spacer-linker moiety a said cell penetrating peptide or cell penetrating Oeotide analogue,- wherein said spacer—linker moiety provides at least a 4 atom chain spacing between said boron rich nanoparticle surface and said cell penetrating peptide or cell penetrating peptide analogue. Suitable spacer moieties include long chain alkylene chains. A long chain dialkylamine may be bonded to a boron carbide or other boron rich nanoparticle by reacting the same with the nanoparticle surface in the presence of alkali metal hydride, e.g. sodium hydride. An alternative way of attaching such a linker compound is to functionalise the particle surface by reaction with tetraethoxysilane or equivalent to form a ^λglass coat' presenting more reactive OH groups which can be reacted with a silane derivative such as an alkylchlorosilane, an alkoxysilane, an aminosilane, an alkoxychlorosilane, or a bromosilane. The use of an aminoalkyl silane such as 3- aminopropylsilane or 3-aminopropyltrie thoxysilane to add amine functionality is preferred. Whilst covalent bonding of cell penetration peptides or analogues via suitable linkers is preferred, another option is to provide a hydrophobic molecule attached covalently or non-covalently to the surface of a boron rich nanoparticle and then to use hydrophobic interaction between that and a hydrophobic moiety attached to said peptide or analogue to retain the cell penetration functionality on the particle. Thus, for instance, a boron carbide nanoparticle having hydroxyl groups may be reacted with a long chain alkyl trichlorosilane such as octadecyltrichlorosilane to provide the particle with a hydrophobic coat. Peptide conjugated to a long chain alkyl group as for instance in TA_T (FITC) -pal (pal indicating palmitic acid) will aggregate to the hydrophobic coat in water, firmly but non-covalently attaching the peptide to the nanoparticle. Other lipophilic moieties may be used in place of palmitic aci .

In a further aspect, the invention includes a process for producing functionalised boron carbide nanoparticles, comprising coating boron carbide nanoparticles with a polymer comprising monomer residues having hydroxyl substituents or other substituents forming stable hydrogen bonds with the surface of the nanoparticles. Said polymer may be a polyvinylalcohol or a vinylalcohol copolymer such as EVOH. It may be a block alkylene (e.g. ethylene) and vinyl alcohol copolymer. It may be an oligosaccharide such as dextran or a similar molecule such as polysorbitol . Said functionalisation process may further comprise bonding a detectable label and/or a cell penetration peptide or analogue thereof to said polymer coating. An example of a suitable method for this is to react hydroxyl groups in the coating with epichlorohydrin, and to open the resulting attached epoxy ring with ammonia to produce an amine functionality. This may be used as an attachment point at which to react suitably functionalised labels or cell penetration peptides. Fluorescent labels such as lissamine and cell penetration peptides such as TAT may be conjugated to amine groups using for instance SMCC . One may form liposomes comprising said functionalised nanoparticles by mixing said polymer coated nanoparticles with a liposome forming lipid, which may be a phospholipid such as distearoylphosphatidylethanolamine (DSPE) or distearoylphosphatidylcholine (DSPC) . A lipidated detectable label or lipidated cell penetration peptide or analogue thereof can then be associated with the liposome by lipid- lipid interaction upon mixing, e.g. by sonication. Similarly, liposomes may be formed using boron rich nanoparticles without a polymer coating. Whilst the use of cell penetrating peptides or peptide analogues is preferred as a means of encouraging uptake of boron containing nanoparticles by cells such as T-cells it is not thought that this is essential. As described in Wilhelm et al, Biomaterials 24 (2003), 1001-1011, nanoparticles bearing negative surface charge can be taken up by cells in an efficient manner without cell penetrating peptides thereon. The same is known to be true of cationic nanoparticles . Nanoparticles according to the invention may be used in methods of boron neutron capture therapy as described in WO 01/70938. In addition to the methods described there in which T-cells are used as a vehicle for delivery of boron to the site of a pathology however, nanoparticles according to the invention may be used in a method of boron neutron capture therapy in which nanoparticles are administered directly to a known site of pathology and are irradiated therein with neutrons. Administration of 1 x 10⁸ or more T-cells, each containing a suitable content of ingested boron containing nanoparticles would be appropriate for boron neutron capture therapy in most cases. From 1 x 10⁸ to 1 x 10⁹ cells would be preferable. This equates to a total boron dosage of from 1 to 20 mg. T-cells of this kind may be administered as an i.v. bolus injection in a volume of say 2 - 5 ml of a suitable liquid carrier, but may also be administered in a large number of alternative methods well known in the art of adoptive T-cell therapy. The invention will be further described and illustrated with reference to the accompanying drawings, in- which:

Figure 1 shows levels of surface amination resulting from milling boron carbide in different atmospheres for different periods;

Figures 2-14 show reaction schemes referred to in the Examples; and

Figures 15 and 16 show results obtained in cell irradiation experiments described in Example 17.

Boron carbide has previously -been regarded as highly inert. The preparation of functionalised boron carbide nanoparticles therefore represents a challenge. However, as illustrated below, we have established several suitable methods. These are based on previously unreported properties of such particles. It has been discovered that boron carbide nanoparticles prepared by ball milling have a significant concentration of oxygen at their surfaces, believed to be in the form of reactive hydroxyl groups . Further, by ball milling in a suitable atmosphere or by reaction with sodium amide, we have produced particles having reactive amine groups thereon. Also, we have observed by gel electrophoresis experiments that milled boron carbide particles bear a negative charge which can be exploited to attract positively charged polymers to form a shell upon which further functionalisation can be based. The following description summarises our findings. Material analysis - Physical properties Physical properties were studied using transmission electron microscopy (TEM) , photon correlation spectroscopy (PCS) , powder x-ray diffraction (XRD) and gel electrophoresis . The morphology of the prepared nanoparticles appeared to be homogeneous and spherical compared to the unmilled material. The overall size distribution was evaluated using PCS and was found to decrease as a function of milling time. The change in particle size (z-average) was used to quantify the effect of milling time. Z-average decreased from 459 nm to 252 nm after 144 hrs of milling. After 144 hrs milling the distribution appeared to be bimodal . By centrifugation the population fraction containing the smaller particles could be isolated. After centrifugation at 300 rcf z-average was found to be 137 nm. At 2400 rcf z-average was reduced to 73 nm and at 14100 rcf z-average was reduced to 50 nm. The starting material was found to be crystalline with the characteristic Bragg peaks of B₄C . However, after ball milling the material was less crystalline and new peaks most likely from tungsten oxide had emerged. The overall particle surface was found to be negatively charged as determined by gel electrophoresis at pH 8.3. Particles prepared in a nitrogen containing milling atmosphere appeared to have the numerical lowest charge-mass ratio. Chemical properties of boron carbide nanoparticles. The chemical properties were studied using X-ray Photoelectron Spectroscopy (XPS) , Fourier Transform Infrared Spe Uu _y

_/ cin-α ci Oiicinxuα _ιl___l_y __ _L I _ αo o αj atomic surface composition (Table 1) was evaluated using XPS The starting material was composed of -60 % boron, ~25 % carbon and ~15 % oxygen with trace amounts of nitrogen, silicon and calcium. The atomic surface composition varied with respect to milling time and atmosphere.

Table 1. The elemental surface composition within each sample by XPS.

In general, the O/B and C/B was found to increase with milling time. The N/B ratio was found to increase as well, however the most profound increase was found in a nitrogen containing atmosphere (i.e. air or nitrogen) . Others have reported the nitriding of ^' elemental boron by milling in an ammonia atmosphere (Ref.: Chen et si. 1999) . A detailed chemical analysis was made by measuring the binding energy shift of B si, C si, 0 si, N si and W 4f with respect to milling time and atmosphere in order: to identify possible chemical states. The C si spectra contained peaks which could be assigned to B₄-C / C-C / C- H species as well as C-OH / C-O-C / O-C-O / C=0 species. The spectral fine structures were similar for all samples, however the peak attributed to boron carbide was found to decrease with respect to milling time. Identical 0 Is spectra were rrecorded for all samples and the peak was attributed to organic species and B-O. The N si spectra contained peaks which could be assigned to B-N / NC₄ ⁺ / NH₄ ⁺ / N-0 (Ref.: Perrone et al. 1998) species. The unmilled - and argon milled spectra could not be fitted due to too low nitrogen content. The B si spectra were assigned to B-C / B-B / B-N / B-C₃.₄ species as well as B-O. The chemical state of boron was significantly changed in the case of milling in air and nitrogen atmosphere. The B-O peak intensity increased with milling time in both an air and nitrogen atmosphere. This was much less profound for the material milled in an argon atmosphe e. The chemical bond structure was also studied using FTIR. A strong vibration at 1076 cm^-1 was attributed to the icosahedral boron carbide vibration (Ref.: Rodriguez et al . 2004). Regardless of atmosphere this peak was less profound in the milled samples. At 1350 cm^-1 a new peak was located after ball milling. The peak was most profound for the material milled in an air and nitrogen atmosphere. The 1300- 1400 cm^-1 region represent B-O and B-N vibrations (Ref.: Pavia et al . 1996) . A broad vibration was observed at 3134 cm^-1. Comparison with literature 3200-2800 cm^"1 has been assigned to primary and secondary amine salts, 3600-3300 cm^""1 has been assigned to hydroxyl and amines, 2800-3700 cm^"1 has been assigned to absorbed moist in boron tri-isopropoxide (Ref.: Rodriguez et al . ) and 3417 cm^"1 has been assigned to NH₂ in a C-N-B hybrid material (Ref. : Huang) . A ninhydrin assay was used to directly detect the presence of amines and quantification was done using UV-VIS absorption spectroscopy. Only boron carbide milled in air or nitrogen gave a positive ninhydrin absorption peak at 566 nm and the positive ninhydrin response was found to be depending on milling time. Milling in argon induced no ninhydrin response. (See fig. 1) The data analysis suggests the presence of protonated nitrogen species in the air and nitrogen milled samples. This is consistent with the low mobility observed in gel electrophoresis. The negative surface charge at pH 8.3 was believed to be a result of boronic surface groups. The following non-limiting examples illustrate the working of the invention.

Example 1: Ball milling of boron carbide particles

Boron carbide particles (5 g) of average largest dimension >300 nm and purity 99% (Alfa Aesar CAS: 1206932-8) were loaded into a clean, dried ball mill (bowl made of tungsten carbide (93.5% W₄C, 6% Co), lid made of tungsten carbide (84.5% WC, 15% Co)) (Fritch-Pulverisette) , dried in air at room temperature. The mill contained 9 tungsten carbide (93.2% WC, 6% Co) milling balls and was set with a counterweight of 5.5 kg. It was run at 200 rpm for a milling time of 6 h, followed by a pause time of 1 h (for cooling and sampling) with reverse milling set to on. A total milling time of 96 hours reduced the average particle size to about 45 nm. It was surprisingly found that the surface of the boron carbide particles had become aminated, as was demonstrable by XPS (X-ray induced photoelectron spectroscopy) detection of nitrogen and by ninhydrin assay for NH₂. Levels of amination found by subjecting solutions of boron carbide particles, (C_f = 1.87 mg/ml) subject to the ninhydrin assay are shown in Figure 1. Surface exposed amino groups produce in this test a magenta complex whose presence is detected by absorbance of light. The absorbance measurements at 566 nm are shown in the figure. The signal seen at t=0h is a turbidity signal with none of the characteristic ninhydrin spectrum. In a positive control reaction of 25 mM ninhydrin with amino silane, a 0.285 mM solution showed an absorbance of 0.703 at 566 nm which corresponds to an apparent molar extinction coefficient of 2500 in our assay (ε₅₆₆,_app = 2500 M^"1 cm^"1) . As shown, amination increased with milling time. The unmilled powder showed no ninhydrin signal, the Α₅₆₅ signal being due to turbidity, whereas 96 h of ball milling resulted in an amination signal of ~ 1.2 μmol/mg particles. Further reduction in particle size is obtainable by more prolonged milling. Alternatively, the smaller fraction of the product of this example can be selected and separated, e.g. by gradient centrifugation. The above procedure was repeated using substantially pure nitrogen as milling atmosphere in place of air. No significant difference in the level of resulting amination was detected. On the other hand, similar milling under an argon atmosphere did not produce detectable amination, showing that atmospheric nitrogen is the source of the introduced amine groups. The surface amination of the boron carbide milled under nitrogen was then further confirmed by chemical negation of ninhydrin signal, using conditions expected to produce the reaction :

B₄C(NH₂) +H₂O₂ (35% (aq)) ^{Neat; r}i B₄C /B₄C( HOH) /B₄C(OH) Boron carbide (50 mg, milled under N₂) was mixecϋ with H₂0₂ (5 ml, 35%), the flask was fitted with a stopper and the black slurry stirred o.n. Excess H₂0₂ was destroyed with Na₂S0₃ (checked with peroxide strips) and some of the slurry was purified by dialysis. A sample was spun and the ninhydrin assay (1 ml ninhydrin added, followed by warmincg to 100°C for 20 rriin.) was performed on both the supernatant a_nd the black precipitate. Both tests were negative.

Example 2: conjugation of Tat peptide to aminated boron carbide nanoparticles . To carry out the reaction scheme shown in figure 2,

Boron carbide (20 mg, milled in air) was dispersed in NaHC0₃ buffer (20 ml, 10 mM pH 8.3), sonicated in a ultrasonic bath (10 min) (Decon F5 minor, Decon ultrasonics Ltd.) and centrifuged (0.8 krpm, 5 min) in a tabletop centrifuge (Eppendorff minispin plus) . The supernatant was collected and applied on a PD-10 desalting column pre-equilibrated with

NaHC0₃ buffer (25 ml, 10 mM, pH 8.3) . An aliquot of the particle solution (10 ml) was then incubated with SMCC (3.9 mg, dissolved in 0.05 ml DMSO followed by brief sonication) for 1 h at room temperature.

The solution was applied on a PD-10 column pre-equilibrated with NaHC0₃ buffer (25 ml, 10 mM, pH 8.3) to ensure mobility in subsequent steps. In order to remove surplus SMCC, a total of 10 ml run-through was applied on four PD-10 columns pre-equilibrated with HEPES buffer (25 ml, 10 mM, pH 7.4) and a total of 14 ml was collected. TAT-FITC (4 mg, dissolved in 0.5 ml 10 mM HEPES buffer) was mixed with the SMCC-conj ugated boron carbide solution (9.5 ml) and incubated protected from light over night at room temperature with magnetic stirring. Surplus TAT was subsequently removed.

TAT was detected in the final particle preparation by fluorescence spectroscopy. As the TAT peptide contains a conjugated FITC fluorophore the detection of FITC in the purified particle batch is interpreted as a successful conjugation of the peptide to the particles.

Example 3: Chemical surface amination of ball milled boron carbide nanoparticles employing NaNH . rxrp _r + B₄C + NaNH₂ '~→- B₄C(NH₂)

Boron carbide (50 mg) was ball milled generally as in Example 1, but under Ar . The resulting particles were mixed with sodium amide (10 mg, 0.26 mmol) in dry THF (2 ml) and the mixture was stirred under Ar for 3 days. The mixture was evaporated in vacuo and the remaining black powder extracted with HC1 (aq: 2 M, 7x0.5 ml) (separated by spinning 10,000 rp for 2 min.). The boron carbide particles showed positive reaction in the ninhydrin assay after this amination procedure. Example 4: Alkylation of aminated boron carbide nanoparticles employing Br-Cχ₈H₃ .

To carry out the reaction scheme shown in Figure 3 , boron carbide 1 (58 mg, milled under N₂) was mixed with reagent 2 (Br- Cι_SH₃ , 0.5 ml) in acetonitrile (30 ml). Compound 3 (triethylamine, 1 ml) was added in one portion and the reaction mixture was refluxed for 60 h under Nitrogen. After cooling and particle precipitation, the supernatant was carefully decanted. The particles were dispersed in ethyl acetate and the resulting black colloid was extracted with water (15 ml) . The particles were observed to remain in the organic phase - hence they are hydrophobic. The organic solvent was removed in vacuo, to produce a yield of 0.40 g (75%) fine black powder. The final particle tested negative in a standard ninhydrin assay.

Example 5 : Acylation of aminated boron carbide nanoparticles employing Cl-CO-C₁₅H₃i .

To carry out the reaction scheme shown in Figure 4, boron carbide, 1 (76 mg, milled under N₂) and reagent 3 (Triethylamine, 1 ml) were suspended in chloroform (20 ml) . Compound 2 (Cl-CO-CιsH₃ι, 0.5ml) was added and the reaction mixture was refluxed mildly o.n. under nitrogen. Another portion of 2 (0.5 ml) was added and the reaction mixture was refluxed for another 16 h. After cooling, a 10 % NaHC03 solution (20 ml) was added and the mixture was stirred for 1 h and left o.n. without stirring. The solvent was removed by evaporation and the precipitate was taken up in ethyl acetate and extracted with water. The organic phase containing the particles was concentrated to dryness (Yield 0.6 g (79%)). The final particles were hydrophobic and tested negative in a standard ninhydrin assay.

Example 6: Chemical amination of boron carbide nanoparticles employing 1, 10-diaminodecane .

To carry out the reaction scheme shown in Figure 5, compound 1 (Boron carbide, 153 mg, milled under Ar) was suspended in toluene (60 ml). Compound 2 (1,10- diaminodecane, 0.816 g) was added, and 3 (NaH (55-60 % susp) , total 0.23 g) was added in portions under nitrogen. The reaction mixture was heated to gentle reflux for 48 h. The reaction mixture was quenched with water (2 ml) . Particles separated to the organic phase. The organic phase was cleaned by consecutive water extractions, by the following protocol :

Add water. Cent. 2 krpm, 5 min. Resuspend pellet in toluene. Cent. 2 krpm, 5 min. Resuspend pellet in chloroform. Cent. 2 krpm, 5 min. Dry by nitrogen flow. The final particles were hydrophobic and tested positive in a standard ninhydrin assay. IR spectra were consistent with the presence of diaminodecane groups.

Example 7 : Chemical silylation of boron carbide nanoparticles employing octadecyltrichlorosilane (OTS) .

To carry out the reaction scheme shown in Figure 6, boron carbide (300 mg, milled in air) was suspended in methylenchloride (20 ml) and subjected to ultrasonication (3x, 10 sec pulse - 20 sec pause, 0.5 amp) . The colloidal solution was poured into a conical glass container with magnet. OTS (2 ml) was added and the container was closed. The reaction was incubated o.n. at RT . The solution was aliquoted into 12 eppendorf tubes and centrifuged (14.5 krpm, 5min) . The supernatant was removed and the rest of the solution was added, followed by centrifugation (14.5 krpm, 5 min) . The particles were washed according to the following procedure repeated three times:

- Add CH₂C1₂ (1 ml) - Mix with glass stick. - Incubate in ultrasonic bath 5 min . - Cent . 14 . 5 krpm, 5 min . - Remove supernatant.

After washing, the pellets were suspended and transferred to a glass container. The liquid was removed by evaporation by nitrogen.

Example 8 : Functionalisation of boron carbide nanoparticles by means of hydrophobic interactions (lipid particle protocol)

To carry out the reaction scheme shown in Figure 7, the following procedure was followed. All steps in the conjugation and subsequent purification were kept as unexposed to light as possible due to the lability of the fluorophore FITC. OTS-B4C (20 mg) powder was placed in a mortar. A mixture of TAT (FITC) -pal (0.5 ml, 1.23 mM) and DDTMAC (2.6 ml, 12,42 mg/ml) was made (final ratio 5:995), followed by grinding into the OTS-B4C by adding to the mortar in several steps (0.1 ml + 0.1 ml 4- 0.1 ml + 0.8 ml + 1 ml + 1 ml). The slurry was subsequently transferred to a glass beaker and subjected to ultrasonication (total time 3 sec, pulse 1 sec, pause 20 sec) Amp 0,5. The suspension was incubated with magnetic stirring o.n wrapped in alufoil. The product was purified as follows. An aliquot of the particle suspension (1.1 ml) was transferred to an eppendorf tube, centrifuged (1 krpm, 10 min) . The supernatant (1 ml) was collected and spun again (1 krpm, 10 min) . The resulting supernatant (800 μl) was collected after which a sample (100 μl) was mixed with Na₂S0₄ (100 μl, 1M) and applied on a Sepharose CL-4B column (approx. vol. 3 ml) pre-equilibrated in MilliQ water (25 ml) . Particles were eluted in MilliQ water and collected in 1 ml fractions. All fractions were subsequently analysed in fluorimeter (Ex. 480 nm, Em. 500-800 nm) for co-elution of fluorescence-label and particles as well as separation from excess fluorescence.

Example 9 : Functionalisation of boron carbide nanoparticles by combining hydrophobic interactions and covalent bonding The reaction scheme of Figure 8 was carried out as follows. The product 1 of Example 6 (B₄C-NH-Cι₀H₂5-NH₂, 70 mg) was ground in a mortar and gradually suspended in chloroform (5 ml) and treated with ultrasonic horn (total time 3 sec, pulse 1 sec, pause 20 sec, level 2) where upon 2 (Lissamine, 175 μg, dissolved in chloroform(50 μg) , 3 (Succinimidyl-4- (N- maleimidomethyl) cyclohexane-1-carboxylate (SMCC Pierce 22360), 1.75 mg) and 4 (ethylamine, 50 μl) was added. In an alternative aqueous procedure one may instead use succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC Sigma M-6035) . The reaction mixture was stirred for 72 h at rt . under nitrogen in the dark. Particles were extracted with water (approx. 3 ml) 8 times (or to clear water phase) to remove excess lissamine, SMCC, amine and salts. The organic solvent was removed by nitrogen flow. The resulting particles were resuspended in MeCN (2 ml) and TAT (FITC) (aq) (140 μl, 3.7 mg/mlθ.5 mg) was added. The mixture was stirred for 24 h after which the solvent was removed by nitrogen flow. The resulting particles were suspended in a Bri30 detergent (1.5 ml, 0.1 mg/ml), sonicated for 10 min and stirred for 3h. The final particles were purified by repetitive centrifugation (3 * 10 krpm, 5 min each washed with fresh milliQ water) and analysed by Dark field microscopy and fluorescence microscopy for correlation of particle, lissamine and FITC signals.

Example 10: Coating and functionalisation of boron carbide nanoparticles by adsorbtion employing PVA and a lipid-TAT conj ugate .

Boron carbide (36 mg, milled in air) was suspended in MilliQ water (1.8 ml), vortexed for 10 sec, ultrasonicated in a waterbath (5 min.) and centrifuged (14.5 krpm, 5 min.). Supernatant was transferred to a new eppendorf tube and centrifuged (14.5 krpm, 5 min.). The pellet was resuspended in MilliQ water to a final particle concentration of 20 mg/ml. An aliquot of the washed particles (0.5 ml) was ultrasonicated (10 sec pulse, 0.5 amp) and mixed with PVA (0.5 ml, 1%) followed by addition of TAT (FITC) -pal (90 μl , 1.23 mg/ml) . The mixture was vortexed for 10 sec. and incubated on the tabletop for 10 min. followed by vortexing(10 sec). An aliquot (200 μl) was subsequently purified by gelfiltration (PD-10 column, pre-equilibrated in 25 ml MilliQ water) and fractions (1ml) were analysed by fluorimetry, dark field- and fluorescence microscopy. Fluorescence was clearly associated with particles. The process is illustrated in Figure 9. The process relies on the negative charge naturally associated with the starting particles .

Example 11: Entrapment and functionalisation of boron carbide nanoparticles by combined adsorption, cross-linking and covalent bonding employing PVA, epichlorohydrin and TAT. The reaction scheme shown in Figure 10 was carried out. The boron carbide particles are shown as having reactive surface hydroxyl groups. These are believed to be present, at least following ball milling in air. A PVA solution (2.5 % (w/v) ) was made by adding PVA (1.26 g) to MilliQ water (50 ml) . The solution was heated to 70-85°C and stirred with a magnet for 1 hr. in a 3-neck bottle with reflux and thermometer using a heating cap. Boron carbide (22 mg, milled under Ar) was mixed with NaOH (0.5 ml, 2 M) and MilliQ water (9.5 ml) . pH of the suspension was ~11. The suspension was added dropwise to the PVA solution. The mixture was incubated with magnetic stirring o.n. at 70 °C after which the suspension was allowed to cool to RT . An aliquot of the suspension (36 ml) was centrifuged (10 krpm, 90 sec.) and the pellet was resuspended in NaOH (10 ml, 0.1 M) followed by sonication in waterbath for 10 min. Epichlorohydrin (5 ml) was added followed by incubation at RT with magnetic stirring o.n. An aliquot (11 ml, 6 eppendorf tubes) was collected from the aqueous phase, centrifugated (10 krpm, 90 sec) and the pellets were resuspended in ammonia (6 ml, 25% (aq) ) . The mixtures were sonicated in waterbath for 2x15 min. After 2 hrs of incubation, the supernatants were removed by centrifugation (10 krpm, 90 sec). The pellets were washed x2 in MilliQ water with sonication in waterbath for 10 min and supernatants removed by centrifugation (10 krpm, 90 sec). The resulting pellets were stored in 6 eppendorf tubes labeled ^λ,B₄C-PVA-NH₂" . Two pellets (B₄C-PVA-NH₂) were washed xl with DMF followed by resuspension of the pellets in acetonitrile (0.8 ml) and sonication in waterbath for 10 min. Et₃N (10 μl) was added. A lissamine solution (2.06 mg lissamine in 100 μl acetonitrile) was added followed by addition of an SMCC solution (0.7 mg SMCC in 100 μl acetonitrile). The mixture was incubated for 1.5 h in the dark at RT after which the supernatant was removed by centrifugation (10 krpm, 90 sec). The particles were washed x2 with acetonitrile and supernatant removed by centrifugation (10 krpm, 90 sec).

The particles were then washed x2 in HEPES buffer (10 mM) and supernatant removed by centrifugation (10 krpm, 90 sec). The resulting pellet was resuspended in HEPES buffer (500μl, 10 mM) and sonicated in waterbath for 5 min. TAT (FITC) was added (500 μl, 10 μM in HEPES buffer) followed by incubation o.n. in the dark at RT . Particles were washed x3 in MilliQ water with supernatants removed by centrifugation (14.5 krpm, 90 sec). The pellet was finally resuspended in MilliQ water (1 ml) . The colloid was analysed by dark field- and fluorescence microscopy. Fluorescence was clearly associated with particles .

Example 12: Preparation and functionalisation of liposomes containing boron carbide nanoparticles. Boron carbide (36 mg, milled in air) was suspended in MilliQ water (1.8 ml) and vortexed for 10 se , ultrasonicated in waterbath (5 min.) and centrifuged (14.5 krpm, 5 min.). Supernatant was transferred to a new eppendorf tube and centrifuged (14.5 krpm, 5 min.). The pellet was resuspended in MilliQ water to a final particle concentration of 20 mg/ml. An aliquot of the washed particles (0.2 ml) with mixed with PVA (0.2 ml, 1% (w/v))and incubated for 10 min at RT . Particles were subsequently purified by gelfiltration (PD-10 column, pre-equilibrated in 25 ml MilliQ water) and fractions (1 ml) containing particles (3 and 4) were collected. DSPC (Product #850365 from Avanti polar-lipids, inc.): 1, 2-Distearoyl-sn-Glycero-3-Phosphocholine (5 ml, 1 mg/ml in CHC1₃) was mixed with TAT (FITC) -pal (0.709 ml, 0.25 mg/ml in MeOH) in a round bottom glass beaker and MeOH (1 ml) was added. The mixture was vortexed (10 sec) and evaporated under nitrogen flow followed by drying in vacuum oven at RT with oil pump for 2h. The PVA-coated particles were added (1.2 ml) and the beaker was vortexed for 10 sec followed by ultrasonication (8x, 10 sec pulse - 20 sec pause, 0.5 amp). The mixture was incubated o.n. at RT in the dark followed by ultrasonication (4x, 10 sec pulse - 20 sec pause, 0.5 amp). The colloid was analysed by dark field- and fluorescence microscopy. Fluorescence was clearly associated with particles . As shown in Figure 11, the TAT-FITC is held in a lipid coating of the boron carbide forming a liposome.

Example 13: Preparation and functionalisation of glass-coated boron carbide nanoparticles . To carry out the reaction scheme of Figure 12, boron carbide (15 mg, milled in Air) was suspended in EtOH (40 ml, Abs.). Triton X (1 ml) and tetraethoxysilane (TEOS) (0.15 ml) were added followed by incubation for 10 min at RT . NH₄0H (1 ml) was added and the mixture incubated o.n.. Solvent was removed by centrifugation (14.5 krpm, 5 min) and the pellet was washed xl in EtOH and x2 in MilliQ water. The pellet was dried in incubator (140 °C, 10 min) . Powder was suspended in acetone (1 ml) and ultrasonicated with U-horn (5 sec, 0.5 amp) . An aliquot (0.5 ml) was mixed with aminosilane (0.5 ml, 10 vol% in acetone) and incubated for 30 min at RT followed by centrifugation (14.5 krpm, 90 sec). The pellet was collected, washed xl in acetone and centrifuged again. The pellet Was heated to 100°C for 10 min on a heating plate. The powder was suspended in DMF (1 ml) and ultrasonicated with U-horn (5 sec, 0.5 amp) . An aliquot (0.5 ml) was mixed with lissamine (0.4 ml, 5.28 mg/ml in DMF) and incubated for 30 min in the dark at RT . The mixture was centrifuged (14.5 krpm / 2 min) and the pellet was resuspended in MilliQ water (0.5 ml). Purification of the particles by gel filtration was attempted, but they stayed on top of the column. The colloid was, however, analysed by dark field- and fluorescence microscopy. Fluorescence was clearly associated with particles with some free fluorescence present as expected.

Example 14: Chemical Functionalisation of boron carbide nanoparticles by click-chemistry employing azides.

As shown in reaction schemes a, b and c in Figure 13, compound al (Boron carbide, 100 mg, milled in Air) was suspended in DCM (10 ml), whereupon compounds a2 (11- bromoundecyltrichloro silane (BUTS) , 1 ml) and a3 (ethylamine) were added. The reaction mixture was incubated with magnetic stirring for 72 hrs under nitrogen. Particles were washed by centrifugation (14.5 krpm, 5 min) and resuspension in water (3x) and chloroform (3x) . The final pellet was dried under nitrogen flow. An aliquot of the powder (50 mg) was suspended in DMF (4 ml) , whereupon b2 sodium azide (50 mg) was added. The reaction mixture was stirred for 72 hrs at ambient conditions. Particles were washed by centrifugation (14.5 krpm, 5 min) and resuspension in chloroform (3x) and DCM (lx) . The final pellet was dried under nitrogen flow. IR spectroscopy shows the appearance of an azide group (strong signal at 2150 cm^-1) in the final product. An aliquot of the powder (20 mg) was suspended in dry toluene (10 ml), whereupon compound c2 (20 mg) was added. The reaction mixture was stirred for 72 h at RT . Particles were washed by centrifugation (14.5 krpm, 5 min) and resuspension in toluene (2x) and chloroform (2x) . The final pellet was dried under nitrogen flow.

Example 15: Entrapment and functionalisation of boron carbide nanoparticles by electrostatic interactions and covalent bonding .

Boron carbide (11.5 mg, milled under argon) was suspended in NaCl (1.15 ml, 50 mM in MilliQ water) and vortexed (30 sec). An aliquot (200 μl) of the colloid was mixed with polyethyleneimine (PEI) (4 ml, 1 mg/ml in 50 mM NaCl adjusted to ~pH 10 with 0.2 M HC1) . The mixture was vortexed (30 sec), transfered to 4 eppendorf ubes and incubated (10 min, RT) . Particles were washed 3x by centrif gation (14.5 krpm, 5 min), resuspension in MilliQ water (1 ml each tube), vortex (10 sec), incubation in ultrasonic waterbath (5 min) . One tube was collected for analysis and precipitate was dissolved in MilliQ water (1 ml). (PSS - poly (sodium 4- styrenesulphonate) - a negatively charged polymer) (1 ml, 1 mg/ml in 50 mM NaCl, pH ~6) was added to each of the three remaining tubes, vortexed (30 sec) and incubated (10 min, RT) . Particles were washed 3x by centrifugation (14.5 krpm, 5 min) , resuspension in MilliQ water (1 ml each tube) , vortex (10 sec), incubation in ultrasonic waterbath (5 min) . One tube was collected for analysis and precipitate was dissolved in MilliQ water (1 ml) . PEI (1 ml, Img/ml in 50 mM NaCl adjusted to ~pH 10 with 0.2 M HC1 ) was added to each of the two remaining tubes, vortexed (30 sec) and incubated (10 min, RT) . Particles were washed 3x by centrifugation (14.5 krpm, 5 rain) , resuspension in MilliQ water (1 ml each tube) , vortex (10 sec), incubation in ultrasonic -waterbath (5 min) . The tubes were collected for analysis and precipitates were dissolved in MilliQ water (1 ml each) . The structure of the resulting particles is shown in Figure 14.

Example 16: Ingestion of Tat conjugated boron carbide nanoparticles by T-cells.

Tat conjugated boron carbide nanoparticles are incorporated into cells by the following protocol.

1. A colloid was produced of Tat conjugated boron carbide nanoparticles in ddH₂0 (or other suitable buffer with physiological pH) .

Nanoparticle suspensions were prepared in various dilutions (typically from 0.1-lOmg/ml) by diluting the stock colloid in ddH20, in order to determine optimal particle concentration for cellular loading. A master mix of 10 x growth media was prepared (containing lOxRPMI, L-glutamine, Penicillin/Streptomycin, Hepes buffer, Foetal calf serum, NaHC0₃ and in some instances β-mercaptoethanol) . The master mix was added to the particle colloids (master mix:particle colloid ratio 1:10) and samples were incubated for 1-2 hours at RT, in the dark.

3. After the incubation period, the supernatant was transferred to a new tube, leaving the precipitated/aggregated particles .

4. The cells to be used (a cell line, naϊve or in vi tro expanded mouse splenic T cells or naϊve or in vitro expanded human peripheral blood T cells) were counted and the appropriate number of cells were washed once in growth media (RPMI-1640) . Typically, the cellular concentration during incubation with particle colloid was approximately 1 x 10⁶ cells/ml. The washed and pelleted cells were resuspended in the particle colloid (obtained in step 3) and the samples were plated in an appropriate multi-well plate.

5. The incubation time should be optimized for each cell type. To define optimal incubation time for a cell type, it is appropriate to test a number of different incubation times between l-24h, at 37°C. 6. The cells were Spun down and the supernatant was discarded.

7. Cells were washed twice in RPMI, at 120Orpm for 5min, and the supernatant was discarded.

8. Cells were resuspended in Hanks Balanced Salt Solution (HBSS) and spun down. Again, the cells were resuspended in HBSS and lympholyte separation was performed in order to remove particles that had not been associated with the cells.

9. For cells to be used for in vivo transfer, the cells were resuspended in appropriate media (typically PBS with autologous serum) .

10. The particles were labelled with one or more fluorochromes (typically FITC and lissamine) . For FACS analysis of loaded cells, cells were transferred to FACS tubes and washed twice in 1 ml of FACS buffer (PBS + 0.5% FCS or BSA) , 1200 rpm, for 5 min.

11. Samples for FACS analysis may be stained with 7-amino- actinomycin D (7-AAD) (BD Pharmingen, product number : 559925) . 7-AAD is a nucleic acid dye that can be used for the exclusion of nonviable cells in flow cytometric assays. 0,25μg/test (1 x 10⁶ cells) were added and incubated for 10-20 minutes at 4°C.

12. The cells were washed once in 1ml of FACS buffer (1200 rpm, 5 min) and re-suspended in 400μl FACS buffer. 13. The samples were then analysed by FACS.

14. For analysis of particle-loaded cells by fluorescence microscopy or confocal microscopy, cells were resuspended in media or FACS buffer after lympholyte separation (step 8) .

15. For determination of the amount of boron associated with the cells after the loading, inductively coupled plasma- atomic emission spectrometry (ICP-AES) was used. Typically 1-2 x 10⁶ cells/sample were analysed. Loadings of up to 3500 ppm have been obtained.

■ Example 17: Irradiation experiment showing irradiation- and boron dependent cellular killing.

Irradiation experiments have been performed after cells have simply been mixed with a medium containing unfunctionalized particles or, after cellular loading of functionalized boron carbide particles (and after the excess of particles have been removed) . The purpose of the first experiment, using unfunctionalized particles, was to investigate whether extracellularly situated particles may induce cellular killing.

Protocol for the experiment involving extracellular particles :

Preparation of media containing various concentration of unfunctionalized boron carbide nanoparticles: 1. Ball-milled Boron Carbide (milled 144h) was weighed out and a lOmg/ml colloid was prepared by resuspending the particles in ddH₂0. 2. Larger particles were pelleted by centrifugation at 2400g, 10 min. 3. Supernatants (stable colloid) were transferred to a new tube and the pellet was discarded. A number of dilutions of the obtained colloid were prepared; 4-fold; 16-fold, 64-fold and 256-fold as well as a negative control containing no particles. 4. To each of the colloids a master mix of 10 x growth media was added.

Preparation of cells:

The B16 F10 cells were cultured using a standard cell culture procedure. Cells were detached from the culture flasks and transferred to 50ml tubes and transported to neutron source. 1. On the day of irradiation, the B16 cells were detached and counted. Appropriate numbers of cells were transferred into six 5 Oml tubes (one for each colloid concentration) . 2. Cells were spun down, 1300rpm and supernatants were discarded. 3. Appropriate volumes of the different colloids, to which xlO culture media had ioeen added, were added to the cells. Final concentration of the cells was 1,4 x 10⁶ cells/ml . 4. 0,65ml (0,91 x 10⁶ cells) of each sample was transferred to small eppendorf tubes. Protocol for the experiment involving cell-loaded nanoparticles : 1. Functionalized boron carbide nanoparticles as produced in Example 9 were generated (¹⁰B-enriched, milled 96h in Argon) and used for particle-loading of B16F10 cells.

2. B16F10 cells were used in exponential growth. The cells were counted and plated at 20 x 10⁶ B16 cells/flask in culture flasks. The cells were allowed to adhere for 5h.

3. The particle colloid and control media (mock-loading) were prepared as follows:

4. The particles were suspended in MilliQ water at lmg/ml and 10 x growth media was added to the colloid.

5. As control, ddH₂0 and 10 x growth media was mixed.

6. The media from the B16F10 cells was removed and the colloid was added to one flask with cells and control media to another flask with cells.

7. The particles and cells were incubated o/n at 37°C.

8. The cells, still attached to the flasks were washed carefully with RPMI .

9. Cells were detached using trypsin solution (washing once with PBS and thereafter allowed to incubate 5min with trypsin at 37°C) . 10. The number of viable cells was determined using trypan blue . 11. Cells were separated from free particles using lympholyte separation.

12. Cells were resuspended in complete media and counted.

13. The mock-loaded and particle-loaded cells were resuspended in 1.55 x 10⁶ cells/ml.

14. 0,65ml (1 x 10⁶ cells) of each sample was transferred to small eppendorf tubes .

The protocol used for irradiation and the following proliferation assay was the same using unfunctionalised or functionalised particles .

Irradiation The 0.65ml samples were irradiated 0 min, 5 min, 10 min or 15 min .

After irradiation samples were spun down (2000rpm, eppendorf centrifuge), supernatant was removed and fresh media (0,65ml) was added.

In vitro proliferation assay of irradiated cells:

1. 200ul of sample (containing 50,000 cells) was plated in row B in a 96-well plate. 2. 100 μl media was added to all other wells. 3. The samples were titrated in 2-fold dilutions, leaving the last row (row H) with media alone 4. Cells were incubated at 37°C for 48 hours. 5.³H—Th^wmidine assay: On day 2 of culture, 0,5 uCi H— thymidine/well was added and the plates were incubated at 37 °C for another 12 hours. The plates were finally put in the freezer until counting was performed. 6. Counting of the plates was done using a semi-automated sample harvester and the ³H-thymidine incorporation was measured in a β-scintillation counter.

Results

As seen in Figure 15, inhibition of the proliferative response following irradiation of B16 cells mixed with a suspension of boron carbide nanoparticles, is boron- concentration dependent. 1 x 10⁶ cells/ sample were resuspended in RPMI-1640 culture media containing various concentrations of boron carbide nanoparticles. The samples were either not irradiated or irradiated for 5, 10 or 15 min. Following irradiation the proliferative capability was determined by analysis of ³H-thymidine incorporation into DNA after 48h incubation (addition of 3H-thymidine during the final 12h) . The data show that the cellular killing following irradiation is boron carbide dose-dependent.

Importantly, boron carbide per se does not seem to influence the proliferative response. The higher the ¹⁰B₄C colloid concentration present, the higher cellular death. Essentially all cells that were mixed with the concentrated ¹⁰BC colloid and thereafter irradiated for 5 min were dead at day 2-3, as determined by H-thymidine incorporation whereas lower colloid concentrations gave intermediate cellular death. At approximately 256-fold dilution of ¹⁰B₄C colloid almost all cells were still alive day 2-3 even if the irradiation was prolonged to up to 15 min. The irradiation per se did not affect the viability of the cells after 5min irradiation. However, a small decrease in viability was detected after 10 min irradiation and a significant decrease in viability was evident after 15 min irradiation compared to non-irradiated cells. As seen in Figure 16, irradiation of B16 cells pre- loaded with boron carbide nanoparticles results in significant cellular death whereas pre-loading of nanoparticles per se (without irradiation) does not seem to influence the proliferative response to any major extent. B16 cells were pre-loaded with the functionalized particles of Example 9 (lmg/ml) by incubation o/n at 37 °C. Thereafter, free nanoparticles that had not been taken up by the cells were removed from the samples using lympholyte separation. Following lympholyte separation the samples were either not irradiated or irradiated for 5 or 10 min. Finally, the proliferative capability was determined by analysis of ³H- thymidine incorporation into DNA after 48h incubation (addition of 3H-thymidine during the final 12h) . The thymidine-assay data revealed that irradiation (both 5 and 10 min) of B16 cells, loaded with boron particles, resulted in proliferative inhibition. This was not seen after irradiation of mock-loaded B16 cells (cells put through the particle loading manipulations but in the absence of particles) , indicating that the cell death is due to the boron particles and not to the irradiation per se . In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of .

References

Chen Y, Gerald JF, Williams JS and Bulcock S (1999) Synthesis of boron nitride nanotubes at low temperature using reactive ball milling. Chemical Physics Letters 299:260-264

Perrone A, Caricato AP, Luches A, Dinescu M, Ghica C, Sandu V and Andrei A (1998) Boron carbonitride films deposited by laser ablation. Applied Surface Science 133:239-242

Rodriguez MG, Kharissova OV and Ortiz-Mendez U (2004)

Formation of boron carbide nanofibers and nanobelts from heated by microwave, Rev. Adv. Mater. Sci. 7:55-60

Goldberg S, Lesch SM and Suarez DL (2000) Predicting Boron Adsorption by Soil Using Chemical Parameters in the Constant Capacitance Model. Soil Sci. Soc. Am. J. 64:1356-1363

Weast CW, Handbook of Chemistry and Physics 52th ed. (1971) THE CHEMICAL RUBBER CO. page dl21

Huang FL, Cao CB, Xiang X, Lv RT and Zhu H S (2004) Synthesis of hexagonal boron carbonitride phase by solvothermal method. Diamond and Related Materials 13:1757-1760

Pavia DL, Lampman GM and Kriz GS (1996) Introduction to Spectroscopy, Saunders College Publishing, 2.nd Ed. SEQUENCE LISTING

<110> T-Cellic A/S

<120> Boron Containing Nanoparticles

<130> P10291WO

<140> GB0404708.0

<141> 2004-03-02

<160> 3

<170> Patentln version 3.1

<210> 1 <211> 14

<212> PRT

<213> hiv-1

<400> 1

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Gly Tyr Lys Cys 1 5 10

<210> 2

<211> 12

<212> PRT

<213> hiv-1

<400> 2

Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro 1 5 10

<210> 3 <211> 27

<212> PRT

<213> chimeric

<400> 3

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys lie Asn

Leu

1 5 10 15

Lys Ala Leu Ala Ala Leu Ala Lys Lys lie Leu 20 25

Claims

1 . Boron compound nanoparticles comprising boron carbide for use as a medicament .

2. Nanoparticles as claimed in claim 1, for use in boron neutron capture therapy.

3. The use of boron carbide nanopa xticles for the preparation of a medicament for use in a method of boron neutron capture therapy.

4. Boron rich nanoparticles comprising elemental boron or boron carbide, having amine groτ_ιps covalently linked to the surface of said particles.

5. Nanoparticles as claimed in claim 4, wherein said active surface groups are prima_ry amine groups .

6. Nanoparticles as claimed in claim 4 or claim 5, having at least 10 active groups per particle.

7. Nanoparticles as claimed in claim 6, having at least 100 active groups per particle.

8. Nanoparticles as claimed in claim 6, having at least 1000 active groups per particle.

9. Boron carbide nanoparticles havi_ng an organic compound on the surface thereof.

10. Nanoparticles as claimed in claim 9, wherein said organic compound is covalently bonded to said nanoparticles .

11. Nanoparticles as claimed in claim 10, wherein said organic compound is covalently bonded to the surface of the nanoparticles via -N- or -0- linkages.

12. Nanoparticles as claimed in claim 10, wherein said organic compound is covalently bonded to the surface of the nanoparticles via B-N- or B-0-, or C-N- or C-O- linkages .

13. Nanoparticles as claimed in claim 9, wherein said organic compound is adsorbed onto the surface of said nanoparticles .

14. Nanoparticles as claimed in claim 13, wherein said organic compound has multiple positively charged sites .

15. Nanoparticles as claimed in claim 14, wherein said organic compound comprises a polyalkyleneimine, polylysine, poly (diallyldialkyl ) ammonium salt, or poly (allylamine) salt.

16. Nanoparticles as claimed in claim 14 or claim 15, wherein said nanoparticles bear alternating layers of positively and negatively charged polymeric material.

17. Nanoparticles as claimed in any one of claims 9 to 16, wherein said organic compound has a chain length of at least 6 atoms .

18. Nanoparticles as claimed in clarm 17, wherein said organic compound has a chain length of at least 10 atoms .

19. Nanoparticles as claimed in any one of claims 9 to 18, wherein said organic compound has a reactive functional group .

20. Nanoparticles as claimed in clai_m 19, wherein said functional group is primary or secondary amine, hydroxyl, carboxylic acid, carboxylic acid anhydride.

21. Nanoparticles as claimed in any one of claims 9 to 20, for use as a medicament.

22. Nanoparticles as claimed in any one of claims 1 to 21, bearing a cell penetrating peptide or cell penetrating peptide analogue.

23. Nanoparticles as claimed in claim 22, wherein the cell penetrating peptide or analogue is a TAT peptide, antennapedia, transportan, or HS V-1 VP22 peptide or an analogue of such a peptide.

24. Nanoparticles as claimed in claim 22 or claim 23, comprising at least 10 cell pene trating peptide molecules per particle.

25. Nanoparticles as claimed in claim 24, comprising at least 100 cell penetrating pepti e molecules per particle.

26. Nanoparticles as claimed in claim 25, comprising at least 1000 cell penetrating peptide molecules per particle.

27. Boron rich nanoparticles comprising a first detectable label covalently bonded to the surface thereof and a second detectable label independently covalently bonded to the surface thereof via a linker comprising a cell penetrating peptide or cell penetrating peptide analogue .

28. Nanoparticles as claimed in any preceding claim, wherein the boron is enriched in ¹⁰B .

29. Nanoparticles as claimed in claim 27, comprising at least 80% ¹⁰B.

30. Biological cells containing ingested nanoparticles as claimed in any preceding claim.

31. Biological cells containing ingested boron carbide nanoparticles .

32. Biological cells as claimed in claim 30 or claim 31, having a predetermined antigen specificity.

33. Biological cells as claimed in any one of claims 30 to claim 32, being T-cells, macrophages, tumour infiltrating lymphocytes, natural killer cells, LAK- cells, or dendritic cells

34. A process for producing boron rich nanoparticles bearing reactive surface groups comprising grinding boron carbide particles to reduce their particle size under an elemental or compound nitrogen containing atmosphere or with alkali metal amide to produce amine groups or under an oxygen containing atmosphere to produce hydroxyl groups.

35. A process for forming boron rich nanoparticles bearing alkyl or alkylene chains comprising forming amine groups on the surface of boron rich nanoparticles and reacting said amine groups with an alkylating agent or an acylating agent.

36. A process for forming boron rich nanoparticles bearing alkyl or alkylene chains comprising reacting hydroxy groups on the surface of boron rich nanoparticles with a reactive silane.

37. A process for forming boron rich nanoparticles bearing a cell penetrating peptide or cell penetrating peptide analogue, comprising forming boron rich nanoparticles having bonded thereto or adsorbed thereon an organic compound providing a spacer-linker moiety, and bonding to said spacer-linker moiety a said a cell penetrating peptide or cell penetrating peptide analogue, wherein said spacer-linker moiety provides at least a 4 atom chain spacing between said boron rich nanoparticle surface and said cell penetrating peptide or cell penetrating peptide analogue .

38. A process for producing functio alised boron carbide nanoparticles, comprising coati g boron carbide nanoparticles with a polymer comprising monomer residues having hydroxyl substi tuents .

39. A process as claimed in claim 38, wherein said polymer is a polyvinylalcohol or vinyla lcohol copolymer or an oligosaccharide or poly sugar alcohol.

40. A process as claimed in claim 38 or claim 39, further comprising bonding a detectable label and/or a cell penetration peptide or analogue thereof to said polymer coating.

41. A process as claimed in any one of claims 38 to 40, further comprising forming lipo somes comprising said functionalised nanoparticles by mixing said polymer coated nanoparticles with a lipid.

42. A process as claimed in claim 41, further comprising non-covalently associating said liposomes with a lipidated detectable label or lipidated cell penetration peptide or analogue thereof.

43. A process for producing functionalised boron rich nanoparticles comprising forming liposomes containing boron rich nanoparticles by mixing said nanoparticles with a lipid and non-covalently associating said liposomes with a lipidated detectable label or lipidated cell penetration peptide or analogue thereof.