Biospecific agents for bone

Abstract

A bone biospecific agent comprises a contrast material core, which is visible using Magnetic Resonance Imaging (MRI) or Computed Tomography (CT). The contrast material core is surrounded by a polymeric shell, which is functionalised with a bone-targeting peptide. In use, the peptide targets the biospecific agent to bone. The bone biospecific agent can be used in diagnostic imaging techniques, such as MRI and CT, and in imaging bone remodelling activities, detecting and treating pathological bone conditions and/or bone repair processes. The invention extends to the diagnosis and/or treatment of bone disease and bone pathologies using the biospecific agents.

Claims

1. A bone biospecific agent comprising a contrast material core, which is visible using Magnetic Resonance Imaging (MRI) or Computed Tomography (CT), the contrast material core being optionally surrounded by a polymeric shell, wherein the core or polymeric shell is functionalised with a bone-targeting peptide, wherein the peptide, in use, targets the biospecific agent to bone, and wherein the peptide mimics proteins participating in the inhibition of osteoclast-osteoclast and/or osteoclast-osteoblast interactions, or recognises the mineral phase of bone, wherein the bone targeting peptide comprises: (i) a peptide selected from SEQ ID Nos: 1 to 8 conjugated to a peptide selected from SEQ ID Nos: 9-11, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), (ii) a peptide of SEQ ID No: 12 and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or (iii) a peptide selected from SEQ ID Nos: 13-14, and wherein the contrast material core comprises a non-magnetic material.

2. A biospecific agent according to claim 1, wherein the contrast material core is both an MM and a CT contrast material.

3. A biospecific agent according to claim 1, wherein the contrast material core comprises gadolinium, gold, iodine or boro-sulphate.

4. A biospecific agent according to claim 1, wherein the contrast material core comprises gadolinium.

5. A biospecific agent according to claim 1, wherein the contrast material core is gadolinium and the bone-targeting peptide is covalently bonded to the core.

6. A biospecific agent according to claim 1, wherein the polymeric shell is functionalised with one, two or more species of bone-targeting peptide, which target the biospecific agent to bone.

7. A biospecific agent according to claim 1, wherein the bone-targeting peptide targets the biospecific agent to: (i) a cell present exclusively in bone and selected from a group consisting of an osteoblast, osteocyte, osteoclast, bone cell progenitor, osteoclast progenitor and a bone lining cell; or (ii) to the bone mineral phase or hydroxyapatite.

8. A biospecific agent according to claim 1, wherein the bone-targeting peptide is attached to the polymeric shell of the bone biospecific agent by covalent bonding.

9. A biospecific agent according to claim 1, wherein the biospecific agent comprises a bioactive compound, which is delivered to the bone due to the presence of the bone-targeting peptide.

10. A biospecific agent according to claim 9, wherein the bioactive compound is selected from a group of molecules consisting of: a dye, electrochemical mediator, protein, peptide, chemical compound, a drug, genetic material, an oligonucleotide, DNA, RNA, small molecule, antibody, and an enzyme.

11. A biospecific agent according to claim 1, wherein the mean diameter of the biospecific agent is 100-450 nm.

12. A biolabel comprising the bone biospecific agent according to claim 1.

13. A pharmaceutical composition comprising the bone biospecific agent according to claim 1, and a pharmaceutically acceptable vehicle.

14. A biospecific agent according to claim 1, wherein the bone-targeting peptide is attached to the polymeric shell of the bone biospecific agent by carbodiimide chemistry.

15. A biospecific agent according to claim 1, wherein the polymeric shell comprises a biocompatible natural or synthetic polymer including chitosan, collagen, gelatine, hyaluronic acid, poly(ethylene glycol) poly(lactic acid), poly(glycolic acid), poly(epsilon-caprolactone) or poly(acrylic acid).

16. A biospecific agent according to claim 1, wherein the polymeric shell is derivatised with succinic anhydride.

Description

(1) For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:—

(2) FIG. 1 shows a scheme of reaction of chitosan (CS) derivatisation by succinic anhydride to form succinate derivatised chitosan (Suc-Chi);

(3) FIG. 2 shows NMR data of underivatised chitosan (CS, A) and derivatised chitosan (Suc-Chi, B). Vertical arrows in (B) show the disappearance of peaks present in the non-modified CS;

(4) FIG. 3 shows the degree of derivatisation of CS with succinic anhydride in suc-chi compared to underivitised CS;

(5) FIG. 4 shows an FTIR spectra of CS derivatisation into Suc-Chi and its functionalization with an OP3-4 peptide which has the following amino acid sequence YCEIEFCYLIR;

(6) FIG. 5A shows Dynamic light scattering (DLS) analysis (A and B) and scanning electron microscopy (SEM) (C and D) of CS beads (A and C) and one embodiment of a biospecific contrast nanoparticle according to the invention (B and D). FIG. 5E is a schematic representation of one embodiment of a nanoparticle of the invention;

(7) FIG. 6 shows the effect of chitosan concentration on nanoparticle size without and with filtration process. (A) 150 molecular weight, (B) 400 molecular weight, (C) 600 molecular weight, (D) Concentration-dependence in different molecular weight Ch (n=4);

(8) FIG. 7 shows the typical structure of a DOTA-OP3-4, i.e. the result of the conjugation of the OPG mimetic peptide, (OP3-4), which specifically targets cytokines expressed on bone cells, to DOTA, which can be used to chelate several metal ions such as gadolinium (Gd.sup.3+) as contrast agents.

(9) FIG. 8 is a schematic representation of the assembly of DOTA-OP3-4. Numbered arrows indicate the order of assembly;

(10) FIG. 9 shows an ion trap MS spectrum of DOTA-OP3-4;

(11) FIG. 10 is a schematic representation of the assembly of G2PL-OP3-1. Numbered arrows indicate the order of assembly;

(12) FIG. 11 shows an ion trap MS spectrum of G2PL-OP3-1;

(13) FIG. 12 shows amino acid analysis of OP3-4 peptide on a nanoparticle and on a nanoparticle-DOTA-Gd-OP3-4 conjugate;

(14) FIG. 13 shows MRI analysis of DOTA-Gd-OP3-4 conjugate and the negative controls DOTA-OP3-4 conjugate and PBS. The peptides were dissolved in PBS at a concentration of 0.4 μg/ml. The scans were performed in the T1-weighted mode with TE 8.7 and TR 550;

(15) FIG. 14 shows an MRI analysis of magnetic nanoparticle-OP3-4 conjugate (mSCB) and nanoparticle-DOTA-Gd-OP3-4 conjugate at 0.4 μg/ml. Phosphate buffered saline (PBS) was used as a negative control. Gadolinium-based and magnetic beads-based contrast agents provide the typical bright and dark imaging respectively;

(16) FIG. 15 shows the effect of peptides on rh RANKL-induced osteoclastogenesis. Monocytes were cultured for 6 days in αMEM medium supplemented with peptides at concentration of 100 μM. The concentration of rh RANKL was 500 ng/ml, of rh M-CSF was 25 ng/ml and rh OPG was 500 ng/ml. The culture medium with respective dosages was replaced after 3 days;

(17) FIG. 16 shows the effect of peptide-tethered nanoparticle on rh RANKL-induced osteoclastogenesis. Monocytes were cultured for 6 days in αMEM medium supplemented with peptides (100 μM) and beads (25 μg/ml). The concentration of rh RANKL was 500 ng/ml, of rh M-CSF was 25 ng/ml and rh OPG was 500 ng/ml. The culture medium with the respective dosages was replaced after 3 days;

(18) FIG. 17 shows the inhibition of rh RANKL-induced osteoclast activity in a monocyte monoculture as determined by analysis of F-actin ring formation using rhodamine-phalloidin and Hoescht 33258 dual stain; and

(19) FIG. 18 are representative SEM micrographs of cells cultured on bone slices. (A) untreated cells (B) M-CSF only (C-D) M-CSF+rh RANK (E-F) representative resorption lacunae (G-H) cells treated with OP3-4 peptide and tethered contrast agents.

EXAMPLES

(20) The inventors were interested in providing improved apparatus and methods for the diagnosis (e.g. by either MRI or CT imaging) or treatment of bone-related conditions. Accordingly, they have designed and developed novel bone specific agents 2 (e.g. nanoparticles, sub-micron particles, and atomic or molecular elements), as illustrated in FIG. 5E, which include a contrasting agent core 4 (e.g., an ion oxide or a gold metallic core), which is coated with a polymer 6 (for example, chitosan), which itself is derivatised or functionalised with one or more peptide(s) 8, which recognise bone cells (such as osteocytes, osteoblasts), or other peptides that are only present in bone, for example hydroxyapatite-specific peptides. Depending on the careful selection of the material in the core 4, of the polymer coating forming the outer shell 6, and of the bone-specific functionalising peptides 8 attached to the polymeric outer shell 6, the nanoparticles 2 etc. can be used in either diagnosis or therapy. For example, the particles 2 may be used in imaging bone remodelling activities, detecting pathological conditions and/or tissue repair processes. The following Examples describe the results of their research.

Example 1—Conjugation of Succinic Anhydride to Chitosan

(21) The inventors have shown that chitosan (CS), a polysaccharide which can be used to coat contrast agents or other active ingredients of a pharmaceutical agent, can be conjugated to succinic anhydride. Chitosan succinate conjugates are known in the art as being both a biocompatible and biodegradable drug delivery agent which may be used in tablets.

(22) 1. Materials and Methods

(23) (i) Chitosan (CS) Derivatisation

(24) CS was derivatised by succinic anhydride (Suc-Chi) using a known ring opening reaction (Yan et al., 2006, Yakugaku Zasshi, 126, 789-793). A 1% (w/v) CS solution (in 1% v/v acetic acid) was filtered through 0.8 μm pore membrane (Millipore) and diluted (1:4) with methanol. Succinic anhydride (>99% GC, Sigma Aldrich) was dissolved in 5 ml acetone at 4% (w/v) was added drop wise under magnetic stirring and left overnight under agitation at room temperature. The gel that formed was removed from excess solution, double diluted in methanol and dialyzed against ultrapure water for 3 days.

(25) The water was changed twice per day and the obtained precipitate was then collected by centrifugation and lyophilised.

(26) (ii) Production of Suc-Chi Submicron Beads (i.e. Nanoparticles)

(27) Suc-Chi beads were produced using an established ionic gelation method (Agnihotri, et al., 2004). Briefly, sodium tripolyphosphate (TPP) solution (1 mg/ml) was added drop wise to a 1 mg/ml Suc-CS solution (as described above) under magnetic stirring at a volume ratio of 1:5 and allowed to react for 45 minutes at room temperature. To produce magnetic resonance imaging (MRI) or CT imaging biospecific contrast agents (i.e. nanoparticles 2 of the invention), iron oxide core 4 particles (Fe.sub.3O.sub.4, 10 nm mean diameter) or gold core 4 particles (<20 nm mean diameter) were first dispersed in the TPP solution using ultrasonication before addition to Suc-CS solutions. The weight of the core particles 4 added was half that of the dissolved polymer 6. The core 4 particles were then washed by centrifugation through ethanol (to sterilise) and then in ultrapure water and reconstituted in sterile PBS.

(28) (iii) Peptide Synthesis

(29) The peptides 8 listed in Table 1 and their corresponding amino acid sequences were synthesised and then used to functionalise the core particles 4.

(30) TABLE-US-00001 TABLE 1 List of typical peptides to be used for contrast agent biofunctionalisation Name Sequence Function GAP.sub.27p SRPTEKTIFII Derived from Cx.sub.43 GAP.sub.27. To be used to block osteoclast-osteoclast  and osteoclast-osteo- blast (Chaytor, et al., 1997, Ilvesaro, et al.,  2001) OP.sub.3-1 YCLEIEFCY Based on OPG residual 113-122. Specifically bind to RANK and in- hibit RANKL induced  osteoclast differenti- ation and activity   (Cheng, et al., 2004,  Shin, et al., 2008, Ta,  et al., 2010) OP.sub.3-4 YCEIEFCYLIR Based on OPG residual 113-122. Specifically  bind to RANK and inhibit RANKL induced osteoclast differen- tiation and activity (Cheng, et al., 2004, Shin, et al., 2008, Ta, et al., 2010) G.sub.1PL K-(KK) Nanosized flexible  G.sub.2PL K-(KK)-(KKKK) carrier of the peptides G.sub.3PL K-(KK)-(KKKK)- with improved solu- (KKKKKKKK) bility in aqueous solu- tion (Lloyd, et al., 2007, Meikle, et al., 2011) G.sub.2PL-OP.sub.3-1 (KKKK)-(KK)-K- Pro-drug: novel OP.sub.3-1  YCLEIEFCY tethered G.sub.2PL DOTA-OP.sub.3-4 DOTA-KGG- Novel DOTA tethered  YCLEIEFCYLIR OP.sub.3-4 peptide for the  chelation of MRI visi- ble Gd.sup.3+ DOTA-Gd- DOTA-KGG- Novel MRI detectable  OP.sub.3-4 YCLEIEFCYLIR OP.sub.3-4 derivative with  a Gd.sup.3+ chelate DOTA-Gd- DOTA-Gd- Novel MRI detectable  FHRRIKA FHRRIKA osteoblast migration   derivative with a Gd.sup.3+  chelate NOTE: G.sub.1PL, G.sub.2PL and G.sub.3PL are not linear molecules, but rather hyperbranched (dendritic). See FIG. 10 where a G.sub.2PL molecule (vertical) is conjugated to OP.sub.3-1 (horizontal).

(31) The peptides 2 were synthesised by solid phase peptide synthesis (SPPS) using the conventional 9-fluorenylmethyloxy carbonyl (Fmoc) protection/deprotection strategy on Tenta Gel S NH.sub.2 resin (0.1 mmol) and dimethylformamide (DMF) as the reaction solvent. An acid-liable Fmoc-Rink-Amide linker (linker) was attached first to the resin for later cleavage of the peptide 8. The peptide 8 was then synthesised by adding the first amino acid from the C-terminal followed by sequential coupling/deprotection steps of subsequent amino acids as per the peptide sequence, as set out in Table 1. The coupling reactions (30 minutes, ×2) were carried out using HBTU (O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate), for amino group activation, and N,N-Diisopropylethylamine (DIPEA) as a tertiary base. The exposure of the protected amino groups was obtained by cleaving the Fmoc protecting group with 20% (v/v) piperidine in DMF (2 minutes, ×3). In all preparations, the resin, linker and amino acids were added in the molar ratio of 1:4:4 respectively. HBTU and DIPEA (diisopropylethylamine) were 1 and 2 times the molar concentration of the amino acids respectively. Each coupling and/or deprotection step was followed by a washing step (×3 with DMF).

(32) Osteoprogeterin (OPG) mimetic peptides, OP3-1 and OP3-4, as listed in Table 1, were cyclised by dimethyl sulfoxide (DMSO) oxidation to form cysteine-cysteine disulfide bonds as described in (Góngora-Benftez, et al., 2011). OP3 is a segment on OPG protein. RANKL on the surface of osteoblast (sometimes release in soluble form) interacts with RANK on osteoclasts, thereby initiating a reaction cascade leading to osteoclast differentiation and increased activity. OPG (released by osteoblasts) is a decoy for RANKL and its binding to RANK inhibits RANKL-RANK interaction, thereby stopping the cascade. Hence, OPG mimetics would act as ligands for the receptors in the bone-associated target cells. After synthesis, the peptides to be cyclised were cleaved from resin in a nitrogen atmosphere for 3 hours. After cleavage, the peptides were collected in cold diethyl ether, isolated by centrifugation and dried over a stream of nitrogen. The peptides were then dissolved in 600 ml of oxidising buffer (100 mM NaH.sub.2PO.sub.4 and 2 mM Gdn.HCl, 5% DMSO, pH 7.0) and shaken for 12 hours. The solution was then acidified with 1 M HCO.sub.2H (250 μl) and purified by LC-MS. The pure fractions were combined and freeze dried. The degree of cyclisation (formation of disulfide bridges) was assessed by the conventional method for quantitation of free thiol groups using Ellman's reagent. The peptides were ultimately characterised by HPLC and MS.

(33) (iv) Production of Peptide-Tethered Biospecific Contrast Agents (i.e. Nanoparticles)

(34) The peptides 2 described above were covalently attached to the core 4 particles by carbodiimide chemistry in order to create nanoparticles 2 of the invention. Non-derivatised particles were first dispersed in 2 ml of 2-(N-morpholino) ethanesulfonic acid (MES) buffer (0.1 M MES, 0.3 M NaCl, pH 6.5) to obtain a 1 mg/ml bead concentration. The carboxyl groups within the core 4 particles were then activated by addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 4 mM) and N-hydroxysuccinimide (NHS, 10 mM). The activation reaction was allowed to proceed at room temperature for 30 minutes. Excess EDC was deactivated by addition 13-mercaptoethanol (2.8 μl) and the core 4 particles were washed through a desalting membrane. A 1 mg/ml solution of the peptide chosen from Table 1 (e.g. OP3-4 peptide having the sequence, YCEIEFCYLIR) in MES buffer was then added to the solution of core 4 particles at a volume ratio of 1:1. The conjugation reaction was allowed to proceed under magnetic stirring for 3 hours at room temperature. The reaction was then quenched by the addition of hydroxylamine to give a final concentration of 5 to 10 mM.

(35) Referring to FIG. 5E, there is shown a schematic representation of one embodiment of the nanoparticle 2 of the invention. The Figure shows the nanoparticle 2 having the inner core 4 (e.g., ion oxide or gold) coated with the polymeric outer shell 6 (e.g. chitosan). The shell 6 is functionalised with a coating of peptides 8, which recognise bone cells, or other peptides that are only present in bone, for example hydroxyapatite. The resultant nanoparticles 2 were then purified using ultrafiltration spin columns (MWCO 100,000), lyophilised and stored at −20° C. FIG. 1 shows the scheme of the derivatisation of CS into Suc-Chi and its subsequent functionalisation with the OP3-4 peptide.

(36) 2. Results

(37) The .sup.1H NMR spectra of CS and Suc-Chi are shown in FIG. 2. Briefly, the signals are assigned as follows: 2.50-2.70 ppm (H—Ac) was attributed acetyl proteins of GlcNAc monomers; 2.50-2.75 ppm (H2D) was attributed to proton 2 of GlcN monomer; 3.95-4.65 ppm (H2-2) was attributed protons 2 to 6 of both GlcNAc and GlcN monomers; 4.65-4.90 ppm (HOD) corresponds to the solvent (HOD); 5.10-5.30 ppm (H1-A) corresponds to proton 1 of GlcNAc monomers; 5.30-5.65 ppm (H1-D) correspond to proton 1 of GlcN monomer. When compared to the spectra of CS, the spectrum of Suc-Chi confirms successful attachment of succinic anhydride to CS. This is seen in the poorly resolved or missing signal for H1(D), H1-A and H2D within the range 5.00-5.70 ppm (see FIG. 2B, arrows, for the first two) and 3.6-3.9 ppm for H2D. The new signals in the range 2.34-2.57 ppm, seen in the spectra of Suc-Chi but absent in the spectra of CS, are attributed to the two methane hydrogen groups (—COCH.sub.2CH.sub.2COOH) of the succinyl group and is in agreement with reports by others (Liang, et al., 2012, Xiangyang, et al., 2007).

(38) The degree of substitution as determined by titration was 25.5% and 30.6% by .sup.1HNMR. Although, less accurate, potentiometric titration analysis allowed for the determination of the molar amounts of the free —NH.sub.2 in both CS and Suc-Chi. The degree of derivatisation (DD) values calculated for CS was 79.92% (+5.85) and for Suc-Chi was 54.4% (±3.7), as shown in FIG. 3. The titrations were repeated 5 times for both polymers. These data suggest a reduction in the amount —NH.sub.2 groups after derivatisation of CS with succinic anhydride. The presence of non-derivatised —NH.sub.2 groups was important to ensure the CS cross-linking by TPP during nanoparticle 2 production.

(39) FTIR results also showed successful derivatisation of chitosan to Suc-Chi and subsequent attachment of OP3-4, as shown in FIG. 4. N—H stretch of primary and secondary amines and O—H stretch are comprised in band 1 (3500-3200 cm.sup.−1). The main contribution to band 1 in the spectra of OP3-4 is from the amines involved in the amide bond. Band 2 (1640-1580 cm.sup.−1) may correspond to the N—H deformation in primary amines, present in the four species; N—H deformation of amides and also carbonyl stretching in secondary amides for the case of OP3-4, Suc-Chi and Suc-Chi-OP3-4. Band 3, (1722 cm.sup.−1), which is present in Suc-Chi and unresolved in Suc-Chi-OP3-4 may be attributed to the carbonyl stretching as result of the linkage of the succinyl group to the polysaccharide through an ester bond, in addition to the linkage via amide bond described earlier (band 3). The aromatic structures present in tyrosine and phenylalanine can be confirmed in Suc-Chi-OP3-4 with band 4 (3000 cm.sup.−1), which originates from C—H stretch in unsaturated species. A large peak at band 5 (500-540 cm.sup.−1) in the spectrum of Suc-Chi-OP3-1, which is weakly present in the OP3-4 sample and absent in Suc-Chi and CS can be attributed to the presence of disulfide bonds.

(40) From the results obtained by .sup.1HNMR and titration, it was concluded that succinyl groups were successfully conjugated to the amine groups in CS. FTIR results further suggest the successful attachment of the OP3-4 peptide to Suc-Chi.

(41) Dynamic light scattering analysis (DLS) and scanning electron microscopy (SEM) showed that the nanoparticles had an average hydrodynamic diameter (Z.sub.average) of 366.4 nm (polydispersity index, PDI of 0.4), which increased to 408.3 nm (PDI of 0.5) after incorporation of Fe.sub.3O.sub.4 core 4 particles (10 nm) to produce MRI detectable nanoparticles 2, see FIGS. 5A-D. Changes in chitosan molecular weight, and concentration without or with the filtration step allowed the tuning of the nanoparticle 2 size and polydispersity index (PDI), see FIGS. 6 A-D.

Example 2—Conjugation of OP3 with DOTA

(42) The inventors then set out to determine whether the protein, osteoprogeterin 3 (OP3), which is specifically expressed on bone cells, such as osteoclasts and oseoblasts, can be conjugated to Dotarem (DOTA), which is a chelator that can be used to coat various contrast agents, including gadolinium. Dotarem is gadoteric acid, a known macrocycle-structured GD-based MRI contrast agent. It consists of the organic acid DOTA as a chelating agent.

(43) 1. Materials and Methods

(44) A novel DOTA-OP3-4 conjugate protein was synthesised by solid phase peptide synthesis using Fmoc chemistry as described in the synthesis of OP3-1 and OP3-4. In this case, however, lysine core amino acid was added first, followed by the coupling of DOTA to the NH.sub.2 group that was protected by Mtt. Two glycine amino acids were then coupled to form a spacer followed by the subsequent assembly of OP3-4 peptide. The introduction of the lysine-glycine-glycine spacer between DOTA molecule and OP3-4 sequence was considered important to avoiding potential steric hindrance during the synthesis and any possible effect on the potency of the peptide. A structure of the DOTA-OP3-4 is shown in FIG. 7.

(45) The same solid phase peptide synthesis method was used to synthesise novel derivatised biospecific peptides with linear (see FIG. 8) or branched (see FIG. 10) ends able to favour the stable binding of the peptides to the contrast agents.

(46) 2. Results

(47) FIGS. 9 and 11 show typical mass spectrometry spectra of a linear (FIG. 9) and a branched (FIG. 11) OP3-4 peptide. These prove the successful synthesis of these peptides that is necessary to the formation of stable binding and functionalisation to the core 4 particle. Purity of above 95% was achieved after purification procedure.

Example 3—Creation of a Biospecific Contrast Agent—DOTA-Coated Gadolinium, which is Conjugated to OP

(48) The inventors next set out to determine whether a gadolinium (Gd)-based contrast agent could be created to form a nanoparticle 2 of the invention by the conjugation of DOTA-coated Gd with the bone-specific protein osteoprogeterin 3 (OP3). The hope was that they could also be used with MRI and/or CT imaging techniques.

(49) 1. Materials and Methods

(50) Novel peptides were used to manufacture biospecific contrast agents (i.e. functionalised nanoparticles 2) for MRI and CT (see Table 1). The chelation of the core 4 particle, Gd.sup.3+, was achieved by incubating DOTA-OP3-4 with GdCl.sub.3 in a buffer system for 15 hours. The DOTA moiety acted as a polydentate ligand and enveloped the metal cations, in this case complexing Gd.sup.3+, to give an MRI-visible peptide. The coordination of the DOTA ligands and metal ion in the complex depends on the conformation of the ligand and geometric tendencies of the metal cation. On its own, DOTA acts as an octadentate ligand, binding the metal through four amino and four carboxylate groups. In this study, the DOTA molecule acts as a septadentate since one of the carboxylate groups is used in the covalent with the peptide. However, a carboxylate group from the amino acid linking DOTA and the peptide provides the eighth ligand and restores the octadentate state, forming a highly stable coordination complex (Viola-Villegas, et al., 2009).

(51) The resultant nanoparticles 2 were obtained through the direct binding of peptides 8 with a linear or branched root to magnetic core 4 particles (e.g. iron oxide) coated with thin films of polymers 6 or ceramics (i.e. MRI contrast agents) or gold core 4 particles (i.e. CT contrast agents). In this case, surface functionalization of polymeric carboxylic groups or hydroxyl groups of polymers 6 and ceramics were activated and derivatised with an amino acid to which a selected biospecific peptide from Table 1 was grafted through covalent binding.

(52) 2. Results

(53) Successful chelation of Gd.sup.3+ was confirmed by LC-MS where the peak with m/z 694.1 for DOTA-OP3-4 peptide was replaced by a peak with m/z 714.8 corresponding to the [M+3H].sup.3+ after chelation of Gd.sup.3+. Amino acid analysis confirmed the successful attachment of OP3-4 to the gadolinium (Gd)-based nanoparticle 2 (FIG. 12). After hydrolysis, the amount of glucosamine units and the amount of amino acids present in the polymer was determined. The amount of peptide 8 attached onto the nanoparticle 2 was calculated by the integration of peak areas. A representative LC profile of the hydrolysis products on the Gd nanoparticle 2 functionalised with the OP3-4 peptide 8 is given in FIG. 12. The glucosamine units per micro gram of material was calculated to be 1.92 nmoles in chitosan-based nanoparticles (CNB), 1.40 nmoles in Gd core 4 particle (on its own) and 0.27 nmoles in Gd nanoparticle 2 functionalised with OP3-4 peptide 8, and Gd nanoparticle-DOTA-Gd-OP3-4.

(54) Importantly, in both nanoparticle-OP3-4 and nanoparticle-DOTA-Gd-OP3-4, the amount of peptide 8 conjugated was calculated to be 4.2 mmoles per gram of nanoparticle 2. Individual amino acids were detected in molar ratios reflective of the amounts in OP3-4 sequence.

Example 4—Testing the Use of the Nanoparticles with MRI

(55) The inventors then tested their biospecific peptide-functionalised nanoparticles 2 for positive MRI signalling under T1 and T2 modes. Biospecific nanoparticles 2 were obtained through the entrapment of Gd core 4 into derivatised peptides 8 and by grafting onto nanoparticles previously functionalised with bioactive peptides.

(56) 1. Materials and Methods

(57) The solutions of the peptides (DOTA-OP3-4 and DOTA-Gd-OP3-4) in PBS buffer were prepared by first dissolving the peptides in a minimum amount of DMSO and then diluted out to give a 20 μg/ml peptide stock solution in PBS (1% DMSO) and the pH adjusted to 7.2 with 0.1M HCl. The various nanoparticles 2 (i.e. core 4 particle alone, nanoparticle-OP3-4 conjugate, nanoparticle-DOTA-Gd-OP3-4 conjugate) were suspended in the same PBS buffer solution. Stock solutions DOTA-Gd.sup.3+ (20 μg/ml) and Fe.sub.3O.sub.4 nanoparticles (10 nm, 20 μg/ml) were prepared and used as a positive controls for gadolinium based and Fe.sub.3O.sub.4 based contrast agents respectively.

(58) A comparison of the nanoparticle-OP3-4 conjugate and nanoparticle-DOTA-Gd-OP3-4 conjugate was carried out first. For this, Whatman filter papers (circular, 15 mm diameter, cat: 1441 150, USA) were soaked in the stock solutions.

(59) In the studies on the effect of the concentration of the nanoparticles 2 and peptides 8, different concentrations of the peptides 8 and nanoparticles 2 were prepared by a series of double dilutions of the stock solutions. The concentrations of DOTA-Gd-OP3-4 was (10, 5, 2.5, 1.25, 0.625, 0.313, 0.078, 0.039, 0.020, 0.010, and 0.005 μg/ml) and for core 4 particle alone was (20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.078, 0.039, 0.020 and 0.010 μg/ml). The analytes were then placed in 24 well culture plates at a volume of 500 μl per well. All MRI imaging was performed on using a Siemens AVANTO 1.5T MRI scanner at the Clinical Imaging Science Centre, Brighton and Sussex Medical School, UK.

(60) 2. Results

(61) FIG. 13 shows a typical MRI scan of a negative control filter impregnated with phosphate buffered saline (PBS), a negative control consisting of DOTA-OP3-4 peptide 8 but with no contrast agent core 4, and a gadolinium-chelating DOTA-OP3-4 nanoparticle 2. The scan clearly show that while the negative control showed only noise signals, the peptide 8 chelating the gadolinium core 4 provided a clear positive signal. A comparative analysis of filters impregnated with either gadolinium-chelating DOTA-OP3-4 and peptide-functionalised magnetic nanoparticle 2 showed the typical bright and dark images expected from the two contrast agents in the chosen mode of detection (FIG. 14).

Example 5—Testing the Inhibitory Effect of OP and OP4-Conjugated Nanoparticles in Vitro

(62) The inventor next determined whether a nanoparticle 2 comprising DOTA-coated gadolinium core 4 conjugated to osteoprogeterin 3 or 4 (OP3 or OP4) peptide 8, would inhibit osteoclastogenesis and osteoclast activity in vitro.

(63) 1. Materials and Methods

(64) Osteoclasts were obtained from mononuclear cells freshly isolated from peripheral blood from healthy human donors according to conventional methods based either on spiking of the cells with RANK and M-CSF or in osteoblast mononuclear cell co-culture systems spiked with M-CSF. Peptides and peptide-tethered nanoparticles 2 (i.e. nanoparticle-OP3-4, magnetic nanoparticle-OP3-4, nanoparticle-OP3-DOTA and nanoparticle-OP3-4-Gd-DOTA) were added to the cells at peptide concentration equivalent of 50 μM as determined by amino acid analysis. The negative controls received no test materials and the positive control received rh OPG (50 ng/ml). Spiking was performed either before or after the differentiation of the mononuclear cells into osteoclasts.

(65) Inhibition of osteoclastogenesis and osteoclast activity was quantitatively assessed by counting the number of TRAP positive multinucleated (MNC) cells using light microscopy and the number of MNC cells presenting F-actin rings using epi-fluorescence microscopy. Osteoclast activity was also assessed qualitatively by analysis of the number of resorption pits formed on the bone slices by SEM. Culture medium was replaced every 3 days with fresh medium supplemented with all the growth factors and test materials.

(66) Three different approaches were used to determine inhibition of osteoclastogenesis and osteoclast activity. These methods were: (1) counting the number of TRAP positive multinucleated cells; (2) counting the number of multinucleated cells possessing the actin rings (MNC-AR+) using Hoechst 33258 and rhodamine phalloidin double stain; and; (3) determining the degree of bone resorption by assessment of resorption pits formed after culturing cells on bone slices. Where cells were too numerous to count microscopically, an image editing software (Image J v1.44P) was used to distinguish and count the cells. The software allows for cells to be tallied according to colour and shape for both osteoclast and non-osteoclast cells and the degree of osteoclastogenesis expressed was a percentage of TRAP positive cells per field (Labno).

(67) 2. Results

(68) The result from the studies on the effect of nanoparticles 2 functionalised with OP3-4 and Gd.sup.3+ chelating derivatives thereof on osteoclastogenesis are shown in FIG. 15. In all formulations, the modified peptides 8 free and bound to nanoparticles 2 and gadolinium reduced the formation of osteoclasts at levels significantly different from the control, but with different degrees of efficacy (FIG. 15).

(69) In addition, peptide-functionalised magnetic nanoparticles 2 appeared to significantly reduce osteoclastogenesis when compared to non-functionalised nanoparticles (see FIG. 16). The number of TRAP positive MNC in culture treated with nanoparticle-OP3-4, nanoparticle-DOTA-OP3-4 and nanoparticle-DOTA-Gd-OP3-4 was not significantly different showing that the various modifications of the peptide and its grafting to nanoparticles 2 did not alter its ability to inhibit osteoclastogenesis. When used on its own, Gd.sup.3+ is toxic both in vitro and in vivo. However, macrocydic chelates such as DOTA tightly trap Gd.sup.3+ improve the ion solubility thus avoiding cytotoxic effects. Indeed Suc-Chi with its many free carboxyl groups and improved solubility would further contain Gd.sup.3+ and improve the biocompatibility of DOTA-Gd-OP3-4.

(70) A clear inhibitory effect on the activity of already differentiated osteoclasts is shown in FIG. 17.

(71) Finally, the inhibition of the activity of differentiated osteoclasts was clearly observed by SEM showing the absence of pits produced by the control cells, as shown in FIG. 18.

SUMMARY

(72) The diagnosis and treatment of bone pathologies (e.g. osteoporosis) with localised injection of agents is widely advocated. The inventors have now developed novel contrast agents for use in MRI and CT imaging, which agents can recognise bone cells, osteoblasts and osteoclasts, as well as the mineralized bone extracellular matrix. These biorecognition properties were obtained through the synthesis of novel derivatised peptides with specificity for various bone cells and the mineral phase of bone. The derivatisation was designed to favour the stable binding with contrast agents of nanoparticulate or ionic composition without affecting their imaging properties. Specific types of contrast agents in the form of magnetised polymeric beads, mainly chitosan nanobeads, were obtained either through methods of coating of the magnetic core or grafting of gadolinium-modified peptides or a dispersion of ions in their cross-linked matrix. This ability to recognise cellular and structural components of the bone was coupled with the ability of controlling the cell behaviour. Biospecific contrast agents able to recognise mononuclear cells during their process of differentiation into osteoclasts as well as to recognise and inhibit the activity of differentiated osteoclasts could be obtained together with agents able to favour osteoblast migration.

(73) In summary:— 1. Surface functionalization of submicron particles, such as Fe.sub.3O.sub.4 nanoparticles (i.e. a MRI contrast agent) and gold nanoparticles (i.e. a CT contrast agent) with osteoblast- and osteoclast-specific peptides as well as with hydroxyapatite-specific peptides are preferred. 2. Gadolinium (i.e. a MRI contrast agent) and iodine or boro-sulphate (i.e. CT contrast agent) entrapped into polymeric beads functionalised with osteoblast- and osteoclast-specific peptides as well as with hydroxyapatite-specific peptides. 3. Gadolinium (i.e. MRI contrast agent) and iodine (i.e. CT contrast agent) complex with osteoblast- and osteoclast-specific peptides as well as with hydroxyapatite-specific peptides. 4. Nanoparticles formed by a coating of synthetic or natural polymers where the morphology and size is determined by the tuned physico-chemical properties of the polymer and where the biorecognition and bioactivity are obtained through its derivatisation with specific peptides capable of recognised tissue cells. 5. Nanoparticles formed by crosslinking (mainly ionically crosslinking) of synthetic and natural polymers (e.g. chitosan) previously derivatised with tissue-specific peptides, where crosslinking and biofunctionalisation are tuned to optimise the stability of the nanoparticle and the presentation of the biospecific/bioactive molecules. These nanoparticles include in their formulation dispersed contrast agents for MRI and CT.

(74) In all cases, these biospecific agents couple the property of contrast agents with combined, built-in biorecognition and bioactivity properties capable of inducing tissue imaging and regeneration.

(75) In vitro mono- and co-culture studies of osteoblasts and osteoclasts demonstrated the ability of bone-specific peptides to the cells. Given the ability of the osteoblast-specific peptides (e.g. FHRRIKA) to encourage cell processes and of the OPG-mimicking peptides to inhibit osteoclastogenesis, these novel material can be also used as theranostic (i.e. combined therapy and diagnostic) agents in the treatment of bone deficiencies.

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