NEURODEGENERATIVE DISORDERS

Abstract

An amyloidogenic peptide biospecific agent comprises a nanoparticle which is visible under near infrared (NIR) and/or using Magnetic Resonance Imaging (MRI) and/or Computed Tomography (CT). The biospecific agent further comprises at least one antibody or antigen binding fragment thereof, which is immunospecific for a transferrin receptor and an amyloidogenic peptide.

Claims

1. An amyloidogenic peptide biospecific agent comprising a nanoparticle which is visible under near infrared (NIR) and/or using Magnetic Resonance Imaging (MRI) and/or Computed Tomography (CT), and at least one antibody or antigen binding fragment thereof, which is immunospecific for a transferrin receptor and an amyloidogenic peptide.

2. A biospecific agent according to claim 1, wherein the at least one antibody or antigen binding fragment comprises an IgG anti-amyloidogenic peptide antibody or antigen binding fragment thereof.

3. A biospecific agent according to claim 1, wherein the at least one antibody or antigen binding fragment binds specifically to oligomers and fibrils of the amylodogenic peptide, but not to amyloidogenic peptide plaques or peptide monomers.

4. A biospecific agent according to claim 1, wherein the immunogen sequence used to create the at least one antibody or antigen binding fragment against the amyloidogenic peptide comprises or consists of the amino acid sequence of A, or a variant or fragment thereof, preferably partially aggregated SEQ ID No:1.

5. A biospecific agent according to claim 1, wherein the immunogen sequence used to create the at least one antibody or antigen binding fragment against the amyloidogenic peptide comprises or consists of the amino acid sequence of Huntingtin, or a variant or fragment thereof, preferably partially aggregated SEQ ID No:2.

6. A biospecific agent according to claim 1, wherein the immunogen sequence used to create the at least one antibody or antigen binding fragment against the amyloidogenic peptide comprises or consists of the amino acid sequence of alpha-synuclein, or a variant or fragment thereof, preferably partially aggregated SEQ ID No:3.

7. (canceled)

8. A biospecific agent according to claim 1, wherein the at least one antibody or antigen binding fragment comprises an IgM anti-transferrin receptor antibody or antigen binding fragment thereof.

9. (canceled)

10. A biospecific agent according to claim 1, wherein the immunogen sequence used to create the at least one antibody or antigen binding fragment against the transferrin receptor comprises or consists of SEQ ID No:4 or a variant or fragment thereof.

11. A biospecific agent according to claim 1, wherein the biospecific agent comprises a plurality of antibodies or antigen binding fragments thereof with immunospecificity for a transferrin receptor, and a plurality of antibodies or antigen binding fragments thereof with immunospecificity for an amyloidogenic peptide.

12.-13. (canceled)

14. A biospecific agent according to claim 1, wherein the at least one antibody or antigen binding fragment thereof comprises an Fab fragment which is immunospecific for a transferrin receptor.

15. A biospecific agent according to claim 1, wherein the at least one antibody or antigen binding fragment thereof comprises an Fab fragment which is immunospecific for an amyloidogenic peptide, and wherein the Fab fragment binds specifically to oligomers and fibrils of amyloidogenic peptide, but not to amyloidogenic peptide plaques or monomers.

16. (canceled)

17. A biospecific agent according to claim 1, wherein the at least one antibody or antigen binding fragment thereof comprises a bispecific F(ab)2 fragment which is immunospecific for an amyloidogenic peptide and a transferrin receptor, and wherein the bispecific F(ab)2 fragment comprises a first Fab fragment exhibiting immunospecificity to a transferrin receptor which is conjugated to a second Fab fragment exhibiting immunospecificity to an amyloidogenic peptide.

18. (canceled)

19. A biospecific agent according to claim 1, wherein the nanoparticle comprises an inner core which is visible under near infrared, and wherein the core comprises cadmium or lead.

20. A biospecific agent according to claim 1, wherein the core comprises a material selected from CdSe, CdTe, CdS, PbS and PbSe.

21. (canceled)

22. A biospecific agent according to claim 1, wherein the nanoparticle comprises a cadmium or zinc shell, which surrounds the core.

23. A biospecific agent according to claim 1, wherein the shell comprises ZnS or CdS.

24. (canceled)

25. A biospecific agent according to claim 1, wherein the nanoparticle comprises a contrast material, which is visible using MRI or CT, wherein the contrast material comprises gadolinium, gold, iodine, boro-sulphate, or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

26.-34. (canceled)

35. An NIR, MRI or CT imaging method comprising the use of the amyloidogenic peptide biospecific agent according to claim 1, optionally wherein the method is for diagnosing a neurodegenerative disorder selected from a group consisting of Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); and Frontotemporal Dementia.

36. (canceled)

37. A kit for diagnosing a subject suffering from a neurodegenerative disorder, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the kit comprising the biospecific agent according to claim 1 configured to detect the concentration of amyloidogenic peptide present in a biological sample from a test subject, wherein presence of peptide in the sample suggests that the subject suffers from neurodegenerative disorder.

38. A method for diagnosing a subject suffering from neurodegenerative disorder, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the method comprising detecting the concentration of amyloidogenic peptide present in a biological sample obtained from a subject, wherein the detection is achieved using the biospecific agent according to claim 1, and wherein presence of antigen in the sample suggests that the subject suffers from neurodegenerative disorder.

39. (canceled)

40. A method of treating, ameliorating, or preventing a neurodegenerative disorder in a subject, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of an amyloidogenic peptide biospecific agent according to claim 1.

41. The method according to claim 40, wherein the neurodegenerative disorder is selected from a group consisting of Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); and Frontotemporal Dementia, and is preferably Alzheimer's disease.

42. (canceled)

43. A pharmaceutical composition comprising a biospecific agent according to claim 1; and optionally a pharmaceutically acceptable vehicle.

44. (canceled)

Description

[0127] 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:

[0128] FIG. 1 is a schematic representation of one embodiment of a nanoparticle or quantum dot according to the invention. The nanoparticle includes a Cadmium selenide core, and a Zinc sulphide shell, which is encapsulated by a Gd-DOTA silica outer shell to which are conjugated bispecific antibodies or antigen-binding fragments thereof with immunospecificity for amyloid beta and the transferrin receptor;

[0129] FIG. 2 shows absorption and emission spectra of Gd-DOTA silica encapsulated CdSe/ZnS nanoparticles conjugated to a bispecific antibody acting as a diagnostic probe;

[0130] FIG. 3 is a barchart showing cell viability following exposure to the nanoparticle of the invention;

[0131] FIG. 4 is a barchart showing neuronal viability following exposure to the nanoparticle;

[0132] FIG. 5 shows the results of surface plasmon resonance employed to determine the affinity of the nanoparticle to transferrin receptors;

[0133] FIG. 6-11 show fluorescent data of the nanoparticle;

[0134] FIG. 12 shows the immunofluorescence staining of C57Bl/Sv129. a) Shows DAPI nuclei stain. b) Shows the localization of insulin. c) amyloid-beta oligomers;

[0135] FIG. 13 shows different immunofluorescence approach was taken; and

[0136] FIG. 14-17 shows the results after amyloid-beta oligomers and the nanoparticle were incubated together for 30+ minutes.

EXAMPLES

Materials and Methods

1) F(ab)2 Fragments

[0137] F(ab)2 fragments were obtained using commercially available kits from Life Technologies:

Monoclonal Antibody (mAB) Anti-A (Oligomer and Fibril Specific) Antibody (IgG1) F(Ab)2 Generation:

[0138] 0.5 mL of the antibody (8 mg/mL) was added to a previously equilibriated immobilised ficin column and incubated (37 C.) for 25 hours. Generated F(ab)2 fragments were purified with NAb Protein A Column and centrifuged (1000g) for 1 minute. Flow-through concentration was determined spectrophotometrically by measuring the absorbance at 280 nm.

Monoclonal Antibody (mAB) Anti-TfR (Transferrin Receptor) Antibody (IgM) F(ab)2 Generation:

[0139] A previously equilibriated immobilised pepsin column was washed with 8 mL IgM F(ab)2 digestion buffer (200 ml, 100 mM sodium acetate, 150 mM NaCl, 0.05% NaN3; pH4.5). The column and 1 mL of antibody (1 mg/mL) were incubated (37 C.) separately for 3 minutes. Antibody was added to column and incubated (37 C.) for 1.5 hours. Generated F(ab)2 fragments were centrifuged in C30 Concentrator and concentration was determined spectrophotometrically by measuring absorbance at 595 nm.

2) Bispecific Antibody Synthesis (Including Fab Generation)

[0140] Antibody synthesis was performed as described by Greg T. Hermanson in Bioconjugate Techniques Second Edition, ISBN: 978-0-12-370501-3.

Fab Generation:

[0141] 1 mL of anti-AB oligomer-specific antibody F(ab)2 (10 mg/mL) was dissolved in 20 mM buffer (sodium phosphate, 0.15M NaCl, 5 mM EDTA, pH7.4). 6 mg of 2-MEA.HCl was added and incubated (37 C.) for 1.5 hours. Excess 2-MEA*HCl was removed by gel-filtration. Protocol was repeated for anti-TfR antibody Fab generation.

[0142] Bispecific Antibody Synthesis:

[0143] Anti-A oligomer-specific antibody Fab(FabA) was added to DTNB (40 mg DTNB, 10 ml 1MTris-HCl, pH7.5) and incubated at room temperature. Equimolar ratios of FabA-DTNB and anti-TfR antibody (FabB) were mixed and incubated (37 C.) for 1.5 hours. Reaction was incubated (4 C.) overnight. Bispecific BsAb fraction was purified with Superdex 200 column equilibriated in PBS.

3) Synthesis of CdSe/ZnS Nanoparticles

[0144] Nanoparticles were synthesised with silica encapsulation based on publications from Yang Xu et aL & B. O. Dabbousi et al. However, the protocol described herein further incorporates gadolinium in the outer shell and allows for carboxyl functionalization to allow antibody fragment conjugation.

CdSe/ZnS Synthesis:

[0145] The preparation of the selenide organometallic precursor (i.e. trioctylphosphine selenide) was achieved by dissolving 0.1 mol of a selenide shot in too ml of trioctylphosphine, thereby resulting in a 1M solution of trioctylphosphine selenide. Dimethylcadmium was used as the other organometallic precursor. The CdSe precursor material (also known as quantum dots) was synthesized via the pyrolysis of dimethylcadmium and trioctylphosphine selenide in the co-ordinating trioctylphosphine oxide solvent. Precursors were injected at 3500 C. and particles/dots were grown at 2900 C. Selective size precipitation was performed with methanol to collect the particles as powders, and then they were redispersed in hexane. 5 g of trioctylphosphine oxide was heated until it reached 1900 C under a vacuum and then it was cooled to 600 C. 0.3 umol of CdSe was dispersed in hexane and transferred into the reaction vessel with the solvent being pumped off.

[0146] Hexamethyldisilathiane and diethylzinc were used as the precursors for zinc and sulphide. The average radius of the CdSe core precursors was determined from TEM, then calculating the appropriate CdSe to ZnS ratio. This was done by considering the ratio of the shell volume to that of the core and assuming a spherical core and shell and taking into account the bulk lattice parameters. Precursors were dissolved in 3 mL trioctylphosphine inside an inert atmospheric glovebox. The precursors were loaded transferred into an addition funnel, attached to a reaction flask with the CdSe cores that were dispersed in trioctylphosphine oxide. The trioctylphosphine was heated under an atmosphere of nitrogen; the precursors were then added dropwise to the reaction mixture for to minutes at a temperature of 1800 C. The mixture was then cooled to 900 C., whilst being left stirring for 3 hours; then 5 mL of butanol was added to inhibit the solidification of the trioctylphosphine oxide upon the cooling period. The nanoparticles were stored in the solution so that their surfaces remained passivated with trioctylphosphine oxide. When recovered, the powder-formed particles were precipitated with methanol and then redispersed in solvents (e.g. hexane, THF etc.).

Chelated Gadolinium (Gd-DOTA) Silica Encapsulation:

[0147] Sodium silicate and mercaptopropyl trimethoxysilane was diluted in deionized water to a final percentage of 0.15% and 0.7%. 0.1 mL of dilute mercaptopropyl trimethoxysilane was added to a 10 mL solution of CdSe/ZnS nanoparticles and then was shaken for 20 minutes. This allows for the linking of the zinc sulphide shell with mercaptopropyl trimethoxysilane through the Zn/thiol bonds to allow for the deposition of the silica coating. 0.2 mL of the previously diluted sodium silicate solution (pH 10) was added, the solution as mixed well and was kept in a dark room at room temperature to allow for the polymerisation of the silica. After 4 hours, the solution was transferred to another vial containing 8 mL ethanol (100%) to allow the growth of a thicker silica coating due to the precipitation of the excessive silicate. The silica encapsulated nanoparticles were then precipitated out. The resulting silica encapsulated nanoparticles were added to 10 umol 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) mono-N-hydroxysuccinimide ester for 24 hours at room temperature. The gadolinium chelation to DOTA was achieved by adding two molar equivalents of the gadolinium precursor (Gd3+; GdCl3) for 24 hours at room temperature. The Gd-DOTA doped silica encapsulated nanoparticles were collected by centrifugation and washing.

Carboxyl-Functionalization of Gd-DOTA Silica Encapsulated Nanoparticles:

[0148] 40 g of Gd-DOTA silica encapsulated nanoparticles were reacted with 0.05 mmol APTES in a 1:2 deionized water-ethanol mixture (12 mL, 4 mL: 12 mL) for 24 hours in room temperature. After being aminated, to convert the terminal amine groups to carboxyl groups, the Gd-DOTA silica encapsulated nanoparticles were twice washed in ethanol and then redispersed in 20 mL of anhydrous dimethylformamide with the addition of succinic anhydride (0.06 mmol) at room temperature overnight followed by another washing with ethanol twice.

4) Conjugation with EDC

[0149] EDC (1-ethyl-3-(3-dimethylamino) propyl carbodiimide hydrochloride) is a water-soluble carbodiimide crosslinker that activates carboxyl groups for spontaneous reaction with primary amines, enabling peptide immobilisation and hapten-carrier protein conjugation. Conjugation involved covalent bonding of bispecific antibody's amine group to carboxyl groups (as described in Wen-Yen Huang et al.)

Bispecific Antibody Conjugation to Carboxyl-Functionalized, Gd-DOTA Silica Encapsulated Nanoparticles:

[0150] 25 mM carboxyl-functionalized Gd-DOTA silica encapsulated nanoparticles were coupled with the bispecific antibody (1 mg/mL). An EDC (20 mM)/sulfo-NHS (50 Mm) was prepared immediately before use. 250 L EDC/sulfo-NHS was added to the solution of carboxyl-functionalized Gd-DOTA silica encapsulated nanoparticles. The reaction was incubated at room temperature for 10 minutes and 7 L of 2-MEA was added to quench any excess EDC. 25 L of the bispecific antibody solution was added to the activated carboxyl-functionalized Gd-DOTA silica encapsulated nanoparticles. The reaction was incubated room temperature for 60 minutes. Excess reactants and sulfo-NHS were removed by dialysis against Tris (pH 7.4, 50 Mm).

5) Aggreation

[0151] The aggregation protocol of amyloid-beta 1-42 was followed as suggested by the manufacturer (abcam http://www.abcam.com/amyloid-beta-peptide-1-42-human-ab120301.html.

[0152] Before use, and prior to opening the vial, it is recommended that the product equilibrates to room temperature for at least 1 hour. Amyloid (1-42) human peptide should be initially dissolved at a concentration of 1 mg/ml in 100% HFIP (1,1,1,3,3,3-hexafluoro-2-propanol). This solution should be incubated at room temperature for 1 hour, with occasional vortexing at a moderate speed. Next, the solution should be sonicated for 10 minutes in a water bath sonicator. The HFIP/peptide solution should then be dried under a gentle stream of nitrogen gas. 100% DMSO should be used to re-suspend the peptide. This solution should be incubated at room temperature for 12 minutes, with occasional vortexing. The final solution should then be aliquoted into smaller volumes and stored at 80 C. For a working solution, add 500-1000 l of D-PBS (depending on the final concentration to be used) to the peptide stock solution and incubate for 2 h at room temperature to allow for peptide aggregation. The molecular weight of the amyloid-beta species was determined by gel electrophoresis.

6) Surface Plasmon Resonance

[0153] Surface Plasmon resonance was performed using GE Healthcare Biacore, the experimental setup was followed as described by the Biacore Assay Handbook and Biacore Sensor Surface Handbook:

Nanoparticle-Probe Affinity:

[0154] Surface plasmon resonance (Biacore) was used to determine the affinity of the bispecific antibody to various targets. A 0.4M EDC/1M NHS solution was added to dextran matrix at flow rate of 10 l/min for 7 min to activate surface. TfR solution (ligand, 50 g/mL, PBS diluent) was added at a flow rate of 10 l/min for 7 min. 1M ethanolamine-HCl (pH8.5) was added at flow rate of 10 l/min for 7 min to deactivate excess reactive groups. Various concentrations of nanoparticle-probe solutions (analyte, PBS diluent) including duplicate-concentrations were used. Unmodified surface was used for reference analysis. Protocol was repeated using AB oligomers of various sizes as ligand.

7) Direct-Fluorescence Assay

[0155] AB monomers, oligomers, fibrils and plaques (100 pg/ml-800 pg/ml) were blocked in PBS (w/5% BSA) in 384 well plates. The probes were added and incubated at room temperature for 1 hour, then washed with PBS-T. Fluorescence was read with a plate reader at 800 nm (488 nm excitation).

8) Assay Kit

[0156] A standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay kit (MTT Cell Proliferation Assay (ATCC 30-1010K) was used to determine the cytotoxicity of the quantum-dot probe against NIH/3T3 cells.

9) Immunofluorescence

[0157] Double immunofluorescence was performed was performed as described by abcam (http://www.abcam.com/ps/pdf/protocols/double %20immunofluorescence %20-simultaneous %20protocol.pdf).

Immunofluorescence Protocol:

[0158] The coverslips were coated with polyethylineimine at room temperature for 1 hour. The coverslips were rinsed well three times with sterile water for 5 minutes each. The coverslips were allowed to dry completely and were then completely sterilized under UV light for 6 hrs. The C57Bl/Sv129 cells were grown on the glass coverslips and then rinsed briefly in phosphate-buffered saline. The cells were incubated for 30 minutes in PBST (w/1% BSA) to reduce unspecific binding. The conjugated primary antibodies (against amyloid beta 1-42 (oligomers and fibrils) and vimentin), which were stored in the dark to avoid photobleaching, were incubated with PBST overnight at 40 C. The solution was decanted and washed thrice for 5 minutes each in PBS. Cells were also incubated with 0.5 g/ml of DAPI for 1 minute and then rinsed in PBS. Mounting medium was dropped onto the coverslip and the coverslip was sealed by applying nail polish to avoid drying. The sample was stored in the dark at 200 C. Confocal microscopy was used to characterise the results of the immunofluorescence.

Example 1The Nanoparticle

[0159] Referring to FIG. 1, there is shown one embodiment of a nanoparticle 2 according to the invention. The nanoparticle 2 is used to detect neurodegenerative disorders, such as Alzheimer's disease or Huntington's disease, by specifically targeting biomarkers prevalent in each disease. In addition, the nanoparticle 2 can be used to treat each disease by blocking and preventing disease development, as discussed below.

[0160] The nanoparticle 2 (also referred to herein as a quantum dot) consists of an inner core 4 made of cadmium selenide (CdSe), which is coated with a Zinc sulphide (ZnS) shell 6, and which is itself encapsulated with Gadolinium(Gd)-DOTA silica forming an outer shell 8. In other embodiments, the core is composed of CdTe, CdS, PbS, or PbSe etc. (instead of CdSe), the shell can be composed of CdS (instead of ZnS), and gold nanoparticles can be employed instead of Gd. A series of bispecific antibodies to or antigen-binding fragments thereof are conjugated to the Gd-DOTA silica shell 8. Each bispecific antibody 10 consists of a first Fab fragment 12 which is immunospecific for the transferrin receptor, i.e. it is the Fab fragment of IgM anti-transferrin receptor antibody.

[0161] In one embodiment, the first Fab fragment 12 is conjugated via its exposed sulfhydryl groups to a second Fab fragment 14 which is immunospecific for the oligomers and fibrils of amyloid beta protein, i.e. it is the Fab fragment of IgG1 anti-amyloid beta (oligomer and fibril specific) antibody; it is not immunospecific for amyloid plaques. In this first embodiment, the bispecific antibody 10 can be use in diagnosing/treating Alzheimer's disease. However, in a second embodiment, the bispecific antibody 10 can be modified for use in diagnosing/treating other neurodegenerative diseases (Parkinson's, Huntington's etc.) by switching the anti-amyloid beta (oligomer- and fibril-specific) Fab fragment 14 in the bispecific antibody 10 to target and thereby identify other biomarkers, e.g. alpha-synuclein oligomers and fibrils for Parkinson's disease, or Huntingtin for Huntington's disease.

[0162] The immunogen used to create the IgG1 anti-amyloid beta (oligomer and fibril specific) antibody fragment 14 was a partially aggregated recombinant peptide corresponding to human amyloid-beta (1-42) having the amino acid sequence: D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A (SEQ ID No:1). The bispecific antibody 2 has a low affinity to transferrin receptors due to the Fab fragment of IgM anti-transferrin receptor antibody, and can cross the blood-brain barrier via receptor-mediated transcytosis. The bispecific antibody 10 is specific to amyloid-beta oligomers and fibrils, whose activity leading to Alzheimer's can be detected a decade before the first symptoms are prevalent, whilst displaying low cross reactivity with amyloid-beta monomers and plaques. Fibrils are significantly larger than oligomers and are also present during the earlier stages of Alzheimer's. Therefore, it is beneficial to detect fibrils and oligomers as opposed to fibrils alone.

[0163] The nanoparticle 2 is created first by forming the inner cadmium selenide core 4, which is surrounded by the zinc sulphide shell 6. The shell 6 is then encapsulated with the carboxyl-functionalized silica shell 8 which incorporates gadolinium. The nanoparticle 2 is carboxyl-functionalized to allow for protein conjugation with the Fab fragments 12, 14 by reacting with their amine group using covalently bonding. The nanoparticle 2 is capable of emit light in the near infrared (NIR II) region and results in reduced autofluorescence with increase photoluminescence with the ability of non-invasive detection via functional NIR I spectroscopy. Maximum emission wavelength of quantum dots is at 845 nm with the maximum absorption wavelength being at 496 nm.

[0164] In addition, the nanoparticle 2 has MRI detection properties due to the Gd-DOTA silica shell 8.

[0165] The nanoparticle 2 with its conjugated bispecific antibody 10 renders the neurotoxic amyloid-beta oligomers insoluble (immobilised), and therefore the bound oligomers are less readily able to enter cells and their toxicity is significantly reduced.

Example 2Assessment of Absorption and Emission Spectra

[0166] Referring to FIG. 2, there is shown the absorption and emission spectra of the Gd-DOTA silica encapsulated CdSe/ZnS nanoparticle 2 conjugated to a bispecific antibody 10. The nanoparticles 2 display a broad absorption spectra whereas a relatively narrow emission spectra with a distinguishable peak, which is highly characteristic of quantum dots. There was also a large Stokes Shift, which would ultimately decrease fluorescence quenching and increase signal, again characteristic of quantum dots. The emission peak was at 850 nm, with the absorption peak being at 496 nm. The emission peak is in the near infrared (NIR), which can penetrate through biological tissue. Furthermore, the use of quantum dots increases the penetration depth to >2 nm as described by Hong et al.

Example 3Cell Viability Tests

[0167] Referring to FIG. 3 there is shown a barchart showing cell viability following exposure to the nanoparticle 2 of the invention. The nanoparticle 2 displayed very little cytotoxicity to neuronal cells (NIH/3T3), which may be credited to the silica encapsulation 8 and the ZnS shell 6 around the cadmium based core 4, with cell viability after a 48 hour incubation period being >90% in comparison to the control sample.

Example 4Neuronal Viability Tests

[0168] Referring to FIG. 4, there is shown a barchart showing neuronal viability following exposure to the nanoparticle 2. Oligomers and fibrils displayed a significant cytotoxicity to neuronal cells (NIH/3T3), with cell viability being at 25% and 42% respectively in comparison to the control sample. However, the bound oligomers and fibrils displayed a decreasing cytotoxicity, with cell viability with bound oligomers being 71% and bound fibrils being 83%. As such, the nanoparticle 2 displays significant therapeutic potential by decreasing the cytotoxicity of amyloid-beta fibrils and oligomers, the most neurotoxic form of amyloid-beta.

Example 5Affinity of the Nanoparticle to Transferrin Receptors

[0169] FIG. 5 shows the results of surface plasmon resonance employed to determine the affinity of the nanoparticle 2 to transferrin receptors. The nanoparticle 2 had a micromolar Kd (disassociation constant) value of 1.3610.sup.4. This high Kd value is a demonstration of a low affinity to transferrin receptors. Due to the low affinity to transferrin receptors, the nanoparticle 2 can cross the blood-brain barrier via receptor-mediated transcytosis. The anti-transferrin receptor Fab12 in the bispecific antibody 10 was an IgM, and IgMs tend to have a naturally low affinity.

Example 6Fluorescence Experiments

[0170] FIG. 6-11 show fluorescent data of the nanoparticle 2. As can be seen, significant fluorescence was emitted from bound fibrils and oligomers. Fluorescence emitted from fibrils stayed constant. However, as can be seen in FIG. 11, fluorescence emitted from bound oligomeric species was dependent on the size (molecular weight) of the oligomers. FIG. 9 shows that bound oligomers of 57 kDa and above resulted in little fluorescence was emitted from monomers and fibrils, therefore demonstrating that the nanoparticle 2 had little cross-reactivity with other amyloid-beta species, thus reducing the chances of misdiagnosis.

Example 7Detection of Amyloid-Beta Oligomers

[0171] Referring to FIG. 12, there is shown the immunofluorescence staining of C57Bl/Sv29. FIG. 12(a) shows DAPI nuclei stain, FIG. 12(b) shows the localization of insulin, and FIG. 12(c) amyloid-beta oligomers. Success of the immunofluorescence demonstrates that the nanoparticle 2 can successfully target intracellular amyloid-beta oligomers.

[0172] FIG. 13 shows different immunofluorescence approach was taken. The nanoparticle was added to the media with the amyloid-beta oligomers and incubated for 15 minutes beforehand. In comparison to FIG. 12, there is a significant decrease in intracellular levels of amyloid-beta oligomers as fewer oligomers were able to enter the cells from the media. This demonstrates that the nanoparticle can hinder the entry of the neurotoxic protein into cells.

Example 8Therapeutic Potential of the Nanoparticle

[0173] FIG. 14-17 shows the results after amyloid-beta oligomers and the nanoparticle 2 were incubated together for 30+ minutes. The media containing the bound oligomers was then introduced to the neuroectodermal cells. There was extremely little intracellular amyloid beta as can be seen by the confocal images. With an incubation period of 15 minutes, as shown in FIG. 13, some amyloid-beta oligomers were still able to enter the cells from the media, however after an incubation period of 30+ minutes, very few oligomers were inside the cells. This shows that the nanoparticle 2 has therapeutic potential by binding to the neurotoxic amyloid-beta oligomers and inhibiting them from entering cells.

DISCUSSION

[0174] The data show that the nanoparticle 2 of the invention consisting of a bispecific antibody 10 conjugated to Gd-DOTA silica outer shell 8 displayed a low cytotoxicity whilst exhibiting the ability to target intracellular amyloid-beta species. The nanoparticle 2 displayed low cross-reactivity with amyloid-beta monomers and plaques, whilst displaying a high affinity to amyloid-beta oligomers and fibrils. The nanoparticle 2 also displayed a low affinity to transferrin receptors, a required characteristic for crossing the blood brain barrier via receptor mediated transcytosis. The outer shell 8 was carboxyl functionalized to allow for direct protein conjugation of the antibody Fab fragments. The nanoparticles were also demonstrated to emit light in the NIR, maximally at 850 nm, due to the CdSe/ZnS composition. The Gd-DOTA silica encapsulation (i.e. the outer shell 8) significantly improves the biocompatibility of the nanoparticles 2 and drastically decreased their toxicity, and the Gd allows for potential MRI detection.

[0175] The inventor was surprised to observe the nanoparticle 2 also displayed therapeutic effects as it bound oligomers which were less readily able to enter the cells due to them being rendered insoluble, as displayed by the immunofluorescence.

[0176] An important feature of the nanoparticle 2 is the IgM anti-transferrin receptor antibody Fab fragment 12, as it allows the nanoparticle 2 to cross the blood-brain barrier. The IgG1 anti-amyloid beta (oligomer and fibril specific) antibody Fab fragment 14 used in the synthesis of the bispecific antibody can however be replaced with antibodies detecting other protein oligomers and fibrils diagnostic for other neurodegenerative diseases. For example, for Parkinson's disease, an antibody such as an anti-alpha synuclein (oligomer and fibril specific) antibody may be used to identify the biomarker characteristic of Parkinson's disease.

REFERENCES

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