Nanocage

Abstract

The invention provides nanocages, and in particular to protein nanocages, and especially ferritin nanocages. The invention extends to variant ferritin polypeptides and their encoding nucleic acids, mutant ferritin nanocages, and their uses in diagnostics and drug delivery, as well as in phenotypic screens in drug development.

Claims

1. A variant ferritin polypeptide comprising a modified amino acid sequence of a wild-type ferritin polypeptide, the modified sequence being in a dimeric subunit interface or the N-terminus of the polypeptide, wherein the variant is incapable of assembling into a ferritin nanocage unless it is contacted with a nucleating agent.

2-10. (canceled)

11. A polypeptide according to claim 1, wherein the variant ferritin polypeptide comprises a variant human heavy chain ferritin.

12. A polypeptide according to claim 11, wherein the variant human heavy chain ferritin comprises one or more modification in the wild-type polypeptide, wherein one or more hydrophobic residue in the heavy chain dimeric subunit interface of the polypeptide is substituted with a small amino acid residue, thereby rendering the variant incapable of forming heavy chain dimers, and hence higher order nanocages, unless it is contacted with a nucleating agent and wherein the heavy chain dimeric subunit interface comprises or consists of amino acid residues as set out in SEQ ID No: 19, 20, 21, 22 or 29.

13. (canceled)

14. A polypeptide according to claim 11, wherein the variant heavy chain ferritin polypeptide comprises at least one, two, three or four modification in amino acids 29, 36, 81 or 83 of SEQ ID No:16.

15. A polypeptide according to claim 11, wherein the variant heavy chain ferritin polypeptide is formed by modification of amino acid residue L29, L36, I81 and/or L83 of SEQ ID No:16, wherein the modification at amino acid L29 comprises a substitution with an alanine, the modification at amino acid L36 comprises a substitution with an alanine, the modification at amino acid I81 comprises a substitution with an alanine, and/or the modification at amino acid L83 comprises a substitution with an alanine.

16. A polypeptide according to claim 11, wherein the variant human heavy chain ferritin polypeptide is encoded by a nucleic acid (SEQ ID No:30) or comprises an amino acid (SEQ ID No:31) sequence, or fragment of variant thereof.

17-32. (canceled)

33. A polypeptide according to claim 1, wherein the variant ferritin comprises an amino acid sequence configured to bind to an antibody or antigen binding fragment thereof, optionally wherein the antibody or antigen binding fragment thereof binding peptide is disposed at or towards the N-terminus of the variant ferritin polypeptide.

34. A polypeptide according to claim 33, wherein the antibody or antigen binding fragment thereof binding amino acid sequence comprises a Z-domain, optionally wherein the Z domain sequence is coded as a repeat so that two tandem domains are disposed adjacent to one another (i.e. ZZ).

35. A polypeptide according to claim 34, wherein the Z-domain is encoded by the nucleic acid sequence (SEQ ID No:48) or comprises the amino acid sequence (SEQ ID No:49), or fragment or variant thereof.

36. A polypeptide according to claim 33, wherein the variant human heavy chain ferritin is encoded by a nucleic acid (SEQ ID No:50) or comprises an amino acid (SEQ ID No:51) sequence, or fragment or variant thereof.

37. (canceled)

38. A fusion protein comprising wild-type ferritin and one or more peptide selected from a group consisting of: an antibody or antigen binding fragment thereof binding peptide; a fluorophore; a His tag; and a nucleating agent binding peptide, wherein the antibody or antigen binding fragment thereof binding peptide is as defined in claim 35.

39. (canceled)

40. (canceled)

41. (canceled)

42. (canceled)

43. A ferritin nanocage comprising the variant ferritin polypeptide according to claim 1 and a nucleating agent.

44. (canceled)

45. A nanocage according to claim 43, wherein the nucleating agent comprises a nanoparticle having an average diameter of about 1-500 nm, 1-100 nm, 2-50 nm, or 3-10 nm.

46. A nanocage according to claim 43, wherein the nucleating agent is metallic, optionally wherein the nucleating agent is gold, iron, or copper.

47. A nanocage according to claim 43, wherein the ferritin nanocage encapsulates a gold nanoparticle.

48. (canceled)

49. A nanocage according to claim 43, wherein the ferritin nanocage comprises or is functionalised with an antibody or antigen binding fragment thereof, optionally wherein the antibody or antigen binding fragment thereof is immunospecific for endocytic receptors or an IgG antibody.

50. A nanocage according to claim 43, wherein the nucleating agent is bound to a payload molecule which is an active agent, such as a drug molecule.

51. (canceled)

52. (canceled)

53. (canceled)

54. A method of encapsulating a payload molecule, preferably a drug molecule, in a ferritin nanocage, the method comprising contacting the variant ferritin polypeptide according to claim 1 with a nucleating agent conjugated to a payload molecule and allowing the polypeptide or protein to self-assemble into a nanocage, thereby encapsulating the payload molecule.

55. A nanocage according to claim 50, wherein the molecular weight of the payload molecule is 50 Da to 10 kDa.

56-65. (canceled)

Description

[0143] 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:

[0144] FIG. 1 shows the results of size exclusion of Bfr. (A.) SEC trace for Bfr with elution peak at 7.13 ml. (B.) SEC trace for Bfr-AuBP with elution peak at 6.97 ml. The black arrow that intersects the x-axis at 5.79 ml shows the elution point of commercial 24-meric horse spleen ferritin. The dark blue and red lines correspond to the absorbance readings at 280 nm and 420 nm respectively. The light blue and red shading corresponds to 1 standard deviation of the mean absorbance readings at 280 nm (protein) and 420 nm (heme), respectively. Each data set is composed of three biological repeats;

[0145] FIG. 2 shows the results of size exclusion chromatography of Bfr with Au nanoparticle. (A) SEC traces for Bfr with and without GNPs shown in red and blue respectively. (B) SEC traces for Bfr-AuBP with and without GNPs shown in red and blue respectively. Peak 1 is the ferritin monomer or dimer, and peak 2 is the 24-mer nanocage. This demonstrates separation of monomer/dimer from nanocage;

[0146] FIG. 3 shows the results of TEM of Bfr with AuNP. (A) Micrograph of Peak 2 (FIG. 2B) showing eight hybrid nanoparticles one of which is highlighted by a blue arrow. The GNPs appear as black circles. The Bfr-AuBP protein component appears as a light halo around each of the encapsulated AuNPs (black circles). A possible protein aggregate is highlighted with a red arrow. (B) Micrograph showing naked GNPs as a control. (C) Micrograph of Peak 1 (FIG. 2B) showing Bfr-AuBP in the absence of AuNPs;

[0147] FIG. 4 shows dimeric interfaces in light chain ferritin (lFTN) and heavy chain ferritin (hFTN). A.lFTN dimer (PDB ID:2FG8 (asymmetric unit) [156]). B. hFTN dimer (PDB ID: 3AJO (biological assembly 1) [158]). For each dimer, one subunit is shown in orange and the other is shown in blue. C.lFTN dimer highlighting the conserved hydrophobic residues in the dimer interface and the list of mutations. D. hFTN dimer highlighting the conserved hydrophobic residues in the dimer interface. E. conserved motifs at the dimer interface for light chain and heavy chain ferritin (lFTN and hFTN) that contain hydrophobic residues and the mutations associated with these conserved domains;

[0148] FIG. 5 the results of destabilisation of lFTN variants by mutagenesis. HPLC SEC chromatograms of (A.) GFP-lFTN, (B.) GFP-lFTN (L32A F36A L67A F79A), (C.) GFP-lFTN-AuBP and (D.) GFP-lFTN (L32A F36A L67A F79A)-AuBP. In all chromatograms, the 24-mer elutes at approximately 5.3 ml and the monomer elutes at approximately 7.1 ml. Constructs containing a mutated version of the hFTN subunit (lFTN (L32A F36A L67A F79A) are seen to elute with a lower proportion of nanocage (panels B. & D.), although a significant degree of 24-mer cage remains and a number of other bands are seen that do not coincide directly with monomer and may be assembly intermediates (>1 and <24 subunits). The dark green line corresponds to the absorbance readings at 497 nm (GFP absorbance). The light green shading corresponds to 1 standard deviation of the mean absorbance readings at 497 nm. Each dataset is comprised of three biological repeats;

[0149] FIG. 6 shows the results of hFTN variants by mutagenesis. HPLC SEC chromatograms of (A.) GFP-hFTN, (B.) GFP-hFTN (L29A L36A I81A L83A), (C.) GFP-hFTN-AuBP and (D.) GFP-hFTN (L29A L36A I81A L83A)-AuBP. In all chromatograms, the 24-mer elutes at approximately 5.3 ml and the monomer elutes at approximately 7.1 ml. Constructs containing a mutated version of the hFTN subunit (hFTN (L29A L36A I81A L83A) are seen to elute primarily as monomers (panels B. & D.) The dark green line corresponds to the absorbance readings at 497 nm (GFP absorbance). The light green shading corresponds to 1 standard deviation of the mean absorbance readings at 497 nm. Each dataset is comprised of three biological repeats;

[0150] FIG. 7 shows ZZ-GFP fusions of hFTN. HPLC SEC chromatograms of (A.) ZZ-GFP-hFTN, (B.) ZZ-GFP-hFTN (L29A L36A I81A L83A). In all chromatograms, the 24-mer elutes at approximately 5.3 ml and the monomer elutes at approximately 6.9 ml. The ZZ-GFP fusion with wt hFTN is seen to elute primarily as 24-mer (panel A), while the mutated hFTN (L29A L36A I81A L83A) is seen to elute primarily as monomer (panel B) The dark green line corresponds to the absorbance readings at 497 nm (GFP absorbance). The light green shading corresponds to 1 standard deviation of the mean absorbance readings at 497 nm. Each dataset is comprised of three biological repeats;

[0151] FIG. 8 shows behaviour of hFTN. HPLC SEC chromatograms of (A.) ZZ-GFP-hFTN, (B.) ZZ-GFP-hFTN with AuNP. In all chromatograms, the 24-mer elutes at approximately 5.3 ml and the monomer elutes at approximately 6.8 ml. The wt hFTN is seen to elute primarily as 24-mer (panel A). In the presence of AuNP, the AuNP co-elutes with the FTN 24-mer. The dark green line corresponds to the absorbance readings at 497 nm (GFP absorbance) and the dark blue line absorbance at 530 nm (AuNP absorbance). The shading in both instances corresponds to 1 standard deviation of the mean absorbance readings. Each dataset is comprised of three biological repeats;

[0152] FIG. 9 shows reassembly of mutant hFTN. HPLC SEC chromatograms of (A.) ZZ-GFP-hFTN (L29A L36A I81A L83A), (B.) ZZ-GFP-hFTN (L29A L36A I81A L83A) with AuNP. In all chromatograms, the 24-mer elutes at approximately 5.3 ml and the monomer elutes at approximately 6.8 ml. The wt hFTN is seen to elute primarily as 24-mer (panel A). In the presence of AuNP, the AuNP co-elutes with the FTN 24-mer. The dark green line corresponds to the absorbance readings at 497 nm (GFP absorbance) and the dark blue line absorbance at 530 nm (AuNP absorbance). The shading in both instances corresponds to 1 standard deviation of the mean absorbance readings. Each dataset is comprised of three biological repeats;

[0153] FIG. 10 shows the results of TEM analysis of hFTN with AuNP. TEM analysis of hFTN with AuNP. (A) wt ZZ-GFP-hFTN with AuNP, blue arrows indicate clusters with AuNP, red arrows indicate isolated nanocages; (B) mutant ZZ-GFP-hFTN (L29A L36A I81A L83A) with AuNP, blue arrows indicate nanocages with encapsulated AuNP, red arrows indicate isolated nanocage fragments, yellow arrows indicate empty nanocages; (C) mutant ZZ-GFP-hFTN (L29A L36A I81A L83A) without AuNP (D) wt ZZ-GFP-hFTN without AuNP, red arrows indicate nanocages;

[0154] FIG. 11 shows the binding of Doxorubicin to gold nanoparticles. The binding of doxorubicin (Dox) to 5 nm gold nanoparticles was monitored from the fluorescence signal of the Dox. A titration of Dox concentration was measured in PBS either in the presence or absence of 5 nm Au nanoparticles. Fluorescence was measured in a BMG Clariostar plate reader (ex: 482-16; emm: 580-30) and intensity plotted after subtraction of background. Binding of the Dox to the Au causes a significant quenching of the Dox fluorescence;

[0155] FIG. 12 shows the interaction of propidium iodide with Au nanoparticles. The binding of propidium iodide (PI) to 5 nm gold nanoparticles was monitored from the fluorescence signal of the PI. A titration of PI concentration was measured in PBS either in the presence or absence of 5 nm Au nanoparticles. Fluorescence was measured in a Fluoromax-4 (ex: 493 nm; emm: 550-750) and emission scans are plotted after subtraction of background. Binding of the PI to the Au causes complete ablation of the PI fluorescence;

[0156] FIG. 13 shows Dox fluorescence in purified nanocage-Au-Dox complexes. Complexes containing hFTN (L29A L36A I81A L83A), Au nanoparticle and Dox were formed by adding the mutant ferritin protein (0.1 M) to different concentrations of Dox (0.1 M to 10.0 M). After 16 h the nanocages formed were purified by HPLC and scanned for Dox fluorescence in a Fluoromax-4 (ex: 482 nm; emm: 500-600);

[0157] FIG. 14 is mass spectrometry analysis of drug encapsulation. Complexes containing hFTN (L29A L36A I81A L83A), Au nanoparticle and Dox were formed by adding the mutant ferritin protein (0.1 M) to different concentrations of Dox (0.1 M to 10.0 M), Au nanoparticle preparations stabilised with either citrate or PBS (phosphate buffered saline) were used to evaluate if this affected the binding of the drug to the gold. After 16 h the nanocages formed were purified by HPLC and analysed by LC-MS (Agilent 6550), data were quantified using a 20 ppm window for Dox and PI based on a calibrated standard;

[0158] FIG. 15 shows antibody directed cell binding of GFP nanocage. Purified wt ZZ-GFP-hFTN (20 g) was mixed with either anti-NK1.1 antibody (1 g) or anti-EGFR antibody (1 g) in 210 l of PBS. For each assay, 50 l of the nanocage-antibody was mixed with 110.sup.6 cells of either HT29 or MNK1.1 in 100 l. Cells were analysed an a BD Fortessa using the FITC channel (ex 488 nm; emm 530-30 nm) to observe GFP fluorescence. Data show cells only (red histogram, all traces) and those with nanocage alone and no antibody for MNK1.1 cells (A) and HT29 cells (C). Nanocage antibody are shown with MNK1.1 cells (B) and HT29 cells (D);

[0159] FIG. 16 shows the fate of the antibody targeted nanocage. Confocal microscopy showing a z-slice. Purified Au-ZZ-GFP-hFTN (L29A L36A I81A L83A) was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. HT-29 cells were seeded on chamber slides (ibidi) in DMEM medium with 10% FBS overnight for cell attachment. Cells were then treated with the purified nanocage-Au complex (20 l) at 37 C. for different times (panels a-c, 2 h, d-f, 24 h). After the incubation, the cells were washed with cold PBS, fixed in 4% cold Paraformaldehyde, and permeabilized with 0.1% Triton X-100. To visualize lysosomes, the cells were further incubated with an anti-Lamp1 (1:100; Biolegend) for 1 h after blocking by 1% BSA . The cells were then washed three times with PBS and incubated with Cy.sub.3 Goat anti mouse IgG (1:500; Biolegend) for 1 h. Nuclei were stained with DAPI (1 g/mL; Sigma) for 2 min at room temperature and then again washed with PBS; cells were covered with mounting media and coverslip and observed under microscope (Brightfield, DAPI ex 405 nm; emm 420-480 nm: CY3 ex 550 nm; emm 560 nm: Dox ex 488 nm; emm 550-590 nm) Zeiss LSM 510 inverted confocal microscope. Images are shown with GFP signal in green, Lmp1 signal in Red and DAPI in blue;

[0160] FIG. 17 shows delivery of Dox to cells by encapsulated nanocage. Confocal microscopy showing a z-slice. Purified Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 30 nM) was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. HT-29 cells were seeded on chamber slides (ibidi) in DMEM medium with 10% FBS overnight for cell attachment. Cells were then treated with the nanocage-antibody complex (100 l) at 37 C. for different times (panels a-c, 2 h, d-f, 24 h). After the incubation, the cells were washed with cold PBS, fixed in 4% cold Paraformaldehyde, and permeabilized with 0.1% Triton X-100. Nuclei were stained with DAPI (1 g/mL; Sigma) for 2 min at room temperature and then again washed with PBS; cells were covered with mounting media and coverslip and observed under microscope (Brightfield: DAPI ex 405 nm; emm 420-480 nm: Dox ex 488 nm; emm 550-590 nm) Zeiss LSM 510 inverted confocal microscope. Images are shown with Dox signal in red, and DAPI in blue;

[0161] FIG. 18 shows delivery of PI to cells by encapsulated nanocage. Confocal microscopy showing a z-slice. Purified Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 30 nM) was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. HT-29 cells were seeded on chamber slides (ibidi) in DMEM medium with 10% FBS overnight for cell attachment. Cells were then treated with the nanocage-antibody complex (100 l) at 37 C. for different times (panels a-c, 2 h, d-f, 24 h). After the incubation, the cells were washed with cold PBS, fixed in 4% cold Paraformaldehyde, and permeabilized with 0.1% Triton X-100. Nuclei were stained with DAPI (1 g/mL; Sigma) for 2 min at room temperature and then again washed with PBS; cells were covered with mounting media 480 nm: PI ex 535 nm; emm 617 nm) Zeiss LSM 510 inverted confocal microscope. Images are shown with Dox signal in red, and DAPI in blue;

[0162] FIG. 19 shows purified Dox/PI-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 30 nM) was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. HT-29 cells were grown in DMEM medium with 10% FBS overnight. Cells were then treated with the nanocage-antibody complex (100 l) at 37 C. for different 48 h and 72 h. After incubation, the cells were washed 3 with cold PBS. Re-suspended cells were analysed by LC-MS (Agilent 6550), data were quantified using a 20 ppm window for Dox and PI based on a calibrated standard;

[0163] FIG. 20 shows phenotypic assays of drug delivery. a) MTT assay. Purified Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 30 nM), prepared by loading with either 0.1 M or 2.0 M DOX, was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. Cells were cultured on a three 96 well plate (5000 cells/well) Then, cells were incubated with the prepared nanocage-antibody complexes. At the indicated time points (24, 48, 72 hours), cells were washed with PBS and then incubated for 3 h at 37 C. with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) stock (5 mg/mL) diluted in PBS ( 1/10th of culture volume typically 20 L). After incubation, MTT solubilizing solution (1:1 of DMSO and isopropyl alcohol) was added to each well to solubilise the MTT formazan crystals Absorbance was read after shaking for 10 minutes at 37 C in a BMG Clariostar at 590 nm and b) ToPro3 assay: cells and nanocages were prepared as above (using 2.0 M DOX). Prior to assay, cells were mixed with ToPro-3 staining solution (1 M) and incubated for 30 min, washed with PBS and analysed on a BD Fortessa (640 nm ex; 670/14 emission), data was analysed using FlowJo;

[0164] FIG. 21 shows a phenotypic cell killing assay using Vybrant staining and flow cytometry for nanocage delivered Paclitaxel (Pac). Purified Pac-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 30 nM), was prepared by loading with 5.0 M Pac and unincorporated drug removed by Zorbax spin column; this was mixed with anti-EGFR antibody (1 g) in PBS and exposed to 510.sup.5 live cells. A shows the degree of dead cells observed after 24 h and 48 h for the drug loaded nanocage in the absence of antibody, in the presence of antibody and for 5 M free drug. B shows the flow cytometry dot plots for cells only, cells with hFtn only, free drug only and Pac loaded nanocage with antibody; the upper left quadrant shows dead cells and the lower left live cells;

[0165] FIG. 22 shows a phenotypic cell killing assay using Vybrant staining and flow cytometry for nanocage delivered Actinomycin-D (Act-D). Purified Act-D-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 30 nM), was prepared by loading with 5.0 M Act-D and unincorporated drug removed by Zorbax spin column; this was mixed with anti-EGFR antibody (1 g) in PBS and exposed to 510.sup.5 live cells. A shows the degree of dead cells observed after 24 h and 48 h for the drug loaded nanocage in the absence of antibody, in the presence of antibody and for 5 M free drug. B shows the flow cytometry dot plots for cells only, cells with hFtn only, free drug only and Act-D loaded nanocage with antibody; the upper left quadrant shows dead cells and the lower left live cells; and

[0166] FIG. 23 shows mass spectrometry results performed to determine quantitation of Act-D as encapsulated within the hFtn nanocage. (A) A calibration curve was performed for the monomer His-ZZ-hFTN(L29A L36A I81A L83A) based on the QNYHQDSEAAINR peptide. (B) A calibration curve was performed for Actinomycin-D bound to Au nanoparticles. (C) HPLC purified Act-D encapsulated nanocage Act-D-His-ZZ-Au-hFTN (L29A L36A I81A L83A) was then analysed by the same method on the same day. Areas for the peptide and Act-D were determined and based on the calibration curves in A and B there were calculated to be 13.3 Act-D molecules per cage.

EXAMPLES

[0167] It has previously been demonstrated that the thermostable ferritin from Archaeoglobus fulgidus (A. fu) is stable in a dimeric form at low salt and reversibly forms nanocage structures on transition to high salt.sup.15, 16. However, while in the destabilised dimeric state, it could interact with a gold nanoparticle to form a ferritin-encapsulated gold nanoparticle. Other efforts to encapsulate either drugs or metal cores into ferritin rely on the fact that it dissociates into its constituent dimers at low pH and can reform the nanocage on transition back to neutral pH.sup.3, 17, 18. However, this pH change is also partially destructive and it impacts the integrity of the reformed nanocage.sup.18. The concept of an ordered disassembly and reassembly under mild conditions that does not damage the ferritin is therefore an attractive option for the creation of nanocages based on ferritin. So far this has not been achieved with anything other than A. fu ferritin. The inventors, therefore, decided to create nanocages that exhibit ordered disassembly and reassembly without the use of harsh denaturation conditions.

[0168] Materials and Methods

[0169] Protein Expression and Purification

[0170] A plasmid encoding the recombinant protein of interest was transformed into E. coli BL21-DE3. Single colonies were suspended in 85 mL LB media containing chloramphenicol (34 g ml.sup.1)and grown overnight at 37 C. and 220 rpm in a shaker incubator. Starter culture inoculated at 1:100 dilution for 2 hours at 37 C. 220 rpm, 10 mL starter culture in 500 mL LB media containing chloramphenicol (34 g ml.sup.1), using two 2-litre conical flasks. Once an OD600 of reached 0.4-0.5 culture induced with 1 mM IPTG, and protein expressed for 6 hours until OD600 reaches 1.7-2.2. Initially culture harvested into 2500 mL centrifuge tubes (5000 rpm, 4 C., 10 mins) pellets were stored at 80 C.

[0171] Pellet cells were thawed on ice in lysis buffer (1PBS, 50 mM imidazole, 100 mM NaCl, pH 7.2) containing 1 protease inhibitor cocktail tablet (Roche). Resuspended cells were sonicated for 210 mins (amplitude 40%, pulse 2 seconds on 2 seconds off) and then centrifuged (15000 rpm, 4 C., 40 min.). Initial purification conducted with immobilized metal ion affinity chromatography (His-tag), His-tag beads (chelating sepharose fast flow, GE healthcare) charged with NiCl.sub.2 were added to the supernatant on ice and mixed every 10 mins for 1 hour. This mix was made up to 50 mL using lysis buffer and centrifuged (3000 rpm, 4 C., 2 mins). This was repeated 2-3 times with lysis buffer until the discarded supernatant was clear. Beads are loaded onto column, washed twice with 10 mL lysis buffer and eluted with 10 mL elution buffer (1 PBS, 300 mM imidazole, 100 mM NaCl, pH 7.4). Eluted protein was dialysed overnight (100 mM NaCl, 1PBS, pH 7.2). Protein was concentrated to 1-2 mL using Amicon ultra-15 centrifugal filter unit (3000 rpm, 4 C., 30 mins). Further purification was conducted using size exclusion chromatography. GE Akta FPLC system combined using a Superdex 200 gel filtration column at a 0.5 mL/min flow rate (buffer 50 mM TRIS, 200 mM NaCl, pH 7.5). Fractions containing protein were combined and concentrated to 1-2 mL (3000 rpm, 4 C., 1 hour). When used for storage mixed equally (by volume) with 80% glycerol.

[0172] HPLC Size Exclusion Chromatography (SEC)

[0173] Once purified, the quaternary structures of our protein samples were analysed using size exclusion chromatography (SEC) on a high performance liquid chromatography (HPLC) platform (Thermo Surveyor with diode array detector). SEC was conducted on a refrigerated (10 c.) TSK-GEL G3000SWXL column (Tosoh Bioscience LLC, Montgomeryville, Pa.) equilibrated with filtered (0.22 m filter) Buffer A (100 mM NaCl, 50 mM HEPES, pH 7.2). Prior to sample injection, protein samples were dialysed overnight against Buffer A, which was also used as the mobile phase in the SEC experiments. For each experiment, 0.2 mg of protein was loaded onto the column. SEC experiments were run for 45 minutes at a flow rate of 0.3 ml/min. A diode array was used to measure the absorbance properties of protein sample as it eluted from the column. Specifically, we combined high frequency (10 Hz) monitoring at three wavelengths (=280 nm, 497 nm, 530 nm) with periodic wavescans (230-700 nm). The column was calibrated using a series of standard commercial proteins, which enabled us to subsequently estimate the molecular masses of our samples. The concentration of protein samples was calculated using absorbance spectroscopy with an extinction coefficient of 15,930 cm.sup.1 M.sup.1 at 280 nm for human light chain ferritin and 18,910 cm.sup.1 M.sup.1 at 280 nm for human heavy chain ferritin. Extinction coefficients for other fusion proteins, extinction coefficients were calculated using the ExPASy ProtParam tool. The ratio of Bfr subunits to heme molecules was calculated using an extinction coefficient for heme of 137,000 cm.sup.1 M.sup.1 at 417 nm.

[0174] Nanocage Fabrication and Drug Encapsulation

[0175] The purified ferritin protein was mixed with 5 nm gold nanoparticles (Sigma Aldrich). Stoichiometry was estimated from protein concentration and stated number of gold particles per unit volume, calculated to give 24 protein monomers per gold nanoparticle. Where drugs were encapsulated, these were added to the Au nanoparticles at the concentration indicated at room temperature, ferritin was added between 1 min and 30 min after. Gold nanoparticles and protein were co-incubated for 12 hours at 4 C. If needed concentrated to 1-2 mL (3000 rpm, 4 C.) and then purified using HPLC size exclusion chromatography, as above. Fractions containing nanocage were combined and concentrated to 1-2 mL (3000 rpm, 4 C., 1 hour). When used for storage mixed equally (by volume) with 80% glycerol.

[0176] Concentrations of gold nanoparticle encapsulated ferritin nanocages were calculated based on the sum of the extinction coefficient at 280 nm of 5 nm gold nanoparticles (1.6610.sup.7 M.sup.cm.sup.1) and the extinction coefficient at 280 nm for the relevant protein components also present.

[0177] Transmission Electron Microscopy Analysis

[0178] Protein samples were mounted on carbon coated copper grids. The grids were prepared in advance using glow discharge. This technique increases the hydrophilicy of the grid allowing the protein sample to adhere to the carbon coating. After the protein sample had been loaded onto the grid, a negative stain was applied (uranyl acetate) to provide contrast.

[0179] Fluorescence Analysis

[0180] Fluorescence measurements were performed either on a Jobin Yvon Fluoromax 4 with a 400 l cuvette using excitation and emission wavelengths as stated and slit widths of 5 nm. Alternatively a BMG Clariostar plate reader was used with filters or monochromator settings as described using clear bottom black wall plates. (Greiner Bio-One).

[0181] LCMS

[0182] Purified protein and cell extract samples were analysed by LCMS on an Agilent 6550. LC separation was achieved using a 1290 Infinity system (Agilent, Santa Clara, Calif.) and a Vydac 214MS C4 column, 2.1150 mm and sum particle size, (Grace, Columbia, Md.) at a temperature of 35 C. with a buffer flow rate of 0.2 ml/min. with a denaturing mobile phase: buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in acetonitrile. Elution of components was achieved using a linear gradient from 3% to 40% buffer B over 18.5 min. On-line mass spectra were accumulated on a 6550 quadrupole time-of-flight instrument with a dual electrospray Jet Stream source (Agilent). Mass spectra were acquired of the m/z range of 100-1700 at a rate of 0.6 spectra per second. Targeted MS/MS were acquired over the range of 100-1700 Da with a 1.3 Da precursor isolation window and a collision energy of 15 eV.

[0183] Proteolytic Digestion of Human Ferritin Mutant Monomer and Nanocage

[0184] The protocol was adapted from the manufacturer's instructions. Nanocage was incubated in 8M Urea in 50 mM Tris-HCl (pH 8) with 4 mM DTT and heated at 95 C. for 20 minutes. After denaturation the reaction mixture was cooled and 50 mM NH.sub.4HCO.sub.3 was added such that the urea concentration is below 1M. Modified Trypsin was then added to a final protease:protein ratio of 1:100 and incubated overnight at 37 C. for complete digestion. Human Ferritin mutant monomer samples did not require urea denaturation and were only digested with Trypsin.

[0185] Standard solutions of varying concentrations (0, 0.05, 0.1, 0.2, 0.5, 1, 2 M) were prepared for the drug in buffer, drug on gold nanoparticles and human Ferritin mutant monomer.

[0186] Targeted LC-MS/MS Measurements

[0187] The targeted LC-MS/MS method was applied using an Agilent 1290 LC system coupled to an Agilent 6550 quadrupole-time-of-flight (Q-ToF) mass spectrometer with electrospray ionization (Agilent, Santa Clara, Calif.). The LC column used was an Agilent Zorbax Extend C-18, 2.150 MM and 1.8 um particle size. The LC buffers were 0.1% formic acid in water and 0.1% formic acid in acetonitrile (v/v). In addition to the target molecule, two diagnostic tryptic peptides for the protein to be measured were selected for the targeted LC-MS/MS method. This was achieved by comparison of the peptides identified from the protein by auto-MS/MS analysis of digested samples with those predicted to be suitable for measurement by LC-MS/MS using Peptide Selector software (Agilent, Santa Clara, Calif.). By combining the recorded LC retention times and target precursor masses, a method to determine the concentration of both the target molecule and protein was developed.

[0188] Quantitation was based on the LC retention times of standards and the area of accurately measured diagnostic precursor or fragment ions. The protonated molecules of each peptide, [M+2H].sup.2+, were targeted and subjected to collision induced dissociation, with product ions accumulated throughout the targeted period. Concentrations were calculated using the integrated area of the peak corresponding to the elution of the molecule or peptide of interest at the retention time of the standards. This was measured from either the response for the precursor ion or for a fragment ion from the product ion spectrum of each entity. Calibration curves generated from the standards were used to calculate concentrations.

[0189] Flow Cytometry

[0190] Flow cytometry was performed on a BD Fortessa using the FITC channel to observe GFP (ex 488 nm; emm 530-30 nm; ToPro-3 was imaged in red channel (640 nm ex; 670/14 emission). Data was analysed using Flow-Jo software.

[0191] Cell Preparation for LCMS Analysis

[0192] Cells were lysed using a bead beading process. Cells were pelleted at 7 k rcf for 10 min. and dissolved in 100 l methanol and vortexed until homogenous. 50 l of acid washed glass beads (Sigma) were added. Cells were then vortexed for 30 s and kept on ice for 30 s four times before centrifugation at 14 krpm at 4 C. for 15 min. Supernatant was then taken for LCMS analysis as above.

[0193] Immunofluorescence

[0194] Cells were washed twice with PBS and fixed with 4% formaldehyde for 10 minutes and then washed 3 with PBS. Cells were then permeabilised with 0.1% TX-boo/PBS for 15-20 minutes and wash 3. Cells were then blocked with 5% normal goat serum/PBS or 1% BSA/PBS for 45 minutes (no washing required). The primary antibody was diluted in blocking solution and applied for 2 h (or overnight at 4 C.). Wash 4 thoroughly to remove unbound primary antibody. Cells were then incubatee with the secondary antibody for 1 h, diluted in blocking solution or wash buffer. The secondary antibody was then aspirated and, if required, incubated with DAPI [1 g/mL] in PBS for 10 minutes and washed 4. Coverslip was then dipped into H.sub.2O to remove residual salts of the wash buffer. A drop of mounting medium was added and the slide sealed. Antibodies used were as stated in Figure legends.

MTT Assay

[0195] Purified Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 31 nM) was prepared by loading with either 0.1 M or 2.0 M DOX, was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. Cells were cultured on a three 96 well plate (5000 cells/well) Then, cells were incubated with nanocage constructs to be tested. At the indicated time points (24, 48, 72 hours), cells were washed with PBS and then incubated for 3 h at 37 C. with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) stock (5 mg/mL) diluted in PBS ( 1/10th of culture Volume typically 20 uL). After incubation, MTT solubilizing solution (1:1 of DMSO and isopropyl alcohol) was added to each well to solubilise the MTT formazan crystals Absorbance was read after shaking for 10 minutes at 37 C. plate shaker at testing wavelength of 590 nm.

[0196] ToPro-3 Assay

[0197] Purified Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) (100 l of 31 nM) was prepared by loading with 2.0 M DOX, was mixed with anti-EGFR antibody (1 g) in 210 l of PBS. Cells were cultured on a three 96 well plate (5000 cells/well) Then, cells were incubated with nanocage constructs to be tested. Prior to assay, cells were mixed with ToPro-3 staining solution (1 M) and incubated for 30 min, washed with PBS and analysed on a BD Fortessa (640 nm ex; 670/14 emission), data was analysed using FlowJo.

[0198] Phenotypic Cell Death Assays Using Vybrant Cell Staining Assay

[0199] HT-29 cells were trypsinised and cell viability was assessed using the Trypan blue exclusion assaycount cells treated with Trypan blue dye using a haemocytometer, and determine the volume of cell suspension that contains 510.sup.5 live cells. Live cells (510.sup.5) seeded into a 12-well plate with a final volume of 500 L. This final 500 L volume will consist of 510.sup.5 cells+medium+drug-Au-ZZ-hFTN+anti-EGFR antibody (0.5-1 g). The plate was incubated in a tissue culture incubator set at 37 C., 5% CO.sub.2, 95% humidity for 24 h or 48 h before the experiment was stopped. Uptake of drug-Au-ZZ-hFTN by cells was stopped by removing the 500 L solution containing test or control compounds, trypsinising cells and pelleting cells in preparation for cytotoxicity assays, e.g. Vybrant cell apoptosis assay. Prior to staining the cells were centrifuged (3000 rpm for 2 min) and then washed in cold PBS before a second centrifugation step at 3000 rpm for 2 min in a microcentrifuge. The supernatant was removed and discarded before the cell pellet was resuspended in 1 mL ice cold sterile 1PBS containing YOPRO and PI stain. The staining solution was prepared by adding 0.25 L YOPRO (Component A) and 0.25 L PI (Component B) stock solutions to 1 mL PBS. Volumes were scaled up for the number of samples that require staining, e.g. 10 samples=2.5 L of each stain in 10 mL PBS. The cells were incubated in staining solution on ice for 20 min. Within 30 min after the incubation period, the stained cells were analysed by flow cytometry, using 488 nm excitation with green fluorescence emission for YOPRO R-1 (i.e., 530/30 bandpass) and red fluorescence emission for propidium iodide (i.e., 610/20 bandpass), gating on cells to exclude debris. Single-color stained cells were used to perform standard compensation. The stained cell population will separate into three groups: live cells show a low level of green fluorescence, apoptotic cells show an incrementally higher level of green fluorescence, and dead cells show both red and green fluorescence.

[0200] Microscopy

[0201] The cellular uptake and distribution of HFn nanocage were studied by confocal microscope (Zeiss LSM 510). Briefly, HT-29 cells were seeded on chamber slides (ibidi) in DMEM medium with 10% FBS overnight for cell attachment. Cells were then treated with HFn at 37 C. for different times. After the incubation, the cells were washed with cold PBS, fixed in 4% cold Paraformaldehyde, and permeabilized with 0.1% Triton X-100. To visualize lysosomes, the cells were further incubated with an anti-Lamp1 (1:100; Biolegend) for 1 h after blocking by 1% BSA. The cells were then washed three times with PBS and incubated with Cy.sub.3 Goat anti mouse IgG (1:500; Biolegend) for 1 h. Nuclei were stained with DAPI (1 g/mL; Sigma) for 2 min at room temperature and then again washed with PBS; cells were covered with mounting media and coverslip and observed under microscope (Brightfield, DAPI ex 405 nm; emm 420-480 nm: CY3 ex 550 nm; emm 560 nm: PI ex 435 nm; emm 617 nm).

[0202] Ferritin

[0203] The inventors have used ferritin from different biological sources: bacterioferritin (Bfr) was isolated from E. coli and contains 24 subunits and 12 heme groups that bind between the dimeric protein interface. Human ferritin (FTN) can be composed of the light chain ferritin subunit (lFTN) or heavy chain ferritin subunit (hFTN), or a combination of both. By expressing either lFTN or hFTN in E. coli it is possible to create ferritin nanocages that consist of only a single protein monomer.

[0204] TEM Method

[0205] Protein samples were mounted on carbon coated copper grids. The grids were prepared in advance using glow discharge. This technique increases the hydrophilicity of the grid allowing the protein sample to adhere to the carbon coating. After the protein sample had been loaded onto the grid, a negative stain was applied (uranyl acetate) to provide contrast. After staining, the samples were imaged using transmission electron microscopy (TEM).

Example 1

Bacterioferritin

[0206] To assess the formation of protein nanocages with E. coli bacterioferritin (Bfr), the bfr gene was amplified from the E. coli genome and cloned into an expressing construct. Two variants of the gene were generated, one (SEQ ID No. 5) included an N-terminal His tag for purification, and the second (SEQ ID No. 9) contained a C-terminal gold binding peptide (AuBP). Metal binding peptides have been shown to provide a mechanism for coordinating the binding of proteins to metallic surfaces.sup.19 and it had been shown that the addition of the Au binding peptide could facilitate the encapsulation of a gold nanoparticle within the ferritin cavity.sup.15.

[0207] Surprisingly, the addition of the N-terminal His-tag meant that the Bfr did not purify in its nanocage composition, but as individual monomers (see FIG. 1). After addition of a 5 nm gold nanoparticle (AuNP) and incubation overnight, the protein containing the AuBP had formed a higher order structure consistent with a nanocage being formed around the Au nanoparticle (see FIG. 2). Transmission electron microscopy (TEM) of the purified nanocage complex demonstrated that the nanocage had indeed formed around the AuNP (see FIG. 3).

[0208] The very subtle modification of the Bfr sequence with an N-terminal purification tag appears to have been sufficient to destabilise the nanocage structure of Bfr under normal conditions. The use of a C-terminal AuBP is sufficient to establish AuBP templated assembly of a nanocage without using harsh denaturation conditions.

Example 2

Human Ferritin Subunit Engineering

[0209] Expression and purification of the human heavy and light chain ferritins (hFTN; lFTN) from E. coli produced stable nanocage structures. The addition of an N-terminal His purification tag to either hFTN or lFTN did not destabilise the higher order cage structure. The inventors therefore sought to destabilise the cage structure based on engineering of the protein amino acid sequence. In forming the higher order 24-mer nanocage structure, the ferritin subunits first assemble into dimers via the symmetrical dimer interface (see FIG. 4). Using considerable inventive endeavour, the inventors conducted detailed structural analysis of the dimers, and demonstrated that this is the most stable interface in the nanocage and so would provide a good basis from which to destabilise the tertiary structure.

[0210] 147 structures of conserved ferritin proteins were analysed to identify evolutionarily conserved hydrophobic residues at the dimer interface of human ferritin proteins that contain at least one hydrophobic residue (see Table 1).

TABLE-US-00036 TABLE 1 Conserved domains at the dimer interface containing at least one hydrophobic residue lFTN hFTN lFTN & hFTN RLLKM (SEQ ID No: 23) GRIFL (SEQ ID No: 19) QDIKK (SEQ ID No: 29) LYLQA (SEQ ID No: 24) LELYA (SEQ ID No: 20) TYLSL (SEQ ID No: 25) VYLSM (SEQ ID No: 21) ALFQD (SEQ ID No: 26) IFLQD (SEQ ID No: 22) LGFYF (SEQ ID No: 27) DEWGK (SEQ ID No: 28)

[0211] Hydrophobic residues within these conserved motifs were then carefully selected for site specific mutagenesis (see FIGS. 4C and 4D). Four mutations were created in the heavy [hFTN (L29A L36A I81A L83A)] and light [lFTN (L32A F36A L67A F79A)] chain variants of FTN according to the conserved motifs identified. These were constructed as N-terminal fusions with GFP (green fluorescent protein) to enable visualisation of the nanocage and either with or without a C-terminal AuBP (SEQ ID No. 7).

[0212] For each heavy and light chain variant of FTN, four protein variants were expressed and purified: [0213] (i) wild type FTN with N-terminal GFP; [0214] (ii) wild type FTN with N-terminal GFP and C-terminal AuBP; [0215] (iii) mutant FTN with N-terminal GFP; and [0216] (iv) mutant FTN with N-terminal GFP and C-terminal AuBP.

[0217] The sequences of these variants (DNA and protein) are provided herein. These four proteins were purified and their quaternary structure analysed by HPLC (see FIGS. 5 and 6). It is evident from analysis of these data that the 4 mutations introduced into the dimer interface of hFTN successfully destabilise the quaternary structure and the mutated protein elutes as a monomer by SEC. While the 4 mutations introduced into lFTN destabilise the quaternary structure to some degree, there is still a large proportion of 24-mer nanocage still present.

[0218] Antibody Binding Domain

[0219] As the destabilisation of hFTN worked well, a domain was added to its N-terminus to facilitate its subsequent binding to antibodies. For this purpose the Z-domain was chosen. This is a derivative of Staphylococcus protein A, and is an engineered version of the IgG binding domain of protein A with greater stability and a higher binding affinity for the Fc antibody domain (Nilsson 1987, ref 21). The Z domain was coded as a repeat so that two tandem domains would be present (ZZ). SEC analysis of hFTN with an N-terminal ZZ and GFP demonstrates that the full length protein is still purified as a nanocage, while the mutated hFTN purifies as a monomer (see FIG. 7).

Example 3

Reassembly of Human Ferritin Nanocages

[0220] Having destabilised the FTN nanocage with the various mutations described in Example 2, the inventors wanted to demonstrate if they could reassemble the nanocage in an ordered manner around a metallic nanoparticle (e.g. gold), as they had done previously with Bfr (see FIG. 3Example 1). The ZZ-GFP-FTN fusions for both wild type hFTN and mutant hFTN (L29A L36A 181A L83A) were incubated with approximately stoichiometric amounts of gold nanoparticle (AuNP), and examined by size exclusion chromatography (SEC). SEC separates proteins and complexes based on their size, where smaller molecules have a longer path through the porous column matrix and elute slower, whereas larger molecules elute quicker as they spend more time in the void volume. This can be used to very effectively separate the ferritin monomer from the cage complexes (see FIG. 2). Both the wild type (see FIG. 8) and the mutant hFTN (L29A L36A 181A L83A) (see FIG. 9) demonstrated a higher order complex containing both protein and AuNP, which appeared to suggest that the AuNP was able to form ordered complexes with both wt and mutated protein.

[0221] Further analysis of the AuNP complexes purified by SEC HPLC was performed by transmission electron microscopy (TEM). These data indicate that the wt ZZ-GFP-hFTN protein forms clusters with the AuNPs, but there is no evidence of the AuNP being encapsulated within the hollow space of the ferritin (see FIG. 10A). The wt ZZ-GFP-hFTN alone readily forms isolated nanocage structures (see FIG. 10D). The ZZ-GFP-hFTN (L29A L36A 181A L83A) mutant does not form nanocages in the absence of AuNP (see FIG. 10C), but in the presence of AuNP there is a high proportion of nanocage structures where the AuNP is clearly encapsulated within the central space of the ferritin nanocage (see FIG. 10B).

[0222] These data clearly demonstrate that the L29A L36A 181A L83A mutations introduced at the dimer interface of hFTN are sufficient to destabilise the protein interface so that it does not form the quaternary nanocage structure. The surprising and unpredicted result is that this destabilised protein will template around a AuNP to form nanocage structures that encapsulate the AuNP with a high degree of efficiency. This is particularly surprising because the template occurred without the need to include a gold binding peptide on the interior C-terminus of the FTN, as was previously required for Bfr (see FIGS. 2 and 3).

Example 4

Encapsulation of Drugs into the Nanocages

[0223] In Example 3, the inventors have demonstrated the ordered assembly of the ferritin nanocages around a gold nanoparticle. They have also used this programmed ordered assembly to enable the direct encapsulation of drugs inside the nanocages. Gold nanoparticles have been considered as stand-alone vectors for drug delivery through the formation of covalent drug-Au conjugates.sup.20. Here they sought to exploit a different approach using passive binding of drug molecules to the highly polarisable Au surface and stabilisation through their subsequent encapsulation in the ferritin nanocage. The inventors evaluated the binding of the anti-cancer drug doxorubicin (Dox) to 5 nm Au nanoparticles through its intrinsic fluorescence. Quenching of the fluorescence in the presence of Au nanoparticles demonstrates an interaction between the Dox and the Au (see FIG. 11). In addition, they demonstrated an interaction between propidium iodide (PI) and Au nanoparticles, and in this instance a complete ablation of fluorescence was observed (see FIG. 12).

[0224] Since small molecules can bind to Au nanoparticles, they hypothesised that this would provide a mechanism for the ordered encapsulation of the drugs into protein nanocages, since they have demonstrated that the nanoparticles can form an ordered structure around the Au nanoparticles. The inventors therefore sought to demonstrate that prior binding of small molecules to Au nanoparticles will lead to their encapsulation within a protein nanocage with the nanocage formation being directed by the Au-drug nanoparticle conjugate. To evaluate this, the mutant hFTN (L29A L36A I81A L83A) protein was added to the Au nanoparticles in the presence of different concentrations of Dox or PI. The nanocages that were formed around the Au nanoparticle were then purified by HPLC (as in FIG. 9). The purified Dox-Au-nanocage complex was then evaluated for Dox by measurement of Dox fluorescence. The clear presence of Dox fluorescence indicated that Dox was present in the purified nanocage complexes (see FIG. 13). Encapsulation of PI by fluorescence could not be monitored due to its complete quenching on binding.

[0225] Further analysis of drug encapsulation was evaluated by mass spectrometry (MS). Complexes of drug-Au-nanocage were purified by HPLC prior to analysis by MS to determine if the drug was present in the complex. Data clearly demonstrate that both PI and Dox were present in the nanocage complex and that encapsulation of the drug occurred with both citrate and PBS stabilised Au nanoparticles (see FIG. 14). Together these data demonstrate that passive binding of small molecules to the Au nanoparticles is sufficient to direct their encapsulation into the ferritin nanocages.

Example 5

Targeting of Ferritin Nanocage to Target Cells

[0226] Ferritin fusions containing an N-terminal ZZ domain, in principle, should be able to bind to IgG isotype antibodies since the Z-domain is a synthetic derivative of an IgG binding domain from Staphylococcus aureus protein A. The inventors evaluated the specificity with which they can direct the targeting of the ferritin nanocage to specific cell types by direct antibody interactions. To establish a fluorescent basis for determining cell binding they used the GFP labelled wt ZZ-GFP-hFTN. Two different cell types and antibodies were used to demonstrate the principle of cell-specific targeting, here they chose MNK1.1 (mouse natural killer cells) and HT29 (colorectal cancer) cell lines, which have known antibodies that can either target the NK1.1 receptor in the case of MNK1.1 or the EGFR receptor in the case of HT29. Flow cytometry studies with wt ZZ-GFP-hFTN in the presence or absence of the appropriate targeting antibody demonstrate no discernible background binding of the nanocage in the absence of antibody, whilst a complete shift in the fluorescence of the population was observed in the presence of the antibody (see FIG. 15).

Example 6

Delivery of Drugs to Target Cells

[0227] Having demonstrated that the nanocage can effectively be targeted to specific cells by prior binding to an antibody exhibiting immunospecificity to such cells, the inventors sought to determine that the drug-loaded nanocage complex could deliver a payload of drugs to cells. Nanocages with GFP were created to monitor the delivery and fate of the nanocage in cells, while ferritin without GFP was used to create nanocages with Au-drug encapsulated so that the fate of the drug could be monitored by fluorescence. Au-ZZ-GFP-hFTN (L29A L36A I81A L83A) and Drug-Au-ZZ-hFTN (L29A L36A I81A L83A) complexes were formed as before and purified by HPLC. They were then mixed with anti-EGFR as before and their interaction with HT29 cells was monitored over time.

[0228] The GFP-labelled nanocages were clearly seen to bind to the cells and after 2 h punctate distributions of nanocages could be observed both on the surface and inside the cells (FIG. 16). Cells were also stained with lamps, a late lysosomal marker. The internalised GFP signal after 2 h can clearly be seen to be punctate but not associate with lysosomes, consistent with early stage endocytosis into endosomes (see FIGS. 16a and 16b). After 24 h, the picture clearly changed, with GFP being dispersed throughout the cell cytoplasm and partly associated with lysosomal signal, consistent with it being broken down and dispersed by the pH drop associated with lysosomes (see FIGS. 16d and 16e).

[0229] The ability of drug-loaded nanocage to deliver drug to cells was monitored by following the fluorescence signal of Dox. Purified Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) was incubated with cells and imaged after 2 h and 24 h for Dox fluorescence with combined DAPI staining of nuclei. After 2 h, there is a weak signal of Dox in the cytoplasm, but Dox bound by the Au-nanoparticle will have significantly reduced fluorescence based on our previous characterisation. After 24 h, there is a clear translocation of Dox signal to the nuclei of cells (see FIG. 17). This is consistent with the fate of the nanocage observed in FIG. 16, with dispersal of the nanocage leading to dispersal of the Dox and its translocation to the nucleus.

[0230] Attempts to observe delivery of PI by confocal microscopy did not successfully observe PI (see FIG. 18). The only signal from the PI channel was also observed with the cell only control and is consistent with auto-fluorescence (note that PI is imaged at a different wavelength to Dox).

[0231] Further evaluation of drug delivery was performed by mass spectrometry. Following the dosing procedure used above, cells were washed prior to lysis and drug presence measured by LC-MS (Agilent 6550). Both PI and Dox delivered by the nanocage were present in the lysed cells (see FIG. 19). It was also possible to see the delivery of drugs alone in the control samples, where free drug concentrations were used that were the same as the concentrations used when making the nanocage-drug conjugates (50 M for PI and 2 M for Dox). Cells that were treated with the nanocage alone did not give any signal by mass spectrometry (not shown).

Example 7

Phenotypic Assay of Drug Delivery to Cells

[0232] The inventors have used phenotypic assays to demonstrate the effective delivery of Dox into cells. The MTT assay measures the metabolic activity of cells via NAD(P)H dependent oxidoreductase enzymes using a tetrazolium dye substrate (MTT) that produces a purple colour on reduction. A reduced numbers of viable cells leads to a loss of activity and hence a reduced colour response. Au-ZZ-GFP-hFTN (L29A L36A I81A L83A) and Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) complexes were formed as before and purified by HPLC. In the case of the Dox loaded nanocages, two concentrations of Dox (0.1 M & 0.2 M) were used when forming the complexes. They were then mixed with anti-EGFR as before and their interaction with HT29 cells was monitored over time prior to measuring viability using the MTT assay. The nanocages that were formed with the higher loading of Dox clearly demonstrated a phenotypic response during the time course of the assay (FIG. 20a). The data also demonstrate a dose response to the different nanocage loading conditions used of Dox (0.1 or 2.0 M). A further phenotypic assay was performed using flow cytometry and the Topro3 dye. Topro3 binds to DNA and preferentially enters non-viable cells. As before, HT29 cells were treated with Au-ZZ-GFP-hFTN (L29A L36A I81A L83A) and Dox-Au-ZZ-hFTN (L29A L36A I81A L83A) complexes pre-bound to the anti-EGFR antibody; a control of Dox only was also performed along with cells only (FIG. 20b). In this assay the drug loaded nanocage demonstrates a clear difference in viability at 24 h. The difference with the control cells becomes less pronounced at longer time points, and this may be due to uptake being triggered by the presence of the anti-EGFR antibody. It is also known that at longer time points this dye becomes less specific as a viability signal, although the cell only control has a low response even after 72 h.

Example 8

Using the Nanocage in a Phenotypic Screening Platform

[0233] The inventors have demonstrated the ability to use the ferritin nanocage as a platform technology for the delivery of small molecule drugs into cells. Because the technology provides a defined process for the encapsulation and assembly of the nanocage complex, it can be envisioned as a generic method for the delivery of compounds into cells. The binding of small molecule compounds to the Au nanoparticle will work for a wide variety of ionic, electrostatic and hydrophobic interactions. The assembly of the mutant nanocage around the drug-bound nanoparticle also appears robust. Further, the binding of the nanocage complex to an antibody by interaction of the ZZ domain with IgG isotype antibodies is fast and effective. This can therefore be applied to a very wide range of commercially available antibodies and so can be used to effectively target a wide range of different cell types.

[0234] Because of the ordered process and versatility of nanocage delivery, it is possible to use this as a platform for screening small molecules for in vivo efficacy. In many instances small molecule drugs fail because of poor cell permeability. Furthermore, during drug development conclusions are frequently made regarding efficacy of classes of compounds in phenotypic cell assays but without any knowledge of cell permeability; the drugs may be highly effective if they can be made to cross the cell membrane. Being able to further delineate the mode of failure, non-cell penetration, or poor biological effectiveness, would be valuable in screening campaigns.

[0235] The ferritin nanocage described herein provides a methodology for the effective delivery of compounds into cells in a phenotypic assay and the ordered assembly process is adaptable to high throughput screening scenarios. Furthermore, nanocages that are made fluorescent, either through chemical labelling, or the fusion of fluorescent proteins, can be used to monitor the uptake of individual cells. When combined with cell sorting methods the phenotypic assays could be correlated to a dose response based on the nanocage fluorescence.

Example 9

Nanocages in the Diagnosis and Treatment of Disease

[0236] The ability to target ferritin nanocages to specific cell types via the binding of antibodies creates possibilities for the diagnosis and treatment of disease. Because the nanocages can be made fluorescent, they can be used in imaging methods to identify specific cell types displaying known epitope disease markers. This creates possibilities for their use in the diagnosis of cancer types in imaging accessible locations. Examples of this are cancers accessible via GI-tract, such as oesophageal, stomach, colorectal, liver, pancreatic, gall bladder. In addition, cancers near to the surface of the body would be accessible for diagnosis including skin cancer and neck and throat cancers.

[0237] The ability to encapsulate drugs into the nanocage also provides the possibility of combined diagnostic and therapy (theranostic) approaches. Furthermore, because the drug encapsulated complex contains an Au nanoparticle, a mechanism for the activated release of drugs is also possible. Au nanoparticles absorb light due to their plasmonic effect and laser irradiation is proven to cause localised heating of the nanoparticle proportional to the intensity of the incident laser irradiation (Honda et al). Following targeting of the nanocage, laser induced heating may therefore be used to activate the release of the encapsulated drug, since localised heating will lead to the thermal disassembly of the nanocage complex. This type of approach can make use of current endoscope technology that can both locally deliver compounds, image and treat using laser light sources. The inventors therefore consider that this type of nanocage device would fit with current therapeutic practices and approaches.

Example 10

Measuring Drug Release by Fluorescence Polarisation

[0238] The principle of laser-induced drug release can be demonstrated by examining the fluorescence polarisation of a fluorescently bound molecule within the nanocage, such as Dox. Anisotropy provides an intensity independent measure of the degree of polarisation within a sample. Briefly, when a fluorescent molecule absorbs plane polarised light, it will be emitted in the same plane as the excitation source. However, during the fluorescence lifetime, between absorption and emission, the molecule may rotate. This means that the emitted light will be relative to the new orientation of the molecule. By measuring the emitted light in both vertical and horizontal planes, it is possible to determine the degree of polarisation (anisotropy). Because large molecules rotate slower than small molecules, the degree of anisotropy will be dependent on the size of the molecule. A fluorescent molecule encapsulated in the nanocage will therefore have a very high anisotropy value. If laser irradiation of the Au nanoparticle leads to breakdown of the nanocage and release of a fluorescent compound, this will be imaged by a significant reduction in the measured anisotropy.

Example 11

Delivery of Paclitaxel to Cells by Ferritin Nanocage

[0239] Paclitaxel (Pac) is a natural product, first isolated from the Pacific yew tree. It is commonly used to treat many types of cancer and is known to have many side effects. It prevents cell division by targeting mitotic spindle assembly. An albumin bound formulation (abraxane) has, to a degree, enhanced the efficacy of the drug in cancer treatment, and alleviated some of the toxicity issues associated with the solvent previously used for administration. Abraxane demonstrates, in principle, the advantages that can be obtained for appropriate drug delivery, but it still has significant toxicity issues.

[0240] The inventors have performed experiments to demonstrate the encapsulation and delivery of Pac by the ferritin nanocage of the invention to a colon tumour cancer cell lineHT-29. Pac (5 M) was encapsulated to create Drug-Au-ZZ-hFtn(L29A L36A L81A L83A) nanocages as described above. Excess free drug was removed using a Zorbax spin column prior to addition to cells. Anti-EGFR antibody (0.5 g) was added to the Pac-Au-ZZ-hFtn(L29A L36A L81A L83A) and the antibody bound cage added to cells (30 nM).

[0241] The unloaded Au-ZZ-hFtn(L29A L36A L81A L83A) delivery vehicle was added to HT29 cells as a control to determine cytotoxic effects of hFtn that only contained gold nanoparticles. Free Pac was added to cells at high concentration (5 M) as a drug only control. The phenotypic effect of the delivery of drugs into cells was assessed via Vybrant fluorescent staining using flow cytometry to measure percentages of dead, apoptotic and live cells.

[0242] After 48 h hFtn-Pac (>70% cell death) can be delivered into cells to release a payload of paclitaxel that causes surprisingly more cell death than the free drug alone (9% cell death) (see FIG. 21). These data demonstrate that the hFtn nanocage is highly efficient at encapsulating and delivering Pac into cells in the presence of an appropriate targeting antibody. Pac causes significant cellular toxicity leading to a strong phenotypic response when delivered, while free Pac, which has poor membrane permeability, has very little effect on cells.

Example 12

Delivery of Actinomycin-D to Cells by Ferritin Nanocage

[0243] Actinomycin-D (Act-D) consists of two cyclic peptides linked via a phenoxazone ring. It is an antibiotic that is also used as a chemotherapy medication to treat a number of types of cancer and is on the WHOs list of essential medicines. It has significant side effects.

[0244] The inventors performed experiments to discover if a cyclic peptide of the size and complexity of Act-D could be encapsulated and delivered to cells by the ferritin nanocage of the invention to a colon tumour cancer cell lineHT-29. Act-D (5 M) was encapsulated to create Drug-Au-ZZ-hFtn(L29A L36A L81A L83A) nanocages as before. Excess free drug was removed using a Zorbax spin column prior to addition to cells. Anti-EGFR antibody (0.5 g) was added to the Act-D-Au-ZZ-hFtn(L29A L36A L81A L83A) and the antibody bound cage added to cells (30 nM).

[0245] The unloaded Au-ZZ-hFtn(L29A L36A L81A L83A) delivery vehicle was added to HT29 cells as a control to determine cytotoxic effects of hFtn that only contained gold nanoparticles. Free Act-D was added to cells at high concentration (5 M) as a drug only control. The phenotypic effect of the delivery of drugs into cells was assessed via Vybrant fluorescent staining using flow cytometry to measure percentages of dead, apoptotic and live cells. After 48 h Act-D-Au-ZZ-hFtn(L29A L36A L81A L83A) (10% cell death) can be delivered into cells to release a payload of Act-D. The free drug alone at high concentration causes a similar degree of cell death to what we have observed with the nanocage delivered drug (see FIG. 22). It is thus evident that the free drug has some cell penetrating properties that lead it to enter into the cell and cause death, although, in the described assay, the degree of cell killing was not as high as that reported in the literature for a similar concentration.sup.22. Treatment with 30 nM Act-D loaded nanocage gave a similar response to high concentrations of free drug demonstrating that a similar level of cellular delivery was achieved with substantially lower concentrations. It appears that Act-D is not as potent at inducing cell death as Pac (compare FIGS. 22 and 21).

Example 13

Compound Nanocages

[0246] Compound nanocages composed of different types of subunit were also created by incubating the Au nanoparticle with His-ZZ-hFtn(L29A L36A L81A L83A) and His-GFP-hFtn(L29A L36A L81A L83A). Since the Au nanoparticle acts as the nucleating agent and the hFtn part of the fusion protein is identical, nanocages formed that contain the ZZ domain on some subunits and the GFP domain on others. These compound nanocages behaved as expected in terms of fluorescence and cellular delivery of drugs.

Example 14

Mass Spectrometry Drug Quantification

[0247] To demonstrate that the loading of drug in the nanocage could be performed, mass spectrometry of the purified Act-D-His-ZZ-hFTN(L29A L36A I81A L83A) nanocage was performed. Calibration curves are necessary for the direct quantitation of samples. For the protein component, a peptide fragment was identified that provided a good readout of hFtn(L29A L36A I81A L83A) monomer concentration; standard dilutions were then used to create a standard curve based on the 50 ppm area of the m/z signal for this peptide. Similarly a standard curve for the Act-D was established based on standard dilutions of Act-D bound to Au nanoparticles in case this affected the ability of the Au-nanoparticle to resolve the Act-D signal.

[0248] Both standard curves provided good linear responses to concentration (see FIG. 23 A&B). Based on these calibration curves a purified ZZ-Au-hFTN (L29A L36A I81A L83A) was analysed. The quantitation of signal arising from ZZ-Au-hFTN (L29A L36A I81A L83A) and Act-D was then calculated based on the standard curves. Following correction for monomer to nanocage formation, the amount of Act-D was calculated as 13.3 molecules per nanocage (see FIG. 23C).

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