BIOCOMPATIBLE MATRICES FOR THE TRANSFER OF BIOLOGICAL MOLECULES

Abstract

There is provided a biocompatible material for delivering a biological molecule to target location, the material comprising: a hydrogel matrix material, a divalent cation-phosphate nanoparticle (in particular Calcium Phosphate), and a biological molecule (in particular a nucleic acid) complexed with the nanoparticle; wherein the nanoparticle is embedded within the hydrogel matrix material. The biocompatible material, particularly when in a 3D form, can be used in the treatment of various diseases. A preferred method of embedding the nanoparticles and biological molecules in the matrix is by electrophoretic transfer.

Claims

1. A biocompatible material for delivering a biological molecule to target location, the material comprising: a) a hydrogel matrix material; b) a divalent cation-phosphate nanoparticle; and c) a biological molecule, wherein the nanoparticle is encompassed within the hydrogel matrix material.

2. The biocompatible material according to claim 1, wherein the nanoparticle is complexed with the biological molecule, wherein the biological molecule is a biologically active molecule.

3. (canceled)

4. The biocompatible material according to claim 1, wherein the hydrogel matrix material comprises a material selected from the group consisting of hyaluronic acid, polyethylene glycol, agarose, collagen, alginate, chitosan, poly(lactic) acid, poly(lactic-co-glycolic) acid, fibrin, platelet-rich plasma gel and combinations thereof.

5.-8. (canceled)

9. The biocompatible material according to claim 1, wherein the biological molecule is selected from the group consisting of a therapeutic agent or precursor thereof, a nucleic acid molecule, a polypeptide and a cell.

10. The biocompatible material according to claim 9, wherein the nucleic acid molecule is a single stranded nucleic acid molecule or a double stranded nucleic acid molecule, wherein the single stranded nucleic acid molecule is selected from the group consisting of a miRNA, an RNA aptamer and a DNA aptamer, and/or wherein the double-stranded nucleic acid molecule is selected from a gene, siRNA, pDNA, a synthetic gene (linear, 5 and 3 end-hairpin ligated expression cassette) and synthetic messenger RNA (mRNA).

11.-13. (canceled)

14. The biocompatible material according to claim 1, wherein the biological molecule is a nucleic acid molecule encoding a polypeptide, or wherein the nucleic acid molecule is a plasmid or vector encoding a plurality of polypeptides.

15. (canceled)

16. The biocompatible material according to claim 1, wherein the polypeptide or plurality of polypeptides is selected from a growth factor, a cytokine, an antibody, an antibody fragment and an extracellular matrix protein, and further wherein, if the polypeptide is a growth factor, it is selected from the group consisting of basic fibroblast growth factor (bFGF, or FGF-2), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), heparin binding growth factor (HBGF), fibroblast growth factor (FGF), vascular endothelium growth factor (VEGF), transforming growth factor, (e.g. TGF-, TGF-, and bone morphogenic proteins such as BMP-2, -3, -4, -6, -7), Wnts, hedgehogs (including sonic, indian and desert hedgehogs), noggin, activins, inhibins, insulin-like growth factor (such as IGF-I and IGF-II), growth and differentiation factors 5, 6, or 7 (GDF 5, 6, 7), leukemia inhibitory factor (LIF/HILDA/DIA), Wnt proteins, platelet-derived growth factors (PDGF), bone sialoprotein (BSP), osteopontin (OPN), CD-RAP/MIA, SDF-1(alpha), HGF and parathyroid hormone related polypeptide (PTHrP).

17-19. (canceled)

20. The biocompatible material according to claim 1, wherein the biological molecule is a cell, and wherein the cell is selected from the group consisting of a neural cell (e.g. a neuron, a oligodendrocytes, a glial cell, an astrocyte), a lung cell, a cell of the eye (e.g. a retinal cell, a retinal pigment epithelial cell, a corneal cell), an epithelial cell, a muscle cell, a bone cell (e.g. a bone marrow stem cell, an osteoblast, an osteoclast or an osteocyte), an endothelial cell, a hepatic cell and a stem cell.

21. The biocompatible material according to claim 1, wherein the divalent cation is selected from Ba.sup.2+, Co.sup.2+, Mg.sup.2+ and Sr.sup.2+.

22. The biocompatible material according to claim 1, wherein the nanoparticle further comprises a branched or linear amine-containing cationic poly-cation, wherein optionally the branched or linear amine-containing cationic poly-cation is poly-ethylene imine (PEI).

23. (canceled)

24. The biocompatible material according to claim 1, which comprises a plurality of divalent cation-phosphate nanoparticles, wherein the plurality of divalent cation-phosphate nanoparticles is dispersed within the hydrogel matrix material, and/or wherein the plurality of divalent cation-phosphate nanoparticles comprises a first set of divalent cation-phosphate nanoparticles having a first predetermined spatial distribution with respect to the hydrogel matrix material and a further set of divalent cation-phosphate nanoparticles having a further pre-determined spatial distribution with respect to the hydrogel matrix material, and/or wherein the first predetermined spatial distribution differs from the further predetermined spatial distribution, and/or wherein the first predetermined spatial distribution and/or the further predetermined spatial distribution each create a concentration gradient of the biological molecule and/or nanoparticle distribution.

25.-27. (canceled)

28. The biocompatible material according to claim 1, wherein the plurality of divalent cation-phosphate nanoparticles comprises a first set of divalent cation-phosphate nanoparticles and a further set of divalent cation-phosphate nanoparticles, wherein the nanoparticles of the first set comprise at least one predetermined characteristic and the nanoparticles of the further set comprise at least one further predetermined Characteristic, and/or wherein the first set of divalent cation-phosphate nanoparticles differs in at least one characteristic from the further set of divalent cation-phosphate nanoparticles, and/or wherein the at least one first characteristic and the at least one further characteristic are independently selected from: a) particle size; b) type of divalent cation; c) type of biological molecule; d) rate of biological molecule release; e) concentration of biological molecule; and f) a combination of (a) to (e).

29.-32. (canceled)

33. The biocompatible material according to claim 1, which comprises a bioactive agent wherein the bioactive agent is a polypeptide selected from the group consisting of an extracellular matrix protein e.g. fibronectin, laminin and/or heparin.

34. A three-dimensional scaffold comprising the biocompatible material according to claim 1, wherein the biocompatible material comprises a plurality of divalent cation-phosphate nanoparticles, wherein the plurality of divalent cation-phosphate nanoparticles comprises a first set of divalent cation-phosphate nanoparticles and a further set of divalent cation-phosphate nanoparticles, further wherein the nanoparticles of the first comprise at least one predetermined characteristic and the nanoparticles of the further set comprise at least one further predetermined characteristic, further wherein the scaffold comprises a first zone and a further zone, said first zone comprising a majority of the first set of divalent cation-phosphate nanoparticles and the second zone comprising a majority of the second set of divalent cation-phosphate nanoparticles, further wherein the first set and the second set differ in at least one predetermined characteristic, further wherein the first zone a first end of the scaffold and the further zone is a further end of the scaffold, further wherein the further zone is a second zone and the scaffold further comprises a third zone, and further wherein the third zone is provided between the first zone and the second zone.

35.-38. (canceled)

39. The three-dimensional scaffold according to claim 1, wherein the three-dimensional scaffold comprises: (i) a first set of divalent cation-phosphate nanoparticles which are associated with a biological molecule which is chondrogenic, wherein the biological molecule is a polypeptide selected from the group consisting of BMP-6, BMP-7, TGF-3, CD-RAP/MIA and combinations thereof or a nucleic acid encoding a polypeptide selected from BMP-6, BMP-7, TGF-3, CD-RAP/MIA and combinations thereof, and/or (ii) first set of divalent cation-phosphate nanoparticles which are associated with a biological molecule which is osteogenic, wherein the biological molecule is a polypeptide selected from the group consisting of BMP-2 and BMP-7 and combinations thereof, and/or heterodimeric BMP e.g. BMP2/6 or BMP4/7 or a nucleic acid molecule encoding a polypeptide selected from BMP-2 and BMP-7 and combinations thereof and/or heterodimeric BMP e.g. BMP2/6 or BMP4/7.

40.-47. (canceled)

48. A vaccine composition comprising the biocompatible material according to claim 1 or the three-dimensional scaffold according to claim 34, wherein the biological molecule is an immunogenic molecule or an antigen encoding nucleic acid molecule.

49.-50. (canceled)

51. A method of preparing a biocompatible material, the biocompatible material comprising: a) a hydrogel matrix material; b) a divalent cation-phosphate nanoparticle; and c) a biological molecule, wherein the nanoparticle and the biological molecule are encompassed within the hydrogel matrix material, wherein the method comprises: i) providing a hydrogel matrix material disposed between a cathode and an anode; ii) supplying phosphate ions to the hydrogel matrix material; iii) supplying a solution comprising a biological molecule to the hydrogel matrix material; iv) supplying a solution comprising a divalent cation to the hydrogel matrix material; and v) applying an electrical field to the hydrogel matrix material between the cathode and the anode such that a divalent cation-phosphate nanoparticle associated with a biological molecule is formed within the hydrogel matrix material.

52. The method according to claim 51, wherein the phosphate ions are comprised in a buffer solution and step (ii) comprises supplying the buffer solution to the hydrogel matrix material, further wherein the method further comprises step (vi) of supplying a buffer solution to the hydrogel matrix material, and wherein steps (i) to (iv) and (vi) may be performed in any order.

53.-54. (canceled)

55. The method according to claim 51, which comprises: (i) supplying a plurality of solutions comprising a biological molecule, wherein at least a first solution of the plurality of solutions comprises a biological molecule which is a different biological molecule to a biological molecule comprised in a further solution of the plurality of solutions; (ii) supplying the first solution comprising a biological molecule to a first target location in the hydrogel matrix material and wherein the method further comprises supplying the further solution comprising a biological molecule to a further target location within the hydrogel matrix material; and (iii) supplying a plurality of solutions comprising a divalent cation to a first target location in the hydrogel matrix material and wherein the method further comprises supplying the further solution comprising a divalent cation to a further target location within the hydrogel matrix material.

56.-59. (canceled)

60. The method according to claim 51, which comprises: supplying a plurality of solutions comprising a biological molecule, wherein at least a first solution of the plurality of solutions comprises a biological molecule which is a different biological molecule to a biological molecule comprised in a further solution of the plurality of solutions; and supplying a plurality of solutions comprising a divalent cation, wherein at least a first solution of the plurality of solutions comprises a divalent cation which is a different divalent cation to a divalent cation comprised in a further solution of the plurality of solutions, wherein each of the plurality of solutions comprising a biological molecule and each of the plurality of solutions comprising a divalent cation are supplied to a common region of the hydrogel matrix material, and further wherein the method further comprises alternating the polarity of the electric field such that each of the divalent cations and each of the biological molecules move to a common target location in the hydrogel matrix material.

61. The method according to claim 52, wherein the buffer solution in the gel and electrophoresis system is a cell and DNA-compatible buffer solution, and wherein the method is carried out under non-denaturing conditions, further optionally wherein the buffer solution is an on-TRIS containing buffer solution, such as HEPES.

62.-65. (canceled)

66. The method according to claim 51, wherein the method further comprises soaking or coating the hydrogel matrix material with an extracellular matrix molecule for example fibronectin and laminin and other RGD-sequence containing peptides to enhance cellular attachment.

67. The method of claim 66, wherein the method further comprises: (i) lyophilising the hydrogel matrix material to form the biocompatible material; (ii) drying the hydrogel matrix material under supercritical drying conditions to form the biocompatible material, wherein the biocompatible material is an aerogel; or (iii) melting the hydrogel matrix material to form an injectable biocompatible material, wherein the biocompatible material forms a hydrogel after implantation.

68.-69. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0144] Certain embodiments of the present invention are described in more detail below, by way of example only, and with reference to the accompanying drawings in which:

[0145] FIG. 1: SEM back-scatter images of lyophilised agarose GAMs and calcium phosphate nanoparticles at a Ca:P ratio of 166.67x. Scale bars represent 20 m (left) and 10 m (right);

[0146] FIG. 2: SEM back-scatter images of aerogel agarose GAMs and calcium phosphate nanoparticles at a Ca:P ratio of 166.67x. Scale bars represent 50 m (left) and 10 m (right).

[0147] FIG. 3: Overlay image of calcium phosphate (light blue) and ethidium-bromide stained plasmid DNA (magenta, loaded for 5 min at 60V) in gels after complexation using different ratios of Ca:P (Ca2+ loaded using 60V and reversed polarity). The extent of co-localisation/co-precipitation is observable in dark blue colour in the overlay image;

[0148] FIG. 4: Migration of 10 g of bovine plasma fibronectin in native agarose gel electrophoresis at 60 Volts for different electrophoresis durations (Coomassie staining);

[0149] FIG. 5: Fluorescent microscopy images of GFP-positive cells transfected by agarose-GAMs without fibronectin (METHOD1) at a calcium to phosphate ratio of 120.37-fold, 1 week post seeding. Scale bars represent 60.8 m (left) and 105 m (right);

[0150] FIG. 6: Metridia luciferase activity of supernatant samples taken from cultures containing lyophilised agarose GAMs using different calcium:phosphate complexation ratios (0, 83.33-fold, 120.37-fold, 157.41-fold, 166.67-fold) taken at 48 hours (A); 1 week (B) and 4 weeks (C) post seeding, comparing samples without (left section of graphs) or with (right section of graphs) the addition of bovine fibronectin. *p<0.5, **p<0.01;

[0151] FIG. 7: Alkaline phosphatase activity in 02012 cells after incubation with recombinant BMP-containing agarose matrices and control matrices using a Ca:P ratio of 166.67-fold. **p0.01 for statistical significance; and

[0152] FIG. 8:

[0153] Representative bioluminescence image of GAM-induced luciferase expression in vivo with quadrants used for quantification of individual implants (4 per animal) outlined (A). (B) Quantification of CBR luciferase activity in different calcium-phosphate containing groups (FN: fibronectin, CaP: calcium phosphate). (C) Comparison of gene transfer efficacy (CBR luciferase activity) of calcium-phosphate (CaP) containing GAMs with magnesium-phosphate (MgP) containing GAMs and GAMs without nanoparticle complexation. *p0.05 (Tukey's multiple comparison test).

[0154] FIG. 9: Confocal laser scanning microscopy image of multiple pDNA gradient within hydrogels. pDNA1, 2, 3 were labelled with cyanine dimer dyes and imaged after sequential loading (pDNA1, 2, 3 in sequence; 5 min loading each, total electrophoresis time indicated below individual images). (A) YOYO1 stained pDNA1, (B) POPO3 stained pDNA2, (C) TOTO3 stained pDNA3; Composite image of all 3 channels (D). Scale bar represents 600 m.

EXAMPLES

Example 1

[0155] Production of Agarose Gene-Activated Matrices and Gene Delivery In Vitro

[0156] In order to demonstrate that electrophoretically-loaded agarose gene-activated matrices (GAMs) can indeed deliver nucleic acids and to investigate the potential beneficial effect of calcium phosphate nanoparticles on gene delivery by the matrix, agarose was loaded with plasmid DNAs (pDNA) encoding luciferase (Metridia luciferase) and green fluorescent protein (GFP) reporter genes using electrophoresis and subsequently agarose-embedded pDNA was complexed with different ratios of calcium (Ca.sup.2+):phosphate (HPO.sub.4.sup.2) ions in order to generate calcium phosphate/DNA co-precipitates during electrophoresis.

[0157] 1.1 Material and Methods

[0158] 1.1.1 Matrix Preparation:

[0159] METHOD 1: 1% (weight/volume-percent, w/v) agarose matrices (NuSieve 3:1 Agarose, Lonza) were prepared using HEPES buffer (25 mM, 70 mM NaCl, pH 7.05) containing 0.75 mM Na.sub.2HPO.sub.4 and left to solidify at room temperature. Subsequently, solidified agarose gels were submerged in HEPES buffer (same as above) and 2.5 g each of Metridia luciferase encoding pDNA (pMetLuc Reporter, Clontech) and green fluorescent protein encoding pDNA (pGFPmax, Amaxa) were loaded to gel slots and electrophoresis was performed for 5 minutes at 60 Volts (constant voltage, variable amperage setting). After DNA loading, different amounts of 500 mM CaCl.sub.2) solution were loaded though the same slots in order to generate a range of different theoretical complexation ratios of buffer phosphate amount (constant 0.75 mM buffer) and Ca.sup.2+ amounts, ranging from 2.25 mol; 3.25 mol, 4.25 mol up to 4.5 mol (resulting in Ca.sup.2+:HPO.sub.4.sup.2 ratios of 83.3-fold, 120.37-fold, 157.41-fold and 166.67-fold). Complexation was performed using reverse polarity for 5 minutes at 60 Volts. After complexation DNA/Calcium phosphate bands were excised using a scalpel and individual agarose scaffolds were frozen at 86 C. and then lyophilised overnight at 0.0010 millibars (Christ Alpha 2-4 LD.sub.Plus lyophiliser). Control samples containing only DNA were obtained in the same way but excised directly after the first loading step and lypohilised as described above for complexed samples. All samples were sterilised by incubation in 70% ethanol for 24 h and lyophilised again to remove ethanol. Given that agarose electrophoresis was carried out without the use of a DNA dye, successful band excision was confirmed by post-staining the remaining gel using 0.5 g/ml Ethidium Bromide containing electrophoresis buffer for staining for 15 min at room temperature and confirming the lack of remaining pDNA at the excision sites.

[0160] METHOD 2: Additionally, agarose matrices containing DNA and bovine plasma fibronectin (Gibco) were prepared by loading 2.5 g Metridia encoding plasmid DNA (pMetLuc Reporter) and 2.5 g green fluorescent protein encoding plasmid DNA (pGFPmax, Amaxa) simultaneously with 10 g fibronectin under non-denaturing conditions. As fibronectin has been shown to be negatively charged under native conditions in agarose electrophoresis using the same conditions as above (see FIG. 4), it was anticipated that fibronectin would co-migrate with the pDNA under the chosen conditions. DNA/fibronectin loading was carried out for 5 min at 60 Volts under the same conditions as standard samples without fibronectin. Complexation with calcium phosphate was performed in parallel to the protocol used for samples without fibronectin used above. After complexation DNA/Calcium phosphate bands and control samples containing only pDNA were excised using a scalpel and individual agarose scaffolds were frozen at 86 C. and lyophilized overnight. Successful excision of DNA/fibronectin containing gel pieces was confirmed as performed previously (see above).

[0161] METHOD 3: For multi-gene distribution imaging purposes, agarose matrices containing multiple different plasmid DNAs were prepared by loading 5 g each of different plasmid DNAs (pMetLuc Reporter, pGFPmax and pCBR) after staining the pDNAs with cyanine dimer dyes (YOYO1, POPO3 and TOTO3 respectively) before loading onto the gels. pDNAs were loaded sequentially (5 min intervals) at 60 Volts under the same conditions as standard samples.

[0162] 1.1.2 Matrix Characterisation In Vitro:

[0163] Scanning Electron Microscopy (SEM)-Characterisation

[0164] For SEM evaluation, agarose matrix samples prepared with calcium:phosphate ratios of 166.67-fold using METHOD1 were either lyophilised to produce lyophilised matrices or supercritical point dried after buffer exchange for acetone using CO.sub.2 to produce aerogels. Samples were sputter coated with gold using an Agar Auto Sputter Coater (approximately 10 nm layer thickness) and then imaged on a Hitachi 53400N scanning electron microscope using dry-stage, back-scattered electron imaging at a beam accelerating voltage of 10 kV, to enable imaging of calcium phosphate precipitates within the matrices (FIG. 1).

[0165] DNA and Calcium Phosphate Co-Precipitation

[0166] Direct observation of DNA/Calcium phosphate precipitation at different complexation-ratios was carried out in parallel using gels prepared with METHOD1 and a wider range of loaded Ca.sup.2+ amounts but additionally post-stained with Ethidium Bromide (0.5 g/ml, 15 min) and imaging of pDNA-localisation was performed after electrophoresis via UV transillumination and calcium phosphate precipitation was imaged using standard VIS imaging (Biorad ChemiDoc MP Imaging System, Image Lab Software). Overlays were produced assigning Ethidium-bromide stained pDNA the red (magenta) and calcium phosphate the blue channel in merged images (FIG. 3).

[0167] Confocal Laser Scanning Microscopy of Multiple pDNA Gradients within Hydrogels

[0168] Hydrogel samples were excised after loading and slices were used for confocal microscopy (CLSM) imaging of the obtained gradients of 3 different pDNAs within the matrices (FIG. 9). DNA bands within gels were detected using CLSM with multi-channel detection at specified wavelengths (YOYO1: 509 nm, POPO3: 574 nm, TOTO3: 660 nm).

[0169] In Vitro Transfection

[0170] The matrices prepared by METHOD1 and METHOD2 for cell culture were preconditioned with 100 l of DMEM for 2 hours prior to seeding. Then 510.sup.4 C2C12 cells were seeded onto the scaffolds in a 96-well plate in 15 l DMEM for 2 hours and subsequently supplemented with 200 l growth medium (DMEM containing 4.5 g/L glucose, 5% fetal bovine serum, 4 mML-glutamine and 1% penicillin/streptomycin) and cultured at 37 C., 5% CO.sub.2, humidified atmosphere in the cell culture incubator for up to 4 weeks. Supernatants containing the secreted luciferase reporter gene were sampled at 48 hours, 1 week and 4 weeks post seeding for gene expression monitoring and where possible microscopic images of GFP fluorescent cells were taken (FIG. 5).

[0171] Metridia luciferase activity was determined using coelenterazine provided as a kit using the manufacturer's instructions (Ready-To-Glow protocol, Clontech) and quantified in a Varioskan Flash plate luminometer using white 96-well plates. Metridia luciferase activity was calculated in fold-activity compared to agarose GAM control matrices containing only DNA without calcium phosphate precipitation (FIG. 6).

[0172] 1.2 Results

[0173] 1.2.1 Matrix Characterisation In Vitro

[0174] SEM-Characterisation

[0175] SEM-imaging of agarose matrices demonstrated the formation of calcium phosphate nanoparticles in pDNA containing gels and the possibility of producing lyophilised gels and aerogels with different surface topologies through different processing routes (FIG. 1).

[0176] DNA and Calcium Phosphate Co-Precipitation

[0177] The complexation study demonstrated that the chosen loading/complexation strategy using pDNA loaded to a phosphate containing gel via electrophoresis and then applying CaCl.sub.2 solution for loading Ca.sup.2+ through the same slots in an electric field of reversed polarity leads to precipitation of calcium phosphate and the co-localisation/co-precipitation of this calcium phosphate with pDNA (FIG. 3). [Ca.sup.2+ ]: [HPO.sub.4.sup.2] ratios 83.33-fold lead to complete immobilisation of the pDNA and co-localisation with the bulk of calcium phosphate precipitate. Very high [Ca.sup.2+]: [HPO.sub.4.sup.2] ratios of 925 lead to an increase in calcium phosphate precipitation in the gel but a marked reduction in co-localisation/co-precipitation of pDNA with the calcium phosphate particles.

[0178] In Vitro Transfection

[0179] The result provided herein demonstrate that it is possible to use the material described herein to deliver biological molecules e.g. nucleic acid molecules and that DNA can be delivered from such systems effectively into cells in vitro as observed by fluorescence microscopy for GFP 1 week post seeding and using detailed quantification of gene delivery efficacies via luciferase measurements. In fact, the complexation of pDNA within the gel with calcium phosphate nanoparticles significantly increased the Metridia luciferase activity-associated gene transfer efficacy 1 week post seeding for both GAM matrix systems with nanoparticles produced by METHOD1 and METHOD2 compared to matrices only containing naked pDNA (FIGS. 4 and 5, from 1.6-fold up to 5.3-fold respectively).

[0180] Furthermore, there was an additional significant enhancement of gene transfer efficacy observed in matrices containing fibronectin (prepared by METHOD2) when compared to matrices at the same calcium:phosphate complexation ratio (prepared by METHOD1) at 4 weeks post seeding (FIG. 5 and FIG. 6C, up 6.13-fold).

[0181] Generally, there was a trend to higher gene delivery efficacies at later timepoints in fibronectin containing matrices, indicating a difference in release/transfection kinetics and beneficial effect of fibronectin on gene delivery in matrices containing nanoparticles. There was however, no beneficial effect observed if fibronectin was added to matrices without calcium phosphate nanoparticles.

[0182] This data clearly demonstrates the capability of the method to produce transfection-capable GAMs and to enhance their transfection efficacy by the additional complexation with calcium phosphate nanoparticles during electrophoresis and demonstrates the beneficial effects of adding fibronectin (compatible with the electrophoretic approach using native electrophoresis of negatively charged fibronectin) to the system.

[0183] Confocal Laser Scanning Microscopy of Multiple pDNA Gradients within Hydrogels

[0184] CLSM showed the establishment of different zones containing different pDNAs within the hydrogel, demonstrating the capability of the developed method to generate matrices with distinct spatial distribution of therapeutic payloads using sequential electrophoretic loading. It was possible to detect each of the 3 different pDNAs within the gels using cyanine dimer labelling and DNA distribution and gradient formation was dependent on the sequence of loading and total loading time for each of the 3 pDNAs (FIG. 9).

Example 2: Production of Agarose Matrices for Recombinant Protein Delivery In Vitro

[0185] The ability of the material described herein to act as a matrix for biologically active recombinant growth factor molecules was investigated. Particularly, it was investigated whether such molecules could also be loaded to agarose matrices, preserving their bioactivity and to use such recombinant growth factor containing matrices for the directed differentiation of target cells in vitro and if the additional formation of calcium phosphate nanoparticles would influence the extend of differentiation of target cells.

[0186] 2.1 Material and Methods

[0187] 2.1.1 Matrix Preparation

[0188] METHOD: Agarose matrices were prepared according to METHOD1 in Example 1 but instead of pDNA, 1 g of recombinant human bone morphogenetic protein 2 (rhBMP2, CHO-derived, PeproTech) was loaded during the first round of electrophoresis (60V, 20 min, standard polarity) after protein loading, samples were either subjected to calcium phosphate particle precipitation (60V, 5 min reversed polarity, [Ca.sup.2]: [HPO.sub.4.sup.2] ratio 166.67-fold) or used without additional nanoparticles. Growth-factor free matrices with or without nanoparticles were used as controls. The matrices were processed as described in Example 1, METHOD1.

[0189] 2.1.2 In Vitro Differentiation Assay

[0190] 24 h post preparation and processing; 510.sup.4 C2C12 cells were seeded onto the scaffolds in a 24-well plate in 200 l DMEM for 2 hours and subsequently supplemented with 1 ml differentiation assay medium (DMEM containing 4.5 g/L glucose, 1% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin) and cultured at 37 C., 5% CO.sub.2, humidified atmosphere in the cell culture incubator for 7 days. On day 7 the matrices were removed and the cell lawn was washed once with 1 phosphate buffered saline (PBS) and then washed once with alkaline-phosphatase (ALP) assay buffer. The cells were lysed with 100 l lysis buffer (ALP-buffer containing 0.25% Triton X-100) on room temperature for 1 h on a plate shaker and then 100 l of ALP-buffer containing 7.4 mg/ml (20 mM) p-Nitrophenyl phosphate (pNPP) was added and the plate was incubated for 20 min in the dark at 37 C. The samples were then transferred to sterile Eppendorf tubes, centrifuged at 13.000 rpm for 2 min and then 100 l of cleared lysate/reaction mix were measured at 405 nm on a plate reader (Varioskan Flash). The obtained optical densities (OD.sub.405) and a standard curve were used to calculate the amount of the released ALP-enzyme reaction product p-Nitrophenol per minute, which gives a direct indication of the extent of osteogenic differentiation induced by rhBMP2 in C2C12 cells.

[0191] 2.2 Results

[0192] 2.2.1 In Vitro Differentiation Assay

[0193] ALP-activity assays demonstrated that it is possible to use the described electrophoretic approach to load bioactive molecules to agarose matrices and that these molecules retain their biological activity even after processing of the gels and thus can be used to deliver growth factors. The recombinant protein rhBMP2 used in this study clearly induced osteogenic differentiation in C2C12 cells after 7 days of exposure to the rhBMP2 containing matrices as observed by significantly elevated ALP-activity. There was no significant increase in ALP activity observable in the growth-factor free controls.

Example 3: Gene Delivery In Vivo Using Agarose Gene-Activated Matrices

[0194] 3.1 Material and Methods

[0195] 3.1.1 GAM Preparation

[0196] GAMs for in vivo implantation were prepared using similar protocols as for in vitro GAMs (see above) but contained an increased amount of pDNA (25 g). The matrices were prepared at a calcium:phosphate ratio of 166.67-fold of loaded Ca2+ to phosphate buffer. Magnesium phosphate containing matrices were also investigated in this study, employing the same complexation ratio and preparation method as described for the calcium-phosphate nanoparticle containing matrices.

[0197] Matrices were loaded using 60V for 5 min for pDNA (for the in vivo studies a red-shifted click beetle luciferase, CBR in the plasmid pCBR Control (Promega) was used) loading and 60V for 5 min reversed polarity for complexation. GAMs were either prepared without addition of fibronectin (METHOD1) or with the addition of 10 g of bovine fibronectin during the pDNA loading step (METHOD2). After complexation DNA/Calcium phosphate or DNA/Magnesium phosphate bands obtained by METHOD1 and METHOD2 were excised using a scalpel and individual agarose scaffolds were frozen at 86 C. and then lyophilised overnight at 0.0010 millibars (Christ Alpha 2-4 LDPlus lyophiliser). All samples were sterilised by incubation in 70% Ethanol for 24 h and lyophilised again to remove ethanol.

[0198] Control samples containing only pDNA were obtained in the same way but excised directly after the first loading step and lypohilised as described above for complexed samples.

[0199] 3.1.2 In Vivo Implantation

[0200] 24 h post preparation the matrices were subcutaneously implanted in the backs of male outbred MF-1 mice (5 weeks, 25-30 g, Charles River) under inhalation anaesthesia (Isoflurane 3% for induction, 1.5% for maintenance, 1 L/min 02) and pockets were closed using resorbable sutures (VICRYL*rapide, polyglactin 910, Ethicon; Johnson & Johnson). 4 samples were implanted per animal (resulting in 4 imaging quadrants) and samples of the different groups (only pDNA, pDNA+fibronectin, pDNA+calcium phosphate, pDNA+calcium phosphate+fibronectin, pDNA+magnesium phosphate) were applied in a randomised, blocked design. Animals received 0.125 mg/kg buprenorphine (Vetergesic, Alstoe Veterinary) for analgesia intraoperatively as subcutaneous injection. Postoperative antibiosis was administered for 1 week using Baytril 0.25 mg/ml (Enrofloxacin, Bayer HealthCare Animal Health Division) in the drinking water provided ad libitum.

[0201] 3.1.3 In Vivo Bioluminescence Imaging

[0202] In vivo CBR-luciferase activity was imaged on a Xenogen IVIS imaging station 2 weeks post implantation. Animals each received a 100 l injection of 5 mg D-luciferin potassium salt (Promega) in physiologic NaCl intraperitoneally prior to imaging and bioluminescence was quantified using the Living Image Software on the imaging station approx. 15 min post injection.

[0203] 3.2 Results

[0204] 3.21. In Vivo Bioluminescence Imaging

[0205] Luciferase imaging 2 weeks post implantation demonstrated luciferase activity for all groups, indicating the potential of agarose to act as a GAM for in vivo gene delivery (FIG. 8 A, B, C). Magnesium-phosphate containing matrices without fibronectin showed a significant enhancement of gene delivery efficacy (FIG. 8C) compared to uncomplexed pCBR pDNA, demonstrating the enhancement of gene delivery in vivo through phosphate salt nanoparticle complexation of the pDNA payloads.

Example 4: Gene Delivery In Vitro Using Magnesium- and Cobalt-Phosphate Nanoparticles

[0206] 4.1 Material and Methods

[0207] 4.1.1. GAM Preparation

[0208] GAMs are prepared using the electrophoretic method adapting above-described protocols for in vitro GAMs (see Example 1, section 1.1.1, above) but divalent calcium-cations are replaced by either magnesium or cobalt ions (provided as magnesium-chloride or cobalt-chloride solutions) in the protocol to lead to the formation of either magnesium-phosphate or cobalt-phosphate precipitates nanoparticles using METHOD1 or METHOD2 (preparation with or without fibronectin) or a modified METHOD1 or METHOD2.

[0209] 4.1.2 In Vitro Transfection

[0210] The matrices prepared by METHOD1 and METHOD2 for cell culture are preconditioned with 100 l of DMEM for 2 hours prior to seeding. Then approximately 510.sup.4 C2C12 cells are seeded onto the scaffolds in a 96-well plate in 15 l DMEM for 2 hours and subsequently supplemented with 200 l growth medium (DMEM containing 4.5 g/L glucose, 5% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin) and cultured at 37 C., 5% CO.sub.2, humidified atmosphere in the cell culture incubator for up to 4 weeks. Supernatants containing the secreted luciferase reporter gene were sampled at 48 hours, 1 week and 4 weeks post seeding for gene expression monitoring and where possible microscopic images of GFP fluorescent cells are taken.

[0211] Metridia luciferase activity is determined using coelenterazine provided as a kit using the manufacturer's instructions (Ready-To-Glow protocol, Clontech) and quantified in a Varioskan Flash plate luminometer using white 96-well plates. Metridia luciferase activity is calculated in fold-activity compared to agarose GAM control matrices containing only DNA without calcium phosphate precipitation.

Example 5: Multi-Gene Delivery In Vitro and In Vivo Using Agarose Gene-Activated Matrices

[0212] 5.1 Material and Methods

[0213] 5.1.1 GAM Preparation

[0214] GAMs for in vivo implantation are prepared using similar protocols as for in vitro GAMs (see above) but containing an increased amount of pDNA (25 g). In order to demonstrate multi-gene delivery capabilities in different areas of the constructs, 2 different luciferase plasmids are employed, a red-shifted luciferase to be encoded in the plasmid pCBR and a green-shifted luciferase to be encoded in the plasmid pCBG99. 25 g of both plasmids are loaded on opposing sides of the matrix, using 2 loading slots at the top and bottom end of the agarose slice using polarity switching and sequential loading. The complexation is carried out at a calcium:phosphate ratio of approximately 166.67-fold of loaded Ca2+ to phosphate buffer for each plasmid, with 60V for 10 min for pDNA1 (pCBR) loading and for 5 min for pDNA2 from the opposing end using reversed polarity. Complexation is carried out at 60V for 5 min for the zone containing pDNA1 and then again using the same parameters but using reversed polarity for complexation in the zone containing pDNA2. GAMs are either prepared without addition of fibronectin (METHOD1) or with the addition of 10 g of bovine fibronectin during the pDNA loading steps (METHOD2). After complexation DNA/Calcium phosphate bands obtainable by METHOD1 and METHOD2 are excised using a scalpel and individual agarose scaffolds frozen at 86 C. and then lyophilised overnight at 0.0010 millibars (Christ Alpha 2-4 LD.sub.Plus lyophiliser). All samples are sterilised by incubation in 70% Ethanol for 24 h and lyophilised again to remove ethanol.

[0215] Control samples containing only pDNA1 and pDNA2 without complexation are obtained in the same way but excised directly after the first loading step and lypohilised as described above for complexed samples. Additional controls containing either only pDNA1 (pCBR) or pDNA2 (pCBG99) as imaging controls are prepared according to the protocol above.

[0216] 5.1.2 In Vitro Evaluation of Dual-Luciferase Activity

[0217] Matrices prepared by METHODS and METHOD2 for cell culture are preconditioned with 100 l of DMEM for 2 hours prior to seeding. Then 510.sup.4 C2C12 cells are seeded onto the scaffolds in a 96-well plate in 15 l DMEM for 2 hours and subsequently supplemented with 200 l growth medium (DMEM containing 4.5 g/L glucose, 5% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin) and cultured at 37 C., 5% CO.sub.2, humidified atmosphere in the cell culture incubator for up to 4 weeks. Luciferase activity is measured at 7 days, 14 days and 4 weeks, 5 min after addition of 1 mM D-luciferin to the wells in a Xenogen IVIS Spectrum imaging system at 37 C. and individual luciferase signals are obtained by spectral unmixing of distinct wavelengths of CBR and CBG99 luciferase.

[0218] 5.1.3 In Vivo Implantation

[0219] 24 h post preparation the matrices are subcutaneously implanted in the backs of male outbred ME-1 mice (5 weeks, 25-30 g, Charles River) under inhalation anaesthesia (Isoflurane 3% for induction, 1.5% for maintenance, 1 L/min O.sub.2) and pockets are closed using resorbable sutures (VICRYL*rapide, polyglactin 910, Ethicon; Johnson & Johnson). 4 samples are implanted per animal (resulting in 4 imaging quadrants) and samples of the 4 groups (only pDNA1+pDNA2, pDNA1+pDNA2+calcium phosphate, pDNA1+pDNA2+calcium phosphate+fibronectin) are applied in a randomised, blocked design. A separate cohort is assigned for the control matrices containing pDNA1+calcium phosphate, pDNA1+calcium phosphate+fibronectin, pDNA2+calcium phosphate, pDNA2+calcium phosphate+fibronectin. Animals receive 0.125 mg/kg buprenorphine (Vetergesic, Alstoe Veterinary) for analgesia intraoperatively as subcutaneous injection. Postoperative antibiosis is administered for 1 week using Baytril 0.25 mg/ml (Enrofloxacin, Bayer HealthCare Animal Health Division) in the drinking water provided ad libitum.

[0220] 5.1.4 In Vivo Bioluminescence Imaging

[0221] In vivo dual-luciferase imaging is imaged on a Xenogen IVIS Spectrum imaging station 2 weeks post implantation. Animals each receive a 100 l injection of 5 mg D-luciferin potassium salt (Promega) in physiologic NaCl intraperitoneally prior to imaging and bioluminescence was quantified using the Living Image Software on the imaging station approx. 15 min post injection. Individual luciferase signals are obtained by spectral unmixing of individual luciferase emission peaks for CBR and CBG luciferase respectively.

Example 6: Delivery of Functional Therapeutic Genes for Bone Formation In Vitro and In Vivo

[0222] 6.1 Material and Methods

[0223] 6.1.1 GAM Preparation

[0224] GAMs for in vivo implantation in functional assays are prepared using similar protocols as for 3.1.1 but containing an osteoinductive bone morphogenetic protein 2 and 7 (BMP2/7) co-expressing plasmid (25 g). The matrices are prepared at a calcium:phosphate ratio of 166.67-fold of loaded Ca2+ to phosphate buffer, with 60V for 5 min for pDNA (for the in vivo studies a red-shifted click beetle luciferase, CBR in the plasmid pCBR Control (Promega) is used), loading and 60V for 5 min reversed polarity for complexation. GAMs are either prepared without addition of fibronectin (METHOD1) or with the addition of 10 g of bovine fibronectin during the pDNA loading step (METHOD2). After complexation DNA/Calcium phosphate bands obtainable by METHOD1 and METHOD2 are excised using a scalpel and individual agarose scaffolds are frozen at 86 C. and then lyophilised overnight at 0.0010 millibars (Christ Alpha 2-4 LD.sub.Plus lyophiliser). All samples are sterilised by incubation in 70% Ethanol for 24 h and lyophilised again to remove ethanol.

[0225] Control samples containing only pDNA are obtained in the same way but excised directly after the first loading step and lypohilised as described above for complexed samples. Additional controls for the osteoconductive background action of calcium-phosphate itself are prepared without the addition of any pDNA and with or without fibronectin in order to be able to appropriately assess the amount of bone formation induced by the therapeutic BMP2/7 plasmid.

[0226] 6.1.2 In Vitro Evaluation of Osteogenic Differentiation

[0227] 24 h post preparation and processing, 510.sup.4 C2C12 cells are seeded onto the scaffolds in a 24-well plate in 200 l DMEM for 2 hours and subsequently supplemented with 1 ml differentiation assay medium (DMEM containing 4.5 g/L glucose, 1% fetal bovine serum, 4 mM L-glutamine and 1% penicillin/streptomycin) and cultured at 37 C., 5% CO.sub.2, humidified atmosphere in the cell culture incubator for 14 days. On day 14 the matrices are removed and the cell lawn washed once with 1 phosphate buffered saline (PBS) and then washed once with alkaline-phosphatase (ALP) assay buffer. The cells are lysed with 100 l lysis buffer (ALP-buffer containing 0.25% Triton X-100) on room temperature for 1 h on a plate shaker and then 100 l of ALP-buffer containing 7.4 mg/ml (20 mM) p-Nitrophenyl phosphate (pNPP) is added and the plate incubated for 20 min in the dark at 37 C. The samples are then transferred to sterile Eppendorf tubes, centrifuged at 13.000 rpm for 2 min and then 100 l of cleared lysate/reaction mix are measured at 405 nm on a plate reader (Varioskan Flash). The obtained optical densities (OD.sub.405) and a standard curve are used to calculate the amount of the released ALP-enzyme reaction product p-Nitrophenol per minute, which gives a direct indication of the extent of osteogenic differentiation induced by rhBMP2 in C2012 cells.

[0228] 6.1.3 In Vivo Implantation

[0229] 24 h post preparation the matrices are intramuscularly implanted in the gastrocnemius muscle in the hindlimbs of male outbred MF-1 mice (5 weeks, 25-30 g, Charles River) under inhalation anaesthesia (Isoflurane 3% for induction, 1.5% for maintenance, 1 L/min O.sub.2) and pockets are closed using resorbable sutures (VICRYL*rapide, polyglactin 910, Ethicon; Johnson & Johnson). 2 samples are implanted per animal and samples of the investigated groups (pDNA alone, pDNA+calcium phosphate, pDNA+calcium phosphate+fibronectin, only calcium phosphate and calcium-phosphate+fibronectin) are applied in a randomised design. Animals receive 0.125 mg/kg buprenorphine (Vetergesic, Alstoe Veterinary) for analgesia intraoperatively as subcutaneous injection. Postoperative antibiosis is administered for 1 week using Baytril 0.25 mg/ml (Enrofloxacin, Bayer HealthCare Animal Health Division) in the drinking water provided ad libitum.

[0230] 6.1.4 CT Analysis of Bone Formation

[0231] 4 weeks post-implantation animals are sacrificed using approved Schedule 1 protocols and hindlimb explants are obtained for in vitro pCT analysis using standard protocols. In order to be able to distinguish pre-formed calcium-phosphate precipitates from endogenously formed bone matrix, a separate, in vitro GAM construct is prepared using only calcium-phosphate at the same concentration as in all other samples to be used as an imaging phantom to define suitable grey-value thresholds. Bone volumes and bone mineral densities are quantified and images rendered using Scanco imaging software.

[0232] 6.1.5 Histological Analysis of Bone Formation

[0233] After CT analysis, explants are additionally investigated using histology to further determine endogenous bone formation using standard protocols. Briefly, ethanol-fixed samples are cut for histological slides and stained for mineralisation using von Kossa staining. A separate set of sections is prepared for immunohistochemistry and stained for osteocalcin in order to define tissue areas with ongoing osteogenic differentiation.

[0234] Throughout the description and claims of this specification, the words comprise and contain and variations of them mean including but not limited to and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0235] Features, integers, characteristics or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and/or steps are mutually exclusive. The invention is not restricted to any details of any foregoing embodiments. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0236] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.