Therapeutic material

Abstract

A bioactive glass composition for use in treating bone cancer includes 0.5-10 mol % gallium oxide or 1.0-20 mol % gallium nitrate/halide; 25 to 75 mol % silicon dioxide; 10 to 30 mol % calcium oxide and/or strontium oxide; up to 30 mol % sodium oxide; and up to 15 mol % phosphorous pentoxide. It may further comprise magnesium and/or potassium oxide. The bioactive glass composition may be positioned within a patient's bone post-surgery to promote apatite formation and to release gallium ions having a toxic effect on any remaining cancerous cells.

Claims

1. A bioactive glass composition comprising: 4 to 10 mol % gallium oxide (Ga.sub.2O.sub.3); 25 to 75 mol % silicon dioxide; 10 to 30 mol % calcium oxide and/or strontium oxide; 1 to 30 mol % sodium oxide; and 1 to 15 mol % phosphorous pentoxide.

2. A bioactive glass composition according to claim 1 comprising between 39.0 and 40.0 mol% silicon dioxide.

3. A bioactive glass composition according to claim 1 further comprising one or more of the following components: K.sub.2O: from 0 to 15 mol % MgO: from 0 to 15 mol % ZnO: from 0 to 10 mol % CaF.sub.2: from 0 to 15 mol % CaCl.sub.2: from 0 to 15 mol % B.sub.2O.sub.3: from 0 to 10 mol % Ag.sub.2O: from 0 to 10 mol %.

4. A bioactive glass composition according to claim 1 comprising from 35 to 55 mol % silicon dioxide.

5. A bioactive glass composition according to claim 1 comprising from 35.5 to 45 mol % silicon dioxide.

6. A bioactive glass composition according to claim 1 comprising from 27.0 to 30.0 mol % calcium oxide.

7. A bioactive glass composition according to claim 1 comprising from 24.5 to 27.0 mol % sodium oxide.

8. A bioactive glass composition according to claim 1 comprising from 2.7 to 3.0 mol % phosphorous pentoxide.

9. A method of treating bone cancer using a bioactive glass composition according to claim 1.

10. A bioactive glass composition comprising (Ga.sub.2O.sub.3).sub.x(SiO.sub.2).sub.46.1-3X(CaO).sub.26.9(Na.sub.2O).sub.24.4(P.sub.2O.sub.5).sub.2.6 wherein x satisfies 1≤×≤10.

11. A bioactive glass composition according to claim 10, wherein x satisfies 4≤×≤10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention will now be described by way of examples with reference to the accompanying drawings in which:

(2) FIG. 1a shows the X-ray diffraction spectra of Ga-doped bioactive glass compositions;

(3) FIG. 1b shows the X-ray diffraction spectra of Ga-doped bioactive glass compositions after exposure to SBF;

(4) FIG. 2 shows release of gallium ions from Ga-doped bioactive glass compositions;

(5) FIG. 3 shows pH of conditioned media containing Ga-doped bioactive glass compositions;

(6) FIGS. 4A-4F shows fluorescence images of live/dead staining of Saos-2 cells cultured for 72 hours in conditioned media containing Ga-doped bioactive glass compositions;

(7) FIGS. 5A-5F shows fluorescence images of live/dead staining of NHOst-osteoblasts cultured for 72 hours in conditioned media containing Ga-doped bioactive glass compositions;

(8) FIG. 6 shows Saos-2 cell viability following treatment with pH neutralised conditioned media containing Ga-doped bioactive glass compositions over 72 hours; and

(9) FIG. 7 shows NHOst cell viability following treatment with pH neutralised conditioned media containing Ga-doped bioactive glass compositions over 72 hours

(10) FIG. 8 shows FT-IR spectra of Ga-doped bioactive glass compositions following immersion in SBF for 7 days.

(11) FIG. 9 shows (a) Phosphorus and (b) calcium XPS spectra of Ga-doped bioactive glass compositions following immersion in SBF for 7 days.

(12) FIGS. 10 A-H show SEM images of Ga-doped bioactive glasses after immersion in SBF for 1 week. (A, C, E, G) 0, 1, 2, and 3% Ga glass surfaces at lower magnification (500×). (B, D, F, H) 0, 1, 2, and 3% Ga glass surfaces at higher magnification (3000×).

EXPERIMENTAL

(13) Glass Preparation and Analysis

(14) Melt-quench derived 45S5 Bioglass, (SiO.sub.2).sub.46.1(CaO).sub.26.9(Na.sub.2O).sub.24.4(P.sub.2O.sub.5).sub.2.6, and gallium doped analogues were prepared using SiO.sub.2 (Alfa Aesar, 99.5%), CaCO.sub.3 (Alfa Aesar, 99.95-100.5%) and Na.sub.2CO.sub.3 (Sigma-Aldrich, 99.5%), NH.sub.4H.sub.2PO.sub.4 (Sigma-Aldrich, 99.5%), and Ga.sub.2O.sub.3 (Alfa Aesar, 99.99%).

(15) The precursors were weighed in the appropriate molar ratio to give (Ga.sub.2O.sub.3).sub.X(SiO.sub.2).sub.46.1-3X(CaO).sub.26.9(Na.sub.2O).sub.24.4(P.sub.2O.sub.5).sub.2.6 where X=1, 2 and 3% as shown in Table 1 below. Figures in brackets show the resulting mass (in grams) of each component in the bioactive glass composition.

(16) TABLE-US-00001 TABLE 1 Control Ex 1 Ex 2 Ex 3 Ex4 Ex5 Ex 6 45S5 Ga1% Ga2% Ga3% Ga4% Ga5% Ga10% Bioglass ™ glass glass glass glass glass glass SiO.sub.2 46.1 43.9 41.7 39.8 37.6 35.5 25.0 (8.97) (8.33) (7.80) (7.24) (6.70) (4.30) Na.sub.2O 24.4 24.9 25.4 25.9 26.4 26.9 29.4 (5.25) (5.25) (5.25) (5.25) (5.25) (5.25) CaO 26.9 27.5 28.1 28.6 29.1 29.7 32.4 (5.23) (5.23) (5.23) (5.23) (5.23) (5.23) P.sub.2O.sub.5 2.6 2.7 2.7 2.8 2.8 2.9 3.1 (1.28) (1.28) (1.28) (1.28) (1.28) (1.28) Ga.sub.2O.sub.3 0 1 2.1 3 4 5 10 (0.65) (1.30) (1.84) (2.40) (2.95) (5.38)

(17) Precursors were thoroughly mixed and placed into a 90% platinum-10% rhodium crucible. The crucible was placed into a furnace at room temperature and heated at a rate of 10° C./min to 1450° C. and held at this temperature for 90 min. The melt was then poured into a graphite mould which had been preheated to 370° C. and annealed at this temperature overnight before being allowed to cool slowly to room temperature.

(18) Glass discs were cut using an IsoMet™ 1000 Precision Diamond Saw (Buehler). The discs, 10 mm diameter and 2 mm thick, were polished using a MetaServ® (Buehler) polishing machine to a finish of 0.06 μm using colloidal silica. Glass particles were prepared using a planetary ball mill (PM100, Retsch) and the particles were sieved to give a size distribution between 40 to 60 microns.

(19) X-ray diffraction experiments were conducted using a Bruker D8 diffractometer operating at the copper k.sub.α wavelength of 1.54 Å. The powdered glass samples were measured over a two theta range of 10 to 80° in 0.02° steps. Measurements were taken at one second per point and no smoothing was undertaken.

(20) As shown in FIG. 1(a) the glasses of Example 1-3 were completely amorphous with no visible signs of Bragg peaks when examined using X-ray diffraction.

(21) Further Ga-doped bioactive glasses were prepared using SiO.sub.2, CaCO.sub.3, Na.sub.2CO.sub.3, NH.sub.4H.sub.2PO.sub.4, MgCO.sub.3, K.sub.2CO.sub.3 and Ga.sub.2O.sub.3 (and ZnO for Example 13) along with an un-doped control having the formula (SiO.sub.2).sub.54.6(CaO).sub.22.1(Na.sub.2O).sub.6(P.sub.2O.sub.5).sub.1.7(MgO).sub.7.7(K.sub.2O).sub.7.9 as shown in Table 2 below.

(22) TABLE-US-00002 TABLE 2 Control Ex 7 Ex8 Ex 9 Ex 10 Ex 11 Ex 12 Ex 13 13-93 Ga1% Ga2% Ga3% Ga4% Ga5% Ga10% Ga3% glass glass glass glass glass glass glass glass SiO.sub.2 54.6 52.5 50.5 48.5 46.4 44.4 34.1 43.0 Na.sub.2O 6.0 6.1 6.3 6.4 6.6 6.7 7.4 3.5 CaO 22.1 22.6 23.1 23.6 24.1 24.6 27.2 34.9 P.sub.2O.sub.5 1.7 1.7 1.8 1.8 1.9 1.9 2.1 1.1 MgO 7.7 8.1 8.3 8.4 8.6 8.8 9.7 7.8 K.sub.2O 7.9 7.9 8.1 8.2 8.4 8.6 9.5 3.5 Ga.sub.2O.sub.3 0 1 2 3 4 5 10 3 ZnO 0 0 0 0 0 0 0 3.2

(23) Precursors were thoroughly mixed and placed into a 90% platinum-10% rhodium crucible. The crucible was placed into a furnace at room temperature and heated at a rate of 10° C./min to 1450° C. and held at this temperature for 90 min. The melt was then poured into a graphite mould which had been preheated to 370° C. and annealed at this temperature overnight before being allowed to cool slowly to room temperature.

(24) Glass discs were cut using an IsoMet™ 1000 Precision Diamond Saw (Buehler). The discs, 10 mm diameter and 2 mm thick, were polished using a MetaServ® (Buehler) polishing machine to a finish of 0.06 μm using colloidal silica. Glass particles were prepared using a planetary ball mill (PM100, Retsch) and the particles were sieved to give a size distribution between 40 to 60 microns.

(25) Apatite Formation

(26) A simulated body fluid (SBF) solution was prepared using the method outlined by Saravanapavan and Hench in Journal of Biomedical Materials Research 54, 608-618. SBF emulates the salt ion concentrations found in human blood plasma and can be used to test for the formation of hydroxyapatite in vitro.

(27) The three gallium containing glasses of Examples 1-3 containing 1%, 2% and 3% together with the control glass (45S5) were placed in separate containers and 40 ml of SBF salt ion solution was added. The samples were sealed and maintained at 37° C. for 7 days. After reacting in SBF the samples were removed and rinsed with distilled water and acetone to remove any residual salts and halt reactions. Samples were dried at 60° C. and then assessed for apatite formation using X-ray diffraction.

(28) A visible layer of hydroxyapatite was observed to have formed on the glass surface. X-ray diffraction confirmed the surface layer was an amorphous calcium phosphate/poorly defined hydroxyapatite (FIG. 1b) which is consistent with results typically observed for bioactive glasses. No reduction in apatite formation was observed for the gallium containing glasses compared to the 45S5 control.

(29) Fourier Transform Infrared Spectroscopy. (FT-IR) spectra were recorded using a Thermo Nicolet IS50 infrared spectrometer fitted with a single bounce diamond ATR crystal. Spectra were recorded from 400 to 4000 cm-1 with a step size of 0.05 cm-1. All measurements were undertaken at room temperature and a total of 64 scans were recorded per sample.

(30) FTIR spectra for the gallium doped bioglasses, after immersion in SBF for 1 week, are shown in FIG. 8 together with a spectra of pure hydroxyapatite for reference. Each of the spectra exhibit the characteristic hydroxyapatite bands at 560, 600, and 1018 cm.sup.−1.

(31) X-ray Photoelectron Spectroscopy. (XPS) measurements were performed using a Kratos axis HSi XP spectrometer fitted with a charge neutralizer and Mg kα anode (1253.6 eV) and a base pressure of 5×10-9 Torr. Samples were loaded via adhesion onto carbon tape and spectra were calibrated to adventitious carbon (284.8 eV). Spectra were fit using CASA v2.3.15 using Gaussian-Lorentz (30) peak shapes and background subtracted with a Shirley background. Calcium peaks were fit with a doublet separation of 3.5 eV and phosphorus peaks with a doublet separation of 0.87 eV.

(32) (SEM) images were recorded using a Carl Zeiss EVO MA10 scanning electron microscope operating at 10 kV and a working distance of 12.5 mm. The bioactive glass discs were immersed in SBF and maintained at 37° C. for 1 week. The samples were then removed, rinsed with water and acetone to remove residual salts and halt any further reactions. The samples were mounted onto SEM stubs using conductive carbon tape and splutter coated with gold. Images were recorded at magnifications of 500× and 3000×.

(33) FIGS. 9a and 9b show XPS analysis of the glass surface following submersion in simulated body fluid for 1 week. This analysis revealed Ca:P ratios consistent with hydroxyapatite (Ca:P atomic ratio≈1.64±0.02) for all samples, corroborating the findings from X-ray diffraction. The Ca 2p3/2 peak binding energy was found to be ˜347.5 eV and phosphorus 2p3/2˜133.5 eV, again consistent with previously reported literature values for Ca2+ and P5+ within an apatite formation.

(34) FIG. 4 shows the glass surface following immersion in SBF for 1 week. As shown there is full surface coverage after 1 week. All samples show similar features; a relatively smooth surface layer composed of smaller fused spherical apatite precipitates is clearly visible. Termination of the surface reaction using acetone caused dehydration and cracking of the apatite layer. Larger fused apatite spheres, as typically observed for bioglass are seen on top of this initial layer.

(35) ICP Analysis of Dissolution Products

(36) To determine the concentration of ions released from the bioactive glasses, ICP analysis was conducted.

(37) Stock solutions of 10 mg/ml of ground bioactive glass (Examples 1-3) in ultra-pure water were prepared for quantitative ionic profile using inductively coupled plasma optical emission spectrometry ICP-OES (iCAP™ 7000 Plus Series). Solutions were maintained at 37° C. for up to 3 days. Reference standards were used to calibrate the concentrations and the amount of each ion was calculated from the linear portion of the generated standard curve.

(38) The concentration of gallium ions released in distilled water as a function of time for 10 mg/ml solutions are given in FIG. 2. A rapid release of ions was observed during the first few hours followed by a slower more gradual increase in ion concentration. As expected the concentration of gallium ions increases in an approximately linear trend with increasing gallium oxide content. At 24 hours the average gallium ion concentrations were 7.3, 18.8 and 30.2 ppm for the Ga 1%, Ga 2% and Ga 3% respectively. The gallium concentration approximately stabilised after 24 hours and minimal increases in concentrations were observed at 48 and 72 hours.

(39) The release rate of Ca, P and Si is essential for upregulating gene expression. The ion concentrations are not significant for altered Ca, P and Si due to the incorporation of gallium oxide into the glasses. For example, at 24 hours the concentration of Si is 65, 70, 61 and 67 ppm for 45S5 (control), Ga 1% (Example 1), Ga 2% (Example 2) and Ga 3% (Example 3) respectively.

(40) Cell Toxicity

(41) Human osteosarcoma (Saos-2) cells were purchased from the American Tissue Culture Collection and maintained in McCoy's 5A medium containing 1.5 mM L-glutamine and 2200 mg/L sodium bicarbonate. Media was supplemented with 1% penicillin, streptomycin and 15% foetal bovine serum (FBS).

(42) Human osteoblasts (NHOst) were purchased from Lonza and cultured in Clonetics OGM Osteoblast growth media supplemented with 10% FBS, 1% L-glutamine and 1% penicillin, streptomycin. Both cell lines were maintained at 37° C. in a humidified atmosphere of 5% CO.sub.2/95% air.

(43) Stock solutions of conditioned media were prepared by dissolving glass particles (from Example 1-3) in complete McCoy's 5A medium to treat Saos-2 cells and Clonetics OGM Osteoblast complete growth media to treat osteoblasts.

(44) Stock solutions were prepared at a concentration of 10 mg/ml and left to incubate in a shaker incubator at 250 rpm at 37° C. for 24 hours. Following the 24 hour incubation period, stock solutions were filtered using 0.2 micron syringe filter. 3 ml samples were excluded to record stock solution pH values following glass dissolution.

(45) As shown in FIG. 3 the conditioned media shows a significant rise in pH as sodium and calcium ions rapidly leach from the glass in the surrounding media. pH readings taken directly after conditioning the media are designated as time point 0 in FIG. 3. Deviations from neutral pH are known to have detrimental effects on cell viability/proliferation, furthermore it is known that the body naturally buffers pH.

(46) Therefore the conditioned media was incubated at 37° C. in a humidified atmosphere of 5% CO.sub.2 for up to 72 hours to buffer the pH. McCoy's media was used as a control and it is clearly evident over the course of 72 hours the pH of the media fluctuated marginally from 7.91 at time point 0 to 7.23 at 72 hours, both readings within the physiological pH range acceptable for cell growth and maintenance.

(47) In contrast 10 mg/ml of 45S5 control glass significantly elevated the pH of the media and at time point 0 a reading of 10.3 was recorded and a gradual decline was observed over the course of 72 hours (1 hour, 9.4, 2 hours, 8.65, 8 hours 7.85). Conditioned media generated by the addition of 10 mg/ml of Ga 1% (Example 1), Ga 2% (Example 2) and Ga 3% (Example 3) gallium doped bioactive glass also exhibited a similar trend, as a high pH was recorded for each glass composition at time point 0 (Ga 1%, pH 9.45, Ga 2% pH 9.26 and Ga 3% pH 9.37). At 24 hours the pH of all gallium containing conditioned media had decreased to physiologically acceptable levels where cell growth is viable (Ga 1%, pH 7.77, Ga 2% pH 7.81 and Ga 3% pH 7.87). Beyond 24 hours negligible changes in pH were observed. Therefore in following experiments the media was conditioned for 24 hours and then placed in the incubator for 24 hours to neutralise before being used. Neutralising the pH in this way eliminated any potential cytotoxic effects of the glass conditioned media due to lethal pH.

(48) Cell cytotoxicity was analysed using MTT viability assay (Thermo Fisher), where 5000 cells per well of both Saos-2 and NHOst cells were cultured using conditioned media containing 10 mg/ml respective glass compositions (Examples 1-3), for a period of 72 hours. 10 μM Etoposide was used as a positive control, while cells grown using normal cell media served as a negative control. For the MTT assay, a 12 mM stock solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was prepared as per manufacturer's instructions and diluted 1/10 in phenol red free media before being added to the cells, which were incubated for 4 hours at 37° C. Following the incubation 75 μl of treatment was removed and 50 μl of Dimethyl sulfoxide (DMSO) was added and left to incubate at 37° C. for 10 minutes. Metabolically active cells reduce MTT to formazan and after formazan extraction the optical density was measured using a spectrophotometer (at 570 nm). This assay was performed in quintuplicate.

(49) No significant reduction in Saos-2 cell viability was observed between the positive control (unconditioned media) and the gallium free control glass (45S5). However a steady but significant decrease in Saos-2 cell viability was observed with increasing gallium oxide content in a dose response manner (Ga 2% (Ex2) p<0.01 and Ga 3% (Ex3) p<0.0001) as shown in FIG. 6. After 72 hours in conditioned Ga 3% (Ex3) conditioned media Saos-2 cell viability was less than 50%. In contrast conditioned media from the glasses showed no cytotoxic effects against normal human osteoblast cells as shown in FIG. 7. In each experiment 10 nm etoposide was used as a positive control and a known inducer of cell death (p<0.0001).

(50) Cell Viability Assay

(51) 10,000 cells per well were seeded of both Saos-2 and NHOst cells and treated for 72 hours with conditioned media containing the dissolution products of 45S5, Ga 1% (Ex 1), Ga 2% (Ex 2) and Ga 3% (Ex 3) at a concentration of 10 mg/ml. Following the incubation period, a positive control of dead cells was established by incubating cells with 70% ethanol for 30 minutes. Cells treated with regular growth media served as a negative control. The polyanionic dye calcein-green is well retained within live cells, producing an intense uniform green fluorescence. Ethidium homodimer-1 (EthD-1) enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells. To stain Saos-2 cells working concentrations of calcein-green at 0.5 μM and EthD-1 at 2.5 μM were combined into one solution and used to treat cells, and to treat NHOst cells both calcein-green and EthD-1 were both prepared at a concentration of 0.5 μM. Cells were overlaid with 100 μl of staining solution and left to incubate for 45 minutes at room temperature. Cells were photographed using a fluorescent microscope at 100× magnification.

(52) It was found that media containing Ga 3% glass significantly increases osteosarcoma (tumorous) cell death relative to the Ga 1% and Ga 2% glasses and 45S5 control as shown in FIG. 4 where FIG. 4A shows cells after treatment with McCoy basal media, FIG. 4B shows cells after treatment with conditioned media containing 45S5 Bioglass™ (control), FIG. 4C shows cells after treatment with conditioned media containing Ga 1% (Ex 1) bioactive glass composition, FIG. 4D shows cells after treatment with conditioned media containing Ga 2% (Ex 2) bioactive glass composition, FIG. 4E shows cells after treatment with conditioned media containing Ga 3% (Ex 3) bioactive glass composition and FIG. 4F shows cells after a 1 nM Etoposide treatment as a positive inducer of cell death. A significant increase in dead cells was observed between the 45S5 control glass and the Ga 1% and Ga 2% glasses whilst with the Ga 3% glass all cells appeared dead. In contrast, the Ga 3% glass remained non-toxic to osteoblast (non-tumorous) cells (see FIG. 5 where FIG. 5A shows cells after treatment with McCoy basal media, FIG. 5B shows cells after treatment with conditioned media containing 45S5 Bioglass™ (control), FIG. 5C shows cells after treatment with conditioned media containing Ga 1% (Ex 1) bioactive glass composition, FIG. 5D shows cells after treatment with conditioned media containing Ga 2% (Ex 2) bioactive glass composition, FIG. 5E shows cells after treatment with conditioned media containing Ga 3% (Ex 3) bioactive glass composition and FIG. 5F shows cells after a 1 nM Etoposide treatment as a positive inducer of cell death).

(53) Experiments described above were performed with at least three independent samples per data point. Data were analysed using SPSS version 21 and GraphPad Prism 6. MTT, ICP and pH results are expressed as the mean±standard deviation. To compare cell viability between different time points and glass compositions Two-way ANOVA and Tukey's multiple comparisons test was conducted to test for significance with statistically significant values defined as P<0.05. Fluorescent microscopy and Live/Dead staining was used to distinguish between viable and non-viable cells results are qualitative and clearly indicate cell cytotoxicity or lack of.

(54) While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

(55) All references referred to above are hereby incorporated by reference.