MESOPOROUS PHOSPHATE BASED GLASS

Abstract

The disclosure provides a method of producing a mesoporous phosphate-based glass. The method comprises (a) contacting a phosphate with an alcohol and/or a glycol ether to create a reaction mixture; (b) contacting the reaction mixture with alkali metal cations and/or alkaline earth metal cations; (c) contacting the alcohol, the glycol ether or the reaction mixture with a surfactant, wherein the surfactant is configured to provide channel-like pores in the resultant mesoporous phosphate-based glass; (d) allowing the reaction mixture to gel; and (e) calcinating the gel to obtain the mesoporous phosphate-based glass.

Claims

1. A method of producing a mesoporous phosphate-based glass, the method comprising: contacting a phosphate with an alcohol and/or a glycol ether to create a reaction mixture, wherein preferably the phosphate is an ormanophosphate; contacting the reaction mixture with alkali metal cations and/or alkaline earth metal cations; contacting the alcohol, the glycol ether or the reaction mixture with a surfactant, wherein the surfactant is configured to provide channel-like pores in the resultant mesoporous phosphate-based glass; allowing the reaction mixture to gel; and calcinating the gel to obtain the mesoporous phosphate-based glass.

2. (canceled)

3. The method of claim 1, wherein the phosphate is an organophosphate and is a compound of formula (I):
PO(OR.sup.1).sub.x(OH).sub.y   (I) where x is an integer between 1 and 3; y is an integer between 0 and 2; the sum of x and y is 3; and each R.sup.1 group is a C.sub.1-C.sub.10 alkyl, a C.sub.1-C.sub.10 alkenyl or a C.sub.1-C.sub.10 alkynyl, and is preferably n-butyl.

4. (canceled)

5. The method of claim 1, wherein the molar ratio of the phosphate to the alcohol and/or glycol ether is between 1:20 and 20:1, between 1:10 and 5:1, between 1:5 and 1:1, between 1:2 and 1:4 or between 1:2.5 and 1:3.5.

6. The method of claim 1, wherein the alkali metal cations and/or alkaline earth metal cations comprise lithium cations, sodium cations, potassium cations, rubidium cations, beryllium cations, magnesium cations, calcium cations and/or strontium cations, wherein preferably the alkali metal cations and/or alkaline earth metal cations comprise sodium cations and calcium cations.

7. (canceled)

8. The method of claim 1, wherein the molar ratio of the alkaline earth metal cation to the phosphate is between 1:50 and 50:1, between 1:25 and 10:1, between 1:15 and 1:1, between 1:10 and 4:10, between 3:15 and 3:8 or between 3:12 and 3:10, or wherein the molar ratio of the alkali metal cation to the phosphate is between 1:1000 and 50:1, between 1:500 and 10:1, between 1:30 and 1:1, between 1:20 and 3:10, between 1:15 and 3:8 or between 1:11 and 3:11.

9. (canceled)

10. The method of claim 1, wherein the method comprises contacting the reaction mixture with an antimicrobial agent, preferably the antimicrobial agent comprises a metal or metal cation, preferably a transition metal a p-block metal or a cation thereof, most preferably the metal or metal cation comprises copper, zinc, strontium, silver, gallium, cerium or a cation thereof.

11. (canceled)

12. (canceled)

13. The method of claim 1, wherein the method comprises contacting the reaction mixture with the surfactant subsequently to contacting the reaction mixture with alkali metal cations and/or alkaline earth metal cations.

14. The method of claim 1, wherein the molar ratio of the phosphate to the surfactant is between 1:1 and 1,000:1, between 5:1 and 500:1, between 10:1 and 100:1, between 12.5:1 and 50:1, between 15:1 and 40:1 or between 17.5:1 and 30:1.

15. The method of claim 1, wherein the surfactant is a copolymer, preferably a triblock copolymer, and most preferably a symmetric triblock copolymer, preferably the copolymer comprises a hydrophobic core and hydrophilic end groups, most preferably the copolymer comprises poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG).

16. (canceled)

17. (canceled)

18. The method of claim 15, wherein the copolymer has a molar mass between 500 and 50,000, between 1,000 and 25,000, between 2,000 and 20,000, between 3,000 and 15,000, between 4,000 and 10,000, and between 4,500 and 9,000, between 5,000 and 7,500 or between 5,500 and 6,000.

19. The method of claim 1, wherein calcinating the gel comprises exposing the gel to an elevated temperature between 100° C. and 1,000° C., between 150° C. and 500° C., between 200° C. and 400° C., between 250° C. and 350° C. or between 275° C. and 325° C.; preferably calcinating the gel comprises increasing the temperature to the elevated temperature at a rate of between 0.01 and 20° C./min, between 0.1 and 10° C./min, between 0.4 and 5° C./min, between 0.6 and 3° C./min or between 0.8 and 1.5° C./min.

20. (canceled)

21. The method of claim 1, wherein, subsequent to calcinating the gel, the method comprises loading the phosphate-based glass with an organic molecule.

22. A mesoporous phosphate-based glass obtained or obtainable by a method comprising: contacting a phosphate with an alcohol and/or a glycol ether to create a reaction mixture; contacting the reaction mixture with alkali metal cations and/or alkaline earth metal cations; contacting the alcohol, the glycol ether or the reaction mixture with a surfactant, wherein the surfactant is configured to provide channel-like pores in the resultant mesoporous phosphate-based glass; allowing the reaction mixture to gel; and calcinating the gel to obtain the mesoporous phosphate-based glass.

23. A phosphate-based glass comprising pores with an average diameter of between 2 and 50 nm, wherein the pores are channel-like.

24. The phosphate-based glass according to claim 22, wherein the pores have an average diameter between 3 and 40 nm, between 4 and 30 nm, between 6 and 25 nm, between 8 and 20 nm, between 10 and 15 nm or between 11 and 13 nm.

25. The phosphate-based glass according to claim 22, wherein the phosphate based glass comprises one or more of: between 10 and 90 mol % phosphorus pentoxide, between 20 and 80 mol % phosphorus pentoxide, between 30 and 75 mol % phosphorus pentoxide, between 40 and 70 mol % phosphorus pentoxide, between 45 and 65 mol % phosphorus pentoxide or between 50 and 60 mol % phosphorus pentoxide; between 5 and 60 mol % alkaline earth metal oxide, between 10 and 50 mol % alkaline earth metal oxide, between 20 and 40 mol % alkaline earth metal oxide or between and 35 mol % alkaline earth metal oxide; between 1 and 50 mol % alkali metal oxide, between 2.5 and 30 mol % alkali metal oxide, between 5 and 20 mol % alkali metal oxide or between 10 and 15 mol % alkali metal oxide.

26. (canceled)

27. (canceled)

28. The phosphate-based glass according to claim 22, wherein, the phosphate-based glass is amorphous.

29. The phosphate-based glass according to claim 22, wherein the phosphate-based glass has a high degree of connectivity.

30. (canceled)

31. (canceled)

32. (canceled)

33. The phosphate-based glass according to claim 23, wherein the pores have an average diameter between 3 and 40 nm, between 4 and 30 nm, between 6 and 25 nm, between 8 and 20 nm, between 10 and 15 nm or between 11 and 13 nm.

34. The phosphate-based glass according to claim 23, wherein the phosphate based glass comprises one or more of: between 10 and 90 mol % phosphorus pentoxide, between 20 and 80 mol % phosphorus pentoxide, between 30 and 75 mol % phosphorus pentoxide, between 40 and 70 mol % phosphorus pentoxide, between 45 and 65 mol % phosphorus pentoxide or between 50 and 60 mol % phosphorus pentoxide; between 5 and 60 mol % alkaline earth metal oxide, between 10 and 50 mol % alkaline earth metal oxide, between 20 and 40 mol % alkaline earth metal oxide or between 25 and 35 mol % alkaline earth metal oxide; between 1 and 50 mol % alkali metal oxide, between 2.5 and 30 mol % alkali metal oxide, between 5 and 20 mol % alkali metal oxide or between 10 and 15 mol % alkali metal oxide.

Description

[0078] 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:—

[0079] FIG. 1 shows X-ray powder diffraction (XRD) patterns of the undoped non porous phosphate-based glass (PG), undoped mesoporous phosphate-based glass (MPG) and mesoporous phosphate-based glasses doped with copper ions 1, 3, 5 mol % (Cu1, Cu3 and Cu5), zinc ions 1, 3, 5 mol % (Zn1, Zn3 and Zn5) and strontium ions 1, 3, 5 mol % (Sr1, Sr3 and Sr5);

[0080] FIG. 2A shows a low angle X-ray diffraction (LA-XRD) pattern of the undoped glasses; and FIG. 2B shows the LA-XRD patterns of the doped glasses;

[0081] FIG. 3 shows N.sub.2 adsorption-desorption isotherms at 77 K for the doped and undoped glasses;

[0082] FIG. 4 shows the pore size distribution for MPG and for the doped glasses;

[0083] FIG. 5 shows Fourier-transform infrared (FTIR) spectra of the undoped and doped glasses;

[0084] FIG. 6 show the 31P-MAS NMR spectra of the undoped and doped glasses;

[0085] FIG. 7 shows SEM images of MPG at two different magnifications, Cu1, Cu3, Cu5, Zn1, Zn3, Zn5, Sr1, Sr3, and Sr5. The SEM images show details of the hexagonal arrangement of mesopores;

[0086] FIGS. 8a-c show the release of (a) P, (b) Ca and (c) Na when the undoped glasses are immersed in deionised water for 7 days, the results are measured using inductively coupled plasma—optical emission spectroscopy (ICP-OES); and FIG. 8d shows a pH study for the undoped glasses when they are immersed in deionised water for 7 days;

[0087] FIG. 9 show the release of P, Ca and Na and the dopant ion (Cu, Zn and Sr, respectively) doped glasses are immersed in deionised water up to 7 days, the results are measured using ICP-OES;

[0088] FIG. 10 shows XRD patterns of the undoped glasses after immersion in simulated body fluid (SBF) for 24 h at 37° C.;

[0089] FIG. 11 shows cell viability assay results after 24 h contact of fibroblast cells with 1 mg/mL of PG and MPG powders, error bars are standard deviation (SD) (n=3);

[0090] FIG. 12 shows Saos-2 cell viability measurement using the Alamar blue fluorescence assay for PG and MPG after 1, 3, 5 and 8 days, error bars are standard deviation (SD) (n=3);

[0091] FIG. 13 shows fluorescent microscopic micrographs of Saos-2 cells stained by DAPI after 8 days for (a) control, (b) PG, and (c) MPG. The green fluorescent stain shows filamentous actin and the blue one shows nuclei. Scale bar=400 μm;

[0092] FIG. 14 shows SEM images showing Saos-2 cell attachment after 8 days for control (cells on cell culture support), PG and MPG;

[0093] FIG. 15 shows Hacat cell viability measurement using the Alamar blue fluorescence assay for PG and MPG after 1, 3, 5 and 7 days. Error bars are SD (n=3);

[0094] FIG. 16 shows the results of an ADM test showing the S. aureus viability when exposed to mesoporous glasses containing increasing concentrations of Cu. The bacterial viability is expressed as log.sub.10 CFU/mL and data are mean f SD with statistical analysis (Two-way ANOVA for each time point);

[0095] FIG. 17 shows the results of an ADM test showing the E. coli viability when exposed to mesoporous glasses containing increasing concentrations of Cu. The bacterial viability is expressed as log.sub.10 CFU/mL and data are mean f SD with statistical analysis (Two-way ANOVA for each time point);

[0096] FIG. 18 shows the results of an ADM test showing the S. aureus viability when exposed to PG and MPG samples loaded with tetracycline hydrochloride (TCH) after 24 h. The bacterial viability is expressed as log.sub.10 CFU/mL and data are mean f SD with statistical analysis (Two-way ANOVA for each time point); and

[0097] FIG. 19 shows the amount of TCH released from loaded PG and MPG sampled when immersed in deionised water for 10, 30 and 120 minutes.

EXAMPLE 1—SYNTHESIS AND CHARACTERISATION OF UNDOPED NON POROUS PHOSPHATE-BASED GLASS (PG), UNDOPED MESOPOROUS PHOSPHATE-BASED GLASS (MPG) AND DOPED MESOPOROUS PHOSPHATE-BASED GLASS

Materials and Methods

Materials

[0098] The following chemical precursors have been used without further purification; n-butyl phosphate (1:1 molar ratio of mono OP(OH).sub.2(OBun) and di-butyl phosphate OP(OH)(OBun).sub.2, Alfa Aesar, 98%), calcium methoxyethoxide (Ca-methoxyethoxide, ABCR, 20% in methoxyethanol), sodium methoxide solution (NaOMe, Aldrich, 30 wt % in methanol), copper(II) acetate (Cu-acetate, Aldrich, 98%), zinc acetate (Zn-acetate, Aldrich, 98%), strontium acetate (Sr-acetate, Aldrich, 97%), ethanol (EtOH, Fisher, 99%), and Pluronic (P123−M.sub.n=5800, Aldrich).

Synthesis Method

[0099] a) Synthesis of Phosphate-Based Glass (PG)

[0100] 1.7 g n-butyl phosphate (PO(OC.sub.4H.sub.9)(OH).sub.2) was diluted in 5 ml EtOH and allowed to react for 10 minutes (the whole reaction being carried out in a dried vessel). Following that 3.5 g Ca-methoxyethoxide and 0.5 g NaOMe were added dropwise into the mixture while it was magnetically stirred, and stirring was continued for about 1 h.

[0101] The reaction was poured into a glass container and allowed to gel at room temperature. The mixture turned to gel after about 10 minutes and was then aged for 1 day at room temperature. Following that the gel was dried by increasing the temperature to 40° C. and holding it for 1 day, the temperature was then increased to 60° C. and held for 2 days, the temperature was then increased to 80° C. and held for 2 days and then to 120° C. and held for 1 day.

[0102] After the drying step, calcination was conducted to remove any remaining surfactant and solvents from the sample. The temperature was increased to 300° C., with the heat being increased at a rate of 1° C./min to prevent the collapse of the porous structure. The sample was held at 300° C. for one hour, and then allowed to cool.

[0103] b) Synthesis of Undoped Mesoporous Phosphate-Based Glass (MPG)

[0104] 1.7 g n-butyl phosphate, comprising a mixture of butyl phosphate (PO(OC.sub.4H.sub.9)(OH).sub.2) and dibutyl phosphate (PO(OC.sub.4H.sub.9).sub.2(OH)) was diluted in 5 ml EtOH and allowed to react for 10 minutes (the whole reaction being carried out in a dried vessel). Following that 3.5 g Ca-methoxyethoxide and 0.5 g NaOMe were added dropwise into the mixture while it was magnetically stirred, and stirring was continued for about 1 h.

[0105] A solution consisting of 3.0 g P123, 5 ml EtOH and 2.5 ml H.sub.2O was then added to the reaction mixture and allowed to react for 10 min. The reaction was stirred continuously for this time.

[0106] The mixture was then poured into a glass container, dried and calcinated as described above.

[0107] c) Synthesis of Doped Mesoporous Phosphate-Based Glasses

[0108] 1.7 g n-butyl phosphate (PO(OC.sub.4H.sub.9)(OH).sub.2) was diluted in 5 ml EtOH and allowed to react for 10 minutes (the whole reaction being carried out in a dried vessel). Following that 3.5 g Ca-methoxyethoxide and NaOMe were added dropwise into the mixture while it was magnetically stirred, and stirring was continued for about 1 h. The quantities of NaOMe used varied for the different samples, and can be calculated from table 1 below.

[0109] Cu, Zn, or Sr-doped glasses were prepared by adding Cu-acetate, Zn-acetate or Sr-acetate, respectively, to the mixture and allowed to react for 10 min. The quantities of Cu-acetate, Zn-acetate or Sr-acetate used to prepare each sample may be calculated from table 1.

TABLE-US-00001 TABLE 1 Composition of undoped and doped phosphate-based glasses in mol % Sample P.sub.2O.sub.5 CaO Na.sub.2O CuO ZnO SrO PG 55 30 15 — — — MPG 55 30 15 — — — Cu1 55 30 14 1 — — Cu3 55 30 12 3 — — Cu5 55 30 10 5 — — Zn1 55 30 14 — 1 — Zn3 55 30 12 — 3 — Zn5 55 30 10 — 5 — Sr1 55 30 14 — — 1 Sr3 55 30 12 — — 3 Sr5 55 30 10 — — 5

[0110] A solution consisting of 3.0 g P123, 5 ml EtOH and 2.5 ml H.sub.2O was then added to the reaction mixture and allowed to react for 10 min.

[0111] The mixture was then poured into a glass container, dried and calcined as described above.

[0112] The obtained sol-gel glasses were ground at 10 Hz to form powders (MM301 milling machine, Retsch GmbH, Hope, UK). The resultant powders were then characterised as described below.

Characterisation

Wide Angle X-Ray Powder Diffraction (WA-XRD)

[0113] WA-XRD (PANalytical X'Pert, Royston, UK) was performed on powdered samples in a flat plate geometry using Ni filtered Cu Kα radiation. Data was collected using a PIXcel-1D detector with a step size of 0.0525° over an angular range of 2θ=10-90° and a count time of 12 S.

Low Angle X-Ray Powder Diffraction (LA-XRD)

[0114] The ordered mesoporous structure was studied by low-angle XRD patterns recorded using Cu Ka radiation in transmission mode on a Panalytical Empyrean diffractometer (PANalytical X'Pert, Royston, UK) equipped with a focusing mirror on the incident beam and a X'Celerator linear detector. The scans were collected within the range of 0.3-6.0 with a step of 0.017.

Surface Area and Pore Size

[0115] Nitrogen (N.sub.2) adsorption-desorption porosimetry was performed on the grounded powders (Gemini V, Micromeritics, Hertfordshire, UK); in particular, the specific surface area (SSA) was assessed by using the Brunauer-Emmet-Teller (BET) method, whereas the pores size distribution was determined from the desorption branch of the isotherm through the Broekhoff-de Boer (BdB) method and the BJH method. Samples were outgassed at 270° C. for 6 h before recording the adsorption measurements.

Fourier-Transform Infrared (FTIR) Spectroscopy

[0116] FTIR was performed using an FTIR-2000 instrument equipped with Timebase software (Perkin Elmer, Seer Green, UK) with attenuated total reflectance accessory (Golden Gate, Specac, Orpington, UK). Measurements were performed at room temperature in absorbance mode in the range of 1400-600 cm.sup.−1.

.SUP.31.P-MAS NMR

[0117] .sup.31P magic angle spinning solid-state nuclear magnetic resonance spectra (AVANCE III, Bruker, Coventry, UK) were recorded and referenced to the resonance of the secondary reference ammonium dihydrogen phosphate (NH.sub.4H.sub.2PO.sub.4) at 0.9 ppm (relative to 85% H.sub.3PO.sub.4 solution at 0 ppm). The spectra were recorded at 161.87 MHz using a 4 mm magic angle spinning probe using direct excitation with a 90° pulse and 60 s recycle delay at ambient probe temperature (˜25° C.) and at a sample spin rate of 12 kHz. Between 20 and 88 repetitions were accumulated and were processed using DM-fit software.

Scanning Electron Microscopy (SEM)

[0118] SEM was performed with a JSM-7100F instrument (Jeol, Welwyn, UK) at an accelerating voltage of 10 kV and working distance of 10 mm. The samples were mounted onto an aluminium stub using carbon conductive tape. The pore size diameters were measured using Image-pro plus software (Media Cybernetics, USA).

Results

Wide Angle X-Ray Powder Diffraction (WA-XRD)

[0119] WA-XRD analysis of the non porous PG and mesoporous doped and undoped phosphate-based glass samples (FIG. 1) showed no Bragg peaks and a broad band centred at about ˜30° due to the phosphate glass network. Accordingly, the inventors concluded that all of the samples are amorphous.

Low Angle X-Ray Diffraction (LA-XRD)

[0120] LA-XRD analysis of the powdered MPG sample showed two peaks at 0.8 and 2.6 degrees which confirmed the short-range ordered mesoporous structure of the prepared sample (FIG. 2A). A similar peak in the range between 0.6-0.9 degrees was observed for the dopes samples (FIG. 2B). No peak was observed for the PG sample between 0.3 and 6.0 degrees.

Surface Area and Pore Size

[0121] N.sub.2 adsorption isotherms for the glasses are shown in FIGS. 3 and 4. All mesoporous glasses show a hysteresis loop typical of mesoporous materials where pores are channel-like. Even if the dopant ion change slightly the shape of the hysteresis loop, the mesoporosity and channel-like structure is preserved.

[0122] The calculated values for surface area and pore sizes of the glasses are given in table 2.

TABLE-US-00002 TABLE 2 Surface are and pore size of phosphate based glasses Surface Area/ Pore Sample m.sup.2 .Math. g.sup.−1 size/nm PG 4.51 — MPG 123 11.8 Cu.sub.1 87 12.6 Cu.sub.3 73 12 Cu.sub.5 70 12.2 Zn.sub.1 92 11.3 Zn.sub.3 82 12.1 Zn.sub.5 76 11.7 Sr.sub.1 112 12.3 Sr.sub.3 94 11.4 Sr.sub.5 73 12

[0123] The surface areas of the MGP and the doped samples are between 70 and 123 m.sup.2.Math.g.sup.−1. These values are significantly higher than the surface area of the PG sample (4.5 m.sup.2.Math.g.sup.−1). The average pore size for the MGP and doped samples is 12 nm.

Fourier-Transform Infrared (FTIR) Spectroscopy

[0124] FIG. 5 shows FTIR data for the MGP and the doped samples. The peak at 730 cm.sup.−1 can be assigned to the symmetrical stretching υs (P—O—P) mode, while the peak at 900 cm.sup.−1 can be assigned to the asymmetrical stretching υas (P—O—P) mode (Q.sup.2 phosphate units). The peaks at 1100 and 1235 cm.sup.−1 can be assigned to asymmetrical υas (PO.sub.3).sup.−2 and υas (PO.sub.2) (Q.sup.1 and Q.sup.2 phosphate units, respectively). [18, 19].

.SUP.31.P-MAS NMR

[0125] The .sup.31P-MAS NMR spectra are presented in FIG. 6. All peaks were fitted by the DM-fit software and the isotropic chemical shifts and relative properties are reported in Table 3. The main peak with chemical shifts in the range of −6.3 to −6.6 ppm is attributed to Q.sup.1 phosphate group [20]. The less intense peak occurring between −23.7 and −23.4 ppm correspond to the Q.sup.2 phosphate groups. Peaks marked with an asterisk are spinning sidebands.

TABLE-US-00003 TABLE 3 .sup.31P-MAS NMR peak parameters of the PG and MPG glasses. Q.sup.n Abundance Sample species (%, ±1) PG Q.sup.1 73 Q.sup.2 27 MPG Q.sup.1 68 Q.sup.2 32 Cu.sub.1 Q.sup.1 65 Q.sup.2 35 Cu.sub.3 Q.sup.1 63 Q.sup.2 37 Cu.sub.5 Q.sup.1 58 Q.sup.2 42 Zn.sub.1 Q.sup.1 67 Q.sup.2 33 Zn.sub.3 Q.sup.1 65 Q.sup.2 35 Zn.sub.5 Q.sup.1 64 Q.sup.2 36 Sr.sub.1 Q.sup.1 62 Q.sup.2 38 Sr.sub.3 Q.sup.1 62 Q.sup.2 38 Sr.sub.5 Q.sup.1 60 Q.sup.2 40

[0126] The presence of Q.sup.2 phosphate groups indicates that the phosphorus has two bridging oxygens, suggesting that there is good connectivity in the glass. This is advantageous, and suggests that the dissolution rate of the composition could be modified for a specific application by making minor modifications to the composition.

Scanning Electron Microscopy (SEM)

[0127] FIG. 7 shows the SEM images of the MPG sol-gel synthesised sample. The hexagonal arrangement of the channel-like pores can be clearly seen in the magnified image.

EXAMPLE 2—DISSOLUTION AND PH STUDIES

Materials and Methods

[0128] The samples prepared in Example 1 where used for dissolution and pH studies.

[0129] For the dissolution study, 10 mg powders of each sample were immersed in 10 mL of deionised water for 1, 3, 5 and 7 days (n=3). The resulting suspensions for each time points were then centrifuged at 4,800 rpm for 10 min to separate the glass particles from the solution. Phosphorus, calcium and sodium content release in the solution were subsequently measured by ICP-OES (720ES-Varian, Crawley, UK) calibrated across the predicted concentration range using standard solutions (ICP multi-element standard solution, VWR). Additionally, content of the ion dopant released (copper, zinc or strontium) was also measured. Both samples and standards were diluted in 1:1 in 4% HNO.sub.3 (Fluka) and analysed in reference to a blank (2% HNO.sub.3) solution.

[0130] For the pH study, 10 mg powders of each sample were immersed in 10 mL deionised water (pH 7.0 f 0.1) and stored at 37° C. for up to 7 days (n=3). The pH of the solution was measured after 0, 1, 3, 5 and 7 days using Orion pH meter (Thermo scientific-Orion star, UK).

Results

[0131] ICP-OES analysis was carried out to determine the ionic release profiles from the dissolution of PG and MPG powders after immersion in deionised water for up to 7 days (FIG. 8). The ICP-OES results show that the ion release occurred fastest in the first 24 hours. The degradation of MPG is higher in comparison with the PG sample. This could be related to the higher surface area of MPG

[0132] pH changes in FIG. 8-d, the initial reduction in pH after 2 h was related to the hydrated phosphate chains dissociating into the solution. However, the pH value increases from 5 to 8 h, which can be related to the presence of Na.sup.+ ions in the solution. These results are in agreement with the degradation results.

[0133] FIG. 9 shows that the addition of copper, zinc, or strontium slightly slows down the degradation of the synthesised glasses.

EXAMPLE 3—CELL VIABILITY

Materials and Methods

[0134] The samples produced in Example 1 where used in the cell viability studies.

[0135] Simulated body fluid (SBF) was prepared according to the methods taught by T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi and T. Yamamuro, “Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W”, J. Biomed. Mater. Res., 24, 721-734 (1990).

Immersion Test

[0136] To determine the biocompatibility of the PG and undoped MPG particles an immersion test in simulated body fluid (SBF) for 24 h at 37° C. was performed. SBF is a solution with an ion concentration close to that of human blood plasma. X-ray powder diffraction (XRD) of the PG and undoped MPG particles was conducted before and after the immersion test.

HaCat Skin Cell Viability Assay

[0137] HaCat skin cells were plated directly into 96-well assay plates at a density of 4000 cells/well and cultured in the presence of ionic release products generated from the dissolution of 1.0 mg/mL PG and MPG in DMEM. After 24 h in culture, cells were placed in contact with the different materials and cultured for 7 days with cell viability assessment during the culture period (after 1, 3, 5, and 7 days) by staining the cells with 1 μM calcein-AM and 2 μM ethidium homodimer-1 (Live/dead assay; Invitrogen) for 15 min. Images of green fluorescent viable cells and red fluorescent dead cell nuclei were acquired in the same well-plates using an Olympus IX81 inverted microscope equipped with a UPlanSApo 10 objective (UIS2 series). All conditions were tested in triplicate.

Fibroblast Cell Viability Assay

[0138] Fibroblast cells were treated with 10 mg/mL of the PG or MPG glass powders. The cell viability was monitored after 24 h and compared with a control.

[0139] Human skin fibroblast cells were procured from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 20% fetal bovine serum (FBS, Gibco, Invitrogen) and 1% antibiotic-antimycotic (Thermo Scientific, UK) in a humidified incubator at 5% CO.sub.2 and 37° C. On reaching 90% confluency, cells were passaged and used for cell viability assay of the glass powders. The cell viability assay was performed using 96-well plates. Fibroblast cells were treated with 10 mg/mL of the PG and MPG powders and MiliQ water was used as a control. Culture media was removed from the well-plate and cells were washed in PBS after 24 h. Following that tetrazolium based powder was dissolved in PBS and added to each well and absorbance at 570 nm was analysed after 3 h using BioTek plate reader.

Saos-2 Cell Viability Assay

[0140] Saos-2, osteosarcoma cells were procured from ATCC and cultured in McCoy's 5a medium (ATCC, UK) with 15% fetal bovine serum (FBS, Gibco, Invitrogen) and 1% antibiotic-antimycotic (Thermo Scientific, UK) in a humidified incubator at 5% CO.sub.2 and 37° C. On reaching 90% confluency, cells were passaged and used for cytocompatibility analysis for the materials. To facilitate the attachment of the cells on to the materials, polycarbonate cell culture inserts with 0.4 μm pore size (Merck Millipore) was used. Materials were placed on the inserts and incubated with medium overnight. 1.2×10.sup.4 cells were placed in each insert with the different materials and cultured for 8 days with cell viability assessment during the culture period. Cells only on inserts was used as control for comparison purposes.

[0141] Alamar Blue assay was carried out on days 1, 3, 5 and 8 to assess cell viability and growth. Cells were incubated for 2 hours in a 10% Alamar Blue (ThermoFisher, UK) solution followed by fluorescence measurement with a BioTek plate reader at 530 nm excitation and 590 nm emission as a direct estimation of cell growth on the different materials.

DAPI-Phalloidin Staining

[0142] At the end of day 8, the cells were fixed using 4% paraformaldehyde and stained with DAPI-Phalloidin for visualisation of the nucleus and actin filaments. Cells were incubated for 20 mins at room temperature in staining solution containing 2.5 μl of DAPI (1 mg/ml stock solution), 4 μl of Phalloidin (200 U/ml stock concentration, Alexa Fluor 488, Phalloidin, Life Technologies) and 20 μl of Triton X per ml of PBS. Cells were then visualised using Cytation5 Cell Imaging Multimode Reader (Biotek).

Results

SBF Immersion Test

[0143] As shown in FIG. 10, XRD analysis of the PG and MPG samples show the formation of hydroxyapatite only on the MPG sample after 24 h immersion. It is noted that the peaks matches with hydroxyapatite standard (ICDD-9-432). This suggests that the MPG has great potential for inducing bone regeneration.

HaCat Skin Cell Viability Assay

[0144] FIG. 14 shows the Hacat cell viability measurement using the Alamar blue fluorescence assay for the PG and MPG after 1, 3, 5 and 7 days. As it can be seen for the MPG sample the fluorescence intensity is higher than the control which confirms the potential application of MPG in soft tissue regeneration and more specifically wound healing.

Fibroblast Cell Viability Assay

[0145] As shown in FIG. 11, the PG (non-porous) glass is similar to the control after 24 h. However, for the MPG powder the cell viability is about two folds higher compared to the control.

Saos-2 Cell Viability Assay

[0146] Saos-2 cell viability measurement using the Alamar blue fluorescence assay the PG and MPG after 1, 3, 5, and 8 days is shown in FIG. 12. The MPG shows higher fluorescence intensity compared with the PG samples. This confirms that the cells attached to the MPGs, and the MPGs could therefore be used for bone tissue regeneration. These results are confirmed by fluorescent microscopic micrographs shown in FIG. 13 and the SEM images of the powders of Saos-2 cells stained by DAPI after 8 days shown in FIG. 14. FIG. 13A shows a mono layer of cells disposed on the culture support. FIGS. 13B and 13C show cells attached on PG and MPG particles respectively. The green fluorescent stain shows filamentous actin and the blue shows nuclei. More actin is observed in FIG. 13C as the cells are attaching to MPG particles.

EXAMPLE 4—ANTIBACTERIAL STUDY

Materials and Methods

[0147] The samples produced in Example 1 where used in the antibacterial studies.

[0148] An additional sample (Cu10) was also prepared. The method used to prepare this sample was the same as described in Example 1, except the amounts of NaOMe and Cu-acetate used were modified to ensure that the final composition comprised 5 mol % Na.sub.2O and 10 mol % CuO.

Antibacterial Study

[0149] Universal tubes with Tryptic Soy Broth (TSB) were inoculated with 50 mg of glass powders containing increasing Cu concentrations up to 10 mol % Cu and an overnight culture of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) at 10.sup.6 CFU/mL. Tubes were incubated at 37° C. for 3 days at 250 rpm and samples were collected after 1, 2 and 3 days to calculate viable CFU/mL of S. aureus and E. coli for each time point. For both antimicrobial tests the overnight bacterial cultures were obtained in TSB at 37° C. for 24 hours, at 250 rpm and the experiments were conducted as two biological replicates. Undoped mesoporous glass (MPG) was used as negative controls (identified as “0%”). No glass was added to samples as a positive control (identified as “control”).

Results

[0150] FIGS. 16 and 17 show bactericidal property of mesoporous glasses containing increasing concentrations of Cu against S. aureus and E. coli. As indicated there is a significant difference between the Cu-doped MPG compared to the control and undoped MPG. In particular, on day 1 the mesoporous glass comprising 10 mol % Cu exhibits a significant difference when compared with both the positive and negative controls, demonstrating a rapid bactericidal effect, and this difference becomes more significant on days 2 and 3.

EXAMPLE 5—DRUG DELIVERY APPLICATIONS

[0151] The samples prepared in Example 1 where used to evaluate the potential drug loading applications of MPG.

Materials and Methods

[0152] A 1 wt % solution of tetracycline hydrochloride (TCH) in ethanol was prepared by dissolving TCH in ethanol at room temperature. PG and MPG samples prepared as described in Example 1, and 100 mg of the PG/MPG samples were then immersed in 10 ml of the solution at 37° C. and the mixture was shaken for 1 hour. The solutions were then centrifuged at 4000 rpm for 10 min to obtain the loaded PG and MPG samples, which were collected and dried in an oven at 60° C. for 2 h.

Antibacterial Study

[0153] Universal tubes with TSB were inoculated with 50 mg of TCH loaded PG and MPG samples and an overnight culture of S. aureus at 10.sup.6 CFU/mL. Tubes were incubated at 37° C. at 250 rpm and samples were collected after 1 day to calculate viable CFU/mL of S. aureus colonies. Unloaded PG and MPG samples were used at the same concentration as negative controls. No glass was added to samples as a positive control (identified as “control”).

TCH Release Study

[0154] TCH release from 50 mg of TCH loaded PG and MPG samples was measured in the region of 300-500 nm using UV-Visible spectrometer (BioChrom Libra, Cambridge, UK) after 10, 30 and 120 min immersion in 5 ml deionised water. The peak at 356 nm (according to the standard calibration for TCH) was used for measuring the amount of loaded TCH for PG and MPG samples.

Results

[0155] The UV-Vis study results show that the MPG can hold about 7 times as much of an active agent compared to the same amount of PG, see FIG. 19. The inventors believe that this is due to the mesoporous structure of the MPG facilitating effective loading. Accordingly, the MPG samples loaded with TCH (a known antibiotic) showed improved antibacterial properties compared to PG loaded with TCH, or positive or negative controls.

CONCLUSIONS

[0156] The inventors have developed a novel-gel synthesis of mesoporous phosphate based glasses for potential biomedical applications. MPGs were successfully synthesised using non-ionic block copolymer EO20 PO70 EO20 (P123). The synthesised glasses were analysed using several characterisation techniques, including wide angle x-ray diffraction (WA-XRD), low angle x-ray diffraction (LA-XRD), .sup.31P magic angle spinning nuclear magnetic resonance (31P-MAS NMR), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and N.sub.2 adsorption surface analysis (BET). The WA-XRD and LA-XRD results confirmed the amorphous structure for the prepared samples. Moreover, the 31P MAS-NMR and FTIR results revealed that the glass structure consist of mainly Q.sup.1 and Q.sup.2 phosphate units. The BET study on MPG confirmed the high specific surface area of around 123 m.sup.2/g with the pore size of 12 nm.

[0157] The degradation was also assessed via inductively coupled plasma-optical emission spectroscopy (ICP-OES). The inventors note that the degradation product can be easily metabolised in the body. This makes the glasses an excellent candidate for both local drug delivery systems and bone tissue regeneration applications.

[0158] Cell studies were performed on Saos-2, HaCat, and fibroblast cells and confirmed the potential applications of these glasses for hard and soft tissue regeneration. The antibacterial properties of Cu doped MPG samples against S. aureus and E. coli were also assessed. The results confirmed the potential for these glasses to deliver antibacterial ions to the site of interest.

[0159] These glasses have also showing great potential application as a carrier in drug delivery applications for more sustained and controlled drug release. The surfaces of the glasses could be functionalised with specific ligands to target a specific site of interest and slow down the release of a drug molecule until the site of interest has been reached. For instance, the compositions could be functionalised to target cancerous cells in cancer treatment.

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