Bioactive Glasses Loaded With Dexamethasone by a Supercritical
Carbon Dioxide Deposition Process
L.T.R. Filipe1, A.P. Piedade2, J.J. Watkins3, M.E.M. Braga1, H.C. de Sousa1*
1
CIEPQPF, Chemical Engineering Department, FCTUC, University of Coimbra, Rua Sílvio Lima, Pólo II –
Pinhal de Marrocos, 3030-790 Coimbra, Portugal.
2
CEMUC, Mechanical Engineering Department, FCTUC, University of Coimbra, Rua Luís Reis Santos, Pólo II
Pinhal de Marrocos, 3030-788 Coimbra, Portugal.
3
Department of Polymer Science and Engineering, University of Massachusetts, 120 Governors Drive, Amherst,
MA 01003, USA.
Corresponding author: [email protected]; Phone: (+351) 239 798 749; Fax: (+351) 239 798 703
ABSTRACT
Bioactive glasses (BGs) have been quite used in implants, fixation devices and hard tissue engineering
applications mostly due to their chemical and physical properties as well as to their bioactivities which allow the
development of an apatite-like layer when in contact with physiological fluids. These inorganic materials are
usually prepared by melting and quenching but they can also be prepared using sol-gel methods. In this work we
prepared BGs (based on SiO2, P2O5, Na2O, CaO) and silica-calcium hydroxyapatite (HA) composites, by a onepot aqueous sol-gel method (at 37 ºC and atmospheric pressure) and using two biomimetic catalysts for the silica
formation from tetraethyl orthosilicate (TEOS): L-glutathione-reduced (GSH) and cysteamine (CYS). Other
precursors included triethyl phosphate (TEP), calcium nitrate tetrahydrate (CaNT) and sodium nitrate (NaNO3).
Additional samples were obtained by replacing TEP, CaNT and NaNO3 by synthetic calcium hydroxyapatite or
by changing the employed amount of water. In addition, some samples were also obtained by adding chitosan
(CHIT) as a porogenic agent. After synthesis, all obtained materials were dried, aged and submitted to a specific
sinterization process (from 60 ºC up to 1000ºC). Obtained materials were characterized by mercury intrusion
porosimetry, nitrogen adsorption, helium pycnometry, simultaneous differential thermal analysis (SDT),
scanning electron microscopy (SEM), infra-red spectroscopy (FTIR) and X-ray diffraction. Obtained materials
were later loaded with dexamethasone (DXMT, a hydrophobic drug with osteogenic properties) by a
supercritical carbon dioxide (scCO2) deposition process or by conventional loading from aqueous solutions.
Drug loading yields were changed by controlling scCO2 densities (755-920 Kg/m3) at a constant depressurization
rate (0.5 MPa/min) and at constant processing times (14 hours). Loading yields and drug release profiles were
obtained and compared to those obtained by the conventional method. The obtained results, in terms of porosity,
surface morphology and drug release profiles, demonstrated the feasibility of the employed sol-gel and
supercritical methods for the development of DXMT-loaded composite materials with potential uses for
pharmaceutical and biomedical applications.
INTRODUCTION
In recent years, bioactive glasses (BGs) have been used as important biomaterials in implants, fixation devices
and other hard tissue engineering applications [1-3]. The most used and best characterized BG is type 45S5,
which is a silicate glass that contains 45% SiO2, 24.5% NaO2, 24.5% CaO and 6% P2O5 (w/w) [1] and that has
been already employed in several FDA-approved implants and devices [2]. BGs present favorable and adequate
chemical, physical and mechanical properties as well as high bioactivities which will induce the development of
a surface apatite-like layer when in contact with physiological fluids [1, 3-6]. This will promote enhanced in vivo
bone bonding and integration without causing inflammation, toxicity or forming fibrous tissues [1, 3]. Once the
layer is formed, a firm connection between BGs and the bone structure (host bone) is allowed, owing to the
similar mineral composition from the layer and bone and to the leaching of Na+ and Ca2+ cations to the
surrounding fluids [1, 3, 4].
These inorganic materials are usually prepared by melting and quenching methods despite the fact that they can
also be prepared using sol-gel methods. However, conventional sol-gel techniques usually require the use of
harsh reaction conditions such as high pressures and/or temperatures, extremes in pH and the use or generation
of caustic chemicals. On the contrary and in nature, the production of amorphous biosilica can be accomplished
under mild conditions.
Recently, we discovered a new biomimetic catalyst for TEOS hydrolysis and condensation: L-glutathionereduced (GSH). Because of its acidic character in aqueous solutions, GSH can be employed alone or titrated
against cysteamine (Cys, basic catalyst already reported in literature) for the formation of silica-based materials
over broad and tunable ranges of pH (including neutral pH). Therefore, these strategies can be used to achieve
the most favorable pH conditions that should be employed for the production of silica-based materials and in
terms of the desired final biological properties (such as biocompatibility, cell toxicity and biodegradability) and
mechanical/morphological properties (such as stability, porosity, nano-rugosity and surface area).
To further improve the osteogenic properties of implanted BGs, several bioactive molecules such as specific
growth factors and dexamethasone (DXMT) can be incorporated into these materials. The supercritical carbon
dioxide (scCO2) deposition method permits to have previously prepared inorganic materials and later deposit
into them a desired scCO2-soluble bioactive molecule, considering the envisaged therapeutic application and
without interfering with materials manufacture and/or processing methods. Moreover, this can be performed
avoiding the use of high temperatures (which may degrade the involved thermo-labile substances) and of organic
solvents (which allow the deposition of those hydrophobic substances that cannot be deposited by aqueous
solution/suspension soaking methods) [7-13].
In this work, we prepared bioactive glasses (BGs) (based on SiO2, P2O5, Na2O, CaO) and silica-calcium
hydroxyapatite (HA) composite materials using a one-pot aqueous sol-gel method at 37 ºC and at atmospheric
pressure [2-4] and using the two above referred biomimetic catalysts (GSH and Cys). Other precursors included
triethyl phosphate (TEP), calcium nitrate tetrahydrate (CaNT) and sodium nitrate (NaNO3). Additional
composite samples were obtained by replacing TEP, CaNT and NaNO3 by synthetic calcium hydroxyapatite
particles or by changing the employed amount of water. In addition, some samples were also obtained by adding
chitosan as an organic porogenic agent. All synthesized wet materials were dried and submitted to a specific
sinterization process (from 60 ºC up to 1000ºC) [2]. Obtained solid materials were later loaded with DXMT by a
scCO2 deposition process or by conventional loading from aqueous dexamethasone solutions. Drug loading
yields were changed by controlling scCO2 densities (755-920 Kg/m3) at a constant depressurization rate (0.5
MPa/min) and processing time (14 hours). Loading yields and drug release profiles were obtained
spectrophotometrically for both drug loading methods All obtained materials were characterized by mercury
intrusion porosimetry, nitrogen adsorption, helium pycnometry, simultaneous differential thermal analysis
(SDT), scanning electron microscopy (SEM), infra-red spectroscopy (FTIR) and X-ray diffraction.
MATERIALS AND METHODS
GSH (Sigma-Aldrich, >99.0%) and Cys (Sigma-Aldrich, >98.0%) were used as biomimetic catalysts. The
following chemical were used as precursors for the sol-gel 45S5 synthesis: tetraethyl orthosilicate (Aldrich,
>99.0%), triethyl phosphate (Aldrich, >99.8%), sodium nitrate (Sigma-Aldrich, >99.0%) and calcium nitrate
tetrahydrate (Sigma-Aldrich, >99.0%). Hydroxyapatite (Agoramat) and chitosan (medium molecular weight,
Sigma-Aldrich), dexamethasone (Sigma-Aldrich, >98.0%) and carbon dioxide (99.998%, Praxair, Spain) were
also used.
Bioactive glasses (BG) (based on SiO2, P2O5, Na2O, CaO) and silica-calcium hydroxyapatite (HA) composite
materials were prepared using a one-pot aqueous sol-gel method at 37 ºC and at atmospheric pressure.
Biomimetic catalysts aqueous solutions were prepared: GSH (0.15M, pH 2.56-2.66) and Cys (0.15M, pH 8.969.20). TEOS, HA, CHIT and other precursors (including TEP, CaNT and NaNO3) were also and the employed
amounts are presented in Table 1. The catalyst solution at neutral pH (GSH+Cys, pH 7.03-7.07, 24.3ºC) was
prepared by the titration of the previously prepared GSH and Cys solutions until neutral pH was attained.
Additional silica-HA composite samples were obtained by replacing TEP, CaNT and NaNO3 by synthetic HA
powders. For other samples, CHIT was added (0.4 g, 2% w/v) to the GSH solution and mixtures were stirred
overnight until complete CHIT dissolution. The final pH of this solution was pH 3.10-3.15.
All reactions were carried for 25 days, for all the employed catalytic systems. After synthesis, obtained materials
were centrifuged (5000 rpm, 10 min), the supernatant was removed and samples were freeze-dried at -47 ºC for
72 hours (Snijders Scientific). Then, samples were grinded and submitted to the sinterization process (at 60, 200,
600 and 1000ºC for 72, 40, 5 and 2h, respectively) [2]. Heating rate was 8ºC/min.
Obtained solid materials were later loaded with dexamethasone by a scCO2 deposition process. Obtained BG or
HA powders (~90 mg) were placed into sealed dialysis bags and then introduced in a high pressure view-cell
which was previously loaded with ~34 mg of DXMT. Drug loading yields were changed by controlling scCO2
densities (755-920 Kg/m3), i.e., by performing experiments at 313K/13.6 MPa, and at 313 K/32.0 MPa.
Employed depressurization rate was 0.5 MPa/min and processing time was 14 hours.
DXMT-loaded BG and HA powders (~10 mg) were placed into sealed dialysis bags and then immersed and
stirred at 310 K in 10 mL of MilliQ water. At pre-determined time intervals, an aliquot was removed and
analyzed in a UV-Vis spectrophotometer at 242 nm.
Table 1 – Synthesized samples and employed chemical amounts.
Catalyst
solution
(ml)
BG
BG + CHIT
BG + water
HAs
HA + CHIT
CHIT
(g)
TEOS
(ml)
TEP
(ml)
Ca(NO3)2.4H2O
(g)
NaNO3
(g)
MilliQ
Water
(ml)
HA (g)
0.40
-
8.95
8.95
8.95
8.95
8.95
0.67
0.67
0.67
-
5.53
5.53
5.53
-
3.73
3.73
3.73
-
10.0
-
2.35
2.35
20.0
20.0
20.0
20.0
20.0
0.40
All obtained materials were chemically and physically characterized. Mercury intrusion porosimetry (Autopore
IV, Micromeritics) was performed to determine pore size distribution while real density was determined by
helium pycnometry (Accupyc 13330, Micromeritics). Surface area, pore diameter and pore volume were
determined nitrogen adsorption (ASAP 2000 V2.04, Micromeritics). Prepared/processed samples were also
characterized by scanning electron microscopy (XL30, Phillips), by FTIR (ATR and transmission modes)
spectroscopy (4000, Jasco) and by X-ray diffraction (XRD) (X’Pert, Phillips). Simultaneous differential thermal
analyses (SDT) were also carried out (Q600, TA Instruments).
RESULTS AND DISCUSSION
It was observed that obtained BGs presented similar chemical compositions as those obtained with conventional
catalysts [2]. In addition, FTIR confirmed the decomposition of the two nitrates during the sinterization thermal
treatment (1042, 820 and 744 cm-1 bands, Figure 1A). A crystalline phase assigned to the P-O deformation
modes in crystalline phosphate is also detected (622 and 617 cm-1 bands) in sinterized samples obtained from
GSH+Cys and from Cys (Figure 1B). On the other hand, the symmetrical Si-O-Si stretching vibration on SiO44of crystalline silicate is detected after sinterization and for samples prepared by using GSH (729 and 697 cm-1
bands). FTIR also confirmed the presence of characteristic silica bands such as hydrogen-bonded water
stretching bands (3470-3450 cm-1), adsorbed water molecules deformation vibrations (1653-1634 cm-1) and
surface silanol groups and silicon-oxygen covalent bonds vibrations (1200-1000 cm-1). Si-O-Si symmetric
stretching vibration (800 cm-1) and the corresponding bending mode (469-466 cm-1) are also present [2].
XRD spectra of prepared BGs (obtained using the different catalytic systems) show that the Na2Ca2Si3O9
crystalline phase could be identified on that BG obtained from GSH (BG-GSH). On samples obtained from Cys
and from GSH+Cys mixtures, it was possible to find a different crystalline phase (mainly Na2Ca3Si6O16) as well
as a specific SiO2 crystalline cristobalite form (and as already found by FTIR). The Na3PO4 phase could also be
identified in the BG-Cys sample (Figure 2).
We recently find that different pH conditions and different biomimetic catalysts (GSH, Cys and GSH+Cys
mixtures) strongly affected the final morphological and mechanical properties of silica-based materials prepared
by the employed aqueous sol-gel methods [13]. This can also be observed in Figure 3 in terms of the obtained
surface areas and real densities of prepared BG and HA samples.
4
BG-GSH
A
Absorbance, u.a.
2
B
BG-Cys
3
Absorbance
BG-Cys without Thermal Treatment
BG-Cys with Thermal Treatment
BG-GSH+Cys
1
0
3400
2400
1400
Wavenumber,
cm-1
400
3400
2400
1400
400
Wavenumber, cm-1
Figure 1. FTIR spectroscopy of some selected BG samples with/without sinterization thermal treatment (A) and sinterized BGs obtained
using different catalysts (B).
BG- GSH
BG-Cys
BG- GSH+Cys
10
20
30
40
50
60
70
80
2θ(o )
Figure 2. XRD patterns of BGs obtained using different catalysts.
Higher surface area and lower real density were obtained for BGs prepared by using a GSH+Cys catalytic
system (Figure 3A). Despite they present similar real densities, these samples presented much lower surface
areas than those prepared with HA (Figure 3B). In fact, the obtained higher surface areas in HA samples is
certainly related to the presence of added calcium hydroxyapatite particles, since there is no HA shrinkage
during the sinterization process (as happens with its silica counterpart). The employed porogenic agent (CHIT)
did not greatly affect the surface areas and real densities of prepared BGs but it dramatically increased the
surface area of prepared HA composites. However, the real density of these materials are quite similar to other
prepared BG and HA composites.
A
GSH
Cys
GSH/Cys
GSH
+ CHIT
GSH
+ H2O
B
GSH/HA
Cys/HA
GSH/Cys/HA
GSH/HA
+ CHIT
Figure 3. Selected morphological characterization results of prepared BGs and HA samples with different catalytic systems: ■ surface area
(m2/g) and □ real density (g/m3).
This can also be verified by SEM results (Figure 4). Biomaterials surface areas, pore sizes and geometries, pore
volumes and densities, as well as surface rugosities are very important parameters to consider in tissue
engineering applications. Despite all prepared samples present quite rugged surfaces, those composites formed
by silica and HA clearly present much higher surface rugosities (for all employed catalytic systems).
Mesoporous materials (1.5 nm < average pore diameters < 15-57 nm) were obtained for all prepared BG and HA
samples (results not presented).
As an example, the thermogravimetric and calorimetric results of a BG sample prepared using GSH as the
catalyst are shown Figure 5. The thermal events associated to the sinterization process (> 600 ºC), to the removal
of water and of un-reacted TEOS (50-180 ºC) and of GSH degradation (200 ºC) can be clearly seen [6, 13]. The
sinterization process started around 700 ºC (with nitrate and silanol removal) and continued by crystallization
(900-1200 ºC), probably leading to the formation of a cristobalite phase, as previously indicated by XRD results.
Cristobalite is a high-temperature silica polymorph that usually forms between 945 °C and 1085 °C [14].
BG-GSH
BG-Cys
BG-GSH+Cys
A
BG-GSH/HA
BG-Cys/HA
BG-GSH+Cys/HA
B
Figure 4. Selected SEM microscopy results of prepared BGs and HA samples with different catalytic systems.
20
8
15
Weight, mg
Catalysts removal
5
0
6
Onset of
crystallization
and silica
polymorphism
5
Nitrate and silanol
removal
4
-5
-10
Heat Flow (mW)
10
7
-15
-20
Water and
chemical residuals
removal
-25
3
-30
0
200
400
600
800
1000
1200
Temperature, ºC
Figure 5. Thermogravimetric and calorimetric SDT profiles for a BG obtained using GSH as catalyst.
DXMT loading was carried out by scCO2 deposition into prepared BG and HA samples. Sustained drug release
profiles were always observed for all prepared and processed BG and HA samples. A selected example is shown
in Figure 6. Even after the presented 10 h release period, it can be observed that scCO2-loaded DXMT was not
completely released from BG samples. It is also clear that samples loaded at lower density conditions can uptake
more DXMT. This can be explained by a higher DXMT affinity for BG than for the scCO2 phase and it is a
common behavior on the impregnation/deposition of those additives presenting relatively low solubilities in
scCO2 and favorable interactions with the material to be loaded.
Dexamethasone/BG, µg/mg
0.25
0.20
0.15
0.10
0.05
0.00
0
2
4
6
8
10
Time, h
Figure 6. Dexamethasone release from BG samples obtained using Cys as catalyst. Samples were processed for 14h and depressurization
rate was 0.5MPa/min. Loading was performed at different scCO2 densities: (○) 755 Kg/m3 (13.6 MPa, 313 K) and (●) 920 Kg/m3 (32.0 MPa,
313 K).
CONCLUSIONS
Different silica formation pH conditions, different biomimetic catalysts (GSH, Cys and GSH+Cys mixtures)
employed at distinct concentrations and the use of chitosan as a porogenic agent, strongly affected the final
morphological and mechanical properties of BGs and of silica-HA composite materials prepared by the
employed aqueous sol-gel method. Consequently, these different properties also had some effect on other
relevant properties for the intended applications such as DXMT loading yields, DXMT release profiles and
materials biological properties (not presented). Nevertheless, these results demonstrated the feasibility and the
associated advantages of the employed aqueous sol-gel method and of the scCO2 deposition process for the
development of dexamethasone-loaded BGs and silica/HA composites for potential biomedical and tissue
engineering applications. However, more work still needs to be performed in order to deeply understand the
already obtained results as well as to optimize the efficiencies of the employed experimental methodologies This
will include the study of other related composite systems and compositions, and the optimization of the scCO2
deposition method operational conditions, of the drug release experimental conditions and methodologies, and of
the hemocompatibility/ biological assays.
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