synthesis of sol-gel mesoporous silica materials providing a slow release of doxorubicin

New XML Template (2007) [6.8.2007–12:21pm] [694–713]{TANDF_REV}TMNC/TMNC_I_24_07/TMNC_A_254625.3d (TMNC) [Revised Proof]

Journal of Microencapsulation, November 2007; 24(7): 694–713

Synthesis of sol-gel mesoporous silica materials providinga slow release of doxorubicin

MAGDALENA PROKOPOWICZ1 & ANDRZEJ PRZYJAZNY2

1Medical Academy of Gdansk, Division of Physical Chemistry, Gdansk, Poland

and 2Chemistry & Biochemistry Department, Kettering University, Flint, MI, USA

(Received 30 August 2006; revised 29 June 2007; accepted 29 June 2007)

AbstractSamples of mesoporous base-catalysed silica xerogel materials made by the sol-gel process wereimpregnated with an anticancer drug—doxorubicin, followed by different times of ageing at roomtemperature. The effect of ageing time on the physical and structural properties as well assorption–desorption of the drug was investigated. The obtained results suggest an inverse relationshipwith a solid density and surface area increasing as the pore size and volume decrease during ageingtime. These results also revealed the effect of ageing time on the efficiency of sorption–desorptionof the drug. An increase in ageing time results in an increase of the efficiency of drug sorption anda decrease in the rate of drug release.

Keywords: Porous silica polymers, sol-gel methods, sorption–desorption process, doxorubicin, controlled release

Introduction

Doxorubicin is an anthracycline cytostatic antibiotic with activity against a wide range

of human cancers, especially in the treatment of bone sarcoma (Coukell and Spencer 1997;

Jain 2001). At present, the treatment is systematic and due to the narrow therapeutic index

of doxorubicin a substantial increase in systematic dose to achieve high concentration of the

drug at the bone sarcomas is not possible. Additionally, the bone is a moderately perfused

organ and the chance of achieving an effective therapeutic efficacy of doxorubicin is likely to

be low (Fan and Dash 2001).

The treatment with doxorubicin is associated with severe toxic side effects, which include

dose-limiting cardiotoxicity and myelosuppression (Rahman et al. 1986; Bally et al. 1990;

Chabner et al. 1996). To reduce the toxicity of doxorubicin and improve delivery to tumour

site, various targeted drug delivery systems such as liposomes (Li et al. 1998), nanoparticles

(Yoo et al. 2000), microspheres (Stolnik et al. 1995), conjugates and polymeric micelles

Correspondence: Magdalena Prokopowicz, Medical Academy of Gdansk, Division of Physical Chemistry, Hallera 107, 80-416

Gdansk, Poland. Fax: 48-58-3493206. E-mail: [email protected]

ISSN 0265–2048 print/ISSN 1464–5246 online � 2007 Informa UK Ltd.

DOI: 10.1080/02652040701547658


(Fan and Dash 2001; Greish et al. 2004) have been used. However, these delivery systems

are usually administered intravenously and are not adequate for the treatment of bone

cancer. Local drug release in the implanted site appears to be a very interesting alternative.

The addition of anticancer drugs to implantable and degradable delivery matrix is a

promising strategy of modifying their biodistribution, of reducing drug toxicity and thus

improving the therapeutic efficacy of anti-tumour agents. The advantage of localized

degradable therapy includes high local drug concentration only at the site of implantation, as

well as obviation of the need for removal of the implant after treatment. Recently,

hydroxyapatite implants (Itokazu et al. 1996) and gelatin cross-linked with glutaraldehyde

implants (Fan and Dash 2001) containing doxorubicin were investigated as a potential,

implantable delivery system for treatment of bone cancer.

In this work, silica biomaterials were synthesized by the sol-gel process because of their

potential application to deliver a drug precisely to a targeted site or to be used as an

implantable drug delivery system (Kortesuo et al. 2001; Prokopowicz et al. 2004;

Czuryszkiewicz et al. 2005; Prokopowicz 2007). The sol-gel process is initiated by adding

water to the alcoholic solution of tetraethylorthosilicate. Following hydrolysis and

polycondensation reactions, a two-phase system is obtained as a result of gel formation.

The process of making silica gel matrices can be modified by varying physicochemical

parameters of gelation, such as temperature, kind of reactants used, their ratio, pH of the

solution and the kind of catalyst used (Iler 1979; Brinker 1996; Meixner et al. 1999).

These manufacturing parameters will have an indirect effect on physicochemical properties

of the silica polymer obtained, including specific surface area, kind, size and distribution of

the pores, degree of cross-linking of the gel, refractive index, hardness, etc. An important

factor influencing the structure of silica xerogels is pH of the solution. In case of an acidic

catalyst, rapidly proceeding hydrolysis causes the formation of linear polymers, resulting in a

solid material with microporous structure. In contrast, basic catalysis favours the formation

of agglomerate consisting of branched colloidal particles, which yields a mesoporous

material with high adsorption capacity (Livage 1997). Authors like Iler (1979) and Brinker

(1996) have elaborated on the whole sol-gel chemistry of silica.

Amorphous biomaterials obtained in this way are characterized by high purity and

homogeneity (Livage 1997), in vivo biocompatibility and bioactivity (Kortesuo et al. 1999,

2001) and slow degradation (Kursawe et al. 1998; Kortesuo et al. 2001; Radin et al. 2002).

The growing interest in these systems is based on their ability to induce in vitro formation

of a hydroxycarbonateapatite-rich layer. This behaviour is considered as an indication

of their in vivo bioactivity (natural, direct bounds with living tissues) (Petruzzeli et al.

1997; Hench et al. 1998). Therefore, the connecting of bioactivity function of these

materials with the function as a drug delivery system—i.e. to bone systems, also seems to be

interesting.

A drug can be introduced into a gel either at the stage of sol formation or after the

formation of a porous xerogel. In the former case (pre-doping), the molecules of a drug are

entrapped within the porous silica gel network as a result of polycondensation (Livage 1997;

Dunn et al. 1998). In the latter case (post-doping), the gel matrix is saturated with a drug

after being stirred for several hours with an excess of drug solution or suspension (Otsuka

et al. 2000; Chen et al. 2004). The important step of preparing drug-loaded silica materials

is their drying, i.e. removing a solvent, such as water. The scommon drying techniques of

wet silica include heating to temperatures above 100�C (Kortesuo et al. 1999), supercritical

drying technique (Hua et al. 1995), under vacuum (Prokopowicz et al. 2005), as well freeze-

drying (Kocklenberg et al. 1998). The choice of an appropriate drying technique of

Synthesis of sol-gel mesoporous silica materials providing a slow release of doxorubicin 695


doxorubicin-loaded xerogels is very important because doxorubicin is thermosensitive and

traditional drying at an elevated temperature is impossible (Prokopowicz et al. 2004).

This paper presents a new approach to the preparation of mesoporous silica materials with

doxorubicin based on using the base-catalysed sol-gel post-doping method. Freeze-drying

was selected as a drying technique for the matrices obtained. Silica xerogels with the desired

porosity were immersed in a doxorubicin solution, followed by different times of ageing at

room temperature. The sorption of the drug on mesoporous silica xerogels was almost

complete and long stirring was not needed. The mechanism used here was based on the

spontaneous deposition of water-soluble molecules (Gao et al. 2002; Liu et al. 2005) and

the deposited substance was either in aggregated or complexed form in the silica xerogels.

This work focuses on the quantitative sorption and subsequent release behaviours of

deposited doxorubicin.

First, the molecular surface structure of silica xerogel was investigated by ATR/FTIR

spectroscopy and SEM microscopy. Some of the key physical properties such as pore size,

pore volume and specific surface by the BET method were studied. Next, the effect of ageing

of xerogels at ambient conditions on the sorption efficiency of the drug was determined

and the drug desorption and re-release examined.

Experimental

All reagents and the drug were obtained from Aldrich-Sigma Chemical Company (Poland)

and used without further purification.

. Reference substance: Doxorubicin hydrochloride 200 mg, 99%, C27H29NO11HCl;

surface area¼ 7.37 nm2, volume¼ 1.34 nm3 (Turker 2002). The molecular structure is

presented in Figure 1

O O OHH3C

O OH

H O

OH

C

O

CH2OH

O

HO

NH3+

Figure 1. Doxorubicin in protonated form.

696 M. Prokopowicz & A. Przyjazny


. tetraethoxysilane (TEOS, 98%); ethanol 98%(v/v); ammonia 25%(v/v); and

. simulated body fluid (SBF) had the following composition: NaCl (136.8 mM), NaHCO3

(4.2 mM), KCl (3.0 mM), K2HPO4 � 3H2O (1.0 mM), MgCl2 � 6 H2O(1.5 mM), CaCl2 � 2

H2O (2.5 mM) and Na2SO4 (0.5 mM). It was buffered at pH 7.4 with tris(hydrox-

ymethyl)aminomethane (TRIS) (50 mM). Deionized water was used for the preparation

of buffers and reagent solutions (pH¼ 6.6).

Preparation of standard solutions of doxorubicin by a static (volumetric) method

A weighed sample of doxorubicin hydrochloride (DOX) 9.2� 0.1 mg was dissolved in

50 mL of deionized water or simulated body fluid (SBF), respectively, in a silanized

polypropylene volumetric flask in the absence of light. Various standard solutions of

doxorubicin (0.368–184.0 mg ml�1) were then prepared from this stock solution after

proper dilution. The solutions were prepared quickly to limit the exposure to light and

carefully due to a high toxicity of doxorubicin. The pH of aqueous standard solutions

ranged from 6.6–6.8. The pH of SBF-based standard solution was 7.4. The pH values of the

DOX solution were determined with a pH meter (Beckman Instrument).

Spectrophotometric measurements

A Jasco V-560 UV/VIS spectrophotometer in the classical mode was used for quantitative

determinations of doxorubicin in various types of solutions (at � close to 480 nm).

Qualitative determinations of doxorubicin-loaded silica particles were made using a UV/VIS

spectrophotometer equipped with an integral sphere and sample holder. Magnesium oxide

and pure silica gel powders were used as the references for doxorubicin-loaded silica

measurements.

Synthesis of porous silica xerogels

The mesoporous xerogels were obtained via the sol-gel process, by hydrolysis and

condensation of TEOS using ammonia as a catalyst. The preparation was performed at

room temperature and under atmospheric pressure in a closed polypropylene flask. Initially,

tetraethoxysilane (6.93 g) with ethanol (27.5 g) were slowly stirred for 15 min. Next, 1 mL

of deionized water with ammonia (pH 10.8) was added. The obtained mixture was sonicated

in a cold water bath until it was clear and homogeneous (8–10 min). The total molar ratio

of TEOS:H2O:C2H5OH:NH4OH was held constant at 1:4:17:0.016.

The solution was allowed to stand undisturbed for gelation at room temperature for

2 days. Next, the resulting wet gels were subjected to ageing in a closed flask at ambient

conditions, for varying times: 24 h (S1), 168 h (S2) and 336 h (S3). After that, the samples

were freeze-dried in the drying chamber of an Alpha 1-2 LD Freeze-Dryer

(Christ, Germany) and cooled to �55�C. Lyophilization was performed at a pressure

of 2 Pa for 48 h.

The resulting gels were crushed using a mortar and then sieved to obtain grains with

a desired diameter 500–700 mm.

Characterization of freeze-dried silica xerogels

The samples were outgassed for 24 h at 25�C under vacuum for determining pore size,

surface area and pore volume by the BET method, using an automatic gas sorption analyser

(Autosorb 1, Quantachrome). Experimental error was �5%. The samples were also



outgassed to a residual pressure of �1 Pa prior to measurement of bulk density calculated

from the following formula:

�1 ¼m1 � �0

ðm1 �m2Þð1Þ

where �1¼ bulk density of the gel, m1¼mass of the dry gel, m2¼mass of the gel immersed

in ethanol, �0¼density of ethanol (0.81 g mL�1 at 20�C). Porosity was calculated assuming

a skeletal density of 2.2 g cm�3) (Iler 1979).

The morphology of the xerogels was observed by scanning electron microscopy (ESEM,

Philips) at an acceleration voltage of 30 kV. Samples for SEM were fixed on carbon tape

and fine gold sputtering was applied for 2 min.

A FTIR spectrometer (Jasco model 410, 4 cm�1 resolution) equipped with a horizontal

attenuated total reflectance accessory (45� angle of incidence, zinc selenide ATR prism) was

used to record HATR/FTIR spectra of the xerogel surface in the range of 1400–750 cm–1.

The thickness of the gel layer was � 1 mm, i.e. much greater than the effective penetration

depth of the IR light passing the ATR crystal and attenuating in the bulk of the sample.

In order to determine the fractions of Si-OH and Si-O– groups in the silica network,

the spectra were deconvoluted between 1400–900 cm–1. The fitting was performed by a

non-linear least squares method, using Gaussian and Lorentzian functions. For a better

comparison, the spectra were normalized to a maximum absorption of the dominant peak at

�1070 cm–1, attributed to the asymmetric stretching of siloxane bands. The proportion

of Si-OH and SiO– groups was calculated by the ratio: (area of SiOH/SiO–1 bands)/[total

area of the �as Si-O-Si band) plus (area of SiOH/SiO–1 bands)] according to Fidalgo and

Ilharco (2004).

Silica xerogel sorption experiments

The sorption experiments were carried out as follows: 20� 0.1 mg of freeze-dried xerogel

grains ranging from 500–700 mm was added to 3 mL of a water-standard DOX solution

(pH close to 6.6), and shaken in a shaking water bath (60 shakes per minute) for a definite

period of time at room temperature (22� 2�C) and kept out of the light. The particle size

and mass of silica were kept constant. The amounts of DOX in the bulk solution were 920,

460 and 230 mg, respectively. At specific time intervals (60, 120, 240 and 300 min), an

aliquot of the solution was centrifuged for 4 min at 10 000 rpm and the supernatant was used

for the spectrophotometric measurement of the amount of drug in the bulk solution. Then,

the xerogels and the supernatant were transferred back into the original vials for further

sorption experiments. The experimental conditions, such as pH and temperature, were kept

constant throughout the entire investigation period because of rapid decomposition of

DOX. According to the literature, doxorubicin hydrochloride is stable in acidic medium in

the pH range 4.5–6.5, but rapid decomposition occurs at a higher pH (6.5–12) (Li et al.

1998). The temperature also affects the degradation of doxorubicin and with its increase

the decomposition of doxorubicin accelerated.

To simplify data analysis, the DOX loading amount (mass) by xerogels was quantified as a

function of mass of drug in bulk solution. The efficiency of doxorubicin sorption by silica

xerogels was calculated from the following formula:

E ¼W0 �We

W0

� �� 100% ð2Þ



where W0 and We are the masses of DOX in a standard solution before (initial) (W0) and

after specific time of sorption (We), respectively.

Quantitative determinations of the amount of DOX in the bulk solution were based on

pre-calibration of the spectrometer using standard solutions of the drug in deionized water.

The theoretical mass of doxorubicin monolayer on 1 g of the silica surface was calculated

using the following formula (Ohta et al. 2005):

W ¼SBET �ms �M

SDOX �NA

� 103 ð3Þ

where W is mg of DOX on the unit mass of silica xerogel, SBET is the specific surface area

of silica xerogels (experimental data in Table I), ms is the mass of silica xerogel¼ 1 g, M is the

molecular weight of DOX in the protonated form¼ 544 g mol�1, SDOX is the surface area

of DOX¼ 7.37 � 10�18 m2, NA is the Avogadro’s number in mol�1.

After sorption experiment, the DOX-loaded silica gels were used in spectrophotometric

measurements to determine the quality of DOX form in the solid.

Desorption studies of doxorubicin hydrochloride under simulated physiological conditions

The desorption of DOX was studied to determine the rate of drug release for this system

under physiological conditions. The release test (each data point is the mean of three

values� confidence interval) was carried out both to compare the rate of drug release from

xerogels aged for various periods of time (with the same amount of DOX loaded) and to

examine the effect of different loading amounts on drug release. In the former case, the

release test was carried out for the xerogels immersed in the bulk solution containing 920 mg

of DOX for 300 min, resulting in the mean total loading of 910 (�5), 900 (�3) and 915

(�9) mg for S1, S2 and S3, respectively. In the latter case, the release studies were carried out

for the same type of xerogels containing various amounts of DOX. In these experiments,

xerogels were immersed in the bulk solutions containing 920, 460 and 230 mg of DOX,

respectively, for 300 min, resulting in the mean DOX loading amounts of 900 (�12),

440 (�8) and 212 (�8) mg for S1, 910 (�10), 400 (�12) and 215 (�10) mg for S2 and

890 (�10), 420 (�5) and 210 (�6) for S3.

First, batches of DOX-loaded xerogels removed from the liquids after sorption

experiment were freeze-dried under the same conditions as mentioned above. The release

test was performed in the SBF solution of pH 7.4 under sink condition of DOX. A volume

of SBF was added in such a way that the calculated maximum DOX concentration of the

solution was the same after complete liberation for all xerogels (in these experiments,

17 mg mL�1 was desired). The mixtures were agitated by shaking in a shaking water bath

(60 shakes per minute) at 37� 0.02�C. At specific time intervals (5 min, 1 h), an aliquot

Table I. Some physical properties of the obtained xerogels: mezopore size (rmp), surface area (SBET), bulk density

(�) and mezopore volume (Vmp). Comparison between structural: ATR/IR position of �as (Si–O–Si) and percentage

of dangling oxygen bridges (Si–OH and Si–O�) and physical properties.

Sample rmp (nm) SBET (m2 g�1) � (g cm�3) Vmp (cm3 g�1)

Dominant mode

of Si–O–Si (cm�1)

Fraction of

Si–OH and Si–O� (%)

S1 10.2 186 0.68 0.99 1071 14

S2 8.5 202 0.72 0.87 1080 11

S3 6.2 248 0.85 0.73 1084 8.8



of the solution was centrifuged for 4 min at 10 000 rpm and the supernatant was used for the

measurement of the amount of released drug. Then, the xerogels and the supernatant were

transferred back into the original vials for further release experiments. The experimental

conditions were kept constant throughout the entire investigation period to avoid

decomposition of DOX, mentioned above.

The experiments were terminated when the equilibrium was reached. The percentage by

mass of desorbed DOX at equilibrium was calculated from the following formula:

Xe

X� 100% ð4Þ

where Xe is the measured mass of desorbed DOX at equilibrium, and X is the calculated

mass of DOX in xerogel taken for release study.

Quantitative determinations of the amount of DOX in the bulk solution were based

on pre-calibration of the spectrometer using standard solutions of the drug in simulated

body fluid.

Effect of release medium change on desorption of drug

The effect of release medium change on the desorption of drug was examined for xerogels

aged for various periods of time and immersed in an initial bulk solution containing 920 mg

of DOX for 300 min, resulting in the calculated mean total loading amount of 890 (�7),

905 (�5) and 900 (�10)mg for S1, S2 and S3, respectively. In order to examine this effect,

after the equilibrium was reached, the release medium (supernatant) was removed and

replaced with an identical volume of the fresh medium. SBF was used as a release medium.

The final parts of experimental procedure and conditions were the same as mentioned

above. The experiments were terminated when the amount of released DOX was below the

detection limit (0.005 mg mL�1).

The percentage by mass of desorbed DOX was calculated from the following formula:

Xe,i

X �Pi�1

i¼0 Xe,i

� 100% ð5Þ

where Xe,i is the measured mass of desorbed DOX at equilibrium, i is the number of changes

of release medium, ðX �Pi�1

i¼0 Xe,iÞ is the actual mass of DOX in the xerogel and X is the

calculated mass of DOX in the xerogel taken for release study, see equation (2).

Influence of temperature of release medium on drug desorption

In order to examine the effect of temperature on drug re-release (each data point is the mean

of three values � confidence interval), xerogels were immersed in the bulk solution

containing 460 mg of DOX for 300 min, resulting in the mean total loading amount of 450

(�5), 440 (�12) and 455 (�4) mg for S1, S2 and S3, respectively. DOX release from the

xerogels was examined at various temperatures (22, 37, 47, 57 and 67� 0.02�C) and

deionized water was used as a release medium. After the equilibrium was reached at a

specific temperature, the temperature was changed and the experiment was continued.

The final parts of experimental procedure and conditions were the same as those in the

desorption study.

The percentage by mass of desorbed DOX was calculated from the following formula:

Xe,i

X� 100% ð6Þ



where Xe,T is the measured mass of desorbed DOX at equilibrium at a specific temperature,

and X is the calculated mass of DOX in the xerogel taken for release study.

Influence of pH of release medium on drug desorption

In order to examine the effect of pH on drug release (each data point is the mean of three

values� confidence interval), xerogels were immersed in the bulk solution containing 230 mg

of DOX for 300 min, resulting in the calculated total loading amount of 215 (�5), 220 (�8)

and 225 (�2) mg for S1, S2 and S3, respectively. DOX release from the xerogels was

examined in acidic or basic aqueous solutions at various pH values (4, 6 and 8, respectively)

at 37� 0.02�C. The final parts of experimental procedure and conditions were the same as

those in the desorption study and standard solutions of DOX with the corresponding

pH values were used for calibration. At the end of the pH study, samples were removed from

the medium, freeze-dried and weighed to determine the weight loss.

Results and discussion

Some of the key physical properties determined for all the xerogel samples are summarized

in Table I: mesopore size (rmp), specific surface area (SBET), bulk density (�) and mesopore

volume (Vmp). The shape of adsorption–desorption isotherms for all the xerogels studied

(data not shown) was of type IV, characteristic of a mesoporous solid, defined as a material

with pores larger than 2.0 nm (Lowell and Shields 1991). A characteristic feature of type IV

isotherm is the hysteresis that is normally attributed to the presence of pore cavities larger in

diameter than the openings (necks) leading into them, forming the so-called bottle-neck or

ink-bottle character of pore system.

In this study, all xerogels were prepared at a low hydrolysis ratio (water to TEOS ratio

4 : 1) and under basic conditions. These conditions favour the formation of highly branched

silica clusters that do not interconnect before gelation and thus behave as discrete species.

These clusters form relatively large and branched polymers that grow by a growing

polymeric fractal structure at the expense of the reformation and shrinkage. During the

ageing process, as polycondensation continues, the degree of cross-linking between

silica particles increases and results in gel shrinkage and expelling solvent from the pores

(Iler 1979; Buckley and Greenblatt 1994). As a result of such gelation and ageing process,

the obtained xerogels after various times of ageing have mesoporous structure and an

increase in ageing time increases bulk density and surface area and decreases pore volume

and slightly pore size, as shown in Table I.

Figure 2 shows the ATR spectra of xerogels in the region characteristic for silica

(1400–750 cm�1). The ATR results show that for all the samples under study there are two

dominant features at �1080 cm�1 and 1170 cm�1 (broad shoulder) that are attributed to

asymmetric stretching vibrations of Si-O-Si in TO (transverse optical) and LO (longitudinal

optical) modes, respectively (Innocenzi 2003). The band around 790 cm�1 is associated with

the symmetric stretching mode of the Si-O-Si group. The band centred at � 950 cm�1

is associated with the stretching mode of non-bridging oxide bands: Si-OH and Si-O�.

The spectra indicated that the SiO2 network at �1071 cm�1 for xerogels aged for 24 h shifts

toward a higher wavenumber and becomes narrower as the ageing time increases. It is also

correlated with a decrease in the wavenumber of stretching mode of the Si–OH and SiO�

groups. The calculated fraction of Si–OH and Si–O�1 and the position of the dominant peak

of �as (Si–O–Si)TO presented in Table I reveal that, with the upshift of dominant mode and

decrease of relative intensities of the non-bridging oxygen, the density and also specific



surface area of xerogels increase. The position of the �as (Si–O–Si)TO mode is highly

dependent on the average Si-O-Si angles and bond lengths, thus reflecting the distribution

of primary silica network units (mostly cyclic siloxane units containing four or six Si atoms)

(Fidalgo and Ilharco 2004). According to the central force network model (Innocenzi 2003),

the upshift in �as (Si–O–Si)TO corresponds to a decrease in the Si–O bond length and an

increase in the Si-O bond strength, resulting in siloxane rings with smaller pores.

Such a decrease leads to increased density of silica. These results also indicate that the

formation of new Si–O–Si bonds and the reduction in number of Si–O–H and Si–O�1 bonds

by further polycondensation reactions continues during ageing and results in an increase in

the structural ordering of the particles. According to Fidalgo and Ilharco, the silanol groups

may undergo further condensation upon ageing of the gels, stiffening the silica network and

resulting in more hydrophobic structure (Fidalgo and Ilharco 2004). Elferink et al. (1996)

state that the formation of a higher ratio of bridging to non-bridging oxygen yields a more

polymerized and more branched structure. Therefore, in this study longer ageing of

xerogels produced the stiffest gels with a higher surface area. In addition, the scanning

electron microscope image of the surface of different ageing xerogels showed the presence

of differences in their microstructure (see Figure 3).

The differences both in molecular structure and physical properties of the aged silica

surface may influence DOX retention by sorption of the protonated DOX molecules.

Figure 4 shows the calculated efficiency of sorption as a function of time of sorption for

the largest initial DOX amount in the bulk solution (920 mg). This relationship demonstrates

that with the increase in time of sorption the efficiency of sorption improves for all types of

xerogels under study and the xerogels aged for 336 h had improved sorption efficiency

compared to 168 h and 24 h-aged xerogels. The xerogels aged for 336 h sorbed the drug

almost completely within 120 min, whereas the xerogels aged for 168 h and 24 h required

200 and 300 min, respectively, for almost complete sorption of DOX. Figure 5 (a–d)

summarizes the efficiency of sorption as a function of the initial amount of DOX in the bulk

solution (920, 460 and 230 mg) for specific times of sorption (60, 120, 180 and 240 min,

respectively).

1400 70080010001200

Abs

(a.

u.)

Wavenumber (cm−1)

vs (Si-O-Si)~800

v (Si-OH/Si-O−) ~950

vas(Si-O-Si)

Figure 2. ATR spectra of xerogels aged for 24 h (dashed line); 168 h (solid line) and 336 h(dotted line). The spectra were normalized to the maximum of the �as Si-O-Si.



S1

S2

S3

Figure 3. SEM images of silica xerogels: S1 (aged for 24 h), S2 (aged for 168 h) and S3 (aged for336 h).



0

20

40

60

80

100

220 460 920

Eff

icie

ncy

of D

OX

sor

ptio

n (%

)

S1 S2 S3

0

20

40

60

80

100

220 460 920

Eff

icie

ncy

of D

OX

sor

ptio

n (%

) S1

(b)

(a)

Initial amount of DOX in solution (µg)


Figure 5. Efficiency of sorption as a function of initial amount of DOX in the bulk solution (920, 460and 230 mg) for specific times of sorption: (a) 60, (b) 120, (c) 180 and (d) 240 min. S1 (aged for 24 h),S2 (aged for 168 h) and S3 (aged for 336 h). Data are given as mean � confidence interval for n¼ 3.

0

20

40

60

80

100

0 100 200 300 400

Eff

icie

ncy

of s

orpt

ion

of D

OX

(%

)

S1

S2

S3

Time of sorption (min)

Figure 4. Efficiency of sorption as a function of time of sorption for the largest initial DOX amount inthe bulk solution (920 mg). S1 (aged for 24 h), S2 (aged for 168 h) and S3 (aged for 336 h). Data aregiven as mean� confidence interval for n¼ 3.



An increase in the initial amount of DOX in the bulk solution results in an increased time

of equilibration between DOX in the bulk solution and in xerogels. According to the

literature, this can be explained by the fact that the mechanism of drug sorption is attributed

both to spontaneous deposition and normal diffusion (Liu et al. 2005). At a smaller initial

amount of DOX, the rate of loading is determined primarily by the deposition mechanism,

whereas at a larger initial amount of DOX, the diffusion of the drug can result in the

increased time of sorption due to saturation of the deposition.

In this study, the sorption of drug was almost complete for all the types of xerogels

under study with different times of sorption for the investigated amounts of DOX

(Figures 4 and 5), indicating that these xerogels have a high sorption capacity and an

increase in time of ageing of the xerogels reduces the time required for complete sorption.

These results are also in good agreement with specific surface areas of xerogels determined

experimentally: an increase in specific surface area resulted in faster sorption of DOX. In

order to estimate the mechanism of drug sorption, the theoretical sorption loading was

calculated from equation (3). The calculated loadings were 24, 26 and 32 mg g�1 for the

xerogels aged for 24, 168 and 336 h, respectively. These values were smaller than the

experimental results obtained for the larger amounts of drug studied (closed to 45 mg g�1).

The higher sorption capacity, regardless of the type of xerogels under study, indicates that

0

20

40

60

80

100

230 460 920

Eff

icie

ncy

of D

OX

sor

ptio

n (%

)

S1 S2 S3

(c)

0

20

40

60

80

100

230 460 920

Eff

icie

ncy

of D

OX

sor

ptio

n (%

) S1 S2

S3

(d)



Figure 5. Continued.



not only liquid sorption between the drug and the xerogel surface occurs, but also drug

deposition within the pores exists. In addition, the sorption results revealed that during the

sorption process doxorubicin can spontaneously diffuse from the bulk solution with a low

concentration of the drug to a high-concentration region existing within the pores of the

system. Therefore, there must exist a force that drives the water-soluble substances from

low- to high-concentration regions. According to the literature, water-soluble substances,

especially positively charged, undergo spontaneous deposition in the matrix and tend to

interact through self-aggregation or complexation. Since a positively charged deposited

substance, such as protonated doxorubicin, can be present in the xerogel pores in an

aggregated or complexed form rather than in a free state, the concentration of the free DOX

within the pores is always lower than in the bulk solution (Liu et al. 2005). Gillies and

Frechet (2005) also showed that doxorubicin molecules are known to self-associate through

stacking interactions and if the molecules are found on the surface of polymers they may

promote aggregation, as was shown for polymer-adsorbed doxorubicin. The comparison of

visible absorption spectrum of silica-loaded doxorubicin and free, protonated doxorubicin in

solution presented in Figure 6 also revealed the red shift of the absorption peak from 481 nm

to �495 nm for all types of xerogels. This red shift can be attributed to the DOX-DOX

interactions, due to �–� stacking (Porumb 1978). Therefore, these results suggest that the

mechanism of doxorubicin retention in silica xerogels could be by spontaneous deposition

both on the surface and within pores.

According to the literature, in addition to spontaneous diffusion, electrostatic attractions

between the oppositely charged drug molecules and the polymer surface are also responsible

for retention of doxorubicin in silica xerogels. In this investigation, xerogels were immersed

in a solution having a pH above 6, at which doxorubicin was in the protonated form and

the silanol groups of xerogels behaved like acids and underwent partial deprotonation

(Atkin et al. 2003). Therefore, the electrostatic interactions could exist. However, the ATR

data presented in Table I suggest that the sorption efficiency of DOX is not correlated with

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

400 450 500 550 600

Abs

orba

nce

(a.u

.)

Wavelength (nm)

Deposited DOX

Free DOX

Figure 6. Visible absorption spectrum of silica-loaded doxorubicin (deposited DOX) and free,protonated doxorubicin in solution.



the amount of residual silanol groups existing on the surface of xerogels. The 336 h-aged

xerogels with the smallest amount of silanol groups (with a decrease in the number

of negatives charges) had an improved sorption efficiency. It also indicates that the physical

properties of silica gels dominated the electrostatic interaction and, when the 336 h-aged

xerogels with the highest specific surface area were immersed in a DOX solution, a larger

amount of DOX was spontaneously deposited inside the mesopores in the shortest time of

sorption.

As the deposition mechanism has been proved and quantified, the question arises whether

the deposited compounds can be released afterwards, which is important for medical

applications, such as drug delivery. The drug release is affected both by the time of ageing of

the xerogels, amount of drug loading, change of medium and also temperature and pH of

release medium. Figure 7 (a–c) shows the DOX release curves during (a) initial time, (b)

prolonged time and (c) square root of initial time in response to different ageing of xerogels.

Figure 8 (a–c) shows the DOX release curves for the xerogels aged for (a) 24 h, (b) 168 h and

(c) 336 h, respectively, in response to different amounts of drug loading. The equilibrium

0

5

10

15

20

25

0 10 20 30 40 50

S1S2S3

0

5

10

15

20

25

0 1 2 3 4

Rel

ease

of

DO

X (

w/w

%)

S1

S2

S3

0

5

10

15

20

25

0 0.5 1 1.5 2

Rel

ease

of

DO

X (

w/w

%)

S1, r=0.986

S2, r= 0.986

S3, r=0.990

Rel

ease

of

DO

X (

w/w

%)

(a) (b)

Initial time (h) Prolonged time (h)

(c)

Square root of initial time (h1/2)

Figure 7. Doxorubicin release curves for silica xerogels aged for: S1 (24 h), S2 (168 h) and S3 (336 h)as a function of (a) initial time, (b) prolonged time and (c) square root of initial time of release test.Release condition: SBF, pH 7.4, temperature 37�C. Xerogels were used with an initial amount ofDOX in solution: 920 mg. Data are given as mean� confidence interval for n¼ 3.



between sorption–desorption process was reached within 4 h for all the xerogels. With an

increased time of ageing both the initial amount and the rate of drug release decrease by 21,

13 and 8% of the initially loaded DOX at equilibrium for the xerogels aged for 24, 168 and

336 h, respectively, for the same load (920 mg). The study of the effect of drug loading on the

drug release revealed that in general both the rate of release of the drug and its initial amount

released decrease significantly with a decrease in drug loading. The higher burst release of

�10 wt% in 30 mins was only seen in the release profiles of xerogels aged for 24 h with the

largest load (920 mg) (Figures 7 and 8).

The DOX release for all types of xerogels was diffusion-controlled and conformed to the

square root of time kinetics for all xerogels (r¼ 0.99), as shown in Figure 7(c).

After changing the release medium, the equilibrium was reached within 4 h for the three

first changes presented in Figure 9(a–c). The equilibrium amount of the drug released

decreases progressively with an increase in the number of change cycles. After eight and

0

5

10

15

20

25

0 1 2 3 4 5

0 1 2 3 4 5

DO

X o

f re

leas

e (w

/w%

)

Loading amount 900 mgLoading amount 415 mgLoading amount 210 mg

0

2

4

6

8

10

12

14

16

0 2 4

DO

X r

elea

se (

w/w

%)


0

1

2

3

4

5

6

7

8

9

DO

X r

elea

se (

w/w

%)


(a)

Time (h)

(b)

Time (h)

Time (h)

(c)

Figure 8. Doxorubicin release curves as a function of time for silica xerogels aged for (a) 24 h,(b) 168 h and (c) 336 h in response to different amounts of drug loading. Release condition: SBF, pH7.4, temperature 37�C. Data are given as mean� confidence interval for n¼ 3.



12 changes for all of the aged xerogels close to 90% of the drug was re-released. Figure 10

shows an example of photographs before and after re-release.

These results are important for the application in chemotherapy with anti-cancer drugs

because further release of DOX takes place only after the drug was consumed. This results in

a constant drug concentration at the implantation site and may help avoid the dose-limiting

toxicity.

An increase in temperature by �10�C accelerates the release process and gradually

decreases the equilibration time by �1 h, as shown in Figure 11. The behaviour of xerogels

was the same at temperatures > 47�C and the maximum amount of drug released at 67�C

within 1 h was �35% of the initially loaded xerogel, regardless of xerogel type. After reaching

room temperature by the xerogel/drug system, spontaneous sorption of the excess drug took

place and the system proceeded to reach sorption–desorption equilibrium.

An increase in pH value increases both the initial amount of drug released and the rate of

drug release, as shown in Figure 12 (a–c). The smaller released amount at lower pH

demonstrates that this is not only a dissolution-driven mechanism, because the solubility of

0

1

2

3

4

5

6

7

8

Rel

ease

of

DO

X (

w/w

%)

S3

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6

Rel

ease

of

DO

X (

w/w

%)

Change 1

Change 2

Change 3

S1

0

2

4

6

8

10

12

14(b)

0 2 4 6

0 2 4 6

Change 1

Change 2

Change 3

Change 1

Change 2

Change 3

Rel

ease

of

DO

X (

w/w

%) S2

(a)

Time (h) Time (h)

(c)

Time (h)

Figure 9. Doxorubicin release curves as a function of time for silica xerogels aged for S1 (24 h), S2(168 h) and S3 (336 h) in response to three changes of medium. Xerogels were used with an initialamount of DOX in solution: 920 mg. Release condition: SBF, pH 7.4, temperature 37�C. Data aregiven as mean� confidence interval for n¼ 3.



DOX at low pH should be larger due to its protonation. Therefore, it is hypothesized that

the electrostatic interactions between positively charged doxorubicin molecules and silanols

groups have an important effect on the retention of DOX. However, comparing the release

profiles in response to different pH values in detail revealed that the 24 h- aged xerogel

containing the largest fraction of silanols groups (Table I) released the drug faster than

others. Thus, these results indicate that the electrostatic attractions do not dominate and

there exists a different cause for an accelerated release of the drug at higher pH values. Iler

(1979) and others workers (Radin et al. 2002) studied the dissolution (erosion and

degradation) process of silica. SiO2 xerogels are hydrophilic polymers with silanol groups on

the surface, so they take up large quantities of water and degrade by hydrolysis of the

siloxane bonds through the entire network (Gopferich 1996). Finally, silica acids are

released, leading to the weight loss of silica xerogels and also an increase in their pore size

0

5

10

15

20

25

30

35

40

45

50

20 30 40 50 60 70

Rel

ease

of

DO

X (

w/w

%)

S1

S2

S3

Temperature (°C)

Figure 11. Doxorubicin release curves for silica xerogels aged for S1 (24 h), S2 (168 h) and S3(336 h) as a function of temperature. Xerogels were used with an initial amount of DOX in solution460 mg. Release condition: SBF, pH 7.4. Data are given as mean � confidence interval for n¼ 3.

(a) (b)

Figure 10. Examples of photographs of (a) silica xerogel after sorption of doxorubicin and (b) silicaxerogel after re-release of doxorubicin.



(Radin et al. 2002). The increase in pore size results in a faster release of the drug molecules

encapsulated in the pores. According to the literature, two parameters affect the rate of

dissolution of silica. The first is associated with pH of solution: with an increase in pH the

silica gels dissolve faster (Iler 1979). The second was attributed to the microstructure of

silica: less condensed and polymerized structures with a larger fraction of silanol groups

hydrolyse faster. These conclusions are in good agreement with the results obtained in this

work: xerogels aged for 24 h with less polymerized structure and a larger amount of silanol

group release the drug faster compared to others both in response to increased pH of

solution and other conditions of this study. In addition, the largest weight loss close to 14%

of the initial mass of xerogel was found for 24 h-aged xerogels after release test at pH 8.

The weight loss of the remaining xerogels was � 9 w/w% and 6 w/w% for the xerogels aged

for 168 h and 336 h, respectively.

Conclusions

The loading process was based on both sorption of doxorubicin from solution onto the

surface area of the base-catalysed xerogel and deposition in the mesopores. The sorption of

doxorubicin occurred by spontaneous sorption. The time of sorption was strongly affected

by the time of ageing of xerogels: an increase in ageing time decreased the time required for

complete sorption of the drug. These results strongly correlated with physical properties of

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4

0 1 2 3 4

Rel

ease

of

DO

X (

w/w

%)

pH 8

pH 6

pH 40

2

4

6

8

10

12

0 1 2 3 4 5

Rel

ease

of

DO

X (

w/w

%)

pH 8

pH 6

pH 4

0123456789

10

Rel

ease

of

DO

X (

w/w

%)

pH 8

pH 6

pH 4

(a)

Time (h)

(b)

Time (h)

(c)

Time (h)

Figure 12. Doxorubicin release curves as a function of time for silica xerogels aged for (a) 24 h,(b) 168 h and (c) 336 h in response to different pH values. Xerogels were used with an initial amount ofDOX in solution 230 mg. Release condition: temperature 37�C. Data are given as mean� confidenceinterval for n¼ 3.



xerogels. With an increase in ageing time the process of structure rebuilding continues and

the longer ageing resulted in an increase in surface area and solid density as well as in the

decrease in pore size and pore volume. The release of doxorubicin was controlled by the

equilibrium between the desorbed drug and the drug deposited within the pores.

The mechanism of desorption was found to be diffusion-controlled. The degree of drug

release was also affected by the time of ageing of xerogels, the loading amount of DOX and

temperature of release. An increase in ageing time decreases both the initial rate of drug

release and the total amount of DOX released. A higher DOX load and also an increase

of temperature and pH of release medium result in a faster release rate at the initial stage and

a larger amount of DOX released. Any changes in the state of equilibrium of the drug results

in its further controlled release.

In summary, mesoporous xerogels loaded with DOX seem to be a promising carrier

material for the precise drug delivery to a targeted site or to be used as an implantable drug

delivery system for long-term bone disease control. In addition, they can find use, e.g. as

supplements in biodegradable materials, such as polylactide and polyglycolide, that are used

as temporary implants in certain bone surgeries (Kursawe et al. 1998).

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synthesis of sol-gel mesoporous silica materials providing a slow release of doxorubicin

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