XEDS

Journal of Molecular Structure 1056–1057 (2014) 262–266

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Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc

The bioactivity studies of drug-loaded mesoporous silica-polydimethylsiloxane xerogels using FTIR and SEM/XEDS Magdalena Prokopowicz ⇑, Adrian Szewczyk, Wiesław Sawicki ´ sk, Department of Physical Chemistry, Hallera 107, 80-416 Gdan ´ sk, Poland Medical University of Gdan

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 DOX-loaded M-silica-PDMS xerogels

were synthesized using sol–gel method.  Obtained xerogels were soaked in SBF with presence or absence of human albumin.  The bioactive properties of xerogels were investigated by FTIR and SEM– XEDS.  Progressive formation of carbonated hydroxyapatite on xerogels surface was observed.  Addition of human albumin slows down the formation process.

a r t i c l e

i n f o

Article history: Received 16 August 2013 Received in revised form 18 October 2013 Accepted 18 October 2013 Available online 28 October 2013 Keywords: Silica xerogels Bioactivity Hydroxyapatite Doxorubicin Simulated body fluid

a b s t r a c t The objective of this study was to evaluate the mineralization potential of bioactive drug-loaded mesoporous silica-polydimethylsiloxane xerogels under in vitro biomimetic condition. In this case, bioactivity is slightly connected with self-formation of carbonate hydroxyapatite (c-HAp) on xerogels surface. Carriers in the form of granules were prepared by using sol–gel method including drug loading – doxorubicin hydrochloride. The drug-loaded carriers were soaked in mineralizing solution with or without content of human albumin for 7, 14, 28 days to produce c-HAp. The mineral formation was measured using Fourier transform infrared spectroscopy and scanning electron microscope with energy dispersive X-ray spectroscopy. The results show that the drug-loaded carriers could significantly mineralize in vitro: the mineralizing rates increasing with the time for which the samples were kept in the mineralizing solution. However, human albumin has significant retardative effect on apatite precipitation from SBF. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Mesoporous silica systems are one of the most progressive biomaterials. Studies about internal conformation of silica carriers can be useful in researches focusing on new forms of drugs and implants, especially in surgery [1]. Progressive medicine concentrates on local drug delivery systems which are less harmful than common treatment. To prevent patient from taking medicine orally different routes of administration are improved [2]. Lesions of bone ⇑ Corresponding author. Tel.: +48 563491653. E-mail address: [email protected] (M. Prokopowicz). 0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molstruc.2013.10.052

tissue, especially cancers, are one of the most difficult to treat locally. However, this kind of treatment can support the proper regeneration of bone tissue. Mesoporous silica xerogels, a type of silica carriers usually obtained in room-temperature by sol–gel method, might be useful in treatment of various bone diseases. One of the common drug using during the bone cancer chemotherapy is doxorubicin (DOX) [3]. That is why DOX-loaded silica xerogels can be studied as a potential bioactive materials which exhibit an anticancer action directly in bones [1,4]. In this case, bioactivity is slightly connected with self-formation of carbonate hydroxyapatite (c-HAp) on xerogels surface. Biological apatite is a mineral component which occurs naturally in

M. Prokopowicz et al. / Journal of Molecular Structure 1056–1057 (2014) 262–266

osseous tissue and provides bones strength and hardness [5]. According to other publications, the main idea of bioactive silica materials is their ability to create a connection: carrier – bone tissue area. It is an effect of chemical interactions, for example ionic exchanges, on silica and osseous surfaces [6]. Carbonate hydroxyapatite is also an important part of bone tissue recovery because it bonds the carrier with the tissue and refills the defects in affected area [7]. On the other hand, the anticancer drug-loaded xerogels might be useful in cancer treatment as a controlled internal drug delivery system which releases the medicine in equal time intervals. An important application of in vitro bioactivity testing is the prediction of in vivo performance of pharmaceutical dosage forms. More and more modern researches try to create the conditions of in vitro studies similar to human body conditions [8]. One of the most popular in vitro factor during the bioactivity studies is simulated body fluid (SBF) by Kokubo et al. [9]. However, standard SBF has became modified. SBF’s modifications like addition of glucose, human albumin etc. give opportunities to create biomimetic conditions which are even more similar to human blood plasma [8,10]. To study the bioactivity of a novel mesoporous silica-polydimethylsiloxane (M-silica-PDMS) xerogels in the granules form and to observe the changes on their surface a Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope analysis (SEM) with energy dispersive X-ray spectroscopy (XEDS) have been chosen. Quick preparation of the samples and short time of analysis make FTIR a basic and still important analytical method [7,11]. In this studies, FTIR method provided many useful information about changes on xerogels surface which appeared after each time of observation. SEM analysis with XEDS gave an elemental composition view of carriers and made an opportunity to observe the formation of carbonate hydroxyapatite during the bioactivity studies. This studies focus mostly on FTIR method. It was important to obtain as many information about silica carriers (characteristic functional groups presence, changes on surface, bioactivity properties) as it was possible using this method. SEM and XEDS analysis were important to prove the validity of FTIR results.

2. Methods 2.1. Mesoporous silica-polydimethylsiloxane xerogels synthesis The innovative M-silica-PDMS carriers were prepared using pre-optimized reactional conditions [12]. Briefly, silica xerogels contained 50 wt% of silica, 20 wt% of polydimethylsiloxane (PDMS), 25 wt% of calcium chloride (CaCl2) and 5 wt% of triethyl phosphate (TEP). In order to obtain silica, the 1.736 g of tetraethyl orthosilicate (TEOS) solution, as a precursor, with 0.146 g of ethyl alcohol and 140 lL of 0.010 M HCl were used in hydrolysis process. At the same time, a PDMS emulsion was obtained by mixing 0.200 g of PDMS with 0.368 g of water and 0.0044 g of sodium dodecyl sulfate under ultrasounds conditions for 15 min. When solution of TEOS became uniform a PDMS emulsion was added and stirred until obtain the clarity. Next, an alcoholic solution of

Fig. 1. Exemplary doxorubicin-loaded M-silica-PDMS xerogels in the granules form.

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CaCl2 (0.151 g of CaCl2 dissolved in 0.428 g of ethyl alcohol) was added. After 1 h of mixing, 0.149 g of TEP was added into solution. Doxorubicin hydrochloride (2 mg) was dissolved in 0.368 g of water and added with 80 lL of 0.2% NH3 (aq.) to the main solution. Finally, 300 lL portions of solution were moved into the forms made of polypropylene by using micropipette. Whole was placed in darken space for 10 days (Fig. 1). 2.2. Bioactivity assay in vitro To study the bioactivity of drug-loaded M-silica-PDMS xerogels, two different simulated body fluids (SBF) were prepared. The first one was a standard inorganic SBF solution by Kokubo et al. [9] which found many applications in bioactivity researchers. The second inorganic–organic SBF solution had the same ion concentration and composition as the basic SBF but was prepared with 30 g addition of human albumin per each 1 L of SBF (SBF-Alb.) [10]. Prepared solutions were poured into the polypropylene containers. Next, drug-loaded xerogels were powdered and divided into 50 mg portions and then were soaked in 50 mL of each SBFs in stirred conditions at 37.4 °C for 28 days. SBFs were exchanged after each 7 days of soaking. 2.3. Fourier transform infrared spectroscopy After each soaking time, powdered drug-loaded M-silica-PDMS xerogels were filtered and dried at 55.0 °C for 2 h. Next, xerogels were weighed and small portion (1.20 ± 0.04 mg) were placed in microcentrifuge tubes prepared for FTIR analysis. Rest part of the xerogels was again soaked in fresh portion of SBF for next 7 days. The bulk structure was studied with FTIR in the operating range of 4000–400 cm 1, using a Jasco model 410 FTIR and a KBr pellet technique [13]. For a better comparison, all the IR spectra were normalized to maximum absorption of a dominant peak at 1090 cm 1. 2.4. SEM and XEDS analysis The morphology and the chemical state of the surface were characterized by scanning electron microscopy, equipped with an energy dispersive X-ray spectroscopy (SEM–XEDS, Philips XL 30, at an acceleration voltage of 3.0 kV or 18 kV, respectively.). SEM and XEDS analysis were carried out only for few selected carriers which gave the clearest FTIR spectra. Samples for SEM were fixed on carbon tape and fine gold sputtering was applied for 2 min. Samples for the XEDS analysis were not gold-coated, since the signal from gold interfered with the phosphorus detection. Three representative surfaces were analysed for each drug-loaded xerogels to calculate an average value of the relative atomic % of C, O, Ca, P and Si. 3. Results and discussion 3.1. Progressive formation of carbonated hydroxyapatite After each soaking time in SBF, characteristic bands for drugloaded M-silica-PDMS xerogels have been noticed (Table 1). It can be easy to observed that all samples have bands characteristic for Si–O–Si and Si–CH3 groups which are connected with basic silica-PDMS structure of the xerogels [14]. However Si–O–Si band, recorded at 1081 cm 1 before soaking, was replaced by band at 1091 cm 1 after 7 days. It means that more energy is required to make a bonds vibrate which probably indicates, that inner structure of carriers became more regular. According to other publications [11,15], bands associated with PO34 and CO23 functional

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Table 1 FTIR characteristic bands obtained from functional groups after each time of soaking in SBF. Wavenumber (cm

1

)

Si–O–Si asymmetric stretching vibrations

Si–O–Si (from PDMS) asymmetric vibrations

Si–CH3/Si–(CH3)2 asymmetric stretching vibrations

v4 PO34 bending vibrations

v3 PO34 asymmetric stretching vibrations

v3 CO23 asymmetric stretching vibrations

v2 CO23 asymmetric stretching vibrations

0 days

1080

800

1264

560

–

–

–

7 days

1091

798

1263

564 603

–

1420

Began formation

14 days

1091

809

1264

563 602

1030

1422

872

28 days

1090

801

1265

567 603

1032

1420

875

Fig. 2. FTIR spectra of drug-loaded M-silica-PDMS samples before and after each 7 days of soaking in SBF.

Fig. 3. FTIR spectra of drug-loaded M-silica-PDMS samples before and after 7 days of soaking in SBF. Calcium chloride was shown as a reference.

groups also appeared and proved the formation of hydroxyapatite on the xerogels surface. Moreover bands at 560–564 cm 1 and 602–601 cm 1 come from regular apatitic PO34 groups.

In reference to characteristic bands in Table 1, the FTIR spectra have been also compared (Fig. 2). It was important to observe the hydroxyapatite formation on xerogels surface which is a gradual process [16]. During the formation time, Si–O–Si band was gradually covered by c-HAp and finally, after 28 days of soaking, Si–O–Si band at 1090 cm 1 became less intensive than v3 PO34 band (1030 cm 1). The similar situation took place at 500–600 cm 1 area. Two bands of absorption at 603–564 cm 1 can be observed progressively with greater intensity after each time of soaking. Band at 560 cm 1 (0 days) became from 5 wt% addition of TEP which accelerates with Ca2+ ions formation of c-HAp [15,16]. What is more, broad bands at 1422 cm 1 and 872 cm 1 have also appeared. Both of them are connected with CO23 functional groups which come from self-carbonated hydroxyapatite forms [11,15]. It was assumed that first 7 days of soaking might be crucial in c-HAp formation process. To observe differences on xerogels surface after this time, exemplary FTIR spectra with CaCl2 pattern, after 7 days of soaking were compared in Fig. 3. A decrease of bands intensity at 3466 cm 1 (which corresponds to hydroxyl group vibrations from adsorbed water), 1637 cm 1 (from molecular water) and 962 cm 1 (from Si–OH groups) can be observed. It means that addition of PDMS during the synthesis increases the hydrophobicity of obtained drug-loaded M-silica-PDMS xerogels [12]. Lower bands characteristic for calcium chloride after 7 days of soaking can be associated with fast exchange of calcium ions from xerogel into solution [16]. What is more, a small bands at 603 cm 1 and 564 cm 1 are able to be seen which means, that cHAp formation has already begun. According to FTIR results (Figs. 2 and 3), it was mentioned above that c-HAp formation on xerogels surface is a gradual process. However, to observe the changes in xerogels morphology SEM analysis with XEDS were also used (Fig. 4). According to SEM–XEDS micrographs a progressive formation of c-HAp might be observed [17]. After 7 days of the bioactivity test in SBF, irregularly shaped flake-like precipitates of calcium phosphate appeared. They formed randomly distributed clusters, indicating a heterogeneous nucleation process. After 14 days, the calcium phosphate precipitates were organized into the larger spherical clusters. After 28 days, the composite surface consisted of a continuous porous apatite layer typical for apatite phosphates [18]. Changes in xerogels elemental composition on their surface have been also compared in Table 2 and Fig. 5. Standard deviations for these measurements varied between 0.5% and 5.0%. In reference to both of them, formation of c-HAp has a dynamic character. It means that during the soaking time more and more molecules of c-HAp accumulate on xerogels surface. It is important to realize that total amount of silicon on carriers surface is constant. However, the amount of self-formed c-HAp progressively increases, that is why the percentage of silicon to whole external surface lowers and became inversely proportional to calcium and phosphorus sum.

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Fig. 4. SEM–XEDS micrographs of drug-loaded M-silica-PDMS xerogels surface after each soaking time in SBF.

Table 2 The percentage of elements on surface of drug-loaded M-silica-PDMS samples. Time of soaking (days)

0 7 14 28 a

Mean element’s surface contenta (%) C

O

Si

Ca

P

Ca + P

10.00 7.94 7.86 7.94

21.15 27.28 22.24 8.88

60.77 21.06 13.83 7.01

6.54 24.08 29.16 43.46

1.54 19.64 26.91 32.71

8.08 43.72 56.07 76.17

Standard deviation varied between 0.5% and 5.0%.

Fig. 6. FTIR spectra of exemplary drug-loaded M-silica-PDMS xerogel before and after 7 days of soaking in SBF-Alb. and re-soaked in SBF for 21 days.

Fig. 5. Changes on external surface of drug-loaded M-silica-PDMS samples between silicon, calcium and phosporus during the soaking time in SBF.

3.2. Bioactivity studies in SBF-Alb. conditions In the next step the behaviour of drug-loaded silica xerogels in the SBF with human albumin was studied. Drug-loaded xerogels were soaked in SBF-Alb. solution for 7 days. After this time FTIR analysis was carried out. Spectrum, which came from solution with

albumin addition SBF-Alb. did not show the bands characteristic for c-HAp. However, bands at 1660 cm 1, 1542 cm 1, 1452– 1407 cm 1 coming from amide I, II, III bands, respectively, have appeared (Fig. 6). All of them are characteristic for NH2 groups connected with molecules of human albumin. It probably means, that protein was adsorbed on mesoporous xerogels surface and then it blocked the centres of c-HAp nucleation [19]. The slow deposition and growth of c-HAp comes also from the fact that the adsorption of albumin reduces the interfacial energy of apatite nuclei with the solution. That deprives the driving force for the growth of the new apatite. It has been suggested that the proteins compete with ions, e.g. Ca2+ and PO34 etc., for the same surface binding sites in the solution [8,10]. In the next step, albuminadsorbed xerogels were soaked once again in standard SBF (without albumin addition) in order to observe the c-HAp

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formation. Bands characteristic for PO34 appeared after 21 days of soaking which means, that adsorbed molecules of human albumin slows down the c-HAp formation process. 4. Conclusions  The bioactivity study of the M-silica-PDMS is able to be observed by FTIR and SEM–XEDS analysis.  According to results a bioactive character of M-silica-PDMS structures has been proved.  There is a dynamic ions exchange between silica surface and solution which is an important part of hydroxyapatite accumulation process.  Different formation rates of carbonate hydroxyapatite in SBF and SBF-Alb. are connected with presence of human albumin. Molecules of proteins might be adsorbed on xerogels surface and they could slow down the formation process.  The progressive formation of self-carbonated hydroxyapatite during soaking time was observed which means that M-silicaPDMS can be a modern bioactive system of drug delivery.  Bioactive character of M-silica-PDMS materials can be a promising aspect in studies of drug distribution under in vivo investigations.

Acknowledgment This work was supported by the National Centre for Science of the Polish State Project No. N N405 024440 awarded to M. Prokopowicz.

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