Materials Science and Engineering C 49 (2015) 66–74
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Piperazine and its carboxylic acid derivatives-functionalized mesoporous silica as nanocarriers for gemcitabine: Adsorption and release study Zohreh Bahrami a, Alireza Badiei a,b,⁎, Fatemeh Atyabi c, Hossein Reza Darabi d, Bita Mehravi e,f a
School of Chemistry, College of Science, University of Tehran, Tehran, Iran Nanobiomedicine Center of Excellence, Nanoscience and Nanotechnology Research Center, University of Tehran, Tehran, Iran Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran d Chemistry and Chemical Engineering Research Center of Iran, Nano and Organic Synthesis Lab, Tehran, Iran e Celullar and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran f Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 30 October 2014 Accepted 17 December 2014 Available online 19 December 2014 Keywords: SBA-15 Nanorods Piperazine Carboxylic acid-functionalization Gemcitabine Cytotoxicity
a b s t r a c t Piperazine-functionalized SBA-15 nanorods were synthesized by post grafting method with methyldimethoxysilylpropylpiperazine (MDSP). The carboxylic acid derivatives of piperazine-functionalized SBA-15 nanorods were obtained using two different kinds of precursors (bromoacetic acid and succinic anhydride). The prepared materials were used as nanocarriers for the anticancer drug (gemcitabine). The obtained samples were characterized by SAXS, N2 adsorption-desorption, SEM, TEM, DLS, thermogravimetric analysis, FTIR, Raman and UV spectroscopies. The adsorption and release properties of all samples were investigated. In vitro study included cell toxicity. It was found that the surface functionalization increases the interaction between the carrier and gemcitabine and results in the loading enhancement of the drug. In addition, the adsorption of gemcitabine on the modified mesoporous matrix depends on the type of the introduced functional groups. The carboxylic acid-modified samples have higher loading content, due to the strong interaction with gemcitabine. The maximum content of deposited drug in the modified SBA-15 nanorods is close to 36 wt.% that it is related to PC2-SBA-15 sample which obtained using succinic anhydride. The obtained results reveal that the surface functionalization leads toward a significant decrease of the drug release rate without any appreciable cytotoxicity. No significant differences are observed among the drug release rate from the modified samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gemcitabine (Gem) is a water-soluble low-molecular-weight anticancer drug that is commonly used for the treatment of several kinds of cancers including colon, pancreatic, lung, breast, ovarian and bladder [1–3]. Gemcitabine passes through the cell membrane with difficulty and requires active transporters localized on the membrane to enter the cells [4,5]. Different drug delivery systems such as liposomes and polymeric nanoparticles were designed in order to protect Gem from rapid metabolization, overcome drug resistance, target drug delivery and improve its anticancer efficacy [6,7]. Since 2001, when MCM-41 was first proposed as a drug delivery system (DDS) [8], silica-based materials have been discussed as drug carriers. Mesoporous silica nanoparticles (MSNs) possess several unique features such as high surface area, large pore volume, uniform and
⁎ Corresponding author at: School of Chemistry, College of Science, University of Tehran, Tehran, Iran. E-mail address: [email protected] (A. Badiei).
http://dx.doi.org/10.1016/j.msec.2014.12.069 0928-4931/© 2014 Elsevier B.V. All rights reserved.
tunable pore sizes, excellent physicochemical stability, controllable morphology, modifiable surfaces, nontoxic nature and well biocompatibility [9–15]. These properties result in the study of application of MSNs in the biomedical field have attracted great attention. MSNs offer the possibility of lodging a variety of drug molecules such as, anti-inflammatory [8,16–20], antibiotic [11,21–23], antihypertensive [24], antiulcer [25,26], anti-osteoporotic [27] and anticancer [23]. It was demonstrated that surface functionalization [16,18,25,28], size and structure of pore [24,29–31], loading conditions [32] and chemical characteristics of the loaded drug [33] can affect both the adsorption and release of the drug into and out of MSNs. The chemical functionalization of the surface is essential because a mesoporous silica surface covered with silanol groups that are not selective enough to adsorb drug molecules with different functionalities [34,35]. In addition, it was shown that the attractive and repulsive electrostatic interactions between the entrapped molecules and the silica surface play an important role in the amount of adsorption of drug molecules and rate of their release. Therefore, the choice of appropriate functionalizing agent can enhance the adsorption capacity of drug and it should also allow modulating its release [35–39]. For example, the modification of MSNs with
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amine and carboxylic acid groups increases the amount of adsorbed drugs with acidic and basic properties, respectively [20,25,38,40,41]. The morphology of MSNs can also affect the drug adsorption and release. Tsai et al. found out that rod-like MSNs have greater potential than that of spherical ones in monitoring the cell trafficking, cancer cell metastasis and drug/DNA delivery [42]. Zhao and co-workers showed that rod-like SBA-15 (about 1–2 μm in length) had a faster adsorption rate and immobilized larger amount of enzymes than rope-like SBA-15 (20 μm in length) [43,44]. The effect of particle shape on the in vivo biological behaviors of MSNs was studied. Huang et al. designed two different shaped fluorescent MSNs, short rod MSN-FITC (ARs of ~1.5, length of ~200 nm) and long rod MSN-FITC (ARs of ~5, length of ~ 800 nm), and investigated this effect. The obtained findings reveal that MSNs actively interact with cells and engage in and mediate in vivo behaviors. It is also demonstrated that MSNs would not cause significant toxicity in vivo [45]. Functionalized SBA-15 nanoparticles with particle sizes of 500–800 nm in length and 300–500 nm in diameter have been investigated as nanocarriers for bleomycin (as an anticancer drug) delivery. The efficient cellular uptake and low cytotoxicity of the functionalized SBA-15 nanoparticles have been confirmed by in vitro cell assays [46]. MCM-41 and SBA-15 as two main conventional mesoporous materials have been applied as excellent carriers for encapsulation of different kinds of drugs. They have ordered hexagonal mesopores and different pore sizes, which are 2–5 nm and 5–10 nm for MCM-41 and SBA-15, respectively. In addition, SBA-15 has thicker pore walls and micropores that interconnect the hexagonal channels [21,40]. It is proved that the large pore size leads to the increasing of the drug loading amount and release rate [17,29,35,47,48]. The influence of the pore morphology and geometry on the drug loading and release properties was investigated. It is shown that the interconnected pore systems result in the easier and faster diffusion process in contrast with the unconnected pore systems [49]. This effect was extensively studied by Alexa et al. on the release profiles of methotrexate, as an anticancer drug. The obtained results reveal that SBA-15 is the better methotrexate delivery system in comparison to MCM-41 [50]. Up to now, to the best of our knowledge, a few articles reported the application of SBA-15 nanoparticles in drug delivery especially for anticancer drugs. This paper describes the adsorption and release of gemcitabine on the piperazine and its carboxylic acid derivatives-functionalized SBA15 nanorods. Investigation of the effect of the surface functionalization on the adsorption capacity and release behavior of Gem is the main aim of this study.
2. Experimental 2.1. Materials Poly(ethylene glycol) block poly(propylene glycol) block poly(ethylene glycol), P123, (EO20PO70EO20, MW = 5800 g/mol) was purchased from Aldrich. Tetraethyl orthosilicate (TEOS), hydrochloric acid (35%), ethanol, N,N′-dimethylformamide (DMF), bromoacetic acid, and succinic anhydride were obtained from Merck (Darmstadt, Germany). Methyldimethoxysilylpropylpiperazine (MDSP) and gemcitabine were purchased from Power Chemical Corporation (Nanjing, China) and Lilly France S.A.S. Company (rue Pagès Suresnes, France), respectively. All chemical reagents were used without further purification. Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), penicillin G (100 U/ml), streptomycin (100 μg/ml), Glutamax and Trypsin-EDTA were purchased from GIBCO-BRL (Grand Island, NY, USA). Human Breast Cancer cell lines (MCF-7) were provided from the National Cell Bank of Pasteur Institute of Iran (Tehran, Iran).
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2.2. Synthesis of SBA-15 nanorods SBA-15 nanorods were synthesized according to the procedure reported in the our previous work [51]. Briefly, 23.4 g of nonionic triblock copolymer (Pluronic P123) was dissolved in the mixture of deionized water (606.8 g) and hydrochloric acid (146.4 g, 35%). 50 g of TEOS was added to the solution while stirring at the rate of 500 rpm at 55 °C. The reaction batch was maintained at static conditions for 24 h at this temperature and then for another 24 h at 100 °C for further condensation. The precipitate was obtained after filtration and then triblock copolymer template was removed by ethanol extraction through a soxhlet extractor. 2.3. Synthesis of piperazine-functionalized SBA-15 nanorods (P-SBA-15) SBA-15 nanorods were functionalized using methyldimethoxysilylpropylpiperazine (MDSP) by post-grafting method [52]. Typically, 0.1 g of SBA-15 nanorods was dispersed in 20 mL of anhydrous ethanol. Following the addition of MDSP (1 mL), the mixture was stirred moderately at 80 °C for 6 h. The precipitate was collected by centrifugation, washed with ethanol and dried at room temperature to give the piperazine-functionalized SBA-15 nanorods as white powder. The obtained modified SBA-15 was denoted as P-SBA-15 (Fig. 1a). 2.4. Synthesis of carboxylic acid-functionalized SBA-15 nanorods (PC1-SBA-15 and PC2-SBA-15) The –COOH functionalization of SBA-15 nanorods was carried out in two ways as follows: 1) 0.1 g of piperazine-functionalized SBA-15 was dispersed in 25 mL ethanol containing 5 mmol bromoacetic acid. The mixture was allowed to react at 80 °C for 4 h. The precipitate was collected by centrifugation, washed with ethanol and dried at room temperature. The synthesized sample was designated as PC1-SBA-15 (Fig. 1b). 2) 0.1 g of piperazine-functionalized SBA-15 nanorods was added in the N,N′-dimethylformamide (DMF) solution of succinic anhydride (50 mL, 2 wt.%). The mixture was stirred for 24 h at room temperature. After that, the product was collected by centrifugation, washed with DMF and dried at room temperature. The obtained sample was denoted as PC2-SBA-15 (Fig. 1b). 2.5. Gemcitabine loading Gemcitabine (Fig. 2) was chosen as a model drug to evaluate the loading and release behavior of the non-modified and functionalized SBA-15 nanorods. SBA-15 nanorods and gemcitabine were dispersed in distilled water to form 5 mg·mL-1 solutions. Then, 2 mL of gemcitabine solution was mixed with 2 mL of SBA-15 nanorods solution. The mixture was stirred at room temperature for 24 h to reach the equilibrium state. The gemcitabine loaded SBA-15 nanorods (G@SBA-15) were collected by centrifugation and washed several times with distilled water to remove the physically adsorbed Gem. A similar procedure was conducted for all the functionalized samples. The prepared samples were labeled as, G@P-SBA-15, G@PC1-SBA-15 and G@PC2-SBA-15. The amount of the loaded drug was determined by a UV/Vis spectrophotometer at 269 nm. The drug loading content and the entrapment efficiency were calculated using the following equations:
Loading contentð%Þ ¼ ðWeight of Gem in mesoporous materialÞ =ðWeight of Gem loaded mesoporous materialÞ Entrapment efficiencyð%Þ ¼ ðWeight of Gem in mesoporous materialÞ =ðInitial weight of GemÞ:
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SBA-15
P-SBA-15
(a)
PC1-SBA-15
(b)
PC2-SBA-15
Fig. 1. Surface functionalization of SBA-15 nanorods: (a) piperazine functionalization and (b) carboxylic acid functionalization.
2.6. Gemcitabine release
Intensity (a. u.)
110 200
100
According to the our previous study [51] and some reports [52,53], the phosphate buffered saline (PBS) solutions with the pH values of 5.6 and 7.4 were chosen for the drug release experiments {since the certain tissues of the body have acidic pH in comparison to the blood (pH = 7.4), it is possible to design the carrier systems, which provide
a safe and efficient way for drug release targeting specific sites in the body, such as endosomes (pH ~ 5.6) [41]}. The certain amount of Gem loaded samples was dispersed in 4 mL of PBS solution and kept at 37 °C using an incubator in order to simulate body temperature. At given time intervals, a suspension was centrifuged at 13000 rpm for 15 min. The amount of released drug was measured by a UV/Vis spectrophotometer at 269 nm.
2θ (degree) Fig. 2. The structure of gemcitabine.
Fig. 3. SAXS patterns of the non-modified and modified SBA-15 nanorods.
Z. Bahrami et al. / Materials Science and Engineering C 49 (2015) 66–74
(a)
(b)
69
(c)
Fig. 4. SBA-15 nanorods : (a) SEM, (b) and (c) TEM images.
2.7. Cell culture Human Breast Cancer (MCF-7) cell lines cultured in flasks and incubated under a humidified atmosphere with 5% CO2 at 37 °C and using standard cell culture media, containing Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin. The complete medium was changed every 3 days. When the cells reached 80% confluence, they were detached and re-plated using 0.25% Trypsin-EDTA. 2.8. Cell viability test For the MTT assay, MCF-7 cell lines (5 × 103 cells per well) were incubated with various amounts of SBA-15 nanorods and G@PC2-SBA-15 (0.1, 0.01, 0.001 mg/mL) in a 96-well micro-plate for 1 h with untreated cells as control. Each concentration was tested in triplicates. The cells were washed with PBS before the addition of the MTT solution (20 μl of 5 mg/mL MTT solution) to each well. After 1 h, absorbance of purple formazone was measured by using BioTek's absorbance microplate readers at 630 nm. 2.9. Inhibitory concentration 50 determination Based on the obtained toxic effects of SBA-15 nanorods and G@PC2SBA-15 synthesized here on MCF-7 cells, the effective half inhibitory dose of the conjugate was calculated based on the linear model method. On the basis of linear regression equation of exposure time of 12 and 24 h, the EC50 or IC50 of SBA-15 nanorods and G@PC2-SBA-15 were obtained to be 21.294 mg/mL and 12.355 mg/mL at 1 h, respectively. 2.10. Characterization The small angle X-ray scattering (SAXS) patterns were recorded with a model Hecus S3-MICROpix SAXS diffractometer with a onedimensional PSD detector using Cu Kα radiation (50 kV, 1 mA) at the
wavelength 1.542 Å. The SEM and TEM images were taken using Oxford LEO 1455 V STEM and Philips EM-208 100 KV, respectively. Nitrogen physisorption isotherms were obtained on a BELSORP mini-II at liquid nitrogen temperature (77 K). The specific surface areas were measured using multiple point Brunauer–Emmett–Teller (BET) method. The pore size distributions were calculated using desorption branches of the isotherms by Barrett–Joyner–Halenda (BJH) method. The mean diameter and size distribution of the nanoparticles were measured by dynamic light scattering (DLS) using Zetasizer (Nano-ZS; Malvern Instruments, Malvern, UK). The FTIR spectra were recorded using Equinox 55 spectrometer in the range between 400 and 4000 cm-1. The Raman spectra were obtained on SENTERRA spectrometer. Thermogravimetric analysis (TGA) was performed on a TA Q50 instrument. The scans were performed between 20 °C and 800 °C at 10 °C/min. The UV/Vis absorption spectra were recorded using a Ray light, UV 1600 spectrophotometer. 3. Results and discussion The small angle X-ray scattering (SAXS) patterns of the nonmodified and modified SBA-15 nanorods were shown in Fig. 3. All the samples have a single intensive reflection at around 2θ = 0.72° similar to the typical SBA-15 materials, which is reported in the literature [54]. Two additional peaks which are related to the higher ordering (110) and (200) reflections are observed in all the patterns. These three peaks are characteristic of the ordered two-dimensional hexagonal (p6mm) structure [55]. The existence of three main diffraction peaks in the patterns of the functionalized samples shows that the hexagonal structure of SBA-15 is stable during the surface functionalization. However, the intensity of the diffraction peaks decreased after the functionalization due to the existence of organic groups in the mesochannels. Scanning electron microscopy (SEM) was used to determine the morphology of the samples. A representative micrograph of the SBA15 matrix is shown in Fig. 4a, where there are particles having a rodlike shape. The transmission electron microscopy (TEM) study was performed to determine the particle size and pore geometry structure. As it
Fig. 5. Size distribution of SBA-15 nanoparticles.
Z. Bahrami et al. / Materials Science and Engineering C 49 (2015) 66–74
dvp/dvr
Va/cm3 (STP) g-1
70
r (nm) p/p0 (a)
(b)
Fig. 6. (a) N2 adsorption/desorption isotherms and (b) pore size distribution of the non-modified SBA-15 nanorods and Gem loaded samples.
Table 1 Specific surface area (SBET) (m2 g-1), pore volume (Vp) (cm3 g-1) and pore diameter (Dp) (nm) from N2 adsorption/desorption for the non-modified SBA-15 nanorods and Gem loaded samples. Sample
Specific surface area (SBET) (m2g-1)
Pore volume (Vp) (cm3g-1)
Pore diameter (Dp) (nm)
SBA-15 nanorods G@SBA-15 G@P-SBA-15 G@PC1-SBA-15 G@PC2-SBA-15
1010 449 263 227 256
1.27 0.811 0.836 0.467 0.552
7.06 6.18 7.06 6.18 5.4
Si-O-Si
Si-O-Si
C=O
C-H
Si-OH
Fig. 7 illustrates the infrared spectra of gemcitabine, non-modified SBA-15 nanorods and Gem loaded samples. All the non-modified and drug loaded samples possess the broad band around 1100 cm-1 which is attributed to the Si-O-Si asymmetric stretching vibration, while the peak at about 800 cm-1 is due to the symmetric stretching vibration of Si-O-Si. In addition, the adsorption band related to the stretching vibrational mode of the silanol groups on the surface was observed around 3400 cm-1 [57,58]. There are new adsorption bands after the surface functionalization and Gem loading. In the FTIR spectrum of G@P-SBA15, the N-H bending peak is shifted to 1470 cm-1, which indicates the interaction between the amine groups and Gem [59]. The presence of electrostatically bonded gemcitabine in G@PC1-SBA-15 and G@PC2SBA-15 was evidenced by the peak at 1555 cm−1, which reveals the formation of COO− -NH+ 3 groups between the carboxyl group of the functionalized samples and amine group of gemcitabine [35,60]. In addition, C-H stretching vibration of methylene groups can be observed around 2850 and 2930 cm-1 [61]. Fig. 8 displays the Raman spectra recorded for the non-modified and functionalized SBA-15 nanorods. The methylene C-H stretching bands are observed at 2800–3100 cm−1 in the Raman spectra of the functionalized samples. In addition, the bands around 1350 and 1650 cm− 1 are related to the C-N and symmetric C_O stretching
Intensity (a. u.)
can be observed in Fig. 4b and c, the width and length of these rods are about 100 nm and 1 μm, respectively. In addition, these nanorods have highly ordered 2D-hexagonal mesochannels along the length of the rods. These observations were in agreement with the results of SAXS patterns. The hydrodynamic diameter and size distribution of the synthesized SBA-15 nanoparticles were measured by DLS. This analysis was done after sonication and filtration of the dispersed nanoparticles in water. Fig. 5 reveals that the diameter of these nanoparticles is about 124 nm with a polydispersity index (PDI) of 0.21. The textural properties of the non-modified SBA-15 nanorods and Gem loaded samples were evaluated by the N2 adsorption–desorption isotherms (Fig. 6a). For all the samples, a typical irreversible type IV nitrogen adsorption isotherm with an H1 hysteresis loop is observed [56]. There is a shift in the hysteresis loop position toward lower relative pressures and a slight decreasing trend in the overall nitrogen adsorption volume in the Gem loaded samples isotherms. This is due to the loaded gemcitabine, which occupies the mesopores of the drug-loaded samples. The shape and the position of hysteresis loop (at p/p0 from 0.60 to 0.85) are correlated to narrow mesopore size distribution [57]. The pore size distribution was calculated by the BJH method based on the desorption branch of N2 adsorption/desorption isotherms. Fig. 6b displays a typical BJH plot with narrow pore size distribution for all the samples. The uniformity of the mesopores in the Gem loaded samples is comparable with the non-modified SBA-15 nanorods. This comparison indicates that the integrity of the original inorganic wall structure of the SBA-15 is retained after the Gem loading. As it can be observed in Table 1, there is a decreasing trend in the specific surface area (SBET), pore volume (Vp) and pore diameter (Dp) of the Gem loaded samples. These results confirm the successful loading of the drug in the mesoporous matrixes.
Wavelength (cm-1) Fig. 7. IR spectra of Gem, non-modified and Gem loaded SBA-15 nanorods samples.
Z. Bahrami et al. / Materials Science and Engineering C 49 (2015) 66–74
2 a. u.
-(CH)2-
15 a. u.
C=O
71
C-N
Intensity (a. u.)
Si-O-Si OH
d c b a
Wavelength (cm-1) Fig. 8. Raman spectra of (a) non-modified SBA-15 nanorods, (b) P-SBA-15, (c) PC1-SBA-15 and (d) PC2-SBA-15.
Weight loss (W%)
vibrations, respectively. These observations confirm the successful functionalization of the mesoporous silica surfaces. Thermogravimetric analysis (TGA) was carried out to verify the grafted organic groups on the surface of SBA-15 nanorods. TGA curves of the samples are shown in Fig. 9. For all the samples, the first weight loss at about 100 °C is related to the desorption of physisorbed water [62]. The weight loss at the temperature range of 200-600 °C is due to the decomposition of the organic groups in all samples. The breakage around 350 °C and the variation of the weight loss are showing two functional groups located on the surface of SBA-15 nanorods. A minor weight loss (about 1%) takes place due to silanol co-condensation at high temperatures [63]. Table 2 presents the drug storage data on four mesoporous silica carriers. In general, the drug adsorption could be divided into two steps: diffusion (to the active sites) and adsorption. SBA-15 nanorods have a large pore size around 7 nm and long pore channels which would be good for the drug diffusion and subsequent adsorption in multilayer. The surface functionalization increases the interaction between the carrier and gemcitabine and results in the better adsorption of the drug. It can be found that the non-modified SBA-15 nanorods can adsorb 7 wt.% of gemcitabine. The modification of SBA-15 nanorods with piperazine and its carboxylic acid derivatives resulted in the higher amount of adsorbed Gem. The increase in Gem loading in the functionalized samples is attributed to the favorable functional groups and drug interaction. In addition, the type of the functional groups also impacts the Gem loading. In contrast with amine groups, carboxylic acid groups have better interaction with Gem. The highest overall Gem loading is achieved for the G@PC2-SBA-15 sample (35.61 wt.%). To the best of our knowledge, this drug loading is high in mesoporous silica-based carrier systems [58,64,65], which may be related to the existence of both
amide (-NH-C_O) and carboxylic acid groups that results in the better and strong interaction between the carrier and drug. The results of the gemcitabine release from all Gem loaded samples are plotted in Fig. 10. The drug release behavior was studied in PBS buffers with different pH values of 5.6 and 7.4 at 37 °C. The drug release could be divided into desorption and diffusion processes. The main influence factor in the desorption of drug from the mesoporous surface, is the superficial functionalization of the mesopores [66]. While for the following diffusion process, the mesoporous structure is a key factor. Surface functionalization leads to an increase in the interaction between the carrier and drug. The interaction between gemcitabine and functional groups (amine and carboxylic acid) in the modified samples is stronger than the interaction between gemcitabine and silanol groups in the non-modified SBA-15 nanorods. Therefore, desorption of gemcitabine from the surface of functionalized samples is more difficult than the non-modified SBA-15 nanorods and the slower drug release rate from the modified samples was observed. SBA-15 nanorods have a large mesoporous size around 7 nm that leads to weak diffusion limitation for drug molecules. After the surface functionalization the pore size of SBA-15 nanorods decreased. Therefore, SBA-15 nanorods have the weaker diffusion limitation in contrast with the modified samples and a faster drug release rate can be observed from SBA-15 nanorods. As shown, the rate of the drug release from G@SBA-15 nanorods is pH dependent and increases with the decrease in pH. The cumulative release amount of Gem could reach up to 60% after 24 h at pH 5.6 in comparison to 27% at pH 7.4. This fact could be explained by taking into account that the hydrogen bonding between gemcitabine and the silanol groups of the non-modified SBA-15 nanorods is relatively weak. In contrast, a slower release rate was observed from the modified samples. It is due to the strong interaction between the functional groups and Gem. The observed decrease of the delivery rate from G@ P-SBA-15 sample is probably because of the strong hydrogen bonding between gemcitabine and amine groups. The formed COO− NH+ 3 bond between Gem and the carboxylic acid groups of PC1-SBA-15 and PC2SBA-15 samples can explain the slower release rates of gemcitabine from carboxylic acid-modified samples. Therefore, the drug release process was governed mainly by the surface functionalization of the carrier.
P-SBA-15 PC2-SBA-15 PC1-SBA-15
Temperature (0C) Fig. 9. TGA curves of the functionalized SBA-15 nanorods.
Table 2 Gemcitabine loading content and entrapment efficiency of the non-modified and modified SBA-15 nanorods. Samples
Loading content (%)
Entrapment efficiency (%)
G@SBA-15 nanorods G@P-SBA-15 G@PC1-SBA-15 G@PC2-SBA-15
7 16.57 24.33 35.61
7.9 19.86 32.16 55.30
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100
100
80
pH=7.4
70
pH=5.6
90
(a) Released Gem (%)
Released Gem (%)
90
60 50 40 30 20 10
(b)
80
pH=7.4
70
pH=5.6
60 50 40 30 20 10
0 0
5
10
15
20
0
25
0
5
10
100 90 80 70 60 50 40 30 20 10 0
15
20
25
Time (h) 100
pH=7.4
90
(c) Released Gem (%)
Released Gem (%)
Time (h)
pH=5.6
80
pH=7.4
70
pH=5.6
(d)
60 50 40 30 20 10
0
5
10
15
20
25
0 0
5
10
Time (h)
15
20
25
Time (h)
Fig. 10. The gemcitabine release profiles of (a) G@SBA-15 nanorods, (b) G@P-SBA, (c) G@PC1-SBA and (d) G@PC2-SBA.
In addition, no significant differences are observed among the drug release rate from G@P-SBA-15, G@PC1-SBA-15 and G@PC2-SBA-15 samples. Although the drug release behavior rate from these samples is similar to each other it should be pointed out that the different amounts of Gem loading content lead to different quantities delivered to the media. From this point of view, it would be possible to choose the amount of drug to be delivered on the basis of the selection of the appropriate functional groups. Fig. 11 shows a comparison of MCF-7 cell line incubated for 1 h at different concentrations (0.1, 0.01, 0.001 mg/mL) of SBA-15 nanorods and G@PC2-SBA-15. The values for two samples were similar to control (untreated cells), indicating that cell viability was not affected at the concentrations range (p b 0.05). As it could be observed from the original graphs for EC50 at 1 h, which are presented in Fig. 12, toxic effects have occurred under different concentrations. The results also showed that when the
concentration is increased (EC50 at 1 h), a significant decrease of cell viability in G@PC2-SBA-15 receptor expressed MCF-7 cell was observed. 3.1. Statistical analysis Multi group comparisons of the means were carried out by oneaway ANOVA. Statistical significance for cell toxicity was set at P b 0.05. Results expressed as mean ± SD. 4. Conclusion We have demonstrated the synthesis of the new functionalized SBA15 nanorods using piperazine and its carboxylic acid derivatives. Bromoacetic acid and succinic anhydride were applied to the preparation of the carboxylic acid-functionalized samples. Gemcitabine, as an anticancer drug, was introduced in the non-modified and functionalized
P
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Piperazine and its carboxylic acid derivatives-functionalized mesoporous silica as nanocarriers for gemcitabine: Adsorption and release study Zohreh Bahrami a, Alireza Badiei a,b,⁎, Fatemeh Atyabi c, Hossein Reza Darabi d, Bita Mehravi e,f a
School of Chemistry, College of Science, University of Tehran, Tehran, Iran Nanobiomedicine Center of Excellence, Nanoscience and Nanotechnology Research Center, University of Tehran, Tehran, Iran Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14174, Iran d Chemistry and Chemical Engineering Research Center of Iran, Nano and Organic Synthesis Lab, Tehran, Iran e Celullar and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran f Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 30 October 2014 Accepted 17 December 2014 Available online 19 December 2014 Keywords: SBA-15 Nanorods Piperazine Carboxylic acid-functionalization Gemcitabine Cytotoxicity
a b s t r a c t Piperazine-functionalized SBA-15 nanorods were synthesized by post grafting method with methyldimethoxysilylpropylpiperazine (MDSP). The carboxylic acid derivatives of piperazine-functionalized SBA-15 nanorods were obtained using two different kinds of precursors (bromoacetic acid and succinic anhydride). The prepared materials were used as nanocarriers for the anticancer drug (gemcitabine). The obtained samples were characterized by SAXS, N2 adsorption-desorption, SEM, TEM, DLS, thermogravimetric analysis, FTIR, Raman and UV spectroscopies. The adsorption and release properties of all samples were investigated. In vitro study included cell toxicity. It was found that the surface functionalization increases the interaction between the carrier and gemcitabine and results in the loading enhancement of the drug. In addition, the adsorption of gemcitabine on the modified mesoporous matrix depends on the type of the introduced functional groups. The carboxylic acid-modified samples have higher loading content, due to the strong interaction with gemcitabine. The maximum content of deposited drug in the modified SBA-15 nanorods is close to 36 wt.% that it is related to PC2-SBA-15 sample which obtained using succinic anhydride. The obtained results reveal that the surface functionalization leads toward a significant decrease of the drug release rate without any appreciable cytotoxicity. No significant differences are observed among the drug release rate from the modified samples. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Gemcitabine (Gem) is a water-soluble low-molecular-weight anticancer drug that is commonly used for the treatment of several kinds of cancers including colon, pancreatic, lung, breast, ovarian and bladder [1–3]. Gemcitabine passes through the cell membrane with difficulty and requires active transporters localized on the membrane to enter the cells [4,5]. Different drug delivery systems such as liposomes and polymeric nanoparticles were designed in order to protect Gem from rapid metabolization, overcome drug resistance, target drug delivery and improve its anticancer efficacy [6,7]. Since 2001, when MCM-41 was first proposed as a drug delivery system (DDS) [8], silica-based materials have been discussed as drug carriers. Mesoporous silica nanoparticles (MSNs) possess several unique features such as high surface area, large pore volume, uniform and
⁎ Corresponding author at: School of Chemistry, College of Science, University of Tehran, Tehran, Iran. E-mail address: [email protected] (A. Badiei).
http://dx.doi.org/10.1016/j.msec.2014.12.069 0928-4931/© 2014 Elsevier B.V. All rights reserved.
tunable pore sizes, excellent physicochemical stability, controllable morphology, modifiable surfaces, nontoxic nature and well biocompatibility [9–15]. These properties result in the study of application of MSNs in the biomedical field have attracted great attention. MSNs offer the possibility of lodging a variety of drug molecules such as, anti-inflammatory [8,16–20], antibiotic [11,21–23], antihypertensive [24], antiulcer [25,26], anti-osteoporotic [27] and anticancer [23]. It was demonstrated that surface functionalization [16,18,25,28], size and structure of pore [24,29–31], loading conditions [32] and chemical characteristics of the loaded drug [33] can affect both the adsorption and release of the drug into and out of MSNs. The chemical functionalization of the surface is essential because a mesoporous silica surface covered with silanol groups that are not selective enough to adsorb drug molecules with different functionalities [34,35]. In addition, it was shown that the attractive and repulsive electrostatic interactions between the entrapped molecules and the silica surface play an important role in the amount of adsorption of drug molecules and rate of their release. Therefore, the choice of appropriate functionalizing agent can enhance the adsorption capacity of drug and it should also allow modulating its release [35–39]. For example, the modification of MSNs with
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amine and carboxylic acid groups increases the amount of adsorbed drugs with acidic and basic properties, respectively [20,25,38,40,41]. The morphology of MSNs can also affect the drug adsorption and release. Tsai et al. found out that rod-like MSNs have greater potential than that of spherical ones in monitoring the cell trafficking, cancer cell metastasis and drug/DNA delivery [42]. Zhao and co-workers showed that rod-like SBA-15 (about 1–2 μm in length) had a faster adsorption rate and immobilized larger amount of enzymes than rope-like SBA-15 (20 μm in length) [43,44]. The effect of particle shape on the in vivo biological behaviors of MSNs was studied. Huang et al. designed two different shaped fluorescent MSNs, short rod MSN-FITC (ARs of ~1.5, length of ~200 nm) and long rod MSN-FITC (ARs of ~5, length of ~ 800 nm), and investigated this effect. The obtained findings reveal that MSNs actively interact with cells and engage in and mediate in vivo behaviors. It is also demonstrated that MSNs would not cause significant toxicity in vivo [45]. Functionalized SBA-15 nanoparticles with particle sizes of 500–800 nm in length and 300–500 nm in diameter have been investigated as nanocarriers for bleomycin (as an anticancer drug) delivery. The efficient cellular uptake and low cytotoxicity of the functionalized SBA-15 nanoparticles have been confirmed by in vitro cell assays [46]. MCM-41 and SBA-15 as two main conventional mesoporous materials have been applied as excellent carriers for encapsulation of different kinds of drugs. They have ordered hexagonal mesopores and different pore sizes, which are 2–5 nm and 5–10 nm for MCM-41 and SBA-15, respectively. In addition, SBA-15 has thicker pore walls and micropores that interconnect the hexagonal channels [21,40]. It is proved that the large pore size leads to the increasing of the drug loading amount and release rate [17,29,35,47,48]. The influence of the pore morphology and geometry on the drug loading and release properties was investigated. It is shown that the interconnected pore systems result in the easier and faster diffusion process in contrast with the unconnected pore systems [49]. This effect was extensively studied by Alexa et al. on the release profiles of methotrexate, as an anticancer drug. The obtained results reveal that SBA-15 is the better methotrexate delivery system in comparison to MCM-41 [50]. Up to now, to the best of our knowledge, a few articles reported the application of SBA-15 nanoparticles in drug delivery especially for anticancer drugs. This paper describes the adsorption and release of gemcitabine on the piperazine and its carboxylic acid derivatives-functionalized SBA15 nanorods. Investigation of the effect of the surface functionalization on the adsorption capacity and release behavior of Gem is the main aim of this study.
2. Experimental 2.1. Materials Poly(ethylene glycol) block poly(propylene glycol) block poly(ethylene glycol), P123, (EO20PO70EO20, MW = 5800 g/mol) was purchased from Aldrich. Tetraethyl orthosilicate (TEOS), hydrochloric acid (35%), ethanol, N,N′-dimethylformamide (DMF), bromoacetic acid, and succinic anhydride were obtained from Merck (Darmstadt, Germany). Methyldimethoxysilylpropylpiperazine (MDSP) and gemcitabine were purchased from Power Chemical Corporation (Nanjing, China) and Lilly France S.A.S. Company (rue Pagès Suresnes, France), respectively. All chemical reagents were used without further purification. Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), penicillin G (100 U/ml), streptomycin (100 μg/ml), Glutamax and Trypsin-EDTA were purchased from GIBCO-BRL (Grand Island, NY, USA). Human Breast Cancer cell lines (MCF-7) were provided from the National Cell Bank of Pasteur Institute of Iran (Tehran, Iran).
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2.2. Synthesis of SBA-15 nanorods SBA-15 nanorods were synthesized according to the procedure reported in the our previous work [51]. Briefly, 23.4 g of nonionic triblock copolymer (Pluronic P123) was dissolved in the mixture of deionized water (606.8 g) and hydrochloric acid (146.4 g, 35%). 50 g of TEOS was added to the solution while stirring at the rate of 500 rpm at 55 °C. The reaction batch was maintained at static conditions for 24 h at this temperature and then for another 24 h at 100 °C for further condensation. The precipitate was obtained after filtration and then triblock copolymer template was removed by ethanol extraction through a soxhlet extractor. 2.3. Synthesis of piperazine-functionalized SBA-15 nanorods (P-SBA-15) SBA-15 nanorods were functionalized using methyldimethoxysilylpropylpiperazine (MDSP) by post-grafting method [52]. Typically, 0.1 g of SBA-15 nanorods was dispersed in 20 mL of anhydrous ethanol. Following the addition of MDSP (1 mL), the mixture was stirred moderately at 80 °C for 6 h. The precipitate was collected by centrifugation, washed with ethanol and dried at room temperature to give the piperazine-functionalized SBA-15 nanorods as white powder. The obtained modified SBA-15 was denoted as P-SBA-15 (Fig. 1a). 2.4. Synthesis of carboxylic acid-functionalized SBA-15 nanorods (PC1-SBA-15 and PC2-SBA-15) The –COOH functionalization of SBA-15 nanorods was carried out in two ways as follows: 1) 0.1 g of piperazine-functionalized SBA-15 was dispersed in 25 mL ethanol containing 5 mmol bromoacetic acid. The mixture was allowed to react at 80 °C for 4 h. The precipitate was collected by centrifugation, washed with ethanol and dried at room temperature. The synthesized sample was designated as PC1-SBA-15 (Fig. 1b). 2) 0.1 g of piperazine-functionalized SBA-15 nanorods was added in the N,N′-dimethylformamide (DMF) solution of succinic anhydride (50 mL, 2 wt.%). The mixture was stirred for 24 h at room temperature. After that, the product was collected by centrifugation, washed with DMF and dried at room temperature. The obtained sample was denoted as PC2-SBA-15 (Fig. 1b). 2.5. Gemcitabine loading Gemcitabine (Fig. 2) was chosen as a model drug to evaluate the loading and release behavior of the non-modified and functionalized SBA-15 nanorods. SBA-15 nanorods and gemcitabine were dispersed in distilled water to form 5 mg·mL-1 solutions. Then, 2 mL of gemcitabine solution was mixed with 2 mL of SBA-15 nanorods solution. The mixture was stirred at room temperature for 24 h to reach the equilibrium state. The gemcitabine loaded SBA-15 nanorods (G@SBA-15) were collected by centrifugation and washed several times with distilled water to remove the physically adsorbed Gem. A similar procedure was conducted for all the functionalized samples. The prepared samples were labeled as, G@P-SBA-15, G@PC1-SBA-15 and G@PC2-SBA-15. The amount of the loaded drug was determined by a UV/Vis spectrophotometer at 269 nm. The drug loading content and the entrapment efficiency were calculated using the following equations:
Loading contentð%Þ ¼ ðWeight of Gem in mesoporous materialÞ =ðWeight of Gem loaded mesoporous materialÞ Entrapment efficiencyð%Þ ¼ ðWeight of Gem in mesoporous materialÞ =ðInitial weight of GemÞ:
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SBA-15
P-SBA-15
(a)
PC1-SBA-15
(b)
PC2-SBA-15
Fig. 1. Surface functionalization of SBA-15 nanorods: (a) piperazine functionalization and (b) carboxylic acid functionalization.
2.6. Gemcitabine release
Intensity (a. u.)
110 200
100
According to the our previous study [51] and some reports [52,53], the phosphate buffered saline (PBS) solutions with the pH values of 5.6 and 7.4 were chosen for the drug release experiments {since the certain tissues of the body have acidic pH in comparison to the blood (pH = 7.4), it is possible to design the carrier systems, which provide
a safe and efficient way for drug release targeting specific sites in the body, such as endosomes (pH ~ 5.6) [41]}. The certain amount of Gem loaded samples was dispersed in 4 mL of PBS solution and kept at 37 °C using an incubator in order to simulate body temperature. At given time intervals, a suspension was centrifuged at 13000 rpm for 15 min. The amount of released drug was measured by a UV/Vis spectrophotometer at 269 nm.
2θ (degree) Fig. 2. The structure of gemcitabine.
Fig. 3. SAXS patterns of the non-modified and modified SBA-15 nanorods.
Z. Bahrami et al. / Materials Science and Engineering C 49 (2015) 66–74
(a)
(b)
69
(c)
Fig. 4. SBA-15 nanorods : (a) SEM, (b) and (c) TEM images.
2.7. Cell culture Human Breast Cancer (MCF-7) cell lines cultured in flasks and incubated under a humidified atmosphere with 5% CO2 at 37 °C and using standard cell culture media, containing Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin. The complete medium was changed every 3 days. When the cells reached 80% confluence, they were detached and re-plated using 0.25% Trypsin-EDTA. 2.8. Cell viability test For the MTT assay, MCF-7 cell lines (5 × 103 cells per well) were incubated with various amounts of SBA-15 nanorods and G@PC2-SBA-15 (0.1, 0.01, 0.001 mg/mL) in a 96-well micro-plate for 1 h with untreated cells as control. Each concentration was tested in triplicates. The cells were washed with PBS before the addition of the MTT solution (20 μl of 5 mg/mL MTT solution) to each well. After 1 h, absorbance of purple formazone was measured by using BioTek's absorbance microplate readers at 630 nm. 2.9. Inhibitory concentration 50 determination Based on the obtained toxic effects of SBA-15 nanorods and G@PC2SBA-15 synthesized here on MCF-7 cells, the effective half inhibitory dose of the conjugate was calculated based on the linear model method. On the basis of linear regression equation of exposure time of 12 and 24 h, the EC50 or IC50 of SBA-15 nanorods and G@PC2-SBA-15 were obtained to be 21.294 mg/mL and 12.355 mg/mL at 1 h, respectively. 2.10. Characterization The small angle X-ray scattering (SAXS) patterns were recorded with a model Hecus S3-MICROpix SAXS diffractometer with a onedimensional PSD detector using Cu Kα radiation (50 kV, 1 mA) at the
wavelength 1.542 Å. The SEM and TEM images were taken using Oxford LEO 1455 V STEM and Philips EM-208 100 KV, respectively. Nitrogen physisorption isotherms were obtained on a BELSORP mini-II at liquid nitrogen temperature (77 K). The specific surface areas were measured using multiple point Brunauer–Emmett–Teller (BET) method. The pore size distributions were calculated using desorption branches of the isotherms by Barrett–Joyner–Halenda (BJH) method. The mean diameter and size distribution of the nanoparticles were measured by dynamic light scattering (DLS) using Zetasizer (Nano-ZS; Malvern Instruments, Malvern, UK). The FTIR spectra were recorded using Equinox 55 spectrometer in the range between 400 and 4000 cm-1. The Raman spectra were obtained on SENTERRA spectrometer. Thermogravimetric analysis (TGA) was performed on a TA Q50 instrument. The scans were performed between 20 °C and 800 °C at 10 °C/min. The UV/Vis absorption spectra were recorded using a Ray light, UV 1600 spectrophotometer. 3. Results and discussion The small angle X-ray scattering (SAXS) patterns of the nonmodified and modified SBA-15 nanorods were shown in Fig. 3. All the samples have a single intensive reflection at around 2θ = 0.72° similar to the typical SBA-15 materials, which is reported in the literature [54]. Two additional peaks which are related to the higher ordering (110) and (200) reflections are observed in all the patterns. These three peaks are characteristic of the ordered two-dimensional hexagonal (p6mm) structure [55]. The existence of three main diffraction peaks in the patterns of the functionalized samples shows that the hexagonal structure of SBA-15 is stable during the surface functionalization. However, the intensity of the diffraction peaks decreased after the functionalization due to the existence of organic groups in the mesochannels. Scanning electron microscopy (SEM) was used to determine the morphology of the samples. A representative micrograph of the SBA15 matrix is shown in Fig. 4a, where there are particles having a rodlike shape. The transmission electron microscopy (TEM) study was performed to determine the particle size and pore geometry structure. As it
Fig. 5. Size distribution of SBA-15 nanoparticles.
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dvp/dvr
Va/cm3 (STP) g-1
70
r (nm) p/p0 (a)
(b)
Fig. 6. (a) N2 adsorption/desorption isotherms and (b) pore size distribution of the non-modified SBA-15 nanorods and Gem loaded samples.
Table 1 Specific surface area (SBET) (m2 g-1), pore volume (Vp) (cm3 g-1) and pore diameter (Dp) (nm) from N2 adsorption/desorption for the non-modified SBA-15 nanorods and Gem loaded samples. Sample
Specific surface area (SBET) (m2g-1)
Pore volume (Vp) (cm3g-1)
Pore diameter (Dp) (nm)
SBA-15 nanorods G@SBA-15 G@P-SBA-15 G@PC1-SBA-15 G@PC2-SBA-15
1010 449 263 227 256
1.27 0.811 0.836 0.467 0.552
7.06 6.18 7.06 6.18 5.4
Si-O-Si
Si-O-Si
C=O
C-H
Si-OH
Fig. 7 illustrates the infrared spectra of gemcitabine, non-modified SBA-15 nanorods and Gem loaded samples. All the non-modified and drug loaded samples possess the broad band around 1100 cm-1 which is attributed to the Si-O-Si asymmetric stretching vibration, while the peak at about 800 cm-1 is due to the symmetric stretching vibration of Si-O-Si. In addition, the adsorption band related to the stretching vibrational mode of the silanol groups on the surface was observed around 3400 cm-1 [57,58]. There are new adsorption bands after the surface functionalization and Gem loading. In the FTIR spectrum of G@P-SBA15, the N-H bending peak is shifted to 1470 cm-1, which indicates the interaction between the amine groups and Gem [59]. The presence of electrostatically bonded gemcitabine in G@PC1-SBA-15 and G@PC2SBA-15 was evidenced by the peak at 1555 cm−1, which reveals the formation of COO− -NH+ 3 groups between the carboxyl group of the functionalized samples and amine group of gemcitabine [35,60]. In addition, C-H stretching vibration of methylene groups can be observed around 2850 and 2930 cm-1 [61]. Fig. 8 displays the Raman spectra recorded for the non-modified and functionalized SBA-15 nanorods. The methylene C-H stretching bands are observed at 2800–3100 cm−1 in the Raman spectra of the functionalized samples. In addition, the bands around 1350 and 1650 cm− 1 are related to the C-N and symmetric C_O stretching
Intensity (a. u.)
can be observed in Fig. 4b and c, the width and length of these rods are about 100 nm and 1 μm, respectively. In addition, these nanorods have highly ordered 2D-hexagonal mesochannels along the length of the rods. These observations were in agreement with the results of SAXS patterns. The hydrodynamic diameter and size distribution of the synthesized SBA-15 nanoparticles were measured by DLS. This analysis was done after sonication and filtration of the dispersed nanoparticles in water. Fig. 5 reveals that the diameter of these nanoparticles is about 124 nm with a polydispersity index (PDI) of 0.21. The textural properties of the non-modified SBA-15 nanorods and Gem loaded samples were evaluated by the N2 adsorption–desorption isotherms (Fig. 6a). For all the samples, a typical irreversible type IV nitrogen adsorption isotherm with an H1 hysteresis loop is observed [56]. There is a shift in the hysteresis loop position toward lower relative pressures and a slight decreasing trend in the overall nitrogen adsorption volume in the Gem loaded samples isotherms. This is due to the loaded gemcitabine, which occupies the mesopores of the drug-loaded samples. The shape and the position of hysteresis loop (at p/p0 from 0.60 to 0.85) are correlated to narrow mesopore size distribution [57]. The pore size distribution was calculated by the BJH method based on the desorption branch of N2 adsorption/desorption isotherms. Fig. 6b displays a typical BJH plot with narrow pore size distribution for all the samples. The uniformity of the mesopores in the Gem loaded samples is comparable with the non-modified SBA-15 nanorods. This comparison indicates that the integrity of the original inorganic wall structure of the SBA-15 is retained after the Gem loading. As it can be observed in Table 1, there is a decreasing trend in the specific surface area (SBET), pore volume (Vp) and pore diameter (Dp) of the Gem loaded samples. These results confirm the successful loading of the drug in the mesoporous matrixes.
Wavelength (cm-1) Fig. 7. IR spectra of Gem, non-modified and Gem loaded SBA-15 nanorods samples.
Z. Bahrami et al. / Materials Science and Engineering C 49 (2015) 66–74
2 a. u.
-(CH)2-
15 a. u.
C=O
71
C-N
Intensity (a. u.)
Si-O-Si OH
d c b a
Wavelength (cm-1) Fig. 8. Raman spectra of (a) non-modified SBA-15 nanorods, (b) P-SBA-15, (c) PC1-SBA-15 and (d) PC2-SBA-15.
Weight loss (W%)
vibrations, respectively. These observations confirm the successful functionalization of the mesoporous silica surfaces. Thermogravimetric analysis (TGA) was carried out to verify the grafted organic groups on the surface of SBA-15 nanorods. TGA curves of the samples are shown in Fig. 9. For all the samples, the first weight loss at about 100 °C is related to the desorption of physisorbed water [62]. The weight loss at the temperature range of 200-600 °C is due to the decomposition of the organic groups in all samples. The breakage around 350 °C and the variation of the weight loss are showing two functional groups located on the surface of SBA-15 nanorods. A minor weight loss (about 1%) takes place due to silanol co-condensation at high temperatures [63]. Table 2 presents the drug storage data on four mesoporous silica carriers. In general, the drug adsorption could be divided into two steps: diffusion (to the active sites) and adsorption. SBA-15 nanorods have a large pore size around 7 nm and long pore channels which would be good for the drug diffusion and subsequent adsorption in multilayer. The surface functionalization increases the interaction between the carrier and gemcitabine and results in the better adsorption of the drug. It can be found that the non-modified SBA-15 nanorods can adsorb 7 wt.% of gemcitabine. The modification of SBA-15 nanorods with piperazine and its carboxylic acid derivatives resulted in the higher amount of adsorbed Gem. The increase in Gem loading in the functionalized samples is attributed to the favorable functional groups and drug interaction. In addition, the type of the functional groups also impacts the Gem loading. In contrast with amine groups, carboxylic acid groups have better interaction with Gem. The highest overall Gem loading is achieved for the G@PC2-SBA-15 sample (35.61 wt.%). To the best of our knowledge, this drug loading is high in mesoporous silica-based carrier systems [58,64,65], which may be related to the existence of both
amide (-NH-C_O) and carboxylic acid groups that results in the better and strong interaction between the carrier and drug. The results of the gemcitabine release from all Gem loaded samples are plotted in Fig. 10. The drug release behavior was studied in PBS buffers with different pH values of 5.6 and 7.4 at 37 °C. The drug release could be divided into desorption and diffusion processes. The main influence factor in the desorption of drug from the mesoporous surface, is the superficial functionalization of the mesopores [66]. While for the following diffusion process, the mesoporous structure is a key factor. Surface functionalization leads to an increase in the interaction between the carrier and drug. The interaction between gemcitabine and functional groups (amine and carboxylic acid) in the modified samples is stronger than the interaction between gemcitabine and silanol groups in the non-modified SBA-15 nanorods. Therefore, desorption of gemcitabine from the surface of functionalized samples is more difficult than the non-modified SBA-15 nanorods and the slower drug release rate from the modified samples was observed. SBA-15 nanorods have a large mesoporous size around 7 nm that leads to weak diffusion limitation for drug molecules. After the surface functionalization the pore size of SBA-15 nanorods decreased. Therefore, SBA-15 nanorods have the weaker diffusion limitation in contrast with the modified samples and a faster drug release rate can be observed from SBA-15 nanorods. As shown, the rate of the drug release from G@SBA-15 nanorods is pH dependent and increases with the decrease in pH. The cumulative release amount of Gem could reach up to 60% after 24 h at pH 5.6 in comparison to 27% at pH 7.4. This fact could be explained by taking into account that the hydrogen bonding between gemcitabine and the silanol groups of the non-modified SBA-15 nanorods is relatively weak. In contrast, a slower release rate was observed from the modified samples. It is due to the strong interaction between the functional groups and Gem. The observed decrease of the delivery rate from G@ P-SBA-15 sample is probably because of the strong hydrogen bonding between gemcitabine and amine groups. The formed COO− NH+ 3 bond between Gem and the carboxylic acid groups of PC1-SBA-15 and PC2SBA-15 samples can explain the slower release rates of gemcitabine from carboxylic acid-modified samples. Therefore, the drug release process was governed mainly by the surface functionalization of the carrier.
P-SBA-15 PC2-SBA-15 PC1-SBA-15
Temperature (0C) Fig. 9. TGA curves of the functionalized SBA-15 nanorods.
Table 2 Gemcitabine loading content and entrapment efficiency of the non-modified and modified SBA-15 nanorods. Samples
Loading content (%)
Entrapment efficiency (%)
G@SBA-15 nanorods G@P-SBA-15 G@PC1-SBA-15 G@PC2-SBA-15
7 16.57 24.33 35.61
7.9 19.86 32.16 55.30
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100
100
80
pH=7.4
70
pH=5.6
90
(a) Released Gem (%)
Released Gem (%)
90
60 50 40 30 20 10
(b)
80
pH=7.4
70
pH=5.6
60 50 40 30 20 10
0 0
5
10
15
20
0
25
0
5
10
100 90 80 70 60 50 40 30 20 10 0
15
20
25
Time (h) 100
pH=7.4
90
(c) Released Gem (%)
Released Gem (%)
Time (h)
pH=5.6
80
pH=7.4
70
pH=5.6
(d)
60 50 40 30 20 10
0
5
10
15
20
25
0 0
5
10
Time (h)
15
20
25
Time (h)
Fig. 10. The gemcitabine release profiles of (a) G@SBA-15 nanorods, (b) G@P-SBA, (c) G@PC1-SBA and (d) G@PC2-SBA.
In addition, no significant differences are observed among the drug release rate from G@P-SBA-15, G@PC1-SBA-15 and G@PC2-SBA-15 samples. Although the drug release behavior rate from these samples is similar to each other it should be pointed out that the different amounts of Gem loading content lead to different quantities delivered to the media. From this point of view, it would be possible to choose the amount of drug to be delivered on the basis of the selection of the appropriate functional groups. Fig. 11 shows a comparison of MCF-7 cell line incubated for 1 h at different concentrations (0.1, 0.01, 0.001 mg/mL) of SBA-15 nanorods and G@PC2-SBA-15. The values for two samples were similar to control (untreated cells), indicating that cell viability was not affected at the concentrations range (p b 0.05). As it could be observed from the original graphs for EC50 at 1 h, which are presented in Fig. 12, toxic effects have occurred under different concentrations. The results also showed that when the
concentration is increased (EC50 at 1 h), a significant decrease of cell viability in G@PC2-SBA-15 receptor expressed MCF-7 cell was observed. 3.1. Statistical analysis Multi group comparisons of the means were carried out by oneaway ANOVA. Statistical significance for cell toxicity was set at P b 0.05. Results expressed as mean ± SD. 4. Conclusion We have demonstrated the synthesis of the new functionalized SBA15 nanorods using piperazine and its carboxylic acid derivatives. Bromoacetic acid and succinic anhydride were applied to the preparation of the carboxylic acid-functionalized samples. Gemcitabine, as an anticancer drug, was introduced in the non-modified and functionalized
P
