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Folate-conjugated hybrid SBA-15 particles for targeted anticancer drug delivery
Journal of Colloid and Interface Science 395 (2013) 31–39
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Folate-conjugated hybrid SBA-15 particles for targeted anticancer drug delivery Jianmei Pang a, Lanxia Zhao a, Longlong Zhang a, Zhonghao Li b, Yuxia Luan a,⇑ a b
School of Pharmaceutical Science, Shandong University, Jinan, Shandong Province 250012, PR China School of Materials Science and Engineering, Shandong University, Jinan, Shandong Province 250061, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2012 Accepted 3 December 2012 Available online 19 December 2012 Keywords: SBA-15 Surface functionalization Targeting Cytotoxicity
a b s t r a c t Surface functionalization is one of the key steps toward the utilization of mesoporous materials in drug delivery system. Here, the folic acid (FA) ligands are conjugated onto poly(ethylene imine) (PEI) modified SBA-15 particles (PEI/SBA-15) via amide reaction, which results in the FA/PEI/SBA-15 particles. Doxorubicin hydrochloride (DOX), an anticancer drug, is successfully loaded into these particles. The in vitro cytotoxicity and cellular uptake of the empty FA/PEI/SBA-15 particles and the DOX-loaded ones are evaluated on two kinds of cancer cells (HeLa cells and A549 cells). Specifically, an excellent cellular uptake using the current anticancer drug delivery vehicles (DOX-loaded FA/PEI/SBA-15 particles) mediated by the FA receptor is demonstrated by fluorescence microscope and flow cytometry. The FA/PEI/SBA-15 particles demonstrate a lower cytotoxicity comparing with the PEI/SBA-15 particles, while the DOX-loaded FA/PEI/SBA-15 particles exhibit much greater inhibition to the studied cancer cells. Furthermore, the in vitro release study shows that the targeted FA/PEI/SBA-15 particles have a typical sustained release behavior. This work therefore demonstrates that drug-loaded FA/PEI/SBA-15 particles have great potential application in targeted anticancer drug delivery for cancer therapy. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction The applications of mesoporous silica particles (MSPs) as drug carriers have drawn growing interest in the past few years [1–7]. MSPs have several attractive features, such as high surface area and large pore volume, stable and ordered pore framework, uniform and tunable pore size, non-toxic and biocompatibility. What’s more, there are silanol groups on both the internal and the external surface, which makes it simple to be modified. In this case, modification of the carrier surface, especially organic and inorganic nanocomposites, is one of the key steps toward the utilization of mesoporous materials [8,9]. Due to the rich Si–OH groups on the surface, the mesoporous silica materials are easy to be modified with desirable functionalities [10]. Among others, hyperbranching surface polymerization is promising for amino functionalization of mesoporous silica as the surface concentration of amino groups is much higher than post-grafting method and co-condensation method [11–15]. Especially, the quantity of the PEI layer can be controlled simply by changing the aziridine/silica ratio in the surface functionalization step. Moreover, it is demonstrated that a hydrophobized mesoporous silica material can be fully redispersed in aqueous media after PEI functionalization of the outer surface [14]. Besides, carboxylic acid functions can be introduced to the PEI layer by amide reactions. ⇑ Corresponding author. Fax: +86 531 88382548. E-mail address: [email protected] (Y. Luan). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.12.016
Doxorubicin hydrochloride (DOX), a potent antibiotic, is the first-line treatment used for a wide range of cancers [16–18]. However, due to the lack of ability to target cancer cells, it presents major drawbacks such as drug resistance and nonselective cytotoxicity, which usually leads to severe side effects. In order to improve the therapeutic efficacy of DOX, various drug delivery systems have been reported, such as liposomes, polymeric particles, and polymer conjugates [11,19–21]. In principle, drug targeting with noninvasive techniques is highly desirable as the preferred system should supply increased local drug concentration with reduced systemic side effects [20]. Still, it is of great challenge to achieve a useful targeted anticancer drug delivery system. Attaching specific ligands to the particle surface to achieve targeting of specific cells is generally studied in drug targeted delivery system. Folic acid (FA), one of the well known receptor mediated targeting moieties, has high binding affinity to the FA receptors on the cell surface of FA-positive tumors. FA receptor can be over expressed on the surfaces of some specific human tumors, including ovarian, brain, endometrial, kidney, and breast cancer cells. Therefore, the covalently conjugated FA to drug carriers could achieve selective targeting to tumors [22–25]. It is known that FA has carboxylic acid thus it can be readily linked by amide reactions if there are amino functions on the outer surface of MSNs [26–28]. Rosenholm et al. prepared PEI-functionalized MSNs by surface hyperbranching polymerization and modified it by introducing fluorescent molecules [15].
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Herein, we load the anticancer drug DOX into the FA-grafted PEI-functionalized SBA-15 particles. The schematic procedure is shown in Scheme 1. The in vitro cytotoxicity and the cellular uptake to the cells demonstrate that the FA-conjugated PEI/SBA-15 particles can effectively target cancer cells and enhance the cellular uptake. Thus, the results indicate that the prepared FA targeted PEI modified mesoporous silica particles would be desirable for its application in cancer therapy.
under dark conditions, the DOX-loaded particles were centrifuged and washed with PBS for 3 times. Finally, the samples were dried at 50 °C for 12 h, sealed, and stored under dark conditions. The DOX loading content was determined by thermogravimetry (TG) and calculated by the following formula:
2. Experimental section
2.4. Characterization
2.1. Preparation of PEI/SBA-15
Scanning electron microscopy (SEM) was performed with a Hitachi SU-70 FESEM scanning electron microscope operated at 3 kV. Samples were sputtered with Au prior to imaging (The thickness of Au was 5 nm.). The Brunauer–Emmett–Teller (BET) surface area and Barrett–Joyner–Halenda (BJH) pore size distribution were measured by nitrogen (N2) adsorption/desorption using a QuadraSorb SI surface area analyzer. Thermogravimetric (TG) analysis was performed using a PerkinElmerPyris6. The Fourier transform infrared (FTIR) spectra were obtained in the range of 400–4000 cm1 using a Nicolet NEXUS 470 FT-IR Spectrometer by dispersing the powder samples in KBr pellets.
2.1.1. Synthesis of aziridine Aziridine was synthesized and purified similar to the reported methods [6]. 25 g of AHS (2-Aminoethyl Hydrogen Sulfate, Tokyo Chemical Industry Co., LTD) was added to NaOH solution (40 wt%). Then, the mixture was heated to boiling in an oil bath. The bath was removed after boiling started and resumed when the mixture stopped boiling. The distillate was quickly collected between 50 and 105 °C. Then, KOH was added to the distillate, which was put in an ice bath. Aziridine was separated in the upper layer which could be collected from the mixture and stored over KOH at 4 °C overnight. After observing that no aqueous layer appeared, the prepared aziridine was sealed and stored at 0 °C. (Caution: Aziridine is a carcinogen and reproductive hazard. It is toxic if swallowed, inhaled, or absorbed through the skin. Aziridine is a carcinogen. Extreme caution is required when handling aziridine.) 2.1.2. Polymerization of aziridine on the surfaces of SBA-15 Hyperbranched PEI was grafted by aziridine polymerization on the surface of SBA-15 (Shenyang shunfeng Paint Co. Ltd.). The synthesis procedure is similar to the previous reported [14]. 1.5 g vacuum-dried SBA-15 was immersed in toluene under nitrogen. Then, 500 ll of aziridine and catalytic amounts of acetic acid were added. The reaction mixture was stirred overnight at 75 °C. The resulting substrate was thoroughly washed with copious amounts of toluene and dried in vacuo at room temperature for 48 h. 2.2. Preparation of FA/PEI/SBA-15 65 mg FA was dissolved in 3 ml dimethylsulfoxide (DMSO) and stirred for 10 h. Then, 34 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 38 mg of NHS (NHydroxysuccinimide) were added to the solution with stirring for 16 h. Subsequently, the amino group terminated PEI/SBA-15 was added to the above solution and allowed to react with stirring for 24 h at room temperature. After the reaction, the mixture was centrifuged and washed with water and ethanol several times to obtain FA/PEI/SBA-15 particles.
Loading Content ¼
Weight of DOX in Particles 100% Weight of the DOX loaded Particles
2.5. Cell culture Two cell lines, HeLa cervical carcinoma cells and A549 cells (human lung adenocarcinoma cell line), were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS, Solarbio), 100 U ml1 penicillin, and 100 lg ml1 streptomycin at 37 °C, in 5% CO2 atmosphere. HeLa and A549 cells were kindly supplied by the Pharmaceutical Department of Shandong University in China. 2.6. Cellular uptake Cellular uptake by HeLa cells and A549 cells were examined using fluorescence microscope and flow cytometry, respectively. The cells were seeded in 6-well culture plates (2 105 cells/well) and grown overnight. Then, the cells were incubated with free DOX or DOX-loaded particles (equivalent DOX concentration: 3 lg ml1) in RPMI1640 supplemented with 10% FBS. For FA competition experiments, the cells were cultured with 2 mM FA prior to addition of DOX-FA/PEI/SBA-15. After 1 h of incubation, the supernatant was carefully removed and the cells were washed three times with 4 °C PBS to remove the extra DOX molecules. The cells were examined by a fluorescence microscope (Olympus, Japan) to visualize cellular uptake. For flow cytometric analysis, the cells (1 104 counts) were harvested and analyzed by a flow cytometry (FACS Vantage, USA) to quantify the fluorescence intensity of DOX within the cell. 2.7. In vitro cytotoxicity
2.3. Loading DOX A total of 100 mg of particles (SBA-15, PEI/SBA-15, or FA/PEI/ SBA-15) were mixed with 20 ml of 1 mg ml1 DOX solution in PBS (phosphate buffered solution, pH 7.4). After stirring for 24 h
Cytotoxicity of free DOX solution, DOX-loaded samples (DOXSBA-15, DOX-FA/PEI/SBA-15), and the samples without DOX (SBA-15, PEI/SBA-15, FA/PEI/SBA-15) were evaluated by measuring the cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
Scheme 1. Schematic procedure for preparation and drug loading of FA/PEI/SBA-15 particles.
J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39
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Fig. 1. SEM images of SBA-15 particles (a), FA/PEI/SBA-15 (b) and N2 adsorption–desorption isotherms of SBA-15 (c), PEI/SBA-15 (d), and FA/PEI/SBA-15 (e), the inserts are the corresponding pore size distributions.
diphenyltetrazolium bromide) assay. The FA-receptor-positive HeLa cells [FR(+)] and FA-receptor-negative A549 cells [FR()] were used for the experiments. Briefly, the HeLa (or A549) cells harvested in a logarithmic growth phase were seeded in 96-well plates at a density of 5000 cells/well and incubated in RPMI1640 for 24 h. Then, the cells were incubated with various concentrations of free DOX or particles. After 24 h, 20 ll of MTT solution in PBS (5 mg ml1) was added and the plates were incubated for another 4 h at 37 °C. After that, the medium containing MTT was removed and 200 ll of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 60 s, and the absorbance of formazan product was measured at 570 nm by a ThermoMax Microplate Reader.
2.8. In vitro release studies In drug release studies, PBS (phosphate buffered solution, pH 7.4) is used, which is a typical medium to mimic blood fluid. The release of samples (pure DOX DOX-SBA-15, DOX-PEI/SBA-15 and DOX-FA/PEI/SBA-15) was performed in 100 ml PBS, respectively. The sample was sealed in a dialysis bag (Solarbio) and suspended in PBS under continuous stirring at a rate of 100 rpm. The in vitro experiments were carried out in a heated bath at 37 °C in triplicate. At predetermined time intervals, 1.5 ml of the resultant release medium was sampled for analysis and then 1.5 ml fresh release medium was immediately added to maintain the original volume. Based on the standard curve, the DOX concentration in the
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sampled release medium was obtained through UV experiment (k = 233 nm). 2.9. Statistical analysis Statistical significance was determined by Tukey’s pairwise comparison with one-way analysis of variance (ANOVA). P < 0.05 was considered as statistically significant. 3. Results and discussion 3.1. Characterization of the particles In order to understand the morphology of the SBA-15 particles used in our experiments, the SEM characterization is performed. Fig. 1a shows the SEM image of the pure SBA-15. It shows that these particles have uniform particle size of 553 ± 110 nm. Fig. 1b shows the SEM image of the FA/PEI/SBA-15, which indicates that the morphology does not have an obvious change comparing with the SBA-15 particles. The average particles size is 557 ± 108 nm. The nitrogen isotherms and pore size distribution of pure SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 are shown in Fig. 1c, d, e, respectively. All the cases of the isotherms can be classified as a type IV isotherm characteristic of mesoporous materials. The pore size distributions are calculated from adsorption branch of the nitrogen isotherms by using the Barrett–Joyner–Halenda (BJH) method. The results show the average pore sizes are 7.21 nm, 6.52 nm, and 6.46 nm for pure SBA-15, PEI/SBA-15, and FA/PEI/SBA-15, respectively. The Brunauer–Emmett–Teller (BET) surface area is calculated to be 400 m2 g1, 306 m2 g1, and 330 m2 g1 for pure SBA-15, PEI/SBA-15, and FA/PEI/SBA-15, respectively. It can be observed that the modification of PEI leads to a decrease in surface area and pore size while the FA molecules conjugated to PEI have no marked influence on surface area and pore size. These results thus evidence the presence of PEI in the mesopores after modification. Zeta potential measurements are used to characterize the surface charges of as-prepared particles. Table 1 shows the zeta potential values of different samples. The mean zeta potential value of SBA-15 particles without modification is 32.81 ± 1.20 mV. This can be easily understood considering the rich silanol groups on the surface of the particles. However, for PEI/SBA-15 and FA/PEI/SBA-15 particles, the values are 39.18 ± 0.62 mV and 31.01 ± 0.82 mV, respectively. The charge type of the modified particles is reversed, which indicates the successfully modification of PEI on the outer surface of SBA-15. Compared with the zeta potential of PEI/SBA-15, the decreased value of FA/PEI/SBA-15 reveals that FA is covalently linked to the amino group of PEI by amide reactions. FTIR spectra are further employed to characterize the as-prepared SBA-15 particles. Fig. 2 shows the FTIR spectra of SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 particles. Compared with pure SBA-15 (Fig. 2a), a new band at 2927 cm1 appears for the PEI/ SBA-15 particles (Fig. 2b), which is assigned to the typical symmetric stretching of CAH of PEI. This indicates that the PEI is successfully grafted onto the SBA-15 particles by hyperbranching surface polymerization. The FTIR spectrum of FA/PEI/SBA-15 particles shows bands at 1511 cm1, belonging to the benzene ring vibration. Specially, there is a new brand at 1698 cm1 assigned to
Table 1 Zeta potential values of the particles. Sample
Zeta potential (mV)
SBA-15 PEI/SBA-15 FA/PEI/SBA-15
32.81 ± 1.20 39.18 ± 0.62 31.01 ± 0.82
Fig. 2. FTIR spectra of (a) pure SBA-15, (b) PEI/SBA-15, and (c) FA/PEI/SBA-15 particles.
Fig. 3. Photograph of (a) SBA-15, (b) PEI/SBA-15, (c) FA/PEI/SBA-15, and (d) DOXFA/PEI/SBA-15 particles dispersed in water.
the typical C@O vibration in the FA-grafted PEI, which indicates that the FA is covalently linked to the PEI by amide reactions. Therefore, the FTIR results demonstrate that hyperbranched PEI and FA ligands have been successfully grafted onto the SBA-15 particles, which agrees well with the results of the zeta potential measurements. Fig. 3 shows the digital photographs of pure SBA-15, PEI/SBA15, FA/PEI/SBA-15, and DOX-FA/PEI/SBA-15 particles dispersed in water. Compared with the white dispersion solutions of pure SBA-15 (Fig. 3a) and PEI/SBA-15 (Fig. 3b), the yellow1 color of FA/ PEI/SBA-15 particle dispersion in Fig. 3c reveals the successful conjugation of FA on PEI/SBA-15. The dark red color of the dispersions in Fig. 3d indicates a high drug loading content of FA/PEI/SBA-15. In order to obtain the amounts of modifiers and DOX in the SBA15, thermogravimetric (TG) analysis is performed. As illustrated in Fig. 4, the curves of SBA-15 and PEI/SBA-15 show weight losses of 5.5 wt% and 13.5 wt%, which are ascribed to the evaporation of water and the decomposition of hyperbranched PEI, respectively [29,30]. The amount of PEI grafted to the SBA-15 particles is 10.8 wt% determined from the weight losses between SBA-15 and PEI/SBA-15. Similarly, a comparison of TG curves between PEI/SBA-15 and FA/PEI/SBA-15 suggests that about 0.5 wt% FA is conjugated to the PEI/SBA-15 particles. For the DOX-loaded samples, the drug loading content of SBA-15, PEI/SBA-15, and FA/PEI/ SBA-15 are 8.7 wt%, 4.0 wt%, and 3.5 wt%, respectively. The decreased drug loading content after modification can be explained
1 For interpretation of color in Fig. 3, the reader is referred to the web version of this article.
J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39
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DOX molecules in the mesopores and on the surface due to the electrostatic interactions. 3.2. Cellular uptake
Fig. 4. TG curves of (a) SBA-15, (b) DOX-SBA-15, (c) FA/PEI/SBA-15 (d) PEI/SBA-15 (e) DOX-PEI/SBA-15, and (f) DOX-FA/PEI/SBA-15.
as follows. On one hand, the surface area and pore size decrease after the modification resulting in the decrease in drug content in the particles. On the other hand, in comparison with the positively charged FA/PEI/SBA-15 particles, the negatively charged surface of the SBA-15 particles can load more positively charged
The cellular uptake property of carriers is important for the delivery efficiency of a drug targeted delivery system. In general, good cellular uptake of carriers usually results in an efficient drug delivery. It is known that the DOX molecule can exhibit inherent fluorescence; therefore, we can directly monitor the cellular uptake of DOX-loaded mesoporous particles by fluorescence microscope. The cellular uptakes are performed on FA-receptornegative A549 cells [FR()] and FA-receptor-positive HeLa cells [FR(+)], respectively. Fig. 5 shows the bright-field and fluorescence microscopy images of cellular uptakes of pure DOX, DOX-SBA-15, DOX-PEI/SBA-15, DOX-FA/PEI/SBA-15, and DOX-FA/PEI/SBA-15 particles with free FA (2 mM) on A549 and HeLa cells. The equivalent DOX concentration is controlled at 3 lg ml1. The bright-field images show that the cells remain attached to the plate and maintain their normal morphology. For the A549 cells incubated with the various studied particles, it is found that the intracellular DOX fluorescence intensity for the DOX-PEI/SBA-15 particles is higher than that for the DOX solution and DOX-SBA-15. However, the intracellular DOX fluorescence intensity for DOX-FA/PEI/SBA15 and DOX-FA/PEI/SBA-15 with free FA (2 mM) exhibit the strongest fluorescence intensity among others. The enhanced DOX fluorescence intensity indicates an increased cellular uptake;
Fig. 5. Bright-field (A–D) and fluorescence microscopy images (a–d) of A549 and HeLa cells after incubation for 2 h with different samples of particles with an equivalent DOX concentration of 3 lg ml1. (A/a) DOX solution, (B/b) DOX-SBA-15, (C/c) DOX- PEI/SBA-15, (D/d) DOX-FA/PEI/SBA-15, and (E/e) DOX-FA/PEI/SBA-15 and free FA solution (2 mM).
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Fig. 6. Flow cytometric analysis of A549 cells and HeLa cells after being incubated with different samples of particles with an equivalent DOX concentration of 3 lg ml1 for 2 h. DOX solution (a), DOX-SBA-15 (b), DOX-FA/PEI/SBA-15 (c), and DOX-FA/PEI/SBA-15 + FA (2 mM) (d) for 2 h.
therefore, the DOX-loaded FA-conjugated particles can significantly enhance the cellular uptake. For the HeLa cells incubated with the various studied particles, the intracellular DOX fluorescence intensity for the DOX-PEI/SBA-15 particles is also higher than the DOX solution and DOX-SBA-15 particles. Among the studied particles, the DOX-FA/PEI/SBA-15 nanoparticles exhibit the obvious enhancement for cellular uptake based on their highest intracellular DOX fluorescence intensity. However, when free FA (2 mM) present in the culture medium, the cellular uptake of the DOX-FA/PEI/SBA-15 nanoparticles decreases. Comparing the results of HeLa cells and A549 cells, it indicates that the presence of FA in the culture medium competitively inhibits the binding of DOX-FA/PEI/SBA-15 to FA-receptor-positive HeLa cells, but not to FA-receptor-negative A549 cells. This demonstrates that FA targeting ligand can significantly enhance the cellular uptake of FA-receptor-positive cells by a FA-receptor-mediated endocytosis process [11]. Flow cytometry is employed to further investigate the selective targeting ability of FA-conjugated particles against A549 cells and HeLa cells. Fig. 6 shows the flow cytometry histograms of DOX fluorescence from A549 cells and HeLa cells incubated with free DOX, DOX-SBA-15, DOX-FA/PEI/SBA-15 and DOX-FA/PEI/SBA-15 with free FA (2 mM) at the same equivalent DOX concentration (3 lg ml1) for 2 h. For both A549 cells and HeLa cells, the flow cytometry analysis exhibits that the DOX-SBA-15 particles have higher intracellular DOX fluorescence intensity than the DOX solution. For A549 cells, the DOX-FA/PEI/SBA-15 particles and the DOX-FA/PEI/SBA-15 nanoparticles with free FA (2 mM) exhibit similar fluorescence intensity, which is much higher than that of DOX and DOX-SBA-15 particles. However, for the HeLa cells, the fluorescence intensity of DOX-FA/PEI/SBA-15 particles without free FA (2 mM) is highest than others. These results agree well with the fluorescence microscopy images in Fig. 5, and further demonstrate that FA targeting ligand can significantly enhance the cellular uptake by the FA-receptor-mediated endocytosis process. Therefore, the FA-conjugated PEI/SBA-15 nanoparticles as anticancer drug delivery carriers can effectively target cancer cells and enhance the cellular uptake. 3.3. In vitro cytotoxicity To understand the cytotoxicity of the FA/PEI/SBA-15 particles, the A549 cells and HeLa cells are employed to evaluate their cytotoxicity by MTT assays. For comparison, the SBA-15 and PEI/SBA15 particles are also characterized. Fig. 7 shows the results of cell inhibition rate of SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 nanoparticles to A549 cells and HeLa cells. It can be seen that all the particles show toxicity to the cells at higher concentrations. The
cytotoxicity decreases with the decrease in the particle concentrations on birth cell lines. Among the studied particle systems, the PEI/SBA-15 exhibit the highest cytotoxicity on both A549 and HeLa cells, while the FA/PEI/SBA-15 particles show lower cytotoxicity on both the cell lines. Especially, the FA/PEI/SBA-15 particles at lower concentration (660 lg ml1) show an inhibition rate less than 10% on both cells. The results reveal that the FA/PEI/SBA-15 particles can decrease the cytotoxicity after FA conjugation and suggest that FA/PEI/SBA-15 particles can be applied as drug delivery carriers for anticancer therapy. To study the anticancer effect of the FA-conjugated particles, the cell inhibition of DOX solution, DOX-SBA-15, and DOX-FA/ PEI/SBA-15 particles are investigated on HeLa cells and A549 cells by MTT assays (Fig. 8). Moreover, to verify the anticancer effect of the DOX-FA/PEI/SBA-15 particles, the cell inhibition of the empty FA/PEI/SBA-15 particles is tested at its corresponding concentrations. The results indicate that the cell inhibition of the empty FA/PEI/SBA-15 particles can be negligible compared with DOXFA/PEI/SBA-15 particles. As shown in Fig. 8, an increased inhibition efficacy of the samples occurs with an increase in their concentrations. For A549 cells, DOX-FA/PEI/SBA-15 particles do not show excellent antitumor effect (0.008 to 5 lg ml1, p > 0.05 between DOX-FA/PEI/SBA-15 and DOX-SBA-15). However, for HeLa cells, the inhibition rate of DOX-FA/PEI/SBA-15 particles is twice as large as DOX-SBA-15 particles and free DOX solution (0.2 to 5 lg ml1, p < 0.05 between DOX-FA/PEI/SBA-15 and DOX-SBA-15). Moreover, the DOX-FA/PEI/SBA-15 particles show much higher inhibition on HeLa cells than that on A549 cells. All these results demonstrate that FA ligands on DOX-FA/PEI/SBA-15 particles play an important role in enhancing the inhibition, which can be attributed to the increased cellular uptake by FA-receptor-mediated endocytosis. Therefore, we conclude that FA/PEI/SBA-15 particles as anticancer drug delivery vehicles can effectively target cancer cells and enhance the anticancer efficacy. 3.4. In vitro DOX release from DOX-loaded particles The in vitro drug release behaviors of pure DOX, DOX-SBA-15, DOX-PEI/SBA-15, and DOX-FA/PEI/SBA-15 particles are studied in phosphate buffered solution (PBS, pH 7.4). As shown in Fig. 9, the pure DOX releases rapidly into the release medium with a release mount of nearly 100% in 12 h. There is burst effect for this sample. In contrast, the DOX-SBA-15 particles show a sustained release profile with a percentage of 30% in 48 h, whereas the release profiles of PEI modified samples and the FA targeted samples are similar with a sustained release percentage of 70% in about 48 h. It is easy to find that all the DOX-loaded particles exhibit the typical sustained release behavior. The main reason is that the DOX
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J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39
100
A549
SBA-15
Inhibition Rate (%)
80
PEI/SBA-15 60
FA/PEI/SBA-15
40 20 0 200 -20
150
100
60
Concentrition of Nanoparticles µg•ml
20
-1
-40 100
HeLa SBA-15
Inhibition Rate (%)
80
PEI/SBA-15 60 FA/PEI/SBA-15 40 20 0 200 -20
150
100
60
20
Concentration of Nanoparticles (µg•ml -1 )
-40 Fig. 7. Cell inhibition rate of SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 nanoparticles to A549 cells and HeLa cells measured by MTT assay.
100
A549
DOX solution
Inhibition Rate (%)
80
DOX-SBA-15 DOX-FA/PEI/SBA-15
60
FA/PEI/SBA-15 40 20 0 5
1
0.2
0.04
0.008
-20 -40
DOX Concentration ( g•ml-1 ) 100
Inhibition Rate (%)
HeLa
DOX solution
80
DOX-SBA-15 DOX-FA/PEI/SBA-15
60
FA/PEI/SBA-15 40 20 0 5
1
0.2
0.04
0.008
-20 -1
DOX Concentration (µg•ml ) Fig. 8. Cell cytotoxicity of DOX solution, DOX-SBA-15, and DOX-FA/PEI/SBA-15 particles to A549 cells and HeLa cells measured by MTT assay.
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Fig. 9. Release profiles of DOX in PBS (pH = 7.4) at 37 °C, (-j-) pure DOX, (-d-) DOX-SBA-15, (-N-) DOX-PEI/SBA-15, and (-.-) DOX-FA/PEI/SBA-15 particles.
molecules have to diffuse from the mesopores into the release fluid. Besides, there is no burst effect for the drug-loaded samples in the release, which can be attributed to the rinsing steps decreasing the amount of DOX adsorbed on the external surfaces of the prepared silica materials. The release rates of DOX-PEI/SBA-15 and DOX-FA/PEI/SBA-15 are similar but faster than DOX-SBA-15. The reasons may be as follows. On the one hand, the modification of PEI increases the hydrophilicity of SBA-15, so it is easy for the release fluid diffuses into the silica mesopores and the water-soluble DOX can release into the release fluid more easily. On the other hand, the zeta potential of negative charged SBA-15 is changed into 39.18 mV for PEI/SBA-15 and 31.01 mV for FA/PEI/SBA-15 and the positively charged DOX is repulsed by the positively charged PEI/ SBA-15 and FA/PEI/SBA-15 and enhances the DOX release [31]. As the FA content in FA/PEI/SBA-15 is too low to influence the hydrophilicity of FA/PEI/SBA-15 particles, thus the DOX release profile for PEI/SBA-15 and FA/PEI/SBA-15 is similar. In general, the particles used in drug delivery could be captured by liver and spleen. However, it is only partly captured. Only drugs released from these particles can play therapeutic roles. If the drug release rate is too high, it will be cleared from plasma and concentrated in
Table 2 The equations for the different models and the regression coefficient (R) of pure DOX and DOX-loaded samples in vitro. Samples
Model
Equation
R
Pure DOX
First-order kinetics
ln(100Q) = 4.358–0.228t
0.987
DOX-SBA-15
First-order kinetics Higuchi equation Ritger–Peppas
ln(100Q) = 0.456–0.0075t
0.928
ln Q = 0.799 ln t + 0.761
0.9726
First-order kinetics Higuchi equation Ritger–Peppas
ln(100Q) = 4.4932–0.022t
0.956
Q = 10.499t1/21.530
0.986
ln Q = 0.732 ln t + 1.758
0.968
First-order kinetics Higuchi equation Ritger–Peppas
ln(100Q) = 4.5420.0269t
0.989
Q = 11.597t1/25.215
0.996
ln Q = 0.869 ln t + 1.365
0.968
DOX-PEI/SBA-15
DOX-FA/PEI/SBA15
Q = 4.913t
1/2
1.438
tissues before it goes to the cancer cells. However, if the release rate is too low, it is difficult to achieve the therapeutic concentration of drugs. The release rate of the DOX-SBA-15 particles is too low to be used in targeted anticancer system, because it is difficult to achieve the therapeutic concentration of DOX. Therefore, the FA targeted PEI modified particles, which can well control the drug release, would be desirable for its application in targeted anticancer therapy. The DOX release kinetics is determined by fitting the curves to First-order, Higuchi and Ritger–Peppas release model. The equations for the different models and the regression coefficient (R) are given in Table 2, and the best-fit model is selected on the basis of R. It is observed that the pure DOX release kinetics fits to the First-order model, while the DOX-loaded particle ones fit to the Higuchi model. Higuchi type of drug release profile indicates the drug diffusion from the mesopores as mechanism of drug release [32]. Drug release from matrices is usually complex and though some processes may be governed by both diffusion and erosion mechanisms. However, the above results may provide a better understanding of the drug controlled release from the drug-loaded particles. 4. Conclusions In this paper, we prepare a targeted anticancer drug delivery system based on FA-conjugated hybrid mesoporous silica particles. Amino-functionalized mesoporous SBA-15 is prepared by surface hyperbranching polymerization of PEI. Subsequently, FA is successfully grafted onto the PEI/SBA-15 mesoporous particles via amide reaction. The cellular uptake and in vitro cytotoxicity of the empty FA/PEI/SBA-15 particles and the DOX-loaded ones to A549 cells and HeLa cells are evaluated. Specifically, an excellent cellular uptake using the current anticancer drug delivery vehicles (DOX-loaded FA/PEI/SBA-15 particles) mediated by the FA receptor is demonstrated by using fluorescence microscope and flow cytometry. In vitro cytotoxicity shows that the empty FA/PEI/SBA-15 particles can be used as drug carriers in drug delivery systems. The DOXloaded FA/PEI/SBA-15 particles exhibit excellent anticancer effect to HeLa cells than the pure DOX and DOX-loaded SBA-15 particles due to its increased cellular uptake mediated by the FA receptors. What’s more, the in vitro release study shows that the FA/PEI/SBA15 particles have a typical sustained release behavior. Therefore, this work demonstrates that the prepared DOX-loaded FA/PEI/ SBA-15 particles might have great potential application in targeted anticancer drug delivery for cancer therapy. Acknowledgments This work is supported by National Natural Science Foundation of China (NSFC, No. 21173127), the Natural Science Foundation of Shandong Province (ZR2011BQ003), and the Independent Innovation Foundation of Shandong University (IIFSDU, 2012TS099).
0.984
References
Note: Q is the fraction of drug release, t is the time, R is the regression coefficient.
[1] J. Pang, Y. Luan, F. Li, X. Cai, Z. Li, Mater. Lett. 64 (2010) 2509. [2] Y. Zhu, Y. Fang, S. Kaskel, J. Phys. Chem. C 114 (2010) 16382. [3] J. Yang, J. Lee, J. Kang, K. Lee, J.S. Suh, H.G. Yoon, Y.M. Huh, S. Haam, Langmuir 24 (2008) 3417. [4] Y. Chen, H. Chen, L. Guo, Q. He, F. Chen, J. Zhou, J. Feng, J. Shi, ACS Nano. 4 (2010) 529. [5] A. Sousa, E.M.B. Sousa, J. Non-Cryst. Solids 352 (2006) 3451. [6] J.M. Rosenholm, M. Linden, Chem. Mater. 19 (2007) 5023. [7] A. Sousa, M.D. Andrada, R.G. Sousa, E.M.B. Sousa, J. Mater. Sci. 45 (2010) 1478. [8] J. Gu, W. Fan, A. Shimojima, T. Okubo, Small 3 (2007) 1740. [9] C.L. Lay, H. Liu, D. Wu, Y. Liu, Chem. Eur. J. 16 (2010) 3001. [10] Y. Zhu, J. Shi, Y. Li, H. Chen, W. Shen, X. Dong, Micropor. Mesopor. Mater. 85 (2005) 75. [11] A. Heidari, H. Younesi, Z. Mehraban, Chem. Eng. J. 153 (2009) 70.
J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39 [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]
Sujandi, E.A. Prasetyanto, S.E. Park, Appl. Catal. A 350 (2008) 244. C.O. Kim, S.J. Cho, J.W. Park, J. Colloid Interface Sci. 260 (2003) 374. J.M. Rosenholm, A. Duchanoy, M. Lindén, Chem. Mater. 20 (2008) 1126. H.J. Kim, J.H. Moon, J.W. Park, J. Colloid Interface Sci. 227 (2000) 247. X. Yang, J.J. Grailer, I.J. Rowland, A. Javadi, S.A. Hurley, D.A. Steeber, S. Gong, Biomaterials 31 (2010) 9065. H.S. Yoo, T.G. Park, J. Controlled Release 96 (2004) 273. C. Sanson, C. Schatz, J.F. Le Meins, A. Soum, J. Thévenot, E. Garanger, S. Lecommandoux, J. Controlled Release 147 (2010) 428. C.J. Ochs, G.K. Such, Y. Yan, M.P. Koeverden, F. Caruso, ACS Nano. 4 (2010) 1653. S. Tinkov, C. Coester, S. Serba, N.A. Geis, H.A. Katus, G. Winter, R. Bekeredjian, J. Controlled Release 148 (2010) 368. H. Zhang, F. Li, J. Yi, C. Gu, L. Fan, Y. Qiao, Y. Tao, C. Cheng, H. Wu, Eur. J. Pharm. Sci. 42 (2011) 517.
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
39
H.S. Yoo, T.G. Park, J. Controlled Release 100 (2004) 247. X. Hu, S. Liu, Y. Huang, X. Chen, X. Jing, Biomacromolecules 11 (2010) 2094. H.S. Yoo, T.G. Park, J. Controlled Release 70 (2001) 63. J. Yang, E.-J. Cho, S. Seo, J.-W. Lee, H.-G. Yoon, J.-S. Suh, Y.-M. Huh, S. Haam, J. Biomed. Mater. Res. A 84 (2008) 273. J.M. Rosenholm, A. Meinander, E. Peuhu, R. Niemi, J.E. Eriksson, C. Sahlgren, M. Linden, ACS Nano. 3 (2009) 197. L. Pasqua, F. Testa, R. Aiello, S. Cundari, J.B. Nagy, Micropor. Mesopor. Mater. 103 (2007) 166. Y. Zhu, T. Ikoma, N. Hanagata, S. Kaskel, Small 6 (2010) 471. L. Chen, Y.M. Wang, M.-Y. He, J. Porous Mater. 18 (2011) 211. X. Li, C.-Y. Hong, C.-Y. Pan, Polymer 51 (2010) 92. Y. Zhu, Y. Fang, L. Borchardt, S. Kaskel, Microporous Mesoporous Mater. 141 (2011) 199. S. Jain, D. Kumar, N.K. Swarnakar, K. Thanki, Biomaterials 33 (2012) 6758.
Contents lists available at SciVerse ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Folate-conjugated hybrid SBA-15 particles for targeted anticancer drug delivery Jianmei Pang a, Lanxia Zhao a, Longlong Zhang a, Zhonghao Li b, Yuxia Luan a,⇑ a b
School of Pharmaceutical Science, Shandong University, Jinan, Shandong Province 250012, PR China School of Materials Science and Engineering, Shandong University, Jinan, Shandong Province 250061, PR China
a r t i c l e
i n f o
Article history: Received 6 October 2012 Accepted 3 December 2012 Available online 19 December 2012 Keywords: SBA-15 Surface functionalization Targeting Cytotoxicity
a b s t r a c t Surface functionalization is one of the key steps toward the utilization of mesoporous materials in drug delivery system. Here, the folic acid (FA) ligands are conjugated onto poly(ethylene imine) (PEI) modified SBA-15 particles (PEI/SBA-15) via amide reaction, which results in the FA/PEI/SBA-15 particles. Doxorubicin hydrochloride (DOX), an anticancer drug, is successfully loaded into these particles. The in vitro cytotoxicity and cellular uptake of the empty FA/PEI/SBA-15 particles and the DOX-loaded ones are evaluated on two kinds of cancer cells (HeLa cells and A549 cells). Specifically, an excellent cellular uptake using the current anticancer drug delivery vehicles (DOX-loaded FA/PEI/SBA-15 particles) mediated by the FA receptor is demonstrated by fluorescence microscope and flow cytometry. The FA/PEI/SBA-15 particles demonstrate a lower cytotoxicity comparing with the PEI/SBA-15 particles, while the DOX-loaded FA/PEI/SBA-15 particles exhibit much greater inhibition to the studied cancer cells. Furthermore, the in vitro release study shows that the targeted FA/PEI/SBA-15 particles have a typical sustained release behavior. This work therefore demonstrates that drug-loaded FA/PEI/SBA-15 particles have great potential application in targeted anticancer drug delivery for cancer therapy. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction The applications of mesoporous silica particles (MSPs) as drug carriers have drawn growing interest in the past few years [1–7]. MSPs have several attractive features, such as high surface area and large pore volume, stable and ordered pore framework, uniform and tunable pore size, non-toxic and biocompatibility. What’s more, there are silanol groups on both the internal and the external surface, which makes it simple to be modified. In this case, modification of the carrier surface, especially organic and inorganic nanocomposites, is one of the key steps toward the utilization of mesoporous materials [8,9]. Due to the rich Si–OH groups on the surface, the mesoporous silica materials are easy to be modified with desirable functionalities [10]. Among others, hyperbranching surface polymerization is promising for amino functionalization of mesoporous silica as the surface concentration of amino groups is much higher than post-grafting method and co-condensation method [11–15]. Especially, the quantity of the PEI layer can be controlled simply by changing the aziridine/silica ratio in the surface functionalization step. Moreover, it is demonstrated that a hydrophobized mesoporous silica material can be fully redispersed in aqueous media after PEI functionalization of the outer surface [14]. Besides, carboxylic acid functions can be introduced to the PEI layer by amide reactions. ⇑ Corresponding author. Fax: +86 531 88382548. E-mail address: [email protected] (Y. Luan). 0021-9797/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2012.12.016
Doxorubicin hydrochloride (DOX), a potent antibiotic, is the first-line treatment used for a wide range of cancers [16–18]. However, due to the lack of ability to target cancer cells, it presents major drawbacks such as drug resistance and nonselective cytotoxicity, which usually leads to severe side effects. In order to improve the therapeutic efficacy of DOX, various drug delivery systems have been reported, such as liposomes, polymeric particles, and polymer conjugates [11,19–21]. In principle, drug targeting with noninvasive techniques is highly desirable as the preferred system should supply increased local drug concentration with reduced systemic side effects [20]. Still, it is of great challenge to achieve a useful targeted anticancer drug delivery system. Attaching specific ligands to the particle surface to achieve targeting of specific cells is generally studied in drug targeted delivery system. Folic acid (FA), one of the well known receptor mediated targeting moieties, has high binding affinity to the FA receptors on the cell surface of FA-positive tumors. FA receptor can be over expressed on the surfaces of some specific human tumors, including ovarian, brain, endometrial, kidney, and breast cancer cells. Therefore, the covalently conjugated FA to drug carriers could achieve selective targeting to tumors [22–25]. It is known that FA has carboxylic acid thus it can be readily linked by amide reactions if there are amino functions on the outer surface of MSNs [26–28]. Rosenholm et al. prepared PEI-functionalized MSNs by surface hyperbranching polymerization and modified it by introducing fluorescent molecules [15].
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Herein, we load the anticancer drug DOX into the FA-grafted PEI-functionalized SBA-15 particles. The schematic procedure is shown in Scheme 1. The in vitro cytotoxicity and the cellular uptake to the cells demonstrate that the FA-conjugated PEI/SBA-15 particles can effectively target cancer cells and enhance the cellular uptake. Thus, the results indicate that the prepared FA targeted PEI modified mesoporous silica particles would be desirable for its application in cancer therapy.
under dark conditions, the DOX-loaded particles were centrifuged and washed with PBS for 3 times. Finally, the samples were dried at 50 °C for 12 h, sealed, and stored under dark conditions. The DOX loading content was determined by thermogravimetry (TG) and calculated by the following formula:
2. Experimental section
2.4. Characterization
2.1. Preparation of PEI/SBA-15
Scanning electron microscopy (SEM) was performed with a Hitachi SU-70 FESEM scanning electron microscope operated at 3 kV. Samples were sputtered with Au prior to imaging (The thickness of Au was 5 nm.). The Brunauer–Emmett–Teller (BET) surface area and Barrett–Joyner–Halenda (BJH) pore size distribution were measured by nitrogen (N2) adsorption/desorption using a QuadraSorb SI surface area analyzer. Thermogravimetric (TG) analysis was performed using a PerkinElmerPyris6. The Fourier transform infrared (FTIR) spectra were obtained in the range of 400–4000 cm1 using a Nicolet NEXUS 470 FT-IR Spectrometer by dispersing the powder samples in KBr pellets.
2.1.1. Synthesis of aziridine Aziridine was synthesized and purified similar to the reported methods [6]. 25 g of AHS (2-Aminoethyl Hydrogen Sulfate, Tokyo Chemical Industry Co., LTD) was added to NaOH solution (40 wt%). Then, the mixture was heated to boiling in an oil bath. The bath was removed after boiling started and resumed when the mixture stopped boiling. The distillate was quickly collected between 50 and 105 °C. Then, KOH was added to the distillate, which was put in an ice bath. Aziridine was separated in the upper layer which could be collected from the mixture and stored over KOH at 4 °C overnight. After observing that no aqueous layer appeared, the prepared aziridine was sealed and stored at 0 °C. (Caution: Aziridine is a carcinogen and reproductive hazard. It is toxic if swallowed, inhaled, or absorbed through the skin. Aziridine is a carcinogen. Extreme caution is required when handling aziridine.) 2.1.2. Polymerization of aziridine on the surfaces of SBA-15 Hyperbranched PEI was grafted by aziridine polymerization on the surface of SBA-15 (Shenyang shunfeng Paint Co. Ltd.). The synthesis procedure is similar to the previous reported [14]. 1.5 g vacuum-dried SBA-15 was immersed in toluene under nitrogen. Then, 500 ll of aziridine and catalytic amounts of acetic acid were added. The reaction mixture was stirred overnight at 75 °C. The resulting substrate was thoroughly washed with copious amounts of toluene and dried in vacuo at room temperature for 48 h. 2.2. Preparation of FA/PEI/SBA-15 65 mg FA was dissolved in 3 ml dimethylsulfoxide (DMSO) and stirred for 10 h. Then, 34 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 38 mg of NHS (NHydroxysuccinimide) were added to the solution with stirring for 16 h. Subsequently, the amino group terminated PEI/SBA-15 was added to the above solution and allowed to react with stirring for 24 h at room temperature. After the reaction, the mixture was centrifuged and washed with water and ethanol several times to obtain FA/PEI/SBA-15 particles.
Loading Content ¼
Weight of DOX in Particles 100% Weight of the DOX loaded Particles
2.5. Cell culture Two cell lines, HeLa cervical carcinoma cells and A549 cells (human lung adenocarcinoma cell line), were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS, Solarbio), 100 U ml1 penicillin, and 100 lg ml1 streptomycin at 37 °C, in 5% CO2 atmosphere. HeLa and A549 cells were kindly supplied by the Pharmaceutical Department of Shandong University in China. 2.6. Cellular uptake Cellular uptake by HeLa cells and A549 cells were examined using fluorescence microscope and flow cytometry, respectively. The cells were seeded in 6-well culture plates (2 105 cells/well) and grown overnight. Then, the cells were incubated with free DOX or DOX-loaded particles (equivalent DOX concentration: 3 lg ml1) in RPMI1640 supplemented with 10% FBS. For FA competition experiments, the cells were cultured with 2 mM FA prior to addition of DOX-FA/PEI/SBA-15. After 1 h of incubation, the supernatant was carefully removed and the cells were washed three times with 4 °C PBS to remove the extra DOX molecules. The cells were examined by a fluorescence microscope (Olympus, Japan) to visualize cellular uptake. For flow cytometric analysis, the cells (1 104 counts) were harvested and analyzed by a flow cytometry (FACS Vantage, USA) to quantify the fluorescence intensity of DOX within the cell. 2.7. In vitro cytotoxicity
2.3. Loading DOX A total of 100 mg of particles (SBA-15, PEI/SBA-15, or FA/PEI/ SBA-15) were mixed with 20 ml of 1 mg ml1 DOX solution in PBS (phosphate buffered solution, pH 7.4). After stirring for 24 h
Cytotoxicity of free DOX solution, DOX-loaded samples (DOXSBA-15, DOX-FA/PEI/SBA-15), and the samples without DOX (SBA-15, PEI/SBA-15, FA/PEI/SBA-15) were evaluated by measuring the cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
Scheme 1. Schematic procedure for preparation and drug loading of FA/PEI/SBA-15 particles.
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Fig. 1. SEM images of SBA-15 particles (a), FA/PEI/SBA-15 (b) and N2 adsorption–desorption isotherms of SBA-15 (c), PEI/SBA-15 (d), and FA/PEI/SBA-15 (e), the inserts are the corresponding pore size distributions.
diphenyltetrazolium bromide) assay. The FA-receptor-positive HeLa cells [FR(+)] and FA-receptor-negative A549 cells [FR()] were used for the experiments. Briefly, the HeLa (or A549) cells harvested in a logarithmic growth phase were seeded in 96-well plates at a density of 5000 cells/well and incubated in RPMI1640 for 24 h. Then, the cells were incubated with various concentrations of free DOX or particles. After 24 h, 20 ll of MTT solution in PBS (5 mg ml1) was added and the plates were incubated for another 4 h at 37 °C. After that, the medium containing MTT was removed and 200 ll of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 60 s, and the absorbance of formazan product was measured at 570 nm by a ThermoMax Microplate Reader.
2.8. In vitro release studies In drug release studies, PBS (phosphate buffered solution, pH 7.4) is used, which is a typical medium to mimic blood fluid. The release of samples (pure DOX DOX-SBA-15, DOX-PEI/SBA-15 and DOX-FA/PEI/SBA-15) was performed in 100 ml PBS, respectively. The sample was sealed in a dialysis bag (Solarbio) and suspended in PBS under continuous stirring at a rate of 100 rpm. The in vitro experiments were carried out in a heated bath at 37 °C in triplicate. At predetermined time intervals, 1.5 ml of the resultant release medium was sampled for analysis and then 1.5 ml fresh release medium was immediately added to maintain the original volume. Based on the standard curve, the DOX concentration in the
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sampled release medium was obtained through UV experiment (k = 233 nm). 2.9. Statistical analysis Statistical significance was determined by Tukey’s pairwise comparison with one-way analysis of variance (ANOVA). P < 0.05 was considered as statistically significant. 3. Results and discussion 3.1. Characterization of the particles In order to understand the morphology of the SBA-15 particles used in our experiments, the SEM characterization is performed. Fig. 1a shows the SEM image of the pure SBA-15. It shows that these particles have uniform particle size of 553 ± 110 nm. Fig. 1b shows the SEM image of the FA/PEI/SBA-15, which indicates that the morphology does not have an obvious change comparing with the SBA-15 particles. The average particles size is 557 ± 108 nm. The nitrogen isotherms and pore size distribution of pure SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 are shown in Fig. 1c, d, e, respectively. All the cases of the isotherms can be classified as a type IV isotherm characteristic of mesoporous materials. The pore size distributions are calculated from adsorption branch of the nitrogen isotherms by using the Barrett–Joyner–Halenda (BJH) method. The results show the average pore sizes are 7.21 nm, 6.52 nm, and 6.46 nm for pure SBA-15, PEI/SBA-15, and FA/PEI/SBA-15, respectively. The Brunauer–Emmett–Teller (BET) surface area is calculated to be 400 m2 g1, 306 m2 g1, and 330 m2 g1 for pure SBA-15, PEI/SBA-15, and FA/PEI/SBA-15, respectively. It can be observed that the modification of PEI leads to a decrease in surface area and pore size while the FA molecules conjugated to PEI have no marked influence on surface area and pore size. These results thus evidence the presence of PEI in the mesopores after modification. Zeta potential measurements are used to characterize the surface charges of as-prepared particles. Table 1 shows the zeta potential values of different samples. The mean zeta potential value of SBA-15 particles without modification is 32.81 ± 1.20 mV. This can be easily understood considering the rich silanol groups on the surface of the particles. However, for PEI/SBA-15 and FA/PEI/SBA-15 particles, the values are 39.18 ± 0.62 mV and 31.01 ± 0.82 mV, respectively. The charge type of the modified particles is reversed, which indicates the successfully modification of PEI on the outer surface of SBA-15. Compared with the zeta potential of PEI/SBA-15, the decreased value of FA/PEI/SBA-15 reveals that FA is covalently linked to the amino group of PEI by amide reactions. FTIR spectra are further employed to characterize the as-prepared SBA-15 particles. Fig. 2 shows the FTIR spectra of SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 particles. Compared with pure SBA-15 (Fig. 2a), a new band at 2927 cm1 appears for the PEI/ SBA-15 particles (Fig. 2b), which is assigned to the typical symmetric stretching of CAH of PEI. This indicates that the PEI is successfully grafted onto the SBA-15 particles by hyperbranching surface polymerization. The FTIR spectrum of FA/PEI/SBA-15 particles shows bands at 1511 cm1, belonging to the benzene ring vibration. Specially, there is a new brand at 1698 cm1 assigned to
Table 1 Zeta potential values of the particles. Sample
Zeta potential (mV)
SBA-15 PEI/SBA-15 FA/PEI/SBA-15
32.81 ± 1.20 39.18 ± 0.62 31.01 ± 0.82
Fig. 2. FTIR spectra of (a) pure SBA-15, (b) PEI/SBA-15, and (c) FA/PEI/SBA-15 particles.
Fig. 3. Photograph of (a) SBA-15, (b) PEI/SBA-15, (c) FA/PEI/SBA-15, and (d) DOXFA/PEI/SBA-15 particles dispersed in water.
the typical C@O vibration in the FA-grafted PEI, which indicates that the FA is covalently linked to the PEI by amide reactions. Therefore, the FTIR results demonstrate that hyperbranched PEI and FA ligands have been successfully grafted onto the SBA-15 particles, which agrees well with the results of the zeta potential measurements. Fig. 3 shows the digital photographs of pure SBA-15, PEI/SBA15, FA/PEI/SBA-15, and DOX-FA/PEI/SBA-15 particles dispersed in water. Compared with the white dispersion solutions of pure SBA-15 (Fig. 3a) and PEI/SBA-15 (Fig. 3b), the yellow1 color of FA/ PEI/SBA-15 particle dispersion in Fig. 3c reveals the successful conjugation of FA on PEI/SBA-15. The dark red color of the dispersions in Fig. 3d indicates a high drug loading content of FA/PEI/SBA-15. In order to obtain the amounts of modifiers and DOX in the SBA15, thermogravimetric (TG) analysis is performed. As illustrated in Fig. 4, the curves of SBA-15 and PEI/SBA-15 show weight losses of 5.5 wt% and 13.5 wt%, which are ascribed to the evaporation of water and the decomposition of hyperbranched PEI, respectively [29,30]. The amount of PEI grafted to the SBA-15 particles is 10.8 wt% determined from the weight losses between SBA-15 and PEI/SBA-15. Similarly, a comparison of TG curves between PEI/SBA-15 and FA/PEI/SBA-15 suggests that about 0.5 wt% FA is conjugated to the PEI/SBA-15 particles. For the DOX-loaded samples, the drug loading content of SBA-15, PEI/SBA-15, and FA/PEI/ SBA-15 are 8.7 wt%, 4.0 wt%, and 3.5 wt%, respectively. The decreased drug loading content after modification can be explained
1 For interpretation of color in Fig. 3, the reader is referred to the web version of this article.
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DOX molecules in the mesopores and on the surface due to the electrostatic interactions. 3.2. Cellular uptake
Fig. 4. TG curves of (a) SBA-15, (b) DOX-SBA-15, (c) FA/PEI/SBA-15 (d) PEI/SBA-15 (e) DOX-PEI/SBA-15, and (f) DOX-FA/PEI/SBA-15.
as follows. On one hand, the surface area and pore size decrease after the modification resulting in the decrease in drug content in the particles. On the other hand, in comparison with the positively charged FA/PEI/SBA-15 particles, the negatively charged surface of the SBA-15 particles can load more positively charged
The cellular uptake property of carriers is important for the delivery efficiency of a drug targeted delivery system. In general, good cellular uptake of carriers usually results in an efficient drug delivery. It is known that the DOX molecule can exhibit inherent fluorescence; therefore, we can directly monitor the cellular uptake of DOX-loaded mesoporous particles by fluorescence microscope. The cellular uptakes are performed on FA-receptornegative A549 cells [FR()] and FA-receptor-positive HeLa cells [FR(+)], respectively. Fig. 5 shows the bright-field and fluorescence microscopy images of cellular uptakes of pure DOX, DOX-SBA-15, DOX-PEI/SBA-15, DOX-FA/PEI/SBA-15, and DOX-FA/PEI/SBA-15 particles with free FA (2 mM) on A549 and HeLa cells. The equivalent DOX concentration is controlled at 3 lg ml1. The bright-field images show that the cells remain attached to the plate and maintain their normal morphology. For the A549 cells incubated with the various studied particles, it is found that the intracellular DOX fluorescence intensity for the DOX-PEI/SBA-15 particles is higher than that for the DOX solution and DOX-SBA-15. However, the intracellular DOX fluorescence intensity for DOX-FA/PEI/SBA15 and DOX-FA/PEI/SBA-15 with free FA (2 mM) exhibit the strongest fluorescence intensity among others. The enhanced DOX fluorescence intensity indicates an increased cellular uptake;
Fig. 5. Bright-field (A–D) and fluorescence microscopy images (a–d) of A549 and HeLa cells after incubation for 2 h with different samples of particles with an equivalent DOX concentration of 3 lg ml1. (A/a) DOX solution, (B/b) DOX-SBA-15, (C/c) DOX- PEI/SBA-15, (D/d) DOX-FA/PEI/SBA-15, and (E/e) DOX-FA/PEI/SBA-15 and free FA solution (2 mM).
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Fig. 6. Flow cytometric analysis of A549 cells and HeLa cells after being incubated with different samples of particles with an equivalent DOX concentration of 3 lg ml1 for 2 h. DOX solution (a), DOX-SBA-15 (b), DOX-FA/PEI/SBA-15 (c), and DOX-FA/PEI/SBA-15 + FA (2 mM) (d) for 2 h.
therefore, the DOX-loaded FA-conjugated particles can significantly enhance the cellular uptake. For the HeLa cells incubated with the various studied particles, the intracellular DOX fluorescence intensity for the DOX-PEI/SBA-15 particles is also higher than the DOX solution and DOX-SBA-15 particles. Among the studied particles, the DOX-FA/PEI/SBA-15 nanoparticles exhibit the obvious enhancement for cellular uptake based on their highest intracellular DOX fluorescence intensity. However, when free FA (2 mM) present in the culture medium, the cellular uptake of the DOX-FA/PEI/SBA-15 nanoparticles decreases. Comparing the results of HeLa cells and A549 cells, it indicates that the presence of FA in the culture medium competitively inhibits the binding of DOX-FA/PEI/SBA-15 to FA-receptor-positive HeLa cells, but not to FA-receptor-negative A549 cells. This demonstrates that FA targeting ligand can significantly enhance the cellular uptake of FA-receptor-positive cells by a FA-receptor-mediated endocytosis process [11]. Flow cytometry is employed to further investigate the selective targeting ability of FA-conjugated particles against A549 cells and HeLa cells. Fig. 6 shows the flow cytometry histograms of DOX fluorescence from A549 cells and HeLa cells incubated with free DOX, DOX-SBA-15, DOX-FA/PEI/SBA-15 and DOX-FA/PEI/SBA-15 with free FA (2 mM) at the same equivalent DOX concentration (3 lg ml1) for 2 h. For both A549 cells and HeLa cells, the flow cytometry analysis exhibits that the DOX-SBA-15 particles have higher intracellular DOX fluorescence intensity than the DOX solution. For A549 cells, the DOX-FA/PEI/SBA-15 particles and the DOX-FA/PEI/SBA-15 nanoparticles with free FA (2 mM) exhibit similar fluorescence intensity, which is much higher than that of DOX and DOX-SBA-15 particles. However, for the HeLa cells, the fluorescence intensity of DOX-FA/PEI/SBA-15 particles without free FA (2 mM) is highest than others. These results agree well with the fluorescence microscopy images in Fig. 5, and further demonstrate that FA targeting ligand can significantly enhance the cellular uptake by the FA-receptor-mediated endocytosis process. Therefore, the FA-conjugated PEI/SBA-15 nanoparticles as anticancer drug delivery carriers can effectively target cancer cells and enhance the cellular uptake. 3.3. In vitro cytotoxicity To understand the cytotoxicity of the FA/PEI/SBA-15 particles, the A549 cells and HeLa cells are employed to evaluate their cytotoxicity by MTT assays. For comparison, the SBA-15 and PEI/SBA15 particles are also characterized. Fig. 7 shows the results of cell inhibition rate of SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 nanoparticles to A549 cells and HeLa cells. It can be seen that all the particles show toxicity to the cells at higher concentrations. The
cytotoxicity decreases with the decrease in the particle concentrations on birth cell lines. Among the studied particle systems, the PEI/SBA-15 exhibit the highest cytotoxicity on both A549 and HeLa cells, while the FA/PEI/SBA-15 particles show lower cytotoxicity on both the cell lines. Especially, the FA/PEI/SBA-15 particles at lower concentration (660 lg ml1) show an inhibition rate less than 10% on both cells. The results reveal that the FA/PEI/SBA-15 particles can decrease the cytotoxicity after FA conjugation and suggest that FA/PEI/SBA-15 particles can be applied as drug delivery carriers for anticancer therapy. To study the anticancer effect of the FA-conjugated particles, the cell inhibition of DOX solution, DOX-SBA-15, and DOX-FA/ PEI/SBA-15 particles are investigated on HeLa cells and A549 cells by MTT assays (Fig. 8). Moreover, to verify the anticancer effect of the DOX-FA/PEI/SBA-15 particles, the cell inhibition of the empty FA/PEI/SBA-15 particles is tested at its corresponding concentrations. The results indicate that the cell inhibition of the empty FA/PEI/SBA-15 particles can be negligible compared with DOXFA/PEI/SBA-15 particles. As shown in Fig. 8, an increased inhibition efficacy of the samples occurs with an increase in their concentrations. For A549 cells, DOX-FA/PEI/SBA-15 particles do not show excellent antitumor effect (0.008 to 5 lg ml1, p > 0.05 between DOX-FA/PEI/SBA-15 and DOX-SBA-15). However, for HeLa cells, the inhibition rate of DOX-FA/PEI/SBA-15 particles is twice as large as DOX-SBA-15 particles and free DOX solution (0.2 to 5 lg ml1, p < 0.05 between DOX-FA/PEI/SBA-15 and DOX-SBA-15). Moreover, the DOX-FA/PEI/SBA-15 particles show much higher inhibition on HeLa cells than that on A549 cells. All these results demonstrate that FA ligands on DOX-FA/PEI/SBA-15 particles play an important role in enhancing the inhibition, which can be attributed to the increased cellular uptake by FA-receptor-mediated endocytosis. Therefore, we conclude that FA/PEI/SBA-15 particles as anticancer drug delivery vehicles can effectively target cancer cells and enhance the anticancer efficacy. 3.4. In vitro DOX release from DOX-loaded particles The in vitro drug release behaviors of pure DOX, DOX-SBA-15, DOX-PEI/SBA-15, and DOX-FA/PEI/SBA-15 particles are studied in phosphate buffered solution (PBS, pH 7.4). As shown in Fig. 9, the pure DOX releases rapidly into the release medium with a release mount of nearly 100% in 12 h. There is burst effect for this sample. In contrast, the DOX-SBA-15 particles show a sustained release profile with a percentage of 30% in 48 h, whereas the release profiles of PEI modified samples and the FA targeted samples are similar with a sustained release percentage of 70% in about 48 h. It is easy to find that all the DOX-loaded particles exhibit the typical sustained release behavior. The main reason is that the DOX
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J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39
100
A549
SBA-15
Inhibition Rate (%)
80
PEI/SBA-15 60
FA/PEI/SBA-15
40 20 0 200 -20
150
100
60
Concentrition of Nanoparticles µg•ml
20
-1
-40 100
HeLa SBA-15
Inhibition Rate (%)
80
PEI/SBA-15 60 FA/PEI/SBA-15 40 20 0 200 -20
150
100
60
20
Concentration of Nanoparticles (µg•ml -1 )
-40 Fig. 7. Cell inhibition rate of SBA-15, PEI/SBA-15, and FA/PEI/SBA-15 nanoparticles to A549 cells and HeLa cells measured by MTT assay.
100
A549
DOX solution
Inhibition Rate (%)
80
DOX-SBA-15 DOX-FA/PEI/SBA-15
60
FA/PEI/SBA-15 40 20 0 5
1
0.2
0.04
0.008
-20 -40
DOX Concentration ( g•ml-1 ) 100
Inhibition Rate (%)
HeLa
DOX solution
80
DOX-SBA-15 DOX-FA/PEI/SBA-15
60
FA/PEI/SBA-15 40 20 0 5
1
0.2
0.04
0.008
-20 -1
DOX Concentration (µg•ml ) Fig. 8. Cell cytotoxicity of DOX solution, DOX-SBA-15, and DOX-FA/PEI/SBA-15 particles to A549 cells and HeLa cells measured by MTT assay.
38
J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39
Fig. 9. Release profiles of DOX in PBS (pH = 7.4) at 37 °C, (-j-) pure DOX, (-d-) DOX-SBA-15, (-N-) DOX-PEI/SBA-15, and (-.-) DOX-FA/PEI/SBA-15 particles.
molecules have to diffuse from the mesopores into the release fluid. Besides, there is no burst effect for the drug-loaded samples in the release, which can be attributed to the rinsing steps decreasing the amount of DOX adsorbed on the external surfaces of the prepared silica materials. The release rates of DOX-PEI/SBA-15 and DOX-FA/PEI/SBA-15 are similar but faster than DOX-SBA-15. The reasons may be as follows. On the one hand, the modification of PEI increases the hydrophilicity of SBA-15, so it is easy for the release fluid diffuses into the silica mesopores and the water-soluble DOX can release into the release fluid more easily. On the other hand, the zeta potential of negative charged SBA-15 is changed into 39.18 mV for PEI/SBA-15 and 31.01 mV for FA/PEI/SBA-15 and the positively charged DOX is repulsed by the positively charged PEI/ SBA-15 and FA/PEI/SBA-15 and enhances the DOX release [31]. As the FA content in FA/PEI/SBA-15 is too low to influence the hydrophilicity of FA/PEI/SBA-15 particles, thus the DOX release profile for PEI/SBA-15 and FA/PEI/SBA-15 is similar. In general, the particles used in drug delivery could be captured by liver and spleen. However, it is only partly captured. Only drugs released from these particles can play therapeutic roles. If the drug release rate is too high, it will be cleared from plasma and concentrated in
Table 2 The equations for the different models and the regression coefficient (R) of pure DOX and DOX-loaded samples in vitro. Samples
Model
Equation
R
Pure DOX
First-order kinetics
ln(100Q) = 4.358–0.228t
0.987
DOX-SBA-15
First-order kinetics Higuchi equation Ritger–Peppas
ln(100Q) = 0.456–0.0075t
0.928
ln Q = 0.799 ln t + 0.761
0.9726
First-order kinetics Higuchi equation Ritger–Peppas
ln(100Q) = 4.4932–0.022t
0.956
Q = 10.499t1/21.530
0.986
ln Q = 0.732 ln t + 1.758
0.968
First-order kinetics Higuchi equation Ritger–Peppas
ln(100Q) = 4.5420.0269t
0.989
Q = 11.597t1/25.215
0.996
ln Q = 0.869 ln t + 1.365
0.968
DOX-PEI/SBA-15
DOX-FA/PEI/SBA15
Q = 4.913t
1/2
1.438
tissues before it goes to the cancer cells. However, if the release rate is too low, it is difficult to achieve the therapeutic concentration of drugs. The release rate of the DOX-SBA-15 particles is too low to be used in targeted anticancer system, because it is difficult to achieve the therapeutic concentration of DOX. Therefore, the FA targeted PEI modified particles, which can well control the drug release, would be desirable for its application in targeted anticancer therapy. The DOX release kinetics is determined by fitting the curves to First-order, Higuchi and Ritger–Peppas release model. The equations for the different models and the regression coefficient (R) are given in Table 2, and the best-fit model is selected on the basis of R. It is observed that the pure DOX release kinetics fits to the First-order model, while the DOX-loaded particle ones fit to the Higuchi model. Higuchi type of drug release profile indicates the drug diffusion from the mesopores as mechanism of drug release [32]. Drug release from matrices is usually complex and though some processes may be governed by both diffusion and erosion mechanisms. However, the above results may provide a better understanding of the drug controlled release from the drug-loaded particles. 4. Conclusions In this paper, we prepare a targeted anticancer drug delivery system based on FA-conjugated hybrid mesoporous silica particles. Amino-functionalized mesoporous SBA-15 is prepared by surface hyperbranching polymerization of PEI. Subsequently, FA is successfully grafted onto the PEI/SBA-15 mesoporous particles via amide reaction. The cellular uptake and in vitro cytotoxicity of the empty FA/PEI/SBA-15 particles and the DOX-loaded ones to A549 cells and HeLa cells are evaluated. Specifically, an excellent cellular uptake using the current anticancer drug delivery vehicles (DOX-loaded FA/PEI/SBA-15 particles) mediated by the FA receptor is demonstrated by using fluorescence microscope and flow cytometry. In vitro cytotoxicity shows that the empty FA/PEI/SBA-15 particles can be used as drug carriers in drug delivery systems. The DOXloaded FA/PEI/SBA-15 particles exhibit excellent anticancer effect to HeLa cells than the pure DOX and DOX-loaded SBA-15 particles due to its increased cellular uptake mediated by the FA receptors. What’s more, the in vitro release study shows that the FA/PEI/SBA15 particles have a typical sustained release behavior. Therefore, this work demonstrates that the prepared DOX-loaded FA/PEI/ SBA-15 particles might have great potential application in targeted anticancer drug delivery for cancer therapy. Acknowledgments This work is supported by National Natural Science Foundation of China (NSFC, No. 21173127), the Natural Science Foundation of Shandong Province (ZR2011BQ003), and the Independent Innovation Foundation of Shandong University (IIFSDU, 2012TS099).
0.984
References
Note: Q is the fraction of drug release, t is the time, R is the regression coefficient.
[1] J. Pang, Y. Luan, F. Li, X. Cai, Z. Li, Mater. Lett. 64 (2010) 2509. [2] Y. Zhu, Y. Fang, S. Kaskel, J. Phys. Chem. C 114 (2010) 16382. [3] J. Yang, J. Lee, J. Kang, K. Lee, J.S. Suh, H.G. Yoon, Y.M. Huh, S. Haam, Langmuir 24 (2008) 3417. [4] Y. Chen, H. Chen, L. Guo, Q. He, F. Chen, J. Zhou, J. Feng, J. Shi, ACS Nano. 4 (2010) 529. [5] A. Sousa, E.M.B. Sousa, J. Non-Cryst. Solids 352 (2006) 3451. [6] J.M. Rosenholm, M. Linden, Chem. Mater. 19 (2007) 5023. [7] A. Sousa, M.D. Andrada, R.G. Sousa, E.M.B. Sousa, J. Mater. Sci. 45 (2010) 1478. [8] J. Gu, W. Fan, A. Shimojima, T. Okubo, Small 3 (2007) 1740. [9] C.L. Lay, H. Liu, D. Wu, Y. Liu, Chem. Eur. J. 16 (2010) 3001. [10] Y. Zhu, J. Shi, Y. Li, H. Chen, W. Shen, X. Dong, Micropor. Mesopor. Mater. 85 (2005) 75. [11] A. Heidari, H. Younesi, Z. Mehraban, Chem. Eng. J. 153 (2009) 70.
J. Pang et al. / Journal of Colloid and Interface Science 395 (2013) 31–39 [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]
Sujandi, E.A. Prasetyanto, S.E. Park, Appl. Catal. A 350 (2008) 244. C.O. Kim, S.J. Cho, J.W. Park, J. Colloid Interface Sci. 260 (2003) 374. J.M. Rosenholm, A. Duchanoy, M. Lindén, Chem. Mater. 20 (2008) 1126. H.J. Kim, J.H. Moon, J.W. Park, J. Colloid Interface Sci. 227 (2000) 247. X. Yang, J.J. Grailer, I.J. Rowland, A. Javadi, S.A. Hurley, D.A. Steeber, S. Gong, Biomaterials 31 (2010) 9065. H.S. Yoo, T.G. Park, J. Controlled Release 96 (2004) 273. C. Sanson, C. Schatz, J.F. Le Meins, A. Soum, J. Thévenot, E. Garanger, S. Lecommandoux, J. Controlled Release 147 (2010) 428. C.J. Ochs, G.K. Such, Y. Yan, M.P. Koeverden, F. Caruso, ACS Nano. 4 (2010) 1653. S. Tinkov, C. Coester, S. Serba, N.A. Geis, H.A. Katus, G. Winter, R. Bekeredjian, J. Controlled Release 148 (2010) 368. H. Zhang, F. Li, J. Yi, C. Gu, L. Fan, Y. Qiao, Y. Tao, C. Cheng, H. Wu, Eur. J. Pharm. Sci. 42 (2011) 517.
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
39
H.S. Yoo, T.G. Park, J. Controlled Release 100 (2004) 247. X. Hu, S. Liu, Y. Huang, X. Chen, X. Jing, Biomacromolecules 11 (2010) 2094. H.S. Yoo, T.G. Park, J. Controlled Release 70 (2001) 63. J. Yang, E.-J. Cho, S. Seo, J.-W. Lee, H.-G. Yoon, J.-S. Suh, Y.-M. Huh, S. Haam, J. Biomed. Mater. Res. A 84 (2008) 273. J.M. Rosenholm, A. Meinander, E. Peuhu, R. Niemi, J.E. Eriksson, C. Sahlgren, M. Linden, ACS Nano. 3 (2009) 197. L. Pasqua, F. Testa, R. Aiello, S. Cundari, J.B. Nagy, Micropor. Mesopor. Mater. 103 (2007) 166. Y. Zhu, T. Ikoma, N. Hanagata, S. Kaskel, Small 6 (2010) 471. L. Chen, Y.M. Wang, M.-Y. He, J. Porous Mater. 18 (2011) 211. X. Li, C.-Y. Hong, C.-Y. Pan, Polymer 51 (2010) 92. Y. Zhu, Y. Fang, L. Borchardt, S. Kaskel, Microporous Mesoporous Mater. 141 (2011) 199. S. Jain, D. Kumar, N.K. Swarnakar, K. Thanki, Biomaterials 33 (2012) 6758.