Drug Delivery System for Emodin Based on Mesoporous Silica SBA-15

In this study mesoporous silica SBA-15 was evaluated as a vehicle for the transport of cytotoxic natural product emodin (EO). SBA-15 was loaded with different quantities of EO (SBA-15|EO1–SBA-15|EO5: 8–36%) and characterized by traditional methods. Several parameters (stabilities) and the in vitro behavior on tumor cell lines (melanoma A375, B16 and B16F10) were investigated. SBA-15 suppresses EO release in extremely acidic milieu, pointing out that EO will not be discharged in the stomach. Furthermore, SBA-15 protects EO from photodecomposition. In vitro studies showed a dose dependent decrease of cellular viability which is directly correlated with an increasing amount of EO in SBA-15 for up to 27% of EO, while a constant activity for 32% and 36% of EO in SBA-15 was observed. Additionally, SBA-15 loaded with EO (SBA-15|EO3) does not disturb viability of peritoneal macrophages. SBA-15|EO3 causes inhibition of tumor cell proliferation and triggers apoptosis, connected with caspase activation, upregulation of Bax, as well as Bcl-2 and Bim downregulation along with amplification of poly-(ADP-ribose)-polymerase (PARP) cleavage fragment. Thus, the mesoporous SBA-15 is a promising carrier of the water-insoluble drug emodin.


Introduction
Quinones represent a large family of compounds having diverse biological properties. Emodin (EO; 1,3,8-trihydroxy-6-methylanthraquinone) belongs to a group of molecules with an anthraquinone core, isolated from the roots and barks of many plants, molds, and lichens [1,2]. Its multiple biological activities can be ascribed to its affinity to modulate different enzymes, but also to intercalate in DNA leading to the inhibition of the action of topoisomerase II [3]. Reports about its genotoxicity/mutagenicity are still controversial [2,4,5]. On the other hand, numerous studies proved its medicinal value such as laxative, antimicrobial, immunosuppressive, hepatoprotective, cardiotonic, vasorelaxant, and antitumor properties [6]. A wide spectrum of its different effects is related to similar signaling pathways involved in the regulation of essential physiological processes in mammalian cells. For example, inhibition of PTK signaling pathways by EO will reflect on the cellular response to different growth factors, cytokines, or hormones [7]. Its antiinflammatory effect is assigned to inhibition of inducible nitric oxide synthase (iNOS), cytokine production, prostaglandin synthesis, and super oxide production [6]. By influencing the redox status, EO indirectly modulates signal transduction [2,6]. However, EO behaves as a scavenger and inducer of oxidative stress retention effect" enables mesoporous silica nanoparticles (MSN) to selectively enter the tumor tissue and to retain within the cells. Their size prevents their extravasation from blood vessels and thus accumulation in normal tissues [37].

Preparation and Characterization of SBA-15 Containing Emodin
SBA-15 is prepared by a standard sol-gel procedure and afterwards calcined [38]. Then, SBA-15 was activated (vacuum, 150 °C, 16 h) and used for loading of different quantities of EO. Initially, loading of EO (ratio: SBA-15, 100 mg; EO, 10 mg) was performed at room temperature (→ SBA-15|EO1) and at 60 °C (→ SBA-15|EO1a) to check the temperature dependency on the loading efficiency. In both cases used conditions showed a high and comparable efficiency of drug loading in SBA-15 (at room temperature: 88.2%, at 60 °C: 90.4%). As similar loading was found for both MSNs all other materials were prepared at room temperature. The ratio of SBA-15 and EO was varied (SBA-15, always 100 mg; EO, 20,30,40,50 or 60 mg; SBA-15|EO1 → SBA-15|EO5, respectively) and materials with different loadings were gained. The content of EO in SBA-15 for these different materials was 8.1-36.4% with efficiency of 88.2%-95.6% (HPLC, see experimental part), contrarily to previously described SBA-15|EO where only 0.7% of EO was loaded into MSNs.
Morphological properties of MSNs were determined using SEM and TEM imaging. The cylindrical shape (ca. 650 ± 70 × 440 ± 50 nm) of materials could be clearly seen with the help of SEM ( Figure 1). A 2D hexagonal structure with an order at the mesoscopic scale could be identified by TEM measurements (Figure 1) [39]. The tubes of the mesoporous material are almost continuous for the full length of the particles. Moreover, these fragile tube systems were not damaged under the loading conditions. SEM and TEM images did not show changes upon loading of SBA-15.  The nitrogen sorption isotherms and SAXS experiments are given in Figure 2. SBA-15 exhibits characteristic type IV isotherm behavior and the hysteresis loop shows type H1 behavior (IUPAC classification) [40] and a narrow size distribution, which indicates a typical mesostructure with open channels. However, upon loading of EO into SBA-15 isotherms and hysteresis loop changed. As expected, the specific surface area (S BET ), determined from nitrogen sorption measurements, was decreased upon loading. SBA-15 loaded with EO showed a decrease of the surface area which is higher in ratio in the case of SBA-15|EO5, as is expected due to the higher amount of EO in its structure (S BET = 651 m 2 ·g −1 , SBA-15; S BET = 79 m 2 ·g −1 , SBA-15|EO5). The mean pore diameter, calculated using the BJH method [41], as well as the pore volume were reduced upon loading. Accordingly, the wall thickness of the MSNs increased. These data evidence that EO has been effectively adsorbed into the SBA-15 interior channels.
SAXS analysis was used for the characterization of MSNs and the obtained patterns are shown in Figure 2b. For all materials, the expected and well-defined structure of SBA-15 was observed and corresponds to a mesoscopic order assigned to mesoporous materials. The SAXS patterns show three peaks, which can be indexed on the 2D hexagonal lattice with the d-spacing values between 43 and 50 Å. MSNs displayed a well resolved pattern, with the highest intensity of diffraction peak at ca. 1.0 • corresponding to the (100) plane. Considerable reduction in the intensity of the diffraction peaks was detected upon loading of EO due to a blocking of the micropores and shrinkage in inner pore dimensions by the organic molecules [42].
IR spectra show typical bands for asymmetric (1061 cm −1 ) [43] and symmetric stretching vibrations of Si-O-Si (ring structures; 806 cm −1 ), as well as symmetric bending vibrations (443 cm −1 ). Nanomaterials containing EO exhibit additional vibrations belonging to C=O (625 cm −1 ) from the active component EO.  The nitrogen sorption isotherms and SAXS experiments are given in Figure 2. SBA-15 exhibits characteristic type IV isotherm behavior and the hysteresis loop shows type H1 behavior (IUPAC classification) [40] and a narrow size distribution, which indicates a typical mesostructure with open channels. However, upon loading of EO into SBA-15 isotherms and hysteresis loop changed. As expected, the specific surface area (SBET), determined from nitrogen sorption measurements, was decreased upon loading. SBA-15 loaded with EO showed a decrease of the surface area which is higher in ratio in the case of SBA-15|EO5, as is expected due to the higher amount of EO in its structure (SBET = 651 m 2 g −1 , SBA-15; SBET = 79 m 2 g −1 , SBA-15|EO5). The mean pore diameter, calculated using the BJH method [41], as well as the pore volume were reduced upon loading. Accordingly, the wall thickness of the MSNs increased. These data evidence that EO has been effectively adsorbed into the SBA-15 interior channels.
SAXS analysis was used for the characterization of MSNs and the obtained patterns are shown in Figure 2b. For all materials, the expected and well-defined structure of SBA-15 was observed and corresponds to a mesoscopic order assigned to mesoporous materials. The SAXS patterns show three peaks, which can be indexed on the 2D hexagonal lattice with the d-spacing values between 43 and 50 Å . MSNs displayed a well resolved pattern, with the highest intensity of diffraction peak at ca. 1.0° corresponding to the (100) plane. Considerable reduction in the intensity of the diffraction peaks was detected upon loading of EO due to a blocking of the micropores and shrinkage in inner pore dimensions by the organic molecules [42].

Stability and Drug Release Studies
Administration of therapeutic drugs is preferably oral because this circumvents discomfort and danger of infection compared to parenteral administration. This enlarges patient compliance [44]. Regularly, drug degradation takes place by hydrolysis, thus acidic conditions (e.g., gastric juice) might have a great impact on drug decay. The stability of EO in acidic media has already been evaluated in the literature [45].
Soaking of SBA-15|EO5 at the physiological pH (7.4) for different times (0, 1, 2, 6 and 24 h) and subsequent quantification of EO revealed that drug release occurs very slowly and in a small amount (Figure 3a). Thus within 24 h only 1.3% of EO is released from SBA-15. Contrary, previously described SBA-15 loaded with low amount of EO (ca. 0.7%) releases ca. 90% of the drug under same

Stability and Drug Release Studies
Administration of therapeutic drugs is preferably oral because this circumvents discomfort and danger of infection compared to parenteral administration. This enlarges patient compliance [44]. Regularly, drug degradation takes place by hydrolysis, thus acidic conditions (e.g., gastric juice) might have a great impact on drug decay. The stability of EO in acidic media has already been evaluated in the literature [45].
Soaking of SBA-15|EO5 at the physiological pH (7.4) for different times (0, 1, 2, 6 and 24 h) and subsequent quantification of EO revealed that drug release occurs very slowly and in a small amount (Figure 3a). Thus within 24 h only 1.3% of EO is released from SBA-15. Contrary, previously described SBA-15 loaded with low amount of EO (ca. 0.7%) releases ca. 90% of the drug under same pH and upon 24 h [46]. In SBA-15 containing ca. 0.7% EO, drug is dispersed as monolayer on the walls of SBA-15 and fast release of drug takes place [46]. Besides, the release profile correlated to the pore diameter and volume. The particles of SBA-15|EO5 having high EO content (36.4%) and those reported previously with low EO content (ca. 0.7%) have 4.36 and 6.45 nm pore diameter and 0.13 and 0.92 cm 3 g −1 pore volume, respectively. Smaller pore diameter and low pore volume of SBA-15|EO5 point out the fact that EO is deposited in mesoporous channels contrary to previously reported EO loaded in SBA-15. Based on results reported herein and literature data, it could be assumed that EO in SBA-15|EO5 is rather deposited as crystalline or amorphous form inside nanopore channels from where a slow release follows [35,47].
In order to assess the effect of acidic conditions present in stomach (pH = 1.5-3.5) on the EO stability, the behavior of the active component as well as SBA-15|EO5 in simulated gastric juice pH as well as under physiological pH (pH = 1.5, 3.5 and 7.4) was tested and analyzed by HPLC (Figure 3b). Soaked solution of EO at pH 1.5 showed the presence of the drug already after 1 h of shaking, along with several degradation products in small quantity ( Figure S1). The amount of solubilized EO increased almost 140 times after 2 h. At pH 3.5 the amount of dissolved EO raised during 2 h by the factor of ca. 1.5. From 3 up to 6 h at pH 1.5 and 3.5 solubilized EO reached concentration of almost 2 mg/mL. The difference in solubilized EO and its solubility (2.7 mg/mL) could be explained by the fact that EO is decomposing at mentioned acidic conditions, which is consistent with the HPLC findings where degradation products were observed. Investigations at physiological pH (7.4) showed that EO is dissolving and reaching ca. 1.5 µg/mL of EO after 6 h. Comparing to experiments at pH 1.5 and 3.5 fewer degradation products could be detected. In contrast, the amounts of released EO from SBA-15|EO5 soaked for 6 h at pH 1.5 and 3.5 were found to be in traces (below the quantification limit of HPLC). Thus, almost all EO loaded remained in the pores of SBA-15, circumventing interaction with acidic media and its degradation. Moreover, EO from SBA-15|EO5 is released very slowly over 6 h at pH 7.4, and fewer degradation products could be detected, in direct comparison to EO used as such. Due to the fact that EO in SBA-15|EO5 is deposited as crystalline or amorphous form inside nanopore channels, the release of EO occurs very slowly. pH and upon 24 h [46]. In SBA-15 containing ca. 0.7% EO, drug is dispersed as monolayer on the walls of SBA-15 and fast release of drug takes place [46]. Besides, the release profile correlated to the pore diameter and volume. The particles of SBA-15|EO5 having high EO content (36.4%) and those reported previously with low EO content (ca. 0.7%) have 4.36 and 6.45 nm pore diameter and 0.13 and 0.92 cm 3 g −1 pore volume, respectively. Smaller pore diameter and low pore volume of SBA-15|EO5 point out the fact that EO is deposited in mesoporous channels contrary to previously reported EO loaded in SBA-15. Based on results reported herein and literature data, it could be assumed that EO in SBA-15|EO5 is rather deposited as crystalline or amorphous form inside nanopore channels from where a slow release follows [35,47]. In order to assess the effect of acidic conditions present in stomach (pH = 1.5-3.5) on the EO stability, the behavior of the active component as well as SBA-15|EO5 in simulated gastric juice pH as well as under physiological pH (pH = 1.5, 3.5 and 7.4) was tested and analyzed by HPLC ( Figure  3b). Soaked solution of EO at pH 1.5 showed the presence of the drug already after 1 h of shaking, along with several degradation products in small quantity ( Figure S1). The amount of solubilized EO increased almost 140 times after 2 h. At pH 3.5 the amount of dissolved EO raised during 2 h by the factor of ca. 1.5. From 3 up to 6 h at pH 1.5 and 3.5 solubilized EO reached concentration of almost 2 mg/mL. The difference in solubilized EO and its solubility (2.7 mg/mL) could be explained by the fact that EO is decomposing at mentioned acidic conditions, which is consistent with the HPLC findings where degradation products were observed. Investigations at physiological pH (7.4) showed that EO is dissolving and reaching ca. 1.5 µ g/mL of EO after 6 h. Comparing to experiments at pH 1.5 and 3.5 fewer degradation products could be detected. In contrast, the amounts of released EO from SBA-15|EO5 soaked for 6 h at pH 1.5 and 3.5 were found to be in traces (below the quantification limit of HPLC). Thus, almost all EO loaded remained in the pores of SBA-15, circumventing interaction with acidic media and its degradation. Moreover, EO from SBA-15|EO5 is released very slowly over 6 h at pH 7.4, and fewer degradation products could be detected, in direct comparison to EO used as such. Due to the fact that EO in SBA-15|EO5 is deposited as crystalline or amorphous form inside nanopore channels, the release of EO occurs very slowly.  Furthermore, it is well known that EO is photolabile when exposed to visible light [48]. In order to investigate if degradation occurs, both EO and SBA-15|EO5 were exposed to light for 2 and 24 h (see Figure S2). The probes with EO showed more degradation products than that of SBA-15|EO5, proving that SBA-15 to some extent protects EO from degradation. Herein, it is proved that EO loaded in SBA-15 is at lower extent degraded than when EO alone exposed to light or to various tested pH as well as forced degradation as described in the literature [45].

In Vitro Studies
To evaluate the efficacy of emodin loaded into SBA-15, human melanoma A375, mouse melanoma B16 and its metastatic subclone B16F10 were exposed to a wide range of doses of original  Furthermore, it is well known that EO is photolabile when exposed to visible light [48]. In order to investigate if degradation occurs, both EO and SBA-15|EO5 were exposed to light for 2 and 24 h (see Figure S2). The probes with EO showed more degradation products than that of SBA-15|EO5, proving that SBA-15 to some extent protects EO from degradation. Herein, it is proved that EO loaded in SBA-15 is at lower extent degraded than when EO alone exposed to light or to various tested pH as well as forced degradation as described in the literature [45].

In Vitro Studies
To evaluate the efficacy of emodin loaded into SBA-15, human melanoma A375, mouse melanoma B16 and its metastatic subclone B16F10 were exposed to a wide range of doses of original drug and six different modalities of SBA-15 with raising concentrations of EO for 48 h (Figure 4). For comparison, SBA-15 alone was tested against the same tumor cell lines (see Figure S3). Materials with the lowest EO content prepared at different temperatures (SBA-15|EO1 and SBA-15|EO1a, r.t. and 60 • C, respectively) showed similar MC 50 values against tested cell lines (results not shown). Dose dependent decrease of cellular viability correlated with increasing amount of EO within SBA-15 carrier with a saturation at ca. 27% (Table 1). Loading of the drug into nanomaterial conserved and even potentiated its activity. EO is a naturally occurring compound whose cellular uptake is easy to follow due to its autofluorescence feature. Therefore, the decrease of cellular viability correlated with the efficient internalization of EO from free as well as loaded form (Figure 4a-c). While tumor cells were highly sensitive to the treatments, the viability of mouse macrophages was not disturbed upon exposure to both EO and SBA-15|EO3 in IC 50 concentrations determined on tumor cells (Figure 4d). It is obvious that the novel nanomaterial shows some selectivity toward malignant cells. The potential of EO to disturb the viability of different tumor cells is well described [2,6,[8][9][10][11][12]47]. However, it is known that MSNs are taken by the cells through endocytosis, so it is clear that macrophages as professional phagocytes uptake MSNs efficiently [49]. Since intracellular targets of EO loaded into SBA-15 are tightly connected with cell ability to proliferate, the viability of nonstimulated primary macrophages, as low proliferative cells, was not diminished.  (Figure 4). For comparison, SBA-15 alone was tested against the same tumor cell lines (see Figure S3). Materials with the lowest EO content prepared at different temperatures (SBA-15|EO1 and SBA-15|EO1a, r.t. and 60 °C, respectively) showed similar MC50 values against tested cell lines (results not shown). Dose dependent decrease of cellular viability correlated with increasing amount of EO within SBA-15 carrier with a saturation at ca. 27% ( Table 1). Loading of the drug into nanomaterial conserved and even potentiated its activity. EO is a naturally occurring compound whose cellular uptake is easy to follow due to its autofluorescence feature. Therefore, the decrease of cellular viability correlated with the efficient internalization of EO from free as well as loaded form (Figure  4a-c). While tumor cells were highly sensitive to the treatments, the viability of mouse macrophages was not disturbed upon exposure to both EO and SBA-15|EO3 in IC50 concentrations determined on tumor cells (Figure 4d). It is obvious that the novel nanomaterial shows some selectivity toward malignant cells. The potential of EO to disturb the viability of different tumor cells is well described [2,6,[8][9][10][11][12]47]. However, it is known that MSNs are taken by the cells through endocytosis, so it is clear that macrophages as professional phagocytes uptake MSNs efficiently [49]. Since intracellular targets of EO loaded into SBA-15 are tightly connected with cell ability to proliferate, the viability of nonstimulated primary macrophages, as low proliferative cells, was not diminished.    Measurement of cellular proliferation revealed that treatment of EO and SBA-15|EO3 influences the division of cells (Figure 5a). The percentage of undivided subpopulations was remarkably higher upon both treatments in comparison to control on A375 and B16 cell lines. As a consequence of inhibited proliferation, triggered apoptotic cell death was detected. After 48 h long treatment with IC 50 /MC 50 doses of both EO and SBA-15|EO3, an elevated percentage of early (Ann + PI -) and late apoptotic (Ann + PI + ) cells was detected (Figure 5b). Concordantly, apoptotic cell death promoted by the agents was visualized by PI staining revealing typical morphological signs of this process ( Figure 5c). Decreased nuclear volume, unregular shape of nuclei as well as condensed chromatine are visible upon the treatment with both EO and SBA-15|EO3. However, these features were more obvious upon the treatment with EO loaded MSN. In parallel, analysis of cell cycle distribution confirmed the presence of hypodiploid cells with fragmented DNA upon the 48 h treatment of A375 and B16 cells with both EO and SBA-15|EO3 ( Figure S4). In compliance with the data from microscopical evaluation of PI stained cells, the effect was stronger in cultures exposed to SBA-15|EO3. Total caspase activity was then analyzed by flow cytometry of Apostat stained cells. Simultaneously, enhanced caspase activity was found (Figure 5d) in both cell types after the treatment with EO in either, free or nanoform. Obtained results clearly indicated that apoptotic process triggered by experimental therapeutics, independently from MSN packaging, is classical caspase dependent form. This finding was in concordance with previously described potential of EO to activate caspase dependent apoptosis [2,6,[8][9][10][11][12].
Having in mind the quantity of EO in SBA-15|EO3 (B16: MC 50 = 19.7 µg/mL → IC 50 = 20.0 µM) and active concentration of EO alone (B16: IC 50 = 42.0 µM), it is clear that loading amplified its activity (for IC 50 /MC 50 values see Table 1). Since the apoptosis is often followed by autophagy, especially in autophagy prone cells such as A375, the presence of autophagosomes in the cytoplasm of cells exposed to both compounds, using acridin orange staining, was evaluated. Treatment with SBA-15|EO3 elevated the percentage of cells with acidic vesicles (8.2%) in A375 but not in B16 cells. Since co-treatment with specific inhibitors of this process, 3-methyl adenine and chloroquine, and SBA-15|EO3 did not result in viability recovery (see Figure S5), it is clear that in this setting autophagy was not cytoprotective but rather slightly contributed to drug mediated toxicity. Induction of autophagy upon the exposure to a similar anthroquinone, aloe emodin was described previously in glioma cells with the same tendency [50]. Obviously, nanopackaging of emodin did not change the mechanism of its action.
As mentioned above apoptosis as the mechanism of EO action is observed in pancreatic, breast, colon, leukemia, lung, hepatocellular carcinoma, tongue squamous, and cervical carcinoma [2,6,[8][9][10][11][12]47]. Therefore, the molecular profile of apoptotic process in response to SBA-15 loaded with EO was evaluated ( Figure 6). The intracellular response of both tested cell lines, independent from differences in their origin and invasiveness, was quite similar in terms of expression of main proapototic and antiapoptotic molecules. As expected, upregulated expression of proapoptotic Bax synchronized with diminished expression of antiapoptotic Bcl-2 in both cell lines was detected. The capability of EO to interfere with the Bax/Bcl-2 ratio is already described in breast and colon cell lines [51,52]. Reduced Bim expression is one of the general features of melanoma cells, and at least partly responsible for the establishment of an apoptosis resistant phenotype [53][54][55].
especially in autophagy prone cells such as A375, the presence of autophagosomes in the cytoplasm of cells exposed to both compounds, using acridin orange staining, was evaluated. Treatment with SBA-15|EO3 elevated the percentage of cells with acidic vesicles (8.2%) in A375 but not in B16 cells. Since co-treatment with specific inhibitors of this process, 3-methyl adenine and chloroquine, and SBA-15|EO3 did not result in viability recovery (see Figure S5), it is clear that in this setting autophagy was not cytoprotective but rather slightly contributed to drug mediated toxicity. Induction of autophagy upon the exposure to a similar anthroquinone, aloe emodin was described previously in glioma cells with the same tendency [50]. Obviously, nanopackaging of emodin did not change the mechanism of its action. As mentioned above apoptosis as the mechanism of EO action is observed in pancreatic, breast, colon, leukemia, lung, hepatocellular carcinoma, tongue squamous, and cervical carcinoma [2,6,[8][9][10][11][12]47]. Therefore, the molecular profile of apoptotic process in response to SBA-15 loaded with EO was evaluated ( Figure 6). The intracellular response of both tested cell lines, independent from differences in their origin and invasiveness, was quite similar in terms of expression of main proapototic and antiapoptotic molecules. As expected, upregulated expression of proapoptotic Bax synchronized with diminished expression of antiapoptotic Bcl-2 in both cell lines was detected. The capability of EO to interfere with the Bax/Bcl-2 ratio is already described in breast and colon cell lines  Upon the exposure of tested cell lines to emodin incorporated in mesoporous silica, Bim additionally decreased but this effect did not compromise the completion of the apoptotic process triggered by the compound. Having in mind that Bax is efficiently upregulated even if Bim is missing, implicated Bim as an independent alternative loop involved in Bax release and transport to the mitochondrial membrane. Together with this, decreased Bcl-2 expression in cells of both cell lines allowed propagation of the apoptotic signal. Finally, amplified presence of cleaved PARP fragment confirmed that apoptosis is realized through a defined route as described in literature [56].

Materials and Methods
Pluronic 123 (P123) and TEOS (Sigma, Taufkirchen, Germany), and EO (TCI chemicals, Eschborn, Germany) were purchased and used as received from the distributor.

Preparation of SBA-15
Pluronic P123 (16.0 g) was dissolved in water (120 mL) and HCl (2 M, 320 g). Afterwards TEOS (35.2 g) was added dropwise. The reaction mixture was stirred for 20 and 24 h at room temperature and 80 • C, respectively [32]. The obtained material was filtered and wasted with water (3 × 100 mL). Subsequently the material was heated (1 K min −1 ) to 500 • C and calcified for 24 h. Yield

Preparation of SBA-15 Loaded with Emodin (EO)
EO was suspended in toluene (20 mL). To the activated SBA-15 (dried under vacuum for 6 h at 160 • C) a suspension of EO was added and the mixture was stirred at room temperature (or in case of SBA-15|EO1a at 60 • C) for 48 h. For the used amount of EO and SBA-15 see details below in appropriate SBA-15|EOn. Afterwards, the suspension was filtered and the isolated material was washed successively with toluene (2 × 10 mL). The obtained materials were dried under vacuum at room temperature and lyophilized for 24 h. Filtrate was evaporated and used for HPLC quantification of non-loaded EO (the difference between the amount of EO used for preparation of nanomaterials and non-loaded amount of EO present the quantity of loaded EO).

Stability and Drug Release Studies
Release of EO from SBA-15|EO5 was assessed suspending SBA-15|EO5 (5 × 5 mg) in solution (1.0 mL) at physiological pH (7.4). Obtained samples were shaken for 0, 1, 2, 6 and 24 h at 25 • C. Afterwards, supernatant was separated by centrifugation and amount of EO was quantified using HPLC as described in Section 3.6.

Preparation of Drug Solutions
EO was dissolved in DMSO at 100 mM and kept at −20 • C until use. Various SBA-15|EO (2 mg/mL) were suspended in culture medium directly before use and working solutions were made in culture medium immediately before treatment.

MTT and CV Test
Cells were cultivated in the presence of different concentrations of EO, SBA-15 alone and various SBA-15|EO for 48 h. For the CV test, cells were fixed with 4% paraformaldehyde for 10 min at RT and subsequently stained for 15 min with 1% CV solution. Then cells were washed with tap water, dried and the dye was dissolved in 33% acetic acid. For the MTT test, viable cells were incubated in MTT staining solution (0.5 mg/mL) for approximately 1 h, the dye was removed and produced formazan was dissolved in DMSO. The absorbance was measured with an automated microplate reader at 540 nm with a reference wavelength of 670 nm. The results are expressed as a percentage of control values obtained in untreated cultures [57].

Annexin V-FITC/PI, Acridin Orange and Apostat Staining
Cells were incubated with IC 50 /MC 50 dose of EO or SBA-15|EO3 for 48 h. Then cells were detached, counted and split for three assays. Cells were stained with AnnV-FITC/PI or Apostat according to the manufacturer's protocol. For AO staining cells were incubated for 15 min at 37 • C in 1 µg/mL of dye solution, washed and resuspended in PBS. Cells were analyzed with a CyFlow ® Space Partec using PartecFloMax ® software.

Carboxyfluorescein Succinimidyl Ester (CFSE) Staining
For detection of cellular proliferation, cells were stained with 1 µM of carboxyfluorescein succinimidyl ester (CFSE) for 10 min at 37 • C, and then exposed to IC 50 /MC 50 dose of EO or SBA-15|EO3 for 48 h. At the end of cultivation, cells were washed, trypsinized, dissolved in PBS and analyzed as mentioned above.

Morphological Assessment of Apoptosis
For morphological assessment of apoptotic cells, cells were seeded at 2 × 10 4 /well on a chamber slide overnight, and incubated for 48 h in the absence or presence of the drugs. After 15 min of fixation with 4% paraformaldehyde at room temperature, cells were stained with 20 µg/mL PI in 0.1% Triton X-100, 0.1 mM EDTA pH 8.0, and 50 µg/mL RNase in PBS, washed in PBS and prepared for fluorescence microscopy by covering with 50% glycerol in PBS.

Cell Cycle Analysis
B16 and A375 cells were seeded in 6-well plates (2.5 × 10 5 /well) and incubated with the IC 50 /MC 50 dose of EO or SBA-15|EO3, respectively for 48 h. After the incubation period, cells were fixed in 70% ethanol and stored at 4 • C overnight. The next day cells were washed twice with PBS and then stained with PI (20 µg/mL) in the presence of RNase (0.1 mg/mL) for 45 min at 37 • C in the dark. The distribution of cells among different cell cycle phases was analyzed by FACSCalibur flow cytometer using Cell Quest Pro software.

Statistical Analysis
The results of cell viability estimation calculated from triplicate observations are presented as mean ± SD from one representative of at least three experiments. Analysis of variance (ANOVA) together with a Student-Newman-Keuls test were used for assessment of significance of the differences between treatments and p values of less than 0.05 were considered as significant.

Conclusions
Herein preparation and characterization of mesoporous silica SBA-15 loaded with various quantities of EO (→ SBA-15|EO1-SBA-15|EO5: 8-36%) is presented. SBA-15 protects EO even in extremely acidic media (pH = 1.5-3.5) and from photodecomposition (24 h). In vitro investigations on human melanoma A375, as well as mouse melanoma B16 and B16F10 cells showed a dose dependent decrease of cell viability which is also in direct relationship with the EO content in SBA-15. SBA-15|EO3, one of the most active mesoporous silica particles loaded with EO, does not influence peritoneal macrophage viability. On the other hand, SBA-15|EO3 hinders tumor cell proliferation. Additionally, the material triggers caspase dependent apoptosis synchronized with overexpression of Bax as well as down-regulation of Bcl-2 and Bim. Consequently, cleaved PARP fragment is amplified. All of this demonstrates positive effects of SBA-15 as drug carrier system. SBA-15 amplifies the activity of EO in vitro and protects the active compound from spontaneous, light promoted inactivation or degradation driven by extremely acidic conditions corresponding to gastric pH.