Biomaterials 32 (2011) 7711e7720
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A pH-responsive mesoporous silica nanoparticles-based multi-drug delivery system for overcoming multi-drug resistance Qianjun He a,1, Yu Gao b,1, Lingxia Zhang a, Zhiwen Zhang b, Fang Gao b, Xiufeng Ji b, Yaping Li b, *, Jianlin Shi a, * a State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Ding-Xi Road, Shanghai 200050, China b Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong-Zhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2011 Accepted 26 June 2011 Available online 4 August 2011
A type of pH-responsive nano multi-drug delivery systems (nano-MDDSs) with uniform particle size (100 13 nm) and excellent monodispersity was developed by in situ co-self-assembly among waterinsoluble anti-cancer drug (doxorubicin, DOX), surfactant micelles (CTAB) as chemosensitiver and silicon species forming drugs/surfactant micelles-co-loaded mesoporous silica nanoparticles (drugs@micelles@MSNs or DOX@CTAB@MSNs) via a micelleseMSNs self-assembly mechanism. The nano-MDDS DOX@CTAB@MSNs had a highly precise pH-responsive drug release behavior both in vitro and in vivo, and exhibited high drug efficiencies against drug-resistant MCF-7/ADR cells as well as drug-sensitive MCF-7 cells by the MSNs-mediated transmembrane delivery, the sustained drug release and the high anti-cancer and multi-drug resistance (MDR)-overcoming efficiencies. The MDR-overcoming mechanism was proved to be a synergistic cell cycle arrest/apoptosis-inducing effect resulted from the chemosensitization of the surfactant CTAB. These results demonstrated a very promising nano-MDDS for the pH-responsive controlled drug release and the cancer MDR overcoming. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Mesoporous silica Nanoparticle Controlled drug release Multi-drug delivery One-pot self-assembly Multi-drug resistance
1. Introduction Cancer is one of the deadliest killers to human lives, and both the incidence and mortality of cancers are continuously rising [1]. Unfortunately, a wholesale and thorough cure for cancers remains elusive for many reasons. One of the well known reason should be strong toxic side effects of the naked drugs or traditional drug delivery systems (DDSs) mainly due to the drug leakage before reaching the cancer focus. Besides, the multi-drug resistance (MDR) in cancers should be responsible for the high recurrence rate and the final failure in cancer chemotherapy: more than 90% of malignant tumor patients die from the certain extent MDR [2]. Therefore, the MDR in cancer become one of the major obstacles in the chemotherapeutic treatment of many human cancers [3]. The overcoming of the MDR in cancer is continuously a hot topic in anti-cancer researches. The current strategies to overcome the tumor MDR generally resort to the multi-drug combined
* Corresponding authors. Tel.: þ86 21 52412714; fax: þ86 21 52413122. E-mail addresses:
[email protected] (Y. Li),
[email protected] (J. Shi). 1 Contributed equally to this work. 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.06.066
chemosensitization, the reconstruction of primary drugs, and bio-/ nano-technologies. The combined use of two or several strategies is being recognized to be a realistic route to the successful chemotherapeutic treatment [4,5]. By integrating the multi-drug chemosensitization with the nanotechnology, some nano drug delivery systems (nano-DDSs) based on organic or inorganic nano-carriers have been designed to overcome the MDR and also enhance the drug efficacies against both drug-sensitive and drug-resistant cancer cells mostly by improving drug bio-accessibility and chemosensitivity [68]. The common organic nano-carriers, such as liposomes, lipid nanocapsules, nano hydrogels, polymer nanoparticles and micelles, can easily solubilize and encapsulate poorly water-soluble drugs into their hydrophobic cores, however are also physicochemically instable and consequently subject to unexpected drug leakage [9]. In contrast, inorganic nano-carriers, such as mesoporous silica nanoparticles (MSNs), carbon nanomaterials and gold nanoparticles, are highly stable physicochemically and biochemically. Especially, MSNs possess some unique features, such as uniform and tunable particle size/pore size/morphology, high surface area and pore volume, facile surface functionalization, etc [1013]. The as-synthesized MSNs before removing the structure directing
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agents, mainly surfactant micelles, which is named as micelles@MSNs here, can be regarded as a kind of organiceinorganic composite nano-carriers. Such a nano-carrier would probably show an almost perfect combination of the water-insoluble drug loading/ delivery, the high physicochemical and biochemical stability, the anti-leaking and the controlled drug release [1416]. In this work, we will demonstrate that such a composite nano-carrier, micelles@MSNs (e.g. CTAB@MSNs when the CTAB is selected as the surfactant micelles), can indeed combine these advantages in one. Further, the multi-drug resistance in cancer is frequently resulted from over low intracellular drug concentrations when administrated with some poorly water-soluble anti-cancer drugs, such as doxorubicin, camptothecin, colchicines, vinblastine, paclitaxel, etc. Therefore, the use of nano-carriers for improving the aqueous solubility of poorly water-soluble drugs and enhancing intracellular drug concentration (bio-accessibility) has attracted great attentions [17], and here we find that the present micelles@MSNs is a good choice of composite nano-carriers thanks to the solubilization effect of the micelles for hydrophobic drugs. In this work, we successfully encapsulated a common poorly watersoluble anti-cancer drug doxorubicin (DOX) into the micelles@MSNs to construct a nano multi-drug delivery system (nanoMDDS) drugs@micelles@MSNs by a facile one-pot co-self-assembly strategy. Noticeably, some surfactants, such as CTAB (cetyl trimethyl ammonium bromide), Tween 80, Triton X-100, P123, F127, Nonidet P-40, DTAB (dodecyl trimethyl ammonium bromide) and SDBS (sodium dodecyl benzene sulfonate), are all well qualified as both anti-cancer active drugs and chemosensitizers for overcoming the MDR in cancer [1822]. Herein, CTAB was chosen as both a structure-directing agent for constructing a drugs@micelles@MSNs nano-MDDS (DOX@CTAB@MSNs) and a chemosensitizer for overcoming the MDR and enhancing the drug efficacies (Scheme 1), because the anti-cancer activity of CTAB has been proved to be exceptionally high [19,20] and the particle size and dispersivity of the CTAB-templated MSNs could be facilely controlled [23]. Compared with the traditional drug post-loading route, in this report the simultaneous loading of multiple drugs including poorly water-soluble drugs and surfactant chemosensitizer was achieved by an in situ drug co-loading strategy, or in another word, a facile one-pot co-self-assembly route, thanks to the hydrophobic nature of the core part of the surfactant micelles in micelles@MSNs, as illustrated by Scheme 1. Moreover, the constructed drugs@micelles@MSNs nano multi-drug delivery system (nano-MDDS) show
a highly desirable precise pH-responsive drug release performance, i.e. loaded drug will not leak in normal tissues where the pH value is usually constant at 7.2e7.5, but will release in a sustained way within cancer cells where the pH value is a little lower through the ion exchanging interaction between Hþ/H3Oþ and positively charged drugs@micelles. Therefore the present nano-MDDS can hopefully resolve the difficulties in the traditional post-loading and uncontrolled release of poorly water-soluble drugs and the low pHresponsivity of the MSNs-based nano-DDSs. Furthermore, this pH value mediated multi-drug co-release is expected to greatly favor the drug accumulation within cancer cells and the multi-drug combined chemosensitization for overcoming MDR in cancer. In addition, in spite of the unavailable cytotoxic specificity of both CTAB and DOX, the present nano-MDDS DOX@CTAB@MSNs is expected not only to show a passive targeting capability via the enhanced permeability and retention (EPR) effect of abnormal tumors, but also to have a specific drug release character: the sustained release of CTAB and DOX within acidic cancer cells but hardly release in normal neutral tissue or cells. 2. Materials and methods 2.1. Preparation of DOX@CTAB@MSNs Poorly water-soluble drug DOX (0.5 mmol, Beijing HuaFeng United Technology Co., Ltd., Beijing) was solubilized in 500 mL of CTAB (Sigma-Aldrich Co.) solution (10 mM) at 80 C under intensive stirring and light-sealed environment. After several minutes, a clear solution was obtained and then NH4F (0.04 mol, Sinopharm Chemical Reagent Co. Ltd., Shanghai) which was used as a catalyzer for accelerating the hydrolyzation and condensation of silicon sources was added. Immediately, tetraethyl orthosilicate (TEOS, 0.04 mol, Sinopharm Chemical Reagent Co. Ltd., Shanghai) was added dropwise for 20 min. After 1 h, the reaction solution in a semitransparent colloidal state was centrifugated for 10 min with the centrifugal force of 18000 g in a high speed refrigerated centrifuge. Nanoparticles were collected and washed with ethanol and deionized water three times in turn for completely removing residual reactants. Finally, products were dispersed in deionized water and the freeze drying power was used for measurements. The control sample CTAB@MSNs without loading drugs was synthesized via the similar method by adding no drugs in the initial reaction solution. To obtain pure MSNs carrier, sample CTAB@MSNs was extracted several times with a mixed solution of ethanol (150 mL) and hydrochloric acid (36e38%, 2 mL) to completely remove CTAB. 2.2. Nanoparticles characterization The UV adsorption spectra of clear reaction solution before adding TEOS and upper clear solution after 1 h reaction and 10 min centrifugation were collected on a Shimadzu UV-3101PC UV-vis absorption spectrophotometer. According to the adsorbance difference, the drug loading capacity of DOX@CTAB@MSNs was calculated by the BeereLambert law. The drug loading capacity of DOX@CTAB@MSNs was
Scheme 1. The methodological comparison between the traditional MSNs-based nano-DDS by the drug post-loading route and the proposed nano-MDDS by the drug in situ co-loading route.
Q. He et al. / Biomaterials 32 (2011) 7711e7720 measured by dissolving DOX@CTAB@MSNs into a clear aqueous solution of HF and then determining the concentration of free drugs (w 2.4 mg DOX and 600 mg CTAB per 1 g MSNs). The morphology and mesostructure of nanoparticles were observed via transmission electron microscopy (TEM). TEM micrographs were obtained on a JEMe2010 electron microscope with an accelerating voltage of 200 kV. The mesostructure ordering was characterized by small-angle X-ray diffraction (SA-XRD). SA-XRD data were recorded on Rigaku D/Max-2550 V diffractometer using Cu Ka radiation (40 kV and 40 mA) at a scanning rate of 0.4 /min over the range of 0.5e6.0 with a step width of 0.002 . The particle size distribution data were collected by a DLS method in a Mastersizer 2000 analyzer (Malvern Instruments Ltd. UK). 2.3. Cytotoxicity against MCF-7 and MCF-7/ADR cells MCF-7 cells were cultured in RPMI 1640 (GIBCO, New York) containing 10% fetal bovine serum (FBS, Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou). MCF-7/ADR cells were cultured in RPMI 1640 containing 10% FBS and 1 mg mL1 of DOX. Cells were maintained at 37 C in a humidified and 5% CO2 incubator. For all experiments, cells were harvested by the use of 0.25% trypsin (Sigma) in D-Hank’s solution (0.40 g KCl, 0.06 g KH2PO4, 8.00 g NaCl, 0.35 g NaHCO3, 0.048 g Na2HPO4, 1000 mL H2O) and resuspended in fresh medium before plating. In vitro cytotoxicity against MCF-7 and MCF-7/ADR cells was assessed by the standard Cell Counting Kit8 (CCK-8, Beyotime Institute of Biotechnology, Jiangsu) assay. The statistical evaluation of data was performed using a two-tailed unpaired Student’s t-test. A p-value of less than 0.05 was considered statistically significant. Each data point is represented as mean standard deviation (SD) of eight independent experiments (n ¼ 8, n indicates the number of wells in a plate for each experimental condition). The time and dose dependences of the cytotoxicity were investigated at different time points of incubation (24 h, 48 h and 72 h) at different concentrations. MCF-7 cells were seeded in 96-well plates at a density of 104 cells per well. After incubation for 24 h at 37 C in 100 mL RPMI 1640 medium containing 10% FBS, culture medium was discarded and then cells were treated with 100 mL pH 7.4 D-Hank’s solution of drugs at different concentrations. At the end of each incubation (24 h, 48 h or 72 h), 10 mL of CCK-8 solution was added into each wells. After cells were incubated for another 4 h, the absorbance was monitored at 450 nm on a microplate reader (Bio-Tek ELx800). A culture medium without nanoparticles was used as the blank control. The cytotoxicity was expressed as the percentage of the cell viability as compared with the blank control. 2.4. Evaluation of MCF-7/ADR cell death mechanisms Annexin V-FITC apoptosis detection kit (Invitrogen, Oregon, USA) was used to quantify the apoptotic and necrotic cells by a standard FACS assay. MCF-7/ADR cells were double stained with Annexin V and PI (propidium iodide) and subject to flow cytometry (FCM). Firstly, MCF-7/ADR cells were seeded into 6-well plates (2 106 cells per well). After incubation for 24 h, cells were treated with 100 mL pH 7.4 DHank’s solution of drugs. The same particle concentration of 100 mg mL1 was used for DOX@CTAB@MSNs and CTAB@MSNs. Correspondingly, the free CTAB solution (60 mg mL1) containing the same drug concentration with the DOX@CTAB@MSNs solution was used for comparison, but the concentration of free DOX was magnified ten times so as to cause cell death, in order to reveal the death mechanisms and cell cycle distributions of MCF-7/ADR cells. At the end of each incubation (1 day, 2 days or 3 days), culture medium was discarded and cells were washed twice with the DHank’s solution. Then the fluorescein isothiocyanate (FITC)-labeled Annexin V (1 mL, 1 mg mL1; Responsif, Erlangen, Germany) was added to each well. Cells were incubated at room temperature on the shaker (100 rpm) for 15 min in darkness. Then the PI stock solution (5 mL, 750 mM) was added to each well. After cells were incubated for another 5 min, cells were washed twice with the D-Hank’s solution again. For each experiment, drug-untreated cells were used as a blank control. Signals collected on a FACSCalibur flow cytometry (Becton Dickinson, Zürich, Switzerland) with a CellQuest software were gated to separate live/apoptotic/ necrotic cells: AV/PI (lower left quadrant), intact live cells; AVþ/PI (lower right quadrant), early apoptotic cells; AVþ/PIþ (upper left quadrant), necrotic cells; AV/ PIþ (upper right quadrant), late apoptotic/secondarily necrotic cells. 2.5. Intracellular ATP level assay MCF-7/ADR cells were seeded into 96-well plates (5000 cells per well), incubated for 24 h at 37 C, and then treated with drugs or carrier alone. Intracellular ATP levels were determined using the luciferin-luciferase-based ATP luminescence assay kit (Beyotime Institute of Biotechnology, Jiangsu) as instructed by the manufacturer.
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twice with ice-cold PBS again, and then incubated with 1 mg mL1 RNase A for 20 min at 37 C, and stained with 10 mg mL1 PI for 30 min in the dark. The DNA content was measured on a FACSCalibur flow cytometry (Becton Dickinson, Zürich, Switzerland), and cell cycle distribution was determined by a ModFit software (Verity Software House, Topsham, ME). 2.7. In vitro drug release behaviors of DOX@CTAB@MSNs in buffer solutions of different pH values Sterilized dialysis bags with dialyzer molecular-weight cut-off 10,000 Da were used to carry out the drug release experiments. These dialysis bags were pretreated prior to use as follows. These dialysis bags were fully immersed into 50% aqueous solution of ethanol and boiled 1 h, and then washed with 50% ethanol, 10 mmol L1 NaHCO3 and 1 mmol L1 EDTA in turn. Phosphate buffered saline (PBS) of pH ¼ 7.4 and acetic buffer solutions (ABS) of pH ¼ 4, 5 and 6.5 were used as the drug release media to simulate normal blood/tissues and tumor environments. The sample DOX@CTAB@MSNs (60 mg) was dispersed into 2 mL release media, and then the solutions were put into pretreated dialysis bags. The sealed dialysis bags were put into brown bottles and then 58 mL release media was added. These bottles were shaken at a speed of 100 rpm at 37 C under a light-sealed condition. At certain time intervals, 3 mL of the release media were taken out for measuring the released drug concentrations by the UV-vis absorption technique, and then were returned to the original release media. The concentrations of released drug were calculated by the BeereLambert law according to the absorbances of the release media at a certain characteristic adsorption wavelength. 2.8. In vitro intracellular uptake and drug release behaviors of DOX@CTAB@MSNs in MCF-7/ADR cells Green emitting fluorescein isothiocyanate (FITC, Sigma-Aldrich Co.) was grafted onto DOX@CTAB@MSNs (denoted by ‘DOX@CTAB@MSNs-FITC’) by a facile cocondensation approach as described in our previous report [24]. In a typical procedure, 104 MCF-7/ADR cells were cultured for 8 h at 37 C in RPMI 1640 medium supplemented with 10% FBS in each well. Then cells were gently washed twice with D-Hank’s solution of pH 7.4, and subsequently D-Hank’s-buffered DOX@CTAB@MSNsFITC at a final concentration of 160 mg mL1 added into petri dishes. After incubation for 4 h, MCF-7/ADR cells were washed for several times with D-Hank’s solution to remove the residual nanoparticles, and then were totally lysed under an ultrasonic cell disruptor and centrifugated for 10 min with the centrifugal force of 18000 g in a high speed refrigerated centrifuge. The intracellular drug-released concentrations were measured by monitoring collected upper clear solutions using a TECAN infinite F200 microplate reader (emission wavelength of 595 nm, excitation wavelength of 480 nm). The intracellular concentrations of all DOX, including non-released and released DOX, were measured by monitoring the dissociated (not lysed) MCF-7/ADR cells by flow cytometry. Besides, the intracellular uptakes and the localizations of DOX@CTAB@MSNs-FITC and released drug DOX were directly visualized via a Leica TCS confocal microscope (Leica Microsystems, Germany). In the assay, all experiments were carried out under a light-sealed condition to avoid photo-bleaching. 2.9. In vivo drug release behaviors of DOX@CTAB@MSNs in tumors The tumor-bearing mouse model was induced by injecting MCF-7/ADR cells into the right infra-axillary dermis of nude mice. MCF-7 tumor-bearing mice were intravenously injected with DOX@CTAB@MSNs at a dose of 5 mg g1 through the tail vein. The administrated mice were maintained daily under a 12 h light/dark cycle at the Animal Care Facility, and fresh distilled water and food for all animals were available ad libitum. After mainline administration for 2 days, mice were sacrificed via the cervical dislocation. The tissues such as liver, spleen, lung and tumor were discretized, rinsed with sterile physiological saline, and then blotted dry with filter paper. All the discretized tissues were cut into slices of 5 mm in thick following polyoxymethylene fixing, paraffine embedding and actin staining (Actin-Tracker Green Kit, Beyotime, Jiangsu) for directly observing the localization and drug release of DOX@CTAB@MSNs on a Leica TCS confocal microscope. The in vivo animal experiments were carried out under the guideline approved by the International Animal Care and Use Committee (IACUC) of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
3. Results and discussion 3.1. Synthesis and characterization of DOX@CTAB@MSNs
2.6. Cell cycle distribution by FCM DNA analysis MCF-7/ADR cells were seeded into 6-well plates (2 106 cells per well), and then treated with drugs for 24 h. After incubation for another 24 h, adherent cells were detached by the addition of trypsin, washed with PBS and collected by refrigerated centrifugation. Then cells were resuspended in ice-cold PBS, and then fixed with 70% precooled ethanol at 4 C overnight. Ethanol-fixed cells were washed
An important feature in the construction of the present nano multi-drug delivery system (nano-MDDS) DOX@CTAB@MSNs is the near neutral reaction conditions. As the co-loaded drug DOX will easily hydrolyze under either basic or acidic conditions, therefore, we used NH4F as a catalyzer for accelerating the hydrolyzation and
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condensation of silicon sources, consequently improving the dispersivity and uniforming the particle size of nano-sized MSNs under a near neutral reaction condition. From transmission electron microscopy (TEM) images, it can be found that both CTAB@MSNs and DOX@CTAB@MSNs have fairly uniform particle sizes (112 14 nm and 101 12 nm, respectively) and almost ideal monodispersity (Fig. 1a and b) in accordance with the dynamic light scattering results (Fig. 1c and d), and show a partially ordered mesoporous structure in conformity with small-angle X-ray diffraction (SA-XRD) measurements (Fig. 1e). Furthermore, a reaction temperature of 80 C was found to be suitable to accelerate the solubilization of DOX without significant damage to DOX under the present near neutral reaction condition. 3.2. In vitro cytotoxicity of the nano-MDDS against MCF-7 and MCF-7/ADR cells Next, we evaluated the cytotoxicity of the synthesized nanoMDDS DOX@CTAB@MSNs against both MCF-7 and MCF-7/ADR cells (human breast cancer cells). As for the drug-sensitive MCF-7 cells, the cytotoxicity of free CTAB and DOX has apparent drug concentration and treatment time dependences (Fig. 2a, b and c), and both free CTAB and free DOX show remarkable cytotoxicity at very low concentrations, and free CTAB is even more toxic than free DOX at the same concentrations, as previously identified as an effective anti-cancer agent [20]. However, this cytotoxicity difference between free CTAB and free DOX become less at the increased drug treatment time. After drug treatment for 3 days, the IC50 (50% inhibiting concentration) values of free CTAB and DOX are about
1.8 mg mL1 and 2.9 mg mL1, respectively. By comparison with free drugs, the cytotoxicity of the constructed nano-MDDS DOX@CTAB@MSNs also show strong drug concentration and treatment time dependences (Fig. 2d e and f). Importantly, the nano-MDDS DOX@CTAB@MSNs has higher cytotoxicity against MCF-7 cells than CTAB@MSNs at the same particle concentrations and for the same treatment time, in spite of lower cytotoxicity of free DOX than free CTAB as above mentioned. This suggests that the nano-MDDS DOX@CTAB@MSNs can be well uptaken by MCF-7 cells, and have remarkably improved the intracellular accessibility of the poorly water-soluble drugs. After drug treatments for 3 days, the IC50 values of CTAB@MSNs and DOX@CTAB@MSNs are about 18.6 mg mL1 and 17.3 mg mL1, respectively, based on particle concentrations. Such low IC50 values of the nano-MDDSs indicate their high drug efficiencies in vitro against MCF-7 cells, and also imply the expected low using amounts of drug and carrier. In addition, the cytotoxicity between free drugs and the nano-MDDS at the same drug concentrations might be incommensurable directly because the drugs were released into cells from the nanoMDDS in a highly sustained way as under-mentioned and the intracellular uptaking fashions of free drug molecules and the nano-MDDS were potentially different. As for drug-resistant MCF-7/ADR cells, both free CTAB and free DOX do not bring visible cytotoxicity in wide ranges of drug concentration and treatment time (Fig. 3a b and c), indicating the representative MDR characteristics of used MCF-7/ADR cells. It is worth noting that compared with free drugs, the synthesized nanoMDDS DOX@CTAB@MSNs and CTAB@MSNs exhibit remarkable cytotoxicity and their cytotoxicities are dependent on the drug
Fig. 1. TEM images of CTAB@MSNs (a) and DOX@CTAB@MSNs (b), and particle diameter distributions of CTAB@MSNs (c) and DOX@CTAB@MSNs (d) measured by the DLS method, and SA-XRD patterns of CTAB@MSNs and DOX@CTAB@MSNs (e).
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Fig. 2. Cytotoxicity of free drugs (aec) and the nano-MDDS (def) against MCF-7 cells after treated for 1 day (a, d), 2 days (b, e) and 3 days (c, f).
concentration and treatment time again (Fig. 3d e and f). This suggests that the nano-MDDS DOX@CTAB@MSNs have significantly improved the intracellular drug accessibility into MCF-7/ADR cells which would be proved further by monitoring the intracellular drug concentrations both qualitatively and quantitatively, as undermentioned. Moreover, CTAB has successfully played a role of chemosensitizer which further enhances the sensitivity of MCF-7/ADR cells to the poorly water-soluble drug DOX, similar to the case of MCF-7 cells as mentioned above. After drug treatments for 3 days, the IC50 values of CTAB@MSNs and DOX@CTAB@MSNs are about 78.5 mg mL1 and 65.2 mg mL1, respectively, for MCF-7/ADR cells based on the concentrations of the nano-MDDS. In addition, the carrier MSNs itself did not bring visible cytotoxicity against both MCF-7 cells and MCF-7/ADR cells in wide ranges of particle concentration and treatment time (Figs. 2 and 3). To sum up, the present nano-MDDS DOX@CTAB@MSNs remarkably enhanced the MCF-7/ADR intracellular accessibility to poorly water-soluble drugs, and had much higher drug efficiencies in vitro against both drug-sensitive MCF-7/ADR cells and drug-resistant MCF-7 cells, as compared with free drugs. 3.3. Death mechanisms of MDR cancer cells Further, the death mechanisms of MCF-7/ADR cells treated with the nano multi-drug delivery system (nano-MDDS) DOX@CTAB@MSNs and free drugs for different time periods were evaluated by flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) protocols (Fig. 4). The carrier MSNs show very little influence
on the apoptosis and necrosis of MCF-7/ADR cells. However, free CTAB at a high enough concentration could simultaneously induce distinct apoptosis and necrosis of MCF-7/ADR cells, and cause preferentially the necrosis in one day and then the major apoptosis and subsequent secondary necrosis in two or three days (Fig. 4). Different from free CTAB, free DOX could only induce the apoptosis of MCF-7/ADR cells slightly although the concentration of free DOX was magnified ten times as compared with DOX encapsulated within DOX@CTAB@MSNs, which is dependent on treatment time. Compared with free CTAB, CTAB@MSNs would induce the apoptosis and subsequent secondary necrosis rather than the necrosis, which could be associated with the low intracellular concentration of CTAB owing to their sustained intracellular release behaviors. Compared with CTAB@MSNs, DOX@CTAB@MSNs remarkably accelerated the apoptosis of MCF-7/ADR cells at the same particle concentrations, which is also dependent on treatment time. Therefore, the nanoMDDS DOX@CTAB@MSNs could induce MCF-7/ADR cell apoptosis by a synergistic apoptosis-accelerating effect between DOX and chemosensitizer CTAB, which should be attributed to the sustained intracellular release of DOX even at a very small DOX-released percentage as under-mentioned, and the synergistic chemosensitization effect between DOX and CTAB as a chemosensitizer (as concluded from Fig. 3). In addition, we further made a preliminary evaluationon the effect of drug and carrier on the intracellular ATP (adenosine triphosphate) level from the energy point of view because CTAB has been identified as a mitochondrial inhibitor for anti-cancer [20]. It is well known that P-glycoprotein (P-gp) protein as a drug efflux pump
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Fig. 3. Cytotoxicity of free drugs (aec) and the nano-MDDS (def) against MCF-7/ADR cells after treated for 1 day (a, d), 2 days (b, e) and 3 days (c, f).
Fig. 4. Evaluation of the death mechanisms of MCF-7/ADR cells treated with free drugs and nano-MDDSs for different time periods (1 day, 2 days and 3 days) by flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) protocols.
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indicates that the nano-MDDS DOX@CTAB@MSNs could disturb/ impair the mitochondrial functions of MCF-7/ADR cells by an enhanced ATP-inhibiting effect, leading to the much reduced drug efflux capability of P-gp. Therefore it is believed that there would exist a synergistic effect in killing MDR cancer cells between the surfactant and the intracellularly released drug: surfactant CTAB plays an important role in inhibiting mitochondrial functions/ATP content [20], and consequently prevents the drug efflux by P-gp and favors the released DOX to diffuse into the nuclei without disturbance, or with much diminished disturbance, from the drug efflux effect of P-gp. Thus the intracellularly released drugs could be accumulated continuously within the MDR cells by this synergistic mechanism and consequently induced the apoptosis of MCF-7/ADR cells, as under mentioned.
3.4. Cell cycle distribution
Fig. 5. Intracellular ATP levels in MCF-7/ADR cells treated with the carrier, free drugs and the nano-MDDS for different time periods.
is over-expressed on the membrane of MCF-7/ADR cells, resulting in the MDR phenotype, and the drug efflux by P-gp is dependent on cell energy [25,26]. The intracellular ATP (adenosine triphosphate) level in MCF-7/ADR cells treated with the carrier MSNs could recover after incubation for 2 days (Fig. 5), though there is a transitory reduction in 1 day as reported by Tao [27]. However, free CTAB at a high concentration could cause the sharp and unrecoverable decrease of the intracellular ATP level. Compared with free CTAB, CTAB@MSNs could advance the slow but ever-increasing inhibition to the intracellular ATP level, owing to the sustained CTAB release. Free DOX can only result in a slight but time-dependent reduction in the intracellular ATP level, although the concentration of free DOX was magnified ten times as compared with DOX encapsulated within DOX@CTAB@MSNs. However, the nano-MDDS DOX@CTAB@MSNs could induce a more intensive reduction in the intracellular ATP level than free DOX and CTAB@MSNs (Fig. 5), which
We next assessed the effects of the nano multi-drug delivery system (nano-MDDS) DOX@CTAB@MSNs on cell cycle progression and cell death by the analysis of DNA content using flow cytometry. After treatment with the carrier MSNs for 3 days, the cell cycle distribution of MCF-7/ADR cells remained constant regardless of incubation time, as compared with the blank control, which indicated that the carrier MSNs itself did not affect the cell cycle (Fig. 6). It is distinct that free CTAB at a high concentration directly caused the cell death, however CTAB@MSNs induced a time-dependent arrest of MCF-7/ADR cells in the G1 phase probably owing to the transmembrane delivery and sustained intracellular release functions of CTAB@MSNs. Different from CTAB, free DOX could cause a time-dependent arrest of MCF-7/ADR cells in the G2 phase untill the concentration of DOX was magnified ten times as compared with DOX encapsulated within DOX@CTAB@MSNs (Fig. 6). The most important is that the nano-MDDS DOX@CTAB@MSNs coloaded with CTAB and DOX could significantly induce the arrest of MCF-7/ADR cells in both G1 and G2 phases and the cell death by a definite and specific synergistic effect between CTAB and DOX. This synergistic cytotoxic and cell cycle blocking effect of
Fig. 6. Cell cycle distribution histograms of MCF-7/ADR cells treated with free drugs and the nano-MDDS for 3 days by flow cytometry evaluation. Four prominent peaks in the last column graphs represent the death cells, G1 phase (DNA pre-synthetic gap phase), S phase (DNA synthesis phase) and G2 phase (DNA post-synthetic phase) from left to right, respectively.
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Fig. 7. In vitro pH-responsive drug release behaviors of the nano-MDDS DOX@CTAB@MSNs in the release media of different pH values which were used to simulate the alkalescent conditions in normal tissues and blood (pH w 7.4) and the acidic conditions in tumor (pH ¼ 4e6.8). The nano-MDDS DOX@CTAB@MSNs hardly released DOX in the pH ¼ 7.4 release medium, but responsively released DOX in pH ¼ 4e6.8 acidic media.
DOX@CTAB@MSNs was further enhanced with the increase of treatment time, owing to the sustained intracellular drug release as under mentioned (Fig. 7). 3.5. In vitro pH-responsive drug release behaviors In addition to the enhancement of drug efficiency, the inhibition of toxic side effects of drugs, especially poorly water-soluble drugs, is also vitally important and significant to anti-cancer and MDR overcoming. One of the widely accepted routes is to endow nano drug delivery systems (nano-DDSs) with the pH-responsive drug release character [2831]. The perfect case is that drugs do not or
hardly release in normal tissues and blood (pH w 7.4), but can responsively release in tumor tissues, or even within cancer cells, to selectively kill cancer cells (pH ¼ 4e6.8). Though some nano-DDSs have been designed to release drugs under in vitro simulated acidic conditions [2931], however, to effectively suppress drug release as slowly as possible in pH ¼ 7.4 normal physiological conditions is still a great challenge, especially under in vivo conditions. Fortunately, we discover that the designed nano-MDDS drugs@CTAB@MSNs has such a desired precise pH-responsive drug release character both in vitro and in vivo. From Fig. 7, it is clear that a very small amount of DOX is released from DOX@CTAB@MSNs in a very slow fashion under pH ¼ 7.4 PBS simulating normal physiological conditions, and only less than 2% of DOX was released after immersion for as long as 14 days which is indeed an extraordinarily low drug-released amount in such a long release time period as compared with previous reports [2931]. When the pH values of release media decreased from 6.5 to 4 for simulating cancer conditions, both the DOX release rates and the DOX-released concentrations became remarkably higher. After immersion for 14 days, the DOX-released percentage can reach about 26.6% in pH ¼ 4 release medium. Therefore, the pH responsivity of in vitro drug release of DOX@CTAB@MSNs is believed to be high enough for the intracellular and in vivo evaluation. Though the low release percentage, the intracellular drug release amounts of DOX@CTAB@MSNs were high enough to kill MCF-7 and MCF-7/ADR cells efficiently by the specific synergistic effect between CTAB and DOX, as concluded above. This means that the nano-MDDS DOX@CTAB@MSNs could work in a relative long time of more than 14 days and in a highly pH-responsive and sustained release way. Further, we think that the pH-responsive drug release mechanism for the nano-MDDS drugs@micelles@MSNs (DOX@CTAB@MSNs) should be highly related to the electrostatic selfassembly mechanism for the synthesis of MSNs. The drugs@micelles@MSNs is formed by the electrostatic attraction between drugs-encapsulated micelles (drugs@micelles) and MSNs [32], therefore positively charged drugs@micelles within drugs@micelles@MSNs are exchangeable with protons in acidic release media, but will be almost inexchangeable in slightly basic pH ¼ 7.4 release media. As a result, surfactant and drugs can be co-released
Fig. 8. The quantitative measurement using a fluorescence microplate reader (a) and the qualitative observation by the confocal microscopic imaging (bee) of intracellular drug release behaviors of the nano-MDDS DOX@CTAB@MSNs-FITC in MCF/ADR cells for representative treatment time periods: (b) 12 h, (c) 24 h, (d) 48 h, and (d) 72 h (see Fig. S2 in the Supporting Information for more detailed drug release processes). Red, green and yellow represent the released drug DOX, the FITC-labeled carrier MSNs and the nano-MDDS DOX@CTAB@MSNs-FITC, represently, and purple from the superposition of blue and red suggests the entry of the released DOX into the nuclei of the MCF-7/ADR cells.
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together under the simulated cancer conditions in a precisely and highly pH-responsive way. 3.6. In vitro intracellular uptake and drug release behaviors Further, we investigated the intracellular DOX release process of DOX@CTAB@MSNs within MCF-7/ADR cells. Firstly, we quantitatively measured the intracellular DOX concentrations in MCF-7/ADR cells at several fixed time points up to 72 h using a fluorescence microplate reader. It can be clearly found that the intracellular DOX concentration in MCF-7/ADR cells increases with the increase of the treatment time on the whole, though could transitorily decrease at certain time points such as at 6 h incubation (Fig. 8a). This suggests that the nano-MDDS DOX@CTAB@MSNs have successfully overcome the drug efflux of P-gp on MCF-7/ADR cells, and favored the continual intracellular accumulation of released DOX molecules, which could be attributed to both the sustained and pH-responsive drug intracellular release from endocytosized nano-MDDS, as concluded above in the simulated cancer conditions (Fig. 7), and the durative transmembrane transport of intercellular DOX@CTAB@MSNs from the outside, as can be concluded by the three-day continual increase of intracellular DOX concentration including
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both non-released and released DOX (Fig. S1). In addition, the qualitative monitoring by the confocal microscopic imaging also reveals that the nano-MDDS DOX@CTAB@MSNs can be uptaken by MCF-7/ADR cells and gradually release DOX into cells (Fig. 8b and Fig. S2). These quantitative and qualitative results demonstrate the reasons why the nano-MDDS DOX@CTAB@MSNs can overcome the MDR of MCF-7/ADR cells in vitro. 3.7. In vivo localization and drug release behaviors in tumors Further, we investigated the in vivo localization and DOX release behavior of the nano-MDDS DOX@CTAB@MSNs in the MCF-7/ADR cells-induced tumor and several normal tissues. It can be found that large numbers of the nano-MDDS DOX@CTAB@MSNs had accumulated in tumor after mainline administration for 1 h (Fig. 9), most probably due to the EPR effect of abnormal tumors. The most important is that DOX was visibly released into the nuclei of MCF-7/ ADR tumor cells after mainline administration for 2 days as shown in Fig. 9, which is very similar to the in vitro intracellular case of MCF-7/ADR cells as mentioned above. Moreover, the density of tumor cells whose nuclei were intruded with released DOX from DOX@CTAB@MSNs seems significantly lower than that of other
Fig. 9. In vivo localization and drug release behaviors of the nano-MDDS DOX@CTAB@MSNs in the MCF-7/ADR cells-induced tumor. Green, blue and red represent actin, nuclei and DOX, respectively, and therefore purple from the superposition of blue and red suggests the entry of the released DOX into the nuclei in the MCF-7/ADR cells-induced tumor, which is especially visible after mainline administration for 2 days. The white circles indicate the zones of both low cell density and high drug content, implying the probable in vivo drug efficiency of the nano-MDDS DOX@CTAB@MSNs against the MCF-7/ADR cells-induced tumor.
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tumor cells (as directed by white circles in Fig. 9), probably implying the efficient in vivo MDR-overcoming action of the nano-MDDS DOX@CTAB@MSNs against the MCF-7/ADR cells-induced tumor. The accurate in vivo MDR-overcoming efficiency of DOX@CTAB@MSNs is under investigation. In addition, DOX@CTAB@MSNs were also found distributed in three normal tissues liver, spleen and lung, however, very interestingly, these DOX@CTAB@MSNs did not release DOX into the nuclei of the normal cells in 2 days after mainline administration (Fig. S3). This suggests that DOX@CTAB@MSNs could release DOX into tumor cells, but virtually not into normal cells in 2 days. We infer that this result can be probably attributed to two causes: one is that DOX@CTAB@MSNs could be more endocytosed by tumor cells owing to their distinctively higher activity (tumor cells proliferate much faster by uptaking more external nutrients) than normal tissues; and the second, more confidently to claim, is that DOX could be released faster in the more acidic condition of tumor cells as compared within normal cells [33,34]. Therefore, such a nano-MDDS shows a favorable in vivo pHresponsive drug release behavior, which is consistent with the in vitro simulated cases as mentioned above. 4. Conclusions In summary, a kind of nano-MDDS drugs@micelles@MSNs has been constructed by the facile co-loading of poorly water-soluble anti-cancer drug and surfactant chemosensitizer into MSNs via a one-pot co-self-assembly strategy among the drugs, surfactant micelles and silicon sources. The nano-MDDS DOX@CTAB@MSNs demonstrated a highly pH-responsive controlled drug release behavior, in favor of precise intracellular drug release, both under in vitro simulated conditions and under in vivo conditions with the tumor-bearing mouse model induced by injecting MCF-7/ADR cells. Meanwhile, the nano-MDDS DOX@CTAB@MSNs also exhibited high drug efficiencies against both drug-sensitive MCF-7 cells and drugresistant MCF-7/ADR cells by a synergistic cell cycle arrest/ apoptosis-inducing effect between poorly water-soluble anticancer drug and surfactant chemosensitizer. Acknowledgements We greatly acknowledge financial supports from the NSFC (Grant Nos. 50823007, 50972154 and 51072212), the National Basic Research Program of China (2011CB707905 and 2010CB934000), the Science and Technology Commission of Shanghai (10430712800), CASKJCX Project (KJCX2-YW-210) and the Science Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures (Grant No. SKL201001). Appendix. Supplementary information Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2011.06.066. References [1] Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidem Biomar 2010;19:1893e907. [2] Lage H. An overview of cancer multidrug resistance: a still unsolved problem. Cell Mol Life Sci 2008;65:3145e67. [3] Ullah MF. Cancer multidrug resistance (MDR): a major impediment to effective chemotherapy. Asian Pac J Cancer Prev 2008;9:1e6. [4] Page R, Takimoto C. Cancer management: a multidisciplinary approach: medical, surgical, and radiation oncology. In: Pazdur R, editor. Principles of chemotherapy. 8. New York: PRR; 2004. p. 21e38.
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