Biomaterials 60 (2015) 111e120

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Multifunctional envelope-type mesoporous silica nanoparticles for pH-responsive drug delivery and magnetic resonance imaging Yan Chen a, b, Kelong Ai a, Jianhua Liu c, Guoying Sun d, Qi Yin d, Lehui Lu a, * a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China b University of Chinese Academy of Sciences, Beijing, 100039, PR China c Department of Radiology, The Second Hospital of Jilin University, Changchun, 130022, PR China d Chemistry and Life Science School, Changchun University of Technology, Changchun, 130022, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 April 2015 Accepted 3 May 2015 Available online xxx

A novel multifunctional envelope-type mesoporous silica nanoparticle (MEMSN) system combining the merits of pH-responsiveness, non-toxicity and biological specificity, is demonstrated for drug delivery and magnetic resonance imaging (MRI). This system is constructed by immobilizing acetals on the surface of mesoporous silica, and then coupling to ultra small lanthanide doped upconverting nanoparticle, which act as a gate keeper. The anticancer drug DOX is thus locked in the pores, and its burst release can be achieved under acidic environment on account of the hydrolyzation reactions of acetals. The nanogated drug release system is highly efficacious for cancer therapy both in vitro and in vivo. Importantly, the nanocomposite could be harmlessly metabolized and degraded into apparently nontoxic products within a few days. The nanoscale effect of the system allows for passive tumor targeting and increased tumor accumulation of the probes via the enhanced permeation and retention (EPR) effect, which is visualized by MRI in vivo. Therefore, such nanosystem should be of great significance in the future development of highly efficient and tumor targeted drug delivery vehicles for cancer chemotherapy. © 2015 Elsevier Ltd. All rights reserved.

Keywords: pH-responsiveness Drug delivery Magnetic resonance imaging EPR effect

1. Introduction The cancer has become one of the world's most devastating diseases [1,2]. There were about 14.1 million new cases of cancer occurred globally in 2012, which have caused about 8.2 million deaths. Unfortunately, most chemotherapeutics, for example, were administered at high doses to compensate for premature degradation and non-specific absorption, which would lead to the development of dose-limited toxicity [3e5]. A holy grail in theranostics lies in the “smart” nanodevices, which would accumulate in tumor tissues and release the drug controllably, making them useful in drug delivery applications [6,7]. Recently, the approach of “multifunctional envelope-type nanodevice (MEND)” has provided an immense potential to revolutionize the tumor treatments by designing a nanocomposite, which is analogous to the envelopetype virus [8,9]. The topology of various functional devices

* Corresponding author. E-mail address: [email protected] (L. Lu). 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

bestows such system with unique advantages in the construction of nanocontainers that exert their functions reasonably, timely and locally according to the predetermined strategy. Inspired by the idea, we developed a multifunctional envelope-type and mesoporous silica nanoparticle (MS)-based nanoplatform as a therapeutic and diagnostic agent. The MS is considered to be a promising nanocontainer for delivery system due to a number of favorable structural features, such as high loading capacity, good biocompatibility, and easy functionalization ability [10,11]. The judicious choice of functional groups on such building blocks could precisely tune the properties of the MS, thus enabling them to respond to external stimuli. As an appealing quest for therapeutic purposes, various actuators, such as temperature, light, redox and enzyme have been reported [12e15]. Despite these sophisticated stimuli-responsive release systems, pH controlled delivery has captured much more attention due to their simple design and universal applicability. In the past decades, MSbased pH-responsive controlled-release systems with various pore blockers have been certified useful for delivering chemotherapeutic drugs [16,17]. However, clinical translations would have been


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impeded owing to concerns about the toxicity from the nanomaterials themselves or degradation by-products [18]. A more desirable design criterion should pay attention to the biocompatibility and possible toxicity of the nanomaterial after they carried out their functions in vivo. Nevertheless, their intracellular biodistribution and metastasis after uncapping, as well as the underlying cell death pathways, which based on the intelligent controlled drug delivery systems, are still issues of concern for potential clinical application. Moreover, there is a growing need to visualize the tumor preoperative diagnosis and intraoperative positioning. Magnetic resonance imaging (MRI) has emerged as one of the most powerful tools for in situ imaging, which offers high spatial resolution with excellent tissue penetration, and does not require ionizing radiation such as used in CT scanning [19,35]. Generally, the contrast agents are widely used in MRI to greatly improve the imaging accuracy and sensitivity, which help to detect and characterize the pathological abnormality. The effective cancer therapy requires cancer diagnosis and treatment simultaneously. For this purpose, “imaging guided therapy” has received significant attention. For example, Haam et al. designed pH-triggered drug-releasing magnetic nanoparticles for cancer therapy guided by MRI [20]. Besides magnetic nanoparticles, lanthanide doped nanoparticles have offered a great potential due to their fascinating features in the fields of imaging and therapy as well [21e23]. To the best of our knowledge, the aforementioned pH-responsive nanogated ensemble that utilized such nanoparticle-capped vessel for controlled drug release and MRI has never been reported previously. With this in mind, an intelligently nanogated multifunctional envelope-type drug release system based on lanthanide doped nanoparticle-capped MS, was designed for pH-responsive drug delivery and MRI. In the present study, the functionalized ultra small lanthanide doped nanoparticles (NaGdF4:Yb/Tm@NaGdF4) with ultra thin TaOx layer (denoted as S-NPs) were grafted onto the orifices of MS with the acid-labile acetals, which have been used as the protecting group for the carbonyl group in organic synthesis. At lower pH, the acetal was hydrolyzed to enable the removal of the SNPs caps, which has allowed the escape of the entrapped drug molecules in a pH-responsive manner. Further in vitro and in vivo experiments demonstrated that such nanogated drug release system was highly efficacious for cancer therapy. More importantly, this nanocomposite could be harmlessly metabolized and degraded into apparently non-toxic products within a few days. Furthermore, the resulted nanocomposite was modified with PEG to achieve longer circulation in blood vessels, and such nanocomposite could accumulate in tumor by taking advantage of their enhanced permeation and retention (EPR) property in the leaky vasculatures of tumor tissues, which was visualized by MRI. 2. Materials and methods 2.1. Materials The rare-earth oxides including gadolinium oxide (Gd2O3, >99.99%), ytterbium oxide (Yb2O3, >99.99%), and thulium oxide (Tm2O3, >99.99%) were purchased from Changchun Hepalink rareearth materials company. The Gd2O3, Yb2O3 and Tm2O3 were reacted with excess hydrochloric acid to form the rare earth chloride compounds, respectively. After the hydrochloric acid and water were evaporated, the resulting powders were re-dispersed in water to yield the GdCl3 (1.0 M), YbCl3 (0.5 M) and TmCl3 (0.1 M) aqueous stocking solutions, separately. Oleic acid (OA, 90%), octadecene (ODE, 90%), polyoxyethylene (5) nonylphenyl ether (Igepal COe520, average Mn ¼ 441), ammonia solution (28.0e30.0% NH3

by weight) and 3e(4,5edimethylthiazole2eyl)e 2,5ediphenyltetrazolium bromide (MTT) were obtained from SigmaeAldrich. Tetraethyl orthosilicate (TEOS) and Ta (CH3CH2O)5 was purchased from Alfa Aesar. Carboxyethylsilanetriol (CTES, sodium salt, 25% in water) was obtained from Gelest Inc. (Morrisville, USA). 3,9eBis(3eaminopropyl)e2,4,8,10etetraoxaspiro[5.5]undecane was purchased from TCI. 2-methoxy(polyethyleneoxy)propyltrimethoxysilane (PEG-silane) was purchased from Meryer Chemical Technology Co., Ltd., (China). Doxorubicin hydrochloride (DOX) was purchased from Sangon (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (New York, USA). All chemicals were analytical grade and used as received without further purification. Water used in the experiment was purified by a Millipore system. 2.2. DOX loading, capping and releasing experiments For DOX loading, the surface-functionalized MS (M-3) (30 mg) was soaked in a solution of DOX (3 mL, 1 mg mL1) in Tris buffer (pH ¼ 7.4) and stirred for 24 h. The solution was then centrifuged. Then the powder was redispersed in Tris buffer, the aqueous solution containing EDC (60 mg), NHS (30 mg) and S-NPs (30 mg) were added. The mixture was stirred for another 6 h. For in vivo application, the nanocomposite was modified with PEG-silane on the outmost surface. In the end the precipitate was centrifuged, washed several times with water (pH 7.0), and the resulted nanocomposites denoted as MS@S-NPs. The control experiments were carried out by simply soaking MS (30 mg) in 3 mL DOX solution (1 mg mL1) in Tris buffer for 24 h before the precipitate was centrifuged and washed extensively with water under the same conditions. The DOX loading was calculated from the difference in concentrations of the initial and left, which was determined to be 0.17 mmol g1 approximately. For DOX release, the nanocomposite was sealed in a dialysis bag (molecular weight cutoff ¼ 8000) and immersed in 5 mL buffer solution at different pH to test the release property. Aliquots were taken from the suspension and the release of DOX from the pore to the buffer solution was monitored via the UVevis absorbance centered at 480 nm (Fig. S9). 2.3. Cytotoxicity assays To evaluate the cytotoxicity of the nanocomposite, the MTT assay was used on the HeLa cell. Briefly, HeLa cells were seeded in a 96-well plates at a density of 6000e7000 cells per well, cultured with fresh DMEM, and supplemented with 20% FBS under a humidified 5% CO2 at 37  C for 24 h. After that, various concentrations of MS@S-NPs, DOX loaded MS@S-NPs, and free DOX were added into the culture wells and the cells were further incubated for 48 h. Subsequently, the cells were washed with culturing medium and treated with 10 mL MTT (5 mg mL1 in PBS) at 37  C for 4 h in an incubator at the same conditions. Finally, the supernatant was removed, and dimethyl sulfoxide (DMSO) was added (100 mL per well) and shaken for 10 min to thoroughly mix the formazan into the solvent. The optical density at 555 nm was measured by the microtiter plate reader. The untreated HeLa cells were used as control samples. The percentage of cell viability was calculated by using the optical densities with respect to the control values. 2.4. Cellular uptake The Hela cells were initially seeded and allowed to adhere for 24 h at 37  C under a humidified 5% CO2. The cells were incubated in culture medium containing DOX loaded MS@S-NPs at 50 mg mL1 for different time periods (0, 0.5, 3, 6 and 12 h). Subsequently, the cells were washed with PBS three times to remove excess

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nanoparticles. Then, the nuclei were stained with 4e6eDiamidinoe2ephenylindole (DAPI) for 10 min. The luminescence signals were detected in the wavelength regions of 435e485 nm and 605e655 nm, respectively. The quantitative evaluation of cellular association and apoptosis were performed by flow cytometry. The HeLa cells (1  105) were seeded in 6-well culture plates and cultured for 48 h. The cells were then treated with DOX loaded MS@S-NPs or free DOX ([DOX] ¼ 5 mg mL1) at 37  C for different time period. The cells were washed with PBS three times before harvested by trypsin treatment. After two cycles of washing and centrifugation, the cells were resuspended in 0.5 mL buffer solution. For apoptosis analysis, cells were stained with Annexin V-FITC and PI for 10 min at room temperature in the dark according to the manufacturer's instruction. Finally, the stained cells were analyzed by using a FACSCalibur flow cytometer (BD Biosciences) system. The uptake efficiency was also monitored by TEM to further validate the cellular uptake in Hela cells. Briefly, the cells were seeded in 6-well culture plates and grown overnight. Then the cells were incubated with medium in 5% CO2 at 37  C, which containing MS@S-NPs and DOX loaded MS@S-NPs at 50 mg mL1 for 12 h. After that, the cells were throughly washed with PBS for three times. Subsequently, cells were trypsinized, collected by centrifugation and fixed in paraformaldehyde. Finally, the cells were embedded in resin and sliced with a thickness of around 50e70 nm for TEM observation. 2.5. Hemolysis assay in vitro Human blood samples stabilized by heparin were obtained from the local hospital. First, 1 mL of blood sample was added to 2 mL of PBS, and then red blood cells were isolated from serum by centrifugation. After being washed several times with PBS solution, the purified blood was diluted with PBS. Second, 0.2 mL of diluted blood cells suspension was mixed with (a) 0.8 mL of PBS as a negative control, (b) 0.8 mL of water as a positive control, (c) 0.8 mL of MS@S-NPs suspensions at concentrations ranging from 12.5 to 1000 mg mL1. And then all the mixtures were kept at room temperature with gentle shaking for 3 h. Finally, the mixtures were centrifuged, and the absorbance of supernatants at 541 nm was determined by UVevis spectra. The hemolysis percent of the red blood cells was calculated as following: Hemolysis Percentage ¼ [(sample absorbance  negative control absorbance)/ (positive control absorbance  negative control absorbance)]  100. 2.6. Toxicology and histology analysis The biodistribution was administrated by intravenously injected the MS@S-NPs nanocomposite (10 mg kg1) into the mice with average weight of 20 g. Animal care and handing procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments. After 2 h, 24 h, 7d and 30d, the mice were sacrificed and the distribution of Gd ions in several organs, including heart, liver, spleen, lung, kidney, blood, muscle, intestine, and stomach, which was monitored by ICP analysis. These organs were lyophilized weighed before digestion in aqua fortis for about 2 h under heat treatment (80  C) for dissolution of the tissues. For histology studies, the mice were sacrificed for 30 days after administration through the tail vein. The main organs (heart, liver, spleen, lung, and kidney) were collected from two groups (control and test groups). And then, they were stained with hematoxylineosin (HE) for histopathological analysis under the optical microscope observation. As for blood analysis, healthy rats were intravenously administered with a single dose of the nanocomposite,


and several other rats were used as the controls. One month later, the rats were anesthetized and the blood was collected by an abdominal aortic blood method for blood biochemistry assay and hematology analysis. 2.7. MRI in vivo The tumor models were achieved by inoculating subcutaneously the 4T1 cells (murine breast cancer cells) to the nude mice. After anesthetized by chloral hydrate solution, 80 mL 3 mg mL1 Gd of MS@S-NPs was administrated intravenously into the tumorbearing mouse. For metabolism analysis, 80 mL 0.5 mg mL1 Gd of the nanocomposite was injected intravenously into the mouse instead. The T1-weighted images were acquired using a 1.5T human clinical scanner. 2.8. Drug delivery in vivo Balb/c nude mice (~20 g) were injected subcutaneously with the cell suspension containing H22 cells (murine hepatocellular carcinoma cells). When the size of tumors reached to 3e4 mm in diameter, the mice were randomly divided into four groups (4 mice per group), minimizing weight and tumor size differences. The mice were injected intravenously with 0.1 mL of saline (control group), MS@S-NPs, free DOX and DOX loaded MS@S-NPs ([DOX] ¼ 5 mg kg1), respectively. The mice were treated via the tail vein at day 1, 3, 7, 9. The tumor size of each group was measured using a caliper, and tumor volume was calculated using the following equation: tumor volume ¼ ab2/2 (a is the maximum diameter of tumor and b is the minimum diameter of tumor). Relative tumor volumes were calculated as V V1 0 (V0 was the tumor volume when the treatment was initiated). 2.9. Characterization The morphology of samples was characterized by a JEOL 2000eFX transmission electron microscopy (TEM). Xeray diffraction patterns (XRD) of the as-prepared samples were collected on a D8 ADVANCE diffractometer (Bruker Co., Germany) using Cu Ka (0.15406 nm) radiation. X-ray photoelectron spectroscopy (XPS) measurements were conducted with an ESCALAB MKII spectrometer (VG Co., United Kingdom). The energy-dispersive Xeray spectra (EDS) and SEM were inspected on a field emission scanning electron microscope (FESEM, S4800, Hitachi Co., Japan) equipped with an EDS (Jeol JXAe840). Zeta potential measurements were carried out on Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Fourier transform infrared (FTIR) spectroscopic analysis was performed on a Bruker Vertex 70 spectrometer. UVevis spectra were recorded on a VARIAN CARY 50 UV-VIS-NIR spectrophotometer. The fluorescence imaging was performed on a reconstructive Nikon TieS fluorescent microscope (Nikon, Tokyo, Japan). 3. Results and discussions 3.1. Synthesis of multifunctional envelope-type mesoporous silica nanoparticle (MEMSN) system Our strategy for the fabrication of the pH-responsive nanogated ensemble was schematically illustrated in Fig. 1. The MS was employed as robust nanocontainers for drug delivery by encapsulating DOX in the pores. To inhibit premature drug release, the outlet of MS was then capped by the S-NPs. Since the linker between the gated MS and S-NPs was the acid-labile acetal, the hydrolysis of the acetal group at acidic pH enabled the removal of the S-NPs, which simultaneously allow the escape of the entrapped


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Fig. 1. Schematic illustration of the MEMSN system for pH-responsive drug delivery and magnetic resonance imaging.

drug. The resultant nanocomposite is expected to show great promise in imaging guided therapy. To construct the nanocomposite, oleic acid-stabilized NaGdF4:Yb/Tm@NaGdF4 nanoparticles with an average size of ~5 nm were firstly synthesized (Figs. S1 and S2). The coreeshell design endows these nanoparticles with higher relaxivity for MRI [24,25]. The resultant nanoparticles were then coated with an ultra thin TaOx layer, and modified with carboxyethylsilanetriol (CTES), which allows

nanoparticles to possess good water solubility, biocompatibility and carboxy groups on their surface. The TaOx is biocompatible, and has been clinically used as stents. The successful coating of TaOx layer on the S-NPs was confirmed by the recorded Ta signals in Xray photoelectron spectrometry (XPS) and energy-dispersive X-ray (EDX) analysis. (Figs. S3 and S4). Secondly, highly dispersed MS with uniform particle size was prepared according to the previously reported method [26]. In this

Fig. 2. TEM images of MS (a) and MS@S-NPs (b). Small-angle powder X-ray diffraction of MS before (yellow) and after (purple) capping (c). Nitrogen adsorptiondesorption isotherms and pore size distribution (inset) curves before and after capping (d). Cumulative release curves of DOX from MS@S-NPs (e) and MS (f) in buffer solutions at different pH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. The UVevis absorption spectra to detect the presence of hemoglobin in the supernatant of MS@S-NPs (A), and the concentration-dependent hemolysis of MS@S-NPs, inset: photographic images for direct observation of hemolysis (B). Viability of HeLa cells in presence of the samples with varied concentrations (C). Evaluation of the death pathways of HeLa cells treated with free DOX (b, c, d) and DOX loaded MS@S-NPs (e, f, g) at different time periods (D): control (a), 0.5 h (b, e), 12 h (c, f) and 48 h (d, g).

case, cetyltrimethylammonium bromide (CTAB) was utilized as a template agent to construct mesophase in the presence of nonionic surfactant F127 [27]. The presence of F127 not only enables the well controlled growth of the nanoparticle grain, but also serves as a dispersion agent to prevent their aggregation (Fig. S7D). Carboxylic groups were grafted onto the outer surfaces of the MS using an established protocol (Fig. S5) [17]. The acetal containing linker was then anchored on the carboxylic acid modified MS through the reaction with excess 3,9-bis(3-aminopropyl)-2,4,8,10-tetraoxaspiro [5.5]undecane. The surface-functionalized MS with void pores can be harnessed as a reservoir to encapsulate the drugs into the pores after the surfactant was extracted via ion exchange. The TEM image (Fig. 2a), small angle X-ray diffraction pattern (Fig. 2c) and nitrogen adsorptionedesorption measurement (Fig. 2d) demonstrated typical mesoporous channels of the MS. The fluorescamine test asserted the existence of free amine from the attached acetal linker (Fig. S7C) [28]. Finally, to create the pH-responsive nanogated ensemble, the SNPs were reacted with the resulted MS through a classic method [29]. It is noted that the S-NPs capping led to an obvious decrease in the intensity of the (100) XRD peak (Fig. 2c), the surface area, as well as the pore diameter of MS (Fig. 2d). The pictures of MS@S-NPs in various solutions showed their excellent colloidal stability (Fig. S6). It is worthy to be noted that the success of such assembled nanocomposite depends strongly on the specific surface properties of MS and S-NPs. On the other hand, the pH value is another key factor requiring consideration to realize the controlled drug delivery. In a control experiment, when CTAB inside the functionalized MS was removed by ion exchange with NH4NO3/C2H5OH solution (pH ¼ 4.2) instead of NaCl in methanol solution [30],

almost no S-NPs could be observed on the surface of MS followed the same experimental protocol after that (Fig. S8b). It indicated that the low pH solution have made the acetal hydrolyzed, hence leading to the absence of anchoring sites for S-NPs. Furthermore, the variation of zeta potential and hydrodynamic radius throughout the process was also investigated (Fig. S7A, B). As a result of PEGylation, the zeta potential of the nanocomposite became slightly negatively charged. The PEG is known to counteract the hydrophobic and electrostatic interaction between the nanoparticles and plasma proteins or macrophages, leading to prolonged blood circulation time, which may increase their probability of reaching the tumor tissues. Moreover, the slightly negatively charged nanoparticles were reported to show the best tumor uptake and penetration ability [31], which would easily target at tumor site via EPR effect. 3.2. pH-responsive drug release The ideal drug delivery systems require “zero release” before reaching the specific position, and thus the product was dispersed in buffer solutions to evaluate their release performance in a biological model at different pH values (Fig. 2e). In the physiological condition (pH ¼ 7.4), there were negligible DOX release, which signified a good capping efficiency of S-NPs for encapsulating the drug molecules against the undesired leaching problem. Notably, the DOX release was markedly increased under acidic conditions, more than 60% and 90% of DOX was released at pH 5.0 and pH 2.0, respectively. Such distinct release profile, which depends strongly on the pH, suggested that DOX molecules were released from the nanocomposite due to the pH-labile linker between the MS and S-


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Fig. 4. Microscopic imaging of HeLa cells after incubated with DOX loaded MS@S-NPs for 0, 0.5, 3, 6 and 12 h at 37  C. Blue DAPI fluorescence (a2, b2, c2, d2, e2) and red DOX fluorescence (a3, b3, c3, d3, e3) were recorded respectively. There are bright-field images (a1, b1, c1, d1, e1), emerged images of dark field (a4, b4, c4, d4, e4), and emerged images of the corresponding cells (a5, b5, c5, d5, e5) as well. All scale bars are 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

NPs, which had unlocked the gate. We further compared the morphology of the nanocomposite after soaked in buffer solutions at different pH (Fig. S8c and S8d). As seen from the images, upon decreasing the pH, S-NPs on the surface was removed from the MS, owing to the hydrolysis of the acetal between MS and S-NPs. In order to further ascertain the above pH-driven controlled delivery, the additional control experiments were carried out by soaking DOX loaded MS without capping in different buffer solutions. The MS exhibited ~10% DOX release at pH 7.4, meanwhile, the release at pH 5.0 was found to be less than 20% (Fig. 2f). The similar release process at different pH indicated that the absorbed drug molecules escaped from the unguarded pores uncontrollably, which is a typical release profile of the ungated MS [17,23]. In comparison, the significantly higher DOX release from the MS@S-NPs at lower pH can be ascribed to the well-reported hydrolyzation of acetal groups at acidic conditions, in which the capped S-NPs have been removed. Prior to using the nanocomposite for intracellular drug delivery and imaging, the hemolytic and MTT assay were carried out to test their potential for therapeutic applications. The hemolytic assay (Fig. 3A and B) demonstrated that extremely low hemolysis of red blood cells could be detected upon the maximal experimental concentration (1000 mg mL1), indicating the good blood compatibility of the nanocomposite and the feasibility for further investigation. Compared with the blank control in the MTT assay (Fig. 3C), the nanocomposite exhibited negligible cytotoxicity against HeLa cells, and the results were in accordance with the hemolytic assay. On the other side, after 48 h incubation, DOX loaded

nanocomposite exhibited comparable cytotoxic effect against cells compared with free DOX. The difference in cell viabilities became accumulatively distinct especially at higher tested doses, in which the DOX loaded nanocomposite showed significant higher cytotoxicity and a clear advantage over free DOX. We attribute the phenomenon to the different cellular uptake route between DOX loaded nanocomposite (endocytosis-mediated) and free DOX (molecule diffusion) [32]. With increasing the concentration, more and more DOX loaded nanocomposite entered the cancer cells released DOX, and consequently killed the cells at enhanced efficacy. Furthermore, in order to evaluate the death mechanism of HeLa cells on chemotherapy, Annexin V-FITC and PI double staining was performed by flow cytometry (FCM) and fluorescenceactivated cell sorting (FACS) approaches (Fig. 3D and S10). Both DOX loaded nanocomposite and free DOX induced obvious apoptosis and necrosis of HeLa cells after incubation for reasonably long time periods. As extending the incubation time to 48 h, the DOX loaded nanocomposite demonstrated considerably enhanced fraction of cellular apoptosis and necrosis effect. This enhancement effect resulted from the integration of the nanocomposite and drug molecules, which can generate synergistic biological effects between the drug and the MS towards cancer cells [33]. Thus, we anticipated that the DOX loaded nanocomposite with pH-responsive property could be a promising nanoplatform for controlled drug delivery in acidic intracellular organelles, such as endosomes and lysosomes, within cancer cells. The release process of DOX from the as-prepared nanocomposite in HeLa cells was

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Fig. 5. TEM images of the HeLa cells after incubated with MS@S-NPs (a1, a, b) and DOX loaded MS@S-NPs (c1, c1a, c, c2), respectively. In the images, blue arrows point the MS and pink arrows point the S-NPs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

monitored by fluorescence microscope (Fig. 4). It can be seen that there was few red fluorescence of DOX in the first 0.5 h. Comparatively, the cells showed highly distributed fluorescence of DOX with growing intensity as time passed, suggesting that the DOX from the nanocomposite was released in a slow and sustained way. The emerged images of the nuclei and DOX, as well as with the bright field, were evident in the whole intracellular region. While prolonging the incubation time to 12 h, the nucleoplasm of the cells became much less compact, and even fragmented compared with the control, which indicated the cell apoptosis owing to the toxicity of the drug released from the nanocomposite. Further, we proved this internalization process by Bio-TEM images with high resolution (Fig. 5). In the beginning, the nanocomposite was phagocytized by cells, and trapped into the intracellular lysosome, where the nanocomposite has been hydrolyzed into individual MS and S-NPs as mentioned above. And then, the resultant nanoparticles escaped from the lysosome into the cytoplasm. Finally, the S-NPs were completely separated from the MS, as observed from the enlarged images, which indicated the pH-responsive property of the acetal linker between MS and S-NPs. These results were in accordance with the above-mentioned morphology changes at different pH. Interestingly, the optical images also showed significant difference in cellular morphology treated separately: the cell treated with drug loaded nanocomposite began to shrink, and nuclear membranes became indistinguishable, showing a signal of cell apoptosis. On the contrary, the cell treated with unloaded nanocomposite was in a shape with more regular dimensions, as the complete and clear structure of cellular organelles can be identified [34]. This phenomenon has

been attributed to the fact that the nanocomposite exhibited negligible cytotoxicity against the cells, however, once loaded with DOX, the released drug from the nanocomposite would induce cell death, well consistent with the fluorescence images. 3.3. Contrast enhanced MRI and chemotherapeutic effect Owing to their paramagnetic property arising from the unpaired electrons, Gd3þ could efficiently alter the relaxation times of surrounding water protons. Thus, introducing gadolinium into the nanocomposite further makes them to be promising T1-MRI contrast agent [35,36]. The T1-weighted magnetic resonance (MR) images were performed at a 1.5 T human clinical scanner, and the images were brightened with the increase in Gd3þ concentration (Fig. 6a). The resultant agent had a relaxivity value of 6.9 mM1 s1 in MR imaging (Fig. 6c), which was comparable with other contrast agents [1]. Despite the contrast enhanced MRI performance, the assembling process was also appealing since it provided a facile way to increase the overall imaging performance per particle. And then, the fluorescence intensity, MR images were introduced at the same concentration (Fig. S11). The signals from MS@S-NPs were significantly amplified than S-NPs. Moreover, the MR images of the cells, which incubated with MS@S-NPs and S-NPs were also demonstrated, respectively. An enhancement of MR signal was observed with MS@S-NPs, which is in agreement with the above-mentioned results. These inspiring observations indicate that the nanocomposite could be applied as an efficient contrast agent for MRI. Combined


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Fig. 6. T1-weighted MR images at varied concentrations (a). In vivo T1-weighted MR images of the tumor-bearing mouse before and after injection intravenously, the white circles point the tumor sites (b). The relaxation rate of MS@S-NPs versus different concentrations of Gd3þ (c). The tumor growth curves (d) and the corresponding survivable mouse (e) of different groups after various treatments.

with the EPR effect, the nanocomposite could be used as a markedly excellent imaging agent at tumor site. The solid tumor has abnormal vasculature with leakiness, which combined with insufficient lymphatic drainage, so that the nanocomposite could have a good chance of accumulating in the tumor tissues via the EPR effect [37]. The EPR effect has been extensively used in the nanomedicine field, for instance, PEGylated liposomal doxorubicin (Doxil), nonPEGylated liposomal doxorubicin (Myocet), and non-PEGylated liposomal daunorubicin (DaunoXome) are routinely used in patients, and many others are currently in (pre-) clinical trials [38]. On the other hand, MRI is considered to be superior for acquiring high spatial resolution anatomical and physiological images in diagnostic radiology to differentiate between normal and abnormal states in tissues. Correspondingly, we evaluated the EPR effect of the nanocomposite by MRI in a tumor-bearing mice model. After intravenous injection, the T1 signals from tumor sites showed a trend of increase and became quite strong at 3 h (Fig. 6b). Driven by the concept of imaging guided therapy, we further evaluated the chemotherapeutic efficacy of the MEMSN system by monitoring the tumor growth rates in terms of tumor volume changes (Fig. 6d and e). Regarding the groups of control, MS@S-NPs and free DOX, the tumor displayed a time-related increase in volume. In striking contrast, the DOX loaded MS@S-NPs group produced remarkable inhibition efficiency of tumor growth, and the treated mice survived over 30 days without any significantly observed tumor growth. The animal studies suggested that fusion of MRI and chemotherapeutics would enable online imaging for detection of diseases, image-guided drug delivery and treatment.

3.4. Metastasis and toxicology in vivo The similar construction has been reported recently [39,40], however, compared with the permanent crosslinking of the nanocomposite in these papers, the pH-responsive nanogated ensemble mentioned here showed more advantages. As silica is generally accepted as a non-toxic and biodegradable agent [41], and the nanoparticle with size less than 10 nm is recently demonstrated to have more efficient clearance ability [42], we anticipated that the decomposition of the nanocomposite under acidic condition would lead to a faster and easier clearance of the nanocomposite, which can effectively reduce potential serious adverse effects in patients. As leaching of free metal ions from the nanocomposite could cause high toxicity towards natural organs and tissues, the toxicology experiment was carried out both in vitro and in vivo. First of all, one-month dialysis experiments against PBS, FBS and DMEM were carried out. Nearly no leaching of free metal ions from the nanocomposite could be detected by ICP analysis. Next, a single dose of the nanocomposite was intravenously injected into the mice, and no abnormalities in eating, drinking, grooming, activity, exploratory behavior, urination, or neurological status were observed. And then the mice were sacrificed at different time period, the content of gadolinium element was monitored by ICP analysis. As seen from Fig. 7a, the accumulation in reticuloendothelial system like liver and spleen were dominant post-injection for 24 h. The accumulation in these organs were noticeably cleared from the body within one week, and then completely cleared in a month. Moreover, the metastasis process was also

Y. Chen et al. / Biomaterials 60 (2015) 111e120


Fig. 7. Time dependent biodistribution of Gd3þ in mice (a). The body weight of the mice versus time with and without intravenously injection of MS@S-NPs (b). Histological changes in heart, liver, spleen, lung and kidney of the mice one month after intravenous injection of a single dose of MS@S-NPs solution. These organs are stained with H&E and observed under microscope. The control shows the organ images of the mouse without injection (c). In vivo MR imaging of the mice after intravenous injection of MS@S-NPs at different time period, the red circles point the liver (d). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

monitored by MRI. In accordance with the results, we found that the MR signal in the organs (mostly in liver) almost got back to preinjection one week later (Fig. 7d). This favorable biocompatibility of the nanocomposite revealed their high potential for further applications. Considering that the degradation of the nanocomposite might induce subsquent damage to the organs, the in vivo long-term toxicity of the nanocomposites was further examined. After intravenous injection of a single dose of the nanocomposite, the mice remained healthy over one-month period, the body weight of the treated group increased gradually similar to that of the control group, indicating that the mice continue to mature without any significant toxic effects (Fig. 7b). One month later, the mice were euthanized and several main organs were stained with hematoxylin and eosin (H&E) for histology analysis (Fig. 7c). There was nearly no distinct tissue damage, inflammation, or lesion post injection compared with the control group. Furthermore, the biochemistry results of the nanocomposite including aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP), blood urea nitrogen (BUN), and creatinine (CRE), which closely related to the liver and kidney function, were demonstrated

(Table S1). Compared with the control group, there was no sign of injury in liver or kidney. In addition, the complete blood tests showed no obvious interference with the physiological regulation of haem or immune response. Although the in vivo metastasis and toxicology results shown here are preliminary, the nanocomposite presented great promise as non-toxic and biocompatibility nanomaterials for applications in biological medicine. 4. Conclusion In summary, we prepared an intelligent MEMSN system for drug delivery and MRI, which combined traits of pH-responsiveness, non-toxicity and biological specificity. The resulted system was believed to be able to efficiently transport DOX into the cancer cells, released rapidly in lysosomes and endosomes due to the acidic situation. The nanogated drug release system was highly efficacious for cancer therapy both in vitro and in vivo. After intravenous injection into the murine model, such nanocomposite, which accumulated in several organs, was degraded into apparently non-toxic products within a few days. Despite the enhanced therapeutic efficacy, the nanocomposite would accumulate at the tumor site


Y. Chen et al. / Biomaterials 60 (2015) 111e120

through the so-called EPR effect, which was visualized by MRI. Therefore, such novel nanosystem should be of great significance in the future development of highly efficient and tumor targeted drug delivery vehicles for cancer chemotherapy. Acknowledgments Financial support acknowledged.






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Multifunctional envelope-type mesoporous silica nanoparticles for pH-responsive drug delivery and magnetic resonance imaging.

A novel multifunctional envelope-type mesoporous silica nanoparticle (MEMSN) system combining the merits of pH-responsiveness, non-toxicity and biolog...
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