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Jul 29, 2014 - The core–shell Fe3O4@mSiO2 nanomaterials are synthesized as the host, and DOX is utilized as a model anticancer drug for convenient ...
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pH-Responsive Magnetic Core−Shell Nanocomposites for Drug Delivery Chunyu Yang, Wei Guo, Liru Cui, Na An, Ting Zhang, Huiming Lin,* and Fengyu Qu* Department of Photoelectric Band Gap Materials, Key Laboratory of Ministry of Education, College of Chemistry and Chemical Engineering, Harbin Normal University, Harbin 150025, China S Supporting Information *

ABSTRACT: Polymer-modified nanoparticles, which can load anticancer drugs such as doxorubicin (DOX), showing the release in response to a specific trigger, have been paid much attention in cancer therapy. In our study, a pH-sensitive drug-delivery system consisting of Fe3O4@mSiO2 core−shell nanocomposite (about 65 nm) and a β-thiopropionatepoly(ethylene glycol) “gatekeeper” (P2) has been successfully synthesized as a drug carrier (Fe3O4@mSiO2@P2). Because of the hydrolysis of the βthiopropionate linker under mildly acidic conditions, Fe3O4@mSiO2@P2 shows a pH-sensitive release performance based on the slight difference between a tumor (weakly acid) and normal tissue (weakly alkaline). And before reaching the tumor site, the drug-delivery system shows good drug retention. Notably, the nanocomposites are quickly taken up by HeLa cells due to their small particle size and the poly(ethylene glycol) modification, which is significant for increasing the drug efficiency as well as the cancer therapy of the drug vehicles. The excellent biocompatibility and selective release performance of the nanocomposites combined with the magnetic targeted ability are expected to be promising in the potential application of cancer treatment.

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supermolecule nanovalves (e.g., β-CD, macrocyclic molecule cucurbituril) have been broadly employed as “gatekeepers”, which show well-controlled release performance.12−18 The controlled-release process can be regulated either by external stimuli such as temperature, light, and electrostatic and magnetic actuation or by internal stimuli such as pH and enzymes. For example, Moon and coworkers have developed a theranostic system based on gold nanocages and phase-change materials (1-tetradecanol) with unique features for photoacoustic imaging and controlled release.19 And Li et al. have successfully demonstrated that hollow mesoporous silica nanoparticles modified with a spiropyran-containing lightresponsive copolymer can be used for light-controlled drug release.20 However, the weak tissue penetration and complicated operation of external stimuli limit their practical applicability.21−24 On the other hand, the internal stimulus seems to be more practical and possible compared to the external stimulus response. For instance, Feng and coworkers have developed a controlled process by using mesoporous silica nanoparticles as hosts capped with acid-labile acetal-grouplinked gold nanoparticles as pH-responsive agents.25 However, a controlled drug release system, which depends only on the internal stimulus, cannot achieve ideal results. With combined internal and external stimuli, the multifunctional drug-delivery

INTRODUCTION Cancer, known as a major cause of mortality worldwide, is a vast group of diseases produced by rapid unregulated cell growth. Chemotherapy as an effective drug treatment is intended to kill cancer cells in individuals with various forms of carcinoma.1 However, chemotherapy always induces a huge side effect besides its efficacy, originating from little specific discrimination between a normal cell and a cancer cell.2 To overcome this problem, a promising approach to effective cancer therapy is systemic nanomedicine using anticancer drugs, which are able to trigger apoptosis by activating key elements of the apoptosis program.3 Currently, multifunctional nanoparticles, including liposomes, dendrimers, polymers, micelles, DNA, ceramics, and even virus capsids, have been employed as platforms to regulate drug release.4−9 In particular, the large surface area, high pore volume, and uniform and tunable pore size of the mesoporous silica nanoparticles (MSNs) have allowed them to be considered as one of the most important candidates for drug carriers. Furthermore, MSNs supply additional biocompatibility and easily modified surface properties, showing a high drug loading amount.10 Furthermore, multiple efforts have been judged to adjust the release process to obtain the desirable controlled release performance. Interestingly, the concept of stimulus-responsive gatekeeping was introduced to regulate the cargo release and to optimize the application of MSNs in nanomedicine.11 At present, some inorganic nanoparticles (e.g., CdS, Fe3O4, and Au), organic polymers (e.g., PAMAM and PNIPAM), and © 2014 American Chemical Society

Received: May 13, 2014 Revised: July 29, 2014 Published: July 29, 2014 9819

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dissolved in 200 g of 1-octadecene (90%) at room temperature. The reaction mixture was heated to 320 °C at a constant heating rate of 3.3 °C min−1 and then kept at that temperature for 30 min. When the reaction temperature reached 320 °C, a severe reaction occurred and the initial transparent solution became turbid and brownish black. The resulting solution containing the nanocrystals was then cooled to room temperature, and 500 mL of ethanol was added to the solution to precipitate the nanocrystals, which were further collected by centrifugation and then dispersed in chloroform. Synthesis of Fe3O4@mSiO2 Nanoparticles. In a typical procedure, 0.5 mL of the Fe3O4 nanocrystals in chloroform (10 mg mL−1) was poured into 8 mL of a 0.2 M aqueous CTAB solution, and the resulting solution was stirred vigorously for 30 min. The formation of an oil-in-water microemulsion resulted in a turbid brown solution. Then, the mixture was held at 60 °C for 30 min to evaporate the chloroform, resulting in a transparent black Fe3O4/CTAB solution. Then, 20 mL of distilled water was added to the obtained black solution, and the pH value of the mixture was adjusted to 8 to 9 by using 0.1 M NaOH. After that, 100 μL of 20% TEOS in ethanol was injected six times in 30 min intervals. The reaction mixture was reacted for 24 h under vigorous stirring. The obtained Fe3O4@mSiO2 NPs were centrifuged and rinsed with ethanol repeatedly to remove the excess precursors and CTAB molecules and then dispersed in ethanol (8 mL). Synthesis of Polymer P2 with Different Molecular Weights. P2 was synthesized following a literature procedure.36 To an ice-cold solution of poly(ethylene glycol) (Mn ≈ 4000/6000) (16.92 g/33.84 g, 0.00846 mol) and triethylamine (3.54 mL, 0.025 mol) in 35 mL/50 mL of dry dichloromethane, a solution of freshly distilled acryloyl chloride (1.71 mL, 0.021 mol) in 10 mL of dry dichloromethane was added dropwise under an inert atmosphere, and then the reaction mixture was stirred at rt for 12 h. The final residue was dried in vacuum at 40 °C overnight. It has been named P1-4000/6000. A solution of monomer MPTMS (0.062 g, 0.3 mmol) in 5 mL of degassed DMAC was added to a 0.1 wt % Me2PPh solution in DMAC (5.0 μL), and the reaction mixture was cooled in an ice bath. Then to the cold solution monomer P1-4000/6000 (0.534 g/0.794 g, 0.13 mmol) was added, and the reaction mixture was allowed to stir under constant Ar flow in the same ice bath for 12 h. Then the product was collected and named P2-4000/6000. Drug Loading. Fe3O4@mSiO2 (60 mg) and DOX (3 mg) were added to the ethanol solution (3 mL) and stirred at 25 °C for 12 h. And then 4.29, 8.58, and 17.16 mmol of P2-4000/6000 were added to the mixed solution. The obtained solids (named DOX-Fe3O4@ mSiO2@P2-4000/6000-1, DOX-Fe3O4@mSiO2@P2-4000/6000-2, and DOX-Fe3O4@mSiO2@P2-4000/6000-3, respectively) were centrifuged and washed several times with ethanol solution. The loading amount of DOX was determined by the UV/vis spectroscope at 480 nm. The loading efficiency (LE wt %) of DOX can be calculated by using eq 1. The experiment was repeated three times.

system, receiving the advantages of the two, would exhibit a more desired release performance as well. Magnetite nanoparticles (Fe3O4), as one of the most important magnetic materials, has aroused great interest in low-field magnetic separation, lithium ion batteries, mimetic enzymes, a dual imaging probe for cancer, and a two-photon fluorescence indicator.26−29 Furthermore, Fe3O4 nanoparticles enable us to induce hyperthermia effects when placed in an alternating magnetic field30−32 for tumor thermotherapy. For example, Wang et al. have synthesized a bicontrollable drugrelease system with PAH/PSS multilayers on Fe3O4/mSiO2, showing pH-controllable and magnetic target behavior.33 Liu et al. have developed a magnetic and reversible pH-responsive MSN-based nanogated ensemble. In the ensemble, superparamagnetic Fe3O4 nanoparticles are used as the gatekeeper capping the outlet of the mesoporous silica via an acid-labile boronate ester linker.34 In the present study, we have developed β-thiopropionatepoly(ethylene glycol)-modified Fe3O4@mSiO2 nanocomposites as the drug-loading system, revealing the controlled release based on the low pH value in cancer/tumors. The core−shell Fe3O4@mSiO2 nanomaterials are synthesized as the host, and DOX is utilized as a model anticancer drug for convenient detection and in vitro experiments. After the drug loading, pHsensitive β-thiopropionate-poly(ethylene glycol) (P2, Figure S1) is employed to graft outside of Fe3O4@mSiO2 as the blocking caps to inhibit premature drug release (DOX-Fe3O4@ mSiO2@P2). Because of the hydrolysis of the ester bond in P2 in an acidic environment, DOX-Fe3O4@mSiO2@P2 is expected to block the pore in a neutral or alkaline environment and to open the pore in an acidic environment (pH 5.8). That makes the nanocarriers respond to the slight difference between the tumor and the normal tissue due to their different physiological environments. Moreover, the small particle size associated with the poly(ethylene glycol) fragment coating causes the DOXFe3O4@mSiO2@P2 nanovalves to show improved dispersion, stability, biocompatibility, and fast uptake by cells for cancer therapy.

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MATERIALS AND METHODS

Materials. Unless specified, all of the chemicals used were analytical grade and used without further purification. Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), doxorubicin hydrochloride (DOX), sodium oleate, oleic acid, 1octadecene, (3-mercaptopropyl)trimethoxysilane (MPTMS), acryloyl chloride, 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi1H-benzimidazole, trihydrochloride (Hoechst 33342), dimethylphenylphosphine, N,N-dimethylacetamide, and poly(ethylene glycol) (Mn ≈ 4000, 6000) were obtained from Aladdin (China). Ferric trichloride hexahydrate (FeCl3·6H2O), ethanol, n-hexane, and triethylamine were purchased from Tianjin Chemical Corp. of China. Synthesis of an Iron−Oleate Complex. In a typical synthesis of an iron−oleate complex, 10.8 g of iron chloride (FeCl3·6H2O, 40 mmol) and 36.5 g of sodium oleate (120 mmol, 95%) were dissolved in a solvent mixture composed of 80 mL of ethanol, 60 mL of distilled water, and 140 mL of hexane. The resulting solution was heated to 70 °C and kept at that temperature for 4 h. When the reaction was completed, the upper organic layer containing the iron−oleate complex was washed three times with 30 mL of distilled water in a separatory funnel. After washing, hexane was evaporated, resulting in an iron−oleate complex in a waxy solid form. Synthesis of Fe3O4 Nanoparticles. Following a literature procedure, Fe3O4 nanoparticles were prepared.35 The iron−oleate complex (36 g, 40 mmol) and oleic acid (5.7 g, 20 mmol, 90%) were

LE wt% = × 100%

m(original DOX) − m(residual DOX) m(Fe3O4 @mSiO2) + m(original DOX) − m(residual DOX) + m(P2)

(1)

Drug Release. The gating protocol was investigated by studying the release profiles of DOX from the DOX-Fe3O4@mSiO2@P2-4000/ 6000 in a pH 5.8 or 7.4 phosphate buffer solution. Briefly, DOXFe3O4@mSiO2@P2-4000/6000 was dispersed in 5 mL of media solution and sealed in a dialysis bag (molecular weight cutoff 8000), which was submerged in 20 mL of media solution. At selected intervals, the solution was taken out to determine the release amount by UV. Cell Culture. HeLa cells (cervical cancer cell line) were grown in a monolayer of Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Tianhang Bioreagent Co., Zhejiang) and penicillin/streptomycin (100 U mL−1 and 100 μg mL−1, respectively, Gibco) in a humidified 5% CO2 atmosphere at 37 °C. 9820

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Confocal Laser Scanning Microscopy (CLSM). To determine the cellular uptake, HeLa cells were cultured in a 12-well chamber slide with one piece of cover glass at the bottom of each chamber in the incubation medium (DMEM) for 24 h. The cell nucleus was labeled with Hoechst 33342. DOX-Fe3O4@mSiO2@P2-4000-3 was added to the incubation medium at a concentration of 100 μg mL−1 for 6 h of incubation in 5% CO2 at 37 °C. After the medium was removed, the cells were washed twice with PBS (pH 7.4) and the cover glass was visualized under a laser scanning confocal microscope (FluoView FV1000, Olympus). Cell Viability. The viability of cells in the presence of nanoparticles was investigated using a 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. The assay was performed in triplicate in the following manner. For the MTT assay, HeLa cells were seeded into 96-well plates at a density of 1 × 104 per well in 100 μL of the medium and grown overnight. The cells were then incubated with various concentrations of Fe3O4@mSiO2s and Fe3O4@mSiO2@P24000-3s for 24 h. Afterwards, cells were incubated in a medium containing 0.5 mg mL−1 MTT for 4 h. The precipitated formazan violet crystals were dissolved in 100 μL of 10% SDS in 10 mmol HCl solution at 37 °C overnight. The absorbance was measured at 570 nm with a multidetection microplate reader (SynergyTM HT, BioTek Instruments Inc, USA). Characterization. Powder X-ray patterns (XRD) were recorded on a Siemens D 5005 X-ray diffractometer with Cu Kα radiation (40 kV, 30 mA). The nitrogen adsorption/desorption, surface areas, and median pore diameters were measured using a Micromeritics ASAP 2010 M sorptometer. The surface area was calculated according to the conventional BET method, and the adsorption branches of the isotherms were used for the calculation of the pore parameters using the BJH method. Fourier transform infrared (FTIR) spectra were recorded on a PerkinElmer 580B infrared spectrophotometer using the KBr pellet technique. A UV−vis spectrum was used to describe the amount of drug released (Shimadzu UV2550 spectrophotometer). Transmission electron microscopy (TEM) images were recorded on a TECNAI F20. Zeta potential and dynamic light scattering (DLS) measurements were carried out with ZetaPALS zeta potential analyzer. The magnetic properties of samples were characterized with a vibrating sample magnetometer (Lake Shore 7410).

an average diameter of about 20 nm in size. Fe3O4@mSiO2 reveals the obvious Fe3O4 core encapsulated by a silica shell (20 nm) with a wormlike porous structure (Figure 1B) which agrees with the corresponding XRD analysis (Figure S2). As can be seen in Figure 1C, the polymer layers of the Fe3O4@ mSiO2@P2-4000-1 surface result in the rough surface and less dispersion of these nanoparticles. Additionally, the hydrodynamic diameter and zeta potential of the samples are measured and summarized in Table 2. As displayed in Table 2, the diameter of Fe3O4@mSiO2 centers at 82.0 nm is larger than that observed from TEM, resulting from the hydrate layer of Fe3O4@mSiO2 in an aqueous environment. After the graft of P2, the diameters of Fe3O4@mSiO2@P2-4000-1, Fe3O4@ mSiO2@P2-4000-2 and Fe3O4@mSiO2@P2-4000-3 add up to 98.5, 110.1, and 148.7 nm, respectively. In addition, the zeta potential was further used to monitor the surface change between Fe3O4@mSiO2 and Fe3O4@mSiO2@P2-4000s. The zeta-potential value of Fe3O4@mSiO2 increases from −15.01 ± 1.17 to −5.45 ± 2.36 mV (Fe3O4@mSiO2@P2-4000-1), −3.09 ± 3.22 mV (Fe3O4@mSiO2@P2-4000-2), and −1.62 ± 3.42 mV (Fe3O4@mSiO2@P2-4000-3), respectively (Table 2), which is possibly attributed to the reduction of surface Si− OH from Fe3O4@mSiO2@P2-4000s substituted by neutral P2. On the basis of the above investigation, it is shown that P2 has been successfully grafted onto the Fe3O4@mSiO2 surface. The X-ray diffraction patterns (XRD) collected from Fe 3 O 4 @mSiO 2 , DOX-Fe 3 O 4 @mSiO 2 @P2-4000-1, DOXFe3O4@mSiO2@P2-4000-2, and DOX-Fe3O4@mSiO2@P24000-3 are shown in Figure S2. As illustrated in Figure S2, all samples reveal only one diffraction peak at about 2 θ = 2.26°, suggesting that they possesses the ordered mesoporous structure. It is clearly observed that the relative intensities of the peaks of the pattern collected from DOX-Fe3O4@mSiO2@ P2-4000s were obviously reduced compared to that of Fe3O4@ mSiO2 without drug loading and P2 grafting. Moreover, the larger the amount of P2 grafted onto Fe3O4@mSiO2, the lower the diffraction intensity of DOX-Fe3O4@mSiO2@P2-4000s, which is consistent with the previous report.2 Figure S3 shows the wide-angle XRD patterns of Fe3O4 and Fe3O4@mSiO2 nanoparticles, respectively. As displayed in Figure S3, all of the diffraction peaks of Fe3O4 nanoparticles are in good agreement with that of standard Fe3O4 (JCPDS card no. 65-3107). The typical diffraction of Fe3O4 also can be found in the XRD pattern of Fe3O4@mSiO2. Moreover, an additional diffraction peak at 22.2° appears due to the amorphous mSiO2 structure. To verify the successful grafting of P2 on Fe3O4@mSiO2, FT-IR spectroscopy was monitored to study the organic and inorganic components of the samples. The corresponding FTIR spectra of PEO-4000, P2-4000, Fe3O4@mSiO2, and Fe3O4@ mSiO2@P2-4000 are shown in Figure 2. For PEO-4000, the absorption band at 1110 cm−1 is assigned to the C−O−C stretching vibrations, and other two peaks at 2945 and 2888 cm−1 are associated with the C−H stretching vibrations (Figure 2A). In addition, the IR bands of 1724 and 691 cm−1 ascribed to v(CO) and v(C−S) appear in P2, demonstrating that P2 has been successfully grafted to P2-4000. As can be seen in Figure 2B, the obvious absorption band at 1086 cm−1 shows the Si−O−Si framework of Fe3O4@mSiO2. After P2-4000 grafting, the absorption peaks at 1724 cm−1 assigned to the CO stretching vibration of P2-4000 also can be found in Fe3O4@ mSiO2@P2-4000, confirming that P2-4000 has been successfully grafted to Fe3O4@mSiO2.

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RESULTS AND DISCUSSION Morphology and Structure. TEM was used to display the structure of the samples. In Figure 1A, Fe3O4 nanoparticles show a uniform and well-dispersed spherical morphology with

Figure 1. TEM images of (A) Fe3O4, (B) Fe3O4@mSiO2, and (C) Fe3O4@mSiO2@P2-1. 9821

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Figure 2. FTIR spectra of (A) PEO-4000 and P2-4000 and (B) Fe3O4@mSiO2 and Fe3O4@mSiO2-P2-4000.

The pore structure and related textural properties of Fe3O4@ mSiO2 and DOX-Fe3O4@mSiO2@P2-4000s were followed by nitrogen adsorption−desorption measurements. The corresponding adsorption−desorption isotherms and the pore size distribution curves are displayed in Figure S4. From Figure S4A, Fe3O4@mSiO2 displays a typical IV adsorption isotherm and a steep capillary condensation step at a relative pressure of P/P0 = 0.2−0.4. The typical H4 hysteresis loop is observed, showing the mesoporous structure of Fe3O4@mSiO2. As depicted in Figure S4A, there are much smaller uptakes of nitrogen for DOX-Fe3O4@mSiO2@P2-4000s if taking its counterpart (Fe3O4@mSiO2) as a comparison. Additionally, the surface area (SBET) and pore volume are reduced from 326 m2 g−1 and 0.285 cm3 g−1 for Fe3O4@mSiO2 to 157 m2 g−1 and 0.161 cm3 g−1 for DOX-Fe3O4@mSiO2@P2-4000-1, 103 m2 g−1 and 0.137 cm3 g−1 for DOX-Fe3O4@mSiO2@P2-4000-2, and 84.0 m2 g−1 and 0.117 cm3 g−1 for DOX-Fe3O4@mSiO2@ P2-4000-3 (Table 1). These results are expected due to the

Figure 3. Magnetization curves of four samples at room temperature: (a) Fe3O4, (b) Fe3O4@mSiO2@P2-4000-1, (c) Fe3O4@mSiO2@P24000-2, and (d) Fe3O4@mSiO2@P2-4000-3.

Table 1. Pore Parameters and Loading Efficiency of the Samples samples Fe3O4@mSiO2 DOX-Fe3O4@mSiO2@ P2-4000-1 DOX-Fe3O4@mSiO2@ P2-4000-2 DOX-Fe3O4@mSiO2@ P2-4000-3

BET (m2 g−1)

Vp (cm3 g−1)

pore size (nm)

LE (wt %)

326 157

0.285 0.161

2.42 2.38

2.74 ± 0.5

103

0.137

2.36

2.71 ± 0.3

84.0

0.117

2.33

2.07 ± 0.6

P2-4000-3 reduced to 67.9, 62.5, and 56.2 emu g−1, respectively, which is ascribed to nonmagnetic mSiO2 and P2. Drug Loading and Release Profiles. To investigate the sensitive controlled release of Fe3O4@mSiO2@P2-4000s systems, DOX was selected as the model drug and the release performances were investigated in detail. The actual loading levels of DOX are calculated to be 2.74 ± 0.5, 2.71 ± 0.3, and 2.07 ± 0.6 wt % for DOX-Fe3O4@mSiO2@P2-4000-1, DOXFe3O4@mSiO2@P2-4000-2, and DOX-Fe3O4@mSiO2@P24000-3, respectively (formula 1). It is known that the drug’s encapsulation ability is related to the surface area of the carriers. With a large surface area, DOX-Fe3O4@mSiO2@P2-4000-1 (157 m2 g−1) shows a high drug-loading amount (2.74 ± 0.5 wt %). The release profiles of DOX-Fe3O4@mSiO2@P2-4000s in different PBS buffers (pH 5.8 and 7.4) are displayed in Figure 4. The fast release behavior can be found at pH 5.8 (Figure 4). As illustrated in Figure 4A, it just takes 4 h to reach 66.6, 45.0, and 31.1% release. And after 24 h, it can reach maximal amounts of 98.0, 95.2 and 93.5% from DOX-Fe3O4@mSiO2@ P2-4000-1, DOX-Fe3 O4@mSiO2@P2-4000-2, and DOXFe3O4@mSiO2@P2-4000-3, respectively. However, in Figure 4B, the corresponding release amounts decrease to below 40.0% at pH 7.4. Moreover, when the amount of P2 is

polymer grafted on the outer surface of Fe3O4@mSiO2 and DOX drug molecules encapsulated in the mSiO2 pores. It is worth mentioning that with the highest packages of P2, DOXFe3O4@mSiO2@P2-4000-3 possesses the lowest surface area and pore volume (Table 1). Figure 3 presents the magnetization characterization of Fe3O4, Fe3O4@mSiO2@P2-4000-1, Fe3O4@mSiO2@P2-40002, and Fe3O4@mSiO2@P2-4000-3 at room temperature. The hysteresis loops (Figure 3) indicate the magnetism of all materials. Furthermore, Fe3O4 nanoparticles possess high saturation magnetizations (Ms) (about 79.9 emu g−1). For comparison, the corresponding Ms values for Fe3O4@mSiO2@ P2-4000-1, Fe3O4@mSiO2@P2-4000-2, and Fe3O4@mSiO2@ 9822

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Figure 4. Release profiles of DOX from (A) DOX-Fe3O4@mSiO2@P2-4000 at pH 5.8, (a) DOX-Fe3O4@mSiO2@P2-1, (b) DOX-Fe3O4@mSiO2@ P2-2, (c) DOX-Fe3O4@mSiO2@P2-3; (B) DOX-Fe3O4@mSiO2@P2-4000 at pH 7.4, (a) DOX-Fe3O4@mSiO2@P2-1, (b) DOX-Fe3O4@mSiO2@ P2-2, and (c) DOX-Fe3O4@mSiO2@P2-3; (C) DOX-Fe3O4@mSiO2@P2-6000 at pH 5.8, (a) DOX-Fe3O4@mSiO2@P2-1, (b) DOX-Fe3O4@ mSiO2@P2-2, (c) DOX-Fe3O4@mSiO2@P2-3; and (D)DOX-Fe3O4@mSiO2@P2-6000 at pH 7.4, (a) DOX-Fe3O4@mSiO2@P2-1, (b) DOXFe3O4@mSiO2@P2-2, and (c) DOX-Fe3O4@mSiO2@P2-3.

increased, the release rates slow down. As depicted in Figure 4, the pH-sensitive release performances of DOX-Fe3O4@ mSiO2@P2-4000s are derived from the hydrolysis of the ester bond in an acidic environment. At pH 5.8, the hydrolysis of the ester bond causes P2 to break down, inducing open pores and drug release. However, at pH 7.4, the P2 chain seems to be stable and prevents drug molecules from escaping. In order to further testify to the controlled mechanism of the drug-delivery system, the hydrodynamic diameter and zeta potential after the drug release of DOX-Fe3O4@mSiO2@P2-4000s were also recorded. As can be seen in Table 2, the hydrodynamic diameters reduced to 89.7, 101.5, and 132.6 nm, as ascribed to the PEO component detached from P2 (Scheme 1). Furthermore, the zeta potentials also decrease to −13.16 ± 2.58, −10.28 ± 1.98, and −8.36 ± 3.01 mV due to the surface carboxyl after the hydrolysis of P2 (Scheme 1). Besides that, P2-6000 (with PEO-6000) was also synthesized as the capping to graft outside the nanoparticles, and the relative release behaviors were also recorded. As can be seen in Figure 4C,D, DOX-Fe3O4@mSiO2@P2-6000s also exhibits acid-enhanced release. Furthermore, when the molecular amount of PEO increases to 6000, the release rate decreases at pH 5.8 as well as at pH 7.4 because the long chain of P26000 slows down the drug release. As displayed in Figure 4C,D, it takes about 24 h to reach the release equilibrium. In short, the

Table 2. Hydrodynamic Size (Diameter) and Zeta Potential of the Samples hydrodynamic size distribution (diameters, nm)

samples Fe3O4@mSiO2 Fe3O4@mSiO2@P2-4000-1 Fe3O4@mSiO2@P2-4000-2 Fe3O4@mSiO2@P2-4000-3 Fe3O4@mSiO2@P2-4000-1 after P2 degraded at pH 5.8 Fe3O4@mSiO2@P2-4000-2 after P2 degraded at pH 5.8 Fe3O4@mSiO2@P2-4000-3 after P2 degraded at pH 5.8

zeta potential test

82.0 98.5 110.1 148.7 89.7

−15.01 −5.45 −3.09 −1.62 −13.16

± ± ± ± ±

101.5

−10.28 ± 1.98

132.6

−8.36 ± 3.01

1.17 2.36 3.22 3.42 2.58

more P2, the slower the release rates of DOX-Fe3O4@mSiO2@ P2-6000s and DOX-Fe3O4@mSiO2@P2-4000s. To learn more about the release behavior, the release data are also analyzed by the Higuchi model.37,38 As we know, drug release kinetics from an insoluble, porous carrier matrix are frequently described by the Higuchi model, and the release rate can be described by eq 2

Q = kt 1/2 9823

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Scheme 1. Schematic Illustration of the pH-Triggered Controlled-Release Drug-Delivery System Based on Fe3O4@mSiO2 Core−Shell Nanoparticles Capped with the β-Thiopropionate-poly(ethylene glycol) “Gatekeeper” (P2)

Figure 5. Higuchi plot for the release of DOX from (A) DOX-Fe3O4@mSiO2@P2-4000 at pH 5.8, (a) DOX-Fe3O4@mSiO2@P2-1, (b) DOXFe3O4@mSiO2@P2-2, and (c) DOX-Fe3O4@mSiO2@P2-3 and (B) DOX-Fe3O4@mSiO2@P2-4000 and DOX-Fe3O4@mSiO2@P2-6000 at pH 5.8, (a) DOX-Fe3O4@mSiO2@P2-4000-3 and (b) DOX-Fe3O4@mSiO2@P2-6000-3.

where Q is the quantity of drug released from the materials, t denotes time, and k is the Higuchi dissolution constant. According to the model, for a purely diffusion-controlled process, the linear relationship is valid for the release of relatively small molecules distributed uniformly throughout the carrier.38 As illustrated in Figure 5A, all of the release behaviors display a two-step release based on the Higuchi model. As can be seen

in Figure 5A, DOX-Fe3O4@mSiO2@P2-4000-1 takes only 8 h for the first-step release, while both DOX-Fe3O4@mSiO2@P24000-2 and DOX-Fe3O4@mSiO2@P2-4000-3 take 12 h for the first-step release. That is because DOX-Fe3O4@mSiO2@P24000-1 possesses the smallest amount of P2, leading to the fastest degradation and the highest dissolution constant k (the slope of the matching lines). It is revealed that the first-step release is ascribed to the degradation of P2. However, in the 9824

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proving the fast cellular uptake ability of the sample, which is ascribed to the small particle size (65 nm) and poly(ethylene glycol) surface of the nanocomposites that are beneficial to entering the cell and enhancing the drug efficacy.39,40 On the basis of previous reports, PEO was just used to improve the cell uptake ability of the host, while in this paper PEO also assists the control release performance. In addition, DOX can also be found in the nucleus after 6 h of incubation, benefitting from the fast cellular uptake ability of these nanocomposites and the low-pH endosomal environment.41 Importantly, the morphology of HeLa cells is not influenced by the addition of DOXFe3O4@mSiO2@P2-3, also illustrating the good biocompatibility of the nanocomposites. The investigation of the cytotoxicity of the synthesized drug carrier is significant for its further biomedical applications. Only nontoxic carriers are appropriate for drug delivery. Here the cellular toxicity of Fe3O4@mSiO2 and Fe3O4@mSiO2@P24000-3 nanoparticles toward HeLa cells was determined by means of a standard MTT cell assay. As illustrated in Figure 7,

second-step release, all of the dissolution constants obviously decrease and tend to be close to each other. This can be explained by the fact that most of the drug molecules have been released after P2 was degraded in the first-step release. As a result, the second-step releases are mainly regulated by the same mesoporous structure (derived from CTAB) and show similar dissolution constants (Figure 5A). To investigate the drug-release behavior of different chain lengths, the release data of DOX-Fe3O4@mSiO2@P2-6000-3 and DOX-Fe3O4@mSiO2@P2-4000-3 are also analyzed by the Higuchi model. As depicted in Figure 5B, DOX-Fe3O4@ mSiO2@P2-6000-3 also exhibits a two-step release based upon the Higuchi model. From the release date, with the longer chain length of P2-6000, DOX-Fe3O4@mSiO2@P2-6000-3 shows a lower dissolution constant in the first several hours. From the above investigation, the first-step release is ascribed to the degradation of P2 so that the short chain of P2-4000 leads to quick H+ diffusion and the fast hydrolysis of P2-4000 as well as the high dissolution constant in the first-step release process for DOX-Fe3O4@mSiO2@P2-4000-3. And in the second step, all of the releases show a similar released dissolution constant that is consistent with the above investigation. In summary, the amounts and chain length of the “gate” (P2) can be used to regulate the release performance of the system. In Vitro Cytotoxic Effect and Cellular Uptake. To investigate the cellular uptake of the sample, DOX-Fe3O4@ mSiO2@P2-4000-3 was incubated with HeLa cells at a concentration of 100 μg mL−1 for 6 h. The cellular uptake and subsequent localization of the sample is shown in Figure 6. As depicted in Figure 6, nanoparticles are localized in the cytoplasm and nucleus after 6 h of incubation with HeLa cells,

Figure 7. Cell viability of HeLa cells incubated with different amounts of Fe3O4@mSiO2 and Fe3O4@mSiO2@P2-4000-3 for 24 h.

pure Fe3O4@mSiO2 and Fe3O4@mSiO2@P2-4000-3 show no significant cytotoxic effect on the HeLa cells over the range of concentration (3.125−50 μg mL−1). When the concentration increases to 50 μg mL−1, the cell viability also attains 88.3% for Fe3O4@mSiO2@P2-4000-3 after 6 h of incubation with HeLa cells. With sound bioactivity, Fe3O4@mSiO2@P2-4000-3 can be regarded as a promising candidate in biomedicine.

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CONCLUSIONS We have designed a novel pH-sensitive release system for promising cancer theranostics. Fe3O4@mSiO2 core−shell nanoparticles are used as the host, and P2 is modified outside the mesoporous silica as the gatekeepers. Owing to the degradation of the “gate” (P2), the cargo-release triggering is pH-sensitive (pH 5.8). The release mechanism is investigated in detail, revealing that the degradation of P2 is associated with the mesoporous structure to determine the release process. Furthermore, the amounts and chain length of P2 can be used to regulate the release performance of the system. The fast cell uptake due to the small particle size (65 nm) and the poly(ethylene glycol)-modified surface combined with the

Figure 6. CLSM images of HeLa cells after incubation with 100 μg mL−1 DOX-Fe3O4@mSiO2@P2-4000-3 for 6 h. (A) Hella cells (bright), (B) DOX fluorescence in cells (red), (C) FITC-labeled DOX-Fe3O4@mSiO2@P2-4000-3 (green), (D) Hoechst 33342 labeled cell nucleus (blue), and (E) merging. 9825

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magnetic targeted and pH-sensitive release lead to promising applications for multifunctional nanocarriers in smart sites and time- and dose-selected drug release.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis route for P2-4000/6000. Low-angle and wide-angle XRD patterns. Nitrogen adsorption−desorption isotherms and pore size distribution. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: (+86) 451-88060653. E-mail: [email protected]. *Tel: (+86) 451-88060653. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this study was provided by the National Natural Science Foundation of China (21171045 and 21101046), the Natural Science Foundation of Heilongjiang Province of China (ZD201214), the Program for Scientific and Technological Innovation Team Construction in Universities of Heilongjiang Province (2011TD010), and the Technology Development Preproject of Harbin Normal University (12XYG-11).

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