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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 Langmuir, Just Accepted Manuscript • DOI: 10.1021/la501833u • Publication Date (Web): 29 Jul 2014 Downloaded from http://pubs.acs.org on August 2, 2014

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pH-Responsive Magnetic Core-Shell Nanocomposites for Drug Delivery Department of Photoelectric Band Gap Materials Key Laboratory of Ministry of Education, College of Chemistry and Chemical Engineering, Harbin Normal University, Harbin 150025, China. Chunyu Yang, Wei Guo, Liru Cui, Na An, Ting Zhang, Huiming Lin* and Fengyu Qu*

*To whom all correspondence should be addressed. Tel: (+86) 451-88060653. E-mail: [email protected] and [email protected]

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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 consisted of Fe3O4@mSiO2 core-shell nanocomposite (about 65 nm) and β-thiopropionate-polyethylene glycol “gatekeeper” (P2) has been successfully synthesized as the drug carriers (Fe3O4@mSiO2@P2). Because of the hydrolysis of β-thiopropionate linker under mild acidic conditions, Fe3O4@mSiO2@P2 shows the pH-sensitive release performance based on the slight difference between tumor (weakly acid) and normal tissue (weakly alkaline). And before reaching tumor site, the drug delivery system shows good drug retention. Notably, the nanocomposites are fastly uptaken by HeLa cells due to their small particle size and the polyethylene 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, is expected to be promising in the potential application of cancer treatment.

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INTRODUCTION Cancer, known as a major cause of mortality world-wide, is a vast group of diseases produced by the 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 the efficacy, originating from little specific discrimination between the normal cell and the cancer cell.2 To overcome this problem, one of promising approaches for 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 the 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 made them to be considered as one of the most important candidates for drug carriers. Further, MSNs supply additional biocompatibility and easily modified surface properties, showing the 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 on nanomedicine.11 At present, some inorganic nanoparticles (e.g. CdS, Fe3O4, and Au), organic polymers (e.g. PAMAM, and PNIPAM), and supermolecule nanovalves (e.g. β-CD, macrocyclic molecule cucurbituril) have been broadly employed as

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“gatekeepers ” , which show the well-controlled release performance.12-18 The controlled-release process can be regulated either by external stimuli such as temperature, light, electrostatic, and magnetic actuation, or by internal stimuli such as pH and enzymes. For example, moon and co-workers 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 spiropyran-containing light-responsive 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 more practical and possible compared with the external stimuli-responsive. For instance, Feng and co-workers have developed a controlled process by using mesoporous silica nanoparticles as hosts capped with acid-labile acetal group linked gold nanoparticles as pH-responsive agents.25 However, a controlled drug release system, which is only depends on the internal stimulus, can’t achieve ideal results. Combined internal and external stimulus, the multifunctional drug delivery system, receiving the advantages of the two, would exhibit the 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 two-photon fluorescence indicator.26-29 Furthermore, Fe3O4 nanoparticles enable to induce hyperthermia effects

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when placed in an alternating magnetic field30-32 for tumor thermotherapy. For example, Wang et al. have synthesized a bicontrollable drug release system with PAH/PSS multilayers on to a Fe3O4/mSiO2, showing the pH-controllable and magnetic target behavior.33 Liu et al. have developed a magnetic and reversible pH-responsive MSNs-based nanogated ensemble. In the ensemble, superparamagnetic Fe3O4 nanoparticles are used as the “gatekeeper” capping onto the outlet of the mesoporous silica via an acid-labile boronate ester linker.34 In the present study, we have developed a β-thiopropionate-polyethylene glycol modified Fe3O4@mSiO2 nanocomposites as the drug loading system, revealing the controlled release based on the low pH value in cancer/tumor. 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, the pH-sensitive β-thiopropionate-polyethylene glycol (P2, Figure S1) is employed to graft outside of the 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 under acid condition, DOX-Fe3O4@mSiO2@P2 is expected to block the pore at neutral or alkaline condition and to open the pore under acid condition (pH 5.8). That makes the nanocarriers respond to the slight difference between the tumor and the normal tissue due to their different physiological environment. Moreover, the small particle size associating with polyethylene glycol fragment coating makes the DOX-Fe3O4@mSiO2@P2 nanovalves show the improved dispersion, stability, biocompatibility and fast uptake by cell 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 and oleic acid, 1-octadecene, (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’-bi-1H-benzimidazole, trihydrochloride (Hoechst 33342), dimethylphenylphosphine, N,N-dimethylacetamide and polyethylene 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 Iron-oleate Complex. In a typical synthesis of iron-oleate complex, 10.8 g of iron chloride (FeCl3•6H2O, 40 mmol) and 36.5 g of sodium oleate (120 mmol, 95%) was dissolved in a mixture solvent composed of 80 mL ethanol, 60 mL distilled water and 140 mL 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 distilled water in a separatory funnel. After washing, hexane was evaporated off, resulting in iron-oleate complex in a waxy solid form. Synthesis of Fe3O4 Nanoparticles. Following a literature procedure Fe3O4 nanoparticles were prepared.35 36 g (40 mmol) of the iron-oleate and 5.7 g of oleic acid (20 mmol, 90%) were dissolved in 200 g of 1-octadecene (90%) at room

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temperature. The reaction mixture was heated to 320 °C with 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 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 heated up to 60 °C for 30 min to evaporate the chloroform, resulting in a transparent black Fe3O4/CTAB solution. Then, 20 mL distilled water was added to the obtained black solution and the pH value of the mixture was adjusted to 8-9 by using 0.1 M NaOH. After that, 100 µL of 20% TEOS in ethanol was injected six times at a 30 min intervals. The reaction mixture was reacted for 24 h under violent 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 Weight. P2 was synthesized following a literature procedure.36 To an ice-cold solution of polyethylene glycol (Mn~4000/6000) (16.92 g/33.84 g, 0.00846 mol) and triethylamine (3.54 mL, 0.025

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mol) in 35 mL/50 mL dry dichloromethane, a solution of freshly distilled acryloyl chloride (1.71 mL, 0.021 mol) in 10 mL dry dichloromethane was added drop-wise under inert atmosphere and then the reaction mixture was stirred at rt for 12 h. Finally, the final residue was dried in vacuum at 40 °C over night. It has been named to P1-4000/6000. A solution of monomer MPTMS (0.062 g, 0.3 mmol) in 5 mL degassed DMAC was added with 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 at the same ice-bath for 12 h. The product is collected. It has been named to 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 was added to the mixed solution. The obtained solid (named as DOX-Fe3O4@mSiO2@P2-4000/6000-1,

DOX-Fe3O4@mSiO2@P2-4000/6000-2,

DOX-Fe3O4@mSiO2@P2-4000/6000-3, respectively) was 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 the formula (1). The experiment repeated three times.

LE wt%=

m（original DOX）− m(residual DOX) ×100% m(Fe O @mSiO2 ) + m（original DOX）− m(residual DOX) + m(P2) 3

(1)

4

Drug Release. Gating protocol was investigated by studying the release profiles of DOX from the DOX-Fe3O4@mSiO2@P2-4000/6000 at pH 5.8 or pH 7.4 phosphate

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buffer solution. Briefly, DOX-Fe3O4@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 interval time, the solution was taken out to determine the release amount by UV. Cell Culture. HeLa cells (cervical cancer cell line) were grown in monolayer in 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. Confocal Laser Scanning Microscopy (CLSM). To check 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 by Hoechst 33342. DOX-Fe3O4@mSiO2@P2-4000-3 was added into the incubation medium at the concentration of 100 µg mL-1 for 6 h 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 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. The assay was performed out in triplicate in the following manner. For MTT assay, HeLa cells were seeded into 96-well plates at a density of 1 × 104 per well in 100 µL of media and grown overnight. The cells were then incubated

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with various concentrations of Fe3O4@mSiO2s and Fe3O4@mSiO2@P2-4000-3s for 24 h. Afterwards, cells were incubated in media containing 0.5 mg mL-1 of 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 by multi-detection microplate reader (SynergyTM HT, BioTek Instruments Inc, USA). Characterization. Powder X-ray patterns (XRD) were recorded on a SIEMENSD 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 2010M sorptometer. 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 Perkin-Elmer 580B Infrared Spectrophotometer using the KBr pellet technique. A UV-vis spectrum was used to describe the amount of the drug release (SHIMADZU UV2550 spectrophotometer). Transmission electron microscopy (TEM) images were recorded on TECNAI F20. Zeta potential and dynamic light scattering (DLS) was carried out with ZetaPALS Zeta Potential Annlyzer. The magnetic properties of samples were characterized with a Vibrating Sample Magnetometer (Lake Shore 7410). RESULTS AND DISCUSSION Morphology and Structure. TEM was used to display the structure of the samples. From Figure 1A, Fe3O4 nanoparticles show the uniform and well dispersed spherical morphology with the average diameter about 20 nm in size. Fe3O4@mSiO2 reveals the

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obvious Fe3O4 core encapsulated by silica shell (20 nm) with worm-like 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 that is larger than that observed from TEM, resulting from the hydrate layer of Fe3O4@mSiO2 in aqueous environment. After the graft of P2, the diameter of Fe3O4@mSiO2@P2-4000-1, Fe3O4@mSiO2@P2-4000-2 and Fe3O4@mSiO2@P2-4000-3 adds 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 mV 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. Based on the above investigation, it is testified that P2 has been successfully grafted on the Fe3O4@mSiO2 surface. The X-ray diffraction patterns (XRD) collected from the Fe3O4@mSiO2, DOX-Fe3O4@mSiO2@P2-4000-1,

DOX-Fe3O4@mSiO2@P2-4000-2

and

DOX-Fe3O4@mSiO2@P2-4000-3 are shown in Figure S2. As illustrated in Figure S2, all samples reveal only one diffraction peak at about 2 θ = 2.26 °, suggesting they

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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 reduced obviously compared to that of Fe3O4@mSiO2 without drug loading and P2 grafted. Moreover, the more amounts P2 is grafted onto the Fe3O4@mSiO2, the lower diffraction intensity the DOX-Fe3O4@mSiO2@P2-4000s have, 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 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

FT-IR

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 the v(C=O) and v(C-S) appear in P2, demonstrating the P2 has been successfully grafted to the P2-4000. As can be seen in Figure 2B, the obvious absorption band at 1086 cm-1 testifies the Si-O-Si framework of the Fe3O4@mSiO2. After P2-4000 grafted, the absorption peaks at 1724 cm-1 assigned to the C=O stretching vibration of P2-4000 also can be found in

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Fe3O4@mSiO2@P2-4000, confirming that P2-4000 has been successfully grafted on Fe3O4@mSiO2. 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, testifying the mesoporous structure of Fe3O4@mSiO2. As depicted in Figure S4A, there is 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 of Fe3O4@mSiO2 to 157 m2 g-1 and 0.161 cm3 g-1 of DOX-Fe3O4@mSiO2@P2-4000-1, 103 m2 g-1 and 0.137 cm3 g-1 of DOX-Fe3O4@mSiO2@P2-4000-2, 84.0 m2 g-1 and 0.117 cm3 g-1 of DOX-Fe3O4@mSiO2@P2-4000-3, respectively (Table 1). These results are expected due to the polymer grafted in the outer surface of Fe3O4@mSiO2 and DOX drug molecules encapsulated in the mSiO2 pores. It is worth mentioned that with the highest packages of P2, DOX-Fe3O4@mSiO2@P2-4000-3 possesses the lowest surface area and pore volume (Table 1). Figure

3

presents

the

magnetization

characterization

of

Fe3O4

and

Fe3O4@mSiO2@P2-4000-1, Fe3O4@mSiO2@P2-4000-2, Fe3O4@mSiO2@P2-4000-3 at room temperature. The hysteresis loops (Figure 3) indicate the magnetism of all

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materials.

Furthermore,

Fe3O4

nanoparticles

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possess

the

high

saturation

magnetizations (Ms) (about 79.9 emu g-1). For comparison, the corresponding Ms of Fe3O4@mSiO2@P2-4000-1,

Fe3O4@mSiO2@P2-4000-2

and

Fe3O4@mSiO2@P2-4000-3 reduced to 67.9, 62.5, and 56.2 emu g-1, respectively, that is ascribed to the non-magnetic 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 level of DOX is calculated to be 2.74 ± 0.5, 2.71 ± 0.3 and 2.07 ± 0.6 wt% for DOX-Fe3O4@mSiO2@P2-4000-1,

DOX-Fe3O4@mSiO2@P2-4000-2

and

DOX-Fe3O4@mSiO2@P2-4000-3, respectively (formula (1)). It is known that, the drug encapsulated ability is related to the surface area of the carriers. With high the large surface area, DOX-Fe3O4@mSiO2@P2-4000-1 (157 m2 g-1) shows the high drug loading amount (2.74 ± 0.5 wt%). The release profiles of DOX-Fe3O4@mSiO2@P2-4000s in different PBS buffer (pH 5.8 and pH 7.4) are displayed in Figure 4. The fast release behavior can be found from 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 the maximal amount 98.0, 95.2 and 93.5% from

DOX-Fe3O4@mSiO2@P2-4000-1,

DOX-Fe3O4@mSiO2@P2-4000-2

and

DOX-Fe3O4@mSiO2@P2-4000-3, respectively. However, from Figure 4B, the corresponding release amounts decrease below 40.0% at pH 7.4. Moreover, increasing the amount of P2, the release rates slow down. As depicted in Figure 4, the

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pH-sensitive release performances of DOX-Fe3O4@mSiO2@P2-4000s are derived from the hydrolysis of the ester bond under acid condition. At pH 5.8, the hydrolysis of the ester bond causes P2 break down, inducing pore open and drug release. While, at pH 7.4, P2 chain seems to stable and prevents drug molecules from escaping. In order to further testify the controlled mechanism of the drug delivery system, the hydrodynamic

diameter

and

zeta

potential

after

drug

release

of

DOX-Fe3O4@mSiO2@P2-4000s were also recorded. As can be seen in Table 2, hydrodynamic diameters reduced to 89.7, 101.5 and 132.6 nm, ascribing to 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 and D, DOX-Fe3O4@mSiO2@P2-6000s also exhibit acid-enhanced release. What’s more, when the molecular amount of PEO increases to 6000, the release rate decreases at pH 5.8 as well as pH 7.4 because the long chain of P2-6000 slows down the drug release. As displayed in 4C and D, it takes about 24 h to reach the release equilibrium. In short, the more amount of P2, the slower release rates of DOX-Fe3O4@mSiO2@P2-6000s as well as DOX-Fe3O4@mSiO2@P2-4000s are. To further learn the release behavior, the release data are also analyzed by Higuchi model.37,38 As we known, drug release kinetics from an insoluble, porous carrier

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matrix are frequently described by the Higuchi model, and the release rate can be described by the follow Equation 2:

Q = k ∗ t1 2

(2)

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 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 only takes 8 h for the first-step release, while both DOX-Fe3O4@mSiO2@P2-4000-2 and DOX-Fe3O4@mSiO2@P2-4000-3 take 12 h for the first-step release. That is because DOX-Fe3O4@mSiO2@P2-4000-1 possesses the lowest amount of P2, making 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 second-step release, all the dissolution constants decrease obviously, which tend to be close to each others. This can be explained by that most of the drug molecules have 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 the similar dissolution constants (Figure 5A). To investigate the drug release behavior of different chain length, the release data of DOX-Fe3O4@mSiO2@P2-6000-3 and DOX-Fe3O4@mSiO2@P2-4000-3 are also

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analyzed

by

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 the 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 the 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 the releases show the similar and released dissolution constant that is consistent with the above investigation. To sum up, the amounts and chain length of “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 the 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 incubated with HeLa cells, proving the fast cellular uptake ability of the sample. That is ascribed to the small particle size (65 nm) and polyethylene glycol surface of the nanocomposites that is benefit to enter into the cell and enhance the drug efficacy.39,40 Based on the 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 be also found in nucleus after 6 h incubated that benefit from the fast cellular uptake ability of these

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nanocomposites and the low-pH endosomal environment.41 Importantly, the morphology

of

HeLa

cells

is

not

influenced

by

the

addition

of

DOX-Fe3O4@mSiO2@P2-3, also illustrating the well 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@P2-4000-3 nanoparticles toward HeLa cells were determined by means of a standard MTT cell assay.

As

illustrated

in

Figure

7,

the

pure

Fe3O4@mSiO2

and

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

CONCLUSION In summary, 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 “gate” (P2), the cargos can release triggered by pH-sensitive (pH 5.8). The release mechanism is investigated in detail, revealing that the degradation of P2 associated with mesoporous structure to determine the release process. Furthermore,

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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 polyethylene glycol modified surface combines with the magnetic targeted and pH-sensitive release make the multi-functional nanocarriers possess a promising application on smart site, time and dose-selected drug release.

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

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Table of Contents Use Only

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The degradation of P2:

Scheme 1

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A

50 nm

B

C

50 nm

50 nm

Figure 1

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Figure 2

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Figure 3

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A

B

C

D

Figure 4

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B

A

Figure 5

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Figure 6

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Figure 7

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Table 1 Pore parameters and loading efficiency of the samples Samples

BET 2

-1

Vp 3

Pore Size

LE

(nm)

(wt%)

-1

(m g )

(cm g )

Fe3O4@mSiO2

326

0.285

2.42

—

DOX-Fe3O4@mSiO2@P2-4000-1

157

0.161

2.38

2.74 ± 0.5

DOX-Fe3O4@mSiO2@P2-4000-2

103

0.137

2.36

2.71 ± 0.3

DOX-Fe3O4@mSiO2@P2-4000-3

84.0

0.117

2.33

2.07 ± 0.6

Table 2 Hydrodynamic size (diameters) and Zeta-potential of the samples Samples

Hydrodynamic size

Zeta-potential test

distribution ( diameters, nm) Fe3O4@mSiO2

82.0

-15.01 ± 1.17

Fe3O4@mSiO2@P2-4000-1

98.5

-5.45 ± 2.36

Fe3O4@mSiO2@P2-4000-2

110.1

-3.09 ± 3.22

Fe3O4@mSiO2@P2-4000-3

148.7

-1.62 ± 3.42

Fe3O4@mSiO2@P2-4000-1 after P2 degraded in pH 5.8

89.7

-13.16 ± 2.58

Fe3O4@mSiO2@P2-4000-2 after P2 degraded in pH 5.8

101.5

-10.28 ± 1.98

Fe3O4@mSiO2@P2-4000-3 after P2 degraded in pH 5.8

132.6

-8.36 ± 3.01

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Captions: Scheme 1. Schematic illustration of the pH triggered controlled-release drug delivery system based on Fe3O4@mSiO2 core-shell nanoparticles capped with β-thiopropionate-polyethylene glycol “gatekeeper” (P2). Figure 1. TEM images of A) Fe3O4, B) Fe3O4@mSiO2, and C) Fe3O4@mSiO2@P2-1. Figure 2. FTIR spectra of (A) PEO-4000 and P2-4000 and (B) Fe3O4@mSiO2 and Fe3O4@mSiO2-P2-4000. Figure 3. Magnetization curves of four samples at room temperature (a) Fe3O4, (b) Fe3O4@mSiO2 @P2-4000-1, (c) Fe3O4@mSiO2@P2-4000-2, and (d) Fe3O4@mSiO2 @P2-4000-3. Figure 4. Release profiles of DOX from A) DOX-Fe3O4@mSiO2 @P2-4000 in 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 in 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 in pH 5.8, (a) DOX-Fe3O4@mSiO2 @P2-1, (b) DOX-Fe3O4@mSiO2 @P2-2, (c) DOX-Fe3O4@mSiO2 @P2-3; D)DOX-Fe3O4@mSiO2@P2-6000 in pH 7.4, (a) DOX-Fe3O4@mSiO2 @P2-1, (b) DOX-Fe3O4@mSiO2 @P2-2, and (c) DOX-Fe3O4@mSiO2 @P2-3. Figure 5. Higuchi plot for the release of DOX from A) DOX-Fe3O4@mSiO2 @P2-4000 in pH 5.8, (a) DOX-Fe3O4@mSiO2@P2-1, (b) DOX-Fe3O4@mSiO2 @P2-2, and (c) DOX-Fe3O4@mSiO2 @P2-3; B) DOX-Fe3O4@mSiO2 @P2-4000 and DOX-Fe3O4@mSiO2@P2-6000 in pH 5.8, (a) DOX-Fe3O4@mSiO2 @P2-4000-3, and (b) DOX-Fe3O4@mSiO2 @P2-6000-3. 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) merged. Figure 7. Cell viability of HeLa cells incubated with different amounts of Fe3O4@mSiO2 and Fe3O4@mSiO2 @P2-4000-3 for 24 h.

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