Janus Nanocage toward Platelet Delivery - ACS Publications

May 2, 2016 - at room temperature in darkness, the product was centrifuged and washed with THF. The product .... is left. The cavity size is determine...
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Janus Nanocage toward Platelet Delivery Lin Tang,† Saina Yang,† Fuxin Liang,*,† Qian Wang,† Xiaozhong Qu,†,‡ and Zhenzhong Yang*,† †

State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: The platelet-shaped Janus nanocages with a mesoporous silica shell are prepared. PEG moiety onto the exterior surface is responsible for good dispersity in water. The graphene sheet inside the cavity is responsible for hydrophobic performance to selectively capture hydrophobic species, and photothermal effect by NIR irradiation. As a biocompatible DOX-loaded Janus platelet delivery, HeLa cell cytotoxicity is greatly enhanced under NIR irradiation. There exists a synergetic effect between the chemotherapy and photothermal therapy. KEYWORDS: graphene, Janus, nanocage, delivery, NIR

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INTRODUCTION Janus materials with two different compositions and performances compartmentalized onto the same object have gained increasing interest due to their diversified promising applications.1−5 Both morphology and composition play significant roles in their performances. Besides extensively reported shapes of particulate, disc, and rod, centrosymmetric hollow spheres with two different compositions segmented onto the interior and exterior surfaces of the shell have gained more concerns. The unique hollow spheres with an asymmetric shell are regarded as Janus.6 When the interior and exterior surfaces are conjugated with different compositions, unique properties would be obtained. For example, when the exterior surface is hydrophilic and the interior surface is hydrophobic, hydrophobic components can be selectively captured into the cavity from their aqueous surroundings.7−9 As for biological concerns, biocompatible compositions like polyethylene glycol (PEG) can be used to conjugate onto the exterior surface. Cavity of the Janus hollow sphere can provide a large space to load increased amount of guest species. After selective grafting stimuli-responsive polymers onto the interior surface, loading and release of desired species could be triggered accordingly.10,11 Janus hollow spheres are promising in smart drug delivery. Mass transportation is required to generate transverse channels within the shell. Mesoporous silica shell is a good candidate because mesoporous silica materials are biocompatible and widely used in drug-delivery systems.12−18 It is expected that a Janus hollow sphere with a mesoporous shell (named Janus cage) could combine the unique features of Janus hollow sphere and mesoporous silica and is of great potential in drug delivery. Another key concern is how to trigger release of drugs from the Janus cage. Controlled release by external stimuli such as © XXXX American Chemical Society

light irradiation, magnetic or electric effects have attracted increasing attention.19,20 Near-infrared (NIR) irradiation has received more interest because an additional hyperthermic effect can be gained for special applications.21−25 Materials with high NIR absorption capability can convert light to thermal energy, which can heat their surroundings while stimulating drug release.23 An additional hyperthermic effect is beneficial to further treat tumor tissues.24,25 Graphene-based materials are promising as photosensitizers due to their high NIR absorption capability. They have shown outstanding performances in NIR triggered release and tumor ablation.25 It will be attractive to combine the NIR-responsive hyperthermic effect of graphene and NIR triggered drug release from Janus cages. Moreover, graphene is potential to fabricate nanomaterials of desirable shapes by bottom-up nanomanufacturing approaches.26,27 In addition, the shape of a drug delivery system is significant in biomedical applications. An anisotropic shape can greatly facilitate cellular uptake.28 Herein, we report on the fabrication of a platelet Janus nanocage with graphene in the cavity. Platelet shape of the nanocage is duplicated from graphene oxide sheet after a favorable surface sol−gel process. PEG moiety onto the exterior surface renders the nanocage good dispersion in water. The transverse mesopores within the silica shell provide transportation channels for drug loading and release. Graphene inside the cavity is responsible for the hydrophobic internal cavity and photothermal effect under NIR irradiation. In our current case, DOX is used as a model hydrophobic drug. NIR laser (808 nm, 1W/cm2) is selected to induce the photothermal effect. Received: March 16, 2016 Accepted: May 2, 2016

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DOI: 10.1021/acsami.6b03208 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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irradiated by NIR laser (808 nm, 1 W/cm2) for 5 min during each interval. The release amount of DOX in the PBS solution was measured by fluorescence spectroscopy at an excitation wavelength of 480 nm. Cell Viability Assay. HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Invitrogen) with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a humidified incubator (37 °C, 5% CO2). The cells were preincubated in a 96-well plate (about 2 × 104 cells/well) for 24 h in a humidified incubator (37 °C, 5% CO2). After each well was washed with PBS, the desired amount of DOX, RGO@mSiO2−PEG Janus nanocages, and DOX-loaded RGO@mSiO2−PEG Janus nanocages were added. After incubation (37 °C, 5% CO2) for 12 h, the corresponding wells were irradiated by NIR laser (808 nm, 1 W/cm2) for 5 min. After incubation for another 12 h, the cell killing efficiency was determined by the CCK-8 assay. Characterization. Morphology of the samples was characterized by transmission electron microscopy (TEM, JEM-1011 operating at 100 kV), and dark-field scanning transmission electron microscopy (DF-STEM, JEM-2100F operating at 200 kV) with energy dispersive spectroscopy (EDS) mapping, and scanning electron microscopy (SEM, S-4800 operating at 15 kV) equipped with an energy dispersive X-ray (EDX) analyzer. Atomic force microscope (AFM) images were recorded under ambient conditions with a Digital Instrument Multimode Nanoscope IIIA at a tapping mode. Nitrogen adsorption isotherm measurement was performed on a Micromeritics ASAP-2020 M porosimeter. Specific surface area was determined based on the Brunauer−Emmett−Teller (BET) equation in the P/P0 range of 0.06−0.20. Pore volume was determined according to the absorption curves using the amount of nitrogen uptake at the P/P0 of 0.975. The surface area and pore volume of the pores with pore diameters from 2.0 to 50 nm were analyzed using the BJH method and the adsorption isotherms. Fourier transform infrared (FTIR) spectra were performed after scanning samples 32 times with a Bruker EQUINOX 55 spectrometer. Ultraviolet−visible spectroscopy (UV−vis, TU-1901) was used to measure the absorbance. The photothermal effect was characterized by an infrared thermal imager (E40, FLIR). Temperature change was recorded each time after an interval of 20 s when 808 nm NIR laser irradiated vertically to a quartz cuvette containing the sample dispersions at varied solid content (total volume of 1 mL). Fluorescence spectra were obtained with a Hitachi F-4500 fluorescence spectrophotometer. Confocal laser scanning microscopy (CLSM) was performed using an Olympus FV1000-IX81 confocal laser biological microscope system.

EXPERIMENTAL METHODS

Materials. Graphene oxide (GO) was purchased from XF NANO (China). 2-Methoxy(polyethyleneoxy)propyl-trimethoxysilane (PEGS), triethylamine (TEA), ascorbic acid, aminopropyltrimethoxysilane (APTMS) were purchased from ACROS Organic. Fluorescein isothiocyanate (FITC) was purchased from Beijing Fanbo Science & Technology (China). Tetrabutyl titanate (TBT), ethylene glycol (EG), ammonia solution (25 wt %), cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), dimethyl sulfoxide (DMSO), Sudan III, tetrahydrofuran (THF) and methyl orange (MO) were purchased from Sinopharm Chemical Reagent Beijing. Doxorubicin hydrochloride (DOX·HCl) was purchased from Wuhan Yuancheng Gongchuang Technology (China). 4′,6-Diamidino-2phenylindole dihydrochloride (DAPI) was purchased from Sigma. Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies. Preparation of GO@TiO2 Composite Platelets. After 3.0 mg of dry GO was dispersed in 4.0 mL of ethanol, 11.5 μL of ammonia was mixed under ultrasonication. 30.0 mg of TBT chelated with 3 mL of EG was dropped under stirring. A sol−gel process was performed at 45 °C for 24 h under stirring. The brown product was collected after centrifugation and washed with ethanol and water. Preparation of GO@TiO2@mSiO2−PEG Composite Platelets. After 20.0 mg of the GO@TiO2 composite platelet was dispersed in 12.0 mL of water, 90.0 μL of ammonia solution was mixed under stirring. After 120.0 mg of CTAB was added and stirred at 40 °C for 2 h, 50.0 μL of TEOS was added. A sol−gel process was conducted at 40 °C for 12 h. The product was collected by centrifugation and washed with ethanol. PEG capped silane was used to modify the exterior silica surface. After the above obtained GO@TiO2@mSiO2 platelets were dispersed in 10.0 mL of ethanol, 25.0 μL of PEG capped silane was added under stirring at 70 °C for 12 h. The modified product (GO@ TiO2@mSiO2−PEG) was collected by centrifugation and washed with ethanol. Preparation of RGO@mSiO2−PEG Janus Nanocages. CTAB was dissolved under refluxing with acetone for 48 h. TiO2 was dissolved with 2 M of HCl. The obtained GO@mSiO2−PEG composite platelet was collected by centrifugation and washed with water until the aqueous solution became neutral. GO was reduced into graphene with ascorbic acid. After the sample was dispersed in 30.0 mL of water, 5.0 mg of ascorbic acid was added. The reduction was conducted under stirring at 95 °C for 3 h. The final product was dialyzed against water, and then collected by centrifugation to obtain the RGO@mSiO2−PEG Janus platelet nanocage. Preparation of FITC-Labeled Janus Nanocages. RGO@ mSiO2−PEG Janus nanocages were further modified with APTMS. After 25.0 mg of the RGO@mSiO2−PEG Janus nanocage was dispersed in 10.0 mL of ethanol, 20.0 μL of APTMS was added. After stirring overnight at 40 °C, the product was collected by centrifugation and wash with ethanol, dried for further use. The as-dried product and 4.0 mg of FITC were mixed in 10.0 mL of THF. After stirring for 4 h at room temperature in darkness, the product was centrifuged and washed with THF. The product was dried to obtain the FITC-labeled RGO@mSiO2−PEG Janus platelet nanocage. Photothermal Effect of RGO@mSiO2−PEG Janus Platelet Nanocages. Dispersions of the RGO@mSiO2−PEG Janus platelet nanocage in phosphate buffer saline (PBS, pH = 7.4) at varied solid content were irradiated by NIR laser (808 nm, 1 W/cm2) for varied time. As the control, GO@mSiO2−PEG composite dispersion is also irradiated under NIR laser. In Vitro Loading and Release of DOX. First, 5.0 mg of DOX· HCl was stirred with 1.5-fold excess of TEA in 5.0 mL of DMSO overnight to obtain DOX. 1.0 mg of the RGO@mSiO2−PEG Janus nanocage was dispersed in 5.0 mL of DMSO containing DOX under stirring for 24 h at room temperature in darkness. The DOX-loaded RGO@mSiO2−PEG Janus delivery was obtained by centrifugation. In vitro release was performed as following. 1.0 mg of the DOXloaded RGO@mSiO2−PEG Janus delivery was mixed with 1.0 mL of PBS solution (10 mM) under stirring at 37 °C. The dispersion was

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RESULTS AND DISCUSSION The preparation route is illustrated in Scheme 1. A sol−gel process of tetrabutyl titanate (TBT) is performed onto the graphene oxide (GO) sheet surface, forming a GO@TiO2 composite platelet with a sandwich structure. Functional groups onto the GO surface are responsible for the favorable Scheme 1. Preparation of Platelet Graphene@mSiO2 Janus Nanocageab

a A GO@TiO2 composite platelet is prepared by a favorable sol−gel process onto the GO sheet surface to form a TiO2 layer. Along step a, GO@TiO2@mSiO2−PEG are prepared by forming a mesostructured silica layer onto the GO@TiO2 surface following a surface modification to graft PEG moiety, the silica mesoporous shell is obtained after dissolution of the surfactant. bAlong step b, after dissolution of TiO2 and reduction of GO into graphene, the platelet RGO@mSiO2−PEG Janus nanocage is achieved.

B

DOI: 10.1021/acsami.6b03208 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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example cetyltrimethylammonium bromide (CTAB).30 A mesostructured silica layer is coated onto the TiO2 surface. Sequentially, PEG is grafted onto the mesostructured silica surface by using a PEG capped silane. After removal of CTAB, a GO@TiO2@mSiO2−PEG composite platelet is formed. The platelet shape is well preserved (Figure 1e). No fracture is found. The composite platelets are ∼35 nm in thickness (Figure 1f). The thickness of mSiO2−PEG layer is ∼ 10.5 nm. Similarly, thickness of the mSiO2 layer is tunable by varying TEOS content. For example, a thicker mSiO2 layer (∼15 nm) can be achieved at a higher level of TEOS content (Figure S1c,d). After dissolution of the intermediate TiO2 layer, a cavity is left. The cavity size is determined by TiO2 layer thickness. The internal cavity becomes hydrophobic after GO is reduced into graphene. The RGO@mSiO2−PEG composite nanocages are Janus. Both shape and thickness of the Janus nanocages (Figures 1g and 1h) remain the same as that of GO@TiO2@ mSiO2−PEG composite platelet. This implies that no collapse occurs after the removal of the intermediate TiO2 layer. No Ti element is detected by EDX in the Janus nanocages (Figure S2), indicating that the removal of TiO2 from the GO@TiO2@ mSiO2−PEG composite platelet is complete. Morphology and internal structural evolution of the composite platelets are characterized by TEM. The GO@ TiO2@mSiO2−PEG composite platelets are irregular with some protruding edges (Figure 2a), which are duplicated from

growth of titania (TiO2). No TiO2 is found in the continuous phase. Along step a, a silica/CTAB composite layer is coated onto the GO@TiO2 composite platelet. PEG is further conjugated onto the silica exterior surface by using 2methoxy(polyethyleneoxy)propyl-trimethoxysilane. After removal of CTAB by dissolution, the silica layer becomes mesoporous. The GO@TiO2@mSiO2−PEG composite platelet is achieved. Along step b, a platelet silica cage is synthesized after dissolving the middle TiO2 layer. GO is encapsulated inside the cavity. After reduction of GO into graphene (RGO), the platelet RGO@mSiO2−PEG Janus nanocage is derived. GO sheets are highly flexible and wrinkled on the surface (Figure 1a). They are as thin as ∼1.1 nm (Figure 1b).29 Polar

Figure 2. (a) TEM and (b) cross-sectional TEM images of the GO@ TiO2@mSiO2−PEG composite platelet; (c) TEM and (d) crosssectional TEM images of the RGO@mSiO2−PEG Janus nanocage.

Figure 1. SEM and AFM images of the representative samples: (a, b) GO nanosheet; (c, d) GO@TiO2 composite platelet; (e, f) GO@ TiO2@mSiO2−PEG composite platelet; (g, h) RGO@mSiO2−PEG Janus platelet nanocage.

the GO sheet contour. A magnified TEM image (inset Figure 2a) shows that the platelets are mesoporous. Cross-sectional TEM image indicates that platelets are solid (Figure 2b). After dissolution of TiO2, the mesoporous structure is well remained (Figure 2c). Cross-sectional TEM image indicates that the Janus nanocage contains a slit-shaped cavity (Figure 2d) and transverse mesoporous channels. A thin graphene sheet is present. The cavity is as thin as ∼15 nm, which is consistent with the TiO2 layer thickness (Figure 1d). No collapse or shrinking is observed after the removal of the TiO2 layer. Multiple microstructure of the samples is further characterized by DF-STEM and EDS mapping. For the GO@TiO2@ mSiO2−PEG composite platelet (Figure 3a), Si and Ti elements are homogeneously distributed at the in-plane surface. C element is present at the whole surface. For the RGO@ mSiO2−PEG Janus nanocage (Figure 3b), nearly no Ti element is detected, while the other two elements are present. Crosssectional DF-STEM and EDS mapping analysis further reveals

groups onto GO surface can induce a favorable surface sol−gel process of TBT. GO sheet is enveloped with TiO2 to form the GO@TiO2 composite platelet. The platelet shape is preserved (Figure 1c), which is as thin as ∼15 nm (Figure 1d). Thickness of the TiO2 layer is cal. ∼ 7 nm. It is noted that the composite platelets are uniform in thickness. This implies that each platelet contains single GO sheet. The TiO2 layer thickness is tunable with TBT feeding content. For example, the TiO2 layer can be further thinner (∼4.5 nm) at lower TBT content (Figures S1a and S1b). The composite platelets appear relatively flat, implying that they become more rigid. The GO@TiO2 composite platelets are negatively charged in alkaline solutions (zeta potential is −42 mV), which facilitates a favorable coassembly of silica and a cationic surfactant for C

DOI: 10.1021/acsami.6b03208 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. DF-STEM images and the corresponding EDS mapping results of (a) the GO@TiO2@mSiO2−PEG composite platelet and (b) RGO@ mSiO2−PEG Janus nanocage. Cross-sectional DF-STEM images and the corresponding EDS mapping results of (c) the GO@TiO2@mSiO2−PEG composite platelet and (d) the RGO@mSiO2−PEG Janus nanocage.

the internal microstructure. For the GO@TiO2@mSiO2−PEG composite platelet (Figure 3c), there exists one peak of Ti element at the middle area, which is overlapped with a C element peak. The C element peak should be corresponded to GO. The C element peak is single, implying individual GO sheet is present. Two symmetric Si element peaks are present, corresponding to the silica double layers onto both sides of the TiO2 layer. Two additional C elements peaks correspond to PEG conjugated onto the silica layers. For the Janus RGO@ mSiO2−PEG composite platelet (Figure 3d), the Ti element peak disappears. The single C element peak remains at the middle area. The mesoporous structure is characterized by nitrogen adsorption/desorption isotherms. Both GO@TiO2@mSiO2− PEG platelet and RGO@mSiO2−PEG Janus nanocage exhibit type-IV isotherm (Figure 4a,b), suggesting a mesoporous structure.31 H3-type hysteresis loops reveal that the pores are slit-shaped.31−33 The GO@TiO2@mSiO2−PEG composite platelet possesses a BET surface area of 487 m2/g and pore volume of 0.68 cm3/g. The average BJH pore size is 2.5 nm. After removal of TiO2 and reducing GO into graphene, the BET surface area and total pore volume increase to 539 m2/g and 0.73 cm3/g, respectively. To obtain graphene, we reduced GO with ascorbic acid. The characteristic absorption peak at 231 nm is significantly redshifted to 269 nm after reduction (Figure 4c).34 Raman spectra

Figure 4. Nitrogen adsorption/desorption isotherms and pore size distribution curves of (a) the GO@TiO2@mSiO2−PEG composite platelet and (b) the RGO@mSiO2−PEG Janus nanocage. (c) UV−vis absorption curves of the GO@mSiO2−PEG composite platelet and the RGO@mSiO2−PEG Janus nanocage dispersions. (d) Raman spectra of the GO@mSiO2−PEG composite platelet and the RGO@ mSiO2−PEG Janus nanocage.

reveal that the D/G band ratio greatly increases after reduction (Figure 4d).35 The reduction is further confirmed by FTIR D

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Figure 5. (a) Dispersion behavior of the RGO@mSiO2−PEG Janus nanocage in water and hexane, respectively; (b) residual amount of different species in the solution after treatment with the RGO@mSiO2−PEG Janus nanocage and (inset) CLSM image of the DOX-loaded RGO@mSiO2− PEG Janus nanocage.

(Figure S3). The peaks at 1727 and 1377 cm−1 of GO are assigned to stretching vibration of CO and C−O bonds. The peak at 1623 cm−1 corresponds to the skeletal vibration of unoxidized graphitic domains.36 After reduction of GO, the above oxygen-containing groups related bands nearly disappear. A new peak appears at 1577 cm−1, which is attributed to the skeletal vibration of the graphene.37 The peak at 1212 cm−1 attributed to the stretching vibration of C−O bond of PEG is less influenced. Because the RGO@mSiO2−PEG Janus nanocage possesses PEG moiety onto the external surface, good dispersion in water is guaranteed (Figure 5a, left). In comparison, the nanocage precipitates in oil such as hexane (Figure 5a, right). RGO is hydrophobic, and it is difficult for the nanocage to capture water-soluble species such as MO. In comparison, the Janus nanocage can easily capture oil-soluble dyes such as Sudan III or drugs such as DOX (Figure 5b). A CLSM image shows that the cavity is fully loaded with DOX (inset Figure 5b). Let us focus on DOX-loaded RGO@mSiO2−PEG Janus nanocages because they are promising as a multiple functional drug delivery. The loading capacity increases with the feed content (Table S1). A maximum capacity can reach 80.5 wt % at a DOX/cage ratio of 2:1. Such high loading level is attributed to the large hydrophobic cavity. The Janus nanocage possesses a strong photothermal effect by NIR irradiation. When the Janus nanocage aqueous dispersion (65 μg/mL) is exposed to NIR, temperature increases rapidly at the beginning (Figure 6a). After 5 min, temperature reaches 48 °C from 25 °C. Later, the increment becomes slower. After another 5 min, temperature reaches 54

°C from 48 °C. The photothermal effect is enhanced at higher content of Janus nanocage (Figure S4). In comparison, temperature of the GO@mSiO2−PEG dispersion (with an equal C content as RGO) slightly increases to 37 from 25 °C after 10 min NIR irradiation. No obvious temperature change is observed for pure water by NIR irradiation. In vitro DOX release process is conducted to assess the NIR irradiation effect. The cumulative release of DOX can reach 13.2 wt % within 8 h after 5 cycles of NIR irradiation (Figure 6b). In comparison, only 5.9 wt % is released without NIR irradiation. The drug release during the irradiation is 20 times faster than that without irradiation. To clearly detect cellular uptake by CLSM, we chemically modified the Janus nanocage with FITC. After the Janus nanocage is modified with a small amount of APTMS, FITC is conjugated via the reaction between amino group and isothiocyano group of FITC.38 The Janus nanocage is thus labeled. New peaks appear at 1655 and 1542 cm −1 , corresponding to N−H stretch and C−N vibration (Figure 7a), indicating the successful conjugation of FITC. The FITClabeled Janus nanocage could be detected in a fluorescence spectrum around 528 nm (Figure 7b). After HeLa cells are

Figure 6. (a) Photothermal curves of three representative samples under NIR laser irradiation (808 nm, 1 W/cm2): the RGO@mSiO2− PEG Janus nanocage dispersion (65 μg/mL), the GO@mSiO2−PEG dispersion, and pure water; (b) cumulative release profiles of DOX from the RGO@mSiO2−PEG Janus nanocage delivery, triggered by NIR laser irradiation, each NIR irradiation time is 5 min.

Figure 7. (a) FTIR spectra of the RGO@mSiO2−PEG Janus nanocage before and after being labeled with FITC; (b) fluorescence spectrum of the labeled Janus nanocages at an excitation wavelength of 488 nm; and (c) CLSM images of the cells uptake of the FITC-labeled Janus nanocages. The nuclei are blue after staining with DAPI, the FITClabeled Janus nanocages are green. E

DOI: 10.1021/acsami.6b03208 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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case of 65 μg/mL of DOX@RGO@mSiO2−PEG Janus delivery, as much as 13.2% of DOX can be released from the drug delivery after 5 cycles NIR irradiation for 8 h. The cell viability from this 13.2% of DOX additionally released should not be lower than 57% in the presence of 17.5% of DOX even under NIR irradiation (Figure S5). On the other hand, 56% of cell viability is achieved by single NIR irradiation. Experimentally, 35% of cell viability (much lower than 56−57%) is achieved from the DOX@RGO@mSiO2−PEG Janus delivery under NIR irradiation. This implies that there are some synergetic therapeutic effects. Further mechanism investigation is in progress.

cultured with the modified Janus nanocage for 5 h, the green cytoplasm is clearly observed (Figure 7c), confirming the successful cellular uptake of the FTIC-labeled Janus nanocage. The synergetic therapeutic effect of the DOX@RGO@ mSiO 2 −PEG Janus nanocage delivery is evaluated by measuring the cell viability under different treatments. The cytoplasm becomes red after uptake of the Janus nanocage delivery (Figure 8a). Influence of different treatments on the

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CONCLUSION In summary, we have fabricated the platelet RGO@mSiO2− PEG Janus nanocages, which possess PEG moiety grafted onto the exterior surface and graphene sheet inside the cavity. The Janus nanocages are well dispersible in water, and can selectively load hydrophobic drugs. The loading capacity is high due to the large cavity. The DOX-loaded RGO@mSiO2− PEG platelet Janus delivery can enter the HeLa cells. Drug release from the delivery is accelerated under NIR. A synergistic effect is achieved by combining the chemotherapy and photothermal therapy. The platelet RGO@mSiO2−PEG Janus nanocages are promising as multiple functional drug delivery.

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Figure 8. (a) CLSM images of HeLa cells after uptaking the DOX@ RGO@mSiO2−PEG Janus nanocage delivery for 12 h, the nucleus are blue after staining with DAPI, the RGO@mSiO2−PEG Janus nanocages are red after loading DOX; (b) viability of HeLa cells under different treatments; the cultivation time for every treatment is 24 h; the NIR laser irradiation time is 5 min. Statistical analysis: *p < 0.05; n.s., not significant.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b03208. Examples of SEM images, AFM images, EDX results, FTIR spectra for some composites; DOX loading capacity of Janus nanocages; Example of photothermal curves; HeLa cell viability under some treatments. (PDF)

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cell viability is systematically characterized. More than 95% cell viability is preserved even at the highest tested solid content (65 μg/mL) of the Janus nanocages (Figure 8b), implying that they have no cell cytotoxicity. NIR irradiation on the Janus nanocage can bring remarkable cell cytotoxicity, which is enhanced with increasing nanocage solid content. At the solid content of 65 μg/mL, cell viability is decreased to 56%. In contrast, the cell viability is not affected by the NIR irradiation when the Janus nanocage is not added (Figure S5). Therefore, the cell cytotoxicity is essentially arisen from the photothermal effect of RGO in the Janus nanocage. DOX@RGO@mSiO2− PEG Janus delivery can release DOX progressively and bring cytotoxicity. The cytotoxicity is increased with increasing the delivery content. The effect of released DOX on cytotoxicity is comparable with the NIR irradiation of RGO@mSiO2−PEG Janus cage. The question is if the cell cytotoxicity of DOX@ RGO@mSiO2−PEG Janus delivery can be enhanced by NIR irradiation. At a concentration of 35 μg/mL of delivery with NIR irradiation, the cell viability is decreased to 55%. In comparison with the cell viability of 72% under a single treatment by either release of DOX or NIR irradiation, NIR irradiation facilitates to greatly enhance cell cytotoxicity. At a high content level, for example, 65 μg/mL of Janus nanocage, the DOX@RGO@mSiO2−PEG Janus delivery can lead a very low cell viability of 35% under NIR irradiation. The cell viability is 56% by single NIR irradiation and 65% by released DOX from the DOX@RGO@mSiO2−PEG Janus delivery without NIR irradiation. Let us analyze the combination effect. In the

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Ministry of Science and Technology of China (2012CB933200) and the National Natural Science Foundation of China (51233007, 51173191).

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REFERENCES

(1) De Gennes, P. G. Soft Matter. Rev. Mod. Phys. 1992, 64, 645− 648. (2) Jiang, S.; Granick, S. Janus Balance of Amphiphilic Colloidal Particles. J. Chem. Phys. 2007, 127, 161102. (3) Walther, A.; Matussek, K.; Müller, A. H. E. Engineering Nanostructured Polymer Blends with Controlled Nanoparticle Location Using Janus Particles. ACS Nano 2008, 2, 1167−1178. (4) Wang, F.; Pauletti, G. M.; Wang, J. T.; Zhang, J. M.; Ewing, R. C.; Wang, Y. L.; Shi, D. L. Dual Surface-Functionalized Janus Nanocomposites of Polystyrene/Fe3O4@SiO2 for Simultaneous Tumor Cell Targeting and Stimulus-Induced Drug Release. Adv. Mater. 2013, 25, 3485−3489. (5) Yoshida, M.; Roh, K.; Mandal, S.; Bhaskar, S.; Lim, D.; Nandivada, H.; Deng, X. P.; Lahann, J. Structurally Controlled BioF

DOI: 10.1021/acsami.6b03208 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces Hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells. Adv. Mater. 2009, 21, 4920−4925. (6) Liang, F. X.; Liu, J. G.; Zhang, C. L.; Qu, X. Z.; Li, J. L.; Yang, Z. Z. Janus Hollow Spheres by Emulsion Interfacial Self-Assembled SolGel process. Chem. Commun. 2011, 47, 1231−1233. (7) Zhao, L. L.; Zhu, L. J.; Chen, Y.; Wang, Q.; Li, J. L.; Zhang, C. L.; Liang, F. X.; Qu, X. Z.; Yang, Z. Z. Janus Micro-Reactors. Chem. Commun. 2013, 49, 6161−6163. (8) Chen, Y.; Yang, H. L.; Zhang, C. L.; Wang, Q.; Qu, X. Z.; Li, J. L.; Liang, F. X.; Yang, Z. Z. Janus Cages of Bilayered Polymer-Inorganic Composites. Macromolecules 2013, 46, 4126−4130. (9) Li, J.; Wang, Y. H.; Zhang, C. L.; Liang, F. X.; Qu, X. Z.; Li, J. L.; Wang, Q.; Qiu, D.; Yang, Z. Z. Janus Polymeric Cages. Polymer 2012, 53, 3712−3718. (10) Zhao, Z. G.; Zhu, F. Y.; Qu, X. Z.; Wu, Q. H.; Wang, Q.; Zhang, G. L.; Liang, F. X. pH-Responsive Polymeric Janus Containers for Controlled Drug Delivery. Polym. Chem. 2015, 6, 4144−4153. (11) Wang, Q. G.; Liang, F. X.; Wang, Q.; Qu, X. Z.; Yang, Z. Z. Responsive Composite Janus Cages. Chin. J. Polym. Sci. 2015, 33, 1462−1469. (12) Tang, F. Q.; Li, L. L.; Chen, D. Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. Adv. Mater. 2012, 24, 1504−1534. (13) Muñoz, B.; Ramila, A.; Perez-Pariente, J.; Díaz, I.; Vallet-Regí, M. MCM-41 Organic Modification as Drug Delivery Rate Regulator. Chem. Mater. 2003, 15, 500−503. (14) Chen, A. M.; Zhang, M.; Wei, D. G.; Stueber, D.; Taratula, O.; Minko, T.; He, H. X. Co-Delivery of Doxorubicin and Bcl-2 siRNA by Mesoporous Silica Nanoparticles Enhances the Efficacy of Chemotherapy in Multidrug-Resistant Cancer Cells. Small 2009, 5, 2673− 2677. (15) Lu, J.; Liong, M.; Zink, J. I.; Tamanoi, F. Mesoporous Silica Nanoparticles as a Delivery System for Hydrophobic Anticancer Drugs. Small 2007, 3, 1341−1346. (16) Kim, H. J.; Matsuda, H.; Zhou, H. S.; Honma, I. UltrasoundTriggered Smart Drug Release from a Poly(dimethylsiloxane)Mesoporous Silica Composite. Adv. Mater. 2006, 18, 3083−3088. (17) Zhu, Y. F.; Shi, J. L.; Shen, W. H.; Dong, X. P.; Feng, J. W.; Ruan, M. L.; Li, Y. S. Stimuli-Responsive Controlled Drug Release from a Hollow Mesoporous Silica Sphere/Polyelectrolyte Multilayer Core-Shell Structure. Angew. Chem., Int. Ed. 2005, 44, 5083−5087. (18) Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.; Jeftinija, S.; Lin, V. S.-Y. A Mesoporous Silica Nanosphere-Based Carrier System with Chemically Removable CdS Nanoparticle Caps for Stimuli-Responsive Controlled Release of Neurotransmitters and Drug Molecules. J. Am. Chem. Soc. 2003, 125, 4451−4459. (19) Timko, B. P.; Dvir, T.; Kohane, D. S. Remotely Triggerable Drug Delivery Systems. Adv. Mater. 2010, 22, 4925−4943. (20) Wu, G. H.; Mikhailovsky, A.; Khant, H. A.; Fu, C.; Chiu, W.; Zasadzinski, J. A. Remotely Triggered Liposome Release by NearInfrared Light Absorption via Hollow Gold Nanoshells. J. Am. Chem. Soc. 2008, 130, 8175−8177. (21) Guardado-Alvarez, T. M.; Sudha Devi, L.; Russell, M. M.; Schwartz, B. J.; Zink, J. I. Activation of Snap-Top Capped Mesoporous Silica Nanocontainers Using Two Near-Infrared Photons. J. Am. Chem. Soc. 2013, 135, 14000−14003. (22) Lin, Q. N.; Huang, Q.; Li, C. Y.; Bao, C. Y.; Liu, Z. Z.; Li, F. Y.; Zhu, L. Y. Anticancer Drug Release from a Mesoporous Silica Based Nanophotocage Regulated by Either a One- or Two-Photon process. J. Am. Chem. Soc. 2010, 132, 10645−10647. (23) Melancon, M. P.; Zhou, M.; Li, C. Cancer Theranostics with Near-Infrared Light-Activatable Multimodal Nanoparticles. Acc. Chem. Res. 2011, 44, 947−956. (24) Yang, K.; Zhang, S.; Zhang, G. X.; Sun, X. M.; Lee, S. T.; Liu, Z. Graphene in Mice: Ultrahigh In Vivo Tumor Uptake and Efficient Photothermal Therapy. Nano Lett. 2010, 10, 3318−3323. (25) Wang, Y.; Wang, K. Y.; Zhao, J. F.; Liu, X. G.; Bu, J.; Yan, X. Y.; Huang, R. Q. Multifunctional Mesoporous Silica-Coated Graphene

Nanosheet Used for Chemo-Photothermal Synergistic Targeted Therapy of Glioma. J. Am. Chem. Soc. 2013, 135, 4799−4804. (26) Zhang, L. Y.; Zeng, X. W.; Wang, X. Q. Programmable Hydrogenation of Graphene for Novel Nanocages. Sci. Rep. 2013, 3, 3162. (27) Zhu, S. Z.; Li, T. Hydrogenation-Assisted Graphene Origami and Its Application in Programmable Molecular Mass Uptake, Storage, and Release. ACS Nano 2014, 8, 2864−2872. (28) Yang, K.; Ma, Y. Q. Computer Simulation of the Translocation of Nanoparticles with Different Shapes Across a Lipid Bilayer. Nat. Nanotechnol. 2010, 5, 579−583. (29) Stankovich, S.; Dikin, D. A.; Dommett, G. H. B.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S. Graphene-Based Composite Materials. Nature 2006, 442, 282−286. (30) Teng, Z. G.; Zheng, G. F.; Dou, Y. Q.; Li, W.; Mou, C. Y.; Zhang, X. H.; Asiri, A. M.; Zhao, D. Y. Highly Ordered Mesoporous Silica Films with Perpendicular Mesochannels by a Simple StöberSolution Growth Approach. Angew. Chem., Int. Ed. 2012, 51, 2173− 2177. (31) Sing, K. S. W; Everett, D.; Haul, H. R. A. W.; Moscou, L.; Pierotti, R. A.; Rouquerol, J.; Siemieniewska, T. Reporting Physisorption Data for Gas/Solid System with Special Reference to the Determination of Surface Area and Porosity. Pure Appl. Chem. 1985, 57, 603−619. (32) Yu, B.; Jiang, X. S.; Yin, J. Silica/Titania Sandwich-Like Mesoporous Nanosheets Embedded with Metal Nanoparticles Templated by Hyperbranched Poly(ether amine) (hPEA). Nanoscale 2013, 5, 5489−5498. (33) Wang, Z. M.; Wang, W. D.; Coombs, N.; Soheilnia, N.; Ozin, G. A. Graphene Oxide Periodic Mesoporous Silica Sandwich Nanocomposites with Vertically Oriented Channels. ACS Nano 2010, 4, 7437−7450. (34) Fernández-Merino, M. J.; Guardia, L.; Paredes, J. I.; Villar-Rodil, S.; Solís-Fernández, P.; Martínez-Alonso, A.; Tascón, J. M. D. Vitamin C is an Ideal Substitute for Hydrazine in the Reduction of Graphene Oxide Suspensions. J. Phys. Chem. C 2010, 114, 6426−6432. (35) Tung, V. C.; Allen, M. J.; Yang, Y.; Kaner, R. B. HighThroughput Solution Processing of Large-Scale Graphene. Nat. Nanotechnol. 2009, 4, 25−29. (36) Xu, Y. X.; Bai, H.; Lu, G. W.; Li, C.; Shi, G. Q. Flexible Graphene Films via the Filtration of Water-Soluble Noncovalent Functionalized Graphene Sheets. J. Am. Chem. Soc. 2008, 130, 5856− 5857. (37) Wu, J. L.; Shen, X. P.; Jiang, L.; Wang, K.; Chen, K. M. Solvothermal Synthesis and Characterization of Sandwich-Like Graphene/ZnO Nanocomposites. Appl. Surf. Sci. 2010, 256, 2826− 2830. (38) Liu, J. W.; Stace-Naughton, A.; Jiang, X. M.; Brinker, C. J. Porous Nanoparticle Supported Lipid Bilayers (Protocells) as Delivery Vehicles. J. Am. Chem. Soc. 2009, 131, 1354−1355.

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DOI: 10.1021/acsami.6b03208 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX