Dual pH-Mediated Mechanized Hollow Zirconia Nanospheres - ACS

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Dual pH-Mediated Mechanized Hollow Zirconia Nanospheres MingDong Wang, GuangCai Gong, Jing Feng, Ting Wang, ChenDi Ding, BaoJing Zhou, Wei Jiang, and JiaJun Fu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07603 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

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ACS Applied Materials & Interfaces

Dual pH-Mediated Mechanized Hollow Zirconia Nanospheres MingDong Wang, † GuangCai Gong, † Jing Feng,† Ting Wang,† ChenDi Ding,† BaoJing Zhou,‡ Wei Jiang§ and JiaJun Fu* † †

School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing,

210094, China ‡

Computational Institute for Molecules and Materials, Nanjing University of Science and

Technology, Nanjing, 210094, China §

National Special Superfine Powder Engineering Research Centre, Nanjing University of

Science and Technology, Nanjing, 210094, China KEYWORDS. Hollow mesoporous zirconia nanospheres, supramolecular switches, mechanized hollow zirconia nanospheres, dual pH-stimuli responsive controlled release, targeted drug delivery

ABSTRACT. We demonstrate for the first time to assemble mechanized hollow zirconia nanospheres (MHZNs), consisting of hollow mesoporous zirconia nanospheres (HMZNs) as nanoscaffolds and supramolecular switches anchored on exterior surface of HMZNs. The remarkable advantage of substitution of HMZNs for conventional mesoporous silica nanoscaffolds is that HMZNs can suffer the hot alkaline reaction environment, which provides a

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novel strategy for functionalization and thus achieve dual pH-mediated controlled release functions by simple and practicable assembly procedure. Under neutral solution, cucurbituril[7] (CB[7]) macrocycles complexed with propanone bis(2-aminoethyl) ketal (PBAEK) to form [2]pseudorotaxanes as supramolecular switches, blocking the pore orifices and preventing the undesirable leakage of cargoes. When solution pH was adjusted to alkaline range, CB[7] macrocycles, acting as caps, disassociated from PBAEK stalks and opened the switches due to the dramatic decrease of ion-dipole interactions. While under acidic conditions, PBAEK stalks were broken on account of the cleavage of ketal groups, resulting in the collapse of supramolecular switches and subsequent release of encapsulated cargoes. MHZNs owning dual pH-mediated controlled release characteristic are expected to apply in many field. In this work, the feasibility of doxorubicin (DOX)-loaded MHZNs as targeted drug delivery systems was evaluated. In vitro cellular studies demonstrate that DOX-loaded MHZNs can be easily taken up by SMMC-7721 cells, rapidly release DOX intracellularly and enhanced cytotoxicity against tumor cells, proving their potential for chemotherapy.

Introduction Molecular switches, categorized into molecular machines, are capable of performing predefined mechanical movements and implementing analogous switching functions at a molecular level.1-5 As the prototypical representative, rotaxane or pseudorotaxane, composed of a linear axle-like component threaded a macrocycle, normally operates by shuttling of a macrocycle along the axle or undirectional dethreading/rethreading of a macrocycle from an axle in response to external energy input. Zink and Stoddart firstly employed [2]pseudorotaxanes as supramolecular switches to assemble mechanized silica nanoparticles, realizing stimuli-responsive controlled release purpose.6 The subsequent numerous mechanized silica nanoparticles that respond to various

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stimuli, such as pH,7-10 light,11-14 redox,15-16 enzyme17-19 or combination of them20-24 have been invented over the past decade and witnessed remarkable achievements in this field. Mesoporous silica nanoparticles (MSNs) were commonly used as nanoscaffolds to accommodate cargoes. Recently, in order to purse more special and superior properties, some other inorganic nanoscaffolds have been put into use. Huskens et al. proposed cyclodextrin-modified zeolites as nanocontainers to encapsulate and deliver drug molecules to targeted regions.25 Yang et al. pioneered the utilization of metal-organic frameworks (MOF) as nanoscaffolds, and fabricated diverse mechanized MOFs to achieve pH/competitive binding, Ca2+/pH/thermo and Zn2+/heating triggered release properties.26-28 Moreover, the inherent weakness, the poor alkaline resistance of MSNs restricts the usage of surface functionalization steps under alkaline environment and influences the further assembly process. Zirconia (zirconium dioxide, ZrO2), as important structural functional material owning three different crystalline phase, has been widely employed in numerous applications, including: catalysis, dye-sensitized solar cells, sensors, heat-insulting coating, implantable bioceramics etc.29-31 Mesoporous zirconia, emerging as promising nanoscaffolds for catalyst supports, drug delivery vehicles and corrosion inhibitor containers,32-34 have been reported and displayed the tendency to substitute for MSNs in certain special areas by virtue of their outstanding advantages in chemical inertness, excellent mechanical, thermal and biocompatible characters. It is not difficult to analyze that mesoporous zirconia have fully qualified for mechanical assembly. First of all, the adsorption capacity of mesoporous zirconia with controllable morphology is not inferior to MSNs. Secondly, the abundant terminal hydroxyl groups occurring on surface of mesoporous zirconia can covalently bind with trialkoxy group of silica coupling agents,35,36 which is the crucial step for the following installation of supramolecular switches. Finally, and

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most importantly, the excellent alkaline resistance of mesoporous zirconia will compensate the drawback of MSNs and make the functionalization steps under hot alkaline solution possible. However, to the best of our knowledge, no mechanized zirconia nanoparticles, consisting of mesoporous zirconia nanoscaffolds and supramolecular switches as gatekeepers have been successfully assembled so far. pH signal, in particular acid signal, has been extensively served as trigger to initiate the stimuli-responsive systems because of the specific pH gradients in human body. It is well-known that extracellular environment of tumor tissues as well as intracellular endosomes/lysosomes in tumor cells present a significantly lower pH environment compared to blood or normal tissue.3739

By

taking

advantage

of

existing

pH

differences,

some

delicately

designed

rotaxanes/pseudorotaxanes, such as, polyethyleneimine⊂α-cyclodextrin (α-CD),40 phenylamine or its derivatives⊂α-CD,41 benzimidazole⊂β-CD,8 rhodamine B/benzidine conjugates⊂β-CD,10 ferrocenecarboxylic acid⊂cucurbit[7]uril (CB[7]),42 viologen dicarboxylic acid⊂CB[6],43 bipyridinium⊂carboxylate-substituted

pillar[6]arene23

etc.

have

been

synthesized

and

immobilized on the exterior surface of mesoporous silica to construct controlled release systems, which are able to avoid undesirable side effects and selectively release drug molecules in tumor regions. Meanwhile, the research and development of alkali-responsive mechanized silica nanoparticles have also been in progress. Their successful application in construction of feedback active anticorrosion coatings has opened up new horizon.44-46 Of particular interest is incorporation of acid and alkali stimuli-responsive characteristics into a single mechanized nanoparticle, which will achieve versatile functionalities and provide a unique opportunity to realize more sensitive, accurate and independent stimuli-release. Up to now, the fabrication of complicated bistable [2]pseudorotaxanes seems to be the only approach to execute controlled

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Scheme 1. Schematic illustration of assembly of MHZNs and dual pH-mediated controlled release of RhB. HMZNs were functionalized with [2]pseudorotaxanes on the exterior surface and the chemical structure of [2]pseudorotaxanes containing CB[7] macrocycles and PBAEK stalks is shown. MHZNs are stable in neutral solution and triggered by acid or alkali stimuli. release activated by dual pH stimuli.47,48 Herein, we report a novel, facile approach for assembling dual pH-mediated mechanized zirconia nanospheres. The whole assembly procedure is illustrated in Scheme 1. The [2]pseudorotaxanes on threading of propanone bis(2-aminoethyl) ketal (PBAEK) based axle into CB[7] macrocycles were anchored onto hollow mesoporous zirconia nanospheres to assemble mechanized hollow zirconia nanospheres (MHZNs). Hollow mesoporous zirconia nanospheres (HMZNs), employed as nanoscaffolds enhance cargo loading capacity in one aspect, and more importantly, they can tolerate the functionalization process under hot alkaline environment and acquire the monolayer coverage of supramolecular switches. Under neutral solution, CB[7] macrocycles encircled on the PBAEK stalks, blocking the pore orifices to prevent undesirable leakage of entrapped cargoes. The release mechanisms of MHZNs

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upon different stimuli were investigated. Under alkali stimulus, the original existing ion-dipole interactions between CB[7] macrocycles and two ends of ammonium groups belonging to PBAEK were disrupted. The dethreading of CB[7] macrocycles from PBAEK stalks unlocked the supramolecular switches and cargoes were released from MHZNs. While exposed to acidic environment, PBAEK stalks were broken due to the existence of acid-degradable ketal groups, which led to the collapse of supramolecular switches and realized acid-responsive controlled release. In view of the potential application of MHZNs in targeted drug delivery systems, in vitro cytotoxicity and intracellular release behaviour were evaluated with doxorubicin (DOX) as model drug. We believe that the rational integration of acid/alkali-responsive release functionalities will broaden applications of MHZNs, which are needed to be explored in the future. Results and Discussion Preparation of Hollow Mesoporous Zirconia Nanospheres (HMZNs) as Nanoscaffolds HMZNs were synthesized by sol-gel protection method reported in our previous literature and the stepwise preparation procedure is schematically in Figure 1A.49 HMZNs fabricated by this approach possess the advantages of both controllable intact morphology and sufficient interfacial terminal hydroxyl groups, which are most desirable for further mechanical assembly. Typically, the monodispersed colloidal silica were synthesized as hard templates by Stöber method. Subsequently, a mixture solution containing polyoxyethylene lauryl ether (Brij) as mesoporesdirecting agents and zirconium butoxide as zirconium source were impregnated and coated on colloidal silica to form sSiO2@ZrO2. After deposition of thin SiO2 protective layer by sol-gel process, the calcination under 500 ℃ was proceeded to remove Brij and yield sSiO2@mZrO2@sSiO2. Finally, silica-based materials, including the hard templates and the

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Figure 1. (A) Schematic diagram of preparation procedure of HMZNs; (B, C) TEM images of HMZNs synthesized by sol-gel protection method, the insert image of EDX line-scan analysis; (D) HMZNsnp synthesized without sol-gel protection; (E) N2 adsorption-desorption isotherm and pore-size distribution of HMZNs; (F) XRD patterns of HMZNs and HMZNsnp; and (G) FTIR spectra of HMZNs (a) and HMZNsnp (b). protective layer, were chemically etched by NaOH solution and the resultant HMZNs were activated by HCl/ethanol mixture. Transmission electron microscopy (TEM) images of HMZNs are shown in Figure 1B and 1C, where the electron contrast between the cores and shells confirms their hollow characteristic, the external diameter as well as shell thickness was

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measured to be about 290±20 nm and 20±5 nm, respectively. Energy dispersive X-ray (EDX) line scan-analysis shows the composition of HMZNs is composed of Zr and O (insert image of Figure 1C). Small angle X-ray diffraction pattern of HMZNs shown in Figure S1 (Supporting Information) reveals one intense peak at around 1.1°, suggesting the oriented mesostructure. N2 adsorption-desorption isotherm of HMZNs is shown in Figure 1E. According to the IUPAC classification, the isotherm displays the type Ⅳ profiles with a H1 hysteresis loop, typical for mesoporous materials. The BET surface area, BJH pore size and pore volume were 345.8 m2 g-1, 2.7 nm and 0.23 cm3 g-1, respectively. The hollow mesoporous structure is the guarantee for high loading capacity. In order to emphasize the merits of sol-gel protection method, the step of deposition of SiO2 protective layer was intentionally skipped and the corresponding resultant products were abbreviated as HMZNsnp. The notable difference is that HMZNsnp can no longer keep the perfect hollow mesoporous structure and the partial broken fragments were observed (Figure 1D), which is in sharp contrast to HMZNs in Figure 1B. It is easy to understand that the intact morphology is the basis for controlled release. Obviously, HMZNsnp cannot execute loading or release tasks well and supramolecular switches installed on broken shells will lose their effectiveness. According to the previous literature, the phenomenon for broken HMZNs may be attributed to the high temperature calcination, where the interior stress, stemming from the growth of crystalline grains, rapidly increase, leading to the collapse of shell structure.50 The wide-angle XRD patterns of HMZNs are amorphous in nature reflected by two evident halos located at 29.8° and 50.1°, whereas HMZNsnp yields metastable tetragonal phase, the diffraction peaks located at 2θ=30.3°, 35.1°, 50.7° and 60.1° can be indexed to the (011), (110), (112) and (121) diffractions of tetragonal, respectively (Figure 1F). Comparative analysis verifies the above hypothesis and reveals that the SiO2 protective layer inhibits the phase transformation,

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makes the zirconia shells avoid suffering high interior stress and thus maintains the intact morphology. Furthermore, the forms and quantities of interfacial hydroxyl groups are another crucial factors to influence the following functionalization processes. Three forms of interfacial hydroxyl groups, including: terminal, bibridged and tribridged can be distinguished in FT-IR spectra.30 In Figure 1G, the terminal hydroxyl groups corresponding to the absorption bands at 3774 cm-1 are predominant in spectrum of HMZNs, while those on HMZNsnp at the band of 3730 cm-1 are mainly of bibridged hydroxyl groups. The terminal hydroxyl groups are more suitable for covalent bonding with silica coupling agent than bibridged ones due to the relatively low steric hindrance. On the whole, HMZNs synthesized through sol-gel protection method are ideal Zr-based nanoscaffolds. Assembly and Characterization of Mechanized Hollow Zirconia Nanospheres (MHZNs) In order to conveniently achieve dual pH-mediated controlled release functionalities, a simple and unique pseudorotaxanes-based supramolecular switches, consisting of two key components: PBAEK stalks and CB[7] macrocycles, were designed and anchored onto surface of HMZNs. The assembly procedure of MHZNs represented in Scheme 1 can be divided into functionalization, loading and capping processes. The functionalization of HMZNs was performed in a three-step procedure. Typically, HMZNs were firstly treated with chloromethyltriethoxysilane (CMTES) to obtain HMZNs-CMTES. After that, [N-(2-(2-(2aminoethoxy) propan-2-yloxy) ethyl)-2,2,2-trifluoroacetamide (APYOT) was synthesized (see Supporting Information for synthetic process in detail, Figure S2-S4) and utilized to react with HMZNs-CMTES through nucleophilic substitution reaction to yield HMZNs-APYOT. Following this, HMZNs-APYOT underwent deprotective reaction under hot alkaline environment to remove trifluoroacetyl groups and form HMZNs-PBAEK. The whole

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Figure 2. (A)

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C SSNMR spectra of (a) HMZNs, (b) HMZNs-CMTES, (c) HMZNs-APYOT,

and (d) HMZNs-PBAEK; (B) XPS wide-scan spectra of functionalized HMZNs materials (a), XPS high-resolution N1s core line spectra of HMZNs-APYOT (b) and HMZNs-PBAEK (c); and (C) HAADF-STEM image of HMZNs-PBAEK (a) and the corresponding elemental mapping images (b). functionalization processes were monitored and characterized by 13C CP/MAS solid state NMR (13C-SSNMR) and X-ray photoelectron spectroscopy (XPS) measurements. The

13

C-SSNMR

spectra of HMZNs, HMZNs-CMTES, HMZNs-APYOT and HMZNs-PBAEK are shown in Figure 2A. The

13

C-SSNMR spectrum of HMZNs-CMTES spectrum displays distinctive

resonance signal at 24.1 ppm (Ca), which corresponds to methylene group of CMTES. Compared

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with HMZNs-CMTES, besides the resonance signal in HMZNs-CMTES, several additional signals at 22.6 (Ce,f), 43.5 (Ch), 54.4 (Cb), 60.1 (Cc,g), 100.6 (Cd), 116.2 (Cj) and 158.3 (Ci) ppm appearing in HMZNs-APYOT spectrum prove the presence of APYOT group covalently coupled with HMZNs-CMTES. After deprotective reaction, the disappearance of signals at 116.2 and 158.3 ppm belonging to trifluoroacetyl group suggests the abscission of protective groups. Figure 2B(a) shows the XPS wide-scan spectra of HMZNs-CMTES, HMZNs-APYOT and HMZNsPBAEK. Except for the expectant Zr, O elemental species, the photoemission peaks at 284 eV, 269 eV, 152 eV, and 101 eV respectively assigned to C1s, Cl1s, Si2s and Si2p were observed in spectrum of HMZNs-CMTES, demonstrating the CMTES functionalization process. The appreciable difference between HMZNs-CMTES and HMZNs-APYOT is the appearance of N1s (399 eV) and F1s (687 eV) signals, which is the strong evidence of chemical attachment of APYOT to HMZNs framework. Meanwhile, the vanishment of Cl1s also collaborates the nucleophilic substitution process. As for HMZNs-PBAEK, the complete disappearance of F1s signals proves the proceeding of deprotective reactions. In addition, from the analysis of high resolution of N1s spectra exhibited in Figure 2B(b and c), the N1s core-level photoelectron spectrum of HMZNs-APYOT is separated into two peak components located at 399.0 and 400.1 eV, corresponding to C-NH-C and C-NH-C(=O) group, while two split peaks at 399.0 eV and 400.6 eV are attributed to C-NH-C and C-NH2 in spectrum of HMZNs-PBAEK. This evolution substantiates the changes of chemical components on surface of HMZNs. The high angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was used to investigate the morphology of HMZNs-PBAEK. STEM image shown in Figure 2C(a) reveals that HMZNsPBAEK still maintain the hollow structure, compared with HMZNs, the diameter and shell thickness substantially unchanged. The STEM-EDS maps of Zr, O, Si, C, and N obtained from

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the selected region are shown in Figure 2C(b). A uniform distribution of Si, C and N proves that the shell walls are homogeneously decorated by the PBAEK stalks. Unfortunately, hollow mesoporous silica nanospheres (HMSNs) cannot experience the same simple functionalization processes. HMSNs were synthesized by hard-template method according to the previous literature51 and the first two functionalization processes (CMTES and APYOT functionalized processes) were carried out smoothly, which can be verified by Fourier Transform Infrared Spectroscopy (FTIR) and 13C-SSNMR (Figure S5, S6 and S7, Supporting Information). However, after the deprotective reaction, a good dispersion of HMSNs-APYOT gradually changed to clear solution, and no resultant products were collected by centrifugation or filtration (Figure S8, Supporting Information), which is undoubtedly ascribed to the dissolution of silica-based nanoscaffolds under hot alkaline solution. Obviously, the HMZNs nanoscaffolds are the appropriate choice to adapt to the specially-designed functionalization processes. Rhodamine B (RhB), is chosen as the model cargo and loaded into HMZNs-PBAEK, and then the shell pores were sealed by CB[7] macrocycles to accomplish the assembly procedure of MHZNs. The thermogravimetric analysis (TGA), N2 adsorption-desorption isotherms and FTIR spectra were utilized to track the loading and capping processes. The amount of CMTES, APYOT and PBAEK grafted onto HMZNs were calculated from mass differences shown in TGA curves (Figure S9, Supporting Information) and determined as 1.27, 0.4 and 0.36 mmol g-1 HMZNs, respectively. Almost the same quantities of APYOT and PBAEK test the complete deprivation of protective groups. After measuring the mass difference between HMZNs-PBAEK and MHZNs without loading RhB, CB[7] macrocycles binding with PBAEK stalks was 0.17 mmol g-1 HMZNs. The molar ratio of CB[7] to PBAEK is about 1:2, which means that not all PBAEK stalks are encircled by CB[7] macrocycles due to the stereo-hindrance effect (Figure

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S10, Supporting Information). The effective loading capacity of RhB was approximately 170 mg g-1 HMZNs, which is in the same level as that for mechanized hollow mesoporous silica nanoparticles.15,48 During the assembly procedure, all the texture parameters persistently decrease, the BET surface area and BJH pore volume of lower to 110 m2 g-1 and 0.05 cm3 g-1 (Figure S11 and Table S1, Supporting Information). It can be inferred that the mesopore structure was dramatically altered by the installation of PBAEK stalks and the adsorption of RhB molecules. The FTIR spectra of HMZNs, HMZNs-CMTES, HMZNs-APYOT, HMZNs-PBAEK and MHZNs are presented in Figure S12 (Supporting Information). Combined with the characteristic adsorption peaks of each material, the whole assembly procedure can be clearly identified and confirmed. The colloidal stability of MHZNs was investigated in phosphate buffer solution (pH 7.0) by simple turbidity tube method,52 no precipitation was observed after 120 min. Controlled Release of RhB from MHZNs by Dual-pH Mediation The dual pH-mediated controlled release of RhB from MHZNs is investigated in water solution. MHZNs were packed into dialysis membrane in the upper of quartz cuvette and then neutral Tris buffer solution (pH 7.0) was carefully added to avoid disturbance. Firstly, the alkali-triggered, real-time release behaviours were continuously monitored by UV-Vis spectroscopy. As shown in Figure 3A, under neutral solution, no obvious UV-Vis absorbance signals were detected at λ=554 nm in the first 2 h, illustrating the closed state of [2]pseudorotaxane-based supramolecular switches and no undesirable leakage of RhB. Upon adjusting the pH to alkaline range, the abrupt increasing of UV-Vis absorbance intensity was observed, demonstrating that the supramolecular switches were opened by alkali stimulus followed by the release of RhB. The release rate, defined as the cumulative release percentage of RhB from MHZNs within 3 h, was directly correlated with the alkalinity of solution. The maximum release rate was reached when pH value

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Figure 3. (A) Alkali-triggered release profiles of RhB from MHZNs under different alkalinity and UV absorbance spectra of the supernatant solution after completion of each release experiment; (B) The probable inclusion configuration between CB[7] and PBAEK under neutral solution calculated by MD/QM/CSM model; (C) 1H NMR spectra (300 MHz, D2O) of (a) 5 mM CB[7], pD=7.0, (b) 5 mM PBAEK, pD=7.0, (c) 5 mM PBAEK and 5 mM CB[7], pD=7.0, (d) 5 mM PBAEK and 5 mM CB[7], pD=12.0, and (e) 5 mM PBAEK, pD=12.0; (D) ITC isotherms for the injection of 1.2 mM solution of CB[7] into 0.2 mM PBAEK solution (pH=7.0) at 298 K (a), and the injection of 2.0 mM solution of CB[7] into 0.2 mM PBAEK solution (pH=12.0) at 298K (b). was tuned to 12.0, at this circumstance, the release rate was 44.9%, whereas only 37.8%, 31.1% and 20.2% was achieved under pH 11.0, 10.0 and 9.0, respectively. The operation of

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supramolecular switches is definitely responsible for the alkali-responsive controlled release of RhB. There have been some reports concerning the construction alkali-responsive mechanized silica nanoparticles by installation of [2]pseudorotaxanes, including: [+NH3-(CH2)6-+NH2⊂CB[6]]

and

[+NH3-(CH2)-(C2N3H)-CH2-+NH2-⊂CB[6]

or

CB[7]]

as

supramolecular

nanovalves, which normally works relying on changes of ion-dipole interactions influenced by solution pH.53,54 In this work, PBAEK stalk, in which the ketal group is in the central position of linear molecule, and the two ends are amine groups, have the analogue structure to these known guest molecules. The structure of host (CB[7])-guest (PBAEK) complex was first predicted by molecular dynamics/quantum chemistry/continuous solution model (MD/QM/CSM, see Supporting Information for the detailed method). The probable supramolecular configurations with high theoretical binding affinity shown in Figure 3B demonstrate PBAEK threads through CB[7], in particular that the ketal group includes into the hydrophobic cavity of CB[7], and the ammonium groups (pKa=9.09) are close to the portal carbonyl groups of CB[7] under neutral solution. For more insight into the work behaviour of supramolecular switches anchored on exterior surface of HMZNs, 1H NMR spectroscopy was carried out. Figure 3C shows the 1H NMR spectra recorded on D2O of PBAEK (see Supporting Information for synthetic process in detail, Figure S13-S15), CB[7] and their complexes under neutral and alkaline solution. As compared to individual PBAEK, when 1.0 equiv CB[7] was added into PBAEK solution, the upfield displacements of Hb (Δδ=-0.42ppm) and Hc (Δδ=-0.62ppm) and the slight downfield displacement of Ha (Δδ=0.22ppm) were observed (Figure 3C(c)). It is well-documented that if the protons are located in the CB cavity, the proton resonances always exhibit upfield shifts due to the shielding effect, whereas the protons are outside of CB cavity and neighbouring the portal

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carbonyl groups of the CB, the proton resonances show downfield shifts ascribing to the deshielding effect.55,56 Based on this, the shifting trends of PBAEK protons confirm the formation of a [2]pseudorotaxane, [PBAEK⊂CB[7]] under neutral solution, where the ketal group completely enters into hydrophobic cavity and the ammonium groups are protrudent and enmeshed in the portal carbonyl groups. This structure is fully consistent with the theoretical configuration described above. The weak hydrophobic interaction and the predominant strong ion-dipole interaction between ammonium groups of PBAEK and carbonyl groups of CB[7] are the main forces to assemble host-guest supramolecular structure. The binding affinity of [PBAEK⊂CB[7]] was quantitatively investigated by means of isothermal titration calorimetry (ITC) and calculated as 1.04×106 M-1 (Figure 3D(a)). Considering the spatial locations of all the components of MHZNs, CB[7] macrocycles, encircling on PBAEK stalks, act as caps to seal the openings, which is accountable for no undesirable leakage of RhB under neutral solution. Once pH of [PBAEK⊂CB[7]] solution was adjusted to 12.0, the 1H NMR spectrum displays the simple superposition of chemical shifts of PBAEK and CB[7] (Figure 3C(d)), suggesting the destruction of original [2]pseudorotaxane. Meanwhile, the binding affinity was measured as only 3.86×104 M-1, which is about two orders of magnitudes lower than that under neutral solution. Under pH 12.0, the deprotonation of ammonium groups destroys the ion-dipole interactions, decreases the binding affinity between host and guest components, and thus results in the dissociation of CB[7] from PBAEK. During release experiment under alkaline solution, the separation of CB[7] from MHZNs means that the supramolecular switches are unlocked and RhB can freely diffuse out. In addition, within the pH range of 9.0 to 12.0, the higher alkalinity, the deeper degree of deprotonation, and the more CB[7] macrocycles leave for MHZNs from the microscopic aspect, which is the principal reason for the differences in release rate.

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Figure 4. (A) Acid-triggered release profiles of RhB from MHZNs under different acidity; (B) 1

H NMR spectra (300 MHz, D2O) of (a) 5 mM PBAEK and 5 mM CB[7], pD=7.0, and (b) 5 mM

PBAEK and 5 mM CB[7], pD=2.0; (C)

13

C SSNMR spectrum of ethanolamine-N-methyl

triethoxysilane functionalized HMZNs.

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The following focus was concentrated on the acid-triggered release characteristic of MHZNs and the cumulative RhB released from MHZNs as a function of time under different acidic pH is shown in Figure 4A. It is natural that only approximately 0.9 % RhB was leaked at pH 7.0 within the initial 2 h. When the pH value decreased from pH 7.0 to acidic range, RhB was released instantly, indicating the activation of supramolecular switches. The release rates show the pronounced acidity dependence. When the release experiment was conducted at pH=2.0, the maximum release rates was obtained as 67.4%, which is ca. 1.9 and 3.5 fold higher than that under pH 4.0 and 6.0, respectively. This phenomenon can be explained by the existence of ketal groups within PBAEK stalks. Some research groups have introduced the fabrication of pH-sensitive polymers or hydrogels by taking advantage of acetal or ketal bonds.57-59 To better understand the release mechanism of MHZNs under acidic conditions, 1H NMR and 13C SSNMR measurements were also adopted. As shown in Figure 4B, the 1H NMR spectrum of [PBAEK⊂CB[7]] at pD=2.0 shows the distinct resonance signal at 2.06 ppm originating from the cleavage product of acetone, and the additional signals at 3.66 and 2.98 ppm are assigned to methylene protons belonging to another hydrolysis product of 2-aminoethanol (Figure S16, Supporting Information). These 1H NMR results validate that PBAEK is completely degraded into acetone and 2-aminoethanol under strongly acidic attack. From the view of the established structure of [PBAEK⊂CB[7]] under neutral solution, the ketal group occupies the certain space of CB[7] cavity, which will stop RhB molecules with relatively large molecular volume pass through CB[7] cavity. However, hydrogen ions can easily penetrate into hydrophobic cavity due to their negligible molecular volume, protonate oxygen atoms of ketal group and thus facilitate the acid-hydrolysis process according to the proven reaction route (insert image of Figure 4A). After accomplishing release

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experiment under pH 2.0, the rest solid products in dialysis membrane were recollected and washed thoroughly with deionized water. The

13

C SSNMR spectrum demonstrates the stalks

remaining on the surface of HMZNs coincide with our expectant chemical structure (Figure 4C). It can be concluded that under strongly acidic attack, the supramolecular switches are disintegrated into free CB[7] macrocycles, acetone and RhB-loaded, ethanolamine-N-methyl triethoxysilane functionalized HMZNs. It is clear that the individual stalks cannot prevent RhB molecules diffusing out of HMZNs, and the disruption of supramolecular switches is responsible for the dramatical increasing of UV-Vis absorbance intensity. The hydrolysis kinetics of ketal bonds have been confirmed to correlate with acidity.60 These hard evidences can be presented to interpret the acidity-dependent release rate of MHZNs. Zheng et al. reported HMZNs as drug delivery in the previous literature.33 From the point of view of controlled release results, although there are certain electrostatic interactions between HMZNs and encapsulated cargoes, the premature leakage of cargoes from HMZNs was clearly observed under pH 7.4. In this work, after installation of supramolecular switches on the exterior surface of HMZNs, the phenomenon of undesirable leakage disappeared, which will help to reduce side effects when MHZNs are applied as drug delivery systems. Two control experiments were designed to further prove the gatekeeper role of CB[7] as well as the necessity of three-step functionalization processes. In the first place, MHZNs-1, prepared following the same route as MHZNs, except for the removement of CB[7] capping process was tested. In this release curve, the UV-Vis absorbance intensity slowly increased and the total amount of RhB in supernatant after 24 h release was only 25 mg g-1 HMZNs (Figure S17, Supporting Information). More importantly, either alkali or acid stimuli could not drastically alter the release rate, meaning that MHZNs-1 basically lost the controlled release characteristics.

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Figure 5. The release profiles of RhB from MHSNs synthesized through two-step functionalization processes. PBAEK stalks were directly linked with HMSNs-CMTES. The insert image illustrates the probable structure of MHSNs and presents the reason for the slow leakage of RhB under neutral solution. Without CB[7] as gatekeepers, the vast majority of RhB molecules were washed out of MHZNs1 during preparation process. No stimuli-responsive release of a tiny amounts of residue RhB molecules also supports that the association/dissociation of CB[7] macrocycles with PBAEK stalks is essential to realize the switch functions. In the next place, we attempted to delete the third-functionalization step of deprotective reaction and graft PBAEK stalks directly onto exterior surface of CMTES functionalized hollow mesoporous nanoscaffolds. HMSNs were chosen as nanoscaffolds due to the abridgement of hot alkaline reaction environment. HMSNs were successively reacted with CMTES and PBAEK to obtain HMSNs-PBAEK. After loading and capping processes, mechanized hollow silica nanoparticles (MHSNs) were constructed (see Supporting Information for detailed synthetic procedure). From the release profiles in Figure 5, a

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gradual release was noticed under neutral solution. Compared with the zero premature release of MHZNs, 10.1% amount of RhB released in supernatant solution within first 2 h indicate the poor blocking effect of supramolecular switches. Upon acid or alkali stimuli, the accelerated RhB release rate was still apparent. Different from the conventional mesoporous materials, the supramolecular switches are required to cover and seal every orifice of HMSNs perfectly, otherwise the entrapped cargoes will leak from the missing orifices. Concretely, the two ends of PBAEK molecule are primary amine groups. During the PBAEK-functionalization step, one PBAEK molecule maybe simultaneously link with two HMSNs by a pair of active amine groups, which will cause that CB[7] macrocycles cannot reside on the PBAEK stalks and make the corresponding pore orifices open for entrapped RhB molecules as shown in the insert image of Figure 5. Although, the acid/alkali stimuli initiated the normal supramolecular switches and provided the more entrances for escape, enhancing the release rate of RhB, the undesirable release phenomenon occurring under neutral solution will influence their practical application. Overall, in this work, the three-step functionalization processes are the essential prerequisite and the choice of HMZNs is crucial. In Vitro Cytotoxicity and Intracellular Release Behaviour One of the potential application of MHZNs owing dual pH-mediated controlled release property is targeted drug delivery system. In order to verify their application feasibility, DOX, as a hydrophilic anticancer drug, was loaded in MHZNs to replace RhB and the in vitro release of DOX was first studied. The total amount of encapsulated DOX was evaluated as 139 mg g-1 MHZNs. In order to simulate the weak acidic micro-environment surrounding tumor cells, the pH stimulus was firstly set as 6.0, which is close to the extracellular environment of tumor cells.61 Subsequently, the in vitro release of DOX was performed under pH 5.5 and 5.0, which

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Figure 6. (A) Acid-triggered release of DOX from DOX-loaded MHZNs; (B) In vitro SMMC7721 cell viability studied by MTT assay. Cells were treated with MHZNs without loading, DOX-loaded MHZNs and free DOX at equiv. concentration for 48 h (DOX concentration: 0.695, 1.39, 3.475, 6.95, and 13.9 μg mL-1); and (C) Fluorescence microscopy images of SMMC-7721 incubated with the DOX-loaded MHZNs (10 μg mL-1). From left to right: Hoechst 33342 (blue), DOX (red) and a merge of the two images. 22


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are near the pH values of endosome and lysosome in tumor cells.62 As depicted in Figure 6A, DOX-loaded MHZNs exhibited the anticipative acid-triggered release of DOX due to the good working of supramolecular switches as well as the similar molecular volume between RhB and DOX. There is the negligible leakage of DOX under psychological pH 7.4 (less than 1.2%). While the pH values decrease to 6.0, 5.5 and 5.0, the release amount of DOX were 52.6%, 72.5% and 91.9% after 48 h, respectively. Before biological tests, the hydrodynamic diameter and zeta potential of DOX-loaded MHZNs were determined. The hydrodynamic diameter of DOX-loaded MHZNs measured by dynamic light scattering (DLS) analysis in PBS (pH 7.4) was 420 nm, which is larger than that observed by TEM (Figure 1B) due to the existence of hydration corona around particles (Figure S18, Supporting Information). There is no obvious difference in hydrodynamic dimeters between the initial stage and after 120 min dispersion, suggesting their high colloidal stability. Zeta potential of DOX-loaded MHZNs at pH 7.4 was measured as +11.2 mV. In vitro cytotoxicity of MHZNs, DOX-loaded MHZNs and free DOX on SMMC-7721 cells was determined by the MTT assay and the corresponding histograms are shown in Figure 6B. SMCC-7721 cells have been used for evaluating the effectiveness of pH-responsive drug delivery systems due to inherent weak acidic micro-environments of cancer cells.63,64 Compared with the blank control, after 48 h incubation, MHZNs without loading DOX demonstrate scarce cytotoxicity against SMMC-7721 cells over the concentration range of 5–100 μg mL-1. In contrast, DOX-loaded MHZNs induced significant anti-tumor performance with a cytotoxicity effect close to that of free DOX with the equivalent dosage. The cell viability decreased with increasing concentration of DOX-loaded MHZNs. Up to the concentration of 100 μg mL-1 (13.9 μg DOX equiv./mL), the cell viability reduced to approximately 17%. Compared with free DOX, DOX-loaded MHZNs show slightly lower cytotoxicity to SMMC-7721 cells at the same dosage

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of DOX. Free DOX moleucles can diffuse into cells and reach the active sites easily. Combined with the in vitro release results in Figure 6A, it can be inferred that DOX molecules entrapped in MHZNs were effectively released inside the cells. The fluorescence microscopy was further used to observe the cellular uptake and intracellular release behaviour. The excitation wavelengths were set as 405 nm for Hoechst and 480 nm for DOX to determine the precise location of SMMC-7721 and DOX-loaded MHZNs. It can be seen from Figure 6C, after being cultured with DOX-loaded MHZNs for 3 h, the cell nuclei stained by Hoechst 33342 were visible, but only very weak red fluorescence emitted from DOX was distinguished. The previous literature has pointed out that when DOX is encapsulated into nanoscaffolds, no fluorescence will be observed due to the self-quenching effect.65 The red fluorescence became stronger as the incubation time went on, indicating that the DOX continued to escape from MHZNs. Notably, the distribution regions of the increased red fluorescence are from cytoplasm (after 12 h incubation) to cell nuclei (after 24 h incubation) in the merged image, which indicates that SMMC-7721 cells were capable of internalizing DOX-loaded MHZNs by endocytosis and DOX was subsequently released activated by weak acidic stimulus arising from endo/lysosomes. The accumulation of DOX in endo/lysosomal compartments gradually transferred into the nuclei of SMMC-7721 cells and finally showed the killing effect. These observations are agreement with the in vitro cytotoxicity results. The low cell viability of incubation with DOX-loaded MHZNs is ascribed to the effective delivery and intracellular release of DOX into the nuclei of SMMC-7721 cells, inhibiting the proliferation of cancer cells. In order to confirm the cytotoxicity to normal cells, HEK 293T cells, as the non-cancer cells, were used for comparison. HEK 293T cells incubated with unloaded MHZNs were almost non-toxic at diverse concentrations (cell viability>90%), presenting the good biocompatibility of MHZNs (Figure S19, Supporting Information).

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Furthermore, with respect to cytotoxicity toward SMMC-7721 cells, DOX-loaded MHZNs exhibited remarkably lower cytotoxicity against HEK 293T cells. These comparable results demonstrate that DOX-loaded MHZNs have selective killing effect and the supramolecular switches are closed under psychological environments of normal cells. Conclusion In conclusion, we have fabricated a novel dual pH-mediated controlled release system, MHZNs, based on HMZNs as nanoscaffolds that were surface-decorated with simple-structured [2]pseudorotaxanes as supramolecular switches. HMZNs, synthesized via sol-gel protection method and as the substitute for available mesoporous silica nanoparticles to accommodate cargoes, overcome the weakness of poor alkaline resistance of silica based nanoscaffolds, and thus make the specially-designed functionalization processes more efficient and feasible. The mechanically interlocked molecules blocked pore orifices and prevent premature release of cargoes under neutral solution. Upon acid or alkali stimuli, the supramolecular switches were opened to cargoes for by disassociation of [PBAEK⊂CB[7]] supramolecular architecture or broken of the functional PBAEK stalks. Taking into consideration of pH gradient in tumor cells, MHZNs have the potential for targeted drug delivery. The in vitro investigations have demonstrated that MHZNs are non-toxic with satisfactory cellular internalization capacity. Effective cellular uptake of MHZNs and acid-responsive controlled release property ensure the delivery of anticancer drugs into SMMC-7721 nuclei for high cytotoxicity toward tumor cells. Furthermore, the alkali-triggered controlled release property widen the application fields of MHZNs, which need to further developed and utilized in our future work. Experimental Section Materials and Methods

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Tetraethyl orthosilicate (TEOS, 99.0%), polyoxyethylene lauryl ether (Brij 30, average Mn～ 362), zirconium(iv) butoxide (80 wt% in 1-butanol), polyvinylpyrrolidone (PVP, average Mn～ 40000), N-(2-hydroxyethyl)-phthalimide (99.0%), chloromethyltriethoxysilane (CMTES, 96%), 2-methoxy propene (97.0%), p-toluenesulfonic acid (pTSA, 98.0%), ethyl trifluoroacetate (99.0%), cetyltrimethylammonium bromide (CTAB, 99.0%), rhodamine B (RhB, 99.0%), [2−(acryloyloxy)ethyl] trimethylammonium chloride solution (AETAC, 80 wt% in water), 2’2azobis(2-methylpropionamidine) dihydrochloride (AMPAD, 97%), tetrazolium bromide (MTT, 98%), cucurbit[7]uril (CB[7]) and doxorubicin hydrochloride (DOX, 98%) were purchased from Sigma-Aldrich. All the other solvents and regents were of analytical grade and used without further purification. Water used in all the experiments was obtained via a Milli-Q water system with a resistivity of 18.0 MΩ cm. Transmission electron microscopy (JEM-2100, JEOL) was used to examine the morphology of the HMZNs and HMSNs. Fourier transform infrared (FTIR) spectra were recorded on a Bruker Tensor 27 FTIR spectrometer. The N2 adsorption-desorption isotherms were obtained at 77 K on a Quanta chrome Nova 1000 Micrometric apparatus by static adsorption procedures. X-ray diffraction (XRD) pattern of the materials was obtained in Bruker D8 Advanced diffractometer using Cu Kα irradiation (λ=0.15406 Å). Thermogravimetric analysis of the materials was performed using a Mettler TGA-SDTA 851e instrument with a heating rate of 10 ℃ min-1 under nitrogen flow as protective gas. Prior to measurements, all the samples were outgassed at 200 ℃ for 12 h in vacuum. 1H and 13C NMR spectra were collected using a Bruker DRX 300 spectrometer at 300 and 75 MHz. The solid state 13C CP-MAS NMR measurements were taken by a Bruker DSX 400 NMR spectrometer, operating at Larmor frequency of 100.6 MHz and the magic angle spin rate was set to 10 kHz. The X-ray photoelectron spectra (XPS) of the powder were obtained on a PHI QUANTERA II X-ray

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photoelectron spectrometer, using a monochromatic Al Kα radiation (λ= 8.4 Å) as the exciting source. All binding energies (BE) were referenced to the adventitious C1s peak at 284.4 eV. Scanning transmission electron microscopy (STEM) imaging and EDS maps were recorded on FEI-Tecnai G2 F30 S-TWIN TEM operated at 200 kV. Isothermal titration calorimetry experiments were conducted on a Malvern MicroCal VP- ITC apparatus. UV/Vis spectroscopy was carried out with a Shimadzu UV-2550 spectrometer. The hydrodynamic diameters were measured by dynamic light scattering (DLS) using a Malvern Zetasizer 90 in PBS (pH 7.4) at 25 ℃, and the zeta potential was also measured by the same instrument at pH 7.4. Preparation of HMZNs Monodispersed silica particles as hard templates were first prepared by the Stöber method. For a typical procedure, ammonium solution (3.5 mL) and H2O (10 mL) were added to ethanol (80 mL). After stirring vigorously for 30 min, TEOS (5 mL) was added, and the mixture was stirred for another 12 h at 30 ℃. The solid SiO2 were obtained by centrifugation and dispersed in ethanol (200 mL) solution containing Brij 30 (0.3 mL) and H2O (0.3 mL). After stirring for 1 h, zirconium butoxide (2 mL) was added dropwise to the mixture and stirred for 12 h. The sSiO2@ZrO2 was collected by centrifugation, washed thoroughly with methanol, dispersed in water (40 mL) and aged for 24 h. For modifying the surface of zirconia, PVP (0.1 g in 10 mL H2O) was added to the mixture and stirred for 12 h. After collecting with centrifugation, the solid was redispersed in ethanol (80 mL) containing ammonium solution (3.5 mL) and H2O (10 mL). Through adding additional TEOS (6 mL), the mixture was stirred for 12 h to form sSiO2@ZrO2@sSiO2. After calcining at 550 ℃ for 6 h, the products, sSiO2@mZrO2@sSiO2, were chemically etched by NaOH (5 M) at 70 ℃ to remove silica based materials. After

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activation in mixture of HCl/methanol (1:50, vol%) at 70 ℃ for 6 h, HMZNs were washed with abundant water and ethanol, and dried under vacuum for further use. Preparation of HMZNs-CMTES HMZNs (200 mg) were suspended in anhydrous toluene (20 mL). CMTES (100 μL, 0.48 mmol) was added dropwise to the mixture and the suspension was stirred under refluxing in the N2 atmosphere overnight. The HMZNs-CMTES were separated by centrifugation, washed with anhydrous toluene and methanol, and dried under vacuum at 60 ℃ overnight. Preparation of HMZNs-APYOT HMZNs-CMTES (200 mg) were suspended in anhydrous toluene (20 mL). APYOT (180 mg, 0.70 mmol) in anhydrous toluene (1 mL) was added dropwise to the mixture and the suspension was stirred under refluxing in the N2 atmosphere overnight. The HMZNs-APYOT were obtained with centrifugation and washed with anhydrous toluene, the products were dried under vacuum at room temperature overnight. Preparation of HMZNs-PBAEK HMZNs-APYOT (200 mg) was suspended in a mixture of methanol (2 mL) and NaOH (6 M, 18 mL). The suspension was stirred under refluxing for 12 h for deprotection of trifluoroacetyl groups. The resulting solid products, HMZNs-PBAEK were isolated by centrifugation, washed thoroughly with abundant methanol, and dried under vacuum at 60 ℃ overnight. Preparation of MHZNs HMZNs-PBAEK (50 mg) was suspended in an ethanol solution of RhB (0.5 g, 5 mL) with the aid of sonication and the suspension was stirred for 24 h at room temperature. The RhB-loaded HMZNs-PBAEK were separated by centrifugation and dried under vacuum overnight. After loading, the PBS solution (pH 7.0, 5 mL) containing CB[7] (50 mg), NaCl (5 mg) and RhB (10

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mg) was prepared. RhB-loaded HMZNs-PBAEK were re-suspended in the above solution. The resulting reaction mixture was stirred for 3 days at room temperature. The MHZNs were collected by centrifugation, washed with excess PBS (pH 7.0), and dried under vacuum overnight. Controlled Release Experiments In order to study the dual pH-mediated controlled release property of MHZNs in aqueous solution, UV/Vis spectroscopy was used to monitor the concentration of RhB in supernatant solution. The RhB-loaded MHZNs (0.5 mg) were placed in the dialysis membrane (MWCO: 1000 Da) and immersed into PBS solution (pH 7.0, 8 mL). The release of RhB from MHZNs was realized by adjusting the aqueous solution to the desired pH with NaOH or HCl solution. The amount of the released RhB from MHZNs was determined the UV-Vis absorbance intensity at λ=554 nm in reference to the standard calibration curve. Similarly, as for DOX-loaded MHZNs, to simulate the intracellular environment, the in vitro release experiments were investigated in different PBS solution (pH=7.4, 5.5, 4.5). For each study, DOX-loaded MHZNs (0.5 mg) packed in dialysis membrane were placed in 8.0 mL PBS solution. The real-time release behaviour of DOX from MHZNs was monitored by UV-Vis spectroscopy at the wavelength of 480 nm, recorded at predetermined times. The amount of released DOX was quantified by comparing the standard calibration curve. Cell Culture Human hepatoma cell lines (SMMC-7721 cells, Institution of Biochemistry and Cell Biology, CAS, China) and human embryonic kidney epithelial cell lines (HEK 293T cells, Shang Hai Haoran Biological Technology Co. Ltd., China) were respectively maintained in Roswell Park Memorial Institute medium (RPMI-1640, GIBCO) and Dulbecco’s Modified Eagle Medium

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(DMEM) containing 10% fetal bovine serum (FBS), 100 U mL-1 penicillin, and 100 μg mL-1 streptomycin. The cells were cultured at 37 ℃ in a 5% CO2 humidified environment. Cytotoxicity Assay MTT assays were performed to evaluate the cytotoxicity. Briefly, SMMC-7721 cells or HEK 293T cells were seeded into 96-well plates at a density of 5 × 104 cells per well and cultured at 37 ℃ under 5% CO2 to allow stabilization. After incubation overnight, free DOX, unloaded MHZNs and DOX-loaded MHZNs with different concentration were added to the medium, and the cells were incubated in 5% CO2 at 37 ℃ for 48 h. Then the cells were washed three times with PBS (pH 7.4) and arranged for the MTT assay. MTT reagent (20 μL, 5 mg mL-1) was added to each well and incubated for another 4 h at 37 ℃ to convert MTT to a purple formazan product. The medium was withdrawn, the MTT-formazan generated by live cells was dissolved in 150 μL of DMSO, and the absorbance at a wavelength of 490 nm of each well was measured using a microplate reader (Bio-Rad model 680). All the tests were performed in triplicate. Intracellular Release of DOX Observed by Fluorescence Microscopy SMMC-7721 cells were first seeded into 12 well plates at 5 × 104 cells per well in RPMI-1640 medium for 24 h. After that, DOX-loaded MHZNs with a final concentration of 10 μg mL-1 were added to each well. The cells were cultured for 1, 3, 12 and 24 h in RPMI 1640 medium at 37 ℃. The cells were washed three times with PBS (pH 7.4) and fixed with fresh 4.0% formaldehyde for 15 min. Finally, Hoechst 33342 (0.5 μg mL-1, 20 μL per well) was used to stain the nuclei of cells. After completely removing excess dye molecules, the intracellular localizations of released DOX from DOX-loaded MHZNs were directly visualized via a fluorescence spectrophotometer (Eclipse Ti-E, Nikon, Japan). ASSOCIATED CONTENT

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Supporting Information. Synthesis procedure, calculation method, small angle XRD, TEM images, preparation and characterization of materials, 1H spectrum and control experiment. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank the Fundamental Research Funds for the Central University, No. 30915011312 and No. 30915012207; QingLan Project, Jiangsu Province, China; the National Science Foundation of Jiangsu Province (No. BK20161496); A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); Industrial Research Project, No. CG1421, Science and Technology Bureau of Lianyungang City; Prospective Joint Research Project, No. BY2015050-01, Jiangsu Province. REFERENCES [1] Song, N.; Yang, Y. W. Molecular and Supramolecular Switches on Mesoporous Silica Nanoparticles. Chem. Soc. Rev. 2015, 44, 3474-3504. [2] Yang, Y. W.; Sun, Y. L.; Song, N. Switchable Host-Guest Systems on Surfaces. Acc. Chem. Res. 2014, 47, 1950-1960.

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Dual pH-Mediated Mechanized Hollow Zirconia Nanospheres - ACS

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