Synthesis of Hollow Mesoporous Silica Nanorods with Controllable

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Synthesis of Hollow Mesoporous Silica Nanorods with Controllable Aspect Ratios for Intracellular Triggered Drug Release in Cancer Cells Xue Yang, Dinggeng He, Xiaoxiao He, Kemin Wang, Jinlu Tang, Zhen Zou, Xing He, Jun Xiong, Liling Li, and Jingfang Shangguan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05065 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 15, 2016

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ACS Applied Materials & Interfaces

Synthesis of Hollow Mesoporous Silica Nanorods with Controllable Aspect Ratios for Intracellular Triggered Drug Release in Cancer Cells

Xue Yang‡, Dinggeng He‡, Xiaoxiao He*, Kemin Wang*, Jinlu Tang, Zhen Zou, Xing He, Jun Xiong, Liling Li, Jingfang Shangguan

College of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082 (China)

* Address correspondence to these authors at: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P.R. China. Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]; [email protected].

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Abstract Here, we have reported a straightforward and effective synthetic strategy for synthesis of aspect-ratios-controllable mesoporous silica nanorods with hollow structure (hMSR) and its application for transcription factor (TFs)-responsive drug delivery intracellular. Templating by an acid-degradable nickel hydrazine nanorods (NHNT), we have first synthesized the hollow dense silica nanorods and then coated on a mesoporous silica layer. Subsequently, the dense silica layer was removed by the surface-protected etching method and the hollow structure of hMSR was finally formed. The aspect ratios of the hMSR can be conveniently controlled by regulating the aspect ratios of NHNT. Four different hMSR with aspect ratios of ca.2.5, ca.5.3, ca.8.1, ca.9.0 has been obtained. It was demonstrated that the as-prepared hMSRs have good stability, high drug loading capacity and fast cell uptake capability, which makes them to be a potential nanocarrier for drug delivery. As the paradigm, hMSR with aspect ratios of ca.8.1 was then applied for TFs-responsive intracellular anticancer drug controlled release by using a Ag+-stabilized molecular switch of triplex DNA (TDNA) as capping agents and probes for TFs recognition. In the presence of TF, the pores of hMSR can be unlocked by the TFs induced disassembly of TDNA, leading to the leakage of DOX. The researches in vitro displayed that this system has a TFs-triggered DOX release, and the cytotoxicity in L02 normal cells was lower than that of HeLa cells. We hope that this developed hMSR-based system will promote the development of cancer therapy in related fields. Keywords: hollow mesoporous silica nanorod, controllable aspect ratios, controlled


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release, intracellular drug delivery, transcription factor (TFs)-responsive Introduction Over the past few decades, hollow-structured controlled release systems have emerged as a wide topic that shows great potential for making the revolutionary impact on drug delivery and cancer therapy.1-5 A large number of hollow-structured carriers, especially for nanocomposite6-8 and inorganic nanoparticles,9-12 have been studied. As one of the most promising candidate, hollow-structured mesoporous silica materials (hMSM), which have the well-defined structures, low density, huge hollow cavities and copious mesoporous channels, have aroused widely attention in many fields.13-16 The large hollow voids within the nanoscale mesoporous channels are highly favorable as the nanocontainer for drug storage, delivery and release after suitable surface modifications, effectively protecting healthy cells from damages of these drugs by nonspecific uptake.17,

18

For instance, Zhao and co-workers have

synthesized the polymer-capped hMSM for DOX delivery through pH, reduction agent and light triggered controlled release.19 Also, Cai and co-workers have designed the polyethylene glycol (PEG) protected hMSM for tumor therapy through pH triggered cascade response in tumor microenvironment.20 However, most of these reported systems have usually employed sphere-like nanoparticles as the carrier. The use of other shaped hMSM-based nanoparticles for drug delivery still in the early stages of development. It has been reported in recent years that the shapes of nanoparticles have play the crucial role in nonspecific uptake behaviors of cells.21-23 Several groups have demonstrated that nanoparticles with rod-like structures and high



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aspect ratios have the increased internalization rates and cell uptake, high drug loading capacities and longer circulation times.24 Among these rod-like nanoparticles, hollow-structured mesoporous silica rods (hMSR) have obtained great attention due to their high aspect ratio and anisotropic structure.25 These results have suggested that hMSR with high aspect ratios can be selected as the promising candidate for drug delivery and cancer therapy. So far, the fabrication routes of hMSR are mainly known as template directed approaches, which has considered to be a simple and scalability synthesis method.26 Various templates, such as polymer nanorods,27 surfactant mixture composed,28 inorganic nanocapsules29, 30 and natural cellulose substance,31 have been applied to determine the final nanostructure of hMSR. However, the synthesis of these templates is complex and high-cost, thus limiting their application in related fields. Moreover, because of the deficiency of appropriate templates, the high-curvature surfaces and the heterogeneity of rod-like templates, most of reported methods usually can not well control the morphologies and aspect ratios of hMSR. The formation of hMSR with controllable aspect ratios still has many challenges. Therefore, it is imperative to find a appropriate template and a straightforward method to synthesize the hMSR with controllable aspect ratios for their applications. Recent studies by Yin’s groups have reported a one-pot synthesis method of silica nanotubes with the precisely controlled dimensions.32 Inspired by this, we here synthesize a series of hMSRs with controllable aspect ratios by a straightforward and effective template-coating-etching approach and then applied for intracellular


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transcription factor-responsive drug release. The synthetic process and application of hMSR is illustrated in Scheme 1. It starts with the formation of nickel hydrazine nanorod template (NHNT) via a simple precipitation reaction of NiCl2 and hydrazine hydrate in a surfactant of Brij C10, followed by silica coating through a sol gel process. The deposited silica layer has replicated the rod shape of NHNT and then produced the hollow silica nanorod (hSNR) after acid etching of NHNT. The obtained hSNR is subsequently employed as the template to direct the deposition of mesoporous silica to form mesoporous silica coated hSNR (MS@hSNR). With the protection of PVP, the silica layer of MS@hSNR has been removed by the method of surface-protected etching while the mesoporous silica layer of MS@hSNR have been retained. Thus, the hMSR can be finally obtained. To show the application of hMSR as the drug carrier, a Ag+-stabilized triplex DNA (TDNA), consisting of a single-stranded DNA (ssDNA) and a nuclear transcription factors κappaB (NF-κB)33-35 specifically recognized double strand DNA (dsDNA),36,

37

has been

selected as the molecular gate to design a intracellular NF-κB triggered drug delivery system. For the synthesis of TDNA-gated system, the ssDNA is grafted on the surface of hMSR to direct the assembly of TDNA in situ via the binding behavior of ssDNA to dsDNA which partially overlapped the homopyrimidine strand with ssDNA by Hoogsteen bonding. The presence of TDNA can blocks the pores of hMSR and strongly inhibits the release of loaded DOX. However, when adding the NF-κB into this system, the dsDNA has been competed away from the mesoporous silica surface by the higher binding capability of NF-κB, leading to the disassembly of TDNA. As a



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result, DOX can be released, and then causes in vitro cell killing, especially for cancer cells which have the increased expression of NF-κB.38, 39 Results and discussion Synthesis of hMSRs with different aspect ratios In order to demonstrate the feasibility of this design, hMSR with different aspect ratios was first synthesized according to the method describe above. The process for the synthesis of hMSR was displayed by the transmission electron microscopy (TEM) images in Figure 1, Figure S1 and Figure S2. As illustrated in the first column of Figure 1, the as-prepared silica coated NHNT (SNR@NHNT) with the fine rod shape has the core/shell structure, which was belonged to the NHNT core and silica shell. The energy-dispersive X-ray spectrum showed that the SNR@NHNT has the element composition of Si, O, Ni and Cl, further proving this successful synthesis (Figure 2A). The length of SNR@NHNT, which has the increased aspect ratios, was about 80, 150, 200, 250 nm respectively. Subsequently, to obtain the hSNR, the cores of NHNT were removed by the selective acid etching. The second column of TEM images in Figure 1 has directly confirmed the formation of hSNRs. For the hSNR, the structure of NHNT was disappeared, meanwhile, a clearly hollow structures and an easily identified silica shell with thickness of ~10 nm were observed, which has proved the formation of hSNR. Moreover, the element composition of hSNR was changed into Si and O, further disclosing this acid etching process (Figure 2B). After that, by the sol-gel method, a mesoporous silica layer was then coated on the as-synthesized hSNR. TEM images in Figure S1 have clearly showed the resulting MS@hSNR with


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a sandwich-like structure. On the surface of obtained hSNR, an obvious mesoporous silica layer with thickness of ~10 nm was appeared. This result has strongly proved the successfully coating behavior of mesoporous silica layer on the hSNR. To eventually achieve the hMSR, this silica layer of MS@hSNR was then removed by a “surface-protected hot water etching” method. As shown in the third column of Figure 1 and Figure S2, the silica layers of MS@hSNR were disappeared, meanwhile, the hollow structure with a ~8 nm thick amorphous mesoporous silica layer was obtained. The average dimensions (length/width) of hMSR were 100/40 nm (hMSR2.5), 160/30 nm (hMSR5.3), 210/26 nm (hMSR8.1) and 270/30 nm (hMSR9.0), which was corresponded to aspect ratios of ~2.5, ~5.3, ~8.1, ~9.0, respectively (Table 1). The zeta potentials of hMSRs were range from -2.38 mV to 6.9 mV (Table 1), which have closed to the corresponding data in conventional mesoporous silica nanoparticles, such as MCM-41 MSN. In addition, dynamic light scattering (DLS) assay displayed that the hMSRs have the average hydrodynamic sizes range from 150 nm to 300 nm, indicating that the hMSRs have the well dispersibility in the aqueous solution without significant aggregation (Table 1). It was worth noticing that hMSRs with small aspect ratios (9.0) was usually instable and easy to fracture (Figure S2). Therefore, we have selected the hMSRs, which have the aspect ratios between 2.5 and 9.0, as the investigated subjects in the following researches. The mesoporous structure of as-prepared hMSRs was further verified by X-ray



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diffraction (XRD) assay. In Figure 2C, the significant small-angle XRD peaks of mesoporous were appeared at 2θ near 2°, further proving the formation of amorphous mesoporous silica shells and their disordered pores. In addition, the peaks from hMSR2.5 to hMSR9.0 were gradually decreased. This phenomenon might be owing to the different aspect ratio of hMSRs. The mesoporous silica shells of hMSRs were also characterized by nitrogen sorption isotherms. As illustrated in Figure 2D, in the relative pressure around 0.4, hMSRs illustrated the sharp, single, strong adsorption steps of typical type IV curve. This typical curve was attributed to the mesoporous structure of hMSRs. The hMSRs samples were also characterized by the pore diameter, surface area and pore volume in Table 1 and Figure S3. Under the model of BJH, the pore sizes of hMSR2.5 and hMSR5.3 were found to be similar, while that of hMSR8.1 and hMSR9.0 appeared to be a little decreased. Moreover, due to the increased aspect ratios of hMSRs, the surface area of hMSRs, obtaining by the application of the BET model, was gradually decreased from hMSR2.5 (641.5 m2 g-1) to hMSR9.0 (294.7 m2 g-1). All of evidences described above have offered the great potential for hMSR to be a promising candidate of drug carrier. The biocompatibility and loading capacity of hMSR with different aspect ratios It has been reported that cells may tend to uptake nanoparticles with large aspect ratios. And the internalized nanoparticles are mainly located in the lysosomes.40, 41 Therefore, to investigate the effect of morphology on the interaction between hMSRs and cells, FITC-labeled hMSRs (hMSR-FITC) were first respectively incubated with HeLa cells for the different periods of time. Subsequently, the lysosomes of cells were


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stained by Lysotracker blue DND-22 to investigate the cell uptake behavior for hMSRs. Figure 3A and Figure S4 have displayed the results of this investigate. With the incubated time increased, more and more fluorescence of FITC was overlapped with the fluorescence of Lysotracker blue DND-22. This phenomenon has suggested that hMSR-FITC was indeed internalized into HeLa cells and the cell uptake of a type of hMSR-FITC was mainly time-dependent. The fluorescence intensity of corresponding images has also displayed this change. As illustrated in Figure S5, the fluorescence intensity of FITC (green line) was overlapped with Lysotracker blue DND-22 (blue line). And this overlapped fluorescence intensity was gradually increased from 1 h incubation to 5 h incubation, which was attributed to the increased hMSR-FITC intracellular. Moreover, the fluorescence of FITC was gradually increased from hMSR2.5-FITC to hMSR9.0-FITC at the same incubated time (Figure 3A, Figure S4 and Figure S5). This result demonstrated that nanoparticles with large aspect ratios have the advantages in cell uptake. Another experiment was also proved this result. Similarly, HeLa cells were first treated with different hMSR at the same Si concentration for 3, 5, 7 h respectively. After that, the free hMSRs were removed by the D-hanks and the remained HeLa cells were subsequently disrupted and solubilized. The concentration of Si in the HeLa cells was then measured by ICP-AES (Figure 3B). With increased aspect ratios from hMSR2.5 to hMSR9.0, an obvious rise of content of Si in HeLa cells was obtained. This result has further proofed that the uptake behavior of HeLa cells to hMSR was shape-dependent, and the hMSR with large aspect ratio has an increased



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cell internalized ratio than the hMSR which has the low aspect ratio. The toxicity of different hMSRs was also studied to guarantee the successful drug delivery intracellular of drug-loaded hMSRs. As Figure 3C showed, the hMSRs exhibited the low cytotoxicity to HeLa cells after incubating hMSRs with HeLa cells for 48 h. In addition, the hemolysis experiment also proved this result. As displayed in Figure 3D, after 12 h blood incubating, there was no obvious hemolysis in all of samples, even at high concentrations of hMSR. Table S4 has listed the hemolysis ratios of hMSR2.5 to 9.0 under different concentrations. As the novel mesoporous silica materials, it was important to further investigate the loading capacity of these hollow mesoporous nanoparticles. Four types of hSNRs and hMSRs (5 mg mL-1) were first treated with model antitumor drug of DOX (5 mM) at pH 7.0. After incubating overnight, the obtain samples were separated via centrifugation and the excess DOX molecules were removed by nanopure water. Based on the characteristic absorbance peak of DOX (λex = 480 nm), the loading capacities of hSNRs and hMSRs were ascertained. As listed in Table 1 and Table S2, the DOX loading amount of hMSRs was almost three times than that in hSNRs. This result was attributed to the mesoporous and hollow structure of hMSRs which can greatly enhance the drug loading capacity. Moreover, with the increased aspect ratios, the DOX loading amount from hMSR2.5 to hMSR9.0 was gradually increased. Notably, hMSR8.1 and hMSR9.0 showed similar DOX loading capacities, which might be attributed to the partially fractured hMSR9.0 (Figure S6), leading to the loss of loaded DOX. Compared with other hMSRs, hMSR8.1 has displayed the better


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rod-like hollow structures, suitable BET surface area and BJH pore size, and higher DOX loading capacity. In addition, hMSR8.1 showed the similar cell internalized ratio to hMSR9.0 after 5 h incubation. Therefore, we have chosen hMSR8.1 as the drug carrier for the following experiments. The preparation of hMSR8.1-TDNA To show the application of hMSR8.1, a transcription factors-responsive nanocarrier was then synthesized according to the method described above. hMSR8.1, employed as the solid supporter, was first functionalized by the chlorine group to form hMSR8.1-Cl. Subsequently, the hMSR8.1-Cl was added into the solution of DMF which containing sodium azide to obtain hMSR8.1-N3. The obtained hMSR8.1-N3 was characterized by N2 adsorption-desorption isotherms, DLS and zeta potential respectively. As shown in Figure S7A and Figure S7B, hMSR8.1-N3 exhibited a characteristic type IV curve of N2 adsorption-desorption isotherms. The surface area and the pore diameter of hMSR8.1-N3 were 471 m2 g-1 and 2.72 nm respectively. These results have strongly proofed that the mesopores structures of hMSR8.1-N3 have not been destroyed by the related modification. The DLS assay demonstrated that the hydrodynamic sizes and the polydispersity index (PDI) of hMSR8.1-N3 were 268.5 nm and 0.012 respectively, disclosing the well dispersibility of hMSR8.1-N3 (Figure S8A and Table S3). In addition, the zeta potential for hMSR8.1 and hMSR8.1-N3 has revealed this successfully modified process. After functionalized with azide group, the zeta potential of hMSR8.1 was changed from -1.02 mV to -36.7 mV, which mainly owing to ionization of azide group on the hMSR8.1-N3 (Figure



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S8B and Table S3). Subsequently, a single-strand DNA (ssDNA), selected as the assembly driver of triplex DNA cap (TDNA), was then grafted onto the surface of hMSR8.1-N3. The as-prepared ssDNA grafted hMSR8.1-N3 (hMSR8.1-ssDNA) was characterized via zeta potential, Fourier Transform Infrared (FTIR) and DLS respectively. The zeta potential of hMSR8.1-ssDNA has directly confirmed this successful grafting process of ssDNA. As shown in Figure S8B and Table S3, because of the increased ssDNA and decreased azide group all around the as-synthesized hMSR8.1, the zeta potential of hMSRc3-ssDNA has became to -22.4 mV. In addition, the FTIR assay also disclosed this successful functionalization on hMSR8.1 (Figure S7C). After modification of azide group, a visible azide stretching signal of assynthesized hMSR8.1-N3 was appeared on 2110 cm-1. While the hMSR8.1 only displayed the framework vibrations of silica. Moreover, the intensity of absorption band of azide group after ssDNA attachment was obvious decreased. These results have indicated that the ssDNA was indeed tethered to hMSR8.1-N3 via covalent binding. By using Biospec-nano UV-vis spectroscopy, the efficiency of immobilized ssDNA was 4.69 µmol g−1 SiO2. In addition, the dispersity of hMSR8.1-ssDNA was also investigated by DLS (Figure S8B and Table S3). Due to the modification of ssDNA, the average diameter of hMSR8.1-ssDNA was increased to 325.3 nm. Moreover, the PDI of hMSR8.1-ssDNA was 0.51, also disclosing a well dispersibility of hMSR8.1-ssDNA in the aqueous solution. In order to ultimately form this transcription factors-triggered drug controlled release system, the ssDNA modified onto hMSR8.1 was then employed as the driver


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to mediate the assembly of Ag+-stabilized TDNA in situ via the binding behavior of ssDNA to dsDNA. Under different conditions, the assembly behavior of ssDNA and dsDNA was first studied through the assay of fluorescence intensity of labeled dsDNA (ldsDNA). In Figure 4A, a strong fluorescence intensity of ldsDNA was observed in sample a (curve a), and this fluorescence intensity has not much changed after treating ldsDNA with Ag+ (curve b). When adding a BHQ-labeled ssDNA (lssDNA) into the ldsDNA solution which has not treated with Ag+, the fluorescence intensity of this sample was slightly decreased (curve c). This result indicated that only a small number of triplex DNA can be formed in the absence of Ag+. However, when adding lssDNA into the sample of ldsDNA which containing Ag+, the fluorescence intensity of this sample was significantly decreased (curve d). This decreased fluorescence intensity was mainly attributed to the formation of copious triplex DNA in the presence of Ag+. The above phenomenon demonstrated that the TDNA can be obtained by the successful assembly of ssDNA and dsDNA and the stability of TDNA can be improved by Ag+. For another control experiment, a BHQ-labeled control single-stranded DNA (lcssDNA) was then added into the ldsDNA solution. As shown in Figure 4B, compared with ldsDNA (curve f), the fluorescence intensity of above sample has a little changed (curve g). When further adding Ag+ into the above solution, the fluorescence intensity of this sample was not changed (curve h), which has indirectly confirmed the conclusions described above. The capping and uncapping behavior of TDNA After proving the successful assembly of TDNA, the capping behavior of this



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TDNA to hMSR8.1 was then investigated. Model antitumor drug of DOX was first loaded into hMSR8.1-TDNA to get hMSR8.1-DOX-TDNA. As illustrated in Figure 4C, the hMSR8.1-DOX-TDNA has the negligible release of DOX. However, hMSR8.1-DOX-TDNA, which has not treated with Ag+ during the synthetic process, was reached a release amount of DOX about 58.0%. This result was mainly due to the stabilization of Ag+ to TDNA. In addition, the release amounts of DOX from hMSR8.1-DOX-ssDNA before and after Ag+ treatment were both reached a high level. The release amounts of DOX were measured to be about 79.5% and 80.8% respectively.

These

excellent

release

percentages

of

DOX

from

hMSR8.1-DOX-ssDNA demonstrated that ssDNA cannot block the pores of hMSRc3 and inhibit the leakage of the entrapped DOX. In order to further prove the blocking behavior of TDNA, a control single-stranded DNA (cssDNA) was then grafted on the hMSR8.1 to obtain hMSR8.1-cssDNA. After DOX loading, the obtained DOX-loaded hMSR8.1-cssDNA (hMSR8.1-DOX-cssDNA) was incubated with dsDNA to get hMSR8.1-DOX-cTDNA. As displayed in Figure 4D, the release amount of DOX from hMSR8.1-DOX-cssDNA was about 80.2%, which was attributed to the unlocked pores

of

hMSR8.1.

Moreover,

the

release

amount

of

DOX

from

hMSR8.1-DOX-cTDNA was as high as that in hMSR8.1-DOX-cssDNA. This result further suggested that only ssDNA can mediate the formation of triplex DNA and subsequently block the pores of hMSR8.1. These results described above have disclosed the strongly storage efficiency of DOX in hMSR8.1 by virtue of the blocking ability of TDNA through the assembly of ssDNA and dsDNA by Hoogsteen


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bonding. The capping behavior of TDNA has motivated us to further study the uncapping behavior of this system. Due to the sequence-specific recognized capability of NF κB to dsDNA (here NF κB p50 has been selected as the model stimuli), the triplex DNA of TDNA can be then disassembled via the strong interaction between NF κB p50 and dsDNA. The specifically responsive experiment under different stimuli was employed to prove this “smart” unlocking behavior of pores. Figure 4E has showed this specific response. The release amount of DOX from hMSR8.1-DOX-TDNA under NF κB p50 treatment was much higher than that in hMSR8.1-DOX-TDNA which was treated with glucose, lactose, glycine, glutamate, cysteine and BSA respectively. In addition, after treating hMSR8.1-DOX-TDNA with the sample which including NF κB p50, glucose, lactose, glycine, glutamate, cysteine and BSA, the release amount of DOX was similar to the hMSR8.1-DOX-TDNA which was only treated with NF-kB p50. Another experiment was used to further investigate the effect of environment intracellular on the TDNA. As shown in Figure S9, when adding TDNA into the cell disruption solution, the fluorescence intensity of both two samples was increased (b). However, when adding TDNA into the cell disruption solution which pretreated with PDTC, the fluorescence intensity of both two samples was small (c). These results have demonstrated that the environment intracellular has no significant effect on the TDNA. The increased fluorescence of samples was mainly attributed to the disassembly of TDNA and subsequently released ldsDNA. According to the finding illustrated above, the NF κB p50-responsive triplex DNA-gated system, which has the



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excellent drug blocking and storing ability, was finally obtained. Subsequently, the release statuses of hMSR8.1-DOX-TDNA under different concentration of NF κB p50 were also carried out (Figure 4F). The hMSR8.1-DOX-TDNA has displayed a NF κB p50-dependent controlled release of DOX. About 6.0%, 59.8%, 69.7% and 85.3% of the DOX were released at different concentration of NF κB p50. Most importantly, the release speed of DOX from hMSR8.1-DOX-TDNA was slower than that from conventional mesoporous silica nanoparticles. The slow release of DOX from hMSR8.1-DOX-TDNA was reached 84.1% within 20 h. And this released percentage of DOX was not changed over hours. The hollow structure of hMSR8.1 and the interference of amorphous mesoporous shell were likely the reasons that result in the slow release of DOX, which was preferred for drug delivery systems to prolong the curative effect. Controlled release behavior of hMSR8.1-DOX-TDNA in vitro The feasibility of controlled release of DOX from hMSR8.1-DOX-TDNA and the excellent selectivity of this system have let us to further investigate the controlled release behavior of hMSR8.1-DOX-TDNA intracellular. Here, a human normal liver cell line (L02 cells) and a human cervical cancer cell line (HeLa cells ) were used as the model cells in this research. It can be seen in Figure S10, after 3 h incubating, most of the fluorescence of DOX (red) was overlapped with the fluorescence of Lysotracker blue DND-22 (blue). This overlapped fluorescence has indicated that the hMSR8.1-DOX-TDNA was internalized into both two cells and mainly located in endosomes and lysosomes.


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As we know, the effective toxicity of DOX to cells is relied on those located DOX in cell nucleus. And these located DOX can subsequently block the replication and transcription of DNA.42,

43

Therefore, we have further investigated the controlled

release behavior of this system in different cells and the cellular distributed sites of those released DOX. Here, blue-fluorescent Hoechst 33342 was used to label the cell nucleus. After incubating hMSR8.1-DOX-TDNA with HeLa cells for 10 h, an obvious red fluorescence, which deriving from the escaped DOX, has overlayed with the fluorescent of Hoechst 33342 (blue) at the nucleus of HeLa cells. However, for the nucleus of L02 cells, only a faint fluorescence of DOX has been observed. The fluorescence intensity of corresponding images has also showed this distinction (Figure S11). The fluorescence intensity of DOX (red line) which overlapped with Hoechst 33342 (blue line) was higher in HeLa cell nucleus than that in L02 cell nucleus. These results suggested that the system of hMSR8.1-DOX-TDNA can effectively store the DOX before endocytosis. Once hMSR8.1-DOX-TDNA was internalized into cells, experically for the cells which containing more NF κB p50, the release of DOX can be obtained due to the disassembled TDNA and those released DOX has subsequently located in cell nucleus. Another experiment has been taken to further proof the irreplaceable role of NF κB p50 on the controlled release behavior of hMSR8.1-DOX-TDNA. The two model cells were first pretreated with PDTC, a NF κB inhibitor. As displayed in Figure 5 and Figure S11, in the cell nucleus of these two cells, the fluorescence of DOX was obvious diminished after pretreating cells with PDTC for 10 h. This finding was



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attributed to the inhibition of PDTC to the NF κB p50 and subsequently decreased amount of released DOX. Conversely, when pretreating cells with NF κB p50 for 10 h, the fluorescence of DOX at the cell nucleus of two model cells was increased, further confirming the unlocking ability of NF κB p50 to hMSR8.1-DOX-TDNA. All of these results described above have provided the strong evidence to prove the unlocking behavior of pores was indeed triggered by the competitive binding of NF κB p50 to dsDNA. The cytotoxicity assay Subsequently, the MTT assay was carried out to study the killing efficacy and cytotoxicity of different samples. As shown in Figure 6A and 6B, hMSR8.1-TDNA displayed the low toxicity to those two model cells under different concentrations. In addition, the hemolysis assay was also studied to evaluate the toxicity of hMSR8.1-TDNA to cells (Figure 6E). After 12 h incubation, no hemolysis was appeared under different concentrations of hMSR8.1-TDNA, indicating the high biocompatibility and low toxicity of hMSR8.1-TDNA. The corresponding hemolysis ratios of hMSR8.1-TDNA were listed in Table S4. However, hMSR8.1-DOX-TDNA showed a significant cytotoxicity for HeLa cells under the same concentration range. And this cytotoxicity of hMSR8.1-DOX-TDNA to HeLa cells was closed to that of DOX only. Moreover, compared with free DOX, the hMSR8.1-DOX-TDNA dramatically showed a relatively low cytotoxicity for L02 cells. About 50.6% of L02 cells were alive, which formd the obvious contrast on the cell viability of HeLa cells. All of these results have indicated that the expression level of NF κB p50 intracellular


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has influenced the release behavior of hMSR8.1-DOX-TDNA. For another research, these two model cells were first pretreated with PDTC and NF κB p50 individually. Subsequently, these cells were further incubated with hMSR8.1-DOX-TDNA to investigate the cell viability of these two cells. The results were illustrated in Figure 6C and 6D. After pretreating with PDTC, the toxicity of hMSR8.1-DOX-TDNA to these two model cells was obviously decreased. However, when the cells were pretreated with NF κB p50, the cytotoxicity of hMSR8.1-DOX-TDNA for L02 cells was increased apparently, which matched well with the cytotoxicity of free DOX (Figure 6B curve DOX). In addition, because of the released balance of DOX, the cytotoxicity of hMSR8.1-DOX-TDNA to HeLa cells before and after NF κB p50 treatment was similar. The difference on the toxicity of hMSR8.1-DOX-TDNA to the two model cells after PDTC and NF κB p50 pretreatment once again demonstrated this NF κB p50-triggered release process of DOX. Moreover, the cytotoxicity of PDTC for HeLa cells and L02 cells was also investigated. As disclosed in Figure S12, no obvious toxicity of PDTC to those two model cells. This result has proofed that the PDTC has little cytotoxicity to cells. Conclusions In summary, a novel type of hollow mesoporous silica nanorods was fabricated and then applied for NF κB-triggered intracellular drug delivery. In this system, by regulating the reacted ratio of NiCl2 and hydrazine hydrate, we can finally obtain the aspect-ratios-controllable mesoporous silica nanorods with hollow structure. hMSR8.1, which has the good stability, high drug loading capacity and fast cell



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uptake capability, was then chosen as the drug delivery carrier. Utilizing triplex DNA capped hMSR8.1, the NF κB p50-based drug delivery can be successfully realized in vitro cellular experiments. The MTT assay confirmed that hMSR8.1-TDNA has a negligible toxicity for those two model cells. However, hMSR8.1-DOX-TDNA exhibited an efficient killing efficacy for HeLa cells than that for L02 cells. With these excellent function and properties, we belive that this system will open a new horizon of drug delivery in the field of cancer therapy. Experimental section Chenicals. Polyoxyethylene(10) cetyl ether (Brij C10), lactose, doxorubicin hydrochloride, bovine serum albumin (BSA)，polyvinylpyrrolidone (PVP, 40.000) and dulbecco’s

phosphate

bufferd

saline

were

getted

from

Sigma-Aldrich.

N-cetyltrimethylammonium bromide (CTAB), fluorescein isothiocyanate (FITC), CuBr (99.9%), hickel chloride anhydrous (NiCl2), hydrazine monohydrate, 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium

bromide

(MTT),

3-aminopropyltriethoxysilane (APTES), 3-chloropropyltrimethoxysilane (ClTMS) were achieved from Alfa Aesar. Ammonium pyrrolidinedithiocarbamate (PDTC) was obtained from Aladdin. The purified recombinant NF-κB p50 protein was obtained from Cayman Chemical (Ann Arbor, MI, USA). Isopropyl alcohol, toluene tetraethylorthosilicate (TEOS, 28%), sodium azide (NaN3, 99%), cyclohexane, dimethyl sulfoxide (DMSO), tert-butyl alcohol (tBuOH), ammonia solution (28 wt %), N, N-dimethylformamide (DMF), sodium nitrate (NaNO3), and trihydroxymethyl aminomethane (Tris-base) were achieved at Dingguo reagent company. Magnesium


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chloride (MgCl2), monopotassium phosphate (KH2PO4), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), magnesium sulfat (Na2SO4) were obtained in Guanghua chemical plants. The water used in this work was nanopure water (18.2 M Ω; Millpore Co., USA). The oligonucleotides used in all of researches of this work were synthesized in Sangon Biotechnology Inc. Table S1 has listed these as-synthsized sequences. All the chemicals were used as received without further purification. Instruments. The zeta potential and size were obtained on the Zetasizer Nano-ZS of Malvern Instruments. FTIR spectra of different samples was detected by the TENSOR 27 spectrometer. TEM image of different samples was collected at accelerating voltage of 100 kV on the JEOL 3010 microscope. The inductively coupled plasma-atomic emission spectrometer (ICP-AES) was used to measure the concentration of Si. The F-7000 FL spectrophotometer (Hitachi) was used to measure the fluorescence spectra of samples. The UV-vis absorption of samples was obtained from the DU-800. CLSM image of samples was collected by the Fluoview FV500, Olympus. The Scintag XDS-2000 powder diffractometer was used to collecte the XRD patterns of different samples by using Cu Kα irradiation (λ = 0.154 nm). Methyl thiazolyl tetrazolium (MTT) assay was measured on the multimode reader M1000 (TECAN). The N2 adsorption-desorption isotherms of samples were collected by the Micromeritics ASAP 2010 sorptometer. The surface area and the pore size distribution of samples were repectively calculated by the Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) method.



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Synthesis of Hollow Silica Nanorod (hSNR). The hSNR was synthesized by a typical template-mediated method. Brij C10 (4.25 g) was first added into cyclohexane (7.5 mL). Then this mixtue was heated up to 50 °C to dissolve the Brij C10. After that, NiCl2 solution (0.8 mL, 0.8 M) was added dropwise. Subsequently, 0.225 mL of hydrazine monohydrate was added into the above mixture to form nickel-hydrazine nanorod template (NHNT). After 3 h of reaction, 500 µL of diethylamine and 1 mL of TEOS were added for silica coating, which was allowed to proceed for 3 h. The result silica@NHNT core/shell structured nanorod (SNR@NHNT) was then collected by centrifugation and washed with isopropanol. Finally, the as-synthesized SNR@NHNT was stored in isopropanol (12.5 mL). By changing the amount of NiCl2 solution in this system, we can obtain different aspect ratios of SNR@NHNT. Here, 0.45 mL, 0.6 mL and 0.7 mL of NiCl2 solution (0.8 M) were selected to synthesize the other three types of SNR@NHNT. Subsequently, the template of NHNT was etched away by the typical method of acid etching. The as-prepared SNR@NHNT was first dispersed in 70 mL of HCl (1 M). After 3 h stirring, the obtained hSNRs (hSNR2.5, hSNR5.3, hSNR8.1 and hSNR9.0) were recovered and washed by ethanol and nanopure water. Synthesis of Hollow Mesoporous Silica Nanorod (hMSR). 20.0 mg of hSNR, 0.22 mL of ammonia solution (28 wt %), 60 mg of CTAB and 12 mL of ethanol were added into 16 mL of nanopure water. After stirring mixture for 30 min, TEOS (0.88 mL, 1.90 mmol) was then carefully added. Subsequently, this mixture was further stirred for 6 h to form mesoporous silica coated hollow silica nanorod (MS@hSNR). In order to remove the surfactant template (CTAB), the the as-synthesized


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MS@hSNR and 0.15 g of NH4Cl was put into 25 mL of ethanol. Then the mixture was stirred for 12 h under 65 °C. After removing CTAB, the as-synthesized MS@hSNRs, which was named [email protected], [email protected], [email protected] and [email protected] respectively, were dispersed in 20 mL nanopure water. According to a typical “surface-protected hot water etching” method,23-25 10 mL of the above solution containing different MS@hSNR was respectively added into 10 mL nanopure water containing 0.2 g of PVP (Mw = 40000). The mixtures were stirred for 0.5 h, and then heated to 95 °C. After etching for 1 h, the hMSRs (hMSR2.5, hMSR5.3, hMSR8.1 and hMSR9.0) were finally obtained. Synthesis of FITC-labeled hMSR (hMSR-FITC). In order to obtain hMSR-FITC, 100 µL of APTES was put into 1.0 mL of absolute EtOH which containing 1.0 mg of FITC. The mixure was reacted in darkness for 12 h. Then the FITC modified APTES (FITC-APTES) was obtained. Subsequently, 20.0 mg of hSNR, 0.22 mL of ammonia solution (28 wt %), 60 mg of CTAB and 12 mL of ethanol were added into 16 mL of nanopure water. After stirring for 30 min, 30 µL of FITC-APTES and 88 µL of TEOS (1.90 mmol) was added into mixture. Then the MS@hSNR-FITC can be formed by further reacting the mixture for 6 h. In order to further remove the CTAB from MS@hSNR-FITC, the mixture containing as-synthesized MS@hSNR-FITC, 0.15 g of NH4Cl and 25 mL of ethanol was reacted for 12 h under 65 °C. After washing with ethanol and water , the obtained MS@hSNR-FITC was then dispersed in nanopure water (20 mL). To further obtain the hMSR-FITC, 10 mL of the above solution containing MS@hSNR-FITC was added into 10 mL nanopure water containing 0.2 g



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of PVP (Mw = 40000). After 0.5 h stirring, this mixture was further etched for 1.5 h under 80 °C to form the final samples. Chemical Modification of the hMSR8.1 Surface. In this process, 0.70 g of as-synthesized hMSR8.1 and 0.70 mL of ClTMS were added into anhydrous toluene ( 60.0 mL). After refluxing this mixture for 20 h, the 3-chloropropyl-functionalized hMSR8.1 (hMSR8.1-Cl) can be obtained. Subsequently, a sodium azide saturated mixture containing 20.0 mL of DMF solution and 200 mg of hMSR8.1-Cl was stirred for 12 h under 70 °C to synthesize azide-functionalized nanoparticles (hMSR8.1-N3). The remained DMF was subsequently removed by PBS buffer solution. Preparation of Triplex DNA-capped hMSR8.1. 2.0 mg of hMSR8.1-N3 nanoparticles were first dispersed into 70 µL of nanopure water. Then tris(benzyltriazolylmethylamine) ligand (1.0 µL, 0.1 M), alkyne-modified ssDNA (130 µL, 100 µM) and CuBr solution (2.0 µL, 0.1 M) was added. The ssDNA-functionalized hMSR8.1 (hMSR8.1-ssDNA) can be formed by shaking above mixture overnight under room temperature. The immobilization amount of ssDNA on the hMSR8.1 was about 4.69 µmol g−1 SiO2. Subsequently, the as-synthesized hMSR8.1-ssDNA was immersed in 200 µL of DOX solution (pH 7.0, 5.0 mM). The DOX-loaded hMSR8.1-ssDNA (hMSR8.1-DOX-ssDNA) was finally obtained by shaking this mixture for 12 h. The loading amount of DOX was determined to be about 120.31 µmol g−1 SiO2. In order to block the pores of hMSR8.1-DOX-ssDNA, the double strand DNA (dsDNA), a specific binding double strand DNA of NF-kB p50 transcription factors,


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was first formed by the corresponding complementary single-strand DNA (DNA1 and DNA2). In this process, DNA1 and DNA2 were mixed in the hybridization buffer at the same molar ratios. After melting for 5 minutes at 95 °C, this sample was subsequently slowly cooled down. To further obtain hMSR8.1-DOX-TDNA, 2.0 mg hMSR8.1-DOX-ssDNA was dispersed into 200 mL mixture containing 20 µL of dsDNA, 5µL of AgNO3 (20 µM) and 175µL of PBS (pH 7.4, 200 mM NaNO3) for 30 minutes at 37 °C. The final concentrations ratio of dsDNA and ssDNA were 1:1. In order to further prove the blocking behavior of this triplex DNA, a control single-stranded DNA (cssDNA) was grafted onto the surface of hMSR8.1-N3 to form cssDNA modified hMSR8.1 (hMSR8.1-cssDNA). The immobilization efficiency of cssDNA was 4.28 µmol g−1 SiO2. Then this hMSR8.1-cssDNA was incubated with DOX

and

dsDNA

to

get

another

triplex

DNA-capped

hMSR8.1-DOX

(hMSR8.1-DOX-cTDNA). The synthetic method was obtained according to the description above mentioned. DOX Release. The capping ability of triplex DNA was first investigated. 0.3 mg of hMSR8.1-DOX-ssDNA,

hMSR8.1-DOX-TDNA,

hMSR8.1-DOX-cssDNA,

hMSR8.1-DOX-cTDNA was respectively added into 200 µL of PBS (pH 7.0, 200 mM NaNO3). The fluorescence intensity of released DOX was monitored every 1 h. The unlocking behavior of hMSR8.1-DOX-TDNA was also investigated. 1.0 mg of hMSR8.1-DOX-TDNA was dispersed into 500 µL PBS (pH 7.0, 200 mM NaNO3), which containing different media (1. PBS: pH 7.0, 200 mM NaNO3, 2-6. glucose, lactose, glycine, glutamate, cysteine: 0.1 M, 7. BSA: 1 mg mL−1, 8. NF κB p50: 5.0



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µg mL-1, 9. mixed sample). Then these samples were incubated for 24 h. Subsequently, all of suspensions, which coming from the above samples, were collected and detected by fluorescence emission spectroscopy via the fluorescence intensity of DOX in the suspensions. Subsequently, the release behavior of hMSR8.1-DOX-TDNA under different concentration of NF κB p50 was further investigated. 200 µL of PBS (pH 7.0, 200 mM NaNO3) containing 0, 1.25, 2.5 and 5 µg mL-1 NF κB p50 respectively was fulled into the cuvette containing 0.3 mg of hMSR8.1-DOX-TDNA. The fluorescence intensity of DOX in the supernatant was detected using F-7000 FL spectrophotometer (λex = 480 nm and λem = 558 nm). CLSM

Images.

For

the

experiment of

cell

uptake

of

hMSRs

and

hMSR8.1-DOX-TDNA, HeLa cells and L02 cells, which have cultivated in RPMI 1640 medium at plastic-bottomed µ-dishes (35 mm), were respectively treated with hMSR-FITC (for HeLa cells only) and hMSR8.1-DOX-TDNA (for the two cells) at the concentration of 60 µg mL-1 for 3 h at 37 °C. After washing with D-hanks, the cells were further stained for 15 min with lysotracker blue. All of samples were imaged at the 100 × oil objective on a confocal microscope. In order to observe this controlled release behavior of hMSR8.1-DOX-TDNA intracellular, these two model cells, which have cultivated in RPMI 1640 medium at plastic-bottomed µ-dishes (35 mm), were respectively treated with 60 µg mL-1 of hMSR8.1-DOX-TDNA for 10 h under 37 °C. Subsequently, the cells were stained with Heochst-33342 in D-hanks for 15 min to stain the cell nuclears. All of samples


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were imaged at the 100 × oil objective on a confocal microscope. In another experiments, NF-κB p50 (2 µg mL-1) and PDTC (20 µM) were respectively pretreating these two cells for 10 h. After washing with D-hanks, the cells were further incubated with 60 µg mL-1 of hMSR8.1-DOX-TDNA for another 10 h. By specific staining of nuclear according to the description above mentioned, the cells were then imaged at the 100 × oil objective on a confocal microscope. In vitro cytotoxicity. The MTT assay was used to evaluate the toxicity of hMSR8.1-DOX-TDNA. HeLa cells and L02 cells (7 × 103 cells per well) were first cultivated at 96-well plates. After that, different samples (DOX, hMSR8.1-TDNA and hMSR8.1-DOX-TDNA) were added into medium and then incubated with cells for 48 h. Then these two model cells were washed with D-hanks and then treated with MTT. The concentration of MTT in the medium was 0.5 mg mL-1. After 4 h treatment, these two cells were further treated with DMSO (150 µL) for 10 min. The UV absorption of final samples were detected through the multi-detection microplate reader at 490 nm. The relative control experiment was also investigated. In this experiment, NF-κB p50 (2 µg mL-1) and PDTC (20 µM) were respectively pretreating these two model cells for 10 h. The cytotoxicity of PDTC was also studied. In this research, the two model cells were respectively treated with PDTC under different concentration for 48 h. Measurement of Fluorescence Spectrum. The formation and disintegration of triplex DNA was confirmed by the fluorescence spectrum of different samples. First, DNA1 and lDNA2, which have the same molar ratios, were added into hybridization buffer at the final concentration of 1 µM. After melting for 5 minutes at 95 °C, this



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sample was subsequently slowly cooled down to form double strand DNA (ldsDNA). Subsequently, 20 µL of ldsDNA (1 µM), 2 µL of lssDNA (10 µM) and 5 µL of AgNO3 (800 µΜ) were added into 173 µL PBS buffer (pH 7.4, 10 mM) which containing 200 mM of NaNO3. Then this mixture was incubated for 30 minutes under 37 °C. Then the triplex DNA (lTDNA) was obtained. For the disintegration of lTDNA, 4 µL NF-κB p50 (25 µg mL-1) was put into the sample of lTDNA above. After that, this mixture was incubated under 37 °C for 10 minutes. In addition, the stabilization of Ag+ to the lTDNA was also investigated. 20 µL of ldsDNA (1 µM) and 2 µL of lssDNA (10 µM) were put into 178µL of PBS buffer which containing 200 mM of NaNO3 at 37 °C for 30 minutes. The effect of Ag+ on ldsDNA was also studied. 20 µL of ldsDNA (1 µM), 5 µL of AgNO3 (800 µΜ) and 175 µL of PBS buffer which containing 200 mM of NaNO3 were mixed at 37 °C for 30 minutes. For the control experiments, a control single-strand DNA (lcssDNA) was further used to indirectly prove the formation of triplex DNA. 20 µL of ldsDNA (1 µM) and 2 µL of lcssDNA (10 µM) were added into 178 µL PBS buffer solution which containing 200 mM of NaNO3 and 20 µM of AgNO3 at 37 °C for 30 minutes. Another control sample was obtained without AgNO3 treatment. The final concentrations were 0.1 µM of ldsDNA, 0.1 µM of lssDNA, 0.1 µM of lcssDNA and 20 µM of AgNO3, respectively. Finally, the fluorescence spectrums were measured by F-7000 FL spectrophotometer (λex = 490 nm and λem = 522 nm). Cell Uptake Experiments. hMSR2.5, hMSR5.3, hMSR8.1 and hMSR9.0 (100 µg mL-1) were respectively added into the 24-well plates which preseeded in the HeLa


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cells and L02 cells (6 × 106 cells). After incubating for different times (3, 5 and 7 h), the cells cultivated in this 24-well plates were subsequently washed by D-hanks and disrupted by ultrasound. The obtained solutions were then respectively added to a mixture containing HClO4 (2 mL), HNO3 (2 mL), HF (100 µL) and HCl (6 mL). Subsequently, these mixtures were reacted under 100 °C for 2 h. The final samples were respectively diluted into 10 mL by nanopure water and then detected through the inductively coupled plasma-atomic emission spectrometry (ICP-AES) to get the concentration of Si. Hemolysis Assay of RBCs. 10 mL of health people blood was first centrifugalized for 10 min under 2000r/min to remove supernatant of sample. Subsequently, by washing those obtained RBCs with PBS, the hemoglobin of the ruptured RBCs was then removed. After that, the RBCs was dispersed in 10 mL of PBS to form the suspension of RBCs. For hemolysis assay, 200 µL of PBS which respectively contening different concentration of hMSRs and hMSR8.1-TDNA was added into 200 µL of RBCs suspension. In addition, 200 µL of PBS and 200 µL of nanopure water were respectively added into 200 µL of RBCs suspension to form negative control sample and positive control sample. All the samples was then shaked in the 37 °C for 12h. After that, the samples was centrifugalized under 2000r/min for 10 min to collect the supernatant. These finally collected supernatant was measured by the UV spectrophotometer. Hemolysis ratio = [(Dt - Dnc) / (Dpc - Dnc)] * 100% Dt: the UV absorption of the supernatant in test sample



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Dnc: the UV absorption of the supernatant in negative control sample Dpc: the UV absorption of the supernatant in positive control sample

AUTHOR INFORMATION Corresponding Author *Phone: +86-731-88823930. Fax: +86-731-88823930. E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ‡ These authors contributed equally. ACKNOWLEDGEMENTS This work was supported in part by the National Natural Science Foundation of China (21190044, 21322509, 21305035, 21305038, and 21221003).

Supporting Information. TEM images of hMSRs, MS@hSNRs and fractured hMSR9.0; pore size distribution plots of hMSRs and hMSR8.1-N3; the CLSM images of cellular uptake of hMSRs-FITC and hMSR8.1-DOX-TDNA; fluorescence intensity of CLSM images; nitrogen sorption isotherms of hMSR8.1-N3; FTIR spectra of hMSR8.1, hMSR8.1-N3 and hMSR8.1-ssDNA; zeta potential and size of different samples; the fluorescence spectra of ldsDNA in the real samples; the cytotoxicity of PDTC; the list of DNA sequences; adsorbing amount of DOX from hSNRs. This material is available free of charge via the Internet at http://pubs.acs.org.


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Cell Death. Cancer Res 2006, 66, 4863-4871.

Scheme 1. Schematic illustration of the synthetic process of hMSR and its application in NF-κB-triggered intracellular drug release.


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Figure 1. TEM images of SNR@NHNT, hSNR and hMSR with different aspect ratios.



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Figure 2. TEM-associated EDX spectra of A) SNR@NHNT and B) hSNR. C) small-angle XRD patterns of hMSR2.5-9.0. D) Nitrogen sorption isotherms of as-synthesized hMSRs.


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Figure 3. A) The CLSM images for cellular uptake of hMSRs-FITC after incubation with HeLa cells for 5 h at a concentration of 60 mg mL-1 respectively. The merged fluorescence was obtained by the overlap of green fluorescence of FITC and blue fluorescence of Lysotracker blue DND-22 (a special straining material for lysosomes). Cells were imaged using a 100 × oil-immersion objective. B) ICP-AES measured Si content per cell for HeLa cells after incubation with hMSRs at the same silica concentration for 3, 5, 7 h respectively. The background Si content per cell has been subtracted from those data. C) The viability of HeLa cells after treated with different concentration of hMSR2.5, hMSR5.3, hMSR8.1 and hMSR9.0 for 48 h respectively. D) Photographs of hemolysis of RBCs incubated with four aspect ratios of hMSRs for 12 h. The concentration of different samples was ranging from 18.75 to 2400 µg mL-1. Red hemoglobin in the supernatant was damaged RBCs. Samples of RBCs incubated with water (+) and PBS (-) was used as positive and negative control, respectively.



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Figure 4. A) The fluorescence spectra of ldsDNA under different conditions: (a) ldsDNA, (b) ldsDNA+Ag+, (c) ldsDNA+lssDNA, (d) ldsDNA+lssDNA+Ag+, (e) ldsDNA+lssDNA+Ag++NF-κB p50. B) The fluorescence spectra of ldsDNA under control

DNA

strand

treatment:

(f)

ldsDNA,

(g)

ldsDNA+lcssDNA,

(h)

ldsDNA+lcssDNA+Ag+. C) Release profiles of DOX from different nanoparticles. D) Release profiles of DOX from hMSR8.1-DOX-cssDNA and hMSR8.1-DOX-cTDNA. E) The specificity response of hMSR8.1-DOX-TDNA under different media (1. PBS: pH 7.0, 200 mM NaNO3, 2-6. glucose, lactose, glycine, glutamate, cysteine: 0.1 M, 7. BSA: 1 mg mL−1, 8. NF κB p50: 5.0 µg mL-1, 9. mixed samples of 2-8). F) Release profiles of DOX from hMSR8.1-DOX-TDNA under different concentrations of NF κB p50.


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Figure 5. The CLSM images of the cellular uptake and controlled release behaviors of hMSR8.1-DOX-TDNA (60 µg mL-1) after incubating with HeLa cells and L02 cells for 10 h under different conditions. Blue-fluorescent Hoechst 33342 was used to stain the nucleus. Cells were imaged using a 100 × oil-immersion objective.



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Figure 6. In vitro cytotoxicity assay curves for A) HeLa cells, and B) L02 cells treated with hMSR8.1-TDNA, hMSR8.1-DOX-TDNA and free DOX respectively. The cytotoxicity of hMSR8.1-DOX-TDNA for C) HeLa cells, and D) L02 cells pretreated with PDTC and NF κB p50 respectively. All the cytotoxicity values were obtained by plotting the cell viability percentage against the concentration of DOX. E) Photographs of RBCs incubated with hMSR8.1-TDNA at different concentrations ranging from 18.75 to 2400 µg mL-1 for 12 h. Red hemoglobin in the supernatant was damaged RBCs. Samples of RBCs incubated with water (+) and PBS (-) was used as positive and negative control, respectively.


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Table 1. Related parameters of different hMSRs Sample

Length (nm)

Width (nm)

aspect ratio

hMSR2.5 hMSR5.3 hMSR8.1 hMSR9.0

100 160 210 270

40 30 26 30

2.5 5.3 8.1 9.0

Zeta Potential (mV) 6.9 4.69 -2.38 -1.02

Size (nm) 122.3 200.9 246.4 297.0

BET surface area SBET (m2 g-1) 641.5 611.2 419.3 294.8


BJH pore Volume VP (cm3 g-1) 0.54 0.51 0.60 0.56

BJH pore diameter Dp (nm) 3.02 3.03 2.46 2.44

Load amount (µmol/g) 76.21 95.39 117.94 126.95


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Synthesis of Hollow Mesoporous Silica Nanorods with Controllable

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