Ru-Decorated Porous MetalâOrganic ... - ACS Publications

Subscriber access provided by University of Colorado Boulder

Article

Se/Ru-decorated Porous Metal-Organic Framework Nanoparticles for The Delivery of Pooled siRNAs to Reversing Multidrug Resistance in Taxol-resistant Breast Cancer Cells Qingchang Chen, Meng Xu, Wenjing Zheng, Taoyuan Xu, Hong Deng, and Jie Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12792 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Se/Ru-decorated Porous Metal-Organic Framework Nanoparticles for The Delivery of Pooled siRNAs to Reversing Multidrug Resistance in Taxol-resistant Breast Cancer Cells Qingchang Chen a, 1, Meng Xu a, 1, Wenjing Zheng a, Taoyuan Xu a, Hong Dengb and Jie Liu a,* a

Department of Chemistry, Jinan University, Guangzhou 510632, China

b

Key Laboratory of Electrochemical Technology on Energy Storage and Power Generation of

Guangdong Higher Education Institutes, South China Normal University, Guangzhou 510006, China * Corresponding authors, E-mail:[email protected]. Tel/Fax : +86-20-85220223 1

These authors contributed equally

Abstract We report here a novel and personalized strategy of selenium/ruthenium nanoparticles modified metal organic frameworks MIL-101(Fe) for delivering pooled small interfering RNAs (siRNAs) to enhance therapy efficacy by silencing multi-drug resistance (MDR) genes and interfere with microtubule (MT) dynamics in MCF-7/T (Taxol-resistance) cell. The existence of coordinatively unsaturated metal sites (CUSs) in MIL-101(Fe) can strongly interactions with the electron-rich functional groups of cysteine, which can be regarded as the linkage between selenium/ruthenium nanoparticles and MIL-101(Fe). Se@MIL-101 and Ru@MIL-101 loaded with MDR gene-silencing siRNAs via surface coordination can significantly enhance protection of siRNAs against nuclease degradation, increase siRNA cellular uptake, and promote siRNA escape from endosomes/lysosome to silence MDR genes in MCF-7/T cell, resulting in enhanced cytotoxicity through the induction of apoptosis with the signaling pathways of phosphorylation of p53, MAPK and PI3K/Akt and the dynamic instability of MTs and disrupting normal mitotic spindle formation. Furthermore, in vivo investigation of the nanoparticles on nude mice bearing MCF-7/T cancer xenografts confirmed that Se@MIL-101-(P+V)siRNA nanoparticles can significantly enhanced cancer therapeutic efficacy and decreased systemic toxicity in vivo. Keywords: selenium/ruthenium nanoparticle, MOF surface modifications, siRNA delivery systems, tubulin polymerization, multidrug resistance cancer therapy 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.Introduction Metal-Organic Frameworks (MOFs) are an emerging class of materials that have attracted increasing attention due to their excellent characteristics of self-assembled, porous and properties can be readily adjusted by varying the organic moleculars blocks. Therefore, porous metal−organic frameworks have been widely studied for varieties of applications.1-3 Recently, MOFs have been scaled down to nano-sizes, and this nanoscale metal organic frameworks (nanoMOFs) possess potential advantages such as structural diversity, excellent loading capacity of drugs, and intrinsic biodegradability.4,5 There have been reported that nanoMOFs could be served as efficient nano-carriers for delivery of agents for chemotherapy and imaging contrast. Lin group have developed two nanoMOF materials to deliver chemotherapeutic drugs and demonstrated that the framework decomposes in physiological medium and then the cisplatin-based prodrug diffuses through the shell structure in a controllable manner.6 Maspoch and co-workers reported that encapsulated some anti-cancer drugs as guest species within Zn2+-based nanoMOFs, and in vitro assays performed with dox-loaded nanoMOFs exhibited enhanced cytotoxicity to that of free dox.7 As these nanoMOFs were utilized as drugs delivery carriers, further research work is needed to demonstrate the real-time monitoring of drug release, uptake and therapeutic efficacy. Among all kinds the MOFs, MIL family is one of the most porous materials, which being a very prominent example that have been studied. For instance, MIL-101 has two different mesoporous cages (~29 Å and 34 Å internal diameters) and large Langmuir surface area, which makes the MIL-101 a unique candidate for storage and delivery of biologically agents. The narrow mesoporous cavities of MIL-101 can be used to load small molecule drugs, while the metal ions on external surfaces can be served to bind biomacromolecule such as siRNA and pDNA. For bioapplication, more and more research revealed that the surface modification of MIL-101 is an important aspect since the effective surface modification could not only avoid the degradation of bio-degrading enzyme to keep the original structure of MIL-101, but also facilitate the loading and release of siRNA or other drugs, thus providing the probability to achieve 2


Page 2 of 43

Page 3 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


targeted delivery.8-10 Despite these advantages, the related studies in the aspect of surface modification are quite rare at the outset of research of MIL-101 in drug and siRNA delivery system. Microtubules(MTs) are a kind of self-assembling protein and biological nanotubes that are essential for many radical cellular activities including cell motility, cell division and intracellular trafficking. For decades, there are many anti-tumor drugs that affect microtubule dynamics, such as paclitaxel and vincaleukoblastinum, have been a mainstay in the clinical treatment of solid tumours and leukemias. Despite the prominent success of these antitumour drugs, their clinical application is severely restricted by intrinsic or acquired drug-resistance in tumour cell.11-13 Nowadays, some mechanisms of tumour drug-resistance have been revealed, such as overexpression of P-glycoprotein (P-gp,MRP4, ABCB1) and the class III β-tubulin.14,15 P-gp is an ATP dependent broad-spectrum drugs efflux protein, which can significantly decrease the intracellular drug accumulation and class III β-tubulin can increase the dynamic instability of MTs, thus counteracting the stabilizati on effect induced by taxanes.16,17 Therefore, combintion of down-regulation P-gp expression and disruption of the dynamics of microtubules may be a efficient approach to inhibit the growth of drug-resistant tumor cells. RNA interference (RNAi) is a powerful and effective tool for sequence-specific suppression of genes, which has potential applications for gene-targeted therapy. Especially for the multidrug resistance mediated by P-gp and class III β-tubulin, which

has become

a

main

obstacle

to

the

therapy efficacy

of

most

microtubule-targeted anti-tumour drugs on clinic. To date, various of co-delivery platforms for nucleic acid drugs and chemotherapy agents have been developed to reverse MDR in tumour cells.18,19 In particular, there has been extensive attention and progress in the nanotechnology engineering of gene/drug delivery systems20, including functionalized mesoporous silica nanoparticles, PEI polymers or metal nanoparticles.21,22 In consideration of the potential advantages of MIL-101 such as high loading capacity, intrinsic biodegradability and the metal ions on the surfaces of nanoMOFs can be used to siRNA bind sites. In this study, we report a simple, tunable, 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 43

and scalable MOF-templated strategy to synthesize a mixed antitomour selenium /ruthenium nano-composite with useful siRNA loading properties. Selenium (Se) is one of the essential trace minerals with important pharmacological functions and many studies including epidemiological investigations, preclinical and clinical intervention have demonstrated that Se supplementation exhibited effective ability in reducing the incidence of cancers.23-25 Particularly, selenium nanoparticles (SeNPs) with excellent anti-tumour activity and low systemic toxicity are drawing increasing attention.26 Also, many ruthenium-based complexes show a remarkable antimetastatic activity

in

the

cancer

treatment.27,28

Therefore,

the

combintion

of

selenium/ruthenium-MOFs nanoparticles and siRNA not only can significantly enhance the therapy efficacy by silencing MDR genes and interfere with microtubule dynamics in MCF-7/T cell, but also possessed high tumour-targeted, enhanced anti-tumour efficacy and decreased systemic toxicity in vivo(scheme 1). These results suggest that selenium/ruthenium-MOFs nanoparticles is a promising vector for the delivery of siRNA and effective therapeutics for treatment of drug-resistant cancers.

Scheme 1. Mechanism of the reversal of drug resistance and induce apoptosis by the disruption of 4


Page 5 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microtubule in MCF-7/T (Taxol- resistance) cancer cells.

2. Materials & methods 2.1 Materials All experiment materials were purchased from commercial companies and used without further additional treatment. FeCl3 ·6H2O, HF, terephthalic acid, cysteine, sodium selenite and Ethidium bromide (EB, 95%) were purchased from Sigma. Agarose and Fetal bovine serum (FBS) were obtained from Shanghai Gene-Tech Co., Ltd and Gibco. The antibodies for VEGF, P-gp, secondary antibody (goat antimouse IgG H&L, Chromeo™488) and DAPI were obtained from UK Abcam. The siRNAs and fluorescent siRNA (FAM-labeled) were obtained from GenePharma Co., Ltd. The sequences

of

pooled

siRNA

5’-AAGAAGGAAAAGAAACCAACUTT-3’,

VEGF

are siRNA

PsiRNA, (VsiRNA,

5’-AUGUGAAUGCAGACCAAAGAATT-3’).

2.2 Synthesis and characterization of Se@MIL-101 and Ru@MIL-101 Firstly, MIL-101(Fe) is synthesized by mixing 0.675 g FeCl3 ·6H2O and a solution of 0.225 g terephthalic acid in 15 mL of Dimethyl Formamide (DMF). After 24 h thermal treatment in a 30 mL stainless steel autoclave at 383 K, and cooling to room temperature. The obtained particles were isolated by centrifuging, and washed with DMF and ethanol. The mixture was filtered first using a large-pore fritted glass filter (n°2, Schott) to remove most of the DMF and carboxylic acid. The MIL-101(Fe) powder and water mixture passed through the filter while the free carboxylic acid stayed in the glass filter. The powder were isolated by centrifuging and washed thoroughly with ethanol and deionized water, and then soaked in a mixture of 95% EtOH and 5% water at 353 K for 24 h. The solid powder was dried overnight at 473 K under vacuum. Secondly, cysteine (Cys) modified MIL-101(Fe) was prepared based on the following process. In brief, 0.5 g of MIL-101(Fe) powder was dried at 473 K for 12 h to remove the water molecular on the CUS sites, then the dehydrated MIL-101(Fe) was 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

suspended in 30 ml anhydrous ethyl alcohol. Cys (0.1 mol ) was added to the suspension and stirred at reflux for 12 h. The mixture was isolated by centrifuging and washed with ethanol/ de-ionized water, and dried at room temperature to obtained cysteine-modified MIL-101(MIL-101-Cys). The contents of sulfur moieties from Cys were determined by an elemental analyzer with a TCD detector (Thermo Fisher, Flash-2000). The amount of Cys is calculated based on the molar masses of S(32.065 g •mol-1), Cys(121.15 g •mol-1). Finally, for selenium and ruthenium nanoparticles incorporation, an appropriate concentration of Na2SeO3 and RuCl3· nH2O aqueous solution was added dropwise to 1.0 g activated MIL-101-Cys under vigorous stirring. MIL-101-Cys was strongly captured SeO32- or Ru3+ by impregnation within a mixture of 120 min. The solid powder was settled down by centrifuging, and filtered with de-ionized water/ethanol solution. The impregnated MIL-101-Cys samples were reduced separately by sodium borohydride-ethanol solution (w/v, 0.7g/mL) at 298 K for 2 h to yield Se@MIL-101 and Ru@MIL-101. The size and morphology were measured using a transmission electronmicroscopy (TEM). The inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to measure the content of selenium and ruthenium in NPs modified MIL-101(Fe). Powder X-ray diffraction patterns (PXRD) of the MIL-101(Fe) and nanoparticles functionalized MIL-101(Fe) were recorded using a CuKɑ line (λ 1.5406 Å) of a Rigaku D/Max 2200 X-ray diffractometer. N2 adsorption-desorption isotherms was performed to measure the BET surface area (p/p0 range from 0.05 to 0.3), pore size (BJH method), and pore volume (at p/p0= 0.99 by single point method). Confocal Raman Spectrum (Renishaw, system 2000) was used to examine MIL-101, Se@MIL-101. Samples were dehydrated by Linkam TS1500 and observed by optical microscope (Leica). Spectra was excitated at 514.5 nm (Power: 2.5 mW; beam size: 1 µm), and collected in the range 100–4000 cm-1.

2.3 Preparation of Se@MIL-101-siRNA and Ru@MIL-101-siRNA Nanoparticle-siRNA composites were prepared by mixing Se@MIL-101 and 6


Page 6 of 43

Page 7 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ru@MIL-101 with 100 nM siRNA for 30 min at room temperature. For the pooled siRNA

groups,

50

nM

P-gp

Se/Ru@MIL-101-(P+V)siRNAs.

siRNA The

and

weight

50

nM

ratio

of

VEGF

siRNA

Se@MIL-101

in and

Ru@MIL-101 are 1:1, 2:1, 4:1, 8:1 and 16:1, respectively. All the nanopaticles and siRNA solutions were diluted with pH 7.4 PBS buffer.

2.4 Gel retardation assay and siRNA release Firstly, 10 µL of Se@MIL-101-siRNA and Ru@MIL-101-siRNA at various ratios (vehicles /siRNA) from 1 to 16 in 80% glycero-HEPES (pH 7.4) buffer were mixed. Electrophoresis was performed on 2% agarose gel containing 0.5 µg/mL ethidium bromide at 100 V for 30 min. To study the protection of siRNA by Se@MIL-101 and Ru@MIL-101, Naked siRNA and Se/Ru@MIL-101-siRNA were incubated in 50% calf serum medium at 37 °C for 0, 1 , 2 , 4 and 6 h. At each time-point, samples were taken directly into 1% SDS loading buffer and frozen at 4 °C. After the final sample was frozen, siRNA were run on a 2% agarose gel containing 0.5 µg/mL ethidium bromide at 100 V for 30 min. To

evaluate

the

release

rate

of

siRNA,

Se@MIL-101-siRNA

and

Ru@MIL-101-siRNA at a mass ratio of 16:1 were cultured in HEPES buffer (10 mM, pH 7.4) at 37 °C. Samples At indicated time points, samples were centrifuged, then using ES-2 spectrophotometer to measure the concentration of siRNA in supernatant. All release experiments were conducted in triplicate.

2.5 Flow cytometric analysis Cellular uptake of nanoparticles was detected with the fluorescent intensity of FITC labeled siRNA (siRNAFAM) in the cells. Paclitaxel resistance MCF-7/T cells (5.0 × 104 cells/well) were cultured in 6-well culture plates. After 24 h incubation, cells were treated with siRNAFAM (100 nM) alone or nanoparticles-siRNAFAM in serum-free medium for 12 h. Then, MCF-7/T cells were collected, washed and analyzed by flow cytometry. 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.6 TEM images of cellular uptake After incubated with Se@MIL-101-siRNA and Ru@MIL-101-siRNA for 24 h, MCF-7/T cells were washed and then fixed. Cells were dehydrated using ethanol and treated with propylene oxide. TEM samples were sectioned with a thickness of 60 nm. The sections examined on a transmission electron microscope (JEOL) at 80 kV.

2.7 Hematoxylin & eosin staining of MCF-7/T cells MCF-7/T cells exposed to Se@MIL-101-siRNA and Ru@MIL-101-siRNA on slides in 6-well culture plate for 6 h, 12 h or 24 hours at 37 °C, 5% CO2, At each time-points, cells was washed three time with PBS and fixed with neutral formalin, then stained with H&E for 6 minutes.

2.8 Confocal Raman Microscopy Confocal Raman Spectrum (Renishaw, system 2000) was used to examine the subcellular localization of nanoparticles in MCF-7/T cells. Samples were dehydrated by Linkam TS1500 and observed by optical microscope (Leica). Spectra was excitated at 514.5 nm (Power: 2.5 mW; beam size: 1 µm), and collected in the range 100–4000 cm-1.

2.9 Endosomal escape Laser confocal microscopy was used to study the cellular uptake and endosomal escape in MCF-7/T cells, Before transfection, 1×104 MCF-7/T cells were seeded in culture dish one day. Then, Se@MIL-101-siRNAFAM and Ru@MIL-101-siRNAFAM (weight ratio of vehicles to siRNAFAM = 16:1, the concentration of Se/Ru@MIL-101 is 10 µg/mL ) were incubated with cells at 37 °C for 60 min and 180 min. After transfection, the culture medium was removed, and the cells were washed with PBS. The samples were stained with LysoTracker solutions and observed by a laser confocal microscope (Leica Microsystems, Wetzlar, Germany). The fluorescence signals are siRNAFAM (green, excition: 488 nm, emission: 490~510 nm ) and LysoTracker (red, excition: 577 nm, emission: 590 nm). 8


Page 8 of 43

Page 9 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.10 In vitro transfection studies Paclitaxel resistance MCF-7/T cells were cultured in 24-well culture plates (5.0×104 cells/well) and incubated at 37 °C, 5% CO2 for 24 h. Briefly, 1.0 µg plasmids (EGFP) mixed with different vehicles at ratios (Se/Ru@MIL-101 : pEGFP) of 4, 8 and 16 for 30 min. The medium was removed and replaced with fresh medium (serum free) before the addition of the nanoparticle-siRNA composites. After incubated with the nanoparticle-siRNA composites for 5 h, the medium was removed and again replaced with flesh 10% FBS medium and post incubated for 48 h. After 48h incubation, the expression of green fluorescence protein was evaluate by a Olympus fluorescence microscope.

2.11 MTT assay and Cell cycle analysis The influences of Se@MIL-101-siRNA and Ru@MIL-101-siRNA nanocomposites on the proliferations of MCF-7/T cells were evaluated by using the MTT assay. Cells were seeded in 96-well plates (4000 cells/well) and incubated for 24 h. The media was removed and exchanged by 100 µL of fresh 10% FBS media. Pooled siRNA solution, paclitaxel solution (1 µg/mL), MIL-101(10 µg/mL), Se/Ru@MIL-101 (10 µg/mL) and Se/Ru@MIL-101-siRNA (weight ratio of vehicles to siRNA = 20:1, the concentration of Se/Ru@MIL-101 is 10 µg/mL ) were added to the culture medium. After 48 h incubation, 10 µL/well MTT was added and further incubated 4 h at 37 °C. Then, the supernatants were removed and replaced with DMSO (150µL/well). Finally, a microplate reader was used to measure the absorbance of 96-well plates at 570 nm. Cell-cycle distribution was measured by flow cytometry (BD, FACS Aria). MCF-7/T cells were cultured in 6-well culture plates with a density of 1 × 106 cells/well. After treated with Se@MIL-101-(P+V)siRNA and Ru@MIL-101-(P+V)siRNA at various concentrations for 24 h, the cells were fixed in 500 µL ethanol (70%) and store at 4 °C overnight. After PBS washed, the cells were stained with PI solution in the dark for 15 min. Then, all samples were measured by flow cytometry.

2.12 Immunofluorescence staining and Western blot 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 43

Paclitaxel resistance MCF-7/T cells (4 × 104 cells/well) were cultured on a samll glass chamber slide in 6-well plates. After 24 h incubation, the drugs and nanocomposites were added into the mediums and further incubated for 12 h. Then, the cells were washed with PBS for three times and fixed in paraformaldehyde (4%) solution for 20 min. Then the cells were permeabilized by using 0.25% Triton X-100 (in PBS) for 5 min. After washed with PBS, the cells were blocked in 1% BSA/ PBS-T solution for 1 h. Then the cells were treated with P-gp and VEGF primary antibody and incubated overnight at 4°C. After washed with PBS three times, the cells further incubated with secondary antibody at 37 °C for 1 h, following by treated with DAPI for 10 min. The cells were washed with PBS and finally observed with a confocal microscope (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany). For western blot, MCF-7/T, MCF-7 cells were cultured in 6-well plates (3 × 105 cells/well) for 24 h, then the cells were incubated with naked-siRNA, Se/Ru@MIL-101,

Se/Ru@MIL-101-PsiRNA,

Se/Ru@MIL-101-VsiRNA,

and

Se/Ru@MIL-101-(P+V)siRNA for another 24 h. The cells were collected and lysed by using a proteins extracted reagent (M-PER, Pierce Inc, USA). The protein concentration of obtained proteins was measured by a standard curve (Novagen Inc, USA, BCA assay kit). Then, SDS-PAGE was performed to separate the proteins and transferred to PVDF membranes, following by blocking with 5% skimmed milk overnight at 4°C. The membranes then treated with primary antibodies (dilution 1:1000), including P-gp, VEGF, Akt, p-AKT, Erk, p-ERK, Bcl-2, Bax, p-p53, Caspase 3, c-Caspase 3 and β-Actin for 1h. Finaly, the membranes incubated with secondary antibody (dilution 1:4000) for 1h. All samples were visualized using the chemiluminescence system (ECL, Pierce Biotech, Rockford, IL).

2.13 Atomic force microscopy (AFM) Atomic force microscope (AFM) was used to detecte the morphology of MCF-7/T cells.

Briefly,

the

cells

were

incubated

with

Se@MIL-101-siRNA

and

Ru@MIL-101-siRNA in 6-well plates for 12 h, 24 hours at 37 °C, 5% CO2. Then, the cells were washed three times with PBS and fixed by paraformaldehyde (10%). The 10


Page 11 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


samples were observed by atomic force microscope (Veeco instruments, USA).

2.14 Tubulin and β-Actin staining MCF-7/T cells (4 × 104 cells/well) were cultured on a samll glass chamber slide in 6-well plates. Then removed the medium and replaced with fresh 10% FBS medium containing Se/Ru@MIL-101-(P+V)siRNA (10 µg/mL). After 12 h and 24 h incubation, the cells were rinsed and stained with β-Actin (Green), Tubulin Tracker (Red) and DAPI (Blue) for 60 min. The samples were observed by a laser confocal microscope.

2.15 MCF-7/T Xenograft mouse model and In vivo antitumor test The animal experiments in this paper were approved by the Institutional Animal Care and Use Committee of Jinan University (NO. SCXK2008-0002). MCF-7/T cells (1×107/mL) were injected into the right subaxillary of 5~6 weeks old nude mice by subcutaneous injection. When the volume of tumor grew to 50 mm3, all the mices were divided into two groups (a control group, a treatment groups, and 5 mices per group). Under administration, the mices in the treatment groups were given with Se@MIL-101-(P+V)siRNA (siRNAs: Se@MIL-101, 1:16 w/w ratio) at dosage of 10 mg/kg by intravenous injection for 15 days (12 µg of siRNA per mouse). Mices in control groups were given with PBS only. The tumour sizes and body weight of mices were recorded everyday, the tumour sizes were calculated as length×width×height by using Vernier caliper measurements. After 15 days treatment, all mices were sacrificed and excised the tumours, then measured the size and photographed. For histological evaluation, visceral organs and tumors were excised. All tissues were fixed with buffered neutral formalin (10 %), dehydration and embedded in paraffin. The sections were cut from each samples and stained primarily with H&E for histopathological study.

2.16 Statistical analysis The statistical analysis was made by the Student t-test or one-way analysis of variance 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(ANOVA). The significant difference was considered *p < 0.05 for significant, and **p < 0.01 for very significant. All data presented were expressed as means ± SD.

3. Results & Discussion 3.1 Synthesis and characterization of Se@MIL-101 and Ru@MIL-101 There have been reported that the existence of CUS in Metal Organic Frameworks porous materials is very beneficial. The presence of unsaturated Fe(III) sites or Cr(III) sites in MIL-101 provides an intrinsic coordination property of chelating with electron-rich functional groups.9,10,29 In this work, we demonstrated that the terminal water molecules possessed by trimeric Fe(III) octahedral clusters of MIL-101, can be removed from the framework through vacuum treatment at 473 K for 12 h, and the providing CUS served as Lewis acid sites in the framework usable for grafting a mild reducing agent Cysteine (Figure 1A). The FTIR spectra of the dehydrated Cys-MIL-101 sample confirmed the grafting. As shown in Figure 1C, in the spectra of the Cys-MIL-101, the ν(N-H), ν(C-H) and ν(C-N) stretching regions were consistent with pure cysteine, which indicated the presence of Cys. Interestingly, the stretching vibrations of C-H are shifted to larger values (2800-3200 cm-1 ), which is consistent with the previous observed when small molecule is grafted to a Lewis acid site by coordination.30 The elemental analysis confirmed the presence of sulfur moieties (3.64 wt.% of the MIL-101) from the functionalization with Cys. So, the amount of Cys in the material is w(Cys)=13.93 wt.%. These results clearly demonstrated that the Cys selective grafting onto CUSs in mesoporous cages of MIL-101(Fe). Furthermore, we propose an important approach for the encapsulation of anti-timour nanoparticles, such as selenium/ruthenium NPs over the Cys-grafted MIL-101 according to in situ reduction of Na2SeO3 and RuCl3. Se@MIL-101 and Ru@MIL-101 nanoparticles were synthesised by an impregnation method using Na2SeO3 and RuCl3 aqueous solution as precursor. Briefly, after the SeO32- and RuCl3 impregnated into the cages of MIL-101, these precursors were gently reduced in situ by NaBH4 to form Se/Ru nanoparticles at a low temperature. TEM images confirmed that SeNPs and RuNPs were modified on the external surface of MIL-101(Fe) with 12


Page 12 of 43

Page 13 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


average diameters of 20 ±2.0 nm and 10±2.0 nm, respectively(Figure 1B). The TEM image of reduced Na2SeO3 in the condition of bare MIL-101 (not grafted with Cys ) also shown for comparison, SeNPs were spread around the MIL-101(Figure S1). The result indicated that the Cys grafted to MIL-101 play a critical role in the strategy of modified SeNPs /RuNPs onto MIL-101. This may attribute to the thiol groups of Cys, it is well known that thiol easily to conjugate to the surface of nanoparticles such as Pd, Pt, or Au by forming metal-S bond.31 Thus, spherical SeNPs and RuNPs were capped with the terminal thiol groups of Cys-grafted MIL-101 through formation of Se-S/Ru-S bond, which led to the highly stable structure of the conjugates. The average particle size of the MIL-101 was approximately 150 nm. After loading with SeNPs or RuNPs, the size of Se@MIL-101 and Ru@MIL-101 increased to 160 nm and 180 nm, respectively. The loading amount of Se and Ru NPs on MIL-101 by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The result showed that the loading amount of selenium and ruthenium (wt%) in NPs modified MIL-101(Fe) are

6.92 wt.% of the Se@MIL-101 and 8.13 wt.% of the Ru@MIL-101, respectively (Table S1). Figure 1D showed the PXRD spectrum of Se/Ru@MIL-101, which are match well with the previous observed XRD spectrum of MIL-101. The results suggested that after the loading of SeNPs and RuNPs, Se@MIL-101 and Ru@MIL-101 showed inconspicuous crystallinity loss in XRD spectrum, and there are no apparent supplementary of Bragg peaks, indicating that the crystal structure of MIL-101 framework is mostly maintained. But the intensities of these XRD peaks of Se@MIL-101 and Ru@MIL-101 are slightly decreased, confirming the introduction of nanoparticles onto the surface of MIL-101. The slight decrease of XRD peaks' intensities of Se@MIL-101 and Ru@MIL-101 may attribute to the Cys grafting and the the inclusion of Se/Ru particles within the framework of MIL-101.32,33

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1.(A)Formation of the Se/Ru Nanoparticles Modified Porous Metal Organic Framework. (B)TEM image of MIL-101,Se@MIL-101 and Ru@MIL-101. (C) FT-IR spectrum of Cys, MIL-101, MIL-101-Cys and (D) XRD pattern of MIL-101, Se@MIL-101 and Ru@MIL-101.

Furthermore, Figure 2A showed that the resulting surface decoration is visible according to the N2 adsorption-desorption isotherms of the nanoparticles loading MIL-101. All adsorption−desorption isotherms show a characteristic of mesoporous materials.34,35 As compared to the MIL-101, the BET surface area, pore volume and size of MIL-101-Cys exhibited a slight decrease after grafted with Cys. The pore size of MIL-101 was decreased from 2.25 nm to 2.18 nm (Figure S2). Se@MIL-101 and Ru@MIL-101 exhibited a significant decrease in the adsorbed amount of N2 and the BET surface areas reduced from 3257 m2·g-1 to 2905 m2·g-1 and 2071 m2·g-1 , respectively. The distribution curves of pore size also suggested that the NPs loading result in a slight decrease of the pore volume and pore sizes (Figure 2B). The pore volume and diameter of pure MIL-101was determined to be 1.49 cm3·g−1 and 2.25 nm. After the loading of Se or Ru nanoparticles, the pore diameter decreased to 2.19 nm, and the pore volume of Se/Ru loaded MIL-101 decreased to 1.39 and 1.37 cm3·g−1, respectively. From the pore size distribution curves of Cys@MIL-101 and NPs@MIL-101, the pore size of Se@MIL-101 and Ru@MIL-101 (2.19 nm and 2.17 nm, respectively) are almost the same as Cys@MIL-101, which clearly indicates that the Se/Ru 14


Page 14 of 43

Page 15 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanoparticles are just modified on external surface of MIL-101(Fe) and not embedded in the pore.

Figure 2. (A) Nitrogen adsorption isotherms 77K and (B) distribution curves of pore volume and pore size (diameter) of the MIL-101, SeNPs@MIL-101 and RuNPs@MIL-101. (C) Agarose gel electrophoresis to evaluate the siRNA loading affinity and protective effect of SeNPs@MIL-101 and RuNPs@MIL-101 at various mass ratios from 1:1 to 16:1. Naked siRNA served as the control group.

3.2 siRNA loading affinity of Se@MIL-101 and Ru@MIL-101 It is well known that the binding affinities between the gene vectors and siRNA is strongly influence the loading capacity, transfection efficiency. Therefore, we firstly investigated the siRNA loading capacity of Se/Ru@MIL-101 by agarose gel electrophoresis. The siRNA was loaded onto Se@MIL-101, Ru@MIL-101 by simply mixing appropriate amount of siRNA and nanoparticles in deionized water to form 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

nanoparticle-siRNA composites. It has been reported that the metal ions on the surface of nanoMOF can be served as the siRNA binding sites. Herein, the internal diameters of mesoporous cages in MIL-101 are ~29 Å and 34 Å, which are too narrow to absorbed siRNA inside. So, the pooled siRNAs is believed to bind to the external surface of Se/Ru@MIL-101, through the formation of multiple coordination bonds between the vacant Fe(III) sites of MIL-101 and phosphate residues on the backbone of siRNA. As shown in Figure 2C(upper), the siRNA binding capabilities of Se/Ru@MIL-101 were evaluated by electrophoresis. After binding to Se@MIL-101 and Ru@MIL-101, siRNA migration band were complete retarded at mass ratio of 16:1 and 4:1 (Se/Ru@MIL-101: siRNA), respectively, indicated that Se@MIL-101 and Ru@MIL-101 can efficiently capture the free siRNA. Furthermore, for in vivo application, it is essential to protect siRNA from degradation by RNase, since free siRNA can be rapidly degraded by RNase during the process of cellular uptake and transfection. We investigated the siRNA protective effect of Se/Ru@MIL-101 in a mimic physiological condition.36 In the serum stability assay, as shown in Figure 2C(bottom), it was clearly observed a siRNA band in the groups of Se/Ru@MIL-101-siRNA incubated in serum for 4 h, while the band of naked siRNA was disappeared (complete degradation) at the same time. These results indicated that Se@MIL-101 and Ru@MIL-101 also exhibited good performance in protecting siRNA from RNase degradation.

16


Page 16 of 43

Page 17 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (A) Flow cytometry of naked siRNAFAM,Se@MIL-101-siRNAFAMand Ru@MIL-101-siRNAFAM uptake in MCF-7/T cells at 12 h post-transfection. (B, C) TEM images of MCF-7/T cells exposed to Se@MIL-101-siRNA and Ru@MIL-101-siRNA nanoparticles. The arrows in the TEM show cellular uptake and localization of Se@MIL-101-siRNA and Ru@MIL-101-siRNA uptake. (D) Morphology of MCF-7/T cells incubated with Se@MIL-101-siRNA and Ru@MIL-101-siRNA at 10 µg/mL for 6，12 or 24 h and stained by H&E. Scale bar represented 50 µm. (E) Raman spectra of Se@MIL-101-siRNA in MCF-7/T cells treated.

3.3 Cellular uptake of Se@MIL-101-siRNA and Ru@MIL-101-siRNA To initiate the processes of transfection and gene silencing, nanoparticle-siRNA composites must be first across cell membranes and uptaked by targeted cells. Therefore, it is important to investigate the efficiency of Se/Ru@MIL-101 to deliver siRNA into MCF-7/T cells. Firstly, MCF-7/T cells were incubated with naked FAM-labeled siRNA, Se@MIL-101-siRNAFAM and Ru@MIL-101-siRNAFAM for 12 h, then the cellular uptake efficiency was evaluated by using flow cytometry to detected the fluorescence intensity of FAM-labeled siRNA in MCF-7/T cells. As shown in Figure 3A, poor cellular uptake was observed in the group of MCF-7/T cells cultured with free siRNA, this may attributed to the negative charge of naked siRNA, which make it difficult to penetrate the cell membrane. However, the cellular uptake of 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Se@MIL-101-siRNAFAM and Ru@MIL-101-siRNAFAM were significantly enhanced as compared to siRNA alone. These results significantly suggested that Se/Ru@MIL-101 can efficiently bind to siRNA and deliver into MCF-7/T cells. Furthermore, the sub-cellular localization of Se/Ru@MIL-101-siRNA in MCF-7/T cells were examined by TEM and Raman microscopy.37 TEM is a powerful technique which can accurately use for intracellular structure characterization. Thus, we made further investigations with TEM to gain more detail information about the cellular uptake proccess inside the cells.38 In this experiment, MCF-7/T cells were incubated with a low concentration of Se/Ru@MIL-101-siRNA (2 µg/mL) to avoid the cell apoptosis inducing by the nanoparticles. As shown in Figure 3B and C, the TEM images clearly showed that Se/Ru@MIL-101-siRNA nanoparticles were uptaked by MCF-7/T cells and demonstrated that the nanoparticles were taken up in membrane bound vesicles of MCF-7/T cells without changing the cell morphology or impacting other intracellular organelles, and the mitochondrial crista and nuclei remained intact after the entry of Se/Ru@MIL-101-siRNA. It is also shown that more secondary lysosomes and vacuoles can be observed in Se@MIL-101-siRNA exposured cells. Intra-cellular localization of nanoparticles are crucial for evaluating their biocompatibility. Therefore, we used H&E staining to examine the influence of higher concentration of Se/Ru@MIL-101-siRNA in the MCF-7/T cells.39,40 As shown in Figure 3D, Se/Ru@MIL-101-siRNA were uptaked through endocytosis and internalized in the cytoplasm after 6 h treated. H&E staining was also used to check the morphology of MCF-7/T cells treated with Se/Ru@MIL-101-siRNA. It is clear that the cells displayed appreciable morphological changes after 24 h treated. As observed under a light microscope, the treated cells became small and round with pyknotic nuclei, the nuclei condensed and fragmented. Overall, the morphology was characteristic of apoptosis. In addition, MTT assay showed that the cytotoxicity of the poold siRNA loaded in Se/Ru@MIL-101 to paclitaxel resistance MCF-7/T cells significantly increased, suggesting that Se/Ru@MIL-101-(P+V)siRNA can enhance the synergistic therapeutic effects of the gene therapeutic and chemotherapy. (Figure S4-5). Furthermore, The existence of the Se@MIL-101-(P+V)siRNA in MCF-7/T 18


Page 18 of 43

Page 19 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cells were primarily recognized by Confocal Raman Spectrum. The Spectrum of the composite material Se@MIL-101 in comparison with that of the isolated MIL-101 was showed in Figure S6, a typical Raman spectrum obtained from isolated MIL-101.41,42 For MIL-101, the characteristic bands at 3070 cm-1 is assigned to the aromatic C-H (Ar-H) vibration of terephthalic. The C=C stretching vibration of C=C -1

-1

-1

(Benzene-body) are found at 1640 cm , 1489 cm and 1389 cm and the peaks at 1560

cm-1 assigned to the symmetric stretching vibration of Ar-CO2- (C=O). However, the Raman spectra of Se@MIL-101 further support the inclusion of the linker Cys and selenium nanoparticles into the MIL-101cavities and surface. Besides the characteristic bands of MIL-101 mentioned above, the spectra of Se@MIL-101 also exhibit new small bands located at approximately 693, 537 and 445 cm−1 assigned to C-S coupled vibration, out-plane rocking vibration and in-plane rocking vibration of L-cysteine, respectively. Furthermore, Confocal Raman Spectrum can also use to confirm the internalization of Se@MIL-101-(P+V)siRNA in MCF-7/T cells. Figure 3E showed the confocal Raman spectrum of MCF-7/T cells exposed to the nanoparticles for 6 h (the sample of H&E). This result confirmed the characteristic bands of MIL-101 in the exposed cells, indicating that the internalized Se@MIL-101-(P+V)siRNA are structurally intact. In addition, these results indicated that the Se/Ru@MIL-101 may facilitates the siRNA internalization via endocytosis pathways.

Figure 4. (A) Time-dependent confocal microscopy of siRNA successfully escaped from endosomes. MCF-7/T cells were incubated with Se@MIL-101- siRNAFAM and Ru@MIL-101-siRNAFAM for 60 min and 180 min. LysoTracker (Red); siRNAFAM(Green). Bar represents 5 µm. (B) Fluorescence microscope images of MCF-7/T cells transfected by 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

Se@MIL-101 and Ru@MIL-101 for 24 h. Green fluorescence represented the expressions of EGFP plasmid in MCF-7/T cells.

3.4 Intracellular release and transfection procedures Following successful internalization into tumour cells, one of the most crucial events for

effective

intracellular

siRNA

delivery

is

the

escape

from

the

endosomal−lysosomal pathway. Therefore, The endosomal escape of siRNA was investigated by confocal laser scanning microscopy. It is clearly observed in Figures 4A, there are amounts of siRNAFAM (Green) were overlapped with the lysosome (Red) in

MCF-7/T

cells

after

60

minutes

incubation,

indicating

that

Se/Ru@MIL-101-siRNAFAM were internalized inside the lysosome of MCF-7/T cells. After incubation for 3 h, most of the green (siRNAFAM) and red (Lysosome tracker) fluorescence in the cytoplasm were separated, suggested that the siRNA escape from the entrapment of endo/lysosome and accumulated in the cytoplasm. For these results, we can infer from the distinctive structure of MIL-101, the vacant Fe sites show a high affinity to phosphate ions. While Se/Ru@MIL-101 internalized and entrapped in the endosome/lysosome, the endogenous high concentrations of phosphate ions will cause the decomposition of MIL-101. After the skeleton of MIL-101 collapsed, the dissociated Fe ions can bind to phosphate-groupenriched lysosome membrane to disturb the stability of lysosome structure, thus to facilitate the siRNA escape and release. We have already demonstrated that the efficient load and release capacity of Se/Ru@MIL-101. Furthermore, the gene transfection efficiency of Se@MIL-101 and Ru@MIL-101 were measured by EGFP transfection assay in MCF-7/T cells. As shown in Figure 4B, the transfection efficiency of EGFP (green fluorescence) significantly increased at mass ratios (Vector/pEGFP) from 4:1 to 16:1. These results strongly imply that Se@MIL-101 and Ru@MIL-101 are efficient gene deliveries vectors. We believe that the large pore volumes and surface area of this two synthesized vectors help to increase the contact area with siRNA, allowing for highly efficient siRNA compaction, which is beneficial for enhanced gene transfection. 20


Page 21 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.5 Overcame multi-drug resistance by Se/Ru@MIL-101-(P+V)siRNA We

further

evaluated

the

efficiency

of

gene

silence

mediated

by

Se/Ru@MIL-101-siRNA in MCF-7/T cells, the expression of p-glycoprotein, VEGF were measured by immunofluorescence and western blot. As shown in Figure 5A and C, a high expression of P-gp, VEGF in Taxol-resistant MCF-7 cells as demonstrated the strong fluorescence intensity detected by CLSM image, revealed that the chemotherapy resistance is caused mainly by the high expression of these drug-resistant related proteins. Figure 5B and D showed that MCF-7/T cells incubated

with

Se/Ru@MIL-101-PsiRNA or

Se/Ru@MIL-101-VsiRNA

can

significantly down-regulated the expression of P-gp or VEGF as compared to the groups of naked Se/Ru@MIL-101 or loading scrambled siRNA (denoted by Se/Ru@MIL-101-XsiRNA). Western blot was also used to evaluate the silencing efficiency of MDR1 gene. P-gp/VEGF expression in MCF-7/T cells exposed to Se@MIL-101-siRNA and Ru@MIL-101-siRNA significantly decreased as compared to the group of naked siRNA, Se/Ru@MIL-101 alone or Se/Ru@MIL-101-XsiRNA. Interestingly, the groups using pooled siRNAs exhibited significantly down-regulation of P-gp and VEGF as compared to single siRNA, suggested the enhanced gene silencing effects of pooled siRNAs ( Figure 5E and F) . Futhermore, we verified that Se/Ru@MIL-101-(P+V)siRNA can enhanced cytotoxicity through the induction of apoptosis with the signaling pathways of phosphorylation of p53, MAPK and PI3K/Akt pathways in the MCF-7/T cells (Figure S7).

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5.(A) The expression of p-glycoprotein (P-gp) in MCF7 and MCF-7/T cells by immunofluorescence staining. (B) Comparison of suppression of P-gp expression in MCF-7/T cells. (C) The expression of VEGF in MCF7 and MCF-7/T cells by Immunofluorescence staining. (D) Comparison of suppression of VEGF expression in MCF-7/T cells. Bar represents 20 µm. (E,F) Comparison of suppression of P-gp and VEGF expression in MCF-7/T cells.1-6: Control, Naked pooled siRNA, Se@MIL-101, Se@MIL-101-PsiRNA, Se@MIL-101-VsiRNA, Se@MIL-101-(P+V)siRNA. a-f: Control, Naked pooled siRNA, Ru@MIL-101, Ru@MIL-101-PsiRNA, Ru@MIL-101-VsiRNA, Ru@MIL-101-(P+V)siRNA. Bars shown are mean ± SE, and differences between control and treated groups were analyzed by one-way ANOVA. P-gp expression of treated group vs. control was indicated by (*) p < 0.05, (**)p < 0.01. VEGF expression of treated group vs. control was indicated by (#) p < 0.05.

3.6 Disruption of the dynamics of microtubules and interfering with interphase in MCF-7/T cells Microtubules is a kind of cytoskeleton proteins, which are closely related to cell viability, cytokinetics and being required for maintaining the relative integrity of cell morphology.43 We studied the change of cellular structure, microtubules and actin in the Se/Ru@MIL-101-(P+V)siRNA treated MCF-7/T cells. The investigation of cell morphology by atomic force microscopy(AFM) upon Se@MIL-101-(P+V)siRNA and 22


Page 22 of 43

Page 23 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ru@MIL-101-(P+V)siRNA treatment exhibited that the periphery of MCF-7/T cells gradually shrinked and the cellular morphology were destroyed after 24 h incubation. (Figure 6A and B). We further determined the inhibiting effect of tubulin sequestered by Se/Ru@MIL-101-(P+V)siRNA nanocomposites on microtubule assembly. MCF-7/T

cells

were

cultured

with

different

concentration

of

Se/Ru@MIL-101-(P+V)siRNA, then immunofluorescent stained with β-tubulin Tracker. CLSM images showed that microtubules in control cells were slender, outstretched and well distributed, exhibiting a normal structure of cytoskeleton. However, it was clearly observed that the peripheral MT were damaged and shrunken moderately in cells that treated with a high concentration of 20 µg/mL(Figure 7B). Fuethermore, we investigated the influence of Se/Ru@MIL-101-(P+V)siRNA on the actin cytoskeleton in MCF-7/T cells. As shown in Figure 7A, MCF-7/T cells in control group exhibited normal structure of actin proteins and single, complete cell nucleus can be observed. In the treated group, Se/Ru@MIL-101-(P+V)siRNA exhibited enhanced damaged and disrupted to the actin, shrinkage of the cytosolic, and leads to a siginificant change in cell morphology.

Figure 6. Confocal fluorescence images of MCF-7/T cells treated with (A) Se@MIL-101-(P+V)siRNA and (B) Se@MIL-101-(P+V)siRNA. Microtubule was stained with its respective Tubulin Tracker (red) and DAPI is used to stain the nucleus. MCF-7/T cells were treated by different nanoparticles for 12h, 24 h. Bar represents 5 µm.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 43

Figure 7. MCF-7/T cells treated with different concentrations of Se@MIL-101-(P+V)siRNA and Ru@MIL-101-(P+V)siRNA showing intracellular damage of (A) Actin and (B) microtubules. Actin is stained with Actin-Tracker (green) and tubulin-red). Microtubule was stained with its respective Tubulin Tracker (red) and DAPI is used to stain the nucleus. MCF-7/T cells were treated by different nanoparticles for 24 h. Bar represents 5 µm.

Microtubules are naturally biopolymer and continuously change their existential state of assembly and disassembly in seconds during the processes of most cellular activities. Especially for cell proliferation, this dynamic behavior will increase to hundreds fold during mitosis.44 As mentioned above, Se@MIL-101-(P+V)siRNA and Ru@MIL-101-(P+V)siRNA interfere with microtubule dynamics in MCF-7/T cell. So, we further study the interference of nanopaticles on the process of mitosis. Immunofluorescence revealed that Se@MIL-101-(P+V)siRNA treatment induced a variety of aberrations occurring at each stage of mitosis. Figure 8A (Upper) displays images of abnormal telophase and anaphase. Asymmetric cell divisions, tripolar telophases or multinuclear are observed. We also confirmed important microtubular defects in the formation of the spindle. As show in Figure 8A (Bottom) , the distributions of chromosome and aberrant spindle microtubules, including acentrosomal, monopolar and multipolar can be found in Se@MIL-101-(P+V)siRNA treated

cells.

In

addition,

cell-cycle

test

comfirmed

that

Se/Ru@MIL-101-(P+V)siRNA induced the arrest of cell cycle at the G0/G1 phase. As showed in Figure 8B, compared to the control group, paclitaxel treated did not significant affect the cell cycle in MCF-7/T cells. WhIile exposed to Se@MIL-101-(P+V)siRNA and Ru@MIL-101-(P+V)siRNA for 24 h, it was clearly observed that cells at G2/M stage decreased, while the cells at stage of G0/G1 phase 24


Page 25 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


significantly rised in MCF-7/T cells. The proportion of cells arrested at G0/G1 phase increased

from

53.58%

Se@MIL-101-(P+V)siRNA

to

57.99%

and

from

in

the

group

43.86%

of to

treated

with

53.83%

in

Ru@MIL-101-(P+V)siRNA treated group. According to the above results, we speculated that the disruption of microtubule dynamics typically lead to the disorder of chromosome malsegregation and abnormal cell division, and finally result in cell cycle arrest and apoptosis.45 This may be attributed to the high binding affinity of SeNPs to critical proteins. Proteomics analysis revealed that the insoluble tubulin are easily sequestrated by SeNPs, and then caused the depolymerization of microtubule.46

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. (A) Confocal fluorescence images of abnormal telophase and aberrant mitotic spindles in MCF-7/T cells incubated with Se@MIL-101-(P+V)siRNA. The distributions of chromosome (Blue) and aberrant spindle microtubules (Red) are observed. (B) cell cycle arrest assay. Paclitaxel resistance MCF-7/T cells were exposed to Se@MIL-101-(P+V)siRNA and Ru@MIL-101-(P+V)siRNA at concentrations of 5, 10, 20 µg/mL for 24 h. Bar represents 5 µm.

3.7 In vivo anti-tumor effect of Se@MIL-101-(P+V)siRNA in xenograft tumor model Furthermore, MCF-7/T cells xenografts model in nude mice was treated with 26


Page 26 of 43

Page 27 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Se@MIL-101-(P+V)siRNA to examine its anti-tumor effects in vivo. When the MCF-7/T tumor xenografts grew to 50 mm3, the mices were treated with Se@MIL-101-(P+V)siRNA every 3 days by intravenous injection and measured the tumor volumes. As shown in Figure 9A and B, the volume of xenografts tumour in the mice treated with Se@MIL-101-(P+V)siRNA was 325±35.4 mm3, exhibited a inhibition rate up to 79.4% as compared to untreated group. These results suggested that Se@MIL-101-(P+V)siRNA can significantly suppress the growth of malignant tumor. Furthermore, in the H&E stain images of tumour tissues in control group, it was clearly observed that hemorrhage, hypercellularity and foci of nuclear polymorphism (Figure 9C). However in the group of Se@MIL-101-(P+V)siRNA treated, a large amount of shrinkage, nuclei fragmentation and chromosome condensation were observed, indicating the high level of induced apoptosis. Moreover, we collected the tissues including liver, spleen, kidneys at the end of the experiments, and stained the tissues with hematoxylin and eosin. All tissue examined exhibited well-organized cellular structure and showed no appreciable abnormalities.

Figure 9. (A) photographs of xenograft tumour from the mice of Se@MIL-101-(P+V)siRNA treated groups and control groups after 15 days administration. (B) Measurement of tumour volumes at time interval of 3day. (C) Ex vivo analysis of the histological characteristics of tumor heart, liver, spleen, lung and kidney tissue by and H&E stain. Scale bar = 50 µm. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4. Conclusions In summary, we present a new approach of selective coordination of cysteine to CUSs in MIL-101, the cysteine served as the linkage and in situ reduction selenite/ ruthenium to form SeNPs/RuNPs functionalized MIL-101. We have shown that the high porosity and surface modification of Se/Ru@MIL-101 can significantly enhance protection of siRNAs against nuclease degradation, increase siRNA cellular uptake, and promote siRNA escape from endosomes/lysosome to silence MDR genes in MCF-7/T cell, resulting in enhanced cytotoxicity through the induction of apoptosis with the signaling pathways of phosphorylation of p53, MAPK and PI3K/Akt and the dynamic instability of MTs and disrupting normal mitotic spindle formation. These results may indicated that NPs-microtubule interaction induced the disruption of microtubule dynamics typically lead to the disorder of chromosome malsegregation and abnormal cell division, and finally result in cell cycle arrest and apoptosis. Since microtubules and actin are required for intracellular organelles trafficking and maintained the cellular morphology during cell migration, cell division and other cellular activities. Therefore, the understanding of the relationship between high-affinity nanoparticle and microtubules could significantly promot the development of biocompatible nanomaterials for chemotherapy.

Supporting Information Additional characterization by TEM, Nitrogen adsorption isotherms, ICP-AES and Raman Spectrum; In vitro release of siRNA; In vitro cytotoxicity; Western Blot.

Acknowledgements This work has been financially supported by the National Natural Science Foundation of China (21171070, 21371075), the Natural Science Foundation of Guangdong Province (2014A030311025) and the Planned Item of Science and Technology of Guangdong Province (2016A020217011).

28


Page 28 of 43

Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Reference (1) Wang, Z.; Cohen, S. M. Postsynthetic Modification of Metal–Organic Frameworks. Chem. Soc. Rev. 2009, 38, 1315-1329. (2) Farha, O. K.; Malliakas, C. D.; Kanatzidis, M. G.; Hupp, J. T. Control Over Catenation in Metal− Organic Frameworks via Rational Design of the Organic Building Block. J. Am. Chem. Soc. 2009, 132, 950-952. (3) Liu, D.; Huxford, R. C.; Lin, W. Phosphorescent Nanoscale Coordination Polymers as Contrast Agents for Optical Imaging. Angew. Chem. Int. Edit. 2011, 123, 3780-3784. (4) He, C.; Lu, K.; Liu, D.; Lin, W. Nanoscale Metal–Organic Frameworks for the Co-Delivery of Cisplatin and Pooled siRNAs to Enhance Therapeutic Efficacy in Drug-Resistant Ovarian Cancer Cells. J. Am. Chem. Soc. 2014, 136, 5181-5184. (5) Horcajada, P.; Chalati, T.; Serre, C.; Gillet, B.; Sebrie, C.; Baati, T.; Eubank, J. F.; Heurtaux, D.; Clayette, P.; Kreuz, C.; Chang, J. S.; Hwang, Y. K.; Marsaud, V.; Bories, P. N.; Cynober, L.; Gil, S.; Ferey, G.; Couvreur, P.; Gref, R. Porous Metal-Organic-Framework Nanoscale Carriers as a Potential Platform for Drug Delivery and Imaging. Nat. Mater. 2010, 9, 172-178. (6) Rieter, W. J.; Pott, K. M.; Taylor, K. M.; Lin, W. Nanoscale Coordination Polymers for Platinum-Based Anticancer Drug Delivery. J. Am. Chem. Soc. 2008, 130, 11584-11585. (7) Imaz, I.; Rubio-Martínez, M.; García-Fernández, L.; García, F.; Ruiz-Molina, D.; Hernando, J.; Puntes, V.; Maspoch, D. Coordination Polymer Particles as Potential Drug Delivery Systems. Chem. Commun. 2010, 46, 4737-4739. (8) Férey, G.; Mellot-Draznieks, C.; Serre, C.; Millange, F.; Dutour, J.; Surblé, S.; Margiolaki, I. A Chromium Terephthalate-Based Solid with Unusually Large Pore Volumes and Surface Area. Science 2005, 309, 2040-2042. (9) Hwang, Y. K.; Hong, D. Y.; Chang, J. S.; Jhung, S. H.; Seo, Y. K.; Kim, J.; Vimont, A.; Daturi, M.; Serre, C.; Férey, G. Amine Grafting on Coordinatively Unsaturated Metal Centers of MOFs: Consequences for Catalysis and Metal Encapsulation. Angew. Chem. Int. Edit. 2008, 47, 4144-4148. (10) Yoon, J. W.; Seo, Y. K.; Hwang, Y. K.; Chang, J. S.; Leclerc, H.; Wuttke, S.; Bazin, P.; Vimont, A.; Daturi, M.; Bloch, E. Controlled Reducibility of a Metal–Organic Framework with Coordinatively Unsaturated Sites for Preferential Gas Sorption. Angew. Chem. Int. Edit. 2010, 49, 5949-5952. (11) Sosnik, A. Reversal of Multidrug Resistance by the Inhibition of ATP-Binding Cassette Pumps Employing “Generally Recognized As Safe”(GRAS) Nanopharmaceuticals: A review. Adv. Drug Delivery Rev. 2013, 65, 1828-1851. (12) Reddy, L. H.; Bazile, D. Drug Delivery Design for Intravenous Route with Integrated Physicochemistry, Pharmacokinetics and Pharmacodynamics: Illustration with the Case of Taxane Therapeutics. Adv. Drug Delivery Rev. 2014, 71, 34-57. (13) Min, Y. Z.; Mao, C. Q.; Chen, S. M.; Ma, G. L.; Wang, J.; Liu, Y. Z. Combating the Drug Resistance of Cisplatin Using a Platinum Prodrug Based Delivery System. Angew. Chem. Int. Edit. 2012, 51, 6742-6747. (14) Kodaira, H.; Kusuhara, H.; Ushiki, J.; Fuse, E.; Sugiyama, Y. Kinetic Analysis of the Cooperation of P-glycoprotein (P-gp/Abcb1) and Breast Cancer Resistance Protein (Bcrp/Abcg2) in limiting the Brain and Testis Penetration of Erlotinib, Flavopiridol, and Mitoxantrone. J. Pharmacol. Exp. Ther. 2010, 333, 788-796. 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 43

(15) Mozzetti, S.; Ferlini, C.; Concolino, P.; Filippetti, F.; Raspaglio, G.; Prislei, S.; Gallo, D.; Martinelli, E.; Ranelletti, F. O.; Ferrandina, G. Class III β-Tubulin Overexpression is a Prominent Mechanism of Paclitaxel Resistance in Ovarian Cancer Patients. Clin. Cancer Res. 2005, 11, 298-305. (16) Yeh, J.; Hsu, W.; Wang, J.; Ho, S.; Kao, A. Predicting Chemotherapy Response to Paclitaxel-Based Therapy in Advanced non-Small-Cell Lung Cancer with P-glycoprotein Expression. Respiration 2003, 70, 32-35. (17) Martínez-Díez, M.; Guillén-Navarro, M. J.; Pera, B.; Bouchet, B. P.; Martínez-Leal, J. F.; Barasoain, I.; Cuevas, C.; Andreu, J. M.; García-Fernández, L. F.; Díaz, J. F. PM060184, A New Tubulin Binding Agent with Potent Antitumor Activity Including P-glycoprotein Over-Expressing Tumors. Biochem. Pharmacol . 2014, 88, 291-302. (18) Li, J.; Yu, X.; Wang, Y.; Yuan, Y.; Xiao, H.; Cheng, D.; Shuai, X. A Reduction and pH Dual‐Sensitive Polymeric Vector for Long‐Circulating and Tumor‐Targeted siRNA Delivery. Adv. Mater. 2014, 26, 8217-8224. (19) Zheng, W.; Cao, C.; Liu, Y.; Yu, Q.; Zheng, C.; Sun, D.; Ren, X.; Liu, J. Multifunctional Polyamidoamine-Modified Selenium Nanoparticles Dual-Delivering siRNA and Cisplatin to A549/DDP Cells for Reversal Multidrug Resistance. Acta Biomater. 2015, 11, 368-380. (20) Li, J. M.; Xue, S. S.; Mao, Z. W. Nanoparticle Delivery Systems for siRNA-Based Therapeutics. J. Mat. Chem. B 2016, 4, 6620-6639. (21) Shen, J. L.; Kim, H. C.; Su, H.; Wang, F.; Wolfram, J.; Kirui, D.; Mai, J. H.; Mu, C. F.; Ji, L. N.; Mao, Z. W.; Shen, H. F. Cyclodextrin and Polyethylenimine Functionalized Mesoporous Silica Nanoparticles for Delivery of siRNA Cancer Therapeutics. Theranostics 2014, 4, 487-497. (22) Shen, J.; Xu, R.; Mai, J.; Kim, H.-C.; Guo, X.; Qin, G.; Yang, Y.; Wolfram, J.; Mu, C.; Xia, X.; Gu, J.; Liu, X.; Mao, Z.-W.; Ferrari, M.; Shen, H. High Capacity Nanoporous Silicon Carrier for Systemic Delivery of Gene Silencing Therapeutics. Acs Nano 2013, 7, 9867-9880. (23) Sinha, R.; Elbayoumy, K. Apoptosis is A Critical Cellular Event in Cancer Chemoprevention and Chemotherapy by Selenium Compounds. Curr. Cancer Drug Tar. 2004, 4, 13-28. (24) Abdulah, R.; Miyazaki, K.; Nakazawa, M.; Koyama, H. Chemical Forms of Selenium for Cancer Prevention. J. Trace Elem. in Med. Bio. 2005, 19, 141. (25) Hatfield, D. L.; Tsuji, P. A.; Carlson, B. A.; Gladyshev, V. N. Selenium and Selenocysteine: Roles in Cancer, Health and Development. Trends Biochem. Sci. 2014, 39, 112. (26) Wang, H.; Zhang, J.; Yu, H. Elemental Selenium at Nano Size Possesses Lower Toxicity without

Compromising

the

Fundamental

Effect

on

Selenoenzymes:

Comparison

with

Selenomethionine in Mice. Free Radical Bio. Med. 2007, 42, 1524. (27) Levina, A.; Mitra, A.; Lay, P. A. Recent Developments in Ruthenium Anticancer Drugs. Metallomics 2009, 1, 458. (28) Bergamo, A.; Gaiddon, C.; Schellens, J. H. M.; Beijnen, J. H.; Sava, G. Approaching Tumour Therapy Beyond Platinum Drugs : Status of the Art and Perspectives of Ruthenium Drug Candidates. J.Inorg. Biochem. 2012, 106, 90-99. (29) Hong, D. Y.; Hwang, Y. K.; Serre, C.; Ferey, G.; Chang, J. S. Porous Chromium Terephthalate

MIL‐101

with

Coordinatively

Unsaturated

Sites:

Surface

Functionalization,

Encapsulation, Sorption and Catalysis. Adv. Funct. Mater. 2009, 19, 1537-1552. (30) Young, D. A.; Freedman, T. B.; Lipp, E. D.; Nafie, L. A. Vibrational Circular Dichroism in Transition-Metal Complexes. 2. Ion Association, Ring Conformation, and Ring Currents of Ethylenediamine ligands. J. Am. Chem. Soc. 1986, 108, 7255-7263. 30


Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31) Azcárate, J. C.; Corthey, G. n.; Pensa, E.; Vericat, C.; Fonticelli, M. H.; Salvarezza, R. C.; Carro, P. Understanding the Surface Chemistry of Thiolate-Protected Metallic Nanoparticles. J. Phys. Chem. Lett. 2013, 4, 3127-3138. (32) Zhang, J.-Y.; Zhang, N.; Zhang, L.; Fang, Y.; Deng, W.; Yu, M.; Wang, Z.; Li, L.; Liu, X.; Li, J. Adsorption of Uranyl ions on Amine-functionalization of MIL-101(Cr) Nanoparticles by a Facile Coordination-based Post-synthetic strategy and X-ray Absorption Spectroscopy Studies. Sci. Rep.-UK 2015, 5. (33) Ertas, I. E.; Gulcan, M.; Bulut, A.; Yurderi, M.; Zahmakiran, M. Metal-Organic Framework (MIL-101) Stabilized Ruthenium Nanoparticles: Highly Efficient Catalytic Material in the Phenol Hydrogenation. Micropor. Mesopor. Mat. 2016, 226, 94-103. (34) He, L.; Liu, Y.; Liu, J.; Xiong, Y.; Zheng, J.; Liu, Y.; Tang, Z. Core–Shell Noble‐Metal@ Metal‐Organic‐Framework Nanoparticles with Highly Selective Sensing Property. Angew. Chem. Int. Edit.2013, 52, 3741-3745. (35) Wu, Y. n.; Zhou, M.; Li, S.; Li, Z.; Li, J.; Wu, B.; Li, G.; Li, F.; Guan, X. Magnetic Metal–Organic Frameworks: γ‐Fe2O3@ MOFs via Confined In Situ Pyrolysis Method for Drug Delivery. Small 2014, 10, 2927-2936. (36) Li, J.-M.; Zhao, M.-X.; Su, H.; Wang, Y.-Y.; Tan, C.-P.; Ji, L.-N.; Mao, Z.-W. Multifunctional Quantum-Dot-Based siRNA Delivery for HPV18 E6 Gene Silence and Intracellular Imaging. Biomaterials 2011, 32, 7978-7987. (37) Wang, X.; Xia, T.; Duch, M. C.; Ji, Z.; Zhang, H.; Li, R.; Sun, B.; Lin, S.; Meng, H.; Liao, Y.-P. Pluronic F108 Coating Decreases the Lung Fibrosis Potential of Multiwall Carbon Nanotubes by Reducing Lysosomal Injury. Nano Lett. 2012, 12, 3050-3061. (38) Sun, D.; Liu, Y.; Yu, Q.; Qin, X.; Yang, L.; Zhou, Y.; Chen, L.; Liu, J. Inhibition of Tumor Growth and Vasculature and Fluorescence Imaging using Functionalized Ruthenium-Thiol Protected Selenium Nanoparticles. Biomaterials 2014, 35, 1572-1583. (39) Meng, L.; Chen, R.; Jiang, A.; Wang, L.; Wang, P.; Li, C. z.; Bai, R.; Zhao, Y.; Autrup, H.; Chen, C. Short Multiwall Carbon Nanotubes Promote Neuronal Differentiation of PC12 Cells via Up‐Regulation of the Neurotrophin Signaling Pathway. Small 2013, 9, 1786-1798. (40) Wang, H.; Zheng, L.; Peng, C.; Shen, M.; Shi, X.; Zhang, G. Folic Acid-Modified Dendrimer-Entrapped Gold Nanoparticles as Nanoprobes for Targeted CT Imaging of Human Lung Adencarcinoma. Biomaterials 2013, 34, 470-480. (41) Sonnauer, A.; Hoffmann, F.; Fröba, M.; Kienle, L.; Duppel, V.; Thommes, M.; Serre, C.; Férey, G.; Stock, N. Giant Pores in a Chromium 2, 6‐Naphthalenedicarboxylate Open‐Framework Structure with MIL‐101 Topology. Angew. Chem. Int. Edit. 2009, 121, 3849-3852. (42) Juan-Alcañiz, J.; Ramos-Fernandez, E. V.; Lafont, U.; Gascon, J.; Kapteijn, F. Building MOF

Bottles

Around

Phosphotungstic

Acid

Ships:

One-Pot

Synthesis

of

bi-Functional

Polyoxometalate-MIL-101 Catalysts. J. Catal. 2010, 269, 229-241. (43) Li, X.; Kolomeisky, A. B. The Role of Multifilament Structures and Lateral Interactions in Dynamics of Cytoskeleton Proteins and Assemblies. J. Phys. Chem. B 2015, 119, 4653-4661. (44) Rodriguez-Fernandez, L.; Valiente, R.; Gonzalez, J.; Villegas, J. C.; Fanarraga, M. n. L. Multiwalled Carbon Nanotubes Display Microtubule Biomimetic Properties In Vivo, Enhancing Microtubule Assembly and Stabilization. ACS nano 2012, 6, 6614-6625. (45) Choudhury, D.; Xavier, P. L.; Chaudhari, K.; John, R.; Dasgupta, A. K.; Pradeep, T.; Chakrabarti, G. Unprecedented Inhibition of Tubulin Polymerization Directed by Gold Nanoparticles 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Inducing Cell Cycle Arrest and Apoptosis. Nanoscale 2013, 5, 4476-4489. (46) Bao, P.; Chen, Z.; Tai, R.-Z.; Shen, H.-M.; Martin, F. L.; Zhu, Y.-G. Selenite-Induced Toxicity in Cancer Cells is Mediated by Metabolic Generation of Endogenous Selenium Nanoparticles. J. Proteome Res. 2015, 14, 1127-1136.

32


Page 32 of 43

Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents

Selenium/Ruthenium nanoparticles modified metal organic frameworks MIL-101(Fe) for the delivery of pooled small interfering RNAs (siRNAs) to enhance therapeutic efficacy by silencing multiple drug resistance (MDR) genes and interfere with microtubule dynamics in MCF-7/T cell.

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Mechanism of the reversal of drug resistance and induce apoptosis by the disruption of microtubule in MCF-7/T (Taxol- resistance) cancer cells. 146x133mm (300 x 300 DPI)


Page 34 of 43

Page 35 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1.(A)Formation of the Se/Ru Nanoparticles Modified Porous Metal Organic Framework. (B)TEM image of MIL-101,Se@MIL-101 and Ru@MIL-101. (C) FT-IR spectrum of Cys, MIL-101, MIL-101-Cys and (D) XRD pattern of MIL-101, Se@MIL-101 and Ru@MIL-101. 123x76mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. (A) Nitrogen adsorption isotherms 77K and (B) distribution curves of pore volume and pore size (diameter) of the MIL-101, SeNPs@MIL-101 and RuNPs@MIL-101. (C) Agarose gel electrophoresis to evaluate the siRNA loading affinity and protective effect of SeNPs@MIL-101 and RuNPs@MIL-101 at various mass ratios from 1:1 to 16:1. Naked siRNA served as the control group. 160x137mm (300 x 300 DPI)


Page 36 of 43

Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (A) Flow cytometry of naked siRNAFAM,Se@MIL-101-siRNAFAMand Ru@MIL-101-siRNAFAM uptake in MCF-7/T cells at 12 h post-transfection. (B, C) TEM images of MCF-7/T cells exposed to Se@MIL-101siRNA and Ru@MIL-101-siRNA nanoparticles. The arrows in the TEM show cellular uptake and localization of Se@MIL-101-siRNA and Ru@MIL-101-siRNA uptake. (D) Morphology of MCF-7/T cells incubated with Se@MIL-101-siRNA and Ru@MIL-101-siRNA at 10 µg/mL for 6，12 or 24 h and stained by H&E. Scale bar represented 50 µm. (E) Raman spectra of Se@MIL-101-siRNA in MCF-7/T cells treated. 109x75mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. (A) Time-dependent confocal microscopy of siRNA successfully escaped from endosomes. MCF-7/T cells were incubated with Se@MIL-101- siRNAFAM and Ru@MIL-101-siRNAFAM for 60 min and 180 min. LysoTracker (Red); siRNAFAM(Green). Bar represents 5 µm. (B) Fluorescence microscope images of MCF7/T cells transfected by Se@MIL-101 and Ru@MIL-101 for 24 h. Green fluorescence represented the expressions of EGFP plasmid in MCF-7/T cells. 250x87mm (300 x 300 DPI)


Page 38 of 43

Page 39 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5.(A) The expression of p-glycoprotein (P-gp) in MCF7 and MCF-7/T cells by immunofluorescence staining. (B) Comparison of suppression of P-gp expression in MCF-7/T cells. (C) The expression of VEGF in MCF7 and MCF-7/T cells by Immunofluorescence staining. (D) Comparison of suppression of VEGF expression in MCF-7/T cells. Bar represents 20 µm. (E,F) Comparison of suppression of P-gp and VEGF expression in MCF-7/T cells.1-6: Control, Naked pooled siRNA, Se@MIL-101, Se@MIL-101-PsiRNA, Se@MIL101-VsiRNA, Se@MIL-101-(P+V)siRNA. a-f: Control, Naked pooled siRNA, Ru@MIL-101, Ru@MIL-101PsiRNA, Ru@MIL-101-VsiRNA, Ru@MIL-101-(P+V)siRNA. Bars shown are mean ± SE, and differences between control and treated groups were analyzed by one-way ANOVA. P-gp expression of treated group vs. control was indicated by (*) p < 0.05, (**)p < 0.01. VEGF expression of treated group vs. control was indicated by (#) p < 0.05. 250x203mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Confocal fluorescence images of MCF-7/T cells treated with (A) Se@MIL-101-(P+V)siRNA and (B) Se@MIL-101-(P+V)siRNA. Microtubule was stained with its respective Tubulin Tracker (red) and DAPI is used to stain the nucleus. MCF-7/T cells were treated by different nanoparticles for 12h, 24 h. Bar represents 5 µm. 250x87mm (300 x 300 DPI)


Page 40 of 43

Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. MCF-7/T cells treated with different concentrations of Se@MIL-101-(P+V)siRNA and Ru@MIL-101(P+V)siRNA showing intracellular damage of (A) Actin and (B) microtubules. Actin is stained with ActinTracker (green) and tubulin-red). Microtubule was stained with its respective Tubulin Tracker (red) and DAPI is used to stain the nucleus. MCF-7/T cells were treated by different nanoparticles for 24 h. Bar represents 5 µm. 299x90mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. (A) Confocal fluorescence images of abnormal telophase and aberrant mitotic spindles in MCF-7/T cells incubated with Se@MIL-101-(P+V)siRNA. The distributions of chromosome (Blue) and aberrant spindle microtubules (Red) are observed. (B) cell cycle arrest assay. Paclitaxel resistance MCF-7/T cells were exposed to Se@MIL-101-(P+V)siRNA and Ru@MIL-101-(P+V)siRNA at concentrations of 5, 10, 20 µg/mL for 24 h. Bar represents 5 µm. 199x249mm (300 x 300 DPI)


Page 42 of 43

Page 43 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. (A) photographs of xenograft tumour from the mice of Se@MIL-101-(P+V)siRNA treated groups and control groups after 15 days administration. (B) Measurement of tumour volumes at time interval of 3day. (C) Ex vivo analysis of the histological characteristics of tumor heart, liver, spleen, lung and kidney tissue by and H&E stain. Scale bar = 50 µm. 160x99mm (300 x 300 DPI)


Ru-Decorated Porous MetalâOrganic ... - ACS Publications

Recommend Documents

Ru-Decorated Porous MetalâOrganic ... - ACS Publications