Enhanced Sensitivity of Cancer Stem Cells to Chemotherapy Using

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Enhanced sensitivity of cancer stem cells to chemotherapy using functionalized mesoporous silica nanoparticles Zhenzhen Chen, Pingping Zhu, Yushun Zhang, Yating Liu, Yuling He, Lifen Zhang, and Yan-Feng Gao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00352 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on June 29, 2016

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Molecular Pharmaceutics

Enhanced sensitivity of cancer stem cells to chemotherapy using functionalized mesoporous silica nanoparticles

Zhenzhen Chen1,4, Pingping Zhu2,4, Yushun Zhang3, Yating Liu1, Yuling He3,Lifen Zhang3*,Yanfeng Gao1* 1

School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China

2

School of Life Sciences, University of Science and Technology of China, Hefei,

Anhui 230027, China. 3

State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous

Metals Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China 4

These two authors contributed equally to this work

*Correspondence to: Lifen Zhang (email: [email protected]);Yanfeng Gao (email: [email protected])

Keywords: Mesoporous silica nanoparticles; dual delivery; cancer stem cells; shABCG2

ACS Paragon Plus Environment


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Abstract Cancer stem cells (CSCs) are responsible for cancer drug resistance with high expression of ABCG2, which pumps the internalized chemotherapeutic out to escape drug-induced cytotoxicity. Here, we established a functionalized mesoporous silica nanoparticle (MSN) system to deliver shABCG2 and doxorubicin (Dox) synergistically. With excellent cell uptake and endosomal escape capacities, the dual-delivery carriers internalized shABCG2 and Dox into CSCs efficiently. ABCG2 depletion increased intracellular and intranuclear Dox enrichment, drove vigorous Dox-induced cell death, and impaired the self-renewal of CSCs. Additionally, the nanoparticles eliminated tumors efficiently and reduced tumor initiation by CSCs in vivo, with negligible side effects. Our findings suggest that well-designed delivery systems for conventional chemotherapeutic agents are promising for CSCs therapy.


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1. Introduction The discovery of cancer stem cells (CSCs) has changed the applicability of chemotherapy1. CSCs evade the anti-cancer effects of standard chemotherapy on a tumor and have emerged as an underestimated biological barrier to the success of systemic chemotherapy2, 3. Small groups of CSCs in tumor tissue play an important role in tumor drug resistance4, 5. Although chemotherapy drugs and chemotherapy regimens are being continuously improved, tumor chemotherapy still aims to reduce the number of cancer cells and shrink the tumor6-8. One solution to the problem of tumor drug resistance is to improve the clinical curative effect of cancer chemotherapy. For example, the treatment of breast cancer with free doxorubicin hydrochloride (Dox) cannot effectively eradicate CSCs because CSCs have high drug resistance 9. The protein ABCG2 is highly expressed in drug-resistant cancer cells and CSCs and largely contributes to their drug resistance10, 11. ABCG2 gene silencing using short hairpin RNA (shRNA) is a promising therapeutic approach for overcoming drug resistance12-14. Nanoparticle-mediated gene delivery has demonstrated enhanced transfection efficacy due to the structure, size, and surface properties of the nanoparticles15, 16, allowing them to readily accumulate in and be retained by solid tumors through the enhanced permeability and retention (EPR) effect17,

18

. We

previously constructed a charge-reversible nanocarrier for delivering shABCG2 into tumor cells and showed that the nanocarrier markedly silenced ABCG2 expression, triggering sensitization of the tumor cells to injected free Dox and resulting in tumor



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cell death19. However, it remains unclear how best to silence the expression of ABCG2 in CSCs and thus provide a tumor therapy effect. Here we constructed bioresponsive functionalized mesoporous silica nanoparticles (MSNs) for co-delivery of Dox and shABCG2 into CSCs in vitro and in vivo (Scheme 1). MSNs were chosen because of their high loading capacity and facile surface modification. The MSNs were modified with disulfide bonds to encapsulate Dox inside the pores and minimize premature drug release. Next, the Dox-loaded MSNs were modified with a low molecular weight cationic polymer, polyethyleneimine (PEI), to enhance the binding of shABCG2. In the cytoplasm, the internalization and release of Dox and shABCG2 was triggered by the cleavage of a disulfide bond with intracellular GSH, which is several times higher in tumor cells than normal cells20. The release of shABCG2 and subsequent depletion of ABCG2 blocked the excretion of cytoplasmic Dox21, thereby maintaining the intracellular Dox concentration, triggering enhanced Dox-induced cell death and increased Dox sensitivity. Sphere formation assays serve as standard experimental procedures to examine CSC self-renewal capacity and isolate CSC. In this paper we establish sphere formation for CSC enrichment and test our hypothesis and strategy for treating CSCs with functionalized MSN nanoparticles in vitro to examine ABCG2 expression levels and cell killing properties. Moreover, we examine the biodistribution, safety, and efficacy of mesoporous silica nanoparticles as a DOX and shABCG2 dual delivery vehicle for cancer treatment in vivo using an orthotropic xenograft human liver cancer model produced by injecting oncosphere cells into BALB/c nude mice.


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Scheme

1

Schematic

showing

MSN-SS-PEI/Dox/shABCG2

and

redox-responsive CSC-targeted drug delivery. PEI was conjugated to MSN with disulfide bond, followed by shRNA adhesion through charge interaction. MSN based Dox/shRNA co-delivery vehicles were disassembled in response to cytoplasmic redox reagents (GSH), releasing Dox and shRNA simultaneously. The nanocarriers co-deliver Dox and shABCG2, and enhance chemotherapy efficacy in CSCs elimination.

Several

procedures

are

needed

for

dual

delivery

by

MSN-SS-PEI/Dox/shABCG2: cell uptake, glutathione-triggered drug release via cleavage of disulfide bond, drug diffusing into the cytoplasm and eventually to the nucleus.

2. Materials and Synthesis



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2.1 Materials Tetraethylorthosilicate (TEOS, 98%), cetyltrimethylammonium bromide (CTAB, 95%),

fluorescein

isothiocyanate

(FITC,

90%),

doxorubicin

hydrochloride

(Dox, >98%), 3-Mercaptopropyl trimethoxysilane (MPTMS), polyethylenimine (PEI, branched, MW 1200 Da), ammonium chloride, were from Sigma (St. Louis, MO). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride

(EDC)

and N-Hydroxysuccinimide (NHS) were from Sinopharm (China). DMEM/F12 medium (11320033) was obtained from Invitrogen (Life Technologies). bFGF (catalog GF446-50UG) was got from Millpore, EGF (catalog E5036-200UG) and DAPI (catalog 28718-90-3) were from Sigma-Aldrich, N2 supplement (catalog 17502-048) and B27 (catalog 17504-044) were purchased from Life Technologies. Anti-β-actin antibody (catalog A1978) was purchased from Sigma-Aldrich. Anti-ABCG2 antibody (10051-1-AP) was obtained from Proteintech Company.

2.2 Synthesis of MSN-SH MSN-SH was synthesized according to the following procedure22. Briefly, 1.0 g CTAB and 280 mg NaOH were dissolved in 480 mL of distilled water. After stirring at room temperature for 20 min, 5.0 g TEOS was added dropwise to the solution. The reaction mixture was vigorously stirred at 80°C. After 15 min 0.97 mL MPTMS was introduced dropwise to the solution. The mixture was continued vigorously stirred for 2 hs to obtain a white precipitate. Finally, the solid product was centrifuged, washed with deionized water and ethanol, and placed under high vacuum. To remove the


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surfactant template (CTAB), the white solid was extracted for 24 hrs in a solution of 9 mL of HCl (37%) and 160 mL of methanol followed by extensive washing with deionized water and ethanol. The resulting nanoparticles were placed under high vacuum to remove the remaining solvent. The obtained MSN-SH were characterized by transmission electron microscopy (TEM, Tecnai G2 F30), scanning electron microscopy (SEM, Hitachi, S-4800) and Brunauer-Emmett-Teller (BET) and Barrett– Joyner–Halenda (BJH) analysis (ASAP 2020, Micromeritics).

2.3 Synthesis of MSN-SS-COOH 100 mg MSN-SH was suspended in 10 mL methanol, and reacted with 100 equivalents of 3-Mercaptopropionic acid and 1 equivalents of H2O2 at room temperature for 24 hrs23. The resulting nanoparticles were separated by centrifugation, washed four times with ethanol and dried under high vacuum.

2.4 Preparation of MSN-SS-COOH/Dox 10 mg MSN-SS-COOH and 5 mg Dox were suspended in 5 mL of DMF and stirred at room temperature in dark for 24 hrs. The free Dox was removed by washing with water until the upper liquid is colorless, and dried under vacuum, to obtain MSN-SS-COOH /Dox. The product was centrifuged and washed with double distilled water 6 times. The solution was collected and combined, and then the amount of free Dox was measured using a fluorescence spectrophotometer. The drug loading efficiency was calculated from the equation as follows: DLE (％) = (amount of



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loaded drug) / amount of feed drug × 100%.

2.5 Synthesis of MSN-SS-PEI 100 mg MSN-SS-COOH solution was ultrasonic for 30 min, 1.5 equivalent of EDC was mixed with the MSN-SS-COOH, followed by mixing with 1.5 equivalent of NHS, 15 min later, 714 mg PEI was added the above solution. This reaction was carried out at room temperature under a nitrogen atmosphere for 24 hrs. The resulting nanoparticles were washed with water, and dried under high vacuum.

2.6 Preparation and characterization of MSN-SS-PEI coating with shABCG2 An agarose gel retardation assay was used to determine the shABCG2 binding to MSN-SS-PEI.1 µg of shABCG2 was mixed with various weight of MSN-SS-PEI. The result MSN-SS-PEI/shABCG2 weight ratios were (2, 5, 7, 10, 12)/1. 10 µL of the polyplex solution was analyzed by 0.8% agarose gel electrophoresis (120 V). After electrophoresis, the gel was stained with ethidium bromide (EB), and the resulting DNA migrate on patterns were analyzed by in vivo imaging instrument (Kodak In-Vivo Imaging System FX Pro).

2.7 MSNs characterization The morphologies and structures of different MSNs were characterized using a scanning electron microscope (SEM) and a transmission electron microscope (TEM). The chemical grafting processes of MSNs were monitored by Fourier transform


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infrared spectroscopy (FT-IR) and zeta potential measurements. The pore size distributions and surface areas of different MSNs were characterized by Brunauer– Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) analyses. The weight loss of different MSNs materials was measured with a Thermal Gravimetric Analysis (TGA). TEM: The samples were carried out on a JEM-2100 instrument operating at an acceleration voltage of 80 KV. Dynamic light scattering (DLS): The hydrodynamic diameter of nanoparticles in DI water were analyzed on a Nano-ZS ZEN3600 particle sizer (Malvern Instruments).

2.8 Release and Uptake

2.8.1 In Vitro Rodox-Induced Drug Release: At a concentration of 0.1 mg/mL, MSN-SS-PEI/Dox capped in 3500 Da dialysis tube and then suspended in different media: PBS (pH = 7.4) and PBS (pH = 7.4) with 10 mM GSH at 37 oC. At the indicated time intervals, the fluorescence intensity of the solution in the release medium was measured using fluorophotometer. The fluorescence standard curve was employed to calculate the cumulative release amounts of Dox. The cumulative release percentage was obtained on the basis of the released and total amounts of Dox.

2.8.2 Sphere formation: Hep3B cells were digested with Trypsin/EDTA and re-suspend with DMEM medium. 1000 single cells were collected and followed by incubation within DMEM/F12 medium supplemented with FGF, EGF, N2 and B27 for 2 weeks. The spheres with cancer stem cells were obtained and used for subsequent experiments.



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2.8.3 CCK8 assay: Cancer stem cells were seeded into 96-well plate, with 1×104 cells per well. Then Dox, MSN/Dox, MSN-SS-PEI/Dox, MSN-SS-PEI/Dox/shCtrl and MSN-SS-PEI/Dox/shABCG2 nanoparticles with a final concentration of 70 µg/mL were added for 48 hrs. Free Dox with the same dose as MSNs loaded was used as the control. Then CCK8 assay was performed according to standard procedure. Briefly speaking, 10 µL CCK8 was added into each well for 4 hrs’ incubation, followed by 450 nm OD detection using Microplate Reader.

2.8.4

FACS:

For

cell

uptake,

Dox,

MSN/Dox,

MSN-SS-PEI/Dox,

MSN-SS-PEI/Dox/shCtrl and MSN-SS-PEI/Dox/shABCG2 nanoparticles with a final concentration of 70 µg/mL treated cancer stem cells were loaded into FACS Calibur (BD). Free Dox with the same dose as MSNs loaded was used as the control. For cell viability, the indicated treated cells were incubated with Annexin V antibody, followed by incubation with FITC conjugated second antibody for 30min on ice. PI was added into the samples and FACS was performed to detect Annexin V and PI signal.

2.9 Gene Silencing Efficiency

2.9.1 shABCG2 construction：ABCG2 shRNA sequence were designed according to online

shRNA

tools

(http://www.clontech.com/CN/Support/xxclt_onlineToolsLoad.jsp?citemId=http://bioi nfo.clontech.com/rnaidesigner/sirnaSequenceDesignInit.do§ion=16260&xxheigh t=1100), and purchased from Sangon Company. The primers were cloned into


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PSiCoRpuro shRNA and transfected into Top10 competent cells, followed by PCR identification and DNA sequencing. Then the shRNA were transfected into 293T cells supplemented with package plasmids (RRE, REV and VSVG). After 2 days, the supernatant was collected and transfected into flesh 293T cells for 2 days incubation, followed by examining ABCG2 knockdown efficiency by realtime PCR and western blot. The confirmed shABCG2 plasmid was used for MSN-SS-PEI/Dox/shABCG2 assembly.

2.9.2 Confocal: The spheres were obtained and fixed with 4% paraformaldehyde (PFA), followed by treatment with 1% triton X-100 overnight at 4 oC. Then the samples were incubated with indicted primary antibodies (Pan-CK, LAMP-1, ABCG2) overnight, followed by washing and subsequent FITC conjugated secondary antibody. DAPI was used for counterstain, and finally the samples were observed using confocal microscope FV1000 (OLYMPUS).

2.9.3 Realtime PCR: Dox, MSN/Dox, MSN-SS-PEI/Dox, MSN-SS-PEI/Dox/shCtrl and MSN-SS-PEI/Dox/shABCG2 nanoparticles with a final concentration of 70 µg/mL treated samples were collected, lyzed with 1mL TRIZOL (Life Company) and added with 200 µL chloroform, followed by 10 mins’ centrifugation at 12000g. Free Dox with the same dose as MSNs loaded was used as the control. The supernatant was collected and added with 0.5 mL isopropyl alcohol, followed by centrifugation at 12000g and washing with 75% ethanol. The RNA samples were used for reverse transcription (RT-PCR) using MMTV (Promega), and the obtained cDNA served as template

for

realtime

PCR.

Primers

were

designed


with

primer

bank


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(http://pga.mgh.harvard.edu/primerbank/) and purchased from Sangon Company. The realtime PCR was performed using ABI7300.

2.9.4 Western Blot: The indicated carriers treated samples were collected and crushed with RIPA buffer, followed by centrifugation at 12000g. The supernatant was obtained and boiled for 10 minutes with loading buffer, then subjected into SDS PAGE for separation. The samples were then transferred onto nitrocellulose filter membrane, followed by incubation with primary antibodies (ABCG2 and β-actin) and corresponding HRP conjugated secondary antibodies, sequentially. Finally the protein levels were visualized with X flim.

2.9.5 ELISA: Mouse IL6 and TNFa Direct ELISA kits were purchased from XinRan Biological Company (Shanghai, China). 24Balb/c nude mice were randomly divided into six groups. No treat for one group; Dox, MSN/Dox, MSN-SS-PEI/Dox, MSN-SS-PEI/Dox/shCtrl and MSN-SS-PEI/Dox/shABCG2 was injected into the tail vein. After 2 days, mouse caudal vein blood was obtained and utilized for the ELISA assay. Briefly, the samples were diluted with coating buffer and then subjected into the coated ELISA plate. After blocking with Blocking Buffer, HRP conjugated antibodies were added for incubation. Finally, TMB reagent was added for detection with Microplate Reader.

2.9.6 Immunohistochemistry: For immunohistochemistry, the indicated treated tumors were obtained followed by paraffin preparation. 5 µL sections were obtained from paraffin-embedded samples, followed by incubation with xylene and graduate ethanol (100%, 100%, 90%, 70%). After 3% H2O2 incubation and subsequent antigen


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retrieval with citric acid buffer, the samples were blotted with ABCG2 antibody overnight, followed by washing with PBS and subsequent incubation with HRP conjugated secondary antibody. Finally ABCG2 expression levels were examined by adding HRP substrate, followed by staining with hematoxyl in and dehydration with graded alcohols (70%, 90%, 100%, 100%) and xylene.

2.10 Mouse model WT or tumor-bearing Balb/c nude mice were treated with free Dox, MSN/Dox, MSN-SS-PEI/Dox, MSN-SS-PEI/Dox/shCtrl and MSN-SS-PEI/Dox/shABCG2 using tail intravenous injection. For in vivo experiments, 1.0 mg indicated nanoparticles were used per mouse (100 µL, 10 mg/mL).

2.11 Statistical analysis

All experiments were repeated at least three times. Data are presented as mean values ± S.D. Statistical significance (p < 0.05) was evaluated by using Student’s t-test when only two groups were compared. While differences between treatments groups (more than two groups) were determined by two-way ANOVA. In all tests, a p value less than 0.05 was considered statistically significant.

3. Results and Discussion 3.1 Preparation and characterization of the MSN-based system The strategy for constructing the MSN-based Dox and shRNA delivery



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nanocarriers is illustrated in Scheme 1. MCM-41 MSNs were synthesized as previously described24. SEM showed that the obtained MSNs were round, had an diameter of~130 nm, and showed good dispersion (Figure 1A). The hydrodynamic diameter of MSN detected by DLS was 142 nm (Table S2). The N2 sorption isotherms of the MSNs further revealed a BET isotherm typical of a MCM-41 structure, with a surface area of 759.2 m2/g and a narrow BJH pore size distribution (average pore diameter: 4.7 nm) (Figure 1B). The TEM images showed that the MSNs had a highly regular mesoporous structure (Figure 1C) and that the surface PEI coating to obtain MSN-SS-PEI. The hydrodynamic diameter of MSN-SS-PEI was 184 nm ((Table S2) detected by DLS. Mesoporous silica nanoparticles with disulfide-linked PEI gatekeeper were generated by first introducing mercaptopropyl groups on the surface of MSNs by treatment with 3-mercaptopropyl-trimethoxysilane to obtain MSN-SH (MSNs with surface thiol groups). Next, the intermediate MSN-SS-COOH was obtained by reacting MSN-SH with 3-mercaptopropionic acid at room temperature for 24 hrs. At the same time, Dox was efficiently loaded into the pores of the silica particles by vigorously stirring a mixture of MSN-SS-COOH nanoparticles and Dox solution for 24 hrs. The drug loading efficiency (DLE) was 58.3%, as determined using a fluorescence spectrophotometer. PEI was immobilized onto Dox-loaded MSN-SS-COOH via disulfide bonds to provide MSN-SS-PEI/Dox. Zeta potential and FT-IR analyses were used in addition to TEM to monitor the modifications. The zeta potential data summarized in Table S2 show that the MCM-41 MSNs prior to derivatization were negatively charged and had


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a zeta potential of –29.9 mV, which jumped to –20.7 mV after modification with 3-mercaptopropyl-trimethoxysilane. After reaction with 3-mercaptopropionic acid, the zeta potential decreased to –37.9 mV. Further conjugation with PEI, which is rich in positively charged amide groups, resulted in a dramatic increase in zeta potential to +64 mV for the MSN-SS-PEI nanoparticles. The result of FT-IR analysis (Figure S1) was consistent with this change in zeta potential: MSN-SS-PEI showed strong peaks at 1635 cm–1 and 1554 cm–1, which were attributed to -CO-NH- groups and indicated that PEI molecules were successfully incorporated into MSN-SS-COOH. The TGA curves show in figure S2, also indicated that PEI was immobilized onto the MSN with success. The small size (

Enhanced Sensitivity of Cancer Stem Cells to Chemotherapy Using

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