An ATP-Responsive Codelivery System of Doxorubicin and MiR-34a

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

An ATP-responsive co-delivery system of doxorubicin and miR-34a to synergistically inhibit the cell proliferation and migration Yudi Wang, Jiawen Chen, Xiao Liang, Haobo Han, Hao Wang, Yan Yang, and Quanshun Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00184 • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 13, 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.

Molecular Pharmaceutics 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 37

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

Molecular Pharmaceutics

An ATP-responsive co-delivery system of doxorubicin and miR-34a to synergistically inhibit the cell proliferation and migration

Yudi Wang,† Jiawen Chen,† Xiao Liang,† Haobo Han,† Hao Wang,‡ Yan Yang,*,† and Quanshun Li*,†

†

Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China ‡

School of Life Sciences, Northeast Normal University, Changchun 130024, China

*

Corresponding author. Tel. and Fax: +86-431-85155200. E-mail: [email protected] (Q. Li); [email protected] (Y. Yang).

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

Page 2 of 37

For Table of Contents Use Only

An ATP-responsive co-delivery system of doxorubicin and miR-34a to synergistically inhibit the cell proliferation and migration

Yudi Wang,† Jiawen Chen,† Xiao Liang,† Haobo Han,† Hao Wang,‡ Yan Yang,*,† and Quanshun Li*,†

2


Page 3 of 37

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


ABSTRACT: Establishing stimuli-responsive nanosystems for the co-delivery of anticancer drug and oligonucleotide is a promising strategy in cancer treatment owing to the combination of chemotherapy and gene therapy in a synergistic manner. Herein, an ATP aptamer and its cDNA sequence were first hybridized to produce the duplex, into which chemotherapeutic agent doxorubicin (DOX) interacted through the GC-rich motif of duplex, and PEI25K was then employed as a carrier to condense the DOX-loading duplex and miR-34a to construct the ternary nanocomplex PEI/DOX-Duplex/miR-34a. The nanocomplex exhibited a favorable drug release profile through the response to high concentration of ATP in the cytosol. The ATP-responsive delivery system was demonstrated to possess higher anti-proliferative effect (cell viability of < 40%) than the single cargo delivery, which could be attributed to the synergistic induction of cell apoptosis and cell cycle arrest from DOX and miR-34a. Furthermore, wound healing and Transwell assay elucidated the higher anti-migration effect of ternary nanocomplex than DOX-Duplex or miR-34a delivery. Overall, the combinatorial delivery of DOX and miR-34a through an ATP-responsive manner could trigger the rapid release of cargos in the cytosol and enhance the inhibition of cell proliferation and migration through the synergistic manner of these two components.

KEYWORDS: ATP response; doxorubicin; miR-34a; co-delivery; anti-proliferation; anti-migration

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 37

1. INTRODUCTION In cancer treatment, surgery, radiation and chemotherapy are the major therapeutic strategies, in which chemotherapy plays an important role despite great advances in the field of surgery and radiation.1 However, severe toxicities and undesirable side effects from anticancer agents can’t be avoided, especially for those non-targeted ones. Additionally, drug resistance is usually produced during the chemotherapeutic process and ultimately leads to the failure of chemotherapy.2,3 In contrast, gene therapy holds a promising prospect to overcome these drawbacks for cancer treatment as it can directly turn off the expression of oncogene, enhance the expression of tumor suppressor, inhibit the neoangiogenesis or activate the antitumor immune response.4-6 MicroRNAs (miRNAs) are endogenous noncoding RNAs of a single strand with 19-25 nucleotides, and the therapy based on miRNAs has been demonstrated to be a promising technique to fulfill the requirements in tumor gene therapy.7,8 They could be transcribed as pre-mRNAs, cleaved by endonuclease and transported to the nucleus, in which mature miRNAs could be produced and then regulate the expression of genes involved in cell proliferation, migration, differentiation and other functions.9-11 Among them, miR-34a is an important mediator of p53 activity which plays a key role in inhibiting the genesis and progression of cancers through decreasing the expression level of Bcl-2, survivin, CD44 and Notch-1.12-15 As miR-34a is common down-regulated in various human tumors, enhancing its expression level through carriers-mediated delivery could be an efficient route for suppressing the proliferation

4


Page 5 of 37

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


and migration of cancer cells.16-18 Additionally, it could overcome the tumors' poor responses to cytotoxic drugs and even exert synergistic effects with them.19-21 Thus, combination of miR-34a and chemotherapy will be an effective strategy as it can improve the overall outcome and reduce the adverse effects of chemotherapeutics through the synergistic effects of these two components. Up to now, the co-delivery of chemotherapeutic agents and miRNA mediated by nanocarriers has been demonstrated to be effective in both in vitro and in vivo studies.19,22-24 To further enhance the biological specificity and therapeutic efficacy, great efforts have been devoted to construct stimuli-responsive delivery systems, which can improve the accumulation of nanocarriers at tumor sites and rapidly release the payloads within tumor cells.25 These stimuli can be external or internal signals,25 such as magnetic field, light, temperature, pH, redox potential and enzymatic activities. However, these stimuli-responsive nanocarriers usually need complex design and construction processes, and accurate experimental conditions.26 Recently, adenosine triphosphate (ATP) responsive nanocarriers for co-delivering drugs and genes have been successfully constructed,26-33 as ATP concentration has been identified to be 1-2 orders higher in the intracellular fluids (1-10 mM) than in the extracellular environment (< 0.4 mM).34,35 For instance, an ATP-triggered nanocarrier for doxorubicin (DOX) release has been constructed through an ATP responsive duplex (the aptamer and its complementary single-stranded DNA), in which DOX could intercalate into the duplex with high GC content in a low ATP environment and release quickly in an ATP-rich condition due to

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

Page 6 of 37

the dissociation of duplex.26 In the present research, an ATP-triggered nanosystem for achieving the co-delivery of DOX and miR-34a was constructed based on an ATP-responsive aptamer duplex, using branched polyethylenimine with a molecular weight of 25 kDa (PEI25K) as the carrier. The nanosystem could achieve the accumulation at tumor sites through the enhanced permeability and retention (EPR) effect,36 and facilitate the rapid release of these two cargos in the cytosol and further enhance their intracellular concentrations in an ATP-responsive manner. Finally, the nanosystem-mediated inhibition of cell proliferation and migration through the synergistic effects of DOX and miR-34a was systematically evaluated.

2. MATERIALS AND METHODS 2.1. Materials. ATP responsive aptamer (5'-ACC TGG GGG AGT ATT GCG GAG GAA GGT-3') and its cDNA (5'-ACC TTC CTC CGC AAT ACT CCC CCA GGT-3') were synthesized by Sangon Biotech. (Shanghai, China). MiR-34a (5’-UGG CAG UGU CUU AGC UGG UUG U-3’ (sense); 5’-AAC CAG CUA AGA CAC UGC CAU U-3’ (antisense)), negative control (NC, 5’-UUC UCC GAA CGU GUC ACG UdTdT-3’ (sense); 5’-ACG UGA CAC GUU CGG AGA AdTdT-3’ (antisense)), and 5-carboxyfluorescein (FAM)-labeled miR-34a (FAM-miR-34a) were obtained from GenePharma (Suzhou, China). Doxorubicin hydrochloride (> 99%) was purchased from Huafeng Biotech. Co. (Beijing, China) and used without further purification. PEI25K,

6


Page 7 of 37

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


crystal violet, heparin and 4,6-diamidino-2-phenylindile (DAPI) were purchased from Sigma-Aldrich (St. Louis, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, USA). Annexin V-FITC/PI apoptosis and cell cycle detection kits were purchased from Bestbio (Shanghai,

China).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT) and polyvinylidene fluoride (PVDF) membrane were purchased from Amersco (Solon,

USA)

and

Millipore

(Billerica,

USA),

respectively.

LIVE/DEAD®

Viability/Cytotoxicity kit for mammalian cells was obtained from ThermoFisher (Grand Island, USA). Diethyl pyrocarbonate (DEPC)-treated water and bovine serum albumin (BSA) were purchased from Dingguo Biotech. Co. (Beijing, China). Antibodies against

β-actin, Bcl-2, Notch-1, PARP, MMP-9, pro-caspase 3, 8, and 9, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were purchased from Abcam (Shanghai, China). The caspase 3, 8 and 9 activity assay kits and BCA protein assay kit were obtained from Promega (Madison, USA). The Golden Transfer agent was kindly provided by Changchun Golden Transfer Science and Technology Co., Ltd. (Changchun, China). All other reagents were purchased with the highest grade commercially available and used as received.

2.2. Construction of DOX-Duplex and ATP-triggered DOX release. First, 160 µL of ATP aptamer (20 µM in DEPC-treated water) and its cDNA with an equal amount were mixed together and incubated at room temperature for 15 min to obtain Duplex.

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

Page 8 of 37

Then 320 µL of DOX solution (20 µM in distilled water) was added into Duplex solution, and the sample was incubated for an additional 15 min to prepare DOX-Duplex. The DOX loading was detected by comparing the fluorescence spectra of 50 µL of DOX-Duplex and 25 µL of DOX solution (20 µM), both of which were diluted to 1 mL with distilled water (equal concentration of DOX). The ATP-triggered DOX release was measured as follows: 100 µL of ATP solution (0.4 or 4.0 mM) was added into 50 µL of DOX-Duplex, and the sample was incubated in the dark for different time, diluted to 1 mL with distilled water and subjected to the fluorescence spectra analysis. The fluorescence spectra were measured using Shimadzu RF-5301 fluorescence spectrometer with excitation and emission wavelength of 480 and 500-700 nm, respectively.

2.3. Construction and characterization of PEI/DOX-Duplex/miR-34a. MiR-34a solution (0.25 µg/µL in DEPC-treated water) and DOX-Duplex were mixed together with predetermined ratios, and then PEI25K was added into the system at different mass ratios. The mixture was incubated at room temperature for 30 min to obtain the nanocomplex PEI/DOX-Duplex/miR-34a. The particle size and zeta potential of nanocomplex were measured by Malvern Nano ZS90 Zetasizer (Malvern, UK). The miR-34a and Duplex condensation ability was detected through gel retardation assay. The nanocomplexes were prepared through the incubation of PEI25K with miR-34a and/or DOX-Duplex at 37 oC for 30 min, and then mixed with appropriate

8


Page 9 of 37

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


amounts of loading buffer and subjected to 1% agarose gel electrophoresis in Tris-acetate-EDTA (TAE) buffer (120 V, 20 min). To determine the stability of complexes in serum, the nanocomplexes were incubated with 50% FBS at 37 oC for 3 h and detected by 1% agarose gel electrophoresis as above, in which 4 mg/mL of heparin solution was used to destroy the nanocomplexes to release oligonucleotides.

2.4. Intracellular distribution of PEI/DOX-Duplex/miR-34a. Human lung adenocarcinoma cells A549 were seeded in 6-well plates harboring 2 mL of 10% FBS-containing DMEM (3.0×105 cells/well), into which the sterilized coverslips were placed. The cells were cultured at 37 oC for 24 h, after which the medium was removed from each well. The serum-free DMEM (1 mL) was then added into each well, and the cells were transfected with PEI/DOX-Duplex/miR-34a nanocomplex at a mass ratio of 4:1.5:2 (miR-34a and DOX concentration of 2 µg/mL and 0.182 µM, respectively) for different time. The medium was discarded after transfection, and the cells were washed with phosphate buffer saline (PBS) three times, fixed with 4% paraformaldehyde solution for 20 min and stained with DAPI solution (1 µg per well) for 3 min. Finally, the coverslips were observed using LSM 710 confocal laser scanning microscope (CLSM, Carl Zeiss Microscopy LLC, Jena, Germany).

2.5. Inhibition of cell proliferation. The MTT assay was used to evaluate the inhibition of cell proliferation by nanocomplex. Briefly, A549 cells were seeded in

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 37

96-well plates with 10% FBS-containing DMEM (200 µL) at a density of 1.0×104 cells/well. After the incubation at 37 oC for 24 h, the cells were transfected with nanocomplex for an additional 24 h. Then 20 µL of MTT solution (5 mg/mL in PBS) was added into each well and removed after 4 h incubation. The formazan crystals in the plates were dissolved using 200 µL DMSO. The plates were incubated at room temperature for 10 min and the absorbance at 492 nm was monitored on a GF-M3000 microplate reader (Shandong, China). The cell viability (%) was calculated as the ratio of absorbance values of the treated and untreated groups.

2.6. Live/Dead staining. Briefly, the A549 cells were seeded in 6-well plates harboring 2 mL of 10% FBS-containing DMEM at a density of 2.0×105 cells/well and incubated at 37 oC for 24 h. The cells were then treated with nanocomplex for 72 h, collected and washed with PBS twice. According to the manufacturer’s instructions, the cells were dyed with live/dead assay reagents for 20 min, washed with 1 mL PBS twice and assayed through IX71 fluorescence microscopy (Olympus, Tokyo, Japan).

2.7. Induction of cell apoptosis. The A549 cells were cultured in 6-well plates at 37 o

C for 24 h with an initial density of 2.0×105 cells/well, and treated with nanocomplexes

for 72 h (miR-34a and DOX concentration of 2 µg/mL and 0.182 µM, respectively). The cells were harvested, washed with PBS twice and suspended in the binding buffer in Annexin V-FITC/PI apoptosis detection kit. According to the manufacturer’s protocol,

10


Page 11 of 37

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


the cells were mixed with Annexin V-FITC and PI and incubated at room temperature for 10 min in the dark. Finally, the induction of cell apoptosis was detected by FACS caliber (BD Bioscience Mountain View, USA).

2.8. Induction of cell cycle arrest. Briefly, the A549 cells were incubated in 6-well plates at 37 oC for 24 h with an initial density of 3.0×105 cells/well, and treated with nanocomplexes for 24 h (miR-34a and DOX concentration of 2 µg/mL and 0.182 µM, respectively). The cells were collected, washed with PBS twice and fixed with 75% cold ethanol at -20 oC for 1 h. According to the manufacturer’s instructions, the cells were treated with 20 µL of RNase A at 37 oC for 30 min and 300 µL of PI at 4 oC for 1 h in the dark, which were provided in the cell cycle detection kit. The cell cycle arrest was then monitored by analyzing 15,000 gated cells through FACS caliber (BD Bioscience Mountain View, USA).

2.9. Western blotting assay. The cell culture and the transfection with nanocomplex were carried out as described in the cell apoptosis assay. The cells were collected, washed with PBS twice and treated with RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride on ice for 2 h. Then the centrifugation at 12,000 rpm for 10 min was performed to obtain the supernatants, and the protein concentration was determined through BCA method. An equal amount of protein was employed in SDS-PAGE and transferred to PVDF membrane by electroblotting. The membrane was

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

Page 12 of 37

blocked with PBS containing 5% nonfat milk for 2 h and incubated with the corresponding primary antibodies at 4 oC overnight. Then the membrane was washed with PBS containing 0.1% Tween-20 (PBST) three times and incubated with HRP-labeled secondary antibody at room temperature for 1 h. Finally, the membrane was washed with PBST twice and subjected to ChemiScope 3600 chemiluminescence imaging system (Clinx Science Instrument, Shanghai, China) to detect the expression level of specific proteins. The relative expression level of proteins was calculated through Tanon 1600 gel-imaging system and GIS 1D 4.1.5 software.

2.10. Wound healing assay. Briefly, the A549 cells were cultured in 6-well plates to 90% confluence, and a mechanical scratch wound was produced on the cell monolayer using a sterile pipette tip. After washing with PBS twice, the cells were treated with different nanocomplexes for different time (2 µg/well miR-34a). The digitized images of wound area were captured with IX71 fluorescence microscopy (Olympus, Tokyo, Japan), and three representative zones were selected to calculate the average length of cell migration at each time point.

2.11. Transwell migration assay. The A549 cells were first cultured in 6-well plates and transfected with different nanocomplexes for 72 h as described above. Then the cells were digested with 0.25% trypsin and added into the upper chamber (8 µm pores) of Transwell chamber (Costar, Corning, NY, USA) containing 200 µL of serum-free

12


Page 13 of 37

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


DMEM at a density of 2.0×104 cells. In the lower chamber, 600 µL of 10% FBS-containing DMEM was added, and meanwhile BSA was added into the upper chamber (final concentration of 1%) to balance the osmotic pressure. After the incubation at 37 °C for 24 h, non-migrating cells on the top of membrane were carefully removed by mechanical wiping, and the cells which have migrated to the lower surface of membrane were fixed with 75% cold ethanol for 20 min. Finally, the cells were stained with 0.2% crystal violet for 20 min, washed with PBS twice and observed through IX71 fluorescence microscopy (Olympus, Tokyo, Japan).

2.12. Statistical analysis. All data were presented as mean value ± SD, and statistical significance of differences between experimental groups and control group were analyzed using one-way ANOVA with SPSS Statistics 23.0 complemented with Student’s t-test (n. s., not significant; *p

An ATP-Responsive Codelivery System of Doxorubicin and MiR-34a

Recommend Documents