Stimuli-Responsive Nanocarrier for Co-delivery of MiR-31 and

Subscriber access provided by TUFTS UNIV

Applications of Polymer, Composite, and Coating Materials

Stimuli-Responsive Nano-Carrier for Co-delivery of MiR-31 and Doxorubicin to Suppress High MtEF4 Cancer Fang Wang, Lingyun Zhang, Xiufeng Bai, Xintao Cao, Xiangyu Jiao, Yan Huang, Yansheng Li, Yan Qin, and Yongqiang Wen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07698 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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 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 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.

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 30 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

Stimuli-Responsive Nano-Carrier for Co-delivery of MiR-31 and Doxorubicin to Suppress High MtEF4 Cancer Fang Wang†‡#, Lingyun Zhang‡§#, Xiufeng Bai‡, Xintao Cao‡§, Xiangyu Jiao†, Yan Huang†, Yansheng Li†, Yan Qin*‡§, and Yongqiang Wen*† †

Research Center for Bioengineering and Sensing Technology, School of Chemistry and

Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China ‡

Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute

of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China §

University of Chinese Academy of Sciences, Beijing 100049, China

KEYWORDS: combined therapy, controlled release, gene interference, microRNA-31, mesoporous silica nanoparticles

ABSTRACT: Gene interference-based therapeutics represents a fascinating challenge and shows enormous potential for cancer treatment, in which microRNA is used to correct abnormal gene. Based on the above, we introduced microRNA-31 to bind to 3’ untranslated region of mtEF4, resulting in the downregulation of its messenger RNA and protein to trigger cancer cells

1 Environment ACS Paragon Plus

ACS Applied Materials & Interfaces 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

apoptosis through mitochondria-related pathway. To achieve better therapeutic effect, a mesoporous silica nanoparticles-based controlled nanoplatform had been developed. This system was fabricated by conjugation of microRNA-31 onto doxorubicin-loaded mesoporous silica nanoparticles with a PEI/HA coating, and drug release was triggered by acidic environment of tumors. By feat of surface functionalization and tumor-specific conjugation to nanoparticles, our drug delivery system could promote intracellular accumulation of drugs via the active transport at tumor site. More importantly, microRNA-31 not only directly targeted to mtEF4 to promote cells death, but had synergistic effects when used in combination with doxorubicin, and achieved excellent superadditive effects. As such, our research might provide new insights towards detecting high mtEF4 cancer and exploiting highly effective anticancer drugs.

INTRODUCTION Cancer is a considerable community health problem globally.1 For this reason, research into its causes and treatment has always been a hot area. In particular, over the past several decades, RNA interference shows great promise in cancer treatment, and various RNA interference-based therapeutics has emerged.2-5 However, relying on the single RNA interference strategy is still suboptimal, which is limited by the undesirable severe side effects, nonspecific delivery.6-7 To push past these obstacles and achieve better therapeutic effect, the combined two or more therapies taking advantage of co-delivery system such as inorganic nanoparticles,8-9 lipoplexes,1011

dendrimers,12-13 polyplexes,14-18 has recently been developed to cooperatively prohibit cancer

development and provide potential synergistic or combined effects with different mechanisms.1922

In recent years, mesoporous silica nanoparticle (MSN) has emerged as potential carrier to codeliver microRNAs (miRNAs) and chemotherapy drugs because of their good biocompatibility,


Page 2 of 30

Page 3 of 30 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


easy synthesis and varieties of surface functionalization.23-25 Further surface modification of the nano-carrier allows to enhance recognition and uptake of the nano-carrier by tumor tissues. For example, branched polyethyleneimine (PEI) has been developed because of its strong endosomal escape capacity.26 And hyaluronic acid (HA) was developed on the external layer of PEI-capped MSN to circumvent the non-specific binding with blood serum,27-28 by targeting the tumor tissue with HA receptor as cluster determinant 44 (CD44), which is over expressed in many cancer types.29-32 In the study of tumorigenesis, mitochondrial elongation factor 4 (mtEF4) is found to overexpress in some tumor tissues comparing to the surrounding normal tissues via Catalogue of Somatic Mutations in Cancer. MtEF4 is a translation factor affecting the synthesis of mitochondria proteins, which is encoded by elongation factor 4 (EF4) gene.33 What’s more, our recent work has verified that mtEF4 knockout leads to mitochondrial dysfunction, thereby affecting mouse reproduction.34 The above relevant studies implies that it is beneficial to inhibit the expression level of mtEF4 for cancer treatment. Here, in vitro experiments are first conducted to reveal the downregulation of mtEF4 levels by microRNA-31(miR-31), which induces cancer cells apoptosis through mitochondria-related pathway. Next, a pH-triggered, MSN-based drug nano-carrier system is fabricated using layerby-layer method to co-deliver miR-31 and DOX aiming at down-regulating the expression of mtEF4 and improving the treatment effect of DOX (Figure 1). Once the nanoparticles are engulfed by cancer cells, miR-31 and DOX are released at the acidic pH due to the breakage of disulfide bond, leading to DOX toxicity and downregulation of gene.35 Finally, an in vivo experiment shows that the co-delivery system markedly inhibits the tumor cells proliferation and promotes tumor cells apoptosis in the tumor-bearing mice. In addition, our study corroborates the



combined effect of gene interference with chemotherapy, and highlights the role of miR-31 not only directly targeting to mtEF4 to promote cells death through mitochondria-related pathway, but achieving excellent superadditive effects when used in combination with doxorubicin.

Figure 1. Schematic representation for construction of MSN/DOX/miR-31/PEI/HA (abbreviated as complex) and the mechanism of tumor-targeted combined therapy. EXPERIMENTAL SECTION Materials. N-Hydroxysuccinimide (NHS), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), Tetraethyl orthosilicate (TEOS, 99.9%), Hexadecyltrimethylammonium bromide (CTAB, 99%), (3- mercaptopropyl)trimethoxysilane (MPTMS) and polyethyleneimine, branched (PEI) was got from Sigma-Aldrich (USA). Hyaluronic Acid (HA) was purchased from Shandong Galaxy Bio-Tech Co., Ltd. Sodium hydroxide (NaOH) was provided by Sinopharm Chemical Reagent Co. Ltd. CCK-8 kit was got from Dojindo Molecular Technologies, Inc. Doxorubicin was purchased from Shanghai Yuanye Biological Technology Co. Ltd. All buffers were made using ultra-pure MilliQ water.


Page 4 of 30

Page 5 of 30 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


RNA Isolation and qPCR. Total RNAs (including microRNA) were got from cells with Trizol reagent following the instruction. RNA was quantitated by NanoDrop ND-2000 spectrophotometer. RNA (0.5 µg) was then reverse transcribed utilizing M-MLV reverse transcriptase (Fermentas). miRNAs were reverse transcribed using special miRNAs primers. qPCR was conducted with specific primers on the ABI-7900HT Fast Real-Time System following construction. We used U6 snRNA to normalize miR-31 expression and β-actin to normalize mtEF4 miRNAs expression. All primers were listed below: miR-31 and U6 snRNA reverse transcription primers: 5’ - CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG AGC TAT GC - 3’ and 5’ - CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG AAA ATA TGG AAC - 3’; miR-31 forward primer: 5’ - ACA CTC CAG CTG GGA GGC AAG ATG CTG GC - 3’; U6 snRNA forward primer: 5’ - ACA CTC CAG CTG GGG TGC TCG CTT CGG CAG CAC A - 3’; reverse primer: 5’ - TGG TGT CGT GGA GTC G-3’; mtEF4 sense: 5’ - CCA GCA ATC AGA TAG CCC AC - 3’; mtEF4 antisense: 5’ - CAA GCC CAA ACT GTA GCA AA - 3’; β-actin sense: 5’ - GGG AAA TCG TGC GTG ACA TTA AG 3’; β-actin antisense: 5’ - TGT GTT GGC GTA CAG GTC TTT G - 3’. Cell Culture and Transfection. HeLa cells (Human cervix cancer) and H1299 cells (nonsmall lung cancer) were maintained in Dulbecco's Modified Eagle's Medium (DMEM, 10% fetal bovine serum and 1% antibiotic). The cells were kept at incubator. MicroRNA-31 mimics (miR31) and mimics control (mimics ctrl) was purchased from Genepharma Company and introduced into cells using LipofectamineTM 2000 reagent (Invitrogen) with a final concentration of 40 nM following instructions. Isolation of Mitochondria Protein and Western Blotting Assay. Mitochondria protein was isolated using Cell Mitochondria Isolation Kit (Beyotime). Protein got from transfected cells was



harvested using RIPA lysis buffer. The proteins were quantitated by BCA method and 50 µg protein was subsequently isolated with a SDS-PAGE gel for analysis of the expression of cytochrome C, mtEF4 and other protein. (loading control: tubulin). The primary antibody: rabbit polyclonal to cytochrome C (CST); rabbit polyclonal to mtEF4 (1:2000), rabbit polyclonal to Bax (1:1000) and rabbit monoclonal to PARP (1:1000) (from Abcam). The secondary antibody: HRP-conjugated goat anti-rabbit antibody. The protein signals were visualized with enhanced chemiluminescence (ECL) following the instruction. Colony Formation Assays. The transfected cells were plated in 12-well plate with 150 cells (per well) and the media were changed every three days. After about 10 days, most colonies had more than 50 cells and then were washed and stained by 5% crystal violet to count. Cell Apoptosis Assay. Cell apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Kit (MbChem) following the instruction. The transfected cells were gathered and then suspended in 400 µl 1×binding buffer, incubation with Annexin V-FITC solution for 15 min at 4℃ avoiding of light. Finally, the cells were incubated with PI for 5 min at 4℃ avoiding of light, followed by measurement of apoptosis using Flow cytometry. Synthesis of Complex. MSN was synthesized following previous method with simple modification.36 The mixture of MilliQ water (48 ml) containing CTAB (500 mg), ethanol (12 ml) and NaOH (2 mol/L) was stirred for 1 h. TEOS (2800 µL) was stirred with the mixture for about 8 h at room temperature. The solution was spun down, washed with ethanol and water several times and dried under vacuum oven (60℃) overnight. To remove the surfactant, 1.0 g pellets were dispersed into ethanol (100 ml) under stirring for 48 h.35-37 The particles without template were washed with ethanol thrice. The removal of template was verified by FTIR (Figure S10a) which showed the weak peak at 724 cm-1 (long carbon chain saturated hydrocarbons). The


Page 6 of 30

Page 7 of 30 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


complex was developed by the layer-by-layer process.38 Briefly, MSN (100 mg) was dispersed into of toluene and added 1ml (3-mercaptopropyl)trimethoxysilane (MPTMS) to stir for 1 h, washed thrice with ethanol, dried under vacuum oven. Then, 50 mg mercapto-modified MSN were dispersed into 1 ml ethanol containing 100 µL of 10 mg/mL DOX by sonication. After sonicating for 2.5 h, MSN/DOX was washed for several minutes and re-dispersed into ethanol thrice. The loading efficiency of MS-DOX was calculated by Equation: Percentage of loading = (total DOX - unloaded DOX) / total DOX. Then, 0.5 mL of MSN/DOX was dispersed to 100 µL of 20 µM miR-31. After stirring for 1 h, the MSN/DOX/miR-31 was cleansed by centrifugal separation several times. The MSN/DOX/miR-31 was re-dispersed in PEI solution. After stirring for 0.5 h, MSN/DOX/miR-31/PEI was purified several times. Finally, adding EDC and NHS (4:1) to HA solution to react for 15 min, followed by adding MSN/DOX/miR-31/PEI. After stirring for 0.5 h, complex was cleansed by centrifugal separation several times. Testing DOX release. Two aliquot of DOX-loaded complex (2 mg) were dispersed in two centrifuge tube containing PBS (1000 µL, pH = 5.2, pH = 7.4), respectively. Taking 150 µL of supernatant at various points, followed by adding 150 µL of new PBS solution. UV-vis absorbance measurements (six replicates) were performed. Cytotoxicity Assay. Plating 5,000 cells (each well) in a 96-well plate (n = 6) to incubate with 0-200 µg/mL MSN dispersed into fresh medium overnight. Viability was performed with CCK-8 kit reagent followed by absorbance readout at 450 nm emission. Flow Cytometry Assay. We incubated HeLa cells in a 6-well plate with the concentration of 250,000 cells and grow overnight, followed by being exposed to control group (only culture media), MSN/DOX/miR-31/PEI, complex (several concentrations), complex + free HA for 1.5 h,



respectively. Cells were then washed thrice with PBS, detached by trypsin, spun down, then dispersed into PBS for FACS analysis. Development of Mice Model. All animal works were agreed by the Institutional Animal Care and Use Committee of the Institute of Biophysics, CAS, following the instruction. Nude mice (≈ 20 g) were got from Beijing Vital River Laboratory Animal Technology Co., Ltd. Tumors were made by injecting about 2 × 106 HeLa cells. Injection started after tumor volume was about 100 mm3 and the mice were randomly divided to receive different treatment (n = 3 each group): (1) Control, mice got 200 µL of PBS injection through caudal vein; (2) MC, mice got 200 µL of MSN/DOX/Mimics Control/PEI/HA (MC) solution injection (200 µg/ml, in PBS); (3) Complex, mice got 200 µL of complex solution injection (200 µg/ml, in PBS). The mice were killed after 10 days injection. Hematoxylin-Eosin (H&E) Assay. Knub section was removed, placed in paraffin, sliced and placed on charged slide. Dewaxed samples were immersed into hematoxylin for several minutes and washed with slowly flowing water for several minutes, then counterstained with eosin for 15 seconds. Finally, the slides were mounted with Permount Mounting Medium (Thermo Scientific). TUNEL Assay. Apoptosis was analyzed with the cell-death detection kit (Selleck, B31112) by instruction. Dewaxed section was placed in blocking solution (3% H2O2 in methanol) for several minutes. Then slide was washed with PBS and placed in the cold mixture of Triton X100 (0.1%) and sodium citrate (0.1%) for 2 minutes, then immersed in reaction mixture (50：l), and placed under 37°C for 60 minutes and kept in dark place. IHC-PCNA assay. Immunohistochemistry was operated with the guideline. Paraffin section of tumor was immersed into the primary antibodies for 0.5 h: anti-PCNA antibodies (Abcam,


Page 8 of 30

Page 9 of 30 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


ab19166). The slides were then washed and placed in Alexa Fluor 488–conjugated secondary antibody (Invitrogen, W10811). Finally, the sample was imaged by microscopy (Nikon, Japan). RESULTS AND DISCUSSIONS MtEF4 is the direct target of miR-31. As shown in Figure 2a, miR-31 has a conserved binding site with the 3’UTR of mtEF4. We cloned the 3’UTR of GUF1 including miR-31 binding sites downstream placing the luciferase-coding gene and created the luciferase reporter system. The luciferase reporter system was performed for luciferase assay in miR-31 transfected cells. As shown in Figure 2b and S1a, the luciferase intensity influenced by mtEF4 3’UTR was decreased to about 50%, but miR-31 did not inpact on the intensity after the binding site was mutated. Next, qPCR assay revealed mtEF4 mRNA levels in both miR-31 transfected Hella cells and H1299 cells was decreased by about 60% and western blotting assay result showed mtEF4 protein levels was significantly reduced (Figure 2c-d and S1b-c). The results indicated that miR31 downregulated mtEF4 expression via binding to its 3’UTR.



Figure 2. (a) Sequence alignment of miR-31 target sites within mtEF4 3’UTR. (b) Luciferase assay: luciferase reporter system including both wild-type and mutant mtEF4 3’UTR in HeLa cell transfected with miR-31. (c) qPCR assay to determine mtEF4 mRNA expression, β-actin (internal control) in miR-31 or mimics ctrl transfected HeLa cells. (d) Western blotting assay to analyze mtEF4 protein level, Tubulin (loading control) (* P < 0.05, ** P < 0.01).

MiR-31 overexpression inhibits cell growth and promotes cell apoptosis. We performed qPCR to verify the successful overexpression of miR-31 by transient transfection (Figure 3a). Some studies verified that the over-representation of miR-31 increased docetaxel-triggered apoptosis and suppressed the proliferation of prostate cancer cells.39-40 MiR-31 also suppressed the G1/S cell cycle transition by down-regulating minichromosome maintenance protein 2 in medulloblastoma,41 serving as a tumor suppressor. MiR-31 promoted cell apoptosis of breast


Page 10 of 30

Page 11 of 30 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


cancer by directly targeting protein kinase C epsilon,42 playing tumor-suppressive functions. Our previous work found mtEF4 knockout can suppress cell growth and promoted cell apoptosis in HeLa and H1299 cells.34 In order to demonstrate whether the effect of miR-31 on cell growth and apoptosis is similar to knocking out mtEF4, we performed MTT assay and cell apoptosis assay. The effect of miR-31 on cell growth and viability in both HeLa and H1299 cells was suppressed by approximately 15%～20% (Figure 3b and S2a). In line with the result of miR-31 effecting on cytoactive, the number of miR-31 transfected HeLa cell colony was reduced by approximately 30% and that of H1299 cell colony by 50% comparing to control groups (Figure 3c and S2b). We further studied the effect of miR-31 over-representation on cell cycle transition. The number of HeLa cells in G1 stage was almost unchanged, while the proportions of cells in S stage decreased by 9.55%, and that in G2 stage increased by 10% (Figure S3a). As shown in Figure S3b, significant inhibition of the G1/S transition was observed in miR-31-transfected H1299 cells, relative to control group (G1 stage increased by 15.04%, S stage decreased by 12.18%). The results indicated that miR-31 might affect cell growth by regulating cell cycle progression. Next, we studied the influence of miR-31 over-representation to apoptosis. The apoptotic rate of HeLa cells transfected with miR-31 increased by 5.17% and the death rate increased by 4.03%. MiR-31-transfected H1299 cells had the higher cell apoptotic rate (7.66%) and death rate (3.72%) (Figure 3d and S2c). MtEF4 is a pivotal factor in the control of mitochondrial translation.43-44 So, we next investigated whether miR-31 promoted cell apoptosis through mitochondria-related pathway. First, we examined mitochondrial membrane potential (MMP) and found it was reduced by



approximately 50% in miR-31 transfected HeLa and H1299 cells (Figure S4a-b). Then, we imaged mitochondria morphology under SIM with mitotracker staining and the mitochondria morphology of miR-31-transfected cells was changed from rod-shape to dot-shape, especially in H1299 cells (Figure S5). All the results indicated that miR-31 caused MMP to decrease and mitochondria morphology to disrupt by promoting cell apoptosis. A previous study had demonstrated that the reduction of MMP was considered as the first step of mitochondria-relevant apoptosis pathway.45 Cytochrome C release can initiate the mitochondria-related apoptosis pathway.46 Mitochondria membrane permeability became increasing with the decreased MMP, and then miR-31 triggered cytochrome C to release from mitochondria to cytoplasm to activate caspase family members and induce apoptosis. Poly ADPribose polymerase (PARP) is the main shear objects of caspase-3 and its cleavage is considered as an important marker of cell apoptosis.47 The expression of Bax and PARP of cancer cells was increased after being transfected with miR-31 (Figure S6). The results indicated that miR-31 enhanced cell apoptosis partially through mitochondria-related pathway.


Page 12 of 30

Page 13 of 30 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) qPCR for the analysis of miR-31 expression. (b) MTT assay druing different period after transfection. (c) Colony formation experiment: miR-31 or mimics ctrl transfected HeLa cells (d) MiR-31 or mimics ctrl transfected HeLa cells for apoptosis analysis, apoptotic cells stained with Annexin-V (right below) and dead cells stained with PI (right upper) (* P < 0.05, ** P < 0.01).

Construction of Nano-carrier. Figure 1 briefly described the preparation process of complex using the layer-by-layer method for cancer therapy.38,

48-49

We made MSN by a

modified sol-gel method.36, 50 As shown in Figure 4a-b, the obtained MSN had a monodisperse diameter ranging from 79 to 110 nm under scanning electron microscopy (SEM). The nanochannels could be clearly observed via transmission electron microscopy (TEM) (Figure 4c-



d and S8). MPTMS was modified on the surface of MSN by cocondensation. DOX was loaded into the modified MSN by sonicating and the loading content of DOX were 6.35 µg/mg. The mercapto-modiefied DOX-loaded MSN and the mercapto group of miR-31 formed disulfide bonds with the loading efficiency of 8.6 nmol/mg, and the electrostatic interaction between MSN/DOX/miR-31 and PEI resulted in the formation of MSN/DOX/miR-31/PEI. Branched polyethyleneimine (PEI) had been developed because of its strong endosomal escape capacity.26 Finally, HA containing a large number of carboxyl was reacted with amino of PEI to get complex. HA was developed to the external layer of PEI-capped MSN to circumvent the nonspecific targeting with blood serum. The formation of complex was verified by Fourier transform infrared spectroscopy (FTIR), which had three special peaks at 1647 cm-1, 1559 cm-1 (amide I, II band) and 560 cm-1 (disulfide bond) (Figure S10b). The release curves of DOX were got under pH 7.4 and pH 5.2 because miR-31 was linked to the nanoparticles via acid-responsive molecules (Figure 4g). Whereas only 1.07% of DOX was measured from supernate at neutral pH for 24 h and about 53.1% of DOX was measured at pH 5.2 after 24 h with the disulfide bonds hydrolyzed under acidic condition. We characterized the formation of MSN/DOX/miR-31 with agarose gel electrophoresis. As shown in Figure 4e, successful assembly of MSN/DOX with miR-31 (lane 4) resulted in retarded mobility of miR-31 in the gel. The left two bands (lane 1, 2) was black because of the absence of miR-31 and the third band with highest mobility were miR31 alone. The successful formation of complex was also verified by the zeta potential analysis, which showed the surface charge of MSN/DOX/miR-31, MSN/DOX/miR-31/PEI, and complex were -11.2 mV, 24.3 mV, -34.1 mV, respectively (Figure 4f and S9). As discussed above, complex was a collaborative platform for gene delivery and chemotherapy drug transport.51 We tested cytotoxicity with the CKK-8 assay by adding different


Page 14 of 30

Page 15 of 30 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


concentrations of MSN to HeLa cells for 24 h. No obvious toxicity was detected with the incubation concentration of MSN under 50 µg/mL. Cell activity still kept above 80% even reaching to highest dose (200 µg/mL), (Figure S7). These data indicated that MSN had negligible cytotoxicity and great biocompatibility.

Figure 4. (a-b) SEM and (c-d) TEM images of MSN. (e) The agarose gel electrophoresis demonstrating the construction procedure of MSN/DOX/miR-31. Lane 1: MSN; lane 2: DOXloaded MSN; lane 3: miR-31; lane 4: MSN/DOX/miR-31; lane 5: marker (f) Zeta potential analysis demonstrating the successful formation of complex in DI water (n = 3). (g) The release profile of DOX from MSN/DOX/miR-31 at pH 7.4 and pH 5.2.

Cellular Uptake. To verify the intracellular uptake of complex, HeLa cells imaged by CLSM. Cells exposed to complex (Figure 5c-d) got a strong fluorescence and the control group (MSN/miR-31/PEI/HA without DOX) (Figure 5a-b) was black. Subsequently, flow cytometry experiment was made to further verify the specific uptake of complex mediated by HA receptor



(Figure 5e, Table S1). For testing the competitive binding, HeLa cells with HA receptor were incubated with or without free HA first and then exposed to complex. The fluorescence intensity of cells cultured with complex and free HA was very low because free HA blocked CD44 receptors and competed with complex in receptor binding. The fluorescence intensity of cells cultured with MSN/DOX/miR-31/PEI was quite lower than that of incubated with complex. This indicated that the nanoparticles carrying the targeting HA had higher cellular uptake than the ones without targeting shell. To further verify the main mechanism of uptake, HeLa cells were exposed to different concentrations of complex. The fluorescence intensity became stronger as the increasing concentrations of complex (Figure 5f, Table S1). The data indicated that the uptake of complex mainly depended on active targeting, which matched the study of Vangara et al about PLGA nanoparticles being used for active transport by modifying tumor-specific ligands (HA) to the surface.52

Figure 5. Evaluation of the intracellular uptake of HeLa cells by CLSM and flow cytometry assay: (a-b) incubated with MSN/miR-31/PEI/HA without DOX, (c-d) incubated with complex; (e) incubated with: cell medium (Ctrl), MSN/DOX/miR-31/PEI, complex + free HA, complex; (f1-f4) incubated with the increasing concentrations (from 1 to 4) of complex.


Page 16 of 30

Page 17 of 30 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


In Vitro Treatment Effect. We measured the influence of combined treatment of miR-31 and chemotherapy drug-DOX to cell growth. As shown in Figure 6a, cells directly exposed to miR-31 in the absence of nanoparticles reduced the cell viability by 13% ~ 19%. After incubated with free DOX (10 ng/ml), the cell viability was decreased by about 25% ~ 39%. However, the complex led significant decrease of the cells viability by 62% ~ 69% after incubation with complex, which was quite less than the effect of DOX or miR-31 alone. Next, we performed the Western blotting assay and CCK-8 assay at the same culture interval to verify whether the effect of DOX on HeLa cells was enhanced via negatively regulating the expression of mtEF4. The mtEF4 level of cells treatment with the miR-31 carried by the nanoparticles was much lower than the control group with mimics control, indicating that the miR-31 maintained their function and affected on mtEF4 expression when conjugated to the MSN nanoparticles (Figure 6b). To some extent, the results validated that introducing miR-31 could significantly boost the treatment effect of DOX by down-regulating the expression of mtEF4. The above in vitro experiments showed that miR-31 could reduce mtEF4 3’UTR intensity, suppress mtEF4 protein and mRNA levels, promote cancer cells apoptosis through mitochondriarelated pathway, and the use of MSN enabled an efficient and specific delivery of miR-31/DOX, further improved the therapeutic efficacy.



Figure 6. Therapeutic effect of nano-composites and the function of miR-31. (a) HeLa cells exposed to cell medium (Ctrl), miR-31, DOX, MSN/DOX/Mimics Control/PEI/HA (MC), complex (100 µg/mL including 10 ng/mL of DOX) (n = 3). (b) Western blotting to test mtEF4 expression in HeLa cells transfected with complex and MC (n = 3).

In Vivo Experiments. Thus far, we had demonstrated the downregulation effect of miR-31 on mtEF4 expression and the effect of combination therapy in in vitro experiments. To inquire whether this effect occurred similarly in vivo results, we examined the therapeutic efficacy of DOX and miR-31 of tumor bearing mice by the histological results. More than 40% tumor size reduction was observed for DOX/miR-31-containing nanoparticles compared with that of only DOX or no DOX/miR-31-containing 10 days after treatment (Figure 7b). This indicated efficient inhibition of the primary tumor progression due to the combined effect of DOX and miR-31 release. The H&E assay showed the necrosis or apoptosis section of the tumor tissues became larger (from left to right). Next, we performed the TUNEL assay for detection of apoptosis of tumor cells. The data showed that the numbers of TUNEL-positive cells (yellow) both of the group with miR-31 and group with mimics control were dramatically increased, especially in the


Page 18 of 30

Page 19 of 30 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


group with miR-31. This result further illustrated that miR-31 could promote tumor cells death. Last, the IHC-PCNA analysis, a cellular marker associated with cell proliferation, revealed that few PCNA positive cells (green-blue area) were observed in tumor tissues treatment with nanoparticles carrying miR-31 or mimics control comparing to control groups without any treatment (Figure 7a).



Figure 7. In vivo experiments. (a) Treated mice (i) Tumor sections (ii) H&E assay (iii) TUNEL assay (iv) IHC-PCNA assay (v) for apoptosis and cell proliferation detection after the treatment for each group to observe resected tumors of different treated mice: (1) Control, mice got 200 µL of PBS injection; (2) MC, mice got 200 µL of MSN/DOX/Mimics Control/PEI/HA (MC) solution injection; (3) Complex, mice got 200 µL of complex solution injection. (b) Tumor volume from mice treated with above three methods (n = 3) (* P < 0.05, *** P < 0.001). The scale bar is 40 µm.

CONCLUSION Here we developed a assembled delivery platform for co-delivery of microRNA and chemotherapy, which was easier to construct and modulate the size for cellular uptake. More importantly, this work achieved the synergistic effect of gene regulation and chemotherapy by introducing miR-31 and DOX to high mtEF4 cancer cells. Especially, the co-delivery of miR-31 with DOX using this platform inhibited cancer cells growth efficiently than delivering miR-31 or DOX alone. Next, we will be centered on the action mechanism between mtEF4 and anti-tumor drugs, which might provide a new idea for the further study of drug resistance. ASSOCIATED CONTENT Supporting Information The Supporting Information is available on the ACS Publications website. Additional methods and experimental data (Figure S1−S10 and Table S1) AUTHOR INFORMATION Corresponding Authors


Page 20 of 30

Page 21 of 30 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


*E-mail: [email protected] *E-mail: [email protected] ORCID Yongqiang Wen: 0000-0002-1924-4166 Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by Beijing Natural Science Foundation (2172039); the National Key R&D Program of China (2018YFA0106901) to Y. Q.; the NSFC (51373023); the Fundamental Research Funds for the Central Universities; Strategic Priority Research Programs (Category A) of CAS (XDA12010313); Key Research Program of Frontier Sciences, CAS, (QYZDB-SSW-SMC028). We are grateful to Guizhi Shi for assistance with H&E assay. REFERENCES (1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2017. CA Cancer J Clin 2017, 67 (1), 7-30.



(2) Zimmermann, T. S.; Lee, A. C.; Akinc, A.; Bramlage, B.; Bumcrot, D.; Fedoruk, M. N.; Harborth, J.; Heyes, J. A.; Jeffs, L. B.; John, M. RNAi-Mediated Gene Silencing in Non-Human Primates. Nature 2006, 441 (7089), 111-114. (3) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W.; Stebbing, D.; Crosley, E. J.; Yaworski, E. Rational Design of Cationic Lipids for SiRNA Delivery. Nat. Biotechnol. 2010, 28 (2), 172-176. (4) Love, K. T.; Mahon, K. P.; Levins, C. G.; Whitehead, K. A.; Querbes, W.; Dorkin, J. R.; Qin, J.; Cantley, W.; Qin, L. L.; Racie, T. Lipid-like Materials for Low-Dose, In Vivo Gene Silencing. Proceedings of the National Academy of Sciences 2010, 107 (5), 1864-1869. (5) Heidel, J. D.; Yu, Z.; Liu, J. Y.-C.; Rele, S. M.; Liang, Y.; Zeidan, R. K.; Kornbrust, D. J.; Davis, M. E. Administration in Non-Human Primates of Escalating Intravenous Doses of Targeted Nanoparticles Containing Ribonucleotide Reductase Subunit M2 SiRNA. Proceedings of the National Academy of Sciences 2007, 104 (14), 5715-5721. (6) Johnstone, R. W.; Ruefli, A. A.; Lowe, S. W. Apoptosis: A Link between Cancer Genetics and Chemotherapy. Cell 2002, 108 (2), 153-164. (7) Chabner, B. A.; Roberts Jr, T. G. Chemotherapy and the War on Cancer. Nat. Rev. Cancer 2005, 5 (1), 65-72. (8) Gao, Y.; Chen, Y.; Ji, X.; He, X.; Yin, Q.; Zhang, Z.; Shi, J.; Li, Y. Controlled Intracellular Release of Doxorubicin in Multidrug-Resistant Cancer Cells by Tuning the Shell-Pore Sizes of Mesoporous Silica Nanoparticles. ACS nano 2011, 5 (12), 9788-9798.


Page 22 of 30

Page 23 of 30 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


(9) Chen, Y.; Chen, H.; Shi, J. Inorganic Nanoparticle-Based Drug Codelivery Nanosystems to Overcome the Multidrug Resistance of Cancer Cells. Mol Pharm 2014, 11 (8), 2495-2510. (10) Sun, T.-M.; Du, J.-Z.; Yao, Y.-D.; Mao, C.-Q.; Dou, S.; Huang, S.-Y.; Zhang, P.-Z.; Leong, K. W.; Song, E.-W.; Wang, J. Simultaneous Delivery of SiRNA and Paclitaxel via a “Two-in-One” Micelleplex Promotes Synergistic Tumor Suppression. ACS nano 2011, 5 (2), 1483-1494. (11) Yu, Y. H.; Kim, E.; Park, D. E.; Shim, G.; Lee, S.; Kim, Y. B.; Kim, C. W.; Oh, Y. K. Cationic Solid Lipid Nanoparticles for Co-delivery of Paclitaxel and SiRNA. Eur J Pharm Biopharm 2012, 80 (2), 268-273. (12) Qian, X.; Long, L.; Shi, Z.; Liu, C.; Qiu, M.; Sheng, J.; Pu, P.; Yuan, X.; Ren, Y.; Kang, C. Star-branched Amphiphilic PLA-b-PDMAEMA Copolymers for Co-delivery of MiR-21 Inhibitor and Doxorubicin to Treat Glioma. Biomaterials 2014, 35 (7), 2322-2335. (13) Cao, N.; Cheng, D.; Zou, S.; Ai, H.; Gao, J.; Shuai, X. The Synergistic Effect of Hierarchical Assemblies of SiRNA and Chemotherapeutic Drugs Co-delivered into Hepatic Cancer Cells. Biomaterials 2011, 32 (8), 2222-2232. (14) Shen, J.; Sun, H.; Meng, Q.; Yin, Q.; Zhang, Z.; Yu, H.; Li, Y. Simultaneous Inhibition of Tumor Growth and Angiogenesis for Resistant Hepatocellular Carcinoma by Co-delivery of Sorafenib and Survivin Small Hairpin RNA. Mol Pharm 2014, 11 (10), 3342-3351. (15) Xu, Z.; Zhang, Z.; Chen, Y.; Chen, L.; Lin, L.; Li, Y. The Characteristics and Performance of a Multifunctional Nanoassembly System for the Co-delivery of Docetaxel and iSur-pDNA in a Mouse Hepatocellular Carcinoma Model. Biomaterials 2010, 31 (5), 916-922.



Page 24 of 30

(16) Shen, J.; Yin, Q.; Chen, L.; Zhang, Z.; Li, Y. Co-delivery of Paclitaxel and Survivin ShRNA by Pluronic P85-PEI/TPGS Complex Nanoparticles to Overcome Drug Resistance in Lung Cancer. Biomaterials 2012, 33 (33), 8613-8624. (17) Guo, X. D.; Tandiono, F.; Wiradharma, N.; Khor, D.; Tan, C. G.; Khan, M.; Qian, Y.; Yang, Y. Y. Cationic Micelles Self-assembled from Cholesterol-conjugated Oligopeptides as an Efficient Gene Delivery Vector. Biomaterials 2008, 29 (36), 4838-4846. (18) Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G.; Watson, N.; Kiziltepe, T.; Sasisekharan, R. Temporal Targeting of Tumour Cells and Neovasculature with a Nanoscale Delivery System. Nature 2005, 436 (7050), 568-572. (19) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nature nanotechnology 2007, 2 (12), 751-760. (20) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle Therapeutics: an Emerging Treatment Modality for Cancer. Nat Rev Drug Discov 2008, 7 (9), 771-782. (21) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat Rev Cancer 2005, 5 (3), 161-171. (22) Liu, C.; Liu, F.; Feng, L.; Li, M.; Zhang, J.; Zhang, N. The Targeted Co-delivery of DNA and Doxorubicin to Tumor Cells via Multifunctional PEI-PEG Based Nanoparticles. Biomaterials 2013, 34 (10), 2547-2564. (23) Climent, E.; Martinez-Manez, R.; Sancenon, F.; Marcos, M. D.; Soto, J.; Maquieira, A.; Amoros,

P.

Controlled

Delivery

Using

Oligonucleotide-Capped

Mesoporous

Nanoparticles. Angewandte Chemie-International Edition 2010, 49 (40), 7281-7283.


Silica

Page 25 of 30 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


(24) Niu, D. C.; Ma, Z.; Li, Y. S.; Shi, J. L. Synthesis of Core-Shell Structured DualMesoporous Silica Spheres with Tunable Pore Size and Controllable Shell Thickness. J Am Chem Soc 2010, 132 (43), 15144-15147. (25) Ashley, C. E.; Carnes, E. C.; Phillips, G. K.; Padilla, D.; Durfee, P. N.; Brown, P. A.; Hanna, T. N.; Liu, J. W.; Phillips, B.; Carter, M. B.; Carroll, N. J.; Jiang, X. M.; Dunphy, D. R.; Willman, C. L.; Petsev, D. N.; Evans, D. G.; Parikh, A. N.; Chackerian, B.; Wharton, W.; Peabody, D. S.; Brinker, C. J. The Targeted Delivery of Multicomponent Cargos to Cancer Cells by Nanoporous Particle-Supported Lipid Bilayers. Nat Mater 2011, 10 (5), 389-397. (26) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Poly(ethylenimine) and Its Role in Gene Delivery. J Control Release 1999, 60 (2-3), 149-160. (27) Park, K.; Lee, M. Y.; Kim, K. S.; Hahn, S. K. Target Specific Tumor Treatment by VEGF SiRNA Complexed with Reducible Polyethyleneimine-Hyaluronic Acid Conjugate. Biomaterials 2010, 31 (19), 5258-5265. (28) Yeom, J.; Bhang, S. H.; Kim, B. S.; Seo, M. S.; Hwang, E. J.; Cho, I. H.; Park, J. K.; Hahn, S. K. Effect of Cross-Linking Reagents for Hyaluronic Acid Hydrogel Dermal Fillers on Tissue Augmentation and Regeneration. Bioconjugate Chem 2010, 21 (2), 240-247. (29) Ito, T.; Iida-Tanaka, N.; Niidome, T.; Kawano, T.; Kubo, K.; Yoshikawa, K.; Sato, T.; Yang, Z. H.; Koyama, Y. Hyaluronic Acid and Its Derivative as a Multi-functional Gene Expression Enhancer: Protection from Non-specific Interactions, Adhesion to Targeted Cells, and Transcriptional Activation. J Control Release 2006, 112 (3), 382-388.



(30) Aruffo, A.; Stamenkovic, I.; Melnick, M.; Underhill, C. B.; Seed, B. Cd44 Is the Principal Cell-Surface Receptor for Hyaluronate. Cell 1990, 61 (7), 1303-1313. (31) Entwistle, J.; Hall, C. L.; Turley, E. A. Receptors: Regulators of Signalling to the Cytoskeleton. J Cell Biochem 1996, 61 (4), 569-577. (32) Zhou, B.; Weigel, J. A.; Fauss, L. A.; Weigel, P. H. Identification of the Hyaluronan Receptor for Endocytosis (HARE). Journal of Biological Chemistry 2000, 275 (48), 3773337741. (33) Alfaiz, A. A.; Muller, V.; Boutry-Kryza, N.; Ville, D.; Guex, N.; de Bellescize, J.; Rivier, C.; Labalme, A.; des Portes, V.; Edery, P.; Till, M.; Xenarios, I.; Sanlaville, D.; Herrmann, J. M.; Lesca, G.; Reymond, A. West Syndrome Caused by Homozygous Variant in the Evolutionary Conserved Gene Encoding the Mitochondrial Elongation Factor GUF1. Eur J Hum Genet 2016, 24 (7), 1001-1008. (34) Gao, Y.; Bai, X.; Zhang, D.; Han, C.; Yuan, J.; Liu, W.; Cao, X.; Chen, Z.; Shangguan, F.; Zhu, Z.; Gao, F.; Qin, Y. Mammalian Elongation Factor 4 Regulates Mitochondrial Translation Essential for Spermatogenesis. Nat Struct Mol Biol 2016, 23 (5), 441-449. (35) Meng, X.; Dai, W.; Zhang, K.; Dong, H.; Zhang, X. Imaging Multiple MicroRNAs in Living Cells using ATP Self-Powered Strand-Displacement Cascade Amplification. Chemical Science 2018, 9 (5), 1184-1190. (36) Wang, W. Q.; Chen, L. F.; Xu, L. P.; Du, H. W.; Wen, Y. Q.; Song, Y. L.; Zhang, X. J. A Free-Blockage Controlled Release System Based on the Hydrophobic/Hydrophilic Conversion of Mesoporous Silica Nanopores. Chem-Eur J 2015, 21 (6), 2680-2685.


Page 26 of 30

Page 27 of 30 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


(37) Cheng, Y.; Jiao, X.; Xu, T.; Wang, W.; Cao, Y.; Wen, Y.; Zhang, X. Free-Blockage Mesoporous Anticancer Nanoparticles Based on ROS-Responsive Wetting Behavior of Nanopores. Small 2017, 13 (40). (38) Lee, M. Y.; Park, S. J.; Park, K.; Kim, K. S.; Lee, H.; Hahn, S. K. Target-Specific Gene Silencing of Layer-by-Layer Assembled Gold-Cysteamine/siRNA/PEI/HA Nanocomplex. Acs Nano 2011, 5 (8), 6138-6147. (39) Zhang, Q.; Padi, S. K. R.; Tindall, D. J.; Guo, B. Polycomb Protein EZH2 Suppresses Apoptosis by Silencing the Proapoptotic MiR-31. Cell Death Dis 2014, 5. (40) Fuse, M.; Kojima, S.; Enokida, H.; Chiyomaru, T.; Yoshino, H.; Nohata, N.; Kinoshita, T.; Sakamoto, S.; Naya, Y.; Nakagawa, M.; Ichikawa, T.; Seki, N. Tumor Suppressive MicroRNAs (miR-222 and miR-31) Regulate Molecular Pathways Based on MicroRNA Expression Signature in Prostate Cancer. J Hum Genet 2012, 57 (11), 691-699. (41) Jin, Y. C.; Xiong, A. W.; Zhang, Z. Y.; Li, S. N.; Huang, H. J.; Yu, T. T.; Cao, X. M.; Cheng, S. Y. MicroRNA-31 Suppresses Medulloblastoma Cell Growth by Inhibiting DNA Replication through Minichromosome Maintenance 2. Oncotarget 2014, 5 (13), 4821-4833. (42) Korner, C.; Keklikoglou, I.; Bender, C.; Worner, A.; Munstermann, E.; Wiemann, S. MicroRNA-31 Sensitizes Human Breast Cells to Apoptosis by Direct Targeting of Protein Kinase C epsilon (PKC epsilon). Journal of Biological Chemistry 2013, 288 (12), 8750-8761. (43) Cao, X.; Qin, Y. Mitochondrial Translation Factors Reflect Coordination between Organelles and Cytoplasmic Translation via mTOR Signaling: Implication in Disease. Free Radic Biol Med 2016, 100, 231-237.



(44) Yang, F.; Gao, Y.; Li, Z.; Chen, L.; Xia, Z.; Xu, T.; Qin, Y. Mitochondrial EF4 Links Respiratory Dysfunction and Cytoplasmic Translation in Caenorhabditis Elegans. Biochim Biophys Acta 2014, 1837 (10), 1674-1683. (45) Schweikl, H.; Petzel, C.; Bolay, C.; Hiller, K. A.; Buchalla, W.; Krifka, S. 2Hydroxyethyl Methacrylate-Induced Apoptosis Through the ATM- and p53-Dependent Intrinsic Mitochondrial Pathway. Biomaterials 2014, 35 (9), 2890-2904. (46) Wang, X. D. The Expanding Role of Mitochondria in Apoptosis. Gene Dev 2001, 15 (22), 2922-2933. (47) A. Hamid Boulares, A. G. Y., Vessela Ivanova, Bogdan A. Stoica, Geping Wang,; Sudha Iyer, a. M. S. Role of Poly(ADP-ribose) Polymerase (PARP) Cleavage in Apoptosis. Journal of Biological Chemistry 1999, 274 (33), 22932–22940. (48) Deng, Z. J.; Morton, S. W.; Ben-Akiva, E.; Dreaden, E. C.; Shopsowitz, K. E.; Hammond, P. T. Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment. Acs Nano 2013, 7 (11), 9571-9584. (49) Ravar, F.; Saadat, E.; Gholami, M.; Dehghankelishadi, P.; Mahdavi, M.; Azami, S.; Dorkoosh, F. A. Hyaluronic Acid-Coated Liposomes for Targeted Delivery of Paclitaxel, Invitro Characterization and In-vivo Evaluation. J Control Release 2016, 229, 10-22. (50) Pan, L. M.; He, Q. J.; Liu, J. N.; Chen, Y.; Ma, M.; Zhang, L. L.; Shi, J. L. NuclearTargeted Drug Delivery of TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles. J Am Chem Soc 2012, 134 (13), 5722-5725.


Page 28 of 30

Page 29 of 30 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


(51) Bai, J.; Liu, Y. W.; Jiang, X. E. Multifunctional PEG-GO/CuS Nanocomposites for NearInfrared Chemo-Photothermal Therapy. Biomaterials 2014, 35 (22), 5805-5813. (52) Vangara, K. K.; Liu, J. L.; Palakurthi, S. Hyaluronic Acid-decorated PLGA-PEG Nanoparticles for Targeted Delivery of SN-38 to Ovarian Cancer. Anticancer Res 2013, 33 (6), 2425-2434.



For TOC only


Page 30 of 30

Stimuli-Responsive Nanocarrier for Co-delivery of MiR-31 and

Recommend Documents