Studying Different Binding and Intracellular Delivery Efficiency of

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Studying different binding and intracellular delivery efficiency of ssDNA -single-walled carbon nanotube and their effects on LC3-related autophagy in renal mesangial cells via miRNA-382 Guobao Wang, Tingting Zhao, Leyu Wang, Bianxiang Hu, Ali Darabi, Jiansheng Lin, Malcolm M.Q. Xing, and Xiaozhong Qiu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b07185 • Publication Date (Web): 01 Sep 2015 Downloaded from http://pubs.acs.org on September 5, 2015

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ACS Applied Materials & Interfaces

Studying Different Binding and Intracellular Delivery Efficiency of ssDNA -single-walled Carbon Nanotube and Their Effects on LC3-related Autophagy in Renal Mesangial Cells via miRNA-382

Guobao Wang,†a,b,c Tingting Zhao,†b Leyu Wang, b Bianxiang Hu,a,b Ali Darabi,c Jiansheng Lin,b Malcolm M.Q. Xing,*c and Xiaozhong Qiu*b a

Division of Nephrology, Nanfang Hospital, Southern Medical University, Key Lab for Organ Failure Research, Ministry of Education, Guangzhou, Guangdong, 510515, China.

b

Department of Anatomy, Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering, Southern Medical University, Guangdong, Guangzhou 510515, China

c

Department of Mechanical, Biochemistry & Medical Genetics, University of Manitoba, and Child Hospital Research Institute of Manitoba, Winnipeg, MB R3T 2N2, Canada. †

Contributed equally to this work

*Corresponding authors: [email protected] (mx), [email protected] (xq)

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Abstract Single walled carbon nanotubes (SWCNTs) have been used to deliver single-stranded (ssDNA). ssDNA in oligonucleotide can act as an inhibitor of microRNA to regulate cellular functions. However, these ssDNA are difficult to bind carbon nanotubes with low transferring efficiency to cells. To this end, we designed ssDNA with regulatory and functional units to form ssDNA-SWCNT hybrids to study their binding effects and transferring efficiency. The functional unit on ssDNA mimics the inhibitor (MI) of miRNA-382, which plays a crucial role in the progress of many diseases such as renal interstitial fibrosis. After verification of over-expression of miRNA-382 in a co-culture system, we designed oligonucleotide sequences (GCG)5-MI, (TAT)5-MI and N23-MI as regulatory units added to the 5’-terminal end of the functional DNA fragment, respectively. These regulatory units lead to different secondary structures and thus exhibit different affinity ability to SWCNTs, and finally decide their deliver efficacy to cells. Autophagy, apoptosis and necrosis were observed in renal mesangial cells.

Key words: single-walled carbon nanotube (SWCNT); single-stranded DNA(ssDNA); autophagy; renal mesangial cell (RMC); miRNA-382; LC3

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Scheme 1. Schematic illustration of the design of ssDNA fragments and the influence of ssDNA-SWCNT hybrids on renal mesangial cells (RMCs).

1. Introduction Small interfering RNA(siRNA) and microRNA(miRNA) are oligonucleotides and have been used as therapeutic effector by inserting them into cells to decrease the mRNA expressions1. microRNAs (miRNAs) are endogenous non-coding RNAs with ~22 nucleotides that negatively control about 30% genes expression in post-transcription2. miRNAs are involved in the modulation of almost all cellular processes. Accordingly, their abnormal expressions are related with a huge majority of human diseases such as breast cancer, renal cell carcinomas, glioblastoma etc3

4 5

. miRNA-382 plays an important role in

the progress of many kinds of diseases, including renal interstitial fibrosis, HIV-1 infection and osteosarcoma6

7 8

. Some studies showed that the high expression of miRNA-382 prompts the cells’

invasion ability and presents its role in the loss of epithelial characteristics. For instance, miRNA-382 expression is significantly elevated in pheochromocytoma and paraganglioma tissues and in the serum of breast cancer patients9

10

. miRNA-382 down-regulates the expression of SOD2, an important enzyme for

the protection against mitochondrial oxidative stress, and induces loss of the epithelial marker, which ultimately results in the development of renal interstitial fibrosis6. Recent research found that miRNA-382 may function as a tumor suppressor and the overexpression of miRNA-382 could be a novel strategy to inhibit tumor metastasis and prevent cancer stem cell-induced relapse in osteosarcoma8.



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However, most ssDNA oligonucleotides have only short half-life due to the digestion of different enzymes in our biological system. Meanwhile, these RNAs can hardly get through cellular membranes and contribute their regulating roles in cellular functions. To this end, quite a few delivery systems have been developed to address these concerns11. Among them, single-walled carbon nanotubes (SWCNTs) are one of a premier candidate due to their high surface-volume ratio, adjustable hydrophobicity and unparalleled electric and mechanical properties. However, the natures of SNCNTs’ hydrophobicity and difficult dispersion compromise their delivery efficacy. Using amphiphilic materials and creating π-π conjugation on SWCNTs can improve these concerns. For example, single-stranded DNA (ssDNA)-SWCNT hybrids can improve the poor nanotube dispersity12, meanwhile, SWCNTs are a vehicle to intracellularly deliver therapeutic agents13. Due to π-stacking between nucleotide bases of nucelotides and nanotube walls, ssDNA can stably bind SWCNT for an efficient transit in vitro and in vivo13

14 15

. As an already known

fact in targeting mRNAs, SWCNT-DNA probe shows better self-delivery capability and intracellular stability compared to free DNA probes13. Besides, SWCNT-siRNA-Murine double minute 2(MDM2) complex could be introduced into breast cancer cells, inhibit the cells growth and induce cell apoptosis by interfering MDM2 gene16. When the sequence-specific ssDNA bound with the appropriate CNT, their binding energy is stable and the CNT displays good electrochemical reaction. Recently, study showed that (TAT)4 forms an ordered right-handed helically wrapped barrel on the (6,5) SWCNT and presents more efficient dispersion ability to SWCNT than (T)12, which formed a left-handed wrap on the (6,5)-SWCNT. It suggests that the DNA secondary structure on nanotubes is important to regulate the properties of the hybrids17. SWCNTs could protect the ssDNA from enzyme digestion when bound together13. The different sequences of ssDNA exhibit different binding strength of ssDNA-SWCNT hybrids. Accordingly, we hypothesise that different secondary structures by designing different sequences of ssDNA influence the DNA-CNT hybrids’ cellular regulating function. In this study, we first tested miRNA-382’s over-expression in co-culture cells of 786-O and BMSC in order to show if miRNA-382 was an important factor in cells’ crosstalking. Herein, we employed a mimic inhibitor complementary to miRNA-382 carried by SWCNTs to regulate human renal mesangial cells (RMCs). We then designed ssDNA fragments composed of two parts: the regulatory DNA and functional DNA (mimic inhibitor of miRNA-382). The regulatory part was a segment of oligonucleotides sequences specially designed for forming the different secondary structure of the DNA. The objective was to study the affinity of ssDNA possessing different secondary structure with SWCNT and their effects on delivery efficacy. The functional part at 3’ terminal was a segment of oligonucleotides designed as mimic inhibitor of miRNA-382. We developed three different regulatory sequences at the 5’ terminal of the mimic inhibitor (Scheme 1). Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were used to observe intracellular delivery of ssDNA-SWCNT and cell morphology. 4 ACS Paragon Plus Environment

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2. Materials and methods 2.1 Cell culture and transwell co-culture system Renal carcinoma cell lines 786-O and mouse Bone Marrow Stromal Cells (BMSCs) were cultured respectively using RPMI1640 medium(GIBCO,USA) and DMEM/F12 medium(GIBCO,USA). Both mediums were supplemented with 10% FBS(GIBCO,USA) and 1% penicillin streptomycin. Cells were incubated at 37℃ in a humidified 95% air/5% CO2 atmosphere and maintained under the same conditions. The mediums were replaced every other day. A transwell co-culture system was used. 786-O cells were seeded in pregelatinised 6 well tissue culture plates. Confluent BMSCs were seeded above transwell 6 well inserts with a pore size of 0.4 µm for incubation. All incubations under the same conditions went on to the seventh day.

2.2 RNA Extraction, Microarray-based miRNA Profiling analysis Total RNA in 786-O cells after been cultured in transwell system or in flask for 7 days were isolated by Trizol reagent kit (Invitrogen) according to the manufacturer’s protocol. miRNA profiling were performed by RIBOBIO company in China. RNA quantity and purity was assessed using NanoDrop K5500. A260/A280 ≥1.5 and A260/A230 ≥1 indicate acceptable RNA purity, while acceptable RIN value ≥5 using Agilent 2200 RNA assay. gDNA contamination was evaluated by gel electrophoresis. To select differentially expressed miRNAs between the two groups, the frequency of miRNAs was normalized to calculate the ratio of Panc-1 to 3T3. A differentially expressed miRNA was indicated by a Panc-1/3T3 ratio>2 and a statistically significant result of t-test with Bonferroni correction.

2.3 Preparation of ssDNA-SWCNT hybrids and the thermodynamics assay The target miRNA-382 sequence is: 5’-GAAGUUGUUCGUGGUGGAUUCG-3’. The sequence of the functional part of ssDNA, which is also known as the mimic inhibitor (MI) of miRNA-382, is:5’GAAGTGCTCGAATCCACCACGAACAACTTC-3’. Then three different oligonucleotides sequences as the regulatory part were designed and added at the 5’-terminal of functional DNA. The three groups of ssDNA were prepared as followings respectively: 5’-(GCG)5-MI-3’(named (GCG)5 -MI); 5’-(TAT)5-MI-3’ (named (TAT)5-MI) and 5’-CAGTGACGGCAATTT-MI-3’(named N23-MI). All three groups were FAM labelled at 5’-terminal for CLSM detection. All of the oligonucleotides were synthesized by SangonBiotech (shanghai, China) Co., Ltd. The secondary structures of ssDNA were predicted on the website of http://kinefold.curie.fr/. The three groups of 200 nM ssDNA and sufficient SWCNT (purity 90%, purchased from Carbon Nanotechnologies) were mixed in aqueous solution respectively, followed by 60 min sonication at a power level of 40 W. The mixture was then centrifuged at 10000g for 30 min to discard the pellet (uncoated



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SWCNT). The supernatant containing DNA-SWCNT compounds were collected and the affinity of DNA and SWCNT were observed. The thermodynamics assay was performed by 100 µL DNA-SWCNT complex and the temperature rose slowly from 4 0C to 90 0C at the rate of 8.6 0C /min.

2.4 The characterization of the DNA-SWCNT hybrids The sizes of (GCG)5-MI-SWCNT and N23-MI-SWCNT hybrids, and surface charge of naked SWCNT, (GCG)5-MI-SWCNT and N23-MI-SWCNT were measured on Malvern Zetasizer apparatus. Atomic Force Microscopy (AFM) measurements were carried out using Nanoscope III atomic force microscope (Digital Instruments Inc.). After centrifugation, the hybrids on supernatant were collected and deposited on mica and AFM imaging was conducted in air at room temperature in contact mode with a resonance frequency of 242 kHz. 2.5 Agarose gel electrophoresis The stability of the ssDNA-SWCNT hybrids was assessed by agarose gel electrophoresis. 10µl samples were loaded to 4% agarose gel with 0.05µg/µL ethidium bromide in a TAE buffer (pH 7.4). The ssDNASWCNT hybrids were run down at 60 V for 20 min. The heparin (1000 units of heparin per µg ssDNA) was added to dissociate the ssDNA from the hybrids. The fluorescent signal intensities were measured and analyzed using FluorChem Q Manager Software (Alpha, USA). 2.6 Cell viability assay The ssDNA sequence, 5’-CAGTGACGGCAATTT-(NNN)3-GCGTAT-3’, was designed as random ssDNA sequence(RAD-oligo, N represents as a random nucleotide). The ssDNA-SWCNT hybrids was synthesized and characterized according to the mentioned methods in manuscript. Cell viability was measured by a Cell Counting Kit-8 system (CCK-8) according to the manufacturer's instruction (Dojindo, Japan). Briefly, cells were seeded in 96-well plates at 1×104 cells per well and cultured overnight. Then, cells were treated with ssDNA-SWCNT hybrids at different concentrations (50 nM, 100 nM, 200 nM, 300 nM) for 24hrs. After that, 10µL of CCK-8 solution was added and incubated for 1 h at 37 0C The absorbance was measured at 450nm using a microplate reader (Bio-Rad 680, Hercules, CA, USA). All data were presented as mean±SEM in triplicate compared to the OD values of untreated cells.

2.7 Cell culture and confocal laser scanning microscopic observation Renal mesangial cells (RMC) were cultured using low glucose DMEM medium (GIBCO, USA). The medium was supplemented with 10% FBS (GIBCO,USA) and 1% penicillin streptomycin. Cells were incubated at 37℃ in a humidified 95% air and 5%CO2 atmosphere. The medium was replaced every other day. RMCs were seeded on glass covers lips and placed in a 24-well plate. The different structures of FAM-labelled ssDNAs and their SWCNT complexes were added with a 200 nM final concentration of ssDNAs when they reached confluence of 80%. After 24 hours, the cover slips were washed with PBS for thrice. Subsequently, 200 nM red fluorescence mitochondrion tracker was added. After 30 min, the cells were incubated with DAPI to stain nuclei and then fixed with 4% paraformaldehyde overnight. The FAM6 ACS Paragon Plus Environment

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DNA (green), mito-tracker (red) and DAPI (blue) were excited at 488 nm, 568 nm and 358 nm using confocal laser scanning microscopy(CLSM) (FV-1000, Japan) respectively.

2.8 Transmission electron microscopy (TEM) For TEM observation, RMCs were incubated with ssDNA (200 nM) or ssDNA-SWCNT (200 nM of DNA) in media for 24 h, then the cells were collected and fixed in 2.5% glutaraldehyde for 4 h at 4℃. The cell pellets were rinsed with PBS and post-fixed in osmic acid, then were washed in DI water and dehydrated in graded acetone. The samples were embedded in epoxy resin. Fifty nanometers ultrathin sections were double stained with uranyl acetate and lead citrate. The sections were then for TEM imaging (H-7500, Japan).

2.9 Immunofluorescence staining assay and confocal laser scanning microscopic observation Immunofluorescence staining assay was performed to observe the nuclear translocation of apoptosis-inducing factor (AIF) and expression of microtubule-associated protein 1 light chain 3 (LC3) in RMCs treated by different ssDNA fragments and their SWCNT complexes. The cells seeded on the coverlips were treated with ssDNAs and ssDNAs-SWCNT hybrids for 24 hours. Then the samples were washed by PBS and fixed by 4% paraformaldehyde overnight at 4 0C. The fixed samples were then rinsed with PBS and permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. After blocking with 1% bovine serum albumin (BSA) at room temperature for 1 hour, samples were incubated in mouse anti-AIF antibody (1 : 100, Santa Cruz), rabbit anti-LC3 antibody (1 : 200, Cell Signaling), overnight at 4°C respectively. Then samples were washed by PBS for 3 times and incubated in in Alexa Fluor® 568 Donkey Anti-Rabbit IgG (H + L) (1 : 500) and Alexa Fluor®568 Donkey Anti-Mouse IgG (H + L) (1 : 500) for 1 hour. The samples were rinsed with PBS, further stained with DAPI for 1 hour and then imaged using a confocal microscope.

2.10 Western blotting Whole proteins were isolated by RIPA buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% SDS, 1% NP-40, 0.25% Sodium deoxy-cholate and 1 mmol/L EDTA). Total protein concentrations were determined by BCA Protein Assay (KeyGEN). Proteins (20 µg/lane) were separated on 12% SDS-PAGE and electrophoretically transferred to PVDF membranes. The membranes were blocked for 2 h at room temperature in TBS-T buffer (10 mmol/L Tris·HCl, pH 7.5, 500 mmol/L NaCl, 0.05% Tween 20), containing 5%(w/v) skim milk, and incubated antibodies against AIF (Santa Cruz), LC3 (Abcam) and βactin (Tianjin Sungene) at 4°C overnight. After incubation with the primary antibody, the membranes were washed three times with TBS buffer and incubated with the appropriate HRP-linked secondary antibodies for 1 h at room temperature. The optical density was determined using the Supersignal Chemiluminescent



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substrate (Invitrogen) according to the manufacturer’s instructions. Chemiluminescence signals were captured by autoradiography and were used to assess protein content.

2.11 Statistical analysis All data was reported as means ± SE. Statistical significance of differences among means was determined by analysis of variance (ANOVA) with post hoc comparison of more than 2 means by the Bonferroni method using SPSS13.0 software. Values of P less than 0.05 were considered significant.

3. Results and discussion 3.1 Overexpression of miRNA-382 in co-cultured cells As shown in Fig. 1, we found that the expression level of miRNA-382 was higher in co-cultured cells of 786-O and BMSC than their single cultured cells of 786-O and BMSC, respectively. The results indicated that miRNA-382 played a critical role in cells’ crosstalking. Hence, we designed ssDNA composed of two parts-the functional unit and the regulatory unit to investigate the affinity ability of ssDNA and SWCNTs and their effects on delivery efficacy and cytotoxicity.

and dispersibility and thus came out in the supernatant. Dynamic light scattering (DLS) further verified that the centralized distribution of (GCG)5-MI-SWCNT and N23-MI-SWCNT hybrids treated with 8 ACS Paragon Plus Environment

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ultrasonication with a single peak at the sizes about 203nm (Fig. 2a) and 57nm (Fig. 2b) respectively. These results indicated that different kinds of ssDNA fragments could wrap on SWCNTs with different efficiency. As reported, ultrasonic dispersing technology could endow SWCNT water dispersions and biocompatibility, meanwhile, ssDNA fragment could be easily absorbed on the surface of SWCNTs by the π-π stacking18

19

. The significant increased surface charge of (GCG)5-MI-SWCNT and N23-MI-SWCNT

hybrids compared with the SWCNTs alone (n=3, P N23-MI > (TAT)5-MI > H2O. The result suggested the thermodynamic stability of ssDNA-SWCNT hybrids at 4℃ in the solution, while degraded after being heated to 90 ℃ .


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Fig. 3. The thermodynamics stability and secondary structures of three ssDNA fragments (a). a1: The thermo dynamic stability of three groups of ssDNA fragments; Lane 1: Double distilled water; Lane 2: N23-MI-SWCNT hybrids; Lane 3: (GCG)5-MI-SWCNT hybrids; Lane 4: (TAT)5-MI-SWCNT hybrids. The upper row was in 4℃ and the lower row was in 90℃; (GCG)5-MI, N23-MI and (TAT)5-MI indicated the secondary structures of different ssDNA fragments respectively. The influence of FAM labelled ssDNA fragments and their related SWCNT hybrids on renal mesangial cells (RMCs) detected by CLSM (b~h). Scale bar=5µm. ssDNA fragments labelled by FAM(green), mitochondria marked by mito-tracker(red), cell nuclei marked by DAPI(blue). b: The control group. c & d: The N23-MI and N23-MI-SWCNT hybrids respectively. e & f: The (GCG)5-MI and (GCG)5-MISWCNT hybrids respectively. g & h: The (TAT)5-MI and (TAT)5-MI-SWCNT hybrids respectively.

3.4 The (GCG)5-MI-SWCNT, N23-MI-SWCNT and (TAT)5-MI-SWCNT hybrids leading to different degree of cell damages In the control group, after treated with double distilled water alone, the cells appeared in normal status with integrated cell shape, distinct boundary of cell membrane and nuclear envelope (Fig. 3b). From the TEM images, the cell boundaries were undamaged and nuclei displayed intact shape and uniform chromatin (Fig. 4a). These results indicated no cell damage resulted from double distilled water. In the experimental groups, after treated with N23-MI ssDNA alone, the cells were found in normal status with intact cell shape, smooth edge of cell membrane and nuclear envelope (Fig. 3c). All organelles observed to be in normal status (Fig. 4b). However, when bound N23-MI ssDNA fragments with SWCNTs, a large amount of green fluorescent N23-MI ssDNA fragments aggregation were found within the cells and a minority passed through the nuclear envelops. The cells and nuclei were swollen with intact cell shape,



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smooth edge of cell membrane and nuclear envelope (Fig. 3d). From TEM, substantial N23-MI-SWCNT hybrids present intracellular (Fig. 4c) and massive multi-membrane autophagosomes appeared in the cytoplasm (Fig 4.d). These results suggested the N23-MI-SWCNT hybrids treatment could induce cell autophagy. After treated with (GCG)5-MI ssDNA alone, only little FAM-labelled (GCG)5-MI (green) appeared in cytoplasm with intact cell shape, smooth edge of cell membrane and nuclear envelope (Fig. 3e). All organelles observed to be in normal status (Fig. 4e). However, when bound with SWCNTs, a large amount of fluorescent (GCG)5-MI were found within the cells and nuclei with atrophied cells, karyopyknosis, and obscure edge of nuclei (Fig. 3f). The cellular normal morphology disappeared and atrophied cells appeared in TEM images (Fig. 4f). Typically, the cell atrophy and karyopyknosis were reported to be the classical presentation of cell apoptosis21

22

. These results revealed (GCG)5-MI-SWCNT hybrids treatment could

induced cell apoptosis. After treated with (TAT)5-MI ssDNA fragments alone, no green fluorescent were found in cells. The cells looked as if in normal status with intact cell shapes and smooth membranes (Fig. 3g and Fig. 4g). When bound with SWCNTs, different from the (GCG)5-MI and N23-MI, only a little (TAT)5-MI could be detected within cells, the cell shape and nuclear condition presented no obvious changes (Fig. 3h and Fig. 4h ).

Fig. 4. The influence of FAM labelled ssDNA fragments and their related SWCNT hybrids on RMCs detected by TEM. Scale bar=500nm. (a): The controlled group. (b): The N23-MI ssDNA fragment. (c & d): The N23-MI-SWCNT hybrids. Red arrow indicating N23-MI-SWCNT hybrids (c) and autophagosomes (d). (e & f): The (GCG)5-MI ssDNA fragment and (GCG)5-MI-SWCNT hybrids separately. Red arrow indicating atrophied cell (f). (g & h): The (TAT)5-MI ssDNA fragment and (TAT)5-MI-SWCNT hybrids separately. To further elucidate the significances of the cell morphology changes and verify the occurrence of cell apoptosis or autophagy after RMCs treated with ssDNA-SWCNT hybrids, immunofluorescence assay was performed to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and the nuclear 12 ACS Paragon Plus Environment

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translocation of apoptosis-inducing factor (AIF). AIF, a marker of cell apoptosis, which is normally confined to mitochondria but translocates to cytoplasm and nucleus when cell apoptosis is induced in response to stimuli23, was labelled to mark apoptosis. LC3, localizing to autophagosomal membranes after post-translational modifications, which has been used as a specific marker to monitor autophagy24

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, was

also traced to verify whether or not autophagy occurred. After treated with double distilled water alone, cells showed in normal status with red fluorescent (AIF positive staining) confined to mitochondria and no obvious green fluorescent spots discovered (Fig. 5a, b and c). After treated with SWCNTs alone, the same as double distilled water treatment, cells displayed normal status with intact cell shape, distinct cell boundary and uniform chromatin. The red fluorescent (AIF positive staining) was confined to mitochondria and no obvious green fluorescent spots discovered (Fig. 5d, e and f). After treated with N23-MI alone, cells showed in normal status with red fluorescent (AIF positive staining) confined to mitochondria and no obvious green fluorescent spots were discovered (Fig 5g, h and i). In contrast, after treated with N23-MI-SWCNT hybrids, cells presented to be swollen with red fluorescent (AIF positive staining) confined to mitochondria and predominant aggregations of LC3 positive green fluorescent spots (autophagosomes) around the swollen nuclei (Fig. 5j, k and l). Red fluorescent (AIF positive staining) distributed uniformly within mitochondria and rarely within the nuclei both in N23-MI alone and N23-MI-SWCNT hybrids treated cells. These results showed that N23-MISWCNT hybrids could only induce autophagy in RMCs. However, there were no cell apoptosis or necrosis in RMCs induced by N23-MI-SWCNT hybrids. After treated with (GCG)5-MI alone, cells displayed with intact cell shape, smooth edge of cell membrane and nuclear envelope. Red fluorescent (AIF positive staining) was observed to be confined to mitochondria, and no green fluorescent spots was found (Fig 5m, n and o). After treated with (GCG)5-MISWCNT hybrids, distinct from (GCG)5-MI alone, atrophied cells were found with massive red fluorescent (AIF positive staining) locating to mitochondria and also within the atrophied nuclei, which verified the cell apoptosis induced by (GCG)5-MI-SWCNT hybrids. Meanwhile, cells seem to be in normal status were also found with substantial aggregations of LC3 positive green fluorescent spots (autophagosomes) around the nuclei (Fig 5p, q and r). The expression level of LC3-Ⅱ protein in (GCG)5-MI-SWCNT hybrids treated cells was significant higher than the other three groups (H2O, SWCNT and (GCG)5-MI ) through western blotting analysis, which further verified the cell autophagy induced by (GCG)5-MI-SWCNT hybrids (Fig. 6A). Besides, after treated with (GCG)5-MI ssDNA alone, the cells appeared in normal status with intact cell shape, smooth edge of cell membrane and nuclear envelope (Fig. 6B1), however, after treated with its hybrids, the cell shape disappeared and karyorrhexis appeared in TEM images (Fig. 6B2), in addition, cell nuclei disintegration could also be observed and scattered nuclear fragments (DAPI marked blue nuclei



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components) even leaked out of cytoplasm in CLSM images (Fig. 6C), which strongly indicated cell necrosis induced by (GCG)5-MI-SWCNT hybrids. Accordingly, these results verified the occurrence of autophagy, apoptosis and necrosis induced by (GCG)5-MI-SWCNT hybrids in RMCs. The immunofluorescence images confirmed that no obvious difference in cell morphology and fluorescent presentation between (TAT)5-MI alone treated groups (Fig 5s, t and u) and (TAT)5-MISWCNT hybrids treated groups (Fig 5v, w and x). In both two groups, the cells presented to be in normal sizes, with red fluorescent (AIF positive staining) confined to mitochondria and no obvious green fluorescent spots aggregated around the cell nuclei. Above all, these results revealed that (GCG)5-MI alone had little influence on RMCs for it failed to across the cell membrane. However, when bound with SWCNT, it could be delivered efficiently into cytoplasm and induce LC3-related cell autophagy, even apoptosis and necrosis via inhibition of miRNA382 function in RMCs. N23-MI-SWCNT hybrids could only induce autophagy in RMCs, neither cell apoptosis nor necrosis, probably due to the aggregation of N23-MI ssDNA fragments in cytoplasm and the less quantity of N23-MI ssDNA fragments passed through the nuclear envelops compared with the (GCG)5MI-SWCNT hybrids treated cells. However, no obvious cell damages were detected after being treated with (TAT)5-MI ssDNA alone and (TAT)5-MI-SWCNT hybrids respectively.

Fig. 5. The immunofluorescence images of the influence of ssDNA fragments and their related SWCNT hybrids on RMCs detected by CLSM. Scale bar=5µm. Blue displayed cell nuclei marked by DAPI. Red indicated AIF proteins. Green indicated LC3 proteins. (b, e, h, k, n, q, t, w) were differential interference contrast (DIC) images. (a & b): The double distilled water; (c) merge image of a and b. (d & e) : The SWCNT alone; (f): merge image of d and e; (g & h): The N23-MI ssDNA fragment alone; (i) merge image of g and h. ( j & k): The N23MI-SWCNT hybrids; (l): merge image of j and k; (m & n): The (GCG)5-MI ssDNA fragment alone; (o): merge image of m and n; ( p & q): The (GCG)5-MI-SWCNT hybrids; (r): merge image of p and q; ( s & t ): The (TAT)5MI ssDNA fragment alone; (u): merge image of s and t; (v & w): The (TAT)5-MI-SWCNT hybrids; (x): merge image of v and w. 14 ACS Paragon Plus Environment

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Fig. 6. The influence of (GCG)5-MI and (GCG)5-MI-SWCNT hybrids on RMC cells. (a) Western blotting analysis of AIF and LC3 after RMCs were challenged with double distilled water, SWCNT, (GCG)5-MI and (GCG)5-MI-SWCNT hybrids respectively for 24hour; (b) The influence of (GCG)5-MI-SWCNT hybrids on RMCs detected by transmission electron microscopy (TEM). Scale bar=2µm. b1: The (GCG)5-MI ssDNA fragment treatment. b2: The (GCG)5-MI-SWCNT hybrids treatment. (c) The influence of (GCG)5-MI-SWCNT hybrids on RMCs detected by confocal laser scanning microscopy (CLSM). Scale bar=10µm. Cell nuclei marked by DAPI (blue), ssDNA fragments labelled by FAM (green), mitochondria marked by mito-tracker (red). DIC: differential interference contrast images. Merge means merge image of DAPI, FAM, mito-tracker and DIC.

4. Conclusion Our study designed three groups of ssDNAs composed of identical functional sequence (MI of miRNA382) and different regulatory sequences to form distinct secondary structures and investigate their influence on RMCs. The (GCG)5-MI-SWCNT and N23-MI-SWCNT hybrids could induce LC3-related autophagy via inhibition of miRNA-382 function in renal mesangial cells, the former even can induce cell apoptosis and necrosis, while (TAT)5-MI-SWCNT hybrids hardly had influence on RMCs. The results proved that different secondary structures of ssDNA fragments possessed different affinity ability to SWCNTs and permeability through cell barriers. Merely different secondary structures caused by different ssDNA sequences can influence the stability and penetrability of ssDNA-SWCNT hybrids. The SWCNTs were non-toxic and the toxic property induced by ssDNA-SWCNT hybrids could be controlled by altering the sequences of the regulatory area of ssDNA fragments.

ASSOCIATED CONTENT Supporting Information Available:



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Fig. S1. The ssDNA dispersion ability to SWCNT. Scale bar= 100 nm; Fig. S2. The stability of N23-MISWCNT, (GCG)5-MI-SWCNT and (TAT)5-MI-SWCNT hybrids(A) and the cell toxicity of random ssDNA-SWCNT hybrids in renal mesangial cells assessed by CCK-8 analysis (B). This material is available free of charge via the Internet athttp://pubs.acs.org.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31371000, 31572343, 51428301 and 31340002) and Guangdong Natural Science Foundation (S2013010016032), Canada NSERC Discovery Grant and CIHR RPP.

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Table of Content Graphic (TOC graphic)


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Different regulatory ssDNA sequence exhibit different affinity ability to SWCNTs leading to different fates of cells


Studying Different Binding and Intracellular Delivery Efficiency of

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