Antisense Oligonucleotide-conjugated Nanostructure Targeting

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Letter

Antisense Oligonucleotide-conjugated Nanostructure Targeting LncRNA MALAT1 Inhibits Cancer Metastasis Ningqiang Gong, Xucong Teng, Jinghong Li, and Xing-Jie Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18288 • Publication Date (Web): 14 Dec 2018 Downloaded from http://pubs.acs.org on December 15, 2018

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ACS Applied Materials & Interfaces

Antisense Oligonucleotide-conjugated Nanostructure Targeting LncRNA MALAT1 Inhibits Cancer Metastasis Ningqiang Gonga,b,c,d, Xucong Tenga, Jinghong Lia,* and Xing-Jie Liangb,c,d,* aDepartment

of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, P.R. China. bChinese

Academy of Sciences (CAS) Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, P.R. China. cLaboratory

of Controllable Nanopharmaceuticals, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, P.R. China. dUniversity

of Chinese Academy of Sciences, Beijing 100049, P.R. China.

*Corresponding authors, Email: [email protected]; [email protected]

Abstract: Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long noncoding RNA (lncRNA) located in the cell nucleus, is a critical regulator of tumor cell migration. Antisense oligonucleotides (ASOs), which can downregulate the expression level of specific RNAs, have been used in clinical for diseases treatment. Herein, we constructed MALAT1-specific ASO and nucleus-targeting TAT peptide co-functionalized Au nanoparticles, namely ASO-Au-TAT NPs, which stabilized the fragile ASOs, enhanced nuclear internalization, and exhibited good biocompatibility. After treated with the ASO-Au-TAT NPs, A549 lung cancer cells showed a greatly reduced MALAT1 expression level and decreased migration ability in vitro. Moreover, the ASO-Au-TAT NPs significantly reduced metastatic tumor nodules formation in vivo. Our results demonstrate that the ASO-Au-TAT NSs have great potential for treatment of cancer metastasis. Keywords: long non-coding RNA, nanostructure, MALAT1, cancer metastasis, gene therapy

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Metastasis is the major cause of cancer relapse and cancer-related mortality.1-3 Presently, surgical resection and chemotherapy are the two most preferred strategies for treating metastatic cancers, but often associated with poor prognosis. 4-6

Therefore, more efficient strategies for metastatic cancer treatment are urgently

needed. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), actively regulates the expression level of many metastasis-related genes.7-10 MALAT1-deficient lung cancer cells are impaired in the formation of lung metastatic foci compared with their wild-type counterparts in vivo, which means that MALAT1 is a potential target of gene therapy for malignant cancers.11-14 However, it is difficult to silence MALAT1 in vivo, because it is located in the cell nucleus and protected by the nucleus envelope.15-16

Scheme 1. Schematic illustration of nucleus-targeting ASO-Au-TAT nanostructures for MALAT1 silencing. TAT promotes nucleus-specific transport of ASO-Au-TAT nanostructures, and then ASO hybridizes to MALAT1 and activates RNase H-dependent RNA degradation.


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Antisense oligonucleotides (ASOs) can downregulate the expression level of specific RNAs within cells. An ASO hybridizes to its complementary RNA to form a DNA–RNA hetero-duplex and triggers RNase H-mediated RNA degradation.14,15 Moreover, ASOs can be chemically modified so that they are resistant to intracellular DNase and do not attenuate the activity of RNase H.17-18 These features make ASOs promising in regulation of nucleus RNA for therapeutic applications. Recently, one ASO-based gene therapy drug, Mipomersen, in which the terminal ribose groups are modified with 2’-O-meth oxyethyl groups, has been approved for treatment of homozygous familial hypercholesterolemia.19 The poor cell membrane permeability and lacking of nucleus-targeting of ASOs restricted their wide application in silicing nucleus RNAs.20 Au nanoparticles (Au NPs) have been used for delivery applications as they are chemically inert, biocompatible and easy of functionalization.21-23 Recently, many DNA-Au NSs have been developed.24-27 DNA-Au composites show enhanced enzyme resistance, cellular uptake and degradation of a cytoplasmic RNA.28 In addition, multiplex ligands can be integrated into a single Au NP to increase the delivery efficiency. As shown in Scheme 1, an antisense oligonucleotide-loaded nanostructure (ASO-Au-TAT) was constructed by coating 2 nm Au NPs with ASOs as well as nucleus-targeting TAT peptide. TAT peptide can transport the cargo across the nuclear pore complex (NPC),29 and after entering the nucleus, the ASO recognizes and binds with MALAT1, and then the resulted RNA-DNA hetero-duplex will be degraded by RNase H.



Figure. 1 TEM image (A), size distribution histogram (B), Zeta potential (C) and UV-Vis spectrum (D) of ASO15-Au-TAT7.5 NSs. (E), In vitro cytotoxicity of Au NPs, ASO15-Au NPs, ASO15-Au-TAT7.5 NSs, ASO15-Au-TAT15 NSs, ASO7.5-Au-TAT15 NSs and Au-TAT15 NPs at various ASO concentrations (0.61, 1.25, 2.5, 5, 10 and 20 nM). (F), Average number of Au NPs in the nuclei after A549 cells treated with Au NPs, ASO15-Au NPs, ASO15-Au-TAT7.5 NSs or ASO15-Au-TAT15 NSs (at ASO concentration of 20 nM) for 2, 4, 6, 8, 10 and 24 h, determined using ICP-MS. We synthesized a series of Au NSs with different ASO:Au:TAT ratio (Tab. S1, Figure. S1). And we then optimized the ASO:Au:TAT ratio of these NSs considering the cell viability (Figure. 1E) and nucleus-targeting efficiency (Figure. 1F). Finally, ASO15-Au-TAT7.5 nanostructure was selected for further study as their relatively non-toxic and nucleus-targeting delivery nature (detailed description and discussions please see supporting information). Transmission electron microscope


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(TEM) image (Figure. 1A) and Dynamic Light Scattering (DLS) (Figure. 1B) results showed that ASO15-Au-TAT7.5 NSs exhibited spherical morphology, with a average diameter of about 3.08 nm and with a negative charge of about -32.4 mV (Table S1). UV-Vis spectrum (Figure. 1D) of the NSs showed no obvious peak above 500 nm, indicating that the NSs were monodispersed in water and without aggregation.

Figure. 2 (A) Subcellular localization of ASO15-Au-TAT7.5 nanostructures visualized by laser scanning confocal microscope. A549 cells were treated with PEG2000-FITC labelled ASO15-Au NPs or ASO15-Au-TAT7.5 nanostructures with an ASO concentration of 20 nM. After 24 h, the cells were fixed and a nuclear speckle marker antibody was employed to profile the nuclear speckles. (B) Line-scan profiles of the fluorescence intensity of ASO15-Au NPs and ASO15-Au-TAT7.5 nanostructures. (C) Average fluorescence intensity in the nucleus and cytoplasm of cells treated with ASO15-Au-TAT7.5 nanostructures or ASO15-Au NPs (100 cells were analysed in each group by Image J software). (D) MALAT1 expression level was measured by RT-qPCR. A549 cells were incubated with non-sense DNA, free ASO, ASO15-Au or ASO15-Au-TAT7.5 at a concentration of 20 nM for 24 h, before total RNA was extracted and analysed.



Cellular uptake of ASO15-Au-TAT7.5 NSs was investigated using ICP-MS, A549 cells were incubated with ASO15-Au-TAT7.5 or ASO15-Au NPs for 24h and cells were digested and average Au atom number in the cells was measured using ICP-MS. As shown in Figure S2, the ASO15-Au-TAT7.5 NSs showed about 9-fold higher cell internalization than the Au NPs with no TAT functionalization. Cellular distribution of ASO15-Au-TAT7.5 NSs was investigated by laser scanning confocal microscopy (LSCM). PEG2000-FITC (10 % of the total number of ASOs and TAT peptides) was conjugated onto the surface of NSs to make them visible by LSCM.30 A549 cells were incubated with ASO15-Au NPs or ASO15-Au-TAT7.5 NSs (with an ASO concentration of 20 nM) for 24 h, and then the cells were fixed. And the nuclear speckles were profiled with a Cy5 conjugated antibody. LSCM images showed that the fluorescence intensity of cells treated with ASO15-Au-TAT7.5 NSs was much higher than that of cells treated with ASO15-Au NPs (Figure. 2). This meant that TAT had great membrane-penetration capacity. ASO15-Au NPs were located mainly in the cytoplasm (Figure. 2A), whereas ASO15-Au-TAT7.5 NSs were located mostly in the nucleus. Line-scan profiles (Figure. 2B) and integral fluorescence intensity analysis (Figure. 2C) further demonstrate that about 67 % of ASO15-Au-TAT7.5 NSs were located in the nucleus while only 11 % of ASO15-Au NPs were located in the nucleus (statistical analysis was performed on 100 cells from each group). The overlapping fluorescence from the nuclear speckles and the ASO15-Au-TAT7.5 NSs indicates that ASO was successfully transported into the nucleus. Though the cytoplasm still showed strong fluorescence at 24h, we found


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that the accumulation of ASO15-Au-TAT7.5 NSs in the nuclear is time-dependent. ASO15-Au-TAT7.5 NSs locates mainly at the nucleus after the cells were incubated with ASO15-Au-TAT7.5 for 36 and 48 h (Figure S3). Bio-TEM was used to further investigate the distribution of the nanoparticles in cells. As shown in Figure S4, ASO15-Au NPs were located mostly in cytoplasm and fewer were observed in the nucleus, while ASO15-Au-TAT7.5 NSs were located mainly in the nucleus with fewer in the cytoplasm. Only a small amount of ASO15-Au NPs entered the nucleus, which could be ascribed to disassembly of the nuclear envelope during cell division, or the smaller size of the NSs compared with the NPC (about 9 nm).

31-32

Both

LSCM and bio-TEM data suggest that the nucleus-targeting ASO15-Au-TAT7.5 nanoparticles are able to efficiently transport the ASO to target MALAT1. To the best of our knowledge, this is the first evidence that distribution of TAT peptide-functionalized ASO-Au NPs can reach to nuclear speckles. This nucleus-targeting strategy may be used for regulation of nuclear speckle-related functions in the future. To investigate the MALAT1 silencing efficiency of ASO15-Au-TAT7.5 NSs, reverse transcription qPCR (RT-qPCR) was performed. A549 cells were incubated with non-sense DNA, ASO, ASO15-Au or ASO15-Au-TAT7.5 NSs with an equal ASO concentration of 20 nM for 24 h, and then the total RNA was extracted. Figure. 2D showed that free ASO, ASO15-Au and ASO15-Au-TAT7.5 NSs reduced MALAT1 expression level by about 49.4 %, 31.3 % and 81.4 %, respectively, whereas non-sense DNA treatment had little effect on the MALAT1 expression level. Free



ASO was less effective than ASO15-Au-TAT7.5, which may be ascribed to attenuated cell uptake and lack of nuclear specificity.33 As nanoparticles can promote cellular uptake of DNA, the ASO15-Au NPs did show a degree of silencing ability. On the other hand, as the NPs are much smaller than the NPC, NPs can passively diffuse into the nucleus.

34

The highest MALAT1 silencing efficiency was

observed by the group treated with ASO15-Au-TAT7.5 NSs, indicating that ASO15-Au-TAT7.5 NSs had higher nucleus-specific delivery efficiency than the other groups, which was consistent with the ICP-MS data. Efficient downregulation of the MALAT1 expression level is crucial for effective inhibition of cell migration ability. The above results demonstrate that the ASO can effectively recognize and bind to MALAT1 when conjugated to Au NPs. And the ASO15-Au-TAT7.5 NSs are efficient in degradation of MALAT1 in vitro.


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Figure. 3 Scratch wound healing assay. Representative images from three independent experiments are shown here. (A) After cell monolayers were scratched, cell migration in the wound areas was observed for 24 and 48 h. (B) Statistical analysis of wound closure. Gap length at 0 h was set to 100 % and the percentage of gap closure was determined 24 h and 48 h after scratching. The efficiency of ASO15-Au-TAT7.5 NSs in inhibiting migration of tumour cells was evaluated. A scratch wound healing assay was conducted.19

A549 cells were

seeded in a 24-well plate. After the cells grew to be confluent, the cell monolayer was scratched with a blunted needle. The wounded monolayer was treated with serum-free culture medium containing non-sense DNA, ASO15-Au NPs, ASO or ASO15-Au-TAT7.5 NSs with an ASO concentration of 20 nM and an equivalent concentration of Au NPs for 24 h and 48 h. Migration of the treated cells was



tracked using a microscope. As shown in Figure 3, control group and the groups treated with Au NPs, ASO15-Au NPs and non-sense DNA achieved almost complete closure of the gap after 48 h. ASO and ASO15-Au-TAT7.5 NSs treatments reduced the gap closure by 69.3 % and 88.7 % respectively after 24 h, and 57.2 % and 81.4 % respectively after 48 h. To further confirm the anti-migration ability of ASO15-Au-TAT7.5 NSs, a transwell-based cell migration assay was performed. 35 As shown in Figure. S5, Au NPs and non-sense DNA showed no notable effect on migration ability compared with the control group, whereas the ASO15-Au, ASO and ASO15-Au-TAT7.5 NSs treatments reduced cell migration by 14.8 %, 35.3 % and 76.1 %, respectively. It has been demonstrated that MALAT1-deficient tumour cells were disabled in the metastatic cascade.

10

The above investigations suggest that

impairment of MALAT1 function by ASO15-Au-TAT7.5 NSs effectively disabled the ability of A549 cells to migrate. ASO15-Au-TAT7.5 NSs were more effective than free ASO at inhibiting cancer cell migration, which can be ascribed to the increased cellular uptake, nucleus targeting effect and ASO stabilization effect mediated by NSs. All these factors ultimately contribute to the ability of ASO15-Au-TAT7.5 NSs to effectively silence MALAT1 and inhibit the migratory properties of A549 cell. Thus, ASO15-Au-TAT7.5 NSs are an effective ASO NSs that can penetrate the nucleus and cause MALAT1 silencing as well as reducing cell migration. This NSs provides a promising strategy for controlling tumour metastasis. Finally, we evaluated the anti-metastasis efficacy of ASO15-Au-TAT7.5 NSs in a metastatic lung cancer model. Firstly, we tested that the ASO15-Au-TAT7.5 NSs has


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good blood biocompatibility that caused no hemolysis of mouse red blood cells (Figure S6) when mix the NSs with mouse blood cells. Mice lung cancer metastatic model was constructed by intravenously (iv) injection of 1 × 106 A549 cells. After that, mice were iv injected with ASO or ASO15-Au-TAT7.5 NSs (with equivalent 100 μg ASO/mice) for 3 times at 3 days’ interval. Mice were sacrificed at day 33 and the lungs were resected for analysis of the formation of lung tumour nodules. As shown in Figure. 4A, the tumour burden was reduced when mice were treated with ASO or ASO15-Au-TAT7.5 NSs, compared with untreated mice. Evidently, mice treated with ASO15-Au-TAT7.5 NSs showed fewer tumour nodules in vivo than the mice treated with

free

ASO,

indicating

that

ASO15-Au-TAT7.5

NSs

had

the

highest

anti-metastasis activity. These observations were consistent with the histological sections stained with hematoxylin and eosin (H&E) (Figure. 4B). For mice injected with ASO15-Au-TAT7.5 NSs, lungs sections stained with H&E showed no obvious growth of tumour cells. It was previously reported that free ASO exhibited limited anti-metastasis capacity in vivo even at very high dose,10 which may be explained by the low stability of ASO and poor penetration into the nucleus. In contrast, ASO15-Au-TAT7.5 NSs can protect ASO from enzyme degradation, improve the nuclear localization efficiency and increase the overall delivery of ASO into the nucleus, which would result in effective inhibition of tumour cell migration, thereby preventing cancer metastasis.



Figure. 4 (A) Formation of lung cancer tumor nodules. Metastatic tumor model was obtained by tail vein injection of A549 cells (1 × 106) at day 0. ASO or ASO15-Au-TAT7.5 nanostructures were iv injected to the mice with equivalent 100 μg ASO per mice for three times at three days’ interval (day 2, day 5 and day 8), and formation of lung tumor nodules on mouse lungs was investigated. The lungs were isolated at day 33, and the tumor area was investigated. Representative pictures of the lungs from each group are shown. Arrows indicate tumor nodules. (B) Histological sections of mice lungs stained with hematoxylin and eosin (H&E) (scale bar, 100 μm). (C) Survival analysis of mice after injection of saline, ASO or ASO15-Au-TAT7.5 nanostructures.

Animal survival was also analysed to evaluate the therapeutic effect of these NSs (Figure 4C). At day 33, the survival rates of the mice treated with ASO or ASO15-Au-TAT7.5 NSs were, 40 % and 100 %, respectively. Mice had a much higher survival rate after injection of ASO or ASO15-Au-TAT7.5 NSs, compared to the untreated mice. Mice treated with free ASO only showed a limited increase in survival. As demonstrated above, ASO15-Au-TAT7.5 NSs demonstrated strong nuclear localization, and this is consistent with their ability to greatly reduce the


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number of tumor nodules in the lung, and also to prolong survival by about 80 % in the xenograft lung cancer mouse model. The above data suggest that ASO-Au-TAT NSs have good performance in controlling tumor cell metastasis. Au NPs have well-characterized favourable properties in terms of chemical inertness, biocompatibility, ease of functionalization and multivalent effect. The DNA-Au composites increased the nuclease resistance of ASO. And the cell-penetrating peptide TAT enabled the cargo to effectively cross cell membranes resulting in enrichment of the ASO in the nucleus. The as-prepared ASO NSs is proved to be suitable for metastasis inhibition in a xenograft lung cancer mouse model. In summary, ASO15-Au-TAT7.5 NSs that target lncRNA MALAT1 have been developed to control cancer metastasis. The NSs can effectively permeate the cell nucleus to reach nucleus speckles where MALAT1 locates. The NSs effectively reduced the MALAT1 expression level in A549 cells and inhibited the cell migration in vitro. In addition, we demonstrated that ASO15-Au-TAT7.5 could not only prevent the formation of tumour nodules effectively, but also prolong animal survival in vivo. We therefore conclude that the ASO15-Au-TAT7.5 NSs have the potential for cancer metastasis treatment in the future.



ASSOCIATED CONTENT Supporting Information Available: Detailed experimental procedures, size distribution and zeta potential of nanostructures, calculation of gold nanoparticle number, Cell uptake of nanoparticels, Bio-TEM analysis of intracellular distribution of ASO15-Au-TAT7.5 and ASO15-Au NSs, transwell-based cell migration assay, Hemolytic analysis.

ORCID Jinghong Li: 0000-0002-0750-7352 Xing-Jie Liang: 0000-0002-4793-1705

Acknowledgements: We thank the financial support from the National Natural Science Foundation of China (No. 21621003, No. 21327806, 31430031, 51373117, 31225009), and the Chinese Academy of Science (No. 121D11KYSB20130006 and No. XDA09030301)

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suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors. Nature 2012, 487, 380-384.

Table of contents

Nucleus-retained long non-coding RNA (lncRNA) MALAT1 contributes to tumor cell metastasis. Herein, a nucleus-targeted antisense oligonucleotide nanocarrier was constructed (ASO-Au-TAT NPs). The nanocarriers can efficiently silence MALAT1 and control tumor cell migration in vitro. Moreover, The nanocarrier treated A549 cells formed fewer metastatic nodules in vivo. The ASO nanocarrier have great potential in treatment of metastatic tumors.




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Antisense Oligonucleotide-conjugated Nanostructure Targeting

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