Uveal melanoma (UM) is a malignant intraocular tumor with a high metastatic risk [1
]. Unfortunately, the treatments for the metastatic UM are not effective [2
] due to the unique characteristics of this tumor and because the visible clinical features only emerge at later stages of the disease [3
]. Drugs developed for other tumors, such as Irinotecan [5
], which is used for colon and lung tumors, have been used for UM. Furthermore, several chemotherapeutics agents are in clinical trials to treat UM, such as Selumetinib or AZD 8055. While these agents are selective against the mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) pathways, respectively; they are not very selective and as a result, healthy cells are also affected.
Therefore, novel approaches to tackle this disease are desirable. In this sense, non-coding genetic elements, such as microRNAs, endogenous small interference RNAs (endo-siRNAs), long non-coding RNAs (lncRNAs), PIWI-associated small RNAs (piRNAs) and small nucleolar RNAs (snoRNAs), are being explored as new therapeutic targets since they play a major role in cellular events such as cell proliferation, differentiation, apoptosis or invasiveness capacity [7
]. Interestingly, treatments based on these molecules are expected to achieve excellent selectivity and reduce side effects as compared to traditional treatments, because of the complementary sequence interactions involved in those approaches.
Among the most studied nucleic acids are microRNAs, which besides their therapeutic potential, can also be used to detect diseases as each tumor presents a unique microRNA profile depending on the tissue and cancer stage [14
]. Additionally, several studies use their particular signature to classify the different diseases and use this information to apply the most suitable treatment [8
MicroRNAs are transcribed initially as primary precursors (pri-microRNAs) from intron coding genes or intergenic regions, which are cleaved by the Drosha complex to produce the precursor hairpin molecule (pre-microRNA). These structures are transported by exportin-5 from the nucleus to the cytoplasm, where the Dicer complex processes pre-microRNAs into the mature microRNAs. Finally, the mature microRNAs guide the RNA-induced silencing complex (RISC) to their corresponding target messenger RNAs (mRNAs), promoting transcript reduction and/or inhibition [16
MicroRNAs are dysregulated in many diseases such as Alzheimer’s, cardiac damage, and cancer [17
]. Particularly in cancers such as sarcoma, glioblastoma, pancreatic cancer, breast and colon cancer, oncogenic microRNAs (e.g., microRNA-20a, microRNA-9, microRNA-21) are overexpressed [23
] and/or tumor suppressors microRNAs (e.g., microRNA-145, microRNA-204) are downregulated [24
In the present study, microRNA-34a, microRNA-182, microRNA-137 and microRNA-144 have been selected since they are downregulated in UM and other cancers [25
]. In particular, they are used in combination as this could provide better results, when compared to the use of individual microRNAs [26
]. Regarding their roles, microRNA-34a is a proapoptotic transcriptional target of the p53 tumor-suppressor gene effector network. Its primary target is the c-Met mRNA [27
], which is a tyrosine-kinase receptor implicated in the pathways of son of sevenless homolog (SOS), phosphatidylinositol 3-kinase (PI3K), and the Ras protein family [28
]. Moreover, overexpression of microRNA-34a can downregulate the Akt protein and cell cycle-related proteins, reducing the metastatic and proliferation potential of the cells [27
MicroRNA-182 participates in the tumor suppression network of p53 in UM, where melanogenesis associated transcription factor (MITF), B-cell lymphoma 2 (BCL2) and cyclin D2 are its main targets. MITF is a master regulator of melanoma oncogenes that mediates the inhibition of Akt and extracellular signal-regulated kinases ERK1/2 pathways, in addition to controlling c-Met. Furthermore, microRNA-182 controls the forkhead box protein O1 (FoxO1), which is related to osteogenesis, T-cell development and tumorigenesis [29
MicroRNA-137 has three major targets: MITF, and the cyclin-dependent kinases CDK2 and CDK6. It is a potent suppressor of the p160 steroid receptor co-activator (SRC) family of transcriptional co-activators, which can inhibit SRC-mediated, steroid receptor-dependent and -independent transcription [30
]. Other studies identified C-terminal binding protein 1 (CTBP1) as a microRNA-137 target. By regulating CTBP1 through microRNA-137, it is possible to avoid E-cadherin suppression [31
MicroRNA-144 acts as a tumor suppressor in UM, inhibiting cell proliferation and migration. As with the previously mentioned microRNAs, c-Met is one of its potential targets [32
To restore the normal levels of microRNAs in tumoral cells, vectors encoding those microRNAs or whole microRNAs can be added to cells. However, modified nucleic acids that mimic the role of the microRNAs (microRNAs mimics) are usually employed at the preclinical and clinical level [26
], such as small interference RNAs (siRNAs), which must be modified to increase their stability in serum and achieve the required activity [34
]. A related strategy employs the use of antisense technologies, which are made of deoxyribonucleotides. Such DNA-based microRNA mimics are more stable and cheaper than their RNA counterparts, and despite their lower activity, they can be used in combination with chemotherapeutic agents with remarkable results [35
]. Despite the enormous potential of oligonucleotides as therapeutics, they present some disadvantages, mainly when based on RNA, as outlined below.
Low stability. They can be degraded by blood circulating enzymes [37
], reducing their therapeutic activity [17
Poor cell internalization. Their negative charge prevents passive endocytosis [17
Poor cellular selectivity. The oligonucleotides cannot discriminate between healthy and cancer cells, leading to a non-specific delivery [37
To overcome these problems, delivery systems such as micelles, liposomes, dendrimers, inorganic particles, nanotubes and carbon nanoparticles, nanoemulsions, viral nanocarriers, polymeric or peptide nanoparticles and solid lipid nanoparticles are being evaluated [38
Among the different systems, gold nanoparticles (AuNPs) are excellent nanomaterials for this purpose, since they are easy to prepare and modify and are considered non-toxic when properly modified. In particular, when functionalized with oligonucleotides these structures do not affect the gene expression or viability of cells. In this case, when the AuNPs are densely modified with oligonucleotides, they are known as spherical nucleic acids (SNAs), which, among other properties, can translocate easily in a wide variety of cell lines [47
Despite the great therapeutic potential of oligonucleotides, chemotherapy-based treatments are usually employed to treat cancers. Unfortunately, the associated lack of selectivity, drug resistance, and high toxicity make this approach far from ideal and limits its use [50
]. Indeed, monotherapy has been proven ineffective for cancer treatment due to the development of resistance and metastasis. For this reason, combination therapies are the preferred choice, since it is possible to reduce the amount of the most toxic agent [51
] and reduce relapse. In this sense, the combination of microRNAs and chemotherapeutic drugs has provided excellent results in different type of cancers, such as human glioma [26
In this study, we have used SN38 (7-ethyl-10-hidroxycamptothecin), a topoisomerase I inhibitor [52
] in combination with oligonucleotides, which are designed to mimic the role of the above-mentioned microRNAs. SN38 has not been tested in UM, although there are reports on Irinotecan, its prodrug, which presents 200–2000-fold less activity [53
]. The principal drawback of SN38 is its poor aqueous solubility (<5 μg/mL), which prevents systemic administration [54
]. To overcome this limitation, drug delivery systems based on liposomes have been reported [53
]. In this regard, we envision that the conjugation of therapeutic oligonucleotides and SN38 on AuNPs can overcome the inherent limitations of these molecules and provide a more effective therapy.
2. Materials and Methods
Solvents and chemical reagents were purchased from Sigma-Aldrich (San Luis, MO, USA), abcr GmbH (Karlsruhe, Germany), Thermo Fisher Scientific (Waltham, MA, USA), Scharlab (Sentmenat, Barcelona, Spain), FluoroChem (Hadfield, UK) and VWR (Radnor, PA, USA).
Oligonucleotides were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).
Roswell Park Memorial Institute (RPMI) medium, streptomycin–penicillin (100X), fetal bovine serum (FBS), l-glutamine (100X), trypsin (10X), phosphate-buffered saline (PBS) and cell culture plasticware were purchased from VWR. Thiazolyl blue tetrazolium bromide, propidium iodide (PI), Hoechst 33342 and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich; and Lipofectamine 2000, RNase H and Opti-MEM from Thermo Fisher Scientific. c-Met and anti-rabbit IgG Alexa Fluor 488 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
2.2. Modified Oligonucleotides
The sequences of the oligonucleotides employed are described in Table 1
. siRNA duplexes were obtained after the annealing of complementary sequences using an annealing buffer described in Section 2.3.1
. The aptamer AS1411 was prepared as previously reported in a Mermade 4 DNA synthesizer [47
2.3. Oligonucleotide Solution Preparation
2.3.1. RNA Annealing (siRNAs)
siRNA duplexes were obtained by mixing the guide and the passenger oligonucleotides in an annealing buffer. The annealing buffer was prepared following the Sigma-Aldrich protocol for annealing oligonucleotides [55
siRNA-34a was obtained from oligonucleotides 9 and 10; siRNA-182 was obtained from 11 and 12; siRNA-137 was obtained from 13 and 14 and siRNA-144 was obtained from 15 and 16 (Table 1
2.3.2. siRNA Mix
A sample containing the four siRNAs previously prepared was obtained by mixing equimolar quantities of each siRNA.
2.3.3. DNA Mix
DNA mix-1 (34.04 μM, final concentration) was obtained from equimolar amounts of oligonucleotides 1, 2, 3 and 4 (Table 1
) in water. This mixture contains unmodified oligonucleotides.
DNA mix-2 (20 μM, final concentration) was obtained from equimolar amounts of oligonucleotides 5, 6, 7 and 8 (Table 1
) in water. This mixture contained thiol-modified oligonucleotides and was used for the functionalization of AuNPs.
2.4. Gold Nanoparticles Synthesis and Characterization
The gold nanoparticles were prepared following standard reported procedures [56
]. The AuNPs concentration was calculated using the Beer–Lambert law [57
] from the absorbance recorded at 520 nm and the extinction coefficient of 2.7 × 108
for 13 nm nanoparticles [58
Particle size and shape were examined by transmission electron microscopy (TEM) in a JEOL JEM 1010 (JOEL, Akhisima, Japan), operating at 100 kV. Samples were prepared by placing one drop of a dilute suspension onto a carbon-coated copper grid and drying the drop with paper after 2 min. The size distributions were determined through manual analysis of ensembles of over 200 particles found in randomly selected areas of the enlarged micrographs, with Image J software [59
] to obtain the mean size and standard deviation (Supplementary Figure S1
Gold Nanoparticle Functionalization
The bare nanoparticles were modified with the different components using the conditions summarized in Table 2
. The oligonucleotides (DNA mix 2 and siRNA mix) were previously treated for 1 h with tris(2-carboxyethyl) phosphine hydrochloride (TCEP), to remove the protecting groups on the thiol moieties. After the addition of each reagent, the solution was incubated for 30 min. The procedure was done as follows: The reagents were added to 1 mL of a solution of AuNPs (10 nM) in the order and quantities detailed in Table 2
. The last step involves the addition of NaCl (70 μL, 5 M) and incubation with agitation at room temperature overnight. The particles were centrifuged down (300× g
) at 4 °C, the supernatant removed and resuspended in water. The process was repeated three times to remove the unbound material [60
The amount of oligonucleotide on the nanoparticle was determined as follows: after purification of the nanoparticle the supernatants were dried in an Eppendorf Concentrator plus (V-AQ mode) (Eppendorf, Hamburg, Germany) for 5 h; then the sample was diluted in water (1 mL) and the concentration of oligonucleotides quantified by absorbance at 260 nm in a plate reader Synergy H4 Hybrid reader (BioTEK, Winooski, VT, USA). This amount was subtracted from the amount added. Concentrations of AuNP–DNA mix and AuNP siRNA mix were 3.9 and 3.28 μM, respectively. The quantification of SN38 on the nanoparticle was carried out in the same way as above, but the absorbance was recorded at 380 nm, resulting in a concentration of 10 μM.
The hydrodynamic size, by dynamic light scattering (DLS), and zeta-potential of the AuNPs was measured on a Zeta Sizer Nano-ZS (Malvern Instruments, Worcestershire, UK). The studies were performed in 12 runs, in a standard cuvette at 25 °C. The AuNPs were diluted in 1 mL water (pH 5.8) to a final concentration of 1 nM. The AuNP’s refraction index for a spherical particle was 1.330. The data presented is the average distribution of these measurements as a function of the number of particles expressed as a percentage.
2.5. Cell Culture and Viability Studies
Mel 202 cells were cultured (Mel202 were donated by Susana Ortiz-Urda at the University of California, San Francisco, CA, USA) in RPMI medium with 10% FBS, 1% streptomycin–penicillin and 1% l-glutamine at 37 °C in a Binder CB210 incubator (5% CO2). All the procedures were performed inside a laminar flow hood Telstar CV-30/70 (Telstar, Terrassa, Spain). Cells were grown in 24-well plates (30,000 cells/well).
The experiments were done when cells reached 60% confluency.
2.5.1. alamarBlue Viability Assay
A stock solution of resazurin sodium salt (Sigma-Aldrich, St. Louis, MO, USA) (1 mg/mL) in PBS was diluted 1% (v/v) in complete RPMI medium and added to the cells. After 3 h in the incubator (37 °C), the fluorescence was measured at 25 °C in a plate reader Synergy H4 Hybrid reader (BioTEK), λex = 550 nm, λem = 590 nm.
The fluorescent intensity measurements were processed using the following Equation:
% Cell viability = ((Sample data − Negative control)/(Positive control − Negative control)) × 100
The positive control corresponds with untreated cells. A resazurin solution without cells was used as negative control.
2.5.2. Oligonucleotide Transfection
A total of 50 μL Opti-MEM was mixed with oligonucleotides (14, 15, 16, 17) or DNA mix 1. Next, it was mixed with 1 μL lipofectamine 2000 in 50 μL Opti-MEM. The final mixture was incubated for 20 min and then 100 μL of the final mixture was added to cells. The final concentration of 14, 15, 16, 17 or DNA mix 1 was 140 nM. After 24 h, the cells were washed three times with PBS and RPMI medium was added.
SN38 stock solution was prepared at 100 μM in DMSO. Then different concentrations of SN38 (100, 50, and 25 nM) were prepared in RPMI medium. It was incubated for 24 h with the cells, then washed three times with PBS and RPMI medium was added. After an additional 24 h, the viability assay was carried out as described in Section 2.5.1
2.5.4. Combination Treatment
In this case, the oligonucleotides were transfected, as indicated in Section 2.5.2
. After 24 h the cells were washed three times with PBS. Then SN38 (25 nM/well) was added and incubated for an additional 24 h. Then, the cells were washed with PBS and RPMI medium was added. The cell viability was evaluated after their incubation for an additional 24 h.
2.5.5. Nanoparticles Treatment
A volume of 100 μL functionalized AuNPs were added in Opti-MEM (500 μL, total volume). The cells were incubated for 24 h, washed with PBS and RPMI medium was added. Twenty-four hours later, the viability was assessed, as described in Section 2.5.1
2.6. c-Met Immunofluorescence
Cells were treated with the oligonucleotides as described in Section 2.5.2
and an immunofluorescence staining was performed as reported elsewhere [59
] using an anti-c-Met antibody (Ab 1003, Cellular Signaling Technology, Danvers, MA, USA) (1:200) as a primary antibody and a goat anti-rabbit IgG Alexa 488 (1:200) as a secondary antibody. The nucleus was stained with Hoechst 33342 and fluorescence was examined using a Leica DMI3000 M inverted microscope (Leica, Wetzlar, Germany) at 274.29 exposure units. Images were collected and analyzed using a customized script for Fiji [61
2.7. Flow Cytometry
Cells were harvested in 6-well plates at a 60% confluency and the treatment was carried out as described above. Then, the samples were trypsinized, fixed with paraformaldehyde 1% (v/v), washed with PBS and centrifuged at 177× g for 5 min in an Eppendorf centrifuge 5804 R (Eppendorf, Hamburg, Germany). Each sample was treated with 10 μg RNAsa A and 20 μg PI. Cell cycle analysis was performed in a Beckman Coulter Cytomics 500 Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA) using 20,000 cells. The acquired data was analyzed with Multicycle software (Perttu Terho, Turku Centre for Biotechnology, Turku, Finland).
These experiments were performed in the Flow Cytometry Service at the CNB-CSIC.
2.8. Synthesis of Modified SN38
All reactions (Scheme 1
) were monitored by thin-layer chromatography (TLC), which was performed on sheets of silica gel 60 F254 (Sigma-Aldrich). The products were purified by flash column chromatography using silica gel (60 Å, 230 × 400 mesh). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Instrument (Bruker, Mannheim, Germany) and reported in MHz as solutions in CDCl3
, the chemical shifts are reported in parts per million (ppm), and the coupling constants are reported in Hz.
2.8.1. α-Lipoic Acid–NHS (1)
Lipoic acid (1 g, 1 equiv) and N
-hydroxysuccinimide (NHS) (667 mg, 1.2 equiv) were dissolved in tetrahydrofuran (THF) (20.8 mL), and the solution was stirred at 0 °C for 10 min. A solution of N
’-dicyclohexylcarbodimide (DCC) (1.2 g, 1.2 equiv) in THF (1.7 mL) was added slowly to the lipoic acid and NHS solution. The reaction was stirred at room temperature for 5 h. The mixture was filtered, and the solution was kept in the freezer overnight, it was filtered again and the solvent was eliminated in vacuum. The compound 1
) was obtained as yellow oil (1.43 g, 99% yield) [62
H NMR (400 MHz, CDCl3): δ = 3.60–3.50 (m, 4H), 3.20–3.06 (m, 1H), 2.81 (s, 4H), 2.61 (t, J
= 7.4 Hz, 2H), 2.50–2.36 (m, 1H), 1.94–1.88 (m, 1H), 1.82–1.72 (m, 2H), 1.72–1.66 (m, 2H), 1.62–1.46 (m, 2H) (Supplementary Figure S2
C NMR (101 MHz, CDCl3): δ = 169.13, 168.42, 67.42, 40.15, 38.52, 34.42, 33.21, 30.79, 25.59, 22.59, 24.36, 23.39.
2.8.2. α-Lipoic Acid–SN38 (2)
(56 mg, 2 equiv), SN38 (36 mg, 1 equiv) and 4-dimethylaminopyridine (DMAP) (3 mg) were dissolved in dimethylformamide (DMF) (3.7 mL), then N
-diisopropylethylamine (DIPEA) was added to the solution. The reaction was stirred at 50 °C for 18 h. The solvent was eliminated in vacuum and the crude was purified by flash chromatography (CH2
/MeOH 40:1) to obtain the compound 2
) as white-yellow solid (21 mg, 40% yield). 1
H-NMR (400 MHz, CDCl3
): δ = 8.24 (d, J
= 9.2 Hz, 1H), 7.83 (d, J
= 2.5 Hz, 1H), 7.65 (s, 1H), 7.55 (dd, J
= 9.2, 2.5 Hz, 1H), 5.76 (d, J
= 16.3 Hz, 1H), 5.35–5.29 (m, 1H), 5.26 (s, 2H), 3.68–3.59 (m, 1H), 3.25–3.10 (m, 4H), 2.69 (t, J
= 7.4 Hz, 2H), 2.55–2.46 (m, 1H), 2.00–1.93 (m, 1H), 1.90–1.83 (m, 3H), 1.83–1.76 (m, 2H), 1.70–1.59 (m, 3H), 1.40 (t, J
= 7.7 Hz, 3H), 1.04 (t, J
= 7.4 Hz, 3H) (Supplementary Figure S3
C-NMR (101 MHz, CDCl3): δ = 173.95, 171.85, 157.67, 151.94, 150.18, 149.64, 147.49, 146.95, 145.27, 132.13, 127.48, 127.27, 125.41, 118.55, 114.59, 97.98, 72.76, 66.38, 56.34, 49.40, 40.29, 38.55, 34.61, 34.21, 31.62, 28.75, 24.58, 23.19, 14.01, 7.83 (Supplementary Figure S4
2.9. Statistical Analysis
The statistical analysis was performed in R Project for Statistical Computing (R-3.2.5) software [63
]. One-way analysis of variance (ANOVA) was used to compare the mean value of each condition vs. control. Significant differences between the means were accepted when the p
-value was lower than 0.001 (***).