Nowadays, cancer is one of the leading causes of death [1
]. Cancer treatments can still be improved, making use of novel technologies and biotechnological drugs. Advances in nanotechnology have shown a prompt impact in numerous applications in cancer, since nano-scaled carriers have the potential for incorporating various kinds of molecules into their architecture, including anticancer drugs and contrast agents, as well as macromolecules, such as proteins, peptides, or oligonucleotides [2
]. Indeed, several nanoformulations, mainly liposomes, have been successfully translated to the clinical practice [4
RNA-based therapeutics, such as small interfering RNA (siRNA) and microRNA (miRNA), provide a promising approach to treat cancer by targeting specific proteins involved in the mechanism of proliferation, invasion, antiapoptosis, drug resistance, and metastasis [6
]. miRNAs are small (17 to 25 nucleotides) non-coding RNA molecules, which specifically interact with target messenger RNA (mRNAs), resulting in inhibited translation or mRNA cleavage and degradation [8
]. It is well known that various miRNAs, for instance, miRNA-10a, miRNA-34b/c, miRNA-137, miRNA-143, and miRNA-145, are downregulated in colorectal cancer cells compared to healthy tissues, and modulation of the corresponding gene expressions is gaining interest for the development of anticancer therapeutics [9
]. However, the major barriers of miRNA delivery are (i) poor systemic stability, (ii) rapid clearance, (iii) degradation by nucleases, (iv) risk of systemic toxicity, (v) elimination by phagocytic immune cells, and (vi) lack of efficient delivery to targeted cells to achieve the desired therapeutic outcome [14
]. For overcoming these constraints, different delivery systems have been proposed to date, such as PLGA/PEI/miRNA/HA nanoparticles, protamine nanoparticles, and magnetic nanoparticles [15
miRNA-nanotherapeutics might represent a promising alternative for the development of cancer therapeutics. For the design of nanostructures, it is possible to use organic biodegradable materials that do not accumulate in the body and do not cause toxicity. Lipidic sphingomyelin-based nanosystems (SNs), of which the main components are sphingomyelin, present in cell membranes, and vitamin E, widely used in different formulations that are of clinical use, are promising carriers due to their high biocompatibility and versatility for the association of a variety of drugs [18
]. In this study, we aimed to optimize the nanoformulations based on SNs, for their specific application in cancer gene therapy, to mediate an efficient association and delivery of oncosuppressor miRNA-145 and treat colorectal cancer. For this, two different strategies were pursued. Firstly, the cationic lipid stearylamine was incorporated into the nanoparticles, in order to obtain cationic SNs and favor the association of miRNA mimics (SNs-ST). Secondly, we proposed the encapsulation of preformed DOTAP-miRNA lipid complexes (Lpx) into SNs (SNs-Lpx) to provide additional protection for the associated miRNA mimics, following a similar approach to that described for plasmid DNA (pDNA), complexed via hydrophobic ion pairing utilizing surfactants and further incorporated into self-nanoemulsifying drug delivery systems [20
]. Intracellular delivery, cell transfection, and functional in vitro assays were accomplished to determine the potential of the proposed formulations to develop new oncosuppressor therapies for colorectal cancer.
2. Materials and Methods
Vitamin E (dl-α-tocopherol) was obtained from Merck (Darmstadt, Germany). Sphingomyelin (SM) and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) were acquired from Lipoid (Ludwigshafen, Germany). Stearylamine (ST), agarose, heparin, Mowiol® 4-88, Dulbecco’s Modified Eagle’s Medium (DMEM), and Dulbecco’s Phosphate Buffered Saline (PBS) were purchased from Sigma Aldrich (St.Louis, MO, USA). PEG12-C18 was supplied by Creative PEGworks (Chapel Hill, NC, USA). N-[11-(dipyrrometheneboron difluoride)undecanoyl]-d-erythro sphingosylphosphoryl choline (SM-TopFluor®) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). RNAs were synthesized by Eurofins Genomics (Ebersberg, Germany) (miRNA145: 5′ GUCCAGUUUUCCCAGGAAUCCCU 3′; miRNA scramble (miRNAscr): 5′ UUCUCCGAACGUUGUCACGUUU 3′; Cy5-modified miRNA145 (miRNA-Cy5): 5’ Cy5- GUCCAGUUUUCCCAGGAAUCCCU 3′). SYBR® Gold Nucleic Acid Gel Stain, RNAse free water, DNA ladder, penicillin, and streptomycin were obtained from Invitrogen (Madrid, Spain). A microRNA Purification Kit was acquired from Norgen Biotek Corporation (Zaragoza, Spain). qScriptTM microRNA cDNA Synthesis kit, PerfeCta® Universal PCR Primer, and PerfeCta® SYBR® Green SuperMix, Low RoxTM were bought from Quantabio, VWR International Eurolab (Barcelona, Spain). Universal primers: hsa-miR145-5p (5′-CGCGCGTTCCAGTTTTCCCAGG-3′), universal reverse PCR primer (5′-GTGCAGGGTCCGAGGT-3′) and the housekeeping small RNA control primer RNU6 (5′CTCGCTTCGGCAGCACA3′, 5′AACGCTTCACGAATTTGCGT3′) were purchased from Fisher Scientific (Madrid, Spain). SW480 cells were obtained from ATCC (ATCC® CCL-228™). Ultrapure Mili-Q water was used all throughout the experiments. Ethanol was of analytical grade and supplied from Thermo Fisher Scientific (Madrid, Spain). All other chemicals used were of reagent grade.
2.2. Preparation and Characterization of SNs
SNs were formulated with vitamin E (VitE), sphingomyelin (SM), and PEG12-C18 (PEG) in a ratio of VitE:SM:PEG (10:1:0.1 w/w), by ethanol injection method. In brief, the components were dissolved in a final volume of 100 μL in ethanol and injected into ultrapure water (1 mL) under magnetic stirring (total lipid concentration 5.05 mg/mL). SNs were isolated from non-interacted compounds by ultracentrifugation (Beckman Coulter, Brea, CA, USA) at 35,000 rpm for 1 h at 15 °C in a 70.1 Ti rotor, obtaining a cream consisting of the isolated SNs on top of the aqueous phase.
2.3. Preparation and Characterization of Cationic Stearylamine SNs (SNs-ST) and Association of miRNA (miR)
Cationic SNs were prepared upon addition of the cationic lipid, stearylamine (ST) to the organic phase at a ratio of VitE:SM:PEG:ST 10:1:0.1:1 (w/w). SNs-ST were fully characterized. miRNA was subsequently associated to SNs-ST by the establishment of electrostatic interactions performing a simple incubation. A total of 10 μL of miRNA (10 μg) were incubated with 90 μL of SNs-ST at different miRNA:ST mass ratios (1:10, 1:7.5, 1:5, 1:2.5, and 1:1) to obtain theoretical loadings ranging from 0.8% to 7.6% (w/w) with respect to the total mass of the nanosystems. The association of miRNA was analyzed by agarose gel electrophoresis (1% w/v in Tris-Acetate-EDTA (TAE) Buffer). Briefly, 0.5 μg of nucleic acids labeled with SYBR® Gold, either in solution, associated to the nanoparticles, or after displacement with an excess of heparin (25-fold heparin with respect to the amount of miRNA for 2 h at 37 °C) were loaded into each well. Gel electrophoresis was run at 100 V, 40 min in a Sub-Cell GT cell 96/192 (Bio-Rad Laboratories Ltd., Deeside, England). Gel images were obtained with a Molecular Imager® Gel DocTM XR System (UV light 302 nm; Bio-Rad, Madrid, Spain).
2.4. Preparation and Characterization of Lipid Complexes of miRNA with Cationic Lipids
Preparation of lipid complexes (Lpx) was attempted with miRNA and cationic lipids (ST or DOTAP). A total of 10 μg of miRNA were incubated with cationic lipid at different miRNA: cationic lipid mass ratios (1:1, 1:5, 1:10, 1:15, and 1:20) in a total solution of 500 μL (H2O 450 μL and EtOH 50 μL). They were characterized by their physicochemical properties.
2.5. Loading of Lpx into SNs (SNs-Lpx)
miRNA:DOTAP Lpx in a ratio of 1:15 were lyophilized using a VirTis GenesisTM 25 EL (Warminster, PA, USA). Lyophilization steps contained thermal treatment, freezing, primary drying, and secondary drying. It was performed at a temperature ranging from −40 °C to +20 °C, applying a progressive vacuum from 200 mTorr to 20 mTorr. Lyophilized Lpx were characterized by DLS. A total of 50 μL of resuspended Lpx in ethanol were diluted in 450 μL of ultrapure water and subsequently analyzed. For preparation of SNs-Lpx, lyophilized Lpx were suspended in 100 μL of the organic phase (containing VitE, SM, and PEG12
, in a ratio of 10:1:0.1 (w
) and immediately injected to water (1 mL). The association efficiency of miRNA was studied by gel agarose gel electrophoresis, following the same protocol as described in the previous Section 2.3
. In this particular case, broken SNs-Lpx (by mixing the formulation with ethanol at 1:5 v
) were additionally incubated with a 25-fold heparin with respect to the amount of miRNA, for 2 h at 37 °C.
2.6. Characterization of Nanosystems (Size and Zeta-Potential Measurements, Transmission Electron Microscopic (TEM), and Nanoparticle Tracking Analysis (NTA))
Nanoparticles were analyzed for their hydrodynamic size and polydispersity index (PDI) by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a standard λ = 633 nm laser as the incident beam. They were diluted in ultrapure water and loaded into a disposable solvent resistant microcuvette (ZEN0040). The obtained data were analyzed based on a cumulative analysis method for determination of mean hydrodynamic diameter and PDI. ζ–potential, a parameter indicative of the surface charge of the nanocarriers, was measured by Laser Doppler Anemometry (LDA) in the same equipment, using a dip-cell (DTS 1060). Each analysis was performed in triplicate at 25 °C.
The morphology of SNs was investigated using TEM (CM12 Philips; Eindhoven, the Netherlands). A total of 10 μL of diluted nanosystems (1/10 in Milli-Q water) were placed on 400-mesh copper grids, incubated for 3 min at room temperature, and stained with 10 μL of 2% phosphotungstic acid solution for 1 min. Excessive solution of phosphotungstic acid was removed with filter paper. The grids were washed five times in water droplets for 1 min (each time) and dried overnight under vacuum.
NTA measurements were performed with a NanoSight NS300 (Malvern Instruments, Worcestershire, UK), equipped with a sample chamber with a 488-nm laser. Samples were diluted 1/1000 in Milli-Q water and then injected in the sample chamber with sterile syringes (Omnifix®-F 1ml, Melsungen, Germany). All measurements were performed at room temperature for 60 s in triplicate. The mean particle size and standard deviation values obtained by the NTA software correspond to the arithmetic values calculated with the sizes of all the particles analyzed by the software.
2.7. In Vitro Cell Uptake
SW480 cells were cultured in DMEM supplemented with 10% (v/v) of FBS and 1% (v/v) of penicillin-streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. All in vitro studies were performed in this setting, unless otherwise stated.
To determine the working conditions, the cytotoxicity of SNs-ST and SNs-Lpx was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Thermo Fisher Scientific) assay. Briefly, cells were seeded at a density of 104 cells/well in a 96-well plate containing 100 μL of fresh culture medium and incubated overnight to allow cell attachment for subsequent study. Then, cells were cultured in the presence of different concentrations for 24 h at 37 °C. After the incubation, MTT (5 mg/mL) was added to medium and further incubated for 4 h, then 100 μL DMSO was added to dissolve the formazan crystals formed in the live cells for 10 min at 37 °C. The absorbance at 570 nm was recorded using a spectrometer.
Confocal laser-scanning microscope (TCS SP5, Leica Microsystems GmbH, Heidelberg, Germany) was used to observe cell uptake of fluorescent nanoparticles. Twenty-four hours before the experiment, SW480 cells (1 × 105 cells/well) were seeded onto 12 mm diameter glass coverslips in a 24-well plate in 0.5 mL of supplemented cell culture medium. SNs-ST and SNs-Lpx were labeled with SM-TopFluor® (0.2 ng/μL) and miRNA-Cy5 (0.2 ng/μL) to allow direct observation of the molecule of interest. After addition of 20 μL of the formulation to the cells, they were incubated for 4 h at 37 °C in the cell incubator. After that, cells were exhaustively washed with PBS and then fixed with paraformaldehyde (PFA; 4% v/v in PBS) in the dark at room temperature for 15 min. Cells were rinsed again with PBS, and cell nuclei were then counterstained with Hoechst 33342 (1:1000 in PBS) for 5 min. They were washed again with PBS. Lastly, 8 μL of Mowiol® 4-88 were used for mounting samples on coverslips. Preparations were conserved in the dark at −20 °C. The confocal laser scanning microscopic images were obtained with a 63× oil immersion objective for Hoechst 33342 (blue), SM-TopFluor® (green), and miRNA-Cy5 (red), respectively (scanning speed 600 Hz, with an image resolution of 1024 × 1024 pixels). The co-localization ratio of miRNA-Cy5 and SM-TopFluor® was determined by LAS AF software (Barcelona, Spain).
Studies were also accomplished by Fluorescence-Activated Cell Sorting (FACScan flow cytometer, BD biosciences, San Jose, CA, USA). For this experiment, the cells were transfected with the same amount of fluorescent formulations as mentioned in the confocal study. They were incubated for 4 h at 37 °C in the cell incubator and washed with PBS. Then, the cells were trypsinized and resuspended in 0.5 mL of PFA (approx. 1 × 105 cells/mL) prior to analysis. The results were analyzed using Flowjo 8.7 (Ashland, OR, USA).
2.8. Transfection Efficiency
SNs-ST, Lpx, and SNs-Lpx were evaluated for their transfection efficacy in SW480 human colorectal cancer cells. All types of nanosystems were formulated with miRNA145 (miR145), and with a scrambled sequence (miRScr). SW480 cells were seeded in 6-well plates (5 × 105 cells/well) and incubated in completed DMEM for 24 h. Formulations were then added (SNs-ST (miR145), SNs-ST (miRScr), Lpx (miR145), Lpx (miRScr), SNs-Lpx (miR145), and SNs-Lpx (miRScr), being the dose 2 μg of miRNA in a final volume of 2 mL of fresh cell culture medium without supplements. The nanocarriers were removed after 4 h of incubation, cells washed, and fresh completed medium added (2 mL). The transfection efficiency was determined 72 h post-transfection by quantitative real-time PCR (qRT-PCR) (Stratagene Mx 3000, Agilent Technologies). According to the manufacturer’s protocol of Norgen Biotek Corporation, microRNA Purification Kit (Thorold, ON, Canada), the total miRNA was extracted from SW480 cells. miRNA concentration and purity were evaluated with UV spectrophotometry (Nanodrop, Spectrophotometer ND-100, Thermo Scientific). Extracted RNA samples were measured and diluted to have the same amount of RNA (120 ng). Next, the reverse RNA transcription to cDNA was carried out using qScript™ microRNA cDNA Synthesis Kits (Quanta Biosciences). The qRT-PCR was performed using PerfeCta® MicroRNA Assays (Quanta Biosciences), with a primer for miRNA 145 (has-miR145-5p). Small nuclear RNA, RNU-6 was employed as an endogenous housekeeping gene to normalize the miRNA amount. PCR cycle consisted of activation at 95 °C (2 min), denaturation at 95 °C (5 s), and annealing at 60 °C (30 s) for 40 cycles. Quantitative data were analyzed by utilizing an AriaMx Real-time PCR System (Agilent Technologies). The relative miRNA145 expression level was calculated based on the comparative 2−ΔΔCT method in relation to RNU6 and normalized to that obtained from non-treated cells.
2.9. Functional Assays
For cell proliferation assay, transfected cells were seeded at 2 × 105 cells/well onto 24-well plate and harvested at 72 h post-transfection. Cells were then counted manually in a Neubauer-improved cell counting chamber, depth 0.100 mm, 0.0025mm2 (Marienfeld, Germany). With respect to the cell migration assay, after transfection, cells were trypsinized and counted. Then, 2 × 105 cells/well were planted onto a 24-well plate, and artificial wounds were created on the confluent cell monolayer using a 200 μL pipette tip. Wounded cells were gently washed PBS for 3 times to remove the detached cells and cultures in 5% CO2 at 37 °C. The wound closure was observed and photographed at time point 0, 24, 48, 72, and 96 h under microscope (Leica type 090-135.00, Wetzlar, Germany). The wound closure area was calculated by analyzing the microscopy images with the software ImageJ (1.48d; National Institutes of Health, Bethesda, MD, USA). For colony-forming assay, transfected cells were seeded at a density of 200 cells/well in 12-well plates and cultured at 37 °C for 2 weeks. Cells were stained with MTT solution (5 mg/mL) and photographed. The images were analyzed using ImageJ software.
2.10. Statistical Analysis
Differences were statistically determined by one-way ANOVA followed by Tukey’s method. All statistical analysis was performed using GraphPad Prism (Version 6.0 software) (GraphPad Software, San Diego, CA, USA). A p value < 0.05 was considered to be significant.