Characterization of the Unloaded and Loaded NPs

The size (diameter), PdI, and surface electrical charge (ζ potential) of the NPs (Figures 2A and 2B) were determined using a Zetasizer Nano ZS (Malvern Panalytical) equipped with a 4 mW HeNe laser beam with a wavelength of 633 nm and a scattering angle of 173° (for size measurements) and 13° (for ζ potential measurements). Size and ζ potential values were automatically calculated through DTS Nano v.6.30 software, using the Stokes-Einstein equation and the Henry equation with the Smoluchowski approximation, respectively. The dispersion solution used was the solvent in which the NPs were dispersed for the DLS, i.e., ultrapure water, and 1 mM KCl for the ELS. Disposable solvent-resistant microcuvettes or folded capillary cells (DLS; Sigma) with gold-plated electrodes (ELS; Malvern) were used. Data were acquired at 25°C, in triplicate.

The morphology of the NPs was observed by a transmission electron microscope (TEM, 80 kV; Tecnai 12; FEI) with a 1 K × 1 K KeenView camera (OSiS). Samples were prepared by placing 3 μL of the NPs suspension onto a 400 μL mesh copper grid coated with carbon (Electron Microscopy Support). One minute after deposition, the grid was tapped with a filter paper. TEM images were obtained at different magnifications (Figure 2C).

The presence of the miR-155-5p was monitored by AGE and qPCR (Figures 3A and 3B, respectively). Five percent agarose gels, prepared in TBE (Tris-Borate-EDTA; Thermo Fisher Scientific) buffer and stained with ethidium bromide, were used to allow detection of nucleic acids within a 10–100 bp range, as well as the GeneRuler Ultra Low Range DNA Ladder (10–300 bp; Thermo Scientific). Samples included unloaded and loaded NPs, plus additional controls from different steps of the miRNA loading: miRNA alone (at the concentration used for the loading), loaded NPs right after miRNA addition, and supernatant from the ultracentrifugation. Samples were run by electrophoresis (50 V, 120 min) and detected through a UV transilluminator smart imaging system (Vilber Lourmat).

As for qPCR, miR-155-5p was detected using the Hs_miR-155_2 miScript Primer Assay (5′UUAAUGCUAAUCGUGAUAGGGGU-3′; QIAGEN), a primer able to detect specifically the human mature miRNA miR-155-5p, following the supplier’s recommendations. For that, NPs were centrifuged (0.7 mL at 4°C, 20,000 g, 30 min), QIAzol lysis reagent (QIAGEN) was added (0.7 mL) to each sample, and the samples were vortexed until homogenization. RNA was isolated using the miRNeasy Mini Kit (QIAGEN). The quality and quantity of total RNA were determined by UV spectrophotometry. The miScript II RT Kit (QIAGEN) with a final volume of 20 μL, containing 90 ng RNA, 4 μL 5× miScript Hiflex buffer, 2 μL 10× miScript Nucleics Mix, and 2 μL miScript Reverse Transcriptase mix were used for cDNA synthesis. The final concentration of cDNA was 4.5 ng/μL. The qPCR analysis was performed using the miScript SYBR Green PCR kit (QIAGEN) for miR-155-5p with StepOnePlus Real-Time PCR System (Applied Biosystems), according to the manufacturer’s recommendations. The amplification was done with a final volume of 25 μL, containing 2× QuantiTect SYBR Green PCR Master Mix, 10× miScript Universal Primer, 10× miScript Primer Assay, and 2.5 μL of each sample at 2 ng/μL. For each DNA amplification, a standard range was also generated from cDNA of a miR-155-5p solution, to determine the efficiency of the primer, as well as the negative control (without the reverse transcriptase). The qPCR conditions included 1 cycle for enzyme activation (95°C for 15 min), followed by 40 cycles of denaturation, annealing, and extension (94°C for 15 s, 55°C for 30 s, and 70°C for 34 s). By means of a standard curve, the expression of the gene encoding the miRNA was calculated using the standard curve and a geometric mean.

Loaded NP stability, while stored at 4°C, was evaluated by DLS (Figures 4A and 4B), ELS (Figure 4C), and AGE (Figure 4D), right after miRNA loading (day 0) and 1, 7, 14, 21, and 28 days after miRNA inclusion. Aliquots for each time point were prepared under sterile and RNase-free conditions at day 0. Furthermore, to assess if all miRNA could be released and desorbed from the NPs under physiological and sterile conditions, loaded NPs were resuspended in PBS at 4°C (instead of the ultrapure water) after centrifugation, transferred into a clear, flat-bottomed, ultra-low attachment plate (Corning) and subjected to strong orbital shaking at 250 rpm at 37°C. At each time point (15 and 30 min and 1, 3, and 6 h), the plate was removed from the orbital shaker and incubator, and one-tenth of the volume of each well was removed and placed at −20°C and replaced by PBS. At the last time point, the remaining content of each well was centrifuged (4°C, 20,000 g, 30 min). Samples were resuspended in 1.5 M NaCl, supernatants collected, and a final run of re-suspension and centrifugation in 1.5 M NaCl was performed to obtain the final NPs. Samples were analyzed by AGE (Figure 5).