Methods

The list of key materials is described in the supplementary section.

LCN preparation

LCN were prepared by a top-down method involving hydration of a bulk mesophase followed by ultrasonication. The precursor gel was obtained at 8:1:91 (w/w/w) MO/OA/aqueous phase ratio, in which molten MO was mixed with OA and subsequently hydrated with 0.01 M sodium phosphate buffer (pH 7.0) containing P407 or P188 (0.5, 1 and 1.5% w/v). For cationic formulations, PAH (0.5%, w/v) was dissolved in the aqueous phase prior to hydration. After 24 h equilibration, the bulk phase was fragmented by ultrasonication (30% amplitude, 1 min, ice bath; Vibra-Cell™ VCX750 Sonics & Materials, Inc.) to yield stable dispersions.

Neutral systems were designated LCNn-P407 and LCNn-P188, while PAH-containing formulations were termed LCNp-P407 and LCNp-P188. For complexation, siRNA was incubated with cationic LCN (2:1 N/P ratio) for 30 min at room temperature.

LCN physical characterization

Hydrodynamic diameter, zeta potential, and particle concentration

The intensity-weighted average hydrodynamic diameter (reported as the z-average) and the particle size distribution (reported as the polydispersity index, PdI) of LCN were analyzed by dynamic light scattering (DLS) using a Malvern Zetasizer Nano (Malvern Instruments, UK). Correlation graphs generated by the software and the correlogram intercept values (>0.9) were used to measure the reliability of the gaussian distributions obtained. Measurements of the electrophoretic mobility of the nanoparticles were expressed as zeta potential using the provided software. The means of three determinations in different batches of the same dispersion type were used in the analysis.

The LCN concentration (particle number per mL) was determined on a NanoSight NS300 (Malvern Instruments, UK). The recorded videos were analyzed using Malvern software (NTA 3.4 Build 3.4.003).

Evaluation of siRNA binding, structural integrity, and nuclease protection in LCNp systems

The RiboGreen® assay was performed to quantify the efficiency of siRNA (20 μM) binding with LCNp, according to the manufacturer's instructions. Samples were analyzed in a spectrofluorometer (λexc 500/ λem 525 nm), and the siRNA binding efficiency was calculated by dividing the quantified free siRNA concentration by the initial siRNA concentration and multiplying the result by 100.

Complex formation (LCNp-P407 or LCNp-P188; N/P 2:1) and siRNA release were further evaluated by agarose gel electrophoresis using GelRed® under standard conditions (TAE buffer, 100 V, 110 mA, 20 min). For release and integrity analysis, complexes were treated with heparin (50 IU, 37 °C, 10 min) prior to electrophoresis, and bands were visualized under UV illumination (Transilluminator, Loccus Biotechnology, Brazil) using Quantity One software.

Protection against enzymatic degradation was investigated via RNase A digestion. LCNp-siRNA complexes (20 μM) were incubated with RNase A (0.5 and 5.0 μg/mL) at 37 °C for 1 and 24 h. Controls included intact siRNA, siRNA with EDTA/heparin, and RNase-treated siRNA. Enzyme activity was inactivated with EDTA (100 mM), followed by heparin-mediated siRNA release and electrophoretic analysis as described above.

Conformational stability of siRNA upon complexation was analyzed by circular dichroism using a Jasco J-810 Spectropolarimeter at 25 °C (200–350 nm, 1 mm cuvette). Spectra (average of four scans) were obtained for free siRNA (20 μM), isolated LCNp (1:50 and 1:100), heparin (50 IU), and LCNp-siRNA complexes prepared immediately prior to analysis.

Small angle X-ray scattering (SAXS) analysis

The LCN internal mesophase was investigated by SAXS using a Xeuss® 2.0 (Xenocs, France) set-up with a Pilatus bidimensional detector, operating at a wavelength of 1.5418 Å (copper tube) and sample-to-detector distance of 0.9 m (q range covers 0.015 Å−1 and 0.43 Å−1). Special glass capillaries (diameter of 2.0 mm) containing the samples were placed in a temperature-controlled (23 ± 1 °C) sample holder. Measurements were performed for 0.01 M sodium phosphate buffer (pH 7.0) as background to obtain the absolute scattering intensity (Silvestrini et al., 2023). The scattering vector, q, was determined from the scattering angle by using the relationship q = (4π/λ)sinθ, with 2θ being the scattering angle, and λ being the X-ray wavelength. To identify the phase type, the q values of the peaks were correlated with Miller indices. Lattice parameters were calculated from peak positions, and variations greater than 0.10 nm were considered significant based on instrument precision.

Cryogenic electron microscopy (Cryo-EM) analysis

Cryo-EM analyses were performed in a Talos F200C microscope (Thermo, USA), operating at 200 kV, with a Ceta 16 M 4 k × 4 k camera (Thermo, USA) for digital image acquisition. The lacey carbon film on a 300-mesh copper grid (Ted Pella®, USA) were previously treated with a load of 25 mA for 50 s, in an EasiGlow (I) equipment (Ted Pella®, USA) and the vitrification of samples Vitrobot Mark IV (Thermo, USA). The sample was applied to each grid, performing the excess draining step (Blot time 3 and Blot force −3) and freezing the grids immediately in liquid ethane. After this step, the grids were kept in liquid nitrogen until insertion under the microscope. ImageJ® software was used to analysis of Cryo-EM micrographs.

Langmuir monolayers

Surface pressure-surface area per molecule isotherms

Langmuir experiments were performed using a KSV-Nima KN2002 trough (Biolin Scientific, Sweden) equipped with symmetric hydrophilic barriers and a Wilhelmy plate (filter paper) as the surface pressure sensor. The trough (243 cm2 surface area) was controlled by dedicated LB software. DPPC monolayers were formed by depositing a chloroform solution (1 mg/mL) at the air–water interface over a PBS subphase (pH 7.2) in the presence or absence of LCN. After 15 min to allow solvent evaporation, the monolayers were compressed at a constant rate of 15 mm/min at 23 ± 1 °C. Surface pressure and molecular area were measured with accuracies of 1 mN/m and 1 Å2, respectively. All experiments were performed at least in triplicate.

Monolayer compressibility was assessed from π–A isotherms by calculating the compressional modulus (Cs−1 = −A(∂π/∂A)). LCN insertion was evaluated by determining changes in molecular area (ΔArea) at defined surface pressures relative to control monolayers.

Surface topography and morphology characterization

The morphology and texture of the DPPC monolayer on the air-water surface in real time as a function of surface pressure were studied by Brewster angle microscopy (BAM) (Roldán-Carmona et al., 2012). Experiments were performed by using KSV NIMA MicroBAM (Biolin Scientific, Sweden) coupled to the KSV-Nima trough.

In vitro cellular studies

Cell line and culture conditions

Human immortalized non-tumorigenic keratinocytes cell line HaCaT and murine immortalized non-tumorigenic monocyte/macrophage cell line Raw264.7 (ATCC TIB-71) were maintained in DMEM supplemented with 10% FBS. Cells were grown at 37 °C and 5% CO2 and were split every 2–4 days and discarded after 15 passages.

Cell viability assay

The cytocompatibility of LCN was measured by the resazurin reduction assay. Briefly, HaCaT and Raw264.7 cells (104 cells/ well) were seeded in a 96-well plate and after an overnight period, the culture medium was replaced with fresh culture medium containing each LCN (concentrations: 50 to 250 μg of MO/mL). After 24 h, a solution of resazurin (25 μg/mL) in fresh culture medium was added to each well and the cells were incubated for another 4 h. Resorufin fluorescence (resazurin reduction) was measured on a BioStack Ready device (BioTek Synergy 2, USA) according to the manufacturer's conditions (λexc 530/ λexc 590 nm). Data are expressed as a percentage of viable cells compared to the untreated control.

Cellular uptake of the LCNp-siRNA complex in 2D culture

Cellular uptake of siRNA mediated by the LCNp-siRNA complex was studied by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) analysis. For FACS analysis, the cells (HaCat cell line) were seeded in a 12-well plate (5 × 105 cells/ well) and after an overnight period, the culture medium was replaced with fresh culture medium containing each sample (LCNp-siRNA AF647 or naked siRNA at 80 μM). After 6, 12, or 24 h, the cells were trypsinized and centrifuged, and the resulting pellet was resuspended in PBS and subjected to BD FACSCanto™ I (BD Biosciences, US).

For CLSM image analyses, cells were seeded onto 35-mm coverglass bottom dishes (105 cells/well), and samples (LCNp-siRNA AF647 or siRNA naked at 80 μM) maintained for 12 h. After that, the cells were washed with PBS, fixed with paraformaldehyde (2% w/v; 10 min) and stained with DAPI solution (0.3 μg/ mL; 10 min) for nucleus labeling. Finally, CLSM images were obtained using a Leica TCS SP8 CLSM microscope (Leica Microsystems Inc., USA) equipped with a 63× oil immersion objective and laser for DAPI (λexc 358/ λexc 461 nm) and for AlexaFluor 647 (λexc 650/ λexc 671 nm).

Proof of concept: TNFα knockdown in LPS-stimulated cells

The gene silencing capacity of siRNA was evaluated in an acute inflammation model using siRNA targeting TNFα. For this purpose, Raw264.7 cells were seeded in a 96-well plate (2.5 × 104 cells/well) and and after an overnight period, the culture medium was replaced with fresh culture medium containing LPS (1 μg/mL) and the formulations for 12, 24, and 48 h. The formulations were used at the following concentrations: naked siTNFα 150 nM; LCNp-P407 or LCNp-P188 at 1.5 or 3 × 108 particles/mL with or without siTNFα 150 nM. At the end of the experiment, the supernatants were collected and TNFα levels were quantified by ELISA, following the manufacturer's instructions. All measurements were performed in quadruplicate.

Cutaneous distribution of the siRNA-LCN complex in porcine skin

The intensity and cutaneous distribution of LCNp-complexed siRNA AF647 (10 μM) in skin were examined by CLSM. Skin samples (dermatomized to a thickness of 500 μm), according to OECD Guideline 428 (Guidelines, 2004), were placed in a Franz Phoenix® automated vertical diffusion cell (Teledyne Hanson Research, USA). The recipient solution consisted of sodium phosphate buffer (DEPC water; pH 7.4 ± 0.2) and was maintained at 32 °C under constant agitation of 400 rpm. The naked siAF647, LCNp-P407-siAF647, and LCNp-P188-siAF647 formulations were applied to the donor compartment. After 2, 6, 12, and 24 h, the skins were collected and cryopreserved for later processing (cryostatic microtome Leica, Germany). The histological cryo-sections were stained with DAPI and CLSM images were obtained using a Leica TCS SP8 CLSM microscope (Leica Microsystems Inc., USA) equipped with a 40× oil immersion objective and laser for DAPI (λexc 358/λem: 461 nm) and for AlexaFluor 647 (λexc 650/ λem 671 nm).

Data analysis

Data are presented as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 9. Comparisons were conducted using one-way or two-way ANOVA followed by appropriate post hoc tests, as indicated in figure legends. Differences were considered significant at p < 0.05.