Materials.

The ionizable cationic lipid O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraem-19-yl)-4-(N,N-dimethylamino)butanoate (DLin-MC3-DMA) was synthesized at AstraZeneca. The 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was obtained from Avanti Polar Lipids, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG₂₀₀₀) was obtained from NOF Corporation, and Cholesterol (Chol) was obtained from Sigma–Aldrich. Deuterated DSPC (d83) and Chol (d7) were obtained from Avanti Polar Lipids. The ³H-labeled DSPC was synthesized at AstraZeneca. Erythropoietin (EPO) mRNA (858 nucleotides) ARCA capped modified with 5-methylcytidine and pseudouridine was purchased from TriLink Biotechnologies. Polyadenylic acid (PolyA) was purchased from Sigma–Aldrich and had a molecular weight range between 600 and 4,000 bases according to analysis by agarose gel electrophoresis in formamide performed by the manufacturer. Hydrogeneous PBS (1 mM KH₂PO₄, 155 mM NaCl, and 3 mM Na₂HPO₄ 0.7H₂O, pH 7.4) was obtained from Life Technologies, whereas deuterated PBS was prepared with D₂O from Sigma–Aldrich. Citrate buffer was purchased from Teknova, and HyClone RNase free water was obtained from GE Healthcare Cell Culture.

LNP Preparation and Characterization.

LNPs were prepared using the microfluidic setup described in detail elsewhere (12). Briefly, stocks of lipids were dissolved in ethanol and mixed in the appropriate molar ratios to obtain a lipid concentration of 12.5 mM (1.85 mg/mL). In the experiments that required radiolabeled DSPC, 1.8 nmol radiolabeled ³H-DSPC was added per formulation, which corresponds to a radioactivity of 5 MBq per formulation. mRNA was diluted in RNase free 50 mM citrate buffer pH 3.0 to obtain a mRNA:lipid weight ratio of 10:1 (CIL:nucleotide 3:1 molar ratio). Empty LNPs were also prepared using 50 mM citrate buffer as the aqueous phase. The aqueous and ethanol solutions were mixed in a 3:1 volume ratio using a microfluidic apparatus NanoAssemblr, from Precision NanoSystems Inc., at a mixing rate of 12 mL/min. LNPs were dialyzed overnight against 500× sample volume using Slide-A-Lyzer G2 dialysis cassettes from Thermo Scientific with a molecular weight cutoff of 10 K. For SAXS, SANS, and cryo-TEM measurements, the particles were concentrated after dialysis to ∼90 mg/mL of lipid using Amicon ultracentrifugation filters.

The size of LNPs was determined by DLS measurements using a Zetasizer Nano ZS from Malvern Instruments Ltd. The number- and volume-weighted particle size distributions were calculated using a particle refractive index of 1.45. The particle concentration (number of LNPs per volume), surface area, and mRNA molecules per LNPs were computed using known molecular volumes (Table S1) and the particle composition, including the water content. The encapsulation and concentration of mRNA were determined using the RiboGreen assay. The encapsulation in all of the samples was typically 90–99%. All of the samples were prepared within a week of use to ensure the chemical stability of the components and no history-dependent in vitro efficacy.

Preparation of Bulk Phases of LNPs.

DLin-MC3-DMA and Chol were dissolved in ethanol and mixed in the appropriate molar ratio to a lipid concentration of 50 mM (23 mg/mL). A total of 1.385 mL of the lipid mixtures were placed in Slide-A-Lyzer G2 dialysis cassettes from Thermo Scientific with a molecular weight cutoff of 10 K with polyA (0.415 mL of 16.4 mg/mL polyA in water, 3:1 CIL:nucleotide molar ratio) to a total final volume of 1.8 mL. The samples were mixed inside the dialysis cassette and placed rapidly in the dialysis buffer. The final concentrations of the lipid mixture and polyA were 21.1 and 2.9 mg/mL, respectively. The samples were dialyzed initially for 2 d against 900 mL (×500 volume of the sample) 50 mM citrate buffer pH 3:ethanol 3:1 by volume mixture, followed by dialysis for 2 d against 900 mL of PBS buffer. The supernatant was removed from the cassettes, and the solid dispersions were extracted and characterized using SAXS.

Patient Consent.

Samples of adipose tissues were collected from patients undergoing elective surgery at Sahlgrenska University Hospital in Gothenburg, Sweden. All study subjects received written and oral information before giving written informed consent for the use of the tissue. The studies were approved by The Regional Ethical Review Board in Gothenburg, Sweden.

Isolation and Culture of Human Adipose-Derived Stem Cells.

Human s.c. white adipose tissue was obtained from healthy women undergoing elective fat removal. Human adipose-derived stem cells (hASCs) were isolated from the stromal vascular fraction as described in Bartesaghi et al. (37) and cultured in a growth medium DMEM/Ham’s F-12 with 10% FBS, 10 mM Hepes, 33 µM biotin, 17 µM pantothenate, 1 nM Fibroblast growth factor (bFGF), all from Sigma–Aldrich, 50 U/mL penicillin and 50 µg/mL streptomycin at 37 °C, 5% CO₂ in air with 80% humidity. For adipocyte differentiation, 90% confluent cells were treated with DMEM/F12 with 3% FCS (PAA; Gold) supplemented with 1 µM dexamethasone (Sigma), 500 µM 3-isobutyl-1-methyxanthine (IBMX; Sigma), and 100 nM insulin. To promote white adipogenesis, 1 µM pioglitazone (Pio) was included in the differentiation medium. Media was changed every other day during proliferation and differentiation, until fully differentiated (day 14). hASCs were tested for and free of mycoplasma.

Culture of iPSC-Derived Hepatocytes.

iPSC-derived hepatocytes (iCell Hepatocytes; Cellular Dynamics International) were cultured in line with the manufacturers protocols. Human hepatocytes were validated by hepatic morphology, hepatic protein staining, and hepatic Cytochrome P450 activity (for details, see https://cellulardynamics.com/). Briefly, cells were thawed rapidly and transferred to prewarmed (37 °C) Kryothaw media (KryoThaw-I-250B; SciKon). Viable cells were isolated by centrifugation at 110 × g for 10 min. Pelleted cells were resuspended in room temperature RPMI medium (Life Technologies) containing 1:50 dilution of B27 (Life Technologies), 20 ng/mL Oncostatin M (R&D Systems), 1 µM dexamethasone (Fisher Scientific), and 25 µg/mL Gentamicin (Life Technologies). Cells were plated at 300,000 cells/cm² in 96-well Biocoat plates (Becton Dickinson) in the above described media at room temperature with the media further supplemented with 250 µg/mL Matrigel (Becton Dickinson) and transferred to an incubator (37 °C, 5% CO₂). Following a 4-h incubation, the medium was substituted to RPMI media containing 1:50 dilution of B27, 20 ng/mL Oncostatin M, 0.1 µM dexamethasone, and 25 µg/mL Gentamicin (Life Technologies) and also containing 250 µg/mL Matrigel as before. Twenty-four hours later, media was substituted to Williams E medium (Life Technologies) containing 1:25 dilution of Hepatocyte maintenance mixture B (Life Technologies) and 0.1 µM Dexamethasone. Media was exchanged every 24 h using the latter described media over the next 4 d to allow maturation before subsequent experimental procedures. The iPSC derived hepatocytes have been extensively characterized based on morphology and protein markers definitive for hepatocytes (including, e.g., albumin and cytochrome P450 enzyme) as well as functionality representative of hepatic biology. iPSC were tested for and free of mycoplasma.

LNP Uptake and hEPO Quantification.

Fully differentiated primary white adipocytes and iPSC-derived hepatocytes were treated with 125 ng of radiolabeled mRNA–LNPs in the presence of 1% human serum. LNP uptakes were quantified using a microbeta luminescence counter (PerkinElmer) as function of time. Uptake of LNPs is expressed as the percent of hEPO mRNA dosed as a function of time. Human Erythropoietin Quantikine IVD ELISA Kit (R&D System) was used to quantify the hEPO levels in culture media collected at several time points post-NP transfection. Data are reported as number of hEPO expressed per mRNA dosed as a function of time or after 48 h of initial dosing. The data correspond to triplicates, which is a standard choice when it comes to biological replicates in in vitro cell biology experiments. The experiments also showed high reproducibility, which enables SD calculations.

Small-Angle X-Ray Scattering.

SAXS experiments were performed using a Ganesha 300XL instrument from SAXSLAB Aps. This instrument is equipped with a Genix 3D microfocus source generating X-rays of a wavelength λ = 0.154 nm. The method measures the scattered intensity on a 2D 300 K Pilatus detector as a function of the momentum transfer, q. The setup employed for the measurements was a two-pinhole system with a beam stop size of 2 mm and a sample-to-detector distance of 350 mm, which leads to a q range of 0.14 < q (nm⁻¹) < 7.53. The measurements of LNPs were carried out with reusable quartz capillaries of 1.5 mm diameter from Hilgenberg GmbH. The concentration of the samples for these measurements was 10–12 mg/mL of lipid. Each sample was measured for 2 h. The data presented are background subtracted, where the background corresponds to the buffer measured in the same capillary. The samples corresponding to the bulk phases were measured for 30 min in a thermostated sandwich holder with disposable mica windows. All of the samples were placed in a thermostated block connected to a circulating water bath to maintain the temperature at 25 °C.

Small-Angle Neutron Scattering.

SANS experiments were performed using the KWS-2 instrument operated by Jülich Centre for Neutron Science (JCNS) at Forschungs-Neutronenquelle Heinz Maier-Leibnitz (38). These measurements are based on the interactions of neutrons with matter, which are scattered by the atomic nuclei. The contrast of a specific material is known as the scattering length density (SLD). Table S1 shows the SLD for neutrons and X-rays of the different lipids and RNA.

Three different instrument setups were employed: 1.2- and 7.8-m sample-to-detector distance with a neutron wavelength λ = 0.5 nm and 20-m sample-to-detector distance with λ = 1 nm. These configurations cover the q range of 0.0133 < q (nm⁻¹) < 4.11. The measurements were done in Hellma quartz disk-shaped (“banjo”) cuvettes of 1- and 2-mm path length. LNPs were diluted in the appropriate solvent ratio of D₂O/H₂O to a final concentration of 3 mg/mL. The samples were placed in a thermostated block to maintain the temperature at 25 °C. The measurement time was adjusted between 0.5 and 3 h depending on the recorded scattering intensity. The data were background subtracted (solvent) and corrected for empty cell and transmission.

Small-Angle Scattering Data Evaluation.

The analyses of the small-angle scattering (SAS) data were performed using the software SasView. The lipid composition across the LNP was determined using the sample with the lipid molar composition DLin-MC3-DMA:DSPC:Chol:DMPE-PEG₂₀₀₀ of 50:10:38.5:1.5, using deuterated DSPC and Chol. The SANS data from the isotopic contrast variation experiments were fitted using the OnionExpShellModel that describes the form factor of a core–multishell sphere where the SLD of the shell can have an exponential decay. The parameters to fit were the thickness of the lipid monolayer and PEG layer, the radius of the core, the SLD of the core and the lipid monolayer, and the exponential decay factor for the SLD of the PEG layer. These parameters were optimized using the simultaneous fitting option with the complex ParkMC fit engine.

Cryogenic Transmission Electron Microscopy of LNPs.

Cryo-TEM experiments were performed as described by Bello et al. (39). A small drop of a concentrated LNP formulation (lipid concentration of 10–20 mg/mL) was placed on a polymer-coated carbon-reinforced copper grid. The grid was bloated with a filter paper to remove the excess of liquid to form a thin film. The samples were vitrified rapidly in liquid ethane at −180 °C to prevent crystallization of water. The preparation was done immediately before examination, and they were kept below −165 °C during the experiment. Cryo-TEM measurements were performed using a Zeiss Libra 120 instrument from Carl Zeiss Nano Technology System operated at 80 kV. The images were recorded with a CCD camera from TRS GmbH and processed with iTEM software from Olympus Soft Imaging Solutions GmbH. The size distributions were calculated from three high-quality images for each LNP sample (>100 particles for each LNP formulation).

Freeze Fracture of Bulk Phases.

Small amounts of the bulk phase samples were placed on small gold cups and rapidly frozen in liquid propane. Three samples were prepared at each freeze fracture preparation, and five freeze fracture preparations were performed, i.e., 15 areas to image. The rapidly frozen bulk phase samples were then transferred into a precooled (−170 °C) vacuum chamber of a Balzer BAF 400 freeze etching system and put under vacuum immediately. The samples were tempered to −105 °C before the freeze fracture, and then they were etched to enhance the structure at the newly fractured surface through sublimation of water by placing a cold trap above the sample. Subsequently, they were rotary shadowed by first platina at an angle of 45° followed by carbon at an angle of 80°. The platina and carbon were evaporated on the fractured surface under vacuum at −105 °C. The thicknesses of the deposited metals were controlled with a quartz crystal monitor, which gave a thickness of the evaporated platina layer of 2 nm. The samples were cleaned from the metal replicas using solvents and acid. The clean replicas were then placed on 400 mesh copper grids before analysis in a transmission electron microscope LEO 906 E at an accelerating voltage of 80 kV.

Solubility of Chol Nanocrystals in DLin-MC3-DMA Emulsion.

A DLin-MC3-DMA emulsion was prepared with a concentration of 11.45 mg/mL in a 50-mM citrate buffer pH 3:ethanol mixture 3:1 by volume with 1.9 mg/mL of DMPE-PEG₂₀₀₀ as stabilizer. The sample was placed on an ultrasonic bath (Elma Transsonic Bath T460/H) for ∼30 min followed by sonication in a focused ultrasonic bath (Covaris S2) at high intensity for 20 min at 18 °C. The emulsion had an average size of 150 ± 10 nm as measured by DLS (zetasizer Nano ZS; Malvern Instruments Ltd). Chol nanocrystals were manufactured by wet milling. A suspension of 9.2 wt % Chol with 2 wt % of DMPE-PEG₂₀₀₀ in milli-Q water was sonicated in an ultrasonic bath for 30 min, stirred over night at room temperature, and sonicated again before milling. Wet milling of 550 μL of sample was performed using a Fritsch Planetary Micromill P7 equipped with 1.2 mL milling bowls and 0.6–0.8 mm milling beads of zirconium oxide, at 44 × g, for 4 × 30 min, with 15 min pauses in between. The sample was extracted from the milling vessel, and the beats were rinsed four times with 550-μL aliquots of milli-Q water. The final concentration of Chol and DMPE-PEG₂₀₀₀ was determined by HPLC. Chol nanosuspension had an average size of 160 nm as measured using a Mastersizer 2000 (Malvern Instruments Ltd.).

The solubility of Chol at 25 °C in the DLin-MC3-DMA emulsion in 50 mM citrate buffer, pH 3:ethanol 3:1 by volume mixture was determined by recording the intensity of the light scattered with a luminescence spectrometer (Perkin–Elmer LS 55) as described by Lindfors et al. (40). The solubility was determined by a extrapolating a linear fit to the high-concentration data to the line of zero intensity. All of the samples were prepared with a fixed concentration of DLin-MC3-DMA (1 mM) and varying concentration of Chol (between 0.05 and 1 mM), and they were left stirring overnight. The mixtures were further diluted by a factor of 100 before the measurements. The emission and excitation wavelengths were set to 700 nm, and the scattered intensity was recorded at an angle of 90°.