Nature-inspired platform nanotechnology for RNA supply to myeloid cells and their bone marrow progenitors

February 4, 2025

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Supplies

POPC (Avanti Polar Lipids, 850457)
DMPC (Avanti Polar Lipids, 850345)
DSPC (Avanti Polar Lipids, 850365)
Ldl cholesterol (Sigma-Aldrich)
Tricaprylin (Sigma-Aldrich)
PEG(2K)-DMG (Avanti Polar Lipids, 880151)
DLin-MC3-DMA (SymoChem)
Sodium acetate (Sigma-Aldrich)
12–14 kDa molecular weight cut-off (MWCO) dialysis membrane, Spectra/Por
Quant-iT RiboGreen reagent (Thermo Fisher Scientific)
TE buffer (10 mM Tris-HCl, 1 mM, EDTA, pH 7.5 in DEPC-treated water)
Apolipoprotein A1 FS assay (DiaSYS)
Spin filters 100,000 Da MWCO (Amicon)
LabAssay phospholipids (Fujifilm, Wako)
Ldl cholesterol FS assay (Diasys)
Twin-Luciferase Reporter Assay System (Promega)
HDL plasma fraction (Medix Biochemica)
RAW 264.7 murine macrophages (ATCC TIB-71)
Lipofectamine RNAiMAX (Thermo Fisher Scientific)
Deferoxamine-DBCO (Macrocyclics)
2 kDa MWCO Dialysis tubing (Sigma-Aldrich)
Corning 70 µm cell strainer (Merck)
CD11b MicroBeads (Miltenyi Biotec)

aNP manufacturing

Apolipoprotein nanoparticles containing nucleic acids have been formulated by speedy T-junction mixing as beforehand described in ref. ²⁸. The lipid molecules (POPC; DMPC or DSPC; ldl cholesterol; tricaprylin (PEG-DMG for LNP particles); and DLin-MC3-DMA) have been dissolved in ethanol at ratios laid out in Supplementary Desk 1. siRNA, mRNA or ASO have been dissolved in 25 mM sodium acetate (pH 4). Subsequent, natural and aqueous phases have been combined via a T-junction at a circulation price ratio of 1:3 (organic-to-aqueous) and picked up in a 12–14 kDa MWCO dialysis membrane (Spectra/Por). Nanoparticle formulations have been dialysed towards 1× PBS (pH 7.4) for 4 h and refreshed for dialysis in a single day at 4 °C whereas stirred at 150 r.p.m. Extra natural and aqueous phases have been saved at 4 °C for physicochemical evaluation. On the following day, samples have been collected from the dialysis baggage and the amount was decided. For the aNP particles, the suitable quantity of apolipoprotein A1 (apoA1) in PBS was diluted to one-third of the nanoparticle pattern quantity. ApoA1 was launched to the nanoparticle formulations by speedy T-junction mixing at a circulation price ratio of 1:3 (apoA1 answer/nanoparticle answer). After apoA1 addition, samples have been incubated at room temperature for 1 h for aNP-siRNA and aNP-ASO, and 20 min for aNP-mRNA. The ensuing product was filtered via a 0.2 μm filter, and concentrated and purified by centrifugal filtration utilizing a 100 kDa MWCO filter. Samples have been diluted to the specified siRNA, ASO or mRNA focus and saved at 4 °C till additional use. Aseptic strategies have been used after 0.2 μm filtration.

aNP physicochemical evaluation

The nanoparticle hydrodynamic diameter (expressed because the number-weighted imply diameter) and zeta potential have been decided by DLS utilizing a Zetasizer Nano ZS (Malvern Devices) geared up with a Zetasizer NanoSampler (Malvern Devices). Dimension dispersity was measured because the PDI. For DLS measurements, 100 µl of the formulation pattern was diluted into 700 µl of 1× PBS and equilibrated at room temperature earlier than evaluation. Every pattern was measured 5 instances (10 runs of 10 s) with out fixing the attenuator and measurement place. For zeta potential measurements, the pattern was diluted 50 instances in MilliQ water, and 700 µl of diluted pattern was loaded in a disposable folded capillary cuvette (DTS1070, Malvern Panalytical). 5 measurements have been carried out at intervals of 300 s, and every measurement comprised 40 runs with 40 V utilized. The Quant-iT RiboGreen assay (Thermo Fisher Scientific) was used to quantify the quantity of RNA loaded inside the formulated particles. A normal formulation pattern with a theoretical RNA focus of 133.3 µg ml^–1, was diluted 200 instances in TE buffer each with and with out 2% Triton X-100 in a black 96-well plate to a complete quantity of 100 µl. The Triton detergent disrupts the lipid-based nanoparticles; subsequently, whole RNA (retained and unretained) turns into accessible for the Quant-iT RiboGreen reagent. Identified RNA controls, saved throughout formulation, have been diluted 53.3 instances in TE buffer each with and with out 2% Triton to a complete quantity of 100 µl. Subsequent, RiboGreen reagent was 200-fold diluted utilizing TE buffer each with and with out Triton X-100; 100 µl of this dilution was added to every nicely containing pattern or management to convey the entire quantity to 200 µl. The fluorescence of the samples was then measured on a Tecan Spark microplate reader at an excitation wavelength of 480 nm and emission wavelength of 520 nm. RNA restoration was decided as: (quantity of RNA (retained + unretained))/(whole quantity of RNA (used within the formulation)) × 100%. The entrapment of RNA was outlined as: (1-((quantity of RNA (retained + unretained))/(whole quantity of RNA (used within the formulation)))) × 100%. RNA retention was decided as: restoration × entrapment. Ldl cholesterol was quantified utilizing the ldl cholesterol FS assays (DiaSYS). Briefly, 240 µl of buffer containing color reagent was added to 10 µl of pattern in a clear 96-well plate and incubated at 37 °C for five min. Absorbance was measured at 500 nm utilizing a Tecan Spark plate reader. Phospholipids have been quantified utilizing the LabAssay Phospholipid (FUJIFILM). For this assay, 190 µl of buffer containing color reagent is added to 10 µl of the pattern in a clear 96-well plate and incubated at 37 °C for 30 min. The absorbance was measured at 600 nm utilizing a Tecan Spark plate reader. The quantity of apoA1 was quantified utilizing the Apolipoprotein A1 FS assay (DiaSYS). In brief, 200 µl of Reagent 1 was added to five µl pattern or management in a clear 96-well plate. After incubating the plate at 37 °C for five min, the absorbance was measured utilizing a Tecan Spark microplate reader at 580 nm. Subsequent, 50 µl of Reagent 2 containing an anti-apoA1 antibody was added and the answer was incubated at 37 °C for five min earlier than measuring the absorbance on the similar setting.

ApoA1 isolation from human plasma

Human HDL plasma fraction was bought from Medix Biochemica (Maryland Heights). Utilizing KBr salt, the density was adjusted to 1.22–1.24 g ml^–1, and the answer was centrifuged for 48 h, 250,000 × g at 4 °C. The gel-like pellets have been rigorously collected and dissolved in a single day in Milli-Q on a curler at 400 r.p.m. and 4 °C to a focus of 37.5 mg ml^–1. Subsequent, the HDL answer was dripped at a 0.4 ml min^–1 into methanol/chloroform (50/50 vol%) on dry ice to separate the lipids from the protein. About 50 mg of HDL precipitated in 240 ml solvent. The solvent containing the protein precipitate was decanted and centrifuged at 300 × g for 10 min at 4 °C. The supernatant was rigorously discarded, and the whitish apoA1 pellets collected. The pellets have been dried within the vacuum oven at 37 °C for two–3 h to take away residual solvent. The ensuing white-yellowish flakes have been redissolved in 6 M guanidine answer (5 ml per 50 mg of protein). Lastly, remoted protein was dialysed towards PBS at 4 °C for one week. Phosphate-buffered saline was refreshed day by day (dialysis ratio = 1:100) and three,500 Da MWCO SnakeSkin dialysis membranes (Thermo Fisher Scientific). The dialysate was collected and high quality was assessed through SDS-page, Nanodrop and Apolipoprotein FS assay. ApoA1 was aliquoted, snap-frozen, and saved at −80 °C.

In vitro gene silencing

In vitro silencing experiments have been carried out in a RAW 264.7 (ATCC TIB-71) cell line transfected with the pmirGLO plasmid (containing Renilla and firefly luciferases expressing gene sequences). Cells have been cultured till 80% confluency, indifferent with trypsin, counted and seeded at 25,000 cells per nicely in a 96-well plate. After in a single day restoration, the cells have been transfected with nanoparticles containing both scrambled siRNA (Built-in DNA Applied sciences, IDT) or anti-firefly luciferase siRNA on the desired focus. After incubating for 48 h, the medium was washed off with 1× PBS and lysate phosphate buffer (Twin-Luciferase Reporter Assay System, Promega) was added to lyse the cells; 10 µl of the cell lysate was transferred to a white 96-well flat-bottomed plate. Subsequently, 40 µl of ONE-Glo reagent (Twin-Luciferase Reporter Assay System, Promega) was added and luminescence was measured with a Tecan Spark microplate reader at an integration time of 500 ms and settle time of 1,000 ms. A luminescence scan was carried out to validate the depth peak at 560 nm for firefly luciferase; 40 µl of Cease and Glo reagent (Twin-Luciferase Reporter Assay System, Promega) was then added to every nicely and the luminescence was measured once more in the identical method. The firefly luminescence was normalized to the Renilla luminescence and subsequently expressed as a proportion of the untreated pattern sign.

In vitro exon skipping

Exon skipping experiments have been carried out on NATURA reporter cells³⁷. Upon ASO-induced exon skipping, the reporter cells swap expression from eGFP to tRFP thereby giving quantitative details about the diploma of skipping. In brief, RAW 264.7_PC2 cells (ATCC TIB-71) have been seeded in a 96-well plate at a density of 30,000 cells per nicely. Cells have been left to connect in a single day. Subsequent, the cells have been transfected with aNPs containing both goal or scrambled ASOs at concentrations starting from 12.5–200 nM ASO; 48 h after transfection, cells have been harvested, washed, stained with DRAQ7 (Thermo Fisher Scientific) and resuspended in FACS buffer (1x PBS + 0.5% BSA + 2 mM EDTA). All information have been acquired utilizing the BD FACSAria III Cell Sorter (BD biosciences), eGFP (FITC), tRFP (PE) and DRAQ7 (APC). As a constructive management, RAW 264.7_PCT cells that repeatedly specific tRFP³⁷ have been utilized. The protein proportion spliced is calculated in line with the next components: pPSI = (0.558 × MFI tRFP)/(MFI eGFP + (0.558 × MFI tRFP)). The relative brightness of tRFP in relation to eGFP is adjusted by multiplying by an element of 0.558.

In vitro mRNA expression

RAW 264.7 (ATCC TIB-71) cells have been seeded in a 96-well plate at a density of 30,000 cells per nicely. The cells have been left to connect in a single day. Cells have been then transfected with aNPs containing eGFP-mRNA (RiboPro) at a focus vary of 0–100 ng per nicely. Cells have been harvested 24 h later and ready for circulation cytometry by washing and marking with DRAQ7 (Thermo Fisher Scientific). Lastly, cells have been resuspended in FACS buffer (1x PBS + 0.5% BSA + 2 mM EDTA). All information have been acquired utilizing the BD FACSAriaTM III Cell Sorter (BD biosciences), eGFP (FITC) and DRAQ7 (APC).

Cryogenic transmission electron microscopy

Simply earlier than the vitrification, the floor of 200-mesh lacey carbon supported copper grids (Electron Microscopy Sciences) was uncovered to plasma for 40 s utilizing a Cressington 208 carbon coater. Subsequently, 3 µl of aNP pattern was utilized on a grid and vitrified into a skinny movie by plunge vitrification in liquid ethane utilizing an automatic robotic (FEI Vitrobot Mark IV). Cryo-EM imaging was carried out on the cryoTITAN (Thermo Fisher Scientific), geared up with a area emission gun, a submit column Gatan imaging filter (mannequin 2002), and a post-GIF 2k × 2k Gatan CCD digicam (mannequin 794). The micrographs have been acquired at both 6,500× (electron dose of 1.64 electrons Å⁻² s⁻¹) or 24,000× magnification (electron dose of 11.8 Å⁻² s⁻¹) at 300 kV acceleration voltage within the bright-field TEM mode with zero-loss vitality filtering and a 1 s acquisition time. The scale was quantified by measuring the nanoparticle diameter utilizing ImageJ (NIH). No less than 150 particles have been analysed for every formulation.

ApoA1-immunogold labelling of apoA1 on siRNA-aNPs

Thirty minutes earlier than staining, a copper grid (carbon assist movie, 200 mesh, Electron Microscopy Sciences) was plasma-discharged and positioned on a 20 µl droplet of the aNP pattern (dilution 1:20) for 3 s with the carbon movie dealing with down. The grid was washed 5 instances by incubating it on a 20 µl droplet of PBS for two min every time. Subsequent, it was positioned on 20 µl of 1% BSA in PBS for 3 min after which on 20 µl of mouse anti-human monoclonal apoA1 antibody in 1% BSA (dilution 1:200, clone: B10, Santa Cruz Biotechnology) for 45 min. After washing, the grid was incubated on 20 µl of the bridging antibody in 1% BSA (1:200 dilution, polyclonal rabbit anti-mouse IgG, Jackson Immunoresearch) for 20 min, washed and positioned on 20 µl of 10 nm protein-A-coated gold nanoparticles (1:25 dilution, Cell Microscopy Core, Division of Cell Biology, Utrecht College Medical Middle) for 20 min. Lastly, the grids have been washed six instances in PBS and ten instances in demi water (every wash was 20 µl and a 3 min incubation). The grid was then positioned on a 20 µl droplet of two% uranyl acetate in water for five min. After these steps, the TEM grids have been air-dried in a single day. The gold-labelled nanoparticles have been visualized at room temperature by electron microscopy. The acquisition parameters have been the identical as for cryo-EM described above.

Cryo-EM evaluation of lipid aggregation

Lipid aggregates have been outlined on cryo-EM photographs at 6,500-times magnification as both spherical lipid buildings, bigger than the principle inhabitants nanoparticles and displaying totally different electron densities, or clusters of nanoparticles (Supplementary Fig. 3). The combination space was manually delineated through the use of a bare-hand drawing device in ImageJ. The lipid aggregation was expressed as the share of aggregates within the whole picture space. No less than ten photographs have been analysed for every formulation.

Radiolabelling of aNP-siRNA

The siRNA-azide had the next sequence:

5′-rArCrCrCrUrGrArArGrUrUrCrArUrCrUrGrCrArCrCrArCCG/3AzideN/-3′.

5′-rCrGrGrUrGrGrUrGrGrArGrArUrGrArArCrUrUrCrArGrGrGrUrCrA-3′.

siRNA-azide and DFO-DBCO have been individually dissolved in Milli-Q water containing 5 vol% DMSO to succeed in concentrations of 0.5 mg ml^–1. The siRNA-azide answer (270 μl, 8 nmol) was combined with the DFO-DBCO answer (129 μl, 7.6 nmol) and incubated in the dead of night and at room temperature for 16 h. The answer was dialysed (2 kDa MWCO dialysis tube, Sigma-Aldrich) towards PBS (1 l). The product was freeze-dried, and product formulation was confirmed by MALDI-TOF-MS. The siRNA-DFO remained steady for at the very least a 12 months when saved at −80 °C. Subsequent, a ⁸⁹Zr oxalate answer in 1 M oxalic acid was neutralized utilizing a 1 M sodium carbonate answer till a pH between 6.8–7.4 was reached (whole quantity < 25 μl). The neutralized ⁸⁹Zr answer was added to DFO-siRNA dissolved in Milli-Q water (0.5 ml) and the combination was incubated at 37 °C utilizing a thermomixer (300 r.p.m.) for 60 min. Completion of the response was confirmed by radio-TLC (Storm 7000IP plate reader, GE Healthcare). The ⁸⁹Zr-siRNA was straight used to formulate the aNP-⁸⁹Zr-siRNA, which resulted in radiochemical yields of >80%, assessed by radio-TLC. Subsequent, the aNP-⁸⁹Zr-siRNA have been analysed by Cryo-EM and DLS, to evaluate their morphology, measurement and dispersity, respectively.

Biodistribution over time

Feminine C57BL/6 mice have been intravenously administered with aNP-⁸⁹Zr-siRNA (8 µCi ⁸⁹Zr correlating to 1–1.5 mg kg^–1 siRNA) in 150–200 µl PBS through tail-vein injection. At numerous time factors after injection (15 m, 1 h, 4 h and after 24 h), mice have been euthanized, perfused with PBS, and tissues (bone marrow, spleen, coronary heart, lung, muscle, tail, blood, mind, liver and kidneys) have been collected and weighed. Subsequent, the emitted ɣ radiation was measured by a gamma counter (2480 WIZARD2 Automated Gamma Counter, PerkinElmer). Radioactivity values have been corrected for decay and normalized to tissue weight to specific radioactivity focus as %ID per gram.

PET–CT imaging acquisition and evaluation

Positron emission tomography imaging was carried out 24 h after aNP-⁸⁹Zr-siRNA injection utilizing an IRIS PET–CT (Inviscan). Mice have been anesthetized with a gasoline combination of two% isoflurane and 5% oxygen. A ten min static whole-body PET scan was performed utilizing an vitality window between 250–750 keV adopted by a 20 s whole-body CT scan (vitality 80 kV, 0.9 mA, 576 projections, voxel measurement 160 µm). Reconstruction of the PET photographs was achieved with the 3D Ordered Subsets Expectation Maximization (3D-OSEM-MC) algorithm (eight subsets and eight iterations) utilizing decay, random and dead-time correction. All PET and CT information have been processed utilizing OsiriX Medical Imaging software program (v.13.0.3) as described earlier than⁵³.

RNA isolation and RT-qPCR

Bone-marrow-derived macrophages cultured in RPMI 1640 medium containing sodium pyruvate (1% P/S, 15% L929 conditioned medium) have been incubated for twenty-four h with aNP-siRNA. The NucleoSpin package (Machery Nagel) was used to isolate RNA from 750,000 BMDMs 48 h after incubation. The RNA was reverse transcribed utilizing the iScript cDNA Synthesis Package (Biorad) at 300 ng per response. The ensuing cDNA was diluted tenfold and amplified in a PowerUp SYBR Inexperienced mastermix (Utilized Biosystems) utilizing a QuantStudio 3 (Utilized Biosystems) thermocycler. A remaining focus of 100 nM for the reverse and ahead primer was used (primer (IDT) particulars are listed in Supplementary Desk 2). Thermocycling was carried out for two min at 95 °C adopted by forty 15 s amplification cycles at 95 °C, and an additional forty 30 s amplification cycles at 60 °C, with fluorescence acquisition on the finish of every cycle. Reactions have been performd in duplicate and every plate included an NTC and a reverse transcriptase management. To confirm the amplification of the supposed product a melting curve evaluation was carried out from 65 °C to 95 °C. All amplification information have been normalized to the ROX dye earlier than additional processing. Every gene of curiosity was normalized to 2 housekeeping genes and RT-qPCR, whereas the ΔCq-expression was normalized to aNP-siCtrl at an equal focus. The share knockdown is calculated in line with the next components: %KD = (1 − ∆∆Cq) × 100.

Making ready single-cell suspensions and marking for circulation cytometry

Single-cell suspensions comprised complete blood, spleen and bone marrow tissue retrieved from euthanized feminine C57BL/6J mice. The whole spleen was straight minced on a Corning 70 µm cell strainer utilizing a syringe plunger and circulation buffer (DPBS, 0.5% BSA, 2 mM EDTA). Single-cell suspensions of the bone marrow have been made by flushing the femurs with 10 ml circulation buffer. The bone marrow was then instantly filtered utilizing a 70 µm cell strainer and centrifugated at 400 × g for five min at 4 °C, aspirating the supernatant. The cell pellets from the entire blood, spleen and bone marrow have been resuspended in 1× purple blood cell lysis (catalogue no. 420302, Biolegend). The samples have been then incubated for 3 × 7 min on ice (blood), or at room temperature for 4 min (spleen) or 1 min (bone marrow). Quenching was achieved with circulation buffer and cells have been centrifuged at 400 × g for five min at 4 °C. Subsequent, the one cells have been transferred to a Corning V-bottom 96-well plate and incubated with 50 µl viakrome 808 (catalogue no. C36628, Beckman Coulter) diluted in PBS for 20 min in the dead of night at room temperature. The cells have been then washed, centrifuged once more and incubated with CD16/32 (Fc-block, 101302, BioLegend) antibodies (apart from the progenitor staining group) for 10 min on ice. The cells have been washed, centrifuged, and good stain buffer (Invitrogen) containing antibodies have been added (whole quantity of fifty µl). The pattern was then incubated on ice for 30 min in the dead of night. All information have been acquired utilizing a 21-colour CytoFLEX LX (Beckman Coulter). The antibodies used for the myeloid cells panel and progenitor cells panel are listed in Supplementary Tables 5–8.

Medical chemistry and cytokine measurements

Feminine C57BL/6J mice have been intravenously administrated with 0.5 mg kg^–1 aNP-siRNA or PBS (n = 4 per group). After 4 h, serum was collected, and cytokine (IL-6 and TNF-α) ranges have been measured utilizing enzyme-linked immunosorbent assay (ELISA) kits (ELISA MAX Deluxe Set Mouse IL-6 (Biolegend) and ELISA MAX Deluxe Set Mouse TNF-α, Biolegend) in line with the producer’s directions. After 24 h and 72 h, complete blood was collected in BD Vacutainer Heparin Tubes (Becton Dickinson) and the serum was separated. The liver perform (ASAT and ALAT) and renal perform (urea and creatinine) have been measured on the Radboudumc Laboratorium voor Diagnostiek core facility.

Liver histology

Mice have been euthanized and perfused with PBS. Entire livers have been collected and stuck in 10% formalin for twenty-four h. The tissue was subsequently processed and embedded in paraffin wax on the diagnostic pathology division. Paraffin sections (5 µm thickness) have been minimize utilizing a Leica RM2235 microtome, mounted on Superfrost Plus Microscope slides (VWR), and stained with haematoxylin and eosin. The slides have been scanned and visualized utilizing CaseViewer software program (3DHISTECH).

Inbred mice pressure

All animal experiments have been performed in compliance with European Union and Dutch pointers in line with the care and use of laboratory mice after approval by the Radboud College Medical Middle’s Dierexperimentencommissie (DEC) (CCD License = AVD10300 2021 15550); 8–12 weeks previous feminine C57BL/6J mice (Mus musculus) obtained from Charles River have been co-housed in climate-controlled circumstances (20–24 °C and a humidity stage of 45–65% RV). An acclimatization interval of 1 week was used and mice had entry to meals and water advert libitum. Mice have been randomized and assigned to regulate and remedy teams. Research have been performed in a blinded method.

Tumour inoculation and remedy routine

Feminine C57BL/6J mice have been inoculated with 5 × 10⁵ MC38 cells in 100 µl PBS through subcutaneous injection in the best flank on day –10. Therapy for all experiments consisted of intravenous administration (7 × 0.5 mg kg^–1 in 14 days, 48 h aside) of aNP₁₈-siCtrl or aNP₁₈-siCCR2. Therapy began on day 0 and lasted till day 14. The examine was stopped at day 14 whereas tumour volumes have been <2,000 mm³ (humane endpoint) to adjust to Dutch laws on animal welfare. Minimal group sizes have been decided by statistical energy calculations (Piface) whereas preserving charges for kind I and II errors <5% and an influence of 80%.

Statistical evaluation

All information are offered as imply ± s.d. or normal error of imply and analysed utilizing GraphPad Prism v.10.0 by one-way ANOVA or Pupil’s t-test, as detailed within the determine captions. P-values < 0.05 have been thought-about vital, with ranges of significance indicated as: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Knowledge distribution was assumed to be regular however this was not formally examined.

Determine design

Schematic figures have been ready utilizing BioRender (BioRender.com) and Servier Medical Artwork (good.servier.com).

Reporting abstract

Additional info on analysis design is obtainable within the Nature Portfolio Reporting Abstract linked to this text.