Expression and purification of Humanized Ferritin
A. fulgidus humanized ferritin (Hum-Ft) was expressed in E. coli and purified (Figure S1) as previously described [37]. Briefly, 100 g of fermented bacterial paste were resuspended and sonicated in 1 L of 20 mM HEPES buffer pH 7.4 containing 50 mM MgCl2 and protease inhibitors. The soluble fraction was treated with 20% (NH4)2SO4. The supernatant was extensively dialyzed versus 10 mM sodium phosphate buffer pH 7.2 containing 20 mM MgCl2 and then digested with 20 mg of deoxyribonuclease I (Sigma Aldrich) for 1 hr at 37°C. The sample was centrifuged and subjected to 2 sequential heat treatments at 62°C and 72°C. At each step, the denatured proteins were removed by centrifugation, and the soluble fraction was filtered under vacuum using 20 g of Sartoclear Dynamics® Lab Filter Aid (Sartorius) containing highly pure diatomaceous earth (Celpure® C300 – pharmaceutical grade). DNA removal was achieved by a crossflow ultrafiltration step using Vivaflow 200 module (Sartorius) with a cut-off of 100 kD. The same device was used in diafiltration mode to exchange the buffer with 20 mM HEPES pH 7.4 containing 50 mM MgCl2.
PAMAM and HumFt labelling
G4 PAMAM dendrimer (Sigma Aldrich) was labelled with fluorescein-isothiocyanide (FITC) (Sigma Aldrich). 17 μL of PAMAM (57 mM) were added to 2 mL of FITC 1 mg/mL (5.14 mM) in methanol [38]. The reaction was stirred in the dark overnight at room temperature. The PAMAM-FITC solution was dried with a nitrogen stream and resuspended in 2 mL of 20 mM HEPES buffer pH 8.0. The unreacted dye was removed by dialysing the sample versus 20 mM HEPES buffer pH 7.4 for 24 hrs in the dark. FITC-labelled PAMAM was characterized by HPLC (Figure S2) and NMR analyses (Figures S3 and S4).
HPLC analysis was performed with an Agilent Infinity 1260 HPLC apparatus equipped with UV and fluorometric detectors. The separation was carried out using a Halo C18 AQ column (3 x 150 mm, 2.7 µm) connected to the C18 AQ guard column (3 x 5 mm, 2.7µm). The elution was performed at a flow rate of 0.8 mL/min, with solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). The mobile phase was linearly increased from 0% to 100% of solvent B in 15 min and then run isocratically for 5 min. Afterwards, buffer A was reintroduced in the mobile phase up to 100%, and the column was allowed to equilibrate for 10 min. The elution profile of G4 PAMAM was monitored by setting the UV detector at 215 nm. G4 PAMAM-FITC elution was followed by setting the fluorometric detector at λex 490 nm and λem 525 nm.
1H NMR spectra were recorded on a Bruker Avance 400 spectrometer equipped with Prodigy cryoprobe, using D2O/H2O buffer pH 8.5 mixture as solvent at 25°C. Water signal was reduced by presaturation during relaxation delay and mixing time using the noesygprpr1D sequence (from Bruker suite of programs).
HumFt was labelled with Alexa Fluor 647 NHS ester (succinimidyl ester) (ThermoFisher Scientific) according to the manufacturer’s standard protocol. 10 mg/mL of purified protein were equilibrated in carbonate buffer 0.1 M pH 9.0 containing 100 mM MgCl2. 0.1 mL of Alexa Fluor 647 1 mg/mL in DMSO were added to 1 mL of HumFt solution and the reaction mix was incubated for 1 hr in the dark at room temperature. To remove the unreacted dye, the labelled protein was dialyzed versus 20 mM HEPES buffer pH 7.4 containing 50 mM MgCl2. Protein labelling was checked by UV-vis spectroscopy and High-Performance Size Exclusion Chromatography (HP-SEC) following Alexa Fluor 647 emission signal (λex 650 nm, λem 665 nm) (Figure S5).
PAMAM mediated HumFt assembly
G3, G4, G5 PAMAM dendrimers were screened for their ability to induce HumFt association at different HumFt:PAMAM molar ratio (1:10, 1:20, 1:30) in the absence of MgCl2. 1 mL of open HumFt 5 mg/mL (10 μM 24-mer) equilibrated in 20 mM HEPES buffer pH 7.4 was added dropwise to 1 mL of dendrimer (100, 200, 300 μM in 20 mM HEPES buffer pH 7.4). Association equilibrium was evaluated by HP-SEC. HP-SEC was performed using an Agilent Infinity 1260 HPLC apparatus equipped with a UV detector. The separation was carried out using an Agilent AdvanceBio SEC 300Å LC column (7.8 x 150 mm, 2.7 µm). The isocratic analysis was carried out with 20 mM HEPES buffer pH 7.4 as mobile phase. The flow rate was 0.7 mL/min over an elution window of 15 min. HumFt assembly was monitored following UV detection at 220 nm.
HumFt-PAMAM-miRNA nanoparticle preparation
1 mL open HumFt 5 mg/mL (10 μM 24-mer) equilibrated in 20 mM HEPES buffer pH 7.4 was added dropwise to 1 mL of G4 PAMAM 4.3 mg/mL (300 μM) in the same buffer. MgCl2 was added at a final concentration of 50 mM and the sample was incubated overnight at room temperature under stirring. To completely remove free PAMAM, HumFt-PAMAM complex was loaded onto a PD-10 Desalting Column (GE Healthcare) equilibrated with 20 mM HEPES buffer pH 7.4 containing 50 mM MgCl2. The protein sample was submitted to a second PD-10 purification step and then concentrated by using Amicon® Ultra-15 Centrifugal Filter Unit with a cut-off of 30 kD to a final protein concentration of 10 μM (Figure S6). Labelled nanoparticles were prepared in the same condition described above by using Alexa Fluor 647 labelled HumFt and fluorescein labelled G4 PAMAM. Both unlabelled and labelled nanoparticles were analysed by HP-SEC as described above. The isocratic analysis was carried out with 20 mM HEPES buffer pH 7.4 containing 50 mM MgCl2 as mobile phase. Nanoparticles containing Alexa Fluor 647 labelled protein or FITC labelled PAMAM were also detected by a fluorometric detector (λex 650 nm and λem 665 nm for Alexa Fluor 647; λex 490 nm and λem 525 nm for FITC).
Protein standard of highly purified HumFt was prepared in the same solution as the mobile phase. HumFt concentration was determined using the theoretical ε280=32430 M−1cm−1.
HumFt-PAMAM-miRNA nanoparticle was prepared adding pre-miRNA-145 (miR-145-5p precursor #AM17100, assay ID PM11480, ThermoFisher Scientific, Waltham, MA USA) or pre-miRNA Precursor Negative Control (#AM17110, ThermoFisher Scientific, Waltham, MA, USA), at a final concentration of 133 nM, to the preassembled HumFt-PAMAM nanoparticle (10 μM) and incubating at 4°C for 5 hours. The formation of the ternary complex with miRNA was monitored by HP-SEC following the absorbance at 260 nm and agarose gel electrophoresis coupled to a GelDoc Imaging System (Biorad). All solutions were prepared by using RNase-free water, tips, and tubes.
Negative staining electron microscopy
Formvar-carbon coated grids were floated onto 20 μL of protein solutions (0.02 mg/mL) for 5 min for adsorption and the excess of sample solution was blotted gently by filter paper. The air-dried grids were stained with an aqueous solution of 2% (w/v) uranyl acetate or 4% ammonium molybdate for 30 s. The excess staining solution was removed carefully. The grids were observed with an EM208S transmission electron microscope (FEI - ThermoFisher Scientific; 208 Eindhoven - The Netherlands) at an acceleration voltage of 100 kV. Electron micrographs were taken with a slow-scan camera (MEGAVIEW III, OLYMPUS).
High magnification protein images were analysed by the freeware software ImageJ (version 1.29, NIH, Bethesda, MD). To calculate the diameter size of the proteins, manual measures of more than 150 particles were taken for each sample and the diameter size distributions were conducted by Excel 2016.
Cell culture, cell death, and differentiation
The NB4 cell line was obtained by DSMZ (Branuschweig, Germany). Cell cultures were routinely tested for mycoplasma contamination and the cells were cultured and treated in RPMI 1640 medium with the addition of penicillin/streptomycin and 10% FCS (Gibco, ThermoFisher Scientific, Waltham, MA, US). NB4 cells were treated with 0.3 μM HumFt, HumFt-PAMAM, and HumFt-PAMAM-pre-miRNA nanoparticles prepared as described above.
Cell death was analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, US) after staining the cells with 2.5 μg/mL propidium iodide (Sigma Aldrich). Cell differentiation was assessed by morphological analysis of cytospin preparations, obtained by spinning about 300,000 cells/slide, stained with Wright-Giemsa (Sigma Aldrich). Slides were examined with a Zeiss Axioskop 2 microscope and images were acquired with the AxioCam HRc and the Axiovision 4.8 software (Zeiss, Oberkochen, Germany).
RNA extraction and Real-Time PCR
Total RNA was isolated by TRIzol (Ambion, ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and the quality of purified RNA was checked by OD 260-280 readings. 250 ng of total RNA was reverse-transcribed with the High-Capacity RNA to cDNA kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, US) in a final volume of 8 μL. cDNA was diluted in RNase-free water and 1 μL of this was used for Real-Time PCR to assess the expression of RARα gene by TaqMan MicroRNA assay (Applied Biosystems, Foster City, CA, US) following the manufacturer’s instructions. The fold increase of RARα was calculated by the ΔΔCt method using the GAPDH gene as the endogenous control for standardization. Reverse transcription of total RNA for detection of miR-145-5p was performed using miScript II RT kit (Qiagen, Chatsworth, CA). Quantification of mature miR-145-5p expression was carried out by miScript SYBR Green PCR kit (Qiagen, Chatsworth, CA, US), using miScript Primer Assay Hs_miR-145_1 (MS00003528 Qiagen, Chatsworth, CA). The fold increase of miRNA-145-5p was calculated by the ΔΔCt method using Hs_SNORD68 RNA (miScript Primer Assay MS00033740 Qiagen, Chatsworth, CA) as the endogenous control for standardization. All reactions were performed in the Quant Studio 7 Real-Time PCR system (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, US). The experiment was performed in biological duplicates and one-way ANOVA statistical analysis was performed using the GraphPad-Prism 6 software (GraphPad Software, La Jolla, CA, US).
Confocal microscopy and FACS analysis
NB4 cells were incubated for 24 hrs with 0.3 μM G4 PAMAM-FITC, or with 0.3 μM HumFt-Alexa Fluor 647, or with 0.3 μM of the double dye-labelled HumFt-PAMAM nanoparticle, prepared as described above. The uptake of the labelled molecules was assessed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, US) and the data were analyzed by the CytExpert Software (Beckman Coulter, Brea, CA, US).
The same cells were examined by confocal microscopy to assess the intracellular localization of the compounds. Cytospin preparations obtained as described above were fixed in 4% PFA for 7 min. DNA was counterstained with Hoechst 33,342. Confocal laser scanning microscopy (CLSM) was performed using a Zeiss LSM 980 with Airyscan2, using the 63x oil objective and excitation spectral laser lines at 405, 488, and 633 nm. Signals from the different fluorescent probes (Hoechst, FITC, and Alexa Fluor 647) were taken in sequential scanning mode to avoid spectral overlap (false-positive signal). CLSM analysis was performed both at the central optical section of each field of observation and as a 3d reconstruction of z-stacks. Co-localization areas were detected in orange. Bright-field images were taken to identify cell morphology. Scale bar are 5 µm.