Prolonged intracellular accumulation of light-inducible nanoparticles in leukemia cells allows their remote activation

Leukaemia cells that are resistant to conventional therapies are thought to reside in protective niches. Here, we describe light-inducible polymeric retinoic acid (RA)-containing nanoparticles (NPs) with the capacity to accumulate in the cytoplasm of leukaemia cells for several days and release their RA payloads within a few minutes upon exposure to blue/UV light. Compared to NPs that are not activated by light exposure, these NPs more efficiently reduce the clonogenicity of bone marrow cancer cells from patients with acute myeloid leukaemia (AML) and induce the differentiation of RA-low sensitive leukaemia cells. Importantly, we show that leukaemia cells transfected with light-inducible NPs containing RA can engraft into bone marrow in vivo in the proximity of other leukaemic cells, differentiate upon exposure to blue light and release paracrine factors that modulate nearby cells. The NPs described here offer a promising strategy for controlling distant cell populations and remotely modulating leukaemic niches.

in the absorbance properties of PEI-DMNC (1 mg/mL in DMSO), PEI (1 mg/mL in DMSO) and DMNC (250 μg/mL, in DMSO). The experimental DS of PEI-DMNC was 10% as determined by spectrophotometry, using a calibration curve of DMNC, and by 1H NMR. For DMNC, the absorption maximum at 355 nm reverted to baseline levels after 10 min of UV exposure, indicating the photo-cleavage of DMNC, and a new absorption peak was observed at 320 nm, due to the formation of 4,5-dimethoxy-2-nitrobenzyl alcohol. For PEI-DMNC, there was a decrease in the intensity of the peak at 355 nm and a concomitant increase in the peak at 320 nm; however our results suggest that not all the attached DMNC molecules were photo-cleaved. (B) 1H NMR spectra of PEI, DMNC and PEI-DMNC. 1H NMR spectra (in DMSO-d6) of (a) PEI-DMNC conjugate, (b) DMNC and (c) PEI. of NPs with (right) and without (left) DMNC photochrome. A suspension of NPs (100 μL, 100 μg, in water) was exposed to a blue laser up to 20 min. Then, the NP suspension was diluted up to 50 μg/ mL in water and the size, zeta potential and number of NPs (Kcps) in the suspension was evaluated by dynamic light scattering. In B results are expressed as Mean ± SEM (n = 5). (C) Confocal images showing light-disassembly of quantum dots 525 (Qdot525)-labeled NPs. A section of a NP aggregate (area delimited in the figure) was bleached continuously by a laser at 405 nm as confocal images were collected every 20 s. The images show the disassembly of the bleached area of the NP aggregate. Fluorescence intensity of the area bleached by the laser and reference area (i.e., not activated by the laser) overtime. Our results show that fluorescence intensity increases after light exposure due to the disassembly of the NP and the decrease in the quenching of Qdot fluorescence after NP disassembly. (D) Confocal imaging of HUVECs after exposure for 4 h to QDot525-labelled NPs. A small section of the cell (region 1, created by a mask) was then exposed to blue light laser cycles (405 nm) in a Zeiss confocal microscope and the intensity of fluorescence at 525 nm monitored. In parallel, the fluorescence of another section of the cell (region 2) not excited with the laser was monitored as a control. Our results show that the fluorescence intensity in region 2 maintains overtime while in region 1 the intensity increases. Blue dots and line presents the blue light laser-exposed area of Qdot525-labelled NPs; orange dots and line presents the control unexposed area of Qdot525-labelled NPs. Dashed areas show cell membrane and nucleus. Nanoparticles (2.5 mg) were placed in PBS (0.5 mL) and incubated under mild agitation at 37 °C. At specific intervals of time, the nanoparticle suspension was centrifuged (at 14.000 g for 3 min) and 0.4 mL of the release medium removed and replaced by a new one. The reserved supernatant was stored at 4°C until the RA content in release samples was assessed by spectrophotometry at 350 nm. Concentrations of RA were determined by comparison to a standard curve. All analyses were conducted in triplicate. SEM is smaller than the symbols. (A) Zeta potential of RA+NPs suspended in water, serum-free RPMI (medium used for leukemia cells) or EBM medium (medium used for HUVECs). It should be noted that the transfection of the cells with RA+NPs was always performed in serum-free media. Diameter (nm) and counts (Kcps) of RA+NPs suspended in water (B), RPMI medium (C) or EBM medium (D). A suspension of RA+NPs (2 mL, 25 μg/mL) was prepared and diameter, counts and zeta potential determined by dynamic light scattering method (DLS) using a Zeta Plus Analyzer (Brookhaven). Results are expressed as Mean ± SEM (n = 3).  Cytotoxicity of RA+NPs against NB4 and U937 cells. Cells were cultured in medium supplemented with light-sensitive RA+NPs for 4 h, washed, exposed or not to a UV light for 10 min, and then cultured for 20 h. Cell cytotoxicity was evaluated by an ATP kit. Results are expressed as Mean ± SEM (n = 3). (B) HUVECs were exposure to 10 min or 60 min of UV light (365 nm, 100 W) (left panel) or blue light (405 nm, 80 mW) (right panel) and allowed to recover for 6 h. Cells were then fixed and stained to identify γH2AX-containing foci, as biomarker for nuclear sites of DNA damage in affected cells. Bar corresponds to 10 μm. (C) Time-dependent increase of γH2AX after UV light (365 nm, 100 W) or blue light (405 nm, 80 mW) irradiation. Quantitative analysis of foci intensity were quantified using imageJ software and normalised to the control condition. Results are expressed as Mean ± SEM. The calculations were performed in 5 different images for a total of ca. 250 cells (ca. 50 cells per image). Cells were cultured in medium supplemented with light-sensitive RA+NPs for 4 h, washed, exposed or not to a UV light for 5 min, and then cultured for 20 h. Cell cytotoxicity was evaluated by an ATP kit (A) or Annexin V/PI staining (B) followed by flow cytometry analyses. In this case, the concentration of RA+NPs was 10 μg/mL. Live cells were negative for Annexin V and PI staining. Results are expressed as Mean ± SEM (n = 3). RA+NP dilution with cell culture was monitored over 6 days by flow cytometry. NB4 cells (500.000 cells/mL) were plated in 6 well plates and incubated in serum-free RPMI-1640 with RA+NPs (20 µg/ mL). After 4 h incubation, cells were washed three times with PBS to remove RA+NPs not internalised and the cells were left to grow at 200.000 cells/mL in complete medium for additional 4 h, 3 days and 6 days. After each incubation, cells were counted, collected by centrifugation and ressuspended in PBS for flow cytometry evaluation. (A) Transport of FITC-labeled transferrin (1 μg/mL) known to selectively enter cells via clathrinmediated endocytosis. Dynasor at concentration of 80 μM inhibits the internalisation of transferrin in U937 cells. Cells were exposed to culture medium with and without dynasor for 30 min, exposed to FITC-labeled transferrin for 3 min, at 4 ºC, and finally characterized by FACS.

Supplementary Figure 12-(A)
Uptake of RA by U937 cells. U937 cells were cultured with soluble [3H]-RA (0.3 or 3 μg/mL) in culture medium for the entire duration of the experiment, or light-activatable [3H]-RA+NPs (1 and 10 μg/mL). NPs were added to cell culture for 4 h. Then, the cells were washed with PBS, and fresh cell medium added and the cells remained in culture for 24/72 h before scintillation counting. (B) Myelocytic differentiation of human THP-1 cells. THP-1 cells were cultured with soluble RA (3 μg of RA per mL) in culture medium for the entire duration of the experiment, or light-activatable RA+NPs (10 μg of NPs/mL, i.e., 1.2 μg of RA per mL), or light-activatable NPs without RA (10 μg of NPs/mL). In the case of cells treated with NPs, cells were treated with RA+NPs for 4 h, washed, activated with UV light (365 nm, 100 Watts) for 5 min, and then cultured up to 24 h or 48 h. In case of cells treated with soluble RA, cells were cultured in media containing soluble RA for the entire period of culture. Results are expressed as Mean ± SEM (n = 3). Statistical analyses were performed by an unpaired t-test. *, ** denotes statistical significance (P<0.05, P<0.01). Cells were treated with RA+NPs for 4 h, washed, activated with UV light (365 nm, 100 W) for 5 min, and then cultured for up to 48 h. In case of cells treated with soluble RA, cells were cultured in media containing soluble RA for the entire period of culture. Cells were then lysed and gene expression profile monitored by qRT-PCR using a Fluidigm equipment. A red-blue color scale was used to reflect standardized gene expression, with red indicating higher expression and blue indicating lower expression. Surprisingly, our clustering analysis indicate that NB4 treated with RA+NPs for the first 24 h were more related to the non-treated cells than soluble RA. Yet this effect is inverted for 48 h. Therefore, our data suggests that the most significant alterations in NB4 cells after treatment with RA+NPs occurs between 24 and 48 h.

Supplementary Figure 14-Heat map of the differentiation pattern of Zn-induced U937
cells after exposure to soluble RA (0.3 or 3 μg/mL) or light-activatable RA+NPs (1 or 10 μg/mL). Cells were treated with RA+NPs for 4 h, washed, activated with UV light (365 nm, 100 Watts) for 5 min, and then cultured for up to 48 h. In case of cells treated with soluble RA, cells were cultured in media containing soluble RA for the entire period of culture. Cells were then lysed and gene expression profile monitored by qRT-PCR using a Fluidigm equipment. A redblue color scale was used to reflect standardized gene expression, with red indicating higher expression and blue indicating lower expression. Our clustering analysis indicate that Zninduced U937 cells treated with 10 μg/mL, but not 1 μg/mL, of RA+NPs were more distant to the non-treated cells than soluble RA. This effect was observed for all the times investigated. Therefore, in contrast to NB4 cells, our clustering analyses indicate that Zn-induced U937 cells were more sensitive to RA released from RA+NPs at least at high concentrations (10 μg/mL). Zn-induced U937-B412, NB4 or NB4-RARE-luciferase reporter cells were cultured with RA+NPs (1 μg/mL) for variable period of times (1 up to 24 h), washed with PBS, resuspended in cell culture media, exposed to UV light (365 nm, 100 Watts) for 5 min, and cultured for 12 h (luciferase measurements) or 72 h (flow cytometry analyses). Results are expressed as Mean ± SEM (n = 3). (B) Zn-induced U937-B412, NB4 or NB4-RARE-luciferase reporter cells were cultured with RA+NPs (1 μg/mL) for 4 h, washed with PBS, resuspended in cell culture media, exposed to UV light for 5 min at variable periods of time (0 up to 44 h), and cultured for 12 h (luciferase measurements) or 72 h (flow cytometry analyses). Results are expressed as Mean ± SEM (n = 3). In NB4-RARE, the activation of RA-dependent signaling pathway was measured by luminescence while in U937 B412 and NB4, cell differentiation was evaluated by the expression of CD11b by flow cytometry.  (3,000,000/mL) were grown in RPMI supplemented with 10% (v/v) FBS, penicillin and streptomycin. Cells were treated with RA+NPs (10 μg/mL) for 4 h, washed with PBS to remove the NPs that were not internalized, activated by a blue laser for 5 min, and then cultured for 18 h under hypoxia conditions (0.5% O2) in exosome-depleted medium. A separate group of cells was treated with RA (3 μg/mL) for 22 h in hypoxia conditions (0.5% O2) in exosome-depleted medium. Then, in both groups, cells were centrifuged at 10.000 g (4ºC; 30 min) and part of the supernatant (named in the plot as "Sup") added to NB4 RARE cells (100,000/mL; 1 mL of the Sup). Alternatively, in both groups, cells were centrifuged in multiple steps (please see Supplementary Information) to remove exosomes (named in the plot as "exo") and added to NB4 RARE cells (100,000/mL; 5 μg/mL de exosomes per mL of media) plated in a 24 well plate. After 24 h, the luciferase assay was performed according to instructions of the luciferase kit (britelite™). (B) Activation of NB4 RARE reporter cell line with the factors secreted by the NB4 cells for 22 h cultured with soluble RA or RA+NPs. Results are expressed as Mean ± SEM (n = 3). Statistical analyses were performed using a One-Way Anova followed by a Newman-Keuls post-test. *, **, *** and **** denotes statistical significance (P<0.05, P<0.01, P<0.001, P<0.0001, respectively). Characterization of the NPs. The diameter of the NPs was measured by photon correlation spectroscopy (PCS) using quasi-elastic light scattering equipment (Zeta-Pals™ Zeta Potential Analyzer, Brookhaven Instruments Corp., Holtsville, NY) and ZetaPlus™ Particle Sizing Software (version 4.03). To measure NP diameter, the NP suspension (2 mL, 50 µg/mL in water for molecular biology) was added to a cuvette and allowed to stabilize for 10 min. The sample was then vortexed for 5 s and subjected to NP size analysis in the ZetaPlus TM for 3 min (3 times; all data were recorded at 90º). After each reading the cuvette was again vortexed for 5 s and exposed to UV light (365 nm) or blue light (405 nm  NP cytotoxicity studies. NPs were suspended in a solution of milli-Q water with PenStrep (5 µL/mL of 10000 U/mL stock solution, Lonza) and Fungizone (2.5 µg/mL, Sigma-Aldrich) for 30 min, centrifuged (14,000 g for 10 min), and finally resuspended in serum free cell culture medium.

Supplementary Figure 14
K562, NB4 and U937 cells (0.1 ⋅ 10 6 cells/condition) were incubated in serum free RPMI-1640 for 4 h in a 96-well plate containing variable amounts of PEI-DMNC:DS NPs. Once the incubations were terminated the cells were washed gently with medium to remove NP excess, and half of the samples were exposed to UV light (365 nm, 100 Watts) for 10 min. The cells were then cultured for 20 h in 100 µL of complete medium (RPMI-1640 medium supplemented with 10 % fetal bovine serum and 100 U/mL PenStrep). A CellTiter-Glo ® luminescent cell viability assay (ATP, Promega, USA) was performed according to the recommendations of the vendor.

Assessment of histone γH2AX phosporylation (DNA damage) induced by UV light or blue light irradiation. HUVEC cells (passage 4) were cultured on 1% gelatin-coated slides until
subconfluency in EGM-2, followed by exposure to UV light (365 nm, 100 Watts) or blue light (405 nm, 80 mW) up to 60 min. Control conditions did not receive any light radiation. Following treatment, the medium was replaced by fresh medium and the cells were incubated for additional 6 h on normal culture conditions. The cells were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature and then washed with PBS. The cells were then permeabilized with 1 % (v/v) Triton-X, blocked with PBS + 2 % BSA and stained for 1 h with anti-human primary γH2AX antibody (clone: N1-431, BD Biosciences). Detection was done with secondary antibody anti-mouse Cy3 conjugate (Jackson ImmunoResearch). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma), and the slides were mounted with mounting medium (Dako) and examined with a Zeiss inverted fluorescence microscope. The NP uptake mechanism was also studied on U937 cells by silencing specific proteins of clathrin-mediated endocytosis (CLTC and LDLR), caveolin-mediated endocytosis (CAV1), GEEC-CCLIC pathways (CDC42) and macropinocytosis (RAC1 and CTBP1) by siRNA (Thermo Fisher).

NP internalization studies. NP internalization was monitored by inductive
Transfection was performed in a 24 well plate with 0.5 ⋅ 10 5 cells in antibiotic-free complete medium with 100 nM siRNA and 1.5 µL of Lipofectamine RNAiMAX (Life Technologies) transfection reagent for 24 h. After this initial period, the transfection medium was replaced by complete medium and the cells incubated for another 48 h. Then, cells were cultured with TRITClabelled NPs (5 µg/mL) for 6 h. Once the incubations were terminated, the cells were centrifuged at 1300 rpm, 20 ºC for 5 min, with PBS, washed one time with cold trypan blue solution (200 µL; 600 µg/mL), re-washed 3 times with cold PBS and then resuspended in PBS containing 2.5 % FBS (500 µL) for flow cytometry analysis. Non-transfected cells or cells transfected with lipofectamine but without siRNAs (MOCK) were used as controls. In all flow cytometry analysis, a total of 10,000 events were recorded per run. All conditions were performed in triplicate.

Intracellular trafficking analyses of NPs. HUVEC cells (passage 4) were cultured on 1 % gelatin-coated slides until subconfluency in EGM-2. The cells were then incubated with FITC-labelled NPs
(1 µg/mL) for 1 or 4 h, washed extensively, exposed or not to UV light (365 nm, 100 Watts) and cultured in normal conditions for up to 12 h. For LysoTracker staining, at time points 2, 6 and 12 h, the cells were incubated with LysoTracker Red DND-99 (50 nM, Invitrogen). After 30 min of incubation, the coverslips were washed extensively with PBS, followed by cell fixation with paraformaldehyde (4%, Electron Microscopy Sciences) for 10 min at room temperature and then washed with PBS. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma), and the slides were mounted with mounting medium (Dako) and examined with a Zeiss LSM 50 confocal microscope.
Co-localization analysis was performed by culturing HUVEC cells (passage 4) on 1 % gelatin-coated slides until subconfluency in EGM-2. Cells were treated with 1 µg/mL FITC-labeled NPs for 1 or 4 hours, washed extensively and cultured in normal conditions for 1 or 1/8 additional hour/s, respectively. Then the cells were fixed with 4 % paraformaldehyde (Electron Microscopy Sciences) for 10 min at room temperature, blocked with 2 % (w/v) BSA, and when necessary, permeabilized with 0.5 % (v/v) Triton-X. Cells were then stained for 1 h with anti-human primary antibodies against EEA1 (clone: C45B10, Cell Signaling), rabankyrin-5 (Rab 5, ANKFY1 (D-15), Santa Cruz Biotechnology), or rabankyrin-7 (Rab 7, clone: D95F2, Cell Signaling). In each immunofluorescence experiment, an isotype-matched IgG control was used. Binding of primary antibodies to specific cells was detected with anti-rabbit or anti-goat IgG Cy3 conjugate (Jackson ImmunoResearch). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma), and the slides were mounted with mounting medium (Dako) and examined with a Zeiss LSM 50 confocal microscope. Co-localization analysis was done in ImageJ through assessment of the percentage of overlapping objects. Two objects are considered to be co-localizing when their intensity profile is overlaping more than 40 %. For this analysis the number (percentage of FITClabeled NPs foci that are positive for EEA-1/Rab-5/Rab-7) and the intensity volume (percentage of FITC-labeled NPs in the EEA-1/Rab-5/Rab-7-positive compartments) were used. This approach was found to be more adequate than classical co-localization tools in ImajeJ or other softwares that measure pixel co-occurrence and correlation analyses, because it allowed us to (i) discriminate between background and vesicle/NP-foci fluorescence and (ii) interpret the results in terms of percentage of NP-foci that are localized to vesicles in another channel of interest.  Time-activation of NPs within cells. NB4 and Zn-induced U937-B412 cells (6.0 ⋅ 10 4 cells/condition) were plated in 24-well plates and transfected with RA + NPs (1 µg/mL) for different time periods (1, 2, 4, 6, 8, 12 and 24 h). The cells were then washed by centrifugation (1300 rpm, 5 min) to remove non-internalized NPs, and immediately exposed to UV light (365 nm, 100 Watts, 5 min). In a second experimental setup, NB4 and Zn-induced U937-B412 cells (6.0 ⋅ 10 4 cells/condition) were plated in 24-well plates and transfected with RA + NPs (1 µg/mL) for 4 h. The cells were then washed by centrifugation (1300 rpm, 5 min) to remove non-internalized NPs, cultured in normal conditions and exposed to UV light (365 nm, 100 Watts, 5 min) at different time points (0, 4, 8, 20 and 44 h). The effect of the intracellular release of RA was evaluated in terms of differentiation of the cells into the myeloid lineage (as assessed by the expression of CD11b) at day 3, as assessed by flow cytometry. All conditions were performed in triplicate.
NB4 cells (between 6.0 ⋅ 10 4 and 10 ⋅ 10 4 cells/condition) were plated in 24-well plates and cultured with soluble RA (3 µg/mL) or light-activatable RA + NPs (10 µg/mL) for 3 days. The NPs were suspended in serum free medium and added to cells for 4 h. The cells were then washed by centrifugation (1300 rpm, 5 min) to remove non-internalized NPs, and half of the samples were exposed to UV light (365 nm, 100 Watts, 5 min). The cells were then cultured up to 3 days in RPMI-1640 medium supplemented with 10 % fetal bovine serum and 100 U/mL PenStrep with half medium changes every 3 days.

NB4 RARE cell line generation. The cignal lenti RARE reporter kit (CLS-016L SABiosciences)
was used for the establishment of a RA reporter NB4 cell line. For that purpose, rectronectin solution (15 µg/cm 2 , 30 µg, 500 µL on PBS, Takara) was plated in a 24-well plate 2 hours prior to cell seeding. The plate was kept at room temperature and was washed one time, immediately before seeding, with PBS. NB4 cells (1 ⋅ 10 5 ) were plated in 175 µL of RPMI-1640 medium (Gibco) supplemented with 0.5 % FBS and 100 U/mL PenStrep and to this condition 125 µL of cignal lentiviral particles were added to a total experimental volume of 300 µL. After a gentle swirl of the plate the cells were incubated 20 hours at 37 ºC in a humidified incubator with 5 % CO 2 atmosphere. In the following day, cells were washed and allowed to recover in the incubator for 24 hours cultured in 500 µL of fresh RPMI-1640 medium supplemented with 10 % FBS and 100 U/mL PenStrep. After that, 2 µg/mL of puromycin (Invitrogen) was added to the culture medium for selection of transduced cells. Evaluation of selection efficiency in puromycin-containing medium was performed every 3 days for a period of 5 weeks.
NB4 RARE luciferase assay. To assess the biological effect of RA in RAR-regulated signalling pathway activity, luciferase reporter assay was performed. NB4-RARE cells (2.5 ⋅ 10 4 cells/condition) were plated in v-shaped 96-well plates and cultured with soluble RA (10 µM) or light-activatable RA + NPs (5 µg/mL). The NPs were suspended in serum free medium and added to cells for 1 h. The cells were then washed by centrifugation (1300 rpm, 5 min) to remove noninternalized NPs, and half of the samples were exposed to blue light (405 nm, 80 mW, 5 min). The cells were then cultured for 12/24 hours in RPMI-1640 medium supplemented with 10 % fetal bovine serum and 100 U/mL PenStrep. After these incubation times, the conditions were NB4 RARE differentiation by cell conditioned medium obtained from NB4 cells exposed to RA or RA + NPs. NB4 cells (3⋅10 6 /mL) were grown in RPMI supplemented with 10% (v/v) FBS, penicillin and streptomycin. Cells were treated with RA + NPs (10 µg/mL) for 4 h, washed with PBS to remove the NPs that were not internalized, activated by a blue laser for 5 min, and then cultured for 18 h under hypoxia conditions (0.5% O 2 ) in exosome-depleted medium. A separate group of cells was treated with RA (3 µg/mL) for 22 h in hypoxia conditions (0.5% O 2 ) in exosome-depleted medium. Then, in both groups, cells were centrifuged at 10.000 g (4ºC; 30 min) and part of the supernatant (named in the plot as "Sup") added to NB4 RARE cells (100,000/mL; 1 mL of the Sup).
Alternatively, in both groups, cells were centrifuged in multiple steps 3 to remove exosomes (named in the plot as "exo") and added to NB4 RARE cells (100,000/mL; 5 µg/mL de exosomes per mL of media) plated in a 24 well plate. After 24 h, the luciferase assay was performed according to instructions of the luciferase kit (britelite™). Briefly, neither patients were PML-RARA: one was standard risk (normal karyotype with a small trisomy 8 subclone), and the other was high risk with complex karyotype. The isolated CD34 + AML cells were maintained in StemSpan SFEM medium (Stemcell Technologies) supplemented with a human cytokine cocktail containing SCF (50 ng/mL, Stemcell Technologies), TPO (15 ng/mL) and Flt-3L (50 ng/mL, PeproTech) plus PenStrep (10,000 U/mL, Lonza) and Fungizone (25 µg/mL, maximum of 0.45 × 10 6 cells were collected for RNA extraction using RNeasy Micro Kit. Afterwards, samples were processed using Fluidigm standard protocols. Briefly, cDNA was obtained from 50 ng of RNA using Reverse Transcription Master Mix (Fluidigm Corporation).
Then samples were pre-amplified for 12 cycles, to increase the number of copies of target DNA, with Fluidigm PreAmp Master Mix (Fluidigm Corporation) and a pool of all the primers tested in the chip. Prior to qPCR reactions the pre-amplification reaction was treated with Exonuclease I to eliminate the carryover of unincorporated primers. Finally, samples and genes pre-mix are prepared separately accordingly to manufactures instructions using SsoFast EvaGreen Supermix with Low ROX (Bio-Rad Laboratories) and loaded into the chip. A pneumatically operated desktop instrument (IFC Controller HX) was used to mix samples and genes pre-mix in the qPCR chamber reaction of the chip. After this procedure, the chip was analyzed using Biomark HD (Fluidigm Corporation) for the thermal cycling and real time fluorescent readings. A melting curve was performed after 30 cycles, for quality control. DeltaCt was calculated using Real Time PCR Analysis Software 4.1.3 (Fluidigm Corporation) and data was further analyzed with Cluster 3.0 and Java Trew View to produced gene expression heatmaps and hierarchical clustering.
Transfection efficiency was evaluated by fluorescence microscopy. After 72 hours viral particles were collected and THP-1 cells were infected (2 x 10 5 cells/ml in a 6-well plate with 4µg/ml polybrene).
In vivo study: subcutaneous implantation. The animal work has been conducted according to relevant national and international guidelines and approved by the Bioethics Committee of University of Salamanca. On the day before injecting the cells, PDMS cylindrical constructs (∅internal = 1.0 cm; ∅external = 1.5 cm) were implanted subcutaneously on NOD/SCID mice (Jackson Laboratory) maintained in pathogen-free conditions with irradiated chow. For the ex-vivo activation studies in the day of the experiment, NB4 cells were suspended in serum free medium with (i) no NPs, (ii) with empty NPs (10 µg/mL) or RA + NPs (10 µg/mL) for 4 h. At the end, cells were washed by centrifugation (1300 rpm, 5 min), and the ones treated with RA + NPs were either activated or not with a blue laser (405 nm, 80 mW) for 5 min. NB4 cells (5 ⋅ 10 6 cells per PDMS construct) were injected subcutaneously in the center of the PDMS construct embedded in Matrigel (200 µL, BD Biosciences). Five days after injection of the cells, animals were sacrificed by cervical dislocation and cells within the cylindrical construct were collected and characterized by flow cytometry. For the in vivo activation studies in the day of the experiment, NB4 cells were suspended in serum free medium with (i) no NPs, (ii) with RA + NPs (10 µg/mL) for 4 h. At the end, cells were washed by centrifugation (1300 rpm, 5 min), and 5 ⋅ 10 6 NB4 cells per PDMS construct were injected subcutaneously in the center of the PDMS construct embedded in Matrigel (200 µL).
One day after injection, experimental groups were either activated or not with a blue optical fiber (405 nm, 80 mW) for 5 min. Three days after injection of the cells, animals were sacrificed by cervical dislocation and cells within the cylindrical construct were collected and characterized by flow cytometry.
In vivo study: bone marrow modulation. NOD.CB17-Prkdcscid /J (NOD/SCID) female mice (n=12) aged 6-8 weeks were employed in this experiment. Before cell injection, mice received 1.5 Gy of total body irradiation from a 137Cs source and were also treated with 200 µg of mouse anti-CD122 monoclonal antibody (NS122) by intraperitoneal injection. Human THP-1 cells were incubated with 20 µg/mL of RA + NPs in RPMI medium for 4 hours, followed by extensive wash with PBS to remove non-internalised NPs. The cells were then resuspended in RPMI medium with 10% FBS and left in the culture incubator overnight. On the following day, the cells loaded with RA + NPs were collected and 1 ⋅ 10 7 cells /mouse in 200 µL PBS were injected into the NOD/SCID mice intravenously, through the tail vein. After 6 days, the mice were randomly divided into two groups: one of the groups was blue laser irradiated (n=6) and the other group was not irradiated (n=6). In the blue laser irradiated group, each mouse was anesthetized with 3.5% chloral hydrate in PBS and the craniums were exposed to a blue laser (405 nm, 80 mWatts) for 5 min. The mice in the non-irradiated group received the same treatment without blue laser activation. After 48 or 72 h, the mice were sacrificed and the long bones/ craniums were collected. The long bones including the femurs, tibias and pelvis were crushed in PBS containing 1% bovine serum albumin (BSA, sigma) and 2 mM EDTA (Invitrogen). The samples were then filtered with 70 µm cell strainer and treated with ACK solution to lyse red blood cells. The resulting cells were stained using PE conjugated anti-human CD45 antibody (eBioscience). The results were measured by flow cytometry and the data were analyzed with FlowJo software.
The mouse craniums were collected for the ex vivo staining examination followed by the traditional protocol. Briefly, the mouse craniums were cut into four pieces along the sutures producing one frontal, two parietal and one occipital bone. Only parietal bones were used in this experiment. Bone pieces were fixed in 4% paraformadehyde for 30 min, washed twice with PBS, blocked with 2% BSA-0.01% Triton X-100 in PBS (BSA buffer) for 1 hr, and incubated with primary antibodies (diluted 1:100 in BSA buffer) overnight at 4ºC. After washing with BSA buffer for 2 hr, bones were incubated with secondary antibodies (diluted 1:200 in BSA buffer) for 2 hr, washed with BSA buffer for 2 hr, and nuclei were stained using DAPI. The primary antibodies included mouse anti-human CD45 antibody (BD) and rabbit anti-human CD11b antibody (Abcam).
The secondary antibodies included goat anti-mouse 555 (invitrogen) and goat anti-rabbit 488 antibodies. The bone pieces were finally examined with a confocal microscope (Nikon Eclipse Ti) under FITC/TRITC/DIC signal channel. Images were analysed in imageJ. Analyse Particles was