Boosting nanomedicine performance by conditioning macrophages with methyl palmitate nanoparticles

Surface PEGylation, biological camouflage, shape and stiffness modulation of nanoparticles as well as liver blockade and macrophage depletion have all improved the blood longevity of nanomedicines. Yet, the mononuclear phagocytic system still recognizes, sequesters, and processes the majority of blood borne particles. Here, the natural fatty acid methyl palmitate is combined with endogenous blood components – albumin – realizing ∼200 nm stable, spherical nanoparticles (MPN) capable of inducing a transient and reversible state of dormancy into macrophages. In primary bone marrow derived monocytes (BMDM), the rate of internalization of 5 different particles, ranging in size from 200 up to 2000 nm, with spherical and discoidal shapes, and made out of lipids and polymers, was almost totally inhibited after an overnight pre-treatment with 0.5 mM MPN. Microscopy analyses revealed that MPN reversibly reduced the extension and branching complexity of the microtubule network in BMDM, thus altering membrane bulging and motility. In immunocompetent mice, a 4 h pre-treatment with MPN was sufficient to redirect 2000 nm rigid particles from the liver to the lungs realizing a lung-to-liver accumulation ratio larger than 2. Also, in mice bearing U87-MG tumor masses, a 4 h pre-treatment with MPN enhanced the therapeutic efficacy of docetaxel-loaded nanoparticles significantly inhibiting tumor growth. The natural liver sequestering function was fully recovered overnight. This data would suggest that MPN pre-treatment could transiently and reversibly inhibit non-specific particle sequestration, thus redirecting nanomedicines towards their specific target tissue while boosting their anti-cancer efficacy and imaging capacity.


SUPPLEMENTARY MATERIALS AND METHODS
Gas chromatography coupled to mass spectrometry for MP quantification in MPN. Methyl palmitate quantification was performed using a GC-ion trap MS system. The instrument consists of a Trace GC Ultra gas chromatograph equipped with an AI1310 autosampler and coupled with an ITQ 1100 ion-trap mass spectrometer (Thermo Fisher Scientific Inc., Austin, Texas, USA). The chromatographic analysis was performed on a TraceGOLD TG-5MS (5% phenyl, 95% methylpolysiloxane) fused-silica capillary column (30m x 0.25 mm x 0.25 um). The oven temperature program was 150°C (held for 4min) to 270°C at 10°C min-1 and to 310°C at 40°C min-1 (held for 4min). Helium was used as carrier gas at a constant flow-rate of 1 ml min-1. The injector temperature was kept at 250°C and samples were injected in the splitless injection mode (1 min). The transfer line and ion source temperatures were kept at 280 and 250° C respectively.
The electron energy and emission current were 70 eV and 250 uA. Parameters such as automatic gain control and multiplier were set by automatic tuning. Precursor ion selected in MS-MS mode was 227 m/z and product ions were in the range 80-227 m/z. The trapping voltages were adjusted setting as automatic the collision energy. For methyl palmitate extraction from MPN 75ul of isopropanol were added to 75ul one MPN batch, the suspension was vortexed for 1 min and 500ul of Heptane were added twice to avoid losing material. Samples were than vortexed again for 1 min and samples were centrifuged at 14800 rpm for 10 minutes. The organic phases were collected and evaporated to dryness under nitrogen. Reconstitution was done with 500uL of Heptane right before injection.
MTT and propidium iodide assay. MTT assay was performed on BMDM cells after treating them with MPN (Supplementary Figure 5a). MPN were used in different equivalent concentrations of methyl palmitate (0; 0.031; 0.062; 0.125; 0.250; 0.50; 1 mM). 20,000 BMDM were seeded into each well of a 96 well plate (Corning, USA). Cells were cultured according to the same condition indicated in the manuscript. 24h after cell seeding, cells were treated with MPN for 24h. At the end of the treatment MTT Formazan (1-(4,5-Dimethylthiazol-2-yl)-3,5diphenylformazan, Thiazolyl blue formazan) (Merk-Sigma, USA) was added to the culture according vendor's indication. Cells were incubated for 4h with the reagent and the plate was than analyzed following vendor's protocol. Propidium Iodide assay (Supplementary Figure 5b) was instead performed following similar indication. 200,000 BMDM were seeded into a 12 multi-wheel plate (Corning, USA) 24h before the treatment.

3D Deconvolution of a Single Cell z-stack:
One single cell z-stack was deconvolved and reconstructed in 3D by using NIS-Software (Nikon) (Supplementary Figure 9). For this experiment the same condition indicated in the section particle internalization analysis via Confocal Microscopy were followed. Waltam, USA). Fixation was performed using a 3.7% solution of paraformaldehyde (Sigma Aldrich, USA) for 10 minutes; 3 washes were performed after cell fixation. A 63X objective was used and a z-stack series was acquired (≥12 steps of 1,000 nm each were acquired per image).

MPN
Images were realized by using a A1-Nikon confocal microscope (Nikon Corporation, Japan).

MPN Biodistribution. MPN biodistribution was analyzed by using a IVIS Spectrum In Vivo
Imaging System (Perkin Elmer -Waltham, USA) (Supplementary Figure 19). 12 8 weeks old female C57BL/6J mice (Charles River, USA) were feed with special diet for 1 week to reduce the fluorescence derived by food. Cy5-MPN were intravenously administered (mass of methyl palmitate: ~1,87 mg/20g). Animals were animals were sacrificed after 2h, 4h and 6h and 24h. Liver, spleen, heart, lungs, brain, kidneys were isolated and images were acquired.
Anesthesia was performed using isoflurane by inhalation and all the procedure were conducted following the guidelines of the Institutional Animal Care and Use Committee of IIT.