500 watt ultrasonic homogenizer

PLA iNPs were prepared using the oil-in-water (o/w) emulsion-solvent evaporation (SE) technique following a similar method as described [10 (link)]. Briefly, 200 mg of PLA was dissolved in ethyl acetate at a concentration of 80 mg/mL, 20 mL of 1% PEMA was added then sonicated at 100% amplitude for 30 s using a Cole-Parmer 500-Watt Ultrasonic Homogenizer to make PLA-PEMA. For PLA-PVA, 200 mg of PLA was dissolved in ethyl acetate at a concentration of 300 mg/mL. To this, 5 mL of 2% PVA was added and sonicated at 40% amplitude for 30 s using the same homogenizer. The resulting o/w emulsion was then poured into 100 mL of magnetically stirred 0.5% PEMA (or 0.5% PVA) overnight to remove ethyl acetate. iNPs were then collected by centrifugation at 12,000× g for 20 min at 4 °C and washed with 40 mL of MilliQ water. The centrifugation and washing steps were repeated two more times. A mixture of sucrose and mannitol were added to the particle suspension as cryoprotectants to achieve a final concentration of 4% and 3% w/v, respectively. The nanoparticles were then frozen at −80 °C and lyophilized for at least 48 h prior to use.
The size and zeta potential of all the particles were determined by dynamic light scattering (DLS) using a Malvern Zetasizer ZSP. Cy5.5-labeled PLA particles were prepared by incorporating 1% w/w of PLA-Cy5.5 into particles as previously described [23 (link)].

In sterile 6‐well plates, Day 8 BMMØs were seeded at 1 × 10⁶ cells/well and incubated at 37°C and 5% CO₂ overnight to allow for cell adherence, as previously described.
¹⁷ (link) BMMØs were treated with 300 μg/mL iNP, 300 μg/mL iNP‐SAHA_Low, or 300 μg/mL iNP‐SAHA_High and incubated for 4, 9, 27, or 48 h. Cells were isolated using RIPA Buffer (#R0278) (Millipore Sigma, St. Louis, MO) containing Halt™ Protease Inhibitor Cocktail (#78429) (Thermo Fisher, Waltham MA) and scraped using a cell scraper (VWR, Radnor, PA). A 1/8″ tip using a Cole‐Parmer 500‐Watt Ultrasonic Homogenizer at 40% amplitude for 10 s on ice was used to sonicate the cell lysates, then centrifuged at 4°C for 20 min at 12,000×g. The supernatant was extracted and frozen at −80°C. A 50/50 sample to 2× SDS/PAGE sample buffer produced protein lysates. SDS/PAGE was used to separate proteins then immunoblotted using Histone H3 (D1H2) (#4499) Rabbit mAb, Acetyl‐Histone H3 (Lys9/Lys14) (#9677) Rabbit mAb, α‐Tubulin (11H10) (#2125) Rabbit mAb, Acetyl‐α‐Tubulin (Lys40) (D20G3) (#5335) Rabbit mAb, and β‐Actin (D6A8) (#8457) primary antibodies (Cell Signaling Technology, Danvers, MA). Enhanced luminol‐based chemiluminescent (ECL) was used for detection of the western blot. Quantification of bands was performed using Image J.