*2.2. iNP Preparation and Characterization*

PLA iNPs were prepared using the oil-in-water (*o*/*w*) emulsion-solvent evaporation (SE) technique following a similar method as described [10]. 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].
