Perchlorate

100 nm polymeric cores were prepared using 0.67 dL g⁻¹ carboxyl-terminated 50:50 poly(lactic-co-glycolic) acid (PLGA) (LACTEL Absorbable Polymers) in a nanoprecipitation process. 1 mL of 10 mg mL⁻¹ PLGA solution in acetone was added dropwise to 3 mL of water. For fluorescently labeled nanoformulations, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (DiD, ex = 644 nm/em = 665 nm, Life Technologies) was loaded into the polymeric cores at 0.1 wt%. The mixture was then stirred in open air for 1 h and placed in vacuum for another 3 h. The resulting nanoparticle solution was filtered with 10 kDa MWCO Amicon Ultra-4 Centrifugal Filters (Millipore). Platelet membrane cloaking was then accomplished by dispersing and fusing platelet membrane vesicles with PLGA particles via sonication using an FS30D bath sonicator at a frequency of 42 kHz and a power of 100 W for 2 min. The size and the surface zeta potential of the replicate PNP samples (n=3) were obtained by DLS measurements using a Malvern ZEN 3600 Zetasizer. PBS stability was examined by mixing 1 mg mL⁻¹ of PNPs in water with 2X PBS at a 1:1 volume ratio. Storability of PNPs was examined by suspending PNPs in 10% sucrose. The nanoparticle solutions were subject to either a freeze-thaw cycle or lyophilization followed by resuspension. The resulting particle solution was then monitored for particle size using DLS. The structure of PNPs was examined with TEM following negative staining with 1 wt% uranyl acetate using an FEI 200 kV Sphera microscope. RBCNPs were prepared using the same polymeric cores and RBC membranes of equivalent total surface area to the platelet membranes using a previously described protocol^{16 (link)}. The RBCNPs were characterized using DLS and had similar size and zeta potential as the PNPs.
Docetaxel-loaded PLGA nanoparticle cores were prepared via a nanoprecipitation process. 10 wt% docetaxel was added to 5 mg PLGA in acetone and precipitated dropwise into 3 mL water. The solvent was evaporated as described above and free docetaxel was removed via repeated wash steps. Vancomycin-loaded nanoparticles were synthesized using a double emulsion process. The inner aqueous phase consisted of 25 µL of vancomycin (Sigma Aldrich) dissolved in 1 M NaOH at 200 mg mL⁻¹. The outer phase consisted of 500 µL of PLGA polymer dissolved in dichloromethane at 50 mg mL⁻¹. The first emulsion was formed via sonication at 70% power pulsed (2 sec on/1 sec off) for 2 min on a Fisher Scientific 150E Sonic Dismembrator. The resulting emulsion was then emulsified in aqueous solution under the same dispersion setting. The final w/o/w emulsion was added to 10 mL of water and the solvent was evaporated in a fume food under gentle stirring for 3 h. The particles were collected via centrifugation at 80,000 × g in a Beckman Coulter Optima L-90K Ultracentrifuge. The particles were washed and resuspended in water. Upon preparation of drug-loaded PLGA cores, cell membrane coating was performed by adding the appropriate surface area equivalent of either platelet or RBC membrane followed by 3 min of sonication in a Fisher Scientific FS30D Bath Sonicator. Particle size, polydispersity (PDI), and surface zeta potential were characterized using DLS. Drug loading yield and release rate of replicate samples (n=3) were quantified by high performance liquid chromatography (HPLC). Drug release was determined by dialyzing 500 µL of particle solution at a concentration of 2.67 mg mL⁻¹ in PBS using 3.5K MWCO Slide-A-Lyzers (Thermo Scientific).