Supplementary Components1. in a focus on the accumulation of particles in whole tumors.1 A range of methods to determine the fraction of the injected dose of the carrier or cargo that accumulates in a whole organ or tumor has driven the assessment of nanoparticle targeting to solid tumors.2C13 However, tumors FGF2 are composed of a variety of cell types, such as fibroblasts and endothelial macrophages and cells and neutrophils, furthermore to tumor cells. The comparative distribution of the cell types varies between tumors.14C17 Whole organ approaches cannot discriminate between accumulation in the intended focus on, cancer cells typically, and additional cells or the extracellular space. For cargo with an intracellular system of action, such as nucleic acids and proteins, delivery to specific cell types is crucial to assessing nanoparticle efficacy and optimizing targeting. Methods for the identification of subtumoral cellular components include microscopy and flow cytometry. Confocal microscopy has been used to determine particle internalization in vivo by analyzing multiple sections of an organ.18 However, meaningful quantification can be challenging. Flow cytometry permits concurrent cellular identification and nanoparticle quantification. Previous studies that have used flow cytometry to examine nanoparticle targeting to organs have not explored the effects of particle characteristics (composition, shape, etc.) or dose on the accumulation in specific cell populations and do not correlate their findings with whole organ assessment.14,19C25 Studies that account KPT276 for both nanocarrier properties as well as intra-organ or intra-tumor distribution have the potential to best inform nanoparticle design and delivery. PRINT is a top-down fabrication strategy that relies on precision molds, offering the advantage of reproducible production of monodisperse particles. This reproducibility eliminates large variation in particle sizes (i.e. PDI) that could influence the association of a subset of the particles with one cell population over another confounding data interpretation. In addition, PRINT also affords homogeneity in the composition of the particles and flexibility in the composition of the desired nanoparticle material. Using flow cytometry, whole organ assessment and live animal in vivo KPT276 confocal microscopy, we analyzed the cell type-specific distribution of PRINT nanoparticles. We identified wide variation in subtumoral cellular association and identify dose and particle properties that influence cellular targeting. Methods Materials Poly(ethylene glycol) diacrylate (Mw 700) (PEG700DA), 2-aminoethyl methacrylate hydrochloride (AEM), diphenyl (2,4,6-trimethylbenzoyl)-phosphine oxide (TPO), and sucrose were purchased from Sigma-Aldrich. Thermo Scientific Dylight 488 maleimide, dimethylformamide (DMF), triethylamine (TEA), pyridine, borate buffer (pH 8.6), acetic anhydride, and methanol were obtained from Fisher Scientific. Conventional filters (2 m) were purchased from Agilent and poly(vinyl alcohol) (Mw 2000) (PVOH) was purchased from Acros Organics. PRINT molds (80 nm80 nm320 nm) were obtained from Liquidia Technologies. Tetraethylene KPT276 glycolmonoacrylate (HP4A) was synthesized in-house as previously described.26 Methoxy-PEG(5k)-succinimidyl carboxy methyl ester (mPEG5k-SCM) was purchased from Creative PEGWorks. Typsin, DPBS, and cell culture media were purchased from Gibco. PRINT nanoparticle fabrication and characterization The PRINT particle fabrication technique has been described previously in detail.27,28 The pre-particle solution was prepared by dissolving 3.5 wt% of the various reactive monomers in methanol. The preparticle solution was comprised of 67.75 wt% HP4A, 20 wt% AEM, 10 wt% PEG700DA, 1 wt% TPO and 1.25 wt% Dylight 488 maleimide. Stock particle concentrations were determined by thermogravimetric analysis (TGA).