(a) Transmission electron micrograph (TEM) pictures of NanoHHI, demonstrating uniformly dispersed nanoparticles with an average size diameter of <100nM. The scale bar on lower right corner equals 100nM.
(b) NanoHHI (right) is a uniform suspension in aqueous media while parental HPI-1 is essentially insoluble, with flakes of drug clearly visible in the suspension. Equivalent amounts of HPI-1 were used in both instances.
(c) In vitro release kinetics of HPI-1 from NanoHHI, performed at 37°C. The Y-axis charts the % cumulative release and X-axis denotes time in days.
(d) Pharmacokinetic disposition of parenteral NanoHHI administered as a single dose (25 mg/kg) compared to that of “free” HPI-1 (25 mg/kg) in corn oil. The experiments were performed in non-tumor bearing CD1 mice, with three mice per cohort. Plasma HPI-1 levels were assessed using LC-MS/MS.

(a) HPI-1 can block Hh signaling in HEK-293 cells expressing human Smo (HsSmo) with a binding site mutation. HEK-293 cells stably expressing either wild type HsSmo (Smo^WT) or mutant HsSmo^D473H were transiently transfected with Gli1-binding site firefly luciferase reporter, and exposed to cyclopamine or HPI-1. Renilla luciferase was used as a control for transfection efficiency. Both cyclopamine and HPI-1 can block Hh signaling in HEK-293 cells expressing HsSmo^WT (left); in contrast, only HPI-1 blocks Hh signaling in HEK-293 cells expressing HsSmo^D473H, while cyclopamine, which is unable to bind to mutant Smo receptor has minimal effect (right). All luciferase assays were performed in triplicate, and the Y-axis denotes relative luciferase units normalized to Renilla.
(b) Murine medulloblastomas arising in Ptch^+/−; Trp53^−/− mice, and harboring either murine Smo^WT (MmSmo^WT) or an acquired MmSmo^D477G mutation, were allografted subcutaneously in athymic mice, and treated with either HhAntag or NanoHHI. Void PLGA-PEG nanoparticles were used as vehicle control. In allografts expressing wild type MmSmo, both HhAntag and NanoHHI demonstrate comparable effects in terms of tumor growth inhibition on day 12 (the experiment had to be terminated due to the rapid increase in growth of vehicle control allografts) (left). In contrast, in allografts expressing the MmSmo^D477G binding site mutation, NanoHHI demonstrates a markedly better efficacy than HhAntag in growth inhibition (right, P<0.01 for HhAntag and P<0.001 for NanoHHI). Representative post-treatment allografts are illustrated for each cohort above the growth curves.
(c) Murine Gli1 (MmGli1) mRNA levels were assessed following treatment in both cohorts, as a measure of Hh pathway inhibition. Mirroring the growth phenotypes, both HhAntag and NanoHHI significantly downregulate MmGli1 levels in MmSmo^WT allografts, while only NanoHHI significantly downregulates MmGli1 expression in MmSmo^D477G expressing allografts. The Y-axis represents relative expression normalized to housekeeping control.
(d) In medulloblastoma allografts expressing the MmSmo^D477G mutation, NanoHHI significantly downregulates bona fide Gli1 target genes, including Akna, Cltb, and Olig2, compared to HhAntag-treated allografts (expression is normalized to the void polymer control treatment arm).

(a) Orthotopic human pancreatic cancer Pa03C xenografts were generated in athymic mice by surgical orthotopic implantation, and treated with vehicle control, NanoHHI, gemcitabine, or the combination. At four weeks of therapy, tumor weights were assessed at necropsy, and demonstrated a significant reduction in weight (in grams) for the combination arm compared to gemcitabine alone (P=0.018). Expectedly, NanoHHI monotherapy had minimal impact on primary tumor weight. Representative post-treatment xenografts are illustrated for each cohort (Left)
(b) NanoHHI depletes aldehyde dehydrogenase (ALDH) expressing cells in Pa03C xenografts. ALDH expression in neoplastic cells was assessed by immunohistochemistry on representative xenografts from each of the four arms (as indicated). Marked reduction in ALDH expressing cells is observed in both NanoHHI monotherapy and combination treatment arms.
(c) Relative expression of stroma-specific murine Gli1 (MmGi1) were assessed in the treated pancreatic cancer Pa03C xenografts. Significant downregulation of MmGli1 was observed in both NanoHHI monotherapy (P<0.05) and combination arms (P<0.001), consistent with downregulation of Hh signaling in the stromal compartment.
(d) Body weight measurements were obtained at five time points in mice bearing orthotopic Pa03C xenografts that were treated with vehicle, NanoHHI or the combination of NanoHHI and gemcitabine over a four week time period. No significant body weight losses are encountered in either NanoHHI arm.