Encapsulation and Delivery of the Kinase Inhibitor PIK-75 by Organic Core High-Density Lipoprotein-Like Nanoparticles Targeting Scavenger Receptor Class B Type 1

Abstract

PIK-75 (F7) is a potent multikinase inhibitor that targets p110α, DNA-PK, and p38γ. PIK-75 has shown potential as a therapy in preclinical cancer models, but it has not been used in the clinic, at least in part, due to limited solubility. We therefore developed a nanoparticle to encapsulate PIK-75 and enable targeted cellular delivery. Scavenger receptor class B type 1 (SR-B1) is often overexpressed in cancer compared with normal cells, which enables targeting by synthetic lipid nanoparticles with some features of native high-density lipoprotein (HDL), the natural ligand of SR-B1. We investigated the use of organic core (oc) molecular platforms to synthesize HDL-like nanoparticles (oc-HDL NP). Employing an oc, we successfully formulated PIK-75 into oc-HDL NPs. The PIK-75 loaded oc-HDL NP (PIK-75 oc-HDL NP), comprising ∼20 PIK-75 molecules/NP, has similar size, surface charge, and surface composition as oc-HDL NP and natural human HDL. Using prostate cancer (PCa) and cutaneous T-cell lymphoma (CTCL) models known to be sensitive to inhibitors of p110α and p38γ, respectively, we found that PIK-75 oc-HDL NPs specifically targeted SR-B1 to deliver PIK-75 and potently induced cell death in vitro in PCa and CTCL and in vivo in a murine PCa model. Additionally, we found that PIK-75 oc-HDL NP, but not free PIK-75 or oc-HDL NP alone, reduced the IC₅₀ in the NCI-60 cell line panel and additional pancreatic cancer cell lines. These data demonstrate the first example of drug-loaded oc-HDL NP that actively target SR-B1 and kill cancer cells in vitro and in vivo, encouraging further development and translation to human patients.

Keywords: PIK-75; cancer; drug encapsulation; lipid nanoparticle; scavenger receptor class B type 1; targeted delivery.

Figure 1

Synthesis scheme and proposed mechanism of PIK-75 oc-HDL NPs. (A) In a single pot synthesis, PIK-75 (DCM), PE-S₄ organic core scaffold (DCM), phospholipids (DCM), and apoA-I (water) are combined to form an emulsion. The DCM is allowed to evaporate, which results in the spontaneous self-assembly and formation of the PIK-75 oc-HDL NPs. (B) Proposed mechanism-of-action of PIK-75 delivery by oc-HDL NPs. PIK-75 oc-HDL NPs bind to SR-B1 and deliver PIK-75, which can then inhibit various intracellular kinases (p110α, p38γ, DNA-PK) leading to cancer cell death.

Figure 2

SR-B1 as a target in CTCL and PCa. Competitive binding assay in THP-1 cells. THP-1 cells were treated with fluorescently labeled Au-HDL NPs (DiI Au-HDL NP), and either unlabeled Au-HDL NP or the PIK-75 oc-HDL NP construct for 2 h, followed by flow cytometric analysis. Reduction in fluorescent signal with addition of the unlabeled Au-HDL NP or PIK-75 oc-HDL NPs indicates binding to SR-B1. (A) Median fluorescent intensities (MFI) graph. ****p < 0.0001. N = 4 per group. Data displayed as mean ± SD. (B) Representative histogram. (C) Flow cytometry analysis of SR-B1 expression in prostate cancer cell lines cells. Ramos and U266B1 were used as positive and negative controls, respectively. (D) Flow cytometry analysis of SR-B1 expression in HH and Hut78 cell lines. Ramos and U266B1 were used as positive and negative controls, respectively. (E) RNA sequencing analysis of 49 primary CTCL samples demonstrates increased expression of SR-B1 in advanced [stage III (T3) and stage IV (T4)] CTCL compared with normal, CD3+ peripheral T cells. Data (mean ± SD) are presented as reads per kilobase per million (RPKM). ***p < 0.05 by one sample t test versus a mean of 1. (F) Comparative analysis of SR-B1 expression in prostate adenocarcinoma sample data were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression database (GTEx). PCa samples (N = 492) had significantly higher SR-B1 expression compared to normal prostate tissue (N = 152). *p < 0.01 vs PCa.

Figure 3

PIK-75 oc-HDL NPs induce cell death in cutaneous T cell lymphoma and prostate cancer cell lines. PIK-75 oc-HDL NPs demonstrated reduced IC₅₀ compared with free PIK-75 against the CTCL cell lines HH (A) and HuT78 (B), and the prostate cancer cell lines CWRR1 Wt (C), CWRR1 EnzR (D), LnCaP Wt (E) and LnCaP EnzR (F). n = 3 per concentration. Data presented as mean ± SD.

Figure 4

SR-B1 mediates uptake of PIK-75 from oc-HDL NPs. HH cells were pulsed with PIK-75 oc-HDL NPs (50 nM) for 2 h. in the presence or absence of BLT-1 (10 μM), an inhibitor of cholesterol flux through SR-B1 (A), or an SR-B1 blocking antibody (1:100 dilution, B). Addition of BLT-1 ameliorated PIK-75 oc-HDL NP induced cell death compared to PIK-75 oc-HDL NPs alone (A). Similar results were obtained using the SR-B1 blocking antibody (B). (A) n = 9 per condition. Data presented as mean ± SD ***p = 0.0001. (B) n = 10 per condition. Data presented as mean ± SD ****p < 0.0001.

Figure 5

PIK-75 oc-HDL NPs reduce p-AKT levels in prostate cancer cells. Western-blot analysis of CWRR1 EnzR cells treated with free PIK-75 (200 nM; on the left) or PIK-75 oc-HDL NPs (10 nM; on the right) for 0 to 24 h. p-AKT levels rapidly decreased with both free and nanoparticle encapsulated PIK-75. The ratio of p-AKT to AKT was calculated using densitometry with ImageJ.

Figure 6

PIK-75 oc-HDL NPs reduce prostate cancer tumor xenograft volumes. CWRR1 EnzR flank tumors were initiated in castrated athymic nude mice, then treated with PBS (200 μL; n = 6 mice), free PIK-75 (50 μM, 200 μL; n = 9), oc-HDL NPs (2.5 μM, 200 μL; n = 7) or PIK-75 oc-HDL NPs (2.5 μM, 200 μL; n = 7) i.p. three times per week for 3 weeks. Measurements were taken at the initiation of treatment and end of the study. Data presented as mean change in tumor volume (volume final–volume initial) ± SD. P values: vehicle control vs free PIK-75 = 0.3042; vehicle control vs empty oc-HDL NPs = 0.9985; *vehicle control vs PIK-75 oc-HDL NPs = 0.0189.