Enhanced efficacy of combined temozolomide and bromodomain inhibitor therapy for gliomas using targeted nanoparticles

Abstract

Effective treatment for glioblastoma (GBM) is limited by the presence of the blood-brain barrier (BBB) and rapid resistance to single agent therapies. To address these issues, we developed a transferrin-functionalized nanoparticle (Tf-NP) that can deliver dual combination therapies. Using intravital imaging, we show the ability of Tf-NPs to traverse intact BBB in mice as well as achieve direct tumor binding in two intracranial orthotopic models of GBM. Treatment of tumor-bearing mice with Tf-NPs loaded with temozolomide and the bromodomain inhibitor JQ1 leads to increased DNA damage and apoptosis that correlates with a 1.5- to 2-fold decrease in tumor burden and corresponding increase in survival compared to equivalent free-drug dosing. Immunocompetent mice treated with Tf-NP-loaded drugs also show protection from the effects of systemic drug toxicity, demonstrating the preclinical potential of this nanoscale platform to deliver novel combination therapies to gliomas and other central nervous system tumors.

Fig. 1

Transferrin-functionalized nanoparticles cross the intact BBB. a Schematic of a PEGylated dual drug-loading liposome that can be functionalized to enhance transport across the BBB and targeting to glioma cells. b Cranial window (black oval delineating region of craniotomy) exposing the brain for in vivo multiphoton imaging. Multiphoton images of: c A brain microvessel showing lack of transport of Hemagglutinin-PEG_2K-Cy5.5 liposomes across the BBB; d Diffusion of transferrin-PEG_2K-Cy5.5 (Tf-NP) liposomes across the endothelium of a brain microvessel (outlined in white) with nanoparticle aggregates in the subarachnoid space (white arrows); and e Composite image showing accumulation of Tf-NP liposomes in the endothelial wall of a brain microvessel (white arrows) with diffusion across the BBB and aggregation of liposomal nanoparticles in the surrounding brain milieu. White outline depicts bony edge of the cranial window with bone second harmonic signal in blue. Images were taken 24 h following a single tail vein injection of nanoparticles. All scale bars = 25 μm

Fig. 2

Transferrin-functionalized liposomes achieve receptor-mediated transcytosis and delivery to intracranial models of GBM. a Immunohistochemistry demonstrates expression of transferrin receptor (α-Tf receptor) in the endothelium of tumor-associated blood vessels and in tumor tissue of U87MG and GL261 glioma brain tumors. Mouse IgG served as a negative control for non-specific antibody staining (IgG control). Scale bar = 20 μm. b Representative western blot and quantification shows ~1.4-fold increased expression of transferrin receptor in U87MG compared to GL261 cells. Data presented as mean ± SEM of three separate experiments. c Immunofluorescence staining demonstrates time-dependent intracellular uptake of Tf-NPs but not PEG_2K-Cy5.5 (PEG-NP) liposomes in U87MG and GL261 cells in vitro. Tf-NPs co-localize to late endosomal/lysosomal compartments (LAMP-1). Nuclei were visualized using DAPI counterstain (DAPI). Scale bar = 10 μm. d Flow cytometry plots and quantification of cellular PEG-NP or Tf-NP signal in U87MG and GL261 cells. Data presented as mean ± SEM of three separate experiments. Statistical analysis performed using Student’s t-test (***p < 0.001). e Multiphoton images of PEG-NP or Tf-NPs (red) at the site of GFP-expressing U87MG and GL261 intracranial gliomas (green). Scale bars = 6.25 μm or 12.5 μm

Fig. 3

The bromodomain inhibitor JQ1 and temozolomide have additive effects in U87MG and GL261 glioma cells. a Representative immunofluorescence images of γH2AX DNA damage foci in U87MG and GL261 cells treated with 500 nM JQ1 and/or 150 μM TMZ for 72 h. Scale bar = 10 μm. b Cell viability plots demonstrating combinatorial effects of JQ1 and TMZ. Combinatorial index (C.I.) values determined using the Chou–Talalay method. Data presented as mean ± SEM of three separate experiments

Fig. 4

Characterization of drug release from JQ1 and TMZ liposomes. a Schematic of liposomes loaded with JQ1 and TMZ. Kinetics of drug release from liposomes loaded with b JQ1 alone, c TMZ alone, or d both JQ1 and TMZ. Data presented as mean ± SEM of three separate experiments

Fig. 5

Tf-NPs loaded with JQ1 and TMZ have superior pharmacodynamic effects in intracranial orthotopic models of glioblastoma. Representative bioluminescent images of a U87MG and b GL261 mice taken on day 0 and day 5 following initiation of treatment with free drug formulations (free drug), drugs loaded in PEG-Cy5.5 liposomes (PEG-NP drug), or transferrin-PEG-Cy5.5 liposomes (Tf-NP drug). Quantification of average tumor bioluminescence values in the different treatment arms throughout the course of treatment for c U87MG and d GL261 mice. Kaplan–Meier survival plots of e U87MG and f GL261 glioma mice in the different treatment arms. Study powered with eight mice per treatment arm for statistical significance. Log-rank (Mantel–Cox) test performed on survival plots. Student t-test used to quantify differences in treatment arms (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 6

U87MG and GL261 mice treated with dual drug-loaded Tf-NPs demonstrate increased DNA damage and apoptosis in tumors. Quantification of number of tumor cells that stained positive for markers of DNA damage (γH2AX), apoptosis (CC3), and proliferation (Ki67) in a U87MG and b GL261 mice that received free drug vs liposome-loaded drug (Tf-NP). Signal intensity was quantified from >300 cells in tumors of three mice per treatment condition using ImageJ. Data presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 7

Immunocompetent GL261 mice treated with drug-loaded Tf-NPs are protected from systemic drug toxicity. Mice treated with drug-loaded Tf-NPs demonstrated relative protection from a leukopenia and b thrombocytopenia caused by JQ1 and TMZ. Mice in either treatment arm did not demonstrate drops in RBC counts (c), which is known to be unaffected by JQ1 or TMZ. d Quantification of average daily body weights of mice at the end of the 96 h treatment course. Study powered with five mice per treatment arm for statistical significance. Student t-test used to quantify changes in hematopoietic profiles between free drug and Tf-NP treatment arms (*p < 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001)