Pharmacokinetics, Biodistribution, and Anti-Angiogenesis Efficacy of Diamino Propane Tetraiodothyroacetic Acid-conjugated Biodegradable Polymeric Nanoparticle

Abstract

The anti-angiogenic agent, diamino propane tetraiodothyroacetic acid (DAT), is a thyro-integrin (integrin αvβ3) antagonist anticancer agent that works via genetic and nongenetic actions. Tetraiodothyroacetic acid (tetrac) and DAT as thyroid hormone derivatives influence gene expression after they transport across cellular membranes. To restrict the action of DAT to the integrin αvβ3 receptors on the cell surface, we used DAT-conjugated PLGA nanoparticles (NDAT) in an active targeting mode to bind to these receptors. Preparation and characterization of NDAT is described, and both in vitro and in vivo experiments were done to compare DAT to NDAT. Intracellular uptake and distribution of DAT and NDAT in U87 glioblastoma cells were evaluated using confocal microscopy and showed that DAT reached the nucleus, but NDAT was restricted from the nucleus. Pharmacokinetic studies using LC-MS/MS analysis in male C57BL/6 mice showed that administration of NDAT improved the area under the drug concentration curve AUC_{(0-48 h)} by 4-fold at a dose of 3 mg/kg when compared with DAT, and C_max of NDAT (4363 ng/mL) was 8-fold greater than that of DAT (548 ng/mL). Biodistribution studies in the mice showed that the concentrations of NDAT were higher than DAT/Cremophor EL micelles in heart, lung, liver, spleen, and kidney. In another mouse model using female NCr nude homozygous mice with U87 xenografts, tumor growth was significantly decreased at doses of 1 and 3 mg/kg of NDAT. In the chick chorioallantoic membrane (CAM) assay used to measure angiogenesis, DAT (500 ng/CAM) resulted in 48% inhibition of angiogenesis levels. In comparison, NDAT at low dose (50 ng/CAM) showed 45% inhibition of angiogenesis levels. Our investigation of NDAT bridges the study of polymeric nanoparticles and anti-angiogenic agents and offers new insight for the rational design of anti-angiogenic agents.

Figure 1

(a) Chemical structure and schematic illustration of the synthesis of diamino tetraiodothyroacetic acid (DAT)-conjugated PLGA (lactide:glycolide 79:21). (b) ¹H NMR spectra in DMSO-D6 and assignments of signals of DAT (bottom) and PLGA-DAT (top).

Figure 2

(a) ¹H-¹³C HSQC/HSQC-DEPT 2D NMR and (b) ¹H-¹³C HMBC 2D NMR of PLGA-DAT in DMSO-D6.

Figure 3

(a) Schematic illustrations of PLGA-DAT polymer self-assembled into NDAT. (b) TEM image of NDAT. (c) Results for NDAT (black dashed line) formulation parameters and physicochemical properties.

Figure 4

Binding affinity of tetrac, DAT, and NDAT to purified αvβ3. Error bars represent standard deviation of the mean, n = 3.

Figure 5

Confocal microscopy images of U87 cells incubated for 4 hours with Cy5 dye-labelled DAT (a) and NDAT (b). DAT reached the nucleus, and NDAT was restricted from going inside the nucleus. Cells were counter-stained with DAPI (4′,6-diamidino-2-phenylindole) to visualize the nuclei: blue for DAPI and red for Cy5. Magnification, 63X.

Figure 6

(a) Mass spectrum of DAT. (b) MRM transitions of PLGA-DAT after an in-source fragmentation. (c) LC-MS/MS chromatogram of DAT (blue) and deuterium-labelled DAT-D7 (red) as internal standard. (d) LC-MS/MS chromatogram of PLGA-DAT (blue) and DAT-D7 (red).

Figure 7

Mouse plasma DAT level versus time profiles. Different formulations of DAT/Cremophor EL micelles (black) and NDAT nanoformulation (red) were administered subcutaneously into mice at dose of 3 mg (DAT-equivalent)/kg body weight. NDAT was purified with dialysis (without using tangential flow filtration, TFF) before injection. Data are shown as mean ± SD (n = 4).

Figure 8

Biodistribution of DAT administered subcutaneously into mice at equivalent DAT amount of 3 mg/kg body weight (a) as NDAT nanoformulation, and (b) as DAT/Cremophor EL micelles formulation. Shown are DAT amounts in plasma and major organs (heart, lung, liver, spleen, and kidney) determined at 4 and 48 hours. Data are shown as mean ± SD (n = 4), and all data have significance (P < 0.001) for NDAT compared to DAT.

Figure 9

Antitumor effect of 27 days daily subcutaneous administration of void PLGA NPs, NDAT at equivalent DAT of 0.1, 0.3, 1, and 3 mg/kg body weight treatment on U87 glioblastoma xenograft weight. Data are shown as mean ± SD (n = 4). For 1 mg/kg dose, *P = 0.0187, for 3 mg.kg dose, *P = 0.01109.

Figure 10

(a) NDAT administered daily for 20 days at 1 mg/kg subcutaneously (s.c.) resulted in greater tumor volume suppression versus DAT administered daily for 20 days at 10 mg/kg, s.c. (b) Images for suppression of b-FGF-induced angiogenesis in the chick chorioallantoic membrane (CAM) model by DAT versus NDAT at 500 ng/CAM. Data illustrate greater anti-angiogenesis efficacy of NDAT versus DAT at the same concentration.