Fluorocoxib A loaded nanoparticles enable targeted visualization of cyclooxygenase-2 in inflammation and cancer

Abstract

Cyclooxygenase-2 (COX-2) is expressed in virtually all solid tumors and its overexpression is a hallmark of inflammation. Thus, it is a potentially powerful biomarker for the early clinical detection of inflammatory disease and human cancers. We report a reactive oxygen species (ROS) responsive micellar nanoparticle, PPS-b-POEGA, that solubilizes the first fluorescent COX-2-selective inhibitor fluorocoxib A (FA) for COX-2 visualization in vivo. Pharmacokinetics and biodistribution of FA-PPS-b-POEGA nanoparticles (FA-NPs) were assessed after a fully-aqueous intravenous (i.v.) administration in wild-type mice and revealed 4-8 h post-injection as an optimal fluorescent imaging window. Carrageenan-induced inflammation in the rat and mouse footpads and 1483 HNSCC tumor xenografts were successfully visualized by FA-NPs with fluorescence up to 10-fold higher than that of normal tissues. The targeted binding of the FA cargo was blocked by pretreatment with the COX-2 inhibitor indomethacin, confirming COX-2-specific binding and local retention of FA at pathological sites. Our collective data indicate that FA-NPs are the first i.v.-ready FA formulation, provide high signal-to-noise in inflamed, premalignant, and malignant tissues, and will uniquely enable clinical translation of the poorly water-soluble FA compound.

Keywords: COX-2; Cancer; Inflammation; Molecular imaging; Nanoparticles; Reactive oxygen species.

Figure 1

Properties and nano-formulation of Fluoroocoxib A (FA). (A) Chemical structure and photophysical properties (λ_ex = 581 nm, λ_em = 605 nm) of FA. (B) Synthesis of PPS₁₀₆-b-POEGA₁₇, FA-NPs and 5-ROX-NPs – Reagents and conditions: (a) DCC, DMAP, CH₂Cl₂ 25 °C, 24 h; (b) POEGA, AIBN, (CH₂)₄O₂, 70 °C, 24 h; (c) CHCl₃, PBS, 25 °C, 24 h; (d) FA or 5-ROX, CHCl₃, PBS, 25 °C, 24 h; (e) solubilization of FA or FA-NPs in PBS – (i) FA (1 mg/mL) and (ii) FA-NPs (1 mg/mL FA).

Figure 2

Physical properties of FA-NPs. (A and B) DLS and TEM characterization confirms NPs are under 100 nm diameter (82.3 ± 8.4 nm hydrodynamic diameter). (C) FA-NPs retain the excitation and emission (λ_ex = 581 nm, λ_em = 605 nm) spectra of the parent FA molecule. (D and E) The critical micelle concentration of NPs was determined as ~ 0.065 mg/mL by nile red assay and observation of size and morphological changes by DLS. (F) NPs release drug dose-dependently in response to increasing H₂O₂.

Figure 3

Targeted in vivo imaging of COX-2 in inflammation and cancer. Female nude mice bearing COX-2-expressing 1483 HNSCC tumor xenografts were dosed with FA-NPs (1 mg/kg FA, i.p.) and imaged at 4 h post-injection of NPs in a Xenogen IVIS200 optical imaging instrument. A subset of animals was pretreated with indomethacin to block the FA binding site on COX-2. (A–B) Fluorescence images of 1483 HNSCC tumor-bearing mice injected with FA-NPs with or without indomethacin pre-treatment 1 h prior to i.p. injection of FA-NPs. (C) Quantification of images showing relative tumor signal (n = 10, p = 0.003). (D–E) Sprague Dawley rats with carrageenan-induced inflammation in their right hind footpads and with or without pre-injection of competitive COX-binding molecule indomethacin (2 mg/kg) were imaged 3-h post injection with FA-NPs (1 mg/kg FA, i.p.). (F) Quantification of signal in images of inflamed (carrageenan injected) versus non inflamed footpads with and without indomethacin pre-treatment (n = 8, p < 0.002).

Figure 4

Pharmacokinetics and biodistribution of IV-injected FA-NPs. (A) FA-NPs have 1.1 h circulation half-life after i.v. administration via tail vein. (B) FA-NPs biodistribute to liver, kidney, and lungs after i.v. administration, but FA is cleared from these organs by 4 h post-injection. (C) FA imaging at 4 h after injection with FA-NPs reveals a significant increase in tumor signal relative to liver, kidney, or lungs. (n = 9, p < 0.004) (D) FA-NP signal is significantly higher in the inflamed footpad of mice over 8 h post-injection (n = 3, p < 0.01, *).

Figure 5

In vivo toxicology of FA-NPs after i.v. administration. (A) Serum chemical markers of liver (ALT and AST) and kidney (BUN) toxicity measured 24 h after i.v. administration of saline, 1 mg/kg, 10 mg/kg, or 20 mg/kg FA-NPs (FA dose). (B) H&E sections of liver and kidney at 20× magnification.