Polymeric Nanoparticles Enable Targeted Visualization of Drug Delivery in Breast Cancer

Abstract

We report the coencapsulation of fluorocoxib Q (FQ) and chemocoxib A (CA) in micellar nanoparticles (FQ-CA-NPs) of a new PPS₁₃₅-b-POEGA₁₇ diblock polymer, which exhibited a hydrodynamic diameter of 109.2 ± 4.1 nm and a zeta potential (ζ) of -1.59 ± 0.3 mV. The uptake of FQ-CA-NPs by 4T1 mouse mammary cancer cells and intracellular cargo release were assessed by fluorescence microscopy that resulted in increased fluorescence in 4T1 cells compared to cells pretreated with celecoxib. The viability of primary human mammary epithelial cells (HMECs) or 4T1 mouse mammary carcinoma cells treated with FQ-CA-NPs were assessed, which showed decreased growth of 4T1 breast cancer cells but showed no effect on the growth of primary human mammary epithelial cells (HMECs). Intravenous dosing of FQ-CA-NPs in mice enabled ROS-induced cargo (FQ and CA) release and fluorescence activation of FQ and resulted in increased fluorescence in breast tumors compared to the tumors of animals pretreated with tempol or celecoxib, and minimum fluorescence was detected in the tumors of animals treated with nothing or empty-NPs. In addition, tumor tissues from treated animals were analyzed ex vivo by liquid chromatography-mass spectrometry (LC-MS)/MS, and identified increased levels of cargo delivery and retention in the tumor compared to tempol- or celecoxib-pretreated animal tumors. These in vivo and ex vivo results confirmed the targeted delivery of loaded NPs followed by ROS-mediated cargo release and fluorescence activation for targeted visualization of drug delivery in breast tumors and CA-induced therapeutic effect in an in vivo tumor growth inhibition assay and an ex vivo hematoxylin and eosin (H&E) staining of tumor tissues. Thus, coencapsulation of FQ and CA into polymeric micellar nanoparticles (FQ-CA-NPs) enabled their ROS-sensitive release followed by fluorescence activation and COX-2-dependent tumor targeting and retention in the visualization of CA delivery in solid breast tumors.

Keywords: chemocoxib A; drug delivery; fluorocoxib Q; micellar nanoparticles; tumor imaging.

Figure 1

(a) Chemical structures of fluorocoxib Q (FQ), chemocoxib A (CA), Tempol, and celecoxib. (b) Formulation of empty-NPs and coencapsulation of FQ and CA using a bulk evaporation method for the formation of micellar nanoparticles (FQ-CA-NPs) with PPS₁₃₅-b-POEGA₁₇ polymer in a 50:50 mixture of CHCl₃/DPBS (v/v) with gentle stirring at 25 °C for 16 h. (c) Dynamic light scattering of empty-NPs and FQ-CA-NPs. (d) Transmission electron microscopy (TEM) of empty-NPs and FQ-CA-NPs. (e) ROS-induced drug release and fluorescence activation of FQ-CA-NPs treated with aqueous hydrogen peroxide (H₂O₂) solutions.

Figure 2

(a) Fluorescence microscopy of vehicle or celecoxib-pretreated 4T1 cells incubated with FQ-CA-NPs for 3 h. (b) Image analysis of vehicle or celecoxib-pretreated 4T1 cells incubated with FQ-CA-NPs using ImageJ software. (c) Viability of primary human mammary epithelial cells (HMECs) or 4T1 mouse mammary carcinoma cells treated with FQ-CA-NPs for 48 h.

Figure 3

(a) In vivo pharmacokinetics of FQ and CA in C57BL/6 mice. (b) In vivo optical imaging of NU/J mice bearing orthotopic 4T1 mammary tumors at 49 h after intravenous administration of FQ-CA-NPs, empty-NPs, and tempol followed by FQ-CA-NPs, and celecoxib followed by FQ-CA-NPs. (c) Measurement of light emission in the normal breast and breast tumors by ImageJ software. (d) Ex vivo optical imaging of the kidney, lung, liver, tumor, muscle, and brain of NU/J mice bearing orthotopic 4T1 mammary tumors at 49 h after intravenous administration of FQ-CA-NPs, empty-NPs, and tempol followed by FQ-CA-NPs, and celecoxib followed by FQ-CA-NPs. (e) Measurement of light emission in the excised kidney, lung, liver, tumor, muscle, and brain by ImageJ software.

Figure 4

Proposed mechanism of ROS-mediated activation of FQ to highly fluorescent FQ-H.