Covalent IR820-PEG-diamine nanoconjugates for theranostic applications in cancer

Abstract

Near-infrared dyes can be used as theranostic agents in cancer management, based on their optical imaging and localized hyperthermia capabilities. However, their clinical translatability is limited by issues such as photobleaching, short circulation times, and nonspecific biodistribution. Nanoconjugate formulations of cyanine dyes, such as IR820, may be able to overcome some of these limitations. We covalently conjugated IR820 with 6 kDa polyethylene glycol (PEG)-diamine to create a nanoconjugate (IRPDcov) with potential for in vivo applications. The conjugation process resulted in nearly spherical, uniformly distributed nanoparticles of approximately 150 nm diameter and zeta potential -0.4±0.3 mV. The IRPDcov formulation retained the ability to fluoresce and to cause hyperthermia-mediated cell-growth inhibition, with enhanced internalization and significantly enhanced cytotoxic hyperthermia effects in cancer cells compared with free dye. Additionally, IRPDcov demonstrated a significantly longer (P<0.05) plasma half-life, elimination half-life, and area under the curve (AUC) value compared with IR820, indicating larger overall exposure to the theranostic agent in mice. The IRPDcov conjugate had different organ localization than did free IR820, with potential reduced accumulation in the kidneys and significantly lower (P<0.05) accumulation in the lungs. Some potential advantages of IR820-PEG-diamine nanoconjugates may include passive targeting of tumor tissue through the enhanced permeability and retention effect, prolonged circulation times resulting in increased windows for combined diagnosis and therapy, and further opportunities for functionalization, targeting, and customization. The conjugation of PEG-diamine with a near-infrared dye provides a multifunctional delivery vector whose localization can be monitored with noninvasive techniques and that may also serve for guided hyperthermia cancer treatments.

Keywords: fluorescent imaging; hyperthermia; image-guided therapy; nanotechnology.

Figure 1

Reaction scheme and formulation of covalent IR820-PEG-diamine nanoconjugate (IRPDcov). Abbreviations: IRPDcov, covalent conjugates of IR820 and PEG-diamine; PEG, polyethylene glycol; TEA, triethylamine.

Figure 2

TEM images of IRPDcov. Abbreviations: IRPDcov, covalent conjugates of IR820 and PEG-diamine; PEG, polyethylene glycol; TEM, transmission electron microscopy.

Figure 3

Absorption spectra of IR820 (solid black), IRPDcov (dashed black), and PEG-diamine (gray) in deionized water. Abbreviations: IRPDcov, covalent conjugates of IR820 and PEG-diamine; PEG, polyethylene glycol; AU, absorbance units.

Figure 4

Fluorescence spectra of IR820 and IRPDcov in phosphate-buffered saline. Abbreviations: IRPDcov, covalent conjugates of IR820 and PEG-diamine; PEG, polyethylene glycol.

Figure 5

Cellular imaging of Dx5 (A and D), MES-SA (B and E), and SKOV-3 cells (C and F) after a 4-hour incubation with 5 μM dye content of IR820 (top) or IRPDcov (bottom); 60×, exposure time 4,000 ms, compared against the pixel intensities scaled from 0 to 255. Abbreviations: IRPDcov, covalent conjugates of IR820 and PEG-diamine; PEG, polyethylene glycol.

Figure 6

Cytotoxicity of IRPDcov or IR820 in cancer cell lines, with or without hyperthermia treatment at 5 μM dye concentration, n=3 experiments, 4 wells/experiment. Notes: All groups are normalized to the control group not exposed to dye or laser. There was no significant effect of laser exposure per se without IRPDcov or IR820, and there was no significant effect of PEG-diamine with or without exposure to laser. *Significant difference (P<0.05) versus nonlaser group; **significant difference (P<0.05) between laser groups. Abbreviations: IRPDcov, covalent conjugates of IR820 and PEG-diamine; PEG, polyethylene glycol.

Figure 7

Images taken 15 minutes and 24 hours after IV injection for IRPDcov (A and D), IR820 (B and E), and ICG (C and F) compared against the pixel intensities scaled from 0 to 255. Note: Mice were shaved for imaging. Abbreviations: ICG, indocyanine green; IRPDcov, covalent conjugates of IR820 and PEG-diamine; IV, intravenous; PEG, polyethylene glycol.

Figure 8

Organ images taken 24 hours after IV injection of IRPDcov, IR820, or ICG compared against the pixel intensities scaled from 0 to 255. Abbreviations: ICG, indocyanine green; IRPDcov, covalent conjugates of IR820 and PEG-diamine; IV, intravenous; PEG, polyethylene glycol.

Figure 9

Quantitative organ content 24 hours after IV injection of IRPDcov, IR820, or ICG. Notes: *Significant difference (P<0.05) with ICG values for same organ; **significant difference (P<0.05) between IRPDcov and IR820 values for same organ. There was no detectable signal for ICG in lungs or kidneys after 24 hours. Abbreviations: ICG, indocyanine green; IRPDcov, covalent conjugates of IR820 and PEG-diamine; IV, intravenous; PEG, polyethylene glycol.

Figure 10

Plasma concentrations (μg/mL) of IRPDcov, IR820, and ICG at different time points after IV injection. Notes: *Significant difference from ICG (P<0.05); **significant difference IRPDcov vs IR820 (P<0.05). Abbreviations: ICG, indocyanine green; IRPDcov, covalent conjugates of IR820 and PEG-diamine; IV, intravenous; PEG, polyethylene glycol.