Evaluation of polymethine dyes as potential probes for near infrared fluorescence imaging of tumors: part - 1

Abstract

Near-infrared (NIR) organic dyes have become important for many biomedical applications, including in vivo optical imaging. Conjugation of NIR fluorescent dyes to photosensitizing molecules (photosensitizers) holds strong potential for NIR fluorescence image guided photodynamic therapy (PDT) of cancer. Therefore, we were interested in investigating the photophysical properties, in vivo tumor-affinity and fluorescence imaging potential of a series of heterocyclic polymethine dyes, which could then be conjugated to certain PDT agents. For our present study, we selected a series of symmetrical polymethine dyes containing a variety of bis-N-substituted indole or benzindole moieties linked by linear conjugation with and without a fused substituted cyclohexene ring. The N-alkyl side chain at the C-terminal position was functionalized with sulfonic, carboxylic acid, methyl ester or hydroxyl groups. Although, among the parent cyanine dyes investigated, the commercially available, cyanine dye IR783 (3) (bis-indole-N-butylsulfonate)-polymethine dye with a cyclic chloro-cyclohexene moiety showed best fluorescence-imaging ability, based on its spectral properties (λAbs=782 nm, λFl=810 nm, ε = 261,000 M(-1)cm(-1), ΦFl≈0.08) and tumor affinity. In addition to 3, parent dyes IR820 and Cypate (6) were also selected and subjected to further modifications by introducing desired functional groups, which could enable further conjugation of the cyanine dyes to an effective photosensitizer HPPH developed in our laboratory. The synthesis and biological studies (tumor-imaging and PDT) of the resulting bifunctional conjugates are discussed in succeeding paper (Part-2 of this study).

Keywords: Fluorescence Quantum Yields.; Near Infrared Fluorescence Imaging; Near Infrared Fluorophores; Photodynamic therapy; Reactive Oxygen Species.

Figure 1

Absorption (A), fluorescence (B) spectra of near infrared fluorophores (NIRFs) 1-4 and ICG in methanol (5 μM).

Figure 2

Absorption (A) and fluorescence (B) spectra of fluorophores 5, 7 and 8 derived from the cyanine dye IR820 in methanol (5µM).

Figure 3

Absorbance (A) and fluorescence (B) spectra of fluorophores 9 and 10 derived from cyanine dye IR783 in methanol (5µM).

Figure 4

A single diode laser with an excitation at 785 nm and an emission at 820 nm long pass (LP) was used to determine the NIR flow uptake of dyes ICG, IR820, 1-10, and 6 (cypate). Figures A and B presents the NIR flow uptake of the dyes (1 μM) in Colon 26 and U87 cells, whereas figures C and D illustrate the fluorescence of the dyes in Colon 26 and U87 media (RPMI and MEM) only.

Figure 5

NIR whole body fluorescence images of BALB/c mice bearing Colon 26 tumors at 24 h post injection (p.i.) of the fluorophores 1-3 (dose: 0.03μmol/kg). The ex vivo image of NIRF 3 at 24 h p.i is shown.

Figure 6

NIR Fluorescence images (no spectral unmixing) of BALB/c mice bearing Colon 26 tumors at 24 h post injection of a non-tumor avid cyanine dye 4 (dose: 0.03μmol/kg).

Figure 7

Fluorescence images of BALB/c mice bearing Colon 26 tumors at 24 h post injection of fluorophores 5-8 (dose: 0.03μmol/kg).

Figure 8

Ex vivo fluorescence biodistribution of NIRFs 1-8. Note: The biodistribution of the cyanine dye 4 is not shown due to its weak in vivo fluorescence intensity.