2.1.

Antibody and Indocyanine Green Labeling

The monoclonal mouse antibody TuBB-9 was produced from hybridoma cells kindly provided by Leibnitz Institute Borstel, Germany.²⁵ Hybridoma cells were cultivated in bioreactor cell culture flasks (INTEGRA Biosciences, Switzerland). The antibodies were purified from the culture supernatant with protein G columns. For labeling with ICG, antibodies ($1\mathrm{mg}/\mathrm{ml}$ in PBS at pH 7.6) were mixed with the ICG-NHS (1.21 mM in PBS at pH 7.6, Intrace medical, Switzerland) in a molar ratio of $1:2$, $1:4$, and $1:8$, respectively. The solution was incubated at room temperature on a shaker for 1 h, and the labeled antibody was purified with a NAP-5 Sephadex column (GE Healthcare). After elution with PBS (pH 7.4), the labeled antibodies were concentrated with Microcon tubes (Millipore) and resuspended in PBS (pH 7.4). The absorption spectrum of free ICG, ICG-NHS and ICG labeled TuBB-9 (TuBB-9-ICG) was measured by UV-VIS spectroscopy (Hitachi, Japan).

2.2.

Conjugation of TuBB-9-ICG with Nuclear Localization Signal Peptides

Cysteine modified NLS (CGGGPKKKVED, Anaspec) was conjugated to TuBB-9-ICG using the linker molecule sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC; Pierce).³⁸ The maleimide groups were introduced to the conjugates by the reaction of 3 mg TuBB-9-ICG with 0.14 mg sulfo-SMCC in phosphate buffered saline (PBS, pH 7.4) in a ratio of $1:15$ at room temperature for 1 h. The maleimide-derivatized TuBB-9-ICG was then purified on a NAP-5 mini-column (exclusion limit 5 kDa; GE Healthcare) eluated with PBS, pH 7.0. The fractions containing maleimide TuBB-9-ICG were transferred to an ultrafiltration device (100 kDa cutoff; Amicon). The device was centrifuged at 800 g for 20 min to concentrate maleimide-TuBB-9-ICG to $2\mathrm{mg}/\mathrm{ml}$, which were then mixed with a 60-fold molar excess of NLS-peptides ($10\mathrm{mg}/\mathrm{ml}$ in PBS, pH 7.0) for 18 h at 4°C. NLS-TuBB-9-ICG conjugates were purified from excess NLS-peptides and concentrated to $2\mathrm{mg}/\mathrm{ml}$ in PBS (pH 7.4) by ultrafiltration.

NLS-TuBB-9-ICG conjugates were analyzed by sodium dodecyl sulfate-2-polyacrylamide gel electrophoresis (SDS–PAGE) under nonreducing conditions on a 5% Tris-HCl ready-minigel (Bio-Rad) stained with Coomassie Brilliant Blue. The migration distance in the gel relative to the bromphenol blue dye front ($R_{\mathrm{f}}$) was measured and the numbers of NLS-peptides bound to the TuBB-9-ICG were estimated. For characterization and concentration measurements the absorption spectra of NLS-TuBB-9-ICG conjugates were measured by UV-VIS spectroscopy (Hitachi, Japan) and the emission spectra were recorded by fluorescence spectroscopy (FluoroMax, SPEX).

2.3.

Cell Culture

Human cervical cancer cell line HeLa and human ovarian adenocarcinoma cell line OVCAR-5 were obtained from American Type Culture Collection. HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM low glucose, Sigma) containing 10% fetal bovine serum (FBS gold, PAA) and 1% Penicillin/Streptomycin (PAA). OVCAR-5 cells were cultured in RPMI-1640 medium (Sigma) containing 10% FBS and 1% Penicillin/Streptomycin. Fibroblast cells were maintained in DMEM medium (High glucose, Sigma) containing 10% FBS and 1% Penicillin/Streptomycin. Cells were incubated in humidified atmosphere at 37°C with 5% ${\mathrm{CO}}_2$.

2.4.

Endosomal Release of TuBB-9-ICG-NLS

HeLa and OVCAR-5 cells were plated in glass bottom imaging dishes ($4\times {10}^4\text{cells}/\mathrm{ml}$) in a medium containing 100 nM BPD (benzoporphyrin derivative monoacid Ring A, Sigma). After 18 to 20 h, cells were washed twice with PBS and incubated for 4 h with ($15\mu \mathrm{g}/\mathrm{ml}$) NLS-TuBB-9-ICG conjugates. Then, the cells were washed once with serum-free DMEM and twice with PBS. Light irradiation for endosomal escape was performed with a 410 nm LED (Roithner Lasertechnik, Vienna, Austria) with $15\mathrm{mW}/{\mathrm{cm}}^2$ for 60 s. Untreated cells, treated cells without irradiation, and treated cells with irradiation were fixed with 4% Paraformaldehyde and permeabilized by 0.25% Triton, then the fixed cells were incubated with $5\mu \mathrm{g}/\mathrm{ml}$ Alexa 546 labeled Goat Anti-Mouse IgG Antibody (Life Technologies) for 45 min. Cells were washed twice with PBS and imaged with a fluorescence microscope (Nikon, Japan).

2.5.

Cell Viability Assay

For viability assay, HeLa and OVCAR-5 cells were seeded with a density of $4\times {10}^4\text{cells}/\mathrm{ml}$ in 96-well plates, 100 nM BPD was added into each well, and incubated for 18 h. Then, cells were incubated for another 4 h with ($15\mu \mathrm{g}/\mathrm{ml}$) NLS-TuBB-9-ICG constructs with different ICG ratios (antibody-ICG ratios: $1:0.6$, $1:1.8$, and $1:5.2$) and for comparison with NLS-TuBB-9-FITC conjugates. After incubation, the cells were washed twice with PBS and wells were divided into four groups: (1) no light irradiation, (2) irradiation with 410 nm laser for endosomal escape ($15\mathrm{mW}/{\mathrm{cm}}^2$, 30 s), (3) incubation for 48 h and irradiation at 690 nm with a LED for photo-inactivation of Ki-67 ($71\mathrm{mW}/{\mathrm{cm}}^2$, 5 min), and (4) irradiation at 410 nm and after 48 h a second irradiation at 690 nm for endosomal escape and photo-inactivation of ICG. After 72 h, cells were washed twice with PBS and $100\mu \mathrm{l}$ MTT-solution ($1\mathrm{mg}/\mathrm{ml}$ in culture medium) was added per well. After an additional incubation for 1 h, culture medium was removed and the samples were incubated in $200\mu \mathrm{l}$ DMSO for 30 min on a shaker to dissolve the formazan crystals. Absorbance was measured on a microplate reader (Spectramax M5, Molecular Devices) at 570 nm. Absorbance changes of treated samples compared to untreated controls were used as a measure for cell viability. Statistical analysis was performed by using SPSS (IBM Deutschland GmbH). The viability data of different groups were expressed as mean value and standard deviation of a total of eight measurements. Student’s $t$-test was used and differences were considered significant at $p<0.05$. To establish the growth curves of the different treatment groups three cell samples were counted each day.

3.1.

Absorption Characteristics of Indocyanine Green and TuBB-9-ICG

For our study, we prepared TuBB-9-ICG conjugates with three different ratios of antibody to ICG as shown in Table 1. The absorption spectrum of ICG-NHS (used for labeling) and different TuBB-9-ICG conjugates are shown in Fig. 1. ICG-NHS and TuBB-9-ICG conjugates show high absorption in the range from 650 to 900 nm, with two peaks around 730 and 800 nm, which fits the optical window of biological tissue. The peak at 280 nm originates from protein absorption of the TuBB-9 antibody.

Table 1

TuBB-9-ICG ratios used for conjugation reaction and the ratios after reaction, determined after absorption measurements.

Fig. 1

Absorption spectra of ICG conjugated in different ratios to the antibody TuBB-9. For measurements, samples were adjusted to the same TuBB-9 concentrations and diluted in PBS. The peak at 280 nm originates from protein absorption of the TuBB-9 antibody.

One of the disadvantages for clinical application of ICG is the nonlinear dependence of light absorption on the concentration, because of aggregation. The ratio of the absorption peaks of free ICG at 700 and 770 nm changes with increasing concentration [Fig. 2(a)]. However, after conjugation to the antibody, the spectral shape remains stable and the two peaks at 730 and 800 nm tend to increase linearly with concentration [Fig. 2(b)].

Fig. 2

Absorption spectra of (a) free ICG and (b) ICG linked to the antibody TuBB-9 (ratio $1:1.8$) in ${\mathrm{H}}_2\mathrm{O}$ at different concentrations. Free ICG shows a characteristic absorption band exhibiting two distinct peaks in the NIR. Conjugation to the antibody leads to a broadening of the absorption peaks. The additional peak at 280 nm originates from protein absorption of the TuBB-9 antibody.

3.2.

Conjugation of TuBB-9-ICG with Nuclear Localization Signal Peptides

In order to increase cellular uptake for intracellular delivery, the three different TuBB-9-ICG conjugates listed in Table 1 were covalently conjugated with the cell penetrating peptide NLS. Figure 3(a) depicts schematically the chemical reaction of the conjugation. The resulting constructs were analyzed by SDS-PAGE to estimate the number of NLS-peptides conjugated to each molecule of TuBB-9-ICG conjugate. As shown in Fig. 3(b) after coomassie blue protein staining, the band of NLS-TuBB-9-ICG has a distinctly longer migration distance than TuBB-9-ICG. This difference in moving distance corresponds to different amounts of NLS-peptides conjugated to TuBB-9-ICG. Absorption spectra of the NLS-TuBB-9-ICG constructs showed no change on the absorption peak position of NLS-TuBB-9-ICG and TuBB-9-ICG. Nevertheless, the molar ratio of TuBB-9 and ICG decreased slightly after the NLS conjugation, since ICG and NLS are both connected via NHS groups to the amino groups of TuBB-9 [Fig. 3(c)]. The characterization results are listed in Table 2. Figure 3(d) shows the fluorescence emission spectrum of ICG-NHS, TuBB-9-ICG 2, and NLS-TuBB-9-ICG 2 excited by 730 nm irradiation. The fluorescence intensity also decreased slightly after the NLS conjugation since the number of ICG molecules per antibody decreased.

Fig. 3

(a) Conjugation scheme of TuBB-9-ICG with NLS. TuBB-9-ICG conjugate was incubated with an SMCC-linker that binds with its NHS group to the amino group of the antibody. In the next step, the maleimide group of the linker reacts with thiols on the NLS peptide to form TuBB-9-ICG-NLS. (b) Characterization of the antibody conjugates with SDS-PAGE and coomassie staining. (c) Absorption spectrum of the NLS-TuBB-9-ICG constructs and (d) fluorescence spectrum of ICG-NHS, TuBB-9-ICG 2, and NLS-TuBB-9-ICG 2. All samples are normalized to the TuBB-9 antibody concentration.

Table 2

Characterization of NLS-TuBB-9-ICG constructs.

3.3.

Nucleolar Localization of NLS-TuBB-9-ICG after Photochemical Internalization Irradiation

After incubation of HeLa and OVCAR-5 cells with NLS-TuBB-9-ICG, constructs are taken up by the cells and are entrapped in endosomal structures (Fig. 4, middle panel). Entrapped constructs were released to the cytosol by the PCI process. Cells were first incubated with BPD and then irradiated at 410 nm. At 410 nm the photosensitizer BPD is efficiently excited without activating ICG, which exhibits a maximum absorption at 700 nm. Figure 4 shows the intracellular distribution of NLS-TuBB-9-ICG before and 48 h after PCI irradiation.

Fig. 4

Nucleolar localization of NLS-TuBB-9-ICG was observed in HeLa and OVCAR-5 cells 48 h after BPD mediated photochemical internalization. Cells were incubated with BPD for 18 h and then for 4 h with NLS-TuBB-9-ICG. Before PCI irradiation, TuBB-9 conjugates located in endosomal structures in the cytoplasm. 48 h after irradiation at 410 nm for activation of BPD conjugates locate also in the nucleoli of the cells at the site of Ki-67. TuBB-9 was stained by the secondary antibody goat-anti-mouse Alexa 546. Arrows in fluorescence images point to the localization of the nucleoli. Size bars: $10\mu \mathrm{m}$.

Without PCI, both HeLa and OVCAR-5 cells showed a dotted fluorescence pattern in the cytoplasm after staining TuBB-9 with a secondary antibody. This indicates the trapping of NLS-TuBB-9-ICG in endosomal structures. In contrast, 48 h after PCI irradiation fluorescence was detected uniformly spread over the cytoplasm and also located in the nucleoli at more distinct sites, which indicates the release of NLS-TuBB-9-ICG into the cytoplasm and binding to the Ki-67 protein. A similar relocalization from the cytosol to the nucleus was observed after cytosolic microinjection of TuBB-9-FITC conjugates in earlier experiments.²⁶ Approximately, 90% of HeLa cells growing as monolayer cell culture are positive for Ki-67.²⁸ This means that at the maximum 90% of the cells can show nucleolar localization of the TuBB-9 constructs. In earlier studies we have shown that with NLS-TuBB-9-FITC nucleolar localization was achieved in 78% of Ki-67 positive cells.²⁸

At the time point of the second irradiation for the Ki-67 inactivation (72 h after incubation start), the BPD fluorescence is not detectable anymore in HeLa or in OVCAR-5 cells (Fig. 5).

Fig. 5

HeLa cells and OVCAR-5 cells incubated with 100 nM BPD. After 24 h incubation, bright BPD fluorescence can be detected under the fluorescence microscope. 72 h after incubation start the BPD fluorescence is not detectable. Illumination, exposure times and objective are identical in all images.

3.4.

In Vitro Ki-67 Photo-Inactivation with Indocyanine Green Conjugates

In previous studies, we demonstrated the ability of NLS-TuBB-9-FITC to kill various proliferating cells after the dual irradiation with wavelengths of 690 and 490 nm.^27,28 The irradiation of NLS-TuBB-9-FITC leads to cell death by the inactivation of the Ki-67 protein or a binding partner. In order to evaluate the efficiency of the ICG constructs exerting their action after the dual irradiation with wavelength of 410 and 690 nm, an in vitro cytotoxicity assay was performed. Figure 6(a) shows the cytotoxic effects of different NLS-TuBB-9-ICG constructs in comparison with NLS-TuBB-9-FITC on HeLa cells. Untreated cells and cells incubated only with BPD served as controls. Due to damaging effects to endosomal membranes, all groups treated with BPD showed a loss in cell viability of approximately 20% to 30% after 410 or 690 nm irradiation alone. However, groups treated with NLS-TuBB-9-FITC or NLS-TuBB-9-ICG and irradiated at 410 and 690 nm showed a considerably higher cell killing compared to the groups of untreated cells and cells incubated with BPD but without irradiation. Differences in cell viability were observed between the different NLS-TuBB-9-ICG constructs and NLS-TuBB-9-FITC. Among the three different ICG constructs, the NLS-TuBB-9-ICG (ratio $1:1.8$) showed the highest cell killing efficiency (cell viability of $13.7\%\pm 3.1\%$), compared to the group treated with the NLS-TuBB-9-ICG (ratio $1:0.6$) ($29.2\%\pm 5.8\%$) or with NLS-TuBB-9-ICG (ratio $1:5.2$) ($22.4\%\pm 2.6\%$). No significant difference was observed between NLS-TuBB-9-ICG (ratio $1:1.8$) and NLS-TuBB-9-FITC.

Fig. 6

NLS-TuBB-9-ICG constructs eliminated effectively proliferating (a) HeLa and (b) OVCAR-5 cells after 410 and 690 nm irradiation. Irradiation at 410 nm caused endosomal release and irradiation at 690 nm lead to the inactivation of Ki-67. The middle columns show cells that got only irradiation with one wavelength and demonstrate unspecific cell killing. All NLS-TuBB-9-ICG constructs showed effective cell elimination, but best results were obtained with NLS-TuBB-9-ICG 2. *** indicates statistically significant difference in cell viability between two linked samples after t-test analysis ($p<0.05$). (B) MTT assay reveals the capacity of the NLS-TuBB-9-ICG 2 for elimination of proliferating OVCAR-5 cells.

The treatment procedure was also applied on ovarian carcinoma cells OVCAR-5 using NLS-TuBB-9-ICG (ratio $1:1.8$) and NLS-TuBB-9-FITC conjugates. Cytotoxic effects were comparable, but slightly lower, compared with HeLa cells [Fig. 6(b)].