LAT1-Targeting Thermoresponsive Fluorescent Polymer Probes for Cancer Cell Imaging

L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[3H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[3H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.


Zeta Potential Measurement
. Zeta potential profiles of prepared polymers. Figure S2. GPC calibration curve using polyethylene glycol standard.
HeLa cells (2.5 × 10 4 cells per well) and HEK 293 cells (1 × 10 5 cells per well) were seeded in BioCoat TM Collagen I Cellware 4-Well Culture Slide in 1 mL of medium. After overnight incubation for HeLa cells and 2-day incubation for HEK 293 cells, the cells were rinsed once with PBS and fixed with 4% paraformaldehyde in PBS for 30 min. The fixed cells were rinsed with PBS and osmotically treated for 30 min at −20 °C in methanol, then incubated with human serum albumin (1:100; Sigma Aldrich) diluted in PBS for 30 min to block non-specific binding sites. Cells were rinsed three times with PBS and the primary antibody, anti-hLAT1 polyclonal antibody rabbit (1:100) was added in PBS at 4 °C overnight. Cells were rinsed three times with PBS and the secondary antibodies, Alexa Fluor ® 488-labeled donkey anti-rabbit IgG antibodies (1:1000; Invitrogen) were added in PBS for 60 min at room temperature. After cells were rinsed three times with PBS, coverslips were mounted over cells in VECTORSHIELD ® HardSet TM Mounting Medium with DAPI (Vector Labolatories, Burlingame, CA). Cells were then observed with a FV1000D (Olympus, Tokyo, Japan) confocal laser-scanning microscope.
As a result, the 38-kDa protein band, attributed to hLAT1, was detected in HeLa cells, while there was no band in HEK 293 cells. Anti-β-actin antibody was using as a loading control ( Figure S3 (a)). The 125-kDa-protein band in HeLa cells is probably a dimer of LAT1 and 4F2hc [48]. Microscopy images of cells after treatment with an anti-hLAT1 polyclonal antibody also showed the expression of LAT1 on the membrane of HeLa cells, while expression was not observed in the fluorescent image of HEK 293 cells ( Figure S3 (b)). These results demonstrate that HeLa cells express LAT1 while HEK 293 cells do not. Figure S3. Western blot analysis was performed on the membrane fraction prepared from HeLa and HEK 293 cells using an anti-hLAT1 antibody (a). For HeLa cells, the 38-kDa-protein band was detected. An anti-β-actin antibody was used as a loading control. Microscopy images of HeLa and HEK 293 cells after treatment with an anti-hLAT1 polyclonal antibody (b). hLAT1 appears green and the nuclei were stained blue (DAPI). Magnification is 60×; scale bar represents 20 μm.

Fluorescence Intensity of Probes
Fluorescence spectra were measured using an FP-6300 spectrofluorometer (Jasco), and the temperature was controlled using an ETC-273T controller (Jasco) and a PT-31 Peltier system (Kruss, Hamburg, Germany). The maxima of the excitation wavelengths (λex) and the emission wavelengths (λem) of the fluorescent probes were measured using concentrations of 1 mg/mL in PBS solution. The effect of temperature on the fluorescence intensity of the probes was evaluated between 25 °C and 45 °C.
The FL modification rate of the probes was determined from the absorption spectrum and the calibration curve of fluorescein-5-maleimide (FL) (Figure S4), and the maximum absorption wavelength values of P(NIPAAm-co-DMAAm20%)-FL and Tyr-P(NIPAAm-co-DMAAm20%)-FL ( Figure S5). The excitation wavelengths and fluorescence wavelengths of the fluorescent probes were examined ( Figure S6). λex of P(NIPAAm-co-DMAAm20%)-FL was 492 nm, λem of (NIPAAm-co-DMAAm20%)-FL was 516 nm, λex of Tyr-P(NIPAAm-co-DMAAm20%)-FL was 492 nm, λem of Tyr-P(NIPAAm-co-DMAAm20%)-FL was 517 nm. The terminal conjugation of fluorescein-5-maleimide to the polymers was possible while maintaining the characteristics of the fluorescent group. Changes in the fluorescence intensity of the fluorescent probes with temperature were observed. Since the polymers are hydrophilic and elongated below the LCST, the fluorescence intensity is presumed to be maintained. In contrast, the fluorescence intensity gradually decreased when the temperature rose by 2-3 °C above the LCST. It is considered that the fluorescent polymer becomes hydrophobic at the LCST or higher and further agglomeration proceeds by raising the temperature ( Figure S7).