Page 24 - GPD-1-1

Gene & Protein in Disease Photothermal therapy of pentamethine cyanine

2.6. Western blot analysis and eosin for 1 min. Morphological changes of the tumor

The expression of HSP70 was detected by Western blotting tissues were observed with an electronic light microscope
according to the standard experimental schemes. As (Olympus, Tokyo, Japan).
previously described , human CRC cells HCT-116 and 2.11. Cell uptake assay
[26]
SW480 were collected and lysed with RIPA buffer in the
[29]
presence of protease inhibitors. The protein was quantified As previously described , HCT-116 and SW480 cells
3
and electrophoresed by sodium dodecyl sulfate– were seeded in 24-well plates at a density of 5 × 10 cells
polyacrylamide gel electrophoresis. After electrophoresis, per well and cultured for 24 h. Different concentrations
the protein was transferred to the polyvinylidene fluoride of CY5-664 were added into culture plates, and the cells
(PVDF) membrane from the gel using the electrospinning were cultured continuously for 2 h. The cells were washed
method. The PVDF membrane was sealed with bovine 3 times with phosphate-buffered solution and detected
serum albumin and coincubated with the primary using a fluorescence inverted microscope (DMI8, Leica,
antibody and the secondary antibody. Finally, the blot Germany).
determination was detected using a chemiluminescence 2.12. PTT by CY5-664
analyzer (Amerisham Biosciences, Boston, MA, U.S.A.).
HCT-116 and SW480 cells were seeded in 96-well plates
2.7. Singlet oxygen detection analysis at a density of 5 × 10 cells per well and cultured for 12 h.
3
The singlet oxygen generation from CY5-664 was Different concentrations of CY5-664 were added to culture
determined by the fluorescent probe SOSG. In brief, plates, and the cells were cultured continuously for 2 h.
2
1.0 × 10 M Cy5-664 and 1.5 × 10 M SOSG were mixed Cells were irradiated for 2 min (660 nm, 0.5 W/cm ) with
-5
-6
in an aqueous solution containing 2% methanol and were or without ice incubation. The cell viability was determined
irradiated with 0.5 W of 660 nm red laser pointer for 2 min by MTT method.
at room temperature. The mixed solution was quickly 2.13. Statistical analysis
transferred to the cuvette after laser irradiation and the
fluorescence intensity was determined using a steady- All statistical analyses were completed by SPSS16.0. The
state transient fluorescence spectrometer. The excitation statistical difference between the treatment and control
wavelength is 494 nm and the emission peak at 530 nm was groups of IMCA was analyzed by Student’s t-test.
used to evaluate singlet oxygen generation.
3. Results and discussion
2.8. In vitro assessment of photothermal effect 3.1. Synthesis and characterization of CY5-664
The photothermal effect of CY5-664 was determined using The synthesis routes are shown in Figure 1A. 2,3,3-trime
a precise thermal imaging instrument in a culture medium. thylindoleninium-5-sulfoacid (1), 1-bromomethyl-3,5-
In brief, different concentrations CY5-664 solutions were bis(2-(2-(2-methoxylethoxyl)ethoxyl)ethoxyl)benzene (3),
prepared according to the predetermined concentrations and 1-(4-carboxybenzyl)-2,3,3-trimethylindoleninium-5-
and were irradiated with 0.5 W of 660 nm red laser sulfonate bromide (5) were synthesized according to the
pointer for 2 min. The temperature of CY5-664 solutions literature [30,31] .
was monitored by a precise thermal imaging instrument
(Fotric 225S#L24, Shanghai, China) every 30 s. A mixture of 2,3,3-trimethylindoleninium-5-sulfoacid
1 (0.8 g, 3.34 mmol) and potassium acetate (0.36 g,
2.9. In vivo photothermal analysis 3.67 mmol) was stirred in 30 mL of methanol at room
BALB/c nude mice bearing human CRC cell HCT-116 were temperature for 0.5 h. After removal of methanol under
injected with NS or CY5-664 through tail vein. At 24 h after vacuum, the resulting potassium salt 2 was heated with
the injection of CY5-664, the tumor was irradiated by laser 1-romomethyl-3,5-bis(2-(2-(2-methoxylethoxyl)ethoxyl)
(660 nm, 0.5 W/cm ) for 2 min, and the local temperature ethoxyl)benzene 3 (1.65 g, 3.33 mmol) in acetonitrile at
2
of the tumor was monitored by a precise thermal imaging 90°C for 24 h under argon. The mixture was cooled to
instrument (Fotric 225S#L24, Shanghai, China) every 30 s. room temperature and the solvent was evaporated. The
pure product 4 (1.11 g) was obtained in 51% yield by the
2.10. Hematoxylin & eosin (H&E) staining analysis purification on silica gel column with CH Cl /CH OH
2
3
2
1
As previously described [27,28] , the tissues from the animal (15:1, V/V) as eluent and the H NMR spectrum is shown
experiments were fixed in 4% paraformaldehyde fixative and in Figure S1 (in Supplementary File).
embedded in paraffin. Formalin-fixed paraffin-embedded The intermediate 4 (374.2 mg, 0.57 mmol)
samples were sliced and stained with hematoxylin for 5 min and malonaldehyde dianilide hydrochloride

Volume 1 Issue 1 (2022) 4 https://doi.org/10.36922/gpd.v1i1.87