Cellular internalization mechanism of novel Raman probes designed for plant cells

Diphenylacetylene derivatives containing different polymeric components, poly(l-lysine) (pLys) or tetra(ethylene glycol) (TEG) were designed as novel Raman imaging probes with high Raman sensitivity and low cytotoxicity in living plant cells. The pLys-conjugated probe is internalized via an endocytosis-dependent pathway, whereas TEG-conjugated probe most likely induces direct penetration into the plant cells.


Synthesis of PhC≡CPh-pLys (1)
P(Boc-Lys) (420.6 mg, 0.3 mmol) and 4-phenylethynylphthalic anhydride (74.47 g, 0.3 mmol) were added to a flask with a stirrer bar under nitrogen at 25 °C. After CH 2 Cl 2 (6 mL) was added, the solution was stirred for 1 h at 25°C and then added to an excess amount of hexane. The precipitate was gathered by filtration. The amide carboxylic acid derivative was obtained as a white solid in 87% yield (431.6 mg, 0.262 mmol). Then, the solid (237 mg, 0.144 mmol) was dissolved in toluene (2.8 mL) under nitrogen at 25°C. The solution was refluxed at 130°C for 1 h, and the solvent was removed under reduced pressure. The crude product was purified by aluminum oxide column chromatography with CHCl 3 /MeOH mixture as the eluent. The imide derivative was obtained as a yellow solid. For the deprotection of Boc groups in the side chain, the obtained yellow solid (90 mg, 0.178 mmol) was placed in a flask. Trifluoroacetic acid (0.272 mL, 3.55 mmol) was added to this solution and stirred at 25°C. After 24 h, the solvent was removed under reduced pressure. The obtained viscous solid was dispersed in water and lyophilized. The title compound was obtained as a white solid in 48% yield (71. 3

Synthesis of PhC≡CPh-PEG (3)
PhCCPh-PEG was synthesized according to the same procedure as PhCCPh-TEG. Amineterminated poly(ethylene glycol) (PEG, degree of polymerization = 24) was used as the starting material instead of 3, 6, 9, 12-tetraoxatridecanamine. The product was obtained as a white powder with a yield of 80%. 1
Arabidopsis thaliana (Col-0) was used as the wild-type. The Arabidopsis var2-1 mutant was provided by Dr W. Sakamoto (Okayama university). 2 The seeds were sterilized in 70% ethanol for 1 min then in 10% NaClO for 15 min, after which they were rinsed three times with sterilized water to remove the NaClO. The seeds were grown on 1/2 Murashige and Skoog medium (MS Basal Medium, M5519; Sigma-Aldrich, St. Louis, MO) containing 2.5 mM MES, 1% sucrose, and 0.8 % (w/v) agarose. After being sown on the medium, the seeds were stratified for two days in the dark at 4 °C, then grown under constant white light (100 µmol m−2 s−1) at 22 °C. In the experiment, 14-day-old seedlings were used.

Raman Microscopy.
All Raman spectra were obtained on a Jasco NRS-4100 laser Raman spectrometer (JASCO, Tokyo, Japan) with excitation at 532 nm. Samples were placed on a microscope slide (SUPERFROST WHITE, S2441, Matsunami, Osaka, Japan) with a covered slip (C218181, Matsunami). The laser output was focused on the sample (UPLSAPO 100XO, Olympus, Tokyo, Japan). The slit size of the spectrograph was 100 x 8000 µm. The light intensity was 33.8 mW/µm 2 .
For each spectrum, the measurement was duplicated with an acquisition time of 60 s and averaged.

Preparation of the Raman Probe-Treated Cells.
Each Raman probe was introduced into BY-2 cells. We prepared 10 mM stock solutions of After the treatments, cells were rinsed with MS medium before observation by Raman microscopy.

Stimulated Raman Scattering (SRS) Imaging of Probe-treated Cells.
The images of the cells were obtained with the SRS imaging system, as previously reported. 3 Briefly, picosecond pump pulses at a wavelength of 843 nm were generated by a Ti:sapphire laser, and wavelength-tunable Stokes pulses at ~1030 nm were generated by an in-house built Yb fiber laser synchronized to the pump laser. The pump and Stokes pulses were combined with a dichroic mirror and sent to a laser scanning SRS microscope, where they were focused by a water immersion objective lens (60´, NA = 1.2) on a sample sandwiched by two cover slips. The optical power of the pump and Stokes pulses were estimated to be ~75 mW and ~55 mW, respectively, at the sample plane. The transmitted pump pulses were detected by a photodiode, and its output was sent to a lock-in amplifier to obtain the SRS signal. SRS images at wavenumbers of 2190 cm −1 , 2215 cm −1 and 2235 cm −1 were acquired successively at a frame rate of 30 frames/sec and averaged over 500 times. The total acquisition time was 50 s. The average image of 2190 and 2235 cm −1 were used as a background image. Because no Raman signal was detected at these regions, the background images were obtained as a difference of irradiated laser intensity reflecting the difference of metabolite concentration which absorbs and/or scatters the laser. The average images of 2190 and 2235 cm −1 were subtracted from the image of 2215 cm −1 as background.

Cell Viability Assay.
The cell viability of BY-2 cells against Raman probes was evaluated by Evans blue (EB) staining.
The method was almost the same as in the previous report. 4 For the assay, the Raman probe (final concentration: 100 µM) was incubated with BY-2 cells (OD 600 : 0.5) at 26°C int he dark. The incubated BY-2 cells were washed with Milli-Q water and mixed with 50 µg mL −1 EB for 10 min.
The stained BY-2 cells were washed with Milli-Q water and treated with methanol/SDS solution for 2 h. The lysates were centrifuged, and the supernatants were measured in OD 600 . As a positive control (100% dead cells), BY-2 cells were incubated for 1 h at 100°C to kill them completely, and then 50 µg mL -1 EB was added for 10 min and washed three times.

Hydrodynamic Size and ζ Potential Measurements
The Raman probes were characterized by a ζ potentiometer (Zetasizer Nano-ZS; Malvern Instrument, Ltd., Worcestershire, UK). Each Raman probe solution was prepared to a final concentration of 100 µM using Milli-Q. The ζ potential and ζ deviation of samples were measured three times by a ζ potentiometer, and the average data were obtained using Zetasizer software ver. 7.12 (Malvern Instruments, Ltd.). Dynamic light scattering (DLS) was performed to determine the hydrodynamic diameter with a ζ nanosizer (Zetasizer software ver. 7.12) using a 633 nm He-Ne laser at 25°C with a backscatter detection angle of 173°.