Investigating Dynamic Molecular Events in Melanoma Cell Nucleus During Photodynamic Therapy by SERS

Photodynamic therapy (PDT) involves the uptake of photosensitizers by cancer cells and the irradiation of a light with a specific wavelength to trigger a series of photochemical reactions based on the generation of reactive oxygen, leading to cancer cell death. PDT has been widely used in various fields of biomedicine. However, the molecular events of the cancer cell nucleus during the PDT process are still unclear. In this work, a nuclear-targeted gold nanorod Raman nanoprobe combined with surface-enhanced Raman scattering spectroscopy (SERS) was exploited to investigate the dynamic intranuclear molecular changes of B16 cells (a murine melanoma cell line) treated with a photosensitizer (Chlorin e6) and the specific light (650 nm). The SERS spectra of the cell nucleus during the PDT treatment were recorded in situ and the spectroscopic analysis of the dynamics of the nucleus uncovered two main events in the therapeutic process: the protein degradation and the DNA fragmentation. We expect that these findings are of vital significance in having a better understanding of the PDT mechanism acting on the cancer cell nucleus and can further help us to design and develop more effective therapeutic platforms and methods.

We employed JEM-2100F field emission transmission electron microscope (TEM, JEOL, Tokyo, Japan) to characterize the morphology of the prepared AuNRs. Ultraviolet-visible (UV-vis) spectroscopy (Ocean Optics, USB4000) and dynamic light scattering (DLS, Malvern Zetasizer Nano ZS) were used to characterize the fabrication of the nanoprobes. And we detected the specific targeting effects of AuNRs-PEG-RGD-NLS by self-built detection platform integrated with both fluorescence microscope (IX71, Olympus) and dark-field microscope (Olympus). SERS spectra were performed on a confocal Raman system (LabRAM Aramis, Horiba JobinYvon, USA) with a 785 nm laser as the excitation source. We used self-built 650nm LED lamp (a Petri dish was placed inside a box with the LED, the distance between LED and the Petri dish is about 25cm.The power at the position of Petri dish is about 18mW/cm -2 .) to produce a therapeutic effect. FV1000 confocal fluorescence microscope (Olympus) was employed to identify the location of Ce6 and prove that AuNRs had a weak PTT effect on the cells under such lamp irradiation. For flow cytometry analysis, the fluorescence intensity was recorded by the FACSCalibur (BD Biosciences, USA).

Synthesis of gold nanorods (AuNRs)
Here, AuNRs which were stabilized by cetyltrimethylammonium bromide (CTAB) were synthesized according to the modified seed-mediated growth method. 120 μL of 15mM HAuCl4 was added to the 5.0 mL of a 0.2 M CTAB solution, then 500 μL 0.01 M of ice-cold NaBH4 was added to the mixture under stirring for 2 min. The mixture was aging 2.5 hrs at the temperature of 30 °C to obtain the Au seeds. Then 500 μL of the seed solution was added to a mixture containing 44.38 mL of a 1.0 M CTAB aqueous solution, 2.5 mL of 15 mM HAuCl4, 2mL of 4.0 mM AgNO3, and 620 μL of 78.8 mM ascorbic acid. The mixture was kept undisturbed for 12 h at 28°C.

Fabrication of nucleus-targeting nanoprobes
AuNRs were modified by mPEG-SH, NLS, and RGD in one step. 150 μL of mPEG-SH solution (0.01 mM), 2.4 μL of RGD (5.0 mM) and 12 μL of NLS (5.0 mM) aqueous solutions were added to 10.0 mL of AuNR solution and then the mixture was allowed to react for 24 h at room temperature. Excess mPEG-SH, NLS and RGD were removed by centrifugation (5000 rpm, 10 min) to achieve the nuclear targeted probes (AuNRs-PEG-RGD-NLS) after 24h. They were redisposed in 1.0 mL deionized water before use. The molar ratio of RGD with AuNRs is 10 3 :1, whereas the molar ratio of NLS is 10 4 :1.

Location of three nanoprobes in cells
Supplementary Figure 2. Fluorescent, dark-field, and overlay images of B16 cells incubated with AuNRs-PEG, AuNRs-PEG-RGD and AuNRs-PEG-NLS for 12 h from top to bottom.

Location of nanoprobes in nucleus
To take the confocal fluorescent images of nanoprobes to ensure the ability of targeting, the FITC-labeled targeted nanoprobes were prepared. The FITC-labeled NLS were used to modify the surface of AuNRs instead of the pure NLS to obtain the FITC-labeled AuNRs-PEG-RGD-NLS. Besides this, the self-built detection platform integrated with both fluorescence microscope (IX71, Olympus) and dark-field microscope (Olympus) were also employed.

Quantification of nanoprobes in cells
In order to quantify the internalization of the nanoprobe into cells, we calculated the amount of AuNRs-PEG-RGD-NLS nanoprobe in cell culture medium before and after incubation with cells via UV-vis spectroscopy based on the plasmonic feature of AuNRs. We co-culture the AuNRs-PEG-RGD-NLS nanoprobe (1.0 nM, 0.6 mL) with B16 cells for 12 h. After that, the culture medium containing uninternalized nanoprobes was collected and centrifuged (6600 rpm, 6 min) to determine the amount of nanoprobes not uptake by the cells (0.3 nM, 1 mL). Therefore, the amount of the nanoprobes that were internalized is 2.102×10 -13 mol, which means there are 1.27×10 11 nanoprobes that entered into all cells (the density is about 4.5×10 6 ). Based on this, we evaluate that about 2820 nanoprobes were internalized or attached on per B16 cell.

Internalization of Ce6
We co-cultured 1.2 μM of Ce6 with B16 cells for different times respectively to determine the proper culture time with the help of fluorescence microscopy. We can easily identify the location of Ce6 because of its red fluorescent emission ( Figure S4). It can be observed that with the increase of the incubation time, the fluorescence intensity gradually increased and then decreased. Since the highest Supplementary Figure 6. Fluorescence images of B16 cells incubated with 1.2μM Ce6 for 2h,4h,6h,8h,10h,12h and 14h respectively. The scale bar is 10 μm. 4h  8h  6h   12h  10h 14h