SERS imaging elucidates the folate receptor-mediated photothermal/photodynamic synergistic anticancer nanodrugs-induced cell apoptosis CURRENT STATUS: UNDER REVIEW

Background: Chemotherapy and radiotherapy are common methods of cancer treatment, but they are accompanied by serious side effects. Actually, many cancer cells have overexpression of folic acid (FA) receptor and by FA receptor-mediated endocytosis, anticancer drugs can be easily internalized into cancer cell, this will greatly improve the curative effect and decrease side effects. Along with the development of nanotechnology, phototherapy, owning advantages in tissue selectivity, process controlling, low toxicity and reproducible treatment, has become very promising, especially for photothermal therapy (PTT) and photodynamic therapy (PDT). Since both PTT and PDT involve the utilization of light energy, so they synergistic treatment should be a good solution by ingenious design. In this paper, based on surface enhanced Raman spectroscopy (SERS) imaging, we hope to construct a FA receptor-mediated PTT/PDT synergistic anticancer nanodrug (nanoprobe), and achieve the intracellular distribution information of the nanodrugs during cell apoptosis, and then elucidate PTT/PDT-induced cell apoptosis and synergistic efficiency. Results: FA receptor-mediated PTT/PDT synergistic anticancer nanodrugs with tracing function are prepared by the chemical synthesis and modification of gold nanorods (GNR), involving protoporphyrin IX (PpIX), 4-mecaptobenzoic acid (MBA), and FA. Based on SERS imaging, it is found that the FA receptor-mediated endocytosis can greatly facilitate the nanodrugs internalization, in which both the number and intracellular dispersion of the PpIX-GNR-MBA-FA nanodrugs are improved relative to the GNR-MBA or PpIX-GNR-MBA compositions, and then enhance the PTT/PDT-induced cell apoptosis. Conclusion: SERS imaging is very suitable for the phototherapy tracing due to its high sensitivity and stability. The FA receptor-mediated way can significantly facilitate nanodrugs internalization, PTT/PDT-induced cell apoptosis and synergistic efficiency.

Importantly, this FA receptor-mediated SERS imaging based PTT/PDT synergistic treatment will provide a novel strategy for the design and application of anticancer phototherapy nanodrugs.

Background
To date, chemotherapy and radiotherapy are common methods of cancer treatment [1], but they are accompanied by serious side effects [2,3]. In fact, folic acid (FA) receptor, having overexpression on the surface of various cancer cells including the brain, breast, lungs and so on, has been widely employed as tumor targeted ligand in selective drug delivery [4][5][6]. By FA receptor-mediated endocytosis, many anti-cancer drugs can be internalized [7,8], this will greatly improve the curative effect and decrease side effects.
Along with rapid development of nanotechnology, phototherapy, owning obvious advantages in tissue selectivity, process controlling, low toxicity and reproducible treatment, has become very promising, especially for the photothermal therapy (PTT) and the photodynamic therapy (PDT) [9,10]. In PTT, by using special materials with high photothermal conversion under the excitation of near infrared light (NIR), the local temperature of cell or tissue can rapidly increase and ablate targeting tumours [11][12][13]. In PDT, under the light irradiation of specific wavelength, the photosensitizer can transfer the absorbed photon energy to surrounding oxygen molecules, and lead to the generation of reactive oxygen species (ROS), SO ( 1 O 2 ) and other strong oxide species, and then induce cell apoptosis [14][15][16].
For the sake of improving treatment efficiency, the synergistic therapy of multi-methods has emerged as an efficient solution. Both PTT and PDT involve the utilization of light energy, and based on the local surface plasmon resonance (LSPR) of noble metal nanoparticles, the efficiency of PDT can be enhanced along with the photothermal effect, 4 so PTT and PDT (PTT/PDT) synergistic treatment should achieve significant efficiency by ingenious design. Current studies have reported good efficiency of PTT/PDT synergistic therapy [17][18][19], but how does FA receptor-mediated endocytosis affect the amount and distribution of intracellular PTT/PDT nanodrugs are still lack.
Till now, a series of photosensitizer agents, including phthalocyanine green (ICG) [35], dihydroporphyrin (Ce6) [36], hematoporphyrin (HP) [37], pheophytin [38], protoporphyrin IX (PpIX) [39][40][41], have been developed. However, the traditional PDT treatment suffers from several drawbacks such as high oxygen dependent, poor water solubility and poor targeting of photosensitizer. Moreover, the generated ROS has very short half-life and limited diffusion distance, so the efficiency of PDT is greatly restricted [42,43]. Due to the advantages in easy modification, strong photodynamic effect, cellular respiration promotion, PpIX is becoming a better candidate of photosensitizer [44,45]. Furthermore, it is found that PpIX is temporarily quenched after conjugation with GNRs, and released when the probe is internalized into cancer cell as a result of avoiding the inconvenience in the dark treatment [18].
For nanodrugs design and application, by the internalized number and distribution information, both fixed-point irradiation and selective treatment can be easily achieved, thereby improving efficiency and saving energy. So far, many methods, including fluorescence imaging [31,32], photoacoustic imaging [46] and magnetic resonance imaging (MRI) [47], have been employed to implement targeted and precise cancer treatment. However, these methods have disadvantages in low stability or low sensitivity.
In view of the above facts, we hope to construct a FA receptor-mediated PTT/PDT synergistic anti-cancer nanodrug with the tracing function based on SERS imaging. Figure 1 shows the synthesis illustration, and the final structure of the nanodrug is named as PpIX-GNR-MBA-FA (Fig. 1C), in which the GNR realize photothermal conversion, but also enhance the efficiency of photodynamic. In order to achieve the conjugation between GNR and PpIX, the thiolation of PpIX with cysteamine hydrochloride is used (Fig. 1A). Raman reports molecules 4-Mercaptobenzoic Acid (MBA) is selected as the bridge between GNR and FA, so not only the FA receptor mediated path is achieved, but also the nanodrug is equipped with SERS signal (Fig. 1B). After that, by using the prepared PpIX-GNR-MBA-FA SERS nanoprobe to image receptor-mediated internalization, we can achieve intracellular distribution information of PTT/PDT nanodrugs during cell apoptosis, and elucidate the FA receptor mediated PTT/PDT synergistic anti-cancer nanodrugs-induced cell apoptosis.

Synthesis and characterization of PTT/PDT nanodrugs
In order to achieve the FA receptor mediated PpIX-GNR-MBA-FA nanoprobe, we first need to prepare PpIX-SH and FA-MBA compositions, respectively. Figure 2A and 2B show the corresponding FTIR spectra. In Fig. 2A, it is observed that two new peaks at 1564 and 1622 cm − 1 , attributed to the amide bond, appear in the PpIX-SH compared with the PpIX, and the characteristic peak of the PpIX-SH at 1988 cm − 1 , corresponding to the stretching 6 vibration of -SH in cysteamine hydrochloride [51], is also observed. Clearly, this result demonstrates that a thiol group has been introduced into PpIX by the conjugation between the carboxyl group of PpIX and the amino group of cysteamine hydrochloride. In addition, a significant blue shift appears in the fluorescence spectrum of PpIX-SH compared with PpIX ( Fig. S1), indicating the strong electronegativity of sulfhydryl group in the cysteine [18].  Also, from the TEM imaging results of GNR and PpIX-GNR-MBA-FA ( Fig. 2E and 2F), we can see that the rod-like GNRs with mean length of 39 ± 2 nm and width of 10 ± 2 nm are observed, in which a gray shell of 2 ~ 6 nm around the GNR surface appears, further indicating that the conjugation among MBA, FA and PpIX is successful. Considering dispersion is an important parameter for nanodrugs application, we also measure the corresponding hydrodynamic size distribution by the dynamic light scattering (DLS) method, as shown in Fig. S4. Although some aggregation occurs, the properties of PTT and PDT properties will not be affected [51].

Cell internalization assays
Photothermal nanodrugs first need to be internalized into cancer cell, and the internalized number is closely related to its efficacy. Using inductively coupled plasma-mass spectrometry (ICP-MS) method [53], Fig. 3 shows the average number of the internalized 8 GNR-MBA, PpIX-GNR-MBA, and PpIX-GNR-MBA-FA by Hela cells were 7.57 × 10 3 , 5.70 × 10 4 , and 3.37 × 10 5 , respectively. This result demonstrates that the FA receptor-mediated mode can greatly facilitate the GNRs internalization for 50-fold increasing.
Also, Raman characterization of the internalized PpIX-GNR-MBA-FA is performed. Figure 4B presents the Raman spectrum of HeLa cells co-cultured with the prepared PpIX-GNR-MBA-

Cytotoxicity and phototoxicity assays
In order to present the efficacy of the PpIX-GNR-MBA-FA nanoprobes, we first employ CCK-8 to assess the bio-compatibility. As shown in Fig. 6A, in the case of no laser irradiation, even if the concentration of PpIX-GNR-MBA-FA nanoprobes reaches 10 µg/mL, the cell viability still maintains more than 85% after co-incubation for 24 h, indicating the low chemical-toxicity of the PpIX-GNR-MBA-FA nanoprobes.
Moreover, to assess the anti-cancer activity of the PpIX-GNR-MBA-FA nanoprobes, the photo-toxicity is tested by the survival rate. Since the photosensitizer PpIX works at wavelength 633 nm and the GNR perform photothermal at wavelength 785 nm, HeLa cells are irradiated with the 633 nm laser (6.54 mW/cm 2 for 20 min) for PDT, the 785 nm laser (177 mW/cm 2 for 10 min) for PTT and the PTT/PDT synergetic treatment with the 785 nm laser then the 633 nm laser. As shown in Fig. 6B, it is found that the viabilities of PTT, PDT and PTT/PDT are decreased with the GNRs concentration increasing, and the high phototoxicity of the PpIX-GNR-MBA-FA nanoprobes is obtained at 10ug/mL. Compared with the cell viability of 31% for PTT and 25% for PDT, the cell viability is reduced to 8% for PTT/PDT treatment. Clearly, this result illustrated the excellent efficiency of PTT/PDT synergistic treatment relative to the individual PTT or PDT. In contrast, the cell viability maintains more than 96% in control group (0ug/mL), indicating that the side effects of laser irradiation can be neglected. Figure 6C shows the corresponding cell morphological changes during laser irradiation by an optical microscope. For PTT treatment, HeLa cells are co-cultured with the PpIX-GNR-MBA-FA nanoprobes, after 785 nm laser irradiation (10 mW for 10 s), the cell membrane becomes blurry, and some small bubbles appear due to the local thermal effect of GNRs leads to the local temperature increasing around the probe. In PDT treatment, it is observed that after laser irradiation, the cell membrane became blurry, some small bubbles appear, and then gradually become large. The possible reason is that the PpIX irradiated by 633 nm laser would lead to the generation of ROS, SO and other strong oxide species, and then rapid oxidation of intracellular macro-molecules, as a result of O 2 production and bubble formation. Actually, the speed of bubble formation is slow due to the PDT process is complicated. Compared with the result of PDT, the speed of bubbles formation in PTT is improved due to photo-effect and heat-effect will cause the rapid increasing of local temperature while the bubble size in PDT is larger than PTT, indicating that the efficiency of PDT is better than PTT. Clearly, this result is consistent with the

Synthesis of PpIX-SH and MBA-FA
According to the previous reports [51], the thiolated photosensitizer was prepared as followings: PpIX (6mg) was added into 2 mL DMSO. After completely dissolved, 4 mg NHS was added to the solution and stirred in N 2 for 2 h at room temperature. The mixture of 7 mg EDC, 3.6 mg cysteamine hydrochloride, and 44.6 µL TEA were stirred under 400rpm in darkness for 24 h. The result product was removed into a dialysis membrane with a molecule weight of 500 and purified by dialysis against deionized water for 3 days, and then freeze-dried to reddish brown powder.
In order to achieve the conjugation of the amino acid (-NH2) of FA and the activated carboxylic acid of MBA, the synthesis of the thiolated FA was also referred to the above 13 method [51]. Briefly, 1mL of MBA (40 μM) activated by EDC/NHS was reacted with 1mL of FA (50 μM) at room temperature for 24 h, following the unconjugated MBA and FA were removed through dialysis.

Synthesis of GNRs
GNRs were synthesized according to the seed-mediated method [18]. Briefly, the seed solution was prepared as following: First, 7.

Characterization of PpIX-GNR-MBA-FA nanodrugs
First, the infrared spectrum of the prepared PpIX-GNR-MBA-FA/SERS nanoprobe is measured by a FTIR spectrometer, and the corresponding ultraviolet-visible absorption spectra is achieved by a ultraviolet-visible spectrophotometer; the fluorescence emission spectrum is performed with a photoluminescence spectrophotometer; the TEM image was implemented with the transmission electron microscope; the cell viability is tested by CCK-8 assay. Specially, to assess quantitative characterization, we measure the intracellular transport and uptake amount of the prepared PpIX-GNR-MBA-FA/SERS nanoprobe by using confocal micro-Raman spectral imaging and inductively coupled plasma-mass spectrometry (ICP-MS) [53], respectively.

Cell culture
HeLa cells, an immortalized cell line with excessive FR expression [18] were cultured in the culture medium containing 90% DMEM, 10% FBS, as well as 1% double resistance (penicillin streptomycin) in 37 ℃ containing 5% CO 2 .

Characterization of nanoprobe internalization and localization
HeLa cells were seeded into 6-well plate of 1.5 mL DMEM (1×10 5 cell/well). After cells attachment, the culture medium was changed to 1. ICP emission spectrometer was also employed for quantitative characterization of the internalized probes, in which HeLa cells (1×10 5 cells/well) were cultured in the 6-well plate of 1.5 mL DMEM for 24 h. After cells attachment, the culture medium was changed as 1.5 mL medium containing GNR-MBA, GNR-MBA-FA, PpIX-GNR-MBA-FA, and co-cultured for 4 h. Subsequently, HeLa cells were digested by trypsin (0.25%) and re-dispersed in PBS after centrifugation; and then the cell suspension was transferred into a 15 mL shrinkable flask, 2 mL HNO 3 and 0.5 mL HCl were added drop by drop to dispel the cells.
Following, the boiling water bath was used to remove the acid until the solution of cell suspension was clarified. Finally, the solution was constant to 10mL and analyzed by ICP emission spectrometer to obtain the content of gold after dissolution, which can be used to calculate the number of GNR. HeLa cells were respectively treated with PpIX-GNR-MBA-FA (0, 2, 5, 10 μg/mL), and incubated for 24 h. And then the cells were irradiated with a 785 nm laser (177 mW/cm 2 , 20 min) for PTT stimulus, a 633 nm laser (6.54 mW/cm 2 , 15 min) for PDT stimulus, and the combination therapy (PTT/PDT) of a 785 nm laser then a 633 nm laser. Finally, the viability was evaluated with CCK-8 assay as described above.

Cytotoxicity and therapeutic efficacy assays
HeLa cells (1×10 5 cells/well) were seeded on 6-well plate with 1.5 mL DMEM and incubated for 24 h. After cells attachment, the culture medium was changed to 1 mL fresh culture medium containing PpIX-GNR-MBA-FA (10 μg/mL) and cultured for 24 h; the in-internalized probes were removed by washing with PBS, and then 1 mL fresh culture solution was added. Finally, HeLa cells were respectively irradiated by a laser of 633 nm (0.37 mW, 10 s) for PDT or 785nm (10 mW, 10 s) for PTT or combined PTT/PDT with the spot diameter of 1.5 μm. The cell morphological changes during laser irradiation were recorded with an optical microscope.

Ethics approval and consent to participate
Not applicable.