Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated

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Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated Photothermal Therapy Limin Pan, Jianan Liu, and Jianlin Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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ACS Applied Materials & Interfaces

Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated Photothermal Therapy Limin Pan, Jianan Liu, and Jianlin Shi*

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Ding-Xi Road, Shanghai 200050, China

KEYWORDS Nuclear targeting, gold nanorods, photothermal therapy, ultralow power density, NIR irradiation, biosafety

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ABSTRACT Photo-related nanomedicine is of particular interest as an emerging paradigm toward precise cancer therapy, as demonstrated by recent developments of photothermal therapy (PTT), an emerging technique employing light converting agents to burn cancerous cells by over-dosed optical energy-converted heat. However, most of the laser irradiations needed for effective PTT significantly exceed the maximal permissible power density in human skin, which is likely to damage surrounding normal tissues. Herein, we report a strategy of intranuclear PTT of cancer enabled by nuclear-targeted delivery of gold nanorods of ~ 10.5 × 40.5 nm in size via conjugation with nuclear location signal peptides (GNRs-NLS) under an extremely low near infrared irradiation of 0.2 W/cm2, much below the maximal permissible exposure of skin. Interestingly, we found that a mild but nuclear-focused temperature increase generated by GNRs-NLS is sufficient to cause damages to intranuclear DNA and the inhibition of DNA repair process, which, interestingly, led to the cancer cell apoptosis, rather than conventional cell necrosis by thermal ablation during PTT. Correspondingly, tumors treated with GNRs-NLS exhibited gradual but significant regressions rather than traditional harsh burning-up of tumors, in comparison with negligible antitumor effect by GNRs without nuclear-targeting under the same ultralow NIR irradiation. This report demonstrates the successful intranuclear efficient photothermal therapy of cancer via cell apoptosis by photoadsorbing agents, e.g., GNRs-NLS in the present case, with largely mitigated sideeffect on normal tissues and therefore substantially improved biosafety. 2


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1. INTRODUCTION Currently, cancer is the biggest threaten to human and may lead to 13.2 million death worldwide by 2030 forecasted by the United Nations.1 At present, the clinically used therapy strategies are mostly limited to surgery, chemotherapy and radiotherapy. However, such approaches will inevitably damage normal tissues, destroy the immune system, and increase incidences of tumor metastasis.2-3 Photothermal therapy (PTT) has attracted great attention in tumour therapy due to its ability of noninvasiveness and high selectivity.4-5 Photoabsorbing agents were used in PTT, converting light energy into heat, and resulting in the ablation of tumours.6-7 Therefore, the PTT effects can only take place in tumour regions under the existence of both lightabsorbing agents and local light irradiation, without killing normal cells.8 In the past decade, numbers of research groups have been devoted to explore near-infrared (NIR) light-absorbing agents because their excitation light is within the optical transparent window in tissues (700-900 nm). For example, numbers of nanostructures, such as carbon nanomaterials (e.g., graphene and carbon nanotubes ),9-12 various gold nanomaterials (e.g., gold nanocages, nanostars, and nanorods),6, nanoparticles,17-18

palladium

nanosheets,19

and

copper-based

13-16

organic

semiconductor

nanoparticles,20-21 have been developed for effective ablation of tumours. Among these nanomaterials that are currently being developed, gold nanorods (GNRs) have been widely used in PTT applications thanks to their advantages of good biocompatibility and favorable optical properties.16, 22-28 In addition, GNRs can also 3



be served as effective contrast agents for tumour diagnosis owing to their tunable and strong absorption in the NIR transparent window.24 Hence, GNRs can be anticipated to be promising therapeutic agents excited by NIR light. However, most of the currently used photothermal agents require laser irradiation with a relatively high power density, which is beyond the maximally permissible exposure (MPE) (e.g., 0.4 W/cm2 for 850 nm) in human skins set by American National Standards Institute.29 High intensity laser would damage adjacent normal tissues due to nonspecific absorption. Moreover, to reach the tumor sites for necessary intratumoral therapeutic dose, enhanced energy input is required, which could cause burning, blistering, and pain on skin and normal tissues in the way of the irradiation. Insufficient intracellular agent delivery, random intra-cellular distribution and low light-heat converting efficacy may be the primary barriers. Consequently, in order to maximize the intratumoral PTT efficacy and compensate for attenuated adverse effects by using as low as possible laser irradiation energy, the exploration of photothermal agents like GNRs for more efficient converting of NIR light energy to heat, especially with enhanced cellular uptake and localized accumulation in subcellular organelles hypersensitive to heat are of great significance.29 With recent developments in nanotechnology, to achieve cell- and site-specific delivery efficiently for the desired PTT treatment, the photothermal agents must be functionalized with cargoes such as peptides, antibodies, and small molecule ligands for targeting.

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It has also been reported that the nuclear matrix is one of the most thermolabile structures in the cells.30 Thermal changes in the nuclear matrix would result in aggregation of proteins to the matrix, disrupting its function by inhibiting DNA supercoiling transformation and changes of specific DNA regions within the nuclear matrix. Therefore, the development of novel nuclear targeting photothermal agents with efficient photothermal conversion property, adequate biocompatibility, easy fabrication and especially intranuclear accumulation is highly desired for subcellular targeted PTT with substantially lowered NIR exposure. Nuclear targeting of nanoparticles is particularly challenging, in which there are multiple barriers existing from the cytoplasmic membrane to the nuclear envelope.3132

Furthermore, the exchange of cytoplasmic and nucleoplasmic has been strictly

restricted by nuclear pore complexes (NPCs) which sized with about 50 nm and serve as gatekeepers on nuclear envelope.33 It has been demonstrated previously that ultrasmall nanoparticles (< 10 nm) can directly cross NPCs and then enter the nucleus.34 To achieve sufficient nuclear targeting of nanoparticles beyond this size, assistance from nuclear localization signals (NLS) by conjugating them onto the nanoparticles, is necessary, which could be those from the simian virus SV 40 large T antigen,35 adenovirus,36 and HIV TAT peptide.37-39 To achieve successful and selective PTT under safe NIR irradiation, it is highly needed to delivery GNRs efficiently to designated tumor cells, and sequentially to thermolabile subcellular organelles. Here, as shown in Figure 1, we present TAT5



peptide functionalized GNRs (denoted as: GNRs-NLS) for both enhanced intracellular uptake and intranuclear delivery, aiming at efficient in vivo photothermal conversion under a NIR laser exposure of as low as 0.2 W/cm2, half of that the MPE of skin. The in vitro mechanisms of intranuclear PTT in the presence of nuclear-targeted GNRs-NLS are explored, followed by the evaluation of therapeutic efficacy of this intranuclear mild hyperthermia in an animal model of human cervical cancer. As far as we know, this is the first study on the intranuclear PTT of cancer under such a low NIR intensity by the nuclear-targeted delivery of GNRs.

2. RESULTS AND DISCUSSION 2.1 Synthesis and Characterization of GNRs-NLS PTT System. In a typical synthesis, GNRs can be obtained via a seed-assisted and surfactantmediated method, and the surfaces were then functionalized with TAT-type NLSs as illustrated in Figure 1a.40 Transmission electron microscopic (TEM) images show that the mean length and width of GNRs are approximate 40.5 ± 2.1 and 10.5 ± 1.7 nm (Figure 2a). The aspect ratio of as-prepared GNRs is ≈ 3.9, and this is of great importance for the efficient NIR-triggered photothermolsis. To fabricate stable and biocompatible GNRs-NLS that will not aggregate with each other in a physiological environment while are equipped with chemical functionality needed, both cysteinecontaining TAT peptide and thiolated poly(ethylene glycol) (SH-PEG) were used for surface modification. TEM and scanning transmission electron microscopy (STEM) images of GNRs-NLS (Figure 2b, c and S1) show that the PEGylation and NLS 6


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conjugation have negligible effects on the original size and morphology of the nanorods, favoring compatible and long blood circulation, accumulation in tumors site via leaky vasculatures and more importantly, nuclear translocation through nuclear pore complexes.41 Moreover, the zeta potential changed from 22.8 to −28.3 (GNRsPEG) and 3.6 mV (GNRs-NLS) (Figure S2). In addition, the transverse and longitudinal typical plasmon resonances peak of GNRs are at 520 nm and 803 nm respectively (Figure 2d). More attractively, the longitudinal band is red-shifted to 815 nm due to the surface PEG and TAT modifications, which provides a great potential for light-mediated therapy. These results also indicate that TAT has been conjugated onto the surface of GNRs. The amounts of TAT peptides on GNRs were also tested by Bradford assay. By the Au-S bonding interaction, 38.4 % of the added TAT ligands were conjugated onto the surfaces of GNRs, which could fulfill the demands for the nuclear translocation and in vivo anti-tumor therapy. Because as-synthesized GNRs coated only with surfactants will aggregate rapidly in phosphate-buffered saline (PBS) and cell culture media, GNRs modification with PEG coating was made and used as non-targeting PTT agents, which was denoted as GNRs for convenience in the following text. In order to examine the photothermolsis effect of GNRs under 808 nm laser irradiation, an infrared thermal imaging camera was applied to monitor the temperature changes of GNRs aqueous solution. As seen in Figure 2e and f, PBS does not show a temperature-rising response to the irradiation even at 2.0 W/cm2, while the temperatures of GNRs and GNRs-NLS aqueous 7



solutions increase in the time course of irradiation, reaching 50.3 and 50.8 °C from 25 o

C in 5 min at the irradiation density of 2.0 W/cm2, respectively, suggesting that the

surface conjugation of PEG and peptides has no significant influence on the photothermal effects. Irradiation to GNRs and GNRs-NLS at 0.2 W/cm2 leads to much less temperature rising by 11.2 and 10.6 °C in 5 min, respectively, implying that temperature increase can be easily tuned by adjusting NIR irradiation density. These results demonstrate that temperature increasement caused by GNRs and GNRs-NLS is sufficient to fulfill the requirement for tumor thermal therapy even at an extremely low light dose of 0.2 W/cm2 for 5 min.

2.2 Nuclear-Targeting in Cancer Cells and In Vitro Cytotoxicity. To have an insight into the nuclear-targeting property of the nanocomposite, we then studied the intracellular distribution of GNRs and GNRs-NLS in cervical cancer cells (HeLa) by using two-photo photoluminescence imaging and Bio-transmission electron microscopy (Bio-TEM). Figure 3a clearly shows that GNRs-NLS distribute in both the nucleoplasm and the perinuclear region while GNRs in the cytoplasm only. The decoration of TAT peptides dose facilitate the intranuclear localization of GNR-NLS in HeLa cells. The Bio-TEM images further confirm the presence of GNRs-NLS in the nuclei (Figure 3b). It can be observed that large numbers of GNRsNLS are inside the nucleus while GNRs without TAT peptide modification on the rods’ surface are outside the nucleus. The nuclear accumulation of GNRs-NLS were further observed by energy-dispersive X-ray (EDX) analysis (Figure S3), which 8


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showing the existence of gold in the nuclei. Moreover, TAT peptides, used as cell penetration peptide (CPPs), also enhances overall intracellular uptake of GNRs-NLS, which could be clearly detected by two-photo photoluminescence microscopy with high spatial resolution. The cellular and nuclear uptakes of GNRs and GNRs-NLS were measured by ICP-AES as shown in Figure 3c. It is clear that TAT conjugation not only enhances the cellular uptakes of GNRs-NLS but also contributes clearly to the intra-nuclear localization of GNRs-NLS. These results agree well with previously published research about the nuclear penetration of nano-sized beads by the facilitation of TAT peptide.38 Encouraged by the high photothermal efficiency of GNRs-NLS, in vitro PTT effects of GNRs-NLS were then studied with a 808 nm laser. After 12 h incubation with GNRs-NLS, HeLa cells were irradiated at two different power densities of 2 and 0.2 W/cm2 for 5 min. For evaluating cell viability visually, the treated cells were incubated with calcien AM (green) and propidium iodide (PI, red) for live and dead cells staining, respectively (Figure 4a). First of all, irradiation-only did not damage the cells even at 2 W/cm2, indicating the bio-safety of 808 nm laser. At 2 W/cm2, cell ablation (red areas) can be distinctively observed after incubated with GNRs or GNRs-NLS followed by immediate irradiation, suggesting that both GNRs and GNRs-NLS are efficient photothermal agents which can lead to photothermal killing of cancer cells instantly when the irradiation power density is high enough to generate severe temperature rising. 9



Furthermore, cells incubated with GNRs were still alive right after irradiation at 0.2 W/cm2, demonstrating that the mild temperature increase by GNRs at such a low light dosage is insufficient to cause instant ablation of cancer cells. In contrast, after irradiation at 0.2 W/cm2, a large portion of cells (empty and red area) incubated with GNRs-NLS were damaged and detached from the dish. These results indicate that PTT based on nuclear-targeted GNRs-NLS could induce gradual cell damage and death at the ultralow irradiation by intranuclear local mild hyperthermia. These results further confirm that the TAT peptide could enhance the nuclear uptake of GNRsNLS, leading to efficient photothermal cytotoxicity in vitro at a NIR irradiance intensity which is safe to skin. This is the first report of subcellular PTT with GNRs at extreme low power density. In addition, the photothermal cytotoxic effects of GNRs and GNRs-NLS with various laser irradiations on HeLa cells was further quantified using a standard thiazolyl blue tetrazolium bromide (MTT) assay (Figure 4b). Cells treated only with laser exposure without GNRs incubation kept alive even at the high power density of 2 W/cm2 (Figure S4), as also indicated in Figure 4a. Although both GNRs and GNRsNLS can act as efficient photothermal agents and cause severe phototoxicity (cell death: 80.8% for GNRs and 89.6% for GNRs-NLS) under irradiation with high light energy input (2 W/cm2), cell viability after treating with GNRs under the low intensity irradiation (0.2 W/cm2) recovered sharply to 96.5%, due to the limited temperature increase which is not enough to kill cells directly by ablation/necrosis. Very 10


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attractively, however, significant enhancement in cell-killing effect (cell death: 81.4%) could be realized by intranuclear delivery of GNRs-NLS under irradiation at 0.2 W/cm2, demonstrating a totally different mechanism of cell-killing from conventional PTT-induced cell thermal ablation/necrosis, which has been achieved by moderate intratumoral heating under the ultra-low optical energy input and the presence of GNRs-NLS within the nuclei. To figure out the underlying mechanism of intranuclear phototherapy, we further investigated possible pathways of cell membrane breaking caused by GNRs-NLS via testing lactate dehydrogenase (LDH) releasing levels,42 which is widely used as a biomarker for monitoring the integrity of cell membrane (Figure 4c). The results show that no significant LDH releasing for GNRs, GNRs-NLS or NIR alone can be observed under the absence of light input (Figure S5). Furthermore, the amounts of released LDH from cells under the irradiation at 2 W/cm2 are much higher than that from untreated ones, indicating severe cell membrane damages caused by efficient hyperthermia effect of GNRs and GNRs-NLS under high intensity irradiation. As expected, by substantially lowering the NIR intensity to 0.2 W/cm2, levels of released LDH remain almost equivalently low compared with untreated control, suggesting no significant cell membrane damage due to the limited and gradual temperature increase induced by either GNRs or GNRs-NLS under the low intensity illumination, though in this case substantial cell death has been induced by GNRs-NLS. These results

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demonstrate that nuclear mild hyperthermia by GNRs-NLS will result in intrinsic cell death while maintain cell membrane integrity. To determine the pathways of cell death caused by nuclear targeted PTT, flow cytometry analysis were operated to confirm whether necrosis or apoptosis that has taken place in the photothemal process. Necrosis will lead to the uptake of nonpermeable dyes (e.g., PI) due to the rupture of cell membrane. In comparison, the membranes of early apoptotic cells can maintain the integrity, thus keeping nonpermeable dyes out of cells. Moreover, a cellular protein annexin V-FITC can be used to stain apoptotic cells because of their specific incorporation with phosphatidylserine, an amino acid that is peculiarly externalized in the membrane of apoptotic cells.42 Consequently, we can easily distinguish healthy cells, apoptotic cells and necrotic cells from each other using flow cytometry analysis of annexin VFITC/PI co-stained cells. Firstly, Figure 4d showed that all cells were alive in the control and only laser irradiation groups without exposure to nanorods regardless of the laser power density, in consistence with MTT assay. After exposure at 2 W/cm2, the percentages of necrosis cells significantly increased to 97.4 % and 94.8 % but with almost 0 % of apoptosis after treated with GNRs and GNRs-NLS, respectively. In contrast, for the GNRs group at the lowered NIR intensity of 0.2 W/cm2, cells underwent neither apoptosis nor necrosis. However, remarkably, the percentage of early and late apoptotic cells was approximately 68.6 % but with 0 % of necrosis after treated with GNRs-NLS under irradiation at 0.2 W/cm2. This result was in accordance 12


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with the data of MTT and LDH release measurements, suggesting the apoptotic pathway of cell death in the case of GNRs-NLS + 0.2 W/cm2 irradiation. Moreover, to clarify the molecular mechanism underlying the enhanced intranuclear PTT effect of GNRs-NLS, ELISA analyses of replication protein A 70 (RPA70) were performed. It has been reported that the RPA70, serving as a primary protein for single-stranded DNA binding, is imperative for the processes associated with DNA replication, repair, recombination, and damage response signaling. The expression level of RPA70 (Figure 4e and Figure S6) indicates that GNRs-NLS can be intra-nuclear-uptaken by HeLa cells to silence the expression of DNA repairrelated protein, which contributes to the significant cell apoptosis of GNRs-NLS by mild nuclear hyperthermia. In addition, it has been reported that heat can impede the repair and recombination processes of DNA-fragmentation rather than induce critical DNA damage indenpendently.43 Thus, the significant suppression of RPA70 in GNRs-NLS-treated cancer cells is closely associated with the inhibited DNA repair and irreversible cell apoptosis due to the intranuclear mild hyperthermia of GNRsNLS under the ultralow dose of NIR irradiation. These data demonstrate that under the ultralow light doses, the non-specifically localized GNRs in whole cancer cells is unlikely to cause irreversible cancer cell damage, but the nuclear-targeted GNRs-NLS that directly leads to mild hyperthermia in the hypersensitive nucleus is capable of inducing effective photo-cytotoxicity for cell apoptosis.

2.3 GNRs-NLS for Nuclear Targeted In Vivo Photothermal Therapy 13



Motivated by the efficient cancer cell DNA disruption by GNRs-NLS, we then analyzed the therapeutic ability of GNRs-NLS in a mouse model bearing HeLa tumor. Firstly, the bio-distributions of nanorods in the tumor and major organs ( heart, liver, spleen, lung, and kidney) were quantified in 3 h, 12 h, and 24 h post intravenous injections of GNRs and GNRs-NLS (Figure S7). Majorities of the injected GNRs and GNRs-NLS were accumulated in reticuloendothelial systems (liver and spleen). The gold content in the tumor reached the maximum value of 7.68 % ID/g in 12 h postinjection of GNRs-NLS, which is much higher than that of GNRs (4.67 % ID/g). Furthermore, the nuclear uptake of GNRs-NLS (Figure S7c) in 24 h post-injection was as high as 2.8 µg/g, while that of GNRs was negligible. Being consistent with in vitro results, both the increased tumor accumulation and substantially enhanced nuclear uptake of GNRs-NLS are beneficial in improving the PTT efficacy. To monitor the PTT effects of GNRs-NLS in vivo, the temperature increase at tumor site under 808 nm irradiation with varied laser power densities was also recorded by IR thermal imaging camera (Figure 5a). After laser irradiation of 2 W/cm2, the temperatures of GNRs-NLS- and GNRs-injected tumors increased rapidly to 55 °C in 5 min of irradiation, which is sufficient to burn the malignant tumors. However, the temperature of tumor increased moderately to 35-38 °C after irradiation with power density of 0.2 W/cm2. In comparison, no significant heating effect was observed on mice injected with PBS under the same irradiation condition. These observations

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certified the efficient heat generation of gold nanorods as light-absorbing agents and the remarkable selectivity of PTT effects of GNRs and GNRs-NLS. Changes in tumor volume are direct indexes to evaluate therapeutic effects. When the sizes of tumors reached to about 50 mm3, 40 mice bearing HeLa tumors were classified into five groups at random. GNRs, GNRs-NLS, or saline were administered by intravenous injections. Tumor growth rates were monitored every two days. As shown in Figure 5b, when irradiated at 2 W/cm2, GNRs exhibit a similar tumor ablation as GNRs-NLS, which indicates that with abundant light absorption, the nonspecific intratumoral localization of GNRs could burn up the tumors efficiently, leaving ugly black scars. As the energy density of the light dose was decreased to 0.2 W/cm2, the tumor growth inhibitions by GNRs and GNRs-NLS become less efficient, which is understandable that less light energy would generate less heat for killing cancer cells at the same concentration of nanorods. However, the antitumor effect of GNRs was remarkably lower than that of GNRs-NLS under the ultralow light energy. The tumors treated by GNRs-NLS exhibit a remarkable regression after nucleartargeted GNRs-NLS delivery by mild PTT (Figure 5c and d), while the tumors treated GNRs demonstrate negligible antitumor effect in comparison with the control PBS group. These studies are in consistence with in vitro results. We attribute the enhanced phototherapy efficiency by nuclear-targeted GNRs-NLS to the gradual but irreversible cell apoptosis due to the intra-nuclear mild hyperthermia. We propose that, once entered the nucleus, the GNRs-NLS will generate moderate heat in-situ 15



within the nuclei and cause gradual damages to the nuclear DNA helix structure under the low intensity irradiation, resulting in the irreversible DNA destruction and malignant can cell apoptosis immediately due to the inhibition of DNA repair process. Furthermore, tumor tissue samples were examined by histological analysis 7 days after the treatments to evaluate the therapeutic effect (Figure 5e and f). In hematoxylin and eosin stained paraffin tumor tissue sections, apparent extensive necrosis of tumor cells can be found on mouse treated with high intensity PTT (2 W/cm2), under the mediation by GNRs and GNRs-NLS, while no significant malignant necrosis areas can be observed in the mouse subjected to GNRs by low intensity irradiation (0.2 W/cm2), as well as in the control group. On the contrary, as expected, the tumors treated with GNRs-NLS show damaged areas in tumor regions even at 0.2 W/cm2, indicating the high efficiency of GNRs-NLS for intra-nuclear mild hyperthermia with ultralow irradiation (Figure 5e). Meanwhile, in order to evaluate cell proliferation, the immunohistochemical staining of tumor slices using antigen Ki67 was performed (Figure 5f). As expected, the treatment with GNRs-NLS resulted in a significantly reduced proliferation index regardless of the irradiation intensity. It was also observed that different treatments showed little influence on the body weights of mice throughout the whole experiment (Figure S8), suggesting that current PTT will not induce acute toxicity or noticeable systemic side effects. In addition, for in vivo and clinical applications, it is favorable to use a minimal amount of energy in PTT so as not to damage surrounding healthy tissue. The average life spans of the mice (Figure 16


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S9) with GNRs-NLS under ultralow irradiation are clearly much longer than those of mice in other groups. Finally, no noticeable tissue abnormalities in the major organs could be observed (Figure S10), which indicates that GNRs-NLS are biologically safe and intranuclear mild hyperthermia has caused negligible side effects. Our results suggest that intra-nuclear GNRs-NLS can generate local heat inside nuclei to obtain higher anti-cancer efficacy than conventional PTT by burning up tumors. It is worthy to emphasize that such enhanced photothermolysis has been obtained upon ultralow NIR irradiance, thus nuclear targeted PTT may offer a much safer and more efficient treatment protocol than conventional PTT for cancer therapy.

3. CONLUDING REMARKS In conclusion, we have demonstrated intranuclear photothermal therapy of cancer for the first time by employing nuclear-targeting GNRs-NLS upon ultralow laser irradiation both in vitro and in vivo. Our results evidence that GNRs-NLS could efficiently accumulate in cell nuclei mediated by the nuclear targeting peptides. Under NIR irradiation at ultralow intensity (0.2 W/cm2), mild intranuclear temperature increase leads to efficient cell death by apoptosis via the inhibition of DNA repair process in comparison with negligible cytotoxicity by GNRs without nucleartargeting under the same NIR irradiation. In contrast with previously reported photothermal therapies by harsh cancer cell burning-up/ablation under relatively high intensity of laser irradiations, our strategy features substantially reduced laser intensity much below the maximum permissible exposure, greatly suppressed 17



damages to surrounding tissues and markedly improved anticancer effect by cancer cell apoptosis based on the intranuclear-delivered GNRs-NLS. Therefore, we believe that this GNRs-NLS is highly potential in the future translation from proof-of-concept to practical application in clinic.

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Figure 1. (a) Schematic illustration of the synthetic procedure of GNRs-NLS. (b) Schematic representation of nuclear targeted photothermal therapy of GNRs-NLS.

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Figure 2. Synthesis and physicochemical characterizations of GNRs and GNRs-NLS. (a, b) Transmission electron microscopic (TEM) images of GNRs and GNRs-NLS. (c) Scanning electron microscopic (SEM) image of GNRs-NLS. (d) UV-vis absorption spectra of GNRs and GNRs-NLS. The longitudinal absorbance band red-shifted from 803 nm to 815 nm after the conjugation of NLS to the surface of GNRs. (e) Photothermal heating curves and (f) corresponding infrared thermal images of PBS, GNRs and GNRs-NLS aqueous solutions (20 ppm) under an 808 nm laser irradiation at varied power densities for 5 min.

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Figure 3. Cellular uptake of GNR-NLS. (a) Confocal laser scanning microscopic (CLSM) images of HeLa cells treated with 60 µg/mL GNRs and GNRs-NLS for 12 h. From left to right, images present DAPI (blue: DAPI = 4',6-diamidino-2phenylindole) stained cell nuclei, two-photon photoluminescence (TPL) from GNRs in cells (white pattern), and overlays of the two images. Images on right column are magnified images of a single selected cell in each red square for the visualization of enhanced intracellular and intranuclear delivery of GNRs-NLS. Scale bar: 50 µm. (b) 21



Bio-TEM images of HeLa cells incubated with 60 µg/mL GNRs and GNRs-NLS for 12 h. The cyan arrows in the images show the nanorods in the cytoplasm and the red one indicates the nuclear distribution of nuclear-targeted nanorods. (c = cytoplasm, n = nucleoplasm, scale bar: 1 µm.) (c) Cellular and nuclear uptake amounts of GNRs and GNRs-NLS by HeLa cells in 12 h of incubations.

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Figure 4. (a) CLSM images of HeLa cells stained with calcein AM and PI after different treatment. HeLa cells were treated with GNRs or GNRs-NLS for 12 h and then radiated for 5 min by an 808 nm laser at the power density of 0.2 or 2 W/cm2. (b) Viabilities of HeLa cells, (c) LDH release from HeLa cells, (d) Flow cytometry analyses of cells, and (e) Relative RPA70 content of HeLa cells after incubations with

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GNRs or GNRs-NLS for 12 h and then irradiated with an 808 nm laser (0.2 or 2 W/cm2, 5 min).

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Figure 5. In vivo PTT effects on xenografted HeLa tumours. (a) Representative infrared thermal images of HeLa tumor-bearing mice were recorded at varied time intervals. Tumors were injected with PBS, GNRs or GNRs-NLS, and then irradiated with an 808 nm laser (0.2 or 2 W/cm2, 5 min). (b) The tumor growth curves of mice after different treatments during PTT period. (c) Tumor weights of mice in different groups in 15 days of PTT. (d) Digital photographs of xenografted tumors taken before (day 0) and on 7th and 15th day after photothermal therapy: control; GNRs; GNRsNLS at a power density of 0.2 or 2 W/cm2 for 5 min. (e) H&E staining and (f) immunohistochemical staining by Ki67 of tumor slices from all of the experimental groups on 7th day post PTT treatment. Scale bare: 100 µm. Ki-67-positive cells are stained brown.

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ASSOCIATED CONTENT Supporting Information. Synthetic procedures and other supplementary figures are enclosed. This material is available free of charge on the ACS Publication website: http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Jianlin Shi, PhD. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was financially supported by National Natural Science Foundation of China (Grant No. 51502326, 51402338), Shanghai Yangfan Program (Grant No. 15YF1413600), Youth Innovation Promotion Association CAS (Grant No. 2017299) and Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures (Grant No. SKL201405). Notes The authors declare no competing financial interest.


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Graphic for manuscript


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Nuclear-Targeting Gold Nanorods for Extremely Low NIR Activated

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