CuS Nanoparticles as a Photodynamic Nanoswitch for Abrogating

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CuS Nanoparticles as a Photodynamic Nanoswitch for Abrogating Bypass Signaling to Overcome Gefitinib Resistance Xiajing Gu, Yuanyuan Qiu, Miao Lin, Kai Cui, Gaoxian Chen, Yingzhi Chen, Chenchen Fan, Yongming Zhang, Lu Xu, Hong-zhuan Chen, Jian-Bo Wan, Wei Lu, and Zeyu Xiao Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b01065 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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CuS Nanoparticles as a Photodynamic Nanoswitch for Abrogating Bypass Signaling to Overcome Gefitinib Resistance Xiajing Gu,†,# Yuanyuan Qiu,†,# Miao Lin, †,# Kai Cui,† Gaoxian Chen,† Yingzhi Chen,† Chenchen Fan,† Yongming Zhang,† Lu Xu,† Hongzhuan Chen,‡ Jian-Bo Wan,§ Wei Lu,¶,* Zeyu Xiao†,* † Department of Nuclear Medicine, Clinical and Fundamental Research Center, Institute of Molecular Medicine, Ren Ji Hospital, & Department of Pharmacology and Chemical Biology, Translational Medicine Collaborative Innovation Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China ¶ Key Laboratory of Smart Drug Delivery, Ministry of Education, & State Key Laboratory of Molecular Engineering of Polymers, School of Pharmacy, Fudan University, Shanghai, China ‡ Institute of Interdisciplinary Integrative Biomedical Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China § State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macao, China

* Corresponding authors: Zeyu Xiao: [email protected] or [email protected] (Z.X.); Tel: (+86)-21-63846590 ext. 776415 Wei Lu: [email protected] (W.L.); Tel: (+86)-21-51980185 1

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ABSTRACT

Bypass signaling activation plays a crucial role in the acquired resistance of gefitinib, the first targeted drug in the clinic to treat advanced non-small cell lung cancer. Although the inactivation of bypass signaling by small-molecule inhibitors or monoclonal antibodies may overcome gefitinib resistance, their clinical use has been limited by the complex production process and off-target toxicity. Here we show CuS nanoparticles (NPs) behaved as a photodynamic nanoswitch to specifically abrogate overactive bypass signaling in resistant tumor cells, without interfering the same signal pathways in normal cells. In representative insulin growth factor-1 receptor (IGF1R) bypass activation-induced gefitinib resistant tumors, CuS NPs upon near-infrared laser irradiation locally elevated reactive oxygen species (ROS) level in tumor cells, leading to the blockage of bypass IGF1R and its downstream AKT/ERK/NF-κB signaling cascades. Consequently, laser-irradiated CuS NPs sensitized tumors to gefitinib treatment and prolonged the survival of mice with no obvious toxicity. Laser-irradiated CuS NPs may serve as a simple and safe nanomedicine strategy to overcome bypass activation-induced gefitinib resistance in a specific and controllable manner, and provide insights into the treatment of a myriad of other resistant tumors in the field of cancer therapy.

KEYWORDS: gefitinib, CuS nanoparticles, non-small cell lung cancer, drug resistance, reactive oxygen species

2


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Lung cancer is the leading cause of cancer-related death throughout the world, and non-small cell lung cancer (NSCLC) accounts for approximate 85% of this disease.

1, 2

The activating mutation

within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) plays a key role in the development and progression of NSCLC.3-6 Serving as a tyrosine kinase inhibitor to inactivate EGFR signal pathway, gefitinib becomes the first FDA-approved targeted drug for the treatment of EGFR-mutant NSCLC.7, 8 Nevertheless, acquired gefitinib resistance universally occurs after 9-12 months of treatment; which significantly hampers the clinical outcome of gefitinib.3, 9, 10 Extensive interest has focused on investigating the molecular mechanisms and finding out solutions of gefitinib resistance.11Among them, bypass signaling activation is one major category engaged in the acquired resistance of gefitinib.12 Bypass activation describes a situation where other parallel signal pathways are activated to compensate gefitinib-induced inhibition of EGFR signals.13 As such, the downstream signaling (e.g., AKT or ERK) can still be aberrantly activated for tumor cell proliferation, leading to the failure of gefitinib treatment.14-16 Representative bypass signaling related to gefitinib resistance includes insulin growth factor-1 receptor (IGF1R) signaling17, mesenchymal epithelial transition (MET) amplification18, HER2 overexpression19, and et al. To combat bypass activation-induced gefitinib resistance, current strategies mainly rely on molecule inhibitors or monoclonal antibodies to block the activated bypass signaling.20,

21, 22

However,

screening or design of specific molecule inhibitors and production of monoclonal antibodies are time-consuming and labor-intensive processes. Systematic administration of these molecule inhibitors or monoclonal antibodies may lead to off-target toxicity, mainly due to the same extent of inactivating these cell proliferation-related signal pathways in normal cells.23, 24 It is highly desirable to develop a simple and safe strategy capable of specifically blocking activated bypass signaling of tumor cells in a controlled manner. To the best of our knowledge, it remains a challenge task and no 3


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reported technologies have achieved this goal till now. Reactive oxygen species (ROS) are widely recognized as signaling molecules regulating intracellular signal pathways25-27, and thus we assume they might be utilized to regulate activated bypass signaling in resistant tumors. In addition, intracellular ROS levels can be controlled in a photodynamic way28, where a specific wavelength of light transforms a photosensitizer (PS) from its ground state to excited single state, and this excited state of PS produces ROS in the presence of oxygen.29 The conventional PSs for anticancer photodynamic therapy mostly rely on small molecules with a tetrapyrrole structure30, such as chlorin e6 (Ce6); however, their usage is challenged by poor pharmacokinetic properties31, short-wavelength (< 700 nm) light excitation32, and compromised ROS producing capability in hypoxia tumor environment33. Despite the integration of nanocarriers improves the tumor accumulation and pharmacokinetics of PSs, the formulation of PSs-loading nanocarriers is a complex and multi-step process.34 In pursuit of choosing an ideal ROS producer for regulating bypass signaling in resistant tumors, we turned our attention to copper sulfide (CuS) nanoparticles (NPs). Unlike PSs-loading nanocarriers, CuS NPs have the ability to generate ROS and behave as PSs by themselves.35 Taking advantage of the enhanced permeability and retention (EPR) effect of the tumor tissue36, CuS NPs can specifically and sufficiently accumulate inside tumor tissues. The water-solubility of CuS NPs contributes to good pharmacokinetics performance with minimized skin accumulation and fast clearance rate

37

.

The photodynamic effect of CuS NPs is activated with a long wavelength laser in near-infrared (NIR) region (780-1100 nm)38, contributing to a deep tumor penetration with minimal damage to normal tissues39. In addition, ROS production by NIR laser-irradiated CuS NPs is considered to derive from accelerated copper ions (Cu2+) release from CuS in tumor acidic environment, and the released Cu2+ ions further react with high concentration of H2O2 in tumor environment via the Haber-Weiss and 4


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Fenton reactions to produce other ROS.40 Therefore, tumor environment with an acidic and high H2O2 condition would specifically promote ROS production. Considering all these features, we choose CuS NPs as the ROS initiator to test our hypothesis. In this work, we depict a laser-irradiated CuS NPs strategy for local and controllable inactivation of bypass signaling in gefitinib-resistant tumors. This strategy simply relies on (i) systematic administration of CuS NPs and subsequent (ii) usage of near infrared (NIR) laser to locally

irradiate

CuS

NPs-harbored

resistant

tumors.

In

two

well-established

IGF1R

activation-induced gefitinib-resistant NSCLC models (HCC827-GR and PC9-GR)41, we show that NIR laser-irradiated CuS NPs result in elevated reactive oxygen species (ROS) level, which further downregulates the expression levels of IGF1R and its downstream AKT/ERK/NF-κB signal proteins in resistant tumor cells (Figure 1A). As such, CuS NPs serve as a photodynamic nanoswitch to inactivate IGF1R bypass signal pathway, thus overcoming gefitinib resistance and prolonging the survival of resistant tumor-bearing mice. Importantly, this CuS-based photo-controllable nanoswitch is able to specifically switch-off overactive bypass signaling in tumor cells, without interfering the same signal pathways in normal cells. Our study provides a simple and safe nanotechnology strategy to overcome bypass activation-related gefitinib resistance in NSCLC, and simultaneously broadens the applications of CuS NPs towards combating tumor drug resistance. RESULTS AND DISCUSSION Synthesizing and characterizing the photodynamic feature of CuS NPs. CuS NPs were prepared by synthesizing Cu2O nanospheres and then reacting with Na2S solution. The CuS NPs consist of numerous ultrasmall CuS particles coated with polyethylene glycol (PEG) to extend blood circulation half-life (Figure 1A). Transmission electron microscopy (TEM) imaging exhibited hollow sphere structure of CuS NPs with diameter about 160 nm (Figure 1B). Dynamic light 5


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scattering demonstrated a uniform NP size distribution at 165.8 ± 3.9 nm with a polydispersity index of 0.179 (Figure 1C). To assess the cellular uptake of CuS NPs in the gefitinib-resistant NSCLC cell lines (HCC827-GR and PC9-GR), cells were incubated with Rhodamine B-labeled CuS NPs for 4 h. Confocal laser scanning microscope (CLSM) images (Figure S1A) exhibited the intracellular ﬂuorescent accumulation, demonstrating the internalization of CuS NPs into the cells. Flow cytometry analysis further verified effective cellular uptake of CuS NPs by both HCC827-GR and PC9-GR cells (Figure S1B). Recently, ROS are extensively reported to act as a second messenger to modulate cellular signaling pathways in cancer therapy.26, 42-44 Previous studies reported that CuS NPs upon the NIR laser irradiation (CuS/Laser) can generate high reactive oxygen species (ROS).35 To evaluate the photodynamic property of CuS/Laser in generating ROS, we incubated CuS NPs with HCC827-GR or

PC9-GR

cells,

followed

by

6-min

NIR

laser

irradiation.

The

ROS

probe,

2',7'-dichlorofluorescein-diacetate (DCFH-DA), was immediately added into the cells. DCFH-DA would diffuse into the cells and be hydrolyzed to DCFH by intracellular esterases. Nonfluorescent DCFH can react with cellular ROS, producing green fluorescent DCF for detection.45 In Figure 1E, cells with CuS/Laser treatment elicited stronger fluorescence signal than untreated control cells, suggesting the production of ROS upon NIR laser irradiation of CuS NPs. In addition, CuS/Laser treatment dramatically enhances cellular ROS level compared to CuS treatment alone (Figure S2), presumably because CuS/Laser-induced hyperthermia (Figure S3) increases mitochondria membrane potential as well as the release of copper ion (a redox active transition metal) from CuS NPs.35, 40 High-resolution single cell analysis with our newly-developed ROS Raman nanoprobe (para-aminothiophenol-decorated gold nanoprobe, Au/PATP)46 further confirmed ROS generation upon laser irradiation of CuS NPs (Figure 1F), as CuS/Laser-treated cells yield higher ROS 6


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characteristic peak intensity at 1142 cm-1 than non-treated control cells (Figure S4). Furthermore, we compared the capability of CuS NPs in producing ROS with that of a conventional small molecule PS (Chlorin e6, Ce6). In Figure S5, ROS level in the laser-irradiated CuS NPs solution was significantly higher than that in the laser-irradiated Ce6 solution, confirming a stronger photodynamic effect of CuS NPs over Ce6. Photodynamic switching off activated IGF1R bypass signaling by laser-irradiated CuS NPs. We next investigated whether elevated ROS level upon laser irradiation of CuS NPs can switch off activated bypass signaling in tumor cells. Extensive research has utilized HCC827-GR and PC9-GR as model cells lines to confirm the involvement of IGF1R bypass activation in the acquired gefitinib resistance of NSCLC.47-49 Western blot analysis confirmed elevated IGF1R expression in these two gefitinib-resistant cell lines (HCC827-GR and PC9-GR) compared to their gefitinib-sensitive counterpart cell lines (HCC827 and PC9) (Figure S6). We then used these two cell lines as representative bypass signal-activated resistant tumor cells to test our assumption. We examined the expression of IGF1R at 24 h post-CuS/Laser treatment. Western blot analysis revealed a significantly lower expression level of IGF1R and the phosphorylated IGF1R after CuS/Laser treatment in these two cell lines (Figure 2A and 2C), indicating downregulation of IGF1R activity upon CuS/Laser treatment. Immunofluorescent imaging further confirmed the reduced IGF1R expression level in CuS/Laser-treated HCC827-GR and PC9-GR cells (Figure 2B and 2D). Subsequently, we examined whether CuS/Laser treatment further downregulated the downstream signals of IGF1R. Two primary signal pathways are involved in the downstream activation of IGF1R, including MAPK/ERK (mitogen activated protein kinase, MAPK, extracellular regulated protein kinases, ERK) pathway and PI3K/AKT (phosphatidylinositol-3-kinase, PI3K) pathway.

50, 51

As ERK is the core protein in MAPK/ERK pathway, phosphorylated ERK indicates 7


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the activation of this signal pathway.52 Similarly, phosphorylated AKT can be an indicator of the activated PI3K/AKT pathway.53,

54

As shown in Figure 2E and 2F, the expression levels of

phosphor-ERK and phosphor-AKT were decreased in cells treated with CuS/Laser compared to untreated cells (control group), suggesting the downregulation of MAPK/ERK and PI3K/AKT signal pathways upon the NIR irradiation of CuS-harbored cells. One of the most significant downstream signaling of MAPK/ERK and PI3K/AKT pathways is NF-κB pathway, which modulates cell proliferation and apoptosis.12,

53

It is also reported to be involved in the IGF1R bypass

activation-related acquired resistance of NSCLC.12,

55

The classic NF-κB signal pathway is

comprised of IκB kinase (IKK), inhibitors of NF-κB (IκB) and p65/p50 heterodimer. IKK is composed of IKKα, IKKβ and IKKγ, and p65/p50 heterodimer forms a complex with IκB (i.e., IκB/p65/p50) in the cytoplasm. A variety of upstream signal pathways (e.g. ERK, AKT) could activate the phosphorylation of IKK, which triggers the phosphorylation of IκB, resulting in the degradation of IκB. Subsequently, p65/p50 heterodimer is released from the IκB/p65/p50 complex, and further be translated to the nucleus for gene regulation. Therefore, to explore whether CuS/Laser treatment inactivates the NF-κB signal pathway, we investigated the phosphorylation of three core proteins including IκB kinase (IKK), inhibitor of NF-κB (IκB), and NF-κB. As shown in Figure 2E and 2F, the expression of phospho-IKK, phospho-NF-κB and phospho-IκB were significantly decreased after CuS/Laser treatment, indicating the inactivation of NF-κB signaling pathway. Taken together, CuS/Laser treatment resulted in the downregulation of IGF1R expression, and subsequent inhibition of MAPK/ERK and PI3K/AKT signaling pathways, leading to the inhibition of cell proliferation followed by NF-κB pathway inactivation. To further verify the elevated ROS level by laser-irradiated CuS NPs is the primary reason for downregulating IGF1R signal pathways, we treated HCC827-GR and PC9-GR cells with H2O2 for 8


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24 h, and then examined the expression of the key signal proteins, including phospho-IGF1R, -ERK, -AKT, -IKK, -NF-κB, and -IκB. As shown in Figure 3, H2O2 treatment significantly decreased the expression of all these signal proteins, indicating the inactivation of IGF1R, PI3K/AKT, MAPK/ERK, and NF-κB signaling pathways. In contrast, when cells were pretreated with the ROS scavenger (N-acetyl-L-cysteine, NAC) before H2O2 treatment, the expression of all these signal proteins were recovered. Collectively, the high ROS level induced by CuS/Laser treatment initially led to the downregulation of IGF1R signal pathway and subsequent PI3K/AKT, MAPK/ERK, NF-κB signaling pathways. Sensitizing resistant cells to gefitinib treatment by laser-irradiated CuS NPs. We next explored whether CuS-based inhibition of bypass signaling could increase the sensitivity of gefitinib treatment in two representative resistant NSCLC cell lines (HCC827-GR and PC9-GR). To achieve this, cells were divided into 6 groups with/without gefitinib (5 μM) treatment. In HCC827-GR cells, gefitinib group showed high cell viability of 84.54 ± 2.91 % due to drug resistance whereas cell viability of CuS/Laser group was decreased to 77.35 ± 4.77 %, resulting from the photothermal efficacy of CuS NPs (Figure S3). In contrast, cell viability in CuS/Laser/gefitinib group was dramatically decreased to 47.14 ± 4.76 % (Figure 4A). Moreover, the inhibition rate in CuS/Laser/gefitinib group (~52.86 %) was higher than the sum of that in CuS/Laser group (~22.65 %) and gefitinib group (~15.46 %); this result suggested laser-irradiated CuS NPs sensitized HCC827-GR cells to gefitinib. Similar results were found in another gefitinib-resistant PC9-GR cells. In Figure 4B, the viability of PC9-GR cells treated with gefitinib, CuS/Laser, CuS/Laser/gefitinib were (81.39 ± 2.48) %, (66.92 ± 3.04) %, (36.53 ± 0.76) %, respectively. Remarkably, the inhibition rate in the CuS/Laser/gefitinib group (~63.47 %) was higher than the sum of that in CuS/Laser treatment (~33.08 %) and gefitinib treatment (~18.61 %). In addition, the cytotoxicity assay with a 9


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gradient concentration of gefitinib demonstrated pretreatment of NIR laser-irradiated CuS NPs could increase the sensitivity of the resistant cells to gefitinib (Figure 4C and 4D). Long-term efficacy was further tested by performing the colony formation assay, in which both cell lines were incubated with gefitinib for up to 10 days after pretreatment with CuS/laser. As shown in Figure 4E, fewer colonies were formed in CuS/Laser/gefitinib group compared with untreated control, gefitinib, and CuS/Laser group. Taken together, our results showed NIR laser-irradiated CuS NPs overcame gefitinib resistance in two NSCLC cell lines. Sensitizing resistant tumors to gefitinib treatment by abrogating IGF1R bypass signaling with laser-irradiated CuS NPs. To investigate whether laser irradiation of CuS NPs could switch off bypass signaling in resistant tumors, HCC827-GR tumor-bearing mice were intravenously administrated with saline, CuS NPs, or Ce6 molecules, respectively. After 24-h blood circulation and tumor accumulation, the tumors were irradiated with an 808 nm laser (CuS NPs group) or a 630 nm laser (Ce6 molecules group) for 6 mins. Tumors were then collected for protein expression analysis at 24 h post-laser irradiation. As shown in Figure 5, CuS/Laser treatment significantly downregulated the expression levels of IGF1R/AKT/ERK/ NF-κB signature proteins and their phosphorylated forms, indicating in vivo inactivation of bypass signaling. In contrast, Ce6/laser treatment had a negligible effect on the expression levels of bypass signaling proteins (Figure S9), presumably because of insufficient tumor accumulation of Ce6 molecules, limited tumor penetration depth of 630 nm laser, or inadequate oxygen supply in hypoxia tumor microenvironment. Collectively, these results verified laser-irradiated CuS NPs can efficiently inactivate bypass signaling in resistant tumors. To elucidate the therapeutic efficacy of CuS-based nanoswitch in sensitizing resistant tumors to gefitinib treatment, we established xenograft NSCLC mice models using HCC827-GR and PC9-GR 10


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cells. Gefitinib was orally administrated into the mice every other day with or without pretreatment with NIR laser-irradiated CuS NPs, mimicking the clinical treatment procedures.56 Saline and CuS/Laser group were set for comparisons. The tumor size was then monitored during the treatment period (Figure 5A and 5E). In the HCC827-GR tumor-bearing mice (Figure 5B and S7A), saline group reached tumor size of 374.4 ± 14.47 mm3; gefitinib treatment alone weakly restrained the growth of resistant tumors with a terminated size of 299.7 ± 19.41 mm3; and CuS/Laser treatment alone demonstrated a modest tumor inhibition with a terminated size of 245.8 ± 18.86 mm3, mainly due to the photothermal ablation effects of CuS NPs upon NIR laser irradiation. Importantly, the combination of NIR laser-irradiated CuS NPs with gefitinib treatment dramatically inhibited tumor growth with a terminated size of 98.02 ± 8.74 mm3. By calculation, the inhibition rate in CuS/Laser/gefitinib group was (71.64 ± 1.03) %, higher than the sum of inhibition rate in CuS/Laser group (25.25 ± 2.97) % and in gefitinib group (18.55 ± 1.88) % (Figure S8A), indicating the enhanced sensitization to gefitinib after pretreatment with NIR laser-irradiated CuS NPs. In the survival analysis, mice in CuS/laser/gefitinib group had been completely alive during the observation, while the other three groups succumbed to death (Figure 5C). The representative photos of collected tumors at the end of treatments were shown in Figure 5D. The efficacy of NIR laser-irradiated CuS approach in sensitizing gefitinib treatment was also verified in gefitinib resistance mouse model of PC9-GR tumor. The combinational CuS/Laser/gefitinib treatment dramatically inhibited tumor growth and prolonged the mice survival, compared to CuS/Laser or gefitinib treatment alone (Figure 5F, 5G, 5H, S7B and S8B). In vitro and in vivo safety evaluation of laser-irradiated CuS NPs. HCC827-GR and PC9-GR cells were incubated with CuS NPs at different concentrations, as shown in Figure S10A and S10B, the cell viability exhibited very high of over 90 % even the concentration up to 200 11


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μg/mL. These results indicated CuS NPs could be favorably internalized into resistant NSCLC cell lines with minimal cytotoxicity. Next, CuS NPs exhibited great biosafety after systematic administration into mice models, as indicated by no noticeable weight loss during the entire treatment period (Figure S10C and 10D), and by no apparent organ damage from hematoxylin and eosin (H&E) analysis of main organs including heart, liver, spleen, lung, and kidney (Figure S11 and S12). Taken together, NIR laser-irradiated CuS NPs can efficiently and safely sensitize resistant NSCLC tumors to gefitinib treatment and improve mice survival rate with minimal systematic toxicity.

Conclusions In summary, we demonstrated that activated IGF1R bypass signaling in gefitinib-resistant tumor cells can be turned off by a CuS NPs-based photodynamic nanoswitch. Principled by the elevated ROS level upon NIR laser irradiation of CuS NPs, the nanoswitch inactivated IGF1R bypass signaling and the downstream ERK/AKT/NF-κB signal pathways, thus eventually restraining cell proliferation and sensitizing gefitinib treatment. Importantly, this CuS-based nanoswitch specifically blocked bypass signaling in tumor cells without interfering the same signal pathway in normal cells; which has obvious advantages over current bypass signaling blockers using small molecules or monoclonal antibodies.

To the best of our knowledge, this is the first time to use a

simple and safe CuS-based nanomedicine strategy to locally abrogate bypass signaling for combating drug resistance. Despite of being utilized for overcoming IGF1R bypass signal-induced gefitinib resistance in current study, this strategy may similarly be applied for the treatment of other bypass signal-induced gefitinib resistance, and may pave the way to overcome bypass activation-induced acquired resistance in other type of cancers. 12


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Authors *Email: [email protected] or [email protected] (Z. X.) *Email: [email protected] (W.L.)

ORCID Zeyu Xiao: 0000-0002-3457-5772 Wei Lu: 0000-0002-1333-0274 Author Contributions # X.G., Y.Q. and M.L. contributed equally to this work. The authors declare no conflict of interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental Section, Cellular uptake of CuS NPs, Intracellular ROS level of HCC827-GR and PC9-GR cells with different treatments, Photothermal effect of CuS NPs, Representative Raman spectra of Au/PATP nanoprobe, ROS levels in CuS NPs and Ce6 solution, IGF1R level in gefitinib-resistant NSCLC cell lines and their non-resistant counterpart cell lines, Tumor growth of every mice with different treatments in HCC827-GR-bearing and PC9-GR-bearing tumor mice, Tumor inhibition rate of various treatments in PC9-GR-bearing and HCC827-GR-bearing tumor mice, Expression levels of IGF1R and its downstream proteins in HCC827-GR-bearing tumors at 24 h posttreatment with Ce6/Laser or CuS/Laser, In vitro and in vivo safety of CuS NPs, H&E staining of main organs collected from tumor bearing mice with different treatments. 13


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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21874092, 31671003, and 81673018), Thousand Young Talents Program, the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. TP2014028), Q.Y. acknowledges the Doctoral Innovation Fellowship from Shanghai Jiao Tong University School of Medicine (No.BXJ201704).

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Figure 1. (A) Schematic illustration of laser-irradiated CuS nanoparticles overcoming gefitinib resistance in NSCLC. After intravenous injection, CuS NPs are accumulated in tumor through EPR effect, and then the tumor was irradiated by NIR laser. NIR laser-irradiated CuS NPs elevate intracellular ROS, which further suppress the activated bypass signal (e.g. IGF1R) and its 17


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downstream ERK, AKT and NF-κB pathways, resulting in enhanced sensitivity to gefitinib in resistant NSCLC. (B) TEM image of CuS nanoparticles (Scale bar: 100 nm). (C) Size distribution of CuS nanoparticles. (D) UV-Vis absorption spectrum of CuS nanoparticles with different concentration. (E) Multiple-cell fluorescence imaging detected by a ROS fluorescence probe DCFH-DA in HCC827-GR and PC9-GR cells (Scale bar: 200 μm). (F) Single-cell confocal Raman imaging detected by a ROS Raman nanoprobe (Au/PATP) in HCC827-GR cells, where green color indicates enhanced ROS level with characteristic peak intensity at 1142cm -1 by using constant peak intensity at 1077cm-1 (red color) as inner standard (Scale bar:10 μm).

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Figure 2. Downregulation of IGF1R and its downstream signal proteins at 24 h posttreatment with laser-irradiated CuS nanoparticles in gefitinib resistant NSCLC cells. Western blotting showing decreased IGF1R, phosphorylated IGF1R (p-IGF1R) at 24 h posttreatment with laser-irradiated CuS nanoparticles in (A) HCC827-GR and (C) PC9-GR cells. Representative immunoﬂuorescence images of IGF1R in (B) HCC827-GR and (D) PC9-GR cells (Scale bar: 250 μm). Western blotting showing decreased phosphorylation levels of downstream AKT, ERK, NF-κB signal proteins at 24 h posttreatment with laser-irradiated CuS nanoparticles in (E) HCC827-GR and (F) PC9-GR cells. Control stands for untreated cells. Quantitative data are relative to β-actin and presented as mean ± SEM (n=3), * p

CuS Nanoparticles as a Photodynamic Nanoswitch for Abrogating

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