Adjuvant Photothermal Therapy Inhibits Local Recurrences after

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Adjuvant Photothermal Therapy Inhibits Local Recurrences after Breast-Conserving Surgery with Little Skin Damage Shouju Wang, Xingqun Ma, Xuhao Hong, Yingxia Chen, Ying Tian, Shuang Zhao, Wenfei Liu, Yuxia Tang, Ruizhi Zhao, Liang Song, Zhaogang Teng, and Guangming Lu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07757 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 23, 2017

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Adjuvant Photothermal Therapy Inhibits Local Recurrences after Breast-Conserving Surgery with Little Skin Damage Shouju Wang1, 2, ‡, *, Xingqun Ma3, ‡, Xuhao Hong4, Yingxia Cheng3, Ying Tian1, Shuang Zhao1, Wenfei Liu1, Yuxia Tang1, Ruizhi Zhao4, Liang Song5, Zhaogang Teng1, 2, Guangming Lu1, 2, * 1. Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, P.R. China 2. State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, P.R. China 3. The affiliated Bayi Hospital of Nanjing University of Chinese Medicine, Nanjing 210002, P.R. China 4. Department of Physics, Nanjing University, Nanjing 210000, P.R. China 5. Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Key Laboratory for Molecular Imaging, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P.R. China

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Email: [email protected], [email protected] Keywords: adjuvant photothermal therapy, breast-conserving surgery, breast cancer, local recurrence, cosmetic outcome

Abstract: Adjuvant treatments followed breast-conserving surgery (BCS) are essential to reduce the risk of local recurrences in patients with breast cancer. However, current adjuvant treatments are based on ionizing radiation, which brings radiation-induced damage and amplifies the risk of death. Here we explore the feasibility of using non-ionizing light to induce photothermal therapy as an adjuvant treatment to BCS. In an orthotopic breast cancer mice model, we demonstrate that adjuvant photothermal therapy (aPTT) decreases the incidence of local recurrences after BCS with no expense of cosmetic outcome. In comparison with conventional photothermal therapy (cPTT), the technique used in aPTT provides more uniformly distributed light energy and less risk of skin burns and local recurrences. Overall, this work represents a departure from the traditional concept of using PTT as an alternative to surgery and reveals the potential of using PTT as an alternative to adjuvant radiation therapy, which is valuable especially for patients susceptible to radiation damage.

Breast cancer is the most common cancer diagnosed in women and 61% of patients are 1

diagnosed at the early stage in the USA. For these patients, breast-conserving surgery (BCS) is strongly recommended by the NCCN guidelines as a good and safe alternative to total mastectomy for its equivalent therapeutic effect and better cosmetic result.

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BCS is usually

followed by whole-breast radiotherapy (WBRT) to lower the incidence of local recurrences after BCS and increase the survival of patients in the long term.

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However, the ionizing radiation


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used in WBRT amplifies the risk of death due to the radiation-induced heart disease and secondary malignant disease.

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For these concerns, up to 50% of patients in the USA who are

clinically eligible for BCS choose to undergo total mastectomy with the goal to omit radiation therapy. 6,7 Since the vast majority of local recurrences that developed in patients who did not receive WBRT are in the vicinity of the primary tumor, WBRT is supposed to exert its primary effect on the residual malignant cells left in the tumor beds.

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Thus, it is reasonable to explore other

modalities of treatment which are more localized to the tumor beds as alternatives to WBRT. Recently, serval phase II and III clinical trials show accelerated partial breast irradiation (APBI), 7,11

a radiation therapy focus specifically on the part of the breast where the tumor is removed, is

as effective as WBRT for patients with early-stage breast cancer. The use of APBI also shortens the course of treatment and reduces the radiation dose to healthy tissues. 12-14 Nonetheless, APBI is still a technique based on ionizing radiation and cannot completely omit the damage to healthy tissues from high-energy radiation. Therefore, it is in great demand to develop adjuvant therapies based on non-ionizing irradiation for patients qualified for the treatment with BCS. Photothermal therapy (PTT) is an emerging therapeutic modality utilizing the heat generated by certain kinds of nanoparticles upon near-infrared (NIR) light irradiation to kill cancers. 15-21

As compared with radiation therapy, the advantages of PTT include: 1) free of ionizing

radiation damage; 2) dual-selectivity from the tumor-targeting accumulation of nanoparticles and the spatially confined delivery of light. It is noted that the penetration depth of the NIR light in tissues is about 1 cm,

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which is close to the margin width required irradiation in the APBI

treatment. 13,23 Thus, PTT may serve as an alternative to the adjuvant APBI treatment to control local recurrences postsurgery with no radiation damage and superior tumor selectivity. However,


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to the best of our knowledge, there are no previous reports to explore the feasibility of using PTT as an adjuvant treatment followed BCS. Herein, we report the application of PEGylated gold nanobipyramids (GBP-PEG) in adjuvant photothermal therapy (aPTT) followed BCS in an orthotopic breast cancer mice model. The production of gold nanobipyramids (GBPs) with high purity is realized by synthetic approaches very recently.

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Previous reports showed GBPs have stronger and narrower localized surface

plasmonic resonance (LSPR) than other shapes of gold nanoparticles, 27,28 thus they are supposed to convert light energy into heat more efficiently. Our results showed that the obtained GBPPEG had superior photothermal conversion efficacy over that of nanorods. More important, we found the GBP-PEG mediated aPTT significantly decreased the likelihood of local recurrences after BCS with no expense of cosmetic outcomes. In contrast, BCS and conventional photothermal therapy (cPTT) alone failed to control the local recurrences. The risk of adverse cosmesis was even escalated in mice treated by cPTT because of the overheating of healthy tissues resulted from maldistributed light energy.

Results First, PEGylated gold nanobipyramids (GBP-PEG) were prepared, purified and characterized. Transmission electronic microscopy (TEM) images display GBP-PEG have a uniform length of 77.2 ± 3.3 nm and width of 23.0 ± 1.8 nm (Figure 1a & 1b). The selective area electronic diffraction (SAED) pattern of GBP-PEG additionally revealed their high crystallinity (Figure S1). UV-vis absorbance showed the LSPR of GBP-PEG peaked at 808-nm. Successful purification was identified by the dramatically reduced absorbance in the spectral region from 500 to 600 nm (Figure 1c). Zeta potential measurement exhibited a decrease of surface charge


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from +39.0 ± 1.4 mV to -2.0 ± 2.1 mV after PEGylation, implying the attached surfactants on crude GBPs were replaced by mPEG-SH molecules. Dynamic light scattering (DLS) measurement showed the hydrodynamic diameter of GBP-PEG is 41.7 ± 1.0. This diameter is not consistent with the size measured on TEM images because of the anisotropic structure of nanobipyramids. The size of GBP-PEG remained stable in PBS for up to one week (Figure S2), suggesting the dense PEG layer protects GBP-PEG from aggregation. Thermal images show the temperature of GBP-PEG solutions increase rapidly upon 808-nm laser irradiation at 1 W·cm-2 and the maximum equilibrium temperature of solutions is correlated directly to the concentration of GBP-PEG. As for the ultrapure water itself, no significant change of temperature was observed during the irradiation (Figure 1d & e). It is noted that the photothermal conversion efficacy of GBP-PEG is higher than PEGylated gold nanorods (GNR-PEG, LSPR peaked at ~810 nm) at the same concentration (Figure S3). The photothermal conversion efficacy of GBPPEG was quantified to be 37.1% following the reported method (Figure S4).

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Furthermore,

the GBP-PEG kept stable through four cycles of laser irradiation (Figure S5). These results indicate that GBP-PEG can produce heat efficiently upon laser irradiation, which encouraged us to apply these nanoparticles to photothermal therapy.


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Figure 1. Characterization of GPB-PEG. (a, b) TEM images of GBP-PEG at low and high magnification. (c) UV-Vis absorbance of crude GBP and GBP-PEG after purification. (d, e) Heating curves and thermal images of various concentrations of GBP-PEG upon laser irradiation for 10 min (808 nm, 1 W·cm-2). Next, the photothermal therapeutic efficacy and biocompatibility of GBP-PEG were investigated. MDA-MB-231 cell, a human triple negative breast cancer cell line, was chosen as the model. The cells were cultured with various concentration of GBP-PEG, washed with cold PBS, irradiated by an 808-nm laser (1 W·cm-2, 10 min) and followed by an MTT assay. The assays showed the viability of MDA-MB-231 cells decreased from 96.7 ± 2.9 to 8.4 ± 1.5% of the blank control after laser irradiation as the concentration of GBP-PEG increased from 2.5 to 40 µg·mL-1 (Figure 2a). It is noted that at concentrations higher than 10 mg·L-1, the therapeutic efficacy of GBP-PEG is significantly higher than that of GNR-PEG at the same concentration. Without laser irradiation, GBP-PEG exhibited little cytotoxicity after 48 h of incubation with


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MDA-MB-231 and MCF 10A (human normal breast epithelial cell line) even at concentrations up to 40 µg·mL-1 (Figure 2b). These results suggested GBP-PEG have superior photothermal therapeutic efficacy over GNR-PEG and excellent biocompatibility in vitro.

Figure 2. GBP-PEG showed superior photothermal therapeutic efficacy and good biocompatibility in vitro. (a) Cell viability of MDA-MB-231 cells after incubation with various concentration of GNR-PEG and GBP-PEG for 48 h and followed by laser irradiation for 5 min (808 nm, 1 W·cm-2). (b) Cell viability of MDA-MB-231 and MCF 10A cells after incubation with various concentration of GBP-PEG for 48 h without laser irradiation. P values less than 0.001 are denoted with three asterisks. To estimate the best time point for photothermal therapy in vivo, the biodistribution of GBPPEG was measured by ICP-AES. As shown in Figure S6, a large amount of gold was accumulated in organs of the reticuloendothelial system (RES) like liver and spleen. Importantly, 4.57 ± 1.27%, 8.61 ± 1.10% and 6.97 ± 0.98% ID/g of gold was found in tumors at 6, 24 and 48 h postinjection, indicating GBP-PEG could gradually accumulate in tumors due to the enhanced permeability and retention (EPR) effect. Since the gold concentration in tumor peaked at 24 h postinjection, this time point was selected for further studies.


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One possible reason for local recurrences after cPTT is the maldistribution of optical intensity in the treatment field.

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As shown in the simulation model, the Gaussian laser beam projects

most of its energy in the center of treatment field (Figure 3a & S7a, the detail of simulation is described in supporting information). This uneven distribution of laser energy lead to insufficient temperature increase and incomplete elimination at the periphery of tumors, but persistent overheating and irreversible burn to the skin at the top of tumors. To avoid these potential side effects, the laser beam was transformed to plane wave with equalized energy distribution for aPTT (Figure 3b & S7b). Since the bulk of tumors is removed by surgery, the laser power and irradiation time for aPTT were lowered than those used for cPTT, thus further reducing the risk of skin burns around the surgical incision. Thermal images showed the temperature of the surgical area increased ~10 oC upon laser irradiation for the mice underwent aPTT. As for the mice underwent cPTT, the temperature of tumor increased ~18 oC due to the higher laser power and longer irradiation time (Figure 3c & d).


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Figure 3. Laser energy distribution and tumor temperature during treatment of cPTT and aPTT. (a, b) simulated laser energy distribution in the irradiated area of cPTT and aPTT. (c, d) The temperature change and corresponding thermal images of the irradiated area during cPTT (2.5 W·cm-2, 10 min) and aPPT (1 W·cm-2, 5 min). To compare the efficacy of different therapeutic strategy, MDA-MB-231/Luc bearing mice were randomly divided into four groups, including control, cPTT, surgery, and surgery plus aPTT. Successful removal of tumors in the surgery and surgery plus aPTT groups was confirmed by the tumor-negative resection margin observed on H&E stained slices (Figure 4a). Bioluminescence signal intensity and volume of tumors were monitored up to 18 days. The


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bioluminescence images are illustrated in Figure 4b and S8-11. The tumor signal intensity and tumor volume of the three treated groups were significantly lower than those of the control group on day 18 posttreatment (p

Adjuvant Photothermal Therapy Inhibits Local Recurrences after

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