European Journal of Radiology 83 (2014) 117–122
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European Journal of Radiology journal homepage: www.elsevier.com/locate/ejrad
Contrast ultrasound-guided photothermal therapy using gold nanoshelled microcapsules in breast cancer Shumin Wang a,b , Zhifei Dai c , Hengte Ke d , Enze Qu a , Xiaoxu Qi e , Kuo Zhang e , Jinrui Wang a,∗ a
Department of Ultrasonography, Peking University Third Hospital, Beijing 100083, China Ordos Center Hospital, Ordos, Inner Mongolia 017000, China c Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China d Nanomedicine and Biosensor Laboratory, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China e Department of Laboratory Animal Science, Peking University Health Science Center, Beijing 100019, China b
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Article history: Received 6 May 2013 Received in revised form 10 August 2013 Accepted 9 September 2013 Keywords: Phototherapy Ultrasonography Breast neoplasms Nanoparticles Laser therapy Thermal ablation Ultrasound guidance
a b s t r a c t Objectives: The purpose of this study was to test whether dual functional gold nano-shelled microcapsules (GNS-MCs) can be used as an ultrasound imaging enhancer and as an optical absorber for photothermal therapy (PTT) in a rodent model of breast cancer. Methods: GNS-MCs were fabricated with an inner air and outer gold nanoshell spherical structure. Photothermal cytotoxicity of GNS-MCs was tested with BT474 cancer cells in vitro and non-obese diabetes-SCID (NOD/SCID) mice with breast cancer. GNS-MCs were injected into the tumor under ultrasound guidance and treated with near-infrared (NIR) laser irradiation. The photothermal ablative effectiveness of GNSMCs was evaluated by measuring the surface and internal temperature of the tumor as well as the size of the tumor using histological confirmation. Results: NIR laser irradiation resulted in significant tumor cell death in GNS-MCs-treated BT474 cells in vitro. GNS-MCs were able to serve as an ultrasound enhancer to guide the intratumoral injection of GNS-MCs and ensure their uniform distribution. In vivo studies revealed that NIR laser irradiation increased the intratumoral temperature to nearly 70 ◦ C for 8 min in GNS-MCs-treated mice. Tumor volumes decreased gradually and tumors were completely ablated in 6 out of 7 mice treated with GNS-MCs and laser irradiation by 17 days after treatment. Conclusion: This study demonstrates that ultrasound-guided PTT with theranostic GNS-MCs is a promising technique for in situ treatment of breast cancer. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Early diagnosis of breast cancer with mammography screening leads to an increased demand for minimally invasive and nonsurgical treatment of patients with small breast cancers [1,2]. Breast-conserving surgical therapy is therefore the treatment of choice for women with relatively small breast cancers [3]. Percutaneous tumor thermal ablation provides an effective alternative to open breast surgery because the breast is a superficial structure without intervening organs. Various techniques of thermal-based ablative therapy, including radiofrequency, microwave, highintensity focused ultrasound, and lasers have been applied for the local treatment of breast cancer [4–7]. Radio frequency ablation
∗ Corresponding author at: Department of Ultrasonography, Peking University Third Hospital, No. 49 Garden Road, Haidian District, Beijing 100083, China. Tel.: +86 010 8226 4486; fax: +86 010 82265851. E-mail address: jinrui [email protected] (J. Wang). 0720-048X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejrad.2013.09.010
(RFA) is considered to be a promising non-surgical technique for the treatment of small breast cancers [8]. However, conventional RFA can cause thermal injury to surrounding normal tissue and requires the insertion of a 14 or 17 gauge electrode probe into the lesion. Furthermore, tissue-dependent electrical characteristics of RFA may cause non-homogeneous deposition of heat, leading to incomplete ablation of tumor cells [9]. Therefore, it is imperative to develop more effective and less invasive treatments for breast cancer. Optical absorbance-mediated NIR laser therapy has the potential to precisely deliver energy to a targeted site by transcutaneous and interstitial approaches, allowing for less or non-invasive local destruction of the cancer in situ [10]. Light in the NIR region (650–900 nm) has minimal human tissue absorption and can penetrate into deep tissue to produce cytotoxic thermal effects, making NIR light an attractive light source for thermal ablation [11]. PTT can induce temperature elevations above 55 ◦ C at the light irradiating site in a very short time and instantaneously cause significant tumor ablation. Nanomaterial-mediated delivery of optical
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absorbers has been investigated and shown promising results for thermal ablation of the tumor using NIR light [12]. Conventional NIR photoabsorbers such as indocyanine Green are susceptible to chemical and thermal denaturation and photobleaching, making NIR light-mediated thermal ablation less efficient [10]. In contrast, gold nanostructures exhibit excellent optical properties and have been investigated as photo-absorbers for NIR laserbased PTT [13–16]. The rigid structure and noble metal surface of gold nanoshells make these particles a much more robust optical absorber than conventional NIR dyes [10,17]. Photothermal therapy against cancer is a viable alternative to surgery, showing both noninvasive and tumor-specific characters. However, for direct clinical translation, there is a pressing need to combine an imaging technique to assess tumor location, size, and internal structures with the ability to detect the distribution of photoabsorbers in the tumor before therapy as well as assess tumor damage after therapy. An appropriate imaging modality is therefore an important aspect for guiding local PTT. A number of imaging techniques such as MRI [18] and SPECT/CT imaging-guided PTT [19] have been used for guiding PTT. However, ultrasound imaging shows a number of advantages over other imaging modalities, including its real-time nature, lack of ionizing radiation, greater availability and portability, ease of use, and far lower cost. More importantly, ultrasound imaging can provide instant feedback and results during thermal ablation procedures [20]. Thus, the combination of ultrasound imaging guidance with PTT could provide a cost-effective, convenient, and non-invasive approach with both diagnostic and therapeutic functions for the treatment of cancer. A dual functioning agent, gold nano-shelled microcapsules (GNS-MCs) with inner air-filled hollow domains, was developed in our lab to be used as an ultrasound enhancer and optical absorber [21]. The purpose of this study was to test whether dual functional GNS-MCs can be used as a contrast agent for ultrasound imaging and, simultaneously, as a photothermal ablative device for treatment of breast cancer in a NOD/SCID mouse model. 2. Materials and methods 2.1. Synthesis and characterization of GNS-MCs Polylactide microcapsules were prepared by the water-inoil-in-water double emulsion method. Citrate-stabilized gold nanoparticles were then deposited on the surface of polylactide microcapsules as seeds with the help of poly (allylamine hydrochloride). Gold nano-shells were formed through the surface seeding method via reduction of tetra-chloroaurate (III) by hydroxylamine hydrochloride. The encapsulated water in the inner aqueous phase of the microcapsules was sublimated, leaving a tiny hollow space with air inside and becoming a nonlinear reflector for enhancing ultrasound imaging. The size of GNS-MCs was determined by static laser scattering to be 2.32 ± 1.07 m. The GNS-MCs showed strong absorption of light at the wavelength of 700–900 nm [21]. 2.2. Photo-cytotoxicity of GNS-MCs in BT474 cells Human breast tumor BT474 cells were seeded in 12-well plates (1 × 106 /well) and incubated for 24 h. GNS-MC (1 mg/mL) was added into cells for 1 h and illuminated with a fiber-coupled diode NIR laser at a wavelength of 808 nm and power intensity of 8 W/cm2 for 10 min (T808F2W, Xi’an Minghui Optoelectronic Technology, China). Cell viability was measured using double-fluorescent staining of calcein-AM (staining live cells) and propidium iodide (PI, staining dead cells) at 24 h after laser irradiation. The tumor cells treated with saline, saline with laser irradiation, and GNS-MCs each
served as a control. Dose-efficacy of GNS-MCs in eliciting BT474 cell death was further analyzed by the MTT assay at the following concentrations: 0, 0.025, 0.05, 0.1, 0.3 and 0.5 mg/mL. 2.3. Animal model of human breast cancer All animal procedures were approved by the Institutional Animal Care and Use Committee at the Institute of Biophysics of Peking University. The 8 week-old female NOD/SCID mice (18–22 g, Institute of Laboratory Animal Science of Chinese Academy of Medical Science, China) were implanted with estrogen pellets (17-estradiol, Innovative Research of America, USA) in the right flank one day before tumor inoculation. Each mouse was inoculated in the right mammary fat pad with BT474 tumor cells (1 × 107 ) in 0.1 mL of PBS with Matrigel (1:1). Twenty-eight NOD/SCID mice with BT474 tumor were randomly divided into four groups (n = 7 per group): saline only, laser only, GNS-MCs only and GNS-MCs+laser. Photothermal treatment of the tumor was performed when the tumor size reached approximately 400 mm3 around 30 days post implantation. 2.4. Ultrasound contrast-guided injection of GNS-MCs In order to ablate the entire tumor, it is critical to make sure that the GNS-MCs infiltrate the entire tumor mass. Because air-filled GNS-MCs can act as an ultrasound contrast agent, we tested whether ultrasound imaging would be able to properly guide the injection of GNS-M Cs and monitor their distribution. With an ultrasound unit with a 10 MHz transducer (Siemens S2000, Siemens Healthcare, WA), contrast ultrasound imaging with cadence-contrast pulse sequence (CPS) technology at a mechanical index of 0.28–0.42 was used to guide and monitor the injection of GNS-MCs into the tumor of the mouse. The CPS imaging mode is a unique pulse sequence processing technology especially designed for contrast microbubbles and has the ability to combine the nonlinear fundamental and higher order harmonic signals to form a highly specific and sensitive contrast agent display in real time. CPS imaging gives the ability to selectively view “tissue only, contrast agent only, or both together” [22]. This imaging mode was chosen for this study because its remarkable differentiation of tissue and GNS-MC signals. GNS-MCs (200 L, 10 mg/mL suspension) or saline injections of the tumor were made with a 27G needle. During the injection, CPS contrast imaging was used to guide the needle position and to display the enhancement of GNS-MCs. The area with no or low GNS-MC enhancement was re-injected under ultrasound guidance to make more uniform distribution of the GNS-MCs throughout the tumor. 2.5. GNS-MCs mediated PTT in vivo The margins of the GNS-MC area of distribution in the tumor was determined by ultrasound imaging and outlined with a marker. The tumors were irradiated with NIR light (808 nm diode laser) at a surface power of 1.3 W/cm2 for 10 min. For the irradiation of the tumor, the laser fiber was set at a fixed distance of 4.0 cm from the tumor to ensure equivalent energy intensity covering the entire tumor area. During the NIR irradiation, an infrared thermographic camera (A40, FLIR Systems Inc., USA) was used to capture the external thermal imaging. The internal temperature of the tumor was measured using a thermometer with a 0.5 m diameter temperature probe (GuangZhou Sun-gun Measurement and Control Technology Co. Ltd.). This computer-connected probe was covered by a plastic sheath and inserted 3–4 mm deep into the tumor through a 23G needle. Data were collected every 10 s during the laser irradiation.
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The tumor volume were measured on day 0, 4, 8, 13 and 17 after laser treatment by calipers and expressed in mm3 using the reported formula [23,24]: V = 0.5 a × b2 (where a and b represent the long and short diameters of the tumor, respectively). Body weight was measured at the same time as an indicator of treatmentinduced toxicity. 2.6. Histological evaluation At the end of the experiment on day 17, mice were sacrificed by CO2 inhalation and cervical dislocation. The gross tumor specimens were measured and fixed in 4% formalin. Slices of the tumor blocks were stained with hematoxylin and eosin (H&E) for pathological examination using a Nikon Eclipse E600 microscope. All evaluations were performed by an investigator blinded to experimental group? 2.7. Statistical analysis The tumor measurements are presented as mean ± SEM. The significance of the difference between mean values was analyzed using statistical software (SPSS 16.0, SPSS Inc., Chicago, IL) by assessment of the variance (ANOVA) followed by post hoc Bonferroni/Dunn tests for multiple comparisons. p ≤ 0.05 was defined as significant. 3. Results 3.1. Photo-cytotoxicity of GNS-MCs on BT474 cells To determine whether GNS-MCs can serve as an optical absorber for NIR wavelengths, we treated breast cancer BT474 cells with GNS-MCs and then irradiated them with NIR laser. NIR irradiation alone (Fig. 1B) or GNS-MC treatment alone (Fig. 1C) did not induce cell death, as reflected by Calcein-AM/PI double staining. In contrast, NIR laser irradiation resulted in significant cell death in the GNS-MC-treated BT474 cells. More importantly, cell death only occurred in the NIR irradiated area (Fig. 1D). These data suggest that BT474 cell death was mediated by NIR light induced photothermal cytotoxicity. In addition, the NIR-mediated cell death was dependent on the dose of GNS-MCs (Fig. 1E), with a concentration of 0.3 mg/mL showing the maximal toxic effect (p = 0.004). Further increases of GNS-MCs to 0.5 mg/mL did not exert additional toxicity to BT474 cells. 3.2. Ultrasound contrast-guided injection of GNS-MCs in the tumors A breast cancer model was established by inoculation of BT474 tumor cells into the right mammary fat pad and GNS-MCs were
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injected into the tumors under ultrasound imaging guidance. As shown in Fig. 2B, no ultrasonographic enhancement was observed before injection. In contrast, the boundary and size of the tumors can be clearly detected in real-time during (Fig. 2C) and after (Fig. 2D) GNS-MC injection, suggesting that ultrasound imaging can help ensure uniform and complete distribution of GNS-MCs into the tumor area. However, the deployment and repositioning of the needle required conventional gray-scale imaging to identify the needle tip and avoid interference by the hyperechogenic background of the tumor tissue. 3.3. Thermal effect of NIR laser-irradiated GNS-MCs in vivo To investigate the thermal effect of GNS-MCs in response to NIR light, the temperature of the tumors in GNS-MC-treated mice was recorded using a thermographic camera during NIR laser irradiation. As shown in Fig. 3A, tumor temperature increased to nearly 60 ◦ C within 2 min and this temperature lasted for about 8 min during the irradiation. Such treatment was long enough to kill the tumor cells. The spatial distribution of temperatures in the tumors and adjacent regions, as detected by a thermal imaging camera, showed that high temperatures only occurred within tumor boundaries, whereas the temperature in surrounding regions was significantly lower (Fig. 3C). In contrast, saline-treated mice failed to show a temperature increase after NIR laser irradiation (Fig. 3A and B). Thermographic cameras can only detect the surface temperature of tumors. To measure the temperature inside the tumors, a thermometer was inserted in the center of the tumors under ultrasound guidance. As shown in Fig. 3D, similar temperature profiles as the thermographic camera were detected by the thermometer. However, the highest temperature of the GNS-MC+laser treated group reached almost 70 ◦ C, approximately 10 ◦ C higher than that measured by the thermographic camera. One reason for this discrepancy could be that the heat produced by the GNS-MC-mediated laser irradiation was trapped inside the tumors and could not diffuse to the surrounding tissue, resulting in higher internal tumor temperatures. 3.4. Efficacy of GNS-MCs-mediated PTT of breast cancer The long-term therapeutic effect of GNS-MCs on tumor viability was tested in a mouse model of breast cancer after NIR irradiation. As shown in Fig. 4A-a–c, the size of the tumors was significantly increased in control mice treated with saline or NIR irradiation or GNS-MCs alone (Fig. 4A-a–c). In contrast, the tumor was completely ablated in mice treated with both GNS-MCs and NIR irradiation (Fig. 4A-d) at 17 days after irradiation. Quantitative
Fig. 1. Photo-cytotoxicity of GNS-MCs on BT474 Cells. (A–D) Fluorescent images of Calcein-AM (green) and PI (red) staining in BT474 cells treated with GNS-MCs and laser irradiation. (A) Saline; (B) saline with laser irradiation; (C) GNS-MCs; (D) GNS-MCs with laser irradiation; (E) cell viability as measured by the MTT assay. Data are shown as mean ± SD, n = 3; **p < 0.01 versus no-laser control.
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Fig. 2. Ultrasound imaging-guided injection of GNS-MCs. (A) Grayscale ultrasound image of tumor. (B–D) CPS and B-mode image of the tumor before, during and after injection of GNS-MCs. The GNS-MC-enhanced areas within the tumors can be seen on the CPS images (arrowheads).
Fig. 3. Temperature profile of tumors after GNS-MC treatment and NIR laser irradiation. (A) Temperature profile of tumors as measured by thermographic camera after laser irradiation in saline or GNS-MC-treated mice. (B) and (C) Representative thermographs of tumors after laser irradiation in saline or GNS-MC-treated mice. Images were taken at a time point of 2 min during NIR laser irradiation. The black dotted circles show the tumor areas. (D) Temperature profiles obtained from the thermometer inserted in the tumors after laser irradiation in saline or GNS-MCs-treated mice.
measurements (Fig. 4B) indicated that tumor volume in the control groups increased from 400 mm3 to approximately 900 mm3 . Strikingly, combined treatment of GNS-MCs and NIR irradiation decreased tumor size gradually and ablated the tumor completely within 17 days after treatment. The body weights of the mice showed no significant difference between groups (Fig. 4C), suggesting that GNS-MCs mediated PTT do not elicit overt systemic toxicity. To further investigate the NIR-mediated photothermal effect of GNS-MCs on tumors at histopathological level, H&E staining was performed. Viable tumor tissues were observed in the control groups (Fig. 5A–C). Combined treatment of GNS-MCs and NIR irradiation (Fig. 5D) resulted in tremendous cellular damage, including karyorrhexis and karyolysis, bleeding and necrosis, as well as the formation of scar tissue, indicating irreversible thermal damage. Notably, no tumor cells whatsoever were found in six out of seven mice. However, cancer cells were found in a small region in one of the PTT-treated mice (Fig. 5E). Possible reasons for this discrepancy may be insufficient laser irradiation and/or no or less GNS-MCs
in that region. Multiple-spot injections of optical absorber and/or longer laser exposure times may be needed for total ablation of tumors and warrant further exploration. 4. Discussion A novel optical absorber, GNS-MCs, with ultrasound enhancing capability was developed in our lab and demonstrated effectiveness for photothermal ablation of breast cancer in a mouse model. The dual functional GNS-MCs were directly injected into the breast tumor under the guidance of contrast-enhanced imaging, which allowed precise delivery and uniform distribution of GNS-MCs throughout the tumor. This gold nanoshelled particle showed potent thermal effects upon NIR irradiation, increasing the local temperature to 60–70 ◦ C for 10 min during irradiation. Importantly, thermal effects were only localized within GNS-MC-injected tumor tissues and no thermal effect was observed in normal tissue surrounding the tumors (Fig. 3). In vitro studies revealed that death of BT474 cells occurred only in NIR-irradiated areas, further
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Fig. 4. Tumor size and body weights after laser irradiation in GNS-MC-treated mice. (A) Representative photographs of tumors in mice treated with saline (a), laser irradiation (b), GNS-MCs (c) and GNS-MCs+laser irradiation (d). The left mice and right mice in each group were photographed before and 17 days after the treatment, respectively. Note that the tumors in GNS-MCs-treated mice are completely ablated 17 days after NIR laser irradiation. (B) Quantitative measurement of tumor volumes after treatments. Data are shown as mean ± SD, *p < 0.05, **p < 0.01 versus saline or irradiation or GNS-MCs treatment. (C) Body weights at the indicated time points after treatment.
supporting the specificity of NIR-initiated thermal effects (Fig. 1). In addition, GNS-MC particles can distribute homogenously and widely within tumors under ultrasound-guided intratumoral injection, which may further contribute to the therapeutic effects observed in our animal model of breast cancer. In this study, a 27G needle was used for injection of GNS-MCs into a superficial target (i.e., breast tumor). This is considered a minimally-invasive procedure compared to RF thermal ablation, as the latter procedure requires 14 or 17G electrodes. The use of contrast ultrasound imaging for guiding the injection can ensure that the GNS-MC only be distributed within the tumor parenchyma and not in the dermis tissue, to avoid any superficial tissue damage. Importantly, GNS-MCs alone showed no obvious systemic toxicity and only exerted cytotoxicity after NIR irradiation. This represents an enormous advantage over traditional chemotherapeutic drugs. There are several components and challenges for the successful application of PTT. First, GNS-MCs must be biocompatible. The
microcapsule agent used in this study is fabricated from poly (lactic acid), which has outstanding biocompatibility and biodegradability. Furthermore, gold has been used in medicine for many years. Our in vitro experiments demonstrated that GNS-MCs alone is not toxic, even at high concentrations (0.5 mg/mL). In vivo experiments further confirmed that GNS-MCs did not exert systemic toxicity. Second, the depth of light penetration in the targeted tissues must be considered. It has been reported that NIR light can penetrate deeper than visible light into tissue [25], making photothermal therapy of breast cancer possible. The third challenge is the delivery approach for the gold nanoshells. The present study demonstrated that direct intratumoral injection is feasible and effective in delivering sufficient gold nanoshells for successful thermal ablation of tumors. Systemically administered nanoparticles can enter tumors passively by passing through the fenestrations of the angiogenic tumor vasculature [26]. However, the anatomy of the fenestration can
Fig. 5. H&E staining of tumors. (A–E) Image of H&E staining under low power (1×, top panel) and high power magnification (20×, bottom panel). Note that viable tumor tissues were seen (blue square) in the slice from mice treated with saline (A) or laser irradiation (B) or GNS-MCs (C). Catastrophic cellular damage was observed in tumors treated with GNS-MCs+laser (D). Viable cancer cells were found in a small region in one of the GNS-MCs+laser-treated mice (E).
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affect the accumulation of nanoparticles, particularly for earlystage tumors that exhibit less neovascularity with fenestrations. This may limit the effectiveness of photothermal therapy after systemic delivery of nanoparticles. Furthermore, systemic administration may cause systemic toxicity or other side effects. In the present study, we have adapted direct intratumoral injection along with contrast ultrasound imaging guidance for effective PTT of breast cancer. Thus, intratumoral injection of the optical absorber for PTT may be a viable alternative for superficially localized cancers, such as breast cancer. Local intratumoral injection can decrease the total dose of photothermal mediator, avoid exposure of adjacent normal tissues to the absorber, and ensure sufficient therapeutic concentrations of the agent within the tumors for PTT. The present study demonstrated the feasibility and effectiveness of photothermal ablation of the superficial tumor by intratumoral delivery of the optical absorber under contrast-enhanced ultrasound imaging. Besides their potent thermal effects, the oscillation of GNS-MCs within the acoustic field renders them strong non-linear enhancers for ultrasound imaging [27]. This advantage allows real-time monitoring of the injection and the distribution of GNS-MCs within the tumors. However, conventional grayscale imaging was still essential for localization of tumors and guidance of the GNS-MC injection, especially for larger and irregular-shape tumors that require multiple injections. There are several limitations in the present investigation. First, this is a proof-of-concept study with limited numbers of animals and short observation timepoints. The dosage and temporal profile of laser irradiation are based on previous studies. Further studies need to optimize the parameters for both the laser and GNS-MC particles. To further evaluate the efficacy of this technique, future studies needs to include larger sample sizes and longer timepoints. Second, although we have demonstrated the feasibility of the technique, the underlying principles and mechanism of the PTT with GNS-MCs need to be further investigated. Third, this method may only target local breast tumors. As breast cancer can metastasize early, this technique may only be effective only in patients without metastasis. Future studies will need to investigate whether systemically administered gold nanoshelled microcapsules can be directionally/specifically delivered to tumors so that both local and distant metastasized cancer can be ablated. Fourth, the only index of systemic toxic side effects is body weight, which is insufficient for a definitive conclusion on safety and toxicity. Finally, intratumoral injection of GNS-MC with surface laser irradiation may not be adequate for deeply-located tumors. In the latter case, interstitial light delivery may be necessary. Nevertheless, intratumoral injection of GNS-MC provides many advantages over tissue-dependent and less-controllable RF ablation. 5. Conclusions This study demonstrated that GNS-MCs act as both an ultrasound contrast agent and an optical absorber for the effective PTT of breast cancer in a mouse tumor model. Ultrasound guided intratumoral injection of GNS-MCs is a feasible method for optical absorber-mediated PTT, as demonstrated by complete ablation of the tumors in 6 out of 7 mice inoculated with BT474 breast cancer. Thus, this study supports the hypothesis that ultrasound-guided PTT with GNS-MCs is a promising technique for the treatment of breast cancer. Conflict of interest There is no conflict of interest or commercial involvement by any of the authors.
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European Journal of Radiology journal homepage: www.elsevier.com/locate/ejrad
Contrast ultrasound-guided photothermal therapy using gold nanoshelled microcapsules in breast cancer Shumin Wang a,b , Zhifei Dai c , Hengte Ke d , Enze Qu a , Xiaoxu Qi e , Kuo Zhang e , Jinrui Wang a,∗ a
Department of Ultrasonography, Peking University Third Hospital, Beijing 100083, China Ordos Center Hospital, Ordos, Inner Mongolia 017000, China c Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China d Nanomedicine and Biosensor Laboratory, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China e Department of Laboratory Animal Science, Peking University Health Science Center, Beijing 100019, China b
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Article history: Received 6 May 2013 Received in revised form 10 August 2013 Accepted 9 September 2013 Keywords: Phototherapy Ultrasonography Breast neoplasms Nanoparticles Laser therapy Thermal ablation Ultrasound guidance
a b s t r a c t Objectives: The purpose of this study was to test whether dual functional gold nano-shelled microcapsules (GNS-MCs) can be used as an ultrasound imaging enhancer and as an optical absorber for photothermal therapy (PTT) in a rodent model of breast cancer. Methods: GNS-MCs were fabricated with an inner air and outer gold nanoshell spherical structure. Photothermal cytotoxicity of GNS-MCs was tested with BT474 cancer cells in vitro and non-obese diabetes-SCID (NOD/SCID) mice with breast cancer. GNS-MCs were injected into the tumor under ultrasound guidance and treated with near-infrared (NIR) laser irradiation. The photothermal ablative effectiveness of GNSMCs was evaluated by measuring the surface and internal temperature of the tumor as well as the size of the tumor using histological confirmation. Results: NIR laser irradiation resulted in significant tumor cell death in GNS-MCs-treated BT474 cells in vitro. GNS-MCs were able to serve as an ultrasound enhancer to guide the intratumoral injection of GNS-MCs and ensure their uniform distribution. In vivo studies revealed that NIR laser irradiation increased the intratumoral temperature to nearly 70 ◦ C for 8 min in GNS-MCs-treated mice. Tumor volumes decreased gradually and tumors were completely ablated in 6 out of 7 mice treated with GNS-MCs and laser irradiation by 17 days after treatment. Conclusion: This study demonstrates that ultrasound-guided PTT with theranostic GNS-MCs is a promising technique for in situ treatment of breast cancer. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Early diagnosis of breast cancer with mammography screening leads to an increased demand for minimally invasive and nonsurgical treatment of patients with small breast cancers [1,2]. Breast-conserving surgical therapy is therefore the treatment of choice for women with relatively small breast cancers [3]. Percutaneous tumor thermal ablation provides an effective alternative to open breast surgery because the breast is a superficial structure without intervening organs. Various techniques of thermal-based ablative therapy, including radiofrequency, microwave, highintensity focused ultrasound, and lasers have been applied for the local treatment of breast cancer [4–7]. Radio frequency ablation
∗ Corresponding author at: Department of Ultrasonography, Peking University Third Hospital, No. 49 Garden Road, Haidian District, Beijing 100083, China. Tel.: +86 010 8226 4486; fax: +86 010 82265851. E-mail address: jinrui [email protected] (J. Wang). 0720-048X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejrad.2013.09.010
(RFA) is considered to be a promising non-surgical technique for the treatment of small breast cancers [8]. However, conventional RFA can cause thermal injury to surrounding normal tissue and requires the insertion of a 14 or 17 gauge electrode probe into the lesion. Furthermore, tissue-dependent electrical characteristics of RFA may cause non-homogeneous deposition of heat, leading to incomplete ablation of tumor cells [9]. Therefore, it is imperative to develop more effective and less invasive treatments for breast cancer. Optical absorbance-mediated NIR laser therapy has the potential to precisely deliver energy to a targeted site by transcutaneous and interstitial approaches, allowing for less or non-invasive local destruction of the cancer in situ [10]. Light in the NIR region (650–900 nm) has minimal human tissue absorption and can penetrate into deep tissue to produce cytotoxic thermal effects, making NIR light an attractive light source for thermal ablation [11]. PTT can induce temperature elevations above 55 ◦ C at the light irradiating site in a very short time and instantaneously cause significant tumor ablation. Nanomaterial-mediated delivery of optical
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absorbers has been investigated and shown promising results for thermal ablation of the tumor using NIR light [12]. Conventional NIR photoabsorbers such as indocyanine Green are susceptible to chemical and thermal denaturation and photobleaching, making NIR light-mediated thermal ablation less efficient [10]. In contrast, gold nanostructures exhibit excellent optical properties and have been investigated as photo-absorbers for NIR laserbased PTT [13–16]. The rigid structure and noble metal surface of gold nanoshells make these particles a much more robust optical absorber than conventional NIR dyes [10,17]. Photothermal therapy against cancer is a viable alternative to surgery, showing both noninvasive and tumor-specific characters. However, for direct clinical translation, there is a pressing need to combine an imaging technique to assess tumor location, size, and internal structures with the ability to detect the distribution of photoabsorbers in the tumor before therapy as well as assess tumor damage after therapy. An appropriate imaging modality is therefore an important aspect for guiding local PTT. A number of imaging techniques such as MRI [18] and SPECT/CT imaging-guided PTT [19] have been used for guiding PTT. However, ultrasound imaging shows a number of advantages over other imaging modalities, including its real-time nature, lack of ionizing radiation, greater availability and portability, ease of use, and far lower cost. More importantly, ultrasound imaging can provide instant feedback and results during thermal ablation procedures [20]. Thus, the combination of ultrasound imaging guidance with PTT could provide a cost-effective, convenient, and non-invasive approach with both diagnostic and therapeutic functions for the treatment of cancer. A dual functioning agent, gold nano-shelled microcapsules (GNS-MCs) with inner air-filled hollow domains, was developed in our lab to be used as an ultrasound enhancer and optical absorber [21]. The purpose of this study was to test whether dual functional GNS-MCs can be used as a contrast agent for ultrasound imaging and, simultaneously, as a photothermal ablative device for treatment of breast cancer in a NOD/SCID mouse model. 2. Materials and methods 2.1. Synthesis and characterization of GNS-MCs Polylactide microcapsules were prepared by the water-inoil-in-water double emulsion method. Citrate-stabilized gold nanoparticles were then deposited on the surface of polylactide microcapsules as seeds with the help of poly (allylamine hydrochloride). Gold nano-shells were formed through the surface seeding method via reduction of tetra-chloroaurate (III) by hydroxylamine hydrochloride. The encapsulated water in the inner aqueous phase of the microcapsules was sublimated, leaving a tiny hollow space with air inside and becoming a nonlinear reflector for enhancing ultrasound imaging. The size of GNS-MCs was determined by static laser scattering to be 2.32 ± 1.07 m. The GNS-MCs showed strong absorption of light at the wavelength of 700–900 nm [21]. 2.2. Photo-cytotoxicity of GNS-MCs in BT474 cells Human breast tumor BT474 cells were seeded in 12-well plates (1 × 106 /well) and incubated for 24 h. GNS-MC (1 mg/mL) was added into cells for 1 h and illuminated with a fiber-coupled diode NIR laser at a wavelength of 808 nm and power intensity of 8 W/cm2 for 10 min (T808F2W, Xi’an Minghui Optoelectronic Technology, China). Cell viability was measured using double-fluorescent staining of calcein-AM (staining live cells) and propidium iodide (PI, staining dead cells) at 24 h after laser irradiation. The tumor cells treated with saline, saline with laser irradiation, and GNS-MCs each
served as a control. Dose-efficacy of GNS-MCs in eliciting BT474 cell death was further analyzed by the MTT assay at the following concentrations: 0, 0.025, 0.05, 0.1, 0.3 and 0.5 mg/mL. 2.3. Animal model of human breast cancer All animal procedures were approved by the Institutional Animal Care and Use Committee at the Institute of Biophysics of Peking University. The 8 week-old female NOD/SCID mice (18–22 g, Institute of Laboratory Animal Science of Chinese Academy of Medical Science, China) were implanted with estrogen pellets (17-estradiol, Innovative Research of America, USA) in the right flank one day before tumor inoculation. Each mouse was inoculated in the right mammary fat pad with BT474 tumor cells (1 × 107 ) in 0.1 mL of PBS with Matrigel (1:1). Twenty-eight NOD/SCID mice with BT474 tumor were randomly divided into four groups (n = 7 per group): saline only, laser only, GNS-MCs only and GNS-MCs+laser. Photothermal treatment of the tumor was performed when the tumor size reached approximately 400 mm3 around 30 days post implantation. 2.4. Ultrasound contrast-guided injection of GNS-MCs In order to ablate the entire tumor, it is critical to make sure that the GNS-MCs infiltrate the entire tumor mass. Because air-filled GNS-MCs can act as an ultrasound contrast agent, we tested whether ultrasound imaging would be able to properly guide the injection of GNS-M Cs and monitor their distribution. With an ultrasound unit with a 10 MHz transducer (Siemens S2000, Siemens Healthcare, WA), contrast ultrasound imaging with cadence-contrast pulse sequence (CPS) technology at a mechanical index of 0.28–0.42 was used to guide and monitor the injection of GNS-MCs into the tumor of the mouse. The CPS imaging mode is a unique pulse sequence processing technology especially designed for contrast microbubbles and has the ability to combine the nonlinear fundamental and higher order harmonic signals to form a highly specific and sensitive contrast agent display in real time. CPS imaging gives the ability to selectively view “tissue only, contrast agent only, or both together” [22]. This imaging mode was chosen for this study because its remarkable differentiation of tissue and GNS-MC signals. GNS-MCs (200 L, 10 mg/mL suspension) or saline injections of the tumor were made with a 27G needle. During the injection, CPS contrast imaging was used to guide the needle position and to display the enhancement of GNS-MCs. The area with no or low GNS-MC enhancement was re-injected under ultrasound guidance to make more uniform distribution of the GNS-MCs throughout the tumor. 2.5. GNS-MCs mediated PTT in vivo The margins of the GNS-MC area of distribution in the tumor was determined by ultrasound imaging and outlined with a marker. The tumors were irradiated with NIR light (808 nm diode laser) at a surface power of 1.3 W/cm2 for 10 min. For the irradiation of the tumor, the laser fiber was set at a fixed distance of 4.0 cm from the tumor to ensure equivalent energy intensity covering the entire tumor area. During the NIR irradiation, an infrared thermographic camera (A40, FLIR Systems Inc., USA) was used to capture the external thermal imaging. The internal temperature of the tumor was measured using a thermometer with a 0.5 m diameter temperature probe (GuangZhou Sun-gun Measurement and Control Technology Co. Ltd.). This computer-connected probe was covered by a plastic sheath and inserted 3–4 mm deep into the tumor through a 23G needle. Data were collected every 10 s during the laser irradiation.
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The tumor volume were measured on day 0, 4, 8, 13 and 17 after laser treatment by calipers and expressed in mm3 using the reported formula [23,24]: V = 0.5 a × b2 (where a and b represent the long and short diameters of the tumor, respectively). Body weight was measured at the same time as an indicator of treatmentinduced toxicity. 2.6. Histological evaluation At the end of the experiment on day 17, mice were sacrificed by CO2 inhalation and cervical dislocation. The gross tumor specimens were measured and fixed in 4% formalin. Slices of the tumor blocks were stained with hematoxylin and eosin (H&E) for pathological examination using a Nikon Eclipse E600 microscope. All evaluations were performed by an investigator blinded to experimental group? 2.7. Statistical analysis The tumor measurements are presented as mean ± SEM. The significance of the difference between mean values was analyzed using statistical software (SPSS 16.0, SPSS Inc., Chicago, IL) by assessment of the variance (ANOVA) followed by post hoc Bonferroni/Dunn tests for multiple comparisons. p ≤ 0.05 was defined as significant. 3. Results 3.1. Photo-cytotoxicity of GNS-MCs on BT474 cells To determine whether GNS-MCs can serve as an optical absorber for NIR wavelengths, we treated breast cancer BT474 cells with GNS-MCs and then irradiated them with NIR laser. NIR irradiation alone (Fig. 1B) or GNS-MC treatment alone (Fig. 1C) did not induce cell death, as reflected by Calcein-AM/PI double staining. In contrast, NIR laser irradiation resulted in significant cell death in the GNS-MC-treated BT474 cells. More importantly, cell death only occurred in the NIR irradiated area (Fig. 1D). These data suggest that BT474 cell death was mediated by NIR light induced photothermal cytotoxicity. In addition, the NIR-mediated cell death was dependent on the dose of GNS-MCs (Fig. 1E), with a concentration of 0.3 mg/mL showing the maximal toxic effect (p = 0.004). Further increases of GNS-MCs to 0.5 mg/mL did not exert additional toxicity to BT474 cells. 3.2. Ultrasound contrast-guided injection of GNS-MCs in the tumors A breast cancer model was established by inoculation of BT474 tumor cells into the right mammary fat pad and GNS-MCs were
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injected into the tumors under ultrasound imaging guidance. As shown in Fig. 2B, no ultrasonographic enhancement was observed before injection. In contrast, the boundary and size of the tumors can be clearly detected in real-time during (Fig. 2C) and after (Fig. 2D) GNS-MC injection, suggesting that ultrasound imaging can help ensure uniform and complete distribution of GNS-MCs into the tumor area. However, the deployment and repositioning of the needle required conventional gray-scale imaging to identify the needle tip and avoid interference by the hyperechogenic background of the tumor tissue. 3.3. Thermal effect of NIR laser-irradiated GNS-MCs in vivo To investigate the thermal effect of GNS-MCs in response to NIR light, the temperature of the tumors in GNS-MC-treated mice was recorded using a thermographic camera during NIR laser irradiation. As shown in Fig. 3A, tumor temperature increased to nearly 60 ◦ C within 2 min and this temperature lasted for about 8 min during the irradiation. Such treatment was long enough to kill the tumor cells. The spatial distribution of temperatures in the tumors and adjacent regions, as detected by a thermal imaging camera, showed that high temperatures only occurred within tumor boundaries, whereas the temperature in surrounding regions was significantly lower (Fig. 3C). In contrast, saline-treated mice failed to show a temperature increase after NIR laser irradiation (Fig. 3A and B). Thermographic cameras can only detect the surface temperature of tumors. To measure the temperature inside the tumors, a thermometer was inserted in the center of the tumors under ultrasound guidance. As shown in Fig. 3D, similar temperature profiles as the thermographic camera were detected by the thermometer. However, the highest temperature of the GNS-MC+laser treated group reached almost 70 ◦ C, approximately 10 ◦ C higher than that measured by the thermographic camera. One reason for this discrepancy could be that the heat produced by the GNS-MC-mediated laser irradiation was trapped inside the tumors and could not diffuse to the surrounding tissue, resulting in higher internal tumor temperatures. 3.4. Efficacy of GNS-MCs-mediated PTT of breast cancer The long-term therapeutic effect of GNS-MCs on tumor viability was tested in a mouse model of breast cancer after NIR irradiation. As shown in Fig. 4A-a–c, the size of the tumors was significantly increased in control mice treated with saline or NIR irradiation or GNS-MCs alone (Fig. 4A-a–c). In contrast, the tumor was completely ablated in mice treated with both GNS-MCs and NIR irradiation (Fig. 4A-d) at 17 days after irradiation. Quantitative
Fig. 1. Photo-cytotoxicity of GNS-MCs on BT474 Cells. (A–D) Fluorescent images of Calcein-AM (green) and PI (red) staining in BT474 cells treated with GNS-MCs and laser irradiation. (A) Saline; (B) saline with laser irradiation; (C) GNS-MCs; (D) GNS-MCs with laser irradiation; (E) cell viability as measured by the MTT assay. Data are shown as mean ± SD, n = 3; **p < 0.01 versus no-laser control.
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Fig. 2. Ultrasound imaging-guided injection of GNS-MCs. (A) Grayscale ultrasound image of tumor. (B–D) CPS and B-mode image of the tumor before, during and after injection of GNS-MCs. The GNS-MC-enhanced areas within the tumors can be seen on the CPS images (arrowheads).
Fig. 3. Temperature profile of tumors after GNS-MC treatment and NIR laser irradiation. (A) Temperature profile of tumors as measured by thermographic camera after laser irradiation in saline or GNS-MC-treated mice. (B) and (C) Representative thermographs of tumors after laser irradiation in saline or GNS-MC-treated mice. Images were taken at a time point of 2 min during NIR laser irradiation. The black dotted circles show the tumor areas. (D) Temperature profiles obtained from the thermometer inserted in the tumors after laser irradiation in saline or GNS-MCs-treated mice.
measurements (Fig. 4B) indicated that tumor volume in the control groups increased from 400 mm3 to approximately 900 mm3 . Strikingly, combined treatment of GNS-MCs and NIR irradiation decreased tumor size gradually and ablated the tumor completely within 17 days after treatment. The body weights of the mice showed no significant difference between groups (Fig. 4C), suggesting that GNS-MCs mediated PTT do not elicit overt systemic toxicity. To further investigate the NIR-mediated photothermal effect of GNS-MCs on tumors at histopathological level, H&E staining was performed. Viable tumor tissues were observed in the control groups (Fig. 5A–C). Combined treatment of GNS-MCs and NIR irradiation (Fig. 5D) resulted in tremendous cellular damage, including karyorrhexis and karyolysis, bleeding and necrosis, as well as the formation of scar tissue, indicating irreversible thermal damage. Notably, no tumor cells whatsoever were found in six out of seven mice. However, cancer cells were found in a small region in one of the PTT-treated mice (Fig. 5E). Possible reasons for this discrepancy may be insufficient laser irradiation and/or no or less GNS-MCs
in that region. Multiple-spot injections of optical absorber and/or longer laser exposure times may be needed for total ablation of tumors and warrant further exploration. 4. Discussion A novel optical absorber, GNS-MCs, with ultrasound enhancing capability was developed in our lab and demonstrated effectiveness for photothermal ablation of breast cancer in a mouse model. The dual functional GNS-MCs were directly injected into the breast tumor under the guidance of contrast-enhanced imaging, which allowed precise delivery and uniform distribution of GNS-MCs throughout the tumor. This gold nanoshelled particle showed potent thermal effects upon NIR irradiation, increasing the local temperature to 60–70 ◦ C for 10 min during irradiation. Importantly, thermal effects were only localized within GNS-MC-injected tumor tissues and no thermal effect was observed in normal tissue surrounding the tumors (Fig. 3). In vitro studies revealed that death of BT474 cells occurred only in NIR-irradiated areas, further
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Fig. 4. Tumor size and body weights after laser irradiation in GNS-MC-treated mice. (A) Representative photographs of tumors in mice treated with saline (a), laser irradiation (b), GNS-MCs (c) and GNS-MCs+laser irradiation (d). The left mice and right mice in each group were photographed before and 17 days after the treatment, respectively. Note that the tumors in GNS-MCs-treated mice are completely ablated 17 days after NIR laser irradiation. (B) Quantitative measurement of tumor volumes after treatments. Data are shown as mean ± SD, *p < 0.05, **p < 0.01 versus saline or irradiation or GNS-MCs treatment. (C) Body weights at the indicated time points after treatment.
supporting the specificity of NIR-initiated thermal effects (Fig. 1). In addition, GNS-MC particles can distribute homogenously and widely within tumors under ultrasound-guided intratumoral injection, which may further contribute to the therapeutic effects observed in our animal model of breast cancer. In this study, a 27G needle was used for injection of GNS-MCs into a superficial target (i.e., breast tumor). This is considered a minimally-invasive procedure compared to RF thermal ablation, as the latter procedure requires 14 or 17G electrodes. The use of contrast ultrasound imaging for guiding the injection can ensure that the GNS-MC only be distributed within the tumor parenchyma and not in the dermis tissue, to avoid any superficial tissue damage. Importantly, GNS-MCs alone showed no obvious systemic toxicity and only exerted cytotoxicity after NIR irradiation. This represents an enormous advantage over traditional chemotherapeutic drugs. There are several components and challenges for the successful application of PTT. First, GNS-MCs must be biocompatible. The
microcapsule agent used in this study is fabricated from poly (lactic acid), which has outstanding biocompatibility and biodegradability. Furthermore, gold has been used in medicine for many years. Our in vitro experiments demonstrated that GNS-MCs alone is not toxic, even at high concentrations (0.5 mg/mL). In vivo experiments further confirmed that GNS-MCs did not exert systemic toxicity. Second, the depth of light penetration in the targeted tissues must be considered. It has been reported that NIR light can penetrate deeper than visible light into tissue [25], making photothermal therapy of breast cancer possible. The third challenge is the delivery approach for the gold nanoshells. The present study demonstrated that direct intratumoral injection is feasible and effective in delivering sufficient gold nanoshells for successful thermal ablation of tumors. Systemically administered nanoparticles can enter tumors passively by passing through the fenestrations of the angiogenic tumor vasculature [26]. However, the anatomy of the fenestration can
Fig. 5. H&E staining of tumors. (A–E) Image of H&E staining under low power (1×, top panel) and high power magnification (20×, bottom panel). Note that viable tumor tissues were seen (blue square) in the slice from mice treated with saline (A) or laser irradiation (B) or GNS-MCs (C). Catastrophic cellular damage was observed in tumors treated with GNS-MCs+laser (D). Viable cancer cells were found in a small region in one of the GNS-MCs+laser-treated mice (E).
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affect the accumulation of nanoparticles, particularly for earlystage tumors that exhibit less neovascularity with fenestrations. This may limit the effectiveness of photothermal therapy after systemic delivery of nanoparticles. Furthermore, systemic administration may cause systemic toxicity or other side effects. In the present study, we have adapted direct intratumoral injection along with contrast ultrasound imaging guidance for effective PTT of breast cancer. Thus, intratumoral injection of the optical absorber for PTT may be a viable alternative for superficially localized cancers, such as breast cancer. Local intratumoral injection can decrease the total dose of photothermal mediator, avoid exposure of adjacent normal tissues to the absorber, and ensure sufficient therapeutic concentrations of the agent within the tumors for PTT. The present study demonstrated the feasibility and effectiveness of photothermal ablation of the superficial tumor by intratumoral delivery of the optical absorber under contrast-enhanced ultrasound imaging. Besides their potent thermal effects, the oscillation of GNS-MCs within the acoustic field renders them strong non-linear enhancers for ultrasound imaging [27]. This advantage allows real-time monitoring of the injection and the distribution of GNS-MCs within the tumors. However, conventional grayscale imaging was still essential for localization of tumors and guidance of the GNS-MC injection, especially for larger and irregular-shape tumors that require multiple injections. There are several limitations in the present investigation. First, this is a proof-of-concept study with limited numbers of animals and short observation timepoints. The dosage and temporal profile of laser irradiation are based on previous studies. Further studies need to optimize the parameters for both the laser and GNS-MC particles. To further evaluate the efficacy of this technique, future studies needs to include larger sample sizes and longer timepoints. Second, although we have demonstrated the feasibility of the technique, the underlying principles and mechanism of the PTT with GNS-MCs need to be further investigated. Third, this method may only target local breast tumors. As breast cancer can metastasize early, this technique may only be effective only in patients without metastasis. Future studies will need to investigate whether systemically administered gold nanoshelled microcapsules can be directionally/specifically delivered to tumors so that both local and distant metastasized cancer can be ablated. Fourth, the only index of systemic toxic side effects is body weight, which is insufficient for a definitive conclusion on safety and toxicity. Finally, intratumoral injection of GNS-MC with surface laser irradiation may not be adequate for deeply-located tumors. In the latter case, interstitial light delivery may be necessary. Nevertheless, intratumoral injection of GNS-MC provides many advantages over tissue-dependent and less-controllable RF ablation. 5. Conclusions This study demonstrated that GNS-MCs act as both an ultrasound contrast agent and an optical absorber for the effective PTT of breast cancer in a mouse tumor model. Ultrasound guided intratumoral injection of GNS-MCs is a feasible method for optical absorber-mediated PTT, as demonstrated by complete ablation of the tumors in 6 out of 7 mice inoculated with BT474 breast cancer. Thus, this study supports the hypothesis that ultrasound-guided PTT with GNS-MCs is a promising technique for the treatment of breast cancer. Conflict of interest There is no conflict of interest or commercial involvement by any of the authors.
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