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hollow mesoporous silica shell particles as a nanoplatform for multimode imaging and photothermal therapy of tumors
Accepted Manuscript Polydopamine-Coated Gold Core/Hollow Mesoporous Silica Shell Particles as a Nanoplatform for Multimode Imaging and Photothermal Therapy of Tumors Chao Cai, Xin Li, Yue Wang, Mengxue Liu, Xiangyang Shi, Jindong Xia, Mingwu Shen PII: DOI: Reference:
S1385-8947(19)30080-4 https://doi.org/10.1016/j.cej.2019.01.072 CEJ 20793
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
31 October 2018 22 December 2018 12 January 2019
Please cite this article as: C. Cai, X. Li, Y. Wang, M. Liu, X. Shi, J. Xia, M. Shen, Polydopamine-Coated Gold Core/ Hollow Mesoporous Silica Shell Particles as a Nanoplatform for Multimode Imaging and Photothermal Therapy of Tumors, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.01.072
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Polydopamine-Coated Gold Core/Hollow Mesoporous Silica Shell Particles as a Nanoplatform for Multimode Imaging and Photothermal Therapy of Tumors
Chao Caia, Xin Lia, Yue Wangb, Mengxue Liua, Xiangyang Shia*, Jindong Xiab*, Mingwu Shena*
a
State Key Laboratory for Modification of Chemical Fiber and Polymer Materials, College of
Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China b
Department of Radiology, Shanghai Songjiang District Central Hospital, Shanghai 201600,
People’s Republic of China
________________________________________________________ * To whom correspondence should be addressed. Tel: +86 21 67792656; fax: +86 21 67792306 804. E-mail addresses: [email protected] (X. Shi), [email protected] (J. Xia) and [email protected] (M. Shen).
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Abstract It is highly desirable to develop a new hybrid nanoplatform that integrates diagnosis and treatment elements for effective theranostics of tumors. Herein, we have skillfully designed a nanoplatform of polydopamine (PDA)-coated and perfluorohexane (PFH)-filled gold core/hollow mesoporous silica shell (Au@mSiO2-PFH-PDA, ASPP for short) particles for photoacoustic (PA)/ultrasound (US)/computed tomography (CT)/thermal imaging and photothermal therapy (PTT) of tumors. In this work, we first synthesized Au seed particles with a diameter of 15.8 nm using a sodium citrate reduction method, and coated Au seeds with polyvinylpyrrolidone for further growth of solid silica shell/mesoporous silica shell onto the Au seeds. After treatment via selective etching to remove solid silica shell, amination of surface of the particles, and filling of PFH into the internal cavity of the spheres with a diameter of 182.1 nm, PDA coating was performed to render the particles with an external shell thickness of 15.1 nm. The formed hybrid particles with a size of 212.2 nm
are
colloidally stable
and
exhibit
good
cytocompatibility,
and
display
excellent
PA/US/CT/thermal imaging property due to the co-presence of PDA, PFH, and Au nanoparticles. Furthermore, the PDA coating renders the platform with a photothermal conversion efficiency of 61.2%, enabling effective photothermal ablation of cancer cells in vitro and a xenografted 4T1 tumor model in vivo under irradiation with an 808 nm laser. More importantly, in the primary 4T1 tumor model, intratumoral injection of the ASPP and irradiation with an 808 nm laser can also completely inhibit the occurrence lung metastasis induced by the 4T1 tumor. The as-prepared hybrid nanoplatform may hold a great promise to be adopted for multimode imaging and PTT of tumors and inhibition of tumor metastasis.
Keywords: Hollow mesoporous silica; Polydopamine NPs; Surface modification; Multimode imaging; Photothermal therapy; Tumor metastasis inhibition
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1. Introduction The precision medicine applications require the nanoplatform to be integrated with both imaging and therapy functions [1, 2]. In general, cancer is difficult to be completely cured once it is not diagnosed in an early stage. This is because cancer cells in the advanced stage usually enter into the blood circulation system to induce metastasis in particular tissues or organs, leading to the cancer recurrence even after treatments [3-5]. Therefore, development of multifunctional theranostic nanoplatforms for accurate diagnosis and efficient treatment as well as inhibition of cancer metastasis is crucial. Currently, there are many different biomedical imaging technologies that have been employed for cancer diagnosis, including magnetic resonance [6, 7], single photon emission computed tomography [8, 9], computed tomography (CT) [10, 11], ultrasound (US) [12, 13], photoacoustic (PA) imaging [14, 15], and thermal imaging [16, 17]. However, each imaging mode has its own advantages and drawbacks. Integrating multiple imaging elements into a single nanosystem to realize dual mode or multimode imaging enables the integration of their merits, making diagnostic results more accurate than single-modality imaging. To date, a wide variety of multimodal imaging agents have been developed to improve the sensitivity and accuracy of cancer diagnosis. For instance, Lei et al. [18] developed polyethylenimine-protected AgBiS2 nanodots for dual-modal CT/PA imaging in vivo. Wang et al. [19] synthesized a heat shock protein-modified and gadolinium-loaded polyethylenimine-entrapped Au nanoparticles (NPs) that can be used for targeted CT/MR dual mode imaging of orthotopic tumors. For tumor treatment, chemotherapy or radiotherapy remains the common option to eliminate the residual cancer cells and prevent the spreading of cancer cells after surgery in clinical manipulation [20, 21]. However, both chemotherapy and radiotherapy treatments lack specificity and generate an adverse effect on normal tissues. In recent years, photothermal therapy (PTT) has been emerging due to its advantages of mild pain, negligible side effects, and benefit to inhibit cancer metastasis [22-25]. The principle of PTT relies on the use of photothermal agents that usually have a strong absorption 3
in the near infrared (NIR) region to convert laser light to thermal energy, causing local ablation of tumors. The commonly reported photothermal agents are gold (Au) nanostars (NSs) [26, 27], MoS2 nanosheets [28], polydopamine (PDA) [29, 30] and CuS superstructures [31]. Among them, PDA has been widely adopted for effective PTT of tumors due to its good biocompatibility and photothermal stability. For instance, in a recent work, Liu et al. [32] prepared PDA-melanin colloidal nanospheres as an efficient PTT agent for cancer therapy in vivo. In another work, Li et al [33] prepared polyethylenimine-stabilized Au NSs that were post-coated with a thin shell of PDA for enhanced PTT of tumors due to the co-presence of Au NSs and PDA. Therefore, PDA has been considered as one of the most promising photothermal agents. In the mean time, most of the PTT agents having an NIR absorption feature also possess the property for PA imaging. Hence, PDA polymer can be used for PA imaging-guided PTT of tumors. In a previous work, we have reported a facile method to prepare hollow mesoporous silica NPs surface decorated with Au NSs and interior filled with perfluorohexane (PFH) for multimode CT/PA/US imaging and PTT of tumors [34]. Furthermore, the Au NS-decorated mesoporous silica hollow spheres can be surface modified with arginine-glycine-aspartic acid (RGD) peptide via a polyethylene glycol spacer and encapsulated with anticancer drug doxorubicin for combination chemotherapy and PTT of tumors in vivo [35]. The major advantages of hollow mesoporous silica NPs lie on the fact that the platform displays a high surface area, allows for facile surface decoration and functionalization, and enables loading of drug or contrast agents within the internal cavity. With the versatility of hollow mesoporous silica NPs and PDA, it is reasonable to hypothesize that new multifunctional nanoplatforms incorporating different components can be developed and used as a “all-in-one” theranostic agent for multimode imaging-guided PTT of tumors. In this work, we designed PDA-coated and PFH-filled gold core/hollow mesoporous silica shell nanoplatform with uniform size and low toxicity for multimodality imaging-guided PTT of tumors and inhibition of the occurrence of tumor metastasis (Scheme 1). Au seed particles with a diameter of 4
15.8 nm were first synthesized using a sodium citrate reduction method, and coated with polyvinylpyrrolidone (PVP) for further growth of solid silica shell/mesoporous silica outer shell. After selective etching to remove the solid silica shell and amination of the particle surfaces, PFH was filled into the internal cavity of the spheres, followed by PDA coating to generate the PFH-filled Au@mSiO2 NPs coated with PDA (for short, Au@mSiO2-PFH-PDA, ASPP). The formed ASPP NPs were systematically characterized using different technologies to validate their structure, composition, morphology, stability, imaging performance, photothermal property, and cytotoxicity. Then, we explored the use of ASPP NPs for in vivo multimode PA/US/CT/thermal imaging and PTT of tumors. In the materials design, the Au cores will be used for CT imaging, while the PDA shell and the filled PFH will be used for PA and US imaging, respectively. Meanwhile, the PDA coating [36] not only improves the colloidal stability and biocompatibility of the nanoplatform, but also afford effective photothermal imaging and PTT of tumors to inhibit the occurrence of tumor metastasis. To our knowledge, this is the first report related to the design of PDA-coated Au core/hollow mesoporous silica shell NPs as a platform for tumor theranostics.
Scheme 1. Schematic illustration of the synthesis of ASPP NPs for multimodal imaging-guided PTT of tumors and for inhibition of lung metastasis occurrence. 5
2. Experimental 2.1. Preparation and surface amination of Au core/hollow mesoporous silica shell nanoparticles Au core/hollow mesoporous silica shell nanoparticles (Au@mSiO2 NPs) were prepared according to a selective etching method [37]. To render the Au@mSiO2 NPs with primary amine groups, the Au@mSiO2 NPs (100 mg) were dispersed in 50 mL of ethanol and 300 µL of APTES was added into above solution under stirring at 50 oC for 6 h. The product was centrifuged (3000 rpm, 5 min), washed with water via repeated centrifugation/water redispersion cycles for 5 times, and lyophilized to get the Au@mSiO2-NH2 NPs. 2.2. Preparation of polydopamine (PDA)-coated Au@mSiO2 NPs filled with PFH PFH was physically filled within the Au@mSiO2-NH2 NPs according to the literature [34]. PDA was coated on the surface of Au@mSiO2-PFH NPs according to a protocol reported in our previous work [33]. In brief, Au@mSiO2-PFH NPs (50 mg) were dispersed in 50 mL of tris buffer (pH 8.5), then dopamine (150 mg) was rapidly added and dissolved into the above solution under stirring for 12 h. After the self-polymerization, the Au@mSiO2-PFH NPs were coated with a thin PDA shell (Au@mSiO2-PFH-PDA, for short, ASPP). See full experimental details in Supporting Information.
3. Results and Discussion 3.1. Synthesis and Characterization of the ASPP NPs According to our materials design (Scheme 1), we first synthesized the core/shell/shell structure of Au@SiO2@mSiO2 NPs, and etched the middle shell of solid silica according to a literature protocol [37] to form Au core hollow mesoporous silica shell NPs (Au@mSiO2 NPs). The Au@mSiO2 NPs were then silanized by APTES to render their surface with primary amines according to the literature [38]. Then the amine-modified Au@mSiO2-NH2 were filled with PFH in the cavity and coated with PDA to form the ASPP NPs. For comparison, we also synthesized PFH-free Au@mSiO2-PDA NPs for easy characterization. By monitoring the solution color, we show that Au seed NPs display a wine red color, the formation of Au@mSiO2 NPs weakens the wine red 6
color, and the PDA coating renders the particles with a dark color (Fig. S1, Supporting Information). The formed Au@SiO2@mSiO2, Au@mSiO2 and ASPP NPs were observed by transmission electron microscopy (TEM). As shown in Fig. 1a, the pristine Au@SiO2@mSiO2 NPs display an Au core size of 15.8 2.9 nm, solid silica shell and mesoporous shell structure with a clear boundary, and a whole size of 186.2 11.5 nm. After etching the solid silica shell, the Au@mSiO2 NPs show a hollow interior with a shell thickness of 22.9 2.2 nm (Fig. 1b). Futher coating of PDA induces the increase of shell thickness of 15.1 1.3 nm (Fig. 1c). Meanwhile, the morphology of Au@SiO2@mSiO2, Au@mSiO2 and ASPP NPs was also observed by scanning electron microscopy (SEM, Fig. S2, Supporting Information), and the synthesized NPs well maintain a regular spherical morphology after different modifications.
Fig. 1. TEM images and diameter distribution histograms of (a) Au seed, (b)Au@SiO2@mSiO2, (c) Au@mSiO2 and (d) ASPP NPs. (e) FTIR spectra of Au@mSiO2 and Au@mSiO2-NH2 NPs. (f) TGA curves of Au@mSiO2, Au@mSiO2-NH2 and Au@mSiO2-PDA NPs. (g) UV-vis spectra of Au seed, 7
Au@SiO2@mSiO2, Au@mSiO2, Au@mSiO2-PDA and ASPP NPs.
FTIR spectra were used to characterize the structure of the Au@mSiO2-NH2 NPs (Fig. 1d). Compared to the Au@mSiO2 NPs, the peaks at 1470 and 2843 cm-1 are associated with the N-H bending and C-H vibrations, respectively, validating the success of the silanization of the NPs by APTES. To quantify the loading of APTES and PDA, TGA analysis was performed (Fig. 1e). When compared to Au@mSiO2 NPs, the weight loss of Au@mSiO2-NH2 and Au@mSiO2-NH2-PDA were estimated to be 13.2% and 46.8%, respectively. By comparison with the weight loss of Au@mSiO2 NPs and Au@mSiO2-NH2 NPs, the loading percentage of APTES and PDA can be estimated to be 13.2% and 33.6%, respectively. The optical property of the Au@mSiO2-PDA NPs was investigated by UV-vis spectrometry (Fig. 1f). In contrast to the Au seed (523 nm), Au@SiO2@mSiO2 (530 nm) and Au@mSiO2 (533 nm) NPs that have a strong absorption in the wavelength range of 500-530 nm, which is associated to the surface plasmon peak of Au NPs, the Au@mSiO2-PDA NPs display a strong wide-band of NIR absorption (600-1000 nm), likely due to the PDA coating. Further loading of PFH within the Au@mSiO2-PDA NPs does not seem to alter significantly the optical property of the particles. The strong NIR absorption feature of the Au@mSiO2-PDA NPs ensures their uses for PA imaging and PTT of cancer cells and tumors (see below). N2 adsorption-desorption technique was used to characterize the structural changes from Au@mSiO2 to Au@mSiO2-NH2 (Fig. S3, Supporting Information). After APTES modification, the BET surface area increases from 175 to 205 m2/g, while the pore volume and average pore size decrease from 0.346 to 0.302 cm3/g and from 4.7 to 3.9 nm, respectively. The surface potential of
8
Au@mSiO2 NPs was negative (-9.8±0.22 mV), while that of the Au@mSiO2-NH2 NPs reverses to be positive (24.4±0.56 mV, Fig. S4, Supporting Information), further elaborating the success of the silanization reaction. After further PDA coating on the surface of Au@mSiO2-NH2 NPs, the surface potential of the Au@mSiO2-PDA reverses back to be negative (-2.6±0.06 mV), which is beneficial for their reduced cytotoxicity (see below). Next, the filling of PFH within the cavity of Au@mSiO2-NH2 for US imaging was proven by looking at the digital photos of the solution picture (Fig. S5). PFH is a volatile organic liquid and was loaded in excess via physical entrapment. Under concentration-based diffusion and the ultrasonic force, PFH can be filled into the cavity of the hollow mesoporous silica shells according to literature protocols [34]. There is a clear phase separation between pure PFH and water, while Au@mSiO2-NH2-PFH NPs do not present the phase separation after shaking and standing for 5 min. It illustrates that PFH is successfully filled into the internal cavity and the PDA coating may be used to prevent PFH leakage and improve the stability of NPs. To test the colloidal stability of the ASPP NPs dispersed in phosphate buffered saline (PBS), we monitored their hydrodynamic size change for over 10 days (Fig. S6, Supporting Information). Clearly, there is no significant change in their hydrodynamic size, validating their desirable colloidal stability. 3.2. PA/US/CT/Thermal Imaging and Photothermal Property of the ASPP NPs Due to the strong NIR absorption feature, the PA imaging performance of the ASPP NPs was first studied. The PA imaging and corresponding PA signal intensity at different Au concentrations was demonstrated. Obviously, as the Au concentration increases, the PA signal intensity increases and the brightness of the PA images enhances (Fig. 2a). Meanwhile, there is a linear correlation between PA value and the Au concentration of ASPP NPs (Fig. 2a1). In addition, the US imaging ability of the PFH-filled ASPP NPs was next tested. B-mode images and the corresponding gray value of ASPP NPs are shown in Fig. 2b. The brightness of B-mode images and the gray value (Fig. 2b1) of the ASPP NP gradually increase with the Au concentration of the ASPP NPs, confirming the 9
potential to use the ASPP NPs for US imaging. Further, we also demonstrated the CT imaging property of the ASPP (Fig. 2c). With the Au concentration, the CT images of the ASPP NPs become brighter and brighter, and the X-ray attenuation intensity gradually increases (Fig. 2c1), demonstrating the potential to use the ASPP for CT imaging applications. We next used the thermal camera to validate the thermal imaging ability of the ASPP NPs after NIR laser irradiation at different time points (Fig. 2d). Clearly, with the laser irradiation time, the thermal images of the ASPP NPs get brighter and brighter for both Au concentrations tested, and the thermal images of the ASPP NPs are much brighter at Au concentration of 48 µg/mL than at Au concentration of 24 µg/mL at the same lase irradtion time points, which corresponds well with the change of ASPP solution temperature as a function of laser irradiation time (Fig. 2d1). Taken together, these results indicate that the ASPP NPs have the potential to be applied for multimodal PA/US/CT/thermal imaging. We further investigated the photothermal conversion properties of the ASPP NPs. The temperature changes of water and ASPP aqueous solutions at different Au concentrations (6-48 µg/mL) after laser irradiation was investigated (Fig. 2e). The temperature changes (ΔTs) of the ASPP solution are Au concentration-dependent, and at the Au concentration of 6, 12, 24 and 48 µg/mL, the ΔTs are 12.6, 15.8, 24.2 and 34.2 °C, respectively (Fig. S7a, Supporting Information). In contrast, the temperature of pure water does not change significantly under the same experimental conditions, similar to our previous study [34]. Based on the results of Fig. 2f and 2g, the photothermal conversion efficiency (η) of the ASPP NPs was calculated to be 61.2%, which is much higher than that of PDA-melanin colloidal nanospheres (40.0%) reported in the literature [32]. The higher η of the ASPP NPs is likely attributed to the higher surface area of PDA coated onto hollow mesoporous silica shell particles than that of PDA after hybridized with melanin colloidal nanospheres. In addition, ASPP NPs also show excellent photothermal stability after four cycles of laser on-off to have a temperature change from 30.3 to 45.6 °C (Fig. S7b), suggesting that ASPP NPs may hold a 10
great promise as an effective tumor PTT agent.
Fig. 2. (a) PA images and (a1) the PA values of the ASPP NPs at different Au concentrations. (b) US images and (b1) the corresponding gray values of water and ASPP NPs at different Au concentrations under B mode. (c) CT phantom images and (c1) X-ray attenuation intensity of the ASPP NPs at different Au concentrations. (d) Thermal images and (d1) the corresponding temperature change profiles of ASPP NPs at different Au concentrations after NIR laser irradiation (808 nm, 1.0 W/cm2) for 5 min. (e) Temperature change of water and the ASPP aqueous solution at varying concentrations under the 808 nm laser irradiation (1.0 W/cm2) for 5 min. (f) Temperature change of the ASPP 11
aqueous solution irradiated by an 808 nm laser (1.0 W/cm2) for 300 s and the laser was turned off for 450 s. (g) Plot of the cooling time vs –lnθ.
3.3. Cytotoxicity and Cellular Uptake Assays We used 4T1 cells to test the cytotoxicity of ASPP NPs by Cell Counting Kit-8 (CCK-8) cell viability assay (Fig. 3a). Clearly, the viability of 4T1 cells exceeds 90% in all tested Au concentrations (0-32 µg/ml), indicating that the designed ASPP NPs possess quite good cytocompatibility in the studied concentration range. Further, the endocytosis of the ASPP NPs by 4T1 cells was also tested. As shown in Fig. S8 (Supporting Information), within a certain range of Au concentration (0-4.8 µg/ml), the Au uptake by 4T1 cells increases with the Au concentration, which may be ascribed to the mechanisms of phagocytosis and diffusion via cell walls, in agreement with the literature [34]. 3.4. Photothermal Therapy of Cancer Cells in Vitro The high photothermal conversion efficiency and low cytotoxicity of ASPP NPs prompted us to assess the potential to use them for PTT of cancer cells in vitro. As shown in Fig. 3b, the cell viability was significantly reduced in the Au concentration range of 0-16 µg/mL under the laser irradiation for 5 min when compared to cells treated with the same concentrations of ASPP NPs without laser showing viability over 90% (p < 0.001). In particular, at the Au concentration of 16 µg/mL, the cell viability can be reduced to as low as 23.3% after 5 min laser irradiation. Furthermore, Calcein-AM/PI staining was carried out to qualitatively evaluate the photothermal ablation effect of 4T1 cells (Fig. 3c). With the increase of Au concentration of the ASPP NPs, laser irradiation induces more and more dead cells (red) under the same laser focular area. In contrast, cells treated with PBS are pretty healthy under the same experimental conditions. It should be noted that PTT of cancer cells may influence the extracellular matrix, and this could be an interesting point deserving further study. 12
Fig. 3. (a) CCK-8 viability assay of 4T1 cells treated with ASPP NPs of different concentrations for 24 h. (b) Cell viability of 4T1 cells treated with ASPP NPs for 6 h and irradiated with an 808 nm laser (1.0 W/cm2) for 5 min or without laser irradiation (0 min). (c) Fluorescence microscopy images of 4T1 cells stained with calcein AM (green, live cells) and PI (red, dead cells) after incubated with PBS and the ASPP NPs with different Au concentrations, followed by an 808 nm laser irradiation (1.0 W/cm2) for 5 min.
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Fig. 4. (a) In vivo PA images and (a1) the corresponding PA values; (b) In vivo US imaging in B mode and (b1) the corresponding gray values; and (c) colored CT images and (c1) the CT values of tumor region. For PA/US/CT imaging, the images were collected before and at different time points post intravenous injection of ASPP NPs ([Au] = 8 mg/mL, in 100 µL PBS for each mouse). (d) In vivo thermal images of 4T1 tumor-bearing mice intratumorally injected with PBS (100 µL) and ASPP NPs ([Au] = 48 µg/mL, in 100 µL PBS) for each mouse, respectively, then irradiated with an 808 nm laser (1.0 W/cm2) and the images were collected at 0 and 5 min, respectively. (d1) The temperature profiles of tumor region after injection of PBS or ASPP NPs as a function of the laser irradiation time.
3.5. In Vivo PA/US/CT/Thermal Imaging of Tumors Then the ASPP NPs were used as a contrast agent for multimode PA/US/CT/thermal imaging of 14
a xenografted tumor model (Fig. 4). For PA imaging (Fig. 4a), the PA signal intensity of the tumor region increases from 0.165 to 0.285 after intravenous injection of ASPP and the maximum PA signal intensity at a peak time of 60 min postinjection is 1.7 times higher than the initial value (Fig. 4a1). For US imaging in B mode (Fig. 4b), the injection of ASPP NPs affords the enhanced contrast in the tumor region at different time points postinjection. At the peak time of 60 min postinjection, the US singal intensity is 5.5 times higher than the intial value (Fig. 4b1). For CT imaging (Fig. 4c and Fig. S9), the CT value increases with the time postinjection and reaches the peak value of 59.97 HU at 60 min postinjection, which is 1.5 times higher than the intial tumor CT value (Fig. 4c1). The peak time for all imaging modes is the same (60 min), hence the ASPP NPs need 60 min to reach the highest concentration in the tumor region. After 60 min, the ASPP NPs start to be slowly metabolized, and hence having a gradually decreased signal intensity. We then used the ASPP NPs for tumor photothermal imaging after intratumoral injection (Fig. 4d). The temperature of the tumor treated with PBS does not have appreciable changes under laser irradiation. In sharp contrast, the temperature of tumor region treated with the ASPP NPs increases rapidly to 60 °C after 5 min laser irradiation (Fig. 4d1). These results suggest that the developed ASPP hold a great promise as an effective PA/US/CT/thermal imaging agent for tumor diagnosis in vivo. 3.6. Photothermal Therapy of Tumors in Vivo To assess the potential of ASPP for PTT of tumors in vivo, nude mice bearing 4T1 tumors were divided into four treatment groups (PBS, PBS+L, ASPP and ASPP+L) to assess the tumor treatment efficacy (Fig. 5). Clearly, the tumors in PBS, PBS+L and ASPP groups keep growing rapidly at a similar speed, while the tumors in the ASPP+L group are able to be ablated after 2 days (Fig. 5a, Fig. S10, Supporting Information). The scar of the tumor region after laser ablation in the ASPP+L group can be completely healed on the fifteen day, validating the excellent tumor PTT efficacy. In addition, the body weight of the mice in the four groups gradually increases (Fig. 5b), indicating that different 15
treatments do not cause potential toxicity to the mice. The tumor-bearing mice treated with the ASPP+L have a survival rate of 100% after 30 days, while the mice in the other three treatment groups are all dead on the day 30 (Fig. 5c). It is worth noting that the application of ASPP NPs is also extremely effective to avoid the recurrence of lung metastasis induced by breast tumor (Fig. 5d). The 4T1 tumors treated by PBS are able to induces multiple metastatic nodules in the lungs, and the treatments of PBS+L and ASPP have no effect on the formation of lung metastases, similar to the PBS group. Importantly, the treatment of ASPP+L is able to effectively inhibit the occurrence of lung metastasis. This was further confirmed by H&E staining of the lung sections (Fig. 5e). The tumor ablation effect after different treatments was also evaluated by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining (Fig. S11, Supporting Information). Obviously, a large number of positive staining of apoptotic cells can be observed in tumor sections of ASPP+L group, while the other three groups have almost no apoptotic cells. Quantitative analysis of TUNEL-stained tumor cells shows that the apoptosis rates of the groups of PBS, PBS+L, ASPP, and ASPP+L are 0.64%, 0.43%, 0.99% and 89.7%, respectively (Fig. S12). To explore the biodistribution of ASPP NPs in various organs in vivo, ICP-OES was used to quantify the Au content in the heart, liver, spleen, lung, kidney and tumor at 1, 24 and 96 h post intravenous injection of ASPP NPs (Fig. S13, Supporting Information). Clearly, the main organs taking Au elements are the lung, liver, and spleen. Among them, lung has the highest uptake of Au element at 1 h post injection of the ASPP. Finally, the ASPP can be gradually metabolized from all the organs and tumor with the time postinjection. It is interesting to note that at 1 h postinjection, Au has the highest tumor uptake among the studied time points, thus enabling effective multimode imaging. In order to evaluate the long-term toxicity and side effect of ASPP NPs in vivo, the primary organs of the mice were removed and H&E stained at 30th day after different treatments. As shown in Fig. S14 (Supporting Information), the main organs of mice in the four groups do not display any 16
obvious morphological changes, inflammatory infiltration and necrosis after different treatments, validating the good biocompatibility of the developed ASPP NPs.
Fig. 5. (a) The relative tumor volume, (b) body weight, and (c) survival rate of the 4T1 tumor-bearing mice after different treatments. (d) Lung images extracted from 4T1 tumor-bearing mice after different treatments. The lung images of PBS group, PBS+L group, ASPP group and ASPP+L group were obtained on the 21st, 22nd, 22nd and 30th day, respectively. (e) H&E staining of the same lung tissues (in (d)) from 4T1 tumor-bearing mice after different treatments.
4. Conclusion Altogether, we have presented a unique nanoplatform based on PFH-filled and PDA-coated Au core/hollow mesoporous silica shell particles for theranostics of tumors. Through the judicious design, multiple imaging and therapeutic components of Au core particles, PFH, and PDA are able to be incorporated within one single platform with a size of 212.2 nm to have a nice colloidal stability, biocompatibility, NIR absorption feature, and a high photothermal conversion efficiency (61.2%) 17
under an 808 nm laser irradiation. These properties enable the use of the developed ASPP NPs for multimode PA/US/CT/thermal imaging and PTT of tumors. More importantly, the ASPP-mediated PTT of tumors enables the complete elimination of solid tumors without damaging normal tissues and inhibition of the occurrence of lung metastasis. The developed ASPP NPs may be used as a promising theranostic nanoplatform for multimode imaging and efficient PTT of other tumor types.
Acknowledgements This study was financially supported by the Science and Technology Commission of Shanghai Municipality (17540712000 and 15520711400), the National Nature Science Foundation of China (21773026 and 81761148028), and the Fundamental Research Funds for the Central Universities (For M. Shen and X. Shi). Dr. Chen Peng was acknowledged for coordinating the experiment of PA imaging.
Author Contributions M. S. and X. S. designed the idea to perform the study. C. C., X. L. and M. L. carried out all materials synthesis, characterization and cell experiments. C. C., Y. W. and J. X. established tumor models and preformed animal experiments. C. C. and X. L. analyzed all data and collected all figures. C. C., X. L., M. S. and X. S. co-wrote the paper. All authors discussed and commented the paper and results.
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Highlights
Au@mSiO2-PFH-PDA NPs can be synthesized via a judicious material design.
PDA coating renders the platform with a photothermal conversion efficiency of 61.2%.
The hybrid particles enables multiple PA/US/CT/thermal imaging of tumors.
The hybrid particles can completely eliminate the solid tumors under laser irradiation.
The ASPP-mediated PTT enables inhibition of the occurrence of lung metastasis.
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S1385-8947(19)30080-4 https://doi.org/10.1016/j.cej.2019.01.072 CEJ 20793
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
31 October 2018 22 December 2018 12 January 2019
Please cite this article as: C. Cai, X. Li, Y. Wang, M. Liu, X. Shi, J. Xia, M. Shen, Polydopamine-Coated Gold Core/ Hollow Mesoporous Silica Shell Particles as a Nanoplatform for Multimode Imaging and Photothermal Therapy of Tumors, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.01.072
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Polydopamine-Coated Gold Core/Hollow Mesoporous Silica Shell Particles as a Nanoplatform for Multimode Imaging and Photothermal Therapy of Tumors
Chao Caia, Xin Lia, Yue Wangb, Mengxue Liua, Xiangyang Shia*, Jindong Xiab*, Mingwu Shena*
a
State Key Laboratory for Modification of Chemical Fiber and Polymer Materials, College of
Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China b
Department of Radiology, Shanghai Songjiang District Central Hospital, Shanghai 201600,
People’s Republic of China
________________________________________________________ * To whom correspondence should be addressed. Tel: +86 21 67792656; fax: +86 21 67792306 804. E-mail addresses: [email protected] (X. Shi), [email protected] (J. Xia) and [email protected] (M. Shen).
1
Abstract It is highly desirable to develop a new hybrid nanoplatform that integrates diagnosis and treatment elements for effective theranostics of tumors. Herein, we have skillfully designed a nanoplatform of polydopamine (PDA)-coated and perfluorohexane (PFH)-filled gold core/hollow mesoporous silica shell (Au@mSiO2-PFH-PDA, ASPP for short) particles for photoacoustic (PA)/ultrasound (US)/computed tomography (CT)/thermal imaging and photothermal therapy (PTT) of tumors. In this work, we first synthesized Au seed particles with a diameter of 15.8 nm using a sodium citrate reduction method, and coated Au seeds with polyvinylpyrrolidone for further growth of solid silica shell/mesoporous silica shell onto the Au seeds. After treatment via selective etching to remove solid silica shell, amination of surface of the particles, and filling of PFH into the internal cavity of the spheres with a diameter of 182.1 nm, PDA coating was performed to render the particles with an external shell thickness of 15.1 nm. The formed hybrid particles with a size of 212.2 nm
are
colloidally stable
and
exhibit
good
cytocompatibility,
and
display
excellent
PA/US/CT/thermal imaging property due to the co-presence of PDA, PFH, and Au nanoparticles. Furthermore, the PDA coating renders the platform with a photothermal conversion efficiency of 61.2%, enabling effective photothermal ablation of cancer cells in vitro and a xenografted 4T1 tumor model in vivo under irradiation with an 808 nm laser. More importantly, in the primary 4T1 tumor model, intratumoral injection of the ASPP and irradiation with an 808 nm laser can also completely inhibit the occurrence lung metastasis induced by the 4T1 tumor. The as-prepared hybrid nanoplatform may hold a great promise to be adopted for multimode imaging and PTT of tumors and inhibition of tumor metastasis.
Keywords: Hollow mesoporous silica; Polydopamine NPs; Surface modification; Multimode imaging; Photothermal therapy; Tumor metastasis inhibition
2
1. Introduction The precision medicine applications require the nanoplatform to be integrated with both imaging and therapy functions [1, 2]. In general, cancer is difficult to be completely cured once it is not diagnosed in an early stage. This is because cancer cells in the advanced stage usually enter into the blood circulation system to induce metastasis in particular tissues or organs, leading to the cancer recurrence even after treatments [3-5]. Therefore, development of multifunctional theranostic nanoplatforms for accurate diagnosis and efficient treatment as well as inhibition of cancer metastasis is crucial. Currently, there are many different biomedical imaging technologies that have been employed for cancer diagnosis, including magnetic resonance [6, 7], single photon emission computed tomography [8, 9], computed tomography (CT) [10, 11], ultrasound (US) [12, 13], photoacoustic (PA) imaging [14, 15], and thermal imaging [16, 17]. However, each imaging mode has its own advantages and drawbacks. Integrating multiple imaging elements into a single nanosystem to realize dual mode or multimode imaging enables the integration of their merits, making diagnostic results more accurate than single-modality imaging. To date, a wide variety of multimodal imaging agents have been developed to improve the sensitivity and accuracy of cancer diagnosis. For instance, Lei et al. [18] developed polyethylenimine-protected AgBiS2 nanodots for dual-modal CT/PA imaging in vivo. Wang et al. [19] synthesized a heat shock protein-modified and gadolinium-loaded polyethylenimine-entrapped Au nanoparticles (NPs) that can be used for targeted CT/MR dual mode imaging of orthotopic tumors. For tumor treatment, chemotherapy or radiotherapy remains the common option to eliminate the residual cancer cells and prevent the spreading of cancer cells after surgery in clinical manipulation [20, 21]. However, both chemotherapy and radiotherapy treatments lack specificity and generate an adverse effect on normal tissues. In recent years, photothermal therapy (PTT) has been emerging due to its advantages of mild pain, negligible side effects, and benefit to inhibit cancer metastasis [22-25]. The principle of PTT relies on the use of photothermal agents that usually have a strong absorption 3
in the near infrared (NIR) region to convert laser light to thermal energy, causing local ablation of tumors. The commonly reported photothermal agents are gold (Au) nanostars (NSs) [26, 27], MoS2 nanosheets [28], polydopamine (PDA) [29, 30] and CuS superstructures [31]. Among them, PDA has been widely adopted for effective PTT of tumors due to its good biocompatibility and photothermal stability. For instance, in a recent work, Liu et al. [32] prepared PDA-melanin colloidal nanospheres as an efficient PTT agent for cancer therapy in vivo. In another work, Li et al [33] prepared polyethylenimine-stabilized Au NSs that were post-coated with a thin shell of PDA for enhanced PTT of tumors due to the co-presence of Au NSs and PDA. Therefore, PDA has been considered as one of the most promising photothermal agents. In the mean time, most of the PTT agents having an NIR absorption feature also possess the property for PA imaging. Hence, PDA polymer can be used for PA imaging-guided PTT of tumors. In a previous work, we have reported a facile method to prepare hollow mesoporous silica NPs surface decorated with Au NSs and interior filled with perfluorohexane (PFH) for multimode CT/PA/US imaging and PTT of tumors [34]. Furthermore, the Au NS-decorated mesoporous silica hollow spheres can be surface modified with arginine-glycine-aspartic acid (RGD) peptide via a polyethylene glycol spacer and encapsulated with anticancer drug doxorubicin for combination chemotherapy and PTT of tumors in vivo [35]. The major advantages of hollow mesoporous silica NPs lie on the fact that the platform displays a high surface area, allows for facile surface decoration and functionalization, and enables loading of drug or contrast agents within the internal cavity. With the versatility of hollow mesoporous silica NPs and PDA, it is reasonable to hypothesize that new multifunctional nanoplatforms incorporating different components can be developed and used as a “all-in-one” theranostic agent for multimode imaging-guided PTT of tumors. In this work, we designed PDA-coated and PFH-filled gold core/hollow mesoporous silica shell nanoplatform with uniform size and low toxicity for multimodality imaging-guided PTT of tumors and inhibition of the occurrence of tumor metastasis (Scheme 1). Au seed particles with a diameter of 4
15.8 nm were first synthesized using a sodium citrate reduction method, and coated with polyvinylpyrrolidone (PVP) for further growth of solid silica shell/mesoporous silica outer shell. After selective etching to remove the solid silica shell and amination of the particle surfaces, PFH was filled into the internal cavity of the spheres, followed by PDA coating to generate the PFH-filled Au@mSiO2 NPs coated with PDA (for short, Au@mSiO2-PFH-PDA, ASPP). The formed ASPP NPs were systematically characterized using different technologies to validate their structure, composition, morphology, stability, imaging performance, photothermal property, and cytotoxicity. Then, we explored the use of ASPP NPs for in vivo multimode PA/US/CT/thermal imaging and PTT of tumors. In the materials design, the Au cores will be used for CT imaging, while the PDA shell and the filled PFH will be used for PA and US imaging, respectively. Meanwhile, the PDA coating [36] not only improves the colloidal stability and biocompatibility of the nanoplatform, but also afford effective photothermal imaging and PTT of tumors to inhibit the occurrence of tumor metastasis. To our knowledge, this is the first report related to the design of PDA-coated Au core/hollow mesoporous silica shell NPs as a platform for tumor theranostics.
Scheme 1. Schematic illustration of the synthesis of ASPP NPs for multimodal imaging-guided PTT of tumors and for inhibition of lung metastasis occurrence. 5
2. Experimental 2.1. Preparation and surface amination of Au core/hollow mesoporous silica shell nanoparticles Au core/hollow mesoporous silica shell nanoparticles (Au@mSiO2 NPs) were prepared according to a selective etching method [37]. To render the Au@mSiO2 NPs with primary amine groups, the Au@mSiO2 NPs (100 mg) were dispersed in 50 mL of ethanol and 300 µL of APTES was added into above solution under stirring at 50 oC for 6 h. The product was centrifuged (3000 rpm, 5 min), washed with water via repeated centrifugation/water redispersion cycles for 5 times, and lyophilized to get the Au@mSiO2-NH2 NPs. 2.2. Preparation of polydopamine (PDA)-coated Au@mSiO2 NPs filled with PFH PFH was physically filled within the Au@mSiO2-NH2 NPs according to the literature [34]. PDA was coated on the surface of Au@mSiO2-PFH NPs according to a protocol reported in our previous work [33]. In brief, Au@mSiO2-PFH NPs (50 mg) were dispersed in 50 mL of tris buffer (pH 8.5), then dopamine (150 mg) was rapidly added and dissolved into the above solution under stirring for 12 h. After the self-polymerization, the Au@mSiO2-PFH NPs were coated with a thin PDA shell (Au@mSiO2-PFH-PDA, for short, ASPP). See full experimental details in Supporting Information.
3. Results and Discussion 3.1. Synthesis and Characterization of the ASPP NPs According to our materials design (Scheme 1), we first synthesized the core/shell/shell structure of Au@SiO2@mSiO2 NPs, and etched the middle shell of solid silica according to a literature protocol [37] to form Au core hollow mesoporous silica shell NPs (Au@mSiO2 NPs). The Au@mSiO2 NPs were then silanized by APTES to render their surface with primary amines according to the literature [38]. Then the amine-modified Au@mSiO2-NH2 were filled with PFH in the cavity and coated with PDA to form the ASPP NPs. For comparison, we also synthesized PFH-free Au@mSiO2-PDA NPs for easy characterization. By monitoring the solution color, we show that Au seed NPs display a wine red color, the formation of Au@mSiO2 NPs weakens the wine red 6
color, and the PDA coating renders the particles with a dark color (Fig. S1, Supporting Information). The formed Au@SiO2@mSiO2, Au@mSiO2 and ASPP NPs were observed by transmission electron microscopy (TEM). As shown in Fig. 1a, the pristine Au@SiO2@mSiO2 NPs display an Au core size of 15.8 2.9 nm, solid silica shell and mesoporous shell structure with a clear boundary, and a whole size of 186.2 11.5 nm. After etching the solid silica shell, the Au@mSiO2 NPs show a hollow interior with a shell thickness of 22.9 2.2 nm (Fig. 1b). Futher coating of PDA induces the increase of shell thickness of 15.1 1.3 nm (Fig. 1c). Meanwhile, the morphology of Au@SiO2@mSiO2, Au@mSiO2 and ASPP NPs was also observed by scanning electron microscopy (SEM, Fig. S2, Supporting Information), and the synthesized NPs well maintain a regular spherical morphology after different modifications.
Fig. 1. TEM images and diameter distribution histograms of (a) Au seed, (b)Au@SiO2@mSiO2, (c) Au@mSiO2 and (d) ASPP NPs. (e) FTIR spectra of Au@mSiO2 and Au@mSiO2-NH2 NPs. (f) TGA curves of Au@mSiO2, Au@mSiO2-NH2 and Au@mSiO2-PDA NPs. (g) UV-vis spectra of Au seed, 7
Au@SiO2@mSiO2, Au@mSiO2, Au@mSiO2-PDA and ASPP NPs.
FTIR spectra were used to characterize the structure of the Au@mSiO2-NH2 NPs (Fig. 1d). Compared to the Au@mSiO2 NPs, the peaks at 1470 and 2843 cm-1 are associated with the N-H bending and C-H vibrations, respectively, validating the success of the silanization of the NPs by APTES. To quantify the loading of APTES and PDA, TGA analysis was performed (Fig. 1e). When compared to Au@mSiO2 NPs, the weight loss of Au@mSiO2-NH2 and Au@mSiO2-NH2-PDA were estimated to be 13.2% and 46.8%, respectively. By comparison with the weight loss of Au@mSiO2 NPs and Au@mSiO2-NH2 NPs, the loading percentage of APTES and PDA can be estimated to be 13.2% and 33.6%, respectively. The optical property of the Au@mSiO2-PDA NPs was investigated by UV-vis spectrometry (Fig. 1f). In contrast to the Au seed (523 nm), Au@SiO2@mSiO2 (530 nm) and Au@mSiO2 (533 nm) NPs that have a strong absorption in the wavelength range of 500-530 nm, which is associated to the surface plasmon peak of Au NPs, the Au@mSiO2-PDA NPs display a strong wide-band of NIR absorption (600-1000 nm), likely due to the PDA coating. Further loading of PFH within the Au@mSiO2-PDA NPs does not seem to alter significantly the optical property of the particles. The strong NIR absorption feature of the Au@mSiO2-PDA NPs ensures their uses for PA imaging and PTT of cancer cells and tumors (see below). N2 adsorption-desorption technique was used to characterize the structural changes from Au@mSiO2 to Au@mSiO2-NH2 (Fig. S3, Supporting Information). After APTES modification, the BET surface area increases from 175 to 205 m2/g, while the pore volume and average pore size decrease from 0.346 to 0.302 cm3/g and from 4.7 to 3.9 nm, respectively. The surface potential of
8
Au@mSiO2 NPs was negative (-9.8±0.22 mV), while that of the Au@mSiO2-NH2 NPs reverses to be positive (24.4±0.56 mV, Fig. S4, Supporting Information), further elaborating the success of the silanization reaction. After further PDA coating on the surface of Au@mSiO2-NH2 NPs, the surface potential of the Au@mSiO2-PDA reverses back to be negative (-2.6±0.06 mV), which is beneficial for their reduced cytotoxicity (see below). Next, the filling of PFH within the cavity of Au@mSiO2-NH2 for US imaging was proven by looking at the digital photos of the solution picture (Fig. S5). PFH is a volatile organic liquid and was loaded in excess via physical entrapment. Under concentration-based diffusion and the ultrasonic force, PFH can be filled into the cavity of the hollow mesoporous silica shells according to literature protocols [34]. There is a clear phase separation between pure PFH and water, while Au@mSiO2-NH2-PFH NPs do not present the phase separation after shaking and standing for 5 min. It illustrates that PFH is successfully filled into the internal cavity and the PDA coating may be used to prevent PFH leakage and improve the stability of NPs. To test the colloidal stability of the ASPP NPs dispersed in phosphate buffered saline (PBS), we monitored their hydrodynamic size change for over 10 days (Fig. S6, Supporting Information). Clearly, there is no significant change in their hydrodynamic size, validating their desirable colloidal stability. 3.2. PA/US/CT/Thermal Imaging and Photothermal Property of the ASPP NPs Due to the strong NIR absorption feature, the PA imaging performance of the ASPP NPs was first studied. The PA imaging and corresponding PA signal intensity at different Au concentrations was demonstrated. Obviously, as the Au concentration increases, the PA signal intensity increases and the brightness of the PA images enhances (Fig. 2a). Meanwhile, there is a linear correlation between PA value and the Au concentration of ASPP NPs (Fig. 2a1). In addition, the US imaging ability of the PFH-filled ASPP NPs was next tested. B-mode images and the corresponding gray value of ASPP NPs are shown in Fig. 2b. The brightness of B-mode images and the gray value (Fig. 2b1) of the ASPP NP gradually increase with the Au concentration of the ASPP NPs, confirming the 9
potential to use the ASPP NPs for US imaging. Further, we also demonstrated the CT imaging property of the ASPP (Fig. 2c). With the Au concentration, the CT images of the ASPP NPs become brighter and brighter, and the X-ray attenuation intensity gradually increases (Fig. 2c1), demonstrating the potential to use the ASPP for CT imaging applications. We next used the thermal camera to validate the thermal imaging ability of the ASPP NPs after NIR laser irradiation at different time points (Fig. 2d). Clearly, with the laser irradiation time, the thermal images of the ASPP NPs get brighter and brighter for both Au concentrations tested, and the thermal images of the ASPP NPs are much brighter at Au concentration of 48 µg/mL than at Au concentration of 24 µg/mL at the same lase irradtion time points, which corresponds well with the change of ASPP solution temperature as a function of laser irradiation time (Fig. 2d1). Taken together, these results indicate that the ASPP NPs have the potential to be applied for multimodal PA/US/CT/thermal imaging. We further investigated the photothermal conversion properties of the ASPP NPs. The temperature changes of water and ASPP aqueous solutions at different Au concentrations (6-48 µg/mL) after laser irradiation was investigated (Fig. 2e). The temperature changes (ΔTs) of the ASPP solution are Au concentration-dependent, and at the Au concentration of 6, 12, 24 and 48 µg/mL, the ΔTs are 12.6, 15.8, 24.2 and 34.2 °C, respectively (Fig. S7a, Supporting Information). In contrast, the temperature of pure water does not change significantly under the same experimental conditions, similar to our previous study [34]. Based on the results of Fig. 2f and 2g, the photothermal conversion efficiency (η) of the ASPP NPs was calculated to be 61.2%, which is much higher than that of PDA-melanin colloidal nanospheres (40.0%) reported in the literature [32]. The higher η of the ASPP NPs is likely attributed to the higher surface area of PDA coated onto hollow mesoporous silica shell particles than that of PDA after hybridized with melanin colloidal nanospheres. In addition, ASPP NPs also show excellent photothermal stability after four cycles of laser on-off to have a temperature change from 30.3 to 45.6 °C (Fig. S7b), suggesting that ASPP NPs may hold a 10
great promise as an effective tumor PTT agent.
Fig. 2. (a) PA images and (a1) the PA values of the ASPP NPs at different Au concentrations. (b) US images and (b1) the corresponding gray values of water and ASPP NPs at different Au concentrations under B mode. (c) CT phantom images and (c1) X-ray attenuation intensity of the ASPP NPs at different Au concentrations. (d) Thermal images and (d1) the corresponding temperature change profiles of ASPP NPs at different Au concentrations after NIR laser irradiation (808 nm, 1.0 W/cm2) for 5 min. (e) Temperature change of water and the ASPP aqueous solution at varying concentrations under the 808 nm laser irradiation (1.0 W/cm2) for 5 min. (f) Temperature change of the ASPP 11
aqueous solution irradiated by an 808 nm laser (1.0 W/cm2) for 300 s and the laser was turned off for 450 s. (g) Plot of the cooling time vs –lnθ.
3.3. Cytotoxicity and Cellular Uptake Assays We used 4T1 cells to test the cytotoxicity of ASPP NPs by Cell Counting Kit-8 (CCK-8) cell viability assay (Fig. 3a). Clearly, the viability of 4T1 cells exceeds 90% in all tested Au concentrations (0-32 µg/ml), indicating that the designed ASPP NPs possess quite good cytocompatibility in the studied concentration range. Further, the endocytosis of the ASPP NPs by 4T1 cells was also tested. As shown in Fig. S8 (Supporting Information), within a certain range of Au concentration (0-4.8 µg/ml), the Au uptake by 4T1 cells increases with the Au concentration, which may be ascribed to the mechanisms of phagocytosis and diffusion via cell walls, in agreement with the literature [34]. 3.4. Photothermal Therapy of Cancer Cells in Vitro The high photothermal conversion efficiency and low cytotoxicity of ASPP NPs prompted us to assess the potential to use them for PTT of cancer cells in vitro. As shown in Fig. 3b, the cell viability was significantly reduced in the Au concentration range of 0-16 µg/mL under the laser irradiation for 5 min when compared to cells treated with the same concentrations of ASPP NPs without laser showing viability over 90% (p < 0.001). In particular, at the Au concentration of 16 µg/mL, the cell viability can be reduced to as low as 23.3% after 5 min laser irradiation. Furthermore, Calcein-AM/PI staining was carried out to qualitatively evaluate the photothermal ablation effect of 4T1 cells (Fig. 3c). With the increase of Au concentration of the ASPP NPs, laser irradiation induces more and more dead cells (red) under the same laser focular area. In contrast, cells treated with PBS are pretty healthy under the same experimental conditions. It should be noted that PTT of cancer cells may influence the extracellular matrix, and this could be an interesting point deserving further study. 12
Fig. 3. (a) CCK-8 viability assay of 4T1 cells treated with ASPP NPs of different concentrations for 24 h. (b) Cell viability of 4T1 cells treated with ASPP NPs for 6 h and irradiated with an 808 nm laser (1.0 W/cm2) for 5 min or without laser irradiation (0 min). (c) Fluorescence microscopy images of 4T1 cells stained with calcein AM (green, live cells) and PI (red, dead cells) after incubated with PBS and the ASPP NPs with different Au concentrations, followed by an 808 nm laser irradiation (1.0 W/cm2) for 5 min.
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Fig. 4. (a) In vivo PA images and (a1) the corresponding PA values; (b) In vivo US imaging in B mode and (b1) the corresponding gray values; and (c) colored CT images and (c1) the CT values of tumor region. For PA/US/CT imaging, the images were collected before and at different time points post intravenous injection of ASPP NPs ([Au] = 8 mg/mL, in 100 µL PBS for each mouse). (d) In vivo thermal images of 4T1 tumor-bearing mice intratumorally injected with PBS (100 µL) and ASPP NPs ([Au] = 48 µg/mL, in 100 µL PBS) for each mouse, respectively, then irradiated with an 808 nm laser (1.0 W/cm2) and the images were collected at 0 and 5 min, respectively. (d1) The temperature profiles of tumor region after injection of PBS or ASPP NPs as a function of the laser irradiation time.
3.5. In Vivo PA/US/CT/Thermal Imaging of Tumors Then the ASPP NPs were used as a contrast agent for multimode PA/US/CT/thermal imaging of 14
a xenografted tumor model (Fig. 4). For PA imaging (Fig. 4a), the PA signal intensity of the tumor region increases from 0.165 to 0.285 after intravenous injection of ASPP and the maximum PA signal intensity at a peak time of 60 min postinjection is 1.7 times higher than the initial value (Fig. 4a1). For US imaging in B mode (Fig. 4b), the injection of ASPP NPs affords the enhanced contrast in the tumor region at different time points postinjection. At the peak time of 60 min postinjection, the US singal intensity is 5.5 times higher than the intial value (Fig. 4b1). For CT imaging (Fig. 4c and Fig. S9), the CT value increases with the time postinjection and reaches the peak value of 59.97 HU at 60 min postinjection, which is 1.5 times higher than the intial tumor CT value (Fig. 4c1). The peak time for all imaging modes is the same (60 min), hence the ASPP NPs need 60 min to reach the highest concentration in the tumor region. After 60 min, the ASPP NPs start to be slowly metabolized, and hence having a gradually decreased signal intensity. We then used the ASPP NPs for tumor photothermal imaging after intratumoral injection (Fig. 4d). The temperature of the tumor treated with PBS does not have appreciable changes under laser irradiation. In sharp contrast, the temperature of tumor region treated with the ASPP NPs increases rapidly to 60 °C after 5 min laser irradiation (Fig. 4d1). These results suggest that the developed ASPP hold a great promise as an effective PA/US/CT/thermal imaging agent for tumor diagnosis in vivo. 3.6. Photothermal Therapy of Tumors in Vivo To assess the potential of ASPP for PTT of tumors in vivo, nude mice bearing 4T1 tumors were divided into four treatment groups (PBS, PBS+L, ASPP and ASPP+L) to assess the tumor treatment efficacy (Fig. 5). Clearly, the tumors in PBS, PBS+L and ASPP groups keep growing rapidly at a similar speed, while the tumors in the ASPP+L group are able to be ablated after 2 days (Fig. 5a, Fig. S10, Supporting Information). The scar of the tumor region after laser ablation in the ASPP+L group can be completely healed on the fifteen day, validating the excellent tumor PTT efficacy. In addition, the body weight of the mice in the four groups gradually increases (Fig. 5b), indicating that different 15
treatments do not cause potential toxicity to the mice. The tumor-bearing mice treated with the ASPP+L have a survival rate of 100% after 30 days, while the mice in the other three treatment groups are all dead on the day 30 (Fig. 5c). It is worth noting that the application of ASPP NPs is also extremely effective to avoid the recurrence of lung metastasis induced by breast tumor (Fig. 5d). The 4T1 tumors treated by PBS are able to induces multiple metastatic nodules in the lungs, and the treatments of PBS+L and ASPP have no effect on the formation of lung metastases, similar to the PBS group. Importantly, the treatment of ASPP+L is able to effectively inhibit the occurrence of lung metastasis. This was further confirmed by H&E staining of the lung sections (Fig. 5e). The tumor ablation effect after different treatments was also evaluated by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining (Fig. S11, Supporting Information). Obviously, a large number of positive staining of apoptotic cells can be observed in tumor sections of ASPP+L group, while the other three groups have almost no apoptotic cells. Quantitative analysis of TUNEL-stained tumor cells shows that the apoptosis rates of the groups of PBS, PBS+L, ASPP, and ASPP+L are 0.64%, 0.43%, 0.99% and 89.7%, respectively (Fig. S12). To explore the biodistribution of ASPP NPs in various organs in vivo, ICP-OES was used to quantify the Au content in the heart, liver, spleen, lung, kidney and tumor at 1, 24 and 96 h post intravenous injection of ASPP NPs (Fig. S13, Supporting Information). Clearly, the main organs taking Au elements are the lung, liver, and spleen. Among them, lung has the highest uptake of Au element at 1 h post injection of the ASPP. Finally, the ASPP can be gradually metabolized from all the organs and tumor with the time postinjection. It is interesting to note that at 1 h postinjection, Au has the highest tumor uptake among the studied time points, thus enabling effective multimode imaging. In order to evaluate the long-term toxicity and side effect of ASPP NPs in vivo, the primary organs of the mice were removed and H&E stained at 30th day after different treatments. As shown in Fig. S14 (Supporting Information), the main organs of mice in the four groups do not display any 16
obvious morphological changes, inflammatory infiltration and necrosis after different treatments, validating the good biocompatibility of the developed ASPP NPs.
Fig. 5. (a) The relative tumor volume, (b) body weight, and (c) survival rate of the 4T1 tumor-bearing mice after different treatments. (d) Lung images extracted from 4T1 tumor-bearing mice after different treatments. The lung images of PBS group, PBS+L group, ASPP group and ASPP+L group were obtained on the 21st, 22nd, 22nd and 30th day, respectively. (e) H&E staining of the same lung tissues (in (d)) from 4T1 tumor-bearing mice after different treatments.
4. Conclusion Altogether, we have presented a unique nanoplatform based on PFH-filled and PDA-coated Au core/hollow mesoporous silica shell particles for theranostics of tumors. Through the judicious design, multiple imaging and therapeutic components of Au core particles, PFH, and PDA are able to be incorporated within one single platform with a size of 212.2 nm to have a nice colloidal stability, biocompatibility, NIR absorption feature, and a high photothermal conversion efficiency (61.2%) 17
under an 808 nm laser irradiation. These properties enable the use of the developed ASPP NPs for multimode PA/US/CT/thermal imaging and PTT of tumors. More importantly, the ASPP-mediated PTT of tumors enables the complete elimination of solid tumors without damaging normal tissues and inhibition of the occurrence of lung metastasis. The developed ASPP NPs may be used as a promising theranostic nanoplatform for multimode imaging and efficient PTT of other tumor types.
Acknowledgements This study was financially supported by the Science and Technology Commission of Shanghai Municipality (17540712000 and 15520711400), the National Nature Science Foundation of China (21773026 and 81761148028), and the Fundamental Research Funds for the Central Universities (For M. Shen and X. Shi). Dr. Chen Peng was acknowledged for coordinating the experiment of PA imaging.
Author Contributions M. S. and X. S. designed the idea to perform the study. C. C., X. L. and M. L. carried out all materials synthesis, characterization and cell experiments. C. C., Y. W. and J. X. established tumor models and preformed animal experiments. C. C. and X. L. analyzed all data and collected all figures. C. C., X. L., M. S. and X. S. co-wrote the paper. All authors discussed and commented the paper and results.
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Highlights
Au@mSiO2-PFH-PDA NPs can be synthesized via a judicious material design.
PDA coating renders the platform with a photothermal conversion efficiency of 61.2%.
The hybrid particles enables multiple PA/US/CT/thermal imaging of tumors.
The hybrid particles can completely eliminate the solid tumors under laser irradiation.
The ASPP-mediated PTT enables inhibition of the occurrence of lung metastasis.
25