Highly Stable Organic Small Molecular Nanoparticles as an Advanced

Jul 10, 2017 - ... application by design and synthesis of an organic small molecule (namely TPA-T-TQ) with intensive absorption in the NIR window...
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Highly Stable Organic Small Molecular Nanoparticles as an Advanced and Biocompatible Phototheranostic Agent of Tumor in Living Mice Ji Qi,†,∥ Yuan Fang,‡,∥ Ryan T. K. Kwok,† Xiaoyan Zhang,‡ Xianglong Hu,† Jacky W. Y. Lam,† Dan Ding,*,‡ and Ben Zhong Tang*,† †

Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Division of Life Science, State Key Laboratory of Molecular Neuroscience, Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China ‡ State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, China S Supporting Information *

ABSTRACT: Near-infrared (NIR)-absorbing organic small molecules hold great promise as the phototheranostic agents for clinical translation by virtue of their intrinsic advantages such as well-defined chemical structure, high purity, and good reproducibility. However, most of the currently available ones face the challenges in varying degrees in terms of photothermal instability, and photobleaching/reactive oxygen nitrogen species (RONS) inresistance, which indeed impair their practical applications in precise diagnosis and treatment of diseases. Herein, we developed highly stable and biocompatible organic nanoparticles (ONPs) for effective phototheranostic application by design and synthesis of an organic small molecule (namely TPA-T-TQ) with intensive absorption in the NIR window. The TPA-T-TQ ONPs with no noticeable in vivo toxicity possess better capacities in photothermal conversion and photoacoustic imaging (PAI), as well as show far higher stabilities including thermal/photothermal stabilities, and photobleaching/RONS resistances, when compared with the clinically popularly used indocyanine green. Thanks to the combined merits, the ONPs can serve as an efficient probe for in vivo PAI in a high-contrast manner, which also significantly causes the stoppage of tumor growth in living mice through PAI-guided photothermal therapy. This study thus provides an insight into the development of advanced NIR-absorbing small molecules for practical phototheranostic applications. KEYWORDS: NIR-absorbing organic small molecule, probe stability, phototheranostics, photoacoustic imaging, photothermal therapy, in vivo toxicity

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combines high resolution and deep tissue penetration of ultrasound imaging with high contrast of optical imaging.7−9 The therapeutic modality naturally accompanied by PAI agents is PTT, as PAI process needs to detect the photothermally generated ultrasound signal.10−12 The most vital prerequisite of PAI/PTT applications is to employ efficient contrast agents

he emergence of phototheranostic agents has opened a new door for cancer research, which are attracting increasing attention as they concurrently and complementarily integrate real-time diagnosis and in situ phototherapeutic capabilities in one platform.1−5 Among versatile light-triggered diagnostic/therapeutic techniques, photoacoustic imaging (PAI) associated with photothermal therapy (PTT) have received more attention because PAI/PTT can accurately probe tumor location, effectively inhibit tumor growth, and minimally cause side effect to normal tissue.4−6 PAI is a very promising noninvasive molecular imaging approach that © 2017 American Chemical Society

Received: May 3, 2017 Accepted: July 10, 2017 Published: July 10, 2017 7177

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ACS Nano Scheme 1. Synthetic Route to Compound TPA-T-TQ

applications in clinic.37−39 For example, indocyanine green (ICG), an ionic compound with strong absorption in the NIR spectral region of 700−850 nm, has been approved by the Food and Drug Administration (FDA) for clinical use, highlighting the potential of organic small molecules for clinical translation and practical applications.40−42 Nevertheless, these cyanine dyes seriously suffer from the problem of poor stability, which significantly leads to untrusted theranostic outcome. One example is that a lot of cyanine dyes are prone to decomposition by reactive oxygen/nitrogen species (RONS), and thus employed to construct sensitive probes for detecting RONS in living body.43−45 This is because the alternatively arranged single and double bonds in cyanine dyes are easily oxidized by the highly reactive RONS, resulting in the decrease or disappearance of featured NIR absorption and fluorescence signals. On the one hand, this demonstrates the smart utilization of the reactive feature of cyanine dyes for ratiometric sensing of RONS. On the other hand, however, this also uncovers that the instability of cyanine dyes would arise serious problems, such as the misleading PAI signal, impaired PTT efficacy and harmful side effect caused by the in vivo decomposition.14,46 Accordingly, the development of highly stable NIR-absorbing organic small molecular agents for effective PAI/PTT applications is desired but remain to be resolved.

with strong absorption in the near-infrared (NIR) interrogation window (700−900 nm), since NIR light is well accepted to penetrate much deeper tissue, and cause less photodamage to living body.13−15 A variety of nanomaterials, such as metal nanomaterials (e.g., gold, and silver nanostructures), carbon nanomaterials (e.g., carbon nanotubes, and graphene), transition metal dichalcogenides (e.g., MoS2, WS2, and Ag2S) and organic material-based nanoparticles, have been tremendously investigated as PAI/ PTT agents.16−22 In comparison with inorganic nanoagents, organic materials, mainly including polymers and small molecules, own salient advantages of outstanding biocompatibility, potential biodegradability, and easy processability, which thus gain extensive interest.23−26 Recently, semiconducting polymer nanoparticles (SPNs) have been explored as administered contrast agents for PAI as well as PTT applications with superb performance.23,24,27−32 In contrast, the development of organic small molecules applicable for PAI/ PTT fairly falls behind, although they possess some intrinsic merits such as well-defined chemical structure, high purity, good reproducibility, facile modification, and easy processability.33−36 However, one serious problem faced by organic small molecules is the awkward unstability issue, which still harasses the clinically available NIR-absorbing organic dyes. Some conventional cyanine dyes have been investigated and used as the intermediates for light-mediated biomedical 7178

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possess remarkable PAI and PTT efficacies, achieving the stoppage of tumor growth in living mice. What’s more, the in vivo toxic side effect of the ONPs was assessed as well by histological examination and blood tests. To the best of our knowledge, most of the currently available NIR-absorbing organic small molecules face the challenges in varying degrees in terms of photothermal instability, photobleaching, and RONS inresistance. Nevertheless, these stability issues of a phototheranostic agent are indeed crucial for precise diagnosis and treatment of diseases. There are few studies, however, focusing on the aforementioned stabilities of NIRabsorbing organic small molecules, although highly stable semiconducting polymers have been extensively investigated due to the urgent pursuit in the field of precise cancer theranosis. Considering that organic small molecules have intrinsic advantages such as well-defined chemical structure, high purity, and good reproducibility, which make them more accessible for industrial production and clinical translation, this work will provide an insight into the development of advanced NIR-absorbing organic small molecules for disease theranosis.

In this contribution, we developed highly stable and biocompatible organic nanoparticles (ONPs) based on a donor−acceptor type organic small molecule 4,4′-((6,7diphenyl-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline) (namely TPA-T-TQ, Scheme 1) with intensive absorption in the NIR biological window of 700−900 nm. Through systemic investigation, the TPA-T-TQ ONPs exhibit strong photoacoustic signal, high thermal and photothermal stabilities, as well as excellent photobleaching and RONS resistances, being far better than clinically popularly used ICG. The in vivo effects of the TPA-T-TQ ONPs on PAI and PAIguided PTT were studied with a xenograft 4T1 tumor-bearing mouse model (Scheme 2). Noteworthy is that the ONPs Scheme 2. TPA-T-TQ ONPs Used for PAI and PAI-Guided PTT Applications

RESULTS AND DISCUSSION The donor−acceptor (D−A) approach or push−pull system, in which the electron-donating and -withdrawing units are alternatively arranged along the molecular backbone, is a useful strategy to obtain low-bandgap organic semiconducting materials.47−49 In this work, we synthesized a D−A type organic small molecule by using triphenylamine (TPA) and thiadiazoloquinoxaline (TQ) as the donor and acceptor, respectively. The planar thiophene (T) ring in between is

Figure 1. (a) UV−vis−NIR absorption spectra of TPA-T-TQ in THF solution (black) and the encapsulated ONPs in water (red). Inset shows photographs of (i) the TPA-T-TQ THF solution, and (ii) the as-prepared ONPs in water. (b) Schematic illustration of the preparation of the TPA-T-TQ ONPs through a nanoprecipitation method. (c) PL spectra of TPA-T-TQ in THF solution (black) and the encapsulated ONPs in water (red). (d) Representative TEM image and (e) DLS profile and of the TPA-T-TQ ONPs. 7179

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Figure 2. (a) IR thermal images of the ONPs, ICG, and ICG NPs in PBS solutions (100 μM) under 808 nm laser irradiation for different times. (b) Photothermal conversion behavior of TPA-T-TQ ONPs at different concentrations (5−100 μM) under 808 nm light irradiation. (c) Comparison of the photothermal conversion behavior of TPA-T-TQ ONPs, ICG, and ICG NPs in PBS solution at the same concentration (100 μM). The 808 nm light was irradiated for 5 min, then the laser was removed, and the samples were naturally cooling down to ambient temperature. (d) Photographs of the ONPs, ICG, and ICG NPs in PBS solutions after 808 nm light irradiation for different time. (e) Plot of I/ I0 versus various irradiation time. I and I0 are the maximal NIR absorption intensity of ONPs/ICG/ICG NPs in PBS solutions after and before laser irradiation, respectively. (f) Antiphotobleaching property of ONPs, ICG, and ICG NPs (100 μM) during five circles of heating−cooling processes. The absorption spectra of (g) TPA-T-TQ ONPs and (h) ICG in PBS solution before and after 400 μM of ONOO− and •OH were added for 1 min. Inset: the photographs of the solutions of ONPs and ICG before and after the addition of RONS. (i) Plot of I/I0 versus RONS (ONOO− and •OH). I and I0 are the maximal NIR absorption intensity of ONPs/ICG in PBS solutions in the presence and absence of RONS, respectively. The power density of the 808 nm laser irradiation is 0.8 W/cm2 for (a−i).

functional as a π-conjugation unit, which facilitates the intramolecular charge transfer (ICT) from TPA to TQ, and therefore leading to a much lower electronic bandgap, or longer absorption spectrum. Meanwhile, another problem may arise in this kind of molecules, i.e., the solubility or processability would be significantly decreased due to the planar molecular structure and strong D−A interaction. In order to endow sufficient processability to the molecule, tert-butyl (tBu) and methyl (Me) groups are introduced into the TPA unit. The final compound (TPA-T-TQ) and the intermediates were fully characterized by 1 H NMR, 13C NMR, and high-resolution mass spectrum (HRMS) (Figure S1−S15 in the Supporting Information) with detailed syntheses and characterizations presented in the

Experimental Section. Key steps include the Stille coupling reaction between 4-(tert-butyl)-N-(p-tolyl)-N-(4-(5(tributylstannyl)thiophen-2-yl)phenyl)aniline and 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole to readily obtain the dinitro compound (10), followed by the acid-assisted iron reduction to the diamine intermediate (11), and subsequent cyclization with benzil to afford the final molecule (Scheme 1). TPA-T-TQ can be facilely soluble in common organic solvents such as tetrahydrofuran (THF), dichloromethane, and chloroform, indicating its good processability and easy modification. The absorption spectrum of TPA-T-TQ in THF is shown in Figure 1a. The compound exhibits strong absorption in the spectral region of 700−900 nm with a peak at ∼780 nm, 7180

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Figure 3. (a) PA images of TPA-T-TQ ONPs and ICG upon excitation at 780 nm at different concentrations based on small molecule. (b) PA amplitudes of TPA-T-TQ ONPs and ICG at 780 nm as a function of concentration based on small molecule. (c) PA images of tumor site after intravenous administration of TPA-T-TQ ONPs for designated time intervals. (d) PA intensity at the tumor site as a function of postinjection time.

matching well with the biological window, and thus illustrating great potential for NIR light-triggered theranosis applications. To explore TPA-T-TQ as a bioavailable phototheranostic agent, we prepared the water-soluble “completely organic components” nanoparticles through nanoprecipitation method in the assistance of amphiphilic biocompatible copolymer 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) as the encapsulation matrix.9,50 The schematic illustration of the encapsulation process is depicted in Figure 1b, and the detailed procedure is presented in the Experimental Section. The TPA-T-TQ ONPs can be homogeneously dispersed in water, and the obtained solution is green-yellow color with high transparency (Inserted photograph in Figure 1a). As shown in Figure 1a, the TPA-TTQ ONPs display strong absorption in the spectral region of 700−900 nm, consistent well with the absorption spectrum of TPA-T-TQ in THF solution. It is interesting to note that the TPA-T-TQ THF solution shows a maximal photoluminescence (PL) at about 1020 nm, whereas nearly no PL signal is observed in the aqueous solution of TPA-T-TQ ONPs (Figure 1c), demonstrating the quenching effect in aggregate state, which significantly increases nonradiative heat generation, and therefore, amplifies the PAI brightness and PTT effect.6,32 The morphology of the ONPs is characterized by transmission electron microscopy (TEM), which shows a kind of uniform spherical structure with an average diameter of ∼56 nm (Figure 1d). The nanostructure of the ONPs is also confirmed by scanning electron microscopy (SEM) observation (Figure S16 in the Supporting Information). Dynamic light scattering (DLS) measurement of the ONPs also reveals a hydrodynamic diameter of ∼68 nm (Figure 1e). The slightly larger diameter measured from DLS relative to TEM result is likely due to the shrinkage of the nanoparticles during the sample preparation of TEM. In addition, the visual appearance of the solution did not change and no precipitation or aggregation was observed after

storage at ambient condition for 50 days, suggesting good colloidal stability of the ONPs. To evaluate the photothermal conversion property, we quantitatively measured the temperature change of TPA-T-TQ ONPs solution at different concentrations as a function of 808 nm laser (0.8 W/cm2) irradiation time. As depicted in Figure 2a and 2b, the temperature increases very fast initially, and reaches to the plateau after 3 min laser irradiation. It is noted that the eventual temperature at the plateau depends on the ONPs concentration (Figure 2b). We further compared the temperature increase of the ONPs and ICG solutions upon exposure to 808 nm laser irradiation. As displayed in Figure 2c, the TPAT-TQ ONPs (ΔT ∼ 53 °C in 5 min) exhibit much higher plateau temperature and faster temperature rise rate than ICG (ΔT ∼ 43 °C in 5 min), revealing superior photothermal conversion behavior of the ONPs. The difference in temperature elevation of the two samples at various irradiation times can be intuitively visualized from the infrared (IR) thermal images (Figure 2a). Interestingly, for the ONPs solution at a concentration of 100 μg/mL, the temperature sharply increases to 80 °C after 808 nm laser irradiation for 5 min, representing one of the best photothermal conversion performance under the same condition.28−30 These results suggest that TPA-T-TQ ONPs is a promising candidate as a light-mediated thermal agent. The stabilities of the contrast agents in terms of thermal and photothermal stabilities as well as photobleaching and RONS resistances are crucial for PAI/PTT applications in vivo, because incorrect or misleading signal, weakened therapeutic efficacy and even harmful side effect would appear if the agent structure is destroyed, especially in living systems.14,46 Thermogravimetric analysis (TGA) was first carried out to measure the thermal stability of TPA-T-TQ, and the decomposition temperature for 5% weight loss of TPA-T-TQ is above 400 °C (Figure S17 in the Supporting Information), suggesting 7181

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Figure 4. (a) IR thermal images of 4T1 tumor-bearing mice under 808 nm laser irradiation (0.5 W/cm2) for different time points. (b) The mean temperature of the tumors as a function of the 808 nm laser (0.5 W/cm2) irradiating time. Laser irradiation was performed post 6 h intravenous administration of TPA-T-TQ ONPs or saline for (a) and (b). (c) Tumor growth curves and (d) body weight changes of mice in different treatment groups. ** represents P < 0.01, in comparison between “ONPs + Laser” group and other treatment groups.

2a and 2c−f), which thus excludes the influence of DSPEPEG2000 on these properties. Another important issue to be concerned for in vivo application is the physiological stability against highly reactive molecules such as RONS. RONS are a kind of signaling molecules not only to regulate a lot of physiological functions, but also to be overproduced in association with many diseases including inflammations, cancers, and cardiovascular diseases.9,51,52 For cancer diagnosis and treatment, it is considerably vital to use RONS-resistant agents to get reliable imaging signal and therapy efficacy. Therefore, we measured and compared the stabilities of TPA-T-TQ ONPs and ICG in the presence of two kinds of RONS, peroxynitrite (ONOO−) and hydroxyl radical (•OH), under physiological condition. The absorption spectra and photographic solutions of TPA-TTQ ONPs and ICG before and after the treatment of RONS reagents are depicted in Figure 2g and 2h, respectively. After addition of ONOO− and •OH, the maximal absorption intensity of ICG (at 780 nm) drops about 40% and 96% relative to the original value, respectively. In marked contrast, there is nearly no change in the absorption spectra and solution appearance of the ONPs after adding each RONS (Figure 2i). These results indicate that TPA-T-TQ ONPs are highly resistant to RONS, which is ideal for stable and accurate in vivo imaging and therapy of RONS-associated diseases (e.g., cancer). After confirming the excellent photothermal conversion property and antiphotobleaching/RONS stabilities of the TPA-T-TQ ONPs, we next investigated its photoacoustic (PA) property. As shown in Figure S18 in the Supporting Information, the PA spectrum of TPA-T-TQ ONPs in PBS solution is measured in the spectral region of 680−900 nm,

excellent thermal stability. Then we evaluated the photothermal stability of TPA-T-TQ ONPs along with ICG under continuous 808 nm laser irradiation (0.8 W/cm2) by recording the apparent colors and absorption spectra after laser irradiation for different times. As shown in Figure 2d and 2e, the colors and absorption spectra of TPA-T-TQ ONPs are nearly unchanged during 15 min NIR light irradiation duration, whereas the blue-green color of ICG solution gradually disappears, and the maximal absorption intensity nearly drops to zero after being exposed to the NIR light for 15 min. Noteworthy is that the optical properties of TPA-T-TQ ONPs are identical to the original state even after continuous laser illumination (0.8 W/cm2) for an hour (Figure 2e). Then we evaluated the antiphotobleaching properties of TPA-T-TQ ONPs and ICG by alternative heating and cooling process (the NIR laser first irradiated the samples for 5 min to heat them up, then the laser was removed, and the samples were naturally cooling down to ambient temperature in 6 min). Interestingly, during five circles of heating and cooling processes, the photothermal conversion ability of the ONPs shows negligible change, while the temperature elevation of ICG dramatically dropped to about 20% (ΔT ∼ 10 °C) of the original value (ΔT ∼ 44 °C) after two circles of heating−cooling processes (Figure 3f). These results provide solid confirmation that TPA-T-TQ ONPs possess superior resistance to photobleaching compared to the existent organic small molecules. Furthermore, we also prepared ICG NPs with DSPE-PEG2000 as the matrix following the same experiment procedure as that for the fabrication of TPA-T-TQ ONPs. It is found that ICG NPs exhibit similar performance to ICG itself in photothermal conversion capacity, photothermal stability and photobleaching resistance (Figure 7182

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Figure 5. (a) Histological H&E, fluorescence TUNEL, and PCNA staining of tumor slices at day 16 after different treatments indicated. (b) Histological H&E staining for livers and spleens on day 16 after different treatments indicated.

nude mice. Before ONPs administration (0 h), there is weak PA signal at 780 nm probably attributed to the absorption of endogenous melanin and hemoglobin in the NIR spectral region.9,14 Compared with the PA image of the tumor at 0 h, the PA brightness of the tumor site after intravenous injection of TPA-T-TQ ONPs significantly increases over time, which reaches to the maximum at 6 h postinjection (Figure 3c), manifesting that 6 h postinjection is the optimized time point for PA imaging and PTT treatment of tumor. The timedependent PA intensities at the tumor site are presented in Figure 3d. The PA signal at 6 h is 2.4-fold higher as compared to that of the tumor background, indicating the prominent enhanced permeability and retention (EPR) effect of the ONPs, which leads to their efficient accumulation in the tumor tissue.53,54 These results reasonably reveal that TPA-T-TQ

with the maximal intensity at about 780 nm, which is in good agreement with the absorption profile (Figure 1a), and reveals that the PA signal is originated from the intense NIR absorption of the organic small molecule. We further performed the PA images and the corresponding PA intensities of the ONPs and ICG, upon excitation at 780 nm at different concentrations (Figure 3a and 3b). The PA amplitudes of the ONPs at 780 nm were determined in a series of concentrations from 0 to 100 μM based on TPA-T-TQ, showing a relatively good linear relationship in a large range. As shown in Figure 3b, in comparison with the clinically used ICG, the ONPs exhibit much better PA conversion performance, which is in accordance with the photothermal conversion property, and would enable superior PAI effect. We then investigated the in vivo PAI by intravenous injection of the TPA-T-TQ ONPs into xenograft 4T1 tumor-bearing 7183

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Figure 6. Blood test parameters in terms of (a) liver function as well as (b) red blood cell, haem regulation and white blood cell count of healthy Balb/c mice intravenously injected with TPA-T-TQ ONPs for 7 days. The untreated mice were used as the control.

and 4b, the tumors from mice in “Saline + Laser” group exhibit little temperature elevation (ΔT ∼ 2.5 °C) upon NIR light exposure for 5 min, implying that laser irradiation alone would have negligible effect on heat-caused tumor inhibition. In comparison, fast temperature elevation is observed from the tumors in “ONPs + Laser”-treated mice, as evidenced by the tumor temperature raising from 36 °C to a plateau of about 64 °C in 3 min light exposure. Such in vivo temperature rise rate and eventual temperature are comparable to the best light-toheat conversion performance of currently available organic photothermal agents.29,34 These results illuminate that the TPA-T-TQ ONPs can result in rapid temperature elevation in tumor site under NIR light irradiation, representing an efficient agent for tumor PTT in living organisms. The in vivo antitumor efficacy of “ONPs + Laser” through a single PTT was studied by monitoring the tumor volumes for 16 days. As presented in Figure 4c, the treatment of “Saline + Laser” totally fails to suppress the tumor growth as compared to the control group (“Only Saline”), indicating that pure 808

ONPs can serve as an effective probe for PAI in a high-contrast manner. The PTT capability of the ONPs was next validated with the xenograft 4T1 tumor mouse model. Tumor-bearing mice were randomly assigned to 4 groups, which were named “Only Saline”, “Saline + Laser”, “Only ONPs”, and “ONPs + Laser”, respectively. For “Only Saline” and “Only ONPs” groups, Saline and TPA-T-TQ ONPs (250 μg/mL based on TPA-TTQ) were injected into 4T1 tumor-bearing mice via the tail vein, respectively, without subsequent laser irradiation. For “Saline + Laser” and “ONPs + Laser” groups, after intravenous injection of saline and TPA-T-TQ ONPs (250 μg/mL based on TPA-T-TQ) for 6 h, respectively, the tumors of mice in each group were continuously irradiated with 808 nm laser (0.5 mW/cm2) for 5 min. First, to verify that TPA-T-TQ ONPs are able to generate heat with laser irradiation in living mice, the tumor temperatures of “ONPs + Laser”-treated and “Saline + Laser”-treated mice were monitored by IR thermography at different laser irradiation time scales. As depicted in Figure 4a 7184

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including white blood cells (WBC), lymphocyte (LYM), hematocrit (HCT), hemoglobin (Hgb), red blood cells (RBC), red cell distribution width (RDW), corpuscular hemoglobin concentration (CHC), platelets (PLT), as well as mean platelet volume (MPV) indicates that there are no statistical differences in these indicators between ONPs and untreated groups (Figure 6b). Further considering the negligible influences of TPA-T-TQ ONPs on mouse body weight (Figure 4d) and the health of important normal organs (Figure 5b), it is reasonable to conclude that the TPA-T-TQ ONPs are a highly biocompatible phototheranostic nanoagent with inducing no noticeable side effect to living mice.

nm laser irradiation does not possess any antitumor effect. Moreover, the tumor growth kinetics from mice in “Only ONPs” group is also similar to that in “Only Saline” group, suggesting that the ONPs themselves have negligible active behavior against cancer. Dramatically, as compared to the other three groups with fast-growing tumor volumes, the “ONPs + Laser” group shows amazing antitumor efficacy. The average tumor volume on day 16 in “ONPs + Laser” group is even slightly smaller than that on day 0, suggesting that the PTT by TPA-T-TQ ONPs is capable of resulting in tumor growth stoppage, which is indeed efficacious on tumor suppression. Besides, the mice in each treatment group were also weighted every other day during 16-day study duration. As shown in Figure 4d, no obvious body weight loss is observed in mice of “Only ONPs”, “Saline + Laser”, and “ONPs + Laser” groups when compared with the control cohort, suggesting the low side toxic effect of the treatment of “ONPs + Laser”. In order to further clarify the tumor inhibition performance of our organic phototheranostic nanoagent, the mice in all the groups were sacrificed on day 16, and then the tumors were collected and sliced for histological hematoxylin and eosin (H&E) staining as well as immunohistochemical analyses including TdT-mediated dUTP-biotin nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA). As presented in Figure 5a, the H&E staining of tumors from various treatment groups clearly demonstrates that as compared to other three treatments, “ONPs + Laser” leads to much more necrotic areas in the tumors. Moreover, the fluorescence TUNEL staining and PCNA staining also reveal that the treatment of “ONPs + Laser” is the most efficacious in inducing the apoptosis and suppressing the proliferation capacity of tumor cells (Figure 5a). These results from the microscopic point of view are pretty consistent with and support the best anticancer activity of “ONPs + Laser” depicted in Figure 4c, which give solid evidence that TPA-T-TQ ONPs are indeed an efficient PTT agent for cancer therapy. To study whether TPA-T-TQ ONPs cause in vivo side toxicity, the livers and spleens of mice in each treatment group were also excised and sectioned for H&E staining at the end time point, as it has been widely accepted that nanomaterials tend to be enriched in reticuloendothelial system (RES) organs including liver and spleen.55,56 As shown in Figure 5b, no noticeable tissue damage and inflammatory lesion can be found in the liver and spleen organs from all the treatment groups of mice. The result suggests the harmlessness of TPA-T-TQ ONPs toward RES organs in spite of significant accumulation. Furthermore, it is also found that the intravenously injected TPA-T-TQ ONPs can be almost completely excreted from the healthy mouse body after 9 days through biliary pathway (Figure S19 and S20 in the Supporting Information). The relatively rapid clearance rate from the body implies the good compatibility and low in vivo toxicity of our ONPs.57 To further investigate the potential toxicology of TPA-T-TQ ONPs, healthy Balb/c mice intravenously administrated with TPA-T-TQ ONPs (250 μg/mL based on TPA-T-TQ) as well as untreated healthy mice were received serum biochemistry assay and complete blood count on day 7 postinjection. The liver function indicators including alanine aminotransferase (ALT), aspartic acid transaminase (AST), albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), and γ-globulin transferase (GGT), are all measured to be normal (Figure 6a), revealing no obvious hepatic disorders of “ONPs + Laser”treated mice. Besides, the assay of complete blood panel

CONCLUSIONS In summary, we have developed a biocompatible organic small molecular nanoparticle with high stabilities for effective PAI and PAI-guided PTT applications. Noteworthy, the TPA-T-TQ ONPs exhibit intense PA signal and super high stabilities in terms of thermal and photothermal stabilities as well as photobleaching and RONS resistances, which are far better than the most popularly used ICG in clinic. The TPA-T-TQ ONPs also show excellent photothermal conversion performance when exposed to NIR light due to the intensive absorption of the organic small molecule in the NIR biological window of 700−900 nm. In vivo imaging study manifests that our ONPs can serve as a biocompatible and effective probe for PAI in a high-contrast manner. In vivo tumor growth kinetics with a xenograft 4T1 tumor-bearing mouse model reveals that the PTT by TPA-T-TQ ONPs gives impressive performance in tumor suppression accompanied by tumor growth stoppage. The superior antitumor efficacy of “ONPs + Laser” is also confirmed in the microscopic level by histological and immunohistochemical staining of tumor slices. This study will inspire more exciting research on the development of useful NIR-absorbing organic small molecules with high stabilities for in vivo phototheranostic applications. EXPERIMENTAL SECTION Chemicals and Methods. All the chemicals and reagents were purchased from chemical sources, and the solvents for chemical reactions were distilled before use. 1H (400 MHz) and 13C (100 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV 400 spectrometer by using CDCl3 or DMSO-d6 as the solvent. High-resolution mass spectra (HRMS) were measured with a GCT premier CAB048 mass spectrometer in MALDI-TOF mode. Thermogravimetric analysis (TGA) measurement was carried out using a TA TGA Q5000 under nitrogen atmosphere with a heating rate of 10 °C/min. The UV−vis−NIR absorption spectra were performed using a PerkinElmer Lambda 365 spectrophotometer. The photoluninescence (PL) spectra were conducted on a Horiba Fluorolog-3 spectrofluorometer. Dynamic light scattering (DLS) was measured on a 90 plus particle size analyzer. Transmission electron microscopy (TEM) images were acquired from a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV. Synthesis of 4,4′-((5,6-Dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(thiophene-5,2-diyl))bis(N-(4-(tert-butyl)phenyl)-N-(p-tolyl)aniline) (10). 4-(tert-Butyl)-N-(p-tolyl)-N-(4-(5-(tributylstannyl)thiophen-2-yl)phenyl)aniline (2.06 g, 3 mmol), 4,7-dibromo-5,6dinitrobenzo[c][1,2,5]thiadiazole (0.46 g, 1.2 mmol), and Pd(PPh3)4 (58 mg, 0.05 mmol) were added into a 100 mL Schlenk flask. The flask was vacuumed and purged with nitrogen three times. Afterward, anhydrous THF (40 mL) was added, and the mixture was stirred under reflux for 24 h. Then the solvent was removed under reduced pressure, and the residue was purified on silica gel chromatography 7185

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ACS Nano

Animal Model. All animal studies were conducted under the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals, and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. The six-week-old female BALB/c mice were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). To establish the xenograft 4T1 tumor-bearing mouse model, one million of murine 4T1 breast cancer cells suspended in 50 μL of RPMI-1640 medium were injected subcutaneously into the right axillary space of the mouse. After about 7 days, mice with tumor volumes at about 80−120 mm3 were used subsequently. In Vivo Photoacoustic Imaging. PA signals or images were acquired on a commercial Endra Nexus 128 photoacoustic tomography system (Endra, Inc., Ann Arbor, Michigan, USA). The 4T1 tumor-bearing nude mice were anesthetized using 2% isoflurane in oxygen, and then TPA-T-TQ ONPs (150 μL, 250 μg/mL based on TPA-T-TQ) were injected into the tumor-bearing mice through tail vein using a microsyringe (n = 4). PA images were subsequently acquired at 780 nm at designated time intervals post ONPs injection. In Vivo Photothermal Therapy. The xenograft 4T1 tumorbearing mice were randomly divided into 4 groups (n = 6 per group), which were named “Only Saline”, “Saline + Laser”, “Only ONPs”, and “ONPs + Laser”, respectively. For “Only Saline” and “Only ONPs” groups, saline and TPA-T-TQ ONPs (250 μg/mL based on TPA-TTQ) were injected into 4T1 tumor-bearing mice through tail vein, respectively, without subsequent laser irradiation. For “Saline + Laser” and “ONPs + Laser” groups, after intravenous injection of saline and TPA-T-TQ ONPs (250 μg/mL based on TPA-T-TQ) for 6 h, respectively, the tumors of mice in each group were continuously irradiated with an 808 nm laser (0.5 mW/cm2) for 5 min. After a variety of treatments, the tumor volumes and mouse body weights were measured every other day for 16 days. The tumor volume was calculated by the following equation: Volume = Width2 × Length/2. Additionally, the temperature changes of ONPs-treated and salinetreated tumors under 808 nm laser irradiation were monitored every 10 s through an IR thermal camera (Fluke Shanghai Inc.). Histological Studies. Sixteen days after the photothermal treatment, the above-mentioned four groups of mice were sacrificed and tumors and important normal organs were excised, sliced and stained. The fluorescent PCNA staining was conducted following common immunohistochemical steps. The fluorescent TUNEL staining was conducted following manual instruction of DeadEnd fluorometric TUNEL system kit (Promega, USA). For hematoxylin and eosin (H&E) staining, the tissues of the mice were fixed in 4% formalin solution, processed into paraffin, and sectioned at 5 μm thickness. The slices were examined with a digital microscope (Leica QWin). Serum Biochemistry Assay and Complete Blood Count. Healthy Balb/c mice were randomly assigned into 2 groups (n = 3 per group). 150 μL of TPA-T-TQ ONPs (250 μg/mL based on TPA-TTQ) was intravenously injected into one group of mice. For the other group, no treatment was performed. After 1 week, blood was collected for all mice and then detected using an automated hematology analyzer. Statistical Analysis. Quantitative data were expressed as mean ± standard deviation (SD). Statistical comparisons were made by ANOVA analysis and two-sample Student’s t-test. P value