Tannic Acid Assembly as a Versatile Photothermal Platform

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Metal Ion/Tannic Acid Assembly as a Versatile Photothermal Platform in Engineering Multimodal Nanotheranostics for Advanced Applications Tao Liu, Mingkang Zhang, Wenlong Liu, Xuan Zeng, Xianlin Song, Xiaoquan Yang, Xian-Zheng Zhang, and Jun Feng ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01456 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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Metal

Ion/Tannic

Photothermal

Acid

Platform

Assembly in

as

Engineering

a

Versatile Multimodal

Nanotheranostics for Advanced Applications

Tao Liu,† Mingkang Zhang,† Wenlong Liu,† Xuan Zeng,† Xianlin Song,‡ Xiaoquan Yang,‡ Xianzheng Zhang,† Jun Feng†,*

†

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

‡

Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics & Moe Key Laboratory of Biomedical Photonics of Ministry of Education, Department of Biomedical Engineering. Huazhong University of Science and Technology Wuhan 430074, China

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ABSTRACT: This study reports a family of photothermal materials, metal ion/tannic acid assemblies (MITAs). MITAs from FeIII, VIII, RuIII afford excellent photothermal efficiency (η ~40%). Sharply differing from the currently existing photothermal agents, MITAs are highlighted by the merits including green synthesis, facile incorporation of diagnostic metal ions and particularly topology-independent adhesion. Owing to the adhesion nature of MITAs, various kinds of MITA-based nanoengineerings are readily available via the self-adhesion of MITAs onto diverse templates, enabling MITAs well suited as a photothermal platform for versatile cooperation with other therapy approaches and imaging techniques. As the proof of concept, polymeric/inorganic nanoparticles/nanovesicles supported FeIII-tannic acid (FeIIITA) are fabricated. The photothermal effect is shown to be not affected by the template origin and type, and FeIIITA thickness on templates. We validate the potency of nanovehiscle-supported FeIIITA (PNV@FeIIITA) for tumor-specific photo-activated utilizations, including NIR photothermal therapy (PTT) with complete tumor elimination, photothermal imaging (PTI) and photoacoustic imaging (PAI) in addition to the T1-MRI imaging. PNV@FeIIITA can be simultaneously equipped with the functionalities, including T2-MRI imaging by additionally doping MnII, and NIR fluorescence imaging by encapsulating hydrophilic NIR fluoroprobe. MITA demonstrate the unparalleled superiority as a photothermal platform in engineering multimodal theranostics for advanced applications.

KEYWORDS: metal/tannic acid complex (MITA), photothermal platform, multimodal imaging, combinational therapy, multimodal theranostic


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The past decades have witnessed the rapid development of photothermal nanomaterials in many fields, particularly for cancer treatments in recent years.1−3 To date, the types of photothermal nanomaterials are still scarce, which are mainly limited to photosensitizer-containing polymers,4, 5

metal nanoparticles,6−8 carbon nanomaterials,9, 10 black phosphorus-based nanosmaterials and

metal chalcogeniders.11−16 Clinical studies show that photothermal therapy (PTT) not only has high selectivity directly on tumors but also can enhance the efficacy of chemotherapy and radiotherapy, improve immunity, and inhibit cancer recurrence and metastasis.9,

17

Based on

PTT’s outstanding merits, its cooperation with other therapy approaches and/or imaging techniques are currently attracting tremendous interest for the advanced applications, such as combinational/synergetic therapy, all-in-one theranostic, multimodal imaging.18 This is very appealing in clinics because such advanced applications can overcome the intrinsic or acquired resistance mechanisms of individual monotherapy, offer more precise diagnosis than single imaging technique, or achieve imaging-guided personalized therapy. For the majority of the currently existing PTT nanomaterials, however, their advanced applications suffer from the poor processing property, the low loading capacity to imaging probes and therapeutics, as well as the complex chemistry and the toxic additives involved in the preparation process. New types of PTT materials have to be sought to surmount these issues and preferably, to be able to function as photothermal platforms that allow the easy access to flexible architectures/geometries and versatile functions in favor of the advanced applications. This study reports a family of photothermal materials, metal ion-crosslinked tannic acid (MITA), which is highlighted by not only the green synthesis pathway but also noticeably, the strong adhesion capability. Metal ions play vital roles in the therapy/diagnosis fields.19 Tannic acid is a naturally occurring polyphenol extracted from plant source and accepted by the FDA with the widespread utilization in food and medicine. The digalloyl groups rich in TA can serve 3 ACS Paragon Plus Environment

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as chelating sites for multivalent metal ions thus to induce rapid self-crosslinking of TA within minutes in water,20,

21

giving stable MITA network with no need of heat treatment, organic

solvents, special instruments and additive chemicals.21 Furthermore, MITA can effectively adsorb aromatic substances and readily undergo chemical modification.22 Of special note, owing to the topology-independent adhesion character of MITAs, it is easily accessible for the generation of a wide array of MITA-adhered materials based on diverse templates (e.g., 2D materials, solid sphere/rod, hollow vesicle, nanowires, nanosheets, nanocubes) originating from different sources (e.g., polymeric and inorganic materials, cells, proteins, virus, bacteria, yeast).23−25 This character can function as an intermediate to link the photothermal ability of MITAs together with the diverse functionality originating from various kinds of templates, such as the desired loading capacity to various cargoes (e.g., hydrophobic/hydrophilic, small/macromolecular, and artificial/natural substances). Compared with the currently existing PTT materials, MITA apparently holds unparalleled advantages to serve as the photothermal platforms that allow the easy access to advanced applications. Herein, different metal ions were screened to explore the photothermal capability of MITAs. Poly(lactic-co-glycolic acid)-based polymeric nanoparticle (PNS) and nanovesicle (PNV),26, 27 and mesoporous silica nanoparticles (MSN) were used as the templates to prepare three kinds of template-supported MITA nanoengineering. Various parameters, including template origin, nanostructure type, MITA thickness and MITA composition, were investigated regarding the influence on the photothermal effect. We validated the potency of MITAs for photo-responsive applications including tumor-specific PTT, photothermal imaging (PTI) and photoacoustic imaging (PAI).18, 28, 29 As the typical example to prove the potential of MITA as a versatile platform for advanced applications, a PNV-supported FeIIITA network (PNV@FeIIITA) was fabricated, which eventually demonstrated the ability of T1,T2-weighted dual-modal MR imaging 4 ACS Paragon Plus Environment

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by additionally doping MnII and of in vivo fluorescence imaging by encapsulating hydrophilic fluoroprobe.21, 30

RESULTS AND DISCUSSION TA, a dendritic polyphenol composed of a central glucose connected with five digalloyl ester groups, can serve as a polydentate ligand for rapid coordination with metal ions into threedimension network within minutes.20, 21 The initiation of this study was inspired by our accidental discovery that FeIIITA NPs exhibited photothermal capability. After simply mixing TA and metal ion in ultrapure water followed by the pH neutralization, FeIIITA was harvested by centrifugation.20,

21

It was found that a low ratio of FeIII/TA failed to enable the network

formation while when the ratio of FeIII/TA was too high, there would appear evident precipitation at the fixed condition.20 Transmission electron microscopy (TEM) observation showed that the mixing of FeIII and TA at the molar ratio 3.5 : 1 afforded nanoscale, albeit irregular nanoparticles (Supporting Information, Figure S1A-C). The mean hydrodynamic diameter (Dh) and the particle size distribution was determined to be ~200 nm and ~0.15 by dynamic light scattering (DLS) technique (Supporting Information, Figure S1D-F). The analysis using inductively coupled plasma-atomic emission spectrometry (ICP-AES) showed that the composition ratio was close to the molar ratio in feed (Supporting Information, Table S1). Upon the irradiation under 808 nm laser, the temperature of FeIIITA dispersion rose rapidly with time, indicating the monotonical enhancement of photothermal effect as a function of MITA concentration and radiant energy (Figure 1A, Supporting Information, Figure S2A). At 10 min post irradiation, the temperature of FeIIITA prepared at the molar ratio 3.5 : 1 (Fe versus TA) was increased by 44.5 °C at a concentration of 200 µg mL-1 (Figure 1A). In contrast, a subtle promotion of 3.8 °C occurred for 5 ACS Paragon Plus Environment

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the blank control. The result indicated that FeIIITA could convert NIR energy into thermal energy rapidly and efficiently. Interestingly, it was found that the photothermal efficiency of FeIIITA was enhanced with the increasing ratio of Fe versus TA (Figure 1B). The positive correlation of heatgeneration capability with the composition ratio might find the explanation based on the fact that the FeIIITAs with higher FeIII content afforded stronger absorption in the near-infrared (NIR) window (the range of wavelengths from 650 to 1350 nm) (Figure 1C).31 Likewise, the stronger absorbance of FeIIITA at 660 nm than at 808 nm correlated well with the better photothermal effect using 660 nm excitation. The former at the power density of 80 mW cm-2 afforded the hyperthermia effect at a similar level as 808 nm irradiation did at 2 W cm-2 (Figure S2B). The unexpected photothermal character of FeIIITA encouraged us to further explore the feasibility of other MITAs including RuIIITA, VIIITA, GdIIITA, CuIITA, MnIITA and NiIITA. These MITAs were prepared in the same way. In all the cases, there occurred distinct color change after mixing for all the investigated ions, indicative of the formation of MITAs. Only RuIIITA, VIIITA exhibit broad absorption in the NIR window, opposite to the negligible absorption detected for the other four samples (Figure 1D). Correspondingly, RuIIITA and VIIITA afforded considerably NIR-activated photothermal conversion while GdIIITA, CuIITA, MnIITA, NiIITA failed (Figure 1E). The photothermal effect of RuIIITA and VIIITA was enhanced with the increasing ratio of metal ions versus TA (Figure 1F, G), which correlated well with the absorbance intensity in NIR area (Supporting Information, Figure S3A, B). Nevertheless, that of GdIIITA, CuIITA, MnIITA, NiIITA remained unchanged at a ultralow level regardless of the composition variation (Figure 1H, Supporting Information, Figure S3C-F). With regard to the photothermal performance, three photothermal MITAs with the identical molar composition (Metal:TA= 3.5:1) were compared. Under irradiation at 808 nm, the ranking 6 ACS Paragon Plus Environment

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is RuIIITA < FeIIITA< VIIITA, which agreed fairly well with the order of the NIR adsorption intensity at 808 nm (Figure 1E). Under 660 nm irradiation, the photothermal performance order was changed to RuIIITA < VIIITA < FeIIITA (Supporting Information, Figure S4). This was also consistent with the order of absorbance intensity at 660 nm. Based on these results, we naturally educed that the photothermal effect of MITAs had strong association with their NIR adsorption intensity. Interestingly, it seemed that the photothermal effect of MITAs correlate well with color darkness (Figure 1I, J). The darker the solution color, the more likely the MITAs are applicable for photothermal conversion, which might be taken as a simple indicator to predict the photothermal effect of MITAs. Further screening of other ions and deeper study is needed in future to probe the mechanisms and factors regarding the photothermal effect of MITAs. Anyway, these results indicated that MITAs can be developed as a family of powerful photothermal agents with green and facile preparation. It is noteworthy to point out that unlike other reported photothermal agents, the photothermal efficiency of MITAs can be easily tailored by merely adjusting the composition, which may favor the practical applications with different demands. As well documented, the shape and size of nanomaterials play important roles in lots of in vivo physiological processes, such as the cellular internalization, circulation duration, passive tumor targeting, tumor-depth infiltration and immunity recognition.32 For the majority of the currently existing photothermal materials, their transformation to various nanostructures with desired shape/size is hardly accessible. The poor processing property hampers their advanced applications. MITAs sharply differs from theses reported materials by the strong topologyindependent adhesion, which perfectly enables the facile and accurate control over the architectures and geometries of the MITA-based materials by aid of template technology. This is an unparalleled advantage compared with the reported photothermal agents. Herein, three kinds of nano-templates with uniform morphology were prepared, including mesoporous silica 7 ACS Paragon Plus Environment

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nanoparticles (MSN), PLGA-based solid nanosphere (PNS) and nanovesicle (PNV). TEM observation clearly indicated the interfacial adhesion of the self-deposited FeIIITA film onto PNS and PNV templates prepared through single-emulsion (O/W) or double-emulsion (W/O/W) method, respectively.26, 27 The parameters, including template origin and type, MITA thickness, were investigated regarding the influences on the photothermal effect. For comparison, three FeIIITA-adhered nanoentities of PNV@FeIIITA, PNS@FeIIITA and MSN@FeIIITA were prepared with the close particle size ~195 nm and film thickness (~30 nm) (Figure 2A-C). All of them had the similar hydrodynamic diameter (225~250 nm) with the narrow size distribution below 0.15 determined by DLS analysis (Figure 2D-F). The 808 nm of NIR irradiation located at biological window affords deep penetration with less damages to normal tissues. Therefore, the irradiation was fixed at 808 nm in the following experiments unless specified otherwise. The hyperthermal curves of PNV@FeIIITA solution under NIR irradiation agreed well with the concentration-dependent variation trend of photothermal effect of FeIIITA (Figure 2G, H). Next, FeIIITA, PNV@FeIIITA, PNS@FeIIITA and MSN@FeIIITA with identical FeIIITA content (100 µg mL-1) were subjected to the irradiation (2 W cm-2). Compared with free FeIIITA without template support, PNS@FeIIITA, PNV@FeIIITA and MSN@FeIIITA showed minimal distinction in the photothermal effect, as evidenced by the hyperthermal curves (Figure 2I). It is suggested that the photothermal effect of FeIIITA was not sensitive to the template introduction, the template origin (organic or inorganic) and the template structure (nanoparticle or nanovesicle). Then, we would like to make clear what influence the film thickness of FeIIITA shell would exert. Identical amount of FeIIITA was self-deposited onto the PNV with different concentrations in aqueous solution, thus providing a series of PNV@FeIIITA ([FeIIITA] = 100 µg mL-1) bearing different shell thickness. As illustrated in Figure 2J, there appeared no evident differences in the photothermal effect among these observed 8 ACS Paragon Plus Environment

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samples, indicating the dependency of photothermal effect with [FeIIITA] rather than FeIIITA thickness. Finally, to exactly assess the photothermal potency of FeIIITA, photothermal conversion efficiency (PCE) was quantified using the index of η value as previously established in references (see Supporting Information for details).33, 34 The η value was determined to be ~45.4% under 808 nm laser (Figure 2K, Supporting Information, Figure S5). This η value of FeIIITA is higher than the currently reported photothermal agents such as Au nanorods (21%), Cu2-xSe (22%), and Cu9S5 (25.7%).35−37 Hence, it is suggested that FeIIITA possess a high capability to convert NIR irradiation to heat. The η value of MITAs from RuIII and VIII was determined to be 44.4% and 37.9% under 808 nm laser, respectively. Next, we studied the photothermal stability of the PNV@FeIIITA. The solution of PNV@FeIIITA was irradiated for 10 min and then naturally cooled for 25 min, this irradiation on/off process was repeated four times. As shown in Figure 2L, the thermal generation ability of PNV@FeIIITA was almost unchanged during the treatment with repeated irradiation on/off cycles, indicating the good photothermal stability that allows repeated PTT treatment. Owing to the identical surface, FeIIITA and PNV@FeIIITA presented the close zeta potential at -26.6 ± 1.6 mV and -25.2 ± 2.1 mV, respectively. The negative potential would contribute to the suppressed the uptake by normal cells and the reduced opsonization by the immune system. In addition, the data demonstrated that the Dh of PNV@FeIIITA remained always steady across 1-month storage (Supporting Information, Figure S6), and SEM images showed minimal morphology changes before and after 1-month storage (Supporting Information, Figure S7). Taken together, these results adumbrated that by aid of template technology, MITAs could be developed as a versatile photothermal platform capable of transforming to diverse nanostructures without compromising the photothermal efficiency. This special character


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together with the photothermal stability and the storage stability offers the basic guarantee to advance MITAs toward the extended applications in vivo based on the photothermal utilization. The in vitro photothermal outcomes of MITAs motivated us to explore their potential for in vivo photo-activated applications, including PTT, photothermal imaging (PTI) and photoacoustic imaging (PAI). To simplify the study, PNV@FeIIITA was intensively explored herein as the typical model. 4T1 tumor-bearing mice were divided into two groups and injected via tail vein with PBS and PNV@FeIIITA solution ([FeIIITA] = 20 mg kg-1) respectively. At 4 h postinjection, the mice were subject to the irradiation for 6 min with a power density of 2 W cm-2. The tumor region harvested apparently much higher temperature than the other regions and the temperature continuously increased with irradiation time, as shown in the in vivo photothermal images (Figure 3A). The temperature at tumor sites was recorded by an IR camera and the data were collected in Figure 3B. It was shown that the temperature in tumor sites rose up rapidly to about 52 °C within 6 min under irradiation. In comparison, PBS treated group displayed only a slight temperature increase by less than 5 °C at tumors. The results suggested that photothermal MITAs could be developed as a PTI contrast agent and permit real-time monitoring over thermal dynamics in PTT process. Considering the strong photothermal effect and the involvement of metal ions, we conjectured that PNV@FeIIITA may be applicable for the in vivo PA imaging. The obtained in vitro and in vivo results verified our surmise (Figure 3C, D). First, PNV@FeIIITA solution with different concentrations ranging from 25 µg mL-1 to 200 µg mL-1 were subject to the scanning by PA imaging system. The intensity of PA signal was positively proportional to the FeIIITA concentration (R2 = 0.996) (Figure 3D, Supporting Information, Figure S8). The in vivo PA imaging was performed at 4 h post the injection with a FeIIITA dosage of 20 mg kg-1 via tail vein. Compared with blank control, intratumoral PA signals were significantly strengthened (Figure 3C). The 3D PAI image even clearly outlined the tumor with distinct tumor-to-background 10 ACS Paragon Plus Environment

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contrast, providing deep tissue spatial information. These results manifested the promise of MITAs as a PAI agent. Because PTT can bypass the inherent barriers of other tumor-specific therapy approaches, such as the multi-drug resistant (MDR) effect frequently occurring for chemotherapy and the hypoxia limitation for photodynamic therapy,38, 39 PTT has gained tremendous interest in tumor treatments. The potential of PNV@FeIIITA for PTT was first investigated in cellular levels using MTT assay. Nearly no dark-cytotoxicity was found after 4T1 cells were co-incubated for 24 h with PNV@FeIIITA ranging from 12.5 to 400 µg mL-1 (Supporting Information, Figure S9A). When the cells were exposed to 5-min irradiation (808nm, 2 W cm-2) before the coincubation, however, there appeared considerable cell death. At 200 µg mL-1, the cell viability sharply dropped below 15% (Supporting Information, Figure S9B). The visual evidence of light-toxicity was additionally offered by live-dead cell staining assay. In the presence of 50 µg mL-1 PNV@FeIIITA, the cells were treated with the irradiation (2 W cm-2) for 5 min followed by 4 h culture. As shown in the confocal laser scanning microscopy (CLSM) images, there emerged a large amount of red fluorescence signals representing dead cells under irradiation, in marked contrast to none of dead cells detected in the blank control, the laser only and the PNV@FeIIITA only treated groups. (Figure 3E). Moreover, almost all of the PNV@FeIIITA-treated cells underwent a striking deformation from fusiform to spherical shape when exposed to irradiation, whereas the cells in the three controls remained always healthy during the treatment. We next tested the PTT potency for in vivo tumor ablation. The mice bearing a ~100 mm3 4T1 tumor near hind limbs were randomly divided into four groups and respectively treated with (I) PBS injection via tail vein, (II) Laser irradiation (808 nm, 2 W cm-2) at 4 h post PBS injection, (III) PNV@FeIIITA injection alone, (IV) Laser irradiation at 4 h post the injection of 11 ACS Paragon Plus Environment

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PNV@FeIIITA. The tumors in group (IV) were gradually shrunken until to complete ablation, and no tumor relapse occurred during the observation period of 14 days (Figure 3F, G). Meanwhile, the body weight remained insignificantly varied in group (IV) (Figure 3H). In sharp contrast, the tumors in other groups kept growing with the similar speed. At 15th day, all the mice were sacrificed, and the tumors and the major organs were harvested for the histological analysis using H&E staining technique. In the histological images of tumor slice acquired from group (IV), there appeared a large number of dead cells together with the corruption of the extracellular matrix (Figure 3I). In addition, it was shown that lots of the tumor cells lost their original morphology with nuclear membrane fragmentation and nuclei shrinkage, indicative of the cellular necrosis or apoptosis. In comparison, abundant live cells were densely packed in the tumor slice of the other groups, and the cell death or the cell deformation was scarcely detectable. Histological examination toward major organs (heart, liver, spleen, kidneys, and lung) was also conducted to preliminarily evaluate the biosafety of PNV@FeIIITA. No discernible pathologic abnormality was observed in the four groups (Supporting Information, Figure S10). Taken together, all these results indicated the favorable tumor-specific PTT efficacy and the minimal systemic toxicity. Compared with the currently existing photothermal agents, MITA holds unparalleled advantages that allow the wide extension toward advanced applications by taking advantage of not only the intrinsic functionality of templates, but also the important role of metal ions in therapy/diagnosis. We herein described two typical examples to evidence this predominant advantage. The possibility of FeIIITA as a T1-weighted MR imaging agent has been described based on the in vitro outcome. Similar to bare FeIIITA as reported in references,[11] PNV@FeIIITA gave the longitudinal relaxivity coefficient (r1) of 4.19 mM-1s-1 (Figure 4A), comparable to that of commercially available Gd-based T1 contrast agents.40 In this study, we further tested the 12 ACS Paragon Plus Environment

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performance of PNV@FeIIITA for in vivo T1-weighted MR imaging. The tumor-bearing mice were injected with PNV@FeIIITA ([FeIIITA] = 20 mg kg-1) via tail vein, leading to the evident intensification of T1-weighted MR contrast at tumor site at 4 h postinjection (Figure 4B). The transverse relaxivity coefficient (r2) value of PNV@FeIIITA was determined to be 12.03 mM-1s-1, which was too low to perform as a T2-weighted contrast agent (Figure 4C). In the same preparation way, MnII was additionally doped to provide PNV@FeIIIMnIITA (FeIII/MnII = 0.82, mol/mol). Its r2 value was elevated by 6 folds (~71.27 mM-1s-1) compared with that of parent PNV@FeIIITA (Figure 4D), which is sufficient to acquire effective in vivo T2-weighted MR imaging of tumors (Figure 4B).41, 42 Interestingly, the addition of MnII concurrently caused the promotion of r1 up to 6.90 mM-1s-1 (Figure 4E) and the enhancement of in vivo T1-weighted MRI contrast at the tumor (Figure 4B). Collectively, all the results indicated that photothermal PNV@FeIIIMnIITA can serve as a double-modal MR imaging contrast agent as well. It is noted that the doping of MnII imparted negligible influences on the photothermal effect of PNV@FeIIITA (Supporting Information, Figure S11). As aforementioned, the import of templates into the MITA-based photothermal materials offers a powerful pathway to introduce the special functionality of templates for the advanced applications. Polymeric vesicles are able to encapsulate the water-soluble substances (e.g., proteins, genes, hydrophilic fluoroprobes) within the inner cavity or embed the hydrophobic agents (e.g., poorly water-soluble drugs) in the polymer domain.43, 44 In this study, hydrophilic Cy 5.5 was encapsulated into the inner core of PNV@FeIIITA to validate the possibility for in vivo NIRF imaging. As shown in Figure 4F, tumor bearing mice were intravenously injected with Cy5.5@PNV@FeIIITA suspension and monitored with time using in vivo fluorescence imaging. The result indicated that Cy5.5@PNV@FeIIITA effectively accumulated at the tumor and the tumor-localized fluorescence signals remained always at a high level during a long observation period of 24 hours, 13 ACS Paragon Plus Environment

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suggesting the excellent tumor-targeted NIRF imaging performance (Figure 4F,G). Though being not investigated, we can image that it is also achievable for the combination of chemotherapy and photothermal therapy by fabricating MITAs onto the templates of mesoporous silicon nanoparticle and polymeric nanoparticle that allow the adsorption or entrapment of hydrophobic drugs into one nanoentity. Collectively, these typical examples adumbrated that in addition to the photo-activated applications as aforedemonstrated, versatile cooperation with other therapy approaches and diagnosis techniques can be easily accessible for this kind of photothermal materials owing to the special merits of MITAs.

CONCLUSIONS In summary, multimodal theranostics based on the cooperation among different therapy approaches, among different imaging techniques, or between therapy approaches and imaging techniques are clinically demanded for advanced applications to offer more precise disease diagnosis and more effective personalized therapy. PTT represents one potent therapy modality particularly for cancer treatments. However, the types of PTT agents are very scarce to date and noticeably, their access to those advanced applications remains significant challenge mainly due to the technical hurdles against the function integration and the poor loading capacity to the desired agents. This study reported a family of photothermal MITA materials with a high photothermal conversion efficiency (η ~40%). MITAs are highlighted by the significant advantages over the currently existing PTT materials, including the green synthesis, the facile incorporation of diagnostic metal ions, and particularly the excellent adhesion capability. Owing to the special adhesion nature of MITAs, various kinds of MITA-based nanostructures are readily available through the self-driven formation of MITAs onto diverse templates, enabling MITAs 14 ACS Paragon Plus Environment

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well suited as a photothermal platform for versatile cooperation with other therapy approaches and diagnosis techniques. Importantly, the introduction of templates would not compromise the performance of photoactivated heat generation. As the proof of concept, we validate the potency of nanovesicle-supported FeIIITA (PNV@FeIIITA) for photo-responsive utilization, including NIR photothermal tumor ablation, photothermal imaging and photoacoustic imaging in addition to the effective T1-MRI imaging. PNV@FeIIITA can be further equipped with other functionalities, such as T2-weighted MR imaging by additionally doping MnII, and in vivo fluorescence imaging by encapsulating hydrophilic NIR fluoroprobe. All the encouraging results suggest that MITAs could be developed as a versatile photothermal platform for various advanced applications.

EXPERIMENTAL SECTION Materials. Tannic acid, Manganese (II) Chloride Tetrahydrate (MnCl2.4H2O), Copper (II) chloride dihydrate (CuCl2.2H2O), Iron (III) chloride hexahydrate (FeCl3.6H2O), Nickel (II) chloride hexahydrate (NiCl2.6H2O), Ruthenium (III) chloride anhydrous (RuCl3), Vanadium (III) chloride (VCl3), Gadolium (III) chloride hexahydrate (GdCl3.6H2O), polyvinyl alcohol (PVA, Mowiol® PVA-105), Poly(D,L-lactide-co-glycolide) (PLGA, lactide:glycolide 50:50, Mw: 38000-54000) and Chloral hydrate were purchased from Aladdin Reagent (Shanghai, China). Dimethyl sulfoxide (DMSO), dichloromethane (DCM), Hexadecyl trimethyl ammonium bromide (CTAB), and tetraethylorthosilicate (TEOS) were purchased from Shanghai Reagent Chemical Co. (China). Dulbecco’s modified Eagle’s medium (DMEM), RMPI medium 1640, penicillin−streptomycin, fetal bovine serum (FBS), trypsin, 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) and Dulbecco’s phosphate buffered saline (PBS) were


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purchased from Invitrogen. calcein acetoxymethyl ester (calcein-AM), and propidium iodide (PI) were purchased from Sigma-Aldrich. Instruments. A probe sonicator (LC-1000) was used for the emulsion process. The hydrodynamic size and zeta potentials of each product was measured by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK). The morphology of each product was observed using transmission electron microscopy (TEM, JEOL-2100, Tokyo, Japan) and Scanning Electron Microscope (SEM, Zeiss Sigma FESEM). Fluorescence images were obtained using a confocal laser scanning microscope (CLSM, C1-Si, Nikon, Tokyo, Japan). The UV-Vis absorption was recorded by UV-Vis Spectrometer (Lambda Bio40, PerkinElmer). The concentration of Fe and Mn was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, IRIS Intrepid II XSP, USA Thermo Elemental). 808 nm and 660nm NIR laser (STL808T1-7.0W, Beijing STONE Laser) were used for NIR irradiation and the temperature change was monitored by a FLIR Ax5 camera (FLIR Systems AB, Sweden). Magnetic Resonance Imaging (MRI) was conducted on a Bruker BioSpec 7T/20 cm system (Bruker, Ettlingen, Germany). The in vivo NIRF imaging was obtained using IVIS imaging systems (PerkinElmer). Photoacoustic (PA) imaging system built by Huazhong University of Science and Technology was used for PA imaging.45, 46 Synthesis

of

PNS@FeIIITA,

PNV@FeIIITA,

Cy5.5@PNV@FeIIITA

and

PNV@FeIIIMnIITA nanoparticles. Prior to the fabrication of PNS@FeIIITA nanoparticles, single emulsion (O/W) method was conducted to provide PLGA nanospheres (PNS). In brief, 50 mg of PLGA was dissolved in 1 mL of dichloromethane (DCM), then 5 mL of PVA (50 mg mL1

) solution in ultrapure water was added and emulsified with a probe sonicator for 5 min under an

ice bath. After sonication, the emulsion was poured into 100 mL of PVA (5 mg mL-1) solution in 16 ACS Paragon Plus Environment

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ultrapure water and stirred overnight to solidify the nanospheres. The PNS were collected by centrifugation (8000 rpm, 20 min) and subsequently rinsed with ultrapure water for 3 times. PNS@FeIIITA was fabricated by dispersing 1 mg of PNS into 2 mL of ultrapure water and subsequently followed by the addition with 10 µL of tannic acid (40 mg mL-1) solution and 20 µL of FeCl3 (10 mg mL-1) solution under vortex. The solution pH was slowly raised to ~7.0 using 0.1 mol L-1 NaOH solution under vortex.20 Finally, PNS@FeIIITA nanoparticles were harvested by centrifugation (8000 rpm, 5 min) and rinsed with ultrapure water thrice to remove unreacted tannic acid and FeIII. Before the preparation of PNV@FeIIITA, double emulsion (W/O/W) method was adopted to prepare PLGA vesicles (PNV). Briefly, the mixture of 1 mL of ultrapure water and 4 mL of PLGA solution in DCM (10 mg mL-1) was emulsified with a probe sonicator for 2 min under ice. The obtained W/O emulsion was then introduced into 15 mL of PVA solution (20 mg mL-1) in water and further emulsified for another 10 min under ice bath to give W/O/W double emulsion. After that, the double emulsion was poured into 100 mL of PVA (5 mg mL-1) solution and stirred overnight to evaporate DCM in order to solidify PNV. Then, PNV were collected by centrifugation (8000 rpm, 20 min) and washing repeatedly. To prepare PNV@FeIIITA, 1mg of PNV was dispersed in 2 mL of ultrapure water and followed by adding 10 µL of tannic acid (40 mg mL-1) solution and 20 µL of FeCl3 (10 mg mL-1) solution under vortex. The pH of the solution was adjusted to ~7.0 with 0.1 mol L-1 NaOH under vortex. PNV@FeIIITA nanoparticles were collected by centrifugation and washed with ultrapure water thrice. Cy5.5@PNV@FeIIITA was fabricated in the similar way, whereas 1 mL of PVA (20 mg mL-1) solution containing 10 mg of hydrophilic Cy5.5 was used instead of

1 mL of ultrapure water in the initial W/O emulsion

procedure.


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To prepare PNV@FeIIITA with different shell thickness, different amount of PNV were dispersed in 2 mL of ultrapure water, then subsequently followed by adding 10 µL of tannic acid (40 mg mL-1) solution and 20 µL of FeCl3 (10 mg mL-1) solution under vortex. After that, the pH was adjusted to ~7.0 with 0.1 mol L-1 NaOH solution under vortex. The products were collected by centrifugation and rinsed with ultrapure water repeatedly. To prepare PNV@FeIIIMnIITA, 1 mg of PNV were dispersed in 2 mL of ultrapure water, then 10 µL of tannic acid (40 mg mL-1) solution, 20 µL of FeCl3 (10 mg mL-1) and 20 µL of MnCl2 (10 mg mL-1) solution were added under vortex. The pH of the solution was adjusted to ~7.0 with 0.1 mol L-1 NaOH solution under vortex. The product was collected by centrifugation and rinsed with ultrapure water repeatedly. Preparation of CuIITA, MnIITA, NiIITA, GdIIITA, RuIIITA, VIIITA, FeIIITA. To prepare different MITAs (Metal/TA = 3.15, mol/mol) complex, 100 µL of tannic acid (40 mg mL-1) was added into 20 mL of ultrapure water, then 200 µL of FeCl3.6H2O (10 mg mL-1), CuCl2.2H2O (6.3 mg mL-1), MnCl2.4H2O (7.33 mg mL-1), NiCl2.6H2O (8.79 mg mL-1), GdCl3.6H2O (13.75 mg mL-1), RuCl3 (7.67 mg mL-1) and VCl3 (5.82 mg mL-1) was added respectively under sonication. The pH of the solutions was raised to ~7.0 by adding 0.1 N NaOH solution under vortex.20, 21 The solution was directly used for the photothermal evaluation. MITAs complexes with different Metal/TA ratio were prepared in the similar way. Preparation of MSN and MSN@FeIIITA. Mesoporous silica nanoparticles (MSN) were prepared using the previously reported method.47 MSN@FeIIITA was prepared using the same method of PNV@FeIIITA preparation by replacing PNV template with MSN. Photothermal Evaluation of MITA Complex. To study the photothermal performance of different metal based MITA complex, 100 µg mL-1 of MITAs (CuIITA, MnIIA, NiIITA, GdIIITA, 18 ACS Paragon Plus Environment

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RuIIITA, VIIITA and FeIIITA) dispersion in water were irradiated with 808 nm or 660 nm laser. The real-time temperature was monitored. To study the relationship between metal content in MITA complex and photothermal efficiency, 100 µg mL-1 of CuIITA, MnIIA, NiIITA, GdIIITA, RuIIITA, VIIITA and FeIIITA suspension prepared with various Metal/TA feeding ratio were used for comparison. Photothermal Evaluation over FeIIITA Nanoentities Supported by Different Templates and PNV@FeIIITA with Different Shell Thickness. To validate the influence of different templates on FeIIITA photothermal performance. FeIIITA complex, PNS@FeIIITA, PNV@FeIIITA and MSN@FeIIITA dispersions with FeIIITA content of 100 µg mL-1 were irradiated with laser (808, 2 W cm-2) for 10 min respectively. To explore the influence of FeIIITA shell thickness of template supported FeIIITA on photothermal performance, PNV@FeIIITA ([FeIIITA] = 100 µg mL-1) with various FeIIITA shell thickness were used under laser irradiation (808, 2 W cm-2) for comparison. Evaluation of Photothermal Performance, Photothermal Conversion Efficiency and Photothermal Stability. To study photothermal performance of FeIIITA and PNV@FeIIITA, 1 mL of FeIIITA and PNV@FeIIITA solutions with different FeIIITA content ([FeIIITA] = 0-200 µg mL-1) were suspended in vials and then irradiated with 808 nm laser (2 W cm-2) for 600 s. The temperature of the solution was recorded by IR camera. To determine photothermal conversion efficiency of FeIIITA, the method established by the previous report was used. 33, 34, 48 Briefly, 1 mL of FeIIITA (100 µg mL-1) solution in vials were irradiated under 808 nm NIR laser for 720 s. Then the laser was turned off and the temperature change of the solution was monitored by an IR camera. The time constant (τs) for heat transfer


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was determined by applying the linear time-dependent data collected during the cooling period. The photothermal conversion efficiency (η) was calculated by following equations:33, 34, 48

η=

hA ∆Tmax – Qs I (1-10-Aλ)

In this equation, η is conversion efficiency value, ∆Tmax is the maximum temperature change when the temperature reaches a steady-state. Qs is relevant to the light absorbance of pure water here. I is the laser power density. Lastly, Aλ is the absorbance of the solution in the UV-Vis spectrum. Only the value of hA was unknown and could be calculated from the following equation.

τs=

mD cD hA

mD is the mass of water used to suspend the nanoparticles, cD here is the heat capacity of water and τs is time constant of the solution and could be calculated according to the temperature cooling curve. Photothermal conversion efficiency of RuIIITA and VIIITA were also calculated by the same method. The photothermal stability of PNV@FeIIITA was evaluated by four circles of laser turn-on and turn-off. Briefly, 1 ml of PNV@FeIIITA dispersion ([FeIIITA] = 100 µg mL-1) was irradiated under NIR laser (808 nm, 2 W cm-2) for 10 min, then the laser was turned off for 25 min. This operation cycle was repeated for four times.


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Cell Culture. 4T1 murine breast cancer was incubated in RMPI 1640 medium containing 10% FBS and 1% antibiotics (penicillin-streptomycin, 10,000 U mL-1) at 37 oC in a humidified atmosphere containing 5% CO2. Live/Dead Cell Staining. 4T1 cells were seeded in 6-well plates at a density of 5 × 105 cells per well and incubated for 24 h, then the medium was replaced with the fresh medium containing 50 µg mL-1 of PNV@FeIIITA and incubated for 4 hours, after that, the medium was removed and washed with PBS then followed by adding fresh medium and irradiated with a 808 nm NIR laser at a power density of 2 W cm-2 for 300 s. The cells that without any treatment, singly performed with NIR laser irradiation and only treated with PNV@FeIIITA incubation were tested as control. Finally, the cells were co-stained with calcein-AM (4 µM) and PI (4 µM) in PBS for 30 min at 37 o

C with 5% CO2. The cells were washed with PBS for 3 times and the fluorescence of the

samples were observed with and observed with CLSM (C1-Si, Nikon, Tokyo, Japan). The excitation wavelength was set up to 488 nm and 543 nm while the emission spectra was collected using 510-540 nm and 570-620 nm respectively. Evaluation of Cytotoxicity. MTT assay was used to evaluate the photothermal cytotoxicity of PNV@FeIIITA. 4T1 cells were seeded in 96-well plate at a density of 5000 cells per well and incubated in 100 µL of RPMI medium 1640 with 10% FBS and 1% antibiotics at 37 oC in a humidified atmosphere containing 5% CO2 for 24 h. Then, another 100 µL of RPMI medium 1640 containing different concentrations of PNV@FeIIITA were added into each well followed by another 24 hours of incubation. After that, 20 µL of MTT solution (5 mg mL-1 in PBS) were added into each well and incubated for another 4 hours. Finally, the medium was removed and replaced with 150 µL of DMSO. The absorbance of the solution was then measured by a microplate reader at the fixed wavelength of 570 nm. 21 ACS Paragon Plus Environment

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To evaluate photothermal induced cytotoxicity of PNV@FeIIITA, 4T1 cells were seeded in 96-well plate at a density of 5000 cells per well and incubated in 100 µL RPMI medium 1640 with 10% FBS and 1% antibiotics at 37 oC in a humidified atmosphere containing 5% CO2 for 24 h. Then, another 100 µL RPMI medium 1640 containing different concentrations of PNV@FeIIITA were added into each well followed by another 4 hours of incubation. Thereafter, the medium were removed and replaced with fresh medium followed by exposing to 300 s laser irradiation (808 nm, 2 W cm-2) and incubated for 20 h. After that, 20 µL of MTT solution (5 mg mL-1 in PBS) were added into each well and incubated for another 4 hours. Finally, the medium was removed and replaced with 150 µL of DMSO. The absorbance of the solution was then measured by a microplate reader at the fixed wavelength of 570 nm. Animals and Tumor Models. 4-5 weeks old female BALB/c mice were purchased from Wuhan University Animal Biosafty Level III Lab. And all research protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University (Wuhan, China). 4T1 murine breast tumor models were obtained by subcutaneously injecting 4T1 tumor cells (1 × 107 cells suspended in 100 µL of PBS) into the flank of each mouse. The tumors were allowed to grow up to 100-150 mm3 for further use. In Vivo NIRF Imaging. 4T1 tumor-bearing BALB/c mice with the tumor size ~150 mm3 were tail vein injected with Cy5.5@PNV@FeIIITA dispersion. Then the mice were placed in IVIS imaging systems to observe NIRF imaging. The excitation wavelength was fixed at 675 nm and the emission wavelength was collected at 720 nm. In Vitro and In Vivo MR Imaging. Both T1-weighed and T2-weighed imaging in vitro and in vivo were conducted on a Bruker BioSpec 7T/20 cm system (Bruker, Ettlingen, Germany). For in vitro imaging, PNV@FeIIITA and PNV@FeIIITAMnII with different concentration gradients of 22 ACS Paragon Plus Environment

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Fe and Mn were prepared and sealed in NMR tubes for test. Concentration of Fe and Mn elements were determined by ICP-AES. For in vivo imaging, 4T1 tumor-bearing BALB/c mice with tumor size ~150 mm3 were intraperitoneal injected with 5% chloral hydrate solution for anesthetization and then scanned before and at 4h after the injection with PNV@FeIIITA and PNV@FeIIIMnIITA suspension via tail vein respectively (at an equivalent dosage of 20 mg [FeIIITA] per kg body weight) for MR imaging. In Vitro and In Vivo PA Imaging. To study PA imaging property of PNV@FeIIITA, PNV@FeIIITA with different FeIIITA concentrations ranging from 25 µg mL-1 to 200µg mL-1 were put in PA imaging system then scanned to obtain in vitro PA imaging. For in vivo imaging, 4T1 tumor-bearing BALB/c mice with tumor size ~150 mm3 were intraperitoneal injected with 5% chloral hydrate solution for anesthetization. Then the tumor-bearing mice were scanned with PA imaging system at tumor sites to obtain PA signals before and after tail vein injection of 200 µL PNV@FeIIITA solution (at an equivalent dosage of 20 mg [FeIIITA] per kg body weight). The wavelength of the laser was fixed at 740 nm, and and the laser energy per pulse was 140 nJ. In Vivo Photothermal Performance and Tumor Therapy. When the tumor volume grew up to ~100 mm3. The tumor-bearing mice were randomly divided into four groups (n = 4) and treated with (1) PBS, (2) PBS+Laser, (3) PNV@FeIIITA, (4) PNV@FeIIITA+Laser respectively by tail vein injection. NIR irradiation (808 nm) last for 6 min with the power intensity of 2 W cm2

. The body weight and tumor volume of each mouse was recorded every 2 days. Tumors size

was measured by a caliper and tumor volume was calculated by the following formula: V=L×W2/2, where L and W represent the maximum diameter and the minimum diameter of the tumor. At 14th day, all the mice were sacrificed and all the tumors and major organs (heart, liver, spleen, kidneys, lung) were harvested and preserved in 4% paraformaldehyde solutions for 23 ACS Paragon Plus Environment

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histological analysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website or from the author.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Jun Feng: 0000-0002-1725-140X Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by the National Key Research and Development Program of China (2016YFC1100703) and the National Natural Science foundation of China (51533006, 21374085).


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Scheme 1. Schematic illustration of the cooperation of adhesive MITAs with diverse templates for the advanced applications, including photothermal imaging (PTI), photoacoustic imaging (PAI), T1-, T2-weighted MR imaging (MRI), and near infrared fluorescence (NIRF) imaging together with photothermal therapy.


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Figure 1. Photothermal evaluation of different MITAs. (A) Photothermal effect of FeIIITA (FeIII/TA = 3.15, mol/mol) solutions at different concentrations under laser irradiation (808 nm, 2 W cm−2); (B) Photothermal effect and (C) UV-Vis-NIR absorbance of FeIIITA (100 µg mL-1) with different FeIII/TA ratios; (D) UV-Vis-NIR absorbance of CuIITA, MnIITA, NiIITA, GdIIITA, RuIIITA, VIIITA and FeIIITA (100 µg mL-1, Metal/TA = 3.15); (E) Photothermal effect of different MITAs (100 µg mL-1, Metal/TA = 3.15) upon 808 nm laser (2 W cm-2) irradiation for 600 s; (F) Photothermal effect of VIIITA, RuIIITA (G) and GdIIITA (H) with different metal/TA ratios; Photographs of (I) CuIITA, MnIITA, NiIITA, GdIIITA dispersion and (J) FeIIITA, RuIIITA, 33 ACS Paragon Plus Environment

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VIIITA dispersion in DI water at a concentration of 100 µg mL-1. The values represent the molar ratio of metal versus of TA.


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Figure 2. Morphology characterization of different templates supported FeIIITA nanoparticles and relative photothermal evaluation. TEM images of (A) PNS@FeIIITA (inset~ PNS), (B) PNV@FeIIITA (inset~ PNV) and (C) MSN@FeIIITA (inset~ MSN). (scale bar: 100 nm); DLS profiles of (D) PNS, PNS@FeIIITA, (E) PNV, PNV@FeIIITA and (F) MSN, MSN@FeIIITA; (G) Thermal images of PNV@FeIIITA solutions treated with laser irradiation (808 nm, 2 W cm-2); (H) Photothermal evaluation of PNV@FeIIITA solutions with different concentrations under laser 35 ACS Paragon Plus Environment

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irradiation (808 nm, 2 W cm-2); (I) Photothermal evaluation of FeIIITA (100 µg mL-1) based on different substrates under laser irradiation (808 nm, 2 W cm-2); (J) Photothermal evaluation of PNV@FeIIITA ([FeIIITA] = 100 µg mL-1) with different thickness of FeIIITA shell (insets show PNV@FeIIITA with shell thickness of ~13 nm and ~56 nm); (K) Temperature change of PNV@FeIIITA solutions (100 µg mL-1) for 600 s laser irradiation (808 nm, 2 W cm-2) followed by the cooling period. The inset shows the time constant (τs) for heat transfer in this system, which was determined to be τs=430 s based on the linear time-dependent data (R2=0.998) collected during the cooling period. (L) Temperature curve of PNV@FeIIITA (100 µg mL-1) with repeated irradiation (808 nm, 2 W cm-2) on/off cycles.


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Figure 3. (A) In vivo photothermal images of 4T1 tumor-bearing mice recorded at 4 h post intravenous injection of PBS and PNV@FeIIITA. (B) Temperature variation at tumor sites irradiated with NIR laser (808 nm, 2 W cm-2). (C) In vivo 2D- and 3D- PA tumor imaging before and at 4 h after intravenous injection of PNV@FeIIITA (744 nm). (D) In vitro PA signals of PNV@FeIIITA solutions with different concentrations. (E) CLSM images of 4T1 cells treated with single laser irradiation (808 nm, 2 W cm-2) for 5 min, 50 µg mL-1 PNV@FeIIITA alone, and 50 µg mL-1 PNV@FeIIITA plus laser irradiation (808 nm, 2 W cm-2) for 5 min, respectively (Scale bar: 100 µm). (F) Photographs of tumors harvested after different treatments after 14 days. (G) Variation of relative tumor volume during 14 days after different treatments. (H) Variation of relative body weight during 14 days after different treatments. (I) Images of H&E stained tumor slices from different treatments after 14 days.


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Figure 4. (A) In vitro T1-weighted images (inset) of PNV@FeIIITA solution with different concentrations and the corresponding T1 relaxation rate (r1). (B) Representative in vivo T1weighted MR images and pseudo-color maps of 4T1 tumor bearing mice before and at 4 h after intravenous injection of PNV@FeIIITA ([FeIIITA] = 20 mg kg-1), and T1, T2-weighted MR images and pseudo-color maps of 4T1 tumor bearing mice before and at 4 h after intravenous injection of PNV@FeIIIMnIITA ([FeIIITA] = 20 mg kg-1), the tumors are marked with yellow circles. In vitro T2-weighted images (inset) of PNV@FeIIITA (C) and PNV@FeIIIMnIITA (D) solutions with different concentrations and the corresponding T2 relaxation rate (r2). (E) In vitro T1-weighted images (inset) of PNV@FeIIIMnIITA solution with different concentrations and the corresponding T1 relaxation rate (r1). (F) In vivo NIRF imaging of 4T1 tumor bearing mice after intravenous injection of Cy5.5@PNV@FeIIITA obtained at different time intervals. (G) Ex vivo fluorescence images of tumor and major organs at 24 h postinjection. 39 ACS Paragon Plus Environment

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Tannic Acid Assembly as a Versatile Photothermal Platform

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