Water-Dispersible Prussian Blue-Hyaluronic Acid ... - ACS Publications

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Functional Inorganic Materials and Devices

Water-Dispersible Prussian Blue-Hyaluronic Acid Nanocubes with Near-Infrared Photoinduced Singlet Oxygen Production and Photothermal Activities for Cancer Theranostics Bo Zhou, Bang-Ping Jiang, Wanying Sun, Fang-Mian Wei, Yun He, Hong Liang, and Xing-Can Shen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01387 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 10, 2018

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ACS Applied Materials & Interfaces

Water-Dispersible Prussian Blue-Hyaluronic Acid Nanocubes with Near-Infrared Photoinduced Singlet Oxygen Production and Photothermal Activities for Cancer Theranostics Bo Zhou,a Bang-Ping Jiang,a Wanying Sun,a Fang-Mian Wei,a Yun He,b Hong Liang,a Xing-Can Shena a

Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of

Chemistry and Pharmaceutical Science, Guangxi Normal University, Guilin, 541004, P. R. China b

College of Physics and Technology, Guangxi Normal University, Guilin, 541004, P. R. China

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ABSTRACT: Design and development of photosensitizers that can efficiently convert energy of near infrared (NIR) laser irradiation are of major importance for cancer photoassisted therapeutics. Herein, for the first time, it is demonstrated that Prussian blue (PB), a classic coordination compound can act as a novel photosensitizer with efficient generation of singlet oxygen and excellent photothermal conversion via a NIR photo irradiation-induced energy-transfer. After modification with hyaluronic acid (HA), the as-prepared HA-modified PB nanocubes (HA@PB) are highly dispersible in aqueous and physiological solutions, as well as shows excellent photothermal/phodynamic activities under NIR (808 nm) photoexcitation. Based on these features, HA@PB are used to study their in vitro and in vivo combined therapeutic effect. Owing to the CD44 ligand of HA, HA@PB have specific uptake by CD44-positive cells in vitro and can be precisely in vivo delivered to the tumor site. HA@PB as one of the synergistically photodynamic/photothermal combination nanoplatforms could achieve excellent therapeutic

efficacy

with

targeted

specificity

under

the

guidance

of

dual-modality

photoacoustic/infrared thermal imaging. Hence, this work is expected to pave the way for using PBbased nanomaterials as a promising multifunctional theranostic nanoplatform in biomedical fields.

KEYWORDS: Prussian blue, hyaluronic acid, theranostics, photothermal therapy, photodynamic therapy

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1. INTRODUCTION Reactive oxygen species (ROS), including hydroxyl radicals (•OH), singlet oxygen (1O2), and superoxide (O2), are highly reactive oxygen intermediates.15 Among them, active 1O2 has strong oxidizing properties, and it can persist for 106103 seconds in solution and about an hour in gaseous phase.1 It plays an vital role in the application for photodynamic therapy (PDT).6 PDT, an intriguing treatment pattern in the field of cancer therapy, which involves photosensitizers (PSs) to effectively transfer the absorbed optical energy (particularly, near infrared (NIR) light) to the surrounding oxygen molecules, generating cytotoxic ROS to kill cancer cells, have been extensively studied as alternative therapy for malignant diseases due to their good therapeutic efficiency, minor injury, remote controllability, as well as low systemic toxicity.7,8 The design and development of NIR mediated PSs for efficient production of 1O2 is a prerequisite for PDT application.9 In addition, from the perspective of practical application, only single-modal PDT cannot completely satisfy the current treatment requirements due to its inherent defects including low ROS production in the hypoxic tumor microenvironment, short permeation distance resulted from excitation wavelength below 700 nm, etc.1012 Based on these, new modalities of synergistic therapy that combines with PDT (such as photothermal therapy (PTT), have been explored intensively to overcome the drawbacks of PDT, which is a promising strategy to take advantage of both therapies while reducing the drawbacks of each and enhancing therapeutic efficiency in a synergistic manner.1317 To construct this kind of combined therapy systems, it is usually required that individual integration of PTT agent and PSs into a nanoplatform.18 For example, photothermal nanomaterials, such as carbon nanotubes,18 gold nanoagents,1921 nanographene oxide,22 and two3



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dimensional (2D) nanoscaled transition-metal dichalcogenides,23 etc., have been used as PSs carriers for the achievement of synergistic PDT/PTT. Despite the great progress have been acquired in this field, cancer treatment based on the integration of PTT and PDT remains a daunting task. On one hand, the integration of different components not only needs tedious synthetic procedures, but also may cause unpredictable mutual interference between the components, together with easy to leak during their circulation in blood.10,11 On the other hand,

at most times

two kinds of different wavelength lasers are needed to respectively activate PTT agent and PSs because of the absorption mismatch of these photoactive agents, causing a comparatively higher treatment costs and longer treatment time.10,24,25 As a consequence, PSs that simultaneously possess the characteristics of production of ROS and local hyperthermia triggered by a single NIR laser will undoubtedly bring about better operability of the synergistic PDT/PTT system and remarkably enhanced therapeutic efficiency in further clinical implementation.26 Recently, plasmonic semiconductor nanomaterials have been used to achieve the synergetic PTT/PDT under a single NIR light excitation by many groups.10,2632 However, such class of ideal synergetic PTT/PDT nanoplatforms is still rarely reported. Thereby, it is greatly anticipated to design and develop biocompatible PSs that intrinsically have both PTT and PDT functions to realize synergistic anticancer effects under excitation of single NIR light. Prussian blue (PB), an empirical formula of Fe4[Fe(CN)6]3 that is one kind of mixed-valence iron hexacyanoferrate, has been extensively studied since its discovery and is approved for radioactive exposure treatment by the US Food and Drug Administration (FDA).3336 PB-based materials have attracted wide attention and great interests from the physicists, chemists, and materials scientists owing to their unique properties such as an intense blue color, magnetism, 4


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zeolitic character, semiconductor behavior, good biocompatibility, excellent stability, etc.3742 To date, PB-based nanomaterials have been extensively studied as a new class of multifunctional nanoplatforms, with multiple promising properties including T1-weighted magnetic resonance,34 photoacoustic (PA) effect,35,37 and PTT after NIR photoexcitation,3337 which exhibited excellent potential application in multimodal-imaging guided PTT. Despite the promise, several factors restrict their practical applications, as following: (1) bare PB are physiologically unstable in absence of surface modification, resulting in unsuitable in vivo application;43 (2) surface-modified substances are usually used to just enhance the water dispersibility of PB, lacking the introduction of other specific functions;44 (3) the introduction of specific functions usually involves multiple synthetic steps and contains a variety of ingredients, which suffers from unpredictable mutual interference between the components.45 Beside that, the phototherapeutic effect of PB-based nanomaterials involves NIR-induced PTT alone, and there is no focus on the NIR-induced PDT application. In this work, we demonstrate that PB shows exceeding capability for the production of 1O2 under NIR photoexcitation for the first time. To resolve the poor colloidal stability and improve the biocompatibility of PB, hyaluronic acid (HA), a water-soluble negatively charged polysaccharide, is modified on the PB surface via a facile one-step approach. It is well-known that besides the good water solubility of HA, HA also has outstanding active the target feature.46 It can selectively bind to the surface of cancer cells that with overexpressed of specific receptors (e.g. CD44), showing great potential for various medical and biological applications, including imaging,46 drug delivery,47 gene delivery,48 etc. Compared to other HA-conjugated PB therapeutic systems,51,52 the surface HA-modified PB nanocubes (HA@PB) are easily prepared, which can 5



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avoid the yield of toxic materials. More importantly, HA@PB not only focus on NIR-induced PTT, but also concern the NIR-induced PDT application under excitation of single NIR light, providing the possibility for synergistically targeted cancer therapy. Moreover, as the contrast agent, HA@PB are also applied for infrared thermal imaging (TI) and photoacoustic (PA) imaging of mice with tumors. Thus, this work opens up a new access to use PB-based nanomaterials as promising multifunctional theranostic nanoplatforms for biomedical applications (Scheme 1).

Scheme 1. Schematic illustration of fabrication of HA@PB and their targeted PTT/PDT of CD44overexpressing cancer cells guided by dual-modality TI/PA imaging. 2. RESULTS AND DISCUSSION 2.1 Preparation and Characterization of HA@PB. Water-dispersible HA@PB were prepared through a facile one-step approach (Scheme 1). The preparation involved the slow addition of a (NH4)2Fe(SO4)2 solution containing HA into an solution of K3[Fe(CN)6] with HA at 90 C under vigorously stirring. As a control, unmodified PB was also synthesized following the same protocol but without adding any HA. After the preparation, the crystallography and phase composition were primarily carried out through X-ray diffraction (XRD) measurement. As can be seen in Figure S1, HA@PB show the presence of peaks at 17.30, 24.62, 35.21, 39.37, 43.45, 50.68, 53.72 and 6


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57.16 which can be respectively allocated to the 200, 220, 400, 420, 422, 440, 600, and 620 crystal planes of PB. This result agrees with the cubic face-centered PB lattice (space group Fm3m).34,36 Moreover, compared to the XRD peak of unmodified PB, that of HA@PB at around 2 = 24.62 is broader due to amorphous HA. It indicates that PB was successfully coated by HA matrix (Figure S1).37 The UV-Vis-NIR spectrum was performed to study the absorption behavior of the asprepared HA@PB in water. HA@PB display a broad 500900 nm absorption, with a centered peak at 697 nm (Figure 1a), which can be assigned to an intervalence charge-transfer of PB.33 For comparison, the UV-Vis-NIR spectrum of unmodified PB was also performed, where insignificant change in absorption is found, except the weaker intensity absorption compared to that of HA@PB (Figure 1a). This observation indicates that the modification of PB with HA does not alter the inherent structure of PB. A visual comparison between water dispersiblility of unmodified PB and HA@PB is showed in the Inset of Figure 1a. HA@PB are highly dispersible in aqueous solutions, whereas unmodified PB settles down in water after 7 days. In particular, the difference between the dispersibility of the two materials is obvious from the different intensity of the colors of the two suspensions, in good agreement with the results of UV-Vis-NIR spectrum. This significant difference in the dispersibility confirms the successful synthesis of HA@PB.

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Figure 1. (a) UV-Vis-NIR spectra of unmodified PB and HA@PB in water. Inset: Photographs of unmodified PB (left) and HA@PB (right). (b) FT-IR data of HA, unmodified PB, and HA@PB. (c) TEM images of HA@PB. (d) DLS data of HA@PB in water.

The interaction between the HA and PB in HA@PB was confirmed through Fourier transform infrared (FT-IR) measurement. As can be found from Figure 1b, the peaks at 2865 and 2940 cm1 are assigned to the (CH) of CH2. Two peaks at 2086 cm1 and at 1045 cm1, which are respectively assigned to the (CN) of Fe2+CNFe3+ moiety PB37 and the (COC) of the HA skeleton, are also clearly observed.49,50 Meanwhile, the band at 1383 cm1 due to theCOO), is shifted to 1409 cm1. Referring to previous studies on the interactions between the carboxyl derivatives and Fe ions, the change can be ascribed to the complexation of the Fe ion with the carboxyl group of HA.5355 These FT-IR results confirm that the formation of HA@PB product driven by the complexation of the Fe ion with the carboxyl group of HA. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements were utilized to characterize size distribution and the morphology of HA@PB. It can be seen that HA@PB are monodisperse with a cube-like shape and 22.2 nm of size (Figure 1c), which is well agreement with the 8


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morphology of unmodified PB (Figure S2). In addition, DLS measurements show that the sizes of the unmodified PB and HA@PB are approximately 58 and 82 nm respectively (Figures 1d, S3a and S3b ). Relative to unmodified PB, the size of HA@PB is slightly larger, as a result of the coating of HA on PB (Figures S3a, S3b, S4a and S4b). Meanwhile, the zeta potentials of PB without and with HA coating decreased from 35 mV to 42 mV, indicating that PB with modification of HA could lead to the increase in negative surface charge of HA@PB (Figure S5). According to previous report, the increase in charge is conducive to the stability of nanomaterials,56 which is consistent with the results that HA@PB exhibit excellent water dispersibility than PB. It should be noted that in general, the negatively charged surface of nanomaterials has negative effect on the surface modification of molecules with the same charge. In HA@PB, the combination of HA and PB is achieved via complex interaction between the iron ion of PB and the carboxyl group of HA, not the charge interaction. Therefore, the negative charge on the surface of PB has a minor effect on the modification of negatively charged HA. To gain insight into the contents of the PB moiety and HA in HA@PB, inductively coupled plasma optical emission spectrometry (ICP-OES) and thermogravimetric analysis (TGA) were performed. Comparable results were obtained with the TGA and ICP-OES show that the average PB content estimated from TGA is 48 wt% (Figure S6) and that derived from ICP-OES being 46 wt% (Table S1).

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Figure 2. Photothermal images of HA@PB solution (25 g mL1) (a) and (b) pure water. (c) The corresponding time-dependent photothermal heating curves of samples. (d) UV-Vis spectra of DPBF+HA@PB with increasing irradiation time. (e) ESR spectra of HA@PB+TEMP in different conditions. Samples were irradiated by an 808-nm laser (1.0 W cm2).

2.2 Photothermal Properties and 1O2 Production by Aqueous HA@PB under 808 nm Light Irradiation. Similar to previous studies, HA@PB comprising PB fragments, display a broad 500900 nm absorption that centers around 700 nm (Figure 1a), which suggests the absorption in NIR range.34,37 To investigate the photothermal properties of HA@PB, the temperature variations of HA@PB in water under continuous exposure to an 808-nm laser with the power of 1.0 W cm2 for 10 min was recorded by a TI camera. As shown in Figure 2a, with increasing exposure time, the temperatures of HA@PB solution (25 g mL1) remarkably increase from 29 to 59 C within 10 min (T  30 C), corresponding to the color change of the photothermal images from purple (a low temperature) to bright red (a high temperature) (Figure 2c). In contrast, pure water exhibits no temperature change when exposed to 808-nm laser (Figure 2b). Such observations illustrate that HA@PB are able to conversion of light energy into thermal energy rapidly and efficiently. 10


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Additionally, obvious dose-dependent photothermal effect further demonstrates that HA@PB can act as an excellent PTT agent (Figure S7). Moreover, HA@PB exhibit remarkable photostability, which show no obvious change in UV-Vis-NIR spectrum of HA@PB even after irradiation for five cycles (Figure S8 and Figure S9). These results demonstrate that HA@PB are stable under irradiation and amenable for their effective PTT applications. It has been reported that PB-based nanomaterials can be excited by light-induced metal-to-metal charge transfer from FeIIINCFeII (the ground state) to FeIINCFeIII (the excited state), and with a broad absorption peak 1.75 eV, which is higher than 0.97 eV of the energy of 1O2.57,58 Thus, when PB in the ground state captures a photon and gets transitioned to the excited state, the excitation energy may be transferred to molecular oxygen to generate 1O2 during PB returns to the ground state. Herein, we expect that PB can serve as an attractive PSs for NIR light-induced ROS generation. Thus, the excellent water dispersibility of HA@PB is an advantage to study the ROS generation of PB materials in aqueous media as a model compound. To examine the capacity of HA@PB to generate ROS induced by light irradiation, the 1,3-diphenylbenzofuran (DPBF) chemical trapping method was used. It is well known that ROS (e.g., 1O2 and O2) can directly oxide DPBF to cause the oxidation product, which leads to decrease in the UV−Vis absorption.5961 The aqueous solution of HA@PB was illuminated using an 808-nm laser, because of its intense absorption shown by HA@PB at 808 nm (Figure 1a). As displayed in Figure 2d, the absorption centered at 425 nm decreased by 40% after 10 min irradiation in the presence of HA@PB. As a control, UV−Vis absorption spectra were also obtained for non-irradiated DPBF in the presence of HA@PB. It can be observed that almost no change is observed in the absorption spectrum upon

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mixing HA@PB and DPBF without irradiation (Figure S10). These results confirm that irradiation by 808 nm laser is essential for generating ROS. Next, to study the nature of the as-obtained ROS, electron spin resonance (ESR) experiments were further implemented, where common spin trap reagents (5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2,2,6,6-tetramethylpiperide (TEMP)) were utilized.29 After irradiated by 808-nm laser, the ESR signal of nitroxide radical is found as a result of the adduct TEMP with 1O2, revealing the effective generation of 1O2 (Figure 2e, blue line); in contrast, no significant signals of radical nitroxide is observed in the absence of laser irradiation (Figure 2e, red line). After DMPO reacts with OH and O2, DMPOOH and DMPO−OOH can be formed, respectively.29 However, in our case, insignificant signals of the DMPO−OH or DMPO−OOH are observed from the ESR spectra (Figure S11). These experimental results indicate that 1O2 was generated in the

aqueous suspension of HA@PB by 808-nm laser irradiation. Moreover, irradiation with a 650 nm laser can also produce 1O2 (Figure S12).

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Figure 3. UV-Vis-NIR spectra of (a) NMB, (b) HA@PB, and (c) unmodified PB in aqueous solution containing DAB irradiated by a 650-nm laser (1 W cm−2) with increasing irradiation time, respectively. (d) The corresponding absorption variations of the DAB solution at 500 nm. [DAB] = 500 μM.

It has been certified that 3,3’-diaminobenzidine (DAB) is highly selective towards 1O2, showing no obvious response toward OH or O2.60 Herein, DAB was used to evaluate the 1O2 quantum yield of HA@PB (HA@PB). It is known that new methylene blue (NMB) has high 1O2 quantum yield under 650 nm laser irradiation, thus, NMB was employed as a standard photosensitizer dye to investigate the 1O2 yield.60 Further, 650-nm laser irradiation was used to evaluate HA@PB. The UV-Vis spectra of a NMB solution containing DAB for various irradiation times with the 650-nm laser are shown in Figure 3a. The absorbance of DAB below 550 nm increases during the 650 nm laser irradiation, as a result of the oxidation of DAB by 1O2 induced by the NMB photosensitizer. The oxidation of DAB by 1O2 using HA@PB was investigated under the same conditions, and it can be observed that HA@PB also caused an increase in the absorbance of DAB (Figure 3b). Moreover, irradiated by 808 nm laser HA@PB can also efficiency produce 1O2 (Figure S13). It is known that in the presence of a photosensitizer, its 1O2 quantum yield is related to the initial reaction rate (Q) of 1O2 with a specific 1O2-captureting dye. Thereby, according to the following equation, we assessed Q of 1O2 with DAB in the presence of NMB or HA@PB by utilization of the above absorbance data:

Q  R/ A Where the value of the initial slope (A/Time) is considered to be equal to the R value (Figure 3d), and A is the absorbance of NMB or HA@PB at 650 nm. Following this protocol, the QHA@PB/QNMB was calculated to be 0.78, indicating the high 1O2 production efficiency of HA@PB, almost equal to that of NMB. As a comparative study, the value of Q unmodified 13


PB/QNMB

was also


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measured to be 1.06 (Figures 3c and 3d), where Qunmodified PB represent the Q of the PB prepared without HA, these results indicate that both HA@PB and unmodified PB can generate 1O2 under 650-nm laser irradiation. Considering the biomedical application, in our work, HA is used to improve the physiological stability and dispersibility of PB. Therefore, as expected, HA@PB can serve as a new NIR laser-induced PS for the generation of 1O2 and is an eligible candidate for further PDT.

Figure 4. XRD (a) and full XPS spectra (b) of HA@PB before and after irradiation using an 808-nm laser. The photoelectron spectroscopy of Fe 2p spectra (c, d), and

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Fe Mössbauer spectra (e, f) of

HA@PB before (c, e) and after (d, f) exposure to an 808-nm laser. The stability of PSs is a vital issue that needs to be taken into account for practical applications

in phototherapy. To get a more insight into the structural stability of HA@PB under irradiation, Xray photoelectron spectra (XPS), XRD, and 57Fe Mӧssbauer measurements were carried out. The XRD patterns of HA@PB at room temperature before and after irradiation are shown in Figure 4a. They are almost the same, and no detectable changes is found in its crystal structure. Other evidences are provided by XPS spectra of HA@PB. From the full XPS spectra (Figure 4b), the 14


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elemental mass fraction of surface C/O/N/Fe in HA@PB were obtained to be 67.79/11.51/17.33/3.38 and 67.56/11.53/17.54/3.40 before and after irradiation, respectively. This result shows that laser irradiation does not lead to any change in the composition of HA@PB. For HA@PB, similar conclusions are also drawn from the photoelectron spectroscopy of Fe 2p spectra, where no obvious changes are observed for the sample before and after irradiation (Figures 4c and 4d). Mӧssbauer spectra of

57

Fe were also carried out to clarify structural changes in HA@PB

during the irradiation process. Both before and after irradiation, a singlet absorption peak due to FeII and a doublet absorption peak attributed to FeIII are observed. The FeIII/FeII ratio is estimated to be 53.3/46.7 before irradiation (Figure 4e). After 808-nm laser irradiation, the FeIII/FeII ratio is about 53.2/46.8 (Figure 4f). Specifically, no significant change in the 57Fe Mössbauer spectra of HA@PB before and after the irradiation also indicates the excellent stability of HA@PB during irradiation. Indeed, ferromagnetic behavior of HA@PB before and after irradiation is found below ca. 4 K. The magnetization values before and after irradiation are almost the same (Figure S14). These results clarify that HA@PB is a stable phototherapeutic agent suitable for recyclable treatment in biological applications.

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Scheme 2. The mechanism of 1O2 generation from excited PB in HA@PB induced by NIR irradiation (S0, singlet ground state; S1, the lowest singlet excited state; ISC, intersystem crossing; T1, the lowest triplet excited state).

It is essential to clearly understand the mechanism of the photoinduced ROS generation for practical applications. In this work, the above-mentioned results reveal that HA@PB can efficiently produce 1O2 upon NIR laser radiation, and that the irradiation does not lead to structure changes in PB of HA@PB. Thus, photoinduced 1O2 generation arises from a reversible reaction between PB and O2. Actually, Arnett et al. have already found that PB can be excited by light induced metal-to-metal charge-transfer from FeIIINCFeII (the ground state) to FeIINCFeIII (the excited state).57 Wojdeł also reported the d-electron charge transfer between the different oxidation states of Fe at neighbouring sites with a broad absorption peak 1.75 eV, which is higher than 0.97 eV of the energy of 1O2.58 Base on the previous reports,62,63 the mechanism of the photoinduced 1

O2 generation from excited PB in HA@PB is proposed there. As illustrated in Scheme 2, the

photoinduced 1O2 generation should be generated form excited PB in HA@PB via a photoinduced energy-transfer process. First, the ground state PB (FeIICNFeIII) is excited to the excited state PB (FeIIICNFeII) by NIR laser irradiation. Subsequently, the photoexcited electron may back to the ground state by two ways: one is vibrational relaxation or radiative relaxation to cause the thermal effect. The other is that a photoexcited electron reaches the triplet state as a result of intersystem crossing, and then O2 becomes 1O2 by an energy-transfer process. The mechanism of 1

O2 generation photoinduced by excited PB in HA@PB is different from the photoassisted Fenton

reaction, which needs to be carried out in the presence of hydrogen peroxide.64 Considering the one-step synthesis and excellent features of HA@PB, we highlight the potential applications of HA@PB for cancer treatment, water treatment, photooxidation catalysis, and so on. 16


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Figure 5. (a) Bright-field and fluorescence images of B16 cells after treated by HA@PB for 6 h. Control group: no HA@PB, no 808-nm laser irradiation. HA@PB group: HA@PB at 100 g mL1, no 808-nm laser irradiation. HA@PB+808-nm laser irradiation group: HA@PB at 100 g mL1 and 808-nm laser (1.0 W cm irradiation for 5 min. Scale bar: 40 m. (b) Time-dependent of HA@PB accumulated inside L929, B16 and 4T1 cells from 0 to 420 min. (c) In vitro photoinduced cell killing; Phototherapeutic effects of PTT and PDT as well as their synergistic effect, 4T1 (d) and B16 (e) cells were incubated with different concentrations of HA@PB solutions (0 100 µg mL1), respectively. *p < 0.05, **p < 0.01.

2.3. In Vitro Cell Experiments. The phototherapeutic effect of HA@PB was further concerned and exploited there. Firstly, it is vital to estimate the stability and intrinsic cytotoxicity of HA@PB for further biological applications. Herein, UV-Vis-NIR measurements were carried out to assess the stabilities of HA@PB under physiological solutions at different pH values, referring to the previously reported study on the stability of PB.65 As can be seen from Figure S15a, almost no change of the maximum absorbance at 697 nm of HA@PB is observed, when they are dispersed in water, phosphate buffered saline (PBS), Dulbecco’s modified eagle medium (DMEM), and 0.9 % NaCl aqueous solution (saline) from pH 5.0 to 8.0, revealing that HA@PB can remain stable at 17



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different pH values in various physiological solutions. Furthermore, time-dependent stabilities of HA@PB in water, saline, PBS and DMEM at pH 7.4 were evaluated by UV-Vis-NIR spectra (Figures S15b and S16) and DLS measurement (Figure S17). It can be found that HA@PB can be stable for at least 7 days. To study the intracellular state of HA@PB, Raman spectra of single cell were carried out, referring to the study reported by Yin et al. on using PB as a background-free and extremely sensitive resonant Raman reporter.66 A peak at 2156 cm, assigned to the (CN) of PB, is clearly seen from both B16 and 4T1 cells treated by HA@PB, while no signal is found for B16 and 4T1 cells without incubation of HA@PB (Figure S18). This result reveals that HA@PB can be effectively delivered into cells. In addition, the intracellular states of HA@PB before and after irradiation were further studied using characteristic Raman peak of PB at 2156 cm. No significant change of characteristic Raman peak is found, even after irradiation for 5 min by a NIR laser (Figure S19). It demonstrates that irradiation does not change the intracellular state of HA@PB. These observations illustrate that the well-dispersed HA@PB have good stability in various physiological solutions and within cells. The cytotoxicity of HA@PB were assessed by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different amounts of HA@PB were added to melanoma (B16) cells, breast cancer (4T1) cells, and normal fibroblast (L929) cells of mouse and incubated for 24 h. Insignificant change of the cell viability is found for HA@PB. In all cases, the cell viability is found to be over 90%, even at the highest concentration of HA@PB (100 μg mL1) (Figure S20). Additionally, the cytotoxicity of PB was also carried out, where PB had negligible cytotoxicity in vitro (Figure S21). These results reveal PB and HA@PB are suitable for further investigation of their phototherapeutic efficacy.

18


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Intracellular ROS production is vital to realize PDT in vitro. Herein, the intracellular production of 1O2 photoinduced by HA@PB was assessed using 2’,7’-dichlorofluorescin diacetate (DCFHDA) on B16 cells. Initially, DCFH-DA is non-fluorescent. However, ROS can fleetly oxidize it to form highly green fluorescence dichlorofluorescein (DCF) that emits at 529 nm.29 As shown in Figure 5a, an intense DCF fluorescence signal is observed with HA@PB under 808 nm laser irradiation, whereas no obvious green fluorescence is observed in cells without HA@PB and 808nm laser irradiation (control group), and little DCF fluorescence signal is found in the HA@PB without 808-nm laser irradiation. Moreover, highly DCF fluorescence is observed in B16 cells when irradiated with 650 nm lase for 5 min (Figure S22). These observations illustrate that the HA@PB have an outstanding ability to produce intracellular 1O2 when irradiated with an NIR laser in vitro. Based on this evidence, the biomedical applications for photodynamic killing of cancer cells were further tested. To investigate the receptor-mediated targeting ability of HA@PB in vitro, L929 (low CD44 expression), B16 (CD44 overexpression), and 4T1 cells (CD44 overexpression) were chosen and incubated with HA@PB for different durations. Then, Fe uptake was quantitatively estimated by ICP-OES. As found from Figure 5b, all the cells incubated with HA@PB lead to a higher Fe uptake. Importantly, the Fe uptake in B16 and 4T1 cancer cells that overexpress the CD44 receptors is remarkably greater than that in L929 in vitro, indicating that HA@PB could accumulate in the tumor through both active targeting of CD44 mediated receptor-ligand interaction and passive targeting of enhanced permeability and retention (EPR) effect mechanisms. This dual-targeting property renders HA@PB immensely promising for cancer theranostics. As a control, in vitro cellular uptake of unmodified PB was further studied. It is found that no significant Fe specific 19



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uptake occurred in all cells (Figure S23). These results further demonstrate that the surface modification of HA is a valid method to increase the uptake of HA@PB by B16 and 4T1 cells via the receptor-ligand interaction mediated by CD44. As a following step, we determined the in vitro phototherapeutic efficacy of HA@PB. L929, B16, and 4T1 cells were treated by a culture medium with different concentrations of HA@PB for 6 h, and further were exposed to an 808-nm laser (1 W cm2) for 5 min. As shown in Figure 5c, the NIR laser irradiation does not results in apparent changes in the normal L929 cells viability with increasing HA@PB concentrations over the range 25100 g mL1. In contrast, after laser irradiation, HA@PB result in a remarkable reduction in B16 and 4T1 cell viabilities, with concentration-dependent phototoxicity. These observations indicate that compared to PB with no cell-killing effect (Figure S24), HA@PB can more effectively destroy B16 and 4T1 than L929 after irradiation, exhibiting a distinctly selective photothermal/photodynamic cell-killing. This significantly targeted specificity may be attributed to the surface of B16 and 4T1 cells with overexpression of HA receptors, which agrees well with the results of Fe uptake, and is similar to already reported HA-targeted nanoplatforms.49,50,55 There are two possible reasons for HA@PB toxicity upon irradiation in the present study: photodynamic effect due to 1O2 and photothermal effect by heat. To confirm whether the photothermal effect or the photodynamic effect leads to the cell death, histidine, as a excellent ROS scavenger, was used to carry out the comparative experiments (denoted as PTT only) or under exposure to 808 nm laser at 4 C (denoted as PDT only). It is known that histidine is an efficient scavenger of 1O2 species, and it can prevent cell damage due to 1O2.60 It has been previously reported that the cell damage induced by heat can be prevented at 4 C.67 As shown in Figures 5d 20


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and 5e, Only partial cell death is observed when there under only either PDT or PTT treatment. These observations reveal that both localized heat and 1O2 generated are responsible for the killing cells with NIR irradiation of HA@PB. Then, to further evaluate the phototherapy efficacy of HA@PB upon irradiation in vitro, Fluorescence microscopy measurements were also carried out. Herein, to distinguish dead cells from living cells, calcein-AM and propidium iodide (PI) were utilized, respectively. HA@PB were firstly used to treat 4T1, B16, and L929 cells for 6 h, and then, PI and calcein-AM were utilized incubated with these cells, respectively. Before irradiation, the green fluorescence of calcein-AM is seen for all types of cells, while no red fluorescence of PI is measured (Figure S25a), suggesting that HA@PB had negligible cytotoxicity and cannot damage cells without irradiation using 808nm laser. With irradiation, intense PI fluorescent is found both in B16 and 4T1 cells. In contrast, in case of L929 cells show little PI fluorescence (Figure S25b). From merging of the fluorescence images, we can find that the PI fluorescence is produced in cells

(Figure S25c). Such results

further indicate that HA@PB can selectively kill the B16 and 4T1 cells rather than L929 cells (Figures 5c). 2.4. In Vivo Dual-Modality Imaging. Thermal imaging (TI) was carried out in vivo on a 4T1tumor bearing on Balb/c nude mice. A total dose 200 µL of saline, HA@PB (1 mg mL1), and PB (1 mg mL1) were injected into 4T1 tumor-bearing mice via intravenous tail, respectively. Then they were exposed to a 650 nm laser or an 808 nm laser at the power of 1.0 W cm2 for 5 min. The temperature changes of the tumor area under irradiation was recorded by an IR thermal camera. As seen from Figure 6a, the temperatures of the mice with injection of HA@PB and exposure to 808-nm laser rapidly increase to 54 C, which is sufficient to kill the 4T1 cells. In contrast, the 21



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tumors of mice treated with saline exhibited moderate increases in their temperatures to approximately 38 °C during 5 min of laser irradiation, demonstrating that PB plays an important part in enhancing the TI. As a control, the temperatures of tumors treated with PB were also measured. The temperatures gradually increased prolonged irradiation time and reached about 45 o

C, but they were less than those of the group injected with HA@PB and irradiation by an 808-nm

laser, which further indicates an efficacious tumor retention of HA@PB because of the EPR effect and receptor-mediated endocytosis of tumors. To further visualize the distribution of the optimal formulation (HA@PB), PA imaging was carried out. The PA signal of HA@PB which significantly boosts up as the concentration increase, as observed in vitro (Figure S26). Encouraged by this positive result, PA imaging of HA@PB was in vivo assessed on a 4T1 tumor xenograft model. As can be seen form Figure 6b, the PA signal at the tumor site is rather week before injection. After intravenous injection, strong PA signals appear quickly and disperse throughout the entire tumor with the progression of time. Such fast accumulation of HA@PB is owing to receptor-mediated endocytosis, where HA can selectively bind to the surface of cancer cells with the overexpression of CD44 receptors. After 24 h following the injection, the tumor tissue still retained its high signal intensity, suggesting a long retention time of HA@PB in the tumor. The NIR TI and PA imaging results show that the HA@PB are also a promising agent for targeting tumor in vivo. In addition, owing to that PB has T1-MR contrasting ability,34,44 our HA@PB may also serve as one kind of effective MRI contrast agent. Thus, in the future work, such MRI feature of PB offers a great possibility to construct a MRI imaging-guided multifunctional diagnostic and therapeutic system by utilization of HA@PB or their analogues.

22


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Figure 6. In vivo dual-modality imaging. (a) TI pictures of 4T1-tumor bearing mice administrated with saline, PB, and HA@PB upon exposure to 808-nm laser (1.0 W cm−2), and administrated with HA@PB exposed to a 650-nm laser at the power of 1.0 W cm−2 for 5 min, respectively. (b) PA images and the corresponding PA values before and after intravenously injecting HA@PB.

23



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Figure 7. Phototherapeutic study in vivo. (A) Photographs of tumor tissues and (B) growth rates of tumor after treated by various methods. The mice was injected with a saline solution of HA@PB via

intravenous tail, which was then exposed to a 650 nm laser (1.0 W cm2) or an 808 nm laser (1.0 W cm2) (HA@PB + 808 nm laser, n = 5; HA@PB + 650 nm laser, n = 5). A saline solution of PB was tail intravenously injected into the mice that was then exposed to an 808 nm laser (PB + 808 nm laser, n = 5). Mice without injection of the HA@PB (Laser Only, n = 5) or in the absence of 808 nm laser irradiation (HA@PB Only, n = 5); blank group with neither laser irradiation nor injection of HA@PB (Blank, n = 5). *p < 0.05, **p < 0.01. (C) H&E stained histological images of tumors in different treatment groups. Scale bar: 100 m.

24


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2.5 In Vivo PTT/PDT of Tumor. Encouraged by the outstanding photoinduced cell-killing efficacy in vitro, the synergistic PTT/PDT effect of HA@PB was further assayed in vivo. The efficacy of the HA@PB mediated phototherapy was evaluated by monitoring the tumor sizes. During the 14-day observation, it is obviously found that the volumes of the tumors in the comparison group and blank group increase with the time, revealing that laser only or the injection of HA@PB without laser irradiation has almost no impact on the tumor growth. Contrarily, in the treatment group, the tumor growth is notablely hindered (Figures 7A and 7B). After the phototherapy, the tumors injected with HA@PB and with an 808 nm laser irradiation almost eliminated on the fourteenth day, which is obviously different from other groups (Figure S27). Moreover, HA@PB + 808 nm laser irradiation shows a more remarkable tumor suppression compared to HA@PB + 650 nm laser irradiation; this difference may be attributed to that the tissue penetration ability of 650 nm laser is less than that of 808 nm laser, thus reducing the formation of heat and 1O2. In addition, the tumors of PB+808 nm laser irradiation group are also suppressed, but less than those of the HA@PB808-nm laser group. This result further indicates that the benign tumor retention of HA@PB is owing to the receptor-mediated endocytosis of tumors. The body weight of the mice remains relatively unchanged in all groups during the 14-day period (Figure S28), implying that HA@PB combined with laser irradiation is not noticeably toxic to mice after systemic administration. Histological examination was further used to assess the synergistic PTT/PDT effect of HA@PB. As seen from Hematoxylin and eosin (H&E) images (Figure 7C), more significant cell damage is found for the tumor tissues of the HA@PB  808 nm laser group; specifically, there were loss of contact, necrosis, and irregular cell shrinkage than those of the other groups, indicating the significantly improved therapeutic efficacy of HA@PB + 808 nm laser 25



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irradiation induced by the combination effect of PTT and PDT. Furthermore, the in vivo biodistribution of HA@PB was studied via monitoring the Fe concentration in the tissues by ICPOES, where after intravenously injecting HA@PB, we surgically excised the major organs and tumor from the mice at various time. It can be seen in Figure S29 that the Fe contents in tumor, lung, liver, and kidney increase during postinjection for 6 h. After that, the Fe elements in the tissues (kidney, spleen, heart, lung, and tumor) were all gradually eliminated as the time prolonged, implying that HA@PB can be gradually cleared. Comparatively speaking, during the whole stage, a relatively greater content of Fe accumulates in the liver. As is known, liver can served as one kind of common clearance pathway, because Kupﬀer cells, which locate inside fenestrated sinusoids of liver blood vessels, are able to take up and damage foreign nanomaterials.10 It indicates that liver can be main metabolic pathway for HA@PB, while other organs slightly metabolize HA@PB, which coincides with previously reported literatures on PB-based on nanomaterials.68 Overall, these results show that HA@PB are one of highly effective and feasible PTT/PDT nanoplatforms for both in vitro and in vivo targeted treatment of cancer. To the best of our knowledge, this work is the first example to use the PB-based nanomaterial as dual-functional PTT/PDT agent for the targeted treatment of cancer. Specifically, HA@PB as one of the synergistically photodynamic/photothermal combination nanoplatforms could achieve high therapeutic

efficacy

for

targeted

treatment

of

cancer

guided

by

dual-modality

photoacoustic/infrared thermal imaging. In addition, it should be pointed out that besides HA, other functional polymer materials can also be applied for the surface modification of PB to solve its stability, such as polydopamine (PDA). It is well-known that PDA have been widely used as surface modifier due to their excellent hydrophilicity, feasible modification of functional group, 26


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good biocompatibility, and strong NIR absorption.69,70 Therefore, the combination of PDA with PB not only can solve the dispersion of PB, but also enhance its biocompatibility as well as photothermal effect. In the future work, we will focus on this direction. 3. CONCLUSION Taken together, we report the efficient generation of 1O2 and the excellent photothermal conversion by PB via a NIR photo irradiation-induced energy-transfer process for the first time. To improve the colloidal stability and active targeting feature of PB, the multifunctional HA@PB have been successfully prepared using HA as a surface modifiers via a simply one-step method. Demonstrated by in vitro and in vivo studies, HA@PB could notably destroy cancer cells with overexpression of CD44 receptors and restrain the growth of tumors owing to the synergistic PTT/PDT effect with targeted specificity. Meanwhile, they could effectively act as dual-modal TI/PA imaging agents for visually monitoring the cancer therapy. More broadly, this work proposed a novel strategy to construct of multifunctional theranostic nanoplatforms. The uniqueness of the metal-coordination centers in PB is of great importance to expand its biological applications as both diagnosis and treatment agents, which is commonly overlooked and rarely explored. Besides, the one-step surface modification of PB with the biopolysaccharide HA contributes to enhance the biocompatible, targeting and theranostic efficacy. Thus, this present study can not only pave the way for the use of HA@PB as a promising multifunctional theranostic agent for biomedical applications, but also provide a new insight into the applications of coordination chemistry in biomaterials.

4. EXPERIMENTAL SECTION 27



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4.1. Materials. K3[Fe(CN)6], 3,3’-Diaminobenzidine (DAB), (NH4)2Fe(SO4)26H2O, 1,3diphenylisobenzofuran (DPBF) and new methylene blue (NMB) were obtained from Sinopharm Chemical Reagent Co., Ltd. 3,6-Di(O-acetyl)-4,5-bis[N,N-bis(carboxymethyl)-aminomethyl] fluorescein, tetraacetoxymethyl ester (Calcein-AM), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 3,8-diamino-5-[3-(diethylmethylammonio) propyl]-6-phenylphenanthridinium diiodide (PI), 2,2,6,6-Tetrame-thylpiperide (TEMP) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (Boston, MA, USA). Hyaluronic acid (HA, MW 8k) was purchased from Freda Company (Shandong, China). Dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Institute of Biotechnol (Jiangsu, China). Water (18.2 MΩ) was produced from a Milli-Q apparatus ( Millipore, Bedford, MA). All of the chemicals were used as received. 4.2. Synthesis of HA@PB, unmodified PB. Typically, 189 mg HA was added into 1.0 mM K3[Fe(CN)6] solution (20 mL) under stirring at 90C. Then, 20 mL 1.0 mM (NH4)2Fe(SO4)2 solution containing HA (189 mg) was slowly dropped into the above mentioned K3[Fe(CN)6] solution, and a blue dispersion was formed immediately, and continued to stirring for another 6 hour. To collect as-prepared HA@PB, the mixture was then centrifuged for 10 min at 10000 r/min. After centrifugation, distilled water was used to wash the precipitation, and separated by centrifuged again. The purification process was repeated for two times. Finally, HA@PB was obtained as blue solid powder. As a control, unmodified PB was synthesized with the same synthetic method of HA@PB, in addition to no need of HA. 4.3. Characterizations and measurements. The morphologies were observed using TEM (JEOL, Japan). FT-IR spectra were performed using a Perkin-Elmer FT-IR spectrometer (Perkin28


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Elmer, U.S.A.). XRD patterns were performed on a Rigaku-D/MAX 2500v/pc diffractometer (Rigaku, Japan). The UV-Vis-NIR spectra were mesured on a UV2600 UV-Vis spectrophotometer (Shimadzu, Japan). Thermogravimetric analysis (TGA, LabsysEvo, Setaram Instruments) was performed in nitrogen gas. The content of PB in HA@PB was assessed using ICP-OES (Flexar/NexlON300X, Perkin-Elmer, U.S.A.). The Mössbauer spectrum (Fast Com Tec PC-mossII) was carried out at room temperature, in constant acceleration mode, and the rays were provided by a

57

Co source in a rhodium matrix. XPS was obtained on an ESCALAB 250Xi X-ray

photoelectron spectrometer (Thermo, U.S.A.). The Mössbauer spectrum and XPS were measured at solid state and performed immediately after NIR light irradiation. 4.4. Spectral detection of 1O2 For ROS evaluation using the DPBF probe, the aqueous solution of HA@PB (50 g mL1) and DPBF (35 M) was illuminated using a 650-nm laser or an 808-nm laser (1.0 W/cm2) for different time intervals. Then, the decomposition of DPBF was monitored by the absorbance at 418 nm. For the DAB method, a 200 L of 10 mM DAB in DMF was mixed with 2.8 mL of aqueous solution of HA@PB (50 g/mL). Then, the solution was exposed to a 650-nm laser. The adsorption spectra were performed at different irradiation time.

4.5. Electron Spin Resonance (ESR) measurements. DMPO and TEMP, as spin-trapping reagents, were employed to detect 1O2, OH or O2 generation by using ESR. 10 L of HA@PB (200 g mL-1), 80 L PBS (pH 7.0), and 10 L DMPO (0.8 M) were mixed to prepare the test solution. Then, the mixed solution was laid up

in a quartz cell and exposure to a 650-nm laser or

an 808-nm laser (1.0 W/cm2) for 5 min, respectively. After that, the treated solution was laid up in 29



Page 30 of 45

the ESR cavity. At 298 K, a Bruker Model A300 spectrometer was used to record the data. The operation parameters were as following: microwave power, 19.87 mW; frequency, 9.8 GHz; sweep width, 200 G; sweep time, 27.65 s; modulation frequency, 100 kHz. The ESR measurement using TEMP was performed with same procedure as described above. 4.6. Photothermal effect measurements of HA@PB in aqueous solution. The photothermal images and the temperature signals of HA@PB and pure water with irradiation of an 808-nm laser (010 min) was noted at different time by an infrared camera (Magnity Electronics, MAG30, China) and handled using Magnity Electronics tools systems. 4.7. Cell lines and culture conditions. Normal fibroblast cells (L929), breast cancer cells (4T1) and melanoma cells (B16) of mouse were obtained from Chinese Academy of Sciences Cells Bank (Shanghai, China). The standard cell medium, approved by American type culture collection (ATCC), was used to culture all the cells under 5% CO2 atmosphere at 37 C. 4.8. Intracellular ROS detection. The generation of ROS intracellular under light irradiation were detected using DCFH-DA. HA@PB (100 μg mL1) were firstly used to incubate the adhered B16 cellsfor 6 h, after that, using PBS buffer washed for twice to remove extracellular HA@PB. Subsequently, to load DCFH-DA into the cells, DCFH-DA (10 M) was incubated with cells in the dark for 1 h , then, washed again with PBS. After 5 min irradiation, imaged using a laser fluorescence microscope (Mshot Model MF50, China). 4.9. Intracellular Raman spectra analysis. The intracellular Raman spectra of the adhered B16 cells or 4T1 cells before and after treatment with HA@PB for 6 h were analyzed by Raman spectroscopy (Renishaw) that used a krypton ion laser at 514 nm, with a laser power of 5%, 30


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exposure times: 10s. Under the same condition, the Raman spectrum of before and after irradiation were performed immediately after NIR light irradiation. 4.10. In vitro cytotoxicity assays of HA@PB. L929, B16 and 4T1 cells seeded into a 96-well were then treated with HA@PB (0, 25, 50, 75, 100 g mL1) for 24 h, respectively. To assess the relative cell viabilities, the standard MTT assay was used. In vitro cytotoxicity assays of unmodified PB was also carried out via using the same method. 4.11. In vitro cellular uptake assay. ICP-OES was used to further confirm the targeted uptake of the HA@PB by cells through quantitatively analysis of the Fe uptake. Briefly, the adhered L929, B16 and 4T1 cells in a 6-well plate were treated with 2 mL of the fresh medium cotaining 100 g mL1 HA@PB for different times (0, 60, 120, 180, 240, 300, 360, 420 min). Subsequently, PBS was used to wash the cells twice, and then, cells were collected. At the last, the Fe concentration in different cells was measured by ICP-OES. As a control, in vitro cellular uptake assay of unmodified PB were also carried out. 4.12. The photodynamic activity and photothermal therapy effect of HA@PB in vitro.

In

a 96-well culture plate, the adhered L929, B16 and 4T1 cells were treated with HA@PB (0, 25, 50, 75, 100 g mL1) for 6 h. Then, the cells were irradiated by an 808-nm laser at the power of 1.0 W cm2 for 5 min, and the relative cell viabilities were evaluated via the standard MTT assay. In vitro photoinduced killing assays of unmodified PB was also carried out under the same condition,. The same procedure was performed to assess

in vitro photothermal cell-killing

efficacy of HA@PB on L929, B16 and 4T1 cell lines in the presence of histidine (10 mM), respectively. 31



Page 32 of 45

Under the same conditions, only in vitro photodynamic cell-killing efficacy of HA@PB was achieved on L929, B16 and 4T1 cell lines by irradiated at 4 oC by NIR an 808-nm laser for 5 min, respectively. 4.13. Fluorescence microscopy measurement. To survey the phototherapy effects of HA@PB on L929, B16 and 4T1 cells, fluorescence microscopy (Mshot Model MF50, China) was carried out. Briefly, three kinds of adhered cells were treated by 2 mL fresh medium with HA@PB (100 g mL1) for another 6 h. To remove the unbound HA@PB, PBS was used to wash the cells for three times. Fresh medium (1 mL) was added again, and then the cells were irradiation by an 808nm laser at the power of 1.0 W cm2 for 5 min. Then, before and after irradiated, 2% PI and 1.6% calcein-AM were used to stain with the cells for 10 min. Finally, the fluorescence images were measured. 4.14. Model of subcutaneous 4T1 breast cancers. The animal protocol was conducted in compliance with the Guideline for the Care and Use of Laboratory Animals (NIH publication 8523), and is approved by the Experimental Animal Ethics Committee of the Guangxi Medical University Laboratory Animal Centre. To generate the tumors, 2 × 106 4T1 cells were d subcutaneously injected into the flank region of nude mice. The mice were stochastically allocated into six groups, when the tumor volume reached about 500 mm3. 4.15. In vivo infrared thermal imaging. The various formulations including saline (200 µL), HA@PB (200 L, 1 mg mL1) and PB (200 L, 1 mg mL1) were injected tail intravenously into the mice, respectively. During laser irradiation (650 nm, 1 W cm2; 808 nm, 1 W cm2) for 5 min, the infrared thermal images and spatial temperature distributions of tumors were taken by an IR 32


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thermal camera (MAG30, Magnity Electronics, China). 4.16. In vitro and in vivo photoacoustic imaging. The signals of various concentrations (0, 2.5, 5, 12.5, 25 and 50 µg/ml) of HA@PB aqueous solutions were acquised for in vitro PA imaging by the multispectral optoacoustic tomography (MSOT) system (iThera Medical GmbH, Neuherberg, Germany. inVision 128). For in vivo PA imaging, a saline of HA@PB solution (200 L, 1 mg mL1) was tail intravenously injected into the 4T1 tumor-bearing mice, and then PA imaging were obtained before and after intravenous injection for 2, 6, 24 hours. 4.17. In vivo tumor growth study. To assess the synergistic phototherapy effect, the mice were stochastically allocated into six groups (n = 5, per group): (i) the test group with tail veins injection of nanomaterials (HA@PB or PB) and exposed to a 650 nm or an 808 nm laser (HA@PB + 650 nm laser, n = 5; HA@PB + 808 nm laser, n = 5; and PB + 808 nm Laser, n = 5), (ii) the control group mice with tail veins injection of HA@PB but no irradiation (HA@PB only, n = 5), and without injection of the HA@PB (Laser Only, n = 5), and (iii) the blank group mice with neither laser irradiation nor injection of HA@PB (Blank, n = 5). During the treatment, the size of the tumors

and the body weights of the mice were monitored daily for two weeks and calculated as volume = (tumor length) × (tumor width)2/2. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volumes when the treatment was initiated). A group of five mice was used to assess the mean and standard deviation of data. Finally, tumors were harvested and collected immediately for H&E staining.

4.18. Biodistribution. A total dose of 200 µL saline solution of HA@PB (1 mg mL1) was injected into 4T1 tumor bearing-female Balb/c mice via tail intravenously. The tissues (lung, heart, spleen, kidneys, liver and tumor) were removed at different time intervals (1 h, 4 h, 6 h, 12 h, 24

33



Page 34 of 45

h, 2 days, 3 days and 7 days) after injection, washed with normal saline solution three times, and then analysis of Fe content by ICP-OES (Flexar/NexlON300X, Perkin-Elmer, U.S.A.). ASSOCIATED CONTENT Supporting Information Additional characterization data including TGA curves, DLS data, XRD patterns, EPR spectra, FCM curves, etc., are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We

thank

the

NSFC

(21671046,

51562001

and

21502028),

Guangxi

NSFGA

(2013GXNSFGA019001, 2017GXNSFGA198004, AD17129007), Ministry of Education Key Laboratory (CMEMR2015-A02), Innovation Project of Guangxi Graduate Education (YCBZ2017028) for financial support. REFERENCES 34


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