Receptor-Mediated and Tumor-Microenvironment Combination

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Biological and Medical Applications of Materials and Interfaces

Receptor-Mediated and Tumor-Microenvironment CombinationResponsive Ru Nanoaggregagtes for Enhanced Cancer Phototheranostics Wen-Long Wang, Zhengxi Guo, Yu Lu, Xing-Can Shen, Ting Chen, RongTao Huang, Bo Zhou, Changchun Wen, Hong Liang, and Bang-Ping Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04531 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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ACS Applied Materials & Interfaces

Receptor-Mediated and Tumor-Microenvironment Combination-Responsive Ru Nanoaggregagtes for Enhanced Cancer Phototheranostics Wen-Long Wang, Zhengxi Guo, Yu Lu, Xing-Can Shen, Ting Chen, Rong-Tao Huang, Bo Zhou, Changchun Wen, Hong Liang, Bang-Ping Jiang

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Science, Guangxi Normal University, Guilin, 541004, P. R. China.

Corresponding Author

E-mail: [email protected]; Tel: +86 773-5846273

ACS Paragon Plus Environment

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ABSTRACT: Although phototherapy has been considered as an emerging and promising technology for cancer therapy, its therapeutic specificity and efficacy are severely limited by nonspecific uptake by normal tissues, tumor hypoxia, etc. Herein, combinationresponsive strategy (CRS) is applied to develop one kind of hyaluronic acid hybridized Ru nanoaggreagtes (HA-Ru NAs) for enhanced cancer phototherapy via the reasonable integration

of

receptor-mediated

targeting

(RMT)

and

tumor-microenvironment

responsiveness (TMR). In this nanosystem, HA component endows HA-Ru NAs with RMT characteristic to selectively recognize CD44-overexpressing cancer cells, while Ru nanocomponent makes that HA-Ru NAs have TMR therapy activity. Specially, Ru nanocomponent not only has near-infrared mediated photothermal and photodynamic functions but also can catalyze H2O2 in tumor tissue to produce O2 for the alleviation of tumor hypoxia and toxic •OH for chemodynamic therapy. Benefitting from these, HA-Ru NAs can be considered as one promising kind of CRS nanoplatform for synergistic photothermal/photodynamic/chemodynamic therapies of cancer, which will not only effectively improve the phototherapeutic specificity and efficacy, but also simplify the


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therapeutic nanosystems. Meanwhile, HA-Ru NAs can serve as a photoacoustic and computed tomography imaging contrast agent to monitor tumor. Such CRS nanoplatforms hold significant potential in improving therapeutic specificity and efficacy for enhanced cancer phototheranostics.

KEYWORDS: phototheranostics, combination-responsive strategy, receptor-mediated targeting, tumor-microenvironment responsiveness, nanoenzyme


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1. INTRODUCTION As an emerging and promising technology for cancer therapy, phototherapy became a very popular research topic of materials and medical science siflds.14 It generally includes photodynamic and photothermal therapies (PDT and PTT, respectively), which depends on photosensitizers (PSs) to convert light energy into heat or reactive oxygen species (ROS) to destroy cancer cells.16 Despite it has made great progress in recent years, phototherapeutic specificity and efficacy are still seriously hindered by its own limitations. For example, most of PSs actually have nonspecific uptake by normal tissues during the phototherapeutic process, which can not only reduce phototherapeutic efficacy but also cause undesired damages to normal tissues.5,6 Hypoxia, described as insuffcient O2 supply, has proven to be one important feature of cancer tissue, largely affecting the therapeutic outcomes in O2-dependent PDT.7,8 To improve phototherapeutic specificity and efficacy, several strategies are proposed. One feasible approach to reduce nonspecific uptake by normal tissues is to precisely deliver PSs to cancer site via receptormediated targeting (RMT),9,10 because several specific receptors (e.g., CD44, folate, glycosylations, and integrins) are overexpressed on the surface of cancer cells.11,12 An available “smart” strategy to alleviate tumor hypoxic is to sophisticatedly exploit tumor-microenvironment responsiveness (TMR), where H2O2 that is overproduced in cancer tissues can act as an endogenous O2 producer toward the enhanced PDT via the catalysis of catalase.13,14 However, RMT or TMR alone can't simultaneously solve the issues of nonspecific uptake by normal tissues and tumor hypoxic.1517 From the practical application point of view, combination of RMT and TMR in one system will serve as a promising strategy to predominantly improve the phototherapeutic specificity and efficacy via simultaneously solving the issues of nonspecific


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uptake by normal tissues and tumor hypoxic. Although the integration of RMT and TMR is essential or enhanced cancer phototherapy.18,19 Yet, combination-responsive strategies (CRS) as the ones mentioned above were seldomly employed in the past. In varieties of tumor-microenvironment characteristics, the overproduction of H2O2 in cancer tissues is an important responsible factor to achieve “smart” treatment of cancer.2022 In addition to produce O2 for the alleviation of tumor hypoxia via the catalysis of catalase, H2O2 can also serve as an endogenous prodrug for chemodynamic therapy (CDT) of cancer, where H2O2 can directly produce toxic •OH via the catalysis of peroxidase.18,19,2325 Inspired by these, the concept of codelivering biomimetic nanoenzymes and PSs to enhance cancer therapy via in situ O2 and •OH production in tumor tissues has been proposed and demonstrated by several groups.2326 However, to develop such H2O2-responsive phototherapy techniques, integration of individual PSs, catalaselike nanoenzyme, and peroxidase-like nanoenzyme into a nano-size platform is needed.14,27,28 The complexity of material design, tedious synthetic procedure, and other drawbacks often make people hesitate.18,19 PSs combining properties such as ROS production, catalase-like and peroxidase-like activities will certainly promote improved operability of the H2O2-responsive phototherapy platform in further practical implementation. Unfortunately, such class of ideal H2O2-responsive phototherapy system is still no reported. Based on the understanding of these, we herein attempt to develop one kind of nanosystem with the reasonable integration of RMT and TMR for enhanced cancer phototherapy, where hyaluronic acid hybridized Ru nano-aggregates (HA-Ru NAs) were constructed in the combination of cysteine-modified HA (Cys-HA) and Ru nanoparticles


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(Ru NPs) using a simple one-step technique. In this nanosystem, HA component has been well-known as an attractive RMT agent. It binds exclusively to CD44 receptors overexpressed on the cancerous cell surface and has been widely used in various biomedical areas such as drug delivery,29 gene delivery,30 as well as imaging.31 The RMT characteristic of HA offers enormous possibility for HA-Ru NAs to achieve highly CD44receptor targeting of cancer. Earlier studies demonstrated near-infrared (NIR) mediated photothermal and photodynamic functions of Ru NPs.3237 Moreover, although biological applications based on enzyme-like activities of Ru-based nanomaterials have not been studied, it has also been proved that they possess catalase-like and peroxidase-like activities to catalyze H2O2 to O2 and toxic •OH.38 In consideration of H2O2 that is overproduced in cancer tissues, Ru NPs in HA-Ru NAs can be considered as one kind of “smart” Ru-based nanoenzyme to realize H2O2-responsive therapy for the first time. Benefitting from the integration of RMT characteristic endowed by HA and TMR therapy activity mediated by Ru NPs, HA-Ru NAs can be considered as one promising kind of CRS nanoplatforms for synergistic PTT/PDT/CDT of cancer (Scheme 1), which will not only effectively improve the phototherapeutic specificity and efficacy, but also simplify the


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therapeutic

nanosystems.

Currently,

ideal

nanomedical

nanosystems

should

simultaneously have diagnostic and therapeutic functions. In addition to diverse therapies of cancer (which include all PTT, PDT and CDT), we also implemented HA-Ru NAs as a contrast agent to image using computed tomography (CT) and photoacoustic (PA) analyses of tumor-bearing mice in vivo, because Ru-based nanomaterials have been already proved to possess both PA and CT capabilities.34,39 By combining PDT, CDT, PTT, PA and CT imaging as well as CRS, one can apply HA-Ru NAs for accurate and exact diagnostics and efficient cancer therapy providing enhanced ability for further multifaceted applications.


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Scheme 1. Schematic illustration of preparation of HA-Ru NAs and their enhanced phototheranostics of tumor via the integration of RMT characteristic endowed by HA and TMR therapy activity mediated by Ru NAs.

2. EXPERIMENTAL SECTION Materials. Ruthenium(III) chloride (RuCl3•xH2O) was purchased from Sigma-Aldrich Chemical

Co.

Hyaluronic

acid,

L-Cysteine

hydrochloride

(L-Cys),

1-(3-

Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), and sodium borohydride (98%) (NaBH4) were purchased from Aladdin Reagents


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(Shanghai, China). HA-Cys was synthesized according to the previous work.40 All commercially purchased reagents and solvents were used without further purification unless specifically noted. Ultrapure Milli-Q water (18.2 MW) was used in all experiments. Apparatus. Low-/high-resolution transmission electron microscopy (TEM) and the measurement of sample composition was carried out on a FEI Tecnai G2S-Twin instrument with a field-emission gun operating at 200 kV. X-ray diffraction (XRD) patterns were acquired on a Rigaku-D/MAX 2500v/pc diffractometer (Rigaku, Japan) equipped with graphite-monochromated CuKα (40 kV, 150 mA) radition at a scanning speed of 5° min1 in the range from 5 to 90°. Thermogravimetric analysis (TGA) was performed on LABSYS evo thermogravimetric analyzer under nitrogen atmosphere at a heating rate of 10 °C min1 up to 1100 °C. A VG ESCALAB MKII spectrometer was used for X-ray photoelectron spectroscopy (XPS) measurements. Fourier transform infrared spectroscopy (FT-IR) was recorded on a PerkinElmer 580B IR spectrophotometer using the KBr pellet technique. Inductivelycoupled plasma optical emission spectroscopy (ICP-OES) was performed using an iCAP 6000 (Thermo Scientific). A Shimandzu UV-3600 spectrophotometer was used record UV-Vis-NIR spectra. The size distribution and zeta potential of the samples was measured by dynamic light scattering (DLS) using Malvern Zetesizer (Nano-ZS90).


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Synthesis of HA-Ru NAs. 0.101 g of HA-Cys and 0.100 g of RuCl3•xH2O (42%) were separately dissolved in 50 mL of Milli-Q water, the solution was then adjusted to pH 8.0 and stirred for 12 hours at room temperature. Next, 0.133 g of NaBH4 solution was added to the above mixture under ultrasonic conditions, and the mixture was sonicated for an additional 3 min. Dialysis was performed on the resulting solution in a dialysis bag with a MWCO of 8000 to 14000 Da for 48 hours in deionized water. The resulting product was freeze dried to produce solid HA-Ru NAs.

3. RESULTS AND DISCUSSION Synthesis and characterization of HA-Ru NAs. HA-Ru NAs were synthesized through a facile one-step reduction of RuCl3 using NaBH4 (Scheme 1). After the synthesis, absorption spectrum of the as-synthesized HA-Ru NAs was carried out to study their absorption behavior in water. HA-Ru NAs displayed strong absorption below 600 nm and a shoulder absorption from 600 to 900 nm like already reported Ru-based NPs,33,34,3941 while the absorption peak of RuCl3 was at 308 nm and no obvious absorption was observed for HA (Figure 1a). The color of RuCl3 was yellowish-brown and the color of HA-Ru NAs was brownish-black (Inset of Figure 1a). Although HA-Ru NAs have no characteristic absorption peak in the NIR region, such notable NIR shoulder absorption is deemed to favor practical phototherapy applications, referring to the


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previous phototherapeutic nanomaterials.39,42,43 It is very necessary to confirm the stability of HA-Ru NAs under physiological conditions for further medical applications. Timedependent Vis-NIR spectra were performed to evaluate the stability of HA-Ru NAs under physiological solutions, including water, PBS, 1640 medium, and DMEM. As shown in Figure S1, almost no absorption change was observed for HA-Ru NAs in solutions from 0 to 7 days. Moreover, there was insignificant flocculation or precipitation in the solution and the bottom of the system after 7 day (Figure S1, Inset). Such results revealed that HA-Ru NAs can be stable under various physiological solutions at least 7 days. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements were utilized to characterize morphology and size distribution of HA-Ru NAs. It can be seen that HARu NAs are aggregated state (40 nm), which are composed of 2~3 nm spherical NPs (Figure 1b). The corresponding size distribution of Ru NPs well agrees with the Gaussian distribution (Figure 1b, Inset). Approximately 2.2 nm is observed from the full-width at half-maximum of the fitted curve, revealing that Ru NPs have a uniform size distribution. DLS measurements showed that HA-Ru NA hydrodynamic diameters were ~50 nm in average (Figure 1c), which agrees well with values obtained from TEM. Zeta potential of HA-Ru NAs was 28.2 eV (see Figure S2) indicating that HA-Ru NAs were stable and negatively charged. Meanwhile, high-resolution TEM (HRTEM) measurements reveal that NPs in HA-Ru NAs have a crystalline structure consisting of parallel crystal planes with lattice spacings of 0.23 nm, which are well in accordance with the


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standard value for the hexagonal close-packed (hcp) crystal structures of Ru crystal (JCPDS 652824),44,45 respectively. To judge the class of Ru species in HA-Ru NAs, X-ray diffraction (XRD) analysis of HA-Ru NAs was performed (Figure S3). Unfortunately, characteristic peaks of Ru-based materials cannot be found. Combining the thermogravimetric analysis (TGA) (Figure S4) of HA-Ru NAs and the previous reports on XRD of HA-based nanomaterials and RuOx,4648 we inferred that the presence of amorphous HA passivates the XRD peak of Ru component. Chemical composition of the sample surface as well as binding states of Ru in HA-Ru NAs were determined using X-ray photoelectron spectroscopy (XPS). As shown in Figure 1d and S5, the binding energies at 280.2, 284.4, 462.5 and 484.3 eV were attributed to zero oxidation state of Ru(0) 3d5/2, Ru(0) 3d3/2, Ru(0) 3p3/2 and Ru(0) 3p1/2, respectively.49,50 In addition, the binding energies at 281.5, 284.8, 286.1, 464.1, and 486.0 eV are respectively corresponding to the high valence state of Ru(Ⅳ) 3d5/2, Ru(Ⅳ) 3d3/2, Ru(Ⅳ) 3d3/2, Ru(Ⅳ) 3p3/2 and Ru(Ⅳ) 3p1/2 in RuO2,4951 as a result of surface oxidized of Ru(0).


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Figure 1. (a) Absorption spectra of HA, RuCl3 and HA-Ru NAs. Inset: photographs of RuCl3 (left) and HA-Ru NAs (right) in water. TEM image (b), DLS data (c), and Ru 3d XPS spectrum (d) of HA-Ru NAs. Inset in (b): HR-TEM image of the crystalline lattice (left) and corresponding size distribution of Ru NPs in (b).

Enzyme-like, photothermal, and photodynamic properties of HA-Ru NAs Although there is no study on the mimetic enzymatic activity of Ru NPs, RuO2 NPs have been reported to exhibit peroxidase-like and catalase-like activities.38 In addition, the mimetic enzymatic activities of other noble metal NPs (e.g., Pt NPs,52 Ag NPs53) have been


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confirmed. Based on these previous,38,52,53 we further explore the potentially enzyme-like catalytic properties of HA-Ru NAs. The catalytic experiments were primarily studied using 3,3,5,5tetramethylbenzidine (TMB) with and without H2O2 (HAc-NaAc buffer solution, pH 4.5) in the presence of HA-Ru NAs. It is known that TMB can react with •OH to form a cation free radical product which is responsible for the blue color (maximum absorbance at 652 nm).54,55 Timedependent absorption spectra were measured as shown in Figure 2a. The results showed that HARu NAs can catalyze the oxidation of TMB by H2O2 to generate a cation free radical product (Figure 2a), suggesting peroxidase-like activity. Electron spin resonance (ESR) measurement proved the production of •OH under the catalysis of H2O2 by HA-Ru NAs (Figure 2c). Besides peroxidase-like activity test, we also confirmed that HA-Ru NAs had good capability to decompose H2O2 to generate O2 with the help of JPBJ608 Portable Dissolved Oxygen Meter. After HA-Ru NAs were added to H2O2 solution, O2 was generated very quickly, whereas there was no O2 production in the absence of HA-Ru NAs or H2O2, indicating that HA-Ru NAs also had catalase-like activity. To simulate in vivo environment, in which H2O2 generation by tumor cells is continuous, amount of generated O2 catalyzed by HA-Ru NAs was measured every 30 minutes while exogenous H2O2 (1 × 10−3 M) was continuously supplied into the system. The results showed stable and continuous generation of O2 (from just one single doze of HA-Ru NAs) for at least four cycles (Figure S6). It is well-known that catalytic activity of enzymes is pH-dependent.56,57 We further measured enzyme-like activities of HA-Ru NAs by variation of the pH from 2 to 12 (Figure 2d). The results confirmed that the enzyme-like activities of HA-Ru NAs are pH-dependent. As the pH increases, the peroxidase-like activity of HA-Ru NAs increased first and then decreased,


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but catalase-like activity had been increasing. From Figure 2d, we can also found that the peroxidase-like and catalase-like capabilities of HA-Ru NAs both maintain high activity around pH 6.5 that is similar to the weak acid environment of tumor. The outstanding pH-dependent enzyme-like characteristics offers enormous possibility for HA-Ru NAs to be used in therapeutic applications of cancer.

Figure 2. (a) Time-dependent absorbance changes at 652 nm of TMB after different treatments. Inset: the corresponding photographs of TMB  HA-Ru NAs and TMB  HA-Ru NAs  H2O2. (b) Time-dependent O2 production of H2O2, HA-Ru NAs, and HA-Ru NAs  H2O2 in water. (c) ESR spectra of DMPO after different treatments. (d) pH-dependent absorbance changes at 652 nm of TMB  HA-Ru NAs  H2O2 (black line) and O2 production of HA-Ru NAs  H2O2 (blue line).


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The understanding of the catalytic mechanism is very crucial for practical biomedical applications. We further explore the catalytic mechanism of H2O2 by HA-Ru NAs. The oxidation potential from Ru3 to Ru and RuO2 to Ru3+ are 0.39 V and 0.87 V,58,59 respectively. In addition, elemental Ru can be oxidized to Run+ by dissolved oxygen having a reduction potential of 1.2 V or by H2O2 having a reduction potential of 1.77 V.53,58 Theoretically, H2O2 can produce •OH via Reactions (1) and (3). To confirm this hypothesis, RuCl3, non-nanosized Ru and RuO2 purchased from commercial resources were used in the comparative study, because pure nanosized Ru NPs and RuO2 NPs cannot be obtained via the preparation method in this work. It was found that both Ru and RuCl3 can catalyze the oxidation of TMB by H2O2 to generate a cation free radical product (Figure 3a), proving the production of •OH as a result of Fenton-like reaction. From the reduction potential of RuO2 to Ru3+ (0.87 V) and the oxidation potential of H2O2 to O2 (1.2 V), we deduce that Reaction (2) can theoretically take place. As shown in Figure 3b, RuO2 can significantly cause H2O2 to produce O2 indicating that RuO2 also have catalase-like activity to decompose H2O2 to generate O2. Moreover, Ru can also cause H2O2 to produce O2 (Figure 3b), whereas in comparison with RuO2, the catalytic capability of Ru is very low. Thus, in HA-Ru NAs, the catalase-like activity is mainly endowed by RuO2 surface layer. Ru + H2O2 + H + → Ru3 + + H2O + OH

(1)

RuO2 + H2O2 → Ru3 + + H + + O2↑ (2) Ru3 + + H2O2 + H + →RuO2 + H2O + OH (3)


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Figure 3. (a) Absorption spectra of TMB  H2O2, TMB  Ru  H2O2, TMB  RuCl3  H2O2, and TMB  HA-Ru NAs  H2O2 in HAc-NaAc buffer solution (pH 4.5). (b) Time-dependent O2 production of H2O2, Ru  H2O2, RuCl3  H2O2, RuO2  H2O2, and HA-Ru NAs  H2O2 in water. Inset: The corresponding photographs of different samples after 23 min of reaction. When considering the relatively high content of H2O2 (concentration range from 100 M to 1 mM) in the tumor microenvironment,13,14 the peroxidase-like and catalase-like activities of HARu NAs is beneficial for practical applications in CDT and enhanced PDT. It makes HA-Ru NAs as one kind of Ru-based nanoenzyme with strong potential to enhance therapeutic efficiency of hypoxic tumors for the first time. Basis for PDT application is formation of cytotoxic ROS as a result of selective light absorption to destroy cancerous cells.16 Capacity of HA-Ru NAs as PSs for PDT was evacuated using 1,3-diphenylbenzofuran (DPBF) as ROS-trapping agent. As shown in Figure 4a and S7, compared to the HA-Ru NAs + 808 nm laser group and HA-Ru NAs  H2O2 group, 808 nm laser irradiation can lead to more obvious diminish of absorption of DPBF when H2O2 was added into the solution of DPBF  HA-Ru NAs. Thus, irradiation of HA-Ru NAs by 808 nm laser could produce ROS. At the same time, H2O2 presence can prominently enhance ROS production of HA-Ru NAs under laser irradiation. Combining the abovementioned enzyme-like


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activity results, we infer that ROS production promoted by H2O2 in HA-Ru NAs  H2O2 + 808 nm laser group can be attributed to two factors: one is that the peroxidase-like activity of HA-Ru NAs leads to •OH production; the other is that the catalase-like activity of HA-Ru NAs catalyzed the O2 production to improve ROS production photoinduced by HA-Ru NAs. The latter was confirmed by ESR results in Figure 4b. It also revealed that the nature of ROS photoinduced by HA-Ru NAs was 1O2, and H2O2 can prominently enhance such 1O2 production owing to the O2 production from H2O2 catalyzed by HA-Ru NAs.

Figure 4. (a) Time-dependent absorbance changes at 652 nm of DPBF after different treatments. (b) ESR spectra of TEMP after different treatments. (c) Confocal images of 4T1 cells with CytoID probe for the detection of hypoxia after different treatments. (d) Confocal images of 4T1 cells with different treatments. The cells were pretreated with 10 mM DCFH-DA for 20 min and washed with PBS for three times. Irradiation was performed using 808 nm laser (1.0 W cm2).


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Photothermal/photodynamic/chemodynamic cell-killing efficacy in vitro Intracellular production of ROS is crucial to realize CDT and PDT.2225 We primarily examined intracellular O2 levels for mouse breast cancer (4T1) cells with different treatments. A Cyto-ID detection kit for hypoxia/oxidative stress was utilized to assess the intracellular hypoxic environment of the cells via confocal microscopy (Figure 4c).27,60 4T1 cells were pretreated with a hypoxia mimetic agent (desferrioxamine, DFO) to provide a hypoxic cellular environment.27 As shown in Figure 4c, in comparison with the control group without DFO treatment, stronger red fluorescence (Cyto-ID hypoxia probe) was found in 4T1 cells after treatment with DFO for 4 h. Notably, the fluorescence intensity significantly decreased in the DFO-pretreated cells after further incubation with HA-Ru NAs (100 g mL1) and H2O2 (100 M) for 12 h. These observations suggested that cellular hypoxia was effectively overcome by the generation of O2 induced by the catalase-like activity of HA-Ru NAs. To further verify the ability of HA-Ru NAs to produce intracellular ROS, the oxidation-sensitive probe 2,7-dichlorofluorescin diacetate (DCFHDA) was employed to determine ROS generation in the 4T1 cells. DCFH-DA, oxidized by ROS, displays green fluorescence.61 After incubation with HA-Ru NAs but no laser


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irradiation, almost no fluorescence was observed in cells (Figure 4d). For the HA-Ru NAs + Laser group (808 nm laser irradiation) and H2O2  HA-Ru NAs group, moderately green fluorescent signal was found. Compared to them, more intense fluorescence was detected in the H2O2  HA-Ru NAs  Laser group. These observations indicate that HARu NAs  H2O2 activated by NIR radiation can generate enough intracellular ROS and that they demonstrate potential to further achieve CDT and PDT with TMR characteristics.

Figure 5. (a) Temperature changes of HA-Ru NAs solution with different concentrations under laser irradiation. (b) Relative viabilities of 7702, 4T1, and Hela cells incubated with


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different concentrations of HA-Ru NAs under laser irradiation. (c) Relative viabilities of 4T1 cells induced by CDT, PDT and PTT as well as their synergistic effect of HA-Ru NAs with different concentrations. (d) Time-dependent Ru content of HA-Ru NAs accumulated inside 7702, 4T1 and Hela cells. Irradiation was performed using 808 nm laser (1.0 W cm2).

HA-Ru NAs have the potential to achieve NIR-mediated photothermal conversion due to their excellent NIR absorption. As shown in Figure 5a, HA-Ru NAs possessed excellent photothermal conversion efficiency under an 808-nm laser irradiation (1.0 W·cm−2), with concentration-dependent photothermal characteristics. In contrast, no noticeable temperature increase rise was observed for pure water. The photothermal conversion efficiency () value of HA-Ru NAs was determined to be 37.4 % using the data obtained from Figure S8. Meanwhile, cyclic irradiation experiment reveals the good photostability of HA-Ru NAs (Figure S9). Such excellent stability and efficient photothermal conversion indicate that HA-Ru NAs have enormous potential as a candidate for PTT.


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These encouraging results prompted us to further determine in vitro cell-killing efficacy of HA-Ru NAs using an assay based on 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT). Two types of cells were selected. First type was cells of human normal liver (7702) with low CD44 expression. Second type was human cervical cancer (Hela) cells with high CD44 expression as well as 4T1 cells. The intrinsic cytotoxicity of HA-Ru NAs was primarily assessed. Insignificant cytotoxicity of HA-Ru NAs was observed for all three kinds of cells, even at concentration as high as 600 µg·mL−1 (Figure 5b), indicating their suitability for further therapeutic efficacy investigation. In vitro cytotoxicity of HA-Ru NAs upon 808 nm laser irradiation was determined next. Upon exposure to laser irradiation, HA-Ru NAs insignificantly decreased the viability of 7702 cells (Figure 5b). However, as shown in Figure 5b, HA-Ru NAs following laser irradiation leaded to obvious concentration-dependent cell death of 4T1 and Hela cells, benefitting from photothermal and photodynamic effects of HA-Ru NAs. These results indicated that HA-Ru NAs following laser irradiation can selectively destroy CD44-overexpressing cancer cells because of CD44 overexpression on the 4T1 and Hela cell surfaces analogous to other RMT systems based on HA.6264 Typically, aberrant metabolism of cancer cells inside


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solid tumors inherently produce H2O2. Therefore, to simulate H2O2-rich tumor microenvironment, the in vitro anticancer efficiency of HA-Ru NAs was further evaluated under the presence of 100 μM H2O2. Without NIR laser irradiation, the cell viabilities of 4T1 and Hela cells slightly decreased as HA-Ru NAs concentration increased. It manifested the feasibility of HA-Ru NAs to kill cancer cells, because HA-Ru NAs catalyzed H2O2 to produce •OH. When 808 nm laser irradiation and H2O2 were combined, much more remarkable decreases in cell viabilities were observed. About 95 % of 4T1 cells and 84 % of Hela cells were killed at the concentration of HA-Ru NAs of 600 g mL1 owing to TMR therapy activity and synergistical anticancer effect of CDT/PDT/PTT (Figure 5c and S10). It was further confirmed by the results of live-dead cell staining method (Figure S11).

To further prove the RMT specificity of HA-Ru NAs, cellular uptake of HA-Ru NAs was observed by measuring intracellular Ru content using ICP-OES. In the case of a cellular uptake being RMT, HA-Ru NAs would accumulate inside the cells as incubation time increases. This would imply that intracellular Ru content will increase as well. Almost no


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change in the Ru content was observed for the 7702 cells with prolonged incubation time, whereas intracellular Ru content for the CD44-overexpressing 4T1 and Hela cells significantly increased as time increased (see Figure 5d). In addition, to prove mediation of HA-Ru NA cellular uptake by CD44 endocytosis, excess HA were utilized to preincubate 4T1 and Hela cells to block their CD44 receptors. Intracellular Ru contents of cells pre-incubated by excess HA were inferior to the untreated cells (Figure 5d). Thus, free HA can competitively bind to surfaces of CD44 cell receptors against HA-Ru NAs. It induces that the cellular uptake of HA-Ru NAs is decreased. These observations confirm RMT being the cellular uptake of HA-Ru NAs for the 4T1 and Hela cells. Such excellent RMT specificity suggests that HA-Ru NAs are one kind of promising candidate to achieve enhanced phototherapeutic effect.


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Figure 6. (a) Concentration of HA-Ru NAs in the major organs, (b-e) hematological index, (f-i) biochemical blood analysis, and (j) corresponding H&E stained organ slices of female Balb/c mice after intravenous administration of HA-Ru NAs (200 µL, 1.0 mg mL1) at different post-injection times. Scale bar: 100 m.


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In vivo biocompatibility and biodegradability Despite above-mentioned in vitro results revealing that HA-Ru NAs possess insignificant cytotoxicity, in vivo biocompatibility and biodegradability of HA-Ru NAs remains widely unknown. Prior to in vivo therapeutic effect evaluation of HA-Ru NAs, female BALB/c mice were chosen for in vivo biological behaviors tests. The results obtained from hematological index, biochemical blood analysis, main organs Ru biodistribution and corresponding slices of H&E stained organa (Figure 6) benign in vivo biodegradability and biocompatibility of HA-Ru NAs, which was promising to be applied for in vivo treatment of cancer. After the investigation on in vivo biocompatibility and biodegradability of HA-Ru NAs, their blood circulation time and accumulation in tumor were studied. Tails of female BALB/c nude mice with 4T1 tumors were intra-venously injected with saline solution of HA-Ru NAs. Half-life of HA-Ru NA blood circulation was measured to be about 3 h (Figure 7a). The relatively long blood circulation time provided the possibility for HA-Ru NAs to effectively enrich at the tumor locations. Biodistribution results revealed relatively high Ru content in the liver and spleen, which is consistent with the results obtained from female Balb/c mice (Figure 6a), indicating that HA-Ru NAs


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mainly accumulated in the reticuloendothelial system. Meanwhile, HA-Ru NAs also showed a gradual and effective tumor enrichment (Figure 7b). The Ru content can reach 15.0 % ID/g in 24 h, possibly owing to the RMT enrichment of HA-Ru NAs by tumor. Such good tumor enrichment of HA-Ru NAs guarantees good therapeutic effect of solid tumors.

Figure 7. Time-dependent Ru content in the blood (a) and biodistribution of Ru in main organs and tumor (b) during 24 h intravenous post-injection of HA-Ru NAs (200 µL, 1.0


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mg mL1). (c) Representative immunofluorescence images of tumor sections after hypoxia staining. The nuclei and hypoxia areas were stained with DAPI (blue) and pimonidazole antibody (green), respectively. Scale bar: 100 m.

In vivo PTT/PDT/CDT of cancer Considering the effective tumor uptake of HA-Ru NAs, the relatively high content of H2O2 in the tumor microenvironment, and that HA-Ru NAs can catalyze H2O2 to generate O2, HA-Ru NAs were expected to significantly increase in situ O2 content in tumor and to improve hypoxic solid tumor conditions. To confirm this, immunofluorescence staining assay was performed using tumors extracted from mice after the injection of HA-Ru NAs for 24 h. Cancerous cell nuclei as well as hypoxia areas were stained with 2-(4amidinophenyl)-6-indolecarbamidinedihydrochloride (DAPI) (blue) and pimonidazole antibody (green), respectively. Green fluorescent signal of pimonidazole is related to O2 content, where stronger green fluorescence intensity indicates more severe hypoxia.26,65 Tumor sections of mice intravenously injected with HA-Ru NAs displayed significantly weakened green fluorescence (Figure 7c) in comparison to the control group not injected


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with HA-Ru NAs. Thus, tumor tissues hypoxia was effectively alleviated after HA-Ru NAs was administered. Such capability of HA-Ru NAs to improve solid tumor hypoxia is be helpful for PDT resistance reduction. In addition, in vivo photothermal effect of HA-Ru NAs was also studied on female Balb/c nude mice bearing 4T1 tumor. In Figure 8a, in

vivo photothermal results illustrated that HA-Ru NAs can serve as an efficient photothermal agent to locally heat the tumor by laser irradiation.


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Figure 8. (a) Infrared thermal images of tumor-bearing mice after intravenous treatment with saline and HA-Ru NAs solution (200 µL, 1.0 mg mL1) at different irradiation time periods (0–8 min). (b) Mean body weights of Balb/c nude mice in different groups. (c) Tumor growth profiles of 4T1 tumors. (d) Representative photograph of the mice in different treatment groups for at different treatment time points. (e) H&E staining of tumor tissues after various treatments. Scale bar: 100 m.

Based on the potential synergistic therapeutic effect and good biocompatibility of HARu NAs, we attempted to further assess their therapeutic efficiency in vivo. Mice with 4T1 tumors were randomly divided into four groups. Relative to the control groups (which were laser- or HA-Ru NA-treated only) and the blank group (Blank), the treatment group (HARu NAs  Laser) exhibited more apparent inhibition of tumor growth during the experimental period (Figure 8c and d), because the synergistical anticancer effect of CDT/PDT/PTT with active targeted specificity. Moreover, the tumors of HA-Ru NAs only group are slightly suppressed but less than those of the treatment group. This slight suppression in HA-Ru NAs only group may be attributed to the fact that HA-Ru NAs can


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catalyze intra-tumoral H2O2 to produce ROS, thus partly inhibiting tumor growth. Moreover, body weights of the tested mice were recorded for fourteen days. As shown in Figure 8b, no obvious fluctuation in body weight is observed, implying presence of negligible signs of toxicity during phototherapy. Furthermore, H&E staining of tumor slices revealed more serious damage of tumors in the treatment group in comparison to other groups (Figure 8e) according to in vivo anticancer activity. Thereby, in vitro as well as in

vivo experiment data proved high efficiency and feasibility of HA-Ru NAs and NIR light combination to realize precisely synergistic enhanced cancer treatment. Besides the RMT specificity of HA-Ru NAs can improve the precision of therapeutic effect, TMR enzymelike activity feature cannot only be dedicated to the precise in vivo treatment but also improve hypoxia environment. This is a typical example of reasonable integration of RMT and TMR into one nanoplatform to achieve enhanced therapeutic effect of cancer via the improvement of therapeutic specificity and hypoxia environment.

In vivo imaging


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In addition to the versatile treatment of cancer, current ideal therapeutic nanosystems should also have imaging functions. We further evaluate the CT and PA imaging abilities of HA-Ru NAs. As shown in Figure 9c, CT images brightness for HA-Ru NAs in water solution gradually strengthens. The corresponding values linearly increased with HA-Ru NA content from 0 to 15.0 mg mL1 (Figure 9a and b). The slope value is ~10.7, which is below value obtained using commercially available Iopromide. However, it still suggested that HA-Ru NAs have a positive CT imaging capability. In vivo CT imaging of BALB/c nude mice bearing 4T1 tumors showed that significant X-ray attenuation signal is highlighted within the tumor site after the injection of HA-Ru NAs (Figure 9d). Similar result is observed for HA-Ru NA-based PA imaging. In vitro solution-state investigation displays that PA signal values increase linearly with increase in the concentration of HARu NAs. In vivo PA imaging indicates that PA signal intensity in the tumor site after injection of HA-Ru NAs is remarkably enhanced. These observations verify that HA-Ru NAs can serve to provide contrast for dual-modality CT/PA imaging.


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Figure 9. (a) The corresponding concentration-dependent CT values of HA-Ru NAs and Iopromide, as well as PA intensity of HA-Ru NAs (b). (c) The corresponding concentration-dependent CT and PA images of intensity of HA-Ru NAs. (d) CT and PA images of the 4T1 tumor-bearing nude mice before and after the injection of HA-Ru NAs.

4. CONCLUSION In conclusion, we successfully developed one kind of CRS nanoplatform to enhance cancer phototherapy via the reasonable integration of RMT and TMR. In this CRS nanoplatform, HA component can selectively recognize CD44-overexpressing cancer cells, while Ru nanocomponent not only has near-infrared mediated photothermal and


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photodynamic functions but also can catalyze H2O2 in tumor tissue to produce O2 for the alleviation of tumor hypoxia and toxic •OH for CDT. As expected, in vitro and in vivo experimental data proved selective killing of CD44-overexpressing cancer cells by HARu NAs. Tumor-specific inhibition by HA-Ru NAs upon 808-nm laser irradiation as well as their remarkable synergistic PTT/PDT/CDT cancer-treatment effects with RMT and TMR characteristics were also confirmed. Besides, HA-Ru NAs can serve as a PA and CT imaging contrast agent to monitor tumor. The combination of PTT, PDT, CDT, PA imaging, CT imaging, and CRS makes that HA-Ru NAs can be applied for precise diagnosis and effective treatment of cancer, thus offering increased potential for multifunctional applications. Therefore, the present work demonstrates very efficient and doable pathway for CRS nanoplatform development with improved specificity and efficacy of phototheranostics for cancer via the reasonable integration of RMT and TMR.

ASSOCIATED CONTENT

Supporting Information.


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Experimental details, zeta potential data, Ru3p XPS spectrum, and other data are described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51562001 and 21671046),

Natural

Science

Foundation

of

Guangxi

(2018GXNSFFA281004,

2017GXNSFGA198004 and AD17129007), and the project of State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (CMEMR2018-C5 and CMEMR2018-C25) for financial support. REFERENCES (1) Kim, H.; Lee, J.; Oh, C.; Park, J. H. Cooperative Tumour Cell Membrane Targeted


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Table of Contents (TOC)


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Receptor-Mediated and Tumor-Microenvironment Combination

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