Intracellular Enzyme-Triggered Assembly of Amino Acid-Modified Gold

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Biological and Medical Applications of Materials and Interfaces

Intracellular Enzyme Triggered Assembly of Amino Acids Modified Gold Nanoparticles for Accurate Cancer Therapy with Multimode Tao Liu, Ronghua Jin, Pingyun Yuan, Yongkang Bai, Bolei Cai, and Xin Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05943 • Publication Date (Web): 11 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Applied Materials & Interfaces

Intracellular Enzyme Triggered Assembly of Amino Acids Modified Gold Nanoparticles for Accurate Cancer Therapy with Multimode Tao Liu,[a] Ronghua Jin,[a] Pingyun Yuan,[a] Yongkang Bai,[a] Bolei Cai,*[b] and Xin Chen*[a]

[a] Dr. T. Liu, R. Jin, P. Yuan, Y. Bai, Prof. X. Chen Department of Chemical Engineering, Shaanxi Key Laboratory of Energy Chemical Process Intensification, Institute of Polymer Science in Chemical Engineering, School of Chemical Engineering and Technology, Shenzhen Research Institute, Xi’an Jiao Tong University, Xi’an 710049, P. R. China E-mail: [email protected] [b] Prof. B. Cai State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University Xi’an 710032, China E-mail: [email protected]

Abstract: Multiple amino acids (glutamine and lysine) modified gold nanoparticles with pH-switchable zwitterionic surface were fabricated through coordination bond using ferrous iron (Fe2+) as bridge ions, which are able to spontaneously and selectively assemble in tumor cells for accurate tumor therapy combining enzyme triggered photothermal therapy and H2O2 dependent catalytic medicine. These gold nanoparticles showed electric neutrality at pH 7.4 (hematological system) to prevent endocytosis of normal cells, which could be positively charged at pH 6.8 (tumor microenvironment) to promote the endocytosis of tumor cells to these nanoparticles, performing great tumor selectivity. After cell uptake, the specific enzyme (transglutaminase) in tumor cells would catalyze the polymerization of glutamine and lysine to cause intracellular assembly of these gold nanoparticles, resulting excellent photothermal property for accurate tumor therapy. Moreover, the Fe2+ could

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decompose excess hydrogen peroxide (H2O2) in tumor cells via Fenton reaction, resulting in a large amount of hydroxyl radicals (·OH). These radicals would also cause tumor cell damage. This synergetic therapy associating with high tumor selectivity generated an eightfold in vitro cytotoxicity against tumor cells compared with normal cells under 48 hours incubation with 10 mins NIR irradiation. Moreover, in vivo data from nude mice bearing tumor models showed that tumors can be completely inhibited and gradually eliminated after multi-mode treatment combining catalytic medicine and photothermal therapy for 3 weeks. This system takes advantage of three tumor microenvironment conditions (low pH, enzyme and H2O2) to trigger the therapeutic actions, which is a promising platform for cancer therapy achieved both prolonged circulation time in blood system, selective cellular uptake and accurate tumor therapy in multiple models.

Keywords: pH-switchable zwitterionic surface; Tumor selectivity; Enzyme triggered photothermal property; Catalytic medicine; Synergetic tumor therapy

1. Introduction Photothermal therapy (PTT) triggered by near-infrared (NIR) light has attracted great attentions for anti-cancer applications because of its high controllability, low toxicity and great therapeutic effect1-3. To perform effective PTT, various photothermal agents including molybdenum disulfide, black phosphorus, polydopamine and infrared dyes has been introduced to cancer treatment, which all presented good antitumor effects 4-7.

Among these photothermal agents, gold nanomaterials such as gold nanoparticles,

nanorods, nanostars and their assemblies has been considered as ideal candidates to facilitate PTT due to their strong NIR absorption and low toxicity

8-11.

However, in

the conventional studies, photothermal therapeutic agents were all designed as sustained heat sources under irradiation regardless of their location


12,13,

which could

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possibly damage the surrounding healthy tissues in treatment meanwhile reduce the efficiency of cancer therapy 14. As to avoid the damages to normal tissues and achieve tumor-specific therapy, gold materials modified by targeting moieties, for example, peptides and antibodies, have been developed

15.

Although this approach enhanced the tumor selectivity for

photothermal therapy, it is still a challenge to achieve the practical application because of the high cost, unstable activity of proteins in bio-system as well as quick recognition/clearance by the immune system. Moreover, according to the previous study, there are only few nanoparticles could reach the site of action even after functionalization by targeting moieties, resulting in the insufficient selectivity 16. The possible off-target effects may cause unexpected biodistribution of most photothermal materials in healthy tissues, which could be seriously harmful to corresponding tissues under NIR. In situ assembly of the small AuNPs in tumor microenvironment to spontaneously form photothermal agent for selective tumor therapy, may offer an effective way to avoid the unexpected damage to healthy tissues or organs. So far, using different stimuli to induce AuNPs assembly at tumor site has attracted wide interests. Notable exception is studies on light-dependent AuNPs aggregation based on dimerization of light-responsive molecules using UV-light or visible light

17-20.

While it is still a

challenge to apply these photothermal therapies in vivo, because of the low penetration of these lights caused by the strong absorption of tissue, body fluid and hemoglobin 21. In recent work of us and other groups, pH-responsive AuNPs, which could aggregrate around tumor tissue owing to the surrounding acidic environment, have been used to achieve tumor selective photothermal therapy22-24. However, the nonspecific acidic extracellular matrix also exists at other locations beside the tumor, which may pose unwanted particle aggregation. Thus, establishing novel ways to accurately manipulate the assembly of AuNPs in tumor is important for effective PTT.



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Herein, we established a simple and effective approach for the preparation of pH-enzyme-H2O2 triple-responsive AuNPs that are able to display tumor selectivity, tumor-specific photothermal property and catalytic medicine. In this work, control of the

photothermal

property

was

achieved

by

the

intracellular

enzyme

(Transglutaminase, TGase) catalyzed polymerization of amino acids (glutamine, Glu and lysine, Lys) on the surface of AuNPs and the following AuNPs assembly, which would serve as photothermal agent for tumor therapy (Scheme 1a). The TGase widely existed in tumor cells 25. As to avoid the unexpected TGase triggered polymerization in normal cells, pH responsive zwitterionic surface was employed based on the different isoelectric points of the selected amino acids, which are able to gradually display resistance to endocytosis in normal tissue (pH 7.4, zwitterionic surface), effective cell uptake and the final TGase triggered AuNPs assembly in tumor tissue (pH 6.8, positive surface) 26. Moreover, the selected Glu and Lys were introduced by forming ion-ligand with L-cysteine modified AuNPs using Fe2+ as the bridging ion. The Fe2+ also serve as a catalyst to utilize the unique intracellular environment of tumor cells containing overexpressed hydrogen peroxide (H2O2)27,28, which could react with Fe2+ to generate cytotoxic hydroxyl radicals (·OH) via Fenton reaction,24,29 resulting in a synergistic effect to enhance photothermal therapy. This AuNPs-Fe-Glu-Lys not only allows the photothermal therapy just happen in the required tumor tissue where the two physiological stimuli are delivered, but integrated the photothermal therapy and catalytic medicine, which would become an effective strategy for cancer therapy (Scheme 1b).

2. Experimental section 2.1 Materials Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4•3H2O) was purchased from Energy Chemical (Shanghai, China). Sodium citrate and ferrous chloride tetrahydrate


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were purchased from Tianjin Tianli Chemical Reagent Co., Ltd. L-cysteine was purchased from Sinopharm Chemical Reagent Co., Ltd (Xi’an, China). Glutamine, lysine and Glutamine transaminase (TGase, 120u/g) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). MilliQ water was prepared using a MilliQ system (Bedford, MA, America).

2.2 Preparation of gold colloidal solutions. All glassware used for the synthesis of gold nanoparticles were rinsed in piranha solution (H2SO4 : H2O2 = 3 : 1, strong oxidizer) and then with Milli-Q water followed by a rinsing in aqua regia (HCl : HNO3 = 3 : 1, highly corrosive) and then Milli-Q water. 500 ul of HAuCl4•3H2O solution (0.01 M) was added in 17 ml MilliQ water then heated to boil under vigorous stirring. After that, 1 mL of sodium citrate solution (0.01M) was added to the solution. The solution turned light red after 10 min reaction with stirring and boiling, indicating the formation of AuNPs. Finally the solution was removed from the hotplate and allowed to cool to get AuNPs solution for future use.

2.3 Synthesis of L-cysteine modified gold nanoparticles (AuNPs-Cys). Synthesis of AuNPs-Cys proceeded by adding L-cysteine to colloidal gold in water. Typically 0.1 mL of a 0.12 mM solution of L-cysteine in water was added to 5 mL of the gold nanoparticle solution (2.5 × 10−4 M Au), mixed well, and stirred for 2 h. The functionalized nanoparticle solution was isolated by multistep centrifugation and re-dispersed in Milli-Q water.

2.4 Synthesis of Glutamine and lysine co-modified gold nanoparticles (AuNPs-Fe-Glu-Lys)



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The as-synthesized AuNPs-Cys was dispersed in 10 ml of Milli-Q water, then 50 ul of ferrous chloride (FeCl2) solution (0.12 mM) was added. The suspension was stirred for 12 h to form unsaturated coordinate bonds between L-cysteine and Fe2+ (AuNPs-Cys-Fe), following with isolation by multistep centrifugation and re-dispersion in Milli-Q water. Then 100 L of Glutamine solution (0.12 mM) and 100 L of lysine solution (0.12 mM) were added in the resulting AuNPs-Cys-Fe suspension under stirring to complete the coordinate bonds via metal−ligand interactions. After 30 min reaction at room temperature under stirring, the generated AuNPs-Fe-Glu-Lys were collected and purified by multistep centrifugation and wash by Milli-Q water.

2.5

Enzyme

(Glutamine

transaminase)

triggered

aggregation

of

AuNPs-Fe-Glu-Lys The as-synthesized AuNPs-Fe-Glu-Lys was dispersed in 10 ml of Milli-Q water, then 100 mg of Glutamine transaminase was added following with 12 h incubation under shaking.

2.6 Measurement of anti-biofouling property of AuNPs-Fe-Glu-Lys Fluorescein

isothiocyanate

labeled

Human

serum

albumin

(FITC-HSA,

Sigma-Aldrich, USA) was used as model protein to investigate the anti-biofouling property of AuNPs-Fe-Glu-Lys by protein adsorption measurements. The AuNPs-Fe-Glu-Lys nanoparticles were first incubated in PBS containing 5 mg/mL of FITC-HSA at pH 7.4 under 37 °C for 24 h, then each sample was centrifuged from the solution. The protein content of supernatants were determined using UV-Vis spectroscopy by measuring the maximal absorbance at 500 nm wavelength. The antifouling ability of AuNPs-Fe-Glu-Lys nanoparticles was calculated by the following formulation.


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Antifouling ability = protein content of nanoparticle centrifugation supernatant /total amount of protein. As control groups, the anti-biofouling property of AuNPs-Fe-Glu-Lys at pH 6.8 and the anti-biofouling property of AuNPs at pH 7.4 were also carried out.

2.7 Hydroxyl radicals detection Hydroxyl radicals produced by the Fenton reaction are detected by UV-Vis spectra. Briefly, we use methyl violet as a hydroxyl radical probe, which could be degradated by hydroxyl radicals, resulting in obvious color change from purple to colorless. As to carry out the experiment, certain amount of AuNPs-Fe-Glu-Lys nanoparticle was dissolved in 1 mL of Ultra-pure water, followed by adding 1 mL methyl violet solution with or without 20 L of hydrogen peroxide (10mM). The mixtures were stirred well, and the supernatants were collected every 10 min to measure the characteristic absorption peak of methyl violet by UV-Vis spectra for calculating the generation rate of hydroxyl radical.

2.8 Cell Culture Human malignant melanoma cells line A375 and human epidermal melanocyte cell line HEM-l were both obtained from the National Institutes of Health (NIH). HEM-l cells and A375 cells were cultured in Dulbecco's modification of Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Gibico, USA) and penicillin-streptomycin (100 U/mL and 100 μg/mL, Gibico, USA), and incubated at 37 o C in 5% CO2.

2.9 Cell uptake experiments



To observe the selective cell uptake of AuNPs-Fe-Glu-Lys nanoparticles, the fluorescence-labeled AuNPs-Fe-Glu-Lys (FITC-AuNPs-Fe-Glu-Lys) were prepared. A375 and HEM-l cells were seeded at 2.5 × 104 per well into 24-well plates with coverglass slides. The FITC-AuNPs-Fe-Glu-Lys were incubated with cells for 24 hours, followed by rinsing with PBS for 5 times, fixed with 4% paraformaldehyde for 20 min, and then stained with 4', 6-diamidino-2-phenylindole (DAPI, Life Technologies, USA). The stained cells were observed under a fluorescence microscope (Olympus BX51, Olympus, Japan). For quantitative analysis of cell uptake of AuNPs-Fe-Glu-Lys nanoparticles, A375 cells and HEM-l cells were seeded in a 6 well plate at a density of 1 × 106/well overnight. Then the FITC-AuNPs-Fe-Glu-Lys were incubated with cells at the Pt concentration of 1 µM at 37 °C for 24 hours. After incubation, the cells were gently washed by PBS 3 times and detached by trypsin (0.25 %). The cell suspensions were spun down and wash by PBS twice. Finally, the cells were counted by flow cytometer (FC500; Beckman Counter, CA, USA) .

2.10 In vitro cytotoxicity analysis A375 cells and HEM-l cells were seeded at 3 × 103 per well in 96-well plate for 24 hours before treatment. Then the cells were exposed to AuNPs-Fe-Glu-Lys for 24 h and 48 h in the absence/presence of 10 min near-infrared (NIR, 808 nm) light irradiation. Cell viability was measured by Cell Counting Kit 8 (CCK-8, Dojindo Co., Ltd. Japan) proliferation assay according to the manufacture’s protocol. The absorbance was read at 450 nm by using Varioskan Flash multimode reader (Thermo Fisher Scientific, USA).

2.11 In vivo Antitumor Efficacy


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All experimental protocols were approved by the Ethics Committee of the Fourth Military Medical University Health Science Center, Xi'an, China. To set up the tumor xenograft model, a total of 5 × 106 A375 cells were injected subcutaneously into the back of BALB/c female nude mice (5 weeks old), and permitted the tumor to reach a size over 50 mm3 in volume. Twenty-four tumor-bearing nude mice were divided into 4 groups (PBS group, AuNPs with NIR group, AuNPs-Fe-Glu-Lys with NIR group and pure AuNPs-Fe-Glu-Lys group). 100 μL of PBS, aqueous solution of AuNPs or AuNPs-Fe-Glu-Lys (2 mg·mL-1) were intravenously injected into the tail vein of the tumor-bearing nude mice. 24 hours post injection, the tumorous areas of AuNPs with NIR group and AuNPs-Fe-Glu-Lys with NIR group were exposed to 808 nm NIR irradiation (0.75 W) for 10 min to investigate the photothermal therapeutic effect in vivo. Tumor size (V=W2×L/2 mm3) was measured and the body weight were recorded every 3 days for 21 days. At day 21, the tumors were collected and fixed in 10% formalin overnight, embedded in paraffin, and sectioned at a thickness of 5 μm. The sections were stained with DeadEnd Fluorometric or Colorimetric TUNEL system (Promega Corporation, Madison, Wis) and hematoxylin and eosin (H&E). The in vivo antitumor activity was further investigated for survival. Another 4 groups (PBS, AuNPs with NIR, AuNPs-Fe-Glu-Lys and AuNPs-Fe-Glu-Lys with NIR) with 6 mice per group were monitored every 3 days until the mice were either naturally died or were sacrificed when the tumor volume grew to 2000 mm3 for survival analysis, according to the animal ethical requirement.

2.12 Characterization Transmission electron microscopy (TEM) images were recorded on a TECNAI G2 spirit BioTwin Transmission electron microscope equipped with energy dispersive spectrometer (EDS). For the TEM observation, samples were obtained by dropping 10



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μL of solution onto carbon-coated copper grids. All the TEM images were visualized without staining. The infrared (IR) spectra were measured by Nicolet iS50 FT-IR using KBr pellets. The ultraviolet-visible (UV-Vis) spectra were measured with dilute aqueous solution in a 2 mm thick quartz cell using a Hitachi U-2910 spectrophotometer. All pH value measurements were carried out on a Sartorius BECKMAN F 34 pH meter. The cellular uptake of FITC labeled AuNPs-Fe-Glu-Lys was monitored by fluorescence microscopy using a Olympus BX51 microscope equipped with a fluorescent lamp; ex = 490 nm, em = 550 nm for FITC.

3. Results and discussion 3.1 Fabrication of the multiple responsive AuNPs-Fe-Glu-Lys As to prepare the pH and enzyme dual responsive AuNPs, L-cysteine modified AuNPs were synthesized using similar method mentioned in the literature

30.

After

that, the resulting AuNPs were grafted by glutamine and lysine through chelation interactions between amino acids and Fe2+ to obtain glutamine and lysine decorated AuNPs (denoted as AuNPs-Fe-Glu-Lys below) 31. Fourier transform infrared (FTIR) spectrometry (Figure S1, Supporting Information) was used to verify these processes of synthesis and functionalization. As can be seen from this figure, obvious signals of COOH, NH2 as well as coordination bond (COOH...Fe2+...NH2) appeared after modification, which indicated the successful formation of L-cysteine, glutamine and lysine modified AuNPs. Moreover, obvious isopeptide bond appeared for the enzyme induced AuNPs aggregates, which demonstrated the polymerization between the amino acids triggered by TGase is the main force for the aggregation. The coordination structure of the AuNPs-Fe-Glu-Lys was also measured by Raman spectra. As can be seen from figure S2, the characteristic vibration peaks of COOH group shifted to short wavelength after the addition of Fe2+. Moreover, the new peaks of Fe...COOH (νFe-O) and Fe...NH2 (νFe-N) appeared at 384 cm-1 and 497 cm-1. These


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results clearly indicated the formation of coordination bond between Fe2+ and COOH/NH2 on AuNPs. The morphology of the AuNPs-Fe-Glu-Lys was also explored by the transmission electron microscopy (TEM, Figure 1a). As shown in this figure, the average size of the as-prepared AuNPs-Fe-Glu-Lys is about 15 nm. Similar size was also observed by the dynamic light scattering (DLS, Figure S3). While the AuNPs-Fe-Glu-Lys largely form particle aggregates after the TGase addition, which presented a size about 300 nm in DLS, suggesting that the interparticle cross-linking generated due to TGase catalyzed polymerization of these amino acids (Figure 1b). In addition to the TEM images, we performed an energy-dispersive X-ray (EDX) spectroscopy analysis of the AuNPs-Fe-Glu-Lys to evaluate their elemental composition. The levels of Au, Fe, S, C and N content, which pointed at the existence of AuNPs, Fe2+ and amino acids in this complex, were determined (Figure 1c). The valence state of the iron in AuNPs-Fe-Glu-Lys was also characterized by X-ray Photoelectron Spectroscopy (XPS). As shown in the figure S4, clear Fe2+ 2p3/2 multiplet peaks and Fe2+ 2p3/2 satellite peak appeared in the range between 709 eV to 714 ev, which directly showed that the iron majorly existed as Fe2+ in AuNPs-Fe-Glu-Lys. To further understand the modification of glutamine and lysine on AuNPs, thermogravimetric analysis (TGA, Figure 1d) and zeta potential measurements (Figure 1e) were performed. These results not only presented the content of L-cysteine (12 wt%) and glutamine/lysine (17 wt%), but exhibited the neutral charge of AuNPs-Fe-Glu-Ly, which were realized by successful combining the glutamine with negative charge and lysine with positive charge. Moreover, the content of ferrous ions in AuNPs-Fe-Glu-Lys nanoparticles was quantified to be 19.4 wt% by phenanthroline chromogenic method, according to the change of absorbance at 500 nm for phenanthroline-Fe2+ complex and the standard curve of phenanthroline-Fe2+ absorbance against the Fe2+ concentration (Figure S5). All these results revealed the successful formation of the AuNPs-Fe-Glu-Lys nanocomposites.



3.2 Enzyme-Triggered Photothermal Conversion Along with the morphological variations, the absorption peak of AuNPs-Fe-Glu-Lys solution shifted to longer wavelength around 800 nm after the TGase addition, which originally appeared at about 520 nm (Figure 1f). In consequence, the corresponding color change of the AuNPs-Fe-Glu-Lys solution from red to purple provided another direct evidence of the formation of AuNPs aggregates, which allows the polymerized AuNPs-Fe-Glu-Lys potentially useful for photothermal therapy of tumors. As to further understand this process, different amount of TGase was chosen to trigger the polymerization. As show in figure S6, the aggregation degree of AuNPs-Fe-Glu-Lys dramatically enhanced with the increase of TGase concentration, indicating that the enzymatic polymerization between amino acids on AuNPs derived the structural and functional transformation of AuNPs-Fe-Glu-Lys, which could be used for selective and effective tumor therapy. Moreover, to guarantee the TGase as only trigger for AuNPs-Fe-Glu-Lys aggregation, the stability of AuNPs-Fe-Glu-Lys nanoparticles in PBS (simulated physiological environment) was measured using UV and DLS after different time points up to 24 h. The results showed no obvious change of the absorption peak and nanoparticle size even after 24 h incubation, which indicated the high stability of the AuNPs-Fe-Glu-Lys without TGase (Figure S7). To directly investigate the TGase-induced AuNPs-Fe-Glu-Lys assembly for selective photothermal treatment, NIR light (800 nm) triggered photothermal conversion of AuNPs-Fe-Glu-Lys was studied before and after adding of TGase (amino acids: TGase = 50 μM per U). The power density of NIR light used in the experiments was 0.75 W/cm2. As we can be seen from Figure 2a, the temperature of the pure AuNPs-Fe-Glu-Lys solution slightly increased about 4 °C even after 15 min NIR irradiation, indicating a poor photothermal effect. By contrast, the temperature of solution containing TGase-induced AuNPs-Fe-Glu-Lys aggregation with same


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amount of AuNPs soared about 20 °C only after NIR irradiation for 5 mins. These results showed that the AuNPs-Fe-Glu-Lys are able to perform two different photothermal functions with and without TGase. This property allows our system to simultaneously achieve effective therapy to tumors (rich of TGase) and avoid unwanted damage to healthy tissues and/or organs (lack of TGase). The photothermal conversion performance of AuNPs-Fe-Glu-Lys treated by different amount TGase was further investigated, which show significant dependence to the TGase concentration

(Figure

S8).

Moreover,

the

photothermal

stability

of

AuNPs-Fe-Glu-Lys was also investigated and shown in figure S9. As shown in the figure, the local temperature could still raise about 20 degree under NIR irradiation even after 5 cycles of photothermal conversion, which indicated the great photothermal stability of AuNPs-Fe-Glu-Lys.

3.3 In vitro Fenton Reaction of AuNPs-Fe-Glu-Lys Considering the high concentration of hydrogen peroxide (H2O2) in tumor cells as well as the existence of Fe2+ ions in our nanocomplex, the AuNPs-Fe-Glu-Lys was expected to trigger Fenton reaction in situ to produce aggressive hydroxyl radicals for tumor therapy.32 As a hydroxyl radical scavenger, methyl violet (MV) was used to visualize the production capacity of hydroxyl radicals. The purple MV could quickly react with generated hydroxyl radical and then shift its color to colorless. 33 Figure 2b presented the UV-Vis spectrum of MV, MV/H2O2, MV/AuNPs-Fe-Glu-Lys, and MV/AuNPs-Fe-Glu-Lys/H2O2 solutions. As can be seen from this figure, both MV/H2O2 and MV/AuNPs-Fe-Glu-Lys solutions exhibited a typical absorption peak of MV at 582 nm, which were same as the untreated MV only with the slight decrease of the peak intensity. However, a remarkable decrease of the MV absorbance appeared after adding both H2O2 and AuNPs-Fe-Glu-Lys into MV solution. These results demonstrated the successful formation of hydroxyl radicals via Fenton reaction,



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which requests both AuNPs-Fe-Glu-Lys and H2O2. The time-dependent catalytic performance of AuNPs-Fe-Glu-Lys to Fenton reaction was investigated and exhibited in the Figure 2c. These results illustrated that the AuNPs-Fe-Glu-Lys is able to perform the H2O2-triggered hydroxyl radical production via Fenton reaction, opening a window of catalytic medicine for anticancer therapy. As biomaterial to treat tumor via Fenton reaction, the constant Fe2+ in AuNPs-Fe-Glu-Lys during therapy is important. Thus the release profile of ferrous ions from the AuNPs-Fe-Glu-Lys in simulated physiological environment was measured by phenanthroline chromogenic method. The results showed that only less than 5% of ferrous ions were released even after 48 h incubation, indicating the sufficient Fe2+ content of our nanoparticles for Fenton reaction during the whole tumor therapy (Figure S10). To further investigate the stability of Fe2+ in AuNPs-Fe-Glu-Lys,

Fenton

reaction

was

carried

out

after

incubating

AuNPs-Fe-Glu-Lys in PBS for different period of time (0-48h). As shown in figure S11, negligible decrease of the catalytic performance were observed even after co-incubation for 48 h. All these data not only provides further evidence that the valence state of iron in AuNPs-Fe-Glu-Lys is Fe2+, but indicated the high stability of Fe2+ in AuNPs-Fe-Glu-Lys under simulated physiological environment for following tumor therapy. 3.4 pH-Responsive Zwitterionic Property of AuNPs-Fe-Glu-Lys Owing to the different isoelectric points of glutamine and lysine, the AuNPs-Fe-Glu-Lys with selected ratio of these two type amino acids should be able to perform a zwitterionic surface under pH 7.4 (normal tissue) and positively charged surface under pH 6.8 (tumor tissue) to further enhance the tumor selectivity. To demonstrate the pH-responsive zwitterionic surface on AuNPs-Fe-Glu-Lys, these nanoparticles were investigated by zeta potential measurements in PBS with pH 7.4 (simulated healthy tissue) and 6.8 (simulated tumor tissue). As shown in figure 1e, the


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AuNPs-Fe-Glu-Lys indeed perform a zwitterionic surface with nearly neutral charge at pH 7.4, which switched to positive layer when the pH value changed to 6.8. As we know, the biomedical function of this switchable zwitterionic surface for resisting nonspecific protein adsorption contributed the sustained blood circulation and subsequent selective endocytosis of our particles into tumor cells. Therefore, the interaction between AuNPs with various compositions and model plasma proteins (bovine serum albumin, BSA) was measured under different pH values to demonstrate this function. As can be seen from Figure 2d, much less BSA absorbed on AuNPs-Fe-Glu-Lys than that on pure AuNPs after incubating in BSA solution with pH 7.4 for 1-7 days. It indicates that the existence of zwitterionic layer on AuNPs effectively improved their resistance to surrounding proteins at normal physiological condition. Moreover, the BSA absorption percentage on AuNPs-Fe-Glu-Lys significantly increased from 10% to 90% only after 1 d incubation, when the environment switch to acidic BSA solution (pH 6.8, simulated extracellular matrix of tumor tissue). These results indicate that the pH-responsive zwitterionic layer could not only prevent the nonspecific protein adsorption and unexpected cell uptake of AuNPs-Fe-Glu-Lys before arriving the tumor tissue, but enhance the endocytosis of these nanoparticles to tumor cells for accurate tumor therapy 26,34. 3.5 In vitro Tumor Therapy of AuNPs-Fe-Glu-Lys To demonstrate the highly selective accumulation of AuNPs-Fe-Glu-Lys in tumor cells, FITC labeled AuNPs-Fe-Glu-Lys (AuNPs-Fe-Glu-Lys-FITC) were incubated with human malignant melanoma cells line A375 (tumor cell) at pH 6.8 and human epidermal melanocyte cell line HEM-l (normal cell) at pH 7.4. After 48 hours incubation, the uptake and distribution of AuNPs-Fe-Glu-Lys-FITC was observed by confocal fluorescence microscope. As can be seen from Figure 3a, green AuNPs-Fe-Glu-Lys-FITC congeries were mainly localized in the cytoplasm of A375 cells, which further demonstrated the happening of the TGase triggered



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AuNPs-Fe-Glu-Lys aggregation in tumor cells after endocytosis. Comparing with A375 cells, negligible and dispersed green dots were observed in HEM-l cells, indicating the highly selective characteristic of AuNPs-Fe-Glu-Lys to tumor cells. The flow cytometry was used to quantitatively study the cell uptake of AuNPs-Fe-Glu-Lys by A375 cells and HEM-l cells (Figure 3b). After the co-incubation of AuNPs-Fe-Glu-Lys-FITC with these cells for 6 h , 4.76±0.59% of HEM-l cells were FITC-positive, while 63.4±2.72% of A375 cells were FITC-positive.

This

result

directly

indicated

the

excellent

selectivity

of

AuNPs-Fe-Glu-Lys for tumor therapy. After demonstrating the tumor selective endocytosis, the intracellular generation of reactive oxygen species (ROS) in tumor cells was also investigated by the reactive oxygen species assay kit. As can be seen from figure S12, A375 tumor cells displayed strong red fluorescence after AuNPs-Fe-Glu-Lys adding. However, no visible fluorescence appeared in cells without AuNPs-Fe-Glu-Lys. These data directly showed the AuNPs-Fe-Glu-Lys induced intracellular generation of ROS. Cell Counting Kit 8 (CCK-8, Dojindo Co., Ltd. Japan) was utilized to evaluate the in vitro

cytotoxicity

of

AuNPs,

AuNPs

with

NIR,

AuNPs-Fe-Glu-Lys,

AuNPs-Fe-Glu-Lys with NIR on A375 cells and HEM-l cells to further investigate the tumor selectivity and final therapeutic efficiency of AuNPs-Fe-Glu-Lys (Figure 3c). After 48 hours incubation, AuNPs-Fe-Glu-Lys exhibited slight toxicity to HEM-l cells with or without NIR irradiation. However, only 40% of A375 cells survived after 48 hours incubation with AuNPs-Fe-Glu-Lys even lack of NIR irradiation, which is attributed to the Fe2+-induced Fenton reaction. The AuNPs-Fe-Glu-Lys with NIR irradiation exhibited the strongest cytotoxicity to A375 cells. Due to the synergistic effect of Fe2+-induced Fenton reaction and photothermal therapy, only 10% of A375 cells survived after 48 hours incubation, which were about one eighth of that to


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HEM-l cells, presenting high efficiency and great selectivity of our device for accurate tumor therapy. As to rule out experimental contingencies, we further tested the cell viability of normal cells and tumor cells at different concentrations of AuNPs-Fe-Glu-Lys with and without NIR irradiation. As shown in figure S13, the AuNPs presented nonselective and negligible cytotoxicity (cell viability over 80%) to both normal cells and tumor cells even the concentration is up to 200 ug/ml, no matter with or without NIR irradiation. However, the AuNPs-Fe-Glu-Lys has showed obvious cytotoxicity at 25 ug/ml, which gradually increased against its concentration. Moreover, the AuNPs-Fe-Glu-Lys always performed high cytotoxicity to tumor cells over normal cells under each concentration, which could be further enhanced by NIR irradiation. 3.6 In vivo Tumor Therapy of AuNPs-Fe-Glu-Lys The current results already indicated that the pH-enzyme-H2O2 multiple responsive AuNPs-Fe-Glu-Lys is promising for tumor therapy in vitro. To exam the synergistic anticancer efficacy in vivo, twenty-four A375 tumor-bearing nude mices were randomly assigned to PBS with NIR group, AuNPs with NIR group, AuNPs-Fe-Glu-Lys group and AuNPs-Fe-Glu-Lys with NIR group, then 100 μL of certain nanoparticle solution with concentration of 2 mg/mL was intravenously injected into these tumor-bearing nude mice. After injecting for 24 h, the tumor of AuNPs-Fe-Glu-Lys with NIR group were irradiated for 10 min using 808 nm NIR laser (0.75 W/cm2) and then the in vivo photothermal therapy of AuNPs-Fe-Glu-Lys was investigated. As we can be seen from Figure 4a, the local temperature of tumors raised to over 60 °C after NIR irradiation for 10 min. However, with the same injection doses, the local tumor temperature of other groups (i: tumors after PBS injection with NIR, ii: tumors after AuNPs injection with NIR and iii: tumors after AuNPs-Fe-Glu-Lys injection without NIR) were all below 42°C under NIR laser irradiation.



As to further prove the aggregation of AuNPs-Fe-Glu-Lys in tumor tissues, the inductively coupled plasma−mass spectrometry (ICP-MS) was used to quantify the Au element in major organs and tumor tissue of mice at 24 h after a single injection via tail vein. As can be seen from figure S14, even some AuNPs-Fe-Glu-Lys appeared in liver and spleen, the tumor tissue still have the highest content of AuNPs-Fe-Glu-Lys. All these results indicated that the AuNPs-Fe-Glu-Lys is able to selectively aggregate in tumor for further therapy. To show the tumor therapy effect in vivo, the average tumor size and body weight of different groups was monitored every 3 days for 21 days. Comparing with the PBS group (295 ± 35 mm3), the AuNPs treated group showed no obvious tumor inhibition effect (243 ± 28 mm3) on the 21st day. However, the single catalytic medicine group (AuNPs-Fe-Glu-Lys without NIR) significantly inhibited the tumor growth (99 ± 7 mm3). Moreover, the dual-modal group combining photothermal therapy and catalytic medicine (AuNPs-Fe-Glu-Lys with NIR) completely inhibited the tumor growth and almost eliminated the tumor (7 ± 3 mm3). Hhematoxylin and eosin (HE) staining and TUNEL staining of tumor sections from all groups were shown in Figure 4c. Compared with the PBS group, the tumor cells under AuNPs-Fe-Glu-Lys with NIR treatment shrunk and lost contact with large necrotic areas. The cell under AuNPs treatment and AuNPs-Fe-Glu-Lys without NIR showed different degrees of damage among tumor cells without necrotic areas. The TUNEL staining showed that, compared with the PBS group and the AuNPs group, higher degree of apoptosis appeared in the tumors treated by AuNPs-Fe-Glu-Lys with and without NIR. These results confirmed that the multi-therapy group has higher therapeutic efficiency than the group with single function, which could be attributed to the synergistic effects as mentioned earlier. In order to further evaluate the therapeutic efficiency, the survival rates of mice treated with different formulations were investigated (Figure 4d). The mice received AuNPs-Fe-Glu-Lys with NIR treatment achieved the longest survival time and the highest survival rate. 66% of the


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mice received AuNPs-Fe-Glu-Lys with NIR treatment survived over 50 days. However, mice receiving other treatments survived for relatively short time. As can be seen from figure 4d, mice treated by PBS and AuNPs with NIR did not survive over 23 d and 28 d, respectively. While 16% of the mice treated by AuNPs-Fe-Glu-Lys without NIR irradiation survived after 50 d, which provide a direct evidence about the efficiency of catalytic medicine beyond photothermal therapy in AuNPs-Fe-Glu-Lys. Moreover, the body weight of mice treated by AuNPs-Fe-Glu-Lys and NIR irradiation had no obvious changes and the H&E stained showed that all the organs had no noticeable pathological abnormalities to the end of the treatment (Figure S15 and Figure S16, Supporting Information). All these results suggested that our multi-functional AuNPs-Fe-Glu-Lys could act as a promising platform for effective tumor treatment, which also perform no visible side effects to the diseased mice.

4. Conclusion In this study, we have developed a simple and novel strategy to fabricate a multiple amino acid modified gold nanoparticles (AuNPs-Fe-Glu-Lys) with pH-dependent zwitterionic surface, enzyme (TGase) responsive photothermal property and H2O2 selective catalytic performance. This design was expected to combine the tumor selectivity, tumor-triggered photothermal therapy as well as intracellular catalytic reaction for efficient and accurate tumor treatment. In the acidic environment of tumor tissue, the well-designed AuNPs-Fe-Glu-Lys would switch from electric neutrality to positive charge, providing efficient cellular uptake only to tumor cells. Then the intracellular TGase started to trigger the polymerization, followed with the AuNPs-Fe-Glu-Lys aggregation and enhanced photothermal property. In addition, the Fe2+ delivery by AuNPs-Fe-Glu-Lys would further cause the H2O2-responsive radicals generation via Fenton reaction, which is able to significantly enhance the



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therapeutic effect. The all-in-one device performed not only an eightfold cytotoxicity against tumor cells compared with normal cells under 48 h after NIR irradiation, but a complete inhibition and gradual elimination of tumor tissue after three weeks of treatment, illustrating its potential for accurate tumor therapy.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: ***. Fourier transform infrared spectroscopy, raman spectra, dynamic light scattering and X-ray photoelectron spectroscopy measurements indicate the chemical composition and physical structure of AuNPs-Fe-Glu-Lys; UV-Vis spectra and dynamic light scattering measurements indicate the stability and enzyme-triggered aggregation of AuNPs-Fe-Glu-Lys;

Thermal

heating

curves

and

thermal

stability

of

AuNPs-Fe-Glu-Lys after enzyme-triggered aggregation; In situ ROS generation in tumor cells; Cytotoxicity of AuNPs-Fe-Glu-Lys with different concentration; Biodistribution of AuNPs-Fe-Glu-Lys; Body weight of tumor-bearing mice after different treatments; Physiological toxicity of AuNPs-Fe-Glu-Lys.

Acknowledgements This work was supported by the National Natural Science Foundation of China (81601606 to X.C.), the “Young Talent Support Plan” of Xi’an Jiaotong University (X.C.), the Technology Foundation for Selected Overseas Chinese Scholar of Shaanxi Province (X.C.), the Fundamental Research Funds for the Central Universities (2016qngz02 to X.C.), the One Hundred Talents Program of Shaanxi Province (X.C.), the Natural Science Foundation of Shaanxi Province (2017JM5023 to X.C.), open


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fund of the State Key Laboratory of Military Stomatology (2017KA02 to X.C.), the Knowledge Innovation Program of Shenzhen (JCYJ20170816100941258 to X.C.)

Scheme 1. (a) Schematic illustration of the synthesis and TGase triggered aggregation of AuNPs-Fe-Glu-Lys. (b) Schematic representation of AuNPs-Fe-Glu-Lys with pH



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switchable zwitterionic surface, TGase induced photothermal property as well as the H2O2 triggered Fenton reaction based on this nanoparticle, which was expected to combine the tumor selectivity, photothermal therapy and catalytic medicine for efficiency and accurate tumor therapy.

Figure 1. Transmission electron microscope images of AuNPs-Fe-Glu-Lys before (a) and after (b) the addition of TGase as well as the corresponding EDS element analysis of AuNPs-Fe-Glu-Lys (c); (d) Thermogravimetric analysis of gold nanoparticles (AuNPs, black curve), L-cysteine modified AuNPs (AuNPs-Cys, red curve) and glutamine/lysine grafted AuNPs-Cys (AuNPs-Fe-Glu-Lys); (e) Zeta-potential changes of gold nanoparticles (AuNPs), L-cysteine modified AuNPs (Au-Cys), glutamine grafted

Au-Cys

(Au-Fe-Glu),

lysine

grafted

Au-Cys

(Au-Fe-Lys)

and

glutamine/lysine grafted Au-Cys (Au-Fe-Glu-Lys) at pH 7.4 and pH 6.8; (f) UV-Vis


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spectra of gold nanoparticles (AuNPs, black curve), L-cysteine modified AuNPs (AuNPs-Cys, red curve), glutamine/lysine grafted AuNPs-Cys (AuNPs-Fe-Glu-Lys) before (blue curve) and after (magenta curve) TGase adding (The inset are photographs of AuNPs-Fe-Glu-Lys solutions before and after TGase adding).

Figure 2. Thermal heating curves (a) and photothermal image (i, ii) of the AuNPs-Fe-Glu-Lys solution with (i) and without (ii) TGase. The local temperatures



were obtained by infrared thermography; ROS generation from AuNPs-Fe-Glu-Lys investigated by UV-vis spectrum (b) and the time-dependent efficiency of AuNPs-Fe-Glu-Lys to catalyze the ROS generation (c); Schematic depiction of pH-switchable zwitterionic layer on AuNPs-Fe-Glu-Lys for resistance of nonspecific protein absorption as well as real BSA adsorption on AuNPs and AuNPs-Fe-Glu-Lys after 1 d, 3 d, and 7 d co-incubation at 37 °C under pH 7.4 or pH 6.8 (d).

Figure 3. Fluorescent microscope images of HEM-l cells (normal cell) and A375 cells (tumor cell) after incubation with FITC labeled AuNPs-Fe-Glu-Lys for 24 h (a);


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Quantitative uptake of FITC labeled AuNPs-Fe-Glu-Lys by A375 cells (tumor cells) and HEM-l cells (healthy cells), which were eveluated by flow cytometry (b); Cell viability of HEM-l cells (normal cell) and A375 cells (tumor cell) incubated with AuNPs and AuNPs-Fe-Glu-Lys for 48 h before and after NIR treatment (c).



Figure 4. In vivo photothermal images of tumor-bearing mice after injection of AuNPs-Fe-Glu-Lys (i, iv), PBS (ii) and AuNPs (iii) with (ii, iii, iv) and without (i) 808 nm irradiation (a); The tumor volume change after treated by different formulations and conditions. The data are shown as mean ± SD, NS means no significance, ***means p

Intracellular Enzyme-Triggered Assembly of Amino Acid-Modified Gold

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