Generation 9 Polyamidoamine Dendrimer Encapsulated Platinum

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Generation 9 Polyamidoamine Dendrimer Encapsulated Platinum Nanoparticle Mimics Catalase Size, Shape and Catalytic Activity Xinyu Wang, Yincong Zhang, Tianfu Li, Wende Tian, Qiang Zhang, and Yiyun Cheng Langmuir, Just Accepted Manuscript • DOI: 10.1021/la3046077 • Publication Date (Web): 01 Apr 2013 Downloaded from http://pubs.acs.org on April 3, 2013

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Generation

9

Polyamidoamine

Dendrimer

Encapsulated

Platinum Nanoparticle Mimics Catalase Size, Shape and Catalytic Activity Xinyu Wang1,†, Yincong Zhang1,†, Tianfu Li2, Wende Tian3, Qiang Zhang1,*, and Yiyun Cheng1,* 1

Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East

China Normal University, Shanghai, 200062, People’s Republic of China 2

China Institute of Atomic Energy, Beijing, 102413, People’s Republic of China

3

Center for Soft Condensed Matter Physics and Interdisciplinary Research, Soochow

University, Suzhou, 215006, People’s Republic of China

Keywords: dendrimer, PAMAM, platinum nanoparticle, catalase mimic

ACS Paragon Plus Environment

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ABSTRACT In the present study, poly(amidoamine) (PAMAM) encapsulated platinum nanoparticles were synthesized and used as catalase mimics. Acetylated generation 9 (Ac-G9) PAMAM dendrimer with a molecular size around 10 nm was used as a template to synthesize platinum nanoparticles. The feeding molar ratio of Pt4+ and Ac-G9 is 2048 and the synthesized platinum nanoparticle (Ac-G9/Pt NP) have an average size of 3.3 nm. Ac-G9/Pt NP has a similar molecular size and globular shape with catalase (~11 nm). The catalytic activity of Ac-G9/Pt NP on the decomposition of H2O2 is approaching that of catalase at 37 °C. Ac-G9/Pt NP shows differential response to the changes of pH and temperature compared with catalase, which can be explained by different catalytic mechanisms of Ac-G9/Pt NP and catalase. Ac-G9/Pt NP also shows horse radish peroxidase activity and is able to scavenge free radicals such

as

di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium

(DPPH).

Furthermore,

Ac-G9/Pt NP shows excellent biocompatibility on different cell lines and can down-regulate H2O2-induced intracellular reactive oxygen species (ROS) in these cells. These results suggest that dendrimers are promising mimics of proteins with different sizes and Ac-G9/Pt NP can be used as an alternative candidate of catalase to decrease oxidation stress in cells.


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INTRODUCTION Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion radical (O2·-) and hydroxyl radical (·OH) are generated as by-products of cellular metabolism.1 They are highly reactive and play critical roles in many biological processes.2 Normal ROS takes responsibilities for cellular signaling, pathogen defense, apoptosis induction, and homeostasis,2-4 but excessive ROS generation can overwhelm protective mechanism and cause significant cellular structure damages, resulting in serious pathological symptoms such as diabetes, inflammatory arthritis, neurodegenerative and cardiovascular diseases.1,5 Generally, cells defense themselves against the ROS damages by enzymes such as catalases, superoxide dismutases (SOD), and peroxiredoxins.1,6 These enzymes are able to eliminate excessive ROS in normal cells. However, cells may cripple or even lost their antioxidant functions under pathological conditions, which can lead to ROS induced cellular damages in term of proteins, DNA, and lipids.5 Recently, artificial enzymes were widely used to promptly eliminate excessive ROS in the cells.7 Molecules such as protein, peptide, cyclodextrin, graphene, and polymers with catalytic active units were developed as artificial enzymes.6-11 Metal and/or metal oxide nanoparticles (NPs) were reported to efficiently eliminate excessive ROS.8-10,12,13 For example, Fe3O4 and Co3O4 NPs possess peroxidase-like and catalase-like activities.12,13 Apoferritin-encapsulated platinum (Pt) NPs are able to efficiently quench H2O2 and O2·-.8 The use of apoferritin as template can not only produce Pt NPs with small size and excellent monodispersity, but also render the Pt NPs with improved biocompatibility, stability, and cellular uptake.8 Apoferritin-encapsulated CeO2 NPs can act as SOD mimics, in which apoferritin can manipulate the electron localization on the surface of CeO2 NPs and improve their ROS scavenging activities.10 These artificial metal and/or metal oxide NPs have shown great promise in the development of antioxidant enzyme mimics and ROS involved disease treatment,6 but their catalytic activities remain to be improved. In addition, protein/peptide-encapsulated NPs are immunogenic and may cause additional risks when used as antioxidant enzyme mimics.14 Furthermore, the sizes of


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the protein encapsulated NPs are distinct from that of natural enzymes, which may lead to different cellular uptake, localization, and ROS generation behaviors.15 Therefore, it is still a great challenge to design and construct artificial enzymes with the comparable catalytic activity, size, shape, and biocompatibility to natural enzymes. Dendrimers, a new class of hyperbranched macromolecules, have been intensively used as substrates for artificial enzymes.16-18 The globular shape, tunable size, and dendritic effect are their unique advantages to construct artificial enzymes.19-24 The interior cavities and surface functionalities offer multiple sites to build the enzyme-like catalytic centers.23,25,26 Though several explorations on enzyme-like properties of dendritic structures have been processed in the past decade, the artificial enzymes are mainly constructed by integrating catalytic residues to dendrimer core, repeated unit, or surface during the synthesis of dendrimers.16,18 Polyamidoamine (PAMAM) dendrimers reported by Tomalia in 1985 are the most used dendrimers to build artificial enzymes.27 The surface amine, interior tertiary amine and amide groups of PAMAM dendrimers have high capacities to bind metal ions such as Pb2+, Cd2+, Pt2+, Pt4+, Ag+, Au3+, Pd3+ through coordination bonds.28,29 These bound metal ions can be chemically reduced, which results in the formation of dendrimer-encapsulated nanoparticles containing up to a few hundred atoms.28,30-36 Up to now, PAMAM dendrimers were used as templates to synthesize monometallic, alloy, core/shell, bimetallic NPs by the Crooks group.30,33,34,37-44 PAMAM encapsulated Pt, Pd, and Au NPs were synthesized and used as catalysts, therapeutics, labeling probes, and X-ray computed tomography contrasts.45,46 However, no report on the use of dendrimer-encapsulated metal NPs as catalase mimics can be found in the references. In this study, generation 9 (G9) PAMAM dendrimer, which possesses a similar size and globular shape as compared to the catalase (~11 nm, Scheme 1), was employed as a template to synthesize Pt NPs. Acetylated G9 PAMAM dendrimer (Ac-G9) was used to improve the biocompatibility of the synthesized dendrimer-encapsulated NPs.47 The feeding molar ratio of Pt4+ and Ac-G9 at 2048 is chosen in the preparation of the artificial catalase. The as-synthesized Pt NPs within Ac-G9 dendrimer have an


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average

size

of

3.3

nm.

The

catalytic

activity

of

Ac-G9

PAMAM

dendrimer-encapsulated Pt NPs (Ac-G9/Pt NP) in decomposing H2O2 approaches that of catalase, but performs a differential response to the changes of temperature and pH. Ac-G9/Pt NP is biocompatible on several cell lines and performs efficiently in the action of ROS scavenging in vitro.

EXPERIMENTAL METHODS Chemicals. G9 ethylenediamine (EDA)-cored and primary amine-terminated PAMAM dendrimers were purchased from Dendritech Inc. (Midland, MI). K2PtCl6, NaBH4, H2O2, Na2HPO4 and NaH2PO4 were purchased from Aladdin Inc. (Shanghai, China). 3,3’,5,5’-tetramethylbenzidine (TMB), 2’,7’-dichlorofluorescein diacetate (DCFH-DA),

and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) were

obtained from Sigma-Aldrich Inc. (St. Louis, MO). Catalase and horse radish peroxidase (HRP) were from Beyotime Institute of Biotechnology (Shanghai, China). Synthesis of Ac-G9 and Ac-G9/Pt NP. Acetylation of G9 PAMAM dendrimer was performed according to a well-established method.48 21.03 mg G9 PAMAM dendrimer was dissolved in 5 mL methanol, followed by the addition of excess amounts of acetic anhydride (5 equivalents per dendrimer surface primary amine to ensure complete acetylation) and triethylamine (1.2 equivalents of acetic anhydride to neutralize the acetic acid generated during acetylation). The mixture was stirred at room temperature for 24 h. The excess acetic anhydride and side products in the mixtures were removed by extensive dialysis (molecular weight cut off is 14000 Da) against phosphate buffer (0.2 M Na2HPO4/NaH2PO4, pH = 7.5) and Millipore water for two days. The purified samples were lyophilized, and white powders were obtained and stored at 4 oC before use. The product of Ac-G9 was characterized by 1H NMR spectroscopy. Ac-G9 dendrimer-encapsulated Pt NPs comprised of 2048 atoms (Ac-G9/Pt NP) were prepared according to the method of Richard M. Crooks with small modifications.28 1 mL, 3.917 µM aqueous solution of Ac-G9 was mixed with 0.802


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mL, 0.010 M K2PtCl6 aqueous solution to process a final Pt4+-to-dendrimer ratio of 2048:1. The mixture solution was then sonicated for 10 min and following magnetic stirring for 12 h, a 5-fold molar excess of aqueous NaBH4 (1 M) was added to reduce Pt4+. This solution was kept capped for 24 h to maximize reduction of Pt4+. Finally, Ac-G9/Pt NP was dialyzed for more than 8 times in 500 mL Millipore water to remove salts and other impurities. Characterization of Ac-G9 and Ac-G9/Pt NP. NMR, TEM, UV-Vis Analysis: 1H NMR experiments on a Varian 699.804 MHz NMR spectrometer at 298.2 ± 0.2 K were conducted for Ac-G9 PAMAM dendrimer in D2O: Ha, 2.302 ppm (-NCH2CH2CONH-); Hb, 2.545 ppm (-CONHCH2CH2N-); Hc, 2.730 ppm (-NCH2CH2CONH-);

Hd,b’,d’,

3.144

ppm

(Hd,

-CONHCH2CH2N-;

Hb’,

-CONHCH2CH2NHCOCH3; Hd’, -CONHCH2CH2NHCOCH3); He, 1.829 ppm (-NHCOCH3).The synthesized Ac-G9/Pt NP is examined by a TEM (JEOL JEM-2100). UV-Vis spectra of the Ac-G9/Pt4+ and Ac-G9/Pt NP were recorded using a UV-2600 spectrophotometer (Techcomp, Shanghai). In Vitro Catalase-like Activity of Ac-G9/Pt NP. The steady-state kinetic assays were carried out at 37 °C in a flask with 10 nM Ac-G9/Pt NP or 2.5 nM catalase in 10 mL reaction solution in the presence of H2O2 (concentration range from 5 mM to 75 mM). The H2O2 concentration can be calculated by C (mM) = 22.94 × A240 according to the protocol of Catalase Assay Kit (Beyotime Institute of Biotechnology, Shanghai), where C is the concentration of H2O2 and A240 is the absorbance of the reaction solution at 240 nm. The reactions were monitored at 240 nm using a spectrophotometer during a period of 10 min. The absorbance was recorded at 2 min, 5 min and 10 min to obtain a reaction rate curve. The initial velocity v0 was calculated by calculating the slope of the tangent at t = 0 min. The apparent kinetic parameters were calculated based on Michaelis equation v0 = Vmax × [S]/( Km + [S]), v0 is the initial velocity, Vmax is the maximal reaction velocity, [S] is the concentration of substrate, and Km is the Michaelis constant.49 Km and Vmax were obtained by Lineweaver-Burk plot method. Temperature- and pH-dependent Assay. The experiments were carried out using


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1 nM catalase or 5 nM Ac-G9/Pt NP in each assay. The H2O2 concentration was 100 mM for temperature-dependent assay and 50 mM for pH-dependent assay. The temperature-dependent assay was performed in aqueous solution while the pH dependent assay was performed in buffers with different pH values at room temperature. In Vitro Peroxidase-like Activity Assay. The peroxidase-like activity assays were performed in 4 mL quartz cells with concentration of Ac-G9/Pt NP and Horse Radish Peroxidase (HRP) at 1 nM. The reaction is carried out in 2 mL buffer with 0.25 mM TMB and 125 mM H2O2. The blue color observed is determined at the absorbance of 655 nm by a UV-Vis spectrophotometer. DPPH Assay. The free radical scavenging activities of Ac-G9/Pt NP was measured by using the method of Brand-Williams et al.50 Briefly, a 0.06 mM solution of the stable free radical DPPH in ethanol was prepared and 1.9 mL of this solution was added to 0.1 mL of Ac-G9/Pt NP aqueous solution. The absorbance of the reaction solution at 517 nm was measured after 60 min. As a control, 0.1 mL of Millipore water was used instead of the experimental sample. Cell Viability Assay. The cells were cultured in DMEM (GIBCO) with 10% fetal bovine serum (GIBCO), 37 °C, 5% CO2. MCF-7 cells and HeLa cells were seeded in a 96-well plate (BD Biosciences) at 1×104 cells/well. For the determination of the cytotoxicity of Ac-G9/Pt NP, an MTT assay was conducted. After the incubation of cells with Ac-G9/Pt NP, the medium was replaced with 0.5% MTT containing DMEM for another 2 h. Then the MTT medium was replaced by 150 µL DMSO. The plate was shaken for more than 10 min until the yielding Formazan was dissolved completely. The absorbance is detected by a microplate reader (MQX200R, BioTek Inc.) at a wavelength of 490 nm and a reference wavelength of 650 nm. The biocompatibility of Ac-G9/Pt NP was further evaluated by an acridine orange (AO)/ ethidium bromide (EB) double-staining experiment. Generally, the cells were cultured in a 24-well plate in DMEM for 24 h before the experiment. The cells were then incubated with 30 nM Ac-G9/Pt NP for 12 h, and the cells were then washed with PBS buffer three times and stained by AO and EB containing PBS (5 µg/mL AO and 5


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µg/mL EB) for 10 min. The stained cells were imaged by a fluorescence microscope (Olympus, Japan). To investigate the protective effect of Ac-G9/Pt NP against the cytotoxicity of H2O2, the cells were plated at a density around 1×104 cells/well in 96-well plates and settled overnight for adherence. Different concentrations of Ac-G9/Pt NP were then added into wells for 12 h incubation. The medium was removed and washed once with PBS buffer, after that 5 mM H2O2 diluted with DMEM without fetal bovine serum was added. The cells were incubated for another 1 h at 37 °C, 5% CO2 prior to being further analyzed. Cell viability was also measured by the MTT assay. For the AO/EB double-staining assay, the cells were plated at a density around 5×104 cells/well in 24-well plates and settled overnight for adherence. And the process is similar to MTT assay with a modification that the H2O2 is incubated for 4 h at a concentration of 1 mM. Intracellular Localization of Ac-G9/Pt NP and Ac-G9. To investigate the intracellular localization of the Ac-G9/Pt NP and Ac-G9, we labeled the materials with a green fluorescent dye fluorescein isothiocyanate (FITC). HeLa cells are incubated on coverslips overnight. The cells are incubated with the FITC-labeled materials for 1 h, and then stained by 100 nM Lysotracker Red DND-99 for 5 min, followed by washing with PBS twice. After that, the cells are fixed by 4% paraformaldehyde for 20 min. The slides were mounted and observed with a confocal laser scan microscope (CLSM, Leica). Determination of intracellular ROS. The intracellular ROS was determined by the DCFH-DA assay. MCF-7, HeLa, and RAW 264.7 cells were seeded in 24-well plates at a density of 5× 104 cells per well and incubated for 12 h at 25 °C. Subsequently, the cells were incubated with 30 nM Ac-G9/Pt NP or Catalase for 6 h. After incubation, the cells were rinsed three times with PBS (pH 7.4), then exposed to 500 µM H2O2 in culture medium for 12 h at 37 °C. Afterwards, the cells were rinsed again, followed by incubation with 20 µM DCFH-DA at 37 °C for 30 min. The green fluorescent intensity within the cells was observed by a fluorescence microscope. The intracellular ROS level in each sample was reflected by the fluorescent intensity of


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the images.

RESULTS AND DISCUSSION Synthesis and Characterization of Ac-G9/Pt NP. Bovine liver catalase is a tetramer consisted of four heme groups that allow the enzyme to react with hydrogen peroxide. It has a globular shape and a nanoscale size around 11 nm (Scheme 1). Catalase is one of the most important antioxidant enzymes in the living organisms and can catalyze the decomposition of millions of H2O2 molecules into water and oxygen per second.51 Here, G9 PAMAM dendrimer with a globular shape and 10 nm size was used as the template to synthesize dendrimer-encapsulated Pt NPs (Scheme 1). Since high generation cationic PAMAM dendrimers were reported with serious cytotoxicity and hemolytic toxicity,47 G9 PAMAM dendrimer was first acetylated to remove the surface amine groups before the synthesis of Pt NPs. Surface acetylation can improve the biocompatibility of cationic dendrimers while maintaining cell membrane permeability.52 Also, surface acetylation can ensure the synthesis of metal NPs within the interior pockets rather on the surface of PAMAM dendrimers. 1H NMR spectra of Ac-G9 indicates successful acetylation of amine groups on G9 PAMAM dendrimer (data not shown). After the addition of Pt4+ ions at a Pt4+/Ac-G9 ratio of 2048 and the reduction of bound Pt4+ ions by sodium borohydrile, Ac-G9/Pt NP was obtained. Transmission electron microscopy (TEM) image in Figure 1A indicates that Ac-G9/Pt NP is sphere-like shape. The average size of Ac-G9/Pt NP is calculated to be 3.3 nm (Figure 1B). The small size of Pt NPs synthesized within Ac-G9 predicts their outstanding catalytic activities on the decomposition of H2O2 molecules. The UV-Vis spectra of Ac-G9/Pt NP is shown in Figure 1C, the mixture of Ac-G9 and K2PtCl6 shows a characteristic peak of Ac-G9/Pt4+ complex at 265 nm. The peak disappears in the absorbance spectra of Ac-G9/Pt NP solution, indicating successful reduction of Ac-G9 bound Pt4+ ions to Ac-G9/Pt NP. No obvious change in Ac-G9/Pt NP aqueous suspension was observed during a period of one month storage at 4 °C, which indicates excellent stability of the synthesized Ac-G9/Pt NP. Catalytic Activity of Ac-G9/Pt NP versus Catalase. The catalytic activities of Pt


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and Fe3O4 nanoparticles can be calculated by the Michaelis-Menten mechanism.9,53 To compare the catalytic activities of Ac-G9/Pt NP and catalase, the Michaelis-Menten curves for both Ac-G9/Pt NP and catalase were obtained within a suitable range of H2O2 concentrations (Figure 2). The fitted data to the Michaelis-Menten model are shown in Table 1. The Michaelis constant, Km, of Ac-G9/Pt NP is significantly higher than that of catalase, which indicates that Ac-G9/Pt NP have a relative lower affinity to H2O2 molecules. However, the Vmax and kcat value of Ac-G9/Pt NP catalytic activity on the decomposition of H2O2 approaches those of catalase. This is due to the fact that Pt NPs synthesized within Ac-G9 dendrimer have a high surface to volume ratio when catalyzing the decomposition of H2O2, while the catalase only has one catalytic center.28,34 Temperature- and pH-dependence on the Catalytic Activity of Ac-G9/Pt NP. The catalytic activity of catalase depends on the three-dimensional structure of the protein. A little bit of changes in the structure and conformation of catalase will lead to significant decrease or even loss of its catalytic activity. In addition, the alteration of cellular microenvironment in the terms of pH value, temperature, and ionic concentration can sensitively influence the catalytic activity of catalase. Therefore, natural enzymes usually suffer from low stability and regeneration problems. Here we tested the catalytic activities of Ac-G9/Pt NP and catalase at temperatures ranging from 4 to 90 °C and at pH values ranging from 2.0 to 11.0, respectively. As shown in Figure 3A, catalase approaches its maximum catalytic activity around 20 oC, and shows gradually decreased catalytic activity at higher temperatures. The decreased catalytic activity of catalase at temperatures above 20 oC should be due to protein denaturation at high temperatures. In comparison, the catalytic activity of Ac-G9/Pt NP on the decomposition of H2O2 continuously increase along with increasing temperatures (Figure 3A), which can be well explained by the Arrhenius equation.54 In the pH assay, the results in Figure 3B show that catalase performs its highest catalytic activity at pH 6.0. The catalytic activity of catalase significantly decreases in the basic media, which is due to the cleavage of disulfide bonds in the protein structure. However, the catalytic activity of Ac-G9/Pt NP on the decomposition of


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H2O2 increases with increasing pH values (Figure 3B), especially above the pH value of 8.0. It is known that H2O2 turns into HO2· in alkaline environments, which is easily decomposed into water and oxygen. Therefore, basic condition facilitates the oxidation process on the surface of Pt NPs.55 Due to different catalysis mechanisms, Ac-G9/Pt NP exhibits distinct temperature- and pH-dependence on catalytic activity as compared to catalase. Cytotoxicity and Antioxidant Activity of Ac-G9/Pt NP. Prior to the in vitro test of ROS scavenging activity of Ac-G9/Pt NP, we firstly evaluated their cytotoxicity by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MCF-7 and HeLa cells were incubated with different concentrations of Ac-G9/Pt NP for 24h. As shown in Figure 4A, cells treated with Ac-G9/Pt NP at concentrations up to 30 nM still kept viability above 90%. This result indicates that Ac-G9/Pt NP have low cytotoxicity on these cells. Similarly, the low cytotoxicity of Ac-G9/Pt NP on HeLa cells was confirmed by an AO/EB double-staining assay. AO is able to penetrate through the cell membranes of both normal and necrotic cells, while EB is only taken by necrotic cells with damaged membranes, resulting in bright green and orange fluorescence on the normal and necrotic cells, respectively.48 As shown in Figure 5, Ac-G9/Pt NP treated HeLa cells were only observed with green fluorescence, suggesting biocompatibility of the synthesized Ac-G9/Pt NP. To investigate the anti-oxidant activity of Ac-G9/Pt NP, protective effect of the NPs on H2O2-induced cytotoxicity on MCF-7 cells was evaluated. Surprisingly, the presence of Ac-G9/Pt NP did not protect MCF-7 cells from H2O2-induced cytotoxicity but increase the cytotoxicity of H2O2 to some extent (Figure 4B). This result is not in accordance with the excellent catalytic activity of Ac-G9/Pt NP on the decomposition of H2O2 as described above. Previous studies have demonstrated that Pt, Au, Ag, and Fe3O4 NPs also have a capability to mimic HRP and HRP splits H2O2 into ·OH under the acidic conditions.12,56-59 Therefore, we identified the HRP-like activity of Ac-G9/Pt NP using TMB as the substrate. The as-generated ·OH can oxide the colorless TMB into blue color. As shown in Figure 6, when the reaction was processed in PBS solution with a physiological pH of 7.4, the absorbances at 655 nm for the reaction solutions of both


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Ac-G9/Pt NP and HRP are scarcely changed, suggesting that no ·OH generated under a neutral pH condition. However, the absorbance monitored at 655 nm for both HRP and Ac-G9/Pt NP gradually increased when the reaction is processed in a buffer solution with a pH value of 4.8 (Figure 6), which respects to the microenvironment of lysosome. The localizations of Ac-G9 and Ac-G9/Pt NP within lysosome after 1 h of incubation are observed in Figure 8. These observations well explains why Ac-G9/Pt NP cannot protect the cells from H2O2-induced cytotoxicity. As shown in Scheme 2, two routes of H2O2 decomposition in the cells are proposed: (1) Ac-G9/Pt NP catalyzes H2O2 splitting into water and oxygen in cytosol; (2) Ac-G9/Pt NP decomposes H2O2 into ·OH in acidic organelle such as lysosome. As ·OH is highly reactive and cannot be eliminated by an enzymatic reaction, it causes serious damages to the cells. Thus Ac-G9/Pt NP increases the cytotoxicity of H2O2 to some extent. Similar results were observed in the AO/EB double-staining assay for H2O2-treated HeLa cells in the presence and absence of Ac-G9/Pt NP (Figure 5). Free Radical and ROS Scavenging Activities of Ac-G9/Pt NP versus Catalase. Free radicals are responsive for aging, cell fitness, and possibly some diseases. They easily bond with biomolecules such as protein, DNA, lipid, and carbohydrate, destroying their normal functions and further continuing the damage process to cells.1 Therefore, we also investigated the free radical scavenging activity of Ac-G9/Pt NP through a di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) assay. The radical DPPH turns from red to yellow after scavenging, which could be detected by monitoring its absorbance at 517 nm. After the treatment of DPPH with increasing concentrations of Ac-G9/Pt NP, a linearly increased DPPH radical scavenging activity is observed (Figure 7). The equation obtained through fitting the data is A = 4.5935C + 1.6804 (R2 = 0.9941), where A and C are the absorbance at 517 nm and the concentration of Ac-G9/Pt NP, respectively. As demonstrated above, Ac-G9/Pt NP has both catalase and HRP activities and are able to scavenge free radicals such as DPPH, suggesting that Ac-G9/Pt NP is a multifunctional artificial enzyme. The synergism of these catalytic activities renders Ac-G9/Pt NP with potential application in the neutralization of oxidative stress in organisms.


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In order to further investigate whether Ac-G9/Pt NP could efficiently mimic catalase-like activity in vitro, we examined their ROS scavenging activity on different cell lines. In this assay, the intracellular ROS was determined by DCFH-DA, a ROS-sensitive fluorescent dye, which can be oxidized into a highly fluorescent compound by intracellular ROS.60 As shown in Figure 9, no green fluorescence was observed in all the three control panels for MCF-7, HeLa, and RAW 264.7 cells. Also no obvious green fluorescence in the cell panels treated with Ac-G9/Pt NP was observed, indicating that Ac-G9/Pt NP at a concentration of 30 nM does not induce cellular ROS enhancement. However, all of three cells treated with H2O2 are observed with a significant enhancement over the green fluorescence, suggesting the increase of intracellular ROS in the three cells. The green fluorescence remarkably decreased and even disappeared in the cells pre-treated with 30 nM Ac-G9/Pt NP or catalase. This result demonstrates that Ac-G9/Pt NP is efficient to mimic the catalase-like activity in vitro. In conclusion, we developed Ac-G9/Pt NP as a high efficient artificial enzyme to mimic catalase. Ac-G9 employed here not only pursues the size and shape mimic of catalase, but also provides precious improvement over the stability and biocompatibility. Pt NPs synthesized within Ac-G9 show an average size of 3.3 nm. The catalytic activity of Ac-G9/Pt NP to decomposing H2O2 surprisingly approaches that of catalase, but perform differential responds to the changes of temperature and pH. Ac-G9/Pt NP has catalase and HRP activities and is able to scavenge free radicals such as DPPH. The in vitro studies indicated that Ac-G9/Pt NP has well biocompatibility and are efficient to scavenge H2O2 induced ROS in different cell lines. We are now modifying the G9 dendrimer with ligands to enhance its endosomal escape ability before the synthesis of Pt NPs thus enhancing the intracellular anti-oxidant activities of the Ac-G9/Pt NP in the current study.


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AUTHOR INFORMATION Corresponding Author *Email : [email protected], [email protected] Author Contributions †

These authors contributed equally on this manuscript.

Notes The authors declare no competing financial interest.

ACKNOMLEDGMENTS We thank financial supports from the Science and Technology of Shanghai Municipality (11DZ2260300), the National Natural Science Foundation of China (No. 21274044), the Innovation Program of Shanghai Municipal Education Commission (No.12ZZ044), and the Program for New Century Excellent Talents in University of Ministry of Education of China (NCET-11-0138) on this project.

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Figures and Schemes

≈11nm

Bovine Liver Catalase

≈10nm

Ac - G9 / Pt NP

Scheme 1. Structure comparison of bovine liver catalase and Ac-G9/Pt NP from molecular simulation. Both bovine liver catalase and Ac-G9/Pt NP have a globular shape with a hydrodynamic size around 11 nm.


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Ac - G9 / Pt NP

Nucleus

Endosome

H2 O+ O 2

H2O 2 Cytosol pH= 7. 4

·OH Lysosome pH= 4. 8

Scheme 2. Schematic illustration of the two proposed routes for H2O2 decomposition in cells in the presence of Ac-G9/Pt NP: (1) decomposing into water and oxygen, and (2) splitting into two hydroxyl radicals in lysosomes.


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B 30

%

20

10

0 2

3

4

5

6

Diameter (nm) 1.6 4+

C

Ac-G9/Pt Ac-G9/Pt NP

1.2 Absorbance

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0.8

0.4

0.0 200

250

300

350

400

Wavelength (nm)

Figure 1. (A) TEM image of Ac-G9/Pt NP which only shows the synthesized Pt NPs within Ac-G9 PAMAM dendrimer. (B) The size distribution histogram of the synthesized Pt NPs. (C) UV-Vis spectra of Ac-G9/Pt4+ and Ac-G9/Pt NP. The characteristic absorption peak for PtCl62- disappeared, replaced by broad absorbances at all wavelengths.


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A 0.022

B

Catalase

0.03

Ac-G9/Pt NP

v (m M· s-1)

v (m M· s- 1)

0.020 0.018 0.016

0.02

0.01 0.014 0.012 0

20

40

0.00

60

20

H2O2 concentration (mM)

40

60

H2O2 concentration (mM)

80

C

D200

Catalase

Ac-G9/Pt NP

1/v (s· mM- 1)

70 1/v (s· mM- 1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60

50

40 0.00

150

100

50

0.05

0.10

1/H2O2 concentration (mM-1)

0.15

0 0.00

0.04

0.08

0.12

1/H 2O2 concentration (mM -1)

Figure 2. Kinetic assay and catalytic mechanism of Ac-G9/Pt NP. (A) and (B) The velocity ( v ) of the reaction was measured using 2.5 nM catalase or 10 nM Ac-G9/Pt NP at 37°C and pH =7.4. Error bars shown represent the standard error obtained from three parallel measurements. (C) and (D) Double-reciprocal plots of activities of catalase and Ac-G9/Pt NP which derived from Michaelis equation.


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A 120

B 120

Catalase Ac-G9/Pt NP

Catalase Ac-G9/Pt NP

100 Relative activity (%)

100 Relative activity (%)

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80 60 40 20 0

80 60 40 20 0

0

20

40

60

80

2

4

Temperature (°C)

6

8

10

pH

Figure 3. The catalase-like activity of Ac-G9/Pt NP is temperature- and pH-dependent (A and B). (A) Catalase shows the maximum catalytic activity around 20 °C, while the catalytic activity of Ac-G9/Pt NP gradually increases with increasing temperatures. (B) Catalase shows the maximum catalytic activity at a pH value around 6.0, while the activity of Ac-G9/Pt NP increases with increasing pH values. The maximum point in each curve was set as 100%.


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120 MCF-7 HeLa

100

B

50 Ac-G9/Pt NP Catalase 40

Cell viability (%)

80 60 40

＊＊

＊＊

30

20

O

30

5

1

Concentration (nM)

20

2

15

H

10

M

5

2

0 Control 1

2

0

n +1 M m En z M H yme 10 2 O nM 2 + 1 m M En z ym H e 30 2 O 2 +1 nM m M Enz H ym 2 O e

10

20

m

A Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4. (A) Cytotoxicity of Ac-G9/Pt NP on HeLa and MCF-7 cells. Cell viability was detected by the MTT assay. The data were normalized to control values (no particle exposure), which were set as 100% cell viability. (B) The effect of Ac-G9/Pt NP and catalase on H2O2-induced cytotoxicity on MCF-7 cells. (＊p

Generation 9 Polyamidoamine Dendrimer Encapsulated Platinum

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