Biocompatible Dendrimer-Encapsulated Palladium Nanoparticles for Oxidation of Morin.

Development of highly efficient catalysts to expedite the degradation of organic dyes has been drawing great attention. The aggregation of catalysts reduces the accessibility of catalytic centers for organic dyes and therefore decreases their catalytic ability. Herein, we report a facile method to prepare highly biocompatible and stable dendrimer-encapsulated palladium nanoparticles (Pd n -G5MCI NPs), which exhibit high catalytic efficiency for oxidation of morin. The biocompatible dendrimers were prepared via surface modification of G5 polyamidoamine (G5 PAMAM) dendrimers using maleic anhydride and l-cysteine. Then, they were incubated with disodium tetrachloropalladate, followed by reduction using sodium borohydride to generate Pd n -G5MCI NPs. Transmission electron microscopy results demonstrated that palladium nanoparticles (Pd NPs) inside Pd n -G5MCI had small diameters (1.77-2.35 nm) and monodisperse states. Dynamic light scattering results confirmed that Pd n -G5MCI NPs had good dispersion and high stability in water. Furthermore, MTT results demonstrated that Pd n -G5MCI NPs had high biocompatibility. More importantly, Pd n -G5MCI NPs successfully catalyzed the decomposition of H2O2 to the hydroxyl radical (•OH), and the generated •OH quickly oxidized morin. This reaction kinetics followed pseudo-first-order kinetics. Apparent rate constant (k app) is an important criterion for evaluating the catalytic rate. The concentrations of Pd n -G5MCI NPs and H2O2 were positively correlated with k app, whereas the correlation between the concentration of morin and k app was negative. The prepared Pd n -G5MCI NPs have great potential to catalyze the degradation of organic dyes in bio-related systems in the future.

Introduction

In recent years, the pollution by organic dyes has been drawing great attention due to the potential toxicity toward humans and other creatures. Morin is a kind of flavonoid dye, which is commonly found in fruits, vegetables, and the chemical industry. Many methods have been developed to treat the organic dyes such as catalytic oxidation,[1] adsorption,[2−4] photodegradation,[5] and electrochemical methods.[6] Catalytic oxidation is one of the most promising methods for removal of organic dyes. Noble-metal nanoparticles such as palladium nanoparticles (Pd NPs) are widely employed in the degradation of pollutants because of their high catalytic activity.[7−9] The smaller sizes of Pd NPs within the size range of 1–10 nm lead to larger specific surface areas and higher catalytic activities. However, Pd NPs are easily aggregated in a thermodynamically unstable state due to their high specific surface energy.

To address this problem, immobilization of Pd NPs by suitable materials such as proteins,[10] polymers,[11] and dendrimers[12] has been used to prepare highly stable and well-dispersed Pd NPs, which was mainly due to the electrostatic and steric effects. Dendrimers have the unique structure and many functional groups both inside and outside the molecule. Poly(amidoamine) (PAMAM) dendrimers were one kind of widely studied dendrimers. Encapsulation of Cu nanoclusters within PAMAM dendrimers were first developed by Crooks and coworkers.[13] Subsequently, many other metal nanoparticles (Au, Pd, Pt) were stabilized/encapsulated by PAMAM dendrimers.[12,14] However, the highly positive charges of PAMAM dendrimers, resulting from the large number of primary amine on their surface, induce the nonspecific interaction with proteins, cells, and bacteria, leading to reduced catalytic ability of noble-metal nanoparticles inside the dendrimers in bio-related medium. Thus, many materials were explored to modify the surface of PAMAM dendrimers to reduce these interactions, including polyethylene glycol, phosphorylcholine,[15] and carboxyl betaine.[16]

In this study, maleic anhydride- and l-cysteine-modified G5 PAMAM dendrimers (G5MCI) have been synthesized. G5MCI was used as the new template to prepare highly biocompatible and stable dendrimer-encapsulated Pd NPs (Pd-G5MCI NPs). The diameter of Pd NPs inside G5MCI was about 2 nm. Pd55-G5MCI-treated HeLa cells showed no noticeable cytotoxicity in vitro. The catalytic oxidation of morin using Pd-G5MCI was used to evaluate their catalytic ability. These results indicated that they had great potential applications as bio-related catalysts.

Results and Discussion

Synthesis and Characterization

In this work, biocompatible and stable Pd-G5MCI NPs were prepared through the method shown in Scheme . Monodispersed metal nanoparticles can be encapsulated in G5 PAMAM dendrimers.[17,18] However, the high density of primary amino groups on the surface of the G5 PAMAM dendrimers leads to highly positive charges, resulting in their strong interactions with proteins, bacteria, and cells. Herein, maleic anhydride and l-cysteine were used to modify the surface of dendrimers to obtain a biocompatible template. The modified process was confirmed by 1H NMR spectroscopy. G5 PAMAM (Figure S1a) had about 128 primary amino groups, which were located on the surface of G5 PAMAM.[19] After the reaction of G5 PAMAM with maleic anhydride, two new peaks at 5.8 and 6.2 ppm appeared (Figure S1b). All primary amines were successfully reacted with maleic anhydride. After the addition of l-cysteine, these peaks completely disappeared and a new peak at 3.2 ppm appeared, indicating the successful preparation of G5MCI.[20] G5MCI and PdCl42– were incubated for 20 min at pH 4, then excess of NaBH4 was added to this solution to obtain Pd55-G5MCI and Pd110-G5MCI. There was slight difference between G5MCI and Pd55-G5MCI (Figure S1d) that occurred in the 1H NMR spectra, indicating that palladium atoms had a weak effect on the spectrum of G5MCI.

Scheme 1

Scheme of Preparation of Dendrimer-Encapsulated Palladium Nanoparticles

In addition, the absorbance of the solution was recorded to monitor the formation of Pd55-G5MCI. As illustrated in Figure , there was one absorption peak at 416 nm. After the addition of NaBH4, a broad absorption band appeared. In addition, the solution color changed from yellow to dark brown. These results were consistent with the previous reports.[18] PdCl42– was reduced to generate Pd NPs stabilized by G5MCI.

Figure 1

(a) UV–vis spectra of solutions: G5MCI, Na2PdCl4, Pd55-G5MCI, Pd110-G5MCI, and G5MCI + Na2PdCl4 and (b) corresponding photos.

The diameters of Pd NPs inside of dendrimers were characterized by transmission electron microscopy (TEM). Figure shows that the average sizes of Pd NPs inside Pd55-G5MCI and Pd110-G5MCI were 1.77 ± 0.37 and 2.35 ± 0.38 nm, respectively. The results indicated that Pd NPs inside G5MCI had a small size and a narrow size distribution. The molar ratio of Na2PdCl4 to G5MCI was used to regulate the size of Pd NPs.[18] The specific surface of Pd55-G5MCI and Pd110-G5MCI were 3.39 and 2.55 nm–1, respectively. The larger specific surface areas resulted in more active centers. These characteristics are beneficial for highly catalytic efficiency degradation of organic dyes.

Figure 2

TEM images and corresponding diameter distribution histogram of Pd NPs inside (a,c) Pd55-G5MCI and (b,d) Pd110-G5MCI.

Stability

Various organic dyes can be commonly found in sewage. High stability of a catalyst in sewage is beneficial for long-term high catalytic ability. Many noble nanoparticles show low stability in sewage with different pH. Here, the effect of pH ranging from 4 to 9 on the stability of Pd55-G5MCI in different solutions was evaluated by the hydrodynamic size change. The hydrodynamic size was monitored by dynamic light scattering. As shown in Figure a, Pd55-G5MCI had similar hydrodynamic size about 10 nm at different pH, indicating that Pd55-G5MCI was well dispersed in water. Pd55-G5MCI maintained their hydrodynamic size without obvious agglomeration within 2 days. Thus, Pd55-G5MCI remained stable within pH 4–9. Figure b shows that the zeta potential of Pd55-G5MCI NPs decreased with the increasing pH. The zeta potential of Pd55-G5MCI changed from −0.95 mV at pH 4 to −7.48 mV at pH 9. This should be due to protonation/deprotonation of primary amine groups and carboxyl groups under different pH conditions. The electrostatic stabilization and steric stabilization affect the stability of nanoparticles.[21] The higher net charge preventing agglomeration often leads to higher stability of NPs due to electrostatic force repulsion. However, the net charge of Pd55-G5MCI was relatively small. The electrostatic stabilization was not the main reason; however, the steric stabilization should be the main reason. Steric stabilization usually occurs between soluble large macromolecules. In short, we obtained the Pd NPs with small particle size and high stability; these properties are ideal for the catalytic oxidation of morin.

Figure 3

(a) Hydrodynamic size and (b) zeta potential of Pd55-G5MCI NPs under different pH.

Biocompatibility

The high biocompatibility of catalysts is good for their usage under bio-related conditions. The cytotoxicity of synthetic Pd55-G5MCI NPs against HeLa cells was evaluated by an MTT assay.[22−24] As depicted in Figure a, the cell viability decreased significantly with increasing concentration of Pd55-G5 NPs. The lowest cell viability treated with Pd55-G5 was 13.7% at the concentration of 200 μg/mL. In contrast, the cell viability treated with Pd55-G5MCI (200 μg/mL) was 96.3%. The cytotoxicity of Pd55-G5 NPs and Pd55-G5MCI NPs was also evaluated by visualizing the morphologies of HeLa cells. Figure c shows obvious shrink morphology for HeLa cells incubated with Pd55-G5 at the concentration of 200 μg/mL, whereas Figure d shows that HeLa cells toward Pd55-G5MCI and control groups had similar cell morphology. The results confirmed that Pd55-G5MCI NPs had no cytotoxicity against HeLa cells within 200 μg/mL. The cytotoxicity of Pd55-G5 should be due to the formation of holes on cell membranes.[25] The enhanced biocompatibility of Pd55-G5MCI was caused by the surface modification using maleic anhydride and l-cysteine,[19] which restricted the entrance of Pd55-G5MCI into the interior of HeLa cells.[26] Hildebrand and coworkers reported the cytotoxicity of palladium/magnetite nanocatalysts, which have been used in catalysis of waste water.[27] The good accessibility between substrates and catalytic sites on catalysts is beneficial for their high catalytic activity. Rotello and coworkers reported that the presence of bacteria inhibited the catalytic oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by Dop-Fe3O4 nanoparticles.[28] The high biocompatibility may be beneficial to good accessibility between substrates and catalytic sites in bio-related degradation.

Figure 4

(a) Cell viabilities of HeLa cells incubated with Pd55-G5 NPs and Pd55-G5MCI NPs; (b–d) cell morphologies for (b) blank, (c) Pd55-G5, or (d) Pd55-G5MCI for 200 μg/mL after 24 h.

Catalytic Kinetics

The molecular formula of morin is C15H10O7·2H2O; the structure of morin has an oxygen-containing heterocyclic ring which connects two aromatic rings. Figure a shows that the maximal absorption wavelength of morin in this buffer was 403 nm. When morin and H2O2 were incubated together, the absorbance at 403 nm only decreased to 98% after 40 min. In addition, the incubation of morin and Pd55-G5MCI had similar results. In contrast, Figure b shows that the absorbance at 403 nm quickly decreased with increasing time when morin, H2O2, and Pd55-G5MCI were mixed together, which was due to the decreased concentration of morin, indicating that catalytic oxidation of morin was obviously accelerated. Meanwhile, a new peak at 318 nm increased, which was due to the formation of intermediate product named benzafuranone. However, the peak at λ 318 nm decreased after about 10 min, indicating that benzafuranone was further oxidized into other products.[29] For the kinetic study, we mainly investigated the initial product in this reaction by controlling the time less than 10 min.

Figure 5

(a) UV–vis spectra of morin oxidation in the presence of (a) H2O2, (b) Pd55-G5MCI + H2O2 (Cmorin = 0.07 mM; CH = 5 mM; CPd = 0.15 μM).

Furthermore, the catalytic kinetics of the oxidation of morin for Pd55-G5MCI and Pd110-G5MCI was investigated. Figure a,b shows that the ln(Ct/C0) was linear with time, indicating that this reaction followed pseudo-first-order kinetics. As shown in Figure c, morin and [morin + Pd55-G5MCI] had similar relationship between ln(Ct/C0) at λ = 403 nm and reaction time (t). In contrast, the addition of Pd55-G5MCI NPs (CPd = 8.05 × 10–6 M) led to an accelerated catalytic rate. Thus, Pd55-G5MCI NPs instead of morin adsorption were the main factor for the catalytic degradation of morin. The apparent rate constant, kapp was calculated from the following eq . Figure d shows that the kapp increased with increasing catalyst concentration. This was due to the increasing surface area. Moreover, the kapp of Pd55-G5MCI is larger than that of Pd110-G5MCI at the same Pd concentration because Pd NPs inside Pd55-G5MCI have smaller size and larger specific surface area, leading to more available catalytic active sites and faster catalytic rate.

Figure 6

The relationship between ln(Ct/C0) at λ = 403 nm and the reaction time (t) for (a) Pd55-G5MCI NPs, (b) Pd110-G5MCI NPs, and (c) control groups (CPd = 8.05 × 10–6 M) (d) Relationship between kapp and the concentration of Pd (Cmorin = 0.07 mM, CH = 5 mM).

Furthermore, the relationship between the different concentrations of morin, H2O2, and kapp were also studied. This kinetic study was measured by determining kapp, while varying the morin concentration and fixing the H2O2 concentration at room temperature. As shown in Figure a, it is obvious that kapp decreased as the morin concentration increased. This result can be attributed to adsorption constants Kmorin > KH.[30] The morin and H2O2 were first adsorbed onto the surface of the catalyst before reaction. As the concentration of morin increased, most of the surface area of Pd NPs inside of Pd55-G5MCI would be covered by morin. This also made it more difficult for H2O2 to be adsorbed onto the active site of the catalyst, and therefore, decreased the reaction rate. Figure b shows that kapp increased as the H2O2 concentration increased. These results were consistent with the previous results that MnO nanoparticles catalyzed the oxidation of morin.[31]

Figure 7

The effect of the concentration of (a) morin and (b) H2O2 on the kapp of Pd55-G5MCI and Pd110-G5MCI (CPd = CPd = 0.16 μM).

The presence of H2O2 was necessary for the oxidation of morin. Terephthalic acid (TA) was used to detect the amount of •OH. TA reacts with •OH to form a strong fluorescence product, and the relative concentration of •OH was determined indirectly by measuring the intensity of the fluorescence after the reaction.[32] Liu and coworkers prepared CeO2-montmorillonites nanocomposites. They found that the generated •OH were from the decomposition of H2O2.[33] Liu and coworkers reported that Ag2S-montmorillonites catalyzed the decomposition of H2O2 into •OH.[134] The presence of free radicals is correlated to the surrounding conditions.[34] As shown in Figure a, the group [TA + H2O2 + Pd55-G5MCI] had the maximal absorption at 430 nm. Figure b shows that the fluorescence intensities of TA, TA + Pd55-G5MCI, TA + H2O2, and TA + H2O2+Pd55-G5MCI were 19.7, 10.5, 406.6, and 1223.0, respectively. Thus, Pd55-G5MCI promoted the production of •OH.

Figure 8

(a) Fluorescence spectra of TA, TA + Pd55-G5MCI, TA + H2O2, and TA + H2O2 + Pd55-G5MCI (CTA = 0.3 mM, CH = 20 mM, CPd = 1.45 μM) and (b) fluorescence intensities of these samples at 430 nm.

The catalytic oxidation of morin by Pd-G5MCI should follow the Langmuir–Hinshelwood model as shown in Figure .[35−37] Both morin and H2O2 were adsorbed onto the surface of Pd NPs. The adsorption of morin and H2O2 were fast and reversible. H2O2 was catalyzed to generate •OH by Pd-G5MCI.[38]Concomitantly, •OH reacted with morin adsorbed on the active sites of Pd NPs of Pd-G5MCI. This step was rate-determining in the whole catalytic cycle. The final product dissociated from Pd NPs surface and the newly formed free active sites were used for new catalytic reaction.[39] The reaction kinetics was controlled by surface reaction kinetics, not diffusion.[40]

Figure 9

Mechanism of the catalytic oxidation of morin by Pd-G5MCI treated with H2O2 based on Langmuir–Hinshelwood model.

Conclusions

In summary, a new method using G5MCI as the template to encapsulate Pd NPs was demonstrated. The prepared Pd-G5MC had high pH sensitivity, stability, and biocompatibility. Pd-G5MCI efficiently catalyzed oxidation of morin in the presence of H2O2, and this reaction followed pseudo-first-order kinetics. The Pd-G5MCI accelerated the decomposition of H2O2 into •OH. The concentration of the catalyst and H2O2 had a positive correlation with kapp, whereas the correlation between morin and kapp was negative. The prepared Pd-G5MC has great potential to be used in the treatment of waste water in the future.

Materials and Methods

Preparation of Pd-G5MCI NPs

G5 PAMAM (8 mg) and maleic anhydride (5.5 mg) were mixed in dimethyl sulfoxide for 24 h and dialyzed against water using a dialysis bag. l-cysteine (128 mg) was added to the obtained solution and reacted for 24 h. The dialysis was carried out to obtain the final product named as G5MCI. Yield: 95%.

G5MCI (13.7 nmol) was dissolved in 1200 μL of deionized water, and the pH of the solution was tuned to 4 by adding 1 M HCl. Then, 381 μL Na2PdCl4 (2 mM) aqueous solution was added for another incubation for 20 min at 20 °C. Five-fold molar excess of NaBH4 dissolved in 0.3 M NaOH was added to yield Pd55-G5MCI NPs. Then, 1 M HCl was added to tune pH of the solution to 7. The solution was dialyzed against water. Pd110-G5MCI NPs were also obtained using this procedure.

Catalytic Properties

Morin was dissolved in pH 9.2 carbonate buffer. Morin (2 mM, 70 μL), carbonate buffer, 50 μL Pd55-G5MCI solution, and 25 μL H2O2 (0.4 M) were added in a cuvette. The final concentration of morin, H2O2, and Pd55-G5MCI was 0.07, 5 mM, and 0.15 μM, respectively. UV–vis spectra were recorded every 2.5 min. The effects of the concentration of Pd-G5MCI (CPd = 8.05–48.3 μM, Cmorin = 0.07 mM, CH = 5 mM) on the kinetics were recorded at λ = 403 nm in a time-dependent mode at room temperature. The catalytic performances at different concentrations of morin (Cmorin = 0.0375–0.285 mM, CPd = 0.16 μM, CH = 0.01 M) and H2O2 (CH = 0.01–0.06 M, CPd = 0.16 μM, Cmorin = 0.055 mM) were also measured at room temperature with the similar procedure. The relationship between concentration of the catalyst, morin, H2O2, and kapp was also calculated.