Synthesis Of Peroxidase Nanozymes And Their Application In Green Synthesis Of Water-Soluble Conducting Polymers

Photothermal therapy (PTT) method, which employs photothermal agents for achieving photothermal damage of tumors, has been explored in the past decade. Currently, available PTT agents mainly comprise noble metal nanoparticles, carbon based materials and semiconductor nanoparticles. Noble metal nanoparticles have low photostability in that their NIR absorbance peak would diminish due to the melting effect after being irradiated for a long period. In addition, the high expense limited the wide use of noble metal nanoparticles. Also these materials are inorganic, which usually are not biodegradable and may remain inside of human body for long time after systemic administration.Alternativly, polymeric materials geared towards application in PTT have been attracting attention. Conducting polymers are non-cytotoxic, low cost, and have good conductivity high absorbance in near infrared (NIR) light.Although conducting polymers have such properties, its polymerization usually depends on chemical and electrochemical methods. Both the methods whether are environmentally hazardous or the synthesized product isn’t water-soluble. So there is increasing interest in environmentally friendly routes to the synthesis of water-soluble conducting polymer. Enzyme-catalyzed polymerization of polymers are attracting great interest since enzymatic approach can overcome many strong drawbacks in traditional chemical process. First of all, enzymatic protocol involves eco-friendly oxidants such as H2O2 and molecular oxygen rather than ammonium peroxydisulfate, which is an environmentally incompatible oxidant, used in traditional process. Secondly, the use of ammonium peroxydisulfate results in formation of a large amount of by-products, whereas in the case of H2O2 the formation of H2O as the only reduction product greatly simplifies post-treatments and recycling. Thirdly, enzymatic approach can offer a higher degree of controlover the kinetics of the reaction. Moreover, enzyme-catalyzed conductive polymers are superior to those synthesized by traditional methods in that they are water-soluble and hence desirable for practical applications. The commonly used natural enzymes possess remarkable advantages such as high substrate specficilities and high efficiency under mild conditions. However, they also bear some serious shortages.For example, their catalytic activity can be easily inhibited and their preparation, purification and storage are usually time-consuming and expensive. In order to conqure or decrease the influence of these shortages, more and more enzyme mimics have been synthesized and applied to catalyze the polymerization of conducting polymer. But these reported enzyme mimics are traditional organic substances which still show low stability in extreme conditions such as high temperature, strongly acidic/alkali, and whose fabrication is also complicated. Inorganic material show advantages to organic substances in better adaptation to extreme environment conditions and lower-cost fabrication, purification and storage.To date, very few reports concentrated on the green synthesis of conducting polymers catalyzed by inorganic enzyme mimics are available.Herein we prepared three kinds of novel peroxidase mimics, black elemental selenium (Se), iron phosphates (FePOs) and nickel phosthates (NiPOs), via solvothermal route and characterized their morphology and component using scanning electron microscope (SEM) and X-ray diffraction (XRD). The redox reaction between tetramethylbenzidine (TMB) and H2O2 was applied to prove peroxidase-like behavior of Se, FePOs and NiPOs. According to this peroxidase-like behavior, Se, FePOs and NiPOs were used to catalyze the polymerization of water-soluble conducting polymers which can be used as photothermal agent.The main contents of this study can be described as follows:1. Synthesis of black elemental selenium peroxidase mimic and it’s application in green synthesis of water-soluble polypyrrole as photothermal agent.Solvothermally synthesized Se was fabricated as peroxidase mimic, which shows potential applications in the field of biocatalyst. The results show that these particles are almost spherical in shape with a diameter of 1-10 μm approximately. The products were found to be capable of catalyzing the oxidation of peroxidase substrate TMB with H2O2, suggesting the peroxidase mimic activity of Se. The catalytic activity of Se was affected deeply by different pH values. When pH values were adjusted to 3 to 6, the Se peroxidase mimic shows high catalytic efficiency and highest in pH 4.5. Either catalysis is inhabited, which adverse the activity of peroxidase of Se particles. Peroxidase-like activity of Se increases with the temperature in the range of 20-70℃, showing a great advantage over HRP which totally losts activity at 70℃.Se particles were used to catalyze the green synthesis of water-soluble polypyrrole (PPy) in the presence of sodium polystyrene sulfonate (PSS) as the template. Using O2 as the oxidant, Se particles could indeed catalyze the polymerization of PPy as the natural enzymes do and the synthetic conditions such as pH, temperature, dosage of Se and cycle times of Se have great impact on the molecular weight and yield of PPy. It is known that, HRP can only reserve 20% of its activity after being scattered into pH 4 solution for 20 min. While our Se particles can catalyze the polymerization of PPy even pH is as low as 2.2, showing a great favor over HRP. As a photothermal agent, the Se-catalyzed PPy exhibited several advantages. First, it displayed excellent biocompatibility when incubated with HeLa cervical cancer (HeLa) cells. That’s because we applied O2 as the oxidant which will not produce byproducts and pollute PPy as the other oxidants do such as ammonium peroxydisulfate, and the Se particles were very easy to remove by centrifugation, so the product is very clean. Second, the Se-catalyzed PPy was monodispersed with a narrow size distribution and an average diameter of 19.7 nm which resulted in excellent water-solubility and stability in biological mediums such as fetal bovine serum and saline, allowing the PPy to be further used in biological systems.After exposure to 808 nm laser light for 10 min at different output, the temperature rise of PPy solutions with different concentration were much higher than that of ultra-pure water proving good photothermal effect of Se-catalyzed PPy which can convert NIR light energy to heat with a photothermal efficiency of 28.4%. HeLa cells were used as a cell model to investigate the anti-tumor effect of the prepared PPy via PTT mechanism. After being incubated with PPy (0.625 mg/mL) and irradiated by 808 nm laser for 10 minutes at an output of 1.5 W, the cell viability of HeLa cells decreased to 11 ±4%, indicating that remarkable anti-tumor effect was observed for Se-catalyzed PPy.2. A green route to water-soluble polyaniline for photothermal therapy catalyzed by iron phosphate peroxidase mimicSolvothermally synthesized FePOs was fabricated as peroxidase mimic, which shows potential applications in the field of biocatalyst. FePOs peroxidase mimic was used to catalyze the green synthesis of water-soluble polyaniline (PANI) in the presence of PSS as the template. Using H2O2 as the oxidant, FePOs peroxidase mimic could indeed catalyze the polymerization of PANI as the natural enzymes do and the synthetic conditions such as pH, temperature, moleur ratio of H2O2 and aniline (H2O2:aniline) have an impact on the molecular weight and conductivity of PANI. The FePOs-catalyzed PANI synthesized at pH 2.2,20℃ and H2O2:aniline=1 shows the largest conductivity of 2.576 ×10-3 S/cm. It is known that, HRP can only reserve 20% of its activity after being scattered into pH 4 solution for 20 min. While our FePOs can catalyze the polymerization of green conducting PANI even pH is as low as 1.5, showing a great favor over HRP. As the photothermal agent, the FePOs-catalyzed PANI exhibited several advantages. First, it displayed excellent biocompatibility when incubated with HeLa cells. That’s because we applied H2O2 as the oxidant which will not produce byproducts and pollute PANI as the other oxidants do such as ammonium peroxydisulfate, so the product is very clean. Second, the FePOs-catalyzed PANI displayed excellent water-solubility and stability, allowing the PANI to be further used in biological systems.After exposure to 808 nm laser light for 10 min at different output, the temperature rise of PANI solutions with different concentration were much higher than that of ultra-pure water proving good photothermal effect of FePOs-catalyzed PANI which can convert NIR light energy to heat with a photothermal efficiency of 39.6%. HeLa cells were used as a cell model to investigate the anti-tumor effect of the prepared PANI via PTT mechanism. After being incubated with PANI (0.4 mg/mL) and irradiated by 808 nm laser for 10 minutes at an output of 1 W, death happened to almost all HeLa cells proving remarkable anti-tumor effect of FePOs-catalyzed PANI.3. Solvothermally synthesized NiPOs was fabricated as photocatalyst, which was used to catalyze the green synthesis of water-soluble poly(3,4-ethylenedioxythiophene) (PEDOT) in the presence of PSS as the template. Using H2O2 as the oxidant, NiPOs could indeed catalyze the polymerization of 3,4-ethylenedioxythiophene (EDOT) under daylight irradiation and the best synthetic conditions were pH 1.5,60℃ and moleur ratio of H2O2 and EDOT (H2O2: EDOT) was 1. The NiPOs-catalyzed PEDOT showed excellent water-solubility.After exposure to 808 nm laser light for 10 min at different output, the temperature rise of PEDOT solutions with different concentration were much higher than that of ultra-pure water proving good photothermal effect of NiPOs-catalyzed PEDOT which can convert NIR light energy to heat with a photothermal efficiency of 16.84%. HeLa cells were used as a cell model to investigate the anti-tumor effect of the prepared PEDOT via PTT mechanism. After being incubated with PEDOT (0.4 mg/mL) and irradiated by 808 nm laser for 10 minutes at an output of 1 W, death happened to almost all HeLa cells, proving remarkable anti-tumor effect of NiPOs-catalyzed PEDOT.