CC BY
55
UDK 541.64
N. V. Kutsevol, V. А. Chumachenko, V. F. Shkodich, D. R. Vadigullin, I. I. Boyoko
GREEN SYNTHESIS OF SILVER NANOPARTICLES USING GLUCOSE AS REDUCING AGENT
Keywords: polyacrylamid, nanopaticles, glucose, synthesis.
Silver sols stabilized by star-like and linear polyacrylamides were synthesized using glucose as reductant. Good biocompatibility and noncytotoxity ofpolymer matrices was proved. Effect ofpolymer matrix type and it's structure on shape, aggregation stability and size distribution of silver nanopaticles (AgNPs) were studied by surface plasmon resonance observation using UV-visible spectroscopy technique and transmition electronic microscopy data analysis.
Ключевые слова: полиакриламид, наночастицы, глюкоза, синтез.
Серебряные золи, для стабилизации звездообразных и линейных полиакриламидов были синтезированы с использованием глюкозы в качестве восстановителя. Была доказана, хорошая биосовместимость полимерных матриц. C использованием УФ-спектроскопии и электронной микроскопии изучалось влияние типа полимерной матрицы и ею структуры на распределение серебряных наночастиц по размерам.
Introduction
Silver nanoparticles (AgNPs) are used in a wide range of applications, including pharmacy, cosmetics, medical devices, food ware, water purification and others uses, due to their antimicrobial properties [1-3]. The use of AgNPs, in comparison with simple Ag salts, seems to have reduced cellular toxicity but not antibacterial efficacy [4-7]. The antibacterial spectrum of AgNPs even extended to antibiotic resistant organisms [4-6]. AgNPs could be used as alternative to antibiotics or as substances enhancing their action at decreasing of their dose [4-6]. To improve the biocompatibility of AgNPs for biomedical application it is obviously preferable to use nontoxic reagents for nanosystem synthesis. Various reducing agents have been reported in the literature, the most being sodium borohydride and hydrazine, which are not convenient for synthesis silver sols for biomedical application [1,79]. In recent years, the research has focused on "green synthesis" to avoid using hazardous effect of the toxic "tails" which can appear in the synthesis route [1,1012]. Monosaccharides, oligosaccharides, polysaccharides or plant extracts can be used as "green" reductants [1, 10-13]. The size and shape of AgNPs are actually controlled by the choice of the reductant, the protection agent, the concentration of the initial Ag salt and the synthesis conditions (Temperature, pH, etc.) [9, 14-15]. Due to high reactivity of AgNPs the preparation of stable nanosystems requires a protection agent preventing a possible aggregation process [6, 7, 16]. Such an aggregation can hinder the production of AgNPs with a small and uniform size and, as a result, reduce their antimicrobial ability [3]. Polymers, including polyvinylpyrrolidone [14], polyethylene glycol [1, 14, 16], starch [6] are commonly used as stabilizing agents. They enhance the stability of nanoparticles by introducing steric and-or electrostatic repulsions between them. Their efficiency however depends on their chemical nature, ionization degree or chemical charge density, and molecular weight. On the other hand, polymers can also play a role in the process of the in-situ nanoparticle formation, namely in the control of their size and morphology as well as the nanosystem stability and aging effect. In our previous
research it was shown that branched polymers are more efficient in this aim in comparison with linear analogs [17-19].
The study presented in this paper describes an synthesis of AgNPs using glucose as reductant and aqueous solutions of branched polymers dextran-graft-polyacrylamide (D-g-PAA), as matrices. The branched polymers play also the role of stabilizers, by preventing any flocculation and sedimentation processes. D-g-PAA copolymers consist of biocompatible and water soluble components (dextran and polyacrylamide), characterized by a high local concentration of functional groups [17], that is why they can be suitable matrices for nanosystem preparation with further their using for targeted delivery of therapeutic agent into tumor cells. The main goal of this study was to synthesize stable biocompatible nanosystems, using host polymer as shape-directing controller of Ag NPs growth and their stabilizer.
Materials and methods
The silver nanoparticles (AgNPs) were synthesized by reduction of the AgNO3 salt using glucose as reductant. The syntheses were carried out in situ into polymer matrices corresponding to dilute aqueous solutions of linear and branched polymers of the same chemical nature.
Glucose was purchased from "Pharma" (Ukraine). AgNO3 (Sigma Aldrich) was used without additional purification. Linear polyacrylamide and dextran-g-polyacrylamide copolymers were obtained according to synthesis routes described elsewhere [17-22].
Polymers
Linear and branched polyacrylamides (PAA) were obtained by free-radical polymerization initiated by ceric ion reducing agent. Branched polyacrylamide, namely dextran-g-polyacrylamide (D-g-PAA) copolymers were synthesized by grafting polyacrylamide chains onto dextran (Mw = 7* 104 g.mol-1) backbone [17]. The redox process initiates free radical sites exclusively on the polysaccharide backbone, thus preventing from the formation of PAA homopolymers [20, 21].
The details in the syntheses, identifications and analysis of the polymer average structure were described in [17, 21]. The theoretical numbers of grafting sites per dextran backbone for the samples we used in the present work are equal to 5 and 20, so the related D-g-PAA copolymers are referred as D70-g-PAA5 and D70-g-PAA20, respectively.
All polymers and copolymers (the nascent and hydrolyzed ones) were precipitated into an excess of aceton, dissolved in bi-distillated water, then freeze-dried and kept under vacuum for preventing them from further hydrolysis.
The biological test for polymer toxicity
The cells culture of murine macrophage J774 was used as the biological object for studying polymer toxicity. The cells culture was obtained from the culture collection of the Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine. The efficiency of absorption of polymer by cell culture was determined by evaluating phagocytic index [23].
AgNPs
Reduction of Ag salt was performed at T=60° C in aqueous solutions of PAA linear chains or D-g-PAA copolymers. Molar ratio of AA monomers to Ag+ cations was equal to 5. The syntheses were carried out in polymer solutions prepared using bi-distilled water. The pH of aqueous solutions of polymer was 5.5, which is the pH of bi-distilled water. The stability of obtained nanosystems was being controlled during 3 months.
2 ml of a 0.1M AgNO3 aqueous solution was first added to 5 ml of aqueous polymer solution (c=1.10-3 g.cm-3) and stirred during 20 min. Then, 2 ml of 0.1 M aqueous solution of glucose was added. The final aqueous solution was heated at T=60°C during 30 min. It turned reddish brown, thus the formation of AgNPs was indicated.
Size-exclusion chromatography
Multidetection SEC analysis of polymers was carried out by using a experimental setup consisting of a LC-10 AD Shimadzu pump (throughput 0.5 ml.min-1; Nakagyo-ku, Kyoto, Japan), an automatic injector WISP 717+ from Waters (Milford, MA, USA), three coupled 30-cm Shodex OH-Pak columns (803HQ, 804HQ, and 806HQ; Munich, Germany), a multi-angle light scattering detector DAWN F from Wyatt Technology (Dernbach, Germany), and a differential refractometer R410 from Waters. Distilled water containing 0.1 M NaNO3 was used as eluent. Dilute polymer solutions (c=1.10-3 g.cm-3< c*=1/[n] (Table 1)) were prepared and injected. The intermolecular correlations were then negligible in the analysis of the light scattering measurements.
UV-vis absorption spectroscopy
UV-visible absorption spectra of silver sols were recorded by Varian Cary 50 scan UV-visible spectrophotometer (Palo Alto, CA, USA). Original silver sols were diluted 25 times before spectral measurements.
Transmission electron microscopy
The identification of AgNPs and their size analysis were carried out using high-resolution transmission electron microscopy (TEM). A Phillips CM 12 (Amsterdam, Netherlands) microscope with an acceleration voltage of 120 kV was used. The samples were prepared by spraying silver sols onto carbon-coated copper grids and then analyzed.
Image processing
The TEM sample images were specifically analyzed using the open-source software ImageJ. This software permitted to calculate the average diameter (d) of the AgNPs from their geometrical characteristics, namely from their area S (d=2V[S/nj) or from their longest and shortest diameters di and d2 (d=[d-i+d2]/2) measured in 2d images. Concerning non-spherical particles or aggregates the S value (black area) was mainly used for this size characterization. For each type of sample several sample images were obviously processed.
Results and discussion
The main characteristics of the polymers used in the matrices for our in-situ AgNPs syntheses are reported in Table 1.
Table 1 - Polymer characteristics determined by SEC and potentiometry: Mw is the weight average molecular weight; I= Mw/Mn, the polydispersity index; Rg, the radius of gyration
SEC analysis indicates that polymer samples possess relatively low polydispersity indexes and display in aqueous solution rather large radii of gyration in agreement with their high average molecular weights. The peculiarities of the molecular structure of the copolymers dextran-graft-polyacrylamide were discussed in [17, 21]. These copolymers are star-like polymers, consisting of a compact dextran core and long polyacrylamide arms. As it was previously reported [17, 21], the average conformation of grafted PAA chains is partially controlled by the grafting ratio. For D70-g-PAA5 the PAA-grafted chains are extended near their tethering point and recover a random conformation far from this point. For D70-g-PAA20 the conformation of PAA-grafts is more extended for a same average molecular weight, as the grafting ratio is increased, and is locally worm-like. Linear polyacrylamide also has a random coil average conformation in aqueous solution. However, its internal structure is drastically different from that of a branched architecture. Thus, the branched polymers, due to their more compact internal structure,
Sample CD '-' сэ"^ t- о XÍE 2 S с 2 ^ 2 - Ü a: JE. n> " E T7 ° *o S s ct ЩсТ- ¿r- E та a: ^ CM та cc <4 TO Q1 II
D70-g-PAA5 2.15 1.72 85 2.1 3.36 0.886
D70-g-PAA20 1.43 1.98 64 4.0 2.87 0.845
PAA 1.40 2.40 68 4.4 - -
have a higher local concentration of functional groups with respect to their linear analogues [17]. The compactness of the branched macromolecules, which can be accessed through the parameter Rg2/Mw or g=Rg2/RgL2, where RgL is the radius of gyration of the linear macromolecule with the same molecular weight, both of them are closely related to the number of grafted chains and their average conformation. Increase in the number of grafted chains leads to higher macromolecule compactness (Table 1).
In situ synthesis of AgNPs into (dilute) aqueous solutions of polymers resulted in rather stable colloids. The pH of solution just after non-ionic polymer dissolving was the same as for bi-distilled water (pH=5.5).
The formation of AgNPs in the polymer solutions was investigated using UV-visible absorption spectroscopy, and was revealed in the related absorption spectra. It is known [25] that the position and shape of the Surface Plasmon Resonance (SPR) band depends on the particle size and shape.
The absorbance of the silver sols synthesized into polymer matrices shows the two well-expressed absorption maxima of the SPR band (Figure 1). The first one in the range of 300 nm may correspond to small AgNPs of 2-4 nm in size or Ag+ ions. Taking in account an excess of reducing agent it can be concluded that this peak deals with the presence in nanosystem only the silver particles less than 4 nm in size. The second one is situated at 430-440 nm and corresponds to the SPR band of AgNPs larger than 10-20 nm. The shoulder in the adsorption band close to 360 nm reveals quadrupole part of the SPR band of polygonal AgNPs with size over 50 nm [26]. The appearance of another shoulder in the range 540-630 nm indicates the existence of lengthening aggregated structures [27].
The maximum intensities of the SPR bands depend on the polymer matrix internal structure. Indeed, syntheses of AgNPs were carried out under the same conditions, thus, only the nature and structure of the polymer matrices affect the particle size and morphology. The most intense SPR band (Figure 1) in complex with the lowest contribution of quadrupole absorption for silver sol in D70-PAA20 matrix suggests that concentration of AgNPs is higher and nanoparticle size is smaller. The increase in compactness of macromolecule (Table 1) leads to an increase in the matrix efficiency for nanosystem preparation. Ag sols synthesised in the aqueous solution of linear PAA reveals a low intensity of the SPR band together with a peak broadening. That testifies to both higher polydispersity and average size increasing in comparison with nanosystems synthesized in branched matrices.
TEM images for sols synthesized in linear and branched polymer matrices are represented in Figure 2 and Figure 3 (a, b). They show that colloids contain both individual AgNPs and clusters of AgNPs. The shape of the individual AgNPs is polygonal. The number of particles seen in each TEM image enables only limited statistical accuracy, and hence for each sample several TEM images were processed and considered for our statistical analysis. The calculated
average diameters obtained by two geometrical characteristics (average diameter d=0.5[d1+d2], where di and d2 are the major and minor ellipse diameters, respectively, and nanoparticle area S) are shown in Figure 4 and Figure 5 (a, b). The statistical analysis of TEM images confirms the higher polydispersity as well as the higher average size of AgNPs for the nanosystem synthesized in the linear PAA matrix in comparison with those synthesized in the branched ones.
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Fig. 1 - UV-visible absorption spectra of silver sols synthesized into polymer matrices: 1- PAA; 2- D70-g-PAA5; 3- D70-g-PAA20
Fig. 2a - TEM image of silver sol synthesized into PAA matrix
Fig. 2b - TEM image of silver sol synthesized into PAA matrix
Fig. 3a - TEM image of silver sol synthesized into D70-g-PAA20 matrix
Fig. 3b - TEM image of silver sol synthesized into D70-g-PAA20 matrix
■ Щ I
20 40 60 80 100 120 140 160 180 d, nm
Fig. 4 - Size distribution of silver sol synthesized into PAA matrix
Conclusions
Thus, using dilute aqueous solutions of branched polymers D-g-PAA it is possible to synthesize rather stable Ag sols with distinct size and shape distributions of AgNPs. The polymer matrix internal structure, the polymer chemical nature and the synthesis conditions actually control the characteristics of these stable Ag sols. It is reasonable to synthesize Ag sols by slow-reduction, using glucose as reductant, in aqueous solutions of Dextran-g-Polyacrylamide in order to
decrease the polydispersity of AgNPs and their aggregation. Green synthesis route for AgNPs fabrication allows to use its for biomedical applications.
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Fig. 5a - Size distribution of silver sol synthesized
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Fig. 5b - Nanoparticle area distribution for sol synthesized into D70-PAA20 matrix
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© N. V. Kutsevol - D.Sc.( Chemistry), professor, Taras Shevchenko National University, Ukraine, kutsevol@ukr.net; V. A. Chumachenko - graduate student, Taras Shevchenko National University, Ukraine; V. F. Shkodich - PhD (Chemistry), assistant professor, Dean of the Faculty of Technology and processing of rubber and elastomers, Kazan National Research Technological University; D. R. Vadigullin - bachelor Department of synthetic rubber technology, Kazan National Research Technological University; I. I. Boyoko - master Kazan National Research Technological University.
© Н. В. Куцевол - д.х.н., ведущий научный сотрудник кафедры химии высокомолекулярных соединений, заместитель декана по научной работе химического факультета, Киевский национальный университет имени Тараса Шевченко, kutsevol@ukr.net; В. А. Чумаченко - аспирант, Киевский национальный университет имени Тараса Шевченко; В. Ф. Шкодич - к.х.н. декан факультета ТПКЭ, доцент каф. ТСК КНИТУ; Д. Р. Вадигуллин - бакалавр кафедры ТСК КНИТУ; И. И. Бойко - магистр, КНИТУ.
