ISSN 19950780, Nanotechnologies in Russia, 2013, Vol. 8, Nos. 5–6, pp. 303–308. © Pleiades Publishing, Ltd., 2013. Original Russian Text © I.A. Mamonova, M.D. Matasov, I.V. Babushkina, O.E. Losev, Ye.G. Chebotareva, E.V. Gladkova, and Ye.V. Borodulina, 2013, published in Rossiiskie Nanotekhnologii, 2013, Vol. 8, Nos. 5–6.
Study of Physical Properties and Biological Activity of Copper Nanoparticles I. A. Mamonovaa, M. D. Matasovb, I. V. Babushkinaa, O. E. Losevc, Ye. G. Chebotarevad, E. V. Gladkovaa, and Ye. V. Borodulinad a Saratov
Research and Development Institute of Traumatic and Orthopedic Surgery under the Ministry of Healthcare and Social Development of Russia, ul. Chernyshevskogo 148, Saratov, Russia b Chernyshevsky State University, ul. Astrakhanskogo 83, Saratov, Russia c Sechenov First Moscow State Medical University under the Ministry of Healthcare and Social Development of Russia, ul. Trubetskaya 8, Moscow, Russia d Razumovsky State Medical University, ul. B. Kazachya 112, Saratov, Russia email:
[email protected] Received September 12, 2012; in final form, February 6, 2013
Abstract—Metals in the form of nanoparticles are one of the longterm challenges for the creation of a new class of antibacterial agents. The biological activity of nanoparticles of metals depends directly on their phys ical and chemical properties. The objective of this work is to study the physicochemical parameters and anti bacterial effects of copper nanoparticles for the standardization of the given nanomaterial during further use as an antibacterial agent. The physical and chemical parameters and antibacterial effects of copper nanopar ticles are investigated. Optical methods, including atomic force microscopy, spectrophotometry, and fluo rometry, are used to determine the sizes of associates of nanoparticles. The size of copper nanoparticles was 75 nm, and the sizes of nanoparticle associates are 481.1–1037 nm. An oxide film without any admixture of organic molecules is detected on the surface of nanoparticles. The antibacterial effects of copper nanoparti cles are studied on the basis of clinical polyantibioticresistant strains of epidermal staphylococcus and gram negative nonfermentative microorganisms (causative agents of purulent flammatory diseases among patients of traumatology and orthopedic inpatient hospital departments). As a result of the investigations, a clear antibacterial effect of copper nanoparticles with specified physical characteristics on grampositive and gramnegative microorganisms of causative agents of wound infection is detected, which gives prospects for the further use of these particles as antibacterial agents. DOI: 10.1134/S1995078013030142
INTRODUCTION Metal nanoparticles are broadly used nowadays as a nanotechnological object. The transition to nanosize level changes the basic properties of matter, such as magnetic characteristics, optical properties, melting point, specific thermal capacity, and superficial and catalytic activity, which results from the manifestation of so called “quantum size effects.” When the size decreases and a transition from a macroscopic body to several hundred or a thousand atoms occurs, the state density in the valency zone and the conductance zone changes considerably, which affects the properties determined by the behavior of electrons, first and fore most, magnetic and electric. The “continuous” state density at macro level is replaced with discrete levels, the distances between which depend on particle size [1]. Metal nanoparticles are typically characterized by the specifics of structure formation and a considerable amount of atoms on their surface. Since connections between atoms in the near surface layers of nanoparti cles are not compensated for, the symmetry in the dis tribution of forces affecting these connections is dis
turbed, which increases the amount of free energy on the surface and activates adsorption and ionic and atomic exchanges [2]. The specific properties of superdispersed metals provide a lot of opportunities for inventing efficient metals and using them in biology and healthcare. Nowadays, one of the topical issues in healthcare is the steady spread of strains of microorganisms with multiple resistances to antibiotics. The development of new medications with antibacterial effects on causative agents of wound infection with different localization is therefore of great theoretical and practical concern. Metal nanopowders manifest clear biological activ ity, including bacteriostatic and bactericide effects. During previous studies the bactericide effect of cop per nanoparticles on clinical strains of Escherichia coli and Staphylococcus aureus was discovered [3, 4]. The biological activity of metal nanoparticles depends directly on their physical and chemical prop erties, which allows one to model their basic charac teristics to obtain necessary results [5]. In several works in which properties of metal nanoparticles are studied, it is shown that antibacterial activity depends
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on the physical characteristics of nanomaterials [6]. The biological effect of metal nanoparticles is con nected directly to their size and high specific area, which ensures intensive chemical activity and the abil ity to penetrate inside the organism. In this regard it is necessary to study the physical and chemical parame ters and actibacterial influence of copper nanoparti cles to standardize this nanomaterial during subse quent use as an antibacterial agent. RESEARCH OBJECTIVE This research is aimed at studying the physical characteristics of copper nanoparticles and the anti bacterial effect of this nanomaterial on clinical strains of nonfermentative gramnegative microorganisms (NFGMs) and St. epidermidis. R, % 28
R = RK(1 + T 2) + T 4Rm, 0
Copper
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20 R = T 4(R − R ) m 0
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MATERIALS AND METHODS The materials used in the study were highly dis persed copper nanopowders synthesized at a plasma chemical complex of a branch of the State Scientific Center of the Russian Federation (State Research Institute for Chemistry and Technology of Organoele ment Compounds) in Moscow. Nanoparticles were obtained from PMS1 GOST 496075 copper with the help of a plasmatic technology based on the evapora tion of raw materials to ultradispersed particles of a required size in a plasmatic flow with a temperature of 5000–60000 K and steam condensation. The optical research was done with the help of a Lambda 950 spectrofotometer (PerkinElmer, United States) and LS55 luminiscent spectrometer (Perki nElmer, United States). The distribution according to particle size was studied using a Zetasizer Nano ZS nanoparticle size analyzer (Malvern Instruments, United States) and sonde nano laboratory NTEGRA Specta (NTMDT ZAO, Russia). The composition and exact size of the particles were determined with the help of a Tescan Mira II LMU electron micro scope (Tescan, Czech Republic). The antibacterial effect of copper nanoparticles was studied on 30 clinical poly antibioticresistant strains of St. epidermidis and 30 strains of NFGMs taken from patients with purulent complications who underwent treatment at the traumatology and ortho pedic inpatient department of the Saratov Research and Development Institute of Traumatic and Ortho pedic Surgery and were resistant to five and more pro file antibiotics. To study its effect on the clinical strains of microor ganisms, the suspension of copper nanopowders was prepared in 900 μL of 0.9% solution of NaCl. The resulting concentrations of nanoparticles were 0.001, 0.01, and 0.1 mg/mL. One hundred microliters of the suspension (100000 CFU/mL) were added to each of the tubes with nanoparticle dilution; then the tubes were shaken and incubated at room temperature for 30, 60, 90, and 120 min. The reference preparation was a bacterial suspension diluted in the 0.9% solution of NaCl. After incubation, each dilution was plated in Petri dishes with 100 mL of solid nutrient medium (GRM agar) and incubated at 37°C for 24 h. The col onies were counted on the next day. Statistical processing of the material was per formed, and average values (M) and their mean square deviations (m) and reliability level (p) were calculated.
0
RESULTS AND DISCUSSION 200
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Fig. 1. Reflection spectrum of a copper nanoparticle pow der: (a) without due account for the holder and (b) with due account for the holder.
To find out whether there were any compounds on particle surfaces and to indirectly define the size of these compounds [7], the luminiscence of copper nanoparticles was studied and the reflection spectrum [8] of the given nanopowder was measured. The results are given in Figs. 1 and 2. The spectrum given in Fig. 1b
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Fig. 2. Luminiscent spectrum of copper nanoparticles.
was marked with an increase in the coefficient of dif fuse reflection in a longwave domain: this coefficient is derived from the Fresnel coefficient of reflection from a separate particle which is multiplied by itself many times. Concurrently, this reflection has a differ ent angle each time. In addition, the dependence of the skin layer on the wave frequency and, therefore, wave length of radiation in the considered wave range may possibly have a considerable effect on copper. The skin layer grows as the wave increases, and, therefore, absorption will increase as well. Peaks of luminiscence were observed in the lumi niscence of copper nanoparticles [9]. This indicated that the particles were covered with an oxide and had semiconductor properties (Figs. 1, 2). The specifics of the copper particles were that they had a fairly bright luminiscence. The following was found: 1. the occurrence of peaks of luminiscence in cop per had nothing to do with diffraction and direct dissi pation and came to the superimposition of other pro cesses on luminiscence [10]; 2. the observed peaks did not depend on the excita tion wavelength (λ@); 3. the proportions between wavelength values λ remained unchanged at λ@ 2 = 1.06–1.15. λ1 4. luminiscence was observed at λ = 484 nm. When the distribution of the particles according to their size was determined with the help of a Zetasizer Nano ZS (Malvern Instruments, United States) it was found that the particles aggregated in large clusters, which proved indirectly that significant chemical NANOTECHNOLOGIES IN RUSSIA
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bonds existed that retained the particles with each other. The aggregates of copper particles were mea sured in three instances: when the aggregates were simply dissolved in dimethyl sulfoxide, when large clusters and conglomerates of these particles were pre cipitated in a centrifuge at g = 18000, and when they were exposed to Xray radiation for 1 min, respectively. The changes in size distribution allowed us to con clude that the particles were retained in significant bonds. The average and the most realistic diameter of the particle conglomerates before precipitation and expo sure was d = 2209 nm and dmax1 = 14 346 nm, dmax2 = 4352 nm; the same indexes after precipitation and exposure were d = 1710 nm and dmax = 1037 nm and after Xray cutting they were d = 557.8 nm and dmax = 481.1 nm. The morphology and physical appearance of the particles were identified with the help of an atom power microscope; a 3d layout of particle surfaces was obtained (Fig. 3). After the resulting images were analyzed, the size of the particles was calculated and appeared to be dmax = 75.7 nm. In addition, the effect of copper particles with specified physical and chemical parameters on bacte rial cells was investigated. The procedure was per formed using 30 clinical strains of NFGMs taken from patients with purulent inflammatory complications who underwent treatment in a traumatology in patient hospital unit. Those strains were causative agents of the severest purulent inflammatory compli 2013
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Table 1. Antibacterial effect of nanoparticles on clinical strains of nonfermentative gramnegative microorganisms Number of colonies in solid nutrient solutions, M ± m Exposure time, minutes
Experimental groups Reference group (n = 30)
1 (n = 30)
2 (n = 30)
3 (n = 30)
0.001 mg/mL
0.01 mg/mL
0.1 mg/mL
381.4 ± 28.8 p < 0.001* 170.2 ± 14.9 p < 0.001* 186.3 ± 26.0 p < 0.001* 59.7 ± 72.5 p < 0.001*
220.7 ± 16.3 p < 0.001* 35.7 ± 12.8 p < 0.001* 92.7 ± 17.4 p < 0.001* 48.3 ± 20.6 p < 0.001*
30
678.6 ± 28.8
560.8 ± 67.5
60
563.5 ± 12.4
90
711.2 ± 23.0
120
539.1 ± 57.4
367.0 ± 28.4 p < 0.001* 289.1 ± 78.5 p < 0.001* 390.8 ± 109.7
Note: * p is for the degree of reliability of differences between the indexes of the experimental group and the reference group.
Table 2. Antibacterial effect of copper nanoparticles in different concentrations on clinical strains of St. epidermidis Number of colonies in solid nutrient solutions, M ± m Exposure time, minutes
Experimental groups Reference group (n = 20)
30
1378.1 ± 120.3
60
1511.3 ± 84.0
90
967.4 ± 92.3
120
871.3 ± 65.2
1 (n = 30)
2 (n = 30)
3 (n = 30)
0.001 mg/mL
0.01 mg/mL
0.1 mg/mL
643.7 ± 33.1 p < 0.001* 105.4 ± 85.7 p < 0.001* 5.8 ± 8.5 p < 0.001* 18.4 ± 17.2 p < 0.001*
326.3 ± 15.2 p < 0.001* Zero growth
Zero growth
Zero growth
Zero growth
Zero growth
Zero growth
Zero growth
Note: * p is for the degree of reliability of differences between the indexes of the experimental group and the reference group.
cations that were very difficult to eliminate with the help of antibiotic therapy. The clinically significant strains were Pseudomonas aeruginosa, as well as Acine tobacter spp., Burkholderia spp., Stenotrophomonas spp., and Chryseobactehum spp. Among the gramneg ative causative agents of the wound infection, the frac tion of Ps. aeruginosa approximated 24%. Concur rently, in 7.4% of investigated cases, this strain was taken from patients of traumatology inpatient depart ments [11, 12]. An important feature of the given agents is the rapid formation of poly antibioticresis tant strains. For the results of calculating the number of colo nies grown on solid nutrient media after exposure to copper nanoparticles in different concentrations and the calculation results in the reference group, see Table 1. The influence of copper nanoparticles with a con centration of 0.001 mg/mL during 30min and 120min incubation did not lead to a statistically reliable decrease in the number of colonies grown in a dense
nutrient medium. During 60min and a 90min expo sure, the amount of viable microorganisms decreased to 65.1% and 40.6%, respectively (p < 0.001). An increase in the concentration of copper nanoparticles to 0.01 mg/mL and 0.1 mg/mL intensified the anti bacterial influence of the studied nanomaterial. The most pronounced antibacterial effect was observed during 120min exposure to copper nanoparticles with a concentration of 0.1 mg/mL; compared to the refer ence figures, the amount of surviving microorganisms was 8.9% (p < 0.001). In addition, the antibacterial influence of copper nanoparticles on 30 poly antibioticresistant clinical strains of St. epidermidis was studied. The representa tives of Staphylococcus spp. are the main causative agents of infectious complications in bones and joints. In addition to St.aureus, an important ethilogical part in the occurrence of purulent inflammatory diseases belongs to coagulasenegative staphylococci and, first and foremost, Staphylococcus epidermidis. For instance, when joints are infected with St. epidermidis,
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Thus, a clear antibacterial effect of copper nano particles on grampositive and gramnegative micro organisms, causative agents of purulent inflammatory complications among the patients of traumatology and orthopedic inpatient units, was discovered.
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Fig. 3. Morphology of the surface of accumulation of cop per nanoparticles.
3–16% of the total amount of Staphylococcus spp. are exuded and, in the case of osteomelitis, these figures are 5–15% [12]. For the results of calculating the number of colo nies grown in solid nutrient media after exposure to copper nanoparticles in different concentrations, see Table 2. A 30min influence of copper nanoparticles with a concentration of 0.001 mg/mL caused a statistically reliable decrease in the amount of viable microorgan isms to 46.6% (p < 0.001). When exposure lasted for 60, 90, and 120 min, the amount of bacterial cells decreased to 6.9, 0.6, and 2.1%, respectively (p < 0.001). The concentration of 0.01 mg/mL during the 30min exposure decreased the amount of bacterial bodies to 23.7% (p < 0.001). Bacterial cells got completely elim inated during the subsequent extension of incubation and concentration of nanoparticles. NANOTECHNOLOGIES IN RUSSIA
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CONCLUSIONS As a result of the above physical and chemical stud ies of copper nanoparticles obtained using a plasmatic chemical approach, the calculated particle size appeared to be 75 nm and the size of the particle asso ciates was 481.1–1037 nm. An oxide film without organic molecules was detected on the surface of the nanoparticles. When the antibacterial influence of the particles was studied, a clear antibacterial effect on clinical poly antibioticresistant strains of epidermal staphylococcus and gramresistant nonfermentative microorganisms was discovered. According to a com parative analysis of the resulting data, strains of St. epi dermidis appeared most sensitive to the influence of copper nanoparticles. For instance, the influence of cop per nanoparticles with a concentration of 0.01 mg/mL during a 60min exposure eliminated St. epidermidis completely, whereas no such effect was observed in the case of NFGMs and the fraction of surviving microor ganisms was 30% (p < 0.001). This fact occurred pos sibly due to radical differences in the structure of cell walls of grampositive and gramnegative microorgan isms. These studies of the physical and chemical param eters and antibacterial effects of copper nanoparticles show that the grampositive and gramnegative micro organisms of the causative agents of the wound infec tion are efficiently affected by this nanomaterial, which makes it possible to use this material as an anti bacterial agent. REFERENCES 1. D. A. Baranov and S. P. Gubin, “Magnetic nanoparti cles. Achievements and problems in chemical synthesis. Radioelectronics,” Nanosist. Inf. Tekhnol., Nos. 1–2, 129–147 (2009). 2. A. P. Shpak, Yu. A. Kunitskii, and V. L. Karbovskii, Cluster and Nanostructured Materials (Akademperi odika, Kiev, 2001), Vol. 1. 3. I. V. Babushkina, V. A. Mart’yanova, E. V. Borodulina, A. L. Borovskii, and M. Sakkala, “Nanosize copper particles impact to clinical strains of gramnegative bacteria,” Vestn. RUDN, No. 4, 162–165 (2010). 4. I. V. Babushkina, V. B. Borodulin, G. V. Korshunov, and D. M. Puchin’yan, “Antibacterial effect of copper nanoparticles onto clinical strains of Staphylococcus Aureus,” Saratov. Nauch.Med. Zh., No. 1, 11–14 (2010). 5. I. N. Andrusishina, “Metallic nanoparticles. Synthesis procedure. Physicochemical properties. Ways for inves tigations and toxicity estimation,” Sovr. Probl. Tok sikol., No. 3, 5–14 (2011). 2013
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6. A. A. Rakhmetova, Extended Abstract of Candidate’s Dissertation in Biology (Moscow, 2011). 7. J. MartinezDuart, R. J. MartinPalmer, and F. Agullo Rueda, Nanotechnology for Microelectronics and Opto electronics (Cambridge Press, 2005). 8. W. Schmidt, Optical Spectroscopy in Chemistry and Life Sciences: An Introduction (WileyVCH, 2005). 9. G. S. Landsberg, Optics (Nauka, Moscow, 1976) [in Russian]. 10. T. L. Maiorova, V. G. Klyuev, V. N. Semenov, N. G. Bol gova, and A. N. Naumov, “Luminescent properties of cadmium polycrystalline films alloyed by firstgroup metals,” Vestn. VGU, No. 2, 38–44 (2005). 11. S. S. Kopenkin and K. A. Talitskii, “Fluroquinolones beforeoperation antibacterial preventive measures in
traumatology and orthopaedy,” Infekts. Antimikrobn. Teropiya, No. 2, 9–13 (2007). 12. T. Ya. Pkhakadze, N. K. Vabishchevich, and G. G. Okroperidze, “Microbe monitoring for infec tious complication in traumatology and orthopaedy,” in Proc. 3rd Russian Sci.Practical Conf. Modern Prob lems in Epidemiology, Diagnosis and Preventing Mea sures of HospitalAcquired Infection (St. Petersburg, 2003), pp. 97–98 [in Russian]. 13. S. A. Bozhkov, “Modern principals of infection diagno sis and antibacterial therapy for prosthetic joints (review of published materials),” Travmatol. Ortoped. Ross., No. 3, 126–136 (2011).
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