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© 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation
Inhibition of Microbial Growth on Chitosan Membranes by Plasma Treatment *Marina de Oliveira Cardoso Macêdo, †Haroldo Reis Alves de Macêdo, ‡Dayanne Lopes Gomes, *Natália de Freitas Daudt, §Hugo Alexandre Oliveira Rocha, and **Clodomiro Alves Jr *Postgraduate Program in Materials Science and Engineering, ‡Postgraduate Program in Biology, §Department of Biochemistry, and **Department of Mechanical Engineering, Federal University of Rio Grande do Norte, Natal; and †Federal Institute of Education, Science and Technology of Piauí, Picos, Brazil
Abstract: The use of polymeric medical devices has stimulated the development of new sterilization methods. The traditional techniques rely on ethylene oxide, but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing. Another common technique is the gamma irradiation process, but it is costly, its safe operation requires an isolated site, and it also affects the bulk properties of the polymers. The use of gas plasma is an elegant alternative sterilization technique. The plasma promotes efficient inactivation of the microorganisms, minimizes damage to the
materials, and presents very little danger for personnel and the environment. In this study we used plasma for microbial inhibition of chitosan membranes. The membranes were treated with oxygen, methane, or argon plasma for different time periods (15, 30, 45, or 60 min). For inhibition of microbial growth with oxygen plasma, the time needed was 60 min. For the methane plasma, samples were successfully treated after 30, 45, and 60 min. For argon plasma, all treatment periods were effective. Key Words: Plasma— Sterilization—Membranes—Chitosan.
Plasma sterilization is fast evolving into a promising alternative to standard sterilizing techniques. Research on plasma sterilization started in 1960. Since then, extensive research has been performed on plasma sterilization. There are numerous parameters involved, such as the pressure and the type of gas used. In an effort to arrive at the optimal mix of parameters, numerous experiments are being performed (1). In modern medical practice, a wide variety of heatsensitive instruments and materials are used that require cold sterilization techniques. Up to now, sterilization of such articles was performed by means of toxic gases—ethylene oxide, pure or mixed with
chlorofluorocarbons—but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing, and this sterilization technique requires a long aeration process (up to 24 h) and, importantly, creates a serious threat for both personnel and the environment. For these reasons, development of new cold sterilization techniques is extremely important (2,3). Plasma-based sterilization techniques do not suffer from the problems described above for the other techniques. Optimal processes are efficient, affect the bulk material only slightly, are environmentally sound, do not produce toxic byproducts, and are fast and cost-effective. The microorganisms are exposed to reactive species, which are produced by applying electromagnetic fields to a gas (normally oxygen) (4,5). There are several mechanisms that may be responsible for the sterilization: interaction of UV radiation with the DNA of the cells, removal of the material of the cells (i.e., etching) by reactive species (oxygen atoms), and the interaction of these two mechanisms (6).
doi:10.1111/aor.12233 Received August 2013. Address correspondence and reprint requests to Dr. Marina de Oliveira Cardoso Macêdo, Postgraduate Program in Materials Science and Engineering, Federal University of Rio Grande do Norte, Natal (RN) 59072-970, Brazil. E-mail: marinalabplasma@ gmail.com Presented in part at the 7th Latin American Congress of Artificial Organs and Biomaterials held August 22–25, 2012 in Natal, Brazil. Artificial Organs 2013, 37(11):998–1002
INHIBITION OF MICROBIAL GROWTH ON CHITOSAN MEMBRANES One of the most promising current alternatives to gaseous sterilization is the use of gas discharge plasma as a sterilizing agent. The plasma technique is advantageous because, as a chemically active medium, plasma is formed by excitation, dissociation, and ionization of any gaseous or vaporous substance, including nontoxic substances and even inert gases. Such active particles exist only while the discharge glows, and they disappear almost immediately after the discharge is turned off. These two circumstances completely solve the problems of safety and environmental hazard (2). For an unbiased estimate of the efficiency and application range of the plasma sterilization technique, it is necessary to first investigate the main sterilizing factors of gas discharge plasma. In recent studies (7,8), the factors determining sterilizing action of direct-current glow discharge plasma (9) in oxygen, air, carbon dioxide gas, hydrogen, argon, nitrogen, and mixtures of these substances were studied both theoretically and experimentally. These investigations show that the main role in plasma sterilization of open surfaces is performed by the plasma’s UV radiation, whereas the sterilization of instruments of complex shape is effected by the action of electrically neutral, chemically active plasma particles (2). The plasma sterilization technique offers several potential advantages over more commonly used methods such as thermal, chemical, and radiation sterilization. For example, it is well suited for materials that are sensitive to heat; it is more cost- and time-effective compared to other techniques; and it eliminates the aeration time necessary in some chemical sterilization methods. Plasma sterilization may also be advantageous in instances where plasma processing is used as a final step in device manufacture (1). The plasma treatment is effective against a range of bacteria, bacterial spores, and fungi, killing these organisms by generating oxygen, hydroxyl radicals, and other reactive species. But these mechanisms are still being investigated. In contrast to other sterilization methods, such as formaldehyde, sterilization by plasma not only kills bacteria and viruses, but also removes the dead bacteria (10). Plasma sterilization is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperature. The use of different gases for this treatment method and the corresponding results were analyzed in this study. Oxygen, argon, and methane were used as plasma source gases. The efficacy of the process was
999
evaluated for different exposure times (15, 30, 45, and 60 min). MATERIALS AND METHODS Preparation of membranes Chitosan powder was dissolved in 2% acetic acid with constant stirring over 24 h. After this period, the chitosan solution was filtered twice, the first time with a nylon filter and the second with a Milli-Q filter (Millipore, Billerica, MA, USA). Then, 30 mL of solution was poured into the petri dishes and placed in the oven for 24 h at 50°C. Subsequently the membranes were neutralized with NaOH, followed by stretching and drying at room temperature for 24 h. Plasma treatment Figure 1 shows the scheme of the reactor used in this study, which consisted of a borosilicate glass tube, closed by two stainless steel flanges. At the top flange, the working gases are introduced. The thermocouple, mechanical vacuum pump, and manometer are located at the lower flange. A mechanical pump was used to evacuate the system to about 0.3 × 102 mbar. The reactor pressure was measured by a capacitive sensor membrane with a digital display. The temperature of the experiment was measured using an alumel–chromel thermocouple located within the cathode. The gas flow was
FIG. 1. Schematic of the plasma reactor used in the treatment of chitosan membranes. Artif Organs, Vol. 37, No. 11, 2013
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regulated by a flow controller and introduced into the reactor by nozzles situated in the upper flange. The sample was placed 5.0 cm away from the cathode (Fig. 1). Oxygen, argon, and methane (White Martins, Rio de Janiero, Brazil) were used as plasma source gases. The efficacy of the processes was evaluated for different exposure times (15, 30, 45, and 60 min). Some parameters were kept constant: pressure (4.5 mbar), current (0.14 A), and gas flow (10 cm3/min). Diagnostic optical spectroscopy was used to investigate the species present in the plasma. The system was composed of an emission spectrometer (Acton Spectrapro 2500i Princeton Instruments, Trenton, NJ, USA) with focal length of 500 mm and minimum spectral resolution of 0.05 nm. This device has three diffraction gratings for different spectral wavelengths. For this study, we used the 1800 g/mm grating, and an optical fiber 5 m in length was used to connect the light from the plasma to the monochromator. A silicon photodiode, 10 mm in diameter with optical response of 200–1100 nm, was used as detector. The optical fiber was positioned near the reactor, pointing directly to the glow discharge. Emission spectra obtained were compared with values found in the database of atomic transition available on the website of the National Institute of Standards and Technology and from selected articles on optical emission spectroscopy. Microbial Control test The microbial test consisted of placing the membranes, treated and untreated, in wells containing Dulbecco’s modified Eagle’s medium (DMEM) at 37°C with 5% CO2 for 7 days. Every day, the wells with the membranes were observed under an optical microscope to check for the emergence of microorganisms on membranes. The assay was performed six times. RESULTS AND DISCUSSION Plasma treatments are currently performed for the sterilization of biomedical materials in order to inhibit or reverse the proliferation of undesirable microorganisms. In Fig. 2, microorganisms can be observed in untreated samples, as well as in treated samples in which the plasma treatment was not able to destroy microorganisms. The treatments carried out with oxygen plasma for 15, 30, and 45 min and methane plasma for 15 min were not effective in eliminating microorganisms present in the samples. Six samples were used for each treatment, and all were contaminated over the 7 days of observation. Artif Organs, Vol. 37, No. 11, 2013
FIG. 2. Images of microorganisms in untreated samples and in treated samples in which the plasma treatment was not effective. (a) Untreated sample. (b) Samples treated with oxygen plasma. (c) Sample treated with methane plasma.
INHIBITION OF MICROBIAL GROWTH ON CHITOSAN MEMBRANES
FIG. 3. Microbial control was successfully demonstrated in a sample treated with argon plasma for 15 min, after 7 days of incubation.
Figure 2 shows the contamination found for each gas, regardless of treatment period used. Argon was efficacious in the inhibition of microbial growth on the membranes at all time periods. Methane gas was effective at periods of 30, 45, and 60 min, and for oxygen, only the 60-min treatment was effective. Figure 3 shows an image of a sample treated with argon plasma (15 min) after 7 days of observation. All other successfully treated samples had the same appearance as that shown in Fig. 3. Plasma can be used for sterilization because sterilizing ultraviolet radiation present during treatment can act on the DNA of microorganisms and also because of the etching process that occurs during the plasma treatment. However, the time required for the ultraviolet radiation from the plasma and the etching process to destroy the microorganism will vary for each gas used (3). Therefore, some gases take less time to complete the sterilization process, and other gases need more time. According to Moreira, some radicals and reactive species and ion bombardment can also decrease the activity of the spores of microorganisms, hence the importance of performing optical emission spectroscopy during the plasma treatment (3). The active species present during the plasma treatment were observed by optical emission spectroscopy. For oxygen treatments, the presence of oxygen ions (O2+) and free oxygen (O) was observed according to Fig. 4. For the argon treatment, free argon, hydrogen molecules (H2), and Hα were found (Fig. 5). During methane treatment, molecules of CH, CN, and N2; ions of carbon (C2+) and nitrogen (N2+); and
1001
FIG. 4. Optical emission spectrum for treatment of chitosan membranes with oxygen plasma.
free hydrogen, Hα, and Hβ were found (Fig. 6). These species present in the plasma during treatment may be acting on the membrane to sterilize it. CONCLUSION Chitosan membranes were prepared by solvent evaporation and then were treated with oxygen, argon, or methane plasma for 15, 30, 45, or 60 min in order to analyze the potential for sterilization of these gases. Treatment with argon plasma was the best, because for all treatment periods, the membranes used remained free of microbial growth after 7 days of incubation in DMEM. The plasma treatment performed with methane was effective at
FIG. 5. Optical emission spectrum for treatment of chitosan membranes with argon plasma. Artif Organs, Vol. 37, No. 11, 2013
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M. DE OLIVEIRA CARDOSO MACÊDO ET AL. REFERENCES
FIG. 6. Optical emission spectrum for treatment of chitosan membranes with methane plasma.
periods of 30, 45, and 60 min, and oxygen plasma treatment was able to inhibit microbial growth on the membranes only after 60 min. The action of ultraviolet radiation, the process of etching, ion bombardment, and the interaction of active species with the chitosan membranes over the treatment period are responsible for the inhibition of microbial growth on the samples. Acknowledgments: Support and funding provided by Labplasma, BioPol, CAPES, and CNPq.
Artif Organs, Vol. 37, No. 11, 2013
1. d’Agostinho R, ed. Plasma Deposition, Treatment, and Etching of Polymers, 1st Edition. San Diego: Academic Press Limited, 1990. 2. Soloshenko A, Tsiolko VV, Khomich VA, et al. Features of sterilization using low-pressure DC-discharge hydrogenperoxide plasma. IEEE Trans Plasma Sci 2002;30:1440–4. 3. Moreira AJ, Mansano RD, Pinto TJA, et al. Sterilization by oxygen plasma. Appl Surf Sci 2004;235:151–5. 4. Lerouge S, Wertheimer MR, Marchand R, et al. Effect of gas composition on spore mortality and etching during lowpressure plasma sterilization. J Biomed Mater Res 2000;51: 129–35. 5. Hermelin IJ, Burtin C, Leverge R, Prugnaud JL. Coût de fonctionnement de deux systèmes de sterilization à basse temperature, centralize et decentralisé. J Pharm Clin 1998;17:138– 44. 6. Moysan M, Barbeau J, Moreau S, et al. Low-temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms. Int J Pharm 2001; 226:1–21. 7. Soloshenko A, Tsiolko VV, Khomich VA, et al. Theoretical and experimental study of the factors of the sterilization of medical articles in low pressure glow discharge plasma. Proceedings of the 14th International Symposium on Plasma Chemistry, Prague, 1999;2551–6. 8. Soloshenko A, Khomich VA, Tsiolko VV, et al. Sterilization of medical products in low-pressure glow discharges. Plasma Phys Rep 2000;26:792–800. 9. Khomich VA, Soloshenko A, Tsiolko VV, et al. Apparatus and process for dry sterilization of medical and dental devices and materials. Alexandria, VA: United States Patent and Trademark Office, 2000: Patent US6113851. 10. Gadri RB, Roth JR, Montie TC, et al. Sterilization and plasma processing of room temperature surfaces with a one atmosphere uniform glow discharge plasma (OAUGDP) . Surf Coat Technol 2000;131:528–41.
© 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation
Inhibition of Microbial Growth on Chitosan Membranes by Plasma Treatment *Marina de Oliveira Cardoso Macêdo, †Haroldo Reis Alves de Macêdo, ‡Dayanne Lopes Gomes, *Natália de Freitas Daudt, §Hugo Alexandre Oliveira Rocha, and **Clodomiro Alves Jr *Postgraduate Program in Materials Science and Engineering, ‡Postgraduate Program in Biology, §Department of Biochemistry, and **Department of Mechanical Engineering, Federal University of Rio Grande do Norte, Natal; and †Federal Institute of Education, Science and Technology of Piauí, Picos, Brazil
Abstract: The use of polymeric medical devices has stimulated the development of new sterilization methods. The traditional techniques rely on ethylene oxide, but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing. Another common technique is the gamma irradiation process, but it is costly, its safe operation requires an isolated site, and it also affects the bulk properties of the polymers. The use of gas plasma is an elegant alternative sterilization technique. The plasma promotes efficient inactivation of the microorganisms, minimizes damage to the
materials, and presents very little danger for personnel and the environment. In this study we used plasma for microbial inhibition of chitosan membranes. The membranes were treated with oxygen, methane, or argon plasma for different time periods (15, 30, 45, or 60 min). For inhibition of microbial growth with oxygen plasma, the time needed was 60 min. For the methane plasma, samples were successfully treated after 30, 45, and 60 min. For argon plasma, all treatment periods were effective. Key Words: Plasma— Sterilization—Membranes—Chitosan.
Plasma sterilization is fast evolving into a promising alternative to standard sterilizing techniques. Research on plasma sterilization started in 1960. Since then, extensive research has been performed on plasma sterilization. There are numerous parameters involved, such as the pressure and the type of gas used. In an effort to arrive at the optimal mix of parameters, numerous experiments are being performed (1). In modern medical practice, a wide variety of heatsensitive instruments and materials are used that require cold sterilization techniques. Up to now, sterilization of such articles was performed by means of toxic gases—ethylene oxide, pure or mixed with
chlorofluorocarbons—but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing, and this sterilization technique requires a long aeration process (up to 24 h) and, importantly, creates a serious threat for both personnel and the environment. For these reasons, development of new cold sterilization techniques is extremely important (2,3). Plasma-based sterilization techniques do not suffer from the problems described above for the other techniques. Optimal processes are efficient, affect the bulk material only slightly, are environmentally sound, do not produce toxic byproducts, and are fast and cost-effective. The microorganisms are exposed to reactive species, which are produced by applying electromagnetic fields to a gas (normally oxygen) (4,5). There are several mechanisms that may be responsible for the sterilization: interaction of UV radiation with the DNA of the cells, removal of the material of the cells (i.e., etching) by reactive species (oxygen atoms), and the interaction of these two mechanisms (6).
doi:10.1111/aor.12233 Received August 2013. Address correspondence and reprint requests to Dr. Marina de Oliveira Cardoso Macêdo, Postgraduate Program in Materials Science and Engineering, Federal University of Rio Grande do Norte, Natal (RN) 59072-970, Brazil. E-mail: marinalabplasma@ gmail.com Presented in part at the 7th Latin American Congress of Artificial Organs and Biomaterials held August 22–25, 2012 in Natal, Brazil. Artificial Organs 2013, 37(11):998–1002
INHIBITION OF MICROBIAL GROWTH ON CHITOSAN MEMBRANES One of the most promising current alternatives to gaseous sterilization is the use of gas discharge plasma as a sterilizing agent. The plasma technique is advantageous because, as a chemically active medium, plasma is formed by excitation, dissociation, and ionization of any gaseous or vaporous substance, including nontoxic substances and even inert gases. Such active particles exist only while the discharge glows, and they disappear almost immediately after the discharge is turned off. These two circumstances completely solve the problems of safety and environmental hazard (2). For an unbiased estimate of the efficiency and application range of the plasma sterilization technique, it is necessary to first investigate the main sterilizing factors of gas discharge plasma. In recent studies (7,8), the factors determining sterilizing action of direct-current glow discharge plasma (9) in oxygen, air, carbon dioxide gas, hydrogen, argon, nitrogen, and mixtures of these substances were studied both theoretically and experimentally. These investigations show that the main role in plasma sterilization of open surfaces is performed by the plasma’s UV radiation, whereas the sterilization of instruments of complex shape is effected by the action of electrically neutral, chemically active plasma particles (2). The plasma sterilization technique offers several potential advantages over more commonly used methods such as thermal, chemical, and radiation sterilization. For example, it is well suited for materials that are sensitive to heat; it is more cost- and time-effective compared to other techniques; and it eliminates the aeration time necessary in some chemical sterilization methods. Plasma sterilization may also be advantageous in instances where plasma processing is used as a final step in device manufacture (1). The plasma treatment is effective against a range of bacteria, bacterial spores, and fungi, killing these organisms by generating oxygen, hydroxyl radicals, and other reactive species. But these mechanisms are still being investigated. In contrast to other sterilization methods, such as formaldehyde, sterilization by plasma not only kills bacteria and viruses, but also removes the dead bacteria (10). Plasma sterilization is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperature. The use of different gases for this treatment method and the corresponding results were analyzed in this study. Oxygen, argon, and methane were used as plasma source gases. The efficacy of the process was
999
evaluated for different exposure times (15, 30, 45, and 60 min). MATERIALS AND METHODS Preparation of membranes Chitosan powder was dissolved in 2% acetic acid with constant stirring over 24 h. After this period, the chitosan solution was filtered twice, the first time with a nylon filter and the second with a Milli-Q filter (Millipore, Billerica, MA, USA). Then, 30 mL of solution was poured into the petri dishes and placed in the oven for 24 h at 50°C. Subsequently the membranes were neutralized with NaOH, followed by stretching and drying at room temperature for 24 h. Plasma treatment Figure 1 shows the scheme of the reactor used in this study, which consisted of a borosilicate glass tube, closed by two stainless steel flanges. At the top flange, the working gases are introduced. The thermocouple, mechanical vacuum pump, and manometer are located at the lower flange. A mechanical pump was used to evacuate the system to about 0.3 × 102 mbar. The reactor pressure was measured by a capacitive sensor membrane with a digital display. The temperature of the experiment was measured using an alumel–chromel thermocouple located within the cathode. The gas flow was
FIG. 1. Schematic of the plasma reactor used in the treatment of chitosan membranes. Artif Organs, Vol. 37, No. 11, 2013
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M. DE OLIVEIRA CARDOSO MACÊDO ET AL.
regulated by a flow controller and introduced into the reactor by nozzles situated in the upper flange. The sample was placed 5.0 cm away from the cathode (Fig. 1). Oxygen, argon, and methane (White Martins, Rio de Janiero, Brazil) were used as plasma source gases. The efficacy of the processes was evaluated for different exposure times (15, 30, 45, and 60 min). Some parameters were kept constant: pressure (4.5 mbar), current (0.14 A), and gas flow (10 cm3/min). Diagnostic optical spectroscopy was used to investigate the species present in the plasma. The system was composed of an emission spectrometer (Acton Spectrapro 2500i Princeton Instruments, Trenton, NJ, USA) with focal length of 500 mm and minimum spectral resolution of 0.05 nm. This device has three diffraction gratings for different spectral wavelengths. For this study, we used the 1800 g/mm grating, and an optical fiber 5 m in length was used to connect the light from the plasma to the monochromator. A silicon photodiode, 10 mm in diameter with optical response of 200–1100 nm, was used as detector. The optical fiber was positioned near the reactor, pointing directly to the glow discharge. Emission spectra obtained were compared with values found in the database of atomic transition available on the website of the National Institute of Standards and Technology and from selected articles on optical emission spectroscopy. Microbial Control test The microbial test consisted of placing the membranes, treated and untreated, in wells containing Dulbecco’s modified Eagle’s medium (DMEM) at 37°C with 5% CO2 for 7 days. Every day, the wells with the membranes were observed under an optical microscope to check for the emergence of microorganisms on membranes. The assay was performed six times. RESULTS AND DISCUSSION Plasma treatments are currently performed for the sterilization of biomedical materials in order to inhibit or reverse the proliferation of undesirable microorganisms. In Fig. 2, microorganisms can be observed in untreated samples, as well as in treated samples in which the plasma treatment was not able to destroy microorganisms. The treatments carried out with oxygen plasma for 15, 30, and 45 min and methane plasma for 15 min were not effective in eliminating microorganisms present in the samples. Six samples were used for each treatment, and all were contaminated over the 7 days of observation. Artif Organs, Vol. 37, No. 11, 2013
FIG. 2. Images of microorganisms in untreated samples and in treated samples in which the plasma treatment was not effective. (a) Untreated sample. (b) Samples treated with oxygen plasma. (c) Sample treated with methane plasma.
INHIBITION OF MICROBIAL GROWTH ON CHITOSAN MEMBRANES
FIG. 3. Microbial control was successfully demonstrated in a sample treated with argon plasma for 15 min, after 7 days of incubation.
Figure 2 shows the contamination found for each gas, regardless of treatment period used. Argon was efficacious in the inhibition of microbial growth on the membranes at all time periods. Methane gas was effective at periods of 30, 45, and 60 min, and for oxygen, only the 60-min treatment was effective. Figure 3 shows an image of a sample treated with argon plasma (15 min) after 7 days of observation. All other successfully treated samples had the same appearance as that shown in Fig. 3. Plasma can be used for sterilization because sterilizing ultraviolet radiation present during treatment can act on the DNA of microorganisms and also because of the etching process that occurs during the plasma treatment. However, the time required for the ultraviolet radiation from the plasma and the etching process to destroy the microorganism will vary for each gas used (3). Therefore, some gases take less time to complete the sterilization process, and other gases need more time. According to Moreira, some radicals and reactive species and ion bombardment can also decrease the activity of the spores of microorganisms, hence the importance of performing optical emission spectroscopy during the plasma treatment (3). The active species present during the plasma treatment were observed by optical emission spectroscopy. For oxygen treatments, the presence of oxygen ions (O2+) and free oxygen (O) was observed according to Fig. 4. For the argon treatment, free argon, hydrogen molecules (H2), and Hα were found (Fig. 5). During methane treatment, molecules of CH, CN, and N2; ions of carbon (C2+) and nitrogen (N2+); and
1001
FIG. 4. Optical emission spectrum for treatment of chitosan membranes with oxygen plasma.
free hydrogen, Hα, and Hβ were found (Fig. 6). These species present in the plasma during treatment may be acting on the membrane to sterilize it. CONCLUSION Chitosan membranes were prepared by solvent evaporation and then were treated with oxygen, argon, or methane plasma for 15, 30, 45, or 60 min in order to analyze the potential for sterilization of these gases. Treatment with argon plasma was the best, because for all treatment periods, the membranes used remained free of microbial growth after 7 days of incubation in DMEM. The plasma treatment performed with methane was effective at
FIG. 5. Optical emission spectrum for treatment of chitosan membranes with argon plasma. Artif Organs, Vol. 37, No. 11, 2013
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M. DE OLIVEIRA CARDOSO MACÊDO ET AL. REFERENCES
FIG. 6. Optical emission spectrum for treatment of chitosan membranes with methane plasma.
periods of 30, 45, and 60 min, and oxygen plasma treatment was able to inhibit microbial growth on the membranes only after 60 min. The action of ultraviolet radiation, the process of etching, ion bombardment, and the interaction of active species with the chitosan membranes over the treatment period are responsible for the inhibition of microbial growth on the samples. Acknowledgments: Support and funding provided by Labplasma, BioPol, CAPES, and CNPq.
Artif Organs, Vol. 37, No. 11, 2013
1. d’Agostinho R, ed. Plasma Deposition, Treatment, and Etching of Polymers, 1st Edition. San Diego: Academic Press Limited, 1990. 2. Soloshenko A, Tsiolko VV, Khomich VA, et al. Features of sterilization using low-pressure DC-discharge hydrogenperoxide plasma. IEEE Trans Plasma Sci 2002;30:1440–4. 3. Moreira AJ, Mansano RD, Pinto TJA, et al. Sterilization by oxygen plasma. Appl Surf Sci 2004;235:151–5. 4. Lerouge S, Wertheimer MR, Marchand R, et al. Effect of gas composition on spore mortality and etching during lowpressure plasma sterilization. J Biomed Mater Res 2000;51: 129–35. 5. Hermelin IJ, Burtin C, Leverge R, Prugnaud JL. Coût de fonctionnement de deux systèmes de sterilization à basse temperature, centralize et decentralisé. J Pharm Clin 1998;17:138– 44. 6. Moysan M, Barbeau J, Moreau S, et al. Low-temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms. Int J Pharm 2001; 226:1–21. 7. Soloshenko A, Tsiolko VV, Khomich VA, et al. Theoretical and experimental study of the factors of the sterilization of medical articles in low pressure glow discharge plasma. Proceedings of the 14th International Symposium on Plasma Chemistry, Prague, 1999;2551–6. 8. Soloshenko A, Khomich VA, Tsiolko VV, et al. Sterilization of medical products in low-pressure glow discharges. Plasma Phys Rep 2000;26:792–800. 9. Khomich VA, Soloshenko A, Tsiolko VV, et al. Apparatus and process for dry sterilization of medical and dental devices and materials. Alexandria, VA: United States Patent and Trademark Office, 2000: Patent US6113851. 10. Gadri RB, Roth JR, Montie TC, et al. Sterilization and plasma processing of room temperature surfaces with a one atmosphere uniform glow discharge plasma (OAUGDP) . Surf Coat Technol 2000;131:528–41.
