rus
Issue #7-8/2019
T.M.Vasilieva, E.V.Kochurova, Е.О.Kudasova, R.A.Akasov, Htet Wai Yan Kyaw, Htet Ko Ko Zaw
Application of low-temperature low pressure plasma in clinical medicine and pharmaceutics
Application of low-temperature low pressure plasma in clinical medicine and pharmaceutics
Described are the generators of electron-beam plasma and hybrid plasma intended for the modification of (bio)polymeric materials used in medicine. Data on surface morphology, chemical structure and biological activity of modified polymers are presented.
Теги: bioactivity biopolymer materials low-temperature plasma modified polymers биологическая активность биополимерные материалы модифицированные полимеры низкотемпературная плазма
INTRODUCTION
In the late 20th century various types of low-temperature plasma (LTP) began to find their numerous practical applications not only for production technologies [1], but also in the modern innovative fields, such as plasmatic medicine. According to VDI Technologiezentrum GmbH, Evaluierung Plasmatechnik (Dusseldorf, Germany), this field will be the most perspective one for the development and implementation of plasma-stimulated processes. Besides, the medicine applications of LTP include the polymer material surface modification in order to increase the biocompatibility with the human body tissues [2]. By changing the surface charge, processing of polymers by LTP increases absorption of adhesive proteins and immobilizes various biologically active molecules at [3]. LTP surface modification affects its morphology and creates various nanoscale structures which serve as the binding points for specific cell membrane proteins thereby improving the cell adhesion to the surface being treated [3].
The low-pressure LTP is the most investigated one from the viewpoint of the polymer materials and articles modification mechanisms and it has been mostly used in the variant of LTP of gas discharges in various frequency regions [4]. Known are the disadvantages of gas discharge reactors which limits their usage for this purpose. First of all, these are the difficulties associated with the formation of huge homogeneous reaction volumes which leads to a significant heating of the plasma-forming medium and objects placed in it. Technical problems connected with the chemical resistance of electrodes may appear because of stability losses in the reagent volume at intensive purging of gas by charging. It is necessary to apply other types of LTP of low pressure, such as e-beam plasma (EBP) and hybrid plasma (HP) in order to overcome these disadvantages. E-beam plasma is generated at the injection of the electron beam into the dense gaseous medium. Geometry, composition, temperature of particles and other characteristics of EBP depend of electron energy Eb, beam power Nb (Nb<1 kWt), pressure Pm (0,1 <Pm<10 kPa), temperature Tm of plasma-forming media and its phase and chemical compositions. It is possible to control the EBP generator modes by changing of EP power at constant Pm (or, in opposite, by changing the gas pressure at stable Nb) and carry out different non-equilibrium plasma-chemical reactions. Also, the sample temperature during the treatment may be fixed at the necessary level, and temperature drop to the room value does not influence high chemical activity of plasma.
Hybrid plasma (HP) is generated when two or more ionizers act together or in sequence on the plasma-forming medium. In this study an e-beam was applied as a main ionizer for the formation of sufficiently large volumes of plasma, and an additional ionization source was used as a HF-discharge at frequency of 13.56 MHz. HP has the very important additional advantages like a possibility of practically inertia-free control of properties and geometry of reaction volume with the help of EP, and the higher stability of this volume to contraction when the pressure grows. Active ions are added to the chemically active stimulated particles in gas-discharge plasma at sufficient concentrations due to the e-beam action in gas. Also, creation of new particles in HP is possible only at combined action of HF-discharge and EP.
The work was aimed at:
Development of technological approaches to the EPP and HP generation and processing of polymer materials of synthetic and natural origin;
Description of the surface morphology, chemical structure and biological properties of plasma-chemically modified (bio)polymers.
METHODS OF RESEARCH
Generation of e-beam and hybrid plasma
Fig.1 presents the general diagram of a plasma-chemical reactor for the EPP and HP generation. A plasma cloud 12 is formed in the reaction camera 11 placed inside of the working chamber 10. Working chamber has a plug 3 for pumping out and plug 4 for feeding the basic plasma-forming gas at a flow rate Gg2. Power from Genesis GHW-12 HF-generator (MKS Instruments, Great Britain, frequency 13.6 MHz) is applied to an active electrode 5 hrough the sealed inlet 8. If necessary, additional gas at flow rate of Gg1 may be supplied through the electrode 5 made of a metal porous pipe. The EP 2 is inserted through the open end of the working chamber. An HF-generator is not used for EPP generation. An electron gun 1 generates an e-beam inside the high-vacuum chamber 14 (∼10–5 Тorr) which is transported into the working chamber through the special outlet device 13. An outlet device is combined with an electrical magnetic deflection system which can decline an EP axis in two orthogonal directions and form a raster.
Stationary EPP and HP clouds and plasma flows may be formed inside the developed reactor using nozzle devices of different design. By using different types of nozzles it is possible to disperse liquids and powders in a plasma cloud or flow and form the working volume in a form of an aerosol. Various types of beam-plasma reactors are described in detail in [5].
The working chamber is equipped with special holders 12 to process small samples. In the experiments considered in this work, were used synthetic organic (polymethyl methacrylate (PMMA), polyethylene terephthalate, silicone rubbers) and natural (chitin, chitosan, cellulose, alginates) polymers. The listed materials are widely used in medical practice, and improving of their biomedical characteristics is an urgent task.
The working chamber is rotated using drive 9 in order to process the powdered materials. In this case the inner accessories of the working chamber are replaced with special ribs to mix powder during treatment in order to provide the homogeneity of material processing across the chamber volume. As will be shown below, it is possible in this way to obtain bioactive compounds with valuable properties from the point of view of their use in pharmaceutics.
The reactor was tested on thin films (thickness of 8±0,5 µm) and powders (diameter of 50 µm) of chitozan, as well as plates made of dental plastic based on PMMA (Villacryl H Plus, Zhermak, Italy). Table 1 shows typical experimental conditions.
RESULTS
EPP stimulated destruction of chitozan powders
Chitozan powders were processed in EPP during for 5 minutes. Plasma-stimulated hydrolysis of polysaccharides led to their rapid depolymerization with the formation of water-soluble oligochitosans mixture with a yield of 85%. The weight average molecular weights of the products of plasmochemical modification of chitozans obtained under optimal conditions varied within 570–2000 kDa, which corresponds to a set of oligomers from dimers to heptamers, with a predominance of trimers.
In this case, two fundamental results of extremely significant practical importance should be noted:
the threshold nature of the relationship correlating the degree of polymer destruction with the duration of the beam-plasma exposure, which allows to optimize the treating process and to eliminate unproductive energy costs;
the probability of appearance of the by-side water-insoluble compounds in the composition of the products of beam-plasma modification of chitozans as an effect occuring due to reverse polymerization of the resulting low molecular weight products when the processing conditions are not optimal.
A study of the properties of chitooligosaccharides obtained by EPP-stimulated hydrolysis of chitosans showed that these products have antibacterial (both in the case of dormant and in the case of multiplying gram-positive and gram-negative microorganisms) and fungicidal (as was shown on a number of yeast-like and mycelial fungi) effects [6].
Modification of (bio)polymers in HP
The possibilities of using HP and hybrid plasma-chemical reactors in the field of biology and medicine were demonstrated in the experiments with thin films obtained from the natural polymer chitozan and plates made from PMMA.
The water contact angle of the HP-modified chitozan films decreased significantly compared with the initial samples (from 96.95 ± 1.89° in the initial films to almost zero when processed in oxygen-containing plasma-forming media), which indicates a radical improvement in hydrophilic properties . This effect was stable for 2 months after treatment.
The values of the wetting contact angle (θW) and diiodomethane (θDM), as well as the total surface energy γtot and its polar γpol (water) and dispersion γdisp (diiodomethane) components for PMMA processed in HP of various plasma gases are given in Table 2. A significant decrease in θW was observed after 2 min of treatment. The free surface energy γtot increased with the duration of the plasma chemical exposure.
The hydrophilicity of PMMA samples treated in HP was compared with the wettability of this polymer after modification in EPP and RF discharge plasma. After 2 min of treatment in the RF high-frequency discharge of oxygen, the edge angle θW of PMMA decreased from 76.00 ± 3.23˚ to 49.00 ± 0.08˚, and with an increase in the modification time to 10 min – to 31.40 ± 0.37˚. Thus, θW values close to θW of PMMA modified in HP were achieved. However, degradation of the hydrophilic properties of the polymer surface after treatment in the HF-discharge occurred much faster than in case of using HP. A similar effect, the explanation of which is given in [5], was also observed in the study of the hydrophilic properties of plasma-chemically modified chitozan films. Processing in oxygen EPP for 10 min reduced θW to 56.7 ± 0.06˚, and degradation of wettability occurred after a week of storage. Thus, HP is the most effective way to control the hydrophilic-hydrophobic properties of the surface in comparison with the EPP and HF-discharge plasma.
The hydrophilicity of a surface is determined by both its relief and chemical composition [7]. Fig.2 shows the surface images of PMMA obtained using atomic force microscopy (AFM), which shows an increase in roughness after modification in hybrid plasma and smoothing of the relief as a result of the action of EPP. It is likely that heavy neutral oxygen particles produced in high concentrations during plasma-chemical reactions in GPs lead to active chemical and mechanical etching of the polymer surface. In contrast, EP high-energy electrons are capable, in certain doses, to facilitate crosslinking of polymer molecules, which causes smoothing of its roughness [8, 9].
With the aid of IR spectroscopy, it was shown that the surface of chitozan films and PMMA plates is formed of polar chemically active oxygen-containing groups (–OH, –OH and –C = O, –COOH). The increase in oxygen content in the LTP-treated samples was also confirmed by XPS data.
Biocompatibility of PMMA modified in oxygen HP was evaluated in experiments on fibroblast cultures in the MTT test, which reflects the ability of cells to grow and reproduce. The results were compared with the bioactivity of PMMA samples treated in EPP and HF-discharge. It was found that the most intensive growth of fibroblasts occurs on the surface of PMMA, modified in HP oxygen. Thus, the modification of the polymer in HP adds the polymer surface the greatest biocompatibility, which is probably due to the large number of polar groups on its surface and, as a consequence, greater hydrophilicity.
The prospects of the developed method for the modification of polymeric materials in practical clinical dentistry were demonstrated by supervising a patient who underwent surgical treatment and gamma therapy for cancer of the buccal mucosa. During the year of monitoring a patient with a tendency to form lichen planus on the background of reduced local and general immunity, a stable remission was achieved, and no pathological elements and new formations appeared in the oral mucosa. The patient herself did not complain of discomfort when wearing the prosthesis and noted an improvement in the quality of life.
CONCLUSIONS
Thus, EPP and HP are the effective and promising tool for modifying natural and synthetic polymers in order to obtain bioactive low molecular weight compounds and materials with improved biocompatibility with body tissues.
Modification products obtained as a result of processing in EPG and HP are potentially interesting for use in practical dentistry, maxillofacial surgery, as well as in pharmaceutics and the cosmetic industry.
ACKNOWLEGEMENTS
The authors are grateful to the candidate of technical sciences, docent, senior research associate of the CCT "Arctic", Northern (Arctic) Federal University (Arkhangelsk) D.G.Chukhchin for his help and performance of the atomic force microscopy of samples. ■
In the late 20th century various types of low-temperature plasma (LTP) began to find their numerous practical applications not only for production technologies [1], but also in the modern innovative fields, such as plasmatic medicine. According to VDI Technologiezentrum GmbH, Evaluierung Plasmatechnik (Dusseldorf, Germany), this field will be the most perspective one for the development and implementation of plasma-stimulated processes. Besides, the medicine applications of LTP include the polymer material surface modification in order to increase the biocompatibility with the human body tissues [2]. By changing the surface charge, processing of polymers by LTP increases absorption of adhesive proteins and immobilizes various biologically active molecules at [3]. LTP surface modification affects its morphology and creates various nanoscale structures which serve as the binding points for specific cell membrane proteins thereby improving the cell adhesion to the surface being treated [3].
The low-pressure LTP is the most investigated one from the viewpoint of the polymer materials and articles modification mechanisms and it has been mostly used in the variant of LTP of gas discharges in various frequency regions [4]. Known are the disadvantages of gas discharge reactors which limits their usage for this purpose. First of all, these are the difficulties associated with the formation of huge homogeneous reaction volumes which leads to a significant heating of the plasma-forming medium and objects placed in it. Technical problems connected with the chemical resistance of electrodes may appear because of stability losses in the reagent volume at intensive purging of gas by charging. It is necessary to apply other types of LTP of low pressure, such as e-beam plasma (EBP) and hybrid plasma (HP) in order to overcome these disadvantages. E-beam plasma is generated at the injection of the electron beam into the dense gaseous medium. Geometry, composition, temperature of particles and other characteristics of EBP depend of electron energy Eb, beam power Nb (Nb<1 kWt), pressure Pm (0,1 <Pm<10 kPa), temperature Tm of plasma-forming media and its phase and chemical compositions. It is possible to control the EBP generator modes by changing of EP power at constant Pm (or, in opposite, by changing the gas pressure at stable Nb) and carry out different non-equilibrium plasma-chemical reactions. Also, the sample temperature during the treatment may be fixed at the necessary level, and temperature drop to the room value does not influence high chemical activity of plasma.
Hybrid plasma (HP) is generated when two or more ionizers act together or in sequence on the plasma-forming medium. In this study an e-beam was applied as a main ionizer for the formation of sufficiently large volumes of plasma, and an additional ionization source was used as a HF-discharge at frequency of 13.56 MHz. HP has the very important additional advantages like a possibility of practically inertia-free control of properties and geometry of reaction volume with the help of EP, and the higher stability of this volume to contraction when the pressure grows. Active ions are added to the chemically active stimulated particles in gas-discharge plasma at sufficient concentrations due to the e-beam action in gas. Also, creation of new particles in HP is possible only at combined action of HF-discharge and EP.
The work was aimed at:
Development of technological approaches to the EPP and HP generation and processing of polymer materials of synthetic and natural origin;
Description of the surface morphology, chemical structure and biological properties of plasma-chemically modified (bio)polymers.
METHODS OF RESEARCH
Generation of e-beam and hybrid plasma
Fig.1 presents the general diagram of a plasma-chemical reactor for the EPP and HP generation. A plasma cloud 12 is formed in the reaction camera 11 placed inside of the working chamber 10. Working chamber has a plug 3 for pumping out and plug 4 for feeding the basic plasma-forming gas at a flow rate Gg2. Power from Genesis GHW-12 HF-generator (MKS Instruments, Great Britain, frequency 13.6 MHz) is applied to an active electrode 5 hrough the sealed inlet 8. If necessary, additional gas at flow rate of Gg1 may be supplied through the electrode 5 made of a metal porous pipe. The EP 2 is inserted through the open end of the working chamber. An HF-generator is not used for EPP generation. An electron gun 1 generates an e-beam inside the high-vacuum chamber 14 (∼10–5 Тorr) which is transported into the working chamber through the special outlet device 13. An outlet device is combined with an electrical magnetic deflection system which can decline an EP axis in two orthogonal directions and form a raster.
Stationary EPP and HP clouds and plasma flows may be formed inside the developed reactor using nozzle devices of different design. By using different types of nozzles it is possible to disperse liquids and powders in a plasma cloud or flow and form the working volume in a form of an aerosol. Various types of beam-plasma reactors are described in detail in [5].
The working chamber is equipped with special holders 12 to process small samples. In the experiments considered in this work, were used synthetic organic (polymethyl methacrylate (PMMA), polyethylene terephthalate, silicone rubbers) and natural (chitin, chitosan, cellulose, alginates) polymers. The listed materials are widely used in medical practice, and improving of their biomedical characteristics is an urgent task.
The working chamber is rotated using drive 9 in order to process the powdered materials. In this case the inner accessories of the working chamber are replaced with special ribs to mix powder during treatment in order to provide the homogeneity of material processing across the chamber volume. As will be shown below, it is possible in this way to obtain bioactive compounds with valuable properties from the point of view of their use in pharmaceutics.
The reactor was tested on thin films (thickness of 8±0,5 µm) and powders (diameter of 50 µm) of chitozan, as well as plates made of dental plastic based on PMMA (Villacryl H Plus, Zhermak, Italy). Table 1 shows typical experimental conditions.
RESULTS
EPP stimulated destruction of chitozan powders
Chitozan powders were processed in EPP during for 5 minutes. Plasma-stimulated hydrolysis of polysaccharides led to their rapid depolymerization with the formation of water-soluble oligochitosans mixture with a yield of 85%. The weight average molecular weights of the products of plasmochemical modification of chitozans obtained under optimal conditions varied within 570–2000 kDa, which corresponds to a set of oligomers from dimers to heptamers, with a predominance of trimers.
In this case, two fundamental results of extremely significant practical importance should be noted:
the threshold nature of the relationship correlating the degree of polymer destruction with the duration of the beam-plasma exposure, which allows to optimize the treating process and to eliminate unproductive energy costs;
the probability of appearance of the by-side water-insoluble compounds in the composition of the products of beam-plasma modification of chitozans as an effect occuring due to reverse polymerization of the resulting low molecular weight products when the processing conditions are not optimal.
A study of the properties of chitooligosaccharides obtained by EPP-stimulated hydrolysis of chitosans showed that these products have antibacterial (both in the case of dormant and in the case of multiplying gram-positive and gram-negative microorganisms) and fungicidal (as was shown on a number of yeast-like and mycelial fungi) effects [6].
Modification of (bio)polymers in HP
The possibilities of using HP and hybrid plasma-chemical reactors in the field of biology and medicine were demonstrated in the experiments with thin films obtained from the natural polymer chitozan and plates made from PMMA.
The water contact angle of the HP-modified chitozan films decreased significantly compared with the initial samples (from 96.95 ± 1.89° in the initial films to almost zero when processed in oxygen-containing plasma-forming media), which indicates a radical improvement in hydrophilic properties . This effect was stable for 2 months after treatment.
The values of the wetting contact angle (θW) and diiodomethane (θDM), as well as the total surface energy γtot and its polar γpol (water) and dispersion γdisp (diiodomethane) components for PMMA processed in HP of various plasma gases are given in Table 2. A significant decrease in θW was observed after 2 min of treatment. The free surface energy γtot increased with the duration of the plasma chemical exposure.
The hydrophilicity of PMMA samples treated in HP was compared with the wettability of this polymer after modification in EPP and RF discharge plasma. After 2 min of treatment in the RF high-frequency discharge of oxygen, the edge angle θW of PMMA decreased from 76.00 ± 3.23˚ to 49.00 ± 0.08˚, and with an increase in the modification time to 10 min – to 31.40 ± 0.37˚. Thus, θW values close to θW of PMMA modified in HP were achieved. However, degradation of the hydrophilic properties of the polymer surface after treatment in the HF-discharge occurred much faster than in case of using HP. A similar effect, the explanation of which is given in [5], was also observed in the study of the hydrophilic properties of plasma-chemically modified chitozan films. Processing in oxygen EPP for 10 min reduced θW to 56.7 ± 0.06˚, and degradation of wettability occurred after a week of storage. Thus, HP is the most effective way to control the hydrophilic-hydrophobic properties of the surface in comparison with the EPP and HF-discharge plasma.
The hydrophilicity of a surface is determined by both its relief and chemical composition [7]. Fig.2 shows the surface images of PMMA obtained using atomic force microscopy (AFM), which shows an increase in roughness after modification in hybrid plasma and smoothing of the relief as a result of the action of EPP. It is likely that heavy neutral oxygen particles produced in high concentrations during plasma-chemical reactions in GPs lead to active chemical and mechanical etching of the polymer surface. In contrast, EP high-energy electrons are capable, in certain doses, to facilitate crosslinking of polymer molecules, which causes smoothing of its roughness [8, 9].
With the aid of IR spectroscopy, it was shown that the surface of chitozan films and PMMA plates is formed of polar chemically active oxygen-containing groups (–OH, –OH and –C = O, –COOH). The increase in oxygen content in the LTP-treated samples was also confirmed by XPS data.
Biocompatibility of PMMA modified in oxygen HP was evaluated in experiments on fibroblast cultures in the MTT test, which reflects the ability of cells to grow and reproduce. The results were compared with the bioactivity of PMMA samples treated in EPP and HF-discharge. It was found that the most intensive growth of fibroblasts occurs on the surface of PMMA, modified in HP oxygen. Thus, the modification of the polymer in HP adds the polymer surface the greatest biocompatibility, which is probably due to the large number of polar groups on its surface and, as a consequence, greater hydrophilicity.
The prospects of the developed method for the modification of polymeric materials in practical clinical dentistry were demonstrated by supervising a patient who underwent surgical treatment and gamma therapy for cancer of the buccal mucosa. During the year of monitoring a patient with a tendency to form lichen planus on the background of reduced local and general immunity, a stable remission was achieved, and no pathological elements and new formations appeared in the oral mucosa. The patient herself did not complain of discomfort when wearing the prosthesis and noted an improvement in the quality of life.
CONCLUSIONS
Thus, EPP and HP are the effective and promising tool for modifying natural and synthetic polymers in order to obtain bioactive low molecular weight compounds and materials with improved biocompatibility with body tissues.
Modification products obtained as a result of processing in EPG and HP are potentially interesting for use in practical dentistry, maxillofacial surgery, as well as in pharmaceutics and the cosmetic industry.
ACKNOWLEGEMENTS
The authors are grateful to the candidate of technical sciences, docent, senior research associate of the CCT "Arctic", Northern (Arctic) Federal University (Arkhangelsk) D.G.Chukhchin for his help and performance of the atomic force microscopy of samples. ■
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