**9. Discussions and conclusions**

302 Practical Applications in Biomedical Engineering

samples.

increase the yield of low molecular weight products with the comparison to the powdered

The PAGE of the BSA samples modified as films in EBP revealed a dramatic decrease in the intensity of the albumin band and the formation of a number of low molecular peptides, of which most have a molecular mass below 14 kDa. The exclusion chromatograms also displayed numerous peaks corresponding to low molecular mass products with elution times of 11,8; 15,1; and 17 min (Figure 12). The concentration of the low molecular mass compounds depended on the plasma treatment time and the nature of the plasma gas: as the treatment time increased, their concentration grew, and BSA degradation being more significant in the water vapor EBP. A decrease in the content of almost all amino acids was observed. The most significant decrease (by 2–3,5 times with respect to untreated BSA) was

in the case of lysine, aspartic and glutamic acids, tyrosine, and cystine (Table 3).

**Figure 12.** The exclusion chromatogram of BSA thing film treated in EBP of oxygen (τ = 10 min)


The following factors influence the biomaterial placed into the plasma cloud:


Bio-Medical Applications of the Electron-Beam Plasma 305

**10. The polysaccharides treatment in the Electron-Beam Plasma** 

their ends if the original materials have been treated in O2 or H2O plasmas.

**Figure 14.** The modification effect in studied polysaccharides as a function of the treatment duration in

The products of the EBP modification of all the substances mentioned above turned out to be bioactive. For instance, modified cellulose and peat are effective substratesfor microorganisms and fungi: the yield of the yeastrel on the plasmachemically treated peat reached 0,14 kg per 1 kg of the peat, whereas the productivity of the untreated substrate was

the EBP of water vapor

only 0,053 kg.

The plasmachemical modifications of cellulose materials, peat, and chitosan in EBP of O2, He, and H2O, are studied. The yield (*S*, %) and the average molecular mass of water-soluble products extracted from the solution were taken as the quantitative criteria for the modification. Note, typical content of water-soluble substances in untreated cellulose materials does not exceed 1–2%.The molecular mass of the cellulose may reach several million. The water-soluble products yield depends upon the processing conditions and has a maximum value *S*max for each plasma-generating gas: for oxygen plasma *S*max ≈ 45%, for plasma of water vapor *S*max ≈ 56%, which is reached during certain time periods of the treatment *τ*0. At first the dependence *S(τ)* increases smoothly, then - steeply in the vicinity of *τ*0 after which the yield of the water-soluble products does not change whatever the time of the treatment could be (Figure 14). The NMR- and IR-spectroscopy analyses showed the final water-soluble products of the cellulose modification to be β-(C1→C4)-tetrasaccharide, the molecule structure and chemical bonds of the products being identified. In particular, low-molecular products (*M* ≈250–800 Da) have attached carbonyl and/or carboxyl groups on


Some new chemical groups might be formed in the molecule structure during the treatment in the EBP. For instance, the ROS generated in the EBP of the water vapor or oxygen can react with the amino acid residues resulting in their oxidation and the formation of various keto-products. The formation of such oxidized substances (e.g. pyruvic acid, kynurenine, glutamic semialdehyde, disulfides, cysteic acid [21]) is possible reason for the changes in the UV-spectra and amino acid composition (see Figure 5 and Tables 2 and 3).

3. the EBPR with the aerosol reaction zone was successfully used to produce compounds with new pharmacological activity and therapeutic effect, e.g. for the production of effective platelet aggregation antagonists. Also the destruction of peptides in the EBP can be used for the effective sterelization of medical instruments, inactivation of pathogenic bateria and prion proteins.

**Figure 13.** The scheme of peptide bound degradation and cross-links formation under ROS action in the EBP of the water vapor or oxygen

#### **10. The polysaccharides treatment in the Electron-Beam Plasma**

304 Practical Applications in Biomedical Engineering

Figure 5 and Tables 2 and 3).

the EBP of the water vapor or oxygen

Peptide bond cleavage

pathogenic bateria and prion proteins.

NH CH R

OH

•

NH C R

O

•

powder;

the fast electrons of the partially degraded EB that bombard the sample;

the EBP-radiation, especially UV one and X-ray (bremsstrahlung);

the secondary electrons of moderate energy produced in the EBP can also act on the

 chemically active heavy particles of the EBP (excited molecules and atoms, ions, radicals). This factor is likely plays the pivotal role for the low molecular peptides formation and for the production of bioactive products [19]. Due to the action of the reactive oxygen species (ROS) the destruction of the peptide bonds occures. The Figure 13 illustrates the possible mechanism of peptide bonds cleavage [20]. The formation of intermolecular cross-links can also occure under the ROS influence and the production of some high molecular weight substances was detected by the exclusion chromatography after the EBP-treament of fibrin-monomer powder (see Figure 8). Some new chemical groups might be formed in the molecule structure during the treatment in the EBP. For instance, the ROS generated in the EBP of the water vapor or oxygen can react with the amino acid residues resulting in their oxidation and the formation of various keto-products. The formation of such oxidized substances (e.g. pyruvic acid, kynurenine, glutamic semialdehyde, disulfides, cysteic acid [21]) is possible reason for the changes in the UV-spectra and amino acid composition (see

3. the EBPR with the aerosol reaction zone was successfully used to produce compounds with new pharmacological activity and therapeutic effect, e.g. for the production of effective platelet aggregation antagonists. Also the destruction of peptides in the EBP can be used for the effective sterelization of medical instruments, inactivation of

~ ~ ~~

CO NH CO

H2O

R

•

NH C R

NH C R

HO2

•

CO

•

O2

Intermolecular cross-links formation

CO O OH

O2

HO2

•

O O

~ ~

**Figure 13.** The scheme of peptide bound degradation and cross-links formation under ROS action in

O2

~ ~ ~ ~

CO

The plasmachemical modifications of cellulose materials, peat, and chitosan in EBP of O2, He, and H2O, are studied. The yield (*S*, %) and the average molecular mass of water-soluble products extracted from the solution were taken as the quantitative criteria for the modification. Note, typical content of water-soluble substances in untreated cellulose materials does not exceed 1–2%.The molecular mass of the cellulose may reach several million. The water-soluble products yield depends upon the processing conditions and has a maximum value *S*max for each plasma-generating gas: for oxygen plasma *S*max ≈ 45%, for plasma of water vapor *S*max ≈ 56%, which is reached during certain time periods of the treatment *τ*0. At first the dependence *S(τ)* increases smoothly, then - steeply in the vicinity of *τ*0 after which the yield of the water-soluble products does not change whatever the time of the treatment could be (Figure 14). The NMR- and IR-spectroscopy analyses showed the final water-soluble products of the cellulose modification to be β-(C1→C4)-tetrasaccharide, the molecule structure and chemical bonds of the products being identified. In particular, low-molecular products (*M* ≈250–800 Da) have attached carbonyl and/or carboxyl groups on their ends if the original materials have been treated in O2 or H2O plasmas.

**Figure 14.** The modification effect in studied polysaccharides as a function of the treatment duration in the EBP of water vapor

The products of the EBP modification of all the substances mentioned above turned out to be bioactive. For instance, modified cellulose and peat are effective substratesfor microorganisms and fungi: the yield of the yeastrel on the plasmachemically treated peat reached 0,14 kg per 1 kg of the peat, whereas the productivity of the untreated substrate was only 0,053 kg.

The original chitosan was not water-soluble while its EBP-treament products became soluble and the effect increased with the prolongation of *τ*. The maximum yield of watersoluble substances was obtained after 10 min and the solubility of these products was up to 95% up at room temperature.

Bio-Medical Applications of the Electron-Beam Plasma 307

EBP-treated chitosan concentration, μg/ml Control

1000 500 250 125


n

NH

.

n

O

NH2

 The possibility of the EBP-stimulated hydrolysis of native polysaccharides and formation of water-soluble low molecular weight bioactive products was proved experimentally. The yield of the water-soluble products depends on the doze of the irradiation by the plasma particles. Other things being equal, the yield of the bioactive products began to increase abruptly at particular duration of treatment 0. At shorter duration < 0 the plasma didn't modify the original substance and the longer treatment

**Figure 16.** The scheme of chitosan degradation under hydroxyl radical action in the EBP of the water

m

O

+ H2O

m

OH

.

HOH2C

NHCOCH <sup>3</sup> <sup>O</sup> HO

O

O

n

O

+ + OH

NHCOCH <sup>3</sup>

n

.

O

HOH2C

O

HO

+ H2O

O

O

HO O HOH2C

O

HO O HOH2C

O

**Table 4.** The microorganism growth under EBP-treated chitosan

O

HOH2C

HOH2C

NHCOCH <sup>3</sup> <sup>O</sup> HO

NHCOCH <sup>3</sup> <sup>O</sup> HO

m

m

NH

**11. Discussions and conclusions** 

.

O

> 0 resulted in an insignificant additional effect.

NH2

*E. coli* ---- ± ± + + *Ps. aeruginosa* + + + + + *S. aureus* ---- ± + + + *C. albicans* ---- ± + + +

Test microorganism

reference sample

HO O HOH2C

HO O HOH2C

vapor [22]

The exclusion chromatography of the EBP-treated chitosan revealed the formation of a number of LMWC with molecular weight varied from 18 kDa to monomeric fragments (Figure 15). The majority of products formed with the EBP-treatment were oligosaccharides with the molecular mass 1 kDa (the elution time 11,3 min).

**Figure 15.** The exclusion chromatogram of chitosan treated in the EBP of water vapor (τ = 10 min)

The degradation of the original polymer is due to the action of free radicals formed in the EBP. Active oxygen particles (O, O•, singlet oxygen) that are produced in plasmachemical processes and the products of the water plasmolisys (e.g. OH•) are likely to be of the most importance. These chemically active particles break the β-1,4 glycosidic bound and decrease the chitosan molecular weight Figure 16 illustrates the possible degradation mechanism [22].

To quantitatively characterize the bioactivity of the EBPproduced LMCW the bacteria growth *in vitro* was studied. The LMCW produced by the EBP-treatment in water vapor at concentration 1000 μg/ml were found to completely suppress the multiplication of colon bacillus, aurococcus and yeast-like fungi. At lower doses the EBP-treatment products were also active and strongly inhibited the microorganism multiplication (Table 4).

.



**Table 4.** The microorganism growth under EBP-treated chitosan

306 Practical Applications in Biomedical Engineering

95% up at room temperature.

mechanism [22].

with the molecular mass 1 kDa (the elution time 11,3 min).

The original chitosan was not water-soluble while its EBP-treament products became soluble and the effect increased with the prolongation of *τ*. The maximum yield of watersoluble substances was obtained after 10 min and the solubility of these products was up to

The exclusion chromatography of the EBP-treated chitosan revealed the formation of a number of LMWC with molecular weight varied from 18 kDa to monomeric fragments (Figure 15). The majority of products formed with the EBP-treatment were oligosaccharides

**Figure 15.** The exclusion chromatogram of chitosan treated in the EBP of water vapor (τ = 10 min)

The degradation of the original polymer is due to the action of free radicals formed in the EBP. Active oxygen particles (O, O•, singlet oxygen) that are produced in plasmachemical processes and the products of the water plasmolisys (e.g. OH•) are likely to be of the most importance. These chemically active particles break the β-1,4 glycosidic bound and decrease the chitosan molecular weight Figure 16 illustrates the possible degradation

To quantitatively characterize the bioactivity of the EBPproduced LMCW the bacteria growth *in vitro* was studied. The LMCW produced by the EBP-treatment in water vapor at concentration 1000 μg/ml were found to completely suppress the multiplication of colon bacillus, aurococcus and yeast-like fungi. At lower doses the EBP-treatment products were

also active and strongly inhibited the microorganism multiplication (Table 4).

**Figure 16.** The scheme of chitosan degradation under hydroxyl radical action in the EBP of the water vapor [22]

#### **11. Discussions and conclusions**

 The possibility of the EBP-stimulated hydrolysis of native polysaccharides and formation of water-soluble low molecular weight bioactive products was proved experimentally. The yield of the water-soluble products depends on the doze of the irradiation by the plasma particles. Other things being equal, the yield of the bioactive products began to increase abruptly at particular duration of treatment 0. At shorter duration < 0 the plasma didn't modify the original substance and the longer treatment > 0 resulted in an insignificant additional effect.

 The 95% yield of low molecular weight EBP-treatment products was attained by optimizing the treatment procedure. The high yields of low molecular weight water soluble products are obtained at treatment time ~10 min whereas the traditional chitosan hydrolysis usually takes several days. The hazardous by-products and toxic wastes are not generated during the EBP-treatment.

Bio-Medical Applications of the Electron-Beam Plasma 309

*Department of General Chemistry, Moscow Institute of Physics and Technology, Dolgoprudny,* 

[1] Solum N.O., Rigollot C., Budzynski A.Z., Marder V.J. A quantitative evaluation of the inhibition of platelet aggregation by low molecular weight degradation products of

[2] Lord S.T. Fibrinogen and fibrin: scaffold proteins in hemostasis. Curr. Opin. Hematol.

[3] Ray S.D. Potential aspects of chitosan as pharmaceutical excipient. Acta Pol. Pharm.

[4] Laurienzo P. Marine polysaccharides in pharmaceutical applications: an overview. Mar.

[5] Aam B.B., Heggset E.B., Norberg A.L., Sorlie M., Varum K.M., Eijsink V.G.H. Production of chitooligosaccharides and their potential applications in medicine. Mar.

[6] Vasiliev M.N. In: Fortov V. E. (ed.) Encyclopedia of the Low-Temperature Plasma.

[7] Vasilieva T., Lysenko S. Factors responsible for biomaterials modification in the

[8] Vasiliev M., Vasilieva T. Electron-beam plasma in the production of bioactive agents

[9] Henderson R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. *1995;* 28(171)

[10] Glaeser R.M. Review: electron crystallography: present excitement, a nod to the past,

[11] Aleksandrov N. L., Vasil'ev M. N., Lysenko S. L., Makhir A. Kh. Experimental and Theoretical Study of a Quasi-Steady Electron-Beam Plasma in Hot Argon. Plasma

[12] Vasilieva T. A beam-plasma source for protein modification technology. IEEE Transac.

[13] Axelsen M., Kroll B., Weeke B. A manual of quantitative immuno-electrophoresis.

[14] Laemmli U.K. Cleavage of structural proteins during the assembly of the head of

[15] Vasilieva T. The controllable production of peptides inhibiting the platelet aggregation by the electron-beam plasma technologies. In: Aimoto S. and Ono S. (ed.) Peptide

electron-beam plasma. J. Phys.: Conf. Ser. 2007; 63(1) 012033.

and drugs. J. Phys.: Conf. Ser. 2006; 44(140) 140-145.

anticipating the future. J. Struct. Biol. 1999; 128(3) 3-14.

bacteriophage T4. Nature 1970; 227(5259) 680-685

Science 2007. The Japanese Peptide Society; 2008. p.35-38.

fibrinogen. Brit. J. Haematol. 1973; 24(4) 419-434.

**Author details** 

*Moscow region, Russia* 

2007; 14(3) 236-241.

2011; 68(5) 619-622.

Drugs 2010; 8(9) 2435-2465.

Drugs 2010; 8(5) 1482-1517.

Nauka: Мoscow; 2001. V. XI p436-44.

Physics Reports 2005; 31(5) 425-435.

Plasma Sci. 2010; 38(8) 1903-1907

Mehtods and applications; 1977.

**13. References** 

171-193.

T. Vasilieva

