**6. Original substances and methods used to investigate products of their EBP-modification**

The plasmachemical modification of the following natural materials was studied in detail:


The original powders of polysaccharides were additionally ground in the laboratory mill before treatment. Average final sizes of the powder particles were within the range 10-40 m. The proteins were treated both in forms of solid powders (BSA and FM) and thing films (BSA). To form a film BSA was dissolved in distilled water and than water evaporation under vacuum was performed. The thickness of the formed film was 1 μm.

The following properties of EBP-modified products were of particular interest:


292 Practical Applications in Biomedical Engineering

for each gas.

function of *x*- and *y*-distances.

some threshold value.

in the EBP of argon excited by the EB at two different values of the beam current *Ib* = 15 and *Ib* =7,5 mA (curves *1* and *2* respectively) are presented in Figure 4 as the example of numerical simulations. In this figure all concentration values *n* are divided by the maximum value of the particles concentration *n*max for *Ib* = 15 mA. The Figure 3 shows that there are optimal values of *Pm* for given *Ib* and *U*, these optimal values being individual

**Figure 3.** a.The incident radiation power of fast electrons on 1 cm2 of the sample surface calculated as a function of *x*- and *y*-distances from the sample center: the treatment in the helium EBP at pressure 40 Torr. b.The incident radiation power of fast electrons on 1 cm2 of the sample surface calculated as a

If optimal values of *Pm* are found the total dose of the powder particles irradiation can be calculated. In particular, the treatment durations required to irradiate the powder by equal dozes at variable gas pressures were calculated and relevant experiments in various gases were planned and carried out. Experiments of this kind showed the modification effect in the organic materials under consideration to appear when the accumulated doze exceeded

The material temperature can be controlled by varying the gas pressure value during the treatment. The lower the gas pressure - the higher the sample temperature since the scattering and absorption of the EB in gaseous media are more intensive at higher pressures than at lower ones. If higher sample temperature is required to obtain the desirable modification effect the gas pressure should be reduced. For this reason the polysaccharides were treated at lower pressures than the proteins because the higher temperature (about 400 K) was optimal for effective polysaccharides modification. On the contrary, when the proteins were treated in helium the higher pressures (*Pm* 40 Torr) were required to prevent the sample from overheating. It was because of the restrictions on the material temperature that the working pressures in the reaction chamber of the EBPR sometimes differed from

their optimal values found as the maximum of the functions *n* = *n*(*Pm*, *Ib*).



*Solubility measurements.* 100±0,1 mg of the preliminary dried sample (*ms*) were placed into a tube and 1,5 ml of distilled water were added to the sample. The resulting mixture was incubated for 24 h at room temperature under periodic mixing. After the incubation the mixture was centrifuged for 5 min and 1 ml of centrifugate was taken and dried. The mass of the dry residue (*mdr*) was measured with an accuracy ±0,1 mg. The sample solubility was calculated as the (*mdr/ ms*)100% ratio.

Bio-Medical Applications of the Electron-Beam Plasma 295

*Biological activity of the EBP-treated alanine derivative and fibrin-monomer.* The EBP-modification products of fibrin-monomer were tested as the platelet aggregation inhibitors [15]. The platelet aggregation *A* (%) was measured by the turbodimetric method [16] and *A* was defined as the ratio of the light transparency of the platelet suspension after ceasing the aggregation process to the initial value of the light transparency. The aggregation was monitored by the aggregometer *Chronolog Corporation* (USA), adenosine diphosphoric acid (ADP, final concentration 110-5 M; *Boechringer Mannheim*, Germany) being used as an aggregation agent.

Some natural amino acids with artificially inserted pirozolidine cycles into their structures were used as original substances and the products of their modification in the EBP of helium and water vapor were tested as inhibitors of the human platelet aggregation. Preliminary analysis showed the substances of this class to be promising as active agents for medical therapy of acute coronary events, and cardiovascular diseases that remain the leading cause of mortality. Their advantages are due to selectivity of the pharmacology action and limited

The powder samples (≈ 50 mg in mass) of the original derivative of alanine were treated in the EBP of water vapor at pressure *Pm* ≈ 9 Torr for variable time duration *τ* = 45-300 s. The typical EB power was *Nb* ≈ 0,1 kW, the sample temperature *Ts* under the treatment could be

The untreated compound was not dissolvable in distilled water at room temperature and the water heating up to 90 °C followed by cooling to 25 °C was required to carry out the control experiments and to study its effect on human platelet aggregation *in vitro*. The treated substance became partially water-soluble at room temperature and the solution at maximum

The untreated derivative decreased human platelet aggregation only to 46±2% with respect to control (56±2%). The water-soluble products of plasma treatment reduced the aggregation degree up to ≈ 30 %, i.e. being treated by the EBP for 5 min the studied substance reduced

> = 90 s, *T*s = 38 C

562% 462% 413% 413% 343% 323% 313%

The effect of the treatment duration on their anti-aggregation activity increased as the treatment prolonged, the anti-aggregation activity rising sharply at 90 < *τ* < 180 s. At shorter durations *τ* < *τ0* the plasma did not modify the original substance and the longer treatment *τ*

**Table 1.** The effect of the plasma modification in the EBP of water vapor on the anti-aggregation activity of the tested alanine derivative (*in vitro*): the aggregation degree as a function of the treatment

and temperature of the substance *T*s under the treatment procedure

ADP + treated amino acid

 = 180 s, *T*s = 38 C

 = 180 s, *T*s = 55 C

 = 300 s, *T*s = 55 C

concentration was added to the platelet suspension to measure the aggregation degree.

the platelet aggregation activity by approximately 45 % (Table 1).

 = 45 s, *T*s = 38 C

**7. The amino-acids treatment in the Electron-Beam Plasma** 

side effects.

ADP

duration 

varied within the range 30-110 °C.

ADP + untreated amino acid

*Molecular mass characterization.* To characterize the molecular masses of the EBP-treatment products the exclusion chromatography was applied. The chromatograph *Staier* (Russia) and the chromatographic column *Phenomenex* BioSep-Sec-S-3000 (USA) with the efficiency of 30000 theoretical plates were used. The analysis conditions were as follows: the elutriating agent – 0,1 M phosphate buffer (pH 6,86) containing 0,05% NaN3; the elution rate - 1 ml/min; temperature - 30оС; UV-detector with the wavelength 280 nm.

The effects of the EBP-treatment on proteins molecular mass and structure were detected also by means of the UV- and IR-spectroscopy, ion-exchange chromatography, immunoelectrophoresis and PAGE-electrophoresis as well [12].

*UV-spectroscopy*. The measurements were performed with the spectrometer *Shimadzu UV-3600* (Japan). The IR-spectra were registered within wavelength λ = 226-418 nm.

*IR-spectroscopy*. The measurements were performed with the IR-spectrometer *Portmann Instruments AG* (Switzerland) equipped with the ZnSe crystal. The IR-spectra were registered within wave numbers *ν* = 500-3600 cm-1. To improve the spectral resolution Fourier analysis was performed.

*Ion-exachange chromatography.* The ion-exchange chromatography (with the preliminary acid hydrolysis of the protein) was performed to reveal the changes in the amino acid composition of proteins due to the EBP-modification. The analyzer *AAA-339 M* (Hungary) was used. To quantitatively analyze the sulfur-containing amino acids (cystine and methionine) the biomaterial was treated with the performic acid before the hydrolysis procedure. Totally 17 basic amino acids contents were measured. The accuracy of the mesuarment was 10%.

*Immunoelectrophoresis.* The electrophoresis was performed in the 1,4% agar gel [13]. The commercial specific antiserum to human fibrinogen was used to characterize and compare antigenic structure of the FM before and after plasmachemical treatment.

PAGE-*electrophoresis* was performed according to Laemmli U.K. [14].

*Biological activity of the EBP-produced low molecular weight chitosans (LMCW).* The inhibition of the bacteria growth *in vitro* was measured to quantitatively characterize the bioactivity of LMCW obtained by the plasma treatment, gram-positive (*S. aureus*), gram-negative (*E. coli, Ps. aeruginosa*) microorganisms and yeast-like fungi (*C. albicans*) being used in these experiments.

*Biological activity of the EBP-treated alanine derivative and fibrin-monomer.* The EBP-modification products of fibrin-monomer were tested as the platelet aggregation inhibitors [15]. The platelet aggregation *A* (%) was measured by the turbodimetric method [16] and *A* was defined as the ratio of the light transparency of the platelet suspension after ceasing the aggregation process to the initial value of the light transparency. The aggregation was monitored by the aggregometer *Chronolog Corporation* (USA), adenosine diphosphoric acid (ADP, final concentration 110-5 M; *Boechringer Mannheim*, Germany) being used as an aggregation agent.
