The first steps on ways to opening the mechanisms cell regulation by nanotechnology preparations

Andrey N. Belousov 

 

      

1.    Introduction 

1.1 The history of nanotechnology

The history of nanotechnology has begun not so long ago, only in 1959 when a Nobelist Richard Freyman delivered literally the following: “As far as I know not a single physical or chemical law doesn’t prevent us from changing relative position of atoms” (Drexler, 1994.).

It has been almost 45 years since the speech of Richard Freyman in front of the Nobel Committee and since then the humanity has passed from words to deeds.

However, nanotechnology still stays the most mysterious and on the other hand the most prospective technology of the twentieth century. So far there is no an exhaustive definition of nanotechnology. By analogy with microtechnology we can say that technology operates with units about the size of a nanometre, i.e. with one billionth of a meter. This is a trace unit which is hundred times smaller than the length of a visible light wave and which is comparable with size of an atom.

Therefore a change from micro to nano is a qualitative change implying the switch from manipulation with substance to a separate atom manipulation. Although not many researches or discoveries have been made in this field of science, most scientists believe that the revolution is to come soon. Nanotechnology is a real breakthrough in science and in life in general.

Nowadays nanotechnology is at the beginning of its development; however it is clear already now that those tiny nanoparticles of size of one million of a pinhead provide great opportunities for various fields of medicine. According to the definition of R. Freyman, a leading scientist in this field, nanomedicine is “Monitoring, correcting, designing, and control over biological systems of a human being at molecular level with the help of nanodevices and nanostructures” (Caruthers S.D., Wickline S.A. & Lanza G.M., 2007).

Metabolic restoration, prolongation of normal function of cells both inside and outside the organism is the main purpose of medical and biological trend in the 21st century. Having solved this task the mankind will closely approach the mystery of longevity, treatment of previously incurable diseases, make a significant advance in the sphere of microbiology, transplantology, growing and storage of cells.             

Will the nearest future allow to purposefully managing metabolism of cells, treating earlier incurable diseases? What should tools and methods be to meet this purpose? All these questions can be answer by the modern trend of science, i.e. nanotechnology.

 

1.2 Nanotechnology is a real breakthrough in the science 

Nanotechnology is a real breakthrough in both the science of the 21st century and life in general. Protection of environment, ozonosphere, manufacture of any fabric, any sort of fuel, physiological immortality of the organism are only a short list of what this branch of science will bring in our life (Chasis J.A. & Mohandas N. J., 1986).

American National Institute of Health (NIH) has included nanomedicine in the top five of priority branches of development of medicine in the 21st century. Scientists from the US National Institute of Cancer consider that nanotechnology will help to treat cancer at its earliest stages and to avoid side-effects.

At now in officially medicine following product nanotechnologies is applied magnet-controlled sorbent (МСS-B) for extracorporeal detoxication of biological liquids. A basis of MCS-B is magnetite nanoparticles (Fe3O4) (Figures 1, 2).

From the standpoint of chemistry of magnetite nanoparticles has the structure of the micelle (Figure 3). The presence adsorption layer provides a nanoparticles sorption activity.

Some results of the sorption capacity of magnet-controlled sorbent (MCS-B) in various biological environments are shown in the Table 1.

 Fig. 1. The study of magnetite nanoparticles with use microscope ion-electronic raster-type Quanta 200 3 D

 

Fig. 2. The study of magnetite nanoparticles with use microscope electronic translucent JEM-2100

Fig 3. The chemical structure of micelle magnetite nanoparticles.

Substance

Biological liquid 

Н2О

Plasma of blood

The blood

Phenol 

1 mcg

0,05 mcg

0,05 mcg

Albumin

Not explore 

Absent

Absent

Creatinin

Not explore

Absent

Absent

Urine

Absent

Absent

Absent

Cholesterol

Not explore

10 mcg

10 mcg

Hormone Т3

Not explore

Absent

Absent

Cu

1,75 mcg

2,5 mcg

1 mcg

Са 

Absent

Absent

Absent

К 

Absent

Absent

Absent

Na

Absent

Absent

Absent

Cl

Absent

Absent

Absent

Mg

Absent

Absent

Absent

Zn

10 mcg

Absent

0,75 mcg

NaNO3 (nitrates)

12,5 mcg

10 mcg

Absent

Cr

2 mcg

0,49 mcg

0,5 mcg

Pb

1,17 mcg

0,3 mcg

0,19 mcg

Cd

0,48 mcg

0,68 mcg

1,55 mcg

Ig A

500 mcmol

300 mcmol

250 mcmol

Ig M

200 mcmol

350 mcmol

250 mcmol

Ig G 

Absent

200 mcmol

250 mcmol

Меdinale

Absent

Absent

Absent

Thiopental Na

Absent

Absent

Absent

Table 1. Some data sorption activity of MCS-B * for a various sort of the substances which are taking place in biological liquid. 

The note: * - at the rate of 30 mg МСS-B per 1 см3 liquids.         

The table 1 demonstrates the sorption activity of magnetite nanoparticles regarding heavy metal salts, nitrates, phenol, and passivity regarding the main electrolytes of blood plasma. This allows using magnet-controlled sorbent MCS-B for cleaning biological body liquids without a threat of electrolytic disorder.

The principle of magnetic phoresis allows magnetite nanoparticles (MCS-B) to restore indices of protein (Figure 4) and lipid blood fractions, to improve albumin-globulin coefficient, ESR, the level of   lipid peroxidation products, regulate the quantity of hormones, of circulating immune complexes, and of lymphocytotoxicity autoantibody.

Fig. 4. Effect selective sorption protein fraction of blood by magnetite nanopaticles (МСS-B).

It is also important to mention that a nanoparticle of magnet-controlled sorbent (MCS-B) has not only a sorption effect but also an indirect effect caused by the influence of a constant magnetic field created by magnetite nanoparticles.

An important advantage MCS-B is that its sorption qualities are highly specific (selective) and that they have a big resemblance with molecular components of blood plasma which stimulate endogenous intoxication syndrome (Fig. 5).

Such selectivity of magnet-controlled sorbent creates prerequisites for indirect sanogenetic effects in the process of therapy.

The presence of a constant magnetic field around magnetite nanoparticles allows magnet-controlled sorbent to not only perform a selective adsorption  of various substances like it is in magnetic phoresis, but also to actively effect intracellular biochemical processes.

So far there is unexplored a mechanism of universality action on homeostasis system alive of organism and metabolism cells of the submitted nanotechnology preparations (NPs).

In this scientific work for the first time are make an attempt investigate mechanisms action of NPs on cell regulation and metabolism as a whole.

Material: nanoparticles of magnet-controlled sorbent (MCS-B). The size of particles is from 6 to 12 nm; the total sorption surface of magnetite nanoparticles is from 800 to 1200 m2/g; magnetization of saturation Is = 2.15 kA/m; volume concentration q = 0.00448; viscosity h = 1.0112 cSt; ζ - potential = - 19 mV. 

Object of research: erythrocytes and leucocytes the patients of blood; the tissue organ of reticuloendothelial system (liver) experimental animals; microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Corynebacterium diphtheria, fungi of Candida type).

 

2.    Effect magnetite nanoparticles MCS-B on functional activity of erythrocytes and parameters of acid-base equilibrium in blood  

 2.1 Material, methods and object of research  

We have selected 50 patients with the marked signs of intoxication who were in intensive care units with various surgical and therapeutic pathologies. The objects of research for this portion of our study were erythrocyte samples from venous blood that had been collected from human subjects.

The influence of МСS-B on functional activity of erythrocytes was studied using the methods of biochemical analysis of the glycolysis processes in erythrocytes. Determination of the bioelectric charge membrane erythrocytes was studied using the method of microelectrophoresis.

2.2 Result of researches glycolysis processes in erythrocytes

As the result of researches it is fixed, that nanoparticles МСS-B causes direct intensification of the glycolysis processes in erythrocytes. It is confirmed by the direct proportional increase (up to double value) of АТPH and 2.3 DPHG quantities 

Glycolysis products 

Stages of the study

 

Normal

Initial date 

After process of MCS-B 

 
 

АТPH (mcmol/mg protein)

 

1.8±0.14 

1.2±0.13

P<0.01

2.3±0.10

P<0.01

P1<0.001

 

2.3 DPHG (mcmol/mg protein)

 

5.1±0.6 

3.3±0.7 

P>0.05 

7.2±0.7

P<0.05

P1<0.001 

 

Table 2. Change of the glycolysis processes in erythrocytes after procedure of MCS-B (n = 50; M±m).

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date. 

2.3 Result of researches the value of charge bioelectric charge of membrane erythrocytes  

The АТPH is a macro energetic combination, which provides preservation of the erythrocytes’ shape, determines the value of charge of their membranes, and volume and duration of circulation of erythrocytes in the blood vessels. Therefore following investigation was study the value of charge bioelectric charge of membrane erythrocytes (Table 3).  

Physical characteristics 

Stages of the study

 

Normal

Initial date 

After process of MCS-B 

 
 

Voltage (V), V

 

0.85±0.02

 

0.83±0.02

P>0.05

 

0.82±0.03

P>0.05

P1>0.05

 

Amperage (I), mА

 

160±13.7

 

225±13.3

P<0.001

 

148±13.2

P>0.05

P1<0.001

 

Table 3. Change of the value of charge bioelectric charge of membrane erythrocytes after procedure of nanoparticles

MCS-B (n = 50; M±m).

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date.

 

Activating the process of oxyhemoglobin dissociation up to 1.5-2 times and raising output of blood oxygen to tissues, magnet-controlled sorbent restores bioelectric potential of erythrocyte membranes improves operation of blood cells, preventing their coagulation and improves motion of blood cells in the vessels. This is in whole normalizes rheology and microcirculation (Figure 6, 7).

2.4 Result of researches the changes state of haemoglobin buffer

Quantity of 2.3 DPHG determines oxygen capacity of the blood through the regulation of oxyhemoglobin dissociation process. Causing changes of functional activity of hemoglobin molecules the MCS-B influence hemoglobin buffer system. The changes on state of hemoglobin buffer determine the acid-alkaline properties of the blood (Table 4).

During the study of рН’s changes and alkaline reserve of the blood it is fixed, that МСS-B is universal corrector of acid-alkaline parameters of the blood.

Thus, MCS-B in vitro produces a marked stimulating effect on processes of glycolysis taking place in the erythrocyte. In the end, this fact causes an increase in the oxygen capacity of blood and normalization of the membrane potential of the erythrocyte charge. The above mentioned circumstance, on the whole, determines the state of activity of the hemoglobin buffer in the venous blood. Finally, this state in the main cause of a rapid and universal correction is acid-alkaline parameters of the blood.

2.5 Result of researches the antiradical protection

Of utmost importance was investigation of antiradical activity enzymes in erythrocytes. The following enzymes were studied antiradical protection: superoxide dismutase (SOD) and catalase (Table 5).

The study found that the antiradical activity of enzymes in red blood cells at an initial stage has been lowered. After treatment of erythrocytes MCS-B revealed a significant increase in the activity of enzymes of antiradical. 

Fig. 6. Initial state of erythrocytes (marked sludge syndrome) of heparinized blood of a patient with K. with toxemia (х 200).

Fig. 7. State of erythrocytes (elimination of sludge syndrome) of heparinized blood of a patient with K. with toxemia after the treatment with magnetite nanoparticles in vitro (х 200).

Indicators

acid-base balance

Stages of the study

Normal 

Initial date

After process of MCS-B 

7.35±0.02

7.29±0.02

Р<0.001 

7.37±0.02

P>0.05

P1<0.001 

ВВ, mmol/l

45±3.2

36±3.0

Р<0.05 

47±1.3

P>0.05

P1<0.01 

Table 4. The influence рН and reserve of alkaline (BB) indexes venous of blood by MCS-B (n=50; М±m).

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date.

Antiradical enzymes 

Stages of the study

Normal 

Initial date

After process of MCS-B 

SОD, IU х106 cel/h 

0.15±0.02

0.07±0.01

P<0.05

0.17±0.02

P<0.001

P1<0.001

Catalase, mccatal х1010 cel 

5.5±0.05 

5.0±0.02

P<0.001

5.5±0.03

P>0.05

P1<0.001

Table 5. The level of enzyme activity of erythrocytes venous blood before and after processing by MCS-B (n=50; М±m)

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date.

 

3. The influence of nanoparticles MCS-B on the hemolysis and activity of transport adenosinetriphosphatese of erythrocytes 

3.1 Material, methods and object of research  

We have selected 20 healthy volunteers.  The age of persons varied from 24 to 40 years. The objects of research for this portion of our study were erythrocyte samples from venous blood that had been collected from human subjects. All researches were performed in vitro.

The researches included 3 stages: stage I - initial condition of erythrocytes; II – condition after processing by nanoparticles of MCS-B; III - condition of erythrocytes on the 21st day of observation.

Research methods: 3 ml intake of venous blood of a patient was performed. For preventing coagulation of blood citrate sodium was introduced. The first test tube was control. Into the second test tube MCS-B was singularly introduced in quantity of 1.5 ml with its following separation by means of a constant magnetic field with the intensity of 200 kA/m. In the third test tube there was the blood processed twice by MCS-B. In the fourth tube there was the blood processed thrice. The suspension of blood cells after performance of the biochemical investigation was stored in the refrigerating chamber at temperature +1ºС. On the 14th day the signs of hemolysis were registered visually.

On stages I and II the activity of transport adenosinetriphosphatese of erythrocytes was studied: Na, K - АТPHese and Ca, Mg – АТPHese by the standard procedure of biochemical analysis (Severina S.E., 1997).

Statistically processing the obtained results was carried out by parametrical method of variation statistics by Student criterion. Processing the obtained data was carried out by means of Excel. 

 

3.2 Result of researches the influence of nanoparticles MCS-B on the hemolysis of erythrocytes

 As a result of the research it was established, that in the control and test tubes where the blood was processed by nanoparticles of MCS-B, on the 1st day of observation visible signs of hemolysis it were not observed (Figure 8).

However the signs of hemolysis on the 21st day were determined in the control and test tubes where the blood was processed thrice by nanoparticles of MCS-B.

On the contrary, in the test tubes where blood was processed by nanoparticles of MCS-B once or twice, hemolysis was practically not observed (Fig. 9).

 

3.3 Result of researches the influence of nanoparticles MCS-B on the activity of adenosinetriphosphateses of erythrocytes

 Results of the research of activity of adenosinetriphosphateses of erythrocytes are presented in table 6.

So, the data of table 1 demonstrate that singular processing of blood by MCS-B reliably reduces (in comparison with the control) activity of Ca, Mg – АТPHese of erythrocytes - by 2.47±0.6 protein mmol/mg in mines (р <0.01), double - by 5.19±0.5 protein mmol/mg in mines (р <0.001), triple - by 6.01±0.5 protein mmol/mg in mines (р <0.001).

On the contrary, reliable differences concerning changes of activity of Na, K - АТPHese in any test tubes (in comparison with the control) were not detected (p> 0.05).

Fig. 8. A visual picture of condition of the erythrocytes on the 1st day of observation (n=20).

Notes: 1 - the control; 2 - after single processing by MCS-B; 3 - after double processing by MCS-B; 4 - after triple processing by MCS-B.   

Fig. 9.  A visual picture of condition of the erythrocytes on the 21st day of observation (n=20).

Notes: 1 - the control; 2 - after single processing by MCS-B; 3 - after double processing by MCS-B; 4 - after triple processing by MCS-B.  

Adenosine-triphosphateses

Control

Frequency rate processing of МСS-B

Single

Double

Triple

Na, К – АТPHese,

protein mmol/mg in mines

 

6.34±0.5

 

6.11±0.6*

 

5.89±0.7*

 

5.93±0.4*

Ca, Mg – АТPHese,

protein mmol/mg

in mines

 

23.64±0.6

 

21.17±0.7**

 

18.45±0.5***

 

17.63±0.3***

Table 6. Results of research of activity of adenosinetriphosphateses before and after processing of erythrocytes by nanoparticles MCS-B (М±m; n=20)

Note: * - p>0.05; ** - p<0.01; *** - p<0.001

 

3.4 Conclusion

 Thus, as a result of the research, the optimum frequency rate of extracorporeal processing of blood by nanoparticles of MCS-B essentially slowing down hemolysis was founded. The minimum value of activity of Ca, Mg - АТPHese erythrocytes was 18.45±0.5 protein mmol/mg in mines. The subsequent depression of activity of Ca, Mg - АТPHese leads to acceleration of hemolysis of erythrocytes.   

This work for the first time describes (in comparison with control) the indices characterizing dependence of time of appearing hemolysis on frequency rate of processing the blood by nanoparticles of MCS-B.

It was established, that extracorporally processing the blood by nanoparticles of MCS-B reliably reduces activity of Ca, Mg - АТPHese of erythrocytes.

 

4.    Effect of magnetite nanoparticles MCS-B on the metabolic processes of leukocytes and free radical oxidation lipid in erythrocytes parameters 

 4.1 Material, methods and object of research  

 From a total of 84 patients, 42 were research exchange of cell products leucocytes, 42 were research free radical oxidation lipid (FRОL) in erythrocytes parameters. The study metabolism of leucocytes included: the research of glycogen, succinate dehydrogenasis activity (SDG) (Asatiani V.S., 1969); protein total, Gl-6PH -DG, lactate degydrogenasis of activity (LDG), creatinine concentration (Melnikiv V.V., 1987); superoxyddysmutase of activity (SОD); catalase activity; glutathione reestablished; diene conjugates (DC) and malonic dialdehyde (МDА) concentration. The erythrocytes hemolysatic for study enzyme activity are received according to technique (Costuk V.A., Potapova A.I. & Covalova T.V, 1990). It is known, that nanoparticles MCS-B have sorption activity, therefore was studied the analysis their sorption activity to superficial protein membranes of erythrocytes.

The object of research: erythrocytes of healthy persons (10) (Chasis J.A. & Mohandas N. J., 1986; Ling E. & Sapirstein V., 1984). The research was included two stages: the studied foregoing of parameters in cells intact and cells after make contact with nanotechnology preparations.

 

4.2 Result of researches metabolic processes in leucocytes at healthy persons

 As a result research of leucocytes the healthy persons after processing blood by colloid magnetite particles was discovered: decrease level the glycogen on 19±0.3 mg/1010 cell; total lipids - on 6.1±0.5 mcg/106 cell; Gl-6PH-DG activity – 302.4±11.2 mmol/1hх1011 cell (basic data 500±10.6 mmol/1hх1011 cell); increase of the phospholipids - on 0.9±0.1 mg/106 cell; total protein - on 8.0±0.5 mcg/1010 cell; creatinine concentration - on 1.29±0.1 mg/109 cell; glutathione (restored) activity – 4.2±0.1 mg/1010 cell. (basic data – 3.2±0.1 mg/1010 cell); gexocenase activity – 11.4±0.2 mmol/1h х1011 cell. (basic data – 9.5±0.2 mmol/1h х1011cell); lactateDG - 611±11.4 mmol/1h х1011 cell (basic data - 542±10.5 mmol/1h х1011 cell); succinateDG – 6.9±0.2 mmol/1h х1011 cell (basic data – 3.8±0.1 mmol/1h х1011 cell); MDA concentration - on 7.7±0.7 nmol/mg; DC - on 66.8±10.5 nmol/mg; SОD activity – 0.18±0.04 IUх106 cell/h (basic data – 0.08±0.02 IUх106 cell/h).

 

4.3 Result of researches metabolic processes in leucocytes at patients with syndrome intoxication

 In leucocytes were discovered depression metabolism processes on the phase starting research at patients with syndrome intoxication. There are phospholipids - on 0.4±0.1mg/106 cell, total protein - on 3.9±0.3 mkg/1010 cell, total lipids - on 8.3±0.6 mcg/106 cell was decreased; creatinine concentration - on 2.09±0.2 mg/109 cell was increased. There are phospholipids - on 1.8±0.1mcg/106 cell, total protein - on 15.3±0.1mcg/1010 cell, creatinine concentration - on 0.7±0.1mg/106 cell was increased; total lipids - on 15.6±0.1mcg/106 cell was decreased after to process by MCS-B of leucocytes. This date was evidenced of metabolism processes restoration in a cell.

 

4.4 Result of researches metabolic processes in leucocytes at the terminal patients

 The similar metabolism processes was decreased in leucocytes at the terminal patients, however, in more expressed form (Table 7). As a result of processing blood by colloide magnetite particles (MCS-B) the restoration of parameters metabolism of cell it wasn’t. So, creatinine concentration did not increase, and was decreased - on 0.4±0.1mg/109 cell; the activity lactateDG was decreased - 330±10.9 mmol/1hх1011 cell (basic data - 348±12.1 mmol/1hх1011cell); succinateDG – 1.6±0.1 mmol/1hх1011cell (basic data – 2.0±0.2 mmol/1hх1011cell); the DC was decreased - on 5.6±10.5 nmol/mg; the SОD activity did not change. The decreased of succinateDG activity is caused by deficiency of oxygen and destabilization of membranes.

 

4.5 Conclusion

 Thus, after processing blood by MCS-B was discovered restoration regulator mechanisms metabolism of cell at the patients with syndrome intoxication. Normalization of a level POL and parameters antioxidation system (АОS) was evidenced of blockade oxidizing stress. On the contrary after processing blood by magnetite particles, restoration of metabolism cell was not discovered at the terminal patients. It is possible this effect explainable of high level intoxication, damage metabolism apparatus of cell, substratum metabolism depletion.

 

4.6 Result of researches the action by magnetite nanoparticles MCS-B on antiradical protection at healthy persons and infectious hepatitis C patients

 The universality of action by colloide magnetic particles on cells regulation processes was confirmed investigations enzymes action of antiradical protection at healthy persons and infectious hepatitis C patients. According to АОS basis parameters all investigated was separated on groups (Table 8).

As a result of study AOS (SОD, catalase and glutathione) of the erythrocytes was discovered to modulation glutathione activity after processing of blood by MCS-B. In I to group of healthy persons after processing by МСS-B of erythrocytes was discovered: increase of glutathione activity – 4.9±0.2 mgх1010 cell. (basic data – 3.7±0.1 mgх1010 cell); SOD activity – 0.26±0.01 IUх106 cell/h (basic data – 0.17±0.01 IUх106 cell/h); catalase activity – 6.2±0.1 mccatal х1010 cell (basic data – 5.4±0.1 mccatal х1010 cell). In II to group the increase of glutathione activity and catalase was not discovered. The SОD activity was decreased – 0.09±0.01 IUх106 cell/h (basic data – 0.11±0.01 IUх106 cell/h). In III to group increase of glutathione activity – 4.5±0.1 mgх1010 cell (3.5±0.1 mgх1010 cell), catalase activity – 5.9±0.1mccatal х1010 cell (basic data – 5.5±0.1mccatal х1010 cell), decrease of SOD activity – 0.07±0.02 IUх106 cell/h (basic data – 0.10±0.02 IUх106 cell/h) was discovered. In IY to group: glutathione activity – 4.4±0.2 mgх1010 cell (basic data – 4.1±0.2 mgх1010 cell), SОD activity – 0.18±0.02 IUх106 cell/h (basic data – 0.15±0.02 IUх106 cell/h) was increase. The catalase activity – 5.3±0.1 mccatal х1010 cell (basic data – 5.7±0.1 mccatalх1010 cell) was decreased. The similar processes correction parameters of level enzyme antiradical protection activity at various initial variants at the infectious hepatitis C patients were discovered (Table 8). Probably, the influence by MCS-B was caused, on the one hand, the properties nanoparticles sorption selectively of molecule proteins from a surface membranes cell; with another, probably, change of conformation structure proteins of the superficial membranes as a result influence of magnetic field which induced by magnetite particles.

 

4.7 Result of researches the sorption activity of MCS-B relative to proteins superficial of erythrocyte membranes

 With the purpose study sorption activity of MCS-B relative to proteins superficial of erythrocyte membranes were investigated healthy persons. As a result of research is established: МСS-B had sorption activity about to spectrine on 7±1 %, ancirine on 3±0.5 % (Figure 10). Thus, MCS-B has sorption activity relative to proteins superficial of cell membranes. However it is necessary to remind, that sorption proteins molecule by MCS-B has selective sorption properties which based on a principle magnetopheresis, and therefore as a whole does not cause destructive of structural cell membranes, and only quantitatively changes structure of molecule proteins. 

 

Fig. 10. The scheme of erythrocyte membrane structure intact and after treatment with the nanoparticles MCS-B

 

The metabolism products of leucocytes

 

Health persons (n=12)

Patients with syndrome intoxication and insufficiency of polyorganic (n=20)

 

The terminal patients (n=10)

Initial

date

After to process

 by МСS-B

 

P

 

Initial

date

After to process

 by МСS-B

 

P

 

Initial

date

After to process

 by МСS-B

 

P

Glycogen, mg/1010 cell

51.4±0.5

32.4±0.3

<0.001

32.3±0.4

21.4±0.5

<0.001

27.6±0.4

23.0±0.3

<0.001

Phospholipines, mgх106 cell

2.5±0.2

3.4 ±0.1

>0.05

2.1±0.2

3,9±0.1

<0.05

1.8±0.1

1.9±0.2

>0.05

Total proteine, mcgх1010 cell

36.2± 0.4

44.2±0.5

<0.001

32.3±0.3

47.6±0.5

<0.001

28.6±0.3

29.3±0.4

>0.05

Total lipides, mcgх106 cell

98.1±0.6

92.0±0.5

<0.001

89.8±0.6

74.2±0.5

<0.001

84.6±0.8

83.1±0.7

>0.05

Creatinine, mgх109 cell

1.01±0.2

2.3±0.1

>0.05

3.1±0.2

3.8±0.1

>0.05

3.4±0.1

3.0±0.2

>0.05

Glutathione (restored), mgх1010 cell

3.2±0.2

4.2±0.1

<0.001

2.7±0.1

4.7±0.2

<0.001

1.0±0.1

1.0±0.2

>0.05

Hexocynase, mmol/1hх1011 cell

9.5±0.2

11.4±0.2

<0.001

7.7±0.1

10.8±0.2

<0.001

6.3±0.1

6.8±0.2

>0.05

Lactat DG,  mmol/1hх1011 cell

542±10.5

611±11.4

<0.001

467±10.6

591±11.1

<0.001

348±12.1

330±10.9

>0.05

Gl-6PH-DG, mmol/1hх1011 cell

500±10.6

302.4±11.2

<0.001

378±10.8

187.3±12.0

<0.001

278.6±10.1

211.5±10.3

<0.001

Succinate DG, mmol/1hх1011 cell

3.8±0.1

6.9±0.2

<0.001

2.7±0.1

6.7±0.2

<0.001

2.0±0.2

1.6±0.1

>0.05

МDA, nmol/mg

22.9±0.5

30.6±0.7

<0.001

32.5±0.5

34.4±0.7

<0.05

36.8±0.5

37.1±0.6

>0.05

DC, nmol/mg

190±10.3

256.8±10.5

<0.001

197±11.2

261.4±12.1

<0.001

205.7±10.3

200.1±10.7

>0.05

SОD, IUх106 cell/h

0.08±0.02

0.18±0.04

>0.05

0.12±0.03

0.28±0.02

>0.05

0.22±0.03

0.22±0.02

>0.05

Таble 7. Parameters of metabolism processes in leucocytes at various persons (M±m)

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date.

 

Variants

 

 FRОL parameters

of  blood in erythrocytes

Health persons (n=21)

Infectious hepatitis C patients (n=21)

Initial

date

After to process

 by МСS-B

P

Initial

date

After to process

 by МСS-B

 

P

 

I

(n=11)

Glutathione, mgх1010 cell

SОD, IUх106 cell/h

Catalase, mccatal х1010 cell

3.7±0.1

0.17±0.01

5.4±0.1

4.9±0.2

0.26±0.01

6.2±0.1

>0.05

<0.001

<0.001

3.9±0.2

0.08±0.01

9.2±0.2

4.8±0.1

0.09±0.01

10.0±0.1

<0.001

>0.05

<0.001

 

II

(n=9)

Glutatione, mgх1010 cell

SОD, IUх106 cell/h

Catalase, mccatal х1010 cell

4.7±0.1

0.11±0.01

5.2±0.1

4.7±0.1

0.09±0.01

5.1±0.1

>0.05

<0.001

>0.05

3.0±0.1

0.11±0.01

10.5±0.1

5.2±0.1

0.07±0.01

10.0±0.1

<0.001

<0.01

>0.05

 

III

(n=12)

Glutathione, mgх1010 cell

SОD, IUх106 cell/h

Catalase, mccatal х1010 cell

3.5±0.1

0.10±0.02

5.5±0.1

4.5±0.1

0.07±0.02

5.9±0.1

<0.001

>0.05

<0.01

2.7±0.1

0.25±0.02

8.7±0.1

5.5±0.1

0.19±0.02

11.0±0.1

<0.001

<0.05

<0.001

 

IY

(n=10)

Glutathione, mgх1010 cell

SОD, IUх106 cell/h

Catalase, mccatal х1010 cell

4.1±0.1

0.15±0.01

5.7±0.1

4.4±0.2

0.18±0.02

5.3±0.1

>0.05

>0.05

<0.01

2.5±0.2

0.09±0.02

9.0±0.1

4.9±0.2

0.14±0.01

8.2±0.1

<0.001

<0.05

<0.001

Таble 8. The result of research free radical oxidation lipids parameters before and after to process of blood by МСS–B in erythrocytes (M±m)

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date.

 

 5. Effect of magnetite nanoparticles MCS-B on phagocytic activity of leucocytes  

 5.1 Result of researches the action by magnetite nanoparticles MCS-B on immune reactivity system

 After the session in vitro the blood was tested for immune reactivity. As a result, a pronounced increase in phagocyte activity of leukocytes was revealed, it testifying about a positive activation of the immunological status against a background of initial immune deficiency (Tab. 9).

Increased phagocytic activity of leucocytes is mainly due to the influence of constant magnetic field, which is induced by the MCS-B nanoparticles (Figure11)

The magnetic field causes excitation (priming) of phagocytes. Activating production of free radicals such as: H2O2, ClO, NO.            Increased phagocytic activity of leukocytes.

Thus, MCS-B nanoparticles are intensively affects the indices of cellular immunity; enhance immune cell resistance to harmful factors.

 

6. Effect of magnetite nanoparticles MCS-B on microorganisms 

6.1 Introduction

Studies of the effects produced by nanoparticles MCS-B magnetic fields on biological objects were started long ago, and now they are urgent. But the majority of experimental data are varied and contradictory, therefore regularities and mechanisms of their action have-not been formulated yet. Recent achievements in the fields of physical and colloidal chemistry as well as medicine have enabled creation of new magnetic structures, ferrocolloidal or magnetic fluids in particular, which are adapted to biological media of a living organism.

Their wide introduction has contributed to development of principally new technological processes and equipment not only in engineering but also for medicobiological researches. Effects of magnetic fields on microorganisms attracted much attention, but such investigations were unidirectional and carried on mostly with Escherichia coli and Staphylococcus. We have not met any reports about effects on bacteria in the home and foreign literature, though a number of authors point out in their researches that a magnetic field can change efficacy of the action of remedies in an organism.

Taking into account all above mentioned, as well as an increased interest to the influence exerted by magnetic fields on a living cell, we have decided to study effects of magnetic fluid of nanoparticles MCS-B on microorganisms causing various suppurative-inflammatory processes in humans. 

Laboratory indices 

Stages of the study 

Normal 

Initial date

After process of MCS-B 

Phagocytosis after 30 minutes:  

 

Phagocytic index, %

 

 

 

Phagocytic indicator, IU 

 

 

 

85.3±4.4 

 

 

 

14.2±0.3 

 

 

44.4±4.2 

P<0.001 

 

 

7.3±0.3 

P<0.001

 

 

88.1±4.3 

P>0.05

P1<0.001 

 

13.6±0.2 

P>0.05

P1<0.001

Phagocytosis after 60 minutes:

 

Phagocytic index, %

 

 

 

Phagocytic indicator, IU 

 

 

 

82.2±4.5 

 

 

 

12.1±0.3 

 

 

 

41.3±4.3

P<0.001

 

 

6.3±0.2 

P<0.001 

 

 

85.8±4.2

P>0.05

P1<0.001

 

12.2±0.2 

P>0.05

P1<0.001 

Completeness of phagocytosis, IU 

3.7±0.2 

 

0.8±0.3

P<0.001

3.1±0.3

P>0.05

P1<0.001

Table 9. Increase in phagocytic activity of leucocytes and in phagocytosis completeness index (M±m; n=50)

Notes: 1. Р – accuracy of differences in comparison with normal; 2. Р1 – accuracy of differences in comparison with initial date.

Fig. 11.  The mechanism of action of nanoparticles MCS-B on the phagocytic activity of blood leukocytes.                                                

 

6.2 Material, methods and object of research

The research was made on the following objects: Staphylococcus aureus, Pseudomonas aeruginosa, Corynebacterium diphtheria, fungi from the genus of Candida.

Method research was method of disks. Method of disks is method for determination of biological activity substances against bacteria test based on a zone of delays or stimulation of growth on solid growth, which placed a paper disk, impregnated researched material (magnetite nanoparticles MCS-B).

 

6.3 Result of researches the bacteria sensitivity

During the study with the method of disks, we revealed increases in diameters of the areas of delayed growth of microorganism strains, treated with the above MCS-B, in comparison with "pure" strains.

Any their characteristic feature was the fact that under the effect of MCS-B the bacteria demonstrated a higher sensitivity to antibiotics.

It is worth mentioning that some strains of microorganisms, that were isolated from patients with pyoinflammatory diseases and possessed resistance to the majority of antibiotics, began to demonstrate sensitivity to these antibiotics, in particular to penicillin, ampicillin, tetracycline and gentamycin, after 24-hours' exposure to the above MCS-B.

Thus three strains of Pseudomonas aeruginosa, isolated from foci of a suppurative wound, were resistant to 24 investigated antibiotics from the groups of penicillins, cephalosporin, aminoglycosides, rifampicins, and others except for zanocin, amicacin and cyproophloxacin. After 24-hours’ treatment with MCS-B these strains were resistant only to 16 antibiotics, but became sensitive even to antibiotics of the penicillin line - carbenicillin and azlocillin.

The minimum inhibitory concentration of antibiotics, producing the effect on pathogenic flora, was significantly decreased. The data are given in Table 10. 

Antibiotic 

S. aureus mg/ml

P. aeruginosa mg/ml

Control

After process of MCS-B

Control

After process

of MCS-B

Carbenicillinum

9.0±0.6

3.0±0.4

P<0.001 

≥100

60.0±10.5

P<0.05 

Gentamicinum

5.0±0.8

2.0±0.9

P<0.05 

12.0±1.2

4.0±1.3

P<0.05 

Riphampicinum

9.0±1.3

3.0±0.7

P<0.001 

14.0±1.4

5.0±1.5

P<0.001 

Ofloxacinum

5.0±1.4

2.0±0.8

P>0.05 

5.0±1.4

3.0±1.1

P>0.05 

Таble 10. Minimal depressing concentration of antibiotics regarding bacteria (mkg/ml) before and after MCS-B (M±m; n=50) exposure.

Note: P – accuracy of differences in comparison with control.

 

6.4 Result of researches the antimicrobial activity to the antibiotics  

Later, our investigations were directed to see changes in the microorganisms under the effect of MCS-B, particularly their antimicrobial activity with respect to the antibiotics which are widely used in clinical practice.

The standard suspension of microorganisms and fungi, amounting to 1 milliard per 1 ml of the physiological solution, was treated with MCS-B at, respectively, 0.9 ml and 0.1 ml of the suspension of the bacteria. The exposure lasted 1 hour, 3 hours, 24 hours, and 48 hours at 37°C. Then cultures of the microorganisms and fungi were inoculated on solid nutrient (agar) media, therewith taking into account how some biological properties changed under the effect of MCS-B.

Exposure of the bacteria and fungi to MCS-B for 1 hour and 3 hours had a negative effect only on growth of cell populations. No cultural, morphological and biochemical properties were changed. Moreover, areas of hemolysis in the microorganisms with hemolytic properties constricted.

Exposure to MCS-B during 24 hours resulted in a significant inhibition of growth properties of the microorganisms (only growth of single colonies was observed), changes in cultural and morphological tests, deceleration of biochemical reactions, in particular fermentation of carbohydrates and utilization of urea. Complete registration of biochemical reactions was performed only after 48 hours.

Exposure to MCS-B for 48 hours resulted in the bacteriostatic effect. Within the first 24 hours any growth of the microorganisms was actually absent, except for growth of single colonies of clinical strains. Growth of microflora on the nutrient media was revealed only on the 2nd day. There was a significant decrease in biochemical activity and changes of cultural and morphological properties there. Studies of antibacterial activity of MCS-B themselves were performed by standard methods (the method of wells or disks). The investigation has revealed that the given magnetic fluid demonstrates only weak activity to reference strains of Staphylococcus and fungi from the genus of Candida. The above MCS-B produced no effect on gram-negative aerobic flora and diphtheria bacilli, as well as on clinical strains of the microorganisms. But in hemolytic colonies their areas of hemolysis were practically absent or very insignificant. Pseudomonas aeruginosa had no pigment. The size of most colonies was diminished. Any other significant differences in biological properties between the reference and clinical strains after their exposure to MCS-B were not observed. Consequently, it should be noted that a prolonged influence of the given MCS-B on the clinical strains did not result in any pronounced bacteriastatic effect. Weak growth of the microorganisms was noticed within the 1st day after inoculation on the nutrient media.

 

6.5 Result of researches the influence of MCS-B on normal microbiocenosia of human mucous membranes

The opportunity of using MCS-B as sorbents has enabled us to study their effects on the microflora, which constitutes normal microbiocenosia of human mucous membranes in humans, in particular on Bifidobacterium and Lactobacterium. As it is known, normoflora takes part in metabolism and synthesis of various acids and vitamins, stimulates the lymphoid apparatus to form immunoglobulins and for phagocytosis, participates in renewal of cells in the mucous membranes thereby forming their resistance.

Therefore, dysfunction of the normal microflora under effect of some substances results in weakening of specific factors of protection of the organism, it enabling invasion of toxins and allergens into the blood stream.

Results of the study have shown that even a prolonged exposure to MCS-B does not cause any changes in biological properties of the normoflora, except for an insignificant inhibition of the growth ones. But the response of inhibition of the normoflora growth was only short-term and 24 hours later the microflora resumed its vitality. A weak response of Bifidobacterium and Lactobacterium to the effects of MCS-B was apparently caused by metabolism of these bacteria, since they represent the anaerobic group of bacteria.

 

7. Ultrastructure of hepatic cells in rabbits after injection of nanoparticles MCS-B

7.1 Material, methods and object of research

In order to determine ultrastructural reconstructions in hepatic cells under effect of magnetite, an experiment was conducted on 45 rabbits. They received a single intravenous injection of nanoparticles MCS-B at a dosage of 75 mg/kg of body weight 24 hours before the investigation.

Organs of intact animals were used as controls. After completion of the experiment the animals were killed and the above organs were taken for electron microscopic examinations.

Necessary areas of sections were photographed on photographic plates which served for subsequent taking of microphotographs.

 

7.2 Result of researches the ultrastructural changes in hepatocyte organelles

Ultrastructural changes in hepatocyte organelles manifested pronounced signs of activation of reparative intracellular processes.

Hepatocyte nuclei held their rounded shape (Figure 12). Nuclear membranes had well-defined contours. Chromatin, in the form of small clods, was evenly distributed throughout the section. Condensation of chromatin on the nuclear membrane was observed only in single hepatocytes. Perinuclear space was not enlarged. Single ribosomes were found on the outer membrane of the nucleus.

Mitochondria were evenly distributed in all parts of the cytoplasm of the hepatic cells (Figure 13). Mitochondrial matrix had a moderate electron density and a fine grane structure. Shape of the mitochondria varied from rounded to stick-like. Many cristae were revealed; they had a pronounced typical orientation. The outer membrane remained integral, without any foci of destruction. In single cells, there were mitochondria having the shape of dumb-bells and with septa. The rough endoplasmic reticulum underwent the most characteristic reconstructions (Figure 14). In the majority of hepatocytes, their rough endoplasmic reticulum was an extensive network of membranes with numerous ribosomes localized on their surfaces. Cisterns of the endoplasmic reticulum were slightly enlarged and their shape resembled flattened vesicles. The substance which filled them was electron transparent. The smooth endoplasmic reticulum was well developed, its vacuoles were mostly localized in basal parts of the cytoplasm. It should be noted that there were great numbers of free ribosomes and granules of glycogen which were evenly distributed throughout the cytoplasm. The laminated cytoplasmic Golgi's complex (Figure 15) was moderately hypertrophic, its membrane part consisted of parallel smooth membranes. Packs of these membranes were surrounded with a great number of large and small vesicles. Single vesicles were filled with a rough fibrous osmiophil substance. There was rather a great number of primary lysosomes in the area of localization of the laminar cytoplasmic Golgi's complex, autophagosomes and small inclusions of lipids being observed in single cells. Bile capillaries were filled with prolonged crimped microvilli and were moderately dilated.

Sinus capillaries and Disse's spaces were dilated rather extensively. Disse's spaces were filled with numerous microvilli. Changes in the ultrastructure of Kupffer cells testified about their functional activity. Nuclei (Figure 16) of Kupffer cells were of irregular shape with deep invaginations of the nuclear membrane. Nuclear matrix had a significant electron density. Karyolemma had no destructive changes.

The cytoplasm of Kupffer cells revealed single and slightly swollen mitochondria which contained a small number of cristae and single cisterns of the rough endoplasmic reticulum. The cytoplasmic membrane did not undergo any changes and held its well-defined bilaminated structure. It should be noted that there was a great number of small electron-transparent micropinocytic vesicles.

 

8. Conclusion

It was established, that activity of Ca, Mg – АТPHese of erythrocytes decreases with increasing frequency rate of processing the blood by nanoparticles of MCS-B. Activity of Na, K - АТPHese of erythrocytes in extracorporally processing the blood by nanoparticles of MCS-B does not change (p> 0.05). The minimum indicator of activity of Ca, Mg - АТPHese is 6.01±1.2 protein mmol/mg in mines which time of appearing hemolysis practically does not differ from the control was found. The optimum frequency rate (1-2 times) of processing the blood by nanoparticles of MCS-B inhibiting hemolysis was found.

The researches has proved that now nanoparticles of MCS-B are able not only to considerably reduce hemolysis, and thereby prolong storage time of the blood, influence activity of adenosinetriphosphateses of erythrocytes, regulate transmembrane exchange, but also to extracorporally influence cellular apoptosis.

The nanoparticles MCS-B are factor modulating metabolism processes in leucocytes of blood healthy persons and patients.

Is established, that action point of nanoparticles MCS –B is protein superficial cells membrane. The selective sorption of protein superficial membrane erythrocytes is result action by MCS-B.

The nanoparticles MCS-B are factor modulating enzyme link АОS activity in erythrocytes healthy persons and infectious hepatitis C patients.

The nanoparticles MCS-B are intensively affects the indices of cellular immunity, enhance immune cell resistance to harmful factors. The performed microbiological studies of the effects produced by nanoparticles MCS-B on microorganisms have revealed the following facts:

1) Pronounced changes in sensitivity of pathogenic microorganisms to antibiotics, widely used in clinical practice, towards increase of their antibacterial activity.

2) A distinct bacteriastatic effect of this MCS-B absence along with of any normoflora pathologies in microbiocenosla of human mucous membranes.

Analysis of the state of submicroscopic architectonics of hepatic cells in rabbits after injection of nanoparticles MCS-B reveals a significant activation of metabolic intracellular processes in liver. Ultrastructural organization of the liver testifies about intensification of synthetic intracellular processes, it being structurally manifested by enlargement of cisterns in the rough endoplasmic reticulum, an increased number of ribosomes and a moderate hypertrophy of the laminar cytoplasmic Goldi's complex. Activation of reparative intracellular processes is another aspect of these reconstructions. It is confirmed by a revealed hyperplasia of the rough endoplasmic reficulum, this hyperplasia testifying about intensive processes of self-renewal in submicroscopic structures. Presence of mitochondria having the shape of dumb-bells and with constrictions in the cytoplasm of hepatic cells enables a statement that a process of an intensive increase in the number of these organelles tares place.

Fig. 12. Ultrastructure of hepatocytes in rabbits after injection of nanoparticles MCS-B х 39.000

Fig. 13. Ultrastructure of hepatocytes in rabbits after injection of nanoparticles MCS-B х 30.000

Fig. 14. Ultrastructure of hepatocytes in rabbits after injection of nanoparticles MCS-B х 42.000

Fig. 15. Ultrastructure of hepatocytes in rabbits after injection of nanoparticles MCS-B х 40.000

Fig. 16. Ultrastructure of hepatocytes in rabbits after injection of nanoparticles MCS-B х 39.000

 

Availability of a great number of mitochondria with unchanged structure and numerous cristae in the cytoplasm of hepatocytes indicates a high activity of redox processes and those of oxidative phosphorylation which satisfy energy needs of synthetic intracellular reactions taking place on the level of membranes and macromolecules. Submicroscopic structure of endotheliocytes in sinus capillaries indicates activation of processes of transcellular transportation of substances and electrolytes by endocytosis, it being confirmed by presence of numerous micropinocytic vesicles in the cytoplasm of these cells.

Thus, we have hypothesis biological action of nanotechnology preparations on protein molecules conformation and structure of cytoplasm and intracellular membranes. The change of protein molecules structure to influences on transport substances of cell and determine intracellular metabolism processes.

 

9. Acknowledgment  

 The authors is thankful to the department of biological chemistry Kharkov National Medical University, the laboratories of mediums and biochemistry of microorganisms and immunology clinic Academy of Medical Sciences of Ukraine Mechnicov Institute of Microbiology and Immunology, urgent laboratory of biochemical Kharkov Region Hospital.

 

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