RU2791817C1 - Method for including biologically active components in erythrocytes by method of flow hypoosmotic dialysis - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: method for incorporating a biologically active component into erythrocytes by the method of flow hypoosmotic dialysis comprises the following steps: adding a biologically active component to a suspension of concentrated erythrocytes washed from whole blood or to a previously obtained erythromass; lysis of erythrocytes in the presence of at least one biologically active component using hypoosmotic flow dialysis; incubating a suspension of erythrocytes lysed in the presence of a biologically active component with a hyperosmotic solution to form sealed carrier erythrocytes containing at least one biologically active component; washing sealed erythrocyte carriers containing at least one biologically active component; concentrating, using a dialyzer, sealed carrier erythrocytes containing at least one biologically active component; washing out the sealed carrier erythrocytes remaining there from the dialyzer and adding them to previously obtained sealed carrier erythrocytes containing at least one biologically active component; while the stage of erythrocyte lysis in the presence of a biologically active component is carried out while maintaining a constant transmembrane pressure in the dialyzer of 160-180 mm Hg and pressure at the inlet to the internal circuit of the dialyzer not exceeding atmospheric by more than 120 mm Hg, and maintaining the temperature from +2°C to +10°C; washing of erythrocytes is carried out at room temperature; the concentration of erythrocytes is carried out while maintaining a constant transmembrane pressure in the dialyzer of 90-120 mm Hg taking into account the measured pressure indicators in the external and internal circuits of the dialyzer and maintaining the temperature from +2°C to +10°C; the stage of sealing lysed in the presence of a biologically active component or components of erythrocytes is carried out at a temperature of +25°C to +40°C; moreover, the stage of concentration of erythrocytes washed from whole blood and the stage of concentration of sealed erythrocyte carriers containing at least one biologically active component are carried out until the haematocrit value of the erythrocyte suspension is 75–80%.

EFFECT: above method allows for high efficiency and reproducible degree of incorporation of various biologically active components into erythrocytes, reducing the loss of intracellular content.

16 cl, 7 dwg, 2 tbl, 2 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to the field of medicine and biology, in particular to methods for creating dosage forms for various enzyme preparations, as well as other biologically active components (BAC), based on erythrocytes, which can be used both in the treatment of a number of diseases and for diagnostic purposes.

BACKGROUND OF THE INVENTION

Enzymes included in erythrocytes can be used both in enzyme replacement and antitumor therapy, and for removing a number of low-molecular toxic compounds from the bloodstream, such as ammonia, cyanide, ethyl and methyl alcohol, acetaldehyde, etc. [1]. Currently, the enzyme L-asparaginase (asparaginase) is an integral part of the complex therapy of acute lymphoblastic leukemia (ALL) in children and adults [2], as well as some other types of cancer [3]. Asparaginase is an enzyme that hydrolyzes the amino acid asparagine to aspartic acid and ammonia. The mechanism of its antitumor action is associated with a decrease in the concentration of asparagine in plasma, since this amino acid is necessary for protein synthesis and its absence leads to a slowdown in the division of tumor cells due to the impossibility of such synthesis. Asparaginase has a particularly effective effect on the cells of some tumors, which, unlike normal cells, cannot independently synthesize asparagine (for example, in ALL) [4]. However, intravenous administration of asparaginase is limited by the existence of many side effects, the main of which are the occurrence of severe allergic reactions, as well as a short lifetime of asparaginase in the bloodstream [5,6]. The creation of a new dosage form of asparaginase (asparaginase included in erythrocytes) improves its pharmacological properties, tk. greatly increases the lifetime of asparaginase in the bloodstream [7,8]. This is because asparaginase conducts its reaction while inside the red blood cells, which protects it from antibodies and proteases in the plasma that can destroy it. The substrate with which asparaginase works - asparagine, is able to penetrate from the plasma into the erythrocyte through the erythrocyte membrane [9]. Asparaginase for therapeutic use is obtained from bacteria (Esherichia coli, or from the phytopathogenic Erwinia chryzanthemi), i.e. it is a foreign protein for the human body. However, if such asparaginase is hidden inside the body's own cells, this prevents the development of allergic reactions to the introduced foreign protein. The situation is similar with other enzyme preparations that are included in erythrocytes and can be used to treat a number of diseases.

The inclusion of other BACs in erythrocytes, for example, the low molecular weight anthracycline antibiotic doxorubicin or the terpene-indole alkaloids vincristine and vinblastine, which are also widely used in the treatment of a number of oncological diseases [10-12], makes it possible to create a depot of these drugs in erythrocytes, which are gradually released from erythrocytes. carriers, maintaining a sufficient therapeutic concentration of the drug in the bloodstream for a long time. This is important because, despite the high efficacy of these drugs when administered directly intravenously, the doses of drugs that can be administered to the body are limited by their high toxicity, for example, their suppressive effect on the bone marrow [12], cardiotoxicity and nephrotoxicity. [13]. With the introduction of these drugs into erythrocytes, it is possible to get rid of their high peak plasma concentrations at the time of administration, which determine, to a large extent, the toxicity of the drugs. Thus, the inclusion of a number of medicinal enzymes and other biologically active components in erythrocytes can improve the pharmacological properties of these BACs (their pharmacokinetics and pharmacodynamics), as well as reduce their toxic effects.

Currently, the most gentle, effective and sparing methods that make it possible to obtain drug-carrying erythrocytes with properties similar to those of the original erythrocytes are methods based on a reversible hypoosmotic effect on cells.

From the current level of technology, various variations of methods for incorporating asparaginase and other enzymes, as well as low-molecular BACs, into erythrocytes are known, which are based on a reversible hypoosmotic effect on cells:

1. Ropars C., Nicolau Y.C., Chassaigne M. Encapsulating biological active substances into erythrocytes, US Patent 4,652,449, March 14, 1987, A61K 35/18 [14].

2. Pages E., Ropars C., Bailleul C. Apparatus for causing medical products to penetrate into red blood cells. US Patent 5,589,389, December 31, 1996, C12M 1/12, C12M 3/06) [15].

3. a) Magnani M., Panzani I., Bigi L., Zanella A. Method of encapsulating biologically active agents within erythrocytes and apparatus thereof (Dideco Sri, Italy). European patent EP 0882448, publication date 01/12/2005, Bulletin 2005/02, A61K 9/50) [16];

b). Mambrini D, Serafini S. Device and kit for encapsulation in erythrocytes of at least one compound for therapeutic and diagnostic applications (EryDel SpA, Italy). Russian patent RU 2595844, publication date of the PCT application 03.11.2011, A61K 9/50, A61M 1/36, B01J 4/00) [17];

V). Mambrini D., Benatti L., Capogrossi D., Mandolini M. Method for producing red blood cells loaded with one or more substances of pharmaceutical interest and thus obtained red blood cells (EryDel SpA, Italy). Russian patent RU 2670070, publication date of the PCT application 11/13/2014, А61К9/50) [18].

4. Godfrin Y. Lisis/resealing process and device for incorporating an active ingredient, in particular asparaginase or inositol hexaphosphate, in erythrocytes (EryTech Pharma, France). US Patent 8,617,840, data of patent 12/31/2013, C12Q 1/24, C12N 13/00, C12M 1/33, C12M 3/08 [19].

All of these methods are used for the inclusion of enzymes and other BAC in erythrocytes various variants of the hypoosmotic method.

The general principle of this technology is that the erythrocytes are brought into contact with a hypoosmotic solution (i.e., a solution having an osmolality below physiological, of 300 mOsm/kg). As a result, there is a difference in osmotic pressure on both sides of the erythrocyte membrane. To balance the osmotic pressure inside and outside the erythrocyte, water begins to flow into it. The erythrocyte gradually swells, as a result of which, after reaching the maximum possible volume (while maintaining a constant surface area of the erythrocyte), bursting defects (pores) with a diameter of 8 to 50 nm appear in its membrane, through which hemoglobin and other compounds can leave the erythrocyte, and components (including asparaginase or other enzymes and low molecular weight compounds, as well as nanoparticles loaded with BAA) present in the external solution can enter the erythrocyte. The pore diameter was measured experimentally and ranged from 8 to 50 nm [20–23], while it was found that the formation of pores with a diameter of about 10 nm is sufficient for the release of hemoglobin from an erythrocyte [23, 24].

The methods disclosed in US patent 4,652,449 (Ropars C., et al.) [14] allow to work with a wide range of volumes of packed erythrocytes (from 200 ml to several ml), which must first be isolated from the corresponding volume of initial blood (from 450 ml to 10 ml).

The substance to be included in the erythrocytes is introduced by means of a pump into a tube through which the erythrocyte suspension is fed into the dialysis element (before the suspension passes through a heat exchange device that provides the temperature required for cell dialysis).

Common features of all variants of the procedure for incorporating a biologically active component into erythrocytes is the use of a dialysis element (dialyzer) for the lysis of erythrocytes, consisting of two compartments (compartments) separated by a dialysis membrane. The device of dialysis elements can be different. They may contain 2 compartments separated by only one flat semi-permeable membrane, or a plurality of semi-permeable membrane hollow fibers surrounded by an outer compartment. In the first compartment, a suspension of erythrocytes circulates, and in the second, a hypoosmotic solution to ensure the lysis of erythrocytes.

The sealing of the resulting carrier erythrocytes can be carried out by sealing the pores in the membranes of these erythrocytes, while restoring normal osmoticity in a suspension of erythrocytes lysed in the presence of the included drug, by incubating them at a temperature of 20°C to 40°C with a hyperosmotic solution (i.e. a solution with an osmolality higher than the physiological one (300 mOsm/kg) both in a separate tank and in the second dialysis element, in the first compartment of which a suspension of lysed erythrocytes circulates, and in the second a hyperosmotic solution.

In some special cases of performing the method [14], the same dialysis element can be used for the purposes of lysis and sealing of erythrocytes, after appropriate replacement of the buffer in the second compartment and setting the temperature corresponding to each of the stages (from about 0°C to 10°C for lysis and about 20°C to 40°C for RBC sealing).

The method of incorporating biologically active substances into erythrocytes, described in the patent [14], is characterized in that a suspension of erythrocytes in an isotonic solution containing a substance (or substances) with biological activity is continuously fed into the first compartment of the dialysis element, and water is continuously fed into the second compartment of the dialysis element. a saline solution that is hypotonic with respect to the erythrocyte suspension and is designed to lyse the erythrocytes. In this case, the erythrocyte lysate is contacted with one or more biologically active substances. Next, an increase in the osmoticity of the erythrocyte lysate is carried out, which makes it possible to reseal the membrane of erythrocytes, in which one or more biologically active component is included.

As a disadvantage of this method, it should be noted that only a suspension of washed erythrocytes can be used as the initial component, which in turn leads to the need for preliminary washing of erythrocytes from whole blood on an external device. The next disadvantage is the ability to work only with large volumes of washed erythrocyte suspension (obtained from blood volumes from 200 to 450 ml), while working with small volumes of erythrocyte suspension (several ml) is complicated, because the method does not provide for the concentration of cells after lysis, and the kinetics of the process changes with a significant dilution of the initial suspension of erythrocytes, which occurs in this case. Strict selection of dialyzer parameters is required (dialysis membrane area and dialyzer throughput, which is determined, among other things, by the rate of flow of erythrocyte suspension and lysing solution through individual compartments of the dialysis element), as well as a strict selection of the ratios of the membrane areas of dialysis elements for cell lysis and for their sealing, which cannot always be provided by currently existing models of dialysis elements.

In the method disclosed in US Pat. No. 5,589,389 (Pages E., Ropars C. and Bailleul C., 1996), which is in fact a variant of the method according to US Pat. No. 4,652,449, a large volume of initial blood (1 transfusion unit, i.e. ~450 ml of blood) [15]. The method includes the step of obtaining a suspension of washed erythrocytes from whole blood. This method is also based on the reversible hypoosmotic lysis of the original erythrocytes washed at 4°C, their incubation with the included substance (at 4°C) and the subsequent sealing of the obtained carrier erythrocytes by returning the erythrocyte suspension of normal osmolality during incubation with a hyperosmotic solution (at 37°C). WITH). An example of a patent contains information about the inclusion of inositol hexaphosphate in erythrocytes, which is added to the medium already at the second and third stages of washing the original erythrocytes. At the stage of lysis of erythrocytes, the temperature is maintained below 10°C (optimally 4°C), the stage of sealing the obtained erythrocyte carriers occurs while maintaining the temperature above 20°C (optimally 37°C).

The methods disclosed in patents EP 0882448 (Dideco Sri, Italy), RU 2595844 and RU 2 670070C2 (EryDel SpA, Italy) make it possible to work with small volumes (usually 50 ml, rarely up to 100 ml) of whole autologous blood of a patient [16,17, 18]. The original blood is centrifuged to separate the erythrocytes from the plasma, and the erythrocytes are washed with iso-osmotic (ie, having a physiological osmolality of 300 mOsm/kg) saline using a centrifuge bell to wash the erythrocytes. To achieve the opening of pores in the erythrocyte membrane, the method of preliminary gradual swelling (pre-swelling) is used, when erythrocytes are placed sequentially in two hypoosmotic solutions (the osmolality of the second solution is lower than the osmolality of the first one). In patents [16, 17], the osmolality of hypoosmotic solutions is chosen so that after contact with the first solution, erythrocytes swell and spherulate, and after adding the second hypoosmotic solution, erythrocytes are lysed (or partially lysed), with the formation of temporary pores in the erythrocyte membrane. In this case, the suspension of erythrocytes is strongly diluted each time with a hypoosmotic solution (up to a final hematocrit of about 4% -2.5%), therefore, after the stage of incubation with hypoosmotic solutions, the suspension of lysed erythrocytes is concentrated. To do this, a dialysis device is used (for example, a cartridge with hollow fibers made of a semipermeable membrane), in which a suspension of lysed (or partially lysed) erythrocytes is pumped along the internal circuit of the dialyzer, and the lysing solution is partially pumped out of the external circuit into an external receiving device. Then, an aqueous solution of the included BAC is added to the concentrated suspension of lysed cells, which begins to enter the erythrocytes through the pores formed in the erythrocyte membrane. After the end of the incubation process, the osmolality of the external environment is restored to a normal value by adding a hyperosmotic solution to the cell suspension, as a result of which the pores in the erythrocyte membrane close and part of the included component remains inside the carrier erythrocytes. Most of the process steps are controlled automatically by a special electronic unit.

The disadvantage of the method according to the invention is a large loss of the intracellular content of erythrocytes, due to the strong dilution of erythrocytes during incubation and lysis in the presence of hypoosmotic solutions, which, in turn, leads to a decrease in the content of intracellular substances in erythrocytes after their membrane becomes partially lysed. And although macromolecules cannot leave the hemofilter (dialyzer), in which concentration is then carried out, the content of low molecular weight glycolysis metabolites (with a molecular weight less than 10,000 daltons) should decrease when the aqueous solution is pumped out of the external circuit of the dialyzer, because these metabolites first enter a large volume of hypoosmotic solutions at the stage of lysis, and then easily pass through the dialysis membrane at the stage of erythrocyte concentration. In addition, the stages of swelling and lysis of erythrocytes take place at room temperature without cooling the solutions and suspensions to about 4°C, which causes a decrease in the yield of inclusion of the drug, because It is known that the lifetime of pores formed in the membrane decreases as the temperature rises to room temperature.

Patent RU 2670070 [18] describes a variant of the method for incorporating BAC into erythrocytes, in which the loss of intracellular content is reduced, and the efficiency of incorporating BAC is increased relative to the method previously described in patents [16,17] due to the fact that the osmoticity of the second hypoosmotic solution is chosen such that lysis of swollen erythrocytes upon contact with the second hypoosmotic solution does not occur. Erythrocytes swollen after two successive stages of treatment with hypoosmotic solutions, where the osmolality of the second solution is lower than the osmolality of the first hypoosmotic solution, are first concentrated, and then an aqueous solution of the included substance is added to them, which leads to the lysis of swollen erythrocytes (already in the presence of BAC). At the same time, the osmoticity of the suspension of swollen erythrocytes after the addition of the first hypoosmotic solution is 250-200 mOsm/kg, the osmoticity of the suspension of swollen erythrocytes after the addition of the second hypoosmotic solution is 200-170 mOsm/kg, and the osmoticity of the suspension of erythrocytes after two stages of swelling and the addition of the included biologically active component is about 150-110 mOsm/kg.

In the known method disclosed in US patent 8,617,840 (EryTech Pharma, France) [19], which we have chosen as the closest analogue, to reduce the osmolality of the medium outside the erythrocytes, the process of dialysis of the erythrocyte suspension against a hypoosmotic solution is used using a dialyzer in which the erythrocyte suspension flows along the inner contour, and the hypoosmotic solution flows countercurrently along its outer contour. The BAC, which should be included in the erythrocytes, is placed either in the initial suspension of washed erythrocytes (ie, in the internal circuit), or in the suspension of already lysed erythrocytes after they exit the dialyzer. Then, the erythrocytes loaded with BAC are sealed, restoring, as in other methods, the osmolality of the external environment.

The features of this method are such that only washed erythromass can be used as the initial component, work with whole blood is not provided. The volume of this erythromass can be from 10 ml to 250 ml of packed erythrocytes.

Shortly before the lysis stage, the osmotic fragility of the erythrocytes is measured, based on the results of which the lysis conditions (osmolarity of the lysing solution and/or the rate of flow of the cell suspension through the dialyzer) are selected.

Measurement of cell fragility is carried out on an external device on the sample, the initial temperature of which is from +1 to +8°C. The process takes 20-30 minutes.

Based on the obtained data, the cell flow rate in the dialyzer or the osmolarity of the dialysate solution is calculated using the following formulas:

where H ₅₀ is the osmolarity at which 50% of the erythrocytes are lysed, V is the volume of the erythrocyte suspension, A and B are variables that are regulated depending on the parameters of the dialyzer and the osmolarity of the lysing solution; K - adjustable constant.

where C and D are variables that are adjusted depending on the parameters of the dialyzer and the flow rate of erythrocytes in the dialyzer; K - adjustable constant.

The calculated parameters of the lysis process in the dialyzer (the flow rate of the erythrocyte suspension along the internal contour of the dialyzer and/or the osmolarity of the lysing solution) are set manually by the operator, because the coefficients in the equations relating various parameters of lysis with individual indicators of the osmotic fragility of the original erythrocytes should be set separately for each type of dialyzers used, because depend on the parameters of the dialyzer itself.

This method of adjusting and setting the parameters of the lysis process is long and laborious, and cannot be easily used in any laboratory. In addition, its use increases the likelihood of establishing incorrect values due to the likelihood of operator error.

DISCLOSURE OF THE INVENTION

The technical problem that the present invention solves is to eliminate the disadvantages inherent in known methods.

The technical problem solved by the present invention is the creation of a highly efficient and reproducible method for incorporating biologically active components into erythrocytes by the method of flow hypoosmotic dialysis.

The technical result of the present invention is to provide a high and reproducible efficiency (degree) of the inclusion of various biologically active components in erythrocytes, reducing the loss of intracellular content. An additional technical result is the ability to use both whole blood and pre-prepared erythromass as a starting material without losing the efficiency of incorporating biologically active components into erythrocytes.

The efficiency of the incorporation of biologically active components into erythrocytes, according to the present method, is superior to that of known methods.

The specified technical result is achieved by creating a method for incorporating at least one biologically active component into erythrocytes by the method of flow hypoosmotic dialysis, comprising the following steps:

- adding at least one biologically active component to a suspension of concentrated erythrocytes washed from whole blood or to a previously obtained erythromass;

- lysis of erythrocytes in the presence of at least one biologically active component using hypoosmotic flow dialysis;

- incubation of a suspension of erythrocytes, lysed in the presence of at least one biologically active component, with a hyperosmotic solution to form sealed carrier erythrocytes containing at least one biologically active component;

- washing sealed erythrocyte carriers containing at least one biologically active component;

- concentration using dialyzer sealed erythrocyte carriers containing at least one biologically active component;

- flushing out of the dialyzer the sealed carrier erythrocytes remaining there and adding them to previously obtained sealed carrier erythrocytes containing at least one biologically active component;

at the same time, the stage of erythrocyte lysis in the presence of at least one biologically active component is carried out while maintaining a constant transmembrane pressure in the dialyzer of 160-180 mm Hg. and pressure at the inlet to the internal circuit of the dialyzer, not exceeding atmospheric by more than 120 mm Hg, and the concentration of erythrocytes is carried out while maintaining a constant transmembrane pressure in the dialyzer of 90-120 mm Hg. taking into account the measured pressure indicators in the external and internal circuits of the dialyzer.

All of the above process steps are necessary to achieve the claimed technical result. The parameters for carrying out each stage were also chosen so as to ensure the achievement of this technical result.

Concentration of the erythrocyte suspension makes it possible to pump out excess liquid from the suspension, preserving the number of whole cells in the sample as much as possible. To select the transmembrane pressure at the stage of concentrating the erythrocyte suspension, it was investigated how an increase in this pressure affects the hemolysis of erythrocytes in the dialyzer. In FIG. 1 shows the results of experiments on erythrocytes from two different blood samples. As can be seen in the presented figure, at a transmembrane pressure above 120 mm Hg. there is already an increase in hemoglobin content in the fluid drained from the dialyzer, which corresponds to an increase in hemolysis during the concentration process. On the other hand, transmembrane pressure is below 90 mm Hg. acceptable, but greatly increases the duration of the concentration procedure. Thus, the transmembrane pressure range of 90-120 mm Hg was chosen as optimal for the stage of concentrating the erythrocyte suspension. For the same reason, in order not to lose erythrocytes as a result of their hemolysis, the pressure at the inlet to the internal circuit of the dialyzer, through which the erythrocyte suspension passes, should not exceed atmospheric pressure by more than 120 mm. Hg

Since erythrocytes lysed as a result of hypoosmotic dialysis have temporary pores in their membrane, they are able to withstand higher transmembrane pressures without complete cell destruction. To select the optimal range of transmembrane pressure in the dialyzer at the stage of lysis, experiments were carried out in which the percentage of asparaginase encapsulation was determined at different transmembrane pressure (Fig. 2). Based on the experiments, it was found that maintaining the transmembrane pressure in the dialyzer during dialysis of 160-180 mm Hg. provides a consistently high percentage of substance incorporation into erythrocytes. A lower pressure is not sufficient for effective incorporation, and too high a pressure leads to a decrease in the efficiency of inclusion, presumably as a result of more severe hemolysis of the cells. Thus, the range of 160-180 mm Hg was chosen as the optimal range of transmembrane pressure during hypoosmotic dialysis of erythrocytes.

The biologically active component (or components) is added to the original erythrocytes or, in the case when whole blood was used as the starting material, to washed from the original whole blood and then concentrated erythrocytes.

If whole blood is used as the starting material, the step of adding at least one biologically active component to the suspension of concentrated erythrocytes washed from whole blood is preceded by the step of washing erythrocytes from the original whole blood and the step of their subsequent concentration. In particular, the concentration of a suspension of erythrocytes washed from whole blood makes it possible to avoid their strong dilution before adding the BAC and, thus, to carry out the process of subsequent hypoosmotic lysis at a higher hematocrit and concentration of the BAC, which further increases the efficiency of the inclusion of the BAC and reduces the loss of intracellular content in during dialysis.

Thus, an additional advantage of the claimed method is the ability to work not only with washed erythrocyte mass, but also with the whole blood of a patient or a donor.

A diagram of the successive stages of the method is shown in Fig. 3, where the following notation is used:

PN1 - peristaltic pump, through which, at different stages of the process, a suspension of erythrocytes or saline enters the internal circuit of the dialyzer; PN2 and PN3 are peristaltic pumps, through which physiological saline or hypoosmotic solution from the respective bags enter and are pumped out of the external circuit of the dialyzer, respectively; PN1 and PN2 - syringe pumps for supplying the included biologically active component (or components) and hyperosmotic solution, respectively; EN - obtained carrier erythrocytes containing BAC.

Each step of the method is indicated by a separate index. In various particular cases of the implementation of the method, either whole blood or pre-washed erythromass is used as the starting material (indicated as steps a1 or a2 in Fig. 3, respectively, which correspond to the introduction of the starting material into the system). The movement of the suspension of erythrocytes along the lines of the device at each stage of the method is indicated as follows: a1 and a2 introduction of the starting material (initial blood or initial washed erythrocytes, respectively) into the system; b - supply of whole blood from the original bag for washing; c - collection of washed erythrocytes in a bag for suspension of washed erythrocytes; d - concentration of washed erythrocytes; e hypoosmotic dialysis and collection of erythrocytes lysed in the presence of BAC into a bag located in the sealing unit, followed by incubation of a suspension of lysed EN in this bag with a hyperosmotic sealing solution; f - submission of sealed EN for laundering; g - collection of sealed carrier erythrocytes after their washing into a bag for EN suspension; h is the concentration of the obtained EN after their washing.

When whole blood is used as the starting material, steps b-h are performed sequentially after step a1; while when pre-washed erythromass is used as starting material, steps e-h are carried out after step a2.

For the method according to the invention, the following temperature regimes are preferred.

The erythrocyte washing step is carried out at room temperature.

The stage of lysis and concentration of the erythrocyte suspension is carried out while maintaining the temperature from +2°C to +10°C, preferably from +4°C to +6°C.

The stage of sealing lysed in the presence of a biologically active component or components of erythrocytes is carried out at a temperature of from +25°C to +40°C.

Such temperature ranges are standard for most methods of BAC incorporation into erythrocytes, and are dictated by the physiological characteristics of the erythrocyte membrane. It is known that the deformability of an erythrocyte changes with temperature [25]. To create stable pores in the membrane at the dialysis stage, it is necessary to ensure a minimum fluidity of the erythrocyte membrane, which is facilitated by low temperature. However, too low a temperature can lead to erythrocyte aggregation [26], so the above range is considered optimal. At the stage of sealing lysed erythrocytes, it is necessary to ensure optimal fluidity of the erythrocyte membrane in order to quickly close the pores in the membrane. Since the fluidity of the erythrocyte membrane increases with increasing temperature, it is logical to create a temperature close to physiological to restore the shape of the erythrocyte. Temperatures above 40°C can lead to denaturation of proteins in the cell membrane and, as a consequence, to hemolysis of erythrocytes.

Preferred, but not limiting, are the following embodiments of the method.

In a particular embodiment of the method, at the stage of erythrocyte lysis using hypoosmotic dialysis, a constant transmembrane pressure in the dialyzer is maintained at 170 mm Hg, and at the stage of concentration, a constant transmembrane pressure of 100 mm Hg is maintained.

In a particular embodiment of the method, the stage of concentration of the washed erythrocytes is carried out until the hematocrit value of the erythrocyte suspension is 75%-80%.

The method is carried out under sterile conditions.

As a biologically active component (BAC) for inclusion in erythrocytes, a number of substances with any medicinal or biological activity can be used. A wide variety of compounds belonging to various classes of substances and microparticles that have biological activity and can be retained inside the erythrocyte can be included in erythrocytes. The specific examples of molecules given here are not intended to limit the method of the invention, but merely demonstrate the possibilities of specific embodiments. A number of useful BAAs include (but are not limited to) the following substances and classes of compounds: various proteins, including enzymes (eg, asparaginase, glutamate dehydrogenase, alanine aminotransferase, or combinations thereof); peptides, oligopeptides and polypeptides, amino acids; nucleic acids, nucleotides, oligonucleotides, nucleotide phosphates, including di- and triphosphates; nucleoside analogues used as therapeutic agents (immunosuppressants and tumor cell growth inhibitors) such as 6-mercaptopurine, azathiopurine, fludarabine phosphate; antiviral agents such as phosphorylated azidothymidine (AZT), dideoxycytosine (ddC), natural and synthetic immunomodulators (activators and suppressors), for example muramyl dipeptide (MDP) derivatives; substances with anticarcinogenic activity (methatrexate, vincristine, vinblastine, doxorubicin, etc.); hormones (eg, corticosteroids and glucocorticosteroids); non-steroidal anti-inflammatory agents; hemoglobin allosteric regulators (eg inositol hexaphosphate or pyridoxal phosphate); substances that protect hemoglobin or red blood cells (for example, cryoprotectants); prostaglandins; leukotrienes (eg leukotriene II); cytokines; antibodies; α-, β- and γ-interferons; glutathione; toxins; and other substances having pharmacological activity.

In addition, various artificial nanoparticles can be included as a biologically active component in erythrocytes, with included biologically active drugs or compounds. This makes it possible, for example, to use a carrier erythrocyte as a depot form in the case of nanoparticles, which, due to the composition of their surface, have a short circulation time in the bloodstream. In addition, such nanoparticles can have, for example, strong fluorescence or magnetic properties, which can be used to create biosensors, concentrate the drug component in the desired organ using magnets, or enhance contrast when creating biological images, for example, in magnetic resonance imaging ( MRI), etc.

The RBC solution can be saline, Bio-Wash, or other iso-osmotic RBC solutions. For lysis of erythrocytes and their subsequent sealing, hypoosmotic and hyperosmotic solutions are used, respectively.

To implement the claimed method, various installation options can be used, containing a device for washing erythrocytes and a dialysis element designed for lysis of erythrocytes. In particular, a dialysis element designed for concentration and hypoosmotic lysis of red blood cells can be used. The installation may include various pumps for pumping solutions and suspensions, as well as appropriate containers for solutions and suspensions of erythrocytes and lines connecting them, on which valves are installed that control the movement of liquids along the lines. As a device for washing erythrocytes, any known device for such washing can be used, for example, a device from Haemonetics ACP-215 (Haemonetics SA, Switzerland), Cell Saver ^® , etc., or a specially designed unit for washing built into the installation containing a centrifuge , a replaceable centrifuged container, the necessary pumps and bags with the initial and spent solution for washing erythrocytes. As a dialysis element, standard dialysis cartridges can be used, for example, standard pediatric dialyzers from Fresenius (FX Paed) with a volume of 18 ml, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

The claimed method can be illustrated by the following figures:

In FIG. Figure 1 shows the dependence of the hemoglobin concentration in the erythrocyte suspension, which passed through the internal circuit of the dialyzer during the stage of concentration of the erythrocyte suspension, on the level of transmembrane pressure in the external and internal circuits of the dialyzer. Shown are the results of experiments on erythrocytes from two different blood samples, in which the concentration of 10% suspension of erythrocytes was carried out for 8 min at different transmembrane pressures in the dialyzer.

In FIG. Figure 2 shows the dependence of the degree of asparaginase encapsulation on the level of transmembrane pressure in the dialyzer at the stage of hypoosmotic dialysis of erythrocytes in the presence of asparaginase.

In FIG. 3 shows a diagram of the successive stages of the proposed method.

In FIG. 4 shows electron micrographs of the original erythrocytes, as well as carrier erythrocytes obtained from them using the proposed method, containing either asparaginase or combined glutamate dehydrogenase and alanine aminotransferase.

In FIG. 5 shows hematological parameters (erythrocyte indices) of initial erythrocytes and carrier erythrocytes loaded with various BAC, obtained using the proposed method.

In FIG. 6 shows the osmotic resistance of the original erythrocytes and carrier erythrocytes loaded with various BAC, obtained using the proposed method.

In FIG. 7 shows the parameters characterizing the filterability of the original erythrocytes and erythrocytes-carriers of various BAC obtained using the proposed method.

Terms and Definitions

BAC biologically active component;

Room temperature - temperature from 20°C to 25°C;

Working solutions - solutions used at different stages of the implementation of the method of incorporating biologically active components into erythrocytes, which include saline solution, Bio-Wash solution or other iso-osmotic solutions for washing erythrocytes, hypoosmotic and hyperosmotic solutions, etc.;

Source material - the whole blood of the patient or the donor's whole blood corresponding to the group (original blood), or pre-washed erythrocytes (original erythrocytes);

Erythrocytes - red blood cells;

Erythrocyte mass (erythromass, packed erythrocytes) - concentrated suspension of washed erythrocytes with high hematocrit (60-80%);

Carrier erythrocytes (EN) - erythrocytes containing included biologically active component (or components);

Lysed erythrocytes - erythrocytes obtained as a result of reversible hypoosmotic lysis, for example, by dialysis of a suspension of erythrocytes against a hypoosmotic buffer using a dialysis device (dialyzer), in the membrane of which temporary pores have formed;

Lysed carrier erythrocytes - erythrocytes lysed in the presence of BAC, for example, by dialysis against a hypoosmotic buffer containing BAC, in the membrane of which temporary pores have formed and part of the BAC has entered the erythrocytes;

Bioreactor erythrocytes are a special case of erythrocytes loaded with BAC. Erythrocytes loaded with an enzyme preparation capable of carrying out its reaction inside the cells in which it is included;

Sealed carrier erythrocytes - erythrocytes loaded with BAC, with restored cell membrane integrity, obtained by restoring osmoticity in a suspension of lysed carrier erythrocytes by adding a hyperosmotic solution to it;

Efficiency (degree or output) of the inclusion of the BAC (E) - the percentage of the total amount of the BAC introduced into the system, which turned out to be sealed inside the carrier erythrocytes as a result of the procedure for incorporating the BAC into the erythrocytes (percentage or encapsulation yield);

Relative efficiency of BAC incorporation (R) - the percentage that is actually obtained in carrier erythrocytes concentration (or, if enzymes are included, activity) of BAC (per 1 ml of carrier erythrocytes at a hematocrit of 100%) of the maximum possible under given conditions , which is taken as the concentration (or activity) of BAC in a suspension of washed from whole blood or initial pre-washed erythrocytes;

Cell yield (C) - the percentage of cells that remained after the procedure for incorporating the BAC into erythrocytes, from the initial number of cells;

Pharmacocytes - erythrocytes with included biologically active component;

Trunks - plastic or silicone flexible tubes connecting various components of the device;

PBS - saline solution containing 10 mm phosphate buffer, having a pH of 7.4.

IMPLEMENTATION OF THE INVENTION

The following information is provided for a better understanding of the principle of the claimed invention. This information is not limiting, but only illustrates the general principles of the method.

To obtain erythrocytes-carriers of a biologically active component (components), both the whole blood of a patient or a donor with a similar blood group, and pre-washed packaged erythrocytes (erythrocyte mass, erythromass) can be used as a starting material.

Below are examples of the inclusion of biologically active components in erythrocytes by the claimed method of hypoosmotic flow dialysis.

Example 1

Inclusion in erythrocytes of L-asparaginase

Asparaginase (L-asparagine-amidohydrolase, ASP) is an enzyme of the hydrolase class that catalyzes the hydrolysis of L-asparagine with the formation of L-aspartic acid and ammonium ion according to reaction (3):

Asparaginase is an integral part of a complex course of therapy for a number of oncological diseases, for example, ALL in adults and children. Since direct intravenous administration of a free enzyme has serious disadvantages (the main of which are severe allergic reactions, a short lifetime of the enzyme in the bloodstream, a decrease in the effectiveness of the drug with repeated injections due to the formation of antibodies to this enzyme in the plasma, the effect of high peak concentrations of the enzyme at the time administration to the blood coagulation system, pancreas, liver and a number of other body systems, etc.), asparaginase included in erythrocytes is used as a dosage form of asparaginase, which has improved pharmacological properties [7].

To incorporate asparaginase into erythrocytes by the proposed method, in this example, whole blood of donors was used as the starting material, which was taken by puncture of the cubital vein into a standard solution of tribasic sodium citrate dihydrate (3.2%, 0.109 M), at a ratio of citrate : blood of 1: 9, as well as lyophilized L-asparaginase from E. coli Vero-asparaginase (VeroPharm OOO, Russia) at 10,000 IU/vial, the substance of which additionally includes mannitol 50 mg and glycine 50 mg (per 10,000 IU). For flow dialysis, standard pediatric dialyzers from Fresenius (FX Paed) with a volume of 18 ml were used. An asparaginase solution was prepared by diluting the lyophilisate of the enzyme preparation with buffer (0.015 M Tris-HCl, 0.015% bovine serum albumin (BSA), pH 7.3) to a final activity of 4000 IU/mL.

In the experiments performed (n=11), the volume of the initial blood was 100 ml, and the volume of the added asparaginase solution was 0.4 ml (with an activity of 4000 IU/ml).

In each experiment, 100 ml of the donor's original whole blood (with a hematocrit of 39.4% to 45.2%) was placed in a bag at room temperature, which was sterilely connected (soldered) to the RBC washer. As a device for washing erythrocytes, an ACP-215 device (Haemonetics, SA, Switzerland) with a centrifuge bell volume of 275 ml was used.

To remove air from the system of highways, the dialyzer was first flushed. Flushing was carried out for 2 min by pumping through the operation of the pumps through the internal and external circuit of the dialyzer at a rate of 100 ml/min, 200 ml of saline from the bags for saline into the bags for collecting waste solutions. To perform RBC washing, the volume of RBC suspension in the starting material bag should be equal to the volume of the centrifuge bell of the device used to wash RBCs (approximately 275 ml). To increase the volume of material in the original blood bag to this volume, after the end of the dialyzer flush, when the valves and pumps on the external dialyzer circuit stopped working, the valve was opened, through which the saline solution from the saline bags was pumped into the bag by the pump on the internal dialyzer circuit. with original blood at a rate of 100 ml / min for 2 minutes.

Further, to carry out the stage of washing erythrocytes from the original whole blood, the blood from the bag with the starting material, through the operation of the internal pump of the device for washing erythrocytes, entered the centrifuge bell, where, through the operation of another internal pump of this device, the solution for washing erythrocytes from the bags for solutions used for washing erythrocytes. The standard Bio-Wash solution was used as a washing solution, which is a saline solution with the addition of glucose (0.2%). The spent solution for washing entered the waste bag, and the erythrocytes washed from the blood entered the bag for the suspension of washed erythrocytes, which was at a temperature of +4°C. The stage of washing erythrocytes continued until the sensor for the presence of liquid in the line recorded the end of the transfer of the suspension from the washing device to the bag for washed erythrocytes.

Since the volume of the obtained suspension of washed erythrocytes is equal to the volume of the centrifuge bell of the device for washing erythrocytes, i.e. the resulting suspension of washed erythrocytes was sufficiently strongly diluted; before the start of the dialysis stage, the stage of concentration of the washed erythrocytes was carried out. To do this, a suspension of erythrocytes from a bag for washed erythrocytes, located on a shaker and constantly stirred, by means of a pump, moved through the open valve along the internal contour of the dialyzer at a rate of 20 ml/min and was collected again in the same bag. Simultaneously, through the operation of two pumps, along the outer contour of the dialyzer, saline was pumped from the bag for saline into the bag for collecting waste solutions. The pump supplying saline to the external circuit of the dialyzer operated at a constant rate of 20 ml/min, while the speed of the second pump pumping the solution out of the external circuit of the dialyzer was initially higher, but changed so that a constant transmembrane pressure of 100 mmHg was maintained in the dialyzer. .st., which was determined by the difference in pressure recorded by pressure sensors on the inner and outer circuits of the dialyzer. As the hematocrit value recorded by the hematocrit sensor approached a value of the order of 70% 75%, a decrease in the speed of the pump on the external circuit of the dialyzer could no longer maintain a constant transmembrane pressure in the dialyzer at the level of 100 mmHg. At the same time, the pressure at the inlet to the internal circuit of the dialyzer began to increase. When the pressure difference between the inlet and outlet of the internal dialyzer circuit (where the pressure is equal to atmospheric pressure) became greater than 120 mmHg, the pump on the internal dialyzer circuit began to decrease in speed to maintain the transmembrane pressure level of 100 mmHg. The suspension of washed erythrocytes was pumped through the dialyzer until the speeds of the pumps on the external circuit of the dialyzer became equal. At the same time, the hematocrit measurement sensor recorded the hematocrit value of the erythrocyte suspension at the outlet of the internal circuit of the dialyzer equal to 75% -80%. After equalizing the speeds of the pumps on the external circuit of the dialyzer, the concentration stage was completed, the pumps on the external circuit of the dialyzer stopped working, and through the internal circuit of the dialyzer, through the operation of the pump on the internal circuit of the dialyzer, another 35 ml of saline solution entered the bag with a suspension of erythrocytes from the bags for physiological solution. to collect the remaining red blood cells in the dialyzer. The speed of the pump on the internal circuit of the dialyzer was automatically maintained as high as possible, at which the pressure at the inlet to the internal circuit of the dialyzer did not exceed atmospheric pressure by more than 120 mm Hg.

At the same time, an asparaginase solution (0.4 ml, 4000 IU/ml) was supplied to the line leading to the bag with the suspension of washed erythrocytes by means of a syringe pump. The valves were then closed.

Next, the lysis stage was carried out, while first, a hypoosmotic solution (5 mM KH ₂ PO ₄ /K ₂ HPO ₄ , 2 mM MgCl ₂ , 5 mM glucose, 37 mM NaCl, 60 mOsm/kg, pH 7.4) at a rate of 30 ml/min from the bag for the hypoosmotic solution to the bag for collecting waste solutions. Then, from the bag with washed erythrocytes (constantly gently agitated with a shaker), the suspension of erythrocytes with asparaginase was pumped through the internal circuit of the dialyzer by means of a pump and collected in a bag for the collection and subsequent sealing of lysed carrier erythrocytes containing BAC (approximately 10–20 min). Simultaneously, in the external circuit of the dialyzer, the hypoosmotic solution was pumped from the bag for the hypoosmotic solution into the bag for collecting waste solutions by means of the operation of pumps on the external circuit of the dialyzer in the countercurrent of the suspension of erythrocytes with asparaginase coming from the bag with erythrocytes. The speed of the pumps on the external circuit of the dialyzer was the same and amounted to 30 ml/min. During dialysis, a constant transmembrane pressure of 170 mmHg was maintained. This was achieved automatically by adjusting the speed of the pump on the internal dialyzer circuit, which was initially set at 6.4 ml/min, but then the pump on the internal circuit of the dialyzer changed the speed, taking into account the readings of the pressure sensors, in order to maintain the transmembrane pressure in the dialyzer at the selected level of 170 mm Hg at the maximum possible speed of the pump on the internal circuit of the dialyzer, at which the pressure at the inlet to the internal circuit of the dialyzer did not exceed atmospheric pressure by more than 120 mm Hg.

It is preferable that the rate of supply of the hypoosmotic solution to the outer circuit of the dialyzer be close to or equal to the rate of pumping this solution out of this circuit, which does not allow excess fluid to enter the inner circuit of the dialyzer and dilute the erythrocyte suspension located there. As a result, an additional increase in the efficiency of BAC inclusion and a decrease in the loss of intracellular content are achieved by reducing the dilution of the erythrocyte suspension during dialysis. The proximity of the velocities implies a discrepancy within 1 rel. %, preferably within 0.5 Rel. %.

After complete passage of the erythrocyte suspension through the dialyzer, the remaining erythrocytes from the dialyzer were collected into a bag for collection and subsequent sealing of the lysed carrier erythrocytes by pumping another 30 ml of saline through the internal circuit of the dialyzer using a pump. The pump on the internal circuit of the dialyzer was running at a rate of 30 ml/min for about 1 minute. Using a syringe pump, 9 ml of a hyperosmotic solution (1 M NaCl, 50 mM KH ₂ PO ₄ /K ₂ HPO ₄ , 5 mM ATP, 50 mM glucose, 50 mM sodium pyruvate, pH 7.4, 2240 mOsm/kg). The calculation of the required volume of the hyperosmotic solution was made in advance in accordance with formula (4):

where V _hyper is the volume of hyperosmotic solution to be added, V _cycn is the volume of the dialyzed erythrocyte suspension, Osm _{dialysis cycn} is the initial osmolality of the dialyzed suspension, Osm _target is the final osmolality of the suspension to be achieved (equal to approximately 300 mOsm/kg) and Osm _{hyper .} - osmolality of the hyperosmotic solution (2240 mOsm/kg).

In order to calculate the volume of the necessary addition of the hyperosmotic solution, in the experiments performed, where the volume of the initial whole blood was 100 ml, the volumes of the erythrocyte suspension obtained under similar conditions after dialysis (V _cycn ) and the osmolality of this suspension (Osm _{dialysis cycn} ) were measured at the osmolality of the hypoosmotic solution 60 mOsm/kg, which averaged 92.8±14.5 ml and 112.11±5.64 mOsm/kg, respectively (n=9). Based on these measurements, it was calculated that in order to restore normal osmolality in the suspension of cells lysed in the presence of asparaginase, it is necessary to use the addition of a hyperosmotic solution (with an osmolality of 2240 mOsm/kg) equal to 9 ml for every 100 ml of the initial blood volume.

Only after the entire suspension of lysed carrier erythrocytes was collected in a bag for collecting and subsequent sealing of lysed carrier erythrocytes, the thermostat was turned on, which provided a temperature of 37 ° C, and the bag with a suspension of erythrocytes lysed in the presence of asparaginase was incubated at 37 °C and constant shaking with a shaker for 30 minutes to ensure sealing of the obtained carrier erythrocytes.

Next, the resulting sealed carrier RBCs containing asparaginase were washed with a Bio-Wash RBC washer and collected in a clean bag for the resulting sealed and washed carrier RBCs. To do this, they first, through the operation of the internal pump ACP-215, entered the centrifuge bell of the device for washing erythrocytes (ACP-215). Simultaneously, an additional 200 ml of saline solution was delivered from the saline bags through the internal dialyzer circuit to the bag for collection and subsequent sealing of lysed carrier erythrocytes by the operation of the pump on the internal circuit of the dialyzer (the speed of the pump on the internal circuit of the dialyzer was 100 ml/min). The sealed carrier RBCs were washed and collected in a clean bag for receiving sealed and washed carrier RBCs. The resulting highly diluted suspension of washed, sealed carrier RBCs was then concentrated by repeating the step described above for the concentration of RBCs washed from the original blood, except that the concentrated carrier RBCs were collected in a bag for the resulting sealed and washed carrier RBCs. The pump pumped the suspension through the inner circuit of the dialyzer at a rate of 20 ml/min, and the pumps on the outer circuit of the dialyzer pumped saline from the saline bag into the waste collection bag (the speed of one pump on the outer circuit of the dialyzer was constant at 20 ml/min). min, and the speed of the other pump on the external circuit of the dialyzer was variable and set at a level that provided a constant transmembrane pressure of 100 mmHg).

In all experiments, the enzyme encapsulation yields (E) and cell yields after the enzyme inclusion procedure (C) were calculated according to formulas (5) and (6), respectively:

where A _ref.cycn.ER and A _cycn.EN are the activities of the enzyme in the initial suspension of washed erythrocytes and in the final suspension of carrier erythrocytes with the included enzyme, respectively, V _ref.cycn.ER and V _cycn.EN are volumes, and Ht _{ref .cycn.ER} and Ht _cycn.EN are the hematocrits of these suspensions, respectively.

In addition, another indicator of incorporation efficiency, the relative incorporation efficiency (R), was estimated as the percentage that the specific activity of the enzyme obtained in erythrocytes is from the maximum possible specific activity of the enzyme that can be obtained under given conditions (formula (7)). Specific activity refers to the amount of enzyme activity per ml of carrier cells (at 100% hematocrit). At the same time, the activity of the included substance in the suspension of initial erythrocytes was taken as the maximum possible included specific activity immediately after asparaginase was introduced into the suspension of erythrocytes washed from the original blood (before the start of the inclusion procedure), because it is this activity that must be established in the cells and the environment if the substance is completely balanced between these phases.

Asparaginase activity was measured by the indooxin method using aspartate-β-hydroxamate (AHA) as the asparaginase substrate and the resulting hydroxylamine was determined spectrophotometrically at a wavelength of 705 nm after its conversion to indooxin in the presence of 8-hydroxyquinoline [27]. To measure the activity of asparaginase incorporated into erythrocytes, the cells were first lysed by diluting 200 times with water, and then the activity of the enzyme in the lysates was determined (for convenient measurement, the activity of asparaginase in solution should be approximately 100 IU/l). The results obtained are presented in Table 1.

Example 2

Joint incorporation of glutamate dehydrogenase and alanine aminotransferase into erythrocytes

The state of hyperammonemia (increased ammonium concentration in the blood) is a dangerous complication of many pathological conditions associated with urea cycle enzyme deficiencies, chronic and acute liver pathologies (cancer, cirrhosis, etc.), as well as some infections of the gastrointestinal tract.

Excess ammonium has a strong neurotoxic effect, which can cause a number of cognitive disorders and even death [28]. Since excess ammonium is highly toxic, it must be quickly removed from the bloodstream, but existing medications are not effective enough. It takes 2 to 7 days to return the ammonium concentration to normal levels (≤60 μM). It was previously established [29] that the joint inclusion of two enzymes, glutamate dehydrogenase (GDH) and alanine aminotransferase (AAT), into erythrocytes makes it possible to create bioreactor erythrocytes that effectively remove excess ammonium from the bloodstream, according to reactions (8) and (9):

- ион аммония, а НАДФ и НАДФН - окисленная и восстановленная формы никотинамидадениндинуклеотидфосфата, соответственно.where AKG - α-ketoglutarate, GLU - glutamate, PIR - pyruvate, ALA - alanine,

- ammonium ion, and NADP and NADPH - oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate, respectively.

To obtain carrier erythrocytes loaded jointly with GDH and AAT, pre-washed packaged erythrocytes obtained in a standard way at a blood transfusion station, as well as solutions with different activities of bacterial GDH from Proteus, sp. (Toyobo Co. Ltd., Osaka, Japan) and porcine heart AAT (Sigma-Aldrich, St. Louis, MO, USA). The initial aqueous solution of GDH had an activity of 1945 IU/mL (at pH 7.4). The volume of his addition was from 0.065 to 0.784 ml. The original AAT was freeze-dried and had an activity of approximately 75 IU/mg of protein. To be added to the system, this enzyme was preliminarily diluted with buffer (0.1 M Tris-HCl, pH 7.4) to obtain a solution with an activity of 250 IU/mL. The volume of addition of this enzyme to the erythrocyte suspension in different experiments ranged from 0.319 to 14.84 ml. Thus, the total volume of the addition of the enzyme mixture varied in different experiments, as a result of which the total volume of the suspension of erythrocytes with the addition of enzymes also varied.

In all experiments (n=17), the flow-through hypoosmotic dialysis method described in Example 1 for asparaginase was used to incorporate enzymes into erythrocytes, with the exception of the first steps of the method, because the pre-washed erythrocyte mass described above was used as starting material in this example.

From 52.5 to 60 ml of a suspension of erythrocytes in physiological saline with a hematocrit of 80%, previously washed by standard centrifugation, was placed in a bag. The bag was then sterilely attached (soldered) to the lines leading to the three channels of the disposable line system of the device in the sealing unit, leading to the hematocrit sensor, the fluid presence sensor in the line, and the valve that allows access to the bag from the internal circuit of the dialyzer.

The dialyzer was flushed for 2 minutes; for this, 200 ml of physiological saline was passed through the operation of the pumps through the internal and external circuits of the dialyzer at a rate of 100 ml/min. At the end of rinsing, a mixture of solutions of included enzymes (in different experiments from 0.384 ml to 15.624 ml) was introduced into the line leading to the bag with erythromass by means of a syringe pump, and after that, by means of pump operation, for 15 sec at a rate of 20 ml/min another 5.0 ml of physiological saline was passed through the internal circuit of the dialyzer into the bag in order to completely flush the introduced mixture of enzymes into the bag. After that, the valves were closed. The calculated hematocrit of the enzyme-added erythrocyte suspension obtained thereafter in the bag ranged from 59.0 to 70% (average 66.6±4.6%, n=17). The activities of the enzymes added to the initial suspension of erythrocytes were also different and ranged from 2.11 to 18.74 IU/ml for GDH suspension and from 1.33 to 45.6 IU/ml for AAT suspension.

All subsequent stages of the method (hypoosmotic dialysis of erythrocytes in the presence of added enzymes, sealing the resulting suspension of erythrocytes lysed in the presence of enzymes, washing the sealed carrier erythrocytes and their subsequent concentration) were carried out strictly according to the protocol described in Example 1 and under the same conditions.

As in Example 1, the incorporation efficiencies E and R were determined (separately for each enzyme) by formulas (5) and (7), as well as the cell yield as a result of the enzyme incorporation procedure (by formula (6)). The results obtained are also presented in Table 1.

Comparison of the results obtained in Examples 1 and 2 showed that the yield of cells when using washed erythromass as a starting material was higher. This can be explained by the fact that all the old and fragile cells present in the original blood in this case were washed out at the stage of preparation of the erythromass. The second fact that can be observed in Example 2 is that the percentage of incorporation of the two enzymes used is different. GDH from Proteus sp. similar to the well-studied bovine liver GDH, which is known to have an ellipsoidal molecule with a molecular weight of 340 kDa, a length of 13.6 nm, and a diameter of 4.3 nm [30,31]. GDH molecule from Proteus sp. also has an elongated shape, but a slightly lower molecular weight (300 kDa) [32]. At the same time, the globular AAT molecule has a molecular weight of only 150 kDa and a diameter of about 4 nm [33]. Thus, the size of the GDH molecule is much larger than that of AAT, which reduces the rate of penetration of this enzyme into the cell through the pores of the erythrocyte membrane, which have a diameter of about 8-10 nm.

The efficiency of BAC incorporation into erythrocytes by the flow-through hypoosmotic dialysis method of the present invention was also compared with the efficiency of BAC incorporation into erythrocytes by reversible hypoosmotic exposure in accordance with other existing methods. Table 2 presents such indicators given in published works or calculated by us independently from published data on the inclusion of the LHC, using each of the known methods.

As an indicator of incorporation in various studies, the authors calculated either the relative incorporation efficiency (K) (i.e., what percentage of the specific activity included in the cells is from the maximum possible under given conditions), or the total percentage of encapsulation of the substance (E) (i.e. the total percentage of the substance included in erythrocytes using the method used), calculated by formulas (7) and (5).

The data presented in Table 2 demonstrates that the efficiency of switching on the LHC using the proposed method is superior to similar efficiencies obtained in other methods.

To assess the quality of carrier erythrocytes obtained by the claimed method, their properties were studied and compared with those of the original erythrocytes.

Properties of carrier erythrocytes obtained by the claimed method

In addition to the effectiveness of BAC incorporation into erythrocytes, to check the quality of BAC-carrier erythrocytes in comparison with the properties of the original erythrocytes, such properties as the shape of cells after the inclusion of BAC, erythrocyte indices of cells, their osmotic resistance and ability to deform were studied.

A. The shape of the cells after the procedure for incorporating the enzyme into erythrocytes

The shape of the resulting carrier erythrocytes can indicate the physical state of the cells after the enzyme is switched on. In the present work, this form was studied by differential interference-contrast (confocal) microscopy. To obtain micrographs, erythrocytes were first fixed. To do this, they were incubated in PBS with albumin (10 mg/ml) and glutaraldehyde (2.5%) for 1 hour at room temperature. The hematocrit of the cell suspension at fixation was 25%. This suspension was then diluted to a hematocrit of about 0.1%. The photographs were taken with a Zeiss Axio Observer Z.1 microscope (Carl Zeiss, Jena, Germany), 100x immersion objective, 1.3 NA, QuantEm 512sc camera.

In FIG. 4 shows micrographs of the original erythrocytes (A), as well as carrier erythrocytes with included asparaginase (B) or GDH from Proteus sp. and AAT from pig heart (B).

From the analysis of photographs, it can be concluded that for both types of carrier erythrocytes containing various BAAs, most of the obtained cells return their biconcave shape after the restoration of the medium osmolality, which favors the ability of erythrocytes to deform and pass through narrow capillary vessels in the body. The size of most erythrocytes with included GDH and AAT practically did not differ from the size of the original erythrocytes, while for erythrocytes loaded with asparaginase, it decreased very slightly.

B. Hematological parameters of carrier erythrocytes obtained by the claimed method

The main standard hematological parameters of cells (erythrocyte indices) were studied for the original erythrocytes (n=9), as well as carrier erythrocytes loaded with glutamate dehydrogenase from Proteus sp. together with porcine heart alanine aminotransferase (GDH+AAT) (n=6) or asparaginase (n=9). As the main indicators, the following were studied: the average erythrocyte volume (MCV, in fl), the average hemoglobin content in the erythrocyte (MCH, in pg) and the average hemoglobin concentration in the erythrocyte (MCHC, in g/dl). Hematological parameters were measured on a Micros OT hematological analyzer (ABX-France, France).

The results obtained are presented in Fig. 5. After the inclusion of various enzymes in erythrocytes, a moderate change in erythrocyte indices was observed. The average cell volume, average content and average concentration of hemoglobin in them always significantly decreased (ANOVA, p<0.05). This is in complete agreement with the data obtained earlier in other works [35]. Despite the fact that the concentration and content of hemoglobin in carrier erythrocytes are reduced compared to the original erythrocytes, and, consequently, their ability to carry oxygen is also reduced, this does not critically affect the performance of the function of gas exchange by erythrocytes as a whole, because the injected volume of carrier erythrocytes will not exceed 10% of the total volume of erythrocytes in human blood. Therefore, even if the concentration of hemoglobin in them is reduced by 50%, the total content of hemoglobin in the body will decrease insignificantly (by about 5%).

Since the size of glycolytic enzyme molecules is close to the size of hemoglobin molecules, it can be assumed that the concentration of these enzymes during hypoosmotic dialysis and opening of pores in the cell membrane will decrease by no more than 30%, as in the case of Hb. Such a decrease cannot affect the rate of glycolytic reactions in the erythrocyte, because all the necessary enzymes are present in it in significant excess. As a result, it can be assumed that the level of cell energy supply will not change significantly, which will allow carrier erythrocytes to survive in the bloodstream for a long time [36].

B. Osmotic resistance of carrier erythrocytes

To measure the osmotic resistance of various erythrocytes, suspensions of the original erythrocytes or obtained carrier erythrocytes were diluted in PBS to a hematocrit of 5%. The measurements were carried out in solutions with different osmolality according to the procedure described in [37]. The curve of osmotic resistance was constructed as a dependence of the percentage of non-lysed erythrocytes on the osmolality of the solutions used. Curves of osmotic resistance of initial erythrocytes (n=13) and carrier erythrocytes obtained by the method of flow hypoosmotic dialysis with GDH from Proteus sp. and AAT from porcine heart (n=9), as well as with included asparaginase (n=13) are presented in Fig. 6 (means ± mean error of the mean, Mean ± SEM) are presented for each point.

The shape of the osmotic resistance curve for carrier erythrocytes of both types was quite different from the curve shape for initial erythrocytes, which is consistent with the results obtained in other studies on the measurement of the osmotic resistance of carrier erythrocytes [35,38]. Osmotic resistance curves for carrier erythrocytes became flatter and shifted towards low osmolality. Thus, carrier erythrocytes are more resistant than initial erythrocytes to changes in the osmolality of the medium in the region of osmolality below 150 mOsm/kg. Lysis of 50% of the original (native) cells occurs already at an osmolality of 135 mOsm/kg, while in carrier erythrocytes at this osmolality, only 25–30% of lysed cells are observed. This can be explained by the fact that erythrocytes resistant to osmotic stress were removed during the procedure of switching on the BAC at the stage of cell washing, and also by the fact that carrier erythrocytes have a reduced hemoglobin content, which affected the overall curve of osmotic resistance. On the other hand, in the region of osmolality close to physiological, the osmotic resistance of carrier erythrocytes almost does not differ from the osmotic resistance of the original erythrocytes.

D. Deformability of obtained erythrocyte-carriers of BAC

The viability of BAC-carrier erythrocytes (pharmacocytes) in the bloodstream is largely determined by their ability to deform and easily pass through narrow capillary vessels. The deformability of erythrocytes can be determined using filtration methods, tk. the rate of filtration of a suspension of erythrocytes or carrier erythrocytes through an artificial membrane filter with pores 3–5 μm in diameter, close to the diameter of erythrocytes (~3–5 μm) and 10 μm long, directly depends on this ability of the cells. The filterability of cells was assessed by the following parameters: the percentage of non-filterable erythrocytes in the suspension (Z) and the filterability index (F), which were determined by successive passage through an artificial polyethylene terphthalate filter (Joint Institute for Nuclear Research, Dubna, RF) with pores 3.5 μm in diameter of a fixed volume (250 μl) first with PBS buffer, and then with a cell suspension with a hematocrit of 0.1%. Then, after washing the filter after passing the suspension, the same volume of PBS was passed through it again. The calculation was carried out according to formulas (10,11) in accordance with [39]:

where t _b is the time of flow through the filter of 250 μl of buffer (PBS), t _s is the time of flow through the same filter of 250 μl of 0.1% cell suspension (initial erythrocytes or carrier erythrocytes containing BAC), t is the total number of cells placed on filter, and N is the number of pores that were blocked by non-filterable cells when the suspension was passed through the filter.

The value of N was determined according to formula (12):

where N ₀ is the known total number of pores on the filter, at _b1 is the re-flow time of 250 μl of buffer through the filter, which was washed after passing the erythrocyte suspension through it.

The filterability parameters of initial and carrier erythrocytes are shown in FIG. 7, which shows the percentage of non-filterable erythrocytes (Z) and the filterability index (F) for the original erythrocytes (1) (n=8), freshly obtained carrier erythrocytes, co-loaded with GDH from Proteus sp. and AAT (2) (n=5), freshly obtained carrier erythrocytes loaded with L-asparaginase (3) (n=3), and carrier erythrocytes loaded with L-asparaginase after incubation in storage buffer for 2 hours at room temperature (4) (n=3). In all cases, mean values ± SD are given.

The percentage of non-filterable cells in the suspension of freshly obtained carrier erythrocytes of both types was significantly higher compared to this parameter of native (initial) erythrocytes (ANOVA, p<0.05). To understand how irreversible these changes are, for asparaginase-loaded erythrocytes, the filterability of not only freshly prepared carrier erythrocytes was measured, but also their filterability after 2 hours of incubation at room temperature in cell storage buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , 1.3 mM CaCl ₂ , 5 mM MgCl ₂ , 10 mM glucose, 5% (by mass) BSA, 30 mM HEPES, 0.28 mM adenine, and 0.02 mg/mL ampicillin (pH 7.4) ). It was shown that after incubation, the percentage of non-filterable cells in the suspension of carrier erythrocytes decreases (relative to the suspension of freshly obtained carrier erythrocytes) and does not differ significantly from the values of this parameter for the initial erythrocytes (ANOVA, p<0.05).

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Claims (27)

1. A method for incorporating at least one biologically active component into erythrocytes by the method of flow hypoosmotic dialysis, comprising the following steps: - adding at least one biologically active component to a suspension of concentrated erythrocytes washed from whole blood or to a previously obtained erythromass; - lysis of erythrocytes in the presence of at least one biologically active component using hypoosmotic flow dialysis; - incubation of a suspension of erythrocytes, lysed in the presence of at least one biologically active component, with a hyperosmotic solution to form sealed carrier erythrocytes containing at least one biologically active component; - washing sealed erythrocyte carriers containing at least one biologically active component; - concentration using dialyzer sealed erythrocyte carriers containing at least one biologically active component; - flushing out of the dialyzer the sealed carrier erythrocytes remaining there and adding them to previously obtained sealed carrier erythrocytes containing at least one biologically active component; at the same time, the stage of erythrocyte lysis in the presence of at least one biologically active component is carried out while maintaining a constant transmembrane pressure in the dialyzer of 160-180 mm Hg. Art. and pressure at the inlet to the internal circuit of the dialyzer, not exceeding atmospheric by more than 120 mm Hg. Art., and maintaining the temperature from +2°C to +10°C; washing of erythrocytes is carried out at room temperature; the concentration of erythrocytes is carried out while maintaining a constant transmembrane pressure in the dialyzer 90-120 mm Hg. Art. taking into account the measured pressure indicators in the external and internal circuits of the dialyzer and maintaining the temperature from +2°С to +10°С; the stage of sealing lysed in the presence of a biologically active component or components of erythrocytes is carried out at a temperature of from +25°C to +40°C; moreover, the stage of concentration of erythrocytes washed from whole blood and the stage of concentration of sealed erythrocyte carriers containing at least one biologically active component are carried out until the hematocrit value of the erythrocyte suspension is 75–80%. 2. The method according to claim 1, further comprising the steps of washing erythrocytes from whole blood and their subsequent concentration, preceding the steps of adding at least one biologically active component to a suspension of concentrated erythrocytes washed from whole blood. 3. The method according to claim 1 or 2, in which at the stage of lysis of erythrocytes by hypoosmotic dialysis, a hypoosmotic solution is passed through the external circuit of the dialyzer. 4. The method according to claim 1 or 2, in which at the stage of erythrocyte lysis by hypoosmotic dialysis, a suspension of erythrocytes with at least one biologically active component is passed through the internal circuit of the dialyzer. 5. The method according to claim 1 or 2, in which at the stage of lysis of erythrocytes maintain a constant transmembrane pressure in the dialyzer 170 mm Hg. Art. 6. The method according to claim 1 or 2, in which at the stage of concentrating the suspension of erythrocytes maintain a constant transmembrane pressure in the dialyzer 100 mm Hg. Art. 7. The method according to claim 1 or 2, in which at least one biologically active component is selected from the group: - proteins, including enzymes, peptides, oligopeptides, polypeptides, amino acids; or - nucleic acids, nucleotides, oligonucleotides, nucleotide phosphates, nucleoside analogs; or - antiviral agents, natural or synthetic immunomodulators, substances with anticarcinogenic activity; hormones; non-steroidal anti-inflammatory agents; allosteric regulators of hemoglobin, substances that protect hemoglobin or erythrocytes, prostaglandins, leukotrienes; cytokines; antibodies, α-, β- or γ-interferons, glutathione, toxins; or - nanoparticles containing components of pharmacological or diagnostic significance. 8. The method of claim 7, wherein the included enzyme is asparaginase, or co-enzyme pairs of glutamate dehydrogenase and alanine aminotransferase, or alcohol dehydrogenase and acetaldehyde dehydrogenase. 9. The method of claim 7, wherein the nucleoside analog is 6-mercaptopurine, azathiopurine, or fludarabine phosphate. 10. The method of claim 7 wherein the antiviral agent is a phosphorylated azidothymidine or dideoxycytosine. 11. The method according to claim 7, wherein the natural or synthetic immunomodulator is a muramyl dipeptide derivative. 12. The method of claim 7, wherein the anticarcinogenic agent is methotrexate, vincristine, vinblastine, or an anthracycline antibiotic. 13. The method of claim 7, wherein the anthracycline antibiotic is doxorubicin, daunorubomycin, or mitoxantrone. 14. The method of claim 7 wherein the hormone is a corticosteroid or a glucocorticosteroid. 15. The method of claim 7 wherein the hemoglobin allosteric regulator is inositol hexaphosphate or pyridoxal phosphate. 16. The method of claim 7 wherein the hemoglobin or erythrocyte protecting agent is a cryoprotectant.

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