RU2369386C2 - Method of production of polyelectrolyte nano- and microcapsules with protein content - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: at first component microspherolites of carbonates of alkaline earth metals and encapsulatable protein are prepared, then capsules are made by sequential adsorption into composite microsperolites of oppositely charged polyelectrolytes to be preselected by sequential examination of each polyelectrolyte effect on encapulatable protein in the solution. The carbonates of alkaline earth metals are removed from capsules by means of EDTA or another chelating agent or by means of acidification. Polyelectrolytes are taken as biodegradable biodegradation deficient complex cations and polyanious of predominantly linear structure. After absorption of required quantity of polyelectrolyte layers the protein activity shall be controlled.

EFFECT: retention of functional properties of protein during its encapsulation.

6 cl, 2 ex, 5 dwg

Description

The present invention relates to the field of biochemistry, mainly polymer nanotechnology.

Significant objects of polymer nanotechnology include polyelectrolyte nano- and microcapsules (PNMK). PNMK have a number of interesting properties. Of these, the most striking is the semi-permeability of the polyelectrolyte shell of the capsules - permeability to low molecular weight compounds and their small aggregates and impermeability to high molecular weight substances and large particles (Antipov AA, Sukhorukov G.V. // Advances in Colloid and Interface Science. 2004. V.111. No. 1-2. P. 49-61 .; Antipov AA, Viera, E., et al. (2002). Controlled and sustained release properties of polyelectrolyte multilayer capsules. WO 0217888).

Available data in the literature indicate the widespread use of PNMK in a number of areas of technology, chemistry, physics, biotechnology and pharmacology (Möhwald H., Donath E. and Sukhorukov G.V. In book: Multilayer Thin Films. Sequential Assembly of Nanocomposite Materials (Ed . By G. Decher and JBSchlenoff). Wiley-VCH, 2002, P.363-392 .; Fery A. Mohwald H., Sukhorukov G.V. // Progr. Polymer. Sci. 2005. V.30 (8 -9) .P.885).

The possibility of using PNMK to obtain prolonged drugs has been shown (Radtchenko IL, Sukhorukov GB, Mohwald H. (2002). A novel method for encapsulation of poorly water-soluble drugs: precipitation in polyelectrolyte multilayer shells. // Intern. J. Pharmaceutics, 242 (1-2): 219-223). The results obtained indicate the advantage of this approach compared, for example, using microgels, micelles, or liposome encapsulation. In contrast to the latter, a spatially organized supramolecular structure resistant to the action of oxygen and other oxidizing agents is formed by the method of layer-by-layer polyelectrolyte encapsulation (PPC). The attractiveness of the PPC method for the preparation of carriers and containers of biologically active substances and, accordingly, prolonged drugs, possibly with targeted delivery, lies in its simplicity. Recently, using the method of layer-by-layer polyelectrolyte encapsulation, the development of new methods for the enzymatic determination of the concentrations of the analyzed low molecular weight substance in a complex biological fluid has begun. This line of research is based on the work on the inclusion of enzymes in polyelectrolyte microcapsules (PE microcapsules).

Currently, there are a number of approaches to loading proteins into polyelectrolyte microparticles. The first of them was proposed in 1999 (Bobreshova M.E., Saburova E.A. et al. // Biophysics. 2000. V.44. No. 5. P.813-820). This method includes the stage of formation of protein microaggregates in the process of salting out and the subsequent stage of alternating adsorption of oppositely charged polyelectrolytes on these microaggregates. The inclusion of the enzyme in the polyelectrolyte microparticle was ~ 70% of the number of protein aggregates, but its activity was noticeably reduced and depended on a number of factors. The main disadvantage of this method is that the microparticles containing the enzyme are irregular in shape and their size cannot be controlled.

There is a method of incorporating protein directly into hollow PNMCs, in the shells of which pores were specially created (the shell was loosened) by adding an organic solvent, changing the salt composition and pH of the medium for protein to penetrate through the shell into the capsule. The amount of protein incorporated in this way into the capsule was less than 10%, while most of it was concentrated in the shell of the microcapsule (Lvov Y., Antipov AA, Mamedov A. et al. // Nanoletters 2001. V. 1. No. 3. P. 125 -128).

Most noteworthy is the recently proposed and adopted as a prototype method of incorporating proteins into a polyelectrolyte microcapsule, the main stages of which are the preliminary formation of the core of the capsule - “core”, in the form of CaCO ₃ microspherulite with a protein incorporated in it, followed by this stage of layer-by-layer sorption of polyelectrolyte on such a box. The final step in protein encapsulation is the removal of CaCO ₃ from the capsule by the addition of EDTA or another chelating agent. This method can include more than 60% of the protein added to the solution. The method is described in detail in the work of A.Petrov, D.Volodkin, G.Sukhorukov. Protein-Calcium Carbonate Coprecipitation: A tool for protein encapsulation. // Biotechnol. Progr., 2005, v. 27, p. 918-925.

In this work, it is assumed that polyelectrolyte shells obtained by the method described above are inert with respect to the proteins contained in them, including enzymes.

However, our studies have shown that many polyelectrolytes cause the inactivation of enzymes, which are protein in nature. From the data obtained by us, it follows that the activity of encapsulated proteins is substantially determined by the nature of the polyelectrolytes that make up the shell, and can decrease up to complete inactivation.

Thus, the method adopted by us as a prototype in the form presented by the authors has a serious drawback, which consists in the fact that the method does not provide for the effect of the polyelectrolyte membrane on protein functions.

The problem to which the invention is directed, is to provide the possibility of maintaining the activity of the protein, in the particular case of the enzyme, when it is encapsulated in a polyelectrolyte shell.

The problem is solved in that a method for producing protein-loaded polyelectrolyte nano- and microcapsules is proposed, which includes the production of composite microspherulites of alkaline earth metal carbonates and encapsulated proteins (enzymes), the formation of capsules by alternately adsorbing oppositely charged polyelectrolytes on said composite microspherolites and removing said alkaline earth metal carbonates these capsules, in which according to the invention pre-conduct the selection of at least howl pair polyelectrolytes alternately examining the impact of each polyelectrolyte in solution Activity on protein encapsulated.

Moreover, as the preferred polyelectrolytes for capsule formation for a specific, predetermined protein, polyelectrolytes are selected that do not affect the concentration of the protein that is optimal for the formation of the capsule or have the effectiveness of inhibiting at least one protein function to the desired limit.

In the particular case of the method, the testing of the polyelectrolyte is carried out by mixing a protein solution with a solution of polyelectrolyte.

In the particular case of the method, an enzyme is taken as a protein.

The effect of the polyelectrolyte on the inhibition of the enzyme is determined when the concentration of this polyelectrolyte is optimal for the formation of the specified capsule, usually 1.5-3 mg / ml

To form the capsule, polyelectrolytes are taken both biodegradable and polycations and polyanions incapable of biodegradation, mainly of linear structure.

For the formation of microspherulite (bark), CaCO _{3 is} predominantly taken.

Alkali metal carbonates from these capsules are removed using EDTA or another chelating agent or by acidification of the medium.

The essence of the invention lies in the fact that it is proposed to form the capsule shell only from selectively selected polyelectrolytes. This selection is especially important for the formation of the first layer of the membrane, since it is in direct contact with the encapsulated protein. This is due to the fact that, as we experimentally established, many polyelectrolytes, when interacting with an enzyme, are capable of inactivating it. Therefore, the technology for producing PNMCs containing proteins and, in particular, enzymes, should include control over the inhibitory effect of polyelectrolytes used to construct the capsule shell.

The non-obviousness of the proposed invention lies in the fact that before performing a complex capsule formation procedure, as a result of which a capsule with an inactive protein can be obtained, we proposed to preliminarily check the effect of each individual polyelectrolyte on the functional activity of the encapsulated protein.

The invention is confirmed by the fact that a large set of polyelectrolytes was studied, both biodegradable and non-biodegradable, in contrast to the prototype, in which capsules composed of only a non-biodegradable pair - polyallylamine (PAA) and polystyrenesulfonate (PSS), or rather capsules with a shell composition, were prepared (PAA / PSS) ₅ .

We have studied the ability to inactivate proteins for 8 different polyelectrolytes, 4 biodegradable and 4 non-biodegradable. Such inactivation studies were carried out mainly on two enzymes: urease and lactate dehydrogenase (LDH). The dependences of the activity of these enzymes on the concentration of polyelectrolytes were obtained.

An analysis of these dependencies led to the conclusion that the simplest criterion for choosing polyelectrolytes for encapsulation is the enzyme activity when polyelectrolyte is added to the medium with a concentration of 1.5-3.0 mg / ml, while the loss of enzyme activity should not exceed 50% of the initial value .

For a better understanding of the invention, the following are examples of specific implementation of the proposed method with reference to the accompanying drawings, where

Figure 1 shows the dependence of the activity of lactate dehydrogenase (LDH) on the concentration of biodegradable polyelectrolytes: dextran sulfate (DS), polyphosphate (PF), polyuridylate (PU) and polyadenylate (PA). The reaction mixture for determining the activity of LDH: pyruvate 1 mm; NADH 0.2 mM; LDH 0.5 μg / ml; 0.05 M Tris-HCl buffer pH 6.2.

Figure 2 shows the dependence of LDH activity on the concentration of non-biodegradable polyelectrolytes: the dependence of LDH activity on the concentration of polyelectrolytes - polystyrene sulfonate (PSS), polyallylamine (PAA), polydiallyldimethylammonium (PDADMA), polymethacrylate (PM). The reaction mixture for determining the activity of LDH: pyruvate 1 mm; NADH 0.2 mM; LDH 0.5 μg / ml; 0.05 M Tris-HCl buffer pH 6.2.

Figure 3 shows the dependence of urease activity on the concentration of non-biodegradable polyelectrolytes: polystyrenesulfonate (PSS), polyallylamine (PAA) and polydiallyldimethylammonium (PDADMA). Conditions for determining the activity of urease: urease 0.5 μg / ml, urea 125 mm, bromcresol violet 0.015 mm, pH 6.2.

Figure 4 is a diagram illustrating the activity of urease enclosed in microcapsules formed from various non-biodegradable polyelectrolytes. The composition of the capsules: PSS (PSS / PAA) ₃ - 1, PSS (PSS / PDADMA) ₃ - 2.

Fig. 5 is a diagram illustrating the activity of lactate dehydrogenase (LDH) encapsulated in microcapsules formed from various biodegradable and non-biodegradable polyelectrolytes. The composition of the capsules: (PAA / PSS) ₃ - 1; (PDADMA / MSS) ₃ - 2; (PAA / DS) (PAA / PSS) ₂ to 3; (PAA / DS) ₂ (PAA / PSA) - 4, (PAA / DS) ₃ - 5.

Examples of carrying out the invention

To confirm the feasibility of the invention, the following reagents were used:

- lactate dehydrogenase (E.C. 1.1.1.1.27) (LDH) from pig skeletal muscle (M ₄ isoform) was isolated according to the method used in the work (Saburova E.A., Khechinashvili N.N. and Elfimova L.I. Poliol- protein interactions Microcalorimetric studies on the denaturation of lactate dehydrogenase Molec. Biol. 1996, v.30. P.1219-1228);

- urease (E.C.3.5.1.5, jack bean) was used by Fluka, No. 94285 with an activity of 97.0 U / mg;

- sodium polystyrenesulfonate (PSS, 70 kDa), polydiallyldimethylammonium chloride (PDADMA, 100-200 kDa) and polyallylamine hydrochloride (PAA, 70 kDa) from Aldrich (Germany);

- sodium polymethacrylate, sodium polyphosphate dextransulfate (DS, 10 kDa) and ethylenediaminetetraacetic acid (EDTA) from Sigma (Germany);

- calcium chloride (CaCl ₂ · 2H ₂ O), carbonate and sodium chloride company Reahim (Russia).

Example 1. The selection of polyelectrolytes to obtain microdiagnosticum containing the enzyme

Hereinafter, a microdiagnostic is understood to mean an enzyme encapsulated in a polyelectrolyte nano- or microsize capsule, suitable for analysis in a multicomponent biologically active medium, such as, for example, blood, urine, cerebrospinal fluid, etc., of a low molecular weight substance - substrate, inhibitor or activator encapsulated enzyme.

1.1. Selection of polyelectrolytes for microdiagnosticum containing lactate dehydrogenase (LDH) as an enzyme

The selection of polyelectrolytes to obtain a microdiagnostic was carried out based on their inactivating effect on the enzyme.

For this, the activity of free lactate dehydrogenase in the presence of a polyelectrolyte was determined by the pyruvate reduction reaction in the presence of NADH by the change in optical density in the NADH absorption band at 340 nm as described in the reference literature (Passonneau JV, Lowry OH In: Methods of Enzymatic Analysis (Edited by Bergmeyer HU) , V.3, NY, Acad. Press, 1974. P.1452).

To determine the activity of LDH, the concentration of LDH in the free (dissolved) state was measured spectrophotometrically using molar extinction coefficients known for LDH: ε _{280 HM} ^{1 mg / ml} = 1.29 according to calculations using the program (http://cn.expasy.org/sprot /).

When studying the degree of LDH inactivation with a polyelectrolyte, the required amount of polyelectrolyte, previously dissolved in water and subsidized to pH 6.2, was added to the reaction mixture.

Figure 1 shows the dependence of LDH activity on the concentration of biodegradable polyelectrolytes: dextran sulfate (DS), polyphosphate (PF), polyuridinylate (PU) and polyadenylate (PA). The reaction mixture for determining the activity of LDH: pyruvate 1 mm; NADH 0.2 mM; LDH 0.5 μg / ml; 0.05 M Tris-HCl buffer pH 6.2.

From the data shown in Fig. 1, it can be seen that among the biodegradable polyelectrolytes studied for LDH, the most potent inhibitor is polyanion polyphosphate, although its concentration at 50% inhibition of LDH (12.5 mg / ml) exceeds the concentration of the polyelectrolyte used in the preparation capsules (2.5 mg / ml). Thus, all these biodegradable polyelectrolytes are candidates for the formation of the capsule shell containing LDH.

Figure 2 shows the dependence of LDH activity on the concentration of non-biodegradable polyelectrolytes: polystyrenesulfonate (PSS), polyallylamine (PAA), polydiallyldimethylammonium (PDADMA), polymethacrylate (PM). The reaction mixture for determining the activity of LDH: pyruvate 1 mm; NADH 0.2 mM; LDH 0.5 μg / ml; 0.05 M Tris-HCl buffer pH 6.2.

As can be seen from figure 2, among the non-biodegradable polyelectrolytes for LDH that we studied, the most powerful inhibitor is the polystyrene sulfonate polyanion, its concentration at 50% inhibition of LDH is 5 μg / ml. The rest of the studied polyelectrolytes are acceptable for the preparation of the shell within the indicated limits of their concentrations. Thus, all these non-biodegradable polyelectrolytes are candidates for the formation of the capsule shell containing LDH, and polystyrene sulfonate requires isolation from direct contact with LDH and, therefore, is only suitable for use in the outer layers of the capsule shell.

Figure 3 shows the dependence of urease activity on the concentration of non-biodegradable polyelectrolytes: polystyrenesulfonate (PSS), polyallylamine (PAA) and polydiallyldimethylammonium (PDADMA). Conditions for determining the activity of urease: urease 0.5 μg / ml, urea 125 mm, bromcresol violet 0.015 mm, pH 6.2.

Figure 3 shows that for urease, unlike LDH, polycation - polyallylamine is the most powerful inhibitor: 50% inhibition of urease occurs at low concentrations of this polyelectrolyte (fractions of μg / ml).

Thus, the experiments performed show that, when constructing the polyelectrolyte shell of the protein PNMC, the best polyelectrolyte pairs are for LDH PAA / DS and PAA / PSS, for urease PSS / PAA and PSS / PDADMA in the indicated layering sequence. Based on this, we have constructed lactate dehydrogenase and urease microcapsules with different polyelectrolyte composition and number of layers.

Example 2. Obtaining microdiagnostic using biodegradable and non-biodegradable polyelectrolytes taking into account their inactivating effect

A) Obtaining microdiagnosticum containing urease

An equal volume of a 0.33 M Na ₂ CO ₃ solution was quickly added to a 0.33 M CaCl ₂ solution containing 2.0 mg / ml urease and intensively stirred on a magnetic stirrer. The mixture was stirred for 30 s, after which the stirring was stopped, and the resulting suspension was kept for 15 min until the supernatant was completely clarified. As a result of this procedure, a composite microspherulite CaCO ₃ urease was obtained. This process of "maturation" of microspherulites was controlled using a light microscope.

Then the supernatant was decanted, the precipitate was washed with water. The amount of the protein-enzyme included in the spherulite was determined spectrophotometrically when comparing the absorbance (λ = 280 nm) of the initial solutions of the protein and the supernatant selected by precipitation of the indicated microspherulites CaCO ₃ urease. The protein content was also determined by the method of Bradford (Anal. Bioch. 1976. V.72. P.248-254) using the dye Coomassie Brilliant Blue G-250 (λ = 595 nm).

Then, on composite CaCO ₃ urease microspherulites, using them as core particles, multilayer polyelectrolyte microcapsules were formed by alternately adsorbing oppositely charged polyelectrolytes onto core particles.

As polyelectrolytes, polystyrenesulfonate (PSS) was used as a polyanion and polyallylamine (PAA) or polydiallyldimethylammonium (PDADMA) polycation, which was prepared at a concentration of 2 mg / ml in a 0.5 M NaCl solution. Based on our data on the inactivating effect of polyelectrolytes on enzymes (Example 1), the adsorption of the first layer was carried out using a non-inactivating polyelectrolyte for urease polystyrenesulfonate (PSS) and then, respectively, its opposite pair - polyallylamine (PAA) or polydiallyldimethylammonium (PDADMA). The number of layers with non-inactivating polyelectrolytes was set in each case individually based on additional tasks.

Then, at the final stage, the inorganic component of the composite microspherulite CaCO _{3 was} dissolved in EDTA.

The formation of microcapsules was carried out using chilled (5-10 ° C) solutions under conditions of maintaining a neutral pH. To prevent particle aggregation during the adsorption of polyelectrolytes, a suspension of microparticles was periodically treated with ultrasound every 5 min for 1-3 s. At the same time, particle aggregation was monitored using a light microscope. After adsorption to the core particle of each polyelectrolyte layer, the suspension was centrifuged at 1 thousand rpm and the precipitate was thoroughly washed from an unbound polyelectrolyte. The washing procedure was repeated three times. Then, after a specified number of layer-by-layer adsorption of polyelectrolytes, the core particle was dissolved in a 0.2 M EDTA solution at pH 7.5 for 12 hours.

4 is a diagram illustrating the activity of V _max encapsulated urease. The composition of the capsules with urease: PSS (PSS / PAA) ₃ - 1, PSS (PSS / PDADMA) ₃ - 2. The reaction conditions for determining the activity of urease are the same as in the description to figure 3.

The titer of capsules containing urease was 1.2 * 10 ⁶ capsules / ml.

B) Obtaining microdiagnosticum containing lactate dehydrogenase

The preparation of microcapsules loaded with LDH was carried out in the same manner as described in part A of this example for urease, except for the selection of oppositely charged polyelectrolyte pairs. The fundamental difference was the inclusion in some layers of the capsule shell as a polyanion of a non-inactivating polyelectrolyte - dextran sulfate. Microdiagnostics with included LDH containing shells were studied, which included both biodegradable and non-biodegradable polyelectrolytes having the following composition: (PAA / PSS) ₃ , (PAA / DS) (PAA / PSS) ₂ , (PAA / DS) ₂ (PAA / PSS), (PAA / DS) ₃ , and also containing the polycation PDADMA instead of PAA: (PDADMA / PSS) ₃ .

5 is a diagram illustrating the activity of V _{max of} encapsulated lactate dehydrogenase (LDH) encapsulated in microcapsules prepared, as described in part B of Example 2, respectively, from different polycations and different polyanions.

The composition of the capsules with LDH: (PAA / PSS) ₃ - 1; (PDADMA / MSS) ₃ - 2; (PAA / DS) (PAA / PSS) ₂ to 3; (PAA / DS) ₂ (PAA / PSA) - 4, (PAA / DS) ₃ - 5.

Conditions for determining the activity of LDH: pyruvate 1 mm, NADH 0.2 mm, 0.05 M phosphate buffer pH 6.2. The titer of LDH-containing capsules was 1.2 * 10 ⁶ capsules / ml.

Among various polyelectrolyte shells, the most optimal for the functioning of the microdiagnosticum with LDH was the shell (PAA / DS) ₂ PAA / PSS.

Such a series of analytical studies can be performed on other microcapsules with any activity of the encapsulated enzyme, which is felt by current methods.

Claims (6)

1. A method of producing protein-loaded polyelectrolyte nano- and microcapsules, comprising obtaining composite microspherulites of alkaline earth metal carbonates and an encapsulated protein, forming capsules by alternately adsorbing oppositely charged polyelectrolytes on said composite microspherulites, and removing said alkaline earth metal carbonates from said capsules, characterized in that pre-selection of at least the first pair of directly in contact with the encapsulated protein ohm polyelectrolytes alternately examining the impact of each polyelectrolyte in solution Activity on protein encapsulated, and polyelectrolytes are selected that do not affect the optimum concentration for the formation of said capsules on the activity of the protein, or having an effectiveness of inhibiting the at least one function of the protein to the desired limit. 2. The method according to claim 1, characterized in that in the particular case the enzyme is taken as a protein, while the loss of enzyme activity should not exceed 50% of the initial value. 3. The method according to claim 1, characterized in that the composite microspherulites of carbonates of alkaline earth metals and encapsulated proteins are obtained mainly by coprecipitation. 4. The method according to claim 1, characterized in that the alkali metal carbonates from these capsules are removed using EDTA or another chelating agent or acidification of the medium. 5. The method according to claim 1, characterized in that for the formation of the capsule take as polyelectrolytes as biodegradable and incapable of biodegradation of polycations and polyanions, mainly of linear structure. 6. The method according to claim 1, characterized in that it further control the activity of the protein after adsorption of the desired number of layers of polyelectrolytes.

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