RU2409384C1 - Method for producing microcapsules for dna delivery to macroorganism - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to biotechnology and genetic engineering. DNA is delivered to macroorganism in biocompatible and biodegradable microcapsules made of multilayer polymeric DEAE-dextrane/k-carrageenan membranes protecting DNA from nuclease splitting while delivered to organism cells. Plasmid DNA is sorbed with porous particles CaCO3 by coprecipitating process. The multilayer membranes are produced by sequential layer of DEAE-dextrane and k-carrageenan on the porous particles CaCO3. The microcapsules are placed in 0.1 M EDTA solution. It is agitated at an intensive rate to dissolve an internal core containing CaCO3. The produced microcapsules are washed with water for three times. They are injected in animal cells. ^ EFFECT: method increases an amount of sorbed DNA to 96,0-99,1 % and provides greater strength of the microcapsule membrane. ^ 3 tbl, 2 ex

Description

The invention relates to biotechnology of DNA vaccines and inherently belongs to somatic gene therapy technologies.

The invention can be used to increase the effectiveness of DNA vaccines in the field of somatic gene therapy.

For microencapsulation of drugs and proteins, calcium carbonate (CaCO ₃ ) microspheres coated with a polymer are used [1]. Calcium alginate and gelatin were used as shells. Such microbeads are ineffective for microencapsulation of organic acids. The authors used this method for the immobilization of proteins; in the process, treatment with acids and the formation of capsules similar to microgel structures were envisaged.

A known method of microencapsulation for the delivery of nucleic acid and adjuvant, which includes microspheres consisting of a soluble polymer. The nucleic acid was introduced into the polymer solution, then the solvent was extracted from the emulsion of the nucleic acid with the polymer, causing the formation of microspheres containing nucleic acid and / or nucleic acid with an adjuvant. The encapsulation process proceeded at 37 ° C. Subsequently, the processes of washing, freezing and drying microspheres were provided. The effectiveness of this method of microcautulation was 50% [2].

However, the known microencapsulation methods are quite complex, ineffective and do not allow to obtain microcapsules with the DNA contained in them, easily degraded in the animal body and unstable during long-term storage, which is a prerequisite for vaccines (DNA vaccines).

The closest analogue is the method of DNA delivery for the development of vaccines, which includes microencapsulation of DNA by sorption of CaCO ₃ microparticles for 20-22 hours at 4 ° C, followed by coating with multilayer coatings: poly-L-lysine - sodium alginate and dissolution of internal CaCO ₃ nuclei [3].

However, the composition of the polyelectrolyte membrane does not provide the necessary conditions for the formation of a tense immune response to the introduction of microencapsulated DNA.

The aim of the invention is to improve the method of obtaining microcapsules for the delivery of DNA into a macroorganism.

This goal is achieved in that the method includes sorption of plasmid DNA by CaCO ₃ microparticles, the formation of layers of a multilayer polymer shell from a modified low molecular weight DEAE-dextran (M 6500) / κ-carrageenan (M 15000-30000, Sigma) by sequentially layering and dissolving the inner core CaCO ₃ , while DNA sorption is carried out simultaneously with the process of synthesis of CaCO ₃ microparticles. To form the first layer of the multilayer shell, the microparticles are centrifuged and transferred to a solution of κ-carrageenan (2 mg / cm ³ in 0.2 M NaCl), incubated for 15 min with shaking and washed twice in physiological saline, and to form the second and subsequent ones, up to the sixth layer, the particles are placed in a solution of DEAE-dextran (2 mg / cm ³ in 0.2 M NaCl), incubated with shaking for 15 minutes, washed twice with distilled water and transferred to a solution of 0.1 M EDTA, intensively mixed and apply the seventh final layer, consisting of and kappa-carrageenan.

Unlike polylysine-alginate microgranules, dextran-kappaginan capsules slowly break down. When checking in cell culture, degradation of polylysine capsules occurs on day 2-3, while the presence of dextran-carrageenan microgranules in the culture fluid is also observed on day 10-12.

EFFECT: increased amount of DNA sorbed by plasmid to 96-99% of the initial amount instead of 90% and its biological properties are improved. The use of a polyelectrolyte pair DEAE-dextran / κ-carrageenan provides a more dense shell, and therefore increases the mechanical strength of the microcapsules. After the formation of the multilayer shell by alternately layering the polymers, the inner core, consisting of CaCO ₃ , is dissolved by placing microcapsules in a solution of 0.1 M EDTA, and intensively mixed until the CaCO ₃ matrix is completely dissolved. Then washed three times with water. Finally, the microcapsules consist of 6-8 layers of polyelectrolytes and the plasmid DNA enclosed in them. The elution of DNA from microcapsules in an aqueous or alcoholic solution does not exceed 1% on the first day and in the future, the exit of DNA from microcapsules ceases (observation period is 20 days). The DNA enclosed in microcapsules is injected into the body of the animal.

Examples of specific performance.

Example 1

As a model for evaluating the proposed method for delivering plasmid DNA to animal cells, pInfNP and pInfHA plasmids are used, encoding nucleoprotein and hemagglutinin of avian influenza virus, respectively.

500 μg of the corresponding plasmid DNA was enclosed in microparticles simultaneously with the preparation of CaCO ₃ particles by coprecipitation. For this, an aqueous solution of plasmid DNA was mixed with a magnetic stirrer with unimolar solutions of calcium chloride and sodium carbonate for 30 seconds. After stirring, the microparticle pellet was separated from the supernatant by centrifugation (1600 g, 1 min). The amount of sorbed DNA was determined spectrophotometrically (λ = 260 nm) by the difference in the adsorption of the initial solutions and the solutions of supernatants taken during precipitation.

The amount of DNA sorbed by the microparticles was 96%.

DNA immobilization in microcapsules (PLL / Alg) ₃ :

To form the 1st layer of the shell, the microparticles are centrifuged and transferred to a solution of low molecular weight (15000-30000) poly-L-lysine (2 mg / cm ³ ) in 0.2 M NaCl, incubated for 15 min with shaking and washed twice in physiological solution. To obtain a second polymer layer, the particles are placed in an alginate solution (1-2 mg / cm ³ in 0.2 M NaCl), incubated with shaking for 15 minutes, washed twice with distilled water and transferred to a solution of 0.1 M EDTA, intensively mixed for 15 minutes. on a shaker with a rotation speed of 60-100 rpm to dissolve the inner core of CaCO ₃ and again washed with water (1000 rpm 5 min). This sequence of operations is repeated until the formation of 7 layers: LL-Alg-PLL-Alg-PLL-Alg-PLL. The last 7th coat is applied in the same way as the previous ones.

DNA immobilization in microcapsules (DEAE-dextran / Car) ₃ :

To form the 1st layer of the shell, the microparticles are centrifuged and transferred to a solution of κ-carrageenan (2 mg / cm ³ ) in 0.2 M NaCl, incubated for 15 min with shaking and washed twice in physiological saline. To obtain a second polymer layer, the particles are placed in a solution of DEAE-dextran (2 mg / cm ³ in 0.2 M NaCl), incubated with shaking for 15 min, washed twice with distilled water and transferred to a solution of 0.1 M EDTA, intensively mixed for 15 minutes on a shaker with a rotation speed of 60-100 rpm to dissolve the inner core of CaCO ₃ and again washed with water (1000 rpm 5 min). This sequence of operations is repeated until the formation of 7 layers: DK-DK-DK-D. After the formation of microcapsules with a multilayer membrane, the concentration of immobilized DNA is 480 μg / cm ³ .

Plasmid DNA preparations at 20 μg / goal. (DNA in aqueous solution and in microcapsules) is administered to mice intramuscularly. On day 14, the highest antibody titer was observed in the blood sera of mice immunized with microcapsules (DEAE-dex-dextran / Car) ₃ . On the 21st day, serum antibody titers during immunization with microcapsules (DEAE-dextran / Car) ₃ were equal to plasmids and were 3-4 times higher than in case of immunization with microcapsules (PLL / Alg) ₃ .

Table 1 Antibody titer during immunization of mice with microcapsules of various compositions containing the plasmid InfHA and InfNP. Blood sampling, day Antibody titer Infha Infnp (PLL / Alg) ₃ (DEAE-dextran / Car) ₃ (PLL / Alg) ₃ (DEAE- (dextran / Car) ₃ fourteen 1: 625 1: 1125 1: 125 1: 2290

Case Study 2

As a model for evaluating the proposed method for delivering plasmid DNA to animal cells, a nucleotide sequence encoding the protective E ₂ protein (pTKShi) of classical swine fever is used.

500 μg of the corresponding plasmid DNA pTKShi was enclosed in microparticles simultaneously with the preparation of CaCO ₃ particles by coprecipitation. For this, an aqueous solution of plasmid DNA is mixed with a magnetic stirrer with unimolar solutions of calcium chloride and sodium carbonate for 30 seconds. After stirring, the microparticle pellet was separated from the supernatant by centrifugation (1600 g, 1 min). The amount of sorbed DNA is determined spectrophotometrically (λ = 260 nm) from the difference in the adsorption of the initial solutions and the solutions of supernatants selected during precipitation.

To form the 1st layer of the shell, the microparticles are centrifuged and transferred to a solution of κ-carrageenan (2 mg / cm ³ ) in 0.2 M NaCl, incubated for 15 min with shaking and washed twice in physiological saline. To obtain a second polymer layer, the particles are placed in a solution of DEAE-dextran (2 mg / cm ³ in 0.2 M NaCl), incubated with shaking for 15 min, washed twice with distilled water and transferred to a solution of 0.1 M EDTA, intensively mixed for 15 minutes on a shaker with a rotation speed of 60-100 rpm to dissolve the inner core of CaCO ₃ and again washed with water (1000 rpm 5 min). This sequence of operations is repeated until the formation of 7 layers: DK-DK-DK-D. The last 7th coat is applied in the same way as the previous ones. After the formation of microcapsules with a multilayer membrane, the concentration of immobilized DNA is 495.5 μg / cm ³ , i.e. 99.1%.

Plasmid DNA preparations at 20 μg / goal. (DNA in aqueous solution and in microcapsules) is administered to mice intramuscularly. On the 14th day, the highest antibody titer was observed in the blood sera of mice immunized with microcapsules (CH-2 (mod) / Car) ₃ . On the 21st day, serum antibody titers during immunization with microcapsules (DEAE-dextran / Car) ₃ were 3-4 times higher than in case of immunization with microcapsules (PLL / Alg) ₃ .

table 2 Antibody titer during immunization of mice with microcapsules of various compositions containing the plasmid pTKShi. Blood sampling, day Antibody titer (PLL / Alg) ₃ (DEAE-dextran / Car) ₃ fourteen 1: 800-1: 1600 1: 6400-1: 12800 21 1: 3200-1: 6400 1: 12800-1: 25600

As can be seen from the table. 2, when the same amount of encapsulated plasmid DNA was introduced, antibody induction depended on the composition of the microcapsules and was 3-4 times higher than in the case of immunization with microcapsules (PLL / Alg) ₃ .

Comparative characteristics of the proposed method of DNA and prototype are presented in table 3.

Table 3 Comparative characteristics of DNA delivery methods Work stages The proposed method Prototype Sorption of DNA by microparticles of CaCO ₃ 2.5 min during the synthesis of microparticles 20-22 hours at 4 ° C The amount of sorbed DNA from the original (%) 96-99.1 80-90 Polyelectrolyte Membrane Composition multilayer coating DEAE-dextran / κ-carrageenan multilayer coating poly-L-lysine (PLL) / alginate (Alg) The formation of antibodies in the blood serum of mice with the introduction of plasmids in microcapsules 3-4 times higher

Using the proposed method for producing microcapsules for the delivery of DNA to a macroorganism in comparison with the existing method has the following advantages:

1 - increases the amount of sorbed DNA;

2 - significantly increases the strength of the multilayer shell of microcapsules through the use of a composite combination of DEAE-dextran / κ-carrageenan;

3 - increases the synthesis of specific antibodies in response to the introduction of DNA in microcapsules.

Information sources

1. Mark E. Johnson, Sally Mossman, Tricia Cecil, Lawrence Evans, US 2002/0032165 A1 Mar. 14, 2002.

2. E.A. Markvicheva, E. Svrshchevskay, D. Volodkin, O. Selina, M. Gerstenberger, A. Batkoviak, G. Sukhorukov. Encapsulated protein and DNA delivery system for the development of vaccines against intracellular pathogens XII International Workshop on bioencapsulation, Vitoria, 2004, p. 319-322.

3. RF patent 2336090, IPC C 12N 15/31. A method for delivering DNA to a macroorganism for the development of vaccines and somatic gene therapy. V.I. Balysheva, E.A. Markvicheva, N.N. Vlasova, Sukhorukov GB, Safiullin N.F., Selina O.E. 2008 year

Claims (1)

A method of producing microcapsules for delivering DNA to a macroorganism, including sorption of DNA with CaCO ₃ microparticles, formation of seven layers of a multilayer polymer shell, dissolution of the internal CaCO ₃ core, characterized in that DNA sorption is carried out simultaneously with the formation of CaCO ₃ particles, and use as a multilayer polymer shell DEAE-dextran / κ-carrageenan, while for the formation of the first layer of the multilayer shell of the microparticle after centrifugation is transferred to a solution of κ-carrageenan, and for the formation of the second, cha a particle placed in DEAE-dextran solution, the process was repeated to form seven layers: KDKDKD seventh and final layer is applied, consisting of kappa-carrageenan.