RU2303064C2 - Molecular complex for transfection of mammalian cells, containing plasmide dna, modified polyethyleneimine, and ligand molecules - Google Patents

Abstract

FIELD: molecular biology.

SUBSTANCE: disclosed is molecular transfecting complex (MTC) comprising of plasmide DNA carrying necessary genetic information, polyethyleneimine, modifies with acylhydrazine groups and being capable to form ion complexes with plasmide DNA, ligand molecules for receptor-mediated MTC addressing to certain cell types and providing prolonged MTC circulation in blood. Ligand molecule contains one or more aldehyde groups, whereby such molecules may be attached after DNA-acylhydrazidopolyethyleneimine complex formation. Invention is useful in medicine for gene therapy.

EFFECT: complexes with prolonged storage life.

5 cl, 8 dwg, 10 ex

Description

The invention relates to molecular biology and can be used in medicine for the purpose of gene therapy, as well as in scientific research.

The main procedure of gene therapy is the introduction into the cells of the corresponding tissues or organs of a patient of normally functioning genes instead of missing or to correct the functioning of defective genes. Currently, molecular constructs based on modified viruses are most often used as carriers (the so-called "vectors") for DNA being introduced. However, due to the high probability of developing very dangerous side effects when using viral vectors, the task of creating non-viral carriers capable of transfection, i.e. DNA delivery to cells, followed by expression of genes integrated into the plasmid, both in vitro and in vivo.

State of the art

Currently, molecular complexes of plasmid DNA with polylysine, polyethyleneimine (PEI), chitosan are most often used as “non-viral vectors” for transfection of eukaryotic cells (that is, introducing plasmids into the cells and subsequent expression of plasmids carrying the desired genetic information into them) and other polycations.

Scientific publications are known (Bioorganic & Medicinal Chemistry Letters 10 (2000) 1233-1235; Gene Ther 1998 Oct; 5 (10): 1425-33; Bioconjug Chem 1997 Nov-Dec; 8 (6): 839-44), in which ligands containing aldehyde groups are attached to PEI due to the formation of unstable azomethine bonds with primary amine groups, and an additional stage of reduction of azomethines to secondary amines is required to obtain stable preparations.

Patents are known (US No. 6420176, No. 6383811), in which covalently modified PEI preparations are used to obtain transmissive plasmid DNA complexes. To our knowledge, none of them uses modification of this cationic polymer by introducing hydrazine residues or acylhydrazide groups.

An important point for efficient in vivo transfection is the ability to attach so-called targeted ligands to carrier molecules, which would be recognized by specific receptors on the surface of certain types of cells and, thereby, would facilitate targeted delivery of the transfection construct to cells of certain organs.

In most published works, targeted ligands are attached to vector molecules covalently before being complexed with plasmid DNA, which in some cases makes it difficult to obtain compact complexes. Therefore, it is desirable to create "universal carriers" in which the possibility of joining the so-called interchangeable ligands after receiving well-compacted complexes with DNA is provided.

The closest analogue is US patent 6635476, which proposes a method for creating molecular transfecting structures based on PEI, which provides the ability to connect the "interchangeable" ligands. However, it uses a fundamentally different than proposed in the present invention, and, in our opinion, more expensive and time-consuming method of ligand attachment, using specially synthesized "chelating" peptides and transition metal ions.

Disclosure of invention

It was decided to use the reaction between hydrazine derivatives (in particular, acylhydrazide derivatives) and ligands containing aldehyde groups to create a new type of non-viral vectors capable of attaching ligands after the formation of compact complexes between carrier molecules and DNA molecules.

For this purpose, the molecules of the polycationic vector containing secondary amine groups, which was chosen as PEI, were first alkylated with acrylamide (for the N-alkylation of polyamines, in addition to acrylamide, esters of acrylic acids can also be used (for example, according to the procedure described in Sashiwa H ; Yaamori N; Sunamoto J; Aiba S "Chemicai modification of chitosan, 17 - Michael reaction of chitosan with acrylic acid water" Macromolecular Biosci 2003, Vol 3, Iss 5, pp 231-233), and then hydrazinolysis of amide groups (hydrazinolysis the amide groups of polyacrylamide is described, for example, in John K. Inman and Howard M. Dintzis "The De rivatization of Cross-Linked Polyacrylamide Beads. Controlled Introduction of Functional Groups for the Preparation of Special-Purpose, Biochemical Adsorbents "Biochemistry v. 8, (N 10) (1969) pp.4074-4082. The conditions for this hydrazinolysis of amide groups), obtaining acylhydrazide "branches" from the polymer chains of the carrier (see figure 1). The PEI-polyhydrazide (PEI-PG) molecules obtained in this way were able to form complexes with plasmid DNA with good transfection activity.

In addition, such complexes bearing acyl hydrazide groups had the ability under mild conditions to covalently attach (with the formation of hydrazone) any ligands containing aldehyde groups (see Fig. 7). The latter circumstance creates unusually broad possibilities to supply the resulting transfecting structures with a wide variety of targeted ligands of oligo- and polysaccharide nature, to attach practically any low-molecular and polymeric substances into which aldehyde groups are introduced using a series of chemical reactions. For example, it is very simple to carry out the attachment of any glycoproteins (after periodical oxidation and dialysis). It is also possible to introduce “shielding polymers” containing aldehyde groups, in particular polyethylene glycol derivatives, by creating so-called “stealth constructions” (Moghimi SM, Szebeni J. "Stealth liposomes and long circulating nanoparticles" Progress Lipid Res. V.42 (N 6 ), pp. 463-478 (2003)).

We confirmed the above possibility experimentally by introducing into the already formed DNA-PEI-PG complexes the residues of succinylated limitedly depolymerized chitosan (succ-ODC) bearing aldehyde groups, and the introduction of these ligands significantly increased the transfection activity of the complexes.

The implementation of the invention

Example 1

Synthesis of a PEI derivative containing acyl hydrazide groups.

20 g of polyethylenimine (BDH, England) was dissolved in 100 ml of 96% ethanol, 11.7 ml of a 2 M solution of acrylamide in ethanol was added, and heated at 90 ° C for 4 hours. After cooling the reaction mixture, it was dialyzed against ethyl alcohol, 10 ml of anhydrous hydrazine hydrate was added and heated for 5 hours at 60 ° C. Then the reaction mixture was cooled and re-dialyzed against deionized water.

Example 2

Determination of the number of hydrazide groups introduced into PEI.

A solution of PEI-PG-HCl (pH = 3.0) was treated with a known excess (vanillin) for 1 hour at room temperature and subjected to exhaustive dialysis, first against 1 N. a solution of sodium chloride containing 0.005 N. HCl and then against deionized water. Since vanillin contains an aldehyde group, even at pH 3 it joins the PEI-PG acyl hydrazide groups, forming acid-stable hydrazone derivatives containing a benzene ring. Thus, the UV absorption spectrum of the polymer varies greatly, a peak appears at 280 nm. This allows us to calculate the approximate amount of vanillin residues per 1 g of PEI-PG based on the molar extinction of vanillin. At the same time, carrying out the reaction and subsequent exhaustive dialysis at a pH of approximately 3 leads to the removal of excess vanillin molecules, since the formation of azomethine derivatives between the aldehyde groups and the amino groups of PEI is in equilibrium (at acidic pH).

According to our calculations, the content of benzene groups in the purified product is 0.097 mol per 1 mol of PEI monomer units, i.e. the degree of substitution with acyl hydrazide groups is approximately 9.7%.

Example 3

Limited depolymerization of chitosan with nitrous acid.

The methodology described in US Pat. No. 5,312,908 was used. Chitosan depolymerization (a preparation of the Sonat Moscow company, containing, according to the manufacturer’s information, 15% N-acetylated glucosamine units) was carried out for 2 hours at a temperature of + 5 ° C, pH = 3 and a molar ratio of HNO ₂ to chitosan amino groups = 0.1. At the end of the incubation, the pH of the mixture was adjusted to 9 with a NaOH solution, the precipitated chitosan was separated by centrifugation and redissolved by adding acetic acid to pH 4.

To completely remove the low molecular weight reaction products after 3 reprecipitation cycles, the ODC was dissolved in 10 ml of 0.05 M acetic acid and dialyzed 2 times against 0.05 M AcOH and 1 time against deionized water using a benzoylated membrane (Sigma USA) passing molecules only less than 2 KDa.

Example 4

Synthesis of the drug "Polyam-K".

In this case, the alkylation of the initial PEI preparation was carried out under milder (compared to Example 1) conditions. 20 g of polyethylenimine (BDH, England) were dissolved in 100 ml of 96% ethanol, 2 ml of a 2 M solution of acrylamide in ethanol was added, and heated at 90 ° C for 2 hours. After cooling the reaction mixture, it was dialyzed against ethyl alcohol, 10 ml of anhydrous hydrazine hydrate was added and heated for 5 hours at 60 ° C. Then the reaction mixture was cooled and re-dialyzed against deionized water. The content of the acyl hydrazide groups was evaluated as described in Example 2. According to our calculations, the degree of substitution of the acyl hydrazide groups was approximately 1.2%.

To the resulting preparation was added ODC in excess of moles of aldehyde groups, and after dialysis against deionized water, the preparation was subjected to freeze drying.

Example 5

Succinylation of ODC.

Acylation of ODC with succinic anhydride was carried out according to the procedure described in US patent No. 5665702, with the difference that after limited depolymerization of chitosan with nitrous acid, the aldehyde group formed at one end of the molecule was not reduced. A solution of succinic anhydride in acetone was added to a solution of depolymerized chitosan. The molar ratio of anhydride to amino groups of ODC was 0.75. The reaction mixture was stirred at room temperature for 24 hours. Succ-ODC was precipitated by adding a large excess of acetone, collected and washed with methanol by centrifugation. The precipitate was dissolved in 0.5 M KOH and dialyzed against deionized water to pH 7. The degree of acylation was estimated to be approximately = 0.45 by quantification of free amino groups with ninhydrin.

Example 6

In vitro transfection technique.

18-24 hours before transfection, the cells were scattered into 6-24 well plates in such a concentration that 40-60% monolayer was formed the next day. DNA polycation complexes were prepared immediately before transfection. To this end, the main solution of plasmid DNA in PBS (pH 7.2), or a 150 mM NaCl solution, or in deionized water was diluted to a concentration of 40 μg / ml. For example: 2 μl DNA (1 μg / ml) + 48 μl PBS. The polycation solution was diluted to such a concentration that, when mixed in equal volumes with DNA, the desired ratio of amine and phosphate groups (N: P) was achieved. The resulting solutions of plasmid DNA and polycation were thoroughly mixed and incubated for 20 minutes at room temperature. In cases where “removable” ligands were used, they were added to the DNA-PEI-PG mixture and incubated for another 20 minutes. Then the transfection mixture was vigorously shaken and introduced into the wells with cells in a volume of 1/10 of the volume of the growth medium. After 4 hours of incubation in the cell wells, the medium was changed. After 48 hours, a transfection efficiency analysis was performed.

Example 7

Modification of DNA-PEI-PG complexes by succ-ODC molecules directly in preparation for transfection.

A transfection mixture of DNA and PEI-PG was prepared as described in Example 5. Then the mixture was vigorously shaken, an aqueous solution of succ-ODC was added to it at the rate of 2 mol of aldehyde groups of succ-ODC per 1 mol of hydrazide groups of PEI-PG and incubated for another 20 minutes . Next, the procedure was carried out as described in example 5.

Example 8

Evaluation of the effectiveness of transfection of various cell lines.

48 hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and fixed with a solution containing 0.25% glutaraldehyde and 2% formaldehyde for 5-10 minutes at 4 ° C. After washing twice, the cells were coated in situ with a staining solution prepared in PBS (pH 7.2) containing 5 mM yellow blood salt, 5 mM red blood salt, 2 mM MgCl ₂ and 0.05% X-gal chromogenic substrate dissolved in the minimum volume of dimethylformamide, and incubated at 37 ° C for 4-24 hours. The expression of the β-galactosidase gene was detected by microscopic examination for the presence of brightly stained blue cells against the background of a colorless monolayer of cells. Transfection efficiency was determined by dividing the number of blue cells by the total number of transfected cells and expressed as a percentage.

Example 9

Quantification of E. coli β-galactosidase in eukaryotic cell extracts.

Monolayer of transfected cells grown on 12-moons. tablets, washed three times with PBS without calcium and magnesium salts. 100 μl of lysis buffer (0.1 M phosphate buffer, pH 7.5, 0.5% Triton X-100 v / v or 0.125% NP-40 v / v) was added to wells with cells. The mixture was incubated by shaking on a shaker for 15 min at 37 ° C. The following reaction mixture was prepared for each test sample (cell well):

100 ^X Mg ₂ + solution (0.1 M MgCl ₂ 4.5 M β-mercaptoethanol) - 6 μl

1 ^X ONPG (4 mg / ml in PBS) - 130 μl

PBS pH 7.5 - 400 μl

The reaction mixture was added to the wells, stirred by shaking, incubated at 37 ° C until a yellow color appeared. If the background was low, the incubation was carried out for 1-2 hours. The time was fixed in the workbook and the reaction was stopped by adding 1 ml of Na ₂ CO ₃ (1 M) to the wells. The optical density of the solution was determined at a wavelength of 420 nm. Β-galactosidase activity was expressed in arbitrary units (optical density of the reaction mixture incubated at 37 ° C for 50 thousand transfected cells, recalculated for 1 hour incubation). The linear plot in the analysis is in the range of 0.2-0.8 OD420. If the values exceeded this limit, the experiment was repeated, incubating the reaction mixture for less time.

Example 10

Comparison of the results obtained by in vitro transfection of various types of cells with different molecular complexes.

A) The transfection efficiency of various cell cultures using Polyam-K and PEI-PG, determined by X-gal staining, was 5-15% (Hela, MA-104), 20-25% (Vero, MeWo, CRFK) and 30- 50% (COS-1), which is 3-15 times more than using the original drug - PEI 90 kD. The optimal N: P ratio, providing maximum transfection efficiency with the least toxicity, ranged from 1:10 to 1:60 and depended on the type of cell. An analysis of such preparations in an electron microscope revealed the presence of compact rounded nanoparticles with a diameter of 20-35 nm.

B) In parallel with the detection of the percentage of cells expressing β-galactosidase, the specific activity of β-galactosidase in cell extracts was determined. The results of experiments on transfection of Vero, MeWo, Hela, and COS-1 cell cultures are presented as histograms in FIGS. 2, 3, 4, and 5. The transfection efficiency of various cell cultures using Polyam-K and PEI-PG, determined by β- activity galactosidase in cell extracts was significantly higher in most experiments than with PEI 90 kD. The difference in the activity of β-galactosidase in cells transfected with Polyam-K and PEI-PG, in comparison with PEI 90 kD, ranged from 5 to 100 times or more.

A generalization of the results showed that the optimal N: P ratio, providing the maximum transfection efficiency at the lowest toxicity, ranged from 1:10 to 1:60 and depended on the type of cells. Long-term storage of the drug at 4 ° C (for several months) and repeated freezing-thawing did not lead to a significant decrease in the efficiency of transfection. The efficiency of transfection did not depend on the presence of cow embryonic serum in the nutrient medium.

B) It was shown that treatment of PEI-PG / plasmid DNA complexes with succ-ODC increases the efficiency of transient transfection of various cell lines.

The efficiency of transient transfection of cell cultures using PEI-PG, determined by X-gal staining, was 2-4% (MeWo, MA-104) and 3-6% (Vero). Treatment of PEI-PG / plasmid DNA complexes with Succ-ODC significantly increased the efficiency of transfection, which amounted to 8-12% (MA-104, Vero) and 15-20% (MeWo). In parallel with the detection of the percentage of cells expressing β-galactosidase, the specific activity of β-galactosidase in cell extracts was determined. The results of experiments on transfection of Vero cell culture are presented in the form of a histogram in Fig.6.

Treatment of the already formed DNA – PEI-PG complexes with succ-ODC, whose molecules contain aldehyde groups at one of its ends, leads to the formation of hydrazone. Thus, succ-ODC molecules containing 15% N-acetylated glucosamine units are attached to the surface of the nanoparticles. It is known that on the surface of many types of eukaryotic cells there are receptors for N-acetylglucosamine molecules (G. G. Krivtsov and R. I. Zhdanov. Targeted delivery of functional genes for gene therapy using carbohydrates containing vectors. Vopr Med Khim 46 (2000) 246-55 Review.). It can be assumed that higher levels of transfection in cases of using complexes containing succ-ODC or ODC (Polyam-K) are explained by the participation of receptor-mediated endocytosis mechanisms in the internalization of compacted nanoparticles.

The above examples do not limit the scope of legal protection of this patent. By analogy with the examples given, any other ligands containing aldehyde groups (for example, periodically oxidized glycoproteins, in particular immunoglobulins), or ligands to which aldehyde-containing residues (for example, polyethylene glycol or highly branched polyhydroxyl-containing polymers) can be used can be used , “Screening” transfection complexes from “capture” by the cells of the reticuloendothelial system).

Description of graphic materials illustrating the invention.

Figure 1.

A) The chemical structure of the linear PEI modified by acyl hydrazide groups (PEI-PG).

B) The chemical structure of PEI-PG after the addition of aldehyde ligands: R1 — CHO, R2 — CHO, etc. to Ri - CHO.

The resulting molecules contain hydrazone groups RiCH = N-N-CO -... The total number of monomer units is N = Σni + Σmi, where i can vary from 0 to i.

Σni is the total number of unmodified monomer units.

Σmi is the total number of modified monomer units.

According to our data (see Example 2), Σni = 0.903 N, and Σmi = 0.097 N.

Figure 2.

Efficiency of temporary transfection of Vero cell culture with complexes of plasmid pCMV-b-GAL-SPORT with different polycations.

* The optical density of the reaction mixture at λ = 420 nm, calculated for 1 hour of color reaction with the chromogenic substrate ONPG at 37 ° C.

Figure 3.

Efficiency of temporary transfection of MeWo cell culture with complexes of plasmid pCMV-b-GAL-SPORT with different polycations.

* The optical density of the reaction mixture at λ = 420 nm, calculated for 1 hour of color reaction with the chromogenic substrate ONPG at 37 ° C.

Figure 4.

Efficiency of transient transfection of Hela cell culture with pCMV-b-GAL-SPORT plasmid complexes with different polycations.

* The optical density of the reaction mixture at λ = 420 nm, calculated for 1 hour of color reaction with the chromogenic substrate ONPG at 37 ° C.

Figure 5.

Efficiency of transient transfection of COS-1 cell culture with complexes of plasmid pCMV-b-GAL-SPORT with different polycations.

* The optical density of the reaction mixture at λ = 420 nm, calculated for 1 hour of color reaction with the chromogenic substrate ONPG at 37 ° C.

6.

Comparison of the effectiveness of transient transfection of Vero cell culture with pCMV-b-GAL-SPORT plasmid complexes with PEI-PG and PEI-PG + succinylated ODC.

* The optical density of the reaction mixture at λ = 420 nm, calculated for 1 hour of color reaction with the chromogenic substrate ONPG at 37 ° C.

Complexes PEI-PG and Polyam-K with DNA were prepared in the ratio N: P = 20: 1.

7. Scheme for obtaining a molecular complex containing plasmid DNA, PEI-PG and ligand molecules.

1) Mixing plasmid DNA and PEI-PG.

2) Addition to the resulting complex of ligand molecules with aldehyde groups.

3) Ready molecular complex containing covalent hydrazone groups (see figure 1).

Claims (5)

1. Molecular complex for transfection of mammalian cells, containing plasmid DNA, modified polyethyleneimine and ligand molecules, characterized in that acyl hydrazide groups are introduced into the polyethyleneimine molecules, and the ligand molecules contain aldehyde groups. 2. The molecular complex according to claim 1, characterized in that the ligand molecules are depolymerized chitosan with a molecular weight in the range of 2-20 kD, carrying an aldehyde group at one end of the molecule. 3. The molecular complex according to claim 1, characterized in that the ligand molecules are N-succinylated depolymerized chitosan with a molecular weight in the range of 2-20 kD, carrying an aldehyde group at one end of the molecule. 4. The molecular complex according to claim 1, characterized in that aldehyde groups are introduced into ligand molecules. 5. The molecular complex according to claim 1, characterized in that the hydrophilic polymers containing aldehyde groups are used as ligands.

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