KR101128108B1 - Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity - Google Patents

Abstract

The present invention relates to an association in which an assembly composed of polyethyleneimine and bile acids, which can efficiently deliver macromolecules, is bound by a reaction between a carboxyl group of the bile acid and an amine group of polyethyleneimine.

Polyethylenimine, Bile Acids, Intracellular Delivery Systems

Description

Amphiphile polymer conjugate consisting of polyehthylenimine and bile acid having cell membrane fusion activity}

The present invention relates to a delivery system that can deliver macromolecules such as nucleic acids or anionic proteins, peptides and anionic nanoparticles using cationic polymers and bile acid conjugates into cells. In particular, the present invention relates to a biocompatible assembly containing polyethyleneimine (PEI) and bile acids, wherein 1) the bile acids are directly linked to the PEI backbone or 2) are linked to the PEI backbone via a crosslinker. .

The limited permeability of cell plasma membranes to biological macromolecules, such as DNA and proteins, is one of the strong barriers to limiting the therapeutic potential of macromolecular drugs, which has led to cell membrane permeation of macromolecules for decades. Promoting delivery systems have been studied. Physical membrane permeability (eg electroporation), direct delivery by membrane fusion with liposomes, and energy dependent cellular uptake of colloidal particles called endocytosis. Many studies have been conducted.

Recently, a series of cationic peptides called cell penetrating peptides (CPPs) have been identified that allow membranes to pass through membrane fusion, lipid structure rearrangement, or pore formation. El-Andaloussi, S .; Holm, T .; Langel, U. Curr Pharm Des 2005, 11, 3597-611). The membrane-active properties of cell penetrating peptides are generally used for intracellular delivery of small molecules, proteins and nucleic acid drugs. Although the mechanism is not clearly identified, peptides are thought to mediate macromolecular membrane permeation of macromolecules through endocytosis-independent and endocytosis-dependent processes (Plank, C .; Zauner, W .; Wagner, E.). Adv Drug Deliv Rev 1998, 34, 21-35)

Most of the peptides have amphiphile properties and typical alpha-helix structures, and the hydrophilic amino acids are parallel to the major axis of the peptide. Such amphiphilicity plays a role in membrane fusion or permeability of peptides (Cheng, Y .; Ho, DM; Gottlieb, GR; Kahne, D .; Bruck, MA J Am Chem Soc 1992, 114, 7319-7320; Legendre, JY; Szoka, FC, Jr. Proc Natl Acad Sci USA 1993, 90, 893-7).

Bile acid series, such as deoxycholic acid, have unusual structural characteristics, which can be explained by the fact that two to three hydrophilic hydroxyl groups are pointing in the same direction and the hydrophobic steroid hydrocarbons are pointing in the opposite direction (Fig. 1) (Axelrod, HR; Sofia, MJ; Walker, S. Adv Drug Deliv Rev 1998, 30, 61-71). Amphiphilic bile acids act as solubilizers and delivery agents for fats and cholesterol. The bile acids and their derivatives are known to interact with the membrane to enhance cellular uptake of polar molecules (Swenson, ES; Curatolo, WJ Adv Drug Deliv Rev 1992, 8, 39-92; Walker; , S .; Sofia, MJ; Kakarla, R .; Kogan, NA; Wierichs, L .; Longley, CB; Bruker, K .; Axelrod, HR; Midha, S .; Babu, S .; Kahne, D. Proc Natl Acad Sci USA 1996, 93, 1585-90)

The present inventors have studied the association of bile acids and polyethyleneimines, which are planar amphiphiles, and confirmed that the macromolecules can be efficiently delivered into cells, thereby completing the present invention.

The present invention is to provide a delivery system capable of delivering macromolecules and the like into the cell.

In order to achieve the above object, the present invention provides a combined association by the reaction of the carboxyl group of the bile acid and the amine group of polyethyleneimine in the association consisting of polyethylenimine (PEI) and bile acid. In particular, the present invention relates to a biocompatible assembly containing the polyethylenimine (PEI) and bile acid. 1) The bile acid is directly connected to the PEI skeleton or 2) to the PEI skeleton through a crosslinker.

Polyethyleneimine used in the present invention has an amine group in the monomer, and has a structure capable of reacting with a carboxyl group of bile acids. The molecular weight of polyethyleneimine used in the present invention is preferably 100 to 100 kDa in consideration of cytotoxicity and smooth clearance during in vivo administration. That is, high molecular weight polyethyleneimine may have a problem of cytotoxicity, and low molecular weight polyethyleneimine has a problem of low transfer efficiency, so that the molecular weight is preferable.

'Bile acid' used in the present invention may be a carboxyl group reacts with an amine group of polyethyleneimine to form an conjugate with polyethyleneimine by amide bond. Bile acids include cholic acid, 5β-cholanic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, and taurocholic acid. A bile acid selected from the group comprising deoxycholic acid, lithocholic acid, 7-oxo-lithocholic acid, and the like may be preferably used. Cholic acid, deoxycholic acid and lithocholine acid can be used.

In the assembly composed of bile acid and polyethyleneimine according to the present invention, it is preferable that the mole ratio of the bile acid and the polyethyleneimine is included in 1 to 50%, more preferably 2 to 15% but preferably limited thereto. It is not. The molar ratio means the number of amine groups in which bile acids are substituted with respect to the total number of amine groups of polyethyleneimine. In theory, the molar ratio of 100% means that bile acids are substituted in all of the amine groups of polyethyleneimine.

Cationic bile acid aggregates of the present invention spontaneously undergoes microphase separation by amphiphilicity in an aqueous solution to form nanoscale self-assembled structures exhibiting regularity of hydrophobic nuclei and hydrophilic shell structures, and critical micelles. Formation concentration is 0.001 to 0.400 g / L and particle size is 70 to 500 nm.

The association consisting of bile acids and polyethyleneimine according to the present invention can be delivered by encapsulating hydrophobic drugs as well as anionic macromolecules or nanoparticles containing nucleic acids in the form of polyelectrolyte complex nanoparticles. That is, the assembly according to the present invention can pass well into the cell, and at this time, the complex encapsulated with the macromolecule or the hydrophobic drug can also pass through the cell, thereby delivering a substance that is difficult to deliver into the cell. The macromolecule may be selected from the group consisting of plasmid DNA, siRNA and antisense oligonucleotide, but is not necessarily limited thereto.

The hydrophobic drugs include hydrophobic anticancer agents such as doxorubicin, adriamycin, adriamycin, cisplatin, paclitaxel or taxol, docetaxel, daunomycin and coenzyme Q10 ( coenzyme Q10), vitamin E and derivatives thereof, and vitamin A and derivatives thereof, and the like, and may be selected from the group consisting of hydrophobic antioxidants.

In addition, the present invention comprises the steps of activating the bile acid with DCC and NHS; And it provides a method for producing an assembly consisting of polyethyleneimine (PEI) and bile acid comprising the step of adding polyethyleneimine to the activated bile acid.

The complex and the complex containing the macromolecule or the hydrophobic drug according to the present invention are characterized as follows.

First, in the case of materials used to deliver macromolecules into conventional cells, the delivery efficiency is not good, and many cases have to go through complicated manufacturing steps to prepare them. However, in the case of the present invention, the assembly can be prepared by a simple method. In addition, the delivery efficiency is very high and may be particularly useful for delivering intracellular nucleic acid drugs.

Second, the association according to the present invention has an amphiphilic property, and thus can efficiently transport macromolecules. In addition, even in the case of hydrophobic drugs can be efficiently encapsulated due to the amphipathic properties can be widely used as a drug delivery system.

Hereinafter, preferred embodiments of the present invention will be presented to assist in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

1. Material used

Deoxycholic acid, branched polyethylenimine (PEI, Mw 25 kDa, 1.8 kDa), dicyclohexyl carbodiimide (DCC), N-hydroxy succinimide (N -hydroxyl succinimide (NHS), and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, Mo.). Cell culture material, Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). Endocytosis inhibitors (chlorpromazine, wortmannin, genistein), metabolic inhibitors (sodium azide, 2-deoxy-glucose), and MTT (3- (4,5-dimethylthyazolyl-2) -2,5-diphenyl tetrazolium bromide) Cell culture grade product, purchased from Sigma (Sigma (St. Louis, Mo.)). The other chemicals and solvents were analytical grade and used without further purification.

2. Synthesis and Characterization of DA-PEI Associations

1) association synthesis

1 shows the synthesis of DA-PEI aggregates.

First, 1 g (2.5 mmol) of DA (deoxycholic acid) dissolved in tetrahydrofuran was activated with DCC (dicyclohexyl carbodiimide, 1.57 g, 7.6 mmol) and NHS (N-hydroxysuccinimide, 0.88 g, 7.6 mmol) (PLGA / DCC / NHS molar ratio = 1: 3: 3). Activated DA was precipitated in n-hexane and dried under reduced pressure. Synthesis of DA-PEI association was performed by adding DA activated in methylene chloride to 1.8 kDa of PEI. An assembly called DA1 and DA3 was prepared, the molar feed ratios of DA1 was 1: 1 (DA / PEI) and the molar feed ratios of DA3 was 1: 3 (DA / PEI). The product was dried by rotary evaporator, dissolved in 0.1 M hydrochloric acid and precipitated in ice-cold acetone / ether (1: 3 mixed) solution. The precipitate was centrifuged and then dried with dry-nitrogen. The product was dissolved in distilled water, filtered through a filter, and lyophilized. The degree of substitution was confirmed by ¹ H-NMR, which is shown in FIG. 2.

2) Hemolysis test

Membrane activity of DA-PEI association was measured by erythrocyte lysis assay. 700 g of blood cultured from SD rats were centrifuged for 10 minutes. The pellet was washed three times with PBS and then redispersed in PBS.

For the hemolytic test, an appropriate amount of association dissolved in PBS was added to the solution containing 1 × 10 ⁸ RBCs. After incubating the test solution at 37 ° C. for 1 hour, the solution was centrifuged at full speed. The degree of hemoglobin release was measured by absorbance at 540 nm of the supernatant, which was confirmed by expressing it in percent of hemolysis. Complete hemolysis to calculate the percent hemolysis was performed by adding 0.1% Triton X-100, which was used as a control (100%).

The results of the assembly synthesis and hemolytic test are shown in Table 1 below, and it can be observed that cell membrane activity increases as the amount of bile acid contained in the assembly increases. Therefore, experiments were performed by selecting associations exhibiting characteristics of cell membrane activity and having low hemolytic properties.

FR ^a DS ^b HL ^c DA1 One 0.7 23.8% DA3 3 2.5 32.9% a: reaction feed ratio (DA / PEI)
b: substituted molar ratio (DA / PEI) as confirmed by ¹ H-NMR
c: Hemolytic in murine erythrocyte at 20 ㎍ / ml

3. Formation and Characterization of High Ion Polyelectrolytes

1) Electrophoretic mobility shift assay (EMSA)

The formation of high ionic complexes between the association and the plasmid DNA was confirmed by EMSA in the DNA band on 0.8% agarose gel. Various molar ratios of DA-PEI associations and 0.3 g of plasmid DNA (pCMVLuc) were each diluted in PBS. Diluted associations and plasmid DNA solutions were mixed at different mass ratios, mixed well and left at room temperature for 15 minutes prior to analysis. Electrophoresis was performed in TAE buffer (40 mM Tris / HCl, 1% (v / v) acetic acid, 1 mM EDTA) at 100 V for 20 minutes. The migration delay of the complex was imaged by ethidium bromide staining (FIG. 3).

Figure 3 shows that the synthesized aggregates lack mobility on agarose gel by forming high ionic complex nanoparticles through ionic interaction with plasmid DNA, which is an anionic macromolecule.

2) Size and surface charge measurements

The hydrodynamic diameter and surface zeta potential for the complex of DA-PEI associations and plasmid DNA were determined using a dynamic light scattering device (Zeta Plus, Brookhaven) with a He-Ne laser at 25 ° C. Instrument Co., NY) was used to measure at 632 nm. All measurements were repeated three times. The measured size and surface charge are shown in Table 2 below.

PS ^a SC ^b DA1 79.8 nm +35.2 mV DA3 73.2 nm +33.2 mV a: complex size (nm, association / DNA mass ratio = 3)
b: complex surface charge (mV, aggregate / DNA mass ratio = 3)

4. Cell culture, transfection and cell viability assays

1) Transfection and reporter gene assay

Human colorectal cancer cells (HCT116) were incubated in DMEM fed with 10% FBS and maintained at 37 ° C. 5% CO ₂ . For transfection, the cultured cells were inoculated into 6-well plates (2 × 10 ⁵ / well) and then incubated for 24 hours under conditions of 5 ° C. CO ₂ at 37 ° C. After replacing the culture medium with a serum-free medium, an appropriate amount of polymer / DNA complex was added. After 4 hours of incubation, the transfection medium was removed and a fresh growth medium containing 10% FBS was supplied. Luciferase activity was measured 24 hours after transfection with a commercially available luciferase assay kit (Promega, Madison, WI) and Lumat FB12 luminometer (Berthold GmbH, Pforzheim, Germany). The transfection efficiency was expressed by the relative light unit (RLU) per gram of cellular protein measured by BCA assay (Pierce, Rockford, IL). The results are shown in FIG. As shown in FIG. 4, the DA3 association showed a 10 times higher transfection efficiency than PEI 25 kDa (w / w = 1) of optimized composition.

2) Endocytosis and metabolic inhibitor studies

The transfection method was performed in the same manner as in 1), except for the addition of the inhibitor and washing with PBS containing heparin sulfate before the reporter gene assay.

For inhibitor assays, cells were treated with appropriate concentrations of various endocytosis inhibitors (10 μg / ml chlorpromazine, 100 nM wortmannin, 200 μM genistein, or a combination of the three inhibitors) and metabolic inhibitors (0.1% sodium azide or 50 mM 2- first incubated in the presence of deoxy-glucose). Next, the polymer / DNA complex was added to cells in a serum-free medium containing the same inhibitor, incubated for 4 hours and then replaced with a medium containing 10% serum. After 24 hours of incubation, the cells were washed with PBS containing 10 mg / ml heparin sulfate and then again with PBS. It was further washed with heparin sulfate to remove the complex remaining on the cell surface. The results are shown in Fig.

As shown in FIG. 5, each inhibitor and combination of inhibitors did not appear to have a significant effect on the transfection of the DA3 association. This suggests that the DA3 assembly utilizes other intracellular uptake pathways independent of endocytosis. In the case of metabolic inhibitors, the transfection efficiency of the DA3 association was reduced to about 73%, whereas for PEI 25 kDa it was reduced to about 99.6%. Thus, it can be seen that the assembly according to the present invention can deliver macromolecules, such as molecules such as plasmid DNA, intracellularly without the help of endocytosis pathways or other energy consuming intracellular uptake pathways.

3) Cytotoxicity Test

Cells were seeded at a density of 5000 cells / well in 96 well plates with 0.2 ml of DMEM growth medium containing 10% FBS. Cells were grown for 24 hours at 37 ° C., the growth medium was removed and a growth medium containing an appropriate amount of polymer was fed. After incubating the cells for 48 hours, the metabolic activity of the cells was measured. 100 μL of growth medium containing 50 μg MTT was added to each well and the cells were incubated for 4 hours. Lysis buffer (10% (w / v) SDS, 45% DMF, pH 4.7) was added and incubated for 24 hours. Absorption at 590 nm was measured with a microplate reader (EL808, Bio-Tek Instrument, Winooski, VT), and survival was calculated by comparison with mock-treated cells (100% survival). The results are shown in FIG.

As shown in FIG. 6, the association shows lower toxicity than PEI25KDa and higher toxicity than PEI1.8, but this may be due to cell membrane activity, which is one of the characteristics of the association.

4) Isolation, Culture and Transfection of Mesenchymal Stem Cells

Bone marrow-derived mesenchimal stem cells (MSCs) were isolated from the femoral and tibia of 4 week Sprague-Dawley rats by magnetic bead-based cell isolation (Song, H.). ; Chang, W .; Lim, S .; Seo, H.-S .; Shim, CY; Park, S .; Yoo, K.-J .; Kim, B.-S .; Min, B.-H Lee, H .; Jang, Y .; Chung, N .; Hwang, K.-C. Stem Cells 2007, 25, 1431-1438). Isolated MSCs were incubated in low glucose DMEM with 10% FBS, maintaining 37 ° C. and 5% CO ₂ conditions. MSCs were characterized by flow cytometry using a series of antibody targeting cellular markers including CD14, CD34, CD71, CD90, CD105, and intracellular adhesion molecules (ICAM-1). . For transfection experiments, cells were trypsinized, inoculated into 6-well plates (1 × 10 ⁵ cells / well), and incubated at 37 ° C. and 5% CO ₂ conditions. After 24 hours of incubation, transfection test using DA3 association, branched PEI 25 kDa (b-PEI25K), linear PEI 25 kDa (l-PEI25K) and Lipofectamine-Plus ™ (Invitrogen, Carlsbad, Calif.), 1 Was carried out in the same manner as). The results are shown in Fig.

As shown in FIG. 7, the DA3 association was 10, 6 and 15 times higher for branched PEI 25 kDa (b-PEI25K), linear PEI 25 kDa (l-PEI25K) and Lipofectamine-Plus ™, respectively. Transfection efficiency is shown. Therefore, the association according to the present invention indicates that it can mediate the delivery of intracellular plasmid DNA in primary cells as well as fusion cells.

Figure 1 shows the synthesis of the DA-PEI assembly of the present invention.

Figure 2 shows the 1H-NMR results to confirm the degree of substitution of DA1 and DA3 of the present invention.

Figure 3 is an image of the migration delay of the complex according to the present invention by ethidium bromide staining.

Figure 4 shows the results of the transfection and reporter gene assay of the assembly according to the present invention.

Figure 5 shows the results of the transfection and reporter gene assay according to the addition of the inhibitor of the association according to the present invention.

Figure 6 shows the results for the cytotoxicity of the assembly according to the present invention.

Figure 7 shows the transfection results of the present invention for mesenchymal stem cells.

Claims (9)

delete delete delete delete delete delete (a) an assembly composed of polyethyleneimine (PEI) and bile acid, in which an amine group of the polyethyleneimine (PEI) is bonded to a carboxy group of bile acid; And, (b) comprises a macromolecule selected from the group consisting of plasmid DNA, siRNA and antisense oligonucleotides, The macromolecule is complex, characterized in that encapsulated in the assembly. According to claim 7, wherein the bile acid (cholic acid), 5β-cholanic acid (5β-cholanic acid), chenodeoxycholic acid (chenodeoxycholic acid), glycocholic acid (glycocholic acid), taurocholic acid ( taurocholic acid), deoxycholic acid (deoxycholic acid), lithocholic acid (lithocholic acid) and 7-oxo-lithocholic acid (7-oxo-lithocholic acid), characterized in that any one selected from the group consisting of Complex. 8. The complex of claim 7, wherein the mole ratio of bile acid and polyethyleneimine is 1-50%.

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