SU1301406A1 - Liposomal vesicula for directional transport of biologically active substances - Google Patents

Abstract

This invention relates to medicine, in particular, to experimental pharmacology, and can be used to transport drugs in the target area. The aim of the invention is to increase the degree of selective absorption of vesicles by macrophages. The shell of liposomal vesicle 1 consists of phospholipids 2 and fibronectin modified by derivatives of fatty acids or phospholipids, the surface protein layer of the shell is formed by protein parts of 3 molecules of the modified fibronectin, and the hydrophobic parts of the molecules (formed by the modifier) are embedded in the shell. 1 dw., 6 tab. p (L with with 4 about O5

Description

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The invention relates to medicine, in particular to experimental pharmacology, and can be used for the transport of drugs in the target area.

The purpose of the invention is to increase the degree of selective absorption of vesicles by macrophages.

The drawing shows the lipo-homogeneous vesicle.

The shell of liposomal vesicle 1 consists of phospholipids 2 and fibronectin modified with dithiodipropionyl phosphatidyl ethanolamine, the surface protein layer of the shell is formed by the protein parts of 3 molecules of the modified fibronectin, and the hydrophobic parts of the molecules formed by modifier 4 are embedded in the shell, 20

Thus, the essential features; The proposed liposomal vesicles are the presence of structural elements — the vesicle shell contains a specific outer surface layer formed by molecules of modified fibronectin; materials, i.e. substances that make up the elements of the vesicles - phospholi13014062

the individual composition of the shell, fibronectin, modified phospholipid derivatives (each of the molecules of which can be divided into protein

15

5, the part formed by the residue of fibronectin itself, and the hydrophobic part formed by the modifier) j is the bond between the elements of the object, characterized by one part of the (hydrophobic) modified fibronectin molecule embedded in the phospholipid shell of the vesicles, and the other (protein), exposed on the surface of the shell, form a surface protein layer.

The fibronectin modification scheme can be conditionally expressed as follows (dithiodipropionylphosphatidylethanolamine - DTDP-FE was used as a modifier),

I.DTPP-FE is activated in an aqueous medium with a water-soluble carbodiimide (pH 3.5),

II, fibronectin in borate buffer (pH 8.5) is added to activated DTDP-PE. As a result, the amino groups of fibronectin are modified by DTDP-FE residues.

carbodiimide

pH 3.5

Np

 .

0-Csh Protein

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pH 8; 5 Fiyoronvntin

cha

The purpose of the invention is achieved by a combination of the above indications relating both to the composition and the structure of the object. At the same time, the structure and structure of the liposomal membrane are no less significant a sign than the composition of the initial components, since it is the presence of the surface protein layer that determines the efficiency of the absorption of vesicles by macrophages,

Liposomal vesicles were prepared as follows.

the part formed by the residue of fibronectin itself, and the hydrophobic part formed by the modifier) j is the bond between the elements of the object, which is characterized by one part of the (hydrophobic) modified fibronectin molecule embedded in the phospholipid shell of the vesicles and the other (protein) exposed on the surface of the shell, form a surface protein layer.

The fibronectin modification scheme can be conditionally expressed as follows (dithiodipropionylphosphatidylethanolamine - DTDP-FE was used as a modifier),

I.DTPP-FE is activated in an aqueous medium with a water-soluble carbodiimide (pH 3.5),

II, fibronectin in borate buffer (pH 8.5) is added to activated DTDP-PE. As a result, the amino groups of fibronectin are modified by DTDP-FE residues.

 ,,

. (P

;five

Belkada part of the molecule

-Protein-T N

Hydro (ris6na part of the molecule

with

6 r

ModisrytsroBanny (rVronectin

Modification of fibronectin. For the binding of fibronectin with liposomes, a preliminary hydro5Q phobic modification of the protein molecules of dithiodipropionyl phosphatidyl ethanolamine was used. For this, 1 mg of the latter is dissolved in 100 ml of dimethyl sulfoxide and 1 ml of 0.15 M NaCl is added, followed by.

With 5 vigorous shaking, 2 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (pH 3.5) was added to the resulting solution. After 10 minutes, 2 mg of fibronectin dissolved in

3130

0.7 ml 0.1 M borate buffer (pH 8.5).

Liposomes are prepared from egg lecithin, total phospholipids and gangliosides of the liver and cholesterol by the method of reversed phases, the ratio of phospholipid and cholesterol is 7: 3. For registration of capture, non-exchanging cholesteryl 14-C-oleate (Amersham company) is inserted into the face-somal membrane. The number of phospholipids assessed by content. Protein is measured by the method of Lowry.

Experience 1, 5 mg of lecithin and 1.44 mg of cholesterol are dissolved in chloroform and evaporated on a rotary evaporator to form a film. The resulting film was dissolved in 3 ml of diethyl ether and 1.6 mg of modified fibronectin in 1 ml of borate buffer was added. The resulting solution is treated with ultrasound. After removal of the ether from the solution on a rotary evaporator, monolayer liposomes are obtained, which is monitored by electron microscopy.

noos- (CO-TSH-w-o-r-o

about

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in the same concentrations and at the same incubation time.

About 5. 5. mg of fibronectin in 20 mg / ml chelate buffer (1 ml) containing 0.15 M NaCf and 0.1 M Na ,,, HPO pH 8.0, is mixed with 10 ml of 10% a solution of palmitoyl chloride in acetone at 4 ° C. After 1 hour, the palmitic acid precipitate is centrifuged (12,000, 15 minutes) and discarded, an equal volume of lipid solution (concentration 10 mg / ml) in the same buffer is added. Incubate for 30 minutes and dialyze for 18 hours against the same buffer that does not contain chelate.

In addition, liposomes - detergent - protein are formed spontaneously from mixed micelles liposomes, the membrane of which contains fibronectin.

The structure of the resulting fibronectin-liposome complexes can be monitored by electron microscopy.

FDI.

Unbound protein can be easily and quickly separated from liposomes by flotation in a stepped ficoll or

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EXAMPLE 2. Fibronectin modified with DTDP-FE is obtained. Mix it with an equal volume of preformed liposomes (concentrate

qip lipid 3 mg / ml) and incubated for 20 hours.

In this case, fibronectin is incorporated by its hydrophobic residues into the membrane of already prepared liposomes due to the hydrophobic interaction of the residues.

phospholipid in a protein molecule with a lipid membrane.

EXAMPLE 3 Conducted in a manner similar to that of 2, but fibronectin is mixed with

a solution of lipid (concentration of 5 mg / ml) in detergent (2% chelate, 0.15 M NaCf, 10 mM, pH 7, -4) and dialyzed for 18 h against a buffer that does not contain detergent. Liposomes are formed that contain fibronectin molecules on the i-iposome membrane, and the protein phospholipid residue is inserted into the liposome membrane. EXAMPLE 4, Conducted Similarly

Experience 2, but glutaryl-PE is used instead of DTDP-FE.

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40

45

„

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gel filtration in a centrifuge at separase CL 4B.

Transformed macrophages of line 1774 are used. A monolayer of cells (7 10) in the well is coated with aliquots of liposomes and incubated for 1 hour. After being removed into JS-8 scintillation liquid, they are counted on a RACKBETA 1215 counter (L KB).

To determine the endocytosis of liposomes by macrophages, cells are incubated for 40 min by a glycolytic endocytosis inhibitor, iodine acetamide (100 µmol).

Example 1: Binding and endocytosis of macrophages (7 x 10 cells) of liposomes from lecithin without fibronectin and coated with fibroectin are compared. The lipid content is 1.5 mg / ml and fibronectin 0.7 mg / ml. The data are given in table. 1 and 2.

PR e and M e p 2. Compare the binding and endocytosis of liposome macrophages from phospholipids and gangliosides

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the liver. The content of the lipid 1.5 mg / ml, gangliosides, fibronectin 0.7 mg / ml The data are given in table, 3 and A,

Example 3: Checking the specific interaction of macrophages with liposomes containing fibronectin, compared with liposomes containing bovine serum albumin.

Liposomes containing lecithin: cholesterol (1: 1) are prepared in which fibronectin and bovine serum albumin (BSA) are inserted through the hydrophobic stem. The weight ratio of the components is the same as in example 1, the amount of albumin is equivalent to the content of TN.

Table 5 gives the number of liposomes bound to the macrophage cell line 1774 (disintegrations / min of C-cholesterol-oleate associated with liposomes)

The kinetics of binding to macrophages of liposomes derived from lecithin and lecithin from fibronectin is investigated.

Table 6 shows data on the binding of liposomes from lecithin and lecithin with fibronectin with macrophages 1774, depending on the incubation time.

Lecithin

0.74 ± 0.18

Note: After homogenizing liposon by

size (4f),

Lecithin 0.37 ± 0.02 Lecithin + Fibronectin

2.74 ± 0.42

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Thus, fibronectin increases the phagocytosis of liposomes by macrophages up to 20 times.

The use of the invention ensures a high degree of capture of liposomes by macrophages, stimulation of phagocytosis of liposomes by macrophages, the possibility of using liposomal vesicles for transporting BAB,

Claims (1)

Invention Formula Liposomal vesicle for directed transport of biologically active substances, containing a membrane consisting of phospholipids and an outer protein layer, characterized in that, in order to increase the degree of selective absorption vesicles by macrophages, the shell additionally contains fibronectin modified by derivatives of fatty acids or phospholipids, and the outer protein layer is formed by protein portions of the modified fibronectin molecules, while the hydrophobic portions of its molecules formed by the modifier are embedded in the phospholipid membrane. Table 1 0.4310.02 0.31 table 2 0,29tO, 03 0.7310.08 0.08 2.01 13014068  Table 3 Capture Capture-Endocytosis LNPOS ass in pri-liposome (nmol absences (test lipid) iodoacetamidalipid) (nmol lipid) 3.67 ± 0.09 1.4410.11 2.23 5.7710.25 2.70 ± 0.45 3-, 07 the After homogenization of liposomes in size (4p). -Table 4 Capture Lipo - Capture Lipo-Endocytosis catfish (nmol catfish in the presence of liposomes lipid) due to iodine- (nmol tamida (lipid lipid) lipid) 1.22 ± 0.08 0.8610.11 0.36 5.6110.59 3.47 + 0.34 2.14 Table 5 and - Decay / min at temperature, ° С one 37 4 211 ± 3297 1 7. + 2967 ± 111304 ± 40 ctin. Lecithin + BSA 600 59 126 ± 6 Editor And, Gorna Compiled by N.Kuzenkova Tehred M.Hodanich Order 1172/4 Circulation 596Signature VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 Production and printing company, Uzhgorod, Projecto st., 4 Table 6 Proofreader S. Shekmar