Delivery of nucleic acids

 

(57) Abstract:

The invention relates to biotechnology and can be used for Introduzione nucleic acids into cells. For delivery of nucleic acids cell incubated with the compound having the formula w...x-y-NH-CR4(CH2OR1)(CH2OR2), in which w represents a nucleic acid, x is a peptide or amino acid, y is a linker having a chain length equivalent to 1-20 carbon atoms or is absent, R4represents N or CH2O - R3; R1, R2and R3are the same or different and are either hydrogen, methyl, ethyl, hydroxyl or acyl group derived from a fatty acid having a carbon chain from 3-24 saturated or unsaturated carbon atoms, with the proviso that at least one of the radicals R1, R2and R3represents an acyl group derived from a fatty acid or a compound having the formula w. . ..xyNHCH2CH2OR5in which w represents a nucleic acid, x is a peptide or amino acid, y is a linker having a chain length equivalent is nuclear biological chemical (NBC acid, having a carbon chain from 3-24 saturated or unsaturated carbon atoms. The invention allows for the delivery of gene therapy medicinal product in the cells. 4 c. and 39 C.p. f-crystals, 24 ill., 5 table.

The present invention relates to a method of inducing compounds, in particular nucleic acids in the cell. In addition, the present invention relates to preparations for use in this way.

There are several situations when it is necessary to deliver specific compounds in cells. One of such operations is the transfection of eukaryotic cells DNA. For that currently use various commercial tools such as Transfectam" (Promega), "DOTAR" (Boehringer Mannheim), "Lipofectin or Lipofectamine" (BRL) or apply a transfection-mediated calcification.

The ability to deliver into cells nucleic acid with other compounds used in drug and gene therapy. Delivery of compounds into cells will vary depending on the formulas of the compounds are presented below. Such changes can be detected in the form of modifications concerning the length of their actions (e.g., slow release or support action), the quantity is within parameters, described below, can also provide target specificity of drugs in the cells or tissues.

The authors of the present invention found that the Association of molecules with compounds, modified acyl derivatives of fatty acids with the General formula below, facilitate the delivery of such compounds in cells.

In accordance with the first objective of the present invention consists of a method of Introduzione nucleic acid in the cell, including the ageing of the cells with a compound having the formula

< / BR>
in which: w represents a nucleic acid;

x is a peptide or amino acid;

y represents a linker having a chain length equivalent to 1-20 carbon atoms, or no;

R4represents H or CH2O - R3; and R1, R2and R3are the same or different and are either hydrogen, methyl, ethyl, hydroxyl or acyl group derived from a fatty acid having a carbon chain from 3-24 saturated or unsaturated carbon atoms, with the proviso that at least one of R1, R2and R3represents ACE consists of a method for introduction of nucleic acid into the cell, which includes a holding cell with a compound having the formula

w...x-y-NH-CH2-CH2O-R5< / BR>
in which: w represents a nucleic acid;

x is a peptide or amino acid;

y represents a linker having a chain length equivalent to 1-20 carbon atoms, or no;

R5represents an acyl group derived from a fatty acid having a carbon chain from 3-24 saturated or unsaturated carbon atoms.

In accordance with the third objective of the present invention includes the connection used to Introduzione nucleic acid into the cell, this compound having the formula

< / BR>
in which: w represents a nucleic acid;

x is a peptide or amino acid;

y represents a chain length equivalent to 1-20 carbon atoms, or no;

R4represents H or CH2O-R3; and R1, R2and R3are the same or different and are either hydrogen, methyl, ethyl, hydroxyl or acyl group derived from a fatty acid having a carbon chain from 3-24 saturated or unsaturated uknow group, derived from fatty acids.

In accordance with the fourth objective of the present invention includes the connection used to Introduzione nucleic acid into the cell, this compound having the formula

w...x-y-NH-CH2-CH2O-R5< / BR>
in which: w represents a nucleic acid;

x is a peptide or amino acid;

y represents a linker having a chain length equivalent to 1-20 carbon atoms, or no;

R5represents an acyl group derived from a fatty acid having a carbon chain from 3-24 saturated or unsaturated carbon atoms.

In the preferred embodiment, each objective of the present invention "y" is present.

In a preferred embodiment of the present invention the nucleic acid is a DNA, RNA or oligonucleotides of DNA or RNA, modified oligonucleotides, or combinations thereof. Nucleic acid may also be chemical additives, such as fluorescein (FITZ), cholesterol, Biotin or radioactive tag.

This method of the present invention can be used for delivery of nucleic acids, including the impact of established cell lines of animal or vegetable origin, primary cell lines of animal or vegetable origin, whole organisms of animals and plants, applied systemically, locally or in aerosol form. On this topic, please refer to the EPO 426688 included in the present description by reference. Nucleic acid may also be chemical additives, such as fluorescein (FITZ), cholesterol, Biotin or radioactive label. This method of the present invention have considered mainly in order to implement the use of compounds mainly in the form of an aqueous mixture on the surface of the respective cells. However, in the case of whole organisms connection can be applied as needed mainly in non-aqueous form, by local or shared injection, topically or by inhalation.

The description of this invention also includes the connection labeled compounds, such as fluorescein (FITZ) or Biotin to the peptide for imaging and tracking in cells and whole organisms. Labelled compound is introduced into cells by any of the above ways or methods.

The peptide can be any length and can include functional domains, such as signals, localized core and/or binding of the nucleic acid. Eoit using lysine. Typically, the peptide will have a total positive charge for the preparation and retention of nucleic acid by attaching, for example, peptide having one or more lysine residues. You can, however, create a peptide bearing total negative charge, if this is an advantage for other forms of delivery, such as for systemic delivery in vivo.

The linker may be any of a variety of molecules, are well known in the art. At present, however, it is preferable that the linker was an amino acid or peptide, such as alanine, leucine, glycine, phenylalanine, lysine or its homologues or heteropolymer. The linker may also include atypical amino acids, allowing thus to increase the length of the linker is greater than when using a standard amino acids. In this respect, particularly preferably, a linker represented aminobutyric, aminocaproic or aminocaproic acid.

In the most preferred embodiment of the present invention R1, R2and R3are the same, but preferably are acyl derivatives of fatty acids comprising the group consisting of palm trees, which Oh by the authors of the application for Australian patent N 649242 disclosed a method for attaching the amino acids or peptides to 1-3 acyl derivatives of fatty acids by tromethamineiv or ethanolamine derived. Using this method, you can get a broad range of peptide/acyl derivatives of fatty acid conjugates 1-3 of acyl derivatives of fatty acid residues. The use of the method disclosed in this application, can be obtained introducing trilinolenin-Tris-tripalmitate connection (CSTRS). The disclosure of that application is incorporated into this invention by reference.

Nucleic acid ("w" connection) can be associated with the rest of this connection using positively charged amino acids such as lysine, arginine, ornithine, etc., in the position "x" in this connection. Particularly preferred groups are monolitiniai, Deliziosa, Telesina, tetraoninae or tantalizingly group. Alternative nucleic acid can be attached covalently to the peptide or the amino acid "x". In addition, the linker group of alanine is possible to replace, for example, on lacinova, glycine, phenylalanine or BOC (without-BOC) - lysine groups and the number of acyl derivatives of fatty acids can vary from 1 to 3. It should be noted that when it is necessary to use one acyl derived fatty acids, Tris can be replaced by ethanolamine.

It is obvious that the technology is over and out of such a lipid, as dioleoylphosphatidylcholine (DOPE), DOPC (DOP-choline) or cholesterol, can increase the efficiency of the invention, contributing to the delivery of compounds into cells. This is most likely when there are 2 aliphatic acyl derivatives. Methods of making liposomes are described, for example, Felgner, P. L. et al 1987 PNAS 84 pp 7413-7417 and Yago, K et al 1993 Biochem and Biophys. Res. Comm. 196(3) pp 1042-1048. It is also shown that the simple addition of DOPE to the connection solutions can significantly enhance their activity (the following experiment 4, VerafectinG2 DOPE). Like other modifications of the conditions of transfection the presence of additives such as salt may enhance transfection. Described changes in ionic strength and the presence of alkaline-earth cations alter the efficiency of transfection (Loeffler and Behr, Methods in Enzimology 1993, H, 599-654).

The main application of the present invention should be the delivery of gene therapy medicinal product. Relatively efficient gene transfer into cells observed so far, in combination with low toxicity (especially when compared to commercial drugs) makes the present invention ideal for therapeutic use of many compounds. Additional information related to the gene is the essence of the present invention more comprehensible, preferred variants are explained by consideration of the following examples and figures, in which:

Figure 1a shows the toxicity Verafectin and DMSO, and 1b shows the survival in % in the presence of Verafectin compared with DMSO.

Cytotoxicity was determined using the standard test with MTT (3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium; Tiazoly blue).

Verafectin is a K3ATP3, and its concentration is indicated in μm, DMSO used in equivalent concentrations, which was diluted Verafectin.

a. The results of cell death with decreasing OP.

b. Cell survival in the presence of one only diluted DMSO, taken as 100%, and survival in the presence of the appropriate concentration Verafectin.

Figure 2a shows the cytotoxicity Verafectin and ethanol, and 2b shows the survival rate in % in the presence of Verafectin compared with ethanol.

Cytotoxicity was determined using the standard test with MTT (3-[4,5,Dimethylthiazol-2-yl]2,5-diphenyltetrazolium; Tiazoly blue).

Verafectin (K3ATP3) was dissolved in 100% hot ethanol at a concentration of 10 mg/ml and then diluted with water to concentrations expressed in microns.

a. In parallel diluted ethanoate cells in the presence of one only of dilute ethanol, taken as 100%, and survival in the presence of the appropriate concentration Verafectin.

Figure 3 shows the expression of CAT 48 hours after transfection pSVLCAT mediated through Verafectin.

The number Verafectin, expressed in microns, was mixed in a 1 µg pSVLCAT, before layering on the cells for transfection. The results presented reflect the levels of CAT expression, measured to 50 μl of cell culture medium 48 hours after transfection.

Figure 4 shows the expression of CAT, and analyzed within 48 hours after transfection of CHO cells, mediated through the tested factors.

The results presented reflect the concentration transfairusa factor, providing the highest level of transfection in the experiment. She was 5 μl in each case. Tests CAT conducted by the method of Neuman J. R., Morency, C. A. and English, K. O. (1987) Biotecniques 5, p 444-447.

Nothing: factor transfection no, TF: Transfectam (Promega), Lfe: Lipofectamine (BRL), VF A3: K3ATP3, VF G2:K3GTP2.

Figure 5 shows the expression of CAT, analyzed 48 hours after transfection of Cos 1 cells, mediated through the tested factors.

The results presented reflect the concentration factor transfection, giving n the em relative cytotoxicity of factors transfection.

Explanatory caption

a. (CHO cells)

Cell viability after transfection of different factors at the time of the collection of cells in the CAT assay.

Nothing: transfairusa reagent no, TF: Transfectam, Lfe: Lipofectamine, VF G2: Verafectin G2 - all 5 µl. The height of the OP reflects the degree of survival of cells.

b. (Cells Cos 1)

The viability of cells as in Fig. a. VF A3: Verafectin A3 - 9 μl, Lfe - 5 ál. The analyses in this experiment was performed in 35 mm cups.

Figures 7a and 7b show the results for peptide x in the experiment "a".

Figure 8 shows the results for peptide x in the experiment "b".

Figure 9 shows the results for peptide x in the experiment "c".

(Explanatory caption of figures 7, 8 and 9; nothing: no factor used with plasmid DNA, rb/reagent: reagent blank, the number following the connection description specifies no. μl of 2 mm stock solution (diluted with water). 7b: 100% cell survival in the cups, where he performed "pseudotranslation" without DNA or transfairusa factor. 0% survival in the absence of cells. Survival was estimated using the dye alamar blue).

Figures 10a and 10b show the results for the linker "y" experiment "a".

Figure 11 parasitaemia "c".

(Explanatory caption of figures 10a, 10b, 11, 12; factor: the test compound was taken from a 2 mm stock solution, except where otherwise stated. (. 4): the mother solution factor of 0.4 mm. 0: no factor. Only DNA under standard conditions of transfection. The empty environment: only the environment in the analysis of CAT. Relative survival (alamar blue): measurement of OP570-595culture medium with alamar blue was added during incubation at 37oC).

Figures 13a and 13b show the results for R1-R4-derived in the experiment "a".

(Explanatory signature: same as explanatory caption to figures 10a, 10b, 11, 12).

Figures 14a and 14b show the results for R1-R4-derived in the experiment "b".

(Explanatory signature: same as explanatory caption for figures 10a, 10b, 11, 12).

Figures 15a and 15b show the results for R1-R4 in the experiment "c".

(Explanatory signature: same as explanatory caption for figures 10a, 10b, 11, 12. Shows the standard deviation).

Figures 16a and 16b show the results for R1-R4-derived in the experiment "d".

Figures 17a and 17b show the results for R1-R4-derived in the experiment "e".

Figures 18a and 18b show the results of 0 ál 48 h culture supernatants were incubated for 5 hours at 37oC for CAT analysis.

Figures 19a and 19b show the results for R1-R4, C10-C16 in the experiment "b". HeLa Cells.

(Explanatory signature: figures 19a and 19b: Lfe: Lipofectamine, (1,39) ratio [lipid] /[DNA], where the optimal transfection. Nothing: no factor, but DNA is included in the transfection. Cytotoxicity was determined using Alamar blue).

Figures 20a and 20b show the results for the linker "y", presents an unusual amino acids, in experiment 1".

(Explanatory signature: LFE: Lipofectamine. The levels of gene expression are shown in "a" units-galactosidase. Relative viability was analyzed in the "b" using dye "alamar blue", and presented as optical density, measured after an incubation period in the presence of the dye. High OP indicates high survival rate of the cells).

Figures 21a and 21b show the results for the linker "y", presents an unusual amino acids, in experiment 2".

(Explanatory signature: lfe: Lipofectamine, vf: Verafectin A3, C3; K3C3TL3, C5; K3C5TL3, C7; K3C7TL3, %% of transfectants was calculated according to the number painted in blue cells among unpainted cells. a: CHO cells, b-cells, Cos 1).

Figures 22a and 22b show the results for the linker "y", pretty "technology of preparation of medicines" experiment "A".

The CAT expression in transfected CHO cells using different formulations of liposomes, ingredients and Lipofectamine (Lfe). Shows the results of a representative experiment.

Figure 23b shows the results of the "technology of preparation of medicines" experiment "b".

The percentage of transfected cells using liposomal compositions of the individual ingredients and commercial products. VF A2: K3ATP2. Liposomes were Packed using VF A2 and DOPE at a certain ratio VF A2: DOPE. Presents the results of a single experiment.

Figure 24 shows the results "- low-tech mixtures" in "experiment 2".

(Explanatory caption: the efficiency of transfection was evaluated by the level of expressive galactosidase units after 48 hours. L3: K3ATL3, M3: K3ATM3. Peak reflects a combination of lipopeptide and DNA, which give a higher reading. [lipid] used reflects the concentration of total lipopeptide).

Examples

Chemistry

The used abbreviations are:

-BOC(-Z-Lys) = -Butyloxycarbonyl--carbobenzoxy-Lysine

AEP = Alanyl-ethanolamine-palmitate

ATP1 = Alanine-Tris-monopalmitate

ATP2 = Alanine-Tris-dipalmitate

Armenian

DIEA = Diisopropylethylamine

DMAP = Dimethylaminopyridine

DMF = Dimethylformamide

DMCO-D6- Sulfoxide-d6< / BR>
DSC = Disuccinimidyl

FATP1 = Fluorescein alanine-Tris-monopalmitate

FATP2 = Fluorescein alanine-Tris-dipalmitate

FATP3 = Fluorescein alanine-Tris-tripalmitate

FITC = Fluoresceinisothiocyanate (isomer I)

HOSU = Hydroxysuccinimide

TEA = Triethylamine

TFA = Triperoxonane acid

THF = Tetrahydrofuran

Tris = 2-amino-2-hydroxy-methyl-1,3-propandiol

Z = N-carbobenzoxy

Materials and methods

-BOC(-Z-Lys) received from the Institute of peptides, Inc. (Osaka, Japan), and DSC was obtained from Tokyo Kasei Kogyo Co. (Tokyo, Japan). All amino acids were L-shaped and purchased from Sigma Chemical (St. Louis, MO), except where otherwise stated. All solvents were trained including D. A. and not subjected to additional treatment.

Thin-layer chromatography

Was performed on plates with Alufolein silica gel 60 F254(Merck) in the following solvent system: Rf1, chloroform/methanol/acetic acid - 95/5/3; Rf2, chloroform/methanol/triethylamine - 95/7/3.

Liquid chromatography high resolution (IHVR)

Ana is surrounding the supply system solvent series 6000A with automatic control of the gradient and the storage device, model 746. Chromatography was performed on a column (h) in reversed phase C18 matrix NOVOPAKTM. The peptides and conjugates Tris-peptide were analyzed in a linear gradient elution 24-80% acetonitrile with 0.1% TFA for 5 min at a flow rate of 2 ml/min (System A). Detection was performed at 260 nm using a Waters Lambda Max 480; (RfA). Conjugates of lipopeptide analyzed on column C18using a linear gradient from 50% water, 50% acetonitrile with 0.1% TFA to 50% acetonitrile, 50% THF with 0.1% TFA for 5 min at a flow rate of 2 ml/min (System B); (RfB). Separation of compounds labeled with fluorescein was performed on prepreparation obraniakowi column (h) with C4-matrix PrepPakRat a flow rate of 6 ml/min

Preparative GHUR

The separation was performed on a Millipore Waters DeltaPrep 4000 HPLC using obratsova column (h mm) with C4matrix PrePak and suirable using a linear gradient of the same eluting buffer systems described above for analytical GHUR, at a flow rate of 20 ml/min

Nuclear magnetic resonance (NMR)

NMR spectra were recorded on a 200 MHz Bruker-spectrophotometer.

Chemical synthesis of

Getting ATP1, ATP2 and ATP3.

2(40 psi) in hydrogenator Parr in the presence of palladium on carbon (10%) in ethanol. Remove benzyloxyaniline group was tracked through GHUR (System B). After removal of catalyst by filtration and evaporation of the solvents, ATP1, ATP2 and ATP3 received in sufficient quantity. Synthesis and purification ZATP1, ZATP2 and ZATP3 and appropriate glycerine compounds described in Whittaker, R. G., Hayes, P. J. and Bender, V. J., 1993), Peptide Research, 6, 125-128, and Whittaker R. W. Patent N 649242, Amino acids, peptides or derivatives thereof, is attached to the fats.

Getting FATP1, FATP2 and FATP3.

To a solution of ATP1 (10 mg, 25 μm) in DCM (500 μl) was added with stirring a solution of FITZ (10 mg, 25 μm) in DMF (500 μl). The exact pH value in the reaction supported the addition of TEA, and the course of the reaction was monitored using GHUR (System B). Education fluorescein-Ala-Tris-palmitate completely ended after 10 min and the product was isolated by purification via preparative GHUR to get FATR1 product to chromatographic clean condition, Rf: 7,08. The solvents were removed under reduced pressure, and FATR1-product liofilizirovanny of tertiary butanol.

FATR2 and FATR3 synthesized in the same manner in the reaction ATP2 (16,3 mg, 25 μm) and ATP3 (22 mg, 25 μm) in DCM (500 m ptx2">

Getting alanyl-ethanolamine-palmitate-labeled with fluorescein.

Alanyl-ethanolamine-palmitate (AEP) was obtained by hydrogenation of Z-Ala-ethanolamine-palmitate in hydrogenator Parr'and in the presence of palladium on coal (10%) in ethanol. Remove benzyloxycarbonyloxy group was tracked through GHUR (System B). After removal of catalyst by filtration and evaporation of the solvent the named compound was obtained in sufficient quantity.

Receiving (Liz)nconnections.

Synthesis and secretion clearance Z-Ala-ethanolamine and the corresponding palmitate described in Whittaker, R. G., Hayes, P. J. and Bender, V. J., 1993), Peptide Research, 6, 125-128, and Whittaker, R. G. Patent N 649242, Amino acids, peptides or derivatives thereof, is attached to the fats.

To a solution of AEP (20 mg, 54 μm) in DMF (500 μl) was added with stirring FITZ (22 mg, 56 μm) and just maintained a pH of 9.0 by the addition of TEA. The reaction proceeded completely in less than 20 min and the product was isolated by purification using preparative GHUR to obtain these compounds in the chromatographic clean condition, Rf: 7,01.

Synthesis of oligo-Liz-compounds [(BOC(-Z-Lys)n] was carried out according to the classical method in the solution (1). The lipopetides BOC(-Z-Lys)n-X-Tris-politicsonline. The implementation of the synthesis consists in joining BOC(-Z-Lys)n-OH to Tris-amino acid with the activated ester and subsequent merger of palmitic acid to this conjugate using a symmetric anhydride. The purity of the intermediate and the final product was controlled by TLC, GHWR and NMR.

1) Bodansky, M. and Bodansky, A. 1984. Fundamentals of peptide synthesis, Springer-Verlag, Berlin.

2) Whittaker, R. G., Hayes, P. J. and Bender, V. J., 1993, Reliable method of joining Tris to amino acids and peptides, Peptide Research, 6, 3 (p. 125-128).

Typical examples of synthesis is shown below.

Stage (I) -BOC(-Z-Lys)2OH

-BOC(-Z-Lys) - OH, (9,9 g, 30 mmol) was dissolved in 100 ml DCM. HOSU (5,2 g, 45 mmol) and DIEA (9.0 g, 15 mm) was added to the solution and cooled to 0oC. into (6.2 g, 30 mmol was dissolved in 50 ml DCM, adding dropwise into the reaction mixture. The resulting solution was stirred at 0oC for 1 h followed by curing at room temperature overnight to produce an activated ester (BOC(-Z-Lys)OSU in the amount of 86% GHUR. The DCU precipitate (Dicyclohexylphosphino) was filtered and the amino (( -Z-Lys) - OH (7,56 g, 27 mm) was added to the reaction mixture and stirred at room temperature overnight. The connection I floor the initial residue was dissolved in ethyl acetate and washed with acid, ground and water. An ethyl acetate phase was dried over sodium sulfate and evaporated to dryness. The residue was ground into powder with diethyl ether to obtain 16.6 g of compound I in the amount of 93%, Rf1: 0,52, RtA: 7,33 min;1NAMR: (DMSO-d6, ppm) of 1.39 (9H, s, BOC(CH3)3), 1,4-1,8 (12H, brs, ,, CH2), to 2.99 (4H, brs, CH2), of 3.95 (1H, m, CH), 4,14 (1H, m, CH ), to 5.03 (4H, s, Ar-CH2), 6,89 (1H, d, urethane NH, J = 7.5 Hz), 7,25 (2H, t, urethane NH), 7,41 (10H, m, Ar(H)), to 7.95 (1H, d, amide NH, J = 8,5 Hz).

Stage (II) H(-Z-Lys)2OH

Compound I (15.6 g, 27 mmol) was dissolved in 50 ml of DCM and cooled to 0oC. TFA (50 ml) was added to the reaction mixture and stirred it at 0oC for 10 min and subsequent 50 min at room temperature. The solvent and excess TFA is evaporated to dryness and the oily residue was ground into powder with diethyl ether. Received of 15.3 g of compound (II); Rf2: 0,19, RtA: 6,07 minutes

Stage (III) -BOC(-Z-Lys)3OH

-BOC(-Z-Lys) - OH (8,96 g, 27 mmol) activated with HOSU and into, as in example I. the DCU was filtered and the filtrate was added to a 15.1 g of compound II. DIEA (6 g, 46 mmol) was added to the reaction mixture and was stirred over night at room temperature. The solvent is evaporated, and the residue was dissolved in dry. The residue was ground into powder with diethyl ether to obtain 20 g of a white precipitate of compound III in the amount of 87%; Rf1: 0,36; RtA: 7,92 min1NAMR: (DMSO-d6, ppm) of 1.39 (9H, s, BOC(CH3)3), 1,4-1,8 (18H, brs, ,, CH2), to 2.99 (6H, brs, CH2), of 3.95 (1H, m, CH ), 4,14 (1H, m, CH ), 4,34 (1H, m, CH ), 5,03 (6H, s, Ar-CH2), 6,8 (1H, d, urethane NH, J = 7.5 Hz), 7,25 (3H, t, urethane NH), 7,41 (15H, m, Ar(H)), 7,80 (1H, d, amide NH, J = 8 Hz), 8,15 (1H, d, amide NH, J = 8 Hz).

Stage (IV) -BOC(-Z-Lys)3-Ala-Tris

-BOC(-Z-Lys)3OH (1 g, 1.2 mmole) was dissolved in 40 ml of DMF and added DSC (0,92 g, 3.6 mmole). After adding DIEA (0.2 ml, 1.2 mmole) and stirring at room temperature for 1 h activated ether three-Liz was formed in the amount of 78%, as determined using GHUR. In the reaction mixture were added Tris-Ala (and 0.46 g, 2.4 mmole) and the pH was brought to 8 by addition of 0.6 ml of DIEA. Obtaining these compounds are continued using GHUR. After 2 h, the activated ester is almost fully utilized either by joining Ala-Tris, with the formation of compound IV, or as a result of hydrolysis of up to three-Liz-connection. The total number of compound IV was 52% GHUR. Preparative GHUR produced 270 mg of purified compounds with RtA: 7,4 min1NAMR: (DMSO-d6, ppm), 1,39 , ,89 (3H, t, OH), 5,08 (6H, s, Ar-CH2), 6,8 (1H, d, urethane NH, J = 7,4 Hz), 7,19 (3H, t, urethane NH), 7,41 (15H, m, Ar(H)), 7,80 (1H, d, amide NH, J = 8 Hz), 8,15 (1H, d, amide NH, J = 8 Hz), 8,35 (1H, d, amide NH, J = 8 Hz).

Stage (V) -BOC(-Z-Lys)3-Ala-Tris - (palmitate)nn = 1, 2, 3

-BOC(-Z-Lys)3-Ala-Tris (173 mg, 0,173 mmole) was dissolved in 3 ml DCM and 1 ml DMF. Palmitic acid and a catalytic amount of DMAP was added to the reaction mixture. It was cooled to 0oC and into (71 mg, 0,346 mmole) was dissolved in 2 ml DCM, drip adding to the reaction mixture. It was stirred at 0oC for 30 min and then kept at room temperature during the night. The ratio of these compounds to mono-, di - and tripalmitin amounted to 17%, 40% and 43% in GHUR (system B).

The solvent was evaporated to dryness and the residue was re-dissolved in DCM. DCU was filtered and the filtrate was washed with sodium bicarbonate (5%) and water. Preparative GHUR this mixture has been purified compounds monopalmitate (17 mg, RtB: 7,63 min), dipalmitate (76 mg, RtB: 8,65 min) and tripalmitate (63 mg, RtB: 9,29 min). 1NAMR connection tripalmitate: (CDCl3, ppm): 0,8-0,95 (9H, t, CH3), 1,3-1,47 (84H, m, BOC (CH3)3), palmitate (CH2), Ala (CH3)), 1,47-1,9 (18H, brs, ,,CH2<, ,04 (6H, s, Ar-CH2), to 5.56 (1H, d, amide NH, J = 7 Hz), 5,7 (1H, d, amide NH, J = 7.5 Hz), 5,78 (1H, d, amide NH, J = 7.5 Hz), 6,92 (1H, d, urethane NH, J = 7,4 Hz), 7,19 (3H, t, urethane NH), 7,35 (15H, m, Ar(H)).

Stage (VI) (Liz)3-Ala-Tris - (palmitate)3< / BR>
Compound V (45 mg) was dissolved in DCM (2 ml) and cooled to 0oC. was Added TFA (2 ml) to remove the Boc-group at 0oC for 10 min and at room temperature for 30 minutes, the Solvent and excess TFA were carefully removed by repeated evaporation with diethyl ether 1NAMR of this compound showed the disappearance of the BOC (CH3) group. Then the residue was dissolved in a solution of DCM/methanol (50/50, 4 ml) and was hydrogenosomal for 2 h at a pressure of 40 pounds per square inch in hydrogenator Parr'and using 10% palladium/carbon for removal of Z-groups. Remove Z-groups confirmed1NAMR-spectroscopy (disappearance of the chemical shift at 5,04 and 7.35 ppm).

Biology

The used abbreviations are:

A = Alanine

DDME = (Incomplete) Modified Dulbecco Wednesday Needle, double the concentration, frozen, Ottana at 37oC, filtered and diluted to 1x before use (Loeffler, J-P and Behr, J-P, Methods in Enzymology (1993) H, p. 599-654.

DME = Modified Dulbecco Wed/BR> FCS = Amniotic calf serum

FLAEP = fluorescein-alanyl-ethanolamine-palmitate

FLATP3 = fluorescein-alanine-Tris-palmitate

G = Glycine

K = Lysine

K3ATP1-3 = trilinolenin-Tris-mono-tripalmitate

L = Leucine

Lfe = Lipofectamine (Gibco BRL)

MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy)-2-(4 - sulfophenyl)-2H-tetrazolium, inner salt; (reagent Owen a)

MTT = 3-[4,5, Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide; tiazoly blue

PBS = phosphate-saline buffer solution

TEM = Transmission electron microscope

VF = Verafectin = K3ATR3

VFA3 = K3ATR3

VFG2 = K3GTP2

A. Cellular uptake and distribution.

To assess the ability to deliver compounds into cells, conducted preliminary experiments with fluorescein-alanyl-ethanolamine-palmitate (FLAEP) and with fluorescein-alanine-Tris-palmitate (FLATP3).

FLAEP and FLATP3-conjugates were diluted with a solution of DMSO in water (PBS) to 10 μm was layered on the washed monolayer almost confluent Cos 1 cells growing on the surface of boiled cover glass. After the incubation period, as specified in the experiment, while keeping up to 24 hours, cells were washed and fixed in formaldehyde for 20 minutes. ZAT is kiravannie for only fifteen minutes, after processing FLAEP or FLATP3, showed intense cytoplasmic staining. Cells are designed equivalent with only one fluorescein, showed very weak total fluorescence of whole cells. It should be particularly emphasized that FLATP3, as it turned out, preferably associated with cellular and nuclear membranes and stored in the cells, preventing their growth within 24 hours, despite the fact that the solution for conjugation was washed with monolayer after 2 hours and replaced with serum containing medium. Such duration of staining was not observed when using FLAEP-conjugate in the same conditions.

Fixation in 4% paraformaldehyde preserved cell morphology better than fixation in formaldehyde. Therefore, the cell has a higher resolution fluorescence images. This suggests that both conjugate localized in very discrete areas of the cytoplasm, and not evenly distributed. Observation in the light microscope show that the areas of localization can be endoplasmic reticulum, Golgi apparatus, and possibly mitochondrial membrane.

The observations performed at low resolution (lens 40x) live, netserver is decomposing the sample color FLAEP-conjugate easily penetrates into the cell, but is concentrated mainly in the nuclei. Perhaps this suggests that it is a generalized cytoplasmic and nuclear distribution depends on the molecules that remain unattached to the components of these compartments. When you commit such molecules will be washed away from the cells, inhibiting only the conjugate, which binds to cellular components. FLATP3-conjugate shows a similar result for live and fixed cells.

In cells treated with any compound at a concentration of 10 μm for up to 2 hours, obvious toxicity was observed. After 72 hours of incubation of Cos 1 cells in the presence of 50 μm FLAEP or more observed significant cytotoxicity in a standard cytotoxic assay with MTT (see below). After the same long 72-hour period keeping FLATP3 caused almost 80% cytotoxicity at a concentration of 12.5 μm (at the same concentration of diluted DMSO) and 70% cytotoxicity is at a concentration of 6.25 μm.

These results clearly indicate that introducing connections, load-bearing lipid acyl derivatives can facilitate their entry into cells. The observed cytotoxicity insignificant in the period neenie. In accordance with this carried out experiments to find out whether these compounds can be used for introduction into cells of other compounds, such as DNA, by linking to them.

B. Experiments for transfection.

(a) Cytotoxicity.

Cytotoxicity was determined using the standard analysis using MTT (3-[4,5, Dimethylthiazol-2-yl] 2,5-diphenyltetrazolium; tiazoly blue). Of Cos 1 cells was inoculated into the wells with 100 ál of culture medium in 96-hole tablet for micrometrology at a concentration of 2104cells/well for their attachment. Then added the test compound at a concentration of 2 to 100 μl of culture medium at the double breeding. Cells were incubated under normal culture conditions at 37oC and 5% CO2within 72 hours, 20 μl of MTT (5 mg/ml PBS) was added for 2.5 hours at 37oC and then all the liquid was removed. Added 100 μl of acidified propanol and the plate was shaken for 10 minutes prior to determination of OD at 570 nm with reference OD 630 nm.

Designed to introduce the connection K3ATP3 (trilinolenin-Tris-tripalmitate), named VerafectinA3, which, as expected, will join the DNA due to its total pologatsa in DMSO. Cytotoxicity was tested by continuous incubation of Cos 1 cells for 72 hours with K3ATP3, in the form of a diluted stock solution (2.5 mg/ml) in 25% DMSO. Watched 70% of surviving cells compared with the same concentration of DMSO, diluted compound at a concentration of 12.5-50 μm (Fig. 1a and b). When the compound was dissolved in ethanol similar concentrations of surviving cells was slightly higher (Fig. 2a and b).

b) delivery of DNA

Experiment 1: to check transfection properties misleading connection K3ATP3 (VerafectinA3), whole uncut plasmid that carries the gene encoding CAT (chloramphenicol-acetyltransferase) under the control of the late promoter of SV40, pSVLCAT (Cameron, F. H. and Jennings, P. A. (1989) PNAS 86, 9139-9143), was mixed with the test factor and put in Cos 1 cells under different conditions. After 48 hours of cultivation was estimated level of secreted CAT in the culture medium.

Way:

Solution I: 1 µg of plasmid DNA in 500 ál of modified Dulbecco eagle medium (DME)

Solution II: 0-20 ál VerafectinA3 (2.5 mg/ml of 25% DMSO) in 500 µl, DME, shake.

Combine the solutions I and II, briefly shake and incubate at room temperature for 10 minutes

Add the mixture to the washed cells in 60 the 37oC, 5% CO2.

Add 2 ml of DME + 10% FCS and leave for 48 hours.

To determine the levels of CAT in tissue culture medium (Sleigh, M. J. (1986) Anal. Biochem. 156.251-256).

The results:

Significant levels of CAT found in samples transfected with 5 μl (12.5 µg) VerafectinA3 (2.5 mg/ml = 1.8 mm) and higher. The peak level of CAT was observed when 10 μl (25 μg) VerafectinA3 (Fig. 3).

Experiment 2:

To compare the levels of transfection in Cos cells 1, used mother liquor VerafectinA3 (10 mg/ml) dissolved in 100% DMSO or in ethanol and within the optimal time stood cells with complex VerafectinA3/DNA.

Way:

a - Compare VerafectinA3 dissolved in DMSO and ethanol.

b - to Compare the effect VerafectinA3 on the cells for 6 hours and during the night. VerafectinA3 at a concentration of 10 mg/ml in 100% solvent diluted with water to 2.5 mg/ml

The transfection was performed as in experiment 1. VerafectinA3 left in contact with the cells for different time.

The results:

i) Significant levels of CAT found in all samples treated with 10 ál (25 mg) VerafectinA3 and above.

ii) Ethanol samples showed reduced efficiency of transfection.

iii) Incubation with Verafecti>/BR>To compare the levels of transfection achieved for CHO cells, used VerafectinA3 and G2, and commercial reagents Transfectam (Promega) and Lipofectamine (BRL).

Way:

Conditions were the same for Verafectin'and for Transfectam'and (see above), except that used 0.5 μg DNA pSVLCAT/60 mm Cup.

Lipofectamine was used according to the instructions for users.

0.5 μg pSVLCAT/60 mm Cup.

The CHO cells were planted at a concentration of 1105/60 mm Cup and left overnight to attach, in the environment of DME/Hans with 10% FCS.

Transfectam 5 or 10 ál of stock solution 1 mg/400 µl.

Lipofectamine 5 or 10 ál of a mixture composed of 3:1 with DOPE) 2 mg/ml

VerafectinA3 (K3ATP3) and VerafectinG2 (K3G[Glycine]TP2) for 5, 10 and 15 µl of each of the 2 mm stock solution (2.8 and 2.3 mg/ml, respectively).

The incubation period without serum in the presence of the tested factors and DNA lasted for 6 hours, then was added 1 ml of DME/Hams + 10% FCS. After 24 hours the medium was replaced with 2 ml of fresh DME/Hams + 10% FCS before collecting cells 48 hours later.

The results:

The CHO cells were literally 48 hours after the start of the operation transfection and tested for CAT activity. In all cases the peak transfection was observed when 5 ál transfection factor. Level the t levels, received for Lipofectamine.

The cat levels obtained using VerafectinG2, equivalent to approximately 64% of the levels obtained for Transfectam and 22% from the levels obtained for Lipofectamine.

See figure 4.

Experiment 4:

To compare the levels of transfection achieved for Cos 1 cells using VerafectinA3 (K3ATP3) and VerafectinG2 (K3GTP2), used commercial Lipofectamine reagent (BRL).

Application conditions described above were the same as for Verafectin'and. Conditions of use of Lipofectamine was in accordance with the instructions for users.

Of Cos 1 cells were sown at a concentration of 5105/60 mm Cup and left to attach overnight in an environment DME with 10% FCS.

Solution A: 0.5 μg pSVLCAT/60 mm Cup.

Solution B: Lipofectamine 10 and 15 µl of a mixture composed of 3:1 with DOPE) 2 mg/ml

or; VerafectinA3 and VerafectinG2 20 ál of 2 mm (respectively 2.8 and 2.3 mg/ml) stock solution.

or VerafectinG2 also tested with equimolar amounts of DOPE. VerafectinG2 and DOPE mixed in DDME and shook for 30 seconds before using.

Solutions A and B were combined before layering on the washed cells.

The incubation period without serum in the presence of the test fact the cells and after 48 hours.

The results:

Samples of tissue culture medium (supernatant) Cos 1 cells were tested for CAT activity after 48 hours after the start of the operation transfection. Of Cos 1 cells were transferrable VerafectinA3 and VerafectinG2 to levels approximately 60% and 30% respectively, which is achieved using Lipofectamine. Adding equimolar quantities of individual DOPE (2 mm in DMSO/H2O) to the sample VerafectinG2 approximately doubles the efficiency of transfection compared with only one VerafectinG2, bringing it up to 60% of the level that was achieved using Lipofectamine and equal action VerafectinA3. Received levels of CAT presented on figure 5.

C. the relative cytotoxicity

Comparing the relative levels of cytotoxicity caused by different factors, using dye experiment "Alamar blue (Alamar, Sacramento CA). Species of the compounds of the present invention typically exhibit much lower cytotoxicity than other factors.

For example, figures 6a and b.

The relative levels of cytotoxicity was determined in subsequent experiments using the dye MTS labelled Cell titer 96, water (Promega).

D. the relative effectiveness of connection options, leading to "TRD concentrations for each transactionname connection. The optimal concentration of a compound that represents the concentration giving the highest level of CAT in 48 hours or the highest percentage lac z (-galactosidase) stained cells after 24 hours was used for comparison of data in table 1. Since all of the compounds have not been tested in one experiment, the absolute values characterizing the transfection, not used. Rather, the values used to characterize the relative efficiency of transfection by Lipofectamine (BRL), which was included in all experiments as a control. Therefore, table 1 reflects the relative efficiency of a number of compounds in mediating transfection at least cells of the same type.

The experiments were carried out as described above in experiment 1, section B; with variations in experiment 3. Presents the results obtained at optimum levels transfairusa factor. All presented results were obtained by these authors; their number does not include commercial agents responsible for the literary execution.

Other variants synthesized and tested compounds described below.

Conclusion

The results presented in table 1, osvidetelstvovaniy level of transfection in the tested cell types. Compounds that did not contain lysine or palmitate, were unable to provide the transfection, indicating needs and DNA, attracting component (K) and the estimated penetrating into the cell component (palmitate). Different linkers have influenced the level of transfection and were both able to facilitate transfection.

Variability of introducing compounds that alter the efficiency of transfection

Compounds with the General formula was considered as having described the basic building blocks of x, y, R1-R4". These components are sequentially changed to determine the optimal building blocks for standard transfection of cell lines in the standard system analysis. The results will be presented through the use of model experiments and are shown as levels of expression of the reporter gene, obtained under standard conditions, or as overall levels of expression (the expression of CAT or-galactosidase) or in the percentage of transfected cells (-galactosidase - blue cells). Representative levels of cytotoxicity will also be described.

Peptide "x".

Introducing the connection VerafectinA3 described above, is 3 lysine residue (K3), prepared from 0+ to 6+.

Purpose: to Evaluate the optimal number of K residues required for efficient transfection.

Method: In a separate experiment conducted comparisons between:

a: K0ATP3, K3ATP3, K5ATP3 and K2ATP2, K5ATP2 (CHO cells)

b: K1ATP2, K2ATP2 (Cos 1 cells).

c. K3ATP3, K4ATP3, K5ATP3 (CHO cells).

All testing lipopeptide reagents initially dissolved at a concentration of 10 mm in 75% DMSO, and then diluted with water to prepare 2 mm mother solution, which was stored at 4oC.

From a 2 mm stock solution selected a number of volumes lipopeptide from 2 μl to 15 μl and diluted in 500 µl of the DDME. 1 µg of plasmid pSVLCAT separately diluted in 500 µl of the DDME. The solutions were combined, passed 10-20 min at room temperature before the addition of 500 ál of DDME 60 mm cups subclinic cells CHO (2105day-1) or Cos cells 1 (5105day-1), laundered DDME-free serum.

Cells were incubated for 6 hours under standard conditions at 37oC for 6 hours in 5% CO2. Over night incubation, the cells were added 1.5 ml DME/Hams + 10% FCS (CHO) or 10% FCS (Cos 1). For the next 24 hours before harvesting the cells, the medium was replaced with fresh - DME/Hams + 10% FCS or 10% FCS. Received cell lysate and determined CAT activity as described above. In the case of mittnik compounds K3ATP3 got a good transfection (mean - 6237 cpm for 25 ál of lysate after 20 min of experimental time at 37oC, 1 ál of C14-acetyl-KoA (the total number of pulses-40-50000 cpm)). K5 connection gave significantly reduced the number of pulses at its optimal level (average 1785 cpm). K0 connection showed moderate transfection in this experimental system, however, when using --Gal experimental system (described below) for CHO cells, the observed low levels of transfection.

Dipalmitate compounds the presence of K2 was significantly improved transfection, giving an average 5418 precision mechanical cpm, compared to the average cpm 132 for K5 connection. The viability of transfected Cup cells are shown in the chart 7b. The highest toxicity (lowest OP) marked in cups, showing the highest levels of transfection.

See figures 7a and 7b.

b. The inclusion of a single K does not significantly affect the course of transfection, medium 12670 cpm (25 μl of lysate, 135 min incubation at 37oC, 2 ál of C14-acetyl-KoA (the total number of pulses)-90-100000 cpm)). The presence of the second lysine residue is about 5 times increases the levels of transfection (average 62215 cpm) relative to the optimal results observed in Tholen decrease transfection with respect to K3-equivalent, giving the average level of the modified CAT activity values respectively 1071, 6363 and 13467 after 2 hours of incubation, 20 μl of the lysate.

Figure 9.

Conclusion

Based on data presented, the growth optimal activity at inclusion 0-5 lysine residues in Kn=1 to 5FNH2/3 can be depicted as next:

K3K2>K5>K4>>K1.

A representative graph of cell viability is shown for experiment b (see Fig. 7b). This shows the inverse relationship between cell viability and transfection. This is the usual pattern.

Linker "y".

Introducing the connection described above, is 3 lysine residue alanine (A) linker at position "y". Experienced options that were carrying the alanine (A), leucine (L), phenylalanine (F), lysine (K) or glycine (G).

Objective: to Determine the optimal linker residue for efficient transfection.

Method: In a separate experiment conducted a comparison between:

a. K2 K/F/L TP2 (Cos 1)

b. K2 A/L TP2 (Cos 1)

c. K3 A/L/G TL3 (Cos 1)

For transfection pSVLCAT and subsequent analysis of the enzyme CAT experiments "a" and "b" was carried out as described above.

In experiment c was transferrable plasmid rotora uses early promoter of SV40 (Sleih, M. J. and Lockett, T. J. (1985). EMBO J. 4: 3831-3837). This experiment and the analysis was modified by Felgner et al J. Biol. Chem. 269(4) 2550-2561, 1994. These conditions made it possible to analyze the 72 different combinations and concentrations of DNA and the test factor on the microtiter plate. As the optimal result for each reagent chose a well-defined level of gene product.

Cytotoxicity was determined using the dye alamar blue, and a representative graph is shown for the experiment "a".

Results.

a: When comparing linkers K, F and L in Deliziosa, dipalmitate connection L showed much better results in comparison with F and was in fact more effective than K-connection. See Fig. 10a. The toxicity of L - and F-compounds was slightly higher than K connections, indicating that high levels of CAT were not visible simply because there were high levels of survival of cells.

Figure 10b.

b: comparison between A-linker used in the experiments compared the peptide, and L is a linker, which showed the highest levels of gene expression in experiment a, the two compounds showed very similar activity.

Figure 11.

c: Two leading linker A and L anaim to obtain transfection compared with other compounds, showing relative levels of transfection: L 57%, G 12% and Lipofectamine 39%.

Figure 12.

Conclusion

When comparing the data presented, the effectiveness of different linkers can be rank

AL>F>K>>g

Lipid acyl derivatives "R1-R4".

I. the Number of acyl derivatives on Tris-linker, 1-3.

The presence of Tris-linker facilitates the connection of one, two or three of the lipid acyl derivatives of the peptide and the linker. In General, these derivatives must be identical to each other. Compounds bearing all identical building blocks, and bearing one, two or three of the lipid acyl derivatives were compared using standard transfection and analysis techniques described above.

Purpose. To compare the ability of the lipid acyl derivatives of peptide compounds bearing one, two or three of the lipid acyl derivatives to transferout mammalian cells.

Way.

Compounds were tested for their ability to transferout or CAT - or --Gal-gene introduced plasmids and analyzed as described above.

Experiments:

a. K2 A T P1/P2/P3 (Palmitate 1, 2 or 3) (Cos 1)

b. K2 F T P1/P2/P3 (Cos 1)

c. K2 L T P1/P2/P3 (Cos 1)

1, 2 or 3) (Cos 1)

g. K4 A T P1/P2/P3 (Cos 1)

Results.

a: comparison of the compounds tested in the experiment "a", showed that the molecules of di - and tripalmitate both give high levels of transfection, whereas the molecule monopalmitate very inefficient.

Figure 13a.

In other experiments K3-monopalmitate connection showed 30% of the activity of molecules K3-tripalmitate (not shown).

The relative cytotoxicity of the compounds shown in the accompanying graph.

Figure 13b.

b: Relative transfection ability mono-, di - and tripalmitate connections phenylalaninol linker were similar to compounds with alanine linker (a).

Figures 14a and b.

c: Relative transfection ability mono-, di - and tripalmitate connections latinoam linker were similar to compounds with alanine linker (a).

Figures 15a and b.

d: In the case of the lysine linker dipalmitate proved to be significantly less active than the molecule tripalmitate. Increase transfectional activity tripalmitate accompanied by an increase in cytotoxicity as seen in the previous cases.

Figures 16a and b.

Figures 17a and b.

f: In this comparison, where the connection with different lipid acyl groups (n = 1-3) were compared on transfection efficiency against 3 different length of the lipid acyl derivatives (palmitate, myristate and laurate), compounds with 3 lipid acyl groups were consistently more efficient in transfection than the connection with 2 groups or with 1 group. In the presence of 3 lipid acyl groups toxicity was generally somewhat higher, but is always less than lipofectamin'ohms. See table 2.

Each presents the result is the highest rate found among the different conditions of 72 wells of microtiter plates. Shows the ratio [lipid]/[DNA], where this maximum was obtained. Shows the experiments carried out on cells, Cos 1, as described above. OP MTS is a measure of the relative viability. *These points are taken from a separate experiment and the small variability between the absolute values of the experiments is expected. Viability in this experiment was measured using the dye alamar blue and therefore not amenable to direct cf is alanine (K4ATP1/2/3). As follows from the previous experiments, tripalmitin was more effective than di - or monopalmitate. When using any one of these compounds activity was moderate. Cytotoxicity was lower, despite the high levels of these reagents required for optimal transfection.

Figures 18a and b.

Conclusion.

The results of experiments on compounds "K3" similar compounds "K4". Then there are three lipid acyl group is better than two or one group, with regard to their ability to transferout cells.

Conclusion for R1-R4.

As shown in the above examples, compounds with three lipid acyl derivatives generally show higher ability to transferout cells than compounds with one or two lipid acyl derivatives. In the optimal points transfection data connections are generally much less toxic to cells than the commercial tool "Lipofectamine". Although much of the data presented in this section are taken from experiments carried out on cells, Cos 1, these results were reflected in the results obtained for CHO-cells. The optimal concentration for different ratio between [lipid] /[DNA] in General did not depend on cell type, and relative transferability compounds remained similar.

Comparison of the presented data allows us to numerically evaluate the effectiveness of different lipid acyl groups (n = 1-3) noncomposite unmixed compounds in the form of a series

3>2>1.

Lipid acylphosphate "R1-R4"

II. The length of the carbon chain.

Objective: to Determine whether lipid acylphosphate different lengths (relative to C16) (palmitate) have greater ability to transferout mammalian cells.

Method: Created connection options Tris-trilinolenin (K3AT), bearing one to three lipid allpresan, characterized by the following data:

C16 Palmitate (P)

C14 Myristate (M)

C12 Laurate (L)

C10 Caproate (C)

Each of these compounds was checked on transfection ability and cytotoxicity in a standard cell lines, using standard conditions, as it is written above.

Results.

a: Comparison of P-, M-, L - and C-derivatives in Cos 1 cells and CHO.

Table 3 shows that the connection laurate and myristate generally able to transferout cells more efficiently than originally used derived odes to table 3.

Each presents the result is the highest indicator for 72 different conditions, wells of microtiter plates. Connection [lipid] /[DNA] , which obtained the maximum reading is presented in parentheses for the two different types of cells in the following order; (1 Cos) (CHO). Presents the experiments carried out on cells, Cos 1, as described above. Viability was measured using the dye alamar blue. Sign # identified compounds analyzed in a separate experiment. Sign*mean viability was determined using the dye MTS. OP is a measure of the relative viability of cells matured with different factors, as well as with the dye alamar blue, a higher value indicates the highest cell viability.

b. R1-R4 as C10-C16, analyzed in HeLa cells.

Objective: to Determine whether the most effective lipid acyl group identified in Cos 1 cells and CHO, is also the most effective and HeLa cells.

Method: Instead of Cos 1 cells and CHO in DME + 10% FCS were seeded HeLa cells at a density of 2104/well and were transferrable their pPGKlaczNLS, as described above.

After 48 hours using the ADC the-Gal-analysis show, that as in the case of Cos 1 cells and CHO, with a decrease in the length of the carbon chain from C16 to C12, transfection capacity increases. When using molecules C10 transfection ability was severely curtailed and was equal in this series peak transfectional efficiency for C12. It was unexpectedly found that the peak level of cell viability was also inherent C12-connection (K3ATL3).

Figures 19a and b.

Conclusion.

Changing the length of the lipid acyl derivatives strongly affects the ability of these compounds to transferout some types of cells. In each of all the analyzed cell types transfection supported approximately at the same level. And myristate, and laurate in this regard to give large levels of transfection than the model palmitate, compared with laurate, which is the best among them.

The linker y is an unusual amino acids

The number of amino acids used as linker groups. It includes, as described above, leucine, glycine, alanine, phenylalanine, -BOC (without -)-lysine. In addition, also analysed unusual amino acids, such as amino, aminocaproic and aminocaproic acid (which create long: to Determine transfection properties of compounds with a longer interval between tricom and charged peptide "x" with an unusual amino acid linkers, changing the length in the position "y".

Method: Synthesized peptide/ipidacrine conjugates using unusual amino acids - amino-propionic (C3), aminocaproic (C5) and aminocaproyl (C7) acid as linker groups, trilinolein domain that binds nucleic acid, and laurate for the three lipid acyl groups. These new conjugates binding to DNA and Tris/ipidacrine components were separated using an additional, respectively, 3, 5 and 7 associated carbon, compared with model molecule. These compounds are then analyzed according to the standard method described above to determine their relative ability to transferout cells.

The results:

K3-y-TL3-connection, where y=C3, C5, C7, A, L, G, compared to transfection efficiency relative to each other and with the commercial tool - Lipofectamine'ω using the above standard system reporter gene-Gal. All connections, except K3GTL3, was more effective for transfection than Lipofectamine. C3-, C5 - and, especially, C7-compounds showed much greater efficiency than the connection using a standard single amino acid linker.

Figure 20a.

Cytotoxic the attention transfection are not accompanied by an increase of cytotoxicity.

Experiment 2.

The proportion of transfected cells.

Objective: to Determine whether the increase in transfection shown by the compounds C3-C7 due to an increase in the proportion of transfected cells.

Method: These conjugates used in transfection of CHO cells and Cos-1 using pPGKlaczNLS to estimate the proportion of transfected cells. The transfection conditions were the same as described above in CAT experiments with cells grown in 35 mm plates for transfection. Cells were sown on the day before transfection at a density 3,4105/Cup (Cos 1) or 6,8104/Cup (CHO). After analysis on the viability after 24 hours of transfection, the cells were washed 2x in PBS, then fixed for 5 min at 4oC in 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. Cells were washed 2x with cold PBS. A freshly made solution for staining (10 ml 0.1 M phosphate buffer, pH 7.3; 1.0 ml of a mixture of 105 mg releasescontact potassium 2.5 ml of H2O and 82 g relatoseroticos potassium 2.5 ml of H2O (1:1); 0.2 ml of 2% X-Gal in dimethylformamide, and 11.2 μl of 1 M MgSO4) (2 ml) was added to each Cup and the cups were incubated at 37oC to the development of painting. Cells are selected from random areas were placed under light microscope and determined who I am when using the test factors are reflected in the increased proportion of transfected cells as shown by the number painted in blue color cells. Figure 21a shows the results obtained with CHO cells, and figure 21b shows the results for Cos 1 cells. On the X-axis of these panels Lfe means Lipofectamine; vf - means model peptide/lepidochelys conjugate K3ATP3; A means K3ATL3, C3, C5 and C7 are amino, aminocaproic and aminocaproyl acid, combining the conjugates.

Figures 21a and b.

Conclusion.

Increasing the length of the linker part of the peptide/ipidacrine conjugates much improve their transfection properties by total levels of expression remoterepo gene, as well as on the fraction of transfected cells. It should be particularly emphasized that the peak levels of transfection using these reagents is achieved at a relatively low level of toxicity. The effect of these factors does not confirm the earlier observation that increased levels of transfection directly associated with increased levels of cytotoxicity.

Improved the ability of the conjugates with long linkers to stimulate the transfection does not depend on cell type. The data presented in figures 22a and b show that K3C7TL3, conjugate, stitched aminocaproic KIS is the prostate cancer cells human T-cell line Jurkat (figure 22b).

Figures 22a and b.

Investigated cell types.

As described above, the conjugates presented the General formula used for successful transfection of several cell types. A more complete list of transfected cells provided at the end of the descriptions in table 5.

Oligonucleotide transfection.

Objective: to Determine whether compounds that have shown the ability to transfer reporter genes in whole plasmids into cells, are also effective for transfection with oligonucleotides.

Method: 18-gauge 5'-fluorescent tiofosfornoy oligonucleotide was transferrable in CHO cells. Compared the ability K3C7ATL3, K3ATL3 without factor to transferout oligonucleotide into cells. Oligonucleotide serially diluted from 121,2 μm solution of nucleotides up to 60.6 and 30.3 μm nucleotide solution. Test lipopetides serially diluted of 84 μm to 42 and 21 μm. These breeding combined in a matrix of 3 x 3, giving, as you can see, half the concentration. In other words, the most dilute the oligonucleotide becomes to 60.6 μm solution of the nucleotide (3,37 18 μm-dimensional oligonucleotide) and 42 μm lipopeptide. The mixture was incubated for 10 min at room temperature and Saudi environment DDME in a total volume of 100 μl. Wells containing mixture with untested factor had three different concentrations of the oligonucleotide, layered under the same conditions.

After 3 hours of incubation under standard conditions at 37oC and 5% CO2the mixture was removed and replaced with 50 μl of DDME. Under the confocal microscope, the cells looked alive. The level of fluorescence was so high that the point aperture for laser, confocal microscope (MRC 500 company Bio Rad, closed to a minimum. All images were collected on the same settings for a quantitative comparison.

After confocal analysis in the wells were again added 100 μl of EMEM + 10% FCS and cells were incubated under standard conditions during the night. Cells are re-examined for fluorescence.

Results.

Results transfection 3 hours after transfection are shown in table 4.

Conclusion.

The use of any transfection factor gives 100% cell survival, absorbing the high levels of the oligonucleotide. A 3-hour time point, as described in the experiment, the cell nucleus intensively stained with a fluorescent oligonucleotide.

Results marked with an asterisk "*"illustrate Naim is and. Without the factor levels of fluorescence is limited to a single spot with the changing intensity of each fluorescent cell at the level of detection used in the experiment.

It is obvious that a large proportion of cells compared with the ones listed in table 4 absorbed oligonucleotide in the absence of transfection factor, however, the absorbed amount was insufficient for registration of the used detection level.

After an overnight incubation in complete medium (EMEM + 10% FCS) levels of fluorescence were reduced in all samples, but especially noticeable change was the loss of fluorescence by the majority of nuclei with characteristic speckled cytoplasmic coloration. The levels of fluorescence in the samples, which used transferowy factor were much higher than those samples that are not used transferowy factor.

The technology of manufacturing of medicines.

Most commercially provided transfection connections made in the form of liposomes with a neutral lipid, dioleoylphosphatidylcholine (DOPE). In addition, from the literature it follows that the connection with two lipid acyl derivatives, probably bol is acyl derivatives, and indeed commercial factors asvariant have two acyl derivatives. In light of the above results, which show that a shorter chain acyl derivatives, especially laurate, has increased the functional activity upon transfection of cells, it is possible that the use of compounds with shorter chain phosphatidylethanolamine also may indicate more efficient transfection. Therefore, the use of DOPE should be viewed in this way to illustrate its ability to form effective liposomal constructs for transfection and is not limited to them.

Objective: to Analyze the effectiveness of liposomal compositions with lipid DOPE.

Method: Verafectin A2 (K3ATP3) was Packed in liposomes using standard methods (K. Yagi et al. Biochem. Biophys. Res. Comm. 196 3., 1993 pp 1042-1048) and placed in water at 2 mm. Compositions were prepared at molar ratios of 1:2, 1:1 and 2:1 with DOPE.

Liposomal compositions were compared with commercial compounds, as described in individual experiments below:

a. Using a standard transfection method described above, and 1 μg plasmid pSVLCAT, these compositions were analyzed in CHO cells when only m on the same type of cell. Education CAT were analyzed 48 hours after transfection.

b. Liposomal compositions were analyzed at the same concentrations used plasmids pPGKlacz that encodes the expression of a-Gal, and compared with Lipofectamine'ohms and DOTAP (Boehringer Mannheim). 24 hours after transfection the cells were fixed and stained to detect the expression of-Gal.

The results:

a. The use of liposomal compositions led to the expansion of CAT expression from CHO cells from 62%to 156% from the optimum Lipofectamine'and that induced expression using three different ratios in two separate experiments. Use VFA2 connection in liposome composition gave levels of about 5% from Lipofectamine'and. DOPE, taken separately, in any form - decomposition or "composite", due to the liposomal method did not give transfection. NB. DOPE, taken separately, does not form a true liposomes, see below EM-section.

Figure 23a.

b. Preliminary counting stained cells among unpainted shows that the best liposomal composition (K3ATP2:DOPE 1:2) gave a double, and DOTAP gave approximately half the number of transfected cells compared to cells obtained with Lipofectamine. The percentage of transfected CHO-cells p is ktora was not optimal for these VerafectinA2/DOPE compositions. And data lipofectamine and DOTAP obtained at optimal concentrations.

Preliminary data suggest that the cytotoxicity of these compositions is roughly equivalent to Lipofectamine and DOTAP (not shown).

Electron microscopy.

Solutions of composite and non-composite connections were analyzed for the presence of membranophones patterns. In separate experiments, analyzed the following samples:

a. Negatively stained samples of composite connections K3ATP:DOPE and non-composite connections K3C7ATL3 and Lipofectamine.

b. Rotary natnennya samples K3C7ATL3 and Lipofectamine were mixed with twice the number of nucleotide (a plasmid).

Way.

Negative staining.

The starting solutions of liposomes with a concentration of 2 mm was diluted 1:10 with filtered distilled water and mixed 1:1 with filtered negative dye (ammonium molybdate 2%, pH 6.5) was layered on a carbon film-substrate coated 200 mesh copper/rhodium mesh for electron microscopy and examined in the electron microscope Jeol JEM 100XC at 60 kV with magnification between 33k and 100k.

Rotary metallovedeniye.

Liposomal ratio is 0.69 and 0.25: 1 mol DNA-nolence-substrate, covered with mesh, and rotation Natinal platinum-palladium metal (60: 40), evaporated at an angle of 7oand examined in the electron microscope at 60 kV with a magnification between 26k and 66k.

Results.

a. To determine the successful formation of liposomes, negatively stained samples composed K3ATP2:DOPE analyzed in TEM. It is established that these samples formed multilamellar liposomal structure, while DOPE, taken separately, in the same conditions they were not formed.

As a composite, showing the ability to active liposome transfection was of interest to determine whether or not pure non-composite connections, which gave an effective transfection had any membranophone structure. Some of these compounds because of negatively stained and re-analyzed in the TEM. It is interesting to note that these compounds, which gave an effective transfection and had 3 lipid acyl derivatives were found spontaneously formed "rafts" or "stack" membrane-bound vesicles with a size of from 10 nm to 0.2 μm.

b. Compounds were mixed with plasmid DNA in suboptimal (0,25) and optimal (0,69) ratios lipopeptide:nucleotide and naturellebella part of the DNA included in the collection of the liposomes. When the ratios when the connection has been represented (0,25:1) a large amount of DNA remained free in solution. This was true for both the analyzed compounds and for K3C7TL3 and Lipofectamine, which showed great similarity under EM when mixed with DNA, while no connection is made from the DNA of their manifestation was different.

Nacompetitie mixture transfection compounds and other lipids.

Noted the discrepancy between the part of the cells transfected with oligonucleotides (100% fluorescent cells) and cells that expressed gene product (<100% blue cells). Since a gene product is needed to DNA found in the nuclei, transcarbamoylase and was broadcast, and taking into account that only the oligonucleotide has the advantage to penetrate through the plasma membrane, in order to implement the "transfection", the result was not unexpected. However, this suggests that the successful transfection and education gene product is a multistep process that could benefit from a combination of compounds that may have different abilities to traverse the various movedinto, bearing various lipid acyl group, differing in the number and in length, and analyzed on-the-Gal Cup in the analysis, as described below.

Improved transfection with the help of some compounds can be seen in the simple addition of neutral lipid DOPE, as shown in the above experiment 4 using VerafectinG2 (K3GTP2).

Objective: Determine whether the mixture lipopeptide compounds with the General formula described in this application have the advantage to improve the transfection of genes, as identified by the detected gene product compared to using net connection.

The mixture of compounds; Experiment 1.

Method: Pre-established that to achieve optimal transfection using the connection K3ATL3 requires 5 μm K3ATL3. K3ATL3 was mixed separately with K3ATL1, K3ATL2, K3ATM3, K3ATP3 and K3ATC3 to obtain a total lipid concentration of 5 μm in 100 μl at a ratio of 0: 5, 1: 4, 2:3, 3:2, 3:1 and 5:0. Similarly K3ATM3 mixed with K3ATM1, K3ATM2 and K3ATP3. Each lipid mixture was combined with a constant amount of DNA pSVGAL (0.5 μg/well) and gave the opportunity for a normal course of transfection.

The results:

Expressioin expression in the holes, containing a combination of K3ATL3 and K3ATM3 taken in the ratio of 4:1, which showed a higher level of expression of a-Gal than taken separately K3ATL3 or K3ATM3 (data not shown).

Experiment 2:

He was devoted to further analysis of the created mixture of two lipopetides K3ATL3 and K3ATM3 (4:1 polyarnosti) and testing them in a standard Cup experience, in which he used twice diluted concentration and lipopeptide, and DNA. This is compared with the expression obtained using pure compounds under the same conditions.

The results:

A small but definite increase in the expression obtained using a mixture of lipopetides. For each analyzed concentration lipopeptide optimal level transfectional factor resulted in higher transfection used for a mixture of L3/M3 (4:1) compared with only one L3, taken in the same concentration.

Figure 24.

Conclusion.

Combination lipopeptide transfection factors can give the best levels of transfection in comparison with any single factor. It is expected that further testing in this area were useful for other combinations that give the best levels of transfection in Estenne effect on their individual transfection characteristics.

Transfection in vivo.

Objective: to Determine whether or not the connection represented by the General formula can be used for efficient delivery of DNA in a body that is manifested in the expression of the delivered gene.

Method: Plasmid pGFP-N1 (Clontech) encoding Green Fluorescent Protein, 10 μg in 10 μl was diluted in 20 μl of 5% dextrose in water and then combined with 2.1 μl of 10 mm K3ATL3 (10% DMSO) 27.9 μl of 5% dextrose in water. After 10 min of incubation at room temperature the mixture were injected with in the leg muscle naked mouse. Mice were bled 24 hours and muscle were rapidly frozen on dry ice. Did tissue slices and analyzed under a confocal microscope for fluorescence.

The results:

In the cytoplasm of muscle cells was observed by fluorescence and scatter very strong fluorescence in the end plates of the motor nerve. In tissue slice penjelasannya feet fluorescence was observed.

Conclusion.

Preliminary experiments show that, using these compounds, it is possible to achieve efficient gene transfer in a whole organism.

The present invention provides an effective method of delivery seandainya traditional-based CaPO4methods of transfection, being in some embodiments, implementation of the present invention is much better to use commercial transfection factors. It is obvious that this method presented in this invention is equally suitable for the delivery of other compounds, such as pharmaceuticals nucleic acids such as ribozymes, antisense sequences, etc.

Professionals in this field should be borne in mind that in the present invention may be a number of changes and/or modifications, as shown in the specific embodiments, its implementation, without going beyond being or volume widely represented of the present invention. Therefore outlines the options considered comprehensively, including illustrative and not limited.

1. How Introduzione nucleic acid into the cell, characterized in that carry out the exposure of the cells with a compound having the formula

< / BR>
in which

w represents a nucleic acid;

x is a peptide or amino acid;

y represents a linker having a chain length equivalent to 1 to 20 carbon atoms, or missing;the same or different and are either hydrogen, methyl, ethyl, hydroxyl or acyl group derived from a fatty acid having a carbon chain of 3 - 24 saturated or unsaturated carbon atoms, provided that at least one of R1, R2and R3represents an acyl group derived from fatty acids.

2. The method according to p. 1, wherein y is present.

3. The method according to p. 1 or 2, wherein R1, R2and R3are the same.

4. The method according to any of paragraphs.1 to 3, wherein R1, R2and/or R3are acyl derivatives of fatty acids selected from the group consisting of palmitate, myristate, laurate, kaproata, oleate or cholesterol.

5. The method according to p. 4, wherein R1, R2and/or R3are acyl derivatives of myristate or laurate.

6. The method according to any of paragraphs. 1 to 5, characterized in that the nucleic acid is a DNA, RNA or oligonucleotides, or DNA, or RNA, modified oligonucleotides, or a combination of both.

7. The method according to any of paragraphs.1 - 6, characterized in that the compound is present in a liposome or mixed with another lipid.

9. The method according to p. 8, wherein y represents amino, or aminocaproic aminocaproyl acid.

10. The method according to any of paragraphs.1 to 9, characterized in that x has a total positive charge.

11. The method according to p. 10, characterized in that x represents monolisan, diligin, drilizen, tetralin or tantalizin.

12. The method according to any of paragraphs.1 - 11, characterized in that w is covalently attached to x.

13. How Introduzione nucleic acid into the cell, characterized in that carry out the exposure of the cells with a compound having the formula

w ... x - y - NH - CH2- CH2O - R5,

in which w represents a nucleic acid;

x is a peptide or amino acid;

y represents a linker having a chain length equivalent to 1 to 20 carbon atoms, or is absent;

R5represents an acyl group derived from a fatty acid having a carbon chain of 3 - 24 saturated or unsaturated carbon atoms.

14. The method according to p. 13, wherein y is present.

15. The method according to p. 13 or 14, wherein R5is an acyl derivative of fatty sour is 16. The method according to p. 15, wherein R5is an acyl derivative of myristate or laurate.

17. The method according to any of paragraphs.13 to 16, characterized in that the nucleic acid is a DNA, RNA or oligonucleotides, or DNA, or RNA, modified oligonucleotides, or a combination of both.

18. The method according to any of paragraphs.13 to 17, characterized in that the compound is present in a liposome or mixed with another lipid.

19. The method according to any of paragraphs.13 to 18, characterized in that the linker group y has a chain length equivalent to 3 to 7 carbon atoms.

20. The method according to p. 19, characterized in that y represents amino, or aminocaproic aminocaproyl acid.

21. The method according to any of paragraphs.13 to 20, characterized in that x has a total positive charge.

22. The method according to p. 21, characterized in that x represents monolisan, diligin, drilizen, tetralin or tantalizin.

23. The method according to any of paragraphs.13 to 22, characterized in that w is covalently attached to x.

24. The compound for use in the introduction of nucleic acid into the cell, and the compound has the formula

< / BR>
in which w represents nucli the length of the chain, the equivalent of 1 to 20 carbon atoms, or is absent;

R4represents H or CH2O - R3;

R1, R2and R3are the same or different and are either hydrogen, methyl, ethyl, hydroxyl or acyl group derived from a fatty acid having a carbon chain of 3 - 24 saturated or unsaturated carbon atoms, provided that at least one of R1, R2and R3represents an acyl group derived from fatty acids.

25. Connection on p. 24, wherein y is present.

26. Connection on p. 24 or 25, characterized in that R1, R2and R3are the same.

27. The compound according to any one of paragraphs.24 to 26, characterized in that R1, R2and/or R3are acyl derivatives of fatty acids selected from the group comprising a palmitate, myristate, laurate, caproate, oleate or cholesterol.

28. Connection on p. 27, wherein R1, R2and/or R3are acyl derivatives of myristate or laurate.

29. The compound according to any one of paragraphs.24 to 28, characterized in that the connection is present A compound contains the linker group y, having a chain length equivalent to 3 to 7 carbon atoms.

31. The compound according to any one of paragraphs.24 to 30, characterized in that w is covalently attached to x.

32. The compound according to any one of paragraphs.24 to 31, wherein w represents a DNA, RNA or oligonucleotides, or DNA, or RNA, modified oligonucleotides, or a combination of both.

33. The compound for use in the introduction of nucleic acid into the cell, and the compound has the formula

w ... x - y - NH - CH2- CH2O - R5< / BR>
in which w represents a nucleic acid;

x is a peptide or amino acid;

y represents a linker having a chain length equivalent to 1 to 20 carbon atoms, or is absent;

R5represents an acyl group derived from a fatty acid having a carbon chain of 3 - 24 saturated or unsaturated carbon atoms.

34. Connection on p. 33, wherein y is present.

35. Connection on p. 33 or 34, characterized in that R5is an acyl derivative of a fatty acid selected from the group consisting of palmitate, myristate, laurate, kaproata, oleate and cholesterol.

36. tx2">

37. The compound according to any one of paragraphs.33 to 36, characterized in that the compound is present in a liposome or mixed with another lipid.

38. The compound according to any one of paragraphs.33 to 37, characterized in that the compound contains the linker group y having a chain length equivalent to 3 to 7 carbon atoms.

39. Connection on p. 38, wherein y represents amino, or aminocaproic aminocaproyl acid.

40. The compound according to any one of paragraphs.33 to 39, characterized in that x has a total positive charge.

41. Connection on p. 40, characterized in that x represents diligin, drilizen, tetralin or tantalizin.

42. The compound according to any one of paragraphs.33 - 41, characterized in that w is covalently attached to x.

43. The compound according to any one of paragraphs.33 - 42, wherein w represents a DNA, RNA or oligonucleotides, or DNA, or RNA, modified oligonucleotides, or a combination of both.

 

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