JPH045226A - Surface-modifiable liposomes and methods for producing surface-modified liposomes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a surface-modifiable liposome used as a drug carrier, an immunoassay reagent, etc., and a method for producing the surface-modified liposome.

(Conventional technology) In recent years, in the medical and pharmaceutical fields, a method (drug delivery system) has been developed to selectively deliver all drugs to cells and tissues that are attached to the affected area without affecting normal cells and tissues. , DDS) are being considered.

To achieve this, liposomes are loaded with hydrophilic or lipophilic drugs, and proteins, antigens,
Surface-modified liposomes with immobilized antibodies, sugars (polysaccharide Kanakuramu), etc. are being investigated.

[Cancer Research (Cancer, Res,) No. ≠
Volume 3, page 13λ1, 13 years ago, same magazine, see! , Toru Da 7/
(/ri17), Liposome Lisa-chi (Liposo
10,000, liposomes are unstable and are particularly susceptible to phagocytosis by phagocytic cells present in reticuloendothelial threads during in vivo administration, or
The problem is that it disappears from the blood very quickly. As a solution to these problems, Japanese Patent Application Laid-Open No. 6J-J/37 discloses a method of modifying the surface of liposomes with a polysaccharide derivative.

Furthermore, in the fields of clinical testing and immunology, various analytical methods using surface-modified liposomes in which physiologically active substances, immunologically active substances (including antibodies and antigens), etc. are immobilized on the liposome surface have been disclosed [JP-A-Sho No. 47-2101!r, JP-A No. 47-1≦26J, JP-A No. 40-/31≠4].

A typical method for modifying the surface of liposomes is N-J-(2-dithiopyridylcopropionyl phos-7atidylethanolamine (DTP-PE)).
N-succinimidyl-3-(λ-pyridyldithio)
A method of combining propionate (8PDP) activated tanba (quality, etc.) [Nature, $21
Volume 1, page 402, / P10], the method using a crosslinking agent (μN-succinimidyl + -(p
-maleimidophenyl)butyrate (8MPB), N-
Succinimidyl-≠-(p-maleimidophenyl)acetate (8MPA), N-succinimidyl-4'-
(p-maleimidophenyl)propionate (8MPP
) [Journal of Biological Chemistry (J, Biol, C
Hem, N, @J7, vol. 7, p. -214, 1995, etc.).

Other methods for immobilizing substrates on the surface of liposomes include
A method in which shield lipids, etc. are mixed in advance during liposome preparation, and the sugars are oxidized to form an aldehyde fund, which is reacted with amino groups such as proteins to form a Schiff salt fund. -Biophys, Act,)
, Vol. tUO, p. 4, /91/) and methods of introducing hydrophobic groups into proteins and incorporating them into separately prepared liposomes [Biochjm, Biophys-Acta]. .

Volume rix, page 114, 79! ! year, etc.).

However, these methods have many steps and are complicated, and the establishment of a method for easily immobilizing and modifying a substrate on the surface of liposomes is a highly needed issue in this field.

10,000, in the field of organic synthetic chemistry, a method for producing aldehydes in good yield by reducing carboxylic acid derivatives using 3-acylthiazolidine-corchion [JP-A-74L-
72271, Chemistry Letters, (Chem, Le
), p. 1aa3, 1977], and a method for selectively producing amides [Tetrahetoy Letters (Tetrahetoy Letters)].
trahedronLett-), Volume 21, 1101
Page, 1yto, Heterocycles
cycles), Volume 77,! 37 pages, /2 years] and method for producing thioester gold [Journal Op.
Chemical Research (J, Chem, Re5search (
S), /213, p. 20] is disclosed. However, there is no example of 3-acylthiazolidine-corchion being used as a surface modification reagent for liposomes.

In addition, in the field of polymer chemistry, activators that have co(3H)-benzoxacilone and 2(JH)-benzothiazolone as leaving groups and chemoselectively form amides [Journal of the Society for Organic Synthesis, Vol. Roll ≠.

Hari page, / year, Bulletin of Salmony
Nnaiatei Shalamin Bull.

Chem-8oc, Jpn, vol. zt, 32ri 1
Page, /f I 7th year, Macro Molecules Macro
molecules, vol. 11, p. 2313 1ytz
] has been disclosed.

However, there are no examples of amide compounds having these leaving groups being used as surface modification reagents for liposomes.

(Problems to be Solved by the Invention) Therefore, one purpose of the present invention is to solve the problems of the conventional method, and to coat the surface of the liposome with peptide (containing amino I12), protein (containing antigen and antibody), The object of the present invention is to provide a method for easily producing surface-modified liposomes on which various substrates such as sugars (including polysaccharides) are immobilized, and to provide surface-modified liposomes on which various substrates are immobilized.

(Means for Solving the Problems) For the purpose of the present invention, (1) be composed of a compound represented by the following general formula CI) and a lipid capable of forming liposomes'fI: characterized surface-modifiable liposomes; (R1 represents a hydrophobic group, R, 2, and R3 are organic groups (Ht
- may be linked to each other to form a ring or a double bond. An oxygen atom or a sulfur atom is shown in X and Y; Production method; (3) At least seven strains of the compound represented by the general formula 1"I) described in (1) and an amino group and/or a thiol group t
- A method for producing surface-modified liposomes, which is characterized by mixing a compound obtained by binding a substrate that binds to a liposome-forming lipid with a lipid capable of forming liposomes.

In other words, in the present invention, a substrate having an amino group or a thiol group is immobilized on the surface of the liposome using a liposome in which a long-chain active amide represented by the general formula CI) is embedded in the ribbon membrane molecular membrane. and general formula C
The main idea is to react the long-chain active amide shown in I) with a substrate containing an amino group or a thiol group to form a substrate derivative containing a long-chain group, and then mix this with a liposome-forming lipid to form a liposome. That is.

A method for forming surface-modified liposomes of the present invention will be explained.

In the first method, a liposome is prepared by mixing a long-chain active amide represented by the general formula [I] with a liposome constituent material.

The constituent materials of the liposome according to the first method of the present invention include lipids used in existing liposome manufacturing methods, excluding phosphatidylethanolamine and phosphatidylserine. (For example, natural lecithins such as egg yolk lecithin and soybean lecithin, di/ξ lumitoylphosphatidylcholine (DPPC), cimilistoylphos-7 atidylcholine (1) MPC), distearoylphos-7 atidylcholine (])8PC), dioleylphos-7 atidylcholine (DOPC) ), dipalmitoylphos-7-acylglycerol (DPPG), simyristoylphosphatidic acid (DMPA), and the like. ) The lipids used may be one type or two or more types.

A mixed system with cholesterol and the like may also be used. Also,
Things other than lipids and cholesterol may be mixed.

In the first method of the present invention, the molar ratio of the long-chain active amide represented by the general formula CI) to the total liposome constituent materials can be freely selected as long as it is within the range that allows liposomes to be constructed, but preferably! Below the OS, more preferably J! Less than 1 is better.

Liposome preparation methods according to the first method of the present invention include portic swing method, ultrasonic method, surfactant removal method, reverse phase evaporation method (CREV method), ethanol injection method, ether injection method, and pre-vesicle method (Pre- Vesicle method),
French press extrusion method, extrusion CExtr
annealing method), annealing method
method), freeze-thaw method, W10/W emulsion H7 method, and 8table Plurilamellar resi
cle method (5PLV method) [Biochemistry (Bio
chemistry), 2nd! Volume, 2Il1 page, lii
s year], etc., all the usual methods are mentioned.

For methods of preparing these liposomes, see Papahad
A review by Jopouls et al. [Ann.

Rev-Biophys, Bioeng, Volume 2, 4
17 pages, 1 year], Nojima, excerpt, bottom set by Bengami et al. [liposome, pp, 2/-30, Nankodo (lrit)
).

Compounds that can be encapsulated in the liposomes according to the first method of the present invention include various drugs, antibiotic dyes, fluorescent substances, and the like.

A substrate having an amino group or a thiol group, or a substrate solution, is added to the surface-modifiable 7Z Q bosome dispersion prepared as described above, and the substrate is immobilized on the liposome by leaving it standing or shaking. Can be manufactured.

The substrate to be immobilized according to the first method of the present invention may have an amino group or a thiol group, preferably a substrate containing an amino group.

Examples of these substrates include oxytocin, vanpressin, thyroid-stimulating hormone secreting hormone (TRH), and other physiologically active substances such as butide and hormones such as butide (containing amino acids), such as IgQ.

Antigens such as immunoglobulins such as IgE and IgM, carcinoembryonic antigen (CEA), pancreatic carcinoembryonic antigen (POA), α
-7 ethoprotein mo7 clonal antibody, immunoglobulin monoclonal antibody, proteins such as bovine serum albumin (B8A), and sugars (including poly-St) such as glucosamine and galactosamine.

Purification of surface-modified or surface-modified liposomes in method 10 of the present invention can be carried out by conventional methods. Examples include gel filtration methods using Sephadex and Sepharose columns, centrifugation methods, and dialysis methods.

The second method is to use a substrate containing an amine group or a thiol group as described in the first method of the present invention and a long-chain active amide represented by the general formula CI) in an organic solvent, water, or a buffer solution. , are mixed to form a substrate derivative having a long chain group. After this is purified, it is mixed with liposome constituent materials to prepare liposomes.

In the second method of the present invention, as the liposome constituent material, in addition to those shown in the first method of the present invention, phos-7 atidylethanolamine, phos-7 atidyl serine, etc. may be used. In the second method of the present invention, the ratio of the liposome constituent material to the substrate derivative containing the long chain group can be freely selected within the range that allows liposome composition, but preferably! O- or less, more preferably 3! Less than 1 is better.

The liposome preparation method according to the second method of the present invention, the compound that can be encapsulated in the liposome, the substrate to be immobilized in the liposome, and the purification of the liposome are the same as the first method of the present invention.

Furthermore, in the second method of the present invention, a substrate derivative having a long chain group may be used without purification depending on the liposome preparation method. Also, water or buffer containing the substrate derivative? +) It may be used as a bonme dispersion.

A hydrophobic group is shown in R1 of the long-chain active amide represented by the general formula CI) used in the present invention. R1 may be single-stranded or double-stranded. - It has a heavy chain and may be linear or branched, and may be saturated or unsaturated.

It may further have a substituent. Rtt! It may have cholesterol as a hydrophobic group.

For example, R5-0-C
H2 (R4 and R5 represent an alkyl group having up to 1 carbon atoms, approximately 4, R5 may be linear or branched, saturated or unsaturated, and may have a substituent group.n
Regarding the chiral carbon exhibiting u/~3, it may be an optically active form or a racemic form.

R1 having a single chain is preferably a straight-chain alkyl group having 1 or more carbon atoms, and more preferably a straight-chain alkyl group having 7 or more carbon atoms.

For R having a double chain, both R4 and R5 are preferably linear alkyl groups having 1 or more carbon atoms, more preferably 12 carbon atoms.
The above straight chain alkyl groups are preferred.

R2,83 of the long chain active amide represented by general formula [I]
represents an organic group (including Hl-). R2 and R3 may be connected to each other to form a ring (saturated hydrocarbon ring, saturated heterocycle, aromatic ring (including heteroffi), etc.), and may also form a double bond.

R2 and R3 are, for example, H, -CH3, -CH2
CH3, -QH, -COOH, -COOCH3, -CH
2-OH, -CH2-COOH.

Φ Φ -CH2-COOCH3, -NH3, -N(CH3)3
,Φ is also H or 10H1-COOHl-N(CH3)3
, -SO4 groups are preferred.

X and Y represent O or 8. Preferably X=Y=8.

Regarding the synthesis of long-chain active amides, see JP-A-14I-7-27! Issue: Tetrahedron Letters
Dron Letters), Volume 1, t
4! / page, year to year; Synthesis
s), /ritko, 933 pages; Maca Molecules (M
acromolecules), Volume 127, page 1,
It can be synthesized using the method described in Litr et al.

Preferred examples of the long-chain activated amide represented by the general formula [I] of the present invention are shown below. However, the present invention is not limited to these.

Synthesis Examples and Examples of the present invention are shown below, but the present invention is not limited thereto.

[Synthesis example]

Synthesis example 1. <Synthesis of long-chain active amide I-/> (general formula (
1], RIC17H35, R+2=R3=)(%
x=y=s) In stearin fl, dissolve dicyclohexylcari diimide (DCC) in '#t dichloromethane joOd.
, ogMJ was stirred. After 30 minutes, 2 = mercaptoco-thiazolinco, coy, dimethylaminase, su, gin (DMAP) 0. Co, pt- added. -Leave it day and night,
After filtration, water was added to the p-liquid, and after drying, dichloromethane was distilled off under reduced pressure. The residue was purified by silica gel chromatography (#! syneresis hexane/ethyl acetate 472) and recrystallized with hexane to form a long-chain active amide t-i<general formula (1) K-OiteR1=Cl7H3
5, R2'''R3=H%

Synthesis example 2. <Synthesis of long-chain active amide l-j> (general formula (
In I) hx=C13H27, R2=R3=H%X
=Y=O) Myristic acid≠, t9, dichloromethane! o.

1. Cooxazolidone/-7j9. DCC≠.

Synthesis Example 1 using 01, 2 g of DMAPO. Similarly, in the long chain active amide I-j (general formula C1),
=C13H27, R2=B 3=H, X=Y=0),
I got a rig.

Synthesis example 3. <Synthesis of long-chain active amide I-t> CH2 R2--C-0-CH3 (8 bodies), R3=H1X=Y-S) Carbobenzoxy-! -Glutamic acid-0j9, dissolved in 300 nl of total tetrahydrofuran, DCC4C.

-I got stronger. Next, cetylamine≠, rp, triethylamine <TEA), /gt was added and left overnight. After evaporation of the solvent, the residue was dissolved in chloroform, the organic layer was washed with water, dried with nitrate, and chloroform was distilled off under reduced pressure. It was purified by silica gel chromatography (fg syneresis hexane/ethyl acetate=r7a), and dialkylamide compound 4 was obtained.
', J#t-obtained.

This was dissolved in total ethyl acetate and j% palladium carbon/,
09t- was used for hydrolysis. (5 hours) The reaction solution was filtered through Celite', and the ethyl acetate in the filtrate was distilled off under reduced pressure to obtain the amine compound [11)J.

Next, glutaric anhydride/, 0111kTHFII
C dissolved. In addition to this, amine body [■] 3.4L9, TE
Ao, tg chloroform solution (100 ydf) was added dropwise. After 2 hours, the solvent was distilled off under reduced pressure, and chloroform was added.

The organic layer was washed with diluted hydrochloric acid (aqueous layer pH=j-l), dried over sulfur and chloroform was completely distilled off under reduced pressure. The residue was dissolved in dichloromethane = 00xl, DCC/Jg was added, and the mixture was stirred for 30 minutes.

≠(R)-Methoxycarbonyl-comel-butot-thiazoliy/, Oji, DMAPO, /9f In addition,
- It was installed in the evening. The reaction solution was filtered, and the filtrate was washed with water and dried over a pycnometer to remove dichloromethane under reduced pressure. The entire residue was purified by silica gel chromatography (eluent: hexane/ethyl acetate = 4/4') to give long-chain active amide I (I M 8-form C) 12 R2=-COCHa CR-form in (general formula CI), X=Y=S)2.3g
I got it.

〔Example〕

Example i, <Method for producing surface-modifiable liposomes> 1)
PPC/j ■, long chain active amide l-/ (general formula CI), R1=C10H3! i, R2=R3=H%X
=Y =8) 2, JMfk 1m1(D/a
After dissolving in a*lum, the solvent was distilled off using a rotary evaporator to form a thin film. After drying this under reduced pressure, R20 and jWLl were added, and the mixture was left to stand for 1 minute in a water bath at 0C. After shaking for 1 minute alternately 3 times using portex mixing, ultrasonic irradiation was performed using a probe-type innicator. Ta. (CojW, 3 minutes) Next, gel filtration was performed using a Sepharose Hiro B column to obtain surface-modifiable liposome gold. (Tc4t10C1 average particle size jj0 nm) Example 2. <Encapsulation test of surface-modifiable fi IJ bomb> Same as Example 1, dispersion liquid gold 200mM! ,(4
)-carpoquine fluorescein (j, (A)-C
F) pHIr,! A liposome was prepared using a borate buffer to obtain a molecule containing j,(4)-CF.

(Average particle size j 00 nm) Example 1 <Production method of surface-modifiable liposomes and their inclusion test> DPPCJOmg, long-chain active amide ■-3 (general formula (I
), R1=C13H27, R2=about 3=)1. X
=Y=0)2Wk,/! Ill (r) Rio
cr form and obtained surface-modifiable liposomes (Tc4c/'C1 average particle size λOnm) t" in the same manner as in Example 10. Furthermore, 200 mM j,
Liposomes were similarly prepared using (≦)-CF pH 7, ≠ phosphate buffer to obtain (4) -CF-containing membranes (average particle size Co/On m)'.

Example 4. <Production method of surface-modifiable liposomes and their inclusion test> Egg PCJoag, long chain activity 7mi)-(-rH2? R2=-C-OCH3 (R form), X=Y=8)λ■5i
Surface-modifiable liposomes (average particle size/20
nm)'. In the same manner as in Example 3, t,
(4) - Obtained CF inclusion name.

Example I <Surface-modifiable liposome and its encapsulation test> DPPCJOda, long-chain active amide ether (general formula CI)
In Example 1, R2=H3=H,
Ir surface-modifiable liposomes (average particle size/!0nrn) were obtained in the same manner as in the method described above.

In the same manner as in Example 3, a j,(j)-CF-encapsulating liposome was obtained.

Example 6. <Production of benzylamine-fixed surface-decorated bonme> Example 1. Surface-modifiable liposome phosphate buffer (p
H7,j) Dispersion 0.4tm (long chain active amide I-/
(In the general formula CI'J, R1=Cl7H35,'&2
=3s=H,X=Y=iS), concentration $X10-'M)
IC reference X10-2M benzylamine phosphate buffer (pH
7,j)a,/ml was added and allowed to stand at 2!0C for 1 hour. As a result, long-chain active amides in the ribbon membrane (R1=ctyHas, R2=R3=H
,X=Y=8)! Obtaining a benzylamine-immobilized surface-modified liposome in which benzylamine was immobilized on Os.

Example 7. <Production of peptide-immobilized surface-modified liposomes> Example 6. Surface-modifiable liposome phosphate buffered N (pH 7-j) dispersion 0.4!1 (long-chain active amide I-/(R1=C17 in general formula (1)) prepared in the same manner as W4.
) (as・32 = R3=H, X=Y=87 density 4AX
/-0"-'M)lC4A×IO-2Mk-)'fdo(
Gly-8er-β-Ala) phosphate buffer (pH 7-
1) 0.1 Wlt was added and left standing under a cot for 72 hours.

As a result, long-chain active amide I-/, (
In general formula (I), 几1=C17H35, R2=R
3=H1X-Y-81) JtTo versus -C peptide (
A peptide-immobilized surface-modified liposome on which Gly-8er-β-Ala) was immobilized was obtained.

Example 8. <Production of protein-immobilized surface-modified liposomes> Example 1. Similarly, DPPC/j■, long chain active amide ■-co (R1=cisHa in general formula (1)
1%R2=Ra=H,X=Y=8)/.

■IJ that can be surface modified using PB8 solution in the dispersion
To obtain fosomes, a microbore filter of fO,-μm was used to obtain an extonosome and a sizing blur in the zooc. The liposomes were sedimented using an ultracentrifuge. (10,000 revolutions, 1 minute) A 31 m phosphate buffer solution was added to this liposome sediment. Of these, 1 m contains human-IgG/■/1 PBS bath solution 1
The mixture was added once and left to stand in Coj'C for 72 hours. This was purified using a PB8 solution using an ultracentrifuge to obtain a protein-immobilized surface-modified liposome on which human IgG was immobilized.

Example 9. <Production of peptide-immobilized surface-modified liposomes> Example 4. Peptide ( G
ly-8er-β-Ala) aqueous solution 'i/- was added and allowed to stand at COJ'C for 72 hours. A liposome fraction was collected by gel filtration to obtain a liposome on which a peptide (Gly-set-β-Ala) was immobilized.

Example 10. <Production of sugar-fixing modified liposomes> Method of Nagachi et al. [Tetrahedron Letters
n Lett, ), Volume 121, 14/L/page, L1
0 years], N-tetradecanoylglucosamine (
III)! Iri.

13H27 N-tetradecanoylglucosamine l■, DPPClj
(2) After dissolving in methanol/chloroform=l/l solution j1, the solvent was distilled off using a rotary evaporator to form a thin film. After drying under reduced pressure, add 31% of PBS solution,
After standing in a to 0c water bath for 1 minute, vortexing and shaking for 1 minute alternately three times, ultrasonic irradiation was performed using a probe-type innicator. (21W, 10 minutes) This was purified by gel filtration using Sephas A's B to obtain a modified liposome with immobilized sugar (glucosamine).

Example 11. <Observation of hydrolysis and amide formation behavior of surface-modifiable liposomes> Example 1. A long-chain active amide I-/(in the general formula [I], 1 = C17H35, R2 = R3
= H,
/, 0) 0. Id gold plus long chain active amide l-/
Decrease in yHPLc (Cs column Shiseido back, CH3CN#
There were syneresis marks due to syneresis.

Similarly μ, 0x10 M hensylamine phosphate buffer (pH 7,7, I=/, 0) 0. ld was added to check for amide formation. The results are shown in Figure 1.

This result shows that long-chain active amide I-/(in general formula (I), R1=Cl7H35, a2=R3=H, X=Y=8
)t! It has been found that in the IJ bombe system, hydrolysis hardly progresses at pH/I, but amide is formed with amines even at pH 7°j.

Comparative example 1. <Measurement of hydrolysis rate and amide formation rate of benzoic acid activated amide or ester> Benzoic acid activated amide or ester shown in Table 1, J,
JJXlo-3M dioxane liquid 1. ! Mix 1 and 0.1M phosphate buffer (pH 7, ≠) J, JllLHPLC
(ODS force 7m, CHaCN-H2o(O, col IA
The hydrolysis rate was measured by tracking the decrease in benzoic acid active amide or ester using cOH-EtsN) eluent). Similarly /3JX10-2M,
Glycine methyl ester hydrochloride phosphate buffer (pH 7-
3) J,! - was used to measure the amide formation rate. The results are shown in Table/.

These results show that long-chain active amides with thiazolidine-corchion as a leaving group are more stable in water than N-hydroxysuccinimide esters, which are conventionally used for immobilization and amide formation, and are also selectively reactive with amino groups. (Effects of the Invention) As described above, by using the present invention, surface-modified liposomes can be produced by immobilizing various substrates on the surface of the ribbon beam under very mild conditions and with very simple operations. can be manufactured. Furthermore, the long-chain active amide used in the present invention has better stability in water and higher operability than h-hydroxysuccinimide ester (for example, 5PDP method), which is often used to immobilize substrates on liposomes.

[Brief explanation of drawings]

Figure 1 shows long-chain active amide I embedded in the ribbon membrane membrane.
−/ (In general formula (1), R1=C17H35,
R2=R3=H, X=Y=8)! nC/Co=/n
Concentration/initial concentration is plotted against time.

Claims (3)

[Claims] (1) A surface-modifiable liposome characterized by being composed of a compound represented by the general formula [I] below and a liposome-formable lipid. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] (R_1 represents a hydrophobic group. R_2 and R_3 are organic groups (
(including H). R_2 and R_3 may be connected to each other to form a ring or a double bond. X and Y represent an oxygen atom or a sulfur atom. (2) A method for producing a surface-modified liposome, which comprises mixing the surface-modifiable liposome according to claim (1) with a substrate having an amino group and/or a thiol group. (3) Mixing a compound obtained by binding at least one compound represented by the general formula [I] according to claim (1) with a substrate having an amino group and/or a thiol group and a lipid capable of forming liposomes. A method for producing surface-modified liposomes, characterized by: