CN103881084A

CN103881084A - Phospholipid derivatives for branching polyethylene glycol, and lipid membrane structural body composed of same

Info

Publication number: CN103881084A
Application number: CN201410096439.9A
Authority: CN
Inventors: 翁文桂; 刘超; 廖金城; 闫策; 林毅
Original assignee: XIAMEN SINOPEG BIOTECH Inc
Current assignee: XIAMEN SINOPEG BIOTECH Inc
Priority date: 2014-03-14
Filing date: 2014-03-14
Publication date: 2014-06-25
Anticipated expiration: 2034-03-14
Also published as: CN105622925B; CN105622925A; CN103881084B

Abstract

The invention discloses phospholipid derivatives for branching polyethylene glycol, and a lipid membrane structural body composed of the same. The general formula of the phospholipid derivatives is as shown in the specification, wherein X1 and X2 are hydroxyl with 1-20 carbon atoms respectively and independently; n1 and n2 are integers ranging from 1 to 1000 respectively and independently, n3 is an integer ranging from 0 to 1000, and n1+n2+n3 is not less than 2 and not greater than 2000; Y is a branching centre with 1-20 carbon atoms, and connected with L1, L2 and L3 by a covalent bond; L1, L2 and L3 are linking groups for Y and polyethylene glycol units; q is 0 or 1; when n3=0, q is 0 and Y is not the polyamino acid residue of 2-10 amino acid residues; L4 is a linking group; M is hydrogen atom or cation; R is the residue of hydrophobic lipids. The lipid membrane structural body composed of the phospholipid derivatives for branching polyethylene glycol is used for wrapping a medicine and capable of effectively prolonging the cycle time of the medicine in a body.

Description

A kind of phospholipid derivative of branched polyethylene glycol and the lipid membrane structure body of composition thereof

Technical field

The present invention relates to Polymer Synthesizing field, relate in particular to a kind of phospholipid derivative of branched polyethylene glycol and the lipid membrane structure body of composition thereof.

Background technology

Liposome is a kind of vesicle of sealing a part of water that lipid (common is phosphatide, cholesterol) is dispersed in water formation, profile be spherical, class is spherical, the shape such as oval, diameter is about tens nanometers to tens micron.Liposome is a kind of new drug carrier, and its stability in blood is the key of performance pharmaceutical carrier effect.In traditional Liposomal formulation, in the time of intravenously administrable, have anelasticity in blood poor, be easy to the problem of being caught by the reticuloendothelial system such as liver, spleen (being designated hereinafter simply as " RES "), utilizing liposome as drug delivery system, useful for drug delivery, to the organ beyond " RES " system or while reagent being trapped in for a long time control drug release in blood, is existed to difficulty.In addition, in blood, there is multiple destructive factor: high-density lipoprotein (HDL) (HDL) is to destroy the main component of liposome, and the exchange of apoA-1 and phosphatide easily occurs for HDL and liposome, and liposome membrane forms hole; Cause drug leakage and water, electrolytically enter in a large number, finally permeate cracking liposome; Serum albumin and liposome phospholipids incorporate form mixture, reduce its stability; Phospholipid hydrolase hydrolyzable phosphatide in blood.These combined factors make the only tens minutes transformation period of traditional liposomal.

In order to extend the transformation period of liposome, avoid catching of RES, mainly utilize at present glycolipid, sugar-protein or hydrophilic polymer modified liposome surface, make liposome there is long cycle characteristics.At present, adopt polyoxyethylene glycol (PEG) coupling and be assembled on liposome membrane, can extend the blood circulation time of liposome.Meanwhile, the prolongation effect of the larger blood circulation time to liposome of the molecular weight of polyoxyethylene glycol is better.Affect pegylated liposomal extend blood circulation time because have: 1. sterically hindered: polyoxyethylene glycol surface of liposome be part extend conformation, this sterically hindered layer is just as one " brush ", close macromole or lipid-protein complex are pushed away to liposome, thereby weaken the effect of various compositions in blood, the particularly regulating effect of plasma proteins and RES picked-up effect subsequently, exchange, the hydrolysis of Phospholipid hydrolase etc. of lipoprotein simultaneously is all subject to effective inhibition.2. improve film surface hydrophilicity: polyoxyethylene glycol has very long polar group, can improve the wetting ability of surface of liposome, thereby improve the energy barrier of mononuclear phygocyte system (MPS) to its absorption destruction, effectively organize the regulating effect of surface of liposome and seralbumin, and reduced the affinity interaction of liposome to MPS.It is generally acknowledged, two factors of wetting ability sterically hindered and raising film surface exist simultaneously, and both actings in conjunction make PEGization liposome become a kind of long-acting liposome.

The pH value fact lower than normal surrounding tissue of utilizing tumor tissues, can design pH susceptibility liposome.Its principle is: when low pH, can cause the protonated of fatty ester carboxyl and cause the formation of hexagonal crystal phase (non-phase layer joint), cause the unstable of liposome membrane, produce and assemble or merge, the material of sealing is imported to endochylema and active target pathological tissues, the target sites such as tumor tissues discharge the bio-related substance being encapsulated in liposome efficiently, avoid RES to remove and lysosomal degraded simultaneously, increase the intake of tissue to medicine.

Summary of the invention

Goal of the invention of the present invention, is for more effective prolong drug cycling time in vivo, and a kind of lipid derivate of branched polyethylene glycol and the lipid membrane structure body of composition thereof are provided.

Above-mentioned purpose of the present invention is achieved by following technical solution:

A phospholipid derivative for branched polyethylene glycol, its general formula as the formula (1):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, n ₃be 0～1000 integer, and 2≤n ₁+ n ₂+ n ₃≤ 2000; Y is branching center, has 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₃be connected; L ₁, L ₂, L ₃for the linking group of branching center Y and polyoxyethylene glycol unit; Q is 0 or 1; Work as n ₃=0 o'clock, q be 0 and Y be not the polyamino acid residue of 2～10 amino-acid residues; L ₄for linking group, it is the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction; M is hydrogen atom or positively charged ion; R is the residue of hydrophobicity lipid.

Said n ₁, n ₂, n ₃preferably meet 1≤n ₁≤ 200,1≤n ₂≤ 200,0≤n ₃≤ 200, and 2≤n ₁+ n ₂+ n ₃≤ 200.

Work as n ₃=0, q be 0 and Y be not the polyamino acid residue of 2～10 amino-acid residues, the phospholipid derivative general formula of described branched polyethylene glycol is as the formula (2):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, and 2≤n ₁+ n ₂≤ 2000; Y is branching center, has 1 to 20 atom, adopts covalent linkage and L ₁, L ₂be connected; L ₁, L ₂for the linking group of branching center Y and polyoxyethylene glycol unit; L ₄for linking group, it is the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction; M is hydrogen atom or positively charged ion; R is the residue of the hydrophobicity lipid that contains reactive group.

Work as n ₃be 1～1000, the phospholipid derivative general formula of described branched polyethylene glycol is as the formula (3):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, n ₃be 1～1000 integer, and 3≤n ₁+ n ₂+ n ₃≤ 2000; Y is branching center, has 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₃be connected, and Y is not the polyamino acid residue of 2～10 amino-acid residues; L ₁, L ₂, L ₃for the linking group of branching center Y and polyoxyethylene glycol unit; Q is 0 or 1; L ₄for linking group, it is the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction; M is hydrogen atom or positively charged ion; R is the residue of hydrophobicity lipid.

Above-mentioned Y is carbon atom branching center, and its representation is as follows:

wherein, R ₁for hydrogen atom, there is the alkyl of 1 to 20 carbon or contain the alkyl with 1 to 20 carbon containing heteroatom group.Described R1 is preferably hydrogen atom or is the alkyl with 1 to 20 carbon, or is the alkyl with 1 to 20 carbon containing ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups.

Above-mentioned Y is nitrogen-atoms branching center, and its representation is as follows:

Above-mentioned R is the residue of hydrophobicity lipid.

This hydrophobicity lipid is not particularly limited, and can be and contains central a kind of or any 2 kinds and the compound lipid of more than two kinds such as aliphatic hydrocarbon, glyceride, sphingolipid, steroid.

When the residue that R is aliphatic hydrocarbon, representation is as follows:

r7 can be saturated or unsaturated, can be the structures such as straight chain shape, a chain, ring-type, pectination, hyperbranched shape, dendroid.Its carbonatoms is 4～50.Residue for this aliphatic hydrocarbon is not particularly limited, can be from butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, isostearoyl base, octadecylene base, 18 carbon dialkylenes, nonadecyl, eicosyl, docosyl, docosane thiazolinyl, tetracosyl, ceryl, octacosyl, triacontyl, dotriacontyl etc.

For the residue of aliphatic hydrocarbon, corresponding aliphatic hydrocarbon is not particularly limited, and can be lipid acid, aliphatic amide, fatty alcohol and derivative thereof, preferred fat alcohol and lipid acid, further preferred fat alcohol.For example, can be selected from butanols, the trimethyl carbinol, amylalcohol, enanthol, 2-Ethylhexyl Alcohol, octanol, decyl alcohol, dodecanol, tetradecanol, cetyl alcohol, heptadecanol, Stearyl alcohol, octadecenyl alcohol, 18 dienols, 18 enols, eicosanol, V-1326, docosene alcohol, Tetracosyl alcohol, n-Hexacosanol, policosanol, triacontanol price quote, n-Dotriacontanol etc. as fatty alcohol, preferably corresponding aliphat single-alcohol.For example, can be selected from butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid etc., preferably corresponding aliphatic monocarboxylic acid as lipid acid.For example, can be selected from butylamine, TERTIARY BUTYL AMINE, amylamine, heptyl amice, 2 ethyl hexylamine, octylame, decyl amine, n-Laurylamine, tetradecylamine, cetylamine, heptadecylamine (HDA), octadecylamine, octadecenyl amine, 18 enamines, 18 enamines, eicosane amine, docosane amine, docosene amine, tetracosane amine, hexacosane amine, octacosane amine, triacontane amine, laccerane amine etc. as aliphatic amide, preferably corresponding aliphatics monoamine.

When the residue that R is glyceride, glyceride can be DG ester.Its representation is as follows:

phosphatide based on this glycerine lipid residue, as glyceryl phosphatide, can be phosphatidylethanolamine, phosphatidic acid, phosphatidyl glycerol, phosphatidyl amino acid etc.In glyceryl phosphatide, the carbon number of multiple acyl groups is independent separately, and carbonatoms is 4～50.This acyl group can be identical or different, can be saturated or unsaturated, can be straight chain shape or a chain.In addition, the kind of acyl group is also not particularly limited, conventionally can preferably derives from the acyl group of lipid acid.Concrete example is as the acyl group of the fatty acid sources such as, butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid.

When the residue that R is glyceride, glyceride can be also monoacylglycerol ester, and its representation is as follows: phosphatide based on this glycerine lipid residue is as lysophospholipid, and the carbon number of the acyl group wherein existing is 4～50.This acyl group can be saturated or unsaturated, can be straight chain shape or a chain.In addition, the kind of acyl group is also not particularly limited, conventionally can preferably uses the acyl group that derives from lipid acid.Concrete example is as the acyl group of the fatty acid sources such as, butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid.

R also can carry out self-structure

or derivative based on this structure, wherein R2, the R3 alkyl for containing 4 to 50 carbon atoms independently of one another.R preferably contains sphingosine skeleton.When R contains sphingosine skeleton, its structure can be expressed as r can be the derivative from above-mentioned sphingosine skeleton.When R is that while having the skeleton of sphingosine or the derivative based on this skeleton, R is sphingolipid residue.Sphingophospholipid based on this sphingolipid residue can be sphingomyelin and derivative thereof, such as ceramide phosphoethanolamine, phospho-glycerol ceramide, phospho-glycerol phosphoric acid ester ceramide etc.For the lipid acid of being combined with sphingosine, can be butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid etc.

When the residue that R is steroid, this steroid can stay alcohol, bilichol, sexual hormoue etc. and derivative thereof for cholesterol, Sitosterol, vitamins D, beta-cholestanol, ergosterol, wool.

Above-mentioned R ₂, R ₃, R ₄, R ₅, R ₆, R ₇be preferably butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, (Z)-9-tetradecene base, (Z)-8-17 thiazolinyls, (Z)-12-bis-hendecene bases, and in same a part, can be the same or different.

The phosphatide forming based on hydrophobicity lipid residue R can be natural phospholipid, can be also artificial synthetic phospholipid.For example, natural phospholipid can be kephalin, Yelkin TTS, sphingophospholipid, lysophospholipid etc., and synthetic phosphatide can be phosphatidylethanolamine, as DSPE, DPPE etc.

Described L ₄be not particularly limited, preferably from

Wherein Z is not particularly limited, preferred alkylidene group or the alkylidene group that contains the groups such as ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups;

G is 0 or 1;

F is 2 to 10 integer.

Described X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom, be preferably methyl, ethyl, propyl group, propenyl, proyl, sec.-propyl, butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, benzyl or butyl phenyl, and in same a part, X ₁, X ₂can be the same or different.

Described L ₁, L ₂, L ₃be the bivalent hydrocarbon radical with 1 to 20 carbon atom of the groups such as the ether, thioether group, amide group, two key, Three key, secondary amine or the tertiary amine groups that contain stable existence under illumination, enzyme, acidity or alkaline condition independently of one another.

Described M is hydrogen atom or positively charged ion, preferably hydrogen atom, sodium ion or NH ₄ ⁺.

In the phospholipid derivative of described branched polyethylene glycol, can contain the residue of phosphatidylethanolamine, the residue forming after phosphatidylethanolamine and other reaction-ity group reaction.

Described phosphatidylethanolamine is not particularly limited; preferably DSPE, two grease acylphosphatidyl ethanolamines, two myristoyl phosphatidylethanolamines, DPPE, two lauroyl phosphatidylethanolamines, two flax acyl phosphatidylethanolamines, two mustard acyl phosphatidylethanolamines or 1-palmityl-2-oleoyl phosphatidylethanolamine.

A kind of lipid membrane structure body of the phospholipid derivative that contains above-mentioned branched polyethylene glycol." lipid membrane structure body " in the present invention refers to by the hydrophilic radical of amphiphilic lipid and/or its derivative and contacts the particle forming with water.Described lipid membrane structure body, can be hollow or solid sealing form, can be also nonocclusive film form.In the time being hollow sealing form, be liposome (liposome), on some documents also referred to as lipid vesicle (lipid vesicle).

In described lipid membrane structure body, the shared mass percent of phospholipid derivative of branched polyethylene glycol is 0.1%～30%, preferably 1%～30%, more preferably 3%～25%, more preferably 3%～20%, and more preferably 3%-15%.

Described lipid membrane structure body, except the phospholipid derivative that branched polyethylene glycol is modified, also contains and does not use polyethyleneglycol modified lipid composition.The lipid composition of described unmodified can be the lipids such as the acyl compounds containing 4-50 carbon atom, fatty acyl glyceryl ester, sphingolipid, glyceryl phosphatide, glycolipid, glyceroglycolipid, steroid.Can be neutral grease matter or synthetic lipid.Describedly not using polyethyleneglycol modified lipid composition, can be the one-component in above-mentioned lipid composition, can be also the mixing lipid of two or more lipid composition.

In described lipid membrane structure body, do not use polyethyleneglycol modified lipid composition, preferably phosphatidylethanolamine.The phosphatidylethanolamine of the preferred fatty family of described phosphatidylethanolamine acyl group.Described aliphatic acyl radical is preferably from the acyl compounds of 8-24 carbon atom, further the preferred acyl compounds of 8-24 carbon atom.For example, described acyl group can be butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, the acyl group of the fatty acid sources such as lacceroic acid, preferably octanoic acid, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, the acyl group in the source such as lignoceric acid, further preferred lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, the acyl group in the source such as lignoceric acid.

When in described lipid membrane structure body not with containing phosphatidylethanolamine in polyethyleneglycol modified lipid composition; moiety using this as film; described phosphatidylethanolamine can be given an example as DSPE, two grease acylphosphatidyl ethanolamines, two myristoyl phosphatidylethanolamines, DPPE, two lauroyl phosphatidylethanolamines, two flax acyl phosphatidylethanolamines, two mustard acyl phosphatidylethanolamines or 1-palmityl-2-oleoyl phosphatidylethanolamine.

The form of described lipid membrane structure body is not particularly limited, and can be the forms such as liposome (or lipid vesicle), micella, mixing micelle, multiple emulsion, laminate structure thing, and preferably its form is liposome.

Described lipid membrane structure body, can further encapsulate bio-related substance.

Described lipid membrane structure body, packaged bio-related substance discharges in the time of low pH, preferably pH≤6.8, more preferably pH≤6.5, further preferred pH≤6.0.

Described its form of lipid membrane structure body is preferably liposome.

Described liposome, can further encapsulate bio-related substance.

Described liposome, packaged bio-related substance discharges in the time of low pH, preferably pH≤6.8, more preferably pH≤6.5, further preferred pH≤6.0.

The bio-related substance encapsulating in described lipid membrane structure body or described liposome is not particularly limited, and includes but not limited to polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein, steroid.

The invention also discloses the phospholipid derivative that one has the branched polyethylene glycol shown in general formula (2):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, and 2≤n ₁+ n ₂≤ 2000; Y is carbon atom branching center or the N atom branching center with 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₄be connected, and Y is not the polyamino acid residue of 2～10 amino-acid residues; L ₁, L ₂independently of one another for connecting branching center Y and polyoxyethylene glycol unit or L ₄the bivalent hydrocarbon radical with 1 to 20 carbon atom of the group such as the ether that contains stable existence under illumination, enzyme, acidity or alkaline condition, thioether group, amide group, two key, Three key, secondary amine or tertiary amine groups; L ₄for linking group, it is the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction; L ₄be not particularly limited, can be selected from one of following group:

Wherein, Z is alkylidene group or the alkylidene group that contains the groups such as ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups;

G is 0 or 1;

F is 2 to 10 integer;

M is hydrogen atom or positively charged ion;

R is hydrophobicity lipid residue, is not particularly limited, and can be aliphatic hydrocarbon residue, DG lipid residue, monoacylglycerol lipid residue, the residue of a kind of or its compound lipid in the lipid residue that contains sphingosinols skeleton or steroid residue.

Also disclose the liposome containing the phospholipid derivative of the described branched polyethylene glycol of general formula (2), the phospholipid derivative of described branched polyethylene glycol accounts for 1%～30% of this liposome total mass, is preferably 3%-20%, more preferably 3%-15%.

Contain in the liposome of phospholipid derivative of the described branched polyethylene glycol of general formula (2), it is packaged with bio-related substance.Described bio-related substance is not particularly limited, include but not limited to polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid etc., can be above-mentioned one or more bio-related substance.

The invention also discloses the phospholipid derivative that one has the branched polyethylene glycol shown in general formula (3):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, n ₃be 1～1000 integer, and 3≤n ₁+ n ₂+ n ₃≤ 2000; Y is carbon atom branching center or the nitrogen-atoms branching center with 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₃be connected; L ₁, L ₂, L ₃independently of one another for connecting the bivalent hydrocarbon radical with 1 to 20 carbon atom of the group such as the ether that contains stable existence under illumination, enzyme, acidity or alkaline condition, thioether group, amide group, two key, Three key, secondary amine or tertiary amine groups of branching center Y and polyoxyethylene glycol unit; L ₄for linking group, be the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction, L ₄be not particularly limited, can be selected from one of following group:

Wherein, Z is alkylidene group or the alkylidene group that contains ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups;

G is 0 or 1;

F is 2 to 10 integer;

M is hydrogen atom or positively charged ion;

Also disclose the liposome containing the phospholipid derivative of the described branched polyethylene glycol of general formula (3), the phospholipid derivative of described branched polyethylene glycol accounts for 1%～30% of this liposome total mass, is preferably 3%-20%, more preferably 3%-15%.

Contain in the liposome of phospholipid derivative of the described branched polyethylene glycol of general formula (3), it is packaged with bio-related substance.Described bio-related substance is not particularly limited, include but not limited to polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid etc., can be above-mentioned one or more bio-related substance.

Compared with prior art, the present invention has following beneficial effect:

(1) the present invention adopts phosphatide to be connected with branched polyethylene glycol, and the lipid derivate of described branched polyethylene glycol still has the characteristic of better formation liposome;

(2) with respect to the linear polyethylene glycol of same molecular amount, owing to thering is special molecular conformation, branched polyoxyethylene glycol can form on the top layer of liposome the protective layer of one deck umbrella shape, increase around sterically hindered, can more effectively stop the attack of other macromolecular complex confrontation medicine in body than linear polyethylene glycol, reduce liposome by RES picked-up, by enzymic hydrolysis, more extended medicine action time in vivo, made the liposome forming there is the longer organism intracellular metabolite transformation period;

(3) reaching in the liposome of identical protection effect, the liposome derivative proportion of the ratio straight chain Pegylation of the lipid derivate of branched polyethylene glycol is little, the stability that is more conducive to the liposome forming, more effectively prevents the leakage of wrapped medicine carrying thing;

(4) bio-related substance encapsulating in lipid membrane structure body of the present invention is in low pH value (preferably pH≤6.8, more preferably pH≤6.5, further preferably pH≤6.0) time discharges, the characteristic of this pH sensitivity, makes lipid membrane structure body optionally to assemble and to discharge at target sites such as tumours, can strengthen the passive targeting of drug delivery, further avoid RES to remove and lysosomal Degradation, increase the intake of tissue to medicine, and then improve result for the treatment of, and reduce toxic side effect.

Embodiment

Wherein, X ₁, X ₂independently of one another for to there is the alkyl of 1 to 20 carbon atom, and can be the same or different at same a part.Wherein including but not limited to methyl, ethyl, propyl group, propenyl, proyl, sec.-propyl, butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, benzyl or butyl phenyl; Be preferably the alkyl with 1 to 10 carbon atom; More preferably there is the alkyl of 1 to 5 carbon atom; Described X ₁, X ₂most preferably be methyl.

Wherein, n ₁, n ₂representing the polymerization degree of two branched chain, is 1～1000 integer independently, wherein, and described n ₁, n ₂be preferably 10～800 integer.Described n ₁, n ₂more preferably 10～200 integer.Described n ₁, n ₂more preferably 10～25 integer.

Wherein, n ₃representing the polymerization degree of main chain, is 0～1000 integer, wherein, and described n ₃be preferably 0 or 10～800 integer.Described n ₃more preferably 0 or 10～200 integer.Wherein, described n ₃more preferably 0 or 10～25 integer.

Wherein, Y is branching center, has 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₃be connected, the branching atom at its center can be carbon atom or nitrogen-atoms.In the time that Y branching center is carbon atom, its representation is as follows:

wherein, R ₁for hydrogen atom, the alkyl with 1 to 20 carbon that there is the alkyl of 1 to 20 carbon or contain heteroatom group.R1 is preferably hydrogen atom or is the alkyl with 1 to 20 carbon, or is the alkyl with 1 to 20 carbon containing ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups.R ₁structure be not particularly limited, can be straight chain, side chain, ring-type or containing ring texture.

Wherein, Y branching center can be also nitrogen-atoms, and its representation is as follows:

Wherein, L ₁, L ₂, L ₃for the linking group of branching center Y and polyoxyethylene glycol unit, be not particularly limited.Described L ₁, L ₂, L ₃can be straight chain or band branched group.Described L ₁, L ₂, L ₃be preferably the alkyl with 1 to 20 carbon atom.

Wherein, described L ₁, L ₂, L ₃be preferably can stable existence group, be preferably the bivalent hydrocarbon radical of the groups such as the ether, thioether group, amide group, two key, Three key, secondary amine or the tertiary amine groups that contain stable existence under illumination, enzyme, acidity or alkaline condition.

Wherein, L ₄for linking group, it is the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction.。L ₄be not particularly limited, preferably from the divalent alkyl of amino, ester group, carbonate group, triazole, isoxzzole, ether, amide group, sub-amide group, imido grpup, secondary amino group, tertiary amine groups, thioester substrate, thioether group, disulfide group, urethane groups, thiocarbonic acid SOH ester group, sulfonate group, sulfoamido, carbamate groups, tyrosine-based, halfcystine base or Histidine base.

Wherein, L ₄be preferably

Wherein Z is alkylidene group or the alkylidene group that contains the groups such as ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups;

G is 0 or 1;

F is 2 to 10 integer.

Wherein, M is hydrogen atom or positively charged ion;

Wherein, the preferred sodium ion of positively charged ion, ammonium radical ion; Most preferably sodium ion.

Wherein, R is the residue of hydrophobicity lipid.

Described hydrophobicity lipid is not particularly limited, and can be central a kind of or its compound lipid such as aliphatic hydrocarbon, glyceride, sphingolipid, glycolipid, glyceroglycolipid, steroid.

When the residue that R is aliphatic hydrocarbon, can be saturated or unsaturated, can be the structures such as straight chain shape, a chain, ring-type, pectination, hyperbranched shape, dendroid.Its carbonatoms is 4～50.Residue for this aliphatic hydrocarbon is not particularly limited, can be from butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, isostearoyl base, octadecylene base, 18 carbon dialkylenes, nonadecyl, eicosyl, docosyl, docosane thiazolinyl, tetracosyl, ceryl, octacosyl, triacontyl, dotriacontyl etc.

For the residue of aliphatic hydrocarbon, corresponding aliphatic hydrocarbon is not particularly limited, for example, can be fatty alcohol, lipid acid, aliphatic amide and derivative thereof, preferred fat alcohol and lipid acid, further preferred fat alcohol.For example, can be selected from butanols, the trimethyl carbinol, amylalcohol, enanthol, 2-Ethylhexyl Alcohol, octanol, decyl alcohol, dodecanol, tetradecanol, cetyl alcohol, heptadecanol, Stearyl alcohol, octadecenyl alcohol, 18 dienols, 18 enols, eicosanol, V-1326, docosene alcohol, Tetracosyl alcohol, n-Hexacosanol, policosanol, triacontanol price quote, n-Dotriacontanol etc. as fatty alcohol, preferably corresponding aliphat single-alcohol.For example, as lipid acid can be selected from butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid,, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid etc., preferably corresponding aliphatic monocarboxylic acid.For example, can be selected from butylamine, TERTIARY BUTYL AMINE, amylamine, heptyl amice, 2 ethyl hexylamine, octylame, decyl amine, n-Laurylamine, tetradecylamine, cetylamine, heptadecylamine (HDA), octadecylamine, octadecenyl amine, 18 enamines, 18 enamines, eicosane amine, docosane amine, docosene amine, tetracosane amine, hexacosane amine, octacosane amine, triacontane amine, laccerane amine etc. as aliphatic amide, preferably corresponding aliphatics monoamine.

For the residue of glyceride, glyceride can be DG fat.Phosphatide based on this glycerine lipid residue as glyceryl phosphatide, for example, can be phosphatidylethanolamine, phosphatidic acid, phosphatidyl glycerol, phosphatidyl amino acid etc.In glyceryl phosphatide, the carbon number of multiple acyl groups is independent separately, and carbonatoms is 4～50.This acyl group can be identical or different, can be saturated or unsaturated, can be straight chain shape or a chain.In addition, the kind of acyl group is also not particularly limited, conventionally can preferably uses the acyl group that derives from lipid acid.Concrete example is as the acyl group of the fatty acid sources such as, butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid.

When the residue that R is glyceride, glyceride can be also monoacylglycerol fat.Phosphatide based on this glycerine lipid residue is as lysophospholipid,, the carbon number of the acyl group wherein existing is 4～50.This acyl group can be saturated or unsaturated, can be straight chain shape or a chain.In addition, the kind of acyl group is also not particularly limited, conventionally can preferably uses the acyl group that derives from lipid acid.Concrete example is as the acyl group of the fatty acid sources such as, butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid.

When the residue that R is sphingolipid, this sphingolipid residue contains sphingosine skeleton.Sphingophospholipid based on this sphingolipid residue can be sphingomyelin and derivative thereof, such as ceramide phosphoethanolamine, phospho-glycerol ceramide, phospho-glycerol phosphoric acid ester ceramide etc.For the lipid acid of being combined with sphingosine, can be butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, lacceroic acid etc.

When the residue that R is steroid, this steroid can stay alcohol, vitamins D, bilichol, sexual hormoue etc. and derivative thereof for cholesterol, Sitosterol, beta-cholestanol, ergosterol, wool.Preferably cholesterol and derivative thereof.

The phosphatide forming based on this hydrophobicity lipid residue can be natural phospholipid, can be also artificial synthetic phospholipid.For example, natural phospholipid can be kephalin, Yelkin TTS, sphingophospholipid, lysophospholipid etc., and synthetic phosphatide can be phosphatidylethanolamine, as DSPE, DPPE etc.

Wherein, work as n ₃be 0, q be 0 and Y be not the polyamino acid residue of 2～10 amino-acid residues, the phospholipid derivative general formula of described branched polyethylene glycol is as the formula (2):

Wherein, X ₁, X ₂, n ₁, n ₂, Y, L ₁, L ₂, L ₄, M, R be same as described above, do not repeat one by one at this.

Wherein, work as n ₃be 1～1000, the phospholipid derivative general formula of described branched polyethylene glycol is as the formula (3):

Wherein, wherein, X ₁, X ₂, n ₁, n ₂, Y, L ₁, L ₂, L ₃, L ₄, M, R be same as described above, do not repeat one by one at this.

Preparation method:

Described single functionalized branched polyethylene glycol (1) can be obtained through a step or polystep reaction by polyethyleneglycol derivative (4) and phosphatide cpd (5).

Wherein, wherein said R ₈, R ₉for functional groups, include but are not limited to following a few class:

Class A:

Class B:

Class C:

Class D:

Class E:

Class F:

Class G:

Class H:

In above-mentioned class A～class H, Z is the covalent linkage linking group between polyoxyethylene glycol and functional groups, is not particularly limited; G is 0 or 1.Wherein, Z can be alkylidene group or the alkylidene group that contains the groups such as ester group, urethane groups, amide group, ether, two key, Three key, carbonate group, secondary amine or tertiary amine groups.Wherein, Z is preferably the alkylidene group of alkylidene group or ether-containing key, amido linkage, secondary amino group.Described alkylidene group is preferably methylene radical, ethylene, trimethylene, propylene, isopropylidene, butylidene, pentylidene and hexylidene.

In above-mentioned class B, Y is the alkyl with 1 to 10 carbon atom that has the alkyl of 1 to 10 carbon atom or comprise fluorine atom.Wherein, described Y is preferably methyl, ethyl, propyl group, sec.-propyl, butyl, the tertiary butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, vinyl, phenyl, benzyl, p-methylphenyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-(trifluoromethoxy) phenyl.Wherein, described Y is preferably methyl, p-methylphenyl, 2,2,2-trifluoroethyl, trifluoromethyl, vinyl.

In above-mentioned class D, described W is halogen atom.Described W is preferably Br or Cl.

In above-mentioned class G, described Q is not particularly limited, as long as contribute to induction, the conjugative effect of unsaturated link(age) electronics.In the time that Q is upper in encircling, can be one or more.Described Q is preferably hydrogen atom, halogen, haloalkane, alkoxyl group, carbonyl compound, nitro-compound.Described Q is preferably hydrogen atom, fluorine atom, trifluoromethyl or methoxyl group.

In above-mentioned class G, described M is the atom of the upper Z of connection of ring, and described M can be carbon atom or nitrogen-atoms.

L ₄for R ₈, R ₉reaction residue.

The preparation method of the phospholipid derivative to branched polyethylene glycol of the present invention describes, the preparation method of the phospholipid derivative of branched polyethylene glycol of the present invention is not particularly limited, and can be prepared by following method as an example: containing the phosphatide cpd of the hydrophobicity lipid residues such as the aliphatic hydrocarbon of reactive group, glyceride, sphingolipid, glycolipid, glyceroglycolipid, steroid and branched polyethylene glycol derivatives reaction containing reactive functional group.

1, work as L ₄in while containing amido linkage (CONH-), can be by synthesizing in the following ways:

1.1 adopt ends to contain phosphatide cpd that amino polyethyleneglycol derivative and end contain carboxylic acid or end contains polyethyleneglycol derivative that amino phosphatide cpd and end contain carboxylic acid and carries out condensation reaction and obtain.

Wherein, be not specially limited condensing agent, but preferred N, N '-dicyclohexyl carbonyl diimine (DCC), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl), 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester (HATU), benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate (HBTU), most preferably is DCC.And the consumption of general condensing agent is 1 to 20 times of carboxylic acid molar equivalent, be preferably 5-10 doubly, this reaction can add suitable catalyzer (as 4-dimethylaminopyridine).

Solvent can be solvent-free or non-protonic solvent, non-protonic solvent comprises toluene, benzene, dimethylbenzene, acetonitrile, ethyl acetate, ether, methyl tertiary butyl ether, tetrahydrofuran (THF), chloroform, methylene dichloride, dimethyl sulfoxide (DMSO), dimethyl formamide or N,N-DIMETHYLACETAMIDE, preferably tetrahydrofuran (THF), methylene dichloride, dimethyl sulfoxide (DMSO), dimethyl formamide.

Alkali comprises and is generally organic bases (as triethylamine, pyridine, 4-dimethylaminopyridine, imidazoles or diisopropyl ethyl amine), preferably triethylamine, pyridine.The consumption of alkali is 1 to 50 times of molar equivalent of carboxylic acid, is preferably 1 to 10 times, more preferably 2 to 3 times.

Temperature of reaction is 0 to 200 ℃, and preferably 0 to 100 ℃, more preferably 25 to 50 ℃, the reaction times is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.The product obtaining can be by addition purifying of the purification process such as extraction, column chromatography, recrystallization, adsorption treatment, precipitation, anti-precipitation, film dialysis or supercritical extraction.

1.2 adopt carboxylic acid derivative that ends contain amino polyethyleneglycol derivative and phosphatide or end to contain amino phosphatide cpd reacts and obtains with polyethylene carboxylic acid derivative.Wherein, carboxylic acid derivative is to react with amido the active intermediate that generates amido linkage, is preferably the succinimide active ester of carboxylic acid halides, carboxylic acid.

General reaction solvent can be solvent-free or non-protonic solvent, non-protonic solvent comprises toluene, benzene, dimethylbenzene, acetonitrile, ethyl acetate, ether, methyl tertiary butyl ether, tetrahydrofuran (THF), chloroform, methylene dichloride, dimethyl sulfoxide (DMSO), dimethyl formamide or N,N-DIMETHYLACETAMIDE, preferably tetrahydrofuran (THF), methylene dichloride, dimethyl sulfoxide (DMSO), dimethyl formamide.

Alkali comprises and is generally organic bases (as triethylamine, pyridine, 4-dimethylaminopyridine, imidazoles or diisopropyl ethyl amine), preferably triethylamine, pyridine.The consumption of alkali is 1 to 50 times of carboxylic acid derivative, is preferably 1 to 10 times, more preferably 2 to 3 times.

Temperature of reaction is 0 to 200 ℃, and preferably 0 to 100 ℃, more preferably 25 to 80 ℃, the reaction times is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.The product obtaining can be by addition purifying of the purification process such as extraction, column chromatography, recrystallization, adsorption treatment, precipitation, anti-precipitation, film dialysis or supercritical extraction.

2, work as L ₄in while containing urethane bond (OCONH-), can be by synthesizing in the following ways: adopt end to contain phosphatide cpd that amino polyethyleneglycol derivative and end contain activated carbon acid esters or end and contain polyethyleneglycol derivative that amino phosphatide cpd and end contain activated carbon acid esters and carry out condensation reaction and obtain.

Wherein active formate can obtain the derivative of urethane bond for reacting with amino, include but not limited to succinimdyl carbonate (SC), p-NP carbonic ether (NPC), 2,4,6-Trichlorophenol carbonic ether, imidazoles carbonic ether, N-hydroxy benzo triazole carbonic ether, preferably succinimdyl carbonate (SC), p-NP carbonic ether (NPC).

3, work as L ₄in while containing ester bond (OCO-), can adopt polyethyleneglycol derivative that phosphatide cpd that phosphatide cpd that polyethyleneglycol derivative that end contains hydroxyl and end contain carboxylic acid or end contain hydroxyl and end contain carboxylic acid to carry out condensation reaction and obtain.

Temperature of reaction is 0 to 200 ℃, and preferably 0 to 100 ℃, more preferably 25 to 80 ℃, the reaction times is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.The product obtaining can be by addition purifying of the purification process such as extraction, column chromatography, recrystallization, adsorption treatment, precipitation, anti-precipitation, film dialysis or supercritical extraction.Work as L ₄in contain secondary amine key (CH ₂nHCH ₂-) time, can adopt that the polyethyleneglycol derivative that phosphatide cpd that phosphatide cpd that polyethyleneglycol derivative that end contains aldehyde radical and end contain amino acid or end contain aldehyde radical and end contain amino acid carries out condensation, reduction reaction obtains.

4, work as L ₄in contain secondary amine key (CH ₂nHCH ₂-) time, can adopt that the polyethyleneglycol derivative that phosphatide cpd that phosphatide cpd that polyethyleneglycol derivative that end contains aldehyde radical and end contain amino acid or end contain aldehyde radical and end contain amino acid carries out condensation, reduction reaction obtains.

Reaction is generally carried out in buffered soln, preferably use acetic acid buffer solution, phosphate buffer solution, Tris acid buffered soln, borate buffer solution etc., in addition for better hydrotropy, in reaction system, further interpolation does not participate in the organic solvent such as acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide, N,N-DIMETHYLACETAMIDE of reaction, the pH value of reaction is 2～8.5, is preferably 3～7.

Temperature of reaction is 0 to 200 ℃, and preferably 0 to 100 ℃, more preferably 25 to 80 ℃, the reaction times is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.In the time not there is not reductive agent, form schiff bases.

Wherein reductive agent is not particularly limited, preferably sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, lithium borohydride, POTASSIUM BOROHYDRIDE etc., and more preferably sodium cyanoborohydride, the consumption of general sodium cyanoborohydride is 1-20 times of aldehyde radical amount of substance, preferably 3-5 is doubly.

The product obtaining can be by addition purifying of the purification process such as extraction, column chromatography, recrystallization, adsorption treatment, precipitation, anti-precipitation, film dialysis or supercritical extraction.

5, work as L ₄in while containing thioether bond (>CHS-), can adopt that the polyethyleneglycol derivative that phosphatide cpd that phosphatide cpd that polyethyleneglycol derivative that end contains sulfydryl and end contain maleimide or end contain sulfydryl and end contain maleimide carries out condensation, reduction reaction obtains.

Temperature of reaction is 0 to 200 ℃, and preferably 0 to 100 ℃, more preferably 25 to 80 ℃, the reaction times is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.

6, work as L ₄in while containing triazole group, can adopt polyethyleneglycol derivative that the phosphatide cpd that the polyethyleneglycol derivative that contains alkynyl contains nitrine with end or the phosphatide cpd that contains alkynyl are contained nitrine with end to react and obtain.

Temperature of reaction is 0 to 200 ℃, preferably 25 to 150 ℃, wherein can adopt illumination, microwave, add the method such as catalyzer, heating to promote the carrying out of reaction.Wherein preferred UV-light, infrared light, the far red light of illumination; The preferred monovalence copper catalyst of catalyzer (I).Reaction times is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.

" lipid membrane structure body " of the present invention refers to by the hydrophilic radical of amphiphilic lipid and/or its derivative and contacts the particle forming with water.

Form for lipid membrane structure of the present invention is not particularly limited.For example, can be hollow or solid sealing form, can be also nonocclusive film form.For example, can be the forms such as liposome (or lipid vesicle), micella, mixing micelle, multiple emulsion, laminate structure thing, preferably its form is liposome.For example, can be for drying regime, can be dispersed in aqueous solvent, also can be for being scattered in after aqueous solvent the further dry or state that freezes.

For dry lipid mixt form, for example, lipid composition first can be dissolved in organic solvent, be then dried by freeze-drying, Rotary Evaporators hypobaric drying method or spray-drying process, described organic solvent can be ether, trichloromethane etc.

For the form being scattered in aqueous solvent, can be vesica or liposome, micella, mixing micelle, multiple emulsion, laminate structure thing etc., preferred liposome, further preferred unilamelar liposome.

For being scattered in the state further freezing after water, can adopt the methods such as freeze-drying, Rotary Evaporators hypobaric drying method or spray-drying process to be dried.For being scattered in the state further freezing after aqueous solvent, can below solvent zero pour, preserve, preferably-20 ℃ and following, more preferably-80 ℃ and following, more preferably in liquid nitrogen, preserve.

For lipid membrane structure body of the present invention, except the phospholipid derivative that branched polyethylene glycol is modified, also contain and do not use polyethyleneglycol modified lipid composition.

Wherein, the mass percent of the phospholipid derivative that branched polyethylene glycol of the present invention is modified is 0.1%～30%, preferably 1%～30%, more preferably 3%～25%, more preferably 3%～20%, and more preferably 3%-15%.

The lipid composition of described unmodified can be the acyl compounds containing 4-50 carbon atom, fatty acyl glyceryl ester, sphingolipid, glyceryl phosphatide, glycolipid, glyceroglycolipid, steroid etc.Can be neutral grease matter or synthetic lipid.Describedly not using polyethyleneglycol modified lipid composition, can be the one-component in above-mentioned lipid composition, can be also the mixing lipid of two or more lipid composition.

For the described acyl compounds containing 4-50 carbon atom, can be saturated or unsaturated.Its structure is not particularly limited, for example, can be the structures such as straight chain shape, a chain, ring-type, pectination, hyperbranched shape, dendroid.

For described fatty acyl glyceryl ester, can be monoglyceride or diacylglycerol (DGDG) or triglyceride, preferably triglyceride, i.e. triglyceride level.

For described sphingolipid, there is sphingosine skeleton, can be for example sphingophospholipid, glycosphingolipid etc.Preferably sphingophospholipid.

For described glyceryl phosphatide, can be DG phosphatide, can be also mono acyl glycero phospholipid.Can be for example kephalin, Yelkin TTS, lysophospholipid, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidyl amino acid etc.Preferably lecithin, phosphatidylethanolamine etc.

Acyl group in the structures such as the above-mentioned acyl compounds containing 4-50 carbon atom, fatty acyl glyceryl ester, sphingolipid, glyceride, glyceryl phosphatide, phosphatidylethanolamine; the preferably acyl compounds of 8-24 carbon atom, the acyl compounds of further preferred 8-24 carbon atom.For example, described acyl group can be butyric acid, tertiary butyric acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, sad, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, myricyl acid, the acyl group of the fatty acid sources such as lacceroic acid, preferably octanoic acid, capric acid, lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, the acyl group in the source such as lignoceric acid, further preferred lauric acid, tetradecanoic acid, palmitinic acid, margaric acid, stearic acid, Unimac 5680, oleic acid, elaidic acid, linolic acid, linolenic acid, eicosanoic acid, arachidonic acid, behenic acid, erucic acid, the acyl group in the source such as lignoceric acid.

For described steroid, for example, can stay alcohol, vitamins D, bilichol, sexual hormoue etc. and derivative thereof for cholesterol, Sitosterol, beta-cholestanol, ergosterol, wool.Preferably cholesterol and derivative thereof, be typically used as membrane stabilizer, such as cholesteryl hemisuccinate etc.Cholesteryl hemisuccinate is a kind of auxiliary material of the pH of having susceptibility, under neutrality and alkaline condition, has the effect of stabilized liposome membrane structure, play slow release effect, and under acidic conditions, as pH≤6.8, more preferably pH≤6.5, further preferred pH≤6.0, promote the release of medicine, demonstrating dashes forward releases effect, can be used to thus increase the selectivity of medicine to target sites such as tumours, intensifier target tropism, improves curative effect.

For described natural lipid, for example, can be kephalin, Yelkin TTS, lysophospholipid, sphingophospholipid, soybean phospholipid acyl thanomin, lecithin acyl thanomin, cholesterol etc.For described synthetic lipid, for example, can be phosphatide, the phosphatidyl amino acid etc. of phosphatidyl glycerol, sphingosine skeleton.Preferably phosphatidyl glycerol, for example, can be phosphatidylethanolamine, as DSPE, DPPE, N-glutaryl--phosphatidylethanolamine, hydrogenation soybean phospholipid acyl thanomin, hydrogenation lecithin acyl thanomin etc.

For the mixing lipid of described two or more lipid composition, for example, it can be the combination of lecithin/cholesterol/phosphatidyl glycerol.The molar ratio of its mixing is 15-85/5-65/1-50 (%mol), preferred 25-70/10-50/10-30 (%mol).

For the lipid membrane structure body being scattered in aqueous solvent, its size is not particularly limited.Describe as an example of the particle diameter of dynamic light scattering determination example.For example, during for liposome or emulsion, particle size range is preferably 20nm～10 μ m, more preferably 50nm～5 μ m.For example, in the time being micellar conformation, particle size range is preferably 5nm～150nm, more preferably 5nm～100nm.For example, in the time being laminate structure thing, thickness in monolayer is preferably 5nm～15nm, more preferably 5nm～10nm.

Kind for aqueous solvent is not particularly limited, as long as can obtain stable lipid membrane structure body when the phospholipid derivative that branched polyethylene glycol of the present invention is modified is scattered in wherein.Can be for example Tris damping fluid, phosphoric acid buffer, citrate buffer solution, borate buffer, phosphate buffer normal saline, physiological saline, cell cultures substratum etc., can add in addition the amino acid solutions such as polyvalent alcohol, L-glutaminate such as the sugar aqueous solutions such as glucose, lactose, sucrose, glycerine, propylene glycol, be dissolvable in water chemokines wherein etc.Employing is scattered in form in aqueous solvent while preserving, for the consideration of the aspects such as physical stability, in order preserving steadily in the long term, preferably to reduce the ionogen in aqueous solvent as far as possible, and removes dissolved oxygen as far as possible, for example, can adopt nitrogen foam-forming method.While adopting drying regime to preserve, preferably the lipid membrane structure body of sugar aqueous solution or polyatomic alcohol water solution dispersion is dried, can adopts freeze-drying, spray-drying process etc., can preserve for a long time.

Concentration to aqueous solvent is not particularly limited.For example, for sugar aqueous solution, be preferably 2%～15% (w/v), more preferably 5%～10% (w/v).For polyhydric alcohol solutions, preferably 1%～8% (w/v), more preferably 2%～3% (w/v).For buffered soln, preferably 5mM～50mM, more preferably 10mM～25mM.

Concentration for lipid membrane structure body in aqueous solvent is not particularly limited, phospholipid derivative and the not modified lipid composition modified through branched polyethylene glycol, be referred to as lipid mixt, its total concn is preferably 0.1mM～500mM, more preferably 1mM～200mM, more preferably 2mM～100mM.

The acquisition pattern that is scattered in aqueous solvent for lipid membrane structure body is not particularly limited.For example can be by above-mentioned dry lipid mixt be joined in aqueous solvent, then carry out emulsification and make, emulsification method can be the modes such as ultrasonic method, refiner method, high pressure spraying mulser method.

The form that is scattered in aqueous solvent for lipid membrane structure body, is not particularly limited, for example, can be the forms such as liposome (or lipid vesicle), micella, mixing micelle, multiple emulsion, laminate structure thing, and preferably its form is liposome.

The described liposome that is scattered in water solvent, its preparation method is not particularly limited, for example, can adopt film dispersion method, reverse phase evaporation, solvent injection method, lyophilization, second emulsifying method, extrusion molding etc.When adopting when extrusion molding, be conducive to obtain little and there is the liposome of homogeneous particle diameter.

For the lipid membrane structure body or the liposome that are scattered in aqueous solvent are further carried out to dry method, be not particularly limited, for example can adopt freeze-drying or spray-drying process.Corresponding aqueous solvent, as mentioned above, can use sugar aqueous solution or polyatomic alcohol water solution, wherein the sugar aqueous solution preferably sucrose aqueous solution, lactose aqueous solution.The lipid membrane structure body that is scattered in aqueous solvent is dried, can preserves for a long time.In addition, in this dry lipid membrane structure body, add the aqueous solution of bio-related substance, can realize the efficient hydration of lipid mixt, can make biologically active substance be held in lipid membrane structure body aspect, there is good efficiency.

Described lipid membrane structure body, is packaged with bio-related substance.

The lipid membrane structure body of described liposome form, is packaged with bio-related substance.

The method of described lipid membrane structure body and liposome encapsulation bio-related substance is not particularly limited, including but not limited to well-known method for packing, the same step that is loaded in of the formation of for example liposome and bio-related substance completes, corresponding passive encapsulation technology, for example also can first form again blank liposome, realize again the loading of bio-related substance by specific gradient (as pH gradient, or ammonium sulphate gradient), corresponding initiatively encapsulation technology.

Described passive encapsulation technology is not particularly limited, for example, and lipid membrane structure body or dried powder that can be based on dry, such as film dispersion method, organic solvent lyophilization, spray-drying process, fluidized bed coating, a single phase soln lyophilization etc.; Can emulsion-based, such as reverse phase evaporation, second emulsifying method etc.; Can such as, based on mixing micelle, cross-stream dialysis method etc.; Can be based on organic solvent (as ethanol), phosphatide or phospholipid derivative, water three-phase mixture, such as technology of preparing, the cross-stream injection technique etc. of alcohol injection, Alza company.

Described active encapsulation technology is not particularly limited.Conventionally can take following steps: the preparation of (1) blank liposome; (2) creation of specific gradient, can realize by modes such as dialysis, column chromatographies; (3), at suitable temperature, hatch having formed the blank liposome of gradient and bio-related substance to be encapsulated inside and outside film, to complete loading.For example, pH value gradient method, ammonium sulphate gradient, calcium acetate gradient method, the outer sour pH value gradient method of interior alkali etc.

Described lipid membrane structure body and liposome, the bio-related substance of its encapsulation discharges in the time of low pH value, preferably pH≤6.8, more preferably pH≤6.5, further preferred pH≤6.0.

For the phospholipid composition used in polyethyleneglycol modified lipid composition of not using that forms lipid membrane structure body and liposome, such as sphingophospholipid, glyceryl phosphatide, phosphatidyl amino acid etc., in the time using acidic phospholipid, under low pH value, cause the disappearance of lipid film surface charge to become unstable due to protonated, give thus lipid membrane structure body pH susceptibility, the bio-related substance that can encapsulate in target spot position especially tumor locus is efficiently released in lipid membrane structure body.For example, the acidic phospholipid that contains terminal carboxyl(group) for hydrophilic segment, when pH≤6.8, preferably pH≤6.5, more preferably pH≤6.0 o'clock, lipid membrane structure body due to protonated cause unstable.Described acidic phospholipid, can be reacted and be prepared with carboxylic acid anhydride by corresponding phosphatide cpd.Described acidic phospholipid preferably glycerine phosphatide, more preferably DOPE (DOPE).

The phospholipid derivative of modifying for the branched polyethylene glycol that forms lipid membrane structure body and liposome, works as L ₄fracture if the phosphatide cpd generating is acidic phospholipid, also can make lipid membrane structure body have pH susceptibility after causing branched polyethylene glycol to depart from.For example; for the Pegylation lipid derivate that adopts the carboxylic phosphatide cpd of end and end to generate containing the branched polyethylene glycol derivatives reaction of amino or hydroxyl; after branched polyethylene glycol part departs from; due to the disappearance of wetting ability protective layer; lipid membrane structure surface produces exposed carboxyl, thereby gives its pH susceptibility.

For the lipid composition without polyethyleneglycol modified; when using glyceryl phosphatide; particularly when DOPE; the phospholipid derivative of modifying for branched polyethylene glycol, the phospholipid derivative that preferably uses branched polyethylene glycol that corresponding DOPE prepared as raw material to modify.When the main composition composition of liposome is DOPE; DOPE is under the condition of low pH; below pH6.8; preferably below pH6.5; more preferably pH6.0 is easy to form hexagonal phase or reversed phase micelle structure below, occurs to assemble or merge, after the protective layer of branched polyethylene glycol departs from; lipid membrane structure body changes hexagonal phase into and discharges medicine, reflects pH responsiveness.

For the liposome form in aqueous solvent, its transformation temperature does not have specific limited, and described transformation temperature is the temperature of liposome membrane generation gel-liquid crystal phase transition.When the transformation temperature of described liposome is higher than normal physiological body temperature, preferred 39-42 ℃ time, can adopt tumor thermotherapy method, utilize physical energy to precipitate in tissue and produce heat effect, making tumor tissues temperature rise on transformation temperature, impelling lipid bilayer to be changed to liquid crystal state by gel state, liposome membrane permeability is increased, and then efficiently discharge medicine at tumor locus, and kill cancer cells and do not damage healthy tissues, strengthen passive target effect.The preferred reverse phase evaporation of method of preparation temperature susceptibility liposome.Generally contain dipalmitoyl phosphatidylcholine (DPPC) for main film material, 40～43 ℃ of corresponding thermotherapy temperatures.Also can add appropriate lysophospholipid MPPC (MPPC) or MSPC (MSPC), phase change temperature of liposome is slightly reduced, preparation is more suitable for 39～42 ℃ of clinical gentle thermotherapy temperatures.

The described bio-related substance that is encapsulated in lipid membrane structure body and liposome, is not particularly limited its hydrophilic and hydrophobic, can be fat-soluble or lipotropy, can be also water-soluble.

The described bio-related substance that is encapsulated in lipid membrane structure body and liposome, its kind is not particularly limited, comprise the biologically active substance of biologically active substance and modification, specifically include but are not limited to following material: polypeptide, protein, enzyme, small-molecule drug, gene-correlation material (nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid), polysaccharide, steroid, steroidal compounds, glycolipid, glycoprotein, lipoid substance, dyestuff, neurotransmission albumen, VITAMIN.

(1) peptide and protein

Proteins and peptides is not particularly limited, and can be exemplified below: hormone, as pituitrin, Triiodothyronine, male hormone, female hormone and suprarenin etc., serum protein, as oxyphorase and blood factor etc., immunoglobulin (Ig), as IgG, IgE, IgM, IgA and IgD etc., cytokine, as interleukin, Interferon, rabbit, granulocyte colony-stimulating factor, macrophage colony stimulating factor, granulocyte-macrophage colony stimutaing factor, platelet derived growth factor, Phospholipid hydrolase activator, Regular Insulin, glucagon, glucagon-like peptide and analogue thereof, lectin, ricin, tumour necrosis factor, epithelical cell growth factor, vascular endothelial growth factor, nerve growth factor, bone growth factor, rhIGF-1, heparin binding growth factor, tumor growth factor, glial cell line derived neurotrophic factor, scavenger cell differentiation factor, differentiation inducing factor, leukaemia inhibitory factor, two regulin, somatomedin, erythropoietin, hemocyte growth hormone, thrombocyte growth hormone and thyrocalcitonin, enzyme, as proteolytic ferment, oxydo-reductase, transferring enzyme, lytic enzyme, lyase, isomerase, ligase enzyme, asparagus fern amine enzyme, arginase, arginine desaminase, adenosine deaminase, superoxide-dismutase, intracellular toxin enzyme, catalase, Chymotrypsin, lipase, uriKoxidase, Proteinase, bone marrow serine, streptokinase, urokinase, uPA, adenosine deaminase, tyrosine oxidase, bilirubin oxidase, glucose oxidase, glucolase and glucuronide enzyme, fiber eliminating enzyme, mono-clonal or polyclonal antibody and fragment thereof, poly propylhomoserin, as polylysine, poly-D-Lys etc., vaccine, antigen and virus, as hepatitis B vaccine, malaria vaccine, Melacine, HIV-1 vaccine etc.

(2) gene-correlation material

Gene-correlation material is not particularly limited, and can be listed below: nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, DNA, RNA etc.

(3) small-molecule drug

Small-molecule drug is not particularly limited, preferably anticancer or antitumor drug and antifungal drug.Anticancer or antitumor drug, preferably taxol and derivative, Zorubicin or doxorubicin hydrochloride, daunorubicin, cis-platinum, daunomycin, mitomycin, vincristine(VCR), vinorelbine, epirubicin, methotrexate, 5 FU 5 fluorouracil, aclacinomycin, Yi Da mycin, bleomycin, pirarubicin, peplomycin, vancomycin, amikacin, camptothecin analogues, hydroxycamptothecine, irinotecan, SN38, topotecan hydrochloride, oxaliplatin, mitoxantrone, all-trans retinoic acid, cytosine arabinoside etc.Antifungal drug, preferably amphotericin B, gentamicin, nystatin, fluoro cytosine(Cyt), miconazole, fluconazole, itraconazole, KETOKONAZOL and polypeptide antifungal drug.

(4) VITAMIN

VITAMIN is that humans and animals is to maintain normal physiological function and the class trace organic substance that must obtain from food, in people's bulk-growth, metabolism, growth course, plays an important role.Specifically include but not limited to vitamin A, vitamins B, vitamins C, vitamin-E and vitamin K etc.

(5) carbohydrate

Carbohydrate is the main component that forms cell and organ, is not particularly limited, and mainly comprises glycolipid, glycoprotein, glycogen etc.Glycolipid is distributed more widely organism, mainly comprises glycosyl acyl glycerine and the large class of glycosphingolipid two, specifically comprises ceramide, cerebroside, sphingosine, Sphingolipids,sialo and glyceryl glycolipid etc.; Glycoprotein is the oligonucleotide chain of branch the formed compounding sugar that is connected with polypeptid covalence, conventionally be secreted in body fluid or the moiety of membranin, specifically comprise Transferrins,iron complexes, Ceruloplasmin, embrane-associated protein, histocompatibility antigen, hormone, carrier, lectin and antibody.

(6) lipid

Lipid mainly comprises grease and the large class of lipoid two.Wherein, the composition of lipid acid is not particularly limited, but preferably has the lipid acid of 12 to 24 carbon atoms, and lipid acid can be saturated fatty acid or unsaturated fatty acids.Lipoid comprises glycolipid, phosphatide, cholesteryl ester, wherein, phosphatide can be that natural phospholipid material is as yolk, soybean etc., can be maybe synthetic phosphate compound, preferably phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, Val, phosphatidylserine, phosphatidylinositols and haemolysis glyceryl phosphatide isomer.The materials such as cholesterol and steroid (steroid) maintain normal metabolism and reproductive process for organism, play important regulating effect, mainly comprise cholesterol, cholic acid, sexual hormoue and vitamins D etc.

(7) other

The bio-related substance that is encapsulated in lipid membrane structure body and liposome is not limited to disease treatment purposes, also can be used for comprising the bio-related substance for other purposes such as biological diagnosis, detection.For example, for the dye molecule of quantitative or semi-quantitative analysis; For example can be used for the fluorine carbon molecule of the purposes such as angiographic diagnosis, blood substitute etc.; Such as anti-parasite medicine is as primaquine etc.; For example can be used as the carrier of toxinicide, as sequestrant ethylenediamine tetraacetic acid (EDTA) (EDTA), PENTETIC ACID (DTPA) etc.

The known bio-related substance of these those skilled in the art such as liposome, cell, micella etc.

Lipid derivate below in conjunction with some embodiments to a kind of branched polyethylene glycol of the present invention and the lipid membrane structure body that contains this derivative are described further.Specific embodiment is for further describing the present invention, non-limiting protection scope of the present invention.

Embodiment 1:L ₄in contain amido linkage preparation

A, method of condensing

L ₄in contain the synthetic of amido linkage compound (A1-1), wherein L ₁=L ₂=L ₃=CH ₂, Y=CH, X ₁=X ₂=CH ₃, q=1, R is two bay acyl glyceride residues, molecular weight polyethylene glycol is about 2000.

In the 1L of dried and clean round-bottomed flask, add (13.9 grams of 40g branched polyethylene glycol acetic acid and DLPE; 1.2 equivalents) after; nitrogen protection, adds after methylene chloride (500mL), after stirring at room temperature 10min; add successively the dichloromethane solution of 20mL triethylamine and 20g bicyclohexane carbodiimide (DCC); under room temperature, react after 24 hours, remove by filter insolubles, concentrated; Virahol recrystallization, obtains containing in L4 amido linkage compound (A1-1).

The hydrogen spectrum data of compd A 1-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.65（CH ₃(CH ₂) ₉-），2.32-2.51(-CH ₂CH ₂COO-,-CH(CH ₂) ₃-)，3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-CHCH ₂O-，-OCH ₂CH ₂NH-)，4.05-4.42(-OCH(CH ₂)CH ₂O-，-OCH ₂CH ₂NH-，-OCH ₂CONH-)，5.16（-OCH(CH ₂)CH ₂O-），8.38（-NHCO-）.

Similarly, L ₄in contain the synthetic of amide compound (A1-2), wherein L ₁=L ₂=L ₃=CH ₂, X ₁=X ₂=CH ₃, Y is C-Bn, q=0,

The preparation method of A1-2 is identical with the preparation method of A1-1, does not repeat one by one at this;

The hydrogen spectrum data of compd A 1-2 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.85-1.62（-CH ₃，-CH ₂-，-CH-），2.15-2.19（-CH ₂C=CH-,-C=CHCH ₂-，-CCH ₂CONH-），2.62（-CH ₂Ph），3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-,-CHO-,-OCH ₂CH ₂NH-)，4.05-4.10(-OCH ₂CH ₂NH-)，5.42(-C=CHCH2-)，7.27-7.42（-CH ₂C ₆H ₅）。

The method of B, active ester

A.L ₄in contain the synthetic of amido linkage compound (A1-3), wherein L ₁=CH ₂cH ₂, L ₂=COCH ₂, L ₃=CH ₂cH ₂, X ₁=X ₂=CH ₃, Y is N, q=1, and R is 1-palmitoyl-2-oleoyl glyceryl ester residue, molecular weight polyethylene glycol is about 4000.

In the 1L of dried and clean round-bottomed flask, add after branched polyethylene glycol propylamine (40g); nitrogen protection; after adding methylene chloride (500mL) to dissolve; after stirring at room temperature 10min, add successively the dichloromethane solution of 20mL triethylamine and 1-palmitoyl-2-oleoyl base phosphatidylethanolamine valeric acid succinimide active ester (10.6 grams, 1.2 equivalents); under room temperature, react after 24 hours; concentrated, Virahol recrystallization, obtains containing in L4 amido linkage compound (A1-2).

The hydrogen spectrum data of compd A 1-3 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.73（-CH ₂-，-NHCH ₂CH ₂CH ₂-），2.15-2.19（-CH ₂CH=CHCH ₂-）2.32-2.41(CH ₂CON-，CH ₂COO-)，3.10(-NHCH ₂CH ₂CH ₂-),3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-CHCH ₂O-，-OCH ₂CH ₂NH-)，4.05-4.42(-OCH ₂CH-，-OCH ₂CH ₂NH-)，5.16（-OCH(CH ₂)CH ₂O-），5.42-5.83（-CH ₂CH=CHCH ₂-,8.38（-NHCO-）.

B.L ₄in contain the synthetic of amido linkage compound (A1-4), wherein L ₁=L ₂=CH ₂, L ₃=CH ₂cH ₂, Y=N, X ₁=X ₂=CH ₃, q=0, R contains stearyl-and sphingosine skeleton, and molecular weight polyethylene glycol is about 2000.

In the 1L of dried and clean round-bottomed flask, add (16.4 grams of 40g branched polyethylene glycol propionic acid succinimide and sphingophospholipid; 1.2 equivalents) after; nitrogen protection; add after solvent anhydrous methylene chloride (500mL); after room temperature reaction 24 hours; concentrated, Virahol recrystallization, obtains containing in L4 amido linkage compound (A1-3).

The hydrogen spectrum data of compd A 1-4 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.65（CH ₃(CH ₂)-），2.05-2.25（-CH=CHCH ₂-,-NHCOCH ₂-）,2.70-2.80(-NCH ₂CH ₂O-)，3.40-3.80(-CH ₂CH ₂O-.-NHCHCH ₂O-,-NHCH ₂CH ₂O-)，4.05-4.42(-CH=CHCH ₂O-,-OCH ₂CHNH-)，5.42-5.83（-CH ₂CH=CHCH ₂-），8.42（-NHCO-）.

Embodiment 2:L ₄in contain amino-formate bond preparation

A, active ester synthetic method:

L ₄in contain the synthetic of carbamate key compound (A2-1), wherein L ₁=L ₂=CH ₂cH ₂,, L ₃=CH ₂cH ₂, X ₁=X ₂=CH ₃, Y is N, q=1, and R is 1-stearyl--2-hydroxyl glyceryl ester residue, molecular weight polyethylene glycol is about 1000.

In the 500mL of dried and clean round-bottomed flask, add (11.6 grams of branched polyethylene glycol carbonic acid succinimide active ester (20g) and 1-stearyl--2-hydroxyl glyceryl phosphatidylethanolamines; 1.2 equivalents) after; nitrogen protection; add after anhydrous methylene chloride (250mL), after stirring at room temperature 10min, add 5mL triethylamine; under room temperature, react after 24 hours; concentrated, Virahol recrystallization, obtains containing in L4 amino original acid ester key compound (A2-1).

The hydrogen spectrum data of compd A 2-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.65（CH ₃(CH ₂) ₁₆-），2.32-2.41(-CH ₂CH ₂COO-)，2.70-2.80(-NCH ₂CH ₂O-)，3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-NCH ₂CH ₂O-)，4.05-4.42(-OCH ₂CH-，-OCH(CH ₂)CH ₂O-，-OCH ₂CH ₂NH-)，5.16（-OCH(CH ₂)CH ₂O-），8.38（-NHCO-）.

B, one-step synthesis

In this example, L ₄in contain the synthetic of carbamate key compound (A2-2), wherein L ₁=CH ₂cH ₂, L ₂=CH ₂cH ₂c ≡ C, L ₃=CH ₂cH ₂cH ₂, X ₁=n-C ₂₀h ₄₁, X ₂=CH ₃, Y is N, q=1, and R is dioleoyl glyceryl ester residue.Total molecular weight is about 3000, wherein L ₁, L ₂the molecular weight of two branched chain that connect is approximately respectively 1000,1000; The molecular weight of main chain is about 1000.

In the 500mL of dried and clean round-bottomed flask, add after branched polyethylene glycol (30g), with anhydrous methylene chloride (250mL) dissolving, under ice bath, slowly at a low price DSC(2.8 gram, 1.1 equivalents) acetonitrile solution, be slowly increased to room temperature, react after 8 hours, add after 3mL triethylamine, slowly drip after the chloroformic solution of DOPE (11 grams, 1.5 equivalents), under room temperature, react after 24 hours, concentrated, Virahol recrystallization, obtains L ₄in contain amino original acid ester key compound (A2-2).

The hydrogen spectrum data of compd A 2-2 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.73（-CH ₂-，-NCH ₂CH ₂CH ₂-），2.15-2.19（-CH ₂CH=CHCH ₂-，-CH ₂C≡C-），2.32-2.84(-CH ₂COO-，-NCH ₂CH ₂-)，3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-OCH ₂CH ₂NH-)，4.05-4.42(-OCH ₂CH-，-OCH(CH ₂)CH ₂O-，-OCH ₂CH ₂NH-)，5.16（-OCH(CH ₂)CH ₂O-），5.42-5.83（-CH ₂CH=CHCH ₂-）,8.38（-NHCO-）.

Embodiment 3:L ₄in contain ester bond (OCO-) preparation

A:L ₄in contain the synthetic of ester bond compound (A3-1), wherein L ₁=L ₂=L ₃=CH ₂, X ₁=X ₂=CH ₃, Y is CH, q=1, and R is the residue of two Semen Myristicae phosphatidyl glycerol esters.Design total molecular weight is about 1500.

In the 1L of dried and clean round-bottomed flask, add (66.6 grams of branched polyethylene glycol acetic acid (30g) and two Semen Myristicae phosphatidyl glycerols; 5 equivalents) after; nitrogen protection; add after methylene chloride (500mL); after stirring at room temperature 10min; add successively the dichloromethane solution of 20mL triethylamine and 20g bicyclohexane carbodiimide (DCC); under room temperature, react after 24 hours; remove by filter insolubles; concentrated, by water dissolution, filter; dialysis, obtains containing in L4 ester bond compound (A3-1).

The hydrogen spectrum data of compound A-13-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.65（-CH ₂-），2.32-2.41(-CH ₂CH ₂COO-)，2.51(-CH(CH ₂) ₃-)，3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-)，4.05-4.42(-OCH ₂CH-，-OCH(CH ₂)CH ₂O-，-OCH ₂CH ₂NH-)，4.90-5.13（-OCH(CH ₂)CH ₂O-）。

Embodiment 4:L ₄in contain secondary amine key (CH ₂nHCH ₂-) preparation

In this example, L ₄in contain the synthetic of secondary amine key compound (A4-1), wherein L ₁=L ₂=L ₃=CH ₂, X ₁=X ₂=CH ₃, Y is CH, q=1, and R is the residue of two Semen Myristicae phosphatidyl glycerol esters.Polyoxyethylene glycol total molecular weight is about 2000.

In the 1L of dried and clean round-bottomed flask, add (16.3 grams of branched polyethylene glycol propionic aldehyde (40g) and two myristoyl phosphatidylserines; 1.2 equivalents) after; nitrogen protection; adding pH value is after 5.0 PBS damping fluid (500mL); after stirring at room temperature 4 hours; add after sodium cyanoborohydride (5 grams); the lower 20 ℃ of reaction 24h of room temperature; add after saturated ammonium chloride solution cancellation; dilute with water, dichloromethane extraction, concentrated; dialysis, obtains containing in L4 secondary amine key compound (A4-1).

The hydrogen spectrum data of compd A 4-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.73（-CH ₂-,-NCH ₂CH ₂CH ₂-），2.32-2.41(-CH ₂CH ₂COO-)，2.51(-CH(CH ₂) ₃-)，2.85(-NHCH ₂-)，3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-CHCH ₂O-)，4.05-4.42(-OCH ₂CH-，-OCH(CH ₂)CH ₂O-)，4.90-5.13（-OCH(CH ₂)CH ₂O-,-NHCH(COOH)-）。

Embodiment 5:L ₄in contain thioether bond (>CHS-) preparation

In this example, L ₄in contain the synthetic of thioether bond (>CHS-) compound (A5-1), wherein L ₁=L ₂=L ₃=CH ₂cH ₂, X ₁=X ₂=CH ₃, Y is N, q=1, the residue that R is distearin.Polyoxyethylene glycol total molecular weight is about 2000.

In the 500mL of dried and clean round-bottomed flask, add 100mL to contain (10 grams of Y type polyoxyethylene glycol mercapto derivatives, phosphate buffer soln (pH=7.4) 5mmol/L), add DSPE-MAL(5 gram, 1.1 equivalents) after, under 4 ℃ of conditions, react after 24 hours, add after distilled water diluting, with dichloromethane extraction, dry, concentrated, after Virahol recrystallization, obtain containing in L4 thioether bond (>CHS-) compound (A5-1).

The hydrogen spectrum data of compound A-45-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.65（CH ₃(CH ₂) ₁₆-），2.32-2.95(-CH ₂COO-，-NCH ₂CH ₂-，-NHCOCH ₂CH ₂-，-OCCH ₂CHS-，)，3.35(CH ₃O-)，3.40-3.80(-OCH ₂CH ₂O-，-NCH ₂CH ₂O-，-CONCH ₂CH-,-OCCHS-)，4.05-4.42(-OCH ₂CHO-，-OCH(CH ₂)CH ₂O-,-OCH ₂CH ₂NHCO-)，5.13（-OCH(CH ₂)CH ₂O-）,8.38（-NHCO-）.

Embodiment 6:L ₄in contain triazole preparation

In this example, L ₄in contain the synthetic of 3-triazole compounds (A5-1), wherein L ₁=L ₂=L ₃=CH ₂, X ₁=X ₂=CH ₃, Y is CH, q=1, the residue that R is distearin.Polyoxyethylene glycol total molecular weight is about 20000.

In the 500mL of dried and clean round-bottomed flask, add after the Y type polyoxyethylene glycol (20 grams, 1mmoL) that contains DIBO group, add acetonitrile, under room temperature, be stirred to after dissolving completely, slowly drip DSPE-N ₃acetonitrile solution (200mL) DSPE-N ₃after (1.55 grams, 2 equivalents), under room temperature, react after 4 hours, concentrated, dialysis obtains containing in L4 triazole (compound (A6-1).

The hydrogen spectrum data of compd A 6-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-1.65（CH ₃(CH ₂) ₁₆-），2.32-2.41(-CH ₂CH ₂COO-)，3.0-3.2（ArCH ₂-），3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-CHCH ₂O-，-OCHCH ₂-，-NCH ₂CH ₂O-)，4.05-4.42(-OCH ₂CH-，-OCH(CH ₂)CH ₂O-)，4.33(-OCCH ₂O-)5.13（-OCH(CH ₂)CH ₂O-），5.62(ArCHO-)，7.31-7.65（C ₆H ₄-）。

Embodiment 7:L ₄in contain 4,5-dihydro-isoxazole

In this example, L ₄in contain the synthetic of 4,5-dihydro-isoxazole (A7-1), wherein L ₁=L ₂=L ₃=CH ₂, X ₁=X ₂=CH ₃, Y is CH, q=1, the residue that R is distearin.Polyoxyethylene glycol total molecular weight is about 5000.

In the 50mL of dried and clean round-bottomed flask, add (2 grams of Y type polyoxyethylene glycol propionic aldehyde, 0.4mmoL), add acetonitrile, under room temperature, be stirred to after dissolving completely, displacement nitrogen, add after hydroxy amine hydrochloric acid salt (4mmol), add sodium-acetate to be adjusted to after PH=8, under room temperature, reaction is spent the night, concentrated, ether sedimentation, after preliminary purification, is directly used in next step reaction.

By the thick product of previous step, after dissolving with DMF (20mL) in the 50mL of dried and clean round-bottomed flask, after displacement nitrogen, add solid NCS(4mmol), after reaction is spent the night under room temperature, add saturated sodium bicarbonate solution (20mL), under room temperature, continue to stir after 4 hours, add after a large amount of methylene dichloride dilutions, use saturated common salt water washing, dry, concentrated, ether sedimentation.

In the 500mL of dried and clean round-bottomed flask, add and walk after the Y type polyoxyethylene glycol cyanogen oxygen compound obtaining, add acetonitrile, under room temperature, be stirred to after dissolving completely, slowly drip after the acetonitrile solution (100mL) of DSPE-Norbornene, under room temperature, react after 4 hours, concentrated, after Virahol recrystallization, obtain containing in L4 triazole (compound (A7-1).

The hydrogen spectrum data of compd A 7-1 are as follows:

¹H?NMR(CDCl ₃)δ(ppm)：0.95（-CH ₂CH ₃），1.21-2.01（CH ₃(CH ₂) ₁₆-，OCH ₂CH ₂CH<，>CHCH ₂CH<，-C(=N)CH ₂CH ₂O-），2.22-2.51(-CH ₂CH ₂COO-，>CHCHC(=N)-)，-CH(CH ₂) ₃-)，3.35(CH ₃O-)，3.40-3.80(-CH ₂CH ₂O-，-CHCH ₂O-)，4.05-4.42(-OCH ₂CH-，-OCH(CH ₂)CH ₂O-)，5.13（-OCH(CH ₂)CH ₂O-）。

Embodiment 8-21: the preparation of liposome and stability test thereof

(1) preparation of liposome

Use gained L in embodiment 1 ₄the A1-3(embodiment 8 of middle amide bond), A1-4(embodiment 9), gained L in embodiment 2 ₄the A2-1(embodiment 10 of middle amido-containing acid ester key), A2-2(embodiment 11), gained L in embodiment 3 ₄in containing the A3-1(embodiment 12 of ester bond), gained L in embodiment 4 ₄in containing the A4-1(embodiment 13 of secondary amine key), prepare liposome with DOPE (DOPE) respectively.

Use gained L in embodiment 1 ₄the A1-2(embodiment 14 of middle amide bond), gained L in embodiment 5 ₄the A5-1(embodiment 15 of middle sulfur-bearing ehter bond), gained L in embodiment 6 ₄in containing the A6-1(embodiment 16 of triazole), gained L in embodiment 7 ₄in containing the A7-1(embodiment 17 of 4,5-dihydro-isoxazole), prepare liposome with DOPE, cholesterol respectively.

Use gained L in embodiment 1 ₄the A1-3(embodiment 18 of middle amide bond), prepare liposome with DOPE and cholesteryl hemisuccinate (CHEMS).

In use embodiment 2, in gained L4, the A2-2 of amido-containing acid ester key prepares liposome with DSPE (DSPE), DPPE (DPPE) respectively, corresponds respectively to embodiment 19-21.

Parameter is as shown in table 1.

Parameters listed in Table 1 were weighed in groups of different instances of the lipid mixture and the Comparative Example 1 DOPE, were fully dissolved in chloroform, and the solvent was removed using a rotary evaporator until a uniform wall bottled lipid film.To the Tris solution 2mL that adds 25mM fluorochrome HPTS in lipid membrane, wherein containing the fluorochrome quencher DPX of 25mM, pH=10.0.After using vortex oscillation device to disperse, use the polycarbonate leaching film of different pore size to filter successively, every kind of aperture filter 23 time, obtains Liposomal dispersion.Adopt dynamic light scattering method to detect particle diameter, the particle diameter of all liposomes is all below 100nm.

Table 1

Numbering	The composition of liposome membrane	Ratio of components (mass ratio)
			Embodiment 8	DOPE/A1-3	99/1
Embodiment 9	DOPE/A1-4	97/3
			Embodiment 10	DOPE/A2-1	95/5
Embodiment 11	DOPE/A2-2	95/5
			Embodiment 12	DOPE/A3-1	90/10
Embodiment 13	DOPE/A4-1	85/15
			Embodiment 14	DOPE/ cholesterol/A1-2	90/7/3
Embodiment 15	DOPE/ cholesterol/A5-1	90/7/3
			Embodiment 16	DOPE/ cholesterol/A6-1	80/17/3
Embodiment 17	DOPE/ cholesterol/A7-1	50/45/5
			Embodiment 18	DOPE/CHEMS/A1-3	80/17/3

Embodiment 19	DSPE/A2-2	70/30
			Embodiment 20	DPPE/A2-2	80/20
Embodiment 21	DPPE/A2-2	99.9/0.1
			Reference examples 1	DOPE	100/0

(2) stability of liposome in damping fluid

Above-mentioned gained Liposomal dispersion is placed 1 month at ambient temperature, and by the naked eye, embodiment 8-20 is still uniform Liposomal dispersion, visually has no variation.The dispersion liquid of embodiment 21 has slight sedimentation.The Liposomal dispersion of reference examples 1 is unstable, visible sedimentation.

(3) concentration-response of liposome and pH susceptibility

Get the Liposomal dispersion of embodiment 8-21, modulate successively pH=5.0, 6.0, 6.5, 6.8, 7.0, 8.0, 9.0, 10.0 dispersion liquid, hatch 1h at 37 ℃, then collecting sample, dilute with the damping fluid of pH10.0, by measuring fluorescence intensity, calculate release rate R (t)=100 × [I (t)-I (0)]/[I (the ∞)-I (0)] of fluorochrome HPTS from liposome, wherein I (t) is the fluorescence intensity in the time of time t, I (0) is initial residual fluorescence intensity, I (∞) is with corresponding maximum fluorescence intensity after 0.1% (w/v) Triton X-100 lipin dissolving body.

Result demonstration, the release of HPTS from liposome has pH susceptibility.When low pH value, in embodiment 8-18, HPTS release rate reduces with the increase of polyethyleneglycol modified lipid derivate content, when pH=6.0, and embodiment 8,9,10,11,12,13 release rate is followed successively by 26%, 18%, and 12%, 10%, 5%, 3%; When pH=6.5, embodiment 8,9,10,11,12,13 release rate is followed successively by 22%, 16%, and 9%, 8%, 4%, 2.5%.In embodiment 11,19,20,21, HPTS release rate increases with the increase of polyethyleneglycol modified lipid derivate content.In reference examples 1, when pH=6.5, release rate, more than 80%, when pH=6.0, reaches more than 90%.Result also shows, being added with of cholesterol is beneficial to the stability that improves liposome membrane, and release rate reduces with the increase of cholesterol level, when pH=6.0, and embodiment 8,14,15,16,17 release rate is followed successively by 20%, 17%, and 16%, 11%, 6%; When pH=6.5, embodiment 8,14,15,16,17 release rate is followed successively by 17%, 15%, and 14%, 10%, 5%.For embodiment 8-17, pH≤7.0 o'clock, release rate reduces with the rising of pH, pH >=7.0 o'clock, release rate is no more than 3%, and for reference examples 1, when pH=7.0, release rate is still more than 20%, and pH >=8.0 o'clock, release rate is no more than 3%.

While adopting CHEMS to do membrane stabilizer, with reference to embodiment 18, under neutrality and alkaline condition, (pH >=7.0) are conducive to the stability of liposome membrane structure, corresponding release rate is no more than 2%, and under acidic conditions, being conducive to accelerate the release of HPTS, when pH=5.0, release rate is 42%, when pH=6.0, when release rate is 30%, pH=6.5, release rate is 21%, when pH=6.8, release rate is 15%.

(4) stability of liposome in serum

Get the Liposomal dispersion of embodiment 8-21, dilute with the substratum of 10% foetal calf serum respectively, hatch at 37 ℃, successively 0,1,6,12,18,24h collecting sample, dilutes with the Tris solution of pH10.0, measures fluorescence intensity, calculates the release rate of HPTS.Result demonstration, the maximum release rate of embodiment 8-21 is followed successively by be and exceedes 5%, is showed no the avalanche of liposome.Visible, lipid membrane structure body is more stable in serum.

Embodiment 22-25: Pharmacokinetic Evaluation and the tissue distribution situation of the liposome that is enclosed with cancer therapy drug (Zorubicin) in blood

(5) preparation of hydrochloric doxorubicin liposome

Use gained L in embodiment 1 ₄the A1-1(embodiment 22 of middle amide bond), A1-4(embodiment 23), gained L in embodiment 3 ₄in containing the A3-1(embodiment 24 of ester bond), gained L in embodiment 5 ₄the A5-1(embodiment 25 of middle sulfur-bearing ehter bond), prepare liposome with DOPE, CHEMS respectively.

Reference examples adopts respectively the corresponding phosphatide of modifying without branched polyethylene glycol; be followed successively by two bay acylglycerol phosphatidylethanolamine (DLPE; reference examples 2), containing the sphingophospholipid (SSL of stearyl-; reference examples 3), DMPG ester (DMPG; reference examples 4), DSPE (DSPE, reference examples 5).

Parameter is as shown in table 2.

Adopt the Liposomal dispersion that is enclosed with doxorubicin hydrochloride of embodiment and reference examples in the method preparation table 2 identical with above-mentioned (1).Replace fluorochrome with doxorubicin hydrochloride, adopt ammonium sulphate gradient that doxorubicin hydrochloride is enclosed in lipid, concentration is 0.3mg doxorubicin hydrochloride/mg liposome.

Table 2

Numbering	The composition of liposome membrane	Ratio of components (mass ratio)
			Embodiment 22	DOPE/CHEMS/A1-1	80/17/3
Reference examples 2	DOPE/CHEMS/DLPE	80/17/3
			Embodiment 23	DOPE/CHEMS/A1-4	65/20/15
Reference examples 3	DOPE/CHEMS/SSL	65/20/15
			Embodiment 24	DOPE/ cholesterol/A3-1	80/17/3
Reference examples 4	DOPE/ cholesterol/DMPG	80/17/3
			Embodiment 25	DOPE/ cholesterol/A5-1	65/25/10
Reference examples 5	DOPE/ cholesterol/DSPE	65/25/10

Table 3

Numbering	The composition of liposome membrane	Ratio of components (mass ratio)	t _1/2γ
				Embodiment 22	DOPE/CHEMS/A1-2	80/17/3	63.4h
Reference examples 2	DOPE/CHEMS/DLPE	80/17/3	9.9h
				Embodiment 23	DOPE/CHEMS/A1-4	65/20/15	61.5h
Reference examples 3	DOPE/CHEMS/SSL	65/20/15	9.2h
				Embodiment 24	DOPE/ cholesterol/A3-1	80/17/3	67.8h
Reference examples 3	DOPE/ cholesterol/DMPG	80/17/3	11.6h
				Embodiment 25	DOPE/ cholesterol/A5-1	65/25/10	65.3h
Reference examples 4	DOPE/ cholesterol/DSPE	65/25/10	10.2h

(6) Pharmacokinetic Evaluation

Select the male Sprague-Dawley rat in 12 week age, with reference to table 2, the hydrochloric doxorubicin liposome dispersion liquid of the dosage such as tail vein injection (15mg liposome/kg), respectively at 1min, 15min, 30min, 1h, 2h, 6h, 12h, 18h, after 24h through rat eye rear vein beard sample of blood, drawn 0.5ml, separation of serum,-20 ℃ of preservations, for measuring Plasma Concentration.Using daunorubicin hydrochloride as interior mark, adopt RP-HPLC method to measure doxorubicin concentration, the medicine-time data of gained meets three compartment models, processes the elimination transformation period t of gained _{1/2 γ}result as shown in table 3.Comparative example 22 with contrast 2, comparative example 23 and reference examples 3, comparative example 24 and reference examples 4, comparative example 25 and reference examples 5, the phospholipid derivative that visible branched polyethylene glycol is modified participates in the transformation period significant prolongation of the liposome (embodiment 22-25) forming, compared with the liposome (reference examples 2-5) of modifying without branched polyethylene glycol, bring up to original 5～10 times.

CHEMS is during as membrane stabilizer, compared with making stablizer with cholesterol, its transformation period is slightly low, this is because CHEMS itself has pH susceptibility, under neutrality and alkaline condition, there is the effect of stabilized liposome membrane structure or liposome, and cause the unstable of lipid membrane structure or liposome acidic conditions is next.

(7) tissue distribution situation

Select the male Sprague-Dawley rat in 12 week age, with reference to the hydrochloric doxorubicin liposome dispersion liquid of the dosage (15mg liposome/kg) such as table 2 intravenous injection, respectively at 5min after administration, 2h, 6h, 12h, 24h, put to death one group of rat by avascularization, and excise brain, the heart, liver, lung, kidney, spleen, stomach and intestine, get a certain amount of tissue, in phosphate buffered saline buffer, under condition of ice bath, be prepared into tissue homogenate through high speed dispersion ,-20 ℃ of preservations, measure for tissue concentration.Using daunorubicin hydrochloride as interior mark, adopt RP-HPLC method to measure doxorubicin concentration.Result shows, the liposome after branched polyethylene glycol is modified, with the liposome phase transformation of not modifying with branched polyethylene glycol, its distribution at heart significantly reduces, greatly reduce cardiac toxic, increase in the abundance at liver, kidney place simultaneously, demonstrate liver, the kidney targeting of enhancing.In addition, adopt embodiment 22-23 and the reference examples 2-3 of CHEMS, than embodiment 24-25 and reference examples 4 and 5, its abundance at stomach enlarges markedly, and targeting improves greatly.

Embodiment 26-29: the antitumous effect evaluation of the liposome of parcel cancer therapy drug (Zorubicin)

Use in embodiment 1 the A1-1(embodiment 26 of amide bond in gained L4), A1-4(embodiment 27), the A3-1(embodiment 28 containing ester bond in gained L4 in embodiment 3) and, gained L in embodiment 5 ₄the A5-1(embodiment 29 of middle sulfur-bearing ehter bond), prepare liposome with DOPE, CHEMS respectively.

Reference examples adopts respectively the corresponding phosphatide of modifying without branched polyethylene glycol; be followed successively by two bay acylglycerol phosphatidylethanolamine (DLPE; reference examples 5), containing the sphingophospholipid (SSL of stearyl-; reference examples 6), DMPG ester (DMPG; reference examples 7), DSPE (DSPE, reference examples 8).

Append a reference examples 9, adopt DLPE-mPEG to replace A1-1, both differences are branched polyethylene glycol to replace with the linear PEG of same equimolecular quantity, and DLPE-mPEG is by reacting and be prepared from DLPE with the mono methoxy polyethylene glycol acetic acid of equimolecular quantity with branched polyethylene glycol.

According to table 4, adopt method in above-mentioned (5) to prepare corresponding hydrochloric doxorubicin liposome dispersion liquid.

Adopt animal transplanting tumor laboratory method, use H ₂₂murine hepatocarcinoma cell is inoculated in the subcutaneous formation solid tumor of right side of mice armpit, after inoculation 2 days, 7 days, carries out tail vein injection administration respectively, and administering mode is single-dose, and dosage is 15mgkg ^-1.Inoculate after 2 weeks, the dislocation of mouse cervical vertebra is put to death, peel off tumour, and weigh.Result shows, (embodiment 26-29 has higher tumour inhibiting rate than not modified reference examples 5-8 to the liposome after branched polyethylene glycol is modified.The embodiment 26-27 that contains CHEMS has higher tumour inhibiting rate than embodiment 28-29.

In addition the liposome (reference examples 9) that the liposome (embodiment 26) that, branched polyethylene glycol is modified is modified compared with linear polyethylene glycol has higher tumour inhibiting rate.

Known by the above results, liposome of the present invention is stable under neutrality and alkaline condition, and be subject to the impact of pH value at tumour isogonic acid sites, the release rate of the bio-related substance of encapsulation obviously improves, the drug distribution amount at these positions also selectivity improves, demonstrate good passive targeting, can avoid because of frequent drug administration or strengthen the toxic side effect that dosage causes.

Table 4

Numbering	The composition of liposome membrane	Ratio of components (mass ratio)
			Embodiment 26	DOPE/CHEMS/A1-1	80/17/3

Reference examples 5	DOPE/CHEMS/DLPE	80/17/3
			Embodiment 27	DOPE/CHEMS/A1-4	65/20/15
Reference examples 6	DOPE/CHEMS/SSL	65/20/15
			Reference examples 9	DOPE/CHEMS/DLPE-mPEG	80/17/3
Embodiment 28	DOPE/ cholesterol/A3-1	80/17/3
			Reference examples 7	DOPE/ cholesterol/DMPG	80/17/3
Embodiment 29	DOPE/ cholesterol/A5-1	65/25/10
			Reference examples 8	DOPE/ cholesterol/DSPE	65/25/10

The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes description of the present invention to do; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.

Claims

1. a phospholipid derivative for branched polyethylene glycol, is characterized in that, the phospholipid derivative general formula of described branched polyethylene glycol as the formula (1):

2. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that n ₁, n ₂, n ₃preferably meet 1≤n ₁≤ 200,1≤n ₂≤ 200,0≤n ₃≤ 200, and 2≤n ₁+ n ₂+ n ₃≤ 200.

3. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that, Y is carbon atom branching center, and its representation is as follows: wherein, R ₁for hydrogen atom, the alkyl with 1 to 20 carbon that there is the alkyl of 1 to 20 carbon or contain heteroatom group.

4. the phospholipid derivative of branched polyethylene glycol according to claim 3, it is characterized in that, described R1 is hydrogen atom or is the alkyl with 1 to 20 carbon, or is the alkyl with 1 to 20 carbon containing ester group, urethane groups, amide group, ether, thioether, two key, Three key, carbonate group, secondary amine or tertiary amine groups.

5. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that, Y is nitrogen-atoms branching center, and its representation is as follows:

6. the phospholipid derivative of branched polyethylene glycol according to claim 1, it is characterized in that, R is hydrophobic fat hydrocarbon residue, or DG lipid residue, or monoacylglycerol lipid residue, or the lipid residue that contains sphingosinols skeleton, or steroid residue, or above-mentioned any two and two or more compound lipid residues;

When R is DG lipid residue, its representation is as follows:

r ₄, R ₅for thering is the alkyl of 4 to 50 carbon atoms;

When R is monoacylglycerol lipid residue, its representation is as follows:

r ₆for thering is the alkyl of 4 to 50 carbon;

When R is the lipid residue that contains sphingosinols skeleton, its representation is as follows:

or be expressed as

r ₂, R ₃independently of one another for thering is the alkyl of 4 to 50 carbon atoms;

When R is hydrophobic fat hydrocarbon residue, its representation is as follows: r ₇for thering is the alkyl of 4 to 50 carbon, can be straight chain, side chain, ring-type or the chain with cyclic group;

When the R residue that is steroid, this steroid is that cholesterol, Sitosterol, vitamins D, beta-cholestanol, ergosterol, wool are stayed alcohol, bilichol or sexual hormoue, or the derivative of above-mentioned steroid;

Described R ₂, R ₃, R ₄, R ₅, R ₆, R ₇for butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, (Z)-9-tetradecene base, (Z)-8-17 thiazolinyls, (Z)-12-bis-hendecene bases, and in same a part, can be the same or different.

7. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that L ₄for

Wherein, Z is alkylidene group or the alkylidene group that contains the groups such as ester group, urethane groups, amide group, ether, thioether, two key, Three key key, carbonate group, secondary amine or tertiary amine groups;

G is 0 or 1;

F is 2 to 10 integer.

8. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that described X ₁, X ₂for methyl, ethyl, propyl group, propenyl, proyl, sec.-propyl, butyl, the tertiary butyl, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, benzyl or butyl phenyl, and in same a part, can be the same or different.

9. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that described L ₁, L ₂, L ₃be the bivalent hydrocarbon radical with 1 to 20 carbon atom of the ether, thioether group, amide group, two key, Three key, secondary amine or the tertiary amine groups that contain stable existence under illumination, enzyme, acidity or alkaline condition independently of one another.

10. the phospholipid derivative of branched polyethylene glycol according to claim 1, is characterized in that, M is hydrogen atom, sodium ion, NH ₄ ⁺.

The 11. phospholipid derivatives of branched polyethylene glycol according to claim 1, is characterized in that the residue that contains phosphatidylethanolamine.

12. according to the phospholipid derivative of branched polyethylene glycol described in claim 11; it is characterized in that, described phosphatidylethanolamine is DSPE, two lauroyl phosphatidylethanolamines, two grease acylphosphatidyl ethanolamines, two myristoyl phosphatidylethanolamines, two flax acyl phosphatidylethanolamines, two mustard acyl phosphatidylethanolamines, 1-palmityl-2-oleoyl phosphatidylethanolamine or DPPE.

13. 1 kinds contain in claim 1～12 the lipid membrane structure body of the phospholipid derivative of branched polyethylene glycol described in any one.

14. according to lipid membrane structure body described in claim 13, it is characterized in that, the phospholipid derivative of branched polyethylene glycol be this lipid membrane structure body total mass 0.1%～30%.

15. according to lipid membrane structure body described in claim 13, it is characterized in that, the phospholipid derivative of branched polyethylene glycol be this lipid membrane structure body total mass 1%～30%, be preferably 3%-20%, more preferably 3%-15%.

16. according to lipid membrane structure body described in claim 13, it is characterized in that, also contains the moiety of phosphatidylethanolamine as film.

17. according to lipid membrane structure body described in claim 16; it is characterized in that; wherein phosphatidylethanolamine is DSPE, two grease acylphosphatidyl ethanolamines, two myristoyl phosphatidylethanolamine or DPPEs, two lauroyl phosphatidylethanolamines, two flax acyl phosphatidylethanolamines, two mustard acyl phosphatidylethanolamines, 1-palmityl-2-oleoyl phosphatidylethanolamine or DPPE.

18. according to lipid membrane structure body described in claim 13, it is characterized in that, it is packaged with bio-related substance.

19. according to lipid membrane structure body described in claim 18, it is characterized in that, described bio-related substance is polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid.

20. according to lipid membrane structure body described in claim 13, it is characterized in that, its form is liposome.

21. according to lipid membrane structure body described in claim 20, it is characterized in that, is packaged with bio-related substance in liposome.

22. according to lipid membrane structure body described in claim 21, it is characterized in that, described bio-related substance is polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid.

23. state lipid membrane structure body according to claim 21, it is characterized in that, described bio-related substance is some release in pH≤6.8, the preferably some release in pH≤6.5, the more preferably some release in pH≤6.0.

The phospholipid derivative of 24. 1 kinds of branched polyethylene glycols, is characterized in that, the phospholipid derivative general formula of described branched polyethylene glycol as the formula (2):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, and 2≤n ₁+ n ₂≤ 2000; Y is carbon atom branching center or the N atom branching center with 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₄be connected, and Y is not the polyamino acid residue of 2～10 amino-acid residues; L ₁, L ₂independently of one another for connecting branching center Y and polyoxyethylene glycol unit or L ₄the bivalent hydrocarbon radical with 1 to 20 carbon atom of the ether that contains stable existence under illumination, enzyme, acidity or alkaline condition, thioether group, amide group, two key, Three key, secondary amine or tertiary amine groups; L ₄for linking group, be the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction, L ₄be selected from one of following group:

G is 0 or 1;

F is 2 to 10 integer;

M is hydrogen atom or positively charged ion;

R is hydrophobicity lipid residue, is selected from aliphatic hydrocarbon residue, DG lipid residue, monoacylglycerol lipid residue, the residue of a kind of or its compound lipid in the lipid residue that contains sphingosinols skeleton or steroid residue.

25. 1 kinds of liposomes that contain the phospholipid derivative of branched polyethylene glycol described in claim 24, is characterized in that, the phospholipid derivative of described branched polyethylene glycol accounts for 1%～30% of this liposome total mass, is preferably 3%-20%, more preferably 3%-15%.

26. according to liposome described in claim 25, it is characterized in that, it is packaged with one or more the bio-related substance such as polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid.

The phospholipid derivative of 27. 1 kinds of branched polyethylene glycols, is characterized in that, the phospholipid derivative general formula of described branched polyethylene glycol as the formula (3):

Wherein, X ₁, X ₂independently of one another for thering is the alkyl of 1 to 20 carbon atom; n ₁, n ₂be 1～1000 integer independently of one another, n ₃be 1～1000 integer, and 3≤n ₁+ n ₂+ n ₃≤ 2000; Y is carbon atom branching center or the nitrogen-atoms branching center with 1 to 20 atom, adopts covalent linkage and L ₁, L ₂, L ₃be connected; L ₁, L ₂, L ₃be the bivalent hydrocarbon radical with 1 to 20 carbon atom that connects the ether that contains stable existence under illumination, enzyme, acidity or alkaline condition, thioether group, amide group, two key, Three key, secondary amine or the tertiary amine groups of branching center Y and polyoxyethylene glycol unit independently of one another; L ₄for linking group, be the residue of polyethyleneglycol derivative and the formation of corresponding phosphatide cpd generation chemical reaction, L ₄be selected from one of following group:

G is 0 or 1;

F is 2 to 10 integer;

M is hydrogen atom or positively charged ion;

28. 1 kinds of liposomes that contain the phospholipid derivative of branched polyethylene glycol described in claim 27, is characterized in that, the phospholipid derivative of described branched polyethylene glycol accounts for 1%～30% of this liposome total mass, is preferably 3%-20%, more preferably 3%-15%.

29. according to liposome described in claim 28, it is characterized in that, it is packaged with one or more the bio-related substance such as polypeptide, protein, enzyme, small-molecule drug, nucleosides, Nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid.