CN103881084B

CN103881084B - The phospholipid derivant of a kind of branched polyethylene glycol and the lipid membrane structure body of composition thereof

Info

Publication number: CN103881084B
Application number: CN201410096439.9A
Authority: CN
Inventors: 翁文桂; 刘超; 廖金城; 闫策; 林毅
Original assignee: Xiamen Sinopeg Biotechnology Co Ltd
Current assignee: Xiamen Sinopeg Biotechnology Co Ltd
Priority date: 2014-03-14
Filing date: 2014-03-14
Publication date: 2016-09-28
Anticipated expiration: 2034-03-14
Also published as: CN105622925B; CN105622925A; CN103881084A

Abstract

The invention discloses the phospholipid derivant of a kind of branched polyethylene glycol and the lipid membrane structure body of composition thereof, its formula is as follows, wherein, and X₁、X₂It is each independently the alkyl of 1 20 carbon atoms；n₁、n₂It is each independently the integer of 1～1000, n₃It is the integer of 0～1000, and 2≤n₁+n₂+n₃≤2000；Y is branch centers, uses covalent bond and L₁、L₂、L₃It is connected；L₁、L₂、L₃Linking group for Y Yu Polyethylene Glycol unit；Q is 0 or 1；Work as n₃When=0, q is 0 and Y not to be the polyamino acid residues of 2～10 amino acid residues；L₄For linking group；M is hydrogen atom or cation；R is the residue of hydrophobic lipid.The lipid membrane structure body using the phospholipid derivant of described branched polyethylene glycol to be formed can effectively extend medicine circulation time in vivo for packaging medicine.

Description

The phospholipid derivant of a kind of branched polyethylene glycol and the lipid membrane structure body of composition thereof

Technical field

The present invention relates to Polymer Synthesizing field, particularly relate to phospholipid derivant and the group thereof of a kind of branched polyethylene glycol The lipid membrane structure body become.

Background technology

Liposome is that lipid (common for phospholipid, cholesterol) is dispersed in water a kind of of formation and encapsulates a part of aqueous phase Vesicle, profile be spherical, class is spherical, the shape such as oval, diameter is about tens nanometers to tens microns.Liposome For a kind of new drug carrier, its stability in blood is the key playing pharmaceutical carrier effect.At traditional liposome In preparation, when intravenously administrable, have that anelasticity in blood is poor, be prone to (following by the reticuloendothelial system such as liver, spleen Referred to as " the RES ") problem captured, utilize liposome as drug delivery system, beyond drug delivery to " RES " system Organ or make reagent be detained for a long time in blood Drug controlled release time, there is difficulty.In addition, blood has many Kind of destructive factor: high density lipoprotein (HDL) is the main component destroying liposome, HDL and liposome easily occur apoA-1 with The exchange of phospholipid, liposome membrane forms hole；Cause drug leakage and water, the entering in a large number of electrolyte, final osmotic lysis fat Plastid；Serum albumin is combined formation complex with liposomal phospholipids, reduces its stability；Phospholipase hydrolyzable phosphorus in blood Fat.These combined factors make a few minutes half-life only ten of traditional liposomal.

In order to extend the half-life of liposome, it is to avoid the capture of RES, currently mainly utilize glycolipid, glycoprotein or hydrophilic Property polymer-modified lipid surface, makes liposome have long circulating characteristic.At present, Polyethylene Glycol (PEG) coupling group are used Install on liposome membrane, the blood circulation time of liposome can be extended.Meanwhile, the molecular weight of Polyethylene Glycol is the biggest to liposome The prolongation effect of blood circulation time is the best.Affect pegylated liposomal extend blood circulation time because of have: 1. solid position Resistance: the conformation that Polyethylene Glycol extends in part at surface of liposome, this sterically hindered layer, just as one " brush ", will be close to Macromole or protein-lipid complex push away liposome, thus weaken the tune of the effect of various compositions, particularly plasma protein in blood Joint effect and RES picked-up effect subsequently, the exchange of lipoprotein simultaneously, the hydrolysis etc. of phospholipase are the most effectively suppressed.② Improve film surface hydrophilicity: Polyethylene Glycol has the longest polar group, can improve the hydrophilic of surface of liposome, thus improve Mononuclear phagocyte system (MPS) absorbs the energy barrier of destruction to it, has effectively organized surface of liposome and blood albumin Regulation effect, and reduce the liposome affinity interaction to MPS.It is generally believed that the sterically hindered and hydrophilic on raising film surface Two factors exist simultaneously, and both jointly act on and make PEGization liposome become a kind of long-acting liposome.

Utilize the fact that pH value is lower than normal surrounding tissue of tumor tissues, pH sensitive liposomes can be designed.It is former Reason is: may result in the protonation of fatty ester carboxyl during low pH and causes the formation of hexagonal crystal phase (non-phase layer joint), causes lipid The instability of body film, produces and assembles or merge, and the material of encapsulating is imported endochylema and active targeting pathological tissues, tumor tissues etc. Target site discharges the bio-related substance being encapsulated in liposome efficiently, avoids RES to remove and lysosomal degraded simultaneously, increases Add the tissue intake to medicine.

Summary of the invention

The goal of the invention of the present invention, is to more effectively extend medicine circulation time in vivo, it is provided that a kind of branched poly- The lipid derivate of ethylene glycol and the lipid membrane structure body of composition thereof.

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

A kind of phospholipid derivant of branched polyethylene glycol, its formula as the formula (1):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂It is each independently 1～1000 Integer, n₃It is the integer of 0～1000, and 2≤n₁+n₂+n₃≤2000；Y is branch centers, for nitrogen-atoms or have 1 to 20 Carbon atom, uses covalent bond and L₁、L₂、L₃It is connected；L₁、L₂、L₃Linking group for branch centers Y Yu Polyethylene Glycol unit；q It is 0 or 1；Work as n₃When=0, q is 0 and Y not to be the polyamino acid residues of 2～10 amino acid residues；L₄For linking group, it is poly- The residue that ethylene glycol derivative is formed with corresponding phosphatide cpd generation chemical reaction；M is hydrogen atom or cation；R is hydrophobic The residue of property lipid.

Above-mentioned n₁,n₂,n₃Preferably meet 1≤n₁≤ 200,1≤n₂≤ 200,0≤n₃≤ 200, and 2≤n₁+n₂+n₃≤200。

Work as n₃=0, q are 0 and Y not to be the polyamino acid residues of 2～10 amino acid residues, the most described branched polyethylene glycol Phospholipid derivant formula as the formula (2):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂It is each independently 1～1000 Integer, and 2≤n₁+n₂≤2000；Y is branch centers, for nitrogen-atoms or have 1 to 20 carbon atom, use covalent bond with L₁、L₂It is connected；L₁、L₂Linking group for branch centers Y Yu Polyethylene Glycol unit；L₄For linking group, spread out for Polyethylene Glycol The biological residue formed with corresponding phosphatide cpd generation chemical reaction；M is hydrogen atom or cation；R is for containing reactive base The residue of the hydrophobic lipid of group.

Work as n₃Be 1～1000, the phospholipid derivant formula of the most described branched polyethylene glycol as the formula (3):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂It is each independently 1～1000 Integer, n₃It is the integer of 1～1000, and 3≤n₁+n₂+n₃≤2000；Y is branch centers, for nitrogen-atoms or have 1 to 20 Carbon atom, uses covalent bond and L₁、L₂、L₃It is connected, and Y is not the polyamino acid residues of 2～10 amino acid residues；L₁、L₂、 L₃Linking group for branch centers Y Yu Polyethylene Glycol unit；Q is 0 or 1；L₄For linking group, for polyethyleneglycol derivative with The residue that corresponding phosphatide cpd generation chemical reaction is formed；M is hydrogen atom or cation；R is the residue of hydrophobic lipid.

Above-mentioned Y is carbon atom branch centers, and its representation is as follows:Wherein, R1 be hydrogen atom, have 1 to The alkyl of 20 carbon or containing the alkyl with 1 to 20 carbon containing heteroatom group.Described R1 is preferably hydrogen atom or for having There is the alkyl of 1 to 20 carbon, or be containing ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonate group, parahelium Base or the alkyl with 1 to 20 carbon of tertiary amino.

Above-mentioned Y is nitrogen-atoms branch centers, and its representation is as follows:

Above-mentioned R is the residue of hydrophobic lipid.

This hydrophobic lipid is not particularly limited, and can be containing in the middle of aliphatic hydrocarbon, glyceride, sphingolipid, steroid etc. Plant or any 2 kinds and complex lipid of more than two kinds.

When R is the residue of aliphatic hydrocarbon, and representation is as follows:R7 can be saturated or unsaturated, can be straight chain The structure such as shape, branched, ring-type, pectination, hyperbranched shape, dendroid.Its carbon number is 4～50.Residual for this aliphatic hydrocarbon Base is not particularly limited, and can come from butyl, the tert-butyl group, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, hendecane Base, dodecyl, tridecyl, myristyl, pentadecyl, cetyl, heptadecyl, octadecyl, isostearoyl base, Octadecylene base, 18 carbon dialkylenes, nonadecyl, eicosyl, docosyl, docosane thiazolinyl, tetracosyl, two Cetyl, octacosyl, melissyl, dotriacontyl etc..

For the residue of aliphatic hydrocarbon, corresponding aliphatic hydrocarbon is not particularly limited, and can be fatty acid, fatty amine, fatty alcohol And derivant, preferred fat alcohol and fatty acid, further preferred fatty alcohol.Such as, as aliphatic alcohol can be selected from butanol, The tert-butyl alcohol, amylalcohol, enanthol, 2-Ethylhexyl Alcohol, capryl alcohol, decanol, dodecanol, tetradecanol, hexadecanol, heptadecanol, ten Eight alkanols, oleic alcohol, 18 dienols, 18 enols, EICOSANOL, tadenan, docosene alcohol, lignocerane Alcohol, hexacosanol, octacosanol, triacontanol, n-Dotriacontanol etc., preferably corresponding aliphat single-alcohol.Such as, as Fatty acid can be selected from butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, Petiolus Trachycarpi Acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenyl Acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid etc., preferably corresponding aliphatic monocarboxylic acid.Such as, Butylamine, tert-butylamine, amylamine, heptyl amice, 2 ethyl hexylamine, octylame, decyl amine, dodecyl amine, the tetradecane can be selected from as fatty amine Amine, cetylamine, heptadecylamine (HDA), octadecylamine, octadecenyl amine, 18 enamines, 18 enamines, eicosane amine, 22 Alkanamine, docosene amine, lignocerane amine, hexacosane amine, octacosane amine, melissane amine, dotriacontane amine etc., preferably phase The aliphatic monoamine answered.

When R is the residue of glyceride, glyceride can be DG ester.Its representation is as follows:Phospholipid based on this glycerol lipid residue, as phosphoglyceride, can be PHOSPHATIDYL ETHANOLAMINE, phospholipid Acid, phosphatidyl glycerol, phosphatidyl aminoacid etc..In phosphoglyceride, the carbon number of multiple acyl groups is each independent, carbon number It is 4～50.This acyl group can be identical or different, can be saturated or unsaturated, can be straight-chain or branched.It addition, it is right The kind of acyl group is also not particularly limited, and generally can be preferably derived from the acyl group of fatty acid.Concrete example such as, butanoic acid, tertiary fourth Acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, different firmly Fat acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, 20 The acyl group of the fatty acid sources such as eight alkanoic acids, melissic acid, lacceroic acid.

When R is the residue of glyceride, and glyceride can also be monoacylglycerol ester, and its representation is as follows:Phospholipid based on this glycerol lipid residue is as lysophosphatide, present in it The carbon number of acyl group is 4～50.This acyl group can be saturated or unsaturated, can be straight-chain or branched.It addition, to acyl group Kind is also not particularly limited, and the acyl group that derive from fatty acid generally can be preferably used.Concrete example such as, butanoic acid, tertiary butanoic acid, Valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, different tristearin Acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, 28 The acyl group of the fatty acid sources such as alkanoic acid, melissic acid, lacceroic acid.

R can be from structureOr derivant of based on this structure, wherein R2, R3 are each independently and contain There is the alkyl of 4 to 50 carbon atoms.R preferably comprises sphingol skeleton.When R contains sphingol skeleton, its structure is represented byR can be the derivant from above-mentioned sphingol skeleton.When R is to have sphingol When skeleton or derivant based on this skeleton, R is sphingolipid residue.Sphingomyelins based on this sphingolipid residue can be sphingomyelin And derivant, such as ceramide phosphoethanolamine, phosphoglycerol ceramide, phosphoglycerol phosphate ester ceramide etc.. For the fatty acid being combined with sphingol, it can be butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, the moon Cinnamic acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, Semen arachidis hypogaeae Acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid etc..

When R is the residue of steroid, this steroid can be cholesterol, sitosterol, vitamin D, beta-cholestanol, Ergota Sterin, Pilus Caprae seu Ovis stay alcohol, bilichol, gonadal hormone etc. and derivant thereof.

Above-mentioned R₂、R₃、R₄、R₅、R₆、R₇Be preferably butyl, the tert-butyl group, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, Decyl, undecyl, dodecyl, tridecyl, myristyl, pentadecyl, cetyl, heptadecyl, octadecyl, Nonadecyl, eicosyl, (Z)-9-tetradecene base, (Z)-8-17 thiazolinyl, (Z)-12-two hendecene base, and at same point In son, can be the same or different.

The phospholipid formed based on hydrophobic lipid residue R can be natural phospholipid, it is also possible to for artificial synthetic phospholipid.Example As, natural phospholipid can be cephalin, lecithin, sphingomyelins, lysophosphatide etc., and synthetic phospholipid can be phosphatidyl ethanol Amine, such as DSPE, DPPE etc..

Described L₄It is not particularly limited, is preferred from

Wherein Z is not particularly limited, preferably alkylidene or containing ester group, urethane groups, amide groups, ether, thioether, double bond, The alkylidene of the groups such as three key, carbonate group, secondary amino group or tertiary amino；

G is 0 or 1；

F is the integer of 2 to 10.

Described X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom, preferably methyl, ethyl, propyl group, propylene Base, propinyl, isopropyl, butyl, the tert-butyl group, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, ten Dialkyl group, tridecyl, myristyl, pentadecyl, cetyl, heptadecyl, octadecyl, nonadecyl, eicosane Base, benzyl or butyl phenyl, and in same a part, X₁、X₂Can be the same or different.

Described L₁、L₂、L₃Be each independently containing the ether existed at illumination, enzyme, acidity or stable under alkaline conditions, The bivalent hydrocarbon radical with 1 to 20 carbon atom of the groups such as thioether group, amide groups, double bond, three key, secondary amino group or tertiary amino.

Described M is hydrogen atom or cation, preferably hydrogen atom, sodium ion or NH₄ ⁺。

In the phospholipid derivative of described branched polyethylene glycol, can be containing the residue of PHOSPHATIDYL ETHANOLAMINE, i.e. phosphatidyl ethanol The residue formed after amine and other reaction-ity group reaction.

Described PHOSPHATIDYL ETHANOLAMINE is not particularly limited, preferably DSPE, two oils and fats acyl group phosphorus Acyl ethanolamine, two myristoyl PHOSPHATIDYL ETHANOLAMINE, DPPE, two lauroyl phosphatidyl ethanol Amine, two Caulis et Folium Lini acyl PHOSPHATIDYL ETHANOLAMINE, two mustard acyl PHOSPHATIDYL ETHANOLAMINE or 1-palmityl-2-oleoylphosphatidyl ethanolamine.

A kind of lipid membrane structure body of the phospholipid derivant containing above-mentioned branched polyethylene glycol." lipid in the present invention Film structure " refer to that the hydrophilic group by amphiphilic lipid and/or its derivant contacts, with aqueous phase, the particle formed.Described lipid Film structure, can be hollow or solid closing form, it is also possible to for nonocclusive film form.When for hollow closing form Time, it is liposome (liposome), some documents are also referred to as lipid vesicle (lipid vesicle).

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

Described lipid membrane structure body, in addition to the phospholipid derivative that branched polyethylene glycol is modified, possibly together with unused Polyethylene Glycol The lipid components modified.The lipid components of described unmodified can be that the acyl compounds containing 4-50 carbon atom, fatty acyl are sweet The lipids such as grease, sphingolipid, phosphoglyceride, glycolipid, glyceroglycolipid, steroid.Can be natural grease matter or synthetic lipid.Institute State unused polyethyleneglycol modified lipid components, can be the one-component in above-mentioned lipid components, it is also possible to be two kinds or two Plant the mixing lipid of above lipid components.

Unused polyethyleneglycol modified lipid components, preferably PHOSPHATIDYL ETHANOLAMINE in described lipid membrane structure body.Described phosphorus The PHOSPHATIDYL ETHANOLAMINE of acyl ethanolamine preferred fatty race acyl group.Described aliphatic acyl radical is preferred from 8-24 carbon atom Acyl compounds, the acyl compounds of further preferred 8-24 carbon atom.Such as, described acyl group can be butanoic acid, tertiary butanoic acid, Valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, different tristearin Acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, 28 The acyl group of the fatty acid sources such as alkanoic acid, melissic acid, lacceroic acid, preferably octanoic acid, capric acid, lauric acid, myristic acid, Palmic acid, Heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, Erucic acid, lignoceric acid etc. source acyl group, further preferred lauric acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, different firmly The acyl in the source such as fat acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid Base.

When the unused polyethyleneglycol modified lipid components in described lipid membrane structure body contains PHOSPHATIDYL ETHANOLAMINE, with This is as the constituent of film, and described PHOSPHATIDYL ETHANOLAMINE can be illustrated such as DSPE, two oils and fats acyl groups PHOSPHATIDYL ETHANOLAMINE, two myristoyl PHOSPHATIDYL ETHANOLAMINE, DPPE, two lauroyl phosphatidyl second Hydramine, two Caulis et Folium Lini acyl PHOSPHATIDYL ETHANOLAMINE, two mustard acyl PHOSPHATIDYL ETHANOLAMINE or 1-palmityl-2-oleoylphosphatidyl ethanolamine.

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

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

Described lipid membrane structure body, packaged bio-related substance discharges when 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 encapsulate bio-related substance further.

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

In described lipid membrane structure body or described liposome encapsulation bio-related substance be not particularly limited, including but not It is limited to polypeptide, protein, enzyme, small-molecule drug, nucleoside, nucleotide, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal Compound, lipoid substance, glycolipid, glycoprotein, steroid.

The invention also discloses one and there is the phospholipid derivant of the branched polyethylene glycol shown in formula (2):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂It is each independently 1～1000 Integer, and 2≤n₁+n₂≤2000；Y is carbon atom branch centers or the atom N branch centers with 1 to 20 atom, uses Covalent bond and L₁、L₂、L₄It is connected, and Y is not the polyamino acid residues of 2～10 amino acid residues；L₁、L₂It is each independently Connect branch centers Y and Polyethylene Glycol unit containing at the ether of illumination, enzyme, acidity or stable under alkaline conditions existence, sulfur The bivalent hydrocarbon radical with 1 to 20 carbon atom of the groups such as ether, amide groups, double bond, key, secondary amine or tertiary amine groups；L₄For Linking group, the residue formed with corresponding phosphatide cpd generation chemical reaction for polyethyleneglycol derivative；L₄Limit the most especially Fixed, it is selected from one of following group:

Wherein, Z be alkylidene or containing ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonate group, The alkylidene of the group such as secondary amino group or tertiary amino；

G is 0 or 1；

F is the integer of 2 to 10；

M is hydrogen atom or cation；

R is hydrophobic lipid residue, is not particularly limited, and can be aliphatic hydrocarbon residue, DG lipid residue, single acyl A kind of or its complex lipid in base glycerol lipid residue, lipid residue containing sphingosinols skeleton or steroidal residues residual Base.

Also disclose the liposome of phospholipid derivant containing formula (2) described branched polyethylene glycol, described branched poly-second The phospholipid derivant of glycol accounts for the 1%～30% of this liposome gross mass, preferably 3%-20%, more preferably 3%-15%.

In liposome containing the phospholipid derivant of formula (2) described branched polyethylene glycol, it is packaged with biological correlative Matter.Described bio-related substance is not particularly limited, and includes but not limited to polypeptide, protein, enzyme, small-molecule drug, nucleoside, core Thuja acid, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid etc., It can be one or more bio-related substance above-mentioned.

The invention also discloses one and there is the phospholipid derivant of the branched polyethylene glycol shown in formula (3):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂It is each independently 1～1000 Integer, n₃It is the integer of 1～1000, and 3≤n₁+n₂+n₃≤2000；Y is that to have the carbon atom of 1 to 20 carbon atom branched Center or nitrogen-atoms branch centers, use covalent bond and L₁、L₂、L₃It is connected；L₁、L₂、L₃Be each independently connection branched in Heart Y and Polyethylene Glycol unit containing at the ether of illumination, enzyme, acidity or stable under alkaline conditions existence, thioether group, amide The bivalent hydrocarbon radical with 1 to 20 carbon atom of the groups such as base, double bond, three key, secondary amino group or tertiary amino；L₄For linking group, The residue formed with corresponding phosphatide cpd generation chemical reaction for polyethyleneglycol derivative,

Wherein, Z be alkylidene or containing ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonate group, Secondary amino group or the alkylidene of tertiary amino；

G is 0 or 1；

F is the integer of 2 to 10；

M is hydrogen atom or cation；

R is hydrophobic lipid residue, is not particularly limited, and can be aliphatic hydrocarbon residue, DG lipid residue, single A kind of or its complex lipid in acylglycerol lipid residue, lipid residue containing sphingosinols skeleton or steroidal residues Residue.

Also disclose the liposome of phospholipid derivant containing formula (3) described branched polyethylene glycol, described branched poly-second The phospholipid derivant of glycol accounts for the 1%～30% of this liposome gross mass, preferably 3%-20%, more preferably 3%-15%.

In liposome containing the phospholipid derivant of formula (3) described branched polyethylene glycol, it is packaged with biological correlative Matter.Described bio-related substance is not particularly limited, and includes but not limited to polypeptide, protein, enzyme, small-molecule drug, nucleoside, core Thuja acid, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycolipid, glycoprotein or steroid etc., It can be one or more bio-related substance above-mentioned.

Compared with prior art, there is advantages that

(1) present invention uses phospholipid to be attached with branched polyethylene glycol, the lipid derivate of described branched polyethylene glycol Still there is the characteristic preferably forming liposome；

(2) relative to the linear polyethylene glycol of same molecular amount, owing to having special molecular conformation, branched poly-second Glycol can form the protective layer of one layer of umbrella shape on the top layer of liposome, increases the sterically hindered of surrounding, than linear poly-second two Alcohol can more effectively stop the attack of other macromolecular complex confrontation medicine internal, decreases liposome by RES picked-up, by enzyme water Solve, more extend medicine action time in vivo, make the liposome of formation have the longer organism intracellular metabolite half-life；

(3) in the liposome reaching identical protected effect, the ratio of the lipid derivate of branched polyethylene glycol compares straight chain The liposome derivatives proportion of Pegylation is little, is more beneficial for the stability of the liposome formed, more effectively prevents institute The leakage of bag medicine carrying thing；

(4) in lipid membrane structure body of the present invention encapsulation bio-related substance low ph value (preferably pH≤6.8, more Preferably pH≤6.5, further preferred pH≤6.0) time release, the characteristic that this pH is sensitive so that lipid membrane structure body can select Property ground assemble release at the target site such as tumor, the passive targeting that medicine delivers can be strengthened, avoid RES to remove and lyase further The Degradation of body, increases the tissue intake to medicine, and then improves therapeutic effect, and reduce toxic and side effects.

Detailed description of the invention

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom, and can identical also may be used with a part With difference.Wherein including but not limited to methyl, ethyl, propyl group, acrylic, propinyl, isopropyl, butyl, the tert-butyl group, penta Base, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, myristyl, pentadecane Base, cetyl, heptadecyl, octadecyl, nonadecyl, eicosyl, benzyl or butyl phenyl；Preferably have 1 to The alkyl of 10 carbon atoms；More preferably there is the alkyl of 1 to 5 carbon atom；Described X₁、X₂Most preferably methyl.

Wherein, n₁、n₂Represent the degree of polymerization of two branched chain, independently be the integer of 1～1000, wherein, described n₁、n₂ It is preferably the integer of 10～800.Described n₁、n₂The integer of more preferably 10～200.Described n₁、n₂More preferably 10～25 whole Number.

Wherein, n₃Represent the degree of polymerization of main chain, be the integer of 0～1000, wherein, described n₃It is preferably 0 or 10～800 Integer.Described n₃More preferably 0 or the integer of 10～200.Wherein, described n₃More preferably 0 or the integer of 10～25.

Wherein, Y is branch centers, for nitrogen-atoms or have 1 to 20 carbon atom, uses covalent bond and L₁、L₂、L₃It is connected Connecing, the branched atom at its center can be carbon atom or nitrogen-atoms.When Y branch centers is carbon atom, its representation is as follows:Wherein, R₁The alkyl that for hydrogen atom, there is 1 to 20 carbon or 1 to 20 carbon that has containing heteroatom group Alkyl.R1 preferably hydrogen atom or be that there is the alkyl of 1 to 20 carbon, or be containing ester group, urethane groups, amide groups, ether, sulfur The alkyl with 1 to 20 carbon of ether, double bond, three key, carbonate group, secondary amino group or tertiary amino.R₁Structure limit the most especially Fixed, can be straight chain, side chain, ring-type or contain circulus.

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

Wherein, L₁、L₂、L₃For the linking group of branch centers Y Yu Polyethylene Glycol unit, it is not particularly limited.Described L₁、 L₂、L₃Can be straight chain or band branched group.Described L₁、L₂、L₃Preferably there is the alkyl of 1 to 20 carbon atom.

Wherein, described L₁、L₂、L₃Preferably can the group of stable existence, be preferably containing in illumination, enzyme, acid or alkaline Under the conditions of the bivalent hydrocarbon radical of the group such as the ether of stable existence, thioether group, amide groups, double bond, three key, secondary amino group or tertiary amino.

Wherein, L₄For linking group, formed for polyethyleneglycol derivative and corresponding phosphatide cpd generation chemical reaction Residue.L₄It is not particularly limited, is preferred from containing amino, ester group, carbonate group, triazole, isoxazole, ether, amide groups, Asia Amide groups, imido grpup, secondary amino group, tertiary amine groups, thioester substrate, thioether group, disulfide group, urethane groups, thiocarbonic acid ester group, sulphonic acid ester Base, sulfoamido, carbamate groups, tyrosine-based, cysteine base or the divalent alkyl of histidine base.

Wherein, L₄It is preferably

Wherein Z is alkylidene or containing ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonate group, secondary The alkylidene of the group such as amino or tertiary amino；

G is 0 or 1；

F is the integer of 2 to 10.

Wherein, M is hydrogen atom or cation；

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

Wherein, R is the residue of hydrophobic lipid.

Described hydrophobic lipid is not particularly limited, and can be that aliphatic hydrocarbon, glyceride, sphingolipid, glycolipid, glyceroglycolipid, class are solid One in the middle of alcohol etc. or its complex lipid.

When R is the residue of aliphatic hydrocarbon, can be saturated or unsaturated, can be straight-chain, branched, ring-type, pectination, super The structures such as branched shape, dendroid.Its carbon number is 4～50.Residue for this aliphatic hydrocarbon is not particularly limited, and can come from Butyl, the tert-butyl group, amyl group, heptyl, 2-ethylhexyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl, ten Tetraalkyl, pentadecyl, cetyl, heptadecyl, octadecyl, isostearoyl base, octadecylene base, 18 carbon dialkylenes, Nonadecyl, eicosyl, docosyl, docosane thiazolinyl, tetracosyl, cerul, octacosyl, three Ten alkyl, dotriacontyl etc..

For the residue of aliphatic hydrocarbon, corresponding aliphatic hydrocarbon is not particularly limited, such as, can be fatty alcohol, fatty acid, fat Fat amine and derivant thereof, preferred fat alcohol and fatty acid, further preferred fatty alcohol.Such as, can be selected from as aliphatic alcohol Butanol, the tert-butyl alcohol, amylalcohol, enanthol, 2-Ethylhexyl Alcohol, capryl alcohol, decanol, dodecanol, tetradecanol, hexadecanol, heptadecane Alcohol, octadecanol, oleic alcohol, 18 dienols, 18 enols, EICOSANOL, tadenan, docosene alcohol, two Tetradecanol, hexacosanol, octacosanol, triacontanol, n-Dotriacontanol etc., preferably corresponding aliphat single-alcohol.Example As, butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, lauric acid, Semen Myristicae can be selected from as fatty acid Acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic Acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid etc., preferably corresponding aliphatic list carboxylic Acid.Such as, butylamine, tert-butylamine, amylamine, heptyl amice, 2 ethyl hexylamine, octylame, decyl amine, dodecane can be selected from as fatty amine Amine, tetradecylamine, cetylamine, heptadecylamine (HDA), octadecylamine, octadecenyl amine, 18 enamines, 18 enamines, eicosane Amine, docosane amine, docosene amine, lignocerane amine, hexacosane amine, octacosane amine, melissane amine, dotriacontane Amine etc., preferably corresponding aliphatic monoamine.

For the residue of glyceride, glyceride can be DG fat.Phospholipid based on this glycerol lipid residue is made For phosphoglyceride, such as, can be PHOSPHATIDYL ETHANOLAMINE, phosphatidic acid, phosphatidyl glycerol, phosphatidyl aminoacid etc..In phosphoglyceride The carbon number of multiple acyl groups is each independent, and carbon number is 4～50.This acyl group can be identical or different, can be saturated Or unsaturated, can be straight-chain or branched.It addition, the kind of acyl group is also not particularly limited, generally can preferably make With the acyl group deriving from fatty acid.Concrete example such as, butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, the moon Cinnamic acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, Semen arachidis hypogaeae The fatty acid sources such as acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid Acyl group.

When R is the residue of glyceride, glyceride can also be monoacylglycerol fat.Phospholipid based on this glycerol lipid residue As lysophosphatide, present in it, the carbon number of acyl group is 4～50.This acyl group can be saturated or unsaturated, can be straight chain Shape or branched.It addition, the kind of acyl group is also not particularly limited, the acyl that derive from fatty acid generally can be preferably used Base.Concrete example such as, butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, lauric acid, myristic acid, Petiolus Trachycarpi Acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenyl The acyl group of the fatty acid sources such as acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid.

When R is the residue of sphingolipid, and this sphingolipid residue contains sphingol skeleton.Sphingomyelins based on this sphingolipid residue can be Sphingomyelin and derivant thereof, such as ceramide phosphoethanolamine, phosphoglycerol ceramide, phosphoglycerol phosphate ester god Through amide etc..For the fatty acid being combined with sphingol, it can be butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, pungent Acid, capric acid, lauric acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, Asia Fiber crops acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid etc..

When R is the residue of steroid, this steroid can be cholesterol, sitosterol, beta-cholestanol, ergosterol, sheep Hair stays alcohol, vitamin D, bilichol, gonadal hormone etc. and derivant thereof.Preferably cholesterol and derivant thereof.

The phospholipid formed based on this hydrophobic lipid residue can be natural phospholipid, it is also possible to for artificial synthetic phospholipid. Such as, natural phospholipid can be cephalin, lecithin, sphingomyelins, lysophosphatide etc., and synthetic phospholipid can be phosphatidyl second Hydramine, such as DSPE, DPPE etc..

Wherein, n is worked as₃Being 0, q is 0 and Y not to be the polyamino acid residues of 2～10 amino acid residues, the most described branched poly- The phospholipid derivant formula of ethylene glycol is as the formula (2):

Wherein, X₁、X₂、n₁、n₂、Y、L₁、L₂、L₄, M, R same as described above, repeat the most one by one at this.

Wherein, n is worked as₃Be 1～1000, the phospholipid derivant formula of the most described branched polyethylene glycol as the formula (3):

Wherein, wherein, X₁、X₂、n₁、n₂、Y、L₁、L₂、L₃、L₄, M, R same as described above, repeat the most one by one at this.

Preparation method:

The branched polyethylene glycol (1) of described single functionalization can be by polyethyleneglycol derivative (4) and phosphatide cpd (5) warp Cross one or multi-step reaction to obtain.

Wherein, wherein said R₈、R₉For 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 bond linking group between Polyethylene Glycol and functional groups, limits the most especially System；G is 0 or 1.Wherein, Z can be alkylidene or contain ester group, urethane groups, amide groups, ether, double bond, three key, carbonic ester The alkylidene of the groups such as base, secondary amino group or ammonia amido.Wherein, Z is preferably alkylidene or ether-containing key, amido link, the Asia of secondary amino group Alkyl.Described alkylidene be preferably methylene, 1,2-ethylidene, 1,3-propylidene, 1,2-propylidene, isopropylidene, butylidene, Pentylidene and hexylidene.

In above-mentioned class B, Y₂For have the alkyl of 1 to 10 carbon atom or include fluorine atom there is 1 to 10 carbon atom Alkyl.Wherein, described Y₂It is preferably methyl, ethyl, propyl group, isopropyl, butyl, the tert-butyl group, amyl group, hexyl, heptyl, pungent Base, nonyl, decyl, vinyl, phenyl, benzyl, p-methylphenyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-(trifluoro methoxy Base) phenyl.Wherein, described Y₂It 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 contributing to the induction of unsaturated bond electronics, conjugation i.e. Can.When Q is on ring, can be one or more.Described Q is preferably hydrogen atom, halogen, alkyl halide, alkoxyl, carbonylation Compound, nitro compound.Described Q is preferably hydrogen atom, fluorine atom, trifluoromethyl or methoxyl group.

In above-mentioned class G, described M₄It is the atom connecting Z on ring, described M₄Can be carbon atom or nitrogen-atoms.

L₄For R₈、R₉Reaction residue.

Illustrating the preparation method of the phospholipid derivant of the branched polyethylene glycol of the present invention, the present invention's is branched poly- The preparation method of the phospholipid derivant of ethylene glycol is not particularly limited, and can be carried out by following method as an example Preparation: the hydrophobic lipid residues such as aliphatic hydrocarbon containing reactive group, glyceride, sphingolipid, glycolipid, glyceroglycolipid, steroid Phosphatide cpd and the branched polyethylene glycol derivatives reaction containing reactive functional group.

1, L is worked as₄In containing amido link (-CONH-) time, can be by synthesizing in the following ways:

Phosphatide cpd or end that 1.1 polyethyleneglycol derivatives using end to contain amino contain carboxylic acid with end contain The polyethyleneglycol derivative having the phosphatide cpd of amino and end to contain carboxylic acid carries out condensation reaction and obtains.

Wherein, it is not specially limited condensing agent, but preferably N, N '-dicyclohexyl carbonyl diimine (DCC), 1-ethyl-(3-bis- Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC HCl), 2-(7-azo BTA)-N, N, N', N'-tetramethyl Urea hexafluorophosphoric acid ester (HATU), BTA-N, N, N', N'-tetramethylurea hexafluorophosphate (HBTU), most preferably DCC. And 1 to 20 times that consumption is carboxylic acid molar equivalent of general condensing agent, preferably 5-10 times, this react can add suitable Catalyst (such as 4-dimethylaminopyridine).

Solvent can be solvent-free or non-protonic solvent, and non-protonic solvent includes toluene, benzene, dimethylbenzene, acetonitrile, second Acetoacetic ester, ether, methyl tertiary butyl ether(MTBE), oxolane, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or diformazan Yl acetamide, preferably oxolane, dichloromethane, dimethyl sulfoxide, dimethylformamide.

Alkali includes that generally organic base is (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazoles or diisopropyl ethyl Amine), preferably triethylamine, pyridine.The consumption of alkali is 1 to 50 times of the molar equivalent of carboxylic acid, preferably 1 to 10 times, more preferably 2 To 3 times.

Reaction temperature is 0 to 200 DEG C, preferably 0 to 100 DEG C, more preferably 25 to 50 DEG C, and the response time is preferably 10 minutes To 48 hours, more preferably 30 minutes to 24 hours.The product obtained can pass through extraction, column chromatography, recrystallization, adsorption treatment, The purification process such as precipitation, anti-precipitation, thin film dialysis or supercritical extraction in addition purification.

The polyethyleneglycol derivative that 1.2 employing ends contain amino contains amino with carboxylic acid derivates or the end of phospholipid Phosphatide cpd and polyethylene carboxylic acid derivant carry out reaction and obtain.Wherein, carboxylic acid derivates is to react generation with amido The reactive intermediate of amido link, the preferably succinimide active ester of carboxylic acid halides, carboxylic acid.

General reactions solvent can be solvent-free or non-protonic solvent, and non-protonic solvent includes toluene, benzene, diformazan Benzene, acetonitrile, ethyl acetate, ether, methyl tertiary butyl ether(MTBE), oxolane, chloroform, dichloromethane, dimethyl sulfoxide, dimethyl methyl Amide or dimethyl acetylamide, preferably oxolane, dichloromethane, dimethyl sulfoxide, dimethylformamide.

Alkali includes that generally organic base is (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazoles or diisopropyl ethyl Amine), preferably triethylamine, pyridine.Consumption is carboxylic acid derivates 1 to 50 times of alkali, preferably 1 to 10 times, more preferably 2 to 3 Times.

Reaction temperature is 0 to 200 DEG C, preferably 0 to 100 DEG C, more preferably 25 to 80 DEG C, and the response time is preferably 10 minutes To 48 hours, more preferably 30 minutes to 24 hours.The product obtained can pass through extraction, column chromatography, recrystallization, adsorption treatment, The purification process such as precipitation, anti-precipitation, thin film dialysis or supercritical extraction in addition purification.

2, L is worked as₄In containing urethane bond (-OCONH-) time, can be by synthesizing in the following ways: use end to contain There are phosphatide cpd that the polyethyleneglycol derivative of amino and end contain activated carbon acid esters or the phospholipid that end contains amino The polyethyleneglycol derivative that compound and end contain activated carbon acid esters carries out condensation reaction and obtains.

Wherein active formate can be can to react with amino to obtain the derivant of urethane bond, includes but not limited to Succinimdyl carbonate (SC), paranitrophenol carbonic ester (NPC), 2,4,6-trichlorophenol, 2,4,6,-T carbonic esters, imidazoles carbonic ester, N- Hydroxy benzo triazole carbonic ester, preferably succinimdyl carbonate (SC), paranitrophenol carbonic ester (NPC).

3, L is worked as₄In containing ester bond (-OCO-) time, polyethyleneglycol derivative and end that end contains hydroxyl can be used The polyethyleneglycol derivative that the phosphatide cpd that phosphatide cpd containing carboxylic acid or end contain hydroxyl and end contain carboxylic acid Carry out condensation reaction to obtain.

Wherein, it is not specially limited condensing agent, but preferably N, N '-dicyclohexyl carbonyl diimine (DCC), 1-ethyl-(3-bis- Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC HCl), 2-(7-azo BTA)-N, N, N', N'-tetramethyl Urea hexafluorophosphoric acid ester (HATU), BTA-N, N, N', N'-tetramethylurea hexafluorophosphate (HBTU), most preferably DCC.And 1 to 20 times that consumption is carboxylic acid molar equivalent of general condensing agent, preferably 5-10 times, this react can add suitable When catalyst (such as 4-dimethylaminopyridine).

Reaction temperature is 0 to 200 DEG C, preferably 0 to 100 DEG C, more preferably 25 to 80 DEG C, and the response time is preferably 10 minutes To 48 hours, more preferably 30 minutes to 24 hours.The product obtained can pass through extraction, column chromatography, recrystallization, adsorption treatment, The purification process such as precipitation, anti-precipitation, thin film dialysis or supercritical extraction in addition purification.Work as L₄In containing secondary amine key (- CH₂NHCH₂-) time, can use phosphatide cpd that the polyethyleneglycol derivative that end contains aldehyde radical contains amino acid with end or The polyethyleneglycol derivative that the phosphatide cpd that end contains aldehyde radical and end contain amino acid carries out being condensed, reduction reaction obtains.

4, L is worked as₄In containing secondary amine key (-CH₂NHCH₂-) time, the polyethyleneglycol derivative that end contains aldehyde radical can be used The phosphatide cpd that the phosphatide cpd containing amino acid with end or end contain aldehyde radical and end contain the Polyethylene Glycol of amino acid Derivant carries out being condensed, reduction reaction obtains.

Reaction is typically carried out in buffer solution, and acetic acid buffer solution, phosphate buffer solution, Tris acid buffering are preferably used Solution, borate buffer solution etc., in addition to preferably hydrotropy, can also add further in reaction system and be not involved in reaction The organic solvents such as acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethyl acetylamide, the pH value of reaction is 2～8.5, is preferably 3～7.

Reaction temperature is 0 to 200 DEG C, preferably 0 to 100 DEG C, more preferably 25 to 80 DEG C, and the response time is preferably 10 minutes To 48 hours, more preferably 30 minutes to 24 hours.When there is not reducing agent, then form schiff bases.

Wherein reducing agent is not particularly limited, preferably sodium borohydride, lithium aluminium hydride reduction, sodium cyanoborohydride, lithium borohydride, boron Hydrofinings etc., more preferably sodium cyanoborohydride, the consumption of general sodium cyanoborohydride is 1-20 times of the amount of aldehyde radical material, preferably 3-5 times.

The product obtained can pass through extraction, column chromatography, recrystallization, adsorption treatment, precipitation, anti-precipitation, thin film dialysis or super The purification process such as critical extraction purification in addition.

5, L is worked as₄In containing thioether bond (> CHS-) time, polyethyleneglycol derivative and end that end contains sulfydryl can be used The phosphatide cpd holding the phosphatide cpd containing maleimide or end to contain sulfydryl contains maleimide with end Polyethyleneglycol derivative carries out being condensed, reduction reaction obtains.

Reaction temperature is 0 to 200 DEG C, preferably 0 to 100 DEG C, more preferably 25 to 80 DEG C, and the response time is preferably 10 points Clock to 48 hours, more preferably 30 minutes to 24 hours.

6, L is worked as₄In containing triazole group time, the polyethyleneglycol derivative containing alkynyl can be used to contain folded with end The polyethyleneglycol derivative that the phosphatide cpd of nitrogen or the phosphatide cpd containing alkynyl and end contain nitrine carries out reacting Arrive.

Reaction temperature is 0 to 200 DEG C, preferably 25 to 150 DEG C, wherein can use illumination, microwave, addition catalyst, add The methods such as heat promote the carrying out of reaction.The wherein preferred ultraviolet light of illumination, infrared light, far red light；Catalyst preferred monovalence copper Catalyst (I).Response time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.

" the lipid membrane structure body " of the present invention refers to be connect with aqueous phase by the hydrophilic group of amphiphilic lipid and/or its derivant Touch the particle formed.

Form for the lipid membrane structure of the present invention is not particularly limited.For example, it is possible to be hollow or solid closing Form, it is also possible to for nonocclusive film form.For example, it is possible to be liposome (or lipid vesicle), micelle, mixed micellazation, be combined The forms, preferably its form such as type Emulsion, layer structure thing are liposome.For example, it is possible to for drying regime, aqueous can be dispersed in In solvent, can also be for the state being further dried or freezing after being scattered in aqueous solvent.

For dry lipid mixture form, such as, lipid components first can be dissolved in organic solvent, then with cold Freezing seasoning, Rotary Evaporators hypobaric drying method or spray drying method to be dried, described organic solvent can be ether, trichlorine Methane etc..

For the form being scattered in aqueous solvent, can be vesicle or liposome, micelle, mixed micellazation, composite milk Agent, layer structure thing etc., preferred liposome, further preferred unilamelar liposome.

For the state freezed further after being scattered in aqueous phase, the decompression of freeze-drying, Rotary Evaporators can be used dry The methods such as dry method or spray drying method are dried.For the state freezed further after being scattered in aqueous solvent, can be at solvent Preserve below freezing point, preferably-20 DEG C and less, more preferably-80 DEG C and less, more preferably liquid nitrogen preserves.

For the lipid membrane structure body of the present invention, in addition to the phospholipid derivative that branched polyethylene glycol is modified, possibly together with unused Polyethyleneglycol modified lipid components.

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

The lipid components of described unmodified can be the acyl compounds containing 4-50 carbon atom, acylglycerol ester, sheath Fat, phosphoglyceride, glycolipid, glyceroglycolipid, steroid etc..Can be natural grease matter or synthetic lipid.Described unused poly-second The lipid components that glycol is modified, can be the one-component in above-mentioned lipid components, it is also possible to for two or more lipid The mixing lipid of composition.

For the described acyl compounds containing 4-50 carbon atom, can be saturated or unsaturated.Its structure is the most especially Limit, such as, can be the structures such as straight-chain, branched, ring-type, pectination, hyperbranched shape, dendroid.

For described acylglycerol ester, can be monoglyceride or diacylglycerol (DGDG) or triglyceride, preferably three Acyl glyceride, i.e. triglyceride.

For described sphingolipid, there is sphingol skeleton, such as, can be sphingomyelins, glycosphingolipid etc..Preferably sphingomyelins.

For described phosphoglyceride, can be DG phospholipid, it is also possible to for mono acyl glycero phospholipid.The most permissible For cephalin, lecithin, lysophosphatide, PHOSPHATIDYL ETHANOLAMINE, phosphatidyl glycerol, phosphatidyl aminoacid etc..Preferably lecithin, phosphorus Acyl ethanolamine etc..

Above-mentioned acyl compounds containing 4-50 carbon atom, acylglycerol ester, sphingolipid, glyceride, phosphoglyceride, phospholipid Acyl group in the structures such as acyl ethanolamine, the acyl compounds of preferably 8-24 carbon atom, further preferred 8-24 carbon atom Acyl compounds.Such as, described acyl group can be butanoic acid, tertiary butanoic acid, valeric acid, enanthic acid, 2 ethyl hexanoic acid, octanoic acid, capric acid, Laurel Acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, The acyl of the fatty acid sources such as arachidonic acid, behenic acid, erucic acid, lignoceric acid, cerinic acid, octocosoic acid, melissic acid, lacceroic acid Base, preferably octanoic acid, capric acid, lauric acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, The acyl group in the source such as linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, erucic acid, lignoceric acid, further preferred Laurel Acid, myristic acid, Palmic acid, heptadecanoic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidic acid, The acyl group in the source such as arachidonic acid, behenic acid, erucic acid, lignoceric acid.

For described steroid, can be such as cholesterol, sitosterol, beta-cholestanol, ergosterol, Pilus Caprae seu Ovis stay alcohol, Vitamin D, bilichol, gonadal hormone etc. and derivant thereof.Preferably cholesterol and derivant thereof, be typically used as membrane stabilizer, such as Cholesteryl hemisuccinate etc..Cholesteryl hemisuccinate is a kind of adjuvant with pH sensitivity, under the conditions of neutrality and alkalescence, There is the effect of stabilized liposome membrane structure, play slow release effect, and in acid condition, such as pH≤6.8, more preferably pH≤6.5, Further preferably pH≤6.0, then promote the release of medicine, and demonstrating dashes forward releases effect, thus can be used to increase drug on tumor etc. The selectivity of target site, intensifier target tropism, improves curative effect.

For described natural lipid, such as, can be cephalin, lecithin, lysophosphatide, sphingomyelins, soybean phospholipid phosphatidyl second Hydramine, lecithin acyl ethanolamine, cholesterol etc..For described synthetic lipid, such as, can be phosphatidyl glycerol, containing sheath The phospholipid of ammonia ol skeleton, phosphatidyl aminoacid etc..Preferably phosphatidyl glycerol, such as, can be PHOSPHATIDYL ETHANOLAMINE, such as distearyl Acylphosphatidyl ethanolamine, DPPE, N-glutaryl-PHOSPHATIDYL ETHANOLAMINE, hydrogen addition soybean phospholipid Acyl ethanolamine, hydrogenation lecithin acyl ethanolamine etc..

For the mixing lipid of the two or two or more lipid components, such as, can be lecithin/cholesterol/phospholipid The combination of acyl 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.With dynamic light scattering Illustrate as a example by the particle diameter that method measures.Such as, during for liposome or emulsion, particle size range is preferably 20nm～10 μm, more excellent Select 50nm～5 μm.Such as, when for micellar conformation, particle size range is preferably 5nm～150nm, more preferably 5nm～100nm.Example As, when for layer structure thing, thickness in monolayer is preferably 5nm～15nm, more preferably 5nm～10nm.

Kind for aqueous solvent is not particularly limited, as long as being spread out by the phospholipid that the branched polyethylene glycol of the present invention is modified Stable lipid membrane structure body can be obtained when biology is dispersed therein.Can be such as Tris buffer, phosphate buffer, Fructus Citri Limoniae Acid buffer, borate buffer, phosphate buffer normal saline, normal saline, cell cultivation culture medium etc., additionally can add Enter the amino acid solutions such as polyhydric alcohol, L-glutaminate such as the sugar aqueous solutions such as glucose, lactose, sucrose, glycerol, propylene glycol, can It is dissolved in chemokines therein etc..When using the form being scattered in aqueous solvent to preserve, for physical stability etc. The consideration of aspect, in order to preserve steadily in the long term, reduce the electrolyte in aqueous solvent preferably as far as possible, and removes molten as far as possible Solve oxygen, such as, can use nitrogen foam-forming method.When using drying regime to preserve, preferably to sugar aqueous solution or polyatomic alcohol water solution Scattered lipid membrane structure body is dried, and can use freeze-drying, spray drying method etc., can preserve for a long time.

The concentration of aqueous solvent is not particularly limited.Such as, for sugar aqueous solution, preferably 2%～15% (w/v), enters One step is preferably 5%～10% (w/v).For polyhydric alcohol solutions, preferably 1%～8% (w/v), more preferably 2%～3% (w/ v).For buffer solution, preferably 5mM～50mM, more preferably 10mM～25mM.

The concentration of lipid membrane structure body in aqueous solvent is not particularly limited, through the phospholipid that branched polyethylene glycol is modified Derivant and the most modified lipid components, be referred to as lipid mixture, and its total concentration is preferably 0.1mM～500mM, more preferably For 1mM～200mM, more preferably 2mM～100mM.

Acquisition pattern lipid membrane structure body being scattered in aqueous solvent is not particularly limited.Such as can be by by upper Stating dry lipid mixture and join in aqueous solvent, then carry out emulsifying and prepare, emulsification method can be ultrasonic method, even The modes such as pulp grinder method, high-pressure injection mulser method.

Lipid membrane structure body is scattered in the form of aqueous solvent, is not particularly limited, can be such as liposome (or Lipid vesicle), micelle, mixed micellazation, multiple emulsion, the form, preferably its form such as layer structure thing be liposome.

The described liposome being scattered in aqueous solvent, its preparation method is not particularly limited, such as, thin film can be used to disperse Method, reverse phase evaporation, solvent injection method, lyophilization, second emulsifying method, extrusion molding etc..When using extrusion molding, be conducive to obtaining Little and there is the liposome of uniform particle size.

For the method lipid membrane structure body or liposome that are scattered in aqueous solvent being dried further, there is no spy Do not limit, such as, can use freeze-drying or spray drying method.Corresponding aqueous solvent, as set forth above, it is possible to use sugar Aqueous solution or polyatomic alcohol water solution, wherein sugar aqueous solution preferably sucrose aqueous solution, lactose aqueous solution.To being scattered in aqueous solvent Lipid membrane structure body is dried, and can preserve for a long time.Additionally, add biological correlative in this lipid membrane structure body being dried The aqueous solution of matter, can realize the efficient hydration of lipid mixture, bioactive substance can be made to be held in lipid membrane structure body side Face, has good efficiency.

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

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

Described lipid membrane structure body and liposome encapsulation bio-related substance method be not particularly limited, including but do not limit Complete due to the formation of well-known method for packing, such as liposome and the same step that is loaded in of bio-related substance, right Should passive encapsulation technology, the most also can be initially formed blank liposome, then by specific gradient (such as pH gradient, or ammonium sulfate Gradient) realize the loading of bio-related substance, corresponding actively encapsulation technology.

Described passive encapsulation technology is not particularly limited, such as, and can be based on dry lipid membrane structure body or dried powder, example Such as film dispersion method, organic solvent lyophilization, spray drying method, fluidized bed coating, single phase soln lyophilization etc.；Can be based on breast Agent, such as reverse phase evaporation, second emulsifying method etc.；Can be based on mixed micellazation, such as cross-flow dialysis method etc.；Can be based on organic molten Agent (such as ethanol), phospholipid or phospholipid derivative, water three-phase mixture, such as alcohol injection, the technology of preparing of Alza company, friendship Distributary injection technique etc..

Described active encapsulation technology is not particularly limited.Generally can take following steps: the preparation of (1) blank liposome； (2) creation of specific gradient, can be realized by modes such as dialysis, column chromatographies；(3) at a suitable temperature, by shape inside and outside film The blank liposome becoming gradient is hatched with bio-related substance to be encapsulated, to complete to load.Such as, pH value gradient method, sulphuric acid The pH value gradient method etc. of the outer acid of ammonium gradient method, Calcium acetate gradient, interior alkali.

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

For constituting phospholipid used in the unused polyethyleneglycol modified lipid components of lipid membrane structure body and liposome Composition, such as sphingomyelins, phosphoglyceride, phosphatidyl aminoacid etc., when using acidic phospholipid, at low ph values, due to proton Change and cause lipid film surface charge to disappear and become unstable, thus give lipid membrane structure body pH sensitivity, can be at target spot The bio-related substance that position especially tumor locus encapsulates in being efficiently released in lipid membrane structure body.Such as, for hydrophilic portion Point containing the acidic phospholipid of terminal carboxyl group, when pH≤6.8, preferably pH≤6.5, more preferably during pH≤6.0, lipid membrane structure body by Instability is caused in protonation.Described acidic phospholipid, can be reacted with carboxylic acid anhydrides by corresponding phosphatide cpd and be prepared. Described acidic phospholipid preferably glycerine phospholipid, more preferably DOPE (DOPE).

For constituting the phospholipid derivative that the branched polyethylene glycol of lipid membrane structure body and liposome is modified, work as L₄Fracture is led After causing branched polyethylene glycol disengaging, if the phosphatide cpd generated is acidic phospholipid, it is possible to so that lipid membrane structure body has pH Sensitivity.Such as, for using the carboxylic phosphatide cpd of end to spread out containing the branched polyethylene glycol of amino or hydroxyl with end The pegylated lipids derivant that biological respinse generates, after branched polyethylene glycol is partially disengaged, due to hydrophilic protective layer Disappearing, lipid membrane structure surface produces exposed carboxyl, thus gives its pH sensitivity.

For without polyethyleneglycol modified lipid components, when using phosphoglyceride, particularly dioleoyl phosphatidyl second During hydramine, the phospholipid derivative that branched polyethylene glycol is modified, corresponding DOPE is preferably used and makees Phospholipid derivative for branched polyethylene glycol modification prepared by raw material.The main composition composition of liposome is dioleoyl phosphatidyl During ethanolamine, DOPE is under conditions of low pH, such as below pH6.8, preferably below pH6.5, more preferably Below pH6.0 is easily formed hexagonal phase or reversed phase micelle structure, occurs to assemble or merge, at the protection pull-up of branched polyethylene glycol From rear, lipid membrane structure body is changed into hexagonal phase and discharges medicine, reflects pH response.

For the liposome in aqueous solvent, its phase transition temperature does not has specific restriction, described phase transition temperature i.e. lipid The temperature of body film generation gel-liquid crystal phase transition.When the phase transition temperature of described liposome is higher than normal physiological body temperature, preferably 39- When 42 DEG C, neoplastic fevers therapy can be used, utilize physical energy to precipitate in the tissue and produce heat effect, make tumor tissues temperature Rise on phase transition temperature, promote lipid bilayer to be changed to liquid crystal state by gel state so that liposome membrane permeability increases Add, and then efficiently discharge medicine at tumor locus, kill cancerous cell and do not damage normal structure, strengthen passive target effect.System The preferred reverse phase evaporation of method of standby temperature sensitivity liposome.It is main for typically containing dipalmitoyl phosphatidyl choline (DPPC) Film material, corresponding thermotherapy temperature 40～43 DEG C.Can also add appropriate lysophosphatide MPPC (MPPC) or MSPC (MSPC) so that phase change temperature of liposome slight decrease, preparation is more suitable for the gentle heat of clinic Treat temperature 39～42 DEG C.

The described bio-related substance being encapsulated in lipid membrane structure body and liposome, is not particularly limited its hydrophilic and hydrophobic, Can be fat-soluble or lipotropy, it is also possible to for water solublity.

The described bio-related substance being encapsulated in lipid membrane structure body and liposome, is not particularly limited its kind, bag Include bioactive substance and modified bioactive substance, specifically include but be not limited only to following material: polypeptide, protein, enzyme, Small-molecule drug, gene-correlation material (nucleoside, nucleotide, oligonucleotide, polynucleotide, nucleic acid), polysaccharide, steroid, steroidal Compound, 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 pituitary hormone, thyroxin, male swash Element, estrogen and epinephrine etc.；Serum albumin, such as hemoglobin and blood factor etc.；Immunoglobulin, as IgG, IgE, IgM, IgA and IgD etc.；Cytokine, such as interleukin, interferon, granulocyte colony-stimulating factor, macrophage colony Stimulating factor, granulocyte-macrophage colony stimutaing factor, platelet derived growth factor, phospholipase activating proteins, insulin, Glucagon, glucagon-like peptide and the like, agglutinin, ricin, tumor necrosis factor, epidermal growth The factor, VEGF, nerve growth factor, skeletal growth factor, insulin like growth factor, Heparin-Binding Growth The factor, tumor growth factor, glial cell line derived neurotrophic factor, the macrophage differentiation factor, differentiation inducing factor, white blood Sick inhibitive factor, double regulin, somatomedin, erythropoietin, blood cell development element, blood platelet auxin and Calcitonin；Enzyme, such as proteolytic enzyme, oxidoreductase, transferring enzyme, hydrolytic enzyme, lyases, isomerase, ligase, Radix Asparagi amine Enzyme, arginase, arginine deaminase, ADA Adenosine deaminase, superoxide dismutase, endotoxin enzyme, catalase, rotten albumen Enzyme, lipase, uricase, Elastase, streptokinase, urokinase, prourokinase, adenosine deaminase, tryrosinase, bilirubin oxygen Change enzyme, glucoseoxidase, glucose enzyme and glucuronide enzyme, Defibrase；Monoclonal or polyclonal antibody and fragment thereof；Many Poly-propylhomoserin, such as polylysine, poly-D-Lys etc.；Vaccine, antigen and virus, such as hepatitis B vaccine, malaria vaccine, black Element tumor vaccine, HIV-1 vaccine etc..

(2) gene-correlation material

Gene-correlation material is not particularly limited, and can be listed below: nucleoside, nucleotide, oligonucleotide, polynucleotide, core Acid, DNA, RNA etc..

(3) small-molecule drug

Small-molecule drug is not particularly limited, the most anticancer or antitumor drug and antifungal drug.Anticancer or antitumor Medicine, preferably paclitaxel and derivant thereof, amycin or doxorubicin hydrochloride, daunorubicin, cisplatin, daunomycin, mitomycin, Vincristine, vinorelbine, epirubicin, methotrexate, 5-fluorouracil, aklavine, Yi Da mycin, bleomycin, Pirarubicin, peplomycin, vancomycin, amikacin, camptothecin analogues, hydroxy camptothecin, irinotecan, SN38, topotecan hydrochloride, oxaliplatin, mitoxantrone, all-trans retinoic acid, cytosine arabinoside etc..Antifungal drug, preferably two Property mycin B, gentamycin, nystatin, fluorocytosin, miconazole, fluconazol, itraconazole, ketoconazole and polypeptide resist Fungi-medicine.

(4) vitamin

Vitamin be humans and animals be to maintain normal physiological function and the class trace that must obtain from food is organic Material, plays an important role in growth in humans, metabolism, growth course.It is specifically including but not limited to vitamin A, dimension life Element B, vitamin C, vitamin E and vitamin K etc..

(5) saccharide

Saccharide is to constitute cell and the main component of organ, is not particularly limited, mainly includes glycolipid, glycoprotein, glycogen Deng.Glycolipid is distributed more widely at organism, mainly comprises glycosyl acyl glycerol and the big class of glycosphingolipid two, specifically comprises ceramide, brain Glycosides fat, sphingol, ganglioside and glyceryl glycolipid etc.；Glycoprotein is that the oligonucleotide chain of branch is connected with polypeptid covalence institute's structure Become glycoconjugates, be generally secreted in body fluid or the constituent of memebrane protein, specifically include transferrins, Ceruloplasmin, Embrane-associated protein, histocompatibility antigen, hormone, carrier, agglutinin and antibody.

(6) lipid

Lipid mainly includes oils and fats and the big class of lipoid two.Wherein, the composition of fatty acid is not particularly limited, but preferably has The fatty acid of 12 to 24 carbon atoms, and fatty acid can be satisfied fatty acid or unsaturated fatty acid.Lipoid includes glycolipid, phosphorus Fat, cholesteryl ester, wherein, phospholipid can be natural phospholipid material such as egg yolk, Semen sojae atricolor etc., can be maybe the Phosphation of synthesis Compound, preferably phosphatidic acid, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, cuorin, Phosphatidylserine, phosphatidylinositols and molten Blood phosphoglyceride isomer.The materials such as cholesterol and steroid (steroid) maintain normal metabolism for organism And reproductive process, play important regulation effect, mainly include cholesterol, cholic acid, gonadal hormone and vitamin D etc..

(7) other

The bio-related substance being encapsulated in lipid membrane structure body and liposome be not limited to disease treatment purposes it can also be used to Including the bio-related substance for other purposes such as biological diagnosis, detection.Such as the dyestuff quantitatively or semi-quantitatively analyzed Molecule；Such as can be used for the fluorine carbon molecule etc. of the purposes such as angiographic diagnosis, blood substitute；Such as anti-parasite medicine such as primaquine Deng；Such as can be used as the carrier of antidote, such as chelating agen ethylenediaminetetraacetic acid (EDTA), Pentetic Acid (DTPA) etc.

The bio-related substance etc. known to this skilled person such as liposome, cell, micelle.

And contain the lipid derivate of a kind of branched polyethylene glycol of the present invention below in conjunction with some detailed description of the invention The lipid membrane structure body having this derivant is described further.Specific embodiment is for further describing the present invention, non-limiting Protection scope of the present invention.

Embodiment 1:L₄In containing the preparation of amido link

A, method of condensing

L₄In containing the synthesis of amido link compound (A1-1), wherein L₁=L₂=L₃=CH₂, Y=CH, X₁=X₂=CH₃, q=1, R are Dilauroyl glyceride residue, molecular weight polyethylene glycol is about 2000.

40g branched polyethylene glycol acetic acid and DLPE (13.9 grams, 1.2 equivalents) is added in the 1L round-bottomed flask of dried and clean After, nitrogen is protected, and after adding methylene chloride (500mL), after 10min is stirred at room temperature, is sequentially added into 20mL triethylamine and 20g The dichloromethane solution of dicyclohexylcarbodiimide (DCC), after reacting 24 hours, is filtered to remove insoluble matter under room temperature, concentrates, different Propanol recrystallization, obtains in L4 containing amido link compound (A1-1).

The hydrogen modal data of compound A1-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.65(CH₃(CH₂)₉-), 2.32-2.51 (- CH₂CH₂COO-,-CH(CH₂)₃-), 3.35 (CH₃O-), 3.40-3.80 (-CH₂CH₂O-,-CHCH₂O-,-OCH₂CH₂NH-), 4.05-4.42(-OCH(CH₂)CH₂O-,-OCH₂CH₂NH-,-OCH₂CONH-), 5.16(-OCH (CH₂)CH₂O-), 8.38(- NHCO-).

It is similar to, L₄In containing the synthesis of amide compound (A1-2), wherein L₁=L₂=L₃=CH₂,X₁=X₂=CH₃, Y is C- Bn, q=0,

The preparation method of A1-2 is identical with the preparation method of A1-1, repeats the most one by one at this；

The hydrogen modal data of compound A1-2 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.85-1.62(-CH₃,-CH₂-,-CH-), 2.15-2.19(-CH₂C=CH-,-C= CHCH₂-,-CCH₂CONH-), 2.62(-CH₂Ph), 3.35 (CH₃O-), 3.40-3.80 (-CH₂CH₂O-,-CHO-,- OCH₂CH₂NH-), 4.05-4.10 (-OCH₂CH₂NH-), 5.42 (-C=CHCH2-), 7.27-7.42(-CH₂C₆H₅).

B, the method for active ester

a.L₄In containing the synthesis of amido link compound (A1-3), wherein L₁=CH₂CH₂,L₂=COCH₂, L₃=CH₂CH₂, X₁=X₂ =CH₃, Y is N, and q=1, R are 1-palmityl-2-oleoyl glycerol ester residue, and molecular weight polyethylene glycol is about 4000.

After adding branched polyethylene glycol propylamine (40g) in the 1L round-bottomed flask of dried and clean, nitrogen is protected, and adds solvent After dichloromethane (500mL) dissolves, after 10min is stirred at room temperature, it is sequentially added into 20mL triethylamine and 1-palmityl-2-oleoyl The dichloromethane solution of base PHOSPHATIDYL ETHANOLAMINE valeric acid succinimide active ester (10.6 grams, 1.2 equivalents), reacts 24 under room temperature After hour, concentrate, recrystallisation from isopropanol, obtain in L4 containing amido link compound (A1-2).

The hydrogen modal data of compound A1-3 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.73(-CH₂-,-NHCH₂CH₂CH₂-), 2.15- 2.19(-CH₂CH=CHCH₂-) 2.32-2.41 (CH₂CON-, CH₂COO-), 3.10 (-NHCH₂CH₂CH₂-),3.35(CH₃O-), 3.40-3.80(-CH₂CH₂O-,-CHCH₂O-,-OCH₂CH₂NH-), 4.05-4.42 (-OCH₂CH-,-OCH₂CH₂NH-), 5.16(- OCH(CH₂)CH₂O-), 5.42-5.83(-CH₂CH=CHCH₂-, 8.38(-NHCO-).

b.L₄In containing the synthesis of amido link compound (A1-4), wherein L₁=L₂=CH₂, L₃=CH₂CH₂, Y=N, X₁=X₂= CH₃, q=0, R contain stearyl and sphingol skeleton, and molecular weight polyethylene glycol is about 2000.

40g branched polyethylene glycol propionic acid succinimidyl and sphingomyelins is added in the 1L round-bottomed flask of dried and clean After (16.4 grams, 1.2 equivalents), nitrogen is protected, and after adding solvent anhydrous methylene chloride (500mL), room temperature reaction is after 24 hours, dense Contracting, recrystallisation from isopropanol, obtain in L4 containing amido link compound (A1-3).

The hydrogen modal data of compound A1-4 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.65(CH₃(CH₂)-), 2.05-2.25(-CH= CHCH₂-,-NHCOCH₂-), 2.70-2.80 (-NCH₂CH₂O-), 3.40-3.80 (-CH₂CH₂O-.-NHCHCH₂O-,- NHCH₂CH₂O-), 4.05-4.42 (-CH=CHCH₂O-,-OCH₂CHNH-), 5.42-5.83(-CH₂CH=CHCH₂-), 8.42(- NHCO-).

Embodiment 2:L₄In containing the preparation of amino-formate bond

A, active ester synthetic method:

L₄In containing the synthesis of carbamate key compound (A2-1), wherein L₁=L₂=CH₂CH₂, L₃=CH₂CH₂, X₁=X₂ =CH₃, Y is N, and q=1, R are 1-stearyl-2-hydroxyl glyceride residue, and molecular weight polyethylene glycol is about 1000.

Branched polyethylene glycol carbonic acid succinimide active ester (20g) is added in the 500mL round-bottomed flask of dried and clean After 1-stearyl-2-hydroxyl glyceryl phosphatidyl ethanolamine (11.6 grams, 1.2 equivalents), nitrogen is protected, and adds anhydrous dichloro After methane (250mL), after 10min is stirred at room temperature, add 5mL triethylamine, after reacting 24 hours under room temperature, concentrate, isopropanol weight Crystallization, obtains in L4 containing amino original acid ester key compound (A2-1).

The hydrogen modal data of compound A2-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.65(CH₃(CH₂)₁₆-), 2.32-2.41 (- CH₂CH₂COO-), 2.70-2.80 (-NCH₂CH₂O-), 3.35 (CH₃O-), 3.40-3.80 (-CH₂CH₂O-,-NCH₂CH₂O-), 4.05-4.42(-OCH₂CH-,-OCH (CH₂)CH₂O-,-OCH₂CH₂NH-), 5.16(-OCH (CH₂)CH₂O-), 8.38(- NHCO-).

B, one-step synthesis

In this example, L₄In containing the synthesis of carbamate key compound (A2-2), wherein L₁=CH₂CH₂,L₂= CH₂CH₂C ≡ C, L₃=CH₂CH₂CH₂,X₁=n-C₂₀H₄₁,X₂=CH₃, Y is N, and q=1, R are dioleoyl glyceride residue.Total molecule Amount is about 3000, wherein L₁、L₂The molecular weight of two branched chain connected about is respectively 1000,1000；The molecular weight of main chain is about 1000。

After adding branched polyethylene glycol (30g) in the 500mL round-bottomed flask of dried and clean, use anhydrous methylene chloride (250mL) dissolve, under ice bath, slowly at a low price DSC(2.8 gram, 1.1 equivalents) acetonitrile solution, be slowly raised to room temperature, react 8 After hour, after adding 3mL triethylamine, after being slowly added dropwise the chloroformic solution of DOPE (11 grams, 1.5 equivalents), After reacting 24 hours under room temperature, concentrate, recrystallisation from isopropanol, obtain L₄In containing amino original acid ester key compound (A2-2).

The hydrogen modal data of compound A2-2 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.73(-CH₂-,-NCH₂CH₂CH₂-), 2.15- 2.19(-CH₂CH=CHCH₂-,-CH₂C ≡ C-), 2.32-2.84 (-CH₂COO-,-NCH₂CH₂-), 3.35 (CH₃O-), 3.40- 3.80(-CH₂CH₂O-,-OCH₂CH₂NH-), 4.05-4.42 (-OCH₂CH-,-OCH (CH₂)CH₂O-,-OCH₂CH₂NH-), 5.16 (-OCH (CH₂)CH₂O-), 5.42-5.83(-CH₂CH=CHCH₂-), 8.38(-NHCO-).

Embodiment 3:L₄In containing the preparation of ester bond (-OCO-)

A:L₄In containing the synthesis of ester bond compound (A3-1), wherein L₁=L₂=L₃=CH₂,X₁=X₂=CH₃, Y is CH, q=1, R It it is the residue of two Semen Myristicae phosphatidyl glycerol esters.Design total molecular weight is about 1500.

Branched polyethylene glycol acetic acid (30g) and two Semen Myristicae phosphatidyl glycerols are added in the 1L round-bottomed flask of dried and clean After (66.6 grams, 5 equivalents), nitrogen is protected, and after adding methylene chloride (500mL), after 10min is stirred at room temperature, is sequentially added into 20mL triethylamine and the dichloromethane solution of 20g dicyclohexylcarbodiimide (DCC), after reacting 24 hours, cross and filter under room temperature Remove insoluble matter, concentrate, with water dissolution, filter, dialysis, obtain in L4 containing ester bond compound (A3-1).

The hydrogen modal data of compound A-13-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.65(-CH₂-), 2.32-2.41 (- CH₂CH₂COO-), 2.51 (-CH (CH₂)₃-), 3.35 (CH₃O-), 3.40-3.80 (-CH₂CH₂O-), 4.05-4.42 (- OCH₂CH-,-OCH (CH₂)CH₂O-,-OCH₂CH₂NH-), 4.90-5.13(-OCH (CH₂)CH₂O-).

Embodiment 4:L₄In containing secondary amine key (-CH₂NHCH₂-) preparation

In this example, L₄In containing the synthesis of secondary amine key compound (A4-1), wherein L₁=L₂=L₃=CH₂,X₁=X₂=CH₃, Y is CH, q=1, R are the residue of two Semen Myristicae phosphatidyl glycerol esters.Polyethylene Glycol total molecular weight is about 2000.

Branched polyethylene glycol propionic aldehyde (40g) and two myristoyl phosphatidyls are added in the 1L round-bottomed flask of dried and clean After serine (16.3 grams, 1.2 equivalents), nitrogen is protected, and after adding the PBS (500mL) that pH value is 5.0, is stirred at room temperature 4 After hour, after adding sodium cyanoborohydride (5 grams), the lower 20 DEG C of reaction 24h of room temperature, after adding saturated ammonium chloride solution cancellation, use Water dilutes, and dichloromethane extracts, and concentrates, dialysis, obtains in L4 containing secondary amine key compound (A4-1).

The hydrogen modal data of compound A4-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.73(-CH₂-,-NCH₂CH₂CH₂-), 2.32- 2.41(-CH₂CH₂COO-), 2.51 (-CH (CH₂)₃-), 2.85 (-NHCH₂-), 3.35 (CH₃O-), 3.40-3.80 (- CH₂CH₂O-,-CHCH₂O-), 4.05-4.42 (-OCH₂CH-,-OCH (CH₂)CH₂O-), 4.90-5.13(-OCH (CH₂)CH₂O-,- NHCH (COOH)-).

Embodiment 5:L₄In containing thioether bond (> CHS-) preparation

In this example, L₄In containing thioether bond (> CHS-) synthesis of compound (A5-1), wherein L₁=L₂=L₃=CH₂CH₂, X₁ =X₂=CH₃, Y is N, and q=1, R are the residue of distearin.Polyethylene Glycol total molecular weight is about 2000.

In the 500mL round-bottomed flask of dried and clean add 100mL contain Y type Polyethylene Glycol mercapto derivatives (10 grams, Phosphate buffered solution (pH=7.4) 5mmol/L), adds DSPE-MAL(5 gram, 1.1 equivalents) after, react 24 under the conditions of 4 DEG C After hour, after adding distilled water diluting, extract with dichloromethane, be dried, concentrate, obtain after recrystallisation from isopropanol in L4 containing sulfur Ehter bond (> CHS-) compound (A5-1).

The hydrogen modal data of compound A-45-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.65(CH₃(CH₂)₁₆-), 2.32-2.95 (- CH₂COO-,-NCH₂CH₂-,-NHCOCH₂CH₂-,-OCCH₂CHS-), 3.35 (CH₃O-), 3.40-3.80 (-OCH₂CH₂O-,- NCH₂CH₂O-,-CONCH₂CH-,-OCCHS-), 4.05-4.42 (-OCH₂CHO-,-OCH (CH₂)CH₂O-,-OCH₂CH₂NHCO-), 5.13(-OCH (CH₂)CH₂O-), 8.38(-NHCO-).

Embodiment 6:L₄In containing the preparation of triazole

In this example, L₄In containing the synthesis of 3-triazole compounds (A5-1), wherein L₁=L₂=L₃=CH₂, X₁=X₂=CH₃, Y is CH, q=1, R are the residue of distearin.Polyethylene Glycol total molecular weight is about 20000.

The Y type Polyethylene Glycol (20 grams, 1mmoL) containing DIBO group is added in the 500mL round-bottomed flask of dried and clean After, add acetonitrile, stir under room temperature to after being completely dissolved, be slowly added dropwise DSPE-N₃Acetonitrile solution (200mL) DSPE-N₃ After (1.55 grams, 2 equivalents), after reacting 4 hours under room temperature, concentrating, dialysis obtains in L4 containing triazole (compound (A6- 1).

The hydrogen modal data of compound A6-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-1.65(CH₃(CH₂)₁₆-), 2.32-2.41 (- CH₂CH₂COO-), 3.0-3.2(ArCH₂-), 3.35 (CH₃O-), 3.40-3.80 (-CH₂CH₂O-,-CHCH₂O-,-OCHCH₂-,- NCH₂CH₂O-), 4.05-4.42 (-OCH₂CH-,-OCH (CH₂)CH₂O-), 4.33 (-OCCH₂O-) 5.13(-OCH (CH₂) CH₂O-), 5.62 (ArCHO-), 7.31-7.65(C₆H₄-).

Embodiment 7:L₄In containing 4,5-dihydro-isoxazole

In this example, L₄In containing 4, the synthesis of 5-dihydro-isoxazole (A7-1), wherein L₁=L₂=L₃=CH₂, X₁=X₂=CH₃, Y For CH, q=1, R are the residue of distearin.Polyethylene Glycol total molecular weight is about 5000.

After adding Y type methoxy PEG-propionaldehyde (2 grams, 0.4mmoL) in the 50mL round-bottomed flask of dried and clean, add second Nitrile, stirs under room temperature to after being completely dissolved, replaces nitrogen, after adding hydroxy amine hydrochloric acid salt (4mmol), adds sodium acetate and regulates extremely After PH=8, react under room temperature overnight, concentrate, ether sedimentation, after preliminary purification, be directly used in next step reaction.

By the thick product of previous step, molten with DMF (20mL) in the 50mL round-bottomed flask of dried and clean Xie Hou, after displacement nitrogen, adds solid NCS(4mmol), after reacting overnight under room temperature, add saturated sodium bicarbonate solution (20mL), after continuing stirring under room temperature 4 hours, after adding substantial amounts of dchloromethane, wash with saturated aqueous common salt, be dried, Concentrate, ether sedimentation.

After walking, on adding in the 500mL round-bottomed flask of dried and clean, the Y type Polyethylene Glycol cyanogen oxygen compound obtained, add Acetonitrile, stirs under room temperature to after being completely dissolved, after being slowly added dropwise the acetonitrile solution (100mL) of DSPE-Norbornene, and room temperature After lower reaction 4 hours, concentrate, obtain after recrystallisation from isopropanol in L4 containing triazole (compound (A7-1).

The hydrogen modal data of compound A7-1 is as follows:

¹H NMR(CDCl₃) δ (ppm): 0.95(-CH₂CH₃), 1.21-2.01(CH₃(CH₂)₁₆-, OCH₂CH₂CH<,> CHCH₂CH < ,-C (=N) CH₂CH₂O-), 2.22-2.51 (-CH₂CH₂COO-, > CHCHC (=N)-) ,-CH (CH₂)₃-), 3.35 (CH₃O-), 3.40-3.80 (-CH₂CH₂O-,-CHCH₂O-), 4.05-4.42 (-OCH₂CH-,-OCH (CH₂)CH₂O-), 5.13(- OCH(CH₂)CH₂O-).

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

(1) preparation of liposome

Use gained L in embodiment 1₄The A1-3(embodiment 8 of middle amide bond), A1-4(embodiment 9), in embodiment 2 Gained L₄The A2-1(embodiment 10 of middle amido-containing acid ester key), A2-2(embodiment 11), gained L in embodiment 3₄In containing ester bond A3-1(embodiment 12), gained L in embodiment 4₄In containing the A4-1(embodiment 13 of secondary amine key), respectively with dioleoyl phospholipid Acyl ethanolamine (DOPE) prepares liposome.

Use gained L in embodiment 1₄The A1-2(embodiment 14 of middle amide bond), gained L in embodiment 5₄Middle Sulfide-containing Hindered The A5-1(embodiment 15 of key), gained L in embodiment 6₄In containing the A6-1(embodiment 16 of triazole), gained L in embodiment 7₄In Containing 4, the A7-1(embodiment 17 of 5-dihydro-isoxazole), prepare liposome with DOPE, cholesterol respectively.

Use gained L in embodiment 1₄The A1-3(embodiment 18 of middle amide bond), with DOPE and cholesteryl hemisuccinate (CHEMS) liposome is prepared.

Use in embodiment 2 in gained L4 the A2-2 of amido-containing acid ester key respectively with DSPE (DSPE), DPPE (DPPE) prepare liposome, correspond respectively to embodiment 19-21.

Parameter is as shown in table 1.

Several groups of different lipid mixtures in example and the DOPE in reference examples 1 is weighed according to parameter listed in table 1, point Fully not dissolving in chloroform, using Rotary Evaporators to remove solvent, until forming uniform lipid membrane on wall.To lipid Thin film adds the Tris solution 2mL of 25mM fluorochrome HPTS, wherein the fluorochrome quencher DPX, pH=containing 25mM 10.0.After using vortex oscillator to disperse, the polycarbonate leaching film of different pore size is used to filter successively, every kind of aperture Filter 23 time, it is thus achieved that Liposomal dispersion.Using dynamic light scattering method to detect particle diameter, the particle diameter of all liposomees all exists 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) liposome stability in buffer

Being placed at ambient temperature by above-mentioned gained Liposomal dispersion 1 month, by the naked eye, embodiment 8-20 is the most all For uniform Liposomal dispersion, visually have no change.The dispersion liquid of embodiment 21 has slight sedimentation.The liposome of reference examples 1 Dispersion liquid is unstable, it is seen that sedimentation.

(3) concentration-response of liposome and pH sensitivity

The Liposomal dispersion of Example 8-21, is sequentially modulated pH=5.0, and 6.0,6.5,6.8,7.0,8.0,9.0, The dispersion liquid of 10.0, hatches 1h at 37 DEG C, then collecting sample, is diluted with the buffer of pH10.0, by measuring fluorescence Intensity, calculates fluorochrome HPTS release rate R (t)=100 × [I (t)-I (0)]/[I (∞)-I (0)] from liposome, its Middle I (t) is the fluorescence intensity when time t, and I (0) is initial residual fluorescence intensity, and I (∞) is with 0.1% (w/v) Triton Maximum fluorescence intensity corresponding after X-100 lipin dissolving body.

Result shows, HPTS release from liposome has pH sensitivity.During low ph value, in embodiment 8-18, HPTS Release rate reduces with the increase of polyethyleneglycol modified lipid derivate content, during pH=6.0, and embodiment 8,9,10,11,12, The release rate of 13 is followed successively by 26%, and 18%, 12%, 10%, 5%, 3%；During pH=6.5, embodiment 8,9, the release rate of 10,11,12,13 It is followed successively by 22%, 16%, 9%, 8%, 4%, 2.5%.Embodiment 11,19, in 20,21, HPTS release rate is with polyethyleneglycol modified fat The increase of matter derivative content and increase.In reference examples 1, during pH=6.5, release rate is more than 80%, during pH=6.0, reach 90% with On.Result it is also shown that cholesterol be added with the stability beneficially improving liposome membrane, release rate is with the increase of cholesterol level And reduce, during pH=6.0, embodiment 8,14, the release rate of 15,16,17 is followed successively by 20%, and 17%, 16%, 11%, 6%;During pH=6.5, The release rate of embodiment 8,14,15,16,17 is followed successively by 17%, and 15%, 14%, 10%, 5%.For embodiment 8-17, during pH≤7.0, Release rate reduces with the rising of pH, and during pH >=7.0, release rate is less than 3%, and during for reference examples 1, pH=7.0, release rate is still More than 20%, and during pH >=8.0, release rate is less than 3%.

When using CHEMS to do membrane stabilizer, with reference to embodiment 18, under the conditions of neutrality and alkalescence, (pH >=7.0) are conducive to fat The stability of plastid membrane structure, corresponding release rate is less than 2%, and in acid condition, then is conducive to accelerating releasing of HPTS Putting, during pH=5.0, release rate is 42%, and during pH=6.0, release rate is 30%, and during pH=6.5, release rate is 21%, during pH=6.8, releases The rate of putting is 15%.

(4) liposome stability in serum

The Liposomal dispersion of Example 8-21, is diluted by the culture medium of 10% hyclone respectively, enters at 37 DEG C Row is hatched, successively 0, and 1,6,12,18,24h collecting sample, it is diluted with the Tris solution of pH10.0, measures fluorescence intensity, Calculate the release rate of HPTS.Result shows, the maximum release rate of embodiment 8-21 is followed successively by and is more than 5%, is showed no liposome Avalanche.Visible, lipid membrane structure body is more stable in serum.

Embodiment 22-25: be enclosed with the liposome of cancer therapy drug (amycin) Pharmacokinetic Evaluation in blood and Tissue distribution patterns

(5) preparation of hydrochloric doxorubicin liposome

Use gained L in embodiment 1₄The A1-1(embodiment 22 of middle amide bond), A1-4(embodiment 23), embodiment 3 Middle gained L₄In containing the A3-1(embodiment 24 of ester bond), gained L in embodiment 5₄The A5-1(embodiment 25 of middle sulfur-bearing ehter bond), point Other and DOPE, CHEMS prepare liposome.

Reference examples is respectively adopted the corresponding phospholipid modified without branched polyethylene glycol, is followed successively by dilauroyl glycerol phosphorus Acyl ethanolamine (DLPE, reference examples 2), the sphingomyelins (SSL, reference examples 3) containing stearyl, two myristoyl phosphatidyls are sweet Grease (DMPG, reference examples 4), DSPE (DSPE, reference examples 5).

Parameter is as shown in table 2.

The method identical with above-mentioned (1) is used to prepare embodiment and the fat being enclosed with doxorubicin hydrochloride of reference examples in table 2 Plastid dispersion liquid.Replace fluorochrome with doxorubicin hydrochloride, use ammonium sulphate gradient to enclose in lipid by doxorubicin hydrochloride, dense Degree 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 of 12 week old, with reference to dosage (15mg lipids such as table 2, tail vein injections Body/kg) hydrochloric doxorubicin liposome dispersion liquid, after 1min, 15min, 30min, 1h, 2h, 6h, 12h, 18h, 24h Gather blood sample 0.5ml through rat eye rear vein beard, separate serum ,-20 DEG C of preservations, be used for measuring blood drug level.With salt Acid daunorubicin, as internal standard, uses RP-HPLC method to measure doxorubicin concentration, and the medicine-time data of gained meets three chambers Model, processes the elimination half-life t of gained_1/2γResult as shown in table 3.Comparative example 22 with compare 2, comparative example 23 With reference examples 3, comparative example 24 and reference examples 4, comparative example 25 and reference examples 5, it is seen that branched polyethylene glycol is modified The half-life of the liposome (embodiment 22-25) that phospholipid derivative participates in composition significantly extends, and repaiies with without branched polyethylene glycol The liposome (reference examples 2-5) of decorations is compared, and brings up to original 5～10 times.

When CHEMS is as membrane stabilizer, make stabilizer with cholesterol compared with, its half-life is slightly lower, this is because CHEMS this Body has pH sensitivity, under the conditions of neutrality and alkalescence, has the effect of stabilized liposome membrane structure or liposome, and at acid bar The instability of lipid membrane structure or liposome is then caused under part.

(7) tissue distribution patterns

Select the male Sprague-Dawley rat of 12 week old, with reference to the dosage such as table 2 intravenous injection (15mg liposome/ Kg) hydrochloric doxorubicin liposome dispersion liquid, respectively at be administered after 5min, 2h, 6h, 12h, 24h, with avascularization put to death one group big Mus, and excise brain, the heart, liver, lung, kidney, spleen, gastrointestinal, take a certain amount of tissue, in phosphate buffer, under condition of ice bath, through height Speed dispersion is prepared as tissue homogenate ,-20 DEG C of preservations, measures for tissue concentration.Using daunorubicin hydrochloride as internal standard, use RP-HPLC method measures doxorubicin concentration.Result shows, the liposome after branched polyethylene glycol is modified, with unused branched poly-second The liposome phase transformation that glycol is modified, it significantly reduces in the distribution of heart, greatly reduces cardiac toxicity, simultaneously at liver, kidney The abundance at dirty place increases, and demonstrates the liver of enhancing, kidney targeting.Additionally, use embodiment 22-23 and the reference examples of CHEMS 2-3, than embodiment 24-25 and reference examples 4 and 5, it significantly increases in the abundance of stomach, and targeting is greatly improved.

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

The A1-1(embodiment 26 of amide bond in gained L4 in use embodiment 1), A1-4(embodiment 27), embodiment 3 Middle gained L4 contains the A3-1(embodiment 28 of ester bond), gained L in embodiment 5₄The A5-1(embodiment 29 of middle sulfur-bearing ehter bond), point Other and DOPE, CHEMS prepare liposome.

Reference examples is respectively adopted the corresponding phospholipid modified without branched polyethylene glycol, is followed successively by dilauroyl glycerol phosphorus Acyl ethanolamine (DLPE, reference examples 5), the sphingomyelins (SSL, reference examples 6) containing stearyl, two myristoyl phosphatidyls are sweet Grease (DMPG, reference examples 7), DSPE (DSPE, reference examples 8).

Adding a reference examples 9, use DLPE-mPEG to replace A1-1, both differences are to replace branched polyethylene glycol It is changed to the linear PEG, DLPE-mPEG of equivalent molecule amount by the mono methoxy polyethylene glycol with branched polyethylene glycol equivalent molecule amount Acetic acid reacts with DLPE and is prepared from.

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

Use animal transplanting tumor laboratory method, use H₂₂Murine hepatocarcinoma cell is inoculated in right side of mice axil subcutaneous formation entity Tumor, respectively inoculation 2 days, after 7 days, carry out tail vein injection administration, administering mode is single-dose, and dosage is 15mg kg^-1.After inoculating 2 weeks, mice cervical dislocation is put to death, peel off tumor, and weigh.Result shows, modifies through branched polyethylene glycol After liposome (embodiment 26-29 has higher tumour inhibiting rate compared to the most modified reference examples 5-8.Containing CHEMS's Embodiment 26-27 has higher tumour inhibiting rate than embodiment 28-29.

Additionally, the liposome that liposome (embodiment 26) the relatively linear polyethylene glycol that branched polyethylene glycol is modified is modified is (right As usual 9) there is higher tumour inhibiting rate.

By the above results, the liposome of the present invention is at neutral and stable under alkaline conditions, and at meta-acids such as tumors Property position is affected by pH value, and the release rate of the bio-related substance of encapsulation significantly improves, the drug distribution amount at these positions Also selectivity improves, and demonstrates preferable passive targeting, and the poison caused because of frequent drug administration or increasing dosage can be avoided secondary Effect.

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 utilize this Equivalent structure or equivalence flow process that bright description is made convert, or are directly or indirectly used in other relevant technology necks Territory, is the most in like manner included in the scope of patent protection of the present invention.

Claims

1. the phospholipid derivant of a branched polyethylene glycol, it is characterised in that the phospholipid of described branched polyethylene glycol derives Shown in thing formula such as formula (1):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂Be each independently 1～1000 whole Number, n₃It is the integer of 0～1000, and 2≤n₁+n₂+n₃≤2000；Y is branch centers, for nitrogen-atoms or to have 1 to 20 carbon former Son, uses covalent bond and L₁、L₂、L₃It is connected；L₁、L₂、L₃Linking group for branch centers Y Yu Polyethylene Glycol unit；Q is 0 Or 1；When Y is the branch centers with 1 to 20 carbon atom, n₃=0；Work as n₃When=0, q is 0 and Y not to be 2～10 amino The polyamino acid residues of acid residue；L₄For linking group, occur chemistry anti-for polyethyleneglycol derivative and corresponding phosphatide cpd The residue that should be formed；M is hydrogen atom or cation；R is the residue of hydrophobic lipid.

The phospholipid derivant of branched polyethylene glycol the most according to claim 1, it is characterised in that described n₁,n₂,n₃Meet 1 ≤n₁≤ 200,1≤n₂≤ 200,0≤n₃≤ 200, and 2≤n₁+n₂+n₃≤200。

The phospholipid derivant of branched polyethylene glycol the most according to claim 1, it is characterised in that described Y is that carbon atom props up Change center, its representation is as follows:Wherein, R₁The alkyl that for hydrogen atom, there is 1 to 20 carbon or containing hetero atom The alkyl with 1 to 20 carbon of group.

The phospholipid derivant of branched polyethylene glycol the most according to claim 3, it is characterised in that described R₁For hydrogen atom, or For having the alkyl of 1 to 20 carbon, or be containing ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonate group, Secondary amino group or the alkyl with 1 to 20 carbon of tertiary amino.

The phospholipid derivant of branched polyethylene glycol the most according to claim 1, it is characterised in that described Y is that nitrogen-atoms props up Change center, its representation is as follows:

The phospholipid derivant of branched polyethylene glycol the most according to claim 1, it is characterised in that described R is hydrophobicity fat Fat hydrocarbon residue, or DG lipid residue, or monoacylglycerol lipid residue, or the lipid containing sphingosinols skeleton is residual Base, or steroidal residues, or above-mentioned any two and two or more complex lipid residues；

When R is DG lipid residue, and its representation is as follows:R₄、R₅For having 4 to 50 carbon The alkyl of atom；

When R is monoacylglycerol lipid residue, and its representation is as follows:R₆For tool There is the alkyl of 4 to 50 carbon；

When R is the lipid residue containing sphingosinols skeleton, and its representation is as follows:Or be expressed asR₂、R₃It is each independently the alkyl with 4 to 50 carbon atoms；

When R is hydrophobic fat hydrocarbon residue, and its representation is as follows:R₇For having the alkyl of 4 to 50 carbon, Ke Yishi Straight chain, side chain, the ring-type or chain of band cyclic group；

When R is the residue of steroid, and this steroid is cholesterol, sitosterol, vitamin D, beta-cholestanol, ergosterol, sheep Hair sterol, bilichol or gonadal hormone, or the derivant of above-mentioned steroid.

The phospholipid derivant of branched polyethylene glycol the most according to claim 6, it is characterised in that described R₂、R₃、R₄、R₅、 R₆、R₇Be each independently selected from butyl, the tert-butyl group, amyl group, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, 13 Alkyl, myristyl, pentadecyl, cetyl, heptadecyl, octadecyl, nonadecyl, eicosyl, (Z)-9-ten Apos, (Z)-8-17 thiazolinyl, (Z)-12-two hendecene base, and in same a part, can be the same or different.

The phospholipid derivant of branched polyethylene glycol the most according to claim 1, it is characterised in that described L₄For

Wherein, Z is alkylidene, or Z is for containing selected from ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonic ester The alkylidene of base, secondary amino group or tertiary amino；

G is 0 or 1；

F is the integer of 2 to 10.

The phospholipid derivant of branched polyethylene glycol the most according to claim 8, it is characterised in that described Z be methylene, 1, 2-ethylidene, 1,3-propylidene, 1,2-propylidene, isopropylidene, butylidene, pentylidene or hexylidene.

The phospholipid derivant of branched polyethylene glycol the most according to claim 1, it is characterised in that described X₁、X₂For methyl, Ethyl, propyl group, acrylic, propinyl, isopropyl, butyl, the tert-butyl group, amyl group, heptyl, octyl group, nonyl, decyl, undecyl, Dodecyl, tridecyl, myristyl, pentadecyl, cetyl, heptadecyl, octadecyl, nonadecyl, 20 Alkyl, benzyl or butyl phenyl, and in same a part, can be the same or different.

The phospholipid derivant of 11. branched polyethylene glycols according to claim 1, it is characterised in that described L₁、L₂、L₃Each Independently be the alkyl with 1 to 20 carbon atom, or be each independently containing under the conditions of illumination, enzyme, acidity or alkalescence The ether of stable existence, thioether group, amide groups, double bond, key, secondary amine or tertiary amine groups there is the two of 1 to 20 carbon atom Valency alkyl.

The phospholipid derivant of 12. branched polyethylene glycols according to claim 1, it is characterised in that described M be hydrogen atom, Sodium ion, NH₄ ⁺。

The phospholipid derivant of 13. branched polyethylene glycols according to claim 1, it is characterised in that containing phosphatidyl ethanol The residue of amine.

14. according to the phospholipid derivant of branched polyethylene glycol described in claim 13, it is characterised in that described phosphatidyl ethanol Amine is DSPE, two lauroyl PHOSPHATIDYL ETHANOLAMINE, two oils and fats acylphosphatidyl ethanolamines, two lima bean Myristoyl PHOSPHATIDYL ETHANOLAMINE, two Caulis et Folium Lini acyl PHOSPHATIDYL ETHANOLAMINE, two mustard acyl PHOSPHATIDYL ETHANOLAMINE, 1-palmityl-2-oleoyl phosphorus Acyl ethanolamine or DPPE.

15. 1 kinds of lipid film knots containing the phospholipid derivant of branched polyethylene glycol according to any one of claim 1～14 Structure body.

16. according to lipid membrane structure body described in claim 15, it is characterised in that the phospholipid derivant of branched polyethylene glycol is The 0.1%～30% of the gross mass of this lipid membrane structure body.

17. according to lipid membrane structure body described in claim 15, it is characterised in that the phospholipid derivant of branched polyethylene glycol is The 1%～30% of the gross mass of this lipid membrane structure body.

18. according to lipid membrane structure body described in claim 15, it is characterised in that the phospholipid derivant of branched polyethylene glycol is The 3%-20% of the gross mass of this lipid membrane structure body.

19. according to lipid membrane structure body described in claim 15, it is characterised in that the phospholipid derivant of branched polyethylene glycol is The 3%-15% of the gross mass of this lipid membrane structure body.

20. according to lipid membrane structure body described in claim 15, it is characterised in that possibly together with PHOSPHATIDYL ETHANOLAMINE as the group of film Become composition.

21. according to lipid membrane structure body described in claim 20, it is characterised in that described PHOSPHATIDYL ETHANOLAMINE is distearyl acyl group PHOSPHATIDYL ETHANOLAMINE, two oils and fats acylphosphatidyl ethanolamines, two myristoyl PHOSPHATIDYL ETHANOLAMINE or two palmityl phospholipid Acyl ethanolamine, two lauroyl PHOSPHATIDYL ETHANOLAMINE, two Caulis et Folium Lini acyl PHOSPHATIDYL ETHANOLAMINE, two mustard acyl PHOSPHATIDYL ETHANOLAMINE, 1-Petiolus Trachycarpi Acyl-2-oleoylphosphatidyl ethanolamine or DPPE.

22. according to lipid membrane structure body described in claim 15, it is characterised in that it is packaged with bio-related substance.

23. according to lipid membrane structure body described in claim 22, it is characterised in that described bio-related substance is polypeptide, albumen In matter, small-molecule drug, nucleoside, nucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycoprotein any one or one Plant above bio-related substance.

24. according to lipid membrane structure body described in claim 23, it is characterised in that described bio-related substance is polypeptide, enzyme, little In molecular medicine, nucleoside, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroid, glycolipid, glycoprotein any one or one with On bio-related substance.

25. according to lipid membrane structure body described in claim 15, it is characterised in that its form is liposome.

26. according to lipid membrane structure body described in claim 25, it is characterised in that be packaged with biological correlative in described liposome Matter.

27. according to lipid membrane structure body described in claim 26, it is characterised in that described bio-related substance is polypeptide, albumen In matter, small-molecule drug, nucleoside, nucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycoprotein any one or one Plant above bio-related substance.

28. according to lipid membrane structure body described in claim 27, it is characterised in that described bio-related substance be polypeptide, enzyme, In small-molecule drug, nucleoside, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroid, glycolipid, glycoprotein any one or a kind of Above bio-related substance.

29. according to lipid membrane structure body described in claim 26, it is characterised in that described bio-related substance is when pH≤6.8 Release.

30. according to lipid membrane structure body described in claim 26, it is characterised in that described bio-related substance is when pH≤6.5 Release.

31. according to lipid membrane structure body described in claim 26, it is characterised in that described bio-related substance is when pH≤6.0 Release.

The phospholipid derivant of 32. 1 kinds of branched polyethylene glycols, it is characterised in that the phospholipid of described branched polyethylene glycol derives Shown in thing formula such as formula (2):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂Be each independently 1～1000 whole Number, and 2≤n₁+n₂≤2000；Y is atom N branch centers or the carbon atom branch centers with 1 to 20 carbon, uses covalent bond With L₁、L₂、L₄It is connected, and Y is not the polyamino acid residues of 2～10 amino acid residues；L₁、L₂For connect branch centers Y with The linking group of Polyethylene Glycol unit, and L₁、L₂It is each independently the alkyl with 1 to 20 carbon atom, or independently of one another For containing the ether existed at illumination, enzyme, acidity or stable under alkaline conditions, thioether group, amide groups, double bond, key, secondary amine Or the bivalent hydrocarbon radical with 1 to 20 carbon atom of tertiary amine groups；L₄For linking group, for polyethyleneglycol derivative and corresponding phospholipid The residue that compound generation chemical reaction is formed；

M is hydrogen atom or cation；R is hydrophobic lipid residue.

33. according to the phospholipid derivant of branched polyethylene glycol described in claim 32, it is characterised in that described L₄For containing amino, Ester group, carbonate group, triazol radical, isoxazolyl, ether, amide groups, sub-amide groups, imido grpup, secondary amino group, tertiary amine groups, sulfur Ester group, thioether group, disulfide group, urethane groups, thiocarbonic acid ester group, sulfonate group, sulfoamido, carbamate groups, tyrosine Base, cysteine base or the divalent alkyl of histidine base.

34. according to the phospholipid derivant of branched polyethylene glycol described in claim 32, it is characterised in that described L₄Selected from following One of group:

Wherein, Z is alkylidene, or for containing selected from ester group, urethane groups, amide groups, ether, thioether, double bond, three key, carbonic ester The alkylidene of base, secondary amino group or tertiary amino；

G is 0 or 1；

F is the integer of 2 to 10.

35. according to the phospholipid derivant of branched polyethylene glycol described in claim 32, it is characterised in that described R is selected from fat Hydrocarbon residue, DG lipid residue, monoacylglycerol lipid residue, lipid residue or class containing sphingosinols skeleton are solid The residue of a kind of or its complex lipid in alcohol residue.

The liposome of 36. 1 kinds of phospholipid derivants containing branched polyethylene glycol described in claim 32, it is characterised in that institute The phospholipid derivant stating branched polyethylene glycol accounts for the 1%～30% of this liposome gross mass.

37. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 36, it is characterised in that described The phospholipid derivant of polyethylene glycol accounts for the 3%-20% of this liposome gross mass.

38. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 36, it is characterised in that described The phospholipid derivant of polyethylene glycol accounts for the 3%-15% of this liposome gross mass.

39. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 36, it is characterised in that its encapsulation Have in polypeptide, protein, small-molecule drug, nucleoside, nucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycoprotein The bio-related substance of any one or more.

40. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 37, it is characterised in that described life Thing related substances be polypeptide, enzyme, small-molecule drug, nucleoside, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroid, glycolipid, The bio-related substance of any one or more in glycoprotein.

The phospholipid derivant of 41. 1 kinds of branched polyethylene glycols, it is characterised in that the phospholipid of described branched polyethylene glycol derives Shown in thing formula such as formula (3):

Wherein, X₁、X₂It is each independently the alkyl with 1 to 20 carbon atom；n₁、n₂Be each independently 1～1000 whole Number, n₃It is the integer of 1～1000, and 3≤n₁+n₂+n₃≤2000；Y is nitrogen-atoms branch centers, uses covalent bond and L₁、L₂、L₃ It is connected；L₁、L₂、L₃It is each independently the linker connecting branch centers Y with Polyethylene Glycol unit, and L₁、L₂、L₃The most solely It is on the spot the alkyl with 1 to 20 carbon atom, or is each independently containing steady under the conditions of illumination, enzyme, acidity or alkalescence The bivalence with 1 to 20 carbon atom of fixed ether, thioether group, amide groups, double bond, three key, secondary amine or the tertiary amine groups existed Alkyl；L₄For linking group, the residue formed with corresponding phosphatide cpd generation chemical reaction for polyethyleneglycol derivative；M is Hydrogen atom or cation；R is hydrophobic lipid residue.

42. according to the phospholipid derivant of branched polyethylene glycol described in claim 41, it is characterised in that described L₄For containing amino, Ester group, carbonate group, triazol radical, isoxazolyl, ether, amide groups, sub-amide groups, imido grpup, secondary amino group, tertiary amine groups, sulfur Ester group, thioether group, disulfide group, urethane groups, thiocarbonic acid ester group, sulfonate group, sulfoamido, carbamate groups, tyrosine Base, cysteine base or the divalent alkyl of histidine base.

43. according to the phospholipid derivant of branched polyethylene glycol described in claim 41, it is characterised in that described L₄Selected from following One of group:

G is 0 or 1；

F is the integer of 2 to 10.

44. according to the phospholipid derivant of branched polyethylene glycol described in claim 41, it is characterised in that described R is selected from fat Hydrocarbon residue, DG lipid residue, monoacylglycerol lipid residue, lipid residue or class containing sphingosinols skeleton are solid The residue of a kind of or its complex lipid in alcohol residue.

The liposome of 45. 1 kinds of phospholipid derivants containing branched polyethylene glycol described in claim 41, it is characterised in that institute The phospholipid derivant stating branched polyethylene glycol accounts for the 1%～30% of this liposome gross mass.

46. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 45, it is characterised in that described The phospholipid derivant of polyethylene glycol accounts for the 3%-20% of this liposome gross mass.

47. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 45, it is characterised in that described The phospholipid derivant of polyethylene glycol accounts for the 3%-15% of this liposome gross mass.

48. according to the liposome of the phospholipid derivant of branched polyethylene glycol described in claim 45, it is characterised in that its encapsulation Have in polypeptide, protein, small-molecule drug, nucleoside, nucleotide, nucleic acid, polysaccharide, steroidal compounds, lipoid substance, glycoprotein The bio-related substance of any one or more.

49. according to the liposome of the phospholipid derivant of claim 48 branched polyethylene glycol, it is characterised in that described biofacies Related substance is polypeptide, enzyme, small-molecule drug, nucleoside, oligonucleotide, polynucleotide, nucleic acid, polysaccharide, steroid, glycolipid, sugar egg The bio-related substance of any one or more in Bai.