CN102604083A - Polymer modified lipid material and application thereof - Google Patents

Polymer modified lipid material and application thereof Download PDF

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CN102604083A
CN102604083A CN2011104083743A CN201110408374A CN102604083A CN 102604083 A CN102604083 A CN 102604083A CN 2011104083743 A CN2011104083743 A CN 2011104083743A CN 201110408374 A CN201110408374 A CN 201110408374A CN 102604083 A CN102604083 A CN 102604083A
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integer
formula
compound
drug administration
administration carrier
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CN102604083B (en
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夏桂民
安智娇
李眉
李子臣
马洁
王玉成
王旸
赵晨
李桂玲
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Institute of Medicinal Biotechnology of CAMS
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Abstract

The invention relates to a polymer modified lipid material and application thereof. The lipid material is a compound shown in the formula I, or a compound shown in the formula 9 or a physiologically acceptable salt thereof, wherein n is an integer in the range of 5-500, m is an integer in the range of 2-12, and p is an integer in the range of 1-8. The invention also relates to an intermediate of the compound shown in the formula I, an application of the compound shown in the formula I and an administration carrier prepared by the compound shown in the formula I. The administration carrier provided by the invention can be used for increasing the concentration of an anti-cancer medicine on a target part, simultaneously reducing the toxin or side effect on a non-target part and increasing the therapeutic index of the medicine.

Description

Polymer-modified matrix material and uses thereof
Technical field
The present invention relates to a kind of matrix material, particularly a kind of polymer-modified matrix material.The purposes that also relates to this matrix material.Matrix material of the present invention can be used as the solid support material of liposome.In addition, the invention still further relates to the midbody of synthetic said matrix material.
Background technology
Liposome is a kind of targeted drug carrier, belongs to a kind of novel form of targeting drug delivery system.Because of it has target property, slow-releasing, characteristics such as tissue affinity, hypotoxicity and high stability receive extensive concern in recent years, and many domestic and international investigators are seeking the liposome vectors material that novel target property is strong and have superperformance.
Folic acid-liposome is a kind ofly through the wetting ability straight chain polymer folate molecule to be connected the receptor type target liposomes that surface of liposome obtains indirectly; Folic acid (in this article; Can be abbreviated as F) have very high tumor-targeting, so folic acid-liposome provides a kind of comparatively practical antitumor drug targeting vector for medicine is imported at high proportion in the tumour cell.Existing at present research is processed the folacin receptor target liposomes with F-PEG-DSPE as fat material parcel antitumor drug, and result of study shows that this liposome has good long Circulation and tumor-targeting.
Gathering (2-ethyl-2-oxazoline) is the polymkeric substance that is obtained by the ring-opening polymerization of 2-ethyl-2-oxazoline (EOz) monomer (PEOz), has characteristics such as good biocompatibility and hypotoxicity.Research shows that the effect of PEOz and PEG is similar, therefore is expected to become the equivalent material of PEG.Discovered in recent years that PEOz had pH susceptibility, its pKa value (4-6) can show different character near the physiological pH value under weak acid environment with under the physiological pH environment.This pH susceptibility is fit to the release of medicine at slightly acidic positions such as tumor tissues and inclusion bodies very much.
Yet people still can not obtain to gather (2-ethyl-2-oxazoline) and phospholipid substance and optional folic acid bonded compounds at present; (for example good water-solubility, biocompatibility and/or hypotoxicity so that obtain to have desirable properties; With optional tumor-targeting) matrix material, particularly can be used as the solid support material of liposome.
Summary of the invention
The purpose of this invention is to provide a kind of polymer-modified matrix material, it has good water-solubility, biocompatibility and/or hypotoxicity and optional tumor-targeting.The inventor finds; Adopt the 2-ethyl-2-oxazoline (in this article; Can be abbreviated as EOz) obtain polymer poly (2-ethyl-2-oxazoline) (in this article, can be abbreviated as PEOz) as monomer through ring-opening polymerization, this PEOz is through specific functional group and DSPE (in this article; Can be abbreviated as DSPE) combine; Randomly further combine with folic acid, can successfully obtain polymer-modified phospholipid substance, these polymer-modified phospholipid substances have the advantageous property of expectation.The present invention is based on above-mentioned discovery and be accomplished.
Put it briefly, the present invention provides with the following formula I compound:
Figure BSA00000632652900021
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.The present invention also provides the midbody of preparation I compound, the purposes of formula I compound and the drug administration carrier that is made by formula I compound.
First aspect present invention provides with the following formula I compound:
Figure BSA00000632652900022
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to the formula I compound of first aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to the formula I compound of first aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to the formula I compound of first aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to the formula I compound of first aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 4 integer.
According to the formula I compound of first aspect present invention, its for following formula Ia compound (be m=6, p=2):
Figure BSA00000632652900031
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, perhaps n is 10 to 250 integer; Perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
Second aspect present invention provides with following formula 9 compounds:
Figure BSA00000632652900032
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to formula 9 compounds of second aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to formula 9 compounds of second aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to formula 9 compounds of second aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to formula 9 compounds of second aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 5 integer.
According to formula 9 compounds of second aspect present invention, it is with following formula 9a compound:
Figure BSA00000632652900033
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, perhaps n is 10 to 250 integer; Perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
Third aspect present invention provides with following formula 4 compounds:
Figure BSA00000632652900041
Or its salt, wherein n is 5 to 500 integer, m is 2 to 12 integer.
According to formula 4 compounds of third aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to formula 4 compounds of third aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to formula 4 compounds of third aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer.
According to formula 4 compounds of third aspect present invention, it is with following formula 4a compound:
Figure BSA00000632652900042
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, perhaps n is 10 to 250 integer; Perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
Fourth aspect present invention provides the method for preparing said formula 9 compounds of second aspect present invention, and it may further comprise the steps:
(i) in the presence of suitable catalyzer (for example DCC), in suitable organic solvent (for example chloroform), make the carboxylic acid and DSPE (DSPE) reaction of the base of Terminal Acetylenes shown in the following formula,
Figure BSA00000632652900051
Obtain containing formula 6 compounds (can be described as Terminal Acetylenes base DSPE) of Terminal Acetylenes base at this paper:
Figure BSA00000632652900052
(ii) make with the reaction of following formula 4 compound step (i) gained formulas 6 compounds,
Figure BSA00000632652900053
Obtain with following formula 9 compounds (can abbreviate PEOz-DSPE as) at this paper:
Wherein, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to the method for fourth aspect present invention, the reaction of wherein said step (i) is under 30-75 ℃ (for example 40-60 ℃, for example about 50 ℃), to carry out 5-25 hour (for example 8-20 hour, for example 10-15 hour, for example about 12 hours).
According to the method for fourth aspect present invention, the reaction (ii) of wherein said step is carried out in the presence of part (for example PMDETA).
According to the method for fourth aspect present invention, the reaction (ii) of wherein said step is carried out in the presence of reagent (for example CuBr).
According to the method for fourth aspect present invention, the reaction (ii) of wherein said step is under 30-75 ℃ (for example 40-60 ℃, for example about 50 ℃), to carry out 15-150 hour (for example 30-120 hour, for example 50-100 hour, for example about 72 hours).
According to the method for fourth aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to the method for fourth aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to the method for fourth aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to the method for fourth aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 5 integer.According to the method for fourth aspect present invention, wherein n is 10 to 100 integer, and m is 4 to 8 integer, and p is 1 to 4 integer.
According to the method for fourth aspect present invention, wherein said formula 4 compounds (can be described as PEOz in this article) are to prepare through the method that may further comprise the steps:
(ia) in the presence of reagent (for example NaI); In organic solvent (for example DMF); Make compound and reaction of sodium azide shown in the formula
Figure BSA00000632652900061
, obtain formula
Figure BSA00000632652900062
compound;
(ib) in the presence of reagent (for example triethylamine and/or trimethylamine hydrochloride); In organic solvent (for example chloroform); Make the reaction of formula
Figure BSA00000632652900063
compound and Tosyl chloride, obtain formula
Figure BSA00000632652900064
compound;
(ic) in organic solvent (for example acetonitrile); Make the monomer reaction of formula
Figure BSA00000632652900065
compound and formula
Figure BSA00000632652900066
; Charge into ammonia then with stopped reaction, obtain with following formula 4 compounds:
Figure BSA00000632652900071
Wherein, X is halogen (for example fluorine, chlorine, bromine, iodine, preferably a chlorine), and n is 5 to 500 integer, and m is 2 to 12 integer.
Fifth aspect present invention provides the method for preparing the said formula I compound of first aspect present invention, and it may further comprise the steps:
(a) in organic solvent (for example triethylamine and/or DMSO), in the presence of catalyzer (for example DCC), make the reaction of folic acid and following formula N-maloyl imines (NHS), obtain folic acid Acibenzolar (can abbreviate F-NHS as) at this paper with following formula 8:
(b) in the presence of alkali (for example triethylamine), in organic solvent (for example DMSO), make formula 8 compounds and react with following formula 9 compounds,
Obtain formula I compound:
Figure BSA00000632652900074
With optional
(c) make gained formula I compound purifying and/or salify,
Wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
According to the method for fifth aspect present invention, wherein n is 10 to 250 integer, and perhaps n is 10 to 200 integer; Perhaps n is 10 to 150 integer, and perhaps n is 10 to 125 integer, and perhaps n is 10 to 100 integer; Perhaps n is 15 to 75 integer, and perhaps n is 15 to 65 integer.
According to the method for fifth aspect present invention, wherein m is 3 to 10 integer, and perhaps m is 4 to 8 integer, and perhaps m is 5 to 8 integer, and perhaps m is 6.
According to the method for fifth aspect present invention, wherein p is 1 to 6 integer, and perhaps p is 1 to 5 integer, and perhaps p is 1 to 4 integer, and perhaps p is 2.
According to the method for fifth aspect present invention, wherein n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 5 integer.
Sixth aspect present invention provides said formula I compound of first aspect present invention or the purposes of said formula 9 compounds of second aspect present invention in the preparation drug administration carrier.
According to the purposes of sixth aspect present invention, wherein said formula I compound or formula 9 compounds are that one of lipid composition as said drug administration carrier is used.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is a pH responsive type drug administration carrier.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is the drug administration carrier with cancer target performance.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is pH responsive type and drug administration carrier with cancer target performance.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is the drug administration carrier that contains or do not contain active constituents of medicine.In one embodiment, said drug administration carrier contains activeconstituents.In one embodiment, said active constituents of medicine is an antitumor drug.
According to the purposes of sixth aspect present invention, wherein said drug administration carrier is a liposome.In one embodiment, said drug administration carrier is an initiatively target liposomes of pH responsive type.In one embodiment, said drug administration carrier is folacin receptor mediated pH responsive type active target liposomes.
Seventh aspect present invention provides a kind of drug administration carrier, wherein comprises said formula 9 compounds of said formula I compound of first aspect present invention or second aspect present invention, and optional active constituents of medicine.
According to the drug administration carrier of seventh aspect present invention, wherein said formula I compound or formula 9 compounds are one of lipid compositions as said drug administration carrier.
According to the drug administration carrier of seventh aspect present invention, it is a pH responsive type drug administration carrier.
According to the drug administration carrier of seventh aspect present invention, it is the drug administration carrier with cancer target performance.
According to the drug administration carrier of seventh aspect present invention, it is pH responsive type and drug administration carrier with cancer target performance.
According to the drug administration carrier of seventh aspect present invention, wherein contain or do not contain active constituents of medicine.In one embodiment, said drug administration carrier contains activeconstituents.In one embodiment, said active constituents of medicine is antitumor drug (a for example Zorubicin).
According to the drug administration carrier of seventh aspect present invention, it is a liposome.In one embodiment, said drug administration carrier is an initiatively target liposomes of pH responsive type.In one embodiment, said drug administration carrier is folacin receptor mediated pH responsive type active target liposomes.
Eighth aspect present invention provides the method that in the experimenter who needs is arranged, treats and/or prevents disease; It comprises to said experimenter uses each described drug administration carrier of seventh aspect present invention, and wherein said drug administration carrier contains the active constituents of medicine of significant quantity.In one embodiment, said disease is tumour or Cancerous disease.In one embodiment, said active constituents of medicine is antitumor drug (a for example Zorubicin).
Of the present invention arbitrary aspect, any one or more technical characterictics in wherein arbitrary embodiment can be combined in another embodiment of this aspect or be combined in arbitrary embodiment of others, as long as this combination can be not conflicting; Certainly when making up each other, necessary words can be done suitably to modify to individual features.In addition, for the term that uses aspect arbitrary in the present invention, its implication is equally applicable to others.
Do further to describe with characteristics to various aspects of the present invention below.
All documents that the present invention quoted from, their full content is incorporated this paper by reference into, and if the expressed implication of these documents and the present invention when inconsistent, be as the criterion with statement of the present invention.In addition; Various terms and phrase that the present invention uses have the general sense of well known to a person skilled in the art; Nonetheless; The present invention still hopes at this more detailed explanation and explanation to be done in these terms and phrase, and term of mentioning and phrase are as the criterion with the implication that the present invention was explained if any inconsistent with known implication.
As described herein, term " pharmaceutical composition ", it can also be meant compsn, is used in to realize treating, prevent, alleviate and/or alleviating disease according to the invention or illness or adverse health situation among the experimenter.It will be apparent to those skilled in the art that; Pharmaceutical composition of the present invention or compsn can be the liposomes that is enclosed with medicine (for example Zorubicin); Also can be the blank liposome that does not comprise medicine, it mixes the pastille liposome with medicine (for example Zorubicin) before clinical application.
As described herein, term " experimenter " also can refer to " patient ", it typically refers to Mammals, for example people, dog, monkey, ox, horse etc.
As described herein, term " significant quantity " is meant the dosage that can in the experimenter, effectively realize treating, preventing, alleviate, alleviate, eliminate relative disease and complication thereof.
As described herein; Term " drug administration carrier "; Also can be described as " drug delivery system ", one of important component of forming this drug administration carrier is a lipid composition, for example the polymer-modified phosphatide with hydrophilic segment and hydrophobic part of the present invention's acquisition; For example the present invention obtains has hydrophilic segment and hydrophobic part and comprises target functional group's polymer-modified phosphatide, and for example well known to a person skilled in the art other phospholipids composition.Said drug administration carrier can be lipid microsphere drug delivery system (for example lipoid microsphere), liposome administration system (for example unilamelar liposome, multilamelar liposome, multiphasic liposomes), lipid vesicle drug delivery system etc.This drug administration carrier can be a pastille, thus can also be not pastille but face the time spent and medicine mixed with this drug administration carrier medicine is mixed in this drug administration carrier.In addition, drug administration carrier of the present invention also can contain other the acceptable vehicle of pharmacy, for example caffolding agent.For example, in one embodiment, drug administration carrier of the present invention is through being similar to following method preparation: make phospholipids, SUV and formula I compound or formula 9 compound formation liposome aqueous suspensions; Add vehicle (for example N.F,USP MANNITOL); Lyophilize obtains the not blank drug administration carrier of pastille; Join in the above-mentioned blank drug administration carrier facing, the medicine parcel is got in liposome, thereby can supply clinical use with the preceding solution that will be dissolved with medicine (for example Zorubicin) (the for example solution of the aqueous solution, sodium chloride injection preparation).
In the present invention, when mentioning Zorubicin, it comprises any pharmacologically acceptable salts of Zorubicin itself and Zorubicin, hydrochloric acid for example, and they all can be expressed as DOX in the present invention.Have under the situation of concrete context at some,, mention that Zorubicin all is meant doxorubicin hydrochloride like not statement in addition.
In the present invention, mention that PEOz-DSPE is meant the binding substances of PEOz and DSPE, promptly like formula 9 compounds of the present invention; Be meant when mentioning F-PEOz-DSPE on the binding substances of above-mentioned PEOz and DSPE to be connected with folic acid, promptly like formula I compound of the present invention; Be meant the binding substances of PEG and DSPE when mentioning PEG-DSPE, for example PEG2000-DSPE is meant the PEG of molecular weight 2000 and the binding substances of DSPE.
As described herein; Term " pH is responsive "; For example when explanation the present invention " drug administration carrier is a pH responsive type drug administration carrier "; Be meant that the material that is referred to has different character under condition of different pH, for example be included under solutions of weak acidity that activeconstituents in this drug administration carrier compares under condition of neutral pH, discharging more easily.
As described herein; Term " cancer target performance "; For example when explanation the present invention " drug administration carrier is the drug administration carrier with cancer target performance "; Be meant that the material that is referred to has the performance that is directed to the related tissue position, for example after drug administration carrier administration, can make this drug administration carrier through the folacin receptor mediated for example tumor locus of relevant histoorgan that initiatively is targeted to because of having folic acid functional group with the formula I compound of the present invention that comprises the folic acid part.
One of purport of the present invention is folacin receptor mediated pH responsive type the Zorubicin initiatively preparation and the application of target liposomes.
In the prior art; The liposome vectors material that some research synthetic contain the PEOz block is confined to its single-ended other functional group that is connected with; And the research that the pH susceptibility of PEOz is applied to liposome seldom, more not about through the particular functional group F-PEOz-DSPE three being coupled together to realize the instruction of the object of the invention.
An object of the present invention is to provide a kind of liposome vectors material folic acid-gather compound method of (2-ethyl-2-oxazoline)-DSPE (F-PEOz-DSPE).
It is the method for the target liposomes of liposome vectors material prepn pastille (for example Zorubicin) with F-PEOz-DSPE that another object of the present invention provides a kind of.
Above-mentioned purpose of the present invention realizes through following technical scheme:
One embodiment of the invention provides the novel lipide solid support material F-PEOz-DSPE as shown in the formula structure shown in the I:
Figure BSA00000632652900111
Wherein n, m, p are as indicated above.
In one embodiment, the invention provides compound with following formula Ia:
Figure BSA00000632652900112
Wherein n, m, p are as indicated above.
Another embodiment of the invention provides the method for preparation I compound.In a specific embodiment, the preparation method with following formula Ia compound is provided:
Figure BSA00000632652900121
Wherein n, m, p are as indicated above.
Particularly, be example with formula Ia compound, the present invention provides the compound method of formula Ia compound may further comprise the steps:
Step 1: prepare initiator 6-nitrine-own ester of 1-tosic acid (compound 3) through two step substitution reactions, shown in the following reaction formula:
Figure BSA00000632652900122
Make 6-chloro-1-hexanol (compound 1) and reaction of sodium azide obtain 6-nitrine-1-hexanol (compound 2), 6-nitrine-1-hexanol obtains 6-nitrine-own ester of 1-tosic acid with the Tosyl chloride reaction again;
Step 2: cause 2-ethyl-2-oxazoline (EOz) cationic ring-opening polymerization with the own ester of 6-nitrine-1-tosic acid, generate that an end is amino, an endlap nitrogen gather (2-ethyl-2-oxazoline) (compound 4a), shown in the following reaction formula:
The feed ratio of control monomer and initiator can generate the polymkeric substance of different polymerization degree, the present invention exemplarily synthesized the polymerization degree (n) be respectively 18,32 and 64 3 kind gather (2-ethyl-2-oxazoline) (PEOz);
Step 3: the condensation acylation reaction through carboxylic acid and amine prepares Terminal Acetylenes base DSPE (compound 6), shown in the following reaction formula:
Figure BSA00000632652900124
Generate Terminal Acetylenes base DSPE (compound 6) with 4-pentynoic acid and DSPE (DSPE, compound 5) reaction;
Step 4: prepare folic acid Acibenzolar (compound 8) through esterification, shown in the following reaction formula:
Make folic acid (compound 7) and N-maloyl imine reaction generate folic acid Acibenzolar (compound 8);
Step 5: through " clicking (Click) " chemical reaction, an end of preparation PEOz amino, an endlap nitrogen is connected with Terminal Acetylenes base DSPE in the performing step two, three, generates PEOz-DSPE (compound 9a), shown in the following reaction formula:
Figure BSA00000632652900132
Step 6: through acylation reaction, the folic acid Acibenzolar of preparation is connected with PEOz-DSPE in the performing step four, five, generate F-PEOz-DSPE (compound I a), shown in the following reaction formula:
Figure BSA00000632652900133
The molecular weight of PEOz is relevant with the moisture in initiator and monomeric ratio, reaction times, temperature of reaction and the system.The inventor earlier carries out pre-reaction under 70 ℃, the condition of reaction 24h, transformation efficiency is about 60%.Feed intake according to three kinds of initiators and monomeric different ratios in view of the above, and the proper extension reaction times, the moisture content in the hierarchy of control has exemplarily been synthesized molecular weight and has been respectively 1900,3300,6,500 three kinds of polymkeric substance (polymerization degree n is respectively 18,32,64).
The solid support material that more existing research synthetic contain the PEOz block is confined to its single-ended other functional group that is connected with.The present invention successfully is connected with the different functions group with the both-end of polymer P EOz, and this has laid important foundation for realizing the object of the invention.The initiator of the at first synthetic end azido-of the present invention; Cause cationic ring-opening polymerization, with ammonia/acetonitrile solution termination reaction, generating an end is that nitrine, an end are amino polymer P EOz; Priority reacts with the DSPE and the folic acid of Terminal Acetylenes base again, generates target compound.Wherein the reaction of PEOz and DSPE has utilized the principle of " click chemistry "; Take place 1 through nitrine and Terminal Acetylenes base, the 3-Dipolar Cycloaddition connects the two, because the molecular weight of PEOz and DSPE is all bigger; Reaction between endlap nitrogen and the Terminal Acetylenes base than the reaction between the small molecules more difficulty carry out; Therefore in an instance of the present invention, temperature of reaction is brought up to 50 ℃, the reaction times extends to 72h, obtains PEOz-DSPE.This reaction has very strong selectivity, to not influence of aminoterminal, has avoided the generation of side reaction.The present invention finds pleasantly surprisedly and can " click chemistry " be applied in synthesizing of liposome material.
In one embodiment, the invention provides the method for preparing Evacet with synthetic F-PEOz-DSPE of the present invention.This method for example can be to carry out in the following manner:
At first, with hydrogenated soy phosphatidyl choline (HSPC), SUV (Chol) and synthetic PEOz-DSPE of the present invention (being the compound of formula 9 or formula 9a) or F-PEOz-DSPE (being the compound of formula I or formula Ia) or the PEG that is used to contrast 2000-DSPE prepares by film dispersion method, can obtain optional pH responsive type lipidosome drug carrier with folic acid target property; Wherein, in this lipidosome drug carrier, preferred, each component can comprise according to the molar part meter:
Figure BSA00000632652900141
Should be appreciated that in this article, more than (1), (2), (3) three can be referred to as " TL " or " lipid mixt "; And above (1), (2), (3) three's mixture or the treated formed lipidosome drug carrier of this three also can be described as " TL " or " lipid mixt ".Certainly; It will be apparent to those skilled in the art that; Phrase " the treated formed lipidosome drug carrier of this three " is meant " lipidosome drug carrier " that is obtained after three kinds of compositions are through the liposome preparation process; Should " lipidosome drug carrier " can also comprise other component in addition, and when mentioning " the treated formed lipidosome drug carrier of this three ", term " TL " is meant above-mentioned (1), (2), (3) three in this liposome.For example phrase " TL amount " is meant above-mentioned in " lipidosome drug carrier " (1), (2), (3) three's amount sum, and other composition in the liposome is not counted in " TL amount ".
Then; To have water miscible Zorubicin and the above-mentioned lipidosome drug carrier that makes (promptly) carries out the preparation of drug-loaded liposome by the ammonium sulfate density gradient method, can obtain optional Evacet or Zorubicin contrast liposome with pH responsive type of folic acid target property.In a preferred embodiment, in the liposome that obtains thus, comprise:
?(a) Antitumor drug (for example Zorubicin) 1～30 weight part and
?(b) TL (promptly above (1), (2), (3) three's sum) 70～99 weight parts;
Preferably, comprise:
?(a) Antitumor drug (for example Zorubicin) 1～20 weight part and
?(b) TL (promptly above (1), (2), (3) three's sum) 80～99 weight parts;
Preferably, comprise:
?(a) Antitumor drug (for example Zorubicin) 2～20 weight parts and
?(b) TL (promptly above (1), (2), (3) three's sum) 80～98 weight parts;
Preferably, comprise:
?(a) Antitumor drug (for example Zorubicin) 2～10 weight parts and
?(b) TL (promptly above (1), (2), (3) three's sum) 90～98 weight parts.
Therefore, for example, in an embodiment of the drug administration carrier of seventh aspect present invention, this drug administration carrier comprises the TL that following mol ratio is formed:
Thus, for example in an embodiment of the drug administration carrier of seventh aspect present invention, this drug administration carrier comprises again:
(a) active constituents of medicine (for example antitumor drug, for example Zorubicin) and
(b) TL of following mol ratio composition:
Figure BSA00000632652900152
And wherein said (a) active constituents of medicine with (b) weight of TL is:
Figure BSA00000632652900153
In one embodiment; Drug administration carrier (for example for drug-loaded liposome) above except comprising (a) active constituents of medicine and (b) TL; Can also comprise other medicines activeconstituents and the acceptable shape agent of other pharmacy, for example water, sodium chloride solution, glucose solution etc.
In one embodiment, the drug administration carrier of seventh aspect present invention is a kind of liposomal pharmaceutical prepn, and wherein each component is formed according to following weight percent:
Figure BSA00000632652900154
Figure BSA00000632652900161
Wherein in said mixture, each component mol ratio is:
Figure BSA00000632652900162
It will be appreciated by those skilled in the art that term " weight part " is a kind of weight unit, it can be microgram, milligram, gram, kilogram etc.Similarly, term " molar part " is a kind of molar weight unit, and it can be micromole, mmole, mole etc.
In one embodiment, the invention provides the preparation method of (initiatively target) liposome of pH responsive type, it can may further comprise the steps substantially:
At first, said each lipid composition (for example phosphatide, SUV, formula I of the present invention or formula 9 compounds) is used dissolved in chloroform, rotary evaporation is removed solvent, adds (NH 4) 2SO 4Solution is to adipose membrane, and ultra-sonic dispersion gets blank liposome turbid liquor, with high pressure homogenizer suspension is passed through polycarbonate leaching film; Promptly get blank liposome;
Then, take by weighing Zorubicin, soluble in water, the aqueous solution is added in the blank liposome, the heating jolting promptly gets (initiatively target) liposome of pH responsive type of the present invention.
In one embodiment, the invention provides the preparation method of (initiatively target) liposome of pH responsive type, it can may further comprise the steps particularly:
(1) gets each component by following weight part: Zorubicin 1-30 weight part (preferred 1-20, preferred 2-20, preferred 2-10 weight part), lipid mixt (hydrogenated soy phosphatidyl choline, SUV and PEOz-DSPE (or F-PEOz-DSPE or PEG 2000-DSPE)) 70-99 weight part (preferred 80-99; Preferred 80-98; Preferred 90-98 weight part); In this lipid mixt, the mol ratio of three constituents is: hydrogenated soy phosphatidyl choline 60～70 molar part, SUV 20～38 molar part, PEOz-DSPE (or F-PEOz-DSPE or PEG-DSPE) 2～10 molar part;
(2) above-mentioned each component is used dissolved in chloroform, rotation vacuum-evaporation film forming (temperature is 10-50 ℃, is preferably 20-40 ℃, more preferably about 35 ℃).With 250mmol/L (NH 4) 2SO 4Solution (pH=4.5-6.5 is preferably pH=5.0-6.0, and preferred pH is about 5.5) adds in the adipose membrane, and (temperature is 40-80 ℃ to ultra-sonic dispersion; Be preferably 50-70 ℃, more preferably about 65 ℃, ultra-sonic dispersion 5-60min; Preferred 10-30min, more preferably from about 20min), get blank liposome turbid liquor.With high pressure homogenizer with the blank liposome suspension continuously through the aperture the polycarbonate leaching film of 200nm, 100nm, 80nm (be preferably and respectively filter 5 times, 65 ℃ of extruding down), ammonium sulfate dissolved blank liposome solution.Adopt the wet method perfusion to fill chromatography column, pour into sephadex G-25 continuously, with 10% sucrose solution elution chromatography post (with 5-20 column volume; Be preferably 10 column volumes); Add ammonium sulfate dissolved blank liposome solution and (be preferably flow velocity=1ml/min), collect the milky liquid that contains liposome; With 5% glucose solution elution chromatography post (, being preferably 10 column volumes) with 5-20 column volume.It is an amount of that precision takes by weighing Zorubicin in addition, is dissolved in 10% sucrose solution, this solution is added to collects in the blank liposome solution; In hot water bath, place (preferred 65 ℃, 30min～60min), jolting frequently; The liposome that has wrapped up medicine is added chromatography column, with 5% glucose solution wash-out, to remove the Zorubicin that is not wrapping to liposome interior; Collect effluent, promptly get Evacet.
Through detecting, the prepared liposome of aforesaid method (adopts three kinds of material PEOz-DSPE or F-PEOz-DSPE or PEG respectively 2000All between 90～200nm, entrapment efficiency is all between 85%～98% for-particle diameter DSPE).
Utilize cationic ring-opening polymerization and " click " chemical reaction of the present invention success have prepared the folic acid of three kinds of different molecular weights-gather (2-ethyl-2-oxazoline)-DSPE (F-PEOz-DSPE) liposome vectors material.Experiment showed, that the liposome that is respectively 18,32,64 PEOz-DSPE and F-PEOz-DSPE preparation with the exemplary synthetic polymerization degree of the present invention has excellent particle size distribution and higher entrapment.In experiment in vitro, the F-PEOz-DSPE liposome shows stronger tumor-targeting than PEOz-DSPE liposome and PEG-DSPE liposome, does not see significant cytotoxicity.
Description of drawings
Fig. 1 is the proton nmr spectra of PEOz
Fig. 2 is the carbon-13 nmr spectra of Terminal Acetylenes base DSPE
Fig. 3 is the high-efficient liquid phase chromatogram of Terminal Acetylenes base DSPE
Fig. 4 is that (Fig. 4 a) and the infared spectrum of PEOz-DSPE (Fig. 4 b) for PEOz
Fig. 5 is the uv-spectrogram of PEOz-DSPE and F-PEOz-DSPE
Fig. 6 is the gel permeation chromatography figure of F-PEOz-DSPE
Fig. 7 is the proton nmr spectra of F-PEOz-DSPE
Fig. 8 has described the cumulative release rate change curve of three kinds of liposomes in the phosphate buffered saline buffer of pH 7.4 and pH 5.0.Each figure in, ordinate zou is cumulative release percentage ratio (%), X-coordinate be time of releasing (hour).Wherein Fig. 8 A is the release profiles of PEG-DSPE liposome, and Fig. 8 B is the release profiles of PEOz-DSPE liposome, and Fig. 8 C is the release profiles of F-PEOz-DSPE liposome.
Fig. 9 has described the external pharmacodynamics evaluation result of three kinds of liposomes to SKOV3 cell (Fig. 9 A) and A549 cell (Fig. 9 B).Each figure in, ordinate zou is inhibiting rate (%), X-coordinate be incubation time (hour).In Fig. 9 A, what the post at left side DOX place was represented is the inhibiting rate without the Zorubicin pair cell of liposome; 0.25,0.5,1,2,4,12,24 or the time point place of 48h, from left to right 3 posts represent to wrap the inhibiting rate of medicine liposome PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE pair cell respectively separately; In Fig. 9 B, each post implication is with identical at Fig. 9 A.
Figure 10 A has described the influence of the liposome pair cell absorption of different film material different concns.
Figure 10 B has described the liposome influence that pair cell is taken under differing temps of different film materials.
Figure 10 C has described the influence that cell is taken in liposome under different incubation times of different film materials.
Figure 10 D has described the absorption of flow cytometer showed cell to different liposome.
Embodiment
Can further describe the present invention through following embodiment, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the TP that are used in the test.Though for realizing that employed many materials of the object of the invention and working method are well known in the art, the present invention still does detailed as far as possible description at this.In the present invention, like not explanation in addition, entrapment efficiency is to measure with classical sephadex column partition method, and the liposome particle diameter uses the particle diameter appearance to measure.
A, embodiment part: preparation F-PEOz-DS
Embodiment 1 to 7 is the synthetic instance of F-PEOz-DSPE (is example with formula Ia compound).
The preparation of embodiment 1:6-nitrine-1-hexanol (compound 2)
Like following formula, in exsiccant 250mL there-necked flask, add 6-chloro-1-hexanol (compound 1) 10g (73mmol), NaN 314.3g (220mmol), NaI 1.1g (7.3mmol) and the dry 100mLDMF that crosses, stirring reaction 18h under 80 ℃ of oil baths.After reacting end, vacuum rotary steam adds ETHYLE ACETATE 200mL dilution, difference water and saturated aqueous common salt washed twice, K to doing 2CO 3Dry organic phase, suction filtration, filtrate decompression is concentrated into dried colourless liquid 10.02g, and yield is 95%.
1H?NMR(300MHz，CDCl 3)δ：1.30-1.71(m，8H)；3.28(t，2H)；3.65(t，2H)。
Embodiment 2: the preparation of initiator 6-nitrine-own ester of 1-tosic acid (compound 3)
Figure BSA00000632652900191
Like following formula; In exsiccant 500mL single port flask, add the dry chloroform of crossing of 6-nitrine-1-hexanol (compound 2) 10g (70mmol), triethylamine 20mL (145mmol), trimethylamine hydrochloride 1.3g (13.5mmol) and 300mL; Stirring and dissolving dropwise adds the dry CHCl of 60mL of Tosyl chloride 20g (105mmol) under the ice bath 3Solution, again in room temperature reaction 1h, the reaction solution vacuum rotary steam concentrates behind 0～5 ℃ of reaction 1h, wash to organic layer colourless, anhydrous CaCl 2Dry organic phase, suction filtration, filtrate decompression is revolved and is steamed to doing, and gets colourless liquid.(eluent is an ETHYLE ACETATE: sherwood oil=1: 30), get pure article is colourless liquid 10.39g to bullion, productive rate 50% through the silica gel chromatography column separating purification.
1H?NMR(300MHz，CDCl 3)δ：1.27(m，4H)；1.41-1.61(m，4H)；2.46(s，3H)；3.23(t，2H)；4.02(t，2H)；7.34(d，2H)；7.79(d，2H)。
Proton nmr spectra (not shown) shows that terminal hydroxy group has changed into p-toluenesulfonic esters.In addition, in infared spectrum (not shown), can see at 2097cm -1There is the charateristic avsorption band of nitrine at the place.Proton nmr spectra is identical with expected structure with infared spectrum explanation synthetic endlap nitrogen initiator structure.
Embodiment 3: the preparation that gathers (2-ethyl-2-oxazoline) (compound 4a)
Like following formula, in initiator (compound 3), add an amount of toluene, twice component distillation of warp is to remove residual moisture.Add EOz monomer 8.5mL (84mmol) and acetonitrile 25mL respectively in three Schlenk bottles on being connected vacuum line; Add initiator (compound 3) 0.620mL (2.5mmol) under the nitrogen respectively; 0.310mL (1.3mmol), 0.155mL (0.63mmol) (by three kinds of different molecular weight designs, is denoted as 1; 2, No. 3 bottles).The Schlenk bottle is sealed back 70 ℃ of reactions down, and 1, No. 2 bottle respectively reacts 24h, No. 3 bottle reaction 48h.1,2,3 bottle be connected in feed high pure nitrogen on the vacuum line, add NH under the ice bath respectively 3/ acetonitrile solution (ammonia is blasted in the acetonitrile, and molecule number ratio is 1: 16) 23.6mL, 11.8mL, 5.9mL react 24h under the room temperature, are used for termination reaction.Add K afterwards respectively 2CO 325g (181mmol) stirs 24h under the room temperature, is used to remove tosic acid.After reaction finished, suction filtration was removed K 2CO 3, rotary evaporation removes and to desolvate, the minimum of chloroform dissolving dropwise is added in the ether precipitated product 2 times, suction filtration, after the vacuum-drying white solid 6.33g, 5.08g, 4.66g, productive rate is respectively 76%, 61%, 56% (being numbered PEOz1, PEOz2 and PEOz3).
The sign of polymer P EOz and the mensuration of molecular weight
Fig. 1 is PEOz's 1H NMR spectrogram, chemical shift δ=1.13 places are the methyl (CH on the side chain 3) proton peak; δ=2.44 places are by methylene radical (O=C-CH on the side chain 2-) proton causes; Methylene radical (CH on the main chain 2-CH 2-N-) proton peak appears at δ=3.57 places.In addition, in infared spectrum, can see at 2099cm -1There are the charateristic avsorption band of nitrine, 1639cm in the place -1Characteristic peak for teritary amide (O=C-N-).More than explanation synthetic polymer architecture conforms to design.
For measuring the molecular weight of polymkeric substance, " Click " (" click ") reaction is taken place with compound 1-ethanoyl-2-nitro-4-proyl oxygen base-5-anisole respectively in three kinds of polymkeric substance (PEOz1, PEOz2 and PEOz3), utilize 1The integration of H on H in the H NMR spectrogram on the phenyl ring and the repeating unit shows than the molecular weight that calculates polymkeric substance with by molecular weight such as table 1 that turnover ratio calculates, uses 1The calculation result of H NMR is as the molecular weight of polymkeric substance, and the polymerization degree is respectively 18,32 and 64.
Figure BSA00000632652900201
" Click " reaction
The mensuration result of table 1 PEOz molecular weight
Figure BSA00000632652900202
Embodiment 4: the preparation of Terminal Acetylenes base DSPE (compound 6)
Figure BSA00000632652900203
Like following formula, take by weighing 4-pentynoic acid 0.8g (8.2mmol) and DCC 1.2g (5.8mmol) and be dissolved in the dry CHCl that crosses of 50mL 3In, activation 4h under the room temperature adds 200 μ L pyridines and DSPE (compound 5) (3g, 50mL CHCl 4mmol) 3Solution, 50 ℃ of reacting by heating 12h, suction filtration is removed by product DCU, organic phase washing 3～5 times, anhydrous Na SO 4Dry 2h, rotary evaporation are to dry, and vacuum-drying 12h gets white solid 2.48g, and productive rate is 75%.
The sign of Terminal Acetylenes base DSPE
ESI-MS:m/z 826.6 [M-H] -, 2300cm in the infared spectrum (not shown) -1The place is the stretching vibration characteristic peak of Terminal Acetylenes base (H-C ≡ C-).
13C NMR (400MHz, CDCl 3) δ: 173.41 (m); 83.12 (l); 69.75 (k); 69.13 (j); 62.11 (i); 35.12 (h); 34.24 (f); 32.75 (g); 31.90 (e); 29.41 (c); 25.12 (d); 22.84 (b); (a) 14.12 (like Fig. 2).
HPLC:Diamond C 18Chromatographic column (250mm * 4.6mm, 5 μ m); Moving phase is water: methyl alcohol=70: 30, flow velocity 1.0mL/min; Detect wavelength 242nm; 30 ℃ of column temperatures; Sample size 20 μ L.High-efficient liquid phase chromatogram is seen Fig. 3, and the t=10.784min place is the chromatographic peak of Terminal Acetylenes base DSPE, and peak area ratio is 96.94%, and the Terminal Acetylenes base DSPE that visible present embodiment obtains has higher degree.
Embodiment 5: folic acid Acibenzolar (compound 8, preparation F-NHS)
Like following formula; Take by weighing folic acid (compound 7)) 5g (11.3mmol), N-maloyl imines (NHS) 1.3g (11.3mmol) places the 100mL reaction flask, adds triethylamine 25mL; DMSO 40mL; Lucifuge is led to nitrogen 10min, treats that folic acid dissolves the back fully and adds DCC 2.5g (12.1mmol), lucifuge room temperature reaction 24h.Suction filtration is removed DCU, and filtrating splashes in the 400mL ETHYLE ACETATE, produces yellow mercury oxide, suction filtration; With washing with alcohol filter cake twice,, splash into precipitated product in the 300mL ether again, suction filtration with 30mL DMSO dissolving filter cake; Vacuum-drying 12h gets red-brown solid 4.51g, and productive rate is 74%.
Folic acid (F) 1H NMR (300MHz, D 2O/NaOD) δ: 8.01 (1H); 7.20 (2H); 6.17 (2H); 3.98 (2H); 3.87 (1H); 2.04 (2H); 1.751.70 (2H).
F-NHS? 1H?NMR(300MHz，D 2O/NaOD)δ：8.16(1H)；7.26(2H)；6.18(2H)；4.15(2H)；4.05(1H)；2.30(4H)；2.10(2H)；1.851.80(2H)。4 corresponding H of δ=2.30 places are the H on the succinimide ring that is connected on the folic acid.1726cm in the infared spectrum -1The place judges to sum up that for the characteristic peak of newly-generated ester (C (=O) O-) folic acid Acibenzolar structure is correct.
The preparation of embodiment 6:PEOz-DSPE (compound 9a)
Figure BSA00000632652900212
Like following formula; Get each 500mg of PEOz1, PEOz2 and PEOz3 (compound 4a) and put into polymerizing pipe; Add Terminal Acetylenes base DSPE (compound 6) 400mg (0.48mmol), 250mg (0.30mmol), 140mg (0.17mmol) respectively; And add part PMDETA (ρ=0.829g/mL) 110 μ L, 69 μ L, 38 μ L, CHCl respectively 3Each 2mL.After twice " cooled with liquid nitrogen-vacuumize-thaw " cyclical operation, feed high pure nitrogen, add CuBr 72mg (0.5mmol), 45mg (0.3mmol), 25mg (0.17mmol) respectively; Vacuumize; Carry out once " cooled with liquid nitrogen-vacuumize " operation again, tube sealing under the vacuum, 50 ℃ are reacted 72h down.After reaction stops, splashing into precipitated product in the ether respectively, filter, solid is used water dissolution, and centrifugal (3800rpm * 5min), repeat 3 times removes insolubles, merges the aqueous solution, freeze-drying.Obtain light green solid (a small amount of mantoquita removes when dialysing at last) 447mg, 447mg, 477mg, productive rate is respectively 63%, 72% and 85%.
Hydrogen spectrum (not shown) and the INFRARED SPECTRUM of the PEOz-DSPE of the above preparation of test. 1In H NMR (400MHz) collection of illustrative plates, there is faint " Click " fignal center at δ=7.96 places, are the proton peak on the newly-generated triazole ring, and δ=1.17 are the proton peak on the DSPE carbon long-chain.2099cm in its infared spectrum (like Fig. 4 b, Fig. 4 a is the infared spectrum of PEOz) -1The nitrine characteristic peak at place disappears, and has explained " Click " reaction has taken place between nitrine and the alkynyl, and promptly through 1, the 3-Dipolar Cycloaddition generates triazole PEOz is connected with DSPE, generates PEOz-DSPE.
Measure through solvability, to be converted to the mole number of DSPE, PEOz1-DSPE, PEOz2-DSPE, the solubleness of PEOz3-DSPE in water (20 ℃) all are more than 10 times of DSPE.
Embodiment 7:F-PEOz-DSPE (compound I preparation a)
Figure BSA00000632652900221
Like following formula; Take by weighing PEOz1-DSPE 300mg (0.11mmol), PEOz2-DSPE 200mg (0.05mmol), PEOz3-DSPE 300mg (0.04mmol) (compound 9a) respectively in three reaction flasks, add F-NHS (compound 8) 180mg (0.33mmol), 80mg (0.15mmol), 66mg (0.12mmol) respectively; Triethylamine 900 μ L, 400 μ L, 240 μ L; Each 1.5mL of DMSO, lucifuge is led to nitrogen 10min, 50 ℃ of lucifuge reaction 72h.After reaction finishes, splash in the ether respectively and be settled out filtration; With chloroform solid is dissolved, filter, get yellow filtrate; Rotary evaporation removes and desolvates, and with the less water dissolving, dialysis (MwCO 1000) is two days in zero(ppm) water; Lyophilize gets faint yellow solid F-PEOz1-DSPE 165mg, F-PEOz2-DSPE 160mg, F-PEOz3-DSPE 184mg respectively, and productive rate is respectively 55%, 80%, 61%.
The sign of F-PEOz-DSPE
Test PEOz-DSPE and the F-PEOz-DSPE uv-absorbing in THF respectively, show that like Fig. 5 the maximum absorption band of PEOz-DSPE is at the 244nm place; The maximum absorption band of F-PEOz-DSPE appears at 256nm and 290nm place, and this also is the charateristic avsorption band of folic acid.Detect its gel permeation chromatography of method survey figure (chromatographic column: Waters Styragel HR post 1 * 10 of (IR) and dual wavelength detection respectively with differential 4, 1 * 10 3And The aperture; Solvent: THF; Flow velocity: 1mL/min; Column temperature: 35 ℃), as shown in Figure 6, the product peak appears at the 22.5min place.
Fig. 7 has shown the proton nmr spectra of F-PEOz-DSPE, 1H NMR (300MHz, DMSO) δ: 8.70 (1H); 7.70 (2H); 6.71 (2H); 4.45 (2H); 4.25 (1H) for being connected the proton peak of the folic acid on the polymkeric substance, δ=8.00 (1H) is the proton peak on the triazole ring, δ=3.35,2.28 and 0.94 are the proton peak of polymer repeat unit, and δ=1.26 are the proton peak of carbon long-chain among the DSPE.More than the product structure of test conforms to the structure of estimating F-PEOz-DSPE.
Measure through solvability, to be converted to the mole number of DSPE, PEOz1-DSPE, PEOz2-DSPE, the solubleness of PEOz3-DSPE in water (20 ℃) all are more than 10 times of DSPE.
Embodiment 8: preparation m is 4 or 8 formula I compound
The method of reference implementation example 1-7; In embodiment 1, be starting substance with 4-chloro-1-butanols and 8-chloro-1-octanol respectively; In embodiment 3, adopt the reaction conditions that obtains n=32, make the formula I compound of n=32, m=4 and p=2 and the formula I compound of n=32, m=8 and p=2.The gained compound characterizes and performance measurement through embodiment 1-7 similar approach, the conforming to of structure structure and design as a result.
Embodiment 9: preparation p is 1 or 4 formula I compound
The method of reference implementation example 1-7; In embodiment 4, be starting substance with 3-butynoic acid and 6-heptynoic acid respectively; In embodiment 3, adopt the reaction conditions that obtains n=18, make the formula I compound of n=18, m=6 and p=1 and the formula I compound of n=18, m=6 and p=4.The gained compound characterizes and performance measurement through embodiment 1-7 similar approach, the conforming to of structure structure and design as a result.
B, preparing carriers example: contain the preparation of the drug administration carrier of active constituents of medicine
Preparation example 1: contain PEG in the film material 2000 The preparation of the Evacet of-DSPE
The consumption of each component: Zorubicin 2-10 weight part; Lipid mixt (hydrogenated soy phosphatidyl choline, SUV and PEG 2000The mixture of-DSPE) 90-98 weight part, in this lipid mixt, hydrogenated soy phosphatidyl choline 60-70 molar part, SUV 20-38 molar part; And polyoxyethylene glycol-DSPE (PEG 2000-DSPE) 2-10 molar part.
Adopt the preparation of ammonium sulfate density gradient method, get blank liposome with high pressure homogenizer control particle diameter.It is an amount of that other takes by weighing Zorubicin, soluble in water, this solution is added to collects in the blank liposome solution; In hot water bath, place (preferred 65 ℃, 30min～60min), jolting frequently; The liposome that has wrapped up medicine is added chromatography column, with 5% glucose solution wash-out, to remove the Zorubicin that is not wrapping to liposome interior; Collect effluent, promptly get and contain PEG 2000-DSPE Evacet.
With Zorubicin 10 weight parts and lipid mixt (hydrogenated soy phosphatidyl choline 70 molar part, SUV 20 molar part and PEG 2000The mixture of-DSPE 10 molar part) 90 weight part feed ratio prepare liposome, with the make an experiment research of example part of this liposome.With Zorubicin 2 weight parts and lipid mixt (hydrogenated soy phosphatidyl choline 60 molar part, SUV 38 molar part and PEG 2000The mixture of-DSPE 2 molar part) 98 weight part feed ratio prepare liposome.With Zorubicin 5 weight parts and lipid mixt (hydrogenated soy phosphatidyl choline 65 molar part, SUV 30 molar part and PEG 2000The mixture of-DSPE 5 molar part) 95 weight part feed ratio prepare liposome.These liposomes with the PEG preparation are analogous to business-like PEGization DSPE liposome.
More than the liposome particle diameter that makes of three kinds of feed ratio between 90～200nm, entrapment efficiency is between 85%～98%.
Preparation example 2: contain the preparation of the Evacet of PEOz-DSPE in the film material
The consumption of each component: Zorubicin 2～20 weight parts; ((embodiment 6 makes lipid mixt for hydrogenated soy phosphatidyl choline, SUV and PEOz-DSPE; Polymerization degree n is respectively 18,32,64) mixture) the 80-98 weight part; In this lipid mixt, hydrogenated soy phosphatidyl choline 60-70 molar part, SUV 20-38 molar part; With gather (2-ethyl-2-oxazoline)-DSPE (PEOz-DSPE, the polymerization degree is respectively 18,32,64) 2-10 molar part.
The preparation method of reference preparation example 1 gets an amount of hydrogenated soy phosphatidyl choline, SUV and PEOz-DSPE (polymerization degree is respectively 18,32,64) with the method operation, makes to contain PEOz-DSPE Evacet (polymerization degree is respectively 18,32,64).
Prepare liposome with Zorubicin 10 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 70 molar part, SUV 20 molar part and PEOz-DSPE (n=18) 10 molar part) 90 weight part feed ratio, with the make an experiment research of example part of this liposome.Prepare liposome with Zorubicin 2 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 60 molar part, SUV 38 molar part and PEOz-DSPE (n=32) 2 molar part) 98 weight part feed ratio.Prepare liposome with Zorubicin 5 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, SUV 30 molar part and PEOz-DSPE (n=64) 5 molar part) 95 weight part feed ratio.Prepare liposome with Zorubicin 20 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, SUV 30 molar part and PEOz-DSPE (n=64) 6 molar part) 80 weight part feed ratio.
More than the liposome particle diameter that makes of various feed ratio between 90～200nm, entrapment efficiency is between 85%～98%.
Preparation example 3: contain the preparation of the Evacet of F-PEOz-DSPE in the film material
The consumption of each component: Zorubicin 2-20 weight part; ((embodiment 7 makes lipid mixt for hydrogenated soy phosphatidyl choline, SUV and F-PEOz-DSPE; Polymerization degree n is respectively 18,32,64) mixture) the 80-98 weight part; In this lipid mixt, hydrogenated soy phosphatidyl choline 60-70 molar part, SUV 20-38 molar part; And folic acid-gather (2-ethyl-2-oxazoline)-DSPE (F-PEOz-DSPE, the polymerization degree is respectively 18,32,64) 2-10 molar part.
The preparation method of reference preparation example 1 gets an amount of hydrogenated soy phosphatidyl choline, SUV and F-PEOz-DSPE (polymerization degree is respectively 18,32,64) with the method operation, makes to contain F-PEOz-DSPE Evacet (polymerization degree is respectively 18,32,64).
Prepare liposome with Zorubicin 10 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 70 molar part, SUV 20 molar part and F-PEOz-DSPE (n=18) 10 molar part) 90 weight part feed ratio, with the make an experiment research of example part of this liposome.Prepare liposome with Zorubicin 2 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 60 molar part, SUV 38 molar part and F-PEOz-DSPE (n=64) 2 molar part) 98 weight part feed ratio.Prepare liposome with Zorubicin 5 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, SUV 30 molar part and F-PEOz-DSPE (n=32) 5 molar part) 95 weight part feed ratio.Prepare liposome with Zorubicin 20 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, SUV 30 molar part and F-PEOz-DSPE (n=32) 6 molar part) 80 weight part feed ratio.Prepare liposome with Zorubicin 30 weight parts and lipid mixt (mixtures of hydrogenated soy phosphatidyl choline 65 molar part, SUV 30 molar part and F-PEOz-DSPE (n=32) 5 molar part) 70 weight part feed ratio.
More than the liposome particle diameter that makes of various feed ratio between 90～200nm, entrapment efficiency is between 85%～98%.
C, Test Example part: the pharmaceutical composition of liposome or the performance of drug administration carrier are investigated
The measuring method of Test Example 1, hydrochloric doxorubicin liposome pH susceptibility
1, reagent: first kind of liposome (promptly by 10 parts of Zorubicins and 90 parts of liposomes that lipid mixt makes) that preceding text preparation example 1,2,3 makes respectively is designated as PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE respectively; The PBS of pH 7.4 (pH 7.4 for potassium primary phosphate, 0.05M), the PBS of pH 5.0 (pH 5.0 for SODIUM PHOSPHATE, MONOBASIC, 0.05M).
2, TP: three kinds of each 1mL of hydrochloric doxorubicin liposome of accurate absorption pack in the dialysis tubing (MWCO:1000); Place the pH 7.4 and pH 5.0 phosphate buffered saline buffers of 100mL respectively; Six beakers are put into rotary constant temperature oscillator, 37 ℃ of following joltings (40r/min).Regularly (15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h 48h) takes out release medium 0.5mL in each beaker, replenishes the fresh release medium of respective amount simultaneously.Measure the medicament contg in each time point release medium with the HPLC method, calculating cumulative release degree is drawn release profiles.
3, test-results: three kinds of liposomes are in the phosphate buffered saline buffer of pH 7.4 and pH 5.0, and cumulative release rate change curve is respectively shown in Fig. 8 A, Fig. 8 B, Fig. 8 C in the 48h.The result shows that the release under two kinds of pH conditions of PEG-DSPE liposome does not have notable difference, and the cumulative release rate reaches about 50% (seeing Fig. 8 A) in the 48h.PEOz-DSPE is similar with the release profiles of PEG-DSPE liposome with F-PEOz-DSPE liposome rate of releasing drug in the release medium of pH 7.4, and the cumulative release rate reaches about 50% in the 48h; But they in the release medium of pH 5.0 rate of releasing drug obviously faster than the rate of releasing drug in pH 7.4 release medium; And the cumulative release rate reaches about 100% (seeing Fig. 8 B and Fig. 8 C) in 48h, and visible PEOz-DSPE and two kinds of liposomes of F-PEOz-DSPE demonstrate tangible pH susceptibility.This character helps medicine and under weak acid environments such as tumor tissues and inclusion body, discharges, and can solve the PEG liposome in target site deposition and discharge problem slowly, improves curative effect of medication, reduces dosage.
Test Example 2, the external evaluating drug effect of liposome
1, reagent: first kind of liposome (promptly by 10 parts of Zorubicins and 90 parts of liposomes that lipid mixt makes) that preceding text preparation example 1,2,3 makes respectively is designated as PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE respectively
2, TP:
For investigating folacin receptor (FR) the target property of liposome of the present invention; In this Test Example; Choosing the ovarian cancer cell line SKOV3 cell of FR (+) and the lung adenocarcinoma cell of FR (-) is the cell in vitro experiment that the A549 cell carries out liposome, with the target property and the validity of checking liposome.Wherein FR (+) expression folacin receptor is positive, i.e. high expression level folacin receptor, and FR (-) expression folacin receptor is negative, the promptly low folacin receptor of expressing.
Because two kinds of cells that use in this test are different to the susceptibility of medicine; Before carrying out to liposome toxicity and evaluating drug effect; Need to measure the effective concentration of Zorubicin (as not indicating in addition, the used reagent Zorubicin of the present invention refers to its hydrochloride, can be expressed as DOX at this paper) to two kinds of cancer cells.This test will be killed the DOX concentration of 80% cancer cells as active drug concentration, promptly will measure the IC of DOX to two kinds of cells 80This result will provide foundation for follow-up toxicity assessment and evaluating drug effect.Warp is measured the doxorubicin hydrochloride of different concns to SKOV3 and A549 two kinds of cell inhibiting rates, IC as a result 80(SKVO3)=12.5 μ g/mL; IC 80(A549)=100 μ g/mL.In addition through measuring; Three kinds of blank liposomes (prepare first kind of liposome preparation that example 1,2,3 makes respectively with reference to preceding text; But do not add the medicine Zorubicin) can not influence the growth of two kinds of cancer cells, it there is not toxic action, to the not influence of drug action evaluation of the liposome of parcel DOX.
According to the drug sensitive test result, select two kinds of cells (SKOV3, IC A549) 80As administration concentration; Detect first kind of liposome (promptly, being designated as PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE respectively) that three kinds of preceding text preparation example 1,2,3 makes respectively fragmentation effect with mtt assay to different tumour cells by 10 parts of Zorubicins and 90 parts of liposomes that lipid mixt makes.
The same drug sensitive test of cell culture processes; Prepare certain density drug-loaded liposome, make contained drug concentration wherein meet the IC of two kinds of clones 80(SKOV3:12.5 μ g/mL; A549:100 μ g/mL); The three kinds of drug-loaded liposomes (PEG-DSPE, PEOz-DSPE, F-PEOz-DSPE) that prepare are hatched with two kinds of clones respectively jointly; After hatching 15min, 30min, 1h, 2h, 4h, 12h, 24h, 48h, the substratum that contains drug-loaded liposome with the ordinary culture medium replacement originally continues culturing cell.Each time period all is provided with 5 parallel control holes, be provided with simultaneously do not contain liposome under the equal drug level naked medicine group as positive control, the blank liposome under the equal liposome concentration is as negative control and the blank of not doing any processing; More than all experimental group after cultivating 48h, unifiedly detect cytoactive with mtt assay, calculate the inhibiting rate of medicine cell growth, and each group data carried out statistical study.
3, test-results: three kinds of drug-loaded liposomes to the fragmentation effect of SKOV3 and two kinds of cancer cells of A549 shown in Fig. 9 A and Fig. 9 B; The cancer cells inhibiting rate of PEOz-DSPE and two kinds of liposomes of F-PEOz-DSPE is apparently higher than business-like PEG-DSPE liposome; And it is particularly evident in initial 1h; This is consistent with three kinds of liposome extracorporeal releasing test results; PEOz-DSPE and F-PEOz-DSPE liposome have under sour environment that medicine is prominent releases phenomenon, have proved that further the PEOz block can produce pH susceptibility.Two kinds of liposomes of PEOz-DSPE and F-PEOz-DSPE do not have notable difference to the fragmentation effect of the A549 cell of FR (-); And for the SKOV3 cell of FR (+); The F-PEOz-DSPE liposome that contains the folic acid group embodies target property behind 12h, fragmentation effect is apparently higher than the PEOz-DSPE liposome that does not have the target group.
More than the experiment of these Test Example show that the growth of three kinds of blank liposome pair cells is not influence almost, do not disturb the drug effect detected result of drug-loaded liposome; Two kinds of liposomes of PEOz-DSPE and F-PEOz-DSPE show the cancer cells restraining effect that is superior to commercialization PEG-DSPE liposome; It is thus clear that the pH susceptibility that the PEOz block embodies helps medicine and in cancer cells, plays a role; Improve the restraining effect of medicine, thereby improve curative effect of medication cancer cells; The F-PEOz-DSPE liposome embodies folic acid target property; The SKOV3 cell inhibiting effect of FR (+) is better than the PEOz-DSPE liposome of no target; A549 cell to FR (-) does not then show target property; This folic acid target property not only can improve the curative effect of medicine to FR (+) tumor tissues, can also reduce the toxic side effect of medicine to other tissue and organ simultaneously.
The biological characteristics of Test Example 3, F-PEOz--DSPE liposome
1, TP: use the SKOV3 cell, its cell to each folic acid liposome of vitro detection is taken in situation.The folic acid liposome utilizes the 25-NBD-SUV to carry out mark, can disperse green fluorescence, under fluorescent microscope, observes.Different with 2 used reagents with Test Example 1, this Test Example 3 is used the method for reference implementations example 1-7 and the routine 1-3 of preparation, prepares three kinds of blank liposomes that do not add activeconstituents as reagent:
(1) P2000 liposome (in this test, can be called for short P2000): with reference to the method for preparing example 3; Do not add Zorubicin, prepare liposome with the feed ratio of hydrogenated soy phosphatidyl choline 70 molar part, SUV 15 molar part, 25-NBD-SUV 5 molar part and PEOz1-DSPE (embodiment 6 preparation) 10 molar part.
(2) F-P2000 liposome (in this test, can be called for short F-P2000): with reference to the method for preparing example 3; Do not add Zorubicin, prepare liposome with the feed ratio of hydrogenated soy phosphatidyl choline 70 molar part, SUV 15 molar part, 25-NBD-SUV 5 molar part and F-PEOz1-DSPE (embodiment 7 preparation) 10 molar part.
(3) F-P3000 liposome (in this test, can be called for short F-P2000): with reference to the method for preparing example 3; Do not add Zorubicin, prepare liposome with the feed ratio of hydrogenated soy phosphatidyl choline 70 molar part, SUV 15 molar part, 25-NBD-SUV 5 molar part and F-PEOz2-DSPE (embodiment 7 preparation) 10 molar part.
2, the influence of the liposome pair cell of different concns absorption: SKOV3 and the various liposome that is mixed with different concns were hatched under 37 ℃ 2 hours, the situation that observation of cell is taken under the fluorescent microscope, the result sees Figure 10 A.Can find out that from last figure the fluorescence intensity of cell (solar flare among the figure) strengthens with the increase of liposome concentration, and the fluorescence intensity of two groups of F-P2000 and F-P3000 is organized apparently higher than P2000 under same concentrations.The increase that liposome concentration is described can strengthen the absorption of cell to liposome, and the liposome of folic acid target also can significantly improve the absorption of cell to liposome under the condition that concentration equates.Subsequent experimental selects for use the liposome of 200uM concentration to carry out.
3, pair cell is taken in the influence of liposome under the condition of different temperatures: the SKOV3 cell was hatched respectively 1 hour under 4 ℃ and 37 ℃ of conditions; Fluorescent microscope is observed down; The result sees Figure 10 B; Find that cell is difficult to take in liposome under 4 ℃ of conditions; And under 37 ℃ of conditions, can normally take in liposome and make cell send green fluorescence (solar flare among the figure), and fluorescence intensity is obvious higher than P2000 in folic acid target liposomes F-P2000 and F-P3000, explains that receptor-mediated folic acid target has increased the absorption of cell to the folic acid liposome.
4, the influence that cell is taken in liposome under the different incubation times: SKOV3 and each liposome are examined under a microscope after hatching different time respectively under 37 ℃, and the result sees Figure 10 C.The fluorescence intensity (solar flare among the figure) of finding cell strengthens the prolongation of incubation time gradually, and F-P2000 is obviously high than the P2000 group with the amount that F-P3000 was taken in by cell after 20 minutes, has shown good receptor-mediated target absorption.
5, the flow cytometer showed cell is to the absorption of different liposome: after SKOV3 is hatched different time with various liposomes respectively; With being dispersed into single cell suspension after the trysinization; The green fluorescence intensity that utilizes the flow cytometry cell to disperse; The result sees Figure 10 D, and 4 of three reagent groups post figure among the figure are respectively the incubation time of 5min, 15min, 30min and 50min from left to right.Visible from figure, fluorescence intensity numerical value strengthens with the prolongation of incubation time, and after hatching 15 minutes, the fluorescence intensity of F-P2000 and F-P3000 group cell is all organized apparently higher than P2000, and along with the prolongation of incubation time significantly strengthens.
What the present invention exemplarily synthesized three kinds of different polymerization degrees gathers (2-ethyl-2-oxazoline) (PEOz); And be connected through chemical bond with folic acid with DSPE (DSPE), be intended to synthesize the highly selective liposome vectors material that has tumor-targeting and pH susceptibility simultaneously.PEOz has well water-soluble and biocompatibility, and toxicity is low and have temperature sensitivity and pH susceptibility, and its pH susceptibility can promote the release of medicine in tumor tissues.The great majority research of existing report be with the PEOz block as the micellar solid support material, study micellar character, confirmed its pH susceptibility, and the research that this character is used for liposome at present also seldom.The present invention through special method with the PEOz block as the hydrophilic parts in the liposome vectors material; One end is connected with the target group; One end is connected with hydrophobic part; Synthesize novel lipide solid support material F-PEOz-DSPE, it has tumor-targeting and pH susceptibility and can be used as a kind of good liposome material.

Claims (15)

1. with the following formula I compound:
Figure FSA00000632652800011
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
2. the compound of claim 1, it is characterized in that following (a) to (d) each or multinomial:
(a) n is 10 to 250 integer.
(b) m is 3 to 10 integer;
(c) p is 1 to 6 integer;
(d) n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 4 integer.
3. the compound of claim 1, it is with following formula Ia compound:
Figure FSA00000632652800012
Or acceptable salt on its physiology, wherein n is 5 to 500 integer.
4. with following formula 9 compounds:
Figure FSA00000632652800013
Or acceptable salt on its physiology, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
5. the compound of claim 4, it is characterized in that following (a) to (d) each or multinomial:
(a) n is 10 to 250 integer.
(b) m is 3 to 10 integer;
(c) p is 1 to 6 integer;
(d) n is 10 to 250 integer, and m is 2 to 8 integer, and p is 1 to 4 integer.
6. the compound of claim 4, it is with following formula 9a compound:
Figure FSA00000632652800021
Or acceptable salt on its physiology, wherein n is 5 to 500 integer.
7. following formula 4 compounds:
Figure FSA00000632652800022
Or its salt, wherein n is 5 to 500 integer, m is 2 to 12 integer.
8. the compound of claim 7, it is characterized in that following (a) to (d) each or multinomial:
(a) n is 10 to 250 integer.
(b) m is 3 to 10 integer;
(c) n is 10 to 250 integer, and m is 2 to 8 integer;
(d) it is with following formula 4a compound:
Figure FSA00000632652800023
Or acceptable salt on its physiology, wherein n is 5 to 500 integer.
9. prepare the method for each said formula 9 compounds of claim 4-6, it may further comprise the steps:
(i) in the presence of suitable catalyzer, in The suitable solvent, make the carboxylic acid and the DSPE reaction of the base of Terminal Acetylenes shown in the following formula,
Figure FSA00000632652800031
Obtain containing formula 6 compounds of Terminal Acetylenes base:
(ii) make with the reaction of following formula 4 compound step (i) gained formulas 6 compounds,
Obtain with following formula 9 compounds:
Figure FSA00000632652800034
Wherein, wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
10. prepare the method for claim 7 or 8 said formula 4 compounds, it may further comprise the steps:
(ia) in the presence of reagent; In solvent; Make compound and reaction of sodium azide shown in the formula
Figure FSA00000632652800035
, obtain formula
Figure FSA00000632652800036
compound;
(ib) in the presence of reagent; In solvent; Make the reaction of formula compound and Tosyl chloride, obtain formula
Figure FSA00000632652800038
compound;
(ic) in solvent; Make the monomer reaction of formula
Figure FSA00000632652800041
compound and formula
Figure FSA00000632652800042
; Charge into ammonia then with stopped reaction, obtain with following formula 4 compounds:
Figure FSA00000632652800043
Wherein, X is a halogen, and n is 5 to 500 integer, and m is 2 to 12 integer.
11. prepare the method for each said formula I compound of claim 1-3, it may further comprise the steps:
(a) in solvent, in the presence of catalyzer, make folic acid and following formula N-maloyl imine reaction, obtain folic acid Acibenzolar with following formula 8:
Figure FSA00000632652800044
(b) in the presence of alkali, in solvent, make formula 8 compounds and react with following formula 9 compounds,
Figure FSA00000632652800045
Obtain formula I compound:
Figure FSA00000632652800046
With optional
(c) make gained formula I compound purifying and/or salify,
Wherein n is 5 to 500 integer, and m is 2 to 12 integer, and p is 1 to 8 integer.
12. the purposes of each said formula 9 compounds of each said formula I compound of claim 1-3 or claim 4-6 in the preparation drug administration carrier.
13. the purposes of claim 12, it is characterized in that following (a) to (g) each or multinomial:
(a) said formula I compound or formula 9 compounds are that one of lipid composition as said drug administration carrier is used;
(b) said drug administration carrier is a pH responsive type drug administration carrier;
(c) said drug administration carrier is the drug administration carrier with cancer target performance;
(d) said drug administration carrier is pH responsive type and drug administration carrier with cancer target performance;
(e) said drug administration carrier is the drug administration carrier that contains or do not contain active constituents of medicine;
(f) said drug administration carrier is a liposome;
(g) said drug administration carrier is an initiatively target liposomes of pH responsive type.
14. a drug administration carrier wherein comprises each said formula 9 compounds of each said formula I compound of claim 1-3 or claim 4-6, and optional active constituents of medicine.
15. the drug administration carrier of claim 12, it is characterized in that following (a) to (g) each or multinomial:
(a) said formula I compound or formula 9 compounds are that one of lipid composition as said drug administration carrier is used;
(b) said drug administration carrier is a pH responsive type drug administration carrier;
(c) said drug administration carrier is the drug administration carrier with cancer target performance;
(d) said drug administration carrier is pH responsive type and drug administration carrier with cancer target performance;
(e) said drug administration carrier is the drug administration carrier that contains or do not contain active constituents of medicine;
(f) said drug administration carrier is a liposome;
(g) said drug administration carrier is an initiatively target liposomes of pH responsive type.
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