CN110540551A

CN110540551A - liposome, preparation method thereof, liposome assembly and loaded liposome complex

Info

Publication number: CN110540551A
Application number: CN201910847775.5A
Authority: CN
Inventors: 于喜飞; 刘三荣; 王文靓; 江桑铌; 李晟冉
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Jilin Siride Biotechnology Co.,Ltd.
Priority date: 2019-09-09
Filing date: 2019-09-09
Publication date: 2019-12-06
Anticipated expiration: 2039-09-09
Also published as: CN110540551B

Description

Liposome, preparation method thereof, liposome assembly and loaded liposome complex

Technical Field

The invention relates to the technical field of drug carriers, in particular to a liposome, a preparation method thereof, a liposome assembly and a loaded liposome complex.

background

currently, liposomes have been widely used as drug carriers, and have been marketed for many years as clinically used doxorubicin-liposomes, daunorubicin-liposomes, paclitaxel-liposomes, and the like. Liposomes, which have long been used as drug carriers, face the following four key problems:

(1) The problem of targeted delivery: the liposome (represented by DPPC and DSPE liposome) compound medicine which is clinically used at present mainly utilizes EPR effect of tumor parts for passive targeted enrichment. However, such passive targeting is not selective and is susceptible to interference from complex physiological environments. In addition, the concentration of the drug on the focus is often low by virtue of a simple EPR effect, in order to achieve an effective treatment purpose, the dosage needs to be increased, so that the utilization efficiency of the drug is low and the side toxic and side effects are caused, and in order to solve the problem, the active targeting modification of a common liposome is often needed.

(2) Long circulation in vivo: the liposome composite medicament clinically used at present is easily and rapidly discharged out of the body by an endothelial reticulum system (RES) through a kidney, a liver and the like in the blood circulation process, so that the problems of short drug half-life period, low medicament utilization rate and the like are caused. Some common methods for prolonging the metabolic half-life of liposome composite drugs, such as modifying polyethylene glycol (PEG) on the surface of liposome, and preparing liposome into "stealth liposomes" (stealth liposomes) by using the anti-protein adsorption property of PEG, although the circulation of liposome in the organism can be prolonged to a certain extent, the repeatability is not good, and the problem of mass production needs to be solved.

(3) chemical modifiability of liposomes: the liposome used clinically at present mainly comprises natural soybean lecithin, yolk lecithin and artificially synthesized liposome, and the main structures of the liposome are alkane long chains and phosphatidylcholine zwitterionic head groups. Due to the lack of effective reaction sites of phosphatidylcholine head groups, the liposome has no chemical modification, and can not be effectively linked with targeting molecules, fluorescent molecules, imaging reagents and the like, thereby greatly limiting the application of the liposome.

(4) The problem of controlled release of drugs: the ideal liposome nano controlled release system requires that the liposome nano controlled release system can effectively interact information with an organism, effectively sense the slight difference between normal tissues and focuses of the organism, amplify the signals of the microscopic difference on a material level, and further promote the targeted aggregation and targeted controlled release of the medicine. In clinical treatment, the traditional liposome does not have environmental stimulation responsiveness (endogenous and exogenous stimulation responses such as pH, oxygen partial pressure, magnetic field, ultrasound, light, enzyme, heat and the like) due to lack of effective chemical modification sites, can only release by depending on the concentration difference of the drugs inside and outside the cavity of the liposome, and does not have controllable release. Therefore, the medicine is urgently needed to be rapidly released around the tumor, a high-concentration gradient is established, and the medicine effect range is enlarged. How to realize the rapid release of the drug at a specific position is a hot topic in liposome research.

Therefore, how to improve the long circulation, the stimulation responsiveness and the targeting property of the liposome and provide more chemically modifiable sites becomes a problem to be solved urgently.

Disclosure of Invention

In view of the above, the present invention provides a liposome, a preparation method thereof, a liposome assembly and a liposome-carried complex. The liposome provided by the invention has excellent long-circulating property, stimulation responsiveness, targeting property and multiple chemically modifiable sites, and is beneficial to improving the practical applicability of the liposome.

The invention provides a liposome, which has a structure shown in a formula (I):

wherein the content of the first and second substances,

a1 and A2 are independently selected from hydrocarbyl groups CxH2x + y; x is an integer of 5-35, y is 1, -3, -5, -7, -9 or-11;

the selection of n1, n2, and L is as follows:

n1 ═ 0, n2 ═ 0, and L is- (CH2) n3 —; wherein n3 is 1-4;

Or

At least one of n1 and n2 is not 0, and L is selected from the following structures:

R is selected from: substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C8 alkenyl, substituted or unsubstituted C3-C8 alkynyl, substituted or unsubstituted C3-C8 epoxy, C3-C8 azido, amino with a protecting group, heteroalicyclic, aryl or heteroaryl.

Preferably, the first and second liquid crystal materials are,

y is 1, a1 and a2 are independently selected from C5 to C35 alkyl;

Or

y-1, a1 and a2 are independently selected from CH3(CH2)5CH ═ CH (CH2)7-, CH3(CH2)5CH ═ CH (CH2)9-, CH3(CH2)7CH ═ CH (CH2)7-, CH3(CH2)5CH ═ CH (CH2)11-, CH3(CH2)7CH ═ CH (CH2) 11-or CH3(CH2)7CH ═ CH (CH2) 13-;

or

y ═ ± 1, a and a are independently selected from CH3CH2 ═ CHCH (CH) 7 ═ CH3CH2 ═ CHCH2 ═ CH (CH) 4 ═ CH3CH2 ═ CHCH (CH) 3 ═ CH2 ═ CHCH (CH) 2 ═ CH (CH) 7 ═ CH (CH) 4 ═ CHCH (CH) 2 ═ CHCH (CH) 4 ═ CH (CH) 2 ═ CH (CH) 4 ═ CH (CH) 2 ═ CH (CH) 4 ═ CH (CH) 2 ═ CH (CH) 4 ═ CH (CH) 2 ═ CH) CH (CH) 2 ═ CH (CH) 2 ═ CH (CH) 4 ═ CH (CH) 2 ═ CH.

Preferably, the n1, n2 and L are selected as follows:

n1 ═ 0, n2 ═ 0, and L is- (CH2) n3 —; wherein n3 is 1-4

or

n 1-1, n 2-2 and L is

Preferably, the a1 and a2 are independently selected from: CH (CH) 8 —, CH (CH) 9 —, CH (CH) 10 —, CH (CH) 11 —, CH (CH) 12 —, CH (CH) 13 —, CH (CH) 14 —, CH (CH) 15 —, CH (CH) 16 —, CH (CH) 17 —, CH (CH) 18 —, CH (CH) 5CH ═ CH (CH) 7 —, CH (CH) 5CH ═ CH (CH) 9 —, CH (CH) 7CH ═ CH (CH) 7 —, CH (CH) 5CH ═ CH (CH) 11 —, CH3CH2 ═ CHCH (CH) 4 ═ CH3CH2 ═ CHCH (CH) 2 ═ CHCH (CH) 4 —, CH (CH) CH2 ═ CHCH (CH) 2 ═ CHCH (CH) 2 —, CH (CH) 2 ═ CHCH (CH), CH3(CH2)4CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2)6 —, CH3(CH2)4CH ═ CHCH2CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2)3 —, CH3(CH2)4CH ═ CHCH ═ CH (CH2)8 —, or CH3(CH2)5CH ═ CHCH ═ CH (CH2)7 —;

the R is selected from: CH3-, CH3CH2-, CH3(CH2)2-, (CH3)2CHCH2-, CH3(CH2)3-, CH2 ═ CH (CH2)2-, CH2 ═ CHCH2-, CH ≡ C (CH2)2-, CH ≡ CCH2, N3- (CH2)2-, N3- (CH2)3-, N3- (CH2)4-, NH2CH2-, NH2(CH2)2-, NH2(CH2)3-, NH2(CH2)3-, BOC-NH (CH2)2-, BOC-NH (CH2)3-, BOC-NH (CH2)3-, (CH 3538) 3-),

Preferably, the structure is selected from one or more of the structures shown in formula I-1 to formula I-7:

wherein the content of the first and second substances,

a1 and A2 are independently selected from: CH3(CH2)8-, CH3(CH2)10-, CH3(CH2)12-, CH3(CH2)14-, CH3(CH2)16-, CH3(CH2)18-, CH3(CH2)5CH ═ CH (CH2)7-, or CH3CH2CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2) 7-;

R is selected from: CH3-, CH3CH2-, CH3(CH2)2-, (CH3)2CHCH2-, CH3(CH2)3-, CH2 ═ CH (CH2)2-, CH ≡ C (CH2)2-, or NH2CH 2-.

The invention also provides a preparation method of the liposome in the technical scheme, which comprises the following steps:

a) Reacting compound A1-COOH and compound A2-COOH with 1, 2-propanediol derivative X-1 to obtain compound Y-1;

b) reacting a phosphine heterocyclic pentanes compound X-2 with a dimethylamine compound X-3 to obtain a compound Y-2;

c) reacting the compound Y-1 with a compound Y-2 to obtain the liposome shown in the formula (I);

The step a) and the step b) are not limited in sequence;

wherein the content of the first and second substances,

the selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4;

or

At least one of n1 and n2 is not 0, and L1 and L2 are independently selected from the following structures:

S1) reacting the compound A1-COOH, the compound A2-COOH and the 1, 2-propanediol derivative X-1 to obtain a compound Y-1;

S2) reacting the compound Y-1 with a dimethylamine compound X-3 to obtain a compound Y-3;

S3) reacting the compound Y-3 with a phosphine heterocyclic pentane compound X-2 to obtain a liposome shown in the formula (I);

wherein the content of the first and second substances,

The selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4;

or

K1) Reacting the 1, 2-propylene glycol derivative X-1 with a dimethylamine compound X-3, and then reacting with a phosphine heterocyclic cyclopentane compound X-2 to obtain a compound Y-4;

K2) Reacting the compound Y-4 with a compound A1-COOH and a compound A2-COOH to obtain a liposome shown as a formula (I);

wherein the content of the first and second substances,

the selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4;

Or

The invention also provides a liposome assembly, wherein the assembly is a micelle, a cluster, a micelle, a vesicle, a lipid bilayer membrane or a lipid multilayer membrane;

the liposome is the liposome in the technical scheme or the liposome prepared by the preparation method in the technical scheme.

The invention also provides a carrier liposome complex, which comprises a liposome carrier and a carrier loaded on the liposome carrier;

The liposome in the liposome carrier is the liposome in the technical scheme or the liposome prepared by the preparation method in the technical scheme;

The load comprises one or more of a drug, a targeting substance, a protein and a nucleic acid.

The invention provides a liposome which has the structure of the formula (1), wherein the left side of an L group is a liposome part, the right side of the L group is a choline phosphate structure, and the L group and the choline phosphate structure are connected by a specific L group to form the liposome with a specific structure. The liposome structure has amphipathy, and rich secondary structures are endowed to the phosphocholine liposome by controlling the length of an alkane chain and a phosphocholine bonding group. Meanwhile, the phosphocholine part and the phosphatidylcholine on the cell surface are in "ectopic orientation", have excellent zwitterion performance, have certain specificity such as transmembrane perturbation (membrane displacement), have protein adsorption resistance and promote cell internalization, and have controllable film forming temperature and the like. Moreover, the liposome has a plurality of modifiable functional sites, such as a hydrocarbon group part in a lipid structure, an R group of choline phosphate and the like, and the abundant modifiable functional groups can endow the choline phosphate liposome with excellent post-modification property, prepare biological agents with various functions and different purposes, thereby providing more convenient choices for diagnosis and treatment of diseases, for example, a diagnosis and treatment integrated liposome compound (the liposome is used as a carrier to load small-molecule drugs, proteins, genes or imaging reagents and the like) is used for cancer treatment, and has extremely wide application prospect. The liposome of the formula (I) provided by the invention has the performances of long circulation, stimulation responsiveness, targeting property and the like, has multiple modifiable sites, can be used for preparing biological agents with various functions, and provides more convenient choices for diagnosis and treatment of diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a transmission electron micrograph of a liposome solution obtained in example 43 of the present invention;

FIG. 2 is a transmission electron micrograph of a liposome solution obtained in example 53 of the present invention;

FIG. 3 is a graph showing the biocompatibility test in example 64 of the present invention;

FIG. 4 is a graph showing the cytotoxicity test in example 64;

FIG. 5 is a graph showing the killing ability of the liposome drug complex to Hela cells in example 64;

FIG. 6 is a graph of the tumor enrichment at various time points for doxorubicin tested in example 64;

FIG. 7 is a test chart of drug enrichment of CP liposomes in example 64 in various organs and tumor sites;

FIG. 8 is a graph showing a test for changes in tumor volume in example 64;

FIG. 9 is a graph showing the test of the change in tumor weight in example 64;

FIG. 10 is a graph showing the staining test for apoptosis of tumor foci in example 64;

FIG. 11 shows the results of H & E staining experiments performed on mouse organs in example 64.

Detailed Description

Wherein the content of the first and second substances,

The selection of n1, n2, and L is as follows:

n1 ═ 0, n2 ═ 0, and L is- (CH2) n3 —; wherein n3 is 1-4;

or

in the present invention, preferably, a1 and a2 are as follows:

when y is 1, a1 and a2 are derived from the hydrocarbyl moiety of a saturated fatty acid, specifically C5 to C35 alkyl;

The saturated fatty acids forming the above a1 and a2 are independently selected from: caproic acid (C6 acid), gluconic acid (C7 acid), caprylic acid (C8 acid), pelargonic acid (C9 acid), capric acid (C10 acid), undecanoic acid (C11 acid), lauric acid (C12 acid), tridecanoic acid (C13 acid), myristic acid (C14 acid), pentadecanoic acid (C15 acid), palmitic acid (C16 acid), pearlitic acid (C16 acid), stearic acid (C16 acid), nonadecanoic acid (C16 acid), arachidic acid (C16 acid), heneicosanoic acid (C16 acid), behenic acid (C16 acid), tricosanic acid (C16 acid), lignoceric acid (xylopyrooleic acid, C16 acid), pentacosanoic acid (C16 acid), cerotic acid (C16 acid), heptacosanoic acid (C16 acid), montanic acid (C16 acid), melissic acid (C16), cerotic acid (C16), tetrapelaidic acid (C16), pentadecanoic acid (C16), melissic acid (C16), melissic 16), meli.

y-1, a1 and a2 are derived from the monoolefinic moiety of an unsaturated fatty acid, in particular, a1 and a2 are independently selected from CH3(CH2)5CH ═ CH (CH2)7-, CH3(CH2)5CH ═ CH (CH2)9-, CH3(CH2)7CH ═ CH (CH2)7-, CH3(CH2)5CH ═ CH (CH2)11-, CH3(CH2)7CH ═ CH (CH2) 11-or CH3(CH2)7CH ═ CH (CH2) 13-;

the unsaturated fatty acids forming the above a1 and a2 are independently selected from: palmitoleic acid, vaccenic acid, oleic acid, trans-oleic acid, eicosenoic acid, erucic acid, nervonic acid (scylleic acid) or 11-eicosenoic acid.

When y ═ ± 1, a and a are derived from the multiolefin or multiolefin moiety of the unsaturated fatty acid, in particular, a and a are independently selected from CH3CH2 ═ CHCH2 ═ CH (CH) 7 ═ CH3CH2 ═ CHCH2 ═ CH (CH) 4 ═ CH3CH2 ═ CHCH (CH) 3 ═ CH (CH) 4 ═ CHCH2 ═ CHCH (CH) 2 ═ CH (CH) 4CH ═ CHCH (CH) 7 ═ CH (CH) 4 ═ CHCH (CH) 2 ═ CH (CH) CH 4 ═ CHCH (CH) 4 ═ CH (CH) CH 4 ═ CH (CH) CH (CH) CH 4 ═ CH (CH) CH (CH) CH (CH 4 ═ CH (CH) CH 4 ═ CHCH (CH) CH 4 ═ CH (CH) CH (CH 4 ═ CH) CH;

The unsaturated fatty acids forming the above a1 and a2 are independently selected from: octadecatrienoic acid (ALA), octadecatetraenoic acid (SDA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), Linoleic Acid (LA), gamma-linolenic acid (GLA), eicosatrienoic acid (DGLA), arachidonic acid (ARA), adrenic acid (Docosateenoic acid) or Conjugated Linoleic Acid (CLA).

In the present invention, more preferably, a1 and a2 are selected from: CH (CH) 8 —, CH (CH) 9 —, CH (CH) 10 —, CH (CH) 11 —, CH (CH) 12 —, CH (CH) 13 —, CH (CH) 14 —, CH (CH) 15 —, CH (CH) 16 —, CH (CH) 17 —, CH (CH) 18 —, CH (CH) 5CH ═ CH (CH) 7 —, CH (CH) 5CH ═ CH (CH) 9 —, CH (CH) 7CH ═ CH (CH) 7 —, CH (CH) 5CH ═ CH (CH) 11 —, CH3CH2 ═ CHCH (CH) 4 ═ CH3CH2 ═ CHCH (CH) 2 ═ CHCH (CH) 4 —, CH (CH) CH2 ═ CHCH (CH) 2 ═ CHCH (CH) 2 —, CH (CH) 2 ═ CHCH (CH), CH3(CH2)4CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2)6 —, CH3(CH2)4CH ═ CHCH2CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2)3 —, CH3(CH2)4CH ═ CHCH ═ CH (CH2)8 —, or CH3(CH2)5CH ═ CHCH ═ CH (CH2) 7-.

In the present invention, preferably, n1, n2 and L are selected as follows:

n1 ═ 0, n2 ═ 0, and L is- (CH2) n3 —; wherein n3 is 1-4;

Or

n 1-1, n 2-2 and L is

in the present invention, preferably, R is selected from: CH3-, CH3CH2-, CH3(CH2)2-, (CH3)2CHCH2-, CH3(CH2)3-, CH2 ═ CH (CH2)2-, CH2 ═ CHCH2-, CH ≡ C (CH2)2-, CH ≡ CCH2, N3- (CH2)2-, N3- (CH2)3-, N3- (CH2)4-, NH2CH2-, NH2(CH2)2-, NH2(CH2)3-, NH2(CH2)3-, BOC-NH (CH2)2-, BOC-NH (CH2)3-, BOC-NH (CH2)3-, (CH 3538) 3-),

In the above group structure of R, in the present invention, the protecting group is preferably benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenyl methoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilethoxycarbonyl (Teoc), methyl (or ethyl) oxycarbonyl, phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), trityl (Trt), dimethoxybenzyl (Dmb), p-methoxybenzyl (PMB), or benzyl (Bn). The protected group is deprotected as appropriate, and the deprotection method is not particularly limited, and may be a deprotection method commonly used in chemical synthesis or specific to a certain protecting group, which is well known to those skilled in the art.

in the present invention, more preferably, the liposome of formula (I) is selected from one or more of the structures shown in formula I-1 to formula I-7:

Wherein the content of the first and second substances,

the invention also provides several preparation methods of the liposome in the technical scheme.

according to the present invention, the first preparation method of the liposome comprises the steps of:

The step a) and the step b) are not limited in sequence;

Wherein the content of the first and second substances,

The selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4. Wherein the types of L1 and L2 are not particularly limited, and they may react with each other to form- (CH2) n3-, for example, L1 is-CH 2Cl, and L2 is-CH 2Cl, and they react with each other by the Wurtz reaction to form- (CH2) 2-; for another example, L1 is-CH 2MgBr and L2 is-CH 2Cl, which react by Grignard reaction to form- (CH2) 2-. L1 and L2 include, but are not limited to, the species described above.

Or

wherein n4 is 1-4; x is halogen; in the above structures, where the curve is a linking position, the terminal unshown group is-CH 3, ═ CH2, or ≡ CH; the selection principle of L1 and L2 is that chemical reaction can occur between the L1 and the L2 to form a stable connection structure; the kind of selection corresponds to the structure of L in the foregoing.

In the invention, in the step a), the reaction route is as follows:

the types of a1 and a2 are the same as those in the foregoing technical solutions, and are not described herein again.

Wherein L1 and L2 are linked to form L in the liposome structure shown in the formula (I) through chemical reaction. Thus, L1 and L2 are selected from groups that react to form the L structure in the above scheme. The types of the above-mentioned components are the same as those in the above-mentioned technical solution, and are not described herein again.

the reaction temperature is not particularly limited, and the reaction can be carried out at room temperature, specifically at 10-40 ℃; the reaction time is 6-14 h.

among them, the reaction is preferably carried out in a solvent medium, and the solvent is preferably an organic solvent, and the kind thereof is not particularly limited, and the raw material may be dissolved. The amount of the solvent is not particularly limited, and the raw materials can be sufficiently dissolved. The reaction is preferably carried out with a catalyst, which in some embodiments of the invention is 4-dimethylaminopyridine (i.e., DMAP) and the water produced by the reaction is absorbed by N, N' -diisopropylcarbodiimide (i.e., DIC), which drives the reaction in the direction of the esterification product. The molar ratio of the catalyst to the 1, 2-propanediol derivative X-1 is preferably (0.005-0.05): 1; the molar ratio of DIC to the 1, 2-propanediol derivative X-1 is preferably (2-2.4): 1. and after the reaction, obtaining a reaction liquid, filtering the reaction liquid, carrying out rotary distillation on the filtrate to obtain a solid crude product, and further crystallizing and purifying to obtain the compound Y-1.

in the invention, in the step b), the reaction route is as follows:

wherein, the kind of R is the same as that described in the above technical solution, and is not described herein again. The reaction is preferably carried out under the protection of inert gas; the inert gas used in the present invention is not particularly limited, and may be a conventional protective gas known to those skilled in the art, such as nitrogen, helium, argon, or the like.

the reaction is preferably carried out in a solvent medium, and the solvent is preferably one or more of acetonitrile, dimethylformamide, dichloromethane, chloroform, tetrahydrofuran, methanol, ethanol, methyl acrylate, methyl methacrylate, ethyl acetate and methyl acetate. In the reaction, preferably, dissolving a phosphine heterocyclic cyclopentane compound X-2 and a dimethylamine compound X-3 in a solvent, and then reacting at 10-90 ℃ for 6-24 h to obtain an intermediate reaction solution; and (3) cooling to room temperature, rotationally evaporating the solvent, and then selecting a corresponding poor solvent to wash the reaction product, wherein the poor solvent is one or more selected from tetrahydrofuran, diethyl ether, petroleum ether and n-hexane. After the poor solvent is used, the product precipitates as a viscous liquid or white powder at the bottom of the reaction flask.

In the invention, in the step c), the reaction route is as follows:

the reaction is preferably carried out in a solvent medium, and the invention is not particularly limited in the kind of the solvent, and the raw materials can be dissolved; specifically, the solvent includes, but is not limited to, one or more of acetonitrile, dimethylsulfoxide (i.e., DMSO), dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methyl acrylate, methyl methacrylate, ethyl acetate, and methyl acetate.

the reaction conditions are different according to the differences of L1 and L2: as previously described, when L1 is an amino group, L2 may alternatively be an acrylate group, corresponding to reaction conditions of base catalysis and heating. When L1 is azido, L2 may be selected as alkynyl, corresponding reaction conditions are oxygen-free reaction catalyzed by bipyridine-cuprous bromide. When L1 is mercapto, L2 is optionally alkenyl or mercapto, and the corresponding reaction conditions are catalysis by a photocatalyst and ultraviolet irradiation. Other options of L1 and L2 may be adjusted as desired.

And (3) obtaining a crude product after the reaction, and further separating and purifying to obtain the liposome shown in the formula (I). The mode of the separation and purification is not particularly limited, and may be any of the purification modes known to those skilled in the art, such as recrystallization, solvent precipitation, solvent extraction, column chromatography, dialysis, or preparative chromatography.

According to the present invention, the second preparation method of the liposome comprises the steps of:

The selection of a1, a2, n1, n2, L1, L2 and R is the same as that in the above technical solution, and is not described in detail herein.

Said step S1) is identical to the reaction conditions in step a) of the above-mentioned first preparation method, and will not be described herein again.

In the step S2), the reaction conditions consistent with those in the step c) of the first preparation method can be flexibly adjusted according to the matching condition of the L1 and L2 groups, and will not be described herein again.

In the step S3), the reaction scheme is as follows:

Wherein the reaction temperature is preferably 50-100 ℃, and the reaction time is preferably 12-36 h. The reaction is preferably carried out in a solvent medium, the type of the solvent is not particularly limited, and the raw materials can be dissolved; specifically, the solvent includes, but is not limited to, one or more of acetonitrile, dimethylsulfoxide (i.e., DMSO), dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, water, methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol. And (3) obtaining a crude product after the reaction, and further separating and purifying to obtain the liposome shown in the formula (I). The mode of the separation and purification is not particularly limited, and may be any of the purification modes known to those skilled in the art, such as recrystallization, solvent precipitation, solvent extraction, column chromatography, preparative chromatography, and the like.

according to the present invention, the third method for preparing the liposome comprises the following steps:

in the step K1), when X-1 reacts with X-3, the reaction conditions can be flexibly adjusted according to the matching condition of the L1 and L2 groups, which is consistent with the reaction conditions in the step c) of the first preparation method and will not be described herein again. When the reaction is continued with the X-2, the reaction temperature is preferably 50-100 ℃, and the reaction time is preferably 12-36 h.

in the step K2), the reaction scheme is as follows:

The reaction temperature is preferably 50-100 ℃, and the reaction time is preferably 6-36 h. The reaction is preferably carried out in a solvent medium, the type of the solvent is not particularly limited, and the raw materials can be dissolved; specifically, the solvent includes, but is not limited to, one or more of acetonitrile, dimethylsulfoxide (i.e., DMSO), dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, water, methanol, ethanol, n-propanol, isopropanol, n-butanol, and isobutanol. And (3) obtaining a crude product after the reaction, and further separating and purifying to obtain the liposome shown in the formula (I). The mode of the separation and purification is not particularly limited, and may be any of the purification modes known to those skilled in the art, such as recrystallization, solvent precipitation, solvent extraction, column chromatography, preparative chromatography, and the like.

The liposome provided by the invention or the liposome prepared by the preparation method in the technical scheme has the following characteristics:

(1) The L group is of a left liposome part and a right phosphocholine structure, wherein the liposome structure part has amphipathy, and the phosphocholine liposome is endowed with rich secondary structures by controlling the length of an alkane chain and a phosphocholine bonding group.

(2) the 'ectopic orientation' exists between the phosphocholine part and the phosphatidylcholine on the cell surface, and the cell surface has excellent zwitterion performance, certain specificity such as transmembrane perturbation (membrane displacement), protein adsorption resistance, promotion of cell internalization, formation of a titanium dioxide support membrane, controllable membrane forming temperature and the like.

(3) The liposome has a plurality of modifiable functional sites, such as unsaturated alkyl parts in a lipid structure, R groups of cholic acid and phosphorus base, reactive sites on L groups and the like, and the abundant modifiable functional groups can endow the cholic acid and phosphorus liposome with excellent post-modification property, and prepare biological agents with various functions and different purposes, thereby providing more convenient choices for diagnosis and treatment of diseases, for example, a diagnosis and treatment integrated liposome compound (the liposome is used as a carrier and is loaded with small-molecule drugs, proteins, genes or imaging reagents and the like) is used for cancer treatment, and has extremely wide application prospects.

(4) During the process of forming L by reacting and linking L1 and L2, a stable link is formed, and a new active reaction site is formed, so that the L can be used for later modification or forming a composition with a specific substance.

(5) the liposome or the modified liposome or the compound formed by the liposome and a specific substance can form various assemblies by utilizing the property that one end of the liposome is hydrophilic and the other end is lipophilic.

the liposome provided by the invention has the performances of long circulation, stimulation responsiveness, targeting property and the like, has multiple modifiable sites, can be used for preparing biological agents with various functions, and provides more convenient choices for diagnosis and treatment of diseases.

the invention also provides a liposome assembly, wherein the assembly is a micelle, a cluster, a micelle, a vesicle, a lipid bilayer membrane or a lipid multilayer membrane; the liposome is the liposome in the technical scheme or the liposome prepared by the preparation method in the technical scheme.

in the present invention, liposomes are self-assembled by an appropriate means to form an assembly. The "suitable manner" refers to a scientifically achievable method of preparing liposome assemblies, including but not limited to self-assembly by hydrophilic-hydrophobic interaction, self-assembly by electrostatic interaction, self-assembly by hydrogen bond interaction, and the like.

In the self-assembly by utilizing the hydrophilic-hydrophobic interaction, the hydrophobic lipid parts of a plurality of liposomes are mutually associated by mainly utilizing the hydrophilic-hydrophobic interaction among liposome molecules, and the hydrophilic phosphocholine part faces to the water phase to form an assembly body.

In the self-assembly by utilizing electrostatic interaction, the choline phosphate part has positive and negative charges, and the liposome can be self-assembled by utilizing the wide electrostatic interaction in and among the molecules.

in the self-assembly by utilizing hydrogen bond interaction, the hydrogen bond interaction which is widely existed in the interior and the intermolecular of the liposome molecule is utilized, so that the self-assembly of the liposome is realized.

In the present invention, when assembling liposomes by the hydrophilic-hydrophobic interaction, the electrostatic interaction, and the hydrogen bond interaction, the interactions are not isolated, and the interactions and the modes of operation are combined to form an assembly.

the size and shape of the assembly formed by the above assembly method are related to the concentration of the liposome, the strength of the interaction and the operation mode of self-assembly, and the shape of the assembly can be spherical, rod-shaped, hexagonal bundle or lamellar. In the present invention, the assemblies having various shapes are stably present in a structure such as a micelle, a cluster, a vesicle, a lipid bilayer membrane, or a lipid multilayer membrane.

The invention also provides a carrier liposome complex, which comprises a liposome carrier and a carrier loaded on the liposome carrier; the liposome in the liposome carrier is the liposome in the technical scheme or the liposome prepared by the preparation method in the technical scheme; the load comprises one or more of a drug, a targeting substance, a protein and a nucleic acid. The invention loads various substances which can be used for treatment, such as pharmaceutically acceptable medicines, target substances, proteins or nucleic acids, and the like on the liposome carrier to form a liposome compound, thereby being used for diagnosis and treatment of diseases. The cargo specifically includes, but is not limited to, doxorubicin, paclitaxel, vincristine, small interfering rna (sirna), a protein (e.g., insulin), a vaccine, or a nucleic acid, and the like.

In the present invention, the above-mentioned loading substance may be added during the self-assembly of the liposome. The mode of adding can be as follows: the liposome carrier and the load are physically blended, and the liposome carrier and the load are chemically reacted, or the physical blending and the chemical reaction are added to act together.

in the invention, when the liposome carrier and the load are physically blended, the components of the liposome carrier and the load can be self-assembled through one or more of hydrophilic-hydrophobic interaction, electrostatic interaction and hydrogen bond interaction. That is, in physical mixing, the various interactions are not isolated, but may be a combination of various interactions, and the individual interactions and the manner of operation may be appropriately adjusted to form the assembly complex.

the liposome carrier and the load are physically combined, and the combining position of the load and the liposome carrier can be the lipid part of the liposome of the formula (I) or the interior of any assembly formed by the liposome in a certain mode. The size and shape of the assembly formed by the above assembly method are related to the concentration of the liposome, the strength of the interaction and the operation mode of self-assembly, and the shape of the assembly can be spherical, rod-shaped, hexagonal bundle or lamellar. In the present invention, the assemblies of various shapes are stably present in a structure such as a micelle, a cluster, a vesicle, a lipid bilayer membrane, or a lipid multilayer membrane, and form a liposome-supported complex.

in the invention, when the liposome carrier and the load are compounded through chemical reaction, the liposome carrier and the load are firstly subjected to chemical reaction and then self-assembly to obtain the compound. Wherein, the chemical reaction refers to the chemical reaction between the corresponding reactive functional group in the load and the reactive group on the liposome of formula (I) (such as the reactive group on the R group, the double bond functional group of the lipid part, and the reactive functional group on the L structure formed after the L1 and the L2 are effectively connected), and after the chemical reactions occur, the load is connected into the liposome molecule by a proper chemical bond to form a stable whole.

after the above chemical reaction, self-assembly is carried out by physical action. Specifically, the self-assembly may be performed by one or more of hydrophilic-hydrophobic interaction, electrostatic interaction, and hydrogen bonding interaction. That is, in physical mixing, the various interactions are not isolated, but may be a combination of various interactions, and the individual interactions and the manner of operation may be appropriately adjusted to form the assembly complex. The size and shape of the assembly formed by the above assembly method are related to the concentration of the liposome, the strength of the interaction and the operation mode of self-assembly, and the shape of the assembly can be spherical, rod-shaped, hexagonal bundle or lamellar. In the present invention, the assemblies of various shapes are stably present in a structure such as a micelle, a cluster, a vesicle, a lipid bilayer membrane, or a lipid multilayer membrane, and form a liposome-supported complex.

In the invention, the liposome carrier and the load can be compounded through two actions of physical mixing and chemical reaction. Specifically, a portion of the material of the load chemically reacts with the liposomes, and another portion of the material physically mixes with the liposomes. Wherein, the physical mixing can be carried out by one or more of hydrophilic-hydrophobic interaction, electrostatic interaction and hydrogen bond interaction, i.e. various interactions are not isolated and can be combined, and the assembly compound can be formed by properly adjusting various interactions and operation modes. The size and shape of the assembly formed by the above assembly method are related to the concentration of the liposome, the strength of the interaction and the operation mode of self-assembly, and the shape of the assembly can be spherical, rod-shaped, hexagonal bundle or lamellar. In the present invention, the assemblies of various shapes are stably present in a structure such as a micelle, a cluster, a vesicle, a lipid bilayer membrane, or a lipid multilayer membrane, and form a liposome-supported complex.

in the invention, the liposome carrier and the loading substance can also be compounded in the following way: independently forming a self-assembly body by the liposome shown in the formula (I), and then adding a load; after the loading substance is added, the loading substance is stably distributed in the cavity, the interior, the surface, the interlayer and other parts of the liposome assembly by utilizing the respective properties to form a liposome-loading substance complex.

the loaded liposome complex provided by the invention has the following effects:

(1) The pH value of the carrying liposome complex provided by the invention in an aqueous solution or body fluid environment is 5.0-8.5, is close to the pH value of an organism, and can better play a role in the organism.

(2) The carrier liposome complex provided by the invention has stimulation responsiveness. The stimulation responsiveness refers to that under the environment of a normal organism, the loaded liposome complex has a certain shape, when reaching a focus part, the assembled shape, the bonding mode and the like of the liposome or the liposome-loaded complex are changed due to the fact that the biological microenvironment of the focus part is different from that of the normal organism, and the loaded matter (such as drug molecules and the like) wrapped or bonded in the liposome or the loaded matter complex is released to achieve the purpose of treating specific diseases. The liposome complex for carrying the medicament has sensitive stimulation responsiveness due to the special structure of the liposome carrier, and can better realize the controllable release of the medicament.

(3) Meanwhile, the loaded liposome complex provided by the invention has the main effect in a living body due to the special structure of the liposome, and can prolong the circulation time of the liposome or the loaded liposome complex (such as a loaded liposome) in the body, thereby achieving the purpose of prolonging the administration. The long-circulating and long-time administration property in vivo is very suitable for treating diseases related to the loaded therapeutic components of organisms, such as cancer.

The loaded liposome complex (such as liposome carrier-drug complex) provided by the invention can be prepared into drugs suitable for intravenous injection, intramuscular and subcutaneous injection, oral administration, ocular administration, pulmonary administration, transdermal administration, nasal administration and other routes, and has wide applicability.

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

EXAMPLES 1-13 preparation of Compound Y-1

Examples 1 to 3

56g of hexadecanoic acid (0.22mol), 12g of N, N-dimethylamino-1, 2-propanediol (0.1mol) were charged into a 500mL reaction flask, 300mL of dichloromethane was added, after stirring sufficiently, 27.8g of N, N' -Diisopropylcarbodiimide (DIC) (0.22mol) and 0.2g of 4-Dimethylaminopyridine (DMAP) were added, after stirring sufficiently at room temperature for 24 hours, filtration was carried out, and the solid obtained after rotary distillation of the filtrate was further recrystallized and purified to obtain 54g of a product, with a yield of 96.8%.

According to the above preparation process, except that 62g of oleic acid and 61g of octadecatrienoic acid (both 0.22mol) were used instead of 56g of hexadecanoic acid in the above preparation process, respectively. The yields of the product obtained and the product structure are shown in table 1.

TABLE 1 yield and structure of examples 1-3

Examples 4 to 6

56g of hexadecanoic acid (0.22mol), 19.1g of N-Boc-2, 3-dihydroxypropylamine (0.1mol) were put into a 500mL reaction flask, 300mL of dichloromethane was added, after stirring sufficiently, 27.8g of N, N' -Diisopropylcarbodiimide (DIC) (0.22mol) and 0.2g of 4-Dimethylaminopyridine (DMAP) were added, after stirring sufficiently at room temperature for 24 hours, the reaction mixture was filtered, and the solid obtained by rotary distillation of the filtrate was further recrystallized, purified and dried to obtain 62g of an intermediate product, which was obtained in 92.8% yield in this step.

All the obtained intermediate products are added into a 250mL reaction bottle, 150mL dichloromethane is added, after full stirring, trifluoroacetic acid with the volume ratio of 20% is added, reaction is carried out for 5h at room temperature, and after the solvent is removed by rotary distillation, recrystallization is further carried out, thus obtaining 51g of final product. The yield of the step is 96.8%.

According to the above preparation process, except that 62g of oleic acid and 61g of octadecatrienoic acid (both 0.22mol) were used instead of 56g of hexadecanoic acid in the above preparation process, the other operations were the same. The yields and molecular structures of the intermediate products and the final products obtained in examples 4-6 are shown in Table 2.

TABLE 2 raw materials and products of examples 4-6

examples 7 to 9

adding 60.5g of hexadecanoyl chloride (0.22mol) and 20g of 3-iodo-1, 2-propanediol (0.1mol) into a 500mL reaction bottle, adding 300mL of trichloromethane, stirring, dropwise adding 22g of triethylamine into the reaction system, after dropwise adding, fully stirring at room temperature for reaction for 24h, filtering, and further recrystallizing and purifying the solid obtained after rotary distillation of the filtrate. 52g of product are obtained, yield 76.6%.

The above preparation was followed except that 66g of oleoyl chloride and 65g of octadecatrienoyl chloride (each 0.22mol) were used in place of 56g of hexadecanoyl chloride in the above preparation, respectively. The yields of the product obtained and the product structure are shown in Table 3.

TABLE 3 raw materials and products of examples 7-9

examples 10 to 12

1.3g of sodium azide (20mmol) and 6.8g of the compound (10mmol) obtained in example 7 were added to a mixed solvent of 60ml of tetrahydrofuran and 40ml of water, heated to 60 ℃ and reacted for 16 hours, then the reaction was stopped, poured into 500ml of water, sufficiently stirred and filtered, and a filter cake was collected to obtain 5.7g of an intermediate product with a yield of 96.0%.

following the above preparation procedures except that 7.9g of the compound obtained in example 8 and 7.2g of the compound obtained in example 9 (each 10mmol) were used instead of 6.8g of the compound obtained in example 7 in the above preparation procedures, respectively, the yields and structures of the obtained series of products are shown in Table 4.

TABLE 4 raw materials and products of examples 10-12

example 13

s1, adding 54g of 1-thioglycerol (0.5mol) and 153g of triphenylchloromethane (0.55mol) into a 1000mL reaction bottle, adding 500mL of dichloromethane, dropwise adding 55g (0.55mol) of triethylamine into the reaction system, fully stirring at room temperature for reacting for 16h, filtering, and carrying out rotary distillation on the filtrate to obtain a solid, and further recrystallizing and purifying the solid. Drying to obtain 156g of intermediate product, wherein the yield of the step is 89.3%, and the intermediate product obtained in the step has the following structure:

S2, adding 35g (0.1mol) of the intermediate product obtained in the previous step into a 500mL reaction bottle, adding 60.5g of hexadecanoyl chloride (0.22mol) and 300mL of trichloromethane, stirring, dropwise adding 22g of triethylamine into the reaction system, after dropwise adding, fully stirring at room temperature for reaction for 24 hours, filtering, and further recrystallizing and purifying the solid obtained after rotary distillation of the filtrate. 64.9g of product are obtained in 78.5% yield, the structure of the product being as follows:

s3, adding the product obtained in the previous step into a 500mL flask, adding 300mL dichloromethane and 8g triethylamine, fully stirring, adding trifluoroacetic acid with the volume ratio of 20%, reacting for 5h at room temperature, removing the solvent by rotary distillation, and then further recrystallizing to obtain 40.3g of the final product of the example. The yield of the step is 87.7%, and the structure of the final product is as follows:

Examples 14-15 preparation of Compound X-2

example 14

Adding 51g of triethylamine (0.51mol) and 37g of anhydrous n-butanol (0.5mol) into a flask with 500ml of anhydrous tetrahydrofuran, mechanically stirring, cooling for 30 minutes in an ice-water bath, dropwise adding 71g of 2-chloro-2-oxo-dioxolane (0.5mol) from a constant-pressure dropping funnel, wherein a reaction system generates a large amount of white precipitates in the dropwise adding process, keeping the ice-water bath reaction for 2 hours, slowly raising the temperature to room temperature, continuing to react for 2 hours, filtering, washing a filter cake twice with the anhydrous tetrahydrofuran, merging and collecting filtrates, removing a solvent under reduced pressure to obtain a crude product, and distilling the crude product with a short neck to obtain 37.2g of pure 2-oxobutyl-2-oxo-dioxolane with the yield of 38%. The product structure is as follows:

Example 15

35g of anhydrous acetylene butanol was used in place of 37g of anhydrous n-butanol in example 14, and the other conditions were not changed to give a crude product, which was subjected to short-neck distillation to give 30.2g of pure 2-oxoalkynylbutyl-2-oxo-dioxaphospholane in 34.3% yield. The product structure is as follows:

examples 16 to 18 preparation of Choline Phosphoric acid Compound Y-2

Examples 16 to 18

Taking 3g N, N-dimethylamino ethyl acrylate (21mmol) and 3.60g of the compound (20mmol) obtained in the example 14, adding 50ml of acetonitrile, electromagnetically stirring, reacting for 16h at 70 ℃, removing the acetonitrile under reduced pressure, adding ether, shaking and washing, precipitating the product at the bottom of a bottle after centrifugation, and repeating for three times to obtain 4.7g of the product with the yield of 83 percent.

the procedure was followed except that 3.52g of the compound obtained in example 15 was used in place of 3.60g of the compound obtained in example 14 in the above procedure, and the same operations were carried out. See table 5, example 17 for product yields and structure.

The procedure was followed except that 2.1g of N, N-dimethylpropylamine was used in place of 3g of ethyl N, N-dimethylaminoacrylate used in the above preparation, and the other operations were the same as above. See table 5, example 18 for yield and molecular structure of the final product.

TABLE 5 products and yields of examples 16-18

examples 19 to 22 preparation of Compound Y-3

examples 19 to 21

1.6g of N, N-dimethylpropylamine (20mol), 3g of the compound (5mmol) obtained in example 10 and 10mg of bipyridine were added to a mixed solvent of 15ml of tetrahydrofuran and 15ml of water, the mixture was frozen with liquid nitrogen, and then, a vacuum-nitrogen cycle was performed three times, after thawing at room temperature, a vacuum-nitrogen cycle was performed three times to complete one cycle, after the above cycle was repeated three times, 5mg of cuprous bromide was added in a nitrogen-charged state, the reaction was performed at room temperature for 24 hours, after the completion of the reaction, the reaction was poured into 200ml of water, and after stirring sufficiently, washing, filtration and drying, 3.3g of the product was obtained, with a yield of 95.6%.

the procedure was followed except that 3.3g of the compound obtained in example 11 and 3.3g of the compound obtained in example 12 (each 10mmol) were used instead of 3.0g of the compound obtained in example 10 in the above procedure, respectively, and the yields and structures of the obtained series of products are shown in Table 6:

TABLE 6 yields and product structures of examples 19-21

example 22

1.7g of N, N-dimethylallylamine (20mol), 2.9g of the compound (5mmol) obtained in example 13 and 5mg of photoinitiator-1173 are added into a mixed solvent formed by 15ml of tetrahydrofuran and 15ml of water, the mixture reacts for 24 hours at room temperature under the irradiation of 365nm ultraviolet light, the mixture is poured into 200ml of water after the reaction is finished, the mixture is fully stirred and washed, then the mixture is filtered, filter cakes are dissolved by dichloromethane, insoluble substances are removed by filtration again, and 3.3g of products are obtained after the solvent is drained, and the yield is 85.3%. The resulting product has the following structure:

Examples 23-42 Synthesis of liposomes of formula I

Examples 23 to 25

Using standard oxygen-free anhydrous procedures, 40ml of dry acetonitrile, 10ml of dichloromethane, 2.4g of the compound (4mmol) from example 1, 1.8g of the compound (10mmol) from example 14 were added sequentially to a well-baked, branched flask, heated to 70 deg.C, reacted for 24 hours, the reaction was stopped, the solvent was removed in vacuo, 50ml of ether was added and washed, filtered to give the crude product, which was isolated by column chromatography to give 2.01g, 57.3% yield.

The procedure was followed except that 2.6g of the compound obtained in example 2 and 2.6g of the compound obtained in example 3 (each 10mmol) were used instead of 2.4g of the compound obtained in example 1 in the above procedure, respectively, and the yields and structures of the obtained series of products are shown in Table 7.

TABLE 7 yields and structures of examples 23-25

Examples 26 to 28

The procedures described in examples 23 to 25 were followed except that 1.8g of the compound obtained in example 15 was used in place of 1.8g of the compound obtained in example 14 in the above procedure, and the yields and structures of the obtained series of products were as shown in Table 8.

TABLE 8 yields and structures of examples 26-28

Examples 29 to 31

Using standard oxygen-free anhydrous procedures, 40ml of dry acetonitrile, 10ml of dichloromethane, 2.3g of the compound obtained in example 4 (4mmol), 3.2g of the compound obtained in example 16 (10mmol), 0.02g of ethylenediamine were added to a well-baked flask in the stated order, heated to 70 ℃ and allowed to react for 24 hours, the reaction was stopped, the solvent was removed in vacuo, 50ml of ether was added and washed, the crude product was filtered and isolated by column chromatography to give 1.54g, 43.2% yield.

following the above preparation procedures except that 2.7g of the compound obtained in example 5 and 2.7g of the compound obtained in example 6 (each 10mmol) were substituted for 2.3g of the compound obtained in example 4 in the above preparation procedures, respectively, the yields and structures of the obtained series of products are shown in Table 9.

TABLE 9 yields and structures of examples 29-31

examples 32 to 34

The procedures described in examples 29 to 31 were followed except that 3.2g of the compound obtained in example 17 was used in place of 3.2g of the compound obtained in example 16 in the above procedures for preparation of examples 29 to 31, and the yields and structures of the obtained series of products were as shown in Table 10.

TABLE 10 yields and structures of examples 32-34

examples 35 to 38

The procedures for preparation of examples 23 to 25 were followed except that 2.76g of the compound obtained in example 19, 2.97g of the compound obtained in example 20, 2.94g of the compound obtained in example 21, 2.68g of the compound obtained in example 22 were used instead of 2.4g of the compound obtained in example 1 in the above preparation of examples 23 to 25, respectively, and the yields and structures of the obtained series of products were found in Table 11.

TABLE 11 yields and structures of examples 35-38

Examples 39 to 42

the procedures as described in examples 23 to 25 were followed except that 2.76g of the compound obtained in example 19, 2.97g of the compound obtained in example 20, 2.94g of the compound obtained in example 21, 2.68g of the compound obtained in example 22 were used in place of 2.4g of the compound obtained in example 1 in the above procedures for preparation of examples 23 to 25 and 1.8g of the compound obtained in example 15 was used in place of 1.8g of the compound obtained in example 14 in examples 23 to 25, respectively, and the yields and structures of the obtained series of products were found in Table 12.

TABLE 12 yields and structures of examples 39-42

EXAMPLES 43-62 Assembly of Liposome drug complexes

examples 43 to 52

5mg of cholesterol and 20mg of the compound obtained in example 23 were dissolved in 10mL of a chloroform-methanol mixed solvent (volume ratio: 4:1) to obtain a homogeneous solution. And then transferring the solution into a 100mL eggplant-shaped bottle, and performing vacuum rotary evaporation at the temperature of 40 ℃ at the rotating speed of 100 r/min for 2h to evaporate the solution to dryness and attach the solution to the inner wall to form the choline phosphate film. Adding 5mL ammonium sulfate solution (200 mM concentration) into the above eggplant-shaped bottle, rotating at 50 deg.C for 10min, ultrasonically dispersing for 20min to obtain liposome with uniform particle diameter, and dialyzing with PBS for 5 hr to obtain liposome solution with inner water phase of ammonium sulfate. Then, 1mL of adriamycin solution (1mg/mL) is dripped into the liposome solution, the mixture is incubated in water bath at 60 ℃ for 10min, the obtained sample is dialyzed for 2h at room temperature by a dialysis bag with molecular cut-off of 3500Da, water is changed every 30min, and then the mixture is filtered by a filter membrane with the diameter of 0.22 mu m, and finally, the choline phosphate liposome solution loaded with the adriamycin is obtained.

according to the above preparation process, except for using 20mg of the compound obtained in example 24, 20mg of the compound obtained in example 25, 20mg of the compound obtained in example 29, 20mg of the compound obtained in example 30, 20mg of the compound obtained in example 31, 20mg of the compound obtained in example 35, 20mg of the compound obtained in example 36, 20mg of the compound obtained in example 37 and 20mg of the compound obtained in example 38, respectively, in place of 20mg of the compound obtained in example 23 in the above preparation process, liposome solutions having uniform particle diameters were obtained.

The particle size and distribution of the liposome solutions were tested using DLS and the particle size and distribution of the resulting liposomes are shown in table 13.

TABLE 13 particle size and distribution of liposome solutions obtained in examples 43 to 52

	Average particle diameter (nm)	Distribution (PDI)
			Example 43	98	0.21
example 44	105	0.19
			Example 45	109	0.18
Example 46	111	0.19
			example 47	95	0.16
Example 48	129	0.21
			example 49	119	0.24
Example 50	123	0.20
			Example 51	117	0.19
Example 52	130	0.20

The results of transmission electron microscopy on the liposome solution obtained in example 43 are shown in FIG. 1, and FIG. 1 is a transmission electron microscopy on the liposome solution obtained in example 43 of the present invention. It can be seen that in the liposome solution, the liposome particles were nanoparticles having a uniform particle size.

Examples 53 to 62

5mg of cholesterol and 20mg of the liposome prepared in example 26 were dissolved in 10mL of a chloroform-methanol mixed solvent (volume ratio: 4:1) to obtain a homogeneous solution. And then transferring the solution into a 100mL eggplant-shaped bottle, and performing vacuum rotary evaporation at the temperature of 40 ℃ at the rotating speed of 100 r/min for 2h to evaporate the solution to dryness and attach the solution to the inner wall to form the choline phosphate film. Adding 5mL PBS into the above eggplant-shaped bottle, rotating at 50 deg.C for 10min, and ultrasonically dispersing for 20min to obtain liposome solution with uniform particle diameter.

Taking 5mL of the prepared liposome solution into a reaction bottle, adding folic acid receptor treated by azide group, then adding 0.5mL of mixed solution of anhydrous copper sulfate and sodium ascorbate (the concentration of copper sulfate is 1mg/mL, and the concentration of sodium ascorbate is 1.5mg/mL), stirring and reacting for 24h at room temperature, and finally dialyzing at room temperature through a dialysis bag with molecular cut-off of 3500Da to remove the catalyst, thus obtaining the folic acid modified choline phosphate liposome solution.

Dripping 1mL adriamycin solution (1mg/mL) into the folic acid modified choline phospholiposome solution, incubating in water bath at 60 ℃ for 10min, dialyzing the obtained sample at room temperature for 2h by a dialysis bag with molecular cut-off of 3500Da, changing water every 30min, filtering with a 0.22 mu m filter membrane, and finally obtaining folic acid modified butynyl choline phospholiposome and adriamycin compound

According to the above preparation process, except for using 20mg of the compound obtained in example 27, 20mg of the compound obtained in example 28, 20mg of the compound obtained in example 32, 20mg of the compound obtained in example 33, 20mg of the compound obtained in example 34, 20mg of the compound obtained in example 39, 20mg of the compound obtained in example 40, 20mg of the compound obtained in example 41 and 20mg of the compound obtained in example 42, respectively, in place of 20mg of the compound obtained in example 26 in the above preparation process, folic acid-modified butynylcholine phosphate liposomes and doxorubicin complex solutions each having a uniform particle size were obtained.

The particle size and distribution of the liposome solutions were tested using DLS and the particle size and distribution of the resulting liposomes are shown in table 14.

TABLE 14 particle size and distribution of liposome solutions obtained in examples 53 to 62

	Average particle diameter (nm)	Distribution (PDI)
			example 53	117	0.24
example 54	105	0.21
			example 55	120	0.22
example 56	127	0.19
			example 57	111	0.15
Example 58	101	0.24
			Example 59	132	0.20
Example 60	133	0.29
			Example 61	118	0.18
example 62	156	0.19

The results of transmission electron microscopy of the liposome solution obtained in example 53 are shown in FIG. 2, and FIG. 2 is a transmission electron microscopy of the liposome solution obtained in example 53 of the present invention. It can be seen that in the liposome solution, the liposome particles were nanoparticles having a uniform particle size.

Example 63 in vivo Long-circulating testing of liposomes

Female ICR mice (body weight: 26g-30g) aged 8 weeks were weighed, and the liposome solution obtained in example 43 was intravenously injected into the tail of the mice in accordance with the standard of 10mg/kg of adriamycin, and blood was collected at the following times to measure the concentration of adriamycin in the blood by HPLC.

The blood sampling time is respectively as follows:

20min、30min、60min、2h、4h、6h、8h、12h、24h、36h、48h、60h、72h。

The mouse is collected in a 1.5mL heparin tube after 0.6mL of blood is collected from the eyeball, the blood is killed after being collected, the blood is centrifuged for 10min by a low-temperature centrifuge at 3000rpm, and the supernatant is taken. Plasma was stored at-20 ℃ until detection. See table 15 for results.

table 15 in vivo long circulation test results for liposomes

Time of sampling	DOX concentration (μ g/mL)
		1h	93.22±5.27
2h	91.8±2.03
		3h	89.43±2.38
6h	83.24±1.24
		9h	77.43±1.13
12h	71.55±1.77
		15h	65.66±1.90
20h	55.46±1.59
		24h	49.76±1.29
36h	38.18±0.38
		48h	29.71±0.12
60h	13.25±0.27
		72h	7.67±0.01

From the above test results it can be seen that: after the liposome drug complex participates in vivo circulation for 48 hours, the drug concentration of 29.71 +/-0.12 mu g/mL in blood can be still measured, and after the liposome drug complex participates in vivo circulation for 72 hours, the drug concentration in blood is still maintained at 7.67 +/-0.01 mu g/mL, so that excellent in vivo long-term circulation effect is shown.

similarly, the liposome solutions obtained in examples 44 to 62 were subjected to in vivo long-circulating test according to the above-described test procedures, and the results were comparable to those in table 16, and the liposome drug complexes obtained in examples 44 to 62 also exhibited excellent in vivo long-circulating effects.

EXAMPLE 64 Liposomal drug complexes for disease treatment

the liposome prepared in example 23, or the liposomal adriamycin pharmaceutical complex prepared in example 43; a mouse model of cervical cancer was constructed using the liposomes prepared in example 26 or the liposome-folate-doxorubicin drug complex prepared in example 53, and the therapeutic effect of the phosphocholine liposomes was studied at the cell level and animal experimental level.

First, various choline phospholiposome doxorubicin complex drugs (DDCP), folic acid modified choline phosphodoxorubicin complex drugs (Floate-DDCP) were prepared using the methods of the above-described examples 43 to 52, examples 53 to 62, phosphatidylcholine doxorubicin complex drug (DDPC) required for a control test was prepared according to the same method, and commercial doxorubicin hydrochloride liposome (domaine, DHLP) was purchased as a positive control group.

1. Characterization of the biocompatibility of the phosphocholine liposomes:

The phosphocholine (CP) liposome prepared in example 23 is prepared into PBS solutions with different concentrations and added into a certain volume of red blood cell suspension; PBS solution without CP liposome and secondary water are respectively used as negative control group and positive control group; after mixing well, incubation was carried out at 37 ℃ for 3 hours, after which centrifugation the supernatant was taken out and tested for UV absorption intensity at 541 nm. The experimental result shows that the CP liposome has excellent biocompatibility, and specifically, referring to fig. 3, fig. 3 is a biocompatibility test chart in example 64 of the present invention; where DMCP-a refers to the liposome sample prepared in example 23 above (corresponding to the left column in each set of histograms), DMCP-b refers to the liposomal adriamycin drug complex sample prepared in example 43 (corresponding to the middle column in each set of histograms), and DMCP-c refers to the liposome-folate-adriamycin drug complex sample prepared in example 53 (corresponding to the right column in each set of histograms).

2. Cytotoxicity testing before and after CP Liposome combination drug

The blank CP liposome prepared in example 23 and the CP liposome loaded with doxorubicin prepared in example 43 and example 53 were prepared into DMEM solutions of different concentrations, and added to a culture plate having a certain HeLa Cell concentration, and after incubation for a certain period of time in a culture form, a cytotoxicity test reagent (Cell-titer Blue) was added, and after continuous culture for 4 hours, fluorescence absorption intensity was measured, thereby testing corresponding cytotoxicity. Results see fig. 4 and 5, fig. 4 is a cytotoxicity test chart; FIG. 5 is a graph showing the killing ability of liposome drug complex to Hela cells;

It can be seen that the blank CP liposomes prepared in example 23 have very low cytotoxicity (see fig. 4), and can be developed for in vivo preparations; after doxorubicin is loaded (example 43), excellent cancer cell killing capacity is shown (see fig. 5), and especially the CP liposome composite drug after folic acid modification of example 53 shows the strongest treatment effect.

Similarly, the above test results of the other examples of examples 44 to 52 and 54 to 62 are similar to the above test results, and the blank CP liposome has low cytotoxicity, and shows excellent cancer cell killing ability after being loaded with adriamycin; the CP lipidosome compound drug modified by folic acid shows excellent targeted treatment effect.

3. drug enrichment test of CP (Cyclopentapeptide) liposome in various organs and tumor parts

According to the relevant procedures of animal experiments, HeLa cells are planted under the skin of nude mice, after the tumor volume is increased to 0.2cm3, the liposome composite drug solutions prepared in examples 43 and 53 are administered through the tail vein (the drug dosage of adriamycin is 5mg/kg), and then the tumor enrichment of adriamycin is tested at different time points, and the tumor enrichment of DDCP, Floate-DDCP and DHLP (control group) liposome composite drugs is tested at 3 hours, 12 hours, 24 hours and 48 hours, respectively, and the results are shown in fig. 6, and fig. 6 is a test chart of the tumor enrichment of adriamycin at different time points in example 64, and it can be seen that the Floate-DDCP liposome can be more effectively enriched in cancer tissues than the DDCP and DHLP liposome.

after 48 hours, the mice were dissected and the organs were imaged by infrared imaging, and the results are shown in fig. 7, which is a test chart of drug enrichment of CP liposomes in example 64 in each organ and tumor site. The Floate-DDCP drug system can be more efficiently enriched in cancer tissues, has lower enrichment degree in other organs, and can effectively improve the utilization efficiency of drugs.

4. CP Liposome tumor inhibition Effect test

according to the relevant operation flow of animal experiments, HeLa cells are planted under the skin of a nude mouse, after the tumor volume is increased to 0.2cm3, the liposome composite medicine solution prepared in example 43 and example 53 is administrated through the tail vein (the dosage of adriamycin is 5mg/kg), the administration period is 2 days, the administration frequency is 2 times (the first administration is recorded as day 0, and the administration is carried out on day 0 and day 2 respectively), and then the change of the tumor volume and the body weight of the mouse are tested in real time. After 14 days, the mice are sacrificed and the viscera are dissected, the viscera are sliced and then subjected to H & E staining, and whether the liposome composite medicament has toxic or side effect on normal viscera and the killing effect on tumor focus cells is observed. The results are shown in FIGS. 8, 9, 10 and 11, respectively, and FIG. 8 is a graph showing the test for the change in tumor volume in example 64; FIG. 9 is a graph showing the test of the change in tumor weight in example 64; FIG. 10 is a graph showing the staining test for apoptosis of tumor foci in example 64; FIG. 11 shows the results of H & E staining experiments performed on mouse organs in example 64.

As can be seen from fig. 8 and 9, the Floate-DDCP liposome composite drug exhibited the best tumor growth inhibition effect, and the tumor volume did not increase significantly in the 14-day treatment period, while the blank group (PBS group) showed the fastest tumor volume increase, and DHLP and DDCP liposomes exhibited similar tumor inhibition effects. Meanwhile, the body weight of the mouse is not obviously reduced in the treatment process, and the fact that the Floate-DDCP liposome composite medicament has no obvious toxic or side effect on organisms is proved.

H & E staining experiments show that tumor focus cells undergo maximum apoptosis compared with other experimental groups after Floate-DDCP liposome composite drug treatment, and further prove that the system has the optimal tumor inhibition effect (see figure 10). Meanwhile, H & E staining experimental results of other organs show that the Floate-DDCP liposome composite drug system has no toxic or side effect on normal tissues and organs, is safe and nontoxic (see figure 11), and shows great commercial application value.

in conclusion, the Floate-DCCP series liposome composite medicine has excellent biological safety and tumor treatment effect, can replace the liposome composite medicine on the current market, and achieves the aim of high-efficiency treatment.

the above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A liposome having the structure of formula (I):

wherein the content of the first and second substances,

The selection of n1, n2, and L is as follows:

n1 ═ 0, n2 ═ 0, and L is- (CH2) n3 —; wherein n3 is 1-4;

or

2. the liposome of claim 1,

y is 1, a1 and a2 are independently selected from C5 to C35 alkyl;

Or

or

3. the liposome of claim 1, wherein n1, n2 and L are selected as follows:

n1 ═ 0, n2 ═ 0, and L is- (CH2) n3 —; wherein n3 is 1-4;

Or

n 1-1, n 2-2 and L is

4. The liposome of claim 2, wherein a1 and a2 are independently selected from: CH (CH) 8 —, CH (CH) 9 —, CH (CH) 10 —, CH (CH) 11 —, CH (CH) 12 —, CH (CH) 13 —, CH (CH) 14 —, CH (CH) 15 —, CH (CH) 16 —, CH (CH) 17 —, CH (CH) 18 —, CH (CH) 5CH ═ CH (CH) 7 —, CH (CH) 5CH ═ CH (CH) 9 —, CH (CH) 7CH ═ CH (CH) 7 —, CH (CH) 5CH ═ CH (CH) 11 —, CH3CH2 ═ CHCH (CH) 4 ═ CH3CH2 ═ CHCH (CH) 2 ═ CHCH (CH) 4 —, CH (CH) CH2 ═ CHCH (CH) 2 ═ CHCH (CH) 2 —, CH (CH) 2 ═ CHCH (CH), CH3(CH2)4CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2)6 —, CH3(CH2)4CH ═ CHCH2CH ═ CHCH2CH ═ CHCH2CH ═ CH (CH2)3 —, CH3(CH2)4CH ═ CHCH ═ CH (CH2)8 —, or CH3(CH2)5CH ═ CHCH ═ CH (CH2)7 —;

5. The liposome of any one of claims 1 to 4, wherein the liposome is selected from one or more of the structures represented by formula I-1 to formula I-7:

wherein the content of the first and second substances,

6. a method of preparing a liposome according to any one of claims 1 to 5, comprising the steps of:

the step a) and the step b) are not limited in sequence;

wherein the content of the first and second substances,

the selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4;

Or

7. A method of preparing a liposome according to any one of claims 1 to 5, comprising the steps of:

Wherein the content of the first and second substances,

The selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4;

Or

8. A method of preparing a liposome according to any one of claims 1 to 5, comprising the steps of:

wherein the content of the first and second substances,

The selection of n1, n2, L1 and L2 is as follows:

n1 is 0, n2 is 0, L1 and L2 are connected to form- (CH2) n3-, and n3 is 1-4;

Or

9. a liposome assembly, wherein the assembly is a micelle, a cluster, a micelle, a vesicle, a lipid bilayer membrane or a lipid multilayer membrane;

The liposome is the liposome according to any one of claims 1 to 5 or the liposome prepared by the preparation method according to any one of claims 6 to 8.

10. A cargo liposome complex, which is characterized by comprising a liposome carrier and a cargo loaded on the liposome carrier;

the liposome in the liposome carrier is the liposome of any one of claims 1 to 5 or the liposome prepared by the preparation method of any one of claims 6 to 8;