CN116874523A - Stimulus-responsive choline phospholipid molecule and preparation method and application thereof - Google Patents

Stimulus-responsive choline phospholipid molecule and preparation method and application thereof Download PDF

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CN116874523A
CN116874523A CN202310528495.4A CN202310528495A CN116874523A CN 116874523 A CN116874523 A CN 116874523A CN 202310528495 A CN202310528495 A CN 202310528495A CN 116874523 A CN116874523 A CN 116874523A
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compound
liposome
integer
responsive
stimulus
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孙伯旺
胡进忠
高志国
李瑶嘉
刘敏
陈剑
蔡卓尔
何小凡
魏宛莹
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Southeast University
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    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl

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Abstract

The invention discloses a stimulus-responsive choline phospholipid molecule, and also discloses a preparation method of the stimulus-responsive choline phospholipid molecule and application of the stimulus-responsive choline phospholipid molecule in preparation of drug-loaded liposome complexes. The liposome of the invention has a hydrophobic chain part on the left side of L and a choline phosphate structure on the right side, and the two are connected by a specific L group to form a lipid molecule with a specific structure, wherein A is 1 And A 2 Introducing thioether, thioketal or disulfide bond stimulus-responsive groups into the hydrophobic double strand, wherein the stimulus-responsive groups are contained in the hydrophobic double strand so as to quickly release the entrapped substances by causing the change of the hydrophobicity (the hydrophobicity is changed into hydrophilicity) of the liposome; the R group of choline phosphate can be selectivelyModification is carried out, so that different kinds of liposomes with different functions can be obtained; the liposome has stimulus responsiveness and tumor targeting, and the prepared drug-loaded liposome compound has obvious inhibition effect on tumor growth, so that the liposome has wide application prospect in the aspect of tumor treatment.

Description

Stimulus-responsive choline phospholipid molecule and preparation method and application thereof
Technical Field
The invention relates to a stimulus-responsive Choline Phosphate (CP) lipid molecule, and also relates to a preparation method of the stimulus-responsive Choline Phosphate (CP) lipid molecule and application of the stimulus-responsive Choline Phosphate (CP) lipid molecule in preparation of drug-loaded liposome complexes.
Background
Since Bangham found liposomes, the study of liposomes as drug carrier formulations has been receiving attention from a large number of pharmaceutical workers, and in the fields of pharmacy and biology, liposomes have been commonly used as cell membrane models and drug delivery carriers because of their amphiphilic bilayer structure properties, and various drug-loaded traditional Phosphorylcholine (PC) liposomes have been marketed and used clinically, but liposome complexes currently used clinically have a number of defects, in particular as follows: (1) The liposome clinically used at present mainly comprises natural and artificial synthesized traditional phospholipids, has a part of structures of alkane long chains and Phosphorylcholine (PC) zwitterions, and PC lipid molecules lack effective modification sites in structure, are difficult to carry out subsequent function assignment and have a narrow application range; (2) The liposome drug-loaded complex used clinically at present has poor targeting property, mainly uses high permeability and retention Effect (EPR) to passively target tumor sites, has poor selectivity, often needs to increase dosage, and causes great toxic and side effects; (3) At present, liposome drug-loaded complexes clinically used do not have environmental stimulus responsiveness (ROS and GSH responses) and have no controllable release, so that the research on controllable responsive release liposomes has important significance; (4) The liposome drug-loaded complex used clinically at present has short blood circulation time and is easy to be rapidly discharged to the outside through liver, kidney and the like, so that the drug utilization rate is low.
Disclosure of Invention
The invention aims to: the invention aims at providing a choline phospholipid molecule with environmental stimulus responsiveness (ROS and GSH responsiveness), modifiable property and targeting property, and another aim of the invention is to provide a preparation method of the choline phospholipid molecule and application of the choline phospholipid molecule in preparation of drug-loaded liposome complexes.
The technical scheme is as follows: the structural formula of the stimulus-responsive choline phospholipid molecule is shown as follows:
l is
When L isWhen A is 1 Is->A 2 Is thatWherein m is 1 And m 2 Is an integer of 0 to 21, n 1 And n 2 Is an integer of 4 to 6; r is C1-C8 alkyl, C3-C8 alkenyl or C3-C8 alkynyl;
when L isWhen A is 1 Is-> A 2 Is-> Wherein x is an integer of 0 to 11; r is C1-C8 alkyl, C3-C8 alkenyl or C3-C8 alkynyl.
Wherein A is 1 And A 2 A group selected from the group consisting of-S-, -S-S-or thioketal; when A is 1 And A 2 Is that Any of the following, including but not limited to: CH (CH) 3 -S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 3 -S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 5 -S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 7 -S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 9 -S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 11 -S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 21 -S-(CH 2 ) 5 -or CH 3 -(CH 2 ) 3 -S-S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 5 -S-S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 7 -S-S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 9 -S-S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 11 -S-S-(CH 2 ) 5 -、CH 3 -(CH 2 ) 21 -S-S-(CH 2 ) 5 -。
When A is 1 And A 2 Is thatAny of the following, including but not limited to: CH (CH) 3 -COO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 3 COO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 5 COO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 7 COO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 19 COO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 11 COO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -OOC-or
CH 3 -COO-(CH 2 ) 2 -S-CH(CH3) 2 -S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 3 COO-(CH 2 ) 2 -S-CH(CH3)2-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 5 COO-(CH 2 ) 2 -S-CH(CH3)2-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 7 COO-(CH 2 ) 2 -S-CH(CH3)2-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 9 COO-(CH 2 ) 2 -S-CH(CH3)2-S-(CH 2 ) 2 -OOC-、CH 3 -(CH 2 ) 11 COO-(CH 2 ) 2 -S-CH(CH3)2-S-(CH 2 ) 2 -OOC-。
R is a C1-C8 alkyl group, a C3-C8 alkenyl group, or a C3-C8 alkynyl group, including but not limited to: CH (CH) 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -、CH≡C-(CH 2 ) 2 -. The invention is thatStimulus-responsive cholinergic lipid molecules when L isWhen A is 1 Is that
A 2 Is->The structural formula is as follows:
the stimulus-responsive choline phosphate lipid molecules of the invention, when L isWhen A is 1 Is that
A 2 Is->The structural formula is as follows:
the stimulus-responsive choline phosphate lipid molecules of the invention, when L isWhen A is 1 Is that
A 2 Is->The structural formula is as follows:
the stimulus-responsive choline phosphate lipid molecules of the invention, when L isWhen A is 1 Is that
A 2 Is->The structural formula is as follows:
wherein m is 1 And m 2 Is an integer of 0 to 21, n 1 And n 2 Is an integer of 4 to 6; x is an integer of 0 to 11; r is a group including but not limited to: CH (CH) 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -、CH≡C-(CH 2 ) 2 -。
The preparation method of the stimulus-responsive choline phospholipid molecule comprises the following steps:
(1) Alkyl bromides with different chain lengths react with ethyl formate after generating Grignard reagent with Mg to obtain a compound X-1;
(2) Condensing the compound X-1 with the compound Y-1 to obtain a compound X-2;
(3) Reacting the compound X-2 with alkyl mercaptans with different chain lengths to obtain a compound X-3;
(4) Reacting a compound X-3 with a compound Y-2 to obtain a liposome molecule of the formula I;
the synthetic route is as follows:
wherein n is 3 Is an integer of 2 to 4, m 1 Is an integer of 0 to 21, n 1 Is an integer of 4 to 6, x is an integer of 0 to 11;
r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
The preparation method of the stimulus-responsive choline phospholipid molecule comprises the following steps:
(1) The compound X-5 is obtained by reacting the compound X-4 with different chain lengths with Mg to form a Grignard reagent and then with ethyl formate;
(2) Condensing the compound X-5 with the compound Y-1 to obtain a compound X-6;
(3) Reacting the compound X-6 with compounds X-7 with different chain lengths to obtain a compound X-8;
(4) Reacting a compound X-8 with Y-2 to obtain a liposome molecule of a formula II;
the synthetic route is as follows:
wherein n is 2 Is an integer of 4 to 6, m 2 Is an integer of 0 to 21;
r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
The preparation method of the stimulus-responsive choline phospholipid molecule comprises the following steps:
(1) Reacting a compound Z-1 or a compound Z-2 with phenyl p-nitrochloroformate and then reacting with a compound Y-3 to obtain a compound Z-4 or a compound Z-5;
(2) Condensing a compound Z-4 or Z-5 with alkyl carboxylic esters with different chain lengths to obtain a compound Z-6 or Z-7;
(3) Reacting a compound Z-6 or Z-7 with a compound Y-2 to obtain a liposome molecule of a formula III or a formula IV;
the synthetic route is as follows:
wherein x is an integer of 0 to 11;
r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
The lipid molecules formed by the two lipid molecules are opposite to the traditional Phosphocholine (PC) in configuration, and the cell structure is the PC structure, so that the CP and the PC with opposite configurations have supermolecular action (through mutual attraction of positive and negative charges), are easier to be absorbed by tumor cells and are enriched at the tumor cells through the EPR effect.
The lipid assembly formed by the lipid molecules has a phospholipid bilayer structure, is spherical nano particles, can be used as a drug delivery system, and can be used for loading drugs, targeting substances, proteins or nucleic acids and the like and delivering the drugs, the targeting substances, the proteins or the nucleic acids to ideal sites.
Preparing a drug-loaded liposome complex based on the lipid assembly, wherein the complex takes liposome as a carrier and further comprises a load loaded on the liposome carrier; the load comprises one or a combination of several of a drug, a targeting substance, a protein or a nucleic acid.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: (1) The liposome of the invention has a hydrophobic chain part on the left side of L and a choline phosphate structure on the right side, and the two are connected by a specific L group to form a lipid molecule with a specific structure, wherein A is 1 And A 2 The introduction of thioether, thioketal or disulfide-bond stimulus-responsive groups (i.e., lipid molecules A1 and A2 contain partially Reactive Oxygen Species (ROS) -responsive thioether or thioketal groups, or Glutathione (GSH) -responsive disulfide bonds) at the hydrophobic duplex, which rapidly release entrapped cargo by causing a change in liposome hydrophobicity (hydrophobicity to hydrophilicity); (2) The R group of choline phosphate can be selectively modified, so that liposomes with different types and different functions can be obtained; (3) The liposome provided by the invention has stimulus responsiveness and tumor targeting, and the prepared drug-loaded liposome compound shows remarkable inhibition effect on tumor growth, so that the liposome has wide application prospect in tumor treatment.
Drawings
FIG. 1 is a transmission electron microscope image of a liposome solution obtained in example 18;
FIG. 2 is a transmission electron microscope image of the drug-loaded liposome solution obtained in example 21;
FIG. 3 is a chart of a biocompatibility test;
FIG. 4 is a graph of a blank liposome cytotoxicity test prepared in example 16;
FIG. 5 is a graph showing the killing ability of 4T1 cells of the drug-loaded liposome complex prepared in example 21;
FIG. 6 is a graph showing enrichment of DSPE-Cy-5.5 labeled liposomes in tumors at different time points;
FIG. 7 is a graph showing drug enrichment test of CP liposomes in various organs and tumor sites;
FIG. 8 is a graph showing tumor volume change test;
FIG. 9 is a graph showing the change in tumor weight;
FIG. 10 is a graph showing apoptosis staining of tumor lesion cells;
FIG. 11 shows the results of H & E staining experiments on mouse organs.
Detailed Description
Example 1: compound X-1 (n) 1 Preparation of =1)
Magnesium metal (792 mg) was added under Ar flow to a three-necked flask equipped with a reflux condenser, followed by 5mL of THF (tetrahydrofuran) and then one shot of iodine (initiator); the suspension was heated to 45℃with stirring, and 0.5g of 5-bromopent-1-ene was added, and after stirring, a THF solution containing 4g of 5-bromopent-1-ene was added at a rate to maintain a gentle reflux state (the volume of THF solution: 40 mL); then stirring the obtained mixture at 40 ℃ for 60min, cooling to room temperature to obtain a Grignard reagent, then dropwise adding a THF solution of anhydrous ethyl formate (the volume of the THF solution is 10mL, and 1.14mL of anhydrous ethyl formate is contained in the 10mL THF solution) into the Grignard reagent at 0 ℃ under Ar gas flow, then heating to room temperature and stirring for 2 hours; the mixture was quenched with ice water (10 mL) and then H 2 O (50 mL) was diluted, and finally extracted with EA (ethyl acetate), and the organic phases were combined, dried, and concentrated to give a yellow oil, which was chromatographed to give compound X-1 as a pale yellow oil (1.2 g, yield 50.93%). 1 H NMR(600MHz,CDCl 3 )δ5.91–5.71(m,2H),5.15–4.83(m,4H),3.60(dd,J＝7.2,4.1Hz,1H),2.21–1.93(m,4H),1.56–1.40(m,8H).
Example 2: preparation of Compound X-2
Compound X-1 (504.84 mg) and 4-dimethylaminobutyrate (603.36 mg) were added to a three-necked round bottle of 100mL volume, followed by 40mL of anhydrous DCM (dichloromethane), then 690.12g of EDCI (carbodiimide), 1.04mL of DIPEA (N, N-diisopropylethylamine) and 91.5mg of DMAP (N, N-dimethyl-4-pyridinamine); the reaction was stirred under nitrogen overnight, after which the mixture was diluted with DCM and saturated NaHCO in turn 3 The solution, water and saturated NaCl solution were washed, the aqueous phases were combined and extracted with DCM, then the organic phases were combined, dried and concentrated to give a yellow oil. Purification by column chromatography gave compound X-2 as a pale yellow oil (570 mg, 67.5% yield).1HNMR(600MHz,CDCl3)δ5.76(ddt,J＝16.9,10.2,6.7Hz,2H),4.98(dd,J＝17.1,1.5Hz,2H),4.92(t,J＝11.7Hz,2H),4.91–4.85(m,1H),2.31(t,J＝7.5Hz,2H),2.29–2.24(m,2H),2.20(s,6H),2.03(dt,J＝13.1,6.7Hz,4H),1.81–1.73(m,2H),1.58–1.46(m,4H),1.44–1.30(m,4H).
Example 3: preparation of Compound X-3
Compound X-2 (562.48 mg), 1-butanethiol (541.14 mg) and azobisisobutyronitrile (164.2 mg) were added to 10mL of ethyl acetate at N 2 The mixture was heated at 80℃under an atmosphere to react for 3 hours, then the solvent was evaporated, and the obtained crude product was purified by a silica gel column to give the product as compound X-2 as a yellow oily liquid (0.66 g, yield 71.46%).
Examples 4 to 7 were each replaced by 709.44mg of 1-hexanethiol, 547.74mg of 1-octanethiol, 1.046g of n-decathiol and 1.2g of n-dodecanethiol in the preparation of example 3, and the yields and structures of the obtained products were shown in Table 1.
Table 1 shows the yields and the structures of the products of the compounds X-3 obtained in examples 3 to 7
Example 8: preparation of Compound Y-2
To an anhydrous THF solution (volume of THF solution: 15 mL) containing 3-butyn-1-ol (1.4 g) at-10℃under argon, 2.92mL of triethylamine was added, and then an anhydrous THF solution (volume of THF solution: 10 mL) containing 2.85g of 2-chloro-2-oxo-1, 3, 2-dioxaphosphorinane was slowly added dropwise at-10℃to warm the mixture to room temperature and stir for 2 hours, after the reaction, filtration was carried out, the filtrate was concentrated by rotary evaporation and vacuum-dried to give a crude product as a yellow oil, and the obtained liquid was purified by distillation under reduced pressure to give the product as compound Y-2 (1.1 g, yield: 31.38%).
The procedure was followed, except that 640.8mg of methanol was used in place of 1.4g of 3-butyn-1-ol in the preparation of example 8 in example 9, and the yield of the obtained product and the structure of the product were shown in Table 2.
Table 2 shows the yields and the structures of the products of the compounds Y-2 obtained in examples 8 to 9
Example 10: synthesis of Liposome represented by formula I
The compound Y-2 (200 mg) obtained in example 8 was dissolved in 3mL of anhydrous acetonitrile under argon, then the compound X-3 (507.45 mg) obtained in example 3 was added thereto, and the mixture was heated to 70℃for 48 hours, and after the reaction, concentrated, and purified by column chromatography to obtain a product (140 mg, yield 19.46%) as a transparent oil.
Examples 11 to 14 were prepared according to the above procedure except that 569.7mg of the product of example 4, 631.4mg of the product of example 5, 693.14mg of the product of example 6 and 754.86mg of the product of example 7 were used in place of 507.45mg of the product of example 3 in the preparation of example 10, respectively, and the yields and structures of the obtained end products are shown in Table 3.
Table 3 shows the yields and the structures of the products of the liposomes of formula I obtained in examples 10 to 14
Example 15: assembly of liposomes
A vial was taken and 5mg of the product from example 10 was dissolved with 1.5mg of cholesterol in 0.5mL of absolute ethanol to give an ethanol solution; taking another small bottle, adding 5mL deionized water or glucose solution with mass fraction of 5%, adding small magneton, dropwise adding the ethanol solution into the bottle under high-speed stirring, performing ultrasonic treatment with a warm water bath probe for 5min after the dropwise addition, removing ethanol by rotary evaporation, and filtering the obtained sample with a 0.22 μm polycarbonate filter membrane to obtain liposome aqueous solution or liposome glucose solution.
According to the above preparation procedure, except that examples 16 to 19 were replaced with 5mg of the product of example 11, 5mg of the product of example 12, 5mg of the product of example 13 and 5mg of the product of example 14, respectively, for 5mg of the product of example 10 in the preparation procedure of example 15, liposome solutions having uniform particle diameters were obtained in examples 15 to 19, and the particle diameters and distribution of the liposome solutions were measured by DLS, and the particle diameters and distribution of the obtained liposomes were shown in Table 4.
Table 4 shows particle diameters and distributions of liposome solutions obtained in examples 15 to 19
Average particle diameter (nm) Distribution (PDI)
Example 15 41.9 0.177
Example 16 44.5 0.202
Example 17 45.8 0.222
Example 18 48.9 0.188
Example 19 52.9 0.210
As a result of transmission electron microscopy observation of the liposome solution obtained in example 18, as shown in FIG. 1, FIG. 1 shows a transmission electron microscopy image of the liposome solution obtained in example 18 of the present invention, and as can be seen from FIG. 1, liposome particles are nanoparticles having a uniform particle size.
Example 20: assembly of drug-loaded liposomes
A vial was taken and 5mg of the product from example 10 was dissolved with 1.5mg of cholesterol in 0.5mL of absolute ethanol to give an ethanol solution; taking another small bottle, adding 5mL deionized water containing 1mg doxorubicin hydrochloride or glucose solution with mass fraction of 5%, adding small magneton, dropwise adding the ethanol solution into the bottle under high-speed stirring, performing ultrasonic treatment for 5min by a warm water bath probe after the dropwise addition, performing rotary evaporation to remove ethanol, dialyzing the obtained sample at room temperature for 6h by a dialysis bag with molecular retention of 3000Da, changing water once every 3h, and filtering by a polycarbonate filter membrane with molecular retention of 0.22 mu m to obtain a liposome aqueous solution or liposome glucose solution loaded by doxorubicin.
According to the above preparation procedure, except that examples 21 to 24 were replaced with 5mg of the product of example 11, 5mg of the product of example 12, 5mg of the product of example 13 and 5mg of the product of example 14, respectively, for 5mg of the product of example 10 in the preparation procedure of example 20, each of examples 20 to 24 gave drug-loaded liposome solutions having uniform particle diameters, and the particle diameters and distributions of the drug-loaded liposome solutions were measured by DLS and are shown in Table 5.
Table 5 shows particle diameters and distributions of drug-loaded liposome solutions obtained in examples 20 to 24
Average particle diameter (nm) Distribution (PDI)
Example 20 106.7 0.138
Example 21 122.3 0.203
Example 22 131.6 0.231
Example 23 138.1 0.198
Example 24 144.3 0.243
As a result of transmission electron microscopy observation of the liposome solution obtained in example 21, FIG. 2 shows, and FIG. 2 shows a transmission electron microscopy image of the drug-loaded liposome solution obtained in example 21 of the present invention, and as can be seen from FIG. 2, liposome particles are nanoparticles having a uniform particle size.
Example 25: in vivo long-circulating test of liposomes
SD rats (weight 180 g-220 g) of 6-8 weeks old were weighed, the drug-loaded liposome solution obtained in example 20 was intravenously injected into the tail of a standard mouse of 10mg/kg of doxorubicin, blood was taken at the following time, and the concentration of doxorubicin in the blood was measured by HPLC method, thereby obtaining the pharmacokinetic percentage content.
The blood taking time is respectively as follows: 30min, 1h, 2h, 4h, 6h, 8h, 10h, 24h,36h, 48h. 0.6mL of orbital blood of the mice is taken and collected in a 1.5mL heparin tube, and the supernatant is taken after centrifugation at 3000rpm in a low-temperature centrifuge for 5 min. The plasma was stored at-20℃and was ready for detection. The results are shown in Table 6.
Table 6 shows the results of in vivo long-circulating test of drug-loaded liposomes
As is clear from Table 6, the drug-loaded liposome complex still measured about 0.68% of the drug concentration in the blood after 24 hours of participation in the in vivo circulation, and the drug concentration in the blood remained about 0.39% after 48 hours of participation in the in vivo circulation, showing excellent in vivo long-circulating effect. Similarly, the drug-loaded liposome solutions obtained in examples 21 to 24 were subjected to the in vivo long-circulating test according to the above test procedure, and the results were comparable to Table 6, and the drug-loaded liposome complexes obtained in examples 21 to 24 also showed excellent in vivo long-circulating effects.
Example 26: preparation of DSPE-Cy-5.5 labeled liposomes
A vial was taken, 5mg of the product obtained in example 10 and 1.5mg of cholesterol were dissolved in 0.5mL of absolute ethanol, and a trace amount of DSPE-Cy-5.5 (8. Mu.L, 1mg/mL in ethanol) was added thereto to obtain a mixed solution; taking another small bottle, adding 5mL deionized water or glucose solution with mass fraction of 5% into the bottle, adding small magneton, dropwise adding the mixed solution into the bottle under high-speed stirring, performing ultrasonic treatment for 5-10 min by a probe after the dropwise addition, removing ethanol by rotary evaporation, and filtering the obtained sample by a polycarbonate filter membrane with the mass fraction of 0.22 mu m to obtain liposome aqueous solution or liposome glucose solution.
According to the above preparation procedure, except that examples 27 to 30 were replaced with 5mg of the product of example 11, 5mg of the product of example 12, 5mg of the product of example 13 and 5mg of the product of example 14, respectively, in the preparation procedure of example 26, each of examples 26 to 30 gave DSPE-Cy-5.5-labeled liposome solutions having a uniform particle size (liposomes themselves were not fluorescent signal, labeled with cyanine-based dye for in vivo imaging identification).
Example 31: liposome drug-loaded complex for in-vitro and in-vivo studies
A mouse model of breast cancer was constructed, and the doxorubicin-loaded liposome complexes prepared in examples 20 to 24 were used as control groups with commercial doxorubicin hydrochloride liposomes (DOX/HSPCs) and hydrogenated soybean lecithin-loaded doxorubicin complexes.
The liposomes prepared in examples 15 to 19 were prepared as 1mL of 5% glucose solutions of liposomes of different concentrations (liposome concentrations of 200. Mu.g/mL, 400. Mu.g/mL, 800. Mu.g/mL, respectively) and added to a mixed solution composed of a red blood cell suspension having a mass fraction of 2% of 1mL and 3mL of physiological saline; taking physiological saline without CP liposome and deionized water without CP liposome as a negative control group and a positive control group respectively; after mixing well, incubation is carried out for 3 hours at 37 ℃, then centrifugation is carried out, and after centrifugation, the supernatant is taken out and tested for ultraviolet absorption intensity at 541 nm. The experimental results show that the CP liposome has excellent biocompatibility, and fig. 3 is a chart for testing the biocompatibility.
The blank liposome prepared in example 16 and the doxorubicin-loaded liposome prepared in example 21 are prepared into medium solutions with different concentrations, the medium solutions are added into a 96-well plate with a certain 4T1 cell concentration, after culturing for 24 hours, the prepared MTT solution is added, after culturing is continued for 4 hours, after dissolving in DMSO, the fluorescence absorption intensity is tested by an enzyme-labeled instrument, and thus the corresponding cytotoxicity is tested. Referring to fig. 4 and 5, fig. 4 is a blank liposome cytotoxicity test chart; FIG. 5 is a graph showing the killing ability of liposome drug complexes on 4T1 cells; as can be seen from fig. 4 to 5, the empty liposome prepared in example 16 has little cytotoxicity and can be used as a carrier; and the drug-loaded liposome loaded with doxorubicin shows excellent cancer cell killing ability.
Drug enrichment test of Cy-5.5 labeled liposomes at tumor site and individual organs:
according to the related operation flow of animal experiment, 4T1 cells are planted under the skin of BALB/c female mouse (18-20 g) until the tumor volume is increased to about 150-200 mm 3 The cy 5.5-labeled liposome solutions prepared in examples 26-30 were then administered through the tail vein (200 μl), after which the cy-5.5-labeled liposomes were tested for tumor enrichment at different time points, the control group was DSPE-cy 5.5-labeled hydrogenated soybean lecithin liposomes, and fluorescence imaging was performed at 3h,6h,9h,12h,24h,36h, and 48h of administration, as shown in fig. 6, which is a graph of the time point test for doxorubicin tumor enrichment, and as can be seen from fig. 6, the cy 5.5-labeled CP liposomes were more effective in cancer tissue enrichment than the cy 5.5-labeled HSPC liposomes in examples 26-30.
After 48h, mice were dissected and the organs were imaged by fluorescence, and the results are shown in FIG. 7, and FIG. 7 is a graph showing drug enrichment test of Cy5.5-labeled liposomes in individual organs and tumor sites. From fig. 7, it can be seen that the lipid systems of examples 26 to 30 can be more efficiently enriched in cancer tissues and less enriched in other organs, so that the prepared CP liposome carrier has tumor targeting.
Drug-loaded liposome tumor inhibition effect test:
according to the related operation flow of animal experiments, 4T1 cells are planted under the skin of a BALB/c female mouse (18-20 g) for 6-7 weeks until the tumor volume is increased to about 150-200 mm 3 The liposomal drug complex solutions prepared in examples 20-24, as well as the control DOX/HSPCs liposomal drug complex, were then administered via the tail vein (5 mg/kg doxorubicin) once every three days, and then tested for changes in tumor volume and body weight every two days. Mice were sacrificed after day 21Dissecting viscera, slicing viscera, and performing H&E, dyeing, and observing whether the liposome composite medicine has toxic and side effects on normal viscera or not and killing effect on tumor focus cells. The results are shown in FIG. 8, FIG. 9, FIG. 10 and FIG. 11, respectively, and FIG. 8 is a graph showing the tumor volume change test after treatment in examples 20 to 24; FIG. 9 is a graph showing the weight change test of mice treated with the drug-loaded liposome complexes of examples 20 to 24; FIG. 10 shows tumor cells H after treatment with the drug-loaded liposome complexes of examples 20 to 24&E and TUNEL staining test patterns; FIG. 11 shows the results of treatment of mice with the drug-loaded liposome complexes of examples 20 to 24&E dyeing test results.
According to fig. 8 and 9, the drug-loaded liposome complex in example 21 exhibited a significant inhibitory effect on tumor growth. The treatment period was 21 days during which the tumor volume did not increase significantly, whereas the tumor volume of the negative control group (PBS group) increased most rapidly. DOX/HSPCs liposomes also exhibit similar anti-tumor effects. Furthermore, there was no significant decrease in mice body weight during the course of treatment, indicating that the drug-loaded liposome complex used in example 21 had no significant toxic side effects.
The drug-loaded liposome complexes used in example 21 exhibited the highest apoptosis rate in treating tumors by H & E and TUNEL staining experiments, compared to the other experimental groups (see fig. 10 for details). In addition, the results of H & E staining experiments on organs such as heart, liver, spleen, lung, kidney, etc., indicate that the drug-loaded liposome complexes prepared in examples 20 to 24 do not exhibit significant toxic and side effects on normal tissues and organs (see fig. 11 for details). Therefore, the drug-loaded liposome compound prepared in examples 20-24 not only has excellent biosafety, but also has remarkable curative effect in tumor treatment, and has wide application prospect.

Claims (10)

1. A stimuli-responsive cholinergic lipid molecule characterized by the following structural formula:
wherein L is
When L isWhen A is 1 Is->A 2 Is thatWherein m is 1 And m 2 Is an integer of 0 to 21, n 1 And n 2 Is an integer of 4 to 6; r is C1-C8 alkyl, C3-C8 alkenyl or C3-C8 alkynyl;
when L isWhen A is 1 Is-> A 2 Is-> Wherein x is an integer of 0 to 11; r is C1-C8 alkyl, C3-C8 alkenyl or C3-C8 alkynyl.
2. The stimuli responsive cholinergic lipid molecule of claim 1, wherein when L isWhen A is 1 Is->A 2 Is->The structural formula is as follows:wherein m is 1 And m 2 Is an integer of 0 to 21, n 1 And n 2 Is an integer of 4 to 6; r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
3. The stimuli responsive cholinergic lipid molecule of claim 1, wherein when L isWhen A is 1 Is->A 2 Is->The structural formula is as follows:wherein m is 1 And m 2 Is an integer of 0 to 21, n 1 And n 2 Is an integer of 4 to 6; r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
4. The stimuli responsive cholinergic lipid molecule of claim 1, wherein when L isWhen A is 1 Is->A 2 Is thatThe structural formula is as follows:wherein x is an integer of 0 to 11; r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
5. The stimuli responsive cholinergic lipid molecule of claim 1, wherein when L isWhen A is 1 Is->A 2 Is thatThe structural formula is as follows: />
Wherein x is an integer of 0 to 11; r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
6. The method for preparing the stimulus-responsive choline phosphate lipid molecule according to claim 2, comprising the steps of:
(1) Alkyl bromides with different chain lengths react with ethyl formate after generating Grignard reagent with Mg to obtain a compound X-1;
(2) Condensing the compound X-1 with the compound Y-1 to obtain a compound X-2;
(3) Reacting the compound X-2 with alkyl mercaptans with different chain lengths to obtain a compound X-3;
(4) Reacting a compound X-3 with a compound Y-2 to obtain a liposome molecule of the formula I;
the synthetic route is as follows:
wherein n is 3 Is an integer of 2 to 4, m 1 Is an integer of 0 to 21, n 1 Is an integer of 4 to 6, x is an integer of 0 to 11;
r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
7. A method for preparing a stimulus-responsive choline phospholipid molecule according to claim 3, comprising the steps of:
(1) The compound X-5 is obtained by reacting the compound X-4 with different chain lengths with Mg to form a Grignard reagent and then with ethyl formate;
(2) Condensing the compound X-5 with the compound Y-1 to obtain a compound X-6;
(3) Reacting the compound X-6 with compounds X-7 with different chain lengths to obtain a compound X-8;
(4) Reacting a compound X-8 with Y-2 to obtain a liposome molecule of a formula II;
the synthetic route is as follows:
wherein n is 2 Is an integer of 4 to 6, m 2 Is an integer of 0 to 21;
r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
8. The method for producing a stimulus-responsive choline phosphate lipid molecule according to claim 4 or 5, comprising the steps of:
(1) Reacting a compound Z-1 or a compound Z-2 with a compound Z-3, and then reacting with a compound Y-3 to obtain a compound Z-4 or a compound Z-5;
(2) Condensing a compound Z-4 or Z-5 with alkyl carboxylic esters with different chain lengths to obtain a compound Z-6 or Z-7;
(3) Reacting a compound Z-6 or Z-7 with a compound Y-2 to obtain a liposome molecule of a formula III or a formula IV;
the synthetic route is as follows:
wherein x is an integer of 0 to 11;
r is CH 3 -、CH 3 CH 2 -、CH 3 (CH 2 ) 2 -、CH 3 (CH 2 ) 3 -、CH 2 ＝CHCH 2 -、CH 2 ＝CH(CH 2 ) 2 -、CH≡C-CH 2 -or CH≡C- (CH) 2 ) 2 -。
9. Use of the stimuli-responsive cholinergic lipid molecule of claim 1 for the preparation of a drug-loaded liposome complex.
10. The use of a stimuli-responsive cholinergic lipid molecule according to claim 9 for the preparation of a drug-loaded liposome complex, characterized in that: the drug-loaded liposome complex comprises a liposome serving as a carrier and a load loaded on the liposome; the load comprises one or a combination of several of a drug, a targeting substance, a protein or a nucleic acid.
CN202310528495.4A 2023-05-11 2023-05-11 Stimulus-responsive choline phospholipid molecule and preparation method and application thereof Pending CN116874523A (en)

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