CN108236603B - Pyrrole imidazole polyamide liposome and preparation method and application thereof - Google Patents
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Abstract
The invention provides a pyrrole imidazole polyamide liposome, a preparation method and application thereof, and particularly discloses a pyrrole imidazole polyamide liposome which is composed of a lipid bilayer and pyrrole imidazole polyamide entrapped in the lipid bilayer, wherein the lipid bilayer comprises phospholipid molecules and cholesterol, the molar ratio of the phospholipid molecules to the cholesterol is (3-4): 1, and the molar ratio of the phospholipid molecules to the pyrrole imidazole polyamide is (20-30): 1. The pyrrole imidazole polyamide liposome provided by the invention uses the cavity in the liposome to encapsulate pyrrole imidazole polyamide, and compared with the encapsulation mode of other carriers, the encapsulation capacity can be effectively improved, so that the bioavailability of pyrrole imidazole polyamide drugs is remarkably improved.
Description
Technical Field
The invention relates to the field of pharmaceutical preparations, in particular to a pyrrole imidazole polyamide liposome and a preparation method thereof.
Background
Cancer treatment taking genes as targets is a new and efficient treatment means, and the development of small molecular compound inhibition drugs for tumor treatment is particularly critical. The pyrrole imidazole polyamide is an artificial micromolecule ligand which is artificially synthesized, mainly comprises five-membered heterocyclic compounds N-methylpyrrole (Py), N-methylimidazole (Im) and N-methyl-3-hydroxypyrrole (Hp) aromatic amino acid and is connected through amido bonds. Pyrrole-imidazole polyamides recognize DNA sequences by forming hydrogen bonds with DNA base pairs to the greatest extent possible: antiparallel paired Py/Py recognizes a T.A or A.T base pair; Im/Py specifically recognizes the G.C base pair, Py/Im specifically recognizes the C.G base pair, Hp/Py specifically recognizes the T.A base pair, and Py/Hp specifically recognizes the A.T base pair. By designing specific pyrrole-imidazole polyamide specifically combined with a key sequence of a target gene promoter, the combination of the transcription regulatory protein and the target gene promoter can be blocked, so that the expression of the gene can be blocked from the gene transcription level. Compared with shRNAi technology widely used in eukaryotic cells at present, the pyrrole-imidazole polyamide mediated gene function inhibition has the remarkable advantages of high stability, good cell permeability and the like while having high specificity, so that the method has great potential in drug research and development aiming at inhibiting gene expression. However, such small molecule drugs (e.g. polyamides) generally have the disadvantages of poor solubility, short half-life in blood, low bioavailability, etc., and the metabolic and kinetic parameters of the drug need to be improved by formulation technology.
The liposome is an ultramicrosphere formed by lipid bilayers, is similar to a biological membrane structure, has a shell layer of single or a plurality of annular lipid bilayers, simultaneously contains a cavity of a water phase inside, and can simultaneously entrap hydrophilic drugs and hydrophobic drugs. The basic component of phospholipid is an inherent component in organisms, is degraded by biotransformation in organisms, has no toxicity and immunogenicity, and therefore, the liposome is considered to be one of the most promising drug carriers.
The group of subjects such as Dervan, Sugiyama and Fukuda, prepares a series of pyrrole-imidazole polyamides and derivatives thereof, and intensively studies the influence of the pyrrole-imidazole polyamides on gene expression in vivo and in vitro, and a remarkable result is obtained in recent years.
Disclosure of Invention
Based on the excellent performance of liposome as a carrier, the invention aims to effectively encapsulate pyrrole-imidazole polyamide by adopting a nano-drug carrier taking liposome as a matrix, and by utilizing the characteristics of tumor growth and a large amount of defective trophoblastic angiogenesis and imperfect lymphatic return system caused by angiogenesis, the passive targeting effect of nanoparticles on tumors by enhancing the permeation and retention effects is promoted. The nano drug delivery carrier with long circulation capability can finally overflow from the vessel, accumulate to tumor tissues and enter cells to release the therapeutic drugs for playing a role.
The invention aims to realize high-specificity DNA binding capacity and excellent membrane penetrating performance of the inhibitor starting from chemical synthesis of the inhibitor through reasonable design; by applying the nano material package and the targeted transportation mechanism, the drug effect and the toxicological characteristics of the polyamide drugs are obviously improved, so that the clinical application prospect of the drugs is improved. The realization of the high-efficiency accumulation of the drug in the tumor tissue is the key point for designing a drug delivery system. In order to realize the purpose, the pyrrole imidazole polyamide is coated by a liposome material with a similar biological membrane structure, so that the solubility of the drug is obviously improved, the transportation mode of the drug is changed, the biocompatibility and the colloidal stability of a drug carrier are improved by a surface modification strategy of a low-immunogenicity coating material (such as long-chain polyethylene glycol), the blood circulation time of the drug carrier is prolonged, and the drug carrier is beneficial to being passively enriched in tumor tissues through an EPR effect.
The invention discloses preparation of a nanoliposome for encapsulating pyrrole imidazole polyamide micromolecule polypeptide drugs capable of identifying Plk1 target genes. The method mainly comprises the following steps: carrying pyrrole imidazole polyamide for specifically recognizing the Plk1 sequence by different phospholipid molecules to prepare nano-liposomes with different surface groups and charges, and obtaining the drug-carrying nano-liposomes by a film dispersion method; polyethylene glycol chains are added on functional groups at the tail ends of phospholipid molecules, and the polyethylene glycol is utilized to improve the blood circulation time of the polyamide liposome after intravenous injection.
The pyrrole imidazole polyamide is an artificially synthesized small molecular compound capable of specifically targeting a critical sequence of a Plk1 promoter. Based on the human Plk1 gene promoter DNA sequence, a highly specific 20-25bp sequence with 2-3 ends positioned around TATA box and GC box is selected as a target sequence. And synthesizing pyrrole imidazole polyamide matched with the selected sequence by a chemical synthesis method. Through reasonable structural design, the benzimidazole fluorescent group is coupled with the pyrrole-imidazole polyamide, so that the membrane penetrating capability of polyamide can be enhanced, and the fluorescence can be generated when the fluorescence-labeled polyamide is combined with a DNA sequence, so that the fluorescence-labeled polyamide can be used as an internal fluorescence probe to monitor the membrane penetrating behavior of the polyamide, the movement of the polyamide in cells and the combination condition of the polyamide and the DNA sequence.
The invention provides a pyrrole imidazole polyamide liposome which is composed of a lipid bilayer and pyrrole imidazole polyamide entrapped in the lipid bilayer, wherein the lipid bilayer comprises phospholipid molecules and cholesterol, the molar ratio of the phospholipid molecules to the cholesterol is (1-10): 1, preferably (3-4): 1, and the molar ratio of the phospholipid molecules to the pyrrole imidazole polyamide is (20-30): 1.
In the technical scheme of the invention, the phospholipid molecule is selected from one or a mixture of more of phospholipid, carboxylated polyethylene glycol-phospholipid, distearoyl phosphatidylcholine, 1, 2-dioleoyl-3-trimethyl ammonium propane and carboxylated polyethylene glycol-distearoyl phosphatidylethanolamine.
Preferably, the polyethylene glycol molecule in the phospholipid molecule is selected from 2000-.
In the technical scheme of the invention, the concentration of the pyrrole imidazole polyamide is 1 μ M-50 μ M, preferably 1 μ M, 2 μ M, 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M, 20 μ M, 30 μ M, 40 μ M and 50 μ M.
In the technical scheme of the invention, the phospholipid molecule is selected from distearoyl phosphatidylcholine, or carboxylated polyethylene glycol-distearoyl phosphatidylethanolamine,
Further, the pyrrole imidazole polyamide liposome is prepared by one of a film dispersion method and a reverse evaporation method.
Further, the pyrrole imidazole polyamide is selected from a conjugate having two or three of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methyl-3-hydroxypyrrole (Hp), or a derivative having a Herster acid modified thereon, preferably a compound having the structure of formula I,
the particle size of the pyrrole imidazole polyamide liposome is 50-300 nm.
The invention also provides a preparation method of the pyrrole imidazole polyamide liposome, which is prepared by a film dispersion method and a reverse evaporation method.
Further, it is prepared by a film dispersion method, comprising the following steps:
1) dissolving phospholipid molecules and cholesterol in an organic solvent to form a membrane material dispersion liquid;
2) under the condition of reduced pressure, removing the organic solvent in the membrane material dispersion liquid, and enabling phospholipid molecules and cholesterol to form a uniform dispersion film on the wall of the container;
3) dissolving pyrrole imidazole polyamide in ultrapure water, dispersing the solution in the container obtained in the step 2), and mechanically dispersing the solution to enable phospholipid molecules and cholesterol to be self-assembled into a liposome encapsulating the pyrrole imidazole polyamide;
optionally, steps 4) and 5) are also included,
4) passing the liposome obtained in step 3) through a 0.22 μm aqueous filter membrane;
5) and (3) treating the pyrrole imidazole polyamide liposome obtained in the step 4) by using an ultrafiltration centrifugal tube to remove free pyrrole imidazole polyamide.
In another aspect, the invention provides the use of a pyrrole imidazole polyamide liposome or a compound of formula I according to the invention for the preparation of a Plk inhibitor, preferably a Plk-1 inhibitor.
In another aspect, the invention provides the use of the pyrrole imidazole polyamide liposomes described in the invention for the preparation of a medicament for the treatment or prevention of a disease associated with a Plk1 inhibitor, said disease associated with a Plk1 inhibitor being selected from the group consisting of a proliferative disease, preferably a tumor, more preferably lung cancer, liver cancer, ovarian cancer, esophageal cancer, cervical cancer, non-small cell lung cancer, skin cancer, leukemia, hodgkin's lymphoma.
Advantageous effects
1) The pyrrole imidazole polyamide liposome provided by the invention uses the cavity in the liposome to encapsulate pyrrole imidazole polyamide, and compared with the encapsulation mode of other carriers, the encapsulation capacity can be effectively improved, so that the bioavailability of pyrrole imidazole polyamide drugs is remarkably improved, and the effective concentration of the pyrrole imidazole polyamide drugs for inhibiting tumor cells can be reduced from 10 mu M to below 1 mu M.
2) The pyrrole imidazole polyamide liposome is coupled with a polyethylene glycol chain on a functional group at the tail end of a phospholipid molecule, and the polyethylene glycol is utilized to improve the blood circulation time of the polyamide liposome after intravenous injection administration and improve the accumulation of a medicament at a tumor part.
Drawings
FIG. 1 is a schematic diagram of the preparation process of pyrrole imidazole polyamide liposome.
FIG. 2 is a transmission electron micrograph of the pyrrole imidazole polyamide liposome of example 1.
FIG. 3 is a particle size diagram of the pyrrole imidazole polyamide liposome of example 3.
FIG. 4 shows that the expression of PlK1 protein is inhibited by DSPC pyrrole imidazole polyamide vector, DSPC-PEG-COOH pyrrole imidazole polyamide vector and free pyrrole imidazole polyamide.
FIG. 5 is a HRMS spectrum of compound of formula I.
Detailed Description
Example 1: preparation method of pyrrole imidazole polyamide liposome
(1) Dissolving 24 μ L of distearoyl phosphatidylcholine (DSPC) with a concentration of 0.1M and 15 μ L of cholesterol with a concentration of 50mM in a mixed solution of 2mL of chloroform and 1mL of methanol;
(2) under the condition of reduced pressure, removing the mixed solvent by rotary evaporation to obtain a layer of uniform film at the bottom of the bottle;
(3) dissolving 0.1 mu mol of pyrrole imidazole polyamide (compound shown in formula I) in 2mL of ultrapure water, adding into the glass bottle covered with the phospholipid membrane, and carrying out ultrasonic treatment for 10-20 min until the solution becomes clear from turbidity;
(4) passing the obtained solution through 0.22 μm water system filter membrane, and repeating for 3 times;
(5) the mixed aqueous solution of pyrrole imidazole and polyamide is transferred into an ultrafiltration centrifugal tube with the molecular cut-off of 50000, and the free medicine is removed by washing more than 3 times with ultrapure water.
Example 2: preparation method of pyrrole imidazole polyamide liposome
(1) Dissolving 240 μ L of distearoyl phosphatidylcholine (DSPC) with a concentration of 0.1M and 150 μ L of cholesterol with a concentration of 50mM in a mixed solution of 20mL of chloroform and 10mL of methanol;
(2) under the condition of reduced pressure, removing the mixed solvent by rotary evaporation to obtain a layer of uniform film at the bottom of the bottle;
(3) dissolving 1 mu mol of pyrrole imidazole polyamide (compound shown in formula I) in 20mL of ultrapure water, adding into the glass bottle covered with the phospholipid membrane, and carrying out ultrasonic treatment for 10-20 min until the solution becomes clear from turbidity;
(4) passing the obtained solution through 0.22 μm water system filter membrane, and repeating for 3 times;
(5) the mixed aqueous solution of pyrrole imidazole and polyamide is transferred into an ultrafiltration centrifugal tube with the molecular cut-off of 50000, and the free medicine is removed by washing more than 3 times with ultrapure water.
Example 3: preparation method of pyrrole imidazole polyamide liposome
(1) Dissolving 24 μ L of carboxylated polyethylene glycol-distearoyl phosphatidyl ethanolamine (DSPC-PEG-COOH) with a concentration of 0.1M and 15 μ L of cholesterol with a concentration of 50mM in a mixed solution of 2mL of chloroform and 1mL of methanol;
(2) under the condition of reduced pressure, removing the mixed solvent by rotary evaporation to obtain a layer of uniform film at the bottom of the bottle;
(3) dissolving 0.1 mu mol of pyrrole imidazole polyamide (compound shown in formula I) in 2mL of ultrapure water, adding into the glass bottle covered with the phospholipid membrane, and carrying out ultrasonic treatment for 10-20 min until the solution becomes clear from turbidity;
(4) passing the obtained solution through 0.22 μm water system filter membrane, and repeating for 3 times;
(5) the mixed aqueous solution of pyrrole imidazole and polyamide is transferred into an ultrafiltration centrifugal tube with the molecular cut-off of 50000, and the free medicine is removed by washing more than 3 times with ultrapure water.
Example 4: preparation method of pyrrole imidazole polyamide liposome
(1) Dissolving 240 μ L of carboxylated polyethylene glycol-distearoyl phosphatidyl ethanolamine (DSPC-PEG-COOH) with a concentration of 0.1M and 150 μ L of cholesterol with a concentration of 50mM in a mixed solution of 20mL of chloroform and 10mL of methanol;
(2) under the condition of reduced pressure, removing the mixed solvent by rotary evaporation to obtain a layer of uniform film at the bottom of the bottle;
(3) dissolving 1 mu mol of pyrrole imidazole polyamide (compound shown in formula I) in 20mL of ultrapure water, adding into the glass bottle covered with the phospholipid membrane, and carrying out ultrasonic treatment for 10-20 min until the solution becomes clear from turbidity;
(4) passing the obtained solution through 0.22 μm water system filter membrane, and repeating for 3 times;
(5) the mixed aqueous solution of pyrrole imidazole and polyamide is transferred into an ultrafiltration centrifugal tube with the molecular cut-off of 50000, and the free medicine is removed by washing more than 3 times with ultrapure water.
EXAMPLE 5 detection of Plk1 protein expression induced by liposomes of the invention by Western blotting
The test cells used human cervical cancer cell line Hela. Hela cells were cultured in complete medium (DMEM supplemented with 10% FBS and antibiotics 10% P/S). After the Hela cells are digested and reselected by pancreatin, the cells are evenly inoculated to a 60mm culture dish, and the test drugs are added after 24 hours. The test drug (examples 1 and 3 and free drug) was a test drug diluted to 1 μ M or 10 μ M with complete medium. After 72 hours of drug action time, the cells were digested with 0.25% pancreatin. The collected cells were transferred to a 1.5ml centrifuge tube, washed once with PBS, added with RIPA lysate, and lysed on ice for 1 hour. Centrifuge at 14000rpm for 15min at 4 degrees, and collect supernatant liquid. After the protein concentration is measured by a BCA protein quantitative method, adding a protein denaturation solution, and diluting to the same protein concentration; incubate with boiling water for 5min to denature the protein. Then separating and purifying the target protein by an SDS polyacrylamide gel electrophoresis method. After completion of the electrophoresis, the proteins on the gel were electrophoretically transferred to a PVDF membrane. Followed by incubation with 5% nonfat dry milk/PBS blocking solution for 1 hour at room temperature. The Plk1 antibody was then added and incubated overnight at 4 degrees. After washing with PBS-T, secondary antibody diluted with blocking solution was added and incubated at room temperature for 1 hour. After PBS cleaning, filter paper is used for sucking out more water on the PVDF membrane, a chemiluminescence substrate is added, and color development and exposure are carried out.
As can be seen from fig. 4, the compound of formula I had inhibitory effect on Plk1, with a strong effect shown in the 10 μ M group, but little inhibitory effect in the 1 μ M group. Whereas, the liposomes of examples 1 and 3 showed strong inhibitory effects in both the 1. mu.M group and the 10. mu.M group, and there was little difference between the 1. mu.M group and the 10. mu.M group. The experiment proves that the liposome preparation effectively improves the bioavailability, and the bioavailability of the compound shown in the formula I can be improved by 10 times, thereby obtaining unexpected effects.
EXAMPLE 6 preparation of Compounds of formula I
1. Synthesis of precursor 1
A50 mL solid phase reactor was charged with hydrazine resin (0.61mmol/g, 400mg, 0.244mmol) and CH2Cl2(3mL) to swell the resin for 20 min. The CH2Cl2 was aspirated, 20% piperidine/DMF solution (3mL) was added to the resin, the resin was bubbled for 5min, the solvent was aspirated, 20% piperidine/DMF solution (3mL) was added, the solvent was aspirated for 5min, the resin was washed with DMF (4X3mL), then NMP (2X3mL) was used to wash the resin for use. While Boc-Py-OH ((176mg,0.732mmol,3eq.) and BOP-Cl (186mg,0.732mmol, 3eq.) were dissolved in NMP (2ml), DIEA (400. mu.L, 2.2mmol, 9eq.) was added to the solution, stirring was continued until the solution was clear, the reaction was transferred to Fmoc-removed hydrazine resin and the resin was transferred to a 50ml EP tube, heating the mixture in a microwave reactor with the heating power of 200w and the heating power of 75 ℃ for 15min (ninhydrin reagent detects the reaction is complete), pumping out the reaction liquid, washing the resin with DMF (4x3mL), then washing the resin with anhydrous DMF (3mL), adding a solution of pivalic anhydride (186. mu.L, 0.96mmol,4eq.) and DIEA (500. mu.L, 2.88mmol, 12eq.) in DMF (2mL) to the resin, reacting for 15min, transferring the resin to a solid phase reactor, pumping out the reaction liquid, washing the resin with DMF (4x3mL), and finally washing the resin with DCM (2x3 mL).
The synthesis of precursor 1 was then completed according to the following procedure
The method A comprises the following steps: deprotection of Boc
A TFA/TIS/H2O solution (95:2.5:2.5,3ml) was added to the resin, the resin was bubbled for 2min, the solvent was purged, a FA/TIS/H2O solution (95:2.5:2.5,3ml) was added, the solvent was bubbled for 20min, the resin was purged, washed first with DCM (2X3mL), then with DMF (4X3mL), and finally with NMP (2X3 mL).
The method B comprises the following steps: coupling of Boc-Im-OH
Boc-Im-OH (176mg,0.732mmol,3eq.) (or Fmoc-D-dab (Boc) -OH (322mg, 0.732mmol, 3eq.)), BOP-Cl (186mg,0.732mmol, 3eq.), HOAT (100mg, 0.732mmol, 3eq.)) were dissolved in NMP (2ml), DIEA (400. mu.L, 2.2mmol, 9eq.) was added to the solution, and the mixture was stirred until the solid was completely dissolved. The reaction solution was transferred to hydrazine resin which had been stripped of Boc and the resin was transferred to a 50ml EP tube and reacted for 15min in a microwave reactor with a heating power of 200w at 75 deg.C (ninhydrin reagent detected to completion). The resin was centrifuged at low speed and the supernatant removed. Then methanol is added into the resin for washing, the resin is centrifuged at low speed, the supernatant is discarded, and the resin is washed for three times by methanol. The methanol washed resin was transferred to a solid phase reactor, which was washed with methanol (2x3mL), then with DMF (4x3mL), and finally with DCM (2x3 mL).
The method C comprises the following steps: coupling of Boc-Py-OH
Boc-Py-OH (176mg,0.732mmol,3eq.) and BOP-Cl (186mg,0.732mmol, 3eq.) were dissolved in NMP (2ml), DIEA (400. mu.L, 2.2mmol, 9eq.) was added to the solution and stirred until the solution was clear. The reaction solution was transferred to hydrazine resin which had been stripped of Boc and the resin was transferred to a 50ml EP tube and reacted for 15min in a microwave reactor with a heating power of 200w at 75 deg.C (ninhydrin reagent detected to completion). The reaction was pumped off, the resin was washed with DMF (4X3mL) and DCM (2X3 mL).
The method D comprises the following steps: coupling of Im-OH
Im-OH (90mg,0.732mmol, 3eq.) and PyBOP (380mg,0.732mmol, 3eq.) were dissolved in anhydrous DMF (2ml), DIEA (400. mu.L, 2.2mmol, 9eq.) was added to the solution, and the solution was stirred until clear. The reaction solution was transferred to hydrazine resin which had been stripped of Boc and the resin was transferred to a 50ml EP tube and reacted for 15min in a microwave reactor with a heating power of 200w at 75 deg.C (ninhydrin reagent detected to completion). The reaction was aspirated and the resin was washed with DMF (4X3 mL).
The method E comprises the following steps: ninhydrin detection
During the coupling reaction, a small amount of resin is taken out, washed twice with DMF, added with two drops of ninhydrin detection solution (15 g of ninhydrin, 3ml of acetic acid and 100ml of n-butanol), heated at 90 ℃ for 3min, and the resin does not generate color change, indicating that the reaction is complete. The resin turned from red to blue indicating the presence of primary ammonia.
2. Preparation of precursor 2
3. The final product Py-Im-Ht
Adding a small amount of DMF, copper acetate and 3-dimethylaminopropylamine into the resin, shaking overnight, purifying by using semi-preparative HPLC, collecting the product, removing acetonitrile by rotary evaporation, and freeze-drying the obtained solution to finally obtain Py-Im-Ht. HRMS (ESI) M/z calcd for C84H95N27O11[ M + H ] +1258.7783, found 1658.7819; [ M +2H ]2+829.8930, found 829.8856; [ M +3H ]3+553.5979, found 553.6090, HRMS spectrum is shown in FIG. 5. The preparation of the compounds of formula I is shown below.
Claims (10)
1. The pyrrole imidazole polyamide liposome is composed of a lipid bilayer and pyrrole imidazole polyamide entrapped in the lipid bilayer, wherein the lipid bilayer comprises phospholipid molecules and cholesterol, the molar ratio of the phospholipid molecules to the cholesterol is (1-10): 1, and the molar ratio of the phospholipid molecules to the pyrrole imidazole polyamide is (20-30): 1;
the pyrrole imidazole polyamide is selected from a compound with a structure shown in formula I
2. The pyrrole imidazole polyamide liposome of claim 1, wherein the molar ratio of phospholipid molecules to cholesterol is (3-4): 1.
3. A pyrrole imidazole polyamide liposome according to claim 1, the phospholipid molecule being selected from the group consisting of a mixture of one or more of phospholipids, carboxylated polyethylene glycol-phospholipids, distearoylphosphatidylcholine, 1, 2-dioleoyl-3-trimethylammonium propane, and carboxylated polyethylene glycol-distearoylphosphatidylethanolamine.
4. The pyrrole imidazole polyamide liposome of claim 3, wherein the polyethylene glycol molecule in the phospholipid molecule is selected from 2000-5000-.
5. A pyrrole imidazole polyamide liposome according to any one of claims 1 to 4, which has a particle size of 50 to 300 nm.
6. A pyrrole imidazole polyamide liposome according to any one of claims 1 to 4, the concentration of pyrrole imidazole polyamide being from 1 μ M to 50 μ M.
7. A method for preparing pyrrole imidazole polyamide liposomes according to any one of claims 1 to 6 selected from the group consisting of thin film dispersion methods and reverse evaporation methods.
8. The method of manufacturing according to claim 7, comprising the steps of:
1) dissolving phospholipid molecules and cholesterol in an organic solvent to form a membrane material dispersion liquid;
2) under the condition of reduced pressure, removing the organic solvent in the membrane material dispersion liquid, and enabling phospholipid molecules and cholesterol to form a uniform dispersion film on the wall of the container;
3) dissolving pyrrole imidazole polyamide in ultrapure water, dispersing the solution in the container obtained in the step 2), and mechanically dispersing the solution to enable phospholipid molecules and cholesterol to be self-assembled into a liposome encapsulating the pyrrole imidazole polyamide;
optionally, steps 4) and 5) are also included,
4) passing the liposome obtained in step 3) through a 0.22 μm aqueous filter membrane;
5) and (3) treating the pyrrole imidazole polyamide liposome obtained in the step 4) by using an ultrafiltration centrifugal tube to remove free pyrrole imidazole polyamide.
9. Use of a pyrrole imidazole polyamide liposome according to any one of claims 1 to 6 for the preparation of a medicament for the treatment or prevention of a disease associated with a Plk1 inhibitor selected from lung cancer, liver cancer, ovarian cancer, esophageal cancer, cervical cancer, skin cancer, leukemia, hodgkin's lymphoma.
10. Use of a pyrrole imidazole polyamide liposome according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment or prevention of a disease associated with a Plk1 inhibitor, the disease associated with a Plk1 inhibitor being non-small cell lung cancer.
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