CN110101665B

CN110101665B - Liposome material and preparation method and application thereof

Info

Publication number: CN110101665B
Application number: CN201910390730.XA
Authority: CN
Inventors: 汪铭; 梁春菁
Original assignee: Beijing Yunxi Zhixiang Biotechnology Co ltd
Current assignee: Beijing Yunxi Zhixiang Biotechnology Co.,Ltd.
Priority date: 2019-05-10
Filing date: 2019-05-10
Publication date: 2021-11-02
Anticipated expiration: 2039-05-10
Also published as: CN110101665A

Abstract

The invention relates to a liposome material and a preparation method and application thereof, wherein the liposome material is a cationic liposome material based on oxidative stress (ROS) response and is prepared by an addition reaction of an organic amine compound and a compound shown in a formula I; the organic amine is aliphatic amine or aromatic amine; the compound of the formula I is shown as follows, wherein m is an integer of 7-20; r₁is-O-, -S-or-NH-; r₂is-S-or-O-; the liposome has cell selectivity in the drug delivery process, not only improves the release efficiency of drugs in disease cells, but also reduces the drug toxicity to normal cells.

Description

Liposome material and preparation method and application thereof

Technical Field

The invention relates to the technical field of biochemistry, and particularly relates to a liposome material as well as a preparation method and application thereof.

Background

Compared with the traditional chemical synthesis medicine, the gene medicine has the advantages of high curative effect, less side effect, strong specificity and the like, and can also obtain better curative effect on some diseases with poor traditional medicine effect, such as various tumors, hereditary diseases and immune-related epidemic diseases. At present, hundreds of gene drugs enter clinical trials, and the search for a carrier which can efficiently transfect the gene drugs into diseased tissues and is nontoxic is one of the main tasks for developing the gene drugs.

In order to transfect gene drugs into cells more efficiently, various gene drug carriers, such as inorganic materials, polymer materials, liposomes, viruses, etc., have been developed and applied. Although viruses can efficiently transfect gene drugs into host cells, viruses are potentially dangerous as vectors, and therefore non-viral vector materials are safer. Inorganic materials are difficult to metabolize in the living body, poor in biocompatibility, and unable to effectively circulate in the living body for a long time, as compared with organic materials. The organic polymer material and the liposome material not only have high transfection efficiency, but also have low toxicity and are easy to be metabolized by organisms. The organic polymer material can significantly affect the transfection efficiency due to the polymerization degree, and the reproducibility is poor compared with that of liposome. The main problems existing in the current liposome delivery gene drugs are that the cell selectivity is poor, and the drug release function in target cells is difficult to realize efficiently and specifically.

Disclosure of Invention

The invention aims to provide a nano-drug carrier, a preparation method and an application thereof, wherein the nano-drug carrier is a cationic liposome based on oxidative stress (ROS) response, has cell selectivity in a drug delivery process, not only improves the release efficiency of drugs in disease cells, but also reduces the drug toxicity to normal cells.

To this end, in a first aspect, the present invention provides a liposome prepared by an addition reaction of an organic amine compound with a compound of formula I,

the organic amine compound is aliphatic amine or aromatic amine;

the structural formula of the compound of the formula I is shown as follows

Wherein m is an integer of 7 to 20;

R₁is-O-, -S-or-NH-;

R₂is-S-or-O-.

Further, in the compounds of formula I, R₁is-O-.

Further, in the compounds of formula I, R₂is-S-.

Further, in the compounds of formula I, m is an integer from 8 to 15, and in one embodiment, m is 12.

Further, the addition reaction is a Michael addition reaction (Michael addition reaction).

Further, the organic amine compound has at least one N-containing group, and the N-containing group is a primary amine or an imino group.

In a specific embodiment, the organic amine compound is one of the following compounds,

in a second aspect, the invention provides intermediates for the preparation of said liposomes, which are compounds of formula I,

wherein m is an integer of 7 to 20;

R₁is-O-, -S-or-NH-;

R₂is-S-or-O-.

Further, in the compounds of formula I, R₁is-O-.

Further, in the compounds of formula I, R₂is-S-.

In a third aspect, the invention provides the use of said liposomes for the preparation of a pharmaceutical carrier.

In a fourth aspect, the present invention provides a nano-drug comprising a) a pharmaceutically active ingredient; b) the liposome of the invention.

Further, the active ingredients of the medicine are one or more of polypeptide, protein and nucleic acid.

In a fifth aspect, the present invention provides a method for preparing the nano-drug, comprising,

s1: mixing the liposome, cholesterol and a chloroform solution of dioleoyl phosphatidylethanolamine, and volatilizing to form a film;

s2: dissolving the membrane obtained in the step S1 in a mixed solution of ethanol and a sodium acetate buffer solution to obtain a liposome solution;

s3: and (3) adding the liposome solution obtained in the step (S2) into a sodium acetate buffer solution containing a medicinal active ingredient and polyethylene glycol, uniformly mixing, and dialyzing by using a PBS buffer solution to obtain the nano-medicament.

Further, in step S3, the polyethylene glycol is PEG-2000, and further, the amount of the polyethylene glycol is 20% to 30%, preferably 25% of the liposome.

Further, in step S3, the concentration of the liposome in the sodium acetate buffer is 0.5 to 5mg/ml, preferably 0.5 to 3mg/ml, more preferably 0.5 to 1mg/ml, and still more preferably 1 mg/ml.

Reactive Oxygen Species (ROS) mainly refers to a kind of free radicals in cells, and they are composed of mitochondria and tend to have high oxidation activity. ROS in cells participate in various biochemical processes, the cells are affected by high or low ROS levels, excessive generated ROS can damage nucleic acid, protein and lipid to cause diseases such as nervous system damage, aging and the like, and the increase of ROS can also stimulate the generation of Hypoxia Inducible Factor (HIF) to promote the generation and development of tumors. Therefore, the ROS content in cells of partial diseases such as tumors, cardiovascular diseases, neurodegenerative diseases, corneal alkali burn and the like is far higher than that of normal cells, and target cells can be subjected to targeted drug delivery by using ROS-responsive drug carriers, so that the drug delivery efficiency and safety are improved.

In the prior art, the hydrophobic end of liposomal drug carriers generally do not contain a responsive group, nor do liposomes containing ROS-responsive groups be disclosed. The cationic liposome provided by the invention is composed of a hydrophobic end, a connecting group and a hydrophilic end, the invention creatively introduces thioketal or ketal groups into the hydrophobic end of the liposome, the groups can be degraded by ROS, and the hydrophobic end is degraded so as to break nanoparticles formed by the liposome, thereby releasing gene drugs. Therefore, the nano-drug carrier provided by the invention has cell selectivity in drug delivery, not only remarkably improves the release efficiency of drugs in disease cells, but also reduces the drug toxicity to normal cells.

The nano-carrier provided by the invention firstly contains the drug to form nano-particles when delivering the drug, then the nano-particles enter a cell lysosome through the endocytosis process of the cell, after the nano-particles escape from the lysosome, as the hydrophobic end of the liposome has a thioketal group, the group can be degraded by ROS to generate sulfhydryl substances and acetone, and the hydrophobic end is degraded to break the nano-particles formed by the liposome, thereby releasing the gene drug.

Compared with the prior art, the invention has the following advantages:

(1) the drug carrier provided by the invention is a liposome and consists of a hydrophobic end, a connecting group and a hydrophilic end, the invention creatively introduces thioketal or ketal groups at the hydrophobic end of the liposome, and the groups can be broken for sensitive response ROS, so that nanoparticles are broken and the entrapped drug is released. Can effectively and specifically realize the drug release function in target cells.

(2) The liposome provided by the invention has extremely high drug delivery efficiency, which is significantly superior to similar drug carriers in the prior art, for example, when the delivered drug is siRNA, the delivery efficiency of the liposome in the preferred embodiment of the invention is the highest known at present; the liposomes provided by the present invention also have extremely high delivery efficiency when delivering other nucleic acid drugs (e.g., mRNA) or protein drugs (e.g., RNase a protein).

(3) The liposome is used as a drug delivery carrier, has the advantages of high safety, high transfection efficiency, easy metabolism by organisms and good reproducibility, further has the advantages of good cell selectivity, specific drug release and high delivery efficiency, and has wide clinical application prospect.

(4) The invention provides a preparation method of liposome, which is characterized in that organic amine compound and acrylate are synthesized into the liposome by a combined chemical method and Michael addition reaction, and the liposome has simple experimental steps and good product stability.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 is a schematic illustration of liposome delivery of a drug;

FIG. 2 is a nuclear magnetic map of ROS-TK-1 liposome;

FIG. 3 is a nuclear magnetic map of ROS-TK-6 liposome;

FIG. 4 is a nuclear magnetic map of ROS-TK-9 liposome;

FIG. 5 is a nuclear magnetic map of ROS-TK-10 liposome;

FIG. 6 is a nuclear magnetic map of ROS-TK-12 liposome;

FIG. 7 is a graph of the efficiency of liposome delivery of siGFP, where the numbers on the horizontal axis represent the n value of ROS-TK-n, control represents control 1, siRNA represents control 2, LPF2K represents control 3;

FIG. 8 is a graph of the efficiency of liposome delivery of siGFP at a gradient concentration of siGFP, where the numbers on the horizontal axis represent the n value of ROS-TK-n;

FIG. 9 is the efficiency of liposome delivery of RFP mRNA, where the numbers on the horizontal axis represent the n-value of ROS-TK-n, and the control represents the control group;

FIG. 10 is the efficiency of liposome delivery of RNase A protein, wherein the number on the horizontal axis represents the n value of ROS-TK-n.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The invention designs and synthesizes a cationic liposome based on oxidative stress (ROS) response to deliver drugs, wherein the liposome consists of hydrophilic end aliphatic amine or aromatic amine, a connecting group ester group and a fatty chain hydrophobic end containing thioketal or ketal group. In a specific embodiment, as shown in fig. 1, the drug is a gene drug, the liposome firstly encapsulates the drug to form nanoparticles when delivering the gene drug, then the nanoparticles enter lysosomes of cells through the endocytosis process of the cells, after the nanoparticles escape from the lysosomes, because the hydrophobic end of the liposome introduces thioketal groups, the groups can be degraded by ROS to generate sulfhydryl substances and acetone, and the hydrophobic end is degraded to break the formed nanoparticles of the liposome, so that the gene drug is released.

In the following examples, liposomes were designated ROS-TK-n (the corresponding structural formula is designated formula II-n), where n represents different organic amines, such as ROS-TK-10, and represents liposomes obtained by addition reaction of organic amine represented by formula 10 with the compound of formula I, and the structural formula is designated formula II-10.

EXAMPLE 1 preparation of Compound b

1mM of Compound a was dissolved in 3ml of 1, 4-dioxane solvent, 1mM of potassium hydroxide powder was added, 1mM of n-dodecylbromide was then added, and the reaction was allowed to proceed overnight at 105 ℃. TLC, extracted with dichloromethane and water after completion of the reaction, dried, rotary evaporated, purified with petroleum ether and ethyl acetate 5: 1, passing through the column to obtain the product, namely the compound b.

EXAMPLE 2 preparation of Compounds of formula I-1

Dissolving the compound b in anhydrous dichloromethane, adding 1.2 equivalents of triethylamine, dropwise adding acryloyl chloride under an ice bath condition, removing the ice bath, reacting at room temperature for 5 hours, adding water after the reaction is finished, quenching, extracting, drying, performing rotary evaporation, and performing reaction on the product by using petroleum ether and ethyl acetate 10: 1 passing through the column. A pale yellow liquid, i.e. the compound of formula I-1, is obtained.

Example 3 liposomes of formula II-1 are prepared: ROS-TK-1

Mixing the compound shown in the formula I-1 and the fatty amine shown in the compound 1 according to a molar ratio of 2.4: 1, mixing, heating to 80 ℃, reacting for 36h, reacting with dichloromethane and methanol 20: 1, passing through a column to prepare the liposome of the formula II-1: the nuclear magnetic spectrum of ROS-TK-1 is shown in figure 2.

Example 4 liposomes of formula II-6 were prepared: ROS-TK-6

Mixing the compound shown as the formula I-1 and the fatty amine shown as the compound 6 according to a molar ratio of 2.4: 1, mixing, heating to 80 ℃, reacting for 36h, reacting with dichloromethane and methanol 20: 1, passing through a column to prepare the liposome of the formula II-6: and the nuclear magnetic spectrum of the ROS-TK-6 is shown in figure 3.

Example 5 preparation of liposomes of formula II-9: ROS-TK-9

Mixing the compound shown as the formula I-1 and the fatty amine shown as the compound 9 according to a molar ratio of 2.4: 1, mixing, heating to 80 ℃, reacting for 36h, reacting with dichloromethane and methanol 20: 1, passing through a column to prepare the liposome of the formula II-9: and the nuclear magnetic spectrum of the ROS-TK-9 is shown in figure 4.

Example 6 liposomes of formula II-10 were prepared: ROS-TK-10

Mixing the compound shown as the formula I-1 and the fatty amine shown as the compound 10 according to a molar ratio of 2.4: 1, mixing, heating to 80 ℃, reacting for 36h, reacting with dichloromethane and methanol 20: 1, passing through a column to prepare the liposome of the formula II-10: the nuclear magnetic spectrum of ROS-TK-10 is shown in figure 5.

Example 7 liposomes of formula II-12 were prepared: ROS-TK-12

Mixing the compound shown as the formula I-1 and the fatty amine shown as the compound 12 according to a molar ratio of 2.4: 1, mixing, heating to 80 ℃, reacting for 36h, reacting with dichloromethane and methanol 20: 1, passing through a column to prepare the liposome of the formula II-12: the nuclear magnetic spectrum of ROS-TK-12 is shown in figure 6.

Example 8

Transfecting small interfering nucleic acid (siGFP) of green fluorescent protein into a human cervical squamous carcinoma cell line (SiHa-GFP) capable of stably expressing the green fluorescent protein by using liposome ROS-TK-2, 3, 5, 7, 8, 9, 10, 11 and 12 as drug carriers respectively, and specifically comprises the following steps:

experimental groups: nucleic acid drug siGFP was added to 25mM sodium acetate buffer solution (pH 7.2) at a final concentration of 5nM, and the liposome molecules were added at a final concentration of 3.33. mu.g/mL, and after mixing, the mixture was allowed to stand for 15 min. And then adding the assembled nano-drugs into SiHa-GFP cells respectively for drug transfection for 8 h.

Three control groups are simultaneously arranged, and each control group is different from the experimental group in that: control group 1, SiHa-GFP cells only, without transfection(ii) a Control 2, only siGFP, no liposomes; control group 3, using Lipofectamine ^@2000 transfection reagents were used to transfect siGFP.

After further incubation for 24h, the change in fluorescence intensity in the cells was detected by flow cytometry. As shown in FIG. 7, compared with the commercial transfection reagent, the liposome provided by the invention can deliver the siGFP with high efficiency, and the efficiency of delivering the siGFP by 5 liposome molecules is 100%, and the three liposome molecules are ROS-TK-3, ROS-TK-5, ROS-TK-9, ROS-TK-11 and ROS-TK-12 respectively.

On this basis, by reducing the concentration of siRNA, changes in liposome delivery efficiency were further examined. Nucleic acid drugs siGFP and liposome molecules were added to 25mM sodium acetate buffer solution (pH 7.2) at different concentrations (0.15nM, 0.3nM, 0.6nM, 1.2nM, 2.5nM, 5nM) to give a final liposome molecule concentration of 3.33ug/mL, mixed well, and then allowed to stand for 15 min. And then adding the assembled nano-drugs into SiHa-GFP cells respectively for drug transfection for 8 h. After further incubation for 24h, the change in fluorescence intensity in the cells was detected by flow cytometry. As shown in fig. 8, the liposome provided by the present invention still has excellent delivery efficiency even when the concentration of siRNA is reduced to nanomolar (nM); when the concentration of siRNA is 5nM, the delivery efficiency of liposome is still above 90%, which is the highest efficiency liposome for delivering siRNA at present.

Example 9

Messenger ribonucleic acid (mRNA) was delivered using liposomal ROS-TK-2, 5, 7, 8, 9, 10, 11, 12, respectively. mRNA is selected from mRNA encoding RFP, which is a red fluorescent protein translated into the red fluorescent protein when entering the cell. The delivery efficiency was evaluated by transfection of HeLa cells with the above liposomes delivering RFP mRNA, the experimental procedure was as follows:

adding RFP mRNA as nucleic acid drug into 25mM sodium acetate buffer solution with pH 7.2, respectively, adding the above liposome molecules, wherein the final concentration of RFP mRNA is 1.7 ng/. mu.L, and the final concentration of liposome is 25 ng/. mu.L, mixing well, standing and assembling for 15 min. And then adding the assembled nano-drugs into HeLa cells respectively for drug transfection for 8 h. Meanwhile, HeLa cells without drug transfection were set as a control group.

After the cells are continuously cultured for 24h, the generation of red fluorescent protein in the cells is detected by a flow cytometer, the detection result is shown in figure 9, and as can be seen from figure 9, the liposome provided by the invention can efficiently deliver RFP mRNA, wherein the delivery efficiency of the liposomes ROS-TK-2, ROS-TK-11 and ROS-TK-12 is more than 70%.

Example 10

Liposomal ROS-TK-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, respectively, are used to deliver protein drugs. The protein drug is RNase A protein. After entering cells, the protein can hydrolyze RNA in the cells to cause apoptosis. The transfection efficiency of liposome delivered RNase A protein was evaluated by the magnitude of cytotoxicity using MTT colorimetry. The experimental procedure was as follows:

experimental groups: adding protein drug RNase A into 25mM sodium acetate buffer solution with pH of 7.2, and adding above liposome molecules, wherein the final concentration of RNase A is 0.5mg/mL and the final concentration of liposome is 0.6mg/mL, mixing well, standing and assembling for 15 min. And then adding the assembled nano-drugs into target cells respectively for drug transfection for 8 h. Meanwhile, cells not subjected to drug transfection were set as a control group.

After the cells are continuously cultured for 48 hours, the OD value of the cells is detected by an MTT method, the detection result is shown in figure 10, and as can be seen from figure 10, the liposomes provided by the invention can efficiently deliver the protein drug RNase A, wherein the delivery efficiency of the liposome ROS-TK-12 to the protein is optimal, and 80% of apoptosis can be caused.

Example 11

The embodiment provides a preparation method of a nano-drug, which comprises the following steps:

s1: mixing liposome ROS-TK-12, cholesterol and a chloroform solution of dioleoyl phosphatidylethanolamine, and volatilizing to form a membrane;

s3: and (3) adding the liposome solution obtained in the step (S2) into a sodium acetate buffer solution containing a medicinal active ingredient and polyethylene glycol (the dosage of the polyethylene glycol is 25 percent of the liposome), so that the final concentration of the liposome is 1mg/ml, uniformly mixing, and dialyzing by using a PBS buffer solution to obtain the nano-drug, wherein the nano-drug can be used for animal experiments.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A cationic liposome material, wherein the liposome material is:

2. the liposomal material of claim 1 wherein the liposomal material is prepared using a compound of formula I as an intermediate,

wherein m is 12;

R₁is-O-.

3. Use of the liposomal material of claim 1 for the preparation of liposomes.