CN114632062A - Neutral liposome for delivering nucleic acid medicament and preparation method and application thereof - Google Patents
Neutral liposome for delivering nucleic acid medicament and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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Abstract
The invention discloses a neutral liposome for delivering nucleic acid drugs and a preparation method and application thereof. A neutral liposome for delivering a nucleic acid drug, said neutral liposome consisting essentially of lecithin, cholesterol, a nucleic acid drug, and a cationic peptide; wherein the negatively charged nucleic acid drug is mixed with the cationic peptide to be electrically neutral. The neutral liposome formed by the components is found to be capable of specifically targeting the mononuclear/macrophage, and an in-vivo and in-vitro experiment is utilized to confirm the characteristic, so that the transfection efficiency of the neutral liposome on the mononuclear/macrophage is fully improved, and the mononuclear/macrophage is modified in vivo and related functions are influenced.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to a neutral liposome for delivering nucleic acid drugs, and a preparation method and application thereof.
Background
Since 2013, tumor immunotherapy is listed as the first ten scientific breakthroughs in the year by the Science journal for three consecutive years, wherein CART is greatly successful in treating blood-type tumors, so that the successful conversion of cell therapy from basic immunology mechanism research to clinical application is realized, and the CART becomes an important milestone in the clinical medical development history of tumors. However, the technology still has significant limitations, such as inability to effectively infiltrate solid tumors, unstable response rate, and cytokine storm, which need to be solved urgently.
The current research shows that a large amount of macrophages infiltrate into the microenvironment of the solid tumor and are closely related to the occurrence, development and development of the tumor. Macrophages are the main effector and regulator of the innate immune system and have the ability to phagocytize, kill cells, secrete proinflammatory factors and present antigens. In addition, macrophages are key effectors of targeted antibody therapy, and as specialized antigen presenting cells, activated macrophages can play an important role in promoting an adaptive anti-tumor immune response. These properties of macrophages have prompted clinical reinfusion of large numbers of autologous macrophages into solid tumor patients, and although these clinical trials have not found significant anti-tumor effects, the feasibility and safety of infusing large numbers of autologous monocyte-derived macrophages has been demonstrated. Based on macrophage biological properties: the tumor has strong infiltration, phagocytic capacity and immunoregulation function, so that the tumor has unique advantages as a new carrier for cellular immunotherapy. However, macrophages do not have the ability to expand in large amounts in vitro, which makes cell preparation difficult. Compared with adoptive cell therapy, namely in vitro gene therapy, in vivo gene therapy can omit tedious operations such as cell collection, gene modification, culture amplification and the like, and has universality.
Liposomes are vesicles of double-layered closed structure formed autonomously by suspending natural lipid compounds in water, and phospholipid compounds mainly synthesized by man-made are currently prepared. Because the liposome can be used as a delivery carrier, the liposome is mainly applied to the fields of medicines, cosmetics, genetic engineering and the like, and especially plays an important role in the delivery of tumor immunotherapy drugs. Compared with viral vectors, the liposome serving as a gene vector has the advantages of high safety, small immunogenicity, small toxicity, easiness in preparation and the like, most of liposomes applied to the market at present are positively charged, mainly combined with negative charge nucleic acid drugs and the like, and are introduced into cells by utilizing the negativity of cell membranes, but the defects of low transfection efficiency, low targeting property and the like limit the clinical application of the liposome.
Disclosure of Invention
The object of the present invention is to address the above-mentioned deficiencies of the prior art by providing a neutral liposome for delivery of nucleic acid drugs.
The invention also aims to provide a preparation method of the neutral liposome.
Still another object of the present invention is to provide the use of the neutral liposome.
The purpose of the invention can be realized by the following technical scheme:
a neutral liposome for delivering a nucleic acid drug, said neutral liposome consisting essentially of lecithin, cholesterol, a nucleic acid drug, and a cationic peptide; wherein the negatively charged nucleic acid drug is mixed with the cationic peptide to be electrically neutral.
As a preferred aspect of the present invention, the ratio of lecithin: the mass ratio of the cholesterol is 5:1-20: 1.
In a preferred embodiment of the present invention, the cationic peptide is selected from antibacterial peptides, and the cationic peptide can be any positively charged cationic peptide, usually antibacterial peptide, or other positively charged safe polypeptide. The cationic peptide is used in an amount to neutralize the negatively charged nucleic acid drug to neutral or near the center of the dot.
The preparation method of the neutral liposome comprises the following steps:
(1) adding lecithin and cholesterol into chloroform, mixing, removing chloroform by a reduced pressure rotary evaporator, and forming a milky-white phospholipid film at the bottom of a bottle;
(2) mixing the nucleic acid drug and the cationic peptide, and detecting the potential to be neutral, wherein the adding amount of the cationic peptide is based on neutralizing the negative electricity of the nucleic acid drug to be neutral;
(3) dispersing the phospholipid film prepared in the step (1) in a water phase gently at the temperature of 25-30 ℃, and vortexing for 10-20min until the phospholipid film is completely resuspended to obtain a white suspension; wherein, the water phase is formed by mixing a liquid A and a liquid B, and the liquid A: PBS, B liquid is the plasmid antibacterial peptide compound prepared in the step (2), and the volume ratio of the A liquid to the B liquid is 1.5-3: 1;
(4) placing the milky white suspension at 25-30 ℃ in N2Neutralizing for 1.5-2 h;
(5) gently shaking the suspension, and then placing on an ultrasonic instrument for constant-temperature ultrasonic treatment for 2-5min at 55 ℃);
(6) the suspension is placed in N at normal temperature2Centrifugation is carried out for 10,000g for 15min after 1.5-2h, and then washing is carried out for 2-3 times by using sterile PBS, and centrifugation is carried out for 30min at 25,000g each time.
Preferably, the mass ratio of the nucleic acid drug to the cationic peptide is 10:1-1: 30.
In a preferred embodiment of the present invention, the mass-to-volume ratio of the phospholipid membrane to the aqueous phase is 1:1 to 1: 10.
The neutral liposome of the invention is applied to the preparation of in vivo or in vitro medicinal preparations for transfecting monocytes/macrophages.
Has the advantages that:
the invention successfully and fully combines the nucleic acid medicament and the neutral liposome to form the neutral liposome medicament preparation for encapsulating the nucleic acid medicament, and the nucleic acid medicament is encapsulated by a bilayer membrane structure formed by the liposome, so that the nucleic acid medicament can be fully protected from being degraded by related nuclease and can be delivered to mononuclear/macrophage. Has sufficient protection effect on nucleic acid medicaments. Meanwhile, the neutral liposome bilayer can also avoid being fused with a charged negative normal cell membrane structure, so that the neutral liposome bilayer has a good protective effect on an organism.
The neutral liposome formed by the components is found to be capable of specifically targeting the mononuclear/macrophage, and an in-vivo and in-vitro experiment is utilized to confirm the characteristic, so that the transfection efficiency of the neutral liposome on the mononuclear/macrophage is fully improved, and the mononuclear/macrophage is modified in vivo and related functions are influenced.
Drawings
FIG. 1 Synthesis and purification of antimicrobial peptide protein
FIG. 2 Synthesis of nucleic acid liposomes and particle size detection
FIG. 3 in vitro cell function analysis of nucleic acid liposomes
FIG. 4 evaluation of peripheral blood cell transfection specificity of nucleic acid liposomes
Detailed Description
The following examples illustrate the technical solution and effects of the present invention by using the antibacterial peptide DEFA3 and GFP plasmid as examples, but should not limit the scope of the present invention. The nucleic acid drug of the present invention may be selected from any electronegative plasmid, and the cationic peptide may be any positively charged cationic peptide, and may be an antibacterial peptide in general, or other positively charged polypeptide having safety.
EXAMPLE 1 protein Synthesis of antimicrobial peptides
The nucleotide sequence of the antibacterial peptide Defa3 obtained from the National Center for Biotechnology Information (NCBI) database is shown in SEQ ID NO. 1:
ATGAAGACACTAGTCCTCCTCTCTGCCCTCGTCCTGCTGGCCTTCCAGGTCCAGGCTGATCCTATCCAAAACACAGATGAAGAGACTAAAACTGAGGAGCAGCCAGGGGAAGACGACCAGGCTGTGTCTGTCTCTTTTGGAGACCCAGAAGGCTCTTCTCTTCAAGAGGAATCGTTGAGAGATCTGGTATGCTATTGTAGAAAAAGAGGCTGCAAAAGAAGAGAACGCATGAATGGGACCTGCAGAAAGGGTCATTTAATGTACACACTCTGCTGTCGCCATCATCACCATCACCACTAA
the sequence is synthesized by a chemical synthesis method, directly recombined and constructed in pIRES2-EGFP plasmid and transfected into competent DH5a escherichia coli.
Plasmids were amplified using LB medium and QIAGEN plasmid extraction kit or plasmid of interest was used.
The plasmid is transferred to 293T cells for eukaryotic expression, and can be processed and released out of cells because the plasmid contains a signal peptide sequence. After 48 hours of transfection, after observing high expression of signal gene EGFP in the plasmid, the released culture solution was collected, lyophilized, then resuspended in PBS containing 8M urea and 20mM imidazole, centrifuged at 10000g for 30min, and the supernatant was collected.
And (3) taking the supernatant, purifying the target protein by adopting a Ni column affinity chromatography, carrying out protein quantification by using a BCA kit, carrying out electrophoresis on SDS-PAGE gel (4% concentrated gel and 15% separation gel), staining with Coomassie brightness, and photographing to record the result. The results are shown in FIG. 1, in which the left lane is crude extract and the right lane is purified antimicrobial peptide protein DEFA 3.
Example 2 nucleic acid liposome Complex preparation
Weighing 86mg of lecithin and 8mg of cholesterol, dissolving in 5ml of chloroform, performing reduced pressure rotary evaporation in a constant-temperature water bath at 40 ℃, and pumping until the chloroform is completely volatilized to form a uniform film. The phospholipid membranes were gently disintegrated (180rpm) at room temperature in 10ml of an aqueous phase (A/B), liquid A: 10ml PBS; and B, liquid B: plasmid antimicrobial peptide complex, 5ml about 3mg, vortex to all heavy suspension (figure 2A). The plasmid antimicrobial peptides were mixed and tested to a neutral state by a potentiostat (FIG. 2B). Placing the milky white suspension in N2Carrying out neutralization for 2 h; gently shaking the suspension, and then placing on an ultrasonic instrument for constant temperature ultrasonic treatment for 3min at 55 ℃; the suspension is placed in N at normal temperature2Centrifuging for 2h at 10,000g for 15min, and washing with PBS for 3 times to obtain GFP neutral liposome. Particle size was detected using a NanoSight instrument (figure 2C).
Example 3 transfection of GFP neutral liposomes into monocytes/macrophages in vitro
The obtained GFP neutral liposome transfected mouse monocyte RAW and breast cancer cell line 4T1 were transfected with 6-well plate, each well was transfected with 10ul, the positive rate of RAW detected by flow cytometry was approximately 37% + -9.8%, and no signal change was detected by breast cancer cell 4T1 (FIG. 3A). Further examination of the expression time of the GFP plasmid in monocytes revealed that a partial fluorescence signal was still detectable at day 30 (FIGS. 3B, C).
Example 4 transfection evaluation of nucleic acid liposomes into monocytes in vivo
Experimental methods
1. Laboratory animals and groups
30 SPF grade C57 mice were kept in a Nanjing university animal rearing room for 3 days with free access to water and diet, and all animals were randomly divided into 6 groups of 5 high, medium and low dose groups of GFP neutral liposomes (example 2), blank liposome groups (blank group), GFP group, normal group, and six weeks old (provided by Hangzhou photon-derived laboratory animal science and technology Co., Ltd., body weight of 22-24 g).
2. Administration of drugs
The administration method is that 100 mu L of each of the prepared GFP liposome suspension (5 mu g/ml,50 mu g/ml and 500 mu g/ml), GFP (50 mu g/ml) and blank liposome suspension is taken and injected by a micro-syringe tail vein. ② the injection is carried out once after 24 hours, and the mice are euthanized after 48 hours. And thirdly, feeding the animals in cages, and freely ingesting food and drinking water.
3. Transfection evaluation method
Peripheral blood of mice was taken, treated with erythrocyte lysate, and then monocytes (CD14), neutrophils (Ly6G) and T lymphocytes (CD4, CD8) were stained, and flow antibodies were derived from eBioscience, and the expression of GFP was detected by flow assay and further detected.
4. Statistical analysis
And mapping the experimental data by using Graphpad8.0 software. Results are expressed as mean + -SEM, and statistical analysis of the set of design data was performed using the t-test. NS is no significant difference; p <0.05 was significantly different; indicates that P <0.001 is very significantly different.
Results
Through observation, the mortality rate is not influenced after the nucleic acid liposome compound is injected into the tail vein of the mouse, which shows that the nucleic acid liposome compound has no mortality rate to the mouse.
Then, flow cytometry is performed to detect the expression of GFP plasmid in mouse peripheral blood, as shown in fig. 4, the results show that there is no significant difference between the blank group and the blank liposome group, i.e. none of them expresses the relevant signal, in the neutrophil, a little signal expression occurs with the addition of high dose of GFP liposome complex, but compared with the signal in the monocyte, the signal is obviously weaker than that of the monocyte, in addition, the monocyte shows a certain dose-dependent effect, with the increase of concentration, the GFP positive signal is as high as about 7.4%, and no GFP signal is detected in the T cell. The above results indicate that our synthesized GFP liposome neutral complex has a certain specificity to monocytes.
Sequence listing
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<120> neutral liposome for delivering nucleic acid drug, preparation method and application thereof
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tgcagaaagg gtcatttaat gtacacactc tgctgtcgcc atcatcacca tcaccactaa 300
Claims (7)
1. A neutral liposome for delivering a nucleic acid drug, characterized in that said neutral liposome consists essentially of lecithin, cholesterol, a nucleic acid drug, and a cationic peptide; wherein the negatively charged nucleic acid drug is mixed with the cationic peptide to be electrically neutral.
2. Neutral liposomes according to claim 1 characterized in that the ratio of lecithin: the mass ratio of the cholesterol is 5:1-20: 1.
3. Neutral liposomes according to claim 1, characterized in that the cationic peptide is selected from the group consisting of antibacterial peptides.
4. A method of preparing neutral liposomes according to any one of claims 1 to 3 characterized by comprising the steps of:
(1) adding lecithin and cholesterol into chloroform, mixing, removing chloroform by a reduced pressure rotary evaporator, and forming a milky-white phospholipid film at the bottom of a bottle;
(2) mixing the nucleic acid drug and the cationic peptide, and detecting the potential to be neutral, wherein the adding amount of the cationic peptide is based on neutralizing the negative electricity of the nucleic acid drug to be neutral;
(3) dispersing the phospholipid film prepared in the step (1) in a water phase gently at the temperature of 25-30 ℃, and vortexing for 10-20min until the phospholipid film is completely resuspended to obtain a white suspension; wherein, the water phase is formed by mixing a liquid A and a liquid B, and the liquid A: PBS, B liquid is the plasmid antibacterial peptide compound prepared in the step (2), and the volume ratio of the A liquid to the B liquid is 1.5-3: 1;
(4) placing the milky white suspension at 25-30 ℃ in N2Neutralizing for 1.5-2 h;
(5) the suspension is gently shaken and then placed on an ultrasonic instrument for constant temperature ultrasonic treatment for 2-5 min;
(6) the suspension is placed in N at normal temperature2Centrifuging for 1.5-2h, washing with sterile PBS for 2-3 times, and centrifuging for 30min at 25,000g after each washing.
5. The method according to claim 4, wherein the mass ratio of the nucleic acid drug to the cationic peptide is 10:1 to 1: 30.
6. The method according to claim 4, wherein the mass-to-volume ratio of the phospholipid membrane to the aqueous phase is 1:1 to 1: 10.
7. Use of a neutral liposome according to any of claims 1-3 for the preparation of an in vivo or in vitro pharmaceutical preparation for transfection of monocytes/macrophages.
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