CN115487150A - Liver-targeted traceable drug delivery carrier, preparation method and application thereof, and diabetes treatment drug - Google Patents
Liver-targeted traceable drug delivery carrier, preparation method and application thereof, and diabetes treatment drug Download PDFInfo
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- CN115487150A CN115487150A CN202211444117.XA CN202211444117A CN115487150A CN 115487150 A CN115487150 A CN 115487150A CN 202211444117 A CN202211444117 A CN 202211444117A CN 115487150 A CN115487150 A CN 115487150A
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- traceable
- drug delivery
- lipid
- liver
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Abstract
In order to overcome the problems of poor stability, short half-life period, low targeting property, low delivery efficiency, incapability of tracking and lack of escape capacity of cell endosomes in-vivo application of siRNA in the prior art, the invention provides a liver-targeted traceable drug delivery carrier, which comprises a lipid assembly, a targeting molecule and a tracer, wherein the targeting molecule comprises galactose modified pyrene-polyethylene glycol. The invention also provides a preparation method and application of the traceable drug delivery carrier and a diabetes treatment drug comprising the traceable drug delivery carrier. The traceable drug delivery carrier provided by the invention realizes the specific delivery of an siRNA delivery system to the liver, and realizes the near infrared fluorescence tracing of the carrier by introducing aggregation-induced emission molecules.
Description
Technical Field
The invention relates to the technical field of siRNA (small interfering ribonucleic acid) targeted drug carriers, in particular to a liver targeted traceable drug delivery carrier, a preparation method and application thereof, and a diabetes treatment drug.
Background
Small interfering RNAs (sirnas) are double-stranded Small nucleic acids that exert RNA interference (RNAi) after transcription, and specifically inhibit the expression of mRNA of disease-related target genes to exert therapeutic effects, and thus research on in vivo application of RNAi is highly regarded. With the elucidation of the biological mechanism of siRNA and the rapid development of siRNA synthesis methods, most of the genes can be silenced by siRNA technology. At present, a plurality of siRNA drugs are approved by FDA to be on the market internationally, and a plurality of siRNA drugs are in different research and development stages domestically. The main problems faced by the current in vivo application of siRNA are: the stability of siRNA is poor, the half-life period is short, the targeting property needs to be improved so as to achieve high-efficiency delivery, the escape capability of cell endosome is lacked, and the like.
Lipid Nanoparticles (LNPs) are lipid vesicles with a homogeneous lipid core, widely used for the delivery of small molecules and nucleic acid drugs. Liposomes have high biocompatibility and biodegradability and can therefore be used to deliver a wide variety of active ingredients. The inside and the outside of the liposome are hydrophilic phases, the phospholipid bilayer is a lipophilic phase, and the medicament can be wrapped in the phospholipid bilayer or the inner and the outer water phases according to the polarity of the medicament.
ASGPR, also known as galactose receptor, was the first mammalian lectin to be identified, being expressed predominantly on the surface of hepatocytes and rarely found on extrahepatic cells, making it an ideal entry route to target hepatocytes. ASGPR recognizes asialoglycoproteins bearing galactose (Gal) residues at their termini, has high affinity, and is also able to promote internalization through clathrin-mediated endocytosis.
Aggregation-induced emission (AIE) refers to a light emission phenomenon in which a type of organic molecules emits little or no light when dissolved in a solution, but emits strong fluorescence when the molecules are aggregated. The AIE molecules can still emit strong fluorescence after forming aggregates in water, and the fluorescence is not quenched due to the aggregation, so the AIE molecules have obvious application advantages in the biological field. The material as a novel organic fluorescent material has a series of unique advantages, including high sensitivity, high aggregation state emission efficiency, good light stability, large Stokes displacement, low background noise, long-term non-wound, strong biological visualization capability and the like, and has been successfully used for a cell fluorescent tracking probe, and is used for clinical treatment of diseases based on image guidance.
Disclosure of Invention
The invention aims to solve the technical problems of poor stability, short half-life period, poor targeting property, lack of escape capacity of a cell endosome and the like of siRNA in the prior art, and provides a liver-targeted traceable drug delivery carrier, a preparation method and application thereof, and a diabetes treatment drug.
The technical scheme adopted by the invention for solving the technical problems is as follows:
in one aspect, the invention provides a liver-targeted traceable drug delivery vehicle comprising a lipid assembly, a targeting molecule and a tracer, the tracer being a near-infrared aggregation-induced emission molecule, the lipid assembly comprising an ionizable lipid comprising a primary ionized lipid molecule and a secondary ionized lipid molecule, and a linked lipid, the targeting molecule comprising a galactose-modified pyrene-polyethylene glycol.
Optionally, the structure of the targeting molecule is as follows:
optionally, the primary ionized lipid molecule is selected from DLin-MC3-DMA, the secondary ionized lipid molecule is selected from DSPC, and the linking lipid is selected from CHOL.
Optionally, the traceable drug delivery vehicle comprises the following components in parts by weight:
38 to 65.5 parts of lipid combination, 2 to 18.5 parts of targeting molecules and 0.1 to 2.5 parts of tracer.
Optionally, the liposome assembly comprises 22 to 36.2 parts by weight of main ionized lipid molecules, 2.5 to 14.5 parts by weight of auxiliary ionized lipid molecules and 5.8 to 19.8 parts by weight of connecting lipid.
Optionally, the tracer comprises at least one of 2TT-C46, 2TT-oC6B, 2TT-oC26B, BTPPA and XA 1.
In a further aspect, the present invention provides the use of a traceable drug delivery vehicle as described above in a liver targeting medicament.
Optionally, the liver targeting drug further comprises a pharmaceutical active ingredient with a diabetes prevention or treatment effect, and the pharmaceutical active ingredient is wrapped in the traceable drug delivery carrier.
Optionally, the diabetes comprises type II diabetes.
Optionally, the pharmaceutically active ingredient comprises siRNA that inhibits expression of LOC157273 gene in liver cells.
In a further aspect, the present invention provides a therapeutic agent for diabetes, comprising a pharmaceutically active ingredient and a traceable drug delivery vehicle as described above, wherein the pharmaceutically active ingredient is encapsulated in the traceable drug delivery vehicle.
Optionally, the pharmaceutically active ingredient comprises siRNA that inhibits expression of LOC157273 gene in liver cells.
Optionally, the diabetes treatment drug further comprises pharmaceutically acceptable auxiliary materials.
Optionally, the auxiliary materials comprise one or more of buffering agent, emulsifying agent, suspending agent, stabilizing agent, preservative, physiological salt, excipient, filling agent, coagulant and blending agent, surfactant, dispersing agent and defoaming agent.
In a further aspect, the present invention provides a method for preparing a traceable drug delivery vehicle as described above, comprising the following operative steps:
weighing main ionized lipid molecules, auxiliary ionized lipid molecules, connecting lipid, a tracer and targeting molecules, dissolving and dispersing in an organic solvent, adding water for hydration, and removing the organic solvent to obtain the traceable drug delivery carrier.
Optionally, the organic solvent is ethanol.
According to the liver-targeted traceable drug delivery carrier provided by the invention, the specific delivery of the siRNA delivery system to the liver is realized by carrying out galactose modification on pyrene-polyethylene glycol; the traceable drug delivery carrier prepared by the invention can deliver siRNA and target-transfect liver cells, and further through cationic liposome delivery effect and target experiment verification, the traceable drug delivery carrier provided by the invention is proved to be capable of efficiently delivering the siRNA drug and simultaneously have specific target effect on the liver cells.
Drawings
FIG. 1 is a graph showing the results of particle size and surface potential measurements of cationic liposome nanoparticles provided in example 1; ( A, detecting a cationic liposome nanoparticle particle size; b cationic liposome nanoparticle surface potential determination diagram )
FIG. 2 is a graph showing the results of particle size and surface potential measurements of cationic liposome nanoparticles provided in example 2; ( A, detecting a particle size detection diagram of cationic liposome nanoparticles; b cationic liposome nanoparticle surface potential determination diagram )
FIG. 3 shows the results of particle size and surface potential measurements of cationic liposome nanoparticles provided in example 3; ( A, detecting a cationic liposome nanoparticle particle size; b cationic liposome nanoparticle surface potential determination diagram )
FIG. 4 is the particle size and surface potential measurements of cationic liposome nanoparticles provided in example 4; ( A, detecting a cationic liposome nanoparticle particle size; b cationic liposome nanoparticle surface potential determination diagram )
FIG. 5 shows the results of particle size and surface potential measurements of cationic liposome nanoparticles provided in example 5; ( A, detecting a cationic liposome nanoparticle particle size; b cationic liposome nanoparticle surface potential determination diagram )
FIG. 6 is a confocal fluorescence diagram of the transfection effect of cationic liposome nanoparticles provided by the present invention; ( A transfection of 24 h; b transfection 48 h; a is white light mode, b synthesis mode, and c fluorescence mode )
Fig. 7 is a graph for detecting the delivery effect of cationic liposome nanoparticles provided by the present invention;
fig. 8 is a confocal fluorescence diagram for verifying the delivery effect of cationic liposome nanoparticles provided by the present invention; (A white light mode; B fluorescent mode)
FIG. 9 is a toxicity test chart of cationic liposome nanoparticles provided by the present invention;
fig. 10 is a diagram for detecting the targeting effect of cationic liposome nanoparticles provided by the invention;
fig. 11 is a verification detection diagram of the targeting effect of the cationic liposome nanoparticle provided by the present invention;
FIG. 12 is a near-infrared fluorescent development of a mouse according to the present invention;
FIG. 13 is a near-infrared fluorescence image of a mouse organ provided by the present invention;
fig. 14 is a schematic view of cationic liposome nanoparticles provided by the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 14, the embodiment of the present invention provides a liver-targeted traceable drug delivery vehicle, comprising a lipid assembly, a targeting molecule and a tracer, wherein the tracer is a near-infrared aggregation-induced emission molecule, the lipid assembly comprises an ionizable lipid and a connecting lipid, the ionizable lipid comprises a main ionized lipid molecule and an auxiliary ionized lipid molecule, and the targeting molecule comprises galactose-modified pyrene-polyethylene glycol.
Specifically, the targeting molecule provided by the invention is prepared by performing Galactose modification on Pyrene-polyethylene glycol to obtain Pyrene-PEG-Galactose (Pyrene-polyethylene glycol-Galactose), and the specific structure of the targeting molecule is as follows:
the specific preparation process for preparing the Pyrene-PEG-Galactose (Pyrene-polyethylene glycol-Galactose) comprises the following steps: dissolving 1-pyrenebutyric acid in a DMF solution, and dissolving amino-polyethylene glycol-galactose in the DMF solution. After the two solutions were mixed well, 1-ethyl-3[3-dimethylaminopropyl ] carbodiimide hydrochloride was added. Refluxing in oil bath overnight, dialyzing for 3 times, lyophilizing, and collecting paste solid to obtain pyrene-PEG-Gal.
The modified Pyrene-PEG-Galactose improves the specificity of combining with ASGPR on the surface of a hepatocyte, is beneficial to improving the liver targeting property of liposome nanoparticles, realizes the specific delivery of a siRNA delivery system to the liver, reduces the damage to other organs of an organism, improves the curative effect of siRNA drugs and provides a more efficient treatment means for the clinical drug application of liver diseases.
The liver targeting molecule can be used for modifying drug delivery carriers such as liposome, emulsion, nanoparticles, microspheres, nanocapsules and micelles, the content of the liver targeting molecule can reach 0.1-100% (mol%) of lipid components, and the liver targeting molecule is used for targeting treatment of anti-liver cancer and anti-hepatitis drugs.
In one embodiment, the primary ionomeric lipid molecule is selected from DLin-MC3-DMA.
Specifically, the DLin-MC3-DMA structure is as follows:
the DLin-MC3-DMA is an efficient, ionizable cationic liposome and has a unique pH-dependent charge-variable property: the nucleic acid is electropositive under acidic condition and neutral under physiological pH condition, so the formed liposome has very small positive charge density in blood, namely very low cytotoxicity, and DLin-MC3-DMA cationic liposome can increase lysosome escape of particles in vivo due to electropositive property, improve transfection efficiency and is not easy to phagocytose by macrophage; by utilizing the characteristics of DLin-MC3-DMA, the liposome/siRNA compound is formed by electrostatic interaction with negatively charged siRNA, and has higher transfection efficiency and low toxicity.
In one embodiment, the auxiliary ionized lipid molecules are selected from DSPC.
Specifically, the structure of the DSPC is as follows:
the DSPC is usually used as a liposome microbubble contrast agent and a drug lipid microsphere, and when the DSPC is used as a membrane material, the DSPC is mostly applied to liposome drugs and has better stability. DSPC is added into the traceable drug delivery carrier, so that the phase transition temperature of the cationic liposome can be increased, the formation of a lamellar lipid bilayer structure is supported, and the structural arrangement of the lamellar lipid bilayer structure is stabilized.
In one embodiment, the linking lipid is selected from CHOL.
Specifically, the CHOL structure is as follows:
the CHOL is one of positive charge lipids commonly used for preparing cationic liposomes, provides positive electricity for particles while stabilizing the liposome membrane structure, and can also efficiently load genes. And CHOL has strong membrane fusion property, and promotes RNA intracellular uptake and cytoplasmic entry.
In some embodiments, the traceable drug delivery vehicle comprises the following components in parts by weight:
38 to 65.5 parts of lipid assembly, 2 to 18.5 parts of targeting molecule and 0.1 to 2.5 parts of tracer.
Specifically, the liposome assembly comprises 22 to 36.2 parts by weight of main ionization lipid molecules, 2.5 to 14.5 parts by weight of auxiliary ionization lipid molecules and 5.8 to 19.8 parts by weight of connecting lipid.
In some embodiments, the tracer comprises at least one of 2TT-C46, 2TT-oC6B, 2TT-oC26B, BTPPA, and XA 1.
In one embodiment, the tracer is selected from 2TT-C46, and 2TT-C46 has the following structure:
the traceable drug delivery carrier provided by another embodiment of the invention is applied to liver targeting drugs.
The main ionized lipid molecules can be combined with hydrogen ions under an acidic environment to form positive electricity, and nucleic acid is wrapped in the lipid nanoparticles by virtue of electrostatic adsorption of the main ionized lipid molecules and the hydrogen ions; the exterior of the wrapped structure is hydrophobic due to the outward hydrophobic end of the cationic lipid, and at the moment, the lipid with PEG modified at one end commonly used in liposome synthesis is added, so that the hydrophobic end of the PEG-lipid is combined with the hydrophobic end of the cationic lipid, and the hydrophilic end (connected with the PEG) of the PEG-lipid outwards forms a nucleic acid lipid nanoparticle shell; in order to increase the stability of the nucleic acid lipid nanoparticle, a proper amount of cholesterol Chol, DSPC and other components can be added, so that the combination of the hydrophobic end of PEG-lipid and the hydrophobic end of cationic lipid is more compact, and finally lipid nanoparticle particles are obtained; specifically, by using the pyrene-polyethylene glycol modified by Galactose, the specific delivery of the siRNA delivery system to the liver is realized, the efficiency of delivering the siRNA medicament to the liver cell is effectively improved, the damage to other organs of the body is reduced, the curative effect of the siRNA medicament is improved, and a more efficient treatment means is provided for the clinical medicament application of the liver disease.
In one embodiment, the liver-targeting drug further comprises a pharmaceutically active ingredient having a diabetes preventing or treating effect, the pharmaceutically active ingredient being encapsulated in the traceable drug delivery vehicle.
The active ingredients of the liver targeting drug are wrapped in a traceable drug delivery carrier, so that the high-efficiency transfer of the drug can be realized, the degradation of the drug is avoided, and the occurrence of toxic and side effects is reduced.
In one embodiment, the diabetes comprises type II diabetes.
In one embodiment, the pharmaceutically active ingredient comprises siRNA that inhibits expression of LOC157273 gene in liver cells.
The active ingredients of the medicine can specifically inhibit the replication and transcription of LOC157273 gene in liver cells, thereby exerting the treatment effect.
Another embodiment of the present invention provides a therapeutic agent for diabetes, wherein the active pharmaceutical ingredient is encapsulated in the traceable drug delivery vehicle.
Specifically, the targeted diabetes treatment drug can be used for directing the drug to the focus position and improving the concentration of the drug entering cells, thereby playing a treatment role.
In one embodiment, the diabetes treatment drug further comprises pharmaceutically acceptable auxiliary materials.
In one embodiment, the adjuvants include one or more of buffers, emulsifiers, suspending agents, stabilizers, preservatives, salts, excipients, fillers, coagulants and blenders, surfactants, dispersing agents, and antifoaming agents.
The pharmaceutical excipients not only have the functions of excipient, carrier and stability improvement, but also have the important functions of solubilization, dissolution assistance, sustained and controlled release and the like, and are important components which may influence the quality, safety and effectiveness of the medicine.
Another embodiment of the present invention further provides a method for preparing a traceable drug delivery vehicle, comprising the following steps:
weighing main ionized lipid molecules, auxiliary ionized lipid molecules, connecting lipid, a tracer and targeting molecules, dissolving and dispersing in an organic solvent, adding water for hydration, and removing the organic solvent to obtain the traceable drug delivery carrier.
In one embodiment, the organic solvent is ethanol.
The present invention is further illustrated by the following examples.
Example 1
This example is intended to illustrate a liver-targeted traceable drug delivery vehicle disclosed in the present invention, comprising the following steps:
preparation of targeting molecule (Pyrene-PEG-Galactose)
288mg of 1-pyrenebutanoic acid was dissolved in 20ml of DMF solution, and 2g of amino-polyethylene glycol-galactose was additionally dissolved in 20ml of DMF solution. After mixing the two solutions evenly, 1.5g of 1-ethyl-3[3-dimethylaminopropyl ] carbodiimide hydrochloride was added. Refluxing in oil bath overnight, dialyzing for 3 times, and freeze-drying to collect pasty solid, i.e. Pyrene-PEG-Galactose.
Preparation of cationic liposome nanoparticles (traceable drug delivery vehicles)
Precisely weighing 32.1mg DLin-MC3-DMA,7.9mg DSPC,14.8mg Chol,2mg Pyrene-PEG-Galactose and 0.5mg 2TT-C46;
dissolving the weighed components in 8ml of organic solvent ethanol, dissolving siRNA in DEPC water, adding the solution into the organic solution, adding the DEPC water, and carrying out low-temperature water bath ultrasound for 20min to fully dissolve the siRNA;
and (3) removing ethanol by blowing 1 h at room temperature in a dark place to obtain the final cationic liposome nano-particles.
Example 2
This example is intended to illustrate a liver-targeted traceable drug delivery vehicle disclosed in the present invention, comprising the following steps:
preparation of targeting molecule (Pyrene-PEG-Galactose)
288mg of 1-pyrenebutanoic acid was dissolved in 20ml of DMF solution, and 2g of amino-polyethylene glycol-galactose was additionally dissolved in 20ml of DMF solution. After mixing the two solutions evenly, 1.5g of 1-ethyl-3[3-dimethylaminopropyl ] carbodiimide hydrochloride was added. Refluxing in oil bath overnight, dialyzing for 3 times, and freeze-drying to collect pasty solid, i.e. Pyrene-PEG-Galactose.
Preparation of cationic liposome nanoparticles
Precisely weighing 32.1mg DLin-MC3-DMA,7.9mg DSPC,15mg Chol,1.8mg Pyrene-PEG-Galactose and 0.9mg 2TT-C46;
dissolving the weighed components in 8ml of organic solvent ethanol, dissolving siRNA in DEPC water, adding the solution into the organic solution, adding DEPC water, and carrying out low-temperature water bath ultrasound for 20min to fully dissolve the siRNA;
and removing ethanol by a nitrogen blowing method 1 h under the conditions of room temperature and light shielding to obtain the final cationic liposome nano-particles.
Example 3
This example is intended to illustrate a liver-targeted traceable drug delivery vehicle disclosed in the present invention, comprising the following steps:
preparation of targeting molecule (Pyrene-PEG-Galactose)
288mg of 1-pyrenebutanoic acid was dissolved in 20ml of DMF solution, and 2g of amino-polyethylene glycol-galactose was additionally dissolved in 20ml of DMF solution. After mixing the two solutions evenly, 1.5g of 1-ethyl-3[3-dimethylaminopropyl ] carbodiimide hydrochloride was added. Refluxing in oil bath overnight, dialyzing for 3 times, and freeze-drying to collect pasty solid, i.e. Pyrene-PEG-Galactose.
Preparation of cationic liposome nanoparticles
Precisely weighing 32.1mg of DLin-MC3-DMA,7.9mg of DSPC,14.3mg of Chol,3.2mg of Pyrene-PEG-Galactose and 1.6mg of 2TT-C46;
dissolving the weighed components in 8ml of organic solvent ethanol, dissolving siRNA in DEPC water, adding the solution into the organic solution, adding DEPC water, and carrying out low-temperature water bath ultrasound for 20min to fully dissolve the siRNA;
and removing ethanol by a nitrogen blowing method 1 h under the conditions of room temperature and light shielding to obtain the final cationic liposome nano-particles.
Example 4
This example is intended to illustrate a liver-targeted traceable drug delivery vehicle disclosed in the present invention, which includes most of the operating steps in example 1, except that:
DSPC and Chol are not added into the components, and weighed DLin-MC3-DMA and Pyrene-PEG-Galactose are dissolved in 2ml of organic solvent ethanol.
Example 5
This example is intended to illustrate a liver-targeted traceable drug delivery vehicle disclosed in the present invention, which includes most of the operating steps of example 3, except that:
no DSPC is added in the components, and the rest components are dissolved in 7ml of organic solvent ethanol.
Performance testing
1. Measurement of particle size and surface potential (Zeta potential) of cationic liposome nanoparticles the results of particle size and surface potential measurements of cationic liposome nanoparticles obtained in examples 1-5 are shown in FIG. 1~5.
From the test results of fig. 1-5, it can be seen that the Zeta potentials of the nanoparticles of examples 4 and 5 are close to 50mV, and the nanoparticles are easy to adsorb with negatively charged biomacromolecules in vivo, easily trigger immune-related reactions, and are not favorable for the nanoparticles to enter cells; the Zeta potential of the nanoparticles prepared in examples 1-3 is reduced by about 10mV compared with the nanoparticles prepared in examples 4 and 5, which not only meets the potential of more than 30mV but also ensures the stability requirement of the nanoparticles, and accordingly, the occurrence of in vivo adsorption reaction can be reduced.
2. Cationic liposome nanoparticle transfection effect verification
The method comprises the following steps: GFP-labeled human hepatoma cell line HepG2
Selecting mRNA of green aequorin fluorescent protein (GFP) of west coast of Victoria of America as a target gene, enabling the human hepatoma cell line HepG2 to stably express the GFP by using the lentivirus particles, then transfecting siRNA targeting the GFP into the HepG2 by using the liposome nanoparticles prepared in the embodiment 1 of the invention, wherein the sequence of 5'-3' is as follows: GCACCAUCUUCUCUCAAGGAdT.
Step two: detection of GFP expression levels in HepG2 cells
The HepG2 cells were as described at 1X 10 4 cells/well density was inoculated into 96-well plates, 100. Mu.L of DMEM medium containing 10% fetal bovine serum and 1% diabesin (ampicillin and kanamycin) was added to each well, and 5% CO was added at 37 ℃ 2 After 24h is cultured in a conditioned incubator, the culture medium is discarded, PBS is washed twice, serum-free DMEM is added, one group is an experimental group, and 20 muL siRNA sequence (5 '-3' is: GCACCAUUCUUCUCAAGGAdTdT) and the cationic liposome nanoparticles prepared in the embodiment 1 are added into each hole; the other group is a control group, the naked siRNA without the liposome carrier is transfected under the same other conditions, and the other group is a blank control group, namely the transfection is carried out under the same other conditions without the liposome carrier and the siRNA; continuously culturing 5h in the incubator, sucking out the culture solution, washing twice with PBS, and adding preheated DMEM culture medium containing 10% fetal calf serum and 1% double antibody; 37 ℃ and 5% CO 2 After 24h and 48h are respectively cultured in the condition incubator, the incubator is placed in a multifunctional microplate reader BioTek, after a plate is vibrated for 10s, excitation is carried out at 488nm, fluorescence intensity is detected by emitting at 510nm, and the detection result is shown in FIGS. 6 and 7.
The test results in fig. 6 show that, when confocal microscope detection and observation are performed, green fluorescence is observed at 24h and 48h, respectively, and fluorescence observed at 48h is relatively weak, that is, the liposome nanoparticle prepared by the invention can deliver siRNA to human hepatoma cells in a wrapping manner, and successfully enter the cells, so that endosome escape of siRNA is realized, and good delivery and release effects in vivo are reflected.
From the test results of fig. 7, it can be known that the cationic liposome nanoparticle provided by the present invention can effectively promote transfection of HepG2 cells with siRNA, compared to naked siRNA without liposome vector.
3. Verification of cationic liposome nanoparticle delivery effect
Selecting a dye cy3 to modify siRNA, transfecting the human liver cancer cell HepG2 by using the liposome nano-particles prepared in the embodiment 2 of the invention, and detecting fluorescence by using a fluorescence inverted microscope after the siRNA transfects the HepG2 cell 24 h.
The test results in fig. 8 show that the liposome nanoparticle prepared by the present invention is transfected into human hepatoma cell HepG2, and after siRNA is transfected into HepG2 cell 24h, the fluorescent inverted microscope is used for observation, green light excites cy3 to emit red light, and cy3 is observed to be distributed in cytoplasm, which indicates that the liposome nanoparticle of the present invention has accurate position targeting and good siRNA delivery effect.
4. Cationic liposome nanoparticle toxicity detection
HepG2 cells in logarithmic growth phase at 1X 10 4 cells/well density was inoculated into 96-well cell culture plates, and after 24h was cultured, 0.5nM, 5nM, and 10nM concentrations of the cationic liposome nanoparticle solution provided in example 3 (diluted with serum-free medium, 0.45 μm water membrane filtration sterilized) were added to each of the three wells in parallel; after 48h was incubated in the dark under the conventional culture conditions, 10. Mu.L of CCK-8 solution was added to each well, 1 h was incubated further, and its absorbance value (A) was measured by a microplate reader.
From the test results of fig. 9, it can be seen that: the effect of cationic liposomal nanoparticles at concentrations of 0.5nM to 10nM on HepG2 cell activity was not very different and killing of cells by cationic liposomal nanoparticles was found to be insignificant.
5. Verification of targeting effect of Galactose modified pyrene-polyethylene glycol
The method comprises the following steps: the HepG2 cells were treated as 1X 10 4 cells/well density was plated in 96-well plates, 100. Mu.L of DMEM medium containing 10% fetal bovine serum and 1% diabody was added to each well, 37 ℃ and 5% CO 2 Culturing 24h in an incubator under the condition;
step two: taking the HepG2, discarding the culture medium, washing with PBS twice, adding serum-free DMEM, and taking the washed solution without adding cationic liposome as a blank control group; a group of LNPs loaded with GFP-siRNA are added; one group was added with the cationic liposome nanoparticles provided in example 3 loaded with GFP-siRNA.
Step three: culturing the above treated samples for 5 hr, removing the culture solution, washing with PBS twice, adding preheated serum containing 10% fetal calf serum and 1% double antibodyDMEM medium, 37 ℃, 5% CO 2 Culturing 24h and 48h in an incubator under the condition respectively;
step four: and placing the sample to be detected in a microplate reader BioTek, vibrating the plate for 10s, setting excitation at 488nm, and emitting fluorescence intensity at 510nm for detection.
From the test results in fig. 10, it can be seen that the fluorescence intensity of the blank control group without the cationic liposome nanoparticle is strongest, the fluorescence intensity of the GFP-siRNA-loaded liposome-modified group is weakest, and the fluorescence intensity of the LNP not modified by the Galactose-loaded group is inferior, which indicates that the transfection effect of the liposome nanoparticle prepared by the present invention is better than that of the liposome delivery without targeted connection and the targeting property.
6. Verification of targeting effect of Galactose modified pyrene-polyethylene glycol
Selecting mRNA of green aequorin fluorescent protein (GFP) of west coast of Victoria of USA as target gene, using lentivirus particles to make human hepatoma cell line HepG2 and HUV human umbilical vein endothelial cell stably express GFP, and respectively making HepG2 human hepatoma cell and HUV human umbilical vein endothelial cell according to 1 × 10 4 cells/well density was plated in 96-well plates, 100. Mu.L of DMEM medium containing 10% fetal bovine serum and 1% diabody was added to each well, 37 ℃ and 5% CO 2 Culturing 24h in an incubator under the condition; taking the HepG2 and the HUV, discarding the culture medium, washing with PBS twice, adding serum-free DMEM, hepG2 and HUV without cationic liposome as blank control group; a group of cationic liposome nanoparticles provided in example 2 loaded with GFP-siRNA were added to HepG2 cells; one group adds the cationic liposome nanoparticles provided in example 2 loaded with GFP-siRNA to HUV cells. Culturing the above differently treated test samples for 5h, aspirating the culture medium, washing twice with PBS, adding preheated DMEM medium containing 10% fetal bovine serum and 1% double antibody, 37 deg.C, 5% 2 Culturing 24h and 48h in an incubator under the condition respectively; and placing the sample to be detected in a microplate reader BioTek, vibrating the plate for 10s, setting excitation at 488nm, and emitting fluorescence intensity at 510nm for detection.
As can be seen from the test results in FIG. 11, compared with HUV human umbilical vein endothelial cells, the liposome nanoparticles prepared by the present invention have better transfection effect on the human hepatoma cell line HepG2, which indicates that they have better liver targeting property.
7. Verification of targeting effect of Galactose modified pyrene-polyethylene glycol
Selecting 6~8 week-old female Balb/c mice for in vivo imaging, and specifically comprising the following experimental steps:
1. shaving all the hairs of the Balb/c mouse except the head, preparing anesthetic chloral hydrate in advance, carrying out intraperitoneal injection on the mouse according to 0.6mL/100g generally, pinching the tail tip of the mouse by using forceps during anesthesia, and judging the anesthesia state of the mouse;
2. after anesthetizing the mice, the cationic liposome nanoparticles provided in example 3 were aspirated by an insulin needle for tail vein injection, and the injection dose per mouse was 100 μ l;
3. the mice were placed in an infrared two-zone animal in vivo imager 24h after injection before injection, photographed using a 808nm laser, and dissected and imaged heart, liver, spleen, lung, and kidney 24h after in vivo imaging, respectively.
As can be seen from the experimental results of fig. 12 and 13, after mice are injected with the cationic liposome nanoparticles of the present invention and imaged, the brightness of the liver is found to be significantly higher than that of other organs, which means that the cationic liposome nanoparticles injected in vivo are mostly enriched in the liver, and the technical effect of targeted liver delivery is verified.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (16)
1. A liver-targeted traceable drug delivery vehicle comprising a lipid assembly, a targeting molecule and a tracer, wherein the tracer is a near-infrared aggregation-induced emission molecule, wherein the lipid assembly comprises an ionizable lipid and a linked lipid, wherein the ionizable lipid comprises a main ionized lipid molecule and an auxiliary ionized lipid molecule, and wherein the targeting molecule comprises a galactose-modified pyrene-polyethylene glycol.
2. The traceable drug delivery vehicle of claim 1, wherein said targeting molecule has the structure shown below:
3. the traceable drug delivery vehicle according to claim 1, wherein said primary ionized lipid molecule is selected from DLin-MC3-DMA, said secondary ionized lipid molecule is selected from DSPC, and said linking lipid is selected from CHOL.
4. The traceable drug delivery vehicle of claim 1, comprising the following components in parts by weight:
38 to 65.5 parts of lipid assembly, 2 to 18.5 parts of targeting molecule and 0.1 to 2.5 parts of tracer.
5. The traceable drug delivery vehicle of claim 4, wherein the liposome assembly comprises 22 to 36.2 parts by weight of main ionized lipid molecules, 2.5 to 14.5 parts by weight of auxiliary ionized lipid molecules and 5.8 to 19.8 parts by weight of connecting lipid.
6. The traceable drug delivery vehicle of any of claim 1~5, wherein said tracer comprises at least one of 2TT-C46, 2TT-oC6B, 2TT-oC26B, BTPPA, and XA 1.
7. Use of the traceable drug delivery vehicle of any of claims 1~6 in a liver-targeted drug.
8. The use of a traceable drug delivery vehicle in a liver-targeted drug according to claim 7, wherein the liver-targeted drug further comprises a pharmaceutically active ingredient having a diabetes preventing or treating effect, said pharmaceutically active ingredient being encapsulated in the traceable drug delivery vehicle.
9. Use of the traceable drug delivery vehicle of claim 8 in a liver-targeted drug, wherein said diabetes comprises type II diabetes.
10. Use of a traceable drug delivery vehicle according to claim 8 in a liver targeted drug, wherein the pharmaceutically active ingredient comprises siRNA that inhibits the expression of LOC157273 gene in liver cells.
11. A therapeutic agent for diabetes comprising a pharmaceutically active ingredient and a traceable drug delivery vehicle according to any of claims 1~6, said pharmaceutically active ingredient being encapsulated in said traceable drug delivery vehicle.
12. The therapeutic agent for diabetes according to claim 11, wherein the pharmaceutically active ingredient comprises siRNA which inhibits the expression of LOC157273 gene in liver cells.
13. The therapeutic agent for diabetes according to claim 11, further comprising a pharmaceutically acceptable excipient.
14. The remedy according to claim 13, wherein the adjuvant comprises one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, a physiological salt, an excipient, a filler, a coagulant and a blender, a surfactant, a dispersing agent and an antifoaming agent.
15. The method of making a traceable drug delivery vehicle of any of claims 1~6 comprising the steps of:
weighing main ionized lipid molecules, auxiliary ionized lipid molecules, connecting lipid, a tracer and targeting molecules, dissolving and dispersing in an organic solvent, adding water for hydration, and removing the organic solvent to obtain the traceable drug delivery carrier.
16. The method of making a traceable drug delivery vehicle of claim 15, wherein the organic solvent is ethanol.
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