CN109589307B - Preparation method of aptamer liposome - Google Patents
Preparation method of aptamer liposome Download PDFInfo
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- CN109589307B CN109589307B CN201811378750.7A CN201811378750A CN109589307B CN 109589307 B CN109589307 B CN 109589307B CN 201811378750 A CN201811378750 A CN 201811378750A CN 109589307 B CN109589307 B CN 109589307B
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
The invention discloses a preparation method of aptamer liposome. The method comprises the following steps: dissolving the compound in a chloroform/ethanol solution at a ratio of 1:1 of the amount of the substance, wherein the reaction needs to be realized in an RNase-free solution and on ice to obtain an RNA aptamer mixture; the reaction system is DOPE, SM and NBD-DSPE, wherein the mass ratio of DOPE, SM and NBD-DOPE is 10: 3:0.5, and reacting at room temperature for 0.3-0.5 hours. The aptamer distribution at the application site is higher than at other sites compared to free aptamer. In the aspect of cell internalization, compared with the previously reported liposome carrier, the liposome carrier has obvious improvement and achieves the double effect of prolonging the targeting action time.
Description
Technical Field
The invention belongs to the technical field of chemical drugs, and relates to a preparation method of aptamer liposome.
Background
The skin is the largest organ of the human body, which protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microbial attacks. The skin of human and higher animals is composed of three layers, epidermis, dermis, and subcutaneous tissue. The problem of skin aging is gradually highlighted as the individual grows and develops. Human skin aging includes exogenous factors (bad lifestyle, environmental pollution, etc.) and endogenous factors (natural deterioration of body functions). After long-term exposure to ultraviolet rays, the oxidative stress balance of fibroblasts of the skin is broken, mitochondria generate a lot of oxygen free radicals, and the defense system of the organism cannot timely clean the oxygen free radicals in the metabolic process, so that the oxygen free radicals are accumulated, and the skin cells are damaged.
The in vivo use of aptamers as therapeutic agents is primarily to minimize off-target effects by enhancing target-specific accumulation. The aptamer is a single-stranded nucleic acid, has small molecular weight, can be combined with small molecules, proteins, virus particles and targets in cells, has high affinity and selectivity, and has the advantages of rapid tissue penetration capability, no immunogenicity, thermal stability, easy chemical modification, low production cost and short production period.
It has been reported that selective mitochondrial delivery of endogenous and exogenous RNAs can be achieved using Ribonuclease P (RP) in mitochondrial RNA aptamers. However, to date, there has been no human demonstration of the problem of whether these aptamers can be used as mitochondrial targeting ligands, since nanocarriers modified with these RNA aptamers are not sufficiently internalized by cells. We invented herein in vivo targeting of light-triggered aptamers to the mitochondrial system, determining the optimal composition of nanocarriers required for efficient cellular uptake and mitochondrial targeting. After light irradiation, the aptamer is released, carries the anti-aging drug wrapped by the liposome, targets the mitochondria and prolongs the time of acting on skin cells. The aptamer distribution at the application site is higher than at other sites compared to free aptamer. In the aspect of cell internalization, compared with the previously reported liposome carrier, the liposome carrier has obvious improvement, and the dual effect of prolonging the target cutting action time is achieved.
Disclosure of Invention
One of the problems to be solved by the present invention is to find a biocompatible, degradable polymer compound in vivo, which must also have the ability to specifically target mitochondria under the illumination condition. In addition, changes to some parameters of the compound can control the release speed of the drug, change the release mode of the drug and hopefully improve the administration dosage form.
The second problem solved by the invention is to provide a drug which can specifically target mitochondria under the condition of illumination and can achieve the effect of slow release in a specific time.
Therefore, the invention discloses a targeted delivery method of an anti-aging drug, which has good targeting property and low cost.
The present invention is realized by the following method.
A preparation method of aptamer liposome comprises the following steps of hybridizing mitochondrial RNA aptamer with photolabile complementary oligonucleotide by using compounds of the following formulas [1], [2], [3], [4] and [5 ]; synthesis of liposome (DOPE + SM + NBD-DSPE) and cross-linking with aptamer; performing post-treatment and purification on the compound to prepare a light-triggered aptamer liposome drug-loading system;
dioleoyl phosphatidylethanolamine (DOPE)
[3]
Sphingomyelin (SM)
[4]
[5] The specific steps of 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- (7-nitro-2-1, 3-benzooxadiazol-4-yl) (NBD-DSPE) are as follows:
(1) dissolving the compounds shown in the formulas [1] and [2] in a chloroform/ethanol solution in a ratio of 1:1, wherein the reaction is realized in an RNase-free solution and on ice to obtain an RNA aptamer mixture;
(2) the reaction system is DOPE, SM and NBD-DSPE, wherein the mass ratio of DOPE, SM and NBD-DOPE is 10: 3:0.5, and reacting at room temperature for 0.3-0.5 hours.
In the method, the step (2) comprises the following specific steps: dissolving DOPE, SM and NBD-DSPE in chloroform/ethanol solution according to the amount ratio of substances, evaporating the chloroform/ethanol solution by a rotary evaporator to form a lipid membrane at the bottom of a glass tube, adding an RNA aptamer mixture of about 3-7% into the lipid mixture, simultaneously adding HEPES (4-hydroxyethylpiperazineethanesulfonic acid) buffer solution with the pH of 5.7 into the lipid membrane, incubating for 30 minutes at room temperature to hydrate the lipid, then ultrasonically treating the lipid membrane by using a bath type ultrasonic instrument for 1-2 minutes, evaporating the organic phase on the rotary evaporator in vacuum, adding 500 mu L of deionized water after 30 minutes, keeping the mixture in vacuum in the rotary evaporator for 30-50 minutes, evaporating the lipid particles (1mL) formed into the organic phase, and extruding for 10-20 times through a polycarbonate membrane with the thickness of 100nm at room temperature.
In the above method, the photolabile bond in 3 '-AGAGGG// ACUCGA// AGUCC-5' is a 1- (2-nitrophenyl) ethyl bond.
In the above method, the liposome compound may be glycerophospholipids (phospholipids) in addition to Dioleoylphosphatidylethanolamine (DOPE), Sphingomyelin (SM), 1, 2-distearoyl-sn-glycerol-3-Phosphoethanolamine-N- (7-nitro-2-1, 3-benzooxadiazol-4-yl) (NBD-DSPE). Glycerophospholipids include mainly Phosphatidylcholine (PC), Phosphatidylserine (PS) and phosphatidylethanolamine (cephalin), Phosphatidylinositol (PI).
In the above method, the crosslinking method is a hydration method.
In the method, the adopted buffer system is HEPES buffer solution, and the incubation time at room temperature is 30 minutes.
In the method, the drug-loading part (DOPE + SM + NBD-DSPE) is an anti-aging drug on the market, and the anti-aging drug is quercetin, vitamin E or clofibrate.
In the above method, the molar ratio of the polymer to the drug is 1:3.2 to 1: 3.5.
In the above method, the purification step is dissolving in deionized water, dialyzing and freeze-drying, or purifying by high-speed centrifugation.
The light-triggered in-vivo targeting system can wrap the medicine and transport the medicine in a liposome in vivo to reach a target site. And the particle size is small, the transdermal absorption is good, the action time is long, and the preparation can be applied to various products.
In the method, the stability of the aptamer part can be ensured by ensuring no RNase in the solution, and the stability of the aptamer in the solution is ensured by complementing the paired double-chain structure. By evaporating the chloroform/ethanol solution, a homogeneous lipid film can be well formed, and the purity of the sample is ensured.
In the process of the present invention, it is necessary to ensure the temperature and light during the preparation process. The single-stranded nucleic acid sequence can better ensure the biological activity under the condition of low temperature, and repeated freeze thawing is avoided.
Compared with the prior art, the invention has the advantages that:
(1) light-triggered in vivo targeting: in the invention, the photolabile nucleotide can be automatically broken under the illumination condition, so that the aptamer of the targeting part can be combined with the target gene, the liposome is delivered to a specific part, the liposome-coated medicine or RNA can be effectively and intensively released at the specific part, and the bioavailability is improved.
(2) Good transdermal absorption: the liposome used in the invention can effectively permeate the skin, and because the liposome has small particle size and is similar to and compatible with the cell membrane of an organism, the liposome can be effectively absorbed by cells and effectively deliver the encapsulated medicine or RNA into the cells. However, sunscreen cream or other skin care products on the market are not easy to absorb and have unobvious action effect.
(3) The action time is long: due to good transdermal absorption, the medicine can be effectively delivered into cells to act on the cells. Compared with surface protection of other skin care products or sunscreen creams, the skin care cream has the advantages that the acting time can be effectively prolonged, and the repeated smearing times are saved.
(4) The application range is wide: the liposome used in the invention can wrap gene drugs such as RNA and the like, and can also wrap small molecule drugs. It can be used in sunscreen cream or skin care product for promoting absorption and resisting ultraviolet injury.
Drawings
FIG. 1 is a particle size diagram of liposomes;
FIG. 2 is a Zeta potential diagram of liposomes;
FIG. 3 is a transmission electron micrograph of liposomes.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Example 1
(1) Hybridization of RNA aptamers to photolabile complementary oligonucleotides
A stock solution was prepared by adding a mitochondrial RNA aptamer (5'-UCUCCCUGAGCUUCAGG-3') and a photolabile complementary oligonucleotide (3 '-AGAGGG// ACUCGA// AGUCC-5') to a PBS (pH 7.4, 150mM NaCl) solution of RNase-free at a ratio of 1:1, and this solution was annealed and stored at 4 ℃ overnight to allow sufficient hybridization.
(2) Synthesis of liposomes and crosslinking with aptamers
Taking DOPE, SM and NBD-DOPE according to a molar ratio of 10: 3:0.5 in chloroform/ethanol solution to prepare lipid stock solution and store at-20 ℃ until use. A lipid film was formed on the bottom of the glass tube by evaporation of the chloroform/ethanol solution and dried under vacuum overnight to remove the organic solvent. The RNA aptamer mixture obtained in (1) was added to a glass tube in a ratio of 3%, 4%, 5%, 6%, and 7% to hydrate the lipid membrane, and HEPES (4-hydroxyethylpiperazineethanesulfonic acid) buffer solution having a pH of 5.7 was added to the lipid membrane, and the mixture was incubated at room temperature for 30 minutes to fully hydrate the lipids. And then ultrasonically treating the lipid membrane for 1-2 minutes by using a bath type ultrasonic instrument to enable the lipid solution to be in a semitransparent liquid state. The organic phase is evaporated in vacuo on a rotary evaporator, after 30 minutes 500. mu.L of deionized water are added and the mixture is kept under vacuum in the rotary evaporator for 30-50 minutes. The lipid particles (1mL) formed into the organic phase were evaporated and extruded through a 100nm polycarbonate film 10-20 times at room temperature.
(3) Post-treatment and purification of the complexes
The aptamer-crosslinked liposome obtained in (2) was centrifuged at 50000g for 45min in a high-speed centrifuge to remove the aptamer that was not linked.
The invention passes the verification of a laboratory, the verification process is carried out according to the steps of the specific embodiment, the particle size of the liposome is measured by a laser particle sizer, the zeta potential is measured by a zeta potential meter, and the morphology and the particle size of the liposome are observed by a transmission electron microscope to obtain a better result. Wherein the particle size of the liposome is about 100nm, the zeta potential is about 54mv, and the positive surface potential can generate electrostatic adsorption with RNA aptamer.
Sequence listing
<110> university of southern China's science
<120> preparation method of aptamer liposome
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA/RNA
<213> human (human)
<400> 1
ucucccugag cuucagg 17
<210> 2
<211> 17
<212> DNA/RNA
<213> human (human)
<400> 2
agagggacuc gaagucc 17
Claims (7)
1. A preparation method of aptamer liposome is characterized in that compounds of the following formulas [1], [2], [3], [4] and [5] are hybridized with photolabile complementary oligonucleotide through mitochondrial RNA aptamer; synthesis of liposome (DOPE + SM + NBD-DSPE) and cross-linking with aptamer; performing post-treatment and purification on the compound to prepare a light-triggered aptamer liposome drug-loading system;
the method comprises the following specific steps:
(1) dissolving the compounds of the formulas [1] and [2] in a chloroform/ethanol solution at a ratio of 1:1 of the amount of the substances, wherein the reaction needs to be realized in an RNase-free solution and on ice to obtain an RNA aptamer mixture;
(2) the reaction system is DOPE, SM and NBD-DSPE, wherein the mass ratio of DOPE, SM and NBD-DOPE is 10: 3:0.5, reacting at room temperature for 0.3-0.5 hours; the term "/in 3 '-AGAGGG// ACUCGA// AGUCC-5' means that the photolabile bond is a 1- (2-nitrophenyl) ethyl bond.
2. The method for preparing aptamer liposome according to claim 1, wherein the specific steps of the step (2) are as follows: dissolving DOPE, SM and NBD-DSPE in chloroform/ethanol solution according to the amount ratio of substances, evaporating the chloroform/ethanol solution by a rotary evaporator to form a lipid membrane at the bottom of a glass tube, adding 3-7% of RNA aptamer mixture into the lipid mixture, simultaneously adding HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) buffer solution with the pH of 5.7 into the lipid membrane, incubating for 30 minutes at room temperature to hydrate the lipid, then ultrasonically treating the lipid membrane by using a bath type ultrasonic instrument for 1-2 minutes, evaporating the organic phase on the rotary evaporator in vacuum, adding 500 mu L of deionized water after 30 minutes, keeping the mixture in vacuum in the rotary evaporator for 30-50 minutes, evaporating the lipid particles (1mL) formed into the organic phase, and extruding for 10-20 times through a 100nm polycarbonate membrane at room temperature.
3. The method for preparing aptamer liposome according to claim 1, wherein the crosslinking method is a hydration method.
4. The method for preparing aptamer liposome of claim 1, wherein the buffer system used is HEPES buffer solution, and the incubation time at room temperature is 30 minutes.
5. The method for preparing aptamer liposome according to claim 1, wherein the drug-loaded fraction (DOPE + SM + NBD-DSPE) is a commercial anti-aging drug, and the anti-aging drug is quercetin, vitamin E or clofibrate.
6. The method for preparing aptamer liposome of claim 5, wherein the molar ratio of the polymer to the drug is 1:3.2-1: 3.5.
7. The method for preparing aptamer liposome of claim 1, wherein the purification step is dissolving in deionized water, dialyzing and freeze-drying, or purifying by high speed centrifugation.
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