CN108187062A - A kind of construction method of the nano-medicament carrier based on nucleic acid - Google Patents
A kind of construction method of the nano-medicament carrier based on nucleic acid Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention discloses a kind of construction method of the nano-medicament carrier based on nucleic acid, the pharmaceutical carrier is 6 groups of column rigid structures for being mutually wound up as double helix, column hole is run through in centre for 1.8 2nm's of diameter, the both ends of carrier are blocked by lock shape single nucleic acid strands lock1 and lock3, a part of the closure sequence lock3 of one end therein is the complementary series of target aptamer mRNA, it is 40 ± 4.3nm by the entire length of carrier that dynamic light scattering measures, when there are during target aptamer mRNA in cell, since the pairing base number of target mRNA and lock3 are much larger than lock3 and the combination number of carrier basic structure port, cause entropy substitution process occurs, one end of carrier is made to be opened, the drug of carrier inside is released out.The present invention regulates and controls the pairing situation between base by engineer, nucleic acid is made to carry out spatial folding formation morphology controllable origami structure, the underlying carrier loaded as drug by the use of itself existing nucleic acid in organism as raw material.
Description
Technical field
The present invention relates to a kind of construction methods of the nano-medicament carrier based on nucleic acid.
Background technology
Nano-carrier excellent penetrates launch performance and drug can control and release as a kind of important nano material, with its
Exoergic power, has broad application prospects on biomedical sector, delays wherein one of most important direction is exactly antineoplastic target
The exploitation of drug release object.
Design medicine transport system aims to solve the problem that drug targeting sex chromosome mosaicism, plurality of chemical drugs in vitro in obtain it is good
Therapeutic effect, but the effect unobvious in human body, reason are that these drugs lack targeting and in human internal environment
Deficient in stability needs to develop special medicament transport technology to overcome as solubility is low, unstable in organism, biological utilisation
The problems such as spending low and normal tissue potential bio-toxicity, good drug delivery systems are the key that solve the problems, such as institute
Core is the design of the pharmaceutical carrier with targeting.
The key problem of nano-medicament carrier research field is mainly grouped as following three aspects at present：
1st, the bioavailability of drug how is improved.There are about 40% in the drug of mankind nowadays exploitation to be insoluble in water,
And the circulatory system in animal body is based primarily upon water phase, water-insoluble medicine is more difficult uniformly to be spread wherein, and then
Cause the reduction of bioavailability.By taking antitumor drug paclitaxel as an example, currently used method mainly uses liposome pair
It is wrapped up, and by the control to its grain size, enabling to be formed in vivo when emulsion ensures it with blood transportation has
Preferable mobility and dispersibility.
But such method still remains the problem of apparent in practical applications, although being wrapped using liposome to drug
It wraps up in, itself and cellular phospholipid bilayer compatibility can be utilized, promote cell that endocytosis occurs, improve the bioavailability of drug,
But due to that can not accomplish targeted delivery, the bio-toxicity of nontarget area biological tissue also will significantly aggravate.
2nd, how to maintain loading drug stablizes release.In clinical treatment, need to ensure that drug can be in certain time
Inherent lesions position maintains enough concentration, often poly- using poly ethylene glycol (PEG), polyethyleneimine (PEI) etc. at present
Close object, with drug molecule be covalently attached or self-assembles formed polymer protective layer, and then packaging medicine make its in vivo not by
Tachymetabolism or removing, avoid by reticuloendothelial system phagocytic, and continual and steady release, to reach sanguimotor for a long time
Purpose.
Although the package of copolymer can promote transhipment and the slow release of drug safety and stability, due to copolymer list
Bio-hormone similarity possessed by body and its degradation in vivo degree and the uncertainty of degradation cycle, lead to such side
Also there are potential hidden danger for the biological safety of method.
3rd, the targeting transport of drug how is realized.It realizes the targeting transport of drug, is to evade drug normal tissue to cause
One of effective means of cytotoxicity, main passing method is that have targeting using SPECIFIC APTAMER, antibody/antigen etc. at present
The component of recognition capability modifies nano-carrier surface, and the carrier after modification can overcome drug to meet in transmission process in vivo
Drug is sent to target position and realizes slow release, for the bioactivity of the half-life shorts such as protein by the various physiologic barriers arrived
Molecule due to the buffer action of carrier, can overcome internal physical chemistry barrier, prevent drug from being dropped before target site is reached
Solution.
Such method is convenient feasible, but due to the uncertainty of intermolecular identification of shaking hands, has phenomenon of missing the target often
Occur, and then the release in advance of loading drug is caused to cause the generation of the adverse reactions such as local necrosis of normal tissue, while such
Targeted approach tends not to the drug-resistant variants of reply target cell, and normal drug delivery cannot be made if target disappears,
Also treatment requirement when advanced tumor diffusion and specific expressed low early stage can not be better met.
Invention content
Technical problem solved by the invention is to provide a large amount of existing, biocompatibilities of institute in a kind of utilization organism
Good nucleic acid is material, by the origami structure to DNA, establishes a kind of new nano-medicament carrier platform, has both routine
The stability of the advantages of liposome compatibility is good and copolymer structure slow release and the targeting of aptamer identification
Property, while solve liposome and lack targeting, the problem of the biological safety of copolymer and single aptamer recognition reaction.
Technical problem solved by the invention is realized using following technical scheme：A kind of Nano medication based on nucleic acid carries
The construction method of body, based on the design principle of DNA origami structures, based on controlling the pairing situation between complementary base, design
Nucleic acid nano carrier structure, main body for 6 groups of column rigid structures for being mutually wound up as double helix (respectively by sequence GJ1,
GJ2, GJ3, GJ4, GJ5, GJ6, mutual spiral hybridizes to be formed 6 nucleic acid sequences two-by-two, and bonding state is as shown in Figure 2), in
Between be diameter 1.8-2nm through column hole, the both ends of carrier are blocked by lock shape single nucleic acid strands (lock1, lock3), wherein
One end closure sequence lock3 a part be target aptamer mRNA complementary series, the load measured by dynamic light scattering
The entire length of body is 40 ± 4.3nm.When in cell there are during target aptamer mRNA, due to the pairing of target mRNA and lock3
Base number causes entropy substitution process occurs, makes one end quilt of carrier much larger than lock3 and the combination number of carrier basic structure port
It opens, the drug of carrier inside is released out.Simultaneously because the slant acidity environment in tumour cell, when one end of carrier is beaten
After opening, rigid structure destroys, and whole stability reduces, and is allowed to generate pH responses, overall skeleton is scattered, and loses original knot
Structure causes the small molecule, anti-tumor drug being intercalated in double-strandednucleic acid structure to be slowly released, and reaches different mechanism medicines
The effect that object delivers jointly improves the efficiency of loading to small molecule nucleic acid interference class drug.On the surface of nano-carrier structure,
Since the nucleic acid sequence for forming structure has carried out carboxylated modification, therefore there are free carboxies, are dissociated using itself and RGD cyclic peptide surface
The condensation of amino modifies RGD cyclic peptide to body structure surface, to realize the combination of nano-carrier and target cell with promoting born of the same parents
Gulp down the progress of effect.
Beneficial effects of the present invention：
1st, using nucleic acid as raw material, using DNA paper folding technologies, a kind of new active transport carrier is created, is drug targeting
Transport provides new carrier fashion and thinking, is different from previous liposome loading technology and lacks targeting and high polymer sustained release
The bio-toxicity of carrier microballoons in Microspheres Technique.Enhance biological safety while bioavailability is improved.
2nd, by the use of itself existing nucleic acid in organism as raw material, the pairing between base is regulated and controled by engineer
Situation makes nucleic acid carry out spatial folding and forms morphology controllable origami structure, the underlying carrier loaded as drug.
3rd, by the use of cell interior specific mrna aptamer as target, and unconventional cell surface aptamer identifies, alleviates
It misses the target, cellular drug resistance variation and reduce the drug as caused by identification mistake and accidentally discharge, caused local tissue necrosis etc. is asked
Topic.
4th, the high-affinity of nano-carrier and cell is realized by the affine cyclic peptide modification of the biology on surface, is improving cell profit
While with rate, it is more prone to control the grain size of finished product relative to liposome, in favor of the medicament transport in organism.
5th, carrier dimensions control is accurately carried out, drug is enable to enter organ and tissue through the gap on capillary,
Therapeutic effect is improved in target site and reduces the toxic side effect of other tissues or organ in vivo.
6th, it is loaded relative to traditional capsule-type, this case is permeated using high concentration, embedding with the nucleic acid of small molecule anticancer drug
Mechanism is inserted, content is realized and loads the double loading effect loaded with structure intercalation, effectively raise carrier medicine carrying efficiency.
7th, inside carrier structure there is uniform duct, can realize a variety of mixing dresses for meeting aperture size drug
The microporous structure that polydispersity size in load rather than previous pharmaceutical carrier differs, is more suitable in clinical treatment instantly
The application of cocktail therapy.
8th, 6 strands of double helix rigid structures can effectively reduce to the degradation of carrier in organism, while have pH
The ability of response, after the inside tumor for entering slant acidity, under the premise of there are target mRNA, one end opens and then bone occurs
Frame disintegrates, and slowly releases institute's intercalation small-molecule drug therein.
Description of the drawings
Fig. 1 is the overall structure figure of nano-carrier
Fig. 2 is that the single-chain nucleic acid of nano-carrier be combined with each other situation schematic diagram
Fig. 3 is the proof diagram for carrying drug carrier transport effect
Fig. 4 is the ultraviolet phenogram for carrying drug carrier surface RGD modifications
Fig. 5 is the Fluorescent Characterization figure of two kinds of drug collaboration release
Fig. 6 is carries drug carrier dynamic light scattering phenogram.
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Conjunction is specifically illustrating, and the present invention is further explained.
Embodiment：To load drug Fluorescent paclitaxel and adriamycin example；
(1) structure of carrier is carrier structure
1st, take respectively a concentration of 10 μM of ol/L of 10 μ L 7 kinds of single nucleic acid strands (GJ1, GJ2, GJ3, GJ4, GJ5, GJ6,
LOCK3 it) in small centrifuge tube, is placed in 95 DEG C of water-bath and heated 4 minutes with blending instrument mixing, then taken out, slow cooling
Carrier is carrier structure is can obtain after to room temperature, is stored in 4 DEG C of environment for use.
(2) modification of body structure surface RGD ring type polypeptides
1st, the single nucleic acid strands (MB) for taking a concentration of 10 μm of ol/L of 50 μ L amido modified then add in 100 μ L in centrifuge tube
The NHS solution of a concentration of 0.2mol/L, it is for use with blending instrument mixing after reacting 30 minutes at room temperature.
2nd, taking the magnetic bead that 10 μ L surface carboxyl groups are modified, the EDC for adding in a concentration of 0.8 mol/L of 100 μ L is molten in centrifuge tube
Liquid, it is for use with blending instrument mixing after reacting 30 minutes at room temperature.
3rd, the 100 above-mentioned solution after reaction of μ L is taken to be placed in the environment of 37 DEG C of shaking table, be protected from light in centrifuge tube respectively
It takes out after oscillating reactions 10h, is washed three times using the Tris-Hcl buffer solutions Magneto separate of pH=7.2, discard supernatant reservation
Magnetic bead is added in after 100 μ LTris-Hcl buffer solutions blending instrument mixing for use.
4th, the carrier is carrier structure obtained in 50 μ L step (1) is taken in centrifuge tube, and it is a concentration of then to add in 100 μ L
The EDC solution of 0.8mol/L, it is for use with blending instrument mixing after reacting 30 minutes at room temperature.
5th, the RGD cyclic peptide solution of a concentration of 25 μm of ol/L of 50 μ L is taken in centrifuge tube, adds in 100 a concentration of 0.2mol/ of μ L
The NHS solution of L, it is for use with blending instrument mixing after reacting 30 minutes at room temperature.
6th, the 50 above-mentioned solution after reaction of μ L is taken to be placed in the environment of 37 DEG C of shaking table, be protected from light in centrifuge tube respectively
It is taken out after oscillating reactions 10h, the magnetic of nucleic acid chains is combined with the surface obtained in step before 100 μ L are added in after blending instrument mixing
Pearl is taken out after being reacted 3 hours in 37 DEG C of shaking table, using the Tris-Hcl buffer solutions of pH=7.2 in 15 DEG C of water-bath ring
In border, Magneto separate washs three times, discards supernatant and retains magnetic bead, adds in 100 μ LTris-Hcl buffer solutions blending instrument mixings
It is for use afterwards.
7th, above-mentioned solution after reaction is heated after five minutes in 60 DEG C of water-baths, the Magneto separate in 50 DEG C of water-bath
Three times, retain supernatant in new centrifuge tube for use to get the nano-carrier structure modified to surface RGD ring type polypeptides.
(3) drug loads
1st, take what is obtained in 50 μ L step (2), surface GD ring type polypeptides modification nano-carrier structure in centrifuge tube,
The Doxorubicin solution of a concentration of 500 μM of 20 μ L and the paclitaxel solution of a concentration of 500 μM of fluorophors modifications of 20 μ L are sequentially added,
The loading for completing drug for use is taken out after oscillating reactions being protected from light in 37 DEG C of shaking table 12 hours.
(4) carrier end is blocked and is purified
1st, be separately added into the solution obtained into step (3) a concentration of 10 μm of ol/L single nucleic acid strands (LOCK1) of 50 μ L,
A concentration of 10 μm of ol/L single nucleic acid strands (LOCK2) of 30 μ L add the surface knot obtained in 50 μ L step (2) in centrifuge tube
Closing has the magnetic bead of nucleic acid chains, is placed in shaking table, is taken out after oscillating reactions being protected from light under conditions of 37 DEG C 3 hours.
2nd, the solution that will be obtained in previous step adds in the Tris-Hcl buffer solutions of pH=7.2 in 15 DEG C of water bath
In, Magneto separate washs three times, discards supernatant and retains magnetic absorption part, adds in 100 μ LTris-Hcl buffer solution mixings
It is for use after instrument mixing.
3rd, above-mentioned solution after reaction is heated after five minutes in 60 DEG C of water-baths, the Magneto separate in 50 DEG C of water-bath
Three times, retain supernatant in new centrifuge tube for use to get the closure of carrier can be completed, obtain from liquid after separating-purifying
Load drug carrier.
(5) pharmaceutical carrier transfection histocyte；
1st, the nano drug-carrying carrier 30 as one kind μ L for having loaded drug are taken in centrifuge tube, add in 1640 cultures containing serum 20%
Liquid 1ml is added in Tissue Culture Dish carry out cell culture after mixing, in 37 DEG C of incubator, 5% carbon dioxide
Under the conditions of, it is mixed 3 hours.
(6) drug release and detection；
1st, the culture dish after being cultivated 3 hours in step (5) is taken out, discards culture solution therein, adds in 1640 fresh trainings
After flushing three times repeatedly, it is for use to add in the culture solution that 1ml contains 20% serum for nutrient solution.
2nd, by culture dish move under laser confocal microscope observe tumour cell in fluorescence distribution situation, using 495nm as
Exciting light collects the transmitting light of 525nm and 585nm respectively, it is observed that its distribution and the mechanism of action one of antitumor drug
It causes, the fluorescent red-orange of adriamycin is assembled in nuclear area, and the green fluorescence of Fluorescent paclitaxel is assembled in cytoplasm, characterizes it
Successful release in cell is antitumor functional with drug.
3rd, the culture dish in previous step with the PBS buffer solution of pH=7.0 is washed 3 times repeatedly, then uses ultrasonic wave
Crush instrument, will be centrifuged after cell pulverization therein, take its supernatant, repeatedly for three times after, take 100 μ L supernatants in micro ratio
In color ware, in sepectrophotofluorometer, using 495nm as excitation wavelength, a situation arises for the fluorescence of scanning 500-700nm ranges,
It is observed that 525nm and two emission peaks of 585nm, correspond to loaded drug Fluorescent paclitaxel and adriamycin, into one respectively
Step determines its successful release in cell.
As shown in Figure 1, the FITC-PTX and DOX shown in it is respectively Fluorescent paclitaxel and two kinds of antineoplastics of adriamycin
Object, RGD be surface modification cyclic peptide, 6 single nucleic acid strands into cylindric skeleton structure, specific combination be Fig. 2
Shown in, LOCK1 is the closure single nucleic acid strands 2 that sequence is identified with target mRNA to block single nucleic acid strands 1, LOCK3.
As shown in Fig. 2, in figure 1,2dock, 3,4,5dock, 6, respectively GJ1, GJ2 in corresponding sequence table, GJ3,
GJ4, GJ5, GJ6, it is each single-stranded in the form of mutually handing in hand and the single-stranded complementary pairing by between base of surrounding forms single-screw knot
Structure forms the main body column hollow structure of nano-carrier.
The nucleic acid sequence of wherein GJ1, GJ2, GJ3, GJ4, GJ5, GJ6, lock1, lock2, lock3 and MB are as follows：
As shown in figure 3, wherein bright is the cell state figure shown in microscope light field, PTX-FITC is fluorescence Japanese yew
Image in alcohol channel, DOX are the image in adriamycin channel, and DAPI is for core transfection reagent confirming nucleus institute
In position, Merge is the superimposed image of above-mentioned channel, visible since adriamycin acts on its distribution of nucleus and DAPI mono- in figure
Directly, therefore after being superimposed in pink colour and Fluorescent paclitaxel is distributed in cytoplasm, act on tubulin, therefore the cytoplasm of surrounding is in green
Color fluorescence.
As shown in figure 4, a in figure, b, tri- curves of c correspond to carrier structure, simple RGD cyclic peptide, have modified RGD rings respectively
The carrier structure of peptide, as seen from the figure, nucleic acid have the UV absorption of feature under 280nm, and RGD cyclic peptide has apparent absorption 215
Peak, after nucleic acid surface modification RGD cyclic peptide, ultraviolet spectra takes into account the two absorption peak and whole blue shift occurs.
As shown in figure 5, a is blank group in figure, b is experimental group, and experimental group is attached with 585nm near 525nm as seen from the figure
Closely there is more strong characteristic emission peak, it is consistent with the launch wavelength of Fluorescent paclitaxel and adriamycin, it was demonstrated that drug is released
It puts and works well.
Shown in Fig. 6, A is the dynamic light scattering phenogram of nano-carrier basic structure in figure, and B is surface modification RGD rings
The carrier dynamic light scattering phenogram of peptide, shown in figure, before RGD cyclic peptide is modified, the particle diameter distribution of carrier is 20nm or so,
The particle diameter distribution for having modified carrier after RGD cyclic peptide becomes 40nm or so, meets the expected design of system, this grain size being capable of organism
The demand of interior diffusive transport.
Basic principle of the invention and main feature and advantages of the present invention, the technology of the industry has been shown and described above
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention, the claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table
<110>Qingdao University of Science and Technology
<120>A kind of construction method of the nano-medicament carrier based on nucleic acid
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Claims (2)
1. a kind of construction method of the nano-medicament carrier based on nucleic acid, it is characterized in that：The pharmaceutical carrier is mutually wound up as 6 groups
Column hole is run through in the column rigid structure of double helix, centre for diameter 1.8-2nm's, and the both ends of carrier are by lock shape single nucleic acid strands
Lock1 and lock3 is blocked, and a part of the closure sequence lock3 of one end therein is the complementary series of target aptamer mRNA, is led to
The entire length of the carrier that dynamic light scattering measures is crossed as 40 ± 4.3nm, when in cell there are during target aptamer mRNA, due to target
The pairing base number of mRNA and lock3 is marked much larger than lock3 and the combination number of carrier basic structure port, causes entropy substitution occurs
Process makes one end of carrier be opened, and the drug of carrier inside is released out.
2. a kind of construction method of nano-medicament carrier based on nucleic acid according to claim 1, it is characterized in that：6 groups of institutes
The double helix stated is respectively GJ1, GJ2, GJ3, GJ4, GJ5, GJ6,6 nucleic acid sequences.
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