CN110368398A - Delivering microRNA nano-carrier and preparation method based on zeolite imidazole skeleton - Google Patents
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Abstract
Step: microRNA (1) is dissolved in sterile no enzyme water, obtains microRNA aqueous solution by the invention discloses a kind of delivering microRNA nano-carrier and preparation method based on zeolite imidazole skeleton；(2) microRNA aqueous solution is mixed with ZIF-8 nanoparticle, obtains the delivering microRNA nano-carrier based on zeolite imidazole skeleton；Method of the invention is simple, and load efficiency is high, simply, at low cost.The delivering microRNA nano-carrier based on zeolite imidazole skeleton in the present invention can effectively protect microRNA from degradation.ZIF-8 can be cracked under weak acid environment, to accelerate quick release of the microRNA at tumour cell, enhance the therapeutic effect of drug.Delivering microRNA nano-carrier good biocompatibility prepared by the present invention based on zeolite imidazole skeleton.
Description
Technical field
The invention belongs to field of biomedical materials, are related to microRNA nanometers of a kind of delivering based on zeolite imidazole skeleton
Carrier and preparation method.
Background technique
Cancer threatens human health, and the main means for the treatment of of cancer at present have: operation, radiotherapy, chemotherapy and exempts from
Epidemic disease therapy etc..Wherein, chemotherapy is main treatment means, but chemotherapy has many toxic side effects, if targeting is poor, medicine
The defects such as object utilization rate is low.
Therefore it needs to develop nano-carrier, the use of the main purpose that nano-carrier is used for drug delivery is to improve medicine for power
Learn and change the bio distribution of drug.To enhance therapeutic efficiency and reduce pair by preferentially accumulating drug in illing tissue
Effect.The nano-carrier has silica, quantum dot, the inorganic nano materials such as metal nanoparticle and organic micella, lipid
Body, the organic nano materials such as dendritic macromole[1].Wherein, ZIF-8 nanoparticle is before kind especially has in zeolite imidazole skeleton
The pharmaceutical carrier of scape is to be coordinated to be formed by the nitrogen-atoms on zinc ion and imidazole ring, under mildly acidic conditions on imidazole ring
Nitrogen-atoms easily protonates and causes the cracking of ZIF-8.It is based on this characteristic, ZIF-8 is widely used in drug delivery nanometer load
Body[2]。
MicroRNA is a kind of highly conserved non-coding endogenous RNA molecule, and length is about 19-23nt, Neng Goutong
It crosses and is incorporated into the end of target gene 3 ' noncoding region downstream, to regulate and control the transcription of target gene, and then influence the biological scholarship and moral conduct of cell
For.Nearest calculating estimation shows that each microRNA adjusts about 200 said target mrnas, it means that more than the egg of one third
White matter encoding gene is controlled by microRNA.Abnormal miroRNA expression is related with tumour generation, and some microRNA can be used as
Real tumor suppressor (microRNA mimics as therapeutics), such as miR-34a, miR-143 and miR-
145 etc.；And other microRNA are then used as oncogene (anti-miRs as therapeutic agents), such as miR-
10b, miR-155 and miR-221 etc.[3].Therefore, microRNA can be used as the potential drug for the treatment of cancer.
In conjunction with the main means of current treating cancer, microRNA nanometers of a kind of delivering based on zeolite imidazole skeleton are developed
Carrier and preparation method and application are necessary.
Bibliography
1.Peer,D.and R.langer,Nanocarriers as an emerging platform for cancer
therapy.nature nanotechnology,2007.2:p.751-760.
2.Wang,H.,etal.,DNAzyme-Loading MOFs for Self-Sufficient Gene
Therapy.Angew Chem Int Ed Engl,2019.
3.Rupaimoole,R.and F.J.Slack,MicroRNA therapeutics:towards a new era
for the management of cancer and other diseases.Nat Rev Drug Discov,2017.16
(3):p.203-222.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of delivering based on zeolite imidazole skeleton
MicroRNA nano-carrier.
The delivering microRNA nano-carrier based on zeolite imidazole skeleton that a second object of the present invention is to provide a kind of
Preparation method.
Technical solution of the present invention is summarized as follows:
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) microRNA is dissolved in sterile no enzyme water, obtains microRNA aqueous solution；
(2) it is carried out with one of following three kinds of modes:
Mode one: the microRNA aqueous solution is mixed with ZIF-8 nanoparticle, makes microRNA and ZIF-8 nanometers
The mass ratio of particle is 1:10-100, obtains the delivering microRNA nano-carrier based on zeolite imidazole skeleton；
Mode two: by the microRNA aqueous solution, zinc nitrate hexahydrate, 2-methylimidazole and solvent, 5- is mixed
20min, the microRNA, zinc nitrate hexahydrate, 2-methylimidazole and solvent ratio be 0.01g:1-5g:3-15g:10-
30mL；Obtain the delivering microRNA nano-carrier based on zeolite imidazole skeleton；
Mode three: by the microRNA aqueous solution, zinc nitrate hexahydrate, imidazoles and solvent, mixing 5-20min, described
MicroRNA, zinc nitrate hexahydrate, imidazoles and solvent ratio be 0.01g:1-5g:2.4-12g:10-30mL；It obtains based on boiling
The delivering microRNA nano-carrier of stone imidazole skeleton；
MicroRNA is miR-34a or miR-122.
Solvent is preferably at least one of water, methanol, ethyl alcohol, isopropanol and N,N-dimethylformamide.
The delivering microRNA nano-carrier based on zeolite imidazole skeleton of above method preparation.
Advantages of the present invention:
(1) microRNA carrying method of the invention is simple, and load efficiency is high, simply, at low cost.
(2) the delivering microRNA nano-carrier based on zeolite imidazole skeleton in the present invention can effectively protect microRNA
From degradation.
(2) in the present invention, ZIF-8 can crack release under weak acid environment, to accelerate microRNA thin in tumour
Quick release at born of the same parents enhances the therapeutic effect of drug.
(3) the delivering microRNA nano-carrier good biocompatibility prepared by the present invention based on zeolite imidazole skeleton, tool
There is extensive biomedical applications prospect.
Detailed description of the invention
Figure 1A is the saturating of the delivering microRNA nano-carrier based on zeolite imidazole skeleton of embodiment 1 (absorption method) preparation
Penetrate electron microscope.
Figure 1B is the saturating of the delivering microRNA nano-carrier based on zeolite imidazole skeleton of embodiment 4 (one kettle way) preparation
Penetrate electron microscope.
Fig. 2 is the cytotoxicity knot of the delivering microRNA nano-carrier based on zeolite imidazole skeleton prepared by embodiment 1
Fruit figure.
Fig. 3 is the delivering microRNA nano-carrier effective protection based on zeolite imidazole skeleton prepared by embodiment 1
MicroRNA schemes from degradation.
Fig. 4 is the delivering microRNA nano-carrier effective protection based on zeolite imidazole skeleton prepared by embodiment 4
MicroRNA schemes from degradation.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
MicroRNA used in the present invention is for miR-34a or miR-122, is to enable those skilled in the art
The present invention is better understood, but is not limited the scope of the invention.
MicroRNA abbreviation miRNA.
MiR-34a and miR-122 is commercially available.
By Zn (NO3)2·6H2O is mixed with 2-methylimidazole with the molar ratio of 1:4, is stirred at room temperature, centrifuge separation, with water,
At least one of methanol, ethyl alcohol sufficiently wash and drying, obtains ZIF-8 nanoparticle.
Embodiment 1
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-34a is dissolved in sterile no enzyme water (sterile no enzyme is appropriate, dissolves miR-34a), obtains miR-34a water
Solution；
(2) the miR-34a aqueous solution is mixed with ZIF-8 nanoparticle, makes miR-34a and ZIF-8 nanoparticle
Mass ratio is 1:10, obtains the delivering microRNA nano-carrier based on zeolite imidazole skeleton, abbreviation ZIF-8/miR-34a.See
Figure 1A transmission electron microscope picture.
Embodiment 2
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-34a is dissolved in sterile no enzyme water (sterile no enzyme is appropriate, dissolves miR-34a), obtains miR-34a water
Solution；
(2) the miR-34a aqueous solution is mixed with ZIF-8 nanoparticle, makes miR-34a and ZIF-8 nanoparticle
Mass ratio is 1:55, obtains the delivering microRNA nano-carrier based on zeolite imidazole skeleton.
Embodiment 3
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-122 is dissolved in sterile no enzyme water (sterile no enzyme is appropriate, dissolves miR-122), obtains miR-122 water
Solution；
(2) the miR-122 aqueous solution is mixed with ZIF-8 nanoparticle, makes miR-122 and ZIF-8 nanoparticle
Mass ratio is 1:100, obtains the delivering microRNA nano-carrier based on zeolite imidazole skeleton.
Embodiment 2 and embodiment 3 delivering microRNA nano-carrier transmission electron microscope picture and reality based on zeolite imidazole skeleton
It is similar to apply delivering microRNA nano-carrier of the example 1 based on zeolite imidazole skeleton.
Embodiment 4
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-34a is dissolved in sterile no enzyme water, obtains miR-34a aqueous solution；
(2) by the miR-34a aqueous solution, zinc nitrate hexahydrate, 2-methylimidazole and water, 10min, the miR- are mixed
34a, zinc nitrate hexahydrate, 2-methylimidazole and water ratio be 0.01g:3g:9g:20mL；It obtains based on zeolite imidazole skeleton
Delivering microRNA nano-carrier, see Figure 1B transmission electron microscope picture.
Embodiment 4 protects microRNA to see Fig. 4 from degradation (method is shown in embodiment 11).
Embodiment 5
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-34a is dissolved in sterile no enzyme water, obtains miR-34a aqueous solution；
(2) by the miR-34a aqueous solution, zinc nitrate hexahydrate, 2-methylimidazole and n,N-Dimethylformamide, mixing
5min, the miR-34a, zinc nitrate hexahydrate, 2-methylimidazole and n,N-Dimethylformamide ratio be 0.01g:1g:
3g:10mL；Obtain the delivering microRNA nano-carrier based on zeolite imidazole skeleton.
Embodiment 6
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-122 is dissolved in sterile no enzyme water, obtains miR-122 aqueous solution；
(2) by the miR-122 aqueous solution, zinc nitrate hexahydrate, 2-methylimidazole and solvent, 20min is mixed, it is described
MiR-122, zinc nitrate hexahydrate, 2-methylimidazole and solvent ratio be 0.01g:5g:15g:30mL；It obtains based on zeolite miaow
The delivering microRNA nano-carrier of azoles skeleton.Wherein solvent is the mixing of methanol, ethyl alcohol and isopropanol that volume ratio is 1:1:1
Object.
Embodiment 5 and embodiment 6 delivering microRNA nano-carrier transmission electron microscope picture and reality based on zeolite imidazole skeleton
It is similar to apply delivering microRNA nano-carrier of the example 4 based on zeolite imidazole skeleton.
Embodiment 7
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-34a is dissolved in sterile no enzyme water, obtains miR-34a aqueous solution；
(2) by the miR-34a aqueous solution, zinc nitrate hexahydrate, imidazoles and water, 10min, the miR-34a, six are mixed
The ratio of nitric hydrate zinc, imidazoles and water is 0.01g:3g:7.2g:20mL；Obtain the delivering based on zeolite imidazole skeleton
MicroRNA nano-carrier.
Embodiment 8
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-34a is dissolved in sterile no enzyme water, obtains miR-34a aqueous solution；
(2) by the miR-34a aqueous solution, zinc nitrate hexahydrate, imidazoles and n,N-Dimethylformamide, 5min is mixed,
The miR-34a, zinc nitrate hexahydrate, imidazoles and N,N-dimethylformamide ratio be 0.01g:1g:2.4g:10mL；?
To the delivering microRNA nano-carrier based on zeolite imidazole skeleton.
Embodiment 9
A kind of preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, includes the following steps:
(1) miR-122 is dissolved in sterile no enzyme water, obtains miR-122 aqueous solution；
(2) by the miR-122 aqueous solution, zinc nitrate hexahydrate, imidazoles and solvent, 20min is mixed, the miR-122,
The ratio of zinc nitrate hexahydrate, imidazoles and solvent is 0.01g:5g:12g:30mL；Obtain the delivering based on zeolite imidazole skeleton
MicroRNA nano-carrier.Wherein solvent is the mixture of methanol, ethyl alcohol and isopropanol that volume ratio is 1:1:1.
Embodiment 10
The cytotoxicity of delivering microRNA nano-carrier based on zeolite imidazole skeleton detects: including the following steps:
(1) ZIF-8 nanoparticle (abbreviation ZIF-8) is dissolved in ultrapure water, is configured to the solution that concentration is 5 μ g/ μ L, and will
It carries out ultraviolet lamp sterilization treatment；It is 5.5 μ g/ μ L that ZIF-8/miR-34a prepared by embodiment 1, which is dissolved in ultrapure water to be made into concentration,
Solution.
(2) MDA-MB-231 cell (Shanghai Inst. of Life Science, CAS) is cultivated, when the cell pastes
When wall grows into 70%-80%, cell is digested and is washed, and rejoins the 1640 culture base weights containing 10% serum
Outstanding cell.Then the cell suspension of 90 μ L is added in the partial hole of 96 orifice plates, contains each hole about 6000-9000 thin
Then cell is put into 5%CO by born of the same parents2, it is incubated overnight in 37 DEG C of cell incubators, keeps cell completely adherent.
Both (3) ZIF-8 solution and ZIF-8/miR-34a solution are diluted to different concentration with ultrapure water respectively, make
The content of middle ZIF-8 is equally, the ZIF-8 solution and ZIF-8/ of the various concentration of 10 μ L to be separately added into the every hole for having cell
MiR-34a solution is as experimental group.Into the other hole for having cell, the ultrapure water of 10 μ L is added as a control group in every hole.
1640 culture mediums that 90 μ L contain 10% serum are added into the hole of not cell, are separately added into corresponding 10 μ L's
ZIF-8 solution, the ZIF-8/miR-34a solution of various concentration, as blank control group.
Experimental group with control group is parallel does 5 multiple holes, blank control group does 1 hole；It is put into 5%CO2, 37 DEG C of incubators
In continue cultivate 48h.
(3) cell is cleaned twice with PBS (pH=7.4).1640 culture mediums containing 10% serum of 90 μ L are added in every hole
With 10 μ L, the MTT aqueous solution of 5 μ g/ μ L continues in 5%CO2, 4h is cultivated in 37 DEG C of incubators.
(4) it discards supernatant, the dimethyl sulfoxide of 110 μ L, jog 10min is added in every hole.Finally with microplate reader in 490nm wave
Strong point surveys each solution absorbance OD value.
The survival rate V of cell of the cell under different materials is calculated according to the following formula:
V=(A-A0)/(AC-A0) * 100%
Wherein, V is the survival rate of cell；A is the OD value of experimental group；A0It is the OD value of blank control group；ACIt is control group
OD value.
Cytotoxicity experiment result is as shown in Figure 2.Available according to fig. 2, the cytotoxicity of ZIF-8 is very small.ZIF-
The toxicity of 8/miR-34a is larger under a certain concentration, shows that ZIF-8/miR-34a has certain killing to MDA-MB-231 cell
Effect.
Embodiment 11
Delivering microRNA nano-carrier based on zeolite imidazole skeleton protects microRNA from degradation.Including walking as follows
It is rapid:
Experimental group: the ratio for being 4:1 by volume ratio, ZIF-8/miR-34a and fetal calf serum prepared by embodiment 1
(FBS) it mixes；
Control group: the ratio for being 4:1 by volume ratio mixes miR-34a and fetal calf serum (FBS)；
It is respectively placed in 37 DEG C of baking oven: 1h, 3h and 5h.The finally PBS (pH=5.5) of addition equivalent, ten minutes
Afterwards, ZIF-8 is made sufficiently to crack release miR-34a.Then glue is run under conditions of 120V, 90min, 20% PAGE, sees Fig. 3.
MiR-34a is in serum with the extension of time, by gradually degrading；ZIF-8/miR-34a in serum with when
Between extension, only small part is degraded.Show that the delivering microRNA nano-carrier based on zeolite imidazole skeleton can be protected
The microRNA loaded is from degradation.
It is demonstrated experimentally that the delivering microRNA nano-carrier based on zeolite imidazole skeleton prepared by embodiment 2 and embodiment 3
Cytotoxicity is similar to Example 1；Protect microRNA similar to Example 1 from degrading.
Experiments have shown that: the delivering microRNA nano-carrier based on zeolite imidazole skeleton prepared by embodiment 5 and embodiment 6
Protect microRNA similar to Example 4 from degrading.
Claims (4)
1. the preparation method of the delivering microRNA nano-carrier based on zeolite imidazole skeleton, it is characterised in that including walking as follows
It is rapid:
(1) microRNA is dissolved in sterile no enzyme water, obtains microRNA aqueous solution；
(2) it is carried out with one of following three kinds of modes:
Mode one: the microRNA aqueous solution is mixed with ZIF-8 nanoparticle, makes microRNA and ZIF-8 nanoparticle
Mass ratio be 1:10-100, obtain the delivering microRNA nano-carrier based on zeolite imidazole skeleton；
Mode two: by the microRNA aqueous solution, zinc nitrate hexahydrate, 2-methylimidazole and solvent, 5-20min, institute are mixed
The ratio for stating microRNA, zinc nitrate hexahydrate, 2-methylimidazole and solvent is 0.01g:1-5g:3-15g:10-30mL；It obtains
Delivering microRNA nano-carrier based on zeolite imidazole skeleton；
Mode three: by the microRNA aqueous solution, zinc nitrate hexahydrate, imidazoles and solvent, mixing 5-20min, described
MicroRNA, zinc nitrate hexahydrate, imidazoles and solvent ratio be 0.01g:1-5g:2.4-12g:10-30mL；It obtains based on boiling
The delivering microRNA nano-carrier of stone imidazole skeleton.
2. according to the method described in claim 1, it is characterized in that microRNA is miR-34a or miR-122.
3. according to the method described in claim 1, it is characterized in that solvent is water, methanol, ethyl alcohol, isopropanol and N, N- dimethyl
At least one of formamide.
4. the delivering microRNA nano-carrier based on zeolite imidazole skeleton prepared by the method for one of claims 1 to 3.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112957343A (en) * | 2021-02-10 | 2021-06-15 | 浙江工业大学 | Protein @ ZIF-8N nano material and preparation and application thereof |
| CN113750246A (en) * | 2020-06-04 | 2021-12-07 | 华南理工大学 | Application of ZIF-8 nano material in degradation of broad-spectrum mutation p53 protein |
| CN114533953A (en) * | 2022-01-24 | 2022-05-27 | 华南理工大学 | Nanoparticle composite hydrogel nerve conduit and preparation method thereof |
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2019
- 2019-07-23 CN CN201910668049.7A patent/CN110368398A/en active Pending
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| QING-SHAN PAN等: "In Situ Synthesis of Ultrathin ZIF‑8 Film-Coated MSNs for Codelivering Bcl 2 siRNA and Doxorubicin to Enhance Chemotherapeutic Efficacy in Drug-Resistant Cancer Cells", 《ACS APPL. MATER. INTERFACES》 * |
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| CN113750246A (en) * | 2020-06-04 | 2021-12-07 | 华南理工大学 | Application of ZIF-8 nano material in degradation of broad-spectrum mutation p53 protein |
| WO2021243925A1 (en) * | 2020-06-04 | 2021-12-09 | 华南理工大学 | Application of zif-8 nano material in degradation of broad-spectrum mutant p53 protein |
| CN113750246B (en) * | 2020-06-04 | 2023-12-05 | 华南理工大学 | Application of ZIF-8 nanomaterial in degradation of broad-spectrum mutant p53 protein |
| CN112957343A (en) * | 2021-02-10 | 2021-06-15 | 浙江工业大学 | Protein @ ZIF-8N nano material and preparation and application thereof |
| CN114533953A (en) * | 2022-01-24 | 2022-05-27 | 华南理工大学 | Nanoparticle composite hydrogel nerve conduit and preparation method thereof |
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Application publication date: 20191025 |