CN107432935A - A kind of cationic polymer/TDNs carries medicine compound and preparation method thereof - Google Patents
A kind of cationic polymer/TDNs carries medicine compound and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of cationic polymer/TDNs to carry medicine compound, and its preparation method comprises the following steps：PEI solution and TDNs solution are mixed according to N/P=0.2 7.5 ratio, are incubated 15 30min at room temperature, is made.This method is mixed the PEI solution of certain concentration with the TDNs of certain concentration according to specific N/P, its gained carries the biocompatibility that medicine compound can effectively improve cation, it can also avoid TDNs from being degraded by DNA enzymatic simultaneously, promote it to enter intracellular, so as to play the purpose for effectively carrying medicine.
Description
Technical field
The invention belongs to DNA drug delivery technologies field, and in particular to a kind of cationic polymer/TDNs carry medicine compound and its
Preparation method.
Background technology
In recent years, the excellent processing characteristics of nucleic acid and biocompatibility are had benefited from, a variety of DNA nanostructures are designed to use
In every field such as load medicine, bioprobes.However, due to the biochemical characteristic of nucleic acid itself, it exists by DNA enzymatic in body circulation
The problems such as degraded, kidney are removed, so as to limit its application in vivo.In addition, although some particular designs in recent years
DNA structure (such as DNA tetrahedrons, TDNs) can enter born of the same parents to a certain extent and enter core, but due to the negative electrical charge property of nucleic acid,
DNA is difficult a large amount of independently through lipid bilayer hence into cell.Therefore, specifically using upper, more using various sides
Method auxiliary nucleic acid enters or transfectional cell.Common method has cationic-liposome, virus, electroporation etc., and its middle-jiao yang, function of the spleen and stomach from
Sub- liposome obtains a wide range of applications because its transfection efficiency is high, speed is fast.Polyethyleneimine (PEI) is a kind of cation
Polymer, it can be combined by electrostatic force with nucleic acid, weaken internal non-specific purge mechanism by wrapping up DNA for taking
Influence with nucleic acid, combined using the positive charge on its surface with cell membrane, hence into cell.Although PEI is with the obvious advantage, application
Extensively, but PEI as cationic polymer under finite concentration it is obvious to cytotoxicity, be also not suitable for internal extensively should
With.
The content of the invention
For the above-mentioned problems in the prior art, it is compound that the present invention provides a kind of cationic polymer/TDNs load medicines
Thing and preparation method thereof, can effectively solve in the prior art that TDNs is easily degraded by DNA enzymatic, and it is few that simple TDNs enters born of the same parents' amount, PEI poison
The problem of property is high.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
A kind of cationic polymer/TDNs carries medicine compound, and its preparation method comprises the following steps：By PEI solution with
TDNs solution mixes according to N/P=0.2-7.5 ratio, is incubated 15-30min at room temperature, is made.
Further, PEI solution and TDNs solution mix according to N/P=1 ratio.
Further, PEI solution concentrations are 1mg/mL.
Further, TDNs solution concentrations are 1 μm of ol/L.
Further, TDNs is prepared by the following method to obtain：
(1) ddH is used respectively by four, DNA tetrahedrons are single-stranded2O dissolves, and it is 1nmol/ μ L to make its concentration, then by ultraviolet fixed
Amount method, measure DNA are the light absorption value at 260nm and 330nm in wavelength, then calculate 100 μ L, 1 μM of body according to below equation
Each single-stranded volume in system：
V=100/ [(A260-A330) × 105/ (15.2 × single-stranded middle adenine number+7.4 × single-stranded middle cytimidine
The number of the number+8.3 of number+11.4 × single-stranded middle guanine × single-stranded middle thymidine)]
(2) according to the result calculated in step (1), four single-stranded solution of DNA tetrahedrons of dissolving are drawn, then with TM
Buffer is mixed, and it is 100 μ L to make its cumulative volume, and whirlpool vibration mixes, is finally placed in PCR instrument, temperature is rapidly brought up into 95 DEG C
Stable 10min, 4 DEG C of stable 20min are cooled to, most after -20 DEG C of preservations.
Further, TM buffer are 5-10mM Tris-HCl, 5-50mM MgCl in step (3)2, pH8.0.
Further, TM buffer are 10mM Tris-HCl, 50mM MgCl in step (3)2, pH8.0.
Tetrahedral four chains of DNA：
S1：
5’-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGA
CGAACATTCCTAAGTCTGAA-3’(SEQ ID NO:1)；
S2：
5’-ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGA
TTCAGACTTAGGAATGTTCG-3’(SEQ ID NO:2)；
S3：
5’-ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGA
CGGGAAGAGCATGCCCATCC-3’(SEQ ID NO:3)；
S4：
5’-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGA
GGATGGGCATGCTCTTCCCG-3’(SEQ ID NO:4)。
A kind of cationic polymer provided by the invention/TDNs carries medicine compound and preparation method thereof, has beneficial below
Effect：
TDNs produced by the present invention, Stability Analysis of Structures, it can individually be combined by cell membrane, PEI by electrostatic force with TDNs,
Weaken internal non-specific purge mechanism by wrapping up TDNs for carrying TDNs influence, using its surface positive charge with
Cell membrane combines, and hence into cell, although PEI can protect common nucleic acid to enter cell, admission velocity is slower, and also
It is to have moiety complex to be blocked in extracellular, and the present invention is interacted by PEI and obtained TDNs, can be effectively improved
Compound enters the speed and percent of pass of cell, and into after cell, PEI can also promote its bag by its " proton sponge " effect
Wrap up in TDNs escape lysosomes.
It can cause too high cytotoxicity when PEI dosages are larger, not have protective effect when too low again, therefore, screening
One suitable PEI and TDNs ratio can control PEI toxicity, maintain translocations of the PEI played in drug-loading system and
TDNs is protected not dissolved by lysosome.
The present invention is mixed the PEI solution of certain concentration with the TDNs of certain concentration according to specific N/P, its gained
Carry medicine compound and can effectively improve the biocompatibility of cation, while can also avoid TDNs from being degraded by DNA enzymatic, promote its entrance
Into the cell, so as to play effectively carry medicine purpose.
Brief description of the drawings
Fig. 1 is the TEM phenograms that cationic polymer made from embodiment 4/TDNs carries medicine compound.
Fig. 2 is the potential energy diagram that cationic polymer made from embodiment 4/TDNs carries medicine compound.
Fig. 3 is the grain-size graph that cationic polymer made from embodiment 4/TDNs carries medicine compound.
After Fig. 4 cultures 24h, cationic polymer/TDNs carries toxicity data of the medicine compound to fibroblast (L929)
Figure.
After Fig. 5 is culture 72h, cationic polymer/TDNs carries toxicity knot of the medicine compound to fibroblast (L929)
Fruit is schemed.
Fig. 6 is the electrophoretogram added in TDNs solution after DNase I.
Fig. 7 be different N/P than compound electrophoretogram.
Fig. 8 is the laser confocal microscope figure of L929 cells and A549 cells.
Fig. 9 is the fluorescence signal intensity figure that cell is detected using flow cytometer；Wherein left side is L929 cells, and right side is
A549 cells.
Embodiment
Embodiment 1
A kind of cationic polymer/TDNs carries medicine compound, and its preparation method comprises the following steps：It is 1mg/mL by concentration
PEI solution mixed with 1 μm of ol/L TDNs according to N/P=0.2 ratio, be incubated 30min at room temperature, be made.
Wherein, 1ug TDNs represent 3nmol phosphoric acid (P), and 1 μ L PEI solution includes 10nmol ammonia nitrogens (N), compound
The TDNs (P) and PEI (N) amount added in solution, determine synthesis TNDs/PEI N/P ratios.
TDNs is prepared by the following method to obtain：
(1) ddH is used respectively by four, DNA tetrahedrons are single-stranded2O dissolves, and it is 1nmol/ μ L to make its concentration, then by ultraviolet fixed
Amount method, measure DNA are the light absorption value at 260nm and 330nm in wavelength, then calculate 100 μ L, 1 μM of body according to below equation
Each single-stranded volume in system：
V=100/ [(A260-A330) × 105/ (15.2 × single-stranded middle adenine number+7.4 × single-stranded middle cytimidine
The number of the number+8.3 of number+11.4 × single-stranded middle guanine × single-stranded middle thymidine)]
(2) according to result of calculation in step (1), four single-stranded solution of DNA tetrahedrons of dissolving are drawn, then with TM
Buffer (10mM Tris-HCl, 50mM MgCl2, pH8.0) and mixing, it is 100 μ L to make its cumulative volume, and whirlpool vibration mixes, most
After be placed in PCR instrument, temperature is rapidly brought up to 95 DEG C of stable 10min, 4 DEG C of stable 20min are cooled to, most after -20 DEG C of guarantors
Deposit.
Embodiment 2
A kind of cationic polymer/TDNs carries medicine compound, and its preparation method comprises the following steps：It is 1mg/mL by concentration
PEI solution mixed with 1 μm of ol/L TDNs according to N/P=5 ratio, be incubated 30min at room temperature, be made.
Wherein, 1ug TDNs represent 3nmol phosphoric acid (P), and 1 μ L PEI solution includes 10nmol ammonia nitrogens (N), compound
The TDNs (P) and PEI (N) amount added in solution, determine synthesis TNDs/PEI N/P ratios.
TDNs preparation method is the same as embodiment 1.
Embodiment 3
A kind of cationic polymer/TDNs carries medicine compound, and its preparation method comprises the following steps：It is 1mg/mL by concentration
PEI solution mixed with 1 μm of ol/L TDNs according to N/P=7.5 ratio, be incubated 30min at room temperature, be made.
Wherein, 1 μ g TDNs represent 3nmol phosphoric acid (P), and 1 μ L PEI solution includes 10nmol ammonia nitrogens (N), compound
The TDNs (P) and PEI (N) amount added in solution, determine synthesis TNDs/PEI N/P ratios.
TDNs preparation method is the same as embodiment 1.
Embodiment 4
A kind of cationic polymer/TDNs carries medicine compound, and its preparation method comprises the following steps：It is 1mg/mL by concentration
PEI solution mixed with 1 μm of ol/L TDNs according to N/P=1 ratio, be incubated 30min at room temperature, be made.
Wherein, 1 μ g TDNs represent 3nmol phosphoric acid (P), and 1 μ L PEI solution includes 10nmol ammonia nitrogens (N), compound
The TDNs (P) and PEI (N) amount added in solution, determine synthesis TNDs/PEI N/P ratios.
TDNs preparation method is the same as embodiment 1.
We have also done Optimal Experimental to PEI solution concentrations and TDNs solution concentrations, and PEI solution concentrations scope is 0.1-
10mg/mL, increased using 0.5mg/mL as gradient；TDNs solution concentrations scope is 0.1-10mg/mL, and ladder is used as using 0.5mg/mL
Degree increase, remaining process is same as Example 4, then carries out orthogonal test, such as toxicity test, to the Protection of nucleic acid and
Enter born of the same parents' experiment, test result indicates that the best results when PEI solution concentrations are 1mg/mL, and TDNs solution concentrations are 1 μm of ol/L.It is right
Toxicity test has similarly been done in embodiment 1-4, to the Protection of nucleic acid and has entered born of the same parents' experiment, the best results of embodiment 4.
We have also done following experiment：PEI and common DNA is combined, proportionate relationship is same as Example 4, although portion
Compound is divided to enter cell, but it is slow into cell speed, and only part can enter, the quantity into cell is low,
Part be blocked in it is extracellular, or enter cell compound be degraded by enzymes because DNA structure is unstable.
Sign and toxicity test to the gained compound of embodiment 4, to the Protection of nucleic acid and enter born of the same parents' experiment, specific mistake
Journey is as follows：
TEM phenograms, potential energy diagram, the particle diameter of medicine compound are carried by cationic polymer made from the above method/TDNs
Figure is shown in Fig. 1, Fig. 2 and Fig. 3 respectively.
As shown in Figure 1, carry medicine compound to be in granular form, apparent diameter has tiny fibre between 200-400 microns, each particle
Dimension connection, is PEI.
As shown in Figure 2, when N/P is 5 or 7.5, it is positive charge that it is electrically charged, which to carry medicine compound institute,；It is compound as N/P=1
Electrically charged thing institute is negative electrical charge.Positively charged particle compares negative electrical charge particle can produce higher toxicity to cell, and pass through
TNDs addition, reverse compound electric charge, it may be possible to the reason that compound cytotoxicity reduces.
From the figure 3, it may be seen that with the increase of PEI ratios, the DLS diameters of composite particles are gradually reduced.It should be noted that
The hydraulic diameter of a diameter of particles of DLS in the solution, its value compare TEM observations and might have difference.
The compound of experimental example 1 is to fibroblastic toxicity test
Using fibroblast (L929) as research object, appropriate fibroblast is taken to be inoculated into (n in 96 orifice plates first
=6, that is, 6 multiple holes are set), 24h is cultivated, serum free culture system is then dropped again, 12h is cultivated when serum-concentration is reduced into 8%, then be reduced to
12h is cultivated when 2%, then is down to after serum content is 0 and balances 1h, according to different grouping, is separately added into blank cultures (RPMI)
100 μ L, blank cultures and 250nM TDNs mixture (blank cultures 75 μ L, TDNs 25 μ L), blank cultures with
The mixture (the μ L of blank cultures 75, the μ L of compound 25) of the compound of different N/P ratios (7.5,5,1,0.2), under the same terms
Culture medium is suctioned out after cultivating 24h, 72h respectively, illustrates to carry out CCK-8 toxicity tests by CCK-8 kits.
After cultivating 24h and 72h, compound is shown in Fig. 4 and Fig. 5 respectively to the toxicity data of fibroblast (L929).
Reported in document, simple PEI has 30min or so visible instantaneous toxicity and 24h or so can for cell
The long term toxicity seen.And from Fig. 4 and Fig. 5, cationic polymer/TDNs carries medicine and answered made from the method provided by us
Compound, at first day of processing and the 3rd day, cell was not influenceed not only fibroblast (L929) by cation toxicity
And suppression is produced, the propagation with significant difference is obtained on the contrary, illustrates that the compound has good biocompatibility.
Protection of the compound of experimental example 2 to nucleic acid
The DNase I (0 unit, 10 units and 20 units) of 250nM TDNs solution various concentrations is digested into 3min, with
Mixed solution is added into 1% Normal Agarose Gel afterwards and carries out electrophoresis experiment, it is bright by the DNase I of 20 units that nucleic acid can be observed
Taken pictures after aobvious degraded.
To different N/P than compound (embodiment 5-8) in add the DNase I of same 20 unit, carried out after reacting 3min
Electrophoresis experiment, its result are shown in Fig. 6 and Fig. 7 respectively.
It will be appreciated from fig. 6 that after simple TDNs hybrid dna enzymes, with the DNA enzymatic of various concentrations different degrees of drop occurs for nucleic acid
Solution, when DNA enzymatic concentration is 20U, nucleic acid can all be degraded.
And after it with the addition of cation, after DNA enzymatic processing, its DNA band throws away the form that can keep good and brightness, can
Know, nucleic acid obtains good protection in compound of the present invention.
The DNA nanostructure of experimental example 3 enters born of the same parents' experiment
Determine that compound of the present invention promotes DNA tetrahedral structures to enter using flow cytometry and Confocal immunofluorescences
Enter the conclusion of cell.
Using L929 cells and A549 cells as object, TDNs is marked using CY5 fluorescence molecules.It is burnt that cell is placed in copolymerization
After cultivating 24h in special glass ware, gradient balances 1h when dropping serum to zero serum, is separately added into containing 100nM TDNs and N/
P=1：The culture medium of 1 compound (embodiment 5), after cultivating 6h, cell is fixed with 4% paraformaldehyde, uses DAPI and Phallus
Cyclic peptide staining cell core and cytoskeleton, finally using laser confocal microscope (TCS SP8；Leica,Wetzlar,
Germany observation film making) is carried out, its result is shown in 8.
After equally carrying out CY5 fluorescence labelings to TDNs, cell is inoculated in 6 orifice plates, then drops serum training again after cultivating 24h
Support, 12h is cultivated when 12h is cultivated when serum-concentration is reduced into 8%, then being reduced to 2%, then be down to after serum content is 0 and balance 1h.Point
Culture medium blank cultures, 100nM TDNs, N/P=1 are not replaced with into:1 compound, digestion centrifugation is thin after cultivating 6h
Born of the same parents, cell suspension is made, the fluorescence signal intensity of cell is detected simultaneously using flow cytometer (FC500Beckman, IL USA)
Quantitative analysis is carried out, as a result sees Fig. 9 (direct streaming figure) and table 1 (positive rate of threshold value).
The fluorescence signal intensity of the cell of table 1
| Control | Simple nucleic acid | Cationic compound | |
| A549 | 0.18±0.02 | 18.00±0.60 | 96.57±1.27 |
| L929 | 0.20±0.03 | 0.19±0.02 | 93.97±0.15 |
The situation of cell is entered using the nucleic acid nano structure of Flow cytometry fluorescence labeling, can by Fig. 9 and table 1
Know, in two kinds of cells of A549 and L929, the ratio that cell is entered using the compound nucleic acid of cation is far longer than simple nucleic acid,
Immunofluorescence can visually see, by cation it is compound after, the nucleic acid of fluorescence labeling enters cell in large quantities.
Claims (8)
1. a kind of cationic polymer/TDNs carries the preparation method of medicine compound, it is characterised in that comprises the following steps：By PEI
Solution and TDNs solution mix according to N/P=0.2-7.5 ratio, are incubated 15-30min at room temperature, are made.
2. cationic polymer according to claim 1/TDNs carries the preparation method of medicine compound, it is characterised in that PEI
Solution and TDNs solution mix according to N/P=1 ratio.
3. cationic polymer according to claim 1/TDNs carries the preparation method of medicine compound, it is characterised in that institute
It is 1mg/mL to state PEI solution concentrations.
4. cationic polymer according to claim 1/TDNs carries the preparation method of medicine compound, it is characterised in that institute
It is 1 μm of ol/L to state TDNs solution concentrations.
5. cationic polymer according to claim 1/TDNs carries the preparation method of medicine compound, it is characterised in that institute
TDNs is stated to be prepared by the following method to obtain：
(1) ddH is used respectively by four, DNA tetrahedrons are single-stranded2O dissolves, and it is 1nmol/ μ L to make its concentration, then by ultraviolet sizing technique,
DNA is determined in the light absorption value that wavelength is 260nm and 330nm places, 100 μ L are then calculated according to below equation, in 1 μM of system respectively
Single-stranded volume：
V=100/ [(A260-A330) × 105/ (number of 15.2 × single-stranded middle adenine number+7.4 × single-stranded middle cytimidine+
The number of number+8.3 × single-stranded middle thymidine of 11.4 × single-stranded middle guanine)]
(2) according to the result calculated in step (1), four single-stranded solution of DNA tetrahedrons of dissolving are drawn, then with TM
Buffer is mixed to 100 μ L, whirlpool vibration and is mixed, be finally placed in PCR instrument, and temperature is rapidly brought up into 95 DEG C of stable 10min, then
4 DEG C of stable 20min are cooled to, most after -20 DEG C of preservations.
6. cationic polymer according to claim 5/TDNs carries the preparation method of medicine compound, it is characterised in that step
Suddenly TM buffer are 5-10mM Tris-HCl, 5-50mM MgCl in (3)2, pH8.0.
7. cationic polymer according to claim 6/TDNs carries the preparation method of medicine compound, it is characterised in that step
Suddenly TM buffer are 10mM Tris-HCl, 50mM MgCl in (3)2, pH8.0.
8. the cationic polymer that the method described in claim any one of 1-7 is prepared/TDNs carries medicine compound.
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| PCT/CN2017/116300 WO2019052049A1 (en) | 2017-09-12 | 2017-12-14 | Cationic polymer/tdns drug-loading complex and preparation method therefor |
| CN201811064385.2A CN109364255B (en) | 2017-09-12 | 2018-09-12 | Cationic polymer/TDNs drug-loaded compound and preparation method and application thereof |
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| CN109364255A (en) * | 2017-09-12 | 2019-02-22 | 四川大学 | A kind of cationic polymer/TDNs carries medicine compound and its preparation method and application |
| CN110665011A (en) * | 2019-11-13 | 2020-01-10 | 北京化工大学 | Nano compound for delivery of small molecule anticancer drugs |
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| CN113373224A (en) * | 2021-06-08 | 2021-09-10 | 王旭耀 | EGFR gene typing detection method and kit based on nucleic acid tetrahedral probe modified printed gold electrode |
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| CN110292644A (en) * | 2019-07-23 | 2019-10-01 | 四川大学 | A kind of drug prevented and treated myocardial ischemia-reperfusion injury or treat heart ischemia disease |
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| US7060498B1 (en) * | 2001-11-28 | 2006-06-13 | Genta Salus Llc | Polycationic water soluble copolymer and method for transferring polyanionic macromolecules across biological barriers |
| CN106540268B (en) * | 2016-11-02 | 2021-01-26 | 四川大学 | TDNs-AS 1411-nucleic acid medicine composite nano material medicine carrying system and preparation method thereof |
| CN106497919A (en) * | 2016-11-02 | 2017-03-15 | 四川大学 | A kind of DNA tetrahedrons of aptamer AS1411 modifications and preparation method thereof |
| CN107432935A (en) * | 2017-09-12 | 2017-12-05 | 四川大学 | A kind of cationic polymer/TDNs carries medicine compound and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109364255A (en) * | 2017-09-12 | 2019-02-22 | 四川大学 | A kind of cationic polymer/TDNs carries medicine compound and its preparation method and application |
| WO2019052049A1 (en) * | 2017-09-12 | 2019-03-21 | 四川大学 | Cationic polymer/tdns drug-loading complex and preparation method therefor |
| CN110665011A (en) * | 2019-11-13 | 2020-01-10 | 北京化工大学 | Nano compound for delivery of small molecule anticancer drugs |
| CN110917121A (en) * | 2019-12-11 | 2020-03-27 | 四川大学 | APD hybrid nano system and construction method and application thereof |
| CN110917121B (en) * | 2019-12-11 | 2021-01-05 | 四川大学 | APD hybrid nano system and construction method and application thereof |
| CN113373224A (en) * | 2021-06-08 | 2021-09-10 | 王旭耀 | EGFR gene typing detection method and kit based on nucleic acid tetrahedral probe modified printed gold electrode |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109364255A (en) | 2019-02-22 |
| WO2019052049A1 (en) | 2019-03-21 |
| CN109364255B (en) | 2022-02-08 |
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