CN108524946A - Ternary complex Nano medication and preparation method thereof and the application in preparing light controlled release nanometer delivery system - Google Patents
Ternary complex Nano medication and preparation method thereof and the application in preparing light controlled release nanometer delivery system Download PDFInfo
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Abstract
Application the invention discloses ternary complex Nano medication and preparation method thereof and in preparing light controlled release nanometer delivery system, ternary complex Nano medication, including Contracting thioketones key crosslinking low molecular weight PEI based polyalcohols, the hyaluronic acid polymer for being connected to photosensitizer, nucleic acid drug.The photosensitizer that the compound of the present invention includes can generate active oxygen under the control of external source feux rouges(ROS), realize photochemistry internalization(PCI)The polymer degradation that the lysosome of mediation is escaped and light promotes, final realize reduce material toxic side effect, improve transfection efficiency, enhance the purpose of therapy of tumor effect.
Description
Technical field
The invention belongs to high polymer material technology and art of pharmacy, it is related to a kind of smooth controlled release nano-delivery system
Preparation method and application.
Background technology
Polyethyleneimine(PEI)As a kind of commercialization genophore, since its structure includes a large amount of positively charged charge ammonia
Base group can effectively contain electronegative nucleic acid, to form stable compound, further deliver into intracellular.But
It is that while macromolecule cation polymer possesses a large amount of positive charges, the cytotoxicity of material be can not ignore, also, be wrapped up
Nucleic acid material wherein is difficult to discharge, and it is limited to eventually lead to transfection.Develop degradable cationic polymer, can solve
Certainly above-mentioned contradiction, while realizing that nucleic acid contains and its intracellular release, and the cytotoxicity for reducing high molecular material itself.Mesh
Preceding stimuli responsive degradable cationic polymer largely uses endogenous stimulus, such as glutathione(GSH), acidic environment
Deng.However, endogenous stimulus Modulatory character is poor.Therefore, research and develop new delivery system, for develop external source sexual stimulus it is controllable,
The macromolecule cation polymer genophore that biological safety is high, toxic side effect is small, transfection efficiency is high is to realize effective ground
Because treating very important means.
Invention content
The present invention provides a kind of ternary complex Nano medications, and the photosensitizer that especially compound includes can be outside
Under the feux rouges control of source, active oxygen is generated(ROS), realize photochemistry internalization(PCI)The lysosome of mediation is escaped and light promotes
Polymer degradation, final realize reduce material toxic side effect, improve transfection efficiency, enhances the mesh of therapy of tumor effect
's.
The present invention adopts the following technical scheme that:
A kind of ternary complex Nano medication, including Contracting thioketones key are crosslinked low molecular weight PEI based polyalcohols, are connected to the saturating of photosensitizer
Bright matter acid polymer, nucleic acid drug.
The present invention provides a kind of Contracting thioketones keys to be crosslinked low molecular weight PEI based polyalcohols, and the Contracting thioketones key is crosslinked low point
Son amount PEI based polyalcohols have structure shown in Formulas I,
Wherein, R is the dendroid PEI that molecular weight is less than 5 kDa;20≤n≤500,4≤m≤24.
Invention additionally discloses a kind of thioketal crosslinking agents, with following chemical structural formula:
The present invention provides the preparation methods that a kind of Contracting thioketones key is crosslinked low molecular weight PEI based polyalcohols, include the following steps:
(1)Using cysteamine, methoxyl group propylene, acryloyl chloride as raw material, reaction prepares thioketal crosslinking agent;
(2)Using thioketal crosslinking agent and PEI as raw material, reaction prepares Contracting thioketones key and is crosslinked low molecular weight PEI based polyalcohols(TK-
PEI).
The present invention provides a kind of preparation methods of thioketal crosslinking agent, include the following steps:
(1)Using cysteamine, methoxyl group propylene, acryloyl chloride as raw material, reaction prepares thioketal crosslinking agent.
In above-mentioned technical proposal, step(1)In, using cysteamine, trifluoroacetic acid as raw material prepare compound 1, then with chemical combination
Object 1 and methoxyl group propylene are raw material prepare compound 2, then the sodium hydroxide of compound 2 is deprotected prepare compound 3, finally
Using compound 3, acryloyl chloride as raw material, thioketal crosslinking agent is prepared.
In above-mentioned technical proposal, step(1)In, cysteamine, trifluoroacetic acid molar ratio be 1: 1.9~2.1;Compound 1
Molar ratio with methoxyl group propylene is 2.5~2.6: 1;Compound 3, the molar ratio of acryloyl chloride are 1: 3.
In above-mentioned technical proposal, step(2)In, Michael addition reaction is passed through as raw material using thioketal crosslinking agent and PEI
Prepare TK-PEI.
In above-mentioned technical proposal, the mass ratio of thioketal crosslinking agent and PEI are 1: 1.3~1.4.
In above-mentioned technical proposal, the PEI that the present invention uses includes the dendroid PEI that molecular weight is less than 5 kDa, such as PEI
(600Da)、PEI (1800Da)。
The method that the present invention prepares TK-PEI can be specifically exemplified below:
(1)By cysteamine(20.0 g, 176.0 mmol, 1 equivalent)It is dissolved in containing triethylamine(35.8 g, 353.9 mmol, 2 work as
Amount)Methanol(400 mL)In.Trifluoroacetic Acid Ethyl Ester is added(26.2 g, 184.8 mmol, 1.05 equivalents), and will react molten
Liquid is stirred at room temperature overnight.Then acetic acid is added, and pH is adjusted to 6.Solution ethyl acetate(3×100 mL)Extraction,
Merge organic layer, with anhydrous MgSO4It is dry.Solution is filtered and rotated.By using n-hexane/ethyl acetate(8/1)Make
Residue is further purified for the silica gel column chromatography of eluent, obtains compound 1, deuterochloroform beats nuclear-magnetism;
(2)By compound 1(14.2 g, 82.0 mmol, 2.5 equivalents)And PTSA(0.2 g, 1.1 mmol, 0.03 equivalent)It is dissolved in
Benzene(250 mL)In, it is stirred at room temperature 10 minutes.Molecular sieve is added(5,100.0 g), and mixture is stirred for
10 minutes.Then 2- methoxyl group propylene is added(2.3 g, 32.6 mmol, 1 equivalent), and mixture is stirred at room temperature 4
h.Solution is filtered and by concentrated by rotary evaporation to obtain compound 2, deuterochloroform beats nuclear-magnetism;
(3)At room temperature by compound 2(10.0 g, 25.8 mmol)In NaOH solution(1 M, 150 mL)Middle deprotection 5 is small
When.Use dichloromethane(5×100 mL)Extraction solution collects water phase, after revolving removes solvent, obtains compound 3, deuterated
DMSO beats nuclear-magnetism;
(4)At 0 DEG C, by compound 3(0.3 g, 1.6 mmol, 1 equivalent)It is dissolved in containing triethylamine(0.9 g, 9.6 mmol, 6
Equivalent)Dichloromethane(30 mL)In.Then by acryloyl chloride at 0 DEG C(0.4 g, 4.8 mmol, 3 equivalents)It is added dropwise to mixed
It closes in object.Use ethyl acetate(3×100 mL)Extraction solution collects water phase and passes through silica gel chromatography(N-hexane/acetic acid second
Ester, 8/1)Purifying, obtains compound 4, i.e. thioketal crosslinking agent, deuterated DMSO beats nuclear-magnetism;
(5)By compound 4(25.0 mg)With PEI (600Da)(33.0 mg)It is dissolved in 1 mL methanol, and nitrogen is protected from light at 45 DEG C
It is stirred 48 hours in compression ring border.To deionized water(Deionized water, MWCO=1kDa)It dialyses and the mixture 2 days and is lyophilized, obtain
To TK-PEI, deuterated heavy water beats nuclear-magnetism, gel permeation chromatography(GPC)Measure molecular weight.
Above-mentioned specific reaction can indicate as follows:
The invention discloses the hyaluronic acid polymers for being connected to photosensitizer(HAP), main chain is hyaluronic acid, and side chain is connected to photosensitive
The molecular weight of agent, the hyaluronic acid is 7~500 kDa, has structure shown in formula II;
Wherein, R2For photosensitizer unit;Preferably, photosensitizer is carboxyl porphyrin derivative, carboxyl chlorophyll corproporphyrin derivative
Or chlorin derivative, structure can indicate as follows:
The invention discloses a kind of preparation methods for the hyaluronic acid polymer being connected to photosensitizer, include the following steps, by acetic acid
Acid anhydride is added in the mixture of hyaluronic acid and pyridine, and acetylation hyaluronic acid is obtained by the reaction;Mixing photosensitizer such as de-magging leaf is green
Plain a (Pha), carbodiimide (EDC), 4-dimethylaminopyridine (DMAP) are added acetylation hyaluronic acid, pass through light after stirring
Quick dose of carboxyl and the hydroxyl reaction of hyaluronic acid obtain the hyaluronic acid polymer for being connected to photosensitizer.
In above-mentioned technical proposal, acetic anhydride, hyaluronic acid molar ratio be 147:1, photosensitizer, acetylation hyaluronic acid
Mass ratio be 20:1.
The method that the present invention prepares HAP can be specifically exemplified below:
By hyaluronic acid(0.5 g, 86.2 μm of ol)And pyridine(1.5mL, 18.6mmol)It is dissolved in 20mL formamides, and will mixing
Object is stirred at room temperature 1 hour.Acetic anhydride is added(1.2 mL, 12.7 mmol), futher stir 24 hours, deionized water is saturating
Analysis(MWCO = 1 kDa)2 days.The HA of acetylation is obtained after freeze-drying;By Pha(1 equivalent), EDC(1 equivalent)And DMAP
(1.5 equivalent)It is dissolved in anhydrous DMSO and stirs the mixture for 6 hours.Then by the HA of acetylation(25.0 mg)It is added above-mentioned
It is stirred in mixture and at room temperature 24 hours.Use deionized water dialysis(MWCO = 1kDa)2 days and be lyophilized after obtain
HAP, ultraviolet-visible(UV-Vis)Spectra methods measures the photosensitive agent content in HAP.
The nucleic acid drug of the present invention is selected from DNA, RNA, oligonucleotide or polynucleotides.
The invention also discloses a kind of preparation methods of ternary complex Nano medication, include the following steps, by thioketal
Key crosslinking low molecular weight PEI based polymer solutions add the hyalomitome for being connected to photosensitizer after mixing incubation with nucleic acid drug solution
Acid polymer solution, is incubated again, obtains ternary complex Nano medication.It is specifically as follows:
TK-PEI is dissolved in DEPC water, and is mixed in different quality ratio with the nucleic acid solution dissolved in advance, and
It is placed in after 37 DEG C of water-baths are incubated 30min and obtains binary complex;It is dissolved in the HAP of DEPC water than being added by different quality again, 37 DEG C
Water-bath is incubated 60min, obtains final Nano medication.
In above-mentioned technical proposal, the nucleic acid drug is selected from DNA, RNA, oligonucleotide or polynucleotides.
In above-mentioned technical proposal, the DNA is p53 Plasmid DNA, can express the protein of p53.
In the ternary complex Nano medication of the present invention, the mass ratio of TK-PEI and nucleic acid drug is(0.1~10): 1, it is excellent
The mass ratio of choosing is(0.5~2.5): 1, preferred mass ratio is 1: 1.
In the ternary complex Nano medication of the present invention, the mass ratio of HAP and nucleic acid drug is(0.1~10): 1, preferably
Mass ratio is(0.5~5): 1, preferred mass ratio is 2.5: 1.
The grain size of the ternary complex Nano medication of the present invention is 100 ~ 1000 nm, and preferred grain size is 100 ~ 300 nm,
Preferred grain size is 100 ~ 150 nm.
The Zeta electric potential of the ternary complex Nano medication of the present invention is -20 ~ 50 mV, and preferred Zeta electric potential is 5 ~ 30
MV, preferred Zeta electric potential are 5 ~ 10 mV.
The invention also discloses applications of the above-mentioned TK-PEI and/or HAP in preparing nucleic acid drug carrier.
The present invention further discloses above-mentioned TK-PEI, HAP or ternary complex Nano medication to prepare genomic medicine
In application, the especially application in preparing light controlled release nano gene drug delivery system.
The main advantage of the present invention is:
(1)TK-PEI contains the binary complex of nucleic acid drug formation as kernel, has the following advantages that and acts on:1. can be steady
Surely nucleic acid drug is contained, the endocytosis of nucleic acid drug is effectively facilitated;2. the structure of polyamino can be improved by proton sponge effect
Lysosome escape capability enhances nucleic acid drug into core amount;3. contained Contracting thioketones key can be broken under environmental stimuli, material sheet is reduced
The toxic side effect of body, and at the same time promoting the release of accounting drug;4. synthetic method is simply controllable;5. raw material is relatively inexpensive, section
About cost;
(2)The tri compound nano particle that the present invention obtains has the following advantages that and acts on:1. the hyaluronic acid of outer layer absorption can
To enhance the serum stability of compound, and pass through the specific endocytosis of active targeting tumour cell raising tumour cell;2. light
Quick dose under short time illumination condition, can generate ROS, further promote the lysosome of nano particle to escape by PCI effects,
The degradation that TK-PEI can also be controlled simultaneously reaches reduction toxic side effect, the effect of enhancing nucleic acid drug release;3. photosensitizer exists
Under long-time illumination condition, lethal dose ROS can be generated, realizes the optical dynamic therapy of tumour(PDT);
(3)Tri compound nano particle disclosed by the invention can play apparent tumour cell or just in different cell models
The effect of normal cell transfecting, and gene transfection is further further increased by illumination;Collaboration may be implemented in vivo
Therapy of tumor and optical dynamic therapy.
Description of the drawings
Fig. 1 is one compound 4 of embodiment1H H NMR spectroscopies;
Fig. 2 is two TK-PEI's of embodiment1H H NMR spectroscopies;
Fig. 3 is the UV-Vis spectrograms of embodiment six;
Fig. 4 is the GPC spectrograms before and after embodiment two, one dioxygen water process of comparative example;
Fig. 5 is that embodiment two and comparative example one wrap up the grain-size graph after DNA;
Fig. 6 is that embodiment two and comparative example one wrap up the potential energy diagram after DNA;
Fig. 7 is embodiment two and comparative example one by the pretreated DNA packages figure of various concentration hydrogen peroxide;
Fig. 8 is the change of size figure of ternary complex and binary complex in serum at any time;
Fig. 9 is embodiment two on B16F10 cells, and comparative example one and PEI 25k wrap up the gene transfection figure of compound after DNA;
Figure 10 is B16F10, HeLa, on COS-7 cells, the gene transfection before and after ternary complex, binary complex illumination
Figure;
Figure 11 is the fluorogram that lysosome is escaped before and after ternary complex illumination on B16F10 cells;
Figure 12 is the fluorogram that DNA discharges before and after ternary complex illumination on B16F10 cells;
Figure 13 is on B16F10 cells, and material is thin after embodiment two, comparative example one, 600 dioxygen water process of PEI 25k and PEI
Cellular toxicity figure;
Figure 14 is on B16F10 tumor models, and ternary complex wraps up pattern DNA respectively(luc)With treatment DNA (p53), short
Tumor suppression curve graph under illumination and long illumination condition.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, or according to the normal condition proposed by manufacturer.In addition, can also be appointed between a variety of different embodiments of the present invention
Meaning combination, as long as it does not violate the idea of the present invention, it should also be regarded as the disclosure of the present invention.
Embodiment one
(1)By cysteamine(20.0 g, 176.0 mmol, 1 equivalent)It is dissolved in containing triethylamine(35.8 g, 353.9 mmol, 2 work as
Amount)Methanol(400 mL)In.Trifluoroacetic Acid Ethyl Ester is added(26.2 g, 184.8 mmol, 1.05 equivalents), and by reaction solution
It is stirred at room temperature overnight.Then acetic acid is added, and pH is adjusted to 6.Solution ethyl acetate(3×100 mL)Extraction is closed
And organic layer, with anhydrous MgSO4It is dry.Solution is filtered and rotated, by using n-hexane/ethyl acetate(8/1)As washing
Residue is further purified in the silica gel column chromatography of de- liquid, obtains compound 1, structural formula is as follows:
(2)By compound 1(14.2 g, 82.0 mmol, 2.5 equivalents)And PTSA(0.2 g, 1.1 mmol, 0.03 equivalent)It is dissolved in
Benzene(250 mL)In, it is stirred at room temperature 10 minutes.Molecular sieve is added(5,100.0 g), and mixture is stirred for
10 minutes.Then 2- methoxyl group propylene is added(2.3 g, 32.6 mmol, 1 equivalent), and 4 h are stirred at room temperature in mixture.
Solution is filtered and by concentrated by rotary evaporation to obtain compound 2, structural formula is as follows:
(3)At room temperature by compound 2(10.0 g, 25.8 mmol)In NaOH solution(1 M, 150 mL)Middle deprotection 5 is small
When.Use dichloromethane(5×100 mL)Extraction solution collects water phase, after revolving removes solvent, obtains compound 3, structural formula
It is as follows:
(4)At 0 DEG C, by compound 3(0.3 g, 1.6 mmol, 1 equivalent)It is dissolved in containing triethylamine(0.9 g, 9.6 mmol, 6
Equivalent)Dichloromethane(30 mL)In.Then by acryloyl chloride at 0 DEG C(0.4 g, 4.8 mmol, 3 equivalents)It is added dropwise to mixed
It closes in object.Use ethyl acetate(3×100 mL)Extraction solution collects water phase and passes through silica gel chromatography(N-hexane/acetic acid second
Ester, 8/1)Purifying, obtains compound 4, structural formula is as follows:
Deuterated DMSO beats nuclear-magnetism, and Fig. 1 is 4 nuclear-magnetism figure of compound.
Embodiment two
By compound 4(25.0 mg)With PEI (600Da)(33.0 mg)It is dissolved in 1 mL methanol, and nitrogen ring is protected from light at 45 DEG C
It is stirred 48 hours in border.To deionized water(Deionized water, MWCO=1kDa)It dialyses and the mixture 2 days and is lyophilized, obtain chemical combination
Object 5(TK-PEI), deuterated heavy water beats nuclear-magnetism, and Fig. 2 is 5 nuclear-magnetism figure of compound, and Fig. 4 is that the GPC of compound 5 schemes, molecular weight 11
kDa。
Embodiment three
By compound 4(25.0 mg)With PEI (1800Da)(33.0 mg)It is dissolved in 1 mL methanol, and nitrogen is protected from light at 45 DEG C
It is stirred 48 hours in environment.To deionized water(Deionized water, MWCO=1kDa)It dialyses and the mixture 2 days and is lyophilized, changed
Close object 5(TK-PEI), molecular weight is 18 kDa.
Example IV
By compound 4(25.0 mg)With PEI (1800Da)(66.0 mg)It is dissolved in 1 mL methanol, and nitrogen is protected from light at 45 DEG C
It is stirred 48 hours in environment.To deionized water(Deionized water, MWCO=1kDa)It dialyses and the mixture 2 days and is lyophilized, changed
Close object 5(TK-PEI), molecular weight is 20 kDa.
Embodiment five
By compound 4(25.0 mg)With PEI (600Da)(66.0 mg)It is dissolved in 1 mL methanol, and nitrogen ring is protected from light at 45 DEG C
It is stirred 48 hours in border.To deionized water(Deionized water, MWCO=1kDa)It dialyses and the mixture 2 days and is lyophilized, obtain chemical combination
Object 5(TK-PEI), molecular weight is 12 kDa.
Comparative example one
(1)At 0 DEG C, by 1,7- heptamethylene diamines(0.3 g, 1.6 mmol, 1 equivalent)It is dissolved in containing triethylamine(0.9 g, 9.6
Mmol, 6 equivalents)Dichloromethane(30 mL)In.Then by acryloyl chloride at 0 DEG C(0.4 g, 4.8 mmol, 3 equivalents)Drop
It adds in mixture.Use ethyl acetate(3×100 mL)Extraction solution collects water phase and passes through silica gel chromatography(N-hexane/
Ethyl acetate, 8/1)Purifying, obtains compound 6, structural formula is as follows:
(2)By compound 6(25.0 mg)With PEI (600Da)(33.0 mg)It is dissolved in 1 mL methanol, and nitrogen is protected from light at 45 DEG C
It is stirred 48 hours in compression ring border.To deionized water(Deionized water, MWCO=1kDa)It dialyses and the mixture 2 days and is lyophilized, obtain
Compound 7(NK-PEI), structural formula is as follows:
Embodiment six
By hyaluronic acid(0.5 g, 86.2 μm of ol)And pyridine(1.5mL, 18.6mmol)It is dissolved in 20mL formamides, and will mixing
Object is stirred at room temperature 1 hour.Acetic anhydride is added(1.2 mL, 12.7 mmol), futher stir 24 hours, deionized water is saturating
Analysis(MWCO = 1 kDa)2 days.The HA of acetylation is obtained after freeze-drying;By Pha(1 equivalent), EDC(1 equivalent)And DMAP
(1.5 equivalent)It is dissolved in anhydrous DMSO and stirs the mixture for 6 hours.Then by the HA of acetylation(25.0 mg)It is added above-mentioned
It is stirred in mixture and at room temperature 24 hours.Use deionized water dialysis(MWCO = 1kDa)2 days and be lyophilized after connect
It is HAP ultraviolet-visible spectrograms to have the hyaluronic acid polymer HAP, Fig. 3 of photosensitizer.
Embodiment seven
By hyaluronic acid(0.5 g, 86.2 μm of ol)And pyridine(1.5mL, 18.6mmol)It is dissolved in 20mL formamides, and will mixing
Object is stirred at room temperature 1 hour.Acetic anhydride is added(1.2 mL, 12.7 mmol), futher stir 24 hours, deionized water is saturating
Analysis(MWCO = 1 kDa)2 days.The HA of acetylation is obtained after freeze-drying;By Pha(1 equivalent), EDC(1 equivalent)And DMAP
(1.5 equivalent)It is dissolved in anhydrous DMSO and stirs the mixture for 6 hours.Then by the HA of acetylation(25.0 mg)It is added above-mentioned
It is stirred in mixture and at room temperature 24 hours.Use deionized water dialysis(MWCO = 1kDa)2 days and be lyophilized after obtain
HAP。
Embodiment eight
By hyaluronic acid(0.5 g, 86.2 μm of ol)And pyridine(1.5mL, 18.6mmol)It is dissolved in 20mL formamides, and will mixing
Object is stirred at room temperature 1 hour.Acetic anhydride is added(1.2 mL, 12.7 mmol), futher stir 24 hours, deionized water is saturating
Analysis(MWCO = 1 kDa)2 days.The HA of acetylation is obtained after freeze-drying;By chlorin Ce6(1 equivalent), EDC(1 works as
Amount)And DMAP(1.5 equivalent)It is dissolved in anhydrous DMSO and stirs the mixture for 6 hours.Then by the HA of acetylation(25.0
mg)It is added in said mixture and is stirred at room temperature 24 hours.Use deionized water dialysis(MWCO = 1kDa)2 days and freeze
HAP is obtained after dry.
Embodiment nine
By hyaluronic acid(0.5 g, 86.2 μm of ol)And pyridine(1.5mL, 18.6mmol)It is dissolved in 20mL formamides, and will mixing
Object is stirred at room temperature 1 hour.Acetic anhydride is added(1.2 mL, 12.7 mmol), futher stir 24 hours, deionized water is saturating
Analysis(MWCO = 1 kDa)2 days.The HA of acetylation is obtained after freeze-drying;By carboxyl porphyrin(1 equivalent), EDC(1 equivalent)With
DMAP(1.5 equivalent)It is dissolved in anhydrous DMSO and stirs the mixture for 6 hours.Then by the HA of acetylation(25.0 mg)It is added
It is stirred in said mixture and at room temperature 24 hours.Use deionized water dialysis(MWCO = 1kDa)2 days and be lyophilized after obtain
Obtain HAP.
Embodiment ten
Using the preparation of the HAP of the TK-PEI of embodiment two, embodiment six Nano medications assembled as carrier and characterization, performance.
Prepare embodiment two TK-PEI and DNA(Plasmid DNA, that is extracted from Escherichia coli contains luciferase or p53
The plasmid of expression)Concentration is respectively the DEPC aqueous solutions of 1 mg/mL and 0.1 mg/mL.According to TK-PEI/DNA different weight ratios
Mixture is vortexed 10 seconds, is then incubated 30 minutes at 37 DEG C, to form TK-PEI/DNA compounds by mixing.
Prepare the DEPC aqueous solutions of a concentration of 1 mg/mL of six HAP of embodiment.It is different according to the above-mentioned binary complexs of HAP/
Weight ratio mixes, and mixture is vortexed 10 seconds, 60 clocks are then incubated at 37 DEG C, to form HAP/TK-PEI/DNA tri compounds
Object.
Utilize dynamic light scattering(DLS)Ternary complex is assessed in the case where different weight is than mixing, the grain size and electricity of compound
Position.
By EB solution and DNA with weight ratio for 10:1 mixing.And it is incubated at room temperature 1 hour.Then according to TK-PEI/
Embodiment is added in EB/DNA mixtures by DNA different weight ratios, is further put mixture and is incubated at room temperature 30 minutes,
Its fluorescence intensity is measured by microplate reader(λ ex=510 nm, λ em=590 nm)It quantitative determines DNA and wraps up efficiency.
B16F10 cells are with every hole 2.5 × 104It is a to be inoculated into 96 orifice plates, then cultivated in the DMEM containing 10%FBS
It is cultivated 24 hours in base.Then culture medium is substituted for the DMEM of serum-free, and the concentration according to 0.3 μ g DNA/ per hole is added two
First compound.After 37 DEG C are incubated 4 hours, culture medium is removed, fresh culture is substituted for and is further incubated for 20 hours later.Make
Luciferase expression is measured with luciferase kit, and uses BCA kit measurement cellular protein concentrations, its gene is evaluated and turns
Contaminate efficiency.
B16F10 cells are with every hole 2.5 × 104It is a to be inoculated into 96 orifice plates, then cultivated in the DMEM containing 10%FBS
It is cultivated 24 hours in base.Ternary complex is added in concentration according to 0.3 μ g DNA/ per hole.After 37 DEG C are incubated 4 hours, remove
Culture medium is substituted for fresh culture, cell is divided into two groups, one group of carry out illumination(661 nm, 8 min), one group without
Illumination is further incubated for 20 hours later.Luciferase expression is measured using luciferase kit, and uses BCA kits
Cellular protein concentration is measured, its efficiency gene transfection is evaluated.
Confocal laser scanning microscope, CLSM can further look at cell internalizing and the distribution of compound.B16F10 cells with
Per hole 1.5 × 104It is a to be inoculated into 24 orifice plates, after then being cultivated 24 hours in the DMEM culture mediums containing 10%FBS, change into
Compound is added according to the concentration in 1 holes μ g YOYO-1-DNA/ in serum free medium.37 DEG C are cultivated 4 hours.With containing heparin sodium
PBS washing three times, use Lysotracker-Red(200 nM)Dyeing 1 hour;After being washed three times with the PBS containing heparin sodium again, use
4% paraformaldehyde fixes 15 minutes;It is then washed with the PBS containing heparin sodium and uses DAPI afterwards three times(5 μg/mL)Dye 10 minutes;Finally
With glycerine mounting.Its fluorescence intensity is observed by laser confocal microscope.
B16F10 cells are with every hole 2.5 × 104It is a to be inoculated into 96 orifice plates, it cultivates 24 hours.Then serum-free training is changed into
Base is supported, the pretreated polymer of hydrogen peroxide of different final concentrations is added.After being incubated 4 hours at 37 DEG C, former culture medium is abandoned, is changed into
After being cultivated 20 hours in the DMEM culture mediums of 10%FBS.Cell survival rate is measured by mtt assay.
The mouse of male C57BL/6 are subcutaneously injected 1 × 10 in left side6A B16F10 cells.When gross tumor volume reaches 80mm3
When, mouse is randomly divided into 4 groups(n = 6), received intratumor injection phosphate buffer at the 1st, 3,5,7 and 9 day or various receive
The grain of rice(3mg DNA/kg, 50 μ L of volume injected).Group 1 receives injection phosphate buffer.TK-PEI is injected in 3rd group of receiving
/ HAP/luc nanoparticles.2nd group and the 4th group receives TK-PEI/HAP/p53 nanoparticles.After injection 4 hours it is right
Animal irradiation in group 2(20mW /cm2, 661nm)8 minutes, and the animal organized in 3 and 4 was irradiated at 4 hours(20mW /cm2,
661nm)After injection in 8 minutes and 30 minutes 24 hours after injection.Every other day monitor tumor size.
The TK-PEI of embodiment two is changed to the NK-PEI of comparative example one, assemble the binary of formation, ternary complex into
Row compares, as a result as follows:
Fig. 4 is the GPC figures before and after one dioxygen water process of embodiment two and comparative example, and the embodiment of the present invention two is found from Fig. 4
There is similar molecular weight with comparative example one(10~12 kDa).Compared to comparative example one, embodiment two is protected after dioxygen water process
It stays the time to be obviously prolonged, illustrates that dioxygen water process leads to the reduction of two molecular weight of embodiment.
Fig. 5 is that embodiment two and comparative example one wrap up the grain-size graph after DNA, finds that the TK-PEI of the present invention makees from Fig. 5
Possess good nanometer particle size distribution, about 100 ~ 150 nm for carrier.
Fig. 6 is that embodiment two and comparative example one wrap up the potential energy diagram after DNA, and embodiment two and comparative example are found from Fig. 6
One becomes positive charge, about 10 ~ 20mV being 0.5 negative electrical charge with nucleic acid drug mass ratio, embodiment two and comparative example one and core
Sour drug is compound good.
Fig. 7 is the DNA package figures that embodiment two and comparative example one wrap up the ethidium bromide processing after DNA, is found from Fig. 7
Embodiment two and comparative example one can be achieved when being 0.5 with nucleic acid drug mass ratio up to nearly 90% and contain rate, it was demonstrated that real
It applies example two and comparative example one can be very good package nucleic acid drug;By using two He of various concentration hydrogen peroxide Processing Example
Comparative example one, it can be found that the DNA of embodiment two contains rate and is decreased obviously in Fig. 7, and one DNA of comparative example contains rate and does not become
Change, illustrates that embodiment two is degraded with hydrogen peroxide response.
Fig. 8 is binary complex(TK-PEI/DNA)And ternary complex(HAP/TK-PEI/DNA)Stabilization in serum
Property figure, from Fig. 8 it can be found that with the extension of time, binary complex grain size significantly increases, ternary complex grain size is kept
Stablize.Illustrate that ternary complex has stronger stability in serum.
Fig. 9 is embodiment two on B16F10 cells, and comparative example one and 25k PEI wrap up the gene turn of compound after DNA
Dye possesses similar it can be found that embodiment two is when with comparative example one in quality ratio identical with nucleic acid drug from Fig. 9
Transfection efficiency.
Figure 10 is on B16F10 cells, and gene transfects before and after the illumination after ternary complex, binary complex package DNA,
It can be found that when carrying out short photoperiod to ternary complex from Figure 10, the transfection of ternary complex has compared to non-illumination
And significantly improve, it was demonstrated that ternary complex have good light can activity, promote light power rupture of membranes, be conducive to compound escape
Endosome.
Figure 11 is the fluorogram of the lysosome escape before and after ternary complex illumination on B16F10 cells, can from Figure 11
After finding illumination, the overlapping of red and green fluorescence is reduced, it was demonstrated that is had more compounds to escape out endosome, be may advantageously facilitate
Gene transfects.
Figure 12 is the fluorogram that the DNA before and after ternary complex illumination is discharged from compound, from figure on B16F10 cells
In 12 it can be found that after illumination, the overlapping of red and green fluorescence is reduced, it was demonstrated that has more DNA to be discharged into cell from compound
In matter, gene transfection may advantageously facilitate.
Figure 13 is embodiment two on B16F10 cells, and comparative example one, 25k PEI and 600 PEI pass through dioxygen water pretreatment
The cytotoxicity of polymer material after 24 hours, it can be found that embodiment two and 600 PEI are with similar thin from Figure 13
Born of the same parents' survival rate, when polymer concentration reaches 0.1mg/mL, cell survival rate can still maintain 90% or more, and comparative example one
Cell survival rate shows apparent cytotoxicity with the increase of polymer concentration.Prove sound of the embodiment two to hydrogen peroxide
Answering property, which is degraded, can improve the biocompatibility of material, reduce toxic side effect.
Figure 14 is on B16F10 tumor models, and ternary complex wraps up pattern DNA(luc)With treatment DNA (p53), short
Tumor suppression curve graph under illumination and long illumination condition.It is seen from figure 14 that compared to single gene therapy or single light
The optical dynamic therapy effect that dynamic therapy, adjunctive therapeutic DNA and long illumination generate can reach the life for best inhibiting tumour
It is long.
Ternary light controlled release nano-delivery system provided by the invention containing photosensitizer and thioketal sensitivity key, design
With feux rouges response ternary complex can be as nucleic acid drug carrier and with good stability, biodegradability
With light can activity, in nucleic acid drug drug delivery system have good application.
Claims (10)
1. a kind of ternary complex Nano medication, including Contracting thioketones key are crosslinked low molecular weight PEI based polyalcohols, are connected to photosensitizer
Hyaluronic acid polymer, nucleic acid drug;
The Contracting thioketones key crosslinking low molecular weight PEI based polyalcohols have structure shown in Formulas I;
Wherein, 20≤n≤500,4≤m≤24;
The hyaluronic acid polymer for being connected to photosensitizer has structure shown in formula II;
Wherein, R2For photosensitizer unit.
2. a kind of Contracting thioketones key is crosslinked low molecular weight PEI based polyalcohols, the Contracting thioketones key is crosslinked low molecular weight PEI based polyalcohols
With structure shown in Formulas I,
Wherein, 20≤n≤500,4≤m≤24.
3. a kind of thioketal crosslinking agent, with following chemical structural formula:
。
4. a kind of preparation method of Contracting thioketones key crosslinking low molecular weight PEI based polyalcohols, includes the following steps:
(1)Using cysteamine, methoxyl group propylene, acryloyl chloride as raw material, reaction prepares thioketal crosslinking agent;
(2)Using thioketal crosslinking agent and PEI as raw material, reaction prepares Contracting thioketones key and is crosslinked low molecular weight PEI based polyalcohols.
5. a kind of preparation method of thioketal crosslinking agent, includes the following steps:
(1)Using cysteamine, methoxyl group propylene, acryloyl chloride as raw material, reaction prepares thioketal crosslinking agent.
6. being connected to the hyaluronic acid polymer of photosensitizer, there is structure shown in formula II;
Wherein, R2For photosensitizer unit.
7. a kind of preparation method for the hyaluronic acid polymer being connected to photosensitizer, includes the following steps, acetic anhydride is added transparent
In the mixture of matter acid and pyridine, acetylation hyaluronic acid is obtained by the reaction;Mixing photosensitizer, carbodiimide, 4- dimethylamino pyrroles
Pyridine is added acetylation hyaluronic acid, the hyaluronic acid polymer for being connected to photosensitizer is obtained by the reaction after stirring.
8. a kind of preparation method of ternary complex Nano medication, includes the following steps, by Contracting thioketones key crosslinking low molecular weight PEI
Based polymer solution adds the hyaluronic acid polymer solution for being connected to photosensitizer after mixing incubation with nucleic acid drug solution, again
It is incubated, obtains ternary complex Nano medication;
The Contracting thioketones key crosslinking low molecular weight PEI based polyalcohols have structure shown in Formulas I;
Wherein, 20≤n≤500,4≤m≤24;
The hyaluronic acid polymer for being connected to photosensitizer has structure shown in formula II;
Wherein, R2For photosensitizer unit.
9. the preparation method of ternary complex Nano medication according to claim 8, which is characterized in that the nucleic acid drug choosing
From DNA, RNA, oligonucleotide or polynucleotides;The Contracting thioketones key crosslinking low molecular weight PEI based polyalcohols and nucleic acid medicine
The mass ratio of object is(0.1~10)∶1;The mass ratio of the hyaluronic acid polymer for being connected to photosensitizer and nucleic acid drug is(0.1
~10)∶1.
10. being connected to described in Contracting thioketones key crosslinking low molecular weight PEI based polyalcohols and/or claim 6 described in claim 2 photosensitive
Application of the hyaluronic acid polymer of agent in preparing nucleic acid drug carrier;Or Contracting thioketones key crosslinking described in claim 2 is low
Be connected to described in molecular weight PEI based polyalcohols, claim 6 photosensitizer hyaluronic acid polymer or claim 1 described in three
Application of first compound Nano medication in preparing genomic medicine is especially preparing light controlled release nano gene drug delivery
Application in system.
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