CN105920618A - Biological targeting nano-gene material and manufacturing method thereof - Google Patents
Biological targeting nano-gene material and manufacturing method thereof Download PDFInfo
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- CN105920618A CN105920618A CN201610420617.8A CN201610420617A CN105920618A CN 105920618 A CN105920618 A CN 105920618A CN 201610420617 A CN201610420617 A CN 201610420617A CN 105920618 A CN105920618 A CN 105920618A
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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
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
Abstract
The invention provides a nano-gene material. The nano-gene material is characterized by comprising a targeting nano-gene substrate, the nano-gene substrate is prepared by amino acid polymer, modified polyethylene glycol and angiotensin II polypeptide, wherein the amino acid polymer and the angiotensin II polypeptide are chemically reacted with modified groups at two ends of the modified polyethylene glycol to produce the targeting nano-gene substrate. The novel nano-gene material provided has targeting to ischemic myocardium and protection for genes, excellent transfection effect and low toxicity.
Description
Technical field
The present invention relates to genophore field, in particular it relates to a kind of biological targeting nano gene material and manufacturer thereof
Method.
Background technology
In recent years, one of disease that in cardiovascular disease has become as world wide, M & M is the highest.Although
Gene therapy has been achieved for gratifying effect, but safely and effectively transmission carrier remains bottleneck place.Commonly use
Cardiac muscle transport vehicle can be divided into viral vector and non-virus carrier.Owing to having higher transfection efficiency, the application of viral vector
Still there is absolute advantages.But, the organism immune response caused due to viral vector is stronger so that it is clinical practice system
About.Effectively supplementing as viral vector, the research of non-virus carrier is the most deep in recent years.
Non-virus carrier possesses without infectiousness, does not has bearer capabilities to limit, and material source is extensive, and chemical constitution can control,
And be prone to prepare in a large number, gene can be loaded, such as: antisense oligonucleotide, have the irreplaceable effect of viral vector.
But, it is low often to there is transfection efficiency in current non-virus carrier, and genes of interest majority can only realize Transient Expression,
The granule of its delivery system is relatively big, easily causes immunoreation and is removed by body.
For this defect, in the existing basic research to non-virus carrier, the carrier (matchmaker that in-vitro transfection is the most frequently used
It is situated between) include cationic-liposome and cationic polymer, such as: Lipo2000, polymine PEI25000, PEI18000 etc.,
This series products is all one of the most generally acknowledged commercialization transfection reagent with relatively high transfection efficiency to various kinds of cell, so often quilt
It is used as evaluating the standard of other siRNA delivery vector effectiveness.
But, research shows, life-time service Lipofectamine 2000TMSeries of products deliver siRNA can increase cell
Autophagy level, induce internal inflammation.In addition the plasma stability of liposome is poor, in blood particle diameter, surface potential and lipid
The trend that composition all changes.And the bio-toxicity of PEI series of products is obvious.These all limit liposome and PEI in clinic
On application.
Thus, it is badly in need of at present a kind of transfection good, good stability, and avirulence or the relatively low carrier of toxicity.
Additionally, currently in the treatment product of cardiovascular disease, for loading the carrier of gene, often when treatment without
Targeting, it is thus impossible to the problem solving the targeted therapy in the case of diseased region enrichment.
Summary of the invention
It is contemplated that overcome drawbacks described above, it is provided that a kind of novel have protective effect, transfection to gene good, malicious
Property is extremely low, realize receiving of targeted therapy effect by being also associated with having the polypeptide A T1 of ischemic myocardium targeting on carrier
Rice genetic material.
The nano gene material that the present invention provides, it is characterised in that: include the nano-gene carrier with targeting；Above-mentioned
Nano-gene carrier is prepared from by amino acid polymer, modified Polyethylene Glycol, the functional sequence polypeptide of angiotensinⅡ；
Wherein, the functional sequence polypeptide of above-mentioned amino acid polymer and angiotensinⅡ respectively with modified Polyethylene Glycol
The nano-gene carrier with targeting is obtained after the modification group generation chemical reaction at two ends.
Further, the nano gene material of the present invention, also have a characteristic that above-mentioned amino acid polymer is for by relying
Propylhomoserin, serine, arginine, histidine, aspartic acid, glutamic acid, alanine, valine, leucine, isoleucine, dried meat ammonia
In acid, phenylalanine, tryptophan, methionine, glycine, threonine, cysteine, tyrosine, agedoite, glutamine
One or more in one or more amino acids formed polymer or copolymer.
It preferably is selected from the polymer prepared by lysine, or includes lysine that mass percentage content is 45-80%
Copolymer.
Further, the nano gene material of the present invention, also have a characteristic that the knot of above-mentioned modified Polyethylene Glycol
Structure is as follows:
Wherein, m is the integer of 80-500；
R1For amide groups, alkylene, cycloalkenyl group, heterocyclic radical with double bond；
The structural formula of above-mentioned amide groups is as follows:
I.e., the structure of modified Polyethylene Glycol is as follows:
R3For alkylene, cycloalkenyl group, heterocyclic radical with double bond；
The structural formula of above-mentioned alkylene is as follows:
I.e., the structure of modified Polyethylene Glycol is as follows:
N1 and n2 is the integer of 0 to 20；When chain length is the longest, the surface activity of this material is the best.
The structural formula of above-mentioned cycloalkenyl group is as follows:
The above-mentioned structural formula with the heterocyclic radical of double bond is as follows:
R2For ester group；
The structural formula of above-mentioned ester group is as follows:
R4For alkyl, aryl, heterocyclic radical；
The structural formula of above-mentioned heterocyclic radical is as follows:
Rx1-20Selected from hydrogen, alkyl, alkoxyl, amino or C-Rx1-20For carbonyl；
Rx1-20Preferably hydrogen, methyl, ethyl, methoxyl group, amino and C-Rx1-20For carbonyl；
Y is nitrogen, oxygen, sulfur；
N is the integer of 0-20, and when chain length is the longest, the surface activity of this material is the best.
Further, the nano gene material of the present invention, also have a characteristic that the polymerization of above-mentioned amino acid polymer
Degree is 50-500；It is preferably 80-160；
Above-mentioned amino acid polymer molecular weight be 10000-100000；It is preferably more than 15000；
The molecular weight of above-mentioned modified Polyethylene Glycol is 1000-10000；It is preferably more than 1500.
Additionally, present invention also offers the preparation method of above-mentioned nano gene material, it is characterised in that:
Step one, configuration reactant:
Amino acid polymer is dissolved in solvent and is configured to solution A；
Modified Polyethylene Glycol is dissolved in solvent and is configured to solution B；
The functional sequence polypeptide of angiotensinⅡ is dissolved in solvent and is configured to solution C；
Step 2, solution B is added dropwise in solution A, at a temperature of 10-50 DEG C, reacts 1-5 hour；
Step 3, protection gas shielded under conditions of, add solution C, at a temperature of 10-50 DEG C, react 8-24 hour；
Step 4, the solution obtained was dialysed after 24-96 hour, lyophilization.
The preparation method of a kind of nano gene material as above-mentioned in claim 5, it is characterised in that:
Above-mentioned solvent selected from pH be the phosphate buffered saline(PBS) of 7-8, dimethylformamide, dimethyl sulfoxide, oxolane,
One or more in alcohol, ether, ester；
Above-mentioned polylysine, the Polyethylene Glycol of modified are 1 with the mol ratio of the functional sequence polypeptide of angiotensinⅡ:
2-100:1-100；
Preferably, the rubbing of the functional sequence polypeptide of Polyethylene Glycol and the angiotensinⅡ of above-mentioned polylysine, modified
That ratio is 1:2-10:2-8.
Further, the preparation method of the above-mentioned nano gene material that the present invention provides, also have a characteristic that i.e.,
Between step three and four, it is additionally provided with neutralization procedure；
Above-mentioned neutralization procedure is: add the neutralization reagent such as cysteamine in the solution of step 3, in the temperature of 10-50 DEG C
Under, react 1-5 hour；
The preparation method of above-mentioned cysteamine is: be dissolved in by Mercaptamine in secondary water, adds organic base concussion mixed
Close 5-20 minute, after cooling, after using organic solvent extraction, after solvent is evaporated off, obtain cysteamine；
Above-mentioned modified Polyethylene Glycol is 1:50-150 with the mol ratio of Mercaptamine；It is preferably 1::80-100；
Above-mentioned organic base is 1:0.1-10 with the mol ratio of Mercaptamine；It is preferably 1:1-1.5.
Further, the nano gene material of the present invention, also have a characteristic that i.e., above-mentioned there is receiving of targeting
Rice genophore loads gene under electrostatic interaction；
Said gene can be selected from the genes such as antisense oligonucleotide, antisense expression plasmid, expression plasmid；
The process of above-mentioned load is: add in gene by the nano-gene carrier with targeting, in 30-after piping and druming uniformly
0.5-2 hour is hatched under conditions of 40 DEG C；
The mass ratio of the above-mentioned nano-gene carrier and gene with targeting is 1:0.01-15；
The mass ratio of the above-mentioned nano-gene carrier and gene with targeting is preferably 1:1-10.
Further, the nano gene material of the present invention, also have a characteristic that i.e., above-mentioned polyamino acid for polymerization
Degree is the dendroid polylysine of 95-135；
The molecular weight of above-mentioned modified Polyethylene Glycol is 1500-2500, and the modification group at its two ends is respectively NHS and MAL.
Further, the nano gene material of the present invention, also have a characteristic that
Said gene is the gene for curing myocardial ischemia；
Said gene is the antisense oligonucleotide of miRNA-1.
Further, the nano gene material of the present invention, also have a characteristic that the above-mentioned nanometer base with targeting
Because carrier loads the antisense oligonucleotide of miRNA-1 under electrostatic interaction；
The process of above-mentioned load is: added by the nano-gene carrier with targeting in the antisense oligonucleotides of miRNA-1,
Hatch under conditions of 30-40 DEG C 0.5-2 hour after piping and druming uniformly.
The mass ratio of this nano-gene carrier and gene with targeting is 0.01-15:1；It is preferably 0.1-10:1；?
It is preferably 0.5-8:1.
Further, the nano gene material of the present invention, also have a characteristic that above-mentioned nano gene material is for having
The nano-gene carrier of ischemic myocardium targeting.
This nano gene material can also be that the nano gene of the functional sequence polypeptide being not connected with angiotensinⅡ carries
Body, i.e., DGL-PEG/AMO-1, its preparation method and material rate, it is same as DGL-PEG-AT1/AMO-1.
The effect of the present invention and effect:
In the present invention, it is provided that a kind of novel nano-gene carrier, this carrier has the work of ischemic myocardium targeting
With, and toxic and side effects is little.
As it is shown in figure 1, in the present invention, the functional sequence polypeptide of amino acid polymer and angiotensinⅡ respectively with repair
The nano-gene carrier with targeting is obtained after the modification group generation chemical reaction at decorations property Polyethylene Glycol two ends, thereafter, logical
Cross to obtain this nano-gene carrier with antisense oligonucleotide, antisense expression plasmid, expression plasmid even load and there is target function
Nano-gene carrier.
Specifically, aminoacid, as constituting the ultimate unit of protein molecule in organism, has with biological vital movement
Close relationship.It has special physiological function in matrix, is one of indispensable nutritional labeling in organism.And
At the polyamino acid selected in the present invention, based on amino acid whose fundamental characteristics, after being applied to carrier, remain to have good
The effects such as biocompatibility, the polyamino acid with electric charge is preferred version in the present invention, due to self with electric charge effect
Really, with the material of reversed charge, intermolecular electrostatic force can occur with other, thus realize being similar to the effect of self assembly
Really.
It addition, in the present invention, the DGL (dendroid polylysine) in the most preferably case that polyamino acid is selected, is one
Planting cationic polymer, lysine (essential amino acid) be polymerized, it has the same of good biocompatibility
Time, its biodegradability and the very excellent capacity of compression DNA, there is substantial amounts of amino on its surface, can be modified, and with positive electricity,
After it is by other base group modifications, due to self with positive charge, can with other with negative charge material occur intermolecular
Electrostatic force, thus realize the effect of load factor or antisense oligonucleotide.
Additionally, in the present invention, selection Polyethylene Glycol is as one of reactive component, owing to itself being a kind of excellent surface
Activating agent, after it reacts with the group generation replacement on polyamino acid etc., can realize forming one layer on the surface of polyamino acid
The effect of hydrated sheath, it is thus possible to reduce the toxicity of polyamino acid, reduces its probability swallowed by macrophage system in vivo.
It addition, in the present invention, the PEG of selection is the PEG that two ends are equipped with modification group, thus is passed to this group
On the material such as active group and polyamino acid react, it is achieved between each component united simultaneously, improve the surface of product
Tension force and compatibility.
It addition, in the most preferably scheme of the present invention, its two ends are connected NHS and MAL group respectively, this NHS group can
Amino generation replacements with polyamino acid surface etc. are reacted, and are coupled together by DGL and PEG；And MAL group can be with polypeptide A T1 (blood
The functional sequence polypeptide of angiotensin II) on sulfydryl-HS there is additive reaction, thus polypeptide A T1 with PEG is coupled together,
And then build the annexation of DGL and AT1.
Additionally, the AT1 used in the present invention, it is: when myocardial infarction (myocardial cell hypoxic-ischemic), cell surface
Angiotensin II type I receptor (AT1R) raise.So one section of functional sequence in the part of chemosynthesis AT1R in vitro
(claim AT1) is connected to nano-material surface, can combine the AT1R of ischemic myocardium by targeting, thus have certain target to ischemic myocardium
To effect.
In the present invention, the AT1 polypeptide of external synthesis, its aminoacid sequence is as follows:
Cys-Gly-Gly-Gly-Gly-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe；
The functional sequence of the part of AT1R is as follows:
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe；
Gly-Gly-Gly-Gly in the middle of it is intervening sequence, and Cys contains sulfydryl-HS in being, is the function being connected chemically
Group.
Additionally, in the present invention, use AMO-1 (antisense oligonucleotide of miRNA-1) as the power supply body of negative charge, with
Above-mentioned synthesis base material realizes the result assembled by electrostatic.
MiRNA-1 raises when myocardial infarction or cell hypoxic-ischemic, can promote the apoptosis of myocardial cell, is that cardiac muscle is thin
The infringement factor of born of the same parents.And AMO-1 refers to the antisense oligonucleotide of miRNA-1, it is the strand complementary with miRNA of chemosynthesis
RNA.It can occur complementary pairing with ripe miRNA-1 in Cytoplasm so that the detrimental effect of miRNA-1 weakens, above-mentioned
The sequence of each compound is as follows:
MiRNA-1 sequence: 5 '-UGGAAUGUAAAGAAGUGUGUAU-3 '
AMO-1 sequence: 5 '-AUACACACUUCUUUACAUUCCA-3 '
In a preferred approach, AMO-1 is electronegative, can pass through electrostatic interaction with positively charged DGL-PEG or DGL-PEG-AT1
Form nano-particle.
Accompanying drawing explanation
Accompanying drawing 1, nano material are formed and the schematic diagram of material structure；
Accompanying drawing 2, nano material nuclear magnetic spectrum；
Particle diameter after accompanying drawing 3, nanomaterial loadings AM0-1 and potential diagram；
Transmission electron microscope picture after accompanying drawing 4, nanomaterial loadings AM0-1；
Accompanying drawing 5, nanomaterial loadings AMO-1 gel electrophoresis figure
Accompanying drawing 6 (a), nano material are to 4 hours comparison diagrams of H9C2 cytotoxicity；
Accompanying drawing 6 (b), nano material are to 8 hours comparison diagrams of H9C2 cytotoxicity；
Accompanying drawing 7 (a1), laser co-focusing observe 4 hours comparison diagrams；
Accompanying drawing 7 (a2), 4 hours comparison diagrams of FCM analysis；
Accompanying drawing 7 (b1), laser co-focusing observe 8 hours comparison diagrams；
Accompanying drawing 7 (b2), 8 hours comparison diagrams of FCM analysis；
Accompanying drawing 8, dead Coloration experiment Comparative result figure of living；
Accompanying drawing 9, primary cardiomyocytes immunofluorescence dyeing comparison diagram；
Accompanying drawing 10, primary cardiomyocytes horizontal stretcher experimental result, the comparison diagram of each process group miRNA-1 amount；
Wherein, matched group: normal cell (normal group) gives DEPC water, hypoxic cell (control group) gives DEPC
Water, hypoxic cell (NC group) give DGL-PEG-AT1/AMO-1-NC；Experimental group: hypoxic cell (PEI group) gives PEI/AMO-
1, hypoxic cell (DGL-PEG group) gives DGL-PEG/AMO-1, hypoxic cell (DGL-PEG-AT1 group) gives DGL-PEG-
AT1/AMO-1。
Detailed description of the invention
1, raw material and the preparation of equipment
1) raw material:
Dendroid polylysine (Dendrigraft poly-L-lysines, DGL)；
End maleimide end N-hydroxy-succinamide Polyethylene Glycol (α-Malemidyl-ω-N-
Hydroxysuccinimidyl polyethylene glycol, NHS-PEG2000-MAL)；
AT1 (target polypeptide sequence:
Cys-Gly-Gly-Gly-Gly-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe)；
Mercaptamine；Dichloromethane, triethylamine.
2) equipment:
Constant volume bottle (1000mL, 2000mL), beaker (500mL, 2L, 5L), 2 flasks (25mL), magneton, magnetic stirring apparatus
(can displays temperature and rotating speed), 1 bottle stopper, 1 rubber lid, 2 syringe needles (length), 2 rubber tubes, MWCO:8000-14000
Bag filter.
2, reagent prepares (needing can also configure the buffer solution of other concentration and pH according to actually used)
1) the PBS stock solution (pH=7.4) of 1000mL 0.2M is configured
Prepare: 250mL graduated cylinder, 500mL beaker, the constant volume bottle of 1000mL, secondary water, Na2HPO4-12H2O,NaH2PO4-
2H2O
Concrete steps:
A) Na is weighed respectively2HPO4-12H2O58.01868g, NaH2PO4-2H2O5.92838g, the burning of 500mL is put in mixing
In Bei, add the secondary water of 500mL, be sufficiently stirred for dissolving.
B) solution in beaker is poured in the constant volume bottle of 1000mL, add secondary water and be settled to 1000mL.(can be by secondary
Water is initially charged in beaker flushing walls of beaker, is then added in constant volume bottle, because flask walls may have the residual of part solute)
C) survey pH with accurate pH test paper, confirm that pH value is at about 7.4 (7.2-7.4)
2) PBS of the 10mM of configuration 500mL
A) taking 2.5mL stock solution, constant volume to 50mL (dilutes 20 times)---and do reaction and use
B) taking 100mL stock solution, the dialysis of constant volume to 2000ml---is used
(note: dialyse below and the PBS concentration reacted is all 10mM)
Embodiment one, the synthesis of DGL-PEG
A) DGL (MW:25000) weighing 22mg (1 μm ol) is dissolved in 5mLPBS buffer (10mM；
PH=7.4) (concentration 0.2 μm ol/mL) (after weighing, solid DGL being placed in 10mLep pipe)；
In the present embodiment, it be also possible to use the DGL of other molecular weight, such as: MW=10000,18000,20000,27000,
30000,50000,80000 or 100000.
B) NHS-PEG2000-MAL (MW:2000) weighing 100mg (50 μm ol) is dissolved in the DMSO of 5mL；
In the present embodiment, it be also possible to use the NHS-PEG2000-MAL of other molecular weight, such as: MW=1000,1800,
2100,2700,3000,4500,6500,8000 or 10000.
In the present embodiment, in the process of scientific research of reaction condition, always according to needing the difference of substituted radical, for DGL
Reaction ratio with NHS-PEG2000-MAL is adjusted, such as: the mol ratio of DGL Yu NHS-PEG2000-MAL is 1:2,1:5,1:
10,1:15,1:25,1:30,1:40,1:45,1:50,1:55,1:60,1:65,1:70,1:80,1:100.
In the present embodiment, in the process of scientific research of reaction condition, always according to the needs of reaction, the concentration to reactant liquor
It is adjusted, such as: concentration is 1 μm ol/ml, 10 μm ol/ml, 50 μm ol/ml, 100 μm ol/ml, 120 μm ol/ml, 150 μm ol/
Ml, 210 μm ol/ml, 650 μm ol/ml, 1000 μm ol/ml, 1200 μm ol/ml, 1500 μm ol/ml.In general, when
In the case of the reaction position more (replacements more than 20) that substitution reaction occurs, the concentration of more than 100 μm ol/ml is used to carry out instead
Should, in the case of the reaction position less (replacements less than 20) that substitution reaction occurs, use the concentration of below 100 μm ol/ml
React；
C) in b) being slowly added dropwise a) (under the mixing condition of 500-1000 rev/min), 55 DEG C of reaction 3h；
In the present embodiment, in the process of scientific research of reaction condition, always according to replacing the difference of quantity, it reacts temperature
Degree can be adjusted to 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, its response time is 0.5 hour, 1 hour,
1.5 hours, 2 hours, 2.5 hours.In general, the reaction position more (replacements more than 20) occurred when substitution reaction
In the case of, the response time is more than 2 hours, in the case of the reaction position less (replacements less than 20) that substitution reaction occurs, instead
Less than 2 hours between Ying Shi, react；
D) weighing 90.88mg (800 μm ol) Mercaptamine (MW:113.61) is in the ep pipe of 20mL, adds 15mL
Secondary water, carries out ultrasonic dissolution at twice；
In the present embodiment, in the process of scientific research of reaction condition, according to needing substituted radical, the difference of replacement quantity,
For NHS-PEG-MAL: the reaction ratio of Mercaptamine can be adjusted, NHS-PEG-MAL: Mercaptamine mole
Ratio is 1:50,1:60,1:70,1:80,1:85,1:95,1:110,1:120,1:130,1:140,1:150.In general,
In the case of the reaction position more (replacements more than 20) that substitution reaction occurs, mol ratio uses the ratio less than 1:85, when
In the case of the reaction position less (replacements less than 20) that substitution reaction occurs, mol ratio uses the ratio more than 1:85；
E) until completely dissolved, triethylamine (MW:101.19 is added；Density: 0.726g/mL) 65.8 μ L, fully shake
5min (shakes with hand, some heating understood by ep pipe).
In the present embodiment, in the process of scientific research of reaction condition, Mercaptamine: the mol ratio of triethylamine is permissible
It is adjusted to 1:0.5,1:0.8,1:1,1:1.5,1:1.9,1:2,1:5,1:10.
F) after question response is complete, adds 10mL dichloromethane, repeatedly extract, obtain after the extract of acquisition is removed under reduced pressure
Solid；The solid obtained is dissolved in 5mLPBS (10mM, pH=7.4), adds in the product of c, react 3h in 55 DEG C.
In the present embodiment, in the process of scientific research of reaction condition, always according to replacing the difference of quantity, it reacts temperature
Degree can be adjusted to 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, its response time is 0.5 hour, 1 hour,
1.5 hours, 2 hours, 2.5,3 hours, 4 hours, 4.5 hours, 5 hours.In general, when the reaction that substitution reaction occurs
In the case of position more (replacements more than 20), the response time is more than 2 hours, and the reaction position occurred when substitution reaction is less (few
In the replacement of 20) in the case of, the response time is less than 2 hours, reacts；
G) solution final in d) is added the 48h that dialyses in a water in the bag filter of MWCO=8000-14000Da.
In the present embodiment, in the process of scientific research of reaction condition, always according to the needs of reaction, the time of dialysis is permissible
It it is 12 hours, 36 hours, 56 hours, 72 hours, 96 hours；
H) lyophilizing.
Embodiment two, the synthesis of DGL-PEG-AT1
Reaction equation is as follows:
A) DGL (MW:25000) weighing 22mg (1 μm ol) is dissolved in 5mLPBS buffer (10mM；PH=7.4) (concentration
0.2 μm ol/mL) (after weighing, solid DGL being placed in 10mLep pipe)；
In the present embodiment, it be also possible to use the DGL of other molecular weight, such as: MW=10000,18000,20000,27000,
30000,50000,80000 or 100000.
B) NHS-PEG2000-MAL (MW:2000) weighing 100mg (50 μm ol) is dissolved in the DMSO of 5mL；
In the present embodiment, it be also possible to use the NHS-PEG2000-MAL of other molecular weight, such as: MW=1000,1800,
2100,2700,3000,4500,6500,8000 or 10000.
In the present embodiment, in the process of scientific research of reaction condition, always according to needing the difference of substituted radical, for DGL
Reaction ratio with NHS-PEG2000-MAL is adjusted, such as: the mol ratio of DGL Yu NHS-PEG2000-MAL is 1:2,1:5,1:
10,1:15,1:25,1:30,1:40,1:45,1:50,1:55,1:60,1:65,1:70,1:80,1:100.
In the present embodiment, in the process of scientific research of reaction condition, always according to the needs of reaction, the concentration to reactant liquor
It is adjusted, such as: concentration is 1 μm ol/ml, 10 μm ol/ml, 50 μm ol/ml, 100 μm ol/ml, 120 μm ol/ml, 150 μm ol/
Ml, 210 μm ol/ml, 650 μm ol/ml, 1000 μm ol/ml, 1200 μm ol/ml, 1500 μm ol/ml.In general, when
In the case of the reaction position more (replacements more than 20) that substitution reaction occurs, the concentration of more than 100 μm ol/ml is used to carry out instead
Should, in the case of the reaction position less (replacements less than 20) that substitution reaction occurs, use the concentration of below 100 μm ol/ml
React；
C) in b) being slowly added dropwise a) (under the mixing condition of 500-1000 rev/min), 55 DEG C of reaction 3h；
In the present embodiment, in the process of scientific research of reaction condition, always according to replacing the difference of quantity, its reaction
Temperature can be adjusted to 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, and its response time is 0.5 hour, 1 little
Time, 1.5 hours, 2 hours, 2.5 hours.In general, when more (the taking more than 20 in reaction position that substitution reaction occurs
Generation) in the case of, the response time is more than 2 hours, when the situation of the reaction position less (replacements less than 20) that substitution reaction occurs
Under, the response time is less than 2 hours, reacts；
D) 35.88mg (25 μm ol) AT is weighed1(MW:1377.55, purity 96%) is dissolved in 500 μ L DMSO；
In the present embodiment, in the process of scientific research of reaction condition, always according to needing the difference of substituted radical, for DGL
Reaction ratio with AT1 is adjusted, such as: the mol ratio of DGL Yu AT1 is 1:1,1:5,1:10,1:15,1:25,1:30,1:40,
1:45,1:50,1:55,1:60,1:65,1:70,1:80,1:100.
In the present embodiment, in the process of scientific research of reaction condition, always according to the needs of reaction, the concentration to reactant liquor
It is adjusted, such as: concentration is 1 μm ol/ml, 10 μm ol/ml, 50 μm ol/ml, 100 μm ol/ml, 120 μm ol/ml, 150 μm ol/
Ml, 210 μm ol/ml, 650 μm ol/ml, 1000 μm ol/ml, 1200 μm ol/ml, 1500 μm ol/ml.In general, when
In the case of the reaction position more (replacements more than 20) that substitution reaction occurs, the concentration of more than 100 μm ol/ml is used to carry out instead
Should, in the case of the reaction position less (replacements less than 20) that substitution reaction occurs, use the concentration of below 100 μm ol/ml
React；
E), in d) adding c), nitrogen (or helium, argon etc.) is protected, room temperature reaction 20h.
In the present embodiment, in the process of scientific research of reaction condition, need its response time is entered always according to react
Row sum-equal matrix, its response time can also be 8 hours, 10 hours, 15 hours, 20 hours, 24 hours.In general, when taking
In the case of the reaction position more (replacements more than 20) that generation reaction occurs, the response time is more than 15 hours, when substitution reaction is sent out
In the case of raw reaction position less (replacements less than 20), the response time is less than 15 hours, reacts；
F) weighing 90.88mg (800 μm ol) Mercaptamine (MW:113.61) is in the ep pipe of 20mL, adds 15mL
Secondary water, carries out ultrasonic dissolution at twice；
In the present embodiment, in the process of scientific research of reaction condition, according to needing substituted radical, the difference of replacement quantity,
For NHS-PEG-MAL: the reaction ratio of Mercaptamine can be adjusted, NHS-PEG-MAL: Mercaptamine mole
Ratio is 1:50,1:60,1:70,1:80,1:85,1:95,1:110,1:120,1:130,1:140,1:150.In general,
In the case of the reaction position more (replacements more than 20) that substitution reaction occurs, mol ratio uses the ratio less than 1:85, when
In the case of the reaction position less (replacements less than 20) that substitution reaction occurs, mol ratio uses the ratio more than 1:85；
G) until completely dissolved, triethylamine (MW:101.19 is added；Density: 0.726g/mL) 65.8 μ L, fully shake
5min (shakes with hand, some heating understood by ep pipe).
In the present embodiment, in the process of scientific research of reaction condition, Mercaptamine: the mol ratio of triethylamine is permissible
It is adjusted to 1:0.5,1:0.8,1:1,1:1.5,1:1.9,1:2,1:5,1:10.
H) after question response is complete, adds 10mL dichloromethane, repeatedly extract, obtain after the extract of acquisition is removed under reduced pressure
Solid；The solid obtained is dissolved in 5mLPBS (10mM, pH=7.4), adds in the product of e, react 3h in 55 DEG C.
In the present embodiment, in the process of scientific research of reaction condition, always according to replacing the difference of quantity, its reaction
Temperature can be adjusted to 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, and its response time is 0.5 hour, 1 little
Time, 1.5 hours, 2 hours, 2.5,3 hours, 4 hours, 4.5 hours, 5 hours.In general, when substitution reaction generation
In the case of reaction position more (replacements more than 20), the response time is more than 2 hours, and the reaction position occurred when substitution reaction is less
In the case of (replacements less than 20), the response time is less than 2 hours, reacts；
I) solution final in f) is added the 48h that dialyses in PBS in the bag filter of MWCO=8000-14000Da, then
Water is dialysed 48 hours.
In the present embodiment, in the process of scientific research of reaction condition, always according to the needs of reaction, the time of twice dialysis
It can be 12 hours, 36 hours, 56 hours, 72 hours, 96 hours, 120 hours；
F) lyophilizing
Note: in a water after dialysis, can produce a little precipitation, take supernatant, lyophilizing after being centrifuged, and precipitation discards.
Embodiment three, load antisense oligonucleotide
In the present embodiment, selecting AMO-1, nano material (NHS-PEG or NHS-PEG-MAL) and AMO-1 are according to quality
Load than (20:1).Nano material and AMO-1 are dissolved in DEPC water, in the ep pipe of 200 μ L, are initially charged AMO-1, add material
Material, piping and druming mixing, vortex 30s is placed in 47 DEG C of incubators and hatches 1 hour.(principle: electrostatic interaction；DGL positively charged, AMO-1 carries
Negative electricity)
(note 1:miRNA-1 raises when Myocytes Anoxia, and the apoptosis to promotion myocardial cell, is the damage of myocardial cell
Noxa.AMO-1 refers to the antisense oligonucleotide of miRNA-1, is the single stranded RNA complementary with miRNA of chemosynthesis.It is carefully
Kytoplasm can occur complementary pairing with ripe miRNA-1 so that the detrimental effect of miRNA-1 weakens.)
(note 2:miRNA-1 sequence: 5 '-UGGAAUGUAAAGAAGUGUGUAU-3 '
AMO-1 sequence: 5 '-AUACACACUUCUUUACAUUCCA-3 ')
In the present embodiment, in the process of scientific research of reaction condition, difference according to demand, for nano material:
The reaction ratio of AMO-1 can be adjusted, the mass ratio of AMO-1: nano material is 1:1,1:5,1:8,1:10,1:15,1:25,1:
30,1:40,1:50,1:60,1:65,1:70,1:80,1:90,1:100.
Embodiment four, physico-chemical analysis and sign
The product of this sign is the carrier using the method for embodiment one and the method for embodiment two to obtain.
Wherein, DGL-PEG is the product that mol ratio is 1:5 of the Polyethylene Glycol selecting polylysine, modified；
DGL-PEG-AT1 be select polylysine, modified Polyethylene Glycol many with the functional sequence of angiotensinⅡ
The mol ratio of peptide is the product of 1:5:2.5；
(1)1HNMR detects collection of illustrative plates
As shown in Figure 2, a (δ=1-1.8ppm) is 3 CH on lysine2On H, be the characteristic peak of DGL.B (δ=
3.6ppm) it is the repetitive sequence O-CH of PEG2-CH2-O, d are the H on MAL for (δ=6.8ppm), and e (δ=2.6ppm) is on NHS
H, be the characteristic peak of MAL-PEG-NHS.C is the H on AT1 phenyl ring, is the characteristic peak of AT1.A in DGL-PEG, b peak exists,
Illustrating that PEG and DGL connects, e peak disappears, and illustrates that NHS reacts；D peak disappears, and illustrates that Mercaptamine and MAL occur anti-
Should.DGL-PEG-AT1 occurs in that c peak, illustrates that AT1 is successfully connected on DGL-PEG-MAL.Thus infer, DGL, NHS-
PEG-MAL, AT1 are successfully connected together.
(2) particle diameter and current potential
After the DGL-PEG-AT1 of synthesis is loaded AMO-1 in DEPC water (mass ratio of nano material/AMO-1 is 8:
1), as it is shown on figure 3, detect its particle diameter about 150nm, current potential about+3mv with particle size analyzer.
(3) Electronic Speculum
As shown in Figure 4, Electronic Speculum result shows: DGL-PEG-AT1/AMO-1 (mass ratio is 8:1) non-hydrated particle diameter is
50nm。
(4) mensuration of material load ability
DGL-PEG-AT1/AMO-1 respectively with mass ratio for 0,0.5,1,2,4,6,8 is supported on together.3% agarose coagulates
Gel electrophoresis runs glue, dyes with SYBR II.(principle: naked AMO-1 is electronegative, can run out of in gel electrophoresis, can be contaminated by SYBR II
Color；When DGL-PEG-AT1 wraps up AMO-1, AMO-1 surface negative electricity reduces, and the minimizing run out of in electrophoresis, if being wrapped completely
Wrapping up in, AMO-1 can not run out of in well.)
As shown in Figure 5: naked AMO-1 has run out of well (white ribbon), when DGL-PEG-AT1/AMO-1 mass ratio is
During 2:1, occur in that traction phenomenon, illustrate that AMO-1 is wrapped up by material, but also do not have fully wrapped around, work as DGL-PEG-AT1/
When AMO-1 mass ratio is 8:1, do not see that AMO-1 runs out of well, well also has no white ribbon, illustrates that AMO-1 is complete
Full parcel.When gel electrophoresis runs 15min, it is seen that the band of naked AMO-1 group disappears (possible AMO-1 has degraded) substantially, but
The hole of 2:1 is still it can be seen that the white ribbon of traction, and illustrative material has certain protective role to AMO-1, can to a certain degree reduce
The degraded of AMO-1.
(5) cytotoxicity experiment
By cladodification PEI (bPEI), DGL, DGL-PEG, DGL-PEG-AT1 are each configured to 6 concentration: 1mg/mL,
500ug/mL, 200ug/mL, 100ug/mL, 50ug/mL, 20ug/mL, acted on H9C2 cell, after 4h or 8h, uses
Mtt assay detects its impact (cytotoxicity) on cell proliferation.
As shown in Fig. 6 (a) and 6 (b): along with material concentration and the increase of action time, cytoactive is gradually lowered.bPEI
Toxicity be significantly greater than DGL, DGL-PEG-MAL, DGL-PEG-AT1.When material concentration is less than 100ug/mL, DGL-PEG-MAL,
The cytoactive of DGL-PEG-AT1 effect is all higher than 80% (toxicity is less), when material concentration is more than 100ug/mL, to cell toxicant
Property substantially increases.Optional 100ug/mL is as successive treatment amount.
(6) cell phagocytosis experiment
Checking H9C2 cell is to PEI (3.9ug/2mL), DGL-PEG (24ug/2mL), DGL-PEG-AT1 (24ug/2mL)
The phagocytosis effect of load AMO-1-FAM (3ug/2mL).
As shown in Fig. 7 (a1), 7 (b1), 7 (a2) and 7 (a2): in A1, B1, blueness is nucleus (DAPI dyeing), and green is
AMO-1-FAM, is positioned at Cytoplasm or nucleus position.Laser co-focusing shows, during effect 8h, and PEI, DGL-PEG, DGL-
PEG-AT1 load AMO-1-FAM group H9C2 cell in, green fluorescence all than 4h time be remarkably reinforced.
During 8h, the green fluorescence of DGL-PEG and DGL-PEG-AT1 group relatively PEI group is strong.FCM analysis result display knot
Fruit is consistent therewith.
Illustrating, the AMO-1 phagocytosis effect relatively PEI that DGL-PEG, DGL-PEG-AT1 are loaded by H9C2 cell is strong, i.e. DGL-
PEG, DGL-PEG-AT1 group is many compared with the AMO-1 that PEI group enters cell.
(7) live dead Coloration experiment
As shown in Figure 8: redness is dead cell (Ethidiumhomodimer-1 dyeing), and green is living cells (Calcein
AM dyes).(owing to Fig. 8 showing, the AMO-1 phagocytosis effect that during cell 8h, DGL-PEG, DGL-PEG-AT1 are loaded by H9C2 cell
Fruit relatively PEI is strong, and the cytotoxicity experiment result further according to Fig. 7 shows that PEI toxicity is big, thus it is speculated that, the AMO-1 that PEI is loaded by cell
Phagocytosis is weak, and to be likely due to PEI toxicity big, in order to verify that this is it is assumed that by the condition same with in material Fig. 8 further
Under, dead dyeing of laboring.) result shows, PEI group and DGL-PEG, DGL-PEG-AT1 compares, and cell is sparse, and red fluorescence
Many compared with green fluorescence, infer the weak reason of cell AMO-1 phagocytosis that PEI is loaded to be that PEI is to cytotoxicity accordingly
Greatly.
(8) cardiac muscle primary cell immunofluorescence dyeing
When myocardial infarction (myocardial cell hypoxic-ischemic), the angiotensin-ii-receptor (AT1R) of cell surface raises.
So one section of functional sequence in the part of chemosynthesis AT1R in vitro is connected to nano-material surface, ischemia can be combined by targeting
The AT1R of cardiac muscle, thus ischemic myocardium is had certain targeting.
Whether raise for AT1R during checking SD Neonatal Rat Primary Cardiomyocytes hypoxic-ischemic, primary cardiomyocytes is done at anoxia
After reason, carry out immunofluorescence dyeing.
As shown in Figure 9: redness represents AT1R, being positioned at surface of cell membrane, blueness represents nucleus.Hypoxic cell surface red
Color fluorescence substantially increases, and the AT1R of the SD Neonatal Rat Primary Cardiomyocytes that thus can be further characterized by anoxia raises.
(9) primary cardiomyocytes horizontal stretcher experiment
With PEI (3.9ug/2mL), DGL-PEG (24ug/2mL), DGL-PEG-AT1 (24ug/2mL) load AMO-1
(3ug/2mL) with the SD Neonatal Rat Primary Cardiomyocytes (1%O of anoxia2, 24h, low sugar DMEM) co-culture 4h after, in normal condition
Under (21%O2, low sugar DMEM) and cultivate 24h, extract total serum IgE, detect the change of its miRNA-1 with Real-time PCR.
As shown in Figure 10: * represents P 0.05, n=3, such as figure, when hypoxic cell (control) group is not treated, miRNA-1
Compared with normal cell (normal) is organized significantly raised；The miRNA-1 of DGL-PEG, DGL-PEG-AT1 group relatively hypoxic cell
(control) group is decreased obviously (having reached therapeutical effect), and DGL-PEG-AT1 group reduces brighter compared with DGL-PEG group miRNA-1
Show and (be likely to be due to the AT1 targeting effect to hypoxic cell so that after DGL-PEG-AT1 load AMO-1, taken the photograph by myocardial cell
Take is more, and therapeutical effect becomes apparent from).But, PEI group does not the most lower miRNA-1, makes miRNA-1 relatively anoxia in cell
Cell (control) group increases (being likely to be due to its cytotoxic effect cause).
The variation of above-described embodiment:
In the above-described embodiments, with polylysine DGL that the degree of polymerization is 123, the terminal modified base of Polyethylene Glycol two as NHS and
MAL is that raw material has carried out the preparation of genophore.
During reality is researched and developed, the experiment also above two raw material replaced respectively, synthesize based on it
Method duplicates with the method in embodiment two to three, the most no longer does concrete discussion, and specific experiment combination is as shown in the table:
Load capacity refers to the mass ratio of nanometer base material and AMO-1；
Toxicity scoring divides for 0-10, is 10 points with PEI, and DGL-PEG-AT1 is 1 point；
Cell phagocytosis scoring divides for 0-10, is 1 point with PEI, and DGL-PEG-AT1 is 10 points；
Therapeutic effect scoring divides for 0-10, is 1 point with PEI, and DGL-PEG-AT1 is 10 points；
From above-described embodiment it can be seen that this targeted nano genophore can be myocardial ischemia or myocardial infarction patient
Gene therapy provides certain experiment basis.This carrier can load AMO-1, it is possible to load is for other base of curing myocardial ischemia
Cause.
Claims (10)
1. a nano gene material, it is characterised in that: include the nano-gene carrier with targeting；
Described nano-gene carrier is by amino acid polymer, modified Polyethylene Glycol, the functional sequence polypeptide of angiotensinⅡ
It is prepared from；
Wherein, the functional sequence polypeptide of described amino acid polymer and angiotensinⅡ respectively with modified Polyethylene Glycol two ends
Modification group generation chemical reaction after obtain there is the nano-gene carrier of targeting.
2. a kind of nano gene material as claimed in claim 1, it is characterised in that:
Described amino acid polymer is by lysine, serine, arginine, histidine, aspartic acid, glutamic acid, alanine, figured silk fabrics
Propylhomoserin, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, threonine, cysteine, cheese
One or more amino acids formed polymer in propylhomoserin, agedoite, glutamine or one or more in copolymer；
Described amino acid polymer is preferably lysine and serine, arginine, histidine, aspartic acid, glutamic acid, the third ammonia
Acid, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, threonine, half Guang ammonia
One or more amino acids formed copolymers in acid, tyrosine, agedoite, glutamine；Wherein, lysine accounts for copolymerization
The mass percentage content of thing is more than 50%；
Described amino acid polymer is preferably cation amino acid polymer.
3. a kind of nano gene material as claimed in claim 1, it is characterised in that:
The structure of described modified Polyethylene Glycol is as follows:
Wherein, m is the integer of 80-500；
R1For amide groups, alkylene, cycloalkenyl group, heterocyclic radical with double bond；
The structural formula of described amide groups is as follows:
R3For alkylene, cycloalkenyl group, heterocyclic radical with double bond；
The structural formula of described alkylene is as follows:
N1 and n2 is the integer of 0 to 20；
The structural formula of described cycloalkenyl group is as follows:
The described structural formula with the heterocyclic radical of double bond is as follows:
R2For ester group；
The structural formula of described ester group is as follows:
R4For alkyl, aryl, heterocyclic radical；
The structural formula of described heterocyclic radical is as follows:
Rx1-20Selected from hydrogen, alkyl, alkoxyl, amino or C-Rx1-20For carbonyl；
Y is nitrogen, oxygen, sulfur；
N is the integer of 0-10.
4. a kind of nano gene material as claimed in claim 1, it is characterised in that:
The degree of polymerization of described amino acid polymer is 50-500；
Described amino acid polymer molecular weight be 10000-100000；
The molecular weight of described modified Polyethylene Glycol is 1000-10000.
5. the preparation method of a nano gene material, it is characterised in that:
Step one, configuration reactant:
Amino acid polymer is dissolved in solvent and is configured to solution A；
Modified Polyethylene Glycol is dissolved in solvent and is configured to solution B；
The functional sequence polypeptide of angiotensinⅡ is dissolved in solvent and is configured to solution C；
Step 2, solution B is added dropwise in solution A, at a temperature of 10-50 DEG C, reacts 1-5 hour；
Step 3, protection gas shielded under conditions of, add solution C, at a temperature of 10-50 DEG C, react 8-24 hour；
Step 4, the solution obtained was dialysed after 24-96 hour, lyophilization.
The preparation method of a kind of nano gene material the most as claimed in claim 5, it is characterised in that:
Described solvent is in phosphate buffered saline(PBS), dimethylformamide, dimethyl sulfoxide, oxolane, alcohol, ether, ester
One or more；
Described polylysine, the Polyethylene Glycol of modified are 1:2-with the mol ratio of the functional sequence polypeptide of angiotensinⅡ
100:1-100；
Preferably, the mol ratio of the functional sequence polypeptide of described polylysine, the Polyethylene Glycol of modified and angiotensinⅡ
For 1:2-10:2-8.
The preparation method of a kind of nano gene material the most as claimed in claim 5, it is characterised in that:
Between step three and four, it is additionally provided with neutralization procedure；
Described neutralization procedure is: adds cysteamine in the solution of step 3, at a temperature of 10-50 DEG C, reacts 1-5 hour；
The preparation method of described cysteamine is: be dissolved in by Mercaptamine in secondary water, adds organic base concussion mixing 5-
20 minutes, after cooling, after using organic solvent extraction, after solvent is evaporated off, obtain cysteamine；
Described modified Polyethylene Glycol is 1:50-150 with the mol ratio of Mercaptamine；
Described organic base is 1:0.1-10 with the mol ratio of Mercaptamine.
8. a kind of nano gene material as described in claim 1-8 is arbitrary, it is characterised in that:
The described nano-gene carrier with targeting loads gene under electrostatic interaction；
The process of described load is: add in gene by the nano-gene carrier with targeting, in 30-40 DEG C after piping and druming uniformly
Under conditions of hatch 0.5-2 hour；
The mass ratio of the described nano-gene carrier and gene with targeting is 0.01-15:1；
The mass ratio of the described nano-gene carrier and gene with targeting is preferably 0.1-10:1.
The mass ratio of the described nano-gene carrier and gene with targeting is most preferably 0.5-8:1.
9. a kind of nano gene material as described in claim 1-8 is arbitrary, it is characterised in that:
Described polyamino acid be the degree of polymerization be the dendroid polylysine of 95-135；
The molecular weight of described modified Polyethylene Glycol is 1500-2500, and the modification group at its two ends is respectively NHS and MAL；
Described gene is the gene for curing myocardial ischemia；
Described gene is preferably antisense oligonucleotide；
Described gene is preferably the antisense oligonucleotide of miRNA-1.
10. a kind of nano gene material as described in claim 1-9 is arbitrary, it is characterised in that:
Described nano gene material is the nano-gene carrier with ischemic myocardium targeting；
Described nano gene material can also be the nano-gene carrier of the functional sequence polypeptide being not connected with angiotensinⅡ.
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| CN111420068A (en) * | 2019-11-13 | 2020-07-17 | 浙江大学 | Polyethylene glycol-dendritic polylysine/anhydride-cisplatin compound and preparation method and application thereof |
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| US20030147958A1 (en) * | 2002-01-29 | 2003-08-07 | Cheol-Hee Ahn | Biodegradable multi-block copolymers of poly(amino acid)s and poly(ethylene glycol) for the delivery of bioactive agents |
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| JP2008542300A (en) * | 2005-05-24 | 2008-11-27 | ウェルルゲン インコーポレーテッド | Compositions and methods for preventing and treating conditions associated with inflammation |
| CN101143894A (en) * | 2007-06-22 | 2008-03-19 | 中国药科大学 | Highly effective polypeptide for inhibiting angiogenesis, physical chemistry modifying method and application thereof |
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| CN109293926A (en) * | 2018-09-29 | 2019-02-01 | 云南师范大学 | Protease responsiveness is linear-dendritic block copolymer and its preparation method and purposes |
| CN109293926B (en) * | 2018-09-29 | 2021-04-23 | 云南师范大学 | Protease responsive linear-dendritic block copolymer and preparation method and application thereof |
| CN111420068A (en) * | 2019-11-13 | 2020-07-17 | 浙江大学 | Polyethylene glycol-dendritic polylysine/anhydride-cisplatin compound and preparation method and application thereof |
| CN111420068B (en) * | 2019-11-13 | 2021-11-09 | 浙江大学 | Polyethylene glycol-dendritic polylysine/anhydride-cisplatin compound and preparation method and application thereof |
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