CN108753829A - Tree form modification transgene carrier, preparation method and the application of Bone targeting peptide and naphthalimide modification - Google Patents
Tree form modification transgene carrier, preparation method and the application of Bone targeting peptide and naphthalimide modification Download PDFInfo
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- CN108753829A CN108753829A CN201810620827.0A CN201810620827A CN108753829A CN 108753829 A CN108753829 A CN 108753829A CN 201810620827 A CN201810620827 A CN 201810620827A CN 108753829 A CN108753829 A CN 108753829A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/028—Polyamidoamines
Abstract
The present invention provides a kind of tree form modification transgene carrier of Bone targeting peptide and naphthalimide modification, it includes tree form modification skeleton, Bone targeting peptide group and functional group containing naphthalimide, and the Bone targeting peptide group and naphthalimide functional group are connected to tree form modification surface with covalent bond；The tree form modification is polyamide-amide tree form modification.The present invention also provides the preparation methods of the Gene transfer vector and tree form modification transgene carrier as the application in nucleic acid molecules delivery vehicles.The present invention modifies tree form modification using completely new method of modifying and functional group, and reaction is simple and efficient, and yield is high, high-efficiency transfection effect is reached during cell transfecting, and have preferable Bone targeting performance.Material production is simple and at low cost, and transfection process cytotoxicity is relatively low, and efficiently and safely gene molecule can be transported in cell, and tree form modification transgene carrier of the invention has the advantages that efficient, less toxic, cheap, synthesis is simple.
Description
Technical field
The invention belongs to polymer chemistry and technical field of biological materials, and in particular to a kind of Bone targeting peptide and naphthalimide
Tree form modification transgene carrier, preparation method and the application of modification.
Background technology
Gene therapy is as a kind of novel treatment means, in major diseases such as cancer, AIDS, osteoporosis
Treatment in show huge application potential.But due to lacking safe and efficient targeting gene carrier, osteoporosis etc.
The gene therapy of skeletal diseases yet there are no breakthrough.
Gene therapy refers to by genophore by the normal channel genes target cell of external source, for correct or compensate because
Gene defect or abnormal caused disease, to achieve the purpose that treatment.However, line styles of the exposed DNA generally to unfold
Spiral form exists, and volume is loose, and DNA molecular sheet is as negatively charged anion, into when cell can with outside cell membrane
Electrostatic repulsion is generated between the anion of layer.These unfavorable factors all make DNA molecular not by other technological means
In the case of, cell membrane can not be independently passed through, is easy to if even if there is a small amount of DNA that can enter cell membrane into after cell
It is degraded by nuclease.Therefore, the exploitation of genophore plays a crucial role during gene therapy.
Genophore includes two class of viral vectors and non-virus carrier.Viral vectors is a kind of efficient transfection reagent, energy
It is enough that the DNA of external source or RNA are efficiently transferred to cell, but it is there are some apparent defects, such as immunogenicity, carcinogenicity,
The size of gene is easy the possibility for being limited by the size that virus is accommodated, being unable to mass production, existing simultaneously variation, safety
Property is poor.Therefore, viral vectors is greatly limited in the application.Compared with viral vectors, structure is easy to the non-disease of modification
Poisonous carrier overcomes the disadvantages mentioned above of viral vectors completely.Non-virus carrier includes cationic-liposome, cationic polymer, metal
Nano-particle and polypeptide etc..Wherein high molecular polymer is with safe, immunity is low, structure is easy to modify and transfects
The features such as efficient, therefore, high molecular polymer became the non-viral gene vector with applications well foreground in recent years.
Tree form modification be it is a kind of it is artificial synthesized have it is tree-like flutter the compound for opening up structure, have controllable molecular weight and
Size, good monodispersity and surface are easy to the features such as polyfunctional group modification.Polycation tree form modification (PAMAM) can
To form nano-complex after being combined with DNA, by DNA or rna transport to intracellular, and can also be protected in transportational process
Protect degradations of the DNA or RNA from nuclease.There are the works that a large amount of tertiary amine plays " proton sponge " inside tree form modification
With nucleic acid complexes endosome being promoted to escape, and efficiently transcribe into cytoplasm, translate and express etc..But without
The tree form modification cytotoxicity of modification is big, and transfection efficiency is low, significantly limits the application of tree form modification.In order to increase
Add biocompatibility, improve transfection efficiency, various functional moleculars are applied to the surface modification of dendrimer, such as ammonia
Base acid, hydrophobic alkyl chain, polypeptide, cyclodextrin, polyethylene glycol (PEG) and chitosan etc..Wherein, there are many branches after modification
Shape molecule has become commercialized transfection reagent such as PolyFect, SuperFect and ProFect etc..But current
The modification of most of tree form modification is more complicated, cytotoxicity is larger after modification, and transfection efficiency is unsatisfactory, these because
Element significantly limits their applications in gene therapy.Therefore, good by straightforward procedure synthesising biological compatibility, transfection effect
The high transgene carrier of rate is the key that solve these problems.Further, since current most of carriers lack targeting group, it is right
The selectivity of cell is poor and transfection efficiency is low.Therefore, exploitation low toxicity efficiently has the non-viral gene of targeting base group modification
Carrier is of great significance.
Invention content
The present invention provides a kind of tree form modification transgene carrier modified based on Bone targeting peptide and naphthalimide and
Preparation method.Naphthoyl imide compounds synthesis is simple and has good structural modification diversity, has big pi bond conjugated system
Chemical structure characteristic, be the intermediate of new function dyestuff.
Naphthoyl imide compounds have special physicochemical property and optical property, have stronger DNA binding abilities, good
The features such as biocompatibility well, excellent internal external imaging performance, it is in the fields such as cancer diagnosis and treatment and gene therapy
Wide application prospect is revealed.Tree form modification synthesis technology based on naphthalimide modification is very ripe, and operates letter
Single, aggregate velocity is fast, and yield is high, and the transgene carrier of high yield can be quickly obtained without cumbersome purification step, is new
Designing and developing for type carrier provides a kind of completely new thinking.
Bone targeting peptide has good close bone, and good biocompatibility, caused immunogenicity is smaller in vivo, will not produce
Adverse reaction in raw body.Secondly, using amido bond as connecting key between Bone targeting peptide molecule inside, property is stablized, and in vivo
Hydrolyzation system can guarantee the release of nucleic acid molecules, to have the function that gene therapy.Moreover, the synthesis of oligopeptides has been provided with
Highly developed method, synthesis cost is low, and combined coefficient is high.
Therefore, such Bone targeting peptide and the tree form modification carrier of naphthalimide modification are low with cytotoxicity, transfect effect
The advantages that rate is high, synthetic method is simple, easily operated can develop for a kind of novel transgenic line and with commercialized
Potentiality.
It is excellent it is an object of the invention to solve at least the above and/or defect, and provide at least to will be described later
Point.
The present invention overcomes the shortcomings of the tree form modification transgenic line of the prior art, innovatively by Bone targeting peptide compounds
With naphthalimide compound simultaneously with polyamide-amide tree form modification by being covalently keyed synthesis transgene carrier, for pacifying
Entirely, the transfection of efficient and less toxic targeting gene, the present invention are with Bone targeting peptide, naphthalimide compound and tree form modification
Raw material, synthesis cost is relatively low, and material is easy to get, can get reaction efficiently, safety, synthesis step it is simple, it is low to obtain cytotoxicity, together
When have the Bone targeting transgene carrier of high transfection efficiency.
It is a still further object of the present invention to provide the tree form modification transgene carriers of Bone targeting peptide and naphthalimide modification
Preparation method and application.
In order to realize these purposes and other advantages according to the present invention, provides a kind of Bone targeting peptide and repaiied with naphthalimide
The structure formula (I) of the tree form modification transgene carrier of decorations, the tree form modification transgene carrier is as follows：
In formula (I), R is tree form modification, and a is the number for the naphthalimide groups being covalently attached with tree form modification surface
Amount, b is the quantity for the Bone targeting peptide being covalently attached with tree form modification surface.
Preferably, wherein the structure formula (II) of R is as follows：
In formula (II), M is ethylenediamine；N=6；M=2.
Preferably, wherein a=1~128, b=1~128.
The purpose of the present invention can also be carried further by the tree form modification transgenosis of Bone targeting peptide and naphthalimide modification
The preparation method of body realizes that the synthetic route of this method is as follows：
Wherein, the method specific steps include：
Step 1: weighing tree form modification R and 2 compound of formula in proportion, solvent dissolving is added, reaction is stirred at room temperature, instead
After answering, it is concentrated under reduced pressure, 3 compound of isolated formula；
Step 2: 3 compound of formula, 4 compound of formula, catalyst, condensing agent and organic base that the step 1 obtains are having
It is reacted in solvent, after reaction, cold 5 compound of isolated formula；
Step 3: 5 compound of formula, p-methyl phenol, trifluoroacetic acid and the dithioglycol that the step 2 obtains are being had
It reacts in solvent or water, after reaction, is concentrated under reduced pressure, the tree-like high score of isolated Bone targeting peptide and naphthalimide modification
Sub- transgene carrier (I).
Preferably, wherein in the step 1, the ratio of the amount of the substance of 2 compound of the tree form modification R and formula
It is 1:10, the reaction time is 8 hours, and solvent is ethyl alcohol, and separation is specially：The second alcohol and water is used to be using molecular weight for solvent
3500 chromatography bag chromatography.
Preferably, wherein in the step 2,3 compound of the formula is respectively with 4 compound mole ratio of the formula
1:20,1:40 or 1:60, the reaction time is 20 hours.
Preferably, wherein in the step 2, the catalyst is I-hydroxybenzotriazole, and the condensing agent is
1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid, the organic base are triethylamine.
Preferably, wherein in the step 3, the trifluoroacetic acid, p-methyl phenol, dithioglycol and water matter
Amount is than being 92.5:2.5:2.5:2.5.
The purpose of the present invention can also be carried further by the tree form modification transgenosis of Bone targeting peptide and naphthalimide modification
Body is being realized as the application in nucleic acid molecules delivery vehicles.
Preferably, wherein nucleic acid molecules miRNA.
The present invention is based on the tree form modification Bone targeting transgene carrier that Bone targeting peptide and naphthalimide compound are modified, naphthalenes
Imide compound is the tree form modification transgene carrier that 4- amino -1,8- naphthalene acid anhydrides are compound-modified shown in formula (Ia).Bone
Targeting peptide compounds are amino, carboxyl and the protected serine-aspartate-Ser-Ser-of hydroxyl shown in formula (Ib)
The tree form modification transgene carrier of aspartic acid pentapeptide modification.
It is including tree-like the present invention is based on the tree form modification transgene carrier that Bone targeting peptide, naphthalimide compound are modified
Macromolecular scaffold, Bone targeting peptide and naphthalimide compound functional group, the Bone targeting peptide and naphthalimide compound work(
Energy group is connected to tree form modification surface by covalent bond.Including polyamide-amide tree form modification, Bone targeting peptide and naphthoyl
The functional group of group with imine moiety modification, the primary amine group on polyamide-amide tree form modification surface and the Bone targeting peptide,
4- amino -1,8- naphthalene acid anhydrides compounds are connected by amidation process with amido bond.
In the present invention, " naphthalimide compound " refers to 4- amino -1,8- naphthalene acid anhydrides, and structure such as (Ia) is shown, Ke Yiyu
Amidation process occurs for the amino on tree form modification surface." Bone targeting peptide " refers to that sequence is serine-aspartate-silk ammonia
Such as shown in (Ib), with the amino on tree form modification surface amidation can occur for the pentapeptide of acid-serine-aspartate, structure
Reaction.
In (I) formula of the invention, led to by polyamide-amide tree form modification and naphthalimide compound, Bone targeting peptide compounds
Covalently key connection to be crossed, and is modified on tree form modification surface, a kind of novel high-molecular bone of synthesis targets transgene carrier,
The polyamide-amide tree form modification synthesizes tree form modification, the polyamide-amide using ethylenediamine and methyl acrylate as monomer
The end of tree form modification is primary amine group, n=6, m=2.
Current tree form modification transgene carrier synthesis technology is complicated, and expensive, cytotoxicity is big, and targeting is poor,
And transfection is undesirable.Tree form modification carrier of the formula (I) of the present invention based on polyamide-amide through naphthalimide compound and
It, can be safely and efficiently by the cell of gene delivery, and to targeting that bone tissue has had after the modification of Bone targeting peptide.This
The advantages that material has synthesis cycle short, and preparation method is simple, and cytotoxicity is low and transfection efficiency is high.
The present invention also provides a kind of tree-like high scores of polyamide-amide modified based on Bone targeting peptide and naphthalimide compound
Sub- genophore and its compound application as the transport agent of nucleic acid molecules in vitro in vivo.The nucleic acid include DNA and
miRNA.
The present invention also provides a kind of compounds, and it includes Bone targeting peptide and naphthalimide compounds as shown in formula (I) to repair
The transgene carrier and nucleic acid of decorations.
The present invention also provides a kind of dendrimers using Bone targeting peptide shown in formula (I) and naphthalimide modification to turn base
The method for conveying nucleic acid because of carrier.That is the tree form modification transgene carrier and core of Bone targeting peptide and naphthalimide compound modification
Acid forms stable nucleic acid complexes after incubation at room temperature, then is incubated altogether with cell, completes gene transfection process.
The present invention modifies tree form modification using completely new method of modifying and functional unit, and raw material is cheap and easy to get, and reaction is high
Effect, product is easy to purify, good to bone tissue targeting to the small toxicity of cell in transfection process, can efficiently carry out gene and turn
Dye is the transgene carrier for having both the advantages that cheap, safe efficient, synthesis is simple.
For using Antagomir-138-5p as the cryptiogene of silence miR-138-5p, carried using transgenosis of the present invention
Body transports miRNA, and experiment shows that the present invention has the following advantages：Genophore not only has good biological compatible in the present invention
Property, and there is efficient efficiency gene transfection.It is found by gene transfection experiments, under optimal conditions to the transfection of RNA
In, transfection efficiency is higher than commercialized transfection reagent Lipofectamine2000.In cytotoxicity experiment, in the present invention
Transgene carrier cytotoxicity it is low, under the concentration tested, cell survival rate be more than 95%, have good bio-compatible
Property.Transgene carrier of the present invention, the required raw material of synthesis is cheap and easy to get, and synthesis step is simple, and sintetics is easy to purify.In cell
In transfection, cytotoxicity is low, and transfection efficiency is high, can be used as a kind of transgenosis load having both the advantages that safe efficient, cheap
Body.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 is the synthesis road for preparing Bone targeting peptide and the tree form modification transgene carrier of naphthalimide modification of the present invention
The structural schematic diagram of line and transgene carrier；
Fig. 2 is present invention prepare compound 5-1 in one embodiment1H NMR figures；
Fig. 3 is present invention prepare compound 5-2 in one embodiment1H NMR figures；
Fig. 4 is present invention prepare compound 5-3 in one embodiment1H NMR figures；
Fig. 5 is that transgene carrier G5-1, G5-2 and G5-3 prepared by the present invention illustrates the absorption result of hydroxyapatite
Figure；
Fig. 6 is the agarose gel electrophoresis of transgene carrier G5-1, G5-2 and G5-3 and RNA compounds prepared by the present invention
Experimental result schematic diagram；
Fig. 7 is that the compound of transgene carrier G5-1, G5-2 and G5-3 and RNA prepared by the present invention are thin in E1 cells
Cellular toxicity figure；
Fig. 8 is that transgene carrier prepared by the present invention is imitated with transfection of the Antagomir-138-5p compounds in E1 cells
Rate figure.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
<Example 1>
It is with the 5th polyamide-amine tree form modification transgene carrier that Bone targeting peptide is modified with naphthalimide compound
Example：It is as shown in Figure 1 the synthesis for preparing Bone targeting peptide and the tree form modification transgene carrier of naphthalimide modification of the present invention
Route,
A kind of tree form modification transgene carrier of Bone targeting peptide and naphthalimide modification, the tree form modification transgenosis
The structure formula (I) of carrier is as follows：
In formula (I), the G5 that R is represented is the 5th polyamide-amine tree form modification, and centronucleus M is ethylenediamine, end primary amine
Group is 128, shown in structure formula (II)：
Wherein, the theory expectation that the theory expectation of a is 10, b is 20；
5th polyamide-amine tree form modification transgene carrier material of Bone targeting peptide and naphthalimide compound modification
Material, synthetic route are as follows：
The method specific steps include：
Step 1: weighing 50 milligrams of tree form modification R and 3.7 milligrams of 2 4- amino -1,8- naphthalene acid anhydrides of compound in proportion
It is dissolved in 5 milliliters of ethyl alcohol.Reaction solution is heated to 70 degree, after stirring 8 hours, is cooled to room temperature.Reaction solution is transferred to again
In the bag filter that molecular cut off is 3500, with ethanol dialysis 10 hours, then three times with water dialysis.It collects sample and freezes dry
It is dry, obtain the 5th polyamide-amine tree form modification material chemical combination modified based on naphthalimide that appearance is orange-yellow powder
Object 3.The ratio between raw materials used tree form modification and 4- amino -1,8- naphthalene acid anhydride compounds mole are 1 in this step:10.
Experimental result：It is compared according to calculating, each tree form modification surface has coupled 8 naphthalimide groups, i.e. formula (3)
Middle a is 8；
Wherein, it is the 5th polyamide-amine tree form modification that the theory expectation of a, which is 10, G5, and centronucleus M is ethylenediamine,
End primary amine group is 128, shown in structure formula (II)：
Step 2: with molar ratio for 1:20 ratio prepares the protected Bone targeting tree form modification of active function groups, institute
State 60 milligrams of 3 compounds of formula, 44 milligrams of 4 compounds of formula, 14 milligrams of I-hydroxybenzotriazole catalyst, 20 that step 1 obtains
Milligram 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid condensing agent and 16 milligrams of organic bases triethylamines, are added to
Dissolved in 10mL dimethyl sulfoxide solvents, room temperature reaction 8 hours after, then by reaction solution be transferred to molecular cut off be 3500 it is saturating
It analyses in bag, with ethanol dialysis 10 hours, then three times with water dialysis.It collects sample and is freeze-dried, it is orange toner to obtain appearance
The compound 5- at end；
With1H NMR methods characterization detects above-mentioned product：The transgene carrier for taking 10 milligrams of above-mentioned steps to synthesize is dissolved in 0.5 milli
It rises in deuterated dimethyl sulfoxide, solution is subjected to nuclear magnetic resonance test, according to1H NMR spectrograms, as shown in Fig. 2, calculating tree-like
High molecular modification efficiency.
Experimental result：It is compared according to calculating, each tree form modification surface has coupled 8 naphthalimide groups, 19 officials
The Bone targeting peptide group that protection can be rolled into a ball, i.e., a is 8, b 19 in formula (5-1).
Wherein, it is the 5th polyamide-amine tree form modification that the theory expectation that the theory expectation of a is 10, b, which is 20, G5, in
Heart core M is ethylenediamine, and end primary amine group is 128, shown in structure formula (II)：
Step 3: being added to 50 milligrams of formula 5-1 compounds that the step 2 obtains containing 3.7 grams of trifluoroacetic acids, 0.1
In the mixed solution of gram p-methyl phenol, 0.1 gram of dithioglycol and 0.1 gram of water, after reaction being stirred at room temperature 10 hours, then will reaction
Liquid is transferred in the bag filter that molecular cut off is 3500, with ethanol dialysis 10 hours, then three times with water dialysis.Collect sample simultaneously
Freeze-drying obtains the 5th polyamide-amine tree modified based on Bone targeting peptide and naphthalimide that appearance is orange-yellow powder
Shape high molecular material compound G5-1, structure are shown below：
Wherein, 8 a, b 19, G5 are the 5th polyamide-amine tree form modification, and centronucleus M is ethylenediamine, terminal primary
Amine groups are 128, shown in structure formula (II)：
<Example 2>
A kind of tree form modification transgene carrier of Bone targeting peptide and naphthalimide modification, the tree form modification transgenosis
The structure formula (I) of carrier is as follows：
In formula (I), the G5 that R is represented is the 5th polyamide-amine tree form modification, and centronucleus M is ethylenediamine, end primary amine
Group is 128, shown in structure formula (II)：
Wherein, the theory expectation that the theory expectation of a is 10, b is 40；
5th polyamide-amine tree form modification transgene carrier material of Bone targeting peptide and naphthalimide compound modification
Material, synthetic route are as follows：
The method specific steps include：
Step 1: weighing 50 milligrams of tree form modification R and 3.7 milligrams of 2 4- amino -1,8- naphthalene acid anhydrides of compound in proportion
It is dissolved in 5 milliliters of ethyl alcohol.Reaction solution is heated to 70 degree, after stirring 8 hours, is cooled to room temperature.Reaction solution is transferred to again
In the bag filter that molecular cut off is 3500, with ethanol dialysis 10 hours, then three times with water dialysis.It collects sample and freezes dry
It is dry, obtain the 5th polyamide-amine tree form modification material chemical combination modified based on naphthalimide that appearance is orange-yellow powder
Object 3.The ratio between raw materials used tree form modification and 4- amino -1,8- naphthalene acid anhydride compounds mole are 1 in this step:10.
Experimental result：It is compared according to calculating, each tree form modification surface has coupled 8 naphthalimide groups, i.e. formula (3)
Middle a is 8；
Wherein, it is the 5th polyamide-amine tree form modification that the theory expectation of a, which is 10, G5, and centronucleus M is ethylenediamine,
End primary amine group is 128, shown in structure formula (II)：
Step 2: with molar ratio for 1:40 ratio prepares the protected Bone targeting tree form modification of active function groups.Institute
State 60 milligrams of 3 compounds of formula, 88 milligrams of 4 compounds of formula, 28 milligrams of I-hydroxybenzotriazole catalyst, 40 that step 1 obtains
Milligram 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid condensing agent and 32 milligrams of organic bases triethylamines, are added to
Dissolved in 10mL dimethyl sulfoxide solvents, room temperature reaction 8 hours after, then by reaction solution be transferred to molecular cut off be 3500 it is saturating
It analyses in bag, with ethanol dialysis 10 hours, then three times with water dialysis.It collects sample and is freeze-dried, it is orange toner to obtain appearance
The compound 5-2 at end.
With1H NMR methods characterization detects above-mentioned product：The transgene carrier for taking middle synthesis in 10 milligrams of above-mentioned steps, is dissolved in
In 0.5 milliliter of deuterated dimethyl sulfoxide, solution is subjected to nuclear magnetic resonance test, according to1H NMR spectras, as shown in figure 3, calculating
The modification efficiency of tree form modification.
Experimental result：It is compared according to calculating, each tree form modification surface has coupled 8 naphthalimide groups, 39 officials
The Bone targeting peptide group that protection can be rolled into a ball, i.e., a is 8, b 39 in formula (5-2).
Wherein, it is the 5th polyamide-amine tree form modification that the theory expectation that the theory expectation of a is 10, b, which is 40, G5, in
Heart core M is ethylenediamine, and end primary amine group is 128, shown in structure formula (II)：
Step 3: being added to 50 milligrams of formula 5-2 compounds that the step 2 obtains containing 3.7 grams of trifluoroacetic acids, 0.1
In the mixed solution of gram p-methyl phenol, 0.1 gram of dithioglycol and 0.1 gram of water, after reaction being stirred at room temperature 10 hours, then will reaction
Liquid is transferred in the bag filter that molecular cut off is 3500, with ethanol dialysis 10 hours, then three times with water dialysis.Collect sample simultaneously
Freeze-drying obtains the 5th polyamide-amine tree modified based on Bone targeting peptide and naphthalimide that appearance is orange-yellow powder
Shape high molecular material compound G5-2, structure are shown below：
Wherein, 8 a, b 39, G5 are the 5th polyamide-amine tree form modification, and centronucleus M is ethylenediamine, terminal primary
Amine groups are 128, shown in structure formula (II)：
<Example 3>
A kind of tree form modification transgene carrier of Bone targeting peptide and naphthalimide modification, the tree form modification transgenosis
The structure formula (I) of carrier is as follows：
In formula (I), the G5 that R is represented is the 5th polyamide-amine tree form modification, and centronucleus M is ethylenediamine, end primary amine
Group is 128, shown in structure formula (II)：
Wherein, the theory expectation that the theory expectation of a is 10, b is 80；
5th polyamide-amine tree form modification transgene carrier material of Bone targeting peptide and naphthalimide compound modification
Material, synthetic route are as follows：
The method specific steps include：
Step 1: weighing 50 milligrams of tree form modification R and 3.7 milligrams of 2 4- amino -1,8- naphthalene acid anhydrides of compound in proportion
It is dissolved in 5 milliliters of ethyl alcohol.Reaction solution is heated to 70 degree, after stirring 8 hours, is cooled to room temperature.Reaction solution is transferred to again
In the bag filter that molecular cut off is 3500, with ethanol dialysis 10 hours, then three times with water dialysis.It collects sample and freezes dry
It is dry, obtain the 5th polyamide-amine tree form modification material chemical combination modified based on naphthalimide that appearance is orange-yellow powder
Object 3.The ratio between raw materials used tree form modification and 4- amino -1,8- naphthalene acid anhydride compounds mole are 1 in this step:10.
Experimental result：It is compared according to calculating, each tree form modification surface has coupled 8 naphthalimide groups, i.e. formula (3)
Middle a is 8；
Wherein, it is the 5th polyamide-amine tree form modification that the theory expectation of a, which is 10, G5, and centronucleus M is ethylenediamine,
End primary amine group is 128, shown in structure formula (II)：
Step 2: with molar ratio for 1:80 ratio prepares the protected Bone targeting tree form modification of active function groups.Institute
State 60 milligrams of 3 compounds of formula, 176 milligrams of 4 compounds of formula, 56 milligrams of I-hydroxybenzotriazole catalyst, 80 that step 1 obtains
Milligram 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid condensing agent and 64 milligrams of organic bases triethylamines, are added to
Dissolved in 10mL dimethyl sulfoxide solvents, room temperature reaction 8 hours after, then by reaction solution be transferred to molecular cut off be 3500 it is saturating
It analyses in bag, with ethanol dialysis 10 hours, then three times with water dialysis.It collects sample and is freeze-dried, it is orange toner to obtain appearance
The compound 5-3 at end.
With1H NMR methods characterization detects above-mentioned product：The transgene carrier synthesized in 10 milligrams of above-mentioned steps is taken, is dissolved in 0.5
In milliliter deuterated dimethyl sulfoxide, solution is subjected to nuclear magnetic resonance test, according to1H NMR spectrograms, as shown in figure 4, calculating tree
The high molecular modification efficiency of shape.
Experimental result：It is compared according to calculating, each tree form modification surface has coupled 8 naphthalimide groups, 59 officials
The Bone targeting peptide group that protection can be rolled into a ball, i.e., a is 8, b 59 in formula (5-3).
Wherein, it is the 5th polyamide-amine tree form modification that the theory expectation that the theory expectation of a is 10, b, which is 80, G5, in
Heart core M is ethylenediamine, and end primary amine group is 128, shown in structure formula (II)：
Step 3: being added to 50 milligrams of formula 5-3 compounds that the step 2 obtains containing 3.7 grams of trifluoroacetic acids, 0.1
In the mixed solution of gram p-methyl phenol, 0.1 gram of dithioglycol and 0.1 gram of water, after reaction being stirred at room temperature 10 hours, then will reaction
Liquid is transferred in the bag filter that molecular cut off is 3500, with ethanol dialysis 10 hours, then three times with water dialysis.Collect sample simultaneously
Freeze-drying obtains the 5th polyamide-amine tree modified based on Bone targeting peptide and naphthalimide that appearance is orange-yellow powder
Shape high molecular material compound G5-3, structure are shown below：
Wherein, 8 a, b 59, G5 are the 5th polyamide-amine tree form modification, and centronucleus M is ethylenediamine, terminal primary
Amine groups are 128, shown in structure formula (II)：
<Example 4>
The detection of following aspect has been carried out to the tree form modification transgene carrier being prepared in embodiment 1,2,3：
(1) agarose gel electrophoresis of transgene carrier and RNA compounds is tested
The preparation of transgene carrier and RNA compounds
At room temperature, transgene carrier and RNA, the wherein quality of transgene carrier G5-1 and RNA are separately added into PE pipes
Than being respectively 0.1:1,0.2:1,1:1,5:The mass ratio of 1, G5-2 and RNA is respectively 40:1,60:1,80:1,100:1, G5-3
Mass ratio with RNA is respectively 40:1,60:1,80:1,100:1, RNA a concentration of 9 μ g/mL.Then, respective volume is added
Ultra-pure water, so that the final volume of reaction solution is maintained at 20 μ L, centrifugation is uniformly mixed.It is put into 37 DEG C of waters bath with thermostatic control, heat preservation 0.5
After hour, 10 × Loading Buffer sample-loading buffers that 2 μ L are added terminate reaction.Wherein, RNA miRNA, herein
The RNA mentioned is miRNA.
The agarose gel electrophoresis of compound is tested
The transgene carrier and 10 μ L of RNA compounds for taking above-mentioned preparation respectively are added in the well on gel.Cover electricity
Swimming is covered, and electrophoresis stops after 30 minutes under the conditions of voltage 100V, takes out gel exposure sampling in gel electrophoresis is at phase system.
As shown in figure 5, being tested using agarose gel electrophoresis, we observe transgene carrier and the effect feelings of RNA
Condition.Transgene carrier G5-1 is best to the flocculating result of RNA, and the mass ratio of G5-1 and RNA are 5:When 1, all RNA are trapped in
Loading hole.As Bone targeting peptide content increases in transgene carrier, the flocculating result of RNA continuously decreased.
(2) adsorption experiment of the transgene carrier to hydroxyapatite
Transgene carrier G5-1, transgene carrier G5-2 and transgene carrier G5-3 are made into the super of a concentration of 30 μ g/mL
Pure water solution, with the initial fluorescence value of fluorescent spectrophotometer assay carrier solution.Then take 1.5mL carrier solutions that 30mg is added
Hydroxyapatite centrifuges after slow magnetic agitation balance 2h under the conditions of room temperature is protected from light, takes supernatant sepectrophotofluorometer
Measure its fluorescent value (EX:447.0nm, EM:536.6nm).With hydroxyapatite balance 2h after supernatant fluorescent value with initially it is glimmering
Absorption percentage of the carrier to hydroxyapatite is calculated in the ratio of light value.
As shown in fig. 6, adsorption rate (G5-1)=△ F/F0=(732-89)/732=88%, adsorption rate (G5-2)=△
F/F0=(604-420)/604=30%, adsorption rate (G5-3)=△ F/F0=(536-437)/536=18%, transgenosis carry
Body G5-1, transgene carrier G5-2 and transgene carrier G5-3 are respectively 88%, 30% to the absorption percentage of hydroxyapatite
With 18%.
(3) transgene carrier cytotoxicity experiment
The preparation of transgene carrier and RNA compounds
At room temperature, transgene carrier is separately added into PE pipes, the mass ratio of wherein transgene carrier and RNA is respectively 1:
1,5:1,10:1,20:1, RNA a concentration of 9 μ g/mL, are added the α-MEM of respective volume, and the final volume of reaction solution is made to keep
In 500 μ L.It is put into 37 DEG C of waters bath with thermostatic control, keeps the temperature 1 hour.
Cytotoxicity experiment
The E1 cells that exponential phase is collected with pancreatin digestion will enter every about 7000, hole cell kind in 96 orifice plates, in
37 DEG C contain 5%CO224 hours of incubator culture, make the fusion for growing to 70%-80% of cell.It discards original in hole
Culture medium is washed 1-2 times with PBS buffer solution, and the transgene carrier and RNA of the above-mentioned various concentration prepared are separately added into every hole
Five parallel holes are arranged in 100 μ L of compound, each concentration；Using the DMEM culture mediums without transgene carrier as blank pair
According to, using commercialization Lipofectamine 2000 be used as positive control, without cell only have α-MEM be used as blank control.4 is small
When after all culture mediums are sucked out, thereto per hole be added 200 μ L contain 10% fetal calf serum α-MEM, cultivated in incubator
After 24 hours, 10 μ L MTT solution are added to every hole, all addition liquid, 150 μ of hyacinthine crystallization of generation are sucked out after 4 hours
L dmso solutions measure absorbance value of each hole at 490nm with microplate reader after ten minutes in being shaken on shaking table.It presses
Following formula calculates cell survival rate (%)=[A490test- blank]/[A490control- blank] × 100.
As shown in fig. 7, the cytotoxicity by measuring transgene carrier in E1 cells, finds the cell of transgene carrier
Toxicity is all relatively low.The survival rate of cell all 95% or more, is higher than commercialized transgene carrier Lipofectamine substantially
2000。
(4) the outer transfection experiment of the composite body of transgene carrier and Antagomir-138-5p
The preparation of transgene carrier and Antagomir-138-5p compounds
At room temperature, it is separately added into transgene carrier G5-1, G5-2, G5-3 and Antagomir-138-5p in PE pipes, turns
The mass ratio of genophore G5-1 and Antagomir-138-5p are 10:1, transgene carrier G5-2 and Antagomir-138-5p
Mass ratio be 1:1, the mass ratio of transgene carrier G5-3 and Antagomir-138-5p are 1:1, Antagomir-138-
α-the MEM of respective volume are added in a concentration of 50nM of 5p, and the final volume of reaction solution is made to be maintained at 1000 μ L, and gently piping and druming is mixed
It is even, it is put into 37 DEG C of waters bath with thermostatic control, keeps the temperature 5 minutes.
In-vitro transfection is tested
The E1 cells that exponential phase is collected with pancreatin digestion will be every about hole 2.0 × 105A cell kind enters 6 hole cells
Culture plate contains 5%CO in 37 DEG C224 hours of incubator culture, make the fusion for growing to 70%-80% of cell.It discards
Original culture medium, is washed 1-2 times with PBS buffer solution in hole, be separately added into every hole the above-mentioned transgene carrier prepared with
1000 μ L of Antagomir-138-5p compounds.All culture mediums are sucked out after 4 hours, 2000 μ L are added per hole thereto contains
α-the MEM of 10% fetal calf serum after being cultivated 48 hours in incubator, take out 24 orifice plates, twice with PBS wash liquids.It is added
200 μ L cell pyrolysis liquids, room temperature shake 15min, and abundant pyrolysis product is transferred to 1.5mL EP pipes, and 12000rpm centrifuges 30s,
The supernatant centrifuged is moved into EP pipes, qPCR detects the expression quantity of miR-138-5p.
According to the compound of above-mentioned method prepare transgenosis carrier Lipofectamine2000 and Antagomir-138-5
Object.
As shown in figure 8, can be obtained by cell transfection assays：In the transfection of E1 cells, transgene carrier G5-1's
Transfection efficiency is higher than the transfection efficiency of other two transgene carriers, meanwhile, also it is better than commercialization transfection reagent
Lipofectamine 2000 can make 95% or more gene miR-138-5p silences.
In conclusion the transgene carrier cytotoxicity in the present invention is relatively low, in the transfection of RNA, all have higher
Transfection efficiency, preparation method is simple, maturation, is easy to control.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily
Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (10)
1. the tree form modification transgene carrier of a kind of Bone targeting peptide and naphthalimide modification, the tree form modification transgenosis carry
The structure formula (I) of body is as follows：
In formula (I), R is tree form modification, and a is the quantity for the naphthalimide groups being covalently attached with tree form modification surface, and b is
The quantity for the Bone targeting peptide being covalently attached with tree form modification surface.
2. the tree form modification transgene carrier of Bone targeting peptide as described in claim 1 and naphthalimide modification, wherein R's
Structure formula (II) is as follows：
In formula (II), M is ethylenediamine；N=6；M=2.
3. the tree form modification transgene carrier of Bone targeting peptide as described in claim 1 and naphthalimide modification, wherein a=1
~128, b=1~128.
4. a kind of tree form modification preparing the modification of claims 1 to 3 any one of them Bone targeting peptide and naphthalimide turns base
Because of the method for carrier, synthetic route is as follows：
Wherein, the method specific steps include：
Step 1: weighing tree form modification R and 2 compound of formula in proportion, solvent dissolving is added, reaction, reaction knot is stirred at room temperature
Shu Hou is concentrated under reduced pressure, 3 compound of isolated formula；
Step 2: 3 compound of formula, 4 compound of formula, catalyst, condensing agent and organic base that the step 1 obtains are organic molten
It is reacted in agent, after reaction, cold 5 compound of isolated formula；
Step 3: 5 compound of formula, p-methyl phenol, trifluoroacetic acid and dithioglycol that the step 2 is obtained are organic molten
It reacts in agent or water, after reaction, is concentrated under reduced pressure, isolated Bone targeting peptide and the tree form modification of naphthalimide modification turn
Genophore (I).
5. method as claimed in claim 4, wherein in the step 1,2 compound of the tree form modification R and formula
The ratio of the amount of substance is 1:10, the reaction time is 8 hours, and solvent is ethyl alcohol, and separation is specially：Use second alcohol and water for solvent profit
The chromatography bag for being 3500 with molecular weight chromatographs.
6. method as claimed in claim 4, wherein in the step 2,3 compound of the formula and 4 compound of the formula
Molar ratio is respectively 1:20,1:40 or 1:80, the reaction time is 20 hours.
7. method as claimed in claim 4, wherein in the step 2, the catalyst is I-hydroxybenzotriazole, institute
It is 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid to state condensing agent, and the organic base is triethylamine.
8. method as claimed in claim 4, wherein in the step 3, the trifluoroacetic acid, p-methyl phenol, second two
The mass ratio of mercaptan and water is 92.5:2.5:2.5:2.5.
9. Bone targeting peptide according to any one of claims 1 to 3 and the tree form modification transgenosis of naphthalimide modification carry
Body is as the application in nucleic acid molecules delivery vehicles.
10. the tree form modification transgene carrier that Bone targeting peptide as claimed in claim 9 is modified with naphthalimide is as core
Application in acid molecule delivery vehicles, wherein nucleic acid molecules miRNA.
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| CN115737914A (en) * | 2022-11-25 | 2023-03-07 | 杭州矩正医疗科技有限公司 | Preparation method of photosensitive material with blood vessel repairing function |
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