CN109364264A - Gene and chemicals corotation carrier and its application method - Google Patents
Gene and chemicals corotation carrier and its application method Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
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- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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Abstract
The present invention proposes a kind of gene and chemicals corotation carrier and its application method, belong to technical field of biological material, the carrier can cotransport genomic medicine and chemicals, and the load of dewatering medicament does not influence the self-assembled structures of polypeptide and the binding ability with DNA, can transport simulation hydrophobic drug simultaneously and DNA enters cell.The technical solution includes that peptide molecule is dissolved in in buffer self assembly is nanofibre-like structure, first sufficiently combined with hydrophobic drug or aids drug molecular solution, obtain polypeptide/drug molecule load liquid solution, the polypeptide/drug molecule load liquid solution is sufficiently mixed in predefined conditions with gene molecule solution and is prepared again, i.e., polypeptide/medicine/gene molecule loads liquid solution.The present invention can be applied to during the cotransporting of gene and chemicals.
Description
Technical field
The invention belongs to technical field of biological material more particularly to a kind of gene and chemicals corotation carrier and its users
Method.
Background technique
Appearance due to organism to chemotherapeutic agent drug resistance, single medicine tend not to obtain in disease treatment
Optimum curative effect, therefore a variety of treated with combined medication become the hot spot of drug research in recent years.Currently, Cocktail treatment mainly wraps
Different chemotherapy combinations are included, wherein small molecule chemotherapeutic drug is received more and more attention with genomic medicine combination therapy.However, its
Develop the obstruction by a principal element, the i.e. building of drug delivery systems, key is to design the medicine that simply cotransports
Object carrier.
Molecule will become gene and drug corotation carrier, it must have, and there are two types of functions: first, as genophore,
It should be a kind of efficient DNA agglomeration reagent.Due to DNA molecular sheet as linear stretch conformation and have negative electrical charge, can with it is thin
The anion of after birth outer layer generates electrostatic repulsion, and DNA molecular is difficult to independently penetrating cell film and enters cell interior, therefore, base
Because DNA can should be agglomerated the particle for small volume by carrier, thus make DNA molecular be easy to by endocytosis or pinocytosis into
Enter intracellular；Second, as dewatering medicament molecular vehicle, need the binding site of dewatering medicament molecule, the hydrophobic medicine of load
Slow release again after object enters in vivo.
Studying more pharmaceutical carrier at present is mostly single medicine carrier, less to the research of corotation carrier, and is mostly
The lipid molecular of surface modification, it can provide hydrophobic cavity and DNA binding site, however these lipid moleculars are carried as drug
Body still has two outstanding problems: first, lipid molecular surface modification process is complicated, and synthesis step is cumbersome；Second, lipid point
The poor biocompatibility of son, is not easy to be decomposed in vivo.Therefore, research and development have compared with high gene transfection efficiency and drug loading efficiencies
Pharmaceutical carrier be expected to provide new selection for disease treatment.
Peptide molecule is made of amino acid residue, has many advantages, such as that biocompatibility is high, degradable in vivo, and short peptide molecules
Designability is strong, and synthetic method is simple, as can designing suitable peptide molecule, introduces more positive charge wherein and makes it have by force
DNA binding ability, while introducing hydrophobic amino acid residues, further assigning the stronger assembling ability of aggregation of molecule makes it can
To provide hydrophobic cavity for the load of dewatering medicament, it will be expected to become excellent gene and chemicals corotation carrier.
Summary of the invention
The present invention proposes a kind of gene and chemicals corotation carrier and its application method, which can cotransport gene
Drug and chemicals, and the load of dewatering medicament does not influence the self-assembled structures of polypeptide and the binding ability with DNA, it can be same
When transport simulation hydrophobic drug and DNA enter cell.
In order to achieve the above object, the present invention provides a kind of genes and chemicals corotation carrier, by peptide molecule
Being dissolved in self assembly in buffer is nanofibre-like structure, is first sufficiently tied with hydrophobic drug or aids drug molecular solution
It closes, obtains polypeptide/drug molecule load liquid solution, then the polypeptide/drug molecule is loaded into liquid solution and gene molecule solution
It is sufficiently mixed and is prepared in predefined conditions, i.e., polypeptide/medicine/gene molecule loads liquid solution.
Preferably, the buffer in above-mentioned technical proposal is the Hepes buffer that concentration is 20mM.The polypeptide is dissolved in slow
It can be self-assembly of fibrous structure in fliud flushing, can use the hydrophobic cavity load dewatering medicament molecule of polypeptide fibers, utilize
The a large amount of positive charge combination loaded gene drug of polypeptide, realization genomic medicine and chemicals transport altogether.
Preferably, the peptide molecule has following sequence:
Ac-IIIIIGPLGLAGGRRRRRRRR-NH2, structural formula are as follows:
Peptide molecule in above-mentioned technical proposal contains 8 positively charged arginine sequences, can tie with electronegative DNA
It closes, therefore, can be used as a kind of efficient gene transfection reagent.
Preferably, the peptide molecule is dissolved in buffer the step of self assembly is nanofibre-like structure includes:
Compound concentration is the Ac-IIIIIGPLGLAGGRRRRRRRR-NH of 2.0-10.0mM2Peptide molecule solution, is dissolved in pH
For in the buffer solution of 7.2-7.4, placement 3 days or more, self assembly was nanofibre-like structure.
Peptide molecule in above-mentioned technical proposal has good self-assembly ability, can be from group when more than 2.0mM concentration
Dress forms nanofibre-like structure, provides hydrophobic cavity for the load of dewatering medicament molecule, therefore, can be used as effective hydrophobicity
Pharmaceutical carrier.
Preferably, the peptide molecule, when concentration reaches 4.0mM or more, self assembly is nanofibre-like structure, receive
Rice fibre diameter is 5-8nm, and length is 500nm-10 μm.
Preferably, the polypeptide/drug molecule load liquid solution preparation steps include:
Hydrophobic drug or aids drug molecule are dissolved in the peptide molecule solution of curing, it is 20-30 minutes ultrasonic, it puts
It sets 6-24 hours, combines drug molecule sufficiently with the nanofiber effect after Study of Self-assembling Peptides, be then centrifuged for, remove molten
The unbonded drug molecule precipitating in liquid bottom, upper solution are polypeptide/drug molecule load liquid solution.
Preferably, the hydrophobic drug is adriamycin, the aids drug molecule is Nile red.
Preferably, the polypeptide/medicine/gene molecule load liquid solution preparation steps include:
Compound concentration is λ-DNA solution of 50 μ g/mL, is dissolved in the buffer solution that pH is 7.2-7.4, obtains gene molecule
Solution；
The polypeptide of above-mentioned curing/drug molecule load liquid solution and gene molecule solution is sufficiently mixed according to predetermined condition
It closes, obtains polypeptide/medicine/gene molecule load liquid solution.
In above-mentioned technical proposal, gene molecule can be DNA or RNA, this sentences Green fluorescent protein fusion vector
PEGFP-N2 is illustrated for representative.
Preferably, the predetermined condition are as follows: gene molecule concentration is 1-50 μ g/mL, peptide molecule positive charge in solution
Ratio with gene molecule negative charge number is 5.0-50.0.
In above-mentioned technical proposal, when the negatively charged number of the positively charged quantity of peptide molecule institute and DNA molecular institute in solution
The ratio of amount can induce DNA molecular and effectively agglomerate at 2 or more, and DNA molecular is protected to degrade from nuclease；When polypeptide point
When the ratio of the negatively charged quantity of the positively charged quantity of sub- institute and DNA molecular institute is 5, DNA molecular can be made by extended conformation turn
Become the cohesion conformation of high compression, and can effective mediated gene transfection.
The present invention also provides a kind of genes as described in any of the above-described technical solution and chemicals corotation carrier
Realize the application method to cotransport, comprising:
Polypeptide/medicine/gene molecule load liquid solution and cell are co-cultured, culture solution is changed to by culture after 4-12 hours
DMEM culture medium containing 10% serum after continuing culture 24-48 hour, can be realized drug and effective transport and base to intracellular
Effective transfection of cause.
In above-mentioned technical proposal, the specific steps of co-cultivation include:
It is 1 × 10 that every hole, which is inoculated with 100 μ L density, in 96 orifice plates5The cell suspension of/mL, culture 24 is small in cell incubator
When cell it is adherent after, culture medium is sucked out, 100 μ L serum-free DMEM are added, it is negative to add 50 μ L polypeptides/medicine/gene molecule
Carrier solution.Culture solution is changed to the DMEM culture medium containing 10% serum after culture 6 hours, continues culture 24-48 hours.
Preferably, the cell is HEK293 cell.
In above-mentioned technical proposal, when peptide molecule positively charged quantity and DNA molecular negatively charged quantity ratio
When being 5, pEGFP-N2 plasmid transfection can be entered to HEK293 cell, efficiency gene transfection is 20% or more.Also, turn in gene
It contaminates under concentration conditions, peptide molecule is little to the toxicity of cell, and cell still can keep good form.
Compared with prior art, the advantages and positive effects of the present invention are:
(1) peptide molecule provided by the present invention has good self-assembly ability, can be from group when more than 2.0mM concentration
Dress forms nanofibre-like structure, can provide hydrophobic cavity for the load of dewatering medicament molecule, be a kind of effective hydrophobicity medicine
Object carrier；Meanwhile containing 8 positively charged arginine sequences, and it can also be in conjunction with electronegative DNA, therefore and one kind has
The gene transfection agent of effect.
(2) corotation carrier synthesis process provided by the present invention is simple, and resulting vehicle can cotransport genomic medicine and change
Drug is learned, and the load of dewatering medicament does not influence the self-assembled structures of polypeptide and the binding ability with DNA, can transport mould simultaneously
Quasi- dewatering medicament molecule Nile red and Plasmid DNA enter cell.
Detailed description of the invention
Fig. 1 (a) EtBr substitution experiment display relative intensity of fluorescence is with the variation of polypeptide/DNA positive and negative charge ratio, (b) TEM
The pattern of DNA condensate when as a result providing polypeptide/DNA positive and negative charge than being 5, (c) gel electrophoresis result is providing polypeptide/DNA just
Electrophoresis result when negative electrical charge is than being respectively 0,3,8,20；
Fig. 2 is that Study of Self-assembling Peptides provided by the embodiment of the present invention is nanofibre-like structure schematic diagram, and wherein a is
Circular dichroism spectra phenogram, b are transmission electron microscope figure, and c is that atomic force microscope characterizes its appearance structure；
Fig. 3 is the transmitted electron after the peptide molecule solution loadings Nile red of 4.0mM provided by the embodiment of the present invention
Microscopic structure；
Fig. 4 be the embodiment of the present invention provided by polypeptide/medicine/gene molecule load with cell complete culture after again
The drug delivery and cell transfecting effect observed under inverted fluorescence microscope, wherein bright after (a) transfected HEK 293
Field imaging, (b) blue light after transfected HEK 293 excites fluorescence imaging, and (c) the green light excitation after transfected HEK 293 is glimmering
Light imaging.
Specific embodiment
In order to become apparent from introduce in detail gene provided by the embodiment of the present invention and chemicals corotation carrier and its
Application method is described below in conjunction with specific embodiment.
Embodiment 1
Peptide molecule solution is prepared:
Compound concentration be 4.0mM peptide molecule Ac-IIIIIGPLGLAGGRRRRRRRR-NH2 solution, be dissolved in pH be 7.4,
Concentration is the Hepes buffer of 20mM, place 3 days or more, self assembly is nanofibre-like structure, is characterized by circular dichroism spectra
Its secondary structure and its appearance structure is characterized by transmission electron microscope and atomic force microscope, as a result as shown in Fig. 2, shape
It is 5-8nm at diameter, length is up to 1 μm or more of fibrous structure；
Polypeptide/drug molecule load liquid solution preparation: the mixed solution of peptide molecule and Nile red molecule, immobilized polypeptide are prepared
Concentration is 4.0mM, the Nile red of molar ratio 5% is added, ultrasound 30 minutes, centrifugation removal solution bottom is unsupported after placing three days
Nile red, calculate determine saturation load capacity be about 1wt%；Polypeptide/drug molecule load transmission electron microscope structure
As shown in figure 3, keeping fibrous structure；
DNA solution is prepared: preparing the solution of Green fluorescent protein fusion vector pEGFP-N2, concentration is 50 μ g/mL, is dissolved in
The Hepes buffer that pH is 7.4, concentration is 20mM；
The load liquid solution preparation of polypeptide/medicine/gene molecule: by aforementioned polypeptides/drug molecule load liquid solution with
PEGFP-N2 solution mixing, so that pEGFP-N2 concentration is 10 μ g/mL, peptide molecule positive charge and gene molecule negative electrical charge in solution
The ratio of number is 5.0；As shown in Figure 1, the combination of peptide molecule and DNA can cause the reduction of fluorescence intensity, charge ratio exists
Reach balance after 3.0, relative intensity of fluorescence is down to 0.1 or less.Transmission electron microscope (TEM) morphology characterization shows that peptide molecule can incite somebody to action
DNA cohesion forms diameter in 500nm fine and close aggregation below.Agarose gel electrophoresis results show that peptide molecule can make
DNA cohesion, the DNA after cohesion cannot be trapped in loading wells by the hole of Ago-Gel.The above results all prove
Peptide molecule can effectively induce the cohesion of DNA molecular.
Genomic medicine and chemicals cotransport: it is 1 × 10 that every hole, which is inoculated with 100 μ L density, in 96 orifice plates5The cell of/mL
Suspension, cultivate in cell incubator 24 hour cells it is adherent after, culture medium is sucked out, 100 μ L serum-free DMEM are added, add
50 μ L polypeptides/medicine/gene molecule loads liquid solution.Culture solution is changed to the culture of the DMEM containing 10% serum after culture 6 hours
Base continues culture 24 hours.It is 5 conditions that peptide molecule and plasmid pEGFP-N2 are observed under inverted fluorescence microscope in charge ratio
Drug delivery and cell transfecting effect after lower transfected HEK 293, as a result as shown in Figure 4.
As shown in figure 4, inversion fluorescence imaging (a) light field after drug delivery and transfected HEK 293, (b) blue light swashs
Hair, (c) green light excites, and a, b, c are common location region.The presence of cell interior red fluorescence illustrates that Nile red molecule successfully transports
To cell interior, the presence of green fluorescence illustrates to transfect successfully, smoothly gives expression to green fluorescent protein.Therefore, this result table
The bright system be successfully realized aids drug molecule (Nile red) and green fluorescent protein plasmid (pEGFP-N2) transport altogether and
Effective gene transfection.
Embodiment 2
With embodiment 1, difference is experimental procedure: by polypeptide point in polypeptide/drug molecule load solution preparation step
Sub- Ac-IIIIIGPLGLAGGRRRRRRRR-NH2Concentration be adjusted to 8.0mM, polypeptide/medicine/gene molecule loads liquid solution
The ratio of peptide molecule positive charge and gene molecule negative charge number is adjusted to 20.0 in preparation step, system can equally succeed
Realize that aids drug molecule (Nile red) and transporting altogether for green fluorescent protein plasmid (pEGFP-N2) are transfected with effective gene.
Embodiment 3
With embodiment 1, difference is experimental procedure: by polypeptide point in polypeptide/drug molecule load solution preparation step
Sub- Ac-IIIIIGPLGLAGGRRRRRRRR-NH2Concentration be adjusted to 2.0mM, polypeptide/medicine/gene molecule loads liquid solution
The ratio of peptide molecule positive charge and gene molecule negative charge number is adjusted to 10.0 in preparation step, system can equally succeed
Realize that aids drug molecule (Nile red) and transporting altogether for green fluorescent protein plasmid (pEGFP-N2) are transfected with effective gene.
Embodiment 4
With embodiment 1, difference is experimental procedure: by polypeptide point in polypeptide/drug molecule load solution preparation step
Sub- Ac-IIIIIGPLGLAGGRRRRRRRR-NH2Concentration be adjusted to 10mM, polypeptide/medicine/gene molecule loads liquid solution system
The ratio of peptide molecule positive charge and gene molecule negative charge number is adjusted to 50.0 in standby step, system can equally succeed real
Transporting altogether for existing aids drug molecule (Nile red) and green fluorescent protein plasmid (pEGFP-N2) is transfected with effective gene.
Claims (10)
1. gene and chemicals corotation carrier, which is characterized in that it is nanometer that peptide molecule, which is dissolved in self assembly in buffer,
Fibrous structure is first sufficiently combined with hydrophobic drug or aids drug molecular solution, and polypeptide/drug molecule load is obtained
Liquid solution, then the polypeptide/drug molecule load liquid solution and gene molecule solution are sufficiently mixed preparation in predefined conditions
It obtains, i.e., polypeptide/medicine/gene molecule loads liquid solution.
2. gene according to claim 1 and chemicals corotation carrier, which is characterized in that the peptide molecule has
Following sequence: Ac-IIIIIGPLGLAGGRRRRRRRR-NH2, structural formula are as follows:
3. gene according to claim 2 and chemicals corotation carrier, which is characterized in that the peptide molecule is dissolved in
In buffer self assembly be nanofibre-like structure the step of include:
Compound concentration is the Ac-IIIIIGPLGLAGGRRRRRRRR-NH of 2.0-10.0mM2Peptide molecule solution, being dissolved in pH is
In the buffer solution of 7.2-7.4, place 3 days or more, self assembly is nanofibre-like structure.
4. gene according to claim 3 and chemicals corotation carrier, which is characterized in that the peptide molecule is dense
When degree reaches 4.0mM or more, self assembly is nanofibre-like structure, and nanofiber diameter 5-8nm, length is 500nm-10 μ
m。
5. gene according to claim 1 and chemicals corotation carrier, which is characterized in that the polypeptide/drug point
Son load liquid solution preparation steps include:
Hydrophobic drug or aids drug molecule are dissolved in the peptide molecule solution of curing, it is 20-30 minutes ultrasonic, place 6-
It 24 hours, combines drug molecule sufficiently with the nanofiber effect after Study of Self-assembling Peptides, is then centrifuged for, removes solution bottom
The unbonded drug molecule precipitating in portion, upper solution are polypeptide/drug molecule load liquid solution.
6. gene according to claim 5 and chemicals corotation carrier, which is characterized in that the hydrophobic drug point
Son is adriamycin, and the aids drug molecule is Nile red.
7. gene according to claim 1 and chemicals corotation carrier, which is characterized in that the polypeptide/drug/base
Because the preparation steps of molecule load liquid solution include:
Compound concentration is λ-DNA solution of 50 μ g/mL, is dissolved in the buffer solution that pH is 7.2-7.4, it is molten to obtain gene molecule
Liquid；
The polypeptide of above-mentioned curing/drug molecule load liquid solution is sufficiently mixed with gene molecule solution according to predetermined condition, is obtained
Liquid solution is loaded to polypeptide/medicine/gene molecule.
8. gene according to claim 7 and chemicals corotation carrier, which is characterized in that the predetermined condition are as follows:
Gene molecule concentration is 1-50 μ g/mL in solution, and the ratio of peptide molecule positive charge and gene molecule negative charge number is 5.0-
50.0。
9. the described in any item genes of claim 1-8 and chemicals corotation carrier realize the application method to cotransport,
It is characterized in that, comprising:
Polypeptide/medicine/gene molecule load liquid solution and cell are co-cultured, culture solution is changed to by culture after 4-12 hours to be contained
The DMEM culture medium of 10% serum after continuing culture 24-48 hour, can be realized drug and effective transport and gene to intracellular
Effective transfection.
10. application method according to claim 9, which is characterized in that the cell is HEK293 cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647626A (en) * | 2020-06-08 | 2020-09-11 | 中国石油大学(华东) | Self-assembly of multi-sulfhydryl peptide and application of gene vector thereof |
| CN111675749A (en) * | 2020-06-09 | 2020-09-18 | 江苏科技大学 | Polypeptide sequence and self-assembly material and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106890343A (en) * | 2017-03-08 | 2017-06-27 | 国家纳米科学中心 | A kind of targeting type polypeptide nano genophore compound |
| CN106916209A (en) * | 2017-03-01 | 2017-07-04 | 中国石油大学(华东) | It is a kind of can be used as the Amphiphilic peptide molecule of genophore |
-
2018
- 2018-11-05 CN CN201811307307.0A patent/CN109364264B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106916209A (en) * | 2017-03-01 | 2017-07-04 | 中国石油大学(华东) | It is a kind of can be used as the Amphiphilic peptide molecule of genophore |
| CN106890343A (en) * | 2017-03-08 | 2017-06-27 | 国家纳米科学中心 | A kind of targeting type polypeptide nano genophore compound |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647626A (en) * | 2020-06-08 | 2020-09-11 | 中国石油大学(华东) | Self-assembly of multi-sulfhydryl peptide and application of gene vector thereof |
| CN111647626B (en) * | 2020-06-08 | 2022-07-22 | 中国石油大学(华东) | Self-assembly of multi-sulfhydryl peptide and application of gene vector thereof |
| CN111675749A (en) * | 2020-06-09 | 2020-09-18 | 江苏科技大学 | Polypeptide sequence and self-assembly material and application thereof |
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