CN107129522A - A kind of intrinsic unordered protein nano carrier of lipoic acid modification and its preparation method and application - Google Patents
A kind of intrinsic unordered protein nano carrier of lipoic acid modification and its preparation method and application Download PDFInfo
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- CN107129522A CN107129522A CN201710202680.9A CN201710202680A CN107129522A CN 107129522 A CN107129522 A CN 107129522A CN 201710202680 A CN201710202680 A CN 201710202680A CN 107129522 A CN107129522 A CN 107129522A
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- lipoic acid
- carrier
- acid modification
- cell
- polypeptide
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- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 230000005186 women's health Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to pharmaceutical technology field, specifically a kind of intrinsic unordered protein nano carrier of lipoic acid modification and its preparation method and application.The disulfide bond that the nano-carrier of the present invention is carried using lipoic acid is crosslinked, the polypeptide polymer formed can be degraded rapidly in the cell, it will not accumulate in the cell, the amino acid for constituting peptide carrier is the amino acid existed in vivo, cell and human body are had no toxic side effect, the method cell proliferation tests of CCK 8 show, the nano-carrier of preparation has very low cytotoxicity, there is the ability for preferably carrying gene and chemotherapeutic altogether again simultaneously, the nano-carrier of present invention energy Successful delivery siRNA in breast cancer treatment suppresses sensitiveness of the autophagy specificity enhancing tumour cell to chemotherapeutic caused by chemotherapeutics, promote the apoptosis of breast cancer cell, so as to a kind of targeting as breast cancer treatment, efficiently, the nanoscale delivery system of low toxicity.
Description
Technical field
It is that a kind of intrinsic unordered protein nano of lipoic acid modification is carried specifically the present invention relates to pharmaceutical technology field
Body and its preparation method and application.
Background technology
Breast cancer is to threaten one of malignant tumour of women's health, and clinic is treated to it still based on operation at present, simultaneously
Radiotherapy, chemotherapeutic drug therapy (abbreviation chemotherapy) is coordinated to carry out complex treatment, wherein chemotherapy plays pass in breast cancer treatment
Key is acted on, but conventional chemotherapy radiotherapy medicine unavoidably injures normal cell without selectivity.Therefore, exploitation is for swollen
The targeting vector of oncocyte treatment is greatly improved the effect to tumour.The introducing of nanometer technology, not only realizes the target of tumour
Tropism treats, can also reduce the toxic side effect of chemotherapeutic.Therefore, a kind of nanometer of energy magnetic target therapy breast cancer is prepared to pass
Medicine system has very important significance.
The appearance of nanometer technology provides opportunity for medicine to intracellular effective conveying.Nanometer skill in art of pharmacy
It is poly- that art product includes but is not limited to nano particle, micella, liposome, nanofiber, nanotube, nano chips and self assembly
Compound nanodevice etc., these nano-carriers have a variety of advantages：Particle diameter is small, and particle diameter distribution is narrow, and target can be carried out after surface modification
To specific localization, protect drug molecule, improve its stability, can be endowed physiological stimulation response and external energy (heat,
Light, sound, magnetic) response, single carrier can contain a variety of therapeutic agents simultaneously, and imaging and treatment can be combined to monitor in real time
Curative effect.
Micella (micelles) is also known as micelle, is the colloidal solution that excessive surfactant is self-assembly of in water,
Least concentration when surfactant molecule associates to form micella is referred to as critical micelle concentration (CMC).Often by micella shape in document
Into process be referred to as " self assembly ".Nano-micelle is in water by the amphiphilic block copolymer with hydrophilic radical and hydrophobic grouping
In the core-shell type micella of nanoscale size that is self-assembly of.
Polypeptide is small molecule as the benefit of carrier.There are multiple polypeptides in human body, these polypeptides be easily metabolized and from
Remove in vivo, non-evident effect.The typically no immunogenicity of polypeptide, can not pass through blood-brain barrier, natural polypeptides maximum
Problem is that half-life is very short, but can be more stable after modification by transforming, and these pass through modification and improved long-acting
Polypeptide can act as effective pharmaceutical carrier.Cell-penetrating peptide (cell penetrating peptides, CPPs) is class tool
There is the small molecule small peptide of stronger cell membrane penetration capacity, a variety of macromolecules into cells can be carried., Green in 1988
Loewenstein et al., which reports I type HIV activating transcription factors TAT, can individually enter cell, and this is CPPs hairs
Open up first milestone in history.1994, the albumen of the report TAT protein energy introducing exogenous such as Fawell entered cell, and
The position for being responsible for transduction is the 47-57 amino acids of TAT protein, referred to as " TAT protein transduction structure " (peptide
Transduction domain, PTD), i.e. protein transduction domain, also as cell-penetrating peptide (cell-penetrating
Peptides, CPPs).This is second milestone in CPPs development histories.This kind of peptide quickly uses the targeting and importing of medicine
In research, effectively it can will include hydrophilic protein matter and polypeptide, nucleotides, small-molecule drug, quantum dot and developer etc.
A variety of cargo molecules inside are imported in various cells, open the new mediated pathways that medicine enters cell.It is in gene therapy
Aspect prepares because of the ability and hypotoxicity of its high efficiency gene delivery material, easily and turned into study hotspot in past more than 20 years.
Research finds that the arginine in polypeptides vector can effectively adsorb negatively charged gene because of the positive charge of its surface enrichment
Material and small, the constitutionally stable carrier/gene composite that forms particle diameter.Such polymer is except shared with non-virus carrier
Advantage outside, its surface is positively charged often with that compared with polyamino, can be protonated in physiological condition, thus compress or
To reach the purpose of parcel gene.In addition cationic polymer can form a kind of core shell structure with DAN or RNA, and shell is hydrophilic
Cationic segment, the core of inner hydrophobic is then the DNA or RNA that part is neutralized, and this structure can improve its stability,
So as to avoid by internal enzyme degraded.
Due to the difficulty and complexity of oncotherapy, the collaboration transmission system of chemotherapeutics and genomic medicine turns into recent years
Carry out the focus of oncotherapy research.Delivery system can improve transfection efficiency altogether, the cooperative effect of curative effect of medication be improved, so as to carry
The curative effect of high oncotherapy.One of challenge of transport of chemotherapeutics and genomic medicine is the design of these common delivery systems
And exploitation.Effective induction system can cross over various obstacles, and medicine delivery is to internal intracellular and produce antitumor action.
Therefore, these transmission systems must have a variety of functions and it is necessary to steady in a long-term, single-minded, and can improve endosome and escape
Ease.
Polypeptides vector is primarily referred to as various cell-penetrating peptides.It is in terms of gene therapy because of its high efficiency gene delivery thing
The ability and hypotoxicity of matter, easily prepare and turned into study hotspot in past more than 20 years.Research is found, in polypeptides vector
Arginine because of the positive charge of its surface enrichment can effectively adsorb negatively charged genetic stew small, the structure that forms particle diameter
Stable carrier/gene composite ([1] Ma, Y.；Gong,C.；Ma,Y.； Fan,F.；et al.Direct cytosolic
delivery of cargoes in vivo by a chimera consisting of D-and L-arginine
residues.J.Controlled Release 2012,162,286-294.[2]Beloor,J.； Choi,C.S.；Nam,
H.Y.；Park,M.；Kim,S.H.；Jackson,A.；Lee,K.Y.；Kim,S.W.； Kumar,P.；Lee,S.-
K.Arginineengrafted biodegradable polymer for the systemic delivery of
therapeutic siRNA.Biomaterials 2012,33,1640-1650.[3]Bagnacani,V.； Franceschi,
V.；Bassi,M.；Lomazzi,M.；Donofrio,G.；Sansone,F.；Casnati,A.； Ungaro,R.Arginine
clustering on calix[4]arene macrocycles for improved cell penetration and DNA
delivery.Nat.Commun.2013,4,1721.[4]Luo,K.；Li,C.；Li, L.；She,W.；Wang,G.；Gu,
Z.Arginine functionalized peptide dendrimers as potential gene delivery
Vehicles.Biomaterials 2012,33,4917-4927) nano-micelle by endocytosis enter cell after quilt
Endocytosis body swallow, and then and lysosome fusion, genomic medicine enter lysosome after easily by lysosome enzyme degrade, because
This, genetic stew only escaped in endosome and lysosome competence exertion effect.
In protein science research field, the normal form thought of " sequence-structure-function " dominates always, but
The intrinsic unordered protein found in recent ten years has broken this golden rule and precious percept.Early in the early 1990s, some protein is found
Although without two grades and tertiary structure, still there is biological function.In latter stage in 20th century, scientist proposes protein
Trinitarian concept (The Protein Trinity), it is believed that under native state, intracellular protein and protein work(
Energy domain exists in the form of one of 3 kinds of thermodynamic states, i.e. ordered state melton-globule state and random coil state, and protein is in this 3 kinds of shapes
Directly function, or functioned by tri-state transformation under state.1999, Wright etc. proposed intrinsic unordered egg
The concept of white matter.
Intrinsic unordered normal albumen (IDPs) is that a class has a uncertain three-dimensional structure under native state, but according to
The protein of normal biological function can so be exercised.There is no the region referred to as region of disorder of rock-steady structure in this proteinoid,
There is the region referred to as order region of rock-steady structure.The amino acid residue of IDPs disordered regions has relatively low hydrophobicity and larger
Electrostatic repulsion, a kind of loose state is presented which results in region of disorder.This existence form has many good qualities, such as solid
There is the contact surface area of unordered albumen larger, conformation is flexible, can be interacted with several parts etc..It is unordered as IDPs
Behind area and ligand binding, due to the reduction of electrostatic charge, IDPs region of disorder can be tended to form folded state.Recent study
Point out, IDPs disordered regions are played an important role in the interaction with biomolecule.IDPs disordered regions can be with
One-to-many or many-to-one interaction is participated in, this is IDPs functional diversities ground major reason.IDPs molecular recognition,
Played a key effect in a variety of physiological activities such as cell cycle regulating, cell signalling, intracellular regulation.
Many CPPs' wears space conformation of the film effect dependent on its alpha-helix and β-pleated sheet, but this space structure one
Aspect accelerates it to enter cell, on the other hand can also cause film fragmentation pathways, and its application is limited to a certain extent.Although
A CPPs cross-film is related to many mechanism, but the CPP carrying medicaments cross-film applied up to now is all with endocytosis cross-film
Based on, and there is the capture of endocytosis body during endocytosis cross-film and the problem of lysosomal degradation.Therefore find a kind of low cracking performance and lead to
New opportunity will be brought into the CPPs of cell membrane to the treatment of tumour by crossing non-phagocytosis.
Lipoic acid is a kind of with intramolecular five-membered ring disulfide bond pattern, the amphiphilic species of terminal carboxyl group, with cell membrane
There is preferable affinity with lipid bilayer, with the effect for preferably containing chemotherapeutics, and disulfide bond can be thin
Cracked under born of the same parents' reductive condition, effective release for medicine in tumor locus provides condition.
Based on above-mentioned theory, the invention provides the IDPs that a kind of non-endocytic mechanism of lipoic acid modification enters cell
(CLIP6) degradable polypeptide gene carrier, overcomes current polypeptides vector and there is the capture of endocytosis body, lysosomal degradation, draws
Enter genetic fragment into cell ability is not strong, efficiency gene transfection is not high, nondegradable defect.
Chinese patent literature CN105727307A discloses a kind of nanometer polypeptide carrier of lipoic acid modification, by arginine,
Histidine, lipoic acid and cysteine composition, the disulfide bond carried using lipoic acid are crosslinked, the polypeptide polymerization formed
Thing can be degraded rapidly in the cell, with very low cytotoxicity, and gene and the energy of chemotherapeutic are preferably carried altogether while have again
Power, to the sensitiveness of chemotherapeutic, can promote breast cancer cell by energy specificity enhancing mdr cell in breast carcinoma resistance treatment
Apoptosis.
A kind of intrinsic unordered protein nano carrier of lipoic acid modification is there is no at present, and being capable of magnetic target therapy mammary gland
The nanometer delivery vector of cancer.
The content of the invention
It is an object of the invention to provide a kind of biodegradable, internal cavities parcel chemotherapeutics, outside contains base
The high polypeptide nano-carrier of the efficiency gene transfection of cause.It is a further object to provide the preparation of the nano-carrier
Method；The third object of the present invention is to provide the nano-carrier in common load chemotherapeutic and the application in gene therapy medicament.
Technical problem underlying to be solved by this invention is：How polypeptide nano carrier guiding gene fragment entrance is improved
The ability of cell, and how to improve polypeptide nano carrier and carry chemotherapeutic and the ability of gene altogether, while ensureing that material has life
Biodegradable characteristic.
The present invention devises a kind of polypeptide nano carrier of the intrinsic unordered albumen of lipoic acid modification, by lysine, figured silk fabrics ammonia
Acid, arginine, proline, threonine, the polypeptide of glutamic acid composition, arginine is positively charged can be with electronegative genetic fragment
With reference to and with membrane penetration effect, wherein anion glutaminic acid residue is to maintain its unordered bioactivity in intrinsic unordered polypeptide
The key amino acid of state, also with its it is unique it is non-in swallow born of the same parents' mechanism and preferable biocompatibility is relevant.Lipoic acid portion
Point can increase the affinity of carrier and cell membrane and chemotherapeutics can be contained and reach common load, and its own disulfide bond crosslinking
And increase and carry medicine and transfection abilities, and the purpose of drug release can be cracked and realized under the conditions of bioreductive.
Compared with the polypeptide LAHR that lipoic acid in Chinese patent literature CN105727307A is modified, the present invention is only to prolong
It is continuous to have used the lipoic acid invented before, realize that tumor microenvironment is sensitive between the disulfide bond of lipoic acid, and institute in the present invention
Cell-penetrating peptide CLIP6 is that a kind of non-endocytic mechanism enters the degradable polypeptide genes of IDPs (CLIP6) of cell and carried
Body, wherein what is played a role is glutamic acid, it is by non-endocytosis body approach, wherein the arginine being rich in can be assigned into cell
It is worn film effect and carries DNA or RNA abilities, and D-PROLINE therein makes its protease hydrolytic stability higher, is expected to
The drug molecule that weak cell permeability and lysosomal degradation can be delivered enters cell.Current polypeptides vector is overcome to exist
The capture of endocytosis body, lysosomal degradation, introducing genetic fragment are into cell ability is not strong, efficiency gene transfection is not high, can not drop
The defect of solution.A kind of CLIP6 for lipoic acid modification that the present invention is provided, laser co-focusing is investigated its internal distribution results and also demonstrate,proved
The siATG7 and hydrophobicity Nile red that the CLIP6 carriers of real lipoic acid modification can effectively mediate FAM to mark enter cell
It is interior.(measured using micella made from the supersound method also critical micelle concentration with very little by fluorescence probe spectroscopic methodology
Critical micelle concentration be 0.00327mg/mL), have good anti-hemodilution ability, its stability in vivo is in fact
The premise of existing passive target.
The first aspect of the present invention is positioned there is provided the polypeptide that a kind of lipoic acid is modified, described polypeptide for IDPs cytoplasm
Internalization peptide 6 (cytosol-localizing internalization peptide 6, CLIP6), the amino of the polypeptide
Acid sequence is as follows：
CLIP6：KVRVRVRVDPTRVRERVK(DP is D-PROLINE) (SEQ ID NO：1)；With peptide bond between amino acid
It is connected, polypeptide can be abbreviated as CLIP6, be abbreviated as CL.
Described lipoic acid modification, refers to that the carboxyl of lipoic acid is connected with the amino of first lysine with amide groups.
The polypeptide of lipoic acid modification of the present invention can be abbreviated as：LA-CLIP6, is abbreviated as LA-CL, and wherein LA is
Lipoic acid, CL is CLIP6 (cytosol-localizing internalization peptide 6).
There is provided the intrinsic unordered protein nano carrier that a kind of lipoic acid is modified, described nanometer for the second aspect of the present invention
The polymer for the polypeptide that carrier is modified for above-mentioned lipoic acid, described polymer is through half Guang ammonia by lipoic acid disulfide bond
What acid was cross-linked to form.
Shown in the chemical structural formula of the monomer of described polymer such as formula (I), the monomer chemistries structure of described polymer
Shown in formula breviaty formula such as formula (II), the chemical constitution such as formula for the intrinsic unordered protein nano carrier that described lipoic acid is modified
(III) shown in：
In formula (II), KVRVRVRVDK in PTRVRERVK:Lysine；V valines；R:Arginine；DP:D-PROLINE；T:
Threonine；E:Glutamic acid.
The peptide C LIP6 of the present invention：KVRVRVRVDK in PTRVRERVK:Lysine；V valines；R:Arginine；DP:D-
Proline；T:Threonine；E:Glutamic acid is constituted to be connected between 17 peptides, amino acid with peptide bond, and english abbreviation is CL；In the N of 17 peptides
End, lipoic acid is connected with amino with amido link, and the polypeptide english abbreviation of lipoic acid modification is LA-CL；The sulfydryl of lipoic acid is through half
The oxidation cross-linked formation polymer of cystine, the english abbreviation of polymer is LA-CLss.
It is preferred that, the molecular weight of described polymer is the polymer discomfort outside 2000-20000Da, the molecular weight
Preferably, the transfection efficiency of genophore can be reduced；Optimal is 15000-20000Da.
It is preferred that, the mole of cysteine is the 10% of the polypeptide that lipoic acid is modified in described polymer.
The preparation side for the intrinsic unordered protein nano carrier that the third aspect of the present invention is modified there is provided above-mentioned lipoic acid
Method, described preparation method comprises the following steps：
(A) the polypeptide LA-CL of above-mentioned lipoic acid modification is synthesized；
(B) preparation of the intrinsic unordered protein nano carrier of lipoic acid modification：The lipoic acid modification that step (A) is synthesized
Polypeptide LA-CL be dissolved in methanol, add cysteine hydrochloride, the mole for making cysteine is the polypeptide that lipoic acid is modified
5%-20%, lucifuge stirring reaction 12h.
In a preferred embodiment of the invention, step (B) is specially：The lipoic acid modification that step (A) is synthesized
Polypeptide LA-CL is dissolved in methanol with cysteine hydrochloride so that the mole of cysteine is the polypeptide that lipoic acid is modified
10%, lucifuge stirring reaction 12h.Reaction temperature is room temperature.
It is preferred that, step (B) reacted solution uses N2Drying.Use N2Sample after drying is in -20 DEG C of preservations.
The reacted solution of step (B) moves into molecular cut off in 1000 bag filter, dialyzate is distilled water, thoroughly
Analysis 12 hours.
To maintain the higher activity of nano carrier material, the solution after dialysis is freeze-dried, and is stored in -20 DEG C, is received
Rice material can be preserved after redissolution 4 DEG C long-term.
The fourth aspect of the present invention is preparing connection there is provided the intrinsic unordered protein nano carrier that above-mentioned lipoic acid is modified
Close the application in chemotherapeutics or genomic medicine.
Further, the intrinsic unordered protein nano carrier of the above-mentioned lipoic acid modification of present invention offer is preparing mammary gland
Application in cancer medicine.
Further, the intrinsic unordered protein nano carrier of the invention for also providing above-mentioned lipoic acid modification carries gene altogether
And chemotherapeutics, the application in breast cancer treatment medicine is prepared.
Described application, refers to that the arginine positively charged in nano-carrier can be combined with electronegative gene.
Described application, refers to that the fat-soluble of the lipoic acid in nano-carrier can contain fat-soluble chemotherapeutics.
Described gene, is DNA or siRNA.
Described chemotherapeutics, is fat-soluble adriamycin or docetaxel, endoxan etc..
Described application is specific as follows：
Nano-carrier and chemotherapeutics are prepared into nano-micelle with ultrasonic emulsification, then it is mixed with DNA, is made and carries altogether
System；
The envelop rate of described chemotherapeutics is 40%-50%, and nano-carrier and DNA N/P ratio are 5:1-80:1.
This nano-carrier is mixed with DNA or siRNA, gene rotaring redyeing system is made.Described nano-carrier and DNA
Or siRNA N/P ratios are 5:1-80:1, in this proportion, described nano carrier material can guide DNA or
SiRNA enters intracellular, there is higher transfection efficiency.
Described nano-carrier contains the ability of taxol, when cysteine ratio is 5%-20%, with preferable
Drugloading rate and envelop rate.
Described nano-carrier is mixed with DNA or siRNA in buffer solution, and pH of buffer is 5.0-7.0, and room temperature is incubated
Educate 20~30 minutes, rational pH value and incubation time ensure that the formation of gene rotaring redyeing system.
The nano-carrier that the present invention is provided is applied to therapeutic DNA or siRNA and chemotherapeutic needed for experiment
Thing.
The invention has the advantages that：
1st, the disulfide bond that nano-carrier of the invention is carried using lipoic acid is crosslinked, the polypeptide polymer formed
It can rapidly be degraded, will not accumulate in the cell in the cell, the amino acid in polypeptide is the amino acid existed in vivo, right
Cell and human body have no toxic side effect, and CCK-8 method cell proliferation tests show, the nano-carrier of preparation has compared with bPEI-25K
There is lower cytotoxicity, while there is the ability for preferably carrying gene and chemotherapeutics altogether again, therefore be very suitable for inside and outside
Chemotherapy and gene therapy study with applying；
2nd, the nano-carrier of the invention energy Successful delivery siRNA in breast cancer treatment suppresses caused by chemotherapeutics certainly
Sensitiveness of the specificity enhancing tumour cell to chemotherapeutic is bitten, promotes the apoptosis of breast cancer cell, so that as breast cancer treatment
A kind of targeting, efficiently, the nanoscale delivery system of low toxicity；
3rd, preparation method of the invention is simple to operate, and reaction reagent and obtained product are non-toxic, and environment will not be produced
Pollution, reaction condition is gentle, and the nano-carrier purifying obtained after reaction is simple, and composition is cheap, and the preparation method can lead to
Cross the ratio of control polypeptide and cysteine to control the degree of cross linking of nano-carrier, beneficial to large-scale promotion is in research and applies
Field.
Brief description of the drawings
Fig. 1 .LA-CLss hydrogen nuclear magnetic resonance spectrogram；
The LA-CLss/pEGFP of different degrees of cross linking grain-size graph under the conditions of Fig. 2 difference N/P ratios；
The potential diagram of LA-CLss/pEGFP nano-micelles under Fig. 3 difference degrees of cross linking；
Different degree of cross linking carrier transfections are investigated under the conditions of Fig. 4 differences N/P；
Fig. 5 cysteines amount is the particle diameter and transmission electron microscope picture of nano-micelle when 10%, N/P is 40；
Fig. 6 cysteines amount is the potential diagram of nano-micelle when 10%, N/P is 40；
DTX release is investigated under the conditions of Fig. 7 difference dissolution mediums；
Fig. 8 .MCF-7 cells to different N/P than LA-CLss/FAM-siRNA intake situation；
Fig. 9 different time points MCF-7 cells are to Nile red (Nile Red) and carry Nile red micella (Nile Red PMs)
Intake situation；
Figure 10 .LA-CLss micellas carry investigation of Nile red and the FAM-siRNA laser co-focusing to its common loading capability；
Carrier is investigated with bPEI-25K to the cytotoxicity of cell under the conditions of Figure 11 various concentrations different times；
To the 24h cytotoxicities of MCF-7 cells under the conditions of Figure 12 investigation various concentrations DTX；
To the 48h cytotoxicities of MCF-7 cells under the conditions of Figure 13 investigation various concentrations DTX；
Figure 14 flow cytometers observe influence of the different dosing group to MCF-7 Apoptosis；
Figure 15 flow cytometers observe influence of the different dosing group to the MCF-7 cell cycles；
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described.It should be understood that following examples are only used
It is used to limit the scope of the present invention in illustrating rather than.
Embodiment 1：The synthesis of 17 peptides of lipoic acid modification
17 peptide amino acid sequences of lipoic acid (LA) modification：Lys Val Arg Val Arg Val Arg ValDPro
Thr Arg Val Arg Gln Arg Val Lys(Lys：Lysine, Val：Valine,DPRO：D-PROLINE, Thr：Soviet Union's ammonia
Acid, Arg：Arginine, Gln：Glutamic acid) (SEQ ID NO：1), LA-CL, by Shanghai gill, biochemical Co., Ltd uses polypeptide
Solid-phase synthesis is synthesized and is named as LA-CL, and the LA-CL of synthesis is purified using preparative high-performance liquid chromatographic, its purity is reached
More than 95%.LA- is lipoic acid, CL：Sequence is KVRVRVRVDPTRVRERVK.Wherein K:Lysine；V valines；R:Smart ammonia
Acid；DP:D-PROLINE；T:Threonine；E:Connect to form 17 peptides with peptide bond between glutamic acid, amino acid.
Embodiment 2：The polypeptide nano-carrier LA-CLss of lipoic acid modification preparation
Take the polypeptide LA-CL that 50mg lipoic acids are modified to be dissolved in different amounts of cysteine hydrochloride in 10ml methanol, stir
Reaction 12 hours is mixed, reaction temperature is 10~30 DEG C.The amount of cysteine is respectively：2.5%th, 5%, 10%, 20%.
As shown in table 1, it is different according to the ratio of LA-CL and cysteine, the LA-CLss of different molecular weight has been made.Institute
Obtain solution to dialyse 12 hours for 1000 bag filter by molecular cut off, dialyzate is distilled water, every 4 hours, changes and dialyses
Liquid.Gained dialysis product freeze-drying, and be stored in -20 DEG C, it is lyophilized after carrier can be preserved for a long time under the conditions of redissolution.Close
Into carrier through proton nmr spectra1H-NMR (600M) detects that molecular weight is determined through gel permeation chromatography (GPC).
The LA-CLss of the different molecular weight of table 1 synthesis
Note:aThe mol ratio of cysteine and LA-CLbDetected by gel permeation chromatography.
As shown in Table 1, add after cysteine, polymer molecular weight is substantially increased, and shows that carrier is successfully crosslinked, and
With the increase of cysteine amount, molecular weight increase.
Embodiment 3：Preparations of the LA-CLss in pDNA nano-micelles
By carrier (LA-CLss) and luciferase expression plasmid pEGFP (Shanghai innovation bio tech ltd) points
It is not soluble in water to prepare the aqueous solution, after being respectively 5,10,20,40,80 configuration nano-complex vortex 10s by N/P ratio (N/P)
30min is stood, nano-micelle is produced.The average grain diameter of nano-micelle is relevant with N/P, and optimum grain-diameter is obtained as N/P=40,
Particle diameter is specifically shown in Fig. 2 between 100-300.The Zeta potential of nano-micelle is raised to N/P increase, when N/P is more than 5
Stabilization is specifically shown in Fig. 3 in 0-30mV.
Embodiment 4：LAHRss/pEGFP transfection efficiency in vitro is investigated
MCF-7 cell strainHJ2mm cell (is purchased from the cell culture of Shanghai bioscience research institute of the Chinese Academy of Sciences
The heart) it is inoculated in respectively on 12 orifice plates according to 30w/ holes, add the DMRM culture mediums that 1ml contains 10%FBS (Gibco companies, the U.S.)
(Gibco companies, the U.S.) cultivates 24 hours, cell confluency degree is reached 70-80%, culture medium is replaced by into serum free medium.
By different degree of cross linking carriers (LA-CL) and luciferase expression plasmid pEGFP difference it is soluble in water prepare it is water-soluble
Liquid, is respectively to stand 30min after 2.5,5,10,20,40,80 configuration nano-complex vortex 10s by N/P ratio (N/P), adds
In orifice plate, after cultivating 4 hours, fresh culture is replaced by, continues to cultivate 24 hours.Fluorescence microscope cell transfecting feelings
Condition, as shown in Figure 4.
By based on the above results, LA-CLss3 has a more suitable molecular weight, less particle diameter, higher positive charge,
And fluorescence intensity is preferably transfected on this condition, therefore take LA-CLss3 to be used to further investigate.
Embodiment 5：The preparation of LA-CLss3/ (DTX/siATG7) nano-micelle
1mg docetaxels are taken to be dissolved in 1ml dichloromethane, by the carrier (LA- after 20mg and cysteine cross
CLss3) it is dissolved in 1ml dichloromethane.
After above two solution is mixed, it is slowly added dropwise with the 10ml pure water containing 1% sodium taurocholate, stirring solvent flashing,
Ultrafiltration removes sodium taurocholate, computational envelope rate and drugloading rate.ATG7siRNA is added by N/P=40, particle diameter and current potential, transmission is determined
Electric Microscopic observation nano-complex form, such as Fig. 5 and 6.
From Fig. 5 and Fig. 6, gained nanometer carries micella particle diameter in 160nm or so altogether, and current potential can guarantee that in more than 30mV
It contains the ability and preferable membrane penetration effect of gene.Fig. 5 transmission electron microscopes show that the form of nano-micelle is complete, subsphaeroidal,
It is uniformly dispersed, preferably can prevents it from assembling.
Embodiment 6：Docetaxel extracorporeal releasing characteristic is investigated
DTX release in vitro situation is investigated using Bag filter method, the bag filter that molecular cut off is 3500, dialysis is chosen
Medium for PH=7.4 PBS solution contain 0.5%w/v Tween-80 respectively contain DTT (50 × 10-3M) and without DTT's
Solution, 37 DEG C are investigated.Take 1mg docetaxels to be dissolved in 1ml dichloromethane, 2mg carriers (LA-CLss3) are dissolved in 1ml
In dichloromethane.8ml water is added after above two solution is mixed, supersound method makes nano-micelle, is slowly added dropwise and contains
In the 10ml pure water of 1% sodium taurocholate, volatile organic solvent is stirred, ultrafiltration removes sodium taurocholate, and LA-CLss3/DTX nanometre glues are made
Beam.By 2ml LA-CLss3/DOX PBS solutions in bag filter, it is positioned in the buffer solution containing 40mlPBS, 100r/min,
37 DEG C, liquid outside 1ml is taken respectively at 2,4,6,8,10,12,24,48,72 time points, and adds 1ml, high performance liquid chromatography is utilized
Determine its concentration.Draw In-vitro release curves as shown in Figure 7.
As shown in Figure 7, the rate of release of docetaxel is significantly faster than that without under the conditions of DTT under the conditions of DTT, and it is 48
During hour, its accumulative release rate nearly 85%.As a result show that the peptide carrier has reductive condition sensitive behavior, it is
Antineoplastic provides condition in tumor microenvironment release.
Embodiment 7：LA-CLss3/Nile Red, LA-CLss3/FAM-siRNA cellular uptake situation
The siRNA (FAM-siRNA) marked using FAM is evaluated as model.By MCF-7 cells according to 30w/ holes point
It is not inoculated on 12 orifice plates, adds DMEM culture medium (Gibco company, U.S. of the 1ml containing 10%FBS (Gibco companies, the U.S.)
State) cultivate 24 hours, cell confluency degree is reached 70-80%, culture medium is replaced by serum free medium.By LA-CLss3/
Nile Red, Nile Red are added in cell hole by 0.1 μ g/ml, culture medium are sucked when 1,2,4h, PBS is washed 3 times, disappeared
Cell is collected by centrifugation in change, with Flow cytometry cellular uptake situation.LA-CLss3/FAM-siRNA is prepared by different N/P
Nano-complex, and add in cell hole, it is incubated 4 hours, sucks culture medium, PBS is washed 3 times, and digestion is collected by centrifugation, and utilizes stream
Formula cell instrument detects intake situation of the cell to FAM-siRNA.
As a result such as Fig. 8 (A：Flow cytometry results figure；B：Niled Red positive MCF- is absorbed after the different dosing time
The ratio of 7 cell numbers) shown in, different time points MCF-7 to Nile Red and LA-CLss3/Nile Red intake difference compared with
Substantially, illustrate to be formed after micella, LA-CLss3/Nile Red intake is significantly more than simple Nile Red, it is seen that carrier
LA-CLss3 can effectively mediate Nile Red to enter cell.LA-CLss3/FAM- of the MCF-7 cells to different N/P
When siRNA intake investigation result is shown in N/P equal to 40 and 80, MCF-7 cells are to LA-CLss3/FAM-siRNA's
Intake is preferable, as a result sees Fig. 9 (A：Flow cytometry results figure；B：The positive cell of intakes of the different N/P than FAM-siRNA
Number).It can be seen that LA-CLss3 can effectively mediate hydrophobic drug and genomic medicine to enter cell by result above.
The laser co-focusing of embodiment 8 observation LA-CLss/FAM-siATG7/ Nile reds carry the intracellular distribution of micella altogether
Experiment is divided into 4 groups：Nile red group (Nile Red), Nile red micella group (Nile Red-PMs), it is loaded with FAM
The siATG7 micellas group (FAM-siATG7-PMs) of mark, altogether load FAM-siATG7 and Nile red group (Co-PMs).MCF is thin
Born of the same parents are according to 1 × 105/ hole is inoculated in be covered with 24 orifice plates of the glass wafer sterilized in advance, cultivates 24h,.Suck culture medium,
Above-mentioned administration group is added, and the culture medium added is the culture medium without serum, continues to cultivate after 4h, is washed with PBS, then
30min is fixed with paraformaldehyde, after washing, the mounting liquid containing DAPI is added, then on laser confocal microscope
Observation.
Carry Nile red altogether by confocal laser scanning microscope and FAM-siATG7 polypeptide micellas are intracellular in MCF-7
Distribution situation, it is the nucleus of DAPI dyes deeply to see in Figure 10, figure what blueness represented, and green represents FAM fluorescence labelings
SiATG7, what red was represented is the Nile red of intracellular distribution.As a result show, compared with simple Nile red, LA-CLss glue
The intake of the Nile red of beam mediation is more, and successfully can carry Nile red altogether and FAM-siATG7 enters cell.
Embodiment 9：LA-CLss3 Study of cytotoxicity
By MCF-7 cells according to 8 × 103/ hole is inoculated in 96 orifice plates, is cultivated 24h, cell confluency degree is reached 50%.
Culture medium is sucked, 100 μ l LA-CLss3 containing various concentrations (5,10,20,40,60,100,120 μ g/ml) are added per hole, is continued
Culture 24,48h, CCK-8 methods detection cytotoxicity, count cell survival rate, with the transfection reagent bPEI (U.S. of commercialization
Sigma-Aldrich companies, molecular weight 25kDa), it is used as control.
As a result as shown in figure 11, the cytotoxicity for compareing bPEI-25K is very strong, and in 40 μ g/ml, cell survival rate is approached
20%, and LA-CLss3 cytotoxicity is relatively low, in 100 μ g/ml, cell survival is barely affected, and cell survival rate is big
In 90%, 80% or so the survival rate in 200 μ g/ml.
Embodiment 10：LA-CLss3/ (DTX/TRAIL) drug efficacy study
Experiment is divided into 3 groups：Simple DTX groups, altogether load DTX micellas group, load DTX and siATG7 groups.MCF-7 cells are pressed
According to 8 × 103/ hole is inoculated in 96 orifice plates, is cultivated 24h, cell confluency degree is reached 50%.Culture medium is sucked, is added per hole
Culture medium (the DTX of 100 μ l concentration containing different pharmaceutical: 0.000050、0.00050、0.0050、0.050、0.50、5.0、50μg/
Ml), continue to cultivate 24,48h, CCK-8 methods detection cytotoxicity counts cell survival rate.
As a result such as Figure 12, shown in 13, for MCF-7 cells, simple DTX groups, carry DTX groups and carry altogether DTX and
The 24h of siATG7 groups IC50Respectively 0.52 ± 1.23 μ g/ml, 0.28 ± 0.74 μ g/ml, 0.043 ± 0.37 μ g/ml；48h
IC50Respectively 0.22 ± 0.98 μ g/ml, 0.11 ± 1.43 μ g/ml, 0.031 ± 0.52 μ g/ml.It can be seen that carrier can be effective
Mediation chemotherapeutic and gene enter cell, and play a role.Chemotherapeutic DTX, which is wrapped up, can increase its drug effect into after micella, and
Altogether after load siATG7, it is suppressed that the protectiveness autophagy that tumour cell is produced under the stimulation of chemotherapeutic, and then enhance cell
To the sensitiveness of chemotherapeutic.
Embodiment 11：LA-CLss3/ (DTX/siATG7) promotees apoptosis research
Experiment is divided into 5 groups：Control group, empty vectors group, simple DTX groups, carry DTX micellas groups, carry DTX altogether and
SiATG7 groups.By MCF cells according to 4 × 105/ hole is inoculated in 12 orifice plates, is cultivated 24h, cell confluency degree is reached 80-
90%.Culture medium is sucked, the culture medium (DTX that 1ml contains certain drug concentration is added per hole：2.5 μ g/ml), continue to cultivate 24h,
Flow cytometer determines Apoptosis.
As a result as shown in figure 14, control group early apoptosis adds the i.e. apoptotic cell toatl proportion of summation of late apoptic to be
6.95%th, empty vectors group is 5.51%, and it is 25.56% that simple DTX groups, which are 12.54%, carry DTX micella groups, carry altogether DTX and
SiATG7 groups are 44.39%.As can be seen here, Apoptosis is promoted after medicine is wrapped up through carrier, and it is brighter to carry effect altogether
It is aobvious.
Embodiment 12：LA-CLss3/DTX Cycle Arrest researchs
Experiment is divided into 4 groups：Control group, empty vectors group, simple DTX groups, load DTX micella groups.By MCF cells according to 4
×105/ hole is inoculated in 12 orifice plates, is cultivated 24h, cell confluency degree is reached 80-90%.Culture medium is sucked, is added per hole
1ml contains the culture medium (DTX of certain drug concentration：2.5 μ g/ml), continue to cultivate 24h, flow cytometer determines Cycle Arrest feelings
Condition.
As a result such as Figure 15 (A:Control group；B：Empty vectors group；C：DTX groups；D：DTX-PMs；) shown in, same dose
Most cells are arrested in the G2/M phases after DTX processing.With control group (G2/M phases cell percentages 14.8% ± 1.3%) and
Empty vectors group (G2/M phases cell percentages 17.4% ± 1.5%) is compared, DTX groups and load DTX micella group G2/M phases cell hundred
Fraction is respectively up to 81.7% ± 2.1% and 92.9% ± 1.1%.As can be seen here, can be substantially by cell after medicine is wrapped up through carrier
The G2/M cycles are arrested in, and it is more obvious to carry DTX micella group effects.
The preferred embodiment to the invention is illustrated above, but the invention is not limited to institute
State embodiment, those skilled in the art can also make a variety of etc. on the premise of without prejudice to the invention spirit
Same modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (10)
1. a kind of polypeptide of lipoic acid modification, it is characterised in that the amino acid sequence of described polypeptide such as SEQ ID NO：1 institute
Show；Described lipoic acid modification, refers to that the carboxyl of lipoic acid is connected with the amino of first lysine with amide groups.
2. a kind of intrinsic unordered protein nano carrier of lipoic acid modification, it is characterised in that described nano-carrier is such as right
It is required that the polymer for the polypeptide that the lipoic acid described in 1 is modified, described polymer is through cysteine by lipoic acid disulfide bond
It is cross-linked to form.
3. the intrinsic unordered protein nano carrier of lipoic acid modification according to claim 2, it is characterised in that described sulphur
Shown in the chemical constitution such as formula (III) of the intrinsic unordered protein nano carrier of octanoic acid modification：
Wherein, K:Lysine；V valines；R:Arginine；DP:D-PROLINE；T:Threonine；E:Glutamic acid.
4. the intrinsic unordered protein nano carrier of lipoic acid modification according to claim 2, it is characterised in that described is poly-
The molecular weight of compound is 2000-20000Da.
5. the intrinsic unordered protein nano carrier of lipoic acid modification according to claim 2, it is characterised in that described is poly-
The mole of cysteine is the 10% of the polypeptide that lipoic acid is modified in compound.
6. a kind of preparation method of the intrinsic unordered protein nano carrier of lipoic acid modification as claimed in claim 2, its feature
It is, described preparation method comprises the following steps：
(A) polypeptide of lipoic acid modification as claimed in claim 1 is synthesized；
(B) preparation of the intrinsic unordered protein nano carrier of lipoic acid modification：The polypeptide for the lipoic acid modification that step (A) is synthesized
Methanol is dissolved in, cysteine hydrochloride is added, it is the 5%-20% of the polypeptide of lipoic acid modification to make the mole of cysteine, is kept away
Light stirring reaction 12h.
7. the preparation method of the intrinsic unordered protein nano carrier of lipoic acid modification according to claim 6, its feature exists
In step (B) is specially：The polypeptide for the lipoic acid modification that step (A) is synthesized is dissolved in methanol with cysteine hydrochloride,
So that the mole of cysteine is the 10% of the polypeptide that lipoic acid is modified, lucifuge stirring reaction 12h.
8. the preparation method of the intrinsic unordered protein nano carrier of the lipoic acid modification according to claim 6 or 7, its feature
It is that step (B) reacted solution uses N2Drying；Use N2Sample after drying is in -20 DEG C of preservations.
9. a kind of intrinsic unordered protein nano carrier of lipoic acid modification as described in claim 2-5 is any is preparing amalgamation
Treat the application in medicine or genomic medicine.
10. the intrinsic unordered protein nano carrier of lipoic acid modification according to claim 9 is preparing combined chemotherapy medicine
Or the application in genomic medicine, it is characterised in that described application is specific as follows：
Nano-carrier and chemotherapeutics are prepared into nano-micelle with ultrasonic emulsification, then it is mixed with DNA or siRNA, is made
Common carrier system；
The envelop rate of described chemotherapeutics is 40%-50%, and nano-carrier and DNA or siRNA N/P ratio are 5:1-80:
1。
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