CN107880137A - A kind of liver targeting small nucleic acids deliver polypeptide - Google Patents
A kind of liver targeting small nucleic acids deliver polypeptide Download PDFInfo
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- CN107880137A CN107880137A CN201711441104.6A CN201711441104A CN107880137A CN 107880137 A CN107880137 A CN 107880137A CN 201711441104 A CN201711441104 A CN 201711441104A CN 107880137 A CN107880137 A CN 107880137A
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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Abstract
The invention discloses a kind of liver targeting block polypeptide as siRNA delivery vectors, it is to combine several functional polypeptides, including positive electricity sequence blocks, the block that can form β-pleated sheet secondary structure and liver targeting block is formed by the amino acid residue of positively charged.Block polypeptide provided by the invention can form that size is suitable, constitutionally stable composite particles with siRNA, realize siRNA liver targeting delivering.
Description
Technical field
The invention belongs to biomedicine field, and in particular to a kind of block type polypeptide for nucleic acid molecules delivering in vivo carries
Body；Further, the present invention relates to the block type peptide carrier and siRNA, oligonucleotide (oligonucleotide),
DNA forms the design and preparation method of nucleic acid/carrier complexes；Further, the present invention relates to nucleic acid/carrier complexes
Size, stability and repeatability.
Background technology
In the late two decades, go deep into genome research, scientists find many disease (genetic disease, cancers
Deng) all related to gene unconventionality.As a kind of revolutionary biomedical scheme, gene therapy exactly develops on the basis of this
Get up.Using delivery vector of different nature, people by normal human gene or with disease treatment act on nucleic acid piece
Section imports target cell, corrects the expression defect of gene, realizes the purpose of disease treatment.1998, scientist has found can be with
Short chain siRNA is medium, suppresses the expression of Disease-causing gene, and this gene therapy for being found to be disease provides one completely newly
Technical scheme.
In gene therapy, current technology bottleneck essentially consist in how efficiently, hypotoxicity by nucleic acid fragment (long-chain
DNA, oligonucleotide, siRNA) it is delivered in target cell.Conventional delivery vector has viral and non-viral two class.Virus type
Carrier has higher gene delivery efficiency, but security is poor, restricted to the size of foreign gene, and easily triggers more tight
The immune response of weight.By comparison, non-virus carrier has higher security, but delivery efficiency is relatively low.
Positively charged cationic compound is the most frequently used non-virus carrier, has polycation, positive electricity phosphatide, shell to gather
The types such as sugar, albumin, dendritic macromole and cationic polypeptide.Utilize positively charged cation group and DNA/RNA
Electrical function between upper negatively charged phosphate group, nucleic acid molecules are compressed, and form nucleic acid/cation carrier compound
Grain.This form that contains enables nucleic acid molecules to pass through multiple barrier cells, completes cell delivery process.Although on
Dispute also be present in the relation between nucleic acid/structure of cation carrier compound and the property of compound and transfection efficiency, but
Substantial amounts of result of study shows that this kind of compound has preferable biocompatibility and higher delivery of nucleic acids efficiency.
It is a prerequisite for carrying out gene therapy to form stable nucleic acid/carrier complexes.The mode being simply mixed
The compound formed is very unstable, such as compound (the Cui Zheng, Lin that oligonucleotide is formed with polylysine
Niu, Jingjing Yan, JieLiu, Ying Luo, Dehai Liang, Structure and stability of the
Complex formed by oligonucleotides Phys.Chem.Chem.Phys.2012,14,7352-7359).It is multiple
The stability of compound also can further influence nucleic acid molecules delivery efficiency (Jihan Zhou, Jie Liu, Tao Shi,
Yuqiong Xia, Ying Luo*, and Dehai Liang*Phase Separation of siRNA/polycation
Complex and Its Effect on Transfection Efficiency, softmatter, 2013,9,2262-
2268).Therefore, it is necessary to design the delivery vector that Stability Analysis of Structures, size suitable complexes can be formed with nucleic acid molecules.
As a kind of important large biological molecule, polypeptide has many unique advantages in gene delivery application aspect：Into
Ripe synthesis in solid state scheme can provide the high-purity peptide molecule that structure is clear and definite, distribution is single；Can be distinct by 20 kinds
The abundant primary sequence of amino acid composition and property；Two with important biomolecule activity such as beta foldings, alpha spirals can be obtained
Level structure；Sequence or molecule coupling labeled easily modified or that there is tissue target function with other.The polypeptide delivery carrier of report is big
Body can be divided into two classes：One kind is simulation, the polypeptide for transforming cell-penetrating peptide (cell penetrating peptide) sequence, and permeable membrane is imitated
Fruit is good, but generally has larger toxicity；Another kind of is to be used in mixed way targeted polypeptide, cell-penetrating peptide and other carriers, this kind of
The structurally and functionally mode of carrier is complex, and druggability is poor.
The content of the invention
The defects of in order to overcome prior art, based on polyelectrolyte composite theory, devise one kind has the present invention
The peptide molecule of block type structure.The polypeptide can form stable nano particle knot by electrostatic interaction with DNA, RNA molecule
Structure, realize liver targeting delivery of nucleic acids.
It is by several polypeptides with difference in functionality provided by the present invention for siRNA or the polypeptide of gene delivery vector
Sequence connects, so as to which the block type polypeptide obtained, including positive electricity sequence blocks, secondary structure block and liver targeting are embedding
Section.
The positive electricity sequence blocks are made up of the amino acid residue of positively charged, and sequence is specially
KKKKKKKKKKKKKKKKKKKK.The effect of the block is the positive charge using its band, is sent out with negatively charged nucleic acid molecules
Raw interaction, form the core of nucleic acid/carrier complexes.
The secondary structure block can form beta sheet structure, play a part of stable compound structure, and sequence is specially
VEVKVEVKVEVKVEVKVEVK。
Above-mentioned positive electricity sequence blocks and secondary structure block can be directly linked together, can also be by a flexible connection chain
Section connects the two.The effect of the flexible connection segment is with flexibility side by positive electricity sequence blocks and secondary structure block
Formula connects, and glycine (G), asparagine (N), glutamine (Q) and the third ammonia may be selected in the amino acid of composition flexible connection segment
One or more in sour (A), preferably glycine (G).The amino acid residue numbers of composition flexible connection segment are usually 1-5
It is individual, preferably 3.It is declined slightly it is worth noting that, lacking flexible connection segment and being likely to result in the effect of block polypeptide, but
It is that this flexible connection segment is not required in that.
The liver target block has the function that targeting liver, and sequence is specially GAGAGSGAGAGSGAG.
Block type polypeptide provided by the invention can be with different N/P ratios (cationic polymer institute's positively charged and nucleic acid
The negatively charged ratio of molecule institute), form with nucleic acid molecules (DNA or RNA) that size is suitable, Stability Analysis of Structures by way of being simply mixed
Nanoscale nucleic acid/carrier complexes, realize nucleic acid molecules Liver targeting delivering and transport.
Beneficial effects of the present invention
Block type polypeptide provided by the invention, the nano particle that size is suitable, stability is strong can be formed with nucleic acid molecules,
It can be not only used for delivering short-chain nucleic acids medicine (antisense oligonucleotide, small RNA, miRNA, other features widow
Poly-ribonucleotide and deoxyribonucleotide), it can also be used to the Liver targeting delivering of long chain DNA.Moreover, the block type polypeptide without
By in vivo or in vitro, less toxicity is respectively provided with.
Brief description of the drawings
SiRNA and the compound of peptide carrier shape granular size in Fig. 1, embodiment two.
The stability study of siRNA/ polypeptide complexes in Fig. 2, embodiment two.
The variation diagram of polypeptide secondary structure is characterized in Fig. 3, embodiment two using circular dichroism spectra.
The liquid AFM figures of siRNA/ polypeptide complex particles in Fig. 4, embodiment two.
Distribution map inside siRNA/ polypeptide complexes in Fig. 5, embodiment three.
The organ distribution figure of siRNA/ polypeptide complexes in Fig. 6, embodiment three.
The quantitative result of siRNA organ distributions in Fig. 7, embodiment three.
SiRNA-3 is acted on liver L amin A/C expression inhibiting in Fig. 8, example IV.
SiRNA-3 cell and toxicity in vivo research in Fig. 9, embodiment five.
Embodiment
Technical scheme and beneficial effect are further illustrated below by embodiment, but is not limited in any way
The scope of the present invention.
Embodiment one：The preparation of laboratory apparatus and sample
1st, laboratory apparatus
In order to study the size of the genophore formed and property, light scattering apparatus is combined using sound state multi-angle
(Brookhaven Instruments Corporation, Holtsville, NY, U.S.A) determines compound size and tracked
Its stability in the solution.Changed with circular dichroism spectra tracking polypeptide secondary structure.
2nd, experiment material
Experimental subjects is used as using the peptide carrier of the double-chain small disturbance RNA (siRNA) of 21 base-pairs and design.SiRNA is purchased
Synthesized from Guangzhou Ribo Bio Co., Ltd., polypeptide by gill biochemistry (Shanghai) Co., Ltd..SiRNA and Liver targeting
Polypeptide L-peptide sequence is as follows, and wherein siRNA-3 is the specific siRNA using mouse Lamin A/C genes as target spot.
SiRNA-1 positive-sense strands (SEQ ID No：1)：5’-GCUACUGAACCACAAGAAUdTclT-3’
SiRNA-1 antisense strands (SEQ ID No：2)：5’-AUUCUUGUGGUUCAGUAGCdTdT-3’
SiRNA-2 positive-sense strands (SEQ lD No：3)：5’-GGUCUUUGCUGAACCUCAAdTdT-3’
SiRNA-2 antisense strands (SEQ ID No：4)：5’-UUGAGGUUCAGCAAAGACCdTdT-3’
SiRNA-3 positive-sense strands (SEQ ID No：5)：5’-CCAGCUAGAGCUGAGCAAAdTdT-3’
SiRNA-3 antisense strands (SEQ ID No：6)：5’-UUUGCUCAGCUCUAGCUGGdTdT-3’
L-peptide(SEQ ID No：7)：Ac-
KKKKKKKKKKKKKKKKKKKKGGGVEVKVEVKVEVKVEVKVEVKNNNGAGAGSGAGAGSGAG-amide
3rd, the preparation of sample
By siRNA-1, siRNA-2 and siRNA-3 positive-sense strand and antisense strand, it is dissolved separately in and removes Ca, Mg ion
In standard phosphate buffer solution (DPBS).The positive-sense strand and antisense strand solution of every siRNA equivalent are taken respectively, are entered according to standard scheme
Row annealing, obtains double-strand siRNA.SiRNA concentration is adjusted to 1x10-4g/mL.By L-peptide polypeptides be dissolved in remove Ca,
In the standard phosphate buffer solution (DPBS) of Mg ions, to final concentration 1x10-4g/mL.Utilize 0.20 μm of aqueous phase filter
(Sartorius stedim Biotech, Goettingen, Germany), is filtered off except the bacterium in siRNA, polypeptide solution
And dust.
Using uniform mixing method, a certain amount of siRNA solution is added in a certain amount of polypeptide solution, with
1200rps/min rotating speed, which is vortexed, to be mixed 30 seconds, is made its fully dispersed, is prepared siRNA/ peptide carrier compounds.Adjust nucleic acid
The usage ratio of solution and polypeptide solution, prepare the siRNA/ polypeptide complexes with different N/P.By the siRNA/ polypeptides of acquisition
Compound stands 30 minutes at room temperature, after its is substantially stabilized, carries out and detects or use in next step.
Embodiment two：The sign of nucleic acid/carrier complexes
According to the method described above, siRNA-1, siRNA-2 or siRNA-3 are utilized respectively to mix with L-peptide polypeptides, is prepared
N/P is than the siRNA/ polypeptide complexes for 5,10,20,40,80.
Using light scattering and AFM, the structure of these compounds is characterized.The measured value of its hydrodynamic radius is such as
Under：
Research finds, the sizes of siRNA/ polypeptide complex particles, stability to N/P than related, with used siRNA
Sequence is unrelated.
Result shown in Fig. 1 is siRNA-1 under conditions of N/P=20, the compound result with L-peptide polypeptides.This
Stability is fine in the solution for one particle, and particle scattered light intensity can be up to hours up to a hundred without changing, corresponding particle chi
Very little also no change (Fig. 2).Follow-up study prepares siRNA/ polypeptide complexes under conditions of N/P=20.
Then, we utilize circular dichroism spectra technology, have detected the architectural feature of compound.Such as Fig. 3 (siRNA-1, N/P=
20) shown in, peptide carrier is with being not random coil before oligonucleotide formation compound, without any secondary structure,
And after forming compound with oligonucleotide, polypeptide presents obvious beta folded signals, and this illustrates that compound is formed
During polypeptide conformation changed.
In addition, the appearance structure of compound is formed using liquid AFM home position observation.By the compound of formation
10 μ L are drawn with liquid-transfering gun and are placed in mica surface, are added PBS cushioning liquid, are directly measured composite structure in the solution, can be with
See that compound size is consistent with the result that light scattering measures, diameter is at 60nm or so (Fig. 4, siRNA-1, N/P=20).
INSTRUMENT MODEL is Nanoscope V.
Embodiment three：Liver targeting siRNA is delivered
The siRNA-2 and siRNA-3 (siRNA-2-cy5, siRNA-3-cy5) of cy5 fluorescence labelings are bought, makes itself and L-
Peptide peptide carriers combine (N/P=20), form the composite particles of fluorescence labeling.By the composite particles of fluorescence labeling,
And the siRNA for the naked fluorescence labeling not combined with polypeptide is expelled in Mice Body by tail vein.The mouse used is hero
Property BALB/c mouse, in 5-7 weeks mouse age, body weight 18-22g, siRNA injection dosage are 1.0mg/kg (1 ×).Put to death after 24 hours
Mouse, carry out the fluorescence imaging observation of whole animal.Compared with having injected naked siRNA-3-cy5 mouse, whole animal imaging
Display L-peptide can improve the distributions of siRNA in vivo, the especially abundance (Fig. 5) in mouse liver.It is used small
Animal imager is Kodak F × Pro, is produced by Carestream Health companies.
Then, the main organs tissue of our separating mouses, including brain, salivary gland, the heart, liver, kidney, spleen and testis.With
Toy phosphorimager observes the fluorescence intensity (Fig. 6) of siRNA-3 in each internal organs.Further, we are in each internal organs
Fluorescence intensity has carried out quantitative analysis, it is found that the fluorescence volume in liver is highest (Fig. 7).
These results show：L-peptide polypeptides can significantly improve enrichments of the siRNA in liver, be that one kind has significantly
The siRNA polypeptide delivery carriers of Liver targeting effect.
Example IV：The expression silencing of the endogenous gene of L-peptide mediations
In order to evaluate whether L-peptide peptide carriers can mediate the expression silencing of endogenous gene, we are selected with Lamin
A/C genes conduct a research for the siRNA-3 of target spot.SiRNA-3 is combined with L-peptide polypeptides, form siRNA-3/L-
Peptide composite particles.Mouse is handled respectively with PBS, naked siRNA-3, siRNA-3/L-peptide compound.Use
Mouse is male BALB/c mouse, and in 5-7 weeks mouse age, body weight 18-22g, siRNA injection dosage are 1.0mg/kg.
Mouse is put to death after 48 hours, obtains the liver organization of experimental group and control group mice, extracts total serum IgE.Utilize
PCR primer special Lamin A/C, analyze the expression of Lamin A/C in the mouse liver Jing Guo different disposal.Contrast
The mouse of PBS and naked siRNA-3 processing, analyze and the expression inhibiting of endogenous gene is made via the siRNA-3 of L-peptide deliverings
With.
Experimental procedure is specific as follows：
1) Total RNAs extraction：Using Trizol reagents, total serum IgE in liver organization is extracted, and quantified.
2) RNA reverse transcriptions：Use Reverse Transcriptase kit (the TIANScript M-MLV, cat of Tiangeng biochemical technology company：
ER104-03 the reverse transcription reaction of RNA samples) is carried out, is specifically carried out according to the method for kit specification.
3) reverse transcription PCR：Use the 2xEasy Taq PCR SuperMix of Beijing Quan Shijin bio tech ltd
Reagent (cat：AS111-01), the expression of gene in sample is detected.1) 1 μ L reverse transcription products are taken, add 12.5 μ
L2xEasyTaq@PCR SuperMix, 0.5 μ L (10pM) sense primer, 0.5 μ L (10pM) anti-sense primer, are eventually adding 10.5 μ
L deionized waters, reaction cumulative volume are 25 μ L.2) of short duration centrifugation after mixing, it is placed in TECHNE PCR instruments and enters performing PCR amplification instead
Should, response parameter is 94 DEG C of pre-degenerations 1 minute, and 94 DEG C are denatured 30 seconds, and 59 DEG C are annealed 30 seconds, and 72 DEG C extend 30 seconds, cycle-index
For 30 circulations；72 DEG C, 5 minutes；It is subsequently placed at 4 DEG C of preservations.
4) glue detection is run：PCR primer is detected with 2% Ago-Gel.
5) RT-PCR data are analyzed, identify the expression of Lamin A/C in each tissue.
Compared with the mouse of PBS or naked siRNA-3 processing, the processing of siRNA-3/L-peptide compounds makes Mouse Liver
Dirty middle Lamin A/C expression have dropped 35% (Fig. 8).This result shows that L-peptide effectively can pass siRNA
Mouse liver is delivered to, and suppresses the expression of its target gene.
Used pcr amplification primer thing sequence is as follows：
LaminA/C sense primers (SEQ ID No：11)：5’-GATGGAGGGCAATGTCAAGT；
LaminA/C anti-sense primers (SEQ ID No：12)：5’-AGAGGTGCAGATGGGAAATG；
β-actin sense primers (SEQ ID No：13)：5’-GAAGAGCTATGAGCTGCCTGA；
β-actin anti-sense primers (SEQ ID No：14)：5’-CTCATCGTACTCCTGCTTGCT.
Embodiment five：The toxicity research of L-peptide polypeptide delivery carriers
In order to assess the cytotoxicity of L-peptide polypeptides, we with different N/P than siRNA-3/L-
In the HEK293 cells of peptide compounds processing culture, control group includes：Without the HEK293 cells of any processing
(CTL), by the compounds of siRNA-3/Lipofectamine 2000 handle HEK293 cells (Lipo), and by not with
The cell (L-peptide) of L-peptide processing compound siRNA.Then CCK-8 kits are used, detect various processing modes
Influence to cell survival rate.
In the previous day of experiment, with 6x103HEK293 cells are inoculated into 96 orifice plates by the inoculum concentration of cells/well.Growth
Overnight after, using with different N/P than siRNA-3/L-peptide composite particles or collating condition handle cell,
SiRNA-3 dosage is 0.08 μ g/ holes.4 hours after transfection, 100 μ LDMEM are added per hole, continue culture 20 hours.
After culture terminates, Cell Counting Kit-8 (CCK-8) reagents (Dojindo is utilized
MolecorTechnologies, Inc.) detection cell activity, concrete operations by kit specification carry out.Experimental result
As shown in figure 9, show when N/P is than controlling between 5-40, to culture cell without significant toxic action.
In embodiment three and example IV, to inject PBS, naked siRNA mouse as control, comparative study injection L-
Toxicity inside peptide complexs.Investigated from mouse weight, food ration, active situation, hair color and hair condition etc.,
It is consistent with control mice, any toxic side effect is not found.This shows that L-peptide peptide carriers have safety inside preferably
Property.
Claims (10)
1. a kind of block polypeptide as liver targeting siRNA deliverings, including positive electricity sequence blocks, secondary structure block and liver
Dirty targeting block, wherein the positive electricity sequence blocks are made up of the amino acid residue of positively charged, the secondary structure block is energy
Form the block of beta sheet, the amino acid residue numbers of the positive electricity sequence blocks are 10-70, the ammonia of the secondary structure block
Base acid number of residues is 10-40.
2. liver targeting siRNA as claimed in claim 1 delivers polypeptide, it is characterised in that forms the positive electricity sequence blocks
Amino acid be lysine and/or arginine.
3. liver targeting siRNA as claimed in claim 1 delivers polypeptide, it is characterised in that the secondary structure block is selected from
One of one sequence：
VEVKVEVKVEVKVEVKVEVK；
IEIKIEIKIEIKIEIKIEIK；
ELKLELKLELKLELKLELK；
GAGAGSGAGAGSGAGAGS；
KSKLALKLALKAWSAALKLA；Or
KLALKLALKALKAALKLA。
4. liver targeting siRNA as claimed in claim 1 delivers polypeptide, it is characterised in that the block polypeptide also includes one
Segment is flexibly connected, the flexible connection segment connects the positive electricity sequence blocks and secondary structure block.
5. liver targeting siRNA as claimed in claim 4 delivers polypeptide, it is characterised in that the ammonia of the flexible connection segment
Base acid number of residues is 1-5；Form it is described flexible connection segment amino acid be selected from glycine, asparagine, glutamine or
One or more in alanine.
6. liver targeting siRNA as claimed in claim 1 delivers polypeptide, it is characterised in that the liver target block is
GAGAGSGAGAGSGAG。
7. liver targeting siRNA as claimed in claim 1 delivers polypeptide, it is characterised in that the block peptide sequence is：
Ac-KKKKKKKKKKKKKKKKKKKKGGGVEVKVEVKVEVKVEVKVEVKNNNGAGAGSGAGAGSGAG-amide。
8. any one of the claim 1-7 liver targeting siRNA deliver polypeptide in the application into hepatic delivery siRNA.
9. any one of the claim 1-7 liver targeting siRNA delivering polypeptides are being prepared into hepatic delivery siRNA medicines
Application.
10. a kind of composition, it includes liver targeting siRNA delivering polypeptides described in claim 1 and effective dose
siRNA。
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| US20040102608A1 (en) * | 2002-05-13 | 2004-05-27 | Cornell Research Foundation, Inc. | Multiblock copolymers having improved mechanical properties |
| CN101405384A (en) * | 2006-03-17 | 2009-04-08 | 三洋化成工业株式会社 | Cell culture substrate |
| JP2012126707A (en) * | 2010-11-04 | 2012-07-05 | Sanyo Chem Ind Ltd | Chemically modified cell-adhesive polypeptide |
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