CN108060176A - A kind of functional form genophore and DNA/ carrier complexes - Google Patents
A kind of functional form genophore and DNA/ carrier complexes Download PDFInfo
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- CN108060176A CN108060176A CN201711181223.2A CN201711181223A CN108060176A CN 108060176 A CN108060176 A CN 108060176A CN 201711181223 A CN201711181223 A CN 201711181223A CN 108060176 A CN108060176 A CN 108060176A
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Abstract
The invention discloses a kind of functional form genophores, it is made of calcium carbonate, sodium alginate and polypeptide, and the mass ratio of the calcium carbonate, sodium alginate and polypeptide is 1~8:1~4:1~12, the invention also discloses a kind of DNA/ carrier complexes, the compound have many advantages, such as low toxicity, efficiently, stablize, preparation process is easy, mild condition, in biomedical sector, as being with a wide range of applications in terms of gene/drug transmission system.
Description
Technical field
The present invention relates to bio-medical carrier material fields, and in particular to a kind of functional form genophore, the present invention also relate to
And a kind of DNA/ carrier complexes and preparation method thereof.
Background technology
With the mankind biomedical sector explore paces quickening, for traditional chemical pharmaceutical means treatment but produce effects
Little part difficult and complicated cases, are also progressively found that new breakthrough method, and gene therapy is exactly the important reason of one of which
By innovation and technological innovation.During gene therapy, functional gene is transported to target cell or target tissue by various carriers
To repair impaired/missing gene, so as to achieve the purpose that cure disease.Wherein, the suitable carrier of construction and screening is to realize base
Effective transmission of cause, it is most important for the success of gene therapy.It is divided into viral vectors and non-disease for the carrier of gene delivery
Two major class of poisonous carrier.Although viral vectors has natural high-efficiency transfection ability, the bio-safety problem in therapeutic process
It still can not be ignored.Therefore, in genophore research field, current related hot spot focus primarily upon such as dendritic macromole,
The organics such as cationic polymer, cationic-liposome and polypeptide non-virus carrier and calcium carbonate and silica-base material etc. are inorganic
Class non-virus carrier.On the premise of safety is guaranteed in application, the transfection efficiency of above-mentioned non-virus carrier how is improved, so that
It can compare favourably with the transfection abilities of viral vectors, always be the focal point of numerous researchers.Wherein, calcium carbonate is as most
One of common inorganic non-virus carrier material, although possess good biocompatibility and biodegradability, and can
Preferably to contain DNA, the transfection of cellular level is realized.But in the preparation, due to raw after calcium ion and carbanion mixing
Process into carrier lacks regulation and control, and it is excessive to be easy to cause diameter of carrier, so as to reduce transfection efficiency.Meanwhile also affect the load
The operability and reappearance of production procedure.
For natural polymer due to deriving from a wealth of sources, biocompatibility is preferable, is also more and more applied to drug and gene
The structure of carrier, such as chitosan and starch.Alginic acid is as a kind of common natural polymer being easy to get, the load developed
Body includes the variforms such as microballoon and gel, but also it is more favourable to assign carrier by electrostatic interaction or chemical modification techniques
Characteristic, be a kind of important bio-medical organic material.In addition, the polypeptide with superior bio compatibility is also a kind of common
Gene vector material.The sequential combination of specific amino acid sequence can be (to carry in each stage of peptide carrier gene transfection process
The gene of body contains, cell-membrane receptor targeting, cross-film enter born of the same parents, inclusion body escape with nuclear location etc.) it extends efficient help, make more
Peptide becomes one of hot spot of current carrier material research.
Preferably organic and inorganic non-virus carrier material of the invention is combined, and exploitation obtains a kind of less toxic, efficient, steady
Organic/inorganic mixing genophore that is fixed, preparing simplicity.
The content of the invention
It is an object of the invention to overcome the shortcomings of in above-mentioned calcium carbonate carrier technology of preparing, a kind of functional form gene is provided
Carrier, the genophore is based on organic and inorganic non-virus carrier material, with toxicity is low, stability is good, transfection efficiency
Height prepares the advantages that easy.
In order to achieve the object of the present invention, technical scheme is as follows：
Present invention firstly provides a kind of functional form genophores, it is made of calcium carbonate, sodium alginate and polypeptide, described
The mass ratio of calcium carbonate, sodium alginate and polypeptide is 1~8:1~4:1~12.
Preferably, the molecular weight of sodium alginate is 50~150kDa.
It is further preferred that the amino acid sequence of the polypeptide such as SEQ ID NO：Shown in 1.
Secondly the present invention provides a kind of DNA/ carrier complexes, it is by functionality described above type genophore and plasmid
DNA is formed.
Preferably, the functional form genophore and the mass ratio of Plasmid DNA are 1~150:1.
The present invention also provides a kind of method for preparing the DNA/ carrier complexes again, it comprises the following steps：
(1) calcium chloride and the mixed aqueous solution of Plasmid DNA are prepared；
(2) mixed aqueous solution of polypeptide, sodium alginate and sodium carbonate is prepared；
(3) step (1) solution is added dropwise in step (2) solution, calcium chloride is made to react with sodium carbonate, and is formed
Zeta potential be -10~50mV, the DNA/ carrier complexes that grain size is about 200~700nm.
The present invention has the following advantages：
(1) present invention makes the genophore being prepared by introducing sodium alginate into inorganic carrier material calcium carbonate
Grain size is controlled, and reduces the increased generation of particle buildup, improves the ability that carrier complexes enter cell, promotes turning for cell
Dye；Simultaneously, moreover it is possible to weaken phagocytosis of the endothelium network to carrier complexes, extend its circulation time in vivo, from
And enhance the stability of carrier complexes, promote the efficiency of gene delivery.
(2) present invention into inorganic carrier material calcium carbonate by introducing containing nuclear localization sequence (PKKKRKV), inclusion body
Escape sequence (H7) and wear film sequence (R8) functional form polypeptide, make the carrier being prepared possess overcome gene transmembrane transport,
Inclusion body is escaped and the ability into obstacles such as nucleus, further improves transfection efficiency.
(3) sodium alginate and polypeptide that the present invention is introduced into inorganic carrier material calcium carbonate, is that biocompatibility is good
Good organic support material, can improve the transfection efficiency to cell in the case where not influencing normal physiological conditions.
(4) present invention incorporates inorganic material calcium carbonate, organic material sodium alginate with polypeptide as the excellent of genophore
Point, the organic/inorganic mixing genophore being prepared have outstanding transfection abilities, and preparation process is simple, mild condition, easily
In operation.
Description of the drawings
Fig. 1 is the DNA/ carrier complexes that are prepared of embodiment 1, embodiment 2 and embodiment 3 to HepG2 cells, COS-
The transfection figure of 7 cells and HeLa cells.
Specific embodiment
Technical scheme is described in detail below in conjunction with specific embodiment, the raw material arrived involved in embodiment
Or reagent is ordinary commercial products.
Embodiment 1
A kind of functional form genophore, is made of calcium carbonate, sodium alginate and polypeptide, the calcium carbonate, sodium alginate and
The mass ratio of polypeptide is 2:1:3, the molecular weight of the sodium alginate is 100kDa, and the polypeptide is PKKKRKVH7R8, amino
Acid sequence such as SEQ ID NO：Shown in 1.
A kind of DNA/ carrier complexes, are made of functional form genophore and Plasmid DNA (luciferase plasmids pGL-3),
The functional form genophore and the mass ratio of Plasmid DNA are 30:1.
The preparation method of the DNA/ carrier complexes is as follows：
(1) 100mg/mL calcium chloride and the mixed aqueous solution of 100 μ g/mL Plasmid DNA (luciferase plasmids pGL-3) are prepared
1mL；
(2) mixed aqueous solution of 1500 μ g/mL polypeptides, 500 μ g/mL sodium alginates and 1.06mg/mL sodium carbonate is prepared
1mL；
(3) step (1) solution is added dropwise in step (2) solution, calcium chloride is made to react with sodium carbonate, and is formed
The suspension aqueous solution of DNA/ carrier complexes.
Current potential and granularmetric analysis are carried out to the DNA/ carrier complexes that embodiment 1 is prepared using dynamic light scattering.
Measurement result shows the Zeta potential of compound as 25mV, average grain diameter 271nm, favorable dispersibility.
Embodiment 2
A kind of functional form genophore, is made of calcium carbonate, sodium alginate and polypeptide, the calcium carbonate, sodium alginate and
The mass ratio of polypeptide is 4:2:5, the molecular weight of the sodium alginate is 50kDa, and the polypeptide is PKKKRKVH7R8.
A kind of DNA/ carrier complexes, are made of functional form genophore and Plasmid DNA (luciferase plasmids pGL-3),
The functional form genophore and the mass ratio of Plasmid DNA are 110:1.
The preparation method of the DNA/ carrier complexes is as follows：
(1) 200mg/mL calcium chloride and the mixed aqueous solution 1mL of 50 μ g/mL Plasmid DNA are prepared；
(2) mixed aqueous solution of 2500 μ g/mL polypeptides, 1000 μ g/mL sodium alginates and 2.12mg/mL sodium carbonate is prepared
1mL；
(3) step (1) solution is added dropwise in step (2) solution, calcium chloride is made to react with sodium carbonate, and is formed
The suspension aqueous solution of DNA/ carrier complexes.
Measurement result shows that compound Zeta potential is 39mV, average grain diameter 409nm.
Embodiment 3
A kind of functional form genophore, is made of calcium carbonate, sodium alginate and polypeptide, the calcium carbonate, sodium alginate and
The mass ratio of polypeptide is 1:1:1, the molecular weight of the sodium alginate is 150kDa, and the polypeptide is PKKKRKVH7R8.
A kind of DNA/ carrier complexes, are made of functional form genophore and Plasmid DNA (luciferase plasmids pGL-3),
The functional form genophore and the mass ratio of Plasmid DNA are 2:1.
The preparation method of the DNA/ carrier complexes is as follows：
(1) 20mg/mL calcium chloride and the mixed aqueous solution 1mL of 300 μ g/mL Plasmid DNA are prepared；
(2) mixed aqueous solution of 200 μ g/mL polypeptides, 200 μ g/mL sodium alginates and 0.212mg/mL sodium carbonate is prepared
1mL；
(3) step (1) solution is added dropwise in step (2) solution, calcium chloride is made to react with sodium carbonate, and is formed
The suspension aqueous solution of DNA/ carrier complexes.
Measurement result shows that compound Zeta potential is -3mV, average grain diameter 532nm.
Test example
(1) cell toxicity test
Suspension made from embodiment 1,2,3 is separately added into and is inoculated with HepG2 cells, COS-7 cells and HeLa cells
96 orifice plates in, DNA/ carrier complexes concentration in each hole is made after co-culturing 48h, to be examined between 0~200mg/L with microplate reader
The absorbance in each hole is surveyed, to evaluate its cytotoxicity.The result shows that DNA/ carrier complexes toxicity produced by the present invention is very low,
The survival rate of three kinds of cells is above 90%, wherein the survival rate highest of embodiment 1.
(2) cell transfecting power is tested
Suspension made from embodiment 1,2,3 is separately added into HepG2 cells, COS-7 cells and HeLa cells, is transfected
After 48h, the expression quantity of luciferase protein in cell is quantitative determined using chemiluminescence detector.Attached result table shown in FIG. 1
Bright, compound is respectively provided with three kinds of cells the transfection energy of preferable transfection, wherein embodiment 1 made from embodiment 1,2,3
Power highest.
Sequence table
<110>Hua Zhong Agriculture University
<120>A kind of functional form genophore and DNA/ carrier complexes
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 1
Pro Lys Lys Lys Arg Lys Val His His His His His His His Arg Arg
1 5 10 15
Arg Arg Arg Arg Arg Arg
20
Claims (6)
1. a kind of functional form genophore, it is characterised in that be made of calcium carbonate, sodium alginate and polypeptide, the calcium carbonate, sea
The mass ratio of mosanom and polypeptide is 1~8:1~4:1~12.
2. functional form genophore as described in claim 1, it is characterised in that：The molecular weight of the sodium alginate for 50~
150kDa。
3. functional form genophore as described in claim 1, it is characterised in that：The amino acid sequence of the polypeptide such as SEQ ID
NO：Shown in 1.
4. a kind of DNA/ carrier complexes, it is characterised in that：It is carried as the functional form gene described in claims 1 to 3 any one
Body and Plasmid DNA composition.
5. DNA/ carrier complexes as claimed in claim 4, it is characterised in that：The functional form genophore and Plasmid DNA
Mass ratio be 1~150:1.
A kind of 6. method for preparing DNA/ carrier complexes described in claim 5, it is characterised in that comprise the following steps：
(1) calcium chloride and the mixed aqueous solution of Plasmid DNA are prepared；
(2) mixed aqueous solution of polypeptide, sodium alginate and sodium carbonate is prepared；
(3) step (1) solution is added dropwise in step (2) solution, calcium chloride is made to react with sodium carbonate, and form Zeta electricity
- 10~50mV is in position, the DNA/ carrier complexes that grain size is about 200~700nm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112089886A (en) * | 2020-09-22 | 2020-12-18 | 青岛大学 | Hydrogel and preparation method thereof |
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| CN101721715A (en) * | 2009-12-15 | 2010-06-09 | 武汉大学 | Compound of tumor targeted nonviral gene vector and plasmid DNA and preparation method thereof |
| CN102286155A (en) * | 2011-05-30 | 2011-12-21 | 武汉大学 | Preparation method of sodium alginate-calcium carbonate hybridized micron particles |
| CN103536934A (en) * | 2013-09-06 | 2014-01-29 | 苏州大学 | Self-assembled nano-gene vector compound based on PAMAM (Poly Amido-Amine) and preparation method thereof |
| CN103665169A (en) * | 2013-11-21 | 2014-03-26 | 上海海洋大学 | Three-function peptide-modified gene carrier as well as preparation method and application thereof |
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| 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 |
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| CN102286155A (en) * | 2011-05-30 | 2011-12-21 | 武汉大学 | Preparation method of sodium alginate-calcium carbonate hybridized micron particles |
| CN103536934A (en) * | 2013-09-06 | 2014-01-29 | 苏州大学 | Self-assembled nano-gene vector compound based on PAMAM (Poly Amido-Amine) and preparation method thereof |
| CN103665169A (en) * | 2013-11-21 | 2014-03-26 | 上海海洋大学 | Three-function peptide-modified gene carrier as well as preparation method and application thereof |
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| 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 |
Non-Patent Citations (3)
| Title |
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| DONG ZHAO等: "Alginate modified nanostructured calcium carbonate with enhanced delivery efficiency for gene and drug delivery", 《MOLECULAR BIOSYSTEMS》 * |
| 王慧媛: "基于功能性多肽的基因载体的制备及其转染性能研究", 《中国博士学位论文全文数据库 工程科技I辑》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112089886A (en) * | 2020-09-22 | 2020-12-18 | 青岛大学 | Hydrogel and preparation method thereof |
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Application publication date: 20180522 |