CN108619487A - Can de-assembly cationic polypeptide nano-fiber material and preparation method and purposes - Google Patents
Can de-assembly cationic polypeptide nano-fiber material and preparation method and purposes Download PDFInfo
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- CN108619487A CN108619487A CN201810356139.8A CN201810356139A CN108619487A CN 108619487 A CN108619487 A CN 108619487A CN 201810356139 A CN201810356139 A CN 201810356139A CN 108619487 A CN108619487 A CN 108619487A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
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Abstract
The invention belongs to polypeptide nano fiber preparation and application fields, and in particular to can de-assembly cationic polypeptide nano-fiber material and preparation method and purposes.In cationic polypeptide aqueous dispersion, control peptide concentration, pH value of solution, incubation temperature T and incubation time t, it is self-assembly of a nanometer long fibre, continue to control pH value of solution, incubation temperature T and incubation time t, nanometer long fibre de-assembly is the nanostructure of nano short fiber and monomer, recycles nanostructure sterilization.
Description
Technical field
The invention belongs to polypeptide nano fiber preparation and application fields, and in particular to can de-assembly cationic polypeptide nanometer
Fibrous material and preparation method and purposes.
Background technology
The pathogenic microorganisms such as bacterium are always one of the arch-criminal of threat people's life security, and not with antibiotic
Reasonable employment, the drug resistance of bacterium to this problem have coverd with one layer of shade according to estimates and have had at present about 70% bacterium at least again
A kind of antibiotic is resistant.Although researcher has made some progress in terms of finding new antiseptic, drug-resistant bacteria
It is constant increase and newly approved antibiotic quantity continue to decline so that find antibacterial agent task quarter do not allow
It is slow.
Antibacterial peptide is the defensive micromolecule polypeptide of a kind of confrontation external source pathogen pathogenic effects generated by organism
(the general 12-50 of amino acid number).Antibacterial peptide is in addition to antibacterial action, also having some fungi and virus or even cancer cell
There is certain lethal effect, there is larger actual application value.
Antibacterial peptide does not depend on specific target site and plays a role, and the negative electrical charge of the positive charge and microbial cell film of institute's band is mutual
Attract, finally makes antibacterial peptide close to microbial cell film surface.Antibacterial peptide is close to after microbial cell film, water-wet side and phosphatide
Molecular layer (cell membrane skeleton) hydrophilic end in contact, so by hydrophobic side be inserted into phospholipid molecule layer hydrophobic interior, interference cell film
Permeability.Under both mechanism, antibacterial peptide finally destroys microbial cell film, keeps microorganism dead.Due to bacterial cell
The amphipathic structure and elecrtonegativity of film can not change, therefore be difficult to develop immunity to drugs.And due to the composition of cell membrane difference, make
Microorganism can selectively be killed without generating significant toxicity to normal cell by obtaining antibacterial peptide, therefore is expected to as antibiotic
One of ideal substitute.
Since traditional antibacterial peptide exists in the solution usually with monomer and bacterial interactions, but monomer is in physiological environment
It is easy down just to lose the ability of sustained anti-microbial after being degraded, and the antibacterial peptide of nanofibrous structures can be dense by changing polypeptide
Degree or pH value of solution and de-assembly are antibacterial peptide monomer, and to have the function that sustained anti-microbial, therefore it is anti-to develop novel de-assembly
Bacterium peptide becomes the hot spot of current research.
Cationic polypeptide FF, in certain environment solution can self assembly be nanofiber.It is template according to this section of sequence
Lysine (K, hydrophilic amino acid) and alanine (A is hydrophobic amino acid) be added thereto by we, comprehensive design amphiphilic
Property polypeptide sequence KKKFAFAFAFAKKK and KKKFAFAFAFAKKK, two segment polypeptides it is positively charged in the solution (pH=1~10,
Potential diagram is shown in Fig. 2), so that it is had antibacterial peptide feature, and be provided simultaneously with the ability of self assembly and de-assembly.
Invention content
Cationic polypeptide nanofiber is prepared, antibacterial is used for after de-assembly.The nanofibrous structures are easy under certain condition
It is degraded, it is strong with bacterial interactions, bacterium can be killed in a short time.
Technical scheme is as follows：
A kind of cationic polypeptide nanofiber carries out as steps described below in the purposes in antibacterial direction：
By 50 μ L bacterial suspensions (~4 × 106cfu mL–1) it is separately added into 500 μ g mL of 2mL–1The solution of cationic polypeptide
Assembling nanostructure solution is placed in co-incubation 6h in 37 DEG C of constant incubators, every 1h, dilutes 1000 with phosphate buffer
Times, it takes the suspension after 100 μ L dilutions to be put into Lysogeny Broth solid mediums, 16h is cultivated at 37 DEG C, calculates bacterium colony
Number.
Cationic polypeptide de-assembly nano-structured solution described in above-mentioned technical proposal, preparation method are as follows：
By a concentration of 200~1000 μ g mL–1Cationic polypeptide be added pH=11~14 solution in, be placed in constant temperature
It is incubated 1.5day~9day on oscillator, forms nanometer long fibre after cationic polypeptide self assembly.
The pH of nanofiber solution is adjusted to 6.5~7.5 again, be placed on constant temperature oscillator continue be incubated 0.5day~
5day, nanofiber de-assembly form nano short fiber and monomer.
Bacterium described in said program is staphylococcus aureus (Staphylococcus aureus), Escherichia coli
(E.coli), one kind in salmonella (salmonella) or bacillus subtilis (Bacillus subtilis).
Cationic polypeptide described in said program is K3(FA)4K3、K6(FA)4、K2(FA)3K2、K4(FA)3、Ac-K3(FA)4K3-NH2、K2(FA)2、K(FA)2K、Ac-K2(FA)3K2-NH2、Ac-K2(FA)2-NH2、Ac-K(FA)2K-NH2、Ac-K2(FA)2-
NH2And Ac-K6(FA)4-NH2In one kind, preferably K3(FA)4K3And K6(FA)4In one kind.
The temperature being incubated on constant temperature oscillator is 37 DEG C, rotating speed 360rpm/min.
A concentration of 200~1000 μ g mL of cationic polypeptide described in said program–1, it is preferable that cationic polypeptide it is dense
Degree is 500 μ g mL–1。
Cationic polypeptide pH value of solution=11~14 described in said program, it is preferable that cationic polypeptide pH value of solution=12.
T=1.5day~the 9day of cationic polypeptide incubation time described in said program, it is preferable that incubation time t=
5day。
The pH of nanofiber solution described in said program is adjusted to 6.5~7.5, it is preferable that pH is adjusted to 7.
Incubation time t=0.5day~5day of nanofiber solution described in said program, it is preferable that incubation time
T=3day.
Nanometer long fiber length described in said program is 1 μm~10 μm, and nano short fiber length is 0.01 μm~1 μm.
Suspension described in said program and solution are aqueous solution, and preferably pure water, phosphate buffer (PB) or phosphate is slow
One kind in fliud flushing (PBS).
Cationic polypeptide nanometer long fibre prepared by the present invention, can by change cationic polypeptide concentration, pH, temperature,
Time, and then it is nano short fiber and polypeptide monomer to regulate and control nanofiber de-assembly, and bacterial interactions, in the solution quickly
Kill bacterium.Nano short fiber and monomer are mixed with bacterial solution, efficiently can quickly kill various bacteria, in 4h or in 60min
Germicidal efficiency is up to 100%.
Description of the drawings
Fig. 1 is cationic polypeptide K3(FA)4K3And K6(FA)4Schematic arrangement.
Fig. 2 is cationic polypeptide K3(FA)4K3And K6(FA)4Potential diagram under difference pH in aqueous solution.
Fig. 3 is cationic polypeptide K3(FA)4K3And K6(FA)4The preparation of de-assembly nanostructure and its flow of antibacterial applications
Figure.
Fig. 4 is cationic polypeptide K3(FA)4K3(a) and K6(FA)4(b) the long stapled transmission electron microscope of nanometer being self-assembly of
(TEM) figure.
Fig. 5 is cationic polypeptide K3(FA)4K3(a) and K6(FA)4(b) transmission electron microscope (TEM) figure of de-assembly nanostructure.
Fig. 6 is cationic polypeptide K3(FA)4K3De-assembly nanostructure with after Escherichia coli effect, scheme by bacterium colony growth.
Fig. 7 is cationic polypeptide K3(FA)4K3And K6(FA)4De-assembly nanostructure with after Escherichia coli effect, deposit by bacterium
Motility rate changes with time figure.
Fig. 8 is to change cationic polypeptide K3(FA)4K3And K6(FA)4In different concentration, the time of the pH of solution and incubation
Under the nanometer long fibre figure that is self-assembly of；(a)K3(FA)4K31000ug/mL, pH=13, self assembly 3day；(b)K6(FA)4
1000ug/mL, pH=13, self assembly 3day.
Fig. 9 is to change cationic polypeptide K3(FA)4K3And K6(FA)4In the pH of different solutions and de-assembly under the time of incubation
Transmission electron microscope (TEM) figure of the nanostructure of formation；(a)K3(FA)4K3500ug/mL, pH=6.5, de-assembly 3day；(b)K6
(FA)4500ug/mL, pH=6.5, de-assembly 3day.
Figure 10 is to change cationic polypeptide K3(FA)4K3And K6(FA)4In the pH of different solutions and the system of solutions under the time of incubation
After filling nanostructure and the salmonella effect formed, Survival probability of bacteria changes with time figure；K3(FA)4K3500ug/mL, pH=6.5, de-assembly 3day；K6(FA)4500ug/mL, pH=6.5, de-assembly 3day.
Specific implementation mode
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, material etc., are commercially available unless otherwise specified.
Embodiment 1
Cationic polypeptide K3(FA)4K3And K6(FA)4Nanofiber prepare：
By a concentration of 500 μ g mL–1Cationic polypeptide be added 37 DEG C (pH=12) water in, be placed on oscillator and incubate
Educate 5day (360rpm/min), cationic polypeptide forms nanofiber after self assembly in the solution, and the shape appearance figure of gained is shown in Fig. 4.
It can be seen that the polypeptide K after incubation3(FA)4K3And K6(FA)4It has been self-assembly of nanometer long fibre.
Embodiment 2
Cationic polypeptide K3(FA)4K3And K6(FA)4The preparation of de-assembly nanostructure：
The pH of the nanofiber solution of above-mentioned preparation is adjusted to 7, is placed on constant temperature oscillator and continues to be incubated 3day (37
DEG C, 360rpm/min), nanofiber de-assembly is nano short fiber and monomer, and the shape appearance figure of gained is shown in Fig. 5.It can be seen that through
Cross the polypeptide K after being incubated3(FA)4K3And K6(FA)4De-assembly forms de-assembly nanostructure (nano short fiber and monomer).Solution
Flow chart prepared by assembling nanostructure is as shown in Figure 3.
Embodiment 3
Cationic polypeptide K3(FA)4K3And K6(FA)4The antibacterial application of de-assembly nanostructure：
By 50 μ L E. coli suspensions (~4 × 106cfu mL–1) it is separately added into the above-mentioned de-assembly nanostructures of 2mL
In solution, 37 DEG C of constant incubator cultures are placed in, every 1h, dilute 1000 times with phosphate buffer, after taking 100 μ L dilutions
Suspension is put into Lysogeny Broth solid mediums, and 16h is cultivated at 37 DEG C, calculates clump count, as shown in fig. 6, gained
Survival probability of bacteria, which changes with time, sees Fig. 7.
Embodiment 4
With embodiment 1, it is respectively 1000 μ g mL to change peptide concentration–1, pH=13 and incubation time are 3day, cation
Nanofiber is formed after self-assembling polypeptide, the shape appearance figure of gained is shown in Fig. 8.It can be seen that the polypeptide K after incubation3(FA)4K3
And K6(FA)4It has been self-assembly of nanometer long fibre.
Embodiment 5
With embodiment 2, it is 3day, nanofiber de-assembly shape to change the pH=6.5 of nanofiber solution and incubation time
Formation of nanostructured (nano short fiber and monomer), the shape appearance figure of gained is shown in Fig. 9.It can be seen that the polypeptide K after incubation3
(FA)4K3And K6(FA)4De-assembly forms nano short fiber and monomer.
Embodiment 6
With embodiment 3, it is the de-assembly nanostructure in embodiment 5 to change cationic polypeptide de-assembly nano-structured solution
Solution, bacterium are salmonella, and the Survival probability of bacteria of gained, which changes with time, sees Figure 10.It can be seen that polypeptide de-assembly shape
At nano short fiber and monomer can quickly kill bacterium, but sterilize rate and be faster than Escherichia coli, be attributable to, different bacteriums
With different biological structure and property.
Claims (10)
1. can de-assembly cationic polypeptide nano-fiber material, which is characterized in that preparation method is as follows：Cationic polypeptide disperses
In solution, control cationic polypeptide concentration, solution ph, incubation temperature T and incubation time t are self-assembly of the long fibre of nanometer
Dimension continues to control pH value of solution, incubation temperature T and incubation time t, and nanometer long fibre de-assembly is nano short fiber and monomer
Nanostructure recycles nanostructure sterilization；Nanometer long fiber length is 1 μm~10 μm, and nano short fiber length is 0.01 μm
~1 μm.
2. it is described in accordance with the claim 1 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that specific steps
It is as follows：By a concentration of 200~1000 μ g mL–1Cationic polypeptide be added pH=11~14 solution in, be placed in constant temperature oscillation
It is incubated 1.5day~9day on device, forms nanometer long fibre after cationic polypeptide self assembly；Again by the pH tune of nanofiber solution
Whole is 6.5~7.5, is placed on constant temperature oscillator and continues to be incubated 0.5day~5day, it is short that nanofiber de-assembly forms nanometer
Fiber and monomer.
3. according to claim 2 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that it is described sun from
Sub- polypeptide is K3(FA)4K3、K6(FA)4、K2(FA)3K2、K4(FA)3、Ac-K3(FA)4K3-NH2、K2(FA)2、K(FA)2K、Ac-K2
(FA)3K2-NH2、Ac-K2(FA)2-NH2、Ac-K(FA)2K-NH2、Ac-K2(FA)2-NH2And Ac-K6(FA)4-NH2In one kind.
4. it is described in accordance with the claim 3 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that it is described sun from
Sub- polypeptide is K3(FA)4K3And K6(FA)4In one kind.
5. according to claim 2 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that constant temperature oscillation
The temperature being incubated on device is 37 DEG C, rotating speed 360rpm/min；The 500 μ g mL of concentration of the cationic polypeptide–1。
6. according to claim 2 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that the pH of solution
Value is 12；Cationic polypeptide incubation time t=5day.
7. according to claim 2 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that described receives
The pH of rice fiber solution is adjusted to 7；The incubation time t=3day of nanofiber solution.
8. it is described in accordance with the claim 1 can de-assembly cationic polypeptide nano-fiber material purposes, which is characterized in that will
50 μ L bacterial suspensions (~4 × 106cfu mL–1) 500 μ g mL of 2mL are added–1The de-assembly nanostructure of cationic polypeptide is molten
Liquid is placed in co-incubation 6h in 37 DEG C of constant incubators, every 1h, dilutes 1000 times with phosphate buffer, takes 100 μ L dilutions
Suspension afterwards is put into Lysogeny Broth solid mediums, and 16h is cultivated at 37 DEG C, calculates clump count.
9. according to claim 8 can de-assembly cationic polypeptide nano-fiber material purposes, which is characterized in that institute
It is staphylococcus aureus (Staphylococcus aureus), Escherichia coli (E.coli), salmonella to state bacterium
(salmonella) or one kind in bacillus subtilis (Bacillus subtilis)；The suspension aqueous solution, specially
One kind in pure water, phosphate buffer (PB) or phosphate buffer (PBS).
10. according to claim 2 can de-assembly cationic polypeptide nano-fiber material, which is characterized in that solution is
One kind in aqueous solution, specially pure water, phosphate buffer (PB) or phosphate buffer (PBS).
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171115A (en) * | 2020-01-06 | 2020-05-19 | 山东大学 | Method for controlling reversible assembly of polypeptide crystal by adjusting pH value |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911258A (en) * | 2012-07-06 | 2013-02-06 | 江苏大学 | Method for forming two-dimensional polypeptide nanosheets by regulating assembly of polypeptides with organic micromolecules |
| CN107216382A (en) * | 2017-06-26 | 2017-09-29 | 江苏大学 | A kind of cationic polypeptide Micelle-like Nano-structure of Two and the purposes in antibacterial direction |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911258A (en) * | 2012-07-06 | 2013-02-06 | 江苏大学 | Method for forming two-dimensional polypeptide nanosheets by regulating assembly of polypeptides with organic micromolecules |
| CN107216382A (en) * | 2017-06-26 | 2017-09-29 | 江苏大学 | A kind of cationic polypeptide Micelle-like Nano-structure of Two and the purposes in antibacterial direction |
Non-Patent Citations (1)
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| LIU L: "A self-assembled nanopatch with peptide-organic multilayers and mechanical properties", 《NANOSCALE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171115A (en) * | 2020-01-06 | 2020-05-19 | 山东大学 | Method for controlling reversible assembly of polypeptide crystal by adjusting pH value |
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