CN110496229B - Nanoparticle-coated antibacterial peptide with slow release property and preparation method thereof - Google Patents
Nanoparticle-coated antibacterial peptide with slow release property and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a nanoparticle-coated antibacterial peptide with slow release property, which comprises a nano drug loading system and an antibacterial peptide loaded on the nano drug loading system, wherein the nano drug loading system is a reduced BSA (bovine serum albumin) molecule, the antibacterial peptide is a cationic antibacterial peptide LL37 modified by a sulfhydryl group, the sulfhydryl group is exposed after the nano drug loading system is reduced, and then the nano drug loading system is connected with the antibacterial peptide through a disulfide bond to synthesize the nanoparticle-coated antibacterial peptide with slow release property. The invention also relates to a preparation method of the antibacterial peptide coated by the nano-particles. The technical method for preparing the nano-particles is simple, short in time consumption and easy to operate, LL37 is successfully coated in a BSA drug-loading system by utilizing the crosslinking among disulfide bonds, LL37 is not easily degraded in the drug delivery process and can play a targeting role by the chemical crosslinking mode, and the method can be applied to the preparation processes of various antibacterial peptides and even different antibiotics, and provides a new idea for preparing various drugs clinically in the future.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a nanoparticle-coated antibacterial peptide with slow release and a preparation method thereof.
Background
The nano material is widely applied to the field of biomedicine due to the advantages of the surface effect, the small-size effect and the like. Among the nano materials, the nano drug-carrying system becomes a hot point for research in the fields of materials and medicine due to a plurality of advantages. The nano drug-loading system refers to that a polymer is used as a carrier, and small-molecule drugs are combined in the carrier in an encapsulation or adsorption mode through a physical or chemical mode to form the drugs with the size of a nano level. The drug loaded nanocarrier will generally be a polymer that is highly biocompatible or can be modified. Such vectors may be synthetic or natural. Different from the traditional drug treatment means, the nano drug loading mainly has the following advantages that firstly, the solubility of the drug is improved, the stability of the drug is enhanced, and the loss of the drug is reduced as much as possible before the drug reaches a treatment part; secondly, the tissue permeability and the membrane permeability of the medicine are improved, and the administration route is increased; thirdly, the targeting property of the medicine is increased, the utilization rate of the medicine is improved, and the nano medicine-carrying system can ensure that the nano medicine can release effective medicine molecules after reaching the pathological change part of an organism to play the medicine effect after being modified; fourthly, the medicine has the function of regulating the slow release and controlling the release.
LL37 is the only cationic antimicrobial peptide found in humans to date. LL37 is an important component of the innate immunity of the body because LL37 has a killing effect on a variety of gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa, Neisseria gonorrhoeae, Klebsiella pneumoniae, Staphylococcus aureus, and the like. LL37 has a broad spectrum of antibacterial activity and multiple biological activities, and is considered to be a new hope for multiple drug resistant bacterial infection. However, the application of the in vitro antibacterial peptide is greatly limited due to various reasons such as high cost, antigenicity and easy inactivation of the in vitro antibacterial peptide in direct transfusion, and the in vitro antibacterial peptide is not used in clinic until now.
Bovine Serum Albumin (BSA) has the advantages of low toxicity, low immunogenicity, strong stability, capability of being combined with various medicaments and the like. BSA has a wide application prospect as a carrier for drug slow release. The studies of Wang Guen bin and the like show that the room temperature stability and the serum stability of paclitaxel loaded by epidermal growth factor coupled bovine serum albumin nanoparticles are higher than those of paclitaxel which is not coated by the nanoparticles. The lung is suitable as a site of administration for local or systemic administration due to its specific physiological structure.
Crosslinking between LL37 molecules is possible using conventional glutaraldehyde crosslinking, but such crosslinking is detrimental to maintaining the biological activity of LL 37. Therefore, we propose a scheme for amplifying charge density in space-time by using cross-linking between BSA molecules while keeping LL37 structurally intact, which can increase the loading rate of LL37 and help stabilize the nanostructure, and no study on this aspect has been reported at present.
Disclosure of Invention
The first purpose of the present invention is to provide a nanoparticle-coated antibacterial peptide with sustained release, which is directed to the deficiencies of the prior art.
The second purpose of the invention is to provide a preparation method of the antibacterial peptide.
In order to achieve the first purpose, the invention adopts the technical scheme that:
the utility model provides an antibiotic peptide of nanoparticle peridium with slowly-releasing nature, includes nanometer drug loading system and the antibiotic peptide of load on nanometer drug loading system, nanometer drug loading system is the BSA molecule by the reduction, antibiotic peptide is the cationic antibiotic peptide LL37 through mercapto modification, nanometer drug loading system exposes mercapto after being reduced, passes through the disulfide bond connection with antibiotic peptide again, and the synthesis has antibiotic peptide of nanoparticle peridium of slowly-releasing nature.
Preferably, the reduced BSA molecule is prepared by: the disulfide bonds of the BSA molecules are cleaved and the thiol groups are exposed by treatment with dithiothreitol, and then the BSA molecules are exposed to more thiol groups by the action of sodium dodecyl sulfate.
Preferably, the nanoparticle-coated antimicrobial peptide is prepared by the following method:
1) dissolving BSA, dithiothreitol and sodium dodecyl sulfate in sterile double distilled water to make the final concentration of BSA be 40mg/ml, and heating the solution in a water bath at 70 ℃ under the magnetic stirring of 250r/min to obtain a reduced BSA solution;
2) diluting the reduced BSA solution with a buffer solution, adding the diluted BSA solution into a 24-well plate, placing the plate in a 37 ℃ oscillator, and reacting for 4.5 hours at 350r/min to synthesize BSA nanoparticles;
3) adding BSA and antimicrobial peptide LL37 into a 24-pore plate, placing the plate in an oscillator with the temperature controlled at 37 ℃, controlling the rotating speed at 350r/min, and synthesizing LL37 nano-particles after 4.5 hours;
4) removing the sodium dodecyl sulfate in the nano particles by using an ultrafiltration method to obtain the nano particle coated antibacterial peptide with slow release property.
More preferably, the mass ratio of the BSA, the dithiothreitol and the sodium dodecyl sulfate in the step 1) is 80: 3: 40.
more preferably, in step 2), the buffer is a 2- (N-morpholino) ethanesulfonic acid solution with a pH of 4.8 and the diluted reduced BSA solution has a concentration of 1 mg/ml.
More preferably, the mass ratio of the BSA and the antimicrobial peptide LL37 in the step 3) is 9: 1.
in order to achieve the second object, the invention adopts the technical scheme that:
the preparation method of the nanoparticle-coated antibacterial peptide comprises the following steps:
1) dissolving BSA, dithiothreitol and sodium dodecyl sulfate in sterile double distilled water, wherein the final concentration of BSA is 40mg/ml, and heating the solution in a water bath at 70 ℃ under the magnetic stirring of 250r/min to obtain a reduced BSA solution;
2) diluting the reduced BSA solution with a buffer solution, adding the diluted BSA solution into a 24-well plate, placing the plate in a 37 ℃ oscillator, and reacting for 4.5 hours at 350r/min to synthesize BSA nanoparticles;
3) adding BSA and antimicrobial peptide LL37 into a 24-pore plate, placing the plate in an oscillator with the temperature controlled at 37 ℃, controlling the rotating speed at 350r/min, and synthesizing LL37 nano-particles after 4.5 hours;
4) removing the sodium dodecyl sulfate in the nano particles by using an ultrafiltration method to obtain the nano particle coated antibacterial peptide with slow release property.
Preferably, the mass ratio of the BSA, the dithiothreitol and the sodium dodecyl sulfate in the step 1) is 80: 3: 40.
preferably, the buffer in step 2) is a 2- (N-morpholino) ethanesulfonic acid solution at pH 4.8, and the concentration of the diluted reduced BSA solution is 1 mg/ml.
Preferably, the mass ratio of BSA to antibacterial peptide LL37 in step 3) is 9: 1.
the invention has the advantages that:
1. it is possible to crosslink antimicrobial peptide LL37 using conventional glutaraldehyde crosslinking, but such crosslinking is not conducive to maintaining the biological activity of antimicrobial peptide LL 37. Therefore, the proposal that the crosslinking between BSA molecules is utilized while the structural integrity of the antimicrobial peptide LL37 is maintained, and a disulfide bond network is formed between BSA and antimicrobial peptide LL37 molecules by a scheme of space-time charge density amplification, so that the LL37 nanoparticles with stable structure are synthesized, the loading rate of the antimicrobial peptide LL37 can be improved, and the nanostructure can be stabilized.
2. The nano drug-carrying system used in the invention is bovine serum albumin BSA which is a naturally existing substance, and compared with an artificially synthesized high molecular polymer, the BSA has higher histocompatibility with mice or human bodies, the probability of immunological rejection of organisms is low, and the reaction is light. In addition, BSA also has the advantages of low toxicity, strong stability, capability of combining various medicaments and the like.
3. The cost for preparing LL37 in vitro is high, LL37 is easy to degrade and inactivate after being fused into plasma, and the amount of effective action in vivo is very small, but in the invention, LL37 is successfully coated in a BSA drug-loading system by utilizing the cross-linking among disulfide bonds, and the chemical cross-linking mode ensures that LL37 is not easy to degrade in the drug delivery process and can play a targeting role.
4. The technical method for preparing the nano-particles is simple, short in time consumption and easy to operate, can be applied to preparation processes of various antibacterial peptides and even different antibiotics, and provides a new idea for preparing various medicines clinically in the future.
Drawings
FIG. 1 is a flow chart of the operation of the preparation of the antimicrobial peptide coated with nanoparticles of the present invention.
FIG. 2 shows the ratio of dead PA after different periods of time (0.5h,1.0h,2.0h,4.0h,8.0h,12.0h,24.0h,48.0h) after cocultivation of LL37 with FITC fluorescence (LL37-FITC40ug/ml,20ug/ml), LL37 nanoparticles with FITC fluorescence (LL37 PNP-FITC 40ug/ml,20ug/ml) with Pseudomonas Aeruginosa (PA), as detected by flow cytometry.
FIG. 3 shows FITC fluorescence intensity detected after different periods of time (0.5h,1.0h,2.0h,4.0h,8.0h,12.0h,24.0h,48.0h) after co-cultivation of LL37 carrying FITC fluorescence (LL37-FITC40ug/ml,20ug/ml), LL37 nanoparticles carrying FITC fluorescence (LL37 PNP-FITC 40ug/ml,20ug/ml) with Pseudomonas Aeruginosa (PA), as detected by flow cytometry.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Substances required in the preparation process comprise Bovine Serum Albumin (BSA), Dithiothreitol (DTT), Sodium Dodecyl Sulfate (SDS), sulfhydryl-modified antibacterial peptide LL37(LL37 peptide), 2- (N-morpholino) ethanesulfonic acid (MES) solution, a water bath heating pot, a temperature-controlled shaking table and a 24-pore plate, wherein the sulfhydryl-modified antibacterial peptide LL37 is purchased from Gill Biochemical Co., Ltd.
EXAMPLE 1 preparation of cationic antimicrobial peptide LL37 coated with BSA nanoparticles
The preparation method comprises the following specific steps:
1) dissolving 40mg BSA, 1.5mg DTT and 20mg SDS in 1mL sterile double distilled water to obtain 40m/mL BSA solution, and heating the solution in a water bath at 70 ℃ under magnetic stirring at 250r/min to obtain a reduced BSA solution;
2) 40mg/ml of reduced BSA solution was diluted to 1mg/ml with MES buffer (pH 4.8). Adding a BSA solution into a 24-pore plate, placing the BSA solution into a 37 ℃ temperature-controlled oscillator, and reacting at 350r/min for 4.5 hours to synthesize BSA nanoparticles;
3) BSA and synthetic LL37 antimicrobial peptide was added at 9: adding the mass ratio of 1 into a 24-hole plate, similarly placing the plate in a 37 ℃ temperature-controlled oscillator at the rotating speed of 350r/min, and synthesizing LL37 nano particles after 4.5 hours;
4) and finally, removing SDS and other substances in the nanoparticles by using an ultrafiltration method to obtain the nanoparticles for subsequent experiments.
Example 2
In order to detect that LL37 nanoparticles are not easily degraded during drug delivery and have sustained release effect, LL37 nanoparticles (LL37 PNP) were synthesized in experiments using FITC labeled LL37 and FITC-loaded LL 37. LL37 with FITC fluorescence (LL37-FITC40ug/ml,20ug/ml), LL37 nanoparticles with FITC fluorescence (LL37 PNP-FITC 40ug/ml,20ug/ml) were co-cultured with Pseudomonas Aeruginosa (PA) at different time periods (0.5h,1.0h,2.0h,4.0h,8.0h,12.0h,24.0h,48.0h), and FITC fluorescence intensity and ratio of dead bacteria to total were detected by flow cytometry.
The streaming results show that: the mortality of PA was positively correlated with the concentration of LL37, LL37PNP added. And the mortality rate of PA increased significantly in the LL37PNP group over time (fig. 2).
In addition, the LL37 group showed a larger number of FITC-fluorescent bacteria at 2.0h and 4.0h of co-cultivation time. However, when the time was extended to 24.0, 48.0h, the amount of bacteria with FITC fluorescence was significantly greater in the LL37PNP group than in the LL37 group (FIG. 3).
Example 3 preparation of cationic antimicrobial peptide LL37 coated with BSA nanoparticles
The preparation method comprises the following specific steps:
1) dissolving 40mg BSA, 1.5mg DTT and 20mg SDS in 1mL sterile double distilled water to obtain 40m/mL BSA solution, and heating the solution in a water bath at 70 ℃ under magnetic stirring at 250r/min to obtain a reduced BSA solution;
2) the 40mg/ml reduced BSA solution was diluted to 1mg/ml with MES buffer (pH 4.8). Adding a BSA solution into a 24-pore plate, placing the BSA solution into a 37 ℃ temperature-controlled oscillator, and reacting at 350r/min for 4.5 hours to synthesize BSA nanoparticles;
3) BSA and synthetic LL37 antimicrobial peptide was added at 8: 2 into a 24-hole plate and similarly placed in a 37 ℃ temperature-controlled oscillator at the rotating speed of 350r/min, and LL37 nano-particles are synthesized after 4.5 hours;
4) and finally, removing SDS and other substances in the nanoparticles by using an ultrafiltration method to obtain the nanoparticles for subsequent experiments.
Example 4 preparation of cationic antimicrobial peptide LL37 coated with BSA nanoparticles
The preparation method comprises the following specific steps:
1) dissolving 40mg BSA, 1.5mg DTT and 20mg SDS in 1mL sterile double distilled water to obtain 40m/mL BSA solution, and heating the solution in a water bath at 70 ℃ under magnetic stirring at 250r/min to obtain a reduced BSA solution;
2) 40mg/ml of reduced BSA solution was diluted to 1mg/ml with MES buffer (pH 4.8). Adding a BSA solution into a 24-pore plate, placing the BSA solution into a 37 ℃ temperature-controlled oscillator, and reacting at 350r/min for 4.5 hours to synthesize BSA nanoparticles;
3) BSA and synthetic LL37 antimicrobial peptide was added at 7: 3 into a 24-hole plate, and similarly placing the plate in a 37 ℃ temperature-controlled oscillator at the rotating speed of 350r/min to synthesize LL37 nano particles after 4.5 hours;
4) and finally, removing SDS and other substances in the nanoparticles by using an ultrafiltration method to obtain the nanoparticles for subsequent experiments.
Example 5 preparation of PLGA nanoparticles coated with cationic antimicrobial peptide LL37
20mg of PLGA polymer was dissolved in 1ml of methylene chloride, 20. mu.l of LL37 were added to the PLGA solution in 20. mu.l of sterile water and sonicated (Branson sonifier, USA) at 70W for 15 seconds to form a W/O primary emulsion. The primary emulsion was further emulsified with 2ml of 1% (W/v) polyvinyl alcohol (PVA, Mw13000-23000, Sigma-Aldrich, DE) dissolved in sterile water and sonicated again at 70W for 15 seconds to produce a W/O/W emulsion. The emulsion was added drop by drop to 50ml of 0.3% (w/v) PVA and stirred on a water bath at 600rpm and 37 ℃ for 1 hour. The nanoparticle suspension was then washed twice for 40 minutes by centrifugation at 22000 Xg and 4 ℃ in sterile water. All particles were then freeze-dried and lyophilized for 24 hours and stored at 4 ℃ until further use. All materials used were sterilized and the procedure was performed under laminar flow.
Example 6 examination of the properties of the particles of examples 1 and 3 to 5
The particle size was measured using a Nano ZS laser particle sizer, the results are shown in table 1; measuring the absorbance of LL37 antibacterial peptide in the drug-loaded particles by adopting a high performance liquid chromatography, and calculating the drug-loading rate of the BSA nanoparticles to the LL37 antibacterial peptide according to a formula, wherein the specific results are shown in Table 1; LL37 nanoparticles were co-cultured with Pseudomonas aeruginosa for 24h using FITC-labeled LL37, LL37 with FITC, and FITC fluorescence intensity was detected by flow cytometry, the results are shown in Table 1.
Table 1 particle size, drug loading rate and sustained release effect of nanoparticles (n ═ 5)
The results show that the cationic antibacterial peptide LL37 coated by the BSA nanoparticles prepared in example 1 has uniform particle size, high drug loading rate and good slow release effect, while the liquids in examples 3 and 4 are obviously cloudy and separated in the mixing process of BSA and the synthesized LL37 antibacterial peptide, the antibacterial peptide LL37 has low utilization rate and the drug loading effect is lower than that in example 1, the difference between the particle sizes is large, the nanoparticles prepared by using PLGA as a carrier have low drug loading rate, and the slow release effect is inferior to that of the cationic antibacterial peptide LL37 coated by the BSA nanoparticles.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (7)
1. The utility model provides an antibiotic peptide of nanoparticle peridium with slowly-releasing nature, includes nanometer drug loading system and the antibiotic peptide of load on nanometer drug loading system, a serial communication port, nanometer drug loading system is the BSA molecule by the reduction, antibiotic peptide is the antibiotic peptide LL37 of cation through mercapto modification, nanometer drug loading system exposes the mercapto after being reduced, passes through the disulfide bond connection with antibiotic peptide again, and the synthesis has the antibiotic peptide of nanoparticle peridium of slowly-releasing nature, BSA and antibiotic peptide LL 37's mass ratio are 9: 1, the reduced BSA molecule prepared by: the disulfide bonds of the BSA molecules are cleaved and the thiol groups are exposed by treatment with dithiothreitol, and then the BSA molecules are exposed to more thiol groups by the action of sodium dodecyl sulfate.
2. The nanoparticle-coated antimicrobial peptide of claim 1, wherein the nanoparticle-coated antimicrobial peptide is prepared by the following method:
1) dissolving BSA, dithiothreitol and sodium dodecyl sulfate in sterile double distilled water to make the final concentration of BSA be 40mg/ml, and heating the solution in a water bath at 70 ℃ under the magnetic stirring of 250r/min to obtain a reduced BSA solution;
2) diluting the reduced BSA solution with a buffer solution, adding the diluted BSA solution into a 24-well plate, placing the plate in a 37 ℃ oscillator, and reacting for 4.5 hours at 350r/min to synthesize BSA nanoparticles;
3) adding BSA and antimicrobial peptide LL37 into a 24-pore plate, placing the plate in an oscillator with the temperature controlled at 37 ℃, controlling the rotating speed at 350r/min, and synthesizing LL37 nano-particles after 4.5 hours;
4) removing the sodium dodecyl sulfate in the nano particles by using an ultrafiltration method to obtain the nano particle coated antibacterial peptide with slow release property.
3. The nanoparticle-coated antimicrobial peptide according to claim 2, wherein the mass ratio of BSA, dithiothreitol and sodium dodecyl sulfate in step 1) is 80: 3: 40.
4. the nanoparticle-coated antimicrobial peptide according to claim 3, wherein the buffer in step 2) is 2- (N-morpholino) ethanesulfonic acid solution with pH =4.8, and the concentration of the diluted reduced BSA solution is 1 mg/ml.
5. The method of preparing the nanoparticle-coated antimicrobial peptide of claim 1, comprising the steps of:
1) dissolving BSA, dithiothreitol and sodium dodecyl sulfate in sterile double distilled water, wherein the final concentration of BSA is 40mg/ml, and heating the solution in a water bath at 70 ℃ under the magnetic stirring of 250r/min to obtain a reduced BSA solution;
2) diluting the reduced BSA solution with a buffer solution, adding the diluted BSA solution into a 24-well plate, placing the plate in a 37 ℃ oscillator, and reacting for 4.5 hours at 350r/min to synthesize BSA nanoparticles;
3) adding BSA and antimicrobial peptide LL37 into a 24-hole plate, placing the plate in an oscillator with the temperature controlled at 37 ℃, controlling the rotating speed at 350r/min, and synthesizing LL37 nano particles after 4.5 hours;
4) removing the sodium dodecyl sulfate in the nano particles by using an ultrafiltration method to obtain the nano particle coated antibacterial peptide with slow release property.
6. The method according to claim 5, wherein the mass ratio of BSA, dithiothreitol and sodium dodecyl sulfate in step 1) is 80: 3: 40.
7. the method according to claim 6, wherein the buffer in step 2) is 2- (N-morpholino) ethanesulfonic acid solution with pH =4.8, and the concentration of the diluted reduced BSA solution is 1 mg/ml.
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| CN109771656A (en) * | 2019-03-12 | 2019-05-21 | 四川载荧生物科技有限公司 | The preparation method of Novel albumin Nano medication under a kind of 37 DEG C of temperate conditions |
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| CN108379228A (en) * | 2018-02-28 | 2018-08-10 | 湖南大学 | A kind of albumin nanoparticle and its preparation method and application of package pharmacological active substance |
| CN108478805A (en) * | 2018-02-28 | 2018-09-04 | 湖南大学 | A kind of Novel albumin-siRNA composite nanometer particles and its preparation method and application |
| CN109771656A (en) * | 2019-03-12 | 2019-05-21 | 四川载荧生物科技有限公司 | The preparation method of Novel albumin Nano medication under a kind of 37 DEG C of temperate conditions |
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