CN116396372A - Peptide-loaded nanoparticle and application thereof in treatment of eye infection of pets - Google Patents
Peptide-loaded nanoparticle and application thereof in treatment of eye infection of pets Download PDFInfo
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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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
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
- A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention provides peptide-carrying nano-particles and application thereof in treatment of eye infection of pets, namely, small molecule cobra derivative peptides are combined on the nano-particles and are used for treating eye bacterial infection of pets; the amino acid sequence of the cobra derived peptide is SEQ ID NO. 2. The PLGA (CATH 20) microsphere prepared by the invention is suitable for bacterial infection of eyes of pets, has a good bactericidal effect, can be attached to mucous membrane, stays in the tissues in front of cornea for a long time without being rapidly cleared, has good biocompatibility, and has good practical value in the biomedical field.
Description
Technical Field
The invention belongs to the technical field of pet medical products, and particularly relates to peptide-carrying nano-particles and application thereof in treatment of eye infection of pets.
Background
Antibacterial peptides are important components of the innate immune defense system of organisms, widely exist in organisms such as microorganisms, animals and plants, and are polypeptide molecules with broad-spectrum antibacterial activity. The antibacterial mechanism of the antibacterial peptide is different from that of the traditional antibiotics, and the antibacterial peptide has a unique membrane targeting action mechanism, so that the molecules are not easy to generate tolerance in the antibacterial process, and are considered to be novel drug-resistant bacteria candidate molecules with great development potential.
The research on nano-carrier coated antibacterial drugs at home and abroad has achieved great results, and various nano-carrier delivery systems such as liposome, nanoemulsion, nanoparticle, nanosuspension, nano micelle and the like are actively explored and researched, so that the nano-carrier coated antibacterial drugs have excellent delivery potential in-vitro and in-vivo animal models, and have the characteristics of prolonged ocular retention time and the like. Studies have shown that the distribution of nanoparticles in the eye is mainly dependent on its size and surface properties, and that nanoparticles of 200-2000 nm can stay in the ocular tissue for at least 2 months. The nano particles can be attached to mucous membrane, stay in the tissue in front of cornea for a long time without being rapidly cleared, and indicate that the nano carrier delivery system has good application prospect in ocular medicine and has the potential of further clinical test development.
Disclosure of Invention
The invention aims to provide peptide-loaded nano-particles and application thereof in treatment of eye infection of pets, namely, small molecule cobra derived peptides are combined on the nano-particles and are used for treating eye bacterial infection of pets.
The invention firstly provides a cobra derivative peptide, which is obtained by modifying a polypeptide with an amino acid sequence of KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPF (SEQ ID NO: 1), wherein the modified cobra derivative peptide is named CATH20, and the amino acid sequence of the cobra derivative peptide is KKRAKKFFKKPRVIGVSQPF (SEQ ID NO: 2).
In a further aspect, the present invention provides a nanoparticle, wherein the nanoparticle is polylactic acid-glycolic acid copolymer PLGA carrying the cobra-derived peptide CATH 20;
the nano particles provided by the invention can be used for preparing products for preventing and treating bacteria on eyes of pets.
The PLGA (CATH 20) microsphere prepared by the invention is suitable for bacterial infection of eyes of pets, has a good bactericidal effect, can be attached to mucous membrane, stays in the tissues in front of cornea for a long time without being rapidly cleared, has good biocompatibility, and has good practical value in the biomedical field.
Drawings
Fig. 1: an amphipathic prediction result diagram measured by the cobra peptide and the cobra derivative peptide;
fig. 2: scanning Electron Microscope (SEM) image of PLGA (CATH 20) microsphere;
fig. 3: in vitro release profile of antibacterial peptide PLGA (CATH 20) microspheres.
Fig. 4: in vitro antibacterial effect comparison graph of antibacterial peptide PLGA (CATH 20) microspheres.
Fig. 5: antibacterial peptide PLGA (CATH 20) microsphere animal test effect diagram.
Detailed Description
The invention will be further described with reference to specific examples to facilitate a better understanding of the invention, but the scope of the invention is not limited to these examples.
Example 1: preparation of cobra derived peptide
The cobra peptide has an amino acid sequence of KRFKKFFKKLKNSVKKRAKKFFKKP RVIGVSIPF, is composed of 34 amino acids, has a molecular weight of 4155.73 daltons, has an isoelectric point of 12.34, and is L-shaped. As the amino acid sequence of the cobra peptide has more hydrophilic functional groups and longer sequence, the applicant discovers that the microsphere prepared by directly using the cobra peptide is unstable and easy to burst, so that the bioavailability of the antibacterial peptide is lower and the antibacterial effect is reduced, and the cobra peptide amino acid sequence is used as a template for transformation.
Specifically, 20 amino acids at the C-terminal of the mature peptide are intercepted, the eighteenth nonpolar hydrophobic amino acid isoleucine (Ile) in the 20 amino acid sequence is replaced by polar hydrophobic amino acid glutamine (Gln), the N-terminal of the sequence is amidated, the derivative peptide CATH20 is obtained, and the sequence is synthesized by biological company.
The modified derivative peptide CATH20 has similar antibacterial activity to that of cobra peptide OH-CATH, has low hemolytic property and toxicity, and retains the amphipathic characteristic. As shown in figure 1, the overall hydrophobic value distribution of the cobra derived peptide and the cobra peptide is continuously increased from the N-terminal to the C-terminal, so that a remarkable amphipathic trend is formed, and the similar antibacterial activity of CATH20 and OH-CATH is ensured.
The invention reforms the Ocular snake derived peptide, retains the characteristics of active fragments and amphipathy of the original peptide, shortens the peptide sequence, replaces amino acids in the sequence, avoids burst release in the later microsphere preparation process, improves the bioavailability of the peptide, thereby improving the antibacterial effect of the prepared microsphere, reducing the difficulty of microsphere embedding and reducing the synthesis cost and purification difficulty of the peptide.
Example 2: preparation of antibacterial peptide (CATH 20) PLGA microspheres
(1) Weighing 10mg of antibacterial peptide (CATH 20) and 40mg of PLGA in a beaker for later use, wherein the molecular weight of the PLGA is 29000;
(2) And (3) preparing an internal water phase: dissolving the weighed CATH20 with 1ml of ddH2O to form a CATH20 solution with the concentration of 10mg/ml;
(3) The preparation method comprises the following steps: dissolving the weighed PLGA with 5ml of dichloromethane to obtain PLGA solution with the concentration of 8mg/ml;
(4) Slowly injecting the water phase in the step (2) into the organic phase in the step (3) by using an injector, wherein the ultrasonic power is 150W, and the time is 90s, so that W/O colostrum is formed;
(5) Injecting the colostrum into 15ml of 1% PVA solution by using a syringe, and performing ultrasonic treatment, wherein the ultrasonic power is 100W, and the ultrasonic treatment time is 60s, so as to form W/O/W compound emulsion;
(6) Pouring the compound emulsion obtained in the step (5) into 20ml of 0.75% PVA solution, stirring at room temperature, solidifying the microspheres and completely volatilizing the organic solvent to obtain PLGA (CATH 20) microsphere mixed solution; centrifuging at 15000rpm and 4deg.C for 10min, discarding supernatant, repeatedly washing with PBS for three times, and freeze-drying at low temperature for 48 hr to obtain antibacterial peptide PLGA (CATH 20) microsphere, sealing, and storing in 4deg.C refrigerator.
Centrifuging 2.0ml suspension in a centrifuge tube at 15000r/min for 15min, collecting supernatant, filtering with 0.22um filter, and measuring free CATH20 content of CATH20 in PLGA by high performance liquid chromatography; the encapsulation efficiency was calculated as the formula = [ (m 1-m 2)/m 1] ×100% respectively. m1: the amount of CATH20 actually put into use, m2: free CATH20. The result shows that the prepared microsphere has high embedding rate up to 87.6%.
Example 3: determination of PLGA (CATH 20) microsphere characterization
(1) Particle size and zeta potential
The average diameters, PDI and zeta potential (n=5) of the three groups of nanoparticles prepared in example 2 were measured by the Zetasizer-nano-ZS dynamic light scattering method, and the results are shown in table 1, and it can be seen that
The microspheres prepared in example 2 were about (728.+ -. 4.87) nm in diameter and had a zeta potential of (-26.36.+ -. 1.81) mv measured at a suitable PDI range. The particle size meets the requirement of ocular administration, and the zeta potential is stable and is not easy to aggregate.
Table 1: zeta potential and particle size measurement results of PLGA (CATH 20) microspheres
(2) Determination of surface topography
Adhering the freeze-dried PLGA microsphere powder to a sample holder by using conductive adhesive, after gold plating, analyzing by using a scanning electron microscope under kilovolts to obtain an electron microscope image of the PLGA (CATH 20) microsphere coated with the antibacterial peptide, wherein FIG. 2 is an electron microscope image of the corresponding PLGA polymer microsphere;
from fig. 2, it can be seen that the electron micrograph of the microspheres prepared in example 2 shows that the antibacterial peptide-loaded PLGA (CATH 20) microspheres are regular spheres, and the microspheres are uniform in size, good in dispersibility, and free from caking.
Example 4: determination of in vitro drug Release Curve
Microspheres of PLGA (OH-CATH) carrying the polypeptide of SEQ ID NO. 1 and PLGA (CATH 20) carrying the polypeptide of SEQ ID NO. 2 were prepared as in example 2. The lyophilized nanoparticles (10 mg) were weighed and resuspended in 1ml deionized water. The suspension was shaken (200 rpm) at 25 ℃. Samples were taken at predetermined time intervals (0.5 h, 1h, 2h, 4h, 6h, 8h, 12h,24h, 48 h), centrifuged at 14,000 rpm for 10 minutes after sampling, and filtered through a 0.22 μm filter to remove remaining particles. The content of free peptide was determined by HPLC method to give fig. 3.
According to fig. 3, it can be seen that the prepared PLGA (CATH 20) drug-loaded microsphere mainly shows two states of primary rapid release and late slow release, and the cumulative release amount at 24 hours is (86.9±2.23)%, so that the sustained release of the drug over a longer period of time can be satisfied.
The PLGA (OH-CATH) microspheres are released rapidly within 2 hours, so that the concentration of peptide in eyes can be increased rapidly after administration, eyes can not absorb the peptide rapidly, and the expected slow-release sterilization effect can not be achieved.
Example 5: determination of in vitro bacteriostasis test
PLGA (OH-CATH) and PLGA (CATH 20) microspheres are used for evaluating in-vitro antibacterial effects of in-vitro escherichia coli, pseudomonas aeruginosa and staphylococcus aureus. Three bacteria were cultured in liquid medium to their log phase and resuspended in PBS to 10 5 CFU/mL bacterial suspension. Bacterial solutions are respectively incubated with PBS, OH-CATH (100 mug/m), PLGA (OH-CATH) (100 mug/ml) and PLGA (CATH 20) (100 mug/ml) for 24 hours, 20 mug of bacterial solution is coated on a flat plate, and the flat plate is placed in an incubator for culturing for 24 hours at 37 ℃ to calculate the bacterial activity.
From fig. 4, it can be seen that the antibacterial effect of the PLGA (OH-CATH) microsphere is significantly lower than that of the PLGA (CATH 20) microsphere, and the antibacterial activity of the microsphere prepared from the modified derivative peptide is higher than that of the cobra peptide alone.
Example 6: single and multiple dosing rabbit eye irritation test
9 healthy rabbits (both male and female) were randomly divided into 3 groups, 100. Mu.l of physiological saline was added dropwise to each group as a control, and 100. Mu.l of physiological saline containing PLGA (CATH 20) microspheres at 200. Mu.g/ml was added dropwise to the right eye. Eyes were examined 1, 2, 4, 24, 48 and 72 hours after administration. Each sample was evaluated for irritation according to the "Draize" ocular irritation test scoring criteria. Eye irritation evaluation criteria no irritation: 0 to 3 minutes; mild irritation: 4-8 minutes; moderate irritation: 9-12 minutes; severe irritation: 13 to 16 minutes.
9 healthy rabbits (both male and female) were selected and randomly divided into 3 groups, 100. Mu.l of physiological saline was added dropwise to each group as a control, and 100. Mu.l of physiological saline containing PLGA (CATH 20) microspheres at 200. Mu.g/ml was added dropwise to the right eye. 3 times per day for 7 consecutive days. Eyes were examined before and 1, 2, 4, 24, 48 and 72 hours after each administration, and each sample was evaluated for irritation according to the "Draize eye irritation test scoring criteria" and the results are shown in Table 2.
The results of single administration showed an average integrated value of 0 score for the saline group and 2 score for the PLGA (CATH 20) solution group, scored according to the scoring criteria.
The results of multiple administrations showed an average integral value of 1 score for the saline group and 4 scores for the PLGA (CATH 20) solution group, as scored by the scoring criteria.
The results of this experiment show that both single and multiple doses of the PLGA (CATH 20) solution prepared in example 2 are not significantly irritating to the eye.
Table 2: antibacterial peptide PLGA (CATH 20) microsphere "Draize" eye irritation test scoring table
Example 7: eye treatment test
6 healthy test rabbits (both male and female) are selected, randomly divided into 3 groups, and treated by staphylococcus aureus and pseudomonas aeruginosa, and then treated by the microspheres prepared in the example 2, and the treatment effect and side effect are observed. Eye changes of rabbits before and after treatment are shown in fig. 5.
From fig. 5, it can be seen that the ocular inflammation of rabbits was significantly reduced after the treatment, and purulent secretion had been significantly reduced on the third day after the treatment. No purulent secretion had occurred on day 7, the cornea was clear and the conjunctiva was pink.
Claims (10)
1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQ ID NO. 2.
2. Use of the polypeptide of claim 1 for the preparation of an antimicrobial article.
3. The use of claim 2, wherein the antimicrobial article is an article for the prophylactic treatment of bacteria in the eye of a pet.
4. Use according to claim 2 or 3, wherein the antimicrobial article is an antimicrobial article prepared using a nanocarrier delivery system.
5. The use according to claim 4, wherein the delivery system with nanocarriers is polylactic acid-glycolic acid copolymer PLGA.
6. A nanoparticle, wherein the nanoparticle is polylactic acid-glycolic acid copolymer PLGA carrying the polypeptide of claim 1.
7. Use of the nanoparticle of claim 6 for the preparation of an article for the prophylactic treatment of ocular bacteria in pets.
8. An article of manufacture for controlling ocular bacteria in pets comprising the nanoparticle of claim 6 and/or the polypeptide of claim 1.
9. A recombinant expression vector carrying a nucleic acid fragment encoding the polypeptide of claim 1.
10. A recombinant engineering bacterium, wherein the recombinant engineering bacterium is used for recombinant expression of the polypeptide of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310135438.XA CN116396372B (en) | 2023-02-18 | 2023-02-18 | Peptide-loaded nanoparticle and application thereof in treatment of eye infection of pets |
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