LU504180B1 - Chitosan nano-conjugate for resisting bacterial biofilm, and preparation method and application thereof - Google Patents

Chitosan nano-conjugate for resisting bacterial biofilm, and preparation method and application thereof Download PDF

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LU504180B1
LU504180B1 LU504180A LU504180A LU504180B1 LU 504180 B1 LU504180 B1 LU 504180B1 LU 504180 A LU504180 A LU 504180A LU 504180 A LU504180 A LU 504180A LU 504180 B1 LU504180 B1 LU 504180B1
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chitosan
cramp
conjugate
biofilm
nano
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LU504180A
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Hongwei Chen
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National Center Of Tech Innovation For Pigs
Univ Southwest
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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
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

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  • Wood Science & Technology (AREA)
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  • Pest Control & Pesticides (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Dispersion Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
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Abstract

This invention provides a chitosan nano-conjugate for resisting bacterial biofilm, and preparation method and application thereof, the preparation method of chitosan nano-conjugate of antibacterial biofilm is as follows: CRAMP is made into solution to activate the carboxyl group of CRAMP, and then chitosan solution is added to couple chitosan with the carboxyl group of CRAMP to form chitosan Cramp conjugate. Then, the amino group of the chitosan -CRAMP conjugate was activated and then added into the graphene oxide colloidal solution for full reaction to obtain the chitosan nano-conjugate with anti-bacterial biofilm. The chitosan nanoconjugate with anti-bacterial biofilm can significantly reduce the amount of biofilm, and has better stability and lower cytotoxicity; the invention is expected to be developed into a new nano-preparation for oral administration to resist biofilm infection, and then to be used to analyze soil components, which has the advantages of on-site, high efficiency and low cost.

Description

1 LU504180
DESCRIPTION
CHITOSAN NANO-CONJUGATE FOR RESISTING BACTERIAL BIOFILM,
AND PREPARATION METHOD AND APPLICATION THEREOF
TECHNICAL FIELD
The invention relates to a chitosan nano-conjugate for resisting bacterial biofilm, a preparation method of polypeptide CRAMP, and an application of the chitosan nano- conjugate with antibacterial biofilm in preparing anti-Pseudomonas aeruginosa drugs.
BACKGROUND
Bacterial biofilm (BF) is an organized microbial colony formed by a plurality of bacteria adhering to abiotic or biological surfaces, secreting extracellular polymeric substances (EPS) and wrapping themselves in it, including polysaccharides, protein, extracellular DNA and lipids, and it is an ecosystem with a three-dimensional spatial structure. With the protection of EPS, bacteria in the form of biofilm can escape the attack of immune system and the killing of antibacterial drugs, and their drug resistance is enhanced. Furthermore, biofilm can continue to release bacteria, causing repeated attacks of infection and long-term treatment. It is reported that bacteria forming biofilm can show up to 1000 times antibiotic resistance than planktonic bacteria, which makes biofilm more likely to cause refractory chronic infection in clinic and seriously threaten human and animal health.
China patent with publication number of CN108752433A discloses an antimicrobial peptide CRAMP and its cyclic peptide, which has a significant scavenging effect on the mature biofilm of Pseudomonas aeruginosa, with a scavenging rate of 50%. However, about 50% of the biofilm has not been removed, and the best time for
CRAMP to act is only 12 hours. When the mature biofilm is intervened for about 4 hours, the biofilm will re-develop to almost the same level as the blank group.
Karin Sauer reported in nature reviews microbiology in 2020 that after the bacteria in the mature biological envelope are dispersed into planktonic bacteria, their metabolic
2 LU504180 activity will be restored, which is beneficial to the killing of sensitive antibiotics.
Therefore, promoting the dispersion of the mature biological envelope will be the most promising strategy to control the infection of the biological envelope at present.
Therefore, there is an urgent need for an optimized modification of CRAMP, which is more conducive to its free shuttle in the "water channel" of mature biofilm, so that it can be released after reaching the root of biofilm, activating its antibacterial effect and providing a basis for antimicrobial peptides to enter clinical application.
SUMMARY
In view of this, the purpose of the present invention is to provide a chitosan nanoconjugate with antibacterial biofilm; The second purpose of the invention is to provide a preparation method of chitosan nanocomposites with antibacterial biofilm;
The third purpose of the present invention is to provide the application of chitosan nanoconjugates with anti-bacterial biofilm in the preparation of drugs for inhibiting
Pseudomonas aeruginosa.
In order to achieve the above purpose, this invention provides technical solution as follows:
A chitosan nano-conjugate for resisting bacterial biofilm, including the chitosan nano-conjugate of the antibacterial biofilm is prepared by coupling polypeptide CRAMP with chitosan and graphene oxide; the amino acid sequence of the polypeptide
CRAMP is GLLRKGGEKIGEKL KKIGQKIKNFFQKLVPQPEQ.
Preferably, the mass ratio of CRAMP to chitosan is 1: 1-1: 3, and the mass ratio of CRAMP to graphene oxide is 1: 1-1: 20.
A preparation method of chitosan nano-conjugate with anti-bacterial biofilm, which is characterized by comprising the following steps: CRAMP is made into solution to activate the carboxyl group of CRAMP, then chitosan solution is added to couple chitosan with the carboxyl group of CRAMP to form a chitosan Cramp conjugate, then the amino group of the chitosan Cramp conjugate is activated, and then the graphene oxide colloid solution is added to fully react to obtain the chitosan nano-conjugate with anti-bacterial biofilm, and dialysis and freeze-drying are carried out to obtain the chitosan nano-conjugate with anti-bacterial biofilm.
3 LU504180
Preferably, the method of activating the carboxyl group of CRAMP is activated by using EDC and NHS.
Preferably, the method of amino activation is that chitosan -CRAMP conjugate is first added into tetraethylenepentamine solution, and then EDC is added to react with
NHS.
Preferably, the concentration of CRAMP solution is 1 mg/ml; The chitosan solution is an acetic acid solution with chitosan concentration of 20mg/mL; The concentration of the graphene oxide colloid is 0.5mg/ml.
Preferably, the condition of activating CRAMP carboxyl group is to stir lucifugally at 4°C for 30min; the coupling condition of chitosan and CRAMP carboxyl group is to stir lucifugally 4°C for 24 hours; the condition of amino activation is to react for 10 hours at 22°C and 250rpm; the reaction condition for preparing the chitosan nano-conjugate of the antibacterial biofilm is 250rpm for 2 hours.
Preferably, the dialysis bag used for dialysis has a pore size of 8000Da.
Preferably, the application of the chitosan nanoconjugate with antibacterial biofilm in preparing a medicine for removing bacterial biofilm.
Preferably, the application of the chitosan nanoconjugate in the preparation of drugs for inhibiting Pseudomonas aeruginosa.
The invention has the beneficial effects that after coupling chitosan -CRAMP- graphene oxide, it is found that the conjugate can significantly reduce the amount of biofilm, and more than 70% of biofilm has been eradicated by laser confocal microscopy. Further study found that the nanoconjugate can freely shuttle in the polar waterway of biofilm, which is more conducive to transporting
CRAMP into the root of biofilm. In addition, it is also found that chitosan-CRAMP-graphene oxide conjugate can promote the biofilm to depolymerize into planktonic bacteria in advance by reducing the level of c-di-GMP and increasing the content of rhamnolipid, and make the biofilm vacuolate, thus truly eradicating the biofilm. Furthermore, the cytotoxicity test and hemolysis test showed that the safety is improved, and the thermal stability, pH stability, salt ion stability, especially the stability of digestive enzymes (pepsin and trypsin) are obviously better than CRAMP, which is expected to be developed into a new nano-preparation for oral administration against biofilm infection.
4 LU504180
BRIEF DESCRIPTION OF THE FIGURES
In order to make the purpose, technical scheme and beneficial effects of the invention clearer, the invention provides the following drawings for explanation:
Fig. 1 shows the characterization of PAO1 mature biofilm by laser confocal microscope;
Fig. 2 shows the bacteriostatic effect of different treatment groups;
Fig. 3 shows the stability and safety test of chitosan nanocomposites with antibacterial biofilm.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be further explained with the attached drawings and specific embodiment, so that those skilled in the art can better understand and implement the invention, but the embodiment given are not taken as limitations of the invention.
Pseudomonas aeruginosa (P.A.) is a common opportunistic pathogen, the main pathogen of chronic lung infections such as Cystic fibrosis (CF), and a common pathogen that causes chronic recurrent infections in immunocompromised organisms.
This bacterium is resistant to many common antibiotics, and its infection is easy to recur, and is difficult to eliminate. The main reason is that it is easy to form biofilms (BF) and can cause widespread infections in animals and plants. It is one of the most known bacteria with obvious ecological significance. Therefore, the embodiment of the invention takes Pseudomonas aeruginosa PAO1 strain as an example to study substances with biological activity on BF.
The amino acid sequence of the antimicrobial peptide CRAMP used in the examples of the present invention is as follows:
GLLRKGGEKIGEKLKK IGQKIKNFFQKLVPQPEQ
Embodiment 1
Preparation of chitosan nano-conjugate for resisting bacterial biofilm
A) preparation of chitosan solution 0.5g of chitosan was added to 25mL of 0.5mol/L acetic acid solution, which was used as the working liquid to make a chitosan solution with a concentration of 20mg/mL, and a blank group solution with the same pH value without chitosan was prepared as the control group.
B) activating carboxyl groups of CRAMP.
Took 2mL of CRAMP solution with the concentration of 1mg/ML, added it to 13.6mg of EDC and 23.8mg of NHS, and stirred lucifugally for 30min at 4°C to activate carboxyl groups.
C) -CRAMP coupling of chitosan
Took 250pL of chitosan solution prepared in step a), added it into CRAMP solution prepared in step b) after carboxyl activation, and stirred lucifugally at 4°C for 24h.
D) activating the amino group of CRAMP:
TEPA solution was diluted with a concentration of 75mM by 25 times, took1mL of it, and added it to the chitosan -CRAMP conjugate solution prepared in step c), then added 10uL of EDC with a concentration of 50mM and 10uL of NHS with a concentration of 100mM, and rotated and reacted the upper solution at 22°C at a rotation speed of 250rpm for 10 hours.
E) diluting the graphene oxide colloid.
The graphene oxide colloid with a concentration of 2mg/ml was diluted to 0.5mg/ml.
F) chitosan -CRAMP- graphene oxide coupling
G) taking the diluted graphene oxide colloidal solution prepared in step e) 200uL was added into the CRAMP solution activated by amino group in step d), and then it was put into a shaking table to react for 2 hours at the rotating speed of 250rpm. After the reaction, the liquid was dialyzed through a dialysis bag with a pore size of 8000Da and freeze-dried to obtain chitosan Cramp graphene oxide conjugate, which was prepared into a suitable concentration for use.
Embodiment 2
Effect of chitosan nanoconjugate with antibacterial biofilm on biofilm quantity and biofilm bacteria number of PAO1 strain. 1. Construct mature biofilm in vitro. 100uL diluted working bacteria solution was added to each test hole, 96-well plate was sealed with plastic wrap and kept it at 37°C for 24 hours. After forming biofilm, discarded the upper culture solution and PBS (pH = 7. 4) solution, washed it for 3 times, and the PBS solution was sucked up. At the same time, different concentrations of nano CRAMP intervention group, different concentrations of CRAMP control group, different concentrations of antibacterial drug enrofloxacin control group, and the blank
6 LU504180 control group were set up, and the intervention time was 1 hour. The optimal concentration was screened by taking the biofilm amount as the inspection index. 2. Detection of biofilm quantity
Took the biofilm prepared in the above step 1, the upper culture solution and PBS (pH = 7.4) solution were discarded, and washed for 3 times, and the PBS solution was sucked up; the methanol solution was fixed for 10min, then 100uL of crystal violet dye solution was added for 20min, and the crystal violet dye solution was sucked up and washed with PBS solution for 3 times.100uL acetic acid solution was added, blew and mixed well, and was transferred to another 96-hole cell culture plate, and measure the absorbance at 630nm of enzyme-labeled instrument. The test was repeated independently for more than 3 times. 3. Biofilm viable bacteria count
Took the biofilm (cultured at 37°C for 14 hours) prepared in the above step, discarded the upper layer culture solution, washed it with PBS solution for three times, 100uL of 0.1% Triton solution was added to dissolve the biofilm, 225pL of MHB (1: 1000) was added to the 96-well cell culture plate, and 25ul was sucked out, and was diluted 10 times to 105 gradients, and took 15jL for culture counting after dropping the plate. The test independently was repeated for more than 3 times.
At the concentration of 2MIC and MIC, the chitosan nanoconjugate (nano
CRAMP) with antibacterial biofilm significantly reduced the number of viable bacteria in PAO1 biofilm, and the Log value decreased by more than 2, indicating that > 99% of biofilm bacteria were killed. CRAMP only significantly reduced the number of viable bacteria in biofilm at the concentration of 2MIC, and the Log value decreased by more than 1, indicating that > 90% of biofilm bacteria were inhibited. Enrofloxacin had almost no effect on the number of viable bacteria in biofilm, as shown in Table 1.
7 LU504180
Table 1. The number of viable bacteria in the biofilm of PAO1 strain (Log10).
Nano CRAMP CRAMP Enrofloxacin
Control 6.919+0.110 6.359+0.744 5.885+0.999 2 MIC 4.574+1.101 5.208+0.613 5.355+0.786
MIC 4.910+0.852 5.795+0.421 5.343+1.453 1/2 MIC 5.978+1.091 5.945+1.023 6.147+0.481 1/4 MIC 6.261+0.907 5.880+0.468 5.915+0.573 1/8 MIC 6.332+0.418 6.027+0.731 5.739+0.374 1/16 MIC 6.499+0.326 6.376+0.521 5.526+0.827
Remarks: The MIC of nano CRAMP and CRAMP is 15.625ug/ml.
The results are shown in Table 2. The results showed that chitosan nanocomposites with antibacterial biofilm significantly reduced the amount of biofilm at 2MIC and MIC concentrations, and significantly reduced the amount of biofilm at 1/2MIC concentration. CRAMP can significantly reduce the amount of biofilm at 2MIC concentration; Enrofloxacin failed to significantly reduce the amount of biofilm.
8 LU504180
Table 2. Biofilm biomass of PAO1 Strain (OD630, x+SD)
Nano CRAMP CRAMP Enrofloxacin
Control 0.411+0.050 0.284+0.058 0.310+0.057 2MIC 0.199+0.054”” 0.179+0.017” 0.226+0.096
MIC 0.272+0.083” 0.261+0.045 0.290+0.093 1/2MIC 0.346+0.015" 0.226+0.056 0.246+0.085 1/4MIC 0.395+0.165 0.267+0.036 0.266+0.086 1/8MIC 0.450+0.185 0.218+0.015 0.290+0.102 1/16MIC 0.474+0.215 0.265+0.013 0.283+0.098
Note: * indicates that there is a significant difference compared with the non- intervention negative control hole (P < 0.05); * * indicates that there is a significant difference (P < 0.01) between the non-intervention negative control wells and the non- intervention negative control wells (P < 0.001).
Embodiment 3. Bacteriostatic effect of chitosan nanoconjugates with anti-bacterial biofilm on PAO1 by laser confocal microscope.
According to the experimental method of Embodiment 2, the PAO1 mature biofilm treated with chitosan nanocomposites (concentration of 4MIC) and the control group (CRAMP, concentration of 4MIC) was dyed with SYTO9/PI, in which SYTO 9 glowed green to represent the number of living bacteria, and PI glowed red to represent the dead bacteria. At least 4 independent repetitions, at least 4 different visual fields were observed for each sample, and one of the representative visual fields of the control hole and the experimental hole was selected. Z-stacks layered scanning was carried out under 10 times and 63 times objective lens respectively.
The fluorescence intensity of all the image files of Z-stack was statistically analyzed, and the results were shown in Figure 1. In Figure. 1, the bottom and right side of each picture respectively showed the cross-sectional and vertical sectional views along the cross. A: CLSM diagram under the 10x objective lens of the control hole; B: CLSM diagram of the control hole under 63x oil mirror, C: CLSM diagram
9 LU504180 under 10x objective lens after the intervention of chitosan nanoconjugate with anti- bacterial biofilm; D: CLSM image of chitosan nanoconjugate with anti-bacterial biofilm after intervention under 63x oil mirror.
The results showed that compared with the control hole of the experimental group, the chitosan nanoconjugate group with anti-bacterial biofilm could significantly reduce the total fluorescence intensity of bacteria, and the total number of bacteria decreased by 72. 69% (P < 0.05). Compared with the control well, the ratio of PI/PI+SYTO (fluorescence intensity of dead bacteria/total fluorescence intensity) in the chitosan nanoconjugate group with anti-bacterial biofilm was significantly increased (P < 0.01), which was significantly higher than that of unmodified Cramp (P < 0.05), as shown in
Figure 2. In Figure. 2, Control: fluorescence diagram of PAO1 strain without any treatment; Nano-CRAMP: fluorescence diagram of PAO1 strain treated with nano- microspheres anti-biofilm peptide CRAMP; CRAMP: fluorescence diagram of PAO1 strain treated with unmodified CRAMP. The results showed that CRAMP, an anti- biofilm peptide of nano-microspheres, had a significant inhibitory effect on the living bacteria in the biofilm of PAO1 strain and a significant scavenging effect on the mature biofilm of PAO1 strain.
In addition, it was also observed that most of the PAO1 biofilms treated with chitosan nanocomposites were removed. When some biofilms were scanned by
Z-stack, it was found that almost all bacteria in each layer were dead, which indicated that nano-CRAMP could effectively enter the biofilms to kill bacteria, and the biofilms were obviously hollow, as shown in Figure 1-D. The results showed that CRAMP can enter the bottom of biofilm after being modified by chitosan and graphene oxide, thus exerting its function of promoting the release and depolymerization of biofilm, and promoting the release of biofilm from the inside, and then cavitation.
Embodiment 4: Safety and Stability of nano-conjugate for resisting bacterial biofilm
The stock solution of protease was prepared according to the instructions. The protease with the final concentration of 1mg/ml, CRAMP and Nano CRAMP with 125ug/ml were heated in water bath at 37°C for 30min, while CRAMP and Nano
CRAMP without enzyme treatment were used as control group. Finally, the size of bacteriostatic circle was determined in nutrient agar by punching method.
The investigation results of thermal stability were shown in figure 3, a. In
Figure.3A, cram: unmodified cram, nano cram: nano microsphere anti-biofilm peptide cram. After being treated at 25°C, 50°C, 75°C and 100°C, the MIC of chitosan nano- conjugate for resisting bacterial biofilm had not changed, and it was more stable than unmodified CRAMP. After being treated with neutral protease, pepsin and trypsin, the antibacterial effect of chitosan nanocomposites with antibacterial biofilm was less different than that without treatment (P<0.05), and the stability was better (shown in
Figure 3-B).In the safety investigation, the hemolysis rate of rabbit red blood cells after chitosan nanoconjugate with antibacterial biofilm was lower (shown in Figure 3-C). The concentration of chitosan CRAMP graphene oxide conjugate was not toxic to
RAW264.7 cells in the range of 0~160ug/mL, while unmodified Cramp had a certain cytotoxicity at 160ug/mL (P<0.05) (shown in Figure 3, D).
The embodiments described above are only the preferred embodiments for fully explaining the present invention, and the protection scope of the present invention is not limited to this. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The scope of protection of the invention is subject to the claims.

Claims (10)

11 LU504180 CLAIMS
1. A chitosan nano-conjugate for resisting bacterial biofilm, including the chitosan nano-conjugate of the antibacterial biofilm is prepared by coupling polypeptide CRAMP with chitosan and graphene oxide; the amino acid sequence of the polypeptide CRAMP is GLLRKGGEKIGEKL KKIGQKIKNFFQKLVPQPEQ.
2. The chitosan nano-conjugate for resisting bacterial biofilm as claimed in claim 1, wherein, the mass ratio of CRAMP to chitosan is 1: 1-1: 3, and the mass ratio of CRAMP to graphene oxide is 1: 1-1: 20.
3. A preparation method of chitosan nano-conjugate with anti-bacterial biofilm, which is characterized by comprising the following steps: CRAMP is made into solution to activate the carboxyl group of CRAMP, then chitosan solution is added to couple chitosan with the carboxyl group of CRAMP to form a chitosan Cramp conjugate, then the amino group of the chitosan Cramp conjugate is activated, and then the graphene oxide colloid solution is added to fully react to obtain the chitosan nano-conjugate with anti-bacterial biofilm, and dialysis and freeze-drying are carried out to obtain the chitosan nano-conjugate with anti-bacterial biofilm.
4. The preparation method of chitosan nano-conjugate with anti-bacterial biofilm according to claim 3 is characterized in that the method of activating the carboxyl group of CRAMP is activated by using EDC and NHS.
5. The preparation method of chitosan nano-conjugate with anti-bacterial biofilm according to claim 3, characterized in that the method of amino activation is that chitosan -CRAMP conjugate is first added into tetraethylenepentamine solution, and then EDC is added to react with NHS.
12 LU504180
6. The preparation method of chitosan nano-conjugate with anti-bacterial biofilm according to claim 3, characterized in that the concentration of CRAMP solution is 1 mg/ml; the chitosan solution is an acetic acid solution with chitosan concentration of 20mg/mL; the concentration of the graphene oxide colloid is 0.5mg/ml.
7. The preparation method of chitosan nano-conjugate with anti-bacterial biofilm according to claim 3 is characterized in that the condition of activating CRAMP carboxyl group is to stir lucifugally at 4°C for 30min; the coupling condition of chitosan and CRAMP carboxyl group is to stir lucifugally 4°C for 24 hours; the condition of amino activation is to react for 10 hours at 22°C and 250rpm; the reaction condition for preparing the chitosan nano-conjugate of the antibacterial biofilm is 250rpm for 2 hours.
8. The preparation method of chitosan nanocomposites with antibacterial biofilm according to claim 3, characterized in that the dialysis bag used for dialysis has a pore size of 8000Da.
9. An application of the chitosan nanoconjugate with antibacterial biofilm according to claim 1 or 2 in preparing a medicine for removing bacterial biofilm.
10. An application of the chitosan nanoconjugate of claim 1 or claim 2 in the preparation of drugs for inhibiting Pseudomonas aeruginosa.
LU504180A 2023-05-10 2023-05-10 Chitosan nano-conjugate for resisting bacterial biofilm, and preparation method and application thereof LU504180B1 (en)

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