CN111973571B

CN111973571B - Antibacterial nanoparticles based on berberine derivatives and rhamnolipid

Info

Publication number: CN111973571B
Application number: CN201910436175.XA
Authority: CN
Inventors: 胡海燕; 申元娜; 邹祎晴; 李彭宇; 陈小楠; 饶义琴
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-05-23
Filing date: 2019-05-23
Publication date: 2022-05-27
Anticipated expiration: 2039-05-23
Also published as: CN111973571A

Abstract

The invention provides an antibacterial nanoparticle which is formed by self-assembling berberine derivatives and rhamnolipid. The invention provides the use of the antibacterial nanoparticles in the preparation of antibacterial formulations, wherein the bacteria are biofilm-producing bacteria, including microaerophilic bacteria (e.g. Microaerobes)H.pylori）、Aerobic bacteria (e.g. Bacillus subtilis)P.aeruginosa) And facultative anaerobes (e.g. anaerobes)E.coliAndS.aureus) Anaerobic bacteria (e.g. Bacillus subtilis)S.mutans) These bacteria encompass gram-negative and gram-positive bacteria. In one embodiment, the biofilm-producing bacterium isH.pylori。

Description

Antibacterial nanoparticles based on berberine derivatives and rhamnolipid

Technical Field

The invention relates to a nano drug delivery system, in particular to an antibacterial nano particle for removing a biological membrane, in particular to a nano preparation for killing or removing bacteria colonized inside or below a mucous layer of epithelial cells.

Background

Helicobacter pylori (h. pylori) is a multi-flagellate, spiral rod-shaped microaerophilic gram-negative bacterium that can cause malignant diseases such as gastritis, gastric ulcer, gastric cancer, and the like. Early triple therapy with amoxicillin, clarithromycin and proton pump inhibitors was used to treat h.pyri, which has increased resistance to antibiotics (including clarithromycin and metronidazole) over the past few years. In order to increase the clearance of h.pyri, quadruple therapy derived from triple therapy is becoming the first line treatment for h.pyri infection. The quadruple therapy is added with bismuth agent based on triple therapy to protect damaged gastric mucosa. Although the four-combination therapy has higher clearance rate to H.pyri than the three-combination therapy, the effect of eradicating H.pyri is gradually reduced, and in addition, the use of antibiotics with large dose for a long time not only increases toxic and side effects, but also increases the drug resistance of H.pyri, so that the treatment of H.pyri faces serious challenges.

H.pyri bifolium has been shown to be the major cause of increased resistance and low clearance of h.pyri against antibiotics. The pyrori is planted at the bottom of the gastric mucus, the gastric mucus is also an important physical barrier, the damage of foreign substances to the gastric tissue can be effectively resisted, and meanwhile, the mucus is continuously secreted and removed, so that some substances can be removed to the outside of the body. Based on this protective barrier, it is difficult for antibiotics to reach effective bactericidal concentrations at sites of h.pyri infection, resulting in a decrease in the clearance of h.pyri by the drug. Therefore, the effective overcoming of the mucus barrier and the sufficient amount of the drug to reach the site of H.pyri infection are also the key to improve the treatment effect of H.pyri.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the obstacle of bacterial biomembrane, improving the antibacterial effect of the medicine and finally achieving the aim of removing bacteria. The invention destroys the biological membrane formed by bacteria by the nanometer preparation, kills the bacteria in the membrane by the released antibacterial drug, and realizes the obvious inhibition, killing and removal of the bacteria.

One aspect of the present invention provides an antibacterial nanoparticle, formed by self-assembly of berberine derivatives and rhamnolipid, wherein the berberine derivatives are modified to have C8-20 alkyl substituents on carbon atoms selected from 8, 12 and 13, or C8-20 alkoxy substituents on carbon atom 9.

In some embodiments, the nanoparticles have a particle size of 100nm to 200nm as determined by a laser particle sizer and a Zeta potential of between-20 mV to-60 mV. In some embodiments, the nanoparticles have a particle size of 120nm to 180nm, preferably 150nm to 180nm, more preferably 160nm to 175nm, more preferably 170nm, 171nm, 172nm, 173nm, 174nm, or 175nm, as determined by laser granulometry. In some embodiments, the Zeta potential of the nanoparticle is between-30 mV and-60 mV, preferably between-40 and-60 mV, more preferably between-30 and-55 mV, more preferably between-35 and-50 mV, more preferably a value of-40 and 50mV, more preferably-40 mV, -41mV, -42, -43, -44, -45, -46, -47, -48, -49 or-50 mV.

In some embodiments, the berberine derivative is substituted with a C10-18 alkoxy group at the 9 th carbon atom. More preferably, the berberine derivative is 9-O-n-decaalkyl berberine, 9-O-n-dodecyl berberine, 9-O-n-hexadecyl berberine or 9-O-n-octadecyl berberine. More preferably, the berberine derivative is 9-O-n-decaalkyl berberine or 9-O-n-dodecyl berberine. Most preferably, the berberine derivative is 9-O-n-decaalkyl berberine.

In some embodiments, in the nanoparticle, the weight ratio of berberine derivative to rhamnolipid is 1:2 to 1: 10. Preferably, in the nano-formulation, the weight ratio of the berberine derivative to the rhamnolipid is 1: 4.

In some embodiments, the nanoparticles are capable of at least partially passing through a mucus layer overlying an epithelial cell layer. In some embodiments, the nanoparticles are capable of passing completely through the mucus layer overlying the epithelial cell layer.

In another aspect, the present invention provides the use of an antibacterial nanoparticle of the present invention in the preparation of an antibacterial formulation, wherein the bacterium is a biofilm-producing bacterium.

In some embodiments, the biofilm-producing bacteria are gram-negative biofilm-producing bacteria. In other embodiments, the biofilm-producing bacteria are gram-positive biofilm-producing bacteria. In some embodiments, the biofilm-producing bacteria are microaerophilic bacteria (e.g., h. pyri), aerobic bacteria (e.g., p. aeruginosa), facultative anaerobic bacteria (e.g., e.coli and s.aureus), or anaerobic bacteria (e.g., s.mutans). In some embodiments, the biofilm-producing bacterium is h. In a preferred embodiment, the biofilm-producing bacteria are bacteria that colonize the interior or below the mucus layer secreted by epithelial cells. In a preferred embodiment, the biofilm-producing bacterium is h.

In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the antibacterial nanoparticle of the present invention and a pharmaceutically acceptable carrier.

In a further aspect of the present invention, there is provided a method of killing bacteria in an organism, the method comprising contacting an antibacterial nanoparticle of the present invention with the bacteria, wherein the bacteria are biofilm-producing bacteria.

The BD and the RHL are prepared into the nano-particles through self-assembly, so that the infection of a biological membrane of bacteria generating a biological membrane (such as H. In the case where the biofilm-producing bacteria are bacteria (e.g., h. pyleri) that colonize inside or below the mucus layer secreted by epithelial cells, the RHL can increase the hydrophilicity of the nanoparticle surface, reduce the electropositivity of the BD, and improve the mucus penetrating ability of the nanoparticle by avoiding interaction with hydrophobic, negatively charged mucins. The nanoparticles penetrate through mucus and reach the infected part of H.pyrori, the EPS is destroyed by RHL to remove the biofilm and expose free bacteria, and the nanoparticles directly play an antibacterial role, so that the drug resistance of H.pyrori is reduced.

Drawings

FIG. 1. Crystal Violet assay to evaluate the clearance of H.pyri bifilm by BD/RHL NPs: (A) C18-BD/RHL NPs (B) C16-BD/RHL NPs (C) C2-BD-RHL NPs (D) C10-BD/RHL NPs.

FIG. 2. evaluation of the clearance of H.pyri bifilm by BD/RHL NPs by fluorescence labeling of dead and alive bacteria.

FIG. 3. effect of BD/RHL NPs on H.pyri activity of dispersed bifilm.

Figure 4.BD and RHL inhibit h. (A) C18-BD (B) C16-BD (C) C12-BD (D) C10-BD (E) RHL.

FIG. 5 BD/RHL NPs inhibit the ability of H.

FIG. 6 BD/RHL NPs inhibited the ability of H.pyrori to adhere to MGC 803 cells.

FIG. 7 Transwell method for determining Papp values for mucus penetration of BD/RHL NPs, # p <0.001vs BD NPs, # p <0.05, # p <0.01vs C18-BD/RHL NPs, & p <0.05vs C16-BD/RHL NPs, & Δ p <0.05vs C12-BD/RHL NPs.

FIG. 8.BD/RHL NPs effect on H.pylori bifilm clearance in the mucus-bifilm model, # p <0.001vs control, # p <0.05vs C18-BD/RHL NPs, & p <0.05vs C16-BD/RHL NPs, & Δ p <0.05vs C12-BD/RHL NPs.

FIG. 9. Crystal Violet assay examines the clearance of SDS, RHL and NAC for five bifilms: (a-C) h.pyri (D-F) e.coli (G-I) p.aeruginosa (J-L) s.aureus (M-O) s.mutans, significance p <0.05, significance p <0.01, significance p < 0.001.

FIG. 10 scanning electron microscopy was used to examine the clearance of SDS, RHL and NAC on five bifilms.

FIG. 11. fluorescent labeling of dead and alive bacteria to examine the clearance of SDS, RHL and NAC on five kinds of bifilm.

Figure 12 inhibition of five bifilms by SDS, RHL and NAC: (a-C) h.pyri (D-F) e.coli (G-I) p.aeruginosa (J-L) s.aureus (M-O) s.mutans, error bars indicate Standard Deviation (SD). significance p <0.05,. significance p <0.01,. significance p < 0.001.

Detailed Description

Definition of

By "berberine derivative" or "BD" is meant herein berberine modified to have a C8-20 alkyl substituent on a carbon atom selected from positions 8, 12 and 13, or a C8-20 alkoxy substituent on a carbon atom at position 9. The term C8-20 alkyl or similar expression refers to a straight or branched alkyl chain having from 8 to 20 carbon atoms. Having C8-20 alkoxy substitution at the 9 th carbon atom means-OCH to the 9 th carbon atom of berberine₃Substituted with C8-20 alkoxy.

In some embodiments, the berberine derivative is substituted with a C10-18 alkoxy group at the 9 th carbon atom. More preferably, the berberine derivative is 9-O-n-decaalkyl berberine, 9-O-n-dodecyl berberine, 9-O-n-hexadecyl berberine or 9-O-n-octadecyl berberine. More preferably, the berberine derivative is 9-O-n-decaalkyl berberine or 9-O-n-dodecyl berberine. Most preferably, the berberine derivative is 9-O-n-decaalkyl berberine. Processes for the preparation of the above berberine derivatives are known from the open literature (e.g. Song, J.et al,2019.Mitochondrial targeting nano-drugs self-assembled from9-O-octade sub-stabilized berberine derivative for cancer treatment by inductive binding miondrial apoptosis pathway. journal of controlled release enzyme 294:27-42, ZL 201310218544.0 or ZL 201310218566.7). For the sake of simplicity, the berberine derivatives used herein are named as substituent + berberine derivatives, which are all 9-O-substituted berberine derivatives, e.g. dodecyl berberine derivative refers to 9-O-n-dodecyl berberine, if not specified.

Biofilm (biofilm) refers to a microbial population in which microorganisms spontaneously adhere to the surface of biological or non-biological materials and are encapsulated by extracellular polymeric matrix (EPS) secreted by themselves. Biofilm is a protection mechanism formed spontaneously for adapting to a severe living environment, and the bacteria in the Biofilm have the characteristics of higher host immune defense property, lower growth rate and the like compared with free bacteria, so that the bacteria in the Biofilm have higher drug resistance, and the drug resistance is improved by 10-1000 times compared with the free bacteria. Resulting in a reduction in the therapeutic effect of the antibiotic.

"biofilm-producing bacteria" refers to bacteria capable of spontaneously forming biofilms (bifilms) in organisms. Under appropriate conditions, the bacteria can form bifilm. In some embodiments, the biofilm-producing bacteria are gram-negative biofilm-producing bacteria. In other embodiments, the biofilm-producing bacteria are gram-positive biofilm-producing bacteria. In some embodiments, the biofilm-producing bacteria are microaerophilic bacteria (e.g., h. pyri), aerobic bacteria (e.g., p. aeruginosa), facultative anaerobic bacteria (e.g., e.coli and s.aureus), or anaerobic bacteria (e.g., s.mutans). In some embodiments, the biofilm-producing bacterium is h. In a preferred embodiment, the biofilm-producing bacteria are bacteria that colonize the interior or below the mucus layer secreted by epithelial cells. In a preferred embodiment, the biofilm-producing bacterium is h.

As used herein, the term "composition" refers to a formulation suitable for administration to a desired animal subject for therapeutic purposes, which contains at least one pharmaceutically active ingredient, e.g., a compound. Optionally, the composition further comprises at least one pharmaceutically acceptable carrier or excipient.

The term "pharmaceutically acceptable" means that the substance does not possess properties that would allow a reasonably prudent medical practitioner to avoid administering the substance to a patient, given the disease or condition to be treated and the respective route of administration. For example, for injectables, it is often desirable that such substances be substantially sterile.

As used herein, "treating" includes administering a compound of the present application, or a pharmaceutically acceptable salt thereof, to alleviate a symptom or complication of a disease or condition, or to eliminate a disease or condition. The term "alleviating" as used herein is used to describe the process of reducing the severity of signs or symptoms of a disorder. Symptoms can be reduced without elimination. In one embodiment, administration of the pharmaceutical composition of the present application results in elimination of the signs or symptoms.

Pharmaceutical composition

In the present invention, a "pharmaceutical composition" refers to a composition comprising the nanoparticles of the present invention and a pharmaceutically acceptable carrier, wherein the nanoparticles and the pharmaceutically acceptable carrier are present in the composition in admixture. The compositions will generally be used for the treatment of human subjects. However, they may also be used to treat similar or identical conditions in other animal subjects. As used herein, the terms "subject," "animal subject," and similar terms refer to humans and non-human vertebrates, e.g., mammals, such as non-human primates, sports and commercial animals, such as horses, cows, pigs, sheep, rodents, and pets, such as dogs and cats.

Suitable dosage forms depend, in part, on the use or route of administration, e.g., oral, transdermal, transmucosal, inhalation, or by injection (parenteral). Such dosage forms should enable the compound to reach the target cell. Other factors are well known in the art, including considerations such as toxicity and the dosage form that delays the compound or composition from exerting its effect.

Carriers or excipients may be used to produce the composition. The carrier or excipient may be selected to facilitate administration of the compound. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (e.g. lactose, glucose or sucrose), or starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile water for injection (WFI), saline solutions and glucose.

The compositions or components of the compositions may be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal or inhalation. In some embodiments, injections or lyophilized injections are preferred. For oral administration, for example, the compounds may be formulated in conventional oral dosage forms such as capsules, tablets, as well as liquid preparations such as syrups, elixirs, and concentrated drops.

Alternatively, injection (parenteral administration), e.g., intramuscular, intravenous, intraperitoneal, and/or subcutaneous, may be used. For injection, the compositions of the invention or components thereof are formulated as sterile liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compositions or components thereof may be formulated in solid form and redissolved or suspended immediately prior to use. Also can be produced in the form of freeze-dried powder.

Administration may also be by transmucosal, topical, or transdermal means. For transmucosal, topical, or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. Additionally, detergents may be used to facilitate penetration. Transmucosal administration, for example, can be through a nasal spray or suppository (rectal or vaginal).

Effective amounts of the various components to be administered can be determined by standard procedures, taking into account the age, size and weight of the subject and the condition associated with the subject. The importance of these and other factors is well known to those of ordinary skill in the art. In general, the dose will be between about 0.01mg/kg and 50mg/kg, preferably between 0.lmg/kg and 20mg/kg of the subject being treated. Multiple doses may be used.

The compositions of the invention or components thereof may also be used in combination with other therapeutic agents for the treatment of the same disease. Such combined use includes administering the compounds and one or more other therapeutic agents at different times, or simultaneously administering the compounds and one or more other therapeutic agents. In some embodiments, the dosage of one or more compounds of the invention or other therapeutic agents used in combination may be modified, for example, by reducing the dosage relative to the compound or therapeutic agent used alone by methods known to those skilled in the art.

Examples

1. Preparation and characterization of self-assembled nanoparticles

Method

The berberine derivative/rhamnolipid self-assembly nanoparticles (BD/RHL NPs) are prepared by respectively weighing decaalkyl berberine derivative (C10-BD), dodecyl berberine derivative (C12-BD), hexadecyl berberine derivative (C16-BD) and octadecyl berberine derivative (C18-BD), adding DMSO, and ultrasonically dissolving to obtain a concentration of 5 mg/mL. An aqueous Rhamnolipid (RHL) solution was prepared at a concentration of 2 mg/mL. Dripping four kinds of Berberine Derivative (BD) solutions into the RHL solution under stirring, and continuing stirring for 2min after the BD solution is dripped, thus forming BD/RHL NPs. BD/RHL NPs were dialyzed in 3500kD dialysis bags for 6h to remove organic solvents. The appearance of the solution was observed and its particle size and potential were measured.

Screening the mass ratio of BD to RHL: precisely sucking a certain amount of BD solution, dropwise adding the BD solution into RHL solutions with different concentrations to enable the mass ratio of BD to RHL to be 1:40, 1:20, 1:10, 1:8, 1:5, 1:4 and 1:2 respectively, preparing BD/RHL NPs, and measuring the particle size and the potential.

As a result, the

As shown in tables 1, 2, 3 and 4, the four BD/RHL NPs have smaller particle sizes and PDI values, uniform particle sizes and negative potentials at a mass ratio of BD to RHL of 1: 4.

TABLE 1 influence of the mass ratio of C10-BD to RHL on the nanoparticle size and potential

TABLE 2 influence of the mass ratio of C12-BD to RHL on the nanoparticle size and potential

TABLE 3 influence of the mass ratio of C16-BD to RHL on the nanoparticle size and potential

TABLE 4 influence of the mass ratio of C18-BD to RHL on the nanoparticle size and potential

2. Method for removing H.pyrori bifilm by self-assembled nanoparticles

Determination of Minimum Inhibitory Concentration (MIC) of pyri

(1) Preparing a sample solution: BD and BD/RHL NPs solutions were diluted with 10% FBS in BHI medium to BD concentrations of 37.5, 18.75, 9.375, 4.69, 2.34, 1.17, 0.6, 0.3, and 0.15. mu.g/mL. (2) Plating and dosing: the bacterial suspension with OD 600 value of 0.1 was diluted 30 times with the medium and added to the well plate at 100. mu.L/well, and then 50. mu.L of the sample solution was added. The plate was incubated at 37 ℃ for 48h with shaking at 100 rpm. (3) Determination of MIC values: after the incubation, the OD 600 values were measured by a microplate reader, and the MIC values of the respective groups were calculated.

Clearance of H.pylori bifilm by BD/RHL NPs:

cultivation of Biofilm: bacterial suspension with OD 600 of 0.1 was added to 48-well plates at 500. mu.L/well, and allowed to stand for 72 hours at 37 ℃ under microaerophilic conditions, to form H.pyri bifilm.

(1) Crystal violet dyeing method: BD and its BD/RHL NPs were diluted by a two-fold dilution method to approximately BD concentrations of 100, 50, 25, 12.5, 6.25, 3.12 and 1.56. mu.g/mL. After the formation of the bifilm, the medium was aspirated off and 500. mu.L of the sample solution was added. Microaerophilic, incubation at 37 ℃ for 24 h. After incubation, the well plate was removed, washed 3 times with sterile PBS, fixed with 500 μ L methanol and stained with 1% crystal violet for 15 min. And adding PBS for cleaning again, adding 500 mu L of 95% ethanol, oscillating and uniformly mixing, measuring the OD 570 value by using an enzyme-labeling instrument, and calculating the bifilm removing capacity of the four BD/RHL NPs.

(2) The dead bacteria and live bacteria fluorescent labeling method comprises the following steps: h. pyri bifilm was washed with sterile PBS, followed by addition of 100 μ g/mL of sample solution. After 24h incubation, the medium was aspirated off, SYTO9/PI solution was added and incubated for 15min in the dark, and then the dishes were placed under laser Confocal (CLSM) to observe the three-dimensional structure of the bifilm and the survival of the bacteria.

Results

The MIC results for BD/RHL NPs versus free H.pyri are shown in Table 5. The C10-BD and C12-BD and the corresponding self-assembled nanoparticles C10-BD/RHL NPs and C12-BD/RHL NPs have smaller MIC values and show stronger free H.pylori resistance.

TABLE 5 MIC values of BD and BD/RHL NPs for free H

The crystal violet method results are shown in figure 1, and the clearance rate of C10-BD/RHL NPs and C12-BD/RHL NPs in the nanoparticles to H.pyriri bifilm is higher, which is related to the stronger antibacterial activity of the nanoparticles to H.pyriri. The result of the fluorescence labeling method of dead bacteria is similar to that of the crystal violet method, and the C10-BD/RHL NPs group has the strongest clearing effect (figure 2).

3. Method for inhibiting reformation of bifilm by self-assembled nanoparticles

Bactericidal capacity of BD/RHL NPs against h.pyri dispersed from biofilm after disruption:

viability of H.pyri dispersed from the bifilm was determined using SYTO9/PI fluorescence labeling. The specific operation steps are as follows: after the formation of the pyri bifilm, the cells were washed 3 times with sterile PBS to remove free bacteria, and incubated for 24h by adding 500. mu.L of a 100. mu.g/mL sample solution. After the incubation, the supernatant of each well was collected and centrifuged to remove the medium, and the h. SYTO9/PI solution was then added for resuspension and incubated for 15min in the dark. Finally, the bacterial suspension was added to a 96-well black plate, the fluorescence intensity of SYTO9 was measured, and the bactericidal activity of BD/RHL NPs on H.pyri dispersed from biofilm was calculated.

Effect of BD/RHL NPs on inhibition of h.pylori bifilm reformation:

according to the MIC values of BD and RHL, a series of sample solutions with different concentrations containing the MIC values are prepared, 250 mu L/well of the sample solutions are added into a 48-well plate, then H.pyri bacterial suspension with the OD 600 value of 0.1 is added, the drug and the bacterial suspension are mixed well and incubated under microaerobic conditions at 37 ℃ for 24 hours, and the formation amount of the bifilm is determined by using a crystal violet method after the incubation is finished. BD/RHL NPs solutions were prepared at concentrations of 3.12, 6.25, 12.5, 25, 50, 100 and 200. mu.g/mL in this order, and the ability of BD/RHL NPs to inhibit the formation of bifilm was determined in the same manner as described above.

BD/RHL NPs inhibit the effect of H.pyrori on cell adhesion:

MGC 803 cells in logarithmic growth phase are cultured at 10 deg.C⁵And inoculating one cell/well into a 24-well laser confocal plate, and culturing for 24 hours to ensure that the cells are completely attached to the wall. After the adherence, the cells are washed for 1 time by PBS, and the cells are marked by adding Hoechst fluorescent dye and incubating for 30min in dark. Bacterial suspensions were collected and labeled with SYTO9 fluorescent dye h. Separately labeled H.pyri and MGC 803 cells were mixed while adding 100. mu.g/mL BD/RHL NPs solution and incubated for 2 h. After incubation, the well plate was washed with PBS and then placed under CLSM for observation and photographing.

Results

The killing ability of BD/RHL NPs to H.pyri dispersed from biofilm is shown in FIG. 3, and the fluorescence intensity of C10-BD and C10-BD/RHL NPs is lowest after treatment, which shows that the two nanoparticles have the strongest killing effect on the dispersed H.pyri.

The results of inhibition of the reformation of bifilm by BD/RHL NPs are shown in FIGS. 4 and 5, and it can be seen from FIG. 4 that several BD and RHL all inhibited the formation of H.pyri bifilm to different degrees below the MIC concentration, while the inhibition of BD/RHL NPs is stronger compared with FIG. 5, and C10-BD/RHL NPs and C12-BD/RHL NPs were able to completely inhibit the formation of H.pyri bifilm at a concentration of 6.25. mu.g/mL, and the inhibition rates were 99.06% and 99.25%, respectively.

The effect of BD/RHL NPs on inhibiting the adhesion of H.pyri on cells is shown in FIG. 6, and the adhesion of H.pyri on cell surfaces is obviously reduced after the treatment of BD/RHL NPs. The above 3 experimental results show that BD/RHL NPs can effectively kill the disperse bacteria and inhibit the re-adhesion thereof to form bifilm, thereby preventing the recurrence of infection.

4. Self-assembled nanoparticle overcoming mucus barrier

Method

Evaluation of transmucosal effect of BD/RHL NPs:

40mg of fresh mucus was weighed into a 24-well Transwell chamber, 200. mu.L of sample solution was added to the upper layer of the mucus, and 500. mu.L of water was added to the receiving chamber. The Transwell plate was then placed at 37 ℃ in an incubator at 100rpm and shaken, and a sample solution in the receiving chamber was taken at a prescribed time to measure the fluorescence intensity, and the apparent permeability coefficient of nanoparticles (Papp) was calculated according to the following formula.

Wherein

Refers to the permeation amount of BD per unit time, A is the effective permeation area, and C is the initial concentration of the drug.

Clearance of biofim in mucus-biofiim mixed model by BD/RHL NPs:

the mucus was added to a Transwell chamber and sterilized by UV irradiation for 6h, and then the chamber was transferred to a well plate containing bifilm to construct a mucus-bifilm mix model. Adding a sample solution into the upper layer of the mucus, incubating for 24h, adding a resazurin solution into the biofilm, incubating for 2h, measuring the fluorescence intensity, and inspecting the effect of the nanoparticles on removing the biofilm under the condition of a mucus barrier.

Results

As shown in FIG. 7, the mucus penetrating ability of BD/RHL NPs was significantly enhanced compared to BD NPs, especially C10-BD/RHL NPs, which had an apparent permeability coefficient 4.33 times that of BD NPs. It can be seen in the mixed model of mucus-bifilm (FIG. 8) that even in the presence of mucus barrier, BD/RHL NPs have a strong clearance for H.pyrori bifilm at the bottom of mucus, the clearance is strongest with C10-BD/RHL NPs, and the clearance rate reaches 83.5%.

5. Self-assembled nanoparticle broad-spectrum anti-bioiflm capacity

Method

MIC values for SDS, RHL, and NAC:

the three substances were diluted in 2-fold gradients to concentrations of SDS and RHL of 30, 15, 7.5, 3.75, 1.86, 0.93, 0.47, 0.24, 0.12 and 0.06mg/mL, respectively. The concentrations of NAC were, in order, 240, 120, 60, 30, 15, 7.5, 3.75, 1.86, 0.93, and 0.47 mg/mL. Five bacterial suspensions with OD 600 values of 0.1 were diluted 30-fold with medium and added to the well plate at 100. mu.L/well, followed by 50. mu.L of sample solution. The plate was incubated at 37 ℃ for 24h with shaking at 100 rpm. After the culture is finished, determining the OD 600 value by using a microplate reader to obtain the MIC values of the three substances.

Clearance of bifilm by SDS, RHL and NAC:

biofilm culture: h, pyri: culturing the suspension of the plate bacteria under a microaerophilic condition with the OD 600 value of 0.1 for 72 h; coli: culturing for 48h when the concentration of the suspension of the bacillus subtilis is 0.1/30; p. aeruginosa: culturing for 24h when the OD 600 of the suspension of the plate bacteria is 0.1; s.aureus: culturing for 24h when the concentration of the suspension of the bacillus subtilis is 0.1/10; s. mutans: the OD 600 value of the suspension of the plate bacteria is 0.13, and the plate bacteria are cultured for 48 hours under an anaerobic condition.

(1) Crystal violet method: preparing SDS, RHL and NAC solutions to make the concentrations of the SDS and the RHL be 10, 5, 2.5, 1.25, 0.62, 0.31 and 0.16 mg/mL; NAC concentrations were 80, 40, 20, 10, 5, 2.5, and 1.25 mg/mL. The five bacteria of the bifilm were washed with PBS, 500. mu.L of sample solution was added, and the plate was incubated for a further 24 h. The clearance of SDS, RHL and NAC for five bacterial maturation bifilm was determined as per the crystal violet method described above.

(2) Scanning electron microscopy: five bacteria, bifilms, were cultured on a cell slide and dosed at a concentration of 4-fold MIC for SDS, RHL, and NAC. After 24h incubation, 2.5% glutaraldehyde was added for fixation overnight and washed 3 times with PBS, followed by dehydration once in 30%, 50%, 70% and 90% ethanol and three times in 100% ethanol for 10min each. Cell slide was dried and after surface gold spraying the bifilm morphology and drug clearance effect were observed by SEM.

(3) The dead bacteria and live bacteria fluorescent labeling method comprises the following steps: pylori bifilm was washed with sterile PBS and dosed at a concentration of 4-fold MIC for SDS, RHL, and NAC. After 24h incubation, SYTO9/PI solution was added in the dark and incubated for 15min, after which the dishes were placed under laser Confocal (CLSM) to observe the three-dimensional structure of the bifilm and the survival of the bacteria.

The effect of SDS, RHL and NAC in inhibiting bifilm reformation:

based on the MIC values of the three substances, a series of sample solutions of different concentrations, including the MIC value, were prepared and added to 48 well plates at 250. mu.L/well, followed by an equal amount of bacterial suspension with an OD 600 value of 0.1, and the plates were incubated at 37 ℃ for 24 h. The inhibition of the formation of the five bacteria, bifilm, by SDS, RHL and NAC was determined according to the crystal violet method described previously.

Results

MIC values for SDS, RHL, and NAC: the MIC values of SDS, RHL and NAC for five bacteria are shown in Table 6, and from the results, it can be seen that SDS has stronger antibacterial ability than RHL and NAC, and has the minimum MIC values for five bacteria. In addition, SDS and RHL have higher antibacterial activity against gram positive bacteria than gram negative bacteria, and RHL has no antibacterial effect even against e. The antibacterial effect of NAC is independent of the type of bacteria, and the MIC value of the NAC to five bacteria is 5-10 mg/mL.

TABLE 6 MIC values (mg/mL) of SDS, RHL and NAC for five bacteria

Clearance of bifilm by SDS, RHL and NAC: to investigate whether the ability of SDS, RHL and NAC to scavenge biofilm is broad-spectrum, we chose five different types of bacteria: respectively belonging to microaerophilic (h.pyri), aerobic (p.aeruginosa), facultative anaerobic (e.coli and s.aureus) and anaerobic (s.microorganisms), which in turn belong to gram-positive (s.aureus and s.microorganisms) and negative (h.pyri, e.coli and p.aeruginosa), by determining the scavenging action on different types of bacteria, bisilm, it is possible to more accurately and comprehensively explore the broad-spectrum anti-bisilm capacity of SDS, RHL and NAC. As shown in fig. 9, both SDS and RHL were effective in scavenging 5 mature biofilms, and therefore it is assumed that RHL anti-biofilm ability has broad-spectrum non-specificity, NAC has no scavenging effect on h. The results of the scanning electron microscope and the staining method of dead and live bacteria (figure 10, figure 11) are consistent with the crystal violet method, and both show that the capacity of SDS and RHL for removing the biofilm has broad spectrum.

The effect of SDS, RHL and NAC in inhibiting bifilm reformation: the ability of SDS, RHL, and NAC to inhibit the bifilm reformation of five bacteria is shown in fig. 12. Both SDS and RHL inhibited the formation of all five bacterial bisofilms with broad-spectrum non-specificity, whereas NAC promoted the formation of four other bacterial bisofilms except p. In conclusion, RHL has broad spectrum on the clearance and inhibition of the bifilm, and in combination with the broad-spectrum antibacterial activity of BD, the BD/RHL NPs constructed by the inventor are also supposed to be suitable for clearing other bacteria of the bifilm, thereby providing a new method for treating the bifilm infection.

The invention combines the effect of RHL on the bifilm and the effect of BD on the H.pyri, and constructs the self-assembled nanoparticles (BD/RHL NPs) formed by RHL and four BDs with different chain lengths. The BD/RHL NPs can penetrate through mucus layers to reach H.pylori infected sites to play a role in eliminating H.pylori bifilm, and can improve the H.pylori clearance rate. In addition, we demonstrate that RHL has broad-spectrum anti-bifilm capability, and since BD has broad-spectrum anti-bifilm capability, it is speculated that BD/RHL NPs in the invention can also eliminate other bacterial bifilm, and provide a new method for treating bifilm infection.

Claims

1. An antibacterial nanoparticle is formed by self-assembling berberine derivatives and rhamnolipid, wherein the berberine derivatives are modified to have C8-20 alkoxy substitution on the 9 th carbon atom.

2. An antibacterial nanoparticle according to claim 1, wherein the particle size of the nanoparticle is 100nm to 200nm as determined by laser granulometry and the Zeta potential is between-20 mV to-40 mV.

3. The antibacterial nanoparticle according to claim 1, wherein the berberine derivative is substituted with a C10-18 alkoxy group at the 9 th carbon atom.

4. An antibacterial nanoparticle according to claim 3, wherein the berberine derivative is 9-O-n-decaalkyl berberine, 9-O-n-dodecyl berberine, 9-O-n-hexadecyl berberine or 9-O-n-octadecyl berberine.

5. An antibacterial nanoparticle according to claim 1, wherein the weight ratio of berberine derivative to rhamnolipid in the nanoparticle is from 1:2 to 1: 10.

6. An antibacterial nanoparticle according to claim 5, wherein the weight ratio of berberine derivative to rhamnolipid in the nanoparticle is 1: 4.

7. The antimicrobial nanoparticle of claim 1, wherein the nanoparticle is capable of at least partially passing through a mucus layer overlying an epithelial cell layer.

8. A pharmaceutical composition comprising the antibacterial nanoparticle of any one of claims 1-7 and a pharmaceutically acceptable carrier.

9. Use of an antibacterial nanoparticle according to any one of claims 1 to 7 in the preparation of an antibacterial formulation, wherein the bacteria are biofilm-producing bacteria.

10. The use of claim 9, wherein the biofilm-producing bacteria are microaerophilic bacteria, aerobic bacteria, facultative anaerobes, or anaerobic bacteria.

11. The use of claim 9, wherein the biofilm-producing bacterium isH. pylori。