CN116392444A - Targeted porphyromonas gingivalis immunoliposome drug, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a targeted porphyromonas gingivalis immunoliposome drug, a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations containing antigens or antibodies. The preparation method comprises the following steps: 1) Preparing PEG modified liposome; 2) Coating antibacterial ginsenoside Rh2 by using PEG modified liposome and purifying; 3) Antibodies with the function of targeting porphyromonas gingivalis are coupled with liposome drugs and purified. The invention utilizes a liposome drug delivery system to carry out PEG modification to prolong the blood circulation time. The liposome drug-carrying system is used for wrapping the multifunctional antibacterial ginsenoside liposome Rh2, so that the cell-entering sterilization effect is achieved, and the liposome drug is coupled with the targeted porphyromonas gingivalis antibody, so that the targeted porphyromonas gingivalis can maintain the stability of flora, and the sterilization effect is enhanced.

Description

A kind of immunoliposome drug targeting Porphyromonas gingivalis and its preparation method and application

technical field

The invention relates to the technical field of pharmaceutical preparations containing antigens or antibodies, in particular to an immunoliposome drug targeting Porphyromonas gingivalis and its preparation method and application.

Background technique

The etiology of periodontitis is very complex, including the joint action of bacteria and cell inflammation. Years of research have found that Porphyromonas gingivalis plays a key role. As the key species of periodontitis, Porphyromonas gingivalis has direct and indirect effects on the occurrence and development of periodontal inflammation. Changes in the microbial community are induced by directly affecting the disruption of host immune responses and indirectly affecting interspecies interactions with other member species. The two effects are mutually reinforcing, with P. gingivalis able to trigger an inflammatory response that induces the generation of inflammatory bacteria and further manipulates the host immune response.

Porphyromonas gingivalis has an immunoregulatory effect on immune cells such as macrophages, and it can enter cells to damage various host defense mechanisms, such as inducing macrophage tolerance and making macrophages present a low immune response. Reactive state. In this state, lysosome production by macrophages is reduced, leading to inhibition of bactericidal activity and enhanced growth of the phagocytosed bacterium, Porphyromonas gingivalis. Even if Porphyromonas gingivalis enters the early phagosome of macrophages, it still has a corresponding immune escape mechanism to protect itself. By activating autophagy, Porphyromonas gingivalis makes itself flow to autophagosomes, and obtains nutrients such as host proteins as its own carbon and nitrogen sources. Since P. gingivalis is then able to leave the host cell and re-enter adjacent cells, macrophage-dependent clearance may instead facilitate immune escape.

At the same time, Porphyromonas gingivalis, as a key species of periodontitis, only needs a low concentration to change the relative abundance of individual members of the bacterial community, thereby affecting disease progression. In recent years, studies have found that periodontitis is not an infectious disease caused by a certain type of microorganisms, but is related to the abundance and influence of each species in the complex microbial community, as well as the influence of the key species of the disease on the healthy flora. Abundance is related to impact. Contrary to the findings of earlier culture-based microbiological studies, most recent studies using molecular approaches such as culture-free sequencing found that P. gingivalis was quantitatively only a minor component of human periodontitis-associated biofilms . Despite its low abundance, P. gingivalis can have a large impact on microbial community structure that is disproportionate to its species abundance. Porphyromonas gingivalis has good adaptability in the human oral cavity, breaking the priority effect of the original microecology. Porphyromonas gingivalis participates in synergy with many other organisms in the oral microbiome by co-accumulating with other bacteria and alters the gene expression patterns of the microbiome. Therefore, Porphyromonas gingivalis can act as a key species of periodontitis, which has a much greater impact on the microbial community than theoretically expected, resulting in the transformation of plaque from healthy flora to pathological flora. A study found that P. gingivalis and total subgingival bacterial loads decreased after P. gingivalis vaccination in nonhuman primates naturally harboring P. gingivalis, suggesting that P. bacteria have a synergistic relationship with the entire disease-associated microbial community.

Chinese patent CN112274638A discloses a specific anti-Porphyromonas gingivalis egg yolk antibody liposome solution and a preparation method, each mL of the egg yolk antibody liposome solution contains 4mg egg yolk antibody liposome, the egg yolk antibody The liposomes are multi-lamellar liposomes; the yolk antibody liposome solution is fluid below room temperature, and forms a gel at a temperature of 37°C, which meets the requirements of injection administration in the periodontal pocket and gelation in vivo Requirements, effectively improve the concentration in the periodontal pocket, reduce the dosage. The invention utilizes lecithin and cholesterol as raw materials, tocopherol as a stabilizer, and adopts a film dispersion method to prepare a liposome film; then wraps the liposome film with yolk antibody to prepare yolk antibody liposome capsules, and finally utilizes a homogenizer The formed liposome vesicles are restructured to obtain a specific anti-Porphyromonas gingivalis egg yolk antibody liposome solution, wherein the egg yolk antibody liposome is a nanoscale liposome.

Chinese patent CN115607511A then provides a kind of resveratrol liposome, its preparation method and its application, and described liposome comprises following raw materials: egg yolk lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and resveratrol; wherein, the ratio of egg yolk lecithin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 is (49～52):(4～6):(2.5～3)(w /w), more preferably 20:2:1 (w/w); wherein, the resveratrol is encapsulated in liposomes. The resveratrol liposome of the invention has good stability in vitro, and has a slow-release effect compared with free drugs, and has clinical application prospects in the field of periodontal inflammatory disease treatment.

In terms of drug selection, it is very important to use drugs that take into account both anti-inflammatory and bactericidal characteristics. The existing technology has a single function, and the ability to enter cells to regulate immune responses and eliminate intracellular bacteria is poor. At the same time, the bioavailability of its active ingredients is low, the water solubility and cell permeability are insufficient, and the efficacy is limited.

Contents of the invention

In view of the above-mentioned defects of the prior art, in the first aspect of the present invention, the technical problem to be solved is to provide a targeted Porphyromonas gingivalis immunoliposome drug, which targets Porphyromonas gingivalis The immunoliposome drug includes PEG modified liposome, ginsenoside Rh2 encapsulated in the PEG modified liposome, and antibodies coupled on the surface of the PEG modified liposome.

Preferably, the PEG-modified liposome is a liposome containing polyethylene glycol (PEG) fragments.

Preferably, the antibody is an antibody with the function of targeting Porphyromonas gingivalis; preferably an antibody against Porphyromonas gingivalis.

In the second aspect of the present invention, the present invention provides a method for preparing the immunoliposome drug targeting Porphyromonas gingivalis described in the first aspect of the present invention, comprising the following steps:

Step 1) preparation of PEG-modified liposomes;

Step 2) using PEG-modified liposomes to coat the antibacterial drug ginsenoside Rh2 and purifying to obtain a liposome-Rh2 complex;

Step 3) Conjugate antibody to liposome-Rh2 complex and purify.

Preferably, the specific method of step 1) is as follows: the liposome raw material is dissolved in a solvent, and the rough liposome is obtained after drying to remove the solvent; the rough liposome is added to a buffer for hydration, and the rough The liposomes are compressed, further shaped, and finally extruded to obtain uniform PEG-modified liposomes for later use.

Further preferably, in the step 1), the liposome raw material is dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE) Preferably, the molar ratio of said dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE) is (1～10): (1 ~1.36): (0.2~5).

Further preferably, in the step 1), the solvent is a mixture of methanol and chloroform at a molar ratio of 1:(0.9-1.1).

Further preferably, in the step 1), the buffer is 10mM Hepes/135mM NaCl (pH7.4) buffer.

Preferably, the specific method of step 2) is as follows: add ginsenoside Rh2 dissolved in a solvent to the PEG-modified liposome and incubate, and obtain a liposome-Rh2 complex after purification, which is ready for use.

Further preferably, in the step 2), the solvent is dimethyl sulfoxide (DMSO).

Further preferably, in the step 2), the molar ratio of the ginsenoside Rh2 to the PEG-modified liposome is (10-18):1.

Preferably, the specific method of step 3) is as follows: the antibody is mixed with a thiol reagent and incubated to form a protective sulfhydryl group; after incubation, it is mixed with the liposome-Rh2 complex and performed overnight in an oxygen-free environment Incubate to form a thioether bond to obtain antibody-coupled liposomes; remove ginsenoside Rh2 and antibodies that have not been successfully encapsulated in the antibody-coupled liposomes to obtain immunity targeting Porphyromonas gingivalis Liposomal drugs.

Further preferably, in the step 3), the molar ratio of the antibody to the thiolation reagent is 1: (80-90); when mixed with the liposome-Rh2 complex after incubation, the antibody and the lipid The DAR value of plastid-Rh2 complex was 25-37.

Further preferably, in the step 3), the thiolating agent is N-succinimidyl-S-acetylthioacetate (SATA).

Preferably, the incubation conditions are independently incubating at 37±5° C. for 45-180 min.

In the third aspect of the present invention, the present invention provides an application of the immunoliposome drug targeting Porphyromonas gingivalis described in the first aspect of the present invention in the preparation of a drug for treating periodontitis.

The introduction and effect of part raw material in the formula of the present invention are as follows:

Dipalmitoylphosphatidylcholine (DPPC): a synthetic phospholipid widely used in the field of pharmaceutical preparations. As an excipient, it can be used in liposomes or temperature-sensitive liposomes, as well as special lipid microspheres, drug complexes and other pharmaceutical preparations.

Cholesterol: A lipophilic substance with both lipophilic and hydrophilic groups. It is more lipophilic than hydrophilic. When it interacts with phospholipids to form liposomes, it can be embedded between phospholipid molecules, adjust the structure and properties of membranes, and improve the function of liposomes. At the same time, it can regulate the fluidity of the phospholipid bimolecular membrane, reduce the permeability of the membrane, and reduce drug leakage. At the same time, it can maintain the flexibility of the lipid membrane and enhance the ability of liposome vesicles to resist changes in external conditions. It protects phospholipids from oxidation. Cholesterol can regulate the phase transition of phospholipids.

Phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE): commonly used in the synthesis of liposomes. Polyethylene glycol (PEG) coupled DSPE is hydrophilic and can be used for drug delivery, gene transfection and biomolecular modification. PEGylation of phospholipids significantly improves the blood circulation time and stability of capsule drugs.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The present invention provides an immunoliposome drug targeting Porphyromonas gingivalis, the active ingredient of which has high bioavailability, good water solubility and cell permeability. Using the liposome drug delivery system, it is modified by PEGylation to prolong the blood circulation time. Using this liposome drug-loading system to encapsulate the multifunctional antibacterial drug ginsenoside liposome Rh2 overcomes the penetration limitation of the cell barrier, can more effectively anti-inflammatory and kill intracellular bacteria, and combine the liposome drug with targeted Porphyromonas gingivalis antibody conjugate can target Porphyromonas gingivalis to maintain the stability of the flora and enhance the bactericidal effect. In addition, the immunoliposome drug targeting Porphyromonas gingivalis uses targeted liposome as the drug carrier, which has slow release, has attenuating effect on cells, and can avoid drug resistance to bacteria.

2. The present invention provides a method for preparing immunoliposome drugs targeting Porphyromonas gingivalis. Through the optimization of liposome preparation, drug coating, antibody coupling and other production and purification steps, a A preparation method of a novel immunoliposome drug targeting Porphyromonas gingivalis, which is safe and efficient, and has good development and application prospects.

3. The present invention provides the application of an immunoliposome drug targeting Porphyromonas gingivalis in the preparation of a drug for treating periodontitis. Compared with free drug, the drug potency of liposome-encapsulated drug will be significantly increased. For extracellular flora-targeted liposomes, flora homeostasis can be maintained, and the curative effect can be improved to achieve the effect of using lower concentrations of highly toxic drugs. Since the abundance of Porphyromonas gingivalis is not high as a key species, reducing it can effectively reduce the harmful bacteria that grow with it and protect the beneficial bacteria at the same time. Targeted therapy can improve the efficiency of controlling the flora, thereby reducing drug toxicity.

Description of drawings

Figure 1 is a schematic diagram of the construction of an immunoliposome drug targeting Porphyromonas gingivalis;

Among Fig. 2, figure A is the Fourier transform infrared detection figure of the pure PEG modified liposome of embodiment 3, and figure B is the Fourier transform infrared detection figure of experimental group 3 in embodiment 1;

Fig. 3 is the A-L-R drug electron microscope characterization figure of experimental group 3 in embodiment 1;

Fig. 4 is pure Rh2 medicine, the A-L-R medicine of experimental group 3 in embodiment 1, the L-R medicine hemolysis experiment detection figure of embodiment 2;

Fig. 5 is the antibody sensitivity and specificity detection result of experimental group 3 in embodiment 1;

Fig. 6 is pure Rh2 medicine, the A-L-R medicine of experimental group 3 in embodiment 1, the antibacterial growth detection result of the L-R medicine of embodiment 2;

Fig. 7 shows the detection results of the bactericidal effect on intracellular bacteria of the pure Rh2 drug, the A-L-R drug of the experimental group 3 in Example 1, the L-R drug of Example 2, and the A-L drug of Example 3.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Example 1

Preparation method of immunoliposome drug targeting Porphyromonas gingivalis:

Step 1) Dissolve dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE) powder in methanol and chloroform at a molar ratio of 1:1 In the solvent prepared by mixing, rough liposomes were obtained after drying to remove the solvent; adding 10mM Hepes/135mM NaCl (pH7.4) buffer solution to the rough liposomes for hydration, and using a 0.45 μm filter membrane after ultrasonic treatment Compress the rough liposomes, then pass through a 0.22 μm filter membrane for further shaping, and finally squeeze through a 0.11 μm filter membrane to obtain uniform PEG-modified liposomes for later use;

Step 2) adding ginsenoside Rh2 dissolved in a solvent to the PEG-modified liposome and incubating at 37±5° C. for 45 min, the molar ratio of ginsenoside Rh2 to PEG-modified liposome is (10-18) : 1, the liposome-Rh2 complex was obtained after purification, for subsequent use;

Step 3) 66 μL of anti-Porphyromonas gingivalis antibody at a concentration of 0.3 nmol/μL was mixed with 8.4 μL of N-succinimidyl-S-acetylthioacetate at a concentration of 200 nmol/μL and incubated, Incubate at 37±5°C for 45min to form a protective sulfhydryl group; mix with 1mL of the liposome-Rh2 complex after incubation, and incubate overnight at 37±5°C in anaerobic environment to form a thioether bond to obtain Antibody-coupled liposomes; ultrafiltration was performed using a 100kDa ultrafiltration tube to remove ginsenoside Rh2 and anti-Porphyromonas gingivalis antibodies that were not successfully encapsulated in the antibody-coupled liposomes to obtain the target Immunoliposome drug delivery to Porphyromonas gingivalis.

In this example, the abbreviation of the immunoliposome drug targeting Porphyromonas gingivalis is marked as A-L-R.

Table 1 Differences in preparation of immunoliposome drugs targeting Porphyromonas gingivalis in experimental groups 1-10

Example 2

The preparation method of liposome-Rh2 complex body:

Step 1) Dissolve the powder of dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE) in methanol and Chloroform is in the solvent of 1:1 mixed preparation with molar ratio, obtains rough liposome after drying and removing solvent; After ultrasonic treatment, use a 0.45 μm filter membrane to compress the rough liposomes, then pass through a 0.22 μm filter membrane for further shaping, and finally squeeze through a 0.11 μm filter membrane to obtain uniform PEG-modified liposomes, which are ready for use;

Step 2) Add ginsenoside Rh2 dissolved in dimethyl sulfoxide to the PEG-modified liposome and incubate at 37±5° C. for 45 min, the molar ratio of ginsenoside Rh2 to PEG-modified liposome is 14 : 1, the liposome-Rh2 complex was obtained after purification.

The liposome-Rh2 complex obtained in this example is abbreviated as L-R.

Example 3

The preparation method of PEG modified liposome:

Dissolve dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE) powder in methanol and chloroform at a molar ratio of 1:1:5 Ratio is 1:1 mixed preparation solvent, after drying and removing solvent, obtain rough liposome; Add 10mM Hepes/135mM NaCl (pH7.4) buffer solution to carry out hydration to described rough liposome, after sonication Rough liposomes were compressed using a 0.45 μm filter membrane, then passed through a 0.22 μm filter membrane for further shaping, and finally extruded through a 0.11 μm filter membrane to obtain uniform PEG-modified liposomes.

The PEG-modified liposomes obtained in this example are abbreviated as A-L.

Example 4

Detection of immunoliposome drugs targeting Porphyromonas gingivalis

1. Detection of liposome drug loading and encapsulation efficiency

Use high-performance liquid chromatography (Shimadzu Instrument Co., Ltd., Japan, LC-20AT) to detect drug loading rate, encapsulation efficiency, DAR value and ensure that free antibodies and drugs have been removed. The calculation formulas of drug loading rate and encapsulation efficiency are respectively: drug loading rate = pure Rh2 drug amount in supernatant/material weight × 100%, encapsulation efficiency = pure Rh2 drug amount in supernatant/total amount of pure Rh2 drug × 100% .

Table 2 Drug loading and DAR of immunoliposome drugs targeting Porphyromonas gingivalis

test group DAR value Drug loading(%) 1 11.593 3.450 2 25.618 76.925 3 30.851 82.491 4 31.655 75.006 5 37.387 64.428 6 38.317 67.115 7 40.371 77.336 8 47.353 77.104

The optimal drug loading can be obtained when the molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol, phospholipid-polyethylene glycol-maleimide (mal-PEG-DSPE) is 1:1:5 And a higher DAR value; at this time, the molar ratio of ginsenoside Rh2 and PEG-modified liposome is preferably 14:1, which can obtain the best drug loading, and the encapsulation efficiency is also relatively high at 20.69%.

2. Liposome infrared spectrum detection

A Fourier transform infrared spectrometer (Mettler-Toledo Instrument Co., Ltd., USA, ReactlR4000) was used to detect the infrared absorption spectrum of the drug. As shown in Figure 2, in the Fourier transform infrared detection images of pure PEG-modified liposomes and ALR drugs, there are antisymmetric and symmetrical stretching vibration peaks of phospholipid fatty phthalein chains at about 2920cm ^-1 and 2850cm ^-1 , Antibodies were successfully coupled to liposomes without damaging the phospholipid fatty acid chains that are characteristic of liposomes.

3. Liposome nanoparticle size detection

The particle size and Zeta value were detected by a nanometer particle size potentiometer (Malvern Instrument Co., Ltd., UK, Zetasizer Nano ZSP). The A-L-R drug has a particle size of less than 100nm in aqueous solution and a Zeta charge of -3.78.

4. Liposome electron microscopy detection

Use a transmission electron microscope (Hitachi H-7000FA, Hitachi Scientific Instrument Co., Ltd., Japan) to observe the particle shape and particle size observation: place it on a copper grid covered with nitrocellulose, control negative staining/positive staining at different times with phosphotungstic acid, and dry at room temperature. Electron microscope characterization of A-L-R drugs, as shown in Figure 3, A-L-R drugs that have been prepared through 100nm filtered and compressed liposomes were negatively stained by transmission electron microscopy and showed a homogeneous circle at about 52.99nm, with slightly rough edges.

5. Liposome Cytotoxicity Detection

When the growth state was good, 1×10 ⁵ /mL macrophage RAW264.7 or human gingival fibroblast HGF were inoculated in 100 μL of 96-well plate to make them evenly adhere to the wall, and then the corresponding concentration of drugs were added for co-culture for 12 hours. Add 10 μL of CCK-8 solution to each well, place the plate in a microplate reader to detect the absorbance at 450 nm, and record the optical density value. Cell viability (cell viability, %) calculation formula: [absorbance of test solution-absorbance of blank medium]/[absorbance of negative control group-absorbance of blank medium]×100. After adding ALR drugs, the cell viability remained above 80%.

6. Liposome hemolysis detection

Sterile blood was collected from the heart of the mouse, and immediately after the blood was drawn, it was carefully poured into a sterile dry glass bottle with glass beads, and fully swirled for 15-20 minutes to remove fibrin and produce anticoagulant blood. Take 2 mL of sterile normal saline and add 0.25 mL of anticoagulant blood, centrifuge at 10,000×g for 15 min, remove the supernatant, and repeat the above centrifugation until the supernatant is transparent to obtain precipitated red blood cells. Red blood cells were resuspended to obtain 2% red blood cells, and 0.5 mL of 2% red blood cells were added to 0.5 mL of the test solution, H ₂ O positive group, and normal saline negative group, respectively, and incubated at 37°C for 24 hours. After centrifugation at 750rpm for 5min, the supernatant was taken to detect the absorbance at 540nm with an ultraviolet spectrometer, and the optical density value was recorded. At this time, the OD ₅₄₀ of the positive control group was 0.8±0.3, and the formula for calculating the hemolysis rate was [(absorbance of the test solution-absorbance of the negative control group)/(absorbance of the positive control group-absorbance of the negative control group)]×100. Through the hemolysis experiment, it can be observed that the hemolysis is strong, and the hemolysis rate of the ALR drug in Figure 4 is less than 5%, and the blood compatibility is good. ALR drugs effectively reduce the toxicity of pure Rh2 drugs and have better biocompatibility.

Example 5

Detection of bactericidal function of immunoliposome drug targeting Porphyromonas gingivalis

Add 1×10 ⁹ Porphyromonas gingivalis, Enterococcus faecalis, and Streptococcus sanguinis to 100 μL per well of a 96-well plate, add 100 μL of 2X ELISA coating solution, and place overnight at 4°C. After removing the coating solution, wash 3 times with PBST, add 200 μL of 5% BSA and place overnight at 4°C. After removing the blocking solution, wash with PBST 3 times, add 100 μL of the antibody/drug to be tested, and incubate at 37°C for 1.5 hours. After washing with PBST for 3 times, 100 μL of diluted HRP-labeled secondary antibody was added and incubated at 37°C for 1 h. After washing with PBST for 3 times, add 100 μL of TMB chromogenic reagent that is currently prepared, and incubate at 37°C for 15 min. Add 100 μL of stop solution, mix well and put it in a microplate reader to detect the absorbance at 450 nm, and record the optical density value. Both MIgG1 (serotype B-specific cell surface antigen antibody) and MIgG2a (anti-gingivalin antibody) can effectively target Porphyromonas gingivalis, and MIgG1 antibody is slightly more sensitive than MIgG2a antibody. Therefore, an ALR drug is prepared by using MIgG1 antibody. As shown in Figure 5, the ELISA test showed that the MIgG1 antibody and the ALR drug prepared using the MIgG1 antibody effectively and specifically bind Porphyromonas gingivalis, and have no binding to healthy commensal bacteria Enterococcus faecalis and Streptococcus sanguis. ALR drugs have good target specificity.

1. Detection of antibacterial growth effect

Take Porphyromonas gingivalis, Enterococcus faecalis, and Streptococcus sanguinis and add 100 μL per well of a 96-well plate, and at the same time add 100 μL of drugs of corresponding concentration, culture them in a complete anaerobic tank at 37°C for 24 hours, and then take samples and put them in a microplate reader Detect the absorbance at 600nm and record the optical density value. Pure Rh2 drugs can effectively remove part of Porphyromonas gingivalis, Streptococcus sanguinis and Enterococcus faecalis. As shown in Figure 6, the A-L-R drug only had a bactericidal effect on Porphyromonas gingivalis, but had no obvious bactericidal effect on Streptococcus sanguinis and Enterococcus faecalis.

2. Detection of bactericidal effect on intracellular bacteria

1×10 ⁶ macrophages were added to a 6-well plate, and Porphyromonas gingivalis was added to co-culture with the cells at an MOI of 100. At the same time, corresponding concentrations of drugs were added, and they were placed in a cell culture incubator for 12 hours. Take 10 μL of extracellular bacteria in the culture medium and smear the plate, and count the CFU. Then remove the medium, wash with preheated PBS for 3 times, add high-sugar DMEN medium containing metronidazole, gentamycin and double antibody (penicillin-streptomycin) and continue to cultivate for 1h to kill extracellular bacteria. Then the culture medium was discarded, washed 3 times with PBS, the cells were lysed with sterile water for 15 min, 10 μL was taken to smear the plate, and the CFU was counted. Porphyromonas gingivalis was co-cultured with macrophages, and bacteria entering the cells were successfully detected and found at 12 hours. Compared with pure Rh2, the co-culture of LR and ALR drugs and cells can effectively kill more intracellular and extracellular bacteria, especially significantly reduce Porphyromonas gingivalis in macrophages. As shown in Figure 7, carrier AL had no effect on bacterial growth, and even promoted bacterial entry into the cell.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (10)

1. A porphyromonas gingivalis-targeted immunoliposome drug, characterized in that: the targeted porphyromonas gingivalis immunoliposome drug comprises PEG modified liposome, ginsenoside Rh2 wrapped in the PEG modified liposome and an antibody coupled to the surface of the PEG modified liposome.

2. The targeted porphyromonas gingivalis immunoliposome drug of claim 1, wherein: the antibody is an anti-Porphyromonas gingivalis antibody.

3. A method for preparing a porphyromonas gingivalis-targeted immunoliposome drug according to claim 1 or 2, comprising the steps of:

step 1) preparation of PEG modified liposome;

step 2), coating antibacterial ginsenoside Rh2 by using PEG modified liposome and purifying to obtain a liposome-Rh 2 complex;

step 3) the antibody is coupled to a liposome-Rh 2 complex and purified.

4. The method for preparing a porphyromonas gingivalis immunoliposome-targeted drug according to claim 3, wherein the specific method of step 1) is as follows:

dissolving liposome raw materials in a solvent, and drying to remove the solvent to obtain coarse liposome; and adding the coarse liposome into a buffer solution for hydration, compressing and further shaping the coarse liposome after uniform dispersion, and finally extruding to obtain uniform PEG modified liposome for later use.

5. The method for preparing the immune liposome drug targeting porphyromonas gingivalis according to claim 4, which is characterized in that: in the step 1), the liposome raw materials are dipalmitoyl phosphatidylcholine, cholesterol, phospholipid-polyethylene glycol-maleimide, and the mole ratio of the dipalmitoyl phosphatidylcholine, the cholesterol and the phospholipid-polyethylene glycol-maleimide is (1-10): (1-1.36): (0.2-5); the solvent is methanol and chloroform with a mole ratio of 1: (0.9-1.1) a mixture; the buffer was 10mM Hepes/135mM NaCl buffer pH=7.4.

6. The method for preparing a porphyromonas gingivalis immunoliposome-targeted drug according to claim 3, wherein the specific method of the step 2) is as follows:

adding ginsenoside Rh2 dissolved in a solvent into the PEG modified liposome, incubating, and purifying to obtain liposome-Rh 2 complex for later use.

7. The method for preparing the immune liposome drug targeting porphyromonas gingivalis according to claim 6, wherein the method comprises the following steps: in the step 2), the solvent is dimethyl sulfoxide; the molar ratio of the ginsenoside Rh2 to the PEG modified liposome is (10-18): 1, a step of; the incubation condition is that the incubation is carried out for 45-180 min at 37+/-5 ℃.

8. The method for preparing a porphyromonas gingivalis immunoliposome-targeted drug according to claim 3, wherein the specific method of the step 3) is as follows:

mixing and incubating the antibody with a thiolating agent to form a protective thiol; mixing the liposome-Rh 2 complex after incubation, and incubating overnight in an anaerobic environment to form thioether bonds to obtain antibody-coupled liposome; and removing the ginsenoside Rh2 which is not successfully wrapped in the antibody-coupled liposome and the antibody to obtain the targeted porphyromonas gingivalis immunoliposome drug.

9. The method for preparing the immune liposome drug targeting porphyromonas gingivalis according to claim 8, wherein the method comprises the following steps: in the step 3), the molar ratio of the antibody to the thiolating agent is 1: (80-90); the thiolating reagent is N-succinimidyl-S-acetylthioacetate; when the antibody is mixed with the liposome-Rh 2 complex after incubation, the DAR value of the antibody and the liposome-Rh 2 complex is 25-37; the incubation condition is that the incubation is carried out for 45-180 min at 37+/-5 ℃.

10. Use of a targeted porphyromonas gingivalis immunoliposome drug according to claim 1 or prepared by the preparation method of any one of claims 2-9 in the preparation of a medicament for treating periodontitis.

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