Preparation method of lauroyl selective antibacterial lipid for probiotic delivery for treating enteritis and colorectal cancer
By encapsulating probiotics with lauroyl lipid molecules to form Cb@S12 nanoparticles, the problem of selectively killing nucleated Clostridium nucleatum was solved, achieving therapeutic effects on colitis and colorectal cancer, while protecting the growth of Clostridium butyricum.
Patent Information
- Application Number
- CN202411506233.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies struggle to selectively kill Clostridium nucleatum (Fn) to treat colitis and colorectal cancer without affecting the growth and activity of beneficial Clostridium butyricum (Cb).
By using lauroyl lipid molecules to encapsulate probiotics through positive and negative charge interactions, forming nanoparticles Cb@S12, selective killing of Clostridium nucleatum and protection of Clostridium butyricum growth are achieved.
It effectively kills Clostridium nucleatum, reduces its adhesion and colonization in colon cancer, reduces inflammation and promotes tumor proliferation, and does not affect the growth of Clostridium butyricum, thus showing significant therapeutic effects.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug carrier materials and relates to a probiotic delivery system encapsulated with lauroyl lipid molecules and its application. Specifically, it relates to the construction and application of a drug delivery system using lauroyl lipid molecules. The lauroyl drug delivery system can be used as a probiotic-encapsulated drug delivery system. Background Technology
[0002] Colitis and colorectal cancer are common intestinal diseases. Studies have shown that the gut microbiota plays an important role in the development and progression of colitis and digestive system malignancies, especially *Fusobacterium nucleatum* (Fn), which can exacerbate colitis symptoms and has been significantly enriched in patients with digestive tract malignancies, including oral cancer, esophageal cancer, and colorectal cancer. It participates in the progression of related digestive system malignancies through various mechanisms, such as adhesion and colonization, inducing inflammation, suppressing immunity, promoting tumor proliferation, and enhancing tumor invasion, and indicates poor prognosis. *Clostridium butyricum* (Cb) is a beneficial gut bacterium that can effectively treat colitis and colorectal cancer. However, if antibiotics are used to suppress the pathogen *Fusobacterium nucleatum*, the beneficial gut bacteria will also be affected by the antibiotics. Therefore, a selective antimicrobial material is needed to deliver probiotics, which can replace antibiotics to eliminate pathogens without interfering with the therapeutic effect of the beneficial gut bacteria. Summary of the Invention
[0003] In view of the above-mentioned technical analysis and existing problems, the present invention provides a probiotic delivery system encapsulated by lauroyl lipid molecules and its application. The preparation method is simple, easy to implement and has low preparation cost.
[0004] The technical solution of this invention:
[0005] A probiotic delivery system encapsulating lauroyl lipid molecules and its application are disclosed. The nanomaterial uses spermine as a backbone and covalently binds with lauric acid to synthesize lauroyl lipid molecules with positively charged amino groups. The probiotics are then encapsulated by the positive and negative charge interactions of the liposomes. The preparation steps are as follows:
[0006] 1) The outer amino group of spermine was protected with ethyl trifluoroacetate, and the unprotected amino group was modified with lauroyl chloride. Further, under alkaline solution conditions, the protection of the outer amino group by the trifluoroacetate group was removed, ultimately preparing N... 4 N 9 -Dilauranamide-spermine (S12). The structural formula of S12 is as follows.
[0007]
[0008] 2) Dissolve S12, cholesterol, distearylphosphatidylcholine, and distearylphosphatidylethanolamine-methoxy polyethylene glycol in methanol or chloroform in an appropriate ratio, and suspend in a glass bottle to obtain a lipid thin layer. Add the bacterial solution of Clostridium butyricum resuspended in PBS to the glass bottle with the lipid thin layer and vortex for 10 minutes. The positive and negative charges of the liposomes interact with the surface of Clostridium butyricum to form a probiotic coating.
[0009] 3) P12 is prepared by amidation of 1,4-bis(3-aminopropyl)piperazine with a lauroyl group. N12 is prepared by amidation of the remaining two amino groups of tris(2-aminoethyl)amine with a Boc-protected monoamino group, followed by lauroyl group linkage and de-Boc protection. The structural formula of N12 is as follows:
[0010]
[0011] The structure of P12 is as follows:
[0012]
[0013] The advantages of this invention are: by utilizing the interaction of positively charged lipids with positive and negative charges to encapsulate probiotics, the probiotics can be delivered orally to the intestinal tract for colonization, thereby achieving an inhibitory effect on enteritis and colon cancer. Furthermore, lauroyl lipids have a significant killing and inhibitory effect on the growth of Fusobacterium nucleatum, effectively killing Fn without affecting the growth of probiotics, thereby reducing the adhesion and colonization of Fusobacterium nucleatum in colon cancer, inducing inflammation, suppressing immunity, promoting tumor proliferation, and enhancing tumor invasion. Attached Figure Description
[0014] 1) Figure 1 This diagram illustrates the design, preparation, and mechanism of action of lipid S12 nanoparticles.
[0015] 2) Figure 2 The 1H NMR spectrum and mass spectrum of S12 are shown.
[0016] 3) Figure 3 The images show the 1H NMR and mass spectra of lipid P12.
[0017] 4) Figure 4 The images show the 1H NMR and mass spectra of lipid P12.
[0018] 5) Figure 5 Data on the selective antimicrobial activity of lipids S12, N12, and P12.
[0019] 6) Figure 6 In vivo imaging of Cb and Cb@S12 in the mouse intestine via IVIS.
[0020] 7) Figure 7 To investigate the changes in colon length and body weight in mice of different treatment groups during an in vivo experiment of Fn-induced aggravated colitis.
[0021] 8) Figure 8 In an in vivo experiment of Fn-associated CRC tumors, tumor images of mice in different treatment groups, as well as changes in mouse weight and survival rate. Detailed Implementation
[0022] Appendix Figure 1 The design and mechanism of action of the nanoparticles of the present invention are given. As can be seen from the figure, the present invention uses Clostridium butyricum as a carrier to load lauroyl lipid molecules S12, which are assembled into Cb@S12 through positive and negative charge attraction interactions.
[0023] The following are specific examples illustrating the present invention:
[0024] 1. Lauroyl lipid molecule N 4 N 9 Synthesis of dilauranamide spermine (S12)
[0025] The primary amino functional group of spermine was protected with ethyl trifluoroacetate (2.2 equivalents) in methanol, and the reaction mixture was stirred at 25°C for 18 hours. After the reaction was complete, the solvent was removed by rotary evaporation. The residue was dissolved in a 1:1 (v / v) mixture of dichloromethane and methanol, and then lauroyl chloride (2.2 equivalents) and triethylamine (2.5 equivalents) were added to the reaction system. The reaction was carried out at low temperature for 4 hours, and the solvent was removed again by rotary evaporation. The protection of the trifluoroacetyl group was removed by treatment with 1 mol / L NaOH in an aqueous methanol solution, followed by rapid column chromatography to obtain S12(N 4 N 9 - Dilauranamide spermine) (Mobile phase: dichloromethane: methanol: concentrated ammonia 25:10:1 v / v / v R f =0.40). Before use, the purified S12 was mixed with a 1 mol / L hydrochloric acid-methanol solution and precipitated at 0 °C. The precipitate was separated by centrifugation at 4000 rpm / min. The target product was dissolved in deuterated chloroform and its 1H NMR spectrum was measured at 25 °C on a 400 MHz spectrometer. Its structure was further confirmed by mass spectrometry. (Appendix) Figure 2 )
[0026] 2. Synthesis of the lauroyl lipid molecule 1,4-bis(3-lauroamidopropyl)piperazine (P12)
[0027] Lauric acid (10 mmol), EDC (10 mmol), and NHS (10 mmol) were dissolved in 20 mL of DCM and stirred at room temperature for 30 minutes. Then, 1,4-bis(3-aminopropyl)piperazine (4.5 mmol) was added. The mixture was stirred at room temperature for 16 hours. The solvent was then removed under vacuum. The crude product P12 was purified by column chromatography using DCM / MeOH (10:1) as eluent. The target product was dissolved in deuterated chloroform and its 1H NMR spectrum was measured at 25 °C on a 400 MHz spectrometer. Its structure was further confirmed by ESI-MS. (Appendix) Figure 3 )
[0028] 3. Synthesis of lauroyl lipid molecule 2-(bis(2-lauroamidoethyl)ethylenediamine (N12)
[0029] Tris-(2-aminoethyl)amine (5.1 mL, 35 mmol) was dissolved in 30 mL of dichloromethane, and a solution of di-tert-butyl dicarbonate (1.2 mL, 5.5 mmol) in DCM (30 mL) was added dropwise over 1 hour at room temperature. The reaction mixture was stirred for 17 hours. The solvent was then removed under vacuum, and the residue was dissolved in water (10 mL). The aqueous solution was extracted with dichloromethane (6 × 15 mL), and the combined organic phases were evaporated to give the intermediate Mono-Boc-tern (1.247 g, 92%). Lauric acid (10 mmol) was dissolved in 20 mL of dichloromethane with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (10 mmol) and N-hydroxysuccinimide (10 mmol), and Mono-Boc-tern (4.5 mmol) was added after stirring for 30 minutes at room temperature. The resulting mixture was stirred at room temperature for 16 hours, and the solvent was concentrated under vacuum. The crude product N12-Boc was purified by column chromatography using DCM / MeOH (20:1) as eluent. 3 mL of a 4 mol / L HCl solution in dioxane was added to the intermediate N12-Boc (0.1 mmol) at 0 °C, and the mixture was stirred at room temperature for 3 hours. The solvent was then removed under vacuum, and the product N12 was purified by column chromatography using DCM / MeOH (10:1) as eluent. The target product was dissolved in deuterated chloroform, and its 1H NMR spectrum was measured at 25 °C using a 400 MHz spectrometer. Its structure was further confirmed by ESI-MS. (Appendix) Figure 4 )
[0030] 4. Selective antibacterial test
[0031] Overnight cultured bacteria Cb and Fn were diluted separately into glass tubes with 1 mL of PBS (pH = 7.4). The OD values of each test bacterium were then calculated. 600 Adjust to 0.01, which corresponds to approximately 10. 7 CFU mL-1 The concentrations were then determined. To test the antibacterial activity of the nanocomposite, aqueous solutions of N12, P12, and S12 were added to the prepared bacterial suspension, respectively. All test tubes were then shaken at 37°C and 170 rpm for 24 hours. Subsequently, the bacterial suspension was removed, its minimum inhibitory concentration (MIC) was determined, and the bacterial suspension was diluted with an appropriate dilution factor. 50 μL of the diluted bacterial suspension was plated on a solid agar plate and incubated at 37°C for 24 hours to determine if the bacteria were completely killed and to determine its minimum bactericidal concentration (MBC). (Appendix) Figure 5 )
[0032] 5. Assembly of nanoparticles
[0033] Purified lipid S12, cholesterol, and DSPE mPEG were added. 2000 Dissolved in 2 mL of chloroform at a molar ratio of 16:4:1. The resulting film containing 50 μg S12 was then dissolved in 1 mL of Clostridium butyricum solution (OD200). 600 =0.5) Hydrate, vortex for 15 minutes, and then store at 4°C for later use.
[0034] 6. In vivo intestinal retention experiment
[0035] To analyze the differences in the in vivo distribution of Cb and Cb@S12 and determine the encapsulation effectiveness of Cb@S12, BLAB / c mice were randomly divided into two groups (n=15 in each group). After a 12-hour starvation period, mice in both the Cb and Cb@S12 groups were fed DiR-labeled Cb (1×10⁻⁶). 8 CFU). Mice in each group were sacrificed at 3, 6, and 12 hours after administration to collect intestinal tissue for IVIS (Perkin-Elmer) in vivo imaging. (Appendix) Figure 6 )
[0036] 7. In vivo treatment of Fn-exacerbated DSS-induced colitis
[0037] Male BLAB / c dogs aged 6-8 weeks were administered 0.2 mL of Fn (1×10⁻⁶) orally via gavage. 8 Mice were given CFU / mouse or 0.2 mL PBS for one week, after which their drinking water was replaced with 3% DSS or sterile drinking water. Mice were then randomly assigned to different treatment groups (n=5 per group) and given PBS, S12, Cb, or Cb@S12 (1×10⁻⁶). B CFU / mouse / day was administered via gavage for 7 days, with mouse weight recorded daily. On the last day of the treatment experiment, colon length was recorded, and the colon and cecum were harvested, fixed in 4% formalin, and subjected to histopathological analysis. (Appendix) Figure 7 )
[0038] 8. In vivo treatment of Fn-associated CRC tumors
[0039] In the in situ implantation model, CT26 cells transfected with luciferase (CT26) Luc Mix 1:1 with matrix gel and inject 40 μL into the cecal wall of 6-8 week old male BALB / c mice. For 4 days prior to the start of any treatment, mice were administered 1 × 10⁻⁶ gavage daily. 8 CFU Fn was administered to mice, who were randomly divided into four groups of five mice each. PBS, S12, Cb, or Cb@S12 were then administered orally every two days. During treatment, the growth of in situ tumors was observed using an IVIS (Perkin-Elmer) in vivo imaging system via bioluminescence of cancer cells. The d-fluorescein concentration used for observation was 10 mg / mL, administered via intraperitoneal injection (100 μL). At the end of treatment, the mice were euthanized, and samples were collected for histological analysis. H&E staining of the heart, liver, spleen, lungs, and kidneys was used to determine the biocompatibility of S12, Cb, and Cb@S12. (Appendix) Figure 7 )
[0040] Figure 1 This diagram illustrates the design, preparation, and mechanism of action of lipid S12 nanoparticles. The diagram is explained as follows: Figure 1 The diagram illustrates the chemical process for the preparation of S12, and how S12 coats Clostridium butyricum through positive and negative electrical interactions, and its therapeutic effect in Fn-related colitis and CRC tumor models.
[0041] Figure 2 The figures show the 1H NMR and mass spectra of lipid S12. This illustrates that NMR and mass spectrometry characterization was performed to demonstrate the successful synthesis of lipid S12.
[0042] Figure 3 The figures show the 1H NMR and mass spectra of lipid P12. This illustrates that NMR and mass spectrometry characterization was performed to demonstrate the successful synthesis of lipid S12.
[0043] Figure 4 The figures show the 1H NMR and mass spectra of lipid N12. This illustrates that NMR and mass spectra were performed to demonstrate the successful synthesis of lipid S12.
[0044] Figure 5 This figure presents the selective antibacterial data for lipids S12, N12, and P12. It illustrates how S12, N12, and P12 were co-incubated with Fn and Cb at different concentrations to determine their respective minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The data show that S12 exhibits the best antibacterial effect against Fn without affecting the activity of Cb.
[0045] Figure 6The image shows in vivo IVIS imaging of Cb and Cb@S12 retention in the mouse intestine. This image illustrates that Cb@S12, compared to the Cb control group, can effectively remain in the intestine, promoting probiotic colonization, indicating that the protective layer of S12 has the effect of prolonging probiotic survival and colonization.
[0046] Figure 7 The figure shows the changes in colon length and body weight in mice of different treatment groups in an in vivo experiment with Fn-exacerbated colitis. This illustrates that, compared to other treatment groups, mice with Fn-exacerbated colitis showed a significant return to normal colon length after Cb@S12 treatment, demonstrating an effective therapeutic effect. Furthermore, the Cb@S12 group exhibited the smallest change in body weight compared to other treatment groups, further indicating the therapeutic effect of Cb@S12 on Fn-exacerbated colitis.
[0047] Figure 8 This figure shows tumor images of mice in different treatment groups, along with changes in mouse body weight and survival rate, in an in vivo experiment of Fn-associated CRC tumors. The figure also illustrates CT26 in mice. Luc The changes in tumor fluorescence at different time points showed a significant decrease in tumor fluorescence in the Cb@S12 treatment group mice. Furthermore, compared to other treatment groups, the Cb@S12 group mice exhibited the highest survival rate and the smallest change in body weight, indicating that Cb@S12 has a therapeutic effect on Fn-induced colitis.
[0048] It should be noted that the above-described embodiments are merely preferred embodiments of the present invention and are only used to further illustrate the present invention, and are not intended to limit the scope of protection of the present invention. Modifications that are obvious from the inventive concept are also within the scope of protection of the present invention.
Claims
1. A method for preparing a lauroyl-based selective antibacterial lipid for the treatment of Clostridium nucleatum (Fn)-associated enteritis and colorectal cancer, comprising encapsulating Clostridium butyricum (Cb) probiotics using a selectively antibacterial lauroyl lipid, the preparation steps of which are as follows: 1) The outer amino group of spermine was protected with ethyl trifluoroacetate, and the unprotected amino group was modified with lauroyl chloride. Further, under alkaline solution conditions, the protection of the outer amino group by the trifluoroacetate group was removed, ultimately preparing N... 4 N 9 -Dilauramidylspermine (S12). The structural formula of S12 is as follows: 2) Add S12, cholesterol, distearate phosphatidylethanolamine-methoxy polyethylene glycol (DSPE-mPEG) 2000 Dissolve the probiotic in ethyl acetate, methanol, or chloroform in an appropriate ratio, suspend it in a glass bottle to obtain a lipid layer, add the probiotic solution to the glass bottle with the lipid layer and vortex for 10 minutes. The probiotic coating is formed by the interaction of positive and negative charges of the liposomes with the surface of Clostridium butyricum. 3) The lipid molecule 1,4-bis(3-lauroamidopropyl)piperazine (P12) is prepared by amidation of 1,4-bis(3-aminopropyl)piperazine with a lauroyl group. The lipid molecule 2-(bis(2-lauroamidoethyl)ethylenediamine) (N12) is prepared by protecting a single amino group on a tris(2-aminoethyl)amine with Boc, followed by amidation of the remaining two amino groups with a lauroyl group, and finally de-Boc protection. The structural formula of N12 is as follows: The structure of P12 is as follows:
2. The method for preparing a probiotic delivery method for treating enteritis and colorectal cancer using a lauroyl selective antibacterial lipid according to claim 1, characterized in that: S12, P12, and N12 are all lauroyl lipids with two tails. Their molecular heads have a positive charge, which can bind to the negative charge on the surface of bacteria to achieve a coating effect. They also have the effect of not killing Clostridium butyricum but selectively killing Clostridium nucleatum.
3. The method for preparing a probiotic delivery method for treating enteritis and colorectal cancer using a lauroyl selective antibacterial lipid according to claim 1, characterized in that: S12, cholesterol, DSPE-mPEG 2000 The ratio is (10~30): (2~10): (0~3).
4. The method for preparing a probiotic delivery method for treating enteritis and colorectal cancer using a lauroyl selective antibacterial lipid according to claim 1, characterized in that: S12 is used at a concentration of less than 100 ug / mL when encapsulating the probiotic Clostridium butyricum.