A selective antimicrobial lipid delivery probiotic system of lauryl quaternary ammonium salt and a method of making the same

Abstract

This invention constructs a selective antibacterial lipid delivery system based on lauroyl quaternary ammonium salt. It targets *Fusobacterium nucleatum* (…). UN ), Anaerobic digestive streptococci ( On ) and Porphyromonas gingivalis ( Because Pathogenic bacteria that promote the progression of colorectal cancer (CRC), such as α1, were used to design and synthesize dilauryl tertiary amine lipid T1, which was then quaternized to obtain broad-spectrum antibacterial lipid Q1 and selective antibacterial lipid Q2, for delivery of active rumenococci ( ). Rg After verifying its selective antibacterial properties using nanoparticles, Q2 was used as the coating to construct... Rg @Q2 and Rg @Q2 / HA delivery system. This system can selectively inhibit harmful bacteria, alleviate immunosuppression, and promote probiotics. Rg This strategy colonizes the gut, thereby enhancing cytotoxicity of immune cells, reshaping the tumor immune microenvironment, and optimizing the microbiota structure. It requires no antibiotics and has promising translational applications in treating CRC and gut microbiota-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for synthesizing a selective antibacterial lipid lauroyl quaternary ammonium salt, a method for preparing a beneficial bacteria delivery system, and the application of the aforementioned beneficial bacteria delivery system in the treatment of colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, and its pathogenesis is closely related to the gut microbiota. Increasing evidence suggests that microbial dysbiosis is a key factor in reshaping the tumor microenvironment, modulating host immunity, and promoting CRC progression. Pathogenic bacteria can promote CRC progression through multiple mechanisms. For example, *Fusobacterium nucleatum* (… Fn ) can activate the β-catenin signaling pathway and mediate immune escape, while anaerobic digestive streptococci ( Pa This organism recruits immunosuppressive myeloid cells (MDSCs) by binding to integrin α2β1 and triggering the PI3K-Akt-NF-κB signaling pathway. Similarly, *Porphyromonas gingivalis* (…) Pg These pathogens participate in CRC progression through similar oncogenic and immunomodulatory mechanisms. They reshape the tumor microenvironment, promote colorectal cancer progression, and enhance treatment resistance. Although antibiotics can eliminate these bacteria, their broad spectrum can disrupt intestinal homeostasis and weaken mucosal immunity.

[0003] Many beneficial bacteria, such as Micrococcus viridans (Micrococcus viridans), are also beneficial. Rg While *C. rubella* is a bacterium that can effectively treat CRC, it, like pathogenic bacteria, cannot tolerate antibiotics and therefore cannot achieve the desired intestinal colonization effect for CRC treatment. Therefore, novel strategies are urgently needed to selectively eliminate tumor-promoting pathogens while preserving beneficial microorganisms. Summary of the Invention

[0004] To address the aforementioned problems in colorectal cancer treatment, this invention screens for selective anti-inflammatory drugs. Fn / Pa / Pg Lauroyl quaternary ammonium salt lipids containing various bacterial pathogens, and as... Rg Bacterial coating, protection Rg It protects against damage to the gastrointestinal environment, thereby achieving intestinal colonization and significantly enhancing the immunotherapy effect of colorectal cancer.

[0005] To achieve selective antibacterial activity, this invention provides a method for preparing a selective antibacterial lipid delivery system for beneficial bacteria using lauroyl quaternary ammonium salts, comprising the following steps: 1) Starting with N-methyl-2,2'-diaminodiethylamine, dilauramide molecule T1 is obtained by amidation of lauric acid and two amino groups. T1 exhibits selective anti-oxidation properties. Fn / Pa / Pg The properties of harmful bacteria; The T1 structure is as follows: ; 2) The T1 molecule obtained above is subjected to a quaternization reaction with bromoethanol to obtain the broad-spectrum antibacterial agent Q1; The Q1 structure is as follows: ; 3) The T1 molecules obtained above are subjected to a quaternization reaction with iodomethane to obtain selective anti-selective ... Fn / Pa / Pg Antibacterial agent Q2 against harmful bacteria; The Q2 structure is as follows: 4) Add Q2, cholesterol, distearate phosphatidylethanolamine-methoxy polyethylene glycol (DSPE-mPEG) 2000 The liposomes are dissolved in ethyl acetate, methanol, or chloroform and suspended in a glass bottle to obtain a thin lipid layer. The bacterial solution is then added to the glass bottle containing the lipid layer and vortexed for 5-15 minutes. The liposomes bind to the surface of active rumenococci through the interaction of their positive and negative charges, forming a bacterial coating. Rg @Q2.

[0006] 5) Dissolve Q2 and cholesterol in ethyl acetate, methanol, or chloroform, suspend in a glass bottle to obtain a lipid thin layer. Add the bacterial solution to the glass bottle containing the lipid thin layer and vortex for 5-15 minutes. The positive and negative charges of the liposomes interact with the surface of active rumenococci to form a bacterial coating. After further coating with an appropriate concentration of hyaluronic acid (HA) and vortexing for 5-15 minutes, the desired result is obtained. Rg @Q2 / HA.

[0007] Furthermore, both T1 and Q2 mentioned above are lauroyl lipids with dual tails. Their molecular heads have a positive charge, enabling them to bind to the negative charge on the bacterial surface, achieving a coating effect. They also exhibit non-killing properties against active rumenococci and are effective against Fusobacterium nucleatum (…). Fn ), Anaerobic digestive streptococci ( Pa ) and Porphyromonas gingivalis ( Pg It exhibits selective killing effects. Furthermore, the Q1 test reveals lipid molecules with broad-spectrum antibacterial properties.

[0008] Furthermore, the above-mentioned Q2, cholesterol, and DSPE-mPEG 2000 The mass ratio is (10-30): (2-10): (0-3).

[0009] Furthermore, the mass ratio of Q2 to cholesterol is (10-30):(2-10), while the concentration range of HA is 50-200 μg / mL.

[0010] Furthermore, the concentration range of Q2 used to encapsulate the probiotic active rumenococcus is 25-100 μg / mL.

[0011] This invention also provides a probiotic delivery system for regulating gut microbiota, comprising: (1) Probiotics Rg; (2) Quaternary ammonium lipids Q2 coated on the surface of the probiotics; (3) Hyaluronic acid (HA) outer layer.

[0012] The beneficial effects of this invention are as follows: via oral delivery Rg @Q2、 Rg @Q2 / HA, Q2 can selectively inhibit pathogens that promote CRC growth in the gut. Fn / Pa / Pg To reduce the proliferation of harmful bacteria and eliminate harmful bacteria that cause CRC, and to relieve... Fn The immunosuppressive effect of the tumor microenvironment caused by the leading harmful bacteria, and its synergistic effect Rg The delivery, making Rg It can better colonize in the intestinal environment. At the same time Rg The colonization of these bacteria can stimulate immune cell toxicity, thereby reshaping the intestinal tumor immune microenvironment, improving the gut microbiota, and enhancing the anti-tumor therapeutic effect by increasing the proportion of anti-cancer bacteria in the gut microbiota.

[0013] Rg @Q2 / HA compared to Rg @Q2, the added HA coating enables a more effective intestinal retention effect, ensuring... Rg Its highly efficient delivery effect. Its tumor-treating efficacy for CRC is superior to... Rg @Q2 is more useful.

[0014] This invention provides a selective antimicrobial lipid delivery system of lauroyl quaternary ammonium salt and its preparation method. Its significance lies in its low cost and good biocompatibility, providing a potent, antibiotic-free treatment strategy. By combining the antitumor effect mediated by probiotics with the selective elimination of pathogenic bacteria, it shows excellent translational potential for the treatment of CRC and other gut microbiota-related diseases. Attached Figure Description

[0015] Figure 1 This is a synthesis route diagram for T1, Q1, and Q2; Figure 2 The 1H NMR spectrum and mass spectrum of T1 in Example 1 are shown. Figure 3 The 1H NMR spectrum and mass spectrum of Q1 in Example 1 are shown. Figure 4 The 1H NMR spectrum and mass spectrum of Q2 in Example 1 are shown below; Figure 5 The images shown are transmission electron microscope images of lipid nanoparticles and DLS particle size distribution diagrams from Example 2. Figure 6 The results of the selective antibacterial performance test of lipid nanoparticles in Example 3; Figure 7 This is a summary of the MIC and MBC results of lipid nanoparticles against different bacteria in Example 3; Figure 8 In Example 4 Rg , Rg @Q2 and Rg Transmission electron microscope image of @Q2 / HA; Figure 9 In Example 5 Rg , Rg @Q2 and Rg Graph showing the effect of intestinal retention in Q2 / HA; Figure 10 The images show tumor fluorescence images, mouse weight, and survival rate changes in mice in different treatment groups in the orthotopic tumor mouse model of Example 6. Detailed Implementation

[0016] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0017] This invention provides a selective antibacterial lipid delivery system of lauroyl quaternary ammonium salt and its preparation method, which relates to the biomedical field.

[0018] For the synthesis method of lauroyl quaternary ammonium salt, please refer to [link / reference]. Figure 1 , Figure 1 This is the synthetic route for lauroyl quaternary ammonium salts.

[0019] The method includes the following steps: Step S1: Using N-methyl-2,2'-diaminodiethylamine as the initial molecule, dilauramide molecule T1 is obtained by amidation of lauric acid with two amino groups; Step S2: The T1 molecule obtained above is subjected to a quaternization reaction with bromoethanol to obtain the broad-spectrum antibacterial agent Q1; Step S3: The obtained T1 molecule is subjected to a quaternization reaction with iodomethane to obtain selective anti-selective ... Fn / Pa / Pg Antibacterial agent Q2 against harmful bacteria; like Figure 2 , Figure 3 , Figure 4 As shown, the 1H NMR and mass spectrometry data confirm that T1, Q1, and Q2 have been successfully synthesized.

[0020] Example 1 This embodiment, in the synthesis of T1, Q1, and Q2, specifically includes the following steps: 2.0 g (10 mmol) of lauric acid was dissolved in 20 mL of anhydrous DMF and premixed with 2.8 g (15 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 1.4 g (12 mmol) of N-hydroxysuccinimide (NHS) for 3 hours. Separately, 586.0 mg (5 mmol) of N-methyl-2,2'-diaminodiethylamine was added to 5 mL of DMF, dissolved, and then added dropwise to the above solvent. The mixture was stirred at room temperature for 16 h, and then concentrated under reduced pressure using an oil pump. After stirring at room temperature for 16 h, the mixture was concentrated under reduced pressure, and the residue was dissolved in DCM (100 mL), washed with saturated NaHCO3 solution (3 × 20 mL), and then concentrated under reduced pressure. The crude product was purified by column chromatography using DCM / MeOH (20:1) eluent. The product can be recrystallized from methanol. The target product T1 was dissolved in CDCl3 and characterized by 1H NMR at 25 °C and 400 MHz. Further verification was then performed by ESI-MS. Figure 2 ).

[0021] Pure T1 (240.7 mg, 0.5 mmol) was dissolved in 5 mL of CHCl3 with stirring at room temperature. Then, 2-bromoethanol (63.75 mg, 0.51 mmol) or iodomethane (78 mg, 0.55 mmol) was added. The solvent was then heated at 50 °C for 24 h, and the solution was concentrated. Products Q1 and Q2 could be recrystallized from methanol. Target product Q1 or Q2, dissolved in CDCl3, was characterized by 400 MHz 1H NMR at 25 °C, and further verified by ESI-MS. Figure 3 and Figure 4 ).

[0022] Example 2 This embodiment takes the preparation of T1, Q1, and Q2 lipid nanoparticles as an example, and specifically includes the following steps: Preparation of lipid nanoparticles: Lipids were dissolved in a mixed solvent of MeOH and CHCl3 (1:3 ratio). The resulting solution was dried using a rotary evaporator at room temperature to form a lipid film. The film was then diluted with PBS to form lipid nanoparticles. The solution was then subjected to pressure filtration through 1.2, 0.8, 0.45, and 0.22 μm filter membranes.

[0023] Figure 5 Transmission electron microscopy images show that the prepared lipid nanoparticles are uniformly spherical, and DLS tests show that the particle size distribution is uniform. The particle sizes of the prepared lipid nanoparticles are NL-T1 197.4 ± 4.7 nm, NL-Q1 107 ± 1.97 nm, and NL-Q2 161.2 ± 6.2 nm.

[0024] Example 3 To investigate the selective antibacterial properties of the lipid nanoparticles, selective antibacterial tests were conducted on the prepared NL-T1, NL-Q1, and NL-Q2 nanoparticles. The tested bacteria were active Ruminococcus. Rg ), Fusobacterium nucleatum ( Fn ), Anaerobic digestive streptococci ( Pa ), Porphyromonas gingivalis ( Pg ).

[0025] MIC and MBC were determined as follows: a bacterial suspension in the logarithmic growth phase (1 × 10⁻⁶) was prepared. 8 CFU / mL, OD 600 (≈ 0.107), diluted to 1 × 10⁻⁶ in BHI broth. 7 The concentration of CFU / mL was determined by placing the microplate in a 48-well plate. 100 μL S8-S16 lipid nanoparticles were added to the bacterial culture to achieve a test concentration gradient of 5-500 μg / mL, followed by anaerobic incubation at 37 ℃ for 18 h. OD6 was then measured using a microplate reader. 00 The values ​​were calculated. BHI broth without bacteria served as a blank control, while BHI fermentation broth containing bacterial suspension served as a positive control. Bacterial cell viability was calculated using the following equation: The MIC (Minimum Intake Capacity) is the minimum concentration at which bacterial survival significantly decreases. After MIC determination, 50 μL of suspension with no obvious bacterial growth pores is spread onto blood agar plates, which are then incubated at 37 °C for 18 h. The final MBC (Medium-to-Bacterial Compatibility) value is determined by examining the bacterial survival on blood agar plates at various lipid nanoparticle concentrations.

[0026] like Figure 6 The figure shows the measured OD. 600 As a result, Figure 7These are the MIC and MBC values ​​summarized from the test results. The test results indicate that T1 and Q2 can inhibit active rumenococci (…). Rg It has a good protective effect and can protect against three pathogens. Fn / Pa / Pg To inflict effective damage.

[0027] Example 4 Using Q2 to target bacteria Rg Package Rg Preparation of Q2: Lipid Q2, cholesterol, and DSPE-mPEG were dissolved in 2 mL of CHCl3 at a mass ratio of 16:4:1. A film containing 50 μg of Q2 was then prepared in 1 mL of... Rg The bacteria were hydrated in PBS solution and vortexed for 15 min. After centrifugation and discarding the supernatant, the solution was resuspended in PBS and stored at 4 °C for subsequent characterization.

[0028] Rg @Q2 / HA Preparation: Purified lipid Q2 and cholesterol were dissolved in 2 mL of CHCl3 at a mass ratio of 16:4. The membrane containing 50 μg of Q2 was hydrated in 1 mL of PBS containing bacteria and vortexed for 15 min. After centrifugation and resuspending, 100 μL of HA solution (1 mg / mL) was added, and the mixture was vortexed again for 15 min. The mixture was centrifuged twice, resuspended in PBS, and stored at 4 °C for subsequent characterization.

[0029] like Figure 8 As shown, the characteristics were determined by transmission electron microscopy. Rg @Q2 and Rg Morphological characteristics of @Q2 / HA after successful preparation.

[0030] Example 5 In order to evaluate Rg @Q2 and Rg The retention effect of Q2 / HA in the intestinal environment was assessed using the following tests: Rg @Q2 and Rg The in vivo distribution of @Q2 / HA was investigated by randomly dividing BLAB / c mice into three groups of nine mice each. After a 12-hour fast, Rg The mice were administered DiR-labeled [substance] via oral gavage. Rg (1 × 10) 8 CFU), while Rg @Q2 and Rg The mice in the Q2 / HA group were administered DiR-labeled coatings of Q2 or Q2 / HA orally via gavage. Rg (1 × 10) 8CFU). Gastrointestinal tissues were collected from mice in each group at 3, 6, and 12 h after drug administration for in vivo IVIS imaging.

[0031] like Figure 9 As shown, the control group of naked bacteria Rg The fluorescence disappeared after 12 hours, while Rg @Q2 and Rg In @Q2 / HA, the fluorescence was particularly strong in the cecum after 12 hours, indicating effective colonization of the delivered bacteria.

[0032] Example 6 Evaluation in a mouse orthotopic tumor model Rg @Q2 and Rg The antitumor effect of @Q2 / HA was specifically demonstrated in the following experiments: In the orthotopic transplantation model, BALB / c mice received an injection of luciferase-labeled CT26 cells (2.0 × 10⁻⁶). 6 Cells were suspended in 50 μL of a 1:1 mixture of PBS and Matrigel, and injected into the cecal wall. In situ tumor growth was monitored by detecting bioluminescence in cancer cells using an IVIS system. Mice were randomly divided into five groups of five each. Subsequently, the following medications were administered orally every two days: PBS, Q2, and... Rg , Rg @Q2 and Rg @Q2 / HA (1 × 10 8 (CFU / animal). Tumor burden was monitored by bioluminescence analysis using an IVIS imaging system combined with intraperitoneal injection of 100 μL d-fluorescein (10 mg / mL).

[0033] like Figure 10 As shown, during treatment, PBS, NL-Q2, Rg The tumor volume in the group was significantly increased. Rg @Q2 and Rg The @Q2 / HA group showed tumor suppression effects. It is noteworthy that... Rg @Q2 / HA exhibited the most significant tumor-suppressive effect. This also confirms... Rg The Q2 / HA double-coating system effectively encapsulates bacteria, resulting in better bacterial colonization and thus a more effective treatment for colorectal cancer.

[0034] This potent, antibiotic-free treatment strategy, by combining probiotic-mediated antitumor effects with selective pathogen clearance, demonstrates remarkable translational potential for the treatment of CRC and other gut microbiota-related diseases.

Claims

1. A method for preparing a selective antibacterial lipid delivery beneficial bacteria system using lauroyl quaternary ammonium salt, characterized in that: Active rumen cocci are encapsulated with selectively antibacterial lipids containing lauroyl quaternary ammonium salts. Rg The preparation steps include the following: 1) Starting with N-methyl-2,2'-diaminodiethylamine, dilauramide molecule T1 is obtained by amidation of lauric acid and two amino groups. The structural formula of T1 is as follows: ； 2) The T1 molecule obtained above is subjected to a quaternization reaction with bromoethanol to obtain a broad-spectrum antibacterial agent Q1, the structural formula of which is as follows: ； 3) The T1 molecule obtained above is subjected to a quaternization reaction with iodomethane to obtain the selective antibacterial lipid Q2, the structural formula of which is as follows: ； 4) Dissolve Q2, cholesterol, and distearate phosphatidylethanolamine-methoxy polyethylene glycol in ethyl acetate, methanol, or chloroform, suspend in a glass bottle to obtain a lipid thin layer, add the bacterial solution to the glass bottle containing the lipid thin layer, vortex for 5-15 minutes, and form a bacterial coating by the positive and negative charge interactions of the liposomes with the surface of active rumenococci. Rg @Q2; 5) Dissolve Q2 and cholesterol in ethyl acetate, methanol, or chloroform, and suspend in a glass bottle to obtain a lipid thin layer. Add the bacterial solution to the glass bottle containing the lipid thin layer and vortex for 5-15 minutes. The liposomes bind to the surface of active rumenococci through the interaction of their positive and negative charges, forming a bacterial coating. After vortexing with hyaluronic acid for 5-15 minutes, the desired result is obtained. Rg @Q2 / HA.

2. The method for preparing a selective antibacterial lipid delivery beneficial bacteria system of lauroyl quaternary ammonium salt according to claim 1, characterized in that: T1 and Q2 are both lauroyl lipids with two tails, while Q1 is a lipid molecule with broad-spectrum antibacterial properties.

3. The method for preparing a selective antibacterial lipid delivery beneficial bacteria system of lauroyl quaternary ammonium salt according to claim 1, characterized in that: Q2. The mass ratio of cholesterol to distearylphosphatidylethanolamine-methoxy polyethylene glycol is (10-30): (2-10): (0-3).

4. The selective antibacterial lipid delivery system for beneficial bacteria using lauroyl quaternary ammonium salt according to claim 1, characterized in that: The mass ratio of Q2 to cholesterol is (10-30):(2-10), while the concentration range of HA is 50-200 μg / mL.

5. The method for preparing a selective antibacterial lipid delivery beneficial bacteria system of lauroyl quaternary ammonium salt according to claim 1, characterized in that: Q2 uses a concentration range of 25-100 μg / mL for encapsulating probiotic active rumenococci.

6. A probiotic delivery system for regulating gut microbiota, characterized in that: include: (1) Probiotics Rg ; (2) Quaternary ammonium lipids Q2 coated on the surface of the probiotics; (3) Hyaluronic acid outer layer.

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