Preparation and application of high-molecular polymer nanoparticles targeting fusobacterium nucleatum biofilm

Abstract

The invention belongs to the field of biomedical materials, and relates to a preparation method and application of a high-molecular nanoparticle targeting a fusobacterium nucleatum biofilm. The material is used for solving the problem that the fusobacterium nucleatum forms a biofilm in colorectal cancer tissues and aggravates immunosuppression. The method comprises the following steps: carrying out ring opening polymerization on an asparagine monomer to generate a polyasparagine main chain, introducing 3, 3 '-diaminodipropylamine through amination, and sequentially grafting lauric acid, FadA recognition peptide and polyethylene glycol to obtain the PLPP capable of being self-assembled into nanoparticles. The material is controllable in structure and simple and convenient in process, has good targeting property, biocompatibility and biological membrane permeability, and can specifically recognize and remove a biological membrane with fusobacterium nucleatum, improve a tumor immune microenvironment and inhibit colorectal cancer progress.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and nanomedicine delivery technology, specifically to the preparation and use of polymer nanoparticles targeting the biofilm of *Fusobacterium nucleatum*. This material is particularly suitable for clearing bacterial infections associated with colorectal cancer and regulating the tumor immune microenvironment, belonging to the interdisciplinary field of antibacterial therapy and adjuvant immunotherapy for tumors. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, with high morbidity and mortality rates. Recent studies have shown that its development and progression are closely related to gut microbiota imbalance, particularly the high accumulation of *F. nucleatum* in CRC tissues. This bacterium can activate the β-catenin signaling pathway by binding to E-cadherin on the surface of host epithelial cells via the adhesin FadA, thereby inducing carcinogenesis. Furthermore, *F. nucleatum* can form robust biofilm structures in the tumor microenvironment, enhancing its colonization ability, resisting antimicrobial drugs, and inducing the recruitment of myeloid-derived suppressor cells (MDSCs) and M2 macrophages, leading to an immunosuppressive state. This suppresses the function of immune effector cells such as T cells, promoting tumor immune escape.

[0003] Traditional antibiotics face numerous challenges in treating *F. nucleatum* infections. On the one hand, biomembrane structures limit drug penetration, leading to low clearance efficiency; on the other hand, broad-spectrum antibacterial agents may disrupt normal gut flora, inducing drug resistance and exacerbating microecological imbalance. Although studies have attempted to deliver antibiotics or immunomodulatory factors using nanocarriers, significant shortcomings remain in terms of targeting, biomembrane penetration, and long-term in vivo circulation. There is also a lack of industrially scalable, targeted, and multifunctional integrated nanomaterial systems. Therefore, developing a therapeutic strategy that can specifically recognize *F. nucleatum*, effectively penetrate biomembranes, and possess immunomodulatory functions has become an important research direction in the interdisciplinary field of tumor microecological regulation and antibacterial therapy. Summary of the Invention

[0004] To address the crucial role of *F. nucleatum* in the development and progression of chronic rheumatoid arthritis (CRC) and the issues of immune escape and drug tolerance caused by its biofilm, this invention designs a polymeric antibacterial nanomaterial, PAsp(Nors)-LA-Pep-PEG (PLPP), possessing targeted recognition, structure-responsive release, and immunomodulatory functions. This material is constructed by sequentially grafting a FadA-specific recognition peptide (Pep), the biofilm-disrupting group lauric acid (LA), and long-cycle polyethylene glycol (PEG) onto the antibacterial PAsp(Nors) backbone, and can self-assemble into nanoparticles in an aqueous phase.

[0005] The PLPP nanoparticles achieve targeted recognition and accumulation in F. nucleatum-rich tumor tissues through the specific binding of the surface-exposed targeting peptide Pep to the F. nucleatum surface adhesin FadA. In the acidic tumor microenvironment, Schiff base structures break down, causing the PEG layer to detach and further exposing Pep, enhancing the interaction between the particles and F. nucleatum. Simultaneously, the LA groups disrupt the biofilm formed by F. nucleatum, thus synergistically achieving efficient removal of the F. nucleatum biofilm.

[0006] In addition, this material can also modulate the tumor immune microenvironment, promote M1 macrophage polarization and CD8+. + T cell infiltration, while inhibiting the recruitment of M2 macrophage phenotype and myeloid-derived suppressor cells (MDSCs), can achieve the goal of inhibiting CRC progression.

[0007] In a first aspect, the present invention provides a method for preparing PLPP nanoparticles, comprising the following steps:

[0008] (1) Using β-benzyloxyaspartic acid NCA as a monomer, ring-opening polymerization was carried out under the action of a tertiary amine initiator to obtain intermediate 1 (PBLA);

[0009]

[0010] (2) PBLA was subjected to an amination reaction with 3,3'-diaminodipropylamine (Nors) to obtain intermediate 2 (PAsp(Nors)) with a primary amine structure;

[0011]

[0012] (3) Using the EDC / NHS activation method, LA and maleimide linking groups (Mal) were grafted onto the PAsp(Nors) main chain in sequence to obtain intermediate 3 (PAsp(Nors)-LA-Mal);

[0013]

[0014] (4) The FadA recognition peptide Pep(CGGASANWTIQYND) was grafted onto the maleimide structure via a thiol-olefin click reaction to obtain intermediate 4(PAsp(Nors)-LA-Pep).

[0015]

[0016] (5) The synthesized methoxy polyethylene glycol benzaldehyde (PEG-CHO) was grafted to the end of the main chain via Schiff base reaction to form a pH-responsive long-cycle structure PLPP.

[0017]

[0018] (6) The obtained polymer is dissolved in the aqueous phase and self-assembled into PLPP nanoparticles under ultrasonic action.

[0019] Secondly, the present invention provides the use of the PLPP nanoparticles in the preparation of a drug for specifically clearing CRC-associated F. nucleatum biofilms.

[0020] Thirdly, the present invention provides that the PLPP nanoparticles achieve precise targeting of F. nucleatum by specifically binding to FadA on the surface of F. nucleatum.

[0021] Fourthly, the present invention also provides that the LA groups in the PLPP nanoparticles have the function of dispersing biological membranes.

[0022] Fifthly, the present invention further provides the application of the PLPP nanoparticles in regulating the tumor immune microenvironment, by inhibiting MDSCs and M2 macrophages, and enhancing M1 macrophages and CD8+. + T cell infiltration inhibits CRC progression.

[0023] In the preparation method, the specific steps for synthesizing intermediate 1 include:

[0024] PBLA was synthesized by ring-opening polymerization of BLA-NCA initiated by n-n-butylamine. The specific steps were as follows: 5 g (20.7 mmol) of BLA-NCA was dissolved in 10 mL of DMF, diluted with 10 mL of dichloromethane, and then 0.19 mL (0.19 mmol) of a mixture of n-n-butylamine and dichloromethane was added. The reaction was stirred at 35 °C under dry nitrogen for 48 h. The resulting product, PBLA, was precipitated in n-hexane / ethyl acetate (6:4), filtered, and dried under vacuum.

[0025] The specific steps for synthesizing intermediate 2 include:

[0026] First, 25 mg of lyophilized PBLA was dissolved in 1 mL of NMP and cooled to 0 °C. Next, 5 mL of Nors was diluted twice with NMP and cooled, and then the PBLA solution was added dropwise to the Nors solution. After stirring at 0 °C for 1 h, the reaction mixture was slowly added to an equimolar amount of aqueous hydrochloric acid in an ice-water bath. The mixture was then transferred to a dialysis bag (molecular weight cutoff 7000 Da) and dialyzed against hydrochloric acid and distilled water for 1-2 days each. After freeze-drying, a white powder, PAsp(Nors), was obtained.

[0027] The specific steps for synthesizing intermediate 3 include:

[0028] LA and PAsp(Nors) were linked via an EDC / NHS coupling method. 3.48 mg of LA was activated by reacting 10 mg of EDC and 6 mg of NHS in 10 mL of DMF for 3 h. Subsequently, 20 mg of PAsp(Nors) was dissolved in 2 mL of dimethyl sulfoxide (DMSO) and slowly added to the above reaction solution, while stirring at 25 °C for 48 h. Finally, the reaction solution was slowly added dropwise to diethyl ether, and the crude product was collected by centrifugation. After dialyzing with distilled water (molecular weight cutoff 3,000 Da), the product was lyophilized to obtain a white powder of PAsp(Nors)-LA.

[0029] The specific steps for synthesizing intermediate 4 include:

[0030] The above experiment first prepared PAsp(Nors)-LA, and then introduced 6-maleimide hexanoic acid (Mal) into PAsp(Nors)-LA via an amidation reaction to prepare PAsp(Nors)-LA-Mal. First, Mal (3.67 mg) was reacted with EDC (10 mg) and NHS (6 mg) in DMSO (10 mL) for 3 h. Next, PAsp(Nors)-LA (20 mg) was dissolved in DMSO (5 mL) and slowly added to the above reaction mixture, and stirred at 25 °C for 48 h. Finally, the reaction mixture was slowly added dropwise to diethyl ether, the crude product was collected by centrifugation, dialyzed against distilled water (molecular weight cutoff 3,000 Da) for 72 h, and lyophilized to obtain a white powder, PAsp(Nors)-LA-Mal. The compound PAsp(Nors)-LA-Pep was prepared via a thiol-maleimide "click" reaction between PAsp(Nors)-LA-Mal and Pep. Pep(CGGASANWTIQYND) (26 mg) and PAsp(Nors)-LA-Mal (50 mg) were dissolved in 4 mL of DMSO. After sonication at room temperature for 20 min, the mixture was washed three times with ether and dialyzed in distilled water for 72 h. The solution was then lyophilized to obtain a pale yellow powder, PAsp(Nors)-LA-Pep.

[0031] The specific steps for synthesizing PLPP include:

[0032] Synthesis of Long-Cycling-Chain Methoxylated Poly(ethylene glycol)benzaldehyde (PEG-CHO). The synthesis method of methoxylated poly(ethylene glycol)benzaldehyde (PEG-CHO) is as follows. First, mPEG2000 (2.5 g, 1.2 mmol) and 4-dimethylaminopyridine (DMAP) (0.16 g, 1.3 mmol) were dissolved in 75 mL of anhydrous dichloromethane (DCM). Next, 4-acetylbenzoic acid (1.5 g, 10.0 mmol) and dicyclohexylcarbodiimide (DCC) (2.1 g, 10.2 mmol) were added, and the mixture was stirred at room temperature for 24 h. The filtrate was concentrated after filtration. The obtained crude product was suspended in 25 mL of deionized water, stirred for 30 min, and filtered again to collect the filtrate. Subsequently, the organic layer was extracted with DCM (100 mL), and dried with anhydrous sodium sulfate. The anhydrous sodium sulfate was removed by filtration, and the clear solution was added dropwise to cold diethyl ether to finally obtain mPEG-CHO.

[0033] 40 mg of PAsp(Nors)-LA-Pep was suspended in 10 mL of pre-dried DMSO. Then, 37 mg (0.03 mol) of mPEG-CHO was slowly added to the solution. The mixture was then stirred at 50 °C for 48 h. Afterward, the reaction solution was transferred to a dialyzer with a molecular weight cutoff of 7,000 Da, dialyzed against anhydrous ethanol for 24 h, followed by dialyzed against 0.01 M phosphate-buffered saline (PBS) at pH 7.4 for 24 h. Finally, PLPP was obtained by freeze-drying.

[0034] The specific steps for preparing PLPP nanoparticles include:

[0035] PLPP was dissolved in 0.5 mL of DMSO and added dropwise to 2.5 mL of deionized water under ultrasonic conditions. After the addition was completed, the mixed solution was ultrasonically treated for another 30 min to allow the polymer PLPP to self-assemble into nanostructures through hydrophilic-hydrophobic interactions. The resulting solution was placed in a dialysis bag and dialyzed with deionized water to remove DMSO, thus obtaining nanomaterials with uniform particle size distribution.

[0036] The ultrasonic treatment is preferably performed at a frequency of 40 kHz and a power of 100 W.

[0037] The beneficial effects of this invention are as follows:

[0038] (1) The PLPP nanomaterial of the present invention has good targeting ability and can bind with high affinity to the FadA protein on the surface of Fusobacterium nucleatum, thereby achieving specific clearance of F. nucleatum;

[0039] (2) The material structure is designed to be pH responsive, which can expose the biomembrane-destructive component LA in the tumor microenvironment and effectively break through the biomembrane barrier;

[0040] (3) This nanosystem can significantly improve the tumor immune microenvironment and enhance immune cell infiltration, providing an innovative strategy for the combined treatment of colorectal cancer. Attached Figure Description

[0041] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which:

[0042] Figure 1 PLPP synthesis and assembly diagram

[0043] Figure 2 This is the NMR spectrum of intermediate 1 prepared in this invention.

[0044] Figure 3 This is the NMR spectrum of intermediate 2 prepared in this invention.

[0045] Figure 4 This is the NMR spectrum of intermediate 3 prepared in this invention.

[0046] Figure 5 This is the NMR spectrum of intermediate 4 prepared in this invention.

[0047] Figure 6 This is the NMR spectrum of the PLPP prepared by this invention.

[0048] Figure 7 This is a scanning electron microscope image of PLPP nanoparticles.

[0049] Figure 8 It represents the potential change caused by the breaking of Schiff base bonds and the shedding of PEG from PLPP nanoparticles under acidic conditions.

[0050] Figure 9 It is a molecular docking model of the interaction between PLPP and FadA adhesin, selectively binding to F. nucleatum.

[0051] Figure 10 This is a quantitative data graph showing how the LA component in PLPP nanoparticles partially disrupts extracellular polysaccharides and external DNA in biological membranes.

[0052] Figure 11 Crystal violet staining is used to specifically remove colorectal cancer-associated Fusobacterium nucleatum biofilms from PLPP nanoparticles.

[0053] Figure 12 This is a quantitative data graph showing how PLPP nanoparticles regulate the tumor immune microenvironment. Detailed Implementation

[0054] This invention provides a method for preparing nanoparticles that specifically scavenge F. nucleatum, comprising the following steps:

[0055] In response to the key role of F. nucleatum in the development and progression of CRC and its immune escape and drug tolerance caused by biofilm, this invention designs a polymeric nano-antibacterial material PAsp(Nors)-LA-Pep-PEG (PLPP) with targeted recognition, structure-responsive release and immunomodulatory functions.

[0056] The PLPP material is constructed by sequentially grafting the FadA-recognizing peptide Pep, the biofilm-disrupting group LA, and PEG onto the antibacterial backbone PAsp(Nors), and then self-assembling into nanoparticles in an aqueous phase to achieve targeted, membrane-penetrating, and synergistic drug efficacy.

[0057] The PLPP nanoparticles bind to the FadA adhesin on the surface of *F. nucleatum* via their surface Pep peptide, targeting the *F. nucleatum*-rich regions. In an acidic microenvironment, the Schiff base structure breaks, promoting PEG shedding and further exposing Pep, enhancing targeted binding; simultaneously, the exposed hydrophobic LA disrupts the bacterial biofilm, achieving highly efficient removal of *F. nucleatum*.

[0058] Furthermore, PLPP nanoparticles can induce tumor-associated macrophages to polarize towards the M1 type and enhance CD8. + T cell infiltration inhibits the recruitment of M2 macrophages and MDSCs, thereby reversing the immunosuppressive microenvironment and synergistically fighting tumors.

[0059] Example 1: 5 g of BLA-NCA was dissolved in 10 mL of DMF, then diluted with 10 mL of DCM, and 0.19 mL of n-n-butylamine (0.19 mmol) was slowly added dropwise. The mixture was stirred at 35 °C under dry argon atmosphere for 48 h. After the reaction was complete, the product was precipitated in n-hexane / ethyl acetate (6:4), filtered, and dried under vacuum to obtain PBLA.

[0060] Example 2: 25 mg of PBLA was dissolved in 1 mL of NMP and cooled to 0°C. 5 mL of Nors was diluted twice with NMP and cooled. The PBLA solution was then added dropwise to the Nors solution, stirred for 1 h, and then added dropwise to an equimolar aqueous solution of hydrochloric acid in an ice-water bath. The mixture was dialyzed against aqueous hydrochloric acid for 1 day, then against distilled water for 2 days, and freeze-dried to obtain PAsp(Nors).

[0061] Example 3: 3.48 mg LA was dissolved in 10 mL DMF and activated by reacting with 10 mg EDC and 6 mg NHS for 3 h. 20 mg PAsp(Nors) was dissolved in 2 mL DMSO and slowly added to the reaction solution, and stirred at 25 °C for 48 h. After the reaction was completed, it was added dropwise to diethyl ether, centrifuged to obtain the crude product, dialyzed for 72 h, and then freeze-dried to obtain PAsp(Nors)-LA.

[0062] Example 4: 3.67 mg Mal was reacted with 10 mg EDC and 6 mg NHS in 10 mL DMSO for 3 h, then 20 mg PAsp(Nors)-LA was added, and the mixture was stirred at 25 °C for 48 h. After the reaction was complete, the mixture was added dropwise to diethyl ether, centrifuged, and dialyzed for 72 h to obtain PAsp(Nors)-LA-Mal. 26 mg Pep and 50 mg PAsp(Nors)-LA-Mal were dissolved together in 4 mL DMSO, sonicated at room temperature for 30 min, washed with diethyl ether, and dialyzed for 72 h to obtain PAsp(Nors)-LA-Pep.

[0063] Example 5: mPEG2000 (2.5 g, 1.2 mmol) and DMAP (0.16 g, 1.3 mmol) were dissolved in 75 mL of DCM, and 4-acetylbenzoic acid (1.5 g, 10 mmol) and DCC (2.1 g, 10.2 mmol) were added. The mixture was reacted at room temperature for 24 h, concentrated, stirred with water, extracted, dried, and ether was added dropwise to obtain mPEG-CHO. 40 mg PAsp(Nors)-LA-Pep and 37 mg mPEG-CHO were dissolved together in DMSO and reacted at 50 °C for 48 h. The mixture was dialyzed against ethanol and PBS for 24 h each, and then freeze-dried to obtain PLPP.

[0064] Example 6: PLPP was dissolved in 0.5 mL of DMSO and ultrasonically added dropwise to 2.5 mL of deionized water. Ultrasonication was continued for 30 min to allow it to self-assemble into PLPP nanoparticles. The solution was placed in a dialysis bag and dialyzed with deionized water to remove DMSO, yielding PLPP nanoparticles with uniform particle size distribution.

[0065] like Figure 6 As shown, the PLPP nanoparticles have a uniform particle size distribution of approximately 200 nm and a regular morphology.

[0066] Example 7: pH responsiveness verification experiment of PLPP nanoparticles (ζ potential change detection)

[0067] This embodiment aims to verify the responsiveness of PLPP nanoparticles under different pH environments, thereby inferring whether the long-cycled protective PEG segments on its surface can be broken down to expose the core under acidic conditions. To verify the pH responsiveness of PLPP nanoparticles under acidic conditions, they were dispersed in PBS buffer (pH 5.5, 1 mg / mL) and incubated at 37°C for 12 h. After incubation, the zeta potential was measured using a Zetasizer Nano ZS dynamic light scattering instrument. The results showed that the zeta potential significantly increased to +40.85 mV, indicating that the PEG segments broke down in the acidic environment, exposing more positively charged groups (such as targeting Pep peptides) on the surface, thereby enhancing the positive charge of the particles. This verifies that PLPP nanoparticles can achieve pH-responsive PEG removal in the acidic tumor microenvironment. Figure 8 ).

[0068] Comparative Example 1:

[0069] This comparative example uses the incubation of PLPP nanoparticles in a PBS solution at pH 7.4 as an example, and includes the following steps:

[0070] PLPP nanoparticles of the same concentration (1 mg / mL) were dispersed in PBS buffer at pH 7.4 and incubated at 37°C for 12 h. The zeta potential was measured using the same method, and the result was +11.67 mV, which was significantly lower than the value under acidic conditions. This indicates that the PEG chain structure is stable under neutral physiological conditions, and the particle surface is still coated with hydrophilic PEG, maintaining a low charge state, thereby ensuring its stability and long-term cycling ability in normal circulatory systems.

[0071] Example 8: Molecular docking experiment of PAsp(Nors)-LA-Pep with F. nucleatum surface FadA, the steps of which are as follows:

[0072] In this embodiment, the molecular structure file of the target protein (PDBID: 3ETW) was located and downloaded from the PDB database (https: / / www1.rcsb.org / ). The downloaded target protein was processed using PyMOL software, including the removal of water molecules and protoligands. Subsequently, the target protein was transformed using the Molecular Operations Environment (MOE v2019, Chemical Computing Group Inc., Montreal, Quebec, Canada) to prepare for docking. Before docking, the implicit solvation model of the force field and reaction field (R-field) of AMBER10:EHT was selected to optimize the protonation state and hydrogen atom orientation of the protein, and energy minimization was performed using default parameters in MOE. 3D conformation generation and energy optimization (MM2 minimization algorithm) of PAsp(Nors)-LA-Pep were performed using Chem3D, and these structures were used for subsequent docking.

[0073] The MOE software performed docking calculations using the molecular-protein docking module in the Dock package. In this module, the parameters were set to select the protein as the acceptor and the molecule as the ligand. The pre-placement count was set to 10,000, the number of optimized models (placements) was 1,000, and the top 100 optimal conformations were output. The conformation with the lowest binding free energy was considered the best binding model. Ligand-acceptor force analysis was performed using the PLIP online server (https: / / plip-tool.biotec.tu-dresden.de / plip-web), and the 3D conformations of the ligand-acceptor were visualized using Pymol software.

[0074] Molecular dynamics simulations show that PAsp(Nors)-LA-Pep can efficiently bind to the adhesion protein FadA of F. nucleatum. Figure 9The docking score of its complex is -56.50 kcal / mol, indicating that it has a strong binding affinity. PAsp(Nors)-LA-Pep forms multiple hydrogen bonds (red dashed lines) with key residues of FadA, including TYR-50, GLU-81 and GLN-61, with bond lengths measured in angstroms.

[0075] In contrast, the ungrafted Pep PAsp(Nors)-LA exhibited a lower binding energy, with a docking score of -42.24 kcal / mol. These results indicate that the addition of the Pep peptide significantly enhances the binding stability between the polymer and FadA.

[0076] Example 9: Verification of the destructive effect of LA in PLPP nanoparticles on EPS and eDNA in F. nucleatum biofilms, the steps of which are as follows:

[0077] To verify the ability of LA in PLPP nanoparticles to disrupt EPS and eDNA in F. nucleatum biofilms, a biofilm model of strains with different material treatments was first established, and a growth control group was set up. The formed biofilms were resuspended in TEN buffer and adjusted to OD. 600 =0.5 to ensure consistency. Subsequently, eDNA was extracted using a bacterial genomic DNA extraction kit (Solarbio, Beijing, China), and the eDNA content was determined using a Nanophotometer NP80 Touch spectrophotometer.

[0078] EPS content was determined using the phenol-sulfuric acid method with a commercially available assay kit (Biosharp, Hefei, China). First, glucose standard solutions of different concentrations were prepared and reacted with 5% phenol and concentrated sulfuric acid, respectively. The absorbance was measured at 490 nm, and a standard curve was plotted followed by linear regression analysis. Subsequently, biofilm samples were collected from each experimental group, treated with the phenol / sulfuric acid reagent, and the absorbance was measured at 490 nm. The corresponding EPS content was calculated based on the standard curve.

[0079] Studies show that under acidic conditions (pH 6.5), 100 μg / mL PLPP nanoparticles can significantly disperse mature *F. nucleatum* biofilms, reducing key matrix components in the biofilm, including EPS and eDNA, thereby effectively weakening the stability of the biofilm structure and leading to a significant reduction in the total volume of the biofilm. Figure 10This result validates that the PLPP nanosystem can intervene in and clear the *F. nucleatum* biofilm by disrupting the biofilm matrix in the slightly acidic environment of tumors. In contrast, the results of PAsp(Nors)-LA treatment were the same as those of the PLPP group, but the amount of EPS and eDNA in the PAsp group did not decrease, indicating that the LA group introduced into the PLPP nanoparticles can have a good disruptive effect on EPS and eDNA.

[0080] Example 10: Test on the selective dispersion performance of PLPP nanoparticles on biofilms of different bacterial strains. The specific experiments are as follows:

[0081] To investigate the selective dispersion effect of PLPP nanoparticles on pre-formed biofilms of *F. nucleatum*, *S. aureus*, *E. coli*, *L. rhamnosus* GG, and *L. reuteri*, the crystal violet staining assay (CV assay) was used for evaluation. First, after culturing each strain to the logarithmic growth phase, they were inoculated into 96-well plates and cultured under suitable conditions for 24 h to form mature biofilms. After removing airborne bacteria and washing with PBS, PLPP nanoparticle solution (100 μg / mL) or a control was added, and incubation continued for 24 h. After discarding the solution, the cells were stained with 0.1% crystal violet for 15 min, washed with PBS, and the dye was dissolved in 95% ethanol. The absorbance (OD) of each well was then measured. 595 The residual biofilm amount was reflected. Normalization was performed using the PBS group as a control, and the relative biofilm residual rate of different treatment groups was calculated. One-way ANOVA was used to assess the differences in dispersion effects of different strains under PLPP treatment.

[0082] like Figure 11 As shown, 100 μg / mL PLPP nanoparticles exhibited a significant dispersing effect on *F. nucleatum* biofilms, with a marked decrease in crystal violet adsorption. In contrast, PLPP nanoparticles showed almost no dispersing effect on biofilms of *S. aureus*, *E. coli*, *L. rhamnosus* GG, and *L. reuteri* at the same concentration. Only when the PLPP concentration increased to above 400 μg / mL were slight disturbances observed in the biofilm structure of these non-target strains observed, and the residual biofilm amount was still significantly higher than that in the *F. nucleatum* group. This result indicates that PLPP nanoparticles have good selectivity and targeting advantages in dispersing *F. nucleatum* biofilms, enabling precise removal of pathogenic bacterial biofilms without disturbing normal flora.

[0083] Example 11: Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells

[0084] Tumor tissue from experimental mice treated with PLPP nanoparticles was used for immune cell infiltration analysis. The specific procedure was as follows: First, tumor tissue was removed and cut into small pieces. The pieces were then incubated at 37°C for 30-45 minutes using a tumor tissue dissociation kit (containing type IV collagenase and DNase I) to obtain a tumor single-cell suspension. The cell suspension was then filtered through a 70μm cell sieve to remove tissue debris and washed twice with cold PBS.

[0085] For subsequent flow cytometry analysis, the obtained cells were resuspended in PBS containing 2% fetal bovine serum (FBS) and incubated at 4°C for 10 min with anti-mouse CD16 / CD32 antibody (Fc receptor blocker) to prevent non-specific Fc receptor-mediated antibody binding. Next, the cells were stained with various fluorescently labeled surface marker monoclonal antibodies for genotyping, including F4 / 80 and CD86 for M1 macrophages, and F4 / 80 and CD206, CD86, etc., for M2 macrophages. + CD3 and CD8 of T cells, and CD11b and Gr-1 of myeloid-derived suppressor cells (MDSCs). After staining for 30 min in the dark at 4°C, the cells were washed again and resuspended in PBS containing 2% FBS.

[0086] Ultimately, the sample was found in BD Accuri TM Data were acquired using a C6 Plus flow cytometer (BD Biosciences, USA), with at least 10,000 live-cell events detected per sample. The resulting data were analyzed and visualized using FlowJo software (Tree Star Inc.) to assess trends and proportions of immune cell populations across different treatment groups.

[0087] The results are as follows Figure 12 As shown, compared with the F. nucleatum(+)+PBS group, the MDSCs (CD11b) in the PLPP nanoparticle treatment group were significantly lower. + Gr-1 + The proportion decreased by 15.5%. Figure 12 A), while T cells (CD3) + CD8 + The proportion increased by 14.43%. Figure 12 B). PLPP nanoparticle therapy significantly increased the proportion of M1 macrophages (F4 / 80). + CD86 + The percentage of M2 macrophages decreased to 14.05% (F4 / 80). + CD206 + ) to 6.48% Figure 12C)。

Claims

1. A method of preparing a high molecular weight polymeric nanoparticle targeted to a biofilm of Fusobacterium nucleatum, comprising: a) providing a high molecular weight polymeric nanoparticle; b) providing a targeting moiety; c) conjugating the targeting moiety to the high molecular weight polymeric nanoparticle; and d) providing a biofilm of Fusobacterium nucleatum. The method comprises the following steps: (1) ring-opening polymerization of β-benzyloxy aspartic acid N-hydroxysuccinimide ester under alkaline conditions to obtain a polyaspartamide backbone; (2) amino reaction of the polyaspartamide with 3,3'-diaminodipropylamine to obtain an intermediate; (3) grafting of the intermediate with lauric acid, 6-maleimide hexanoic acid, FadA specific peptide and polyethylene glycol CHO-mPEG through covalent bonds to obtain a functional polymer PLPP; (4) self-assembly of the functional polymer into PLPP nanoparticles through hydrophilic-hydrophobic interaction to obtain a nanometer antibacterial material targeting F.nucleatum biofilm.

2. The production method according to claim 1, characterized by, The ring-opening polymerization reaction uses n-decylamine as a catalyst, The reaction temperature is 25 DEG C, and the reaction time is 24 h.

3. The preparation method according to claim 1, characterized in that, The amino reaction of lauric acid, 6-maleimide hexanoic acid and polyaspartamide with 3,3'-diaminodipropylamine obtains an intermediate, and the molar ratio is 0.5:1-1.5:1, the grafting reaction is carried out in N,N-dimethylformamide or dimethyl sulfoxide, and EDC / NHS is used as an activator system.

4. The method of claim 1, wherein, The FadA specific peptide Pep forms a stable connection with the maleimide group on the polymer backbone through click chemistry, and the reaction time is 0.5 h.

5. The preparation method according to claim 1, characterized in that, PEG is aldehyde-terminated CHO-mPEG with a molecular weight of 2 kDa, and the grafting can be achieved by forming a Schiff base bond between the aldehyde group of polyethylene glycol and the amino group on the polymer.

6. The method of claim 1, wherein, The self-assembly process of the nanoparticles adopts a dropwise addition method or a solvent replacement method, and is carried out under ultrasonic assistance, and the formed nanoparticles have a particle size of 150-200 nm.

7. A polymeric nanoparticle targeting a biofilm of Fusobacterium nucleatum, characterized in that, The polymer nanoparticle is prepared by the preparation method of any one of claims 1-6, has a spherical nanoparticle structure, and comprises a hydrophilic shell layer and a hydrophobic core layer.

8. Use of the polymer nanoparticle targeting F.nucleatum biofilm according to claim 7 in the preparation of a medicament for selectively eliminating F.nucleatum biofilm associated with colorectal cancer.

9. Use according to claim 8, characterized in that, The nanoparticle specifically binds to the FadA protein on the surface of F.nucleatum through the FadA specific peptide Pep, and realizes targeted recognition and adhesion. ​ 10. Use according to claim 8, characterized in that, Nanometer antibacterial material can induce the reprogramming of tumor immune microenvironment while eliminating F. nucleatum biofilm, which is manifested by significantly reducing myeloid-derived suppressor cells and M2 macrophages, and promoting M1 macrophages and CD8 + T cell infiltration, thereby reversing immune suppression, enhancing anti-tumor immunity, and ultimately achieving the effect of inhibiting the progression of colorectal cancer.

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