A bioactive assembly, pharmaceutical composition and preparation method

Abstract

The present invention discloses a bioactive assembly, a pharmaceutical composition, and a preparation method. The present invention prepares a bacterial assembly that, by loading a drug (such as, but not limited to, an antibody-drug conjugate) onto the bacterial surface and subsequently using it for tumor treatment, enables the bacterial assembly to colonize hypoxic regions within tumors and provide long-term tumor treatment through the loaded drug and the bacterial metabolites. Furthermore, the bacterial assembly of the present invention has simple synthesis steps, is easy to operate, and has good biosafety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bioactive assembly, a pharmaceutical composition and a preparation method. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is a common cancer that occurs in the head and neck, often seriously affecting the patient's important physiological functions such as voice, breathing, chewing, and swallowing, and even threatening the patient's life. There are many methods to treat oral cancer, including surgery, radiotherapy, chemotherapy, and new targeted therapies and immunotherapy. Currently, monoclonal antibodies have been widely used in clinical practice due to their high specificity and low side effects. Among them, cetuximab is approved by the FDA for the treatment of OSCC patients. However, only a small number of patients respond to this drug, which limits its therapeutic ability. Therefore, it is urgent to develop new and more effective treatments to enhance the effect of drugs and improve the clinical survival rate and treatment effect of oral cancer patients.

[0003] Over the past few years, antibody-drug conjugates (ADCs), a rapidly developing biotherapy consisting of a specific monoclonal antibody chemically linked to a cytotoxic drug, have shown promising results in the treatment of various cancer types. However, due to the complex local tumor microenvironment, ADCs have difficulty penetrating the hypoxic regions within the tumor, and their rapid metabolism compromises their anti-tumor efficacy. Therefore, it is necessary to develop an ADC drug carrier that allows it to penetrate and exert its effect within the tumor for a prolonged period of time, as an innovative therapeutic approach for the treatment of OSCC.

[0004] Furthermore, bacteria have formed complex interactions with their human hosts, and studies have shown that bacteria can be used to treat tumors—a practice known as bacterial therapy. Currently, common bacterial therapies include the use of oncolytic bacteria, bacterial-mediated immunotherapy, and gene editing techniques to engineer bacteria to express small anti-tumor molecules. However, the therapeutic potential of bacteria alone is unsatisfactory, and gene editing techniques are complex and have limited bacterial drug release capabilities. Therefore, improvements to traditional bacterial therapies are needed. Summary of the Invention

[0005] To solve at least some of the problems in the prior art, the present invention provides a bioactive assembly, a pharmaceutical composition, and a preparation method. Specifically, the present invention includes the following contents.

[0006] In a first aspect, the present invention provides a bioactive assembly comprising active particles having peptidoglycan on the surface, a deposition layer coating the active particles, and a linker connected to the deposition layer.

[0007] In certain embodiments, the bioactive assembly according to the present invention further comprises a first drug, which is connected to the assembly via the connector, and the drug is at least one of an antibody or a functional fragment thereof, an antibody derivative, and an antibody-drug conjugate.

[0008] In certain embodiments, according to the bioactive assembly of the present invention, the active particles are probiotics or particles containing a lipid layer, and the particles further contain a second drug or the active particles are capable of producing a second drug.

[0009] In certain embodiments, according to the bioactive assembly of the present invention, the deposition layer comprises an artificially synthesized polymer material or a natural polymer material having adhesive properties, which is selected from at least one of polydopamine, sodium alginate, hyaluronic acid, heparin, chondroitin sulfate, carrageenan, chitosan, chitin and cationic starch.

[0010] In certain embodiments, according to the bioactive assembly of the present invention, the deposition layer comprises an adhesive protein, and the adhesive protein is selected from at least one of mussel foot protein, fibrinogen and collagen.

[0011] In certain embodiments, according to the bioactive assembly of the present invention, the linker comprises Staphylococcus aureus protein A or IgG binding protein, and the IgG binding protein comprises Streptococcus protein G or Peptostreptococcus magnus protein L.

[0012] The second aspect of the present invention provides a pharmaceutical composition comprising the bioactive assembly described in the first aspect and a pharmaceutically acceptable carrier.

[0013] In certain embodiments, the pharmaceutical composition according to the present invention is a topical preparation.

[0014] In certain embodiments, the pharmaceutical composition according to the present invention is an injection.

[0015] A third aspect of the present invention provides a method for preparing a bioactive assembly, comprising:

[0016] (1) Preparing particles with peptidoglycan on the surface;

[0017] (2) coating the surface of the particles with a deposition layer, and connecting a linker for binding to the drug to the deposition layer.

[0018] The bioactive assembly of the present invention is a modification of probiotics by treating the surface of lipid-containing particles, especially probiotics, so that the drug components can be further specifically introduced while maintaining their original activity. In certain embodiments, the bioactive assembly of the present invention is a modification of probiotics, thereby solving the technical problems of poor therapeutic effect of simple bacterial anti-tumor therapy, difficulty of ADC drugs in entering the hypoxic zone inside the tumor, and fast drug metabolism time. In certain embodiments, the present invention designs and prepares a bioactive assembly based on Lactobacillus reuteri, and loads ADC drugs on the surface of bacteria by chemical methods and uses them for subsequent tumor treatment, so that the bacterial assembly can colonize in the hypoxic zone inside the tumor and provide long-term tumor treatment through the loaded ADC and the bacteria's own metabolites (reuterin). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron micrographs of the Lr, Lr-D, Lr-DS, and Lr-DSA bacterial assemblies prepared in the examples are shown.

[0020] Figure 2 Shown are fluorescence confocal images of Lr-DSA bacterial assemblies prepared in the Examples.

[0021] Figure 3 Further shown are the zeta potential measurement results of different bacterial assemblies.

[0022] Figure 4 Shown are the bacterial plating results of different bacterial assemblies.

[0023] Figure 5 The figure shows the results of liquid chromatography-mass spectrometry of reuterin secretion by simple Lactobacillus reuteri and Lr-DSA prepared in the examples. Figure 5 A is the result of Lr-DSA secreting reuterin, B is the result of simple Lactobacillus reuteri secreting reuterin, and C is the spectrum of reuterin standard.

[0024] Figure 6 The flow cytometry results of the bacterial assembly (Lr-DSC) and the control group (Lr-DC) without adding SPA during the synthesis process were shown for co-culture with tumor cells.

[0025] Figure 7 The results of CCK8 assays showing the cell activity of CAL27 tumor cells stimulated by different bacterial assemblies.

[0026] Figure 8 The images (A) and statistical graphs (B) of the Lr-DSA bacterial assembly prepared in the examples and the ADC alone in the treatment of CAL27 tumor spheres are shown.

[0027] Figure 9 Shown are the small animal imaging images (A) and the local mean fluorescence intensity statistical graph (B) of the Lr-DSA bacterial assembly prepared in the example after injection into tumor-bearing mice.

[0028] Figure 10 The graph shows the bacterial smear statistics of the main organs of tumor-bearing mice after the Lr, Lr-D, Lr-DS and Lr-DSA bacterial assemblies prepared in the examples were injected into the mice.

[0029] Figure 11 The results of the safety evaluation of the Lr-DSA bacterial assembly prepared in the Examples on mouse organs are shown.

[0030] Figure 12 The results of the effects of the Lr-DSA bacterial assembly prepared in the examples on the blood routine and biochemical indicators of mice are shown.

[0031] Figure 13 The comparative results of tumor growth in tumor-bearing mice using the Lr, Lr-D, Lr-DS and Lr-DSA bacterial assemblies prepared in the examples are shown.

[0032] Figure 14 The comparative results of the survival of tumor-bearing mice using the Lr, Lr-D, Lr-DS and Lr-DSA bacterial assemblies prepared in the examples are shown. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0036] Bioactive assemblies

[0037] One aspect of the present invention provides a bioactive assembly (hereinafter referred to as the assembly) comprising active particles having peptidoglycan on their surfaces, a sedimentary layer coating the active particles, and a linker attached to the sedimentary layer for drug binding. This is described in detail below.

[0038] Active particles with peptidoglycan on their surface

[0039] In the present invention, particle does not make any restriction, as long as its surface has peptidoglycan.Peptidoglycan structure is important for subsequent introduction of sedimentary layer and does not affect particle (particularly thalline) itself activity.The particle that surface has peptidoglycan can derive from naturally occurring structure (for example, there is the membrane structure of peptidoglycan) or introduce the assembly that peptidoglycan structure forms on particle by suitable method.Particle size is not particularly limited, can be the particle with nanometer level or micron size particle diameter, can have the diameter of 0.1-600 μ m usually.

[0040] The method of introducing the peptidoglycan structure is not particularly limited and can be prepared by biosynthesis or chemical cross-linking. For example, peptidoglycan monomers (such as N-acetylglucosamine, N-acetylmuramic acid and short peptide chains) can be spontaneously assembled under appropriate conditions, or glutaraldehyde, carbodiimide (EDC) or click chemistry (such as DBCO-azide) can be used to achieve covalent cross-linking to connect peptidoglycan monomers or short chains into a stable structure.

[0041] In a preferred embodiment, the particles include bacteria capable of secreting active substances, including Gram-positive bacteria. In a specific embodiment, the particles are Lactobacillus reuteri ( Lactobacillus reuteri ).

[0042] Those skilled in the art will understand that in addition to the Lactobacillus reuteri mentioned above, probiotics such as lactobacilli (including but not limited to Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus salivarius, etc.), bifidobacteria (including but not limited to Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium angularis, Bifidobacterium pseudocatenulatum, etc.) can also be used as particles in the assembly of the present invention.

[0043] Sedimentary layer

[0044] In the present invention, the deposition layer is an artificially synthesized polymer material or a natural polymer material, or an adhesive protein, that can coat the active particles and has reactive groups on its surface. As long as the deposition layer of the present invention can achieve the purpose of not affecting the activity of the particles, especially the bacteria (such as secretory activity or growth, proliferation activity), it can be selected within the above-mentioned material range. In certain embodiments, after coating the deposition layer and subsequently connecting the drug through a connector, the assembly still retains the activity or biological function of the particles, especially the bacteria, in particular, the secretion of active ingredients or functional factors, such as the secretion of reuterin. "Retaining the activity or biological function of the bacteria" means that compared with the untreated bacteria, the amount of reuterin secreted by the bacteria after treatment of the present invention retains at least 50% of the secreted amount, such as 60%, 70%, 80%, 90%, 99%, or even more than 99%.

[0045] To facilitate subsequent connection with a linker (including covalent and / or non-covalent bonding), the surface of the deposited layer preferably has at least one reactive group selected from oxygen-containing groups (hydroxyl, carboxylic acid, ester), nitrogen-containing groups (amino, amide), sulfur-containing groups (sulfonic acid, thiol), and unsaturated double bonds. Alternatively, an adhesive protein may be used, examples of which include, but are not limited to, at least one of mussel foot protein, fibrinogen, and collagen.

[0046] Connectors

[0047] In the present invention, the connector is connected to the deposited layer on the one hand and to the first drug to be loaded on the other hand, and this connection can be carried out in a covalent and / or non-covalent manner. The first drug is not particularly limited, and its examples include but are not limited to at least one of an antibody or its functional fragment, an antibody derivative, and an antibody drug conjugate, in particular an antibody drug conjugate (or conjugate, i.e., ADC). In a preferred embodiment, the first drug is an antibody drug conjugate. In certain embodiments, the first drug is directionally (i.e., not randomly) connected to the particle.

[0048] In the present invention, antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, or murine antibodies. Functional fragments of antibodies include those containing an Fc fragment, such as scFv-Fc. Antibody derivatives are also within the scope of protection of the present invention, and Fc fusion proteins, for example, can be Fc fusion proteins.

[0049] In a preferred embodiment, the linker has a structure capable of specifically binding to the Fc fragment in the above-mentioned substance. In a further preferred embodiment, the linker is an antibody binding protein, including but not limited to Staphylococcus aureus protein A or IgG binding protein, wherein the IgG binding protein is preferably Streptococcus protein G or Peptostreptococcus magnus protein L.

[0050] In the present invention, the lack of a deposition layer prevents the linker (for example, but not limited to, Staphylococcus aureus protein A) from being deposited on the microbial surface, thereby affecting the subsequent binding of the first drug (for example, but not limited to, ADC). The lack of a linker significantly reduces the loading amount of the first drug (for example, but not limited to, ADC). The lack of the first drug (for example, but not limited to, ADC) results in the lack of a key therapeutic drug, thereby affecting the drug treatment or improvement effect (for example, but not limited to, anti-tumor effect) of the entire drug delivery system.

[0051] In the present invention, when the active particle is a particle comprising a lipid layer, the particle further comprises a second drug, which can be any therapeutic substance, such as an active small molecule compound, a natural small molecule drug, an antibiotic, a tumor therapeutic agent, a therapeutic protein, etc. In a preferred embodiment, the second drug is reuterin.

[0052] Pharmaceutical composition

[0053] In one aspect of the present invention, a pharmaceutical composition is provided, which comprises the bioactive assembly and a pharmaceutically acceptable excipient or carrier.

[0054] Pharmaceutically acceptable excipients or carriers are well known in the art and include any excipient that does not itself induce harmful effects on the subject receiving the composition. Those of ordinary skill in the art will be able to determine whether they meet clinical criteria. Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, excipients, wetting agents, emulsifiers, pH buffers, and the like. Diluents include, but are not limited to, water, sterile pyrogen-free water, saline, phosphate-buffered saline, glycerol, and the like.

[0055] The pharmaceutical composition of the present invention can be in any suitable dosage form. For example, an injection, a suspension, an emulsifier, etc. The pharmaceutical composition of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by intramuscular injection, optionally administered intravenously, transdermally, intranasally, orally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered herein can vary to a large extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0056] In a specific embodiment, the pharmaceutical composition of the present invention is a topical formulation.

[0057] In a specific embodiment, the pharmaceutical composition of the present invention is an injection.

[0058] Preparation method

[0059] In one aspect of the present invention, a method for preparing a bioactive assembly is provided, which comprises (1) preparing active particles having peptidoglycan on the surface; (2) coating the surface of the active particles with a deposition layer and connecting a linker to the deposition layer; and optionally (3) connecting a first drug to the linker.

[0060] In step (1) of the present invention, the particles with peptidoglycan on the surface can be isolated and cultured Lactobacillus reuteri, or can be commercially available active Lactobacillus reuteri. The number of Lactobacillus reuteri is not particularly limited and can be (0.1-10)×10 (4-9) CFU, for example (1-5) × 10 (5-9) 、(1-5)×10 (6-9) 、(1-5)×10 (7-9) 、(1-5)×10 8 , for example 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 CFU.

[0061] Step (2) of the present invention is a step of coating the surface of the active particles with a deposition layer and connecting a linker to the deposition layer. The weight ratio of the raw materials from the deposition layer to the linker is 0.1-1:1, preferably 0.1-9:1, and more preferably 0.1-8:1, such as 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, and 0.8:1. The reaction temperature is 20-50°C, preferably 30-45°C, and more preferably 30-40°C, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40°C. The reaction is carried out at a low speed, which means less than 500 rpm, preferably less than 400 rpm, and more preferably less than 300 rpm, such as 280, 260, 240, 220, 200, 150, and 100 rpm. The reaction pH is alkaline, preferably 8-9, such as 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. The reaction time is 5-120 min, preferably 10-100 min, and more preferably 20-60 min, such as 20, 30, 40, 50, or 60 min.

[0062] In step (3) of the preparation method of the present invention, the weight ratio of the raw materials from the drug to the linker is 0.5-2:1, preferably 0.6-1.8:1, for example, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, and 1.8:1. The reaction temperature and the rotation speed during the reaction are as described in step (2) and are not further described here.

[0063] application

[0064] One aspect of the present invention provides pharmaceutical uses, in particular, uses of the assembly of the present invention in the preparation of a medicament for treating or ameliorating a disease in a subject in need thereof.

[0065] As used herein, the terms "treat or prevent" refer to ameliorating a disease or disorder, either before or after it develops. This reduction or prevention is achieved by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique compared to an untreated control group under the same conditions. As used herein, the term "treat" refers to both therapeutic treatment and prophylactic or preventative measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as tumor progression. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and palliation (whether partial or complete), whether detectable or undetectable. "Treatment" also means prolonging survival compared to the expected survival in the absence of treatment. Those in need of treatment include those already suffering from the disease or disorder, as well as those susceptible to developing the disease or disorder, or those in need of prevention of the disease or disorder.

[0066] In the present invention, the disease is a tumor, which includes solid tumors and non-solid tumors. The solid tumor is selected from the group consisting of oral squamous cell carcinoma, breast cancer, prostate cancer, melanoma, osteosarcoma, neuroblastoma, pancreatic cancer, lung cancer, rhabdomyosarcoma, Ewing's sarcoma, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, kidney cancer, head and neck cancer, esophageal cancer, testicular cancer, thyroid cancer, or brain cancer. Non-solid tumors include hematologic tumors such as leukemia and lymphoma, as well as tumors of the nervous system.

[0067] Example

[0068] 1. Experimental Methods

[0069] 1. Preparation of Lr-D and Lr-DS

[0070] Lr-D and Lr-DS were prepared as follows: 1×10 8 Colony-forming units (CFU) of Lactobacillus reuteri were collected by centrifugation at 5000 rpm for 5 minutes and then dispersed in 1 mL of 0.01 M Tris-HCl buffer (pH 8.8) containing 0.5 mg of dopamine hydrochloride or 0.5 mg of dopamine hydrochloride and 2 mg of Protein A (Sino Biological). After shaking at 200 rpm at 37°C for 30 minutes, the mixture was centrifuged at 5000 rpm for 5 minutes and washed three times with PBS. Finally, Lr-D and Lr-DS were resuspended in PBS for subsequent experiments.

[0071] 2. Preparation of Lr-DSC and Lr-DSA

[0072] Lr-DSC and Lr-DSA were prepared in two steps. First, 1×10 8 CFU of Lactobacillus reuteri were dispersed in 1 mL of 0.01 M Tris-HCl (pH 8.8) containing 0.5 mg of dopamine hydrochloride and 2 mg of protein A and shaken at 37°C and 200 rpm for 30 minutes. After the reaction was complete, 1 mg of cetuximab or ADC was added and stirred under the same conditions. The final product was centrifuged at 5000 rpm for 5 minutes and washed three times with PBS to remove free dopamine, protein A, cetuximab, and ADC. Finally, Lr-DSC and Lr-DSA were resuspended in PBS for subsequent experiments.

[0073] 3. Preparation of Lr-DC

[0074] The preparation of Lr-DC refers to the above method. First, 1×10 8 CFU of Lactobacillus reuteri were dispersed in 1 mL of 0.01 M Tris-HCl (pH 8.8) containing 0.5 mg of dopamine hydrochloride and shaken at 37°C and 200 rpm for 30 minutes. After the reaction was complete, 1 mg of cetuximab was added and stirred under the same conditions. The final product was centrifuged at 5000 rpm for 5 minutes and washed three times with PBS to remove free dopamine and cetuximab. Finally, Lr-DCs were resuspended in PBS for subsequent experiments.

[0075] 4. Conjugation of Cetuximab to SMCC-DM1 (ADC Preparation)

[0076] Purified cetuximab (Merck KGaA) was buffer-exchanged into coupling buffer (2 mM EDTA, 50 mM NaCl, 50 mM KPi (potassium phosphate), pH 7.4). SMCC-DM1 (Shanghai Yuanye Biotechnology Co., Ltd.) was dissolved in dimethylacetamide (DMA), and the antibody solution was added to a final SMCC-DM1 / antibody molar ratio of 7:1. The mixture was then gently stirred (50 rpm) at room temperature overnight. Unreacted SMCC-DM1 was removed using an Amicon Ultra-0.5 mL, 10 kDa filter. The prepared ADCs were stored at 4°C for further analysis.

[0077] 5. Identification of reuterin in Lr-DSA culture supernatant

[0078] Reuterin was identified in culture supernatants or 3-hydroxypropionaldehyde using LC-MS. Briefly, lyophilized supernatants were derivatized and analyzed using an LC-MS / MS instrument consisting of an Acquity I-class UPLC system (Waters) and an AB 4500 triple quadrupole mass spectrometer (ABSciex) equipped with an electrospray ionization source. Data acquisition and analysis were performed using MultiQuant 3.0.3 software. Chromatographic separation was performed on a 100 mm × 2.1 mm i.d., 1.7 μm UPLCBEHC18 column (Waters) with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B. The column temperature was 40°C, and the injection volume was 1 μl.

[0079] 6. Characterization of Conjugated Bacteria

[0080] The morphology of L. reuteri and encapsulated L. reuteri was observed using a transmission electron microscope (TEM, JEM-1400, JEOL) at 100 kV. The zeta potential of the five L. reuteri strains was measured using a Zetasizer Nano ZS90 (Malvern). Binding of rhodamine B-labeled protein A and FITC-labeled cetuximab to the bacterial surface was confirmed using laser scanning confocal microscopy (Leica TCSSP8STED).

[0081] 7. Cell Lines and Culture

[0082] The CAL 27 cell line was purchased from the American Type Culture Collection (ATCC), and RFP-expressing CAL 27 cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (Ningbo, China) and cultured in complete Dulbecco's modified Eagle's medium (Procell Life Science & Technology Co., Ltd.) containing 10% fetal bovine serum (Procell Life Science & Technology Co., Ltd.) with or without the addition of 100 μg / ml streptomycin and 100 units / ml penicillin at 37°C in a humidified atmosphere with 5% CO .

[0083] 8. Cell Viability Assay

[0084] Cell viability was measured using a CCK8 assay kit (BS350A, Biosharp) according to the manufacturer's instructions. CAL 27 cells were plated at 1 × 10 3 Cells were seeded in 96-well plates and cultured for 24 hours before treatment. 6 or 1×10 7CFU of bacteria were added to the inoculated cells and cultured for 6 hours in Dulbecco's modified Eagle's medium without antibiotics, followed by washing with PBS. After 24 or 48 hours of culture in Dulbecco's modified Eagle's medium supplemented with 100 μg / ml streptomycin and 100 units / ml penicillin, cell viability was measured using CCK8.

[0085] 9. Survival rate of coupled bacteria

[0086] The survival rate of the coupled bacteria was calculated by counting colonies on MRS agar plates, which is the ratio of viable encapsulated bacteria to unencapsulated bacteria. Equal volumes of a suspension of encapsulated or unencapsulated Lactobacillus reuteri were serially diluted and plated onto MRS agar plates and incubated at 37°C in an anaerobic environment. When colonies were visible on the plates, they were counted using an automated colony counter (ProSource Scientific).

[0087] 10. Affinity of coupled bacteria and cancer cells (effect of adding SPA)

[0088] The affinity of protein A-linked cetuximab-conjugated Lactobacillus reuteri for cancer cells was measured by flow cytometry. Briefly, CAL 27 cells expressing RFP were seeded in well plates and cultured for 24 hours before treatment. 6 CFULr-DC and Lr-DSC (containing FITC-labeled cetuximab) were co-cultured for 4 hours at 37°C. Subsequently, the cells in the well plate were washed and collected into flow tubes, and the fluorescence intensity of the FITC channel was detected by flow cytometer (Cytoflex).

[0089] 11. Establishment of the Bacteria-Spheroid Co-culture (BSCC) System

[0090] The bacteria-spheroid co-culture (BSCC) system was established as follows: 30,000 RFP-expressing CAL 27 cells were seeded in 200 µl of antibiotic-free DMEM medium in a microplate (Corning). The microplate was centrifuged at 100 g for 5 minutes to allow the cells to aggregate at the bottom of the wells. After 4 days of culture, the cells were co-cultured with bacteria. Subsequently, 1×10 FITC-labeled 6 CFULr-DSA or 0.66 µg of ADC was inoculated into wells containing tumor spheroids. After 2 hours of co-culture, the tumor spheroids were repeatedly washed with PBS. Subsequently, 200 µl of DMEM medium containing 10 µg / ml gentamicin was added, and 0.66 µg of FITC-labeled ADC was added back to the treated wells. Tumor spheroids were cultured at 37°C and 5% CO2 in a standard humidified incubator and imaged continuously for 3 days using the IncucyteSX5 Live Cell Imaging and Analysis System using standard brightfield, green, and orange color modules.

[0091] 12. Establishing Animal Models

[0092] All animal experiments were approved by the Institutional Animal Care and Development Committee (IACUC) of Peking University Health Science Center (approval number PUIRB-LA2023108). Six- to eight-week-old female BALB / c nude mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were randomly divided into different groups. A subcutaneous tumor model was established using CAL 27 cells. Each mouse was subcutaneously injected in the right flank with 100 μl of 5 × 10 6 Tumors grew to an average size of approximately 100 mm 3 Then start the experiment.

[0093] 12.1 In vivo anti-tumor efficacy of Lr-ADC

[0094] CAL 27 tumor-bearing mice were injected intratumorally with 50 μl Lr-ADC (1×10 8 CFU / each), Lactobacillus reuteri (1×10 8 CFU / mouse), free ADC (66 μg / mouse), and PBS. Mouse body weight and tumor volume were recorded every other day after treatment. Tumor volume was calculated according to the formula 0.5 × length × (width) 2 Calculation. Survival rate was evaluated based on the tumor volume reaching 1000 mm 3 The end point of death.

[0095] 12.2 Biodistribution and in vivo animal imaging

[0096] Bacterial strains were labeled with DiR (MedChemexpress) membrane staining dye and ADCs were labeled with Cy7-NHS (Aladdin), visualized using an IVIS spectral imaging system (PerkinElmer), and quantified using Living Image software. Images of mice were acquired 24, 48, and 72 hours after dosing. On day 3, mice were euthanized by carbon dioxide, and tumors and organs (heart, liver, spleen, lungs, and kidneys) were removed for imaging, then weighed and homogenized. The homogenate was serially diluted with sterile PBS and plated on MRS agar plates, which were then incubated at 37°C in an anaerobic environment. Colonies were counted when visible on the plates.

[0097] 12.3 Biosafety Testing

[0098] Biosafety testing was performed on 6-8 week old female BALB / c nude mice. The animals were injected with 50 μl of Lr-ADC (1×10 8 CFU / each), Lactobacillus reuteri (1×10 8CFU / mouse), free ADC (66 μg / mouse), and PBS. After 14 days, blood was collected under anesthesia for routine blood tests, and serum was collected for biochemical analysis. Meanwhile, major organs (heart, liver, spleen, lungs, and kidneys) were obtained for H&E staining.

[0099] 2. Experimental Results

[0100] Figure 1 Transmission electron micrographs of Lr, Lr-D, Lr-DS, and Lr-DSA bacterial assemblies prepared in the Examples are shown. It can be seen that after dopamine is coated on the bacteria, a layer of deposit approximately 100-150 nm thick forms on the surface. Further attachment of the ADC drug results in distinct drug particles (scale bar = 500 nm).

[0101] Figure 2 The fluorescence confocal image of the Lr-DSA bacterial assembly prepared in the example is shown, in which rhodamine B-labeled SPA and FITC-labeled ADC can be co-localized on the bacterial surface, proving the successful synthesis of the bacterial assembly. Figure 3 Further shown are the zeta potential measurement results of different bacterial assemblies.

[0102] Figure 4 The bacterial plating results of different bacterial assemblies are shown, and the plate images show that the effect of the preparation process on bacterial viability is negligible. Figure 5 Figures AC further show the liquid chromatography-mass spectrometry results of reuterin secretion by Lactobacillus reuteri alone and Lr-DSA prepared in the examples. The results indicate that the synthesis steps did not significantly affect the bacteria's ability to secrete reuterin. Therefore, the preparation process did not significantly impair bacterial viability, thereby ensuring the functional integrity of the bacteria.

[0103] Figure 6 Flow cytometry results are shown for the co-culture of tumor cells with bacterial assemblies (Lr-DSC) and a control group (Lr-DC) without the addition of SPA during synthesis, in which cetuximab was conjugated to FITC. The results showed a decrease in tumor cell surface fluorescence, demonstrating that the absence of SPA reduced the cetuximab loading capacity on the bacterial assemblies while significantly improving the efficiency of the coated bacteria in targeting oral cancer cells.

[0104] Figure 7 The results of CCK8 assays showing the cell activity of CAL27 tumor cells stimulated by different bacterial assemblies.

[0105] Figure 8Images (A) and statistical graphs (B) of CAL27 tumor spheres treated with Lr-DSA bacterial assemblies prepared in the Examples and ADC alone are shown. The results demonstrate that Lr-DSA bacterial assemblies can rapidly invade tumors and colonize them for long periods, effectively inhibiting tumor sphere growth (scale bar, 400 μm).

[0106] Figure 9 Figure 1 shows images of tumor-bearing mice after injection of the Lr-DSA bacterial assembly prepared in the examples (A) and statistical graphs of the local mean fluorescence intensity (B). All injected materials were linked to fluorescent groups, and the Lr-DSA group showed the slowest fluorescence decay at the tumor site and the longest retention time.

[0107] The viability of CAL27 cell line was detected after 24 and 48 hours, and the conjugated Lr was confirmed at 5×10 6 and 1×10 7 Antiproliferative effect at CFU level. Compared with the control group and the other three Lr groups, Lr-DSA treatment significantly reduced the viability of oral cancer cell lines. Furthermore, almost no surviving cancer cells remained after 48 hours, demonstrating the highly effective tumor-killing ability of Lr-DSA.

[0108] The 3D tumor spheroid model provides an ideal platform for analyzing drug distribution and quantitatively monitoring bacterial positioning. In this example, a bacteria-spheroid co-culture (BSCC) system was established to compare the effects of Lr-DSA and a single ADC drug. In order to study the spatiotemporal dynamics of modified bacteria and drugs in detail, FITC-labeled ADC and Lr-DSA, a CAL27 cell line expressing RFP, and an automated imaging analysis platform were used to quantitatively analyze the distribution of fluorescence intensity in tumor spheroids over time. Both ADC-carrying bacteria and single ADC drugs can infiltrate the spheroids, but ADC-modified bacteria infiltrate faster. Interestingly, ADC-coupled Lr achieved stable colonization for three days, and the spheroid area was reduced by about 20% compared to single ADC treatment. In addition, it was found that ADC-loaded bacteria were located in the necrotic and hypoxic areas of the spheroid core (displayed by hypoxia probe dye), while ADC was mainly distributed at the edge of the spheroid.

[0109] Figure 10 Figure 2 shows bacterial colonization statistics of major organs of tumor-bearing mice after injection of Lr, Lr-D, Lr-DS, and Lr-DSA bacterial assemblies prepared in the Examples. The results show that the bacteria did not migrate to major organs but were enriched in the tumor area. Figure 11 The results of the safety evaluation of the Lr-DSA bacterial assembly prepared in the Examples on mouse organs are shown. Figure 12 The results of the effects of the Lr-DSA bacterial assembly prepared in the examples on the blood routine and biochemical indicators of mice are shown. Figure 13The comparative results of tumor growth in tumor-bearing mice using the Lr, Lr-D, Lr-DS and Lr-DSA bacterial assemblies prepared in the examples are shown. Figure 14 The comparative results of the survival of tumor-bearing mice using the Lr, Lr-D, Lr-DS and Lr-DSA bacterial assemblies prepared in the examples are shown.

[0110] To investigate the biodistribution and retention of bacteria or drugs, imaging and plate counts were performed in CAL27 tumor-bearing mice via intratumoral injection. All bacterial groups were administered with a dose of 1 × 10 8 CFU, the equivalent dose of the ADC group was 66 μg. The fluorescence intensity of the ADC-treated group in the tumor model decayed rapidly over time, while that of the bacterial group (including ADC-coated bacteria) decayed more slowly. Fluorescence intensity statistics showed that the ADC group dropped sharply by approximately 75% within 24 hours, while the bacterial group decreased slightly. Immunofluorescence staining images were used to further investigate the intratumoral distribution of ADC or Lr-DSA at 72 hours. These results indicate that the low decay efficiency, long residence time, and excellent colonization ability of the bacterial strain together give bacterial cancer therapy significant therapeutic potential. In addition, after homogenization of major organs and tumor tissues and smearing on MRS agar plates for CFU counts, colony formation was observed only in tumor tissues. This indicates that the bacteria targeted tumor tissues with necrotic and hypoxic microenvironments.

[0111] Although Lr as a probiotic does not cause tissue irritation, drug-loaded Lr may raise concerns. Therefore, a series of biosafety experiments were conducted by tail vein injection of equal doses. After 14 days, H&E staining of the heart, liver, spleen, lungs, and kidneys revealed no significant organ damage. Peripheral blood was also collected for hematological examination, revealing no significant differences in white blood cell (WBC) counts, lymphocytes, granulocytes, ALT, and AST.

[0112] The therapeutic effect of Lr-DSA was evaluated by subcutaneously implanting CAL27 oral cancer xenograft tumors in nude mice. 3 At day 0 and day 7, mice were injected intratumorally with 50 μl Lr-ADC (1×10 8 CFU / each), Lactobacillus reuteri (1×10 8 CFU / mouse), free ADC (66 μg / mouse), and PBS. Body weight and tumor size were recorded every other day. Results showed that tumor growth was significantly inhibited in the Lr-DSA group, and even cases of tumor disappearance were observed. In addition, Lr-DSA treatment significantly prolonged the survival of CAL27 tumor-bearing mice. Therefore, Lr-DSA provides an effective strategy for in vivo tumor treatment.

[0113] In summary, while Lr microbes (i.e., Lactobacillus reuteri without any drug attached) can produce anti-tumor metabolites, they do not exhibit strong anti-tumor effects when acting alone on cancer cells. The Lr-D microbial system, in which only dopamine is coated on the surface of Lactobacillus reuteri, exhibits similar effects to Lactobacillus reuteri alone, but Lr-D also fails to demonstrate strong anti-tumor effects. The Lr-DS microbial system, in which only dopamine and protein A are coated on the surface of Lactobacillus reuteri, also exhibits similar effects to Lactobacillus reuteri alone, but Lr-DS also fails to demonstrate strong anti-tumor effects. ADC drugs, in which the drug is pure and without a microbial carrier, exhibit short retention times and difficulty penetrating hypoxic areas within tumors. While ADC drugs alone can exhibit some anti-tumor effects, their efficacy is significantly reduced compared to Lr-DSA.

[0114] In this invention, Lactobacillus reuteri is a probiotic that bypasses the immune system and is difficult to eliminate. This overcomes the drawbacks of ADC drugs, which have difficulty penetrating the hypoxic zone at the center of tumors and are eliminated too quickly. As an anaerobic bacterium, Lactobacillus reuteri can carry anti-tumor drugs and colonize in the hypoxic zone at the center of tumors, providing long-term ADC release. Metabolites secreted by Lactobacillus reuteri can also assist in killing tumor cells. Furthermore, the Lr-DSA bacterial assembly has simple synthesis steps, is easy to operate, and is biosafe.

[0115] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A bioactive assembly with anti-tumor effect, characterized in that: The assembly comprises active particles having peptidoglycan on the surface, a sediment layer coating the active particles, a connector connected to the sediment layer, and a first drug, wherein the first drug is connected to the assembly via the connector, and the drug is an antibody-drug conjugate, and the particles are Lactobacillus reuteri capable of secreting a second drug, reuterin; The deposited layer is polydopamine; The linker is Staphylococcus aureus protein A.

2. A pharmaceutical composition, characterized in that The invention comprises the bioactive assembly according to claim 1 and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that It is a preparation for topical administration.

4. The pharmaceutical composition according to claim 2, characterized in that It is an injection.

5. A method for preparing a bioactive assembly with anti-tumor effect, characterized in that: include: (1) preparing active particles having peptidoglycan on the surface, wherein the particles are Lactobacillus reuteri capable of secreting a second drug, reuterin; (2) coating the surface of the active particles with a deposition layer, and connecting a linker to the deposition layer, wherein the deposition layer is polydopamine and the linker is Staphylococcus aureus protein A; and (3) Connecting a first drug to the linker, wherein the drug is an antibody-drug conjugate.

6. The method for preparing a bioactive assembly with anti-tumor effect according to claim 5, characterized in that: The weight ratio of the raw materials of the deposited layer to the connecting object is 0.1-1:

1.

7. The method for preparing a bioactive assembly with anti-tumor effect according to claim 5, characterized in that: The reaction temperature of step (2) is 20-50°C.

8. The method for preparing a bioactive assembly with anti-tumor effect according to claim 5, characterized in that: The pH of the reaction in step (2) is alkaline.

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