Lanthanum protein engineered bacterial embolism microspheres as well as preparation method and application thereof

Abstract

The invention discloses lanthanum protein engineered bacterial embolism microspheres as well as a preparation method and application thereof, and belongs to the technical field of biological medicine agents. The lanthanum protein engineered bacterial embolism microsphere is prepared by the following steps: preparing a microemulsion from a functional water phase containing a microsphere matrix, functional bacteria and a photo-crosslinking agent and an oil phase containing an emulsifier and an oil phase component, and carrying out a cross-linking reaction. According to the lanthanum protein engineered bacterial embolism microspheres prepared by the method, lanthanum protein is specifically combined with radionuclide, so that rapid and efficient radioactive labeling with LanM protein on the surfaces of bacteria is realized, and labeling is realized at any time when the lanthanum protein engineered bacterial embolism microspheres are used. The radionuclide-containing microspheres can be accurately delivered to a tumor blood supply artery through an intervention means, and after the radionuclide-containing microspheres are retained in a tumor area, multiple treatment functions are achieved: PEGDA hydrogel provides physical embolism, slowly releases bacteria PAMPs and activates immunity, meanwhile, close-range radiotherapy is achieved through lanthanum protein anchoring 90Y, and finally the'embolism-biology-radiotherapy 'triple synergistic anti-tumor effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and particularly relates to a lanthanum protein-engineered bacterial embolization microsphere, its preparation method, and its application. Background Technology

[0002] Bacterial-mediated tumor therapy utilizes the ability of certain anaerobic or facultative anaerobic bacteria to target and colonize hypoxic necrotic areas within solid tumors, transforming them into living "drug factories" for targeted tumor therapy. With advancements in synthetic biology, engineered bacteria can now efficiently express various therapeutic molecules (such as cytotoxins, immunomodulatory factors, and oncolytic enzymes), demonstrating potential surpassing traditional chemotherapy and radiotherapy. However, intravenous injection of free engineered bacteria faces two major challenges: systemic biosafety risks (such as sepsis) and low tumor-targeting enrichment efficiency.

[0003] To address these challenges, encapsulating engineered bacteria in microspheres, especially those for transarterial embolization, has emerged as a new research direction. This "bacterial embolization microsphere" strategy aims to physically confine bacteria to the tumor region while utilizing the microsphere's carrier function to achieve controlled drug release. Achieving precise temporal and tertiary control of the encapsulated bacterial life activities is crucial for improving therapeutic efficacy and safety. While currently available glass and resin microspheres (such as TheraSphere® and SIR-Spheres®) offer proven efficacy and mature technology, their radionuclide labeling relies on matrix embedding or ion exchange, requiring specialized equipment and complex processes. Furthermore, labeling efficiency is easily affected by factors such as pH and dosage. These microspheres also present limitations due to high cost, high technical barriers, strict indications, significant side effects, and inherent material properties. 90 The main reason why radiation embolization therapy is not widely adopted is Y-radioembolization. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a lanthanum protein-engineered bacterial embolization microsphere, its preparation method and application, wherein the lanthanum protein in the microsphere specifically binds to a series of radionuclides for precise delivery to the tumor, and after remaining in the tumor area, it achieves multiple therapeutic functions.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides lanthanum protein-engineered bacterial embolization microspheres, which are formed by cross-linking a microemulsion made from a functional aqueous phase containing a microsphere matrix, functional bacteria, and a photocrosslinking agent and an oil phase containing an emulsifier and an oil phase component; the functional bacteria are lanthanum protein-engineered bacteria; the target gene sequence of the lanthanum protein-engineered bacteria is shown in SEQ ID NO.1.

[0006] Preferably, the microsphere matrix comprises polyethylene glycol diacrylate (PEGDA); the photocrosslinking agent comprises TPO-L photoinitiator.

[0007] Preferably, the emulsifier includes Triton X-100, and the oil phase component includes polydimethylsiloxane oil (PDMS).

[0008] The present invention provides a method for preparing lanthanum protein-engineered bacterial embolization microspheres, comprising the following steps: mixing a microsphere matrix with a photocrosslinking agent to obtain a polymer precursor, and then mixing it with a lanthanum protein-engineered bacterial suspension to obtain a functional aqueous phase; mixing an emulsifier with an oil phase component to obtain an oil phase; mixing the functional aqueous phase and the oil phase to obtain an emulsion; and irradiating the emulsion with light to form hydrogel microspheres.

[0009] Preferably, the mass ratio of the microsphere matrix to the photocrosslinking agent is 95-103:1; the volume ratio of the polymer precursor to the lanthanum protein-engineered bacterial suspension is 20-30:70-80; and the concentration of the lanthanum protein-engineered bacterial suspension is 0.05-0.15 × 10⁻⁶. 11 Cells / mL; the mass ratio of emulsifier to oil phase component is 1:95-103.

[0010] Preferably, the illumination conditions include: irradiation by a 360-370nm ultraviolet lamp and an irradiation power density of 2-6W / cm². 2 Irradiation time: 110-130s.

[0011] This invention provides a functionalized radioactive bacterial embolization microsphere, which is prepared by combining the lanthanum protein-engineered bacterial embolization microsphere with a radionuclide.

[0012] Preferably, the radionuclide includes 90 Y, lanthanide radionuclides and actinide radionuclides.

[0013] The present invention provides a method for preparing the functionalized radioactive bacterial embolization microspheres, comprising the following steps: mixing lanthanum protein-engineered bacterial embolization microspheres with a radionuclide.

[0014] This invention provides the application of the lanthanum protein-engineered bacterial embolization microspheres or the functionalized radioactive bacterial embolization microspheres in the preparation of antitumor products.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides lanthanum protein-engineered bacterial embolization microspheres. The core of this invention involves encapsulating lanthanum protein-engineered bacteria within a microemulsion matrix hydrogel microsphere using a bulk emulsion method. These microspheres specifically bind to radionuclides, forming a microsphere carrier that synergistically combines physical embolization, radiotherapy, and biotherapy. These functional microspheres can be precisely delivered to the tumor-feeding artery via interventional methods (such as through an arterial catheter). Once stationary in the tumor area, they achieve multiple therapeutic functions: the PEGDA hydrogel provides physical embolization and sustainably releases bacterial PAMPs, activating the immune system, while simultaneously anchoring via lanthanum protein. 90 Y achieves brachytherapy, ultimately achieving a triple synergistic anti-tumor effect of "embolization-biological-radiotherapy".

[0016] Furthermore, the functionalized radioactive bacterial embolization microspheres prepared in this invention achieve a triple synergy of embolization, radiotherapy, and immunotherapy, surpassing existing single-function microspheres that only possess the dual functions of physical embolization and radiotherapy; the microspheres of this invention simultaneously achieve: physical embolization (PEGDA hydrogel blocking tumor blood supply) and targeted radiotherapy (LanM anchoring). 90 The combination of gamma and beta rays for precise tumor killing, and immune activation (microspheres release bacterial PAMPs to activate local tumor immunity) provides a triple synergistic effect. Compared to the single / dual functions of traditional microspheres, this results in a more significant anti-tumor effect, achieving a comprehensive advantage of efficient labeling, synergistic therapy, safe targeting, and low-cost preparation. This represents a breakthrough improvement in the field of radioembolization therapy.

[0017] Furthermore, the technical solution of this invention addresses the safety and targeting issues of free engineered bacteria. Intravenous injection of free engineered bacteria easily induces sepsis and systemic immune storms, and the tumor-targeting enrichment rate is low (usually <10%). This invention physically encapsulates engineered bacteria with PEGDA microspheres and precisely delivers them to the tumor area via arterial intervention. This not only restricts the release of bacteria to the tumor site (avoiding systemic spread), but also improves the bacterial retention rate at the tumor site through microsphere embolization, while simultaneously achieving LanM anchoring. 90 Y further enhances tumor targeting (tumor uptake rate >90% in mouse models).

[0018] The technical solution of this invention has a simple preparation process and controllable cost, which lowers the technical threshold. Existing glass / resin microspheres rely on precise synthesis processes (such as glass melting / resin porosification), and the production equipment is expensive. This invention adopts the bulk emulsion method and photocrosslinking, the raw materials (PEGDA, TPO-L) are cheap and readily available, the process steps are short (mixing-emulsification-UV crosslinking <10min), and it is easy to scale up production, which helps to reduce treatment costs and expand the coverage of indications. Attached Figure Description

[0019] Figure 1 This is a map of the recombinant plasmid.

[0020] Figure 2Characterization results of lanthanum protein-engineered bacterial embolization microspheres.

[0021] Figure 3 Labeling of lanthanum protein-engineered bacterial embolism microspheres under different conditions 90 The effect of Y is shown in the figure.

[0022] Figure 4 For containing 90 The effect of each group of drugs on tumors in tumor-bearing mice is shown in the figure.

[0023] Figure 5 For containing 90 Effects of Y-functionalized radioactive bacterial embolization microspheres on tumor volume changes and mouse survival curves in mouse models.

[0024] Figure 6 For containing 90 Figure 1. Functionalized radioactive bacterial embolization microspheres of Y for interventional embolization therapy of in situ hepatocellular carcinoma in rabbits. Detailed Implementation

[0025] This invention provides lanthanum protein-engineered bacterial embolization microspheres. These microspheres are formed by cross-linking a functional aqueous phase containing a microsphere matrix, functional bacteria, and a photocrosslinking agent with an oil phase containing an emulsifier and oil phase components. The functional bacteria are lanthanum protein-engineered bacteria. The target gene sequence of the lanthanum protein-engineered bacteria is shown in SEQ ID NO.1. The genetically engineered lanthanum protein-engineered bacteria of this invention express lanthanum ion-binding protein (LanM), which can specifically bind to radioactive nuclides. 90 Y enables in-situ enrichment and stable anchoring of radionuclides within the microspheres.

[0026] In this invention, the microsphere matrix comprises polyethylene glycol diacrylate; the photocrosslinking agent comprises TPO-L photoinitiator; the emulsifier comprises Triton X-100; and the oil phase component comprises polydimethylsiloxane oil. The PEGDA in this invention has a molecular weight of 565-585 Da, preferably 570-580 Da, and more preferably 572 Da, and serves as a biocompatible carrier for encapsulating bacteria, providing physical embolization and controlling the bacterial release rate. The viscosity of the polydimethylsiloxane oil in this invention is 5-15 cSt (25°C), preferably 8-13 cSt, and more preferably 10 cSt.

[0027] The present invention also provides a method for preparing the lanthanum protein-engineered bacterial embolization microspheres, comprising the following steps: mixing the microsphere matrix with a photocrosslinking agent to obtain a polymer precursor, and then mixing it with a lanthanum protein-engineered bacterial suspension to obtain a functional aqueous phase; mixing an emulsifier with an oil phase component to obtain an oil phase; mixing the functional aqueous phase and the oil phase to obtain an emulsion; and irradiating the emulsion with light to form hydrogel microspheres.

[0028] In this invention, the mass ratio of the microsphere matrix to the photocrosslinking agent is 95-103:1, preferably 98-101:1, and more preferably 99:1; the volume ratio of the polymer precursor to the lanthanum protein-engineered bacterial suspension is 20-30:70-80, preferably 22-28:72-78, and more preferably 25:75; the concentration of the lanthanum protein-engineered bacterial suspension is 0.05-0.15 × 10⁻⁶. 11 Cells / mL, preferably 0.08-0.12 × 10⁻⁶. 11 Cells / mL, more preferably 0.1 × 10⁻⁶ cells / mL. 11 Cells / mL; the mass ratio of emulsifier to oil phase component is 1:95-103, preferably 1:98-101, and more preferably 1:99.

[0029] In this invention, the illumination conditions include: irradiation by a 360-370nm ultraviolet lamp and an irradiation power density of 2-6W / cm². 2 Irradiation time: 110-130s; preferably irradiation with a wavelength of 363-367nm ultraviolet lamp; irradiation power density: 3-5W / cm². 2 Irradiation time 113-127s; more preferably, irradiation with a 365nm wavelength ultraviolet lamp and an irradiation power density of 4W / cm². 2 The irradiation time is 120 seconds. After irradiation by the ultraviolet lamp, the acrylate double bond cross-linking reaction between PEGDA molecules is initiated, forming solid PEGDA hydrogel microspheres, in which bacteria are encapsulated.

[0030] This invention provides a functionalized radioactive bacterial embolization microsphere, prepared by combining the aforementioned lanthanum protein-engineered bacterial embolization microsphere with a radionuclide. The radionuclide of this invention includes... 90 Y, lanthanide radionuclides and actinide radionuclides, preferably 90 Y. This invention utilizes the lanthanum binding sites on the surface of lanthanum-engineered bacterial embolization microspheres after (or during) their preparation (or use), to... 90 The specific binding of Y and lanthanum ions makes... 90 Y is specifically anchored to the surface of bacteria inside the microspheres through lanthanum ion mediation, forming radioactive embolized microspheres. 90 The beta rays released during the decay of Y can directly kill surrounding tumor cells. At the same time, bacterial PAMPs are released in the tumor microenvironment and activate the immune system, achieving a synergistic effect between radiotherapy and immunotherapy.

[0031] This invention also provides a method for preparing the functionalized radioactive bacterial embolization microspheres, comprising the following steps: mixing lanthanum protein-engineered bacterial embolization microspheres with a radionuclide. In this invention, the lanthanum protein-engineered bacterial embolization microspheres are dissolved in a sodium acetate buffer solution, and then yttrium trichloride is added. 90 Incubate solution Y, shake at room temperature for 10-30 min, centrifuge at 2500-3500 rpm, and remove unbound molecules from the supernatant. 90 Y, the precipitate obtained is containing 90 Y-functionalized radial bacterial embolization microspheres. The sodium acetate buffer solution of this invention has a pH of 3-8, preferably 3.5-7.5, more preferably 4-7. This invention utilizes lanthanum protein-mediated binding to... 90 The γ protein is anchored to the bacterial surface, ultimately yielding microspheres capable of simultaneously releasing radiotherapy and immunotherapy effects. This invention utilizes lanthanum protein-engineered LanM protein for specific chelation of the bacterial surface. 90 Y, only microspheres and 90 Y is easy to mix (nearly 100% labeling rate within 1 minute), enabling labeling on demand, while exhibiting strong pH adaptability (maintaining a labeling rate of over 85% even at pH 3-8) and good dose tolerance (high doses). 90 The labeling rate is stable under Y, which greatly reduces labeling costs and operational barriers.

[0032] The present invention also provides the application of the lanthanum protein-engineered bacterial embolization microspheres or the functionalized radioactive bacterial embolization microspheres in the preparation of antitumor products.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the following embodiments are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] Example 1 1. A method for preparing lanthanum protein-engineered bacterial embolization microspheres, comprising the following steps: Step 1: Preparation of polymer precursor (aqueous phase basis) Weigh 99% w / w polyethylene glycol diacrylate (PEGDA, average molecular weight 575, purity 99%) and 1% w / w TPO-L photoinitiator as polymer precursors, and stir magnetically (300 rpm, room temperature) for 30 minutes in the dark to form a homogeneous and transparent polymer precursor solution, which is denoted as the aqueous precursor.

[0037] Step 2: Preparation of concentrated bacterial suspension (functional phase) (1) The construction process of lanthanum protein engineered bacteria is as follows: The target protein Clya-His-HA-LanM sequence was designed as shown in SEQ ID NO.1. Using Ncol and EcoRI double restriction sites, the target protein sequence was inserted into the plasmid vector pBAD (obtained from Suzhou Genewise Biotechnology Co., Ltd.) via homologous recombination to construct a complete recombinant plasmid. The recombinant plasmid map is shown below. Figure 1 As shown. The recombinant plasmid constructed above was transformed into 200 μL of VNP20009 competent bacteria (the ratio of plasmid to competent cells was less than 1:10), incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, then incubated on ice for 5 min. After adding 800 μL of antibiotic-free medium and culturing for 1 h, 200 μL was spread on ampicillin (Amp) resistant solid medium and cultured overnight at 37℃. The lanthanum protein-engineered VNP20009 was obtained by screening. Single colonies were picked and cultured in LB medium containing Amp at 37℃ and 200 rpm. When the bacterial OD=0.4-0.6, the culture was preserved with glycerol and stored at -80℃.

[0038] (2) Lanthanum protein-engineered bacteria (VNP20009 expressing LanM protein) were inoculated 1:1000 into LB medium (containing Amp antibiotic for screening), and cultured overnight at 37°C with shaking at 200 rpm. The next day, they were inoculated 1:100 into LB medium (containing Amp antibiotic) until the logarithmic growth phase (OD200). 600 =0.6-0.8), add 1:100 of 20% Ara to induce expression of the target protein for 4 h, centrifuge (5000 rpm, 5 min) to collect the cell pellet, wash twice with MES (2-morpholinoethanesulfonic acid, pH 6, 10 mM) solution and resuspend, finally adjust to 0.1 × 10⁻⁸ with MES. 11 A concentrated suspension of cells / mL is denoted as the bacterial phase.

[0039] Step 3: Mix the aqueous phase and the bacterial phase Mix the polymer precursor (25% v / v, 1 mL) from step 1 with the bacterial suspension (75% v / v, 3 mL) from step 2 at a volume ratio, and stir in the dark (200 rpm, room temperature) for 10 minutes to ensure that the bacteria are uniformly dispersed in the PEGDA hydrogel precursor. The mixture is recorded as the functional aqueous phase (4 mL).

[0040] Step 4: Preparation of microspheres using bulk emulsion method (1) Preparation of oil phase: In a 40mL vial of silanized borosilicate, 1% w / w Triton X-100 surfactant (0.04g) and 99% w / w polydimethylsiloxane oil (10cSt PDMS oil at 25℃, 3.46g) were mixed evenly to form an oil phase.

[0041] (2) Emulsion formation: According to the volume ratio of functional aqueous phase to oil phase of 1:7, the functional aqueous phase (total volume adjusted according to the bottle capacity, usually 5-10mL) from step 3 is quickly added dropwise to the oil phase, and immediately stirred with a glass rod or magnetic stirrer for 15 seconds (speed about 1000rpm) to form a uniform W / O (water / oil) emulsion (droplet diameter about 50-200μm).

[0042] (3) Photocrosslinking curing: Immediately expose the vial containing the emulsion to a UV lamp (4W / cm²). 2 Irradiation at 365nm for 120 seconds induces acrylate double bond cross-linking between PEGDA molecules by TPO-L, forming solid PEGDA hydrogel microspheres, in which bacteria are encapsulated.

[0043] Step 5: Post-processing and purification (1) Removal of residual oil phase: The polymerized microsphere suspension was vacuum filtered through a 20μm nylon mesh filter to remove unreacted PDMS oil and large oil droplets.

[0044] (2) Washing: The microspheres were rinsed repeatedly with 0.9% w / v NaCl solution 10 times (stirring for 5 minutes each time, centrifuging and discarding the supernatant) until the filtrate was free of oil stains.

[0045] (3) Storage: The final microspheres were stored in 10mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.4) at 4°C in the dark for later use.

[0046] 2. A kind containing 90 The preparation of functionalized radioactive bacterial embolization microspheres of Y is as follows: 1 mg of the lanthanum protein-engineered bacterial embolization microspheres prepared above was dissolved in 100 μL of 0.5 M sodium acetate buffer solution (pH 5.5), and then 100 μCi of yttrium trichloride was added. 90 [Y] solution (purchased from Chengdu Newrite Medical Technology Co., Ltd., item number: 2024070601), shaken at room temperature for 10 min, centrifuged at 3000 rpm to remove unbound molecules from the supernatant. 90 Y, the precipitate obtained is containing 90 Y-functionalized radioactive bacterial embolization microspheres.

[0047] Example 2 A method for preparing lanthanum protein-engineered bacterial embolization microspheres, comprising the following steps: Step 1: Preparation of polymer precursor (aqueous phase basis) Weigh 98% w / w polyethylene glycol diacrylate (PEGDA, average molecular weight 575, purity 99%) and 2% w / w TPO-L photoinitiator as polymer precursors, and stir magnetically (250 rpm, room temperature) for 25 minutes in the dark to form a homogeneous and transparent polymer precursor solution, which is denoted as the aqueous precursor.

[0048] Step 2: Preparation of concentrated bacterial suspension (functional phase) Finally, it was adjusted to 0.05×10 using MES. 11 The concentrated suspension was prepared at 1 cell / mL, and the remaining steps were the same as in Example 1.

[0049] Step 3: Mix the aqueous phase and the bacterial phase The polymer precursor (20% v / v) from step 1 and the bacterial suspension (80% v / v) from step 2 were mixed at a volume ratio and stirred in the dark (150 rpm, room temperature) for 8 minutes to ensure that the bacteria were uniformly dispersed in the PEGDA hydrogel precursor. The mixture was designated as the functional aqueous phase.

[0050] Step 4: Preparation of microspheres using bulk emulsion method (1) Preparation of oil phase: In a 40 mL vial of silanized borosilicate, mix 0.5% w / w Triton X-100 surfactant and 99.5% w / w polydimethylsiloxane oil (10 cSt PDMS oil) evenly to form an oil phase.

[0051] (2) Emulsion formation: The functional aqueous phase in step 3 is rapidly added to the oil phase at a volume ratio of 1:5. The mixture is then immediately stirred with a glass rod or magnetic stirrer for 10 seconds (approximately 900 rpm) to form a uniform W / O (water / oil) emulsion (droplet diameter approximately 50-200 μm).

[0052] (3) Photocrosslinking curing: Immediately expose the vial containing the emulsion to a UV lamp (2W / cm²). 2 Irradiation at 355nm for 110 seconds induces acrylate double bond cross-linking between PEGDA molecules by TPO-L, forming solid PEGDA hydrogel microspheres, in which bacteria are encapsulated.

[0053] Step 5: Post-processing and purification steps are the same as in Example 1.

[0054] A kind of 90 The preparation of functionalized radioactive bacterial embolization microspheres of Y is as follows: 0.5 mg of the prepared lanthanum protein-engineered bacterial embolization microspheres were dissolved in 100 μL of 0.5 M sodium acetate buffer (pH 4.5), and then 90 μCi of yttrium trichloride was added. 90[Y] solution (purchased from Chengdu Newrite Medical Technology Co., Ltd., item number: 2024070601), shaken at room temperature for 8 min, centrifuged at 2500 rpm to remove unbound molecules from the supernatant. 90 Y, the precipitate obtained is containing 90 Y-functionalized radioactive bacterial embolization microspheres.

[0055] Example 3 A method for preparing lanthanum protein-engineered bacterial embolization microspheres, comprising the following steps: Step 1: Preparation of polymer precursor (aqueous phase basis) Weigh 99.5% w / w polyethylene glycol diacrylate (PEGDA, average molecular weight 575, purity 99%) and 0.5% w / w TPO-L photoinitiator as polymer precursors, and stir magnetically (350 rpm, room temperature) for 35 minutes in the dark to form a homogeneous and transparent polymer precursor solution, which is denoted as the aqueous precursor.

[0056] Step 2: Preparation of concentrated bacterial suspension (functional phase) Finally, it was adjusted to 0.15×10 using MES. 11 The concentrated suspension was prepared at 1 cell / mL, and the remaining steps were the same as in Example 1.

[0057] Step 3: Mix the aqueous phase and the bacterial phase The polymer precursor (30% v / v) from step 1 and the bacterial suspension (70% v / v) from step 2 were mixed at a volume ratio and stirred in the dark (250 rpm, room temperature) for 12 minutes to ensure that the bacteria were uniformly dispersed in the PEGDA hydrogel precursor. The mixture was designated as the functional aqueous phase.

[0058] Step 4: Preparation of microspheres using bulk emulsion method (1) Preparation of oil phase: In a 40 mL vial of silanized borosilicate, 1.5% w / w Triton X-100 surfactant and 98.5% w / w polydimethylsiloxane oil (10 cSt PDMS oil) were mixed evenly to form an oil phase.

[0059] (2) Emulsion formation: The functional aqueous phase in step 3 is rapidly added to the oil phase at a volume ratio of 1:8. Immediately stir with a glass rod or magnetic stirrer for 20 seconds (about 1100 rpm) to form a uniform W / O (water / oil) emulsion (droplet diameter about 50-200 μm).

[0060] (3) Photocrosslinking curing: Immediately expose the vial containing the emulsion to a UV lamp (6W / cm²). 2Irradiation at 375nm for 130 seconds induces acrylate double bond cross-linking between PEGDA molecules by TPO-L, forming solid PEGDA hydrogel microspheres, in which bacteria are encapsulated.

[0061] Step 5: Post-processing and purification steps are the same as in Example 1.

[0062] A kind of 90 The preparation of functionalized radioactive bacterial embolization microspheres of Y is as follows: 1.5 mg of the prepared lanthanum protein-engineered bacterial embolization microspheres were dissolved in 100 μL of 0.5 M sodium acetate buffer (pH 4.5), and then 300 μCi of yttrium trichloride was added. 90 [Y] solution (purchased from Chengdu Newrite Medical Technology Co., Ltd., item number: 2024070601), shaken at room temperature for 12 min, centrifuged at 3500 rpm to remove unbound molecules from the supernatant. 90 Y, the precipitate obtained is containing 90 Y-functionalized radioactive bacterial embolization microspheres.

[0063] Example 1: Characterization and determination of lanthanum protein-engineered bacterial embolization microspheres Grouping: -VNP LanM Group: Engineered bacteria without Ara induction; samples were obtained from the concentrated cell suspension prepared in step 2 of Example 1 without Ara induction. +VNP LanM Group: Engineered bacteria induced by Ara, samples were obtained by inducing Ara in the concentrated cell suspension prepared in step 2 of Example 1. PEGDA@VNP LanM Group: Lanthanum protein-engineered bacterial embolization microspheres, prepared in Example 1.

[0064] Western blot was used to characterize the target protein in the three groups of samples. The results are as follows: Figure 2 As shown in a, the target protein has a molecular weight of 47 kDa, and engineered bacteria without Ara-induced expression do not express the target protein.

[0065] The morphology of the prepared protein-engineered bacterial embolic microspheres was characterized under an optical microscope, and the results are as follows: Figure 2 As shown in b, the size is appropriate, the morphology is uniform, and the average particle size is 40 μm.

[0066] The prepared lanthanum protein-engineered bacterial embolization microspheres were characterized by transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown in Figure c, engineered bacteria were observed inside the microspheres.

[0067] The prepared lanthanum protein-engineered bacterial embolization microspheres were stained with propidium iodide (PI), and the results are as follows: Figure 2d indicates that the bacteria are all dead, not alive, indicating that the prepared microspheres have good safety.

[0068] The prepared lanthanum protein-engineered bacterial embolization microspheres were characterized by scanning electron microscopy (SEM), and the results are as follows: Figure 2 e indicates that the morphology is uniform and the size is around 40μm, which is suitable for embolization.

[0069] Experimental Example 2: Labeling of Lanthanum Protein-Engineered Bacterial Embolism Microspheres under Different Conditions 90 Determination of the influence of Y In step 3 of Example 1, instead of adding 75% bacterial suspension, 75% MES buffer was added. The remaining steps were the same as in Example 1, and microspheres without added bacteria were prepared, which were designated as the PEGDA group.

[0070] The engineered bacteria in Example 1 were replaced with wild-type WT bacteria VNP20009. The step of constructing the engineered bacteria was omitted, while the remaining steps were the same as in Example 1. Microspheres containing wild-type bacteria were prepared and denoted as PEGDA@VNP. WT Group.

[0071] The lanthanum protein-engineered bacterial embolization microspheres prepared in Example 1 are designated as PEGDA@VNP. LanM Group.

[0072] The three types of microspheres were subjected to different pH values ​​(pH 3, 4, 5, 6, 7, and 8) in buffer solutions. 90 Labeling with the radioactive nuclide Y, the labeling conditions being: yttrium trichloride [ 90 The amount of γ-solution was 200 µCi, the labeling time was 10 min, and the labeling method was the same as in Example 1. The labeling rate of each group was measured using a γ-counter. Figure 3 It can be seen that within the pH range of 3-8, PEGDA@VNP LanM Maintaining a high labeling rate of over 85% consistently; while PEGDA and PEGDA@VNP WT The labeling rate decreased with increasing pH, and was significantly lower than PEGDA@VNPLanM at low pH. Even at low pH=3, compared to the other two control groups, PEGDA@VNP... LanM The group also had a labeling rate of over 85%.

[0073] Different doses of yttrium trichloride were set (0 μCi, 50 μCi, 100 μCi, 150 μCi, 200 μCi, 400 μCi). 90 Y] solution, respectively, was used to treat 1 mg of three types of microspheres. 90The gamma-ray radionuclide was labeled under the following conditions: pH = 5.5, labeling time 10 min, and labeling method as in Example 1. The labeling rate of each group was determined using a gamma counter. Figure 3 b shows that low-dose yttrium trichloride [ 90 In the Y] solution, PEGDA@VNP WT Due to its non-specific adsorption, it has a high labeling rate, but with the adsorption of yttrium trichloride [ 90 With increasing solution dosage, the labeling rate of PEGDA@VNP decreased rapidly. LanM Its labeling rate has consistently maintained an advantage.

[0074] Different labeling times (1, 2, 10, 20, 30, 60 min) were set to treat the three types of microspheres respectively. 90 Labeling with the radioactive nuclide Y, under the following conditions: pH = 5.5, and a labeling dose of yttrium trichloride. 90 The Y] solution was 100 μCi, and the labeling method was the same as in Example 1. The labeling rate of each group was determined using a γ-counter. Figure 3 c shows that PEGDA@VNP LanM Nearly 100% labeling rate can be achieved within 1 minute of labeling, and it remains stable over a long period; PEGDA, PEGDA@VNP WT The labeling rate is significantly lower and it lacks the characteristics of efficient and rapid labeling. This indicates that PEGDA@VNP... LanM It has high marking efficiency and can mark items as needed.

[0075] The above results indicate that PEGDA@VNP LanM (Final microsphere material) on 90 The labeling of Y has the advantages of strong pH adaptability, good dose tolerance, high labeling efficiency and speed. Its labeling performance is far superior to that of sterile PEGDA and PEGDA@VNP loaded with non-engineered bacteria. WT It can achieve 90 Efficient, fast, and stable labeling of Y.

[0076] Experimental Example 3 includes 90 Effects of Y-functionalized radioactive bacterial embolization microspheres on tumor-bearing mice Materials used by each group: Free 90 Y (free) 90 Group Y): Yttrium trichloride as described in Example 1 [ 90 Y] solution; PEGDA@WT@ 90 Y (loaded with non-engineered bacteria + 90 Group Y: The engineered bacteria in Example 1 were replaced with wild-type WT bacteria VNP20009, and the other preparation methods for the microspheres were the same as in Example 1; PEGDA@ 90 Y (sterile +) 90Group Y: The 75% bacterial suspension in Example 1 was replaced with 75% MES buffer, and all other steps were the same as those for the microspheres prepared in Example 1; PEGDA@LanM@ 90 Y (loaded engineered bacteria +) 90 Group Y: The product prepared in Example 1 containing... 90 Y-functionalized radioactive bacterial embolization microspheres (1 mg microspheres labeled with 100 μCi) 90 Y).

[0077] Select 7-8 week old female Balb / c mice with a standard weight (18g) and subcutaneously inject 50μl of a solution containing 10 mg of iodine into the hind limb on the back. 6 CT26 cells were used to construct a tumor model. After 7 days, when the tumor volume reached 150 mm... 3 At that time, each group of materials was injected into the tumor in a volume of 25 μl per animal, and imaging was performed using a small animal in vivo imaging system after injection.

[0078] The distribution of radioactive signals in tumor-bearing mice over 1-6 days is shown as follows: Figure 4 As shown in a, PEGDA@LanM@ 90 Group Y: The radioactive signal at the tumor site (highlighted in blue) was persistently strong and concentrated, while the signal in other tissues was weak; other groups (especially Free) 90 Y、PEGDA@ 90 Y) Radioactive signals are dispersed throughout the body, but the signal is weak and decays rapidly over time at the tumor site.

[0079] The radioactive uptake rate of tumors in tumor-bearing mice was measured within 1-6 days, such as... Figure 4 As shown in b, the quantitative representation of the tumor's effect on... 90 Y intake percentage (%), PEGDA@LanM@ 90 The tumor uptake rate in group Y remained consistently above 90% and remained stable over the long term; other groups (Free) 90 Y、PEGDA@ 90 (e.g., Y) tumor uptake rates were significantly lower and declined rapidly over time, such as Free 90 Y intake rate was only about 40% over 6 days.

[0080] Measurements were taken in various tissues at 6 days. 90 The distribution ratio of Y (percentage of injected dose per gram of tissue), such as Figure 4 As shown in c, PEGDA@LanM@ 90 Group Y was highly prevalent only in primary tumors and distant metastases, with very little distribution in other normal tissues (heart, liver, spleen, etc.); other groups 90 Y is more abundant in normal tissues (such as the liver and spleen), but has a weaker specific distribution advantage in tumor tissues.

[0081] Example 4 includes 90 Y-functionalized radioactive bacterial embolization microsphere tumor therapy Grouping and the four materials involved in each group: PBS group; Free 90 Y (free) 90 Y): Yttrium trichloride as described in Example 1 [ 90 [Y] Solution; PEGDA group: Replace the 75% bacterial suspension in Example 1 with 75% MES buffer, and follow the same steps as the microspheres prepared in step 1 of Example 1; PEGDA@LanM@ 90 Y (loaded engineered bacteria +) 90 Y): The product prepared in Example 1 containing 90 Y-functionalized radioactive bacterial embolization microspheres.

[0082] The tumor-bearing method was the same as in Example 3, with 100 μCi and 1 mg / mouse of the same drug injected intratumorally into CT26 or 4T1 mouse models, respectively. 90 Functionalized radioactive bacterial embolization microspheres of Y and corresponding amounts of other drugs were used to observe changes in tumor volume and mouse survival curves. Figure 5 From the perspective of the treatment results, 90 The tumor volume in the functionalized radioactive bacterial embolization microsphere group Y was significantly reduced and could disappear completely. The mice survived longer than those in the other groups, and no mice died.

[0083] Example 5 includes 90 Y-functionalized radioactive bacterial embolization microspheres applied to interventional embolization therapy for in situ hepatocellular carcinoma in rabbits. The construction of rabbit in situ hepatocellular carcinoma and the embolization interventional treatment steps are as follows: ① A 2.5kg New Zealand white rabbit was placed under general anesthesia and secured to a rabbit board. ② The abdomen was shaved, disinfected, and draped. Under aseptic conditions, a 2cm incision was made along the midline of the abdomen. The left lobe of the liver was exposed by dissecting layer by layer (the left lobe was chosen as the tumor implantation site to avoid the cystic artery during embolization). The liver was grasped with the tail of a cotton swab, and the liver was secured with sterile gauze in the left hand. If the search for the left lobe took too long, the liver should be returned to the abdominal cavity to avoid ischemic damage. ③ The liver capsule was punctured with sterile ophthalmic forceps, and the prepared tumor fragment (0.5mm×0.5mm×0.5mm) was inserted into the liver to a depth of 1cm through the puncture point. Gelatin sponge was used for local hemostasis to ensure the tumor fragment was fixed in the liver. ④ After confirming no active bleeding, the abdomen was closed by suturing layer by layer. The rabbit was returned to the animal room and cared for by animal keepers until it regained consciousness. At the same time, penicillin 50,000 units / kg was administered intramuscularly for 3 consecutive days. ⑤ 14 days later, the rabbit was injected with 2 mCi via the marginal ear vein. 18 F-FDG was performed, and PET-CT was used 1 hour later to observe that the liver tumor had reached 500mm. 3 One ml of femoral artery embolization can be used for treatment. 90Y-functionalized radioactive bacterial embolization microspheres, configured as PEGDA@Lanm@ 90 Group Y. The preparation of this functionalized microsphere is the same as in Example 1, except that the lanthanum protein-engineered bacterial embolization microspheres are 10 mg and labeled with 2 mCi yttrium trichloride. 90 [Y] solution. The control group was embolized without microspheres, and PBS was administered as a control. ⑥ PET-CT imaging was performed at 6 and 12 days after treatment to observe changes in tumor volume.

[0084] The results are as follows Figure 6 As shown, the red circle indicates the tumor site, showing that the tumors in the Control group are growing larger, while those in the PEGDA@Lanm@ group are growing larger. 90 The tumors in group Y are getting smaller and smaller, indicating that... 90 Functionalized radioactive bacterial embolization microspheres of Y can effectively eliminate tumors. Figure a shows the treatment time, Figure b shows the contrast imaging of the liver tumor before and after interventional embolization to confirm the tumor location, and Figure c shows PET-CT images one day before embolization, 6 days after embolization, and 12 days after embolization.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lanthanum protein-engineered bacterial embolization microsphere, characterized in that, The lanthanum protein-engineered bacterial embolization microspheres are formed by cross-linking after a microemulsion is prepared by mixing a functional aqueous phase containing a microsphere matrix, functional bacteria, and a photocrosslinking agent with an oil phase containing an emulsifier and an oil phase component. The functional bacteria are lanthanum protein-engineered bacteria; the target gene sequence of the lanthanum protein-engineered bacteria is shown in SEQ ID NO.

1.

2. The lanthanum protein-engineered bacterial embolization microspheres as described in claim 1, characterized in that, The microsphere matrix comprises polyethylene glycol diacrylate; the photocrosslinking agent comprises TPO-L photoinitiator.

3. The lanthanum protein-engineered bacterial embolization microspheres as described in claim 1, characterized in that, The emulsifier includes Triton X-100, and the oil phase component includes polydimethylsiloxane oil.

4. The method for preparing lanthanum protein-engineered bacterial embolic microspheres according to any one of claims 1-3, characterized in that, Includes the following steps: The microsphere matrix was mixed with a photocrosslinking agent to obtain a polymer precursor, which was then mixed with a lanthanum protein-engineered bacterial suspension to obtain a functional aqueous phase. The emulsifier was mixed with the oil phase component to obtain an oil phase. The functional aqueous phase and the oil phase were mixed to obtain an emulsion. The emulsion was irradiated with light to form hydrogel microspheres.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the microsphere matrix to the photocrosslinking agent is 95-103:1; the volume ratio of the polymer precursor to the lanthanum protein-engineered bacterial suspension is 20-30:70-80; and the concentration of the lanthanum protein-engineered bacterial suspension is 0.05-0.15 × 10⁻⁶. 11 Cells / mL; the mass ratio of the emulsifier to the oil phase component is 1:95-103.

6. The preparation method according to claim 4, characterized in that, The illumination conditions include: ultraviolet lamp irradiation with a wavelength of 360-370nm and an irradiation power density of 2-6W / cm². 2 Irradiation time: 110-130s.

7. A functionalized radioactive bacterial embolization microsphere, characterized in that, It is prepared by combining lanthanum protein-engineered bacterial embolization microspheres with radionuclides as described in any one of claims 1-3.

8. The functionalized radioactive bacterial embolization microspheres as described in claim 7, characterized in that, The radionuclides include 90 Y, lanthanide radionuclides and actinide radionuclides.

9. The method for preparing functionalized radioactive bacterial embolization microspheres as described in claim 7 or 8, characterized in that, The process includes the following steps: mixing lanthanum protein-engineered bacterial embolization microspheres with a radionuclide.

10. The use of the lanthanum protein-engineered bacterial embolization microspheres according to any one of claims 1-3 or the functionalized radioactive bacterial embolization microspheres according to claim 7 or 8 in the preparation of antitumor products.

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