Inhalable oncolytic virus-bacterium coupling delivery system as well as preparation method and application thereof

Abstract

The invention discloses an inhalable oncolytic virus-bacterium coupling delivery system as well as a preparation method and application thereof, and belongs to the field of medical biotechnology and tumor immunotherapy. The delivery system is characterized in that a bacterial carrier with the capability of actively moving and competitively depriving tumor interstitial calcium ions, an oncolytic virus and a tumor microenvironment response type connexon subjected to functional modification are subjected to click coupling to form an oncolytic virus-bacterial conjugate. The preparation method comprises the following steps: reacting an oncolytic virus with a functional modifier containing a disulfide bond and diphenyl cyclooctyne to construct a functional oncolytic virus, and reacting a bacterial vector with a functional modifier containing an azide group to construct a functional bacterial vector, and carrying out click reaction on the functionalized oncolytic virus and the functionalized bacterial carrier to obtain the inhalable oncolytic virus-bacterial conjugate. According to the invention, the problem that the traditional oncolytic virus preparation in the deep treatment field of lung cancer is poor in permeability and easy to neutralize and remove during respiratory tract administration is solved, and the synergistic effect of physical penetration and immune remodeling is realized.

Description

An inhalable oncolytic virus-bacterial conjugate delivery system, its preparation method and application Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology and tumor immunotherapy, specifically relating to an inhalable oncolytic virus-bacteria conjugate delivery system, its preparation method, and its application. Background Technology

[0002] Lung cancer is a prevalent malignant tumor worldwide, with a high mortality rate and a tendency to develop multiple lesions. Current treatments such as chemotherapy and immune checkpoint inhibitors (ICIs) still have limited response rates when dealing with the highly heterogeneous and "cold tumor" characteristics of lung cancer. Oncolytic viruses (OVs) offer a new approach to lung cancer treatment by lysing tumor cells and activating anti-tumor immunity.

[0003] However, the clinical translation of oral viral vectors (OVs) is constrained by multiple physiological and pathological barriers: First, delivery challenges: systemic administration is easily cleared by neutralizing antibodies; while inhalation administration can bypass systemic circulation, free viruses lack active navigation capabilities and struggle to penetrate the thick respiratory mucus layer and tumor stroma barrier. Second, tissue penetration challenges: E-cadherin, a common protein in lung cancer tissue, is highly expressed, forming dense intercellular junctions that limit drug diffusion to the tumor core. Third, limitations of existing technologies: although there is research on bacterial vectors, most rely on flagellar movement, are easily entangled by mucus fibers, and some strains pose pathogenicity and safety risks.

[0004] Therefore, there is an urgent need in this field for a novel oncolytic virus delivery method that has efficient barrier penetration capability, good biosafety, and can enhance the permeability of tumor tissue. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide an inhalable oncolytic virus-bacterial conjugated delivery system and its preparation method. By selecting a bacterial carrier with active motility and conjugating it with an oncolytic virus, the bacterial carrier achieves efficient delivery and immune enhancement of the oncolytic virus in deep tumor tissues through the synergistic effect of active motility and competitive deprivation of calcium ions from the tumor stroma.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides an inhalable oncolytic virus-bacterial conjugate delivery system, wherein the delivery system comprises a bacterial carrier with the ability to actively move and competitively deprive the tumor stroma of calcium ions and an oncolytic virus, and a tumor microenvironment-responsive linker after functionalization modification, to click-couple the oncolytic virus to form an oncolytic virus-bacterial conjugate.

[0008] Furthermore, the bacterial carriers capable of active motility and competitive deprivation of calcium ions from the tumor stroma are selected from one or more of the genera *Synechococcus*, *Prochlorococcus*, *Salmonella VNP20009*, or *Escherichia coli* k12.

[0009] Oncolytic viruses are selected from one or more of the following: oncolytic adenovirus, herpes simplex virus, vaccinia virus, reovirus, Malaba virus, vesicular stomatitis virus, or Newcastle disease virus.

[0010] Preferably, the bacterial carrier with the ability to actively move and competitively deprive the tumor stroma of calcium ions is Synechococcus sp. WH8102, and the oncolytic virus is oncolytic adenovirus (Ads).

[0011] Furthermore, tumor microenvironment-responsive linkers contain redox-sensitive disulfide bonds. Bacterial vectors are functionalized to form linkers containing azide groups, and oncolytic viruses are functionalized to form tumor microenvironment-responsive linkers containing disulfide bonds and diphenylcyclooctylene. The azide groups and diphenylcyclooctylene undergo click chemical reactions.

[0012] The ratio of oncolytic virus titer (PFU) to bacterial vector quantity (CFU) was (5-20):1.

[0013] Furthermore, the proportion of oncolytic virus-bacterial conjugate particles with an aerodynamic particle size distribution in the range of 0.94 μm-4.46 μm is ≥50 wt.%.

[0014] In a second aspect, the present invention provides a method for preparing an inhalable oncolytic virus-bacteria conjugate delivery system, comprising the following steps:

[0015] Construction of functionalized oncolytic viruses:

[0016] 1) Incubate oncolytic viruses with functionalized modifiers containing disulfide bonds and diphenylcyclooctylene;

[0017] 2) After the reaction is complete, unreacted small molecules are removed by ultrafiltration concentration to obtain functionalized oncolytic viruses with alkyne groups on their surface;

[0018] Construction of functionalized bacterial vectors:

[0019] 1) Incubate bacterial vectors with functionalized modifiers containing azide groups;

[0020] 2) After the reaction is complete, the free modifier is removed by centrifugation and washing to obtain a functionalized bacterial vector with azide groups on its surface;

[0021] Assembly of oncolytic virus-bacterial conjugates:

[0022] 1) Disperse the functionalized oncolytic virus in a buffer solution containing a functionalized bacterial vector and carry out a copper-free click chemical reaction by stirring;

[0023] 2) Collect the generated conjugate precipitate by centrifugation and resuspend it. Repeat washing removes unbound viruses to obtain inhalable oncolytic virus-bacterial conjugates.

[0024] Furthermore, in step 1) of constructing the functionalized oncolytic virus, the functionalizing agent containing disulfide bonds and diphenylcyclooctyne is DBCO-PEG2000-SS-NHS; the ratio of the functionalizing agent containing disulfide bonds and diphenylcyclooctyne to the oncolytic virus is 1.124 × 10⁻⁶. 8 PFU oncolytic virus corresponds to 0.8mg-2mg of modifier; incubation time is 30min-60min;

[0025] In step 2), the ultrafiltration concentration method involves centrifuging and then repeatedly washing the ultrafiltration tube. The ultrafiltration tube has a molecular weight cutoff of 100 kDa, the centrifugation speed is 2500 rpm-4000 rpm, and the centrifugation time is 3 min-8 min.

[0026] Furthermore, in step 1) of constructing the functionalized bacterial vector, the functionalizing agent containing an azide group is Azido-PEG2000-NHS; the ratio of the functionalizing agent containing an azide group to the bacterial vector is 1.124 × 10⁻⁶. 7 CFU bacteria correspond to 1.2mg-4.2mg of modifier; incubation time is 30min-60min;

[0027] In step 2), the centrifugation speed is 2500rpm-4000rpm and the centrifugation time is 3min-8min.

[0028] Further, in step 1) of assembling the oncolytic virus-bacterial conjugate, the functionalized oncolytic virus is dispersed in the buffer of the functionalized bacterial vector at a ratio of PFU:CFU=(5-20):1; the stirring speed is 100rpm-200rpm and the stirring time is 30min-90min.

[0029] In step 2), the centrifugation speed is 2500rpm-4000rpm and the centrifugation time is 3min-8min.

[0030] Thirdly, the present invention provides the application of an inhalable oncolytic virus-bacteria conjugate delivery system in the preparation of antitumor drugs.

[0031] Furthermore, antitumor drugs are drugs for treating lung cancer in situ or inhibiting lung metastases, and can be formulated as respiratory preparations for intratracheal nebulization.

[0032] Advantages and effects of the present invention:

[0033] This invention focuses on the deep treatment of lung cancer, solving the problems of poor permeability and easy neutralization and clearance of traditional oncolytic virus preparations when administered via the respiratory tract. By introducing a bacterial carrier with active motility, this invention achieves active drug navigation and mucus evasion; and proposes a novel strategy of competitively depriving the tumor's physical barrier of calcium ions, providing strong support for enhancing the infiltration of oncolytic viruses within solid tumors. This invention achieves a synergistic effect of physical penetration and immune remodeling, providing a scientific basis for the design of novel, efficient, and low-toxicity oncolytic virus-bacteria conjugate delivery systems, and possesses significant clinical translational and research value. Attached Figure Description

[0034] Figure 1 is a process flow diagram of the preparation of the inhalable oncolytic virus-bacteria conjugate delivery system in Example 1;

[0035] Figure 2 shows the qPCR detection results of Ads virus connectivity in Examples 1, 2, and 3.

[0036] Figure 3 shows the loading efficiency of Synechococcus on Ads virus in Examples 1, 2, and 3;

[0037] Figure 4 is a laser confocal fluorescence colocalization diagram of the oncolytic virus-bacterial conjugate prepared in Example 1;

[0038] Figure 5 is a transmission electron microscope image of Ads, Synechococcus, and oncolytic virus-bacterial conjugate in Example 1.

[0039] Figure 6 shows the dynamic light scattering particle size distribution of Ads, functionalized Ads, Polychondria, and oncolytic virus-bacterial conjugate in Example 1.

[0040] Figure 7 shows the surface zeta potential of Ads, functionalized Ads, Polychondria, and oncolytic virus-bacterial conjugate in Example 1.

[0041] Figure 8 is a curve showing the virus release kinetics of the oncolytic virus-bacterial conjugate prepared in Example 1 under simulated lung fluid environment.

[0042] Figure 9 shows the in vitro cytotoxicity evaluation of the oncolytic virus-bacterial conjugate prepared in Example 1 against TC-1-hCD46 cells.

[0043] Figure 10 shows the chemotactic trajectory and relative velocity quantification of the oncolytic virus-bacterial conjugate prepared in Example 1 within 20 s, where: (a) is the chemotactic trajectory, and (b) is the relative velocity quantification.

[0044] Figure 11 is a quantitative graph of the penetration percentage of the oncolytic virus-bacterial conjugate prepared in Example 1 in the artificial mucus layer, where: (a) is a three-dimensional confocal imaging image of artificial mucus penetration, and (b) is a quantitative statistical graph of fluorescence intensity percentage.

[0045] Figure 12 shows the clearance resistance assessment of the oncolytic virus-bacterial conjugate prepared in Example 1 during co-culture with macrophages;

[0046] Figure 13 shows the three-dimensional diffusion distribution of the oncolytic virus-bacterial conjugate prepared in Example 1 in a simulated matrix gel model;

[0047] Figure 14 is an evaluation diagram of the adhesion ability of the oncolytic virus-bacterial conjugate prepared in Example 1 to tumor cells.

[0048] Figure 15 shows the deep penetration behavior of the oncolytic virus-bacterial conjugate prepared in Example 1 in a tumor multicellular sphere model.

[0049] Figure 16 shows the fluorescence imaging and quantitative distribution of the lungs of mice with orthotopic lung cancer in Example 1 at 2h, 6h and 12h after inhalation administration, where: (a) is fluorescence imaging and (b) is quantitative distribution of fluorescence intensity.

[0050] Figure 17 shows the in vitro imaging and Ads copy number quantification of major organs 24 hours after drug administration in Example 1, where: (a) is the in vitro imaging and fluorescence quantification of major organs, and (b) is the statistical graph of Ads copy number qPCR quantification in lung tissue;

[0051] Figure 18 shows the fluorescence distribution of lung tissue sections in Example 1;

[0052] Figure 19 shows the tumor bioluminescence monitoring and growth curve of the orthotopic lung cancer model mice during treatment in Example 1, where: (a) is a schematic diagram of the treatment process, (b) is a bioluminescence imaging image of the tumor in vivo, and (c) is a tumor growth curve represented by fluorescence intensity.

[0053] Figure 20 shows the CD8 concentration in blood and lungs after treatment in Example 1. + Flow cytometry plot of T cell proportion;

[0054] Figure 21 is a flow cytometry analysis of the proportions of Treg cells and MDSCs in blood and lungs in Example 1;

[0055] Figure 22 is a flow cytometry analysis of the maturity of DC cells and the polarization typing of macrophages in lung tissue in Example 1;

[0056] Figure 23 shows the detection levels of various cytokines IFN-γ, IL-6, and TNF-α in serum and lung tissue in Example 1.

[0057] Figure 24 shows the tumor growth inhibition of recipient mice after the adoptive T cell transfer experiment (ACT) in Example 1, where: (a) is a schematic diagram of the ACT experimental protocol, and (b) is a comparison of bioluminescence imaging of lung tumors in recipient mice;

[0058] Figure 25 shows the number of lung nodules and the quantitative volume of in situ tumors in mice in the distant metastatic lung cancer model in Example 1, where: (a) is a schematic diagram of the experimental procedure, (b) is a macroscopic photograph of lung tissue nodules, (c) is a macroscopic photograph of distant subcutaneous tumors, (d) is a statistical chart of the number of lung nodules, (e) is a growth curve of distant tumor volume, (f) is a statistical chart of distant tumor weight, and (g) is a survival curve of mice in each group.

[0059] Figure 26 shows microCT imaging of the lungs of the mice tested in Example 1, where: (a) is a representative image of the lungs in axial, coronal and sagittal planes, and (b) is a comparison analysis of H&E staining between healthy mice and the treatment group mice;

[0060] Figure 27 shows the tumor growth inhibition in the recipient mice of the human tumor xenograft model in Example 1, including: (a) a schematic diagram of the experimental protocol, and (b) a fluorescence imaging diagram of tumor growth.

[0061] Figure 28 shows the human CD8 in the humanized NOG mouse model in Example 1. + Flow cytometry validation plot of T cell proportion;

[0062] Figure 29 shows the competitive uptake of calcium ions (Ca) by Synechococcus in Example 1. 2+ ) and its effect on intracellular Ca in tumor cells 2+ Fluorescence and flow cytometry plots showing the effect of content on the content, where: (a) represents the competitive uptake of Ca by algae. 2+ Schematic diagram of the mechanism, (b) shows Ca in tumor cells. 2+ Fluorescence imaging, (c) is a statistical graph of quantitative detection by flow cytometry;

[0063] Figure 30 is a Western blot diagram showing the expression level of E-cadherin protein in tumor cells after treatment with different components in Example 1 of the present invention. Detailed Implementation

[0064] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples.

[0065] An inhalable oncolytic virus-bacterial conjugated delivery system is disclosed. This system combines a bacterial vector with the ability to actively motility and competitively deprive the tumor stroma of calcium ions with an oncolytic virus. The bacterial vector is selected from one or more of the genera *Synechococcus*, *Prochlorococcus*, *Salmonella VNP20009*, or *Escherichia coli* K12. The oncolytic virus is selected from oncolytic adenovirus, herpes simplex virus, vaccinia virus, reovirus, Malaba virus, vesicular stomatitis virus, or other novel coronaviruses. One or more of the urban epidemic viruses are used. Bacterial vectors are functionalized to form linkers containing azide groups, and oncolytic viruses are functionalized to form tumor microenvironment-responsive linkers containing disulfide bonds and diphenylcyclooctylene. The azide groups and diphenylcyclooctylene undergo a click chemical reaction. The titer of the oncolytic virus and the number of bacterial vectors (PFU:CFU) are calculated as (5-20):1, forming an oncolytic virus-bacterial conjugate. The proportion of particles with aerodynamic particle size distribution in the range of 0.94 μm-4.46 μm is ≥50 wt.%. The bacterial vector with active motility and competitive deprivation of calcium ions from the tumor stroma is preferably *Synechococcus* sp. WH8102, which has non-flagellated swimming ability. The oncolytic virus is preferably an oncolytic adenovirus (Ads).

[0066] A method for preparing an inhalable oncolytic virus-bacterial conjugated delivery system includes the following steps:

[0067] Construction of functionalized oncolytic viruses:

[0068] 1) Incubate the oncolytic virus with a functionalized modifier containing disulfide bonds and diphenylcyclooctylene, DBCO-PEG2000-SS-NHS, for 30-60 minutes, at a concentration of 1.124 × 10⁻⁶ mg / L. 8 PFU oncolytic virus corresponds to 0.8mg-2mg of functionalized modifiers containing disulfide bonds and diphenylcyclooctylene;

[0069] 2) After the reaction is complete, the virus is centrifuged at 2500 rpm to 4000 rpm for 3 min to 8 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa. The washing is repeated 3 times to remove unreacted small molecules and obtain functionalized oncolytic viruses with alkyne groups on the surface.

[0070] Construction of functionalized bacterial vectors:

[0071] 1) Incubate the bacterial vector with Azido-PEG2000-NHS, a functionalized modifier containing azide groups, for 30-60 minutes, at a concentration of 1.124 × 10⁻⁶ mg / L. 7 CFU bacteria correspond to 1.2mg-4.2mg of functionalized modifiers containing azide groups;

[0072] 2) After the reaction is complete, centrifuge at 2500rpm-4000rpm for 3min-8min, wash to remove free modifier, and obtain functionalized bacterial vectors with azide groups on the surface.

[0073] Assembly of oncolytic virus-bacterial conjugates:

[0074] 1) Disperse the functionalized oncolytic virus in a buffer solution containing a functionalized bacterial vector at a ratio of PFU:CFU=(5-20):1, and stir at 100rpm-200rpm for 30min-90min at room temperature to carry out a copper-free click chemical reaction.

[0075] 2) Centrifuge at 2500rpm-4000rpm for 3min-8min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound viruses to obtain inhalable oncolytic virus-bacterial conjugates.

[0076] Application of an inhalable oncolytic virus-bacterial conjugate delivery system in the preparation of antitumor drugs, which are drugs for treating lung cancer in situ or inhibiting lung metastases, and can be made into respiratory preparations for intratracheal nebulization.

[0077] Example 1

[0078] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0079] Construction of functionalized oncolytic viruses:

[0080] 1) 1.124 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 1.2 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 60 min; the viral titer was determined using the Reed-Muench method.

[0081] 2) After the reaction is complete, the virus is centrifuged at 3000 rpm for 5 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0082] Construction of functionalized bacterial vectors:

[0083] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7CFU of Synechococcus polyphylla WH8102 was mixed with 2.4 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 60 min.

[0084] 2) After the reaction is complete, centrifuge at 3000 rpm for 5 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0085] Assembly of oncolytic virus-bacterial conjugates:

[0086] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 10:1 and stirred at 120 rpm for 60 min at room temperature to carry out a copper-free click chemical reaction.

[0087] 2) Centrifuge at 3000 rpm for 5 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0088] The inhalable oncolytic virus-bacterial conjugate delivery system prepared in Example 1, as analyzed by NGI (flow rate 60 mL / min, pressure 4 kPa), showed good aerodynamic particle size distribution characteristics, with 57.53% of the particles distributed within the ideal alveolar deposition range of 0.94 μm-4.46 μm.

[0089] 1. Performance Evaluation:

[0090] (1) Connection rate and load analysis:

[0091] Quantitative analysis by qPCR: As shown in Figure 2, the copy number (%) of Ads in the conjugate precipitate and the supernatant (unbound virus) were detected respectively. The calculation showed that the Ads ligation rate reached 77.83% in this Example 1 at a PFU:CFU ratio of 10:1.

[0092] Flow cytometry detection: The viral load on the surface of Synechococcus was observed using FITC-labeled Ads, as shown in Figure 3. In this example 1, the positive rate of Ads load on Synechococcus was as high as 95.8% at a PFU:CFU ratio of 10:1.

[0093] (2) Physical property characterization:

[0094] Morphological observation: As shown in Figure 4, the FITC-labeled Ads virus (green) and DAPI-stained Synechococcus (red) were highly colocalized. As shown in Figure 5, the Ads virus particles were uniformly distributed on the surface of Synechococcus.

[0095] Particle size and potential: Dynamic light scattering (DLS) analysis, as shown in Figures 6 and 7, shows that after assembly into oncolytic virus-bacterial conjugates, the average particle size increases significantly (approximately 2000 nm), and the surface potential shifts from negative to near neutral, confirming the success of covalent linkage.

[0096] Aerodynamic particle size distribution: Next-generation impactor (NGI) analysis showed that the proportion of particles in the complex in the range of 0.94-4.46 μm was 57.53%, which meets the requirements for inhaled formulations delivered deep into the lungs.

[0097] (3) Environmental response release performance analysis:

[0098] The conjugate of Example 1 was placed in simulated lung fluid (SLF, containing 0.2 mM glutathione GSH), with the oncolytic virus-bacterial conjugate formed by ordinary linkers without disulfide bonds as the control group. As shown in Figure 8, it can be seen that the oncolytic virus-bacterial conjugate containing disulfide bonds achieved 100% release of Ads within 12 hours, while the ordinary linker group only released about 40%, which proves the precise response and release capability of the conjugate of the present invention in the lung microenvironment.

[0099] (4) Preliminary study on in vitro cytotoxicity:

[0100] In an environment containing 0.2 mM GSH, the toxicity of TC-1-hCD46 cells was evaluated. As shown in Figure 9, the PEG-functionalized Ads maintained good infectivity and killing activity, and the conjugate of Example 1 showed better antitumor tendency than the single-component controls in vitro.

[0101] (5) Analysis of in vitro tumor kinetics and active motility:

[0102] Methods: The conjugate and control group of Example 1 were monitored in real time using the pendant drop method combined with laser confocal microscopy (CLSM). 5 μL of oncolytic virus-bacterial conjugate suspension labeled with DiI, WH8102 and non-motile strain PCC7942 of the control group were placed in the center of a coverslip and fixed with Vaseline to form an observation cavity.

[0103] Tracking analysis: Record continuous video frames within 20 seconds using a 100× oil mirror and perform trajectory tracking using ImageJ's MTrackJ plugin.

[0104] Results: As shown in Figure 10, quantitative calculations show that the bacterial vector WH8102 exhibits strong swimming ability, with a speed more than twice that of the non-motorized strain PCC7942. Crucially, after loading Ads virus particles, the motility of the conjugate in Example 1 was not significantly impaired, and it still maintained excellent relative speed and displacement, confirming its propulsion efficiency as an active motorized carrier in complex liquid environments.

[0105] (6) In vitro anti-mucus clearance ability analysis:

[0106] Model construction: A mucus barrier model of artificial airway was constructed with reference to relevant literature; a 4 mm thick 10% gelatin layer was laid at the bottom of a 24-well plate, and a 2 mm thick artificial mucus layer (containing mucin, DNA, DTPA, egg yolk emulsion and other components) was superimposed on it.

[0107] Permeability assay: Equal amounts of the DiI-labeled conjugate and the control group were added to the surface of the mucus layer and incubated at 37°C for 12 hours. The fluorescence intensity in the gelatin layer was measured using a multi-functional microplate reader, and the relative penetration percentage was calculated.

[0108] Results: As shown in Figure 11, compared with the control group constructed by the non-motile strain PCC7942, the retention rate (permeability) of the conjugate constructed by WH8102 in the gelatin layer reached 34%, which was significantly better than the 11% of the control group without motility. This result shows that the conjugate of Example 1 can effectively avoid the physical entanglement of mucus fibers by utilizing the non-flagellated characteristics of Synechococcus, and achieve deep penetration of the respiratory mucus barrier.

[0109] (7) Analysis of macrophage clearance and escape capabilities:

[0110] Experimental protocol: The conjugate of Example 1 was co-cultured with MH-S mouse alveolar macrophages at a 1:1 ratio to simulate the clearance pressure in the lung immune environment; the concentration of residual carrier was evaluated by measuring the absorbance (OD680) of the culture supernatant at 680 nm at time points of 0 h, 4 h, 12 h, 24 h, and 48 h.

[0111] Results: Using the non-motile control strain PCC7942 as the control group, as shown in Figure 12, the non-motile control strain PCC7942 was rapidly cleared by macrophages within 4 hours; while the conjugate of Example 1 with active motility still retained 47.10% of the initial load after 48 hours, and the original strain WH8102 had a retention rate of 56.04%. This data confirms that active motility not only enhances penetration but also endows the conjugate with the ability to evade phagocytosis and clearance in the complex immune microenvironment of the lungs, thereby prolonging the drug's retention time in the lungs.

[0112] (8) Analysis of the penetration ability of simulated tissue barrier (matrix):

[0113] The matrix adhesive dispersion and penetration experiments are detailed below:

[0114] Model construction: TC-1-hCD46 cells labeled with DiI were mixed into Matrigel (×10⁻⁶). 6 (cells / mL), injected into 24-well plates and used to form a simulated epithelial barrier at physiological temperature.

[0115] Sample injection: Using a microsyringe, inject 4 μL of sample containing 1×10⁻⁶ mol / L of sample. 7 The conjugate of CFU labeled with Cy7, WH8102, and non-motile strain PCC7942 (control group) was injected into the center of the matrix gel.

[0116] Follow-up observation: 3D scanning imaging was performed using laser confocal microscopy to monitor the spatial distribution of the carrier at 2h and 12h, as shown in Figure 13. It can be seen that the WH8102 conjugate with active motility can be uniformly diffused throughout the entire matrix gel area after 12h, while the PCC7942 control group mainly aggregated in clumps at the injection point. This proves that active motility plays a key role in overcoming the dense physical barrier similar to the lung matrix.

[0117] (9) Tumor cell adhesion performance analysis:

[0118] Culture: Free Ads, functionalized Ads (PEG-Ads) and the conjugate of Example 1 were co-cultured with TC-1-hCD46 cells plated on a glass dish for 60 min.

[0119] Elution and fixation: The sample was gently rinsed three times with PBS (pH 7.2) to remove unbound samples, then fixed with 4% paraformaldehyde and stained with DAPI.

[0120] Quantitative analysis: Fluorescence images were acquired using CLSM and the average fluorescence intensity per unit cell area was calculated using ImageJ; as shown in Figure 14, it can be seen that the amount of conjugate in Example 1 on the cell surface was significantly higher than that in the free virus group and the single-modification group, indicating that the Synechococcus carrier endows the system with excellent bioadhesion, which helps to resist physical rejection caused by coughing after inhalation administration to the lungs.

[0121] (10) Analysis of the deep penetration behavior of tumor multicellular spheres (3DSpheroids):

[0122] Tumor sphere preparation: TC-1-hCD46 cells were cultured in a complete medium containing methylcellulose for 24 h using the hanging drop method to form a 3D tumor multicellular sphere model with uniform particle size.

[0123] Osmotic drug delivery: After 12 hours of culture, FITC-labeled free Ads virus, Synechococcus, and the conjugate of Example 1 were added respectively.

[0124] Dynamic scanning observation: Depth-direction (Z-stack) scanning was performed using a laser confocal microscope, as shown in Figure 15, with a layer spacing of 10 μm and a depth of 200-250 μm.

[0125] Experimental Results: As shown in Figure 15, observations at 6h and 12h revealed that the free Ads group remained only on the outermost cell surface of the tumor sphere; while the conjugate of Example 1 exhibited superior inward propulsion. Synergistically with its competitive calcium ion uptake and disruption of tumor cell tight junctions, the conjugate of Example 1 successfully penetrated into the core region of the tumor sphere, achieving deep spatial coverage of the virus-loaded cells. This provides an efficient engineered solution to address the problem of insufficient viral infection within solid tumors.

[0126] (11) In vivo biodistribution, lesion targeting, and retention analysis in the orthotopic lung cancer model:

[0127] 1) Experimental design and model establishment:

[0128] Establishment of a mouse orthotopic lung cancer model: 1.66 × 10⁻⁶ mcg was injected via the tail vein. 6 TC-1-hCD46-Luc cells, which co-express human CD46 receptor and luciferase, were used to confirm lung tumor formation on day 7 post-tumor inoculation using a small animal in vivo imaging system (IVIS).

[0129] The experiment consisted of four treatment groups: free Ads group, PEG-Ads group, conjugate group (Cy7 fluorescently labeled Ads) as described in Example 1, and an intravenous injection control group; the dosage for each group was uniformly set at 1×10⁻⁶. 7 PFUAds equivalent.

[0130] Administration regimen: Intratracheal microneedling was used for drug delivery via a visual small animal spray device; real-time IVIS scans were performed at 2h, 6h and 12h after drug administration to monitor the dynamic distribution of fluorescence signals in the lungs.

[0131] 2) Observation of results:

[0132] Dynamic distribution in the lungs: As shown in Figure 16, 2 hours after administration, the initial deposition in the lungs of the conjugate group in Example 1 was significantly higher than that of the free Ads group; 12 hours after administration, the retention rate of the conjugate group in the lungs remained at 75.18%, while the fluorescence signals of the free Ads and PEG-Ads groups had been basically cleared or degraded by respiratory mucus.

[0133] Distribution of organs outside the body: Organs were dissected and imaged 24 hours after administration, as shown in Figure 17. In the inhalation administration group of Example 1, the conjugates were mainly concentrated in the lungs and very little entered the systemic circulation; while in the intravenous administration group, they were mainly accumulated in the liver and there was almost no distribution in the lungs.

[0134] qPCR quantitative verification: The copy number of Ads in lung tissue was detected, as shown in Figure 17. The results showed that the viral load in the lungs of the inhaled combination group was 638.59% of that in the free inhalation group.

[0135] Lesion targeting: Fluorescence analysis of lung tissue sections was conducted to observe the enrichment of Ads at the tumor lesion site. As shown in Figure 18, the green fluorescence signal of Ads was precisely distributed in the lung tumor nodule area, rather than in the normal alveolar tissue, confirming the system's good lesion targeting.

[0136] 3) Conclusion Summary:

[0137] Experimental results show that the conjugate in Example 1 utilizes the active motility and chemotactic properties of the Synechococcus carrier to effectively overcome the physical clearance barrier of the respiratory tract, significantly prolonging the retention time of the drug in the tumor lesion, and laying a physical basis for subsequent efficient treatment.

[0138] (12) In vivo antitumor efficacy analysis in an orthotopic lung cancer model:

[0139] 1) Pharmacodynamic design:

[0140] A TC-1-hCD46-Luc mouse orthotopic lung cancer model was established: the mice were randomly divided into 5 groups (6 mice in each group): PBS group, Algae simple group, Ads simple virus group, PEGylated virus (PEG-Ads) group and Consortium group of Example 1.

[0141] Treatment regimen: As shown in Figure 19, medication was administered starting on day 7 after tumor inoculation, once every 3 days via intratracheal microneedling, for a total of 4 treatments; the viral dose was 1×10⁻⁶. 7 PFU / animal, Synechococcus dosage 1×10 6 CFU / each.

[0142] Evaluation indicators: Tumor bioluminescence intensity was monitored regularly using IVIS, weight changes in mice in each group were recorded, and survival time was monitored over a long period.

[0143] 2) Observation of results:

[0144] Tumor growth inhibition: As shown in Figure 19, the tumor luminescence intensity of the PBS group and the simple Synechococcus group increased exponentially over time, indicating a heavy tumor burden; although the simple virus group and the PEGylated virus group had a certain tumor-suppressing effect, they could not prevent the overall expansion of the tumor.

[0145] Efficacy of the conjugate group in Example 1: The conjugate group in Example 1 showed excellent therapeutic effects. On the 9th day of monitoring, the luminescent signal of the lung tumor decreased to the baseline background level, with an inhibition rate of more than 99%. Anatomical observation showed that there were basically no visible masses in the lungs of the mice in this group.

[0146] Improved survival: In subsequent long-term monitoring, the survival rate of mice in the conjugate group of Example 1 was 100% at the observation endpoint, which was significantly better than that of other control groups;

[0147] Safety performance: Throughout the entire treatment monitoring period, the weight gain curve of mice in the conjugate group of Example 1 remained stable, and no rapid weight loss due to the toxicity of the preparation was observed. No pathological damage was found in the sections of major organs.

[0148] 3) Conclusion Summary:

[0149] Experiments have shown that the inhalable oncolytic virus-bacterial conjugate of this invention can significantly improve the killing efficiency of oncolytic viruses against in situ lung cancer; through an active penetration and responsive release scheme, near-complete tumor eradication can be achieved at conventional doses, demonstrating significant technological advancement and broad clinical application prospects.

[0150] (13) Analysis of antitumor immune activation, microenvironment remodeling and cytokine levels induced by coupling:

[0151] 1) Experimental protocol and immunophenotypic analysis:

[0152] ① On the 10th day after the end of treatment of the orthotopic lung cancer model, peripheral blood and lung tissue samples were collected from mice in each treatment group;

[0153] ② Lung tissue was digested with 1.8 mg / mL collagenase IV for 1 hour to prepare a single-cell suspension; lymphocytes were obtained from peripheral blood using mouse lymphocyte separation medium.

[0154] ③ Use fluorescently labeled antibodies to stain the surface or intracellular cells of markers such as CD45, CD3, CD4, CD8, CD25, FOXP3, CD11b, GR-1 (Ly6G / Ly6C), CD80, CD206, CD11c, and CD86.

[0155] ④ The detection was performed using flow cytometry, and the levels of cytokines IFN-γ, IL-6 and TNF-α in serum and lung tissue homogenates were quantitatively detected using an ELISA kit.

[0156] 2) Observation of results:

[0157] Effector T cell infiltration: As shown in Figure 20, the conjugate treatment group induced the strongest CD8 cell infiltration. + T cell responses were observed in lung tissue and peripheral blood at rates of 33.4% and 32.9%, respectively, which were significantly higher than those in the PBS group (13.8% and 14.4%) and other single-component control groups.

[0158] Downregulation of suppressor cells: As shown in Figure 21, compared with other groups, the combination group significantly reduced the infiltration rate of lung Treg cells (down to 1.73%) and MDSCs cells (down to 7.36%), effectively alleviating the immunosuppressive state of the tumor microenvironment.

[0159] Myeloid cell remodeling: As shown in Figure 22, the consortium induced significant maturation of lung dendritic cells (CD80+ / CD86+ ratio 20.7%). Furthermore, macrophages exhibited a marked shift towards the M1 morphology (CD80+ / CD86+). + The polarization tendency was 39.9%, while the M2 type (CD206) accounted for 39.9%. + The polarization ratio was suppressed to 1.27%.

[0160] Pro-inflammatory cytokine secretion: As shown in Figure 23, the levels of IL-6 and TNF-α in the lungs and serum of mice in the consortium group were significantly increased, confirming the successful construction of a pro-inflammatory tumor microenvironment. IFN-γ levels remained within a moderate range, indicating that the systemic antiviral response was within a safe and controlled range.

[0161] (3) Conclusion Summary:

[0162] Experimental results show that the conjugate in Example 1 can reshape the "cold" tumor microenvironment into a "hot" environment through the synergistic effect of bacterial vector and oncolytic virus, and achieve a deep reversal of the immunosuppressive state of lung cancer by activating the cascade reaction of innate and acquired immunity.

[0163] (14) Validation analysis of the systemic antitumor immune response induced by the consortium through adoptive T-cell transfer (ACT):

[0164] 1) ACT Experiment Design:

[0165] Donor mouse pretreatment: Tumor-bearing donor mice were treated with endotracheal nebulization of the combination of the present invention. After treatment, CD8 was isolated and purified from the peripheral blood of the donor mice. + T cells.

[0166] Effector cell reinfusion: A mouse model of TC-1-hCD46-Luc lung tumor receptor was established, and 1×10-1 effector cells were infused. 6Individual effect CD8 + T cells were reinfused into the recipient mouse via tail vein injection.

[0167] Monitoring and Evaluation: The bioluminescence intensity of lung tumors in recipient mice was monitored on days 0, 7, and 14 after infusion using the IVIS system to evaluate the distal tumor suppression ability of adoptive T cells.

[0168] 2) Observation of results:

[0169] Systemic tumor suppression effect: As shown in Figure 24, the growth rate of lung tumor fluorescence signal in mice that received T cells derived from the "Example 1 Coupler Group" was significantly slower than that in mice that received T cells derived from the "PBS Group" or "Single Drug Group".

[0170] Long-term immune surveillance: Quantitative statistics showed that T cells derived from the conjugate group in Example 1 exhibited stronger amplification capacity and more durable tumor clearance activity in recipient mice, effectively inhibiting the expansion of lung lesions.

[0171] Safety record: All recipient mice that received cell reinfusion maintained normal body weight during monitoring, and no graft-versus-host disease or other significant systemic toxicity was observed.

[0172] 3) Conclusion Summary:

[0173] This experiment directly confirms that the anti-tumor immunity induced by the conjugate of this invention is systemic. Effector T cells stimulated by local respiratory administration can enter the bloodstream and home to the tumor site to exert a killing effect, providing a solid immunological basis for the treatment of multiple lung lesions and micrometastases.

[0174] (15) Therapeutic effect and immune memory verification in a model of distant lung metastases:

[0175] 1) Experimental Design and Model Establishment

[0176] Establishing a dual-lesion model of a lung tumor in the anteroposterior position and a distant metastatic tumor in the lateral abdomen: First, inject 1.66 × 10⁻⁶ mmol / L via the tail vein. 6 In situ lung cancer was established using TC-1 cells, and then 4 × 10⁴ cells were subcutaneously injected into the right ventral region of mice on day 9. 6 TC-1 cells were used to mimic distant metastatic lesions in clinical lung cancer.

[0177] Treatment plan: Intratracheal nebulization inhalation of the conjugate of this invention is administered only to the lung lesions (dose is 1×10). 7 PFU Ads equivalent, once every 2 days for a total of 4 times), no local treatment was performed on the lateral abdominal metastatic tumor site.

[0178] Evaluation indicators: Half of the mice were sacrificed on day 21, the lungs were fixed with Bouin solution and the number of tumor nodules was counted; the volume and weight of the flank metastatic tumors were measured; and the survival of the remaining mice was monitored.

[0179] 2) Observation of results:

[0180] Suppression of lung lesions: As shown in Figure 25, the lungs of the conjugate group in Example 1 remained basically pink and moist, with very few tumor nodules and small size, which was significantly better than the PBS group and other single-drug control groups (p < 0.0001).

[0181] Distant metastatic tumor suppression: As shown in Figure 25, despite no direct treatment of the flank tumor, the volume and weight of the distal tumor in the combined group mice were significantly suppressed. This indicates that local inhalation administration induced a potent systemic antitumor response.

[0182] Survival benefit: As shown in Figure 25, the mice in the consortium group exhibited the longest survival time and maintained a very high survival rate at the observation endpoint, demonstrating excellent immune memory protection.

[0183] 3) Conclusion Summary:

[0184] The results confirm that the inhalable oncolytic virus-bacterial conjugate provided by this invention can not only efficiently clear in situ lung tumors, but also simultaneously inhibit distant metastatic lesions outside the lungs by activating systemic anti-tumor immunity, providing an innovative immunotherapy strategy for multiple and metastatic lung cancer.

[0185] (16) In vivo safety analysis in the lungs:

[0186] 1) Experimental design:

[0187] Respiratory system imaging assessment: Healthy mice were treated with the clinical dose equivalent of the conjugate of Example 1 by inhalation. Four days after administration, iron oxide nanoparticles were introduced into the lungs as a contrast agent, and the lungs were scanned in axial, coronal and sagittal three-dimensional shapes using a micro-CT system.

[0188] Systemic toxicology evaluation: After the long-term efficacy experiment, the heart, liver, spleen, lungs, kidneys and other major organs of the test mice were collected, fixed in 4% paraformaldehyde and embedded in paraffin.

[0189] Histological staining: Routine H&E staining was performed, and the integrity of the tissue structure and the extent of inflammatory infiltration were observed using a digital slide scanner.

[0190] 2) Observation of results:

[0191] Imaging findings: CT images were used to assess the safety of lung inflammation. As shown in Figure 26, the lung contours of mice in each treatment group were clear, the translucency of the lung parenchyma was normal, and no obvious pulmonary exudation, pulmonary consolidation or ground-glass opacity were observed.

[0192] Organ histological analysis: H&E staining showed that the myocardial fibers of the mice in the conjugate group of Example 1 were neatly arranged, the liver lobule structure was intact, and the glomeruli were morphologically normal. Except for the tumor clearance area, no significant immune damage or interstitial pneumonia was observed in the lung tissue.

[0193] Weight monitoring: Throughout the treatment period, the weight curve of the mice in the combined group remained stable without significant fluctuations.

[0194] 3) Conclusion Summary:

[0195] This experiment demonstrates that the conjugate of this invention, while exerting a potent antitumor effect, possesses excellent organ compatibility and local respiratory safety; through PEG modification and carrier selection strategies, the cytotoxicity and biosafety of oncolytic viruses are effectively balanced, providing a good foundation for clinical translation.

[0196] (17) Analysis of cross-species tumor suppression effect in human tumor xenograft model (BALB / c nude mice):

[0197] 1) Experimental Design:

[0198] Model establishment: Male BALB / c nude mice aged 6-8 weeks were selected and injected with 2×10⁻⁶ mice via the tail vein. 6 A human tumor model was established using A549-Luc human lung cancer cells.

[0199] Treatment regimen: Tumor-bearing nude mice were randomly divided into PBS (pH 7.2), simple Synechococcus, simple virus, PEGylated virus and conjugate group of Example 1; intratracheal nebulized inhalation was administered on days 0, 3, 6 and 9 after tumor inoculation.

[0200] Monitoring indicators: The bioluminescence intensity of tumors was monitored regularly using the IVIS system. Mice were sacrificed on day 10, and the number of visible tumor nodules in the lungs was counted.

[0201] (2) Observation of results:

[0202] Tumor inhibition rate evaluation: As shown in Figure 27, compared with the PBS group, the conjugate group in Example 1 showed the most significant tumor inhibition effect, with a decrease in fluorescence intensity of about 95% and a significant reduction in the number of lung metastatic nodules.

[0203] Mechanism verification: Since nude mice lack mature T cells, the experimental results directly prove that the combination of the present invention has cross-species direct killing and oncolytic efficacy against human lung cancer cells, which does not completely depend on T cell immunity.

[0204] (3) Conclusion Summary:

[0205] Experimental results show that the conjugate group in Example 1 still maintains efficient delivery and killing functions in the context of human tumors, and has good cross-species applicability.

[0206] (18) Analysis of human immune activation in humanized NOG mouse model:

[0207] 1) Experimental Design:

[0208] Immune reconstitution: In NOG mice, 2×10⁻⁶ cells were intravenously reinfused. 6 Human peripheral blood mononuclear cells (PBMCs) amplified by IL-2 were used to rebuild the human immune system.

[0209] Tumor inoculation and treatment: A549-Luc cells were inoculated while rebuilding the immune system, and on day 5, inhalation therapy with the conjugate from Example 1 was administered at a dose of 1×10⁻⁶. 7 PFU equivalent.

[0210] Immunological assay: Lung tissue and blood were collected 48 hours after drug administration (day 7), and human CD8 was detected by flow cytometry. + The proportion of T cells.

[0211] 2) Observation of results:

[0212] Human T cell activation: As shown in Figure 28, the inhalation conjugate group induced extremely strong human CD8 activation. + T cell infiltration; human CD8 in lung tissue + The proportion of T cells was as high as 90.8%, and the proportion in peripheral blood was 67.5%, which was significantly better than the free Ads group (76.2% in the lungs and 40.8% in the blood) and other control groups.

[0213] Immune synergistic effect: The results confirm that the consortium can effectively activate and recruit human effector T cells into the tumor region by releasing PAMPs and tumor antigens.

[0214] 3) Conclusion Summary:

[0215] Experiments have shown that the conjugate of this invention can effectively activate the anti-tumor response generated by the human immune system, demonstrating its high translational potential in the clinical treatment of lung cancer patients.

[0216] 2. Mechanism verification:

[0217] (1) Calcium ions (Ca 2+ ) Verification of competitive intake mechanism:

[0218] Experimental protocol: The WH8102 Synechococcus was pretreated in calcium and magnesium-free D-PBS (pH 7.2) for 8 h, and then incubated for 12 h in simple buffer and culture system containing TC-1-hCD46 cells, respectively.

[0219] Fluorescence detection: Intracellular calcium concentration was detected using the Fluo-4,AM calcium ion probe.

[0220] Results Analysis: As shown in Figure 29, when WH8102 was co-cultured with tumor cells, Synechococcus exhibited a very strong green fluorescence signal inside, while the calcium fluorescence signal inside the tumor cells was significantly weakened. Quantitative analysis by flow cytometry further confirmed that WH8102 absorbed Ca... 2+ The amount was significantly higher than that of the non-chemotactic strain PCC7942; this indicates that Synechococcus can competitively deprive extracellular calcium from the tumor microenvironment using its surface calcium-binding groups.

[0221] (2) Evaluation of E-cadherin expression, a tight junction protein:

[0222] Protein extraction: After co-culturing TC-1-hCD46 cells with each component for 12 h, total cell protein was extracted using RIPA lysis buffer.

[0223] Western blotting analysis: Electrophoresis and imaging were performed to detect E-cadherin and the internal control beta-actin.

[0224] Conclusion: As shown in Figure 30, co-culturing with the WH8102 conjugate significantly downregulated E-cadherin expression in tumor cells; this mechanism demonstrates that the conjugate of this invention deprives C-cadherin of its Ca2+ expression level. 2+ Induced Ca 2+ The conformational breakdown of E-cadherin-dependent tumor tissues loosens the tight junctions between tumor cells at a biochemical level.

[0225] Example 2

[0226] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0227] Construction of functionalized oncolytic viruses:

[0228] 1) 5.62 × 10 7PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 1.2 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 60 min; the viral titer was determined using the Reed-Muench method.

[0229] 2) After the reaction is complete, the virus is centrifuged at 3000 rpm for 5 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0230] Construction of functionalized bacterial vectors:

[0231] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 2.4 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 60 min.

[0232] 2) After the reaction is complete, centrifuge at 3000 rpm for 5 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0233] Assembly of oncolytic virus-bacterial conjugates:

[0234] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 5:1 and stirred at 120 rpm for 60 min at room temperature to carry out a copper-free click chemical reaction.

[0235] 2) Centrifuge at 3000 rpm for 5 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0236] Connection rate and load analysis:

[0237] Quantitative analysis by qPCR: As shown in Figure 2, the copy number (%) of Ads in the conjugate precipitate and the supernatant (unbound virus) were detected respectively. The calculation showed that the Ads ligation rate in this Example 2 was 85.26% at a PFU:CFU ratio of 5:1.

[0238] Flow cytometry detection: The viral load on the surface of Synechococcus was observed using FITC-labeled Ads, as shown in Figure 3. In this Example 2, the positive rate of Ads load on Synechococcus was 71.0% at a PFU:CFU ratio of 5:1.

[0239] Example 3

[0240] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0241] Construction of functionalized oncolytic viruses:

[0242] 1) 2.248 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 1.2 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 60 min; the viral titer was determined using the Reed-Muench method.

[0243] 2) After the reaction is complete, the virus is centrifuged at 3000 rpm for 5 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0244] Construction of functionalized bacterial vectors:

[0245] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 2.4 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 60 min.

[0246] 2) After the reaction is complete, centrifuge at 3000 rpm for 5 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0247] Assembly of oncolytic virus-bacterial conjugates:

[0248] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 20:1 and stirred at 120 rpm for 60 min at room temperature to carry out a copper-free click chemical reaction.

[0249] 2) Centrifuge at 3000 rpm for 5 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0250] Connection rate analysis:

[0251] Quantitative analysis by qPCR: As shown in Figure 2, the copy number (%) of Ads in the conjugate precipitate and the supernatant (unbound virus) were detected respectively. The calculation showed that the Ads conjugation rate in this Example 3 was 53.43% at a PFU:CFU ratio of 20:1.

[0252] Example 4

[0253] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0254] Construction of functionalized oncolytic viruses:

[0255] 1) 1.124 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 0.8 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 30 min; the viral titer was determined using the Reed-Muench method.

[0256] 2) After the reaction is complete, the virus is centrifuged at 2500 rpm for 3 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed 3 times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0257] Construction of functionalized bacterial vectors:

[0258] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 1.2 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 30 min.

[0259] 2) After the reaction is complete, centrifuge at 2500 rpm for 3 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0260] Assembly of oncolytic virus-bacterial conjugates:

[0261] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 10:1 and stirred at 120 rpm for 60 min at room temperature to carry out a copper-free click chemical reaction.

[0262] 2) Centrifuge at 2500 rpm for 3 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0263] Example 5

[0264] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0265] Construction of functionalized oncolytic viruses:

[0266] 1) 1.124 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 2.0 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 60 min; the viral titer was determined using the Reed-Muench method.

[0267] 2) After the reaction was completed, the virus was centrifuged at 4000 rpm for 8 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0268] Construction of functionalized bacterial vectors:

[0269] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 4.2 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 60 min.

[0270] 2) After the reaction was completed, the mixture was centrifuged at 4000 rpm for 8 min, and the free modifier was washed to remove it, thus obtaining functionalized Synechococcus with azide groups on its surface.

[0271] Assembly of oncolytic virus-bacterial conjugates:

[0272] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 10:1 and stirred at 120 rpm for 60 min at room temperature to carry out a copper-free click chemical reaction.

[0273] 2) Centrifuge at 4000 rpm for 8 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0274] Example 6

[0275] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0276] Construction of functionalized oncolytic viruses:

[0277] 1) 1.124 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 1.5 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 45 min; the viral titer was determined using the Reed-Muench method.

[0278] 2) After the reaction is complete, the virus is centrifuged at 3000 rpm for 5 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0279] Construction of functionalized bacterial vectors:

[0280] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 2.8 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 45 min.

[0281] 2) After the reaction is complete, centrifuge at 3000 rpm for 5 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0282] Assembly of oncolytic virus-bacterial conjugates:

[0283] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 10:1 and stirred at 150 rpm for 60 min at room temperature to carry out a copper-free click chemical reaction.

[0284] 2) Centrifuge at 3000 rpm for 5 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0285] Example 7

[0286] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0287] Construction of functionalized oncolytic viruses:

[0288] 1) 1.124 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 1.2 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 60 min; the viral titer was determined using the Reed-Muench method.

[0289] 2) After the reaction is complete, the virus is centrifuged at 3000 rpm for 5 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0290] Construction of functionalized bacterial vectors:

[0291] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 2.4 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 60 min.

[0292] 2) After the reaction is complete, centrifuge at 3000 rpm for 5 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0293] Assembly of oncolytic virus-bacterial conjugates:

[0294] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 10:1 and stirred at 100 rpm for 30 min at room temperature to carry out a copper-free click chemical reaction.

[0295] 2) Centrifuge at 3000 rpm for 5 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0296] Example 8

[0297] A method for preparing an inhalable oncolytic virus-bacteria conjugated delivery system, the process flow of which is shown in Figure 1, includes the following steps:

[0298] Construction of functionalized oncolytic viruses:

[0299] 1) 1.124 × 10 8 PFU-containing oncolytic adenovirus Ad11-5ETel-GFP (containing the E1A gene driven by the hTERT promoter and the EGFP expression cassette) was mixed with 1.2 mg of the responsive linker DBCO-PEG2000-SS-NHS ester in 1 mL of PBS (pH 7.2) buffer and incubated at room temperature for 60 min; the viral titer was determined using the Reed-Muench method.

[0300] 2) After the reaction is complete, the virus is centrifuged at 3000 rpm for 5 min through an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and washed three times to remove unreacted small molecules, thus obtaining a functionalized oncolytic virus with alkyne groups on its surface.

[0301] Construction of functionalized bacterial vectors:

[0302] 1) Synechococcus hydrophila WH8102 was cultured in PCR-S11-RedSea medium at 20°C for a 12-hour light / dark cycle, and then 1.124×10⁻⁶ ppm was added. 7 CFU of Synechococcus polyphylla WH8102 was mixed with 2.4 mg of the non-responsive linker Azido-PEG2000-NHS ester and incubated at room temperature for 60 min.

[0303] 2) After the reaction is complete, centrifuge at 3000 rpm for 5 min, wash to remove free modifier, and obtain functionalized Synechococcus with azide groups on the surface.

[0304] Assembly of oncolytic virus-bacterial conjugates:

[0305] 1) The functionalized oncolytic virus was dispersed in PBS (pH 7.2) buffer containing the functionalized bacterial vector at a ratio of PFU:CFU = 10:1 and stirred at 200 rpm for 90 min at room temperature to carry out a copper-free click chemical reaction.

[0306] 2) Centrifuge at 3000 rpm for 5 min, collect the generated conjugate precipitate and resuspend it, and wash repeatedly to remove unbound virus particles to obtain inhalable oncolytic virus-bacterial conjugate.

[0307] In summary, the inhalable oncolytic virus-bacterial conjugate delivery system of this invention utilizes a natural cyanobacterium (Synechococcus WH8102) with active chemotactic motility as a carrier. Through bioorthogonal click chemistry, oncolytic adenoviruses (Ads) modified with tumor microenvironment-responsive polyethylene glycol (PEG) are conjugated to the surface of the cyanobacterium, constructing an inhalable oncolytic virus-bacterial conjugate. This conjugate delivery system, based on an actively motile carrier, utilizes the active navigation ability of cyanobacteria to circumvent respiratory mucus clearance and penetrate the epithelial barrier after drug administration, achieving efficient accumulation of oncolytic viruses at lung tumor lesions. Furthermore, the competitive deprivation of tumor interstitial calcium ions (Ca) by cyanobacteria... 2+ The action of [a specific substance] causes conformational collapse of the Ca2+-dependent E-cadherin domain, reducing intercellular adhesion and disrupting tight junctions in tumor tissue, thereby enhancing the penetration depth of Ads in the tumor core region. Oncolytic viruses and cyanobacteria synergistically remodel the tumor immune microenvironment, activating dendritic cell and macrophage maturation through pathogen-associated molecular patterns (PAMPs), enhancing the efficiency of Ads-mediated immunotherapy.

Claims

1. An inhalable oncolytic virus-bacteria conjugate delivery system, characterized in that, This delivery system involves clicking-coupled a bacterial carrier with the ability to actively move and competitively deprive the tumor stroma of calcium ions with an oncolytic virus and a tumor microenvironment-responsive linker that has been functionalized to form an oncolytic virus-bacterial conjugate.

2. The inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 1, characterized in that, The bacterial vectors capable of active motility and competitive deprivation of calcium ions from the tumor stroma are selected from one or more of the genera *Synechococcus*, *Prochlorococcus*, *Salmonella VNP20009*, or *Escherichia coli* K12; the oncolytic virus is selected from one or more of the genera oncolytic adenovirus, herpes simplex virus, vaccinia virus, reovirus, Malaba virus, vesicular stomatitis virus, or Newcastle disease virus.

3. The inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 1, characterized in that, Tumor microenvironment-responsive linkers contain redox-sensitive disulfide bonds. Bacterial vectors are functionalized to form linkers containing azide groups. Oncolytic viruses are functionalized to form tumor microenvironment-responsive linkers containing disulfide bonds and diphenylcyclooctylene. The azide groups and diphenylcyclooctylene undergo click chemistry. The ratio of oncolytic virus titer (PFU) to bacterial vector CFU is (5-20):

1.

4. The inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 1, characterized in that, The proportion of particles with an aerodynamic particle size distribution of oncolytic virus-bacterial conjugate in the range of 0.94 μm-4.46 μm is ≥50 wt.%.

5. A method for preparing the inhalable oncolytic virus-bacteria conjugate delivery system according to claim 1, characterized in that, Includes the following steps: Construction of functionalized oncolytic viruses: 1) Oncolytic viruses were mixed and incubated with functionalized modifiers containing disulfide bonds and diphenylcyclooctylene; 2) After the reaction was completed, unreacted small molecules were removed by ultrafiltration concentration to obtain functionalized oncolytic viruses with alkyne groups on their surface; Construction of functionalized bacterial vectors: 1) Bacterial vectors were mixed and incubated with functionalized modifiers containing azide groups; 2) After the reaction was completed, free modifiers were removed by centrifugation and washing to obtain functionalized bacterial vectors with azide groups on their surface; Assembly of oncolytic virus-bacterial conjugates: 1) Functionalized oncolytic viruses were dispersed in a buffer solution containing functionalized bacterial vectors and stirred to carry out a copper-free click chemistry reaction; 2) The generated conjugate precipitate was collected by centrifugation and resuspended, and unbound viruses were repeatedly washed to obtain inhalable oncolytic virus-bacterial conjugates.

6. The method for preparing the inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 5, characterized in that, In step 1) of constructing the functionalized oncolytic virus, the functionalizing agent containing disulfide bonds and diphenylcyclooctylene is DBCO-PEG2000-SS-NHS; the ratio of the functionalizing agent containing disulfide bonds and diphenylcyclooctylene to the oncolytic virus is 1.124 × 10⁻⁶. 8 PFU oncolytic virus corresponds to 0.8mg-2mg of modifier; the incubation time is 30min-60min; in step 2), the ultrafiltration concentration method is to use an ultrafiltration tube that is centrifuged and then repeatedly washed, wherein the molecular weight cutoff of the ultrafiltration tube is 100kDa, the centrifugation speed is 2500rpm-4000rpm, and the centrifugation time is 3min-8min.

7. The method for preparing the inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 5, characterized in that, In step 1) of constructing the functionalized bacterial vector, the functionalizing agent containing an azide group was Azido-PEG2000-NHS; the ratio of the functionalizing agent containing an azide group to the bacterial vector was 1.124 × 10⁻⁶. 7 CFU bacteria correspond to 1.2mg-4.2mg of modifier; incubation time is 30min-60min; in step 2), the centrifugation speed is 2500rpm-4000rpm and the centrifugation time is 3min-8min.

8. The method for preparing the inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 5, characterized in that, In step 1) of the assembly of the oncolytic virus-bacterial conjugate, the functionalized oncolytic virus is dispersed in the buffer of the functionalized bacterial vector at a ratio of PFU:CFU=(5-20):1; the stirring speed is 100rpm-200rpm and the stirring time is 30min-90min; in step 2), the centrifugation speed is 2500rpm-4000rpm and the centrifugation time is 3min-8min.

9. The use of the inhalable oncolytic virus-bacterial conjugate delivery system of claim 1 in the preparation of antitumor drugs.

10. The application of the inhalable oncolytic virus-bacteria conjugate delivery system as described in claim 9 in the preparation of an antitumor drug, wherein the antitumor drug is a drug for treating in situ lung cancer or inhibiting lung metastases, and can be prepared as a respiratory preparation for intratracheal nebulization inhalation.

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