A method for assessing the targeting function of a cell association delivery system

Abstract

The application discloses a detection method for evaluating the targeting function of a cell combined delivery system. The method is aimed at a combined system containing navigation cells and drug-loaded units. Three groups of control systems are constructed, i.e. a system only containing labeled navigation cells, a system containing unlabeled navigation cells and labeled drug-loaded units in physical mixing, and a system containing unlabeled navigation cells and labeled drug-loaded units in chemical connection, and then the systems are injected into animal models of diseases. The fluorescence signals of target tissues are detected at preset time points. By comparing the signal differences of the systems, the evaluation method for distinguishing between cell "active delivery" and "passive retention" can be effectively obtained. The method is scientific in design and clear in steps, is suitable for the combined delivery system of various cell combinations, provides a reliable and fine analysis scheme for the standardized in-vivo targeting function evaluation, and can be used for system optimization, comparison of targeting ability and preclinical pharmacokinetic research.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and more specifically, to a detection method for quantitatively evaluating the in vivo targeting function of a cell-based drug delivery system, particularly suitable for evaluating the targeting efficiency and specificity of a combined delivery system formed by physical or chemical connections of different types of cells (such as immune cells and platelets). Background Technology

[0002] Combined delivery systems using cells as carriers or components, such as those combining chemotactic immune cells (e.g., macrophages, mesenchymal stem cells) with drug-carrying blood cells (e.g., platelets), have shown great potential in targeted therapy. The core advantage of these systems lies in their ability to actively deliver drug-carrying units to lesion areas where conventional drugs are difficult to accumulate, leveraging the homing capabilities of "navigation cells," thereby enhancing efficacy and reducing systemic side effects.

[0003] However, accurately and objectively assessing the in vivo targeting function of such complex living systems remains a significant technological challenge. Traditional assessment methods primarily rely on in vivo fluorescence imaging to observe the fluorescence signal intensity of whole organs or tissues. While intuitive, this method has significant drawbacks: it cannot distinguish whether the observed signal originates from the targeting of the carrier cells themselves, the targeting of the delivery unit they carry, or the result of their independent behavior after a simple mixture; it also cannot assess the improvement in targeting efficiency of the combined system compared to a simple mixture; finally, traditional methods are mostly qualitative or semi-quantitative, lacking standardized quantitative indicators, making it difficult to compare results from different studies and hindering the fine-grained optimization of delivery systems.

[0004] Therefore, developing a detection method capable of accurately quantifying the targeting behavior of each component in a cell-co-delivery system, particularly one that can distinguish and quantify the effects of "active carrying" and "passive coexistence," is of paramount importance for the research, optimization, and evaluation of such systems. Currently, there are no reported standardized detection methods specifically designed for the precise evaluation of the targeting function of cell-co-delivery systems. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing evaluation techniques, such as the inability to analyze signal sources and the lack of spatial fine-grained analysis capabilities, and to provide a novel detection method for evaluating the in vivo targeting function of cell-based combined delivery systems. This method can clearly distinguish the targeting contribution of each component in the combined system and provide its spatial distribution information within the target tissue.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for evaluating the targeting function of a cell-based combined delivery system, the system comprising a first type of navigation cells and a second type of drug delivery unit; characterized in that the method includes the following core steps: (1) Cell fluorescence labeling: The first type of navigation cells and the second type of drug-loaded cells were labeled with fluorescent markers respectively; (2) Construction of control systems: Construct at least three sets of test systems as follows: Group A: Contains only labeled Class I navigation cells; Group B: A physical mixture containing unlabeled Type I navigation cells and labeled Type II drug delivery units; Group C: A cell-based joint delivery system consisting of unlabeled Type I navigation cells and labeled Type II drug delivery units connected together; (3) In vivo delivery: The A, B and C systems were respectively introduced into the animal models of the disease; (4) Target tissue acquisition and signal detection: At preset time points, target tissues of model animals are acquired, and the signal intensity of the fluorescent markers in the target tissues is detected respectively; Preferably, the first type of navigation cell is one of macrophages, mesenchymal stem cells, or neutrophils.

[0007] Preferably, the second type of drug-carrying unit is one of platelets, red blood cells, or engineered microvesicles.

[0008] Preferably, the fluorescent label is one of DiO, DiI, DiD or DiR.

[0009] Preferably, the connection method is a bioorthogonal chemical reaction connection, an antibody-antigen mediated connection, or a biotin-avidin based connection.

[0010] Preferably, the signal detection method is quantitative analysis by fluorescence microscopy after in vivo imaging of small animals or frozen sections.

[0011] Preferably, the animal model of the disease is one of a spinal cord injury model, a myocardial infarction model, or a bone defect model.

[0012] This invention also provides a method for preparing a cell-based co-delivery system for macrophages (navigation cells) coupled with platelet drugs (drug delivery units), comprising the following steps: (1) Platelet isolation and purification: Peripheral blood or cardiac blood was collected from healthy mammals, using acid-citrate-glucose solution A (ACD-A) as an anticoagulant. The anticoagulated whole blood was centrifuged at 150 x g for 10–15 minutes at room temperature (approximately 25°C). The supernatant platelet-rich plasma (PRP) was carefully collected. Subsequently, the PRP was centrifuged again at 800 x g for 20 minutes at room temperature to precipitate the platelets. The supernatant was gently discarded, and the platelet pellet was resuspended in phosphate-buffered saline (PBS, pH 7.4) containing 1 μM prostaglandin E1 (PGE1) to inhibit pre-activation of platelets. Platelets were counted using a cell counter and stored at room temperature for later use.

[0013] (2) Preparation of gene transfection nanocomposites: A cationic polymer with reactive oxygen species (ROS) responsive properties (e.g., polymer CBP5 containing borate ester bonds) was dissolved in 10 mM HEPES buffer (pH 7.4) and diluted to 0.642–1.925 mg / mL. Simultaneously, the desired target gene (e.g., therapeutic plasmid DNA) was dissolved in the same HEPES buffer and diluted to 40 μg / mL as a nucleic acid drug. The polymer solution was rapidly added to the DNA solution at a 1:1 volume ratio of nucleic acid drug to cationic polymer, and immediately vortexed for 20 seconds. The mixture was then incubated at room temperature for 30 minutes to obtain gene transfection nanocomposites with a nitrogen-phosphorus molar ratio of 10–30.

[0014] (3) Preparation of drug-loaded platelets: The platelet suspension prepared in step (1) and the gene transfection nanocomplex solution prepared in step (2) were mixed at a volume ratio of 1:2 to 1:4. The mixture was placed in a constant temperature shaker at 37°C and gently incubated in the dark at 60 rpm for 1 to 2 hours. After incubation, the mixture was centrifuged at 1000 x g for 5 minutes, and the supernatant was discarded to remove unloaded free nanocomplexes. The precipitate was washed twice with PBS buffer containing 1 μM PGE1, and finally resuspended to obtain drug-loaded platelets.

[0015] (4) Obtaining macrophages: Mononuclear cells were isolated from mammalian bone marrow and induced to differentiate for 5-7 days using complete culture medium containing 25 ng / mL M-CSF to obtain bone marrow-derived macrophages.

[0016] (5) Cell covalent linkage: The macrophages obtained in step (4) were co-incubated with a medium containing 40 μM N-azylacetylgalactosamine (Ac4GalNAz) for 48 to 72 hours. After incubation, the cells were gently washed three times with PBS to remove uninvolved markers. The drug-loaded platelets prepared in step (3) were incubated with 20 μM dibenzocyclooctyne-polyethylene glycol-active ester (DBCO-PEG4-NHS ester) at room temperature in the dark for 30 minutes. The cells were then washed twice with PBS by centrifugation to remove excess DBCO reagent. The azide-modified macrophages and DBCO-modified drug-loaded platelets were mixed at a cell ratio of 1:1 to 1:4 and gently incubated at room temperature for 30 to 45 minutes. The final product was collected by centrifugation at 300 x g for 5 minutes to obtain the macrophage-platelet drug delivery system.

[0017] Preferably, the mammalian platelets are platelets extracted from the heart blood of 6-8 week old C57BL / 6J mice.

[0018] Preferably, the mammalian macrophages are macrophages derived from mononuclear cells of the femur and tibia of 6-8 week old C57BL / 6J mice.

[0019] Preferably, the nucleic acid drug is one of DNA, microRNA, or siRNA.

[0020] Preferably, the nitrogen-to-phosphorus ratio of the nucleic acid drug to the polymer in step (2) is 20.

[0021] Preferably, the gene transfection nanoparticles have a diameter range of 50–100 nm.

[0022] Preferably, the platelet suspension prepared in step (1) and the gene transfection nanocomplex solution prepared in step (2) are mixed at a volume ratio of 1:4. Preferably, the azide modification in step (5) is achieved by co-incubating macrophages with Ac4GalNAz for 72 hours.

[0023] Preferably, the azide-modified macrophages and DBCO-modified drug-loaded platelets are mixed at a cell number ratio of 1:1 at room temperature in step (5).

[0024] Preferably, the azide-modified macrophages and DBCO-modified drug-loaded platelets described in step (5) are gently incubated at room temperature for 45 minutes.

[0025] This invention also claims protection for the application of the detection method in any of the following aspects: (1) Used to screen and optimize the optimal ratio and connection strategy of two types of cells in a cell co-delivery system; (2) Used to compare the targeting guidance capabilities of first-class carrier cells from different sources or under different treatment states; (3) As a core method for evaluating the pharmacokinetic and tissue distribution characteristics of cell-based combined delivery systems in preclinical studies; (4) Used to evaluate the performance of other complex biological delivery systems that require deconstructive analysis of the targeting behavior of different components.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention is the first to propose a dual control by setting up a "physical hybrid group" and a "combined system group" to deconstruct the active contribution rate of carrier cells to the targeting of the delivery unit in a cell-combined delivery system. The concept is novel and the design is ingenious.

[0027] 2) It effectively distinguishes between two different situations: the enrichment of the carrier unit due to the "carrying" of the carrier cell and the enrichment due to its own "non-specific retention", which truly reflects the construction value of the joint system.

[0028] 3) The method has clear steps, requires conventional reagents and equipment, and is easy to implement and promote in ordinary biological laboratories, providing a feasible solution for standardized evaluation of targeted functions.

[0029] 4) It is not only applicable to the macrophage-platelet system, but its methodology can also be extended to other combined delivery systems of various cell combinations, and has broad applicability. Attached image description: Figure 1 This is a laser confocal image of platelet-loaded gene transfection complexes.

[0030] Figure 2 It is a particle size and potential distribution diagram of gene transfection complex, platelets, and drug-loaded platelets.

[0031] Figure 3 These are laser confocal microscopy and electron microscopy images of the macrophage-platelet drug delivery system.

[0032] Figure 4 This is a graph showing the fluorescence distribution and statistical distribution of various tissues in in vivo imaging of a macrophage-platelet drug delivery system in mice with spinal cord injury.

[0033] Figure 5 This is a laser confocal fluorescence colocalization map of the macrophage-platelet drug delivery system and macrophages in the spinal cord tissue of mice. Detailed Implementation

[0034] The following description, in conjunction with embodiments, further illustrates a macrophage-platelet drug delivery system and its preparation method provided by the present invention. However, the following embodiments should not be considered as limiting the scope of protection of the present invention.

[0035] Example 1: Cell fluorescent labeling Macrophage BMMs and platelet (PLT) cells were co-incubated with the fluorescent dye 1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarbonylcyanine perchlorate (DiD, 1 μM) for 10 minutes. After incubation, the labeled BMMs were centrifuged at 400 x g for 5 minutes and the labeled PLTs at 800 x g for 10 minutes to remove unbound free dye. Washing was performed twice with PBS buffer to obtain fluorescently labeled BMMs. DiD and PLT DiD .

[0036] Example 2: Cell fluorescent labeling Macrophage BMMs and platelet-derived PLTs were co-incubated with the fluorescent dye 1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarbonylcyanine perchlorate (DiD, 1 μM) for 15 minutes. After incubation, the labeled BMMs were centrifuged at 400 x g for 5 minutes and the labeled PLTs at 800 x g for 10 minutes to remove unbound free dye. Washing was performed twice with PBS buffer to obtain fluorescently labeled BMMs. DiD and PLT DiD .

[0037] Example 3: Cell fluorescent labeling Macrophage BMMs and platelet-derived PLTs were co-incubated with the fluorescent dye 1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarbonylcyanine perchlorate (DiD, 1 μM) for 20 minutes. After incubation, the labeled BMMs were centrifuged at 400 x g for 5 minutes and the labeled PLTs at 800 x g for 10 minutes to remove unbound free dye. Washing was performed twice with PBS buffer to obtain fluorescently labeled BMMs. DiD and PLT DiD .

[0038] Example 4: Construction of a control system and in vivo targeted detection Construct at least the following three test systems: Group A: Label-only BMMs (BMMs) DiD 1×10 6 Group A: contains unlabeled BMMs and PLTs (cells); Group B: contains unlabeled BMMs and PLTs. DiD Physical mixture (BMM+PLT) DiD 1×10 6Group C: Unlabeled BMMs and PLTs (cells); DiD The cells were joined using the aforementioned "cell covalent linking" method (i.e., conjugate reaction based on click chemistry) to prepare a combined delivery system (BMM-PLT). DiD 1×10 6 On day 3 after spinal cord injury (SCI) modeling was completed in 8-12 week old C57BL / 6 mice, the cells were administered via tail vein, with a total cell suspension volume of 200 μL per mouse. In vivo imaging was performed at 6 hours and 24 hours post-injection. The IVIS Spectrum in vivo imaging system was used, with an excitation wavelength of 644 nm and an emission filter of 663 nm to specifically detect DiD fluorescence signals. In vitro imaging was performed on major organs (heart, liver, spleen, lung, kidney) and the target tissue (spinal cord) to systematically analyze the in vivo distribution of DiD-labeled cells. High-resolution imaging of tissue sections was further performed using laser confocal microscopy to achieve specific targeted localization analysis at the cellular level.

[0039] Example 5: Construction of a control system and in vivo targeted detection Construct at least the following three test systems: Group A: Label-only BMMs (BMMs) DiD 5×10 6 Group A: contains unlabeled BMMs and PLTs (cells); Group B: contains unlabeled BMMs and PLTs. DiD Physical mixture (BMM+PLT) DiD 5×10 6 Group C: Unlabeled BMMs and PLTs (cells); DiD The cells were joined using the aforementioned "cell covalent linking" method (i.e., conjugate reaction based on click chemistry) to prepare a combined delivery system (BMM-PLT). DiD 5×10 6 On day 3 after spinal cord injury (SCI) modeling was completed in 8-12 week old C57BL / 6 mice, the cells were administered via tail vein, with a total cell suspension volume of 200 μL per mouse. In vivo imaging was performed at 6 hours and 24 hours post-injection. The IVIS Spectrum in vivo imaging system was used, with an excitation wavelength of 644 nm and an emission filter of 663 nm to specifically detect DiD fluorescence signals. In vitro imaging was performed on major organs (heart, liver, spleen, lung, kidney) and the target tissue (spinal cord) to systematically analyze the in vivo distribution of DiD-labeled cells. High-resolution imaging of tissue sections was further performed using laser confocal microscopy to achieve specific targeted localization analysis at the cellular level.

[0040] Example 6: Construction of a control system and in vivo targeted detection Construct at least the following three test systems: Group A: Label-only BMMs (BMMs) DiD 1×10 7 Group A: contains unlabeled BMMs and PLTs (cells); Group B: contains unlabeled BMMs and PLTs. DiD Physical mixture (BMM+PLT) DiD 1×10 7 Group C: Unlabeled BMMs and PLTs (cells); DiD The cells were joined using the aforementioned "cell covalent linking" method (i.e., conjugate reaction based on click chemistry) to prepare a combined delivery system (BMM-PLT). DiD 1×10 7 On day 3 after spinal cord injury (SCI) modeling was completed in 8-12 week old C57BL / 6 mice, the cells were administered via tail vein, with a total cell suspension volume of 200 μL per mouse. In vivo imaging was performed at 6 hours and 24 hours post-injection. The IVIS Spectrum in vivo imaging system was used, with an excitation wavelength of 644 nm and an emission filter of 663 nm to specifically detect DiD fluorescence signals. In vitro imaging was performed on major organs (heart, liver, spleen, lung, kidney) and the target tissue (spinal cord) to systematically analyze the in vivo distribution of DiD-labeled cells. High-resolution imaging of tissue sections was further performed using laser confocal microscopy to achieve specific targeted localization analysis at the cellular level.

[0041] The fluorescently labeled cell delivery system prepared according to the aforementioned method can be used for targeted drug delivery detection and analysis in tissue damage. Relevant confirmatory experimental data are as follows: Experimental Example 1: Laser confocal microscopy assay of platelet-loaded gene transfection complexes Following the cell-based combined delivery system preparation method, plasmid DNA was labeled with Cy5 fluorescent dye (red) to prepare drug-loaded platelets (P-NPs@DNA). Cy5 P-NPs@DNA Cy5 Short-term staining with the membrane dye WGA-FITC (green) was used to mark cell outlines, followed by observation of the slide. Figure 1 As shown, under fluorescence, a clear red Cy5 signal can be observed distributed within the platelet outline delineated by WGA-FITC green fluorescence. The red signal is highly co-localized with the green outline and is located inside the cell, rather than simply adsorbed on the membrane surface. This result directly confirms that the DNA nanocomposite is efficiently encapsulated in the cytoplasm of platelets, successfully preparing drug-loaded platelets.

[0042] Experimental Example 2: Particle size and potential analysis of gene transfection complex, platelets, and drug-loaded platelets Gene transfection complexes, platelets, and drug-loaded platelets were prepared according to the cell-based combined delivery system preparation method. For example... Figure 2 As shown, the gene-transfected nanocomposite has a particle size of approximately 65.2 nm and a potential of 12.1 mV. Natural platelets have a particle size of approximately 2.5 μm and a potential of -8.6 mV. In contrast, the drug-loaded platelets (P-NPs) have a particle size of approximately 2.7 μm, only slightly larger than natural platelets, and their potential (-7.9 mV) is very close to that of natural platelets. This further demonstrates that the gene-transfected nanocomposite was successfully encapsulated on platelets, while the drug loading process had minimal impact on the size and surface properties of the platelets themselves.

[0043] Morphological characterization of the macrophage-platelet drug delivery system in Experiment Example 3 The macrophage-platelet cell co-delivery system (MP-NPs@DNA), prepared according to the cell co-delivery system preparation method, was observed using laser confocal microscopy and scanning electron microscopy. For example... Figure 3 As shown in Figure A, the confocal image reveals that after click chemical bonding, Rhodamine B-labeled drug-loaded platelets (red) bind tightly to bright-field macrophages (white), and platelet pseudopodia growth is observed after Th activation, suggesting that the bonding process preserves platelet functional characteristics. Figure 3 As shown in Figure B, electron microscopy further confirmed the ultrastructural characteristics of platelets attached to the macrophage surface, with both cell morphologies well preserved. The platelets retained their typical disc-shaped or activated spheroidal morphology. This indicates that we have successfully constructed a structurally stable macrophage-platelet drug delivery system.

[0044] Experimental Example 4: In vivo targeted distribution analysis of macrophage-platelet drug delivery system Establishing a mouse model of spinal cord injury: 8-12 week old C57BL / 6 mice were anesthetized and fixed in a prone position. Hair was shaved from the T8-T12 region of the back and the area was disinfected. The skin was incised along the midline, and the muscle tissue was bluntly dissected. A T10 laminectomy was performed to fully expose the spinal cord. An impactor (such as an IH Impactor) was used to induce contusion of the exposed spinal cord with a specific impact force (typically 50-70 kdyn). The muscle and skin incisions were sutured layer by layer. Indicators of a successful model include: post-modeling paralysis of both hind limbs and a significant decrease in the BassoMouseScale (BMS) score.

[0045] In vivo targeting ability test of macrophage-platelet drug delivery system: BMM-PLT was prepared according to the methods in Examples 2 and 4. DiD And a simple combination of the two (BMM+PLT) DiD ), BMM DiD BMM+PLT DiDBMM-PLT DiD (1 x 10 each) 6 (200 µL per mouse) was injected via the tail vein into mice 3 days after spinal cord injury. Figure 4 As shown, at 6 hours and 24 hours post-injection, the simple mixture BMM+PLT was compared with... DiD In comparison, BMM DiD BMM-PLT with group and covalent connection DiD The groups all produced stronger fluorescence signals in the spinal cord region. Furthermore, the simple mixture BMM+PLT... DiD No PLT added DiD Enrichment in the spinal cord. This indicates that the transport of covalently linked platelets to the site of injury is primarily mediated by the targeting capabilities of macrophages, a capability that platelets cannot achieve in the subacute phase.

[0046] Experimental Example 5: Cellular Localization Analysis of Macrophage-Platelet Drug Delivery System in Tissues To further validate in vivo cell-targeting capabilities, a macrophage-platelet drug delivery system (MP-NPs@DNA) was prepared by labeling plasmid DNA with Cy5 fluorescent dye (red) according to the cell-based combined delivery system preparation method. Cy5 After the in vivo experiments were completed, mouse spinal cord tissue was taken for frozen sectioning and immunofluorescence staining analysis. Figure 5 As shown, in sections of the spinal cord injury area, we stained for the macrophage marker CD11b (green) and the system-carried Cy5-DNA (red). Confocal images revealed a high degree of co-localization between the red Cy5 fluorescence signal and the green CD11b-positive macrophages, indicating that the gene therapy delivered to the injured spinal cord was mainly distributed within the infiltrating macrophages. This directly demonstrates that the MP-NPs system constructed in this invention can be carried by macrophages, successfully cross complex in vivo barriers, and precisely deliver therapeutic genes to effector cells in the target region, achieving precise localization at the cellular level.

[0047] The present invention, through the above embodiments, verifies that the proposed detection method can achieve multi-dimensional and refined evaluation of the targeting function of cell-assisted delivery systems. This method, combining relative quantitative analysis and distribution characteristic analysis, can effectively evaluate targeting efficiency, cell synergy, and spatial distribution characteristics within tissues, providing a systematic experimental analysis framework for the research and optimization of related delivery systems.

[0048] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any improvements and modifications made by those skilled in the art within the scope of the technical principles disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for evaluating the targeting function of a cell-based combined delivery system, the delivery system comprising a first type of navigation cells and a second type of drug-carrying unit, characterized in that, The detection method includes the following steps: (1) The first type of navigation cells and the second type of drug delivery units were labeled with fluorescent markers, respectively; (2) Construct at least three sets of test systems: Group A: Contains only labeled Class I navigation cells; Group B: A physical mixture comprising unlabeled Type I navigation cells and labeled Type II drug delivery units; Group C: A cell-based co-delivery system comprising unlabeled Type I navigation cells and labeled Type II drug delivery units formed by chemical bonding; (3) Inject the A, B and C systems constructed in step (2) into the animal models of the disease respectively; (4) At a preset time point, target tissues of the model animal are collected, and the signal intensity of the fluorescent marker in each target tissue is detected. By comparing and analyzing the differences in signal intensity of each group in the target tissue, the active contribution of the first type of navigation cells to the targeted enrichment of the second type of drug delivery unit is evaluated.

2. The detection method according to claim 1, characterized in that, The first type of navigation cells is selected from macrophages, mesenchymal stem cells, or neutrophils.

3. The detection method according to claim 1, characterized in that, The second type of drug delivery unit is selected from platelets, erythrocytes, or engineered microvesicles.

4. The detection method according to claim 1, characterized in that, The fluorescent marker is selected from one of DiO, DiI, DiD or DiR.

5. The detection method according to claim 1, characterized in that, The chemical connection method is selected from one of click chemical reaction connection, antibody-antigen mediated connection, or biotin-avidin based connection.

6. The detection method according to claim 1, characterized in that, The signal detection method described in step (4) is selected from small animal in vivo imaging or quantitative analysis by fluorescence microscopy after frozen sectioning.

7. The detection method according to claim 1, characterized in that, The animal model for the disease is selected from one of the following: spinal cord injury model, myocardial infarction model, or bone defect model.

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