High-drug-loading-capacity living cell preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a high-drug-loading-capacity living cell preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: co-incubating drug carrier microspheres and carrier cells to enable the carrier cells to phagocytize the drug carrier microspheres, and removing the drug carrier microspheres which are not phagocytized to obtain the living cell preparation. The invention discloses a high-drug-loading-capacity living cell preparation as well as a preparation method and application thereof, solves the technical problems that an existing living cell drug delivery system is low in drug loading capacity, uncontrollable in release, easy to damage cell functions and the like, and brings new prospects for treatment of diseases such as tumors, autoimmunity and chronic inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a high-drug-load live cell preparation, its preparation method, and its application. Background Technology

[0002] With the development of cell therapy technology, utilizing live cells with natural chemotactic migration capabilities, such as macrophages and mesenchymal stem cells, as drug carriers has become an important strategy for targeted delivery. Current technologies typically employ biodegradable polymer microspheres and nanoparticles as carriers to load drugs via phagocytosis, hoping to leverage the cell's navigation function to increase drug concentration at the lesion site. However, this field still faces significant limitations: on the one hand, the drug loading ratio of traditional carriers is relatively low, typically not exceeding 10 wt%; on the other hand, drug carriers entering cells often exhibit significant initial burst release and are easily degraded by lysosomes or rapidly expelled via exocytosis, making it difficult to maintain long-term stable release behavior. Furthermore, attempts to increase drug loading by increasing carrier particle size or improving the drug loading ratio often result in problems such as drug crystallization and structural instability, which can adversely affect cell viability and migration function. Therefore, current technologies have not yet been able to simultaneously achieve high particle drug loading, high single-cell drug loading, and long-term controllable intracellular release while ensuring cell viability and function, thus limiting the clinical translational potential of live cell drug delivery technology. Summary of the Invention

[0003] This invention aims to provide a high-drug-load live cell preparation, its preparation method, and its application, solving the technical problems of low drug loading, uncontrollable release, and easy damage to cell function in existing live cell drug delivery systems, and bringing new prospects for the treatment of diseases such as tumors, autoimmune diseases, and chronic inflammation.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a high-drug-loaded live cell preparation includes the following steps: Drug carrier microspheres are co-incubated with carrier cells, allowing the carrier cells to engulf the drug carrier microspheres. Unengulfed drug carrier microspheres are then removed to obtain a live cell preparation.

[0005] Preferably, the mass fraction of the drug in the drug carrier microspheres is ≥20wt%, and the particle size of the drug carrier microspheres is 1-5μm.

[0006] More preferably, the mass fraction of the drug in the drug carrier microspheres is ≥50wt%, and the particle size of the drug carrier microspheres is 1-5μm.

[0007] More preferably, the mass fraction of the drug in the drug carrier microspheres is 50wt%-80wt%.

[0008] Preferably, the single-cell drug loading obtained after the carrier cells engulf the drug carrier microspheres is ≥50 pg / cell.

[0009] Preferably, the carrier cells are selected from one or two of the following: macrophages, macrophages differentiated from monocytes, bone marrow-derived macrophages, tissue macrophages, peritoneal macrophages, embryonic stem cells, pluripotent stem cells, dendritic cells, or neutrophils.

[0010] Preferably, the drug carrier microspheres comprise one or both of biodegradable polyester materials and fatty acid prodrug materials.

[0011] Preferably, the biodegradable polyester material includes one or two of polylactic acid-glycolic acid copolymer, polylactic acid, or polycaprolactone.

[0012] Preferably, the fatty acid prodrug includes one or both of saturated fatty acid prodrugs and unsaturated fatty acid prodrugs.

[0013] The present invention also provides a high-drug-loaded live cell preparation prepared by the method described above.

[0014] The present invention also provides the use of the high-drug-loaded live cell preparation in the preparation of a medicament for treating inflammatory diseases, tumor diseases or autoimmune diseases.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a high-drug-load viable cell formulation, its preparation method, and its applications. The drug carrier microspheres have a drug loading capacity of over 50 wt%, enabling single-cell drug loading to reach over 50 pg, significantly reducing the required cell number. This high-drug-load viable cell formulation exhibits stable near-zero-order release kinetics within 12-72 hours, with a low initial burst release and a constant release rate. Simultaneously, cell viability and migration function are well preserved. This technology ultimately demonstrated excellent lesion-targeting enrichment ability and long-lasting local drug release in animal models, significantly reducing systemic toxicity while ensuring therapeutic efficacy.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 Scanning electron microscope image of 3µm dexamethasone PLGA particles; Figure 2 Scanning electron microscope image of 3µm dexamethasone palmitate microspheres; Figure 3 This is a schematic diagram showing the drug loading capacity within macrophages. Figure 4This is a schematic diagram of macrophages endocytizing 3µm dexamethasone palmitate microspheres, where red represents the cell membrane and yellow represents the dexamethasone palmitate microspheres. Figure 5 The drug release curve of dexamethasone palmitate microspheres in macrophages ( n = 3); Figure 6 The percentage of migration ability of blank macrophages and macrophages loaded with 3-micron PLGA particles in the in vitro cell transwell assay. n = 6); Figure 7 A comparison was made between blank macrophages and macrophages loaded with 3-micron PLGA particles targeting inflamed muscle. Figure 7 In figure 'a', the image shows the in vivo imaging results of adoptive macrophages (including blank macrophages and macrophages loaded with PLGA3 particles) in a mouse inflammatory muscle model, using a whole-body in vivo imaging system. Figure 7 In Figure a, b is a schematic diagram of the quantitative analysis of the fluorescence intensity of infiltrating macrophages in muscle measured at 48 hours. n = 3), Figure 7 In the diagram, 'c' represents the targeting efficiency of blank macrophages and macrophages loaded with PLGA3 particles in bilateral skeletal muscle as measured by flow cytometry. n = 3). Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0021] Example 1 A drug carrier microsphere is prepared by the following steps: S1. Dissolve 5 mg of dexamethasone and 10 mg of polylactic acid-glycolic acid copolymer (PLGA) in 1 mL of dichloromethane, and stir magnetically or vortex at room temperature until completely dissolved to obtain a uniform and transparent dispersed phase.

[0022] S2. 10 mL of an aqueous solution containing 1% (by mass / volume) polyoxyethylene-polyoxypropylene block copolymer P407 was used as the continuous phase. This solution, along with the dispersed phase obtained in S1, was introduced into the microfluidic chip via a syringe pump. At the chip junction, a stable flow rate ratio (dispersed phase flow rate: continuous phase flow rate = 1:15) was used for shear dispersion, forming an oil-in-water droplet structure. By adjusting the flow rates of the two phases and the microfluidic channel structure, the droplet size could be controlled within the range of 1–5 μm. S3. The droplets obtained in S2 were directly collected in a container containing a large amount of continuous phase of the same composition. The mixture was magnetically stirred at 250 rpm for 3 hours at room temperature to allow the organic solvent to fully diffuse or evaporate, and the droplets solidified to form drug carrier microspheres. After solidification, the suspension was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended in an appropriate amount of deionized water and washed three times to remove free drug and excess surfactant, finally obtaining a microsphere suspension. The suspension was then freeze-dried at -80℃ for 12 hours to obtain dried polylactic acid-glycolic acid copolymer microspheres (PLGA MPs).

[0023] The resulting microspheres are white or off-white powders with good flowability, making them suitable as carrier materials for subsequent cell loading experiments.

[0024] The above-mentioned drug carrier microspheres were characterized as follows: Scanning electron microscopy (SEM) was used to observe and measure the particle size and morphology, obtaining representative images of microspheres. Image analysis software was used to measure the diameter of each microsphere in the SEM images, with at least 100 particles selected for statistical analysis to calculate the average particle size and particle size distribution range. The results are as follows: Figure 1 As shown.

[0025] Depend on Figure 1 It can be seen that the obtained microspheres are regular spherical or near-spherical in shape, with smooth surfaces, and no obvious aggregation or deformed particles were observed. The particle size can be adjusted within the range of 1~5μm depending on the preparation parameters, and the coefficient of variation of the particle size of the microspheres does not exceed 10%.

[0026] Drug loading was determined by HPLC. Chromatographic conditions were selected using a C18 column, a mobile phase composition and ratio of acetonitrile:water = 60%:40%, a flow rate of 1 mL / min, and a detection wavelength of 236 nm. The drug content in the sample solution was calculated using the standard curve method, and then the drug loading (wt%) and / or encapsulation efficiency were calculated by combining the total mass of the sample.

[0027] In this embodiment, the mass fraction of the drug in the microspheres, as determined by HPLC, can stably reach 50-80 wt%.

[0028] Based on the combined results of particle size, morphology, and HPLC, the drug carrier microspheres prepared in this embodiment have uniform particle size, intact structure, and high drug loading capacity. No obvious drug crystal precipitation was observed under microscopy and scanning electron microscopy. The lyophilized powder has good stability under normal storage conditions and can be used for further cell phagocytosis loading and in vitro and in vivo drug release experiments.

[0029] Example 2 A drug carrier microsphere is prepared by the following steps: S1. Dissolve 10 mg of dexamethasone palmitate (DexP) in 1 mL of dichloromethane and stir magnetically or vortex at room temperature until completely dissolved to obtain a homogeneous and transparent dispersion. S2. 10 mL of an aqueous phase containing 1% polyoxyethylene-polyoxypropylene block copolymer P407 was used as the continuous phase material. This phase, along with the dispersed phase obtained in S1, was introduced into the microfluidic chip via a syringe pump. At the chip junction, a stable flow rate ratio (dispersed phase flow rate: continuous phase flow rate = 1:15) was used for shear dispersion, forming an oil-in-water droplet structure. By adjusting the flow rates of the two phases and the microfluidic channel structure, the droplet size could be controlled within the range of 1–5 μm. S3. The droplets obtained in S2 were directly collected in a container containing a large amount of continuous phase of the same composition. The mixture was magnetically stirred at 250 rpm for 3 hours at room temperature to allow the organic solvent to fully diffuse or evaporate, and the droplets solidified to form drug carrier microspheres. After solidification, the suspension was centrifuged at 4000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended in an appropriate amount of deionized water and washed three times to remove free drug and excess surfactant, finally obtaining a microsphere suspension. The suspension was then freeze-dried at -80℃ for 12 hours to obtain dried dexamethasone palmitate microspheres (DexP MPs).

[0030] The resulting microspheres are white or off-white powders with good flowability, making them suitable as carrier materials for subsequent cell loading experiments.

[0031] The above-mentioned drug carrier microspheres were characterized as follows: The particle size and morphology were characterized using the same method as in Example 1, and the results are as follows: Figure 2 As shown.

[0032] Depend on Figure 2 It can be seen that the obtained microspheres are regular spherical or near-spherical in shape, with smooth surfaces, and no obvious aggregation or deformed particles were observed. The particle size can be adjusted within the range of 1~5μm depending on the preparation parameters, and the coefficient of variation of the particle size of the microspheres does not exceed 10%.

[0033] Drug loading was determined by HPLC. Chromatographic conditions were selected using a C18 column, a mobile phase composition and ratio of acetonitrile:isopropanol = 85%:15%, a flow rate of 1 mL / min, and a detection wavelength of 239 nm. The drug content in the sample solution was calculated using the standard curve method, and then the total mass of the sample was determined to calculate the drug loading (wt%) and / or encapsulation efficiency.

[0034] In this embodiment, the mass fraction of the drug in the microspheres, as determined by HPLC, can stably reach 50-80 wt%.

[0035] Based on the combined results of particle size, morphology, and HPLC, the drug carrier microspheres prepared in this embodiment have uniform particle size, intact structure, and high drug loading capacity. No obvious drug crystal precipitation was observed under microscopy and scanning electron microscopy. The lyophilized powder has good stability under normal storage conditions and can be used for further cell phagocytosis loading and in vitro and in vivo drug release experiments.

[0036] Example 3 This embodiment provides a method for preparing a high-drug-load live cell preparation, and verifies the loading capacity of drug carrier microspheres in macrophages and the drug release kinetics characteristics through in vitro cell experiments.

[0037] First, bone marrow-derived macrophages were cultured to maturity in a complete culture medium containing macrophage colony-stimulating factor (M-CSF). The resulting cells exhibited stable morphology and good adherent growth, and were used as carrier cells for future use.

[0038] The drug carrier microspheres prepared in Example 1 were added to the above-described carrier cell culture system, with the final concentration of microspheres in the culture system controlled within the range of 50-200 μg / mL. Co-incubation was then performed at 37°C and 5% CO2. During co-incubation, the carrier cells gradually took up the drug carrier microspheres, and the phagocytosis process was completed within 1-4 hours. After incubation, unphagocytosed free microspheres were gently washed with PBS to remove them, and the drug-loaded macrophages carrying the drug carrier microspheres were collected by centrifugation, thus obtaining a high-drug-loaded live cell formulation.

[0039] To quantitatively analyze drug loading in single cells, this embodiment employs a "cell counting-lysis extraction-HPLC, quantification-conversion to single cells" method. The specific experimental protocol is as follows: Drug-loaded macrophages were collected after phagocytizing drug carrier microspheres and washing to remove free microspheres. The cell count was performed using a cell counter. Subsequently, the cells were counted at a predetermined number of 1×10⁶ cells. 6Aliquots were placed into centrifuge tubes and gently washed twice with PBS to remove as much culture medium residue as possible. The supernatant was discarded. 0.2 mL of DMSO was added to each cell pellet to ensure complete coverage and drug dissolution. The pellets were thoroughly vortexed and then sonicated to promote cell lysis and drug release. Intermittent sonication in an ice bath (100 W power, 3 s intervals, 5 s intervals, total 1 min) was used to avoid sample temperature rise leading to drug degradation. After sonication, the samples were centrifuged at 13,000 rpm for 10 min to precipitate cell debris, microsphere residue, and insoluble impurities. The supernatant was used as the test sample and filtered through a 0.22 μm organic phase filter before injection. The supernatant was analyzed using HPLC under the same chromatographic conditions as in Example 1 or Example 2. A drug standard curve (peak area-concentration relationship) was established using the external standard method. The peak area was converted to drug concentration to obtain the total amount of drug in the sample.

[0040] The average drug load per cell can be calculated using the following formula: set up: The total amount of drug in the sample (μg); This represents the total number of cells for that sample. The average drug load per single cell (pg / cell) is: ; Total amount of drug in the sample The concentration can be calculated from HPLC quantitative results. If the drug concentration in the supernatant is obtained by conversion using a standard curve, then... (ng / mL), total supernatant volume is (mL), and dilution factors exist during the measurement process. (If there is no dilution, then DF=1), then: ; Substituting, we get: ; To ensure reliable results, parallel samples (n=3) can be set up under the same conditions. Blank cells (unloaded macrophages) are added in each measurement and extracted using the same DMSO process as a matrix control. If necessary, the peak area of ​​the blank control is subtracted. Simultaneously, the extraction efficiency can be evaluated through recovery verification. This involves adding a known amount of drug to the blank cell precipitate, extracting and measuring using the same process, and calculating the recovery rate to ensure that DMSO ultrasonic extraction can fully release the intracellular drug and meet the quantification requirements. This method can accurately convert the total amount of drug in drug-loaded cells to the single-cell level, thereby obtaining the average drug load per cell, which can be used to evaluate the cell loading effect of different microsphere systems and under different incubation conditions. Results are as follows: Figure 3As shown.

[0041] Depend on Figure 3 It can be seen that in the live cell preparation prepared in Example 1, the drug loading per cell can stably reach more than 50 pg; when the drug carrier microspheres are fatty acid prodrug microspheres, the drug loading per cell can be further increased to more than 80-100 pg.

[0042] Simultaneously, the intracellular phagocytic localization of the microspheres was verified by imaging the drug-loaded cells using laser confocal microscopy. To facilitate fluorescence tracking, 1% PLGA-RhB was added during the preparation of the PLGA microspheres described in Example 1, resulting in yellow fluorescence in the microspheres during confocal imaging; similarly, 1% DiI was added as a hydrophobic fluorescent tracer molecule during the preparation of the fatty acid prodrug microspheres described in Example 2, also resulting in yellow fluorescence in the microspheres during confocal imaging. Macrophages were stained with DiD membrane dye to mark cell outlines. After the obtained drug-loaded macrophages were cultured and adhered, fluorescence signals from the cell channels and microsphere channels were acquired using confocal microscopy. The results are as follows: Figure 4 As shown.

[0043] Depend on Figure 4 It can be seen that the fluorescent microsphere signal is mainly located within the DiD-labeled cell boundary and is dispersed in the cytoplasm, suggesting that macrophages can engulf multiple drug carrier microspheres; at the same time, the drug-loaded cells maintain normal adherence morphology, with intact cell outlines and clear structures, and no obvious cell fragmentation or abnormal shrinkage is observed.

[0044] The migration ability of drug-loaded cells was further evaluated using a Transwell chemotactic migration assay to verify whether the microsphere loading process affected macrophage migration function. Specifically, drug-loaded macrophages and unloaded control macrophages were digested and resuspended in serum-free or low-serum culture media, respectively, and adjusted to the same cell density before being added to the upper chamber of a Transwell (an insertable chamber with an 8 μm pore size). Macrophages were pre-seeded in the lower chamber and stimulated with LPS for a certain period of time to establish an inflammatory chemotactic microenvironment, thus serving as a source of chemotactic signals. The upper and lower chambers were assembled and incubated at 37°C and 5% CO2 for 24 h, allowing macrophages in the upper chamber to migrate towards the lower chamber under the influence of chemotactic signals. After incubation, unmigrated cells in the upper chamber were removed, and the membrane surface was gently wiped. Migrated cells on the lower membrane surface were fixed and stained with DAPI. The number of migrating cells per unit field of view was counted under a microscope, or the staining area / fluorescence intensity was used to quantify the migration ability. The results are as follows: Figure 5 As shown.

[0045] Depend on Figure 5It can be seen that, compared with the unloaded control cells, the migration ability of drug-loaded macrophages to the chemotactic environment formed by LPS-stimulated macrophages did not decrease significantly, indicating that the loading process of highly drug-loaded microspheres did not have a significant adverse effect on the chemotactic migration function of macrophages. Here, MΦ represents macrophages; PLGA3@MΦ represents macrophages loaded with polylactic acid-glycolic acid copolymer microspheres.

[0046] Based on the above, in vitro cell experiments were used to further verify the drug release kinetics of the drug carrier microspheres in macrophages. The obtained high-drug-loaded live cell formulation was cultured at 37℃ and 5% CO2, and cell culture supernatant and cell pellet were collected at preset time points (0, 1, 2, 3, 4, 5, 6, and 7 days). At each time point, the culture supernatant was first collected to detect the amount of drug released extracellularly; the cell pellet was washed with PBS to remove residual supernatant and then lysed, and the residual drug content in the cells was determined by high-performance liquid chromatography (HPLC). Based on the initial single-cell drug load and the total amount of intracellular and extracellular drug at each time point, the rate of drug release from the cells per unit time was calculated, and cumulative release-time curves and / or intracellular residual amount-time curves were plotted to characterize the intracellular release behavior. Results are as follows: Figure 6 As shown.

[0047] Depend on Figure 6 It can be seen that under the conditions of the drug carrier microspheres, the release of the drug in macrophages shows an approximately linear trend over a relatively long period of time. The decrease in intracellular drug concentration over time basically conforms to the zero-order or near-zero-order release kinetics, that is, the amount of drug released per unit time changes little within the range of 0 to 7 days, and the initial burst release ratio is significantly reduced.

[0048] In addition, the viability and phenotype of drug-loaded macrophages were monitored simultaneously within the above-mentioned culture time range. The results showed that no significant increase in apoptosis or necrosis was observed, the overall cell survival status was good, and the anti-inflammatory phenotype could be maintained within 5 days, indicating that macrophages can still maintain good survival ability and basic phenotypic stability under high drug loading conditions.

[0049] Through the above-mentioned confocal imaging, migration ability assessment, in vitro release kinetics determination and cell phenotype maintenance experiments, it was demonstrated at the cellular level that the drug carrier microspheres can form a stable "intracellular drug library" in macrophages and achieve continuous and stable drug output through near-zero-order drug release kinetics, thereby maintaining the anti-inflammatory phenotype of the carrier macrophages, providing experimental evidence for subsequent in vivo targeted delivery and disease treatment.

[0050] Example 4 This embodiment is used to verify the ability of the live cell preparation to target and aggregate in inflammatory lesions in vivo.

[0051] An acute muscle inflammation model was selected as a representative inflammatory model for evaluation. Experimental animals were 6-10 week old mice. The acute muscle inflammation model was established by injecting 100 μL of the inflammatory agent lipopolysaccharide (LPS) (1 mg / mL) into the muscle of one unilateral hind limb of the mouse to induce a local acute inflammatory response; the IBD model could be established using DSS-induced colitis or other commonly used methods in the field. After successful modeling, the cell-based drug delivery phase began, and healthy muscle from the contralateral hind limb of the mouse was used as a non-inflammatory control to assess non-specific distribution.

[0052] The preferred carrier cells for targeted detection are bone marrow-derived macrophages (BMDM). Macrophages can be fluorescently labeled using a DiD labeling method before taking up the drug carrier microspheres. After labeling, the cells are washed to remove free dye, and cell viability is measured to ensure that the labeling process does not significantly affect the cell state. Following the method described in Example 1 above, macrophages are allowed to take up the drug carrier microspheres to obtain drug-loaded macrophages; simultaneously, unloaded macrophages that have not taken up the microspheres but have undergone the same labeling treatment serve as a control. To ensure consistency, both groups of cells are adjusted to the same cell concentration before injection, and cell viability and cell count are measured again before injection to ensure accurate dosage.

[0053] Drug-loaded or unloaded macrophages were injected intravenously into mice via the tail vein, with a dose of 2 × 10⁶ cells per mouse. 6 One cell was injected, with a volume of 100 μL of physiological saline. In vivo fluorescence imaging was performed at 24 h and 48 h to observe the migration and enrichment trends of the tracer signal over time. Animals were euthanized at the endpoint, and inflamed tissue was isolated (in an acute muscle inflammation model, inflamed muscle and contralateral normal muscle were collected) for quantitative analysis using in vitro fluorescence imaging. In in vitro imaging, by standardizing exposure conditions, setting background subtraction, and normalizing fluorescence intensity using the ROI (region of interest) method, the enrichment intensity comparison between the drug-loaded and unloaded groups in inflamed tissue could be obtained.

[0054] Alternatively, tissue can be digested to prepare a single-cell suspension, and the proportion of tracer-positive cells in inflamed tissue can be detected by flow cytometry, thereby further quantifying the targeted enrichment ability of live cell preparations. Results are as follows... Figure 7 As shown.

[0055] Depend on Figure 7It was observed that in the acute muscle inflammation model, drug-loaded macrophages were able to migrate and enter the inflamed tissue, showing significant enrichment in the inflamed muscle region. Comparison of the in vivo distribution signals of drug-loaded and unloaded macrophages revealed no significant difference in enrichment efficiency at the inflammatory site, suggesting that the uptake of drug carrier microspheres did not weaken the macrophages' autonomous chemotaxis and migration capabilities. In vivo imaging, in vitro imaging, and single-cell statistical results were consistent, showing a high degree of overlap between the tracer signal and the inflammatory area, and maintaining a certain level of retention and enrichment within the observation timeframe.

[0056] Combining the quasi-zero-order release characteristics of the drug carrier microspheres within cells, drug-loaded macrophages can form a mode of action with high local drug concentration and continuous output in the inflammatory area, thereby improving the effective drug exposure at the lesion site without increasing systemic exposure. This provides a more stable and controllable targeted delivery method for the treatment of inflammation-related diseases and tumor microenvironment with inflammatory microenvironment characteristics.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a high-drug-loaded live cell preparation, characterized in that, Includes the following steps: Drug carrier microspheres are co-incubated with carrier cells, allowing the carrier cells to engulf the drug carrier microspheres. Unengulfed drug carrier microspheres are then removed to obtain a live cell preparation.

2. The preparation method according to claim 1, characterized in that, The drug carrier microspheres contain a drug mass fraction of ≥20wt% and have a particle size of 1-5μm.

3. The preparation method according to claim 1, characterized in that, The single-cell drug loading obtained after the carrier cells engulf the drug carrier microspheres is ≥50 pg drug / cell.

4. The preparation method according to claim 1, characterized in that, The carrier cells are selected from macrophages, macrophages differentiated from monocytes, macrophages derived from bone marrow, tissue macrophages, peritoneal macrophages, embryonic stem cells, pluripotent stem cells, dendritic cells, or neutrophils.

5. The preparation method according to claim 1, characterized in that, The drug carrier microspheres include one or both of biodegradable polyester materials and fatty acid prodrug materials.

6. The preparation method according to claim 5, characterized in that, The biodegradable polyester material includes one or two of polylactic acid-glycolic acid copolymer, polylactic acid, or polycaprolactone.

7. The preparation method according to claim 5, characterized in that, The fatty acid prodrug includes one or both of saturated fatty acid prodrugs and unsaturated fatty acid prodrugs.

8. A high-drug-loaded live cell preparation obtained by the preparation method according to any one of claims 1-7.

9. The use of the high-drug-load live cell preparation as described in claim 8 in the preparation of a medicament for treating inflammatory diseases, tumor diseases or autoimmune diseases.

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