Method for preparing drug-loaded neutrophil

Abstract

Provided in the present invention is a method for preparing a drug-loaded neutrophil. The method comprises the step of co-incubating, in a buffer containing nicotinamide, Q-VD-Oph and vitamin C, a neutrophil and a drug to be loaded. Further provided in the present invention are a drug-loaded neutrophil prepared using the method, and the use of the neutrophil in the preparation of a drug. The method provided in the present invention can significantly reduce the apoptosis ratio of neutrophils, reduce the in-vitro activation level of neutrophils, improve neutrophil viability and chemotactic efficiency under inflammatory conditions, and also improve the drug-loading efficiency of neutrophils, thereby realizing the efficient release of a drug at a disease site, and further improving the in-vivo survival time and efficacy of the drug.

Description

A method for preparing drug-loaded neutrophils TECHNICAL FIELD

[0001] The application discloses a method for preparing drug-loaded cells and belongs to the field of cell biology. BACKGROUND

[0002] A major challenge in cancer therapy is how to effectively deliver antitumor drugs into tumor tissues while minimizing drug-related adverse reactions. Drug delivery systems such as liposomes can change the pharmacokinetics and biodistribution of drugs, thereby improving the overall pharmacological properties of commonly used small molecule drugs, improving the circulation time of drugs, and reducing the toxic side effects of drugs. However, liposome drugs are affected by multiple biological steps during systemic delivery, such as liposome-protein interaction, blood circulation, extravasation, and interaction with the tumor microenvironment around blood vessels, tumor tissue penetration, immunogenicity and off-target effects, resulting in little benefit in clinical trials. Moreover, nanocarriers cannot actively cross the blood-brain barrier, so it is urgent to develop more efficient drug delivery carriers to achieve precise targeted drug delivery, reduce drug immunogenicity, improve drug bioavailability, and thus improve drug efficacy and reduce drug side effects.

[0003] Active targeting delivery strategy often has higher delivery efficiency than passive accumulation, and using living cells as carriers to achieve targeted delivery to target tissues and organs has a more broad prospect. Endogenous cells have the natural advantage of active targeting delivery, can actively penetrate various biological barriers, shield the immunogenicity of liposome drugs, achieve long circulation and targeted delivery of drugs, and thus improve the bioavailability of drugs and the drug concentration at the tumor and disease site. Among them, neutrophils are the first line of defense of the human innate immune system, and have natural advantages as drug delivery carriers. Neutrophils are the most rapid inflammatory chemotactic cells, can efficiently uptake and load drugs, and efficiently release drugs inside the cells at inflammatory sites; neutrophils are the most abundant white blood cells in the human blood circulation, accounting for 50%-70% of human white blood cells, and are extremely easy to obtain; neutrophils can penetrate physiological barriers, including vascular barriers, blood-brain barriers, blood-brain tumor barriers, etc.; Li et al. used neutrophils loaded with paclitaxel liposomes to inhibit the recurrence of postoperative glioma, and the results showed that neutrophils carrying paclitaxel liposomes can penetrate the blood-brain barrier and inhibit the recurrence of glioma in mice whose tumors have been surgically removed (Nature Nanotechnology. 2017, 12(7), 692-700). Another study confirmed the in vivo directional migration behavior of neutrophils carrying liposome-loaded drugs by delivering drug-loaded liposomes to the inflamed skeletal muscle of mice (Advanced Materials. 2020, 32(48), 2003598). Deprez et al. studied in a collagen-induced arthritis model and the results showed that neutrophils can transport liposomes to inflammatory sites, and that medullary cell-mediated liposome drug transport accounts for more than half of the accumulation of liposomes in the inflammatory area (Nature Nanotechnology. 2023, 18(11), 1341-1350). In summary, neutrophils can be developed as a general platform for drug delivery (Therapeutic Delivery. 2018, 9(1), 29-35; Translational Oncology. 2024, 39, 101813) as they can efficiently load and release drugs and cross the blood-brain barrier, making them an excellent drug delivery carrier.

[0004] Neutrophils are easily activated in vitro, which in turn causes neutrophil apoptosis or death, reduces drug loading efficiency, shortens in vivo survival time, and reduces chemotaxis rate, which undoubtedly brings great challenges to the development of drug-loaded cell therapy products. Therefore, it is urgent to further research and develop an efficient neutrophil drug loading technology to improve the drug loading efficiency of neutrophils while reducing the activation level of neutrophils, thereby improving the survival rate of neutrophil drugs and prolonging the survival time of drugs in the body. In addition, the in vitro drug loading technology further improves the inflammatory chemotaxis of neutrophils and improves the enrichment efficiency of neutrophils at the disease site, which can greatly improve the clinical efficacy of drug-loaded drugs. TECHNICAL PROBLEM

[0005] The existing method of loading liposome drugs in neutrophils is obtained by incubating in PBS or serum-free medium at a specific temperature (Nature Communications. 2023, 14(1); Nature Nanotechnology. 2017, 18(11), 1341-1350). This method ignores the problems of neutrophil apoptosis and activation during in vitro processing, and at the same time reduces the loading efficiency of neutrophils and causes the premature release of drug-loaded drugs.

[0006] Based on the bottleneck problem of developing cell drugs using existing neutrophils as delivery carriers, the purpose of the present application is to provide a method for preparing neutrophil drug-loaded preparations and applications. TECHNICAL SOLUTION

[0007] Based on the above purpose, the present application first provides a method for preparing drug-loaded neutrophils, which comprises the step of co-incubating neutrophils with a drug to be loaded in a buffer containing nicotinamide, Q-VD-Oph and vitamin C. Through co-incubation, the drug to be loaded enters the carrier through receptor-mediated endocytosis, macropinocytosis or phagocytosis on the cell membrane of neutrophils, forming a neutrophil drug-loaded preparation using neutrophils as carriers, i.e. the drug-loaded neutrophils of the present application.

[0008] In a preferred technical solution of the present application, the concentration of nicotinamide is 0.05-60 mM, the concentration of Q-VD-Oph is 0.05-60 µM, the concentration of vitamin C is 0.05-60 µM, the concentration of neutrophils is 2.5×10 6 -1×10 7 / mL, and the concentration of the drug to be loaded is 5-30 ug / mL.

[0009] In a more preferred embodiment, the concentration of said nicotinamide is 0.5-50 mM, the concentration of Q-VD-Oph is 0.5-50 µM, and the concentration of vitamin C is 0.5-50 µM.

[0010] In a preferred embodiment, the drug comprises one or more of a compound molecule, a polypeptide, a nucleic acid, a nanoparticle, a liposome or a micelle. The compound molecule of the present application refers to a free chemical drug molecule with a specific chemical structure and pharmacological effect, the polypeptide refers to a protein, an enzyme, an antibody, a receptor, a ligand and other multimeric amino acid molecules with a specific sequence and spatial structure, the nucleic acid refers to a DNA molecule and an RNA molecule with a specific sequence and spatial structure; the nanoparticle refers to a nanoscale composite particle encapsulating or loading the above-mentioned compound molecule, polypeptide and nucleic acid, the liposome refers to a micro-sized bubble body formed by encapsulating or loading the above-mentioned compound molecule, polypeptide and nucleic acid in a lipid bilayer, and the micelle refers to a colloidal aggregate of surfactant molecules encapsulating or loading the above-mentioned compound molecule, polypeptide and nucleic acid.

[0011] In a more preferred embodiment, the concentration of said drug loaded is 6.25-25 ug / mL. In a specific embodiment of the present application, the concentration of doxorubicin hydrochloride loaded liposome is 25 ug / mL, and in another specific embodiment of the present application, the concentration of lip-SG3199 loaded is 25 ug / mL.

[0012] In a preferred embodiment of the present application, the drug loaded by the neutrophil is a liposome, and in a specific embodiment, the concentration of said liposome is 25 ug / mL, the concentration of nicotinamide is 5 mM, the concentration of Q-VD-Oph is 5 µM, the concentration of vitamin C is 5 µM, the co-incubation time is 10-60 minutes, and the incubation temperature is 2-28℃.

[0013] More preferably, the co-incubation time is 30 minutes, and the incubation temperature is 22-25℃.

[0014] In one embodiment of the present application, doxorubicin hydrochloride (Doxil, Sequus) is selected as the drug loaded by the neutrophil, and in another embodiment of the present application, lip-SG3199 (unilamellar nanoliposome loaded with SG3199 small molecule drug, composed of DSPE-PEG2000-Cy5, DSPC, cholesterol and SG3199) is selected as the drug loaded by the neutrophil. According to the specific embodiments of the present application, it is obvious to those skilled in the art that other drugs in the prior art, such as compound molecule drugs, polypeptide drugs, nucleic acid drugs, nanoparticle drugs or micellar drugs, for example, for compound molecule drugs, other tumor drugs, pyrrolobenzodiazepine (PBD) dimers, monomethyl auristatin E (MMAE), deruxtecan (DXD), 7-ethyl-10-hydroxycamptothecin (SN38), monomethyl auristatin F (MMAF), calicheamicin and paclitaxel are also suitable for the implementation of the present application; for example, other liposome drugs in the prior art, PBD dimmer-loaded liposomes or MMAE-loaded liposomes are also suitable for the implementation of the present application.

[0015] Secondly, the present application provides a drug-loaded neutrophil prepared according to the above method. The neutrophil contains a drug molecule with therapeutic activity or a complex containing a drug molecule, such as a nanoparticle, a micelle or a liposome. In one embodiment of the present application, the neutrophil is loaded with doxorubicin hydrochloride liposomes, and in another embodiment of the present application, the neutrophil is loaded with lip-SG3199. As described above, it is obvious to those skilled in the art that the neutrophil can be loaded with other drugs in the prior art, such as compound molecule drugs, polypeptide drugs, nucleic acid drugs, nanoparticle drugs or micellar drugs, or loaded with other liposome drugs in the prior art.

[0016] Finally, the present application provides the use of the neutrophil according to the above in the preparation of a drug.

[0017] In a preferred embodiment, the drug includes an anti-tumor therapeutic drug, an anti-inflammatory therapeutic drug, an anti-infective drug, an autoimmune disease therapeutic drug or a neurological disease therapeutic drug.

[0018] In a preferred embodiment, the drug-loaded neutrophil preparation can be selected to treat the corresponding disease, for example, the tumor treatment drug PBD dimmer, MMAE, DXD, SN38, MMAF, Calicheamicin, paclitaxel or doxorubicin can be selected to prepare the corresponding tumor treatment drug, and the drug molecule can be a free molecule, a nanoparticle complex, a micelle complex and a liposome complex, and a biological macromolecule drug such as a polypeptide drug or a nucleic acid drug can be selected to be loaded into a neutrophil through a liposome or a nanoparticle complex, a micelle complex to prepare a corresponding disease treatment drug.

[0019] In a specific embodiment of the present application, the drug is doxorubicin hydrochloride liposome. In another specific embodiment of the present application, the drug is lip-SG3199. Advantages

[0020] The present application uses the activity, drug loading amount, activation phenotype, chemotactic efficiency and killing efficiency of drug-loaded neutrophils as evaluation indexes to evaluate the activity and physiological function of drug-loaded cells. First, the drug loading conditions of neutrophils are optimized, including drug loading temperature, drug loading time, cell density and drug concentration. At the same time, after adding different concentrations and different components during incubation, the activity, activation and apoptosis level of neutrophil drugs, the drug loading amount of neutrophils and the chemotactic efficiency of drug-loaded neutrophils are compared. The comprehensive indexes finally determine the optimal method of drug loading of neutrophils. Incubation is carried out under specific conditions, and the addition of specific concentrations of nicotinamide, Q-VD-Oph and vitamin C components during incubation can significantly reduce the apoptosis rate of neutrophils, reduce the in vitro activation level of neutrophils, improve the activity of neutrophils and the chemotactic efficiency under inflammatory conditions, and improve the drug loading efficiency of neutrophils. The drug is efficiently released at the disease site, further improving the in vivo survival time and efficacy of the drug. The drug-loaded neutrophils prepared by the developed drug loading method have a longer survival time and higher tumor inhibition efficiency after being reinfused into tumor-bearing mice. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a statistical diagram of the cell activity and drug loading amount of neutrophils under different drug concentrations, cell densities, incubation times and temperatures;

[0022] Figure 2 is a statistical diagram of the cell drug loading amount, cell activity and activation phenotype of neutrophils under different buffer conditions;

[0023] Figure 3 is a statistical diagram of the chemotactic efficiency and killing efficiency of drug-loaded neutrophils prepared under different buffer conditions;

[0024] Figure 4 is a graph showing the tumor inhibition efficiency of drug-loaded neutrophils prepared under different buffer conditions in vivo;

[0025] Figure 5 is a graph showing the tumor site accumulation and survival time of drug-loaded neutrophils prepared under different buffer conditions. Embodiments of the present application

[0026] The present application will be further described with reference to the following specific examples. The advantages and features of the present application will become apparent with the description.

[0027] Example 1: Cell viability and drug loading of neutrophils under different drug concentrations, cell densities, incubation times and temperatures

[0028] 1. Isolation of neutrophils

[0029] Mouse bone marrow neutrophil isolation kit (Tianjin Haoyang Biological, Item No: TBD2013NM) was used to isolate mouse bone marrow neutrophils, which is briefly described as follows:

[0030] The animals were sacrificed in an appropriate manner, and the femur or tibia was stripped out. The bone was cut with scissors to expose the inner cavity. An appropriate amount (according to the size of the animal) of homogenate rinse solution (product number: F2013TBD) was sucked into a syringe to rinse the bone marrow in the inner cavity. The bone marrow suspension was collected into a suitable centrifuge tube, and repeatedly blown into a single cell suspension with a 70 μm cell screen (product number: TBDTM-SC). Centrifugation at 450g for 10 min, and discard the supernatant. Resuspend the bone marrow cells with 1 mL of erythrocyte sedimentation liquid, and the cell concentration was 2×10 8 / mL-1×10 9 / mL of single cell suspension for later use. Take a 5 mL sterile glass centrifuge tube (TUB2016), first add 2 mL of separation solution 1, then slowly add 1 mL of 80% concentration of separation solution (4:1 mixture of separation solution 1 and tissue sample diluent), to form a gradient interface. Slowly add 0.5-1.0 mL of sample cell suspension. Carefully add the sample cell suspension above the separation solution interface. Each liquid interface must be clearly layered (the total amount of separation solution should not be less than 2 mL, and the sample cell suspension should not be less than 0.5 mL). Centrifuge at 450g for 30 min, with 9 acceleration and deceleration. After centrifugation, the centrifuge tube is divided into five layers from top to bottom. The first layer is the tissue diluent layer. The second layer is the annular milky white mononuclear cell layer. The third layer is the annular milky white neutrophil layer (may have a small amount of red blood cells mixed). The fourth layer is the transparent separation solution layer. The fifth layer is the red blood cell layer. Carefully aspirate the annular milky white neutrophil layer from the centrifuge tube and transfer it to a new centrifuge tube. Add 10 mL of washing solution (product number: 2010X1118) to the centrifuge tube containing the neutrophils, mix the cells. Centrifuge at 400g for 10 min, discard the supernatant. Use a pipette to aspirate 5 mL of washing solution (product number: 2010X1118) to resuspend the resulting cells. Centrifuge at 250g for 10 min, discard the supernatant. Repeat the washing twice, discard the supernatant, and add the corresponding buffer according to the next experimental requirement to resuspend the resulting cells.

[0031] 2. Drug loading at different drug concentrations

[0032] First, dilute the isolated neutrophils to 1×10 7 / mL with PBS, and divide them into 5 groups. Add 1 mL of cell suspension to the corresponding centrifuge tube in each group, and add doxorubicin hydrochloride liposomes (Sequus Doxil) with a final concentration of 6.25, 12.5, 25, 50, and 100 ug / mL, respectively. Incubate at room temperature for 30 min, then centrifuge at 300g for 5 min to remove free drugs, and collect the cells. Resuspend the cells with 1 mL of PBS with a pH of 7.4, and centrifuge at 300g for 5 min to discard the supernatant. Repeat the washing step 2 times. After the last washing, resuspend the cell pellet with PBS, and adjust the cells to 1×10 7 / mL, the viability of drug-loaded neutrophils was detected by cell counter (Thermo, instrument model: Countess 3 FL), and the drug loading of cells was detected by microplate reader. The results (Fig. 1 A, B) show that in the concentration range of 6.25, 12.5, 25 ug / mL, the drug loading gradually increased with the increase of concentration, and the cell viability did not change significantly; in the concentration range of 50, 100 ug / mL, the drug loading did not change significantly, and the cell viability decreased.

[0033] 3. Drug loading of neutrophils with different densities

[0034] The isolated neutrophils were diluted to 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 , 2 x 10 7 / mL with PBS, and divided into 4 groups. 1 mL of cell suspension was taken into the corresponding centrifuge tube in each group, and Doxil drug with a final concentration of 25 ug / mL was added. After incubation at room temperature for 30 min, 300 g centrifugation was performed for 5 min, the supernatant was discarded, and the free drug was removed. The cells were collected. 1 mL of PBS with pH value of 7.4 was added to resuspend the cells, 300 g centrifugation was performed for 5 min, and the supernatant was discarded. The washing step was repeated twice. After the last washing, the cell pellet was resuspended with PBS, and the cells were adjusted to 1 x 10 7 / mL. The viability of drug-loaded neutrophils was detected by cell counter, and the drug loading of cells was detected by microplate reader. The results (Fig. 1 C, D) show that in the concentration range of 2.5 x 10 6 , 5 x 10 6 , 1 x 10 7 / mL, the drug loading did not change significantly, and the cell viability did not change significantly; in the concentration range of 2 x 10 7 / mL, the drug loading decreased, and the cell viability decreased.

[0035] 4. Drug loading with different incubation times

[0036] The isolated neutrophils were diluted to 1 x 10 7 / mL with PBS, and Doxil drug with a final concentration of 25 ug / mL was added. 1 mL of cell suspension was taken into the corresponding centrifuge tube in each group, and incubation was performed at room temperature for 10, 30, 60, and 120 min, respectively. After 300 g centrifugation for 5 min, the supernatant was discarded, and the free drug was removed. The cells were collected. 1 mL of PBS with pH value of 7.4 was added to resuspend the cells, 300 g centrifugation was performed for 5 min, and the supernatant was discarded. The washing step was repeated twice. After the last washing, the cell pellet was resuspended with PBS, and the cells were adjusted to 1 x 10 7 / mL, the viability of the drug-loaded neutrophils was detected using a cell counter, and the drug loading of the cells was detected using a microplate reader. The results (Fig. 1, E and F) show that, in the 10-30 min time range, the drug loading gradually increased with time, and the cell viability did not change significantly; in the 30-60-120 min time range, the drug loading did not change significantly, and the cell viability decreased.

[0037] 5. Drug loading under different temperature conditions

[0038] The isolated neutrophils were diluted to 1 x 10 7 / mL using PBS, and 25 ug / mL of Doxil was added. The cells were divided into three groups, and 1 mL of the cell suspension was added to a centrifuge tube of the corresponding group. The tubes were placed in a 2-8°C, 22-25°C, or 36-38°C environment, respectively, and incubated for 30 min. The cells were then centrifuged at 300 g for 5 min, and the supernatant was discarded to remove the free drug. The cells were resuspended in 1 mL of PBS with a pH of 7.4, centrifuged at 300 g for 5 min, and the supernatant was discarded. The washing step was repeated twice. After the final washing step, the cell pellet was resuspended using PBS, and the cell density was adjusted to 1 x 10 7 / mL. The viability of the drug-loaded neutrophils was detected using a cell counter, and the drug loading of the cells was detected using a microplate reader.

[0039] The specific drug loading of the cells was determined as follows: Doxil was diluted to a concentration of 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16, 0.08, 0.04, or 0 ug / mL, and 100 uL of each was added to a black microplate. Triton X-100 was added to a final concentration of 0.1% for lysis, and the plate was incubated at room temperature for 1 h. The fluorescence of Doxil was detected at excitation and emission wavelengths of 544 and 585, respectively, and a standard curve was drawn. The drug-loaded neutrophils were diluted to a density of 5 x 10 6 / mL, and 100 uL of the diluted cell suspension was added to a 96-well black microplate. Triton X-100 was added to a final concentration of 0.1% for lysis, and the plate was incubated at room temperature for 1 h. The fluorescence of Doxil was detected at excitation and emission wavelengths of 544 and 585, respectively. The drug loading of the cells was calculated according to the standard curve based on the detected fluorescence value. The results (Fig. 1, G and H) show that, in the 2-8°C and 22-25°C temperature range, the drug loading gradually increased with increasing temperature, and the cell viability did not change significantly; in the 22-25°C and 36-38°C range, the drug loading did not change significantly, and the cell viability decreased.

[0040] Example 2: Detection of the drug loading, cell viability, and activated phenotype of neutrophils under different buffer conditions

[0041] Isolate mouse bone marrow neutrophils, refer to Example 1 for isolation method, before the last time of washing cells, divide the cells into 6 groups, add into the corresponding numbered centrifuge tube, 300 g centrifugation for 5 min, discard the supernatant, resuspend the cell pellet with different buffers (see Table 1) containing or not containing different concentrations of nicotinamide (purchased from MedChemExpress, Catalog No: HY-B0150), quinoline-aspartate-valine-difluorophenoxymethyl ketone (Q-VD-Oph, a pan-caspase inhibitor acting on caspases 1, 3, 8 and 9, purchased from MedChemExpress, Catalog No: HY-12305), Vitamin C (purchased from MedChemExpress, Catalog No: HY-B0166). Adjust the density of neutrophils to 1×10 7 / mL, take 1 mL from each group, add Doxil drug with a final concentration of 25 ug / mL, place in 22-25℃ conditions, incubate for 30 min, 300 g centrifugation for 5 min, discard the supernatant, remove free drugs, collect cells. Add 1 mL PBS with pH value of 7.4 to resuspend the cells, 300 g centrifugation for 5 min, discard the supernatant. Repeat the washing step 2 times, after the last time of washing, resuspend the cell pellet with PBS, adjust the cells to 1×10 7 / mL, use a cell counter to detect the viability of drug-loaded neutrophils, use a microplate reader to detect the drug loading of cells, and use a flow cytometer to detect the activated phenotype CD62L and CD11b of cells.

[0042] Table 1. Composition of different buffers

[0043]

[0044] The specific flow cytometry method for detecting cell activation phenotype is briefly described as follows:

[0045] (1) Take 2×10 5 Drug-loaded neutrophils, wash the cells with 1 mL PBS once; after washing, resuspend the cells with 100 μL PBS;

[0046] (2) Add the corresponding volume of CD62L-APC, CD11b-APC / Cy7 and Ly6G-FITC flow cytometry antibodies (see Table 2), incubate at 4℃ for 30 min;

[0047] Table 2. Antibody information for phenotype detection

[0048]

[0049] (3) After incubation, wash the cells with 1 mL flow buffer;

[0050] (4) Resuspend the cells with 200 μL flow buffer;

[0051] (5) Detect with flow cytometry (Agilent, model NovoCyte) and analyze with NovoExpress software.

[0052] The results (Fig. 2 A, B, C, D) show that the drug loading of buffers 1-6 (ug / 1*10^6 cells) were 4.83±0.25, 6.57±0.31, 7.50±0.30, 5.47±0.15, 4.93±0.15, 4.87±0.15, respectively, and the cell viability (%) were 92.40±0.46, 94.83±0.32, 97.97±0.21, 92.27±0.76, 90.83±0.87, 89.60±0.62, respectively; the MFI values of activated CD62L were 185461±1925.53, 232713±1509.64, 236087±3435.41, 217480±1713.12, 201329±901.70, 186892±2213.12, respectively; the MFI values of activated CD11b were 144218±1696.77, 122407±983.34, 112734±728.62, 133916±1907.89, 136207±1697.08, 148881±990.00, respectively. Under the conditions of buffer 2 and buffer 3, the drug loading of the cells was the highest, the cell activation efficiency was the lowest, and the cell viability was the highest; more preferably, under the condition of buffer 3, the drug loading of the neutrophils was the highest, the cell activation level was the lowest, and the cell viability was the highest.

[0053] Example 3: Chemotactic efficiency and killing efficiency of drug-loaded neutrophils prepared under different buffer conditions

[0054] Isolate mouse bone marrow neutrophils, refer to the isolation method in Example 1, before the last washing of the cells, divide the cells into 6 groups, add them into the corresponding numbered centrifuge tubes, centrifuge at 300g for 5 min, discard the supernatant, and resuspend the cell pellets with different buffers (see Table 1), adjust the density of the neutrophils to 1*10 7 / mL, take 1 mL, add lip-SG3199 drug (single-layer nanoliposome loaded with SG3199 small molecule drug composed of DSPE-PEG2000, DSPC, cholesterol and SG3199) with a final concentration of 25 ug / mL, place at 22-25°C, incubate for 30 min, centrifuge at 300 g for 5 min, discard the supernatant, remove the free drug, collect the cells. Add 1 mL of PBS with pH value of 7.4 to resuspend the cells, centrifuge at 300 g for 5 min, discard the supernatant. Repeat the washing step 2 times, after the last washing, resuspend the cell pellet with the corresponding buffer, adjust the cells to 1×10 7 / mL, detect the chemotactic efficiency and killing efficiency of drug-loaded neutrophils.

[0055] The method for detecting the chemotactic efficiency of drug-loaded neutrophils is briefly described as follows:

[0056] (1) Adjust the density of drug-loaded neutrophils after washing to 1.5×10 6 / mL using DMEM medium (Gibco, Cat No: C11995500BT);

[0057] (2) Add 600 μL of DMEM medium containing 100 nM N-Formyl-Met-Leu-Phe (fMLP, MCE, Cat No: HY-P0224) to the bottom of each well, and add 200 μL of cell preparation prepared in step (1) to the top of each filter (set 3 replicate wells for each sample);

[0058] (3) Place the transwell culture plate (corning, Cat No: 3472) with added cell suspension and medium at 37°C and 5% CO2 for 1 hour;

[0059] (4) Then take out the upper chamber of the transwell culture plate, collect the cells in the lower chamber into a centrifuge tube, and use a flow cytometer (Agilent, Model NovoCyte) to detect the absolute count of the collected cells, and use NovoExpress software for analysis.

[0060] The method for detecting the killing efficiency of drug-loaded neutrophils is briefly described as follows:

[0061] (1) Dilute the digested U87MG-luc target cells to 5×10 4 / mL with DMEM+10% FBS medium, take 100 μL of cell suspension and inoculate into a 96-well flat-bottom culture plate (corning, Cat No: 3599);

[0062] (2) 24 h after U87MG-luc tumor cell line was inoculated, the prepared drug-loaded neutrophils were resuspended in DMEM + 10% FBS medium to 1.25 x 10 5 / ml, 100 μL was added to each well, and the effector-target ratio was 2.5:1;

[0063] (3) The 96-well culture plate (corning, item number: 3599) co-incubated with drug-loaded neutrophils and tumor cells was incubated at 37°C, 5% CO2 for 48 h;

[0064] (4) After incubation, the medium in the detection well was aspirated, and 100 μL of PBS was added to each well;

[0065] (5) 100 μL of One-Lite Luciferase Assay System detection reagent (vazyme, item number: DD1203) was added to each corresponding detection well, and incubated at room temperature for 10 min in the dark;

[0066] (6) 100 μL of the reaction solution was transferred to a 96-well flat-bottom white plate (white 96-well Immuno plate, item number: 7905);

[0067] (7) The luc value was read using an enzyme marker (MD, model: paradigm);

[0068] (8) The killing efficiency calculation formula: Cytotoxicity (%) = 100 x (Total Target cell LUC-remaining reaction cells LUC) / Total Target cell LUC.

[0069] Results (Figure 3 A, B) show that the cell chemotaxis efficiency (%) of buffer 1-6 was 46.1 ± 2.52, 56.6 ± 0.81, 58.6 ± 2.70, 52.4 ± 1.70, 48.6 ± 1.12, and 43.4 ± 1.33, respectively. The killing efficiency (%) of buffer 1-6 was 57.17 ± 5.52, 70.57 ± 0.31, 80.50 ± 0.30, 60.47 ± 0.15, 53.93 ± 5.34, and 53.87 ± 5.31, respectively. The cell chemotaxis efficiency and killing efficiency were higher under buffer 2 and buffer 3 conditions, and the chemotaxis efficiency of drug-loaded neutrophils prepared under buffer 3 conditions was the highest.

[0070] Example 4: In vivo drug efficacy of drug-loaded neutrophils prepared under different buffer conditions

[0071] U87MG tumor-bearing NPG (NOD.Cg-Prkdc scid il2rg tm1Vst / Vst) mice (Vivida) model to evaluate the in vivo anti-tumor effect of NE-SG3199 (SG3199-loaded neutrophils), U87MG cells in the logarithmic growth phase were digested, counted by a cell counter, and then diluted with PBS to 5 × 10 7 cells / mL. 100 μL of the cell suspension was injected subcutaneously into the right scapular of each NPG mouse using a 1 mL sterile syringe to establish a human glioma U87MG cell subcutaneous tumor-bearing tumor model.

[0072] After inoculation, wait for the average tumor volume to grow to 100 mm 3 left, the tumor-bearing mice were randomly divided into 5 groups (n = 6). On the day of administration, drug-loaded neutrophils were prepared, the preparation method is described in Example 3, and the buffer is described in Table 1. On the day of administration, the mice were subjected to radiotherapy locally at the tumor site at a dose of 0.5 Gy. Intravenous administration of 100 uL was performed 5 hours after radiotherapy. The specific groups are shown in Table 3. Subsequent administration was performed once a week, and 0.5 Gy radiotherapy was performed locally at the tumor site before each administration. Intravenous administration was performed 5 hours after radiotherapy. Tumor volume and body weight were measured twice a week after administration. The tumor volume calculation formula is: V = L × S 2 × 0.5, where V represents the tumor volume, L represents the longest diameter of the tumor, and S represents the shortest diameter of the tumor.

[0073] Table 3. Information on the grouping of drug-loaded neutrophils in vivo

[0074]

[0075] The results (Figure 4) show that drug-loaded neutrophils prepared under buffer 2 and buffer 3 conditions have more significant tumor inhibition efficiency. On day 27, the tumor volumes of the 5 groups were 2048.07, 1131.80 ± 399.38, 380.62 ± 87.15, 92.75 ± 6.20, and 680.56 ± 47.68 mm 3 , respectively, with the highest in vivo efficacy of drug-loaded neutrophils prepared under buffer 3 conditions.

[0076] Example 5: Tumor site accumulation of drug-loaded neutrophils prepared under different buffer conditions

[0077] A U87MG tumor-bearing NPG mouse (Vivida) model was established to evaluate the in vivo survival time and tumor site accumulation. The tumor modeling method is described in Example 4.

[0078] After inoculation, wait for the average tumor volume to grow to 100 mm3 When the tumor-bearing mice were randomly divided into 5 groups (n = 3), the drug-loaded neutrophil drug (NE-SG3199) was prepared on the administration day, the preparation method was referred to Example 3, the buffer was prepared according to Table 1, and the prepared neutrophil drug was labeled with DIR. The specific labeling method is as follows: the drug-loaded neutrophil was diluted to a density of 1 x 10 7 / mL with PBS, and 5 µM of DIR dye (MCE, item number: HY-D1048) was added. Incubate at room temperature for 20 min. After incubation, wash the cells twice with PBS buffer, centrifuge at 300g for 5 min, discard the supernatant, and resuspend the washed cells in PBS buffer to a concentration of 5 x 10 7 / mL; on the administration day, the mice were subjected to local radiotherapy with a dose of 0.5 Gy, and the neutrophil drug was administered 5 h after radiotherapy. The specific groups are shown in Table 4. The small animal live imaging system (PE, instrument model: IVIS Spectrum) was used for imaging at 1, 24, 48, and 72 h after administration, and the Living Image software was used for data analysis. The Local Luc radiance value was calculated.

[0079] Table 4. Information on the grouping of mice for the accumulation of drug-loaded neutrophil tumor site experiments

[0080]

[0081] The results (Figure 5) show that at 1 h, the radiation values (p / s) of groups 1-4 were 7.00 x 10 8 ± 7.29 x 10 7 , 9.00 x 10 8 ± 7.29 x 10 7 , 9.67 x 10 8 ± 1.16 x 10 8 , and 7.67 x 10 8 ± 2.03 x 10 7 , respectively. At 24 h, the radiation values of groups 1-4 were 1.83 x 10 9 ± 2.69 x 10 8 , 3.23 x 10 9 ± 2.60 x 10 8 , 3.83 x 10 9 ± 9.74 x 10 8 , and 2.23 x 10 9 ± 2.60 x 10 8 , respectively. At 48 h, the radiation values of groups 1-4 were 2.45 x 10 9 ± 6.94 x 10 84.78×10 9 ±1.02×10 9 6.45×10 9 ±3.90×10 8 3.95×10 9 ±3.90×10 8 The radiation values ​​of groups 1-4 at 72 hours were 2.53 × 10⁻⁶. 9 ±5.53×10 8 4.53×10 9 ±5.53×10 8 5.86×10 9 ±5.95×10 7 3.53×10 9 ±5.53×10 8 Drug-loaded neutrophils prepared under buffer 2 and buffer 3 conditions showed a more significant cumulative number of tumor sites, with drug-loaded neutrophils prepared under buffer 3 showing a more significant cumulative number of tumor sites.

Claims

1. A method for preparing drug-loaded neutrophils, characterized by, The method comprises the step of co-incubating the neutrophil with the drug to be loaded in a buffer containing nicotinamide, Q-VD-Oph and vitamin C.

2. The method of claim 1, wherein the drug-loaded neutrophils are prepared by the method comprising the steps of: The concentration of the nicotinamide is 0.05-60 mM, the concentration of Q-VD-Oph is 0.05-60 µM, the concentration of vitamin C is 0.05-60 µM, the concentration of the neutrophils is 2.5×10 6 -1×10 7 / mL, and the concentration of the drug to be loaded is 5-30 ug / mL.

3. The method of claim 2, wherein the drug-loaded neutrophils are prepared by the method comprising the steps of: The concentration of the nicotinamide is 0.5-50 mM, the concentration of Q-VD-Oph is 0.5-50 µM, and the concentration of vitamin C is 0.5-50 µM.

4. The method of claim 1, wherein the drug-loaded neutrophils are prepared by the method comprising: (a) obtaining a sample of neutrophils from a subject; (b) incubating the neutrophils with a drug; and (c) isolating the drug-loaded neutrophils from the sample. The drug comprises one or more of a compound molecule, a polypeptide, a nucleic acid, a nanoparticle, a liposome or a micelle.

5. The method for preparing drug-loaded neutrophils according to claim 3, characterized in that, The concentration of the drug to be loaded is 6.25-25 ug / mL.

6. The method for preparing drug-loaded neutrophils according to claim 5, characterized in that, The drug is a liposome, the concentration of the liposome is 25 µg / mL, the concentration of the nicotinamide is 5 mM, the concentration of Q-VD-Oph is 5 µM, the concentration of vitamin C is 5 µM, the co-incubation time is 10-60 minutes, and the co-incubation temperature is 2-28℃.

7. The method for preparing drug-loaded neutrophils according to claim 6, characterized in that, The co-incubation time is 30 minutes, and the co-incubation temperature is 22-25℃.

8. A drug-loaded neutrophil prepared according to any one of claims 1-7.

9. Use of the neutrophil of claim 8 in the preparation of a medicament.

10. Use according to claim 9, characterized in that, The medicament comprises an anti-tumor therapeutic drug, an anti-inflammatory therapeutic drug, an anti-infective drug, an autoimmune disease therapeutic drug or a neurological disease therapeutic drug.

11. Use according to claim 9, characterized in that, The drug is a neutrophil loaded with PBD dimmer, MMAE, DXD, SN38, MMAF, Calicheamicin, paclitaxel or doxorubicin.

12. Use according to claim 11, characterized in that, The PBD dimmer or doxorubicin is loaded in a liposome.

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