A method of administration using in vitro differentiated platelets as delivery vehicles

Abstract

The invention belongs to the field of medicine and health, and particularly provides a drug delivery method using in-vitro differentiated platelets as a delivery carrier, and the platelets obtained by redifferentiation can still efficiently deliver a drug even after being frozen and preserved by expressing a loaded drug at a precursor cell stage of the platelets. The platelet delivery system has important significance on marketization of a drug delivery method of the platelet serving as the delivery system.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610455785.4 (22) Application Date 2026.04.08 (71) Applicant Xueji Biotechnology (Shanghai) Co., Ltd. Address 1-2 Floor, No. 11, Lane 889, Ziping Road, Pudong New Area, Shanghai 201318 (72) Inventors Zhu Fangfang Zhang Pengbo (74) Patent Agency Shanghai Yiping Intellectual Property Agency Co., Ltd. 31266 Patent Attorney Xu Xun Cui Jiajia (51) Int.Cl. A61K 47 / 46 (2006.01) (54) Invention Title: A Drug Delivery Method Using In Vitro Differentiated Platelets as Delivery Carriers (57) Abstract: This invention belongs to the field of medicine and health. Specifically, this invention provides a drug delivery method using in vitro differentiated platelets as delivery carriers. By expressing drug-loaded substances at the platelet precursor cell stage, the redifferentiated platelets can still efficiently deliver drugs even after cryopreservation. This is of great significance for the marketization of drug delivery methods using platelets as delivery systems. Claims 1 page, Description 9 pages, Sequence List (electronic publication), Drawings 3 pages, CN 122297700 A 2026.06.30 CN 1 22 29 77 00 A 1. A drug delivery device using platelets as a delivery carrier, characterized in that the device comprises the following modules: (Z1) a drug-loaded platelet providing module, the drug-loaded platelet providing module being configured to: provide frozen drug-loaded platelets formed in vitro from platelet precursor cells expressing a drug; (Z2) a thawing and elution module, the thawing and elution module being configured to: thaw and elute the frozen drug-loaded platelets to obtain a drug-loaded platelet solution; (Z3) a drug delivery module, the drug delivery module being configured to: administer the drug-loaded platelet solution to a test subject, thereby delivering the drug into the body of the test subject. 2. The drug delivery device of claim 1, wherein the drug delivery module is configured to administer 5 × 10⁶ to 5 × 10⁷ drug-loaded platelets to the subject each time. 3. The drug delivery device of claim 1, wherein the drug delivery module is configured to administer the drug-loaded platelet solution to the subject multiple times at specific time intervals; wherein the multiple times is ≥2 times; and the specific time interval is 1-8 days. 4. The drug delivery device of claim 1, wherein the platelet precursor cells are selected from the group consisting of: induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem-progenitor cells, megakaryocyte progenitor cells, or megakaryocytes. 5. The drug delivery device of claim 1, wherein the drug comprises: a protein drug, a peptide drug, an antibody drug, or a combination thereof.6. The drug delivery device of claim 1, wherein the peripheral blood platelet level of the test subject is comparable to that of a healthy control. 7. A formulation comprising: (a) platelet precursor cells; (b) a gene-editing reagent that enables the platelet precursor cells to express a drug; (c) a platelet cryopreservation solution; (d) a platelet eluent; and (e) a culture medium environment suitable for the differentiation of the drug-expressing platelet precursor cells into platelets. 8. Use of the formulation of claim 7, wherein it is used to prepare a kit for delivering the drug into a test subject. 9. A platelet-based drug delivery method for non-diagnostic and non-therapeutic purposes, characterized by comprising the following steps: (S1) providing frozen drug-loaded platelets differentiated in vitro from platelet precursor cells expressing a drug; (S2) thawing and eluting the frozen drug-loaded platelets to obtain a drug-loaded platelet solution; and (S3) administering the drug-loaded platelet solution to a test subject, thereby delivering the drug into the test subject; wherein the test subject is a non-human mammal. 10. The method of claim 9, characterized in that the test subject is a mouse with a humanized immune system. Claims 1 / 1 page 2 CN 122297700 A Drug delivery method using in vitro differentiated platelets as a delivery carrier Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to a drug delivery method using in vitro differentiated platelets as a delivery carrier. Background Art

[0002] Targeted drug delivery systems are a research hotspot in the biomedical field. Platelets, as naturally occurring anucleate cells in the blood, are expected to become ideal drug delivery carriers due to their excellent biocompatibility, targeting of inflammatory sites, and immune evasion capabilities.

[0003] Existing technologies have confirmed that platelet-derived exosomes can be loaded with hydrophilic and hydrophobic drugs, achieving precise drug delivery through specific receptor mediation, showing significant advantages in the treatment of diseases such as rheumatoid arthritis and ischemic heart disease.

[0004] However, current platelet delivery systems still face three major technological bottlenecks: First, traditional carriers mostly use mature platelets for direct drug delivery, which, due to cell structure characteristics, generally results in low drug delivery efficiency and unstable loading, making it easy for drugs to leak prematurely in vivo, affecting therapeutic effects and increasing the risk of side effects; second, platelets can only be stored at room temperature for short periods, failing to meet the storage requirements for large-scale clinical applications; third, there is a lack of unified optimization standards for clinical dosage and frequency of administration, autologous platelet preparation is costly and time-consuming, allogeneic platelet transfusion is prone to triggering immune reactions, and improper dosage may lead to coagulation disorders, severely restricting technology transfer. Therefore, existing technologies struggle to balance drug delivery efficiency, storage stability, and clinical safety.

[0005] Therefore, developing a drug delivery method using platelets as a delivery carrier has become a key requirement for overcoming current technological bottlenecks. Summary of the Invention

[0006] This invention provides a drug delivery method using in vitro differentiated platelets as a delivery carrier and its application.

[0007] In a first aspect of this invention, a drug delivery device using platelets as a delivery carrier is provided, the device comprising the following modules: (Z1) a drug-loaded platelet providing module, the drug-loaded platelet providing module being configured to: provide frozen drug-loaded platelets formed in vitro from platelet precursor cells expressing a drug; (Z2) a thawing and elution module, the thawing and elution module being configured to: thaw and elute the frozen drug-loaded platelets to obtain a drug-loaded platelet solution; (Z3) a drug delivery module, the drug delivery module being configured to: administer the drug-loaded platelet solution to a test subject, thereby delivering the drug into the test subject's body.

[0008] In another preferred embodiment, "in vivo" refers to: peripheral blood, bone marrow, organs, or a combination thereof.

[0009] In another preferred embodiment, "in vivo" refers to: tumor cell environment, thrombus site, inflammatory site, or a combination thereof.

[0010] In another preferred embodiment, the organ includes: lung, heart, pancreas, ovary, kidney, bladder, brain, or a combination thereof.

[0011] In another preferred embodiment, the test subject is a human or a non-human mammal.

[0012] In another preferred embodiment, the non-human mammal is selected from the group consisting of: rats, mice, rabbits, dogs, pigs, and non-human primates.

[0013] In another preferred embodiment, the test subject is a mouse.

[0014] In another preferred embodiment, the drug-loaded platelet providing module is configured to: provide drug-expressing platelet precursor cells, differentiate the drug-expressing platelet precursor cells into platelets in vitro, thereby providing frozen drug-loaded platelets.

[0015] In another preferred embodiment, the drug delivery module is configured to: administer 5 × 10⁶ to 5 × 10⁷ drug-loaded platelets to the subject each time.

[0016] In another preferred embodiment, the dosage of the drug-loaded platelets is 1 × 10⁷ per administration.

[0017] In another preferred embodiment, the administration refers to a single administration.

[0018] In another preferred embodiment, the drug delivery module is configured to: administer the drug-loaded platelet solution to the subject multiple times at specific time intervals; wherein, the multiple times is ≥2 times; the specific time interval is 1-8 days.

[0019] In another preferred embodiment, the multiple times is ≥3 times, preferably ≥4 times.

[0020] In another preferred embodiment, the specific time interval is preferably 2-6 days, more preferably 2-4 days, and most preferably 3-4 days.

[0021] In another preferred embodiment, the drug-loaded platelet solution is administered to the subject on day 0, day 4, day 7, and day 11, respectively.

[0022] In another preferred embodiment, the platelet precursor cells are selected from the group consisting of: induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem progenitor cells, megakaryocyte progenitor cells, or megakaryocytes.

[0023] In another preferred embodiment, the drug-loaded platelet delivery module is configured to: express a drug in platelet precursor cells using gene editing technology, thereby providing drug-expressing platelet precursor cells; differentiate the drug-expressing platelet precursor cells into platelets in vitro, thereby providing frozen drug-loaded platelets.

[0024] In another preferred embodiment, differentiating drug-expressing platelet precursor cells into platelets in vitro means: differentiating drug-expressing platelet precursor cells into platelets in vitro under a suitable culture medium environment for differentiation, thereby providing frozen drug-loaded platelets.

[0025] In another preferred embodiment, the frozen drug-loaded platelets refer to drug-loaded platelets stored in liquid nitrogen.

[0026] In another preferred embodiment, providing frozen drug-loaded platelets means: cooling the differentiated drug-loaded platelets to -140°C ± 10°C and transferring them to a liquid nitrogen tank for storage, thereby providing frozen drug-loaded platelets.

[0027] In another preferred embodiment, the cooling is performed using a programmable cooling device.

[0028] In another preferred embodiment, the culture medium environment for the differentiation of the drug-expressing platelet precursor cells into platelets comprises: a first-stage culture medium for differentiating iPSC stage cells into mesodermal stage cells.

[0029] In another preferred embodiment, the first-stage culture medium comprises: basal medium, BMP4, activin A, VEGF, CHIR99021, or combinations thereof.

[0030] In another preferred embodiment, the culture medium environment for the differentiation of the drug-expressing platelet precursor cells into platelets comprises: a second-stage culture medium for differentiating mesodermal stage cells into hematopoietic endothelial cell stage cells.

[0031] In another preferred embodiment, the second-stage culture medium comprises: basal medium, BMP4, bFGF, VEGF, or combinations thereof. Instructions for Use, Page 2 / 9, CN 122297700 A

[0032] In another preferred embodiment, the culture medium environment for the differentiation of the platelet precursor cells suitable for expressing the drug into platelets includes: a third-stage culture medium for differentiating cells in the hematopoietic endothelial cell stage into cells in the megakaryocyte progenitor cell stage.

[0033] In another preferred embodiment, the third-stage culture medium comprises: basal culture medium, TPO, SCF, Flt3L, IL-3, IL-6, 5% PFHM-II, heparin, or a combination thereof.

[0034] In another preferred embodiment, the culture medium environment suitable for differentiating platelet progenitor cells expressing the drug into platelets comprises: a fourth-stage culture medium, the fourth-stage culture medium being used to differentiate megakaryocyte progenitor cells into mature megakaryocytes and platelet cells.

[0035] In another preferred embodiment, the fourth-stage culture medium comprises: basal culture medium, SCF, TPO, IL-6, GNF351, KP457, Y39983, or a combination thereof.

[0036] In another preferred embodiment, the drug comprises: a protein drug, a peptide drug, an antibody drug, or a combination thereof.

[0037] In another preferred embodiment, the drug comprises: an antibody targeting a tumor antigen, a T cell co-stimulatory signaling ligand protein, a cytokine protein, or a combination thereof.

[0038] In another preferred embodiment, the thawing refers to thawing at 37℃±2℃.

[0039] In another preferred embodiment, the thawing continues until the ice crystals in the frozen drug-loaded platelets decrease in size but do not completely disappear.

[0040] In another preferred embodiment, the thawing continues until the diameter of the ice crystals in the frozen drug-loaded platelets is 3-7 mm.

[0041] In another preferred embodiment, during elution, the centrifugation speed is 500-1500 g, preferably 800-1200 g, and most preferably 1000 g.

[0042] In another preferred embodiment, during elution, the centrifugation time is 5-15 min, preferably 8-12 min, and most preferably 10 min.

[0043] In another preferred embodiment, during elution, the centrifugation rate at the rising speed is 3-8.

[0044] In another preferred embodiment, during elution, the centrifugation rate at the falling speed is 3-6.

[0045] In another preferred embodiment, the administration comprises: intravenous injection, intra-organ administration.

[0046] In another preferred embodiment, the peripheral blood platelet level of the test subject is comparable to that of the peripheral blood platelet level of a healthy control.

[0047] In another preferred embodiment, "comparable" means that the ratio of the peripheral blood platelet level Y1 of the test subject to the peripheral blood platelet level Y0 of the healthy control is 0.8 ≤ Y1 / Y0 ≤ 1.2, preferably 0.9 ≤ Y1 / Y0 ≤ 1.1.

[0048] In a second aspect of the invention, a formulation is provided, the formulation comprising: (a) platelet precursor cells; (b) a gene-editing reagent that causes the platelet precursor cells to express a drug; (c) a platelet cryopreservation solution; (d) a platelet eluent; and (e) a culture medium environment suitable for the differentiation of the drug-expressing platelet precursor cells into platelets.

[0049] In another preferred embodiment, the platelet cryopreservation solution comprises: sodium bicarbonate Ringer's injection, blood preservation solution, trehalose, human serum albumin, and CryoSure-DMSO.

[0050] In another preferred embodiment, the platelet eluent comprises: sodium bicarbonate Ringer's injection or sodium bicarbonate Ringer's injection, blood preservation solution, and human serum albumin.

[0051] In a third aspect of the invention, use of the formulation combination described in the second aspect of the invention is provided for preparing a kit for delivering the drug into a test subject. Specification 3 / 9 pages 5 CN 122297700 A

[0052] In another preferred embodiment, the kit is used for tumor-targeted release of a drug.

[0053] In another preferred embodiment, the kit is used for treating tumors.

[0054] In a fourth aspect of the invention, a platelet-based drug delivery method for non-diagnostic and non-therapeutic purposes is provided, comprising the following steps: (S1) providing frozen drug-loaded platelets differentiated in vitro from drug-expressing platelet precursor cells; (S2) thawing and eluting the frozen drug-loaded platelets to obtain a drug-loaded platelet solution; and (S3) administering the drug-loaded platelet solution to a test subject to deliver the drug into the test subject; wherein the test subject is a non-human mammal.

[0055] In another preferred embodiment, step (S1) comprises: providing drug-expressing platelet precursor cells, differentiating the drug-expressing platelet precursor cells into platelets in vitro to provide frozen drug-loaded platelets.

[0056] In another preferred embodiment, the test subject is a mouse with a humanized immune system.

[0057] It should be understood that, within the scope of this invention, the above-mentioned technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Brief Description of the Drawings

[0058] Figure 1 shows the reconstitution effect of the two selected donors (0336 and 0178) in an immunodeficient mouse model. The hCD45 ratio represents the proportion of human blood cells in the peripheral blood of mice.

[0059] Figure 2 shows that drug-loaded platelets can still be detected in a leukemia mouse model more than 24 hours after infusion. G1 is the injection of control platelets, and G2 is the injection of drug-loaded platelets.

[0060] Figure 3 shows the T cell proliferation after PBMCs are co-incubated with drug-loaded or control platelets in vitro for 5 days. The results show that drug-loaded platelets can induce more T cell proliferation compared with control platelets.

[0061] Figure 4 shows that after drug-loaded or control platelets were co-incubated with PBMCs in vitro, the activated PBMCs were then co-cultured with Nalm6 tumor cells to observe their killing ability.The results showed that drug-loaded platelets significantly enhanced the killing ability of PBMCs against tumor cells compared with control platelets. The E:T ratio refers to the ratio of PBMCs to Nalm6 tumor cells.

[0062] Figure 5 shows that after drug-loaded platelets were infused into a humanized mouse tumor model, no changes in ALT and AST were observed, indicating that drug-loaded platelets did not cause liver damage caused by CRS.

[0063] Figure 6 shows that after drug-loaded platelets were infused into a humanized mouse tumor model, the release of cytokines caused by them remained at a low level, indicating that drug-loaded platelets did not cause CRS.

[0064] Figure 7 shows the distribution of the drug when the platelets are used as a delivery carrier for drug administration according to the present invention. Detailed Embodiments

[0065] Through extensive and in-depth research, the inventors of the present invention have unexpectedly discovered a drug administration method using in vitro differentiated platelets as a delivery carrier. By expressing drug-loaded platelets at the platelet precursor cell stage, the redifferentiated platelets can still efficiently deliver drugs even after cryopreservation. This is of great significance for the marketization of platelet-based drug delivery systems. Surprisingly, drug-loaded platelets delivered in this manner have higher delivery efficiency and better stability than ordinary platelets. The present invention was completed based on this.

[0066] Terminology To facilitate a better understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified in this specification (page 4 / 9, CN 122297700 A), each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0067] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the associated listed items.

[0068] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the term includes “consisting of” or “substantially consisting of”.

[0069] As used herein, the term “or a combination thereof” means “any combination thereof”.

[0070] As used herein, in multiple administrations of the drug-loaded platelet solution to a subject at specific time intervals, the time interval between any two administrations is not necessarily the same. For example, the time interval between the first and second administrations may be 4 days, and the time interval between the second and third administrations may be 3 days or 4 days. However, in specific embodiments, the time interval between any two administrations is 1-8 days, preferably 2-6 days, more preferably 2-4 days, and most preferably 3-4 days.

[0071] Platelets are anucleate cytoplasmic fragments shed from megakaryocytes in the bone marrow. The normal reference value for platelets in healthy controls is (100–300) × 10⁹ / L. Their core functions are hemostasis, coagulation, and vascular repair, and they also participate in inflammation and immune regulation.

[0072] Platelet-based delivery systems have advantages such as high biocompatibility and low immunogenicity. Therefore, platelets are expected to be ideal delivery carriers, and drug loading can be achieved through various methods, including direct phagocytosis, surface modification, membrane coating, and gene editing at the progenitor cell stage.

[0073] The in vivo circulation cycle of platelets is relatively short (about 5–7 days), so when used as a drug carrier, the duration of stimulation of immune cells is more controllable, which helps to reduce the potential toxic risks from long-term exposure. In addition, platelets have a natural tendency to adhere to tumors during tumorigenesis and development. For example, platelets can interact with tumor cells through adhesion axes such as CD44 / P-selectin and CD40 / CD40L, adhering to their surface to form complexes; at the same time, platelet integrin αIIbβ3 (GPIIb / IIIa) can mediate stable binding with integrins such as αvβ3 or fibrinogen bridging structures on the surface of tumor cells. Platelet infiltration has been observed in clinical samples from patients with various solid tumors, suggesting that they play a wide role in the tumor microenvironment. Therefore, platelets have a more solid biological basis as a delivery carrier in tumor targeting and immune regulation applications.

[0074] Platelet-based drug delivery systems (PDSs) have received widespread attention in the field of tumor treatment in recent years. However, their wider application is limited by factors such as the limited source of platelets and the inability to edit genes.

[0075] In addition, current research mostly uses mouse platelets, and a few use human platelets, but they all come from blood donors, which face the problem of extremely limited sources and large batch-to-batch differences in clinical applications.

[0076] The therapeutic effect and safety of drugs are greatly affected by the type of delivery carrier, because the carrier determines the in vivo circulation time, biodistribution, and target specificity of the drug. Therefore, this invention provides a drug delivery method using in vitro differentiated platelets as a carrier.

[0077] The drug delivery method of the present invention aims to overcome the limitations of platelet donor dependence and accelerate the clinical translation of platelet delivery systems. The present invention provides a renewable and programmable platelet drug delivery platform based on human induced pluripotent stem cell (hiPSC) derived platelets (iPLTs).

[0078] hiPSCs provide a nearly unlimited and genetically engineerable cell source for the large-scale production of platelets, making it possible to construct uniform and sustainably updated drug delivery vectors.

[0079] The present invention performs stable drug expression modification on hiPSCs and differentiates them into drug-loaded iPLTs.In summary, this study has established a regenerative, gene-editable platelet delivery platform with clinical translational potential, providing a new technical pathway for next-generation tumor immunotherapy and broader biomedical applications.

[0080] It should be understood that the differentiation of induced pluripotent stem cells (iPSCs) into platelets is a complex process. iPSCs are in a highly plastic state with active epigenetic remodeling. Gene editing may affect their proliferation capacity, differentiation potential, and lineage bias. The platelet differentiation process involves multiple stages of fate determination, and any interference may lead to a decrease in differentiation efficiency or maturation obstacles, thereby affecting the in vitro generation of platelets. However, in this invention, it was unexpectedly discovered that platelet drug delivery obtained by differentiating drug-expressing platelet precursor cells has high drug delivery efficiency, requires a small dose of platelets, has high safety, and the targeted release delivery method of this invention can achieve simultaneous loading of multiple drugs and good therapeutic effects. Drugs loaded on the surface of platelets can directly act on target cells after entering the body and reaching the target site. Meanwhile, cells loaded inside platelets can be activated in the targeted microenvironment, releasing drugs to act on the target cells.

[0081] In a specific embodiment, compared to directly modifying mature platelets (e.g., through click chemical reactions), this invention employs a strategy of gene transduction at the iPSC stage followed by differentiation to obtain engineered platelets. This avoids potential protein shedding or unstable linkage problems associated with exogenous modifications, resulting in more persistent and reliable expression of exogenous drugs. Simultaneously, since genetic modification occurs at the iPSC cell origin stage, after monoclonal screening or the establishment of stable lines, a cell population with relatively consistent expression levels can be obtained, significantly reducing drug loading fluctuations between different batches. This approach does not rely on later conjugation efficiency or reaction condition control, improving batch-to-batch consistency and reproducibility. Furthermore, this strategy only requires routine purification and collection steps after obtaining platelets, avoiding the potential impact of cross-linking reactions, surface modifications, or exposure of active groups on platelet membrane structure, membrane protein spatial conformation, and integrin functional state.

[0082] When this drug delivery method is used in humans, it is equivalent to a treatment method. Therefore, this invention provides a drug delivery device (or drug delivery equipment). This device is essentially a genetically engineered drug-loaded platelet + standardized preparation / freezing / thawing / infusion integrated drug delivery system. It can be a bedside integrated fully automated drug-loaded platelet drug delivery device, internally divided into three functional areas (but in the same machine, in the same shell, or in the same control system). In fact, such integrated devices already exist, such as Fresenius / Termall platelet washing machine + transfusion pump integrated machine, Lonza Cocoon fully automated cell preparation system, etc. In use, a pharmaceutically effective amount of frozen drug-loaded platelets can be provided as needed.

[0083] When this drug delivery method is used in animals, it is not a treatment method.When this drug delivery method is used in animals, it can be used for scientific research on platelet drug delivery, such as further optimization of the drug delivery regimen and further optimization of the targeting performance; it can also be used to study the release and distribution of platelet drugs. In short, those skilled in the art can use the treatment method of the present invention for non-diagnostic and non-therapeutic purposes as needed.

[0084] The main advantages of the present invention include: (a) Synergistic improvement of drug loading efficiency and targeting: The drug delivery method of the present invention expresses drugs through the platelet precursor cell stage, and uses its intracellular synthesis and sorting mechanism to achieve efficient encapsulation of drugs, solving the problems of low drug loading efficiency, drug instability and easy leakage of mature platelets; differentiated platelets inherit natural targeting characteristics and can accurately target inflammation or lesion sites, significantly increasing the drug concentration at the lesion site.

[0085] (b) Breakthrough in cryopreservation stability: The drug delivery method of the present invention breaks the limitation of traditional platelet drug delivery requiring short-term storage at room temperature, extends the product shelf life, and meets the needs of clinical emergency transfusion and large-scale supply.

[0086] (c) Optimization of clinical application safety and efficacy: The optimal dosage and dosing interval are determined through systematic studies to avoid immune reactions or coagulation abnormalities caused by excessive dosage, while rationalizing the number of administrations, reducing the treatment cost and body burden for patients, taking into account both therapeutic effect and clinical safety, and promoting the clinical translation of technology.

[0087] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0088] Example 1: Preparation and cryopreservation of drug-loaded platelets.

[0089] In this embodiment, drug-loaded platelets differentiated in vitro were prepared (hereinafter referred to as drug-loaded platelets). Specifically, in its precursor cell stage (e.g., iPSC stage), the drug was expressed in iPSCs through gene editing, so that the platelets produced by iPSC differentiation would carry the drug.

[0090] Taking simultaneous loading of 4-1BBL and IL-12 as an example, the vector expressing these two proteins was inserted into the genome in the iPSC stage through site-directed gene editing. Then, through a four-stage directed induction differentiation process of about 21 days, it differentiated into platelets. The first stage was mesodermal induction, and the differentiation conditions were BMP4, activin A, VEGF, and CHIR99021 added to the basal culture medium.The second stage is the differentiation of hematopoietic endothelial cells, with the basal medium supplemented with BMP4, bFGF, and VEGF. The third stage is the generation of megakaryocyte progenitor cells, with the basal medium supplemented with TPO, SCF, Flt3L, IL-3, IL-6, 5% PFHM-II, and heparin. The fourth stage is the release of platelets, with the basal medium supplemented with SCF, TPO, IL-6, GNF351, KP457, and Y39983, thereby obtaining drug-loaded platelets.

[0091] The differentiated drug-loaded platelets were cooled to -140°C using a programmable cooling device and transferred to a liquid nitrogen tank for storage, thereby providing frozen drug-loaded platelets.

[0092] Example 2: Recovery and counting of frozen platelets.

[0093] Remove the platelet cryopreservation tube from the liquid nitrogen tank. Immediately use hemostatic forceps to gently clamp the base of the pressure buffer tube of the cryopreservation tube, keeping the rubber stopper facing down and not inverted. Completely submerge the cryopreservation tube below the water surface in the water bath and continuously shake it in a circular motion to ensure even heating. After about 30 seconds, briefly remove the tube every 5-10 seconds to observe the size of the ice crystals. Stop the water bath when the ice crystals are slightly smaller than a mung bean. Wipe dry with sterile gauze, spray with 75% ethanol, and then transfer to a biosafety cabinet. Remove the aluminum foil from the blue rubber stopper at the bottom of the tube, cut the pressure buffer tube, and use a 1 mL syringe to puncture the blue rubber stopper at the bottom of the tube. While shaking, extract all samples and transfer them to a new 1.5 mL centrifuge tube. After thoroughly mixing with a 1000 μL pipette, immediately aspirate as much of the sample as possible and transfer it to a 15 mL centrifuge tube. Then add frozen platelet diluent so that the volume ratio of the extracted frozen platelet concentrate to the frozen platelet diluent is 1:1.5.

[0094] Thoroughly mix the above platelet suspension with a 1000 μL pipette, and immediately take two 30 μL samples from the middle layer and transfer them to 0.5 mL centrifuge tubes for formulation analysis (cell counting by flow cytometry). Store the remaining platelet suspension at room temperature.

[0095] The principle of platelet counting is as follows: when flow cytometry antibody (PE-CD41) is added to the test sample, the fluorescently labeled antibody specifically binds to the platelet surface antigen. Before testing, an absolute counting microsphere of the same volume as the test sample is added and thoroughly mixed. The number of cells in a certain volume of sample can be calculated based on the known number of absolute counting microspheres. Combined with the flow cytometry antibody clustering method, the absolute number of a certain group of cells in a certain volume can be calculated. Specific experimental method: Platelets to be tested are sampled into a 1.5 mL sterile centrifuge tube and should be tested immediately after sampling. 50 μL of platelet sample and 20 μL of diluted PE-CD41 antibody are added to the top of the stainless steel holder of the absolute counting tube (Note: Do not touch the microspheres or the tube wall) using the reverse pipetting method. The tube is capped, gently vortexed to mix, and reacted at room temperature in the dark for 15 min.Then, 4.3.5. Add 400 μL of diluent to each tube, cap the tubes, gently vortex to mix, and react at room temperature in the dark for 15 min. Before analysis, the prepared samples should be stored at 4~10℃ in the dark and should be analyzed within 1 h. Before running the flow cytometer, the counting tubes should be vortexed at low speed to resuspend the microspheres and reduce cell aggregation.

[0096] After counting, thoroughly pipette the platelet suspension, accurately aspirate a certain volume (as much as possible) of the platelet suspension, centrifuge at 1000 g for 10 min at room temperature, with an acceleration of 5 and a deceleration of 4. After disinfection by spraying with 75% ethanol, transfer to a biosafety cabinet. According to the results of the formulation analysis, carefully aspirate a certain volume of supernatant with a pipette, so that the remaining volume meets the required platelet concentration after resuspension.

[0097] Example 3: Construction of a humanized mouse Nalm6 tumor model and platelet drug delivery.

[0098] NPG-dKO mice are severely immunodeficient mice. 1E7 human peripheral blood mononuclear cells (PBMCs) were transplanted into 6-week-old NPG-dKO mice via tail vein injection, thereby implanting human immune cells (mainly human T cells, monocytes, and B cells) into the mice; this created human immune surveillance / immune response in the mice, simulating the immune environment in the human body.

[0099] NALM6-Luc tumor cells were resuscitated and passaged. Eight days after NPG-dKO mice were inoculated with huPBMCs, NALM6-Luc cells in the logarithmic growth phase were collected. The culture medium was removed, and the cells were washed twice with DPBS. Then, 5E5 tumor cells were injected into each mouse via tail vein injection.

[0100] On day 13 after inoculation with huPBMCs, the humanization reconstitution rate was detected by flow cytometry. The results are shown in Figure 1. The reconstitution effect of transplanting 5M or 10M cells of PBMCs from different donors (0336 and 0178) into an immunodeficient mouse model via tail vein injection (IV) was shown. The hCD45 ratio represents the proportion of human blood cells in the peripheral blood of mice. Among them, 5M and 10M represent the number of PBMCs injected (5E6 and 10E6), respectively, and iv represents intravenous injection. This figure shows that when testing different PBMC injection doses, the reconstitution effect of 10E6 cells was better. In addition, the reconstitution effect of PBMCs from different donors after injection into mice varied, but all were above 10%.

[0101] On day 15 after inoculation with huPBMCs (i.e., day 7 after inoculation with NALM6-Luc), the average tumor photon count was detected by in vivo imaging, which reached an average of 2.80E+04.

[0102] Nineteen mice were randomly assigned to groups according to the experimental design based on reconstruction rate and tumor photon count. The day of grouping was defined as D0.Clodronate liposomes were injected intraperitoneally on D-1, D3, D6, and D10 to clear residual macrophages in mice. Platelets (drug-loaded platelets and control platelets) were injected on D0, D4, D7, and D11 at a dose of 1E7 platelets per mouse.

[0103] Example 4: Effect of the platelet delivery method of the present invention.

[0104] The resuscitated platelets were diluted and transferred to EP tubes, delivered to the animal facility at room temperature, and stored at room temperature for inoculation in the humanized mouse Nalm6 tumor model. 5E6-1E7 platelets were injected into each mouse at inoculation. The proportion of human platelets in the peripheral blood of mice was detected by flow cytometry 25 hours after inoculation.

[0105] The results are shown in Figure 2. G1 represents unloaded control platelets, and G2 represents drug-loaded platelets. Drug-loaded platelets were still detectable in the leukemia mouse model more than 24 hours after inoculation.

[0106] Furthermore, the function of the drug-loaded platelets obtained in Further Example 3 was identified, and the results are shown in Figures 3 and 4. The results show that the drug-loaded platelets can induce more T cell proliferation and significantly enhance the killing ability of PBMCs against tumor cells, which confirms the effectiveness of this platelet drug delivery method.

[0107] In fact, the drug delivery effect of this delivery method is significantly improved compared to directly incubating the drug and platelets and then delivering it.

[0108] Example 5: Platelet tracing and distribution detection.

[0109] The content in organs is generally lower than that in peripheral blood. Therefore, the requirements for platelet tracing methods in organs are relatively high (such as the precision and accuracy of the method, the detection limit and stability, etc.). Instructions for Use, Pages 8 / 9, CN 122297700 A

[0110] In this embodiment, platelets were transplanted into NPG severely immunodeficient mice via tail vein injection. The mice were euthanized at 30 min (±5 min), 2 h (±10 min), 6 h (±10 min), 12 h (±15 min), 24 h (±30 min), 72 h (±30 min), and 168 h (±30 min) after administration. Whole blood and various tissues (brain, quadriceps femoris, ovary, testis, bladder, jejunum, stomach, pancreas, inguinal lymph nodes, kidney, adrenal gland, femoral bone marrow, heart, liver, spinal cord (thoracic vertebrae), lung, and spleen) were collected.

[0111] Genomic DNA was then extracted from the whole blood and various tissues using a magnetic bead method. The OD value of the nucleic acid was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the concentration was calculated.

[0112] Primers specific to human platelet mitochondrial genes were designed (see Table 1), and detection was performed using a real-time quantitative PCR instrument. The raw data were output using relevant software, and the content of the sample was calculated. The results are shown in Figure 7.

[0113] Table 1 Primers specific to human platelet mitochondrial genes. This embodiment also detected cytokine release and blood biochemistry ALT / AST in mouse peripheral blood. The results are shown in Figures 5 and 6, which preliminarily demonstrated the risk of platelet drug delivery without cytokine release syndrome (CRS).

[0114] All references mentioned in this invention are incorporated herein by reference as if each reference were individually incorporated by reference. Furthermore, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. This invention belongs to the field of pharmaceuticals and healthcare. Specifically, it provides a drug delivery method using in vitro differentiated platelets as a delivery carrier. By expressing drug-loaded cells at the platelet precursor cell stage, the redifferentiated platelets can still efficiently deliver drugs even after cryopreservation. This is of great significance for the commercialization of drug delivery methods using platelets as a delivery system.

Claims

1. A drug delivery device using platelets as a delivery carrier, characterized in that, The device includes the following modules: (Z1) Drug-loaded platelet delivery module, the drug-loaded platelet delivery module being configured to: deliver frozen drug-loaded platelets formed in vitro from platelet precursor cells expressing a drug; (Z2) Thawing and elution module, wherein the thawing and elution module is configured to thaw and elute frozen drug-loaded platelets to obtain a drug-loaded platelet solution; (Z3) Drug delivery module, the drug delivery module being configured to administer the drug-loaded platelet solution to the test subject, thereby delivering the drug into the test subject's body.

2. The drug delivery device as claimed in claim 1, characterized in that, The drug delivery module is configured to administer 5 × 10 doses to the test subject each time. 6 -5×10 7 One drug-loaded platelet.

3. The drug delivery device as claimed in claim 1, characterized in that, The drug delivery module is configured to administer the drug-loaded platelet solution to the test subject multiple times at specific time intervals. Wherein, "multiple times" means ≥2 times; The specific time interval is 1-8 days.

4. The drug delivery device as claimed in claim 1, characterized in that, The platelet precursor cells are selected from the following group: induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells, hematopoietic progenitor cells, hematopoietic stem-progenitor cells, megakaryocyte progenitor cells, or megakaryocytes.

5. The drug delivery device as claimed in claim 1, characterized in that, The drug includes: protein drugs, peptide drugs, antibody drugs, or combinations thereof.

6. The drug delivery device as claimed in claim 1, characterized in that, The platelet levels in the peripheral blood of the subjects were comparable to those in healthy controls.

7. A formulation combination, characterized in that, The formulation combination comprises: (a) Platelet precursor cells; (b) A gene-editing reagent that enables the platelet precursor cells to express the drug; (c) Platelet cryopreservation solution; (d) Platelet eluate; (e) A culture medium environment suitable for the differentiation of platelet precursor cells expressing the drug into platelets.

8. The use of the formulation combination according to claim 7, characterized in that, This is used to prepare a kit for delivering the drug into a test subject.

9. A platelet-based drug delivery method for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: (S1) Provides frozen drug-loaded platelets differentiated in vitro from drug-expressing platelet precursor cells; (S2) Thaw and elute the frozen drug-loaded platelets to obtain a drug-loaded platelet solution; and (S3) The drug-loaded platelet solution is administered to the test subject to deliver the drug into the test subject's body; The subjects in this study were non-human mammals.

10. The method as described in claim 9, characterized in that, The test subjects were mice with humanized immune systems.

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