Engineered vesicle, microneedle and microneedle patch for treating acute radiation sickness

Abstract

The invention discloses a vesicle, a microneedle loaded with the vesicle and a microneedle patch. An activated platelet derivatization vesicle is used as a bone marrow targeting carrier to prepare an engineered vesicle of which the outer layer is crosslinked with an antioxidant and the inner part is entrapped with an anti-radiation drug; then, a soluble microneedle drug delivery system with the engineered vesicles entrapped on the needle tip is constructed in combination with a soluble microneedle technology. The microneedle patch prepared from the microneedle drug delivery system has the characteristics of convenient drug delivery and targeted therapy, and can meet treatment scenes under various irradiation conditions.

Description

Technical Field

[0001] The present invention relates to a drug delivery system, and in particular to a soluble microneedle patch drug delivery system constructed using soluble microneedle technology and encapsulating engineered vesicles, which achieves the treatment of acute radiation sickness, has the characteristics of convenient drug delivery and targeted treatment, and can meet the treatment scenarios under various irradiation conditions. Background Art

[0002] Treatment of acute radiation sickness is a key component of nuclear accident emergency medical rescue and a crucial area of ​​radiation medicine research. Based on its clinical characteristics and underlying pathological symptoms, acute radiation sickness is categorized into three types: myeloid, intestinal, and cerebral. Hematopoietic stem and progenitor cells in the bone marrow are extremely sensitive to radiation. A dose of just 1 Gy can produce significant radiation damage symptoms, such as anemia, bleeding, and infection, posing a serious threat to the patient's life. Therefore, bone marrow acute radiation sickness (BM-ARS) is the most common and clinically significant radiation-induced illness. The main challenge facing this field is the lack of highly effective, low-toxic anti-radiation drugs.

[0003] Currently, the clinical treatment for BM-ARS involves continuous subcutaneous injection of hematopoietic growth factors, such as recombinant granulocyte colony-stimulating factor (G-CSF), thrombopoietin peptidomimetics, and thrombopoietin (TPO). However, continuous administration can easily cause immunogenicity, resulting in poor therapeutic effects on the hematopoietic system. Therefore, how to construct safe, stable, and efficient drug-loaded targeted formulations to improve the efficacy and safety of drugs in treating BM-ARS is a key scientific issue that needs to be addressed urgently.

[0004] Extracellular vesicles are a type of non-uniform nanoscale vesicles secreted by cells. They are divided into exosomes (30-120 nm) and microparticles (100 nm-1 mm) based on their size. Vesicles have the advantages of being similar to cell membranes, small in size, negatively charged, able to avoid phagocytosis, produce immune escape, have a long circulation time, good biocompatibility, and can cross natural tissue barriers such as the blood-brain barrier and the bone marrow barrier. Therefore, vesicles can be used as an ideal natural nanocarrier for drug delivery. In addition, traditional nanocarriers have a low encapsulation rate for hydrophilic drugs and have certain limitations in delivering protein and nucleic acid drugs. Vesicles have better affinity with the above two types of drugs, which can significantly improve the encapsulation rate of drugs and achieve efficient targeted delivery.

[0005] Therefore, extracellular vesicles as a new drug delivery technology are expected to promote the development of highly effective and low-toxic anti-radiation drug preparations.

[0006] Platelet-derived vesicles (PDVs) are vesicles secreted by activated platelets. They can carry large molecular proteins and RNA to penetrate the bone marrow-blood barrier, adhere to bone marrow hematopoietic progenitor cells (HPCs) and bone marrow hematopoietic stem cells (HSCs), and be absorbed by the cells through endocytosis. Therefore, PDVs can be used as drug delivery vehicles targeting bone marrow HPCs / HSCs, and are expected to achieve targeted treatment of BM-ARS. However, single drug-loaded PDV injections have the following drawbacks:

[0007] (1) In vivo, the highly reactive oxygen species (ROS, such as OH·, H·, and H2O2) generated by radiation will oxidize and destroy the phospholipid bilayer structure of the vesicles, causing premature release of the drug and reducing the efficiency of targeted drug delivery; (2) In vitro, the drug contained in the vesicles is prone to leakage, and low-temperature or freeze-dried powders cannot be stored for a long time and need to be prepared and used immediately.

[0008] Therefore, it is urgent to combine other formulation technologies to maintain the long-term stability of vesicle biologics. Summary of the Invention

[0009] The present invention combines activated platelet-derived vesicles with microneedle technology to prepare an engineered vesicle-loaded microneedle patch with convenient drug delivery and targeted treatment for acute radiation sickness. Currently, no related reports have been published.

[0010] One object of the present invention is to provide a vesicle for the clinical treatment of acute radiation sickness. Specifically, the vesicle is composed of activated platelets, a cross-linking agent, an antioxidant, and an anti-radiation substance, and is produced through engineering.

[0011] An engineered vesicle for the treatment of acute radiation sickness begins by covalently crosslinking antioxidants to the surface of PDVs and purifying them to obtain anti-ROS vesicles. These vesicles are then used as carriers in an extrusion process to create an engineered vesicle delivery system containing an anti-acute radiation sickness drug. This system not only enables bone marrow-targeted drug delivery but also utilizes the cross-linked antioxidants in the outer layer to reduce ROS damage in the body while maintaining vesicle stability. This system offers convenient drug delivery and targeted therapy, meeting the needs of various treatment scenarios under various irradiation conditions.

[0012] According to some embodiments, the entrapped antiradioactive drug is selected from one or more of recombinant granulocyte colony stimulating factor, thrombopoietin and thrombopoietin peptidomimetics, amifostine, superoxide dismutase, and catalase.

[0013] According to some embodiments, the antioxidant is selected from one or more of glutathione and its derivative glutathione ethyl ester, N-acetylcysteine, lipoic acid and its derivative dihydroxylipoic acid, superoxide dismutase, and glutathione peroxidase.

[0014] According to some embodiments, the cross-linking agent is selected from one or more of bissuberate, succinimide ester-polyethylene glycol-maleimide.

[0015] Microneedles are a novel drug delivery vehicle proposed in the 1970s. Compared to traditional transdermal drug delivery systems, microneedles offer the advantage of rapid delivery of biomacromolecules that are poorly absorbed by the gastrointestinal tract, such as proteins, peptides, antibodies, vaccines, RNA, and DNA. Based on the manufacturing process and material properties, microneedles can be categorized into four types: solid microneedles, coated microneedles, soluble microneedles, and hollow microneedles. These microneedles achieve convenient and efficient drug delivery through minimally invasive procedures in the stratum corneum. In 2019, Zosano Pharma submitted a marketing application to the FDA for Qtrypta, the world's first microneedle drug delivery system for the treatment of migraines. Currently, soluble microneedles are widely used in the cosmetics industry, resulting in a variety of star products, including wrinkle-removing patches for the eyes, acne patches, and freckle patches.

[0016] The second object of the present invention is to provide an engineered vesicle microneedle for treating acute radiation sickness and maintain the long-term stability of the vesicle biological preparation.

[0017] The third object of the present invention is to provide an engineered vesicle microneedle patch for treating acute radiation sickness, which has the characteristics of convenient drug delivery and targeted treatment.

[0018] The second object of the present invention is to provide an application of a microneedle or a microneedle patch in the preparation of a medical device for treating acute radiation sickness.

[0019] A third object of the present invention is to provide a medical device comprising an engineered vesicle microneedle or microneedle patch for treating acute radiation sickness.

[0020] The engineered vesicle microneedle of the present invention comprises an engineered vesicle.

[0021] According to some embodiments, the microneedle tip matrix material is made of a polymer. Suitable polymers include, but are not limited to, polyvinyl alcohol, polylactic acid, silk fibroin, sodium carboxymethyl cellulose, chitosan, alginate, hyaluronate, and polyvinyl pyrrolidone, as well as methacrylic silk fibroin, methacrylic chitosan, methacrylic gelatin, and methacrylic hyaluronic acid. These polymers can be used alone or in combination in the present invention, with hyaluronic acid and trehalose being preferred.

[0022] Each microneedle is disposed on a base layer to form a microneedle patch. The base layer is also composed of polymers, including but not limited to polyvinyl alcohol, polylactic acid, silk fibroin, sodium carboxymethyl cellulose, chitosan, alginate, hyaluronate, and polyvinyl pyrrolidone. These polymers are used alone or in combination in the present invention, with polyvinyl pyrrolidone being preferred.

[0023] A fourth object of the present invention is to provide a microneedle patch made of a plurality of engineered vesicle microneedles arranged in a regular pattern.

[0024] According to some embodiments, the distance between each microneedle is equal. The microneedles may have shapes such as, but not limited to, conical and prismatic. Conical shapes are preferred. The needle length ranges from 100 μm to 1000 μm. The diameter of the needle base ranges from 30 μm to 500 μm.

[0025] According to some preferred embodiments, the length of the needle body ranges from 100 μm to 1000 μm.

[0026] According to some preferred embodiments, the diameter of the base surface of the needle body ranges from 30 μm to 500 μm.

[0027] According to some preferred embodiments, 1 to 3 microneedles are provided within a length range of 1 mm, with 1 to 2 microneedles being preferred.

[0028] According to some preferred embodiments, in an area of ​​1cm 2 Within the range of , 10 to 100 microneedles are set, with 50 to 100 microneedles being preferred.

[0029] A fifth object of the present invention is to provide a method for preparing a microneedle. To facilitate those skilled in the art to understand the method for preparing the microneedle patch, the present invention lists the following preparation process by way of example, but not limitation:

[0030] (1) Preparation of anti-free radical vesicles

[0031] Whole blood was collected from the mouse heart, and plasma-free platelets were collected and activated with thrombin. Gradient centrifugation was then performed, and the precipitate was resuspended in PBS to obtain the desired activated platelet-derivatized vesicle solution. A PBS aqueous solution (pH 7.4) containing activated platelet-derivatized vesicles was prepared, 1 ml of an aqueous solution containing a crosslinker was added, and the reaction was vortexed at room temperature for 30 minutes. The crosslinked vesicles were isolated and purified using a dextran gel column method. The purified PBS solution of crosslinked vesicles was then mixed with a PBS solution containing an antioxidant and vortexed at room temperature for 30 minutes. The anti-free radical vesicles were isolated and purified using a dextran gel column method.

[0032] (2) Preparation of engineered vesicles loaded with drugs for treating acute radiation sickness

[0033] Different amounts of anti-radiation sickness drugs were added to the above-mentioned anti-free radical vesicle solution, and drug-loaded engineered vesicles were prepared using a liposome extruder. The drug-loaded engineered vesicles and free drugs were separated using a dextran gel column method, and the drug loading amount and encapsulation efficiency were calculated.

[0034] (3) Preparation of engineered vesicle microneedles loaded with anti-acute radiation sickness drugs

[0035] Polymer 1 is selected as the needle tip matrix material of the microneedle, and a microneedle matrix solution containing engineered vesicles loaded with anti-radiation sickness drugs is prepared; the microneedle tip of the drug-loaded engineered vesicles is prepared by pouring into the mold and centrifuging and air-drying. The microneedle base solution containing polymer 2 is dropped into the above-mentioned mold carrying the needle tip and centrifuged and air-dried again to obtain the final engineered vesicle microneedle patch.

[0036] According to some embodiments, the activated platelet-derived vesicle solution has a density of 1 mg / ml to 5000 mg / ml of activated platelet-derived vesicles, calculated based on the total protein content.

[0037] According to some embodiments, the cross-linking agent solution contains a mass fraction of 0.01% to 10%.

[0038] According to some embodiments, the mass fraction of the anti-radiation drug in the microneedle matrix solution of the engineered vesicles carrying the anti-radiation drug is 0.1% to 10%.

[0039] According to some embodiments, the mass fraction of polymer 1 in the microneedle matrix solution of engineered vesicles carrying anti-radiation sickness drugs is 5% to 50%, and the mass fraction of polymer 2 in the microneedle base solution is 5% to 50%.

[0040] Compared with the prior art, the technical effects of the present invention are:

[0041] The microneedle patch of this invention not only delivers targeted drugs to the bone marrow but also utilizes a cross-linked antioxidant in its outer layer to reduce ROS damage in the body while maintaining the stability of the vesicles within the body. This convenient, targeted approach addresses the challenges of various irradiation treatment scenarios, filling a gap in the field of engineered vesicle-based microneedle patch systems for the treatment of acute radiation sickness and holds broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure is a schematic diagram of the preparation of the engineered vesicle microneedle of the present invention and its application in the treatment of acute radiation sickness;

[0043] Figure 2Figures 2 and 3 are the characterization and functional confirmation of anti-ROS vesicles, where A is an electron microscopy image of anti-ROS, scale bar 100 μm; B is the particle size distribution and potential; C is the DTNB-UV spectrum curve; D is a western blot image;

[0044] Figure 3 Characterization diagram of TPO-loaded engineered vesicles, where A is the electron microscope image of TPO-loaded engineered vesicles, with a scale of 100 μm; B is the particle size distribution and potential;

[0045] Figure 4 Figure 1 is a validation diagram of the anti-ROS function of TPO-loaded engineered vesicles, where A is the experiment of eliminating H2O2 by TPO-loaded engineered vesicles; B is an electron micrograph of TPO-loaded platelet-derivatized vesicles after 2 hours of reaction; C is an electron micrograph of TPO-loaded engineered vesicles after 2 hours of reaction, with a scale of 200 μm.

[0046] Figure 5 Characterization diagram of TPO-loaded engineered vesicle microneedles, where A is a scanning electron microscope image; B is a stereomicroscope image; C is a confocal image; D is a pressure-displacement curve; E is a stability study of the TPO preparation; F is an in vitro transdermal permeation curve; all scales are 200 μm.

[0047] Figure 6 Figure 2 is a study on the distribution of TPO in the bone marrow group, where A is the bone marrow immunohistochemistry image of healthy mice; B is the bone marrow immunohistochemistry image 24 hours after subcutaneous injection of TPO 2 hours after irradiation; C is the bone marrow immunohistochemistry image 24 hours after microneedle administration of TPO 2 hours after irradiation; D is the bone marrow immunohistochemistry image 24 hours after microneedle administration of TPO-loaded platelet-derived vesicles 2 hours after irradiation; E is the bone marrow immunohistochemistry image 24 hours after microneedle administration of a physical mixture of TPO-loaded platelet-derived vesicles and glutathione 2 hours after irradiation; F is the bone marrow immunohistochemistry image 24 hours after microneedle administration of TPO-loaded engineered vesicles, with a scale of 100 μm.

[0048] Figure 7 Figure 3 is a peripheral blood recovery evaluation chart, where A represents leukocytes; B represents platelets. Between the TPO-loaded engineered vesicle microneedle group and the model group, # represents P < 0.05, and ## represents P < 0.01; between the TPO subcutaneous injection group and the model group, * represents P < 0.05; between the TPO-loaded platelet-derived vesicle microneedle group and the model group, @ represents P < 0.05; between the TPO-loaded platelet-derived vesicle and glutathione physical combination microneedle group and the model group, & represents P < 0.05, and && represents P < 0.01; between the TPO-loaded engineered vesicle microneedle group and the TPO subcutaneous injection group, $ represents P < 0.05. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are intended only to illustrate the technical solution of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solution of the present invention, and all such modifications or equivalents should be included in the scope of the claims of the present invention.

[0050] Figure 1 The following diagram illustrates the principle of preparing the engineered vesicle microneedles of the present invention and their application in treating acute radiation sickness. The following examples of the present invention all utilize the pathways shown in the diagram to prepare engineered vesicles, their microneedles, and microneedle patches.

[0051] Example 1 Preparation of TPO-loaded engineered vesicle hyaluronic acid microneedles

[0052] (1) Activated PDVs extraction

[0053] Whole blood was collected from C57BL / 6J mice and added to a 9:1 solution of acidic citrate dextrose. After centrifugation (150 g, 23°C for 15 min) to remove leukocytes, plasma-free platelets were collected by further centrifugation for 15 min and resuspended in 1 mL of PBS. Platelets were activated by the addition of 0.1 U / mL thrombin (10602400001, Sigma-Aldrich, St. Louis, USA) and incubated at 37°C for 30 min. Gradient centrifugation was then performed as follows: 300 g for 10 min at 4°C to remove live cells; 1500 g for 15 min to remove dead cells; and 20,000 g for 30 min to remove cell debris. The supernatant from all gradient centrifugations was then collected. Finally, ultracentrifugation was performed at 100,000 g for 90 min, the supernatant removed, and the pellet resuspended in PBS to obtain the desired PDV solution.

[0054] (2) Preparation and structural confirmation of anti-ROS vesicles

[0055] Prepare 10 ml of PBS (pH 7.4) containing 10 mg / ml of PDVs, add 1 ml of 10 mM bis(sulfosuccinimidyl) suberate crosslinker, and vortex at room temperature for 30 minutes. Separate and purify the crosslinker-attached vesicles using a Sephadex column. Next, mix the purified crosslinker-attached vesicles in PBS with 10 mM glutathione and vortex at room temperature for 30 minutes. Separate and purify the vesicles using a Sephadex column to obtain a 10 mg / ml ROS-resistant vesicle solution.

[0056] (3) Preparation of TPO-loaded engineered vesicles

[0057] An equal volume of 1 mg / ml TPO solution was mixed with the above-mentioned anti-ROS vesicle solution at a volume ratio of 1:1, and TPO-loaded engineered vesicles were prepared using a liposome extruder; the TPO-loaded engineered vesicles and free TPO were separated using a dextran gel column method.

[0058] (4) Preparation of TPO-loaded engineered vesicle microneedles

[0059] Hyaluronic acid and trehalose were selected as the needle tip matrix materials of the microneedle, and a microneedle matrix solution containing TPO engineered vesicles was prepared at a concentration of 1 mg / ml (the solution contained 6% (w / v) hyaluronic acid and 5% (w / v) trehalose); the microneedle tip loaded with TPO engineered vesicles was prepared by centrifugation-air drying method (2000g, 10min) through a mold (10×10 array, conical hollow, pore diameter 300μm, pore depth 800μm, provided by Taizhou Microchip Pharmaceutical Technology Co., Ltd.); the microneedle base solution containing 15% (w / v) polyvinylpyrrolidone K90 was dropped into the above-mentioned mold carrying the needle tip and centrifuged again (2000g, 10min) and air-dried to obtain the final TPO engineered vesicle-loaded microneedle patch.

[0060] Example 2 Preparation of G-CSF-loaded Engineered Vesicle Polyvinylpyrrolidone Microneedles

[0061] (1) Activated PDVs extraction

[0062] Whole blood was collected from C57BL / 6J mice and added to a 9:1 solution of acidic citrate dextrose. After centrifugation (150 g, 23°C for 15 min) to remove leukocytes, plasma-free platelets were collected by further centrifugation for 15 min and resuspended in 1 mL of PBS. Platelets were activated by the addition of 0.1 U / mL thrombin (10602400001, Sigma-Aldrich, St. Louis, USA) and incubated at 37°C for 30 min. Gradient centrifugation was then performed as follows: 300 g for 10 min at 4°C to remove live cells; 1500 g for 15 min to remove dead cells; and 20,000 g for 30 min to remove cell debris. The supernatant from all gradient centrifugations was then collected. Finally, ultracentrifugation was performed at 100,000 g for 90 min, the supernatant removed, and the pellet resuspended in PBS to obtain the desired PDV solution.

[0063] (2) Preparation and structural confirmation of anti-ROS vesicles

[0064] Prepare a 10ml PBS solution (pH 7.4) containing 10mg / ml PDVs, add 1ml of a 10mM bis(sulfosuccinimidyl) suberate crosslinker, and vortex at room temperature for 30 minutes. Separate and purify the crosslinker-attached vesicles using a Sephadex column. Next, mix the purified crosslinker-attached vesicles in PBS with 10mM superoxide dismutase and vortex at room temperature for 30 minutes. Separate and purify the vesicles using a Sephadex column to obtain a 10mg / ml ROS-resistant vesicle solution.

[0065] (3) Preparation of G-CSF-loaded engineered vesicles

[0066] An equal volume of 2 mg / ml G-CSF solution was mixed with the above-mentioned anti-ROS vesicle solution in a volume ratio of 1:1, and G-CSF-loaded engineered vesicles were prepared using a liposome extruder; the G-CSF-loaded engineered vesicles and free G-CSF were separated using a dextran gel column method.

[0067] (4) Preparation of G-CSF-loaded engineered vesicle microneedles

[0068] Polyvinyl pyrrolidone K90 was selected as the needle tip matrix material of the microneedle, and a microneedle matrix solution containing G-CSF engineered vesicles at a concentration of 1 mg / ml was prepared (the solution contained 15% (w / v) polyvinyl pyrrolidone K90); a mold (10×10 array, conical hollow, pore diameter 300 μm, pore depth 800 μm, provided by Taizhou Microchip Pharmaceutical Technology Co., Ltd.) was filled with centrifugation-air drying method (2000g, 10 min) to prepare microneedle tips loaded with G-CSF engineered vesicles; the microneedle base solution containing 15% (w / v) polyvinyl pyrrolidone K90 was dropped into the above-mentioned mold containing the needle tip and centrifuged again (2000g, 10 min) and air-dried to obtain the final G-CSF engineered vesicle microneedle patch.

[0069] Example 3 Preparation of Polyvinylpyrrolidone Microneedles Loaded with Thrombopoietin Peptide Mimetic Engineering Vesicles

[0070] (1) Activated PDVs extraction

[0071] Whole blood was collected from C57BL / 6J mice and added to a 9:1 solution of acidic citrate dextrose. After centrifugation (150 g, 23°C for 15 min) to remove leukocytes, plasma-free platelets were collected by further centrifugation for 15 min and resuspended in 1 mL of PBS. Platelets were activated by the addition of 0.1 U / mL thrombin (10602400001, Sigma-Aldrich, St. Louis, USA) and incubated at 37°C for 30 min. Gradient centrifugation was then performed as follows: 300 g for 10 min at 4°C to remove live cells; 1500 g for 15 min to remove dead cells; and 20,000 g for 30 min to remove cell debris. The supernatant from all gradient centrifugations was then collected. Finally, ultracentrifugation was performed at 100,000 g for 90 min, the supernatant removed, and the pellet resuspended in PBS to obtain the desired PDV solution.

[0072] (2) Preparation and structural confirmation of anti-ROS vesicles

[0073] Prepare 10 ml of PBS (pH 7.4) containing 10 mg / ml of PDVs, add 1 ml of 10 mM bis(sulfosuccinimidyl) suberate crosslinker, and vortex at room temperature for 30 minutes. Separate and purify the crosslinker-attached vesicles using a Sephadex column. Next, mix the purified crosslinker-attached vesicles in PBS with 10 mM glutathione and vortex at room temperature for 30 minutes. Separate and purify the vesicles using a Sephadex column to obtain a 10 mg / ml ROS-resistant vesicle solution.

[0074] (3) Preparation of thrombopoietin-loaded peptidomimetic engineered vesicles

[0075] An equal volume of 2 mg / ml thrombopoietin mimetic solution was mixed with the above-mentioned anti-ROS vesicle solution in a volume ratio of 1:1, and thrombopoietin mimetic engineered vesicles were prepared using a liposome extruder; the thrombopoietin mimetic engineered vesicles and free thrombopoietin mimetic were separated using a dextran gel column method.

[0076] (4) Preparation of thrombopoietin-loaded peptidomimetic engineered vesicle microneedles

[0077] Polyvinyl pyrrolidone K90 was selected as the needle tip matrix material of the microneedle, and a microneedle matrix solution of thrombopoietin-loaded peptoid engineered vesicles was prepared at a concentration of 1 mg / ml (the solution contained 15% (w / v) polyvinyl pyrrolidone K90); a mold (10×10 array, conical hollow, pore size 300 μm, pore depth 800 μm, provided by Taizhou Microchip Pharmaceutical Technology Co., Ltd.) was filled with centrifugation-air drying method (2000 g, 10 min) to prepare the microneedle tip of the thrombopoietin-loaded peptoid engineered vesicle; the microneedle base solution containing 15% (w / v) polyvinyl pyrrolidone K90 was dripped into the above-mentioned mold containing the needle tip and centrifuged again (2000 g, 10 min) and air-dried to obtain the final thrombopoietin-loaded peptoid engineered vesicle microneedle patch.

[0078] Example 4 Preparation of Superoxide Dismutase-Loaded Engineered Vesicle Hyaluronic Acid Microneedles

[0079] (1) Activated PDVs extraction

[0080] Whole blood was collected from C57BL / 6J mice and added to a 9:1 solution of acidic citrate dextrose. After centrifugation (150 g, 23°C for 15 min) to remove leukocytes, plasma-free platelets were collected by further centrifugation for 15 min and resuspended in 1 mL of PBS. Platelets were activated by the addition of 0.1 U / mL thrombin (10602400001, Sigma-Aldrich, St. Louis, USA) and incubated at 37°C for 30 min. Gradient centrifugation was then performed as follows: 300 g for 10 min at 4°C to remove live cells; 1500 g for 15 min to remove dead cells; and 20,000 g for 30 min to remove cell debris. The supernatant from all gradient centrifugations was then collected. Finally, ultracentrifugation was performed at 100,000 g for 90 min, the supernatant removed, and the pellet resuspended in PBS to obtain the desired PDV solution.

[0081] (2) Preparation and structural confirmation of anti-ROS vesicles

[0082] Prepare 10 ml of PBS (pH 7.4) containing 10 mg / ml of PDVs, add 1 ml of 10 mM bis(sulfosuccinimidyl) suberate crosslinker, and vortex at room temperature for 30 minutes. Separate and purify the crosslinker-attached vesicles using a Sephadex column. Next, mix the purified crosslinker-attached vesicles in PBS with 10 mM glutathione and vortex at room temperature for 30 minutes. Separate and purify the vesicles using a Sephadex column to obtain a 10 mg / ml ROS-resistant vesicle solution.

[0083] (3) Preparation of superoxide dismutase-loaded engineered vesicles

[0084] An equal volume of 1 mg / ml superoxide dismutase solution was mixed with the above-mentioned anti-ROS vesicle solution in a volume ratio of 1:1, and superoxide dismutase-loaded engineered vesicles were prepared using a liposome extruder; the superoxide dismutase-loaded engineered vesicles and free superoxide dismutase were separated using a dextran gel column method.

[0085] (4) Preparation of superoxide dismutase-loaded engineered vesicle microneedles

[0086] Hyaluronic acid and trehalose were selected as the needle tip matrix materials of the microneedle, and a microneedle matrix solution containing superoxide dismutase engineered vesicles was prepared at a concentration of 1 mg / ml (the solution contained 6% (w / v) hyaluronic acid and 5% (w / v) trehalose); the microneedle tip containing superoxide dismutase engineered vesicles was prepared by centrifugation-air drying method (2000g, 10min) through a mold (10×10 array, conical hollow, pore diameter 300μm, pore depth 800μm, provided by Taizhou Microchip Pharmaceutical Technology Co., Ltd.); the microneedle base solution containing 15% (w / v) polyvinylpyrrolidone K90 was dripped into the above-mentioned mold carrying the needle tip and centrifuged again (2000g, 10min) and air-dried to obtain the final superoxide dismutase engineered vesicle microneedle patch.

[0087] Example 5 Preparation of Catalase-Loaded Engineered Hyaluronic Acid Vesicle Microneedles

[0088] (1) Activated PDVs extraction

[0089] Whole blood was collected from C57BL / 6J mice and added to a 9:1 solution of acidic citrate dextrose. After centrifugation (150 g, 23°C for 15 min) to remove leukocytes, plasma-free platelets were collected by further centrifugation for 15 min and resuspended in 1 mL of PBS. Platelets were activated by the addition of 0.1 U / mL thrombin (10602400001, Sigma-Aldrich, St. Louis, USA) and incubated at 37°C for 30 min. Gradient centrifugation was then performed as follows: 300 g for 10 min at 4°C to remove live cells; 1500 g for 15 min to remove dead cells; and 20,000 g for 30 min to remove cell debris. The supernatant from all gradient centrifugations was then collected. Finally, ultracentrifugation was performed at 100,000 g for 90 min, the supernatant removed, and the pellet resuspended in PBS to obtain the desired PDV solution.

[0090] (2) Preparation and structural confirmation of anti-ROS vesicles

[0091] Prepare 10 ml of PBS (pH 7.4) containing 10 mg / ml of PDVs, add 1 ml of 10 mM bis(sulfosuccinimidyl) suberate crosslinker, and vortex at room temperature for 30 minutes. Separate and purify the crosslinker-attached vesicles using a Sephadex column. Next, mix the purified crosslinker-attached vesicles in PBS with 10 mM glutathione and vortex at room temperature for 30 minutes. Separate and purify the vesicles using a Sephadex column to obtain a 10 mg / ml ROS-resistant vesicle solution.

[0092] (3) Preparation of superoxide dismutase-loaded engineered vesicles

[0093] An equal volume of 1 mg / ml catalase solution was mixed with the above-mentioned anti-ROS vesicle solution in a volume ratio of 1:1, and catalase-loaded engineered vesicles were prepared using a liposome extruder; the catalase-loaded engineered vesicles and free catalase were separated using a dextran gel column method.

[0094] (4) Preparation of catalase-loaded engineered vesicle microneedles

[0095] Hyaluronic acid and trehalose were selected as the needle tip matrix materials of the microneedle, and a microneedle matrix solution of catalase engineered vesicles was prepared at a concentration of 1 mg / ml (the solution contained 6% (w / v) hyaluronic acid and 5% (w / v) trehalose); a mold (10×10 array, conical hollow, pore diameter 300 μm, pore depth 800 μm, provided by Taizhou Microchip Pharmaceutical Technology Co., Ltd.) was filled with centrifugation-air drying method (2000g, 10 min) to prepare microneedle tips loaded with catalase engineered vesicles; a microneedle base solution containing 15% (w / v) polyvinyl pyrrolidone K90 was dripped into the above-mentioned mold carrying the needle tip and centrifuged again (2000g, 10 min) and air-dried to obtain the final catalase engineered vesicle microneedle patch.

[0096] Experimental Example 1 Structural confirmation of anti-ROS vesicles

[0097] The specific function and structure of the anti-ROS vesicles were determined using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB)-UV spectroscopy and western blot. Finally, dynamic light scattering and transmission electron microscopy were used to examine the particle size and distribution, zeta potential, and morphology of the anti-ROS vesicles. The preparation process was optimized.

[0098] Experimental results:

[0099] Glutathione was used as an anti-ROS agent and bis(sulfosuccinimidyl) suberate crosslinker was used to bridge glutathione and activated PDVs through covalent crosslinking to synthesize anti-ROS vesicles. Figure 2 A), the particle size is about 146nm, and the Zeta potential is -15mV ( Figure 2 B). The product generated by the reaction between DTNB and the sulfhydryl group of glutathione has an absorbance of ( Figure 2 C). DTNB-UV spectroscopy experiments showed that conventional platelet vesicle solutions had no absorbance beyond 400 nm, while the purified anti-ROS vesicle solution had significant absorption beyond 400 nm ( Figure 2 C), proving that glutathione has been cross-linked to the vesicles. Western blot again proved that anti-ROS vesicles with glutathione attached to the surface have been obtained ( Figure 2 D).

[0100] Experimental Example 2 In vitro characterization of TPO-loaded engineered vesicles

[0101] Dynamic light scattering and transmission electron microscopy were used to examine the particle size and distribution, zeta potential, and morphology of the TPO-loaded engineered vesicles. A solution of TPO-loaded engineered vesicles was mixed with a 1% Tween solution (to disrupt the vesicles) at a volume ratio of 1:100, and the glutathione and TPO contents in the TPO-loaded engineered vesicles were measured using ELISA kits. Release experiments were conducted to investigate the drug release behavior of the TPO-loaded engineered vesicles and the changes in H2O2 concentrations, and to comprehensively analyze the anti-ROS activity of the TPO-loaded engineered vesicles.

[0102] Experimental results:

[0103] The above-mentioned anti-ROS vesicles were used as targeting carriers to prepare TPO-loaded engineered vesicles by extrusion method. The results showed that the vesicles were still in the shape of a saucer ( Figure 3 A), the particle size increased to about 165nm, and the Zeta potential was -23.5mV ( Figure 3 B).

[0104] 1 ml of TPO-loaded engineered vesicle solution (containing approximately 81 mg of glutathione and 213 μg of TPO) and 1 ml of TPO-loaded platelet-derived vesicle solution (containing approximately 210 μg of TPO) were added dropwise to 10 ml of 0.5 mM H2O2. After 2 h, the residual H2O2 content of the TPO-loaded platelet-derived vesicle was 46 ± 3.2% ( Figure 4 A). The electron microscopy photograph of the solution remaining after 2 hours showed that the vesicles were completely oxidized and broken ( Figure 4 B). In the TPO-loaded engineered vesicle solution group, only 9.1±2.8% of H2O2 remained. However, the electron microscopy photograph of the remaining solution after 2 hours showed that the TPO-loaded engineered vesicles maintained their complete morphology ( Figure 4C) This experiment shows that the TPO-loaded engineered vesicle solution not only has the function of resisting ROS, but also can maintain its own structural stability in the ROS solution.

[0105] Experimental Example 3 In vitro property evaluation and stability investigation of TPO-loaded engineered vesicle microneedles

[0106] The morphology and size of the microneedles were observed using a scanning electron microscope and a stereoscope. TPO-FITC vesicle microneedle patches were prepared according to the above preparation method, and the drug distribution at the needle tip was examined using a confocal microscope. The mechanical strength of the microneedles was evaluated using a tension-pressure tester. The drug loading at the needle tip was determined using an ELISA kit. The transdermal drug release ability of the microneedles was investigated using a Franz diffusion cell technique using a 1% (v / v) Tween solution as the release medium.

[0107] The soluble microneedles loaded with TPO engineered vesicles were dissolved, and the encapsulation efficiency of TPO engineered vesicles was measured by dextran gel column and ELISA method at 2 months, 4 months, and 6 months, respectively, to evaluate the long-term stability of the preparation.

[0108] Experimental results:

[0109] Scanning electron microscopy showed that the prepared microneedles were cone-shaped arrays ( Figure 5 A), stereomicroscope showed that the microneedle was about 812 μm long and had a bottom diameter of about 304 μm ( Figure 5 B). Confocal microscopy shows that the drug-loaded engineered vesicles are mainly concentrated at the needle tip ( Figure 5 C). The mechanical strength of the microneedle was determined by the pressure-tension test. When the deformation was 0.8 mm, the maximum pressure the needle could withstand was 0.12 N ( Figure 5 D). The encapsulation efficiency of the microneedle group did not change significantly within 30 days, while the encapsulation efficiency of the vesicle solution group decreased from the initial 82±5% to 8±4% within 30 days ( Figure 5 E). The results show that the stability of the vesicle preparation can be well maintained at room temperature. In vitro transdermal experiments show that drugs can be delivered through the skin via microneedles ( Figure 5 F)

[0110] Experimental Example 4: Study on the targeting of TPO-loaded engineered vesicles to the bone marrow hematopoietic system

[0111] SPF grade C57BL / 6J male mice (20±1g) were selected as model mice and sent to the cobalt source room for 60Models were established with a single whole-body irradiation of 6.5 Gy of Co irradiation (dose rate 60 cGy / min) (the conventional modeling dose). Blood concentrations of TPO were measured using ELISA kits in four groups: those injected subcutaneously with TPO, a microneedle patch containing TPO, a microneedle patch containing a physical mixture of TPO vesicles and glutathione, and a microneedle patch containing engineered TPO vesicles. Pharmacokinetic curves were plotted, and drug distribution in the bone marrow, heart, liver, spleen, lung, and kidney was calculated. Immunohistochemistry was used to further investigate the distribution of TPO in bone marrow tissue.

[0112] Experimental results:

[0113] TPO was barely detected in the bone marrow of healthy mice by immunohistochemical analysis. Figure 6 A). The drug was administered 2 hours after irradiation and mice were killed 24 hours later. Immunohistochemical analysis showed that TPO subcutaneous injection group ( Figure 6 B), TPO-loaded microneedle group ( Figure 6 C), TPO-loaded platelet-derived vesicle microneedle group ( Figure 6 D) Microneedle group containing a physical mixture of TPO-loaded platelet-derived vesicles and glutathione ( Figure 6 E) can deliver TPO to the bone marrow, and the drug content in the bone marrow tissue of the TPO-loaded engineered vesicle microneedle group is significantly greater than that of the above groups ( Figure 6 F), the dosage of each group was 100 μg / kg. This experiment demonstrated that the TPO-loaded engineered vesicle microneedles have the function of bone marrow-targeted drug delivery.

[0114] Experimental Example 5 Pharmacodynamic Evaluation of TPO-Loaded Engineered Vesicle Soluble Microneedles

[0115] (1) Establishment of animal model

[0116] SPF-grade C57BL / 6J male mice (20±1g) were selected as model mice and divided into 6 groups (20 in each group), including model control group, TPO subcutaneous injection group, anti-ROS vesicle microneedle patch, TPO microneedle patch, microneedle patch containing a physical mixture of TPO vesicles and glutathione, and TPO engineered vesicle microneedle patch. The treatment group was given a dose of 100μg / kg. The above 6 groups of mice were sent to the cobalt source room for treatment. 60 The whole body was irradiated with Co ray 6.5Gy (conventional modeling dose) once (dose rate 60cGy / min).

[0117] (2) Evaluation of the effect of TPO-loaded engineered vesicle microneedles on hematopoietic system reconstruction

[0118] Peripheral blood recovery: Blood was collected from the tail vein, and the changes in peripheral blood cells (such as white blood cells, platelets, red blood cells, etc.) of each group of animals (10 animals / group) within 30 days after irradiation were recorded using a peripheral blood cell counter.

[0119] Experimental results:

[0120] The number of peripheral leukocytes showed that the TPO injection group, the TPO-loaded platelet-derived vesicles and glutathione physical mixture microneedle group, and the TPO-loaded engineered vesicles microneedle group were significantly different from the model group starting from the fourth day after irradiation, while there was no statistical difference between the TPO-loaded platelet-derived vesicles microneedle group and the model group. In addition, there was also a significant difference between the TPO-loaded engineered vesicles microneedle group and the TPO injection group ( Figure 7 A). The platelet count also showed significant differences between the TPO injection group, TPO-loaded platelet-derived vesicle microneedle group, TPO-loaded platelet-derived vesicle and glutathione physical mixture microneedle group, and TPO-loaded engineered vesicle microneedle group compared with the model group. Similarly, the TPO-loaded engineered vesicle microneedle group also showed significant differences with the TPO injection group ( Figure 7 B) These experimental results preliminarily demonstrate that TPO-loaded engineered vesicle microneedles are more effective than TPO injections. Furthermore, they demonstrate that TPO-loaded engineered vesicles are more effective in treating BM-ARS than a simple physical combination of TPO-loaded platelet-derived vesicles and glutathione.

Claims

1. A vesicle, characterized in that The vesicle is composed of activated platelets, a cross-linking agent, an antioxidant and an anti-radiation substance, and is prepared through engineering.

2. The vesicle according to claim 1, characterized in that The engineering method involves covalently cross-linking antioxidants to the surface of PDVs to purify and obtain anti-ROS vesicles. Subsequently, the vesicles are used as carriers to prepare engineered vesicles encapsulating anti-acute radiation sickness drugs through extrusion technology as a drug delivery system.

3. The vesicle according to claim 1, characterized in that The antioxidant is selected from one or more of glutathione and its derivative glutathione ethyl ester, N-acetylcysteine, lipoic acid and its derivative dihydroxylipoic acid, superoxide dismutase, and glutathione peroxidase.

4. The vesicle according to claim 1, characterized in that The anti-radiation substance is selected from one or more of recombinant granulocyte colony stimulating factor, thrombopoietin and thrombopoietin peptidomimetic, amifostine, superoxide dismutase and catalase.

5. The microneedle patch according to claim 1, characterized in that The cross-linking agent is selected from one or more of bissuberate, succinimide ester-polyethylene glycol-maleimide.

6. A microneedle, characterized in that: Comprising the vesicle according to claim 1.

7. A microneedle patch, characterized in that The microneedle according to claim 6 is made of a polymer as a matrix material, the needle body length ranges from 100 μm to 1000 μm, the needle body base surface diameter ranges from 30 μm to 500 μm, and the area is 1 cm 2 Within the range of , 10 to 100 of the microneedles are arranged; The polymer is selected from one or more of polyvinyl alcohol, polylactic acid, silk fibroin, sodium carboxymethyl cellulose, chitosan, alginate, hyaluronate, polyvinyl pyrrolidone, methacrylic silk fibroin, methacrylic chitosan, methacrylic gelatin, and methacrylic hyaluronic acid.

8. A method for preparing the microneedle patch according to claim 7, characterized in that: include: (1) Preparation of anti-free radical vesicles Plasma-free platelets were collected and activated with thrombin; Then, gradient centrifugation was performed, and the precipitate was resuspended in PBS to obtain the desired activated platelet-derived vesicle solution. A PBS aqueous solution containing activated platelet-derived vesicles was prepared, and a crosslinker solution was added. The mixture was stirred at room temperature for 30 minutes, and the vesicles connected to the crosslinker were isolated and purified. Then, the purified PBS solution of the vesicles with the cross-linking agent is mixed with a PBS solution containing an antioxidant, stirred for 30 minutes, and separated and purified by a gel column method to obtain anti-free radical vesicles, wherein the activated platelet-derived vesicle solution has a density of 1 mg / ml to 5000 mg / ml of activated platelet-derived vesicles calculated based on the total protein amount, and the mass fraction of the activated platelet-derived vesicles in the cross-linking agent solution is 0.01% to 10%; (2) Preparation of engineered vesicles loaded with drugs for treating acute radiation sickness The anti-radiation sickness drug is added to the anti-free radical vesicle solution, and the drug-loaded engineered vesicles are prepared using a liposome extruder; the drug-loaded engineered vesicles and free drugs are separated using a gel column method; (3) Preparation of engineered vesicle microneedles loaded with anti-acute radiation sickness drugs Polymer 1 is used as the needle tip matrix material of the microneedle, and a microneedle matrix solution containing engineered vesicles loaded with anti-radiation sickness drugs is prepared; the microneedle tips of the drug-loaded engineered vesicles are prepared by mold pouring, centrifugation and air drying; a microneedle base solution containing polymer 2 is dripped into the mold loaded with the needle tip and centrifuged and air-dried again to obtain the final engineered vesicle microneedle patch, wherein the mass fraction of the anti-radiation drug in the microneedle matrix solution of the engineered vesicles loaded with anti-radiation sickness drugs is 0.1% to 10%, the mass fraction of polymer 1 in the microneedle matrix solution of the engineered vesicles loaded with anti-radiation sickness drugs is 5% to 50%, and the mass fraction of polymer 2 in the microneedle base solution is 5% to 50%.

9. Use of the microneedle according to any one of claims 1 to 6 in the preparation of a medical device for treating acute radiation sickness.

10. A medical device, characterized in that The microneedle comprises the microneedle according to any one of claims 1 to 6.

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