A method of lyophilizing stored platelets

Abstract

The application discloses a method for freeze-drying and storing platelets, and belongs to the technical field of biology. The method is characterized by the following steps: fusing liposomes loaded with bovine serum albumin with fresh platelets, and then performing freeze-drying to obtain platelet freeze-drying powder. The liposomes are used to efficiently and gently deliver the bovine serum albumin with the function of protecting platelets into the platelet cells. The freeze-drying powder can be stored for more than 30 days under low-temperature conditions, and the biochemical characteristics and biological activity of the rehydrated platelets are consistent with those of fresh platelets. Therefore, the method for freeze-drying and storing platelets is simple, time-saving and efficient, and has a huge application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for freeze-drying and preserving platelets. Background Technology

[0002] Over the past few decades, cell-based systems (such as those based on erythrocytes, platelets, stem cells, and dendritic cells) have become important drug carriers. These cells possess unique advantages, such as long circulation time, low immunogenicity, high targeted delivery specificity, and strong ability to penetrate natural physiological barriers. Among them, platelets produced by megakaryocytes have the natural ability to target damaged and inflamed tissues and circulating tumor cells, and various platelet-based drug delivery systems have been used in the treatment of metastatic and postoperative cancers.

[0003] With the deepening of research on platelet function, the application of platelets is no longer limited to achieving hemostasis and coagulation through platelet supplementation. Platelet components and derivatives can also be used in the diagnosis, treatment, and cosmetic procedures of clinical diseases. However, current conventional methods for preserving platelets have limited shelf life, and there is a lack of standardized and mature long-term preservation protocols. Furthermore, the protection of personalized platelet components has not yet received sufficient attention.

[0004] Existing platelet storage technologies include room temperature shaking, refrigeration at 2℃-6℃, low-temperature storage with dimethyl sulfoxide (DMSO) (-80℃), freeze-drying, PRP freezing (-80℃), and washed platelet storage. The ambient temperature shaking method is widely used for the preservation of blood for clinical transfusions, but this method has a short preservation time, with an effective period of only 5 days. Furthermore, with prolonged preservation, platelets not only face the risk of bacterial growth, but their physicochemical indicators and various functions also tend to decline. According to the article by Wang et al. (DOI: 10.13303 / j.cjbt.issn.1004-549x.2022.05.028), although platelets have a higher activation rate at low temperatures (<15℃), their morphology changes from disc-shaped to spherical and they extend pseudopodia. Consequently, they are phagocytosed after entering the human circulation but before they can perform their functions. Therefore, the 2℃~6℃ refrigeration method is not very suitable for platelet preservation. According to the article by Zhao et al. (DOI: 10.3969 / j...), ... According to ISSN 1671-8348.2019.01.001, before using the DMSO low-temperature (-80℃) preservation method for platelets, they need to be washed with a solution containing 0.9% sodium chloride, 0.2% glucose, and 40 mg / L inorganic phosphate (pH=5.0) to remove DMSO. This method is cumbersome to operate before use and increases the risk of platelet contamination. The PRP direct freezing preservation method is only used when the function of platelets needs to be preserved, but there are no requirements for platelet activity and morphology. The washed platelet preservation method is mainly used for the preservation and research of RNA in platelets. In contrast, freeze-dried platelets have low requirements for storage temperature, and freeze-dried platelets are easy to carry, easy to inactivate pathogens, can be quickly dissolved by adding rehydration solution, and can be used directly after rehydration. According to the article by Wei et al. (DOI: 10.13303 / j.cjbt.issn.1004-549x.2018.04.007), the quality control of various preservation processes, such as the type and concentration of lyophilization protectant, the freezing process, and rehydration conditions, will affect the recovery rate and function of platelets after rehydration. There is a lot of research on this topic, but there is still no mature preservation method.

[0005] Therefore, there is an urgent need for a convenient, efficient, and mature method for freeze-drying platelets. Summary of the Invention

[0006] Platelets are used in surgical hemostasis and the treatment of diseases such as aplastic anemia, thrombocytopenia, hemophilia, cirrhosis, myelodysplastic syndrome, acute leukemia, and lymphoma; for wound healing, including acute wounds, chronic wounds, and burn wounds; for the treatment of inflammation, including tendinitis, bursitis, and rheumatoid arthritis; and for the repair of soft tissue (muscles, tendons, ligaments, menisci) injuries and nerve function damage. The technical problem this invention aims to solve is to overcome the shortcomings of existing platelet freeze-drying technologies, such as low recovery rate, low aggregation rate, poor activity, and easy breakage of platelets. Therefore, this invention provides a method for long-term storage of freeze-dried platelets, belonging to the field of biotechnology. The method involves fusing liposomes loaded with bovine serum albumin with fresh platelets, followed by freeze-drying to prepare freeze-dried platelet powder. The liposomes efficiently and gently deliver bovine serum albumin, which has platelet-protective functions, into the platelet cells. The freeze-dried powder can be stored at low temperatures for more than 30 days, and the rehydrated platelets exhibit the same biochemical characteristics and biological activity as fresh platelets. In summary, this invention provides a simple, time-saving, and efficient method for freeze-drying and preserving platelets, with significant application potential.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] On one hand, the present invention provides a lyophilization protectant for platelets, the lyophilization protectant comprising liposomes.

[0009] The liposomes function to deliver other protective substances from the lyophilized protectant into platelets. In some embodiments, the substance is BSA, and intracellular BSA provides stronger protection to platelets than extracellular BSA. In some embodiments, the bovine serum albumin has a molecular weight of 68,000; in use, 50 g of BSA is dissolved in PBS buffer, with the solvent pH at fusion being 5.7-7.4, preferably 7.4.

[0010] Further, the liposomes comprise any one or more of DOPE (dioleoylphosphatidylethanolamine), cholesterol, DPPC (dispalmitoylphosphatidylcholine), DMPC (dimyristicoylphosphatidylcholine), DOSPA (dioleoyl-3-succinimideoxypropyltrimethylammonium), DLin-MC3-DMA, DOTMA (dioleoyloxypropyltrimethylammonium), DOTAP (dioleoyloxypropyltrimethylammonium propane), and DSPE-PEG2000 (distearylphosphatidylethanolamine-polyethylene glycol 2000).

[0011] Furthermore, the liposomes are composed of DMPC, DOTAP, and DSPE-PEG2000; DMPC is a neutral phospholipid commonly used to form lipid bilayer structures and provide stability; DOTAP is a cationic lipid that can impart a positive charge to the liposomes, facilitating their binding to negatively charged cell membranes, nucleic acids, or proteins; DSPE-PEG2000 is a PEG-modified phospholipid that can increase the circulation time of the liposomes and reduce their clearance by the immune system.

[0012] In some embodiments, the molar ratio of DMPC, DOTAP and DSPE-PEG2000 is (2-10):(0.5-3):(0.05-2), preferably 5:1:0.1.

[0013] Furthermore, the freeze-drying protectant also comprises any one or more of bovine serum albumin (BSA), HAS, gelatin, casein, trehalose, and platelet-free plasma.

[0014] In some embodiments, the lyophilization protectant comprises the aforementioned liposomes, bovine serum albumin, trehalose, and platelet-free plasma. Trehalose can form an amorphous glassy state (rather than ice crystals) during freezing, reducing mechanical damage to cell membranes or biomolecules (such as proteins and lipids) caused by ice crystals, and is non-immunogenic, making it suitable for clinical-grade formulations. Albumin and fibrinogen in platelet-free plasma reduce denaturation during lyophilization by coating the surface of biomolecules; furthermore, natural components in plasma (such as growth factors and amino acids) can maintain cellular metabolic balance and buffer pH fluctuations; colloidal components in plasma can inhibit ice crystal growth; and autologous plasma poses no risk of immune rejection.

[0015] Further, the mass ratio of the liposomes to bovine serum albumin is 20:(50-200), preferably 20:100.

[0016] In some methods, the mass ratio of liposomes loaded with bovine serum albumin to platelets is (1-10):(1-10), preferably 1:1.

[0017] On the other hand, the present invention provides the use of liposomes in the preparation of formulations that prolong the storage time of platelets and / or improve the performance of lyophilized platelets, wherein the liposomes are as described above, and the performance includes, but is not limited to, platelet morphology, adhesion, recovery rate, survival rate, and activation rate.

[0018] In some methods, freeze-dried platelet powder can be stored at low temperatures for more than 30 days, and the rehydrated platelets still exhibit good biochemical activity (activation rate and survival rate).

[0019] On the other hand, the present invention provides a method for freeze-drying platelets, wherein the method refers to mixing the freeze-drying protectant with platelets, and then freeze-drying the platelets to prepare freeze-dried platelet powder.

[0020] On the other hand, the present invention provides a method for freeze-drying protected platelets, wherein a protective substance is delivered into the platelet, and the platelet is then freeze-dried to obtain freeze-dried platelet powder.

[0021] The protective substance refers to a substance that can protect platelets from damage caused by the freeze-drying process and / or is beneficial for the long-term preservation of platelets after freeze-drying.

[0022] In some embodiments, the protective substance includes, but is not limited to, bovine serum albumin, human serum albumin, casein, gelatin, ferritin, and heat shock proteins.

[0023] In some specific embodiments, the protective substance is bovine serum albumin (BSAL); in some methods, the higher the BSAL content, the stronger the protective effect on platelets, wherein the BSAL content is 1 μg BSAL / 10 8 One platelet count ~ 50 μg bovine serum albumin / 10 8 Platelets; preferably 6 μg bovine serum albumin / 10 8 One platelet count ~ 8 μg bovine serum albumin / 10 8 Platelets, more preferably, 6 μg bovine serum albumin / 10 8 One platelet.

[0024] Existing technologies rely on the protective substance to function outside the cell, as the physicochemical processes of the protective substance within the cell cannot be guaranteed. This invention hypothesizes and demonstrates that the protective substance can be delivered into cells through various means, which helps protect the biochemical function of platelets and prolongs the storage time of freeze-dried platelets.

[0025] Furthermore, the protective substance is delivered into the platelets using one or more of the following methods: permeabilizers, electroporators, liposomes, and nanoparticles.

[0026] In some specific embodiments, liposomes are used to deliver the protective substance.

[0027] Specifically, the steps of the method are as follows:

[0028] (1) Empty liposomes are prepared by thin-film hydration, ultrasonic dispersion, reverse evaporation, solvent injection, detergent removal, freeze-thaw, or microfluidic methods (preferably thin-film hydration). The liposomes are composed of dimyristoyl phosphatidylcholine (DMPC), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), and distearate phosphatidylethanolamine-polyethylene glycol maleimide (DSPE-PEG2000), with a preferred molar ratio of (3-5):(0.8-1.5):(0.05-0.15), and more preferably 5:1:0.1.

[0029] (2) Bovine serum albumin was loaded into empty liposomes using thin-film hydration and ultrasonic treatment to form liposomes loaded with bovine serum albumin.

[0030] (3) Platelets are obtained by differential centrifugation, wherein the platelets are derived from living mammalian cells, preferably rodents or primates, more preferably mice, rabbits, or humans, and even more preferably mice.

[0031] (4) The liposomes loaded with bovine serum albumin prepared in step (2) and the platelets prepared in step (3) are placed in a trehalose solution, mixed directly for 2 hours, and then freeze-dried. The freeze-drying process of the platelets is carried out in a mixed solution of 70% trehalose solution and 30% anemic platelet plasma.

[0032] In some methods, platelets prepared by the above-mentioned freeze-drying technology are stored at 4°C for a long period of time.

[0033] In some methods, lyophilized platelet powder is resuscitated (i.e., rehydrated) before being used clinically, wherein the resuscitation conditions are to resuscitate lyophilized platelets stored at 4°C using PBS solution preheated to 37°C.

[0034] The beneficial effects of this invention include:

[0035] 1. Liposomes loaded with bovine serum albumin are fused with live platelets. The bovine serum albumin in the liposomes after fusion with platelets can reduce or prevent damage to platelets caused by freeze-drying or rehydration during the freeze-drying process. Furthermore, platelets that have been freeze-dried and stored for a long time (>30 days) can be revived under mild conditions to maximize the preservation of the original physiological and biochemical characteristics and biological activities of platelets.

[0036] 2. A lyophilization protectant comprising liposomes is provided, wherein the lyophilization protectant is capable of delivering a protective substance into the platelet, thereby maximizing the protective effect of the substance;

[0037] 3. This invention uses bovine serum albumin liposomes and live platelets to prepare a platelet freeze-drying system, which is simple, time-saving, and efficient.

[0038] 4. The freeze-dried platelets prepared by this invention do not have high requirements for storage temperature, and the freeze-dried platelets are easy to carry and easy to inactivate pathogens; they can be quickly dissolved and rehydrated by adding rehydration solution, and the rehydrated platelets can be used directly, and their activity is comparable to that of fresh platelets. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The image is a confocal microscope image of the platelets fused with liposomes containing FITC-labeled DSPE-PEG2000 on the surface and Cy5-labeled bovine serum albumin as described in Example 2. The scale bar is 10 micrometers and the n=3.

[0041] Figure 2 The statistics of platelet recovery rate and platelet activation rate after freeze-drying using the method provided by the present invention as described in Example 2, n=4;

[0042] Figure 3 For Example 2, the collagen adhesion ability of platelets freeze-dried using the method provided by the present invention under different activation conditions was tested, n=3;

[0043] Figure 4 For the hemostatic ability test of platelets freeze-dried using the method provided by the present invention as described in Example 2, n=6;

[0044] Figure 5 The effect of intracellular BSA content on platelet recovery rate, survival rate and activation rate as described in Example 3, n=4.

[0045] Figure 6 The effect of the low-temperature storage time described in Example 7 on the survival rate and activation rate of platelets after freeze-drying using the method provided by the present invention is n=4. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; each experimental group has more than 3 replicates.

[0048] Example 1: A method for freeze-drying platelets

[0049] The method for preparing platelets for lyophilization includes the following steps:

[0050] 1.1 Platelets were separated from mouse whole blood by differential centrifugation.

[0051] Under anesthesia, orbital venous blood was collected from mice and added to centrifuge tubes containing an ACD anticoagulant (16 mmol / L citric acid, 90 mmol / L sodium citrate, 16 mmol / L sodium dihydrogen phosphate, and 142 mmol / L glucose, prepared, filtered through a 0.22 μm filter, aliquoted, and stored at 4°C). The volume ratio of whole blood to ACD anticoagulant was approximately 5:1. The tubes were centrifuged at 150 g for 10 minutes at room temperature, and the supernatant plasma rich in platelets was collected. This was then centrifuged at 1000 g for 5 minutes to separate platelets. Approximately 5–7 × 10⁶ platelets were obtained per mL of whole blood. 8 10 platelets (preferably 6 × 10⁶ in this embodiment) 8 ).

[0052] 1.2 Preparation of bovine serum albumin-liposomes by thin-film hydration method

[0053] Dimyristic phosphatidylcholine (DMPC, Sigma-Aldrich, catalog number 850345P-1G), (2,3-dioleoyl-propyl)trimethylamine (DOTAP, Aladdin, D292990-50mg), and distearate phosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000, Yusi Pharmaceutical, YS-DM201) were dissolved in anhydrous ethanol at a molar ratio of 5:1:0.1. 355 μL of anhydrous ethanol was used to dissolve every 20 mg of total lipids. Specifically, 12.8 mg DMPC, 3.5 mg DOTAP, and 1.44 mg... DSPE-PEG2000 was dissolved in 355 μL of anhydrous ethanol to obtain a lipid-ethanol solution. The solvent (anhydrous ethanol) in the 355 μL lipid-ethanol solution was evaporated by slowly rotating the rotary evaporator (Dalong Instruments). After the water pump evacuated the vacuum, 1 mL of PBS solution (pH=7.4) containing 50 g / L bovine serum albumin (Solepro, A8010-10 g) was added to initially obtain bovine serum albumin-liposomes. The above liposome mixture was hydrated at 37 °C for 10 min. After the liposomes were briefly sonicated in a 37 °C water bath, the liposomes were squeezed 10 times using a polycarbonate membrane (Whatman, 10417004) with a pore size of 200 nm.

[0054] 1.3 Preparation of bovine serum albumin-liposome platelet solution using cationic liposome fusion method

[0055] The 25 μL of liposomes loaded with bovine serum albumin prepared in step 1.2 were mixed with 25 μL of a total volume of 1×10⁻⁶. 8 Platelets were fused for 2 hours in PBS solution (pH=7.4) containing 50 mM trehalose (Solepro, G8570-100g) sterilized through a 0.22 μm filter (Millipore, SLGPR33RB). Subsequently, the mixture was centrifuged at 1,000g for 5 min, and the supernatant was discarded to remove unreacted liposomes. 100 μL of a mixture of 50 mM trehalose solution and anemic platelet-plasma (APP) at a mass ratio of 7:3 was added. This mixture was then placed at -80°C and frozen for 1 hour. The anemic platelet-plasma refers to the supernatant solution obtained after centrifugation at 1,000g for 5 min in step 1.1, after separating the platelets. It should be noted that in this embodiment, the platelet-to-BSA mass ratio is 1:1; specifically, 6 μg bovine serum albumin corresponds to 10... 8 One platelet.

[0056] 1.4 Freeze-dried platelet solution

[0057] The platelet solution frozen in step 1.3 was quickly transferred to a freeze dryer (LABCONCO, 70020) and freeze-dried for 12 hours under conditions of cold trap temperature below -40°C and vacuum degree below 20 Pa to obtain platelet freeze-dried powder. The powder was then sealed and transferred to a 4°C freezer for storage and rehydrated before use.

[0058] 1.5 Platelet rehydration

[0059] The lyophilized platelet powder prepared in step 1.4 was added to Tyrode's solution (Yuan Ye, R27812-500mL) that was preheated at 37°C and had the same volume as the platelet solution before lyophilization (i.e., 100μL) until the solid disappeared and a turbid liquid was formed. The solution was centrifuged at 1,000g for 5 minutes and the platelets were collected for subsequent performance tests.

[0060] Example 2: Performance testing of freeze-dried platelets

[0061] The freeze-drying method provided by this invention is designed based on the idea that delivering a protective substance (preferably bovine serum albumin in this embodiment) into the platelet can improve the protective effect on the platelet. In order to explore the overall effect of the freeze-drying method, this embodiment conducts performance tests on the freeze-dried and rehydrated platelets prepared by the method described in Example 1.

[0062] 2.1 Platelet morphology observation

[0063] To visually verify whether liposomes can deliver BSA into platelets as expected, this embodiment observed the fusion process of liposomes and platelets under a fluorescence microscope. The specific operation steps are as follows: FITC-conjugated DSPE-PEG2000 (DSPE) was used as the fluorescent material to label the liposome membrane, and Cy5-conjugated bovine serum albumin was used as the encapsulating material inside the liposomes. Labeled bovine serum albumin-liposomes were prepared according to step 1.2; 25 μL of the liposomes were then mixed with 25 μL of 1×10⁻⁶ BSA. 8 Platelets were fused for 2 hours in PBS solution (pH=7.4) containing 50 mM trehalose (Aladdin) and sterilized through a 0.22 μm filter membrane (Millipore, SLGPR33RB). Then, after washing twice with 100 μL of PBS solution (pH=7.4) containing 1 μM PGE1 (alprostaglandin E1, Sigma-Aldrich, 900100P-1MG), a drop of bovine serum albumin-liposome platelet solution was placed on a glass-bottomed culture dish. The morphology of the fused platelets was observed under a confocal microscope (Nikon AX), and the fluorescence gray values ​​emitted by the FITC and Cy5 groups were quantified using ImageJ software.

[0064] like Figure 1As shown, most Cy5-labeled bovine serum albumin (BSAL) is present inside platelets, while FITC-labeled DSPE is distributed on the platelet surface. This result demonstrates the fusion process of BSAL-liposomes with platelets and indicates that BSAL was successfully delivered into the platelets by liposomes. Furthermore, the grayscale analysis results are consistent with those observed under a fluorescence microscope.

[0065] 2.2 Platelet Count Statistics

[0066] Platelet counts were performed using a hemocytometer (Qiujing Biochemical Reagent Instrument Co., Ltd.) in 100 μL platelet solutions before and after lyophilization. The platelet solution before lyophilization was the one prepared in step 1.3, and the platelet solution after lyophilization was the one prepared in step 1.5. Platelet recovery rate was calculated as: Platelet recovery rate = (Number of platelets after lyophilization / Number of platelets before lyophilization) × 100%. Furthermore, the lyophilized platelets were stained with APC-conjugated CD62P antibody (Biolegend, 148304). The platelets were resuspended in 100 μL of cell staining buffer (Biolegend, 420201), 1.25 μL of APC-conjugated CD62P antibody was added, and the platelets were stained in the dark at room temperature for 15 minutes, centrifuged at 1000g for 5 minutes, washed once with cell staining buffer, resuspended in 200 μL of staining solution, and then analyzed by flow cytometry (BD FACS). Celesta), where platelet activation rate = number of CD62P positive platelets / total number of platelets involved in flow cytometry analysis × 100%. This example includes two controls: platelets are lyophilized and preserved using phosphate buffer or a 50mM trehalose solution containing the same concentration of bovine serum albumin but without liposomes instead of the bovine serum albumin-liposome solution (i.e., bovine serum albumin is outside the platelet cells). The specific experimental steps are the same as described in Example 1.

[0067] The results show that ( Figure 2 Compared to phosphate buffer, a protective agent containing bovine serum albumin (BSA) Figure 2Both the trehalose solution containing the same concentration of albumin and the method described above significantly improved platelet recovery and reduced platelet activation after lyophilization and rehydration. The lower activation rate indicates that lyophilization preserves platelet activity to a greater extent, ensuring normal platelet function during subsequent use. This experimental result demonstrates that bovine serum albumin (BSA) does indeed protect platelets. Furthermore, compared to BSA's extracellular effect, delivering albumin intracellularly via liposomes significantly improved the overall physicochemical properties (recovery rate and activation rate) of lyophilized and rehydrated platelets, indicating that BSA plays a more effective protective role within platelets. In summary, during lyophilization, compared to conventional protectants (phosphate buffer and trehalose solution containing only BSA), the BSA-liposome solution provides higher and gentler protection for platelets, specifically manifested in a higher platelet count and lower activation rate after lyophilization using the BSA-liposome solution. This is because trehalose readily enters platelets, while large molecules like BSA have difficulty doing so, resulting in a higher intracellular trehalose / (intracellular trehalose + intracellular albumin) ratio. Studies have shown that a higher value corresponds to a lower glass transition temperature, and increasing the glass transition temperature of the system is beneficial for lyophilization. Therefore, delivering BSA into platelets can enhance the lyophilization protection effect.

[0068] 2.3 Platelet Adhesion Performance Test

[0069] Platelet adhesion is a key step in the hemostatic function of platelets during vascular injury, referring to the ability of platelets to adhere to the damaged blood vessel wall or other foreign body surfaces. Adequate adhesion is crucial for maintaining normal physiological function. Therefore, to investigate whether lyophilized platelets still retain normal biological function, this method used 100 μL of 50 μM ADP (adenosine diphosphate, Solarbio, A0180) and 100 μL of 0.5 U / mL thrombin (Maclean, T832140-1KU) to analyze platelets with a total platelet count of 1×10⁻⁶. 8Fresh and lyophilized platelets were activated, followed by a platelet collagen adhesion assay. Specifically, 1 mL of rat tail collagen type I solution (Solepro, C8062, 2.0 mg / mL) was added to a 24-well plate and incubated overnight at 4°C. Fresh or lyophilized platelets were pre-stained with 100 μL of 25 μg / mL Cy5-NHS (Meilun Biotechnology, MB12193-1) for 10 min, followed by washing twice with PBS solution containing 1 μM PGE1. The stained or lyophilized platelets were then resuspended in 2% BSA solution and incubated at room temperature for 30 min to block non-specific adsorption. Platelets were then treated with PBS solution containing 0.5 U / mL thrombin and PBS solution containing 50 μM ADP for 5 min before being added to collagen-coated 24-well plates (5 × 10⁻⁶ wells). 7 Platelets were incubated per well for 30 seconds. After washing with PBS, the bound platelets were detected under a confocal microscope (Nikon) and quantified using ImageJ software. The fresh platelets were those obtained in step 1.1, and a negative control (i.e., platelets activated using PBS) was also included.

[0070] Fluorescence intensity analysis results showed that ( Figure 3 Regardless of whether ADP or thrombin is used to activate platelets, fresh platelets and lyophilized platelets exhibit similar adhesion behavior on collagen-coated cell culture plates. Furthermore, when thrombin is used as the activator, the lyophilized platelets emit stronger fluorescence, indicating better adhesion under these conditions. In conclusion, the collagen adhesion experiment demonstrates that platelets lyophilized using the method described in this invention retain their original biological activity.

[0071] 2.4 Coagulation Test

[0072] The primary function of platelets is blood clotting. To further investigate whether freeze-dried platelets still retain normal biological functions, 1×10⁻⁶ platelets were separately... 8 Freeze-dried platelets and fresh platelets were intravenously injected into healthy 10-week-old female Balb / c mice weighing approximately 20g. After 30 minutes, the tails of the mice were cut off 3mm from the tip, and the tails of the mice with the severed tail tip were placed in PBS solution preheated at 37°C. The bleeding time of the mouse tail was recorded, and the time point at which no obvious bleeding was observed in the mouse tail and this state could be maintained for 1 minute was set as the clotting time.

[0073] Depend on Figure 4 It can be seen that the clotting time of freeze-dried platelets and fresh platelets is approximately 150 seconds, both of which significantly reduced the tail bleeding time in mice.

[0074] In summary, this embodiment demonstrates that the number and activation rate of platelets rehydrated after freeze-drying in Example 1 are superior to those of conventional methods, and their adhesion and coagulation properties are consistent with those of fresh platelets. These results collectively illustrate the superiority of the freeze-drying preservation method provided by this invention (i.e., using bovine serum albumin-liposomes to freeze-dry platelets together), providing a new option for platelet preservation and showing great application potential.

[0075] Example 3: Effect of BSA content on platelet performance

[0076] Examples 1 and 2 confirmed that BSA can be delivered into platelet cells using liposomes, and that intracellular BSA provides better protection. Specifically, compared to protectants containing only BSA, the bovine serum albumin-liposome protectant maintains platelet morphology and number after freeze-drying while preserving platelet physiological function. Bovine serum albumin is one of the main components of this protectant. Therefore, this method explored the relationship between intracellular BSA content and platelet survival rate, platelet recovery rate, and platelet activation rate. Specifically, the platelet-to-BSA mass ratio was adjusted to 1:0.2 (i.e., 2 μg BSA / 10 μL). 8 (platelets), 1:0.5 (i.e., 4μg BSA / 10) 8 (platelets), 1:1 (i.e., 6μg BSA / 10) 8 (platelets), 1:2 (i.e., 8μg BSA / 10) 8 (platelets), 1:5 (i.e., 10 μg BSA / 10 8 Platelets were collected and fused. The platelet recovery rate and platelet activation rate were calculated using the same methods as described in Example 2. Simultaneously, calcein (Beyotime, C2015M) was used to stain the lyophilized platelets. The calcein stock solution in the Beyotime kit was diluted 1,000 times with PBS solution as the staining working solution. The lyophilized platelets were then resuspended in 100 μL of the working solution and stained for 15 minutes. Flow cytometry analysis was then performed. Platelet survival rate = number of calcein-positive platelets / total number of platelets analyzed × 100%.

[0077] The results show that ( Figure 5 Platelet survival rate and recovery rate were generally positively correlated with the BSA content, while platelet activation rate was negatively correlated with the BSA content, indicating that within a certain range (1 μg BSA / 10⁻⁶), platelet survival rate and recovery rate were positively correlated with the BSA content. 8 10 μg BSA per platelet count 8 The higher the BSA content (per platelet), the better the protective effect of bovine serum albumin-liposome solution on platelets; 6 μg / 10⁻⁶ is preferred. 8 BSA per 10 μg platelets BSA / 108 Platelets, of which BSA content is greater than 6 μg / 10 8 When platelet counts are small, platelet performance is not significantly different, so 6 μg / 10 is preferred. 8 One platelet, meaning the mass ratio of platelets to BSA is 1:1.

[0078] Example 4: Effects of BSA delivery method on platelet performance

[0079] Examples 2-3 demonstrate that liposomes can deliver BSA into platelets, thereby enhancing the protective effect of BSA on lyophilized platelets. The higher the BSA content within the platelets, the better the protective effect. Because BSA itself has a large molecular weight (approximately 66 kDa) and is uncharged, it is difficult to cross the cell membrane via passive diffusion. Therefore, the delivery of bovine serum albumin into cells usually requires specific methods. Currently, besides liposomes, other methods for delivering BSA into cells include direct mixing, electroporation, and nanoparticle delivery. However, the efficiency of these methods in delivering BSA and whether they affect platelet performance are still unclear. Therefore, this example uses different methods to deliver 8 μg of FITC-labeled BSA into 1 × 10⁻⁶ platelets. 8 In a sample of platelets, the delivery efficiency, platelet survival rate, recovery rate, and activation rate were determined after freeze-drying and rehydration. The experimental procedures are as follows:

[0080] 4.1 Direct Mixing Method

[0081] Before the experiment, the platelets were washed twice with PBS and then replaced with serum-free culture medium. The platelet density was 1×10⁻⁶. 9 Platelets / mL; Dissolve BSA in serum-free culture medium to a final concentration of 50 mg / mL, add Saponin to a final concentration of 0.05%; Add the above BSA mixture to platelets, incubate at 37°C for 10-30 minutes (preferably 15 minutes in this example), discard the mixture, wash platelets 3 times with PBS to remove residual Saponin permeabilizing agent, replenish with complete culture medium, and continue culturing for 1 hour to restore cell membrane integrity.

[0082] 4.2 Electroporation

[0083] Platelets were washed twice with pre-cooled electroporation buffer (Bio-Rad Gene Pulser) and resuspended to a density of 1×10⁻⁶. 9Cells / mL; Dissolve BSA in electroporation buffer to a final concentration of 50 mg / mL; Mix cell suspension and BSA solution at a 1:1 volume ratio (final volume 100 μL); Transfer the mixture to an electroporation cuvette, avoiding air bubbles; Electroporation parameters: voltage 150 V, capacitance 500 μF, pulse duration 10 ms; Start the electroporator (Bio-Rad Gene Pulser Xcell); Apply a single pulse; Immediately after electroporation, transfer the cell suspension to preheated complete culture medium and incubate at 37°C for 10 minutes; After completion, seed cells onto a culture plate and incubate at 37°C for 4 hours.

[0084] 4.3 Nanoparticle Delivery Method

[0085] Dissolve 50 mg PLGA in 5 mL of dichloromethane, and dissolve 100 mg BSA in 2 mL of 1% PVA aqueous solution (aqueous phase). Slowly add the aqueous phase to the organic phase and sonicate (ice bath, 100 W, 2 min) to form a W / O promulgation. Pour the promulgation into 50 mL of 0.3% PVA aqueous solution and stir magnetically for 2 hours to evaporate the organic solvent. Centrifuge (15,000 rpm, 20 min) to collect the nanoparticles, wash three times with ultrapure water, freeze-dry and store at 4 °C. When reconstituted, disperse with PBS using sonication. Co-incubate 100 μg / mL PLGA-BSA nanoparticles with cells for 8 hours.

[0086] 4.4 Liposome Delivery Method

[0087] Same as described in Example 1.

[0088] Platelets prepared by the above four methods were freeze-dried and rehydrated, with the specific steps as described in Example 1; the methods for detecting platelet viability, recovery rate and activation rate were the same as described in Examples 2 and 3; the delivery efficiency was determined by flow cytometry, and the calculation formula was: delivery efficiency = number of FITC-positive platelets / total number of platelets involved in flow cytometry analysis × 100%; the results are shown in Table 1.

[0089] Table 1. Delivery efficiency of different delivery methods and their impact on platelet performance after freeze-drying.

[0090] Delivery method Delivery efficiency Survival rate Recovery rate Activation rate Direct mixing method 48％ 65％ 48％ 30％ Electroporation 96％ 40％ 35％ 40％ Nanoparticle delivery method 75％ 85％ 60％ 15％ Liposome delivery method 95％ 98％ 78％ 7％

[0091] Because platelets have a weak phagocytic capacity, direct mixing and nanoparticle delivery methods are not very efficient, resulting in a decrease in intracellular BSA content and consequently a significant decline in platelet performance after lyophilization. Conversely, although electroporation has a delivery efficiency comparable to liposomes, it causes greater cell damage and affects platelet function. Liposomes, on the other hand, have the advantage of being a gentler process and can efficiently deliver BSA into platelets via membrane fusion, thereby maintaining the activity and physiological function of lyophilized platelets. Therefore, liposomes are the preferred method for delivering BSA into platelets.

[0092] Example 5: Effects of liposome delivery on platelet performance

[0093] Example 4 demonstrates that liposomes can gently and efficiently deliver protective BSA to platelets, thereby maximizing the preservation of the physicochemical properties of lyophilized platelets. Furthermore, besides BSA, HAS, casein, and gelatin are also commonly used lyophilization protective components. Therefore, this example uses liposomes to deliver these chemicals to platelets and evaluates their protective effects on platelets. BSA, HAS, casein, and gelatin were all purchased from Sigma-Aldrich, all FITC-labeled, and the total amount was 8 μg. The total platelet count was 1 × 10⁻⁶. 8 The remaining experimental steps are the same as those described in Examples 1 to 4, and the specific experimental results are shown in Table 2.

[0094] Table 2. Effects of different protective components on platelet properties after freeze-drying

[0095]

[0096]

[0097] The results show that liposomes can efficiently deliver the above four compounds to platelets, but the degree of protection of platelets varies among different substances. In general, BSA is the most effective, followed by HAS. Therefore, the liposome-BSA combination is preferred to protect platelets from damage during freeze-drying.

[0098] Example 6: Effects of liposome components on platelet performance

[0099] Example 5 demonstrated that the liposome-BSA combination had the best protective effect on platelets. In order to further improve the efficiency of BSA delivery by liposomes and the protective effect of the combination, the composition of the liposomes was optimized in this example. The experimental steps were the same as those in Examples 1 to 4, and the results are shown in Table 3.

[0100] Table 3. Effects of liposome components on platelet properties after lyophilization

[0101]

[0102] As shown in Table 3, when the added neutral phospholipids (DOPE, cholesterol, DPPC, DMPC): cationic lipids (DOSPA, DLin-MC3-DMA, DOTMA, DOTAP):DSPE-PEG2000 molar ratio is the same, the liposomes composed of DMPC, DOTAP, and DSPE-PEG2000 have the highest efficiency in delivering BSA and this combination best protects platelets from damage caused by the freeze-drying process, so that the rehydrated platelets after freeze-drying still maintain the best overall performance. Therefore, the combination of liposomes prepared from DMPC, DOTAP, and DSPE-PEG2000 with BSA is preferred for use in freeze-dried platelet preservation.

[0103] Example 7: Effect of storage time on platelet performance

[0104] This embodiment investigated the changes in platelet activation rate and survival rate after storing the freeze-dried platelet powder described in Example 1 at 4℃ for different times and after rehydration. Specifically, 50 mg of freeze-dried platelet powder prepared in step 1.4 was stored at 4℃ in the dark for 0, 10, 20 and 30 days respectively. After storage, the freeze-dried platelet powder was rehydrated according to step 1.5, and then the platelet activation rate and survival rate were measured according to the steps described in Examples 2 and 3.

[0105] The results show that ( Figure 6 The survival rate of platelets that were freeze-dried at 4°C for 30 days was still higher than 90%, and the activation rate was still lower than 10%. These results indicate that the freeze-dried platelet powder prepared by the method provided in this invention can be stored for a long time, and the platelets can still maintain high activity after rehydration.

[0106] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A lyophilization protectant for platelets, characterized in that, The lyophilization protectant contains liposomes.

2. The freeze-drying protectant as described in claim 1, characterized in that, The liposomes comprise any one or more of DOPE, cholesterol, DPPC, DMPC, DOSPA, DLin-MC3-DMA, DOTMA, DOTAP, and DSPE-PEG2000.

3. The freeze-drying protectant as described in claim 2, characterized in that, The liposomes are composed of DMPC, DOTAP and DSPE-PEG2000; the molar ratio of DMPC, DOTAP and DSPE-PEG2000 is (2-10):(0.5-3):(0.05-2).

4. The freeze-drying protectant as described in claim 1, characterized in that, The freeze-drying protectant also includes any one or more of bovine serum albumin, HAS, gelatin, casein, trehalose, and platelet-free plasma.

5. The freeze-drying protectant as described in claim 4, characterized in that, The mass ratio of the liposomes to bovine serum albumin is 20:(50-200).

6. The use of liposomes in the preparation of formulations that prolong platelet storage time and / or improve platelet performance after lyophilization, characterized in that, The liposomes are as described in any one of claims 2 to 3.

7. A method for freeze-drying and protecting platelets, characterized in that, The protective substance is delivered into the platelets, and then the platelets are freeze-dried to obtain freeze-dried platelet powder.

8. The method as described in claim 7, characterized in that, The protective substance is delivered into the platelet using one or more of the following methods: permeabilizer, electroporator, liposome, and nanoparticle.

9. The method as described in claim 7, characterized in that, The protective substances include, but are not limited to, bovine serum albumin, human serum albumin, casein, gelatin, ferritin, and heat shock proteins.

10. The method as described in claim 9, characterized in that, The protective substance is bovine serum albumin; the content of bovine serum albumin is 1 μg bovine serum albumin / 10 8 One platelet count ~ 50 μg bovine serum albumin / 10 8 One platelet.

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