Nanocrystallized platelets as well as preparation process and application thereof

Abstract

The invention relates to the technical field of platelet biological materials, aims at solving the problems of limited biological distribution, insufficient in-vivo stability and limited mitochondrial function protection effect caused by overlarge particle size of existing platelet-rich preparations such as PRP (platelet rich protein) and the like, and particularly discloses a nanocrystallization platelet as well as a preparation process and application thereof. Comprising the following steps: preparing platelet-rich plasma, adding an extracting solution A, uniformly mixing, centrifuging, and collecting supernate a; adding the extracting solution B, uniformly mixing, centrifuging, and collecting supernate b; centrifuging and collecting a precipitate m; adding a platelet washing solution C, centrifuging, collecting a precipitate n, and resuspending; and taking the resuspended platelets, activating, and ultrasonically crushing to obtain the nanocrystallized platelets. Through nanocrystallization processing, the particle size is significantly reduced, the surface charge characteristics are optimized and the like, the stability and the tissue penetrating ability in vivo are improved, the circulation time is prolonged, the interaction with cell membranes is enhanced, and the application in the aspects of drug delivery, mitochondrial function protection, oxidative stress resistance, inflammation regulation and control and the like is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of platelet biomaterials, in particular to a nano-platelet and a preparation process and application thereof. BACKGROUND

[0002] Acute kidney ischemia-reperfusion injury (IRI), inflammation associated with tissue aging, and severe skin trauma (such as burns) are common and harmful pathological conditions in clinical practice. These disease processes are often accompanied by mitochondrial dysfunction, increased oxidative stress, excessive activation of inflammatory responses, and decreased tissue repair capacity, ultimately leading to cell damage, organ dysfunction, and even failure.

[0003] In acute kidney injury, energy metabolism disorders, excessive production of reactive oxygen species (ROS), and release of pro-inflammatory factors induced by ischemia-reperfusion are key factors leading to renal tubular epithelial cell necrosis and renal failure. Current clinical treatment methods mainly focus on supportive treatment and symptomatic treatment, and there is a lack of effective drugs that can simultaneously improve mitochondrial function, inhibit oxidative stress, and regulate inflammatory responses.

[0004] In the field of severe wound repair, especially burns and scalds, there is obvious inflammation and exudation in the early stage of wound repair, rapid generation of granulation tissue and promotion of angiogenesis are required in the middle stage, and epithelialization and tissue remodeling need to be accelerated in the late stage. Most existing dressings or drugs can only play a role in a certain stage of healing, and there is a lack of comprehensive treatment methods for promoting repair throughout the process.

[0005] For the treatment of diseases such as IRI and severe skin trauma repair, a variety of treatment methods have been proposed and have achieved some results to some extent.

[0006] In the treatment of acute kidney ischemia-reperfusion injury, common clinical interventions include antioxidants (such as N-acetylcysteine), anti-inflammatory drugs (such as glucocorticoids), and supportive treatment (such as fluid management, dialysis, etc.). In addition, some studies have reported attempts to use platelet-rich plasma (PRP) to improve kidney injury. PRP can release a variety of growth factors to promote angiogenesis and tissue repair. However, traditional PRP and platelet preparations have limitations such as large particle size, short circulation time, low enrichment efficiency in target tissues, and limited effects on improving mitochondrial function and systemic anti-inflammatory effects.

[0007] In the aspect of wound repair, the existing treatment methods include functional dressings, growth factor preparations, stem cell transplantation, etc. These solutions can usually play a role in a certain stage of wound healing, such as inhibiting infection or promoting granulation tissue formation, but there are still obvious deficiencies in continuously promoting wound repair at multiple stages while controlling inflammation and accelerating tissue remodeling. In addition, the poor in vivo stability and short local retention time of growth factor or cell preparations also limit their clinical effect.

[0008] Among them, for example, PRP and other platelet-rich preparations can release a variety of pro-repair factors, but there are problems such as limited biodistribution due to too large particle size, insufficient in vivo stability, etc., and there are still deficiencies in comprehensive intervention at multiple targets such as anti-inflammatory, antioxidant and promotion of tissue regeneration. Therefore, there is an urgent need for a drug preparation with better particle size distribution, longer circulation time, stronger tissue penetration ability, better targeting, and can simultaneously achieve anti-inflammatory, antioxidant and tissue repair promotion, to meet the multiple clinical needs of acute organ injury and wound repair. SUMMARY

[0009] The purpose of the present application is to solve the problem of existing platelet-rich preparations such as PRP, which have a particle size that is too large, resulting in limited biodistribution and insufficient in vivo stability.

[0010] The present application is achieved by the following technical solutions: The present application provides a preparation method of nanoscale platelets, comprising the following steps: S1 Preparation of anticoagulated blood; S2 Extraction of platelet-rich plasma from anticoagulated blood; S3 Take the platelet-rich plasma, add platelet extraction solution A, mix well, centrifuge, collect supernatant a; S4 Take supernatant a, add platelet extraction solution B, mix well, centrifuge, collect supernatant b; S5 Take supernatant b, centrifuge, collect precipitate m; S6 Take precipitate m, add platelet washing solution C, centrifuge, collect precipitate n, resuspend to obtain resuspended platelets; S7 Take the resuspended platelets, perform activation treatment, and ultrasonic crushing to obtain nanoscale platelets.

[0011] Preferably, in step S7, the method of activation treatment is as follows: Place the resuspended platelets in liquid nitrogen, freeze for 5-20s, then thaw at room temperature, repeat 2-5 times, and the platelets can be activated.

[0012] Preferably, in step S7, the ultrasonic crushing time is 8-12min, and each time it is turned on for 2-3s and then turned off for 3-5s.

[0013] The nanoplatelets prepared by the above-mentioned preparation method proposed in this invention have an average particle size of 225±10nm and a surface Zeta potential of -10.5±2mV.

[0014] The above-mentioned nano-platelets of the present invention can be used as a drug component for the treatment of acute renal ischemia-reperfusion injury, and can also be used as a drug component for the treatment of skin trauma.

[0015] The technical solution of the present invention has the following beneficial effects: The nano-platelet (N-PLT) proposed in this invention significantly reduces particle size and optimizes surface charge properties through nano-processing. Its particle size is approximately 224.88 nm, and its Zeta potential is approximately -10.5 mV. This not only improves its stability and tissue penetration in vivo but also prolongs circulation time and enhances its interaction with cell membranes, enabling its application in drug delivery, anti-oxidative stress, and inflammation regulation. Specifically, through the synergistic action of multiple mechanisms, it achieves tissue protection, functional recovery, and regenerative repair effects under various pathological conditions, demonstrating broad clinical application prospects and market value: (1) Optimization of particle size and surface properties The N-PLT of this invention has an average particle size of 225±10nm and a surface Zeta potential of -10.5±2mV. It has a concentrated particle size distribution and good stability, which can significantly prolong the in vivo circulation time, improve tissue penetration and targeted delivery efficiency, avoid particle aggregation, and improve bioavailability in complex pathological environments.

[0016] (2) Broad-spectrum tissue protection under multiple pathological conditions The N-PLT of the present invention has shown significant efficacy in various animal disease models, including but not limited to renal ischemia-reperfusion injury and full-thickness skin defect repair, and has broad-spectrum adaptability and cross-system therapeutic potential.

[0017] (3) Enhance antioxidant defense The N-PLT of this invention can significantly increase superoxide dismutase (SOD) while reducing the level of malondialdehyde (MDA), a lipid peroxidation product, thereby reducing the damage of oxidative stress to tissues.

[0018] (4) Inhibit the inflammatory response The N-PLT of this invention can reduce the expression level of the pro-inflammatory factor TNF-α, effectively alleviate acute and chronic inflammatory responses, and improve inflammation-mediated tissue damage.

[0019] (5) Promote tissue repair and regeneration In a full-thickness skin defect model, the N-PLT of the present invention can significantly accelerate wound contraction and closure, promote granulation tissue formation and remodeling, shorten the healing cycle, and improve the quality of tissue structure restoration. Attached Figure Description

[0020] Figure 1 The particle size distribution of the nano-sized platelet N-PLT in Experimental Example 1.1 is shown. Figure 2 The graph shows the surface potential (Zeta potential) analysis results of the nano-platelet N-PLT in Experimental Example 1.2; Figure 3 A comparison chart of the kidney index of rats in Experiment 2.2; Figure 4 A comparative graph showing the serum creatinine (CRE) levels in the kidneys of rats in Experiment 2.3; Figure 5 A comparison chart of blood urea nitrogen (BUN) levels in the kidneys of rats in Experiment 2.3; Figure 6 This is a comparison chart of urinary protein levels in rats from Experiment 2.4; Figure 7 A comparison of reactive oxygen species (ROS) levels in renal tissue from Experiment 2.5; Figure 8 A comparative diagram of TNF-α levels in rat kidney tissue from Experiment 2.6; Figure 9 This is a comparison of cell viability after P-NV and N-PLT treatment in Experiment 3.1; Figure 10 HSF micrographs of different experimental groups in Experiment 3.1; Figure 11 Microscopic comparison of HSF cell migration in different experimental groups in Experiment 3.2; Figure 12 This is a schematic diagram of the wound healing process in the rat full-thickness skin injury model of Experiment 3.3; Figure 13 This is a comparison chart of the healing rate changes at different times for full-thickness skin injuries in rats in Experiment 3.3; Figure 14 This is a graph showing the quantitative change in wound area of ​​full-thickness skin injury in rats in Experiment 3.3; Figure 15 This is a comparison of HE and Masson staining results of rat kidney tissue from Experiment 3.4. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer; where the manufacturers of the instruments, equipment, reagents, or raw materials used are not specified, they are all conventional products that can be purchased commercially.

[0022] This invention provides a nano-sized platelet with an average particle size of approximately 225±10 nm and a surface Zeta potential of approximately -10.5±2 mV. Its preparation process includes the following steps: (1) Take sodium citrate anticoagulant and mix it with fresh blood to obtain anticoagulant blood.

[0023] (2) Place the anticoagulated blood in a centrifuge and centrifuge at room temperature. Collect the upper plasma layer (including the white membrane layer) and discard the lower blood cell precipitate to obtain platelet-rich plasma.

[0024] (3) Add platelet extract A to platelet-rich plasma, mix well, centrifuge at room temperature, and collect the supernatant a.

[0025] (4) Add platelet extract B to supernatant a, mix well, centrifuge at room temperature, and collect supernatant b.

[0026] (5) Centrifuge the supernatant b at room temperature, discard the supernatant, and collect the precipitate m.

[0027] (6) Add platelet washing solution C to the precipitate m, centrifuge at room temperature, discard the supernatant, collect the precipitate n, which is the platelet, and resuspend the precipitate n with an appropriate amount of PBS.

[0028] (7) Platelets are activated by repeated freeze-thaw cycles. The resuspended platelets are placed in liquid nitrogen and frozen for 5-20 seconds, then thawed at room temperature. This process is repeated 2-5 times to activate the platelets. The activated platelets are then broken up using a cell sonicator with the power set to 70-90%. The sonicator is turned on for 2-3 seconds and then turned off for 3-5 seconds each time. After sonication for 8-12 minutes, nano-sized platelets are obtained.

[0029] The nano-sized platelets proposed in this invention can be used as drug components in the clinical treatment of diseases such as acute renal ischemia-reperfusion injury, tissue aging-related inflammatory response, and severe skin trauma.

[0030] Example 1 Platelet extraction kits purchased from Beijing Bio-Labs Technology Co., Ltd., including platelet extract A, platelet extract B, and platelet washing solution C, were used for the following processing: (1) Take a 15mL sterile centrifuge tube, add 1mL of sodium citrate anticoagulant, then add 10mL of fresh rat orbital blood, shake gently to mix the sodium citrate anticoagulant and blood evenly to obtain anticoagulated blood.

[0031] (2) Place the anticoagulated blood in a centrifuge and centrifuge at 400×g for 8 minutes at room temperature. Collect the upper plasma layer (including the white membrane layer) and discard the lower blood cell precipitate to obtain platelet-rich plasma.

[0032] (3) Add 1 mL of platelet extract A to platelet-rich plasma, mix well, centrifuge at 400×g for 8 min at room temperature, and collect the supernatant a.

[0033] (4) Add 1 mL of platelet extract B to supernatant a, mix well, centrifuge at 400×g for 8 min at room temperature, and collect supernatant b.

[0034] (5) Centrifuge the supernatant b at 2600×g for 10 min at room temperature, discard the supernatant, and collect the precipitate m.

[0035] (6) Add 1 mL of platelet washing solution C to the precipitate m, centrifuge at 2600×g for 10 min at room temperature, discard the supernatant, collect the precipitate n, which is the platelet, and resuspend the precipitate n with an appropriate amount of PBS.

[0036] (7) Place the resuspended platelets in liquid nitrogen, freeze for 10 seconds and then thaw at room temperature. Repeat this process 3 times to activate the platelets. Then break up the activated platelets using a cell sonicator with the power set to 80%. Turn it on for 2 seconds and then turn it off for 3 seconds each time. After sonicating for 10 minutes, nano-sized platelets are obtained and denoted as N-PLT.

[0037] Test case Sample: Example 1 (1) Performance test of nano-sized platelets 1.1 Dynamic light scattering (DLS) technique was used to measure the particle size of the sample.

[0038] The specific measurement steps are as follows: First, preheat the particle size analyzer for 30 minutes; then, dilute the nano-platelet sample to 1 mg / mL with PBS, take 1.5 mL of the diluted sample and place it in a test dish. Gently tap the dish wall to distribute the sample evenly and reduce measurement error, then place it in the sample cell for detection. Each sample is measured three times. Rinse the transparent quartz dish with ultrapure water to ensure it is clean and free of impurities, then add a small amount of sample to the quartz dish and rinse 1-2 times. Add 2-2.5 mL of sample, place it in the particle size analyzer, and immediately close the lid for detection.

[0039] The results are as follows Figure 1 The image shown is a particle size distribution of the nano-sized platelet N-PLT from Example 1. Figure 1As can be seen, the average particle size of the sample is 224.88 nm, and the particle size distribution is concentrated in a relatively small range, exhibiting a single-peak distribution. The fact that the sample is within this particle size range indicates that it possesses good biocompatibility and tissue penetration ability in vivo, making it suitable for nanomedicine delivery systems.

[0040] 1.2 Electrophoretic light scattering (ELS) was used to measure the surface zeta potential of nano-sized platelets.

[0041] Take 1.5 mL of nano-sized platelet sample, place it in a test dish, put it into the sample cell, insert the electrode pad, and measure the Zeta potential on the sample surface.

[0042] The results are as follows Figure 2 The image shown is a graph illustrating the surface potential (Zeta potential) analysis results of the nano-platelet N-PLT in Example 1. From... Figure 2 As can be seen, the zeta potential of N-PLT is -10.5mV. This negative potential value indicates that N-PLT has good stability because the negative charge can effectively prevent the aggregation between nanoparticles and prolong their blood circulation time in the body. In addition, the negative potential also helps to improve the interaction between N-PLT and the cell membrane, thereby enhancing its efficiency in drug delivery.

[0043] (2) Therapeutic effect of nanoplatelets on renal ischemia-reperfusion in rats 2.1 Establish a rat model of renal ischemia-reperfusion.

[0044] Sixteen healthy male 7-week-old SD rats were fasted for 12 hours and then randomly divided into four groups: ①Sham surgery group (Sham group): saline was injected into the tail vein; ② Model group: saline was injected via the tail vein; ③PRP group: local injection of 1 mg / kg PRP into the kidney; ④ Nanoparticle platelet group (N-PLT group): 1 mg / kg N-PLT was injected via the tail vein.

[0045] All animals were anesthetized by intraperitoneal injection of 5% chloral hydrate. After shaving the backs of rats, the following procedures were performed: Sham group: A 2cm strip incision was made on each side of the kidney on the back of the rat, then sutured with surgical sutures, and the wound was disinfected with povidone-iodine; Model group: After shaving the backs of rats and disinfecting with povidone-iodine, the left kidney was removed, and the right kidney was clamped with an arterial clamp for 1 hour of ischemia. At 55 minutes of ischemia, 300μL of physiological saline was injected via the tail vein, then the arterial clamp was removed, and the wound was sutured with surgical sutures, and the wound was disinfected with povidone-iodine; PRP group: At 55 minutes of ischemia, 300μL of 1mg / kg PRP was injected locally into the kidney; N-PLT group: After shaving the backs of rats and disinfecting with povidone-iodine, the left kidney was removed, and the right kidney was clamped with an arterial clamp for 1 hour of ischemia. At 55 minutes of ischemia, 300μL of 1mg / kg N-PLT was injected via the tail vein. The drugs were administered on days 3, 7, and 10, and the rats were dissected on day 14.

[0046] 2.2 Determine the kidney index in rats.

[0047] After successful model establishment, rats were treated with N-PLT for 14 days. On day 14, the weight of rats in different experimental groups was measured, and then the kidneys were dissected, washed with PBS, and dried with filter paper before being weighed. The rat kidney index was calculated as (kidney / body weight × 100%). The results are summarized as follows. Figure 3 As shown.

[0048] Depend on Figure 3 It can be seen that the renal organ index of the N-PLT group was significantly lower than that of the PRP group and the Model group, and close to that of the Sham group, indicating that the N-PLT group can show better effects in reducing kidney damage and restoring kidney function.

[0049] 2.3 Detection of serum creatinine (CRE) and blood urea nitrogen (BUN) levels in rats to assess renal function. The CRE and BUN kits from Nanjing Jiancheng Biotechnology Institute were used to detect the effects of centrifuged rat serum samples. Serum creatinine (CRE) determination: The CRE value of each sample was determined and calculated using the sarcosine oxidase method according to the formula. The sample preparation steps are shown in Table 1 below: Table 1 Sample preparation steps for serum creatinine (CRE) measurement

[0050] Blood urea nitrogen (BUN) determination: The urease method was used to determine and calculate the BUN value of each sample according to the formula. The sample preparation steps are shown in Table 2 below: Table 2 Sample preparation steps for blood urea nitrogen (BUN) determination

[0051] 2.4 Detection of urinary protein in rats Collect 24-hour fresh urine and centrifuge at 3000 rpm for 5 minutes to remove impurities and precipitates; prepare CBB reagent at a ratio of CBB reagent to distilled water of 1:4 (i.e., 5-fold dilution), and use immediately. The sample addition and processing steps are shown in Table 3 below: Table 3 Sample preparation steps for rat urinary protein assay

[0052] Take the above test measurements, and then calculate the urine protein concentration (mg / L) according to (OD). 标准值 OD 空白值 ) / (OD 测定值 OD 空白值 Calculate the rat urine protein concentration by multiplying 524 by the dilution factor.

[0053] Based on Experiments 2.3 and 2.4, using a rat model of renal ischemia-reperfusion injury, and combining indicators such as serum creatinine, blood urea nitrogen, and urinary protein, the effect of N-PLT in alleviating renal injury caused by IRI was evaluated. The results are as follows: Figure 4 to Figure 6 As shown.

[0054] Serum creatinine is a routine biomarker for assessing kidney function. In renal insufficiency, serum creatinine levels are often elevated. Elevated creatinine reflects suppressed renal excretion function and is an early sign of kidney damage. Figure 4 The image shows a comparison of serum creatinine (CRE) levels in the kidneys of rats in Experiment 2.3. Figure 4 As can be seen, the serum creatinine level in the N-PLT group was significantly lower than that in the Model group and close to that in the Sham group. This indicates that N-PLT plays a protective role in the recovery of kidney function and can effectively reduce the increase in serum creatinine after kidney function impairment. In contrast, the serum creatinine level in the Model group was significantly higher, suggesting that kidney function was injured by ischemia-reperfusion injury.

[0055] Blood urea nitrogen (BUN) is a waste product in the blood, commonly used to measure the kidneys' ability to excrete it. Kidney damage caused by renal infarction (IRI) leads to the accumulation of BUN; therefore, BUN is also an important indicator of kidney function impairment. Figure 5 The image shows a comparison of blood urea nitrogen (BUN) levels in the kidneys of rats in Experiment 2.3. Figure 5 It can be seen that the BUN level in the N-PLT group was significantly lower than that in the Model group, indicating that N-PLT can effectively reduce urea nitrogen accumulation caused by kidney damage; in contrast, the BUN level in the Model group was significantly higher, indicating that kidney function was impaired and could not effectively eliminate waste products from the body.

[0056] The presence of proteinuria usually indicates structural damage to the kidneys. During renal tubule injury (IRI), damage to the renal tubules leads to protein leakage into the urine. The detection of proteinuria is clinically used to assess the degree of kidney function impairment and is a sensitive indicator of kidney damage. Elevated proteinuria is generally one of the hallmarks of kidney damage; therefore, the therapeutic effect of N-PLT lies in reducing structural damage to the kidneys. Figure 6 The image shown is a comparison of urinary protein levels in rats from Experiment 2.4. Figure 6 It can be seen that the urinary protein level in the N-PLT group was significantly lower than that in the Model group, indicating that N-PLT can reduce urinary protein leakage caused by kidney damage.

[0057] 2.5 Detect the level of reactive oxygen species (ROS) in kidney tissue.

[0058] Preparation of single-cell suspension of kidney tissue: Freshly extracted kidney tissue specimens were immediately transferred to pre-chilled centrifuge tubes containing 4°C phosphate-buffered saline (PBS, pH 7.4) and washed three times, with fresh buffer after each wash. The tissue was then transferred to pre-chilled culture dishes and mechanically dispersed using ophthalmic scissors under a PBS film. The fragments were then collected by centrifugation at 1000 rpm for 5 min at 4°C. Five volumes of trypsin-EDTA working solution were added, and the mixture was placed in a cell culture incubator and resuspended every 5 min. After digestion was terminated with complete culture medium (stop solution containing 10% FBS), the mixture was filtered through a 70 μm nylon cell filter. The filtrate was then subjected to gradient centrifugation (4°C, 1000 rpm, 10 min) to obtain primary cell pellets. The supernatant was discarded, and the cells were collected. The process of centrifugation with PBS was repeated twice, and the cells were collected again.

[0059] Preparation of DCFH-DA working solution: Dilute 10mM DCFH-DA stock solution with sterile PBS buffer or culture medium at a ratio of 1:1000 to prepare a 10μM DCFH-DA working solution.

[0060] Add the DCFH-DA probe working solution to the collected cells and incubate for 20 min in a cell culture incubator at 37°C in the dark. After staining, centrifuge at 600g for 5 min at 4°C, discard the supernatant, wash the cells twice with serum-free medium, and resuspend the cells in a certain amount of DMEM basal medium. Then, add the cell suspensions to each group into opaque 96-well plates and detect the fluorescence intensity at 488nm excitation and 525nm emission wavelength using an ELISA reader.

[0061] In ischemia-reperfusion injury (IRI), dysregulation of cellular energy metabolism is a significant factor leading to kidney damage. ATP (adenosine triphosphate), the main energy molecule in cells, directly reflects abnormal cellular energy metabolism. After ischemia, decreased ATP synthesis capacity leads to the inability of cells to maintain basic functions, ultimately causing cell death. Therefore, ATP level is a crucial indicator for evaluating kidney damage and is of great significance in IRI research. Furthermore, changes in mitochondrial function play a key role in IRI. JC-1 dye can be used to monitor changes in mitochondrial membrane potential, thereby assessing the functional status of mitochondria. In IRI, loss of mitochondrial membrane potential signifies mitochondrial dysfunction, leading to cell death. Therefore, the JC-1 ratio is an important indicator of cellular damage after IRI. Oxidative stress is another important process in IRI. After ischemia-reperfusion recovery, intracellular reactive oxygen species (ROS) levels increase sharply. Excessive ROS accumulation damages cell structure, leading to cell membrane disruption, protein denaturation, and DNA damage. Elevated ROS levels are an important marker of IRI, and quantitative analysis helps assess the severity of IRI and the effectiveness of treatment measures.

[0062] like Figure 7 The image shows a comparison of reactive oxygen species (ROS) levels in kidney tissue from Experiment Example 2.5 above.

[0063] Depend on Figure 7 It can be seen that the ROS level in the N-PLT group was significantly lower than that in the Model group and close to that in the Sham group. This indicates that N-PLT can significantly reduce the level of peroxides generated in renal ischemia-reperfusion injury through its antioxidant effect; in contrast, the ROS level in the Model group was significantly increased, suggesting that ischemia-reperfusion caused oxidative stress.

[0064] 2.6 The inflammatory factor TNF-α (tumor necrosis factor α) was detected using the ELISA method.

[0065] Serum preparation: Before dissection, 1 mL of blood was collected from the orbit of each rat and placed in a 1.5 mL centrifuge tube. The tube was allowed to stand at room temperature for 2 hours, then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was separated and aliquoted into 200 μL centrifuge tubes. 20 minutes before the experiment, the reagent kit was removed from the refrigerator and allowed to equilibrate to room temperature.

[0066] Preparation of standard solutions: Dissolve the lyophilized standard in 0.6 mL of diluent, and dilute it serially to a concentration of 2000 pg / mL to obtain standard solutions of 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL.

[0067] Add standard diluent to blank wells and sample or standard solution to the remaining wells, 100 μL / well. After sealing, incubate at 37°C in the dark for 90 min.

[0068] 20 minutes before the experiment, prepare the working solution for the biotinylated antibody: dilute the 30× concentrated biotinylated antibody with diluent to make a 1× working solution; prepare the washing solution: dilute the 20× concentrated washing solution with double-distilled water to make a 1× working solution.

[0069] Washing: After incubation, discard the liquid, add 350 μL of washing buffer to each well, let stand for 30 seconds, shake dry, and repeat 5 times. After washing, add biotinylated antibody diluent to the blank wells and biotinylated antibody working solution to the remaining wells, 100 μL / well. After sealing, incubate at 37°C in the dark for 60 min.

[0070] 20 minutes before the experiment, prepare the enzyme conjugation working solution: dilute the 30× concentrated enzyme conjugate with diluent to make a 1× working solution.

[0071] Washing: After incubation, discard the liquid, add 350 μL of washing buffer to each well, let stand for 30 seconds, shake dry, and repeat 5 times. After washing, add enzyme conjugate dilution to the blank wells and enzyme conjugate working solution to the remaining wells, 100 μL / well. After sealing, incubate at 37°C in the dark for 30 min.

[0072] Washing: After incubation, discard the liquid, add 350 μL of washing buffer to each well, let stand for 30 seconds, spin dry, and repeat 5 times. After washing, add 100 μL of TMB substrate to each well, seal the wells, and incubate at 37°C in the dark for 15 minutes.

[0073] After the reaction is complete, add 100 μL of stop solution to each well and measure the OD value at 450 nm using an ELISA reader within 3 minutes.

[0074] During ischemia-reperfusion injury (IRI), activation of the inflammatory response is often an early manifestation of kidney damage. Excessive release of inflammatory factors can lead to apoptosis, tissue damage, and organ dysfunction. Tumor necrosis factor-α (TNF-α) is an important pro-inflammatory cytokine in the inflammatory response, playing a crucial role in ischemia-reperfusion injury. Excessive release of TNF-α can lead to cell death and tissue necrosis, impairing the structure and function of the kidney.

[0075] like Figure 8 The image shown is a comparison of TNF-α levels in rat kidney tissue from Experiment 2.6. Figure 8It can be seen that the TNF-α level in the model group was significantly higher than that in the Sham group, while the TNF-α level in the N-PLT group was significantly lower than that in the model group, approaching the level in the Sham group. This indicates that N-PLT can effectively inhibit the inflammatory response induced by ischemia-reperfusion injury, reduce the release of TNF-α, and thus reduce kidney damage.

[0076] IRI (Intracranial Renal Injury) is a common clinical acute kidney injury with a complex mechanism involving multiple factors such as oxidative stress, inflammatory response, and apoptosis. Oxidative stress is one of the main pathological processes in IRI. After ischemia-reperfusion recovery, the level of reactive oxygen species (ROS) in renal cells increases sharply, leading to lipid peroxidation, protein oxidation, and DNA damage, thereby damaging kidney tissue. Malondialdehyde (MDA), a product of lipid peroxidation, is often used as a marker of oxidative stress. Excessive oxidative stress leads to the accumulation of MDA, further exacerbating kidney damage. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) are important intracellular antioxidant enzymes that can effectively scavenge excess ROS and reduce oxidative damage. SOD catalyzes the conversion of superoxide anion radicals into hydrogen peroxide, while GSH-PX maintains cellular redox balance by decomposing hydrogen peroxide and toxic organic peroxides. In IRI, decreased antioxidant enzyme activity and increased MDA levels are important factors leading to kidney damage. Therefore, regulating oxidative stress response and increasing the activity of antioxidant enzymes are important means of protecting kidney function.

[0077] (3) Treatment of rat skin lesions by nano-sized platelets 3.1 HSF cell proliferation assay 5000 HSF cells were seeded in 96-well plates and divided into three groups: Control group, P-NV group, and N-PLT group. After seeding for 24 hours, CCK-8 solution was added: after the cell drug treatment time was over, 10 μL of CCK-8 solution was added to each well and treated for 2 hours. The absorbance was measured at 450 nm.

[0078] like Figure 9 The image shown is a comparison of cell viability after P-NV and N-PLT treatment in Experiment 3.1. Figure 9 It can be seen that both P-NV and N-PLT treatments significantly improved HSF cell viability, with P-NV increasing by approximately 160-170% and N-PLT by approximately 180-190%. Compared with the control, the two treatments showed extremely significant differences, and N-PLT further increased the viability compared with P-NV, suggesting that it has a stronger proliferative capacity.

[0079] like Figure 10 The image shown is a micrograph of the HSF from different experimental groups in Example 3.1. Figure 10As can be seen, under the same inoculation density and culture time, the microscopic images of the three groups of HSF showed that: the control group had sparse cells, a small spreading area, and a large intercellular distance; the P-NV group had a significantly increased cell number compared to the control group, with a good fibrous spindle-shaped morphology; the N-PLT group had the most dense cells, spread sufficiently, and tended to have a higher degree of confluence, with no obvious signs of cytotoxicity such as rounding or fragmentation. These morphological changes are consistent with the increasing trend of CCK-8 activity, suggesting that both P-NV and N-PLT can promote HSF proliferation, with N-PLT having the strongest effect.

[0080] 3.2 HSF cell migration assay An 8.0 μm pore size transwell chamber was placed in a 12-well plate; 1×10 5 One HSF was placed in the upper chamber of the transwell insert along with P-NV and N-PLT and cultured, while the lower chamber contained 700 μL of culture medium. After incubation for 24 h, the cells in the upper chamber were fixed with crystal violet solution (0.5% methanol solution) for 2 h. The transwell chamber was washed twice with PBS and any non-migrating cells were removed using sterile cotton swabs. Migrating cells were observed under a JASCO microscope (Tokyo, Japan).

[0081] like Figure 11 The image shown is a microscopic comparison of HSF cell migration in different experimental groups in Experiment 3.2. Figure 11 As can be seen, the number of migrating cells (purple staining) in the three groups from fewest to most is: Control < P-NV < N-PLT. Only scattered cells were observed to migrate through the membrane in the control group; the number of migrating cells in the P-NV group was significantly increased, and the cells were spindle-shaped fibroblasts with a relatively uniform distribution; the subcellular cells in the N-PLT group were the most dense, almost filling the entire field of view, indicating that it had the strongest promoting effect on HSF migration, and no obvious morphological abnormalities or necrotic fragments were observed.

[0082] 3.3 Animal modeling and drug administration experiments To evaluate the therapeutic potential of N-PLT for skin wound repair, a rat model of full-thickness skin defect was established, and rats were treated with saline, P-NV, and N-PLT respectively. The wound healing process was then systematically observed and quantitatively analyzed.

[0083] Healthy SD rats weighing 140-160g at 6-7 weeks of age were randomly divided into three groups: Saline group, P-NV group, and N-PLT group, n=3. (P-NV refers to platelet lysis buffer, which is used to break activated platelets using a cell sonicator at 80% power for 10 minutes, followed by centrifugation at 4000g for 10 minutes at 4℃; the supernatant is P-NV). Rats were injected intraperitoneally with 5% chloral hydrate. After anesthesia, the rats' backs were shaved, and two full-thickness wounds (1.2cm in diameter) were made on either side of the midline using a punch, and disinfected with povidone-iodine. 200μL of 2mg / kg N-PLT was evenly applied to the wounds using a pipette and allowed to be fully absorbed. The medication was administered and photographed on days 0, 3, 5, and 7. The wound area was analyzed using ImageJ software, and the healing rate was calculated using the following formula: Healing rate = (A0 - A) n ) / A0×100% like Figure 12 The image shown is a schematic diagram illustrating the wound healing process in the rat full-thickness skin injury model from Experiment 3.3. Figure 12 As can be seen, during the 9-day wound healing observation, the wound contraction rate in the N-PLT group was significantly faster than that in the control group. This indicates that the healing rate in the saline group was slower, with the wound not fully healed by day 9, suggesting that saline did not significantly promote wound healing. The P-NV group showed a faster healing rate, with noticeable healing by day 7 and most of the wound healed by day 9, suggesting that P-NV may have a certain promoting effect on skin damage. However, the N-PLT group showed the most significant healing effect, with a wound healing rate far exceeding the other two groups, and the wound almost completely healed by day 9. The rapid healing of the N-PLT group may be related to its properties of promoting cell proliferation and repair. Therefore, N-PLT is considered the most effective treatment for promoting skin healing in this experiment. In conclusion, the N-PLT group showed the strongest effect in promoting the healing of skin damage in rats, providing an effective treatment option.

[0084] like Figure 13 The image shown is a comparison of the healing rate changes at different times for full-thickness skin injuries in rats in Experiment 3.3; Figure 14 The figure shown is a quantitative change curve of the wound area of ​​full-thickness skin injury in rats in Experiment 3.3. Figure 13 and Figure 14It can be seen that at different time points of 3, 5, 7, and 9 days, the skin lesion healing rate of the N-PLT group was significantly higher than that of the P-NV group and the saline group, indicating that N-PLT has a significant advantage in promoting skin healing. Specifically, the healing rate of the N-PLT group was significantly higher than that of the other two groups at 3, 5, and 7 days, and maintained a high healing rate at 9 days; the healing rate of the P-NV group was also better than that of the saline group at all time points, but its effect was not as good as that of the N-PLT group. Furthermore, Figure 14 The changes in wound diameter in each group were visualized using heat maps. Red represents the core area of ​​the wound, with green, blue, and yellow representing different healing levels outwards. Figure 14 As can be seen, the central red area of ​​the N-PLT group was significantly reduced and the healing ring was significantly narrowed, verifying its effect of accelerating wound contraction and tissue regeneration.

[0085] In conclusion, N-PLT can significantly promote the healing of burn wounds by regulating local inflammatory responses and promoting granulation tissue formation and remodeling, and has good prospects for clinical translation.

[0086] 3.4 Tissue staining Perform HE and Masson staining: 3.4.1 Preparation of paraffin sections Take a tissue block, fix the tissue specimen with 10% formalin, cut the tissue block into 4-5μm thick sections using a microtome, mount the sections on a glass slide, and set aside for later use.

[0087] 3.4.2 Dewaxing and Hydration The sections were immersed in xylene for 2×5 min to remove paraffin; then the sections were immersed in alcohol solutions of different concentrations in turn to achieve hydration: 100% ethanol for 2×5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and deionized water for 2×5 min.

[0088] 3.4.3 HE staining Place the slides in Hematoxylin staining solution for 10-15 minutes until the cell nuclei are stained deep blue. Rinse the slides with running water and differentiate the cell nuclei, usually by rapid differentiation with 1% hydrochloric acid ethanol for 1-2 seconds, then rinse immediately with water. Finally, immerse the slides in Eosin staining solution for 2-5 minutes until the cytoplasm turns pink.

[0089] 3.4.4 Masson's trichrome staining Immerse the sections in a Weigert blue solution for 5-10 minutes for staining, then rinse with deionized water. Next, immerse the sections in Picrosirius Red staining solution for 20-30 minutes for staining, primarily to mark collagen fibers. Then, dehydrate the sections using 95% ethanol and 100% ethanol, respectively, and clear them with xylene. Finally, mount the sections with neutral resin and observe them under a microscope.

[0090] like Figure 15 The image shown is a comparison of HE and Masson staining results of rat kidney tissue in Experiment 3.4.

[0091] Depend on Figure 15 It can be seen that, through HE staining, the morphological changes of the tissue were observed as follows: in the saline group (NS), the wound area healed poorly and there was a significant inflammatory response; the P-NV group and the N-PLT group healed better, and the wound in the N-PLT group healed more completely and the cells were arranged more regularly, indicating its advantage in promoting tissue repair.

[0092] Masson staining is mainly used to observe the formation of collagen fibers: in the N-PLT group, the distribution and arrangement of collagen fibers are the most compact and regular, indicating that it has a good effect on promoting collagen synthesis; the P-NV group shows a moderate degree of collagen fiber formation, while the saline group has poor collagen synthesis and a slow repair process.

[0093] The above experiments show that the nanoplatelets proposed in this invention have significantly smaller particle size and excellent surface charge properties. Under the synergistic effect of multiple mechanisms, they have good stability and tissue penetration ability in vivo, can prolong circulation time and enhance their interaction with cell membranes, and realize their application in drug delivery, mitochondrial function protection, anti-oxidative stress and inflammation regulation.

[0094] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing nano-sized platelets, characterized in that, Includes the following steps: S1 is used to prepare anticoagulants; S2 extracts platelet-rich plasma from anticoagulated blood; S3 Take platelet-rich plasma, add extraction solution A, mix well, centrifuge, and collect the supernatant a; S4 Take the supernatant a, add the extract B, mix well, centrifuge, and collect the supernatant b; S5 Take the supernatant b, centrifuge, and collect the precipitate m; S6 Take the precipitate m, add platelet washing solution C, centrifuge, collect the precipitate n, resuspend, and obtain resuspended platelets; S7 platelets were resuspended, activated, and then ultrasonically broken to obtain nano-sized platelets.

2. The method for preparing nano-sized platelets according to claim 1, characterized in that, In step S7, the activation process is as follows: Platelets can be activated by placing resuspended platelets in liquid nitrogen, freezing for 5-20 seconds, and then thawing at room temperature. This process can be repeated 2-5 times.

3. The method for preparing nano-sized platelets according to claim 1, characterized in that, In step S7, the ultrasonic disruption time is 8-12 minutes, and it is turned on for 2-3 seconds and then off for 3-5 seconds each time.

4. A nano-sized platelet prepared by the preparation method according to any one of claims 1 to 3, characterized in that, The average particle size of the nano-platelets is 225±10nm, and the surface zeta potential is -10.5±2mV.

5. The application of nano-sized platelets as described in claim 4, characterized in that, Drug components used for the treatment of acute renal ischemia-reperfusion injury.

6. The application of nano-sized platelets as described in claim 4, characterized in that, Drug components used for the treatment of tissue aging-related inflammation.

7. The application of nano-sized platelets as described in claim 4, characterized in that, Drug components used for the treatment of skin injuries.

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