Nanocrystallized platelet particles, and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a nanocrystallized platelet particle and a preparation method and application thereof, and the preparation method comprises the following steps: (a) separating and purifying platelets; (b) nanocrystallization treatment of the platelets; and (c) mixing of the nanoparticles. According to the preparation method disclosed by the invention, the nanocrystallized platelet-PRP composite particles are efficiently and controllably prepared by innovatively combining a repeated freezing and thawing technology and a micro-fluidic chip shearing treatment technology, and the particles have high permeability of a nano material and a regeneration and repair function of a PRP growth factor; the compound is applied to treatment of renal ischemia reperfusion injury through intravenous injection, the renal function can be remarkably improved, the mitochondrial function can be recovered, oxidative stress and inflammatory reaction can be effectively relieved, and therefore a new treatment strategy with the advanced process and the exact curative effect is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a nanoscale platelet particle, a preparation method and application thereof. BACKGROUND

[0002] Renal ischemia-reperfusion injury (RIRI) is one of the main causes of hospital-acquired acute kidney injury (AKI), and its pathological mechanism is complex and the mortality rate is high. Although the clinical support means is constantly improving, specific treatment means for the cause is still scarce. As a high-oxygen-consuming organ, the kidney is rich in mitochondria and is abnormally sensitive to ischemia and hypoxia. When RIRI occurs, mitochondrial dysfunction becomes a core link, leading to the interruption of ATP synthesis, the explosive generation of reactive oxygen species (ROS), and the subsequent apoptosis, necrosis and severe inflammatory response, ultimately leading to serious damage to kidney function.

[0003] Platelet-rich plasma (PRP) has shown great potential in the field of tissue repair and regenerative medicine due to its rich growth factors (such as PDGF, VEGF, TGF-β, etc.) and bioactive proteins. In theory, the anti-inflammatory, antioxidant and cell proliferation-promoting abilities of PRP make it a potential candidate strategy for treating RIRI. However, the clinical application of PRP faces two key technical bottlenecks: first, the lack of standardization in the preparation process leads to significant batch-to-batch differences in the concentration and potency of key active ingredients in the product, affecting the repeatability of treatment effects and posing potential safety risks; second, and more importantly, is the dilemma of choosing the administration route.

[0004] In the prior art, there have been studies attempting to use PRP for local treatment of the kidney. For example, the research results disclosed in PloS One, “Effects of Platelet-Rich Plasma (PRP) on a Model of Renal Ischemia-Reperfusion in Rats”, show that by giving PRP through subcapsular injection, not only did it fail to alleviate the injury, but it also led to worse kidney blood flow and more severe histopathological damage, which reveals that injecting in the local part of the already damaged kidney may pose the risk of exacerbating local inflammation, microvascular embolism or mechanical compression, thereby limiting the application of PRP in the treatment of RIRI.

[0005] On the other hand, in order to improve the targeting and efficacy of drugs, nanotechnology is introduced into this field. Prior art one (Chinese patent CN111671773A) discloses "Application of platelet-rich plasma stimulated umbilical cord mesenchymal stem cell exosome in preparation of drug for improving repair of acute kidney injury". This technology needs complex cell culture and exosome extraction process, long preparation period (45-50 hours), complicated steps (multiple centrifugation), high cost, and is difficult to meet the clinical needs of rapid treatment of acute kidney injury. Prior art two (Chinese patent CN116083357A) discloses "A nano-enriched platelet vesicle preparation, its preparation method and application", which realizes nanocrystallization by physically extruding the enriched platelets through different pore size membranes in turn. This method (i.e. physical extrusion method) requires high equipment, time-consuming process and may cause inactivation of platelet active ingredients due to high shear force, while the loss and residue of membrane material also introduce additional impurity risk and process complexity.

[0006] In summary, it is necessary to propose a kind of nanometered platelet particles, its preparation method and application, which can overcome the above-mentioned defects, optimize and standardize the preparation process, improve the stability and targeting of drugs, and realize safe and effective systemic drug delivery. SUMMARY

[0007] The purpose of the present application is to provide a kind of nanometered platelet particles, its preparation method and application, which can efficiently and controllably prepare nanometered platelet-PRP composite particles by innovative combination of repeated freeze-thaw and microfluidic chip shear processing technology. The particles have high permeability of nanomaterials and regenerative repair function of PRP growth factors. When applied to renal ischemia-reperfusion injury treatment through intravenous injection, it can significantly improve renal function, restore mitochondrial function, and effectively relieve oxidative stress and inflammatory response, thereby providing a new treatment strategy with advanced process and definite efficacy.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: A preparation method of nanometered platelet particles, comprising the following steps: (a) separation and purification of platelets: platelet-rich plasma is separated from anticoagulated blood, and platelet sediment and PRP growth factor concentrate are further separated and purified; (b) nanometerization treatment of platelets: after resuspension of the platelet sediment, repeated freeze-thaw cycle treatment and microfluidic chip shear treatment are carried out in turn to obtain nanometerized platelet suspension; (c) mixing of nanoparticles: the nanometerized platelet suspension and the PRP growth factor concentrate are mixed in a volume ratio of (4-6):1, and the supernatant is taken after centrifugation, thereby obtaining the nanometerized platelet-PRP composite particles.

[0009] The present application efficiently separates PRP supernatant (i.e. PRP growth factor concentrate) and intact platelet cells from whole blood by gradient centrifugation. Further, the cell membrane structure of platelets is preliminarily destroyed by repeated freeze-thawing, and then combined with high-precision shear force of a microfluidic chip to achieve controllable and uniform nanometerization of platelets, i.e. to break the micro cells into nanometer-sized particles. Finally, the nanometerized platelet particles are compounded with the PRP growth factor concentrate at an optimized ratio, so that the active ingredients from the inside of the platelets are synergistically integrated with the free growth factors in the PRP to construct a functional nanocomposite preparation.

[0010] Further, in step (a), the separation and purification of the PRP growth factor concentrate is performed at room temperature, first centrifuging at 450xg for 10 min to collect the upper plasma and buffy coat layer, i.e. crude PRP; then centrifuging the crude PRP at 1200xg for 8 min to obtain the supernatant, i.e. the PRP growth factor concentrate.

[0011] Further, in step (b), the repeated freeze-thawing cycle is a cycle of rapid freezing in liquid nitrogen and recovery in a 37°C water bath, and the cycle is repeated 3-7 times; the flow rate of the microfluidic chip for shear treatment is 80-120 μL / min, and the working pressure is 25-35 psi.

[0012] Further, in step (c), after the nanometerized platelet suspension is mixed with the PRP growth factor concentrate, centrifugation at 10000xg for 15 min is performed to remove the unbroken fragments, and the nanometerized PRP composite particle suspension is obtained.

[0013] Further, in step (b), the microfluidic chip for shear treatment comprises: an inlet zone provided with double independent inlet channels, and a filter membrane built-in the inlet zone for separately introducing the platelet suspension and the PRP growth factor concentrate; a breaking zone in communication with the inlet zone, and a contraction-expansion type microchannel array built-in the breaking zone for nanometerizing and breaking the platelets; a mixing zone in communication with the breaking zone, and a static mixer built-in the mixing zone for mixing the nanometerized platelet suspension and the PRP growth factor concentrate.

[0014] Further, in the contraction-expansion type microchannel array of the breaking zone, the width of the contraction section gradually changes from 500 μm to 50 μm, and the width of the expansion section gradually changes from 50 μm to 200 μm.

[0015] Further, the mixing zone comprises a static mixer having not less than 10 groups of staggered fish teeth structures for realizing uniform mixing of the nanometerized platelet suspension and the PRP growth factor concentrate.

[0016] The application further provides a nanometerized platelet particle prepared by the preparation method.

[0017] Meanwhile, the application further provides application of the nanometerized platelet particle in preparation of a medicine for preventing and / or treating renal ischemia-reperfusion injury.

[0018] The medicine is an intravenous injection medicine, and the medicine comprises medicines for an ischemic preconditioning period, a reperfusion injury period, an acute inflammation period, an early repair period and a late repair period.

[0019] The application has the following beneficial effects: (1) The application combines a synergistic nanometerization strategy of repeated freeze-thawing and microfluidic shearing, and overcomes the defects of easy blockage, large activity loss of a traditional physical extrusion method and poor uniformity of a single ultrasonic method. The microfluidic technology ensures uniform particle size and good reproducibility of the nanometer particles, and lays a foundation for standardized production; the prepared nanometerized platelet-PRP composite particles have both the physical advantage of easy penetration of the nanometer particles through a tissue barrier and the biochemical treatment advantage of multiple growth factors of PRP, multiple synergistic treatment effects are achieved, and target specificity and bioavailability are significantly improved.

[0020] (2) The special microfluidic chip designed in the application synchronously completes nanometerization breaking of the platelets and instant mixing of the active ingredients in a closed and continuous system through integrated sampling, breaking and mixing areas. The integrated process not only greatly improves the preparation efficiency, avoids pollution and activity loss that may be caused by traditional multi-step operations, but also ensures uniformity of particle size of the final product nanometer particles and batch stability through precise fluid control.

[0021] (3) The double channels and the built-in filter membrane arranged in the sampling area can respectively introduce components and filter out potential impurities or large aggregates at the source, and provide pure raw materials for subsequent processing.

[0022] (4) The contraction-dilation type microchannel array used in the breaking area realizes physical nanometerization through controllable fluid shearing force, and compared with ultrasonic or repeated freeze-thawing methods, the action is more uniform, the thermal effect is lower, and the integrity of the biological active substances contained in the platelets can be better protected.

[0023] (5) The staggered fishbone tooth static mixer built in the mixing area can realize rapid and sufficient mixing of the nanometer particles and the growth factors under the condition of no external power through the microfluid channel structure, and ensures homogeneity and functional consistency of the composite particle components.

[0024] (6) The prepared nanometerized platelet-PRP composite particles are applied to the prevention and / or treatment of renal ischemia-reperfusion injury, and systemic administration is achieved through intravenous injection, overcoming the risk of local injection. Further, a staged dose adjustment administration scheme is implemented according to the disease pathological process (such as the ischemic preconditioning period, the reperfusion injury period, etc.), realizing the transition from passive treatment to active intervention and dynamic regulation, making the drug treatment window wider and the efficacy better, while potentially reducing the risk of side effects.

[0025] (7) The prepared nanometerized platelet-PRP composite particles can effectively act on the kidney through intravenous injection, and through multiple mechanisms such as improving mitochondrial function, reducing oxidative stress and inhibiting inflammatory response, synergistically promoting the repair of renal ischemia-reperfusion injury, and significantly improving renal function indicators. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a flowchart of the preparation method of the nanometerized platelet particles of embodiment 1 of the present application.

[0027] Figure 2 It is an axonometric view of the microfluidic chip used in the preparation method of the nanometerized platelet particles of embodiment 1 of the present application.

[0028] Figure 3 It is a front view of the microfluidic chip used in the preparation method of the nanometerized platelet particles of embodiment 1 of the present application.

[0029] Figure 4 It is the JC-1 and ROS level change of PRP treatment of renal ischemia-reperfusion injury in rats of experimental example 1, P<0.05, **, P<0.01, ***, P<0.001.

[0030] Figure 5 It is the serum creatinine (CRE) and urea nitrogen (BUN) level change of PRP treatment of renal ischemia-reperfusion injury in rats of experimental example 1, P<0.05, **, P<0.01, ***, P<0.001.

[0031] Figure 6 It is the SOD and MDA level change of PRP treatment of renal ischemia-reperfusion injury in rats of experimental example 1, P<0.05, **, P<0.01, ***, P<0.001.

[0032] Figure 7 It is the TNF-α, IL-6 and IL-1β level change of PRP treatment of renal ischemia-reperfusion injury in rats of experimental example 1, P<0.05, **, P<0.01, ***, P<0.001.

[0033] Reference signs: 1, microfluidic chip; 2, filter membrane; 3, sample inlet channel; 4, microchannel array; 5, static mixer. DETAILED DESCRIPTION

[0034] The specific embodiments are described below with reference to the accompanying drawings.

[0035] Example 1 The present embodiment provides a preparation method of nanoscale platelet particles, and the preparation process is shown in the accompanying drawing Figure 1 The preparation method comprises the following steps: (a) Isolation and purification of platelets: platelet-rich plasma is obtained by separation from anticoagulated blood, and platelet precipitate and PRP growth factor concentrate are further separated and purified. The separation and purification of the PRP growth factor concentrate is carried out at room temperature, first centrifuged at 450 x g for 10 min, and the upper plasma and white membrane layer, i.e. crude PRP, are collected; then the crude PRP is centrifuged at 1200 x g for 8 min, and the supernatant is taken, i.e. the PRP growth factor concentrate.

[0036] (b) Nanoscale treatment of platelets: after resuspension of the platelet precipitate, repeated freeze-thaw cycle treatment and microfluidic chip 1 shearing treatment are sequentially carried out to obtain a nanoscale platelet suspension. The repeated freeze-thaw cycle treatment is a cycle of liquid nitrogen quick freezing and 37°C water bath recovery, and the cycle number is 3-7 times. The flow rate of the microfluidic chip 1 shearing treatment is 80-120 μL / min, and the working pressure is 25-35 psi.

[0037] (c) Mixing of nanoparticles: the nanoscale platelet suspension and the PRP growth factor concentrate are mixed in a volume ratio of (4-6):1, and after centrifugation, the supernatant is taken, i.e. the nanoscale platelet-PRP composite particles are obtained. After mixing of the nanoscale platelet suspension and the PRP growth factor concentrate, centrifugation at 10000 x g for 15 min is carried out to remove unbroken fragments, and a nanoscale PRP composite particle suspension is obtained.

[0038] Specifically, the specific operation of step (a) of the present embodiment for isolation and purification of platelets comprises the following steps: (1) Animal preparation and blood collection: healthy adult SD rats are taken, and anesthesia is performed by intraperitoneal injection of sodium pentobarbital (40 mg / kg). After the anesthesia takes effect, the facial hair is cut off, and 4 mL of blood is collected from the retro-orbital plexus using a heparin sodium washed capillary glass tube, and is quickly transferred to a 10 mL centrifuge tube containing 450 μL of sodium citrate anticoagulant. Immediately, the centrifuge tube is inverted up and down 8-10 times gently to ensure that the blood and anticoagulant are thoroughly mixed to prevent coagulation.

[0039] (2) Obtain crude PRP: Anticoagulated whole blood is centrifuged at 450 x g for 10 minutes at room temperature (22 ± 2 °C). After centrifugation, the blood separates into three layers: the bottom layer is red blood cells, the middle thin layer is white blood cells (buffy coat), and the top layer is plasma. The plasma and the immediately adjacent buffy coat layer are carefully pipetted and transferred to a new sterile centrifuge tube. This is the crude PRP.

[0040] (3) Isolate platelet pellet and PRP growth factor concentrate: The crude PRP obtained above is centrifuged again at 1200 x g for 8 minutes at room temperature. After centrifugation, the platelets will pellet at the bottom of the tube, and the supernatant is the PRP growth factor concentrate. The supernatant is carefully pipetted into another sterile tube and set aside. The creamy white pellet at the bottom of the tube is the platelet pellet.

[0041] (4) Wash platelets: Add 5 mL of pre-chilled phosphate buffered saline (PBS) to the platelet pellet and gently resuspend by pipetting. After resuspension, centrifuge at 400 x g for 8 minutes at room temperature, and discard the supernatant. This step is intended to remove residual plasma proteins. Subsequently, add 2 mL of red blood cell lysing solution to the pellet and incubate on ice for 10 minutes to completely remove any contaminating red blood cells. After incubation, centrifuge at 2600 x g for 10 minutes, and carefully discard the supernatant to obtain a pure platelet pellet.

[0042] (5) Resuspend platelets: Add 1 mL of PBS to the washed platelet pellet and gently resuspend by pipetting. Count the platelets using a hemocytometer and adjust the platelet concentration to 1 x 109cells / mL with PBS. The platelets are ready for use.

[0043] Specifically, the specific operation of step (b) of the nanofication treatment of platelets of the present embodiment includes the following steps: (1) Repeated freeze-thawing for initial disruption: The resuspended platelet suspension is aliquoted into 1.5 mL cryovials. Directly immerse the cryovials in liquid nitrogen for rapid freezing for 2 minutes, then quickly transfer to a 37 °C water bath for 3 minutes of shaking recovery. This is one freeze-thaw cycle. Repeat this process for a total of 5 cycles. This step effectively disrupts the cell membrane and organelle structure of the platelets, laying the foundation for subsequent nanofication.

[0044] (2) Fine shearing by microfluidic chip 1: The freeze-thawed platelet suspension is subjected to shearing treatment by microfluidic chip 1.

[0045] Specifically, the specific operation of step (c) of the mixing and purification of nanoparticles of the present embodiment includes the following steps: The PRP growth factor concentrate prepared in step (a) is introduced into the microfluidic chip 1 through another inlet channel 3, and is combined with the platelet suspension after nanofraction in the breaking zone. The two are mixed in the mixing zone according to a volume ratio of 5:1. The mixed suspension is collected from the outlet of the chip.

[0046] The collected composite suspension is centrifuged at 10,000 x g for 15 minutes at 4°C. After centrifugation, the supernatant is carefully aspirated, which is the final nanofractioned platelet-PRP composite particle suspension. A small amount of precipitate of incompletely broken large fragments that may exist at the bottom of the tube is discarded. The resulting nanosuspension can be stored for short-term at 4°C or long-term at -80°C for later use.

[0047] In this embodiment, the microfluidic chip 1 used for shearing treatment in step (b) comprises: An inlet zone is provided with two independent inlet channels 3 for introducing platelet suspension and PRP growth factor concentrate, respectively. The channel in the inlet zone has an inner diameter of 1000 μm and is internally provided with a 0.22 μm filter membrane 2. A breaking zone in communication with the inlet zone is provided with a contraction-expansion type microchannel array 4 for nanofractioning platelets. In the contraction-expansion type microchannel array 4 of the breaking zone, the width of the contraction section gradually changes from 500 μm to 50 μm, and the width of the expansion section gradually changes from 50 μm to 200 μm. A mixing zone in communication with the breaking zone is provided with a static mixer 5 for mixing the nanofractioned platelet suspension with the PRP growth factor concentrate. The mixing zone comprises a static mixer 5, which has not less than 10 groups of staggered fish teeth structures for realizing uniform mixing of the nanofractioned platelet suspension and the PRP growth factor concentrate.

[0048] Specific implementation process: In this embodiment, a specially designed microfluidic chip 1 is used for nanofractionation and compounding of platelets. The inlet zone is responsible for accurately and controllably introducing the pretreated sample into the chip system and performing preliminary purification. The zone is provided with two independent inlet channels 3. One channel is used to introduce the platelet suspension pretreated by repeated freezing and thawing, and the other channel is used to introduce the PRP growth factor concentrate prepared in Example 1. The channel has an inner diameter of 1000 μm to ensure smooth flow of the fluid and reduce the risk of blockage. The key is that a 0.22 μm filter membrane 2 is internally provided at the inlet of each inlet channel 3. The filter membrane 2 filters out possible small impurities, protein aggregates or incompletely broken large cell fragments in the sample, ensuring that the material entering the subsequent precision processing zone is pure and protecting the downstream microstructure from blockage.

[0049] The breaking zone utilizes fluid shear force to break the pretreated platelets into nanoscale particles efficiently and uniformly. The zone is designed with a contraction-expansion microchannel array 4. The structural feature is that the channel width periodically changes from 500 μm (the beginning of the contraction section) to 50 μm (the narrowest part of the contraction section), and then from 50 μm (the beginning of the expansion section) to 200 μm (the widest part of the expansion section). When the platelet suspension flows through these narrow contraction sections under pressure driving, the fluid velocity increases sharply, generating strong shear force; when entering the expansion section, the fluid undergoes cavitation effect and turbulent flow. This periodic acceleration-deceleration change exerts repeated tensile and extrusion stress on the platelet cells, thereby mechanically breaking them into nanoparticles. By precisely controlling the flow rate (80-120 μL / min) and working pressure (25-35 psi), the particle size of the nanoparticles can be precisely controlled.

[0050] The mixing zone rapidly, fully and uniformly mixes the nanofied platelet suspension with the PRP growth factor concentrate. The zone integrates a static mixer 5 with more than 10 groups of staggered fishbone teeth structures inside. When the two liquid streams enter this region, the fishbone teeth structures will continuously divide, twist and recombine the liquid streams, generating strong chaotic convection at the microscale. This design does not require any moving parts, and the two liquids can be efficiently mixed by the microchannel structure itself, ensuring that each nanofied platelet particle can fully contact the growth factors in the PRP to form a composite preparation with uniform composition.

[0051] The product prepared in this embodiment is a nanofied platelet-PRP composite particle suspension. The suspension appears translucent and milky white, and under laser pen irradiation, a clear Tyndall effect can be observed, indicating that it forms a stable nanoscale colloidal dispersion system. The average particle size is 152.3±12.7 nm, and the polydispersity index (PDI) is 0.18, indicating that the particle size is uniform and the distribution range is narrow. The Zeta potential is -25.4±2.1 mV, indicating that the system has good electrostatic stability. The total protein concentration of the particle suspension is 4.5 mg / mL as determined by the BCA method. The product is rich in platelet-derived growth factors (such as PDGF-BB, TGF-β1, VEGF, etc.), and maintains the biological activity of these growth factors.

[0052] Example 2 This embodiment provides an intravenous injection drug for treating renal ischemia-reperfusion injury, which is composed of the nanofied platelet-PRP composite particles prepared in Example 1 and a drug carrier; the drug carrier is sterile normal saline (0.9% NaCl solution).

[0053] The weight fraction of the nanofied platelet-PRP composite particles and the drug carrier is: the concentration of the nanofied platelet-PRP composite particles (total protein) in the final drug is 1 mg / mL. That is, in every 1 mL of intravenous injection drug, 1 mg of nanofied platelet-PRP composite particles (total protein) is contained, and the rest is sterile normal saline.

[0054] The preparation method is: under sterile conditions, take an appropriate amount of the nanofied platelet-PRP composite particle suspension prepared in Example 1, dilute with sterile normal saline, mix gently, filter through a 0.22 μm microporous filter, and then divide into sterile vials to obtain the product.

[0055] Comparative Example 1 The difference between Example 1 and Comparative Example 1 is that only the repeated freeze-thaw method is used for nanofication, and the microfluidic chip shearing treatment is not used.

[0056] The specific steps are: after completing the step (a) of separating and purifying the platelets in Example 1, the resuspended platelet suspension is subjected to 5 cycles of liquid nitrogen quick freezing and 37°C water bath recovery. Subsequently, without microfluidic chip treatment, the freeze-thawed suspension is directly mixed with the PRP growth factor concentrate at a volume ratio of 5:1, and then centrifuged at 10,000 x g for 15 minutes. The supernatant is taken to obtain the product of Comparative Example 1.

[0057] The results of the comparative experiment are shown in Table 1 below: the average particle size of the product of Comparative Example 1 is 485.6 ± 105.4 nm, and the polydispersity index (PDI) is 0.45. The results show that the particle size obtained by only using the repeated freeze-thaw method is large and the distribution is extremely uneven, there are a large number of sub-micron vesicles and fragments, and the nanofication effect is far inferior to the synergistic method of Example 1 of the present application.

[0058] Table 1. Physicochemical properties of nanofied platelet-PRP composite particles

[0059] Experimental Example In vivo administration experiment for treating renal ischemia-reperfusion injury 1. Establishment of animal model: (1) Grouping and preparation: take several healthy male SD rats, and randomly divide them into 3 groups: sham operation group (Sham group), model group (Model group) and nanometer particle treatment group (NP-PRP group). All rats were fasted for 12 hours before the experiment, and free water was provided.

[0060] (2) Modeling surgery: All rats except the Sham group were subjected to renal ischemia-reperfusion surgery. After anesthesia with pentobarbital sodium, the rats were fixed in a supine position, and the skin was prepared and disinfected. The skin and muscle layer were incised along the midline of the abdomen, and the kidney was exposed. First, the left kidney was ligated and removed. Then, the right kidney peduncle (renal artery and vein) was clamped using a non-traumatic artery clamp to completely block the blood flow. At this time, the right kidney color changed from bright red to dark red, indicating the start of ischemia. Ischemia lasted for 60 minutes.

[0061] 2. Drug administration scheme: (1) Administration route: All injections were performed by tail vein injection. Before injection, the mouse tail was wiped with alcohol cotton ball to dilate the tail vein. The middle segment of the tail vein was selected, and the needle was inserted at an angle of about 5-15 degrees. After confirming that the needle was in the vein, the drug was slowly injected.

[0062] (2) Dose and time point of administration: The drug administration scheme was adjusted in stages in this experiment.

[0063] PRP group: 5 minutes before reperfusion (i.e., at the end of ischemia), the nanoscale platelet-PRP composite particle suspension prepared in Example 1 was injected into the tail vein, with a dose of 1 mg / kg (based on the total protein of the particles). Thereafter, the same dose of nanoparticles was administered again on the 3rd, 7th, and 10th days after reperfusion.

[0064] Sham group and Model group: At the same time point, an equal volume of normal saline was injected into the tail vein as a control.

[0065] (3) Reperfusion: After the first administration, the artery clamp on the right kidney was loosened to restore blood perfusion, and the color of the kidney quickly returned to bright red. Then, the abdominal incision was sutured layer by layer.

[0066] On the 14th day after reperfusion, all rats were anesthetized, weighed, and sampled. Blood was collected for serum separation, and kidney tissue was removed.

[0067] Renal function assessment: The levels of serum creatinine (CRE) and urea nitrogen (BUN) were detected.

[0068] Histological and molecular biological assessment: The kidney tissue was subjected to JC-1 staining to detect mitochondrial membrane potential, DHE staining to detect reactive oxygen species (ROS) levels, and detection of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. At the same time, the levels of inflammatory factors TNF-a, IL-6, and IL-1b in the serum were detected by ELISA kit.

[0069] The results are shown in Figure 4 The mitochondrial membrane potential of the PRP group was significantly restored compared with the Model group. Figure 5It is shown that the creatinine (CRE) level of the PRP group is significantly lower than that of the Model group (P<0.01) and the urea nitrogen (BUN) level is higher than that of the Model group (p<0.05), which indicates that the renal function is recovered.

[0070] Figure 6 It is shown that the SOD level of the PRP group is higher than that of the Model group, and the MDA level is consistent with that of the Sham group and is higher than that of the Model group (P<0.05), which indicates that the renal oxidative stress is alleviated, Figure 7 It is shown that the levels of inflammatory factors TNF-α, IL-6 and IL-1β are decreased, which indicates that the renal inflammation is alleviated.

[0071] These data comprehensively show that the nanometerized platelet-PRP composite particles prepared in the application can effectively alleviate the renal ischemia-reperfusion injury through intravenous injection, and promote the recovery of renal function and tissue structure.

Claims

1. A method for preparing nanosized platelet particles, characterized in that, The method comprises the following steps: (a) Separation and purification of platelets: platelet-rich plasma is separated from anticoagulated blood, and platelet pellets and PRP growth factor concentrate are further separated and purified; (b) Nanoparticle treatment of platelets: after resuspension of the platelet pellets, repeated freeze-thaw cycle treatment and microfluidic chip (1) shear treatment are sequentially performed to obtain a nanoparticle platelet suspension; (c) Mixing of nanoparticles: the nanoparticle platelet suspension and the PRP growth factor concentrate are mixed at a volume ratio of (4-6):1, and the supernatant is obtained after centrifugation, thereby obtaining the nanoparticle PRP composite particles.

2. The method of claim 1, wherein the nanofϊcated platelet particles are prepared by a method comprising the steps of: In step (a), the separation and purification of the PRP growth factor concentrate is performed at room temperature, first centrifuged at 450xg for 10 min, and the upper plasma and white membrane layer, i.e. crude PRP, is collected; Then the crude PRP is centrifuged at 1200xg for 8 min, and the supernatant is collected, which is the PRP growth factor concentrate.

3. The method for preparing nano-sized platelet particles according to claim 1, characterized in that, In step (b), the repeated freeze-thaw cycle treatment is a cycle of liquid nitrogen freezing and 37°C water bath recovery, and the cycle number is 3-7 times; the flow rate of the microfluidic chip (1) shear treatment is 80-120 μL / min, and the working pressure is 25-35 psi.

4. The method for preparing nano-sized platelet particles according to claim 3, characterized in that, The microfluidic chip (1) used for shear treatment in step (b) comprises: a sample inlet area provided with a double-path independent sample inlet channel (3), a filter membrane (2) built-in the sample inlet area for separately introducing platelet suspension and PRP growth factor concentrate; a crushing area in communication with the sample inlet area, the crushing area is provided with a contraction-expansion type microchannel array (4) for nanoparticle crushing of platelets; a mixing area in communication with the crushing area, the mixing area is provided with a static mixer (5) for mixing the nanoparticle platelet suspension and the PRP growth factor concentrate.

5. The method for preparing nano-sized platelet particles according to claim 4, characterized in that, In the contraction-expansion type microchannel array (4) of the crushing area, the width of the contraction section gradually changes from 500 μm to 50 μm, and the width of the expansion section gradually changes from 50 μm to 200 μm.

6. The method for preparing nano-sized platelet particles according to claim 4, characterized in that, The mixing area comprises a static mixer (5) having not less than 10 groups of staggered fish teeth structures for realizing uniform mixing of the nanoparticle platelet suspension and the PRP growth factor concentrate.

7. The method for preparing nano-sized platelet particles according to claim 1, characterized in that, In step (c), after mixing the nanoparticle platelet suspension and the PRP growth factor concentrate, centrifugation is performed at 10000xg for 15 min to remove unbroken fragments, and a nanoparticle PRP composite particle suspension is obtained.

8. A nanoparticle platelet particle prepared by the nanoparticle platelet particle preparation method of any one of claims 1-7.

9. Use of the nanoparticle platelet particle of claim 9 and / or the nanoparticle platelet particle preparation method of any one of claims 1-8 in the preparation of a medicament for preventing and / or treating renal ischemia-reperfusion injury.

10. Use according to claim 9, characterized in that, The medicament is an intravenous injection medicament, and the medicament comprises medicaments for ischemic preconditioning period, reperfusion injury period, acute inflammation period, early repair period and late repair period.

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