Drug-loaded system, preparation method and application thereof

Abstract

The application discloses a drug-loaded system and a preparation method and application thereof, and belongs to the technical field of medicines. The drug-loaded system is a drug system in which a nano drug-loaded material is carried on a supermolecular engineering modified frozen platelet; the nano drug-loaded material is a drug-loaded nanomaterial modified by a first modifier; the supermolecular engineering modified frozen platelet is a liquid nitrogen quick-frozen activated platelet modified by a second modifier; and the first and second modifiers can be self-assembled and combined. The frozen platelet retains thrombus targeting, and after the supermolecular system is engineered to carry the nano drug system, the targeted delivery and rapid release of drugs to a thrombus site can be realized, the drug-loaded system has high safety and specificity, and therefore has high broad-spectrum and promotion values. The preparation method of the drug-loaded system is simple, and the drug-loaded system can be used for preparing a medicine for treating thromboembolic diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a drug delivery system, a preparation method and application thereof. BACKGROUND

[0002] Thromboembolic disease is a vascular event with a mortality rate of more than 25%, which usually occurs during or after thrombosis and vascular occlusion, mainly caused by arterial or venous thrombosis, such as ischemic stroke, pulmonary embolism and mesenteric embolism. Thromboembolic disease has a rapid onset and progression, and the treatment time window is narrow, so timely treatment is particularly important.

[0003] However, the therapeutic drugs lack target selectivity, have a short half-life, and secondary bleeding after treatment greatly reduces the therapeutic effect of the drugs. Traditional nano-preparations such as liposomes, micelles, polymeric nanoparticles, inorganic nanomaterials, nanogels and nanocapsules have low targeting efficiency for thrombus sites, require additional modification of targeting proteins, have complex preparation methods, and have potential toxic effects, which limit their wide application. Biomimetic nano- or micro-preparations usually have natural targeting, good immunocompatibility, and enhanced targeted delivery of drugs to disease sites.

[0004] Platelets are widely used as biomimetic carriers for drug delivery for thrombus-related diseases due to their natural wound targeting. However, due to the strong membrane fluidity of living platelets, active endocytosis and exocytosis, and the stability of modified components after non-covalent modification of the surface, it is difficult to maintain the characteristics of the platelets during the modification process.

[0005] The current common method is to strictly control the modification conditions and limit the storage time after modification, but maintaining the long-term stability after modification still faces great challenges.

[0006] In addition, for diseases related to thromboembolism, exploring new delivery strategies and developing a nano-drug delivery platform with biocompatibility, low biological toxicity and strong biological targeting is an important direction for preclinical research.

[0007] In view of this, the present application is proposed. SUMMARY

[0008] One of the purposes of the present application is to provide a drug delivery system which can achieve targeted delivery of drugs to thrombus sites and rapid release of drugs at thrombus sites, has high safety and specificity, and thus has high broad-spectrum and promotional value.

[0009] The second purpose of the present application is to provide a preparation method of the drug delivery system.

[0010] The third purpose of the present application is to provide an application of the drug delivery system.

[0011] The application can be implemented as follows:

[0012] In a first aspect, the application provides a drug delivery system, which is a drug delivery system of a supramolecular engineered modified frozen platelet carrying a nanometer drug delivery material.

[0013] The nanometer drug delivery material is a nanometer material carrying a drug; the nanometer material is modified by a first modifier, and the supramolecular engineered modified frozen platelet is a liquid nitrogen flash frozen activated platelet modified by a second modifier.

[0014] The first modifier comprises a first host molecule and / or a derivative thereof, or the first modifier comprises a first guest molecule and / or a derivative thereof; the second modifier comprises a second host molecule and / or a derivative thereof, or the second modifier comprises a second guest molecule and / or a derivative thereof.

[0015] The first modifier and the second modifier can self-assemble and combine.

[0016] In an optional embodiment, the first modifier is a first host molecule and / or a derivative thereof, and the second modifier is a second guest molecule and / or a derivative thereof that can self-assemble and combine with the first modifier.

[0017] In an optional embodiment, the modification of the nanometer drug delivery material by the first modifier is covalent bond modification.

[0018] In an optional embodiment, the modification of the frozen platelet by the second modifier is non-covalent bond modification.

[0019] In an optional embodiment, the nanometer drug delivery material comprises a nanometer material and a drug loaded on the nanometer material.

[0020] In an optional embodiment, the nanometer material is a drug-releasable material.

[0021] In an optional embodiment, the nanometer material is in the form of particles.

[0022] In an optional embodiment, the nanometer material comprises at least one of a liposome, a micelle, a polymer nanoparticle, an inorganic nanometer material, a nanogel, and a nanocapsule.

[0023] In an optional embodiment, the nanometer material comprises at least one of a liposome and a micelle.

[0024] In an optional embodiment, the nanometer material comprises at least one of a liposome capable of simultaneously loading a fat-soluble drug and a water-soluble drug, and a micelle capable of simultaneously loading a fat-soluble drug and a water-soluble drug.

[0025] In optional embodiments, the liposome and / or the micelle is a pegylated normal or responsive liposome and / or micelle.

[0026] In optional embodiments, the drug comprises at least one of a thrombolytic drug, an anti-platelet aggregation drug, an anticoagulant, a vasodilator, and a nerve cell protective agent.

[0027] In optional embodiments, the thrombolytic drug comprises at least one of urokinase, streptokinase, alteplase, and tenecteplase;

[0028] and / or, the anti-platelet aggregation drug comprises at least one of aspirin, clopidogrel, and dipyridamole;

[0029] and / or, the anticoagulant comprises at least one of sodium heparin, sodium warfarin, and alglucosidase alfa;

[0030] and / or, the vasodilator comprises at least one of flunarizine, betahistine, and dihydroergotoxin;

[0031] and / or, the nerve cell protective agent comprises at least one of nimodipine and mannitol.

[0032] In optional embodiments, the drug is in free form in the nanomaterial.

[0033] In optional embodiments, the first host molecule and the second host molecule are independently a macrocyclic molecule with an external hydrophilic and an internal hydrophobic, and / or, the first guest molecule and the second guest molecule are independently a hydrophobic molecule.

[0034] In optional embodiments, the first guest molecule and the second guest molecule are independently capable of binding to the hydrophobic site of the macrocyclic molecule and forming an inclusion structure in an aqueous solution.

[0035] In optional embodiments, the macrocyclic molecule comprises at least one of a cyclodextrin, a cucurbituril, a pillararene, a calixarene, and a crown ether;

[0036] In optional embodiments, the cyclodextrin comprises a β-cyclodextrin;

[0037] and / or, the cucurbituril comprises at least one of a cucurbit[6]uril and a cucurbit[7]uril;

[0038] and / or, the pillararene comprises at least one of a pillar[5]arene or a pillar[6]arene;

[0039] and / or, the crown ether comprises at least one of a bicyclic crown ether, a tricyclic crown ether, a polycyclic crown ether, and a heterocyclic crown ether.

[0040] In optional embodiments, the first guest molecule and the second guest molecule independently comprise at least one of adamantane, ferrocene, spermine, and azobenzene.

[0041] In an alternative embodiment, the first guest molecule and the second guest molecule independently comprise adamantane.

[0042] In an alternative embodiment, the first host molecule and the second host molecule independently comprise cucurbituril, and the second guest molecule and the second guest molecule independently comprise at least one of adamantane and ferrocene.

[0043] In an alternative embodiment, the molar ratio of the host molecule and / or its derivative to the guest molecule and / or its derivative in the drug delivery system is 1-100:1-100.

[0044] In an alternative embodiment, the molar ratio of the host molecule and / or its derivative to the guest molecule and / or its derivative in the drug delivery system is 1:1.

[0045] In an alternative embodiment, the size of the platelets in the frozen platelets is micrometer size.

[0046] In an alternative embodiment, the frozen platelets are obtained by subjecting live platelets to quick-freezing in liquid nitrogen, thawing, and modifying with a second modifier.

[0047] In an alternative embodiment, the molar ratio of the host molecule and / or its derivative to the guest molecule and / or its derivative in the drug delivery system is 1-100:1-100.

[0048] In an alternative embodiment, the incubation temperature is 20-37°C, and / or the incubation time is 1 min-1 h.

[0049] In an alternative embodiment, the incubation time is 15 min.

[0050] In an alternative embodiment, the incubation time is 15 min.

[0051] The beneficial effects of the present application include:

[0052] The frozen platelets in the drug delivery system provided by the present application can retain thrombus targeting, can significantly inhibit thrombus aggravation caused by live activated platelet transfusion, and can achieve targeted delivery of drugs to the thrombus site after the frozen platelets are modified by the supramolecular system and loaded with the nanodrug system, thereby having high safety and specificity. In other words, the frozen platelets used in the present application have almost lost activity, but retain the tendency to the thrombus site, the supramolecular surface modification has no significant effect on the thrombus tendency of the platelets, and the loaded drug-loaded nanomaterials can achieve rapid release of drugs at the thrombus site, thereby having high broad-spectrum and promotional value. BRIEF DESCRIPTION OF DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0054] Figure 1 Scanning electron microscope images of pre-activated living platelets (A) and liquid nitrogen snap-frozen platelets (B) in Example 1 of the present application;

[0055] Figure 2 Flow cytometry detection statistical graph of supermolecular engineering modified frozen platelets in Example 2 of the present application;

[0056] Figure 3 Targeting imaging graph of cerebral thrombosis site before and after supermolecular engineering modification in Example 3 of the present application;

[0057] Figure 4 Flow cytometry detection statistical graph of the content of fluorescent reactive oxygen species responsive liposomes on the surface of the frozen platelets in Example 4 of the present application;

[0058] Figure 5 Transmission electron microscope imaging graph of the host-guest self-assembly after liquid nitrogen snap-frozen platelets and reactive oxygen species responsive liposomes are incubated at room temperature for 15 minutes in Example 4 of the present application;

[0059] Figure 6 Statistical graph of the binding characteristics of reactive oxygen species responsive nanoparticles and supermolecular engineering modified frozen platelets carrying nanoparticles with activated platelets in vitro in Example 5 of the present application;

[0060] Figure 7 Targeting imaging graph of the stroke site of the responsive nanoparticles, the frozen platelets carrying reactive oxygen species responsive liposomes, and the supermolecular engineering modified frozen platelets in Example 6 of the present application. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described as follows. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be obtained by market purchase.

[0062] The drug-loaded system provided by the present application and the preparation method and application thereof will be specifically described as follows.

[0063] The present application provides a drug delivery system, which is a drug delivery system of supermolecular engineering modified frozen platelets carrying nanometer drug delivery materials.

[0064] In the drug delivery system, the nanometer drug delivery material is a drug-loaded nanomaterial modified by a first modifier, and the supermolecular engineering modified frozen platelet is a liquid nitrogen flash frozen activated platelet modified by a second modifier.

[0065] The first modifier comprises a first host molecule and / or a derivative thereof, or the first modifier comprises a first guest molecule and / or a derivative thereof; and the second modifier comprises a second host molecule and / or a derivative thereof, or the second modifier comprises a second guest molecule and / or a derivative thereof.

[0066] The first modifier and the second modifier can self-assemble and combine.

[0067] That is, it can be understood that the first modifier and the second modifier are in a host-guest interaction relationship. If the first modifier is a first host molecule and / or a derivative thereof, the second modifier is a second guest molecule (and / or a derivative thereof) capable of self-assembling and combining with the first host molecule (and / or a derivative thereof). Correspondingly, the drug delivery system is obtained by self-assembly of the drug-loaded nanomaterial modified by the host molecule (and / or a derivative thereof) and the liquid nitrogen flash frozen activated platelet modified by the guest molecule (and / or a derivative thereof).

[0068] Similarly, if the first modifier is a first guest molecule and / or a derivative thereof, the second modifier is a second host molecule (and / or a derivative thereof) capable of self-assembling and combining with the first guest molecule (and / or a derivative thereof). Correspondingly, the drug delivery system is obtained by self-assembly of the drug-loaded nanomaterial modified by the guest molecule (and / or a derivative thereof) and the frozen platelet modified by the host molecule (and / or a derivative thereof).

[0069] In some preferred embodiments, the first modifier is a first host molecule and / or a derivative thereof, and the second modifier is a second guest molecule (and / or a derivative thereof) capable of self-assembling and combining with the first modifier. Correspondingly, the drug delivery system is obtained by self-assembly of the frozen platelet modified by the host molecule (and / or a derivative thereof) and the drug-loaded nanomaterial modified by the guest molecule (and / or a derivative thereof), which facilitates the determination of the quantity and stability of the modified host molecule (and / or a derivative thereof) on the frozen platelet.

[0070] In the above drug delivery system, the frozen platelet serves as a targeting carrier, and the drug-loaded nanomaterial is the main component for thrombus treatment.

[0071] It should be noted that platelets are one of the essential components in blood, and the main function is to participate in the formation of thrombus and promote blood coagulation. Activated platelets are one of the indispensable pathological factors of thromboembolic diseases, and its characteristics are the activation of integrin αIIbβ3, the overexpression of P-selectin (CD62P), the exposure of phosphatidylserine (PS), etc. Due to its unique characteristics, platelets and their membranes have natural advantages as carriers to deliver nanomedicines to the damaged vascular site. At present, most of the construction methods using platelets as carriers have certain limitations. The functionalization modification process of live platelets on the surface is easy to cause platelet activation, especially when the functionalization method involves chemical reaction or shear stress. At the same time, due to the membrane fluidity and endocytosis and exocytosis process of live platelets, the stability of the functionalized molecules will be reduced. In addition, the directionality of the platelet membrane biomimetic nanomaterial is difficult to be stably maintained during the preparation process, which will reduce the targeting of the damaged vascular site.

[0072] The above drug delivery system provided by the present application, wherein the frozen platelets carry nanomedicine materials for drug delivery in the treatment of thromboembolic diseases. The activated platelets treated by freezing are in a rigid state, and the membrane fluidity is lost, so that the surface modified supramolecules can exist stably, and the directionality of the membrane can be fixed to maintain strong targeting to the damaged vascular site. By self-assembly between the host molecules (and / or their derivatives) and the guest molecules (and / or their derivatives), nanomedicines can be carried to quickly deliver therapeutic drugs. This drug delivery system avoids the change of the properties of live platelets during the modification process, so that the modified molecules can exist stably for a long time, and the directionality of the platelet membrane is ensured, which reduces the influence on the targeting. In addition, the carried nanomedicine materials can achieve rapid release and treatment, thereby improving the drug delivery efficiency.

[0073] In the present application, the modification method of the first modification agent (the first host molecule (and / or its derivative) and the first guest molecule (and / or its derivative)) to the nanomedicine material can be covalent bond modification or non-covalent bond modification, preferably covalent bond modification.

[0074] The modification method of the second modification agent (the second host molecule (and / or its derivative) and the second guest molecule (and / or its derivative)) to the frozen platelets can be covalent bond modification or non-covalent bond modification, preferably non-covalent bond modification.

[0075] In the present application, the nanomedicine material includes a nanomaterial and a drug loaded on the nanomaterial.

[0076] For reference, the nanomaterial is a nanomaterial that can easily release drugs, which can be in the form of particles, and other forms are not excluded.

[0077] In some embodiments, the nanomaterials can include at least one of liposomes, micelles, polymeric nanoparticles, inorganic nanomaterials, nanogels, and nanocapsules.

[0078] The nanomaterials described above can release the loaded drugs rapidly.

[0079] In some preferred embodiments, the nanomaterials include at least one of liposomes and micelles. This type of liposomes, micelles, etc. can simultaneously load both lipid-soluble and water-soluble drugs, and after being carried by the frozen platelets, can reach the thrombus site to promote rapid release of the drugs.

[0080] In some more preferred embodiments, the nanomaterials include at least one of liposomes capable of simultaneously loading lipid-soluble drugs and water-soluble drugs and micelles capable of simultaneously loading lipid-soluble drugs and water-soluble drugs.

[0081] In addition to the effect of rapid drug release, the preferred nanomaterials described above have no significant impact on the targeting of the frozen platelets after modification.

[0082] For example, the liposomes are PEGylated normal or responsive liposomes, and the micelles are PEGylated normal or responsive micelles.

[0083] The nanomaterials described above, such as polymeric nanoparticles, inorganic nanomaterials, nanogels, and nanocapsules, which are easily recognized and removed by the immune system, can also enhance tissue compatibility, reduce the removal of immune cells, and reduce potential toxicity under the carrying effect of the frozen platelets.

[0084] For reference, the drugs can include at least one of thrombolytic drugs, anti-platelet aggregation drugs, anticoagulants, vasodilators, and nerve cell protective agents.

[0085] For example, but not limited to, the thrombolytic drugs can include at least one of urokinase, streptokinase, alteplase, and tenecteplase. For example, but not limited to, the anti-platelet aggregation drugs can include at least one of aspirin, clopidogrel, and dipyridamole. For example, but not limited to, the anticoagulants can include at least one of heparin sodium, warfarin sodium, and alginic acid bis esterase. For example, but not limited to, the vasodilators can include at least one of flunarizine, betahistine, and buflomedil. For example, but not limited to, the nerve cell protective agents can include at least one of nimodipine and mannitol.

[0086] Preferably, the drugs described above are in free form in the nanomaterials. By loading in free form in the nanomaterials, the stability of the drugs can be improved, and the in vivo half-life of the drugs can be prolonged.

[0087] In the present application, the binding force between the interacting host-guest molecules is essentially non-covalent bond, and its strength depends on the hydrophilic-hydrophobic interaction strength between the host-guest molecules.

[0088] Exemplarily, the host molecule (whether the first host molecule or the second host molecule) can be an external hydrophilic and internal hydrophobic macrocyclic molecule, and the guest molecule (whether the first guest molecule or the second guest molecule) can be a hydrophobic molecule. In some embodiments, the guest molecule (whether the first guest molecule or the second guest molecule) can bind to the hydrophobic position of the macrocyclic molecule with which it interacts in an aqueous solution and form a (stable) inclusion structure.

[0089] It should be noted that the above action principle can refer to: the macrocyclic host molecule (and / or its derivative) and the guest molecule (and / or its derivative) interacting therewith are connected to the active sites on the surface of the nanomaterial through non-covalent bonds to form a stable modified structure on the surface of the nanomaterial. For nanomaterials such as liposomes or micelles, the host molecule (and / or its derivative) or the guest molecule (and / or its derivative) can be modified first, and then the drug can be loaded.

[0090] For reference, the above macrocyclic molecule exemplarily but not limitatively can include at least one of cyclodextrin, cucurbituril, pillararene, calixarene and crown ether, and preferably includes cucurbituril.

[0091] Preferably, the cyclodextrin can include (be) β-cyclodextrin. The cucurbituril can include at least one of cucurbit[6]uril and cucurbit[7]uril. The pillararene can include at least one of pillar[5]arene or pillar[6]arene. The crown ether can include at least one of bicyclic crown ether, tricyclic crown ether, polycyclic crown ether and heterocrown ether.

[0092] In the present application, the guest molecule (whether the first guest molecule or the second guest molecule) can include at least one of adamantane, ferrocene, spermine and azobenzene. Preferably, the guest molecule (whether the first guest molecule or the second guest molecule) includes adamantane.

[0093] In some preferred embodiments, the host molecule (whether the first host molecule or the second host molecule) includes cucurbituril, and the guest molecule (whether the first host molecule or the second host molecule) includes at least one of adamantane and ferrocene. In some specific embodiments, the host-guest binding can be the binding of cucurbituril and adamantane; in other specific embodiments, the host-guest binding can be the binding of cucurbituril and ferrocene.

[0094] In addition, the host molecule (and / or its derivative) capable of interacting with the guest molecule (and / or its derivative) provided in the application has a high binding constant, and the corresponding binding force has a microenvironment-stimulated responsive change, so that the rapid release of the nanodrug in the thromboembolic environment can be realized.

[0095] For reference, the molar ratio of the host molecule (and / or its derivative) and the guest molecule (and / or its derivative) in the drug-loaded system described above can be 1-100:1-100, such as 1:1, 1:2, 1:5, 1:10, 1:50, 1:100, 5:1, 5:10, 5:20, 5:50, 5:80, 5:100, 10:1, 20:50, 20:80, 30:1, 30:5, 30:50, 40:1, 40:5, 40:10, 40:50, 50:1, 50:20, 50:80, 60:1, 60:10, 60:20, 60:50, 60:80, 70:1, 70:10, 70:30, 70:50, 70:80, 80:1, 80:5, 80:10, 80:50, 90:1, 90:10, 90:20, 90:50, 90:80, 100:1, 100:50, or 100:80, etc., and can also be any other value within the range of 1-100:1-100. The ratio of the host molecule and the guest molecule can be adjusted according to different needs.

[0096] In some preferred embodiments, the molar ratio of the host molecule (and / or its derivative) and the guest molecule (and / or its derivative) in the drug-loaded system is 1:1, so as to obtain a higher drug loading efficiency without affecting the targeting of platelets.

[0097] In the application, the size of the platelets in the frozen platelets is micrometers.

[0098] For reference, the frozen platelets are obtained by rapidly freezing the living platelets in liquid nitrogen, thawing, and modifying with a second modifier.

[0099] Specifically, the platelets can be separated from fresh blood containing an anticoagulant, pretreated and activated by using thrombin, then rapidly frozen in liquid nitrogen, thawed before use, and modified by a second modifier.

[0100] It should be noted that the platelets to be pretreated and activated can also be other living platelets stored at room temperature, stored at 4°C, gradiently cooled and stored, and freeze-dried and stored. In addition, the activation state of the platelets in the application can be selected according to different thrombotic diseases, which is not limited here.

[0101] For reference, the supramolecular engineered modified cryoplatelets can be obtained by incubating the cryoplatelets modified by the second host molecule (and / or its derivative) or the second guest molecule (and / or its derivative) at room temperature. The incubation time is preferably 2 hours, and the corresponding second modifier can be stably present on the surface of the platelets within 16 hours.

[0102] The supramolecular engineered cryoplatelet modification process provided in the present application is simple, has no significant effect on the properties of platelets, and the obtained product has high biocompatibility and high targeting efficiency. In addition, it has broad spectrum for various nanomaterials and thrombus-targeting cells, and thus can be considered as a potential anti-thrombus treatment strategy.

[0103] Correspondingly, the present application also provides a preparation method of the above drug-loaded system, which can include the following steps: co-incubating the nanodrug-loaded material modified by the first modifier and the cryoplatelets modified by the second modifier to allow the first modifier and the second modifier to self-assemble and combine.

[0104] That is, co-incubating the nanodrug-loaded material modified by the first host molecule and / or its derivative with the cryoplatelets modified by the second guest molecule and / or its derivative, or co-incubating the nanodrug-loaded material modified by the first guest molecule and / or its derivative with the cryoplatelets modified by the second host molecule and / or its derivative.

[0105] For reference, the incubation temperature of the nanodrug-loaded material modified by the first modifier and the cryoplatelets modified by the second modifier can be room temperature, such as 20-37℃, and can be 20℃, 25℃, 30℃, 35℃ or 37℃, etc., or any other value within the range of 20-37℃.

[0106] The incubation time of the nanodrug-loaded material modified by the first modifier and the cryoplatelets modified by the second modifier can be 1min-1h, such as 1min, 2min, 5min, 10min, 20min, 30min, 40min, 50min or 60min, etc., or any other value within the range of 1min-1h, and is preferably 15min.

[0107] In addition, the present application also provides the application of the above drug-loaded system, which can be used for preparing a drug for treating thromboembolic diseases.

[0108] Correspondingly, the present application also relates to a drug, which includes the above drug-loaded system. The drug can be a drug for treating thromboembolic diseases.

[0109] The drug release mechanisms involved in the aforementioned drug delivery systems can be simple release from the drug-loaded nanomaterials, responsive release after microenvironmental stimulation, or simultaneous response of the host and the drug-loaded nanomaterials, promoting rapid and large-scale drug release at the lesion site and enhancing deep penetration of the thrombus site.

[0110] The mechanism of action of the drug involved in this application includes: upon reaching the thrombus site, the frozen platelets carrying nanomedicine occupy binding sites on activated platelets at the thrombus site, inhibiting further activation of resting platelets in circulation. Drug-loaded nanomaterials can rapidly release drugs for thrombolysis, anti-inflammation, anti-platelet activation, or neuroprotective treatments.

[0111] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0112] Example 1

[0113] This embodiment provides a method for preparing frozen platelets.

[0114] Thrombin, phosphate buffer, and other reagents used in the preparation process were purchased from Beijing Solarbio Science & Technology Co., Ltd. Glutaraldehyde was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0115] The preparation method includes: platelets are isolated from fresh blood containing anticoagulant from C57BL / 6J mice by low-speed centrifugation (100g centrifugation for 15 minutes), washed 3 times with phosphate-buffered saline (PBS), and pre-activated by stimulation with thrombin (2U / mL) and calcium chloride (8nM) for 10 minutes to obtain activated platelets.

[0116] Subsequently, activated platelets were resuspended in PBS solution containing 2% dimethyl sulfoxide, rapidly immersed in liquid nitrogen, and soaked in liquid nitrogen for 12 hours to obtain frozen platelets. The frozen platelets were then thawed at 37°C and centrifuged at 3000 rpm for 2 minutes. They were collected after washing with PBS at least three times.

[0117] The live and frozen platelets obtained above were fixed with 2.5% glutaraldehyde for 15 minutes, washed three times with pure water, and then collected. The scanning electron microscopy imaging results are as follows. Figure 1 As shown (A represents live platelets; B represents frozen platelets).

[0118] Depend on Figure 1 It can be seen that the surface of frozen platelets is rougher than that of live platelets, and they lose membrane fluidity, but their basic morphology remains unchanged.

[0119] Example 2

[0120] This embodiment provides a supramolecularly engineered frozen platelet and its preparation method.

[0121] The DSPE-PEG2000-CB[7] used in the preparation process was synthesized from DSPE-PEG2000-SH and monoallyloxy CB[7] synthesized in the laboratory. The fluorescent dye fluorescein isothiocyanate FITC-coupled adamantylamine (Ada-FITC) was purchased from Xi'an Ruishi Biological Technology Co., Ltd. (China).

[0122] The frozen platelets obtained in Example 1 were incubated with DSPE-PEG-CB[7] at 37°C for 1h, 2h, 4h, 6h, 8h and 12h respectively, and then the free DSPE-PEG-CB[7] was removed to obtain CB[7] membrane-inserted modified frozen platelets. Subsequently, Ada-FITC was used to detect the CB[7] modified on the surface of the frozen platelets, and the flow cytometry detection results (as shown in Figure 2 The relative qualitative statistical chart shows that 2 hours of incubation is the best time. Statistical significance: *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0123] Example 3

[0124] This example provides a kind of supramolecular engineering modified frozen platelet and its mouse brain stroke site targeting property.

[0125] The DSPE-PEG2000-CB[7] used in the preparation process was synthesized from DSPE-PEG2000-SH and monoallyloxy CB[7] synthesized in the laboratory. Cy5.5-NHS fluorescent molecules were purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.

[0126] According to the method in Example 2, the frozen platelets with surface modified CB[7] were obtained by incubating for 2 hours, and then a mouse acute ischemic stroke model was established by photochemical induction. At 1.5 hours after the model was established, fluorescent Cy5.5-labeled frozen platelets and supramolecular engineering modified frozen platelets were injected intravenously, respectively. At 0.5h, 1h, 1.5h and 2h after intravenous injection, the stroke brain tissue was taken for fluorescence imaging.

[0127] As shown in Figure 3 , there was no significant difference in the ability of frozen platelets to target the thrombus site of brain stroke before and after supramolecular engineering modification (CsPLT represents frozen platelets; CsPLT-CB[7] represents frozen platelets with surface modified CB[7]).

[0128] Example 4

[0129] This example provides a kind of supramolecular engineering modified frozen platelet carrying active oxygen responsive liposome and a preparation method thereof.

[0130] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Se-Se-polyethylene glycol-N-hydroxysuccinimide (DSPE-Se-Se-PEG-NHS) used in the preparation process was purchased from Xi'an Ruishi Biological Technology Co., Ltd. DSPE-PEG2000-CB[7] was synthesized by DSPE-PEG2000-SH and monoallyloxy CB[7] synthesized in the laboratory. Soybean lecithin, cholesterol, adamantylamine, fluorescent dye fluorescein isothiocyanate (FITC) and other chemical reagents were from Shanghai Aladdin Biochem Technology Co., Ltd. (China).

[0131] After DSPE-Se-Se-PEG-NHS reacted with adamantylamine at room temperature, active oxygen-responsive 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Se-Se-polyethylene glycol-adamantylamine (DSPE-Se-Se-PEG-Ada) was obtained by purification.

[0132] The active oxygen-responsive liposome cross-linked with adamantylamine was prepared by the thin film-hydration method according to the molar ratio of lecithin: cholesterol: DSPE-Se-Se-PEG-Ada of 34: 16: 14, and was repeatedly extruded 20 times by filter membranes with pore sizes of 400 nm and 200 nm, respectively.

[0133] According to the method in Example 2, the CB[7] interlayer modified frozen platelets and the fluorescent dye FITC labeled active oxygen-responsive liposome (nanoparticle) cross-linked with adamantylamine were obtained. According to the molar ratio of 1:1, they were incubated at room temperature for 1 min, 2 min, 5 min, 15 min, 30 min and 60 min, respectively. Then, the content of the fluorescent active oxygen-responsive liposome modified on the surface of the frozen platelets was detected by flow cytometry. As shown in Figure 4 (relative statistical chart), the best time was 15 minutes. Statistical significance: *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0134] At the same time, the transmission electron microscopy imaging results of the supermolecular engineering modified frozen platelets carrying active oxygen-responsive liposome system are shown in Figure 5 When incubated according to the molar ratio of 1:1, the liposomes (arrows) bound to the surface of the frozen platelets can be clearly seen.

[0135] Example 5

[0136] This example provides a supermolecular engineering modified frozen platelet carrying active oxygen-responsive liposome system and its in vitro binding characteristics with activated platelets.

[0137] In this embodiment, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Se-Se-polyethylene glycol-N-hydroxysuccinimide (DSPE-Se-Se-PEG-NHS), DSPE-PEG2000-CB[7], soybean lecithin, cholesterol, adamantylamine, fluorescent dye fluorescein isothiocyanate (FITC) and other chemical reagents are all from the same source as in Example 4. Flow cytometry antibody PE-CD62P is purchased from Biolegend.

[0138] According to the method in Example 1, frozen platelets were prepared.

[0139] According to the method in Example 2, super-molecular engineering modified frozen platelets were prepared.

[0140] According to the method in Example 4, a system of responsive nanoparticles and frozen platelets carrying responsive liposomes was prepared.

[0141] The obtained responsive nanoparticles and frozen platelets carrying responsive nanoparticles were respectively labeled with fluorescent dye FITC at room temperature, with a final concentration of 0.5 mg / mL.

[0142] According to the method in Example 1, activated platelets were extracted and obtained, and were incubated with PE-CD62P antibody for 20 minutes for labeling; then were incubated with FITC-labeled responsive nanoparticles and frozen platelets carrying responsive nanoparticles at room temperature for 30 minutes, respectively, and then flow cytometry was used to detect the activated platelets combined with responsive nanoparticles and frozen platelets carrying responsive nanoparticles. As shown in FIG. 1 (PE+ represents activated platelets with high expression of CD62P), frozen platelets carrying responsive nanoparticles can be significantly combined with activated platelets (right), while responsive nanoparticles are hardly combined with activated platelets (left). Figure 6

[0143] Example 6

[0144] This embodiment provides a system of super-molecular engineering modified frozen platelets carrying active oxygen responsive liposomes and its targeting properties in mouse brain stroke site.

[0145] In this embodiment, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Se-Se-polyethylene glycol-N-hydroxysuccinimide (DSPE-Se-Se-PEG-NHS), DSPE-PEG2000-CB[7], soybean lecithin, cholesterol, adamantylamine and other chemical reagents are all from the same source as in Example 4. Cy5.5-NHS fluorescent molecule is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.

[0146] According to the method in Example 1, frozen platelets were prepared.​

[0147] According to the method in Example 2, the supermolecular engineering modified frozen platelets were prepared.

[0148] According to the method in Example 4, the responsive nanoparticle and frozen platelet loaded responsive liposome system were prepared.

[0149] The obtained responsive nanoparticle, frozen platelet loaded responsive liposome and supermolecular engineering modified frozen platelets were respectively labeled with Cy5.5-NHS at room temperature, and the final concentration was 0.5 mg / mL.

[0150] According to the method in Example 3, the mouse acute ischemic stroke model was established by photochemical induction, and 1.5 hours after the model was established, the Cy5.5 fluorescently labeled responsive nanoparticle, frozen platelet loaded responsive liposome and supermolecular engineering modified frozen platelets were respectively injected intravenously, and the mice were sacrificed 24 hours after injection, and the brain tissue was taken for fluorescence imaging.

[0151] The results are shown in Figure 7 , as shown in the figure. Figure 7 The left corresponds to the responsive nanoparticle, the middle corresponds to the frozen platelet loaded responsive liposome, and the right corresponds to the supermolecular engineering modified frozen platelet. As can be seen from the figure, the supermolecular engineering modified frozen platelet has no significant effect on the targeting of platelets.

[0152] In summary, the present application provides a frozen platelet loaded with nanodrug system based on supermolecular engineering modification for drug delivery in thromboembolic therapy. The pre-activated platelets are rapidly frozen in liquid nitrogen, which are in a fixed state, which is conducive to surface modification and can maintain long-term stability. At the same time, the freezing treatment will fix the directionality of the platelet membrane to maintain strong targeting effect on the injured blood vessel site. Subsequently, the nanodrug is loaded by host-guest self-assembly, which can facilitate rapid drug delivery at the thrombus site. The scheme provided by the present application avoids the change of the nature of the live platelets during the modification process, so that the modified molecules can exist stably for a long time, while ensuring the directionality of the platelet membrane and reducing the influence on the targeting. In addition, due to the good biocompatibility of platelets and the targeting of thrombus site, the rapid clearance of nanodrug is reduced, and the drug delivery efficiency is improved. At the same time, this method is suitable for resting or activated platelets and various nanodrugs, which maximizes the retention of platelet morphology and targeting, has high universality and transformation potential.

[0153] The above merely describes the preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A drug delivery system, characterized in that, The drug delivery system is a supramolecularly engineered frozen platelet-loaded drug delivery system. Wherein, the drug-carrying nanomaterial is a drug-loaded nanomaterial modified with a first modifier; the supramolecularly engineered frozen platelets are activated platelets flash-frozen in liquid nitrogen modified with a second modifier; the frozen platelets are obtained by flash-freezing activated platelets in liquid nitrogen, thawing them, and then modifying them with the second modifier. The first modifier comprises a first host molecule and / or its derivatives, or the first modifier comprises a first guest molecule and / or its derivatives; the second modifier comprises a second host molecule and / or its derivatives, or the second modifier comprises a second guest molecule and / or its derivatives. The first modifier and the second modifier can self-assemble and bond together; The first host molecule and the second host molecule are independently macrocyclic molecules that are hydrophilic on the outside and hydrophobic on the inside, and the macrocyclic molecules include cucurbitaureus; the first guest molecule and the second guest molecule are independently adamantane. The drug-carrying nanomaterial includes nanomaterials and drugs loaded onto the nanomaterials; the nanomaterials include PEGylated reactive oxygen species-responsive liposomes; The second modifier modifies the frozen platelets in a non-covalent manner; The drug includes at least one of thrombolytic drugs, antiplatelet aggregation drugs, anticoagulants, vasodilators, and neuroprotective agents; The drug is in a free form within the nanomaterial.

2. The drug delivery system according to claim 1, characterized in that, The nanomaterials include liposomes capable of simultaneously loading lipid-soluble and water-soluble drugs.

3. The drug delivery system according to claim 1, characterized in that, The thrombolytic drug includes at least one of urokinase, streptokinase, alteplase, and tenecteplase; And / or, the antiplatelet aggregation drug includes at least one of aspirin, clopidogrel, and dipyridamole; And / or, the anticoagulant includes at least one of heparin sodium, warfarin sodium, and alginate diesterase; And / or, the vasodilator includes at least one of flunarizine, betahistine, and buprofen; And / or, the neuroprotective agent includes at least one of nimodipine and mannitol.

4. The drug delivery system according to claim 1, characterized in that, The molar ratio of the host molecule and / or its derivatives to the guest molecule and / or its derivatives in the drug delivery system is 1-100:1-100.

5. The drug delivery system according to claim 4, characterized in that, The molar ratio of the host molecule and / or its derivatives to the guest molecule and / or its derivatives in the drug delivery system is 1:

1.

6. The drug delivery system according to claim 1, characterized in that, The size of the frozen platelets is in the micrometer range.

7. The method for preparing the drug-loaded system according to any one of claims 1-6, characterized in that, Includes the following steps: The drug-carrying nanomaterial modified with the first modifier was co-incubated with frozen platelets modified with the second modifier to allow the first and second modifiers to self-assemble and combine.

8. The preparation method according to claim 7, characterized in that, The incubation temperature is 20-37℃, and / or the incubation time is 1 min-1 h.

9. The preparation method according to claim 7, characterized in that, The incubation time is 15 minutes.

10. The application of the drug delivery system according to any one of claims 1-6, characterized in that, The drug delivery system is used to prepare drugs for treating thromboembolic diseases.

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