Targeting nano-particle as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a targeting nanoparticle and a preparation method and application thereof. The targeted nano-particle designed and synthesized by the invention comprises a nano-liposome and low-molecular heparin entrapped in the nano-liposome, wherein the surface of the nano-liposome is modified with CREKA peptide. Wherein the CREKA peptide can target fibrinogen in thrombus and specifically recognize FN1 at the thrombus part, so that LMWH is delivered to the thrombus part in a targeted manner, the local antithrombotic effect is improved, meanwhile, the risks of extra bleeding and toxicity are reduced to the maximum extent, and the limitation of traditional LMWH is solved.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular to a targeted nanoparticle and a preparation method and application thereof. Background Art

[0002] Venous thrombosis, including deep vein thrombosis and pulmonary embolism, is associated with significant morbidity and mortality. Among them, deep vein thrombosis includes cerebral venous thrombosis (CVT), which is a unique subtype of deep vein thrombosis that can lead to pathological damage characterized by brain tissue infarction and cerebral hemorrhage. Although clinical guidelines recognize the use of anticoagulant therapy in the setting of cerebral hemorrhage, there are inherent risks in the use of anticoagulant therapy in certain clinical situations. In contrast, patients with chronic venous thrombosis often require extended anticoagulant therapy to prevent recurrence. However, a significant proportion of these patients have comorbidities, including digestive system diseases and intracranial aneurysms. These complexities emphasize the critical need to develop targeted anticoagulants.

[0003] Thrombosis is mainly initiated by collagen, tissue factor, thrombin, fibrin and other elements, which stimulate platelet activation and the formation of fibronectin (FN1) complex, ultimately leading to thrombosis. According to the guidelines, anticoagulant therapy is the main strategy for the treatment of venous thrombosis. Low molecular weight heparin (LMWH) is the first choice for the treatment of venous thrombosis and has been widely used in clinical practice. Systemic application of LMWH can prevent thrombus growth in the acute phase and prevent thrombus recurrence in the chronic phase; however, the application of LMWH in the treatment of venous thrombotic diseases has limitations, including short half-life, non-targetedness and bleeding complications, so it is urgent to study targeted drug delivery methods for the treatment of venous thrombosis. Summary of the invention

[0004] In order to solve the above technical problems, the present disclosure provides a targeted nanoparticle and a preparation method and application thereof.

[0005] In a first aspect, the present disclosure provides a targeted nanoparticle, comprising a nanoliposome and a low molecular weight heparin encapsulated in the nanoliposome, wherein the surface of the nanoliposome is modified with a CREKA peptide.

[0006] Among them, the amino acid sequence of CREKA peptide is Cys-Arg-Glu-Lys-Ala (SEQ ID NO: 1).

[0007] The following are preferred technical solutions of the present disclosure, but are not intended to be limitations of the technical solutions provided by the present disclosure. Through the following technical solutions, the technical objectives and beneficial effects of the present disclosure can be better achieved and realized.

[0008] As a preferred technical solution of the present disclosure, in the targeted nanoparticles, the content of CREKA peptide is 8 to 12 wt %.

[0009] As a preferred technical solution of the present disclosure, the content of low molecular weight heparin in the targeted nanoparticles is 5 to 7 wt %.

[0010] As a preferred technical solution of the present disclosure, the materials of the nanoliposome include soybean lecithin, cholesterol and DSPE-PEG-CREKA.

[0011] As a preferred technical solution of the present disclosure, the particle size of the targeted nanoparticles is 125 to 138 nm.

[0012] In a second aspect, the present disclosure provides a method for preparing the targeted nanoparticles according to the first aspect, the preparation method comprising the following steps:

[0013] DSPE-PEG-2K-NHS is mixed with CREKA peptide, and CREKA peptide is modified onto DSPE-PEG-2K-NHS through amide condensation reaction under the action of a catalyst to obtain DSPE-PEG-CREKA;

[0014] Soy lecithin, cholesterol and DSPE-PEG-CREKA are mixed with a solvent and evaporated under reduced pressure to form a film, and then low molecular weight heparin is added. The film is extruded, purified and freeze-dried in sequence to obtain targeted nanoparticles.

[0015] As a preferred technical solution of the present disclosure, the catalyst includes triethylamine;

[0016] Preferably, the solvent comprises chloroform.

[0017] In a third aspect, the present disclosure provides a use of the targeted nanoparticles as described in the first aspect or the targeted nanoparticles prepared by the preparation method as described in the second aspect in the preparation of a drug for treating venous thrombosis.

[0018] In a fourth aspect, the present disclosure provides a drug for treating venous thrombosis, wherein the drug for treating venous thrombosis comprises the targeted nanoparticles as described in the first aspect or the targeted nanoparticles prepared by the preparation method described in the second aspect, and one or more pharmaceutically acceptable excipients.

[0019] As a preferred technical solution of the present disclosure, the pharmaceutically acceptable excipients include solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives. One or more.

[0020] Compared with the prior art, the technical solution provided by the embodiments of the present disclosure has the following advantages:

[0021] The present invention discloses a designed and synthesized nanoliposome with a surface modified with CREKA peptide and loaded with low molecular weight heparin. The nanoliposome targets fibrinogen in the thrombus through the CREKA peptide, specifically recognizes FN1 at the thrombus site, and delivers LMWH to the thrombus site in a targeted manner, thereby improving the local anti-thrombotic effect and minimizing the risk of additional bleeding and toxicity, thereby overcoming the limitations of traditional LMWH. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0024] Figure 1 This is a graph showing the kinetic diameter results of the CREKA-LMWH NPs described in Example 4 of the present disclosure after being dissolved in physiological saline and placed at 37° C. for 7 days;

[0025] Figure 2 This is a graph showing the survival rate of HUVEC cells treated with different groups of drugs using an MTT experiment in Example 5 of the present disclosure;

[0026] Figure 3 The fluorescence images of the thrombus in the superior sagittal sinus of CVT model mice at 0h, 6h and 24h after LMWH NPs and CREKA-LMWHNPs were injected into the tail vein of the mice in Example 6 of the present disclosure; wherein LMWH was labeled with Cy5;

[0027] Figure 4 This is a quantitative graph of the average fluorescence intensity of thrombus in the superior sagittal sinus of CVT model mice at 0h, 6h and 24h after LMWH NPs and CREKA-LMWHNPs were injected into the tail vein of the mice in Example 6 of the present disclosure; wherein LMWH was labeled with Cy5;

[0028] Figure 5 The fluorescence images of Example 6 of the present disclosure in vitro incubation of LMWH NPs and CREKA-LMWH NPs with external jugular vein thrombus, respectively; wherein LMWH are labeled with Cy5;

[0029] Figure 6This is a quantitative graph of the average fluorescence intensity of LMWH NPs and CREKA-LMWH NPs binding to external jugular vein thrombus in vitro in Example 6 of the present disclosure; wherein LMWH is labeled with Cy5;

[0030] Figure 7 The in vitro fluorescence images of Example 6 of the present disclosure in which LMWH NPs and CREKA-LMWH NPs were incubated with femoral vein thrombus in vitro; wherein LMWH was labeled with Cy5;

[0031] Figure 8 This is a quantitative graph of the average fluorescence intensity of LMWH NPs and CREKA-LMWH NPs binding to femoral vein thrombus in vitro in Example 6 of the present disclosure; wherein LMWH is labeled with Cy5;

[0032] Fig. 9 In Example 7 of the present disclosure, free LMWH, LMWH NPs and CREKA-LMWH NPs were injected into CVT model mice through the tail vein, and the superior sagittal sinus containing thrombus was removed 24 hours later, and the fluorescent images after frozen section and labeling of the vascular wall with Lectin were obtained; wherein LMWH was labeled with Cy5;

[0033] Fig.10 In Example 7 of the present disclosure, LMWH NPs and CREKA-LMWHNPs were injected into CVT model mice through the tail vein, and the superior sagittal sinus containing thrombus was removed 24 hours later. The fluorescence intensity quantitative graph after frozen section and labeling of the vascular wall with Lectin was obtained; wherein LMWH was labeled with Cy5;

[0034] Fig.11 In Example 7 of the present disclosure, free LMWH, LMWH NPs and CREKA-LMWH NPs were injected into the external jugular vein thrombosis model mice through the tail vein, and the external jugular vein containing thrombus was removed 24 hours later. Fluorescence images after frozen section and labeling of the vascular wall with Lectin were obtained; wherein LMWH was labeled with Cy5;

[0035] Fig.12 In Example 7 of the present disclosure, LMWH NPs and CREKA-LMWH NPs were injected into the external jugular vein thrombosis model mice through the tail vein, and the external jugular vein containing thrombus was removed 24 hours later. The fluorescence intensity quantitative graph after frozen section and labeling of the vascular wall with Lectin; wherein, LMWH was labeled with Cy5;

[0036] Fig.13In Example 7 of the present disclosure, free LMWH, LMWHNPs and CREKA-LMWH NPs were injected into the femoral vein thrombosis model mice through the tail vein, and the femoral vein containing thrombus was removed 24 hours later. The fluorescent images after frozen section and labeling of the vascular wall with Lectin were obtained; wherein LMWH was labeled with Cy5;

[0037] Fig.14 In Example 7 of the present disclosure, LMWH NPs and CREKA-LMWH NPs were injected into the femoral vein thrombosis model mice through the tail vein, and the femoral vein containing thrombus was removed 24 hours later. The fluorescence intensity quantitative graph after frozen section and labeling of the vascular wall with Lectin; wherein LMWH was labeled with Cy5;

[0038] Fig.15 HE staining images of superior sagittal sinus thrombus in mice after the CVT model mice in Example 8 of the present disclosure were treated with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs respectively;

[0039] Fig.16 The statistical graph of the degree of thrombus formation after the CVT model mice in Example 8 of the present disclosure were treated with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs respectively;

[0040] Fig.17 HE staining images of femoral vein thrombosis in mice with femoral vein thrombosis model in Example 8 of the present disclosure after treatment with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs, respectively; wherein A in the figure represents artery, and V represents vein;

[0041] Fig.18 This is a statistical graph of the degree of thrombus formation after the femoral vein thrombosis model mice in Example 8 of the present disclosure were treated with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs respectively. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] Unless otherwise specified, the significance symbols of P values ​​in the examples and drawings of the present invention are defined as follows: ns means not significant, **P<0.01, ***P<0.001.

[0044] The disclosed embodiment provides a targeted nanoparticle, which includes a nanoliposome, the surface of the nanoliposome is modified with CREKA peptide and the interior is loaded with low molecular weight heparin.

[0045] The present disclosure confirms through proteomics experimental results that fibronectin (FN1) is significantly enriched in cerebral venous thrombosis, and its expression level is 9.59 times higher than that in plasma. Based on the above results, the present disclosure utilizes the obvious targeting effect of CREKA peptide on fibrin with high expression abundance in tumor, myocardial ischemia-reperfusion and atherosclerosis models, and modifies it onto a drug carrier, so that the carrier containing low molecular weight heparin (LMWH) can be accurately targeted and delivered to the site of venous thrombosis, thereby improving the therapeutic effect of the drug on patients with venous thrombosis.

[0046] As a preferred implementation of the disclosed embodiments, in the targeted nanoparticles, the content of CREKA peptide is 8 to 12 wt%, for example, 8 wt%, 9 wt%, 10 wt%, 11 wt% or 12 wt%, etc., but is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0047] As a preferred implementation of the disclosed embodiment, in the targeted nanoparticles, the content of low molecular weight heparin is 5 to 7 wt%, for example, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt% or 7 wt%, etc., but is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0048] As a preferred implementation of the disclosed embodiment, the materials of the nanoliposomes include soybean lecithin, cholesterol and DSPE-PEG-CREKA.

[0049] As a preferred implementation of the disclosed embodiment, the particle size of the targeted nanoparticles is 125 to 138 nm, for example, 125 nm, 130 nm, 135 nm or 138 nm, but is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0050] The second aspect of the disclosed embodiment further proposes a method for preparing the targeted nanoparticles, comprising:

[0051] DSPE-PEG-2K-NHS is mixed with CREKA peptide, and CREKA peptide is modified onto DSPE-PEG-2K-NHS through amide condensation reaction under the action of a catalyst to obtain DSPE-PEG-CREKA;

[0052] Soy lecithin, cholesterol and DSPE-PEG-CREKA are mixed with a solvent and evaporated under reduced pressure to form a film, and then a low molecular weight heparin solution is added. The film is extruded, purified and freeze-dried in sequence to obtain targeted nanoparticles.

[0053] Among them, DSPE-PEG-2K-NHS is a linear heterobifunctional PEGylation reagent composed of DSPE phospholipids, PEG chains and NHS (N-hydroxysuccinimide) activation groups, and "2K" represents that the molecular weight of polyethylene glycol (PEG) is 2000 Daltons.

[0054] The added mass ratio of soybean lecithin, cholesterol and DSPE-PEG-CREKA is (9-11):(0.5-1.5):(0.5-1.5).

[0055] As a preferred implementation of the embodiments of the present disclosure, the catalyst includes triethylamine.

[0056] As a preferred implementation of the embodiment of the present disclosure, the solvent includes chloroform.

[0057] The third aspect of the disclosed embodiments further proposes the use of the targeted nanoparticles in the preparation of drugs for treating venous thrombosis.

[0058] Furthermore, in some preferred embodiments, the present disclosure also proposes the use of the targeted nanoparticles in the preparation of drugs for treating cerebral venous thrombosis.

[0059] The fourth aspect of the disclosed embodiments further proposes a drug for treating venous thrombosis, wherein the drug for treating venous thrombosis comprises the targeted nanoparticles and one or more pharmaceutically acceptable excipients.

[0060] As a preferred implementation of the embodiments of the present disclosure, the pharmaceutically acceptable excipients include solubilizers, co-solvents, emulsifiers, flavoring agents, olfactory agents, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives. One or more.

[0061] The embodiments of the present disclosure will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0062] In the following examples and comparative examples, the low molecular weight heparin used is low molecular weight heparin sodium.

[0063] Example 1

[0064] This embodiment provides a targeted nanoparticle and a preparation method thereof. The targeted nanoparticle comprises a nanoliposome. The surface of the nanoliposome is modified with CREKA peptide and low molecular weight heparin (LMWH) is encapsulated inside.

[0065] Among them, the content of LMWH is 5.67wt%, and the content of CREKA peptide is 10wt%.

[0066] The preparation method comprises the following steps:

[0067] Weigh 100 mg of DSPE-PEG-2K-NHS and dissolve it in 3 mL of DMF, add CREKA peptide (the molar amount added is 1.1 times that of DSPE-PEG-2K-NHS) and triethylamine (the molar amount added is 1.1 times that of DSPE-PEG-2K-NHS), and react at room temperature for 12 hours; then transfer the reaction solution to a dialysis bag (molecular weight cutoff is 1000 Da), dialyze it with pure water for 24 hours, collect the dialysate and freeze-dry it to obtain DSPE-PEG-CREKA;

[0068] 100 mg of soybean lecithin, 10 mg of cholesterol and 10 mg of DSPE-PEG-CREK were dissolved in 3 mL of chloroform and evaporated under reduced pressure to form a film. 12 mg of LMWH was then added, followed by ultrasound, liposome extrusion (wherein the pore size of the polycarbonate membrane was 100 nm), dialysis by a nanodialysis device (wherein the pore size of the polycarbonate membrane was 30 nm), and freeze-drying to obtain targeted nanoparticles (CREKA-LMWH NPs) with a particle size of 131.033±5.98 nm.

[0069] Example 2

[0070] This embodiment provides a targeted nanoparticle, which refers to the targeted nanoparticle in Example 1, except that the content of LMWH is 7wt% and the content of CREKA peptide is 12wt%.

[0071] Example 3

[0072] This embodiment provides a targeted nanoparticle, which refers to the targeted nanoparticle in Example 1, except that the content of LMWH is 5wt% and the content of CREKA peptide is 8wt%.

[0073] Comparative Example 1

[0074] This comparative example provides a nanoparticle and a preparation method thereof, referring to Example 1, except that: during the preparation process, the surface of the nanoliposome is not modified with CREKA peptide, and DSPE-PEG-CREK is replaced with DSPE-PEG-2K to obtain LMWH NPs.

[0075] Example 4

[0076] Evaluation of the stability of targeted nanoparticles

[0077] 1. Experimental methods

[0078] The CREKA-LMWH NPs obtained in Example 1 were mixed with physiological saline and placed at 37°C for one week, wherein the LMWH was 1 IU / ml. The kinetic diameter of the targeted nanoparticles was then measured using a NanoBrook 90plus PALS nanoparticle size potentiometer from Brookhaven, USA. The results are shown in FIG. Figure 1 shown.

[0079] 2. Experimental results

[0080] Depend on Figure 1 It can be seen that the particle size of CREKA-LMWH NPs remains relatively stable, indicating that the targeted nanoparticles have good stability.

[0081] Example 5

[0082] Evaluating the biocompatibility of targeted nanoparticles

[0083] 1. Create groups

[0084] Control group (denoted as Ctrl): normal saline;

[0085] LMWH group (denoted as LMWH): physiological saline solution containing free LMWH;

[0086] LMWH NPs group (denoted as LMWH NPs): physiological saline solution containing LMWH NPs obtained in Comparative Example 1;

[0087] CREKA-LMWH NPs group (referred to as CREKA-LMWH NPs): physiological saline solution containing CREKA-LMWH NPs obtained in Example 1.

[0088] 2. Experimental method: MTT experiment was used to detect the cytotoxicity of different groups of drugs on HUVEC cells:

[0089] HUVEC cells were seeded in a 96-well plate with a seeding density of approximately 8,000 cells / well and cultured for 24 hours. They were divided into a control group, a LMWH group, a LMWH NPs group, and a CREKA-LMWH NPs group, where the concentration of LMWH was 1 IU / ml. Complete culture medium (containing 10% fetal bovine serum) was added and cultured for another 24 hours. A blank group (without cells and without drugs) was also set up. After the culture was completed, the old culture medium was removed, and the complete culture medium and MTT solution were mixed at a volume ratio of 1:9. After thorough mixing, the wells were added to the 96-well plate and incubated for 4 hours. 100 microliters of Formazan solution was added to each well, mixed appropriately, and continued to incubate in a 37°C incubator for 4 hours. The OD value of each well was measured at 570 nm using an enzyme reader, and the results were recorded. Cell viability was plotted against dose, and the results are shown in Figure 2. Figure 2 shown.

[0090] Cell survival rate = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0091] 3. Experimental results:

[0092] Depend on Figure 2 It can be seen that the results show that the prepared CREKA-LMWH NPs have no obvious toxicity when LMWH is 1 IU / ml, indicating that CREKA-LMWH NPs have good biocompatibility.

[0093] Example 6

[0094] Assessing the targeting properties of targeted nanoparticles

[0095] 1. Experimental animals:

[0096] For each disease model, mice were prepared at 12 weeks of age and randomly divided into 2 groups with the same number of mice in each group: LMWHNPs group and CREKA-LMWH NPs group.

[0097] 2. Model establishment:

[0098] Establishment of mouse cerebral venous sinus thrombosis (CVT) model:

[0099] Mice were anesthetized with 2% isoflurane and fixed on a stereotaxic apparatus. A 1.5 cm incision was made along the midline of each mouse's head to expose the skull. A longitudinal cranial window (10×2 mm) was opened between the anterior and posterior bregma, and the superior sagittal sinus and bilateral cortex were exposed with an animal skull drill. Subsequently, rose bengal was injected through the tail vein. After 2 minutes, a laser was used to irradiate the exposed superior sagittal sinus with 532 nm green light vertically for 2 minutes, and then the mouse wound was sutured; the mice were subcutaneously injected with ketoprofen for analgesia.

[0100] Establishment of mouse external jugular vein thrombosis model:

[0101] The mice were anesthetized with 2% isoflurane, and a 1.5-cm incision was made in the neck skin to expose the external jugular vein. Rose Bengal was injected through the tail vein, and then a laser was used to irradiate the exposed external jugular vein vertically with 532 nm green light for 2 minutes, after which the wound was sutured. Ketoprofen was injected subcutaneously into the mice for analgesia.

[0102] Establishment of mouse femoral vein thrombosis model:

[0103] The mice were anesthetized with 2% isoflurane, and a 1.5-cm incision was made in the skin at the groin to expose the femoral vein. Rose Bengal was injected through the tail vein, and then a laser was used to irradiate the exposed femoral vein vertically with 532 nm green light for 2 minutes, after which the wound was sutured. Ketoprofen was injected subcutaneously into the mice for analgesia.

[0104] 3. Experimental methods:

[0105] Mouse cerebral venous sinus thrombosis (CVT) model:

[0106] LMWH NPs group: 48 hours after successful modeling, 100 μl of physiological saline solution containing LMWH NPs (obtained in Comparative Example 1) was injected once through the tail vein, and then administered again after 12 hours, wherein LMWH: 1 IU, and LMWH was labeled with Cy5;

[0107] CREKA-LMWH NPs group: 48 hours after successful modeling, 100 μl of physiological saline solution containing CREKA-LMWH NPs (obtained in Example 1) was injected once through the tail vein, and then administered again after 12 hours, wherein LMWH: 1 IU, and LMWH was labeled with Cy5;

[0108] After the administration, in vivo fluorescence imaging was used to record the changes of thrombus in the superior sagittal sinus (SSS) of mice over time.

[0109] Among them, after the two groups of mice were injected with LMWH NPs and CREKA-LMWH NPs through the tail vein, the fluorescence images of the thrombus in the superior sagittal sinus of the mice at 0h, 6h and 24h were as follows Figure 3 As shown in the figure; After the two groups of mice were injected with LMWH NPs and CREKA-LMWH NPs through the tail vein, the average fluorescence intensity of the thrombus in the superior sagittal sinus of the mice at 0h, 6h and 24h was quantified as shown in the figure Figure 4 shown.

[0110] Mouse external jugular vein thrombosis model and mouse femoral vein thrombosis model:

[0111] 48 hours after the successful establishment of the mouse external jugular vein thrombosis model and femoral vein model, the external jugular vein thrombosis or femoral vein thrombosis of the mouse was removed and placed in a black 96-well plate. 100 μl of LMWH NPs (wherein LMWH: 1 IU, and labeled with Cy5) and CREKA-LMWH NPs (wherein LMWH: 1 IU, and labeled with Cy5) were added to the wells, respectively, and incubated at 37°C for 30 minutes. After that, the plates were washed three times with PBS solution, and the fluorescence imaging results of mouse venous thrombosis were recorded using in vitro fluorescence microscopy.

[0112] Among them, the fluorescence images of the two groups of mice incubated with LMWH NPs and CREKA-LMWH NPs combined with external jugular vein thrombus in vitro are shown in Figure 2. Figure 5 As shown; the average fluorescence intensity of the two groups of mice incubated with LMWH NPs and CREKA-LMWH NPs and external jugular vein thrombus in vitro was quantified as shown Figure 6 shown.

[0113] In vitro fluorescence images of two groups of mice incubated with LMWH NPs and CREKA-LMWH NPs combined with femoral vein thrombus in vitro Figure 7 As shown; the average fluorescence intensity quantitative graph of the two groups of mice binding to femoral vein thrombus in vitro with LMWH NPs and CREKA-LMWH NPs is shown in Figure 8 shown.

[0114] 4. Experimental results

[0115] 4.1 Cerebral venous sinus thrombosis (CVT) mice

[0116] from Figure 3 and Figure 4 It can be seen that just after the intravenous injection, the thrombi in the SSS of mice in the LMWH NPs group and the CREKA-LMWH NPs group showed very strong fluorescence signals; 6 hours after the injection, the fluorescence intensity of the thrombi in the superior sagittal sinus of the two groups of mice gradually weakened, among which the signal in the superior sagittal sinus of the CREKA-LMWH NPs group was slightly stronger; 24 hours after the injection, the fluorescence signal intensity continued to weaken. Importantly, the fluorescence intensity of the superior sagittal sinus of mice in the CREKA-LMWH NPs-treated group was twice that of the LMWH NPs-treated group, indicating that CREKA-LMWH NPs can target FN1 through CREKA, and therefore, more circulating CREKA-LMWH NPs bind to fibrin and aggregate at the thrombus site.

[0117] 4.2 External jugular vein thrombosis in mice

[0118] from Figure 5 and Figure 6It can be seen that the fluorescence signal of the CREKA-LMWH NPs group binding to the thrombus is stronger than the fluorescence signal of the LMWH NPs group binding to the thrombus, indicating that CREKA-LMWH NPs have a good binding ability with the external jugular vein thrombus.

[0119] 4.3 Femoral vein thrombosis in mice

[0120] from Figure 7 and Figure 8 It can be seen that the fluorescence signal of the CREKA-LMWH NPs group binding to the thrombus is stronger than the fluorescence signal of the LMWH NPs group binding to the thrombus, indicating that CREKA-LMWH NPs have a good binding ability with the femoral vein thrombus.

[0121] According to the experimental results of 4.1 to 4.3, it was found that the half-life and targeting of the targeted nanoparticle delivery system were significantly improved.

[0122] Example 7

[0123] Evaluation of the affinity of targeted nanoparticles for thrombus

[0124] 1. Experimental animals:

[0125] For each disease model, mice were prepared at 12 weeks of age and randomly divided into three groups with the same number of mice in each group, namely:

[0126] LMWH group, LMWH NPs group, and CREKA-LMWH NPs group.

[0127] 2. Model establishment: Referring to the method in Example 3, a mouse cerebral venous sinus thrombosis (CVT) model, a mouse external jugular vein thrombosis model, and a mouse femoral vein thrombosis model were established;

[0128] 3. Experimental methods:

[0129] LMWH group: 48 hours after successful modeling, 100 μl of physiological saline solution containing free LMWH was injected once through the tail vein, and then injected again after 12 hours, of which free LMWH: 1 IU;

[0130] LMWH NPs group: 48 hours after successful modeling, 100 μl of physiological saline solution containing LMWH NPs (obtained in Comparative Example 1) was injected once through the tail vein, and then administered again after 12 hours, wherein LMWH: 1 IU, and LMWH was labeled with Cy5;

[0131] CREKA-LMWH NPs group: 48 hours after successful modeling, 100 μl of physiological saline solution containing CREKA-LMWH NPs (obtained in Example 1) was injected once through the tail vein, and then administered again after 12 hours, wherein LMWH: 1 IU, and LMWH was labeled with Cy5;

[0132] After the administration, the mice were euthanized, and the venous thrombus was fixed with OCT. The frozen sections of the mouse venous thrombus were 30 μm thick, and the blood vessels were labeled with Lectin. The fluorescence imaging results of the mouse venous thrombus sections were recorded using in vitro fluorescence microscopy.

[0133] Among them, free LMWH, LMWH NPs and CREKA-LMWH NPs were injected into CVT model mice through the tail vein, and the superior sagittal sinus containing thrombus was removed 24 hours later. The fluorescence images after frozen section and labeling of the vascular wall with Lectin are shown in Figure 2. Fig. 9 As shown;

[0134] CVT model mice were injected with LMWH NPs and CREKA-LMWH NPs via the tail vein. After 24 hours, the superior sagittal sinus containing thrombus was removed, frozen sectioned and the vascular wall was labeled with Lectin for quantitative fluorescence intensity. Fig.10 As shown;

[0135] The external jugular vein thrombosis model mice were injected with free LMWH, LMWH NPs and CREKA-LMWHNPs through the tail vein. After 24 hours, the external jugular vein containing thrombus was removed, and the fluorescence images after frozen section and lectin labeling of the vascular wall were shown as follows: Fig.11 As shown;

[0136] The external jugular vein thrombosis model mice were injected with LMWH NPs and CREKA-LMWH NPs through the tail vein. After 24 hours, the external jugular vein containing thrombus was removed, frozen sectioned and the vascular wall was labeled with Lectin for quantitative fluorescence intensity. Fig.12 As shown;

[0137] The femoral vein thrombosis model mice were injected with free LMWH, LMWH NPs and CREKA-LMWHNPs through the tail vein. After 24 hours, the femoral vein containing thrombus was removed and the fluorescence images after frozen section and lectin labeling of the vascular wall were shown in the figure. Fig.13 As shown;

[0138] Mice with femoral vein thrombosis were injected with LMWH NPs and CREKA-LMWH NPs via the tail vein. After 24 hours, the femoral vein containing thrombus was removed, and the fluorescence intensity was quantified after frozen section and labeling of the vascular wall with Lectin. Fig.14 shown.

[0139] 4. Experimental results

[0140] Depend on Figures 9 to 14 It can be seen that the fluorescence intensity of CREKA-LMWH NPs binding to thrombi in the superior sagittal sinus, external jugular vein thrombi and femoral vein thrombi is higher than that of LMWH NPs, indicating its good thrombus affinity.

[0141] Example 8

[0142] Evaluating the antithrombotic effect of targeted nanoparticles

[0143] 1. Experimental animals:

[0144] For each disease model, mice were prepared at 12 weeks of age and randomly divided into 4 groups with the same number of mice in each group, namely:

[0145] Control group, LMWH group, LMWH NPs group, and CREKA-LMWH NPs group.

[0146] 2. Model establishment: Referring to the method in Example 3, a mouse cerebral venous sinus thrombosis (CVT) model and a mouse femoral vein thrombosis model were established;

[0147] 3. Experimental methods:

[0148] Control group: 100 μl of saline solution was injected via tail vein;

[0149] LMWH group: 48 hours after successful modeling, 100 μl of physiological saline solution containing free LMWH was injected once through the tail vein, and then administered again after 12 hours, where LMWH: 1 IU;

[0150] LMWH NPs group: 48 hours after successful modeling, 100 μl of physiological saline solution containing LMWH NPs (obtained in Comparative Example 1) was injected once through the tail vein, and then administered again after 12 hours, wherein LMWH: 1 IU;

[0151] CREKA-LMWH NPs group: 48 hours after successful modeling, 100 μl of physiological saline solution containing CREKA-LMWH NPs (obtained in Example 1) was injected once through the tail vein, and then administered again after 12 hours, wherein LMWH: 1 IU;

[0152] After the administration, the mice were euthanized, the thrombi were harvested, fixed with paraformaldehyde, dehydrated and embedded, and the mouse thrombi were sliced ​​with a thickness of 10 μm. The slices were stained with HE and observed under a microscope.

[0153] Among them, the HE staining images of the superior sagittal sinus thrombus of CVT model mice after they were treated with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs were shown in Figure 2. Fig.15 As shown in the figure; after CVT model mice were treated with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs, the statistical diagram of the degree of thrombus formation is shown in the figure Fig.16 shown.

[0154] The HE staining images of the femoral vein thrombosis model mice were shown in Figure 2 after they were treated with normal saline, free LMWH, LMWH NPs, and CREKA-LMWH NPs. Fig.17 As shown in the figure; after the femoral vein thrombosis model mice were treated with normal saline, free LMWH, LMWH NPs and CREKA-LMWH NPs, the statistical diagram of the degree of thrombosis formation is shown in the figure Fig.18 shown.

[0155] 4. Experimental results

[0156] from Fig.15 and Fig.16 It can be seen that the degree of thrombosis in the untreated control group mice was close to 100%, the degree of thrombosis in the free LMWH group was 68.6%, the LMWH NPs group was 69%, and the CREKA-LMWH NPs group was only 36.2%, indicating that the CREKA-LMWH NPs group had a better anti-thrombotic effect. Fig.17 and Fig.18 The degree of femoral vein thrombosis gradually decreased from 91.8% in the control group to 64.2% (LMWH group), 59.8% (LMWH NPs group), and 39.4% (CREKA-LMWHNPs group), further confirming that CREKA-LMWH NPs have stronger targeting and therapeutic effects on venous thrombosis.

[0157] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A targeted nanoparticle, characterized in that: The targeted nanoparticles include nanoliposomes and low molecular weight heparin contained in the nanoliposomes, and the surface of the nanoliposomes is modified with CREKA peptide.

2. The targeted nanoparticle according to claim 1, characterized in that In the targeted nanoparticles, the content of CREKA peptide is 8-12 wt %.

3. The targeted nanoparticle according to claim 1 or 2, characterized in that: In the targeted nanoparticles, the content of low molecular weight heparin is 5-7wt%.

4. The targeted nanoparticle according to any one of claims 1 to 3, characterized in that: The components of the nanoliposome include soybean lecithin, cholesterol and DSPE-PEG-CREKA.

5. The targeted nanoparticle according to any one of claims 1 to 4, characterized in that: The particle size of the targeted nanoparticles is 125-138 nm.

6. A method for preparing the targeted nanoparticles according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: DSPE-PEG-2K-NHS is mixed with CREKA peptide, and CREKA peptide is modified onto DSPE-PEG-2K-NHS through amide condensation reaction under the action of a catalyst to obtain DSPE-PEG-CREKA; Soy lecithin, cholesterol and DSPE-PEG-CREKA are mixed with a solvent and evaporated under reduced pressure to form a film, and then low molecular weight heparin is added. The film is extruded, purified and freeze-dried in sequence to obtain targeted nanoparticles.

7. The preparation method according to claim 6, characterized in that: The catalyst includes triethylamine; Preferably, the solvent comprises chloroform.

8. Use of the targeted nanoparticles according to any one of claims 1 to 5 or the targeted nanoparticles prepared by the preparation method according to claim 6 or 7 in the preparation of a drug for treating venous thrombosis.

9. A drug for treating venous thrombosis, characterized in that: The drug for treating venous thrombosis includes the targeted nanoparticles according to any one of claims 1 to 5 or the targeted nanoparticles prepared by the preparation method according to claim 6 or 7, and one or more pharmaceutically acceptable excipients.

10. The drug for treating venous thrombosis according to claim 9, characterized in that: The pharmaceutically acceptable excipients include one or more of solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives.

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