Nanometer delivery system, preparation method thereof and application of nanometer delivery system in preparation of medicine for treating cerebral apoplexy

Abstract

The invention relates to the technical field of medicines, in particular to a nano delivery system, a preparation method thereof and application of the nano delivery system in preparation of drugs for treating cerebral apoplexy. The nano-delivery system comprises a liposome containing an antioxidant drug and a thrombolytic drug, and a platelet membrane hybridized and combined with the liposome. The nano delivery system provided by the invention can be gathered at the focus of cerebral apoplexy in a targeted manner, and the distribution of drugs at non-focus parts is remarkably reduced, so that the systemic side effect is reduced, the synergistic treatment effect of thrombolysis and oxidation resistance is realized, the oxidative stress injury is effectively relieved, and the blood flow of the brain is recovered. In addition, the hybrid structure improves the biocompatibility and delivery stability of the carrier, and ensures efficient delivery and controllable release of the drug in vivo. Through the nano delivery system, a brand new thought and technical support are provided for cerebral apoplexy treatment, the defects of an existing treatment method are expected to be overcome, and a safer and more efficient treatment scheme is brought to a patient.

Description

Technical Field

[0001] The present application relates to the technical field of drugs for treating stroke, and in particular to a nano delivery system and a preparation method thereof, and application of the nano delivery system in preparing drugs for treating stroke. Background Art

[0002] Ischemic stroke is an acute cerebrovascular disease with high disability and mortality rates. In ischemic stroke, thrombus formation is a key pathological process. Thrombus formation begins with vascular endothelial damage. The exposed collagen fibers and tissue factor trigger platelet adhesion and activation, which then triggers a cascade of coagulation factors to generate a large amount of fibrin and reinforce the thrombus. Eventually, the thrombus increases in size and completely blocks the blood vessel, leading to ischemia and hypoxia of downstream tissues. Under normal circumstances, the fibrinolytic system inhibits thrombus expansion by activating plasmin to dissolve fibrin, but under pathological conditions, the functional imbalance of this system exacerbates thrombus formation.

[0003] In view of this pathological mechanism, the main clinical treatment method is intravenous injection of the thrombolytic drug recombinant tissue plasminogen activator (rt-PA), which activates plasminogen to plasmin, thereby degrading fibrin, dissolving thrombus and restoring blood flow. However, the clinical application of rt-PA is significantly limited.

[0004] First, the optimal treatment time window for rt-PA is only within 4.5 hours after onset. Patients who exceed this time window cannot benefit due to increased risk of reperfusion injury and cerebral hemorrhage. Second, rt-PA treatment has a high risk of bleeding, especially in patients with hypertension or cerebral arteriosclerosis, whose vascular endothelial integrity may be damaged by thrombolysis, thus causing severe intracranial hemorrhage. In addition, the destruction of the blood-brain barrier in severe ischemic conditions may aggravate the neurotoxicity of rt-PA, further affecting the therapeutic effect.

[0005] Although the antioxidant drug edaravone (EDA) can remove reactive oxygen species (ROS) to reduce ischemia-reperfusion injury, it also has obvious limitations. Edaravone lacks targeting in the body and is rapidly distributed throughout the body after injection, resulting in low concentrations in the lesion site, which limits the therapeutic effect and may cause side effects such as abnormal liver and kidney function. In addition, edaravone has a short half-life and requires frequent administration to maintain effective concentrations, which increases the burden of treatment, and the hypoxic microenvironment of stroke lesions may also limit the antioxidant effect. Edaravone has a narrow therapeutic window and must be administered in time during the acute phase. Exceeding this time window may make it difficult to improve neurological deficits. These limitations highlight the shortcomings of existing thrombolytic and antioxidant therapies, and also expose the needs of stroke treatment in terms of targeting, drug delivery efficiency, and safety. Although studies have attempted to use liposomes or cell membrane-modified nanocarriers to improve drug delivery, these schemes still have deficiencies in targeting, biocompatibility, and drug release control. Summary of the invention

[0006] In order to solve at least one of the above technical problems, the present application proposes a nano delivery system (PLEA) and a preparation method thereof and application thereof in the preparation of a drug for treating stroke.

[0007] In a first aspect, a nano-delivery system is provided, comprising a liposome containing an antioxidant drug and a thrombolytic drug, and a platelet membrane hybridized with the liposome.

[0008] In some possible embodiments, the antioxidant drug is edaravone, and the thrombolytic drug is rt-PA.

[0009] In a second aspect, a method for preparing the nano delivery system according to the first aspect is provided, comprising:

[0010] Providing liposomes containing edaravone and rt-PA;

[0011] Provides platelet membranes;

[0012] The platelet membrane and the liposome are hybridized and combined.

[0013] In some possible embodiments, providing liposomes specifically includes:

[0014] 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl sn-glycero-3-phosphoacetamide-N-[biotin(polyethylene glycol) 2000] (DSPE-PEG 2000), 1,2-dipalmitoyl sn-glycol-3-phosphate (DPPA) and edaravone were dissolved in chloroform to obtain a solution;

[0015] evaporating the solution to obtain a lipid film;

[0016] Dissolving rt-PA in double distilled water, and resuspending the lipid film using the double distilled water containing the rt-PA;

[0017] The resuspended suspension was subjected to ultrasonic treatment and then passed through a filter membrane to obtain liposomes as a filtrate.

[0018] In some possible embodiments, providing liposomes specifically includes:

[0019] Dissolve 18 parts by mass of DPPC, 3.5 parts by mass of DSPE-PEG 2000, 1 part by mass of DPPA and 5 parts by mass of edaravone in 5 parts by volume of chloroform to obtain a solution, wherein the ratio of the parts by mass to the parts by volume is mg / ml;

[0020] evaporating the solution to obtain a lipid film;

[0021] Dissolving 5 parts by mass of rt-PA in 5 parts by volume of double distilled water, and resuspending the lipid film using the double distilled water containing the rt-PA;

[0022] The resuspended suspension was sonicated in an ice bath and then passed through 400 nm and 200 nm polycarbonate membranes in sequence to obtain liposomes as a filtrate.

[0023] In some possible embodiments, providing a platelet membrane specifically includes:

[0024] Whole blood was obtained from the orbital vein of mice;

[0025] Centrifuging the blood to obtain an upper layer of platelet-rich plasma;

[0026] The plasma was centrifuged, the supernatant was removed, and the obtained platelets were resuspended in a solution containing 1×10 -3 M ethylenediaminetetraacetic acid and protease inhibitors in PBS to obtain a suspension;

[0027] The suspension is rapidly frozen using liquid nitrogen and then thawed to room temperature, and this is repeated at least three times;

[0028] The suspension thawed for the last time was subjected to ultrasonic treatment and then to centrifugation to obtain platelet membrane.

[0029] In some possible embodiments, providing a platelet membrane specifically includes:

[0030] Whole blood was obtained from the orbital vein of C57BL / 6J mice using a capillary tube, and 5×103 M EDTA;

[0031] The blood to which the EDTA was added was centrifuged at room temperature to obtain the upper platelet-rich plasma, which was transferred to another container and 1×10 -6 M PGE1;

[0032] The plasma to which the prostaglandin E1 was added was centrifuged, the supernatant was removed, and the obtained platelets were resuspended in a solution containing 1×10 -3 M ethylenediaminetetraacetic acid and protease inhibitors in PBS to obtain a suspension;

[0033] The suspension is rapidly frozen using liquid nitrogen and then thawed to room temperature, and this is repeated at least three times;

[0034] The suspension thawed for the last time was sonicated and centrifuged to obtain platelet membranes, which were then washed with PBS containing protease inhibitors.

[0035] In some possible embodiments, the hybridization of the platelet membrane and the liposome specifically includes:

[0036] subjecting the mixture of the liposome and the platelet membrane to ultrasonic treatment on ice;

[0037] The composition of the liposome and the platelet after the ultrasonic treatment is passed through a filtration membrane to obtain a nano-delivery system as a filtrate.

[0038] In some possible embodiments, the hybridization of the platelet membrane and the liposome specifically includes:

[0039] The liposome and the platelet membrane are mixed in a container at a mass ratio of 50:1, and the container is subjected to ultrasonic treatment on ice using an ultrasonic disruptor;

[0040] The volume of the composition of the liposome and the platelet after the ultrasonic treatment is fixed to 1 ml using double distilled water;

[0041] The composition adjusted to 1 ml was passed through a 200 nm polycarbonate membrane to obtain the nanodelivery system as a filtrate.

[0042] In a third aspect, a method for preparing a drug for treating stroke is proposed.

[0043] According to the nano delivery system provided by the present application, by utilizing the natural targeting ability of the platelet membrane, it is functionalized for identifying and targeting the stroke lesion site, and at the same time, combined with the high drug loading and stability of the liposome, the antioxidant drug edaravone and the thrombolytic drug rt-PA are wrapped therein. The system can be targeted and aggregated in the stroke lesion, significantly reducing the distribution of the drug in the non-lesion site, thereby reducing systemic side effects, achieving the synergistic therapeutic effect of thrombolysis and anti-oxidation, effectively reducing oxidative stress damage, and restoring cerebral blood flow. In addition, the hybrid structure improves the biocompatibility and delivery stability of the carrier, ensuring efficient delivery and controlled release of the drug in vivo. Through the nano delivery system of the present application, a new idea and technical support are provided for the treatment of stroke, which is expected to overcome the shortcomings of existing treatment methods and bring safer and more efficient treatment plans to patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present application, rather than limiting the present application.

[0045] Figure 1 It is a flow chart of the preparation method of the nano delivery system provided in the examples of the present application.

[0046] Figure 2 This is the TEM image of the nanodelivery system prepared in this example.

[0047] Figure 3 The particle size distribution of the nano-delivery system prepared in this example is shown in the form of a bar graph.

[0048] Figure 4 This is the spectral absorption diagram of the nanodelivery system prepared in this example.

[0049] Figure 5 3 is a comparison chart of the ROS scavenging ability of EDA (edaravone) and the nano delivery system prepared in this example, wherein (a) corresponds to the measurement result of the DPPH method, and (b) corresponds to the measurement result of the ABTS method.

[0050] Figure 6 The figures are comparative diagrams for verifying the thrombolytic effect of the nano-delivery system prepared in this example, rt-PA and PBS, wherein (a) shows the dissolving effect of different drugs on blood clots in the form of pictures, and (b) shows the spectral absorption intensity results of each supernatant in (a) in the form of data curves. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the embodiment of the present application will be clearly and completely described below in conjunction with the drawings of the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all the embodiments. Based on the described embodiment of the present application, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of this application. It can be understood that, in the absence of conflict, some technical means of the various embodiments described herein can be replaced or combined with each other.

[0052] In the description of the present application, reference to "one embodiment" or "some embodiments" etc. means that one or more embodiments of the present application include a particular feature, structure or characteristic described in conjunction with the embodiment. Thus, the phrases "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. appearing in different places in the present specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways.

[0053] See also Figure 1 The preparation method of the nano delivery system provided in the embodiment of the present application includes S100 to S300:

[0054] S100, providing liposomes containing edaravone and rt-PA. Specifically, the liposomes containing edaravone and rt-PA are prepared by thin film hydration extrusion method.

[0055] More specifically, step S100 includes steps S101 to S104:

[0056] S101, 18 mg of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 3.5 mg of 1,2-distearoyl sn-glycero-3-phosphoacetamide-N-[biotin(polyethylene glycol) 2000] (DSPE-PEG 2000), 1 mg of 1,2-dipalmitoyl sn-glycol-3-phosphate (DPPA) and 5 mg of edaravone were dissolved in 5.0 ml of chloroform to obtain a solution.

[0057] S102, evaporating the solution obtained in step S101 at 65°C using a rotary evaporation system to obtain a lipid film.

[0058] S103, dissolving 5 mg of rt-PA in 5 ml of double distilled water, and resuspending the lipid film obtained in step S102 using the double distilled water containing rt-PA.

[0059] S104, after the resuspended suspension is subjected to ultrasonic treatment in an ice bath for 20 minutes, the suspension (i.e., the suspension after 20 minutes of ultrasonic treatment) is sequentially passed through a 400 nm and a 200 nm polycarbonate membrane using an extruder, thereby obtaining liposomes as a filtrate of the polycarbonate membrane and having an average particle size not exceeding 200 nm.

[0060] S200, providing platelet membrane.

[0061] More specifically, step S200 includes steps S201 to S204:

[0062] S201, whole blood was obtained from the orbital vein of C57BL / 6J mice using a 0.2 μm capillary tube, and 5×10 3 M ethylenediaminetetraacetic acid (EDTA) was used for anticoagulation.

[0063] As we all know, the aforementioned 5×10 3 M represents the molar concentration of EDTA.

[0064] S202, the blood to which EDTA was added was centrifuged at 100 g for 20 min at room temperature to obtain the upper platelet-rich plasma, and the obtained plasma was transferred to another container (such as a test tube). In addition, 1×10 -6 M prostaglandin E1.

[0065] As is known to all, the aforementioned 100g indicates the magnitude of the centrifugal force.

[0066] S203, the plasma to which prostaglandin E1 was added was centrifuged at 850 g for 20 min, the supernatant was removed, and the obtained platelets were resuspended in a solution containing 1×10 -3 The suspension was obtained by adding PBS (phosphate buffer solution) containing 1 M ethylenediaminetetraacetic acid and protease inhibitors.

[0067] S204, using liquid nitrogen to quickly freeze the suspension obtained in step S203, and then thawing it to room temperature, and repeating this process three times.

[0068] In some examples, the operation of step S204 may be repeated more than three times, such as four times or five times.

[0069] S205, using an ultrasonic disruptor to ultrasonically treat the suspension that has been thawed for the last time at 70 W power and 20 kHz frequency for 45 seconds, and then centrifuge it at 4000 g for 5 minutes to obtain platelet membranes, and wash the obtained platelet membranes three times with PBS containing protease inhibitors to improve their purity.

[0070] The platelet membranes purified by washing can be stored at -80°C.

[0071] S300, hybrid combination of platelet membrane and liposome.

[0072] More specifically, step S300 includes steps S301 to S303:

[0073] S301, liposomes and platelet membranes at a mass ratio of 50:1 are mixed into a vial, and the vial containing the mixture of liposomes and platelets is subjected to ultrasonic treatment on ice for 30 seconds using an ultrasonic disruptor.

[0074] S302, using double distilled water to adjust the volume of the composition of liposomes and platelets treated in step S301 to 1 ml.

[0075] S303 , using an extruder to allow the composition adjusted to 1 ml in step S302 to pass through a 200 nm polycarbonate membrane, to obtain a nano delivery system as a filtrate of the polycarbonate membrane with an average particle size not exceeding 200 nm.

[0076] By controlling the particle size of the nanodelivery system to below 200 nm, it is called nanoparticles with nanoscale dimensions, which can optimize its stability in blood circulation and its ability to cross the blood-brain barrier.

[0077] Figure 2 is a TEM image of the nano-delivery system prepared in this example, and Figure 3 The particle size distribution of the nano-delivery system prepared in this example is shown. Figure 2 and Figure 3 It can be seen that the particle size of the obtained nanodelivery system is mainly distributed around 200 nm.

[0078] Figure 4 This is the spectral absorption diagram of the nano-delivery system prepared in this example. It can be seen from the diagram that the nano-delivery system is successfully loaded with edaravone (EDA).

[0079] Figure 5 The figure is a comparison of the ROS scavenging ability of EDA and the nano-delivery system prepared in this example, wherein (a) corresponds to the measurement result of the DPPH method, and (b) corresponds to the measurement result of the ABTS method. Figure 5 It can be seen that the nanodelivery system prepared in this example has a ROS scavenging ability similar to that of EDA.

[0080] Figure 6The comparison diagrams of the thrombolytic effect verification of the nano-delivery system prepared in this example and rt-PA and PBS, wherein (a) shows the dissolving effect of different drugs on blood clots in the form of pictures, and (b) shows the spectral absorption intensity results of each supernatant in (a) in the form of data curves. It can be seen from the figure that the nano-delivery system prepared in this example has a thrombolytic ability similar to that of rt-PA.

[0081] According to the introduction of the foregoing, it can be known that the nano delivery system prepared according to the above-mentioned embodiment includes liposomes containing antioxidant drug edaravone and thrombolytic drug rt-PA and platelet membranes hybridized with the liposomes. In this way, the nano delivery system can identify and target the stroke lesion site by utilizing the natural targeting ability of the platelet membrane, and at the same time, the high drug loading and stability of the liposome are combined, and the antioxidant drug edaravone and the thrombolytic drug rt-PA are wrapped therein, and the target aggregation can be achieved in the stroke lesion, and the distribution of the drug in the non-lesion site is significantly reduced, thereby reducing systemic side effects, achieving the synergistic therapeutic effect of thrombolysis and anti-oxidation, effectively reducing oxidative stress damage, and restoring brain blood flow. In addition, the hybrid structure improves the biocompatibility and delivery stability of the carrier, and ensures the efficient transmission and controlled release of the drug in vivo. Therefore, the nano delivery system can be applied to the preparation of drugs for the treatment of stroke.

Claims

1. A nano delivery system, characterized in that: The nano delivery system comprises liposomes containing antioxidant drugs and thrombolytic drugs, and platelet membranes hybridized and combined with the liposomes.

2. The nano delivery system according to claim 1, characterized in that The antioxidant drug is edaravone, and the thrombolytic drug is rt-PA.

3. A method for preparing the nano delivery system according to claim 1 or 2, characterized in that: include: Providing liposomes containing edaravone and rt-PA; Provides platelet membranes; The platelet membrane and the liposome are hybridized and combined.

4. The preparation method according to claim 3, characterized in that: The provision of liposomes specifically comprises: 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl sn-glycero-3-phosphoacetamide-N-[biotin(polyethylene glycol) 2000] (DSPE-PEG 2000), 1,2-dipalmitoyl sn-glycol-3-phosphate (DPPA) and edaravone were dissolved in chloroform to obtain a solution; evaporating the solution to obtain a lipid film; Dissolving rt-PA in double distilled water, and resuspending the lipid film using the double distilled water containing the rt-PA; The resuspended suspension was subjected to ultrasonic treatment and then passed through a filter membrane to obtain liposomes as a filtrate.

5. The preparation method according to claim 4, characterized in that: The provision of liposomes specifically comprises: Dissolve 18 parts by mass of DPPC, 3.5 parts by mass of DSPE-PEG 2000, 1 part by mass of DPPA and 5 parts by mass of edaravone in 5 parts by volume of chloroform to obtain a solution, wherein the ratio of the parts by mass to the parts by volume is mg / ml; evaporating the solution to obtain a lipid film; Dissolving 5 parts by mass of rt-PA in 5 parts by volume of double distilled water, and resuspending the lipid film using the double distilled water containing the rt-PA; The resuspended suspension was sonicated in an ice bath and then passed through 400 nm and 200 nm polycarbonate membranes in sequence to obtain liposomes as a filtrate.

6. The preparation method according to claim 3, characterized in that: The providing of platelet membrane specifically comprises: Whole blood was obtained from the orbital vein of mice; Centrifuging the blood to obtain an upper layer of platelet-rich plasma; The plasma was centrifuged, the supernatant was removed, and the obtained platelets were resuspended in a solution containing 1×10 -3 M ethylenediaminetetraacetic acid and protease inhibitors in PBS to obtain a suspension; The suspension is rapidly frozen using liquid nitrogen and then thawed to room temperature, and this is repeated at least three times; The suspension thawed for the last time was subjected to ultrasonic treatment and then to centrifugation to obtain platelet membrane.

7. The preparation method according to claim 6, characterized in that: The providing of platelet membrane specifically comprises: Whole blood was obtained from the orbital vein of C57BL / 6J mice using a capillary tube, and 5×10 3 M EDTA; The blood to which the EDTA was added was centrifuged at room temperature to obtain the upper platelet-rich plasma, which was transferred to another container and 1×10 -6 M PGE1; The plasma to which the prostaglandin E1 was added was centrifuged, the supernatant was removed, and the obtained platelets were resuspended in a solution containing 1×10 -3 M ethylenediaminetetraacetic acid and protease inhibitors in PBS to obtain a suspension; The suspension is rapidly frozen using liquid nitrogen and then thawed to room temperature, and this is repeated at least three times; The suspension thawed for the last time was sonicated and centrifuged to obtain platelet membranes, which were then washed with PBS containing protease inhibitors.

8. The preparation method according to claim 3, characterized in that: The hybridization of the platelet membrane and the liposome specifically comprises: subjecting the mixture of the liposome and the platelet membrane to ultrasonic treatment on ice; The composition of the liposome and the platelet after the ultrasonic treatment is passed through a filtration membrane to obtain a nano-delivery system as a filtrate.

9. The preparation method according to claim 8, characterized in that: The hybridization of the platelet membrane and the liposome specifically comprises: The liposome and the platelet membrane are mixed in a container at a mass ratio of 50:1, and the container is subjected to ultrasonic treatment on ice using an ultrasonic disruptor; The volume of the composition of the liposome and the platelet after the ultrasonic treatment is fixed to 1 ml using double distilled water; The composition adjusted to 1 ml was passed through a 200 nm polycarbonate membrane to obtain the nanodelivery system as a filtrate.

10. Use of the nano delivery system according to claim 1 or 2 in the preparation of a drug for treating cerebral stroke.

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