CN115025219B - Ultrasonic response urokinase thrombolysis nanoliposome and preparation and application thereof - Google Patents
Ultrasonic response urokinase thrombolysis nanoliposome and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of intelligent medicaments, in particular to an ultrasonic response urokinase thrombolysis nanoliposome and preparation and application thereof. First EDC, NHS, ppIX and DSPE-NH 2 Amidation reaction to obtain DSPE-PpIX; EDC, NHS, DSPE ROS Linker-COOH and PEG 2k ‑NH 2 Amidation reaction to obtain DSPE-ROS Linker-PEG 2k The method comprises the steps of carrying out a first treatment on the surface of the Then DSPE-PpIX is reacted with DSPE-ROS Linker-PEG 2k Dissolving in solvent, and post-treating to obtain precursor liquid; finally, urokinase and precursor liquid are uniformly mixed, and the ultrasonic response urokinase nanoliposome is obtained after self-assembly and post-treatment. The ultrasonic response urokinase thrombolysis nanoliposome prepared by the invention has good application prospect in preparing medicines loaded with urokinase, thrombin and hydrophilic and hydrophobic chemotherapeutics.
Description
Technical Field
The invention relates to the technical field of intelligent medicaments, in particular to an ultrasonic response urokinase thrombolysis nanoliposome and preparation and application thereof.
Background
Pulmonary embolism (pulmonary embolism, PE) refers to diseases or clinical syndromes caused by various emboli blocking pulmonary arteries or branches thereof, including pulmonary thromboembolism (pulmonary thromboembolism, PTE), fat embolism, amniotic fluid embolism, tumor embolism, etc., wherein pulmonary thromboembolism is the most common type, i.e. we refer to acute pulmonary embolism (acute pulmonary embolism, APE). Pulmonary embolism is often secondary to deep vein thrombosis (deep venous thrombosis, DVT), a clinical manifestation of essentially the same disease at different stages, collectively known as venous thromboembolism (venous thromboembolism, VTE). APE also has a higher mortality rate and is located in the third most western world after myocardial infarction and malignancy. It is estimated that APE dies within 1h after onset to about 10%, the mortality rate of untreated APE can reach 30%, and if early intervention and treatment are performed, the mortality rate can be reduced to 2% -8%. Therefore, APE is a disease with high morbidity, high misdiagnosis rate and high mortality rate, and needs to be paid attention to the disease by clinicians. The incidence of APE increases with age, and ages above 40 are considered as high risk factors for APE. PE patients under 40 years old have much lower morbidity and mortality than the elderly
The thrombus in the blood vessel can be effectively removed by the surgical operation, but the operation has high difficulty and high risk, and has high requirements on medical equipment of hospitals and medical level of doctors; requiring complex preoperative preparation; postoperative complications are more frequent; the cost is also high. Besides surgery, thrombolysis can also be used. The thrombolytic drugs used clinically to date have been mainly Urokinase (UK). While thrombolysis must be considered, the risk of bleeding caused by overdose is considered, and the balance of thrombolysis is not achieved due to small dose. Therefore, there is an urgent need to explore and develop a new medicament or new technique for acute thromboembolism with reliable curative effect, simple operation, little side effect and few complications.
Disclosure of Invention
In order to solve the problem of intelligent controlled release of urokinase and avoid bleeding risk caused by thrombolysis, the invention aims to provide an ultrasonic response urokinase thrombolysis nanoliposome and preparation and application thereof.
The acoustic power regulation and control drug controlled release system developed in recent years is a novel technology for realizing accurate release of drugs based on the fact that ultrasonic wave excitation of a sensitizer triggers an sonochemical reaction and generates Reactive Oxygen Species (ROS) to activate or destroy chemical bonds and spatial structures of the drug controlled release system. Compared with the technology of regulating and controlling drug release by using laser as a medium, the ultrasonic wave has deeper soft tissue penetration depth (more than or equal to 10 cm) than the laser, and has better clinical application and conversion potential.
The invention aims at combining deep tissue penetration of ultrasonic wave and high-efficiency thrombolysis advantage of urokinase, developing an advanced, safe and high-efficiency intelligent thrombolytic drug and being used for treating acute pulmonary embolism. The invention firstly carries out structural modification on nano liposome, and uses ROS Linker sensitive to ROS (singlet oxygen sensitive) for connecting hydrophilic end (PEG) of the liposome 2k -NH 2 ) And a hydrophobic end (DSPE-ROS Linker-COOH) to obtain amphiphilic liposome fragments to obtain intelligent "nanocapsules" of ultrasound response. The sonosensitizer protoporphyrin in the main component of the nanoliposome generates singlet oxygen under the action of ultrasound to break the connection between the hydrophilic segment and the hydrophobic segment, and finally the nanoliposome structure collapses. On one hand, the precise release of urokinase at the focus embolism position is realized, the local medicine concentration of the embolism position is improved, and the thrombus is dissolved efficiently; on the other hand, ULR loaded with thrombolytic drug urokinase is not stimulated by exogenous ultrasound in other organs or tissues, the capsule structure is kept intact, and urokinase is still 'blocked' in the nano-liposome, so that the bleeding risk caused by overdose of the traditional intravenous urokinase is avoided. The invention can be applied to intelligent delivery of ultrasonic response of urokinase, thrombin and hydrophilic and hydrophobic therapeutic drugs, and provides a new strategy and reference for development of high-efficiency and safe therapeutic drugs for acute pulmonary embolism.
The aim of the invention can be achieved by the following technical scheme:
the first object of the invention is to provide a preparation method of ultrasonic response urokinase thrombolysis nanoliposome, which comprises the following steps:
(1) EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), ppIX (protoporphyrin) and DSPE-NH 2 Carrying out amidation reaction on the (poly (beta-amino ester) -amino group), and carrying out post-treatment to obtain DSPE-PpIX (hydrophobic phospholipid fragment);
(2)EDC、NHS、DSPE-ROS Linker-COOH(COOH-S-C(CH 3 ) 2 -S-COOH) and PEG 2k -NH 2 Amidation reaction of (polyethylene glycol) and post-treatment to obtain DSPE-ROS Linker-PEG 2k (amphipathic phospholipid fragment);
(3) The DSPE-PpIX prepared in the step (1) and the DSPE-ROS Linker-PEG prepared in the step (2) are mixed 2k Dissolving in solvent, and post-treating to obtain precursor liquid;
(4) And (3) uniformly mixing urokinase with the precursor liquid prepared in the step (3), and performing post-treatment after self-assembly to obtain the ultrasonic response urokinase nanoliposome.
In one embodiment of the invention EDC, NHS, ppIX, DSPE-NH 2 Respectively dissolved in methanol solution.
In one embodiment of the present invention, in step (1), EDC, NHS, ppIX is reacted with DSPE-NH 2 The molar ratio of (2) is 5mmol:5mmol:1mmol:1-2mmol;
in the amidation reaction process, the reaction temperature is 20-30 ℃ and the reaction time is 12-24h.
In one embodiment of the present invention, EDC, NHS, DSPE-NH is added sequentially to PpIX during the reaction 2 The method comprises the steps of carrying out a first treatment on the surface of the The whole reaction process is under a light-proof condition.
In one embodiment of the invention, in step (1), the post-treatment is to subject the resulting solution after the reaction to dialysis in distilled water (molecular weight cut-off of 3000 daltons), and freeze-drying for use.
In one embodiment of the invention, EDC, NHS, DSPE-ROS Linker-COOH is combined with PEG 2k -NH 2 Respectively dissolving in chloroform.
In one embodiment of the present invention, in step (2), EDC, NHS, DSPE-ROS Linker-COOH is mixed with PEG 2k -NH 2 The molar ratio of (2) is 5mmol:5mmol:1mmol:1-2mmol;
in the amidation reaction process, the reaction temperature is 20-30 ℃ and the reaction time is 12-24h.
In one embodiment of the present invention, EDC, NHS, PEG is added sequentially to DSPE-ROS Linker-COOH during the reaction 2k -NH 2 。
In one embodiment of the invention, in the step (2), the post-treatment is to remove chloroform from the solution obtained after the reaction by reduced pressure rotary evaporation, then dialyze the solution in distilled water (molecular weight cut-off of 3000 daltons), and freeze-dry the solution for later use.
In one embodiment of the present invention, in step (3), the solvent is selected from one or more of methanol, dimethyl sulfoxide or chloroform.
In one embodiment of the present invention, in step (3), the post-treatment is rotary evaporation to remove the solvent to form a film, and after ultrasonic hydration, cooling to 30-40 ℃.
In one embodiment of the invention, in the ultrasonic hydration process, the power of ultrasonic waves is 300-350W, the hydration temperature is 60-65 ℃ and the hydration time is 30-60min.
In one embodiment of the present invention, in step (4), urokinase is combined with the hydrophobic fragment DSPE-PpIX, the amphiphilic fragment DSPE-ROS Linker-PEG in the precursor fluid 2k The dosage ratio of (2) is 2000-4000U:10mg;50mg.
In one embodiment of the invention, the self-assembly process is in particular ultrafiltration after phacoemulsification (ultrafiltration to remove free urokinase, hydrophobic phospholipid fragments and amphipathic phospholipid fragments);
in the ultrasonic emulsification process, the power of ultrasonic waves is 300-350W, the emulsification time is 30-60min, and the emulsification temperature is 0-4 ℃;
during ultrafiltration, the molecular weight cut-off was 100000 daltons.
The second object of the invention is to provide an ultrasonic response urokinase thrombolysis nanoliposome prepared by the method.
The third object of the invention is to provide an application of ultrasonic response urokinase thrombolysis nanoliposome in preparing medicines loaded with urokinase, thrombin and hydrophilic and hydrophobic therapeutic drugs.
Compared with the prior art, the invention has the following beneficial effects:
(1) The preparation method has simple and convenient reaction, is easy to control and can easily obtain the target product.
(2) The preparation method can well maintain the biological activity of urokinase, so that the efficient thrombolysis function of urokinase is maintained, meanwhile, the urokinase is sealed in the nanocapsule through the liposome responded by ultrasound, and the release behavior of the urokinase is strictly controlled by ultrasound, so that the potential bleeding risk of direct administration of the tail vein in clinic is avoided, and the thrombolysis efficiency is ensured while the use safety is ensured.
(3) The nano liposome prepared by the invention can effectively wrap urokinase, and the release of enzyme is strictly controlled by ultrasonic waves, so that the safe and efficient thrombolytic treatment of acute pulmonary embolism is realized.
Drawings
FIG. 1 shows DSPE-PpIX in example 1 1 H NMR chart.
FIG. 2 is a DSPE-ROS Linker-PEG of example 2 2k A kind of electronic device 1 H NMR chart.
FIG. 3 is a TEM image of the ULU of example 3.
FIG. 4 is a UV-Vis spectrum of the ULU of example 3.
FIG. 5 is a circular dichroism spectrum of ULU and urokinase in example 3.
FIG. 6 is the results of ESR testing of ULU of example 3 for singlet oxygen production under ultrasonic stimulation.
FIG. 7 shows the results of the characterization of nanoliposome vesicle structure changes by TEM after ultrasound stimulation of ULU in example 3.
FIG. 8 is a graph showing the UV absorption profile of ULU release of urokinase after various long ultrasound stimuli in example 3.
FIG. 9 is a graph showing cell viability after 24h (FIG. 9 a) and 48h (FIG. 9 b) of incubation of vascular endothelial cells with varying concentrations of ULU of example 3.
FIG. 10 shows the results of digital subtraction tests of animals of each group (saline treatment: sample; pure ultrasound treatment: +US; ULU treatment: ULU-US; urokinase UK treatment: UK; ULU and ultrasound stimulation co-treatment: ULU+UK) after receiving different treatments/drugs in New Zealand rabbits successfully constructed for acute pulmonary embolism.
Detailed Description
The invention provides a preparation method of an ultrasonic response urokinase thrombolysis nanoliposome, which comprises the following steps:
(1) EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), ppIX (protoporphyrin) and DSPE-NH 2 Carrying out amidation reaction on the (poly (beta-amino ester) -amino group), and carrying out post-treatment to obtain DSPE-PpIX (hydrophobic phospholipid fragment);
(2)EDC、NHS、DSPE-ROS Linker-COOH(COOH-S-C(CH 3 ) 2 -S-COOH) and PEG 2k -NH 2 Amidation reaction of (polyethylene glycol) and post-treatment to obtain DSPE-ROS Linker-PEG 2k (amphipathic phospholipid fragment);
(3) The DSPE-PpIX prepared in the step (1) and the DSPE-ROS Linker-PEG prepared in the step (2) are mixed 2k Dissolving in solvent, and post-treating to obtain precursor liquid;
(4) And (3) uniformly mixing urokinase with the precursor liquid prepared in the step (3), and performing post-treatment after self-assembly to obtain the ultrasonic response urokinase nanoliposome.
In one embodiment of the invention EDC, NHS, ppIX, DSPE-NH 2 Respectively dissolved in methanol solution.
In one embodiment of the present invention, in step (1), EDC, NHS, ppIX is reacted with DSPE-NH 2 The molar ratio of (2) is 5mmol:5mmol:1mmol:1-2mmol;
in the amidation reaction process, the reaction temperature is 20-30 ℃ and the reaction time is 12-24h.
In one embodiment of the present invention, EDC, NHS, DSPE-NH is added sequentially to PpIX during the reaction 2 The method comprises the steps of carrying out a first treatment on the surface of the Is in light-proof state during the whole reaction processUnder the condition that.
In one embodiment of the invention, in step (1), the post-treatment is to subject the resulting solution after the reaction to dialysis in distilled water (molecular weight cut-off of 3000 daltons), and freeze-drying for use.
In one embodiment of the invention, EDC, NHS, DSPE-ROS Linker-COOH is combined with PEG 2k -NH 2 Respectively dissolving in chloroform.
In one embodiment of the present invention, in step (2), EDC, NHS, DSPE-ROS Linker-COOH is mixed with PEG 2k -NH 2 The molar ratio of (2) is 5mmol:5mmol:1mmol:1-2mmol;
in the amidation reaction process, the reaction temperature is 20-30 ℃ and the reaction time is 12-24h.
In one embodiment of the present invention, EDC, NHS, PEG is added sequentially to DSPE-ROS Linker-COOH during the reaction 2k -NH 2 。
In one embodiment of the invention, in the step (2), the post-treatment is to remove chloroform from the solution obtained after the reaction by reduced pressure rotary evaporation, then dialyze the solution in distilled water (molecular weight cut-off of 3000 daltons), and freeze-dry the solution for later use.
In one embodiment of the present invention, in step (3), the solvent is selected from one or more of methanol, dimethyl sulfoxide or chloroform.
In one embodiment of the present invention, in step (3), the post-treatment is rotary evaporation to remove the solvent to form a film, and after ultrasonic hydration, cooling to 30-40 ℃.
In one embodiment of the invention, in the ultrasonic hydration process, the power of ultrasonic waves is 300-350W, the hydration temperature is 60-65 ℃ and the hydration time is 30-60min.
In one embodiment of the present invention, in step (4), urokinase is combined with the hydrophobic fragment DSPE-PpIX, the amphiphilic fragment DSPE-ROS Linker-PEG in the precursor fluid 2k The dosage ratio of (2) is 2000-4000U:10mg;50mg.
In one embodiment of the invention, the self-assembly process is in particular ultrafiltration after phacoemulsification (ultrafiltration to remove free urokinase, hydrophobic phospholipid fragments and amphipathic phospholipid fragments);
in the ultrasonic emulsification process, the power of ultrasonic waves is 300-350W, the emulsification time is 30-60min, and the emulsification temperature is 0-4 ℃;
during ultrafiltration, the molecular weight cut-off was 100000 daltons.
The invention provides an ultrasonic response urokinase thrombolysis nanoliposome prepared by the method.
The invention provides an application of ultrasonic response urokinase thrombolysis nanoliposome in preparing a drug loaded with urokinase, thrombin and hydrophilic and hydrophobic therapeutic drugs.
The invention will now be described in detail with reference to the drawings and specific examples.
In the examples below, the reagents used were all commercially available unless otherwise specified; the detection method and means are all conventional detection methods and means in the field.
Example 1
This example provides a method for preparing DSPE-PpIX.
Weighing 0.1mmol PpIX and 0.1mmol DSPE-NH respectively 2 Dissolved in 10mL of methanol solution, 0.5mmol of EDC and 0.5mmol of NHS were weighed separately and dissolved in 5mL of methanol. At 20 ℃, dropwise adding EDC/methanol solution into PpIX/methanol solution under stirring, stirring in a dark place for reaction for 30min, dropwise adding NHS/methanol solution into the PpIX/EDC/methanol solution, and continuously stirring in a dark place for reaction for 3h; dropwise adding the mixed solution into DSPE-NH 2 In methanol solution, the reaction is carried out for 24 hours under continuous magnetic stirring in dark place. After the reaction, the solution obtained by the reaction was dialyzed in distilled water for 24 hours (dialysis bag cut-off molecular weight 3000 daltons), and water was changed for 4 times during the period to obtain DSPE-PpIX, which was freeze-dried for use.
FIG. 1 shows the DSPE-PpIX obtained in this example 1 H NMR spectra, as can be seen from the figure, ppIX was modified to DSPE.
Example 2
The present embodiment provides DSPE-ROS Linker-PEG 2k Is prepared by the preparation method of (1).
0.1mmol DSPE-ROS Linker-COOH and 0.1mmol PEG were weighed out separately 2k -NH 2 Dissolved in 10mL of chloroform, 0.5mmol of EDC and 0.5mmol of NHS were weighed and dissolved in 5mL of chloroform, respectively. At 20 ℃, dropwise adding EDC/chloroform solution into DSPE-ROS Linker-COOH/chloroform solution under stirring, magnetically stirring for reaction for 30min, dropwise adding NHS/chloroform solution into the DSPE-ROS Linker-COOH/EDC/chloroform solution, and magnetically stirring for reaction for 3h; dropwise adding the mixed solution to PEG 2k -NH 2 In the chloroform solution, the reaction is carried out for 24 hours by continuous magnetic stirring. After the reaction is finished, the solution obtained by the reaction is subjected to reduced pressure rotary evaporation to remove the chloroform, and then the liquid after the removal of the chloroform is dialyzed for 24 hours (dialysis bag with a cut-off molecular weight of 3000 daltons) in distilled water, and water is changed for 4 times during the period to obtain DSPE-ROS Linker-PEG 2k And then freeze-drying for later use.
FIG. 2 shows DSPE-ROS Linker-PEG prepared in this example 2k (amphipathic phospholipid fragment) 1 From the H NMR spectra, it can be seen that DSPE-ROS Linker was modified to PEG 2k And (3) upper part.
Example 3
The embodiment provides a preparation method of an ultrasonic response urokinase thrombolysis nanoliposome.
10mg of DSPE-PpIX prepared in example 1 and 50mg of DSPE-ROS Linker-PEG prepared in example 2 were weighed out respectively 2k Dissolving in 20mL of chloroform, completely dissolving by ultrasonic, and then removing solvent chloroform by normal temperature and reduced pressure distillation to obtain a uniform film. 30mL of ultrapure water was added to the round-necked flask, and the mixture was hydrated by magnetic force (ultrasonic power: 350W) at 65℃for 30 minutes with stirring. After hydration, heating is stopped, the room temperature is cooled to 30 ℃, 2000U of urokinase is weighed and dispersed in 1mL of ultrapure water, and the completely dissolved urokinase solution is added into the hydrated liposome fragments to continue magnetic stirring and mixing for 30min. The well mixed solution was transferred to a 50mL centrifuge tube and sonicated under ice bath protection for 60min (ultrasonic power: 300W; ultrasonic interval: 2 seconds on, 2 seconds off). Finally, the dispersion liquid after self-assembly is adoptedRemoving free urokinase and phospholipid fragment by ultrafiltration (dialysis bag cut-off molecular weight 10000 daltons) to obtain ultrasonic response urokinase thrombolysis nanoliposome (ULU).
Fig. 3 is a TEM image of the ULU prepared in this example. As can be seen from the figure, the prepared ULU capsules are uniform in size distribution and about 30nm in size.
FIG. 4 is a graph showing the UV absorption curve of ULU prepared in this example, wherein the ULU has a characteristic absorption peak at 280nm attributed to urokinase protein, and the result shows that urokinase is successfully encapsulated in nanoliposomes.
FIG. 5 shows the circular dichroism spectrum of ULRU and free urokinase prepared in this example, and from the figure, it can be seen that the circular dichroism spectrum of ULRU and the initial material urokinase are basically the same, which indicates that the protein structure is not destroyed in the synthesis process, thereby well preserving the biological activity of urokinase and laying a foundation for subsequent in vivo thrombolysis.
FIG. 6 shows the results of ESR tests of the ULU prepared in this example under ultrasonic stimulation (ULU+US), showing that the prepared ULU shows a typical 1 under ultrasonic stimulation with TEMPO as singlet oxygen scavenger: 1:1, which shows that PpIX generates a large amount of ROS under the stimulation of ultrasonic waves, can be used for cutting off ROS Linker so as to destroy the vesicle structure of nano-liposome and control the release of urokinase.
Fig. 7 is a TEM image of the ULU prepared in this example after ultrasonic stimulation, and it can be seen from the image that the liposome nanocapsule structure is damaged after ultrasonic stimulation, and fragmentation is presented, and the result shows that the ultrasonic response nanoliposome controlled release system prepared in this example is feasible, which lays a foundation for controlling urokinase release of the subsequent ULU under ultrasonic stimulation in an animal body.
FIG. 8 shows the ULU prepared in this example, after being stimulated by ultrasonic waves of different durations, is subjected to UV-Vis to determine the urokinase release curve, and the result shows that the urokinase is rapidly released from the nanocapsules (release rate reaches 45%) after being stimulated by ultrasonic waves for 1min, and the release rate of the urokinase reaches 63% after being subjected to ultrasonic treatment for 5min, which indicates that the prepared ULU has sensitive response release to ultrasonic waves.
FIG. 9 shows the cell viability of ULU prepared in this example after 24h and 48h incubation with vascular endothelial cells, as measured by CCK8 method. The results showed that the cell viability of the ULU after 24h (fig. 9 a) and 48h (fig. 9 b) co-culture with vascular endothelial cells at concentrations of 0-500 μg/mL exceeded 90%, suggesting that the ULU has good cell compatibility, further demonstrating the safety of the ULU.
Example 4
The rabbit is weighed, the anesthetized and then is fixed on an operating table in a supine position, and the hair pusher shears off neck rabbit hair; and then sterilized by alcohol. A 1cm longitudinal incision was made beside the right side of the center of the trachea, the skin was cut, and the blunt free subcutaneous tissue was left, and the right jugular vein was completely clearly exposed. Puncturing the right jugular vein by using a Seldinger puncture method, then placing a 5F sheath tube, and implanting the sheath tube to a depth of 6-8cm; and (5) taking a blood sample, and checking blood cells, urea nitrogen, creatinine, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, bilirubin and blood coagulation functions. Then fixing the catheter by using a suture, and then suturing the neck incision; 2mL of venous blood was withdrawn through the sheath and injected into the 6F vascular sheath. Standing for 10min to naturally solidify, and placing into water bath at 60deg.C for 10min to promote thrombosis. Flushing the indwelling sheath tube with physiological saline to prevent coagulation in the sheath tube; the syringe then withdraws saline, flushes the 6F sheath, and beats out the thrombus inside it in a sterile tray. Washing with physiological saline to obtain uniform autologous thrombus with diameter of 2 mm. The length of the blood clot was measured using a vernier caliper and the autologous thrombus was cut into 10mm long thrombus strips.
Rabbits were transported to the DSA room and pulmonary angiography was performed using a philips digital subtraction machine. The rabbit was placed in a supine position, with the 4F single-curve catheter placed in the right atrium under fluoroscopy with the guidewire fitted through the vascular sheath left in the right jugular vein. Pulmonary artery angiography is performed prior to embolus injection. Contrast condition setting: the total amount is 12-18mL,6-8mL/s, and the pressure is 100mmhg. The digital subtraction radiography technique is adopted, and the front perspective is 15 frames/second. And observing the normal pulmonary artery vessel images from the pulmonary artery phase, the pulmonary parenchyma phase to the pulmonary vein reflux phase. The syringe draws out normal saline and autologous thrombus, slowly injects into the catheter through the indwelling catheter, and delivers the autologous thrombus to the right atrium. And then, carrying out pulmonary artery radiography examination again, comparing and observing with the radiography examination result before the embolic injection, and recording the pulmonary artery position and the embolic scope of the embolic embolism.
New Zealand rabbits were used to build animal models of pulmonary artery embolism according to the method described above. A total of 15 healthy new zealand rabbits were included in the study, divided into 5 groups of 3 according to different treatment intervention patterns.
(1) Control group: after the rabbit pulmonary embolism model is established, physiological saline is injected into the ear margin by intravenous injection.
(2) Ultrasound treatment group: after the rabbit pulmonary embolism model is established, pulmonary artery radiography locates pulmonary embolism parts. Using ultrasonic intervention, an ultrasonic probe is irradiated under fluoroscopy against the pulmonary artery embolism site.
The ultrasonic irradiation conditions are as follows: the frequency is 1MHz, the intensity is 1.0W/cm 2 Duty cycle: 95%, ultrasonic treatment time: 5min.
(3) Urokinase nanocapsules: after the rabbit pulmonary embolism model is established, the ultrasonic response urokinase thrombolysis nanoliposome prepared in example 3 is injected by ear margin vein, and the injection dosage is as follows: 2000U/kg.
(4) Urokinase treatment group: after the rabbit pulmonary embolism model is established, urokinase solution is injected through the auricular vein, and the injection dosage is that: 2000U/kg.
(5) Ultrasound activated urokinase nanocapsules: after the rabbit pulmonary embolism model is established, pulmonary artery radiography is performed first, and pulmonary artery embolism is located. Then, 500U/kg of the ultrasonic response urokinase thrombolysis nanoliposome prepared in example 3 was injected, and then an ultrasonic probe was applied to the pulmonary artery embolism site under fluoroscopy for ultrasonic irradiation.
The ultrasonic irradiation conditions are as follows: the frequency is 1MHz, the intensity is 1.0W/cm 2 Duty cycle: 95%, ultrasonic treatment time: 5min.
Thrombolytic therapy was then performed according to the above group and rabbit response was observed, and the experimental results are shown in fig. 10.
Saline refers to the control group in fig. 10; +us refers to the ultrasound treatment group; ULU-US refers to urokinase nanocapsules; UK refers to the urokinase treatment group; ulu+us refers to the group of ultrasonically activated urokinase nanocapsules. The results show that the acute pulmonary embolism animal model successfully prepared by operation does not improve or dissolve pulmonary embolism under treatment by normal saline, pure ultrasonic stimulation and pure material; the ULU body prepared in the embodiment shows superior pulmonary embolism thrombolysis effect than that of pure urokinase at the same dosage under ultrasonic stimulation, and further illustrates the thrombolysis effectiveness of the ULU.
Example 5
This example provides a method for preparing DSPE-PpIX.
Weighing 0.1mmol PpIX and 0.15mmol DSPE-NH respectively 2 Dissolved in 10mL of methanol solution, 0.5mmol of EDC and 0.5mmol of NHS were weighed separately and dissolved in 5mL of methanol. At 25 ℃, dropwise adding EDC/methanol solution into PpIX/methanol solution under stirring, stirring in a dark place for reaction for 30min, dropwise adding NHS/methanol solution into the PpIX/EDC/methanol solution, and continuously stirring in a dark place for reaction for 3h; dropwise adding the mixed solution into DSPE-NH 2 In methanol solution, the reaction is carried out for 24 hours under continuous magnetic stirring in dark place. After the reaction, the solution obtained by the reaction was dialyzed in distilled water for 30 hours (dialysis bag cut-off molecular weight 3000 daltons), water was changed 5 times during the period, to obtain DSPE-PpIX, and freeze-dried for use.
Example 6
The present embodiment provides DSPE-ROS Linker-PEG 2k Is prepared by the preparation method of (1).
0.1mmol DSPE-ROS Linker-COOH and 0.15mmol PEG were weighed out separately 2k -NH 2 Dissolved in 10mL of chloroform, 0.5mmol of EDC and 0.5mmol of NHS were weighed and dissolved in 5mL of chloroform, respectively. At 25 ℃, dropwise adding EDC/chloroform solution into DSPE-ROS Linker-COOH/chloroform solution under stirring, magnetically stirring for reaction for 30min, dropwise adding NHS/chloroform solution into the DSPE-ROS Linker-COOH/EDC/chloroform solution, and magnetically stirring for reaction for 3h; dropwise adding the mixed solution to PEG 2k -NH 2 And (3) in the chloroform solution, continuously stirring by magnetic force for reaction for 12-24h. After the reaction is finishedRemoving chloroform by rotary evaporation under reduced pressure, dialyzing the solution after removing chloroform in distilled water for 30 hr (dialysis bag with molecular weight cut-off of 3000 daltons), and changing water for 5 times to obtain DSPE-ROS Linker-PEG 2k And then freeze-drying for later use.
Example 7
The embodiment provides a preparation method of an ultrasonic response urokinase thrombolysis nanoliposome.
10mg of DSPE-PpIX prepared in example 5 and 50mg of DSPE-ROS Linker-PEG prepared in example 6 were weighed out separately 2k Dissolving in 20mL of chloroform, completely dissolving by ultrasonic, and then removing solvent chloroform by normal temperature and reduced pressure distillation to obtain a uniform film. 30mL of ultrapure water was added to the round-necked flask, and the mixture was hydrated by stirring with a magnetic force (ultrasonic power: 320W) at 63℃for 45 minutes. After hydration, heating is stopped, the room temperature is cooled to 35 ℃, 3000U of urokinase is weighed and dispersed in 1mL of ultrapure water, and the completely dissolved urokinase solution is added into the hydrated liposome fragments to continue magnetic stirring and mixing for 30min. The well mixed solution was transferred to a 50mL centrifuge tube and sonicated for 45min under ice bath protection (ultrasonic power: 320W; ultrasonic interval: 2 seconds on, 2 seconds off). Finally, removing free urokinase and phospholipid fragments from the dispersion liquid after self-assembly by adopting an ultrafiltration mode (dialysis bag cut-off molecular weight 10000 daltons) to obtain ultrasonic response urokinase thrombolysis nano-liposome (ULU).
Example 8
This example provides a method for preparing DSPE-PpIX.
Weighing 0.1mmol PpIX and 0.2mmol DSPE-NH respectively 2 Dissolved in 10mL of methanol solution, 0.5mmol of EDC and 0.5mmol of NHS were weighed separately and dissolved in 5mL of methanol. At 30 ℃, dropwise adding EDC/methanol solution into PpIX/methanol solution under stirring, stirring in a dark place for reaction for 30min, dropwise adding NHS/methanol solution into the PpIX/EDC/methanol solution, and continuously stirring in a dark place for reaction for 3h; dropwise adding the mixed solution into DSPE-NH 2 In methanol solution, the reaction is carried out for 24 hours under continuous magnetic stirring in dark place. After the reaction, the solution obtained by the reaction is treated in distilled waterAnd (3) dialyzing for 36h (dialysis bag with molecular weight cut-off of 3000 daltons), changing water for 6 times to obtain DSPE-PpIX, and freeze-drying for later use.
Example 9
The present embodiment provides DSPE-ROS Linker-PEG 2k Is prepared by the preparation method of (1).
Weighing 0.1mmol DSPE-ROS Linker-COOH and 0.2mmol PEG respectively 2k -NH 2 Dissolved in 10mL of chloroform, 0.5mmol of EDC and 0.5mmol of NHS were weighed and dissolved in 5mL of chloroform, respectively. At 30 ℃, dropwise adding EDC/chloroform solution into DSPE-ROS Linker-COOH/chloroform solution under stirring, magnetically stirring for reacting for 30min, dropwise adding NHS/chloroform solution into the DSPE-ROS Linker-COOH/EDC/chloroform solution, and magnetically stirring for reacting for 3h; dropwise adding the mixed solution to PEG 2k -NH 2 In the chloroform solution, the reaction is carried out for 24 hours by continuous magnetic stirring. After the reaction is finished, the solution obtained by the reaction is subjected to reduced pressure rotary evaporation to remove the chloroform, and then the liquid after the removal of the chloroform is dialyzed for 36 hours (dialysis bag with a cut-off molecular weight of 3000 daltons) in distilled water, and water is changed for 6 times during the period to obtain DSPE-ROS Linker-PEG 2k And then freeze-drying for later use.
Example 10
The embodiment provides a preparation method of an ultrasonic response urokinase thrombolysis nanoliposome.
10mg of DSPE-PpIX prepared in example 8 and 50mg of DSPE-ROS Linker-PEG prepared in example 9 were weighed out respectively 2k Dissolving in 20mL of chloroform, completely dissolving by ultrasonic, and then removing solvent chloroform by normal temperature and reduced pressure distillation to obtain a uniform film. 30mL of ultrapure water was added to the round-necked flask, and the mixture was hydrated by stirring with a magnetic force (ultrasonic power: 300W) at 60℃for 60 minutes. After hydration, heating is stopped, the room temperature is cooled to 40 ℃, 4000U of urokinase is weighed and dispersed in 1mL of ultrapure water, and the completely dissolved urokinase solution is added into the hydrated liposome fragments to continue magnetic stirring and mixing for 30min. Transferring the solution after uniform mixing into a 50mL centrifuge tube, and performing ultrasonic emulsification under ice bath protection for 30min (ultrasonic power: 350W; ultrasonic interval: 2 seconds apart, 2 seconds stop)). Finally, removing free urokinase and phospholipid fragments from the dispersion liquid after self-assembly by adopting an ultrafiltration mode (dialysis bag cut-off molecular weight 10000 daltons) to obtain ultrasonic response urokinase thrombolysis nano-liposome (ULU).
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The preparation method of the ultrasonic response urokinase thrombolysis nanoliposome is characterized by comprising the following steps of:
(1) EDC, NHS, ppIX and DSPE-NH 2 Amidation reaction to obtain DSPE-PpIX;
(2) EDC, NHS, DSPE ROS Linker-COOH and PEG 2k -NH 2 Amidation reaction to obtain DSPE-ROS Linker-PEG 2k ；
(3) The DSPE-PpIX prepared in the step (1) and the DSPE-ROS Linker-PEG prepared in the step (2) are mixed 2k Dissolving in solvent, and post-treating to obtain precursor liquid;
(4) And (3) uniformly mixing urokinase with the precursor liquid prepared in the step (3), and performing post-treatment after self-assembly to obtain the ultrasonic response urokinase nanoliposome.
2. The method for preparing ultrasonic response urokinase thrombolysis nanoliposome according to claim 1, wherein EDC, NHS, ppIX and DSPE-NH are mixed in step (1) 2 The molar ratio of (2) is 5mmol:5mmol:1mmol:1-2mmol;
in the amidation reaction process, the reaction temperature is 20-30 ℃ and the reaction time is 12-24h.
3. The method for preparing the ultrasonic response urokinase thrombolysis nanoliposome according to claim 1, wherein in the step (2), EDC, NHS, DSPE-ROS Linker-COOH and PEG are mixed 2k -NH 2 The molar ratio of (2) is 5mmol:5mmol:1mmol:1-2mmol;
in the amidation reaction process, the reaction temperature is 20-30 ℃ and the reaction time is 12-24h.
4. The method for preparing an ultrasonic response urokinase thrombolysis nanoliposome according to claim 1, wherein in the step (3), the solvent is one or more selected from methanol, dimethyl sulfoxide and chloroform.
5. The method for preparing the ultrasonic response urokinase thrombolytic nanoliposome according to claim 1, wherein in the step (3), the post-treatment is to remove the solvent by rotary evaporation to form a film, and the film is cooled to 30-40 ℃ after ultrasonic hydration.
6. The method for preparing the ultrasonic response urokinase thrombolysis nanoliposome according to claim 5, wherein the ultrasonic power is 300-350W, the hydration temperature is 60-65 ℃ and the hydration time is 30-60min in the ultrasonic hydration process.
7. The method for preparing an ultrasonic response urokinase thrombolysis nanoliposome according to claim 1, wherein in the step (4), urokinase and DSPE-PpIX and DSPE-ROS Linker-PEG in the precursor solution 2k The dosage ratio of (2) is 2000-4000U:10mg;50mg.
8. The method for preparing the ultrasonic response urokinase thrombolysis nanoliposome according to claim 1, wherein the self-assembly process is ultrafiltration after ultrasonic emulsification;
in the ultrasonic emulsification process, the power of ultrasonic waves is 300-350W, the emulsification time is 30-60min, and the emulsification temperature is 0-4 ℃;
during ultrafiltration, the molecular weight cut-off was 100000 daltons.
9. An ultrasonically responsive urokinase thrombolytic nanoliposome prepared according to the method of any one of claims 1-8.
10. Use of the ultrasonically responsive urokinase thrombolytic nanoliposome of claim 9 in the preparation of a medicament for the treatment of acute pulmonary embolism.
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