A nano-lipid preparation for encapsulating perfluorohexane and antioxidant, its preparation method and application for preparing a medicine for treating myocardial infarction

Abstract

The application belongs to the field of biological medicine, and discloses a nano-lipid preparation for loading perfluorohexane and an antioxidant, wherein the nano-lipid preparation is a core-shell structure, the core-shell structure takes a phospholipid liposome membrane layer as an outer shell, and perfluorohexane and the antioxidant loaded in the outer shell serve as an inner core; the phospholipid liposome membrane layer is made of hydrogenated lecithin, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and cholesterol; and the application discloses an application of the nano-lipid preparation for loading perfluorohexane and the antioxidant in preparation of a medicine for treating myocardial infarction. The nano-lipid preparation has a suitable particle size, can be passively targeted and enriched to a myocardial infarction site, is stable, has a high encapsulation efficiency, and has a simple administration mode; after the lipid preparation is taken by myocardial cells, oxygen and the antioxidant are released, the oxygen can relieve an anoxic condition of a myocardial ischemia site, and the antioxidant can reduce ROS damage, both of which play a synergistic role, improve the survival rate of ischemic myocardial cells, and improve heart function.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a nanolipid formulation loaded with perfluorohexane and antioxidants, its preparation method, and its application in the preparation of drugs for treating myocardial infarction. Background Technology

[0002] Acute myocardial infarction (AMI) is a serious ischemic heart disease with extremely high morbidity and mortality, and the number of cases is increasing year by year. Its pathogenesis mainly involves coronary artery disease and blockage leading to a sharp reduction or interruption of blood flow, resulting in severe and prolonged acute ischemia and hypoxia of the myocardium, ultimately causing irreversible myocardial damage. Following myocardial ischemia and hypoxia, ATP is rapidly depleted, and contractile function is inhibited within seconds. Early ultrastructural changes in cardiomyocytes are visible within minutes. As the ischemic time prolongs, the number of apoptotic and necrotic cardiomyocytes in the subendocardial region increases. Currently, reperfusion interventions such as coronary artery bypass grafting and percutaneous coronary intervention are standard clinical treatments. However, when myocardial blood flow is restored through reperfusion, the myocardium, which has been in a state of ischemia for a long time, will experience abnormal oxidative stress. The large burst of reactive oxygen species (ROS) during perfusion can damage mitochondria and induce cardiomyocyte apoptosis, leading to more severe myocardial ischemia-reperfusion injury. Because mature cardiomyocytes are highly differentiated terminal cells, reperfusion injury can permanently impair cardiac function. Therefore, finding new strategies to reduce cardiomyocyte death and cardiac function impairment has become particularly important.

[0003] Oxygen therapy is a routine treatment for ischemic diseases. Oxygen is believed to increase oxygen supply to ischemic myocardium, thereby improving cardiac metabolism, increasing arterial blood oxygen tension, and reducing myocardial cell apoptosis and infarct size. However, inhaled oxygen delivery lacks targeting, leading to increased systemic blood oxygen concentration, stimulating vascular endothelial cells to produce more reactive oxygen species (ROS), further aggravating damage at the infarct site. Perfluorohexane (PFH) is a short-chain perfluorocarbon compound with biological inertness and chemical stability. PFH has a high oxygen-binding capacity, approximately 20 times that of dissolved oxygen in water, enabling targeted oxygen delivery. Currently, PFH is often studied as an oxygen donor in photodynamic therapy for anti-tumor and antibacterial effects, and rarely used to treat myocardial infarction. If targeted oxygen delivery to ischemic myocardium can be achieved, the side effects of oxygen therapy can be reduced, myocardial hypoxia can be alleviated, and metabolic capacity can be restored. Excessive ROS production after myocardial ischemia and hypoxia can lead to myocardial stunning, damage to myocardial cell nuclear and mitochondrial DNA, intracellular protein denaturation, lipid peroxidation, and inflammatory responses. Therefore, reducing oxidative stress in damaged myocardial sites is beneficial for improving cardiomyocyte survival and restoring cardiac function. Combining oxygen-carrying PFHs with antioxidants can alleviate the hypoxic microenvironment of ischemic myocardium and reduce oxidative stress, while the antioxidants can eliminate ROS generated in ischemic sites.

[0004] Nanoparticle delivery systems have been extensively studied in the field of targeted drug delivery. Following myocardial infarction, vascular endothelial cells become disordered, and permeability increases, allowing nanomaterials to passively target the infarcted myocardium through the enhanced permibility and retention (EPR) effect. Liposomes, typically composed of phospholipids, cholesterol, and phospholipid derivatives assembled in an aqueous phase, are cell-membrane-like bilayer structures. Due to their simple preparation and good biocompatibility, they are widely used in cancer treatment as an important nanoparticle drug delivery system. Summary of the Invention

[0005] The purpose of this invention is to provide a nanolipid formulation encapsulating oxygen-carrying PFH and antioxidants. This nanolipid formulation is prepared by thin-film dispersion, which is simple to prepare and has good biocompatibility. It delivers oxygen and antioxidant drugs to damaged myocardial cells, which helps to promote the recovery of myocardial cells and improve the treatment effect of myocardial infarction.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0007] A nanolipid formulation loaded with perfluorohexane and antioxidants, wherein the nanolipid formulation has a core-shell structure, with a phospholipid liposome membrane layer as the outer shell and perfluorohexane and antioxidants loaded on the outer shell as the core; wherein the phospholipid liposome membrane layer is made of hydrogenated lecithin (HSPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and cholesterol (Cho).

[0008] The hydrogenated lecithin mentioned above is selected from hydrogenated soybean lecithin.

[0009] The distearyl phosphatidylethanolamine-polyethylene glycol 2000 (liposome for prolonging blood circulation) is selected from any one or more of the following: distearyl phosphatidylethanolamine-polyethylene glycol 2000, distearyl phosphatidylethanolamine-polyethylene glycol 5000, distearyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and distearyl phosphatidylethanolamine-polyethylene glycol 5000-maleimide.

[0010] The molar ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol is 65:(4-8):(27-31), preferably 13:1:6.

[0011] The total mass ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol to perfluorohexane is 1:6 to 1:20, preferably 1:20.

[0012] The antioxidant is selected from any one of resveratrol, pterostilbene, quercetin, lutein, etc.

[0013] The ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol to the molar amount of antioxidant is ≤25:0.002 mg / mmol, preferably 25:0.002 mg / mmol.

[0014] Specifically, when the antioxidant is resveratrol, the ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol to the molar amount of the antioxidant can be 25:0.002 mg / mmol.

[0015] The present invention relates to a nanolipid formulation loaded with oxygen-carrying perfluorohexane and an antioxidant. The nanolipid formulation loading perfluorohexane and antioxidant has a particle size of 50–300 nm, preferably 110–130 nm. This nanolipid formulation can passively target and accumulate at the site of myocardial infarction, releasing oxygen and drugs.

[0016] Another object of the present invention is to provide a method for preparing a nanolipid formulation loaded with perfluorohexane and an antioxidant, comprising the following steps:

[0017] Step (1): Dissolve hydrogenated lecithin, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000 in an organic solvent to form a lipid solution, and dissolve the antioxidant in methanol to form a solution;

[0018] Step (2): Mix the lipid solution and the antioxidant solution, and remove the solvent by rotary evaporation to obtain a lipid film;

[0019] Step (3): Add pure water and hydrate with ultrasound to obtain crude liposomes. Then add perfluorohexane and use an ultrasonic cell disruptor to obtain a uniform nanoliposome preparation.

[0020] In step (1), the organic solvent is one of chloroform and dichloromethane.

[0021] The ratio of organic solvent to methanol has no significant effect on the particle size of liposomes. Generally, the volume ratio of organic solvent to methanol is 8:1 to 10:1, preferably 10:1.

[0022] In step (2), the temperature of rotary evaporation is 37-40 °C and the time of rotary evaporation is 5-10 min.

[0023] In step (3), during hydration, the ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol to the volume of pure water is 25 mg: 2 to 8 mL, preferably 25 mg: 2.7 mL.

[0024] The ultrasonic cell disruptor has an ultrasonic power of 350 W and an ultrasonic time of 2–8 min.

[0025] Another object of the present invention is to provide the use of the nanolipid formulation containing perfluorohexane and antioxidants in the preparation of drugs for treating myocardial infarction.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The nanolipid formulation of this invention has a suitable particle size, which can be passively targeted and enriched at the site of myocardial infarction. The nanolipid formulation is stable, has a high encapsulation rate, and is easy to administer. After being taken up by myocardial cells, the lipid formulation releases oxygen and antioxidants, which can alleviate hypoxia in the ischemic area of ​​myocardium and reduce ROS damage. The two work synergistically to improve the survival rate of ischemic myocardial cells and improve cardiac function. Attached Figure Description

[0028] Figure 1This is a flowchart illustrating the preparation process of nanolipid formulations loaded with perfluorohexane and resveratrol.

[0029] Figure 2 The particle size of nanolipid formulations prepared under different ultrasonic powers.

[0030] Figure 3 The particle size of the nanolipid formulations prepared under different amounts of pure water during hydration is shown.

[0031] Figure 4 The particle size of nanolipid formulations prepared with different amounts of perfluorohexane.

[0032] Figure 5 The effect of the ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol on the particle size of nanolipid formulations.

[0033] Figure 6 TEM (a), particle size distribution (b), and stability diagram (c) of the nanolipid formulation Lip(PFH, Res) prepared in Example 5.

[0034] Figure 7 Particle size distribution (a) of Lip(PFH, Res)-d1 prepared for Comparative Example 3 and its passive targeting capability at the site of myocardial infarction (b).

[0035] Figure 8 The passive targeting ability of the nanolipid formulation Lip(PFH, Res) prepared in Example 5 at the site of myocardial infarction.

[0036] Figure 9 The graph shows the evaluation of the antioxidant capacity of the nanolipid formulation Lip(PFH, Res) prepared in Example 5 at the site of myocardial infarction.

[0037] Figure 10 This is an evaluation graph showing the ability of the nanolipid formulation Lip(PFH, Res) prepared in Example 5 to relieve hypoxia at the site of myocardial infarction.

[0038] Figure 11 The graph shows the evaluation of the reduction of myocardial infarction markers by the nanolipid formulation Lip(PFH, Res) prepared in Example 5 at the site of myocardial infarction.

[0039] Figure 12 The nanolipid formulation Lip(PFH, Res) prepared in Example 5 improved cardiac function in patients with myocardial infarction. Detailed Implementation

[0040] The technical solution of the present invention will be further illustrated below through embodiments, the purpose of which is only to better understand the content of the present invention. Therefore, the listed embodiments do not limit the scope of protection of the present invention.

[0041] The room temperature in the examples was 25–28 °C; all raw materials and reagents used were commercially available.

[0042] Example 1

[0043] Investigating the effect of ultrasonic power on the particle size of nanolipid formulations

[0044] like Figure 1 As shown, nanolipid formulations loaded with perfluorohexane and resveratrol (Res) were prepared under different ultrasonic powers. The preparation methods are as follows:

[0045] Step (1): Weigh 16.6 mg of hydrogenated soybean lecithin, 4.6 mg of distearylphosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg of cholesterol (the molar ratio of hydrogenated lecithin, distearylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:1:6) and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg of resveratrol in 0.5 mL of methanol to form a resveratrol solution.

[0046] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film.

[0047] Step (3): Add 2.7 mL of pure water to the eggplant-shaped flask, and obtain crude liposomes by ultrasonic hydration and peeling off the lipid membrane. Add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min (ultrasonic power of 150 W, 250 W, and 350 W respectively) to obtain a uniform nanoliposome preparation.

[0048] Depend on Figure 2 It can be seen that when the ultrasonic power is 150 W, 250 W and 350 W respectively, the corresponding particle sizes of the nanolipid formulation are 267 nm, 174 nm and 127 nm respectively.

[0049] Example 2

[0050] Investigating the effect of pure water dosage during hydration on the particle size of nanolipid formulations

[0051] Nanolipid formulations loaded with perfluorohexane and resveratrol were prepared under different pure water dosages. The preparation methods are as follows:

[0052] Step (1): Weigh out three portions of 16.6 mg hydrogenated soybean lecithin, 4.6 mg distearyl phosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg cholesterol (the molar ratio of hydrogenated lecithin, distearyl phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:1:6) and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg resveratrol in 0.5 mL of methanol to form a resveratrol solution.

[0053] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film.

[0054] Step (3): Add pure water (1.7 mL, 2.7 mL, and 7.7 mL respectively) to the eggplant-shaped flask, and obtain crude liposomes by ultrasonic hydration and peeling off the lipid membrane. Add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min at a power of 350 W to obtain a uniform nanoliposome preparation.

[0055] Depend on Figure 3 It can be seen that when the amount of pure water used during hydration is 1.7 mL, 2.7 mL, and 7.7 mL, the corresponding particle sizes of the nanolipid formulations are 214 nm, 127 nm, and 120 nm, respectively.

[0056] Example 3

[0057] The effect of perfluorohexane dosage on the particle size of nanolipid formulations was investigated.

[0058] Nanolipid formulations loaded with perfluorohexane and resveratrol (Res) were prepared under different amounts of perfluorohexane. The preparation methods are as follows:

[0059] Step (1): Weigh 16.6 mg of hydrogenated soybean lecithin, 4.6 mg of distearylphosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg of cholesterol (the molar ratio of hydrogenated lecithin, distearylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:1:6) and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg of resveratrol in 0.5 mL of methanol to form a resveratrol solution.

[0060] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film.

[0061] Step (3): Add 2.9, 2.7, and 2.6 mL of pure water to the eggplant-shaped flasks respectively, and obtain crude liposomes by ultrasonic hydration and stripping of the lipid membrane. Add 0.1, 0.3, and 0.4 mL of PFH respectively to make the final volume of the nanoliposome preparation the same. Insert the probe of the ultrasonic cell disruptor below the liquid surface and sonicate at a power of 350 W for 5 min to obtain a uniform nanoliposome preparation.

[0062] Depend on Figure 4 It can be seen that when the amount of perfluorohexane used is 0.1, 0.3, and 0.4 mL, the corresponding particle sizes of the nanolipid formulations are 113 nm, 127 nm, and 236 nm, respectively.

[0063] Example 4

[0064] The effects of hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol ratio on the particle size of nanolipid formulations were investigated.

[0065] Nanolipid formulations loaded with perfluorohexane and resveratrol were prepared under different amounts of hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol (a: 65%, 2%, and 33% molar percentages of hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol; b: 65%, 5%, and 30% molar percentages of hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol; 65%, 10%, and 25% molar percentages of hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol; 65%, 15%, and 20% molar percentages of hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol). The preparation methods are as follows:

[0066] Step (1): Weigh out hydrogenated soybean lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol, respectively:

[0067] Dosage a: 18.4 mg hydrogenated soybean lecithin, 2 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, 4.6 mg cholesterol;

[0068] Dosage b: 16.6 mg hydrogenated soybean lecithin, 4.6 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, 3.8 mg cholesterol;

[0069] Dosage c: 14.4 mg hydrogenated soybean lecithin, 7.9 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, 2.7 mg cholesterol;

[0070] Dosage d: 12.6 mg hydrogenated soybean lecithin, 10.4 mg distearate phosphatidylethanolamine-polyethylene glycol 2000, 1.9 mg cholesterol;

[0071] Each was dissolved in 5 mL of chloroform to form a lipid solution, and 0.5 mg of resveratrol was dissolved in 0.5 mL of methanol to form a resveratrol solution.

[0072] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film.

[0073] Step (3): Add 2.7 mL of pure water to the eggplant-shaped flask, and obtain crude liposomes by ultrasonic hydration and peeling off the lipid membrane. Add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min at a power of 350 W to obtain a uniform nanoliposome preparation.

[0074] Depend on Figure 5 It can be seen that the particle sizes of the nanolipid formulations prepared by dosages a, b, c, and d are 147 nm, 127 nm, 193 nm, and 157 nm, respectively.

[0075] Example 5

[0076] A method for preparing a nanolipid formulation loaded with perfluorohexane and resveratrol includes the following steps:

[0077] Step (1): Weigh 16.6 mg of hydrogenated soybean lecithin, 4.6 mg of distearylphosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg of cholesterol (the molar ratio of hydrogenated soybean lecithin, distearylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:1:6), dissolve them in 5 mL of chloroform to form a lipid solution, and dissolve 0.5 mg of resveratrol in 0.5 mL of methanol to form a resveratrol solution;

[0078] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film.

[0079] Step (3): Add 2.7 mL of pure water to the eggplant-shaped flask, and obtain crude liposomes by ultrasonic hydration and peeling off the lipid membrane. Add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min at a power of 350 W to obtain a uniform nanoliposome preparation Lip(PFH, Res).

[0080] The Lip(PFH, Res) prepared in this embodiment was characterized by TEM (exposure profilometry). Figure 6 a) It can be seen that the morphology of Lip(PFH, Res) is spherical with a particle size of approximately 127 nm. Since the lipids are loaded with PFH and Res, the color of the lipid center is darker than that of the lipid membrane, indicating that Lip(PFH, Res) was successfully synthesized. The hydrated particle size of Lip(PFH, Res) was determined using a laser particle size analyzer, see [link to data]. Figure 6 b. The size of the Lip(PFH, Res) nanoparticles is approximately 127 nm. The stability of Lip(PFH, Res) in PBS (pH=7.4) or DMEM + 10% FBS was assessed for seven consecutive days. The results are shown in [Figure number missing]. Figure 6 c. The particle size of Lip(PFH, Res) did not change much in PBS and DMEM+10% FBS, indicating that Lip(PFH, Res) has good in vitro stability.

[0081] Example 6

[0082] To investigate the passive targeting performance of Lip(PFH, Res) in Example 5, succinimide ester (Cyanine5.5 NHS ester, Cy5.5) was introduced to prepare Lip(PFH, Res, Cy5.5) with a fluorescent dye. The preparation method is as follows:

[0083] Step (1): Weigh 16.6 mg of hydrogenated soybean lecithin, 4.6 mg of distearylphosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg of cholesterol (the molar ratio of hydrogenated soybean lecithin, distearylphosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:1:6) and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg of resveratrol in 0.5 mL of methanol to form a resveratrol solution; and dissolve 2 mg of Cy5.5 in 1 mL of DMSO to form a Cy5.5 solution with a concentration of approximately 2 mg / mL.

[0084] Step (2): Place the above lipid solution, resveratrol solution and 20 μL Cy5.5 solution into a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and rotary evaporate at 37 ℃ for 5 min to remove the solvent, and obtain a lipid film.

[0085] Step (3): Add 2.7 mL of pure water to the eggplant-shaped flask, and obtain crude liposomes by ultrasonic hydration and peeling off the lipid membrane. Add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate at 350 W for 5 min to obtain a uniform nanoliposome preparation Lip(PFH, Res, Cy5.5).

[0086] Comparative Example 1

[0087] A method for preparing a nanolipid formulation loaded with perfluorohexane includes the following steps:

[0088] Step (1): Weigh 16.6 mg of hydrogenated soybean lecithin, 4.6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg of cholesterol and dissolve them in 5 mL of chloroform to form a lipid solution.

[0089] Step (2): Place the above lipid solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and rotary evaporate at 37 ℃ for 5 min to obtain a lipid film.

[0090] Step (3): Add 2.7 mL of pure water to the eggplant-shaped flask, and after obtaining crude liposomes by ultrasonic hydration and peeling of the lipid membrane, add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min at a power of 350 W to obtain the nanolipid formulation Lip(PFH).

[0091] Comparative Example 2

[0092] A method for preparing a nanolipid formulation loaded with resveratrol includes the following steps:

[0093] Step (1): Weigh 16.6 mg of hydrogenated soybean lecithin, 4.6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, and 3.8 mg of cholesterol and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg of Res resveratrol in 0.5 mL of methanol to form a resveratrol solution.

[0094] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and rotary evaporate at 37 ℃ for 5 min to obtain a lipid film.

[0095] Step (3): Add 2.7 mL of pure water to the eggplant-shaped bottle, and after ultrasonic hydration and stripping of the lipid membrane to obtain crude liposomes, add 0.3 mL of pure water, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min at a power of 350 W to obtain the nanoliposome preparation Lip(Res).

[0096] Comparative Example 3

[0097] A method for preparing a nanolipid formulation loaded with perfluorohexane and resveratrol includes the following steps:

[0098] Step (1): Weigh 14.4 mg of hydrogenated soybean lecithin, 7.9 mg of distearyl phosphatidylethanolamine-polyethylene glycol 2000, and 2.7 mg of cholesterol (the molar ratio of hydrogenated soybean lecithin, distearyl phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:2:5) and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg of resveratrol in 0.5 mL of methanol to form a resveratrol solution.

[0099] Step (2): Place the above lipid solution and resveratrol solution in a 25 mL eggplant-shaped flask, sonicate for 15 s to mix, place the eggplant-shaped flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film.

[0100] Step (3): Add 2.7 mL of pure water to a flask, and obtain crude liposomes by ultrasonic hydration and stripping of the lipid membrane. Add 0.3 mL of PFH, insert the probe of an ultrasonic cell disruptor below the liquid surface, and sonicate at 350 W for 5 min to obtain a homogeneous nanoliposome formulation, denoted as Lip(PFH, Res)-d1, with a particle size of 193 nm. Figure 7 a).

[0101] Comparative Example 4

[0102] To investigate the passive targeting of comparative example 3 Lip(PFH, Res)-d1, succinimide ester (Cyanine5.5 NHS ester, Cy5.5) was introduced to prepare Lip(PFH, Res, Cy5.5)-d1 with a fluorescent dye. The preparation method is as follows:

[0103] Step (1): Weigh 14.4 mg of hydrogenated soybean lecithin, 7.9 mg of distearyl phosphatidylethanolamine-polyethylene glycol 2000, and 2.7 mg of cholesterol (the molar ratio of hydrogenated soybean lecithin, distearyl phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 13:2:5) and dissolve them in 5 mL of chloroform to form a lipid solution; dissolve 0.5 mg of resveratrol in 0.5 mL of methanol to form a resveratrol solution.

[0104] Step (2): Dissolve 2 mg Cy5.5 in 1 mL DMSO to form a Cy5.5 solution of about 2 mg / mL; place the above lipid solution, resveratrol solution and 20 μL Cy5.5 solution in a 25 mL flask, sonicate for 15 s to mix, place the flask on a rotary evaporator, and remove the solvent by rotary evaporation at 37 ℃ for 5 min to obtain a lipid film;

[0105] Step (3): Add 2.7 mL of pure water to the eggplant-shaped flask, and obtain crude liposomes by ultrasonic hydration and peeling off the lipid membrane. Add 0.3 mL of PFH, insert the probe of the ultrasonic cell disruptor below the liquid surface, and sonicate for 5 min at a power of 350 W to obtain a uniform nanoliposome preparation, denoted as Lip(PFH, Res, Cy5.5)-d1.

[0106] Example 7

[0107] (1) Experimental groups: normal control group (Healthy / Control), myocardial infarction model group (MI group), MI+Lip(PFH) group (Lip(PFH) group, 666.7 mg / kg), MI+Lip(Res) group (Lip(Res) group, 0.67 mg / kg), MI+Lip(PFH, Res) group (Lip(PFH, Res) group, PFH: 666.7 mg / kg, Res: 0.67 mg / kg), MI+Lip(PFH, Res)-d1 group (Lip(PFH, Res)-d1 group, PFH: 333.3 mg / kg, Res: 0.67 mg / kg), all were prepared with physiological saline and administered to rats in each group via tail vein.

[0108] For the passive targeting experiment (biodistribution), Lip(PFH, Res, Cy5.5) with fluorescent dye prepared in Example 6 and Lip(PFH, Res, Cy5.5)-d1 prepared in Comparative Example 4 were used. For the other experiments, Lip(PFH, Res) prepared in Example 5 was used.

[0109] (2) Establishment of a myocardial infarction model:

[0110] All SD rats underwent a one-week acclimatization period, and were fasted for 12 hours prior to the experiment. After anesthesia, the rats were fixed in a supine position on a rat board. Hair was removed from the neck and left chest, and the area was disinfected with 75% alcohol swabs. The neck skin was centrally incised with surgical scissors, and the trachea was bluntly dissected. After carefully incising the trachea, a small animal ventilator was connected (respiratory rate 80 breaths / min, respiratory ratio 5:4, tidal volume 8 ml), and the rat was intubated. The left chest skin was incised with surgical scissors, and the muscles were bluntly dissected. An opening was made in the left third intercostal space, and the opening was widened with surgical forceps to fully expose the heart. The left atrial appendage was located, the pericardium was dissected, and the left anterior descending coronary artery was identified. Myocardial ischemia was induced by ligation with 6-0 surgical sutures 2 mm below the lower edge of the left atrial appendage. A pale white appearance of the left ventricular anterior wall below the ligation site indicated successful MI modeling. The muscles and skin were then carefully sutured, and the rat was placed on an electric blanket and observed until it woke up. Forty-five minutes after modeling, rats were injected with the drug via the tail vein, while the MI group was injected with an equal volume of physiological saline.

[0111] (3) Evaluation of passive targeting ability: 45 min after modeling, rats in the MI+Lip(PFH, Res) group were given Lip(PFH, Res,Cy5.5) prepared in Example 6 via tail vein injection, rats in the MI+Lip(PFH, Res)-d1 group were given Lip(PFH, Res, Cy5.5)-d1 prepared in Comparative Example 4 via tail vein injection, and rats in the Control group were given an equal volume of physiological saline via tail vein injection. 24 h after myocardial infarction, the rats were dissected, and the main organs (heart, liver, spleen, lung, kidney) were removed, washed, dried, and imaged using small animal in vivo imaging.

[0112] Experimental results: such as Figure 7 As shown in b, the cardiac fluorescence intensity of rats in the Lip(PFH, Res)-d1 group was not significantly different from that in the Control group, indicating that Lip(PFH, Res)-d1 with a particle size of 193 nm is difficult to target the myocardial infarction site. Figure 8 As shown, compared to the Control group, the hearts of rats in the Lip(PFH, Res) group exhibited stronger fluorescence signals, indicating a higher distribution of Lip(PFH, Res) in the hearts of rats in the Lip(PFH, Res) group. This suggests that Lip(PFH, Res) can be passively targeted and enriched at the site of myocardial infarction. Other organs of rats in the Lip(PFH, Res) group were similar to those in the Control group, with a higher distribution of Lip(PFH, Res) in the liver compared to the Control group. This is likely related to the fact that Lip(PFH, Res) is metabolized by the liver.

[0113] (4) Evaluate the ROS clearance capacity and oxygen delivery capacity of the myocardial infarction site separately:

[0114] Forty-five minutes after successful modeling, the following drugs were administered via tail vein injection: the Lip(PFH) group received Lip(PFH) prepared in Comparative Example 1 at 666.7 mg / kg; the Lip(Res) group received Lip(Res) prepared in Comparative Example 2 at 0.67 mg / kg; the Lip(PFH, Res) group received Lip(PFH, Res) prepared in Example 5, with PFH at 666.7 mg / kg and Res at 0.67 mg / kg; healthy rats in the Control group and rats in the myocardial infarction model group (MI group) received an equal volume of physiological saline. Twenty-four hours after myocardial infarction, the rats were sacrificed, and their ROS clearance capacity and oxygen-relieving capacity at the myocardial infarction site were evaluated.

[0115] Evaluation of ROS clearance capacity in myocardial infarction sites: After euthanizing rats, fresh hearts were immediately harvested, and the left ventricle portion was removed. The tissue was washed in pre-cooled PBS until no residual blood remained. The tissue block was placed in an embedding cassette containing OCT embedding medium, and an appropriate amount of OCT embedding medium was added until the tissue block was completely embedded. The tissue block was frozen in liquid nitrogen for approximately 30 seconds for cryo-embedding. Sections were prepared, and an appropriate amount of 10 μM DCFH-DA staining solution was added. The sections were incubated at 37 ℃ in the dark for 30 min, washed three times with PBS, and then DAPI staining solution was added. The sections were incubated at room temperature in the dark for 10 min, washed three times with PBS, and then mounted with anti-fluorescence quenching mounting solution. Finally, the sections were observed and photographed under a confocal fluorescence microscope. Statistical analysis was performed using ImageJ, and a statistical graph was obtained.

[0116] Experimental results are as follows Figure 9 As shown, the hearts of rats in the MI group injected with physiological saline exhibited large areas of strong green fluorescence, indicating the production of a large amount of ROS after myocardial infarction. After injection of Lip(PFH), the fluorescence intensity further increased, indicating that the presence of oxygen-carrying PFH in Lip(PFH) could induce more ROS production in the infarcted myocardial tissue. After treatment with Lip(Res) and Lip(PFH, Res), the fluorescence intensity significantly decreased and was similar to that of healthy rats in the Control group, indicating that the Res-loaded lipid nanoparticle formulation can effectively scavenge oxidative stress at the myocardial infarction site and eliminate ROS generated by oxygen-carrying PFH. Therefore, Lip(PFH, Res) can effectively reduce the ROS content at the myocardial infarction site and can reduce the side effects of administering Lip(PFH) alone.

[0117] Evaluation of oxygen-relieving capacity of myocardial infarction sites: After euthanizing rats, fresh hearts were immediately harvested, and the left ventricle was excised, cleaned, fixed, dehydrated, embedded in paraffin, and sectioned. Paraffin sections were dewaxed to water and placed in 0.5% Triton X-100 solution for antigen retrieval. The sections were then blocked in bovine serum albumin (BSA) solution for 1 h. After blocking, diluted murine HIF-1α antibody and tissue sections were incubated overnight at 4 °C. After washing three times with PBS, a FITC-labeled secondary antibody solution was added and incubated at 37 °C for 2 h. Excess staining solution was washed away with PBS, and anti-fluorescence quenching mounting solution was added for mounting. Finally, the sections were observed and photographed under a co-fluorescence microscope. Statistical analysis was performed using ImageJ, and statistical graphs were obtained.

[0118] Experimental results are as follows Figure 10 As shown, after Lip(Res) administration, the expression level of HIF-1α was similar to that in the MI group. However, the expression level of HIF-1α in myocardial tissue after injection of Lip(PFH) and Lip(PFH, Res) was not significantly different from that in the control group of healthy rats, indicating that Lip(PFH) and Lip(PFH, Res) can improve the hypoxic environment of myocardial tissue.

[0119] (5) Evaluation of myocardial infarction markers and echocardiographic examination in myocardial infarction tissue:

[0120] The drugs were administered via tail vein injection 45 min after successful modeling, on day 2, and on day 4: the Lip(PFH) group was given Lip(PFH) prepared in Comparative Example 1 at 666.7 mg / kg; the Lip(Res) group was given Lip(Res) prepared in Comparative Example 2 at 0.67 mg / kg; the Lip(PFH, Res) group was given Lip(PFH, Res) prepared in Example 5, with PFH: 666.7 mg / kg and Res: 0.67 mg / kg; healthy rats in the Control group and rats in the myocardial infarction model group (MI group) were given an equal volume of physiological saline.

[0121] Evaluation of reduced myocardial infarction markers in myocardial infarction tissue: Blood was collected from the canthus of rats on day 4, left at room temperature for 1 h, centrifuged at 3000 rpm for 10 min at 4 ℃, and the supernatant was collected and detected according to the corresponding detection kits for CKMB and LDH.

[0122] Experimental results are as follows Figure 11As shown, compared to the model group, administration of Lip(PFH) and Lip(Res) reduced CKMB and LDH levels, with similar reductions in both groups. However, treatment with Lip(PFH, Res) significantly reduced CKMB and LDH levels, similar to the control group, indicating that PFH and Res in Lip(PFH, Res) act synergistically to alleviate the severity of myocardial infarction.

[0123] Cardiac ultrasound examination: After MI injury, cardiac function was examined using an ultra-high resolution small animal ultrasound imaging system. Vevo LAB 3.2.0 software was used to calculate the cardiac function of rats, including left ventricular shortening fraction, left ventricular ejection fraction, and cardiac output.

[0124] Experimental results are as follows Figure 12 As shown in the echocardiogram (a), compared to the healthy rat control group, the anterior wall of the left ventricle was thinned, the left ventricular cavity was enlarged, and the cardiac amplitude was reduced after myocardial infarction (MI). Administration of Lip(PFH) and Lip(Res) both improved the systolic function of the infarcted rats and alleviated ventricular enlargement starting from day 3. Administration of Lip(PFH, Res) significantly improved the systolic function of the rats from day 3, increased the cardiac amplitude, and significantly reduced the left ventricular cavity enlargement. As shown in the statistical chart (b), the analysis of left ventricular ejection fraction (EF), left ventricular shortening fraction (FS), and cardiac output (CO) of rats in each group from day 1 to day 14 showed that Lip(PFH) and Lip(Res) began to improve cardiac function on day 3, maintained cardiac function on day 7, but both further improved cardiac function on day 14 and the degree of improvement was similar. However, Lip(PFH, Res) could significantly improve cardiac function on day 3 and reached a level of cardiac function similar to the normal group on day 14.

[0125] result

[0126] Small animal in vivo imaging results showed that, compared with the control group, rats with successful MI modeling showed significantly enhanced cardiac fluorescence intensity after administration of Lip(PFH, Res, Cy5.5), indicating that Lip(PFH, Res) can passively target the infarcted heart. DCFH-DA detection results showed that Lip(PFH, Res) significantly reduced ROS generated after myocardial infarction. HIF-1α detection results showed that Lip(PFH, Res) alleviated the hypoxic microenvironment after myocardial infarction. CKMB and LDH assay results showed that Lip(PFH, Res) significantly reduced the myocardial infarction markers CKMB and LDH after myocardial infarction. Echocardiography results showed that Lip(PFH, Res) significantly improved systolic function, ejection fraction, and cardiac output after myocardial infarction.

[0127] In summary, the nanolipid formulation of this invention exhibits good in vitro stability. This invention uses SD rats as experimental subjects to establish a myocardial infarction model by ligating the left anterior descending coronary artery. Treatment of myocardial infarction rats with Lip(PFH, Res) showed good passive targeting, and the nanolipid formulation can alleviate the hypoxic and oxidative stress microenvironment at the myocardial infarction site through oxygen release and antioxidant drugs, respectively, significantly reducing myocardial infarction indices and improving cardiac function. Therefore, Lip(PFH, Res) can have a protective effect against myocardial infarction and can be used as a nanolipid formulation for the preparation of myocardial infarction relief or treatment.

[0128] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A nanolipid formulation encapsulating perfluorohexane and an antioxidant for treating myocardial infarction, characterized in that: The nanoliposome formulation has a core-shell structure, with a phospholipid liposome membrane layer as the outer shell and perfluorohexane and an antioxidant encapsulated in the outer shell as the core. The phospholipid liposome membrane layer is made of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol. The molar ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol is 65:(4-8):(27-31). The mass ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol to the mass of perfluorohexane is 1:6 to 1:

20. The molar ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol to the amount of antioxidant is ≤25:0.002 mg / mmol. The antioxidant is selected from resveratrol.

2. The nanolipid formulation loaded with perfluorohexane and an antioxidant according to claim 1, characterized in that: The hydrogenated lecithin mentioned above is selected from hydrogenated soybean lecithin.

3. The nanolipid formulation loaded with perfluorohexane and an antioxidant according to claim 1, characterized in that: The molar ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol is 13:1:6; The total mass ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol to the mass ratio of perfluorohexane is 1:

20. The total mass ratio of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol to the molar amount of antioxidants was 25:0.002 mg / mmol.

4. The nanolipid formulation loaded with perfluorohexane and an antioxidant according to claim 1, characterized in that: The nanolipid formulation containing perfluorohexane and antioxidants has a particle size of 110–130 nm.

5. A method for preparing a nanolipid formulation loaded with perfluorohexane and an antioxidant as described in claim 1, characterized in that: Includes the following steps: Step (1): Dissolve hydrogenated lecithin, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000 in an organic solvent to form a lipid solution, and dissolve the antioxidant in methanol to form a solution; Step (2): Mix the lipid solution and the antioxidant solution, and remove the solvent by rotary evaporation to obtain a lipid film; Step (3): Add pure water and hydrate with ultrasound to obtain crude liposomes. Then add perfluorohexane and use an ultrasonic cell disruptor to obtain a uniform nanoliposome preparation.

6. The method for preparing the nanolipid formulation loaded with perfluorohexane and antioxidant according to claim 5, characterized in that: In step (1), the organic solvent is one of chloroform and dichloromethane.

7. The method for preparing the nanolipid formulation loaded with perfluorohexane and antioxidant according to claim 5, characterized in that: In step (2), the temperature of rotary evaporation is 37–40 °C.

8. The method for preparing the nanolipid formulation loaded with perfluorohexane and antioxidant according to claim 5, characterized in that: In step (3), during hydration, the ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol to the volume of pure water is 25 mg: 2-8 mL.

9. The method for preparing the nanolipid formulation loaded with perfluorohexane and antioxidant according to claim 8, characterized in that: In step (3), during hydration, the ratio of the total mass of hydrogenated lecithin, distearate phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol to the volume of pure water is 25 mg: 2.7 mL.

10. The use of the nanolipid formulation containing perfluorohexane and antioxidant as described in claim 1 in the preparation of a drug for treating myocardial infarction.

Images