Triprostinil multivesicular liposomes for treating pulmonary arterial hypertension as well as preparation method and application of triprostinil multivesicular liposomes

Abstract

The invention discloses treprostinil multivesicular liposome for treating pulmonary arterial hypertension. The multivesicular liposome is prepared from an inner water phase, an oil phase and an outer water phase through a double emulsification method, the treprostinil is dissolved in the inner water phase; the oil phase comprises soybean lecithin, cholesterol, glyceryl trioleate and electronegative lipid; the inner water phase and the outer water phase are body fluid isotonic solutions. The treprostinil multivesicular liposome disclosed by the invention has good stability and high encapsulation efficiency, obviously reduces burst release effect, shows obvious slow release characteristic, is beneficial to prolonging in-vivo drug half-life period, and can realize 48-hour treatment, reduce administration frequency, simplify administration method and improve patient compliance through simple subcutaneous injection. After the treprostinil multivesicular liposome is injected once, the inflammatory reaction of the injection part can be eliminated within 7 days, and the treprostinil multivesicular liposome has good safety.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations and relates to a treprostinil polycystic liposome for the treatment of pulmonary hypertension, its preparation method and application. Background Technology

[0002] Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disease whose occurrence and development involve multiple molecular mechanisms and diseases. The main pathological features of PAH are abnormally high mean pulmonary artery pressure, pulmonary vascular remodeling, and compensatory hypertrophy of the right heart, ultimately leading to right heart failure. Due to its complex pathogenesis, insidious clinical symptoms, and high treatment costs, PAH treatment faces challenges such as difficult clinical diagnosis, short patient survival, and high mortality.

[0003] Currently, clinical treatment for PAH primarily focuses on vasodilation to reduce mean pulmonary artery pressure and improve patient mobility. Treprostaniel (TRE) is a stable prostacyclin analog that dilates blood vessels by upregulating cyclic adenosine monophosphate (cAMP) levels and inhibits pulmonary hypertension-related pulmonary artery smooth muscle cell proliferation and platelet aggregation. Treprostaniel is mainly administered subcutaneously, intravenously, and orally, but this approach has drawbacks such as poor patient compliance, injection site pain, risk of bloodstream infection, and high treatment costs. For patients with intermediate to advanced PAH, intravenous infusion of treprostaniel has become a necessary treatment method, but this brings problems such as frequent changes in medication, difficulties in catheter and pump management, and the risk of bloodstream infection.

[0004] Liposome formulations have been extensively studied as a novel drug carrier. Liposomes are bilayered vesicle structures composed of phospholipids and other substances, capable of encapsulating drugs in their hydrophilic or hydrophobic regions, achieving targeted delivery and sustained-release. Multivesicular liposomes (MVLs) are complex liposome systems composed of multiple small vesicles, each encapsulated by an independent lipid bilayer membrane and containing an aqueous chamber. This unique structure gives them high drug encapsulation efficiency and long drug release time, allowing for better control of the drug release rate, reducing burst release, and improving the safety and efficacy of drug therapy. Furthermore, MVLs are a clinically proven long-acting sustained-release delivery technology with high drug loading capacity and sustained release characteristics.

[0005] Therefore, if treprostinil can be prepared into multi-cystic liposomes, it can be used for long-term treatment of pulmonary hypertension via subcutaneous injection. Summary of the Invention

[0006] One of the objectives of this invention is to provide a long-acting formulation of treprostinil multicystic liposomes (TRE MVLs) for the treatment of pulmonary hypertension.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] A treprostene multicystic liposome for treating pulmonary hypertension comprises treprostene. The multicystic liposome is prepared by a double emulsification method consisting of an inner aqueous phase (W1 phase), an oil phase (O phase), and an outer aqueous phase (W2 phase). The treprostene is dissolved in the inner aqueous phase. The oil phase comprises soybean lecithin, cholesterol, trioleic acid glycerides, and negatively charged lipids. Both the inner and outer aqueous phases are isotonic solutions of body fluids.

[0009] Preferably, the treprostinil is dissolved in the inner aqueous phase, and the pH of the outer aqueous phase is adjusted to alkaline.

[0010] The negatively charged lipid is selected from one of stearic acid, vitamin E succinate, and dipalmitoyl phosphatidylglycerol (DPPG), preferably vitamin E succinate or dipalmitoyl phosphatidylglycerol, and more preferably dipalmitoyl phosphatidylglycerol.

[0011] Soy lecithin is the main component of liposome membranes; cholesterol can effectively regulate the fluidity and rigidity of the membrane; the role of auxiliary lipids (trioleylglycerol and negatively charged lipids) is to introduce charges or functional groups, thereby regulating the surface properties of liposomes.

[0012] The internal aqueous phase is selected from a 5% (w / v) glucose (Glu) solution or a 2.1% (w / v) glycine (Gly) solution that is isotonic with body fluids, preferably a 5% glucose solution.

[0013] The external aqueous phase is selected from a 5% (w / v) glucose solution containing 40 mmol / L lysine (Lys) that is isotonic with body fluids or a 2.1% (w / v) glycine solution containing 40 mmol / L lysine, preferably a 5% (w / v) glucose solution containing 40 mmol / L lysine.

[0014] w / v is the mass-volume concentration. For example, 5% (w / v) means that 100 mL of solution contains 5 g of solute.

[0015] The isotonic solution of body fluids described in this invention is prepared using pure water, and the preparation method is a method known to those skilled in the art.

[0016] The double emulsification method is as follows: soybean lecithin, cholesterol, trioleic acid glycerides, and negatively charged lipids are dissolved in an organic solvent to obtain an oil phase (O phase); treprostrin is dissolved in an inner aqueous phase (W1 phase), and the pH is adjusted to alkaline with sodium hydroxide solution to obtain an inner aqueous phase containing treprostrin sodium salt; the inner aqueous phase containing treprostrin sodium salt is added to the oil phase and ultrasonically emulsified to form a W1 / O primary emulsion; the W1 / O primary emulsion and the outer aqueous phase (W2 phase) are vortex-mixed to form a W1 / O / W2 secondary emulsion; the W1 / O / W2 secondary emulsion is centrifuged, the precipitate is collected, and washed with the outer aqueous phase; the washed sample is resuspended in the outer aqueous phase, the organic solvent is removed by nitrogen purging, and then centrifuged again, and the precipitate is resuspended in the outer aqueous phase to obtain treprostrin polycystic liposomes.

[0017] The treprostone multicystic liposome exhibits a multicystic structure, with treprostone encapsulated in the inner aqueous chamber of the multicystic liposome. Multiple non-concentric inner aqueous chambers (i.e., small vesicles) are separated by a lipid bilayer (formed by the precipitation of phospholipids and other substances in the oil phase due to solvent evaporation), and the whole is suspended in the outer aqueous phase.

[0018] Another object of the present invention is to provide a method for preparing the aforementioned treprostone polycystic liposomes, comprising the following steps:

[0019] Step (1): Dissolve soybean lecithin, cholesterol, trioleic acid glycerides and negatively charged lipids in an organic solvent to obtain the oil phase (O phase);

[0020] Step (2): Dissolve treprostrin in the inner aqueous phase (W1 phase), adjust the pH to alkaline with sodium hydroxide solution to obtain an inner aqueous phase containing treprostrin sodium salt;

[0021] Step (3): Add the aqueous phase containing treprostyl sodium salt to the oil phase and perform ultrasonic emulsification to form W1 / O primary emulsion;

[0022] Step (4): Vortex mix the W1 / O primary emulsion and the external aqueous phase (W2 phase) to form a W1 / O / W2 double emulsion;

[0023] Step (5): Centrifuge the W1 / O / W2 double emulsion, collect the precipitate, and wash it with an external aqueous phase;

[0024] Step (6): The washed sample is resuspended in an external aqueous phase, the organic solvent is removed by nitrogen purging, and then centrifuged. The precipitate is resuspended in an external aqueous phase to obtain treprostone nifedipine liposomes.

[0025] In step (1), the organic solvent is a mixture of chloroform and methanol in a volume ratio of 8.5:1. The organic solvent in the oil phase must meet the requirements of "dissolving lipids, being immiscible with water, and being easily removed". The mixed solvent of this invention can ensure that negatively charged lipids such as DPPG can be completely dissolved, and can also minimize the amount of methanol used, reduce its impact on the stability of vesicle structure, and avoid vesicle rupture.

[0026] In the oil phase, the concentration of soybean lecithin is 16-24 mg / mL, preferably 24 mg / mL; the concentration of cholesterol is 6-12 mg / mL, preferably 8-10 mg / mL, and most preferably 8 mg / mL; the concentration of trioleic acid glyceride is 4-12 mg / mL, preferably 6-12 mg / mL, more preferably 6-8 mg / mL, and most preferably 8 mg / mL; and the concentration of negatively charged lipids is 1-2.5 mg / mL, preferably 1.5 mg / mL.

[0027] Specifically, the concentration of soybean lecithin in the oil phase is 16, 20, or 24 mg / mL.

[0028] Specifically, in the oil phase, the concentration of cholesterol is 6, 8, 10, or 12 mg / mL.

[0029] Specifically, in the oil phase, the concentration of trioleic acid glyceride is 4, 6, 8, or 12 mg / mL.

[0030] Specifically, in the oil phase, the concentration of the negatively charged lipid is 1, 1.5, 2, or 2.5 mg / mL.

[0031] In step (2), the concentration of treprostyl in the internal aqueous phase is 2-8 mg / mL, preferably 2-5 mg / mL, more preferably 2-3 mg / mL, and most preferably 3 mg / mL.

[0032] Specifically, the concentration of treprostyl in the internal aqueous phase is 2, 3, 5, or 8 mg / mL.

[0033] Treprostl is an acidic, poorly soluble drug with a pKa of 3.5 and log P of 4. Therefore, this invention adjusts the internal aqueous phase to alkaline to form treprostl sodium salt, thereby improving the solubility of treprostl. Preferably, the pH of the internal aqueous phase is adjusted to 10 using sodium hydroxide solution.

[0034] The concentration of the sodium hydroxide solution is 0.1 mol / L.

[0035] In step (3), the volume ratio of the internal aqueous phase to the oil phase is 1:1 to 1:2, preferably 1:1. By controlling the proportion of the internal aqueous phase, this invention avoids the initial emulsion from becoming too viscous due to an excessively high proportion of the internal aqueous phase, which would affect the subsequent preparation of the re-emulsion; it also avoids the encapsulation rate from decreasing due to an excessively low proportion of the internal aqueous phase.

[0036] Specifically, the volume ratio of the internal aqueous phase to the oil phase is 1:1, 1:1.5, or 1:2.

[0037] The device used for ultrasonic emulsification is an ultrasonic cell disruptor.

[0038] The ultrasonic emulsification power is 150-200W, and the ultrasonic emulsification time is 15-25 minutes; preferably, the ultrasonic emulsification power is 180-200W, and the ultrasonic emulsification time is 15-20 minutes; most preferably, the ultrasonic emulsification power is 180W, and the ultrasonic emulsification time is 20 minutes.

[0039] Specifically, the conditions for ultrasonic emulsification are: ultrasonic power of 150W for 25 minutes; ultrasonic power of 180W for 20 minutes; and ultrasonic power of 200W for 15 minutes.

[0040] In step (4), the external aqueous phase needs to be kept in excess to dilute the W1 / O pre-emulsion. Usually, the volume of the external aqueous phase is larger than that of the internal aqueous phase to ensure uniform dispersion of the re-emulsion. The volume ratio of the W1 / O pre-emulsion (based on the sum of the volumes of the internal aqueous phase and the oil phase) to the external aqueous phase (W2 phase) is 1:0.75 to 1:1.25, preferably 1:1.

[0041] Specifically, the volume ratio of the W1 / O colostrum to the W2 phase is 1:0.75, 1:1, or 1:1.25.

[0042] The device used to generate the vortex is a vortex generator.

[0043] The rotational speed of the vortex is 2500-3000 rpm, preferably 2800 rpm; the vortex duration is 40-80 s, preferably 60 s.

[0044] Specifically, the vortex time is 40s, 60s, and 80s.

[0045] In step (5), the temperature of the W1 / O / W2 re-emulsion centrifugation is 4-10℃, preferably 4℃, the centrifugation speed is 2000-3000rpm, preferably 3000rpm, and the centrifugation time is 5-10min, preferably 5min.

[0046] Preferably, the W1 / O / W2 double emulsion is centrifuged, the precipitate is collected, and washed with an external aqueous phase. Each time the precipitate is washed, an external aqueous phase of equal volume to the supernatant is added for resuspension and centrifugation, for a total of three washes.

[0047] The volume of external aqueous phase added in each wash is the same as the volume of supernatant obtained from the previous centrifugation.

[0048] For each washing cycle, the centrifugation temperature is 4–10°C, preferably 4°C, the centrifugation speed is 2000–3000 rpm, preferably 3000 rpm, and the centrifugation time is 5–10 min, preferably 5 min.

[0049] In step (6), preferably, the washed sample is resuspended in an external aqueous phase of the same volume as the supernatant obtained from the previous centrifugation in step (5).

[0050] The method for removing organic solvents by nitrogen purging is as follows: in a water bath at 30-37°C, purging is performed transversely with nitrogen for 30-40 minutes.

[0051] Preferably, the method for removing organic solvents by nitrogen purging is as follows: purging with nitrogen in a horizontal direction for 40 minutes in a water bath at 37°C.

[0052] The centrifugation temperature is 4-10℃, preferably 4℃; the centrifugation speed is 2000-3000 rpm, preferably 3000 rpm; and the centrifugation time is 5-10 min, preferably 5 min.

[0053] This invention utilizes a dual emulsification method to prepare treprostrin multivesicular liposomes, which form a drug reservoir at the injection site after subcutaneous injection. Based on the permeation and fusion of the vesicle membrane, treprostrin is slowly released from the vesicles and enters the systemic circulation through capillary permeation, providing a long-lasting effect on PAH lesions in the lungs, thus achieving effective delivery of treprostrin.

[0054] In vitro release results showed that the cumulative release rate of free treprostone was 22.53±1.83% at 0.5h and reached 80.35% at 9h; while the release rate of treprostone polycystic liposomes was only 6.75±1.05% at 0.5h, significantly reducing the burst release effect, and the cumulative release rate reached 85.37% at 48h, showing obvious sustained-release characteristics, which helps to prolong the drug half-life in vivo.

[0055] Pharmacokinetic data showed that after a single subcutaneous injection, the peak concentration of free treprostolin was (C0). max The concentration was 1048.70 ng / mL, while the C of treprostinil polycystic liposomes was... max The concentration was 132.62 ng / mL, a significant difference. Compared with the free drug, multi-capsule liposomes significantly reduced C... maxTo avoid side effects caused by high drug concentrations. The drug half-life (t) of the multi-cyst liposome group. 1 / 2 The duration of action was extended to 10.81 hours, five times longer than that of the free drug. Seven hours after subcutaneous injection of the free drug, the plasma concentration was below 1 ng / mL (minimum effective concentration), while the plasma concentration of the multicystic liposomes remained at 6.77 ng / mL after 48 hours, significantly prolonging the drug's circulation and treatment duration. The area under the curve (AUC) of treprostinil multicystic liposomes was 1427.22 h·ng·mL. -1 It increased by approximately 47% compared to free drug (P<0.05), thus enhancing the therapeutic effect.

[0056] Another object of the present invention is to provide the use of the described treprostinil polycystic liposome in the preparation of a medicament for treating pulmonary hypertension.

[0057] The drug is in the form of an injection.

[0058] Preferably, the dosage form of the drug is a subcutaneous injection.

[0059] The beneficial effects of this invention are:

[0060] This invention encapsulates treprostrin within the aqueous chamber of a multi-capsule liposome. The treprostrin multi-capsule liposome exhibits good stability and high encapsulation efficiency, significantly reducing burst release effects and demonstrating pronounced sustained-release characteristics, achieving long-term drug release and helping to prolong the drug's half-life in vivo. It can achieve a 48-hour treatment duration through simple subcutaneous injection, reducing dosing frequency, simplifying the administration method, lowering the risk of side effects, and improving patient compliance and quality of life. Furthermore, the inflammatory response at the injection site after a single injection of treprostrin multi-capsule liposomes can subside within 7 days, demonstrating good safety and facilitating long-term use of treprostrin multi-capsule liposomes for the treatment of pulmonary hypertension.

[0061] The preparation method of treprostinil polycystic liposomes is simple to operate, suitable for large-scale production, and has good application prospects. Attached Figure Description

[0062] Figure 1 This is a histogram of the particle size distribution of TRE MVLs.

[0063] Figure 2 This is an upright microscope image of TRE MVLs.

[0064] Figure 3 This is a cryo-scanning electron microscope image of TRE MVLs.

[0065] Figure 4 The in vitro release curves of TRE MVLs are shown (n=3).

[0066] Figure 5 The changes in particle size and encapsulation efficiency of TRE MVLs over 28 days (n=3).

[0067] Figure 6 The in vitro long-term antiproliferative effect of TRE MVLs on PAH-PASMCs (n=6); where *P<0.05, ****P<0.0001, and ns indicate no significant difference.

[0068] Figure 7 The in vivo retention effect after subcutaneous injection of TRE MVLs (n=3).

[0069] Figure 8 Semi-quantitative results of fluorescence intensity after subcutaneous injection of TRE MVLs (n=3); among them, compared with Cy5.5-TRE, *P<0.05, ****P<0.001, ns indicates no significant difference; in the Cy5.5-TRE MVLs group, compared with adjacent time points, ####P<0.001, ns indicates no significant difference.

[0070] Figure 9 The drug concentration-time curves for TRE MVLs (n=4).

[0071] Figure 10 The mean pulmonary artery pressure after treatment in each group (n=5) is shown below. Among them, compared with MCT, ***P<0.001 and ****P<0.0001 were observed; compared with TRE MVLs, ##P<0.01 were observed, and ns indicates no significant difference.

[0072] Figure 11 Mean pulmonary artery pressure at each time point after subcutaneous injection of TRE MVLs (n=3).

[0073] Figure 12 HE staining image of pulmonary vessels (scale bar 20 μm).

[0074] Figure 13 Semi-quantitative results of HE staining of pulmonary vessels (n=5); among them, compared with MCT, *P<0.05, ****P<0.0001; compared with TRE MVLs, #####P<0.001, ns indicates no significant difference.

[0075] Figure 14 Immunohistochemical results of pulmonary vascular α-SMA (scale bar 50 μm).

[0076] Figure 15Semi-quantitative results of immunohistochemistry for pulmonary vascular α-SMA (n=5); among them, compared with MCT, ***P<0.001, ****P<0.0001; compared with TRE MVLs, ##P<0.01, ns indicates no significant difference.

[0077] Figure 16 The results of Ki67 immunohistochemistry are shown (scale bar is 50 μm).

[0078] Figure 17 The results of Ki67 immunohistochemistry are semi-quantitative (n=5); among them, compared with MCT, ***P<0.001, ****P<0.0001, ns indicates no significant difference; compared with TRE MVLs, #P<0.05.

[0079] Figure 18 The results are from TUNEL immunohistochemistry (scale bar is 50 μm).

[0080] Figure 19 The results of TUNEL immunohistochemistry are semi-quantitative (n=5); among them, compared with MCT, ****P<0.0001, ns indicates no significant difference; compared with TRE MVLs, ###P<0.001, ####P<0.0001.

[0081] Figure 20 HE staining image of right heart tissue (scale bar 50 μm).

[0082] Figure 21 Semi-quantitative results of HE staining of right heart tissue (n=5); among them, compared with MCT, ****P<0.0001, ns indicates no significant difference; compared with TRE MVLs, #P<0.05, ###P<0.001.

[0083] Figure 22 The right ventricular hypertrophy index (n=5) of PAH rats after treatment was calculated. Among them, compared with MCT, ****P<0.001, ns indicates no significant difference; compared with TRE MVLs, #P<0.05, ##P<0.05, ns indicates no significant difference.

[0084] Figure 23 The cytotoxicity of different concentrations of TRE to PAH-PASMCs was evaluated (n=6); where ***P<0.001, ns indicates no significant difference.

[0085] Figure 24 HE staining results at the injection site after subcutaneous injection of TRE MVLs in rats. Detailed Implementation

[0086] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0087] The active pharmaceutical ingredients or reagents used in this invention are all commercially available.

[0088] Abbreviations: TRE: Treprostene; PAH: Pulmonary hypertension; MVLs: Multicystic liposomes; DPPG: Dipalmitoylphosphatidylglycerol; Glu: Glucose; Lys: Lysine; Gly: Glycine; C max Peak concentration; FBS: Fetal bovine serum; LC-MS: Liquid chromatography-tandem mass spectrometry; mPAP: Mean pulmonary artery pressure; MCT: Lilidinyl alkaloid; MTT: Tetramethylazazole blue; PAH-PASMCs: Pulmonary hypertension-pulmonary artery smooth muscle cells; PASMCs: Pulmonary artery smooth muscle cells; PBS: Phosphate-buffered saline; PC: Phosphatidylcholine; PVR: Pulmonary vascular resistance; PGI2: Prostacyclin; PDK: Pyruvate dehydrogenase; RVHI: Right ventricular hypertrophy index; sGC: Soluble guanylate cyclase; SMCs: Smooth muscle cells; Tunel: Deoxyribonucleotide terminal transferase-mediated nick-end labeling.

[0089] Example 1

[0090] Preparation of TRE MVLs

[0091] 48 mg of soybean lecithin, 16 mg of cholesterol, 12 mg of trioleic acid glyceride, and 3 mg of DPPG were weighed and placed in a vial. 2 mL of a chloroform-methanol mixture (chloroform:methanol = 8.5:1 v / v) was added to dissolve the lecithin, yielding the oil phase (O phase). 4 mg of TRE was weighed and placed in another vial. 2 mL of 5% Glu solution was added to dissolve the TRE, and the pH was adjusted to 10 with 0.1 mol / L NaOH to form treprostene sodium salt, which served as the internal aqueous phase (W1 phase) containing TRE sodium salt. The W1 phase containing TRE sodium salt was slowly added dropwise to the O phase. Using an ultrasonic cell disruptor, the probe was inserted into the material, and probe-type ultrasonic emulsification was performed at 180 W for 20 min to obtain the W1 / O colostrum. The W1 / O colostrum was then rapidly injected into 4 mL of a 5% Glu solution containing 40 mmol / L lysine (W2 phase), and vortexed at 2800 rpm for 60 s to obtain the W1 / O / W2 double emulsion. The W1 / O / W2 double emulsion was centrifuged at 4°C and 3000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed. Each wash involved resuspending the precipitate in an equal volume of 5% Glu solution containing 40 mmol / L lysine, followed by centrifugation at 4°C and 3000 rpm for 5 min, for a total of three washes. The washed sample was resuspended in an equal volume of 5% Glu solution containing 40 mmol / L lysine, followed by horizontal nitrogen purging in a 37°C water bath for 40 min, and then centrifuged at 4°C and 3000 rpm for 5 min. The precipitate was then resuspended in a 5% Glu solution containing 40 mmol / L lysine to obtain the TRE MVLs.

[0092] Example 2

[0093] Formulation and process screening of TRE MVLs

[0094] (I) Screening of soybean lecithin concentration

[0095] TRE MVLs were prepared according to the method of Example 1, except that the concentration of soybean lecithin in phase O was adjusted to 16, 20, and 24 mg / mL (corresponding to soybean lecithin masses of 32, 40, and 48 mg, respectively). All other aspects were the same as in Example 1. The optimal concentration of soybean lecithin was screened by measuring the encapsulation efficiency, and the results are shown in Table 1.

[0096] As shown in Table 1, the encapsulation efficiency of TRE MVLs is the highest when the soybean lecithin concentration is 24 mg / mL.

[0097] Table 1: Effect of different soybean lecithin concentrations on the encapsulation efficiency of TRE MVLs

[0098]

[0099]

[0100] (II) Screening for cholesterol concentration

[0101] TRE MVLs were prepared according to the method of Example 1, except that the cholesterol concentration in the O phase was adjusted to 6, 8, 10, and 12 mg / mL (corresponding to cholesterol masses of 12, 16, 20, and 24 mg, respectively). All other parameters were the same as in Example 1. The optimal cholesterol concentration was screened by measuring the encapsulation efficiency, and the results are shown in Table 2.

[0102] As shown in Table 2, TRE MVLs can achieve a good encapsulation rate when the cholesterol concentration is 8–10 mg / mL, and the encapsulation rate is the highest when the cholesterol concentration is 8 mg / mL.

[0103] Table 2: Effect of different cholesterol concentrations on the encapsulation efficiency of TRE MVLs

[0104]

[0105] (III) Screening of trioleic acid glyceride concentration

[0106] TRE MVLs were prepared according to the method in Example 1, except that the concentration of trioleic acid glyceride in the O phase was adjusted to 4, 6, 8, and 12 mg / mL (corresponding to the mass of trioleic acid glyceride of 8, 12, 16, and 24 mg), and all other parameters were the same as in Example 1. The optimal concentration of trioleic acid glyceride was determined by measuring the encapsulation efficiency, and the results are shown in Table 3.

[0107] As shown in Table 3, TRE MVLs can achieve good encapsulation efficiency when the trioleic acid glyceride concentration is 6–12 mg / mL. When the trioleic acid glyceride concentration is 6–8 mg / mL, the encapsulation efficiency of TRE MVLs is above 80%. In particular, the encapsulation efficiency of TRE MVLs is the highest when the trioleic acid glyceride concentration is 8 mg / mL.

[0108] Table 3: Effect of different trioleic acid glyceride concentrations on the encapsulation efficiency of TRE MVLs

[0109]

[0110] (iv) Screening of negatively charged lipids

[0111] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in the O phase was adjusted to the optimal concentration (8 mg / mL). The types of negatively charged lipids were then adjusted to stearic acid, vitamin E succinate, and DPPG, respectively. All other aspects were the same as in Example 1. The optimal type of negatively charged lipid was determined by measuring the encapsulation efficiency. The results are shown in Table 4.

[0112] As shown in Table 4, the encapsulation efficiency of TRE MVLs is the highest when the negatively charged lipid is DPPG.

[0113] Table 4: Effect of different negatively charged lipids on the encapsulation efficiency of TRE MVLs

[0114]

[0115] (V) Screening of negatively charged lipid concentrations

[0116] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of DPPG in phase O was adjusted to 1, 1.5, 2, and 2.5 mg / mL, respectively. All other parameters were the same as in Example 1. The optimal concentration of negatively charged lipids was determined by measuring the encapsulation efficiency. The results are shown in Table 5.

[0117] As shown in Table 5, the encapsulation efficiency of TRE MVLs is the highest when DPPG is 1.5 mg / mL.

[0118] Table 5: Effect of different DPPG concentrations on the encapsulation efficiency of TRE MVLs

[0119]

[0120] (vi) Screening of TRE concentration

[0121] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of TRE in phase W1 was adjusted to 2, 3, 5, and 8 mg / mL (corresponding to TRE masses of 4, 6, 10, and 16 mg). All other parameters were the same as in Example 1. The optimal concentration of TRE was determined by measuring the encapsulation efficiency, and the results are shown in Table 6.

[0122] As shown in Table 6, TRE MVLs can achieve good encapsulation efficiency when the TRE concentration is 2–8 mg / mL. When the TRE concentration is 2–3 mg / mL, the encapsulation efficiency of TRE MVLs is above 90%. In particular, the encapsulation efficiency of TRE MVLs is the highest when the TRE concentration is 3 mg / mL.

[0123] Table 6: Effect of different TRE concentrations on the encapsulation efficiency of TRE MVLs

[0124]

[0125] (vii) Screening of the volume ratio of W1 phase to O phase

[0126] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of TRE in phase W1 was adjusted to the optimal concentration (3 mg / mL). The volume ratio of phase W1 to phase O was then adjusted to 1:1, 1:1.5, and 1:2, respectively. The optimal volume ratio of phase W1 to phase O was determined by measuring the encapsulation efficiency. The results are shown in Table 7.

[0127] As shown in Table 7, when the volume ratio of W1 phase to O phase is 1:1 to 1:2, the encapsulation efficiency of TRE MVLs is above 85%, and the encapsulation efficiency of TRE MVLs is the highest when the volume ratio of W1 phase to O phase is 1:1.

[0128] Table 7: Effect of different W1 phase to O phase volume ratios on the encapsulation efficiency of TRE MVLs

[0129]

[0130] (viii) Screening of power and time for ultrasonic emulsification

[0131] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of TRE in phase W1 was adjusted to the optimal concentration (3 mg / mL). The ultrasonic conditions of the probe during the preparation of W1 / O primary emulsion were then adjusted to 150 W / 25 min (ultrasonic power / ultrasonic time), 180 W / 20 min, and 200 W / 15 min, respectively. The optimal ultrasonic emulsification conditions were screened by measuring the encapsulation rate. The results are shown in Table 8.

[0132] As shown in Table 8, when the ultrasonic power is 180-200W and the ultrasonic time is 15-20min, the encapsulation rate of TRE MVLs is above 80%, especially when the probe ultrasonic conditions are 180W and 20min, the encapsulation rate of TRE MVLs is the highest.

[0133] Table 8: Effect of different ultrasound conditions on the encapsulation efficiency of TRE MVLs

[0134]

[0135] (ix) Screening of the components of W1 and W2 phases

[0136] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of TRE in phase W1 was adjusted to the optimal concentration (3 mg / mL). The types of phases W1 and W2 were then adjusted. The W1 / W2 combinations were: 5% Glu / (5% Glu + 40 mmol / L Lys), 2.1% Gly / (2.1% Gly + 40 mmol / L Lys), 2.1% Gly / (5% Glu + 40 mmol / L Lys), and 5% Glu / (2.1% Gly + 40 mmol / L Lys). The optimal combination of phases W1 / W2 was determined by measuring the encapsulation efficiency, and the results are shown in Table 9.

[0137] Table 9 shows that the encapsulation efficiency of TRE MVLs is highest when the combination of W1 and W2 phases is 5% Glu / (5% Glu + 40 mmol / L Lys) and 5% Glu / (2.1% Gly + 40 mmol / L Lys). Under alkaline conditions, Glu is electroneutrally neutral, while Gly is negatively charged. Considering the charge balance requirements of the TRE MVLs system, 5% Glu / (5% Glu + 40 mmol / L Lys) is preferentially chosen as the W1 and W2 phases.

[0138] Table 9: Effect of different W1 / W2 combinations on the encapsulation efficiency of TRE MVLs

[0139]

[0140]

[0141] (x) Screening of the volume ratio of W1 / O colostrum and W2 phase

[0142] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of TRE in phase W1 was adjusted to the optimal concentration (3 mg / mL). The volume ratios of W1 / O / W2 were then adjusted to 1:1:1.5, 1:1:2, and 1:1:2.5 (i.e., the sum of the volumes of phases W1 and O and the volume ratio of W2 = 1:0.75, 1:1, and 1:1.25). The optimal volume ratio of phases W1 / O to W2 was determined by measuring the encapsulation efficiency. The results are shown in Table 10.

[0143] According to Table 10, when the volume ratio of W1 / O / W2 is 1:1:(1.5~2.5), the encapsulation efficiency of TRE MVLs is above 80%, and when the volume ratio of W1 / O / W2 is 1:1:2, the encapsulation efficiency of TRE MVLs is the highest.

[0144] Table 10: Effect of different W1 / O / W2 volume ratios on the encapsulation efficiency of TRE MVLs

[0145]

[0146] (xi) Screening of vortex time

[0147] TRE MVLs were prepared according to the method in Example 1. The concentration of trioleic acid glyceride in phase O was adjusted to the optimal concentration (8 mg / mL), and the concentration of TRE in phase W1 was adjusted to the optimal concentration (3 mg / mL). The vortexing time for preparing W1 / O / W2 double emulsion was then adjusted to 40 s, 60 s, and 80 s, respectively. The optimal vortexing time was determined by measuring the encapsulation efficiency. The results are shown in Table 11.

[0148] As shown in Table 11, the encapsulation efficiency of TRE MVLs is the highest when the vortex time is 60s.

[0149] Table 11: Effect of different vortex times on the encapsulation efficiency of TRE MVLs

[0150]

[0151] In summary, the optimal formulation for TRE MVLs is: soy lecithin: cholesterol: triolein: DPPG: TRE = 24:8:8:1.5:3 (w / w), with W1 phase and W2 phase containing 5% Glu and 5% Glu containing 40 mmol / L Lys, respectively, and W1 / O / W2 = 1:1:2 (v / v); the optimal parameters for ultrasonic emulsification are: 180W, 20 min; and the optimal parameters for vortexing time are: vortexing for 60 s.

[0152] Example 3

[0153] Preparation of treprostone polycystic liposomes:

[0154] 48 mg of soybean lecithin, 16 mg of cholesterol, 16 mg of trioleic acid glyceride, and 3 mg of DPPG were weighed and placed in a vial. 2 mL of a chloroform-methanol mixture (chloroform:methanol = 8.5:1 v / v) was added to dissolve the lecithin, yielding the oil phase (O phase). 6 mg of TRE was weighed and placed in another vial. 2 mL of 5% Glu solution was added to dissolve the TRE, and the pH was adjusted to 10 with 0.1 mol / L NaOH to form treprostene sodium salt, which served as the internal aqueous phase (W1 phase) containing TRE sodium salt. The W1 phase containing TRE sodium salt was slowly added dropwise to the O phase. Using an ultrasonic cell disruptor, the probe was inserted into the material, and probe-type ultrasonic emulsification was performed at 180 W for 20 min to obtain the W1 / O colostrum. The W1 / O colostrum was then rapidly injected into 4 mL of a 5% Glu solution containing 40 mmol / L Lys (W2 phase), and vortexed at 2800 rpm for 60 s to obtain the W1 / O / W2 double emulsion. The W1 / O / W2 double emulsion was centrifuged at 4 °C and 3000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed. Each wash involved resuspending the precipitate in a 5% Glu solution containing 40 mmol / L Lysine with an equal volume of the supernatant obtained from the previous centrifugation, and then centrifuged again at 4 °C and 3000 rpm for 5 min, for a total of three washes. The washed sample was resuspended in a 5% Glu solution containing 40 mmol / L lysine, with an equal volume of the supernatant obtained from the previous centrifugation. The sample was then purged with nitrogen in a horizontal direction in a 37°C water bath for 40 min, followed by centrifugation at 4°C and 3000 rpm for 5 min. The precipitate was resuspended in a 5% Glu solution containing 40 mmol / L Lys to obtain TRE MVLs with a concentration (based on the ratio of TRE to external aqueous phase) of 4.8 mg / mL or 1.2 mg / mL.

[0155] Characterization of treprostone multicystic liposomes

[0156] (I) Encapsulation efficiency and particle size distribution

[0157] The encapsulation efficiency (EE) of the three groups of samples was determined, and the particle size of the three groups of samples was measured using a laser particle size analyzer. The results are shown in Table 12 and 13. Figure 1 .

[0158] According to Table 12, the encapsulation efficiency of TRE MVLs is >80%, specifically 86.24 ± 2.93%, which meets the requirements. The particle size (D90) of TRE MVLs is 74.89 ± 0.47 μm. Figure 1 It can be seen that the particle size of TRE MVLs exhibits a unimodal normal distribution, concentrated in the range of 10–100 μm, indicating that the particle size distribution is uniform and there is no obvious polydispersity.

[0159] Table 12: Encapsulation efficiency and particle size of TRE MVLs

[0160]

[0161] (II) Morphological Examination

[0162] The morphology of TRE MVLs was observed using a bright-field microscope at 40×40 magnification. The results are as follows: Figure 2 As shown, TRE MVLs exhibit a multi-cystic structure under an upright microscope, with a particle size of approximately 70 μm.

[0163] The morphology of TRE MVLs was observed using cryo-scanning electron microscopy. The results are as follows: Figure 3 As shown, TRE MVLs have large internal vesicles surrounding multiple small vesicles, forming a non-concentric structure with a rough and porous surface.

[0164] Example 4

[0165] In vitro release behavior study of TRE MVLs

[0166] One mL of free TRE solution (TRE dissolved in a 5% Glu solution containing 40 mmol / L Lys) and one mL of TRE MVLs (Example 3) were placed in dialysis bags (MWCO 3500 Da). Air bubbles were removed from the dialysis bags, and both ends were sealed. The bags were then placed in EP tubes containing 30 mL of release medium (a 5% Glu solution containing 40 mmol / L Lys at pH 7.4). The EP tubes were placed in a 37°C water bath with shaking at 50 rpm. One mL of release medium was collected at 0.5, 1, 2, 4, 6, 9, 12, 24, 36, and 48 hours, and an equal volume of fresh release medium was added. After passing the samples through a 0.22 μm aqueous filter membrane, the drug concentration of the samples was determined by high-performance liquid chromatography (HPLC), and the cumulative drug release percentage was calculated. A release curve was plotted with time on the x-axis and the cumulative drug release percentage on the y-axis.

[0167] The results are as follows Figure 4 As shown, the cumulative release rate of free TREs at 0.5h was 22.53±1.83%, reaching 80.35% at 9h, and completely released at 24h; while the release rate of TRE MVLs at 0.5h was only 6.75±1.05%, significantly reducing the burst release effect, with a cumulative release rate of 85.37% at 48h, exhibiting obvious sustained-release characteristics, which helps to prolong the half-life of drugs in vivo.

[0168] Example 5

[0169] Physical stability of TRE MVLs

[0170] TRE MVLs (Example 3) were stored at 4°C for 28 days. The encapsulation efficiency and particle size of TRE MVLs were measured on days 1, 4, 7, 14, 21, and 28 to investigate their short-term stability.

[0171] The results are as follows Figure 5 As shown, after being placed at 4°C for 28 days, the encapsulation efficiency and particle size of TRE MVLs did not change significantly, and the particle size distribution was uniform. This indicates that TRE will not leak from the water core inside the TRE MVLs in a short period of time.

[0172] Example 6

[0173] Long-lasting in vitro antiproliferative effect of TRE MVLs

[0174] 1 mL of 4.8 mg / mL TRE MVLs (Example 3) and 1 mL of 5% Glu solution were added to a 6-well plate, gently vortexed to mix, and incubated at 37°C and 5% CO2 for 12 h and 24 h, respectively. After centrifugation at 2000 rpm for 3 min, the supernatant was collected. RPMI 1640 medium containing 10% fetal bovine serum was added, and the volume of the supernatant was diluted by 6, 8, 12, 24, 48, and 240 times, respectively, to obtain TRE MVLs extracts with concentrations of 400, 300, 200, 100, 50, and 10 μg / L.

[0175] Dissolve 9.6 mg TRE in 1 mL DMSO and dilute with RPMI 1640 medium containing 10% fetal bovine serum to obtain free TRE solutions of 400, 300, 200, 100, 50, and 10 μg / mL.

[0176] PAH-PASMCs in the exponential growth phase were taken, and 1.5 mL of 0.25% trypsin was added. After shaking well, the cells were digested in an incubator for 6 min. Digestion was terminated by adding 4.5 mL of RPMI 1640 medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum to obtain a cell suspension. 200 μL of cell suspension was added to each well of a 96-well plate at a density of 6500 cells / well and cultured for 24 h. The supernatant was discarded, and 200 μL of different concentrations of TRE MVLs extract or Free TRE solution were added, with 6 replicates for each concentration. Cells without the drug solution served as the control group, and cells without the drug solution in RPMI 1640 medium containing 10% fetal bovine serum served as the blank group. The cells were incubated for another 24 h, the supernatant was discarded, and 20 μL of 5 mg / mL MTT solution was added to each well. The cells were incubated in an incubator for 4 h. Discard the upper culture medium, add 200 μL LDMSO to each well, and shake thoroughly in a shaker under dark conditions. Measure the absorbance (Abs) of each well at 570 nm using a microplate reader. Calculate the cell viability using the formula.

[0177]

[0178] In this study, the experimental group Abs represents the absorbance of cells cultured with TRE MVLs extract or free TRE solution at 570 nm, the control well Abs represents the absorbance of cells cultured without the drug solution at 570 nm, and the blank well Abs represents the absorbance of cell-free RPMI 1640 medium containing 10% fetal bovine serum at 570 nm.

[0179] The results are as follows Figure 6 As shown, TRE MVLs-12h and TRE MVLs-24h represent TRE MVLs solution incubated with 1 mL of 5% Glu solution for 12h and 24h, respectively. When the TRE concentration was 400 μg / mL, the cell survival rate of the TRE-MVLs-12h group was the highest (82.63±5.72%), which was significantly different from that of the TRE-MVLs-24h group (56.52±5.33%) (P<0.0001). At a concentration of 300 μg / mL, the cell survival rate of the TRE-MVLs-24h group was lower than that of the TRE-MVLs-12h group. When the concentration did not exceed 200 μg / mL, there was no significant difference in cell survival rate among the different formulation groups. The results showed that the antiproliferative effect of TRE-MVLs extract and Free TRE on PAH-PASMCs was concentration-dependent; at the same concentration, the cell survival rate of the TRE-MVLs-24h group was lower than that of the TRE-MVLs-12h group, suggesting that TRE-MVLs maintains a long-term effect through a slow release mechanism, and the antiproliferative effect is time-dependent.

[0180] Example 7

[0181] TRE MVLs in vivo retention time

[0182] 10 mg of TRE and 14 mg of HATU were weighed and dissolved in DMF. After stirring for 2 hours, 7.7 mg of triethylamine and 5 mg of sulfonated Cy5.5-NH2 (Sulfo-Cyanine 5.5Amine) were added. The mixture was heated at 60 °C for 6 hours and dried under reduced pressure to obtain Cy5.5-TRE. The TRE in Example 3 was replaced with an equal amount of Cy5.5-TRE (the amount of TRE in Cy5.5-TRE was equal to the amount of TRE in Example 3), and Cy5.5-TRE MVLs were prepared according to the method of Example 3.

[0183] Six healthy SD rats were randomly divided into two groups: the Cy5.5-TRE group and the Cy5.5-TRE MVLs group. The Cy5.5-TRE group (Cy5.5-TRE dissolved in PBS buffer) and the Cy5.5-TRE MVLs group (TRE dose 1.2 mg / kg) were subcutaneously injected, respectively. On days 1, 2, 3, 4, 5, 6, 7 and 8 after administration, the rats were anesthetized with isoflurane for in vivo imaging (excitation wavelength of sulfonated Cy5.5-NH2 was 678 nm, and emission wavelength was 706 nm).

[0184] The results are as follows Figure 7 , Figure 8 As shown, the fluorescence in the Cy5.5-TRE group rats was maintained for only 3 days, decaying to normal levels on the 4th day, indicating that it was rapidly metabolized and eliminated by the liver in systemic circulation; the fluorescence in the Cy5.5-TRE MVLs group rats could be maintained until the 5th day. Quantitative fluorescence analysis showed that the fluorescence intensity in the Cy5.5-TRE group rats decreased exponentially by 65% ​​on the 2nd day (P<0.001), while the fluorescence intensity in the Cy5.5-TRE MVLs group rats decreased by approximately 37% exponentially (P<0.01). In conclusion, TRE MVLs can prolong the drug retention time at the injection site and reduce the C-level concentration of TREs in vivo. max To avoid side effects such as facial flushing and diarrhea, while significantly slowing down the release rate, prolonging the in vivo half-life and improving bioavailability.

[0185] Example 8

[0186] In vivo pharmacokinetic studies

[0187] Eight healthy SD rats weighing approximately 220g were fasted for 12 hours and randomly divided into two groups. The Free TRE group consisted of 1.2mg TRE, 5.3mg NaCl, 3mg m-cresol, and 6.3mg trisodium citrate dissolved in 1mL of ultrapure water. The TRE MVLs group prepared 1.2mg / mL TRE MVLs according to the method in Example 3. Rats were weighed, tagged, and their whiskers were trimmed. The corresponding drug solutions were injected subcutaneously into the neck and back. The treatment dose for both groups was 1.2mg / kg (based on TRE). At 5min, 15min, 30min, 1h, 3h, 5h, 7h, 8h, 12h, 24h, and 48h after administration, 0.5mL of blood was collected from the orbital sinus into 1.5mL EP tubes (pre-filled with 50μL of 3.3% sodium citrate solution) and stored at 4℃.

[0188] Plasma sample pretreatment: TRE plasma samples were pretreated using the protein precipitation method: 0.5 mL of whole blood was centrifuged at 2000 rpm for 20 min at room temperature. 100 μL of the supernatant plasma was collected, and 10 μL of 500 ng / mL 1-naphthoxyacetic acid (IS) (500 ng 1-naphthoxyacetic acid dissolved in 1 mL anhydrous ethanol) and 20 μL of isopropanol were added. The mixture was vortexed, and then 300 μL of acetonitrile was added. The mixture was vortexed for 5 min, centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected and injected. The TRE concentration in the plasma was detected using LC-MS, and a concentration-time curve was plotted. Pharmacokinetic parameters were calculated using a non-compartmental, non-oral model with PKsolver software.

[0189] The results are as follows Figure 9 As shown in Table 13, after a single subcutaneous injection, the C in the Free TRE group max The C-cell concentration was 1048.70 ng / mL in the control group and 132.62 ng / mL in the TREMVLs group, with a significant difference (P<0.0001), indicating that TREMVLs can significantly reduce C-cell concentration. max To avoid side effects caused by high concentrations of TRE. The half-life (t) of the TRE MVLs group... 1 / 2 The time to TRE administration was 10.81 h, which was 5 times longer than that in the Free TRE group (P<0.05). In the Free TRE group, the plasma TRE concentration was below 1 ng / mL (lowest effective plasma concentration) 7 h after subcutaneous injection; in the TRE MVLs group, the plasma concentration was 6.77 ng / mL at 48 h, indicating that TRE MVLs lower C-cell concentration. max , extend t 1 / 2 This significantly prolonged in vivo circulation and treatment time. Furthermore, the area under the curve (AUC) in the TRE MVLs group was 1427.22 h·ng·mL. -1 The efficacy was improved by approximately 47% compared to the Free TRE group (P<0.05), enhancing the therapeutic effect of the drug.

[0190] Table 13: Pharmacokinetic parameters of TRE MVLs in vivo (n=4)

[0191]

[0192] Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0193] Example 9

[0194] Pharmacodynamic studies of TRE MVLs

[0195] (I) Pulmonary artery pressure measurement

[0196] 1. Measurement of mean pulmonary artery pressure

[0197] 33 mg of MCT was dissolved in 1 mL of DMSO and sonicated to obtain an MCT solution, which was stored in the dark. SD rats were subcutaneously injected with this MCT solution at a dose of 60 mg / kg. After 3 weeks, a PAH model was successfully established. PAH rats were weighed, marked, and randomly divided into three groups: MCT group, Free TRE group (1.2 mg TRE, 5.3 mg NaCl, 3 mg m-cresol, and 6.3 mg trisodium citrate dissolved in 1 mL of ultrapure water), and TRE MVLs group (administered the TRE MVLs from Example 3). A WT group (normal control group) was also included, with 5 rats in each group. The single dose of TRE in the Free TRE group and TRE MVLs group was 1.2 mg / kg (subcutaneous injection). Rats in the MCT group and WT group were subcutaneously injected with an equal volume of physiological saline. The administration was repeated every 3 days for 2 weeks.

[0198] Two weeks after drug administration, mPAP in each group was recorded using a direct measurement method. Rats were deeply anesthetized with isoflurane, fixed on an operating table, shaved, and had their trachea incised. A tracheal tube was inserted and connected to a small animal ventilator. A mini-catheter filled with heparinized sodium saline was connected to a pressure sensor and calibrated. Thoracotomy was performed to expose the heart, and the pulmonary artery was accessed via right pericardial puncture. mPAP was recorded after the pressure sensor data stabilized. The heart and lungs were then removed, rinsed with saline, blotted dry with filter paper, and placed in EP tubes for storage at 4°C for later use.

[0199] Mean pulmonary artery pressure results as follows Figure 10As shown, compared with the WT group, the mPAP of the MCT group was significantly increased to 35 mmHg (P<0.0001). This may be due to MCT-induced pulmonary vascular endothelial cell damage, triggering oxidative stress and inflammatory response, leading to thickening of the pulmonary vascular intima and increased resistance. Compared with the MCT group, the mPAP of both the Free TRE group and the TRE MVLs group was decreased, suggesting that TREs exert their effects by selectively activating pulmonary vascular IP receptors, dilating blood vessels, and reducing resistance. TRE MVLs showed the best antihypertensive effect and the lowest mPAP value (P<0.01), with no significant difference compared to the WT group. Combined with the experimental results showing that TRE MVLs can prolong the in vivo TRE half-life, this suggests that they have the potential to reduce mPAP in the long term and may improve the exercise capacity of PAH patients.

[0200] 2. Long-term pulmonary artery pressure measurement

[0201] Another 24 PAH rats were randomly divided into 8 groups, designated as groups for treatment at 1h, 3h, 6h, 1d, 2d, 3d, 4d, and 5d. Each of the 24 PAH rats received a single subcutaneous injection of TRE MVLs (Example 3), with a TRE dosage of 1.2 mg / kg. At 1h, 3h, 6h, 1d, 2d, 3d, 4d, and 5d post-treatment, rats in the corresponding groups were harvested, and mPAP was measured at different time points using a direct measurement method, referring to "1. Measurement of Mean Pulmonary Artery Pressure," to evaluate the long-term antihypertensive effect of TRE MVLs.

[0202] Another 24 PAH rats were randomly divided into 8 groups, corresponding to time points of 1h, 3h, 6h, 1d, 2d, 3d, 4d, and 5d after treatment. Each group received a single subcutaneous injection of TRE MVLs (Example 3) at a dose of 1.2mg / kg. Rats were harvested at the corresponding time points, and mPAP was measured using the direct measurement method as described in "1. Measurement of Mean Pulmonary Artery Pressure" to evaluate its long-term antihypertensive effect.

[0203] Long-term pulmonary artery pressure such as Figure 11 As shown, a single subcutaneous injection of TRE MVLs can rapidly reduce mPAP from 33 mmHg to 20 mmHg within 1 hour and maintain it at a normal level for 3 days. On day 4 after a single subcutaneous injection, mPAP was 27.67 ± 4.63 mmHg, a 16% reduction compared to pre-administration levels (33.0 ± 1.00 mmHg). This indicates that TRE MVLs can effectively reduce mPAP in PAH rats and maintain it at a normal level for a longer period, improving the patient's motor function. Its effect is similar to that of commercially available subcutaneous infusion pumps administered once every 3 days.

[0204] (II) Evaluation of Pulmonary Artery Remodeling Inhibition

[0205] 1. In the "1. Mean Pulmonary Artery Pressure Measurement" section, the lung tissue of each group of rats was fixed with 4% paraformaldehyde and then processed according to the following steps:

[0206] (1) Preparation of paraffin sections: The tissue was completely immersed in 4% paraformaldehyde for fixation for more than 24 hours, and then dehydrated sequentially with 70%, 80%, 90%, 95% and 100% ethanol (30 min each), dehydrated twice with xylene (60 min each), and then dehydrated three times with paraffin (60 min each). After dehydration, the tissue was embedded in paraffin and sections with a thickness of 5 μm were prepared using a microtome.

[0207] (2) Dewaxing and hydration: The sections were dewaxed twice with xylene (5-10 min / time), and then hydrated in a gradient of 100%, 95%, 80%, and 70% ethanol (2-3 min / stage), followed by rinsing with distilled water for 1-2 min;

[0208] (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-10 min, rinse with distilled water for 5-10 min to remove the surface stain; differentiate with 1% hydrochloric acid alcohol for 10-30 s until the cell nuclei are clear and the background is colorless, wash with distilled water to reverse the blue stain for 10-15 min;

[0209] (4) Eosin staining: Immerse the sections in eosin staining solution for 30-120 seconds, then rinse quickly with distilled water to remove excess staining solution;

[0210] (5) Dehydration and clearing: The tissue is dehydrated sequentially with 70%, 80%, 95% and 100% ethanol (1-2 min / stage), and dehydrated twice with xylene (2-5 min / time) until the tissue is clear;

[0211] (6) Mounting and observation: After mounting with neutral resin, the slides are air-dried at room temperature and observed and photographed under a bright-field fluorescence microscope. The pulmonary artery media wall thickness is calculated.

[0212]

[0213] Results of vascular morphology analysis, such as Figure 12 , Figure 13As shown, the pulmonary artery walls of rats in the WT group were thin and the lumen was patent; while the pulmonary artery walls of rats in the MCT group were significantly thickened, leading to stenosis and even occlusion. This may be related to MCT-induced damage to vascular endothelial cells, the release of vascular endothelial growth factor, and the stimulation of abnormal proliferation and migration of smooth muscle cells, thereby promoting intimal thickening and remodeling. After two weeks of treatment with the drug administered once every three days, the pulmonary artery media thickness of rats in the Free TRE group and the TRE MVLs group decreased by 20% and 64% respectively compared with the MCT group, suggesting that TRE can inhibit abnormal proliferation and migration of smooth muscle cells to alleviate intimal thickening. The media thickness in the TREMVLs group was not significantly different from that in the WT group, indicating that it enhanced the inhibitory effect on MCT-induced vascular remodeling by delaying drug release and prolonging the half-life.

[0214] 2. Immunohistochemistry was used to detect α-SMA expression in lung vascular cells. The specific steps are as follows:

[0215] (1) The lung tissue was embedded in paraffin, sectioned and dewaxed and hydrated.

[0216] (2) Antigen retrieval: Place the slide in sodium citrate buffer (pH 6.0), boil at 95°C or above for 10-20 minutes, and then cool naturally to room temperature.

[0217] (3) Then block endogenous peroxidase and incubate with 3% H2O2 at room temperature in the dark for 10 min.

[0218] (4) Blocking and primary antibody incubation: Block with 5% BSA at room temperature for 30 min, discard the blocking solution, add 1:200 diluted α-SMA monoclonal antibody, and incubate overnight at 4°C. Wash 3 times with PBS, 5 min each time.

[0219] (5) Secondary antibody incubation: Add biotin-labeled secondary antibody and incubate at room temperature for 30-60 min. Wash with PBS in the same way.

[0220] (6) Color development and restaining: Develop color with DAB working solution for 1-5 min, then stop the reaction with running water; soak in hematoxylin dye solution for 30-60 s, then rinse with running water to reverse the blue color.

[0221] (7) Dehydration and clearing: Dehydrated with 70%, 80%, 95%, and 100% ethanol for 1-2 min each, and treated with xylene twice (2-5 min each time) until the tissue is clear.

[0222] (8) Mounting and observation: Add neutral resin to mount the slide, let it dry at room temperature, and then observe and photograph it under a bright field fluorescence microscope.

[0223] Results of pulmonary vascular α-SMA expression investigation Figure 14 , Figure 15As shown, compared with the WT group, the area of ​​α-SMA-positive regions (i.e., brown areas) in the pulmonary vessels of rats in the MCT group was significantly increased, suggesting that MCT promotes vascular intimal thickening and remodeling by inducing abnormal proliferation of PASMACs. After treatment, the area of ​​positive regions in rats in both the Free TRE group and the TRE MVLs group decreased, with the average optical density (AOD) of α-SMA decreasing by approximately 5% and 30% respectively compared to the MCT group, indicating that TRE can inhibit abnormal proliferation of PASMACs and alleviate vascular intimal thickening to relieve remodeling. Notably, the α-SMA expression level was lowest in the TRE MVLs group, with no significant difference from the WT group, and at the same dose, its long-acting formulation significantly enhanced the inhibitory effect on abnormal proliferation of PASMACs by prolonging the duration of action.

[0224] 3. Following the above-described immunohistochemical method for detecting α-SMA expression in pulmonary vascular cells, Ki67 monoclonal antibody (1:1000 dilution) was used to detect lung cell proliferation.

[0225] Lung cell apoptosis detection (Tunnel method) steps:

[0226] (1) Tissue pretreatment: Paraffin embedding, sectioning and dewaxing hydration were completed;

[0227] (2) Protease digestion: Tissue sections were treated with 20 μg / mL proteinase K at room temperature for 20 min, washed with 1×TBS buffer, fixed with 4% paraformaldehyde at room temperature for 5 min, and then washed with TBS 3 times (5 min / time).

[0228] (3) End labeling reaction: Add 100 μL of equilibration buffer and incubate at room temperature for 10-30 min. After removing excess liquid, add 57 μL of TdT labeling reaction solution and 3 μL of TdT enzyme mixture prepared on ice and incubate at 37°C for 1.5 h.

[0229] (4) Reaction termination and washing: After washing with 1×TBS, add the termination solution and incubate at 37℃ for 5 min to terminate the reaction, and then wash with TBS again.

[0230] (5) Blocking and color development: Blocking buffer at room temperature for 10 min, incubate for 30 min, wash with TBS, stain with DAB color development solution for 10-15 min, stop color development with tap water, counterstain with hematoxylin and mount with neutral resin.

[0231] (6) Observation and recording: Observation and image acquisition were performed using an upright fluorescence microscope in bright field.

[0232] Lung antiproliferative results as follows Figure 16 , Figure 17As shown, the area of ​​the Ki67-positive expression region (i.e., the area of ​​the brown region) is positively correlated with cell proliferation activity. Compared with the WT group, Ki67 expression was significantly increased in the MCT group, indicating abnormal proliferation of PASMCs; after treatment with FreeTRE and TRE MVLs, Ki67 expression levels were downregulated. The AOD of Ki67 in the Free TRE group and the TRE MVLs group was reduced by 15% and 33% compared with the MCT group, respectively. Among them, the expression level in the TRE MVLs group was close to that in the WT group, indicating that TRE MVLs had the strongest inhibitory effect on abnormal cell proliferation.

[0233] Apoptosis analysis such as Figure 18 , Figure 19 As shown, the apoptosis rate in the MCT group was significantly lower than that in the WT group, while Free TRE and TREMVLs increased the apoptosis rate by 10% and 28%, respectively. Among them, the apoptosis rate in the TREMVLs group was significantly higher than that in the MCT group (P<0.0001), showing the best pro-apoptotic effect.

[0234] In summary, long-acting TRE MVLs can inhibit pulmonary vascular intima thickening and alleviate pulmonary vascular remodeling by enhancing the inhibition of abnormal proliferation and promoting apoptosis of PASMCs. This mechanism can further reduce pulmonary vascular resistance and mPAP, improving the exercise capacity and quality of life of PAH patients.

[0235] (III) Evaluation of Right Heart Remodeling

[0236] 1. Examination of the degree of right ventricular myocardial hypertrophy

[0237] In the "1. Mean Pulmonary Artery Pressure Measurement" study, right heart tissue from each group was fixed with 4% paraformaldehyde, then dehydrated, cleared, embedded in paraffin, and sectioned for H&E staining and observation. The specific steps are as follows:

[0238] (1) Paraffin section preparation: Lung tissue was completely immersed in 4% paraformaldehyde for fixation for more than 24 hours, then dehydrated in a gradient of 70%, 80%, 90%, 95% and 100% ethanol (30 min each), cleared twice with xylene (60 min each), and then infiltrated with paraffin three times (60 min each). After dehydration, the tissue was embedded in paraffin and prepared into 5 μm thick tissue sections using a microtome.

[0239] (2) Dewaxing to hydration: The sections were dewaxed twice with xylene (5-10 min / time), and then hydrated in a gradient of 100%, 95%, 80%, and 70% ethanol solutions (2-3 min / stage), and rinsed with distilled water for 1-2 min.

[0240] (3) H&E staining: Immerse the sections in hematoxylin staining solution for 3-10 min, rinse with distilled water for 5-10 min; differentiate with 1% hydrochloric acid alcohol for 10-30 s until the cell nuclei are clear and the background is colorless, then reflect blue with distilled water for 10-15 min; stain with eosin solution for 30-120 s, then rinse quickly with distilled water to remove excess staining solution.

[0241] (4) Dehydration, clearing and mounting: After staining, the sections were dehydrated by gradients of 70%, 80%, 95% and 100% ethanol (1-2 min / grade), cleared twice with xylene (2-5 min / time), mounted with neutral resin, and air-dried at room temperature. They were then observed and photographed under a bright field fluorescence microscope and the cross-sectional area (CSA) of cardiomyocytes was quantitatively analyzed using ImageJ software.

[0242] The results of the right ventricular cardiomyocyte hypertrophy test are as follows: Figure 20 , Figure 21 As shown, compared with the WT group, the cross-sectional area (CSA) of cardiomyocytes in the MCT group was significantly increased. After treatment, the CSA of both the Free TRE group and the TRE MVLs group decreased, with the TREMVLs group showing the lowest CSA value, close to the level of normal rats, suggesting that TRE MVLs can effectively inhibit cardiomyocyte hypertrophy. This effect may be related to its ability to alleviate pulmonary vascular remodeling, reduce mean pulmonary artery pressure (mPAP), and reduce right ventricular load.

[0243] 2. Right ventricular hypertrophy index (RVHI) testing

[0244] 33 mg of MCT was dissolved in 1 mL of DMSO and sonicated to obtain an MCT solution, which was stored in the dark. SD rats were subcutaneously injected with this MCT solution at a dose of 60 mg / kg. After 3 weeks, a PAH rat model was successfully established. The PAH rats were weighed, marked, and randomly divided into 4 groups: MCT group, Free TRE group (1.2 mg TRE, 5.3 mg NaCl, 3 mg m-cresol, and 6.3 mg trisodium citrate dissolved in 1 mL of ultrapure water), low-dose TRE MVLs group (administered TRE MVLs from Example 3, TRE MVLs (Low) group), and high-dose TRE MVLs group (administered TRE MVLs from Example 3, TRE MVLs (High) group), and a WT group (normal control group) was also set up, with 5 rats in each group. All five groups were administered subcutaneously. The single dose of TRE in the Free TRE group and the TRE MVLs (Low) group was 1.2 mg / kg, and the single dose of TRE in the TRE MVLs (High) group was 1.6 mg / kg. Rats in the MCT group and the WT group were given an equal volume of physiological saline. The administration was carried out once every 3 days for 2 weeks.

[0245] Two weeks after administration, the rats were sacrificed, their hearts were removed, washed with saline, and cut along the junction of the right ventricle and interventricular septum to separate the free wall of the right ventricle from the left ventricle and interventricular septum tissue, which were then weighed separately. The RVHI was calculated as the ratio of the right ventricle weight (RV) to the left ventricle and interventricular septum weight (LV+S).

[0246]

[0247] Assessment of right ventricular hypertrophy Figure 22 As shown, MCT induction significantly increased the right ventricular hypertrophy index (RVHI, P<0.0001); the RVHI of rats treated with Free TRE, TRE MVLs (Low), and TRE MVLs (High) groups decreased to varying degrees. Furthermore, the RVHI value was lower with increasing treatment dose. Among these, the TRE MVLs (High) group was similar to the WT group, indicating that it had the best inhibitory effect on right ventricular hypertrophy.

[0248] Comprehensive analysis suggests that long-acting TRE MVLs, by prolonging the duration of drug action, enhance the inhibitory effect on pulmonary vascular remodeling, further reducing mPAP and right ventricular load, thereby curbing compensatory right ventricular hypertrophy. Given that current clinical treatment for PAH primarily focuses on symptomatic relief to improve exercise capacity, which is unlikely to prolong patient survival, long-acting TRE MVLs, while improving exercise function, can reduce the risk of death from right heart failure by inhibiting pulmonary vascular and right ventricular remodeling, potentially extending the survival of PAH patients.

[0249] Example 10

[0250] TRE MVLs Security Study

[0251] (I) Cytotoxicity of TRE and TRE MVLs to PASMCs

[0252] TRE MVLs with a concentration of 4.8 mg / mL were prepared according to Example 3. The solutions were diluted with RPMI 1640 medium containing 10% fetal bovine serum to obtain TRE MVLs solutions with concentration gradients of 400, 300, 200, 100, 50 and 10 μg / mL.

[0253] TRE solutions with the same concentration gradient were prepared according to Example 6.

[0254] PAH-PASMCs in the exponential growth phase were harvested, and 1.5 mL of 0.25% trypsin was added. After shaking, the cells were incubated for 6 min. Digestion was terminated by adding 4.5 mL of RPMI 1640 medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum to obtain a cell suspension. 200 μL of the cell suspension was added to each well of a 96-well plate at a density of 6500 cells / well and cultured for 24 h. The supernatant was discarded, and 200 μL of different concentrations of TREMVLs and Free TRE solution were added, respectively. Cells without the drug solution served as the control group, and cells without the drug solution served as the blank group in RPMI 1640 medium containing 10% fetal bovine serum. Each concentration was used in 6 replicates. Incubation continued for 24 h, the supernatant was discarded, and 20 μL of 5 mg / mL LMT solution was added to each well. The cells were incubated for 4 h. Discard the upper culture medium, add 200 μL DMSO to each well, and shake thoroughly in a shaker under dark conditions. Measure the absorbance (Abs) of each well at 570 nm using a microplate reader. Use cells without the drug solution as the control group and cell-free RPMI 1640 medium containing 10% fetal bovine serum as the blank group. Set up 6 replicates for each concentration and calculate the cell viability using the formula.

[0255]

[0256] In this study, the absorbance of cells cultured with TRE MVLs or Free TRE at 570 nm was measured in the experimental group (Abs), the absorbance of cells cultured without the drug at 570 nm was measured in the control group (Abs), and the absorbance of cells cultured without the drug at 570 nm was measured in cell-free RPMI 1640 medium containing 10% fetal bovine serum at 570 nm.

[0257] The results are as follows Figure 23 As shown, when the TRE concentration did not exceed 200 μg / mL, the cell survival rate in both the TRE MVLs group and the Free TRE group was greater than 75%; when the TRE concentration was 400 μg / mL, the cell survival rate in the TRE MVLs group was only 40%, while that in the Free TRE group was 64.00%. The experiment indicates that the toxicity of TRE to PASMCs is concentration-dependent, and that TRE MVLs have a stronger ability to inhibit the proliferation of PAH-PASMCs at higher concentrations.

[0258] (II) Organizational Security Assessment of TRE MVLs

[0259] This study used H&E staining to observe inflammatory cells in subcutaneous tissue and assess the tissue safety of TRE MVLs at the injection site. A PAH rat model was constructed according to Example 9, "1. Measurement of Mean Pulmonary Artery Pressure". Twenty-four PAH rats were randomly divided into two groups: a control group and a TRE MVLs group. The control group received subcutaneous injection of physiological saline, while the TRE MVLs group received subcutaneous injection of 1.2 mg / kg TREMVLs (Example 3). Three rats from each group were sacrificed on days 1, 3, 7, and 14 after administration. Subcutaneous tissue at the injection site was isolated, fixed with paraformaldehyde, and prepared into paraffin sections for H&E staining analysis. The sections were observed and photographed under a bright-field fluorescence microscope.

[0260] The results are as follows Figure 24 As shown, local inflammatory cell infiltration occurred one day after TRE MVLs injection, while the subcutaneous tissue morphology showed no significant difference from the saline control group within seven days. H&E staining results indicated that the inflammation at the injection site induced by TRE MVLs could subside rapidly in a short period, suggesting that this administration method has high safety in long-term treatment.

[0261] In summary, the treprostinil polycystic liposome of this invention has a high drug loading capacity and good stability. Subcutaneous injection reduces the frequency of administration, improves patient compliance, and lowers treatment costs. Furthermore, the treprostinil polycystic liposome can improve patients' motor function within three days by reducing mPAP, avoiding three times a day oral administration, and achieving a therapeutic effect similar to subcutaneous infusion once every three days.

Claims

1. A treprostinil polycystic liposome for treating pulmonary hypertension, characterized in that: The multi-capsule liposomes are prepared by a double emulsification method using an inner aqueous phase, an oil phase, and an outer aqueous phase; the treprostinil is dissolved in the inner aqueous phase; the oil phase contains soybean lecithin, cholesterol, trioleic acid glycerides, and negatively charged lipids; both the inner and outer aqueous phases are isotonic solutions of body fluids.

2. The treprostone multivesicular liposome according to claim 1, characterized in that: The negatively charged lipids are selected from one of stearic acid, vitamin E succinate, and dipalmitoylphosphatidylglycerol; The internal aqueous phase is selected from a 5% glucose solution or a 2.1% glycine solution that is isotonic with body fluids; The external aqueous phase is selected from a 5% glucose solution containing 40 mmol / L lysine or a 2.1% glycine solution containing 40 mmol / L lysine, which is isotonic with body fluids.

3. The treprostone multivesicular liposome according to claim 2, characterized in that: The negatively charged lipid is vitamin E succinate or dipalmitoylphosphatidylglycerol, preferably dipalmitoylphosphatidylglycerol; The internal aqueous phase is a 5% glucose solution that is isotonic with body fluids; The external aqueous phase is a 5% glucose solution containing 40 mmol / L lysine, which is isotonic with body fluids.

4. The treprostone liposome according to claim 1, characterized in that: The aforementioned treprostone multicystic liposome exhibits a multicystic structure, with multiple inner aqueous chambers separated by a lipid bilayer, and the entire structure is suspended in the outer aqueous phase.

5. A method for preparing treprostone liposomes according to claim 1, characterized in that: Includes the following steps: Step (1): Dissolve soybean lecithin, cholesterol, trioleic acid glycerides, and negatively charged lipids in an organic solvent to obtain the oil phase; Step (2): Dissolve treprostrin in the internal aqueous phase, adjust the pH to alkaline with sodium hydroxide solution to obtain an internal aqueous phase containing treprostrin sodium salt; Step (3): Add the aqueous phase containing treprostyl sodium salt to the oil phase and perform ultrasonic emulsification to form W1 / O primary emulsion; Step (4): Vortex mix the W1 / O primary emulsion and the external aqueous phase to form a W1 / O / W2 double emulsion; Step (5): Centrifuge the W1 / O / W2 double emulsion, collect the precipitate, and wash it with an external aqueous phase; Step (6): The washed sample is resuspended in an external aqueous phase, the organic solvent is removed by nitrogen purging, and then centrifuged. The precipitate is resuspended in an external aqueous phase to obtain treprostone nifedipine liposomes.

6. The method for preparing treprostone multivesicular liposomes according to claim 5, characterized in that: In step (1), the organic solvent is a mixed solvent of chloroform and methanol in a volume ratio of 8.5:1; in the oil phase, the concentration of soybean lecithin is 16-24 mg / mL, preferably 24 mg / mL; the concentration of cholesterol is 6-12 mg / mL, preferably 8-10 mg / mL, and most preferably 8 mg / mL; the concentration of trioleic acid glyceride is 4-12 mg / mL, preferably 6-12 mg / mL, more preferably 6-8 mg / mL, and most preferably 8 mg / mL; and the concentration of the negatively charged lipid is 1-2.5 mg / mL, preferably 1.5 mg / mL.

7. The method for preparing treprostone liposomes according to claim 5, characterized in that: In step (2), the concentration of treprostyl in the inner aqueous phase is 2-8 mg / mL, preferably 2-5 mg / mL, more preferably 2-3 mg / mL, and most preferably 3 mg / mL; the pH of the inner aqueous phase is adjusted to 10 using sodium hydroxide solution.

8. The method for preparing treprostone multicystic liposomes according to claim 5, characterized in that: In step (3), the volume ratio of the internal aqueous phase to the oil phase is 1:1 to 1:2, preferably 1:1; the ultrasonic power of the ultrasonic emulsification is 150 to 200 W, and the ultrasonic emulsification time is 15 min to 25 min; preferably, the ultrasonic power of the ultrasonic emulsification is 180 to 200 W, and the ultrasonic emulsification time is 15 min to 20 min; most preferably, the ultrasonic power of the ultrasonic emulsification is 180 W, and the ultrasonic emulsification time is 20 min. In step (4), the volume ratio of the W1 / O pre-emulsion to the external aqueous phase is 1:0.75 to 1:1.25, preferably 1:1; the rotational speed of the vortex is 2500 to 3000 rpm, preferably 2800 rpm; and the vortexing time is 40 to 80 s, preferably 60 s. In step (5), the W1 / O / W2 double emulsion is centrifuged, the precipitate is taken, and it is washed with an external aqueous phase. Each time it is washed, an external aqueous phase of the same volume as the supernatant is added for resuspension and centrifugation. The washing is repeated three times in total. The centrifugation temperature for W1 / O / W2 re-emulsion is 4-10℃, the centrifugation speed is 2000-3000 rpm, and the centrifugation time is 5-10 min; For each wash, the centrifugation temperature is 4–10℃, the centrifugation speed is 2000–3000 rpm, and the centrifugation time is 5–10 min; In step (6), the method of removing organic solvent by nitrogen purging is to purge with nitrogen in a horizontal direction for 30 to 40 minutes in a water bath at 30 to 37°C.

9. The use of the treprostinil polycystic liposome according to claim 1 in the preparation of a medicament for treating pulmonary hypertension.

10. The application according to claim 9, characterized in that: The drug is in the form of an injection.

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