Targeted Atherosclerosis Liposome Nanocarrier Delivery System and Its Preparation Method

Abstract

The present disclosure provides an active encapsulation liposome nanocarrier delivery system, and the liposome nanocarrier delivery system includes a liposome carrier and a substance actively encapsulated therein for preventing and / or treating atherosclerosis or diseases related to atherosclerosis; wherein the preparation of the nanocarrier delivery system includes a step of hydrating the liposome carrier by adding a salt solution that creates an acidity gradient between the inside and outside of the liposome to actively encapsulate the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis, and also provides a preparation method and applications thereof.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an active encapsulation liposome nanocarrier delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Cardiovascular and cerebrovascular diseases are the number one chronic diseases with high disability rate, high mortality rate, high medical risk and high medical expenses, and are the leading cause of death globally. Cardiovascular and cerebrovascular diseases account for more than 40% of all deaths of residents, and atherosclerotic-related diseases are the top priority of cardiovascular and cerebrovascular diseases. According to statistics, there are 466 million atherosclerotic patients globally, including 154 million coronary heart disease patients, with 27.3 million new cases annually. There are 19 million new cases of acute myocardial infarction globally every year, and the mortality rate of patients is 34% to 42%. Even if the patient is lucky enough to survive, the relative risk of all-cause death and cardiovascular events within 1-5 years after myocardial infarction is at least 30% higher than that of the general population.

[0003] The "Report on Cardiovascular Diseases in China 2018" shows that: According to the data of the sixth national census in China in 2010, the number of coronary heart disease patients in China has reached 11 million, with more than 1 million new patients every year, and as many as 3.9 million patients die of cardiovascular diseases every year, accounting for more than 40% of the composition of disease deaths of residents. A large number of studies have confirmed that atherosclerosis is the main cause of myocardial infarction and ischemic stroke in patients, and such patients are also most likely to have serious adverse events such as sudden death, heart failure, and sequelae of cerebral infarction, bringing a heavy medical burden to families and society.

[0004] However, in the face of such a major disease, there is no good prevention and treatment method globally. The current treatment for atherosclerosis is still systemic medication. Oral drugs can only delay the progression of plaques, but cannot significantly reverse plaques, let alone cure the disease completely. Moreover, Chinese people have poor tolerance to drugs such as statins and are prone to induce complications such as abnormal liver function, new-onset diabetes, rhabdomyolysis, and cognitive impairment. At present, the treatment strategy for patients is still lifelong drug treatment, which cannot cure the disease and also faces many drug side effects. For example, oral statin drugs need to be used in high doses to have the effect of stabilizing plaques in the treatment of atherosclerosis, and the systemic use of high-dose statin drugs also has the risk of increased incidence of serious side effects (such as abnormal liver function, rhabdomyolysis, type II diabetes, etc.).

[0005] For existing systemic drug administration, usually only a very small part of the active ingredients can truly act on the diseased site after the drug enters the body. Especially for atherosclerosis, which is located in the blood vessel wall where the blood flow is fast, the drug cannot enter the plaque, let alone accumulate in the plaque. This is the fundamental reason that restricts the drug efficacy and causes drug side effects. A targeted drug delivery system refers to a drug delivery system with the ability of targeted drug delivery. After being administered through a certain route, the drugs contained in the targeted drug delivery system will specifically accumulate in the target site due to specific properties such as particle size or electrical properties, or specifically accumulate in the target site through a carrier with a targeting probe. The targeted drug delivery system can enable the drug to target a specific diseased site and release the active ingredients at the target diseased site. Therefore, the targeted drug delivery system can form a relatively high concentration of the drug at the target diseased site and reduce the amount of drug in the blood circulation, thereby improving the drug efficacy while inhibiting side effects and reducing the damage to normal tissues and cells.

[0006] Liposomes are commonly used targeted drug delivery systems. Liposomes have obvious advantages such as improving drug efficacy, reducing drug side effects, enhancing the bioavailability of drugs, and strong biological safety. ZL201980001843.4 discloses a liposome nanodrug targeting CD44 prepared by the passive encapsulation method. It can utilize the expression state of CD44 on the surfaces of macrophages, monocytes, endothelial cells, lymphocytes, and smooth muscle cells mainly present in vulnerable plaques and their affinity with hyaluronic acid (HA) to construct a liposome drug delivery system for targeting plaques or diseases related to vulnerable plaques that can achieve drug delivery for diagnosing or treating vulnerable plaques and continuously release, but the current method is limited to the passive drug loading method. ZL201980001833.0 discloses a two-step method of film hydration and probe sonication to coat a series of hydrophilic or hydrophobic therapeutic drugs and contrast agents in the hydrophilic inner cavity or hydrophobic lipid layer, and further conjugate with a targeting ligand. This strategy belongs to passive encapsulation. Although it has strong universality, the encapsulation efficiency is often not high enough, and there is still room for further improvement in the treatment effect.

[0007] In addition, studies on preparing liposomes from highly lipophilic statin drugs have shown that by using the film hydration or reverse evaporation method for highly lipophilic statin drugs, the drugs can be encapsulated in the liposome bilayer membrane to achieve the purpose of increasing the metabolism time and enhancing the bioavailability. However, these methods are not applicable to statin drugs with a certain water solubility. To solve these problems, the inventors of this patent application discovered and disclosed a method of actively encapsulating statin drugs with a certain water solubility through the active drug loading method to achieve targeted drug delivery.

[0008] In the field of targeted nanomedicines, the effects of formulation particle size and the conjugation density of targeting ligands on the therapeutic effect are closely related, but the conclusions are not unified. That is, there is no unified standard for the size and surface ligand conjugation concentration of nanomedicines for different indications, and there are significant differences for different indications. For the disease atherosclerosis, exploring appropriate particle sizes and ligand concentrations is crucial for the exertion of drug efficacy. This patent application discloses a new drug-loading process, as well as a formulation for treating atherosclerosis using specific nanostructures and specific ligand densities. Summary of the Invention

[0009] The object of the present invention is to overcome the defects in the prior art and provide an active-encapsulation liposome nanocarrier delivery system, its preparation method and application. At the same time, the present invention discloses the effects of specific particle sizes and surface ligand densities on drug efficacy in targeted nanodrugs.

[0010] Before elaborating on the content of the present invention, the terms used in this application are defined as follows:

[0011] The term "targeted drug delivery system" refers to: a drug delivery system with the ability of targeted drug delivery. After administration through a certain route, the drug contained in the targeted drug delivery system will specifically accumulate in the target site through the action of a special carrier or targeting warhead (for example, a targeting ligand). Currently known means for achieving targeted drug delivery include utilizing the passive targeting properties of various particulate drug delivery systems, chemically modifying the surface of particulate drug delivery systems, utilizing some special physical and chemical properties, utilizing antibody-mediated targeted drug delivery, utilizing ligand-mediated targeted drug delivery, utilizing prodrug targeted drug delivery, etc.

[0012] The term "atherosclerosis" refers to: a common and most important one among a group of vascular diseases called arteriosclerosis, characterized by the involvement of arterial lesions starting from the intima, followed by the accumulation of lipids and complex carbohydrates, bleeding and thrombosis, fibrotic tissue hyperplasia and calcinosis, and the gradual degeneration and calcification of the arterial media. Since the lipids accumulated in the arterial intima appear yellow and porridge-like, it is called atherosclerosis.

[0013] The term "active-encapsulation liposome" refers to: using a transmembrane pH gradient or ion gradient to load certain hydrophilic or amphiphilic compounds into prefabricated liposomes. This technique is called active or remote encapsulation.

[0014] To achieve the above object, the first aspect of the present invention provides an active-encapsulation liposome nanocarrier delivery system. The liposome nanocarrier delivery system includes a liposome carrier and a substance actively encapsulated therein for preventing and / or treating atherosclerosis or diseases related to atherosclerosis.

[0015] The liposome nanocarrier delivery system according to the first aspect of the present invention, wherein the material of the liposome carrier comprises phospholipids and cholesterol;

[0016] Preferably, the phospholipids are selected from one or more of the following: hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, polyethylene glycol-distearoyl phosphatidylethanolamine, distearoyl phosphatidylglycerol, distearoyl phosphatidylserine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylserine, dioleoyl phosphatidylethanolamine-polyethylene glycol, dicrotonoyl phosphatidylcholine, dicrotonoyl phosphatidylethanolamine, dicrotonoyl phosphatidylserine, dicrotonoyl phosphatidylethanolamine-polyethylene glycol, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, erucoyl phosphatidylcholine, erucoyl phosphatidylethanolamine, erucoyl phosphatidylserine, erucoyl phosphatidylethanolamine-polyethylene glycol, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol.

[0017] The liposome nanocarrier delivery system according to the first aspect of the present invention, wherein the mass ratio of the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis to the liposome carrier is 1:0.1 to 50, preferably 1:0.2 to 30, more preferably 1:0.5 to 20. Optionally, the preparation of the nanocarrier delivery system further comprises the step of actively encapsulating the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis with the liposome carrier hydrated with a salt solution.

[0018] The liposome nanocarrier delivery system according to the first aspect of the present invention, wherein the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is a weakly acidic substance;

[0019] Preferably, the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is selected from one or more of the following: statins, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulants, angiotensin-converting enzyme inhibitors, calcium antagonists, MMPs inhibitors, β-blockers, and glucocorticoids, and pharmaceutically acceptable salts thereof, as well as active preparations of the drugs or substances;

[0020] More preferably, the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is selected from one or more of the following: lovastatin, atorvastatin, rosuvastatin, simvastatin, pitavastatin, pravastatin, bezafibrate, ciprofibrate, gemfibrozil, aspirin, acemetacin, ozagrel sodium, tirofiban, and their pharmacodynamic fragments or pharmaceutically acceptable salts.

[0021] The liposome nanocarrier delivery system according to the first aspect of the present invention, wherein the liposome nanocarrier delivery system can be modified by targeting ligands, such as GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, and hyaluronic acid or derivatives of hyaluronic acid;

[0022] Preferably, the targeting ligand is selected from hyaluronic acid, serglycin, collagen, fibronectin, selectin, osteopontin (OPN), monoclonal antibodies HI44a, and IM7.

[0023] The liposome nanocarrier delivery system according to the first aspect of the present invention, wherein the liposome carrier is selected from small unilamellar liposomes, large unilamellar liposomes, and multilamellar liposomes.

[0024] The second aspect of the present invention provides a method for preparing the liposome nanocarrier delivery system described in the first aspect, including the steps of hydrating the liposome carrier by adding a salt solution and separating to create an acidity gradient between the inner and outer solutions of the liposome.

[0025] According to the liposome nanocarrier delivery system of the second aspect of the present invention, wherein the salt is a salt of a weak acid and a strong base;

[0026] Preferably, the anionic part of the salt is selected from one or more of the following: acetate, edetate, bicarbonate, citrate, benzoate, and gluconate; and / or

[0027] The cationic part of the salt is selected from one or more of the following: calcium ion, copper ion, nickel ion, barium ion, magnesium ion, and zinc ion;

[0028] More preferably, the salt is selected from one or more of the following: calcium acetate, calcium bicarbonate, magnesium citrate, and copper gluconate.

[0029] According to the preparation method of the second aspect of the present invention, wherein the preparation method may include the following steps:

[0030] (1) Dissolve the liposome carrier material in a good solvent;

[0031] (2) Add a salt solution that creates an acidity gradient between the inside and outside of the liposome to the product obtained in step (1) for hydration to disperse it into a crude lipid solution;

[0032] (3) Reduce the particle size of the crude lipid solution obtained in step (2) to obtain refined liposomes;

[0033] (4) Purify the refined liposome solution obtained in step (3) to remove external ions, forming an acidity gradient between the inside and outside of the liposome;

[0034] (5) Add the solution of the substance to be coated to the liposome solution obtained in step (4), and incubate to allow the drug to enter the interior of the liposome to obtain the liposome nanocarrier delivery system.

[0035] According to the preparation method of the second aspect of the present invention, wherein, in step (1), there is also a step of evaporating the solvent to form a lipid film of the liposome;

[0036] Preferably, the evaporation temperature is 40 - 80 °C, preferably 50 - 70 °C, more preferably 50 - 60 °C;

[0037] More preferably, the evaporation method is rotary evaporation;

[0038] Further preferably, the rotary evaporation speed is 50 - 200 r / min, preferably 70 - 120 r / min, more preferably 80 - 100 r / min.

[0039] According to the preparation method of the second aspect of the present invention, wherein, in step (1), the good solvent is selected from one or more of the following: chloroform, ethanol, methanol, dichloromethane, acetone, toluene, ether, ethyl acetate, methyl acetate, acetonitrile, and dichloroethane.

[0040] According to the preparation method of the second aspect of the present invention, wherein, in step (1), the liposome carrier material contains phospholipid and cholesterol;

[0041] Preferably, the phospholipid concentration is 0.1 - 500 mg / mL, preferably 0.1 - 200 mg / mL, more preferably 0.2 - 100 mg / mL; and / or

[0042] The cholesterol concentration is 0.05 - 200 mg / mL, preferably 0.1 - 100 mg / mL, more preferably 0.1 - 50 mg / mL.

[0043] According to the preparation method of the second aspect of the present invention, wherein, in step (2), the concentration of the salt solution is 100 - 500 mM, preferably 200 - 300 mM, more preferably 200 - 250 mM; and / or

[0044] The hydration temperature is 30 to 90 °C, preferably 40 to 80 °C, more preferably 45 to 65 °C.

[0045] According to the preparation method of the second aspect of the present invention, in step (3), the method for reducing the particle size is to extrude the crude lipid solution through a filter membrane;

[0046] Preferably, the filter membrane is selected as a polycarbonate membrane;

[0047] More preferably, the pore size of the filter membrane is 30 to 800 nm, such as 50 to 400 nm, such as 100 to 200 nm, such as 30 nm, 50 nm, 100 nm, 200 nm, 400 nm, 600 nm or 800 nm, preferably 100 - 150 nm.

[0048] According to the preparation method of the second aspect of the present invention, in step (4), the method for removing external ions is selected from one or more of the following: ultrafiltration membrane package separation, dialysis separation and ion exchange.

[0049] According to the preparation method of the second aspect of the present invention, in step (5), the concentration of the coating substance solution is 0.01 to 100 mg / mL, preferably 0.05 to 50 mg / mL, more preferably 0.1 to 20 mg / mL, and further preferably 0.1 - 10 mg / mL.

[0050] According to the preparation method of the second aspect of the present invention, in step (5), the incubation temperature is 20 to 80 °C, preferably 25 to 70 °C

[0051] According to the preparation method of the second aspect of the present invention, when the liposome nanocarrier delivery system is modified by a targeting ligand, the method further includes the following steps:

[0052] (6) Co - incubate the targeting ligand with the liposome nanocarrier delivery system obtained in step (5) to obtain the modified liposome nanocarrier delivery system.

[0053] The third aspect of the present invention provides a drug, which comprises the nanocarrier delivery system described in the first aspect or the nanocarrier delivery system prepared according to the preparation method described in the second aspect, and at least one pharmaceutically acceptable carrier.

[0054] The fourth aspect of the present invention provides the application of the nanocarrier delivery system described in the first aspect, the nanocarrier delivery system prepared according to the preparation method described in the second aspect, and the drug described in the third aspect in the preparation of a drug for preventing and / or treating atherosclerosis or diseases related to atherosclerosis.

[0055] Preferably, the atherosclerosis-related diseases are selected from one or more of the following: atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia-latent coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including ischemic stroke, hemorrhagic stroke), peripheral vascular atherosclerosis (including carotid atherosclerosis, vertebral atherosclerosis, subclavian atherosclerosis, occlusive peripheral atherosclerosis, retinal atherosclerosis, renal artery atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, mesenteric atherosclerosis, atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure and cardiogenic shock.

[0056] The fifth aspect of the present invention provides the use of the nanocarrier delivery system described in the first aspect, the nanocarrier delivery system prepared according to the preparation method described in the second aspect, and the drug described in the third aspect in the preparation of a drug for treating vascular plaques;

[0057] Preferably, the vascular plaque is an arterial plaque;

[0058] More preferably, the medicament is used to arrest the progression of arterial plaque, reverse arterial plaque and / or reduce the volume of arterial plaque.

[0059] The present invention belongs to the field of targeted preparations, and relates to an active targeted liposome nanocarrier delivery system and a preparation method and application thereof, in particular to an innovative preparation method and application of an actively encapsulated liposome nanomedicine targeting atherosclerosis, such as application in the diagnosis, prevention and treatment of atherosclerosis or diseases related to atherosclerosis.

[0060] In order to further improve the therapeutic effect of nano drug delivery system, increase the drug loading and encapsulation rate of nano carriers, and enhance the clinical transformation ability of targeted nano drugs, this patent application discloses a process route for preparing targeted liposome nano drugs by active drug loading, and the inventor of this patent unexpectedly found that the appropriate particle size and ligand concentration play a vital role in drug efficacy. This patent application discloses drug loading technology, specific carrier nanostructure and specific ligand density, as well as a new method for the treatment of atherosclerotic diseases, and realizes the efficient enrichment of a variety of atherosclerotic, anti-inflammatory, anti-platelet and other drugs in atherosclerotic sites.

[0061] This patent application discloses a series of new systems, methods and processes for active drug delivery of targeted liposome drugs, which enable the drug encapsulation rate to reach more than 90%, reflecting its obvious advantages as a drug carrier, and further verifying its excellent therapeutic effect in model animals, expanding the application of targeted atherosclerosis liposome drug carriers.

[0062] Preferably, the formulation of the liposome comprises: phospholipid 0.2 - 100 mg / mL, cholesterol 0.1 - 50 mg / mL, drug 0.1 - 10 mg / mL, wherein the mass ratio of the drug to the lipid material is between 1:0.5 and 1:20.

[0063] According to an embodiment of the present invention, the present patent application provides a liposome preparation for treating atherosclerosis by active encapsulation of a drug, with a particle size of 50 - 300 nm, phospholipid 0.2 - 100 mg / mL, cholesterol 0.1 - 50 mg / mL, drug 0.1 - 10 mg / mL, wherein the mass ratio of the drug to the lipid material is between 1:0.5 and 1:20. Preferably, the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is selected from statins, fibrates, antiplatelet drugs, PCSK9 inhibitors, anticoagulants, angiotensin-converting enzyme inhibitors, calcium antagonists, MMPs inhibitors, β-blockers, and glucocorticoids, and one or more of their pharmaceutically acceptable salts, including active preparations of these types of drugs or substances.

[0064] More preferably, the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is selected from lovastatin, atorvastatin, rosuvastatin, simvastatin, pitavastatin, pravastatin, bezafibrate, ciprofibrate, gemfibrozil, aspirin, acemetacin, sodium ozagrel, and tirofiban, and one or more of their pharmacodynamic fragments or pharmaceutically acceptable salts, and one or more of their pharmaceutically acceptable salts.

[0065] An embodiment of the present invention provides a preparation method of a nanocarrier delivery system, and the method comprises the following steps:

[0066] (1) Dissolve the phospholipid membrane material in a good solvent, and the solvent can be evaporated or not removed;

[0067] (2) Add a salt solution that creates an acidity gradient inside and outside the liposome to hydrate the solution or dried residue obtained in step (1), and preliminarily disperse it into a crude lipid solution;

[0068] (3) Reduce the particle size of the crude lipid solution to obtain a refined liposome;

[0069] (4) Purify the liposome solution obtained in step (3) to remove external ions, and form an acidity gradient inside and outside the liposome;

[0070] (5) Add the drug solution to be encapsulated to the liposome solution obtained in step (4) to allow the drug to enter the inside of the liposome;

[0071] (6) Assemble the targeting ligand on the surface of the liposome based on the insertion method.

[0072] The present invention provides an application of actively encapsulated liposomes in the preparation of products for the diagnosis, prevention, and treatment of atherosclerosis or diseases related to atherosclerosis.

[0073] The nano - carrier delivery system of the present invention may have, but is not limited to, the following beneficial effects:

[0074] The present invention provides a process route for preparing an actively encapsulated liposome nano - carrier delivery system and its optimization process, and simultaneously verifies the ability of the obtained targeted delivery system to reverse plaques. This system has a high encapsulation rate and drug - loading capacity, strong protection for the encapsulated drug, the drug will not be released during long - term storage, the drug is slowly released in the body, can improve the enrichment concentration of the drug at the local lesion, and has the advantages of low toxicity and high efficiency. The method of the present invention is simple, green, controllable, has a high drug - loading capacity, good compatibility with various drugs, good biocompatibility, high safety, is easy to perform targeted ligand modification, and the obtained targeted drug has a good effect on the treatment of atherosclerosis. Brief Description of the Drawings

[0075] When read in conjunction with the attached Figure 1 The foregoing invention content and the following detailed description of the invention will be better understood. To illustrate the present disclosure, the drawings illustrate some but not all of the alternative embodiments. However, it should be understood that the present disclosure is not limited to the precise arrangements and means shown. These figures, which are incorporated into and constitute a part of this specification, help to explain the principles of the present disclosure.

[0076] Figure 1 Illustrate the infrared spectra of sodium hyaluronate (HA) and hyaluronic acid plaque target (HPD).

[0077] Figure 2 Illustrate the cryo - electron microscopy (A, B, C) and hydrated particle size results (D, E, F) of actively encapsulated rosuvastatin nano - liposomes LP / R A90 、LP / R A120 and LP / R A150

[0078] Figure 3 Illustrate the cryo - electron microscopy (A, B, C) and hydrated particle size results (D, E, F) of actively encapsulated rosuvastatin nano - liposomes LP / R B90 、LP / R B120 and LP / R B150

[0079] Figure 4 Illustrate actively encapsulated rosuvastatin nano - liposome HA H -LP / RA150 , HA M -LP / R A150 and HA L -LP / R A150 Cryo-EM (A, B, C) and hydrated particle size results (D, E, F).

[0080] Figure 5 Illustration of the targeted rosuvastatin actively encapsulated liposomal nanocarrier delivery system HA@LP / R C Cryo-EM (A) and hydrated particle size results (B).

[0081] Figure 6 Illustration of the targeted atorvastatin actively encapsulated liposome HA-LP / A C Cryo-EM and Dynamic Light Scattering (DLS).

[0082] Figure 7 Illustration of the targeted aspirin actively encapsulated liposome HA-LP / Asp C Cryo-EM and DLS.

[0083] Figure 8 Illustration of the targeted pitavastatin actively encapsulated liposome HA-LP / P C Cryo-EM and DLS.

[0084] Figure 9 Illustration of the targeted lovastatin actively encapsulated liposome HA-LP / L C Cryo-EM and DLS.

[0085] Figure 10 Illustration of the targeted simvastatin actively encapsulated liposome HA-LP / S C Cryo-EM and DLS.

[0086] Figure 11 Illustration of the targeted pravastatin actively encapsulated liposome HA-LP / P C Cryo-EM and DLS.

[0087] Figure 12 Illustration of the targeted bezafibrate actively encapsulated liposome HA-LP / B C Cryo-EM and DLS.

[0088] Figure 13 Illustration of the targeted ciprofibrate actively encapsulated liposome HA-LP / C C Cryo-EM and DLS.

[0089] Figure 14 Illustration of the targeted acemetacin actively encapsulated liposome HA-LP / Am C Cryo-EM and DLS.

[0090] Figure 15 Exemplified actively encapsulated liposome HA-LP / O targeting ozagrel C Cryo-electron microscopy and DLS.

[0091] Figure 16 Exemplified actively encapsulated liposome HA-LP / T targeting tirofiban C Cryo-electron microscopy and DLS.

[0092] Figure 17 Exemplified actively encapsulated liposome HA@LP / R targeting rosuvastatin d Cryo-electron microscopy and DLS.

[0093] Figure 18 Exemplified actively encapsulated liposome HA@LP / A targeting atorvastatin d Cryo-electron microscopy and DLS.

[0094] Figure 19 Exemplified actively encapsulated liposome HA@LP / C targeting ciprofibrate d Cryo-electron microscopy and DLS.

[0095] Figure 20 Exemplified actively encapsulated liposome HA-LP / B targeting bezafibrate d Cryo-electron microscopy and DLS.

[0096] Figure 21 Exemplified results of the long-term stability investigation of the nano-delivery system of the present invention.

[0097] Figure 22 Exemplified percentage of plaque volume progression of the HA-LP / R nano-drug delivery system of the present invention and each control group.

[0098] Figure 23 Exemplified comparison of the enrichment degree of the aorta for different therapeutic drugs

[0099] Figure 24a and Figure 24b Exemplified comparison of the drug effects at different HA / lipid material mass ratios.

[0100] Figure 25 Exemplified comparison of the fluorescence values of the cell lysates in the cell endocytosis experiment.

[0101] Figure 26a Exemplified LP / R C Fluorescence microscope photos of live cell detection in the liposome cell endocytosis experiment; Figure 26b Exemplified HA@LP / R CFluorescence microscopy images of intracellular uptake experiments of the nano - carrier delivery system in living cells; the blue color represents the cell nuclei stained with Hoechst33342, and the red color represents the Cy5.5 - labeled liposomes inside the cells.

[0102] Figure 27 Illustrating HA@LP / R labeled with fluorescence under different time conditions C and LP / R C In - vivo distribution and metabolism results.

[0103] Figure 28 Illustrating the body weight changes of animals after administration of HA@LP / R C

[0104] Figure 29 Illustrating the reference standard curve for HPD determination Detailed implementation manners

[0105] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only used for more detailed and specific illustration, and should not be construed as limiting the present invention in any form.

[0106] This part describes the materials and test methods used in the experiments of the present invention. Those skilled in the art are aware that, in the context, if not otherwise specified, the materials and operation methods used in the present invention are well - known in the art.

[0107] The reagents and instruments used in the following examples are as follows:

[0108] Reagents:

[0109] DSPC, DSPE, cholesterol, DSPE - PEG2000, purchased from AVT Corporation;

[0110] Chloroform, absolute ethanol, sodium bicarbonate, EDC, NHS, calcium acetate, rosuvastatin calcium, purchased from J&K Scientific;

[0111] HA, purchased from Bloomage Biotechnology; DSPE - PEG2000 - NH2, purchased from Sigma;

[0112] Ultrafiltration membrane packages, purchased from Merck; polycarbonate membranes, purchased from Whatman;

[0113] Atorvastatin, purchased from Larexen;

[0114] Aspirin, pitavastatin, lovastatin, simvastatin, pravastatin, bezafibrate, ciprofibrate, acemetacin, ozagrel, tirofiban were purchased from J&K Scientific. 2 - hydroxypropyl - β - cyclodextrin was purchased from Sigma - Aldrich.

[0115] Instruments:​

[0116] Extrusion instrument, ATS, AE001;

[0117] Laser particle size analyzer, purchased from Malvern Instruments, model Nano-ZS.

[0118] HPLC, purchased from Waters Corporation, model e2695.

[0119] Example 1: Construction of a Targeted Plaque Hyaluronic Acid Target (HPD) Synthesis

[0120] In this example, sodium hyaluronate (HA) with an average molecular weight of 12,000 - 16,000 and DSPE-PEG2000-NH2 (DPN) were used as raw materials. Under the action of EDC and NHSS, the two were condensed through an amide bond, and after crystallization and ethanol washing, the finished HPD targeting material targeting CD44 was obtained.

[0121] The specific method was to weigh 75 g of HA and dissolve it by stirring with purified water or water for injection. DPN or a DMF solution of DPN was added, and the feeding mass ratio of DPN to HA was controlled at 1:1 or greater than 1:1. EDC or EDC and NHSS were added, and the mixture was stirred evenly and reacted at 25 - 40 °C for ≥ 10 hours. After the reaction, an equal volume or more than double the volume of absolute ethanol was added to the reaction solution, and the mixture was stirred evenly and then allowed to stand for more than 20 minutes. The precipitate was collected by centrifugation of the standing solution or after concentration by membrane package and then centrifugation. The precipitate was resuspended with ethanol, washed, and centrifuged, and the washing was repeated more than 2 times. The centrifuged precipitate obtained after washing was vacuum dried or freeze-dried, and the water content in the final product was controlled to be less than 10%.

[0122] From Figure 1 it is possible to see the infrared spectra of HA and HPD.

[0123] Example 2: Preparation of Rosuvastatin-Loaded Nano-Liposomes by Ethanol Injection Method

[0124] In this example, the nano-liposomes loaded with rosuvastatin were prepared by the ethanol injection & active encapsulation method.

[0125] Weigh DSPC, cholesterol, and DSPE-PEG2000 (mass ratio is 3:1:1 or 3:1:0.5). Dissolve the above lipid materials in ethanol. Add an aqueous calcium acetate solution (greater than or equal to 150 mM) and inject the ethanol phase into the aqueous calcium acetate solution under the condition of heat preservation and stirring at 40 - 60 °C to fully hydrate the lipid material solution, forming a crude liposome suspension (the volume ratio of the aqueous calcium acetate phase to the ethanol phase solution is greater than 2). Use an extruder to extrude through a polycarbonate membrane with a pore size of 600 nm, 400 nm, 200 nm, and 100 nm or a multi-layer composite membrane thereof more than 3 times. Dilute the extruded nano-liposome solution with purified water by 20 times or more to obtain three different sizes of nanoparticles with a particle size of approximately 90 nm, 120 nm, and 150 nm (LP / R A90 , LP / R A120 , LP / R A150 ), and the PDI is less than 0.2. Use a dialysis bag or ultrafiltration membrane package to dialyze and separate the unencapsulated calcium acetate, collect the inner solution, mix it with rosuvastatin calcium solution or a mixed solution of rosuvastatin calcium and sucrose (the mass ratio of rosuvastatin calcium to the total lipid material is less than 0.25), and incubate at 4 - 66 °C for 15 minutes or more to obtain nano-liposomes with rosuvastatin calcium actively encapsulated. The encapsulation efficiency of the prepared nano-liposomes for API is greater than 80%, and the concentration of API in the final preparation is less than or equal to 20 mg / mL.

[0126] From Figure 2 , it can be seen the cryo-electron microscopy (A, B, C) and hydrated particle size results (D, E, F) of the nano-liposomes LP / R A90 , LP / R A120 and LP / R A150 with rosuvastatin actively encapsulated by the ethanol injection method in this example.

[0127] Example 3: Preparation of Rosuvastatin-Loaded Nano-Liposomes by Mixed Hydration Method

[0128] In this embodiment, nano-liposomes of rosuvastatin are prepared by the method of hybrid hydration & active encapsulation. Weigh DSPC, cholesterol, and DSPE-PEG2000 (mass ratio is 3:1:1 - 0.5), and dissolve the above lipid materials in ethanol. Add an aqueous calcium acetate solution (150 mM or greater than 150 mM), and under the condition of keeping warm at 40 - 60 °C, use a three-way tube, microfluidics or other mixers to mix the ethanol phase and the aqueous phase in a constant ratio, so that the lipid materials are fully hydrated to form a crude liposome suspension (the volume ratio of the aqueous calcium acetate phase to the ethanol phase solution is greater than 2). Use an extruder to extrude through a polycarbonate membrane with pore sizes of 600 nm, 400 nm, 200 nm, and 100 nm or a multi-layer composite membrane thereof more than 3 times. The extruded liposome solution is diluted 20 times or more with purified water, and by controlling the membrane pore size and the number of extrusion times, three different-sized nanoparticles (LP / R B90 , LP / R B120 , LP / R B150 ) with particle sizes of approximately 90 nm, 120 nm, and 150 nm are finally obtained, and the PDI is less than 0.2. Use a dialysis bag or ultrafiltration membrane to dialyze and separate the unencapsulated calcium acetate, collect the inner liquid, mix it with a rosuvastatin calcium solution or a mixed solution of rosuvastatin calcium and sucrose (the mass ratio of rosuvastatin calcium to the total lipid materials is less than 0.25), and incubate at 4 - 66 °C for 15 minutes or more to obtain nano-liposomes with rosuvastatin calcium actively encapsulated. The encapsulation efficiency of the prepared nano-liposomes for the API is greater than 80%, and the concentration of the API in the final preparation is less than or equal to 20 mg / mL.

[0129] It can be seen from Figure 3 that in this embodiment, the cryo-electron microscopy (A, B, C) and hydrated particle size results (D, E, F) of the nano-liposomes LP / R B90 , LP / R B120 and LP / R B150 prepared by hybrid hydration and active encapsulation of rosuvastatin.

[0130] Example 4: Preparation of a Targeted Rosuvastatin-Loaded Nano-Liposome Preparation

[0131] As the targeting material HPD prepared in the above-mentioned Example 1 is inserted into the 150-nm drug-loaded nano-liposomes LP / R A150 prepared in Example 2, targeting drug-loaded nano-liposomes are obtained. The specific insertion method is as follows: Mix the nano-liposome solution loaded with rosuvastatin calcium prepared in Example 2 and the pre-dissolved aqueous HPD solution, and incubate at 25 °C or greater than 25 °C for more than 5 minutes. After the incubation, use a dialysis bag or ultrafiltration membrane to dialyze and separate the uninserted HPD, and obtain a high-concentration targeting ligand liposome (HA H -LP / R A150) Medium concentration targeted ligand liposome 100 ± 24 μg / mL (HA M -LP / R A150 ) Low concentration targeted drug-loaded liposome 10 ± 5 μg / mL (HA L -LP / R A150 ).

[0132] From Figure 4 it can be seen that the actively encapsulated rosuvastatin nano-liposome HA H -LP / R A150 、HA M -LP / R A150 and HA L -LP / R A150 in this example, as well as the cryo-electron microscopy (A, B, C) and hydrated particle size results (D, E, F).

[0133] The kit uses competitive enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with hyaluronic acid derivative (HA), specimens, standards, HA-binding protein (HABP) and anti-HABP antibody are added in sequence. After incubation and thorough washing to remove unbound components, HRP-labeled anti-mouse immunoglobulin G (enzyme-labeled secondary antibody) is added. After incubation and thorough washing to remove unbound components, an immune complex of solid-phase antigen-HA-binding protein-HABP antibody-enzyme-labeled antibody is formed on the solid surface of the microwell plate. Substrates A and B are added. Under the catalysis of HRP, the substrates produce a blue product, which is finally converted to yellow under the action of the stop solution (2M sulfuric acid). The intensity of the color is negatively correlated with the hyaluronic acid (HA) in the sample. The absorbance (OD value) is measured at a wavelength of 450 nm using an enzyme-labeled instrument, and by fitting the standard curve, the concentration of hyaluronic acid (HA) in the sample can be calculated.

[0134] Preparation of the targeted rosuvastatin actively encapsulated liposome nanocarrier delivery system (HA@LP / R C )

[0135] In this example, the liposome HA@LP / R loaded with a therapeutic agent is prepared by thin film dispersion method, active drug loading and chemical coupling process C .

[0136] Weigh distearoyl phosphatidylcholine (DSPC), DPN, cholesterol, and polyethylene glycol - distearoyl phosphatidylethanolamine (DSPE - PEG2000) (mass ratio is 3:0.5:1:1). Dissolve the above lipid materials in chloroform. Remove the organic solvent by slow rotary evaporation (55 °C water bath, 90 r / min, 30 min) to form a lipid film on the container wall. Add an aqueous calcium acetate solution (200 mM) and fully hydrate the lipid film in a constant temperature water bath at 45 °C to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, and then mix it with 25 mL of rosuvastatin calcium (133 mg), and incubate at 65 °C for half an hour to obtain the actively encapsulated drug liposome LP / R with a particle size of 160 nm C . Figure 5 shows the particle size distribution diagram of HA@LP / R in Example 5 of the present invention C .

[0137] Mix the prepared rosuvastatin calcium nano - liposome solution and the pre - dissolved HA aqueous solution, add the coupling agents EDC and NHSS, and react for more than 2 hours under the insulation condition at 25 °C or above 25 °C. After the incubation ends, use a dialysis bag or an ultrafiltration membrane package to dialyze and separate the uncoupled HA to obtain the targeted drug - loaded liposome HA@LP / R with an HA concentration of 10 - 200 μg / mL in the final product C .

[0138] Example 6: Preparation of a Targeted Atorvastatin-Loaded Liposome Nanocarrier

[0139] In this example, the ethanol injection - active encapsulation method and the insertion method are used to prepare the liposome HA - LP / A loaded with a therapeutic agent C .

[0140] Weigh DSPC, cholesterol, and DSPE - PEG2000 (mass ratio is 3:1:1), and dissolve them in ethanol. Add an aqueous calcium acetate solution (250 mM) and fully hydrate the lipid material solution in a constant temperature water bath at 65 °C to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively to control the final particle size to be 130 nm. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, take 1 mL and mix it with 2 mL of atorvastatin calcium (2.5 mg), and incubate at 60 °C for 10 min to obtain the actively encapsulated drug liposome LP / A C .

[0141] The prepared nano-liposome solution loaded with atorvastatin calcium is mixed with the pre-dissolved aqueous HPD solution, incubated for more than 5 minutes under the condition of keeping warm at 25 °C or above 25 °C, and after the incubation, the uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package to obtain a targeted drug-loaded liposome with an HPD concentration of 10 - 200 μg / mL in the final product.

[0142] From Figure 6 it can be seen that the targeted atorvastatin actively encapsulated liposome HA-LP / A in this example C by cryo-electron microscopy and DLS.

[0143] Example 7: Preparation of a Targeted Aspirin-Loaded Liposome Nanocarrier

[0144] In this example, HA-LP / Asp was prepared by the ethanol injection-active encapsulation method and the post-insertion method C . The aqueous aspirin solution (2 mg / mL) was adjusted to dissolve by NaOH. DSPC, cholesterol, and DSPE-PEG2000 (mass ratio 3:1:1) were weighed and dissolved in ethanol. An aqueous calcium acetate solution (250 mM) was added and the lipid solution was fully hydrated in a constant temperature water bath at 65 °C to form a crude liposome suspension. It was extruded through polycarbonate membranes with pore sizes of 200 nm and 100 nm using an extruder. The unencapsulated calcium acetate was separated using an ultrafiltration membrane package, the inner solution was collected, 2 mL of it was mixed with 1 mL of the aqueous aspirin solution, and then incubated at 40 °C for 10 min to obtain the actively encapsulated drug liposome LP / Asp C .

[0145] The prepared nano-liposome solution loaded with aspirin is mixed with the pre-dissolved aqueous HPD solution, incubated for more than 5 minutes under the condition of keeping warm at 25 °C or above 25 °C, and after the incubation, the uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package to obtain a targeted drug-loaded liposome with an HPD concentration of 10 - 200 μg / mL in the final product.

[0146] Figure 7 shows the targeted aspirin actively encapsulated drug liposome HA-LP / Asp in this example C by cryo-electron microscopy and DLS.

[0147] Example 8: Preparation of a Targeted Pitavastatin-Loaded Liposome Nanocarrier

[0148] In this example, HA-LP / P was prepared by the ethanol injection-active encapsulation method and the post-insertion method C .

[0149] Pitavastatin aqueous solution (1 mg / mL). Weigh DSPC, cholesterol, and DSPE-PEG2000 (mass ratio 3:1:1), dissolve in ethanol. Add calcium acetate aqueous solution (250 mM), and fully hydrate the lipid material solution in a 65°C constant temperature water bath to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, take 2 mL and mix it with 1 mL of pitavastatin aqueous solution, and incubate at room temperature for 10 min to obtain actively encapsulated drug liposomes LP / P C .

[0150] The prepared pitavastatin-loaded nano-liposome solution is mixed with the pre-dissolved HPD aqueous solution, incubated for more than 5 minutes under the condition of keeping warm at 25°C or above 25°C. After the incubation, use a dialysis bag or an ultrafiltration membrane package to dialyze and separate the uninserted HPD, and obtain the targeted drug-loaded liposomes (HA-LP / P) with an HPD concentration of 10 - 200 μg / mL in the final product C ).

[0151] Figure 8 Shows the targeted pitavastatin actively encapsulated liposomes HA-LP / P in this example C Cryo-electron microscopy and DLS

[0152] Example 9: Preparation of a Targeted Lovastatin-Loaded Liposome Nanocarrier

[0153] In this example, HA-LP / L was prepared by ethanol injection - active encapsulation and post-insertion method C . The lovastatin aqueous solution (Lova, 1 mg / mL) was dissolved by adjusting the pH with NaOH. Weigh DSPC, cholesterol, and DSPE-PEG (mass ratio 3:1:1), dissolve in ethanol. Add calcium acetate aqueous solution (250 mM), and fully hydrate the lipid material solution in a 65°C constant temperature water bath to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively for 10 times. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, take 2 mL and mix it with 1 mL of lovastatin aqueous solution, and incubate at 65°C for 10 min to obtain actively encapsulated drug liposomes LP / L C .

[0154] The prepared lovastatin-loaded nano-liposome solution is mixed with the pre-dissolved HPD aqueous solution, incubated for more than 5 minutes under the condition of keeping warm at 25°C or above 25°C. After the incubation, use a dialysis bag or an ultrafiltration membrane package to dialyze and separate the uninserted HPD, and obtain the targeted drug-loaded liposomes (HA-LP / L) with an HPD concentration of 10 - 200 μg / mL in the final product C ).

[0155] Figure 9 shows the targeted lovastatin actively encapsulated liposome HA-LP / L in this example C Cryo-electron microscopy and DLS.

[0156] Example 10: Preparation of a Targeted Simvastatin-Loaded Liposome Nanocarrier

[0157] In this example, HA-LP / S was prepared by ethanol injection, active encapsulation method, and post-insertion method C . Simvastatin aqueous solution (Simv, 1 mg / mL) was adjusted to pH with NaOH and 10% 2-hydroxypropyl-β-cyclodextrin was added to dissolve it. DSPC, cholesterol, and polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio 3:1:1) were weighed and dissolved in ethanol. An aqueous calcium acetate solution (250 mM) was added and the lipid material solution was fully hydrated in a constant temperature water bath at 65 °C to form a crude liposome suspension. The suspension was extruded 10 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm using an extruder. The unencapsulated calcium acetate was separated using an ultrafiltration membrane package, the inner solution was collected, 2 mL of it was mixed with 1 mL of simvastatin aqueous solution, and incubated at room temperature for 30 min to obtain the actively encapsulated drug liposome LP / S C .

[0158] The prepared simvastatin-loaded nanoliposome solution was mixed with the pre-dissolved HPD aqueous solution, incubated for more than 5 minutes under a warming condition at 25 °C or above 25 °C, and after incubation, dialysis was performed using a dialysis bag or an ultrafiltration membrane package to separate the uninserted HPD, obtaining the targeted drug-loaded liposome (HA-LP / S) with an HPD concentration of 10 - 200 μg / mL in the final product C ).

[0159] Figure 10 shows the targeted simvastatin actively encapsulated liposome HA-LP / S in this example C Cryo-electron microscopy and DLS.

[0160] Example 11: Preparation of a Targeted Pravastatin-Loaded Liposome Nanocarrier

[0161] In this example, HA-LP / P was prepared by ethanol injection-active encapsulation method and post-insertion method C。Pravastatin aqueous solution (Pravas, 1 mg / mL). Weigh DSPC, cholesterol, polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio 3:1:1), dissolve in ethanol. Add calcium acetate aqueous solution (250 mM) and fully hydrate the lipid material solution in a constant temperature water bath at 65 °C to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes of 200 nm and 100 nm 10 times respectively. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, take 2 mL and mix it with 1 mL of pravastatin aqueous solution, and incubate at 40 °C for 30 min to obtain actively encapsulated drug liposomes LP / P C 。

[0162] Mix the prepared pravastatin-loaded nanoliposome solution with the pre-dissolved HPD aqueous solution, incubate for more than 5 minutes under the condition of keeping warm at 25 °C or higher than 25 °C. After the incubation, use a dialysis bag or an ultrafiltration membrane package to dialyze and separate the uninserted HPD to obtain targeted drug-loaded liposomes (HA-LP / P) with an HPD concentration of 10 - 200 μg / mL in the final product C )。

[0163] Figure 11 Shows the targeted pravastatin actively encapsulated liposome HA-LP / P in this example C Cryo-electron microscopy and DLS.

[0164] Example 12: Preparation of a Targeted Bezafibrate-Loaded Liposome Nanocarrier

[0165] In this example, HA-LP / B was prepared by ethanol injection - active encapsulation and post-insertion method C 。Bezafibrate aqueous solution (Beza, 1 mg / mL) was adjusted to the appropriate pH with NaOH and 10% 2-hydroxypropyl-β-cyclodextrin was added to dissolve it. Weigh DSPC, cholesterol, polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio 3:1:1), dissolve the lipid material in ethanol. Add calcium acetate aqueous solution (250 mM) and fully hydrate the lipid material solution in a constant temperature water bath at 65 °C to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes of 200 nm and 100 nm 10 times respectively. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, take 2 mL and mix it with 1 mL of bezafibrate aqueous solution, and incubate at 40 °C for 30 min to obtain actively encapsulated drug liposomes LP / B C130 。

[0166] The prepared bezafibrate nanoliposome solution is mixed with the pre-dissolved HPD aqueous solution and incubated for more than 5 minutes under the condition of keeping warm at 25 °C or higher than 25 °C. After the incubation, the uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package to obtain the targeted drug-loaded liposome (HA-LP / B with an HPD concentration of 10 - 200 μg / mL in the final product C ).

[0167] Figure 12 Shows the targeted bezafibrate actively encapsulated liposome HA-LP / B in this example C Cryo-electron microscopy and DLS.

[0168] Example 13: Preparation of a Targeted Ciprofibrate-Loaded Liposome Nanocarrier

[0169] In this example, HA-LP / C was prepared by the ethanol injection, active encapsulation, and post-insertion method C . The ciprofibrate aqueous solution (Cipro, 1.5 mg / mL) was adjusted to pH 6.0 with NaOH for dissolution. Weighed amounts of distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG2000) (mass ratio 3:1:1) lipid materials were dissolved in ethanol. An aqueous calcium acetate solution (250 mM) was added to fully hydrate the lipid material solution in a constant temperature water bath at 65 °C to form a crude liposome suspension. The suspension was extruded 10 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm using an extruder. The unencapsulated calcium acetate was separated using an ultrafiltration membrane package, and the inner solution was collected. 2 mL of the inner solution was mixed with 1 mL of the ciprofibrate aqueous solution and incubated at room temperature for 30 min to obtain the actively encapsulated drug liposome LP / C C .

[0170] The prepared ciprofibrate nanoliposome solution is mixed with the pre-dissolved HPD aqueous solution and incubated for more than 5 minutes under the condition of keeping warm at 25 °C or higher than 25 °C. After the incubation, the uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package to obtain the targeted drug-loaded liposome (HA-LP / C with an HPD concentration of 10 - 200 μg / mL in the final product C ).

[0171] Figure 13 Shows the targeted ciprofibrate actively encapsulated liposome HA-LP / C in this example C Cryo-electron microscopy and DLS.

[0172] Example 14: Preparation of a Targeted Acemetacin-Loaded Liposome Nanocarrier

[0173] In this example, HA-LP / Am was prepared by the ethanol injection, active encapsulation, and post-insertion method CThe aqueous solution of acemetacin (Acemet, 1 mg / mL) was added with 10% 2-hydroxypropyl-β-cyclodextrin to dissolve it. Weighed distearoyl phosphatidylcholine (DSPC), cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) (mass ratio 3:1:1), and the lipid materials were dissolved in ethanol. An aqueous solution of calcium acetate (250 mM) was added, and the lipid material solution was fully hydrated in a constant temperature water bath at 65 °C to form a crude liposome suspension. The suspension was extruded 10 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm using an extruder. The unencapsulated calcium acetate was separated using an ultrafiltration membrane package, and the inner solution was collected. 2 mL of the inner solution was mixed with 1 mL of the aqueous solution of acemetacin and incubated at room temperature for 30 min to obtain the actively encapsulated drug liposome LP / Am C 。

[0174] The prepared acemetacin nano-liposome solution was mixed with the pre-dissolved aqueous solution of HPD and incubated for more than 5 minutes under the condition of keeping warm at 25 °C or higher than 25 °C. After incubation, dialysis was used with a dialysis bag or an ultrafiltration membrane package to separate the uninserted HPD, and the targeted drug-loaded liposome (HA-LP / Am) with an HPD concentration of 10 - 200 μg / mL in the final product was obtained C )。

[0175] Figure 14 showed the targeted acemetacin actively encapsulated liposome HA-LP / Am in this example C Cryo-electron microscopy and DLS.

[0176] Example 15: Preparation of a Targeted Ozagrel-Loaded Liposome Nanocarrier

[0177] In this example, LP / O was prepared by ethanol injection, active encapsulation, and post-insertion method C 。The aqueous solution of ozagrel (Ozagrel, 2 mg / mL) was added with 10% 2-hydroxypropyl-β-cyclodextrin to dissolve it. Weighed distearoyl phosphatidylcholine (DSPC), cholesterol, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) (mass ratio 3:1:1), and the lipid materials were dissolved in ethanol. An aqueous solution of calcium acetate (250 mM) was added, and the lipid material solution was fully hydrated in a constant temperature water bath at 65 °C to form a crude liposome suspension. The suspension was extruded 10 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm using an extruder. The unencapsulated calcium acetate was separated using an ultrafiltration membrane package, and the inner solution was collected. 2 mL of the inner solution was mixed with 1 mL of the aqueous solution of Ozagrel and incubated at room temperature for 30 min to obtain the actively encapsulated drug liposome LP / O C 。

[0178] The prepared ozagrel nano-liposome solution is mixed with the pre-dissolved HPD aqueous solution, incubated for more than 5 minutes under the condition of keeping warm at 25 °C or above 25 °C. After the incubation, the uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package, and the targeted drug-loaded liposome (HA-LP / O) with an HPD concentration of 10 - 200 μg / mL in the final product is obtained. C )

[0179] Figure 15 The targeted ozagrel actively encapsulated liposome HA-LP / O in this example is shown. C Cryo-electron microscopy and DLS.

[0180] Example 16: Preparation of a Targeted Tirofiban-Loaded Liposome Nanocarrier

[0181] In this example, LP / T was prepared by ethanol injection, active encapsulation, and post-insertion methods. C Tirofiban aqueous solution (Tirofiban, 2 mg / mL) was added with 10% 2-hydroxypropyl-β-cyclodextrin to dissolve it. Weighed distearoyl phosphatidylcholine (DSPC), cholesterol, polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio 3:1:1), and the lipid materials were dissolved in ethanol. An aqueous calcium acetate solution (250 mM) was added to fully hydrate the lipid material solution in a constant temperature water bath at 65 °C to form a crude liposome suspension. The suspension was extruded 10 times through polycarbonate membranes with pore sizes of 200 nm and 100 nm using an extruder. The unencapsulated calcium acetate was separated using an ultrafiltration membrane package, and the inner liquid was collected. 2 mL of the inner liquid was mixed with 1 mL of the Tirofiban aqueous solution and incubated at 65 °C for 30 min to obtain the actively encapsulated drug liposome LP / T. C130

[0182] The prepared tirofiban nano-liposome solution is mixed with the pre-dissolved HPD aqueous solution, incubated for more than 5 minutes under the condition of keeping warm at 25 °C or above 25 °C. After the incubation, the uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package, and the targeted drug-loaded liposome (HA-LP / T) with an HPD concentration of 10 - 200 μg / mL in the final product is obtained. C )

[0183] Figure 16 The targeted tirofiban actively encapsulated liposome HA-LP / T in this example is shown. C Cryo-electron microscopy and DLS.

[0184] Example 17 Targeted Rosuvastatin Active Encapsulation Liposome Nanocarrier Delivery System (HA@LP / R d ) Preparation Preparation

[0185] Weigh hydrogenated soy phosphatidylcholine, DPN, cholesterol, polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio is 3:0.5:1:1), and dissolve the above lipid materials with chloroform. Remove the organic solvent by slow rotary evaporation (55 °C water bath, 90 r / min, 30 min), so as to form a lipid film on the container wall. Add an aqueous solution of copper gluconate (200 mM) and fully hydrate the lipid film in a constant temperature water bath at 45 °C to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, and then mix it with 25 mL of rosuvastatin calcium (133 mg), and incubate at 65 °C for half an hour to obtain the actively encapsulated drug liposome LP / R d 。

[0186] Mix the HA aqueous solution with the coupling agents EDC and NHSS, and couple it with LP / R d Mix the lipid solution to achieve coupling, react for more than 2 hours under the insulation condition at 25 °C or above 25 °C. After completion, use a dialysis bag or an ultrafiltration membrane package to dialyze and separate the uncoupled HA, and obtain the targeted drug-loaded liposome HA@LP / R with an HA concentration of 10 - 200 μg / mL in the final product d 。

[0187] Figure 17 Shows the targeted rosuvastatin actively encapsulated liposome HA@LP / R in this example d Cryo-electron microscopy and DLS

[0188] Example 18 Targeted Atorvastatin Active Encapsulation Liposome Nanocarrier Delivery System (HA@LP / A d ) Preparation Preparation

[0189] Weigh dipalmitoyl phosphatidylcholine, DPN, cholesterol, polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio is 3:0.5:1:1), and dissolve the above lipid materials with chloroform. Remove the organic solvent by slow rotary evaporation (55 °C water bath, 90 r / min, 30 min), so as to form a lipid film on the container wall. Add an aqueous solution of calcium acetate (300 mM) and fully hydrate the lipid film in a constant temperature water bath at 45 °C to form a crude liposome suspension. Use an extruder to extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively. Use an ultrafiltration membrane package to separate the unencapsulated calcium acetate, collect the inner liquid, and then mix it with 25 mL of atorvastatin calcium, and incubate at 65 °C for half an hour to obtain the actively encapsulated drug liposome LP / A with a particle size of 130 nm d 。

[0190] Mix the HA aqueous solution with the coupling agents EDC and NHSS, and couple it with LP / A dCoupling is achieved by mixing lipids and the reaction is carried out for more than 2 hours under the condition of keeping warm at 25 °C or higher than 25 °C. After the reaction, uncoupled HA is separated by dialysis using a dialysis bag or an ultrafiltration membrane package, and the targeted drug-loaded liposome HA@LP / A with an HA concentration of 10 - 200 μg / mL in the final product is obtained. d 。

[0191] Figure 18 The targeted atorvastatin actively encapsulated liposome HA@LP / A in this example is shown. d Cryo-electron microscopy and DLS.

[0192] Example 19 Preparation of a Targeted Clofibrate Active Encapsulation Liposome Nanocarrier Delivery System (HA@LP / C d )

[0193] Weigh sphingomyelin, cholesterol, polyethylene glycol-distearoyl phosphatidylethanolamine (DSPE-PEG2000) (mass ratio 3:1:1), and dissolve the above lipid materials in chloroform. Remove the organic solvent by slow rotary evaporation (55 °C water bath, 90 r / min, 30 min), thereby forming a lipid film on the container wall. Add an aqueous calcium bicarbonate solution (250 mM) and fully hydrate the lipid film in a constant temperature water bath at 45 °C to form a crude liposome suspension. Extrude through polycarbonate membranes with pore sizes of 200 nm and 100 nm respectively using an extruder. Separate the unencapsulated calcium bicarbonate using an ultrafiltration membrane package, collect the inner liquid, and then mix it with 25 mL of ciprofibrate and incubate at 65 °C for half an hour to obtain the actively encapsulated drug liposome LP / C with a particle size of 130 nm. d 。

[0194] Mix with the HPD aqueous solution prepared in Example 1 and incubate for more than 30 minutes under the condition of keeping warm at 25 °C or higher than 25 °C. After incubation, uninserted HPD is separated by dialysis using a dialysis bag or an ultrafiltration membrane package to obtain a high-concentration targeted ligand liposome with an HPD concentration of 200 μg / mL in the final product, and a targeted drug-loaded liposome HA@LP / C with an HA concentration of 10 - 200 μg / mL in the final product. d 。

[0195] Figure 19 The targeted ciprofibrate actively encapsulated liposome HA@LP / C in this example is shown. d Cryo-electron microscopy and DLS.

[0196] Example 20 Preparation of a targeted bezafibrate actively encapsulated liposome nanocarrier delivery system (HA-LP / B d )

[0197] In this example, ethanol injection & active encapsulation method is used to prepare nanoliposomes loaded with bezafibrate.

[0198] Weigh distearoyl phosphatidylserine (DOPS), cholesterol, and DSPE-PEG2000 (mass ratio is 3:1:1). Dissolve the above lipid materials in ethanol. Add magnesium citrate aqueous solution (200 mM). Under the condition of keeping warm and stirring at 40 - 60 °C, inject the ethanol phase into the magnesium citrate aqueous solution to fully hydrate the lipid material solution, forming a crude liposome suspension (the volume ratio of the magnesium citrate aqueous phase to the ethanol phase solution is greater than 2). Use a dialysis bag or ultrafiltration membrane package to dialyze and separate the unencapsulated magnesium citrate. Collect the inner liquid, mix it with bezafibrate solution (the mass ratio of bezafibrate to the total lipid materials is less than 0.25), and incubate at 4 - 66 °C for 15 minutes or more to obtain nanoliposomes with actively encapsulated bezafibrate. The encapsulation efficiency of the prepared nanoliposomes for API is greater than 20%, and the concentration of API in the final preparation is less than or equal to 20 mg / mL.

[0199] Mix with the HPD aqueous solution prepared in Example 1, incubate at 25 °C or above the temperature of 25 °C for more than 30 minutes. After the incubation, use a dialysis bag or ultrafiltration membrane package to dialyze and separate the uninserted HPD to obtain high-concentration targeting ligand liposomes with an HPD concentration of 200 μg / mL in the final product, and targeting drug-loaded liposomes HA-LP / B with an HA concentration of 10 - 200 μg / mL in the final product d 。

[0200] Figure 20 Shows the targeted bezafibrate actively encapsulated liposomes HA-LP / B in this example d Cryo-electron microscopy and DLS.

[0201] Test Example 1: Stability and Controllability of the Nanocarrier Delivery System of the Invention

[0202] Taking the nanodelivery system loaded with therapeutic agents prepared according to the present invention as an example, the delivery system of the present invention has stable and controllable properties, thus being suitable for the treatment of atherosclerosis or diseases related to atherosclerosis.

[0203] 1. Drug concentration determination method:

[0204] The carrier drugs rosuvastatin, tirofiban, ozagrel, acemetacin, ciprofibrate, bezafibrate, pravastatin, aspirin, pitavastatin calcium, lovastatin, and simvastatin have strong ultraviolet absorption characteristics. Therefore, a standard quantitative equation can be established by using the HPLC-UV method for the concentration (X) of the drug solution and the peak area (Y) of the HPLC chromatographic peak.

[0205] The HPLC analysis method is as follows: Mobile phase: A: 1.0% trifluoroacetic acid: acetonitrile: water (1:29:70); B: 1.0% trifluoroacetic acid: water: acetonitrile (1:24:75). Column temperature: 40 °C, model: Inertsil ODS-3 (150*3.0) mm. Equilibrate the system and inject the sample after the baseline is stable.

[0206] 2. Determination of the hydrated particle size:

[0207] The particle size of the delivery system of the present invention was determined by a Malvern intelligent laser particle size analyzer. The results are shown in Table 1.

[0208] 3. Determination of HPD in the preparation:

[0209] The kit uses a competitive enzyme-linked immunosorbent assay (ELISA). To the pre-coated microwells with hyaluronic acid derivative (HA), add the specimen, standard, HA-binding protein (HABP), and anti-HABP antibody in sequence. After incubation and thorough washing to remove the unbound components, add HRP-labeled anti-mouse immunoglobulin G (enzyme-labeled secondary antibody). After incubation and thorough washing to remove the unbound components, an immune complex of solid-phase antigen - HA-binding protein - HABP antibody - enzyme-labeled antibody is formed on the solid surface of the microwell plate. Add substrates A and B. The substrates are catalyzed by HRP to produce a blue product, which is finally converted to yellow under the action of the stop solution (2M sulfuric acid). The intensity of the color is negatively correlated with the hyaluronic acid (HA) in the sample. Measure the absorbance (OD value) at a wavelength of 450 nm with an enzyme-labeled instrument, fit the standard curve of the standard, and the concentration of hyaluronic acid (HA) in the sample can be calculated.

[0210] Detection was carried out using a hyaluronic acid (HA) quantitative detection ELISA kit. Weigh an appropriate amount of HPD accurately, dissolve it in water to prepare a control stock solution of about 2.5 mg / mL, and prepare a linear working solution (0 - 1.25 mg / mL) by diluting with water in equal proportions. Take an appropriate amount of the preparation solution without HPD, mix it with each linear working solution respectively as the standard linear solution; take an appropriate amount of the preparation solution, dilute it with the preparation solution without HPD as needed, and add an appropriate amount of water to make the liposome ratio in the preparation test solution the same as that in the standard linear solution as the test sample solution; before the ELISA kit determination, the standard linear solution and the test sample solution are respectively added with 10% Triton solution for dissolution, mixed evenly, and carried out according to the operation procedure of the ELISA kit. Measure the absorbance (OD value) at a wavelength of 450 nm with an enzyme-labeled instrument, fit the standard curve with four parameters, and calculate the concentration of HPD in the test sample.

[0211] The reference standard curve graph is as Figure 29As shown, the fitting equation is: y = (A - D) / [1 + (x / C)^B] + D, where: A = 2.763; B = 1.777; C = 3.681; D = 0.146; r2 = 0.999.

[0212] 4. Determination of encapsulation efficiency:

[0213] Take a certain amount of nanocarriers, measure the amount of free drug and total drug by HPLC method, and calculate the encapsulation efficiency through the following formula 1. The results are shown in Table 1.

[0214] Encapsulation efficiency (%) = (1 - M_free drug amount / M_total drug amount) * 100% …………… Formula 1 Table 1 Process description, average particle size and encapsulation efficiency results of the nanocarrier delivery system of the present invention

[0215]

[0216]

[0217] Note: The above data are all expressed in the form of "average value + standard deviation" of the results of parallel determination 3 times.

[0218] 5. Investigation of long-term stability

[0219] Store the nanodelivery system of the present invention at 4°C, sample at different time points, and detect the change of its hydrated particle size by a laser particle size analyzer. Investigate the change of its content and encapsulation efficiency by HPLC method. The results are as Figure 21 shown.

[0220] Test Example 2: Verification of the Targeting Ability of the Targeted Liposome Nanocarrier Delivery System of the Invention

[0221] The purpose of this experimental example is to verify the targeting ability of the targeted liposome nanodrug carrier delivery system described in the present invention, such as HA@LP / R C carrier delivery system, for atherosclerotic plaques. Specifically, since atherosclerotic plaques are rich in macrophages and highly express CD44 receptors, THP1 macrophages were selected for this study and activated with lipopolysaccharide. By incubating with fluorescently labeled targeted HA@LP / R C or non-targeted LP / R C together, and then performing live cell microscopy and fluorescence value detection. By comparing the results of the HA-targeted and non-targeted liposome groups, the targeting of HA-targeted liposomes to macrophages highly expressing CD44 receptors was evaluated.

[0222] 1. Experimental reagents and instruments:

[0223] Table 2 List of experimental reagents used

[0224]

[0225] Table 3 List of Instruments Used in the Experiment

[0226] Name Manufacturer Model Fluorescence Microplate Reader Perkin Elmer EnVision2105 Intelligent Live Cell Detection System BioTek Lionheart FX Medical Low-Speed Horizontal Rotor Centrifuge Beijing Baiyang 52A

[0227] 2. Experimental Procedures

[0228] 2.1 Fluorescent Labeling of Test Substances

[0229] Accurately weigh 1.0 mg of Cy5.5 dye and dissolve it in 1 ml of absolute ethanol to prepare a 1 mg / ml Cy5.5 solution. Take 5 ml of targeted HA@LP / R C or non-targeted LP / R C liposomes, add 0.1 mL of the 1 mg / ml Cy5.5 solution, stir at 150 rpm for 12 h for fluorescent labeling. Then filter with an ultrafiltration tube to remove unreacted fluorescent molecules. Use a fluorescence microplate reader to perform fluorescence detection on the fluorescently labeled liposomes obtained after filtration. The fluorescently labeled liposomes after detection are used for subsequent intracellular uptake experiments.

[0230] 2.2 Intracellular Uptake Experiment

[0231] The operation steps refer to the cell resuscitation operation process of this platform, and the culture medium uses the conventional THP1 cell culture medium (RPMI1640 + 10% FBS) of this platform. After cell resuscitation, the resuspension density is 5×10 5 / ml, and inoculate into a T75 culture flask. Transfer 10 ml of suspended THP1 cells to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and resuspend in 1 ml of culture medium and perform cell counting to prepare a cell suspension with a concentration of 1×10 5 / 200 μl. Add PMA to the cell suspension of 1×10 5 / 200 μl to a final concentration of 100 ng / ml. After mixing evenly, inoculate the cell suspension into a 96-well plate, 200 μl / well, and continuously stimulate for 72 h. After stimulating for 72 hours, discard the original culture medium, and add fresh culture medium containing lipopolysaccharide (LPS) (the LPS solution is diluted 1000-fold in fresh culture medium, and the prepared final concentration is 2.5 μg / mL). Treat with LPS for 2 hours.

[0232] Set up two groups in the experiment and add them separately. Take 1 ml of fluorescently labeled liposomes, dilute them 10-fold by volume in fresh culture medium, and at this time the rosuvastatin concentration is 0.2 mg / ml. Discard the culture solution containing LPS in the 96-well plate, add the diluted fluorescently labeled test substance, with three replicates in each group, and continue to incubate at 37°C for 30 min.

[0233] Wash the plate once with PBS. Dilute 2 μl of Hoechst33342 stock solution in 10 ml of fresh medium to prepare a diluted Hoechst33342 solution, and incubate the diluted Hoechst33342 solution with the cells for 10 min for nuclear staining. Take pictures using the Lionheart FX intelligent live cell imaging analysis system. For the well plate after taking pictures, discard the staining solution, add 100 μl of RIPA cell lysate, and detect the fluorescence intensity in the well plate using a fluorescence microplate reader. The obtained experimental data were used to calculate the mean and standard deviation using Graph Pad Prism 8 software and perform statistical analysis of t-test. p < 0.05 was considered statistically significant.

[0234] 3. Experimental results

[0235] The intracellular uptake experiment compared the uptake efficiency of THP1 cells after LPS stimulation for HA@LP / R C and LP / R C . The experimental results are shown in Figure 25 . Compared with the non-targeted LP / R C liposome group, the fluorescence value of the HA@LP / R C group increased significantly, which was 3 times that of the non-targeted liposome group. Fluorescence microscopy observation also showed (see Figure 26) that the intracellular red fluorescence of the HA@LP / R C group was significantly increased compared with that of the non-targeted LP / R C group. It was shown that the uptake efficiency of THP1 cells after LPS stimulation for HA@LP / R C was significantly higher than that for non-targeted liposomes.

[0236] The results showed that the number of cells showing red fluorescence in the targeted group was significantly more than that in the non-targeted liposome group. And compared with the non-targeted liposome group, the fluorescence value in the targeted group also increased significantly. It can be concluded that liposomes containing HA targeting heads can be specifically taken up by activated macrophages highly expressing CD44 receptors.

[0237] Test Example 3: In Vivo Distribution and Metabolism Study of the Targeted Liposome Nanocarrier Delivery System of the Invention

[0238] The purpose of this experimental example was to preliminarily reveal the distribution and metabolism of the targeted liposome nano-drug carrier delivery system described in the present invention in animals. In this experiment, the HA@LP / R C and LP / R C liposomes from the intracellular uptake experiment in Experimental Example 2 were continued to be used for preliminary studies on the distribution and metabolism in animals.

[0239] Specifically, 6-week-old ApoE- / - mice were fed a high-fat diet for 28 weeks to establish an AS animal model with aortic plaque formation. The targeted nano-statin preparation (HA@LP / RC , the tissue distribution and metabolism after intravenous administration of the targeted nanon preparation (dose 5 mg / kg) and the non-targeted nanon preparation to the model animals. As Figure 27 shown, it can be seen that the nanon preparation is mainly enriched in the liver in vivo and cannot cross the blood-brain barrier. There is no obvious difference in the distribution and metabolism of the targeted and non-targeted drugs in vivo.

[0240] Furthermore, the experimental animals were randomly divided into the following groups, with 6 animals in each group:

[0241] Rosuvastatin oral group: Oral administration was carried out at a dose of 0.5 mg rosuvastatin / kg body weight;

[0242] Rosuvastatin intravenous injection group: Intravenous injection was carried out at a dose of 0.5 mg rosuvastatin HA@LP / R C / kg body weight;

[0243] The treatment of the treatment group was carried out once every other day for a total of 11 administrations.

[0244] The curve of the animal body weight changing with the treatment time is as Figure 28 shown. It can be Figure 28 seen that the animal body weight increased during the treatment period and no death occurred, indicating that the drug has good safety.

[0245] Test Example 4: In Vivo Experiment on the Effect of the Targeted Liposome Nanocarrier Delivery System of the Invention on Atherosclerosis Experiment

[0246] The purpose of this test example is to verify the in vivo therapeutic effect on arterial plaques of the targeted liposome nanodrug carrier delivery system described in the present invention, such as HA M -LP / R A150 carrier delivery system.

[0247] 1. Establishment of animal models and grouping of experimental animals

[0248] (1) Prepare a physiological saline solution of free rosuvastatin, and prepare a nanodelivery system loaded with a therapeutic agent by the method described in Example 2 above.

[0249] (2) Establishment of an ApoE- / - mouse atherosclerosis model:

[0250] SPF-grade ApoE- / - mice (42 mice, 5 - 6 weeks old, body weight 20 ± 1 g) were used as experimental animals. After 4 weeks of feeding the mice with an adaptive high-fat diet (10% (w / w) fat, 2% (w / w) cholesterol, 0.5% (w / w) sodium cholate, and the rest being ordinary mouse feed), they were anesthetized by intraperitoneal injection with 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 mL of physiological saline) at a dose of 40 mg / kg. Then, the mice were fixed on the operating board in the supine position, disinfected with 75% (v / v) alcohol centered on the neck, the neck skin was longitudinally incised, the anterior cervical gland was bluntly dissected, and the pulsating left common carotid artery could be seen on the left side of the trachea. The common carotid artery was carefully dissected to the bifurcation, and a silicone tube with a length of 2.5 mm and an inner diameter of 0.3 mm was sleeved around the left common carotid artery, and both the proximal and distal segments of the cannula were constricted and fixed with fine silk threads. Local constriction caused turbulent blood flow in the proximal end, increased shear stress, and caused vascular intimal injury. The carotid artery was reset, and the anterior cervical skin was intermittently sutured. All operations were performed under a 10-fold stereomicroscope. After the mice woke up postoperatively, they were returned to the cage, and the environmental temperature was maintained at 20 - 25 °C, with the light on and off for 12 h each. Starting from the 4th week after surgery, lipopolysaccharide (LPS) (1 mg / kg, in 0.2 mL of phosphate-buffered saline, Sigma, USA) was intraperitoneally injected twice a week for 10 weeks to induce chronic inflammation. At 8 weeks after surgery, the mice were placed in a 50 mL syringe (with sufficient ventilation holes reserved) to cause restrictive mental stress, 6 hours / day, 5 days / week, for a total of 6 weeks. The atherosclerotic mouse model was completed 14 weeks after surgery.

[0251] (3) Grouping and treatment of experimental animals:

[0252] The experimental animals were randomly divided into the following groups, with 6 mice in each group:

[0253] Atherosclerosis model control group: The animals in this group were not given any therapeutic treatment;

[0254] Rosuvastatin oral group: Oral administration was carried out at a dose of 0.66 mg of rosuvastatin / kg body weight;

[0255] Rosuvastatin intravenous injection group: Intravenous injection was carried out at a dose of 0.66 mg of rosuvastatin / kg body weight;

[0256] Nanopreparation administration (HA M -LP / R A150 ) group: Intravenous injection was carried out at a dose of 0.66 mg of rosuvastatin / kg body weight;

[0257] Except for the atherosclerotic model control group, the treatment in the treatment group was carried out once every other day for a total of 4 weeks of drug administration. For each group of animals, carotid artery MRI scans were performed weekly before and after treatment to detect plaque and lumen area, and the percentage of plaque volume progression was calculated by combining the scan thickness.

[0258] Percentage of plaque volume progression = (Plaque volume after treatment - Plaque volume before treatment) / Plaque volume before treatment.

[0259] Figure 22 and 23 shows the in vivo therapeutic effect of the HA M -LP / R A150 vector delivery system on atherosclerosis. As Figure 22 shown, oral and intravenous injection of free statin can only delay plaque progression, but cannot regress existing plaques. Targeted nano-drug therapy not only significantly curbed plaque progression, but also showed a significant 30.3% reversal and regression of plaque volume. As Figure 23 shown, compared with oral and injection of statin drugs, the nano-delivery system showed its extremely significant aortic enrichment function. Compared with the non-targeted delivery system (LP / R A150 ), this enrichment effect was more obvious under the action of the targeted nano-preparation (HA M -LP / R A150 ). In summary, for atherosclerosis in mice in vivo, oral and intravenous administration of free rosuvastatin did not show the effect of reversing plaques. However, when statin was loaded in the nano-delivery system described in the present invention, its therapeutic effect on atherosclerosis was significantly improved, and it played a therapeutic effect of reducing plaque, and the nano-system with functional modification had a better effect.

[0260] 2. Pharmacodynamic comparison of different HA / lipid material mass ratios

[0261] 6-week-old ApoE- / - mice were fed a high-fat diet for 28 weeks to establish an AS animal model with aortic plaque formation. The successfully modeled animals were randomly divided into a plaque model control group, a non-targeted nano-statin preparation group (LP / R A150 , 5 mg / kg), a targeted nano-statin preparation-1 (HA L -LP / R A150 , dose 5 mg / kg), a targeted nano-statin preparation-2 (HA M -LP / R A150 , dose 5 mg / kg, iv), a targeted nano-statin preparation-3 (HA H -LP / R A150, at a dose of 5 mg / kg, iv), with 10 animals in each group. The treatment was administered once a day for a total of 28 days, with a treatment course of 4 weeks. After the treatment course, en-face gross pathology of aortic plaques was performed. The targeted preparation could achieve the reversal of aortic plaques in AS model mice in a short time, and in HA M -LP / R A150 and HA L -LP / R A150 The preparation group showed a significant plaque reversal.

[0262] The above research shows that when developing a ligand-conjugated active targeting nanodelivery system, attention needs to be paid to the conjugation density of the ligand on the surface of the nanocarrier. As a ligand molecule with special physiological functions, neither low-density nor excessive conjugation can achieve a good targeting effect, suggesting that we especially need to achieve a controllable conjugation process when developing such products.

[0263] Figure 24a and Figure 24b showed a comparison of the drug effects at different HA / lipid material mass ratios.

Claims

1. An active encapsulation liposome nanocarrier delivery system, characterized in that, The liposome nanocarrier delivery system includes a liposome carrier and a substance actively encapsulated therein for preventing and / or treating atherosclerosis or diseases related to atherosclerosis; Wherein, The diseases related to atherosclerosis are selected from one or more of the following: coronary atherosclerotic heart disease, cerebral atherosclerosis, and peripheral vascular atherosclerosis; The substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is selected from one or more of the following: statins, fibrates, antiplatelet drugs, anticoagulants, angiotensin-converting enzyme inhibitors, calcium antagonists, β-blockers, and glucocorticoids, and pharmaceutically acceptable salts thereof, as well as active preparations of the substances; The material of the liposome carrier comprises phospholipids, cholesterol, and DSPE-PEG2000; The liposome nanocarrier delivery system further includes a targeting ligand, which is modified by co-incubation with the liposome carrier, and the targeting ligand is a compound formed by condensation of sodium hyaluronate and DSPE-PEG2000-NH2 through an amide bond; In the liposome nanocarrier delivery system, the final concentration of the targeting ligand is 10 - 100 μg / mL.

2. The liposome nanocarrier delivery system according to claim 1, wherein The phospholipids are selected from one or more of the following: hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine, distearoyl phosphatidylethanolamine, polyethylene glycol-distearoyl phosphatidylethanolamine, distearoyl phosphatidylglycerol, distearoyl phosphatidylserine, dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylserine, dioleoyl phosphatidylethanolamine-polyethylene glycol, dicitraoyl phosphatidylcholine, dicitraoyl phosphatidylethanolamine, dicitraoyl phosphatidylserine, dicitraoyl phosphatidylethanolamine-polyethylene glycol, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, dierucoyl phosphatidylcholine, dierucoyl phosphatidylethanolamine, dierucoyl phosphatidylserine, dierucoyl phosphatidylethanolamine-polyethylene glycol, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol.

3. The liposome nanocarrier delivery system according to claim 1, wherein The mass ratio of the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis to the liposome carrier is 1:0.1 - 50.

4. The liposome nanocarrier delivery system according to claim 3, characterized in that, The mass ratio of the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis to the liposome carrier is 1:0.2 - 30.

5. The liposome nanocarrier delivery system according to claim 4, characterized in that, The mass ratio of the substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis to the liposome carrier is 1:0.5 - 20.

6. The liposome nanocarrier delivery system according to any one of claims 1 to 5, characterized in that, The average molecular weight of the sodium hyaluronate is 12,000 - 16,000.

7. The liposome nanocarrier delivery system according to claim 6, characterized in that, The substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is selected from one or more of the following: lovastatin, atorvastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, pravastatin, bezafibrate, ciprofibrate, gemfibrozil, aspirin, acemetacin, ozagrel sodium, beraprost sodium, and tirofiban, as well as their pharmacodynamic fragments or pharmaceutically acceptable salts.

8. The liposome nanocarrier delivery system according to claim 6, wherein The substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is a water-soluble statin.

9. The liposome nanocarrier delivery system according to claim 6, wherein, The substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is rosuvastatin, pravastatin, or atorvastatin.

10. The liposome nanocarrier delivery system according to any one of claims 1 to 5, characterized in that, The substance for preventing and / or treating atherosclerosis or diseases related to atherosclerosis is rosuvastatin, and the concentration of the targeting ligand is 100 ± 24 μg / mL.

11. The liposome nanocarrier delivery system according to any one of claims 1 to 5, characterized in that, The liposome carrier is selected from small unilamellar liposomes, large unilamellar liposomes, or multilamellar liposomes.

12. A method for preparing the liposome nanocarrier delivery system according to any one of claims 1 to 11, comprising the steps of hydrating the liposome carrier by adding a salt solution and separating to create an acidity gradient between the internal and external solutions of the liposome.

13. The method for preparing a liposome nanocarrier delivery system according to claim 12, characterized in that, The salt is a salt of a weak acid and a strong base.

14. The method for preparing a liposome nanocarrier delivery system according to claim 12, characterized in that, The anionic part of the salt is selected from one or more of the following: acetate, edetate, bicarbonate, hypochlorite, citrate, benzoate, and gluconate; and / or The cationic part of the salt is selected from one or more of the following: calcium ion, copper ion, nickel ion, barium ion, magnesium ion, and zinc ion.

15. The method for preparing a liposome nanocarrier delivery system according to claim 12, wherein The salt is selected from one or more of the following: calcium acetate, calcium bicarbonate, magnesium citrate, and copper gluconate.

16. The preparation method of the liposome nanocarrier delivery system according to claim 12, characterized in that, The method comprises the following steps: (1) Dissolving the liposome carrier material in a good solvent; (2) Adding a salt solution that creates an acidity gradient between the internal and external solutions of the liposome to hydrate the product obtained in step (1) and dispersing it into a crude lipid solution; (3) Reducing the particle size of the crude lipid solution obtained in step (2) to obtain refined liposomes; (4) Removing external ions from the refined liposome solution obtained in step (3) by purification to form an acidity gradient between the inside and outside of the liposome; (5) Adding the solution of the substance to be coated to the liposome solution obtained in step (4) and incubating to allow the drug to enter the interior of the liposome to obtain the liposome nanocarrier delivery system.

17. The method according to claim 16, characterized in that, Step (1) further includes a step of evaporating the solvent to form a lipid film of the liposome.

18. The method according to claim 17, characterized in that, The evaporation temperature is 40 - 80°C.

19. The method according to claim 17, wherein The evaporation temperature is 50 - 70°C.

20. The method according to claim 19, characterized in that, The evaporation temperature is 50 - 60°C.

21. The method according to any one of claims 17 to 20, characterized in that, The evaporation method is rotary evaporation.

22. The method according to claim 21, characterized in that, The rotary evaporation speed is 50 - 200 r / min.

23. The method according to claim 22, characterized in that, The rotary evaporation speed is 70 - 120 r / min.

24. The method according to claim 22, wherein The rotary evaporation speed is 80 - 100 r / min.

25. The method according to claim 16 or 17, characterized in that In step (1), the good solvent is selected from one or more of the following: chloroform, ethanol, methanol, dichloromethane, acetone, toluene, ether, ethyl acetate, methyl acetate, acetonitrile, and dichloroethane.

26. The method according to claim 16 or 17, characterized in that, In step (1), in the liposome carrier material, the phospholipid concentration is 0.1 - 500 mg / mL; and / or The cholesterol concentration is 0.05 to 200 mg / mL.

27. The method according to claim 26, wherein In step (1), in the liposome carrier material, the phospholipid concentration is 0.1 to 200 mg / mL; and / or The cholesterol concentration is 0.1 to 100 mg / mL.

28. The method according to claim 26, wherein In step (1), in the liposome carrier material, the phospholipid concentration is 0.2 - 100 mg / mL; and / or The cholesterol concentration is 0.1 - 50 mg / mL.

29. The method according to claim 16 or 17, characterized in that, In step (2), the concentration of the salt solution is 100 to 500 mM; and / or The hydration temperature is 30 to 90 °C.

30. The method according to claim 29, wherein In step (2), in the liposome carrier material, the concentration of the salt solution is 200 to 300 mM; and / or The hydration temperature is 40 to 80 °C.

31. The method according to claim 29, characterized in that, In step (2), in the liposome carrier material, the concentration of the salt solution is 200 to 250 mM; and / or The hydration temperature is 45 to 65 °C.

32. The method according to claim 16 or 17, characterized in that, In step (3), the method for reducing the particle size is to extrude the crude lipid solution through a filter membrane.

33. The method according to claim 32, wherein The filter membrane is a polycarbonate membrane.

34. The method according to claim 32, wherein The pore size of the filter membrane is 30 to 800 nm.

35. The method according to claim 34, wherein The pore size of the filter membrane is 50 to 400 nm.

36. The method according to claim 34, wherein The pore size of the filter membrane is 100 to 200 nm.

37. The method according to claim 34, characterized in that, The pore size of the filter membrane is 30 nm, 50 nm, 100 nm, 200 nm, 400 nm, 600 nm or 800 nm.

38. The method according to claim 34, wherein The pore size of the filter membrane is 100 - 150 nm.

39. The method according to claim 16 or 17, characterized in that, In step (4), the method for removing external ions is selected from one or more of the following: ultrafiltration membrane package separation, dialysis separation and ion exchange.

40. The method according to claim 16 or 17, characterized in that, In step (5), the concentration of the coating substance solution is 0.01 to 100 mg / mL.

41. The method according to claim 40, wherein In step (5), the concentration of the coating substance solution is 0.05 to 50 mg / mL.

42. The method according to claim 40, characterized in that, In step (5), the concentration of the coating substance solution is 0.1 to 20 mg / mL.

43. The method according to claim 40, wherein In step (5), the concentration of the coating substance solution is 0.1 - 10 mg / mL.

44. The method according to claim 16 or 17, characterized in that, In step (5), the incubation temperature is 20 to 80 °C.

45. The method according to claim 44, wherein In step (5), the incubation temperature is 40 to 70 °C.

46. The method according to claim 44, wherein, In step (5), the incubation temperature is 55 to 65 °C.

47. The method according to claim 16 or 17, characterized in that, When the liposome nanocarrier delivery system is modified by a targeting ligand, the method further includes the following steps: (6) Co-incubating the targeting ligand with the liposome nanocarrier delivery system obtained in step (5) to obtain the modified liposome nanocarrier delivery system.

48. A drug, characterized in that, The drug comprises the nanocarrier delivery system according to any one of claims 1 to 11 or the nanocarrier delivery system prepared by the preparation method according to any one of claims 12 to 47, and a pharmaceutically acceptable carrier.

49. Use of the nanocarrier delivery system according to any one of claims 1 to 11, the nanocarrier delivery system prepared by the preparation method according to any one of claims 12 to 47, and the drug according to claim 48 in the preparation of a drug for preventing and / or treating atherosclerosis or a disease related to atherosclerosis; Among them, The atherosclerosis-related disease is selected from one or more of the following: coronary atherosclerotic heart disease, cerebral atherosclerosis and peripheral vascular atherosclerosis. The use according to claim 49, wherein, The coronary atherosclerotic heart disease is selected from acute coronary syndrome, asymptomatic myocardial ischemia-latent coronary heart disease, angina pectoris, myocardial infarction, ischemic heart disease, sudden death and in-stent restenosis. The use according to claim 49, wherein, The cerebral atherosclerosis is selected from ischemic stroke and hemorrhagic stroke. The use according to claim 49, wherein, The peripheral vascular atherosclerosis is selected from carotid atherosclerosis, vertebral atherosclerosis, subclavian atherosclerosis, occlusive peripheral atherosclerosis, retinal atherosclerosis, renal artery atherosclerosis, lower limb atherosclerosis, upper limb atherosclerosis, mesenteric atherosclerosis and atherosclerotic impotence.

53. Use of the nanocarrier delivery system of any one of claims 1 to 11, the nanocarrier delivery system prepared by the preparation method of any one of claims 12 to 47, and the drug of claim 48 in the preparation of a drug for treating arterial plaque. The use according to claim 53, wherein, The medicament is used to arrest the progression of arterial plaque, reverse arterial plaque and / or reduce the volume of arterial plaque.

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