Lipid-polymer nano-drug and preparation method thereof

Abstract

The invention discloses a lipid-polymer nano-drug, which comprises polymer nanoparticles and a shell layer coated on the surfaces of the polymer nanoparticles, the polymer nanoparticles comprise a block copolymer methoxy poly (ethylene glycol)-b-poly (L-glutamic acid-co-L-phenylalanine) and a small molecule vascular blocker, the shell layer comprises phospholipid and cholesterol. The invention also provides a preparation method of the lipid-polymer nano-drug. A shell layer is composed of phospholipid and cholesterol, the shell layer and the polymer nanoparticles jointly form a lipid-polymer nano-drug, the shell layer can maintain the stability of the polymer nanoparticle structure and prevent burst release of the small-molecular vascular blocker in the polymer nanoparticles, the toxic and side effects of the small-molecular vascular blocker can be effectively reduced, and the lipid-polymer nano-drug can be used for preparing the lipid-polymer nano-drug. And the tumor blood vessels can be efficiently and selectively destroyed by utilizing the characteristic that the blood vessel blocking agent nano-drug is enriched around the tumor blood vessels, so that the anti-tumor effect of the small-molecule blood vessel blocking agent is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of nanomedicine, and in particular to a lipid-polymer nanomedicine and a preparation method thereof. Background Art

[0002] Malignant tumors have been seriously affecting the health and quality of life of many people. In order to eliminate the threat posed by cancer, most clinical treatment options are based on surgery, chemotherapy and radiotherapy, which can also be combined with targeted therapy to improve prognosis. The tumor vascular system is essential for tumor growth because it can provide tumor cells with sufficient oxygen and nutrients, remove the metabolic waste produced, and provide a convenient channel for metastasis and spread. The tumor vascular system is formed as the tumor occurs and develops, and is often described as immature and abnormal. For example, their vascular walls lack development and often have discontinuous endothelial cell linings and irregular, structurally abnormal basement membranes. The obvious characteristic differences between tumor blood vessels and normal tissue blood vessels make tumor blood vessels a special target for tumor treatment. Therefore, the research focus began to shift to targeting tumor blood vessels, hoping to prevent the development of tumors by destroying the function of blood vessels.

[0003] By using vascular disrupting agents (VDAs) to destroy rapidly proliferating tumor vascular endothelial cells, the established tumor blood vessels are selectively closed, resulting in secondary tumor cell necrosis, which has a significant anti-tumor effect on most solid tumors. This vascular targeted treatment mode takes advantage of the differences between normal tissue vascular endothelium and tumor vascular endothelium. One of the more famous VDAs, Combretastain A-4 (CA4), has the same mechanism of action as colchicine, interacts with the colchicine site of tubulin, and disrupts the dynamic balance of tubulin polymerization / depolymerization in endothelial cells, thereby destroying tumor blood vessels. They selectively destroy immature and rapidly proliferating tumor vascular endothelial cells, blocking the supply of nutrients and oxygen to the tumor site. However, this vascular destruction effect is dose-related. At the same time, the higher the dose used, the greater the systemic toxicity. This also leads to poor tumor suppression after low-dose treatment, with tumor cell necrosis limited to the center of the tumor, while tumor cells at the edge still survive.

[0004] In view of this, a water-soluble serine CA4 derivative prodrug, Ombrabulin (Omb), came into being to enhance the anti-tumor effect and reduce systemic toxicity. However, although this small molecule drug has been approved for clinical trials, many patients still experience strong dose-limiting toxicity when using the recommended clinical dose, including transient hypertension, nausea and diarrhea.

[0005] Therefore, how to utilize small molecule vascular blockers more safely and effectively, reduce systemic toxicity, and retain their maximum therapeutic effect is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present application provides a lipid-polymer nanomedicine that can effectively reduce the toxic side effects of small molecule vascular blockers and increase their enrichment in tumor sites to enhance the anti-tumor effect of small molecule vascular blockers.

[0007] An ideal drug delivery system needs to effectively encapsulate drugs and avoid large amounts of drug leakage, thereby improving drug stability and bioavailability and achieving efficient and safe drug delivery. As an efficient drug delivery system, lipid nanoparticles are able to encapsulate therapeutic drugs and deliver them to target locations in the body. Many lipid nanodrug preparations have been approved and successfully used in clinical tumor treatment practices. In addition to being able to load therapeutic small molecules, lipid nanodrugs are also an ideal platform for loading macromolecules (including proteins, nucleic acids, etc.) for tumor treatment. Since the composition of lipid nanoparticles is similar to that of cell membranes, they have high biocompatibility, and they can hide therapeutic drugs inside, thereby exhibiting excellent in vivo stealth pharmacokinetic behavior. At the same time, compared with free anti-tumor drugs, lipid nanoparticles encapsulated anti-tumor drugs have higher bioavailability and selectivity.

[0008] In order to reduce the toxic side effects of free drugs, many studies have shown that lipid nanocarriers can reduce the toxicity of drugs to normal tissues, prolong the time of drug circulation in the body, and improve drug release behavior. Most non-targeted modified nanoparticles rely on the classic enhanced permeability and retention (EPR) effect to obtain the ability to passively transport to tumor sites. Even if these nanoparticles pass through the tumor vascular endothelial barrier, they still encounter the basement membrane barrier, which makes it difficult for them to truly and effectively pass through tumor blood vessels. In contrast, the target of tumor VDAs happens to be the rapidly proliferating tumor vascular endothelial cells. Therefore, the difficult-to-penetrate characteristics of nanoparticles can be utilized to increase the enrichment in the tumor vascular site and achieve a long-term tumor vascular destruction effect. By designing a nanoplatform, not only can the systemic toxicity of small molecule VDAs original drugs be reduced, but also the strong destruction of tumor blood vessels by VDAs can be enhanced.

[0009] There are some problems in the current technical solution. Although the toxic side effects can be reduced by loading small molecule drugs with polymer carriers, there are still problems such as poor stability of nano drugs, sudden release of small molecule drugs and short circulation time in the body. In addition, the degree of reduction of active drugs in the body formed by the polymer carrier grafted with small molecule drugs is insufficient, resulting in poor therapeutic effect. At the same time, this chemical reduction process in the body will greatly affect its safety and future clinical application.

[0010] Anti-tumor therapeutics and imaging agents based on small molecules, antibodies, nucleic acids and proteins generally do not exhibit ideal pharmacokinetic and pharmacodynamic properties in their natural form. These drugs may be toxic, unstable, rapidly metabolized or lack specificity. In order to protect healthy tissues from being affected and optimize their biodistribution to improve tumor delivery, methods of packaging these drugs into nanoparticles have been adopted. However, only a few cancer nanomedicines have been approved for clinical use worldwide. The vast majority (86%) of cancer nanomedicines failed in Phase III clinical trials, mainly because the nanomedicines did not penetrate sufficiently at the tumor site and could not effectively pass through the tumor vascular endothelial cells, so that only a very small dose of injected nanoparticles was delivered to the tumor, and the enrichment of the drug at the tumor site was reduced. This application mainly solves the problems of short half-life of small molecule vascular blockers, systemic distribution in patients after use, large toxic side effects and poor therapeutic effects.

[0011] Based on this, the present application provides a lipid-polymer nanomedicine, comprising polymer nanoparticles and an outer shell layer coated on the surface of the polymer nanoparticles, wherein the polymer nanoparticles comprise a block copolymer methoxypoly(ethylene glycol)-b-poly(L-glutamic acid-co-L-phenylalanine) and a small molecule vascular blocker, and the outer shell layer comprises phospholipids and cholesterol.

[0012] In order to reduce the toxic side effects of orebrine, the present application constructs a lipid-polymer nanodrug based on lipids and polymers to achieve efficient and safe tumor vascular destruction. The lipid-polymer nanodrug has an eggshell structure with a particle size of 143nm to 331nm. It is first self-assembled by electrostatic and hydrophobic interactions between a biocompatible and biodegradable block copolymer methoxypoly(ethylene glycol)-b-poly(L-glutamic acid-co-L-phenylalanine) (mPEG-bP(Glu-co-Phe)) and a small molecule vascular blocker to form polymer core nanoparticles. In some specific implementations, the small molecule vascular blocker includes orebrine (Omb), which is an amino acid prodrug conjugate in which the primary amine can be protonated under acidic conditions to form NH 3 +, the phospholipids include one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phospholipids, phosphatidylglycerol or sphingomyelin. In some specific implementations, the phosphatidylcholine includes 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In some specific implementations, the number average molecular weight of the block copolymer methoxy poly (ethylene glycol) -b- poly (L-glutamic acid -co-L-phenylalanine) is 6000g / mol to 15000g / mol, preferably 7000g / mol to 10000g / mol, more preferably 7760g / mol. Then, the outer shell is composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate (DPPC) and cholesterol (Cholesterol). DPPC is a pharmaceutical phospholipid excipient used for drug delivery, which helps the formation of lipid-polymer nanomedicines and prolongs their circulation time in the blood, while cholesterol can improve their stability and promote membrane fusion. The lipid shell layer in lipid-polymer nanomedicines can maintain the stability of the polymer nanoparticle structure and prevent the burst release of small molecule vascular blockers in polymer core polymer nanoparticles. Lipid-polymer nanomedicines based on lipids and polymers can reduce toxic side effects while causing extensive tumor vascular damage and reduced perfusion, ultimately leading to sustained tumor regression and enhanced anti-tumor effects.

[0013] In some specific implementations, the mass ratio of the block copolymer methoxy poly(ethylene glycol)-b-poly(L-glutamic acid-co-L-phenylalanine) to the small molecule vascular blocking agent is 1:1 to 15:1, and can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, preferably 2.5:1 to 8:1, and more preferably 2.5:1 to 5:1.

[0014] In some specific implementations, the molar ratio of phospholipid to cholesterol is 0.5:1 to 9:1, and can be 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, preferably 0.5:1 to 4:1, and more preferably 1.5:1 to 3:1.

[0015] In some specific implementations, the mass ratio of the polymer nanoparticles to the outer shell layer is 5:1 to 3:1, and can be 5:1, 4.5:1, 4:1, 3.5:1, 3:1, preferably 4.5:1 to 3.5:1, and more preferably 4:1.

[0016] The present application constructs a lipid-polymer nanomedicine with a stable lipid shell-polymer core and an eggshell-like structure of a vascular blocker, which not only effectively reduces the toxic side effects caused by small molecule Omb, but also selectively destroys rapidly proliferating tumor blood vessels, enhancing the enrichment and anti-tumor effect of Omb nanomedicine in tumors. Lipid-polymer nanomedicines selectively destroy rapidly proliferating tumor blood vessels, achieving selective killing of tumors. It shows that the addition of lipid components has an auxiliary effect on the blood stability and tumor enrichment effect of Omb nanomedicines, which improves the anti-tumor effect of Omb and reduces its toxic side effects.

[0017] The present application also provides a method for preparing a lipid-polymer nanomedicine, comprising:

[0018] a) mixing a block copolymer methoxy poly(ethylene glycol)-b-poly(L-glutamic acid-co-L-phenylalanine) with a small molecule vascular blocking agent, dialyzing, and freeze-drying to obtain polymer nanoparticles;

[0019] b) dissolving phospholipids and cholesterol in an organic solvent to form a lipid membrane, using polymer nanoparticles to hydrate the dried lipid membrane, and extruding the polymer nanoparticles so that the lipid membrane wraps the polymer nanoparticles to obtain a lipid-polymer nanomedicine.

[0020] The present application first dissolves mPEG-bP (Glu-co-Phe) in water, then adjusts the pH value of the solution to 7.5 to 8.5 to obtain solution a, dissolves Omb in 0.02M HCl aqueous solution, adds it dropwise to solution a, stirs vigorously for 12 hours in the dark at room temperature, dialyzes with deionized water (MWCO 3500Da) for about 40 to 50 hours, preferably 48 hours, to remove free small molecule drugs, and then freeze-drying to obtain P-Omb. In some specific implementations, the mixing and stirring in step a is performed under weakly alkaline conditions.

[0021] The present application then dissolves DPPC and cholesterol in an organic solvent at different molar ratios in a round-bottom flask to obtain solution b. The present application has no special restrictions on the selection of organic solvents. In some specific implementations, the organic solvent includes dichloromethane. The solution b is rotary evaporated at 35°C to 45°C for 0.5 to 1 hour to remove the organic solvent to form a thin lipid film, which is dried under vacuum for 4 to 6 hours to obtain a dry lipid film; the dried lipid film is stirred and hydrated with a certain amount of P-Omb aqueous solution at 50°C to 60°C for 30 to 40 minutes, and then the solution is extruded 15 to 25 times with a 200nm polycarbonate membrane to wrap the polymer nanoparticles to obtain lipid-polymer nanomedicine (PP-Omb). In some specific implementations, the extrusion in step b includes extrusion using a polycarbonate membrane, and the number of extrusions is 15 to 25 times. In some specific implementations, the usage amount of the P-Omb aqueous solution is 10 mg / mL to 30 mg / mL, preferably 20 mg / mL.

[0022] The present application realizes the successful loading of the vascular blocking agent Omb by the carrier material through electrostatic and hydrophobic interactions.

[0023] The lipid-polymer nanomedicine disclosed in the present application includes polymer nanoparticles and an outer shell layer coated on the surface of the polymer nanoparticles. The polymer nanoparticles are formed by block copolymer methoxy poly (ethylene glycol) -b-poly (L-glutamic acid-co-L-phenylalanine) and a small molecule vascular blocker through electrostatic and hydrophobic effects, and the components of the outer shell layer include a combination of phospholipids and cholesterol. The lipid outer shell layer is composed of phospholipids and cholesterol, and together with P-Omb, it constitutes a lipid-polymer nanomedicine. The lipid outer shell layer can maintain the stability of the polymer nanoparticle structure and prevent the burst release of the small molecule vascular blocker in the polymer nanoparticle, which can not only effectively reduce the toxic and side effects of the small molecule vascular blocker, but also can utilize the characteristics of the vascular blocker nanomedicine enriched around the tumor blood vessels to efficiently and selectively destroy the tumor blood vessels, thereby enhancing the anti-tumor effect of the small molecule vascular blocker. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The H NMR spectrum of the block copolymer mPEG-bP (Glu-co-Phe) provided in Example 1 of the present application;

[0025] Figure 2 This is a particle size distribution diagram of the polymer nanoparticles prepared in Example 3 of the present application;

[0026] Figure 3 This is a cryo-transmission electron microscope microscopic morphology image of the polymer nanoparticles prepared in Example 3 of the present application;

[0027] Figure 4 The particle size distribution diagram of the lipid-polymer nanomedicine prepared in Example 5 of the present application with a molar ratio of DPPC to cholesterol of 0.75:1;

[0028] Figure 5 The particle size distribution diagram of the lipid-polymer nanomedicine prepared in Example 6 of the present application with a molar ratio of DPPC to cholesterol of 1.5:1;

[0029] Figure 6 This is a particle size distribution diagram of the lipid-polymer nanomedicine prepared in Example 7 of the present application with a molar ratio of DPPC to cholesterol of 3:1;

[0030] Figure 7 This is a particle size bar graph of the lipid-polymer nanomedicines prepared in Examples 5 to 7 of the present application;

[0031] Figure 8 The polydispersity coefficient bar graph of the lipid-polymer nanomedicines prepared in Examples 5 to 7 of the present application;

[0032] Fig. 9 This is a particle size distribution diagram of the lipid-polymer nanomedicine used in Example 6 of the present application;

[0033] Fig.10 This is a cryo-TEM microscopic morphology of the lipid-polymer nanomedicine used in Example 6 of the present application;

[0034] Fig.11 Zeta potential value diagram of mPEG-bP(Glu-co-Phe), Omb, P-Omb and PP-Omb solutions;

[0035] Fig.12 The particle size diagram of PP-Omb in PBS pH 7.4 or PBS pH 7.4 solution containing 10% FBS within one week;

[0036] Fig.13 Cell viability diagram after co-incubation of different concentrations of lipid-polymer nanomedicine with simulated fusion (mature) / non-fusion (immature) HUVECs for 24 hours;

[0037] Fig.14 The cell viability diagram after different concentrations of lipid-polymer nanomedicines were co-incubated with simulated fusion (mature) / non-fusion (immature) HUVECs for 48 hours;

[0038] Fig.15 The cell morphology changes after different concentrations of lipid-polymer nanomedicines were co-incubated with simulated fusion (mature) / non-fusion (immature) HUVECs for 48 hours;

[0039] Fig.16 H&E staining images of the main organs of tumor-bearing mice after treatment with PBS, Omb, P-Omb and PP-Omb;

[0040] Fig.17 H&E staining of tumors in tumor-bearing mice treated with phosphate buffered saline (PBS), Omb, P-Omb, and PP-Omb;

[0041] Fig.18 The tumor appearance of tumor-bearing mice treated with PBS, Omb, P-Omb and PP-Omb for 24 hours, respectively, after intravenous injection of Evans blue 3 hours before tumor resection;

[0042] Fig.19 The Evans blue content in tumor tissue after PBS, Omb, P-Omb and PP-Omb treatment;

[0043] Fig. 20 Immunohistochemical staining of microvessels in tumor tissue after treatment with PBS, Omb, P-Omb, and PP-Omb;

[0044] Fig.21 The diagram shows the experimental process of treating CT26 tumor-bearing mice with PBS, Omb, P-Omb and PP-Omb;

[0045] Fig. 22 This is the average tumor growth curve of tumor-bearing mice during different drug treatments;

[0046] Fig.23 Figure 2 is a graph of the tumor inhibition rate of the treatment group during the treatment period;

[0047] Fig.24 Figure 2 is the weight change of mice in each treatment group during the treatment period;

[0048] Fig.25 This is a graph showing the survival rate of CT26 tumor-bearing mice within 60 days after treatment;

[0049] Fig.26 Figure 2 shows the appearance of mouse tumors during treatment. DETAILED DESCRIPTION

[0050] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0051] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0052] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0053] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0054] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0055] At present, there are still some problems in loading small molecule drugs through polymer carriers. First, the stability of nano drugs is poor, resulting in sudden release of small molecule drugs and short circulation time in the body. In addition, this method does not effectively reduce the toxic side effects of small molecule drugs, and the bioavailability of nano drugs is still insufficient. In addition, the nano drugs formed by grafting small molecule drugs with polymer carriers do not reduce the active drugs in the body to an insufficient extent, resulting in poor therapeutic effects. At the same time, this chemical reduction process in the body may affect its safety and future clinical application. It is worth noting that only a few cancer nano drugs have been approved for clinical use worldwide, and the vast majority of them (86%) failed in Phase III clinical trials due to lack of therapeutic effect. The main reason is that nano drugs have insufficient penetration in the tumor site and cannot effectively pass through the tumor vascular endothelial cells, resulting in only a very small amount of nanoparticles being delivered to the tumor, thereby reducing the enrichment of drugs at the tumor site.

[0056] This application aims to transform the difficulty of nano-drugs to effectively penetrate tumor sites and the characteristics of VDAs to selectively destroy rapidly proliferating tumor vascular endothelial cells into advantages. By designing structurally stable eggshell-shaped vascular blocker nano-drugs based on lipids and polymers, not only can the toxic side effects of small molecule vascular blockers be effectively reduced, but also the characteristics of vascular blocker nano-drugs enriched around tumor blood vessels can be utilized to efficiently and selectively destroy tumor blood vessels, thereby enhancing the anti-tumor effect of small molecule vascular blockers.

[0057] The present invention is further described below in conjunction with the following examples. The protection scope of the present invention is not limited by the following examples.

[0058] Example 1

[0059] Preparation method of block copolymer mPEG-bP (Glu-co-Phe): By using mPEG-NH 2 As an initiator, Phe-NCA and BLG-NCA monomers were subjected to ring-opening polymerization (ROP) and benzyl deprotection to synthesize methoxy poly (ethylene glycol) -b-poly (L-glutamic acid co-L-phenylalanine) (mPEG-bP (Glu-co-Phe)) copolymers. The specific preparation method is referred to S. Lv, W. Song, Z. Tang, M. Li, H. Yu, H. Hong, X. Chen, Charge-Conversional PEG-Polypeptide Polyionic Complex Nanoparticles from Simple Blending of a Pair of Oppositely Charged Block Copolymers as an Intelligent Vehicle for Efficient Antitumor Drug Delivery, Molecular Pharmaceutics 11 (5) (2014) 1562-1574. The obtained block copolymer mPEG-bP (Glu-co-Phe) was tested by nuclear magnetic resonance, and the nuclear magnetic hydrogen spectrum is shown as follows Figure 1 shown.

[0060] Example 2

[0061] The mPEG-bP (Glu-co-Phe) powder was dissolved in water, and the pH value of the solution was adjusted to 8.0. Omb was dissolved in 0.02M HCl aqueous solution, and then added dropwise to the above solution, and stirred vigorously at room temperature in the dark for 12 hours, wherein the mass ratio of mPEG-bP (Glu-co-Phe) powder to Omb was 9:1. Subsequently, it was dialyzed with deionized water (MWCO 3500Da) for about 48 hours to remove free small molecule drugs, and then lyophilized to obtain P-Omb, whose drug loading rate was 8.2% and encapsulation efficiency was 82.2%.

[0062] Example 3

[0063] The difference from Example 2 is that the mass ratio of mPEG-bP (Glu-co-Phe) powder to Omb is 5:1. The particle size distribution of the prepared P-Omb is shown in Figure 2 As shown in Figure 1, the particle size distribution is uniform, and the particle size is 122.3±4.7nm. The cryo-TEM microscopic morphology of the prepared P-Omb is shown in Figure 1. Figure 3 As shown, cryo-transmission electron microscopy (cryo-TEM) confirmed that it had a spherical morphology, a drug loading rate of 15.7%, and an encapsulation efficiency of 94.4%.

[0064] Example 4

[0065] The only difference from Example 2 is that the mass ratio of mPEG-bP (Glu-co-Phe) powder to Omb is 2.5:1, the drug loading rate is 24.5%, and the encapsulation efficiency is 85.6%.

[0066] Example 5

[0067] The lipid-polymer nanodrug (PP-Omb) was prepared by a method of thin film hydration followed by extrusion. First, DPPC and cholesterol were dissolved in chloroform at a molar ratio of 0.75:1 in a round-bottom flask; then, the organic solvent was removed by rotary evaporation at 35°C for 0.5 hours to form a thin lipid film, which was dried under vacuum for 4 hours. The dried lipid film was stirred and hydrated with a certain amount of the P-Omb aqueous solution provided by Example 2-4 at 50°C for 30 minutes. The solution was then extruded 20 times with a 200nm polycarbonate membrane to obtain PP-Omb. Unencapsulated Omb was removed by ultrafiltration (MWCO 3000Da), and the obtained solution was stored at 4°C for further use. The particle size distribution of the prepared lipid-polymer nanodrug is shown in the figure. Figure 4 The particle size bar graph of lipid-polymer nanomedicine measured by DLS is shown in Figure 7 The polydispersity coefficient histogram is shown in Figure 8 shown.

[0068] Example 6

[0069] The only difference from Example 2 is that the molar ratio of DPPC to cholesterol is 1.5:1.

[0070] The particle size distribution of lipid-polymer nanomedicine prepared by DLS measurement with a molar ratio of DPPC to cholesterol of 1.5:1 is shown in the figure below. Fig. 9 As shown in the figure, the particle size of the prepared PP-Omb is 163.2±1.7nm, which is larger than the particle size of the Omb core nanoparticle P-Omb. After the introduction of the phospholipid shell, the average particle size of the polymer nanoparticles increased from 122.3nm to 163.2nm, which shows that the lipid-polymer nanomedicine has been successfully obtained. The cryo-TEM microscopic morphology of PP-Omb in aqueous solution is shown in Fig.10 The characterization further proved its eggshell-like structure. In addition, the Zeta potential of the nanoparticles was measured. The Zeta potential values ​​of mPEG-bP(Glu-co-Phe), Omb, P-Omb and PP-Omb solutions are shown in the figure Fig.11 As shown. The average Zeta potential of the mPEG-bP (Glu-co-Phe) copolymer was measured to be -30.47 mV. When it was loaded with protonated Omb, the surface charge increased to -19.87 mV. In addition, compared with P-Omb, the surface charge further increased to -13.47 mV with the introduction of the surface lipid shell, which confirmed the successful lipid membrane coating in PP-Omb. The high negative charge not only improves the stability of the polymer nanoparticles themselves, but also has the potential to resist protein and prolong blood circulation time when used in vivo. Therefore, PP-Omb was incubated with phosphate buffered solution (PBS) or phosphate buffered solution containing 10% fetal bovine serum (FBS) to detect the stability of PP-Omb. The lipid-polymer nanomedicine was dissolved in PBS pH 7.4 buffer with or without 10% FBS and then incubated at 37°C and an oscillation rate of 100 rpm. At specific time intervals, the sample particle size was analyzed by DLS. The data showed that after 7 days of incubation in PBS at pH 7.4, the particle size of PP-Omb did not change significantly. When serum was present, the particle size increased slightly compared to that in PBS, but still did not change significantly. Within one week, the particle size of PP-Omb in PBS pH 7.4 or PBS pH 7.4 solution containing 10% FBS was similar to that in the control group. Fig.12 The particle size distribution of the prepared lipid-polymer nanomedicine is shown in Figure 5 The particle size bar graph of lipid-polymer nanomedicine measured by DLS is shown in Figure 7 The polydispersity coefficient histogram is shown in Figure 8 shown.

[0071] Example 7

[0072] The only difference from Example 2 is that the molar ratio of DPPC to cholesterol is 3:1.

[0073] Different molar ratios of DPPC and cholesterol have a certain effect on the particle size of the formed PP-Omb. The particle size distribution diagram of the prepared lipid-polymer nanomedicine is shown in Figure 6 As shown in the figure, with the increase of DPPC content, the particle size of PP-Omb gradually increased, and the particle sizes of PP-Omb at different molar ratios of 0.75:1, 1.5:1 and 3:1 with cholesterol were 154.1±11.1nm, 169.6±2.1nm and 281.0±5.0nm respectively. The particle size bar chart of lipid-polymer nanomedicine measured by DLS is shown in the figure. Figure 7 The polydispersity coefficient histogram is shown in Figure 8 As shown, the average PDI (Polydispersity index) values ​​are 0.46, 0.16 and 0.41, respectively. It can be seen that when the molar ratio of DPPC to cholesterol is 1.5:1, the prepared PP-Omb particle size is uniform and the distribution is concentrated.

[0074] Example 8

[0075] Preparation of confluent and non-confluent HUVECs. Confluent or non-confluent HUVECs were prepared by seeding 10,000 or 5,000 cells per well in a 96-well plate and incubating in a 5% CO atmosphere. 2 The cells were incubated at 37°C in a moist environment until close contact and fusion between cells occurred, which was considered as fusion-type HUVECs.

[0076] Confluent and non-confluent HUVECs (Human Umbilical Vein Endothelial Cells) were used to simulate mature and immature tumor vascular endothelial cells, and then the selectivity was evaluated by measuring the effect of PP-Omb on their proliferation activity after 24 hours and 48 hours of co-incubation. The cells were co-incubated with a series of concentrations of PP-Omb (10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 μM) for 24 or 48 hours, after which 10% CCK-8 solution was added to each well. After incubation with CCK-8 solution for a certain period of time, the optical density (OD) value of each sample at 450 nm was measured using a microplate reader to calculate cell viability.

[0077] Cell viability after 24 h of co-incubation of different concentrations of PP-Omb with simulated fusion (mature) / non-fusion (immature) HUVECs Fig.13 As shown, the cell viability after 48 hours is shown in Fig.14 As shown in the figure, the proliferation activity of non-fused HUVECs decreased significantly with the increase of PP-Omb concentration, while the proliferation activity of fused HUVECs was affected only when the concentration of PP-Omb increased to a certain extent. Compared with mature HUVECs, immature HUVECs are more sensitive to PP-Omb, indicating that PP-Omb has a selective effect on rapidly proliferating tumor vascular endothelial cells. The half-maximal inhibitory concentration (IC) of PP-Omb on fused or non-fused HUVECs after 24 or 48 hours was measured. 50 ) value, IC value of PP-Omb on mature cells at 24 h 50 The value is 8.74 μM, which is the IC 50 The difference reached a maximum of 21.6 times at 48 h. The IC value of PP-Omb for mature or immature cells was 50 The values ​​were 0.16μM and 3.45μM, respectively. Inhibiting the polymerization of endothelial cell microtubules will cause changes in the morphology of endothelial cells themselves. The cell morphology changes after 48 hours of co-incubation of different concentrations of PP-Omb with simulated fusion (mature) / unfusion (immature) HUVECs are shown in the figure below. Fig.15 As shown. In the control group, the cell morphology can be seen to be cobblestone-like with translucent edges. There is extensive contact and fusion between cells in the simulated mature endothelial cells. When HUVECs were co-incubated with PP-Omb, as the concentration increased, HUVECs gradually shrank and spherical cells began to appear. For the simulated immature HUVECs, this change began to appear at a lower concentration because they are more sensitive to the drug. In general, these results demonstrate that immature and proliferating endothelial cells are more sensitive to PP-Omb, suggesting that PP-Omb can selectively destroy rapidly proliferating tumor vascular endothelial cells. At the same time, this result shows that PP-Omb can target tumor blood vessels with high selectivity, thereby reducing the impact on normal tissue blood vessels.

[0078] Example 9

[0079] To investigate the effect of PP-Omb on tumor angiogenesis, female BALB / c mice were subcutaneously injected with 2×10 6CT26 cells were used to establish a subcutaneous CT26 tumor model, followed by treatment and histological examination. Mice were injected with PBS, Omb, P-Omb and PP-Omb, respectively. Tumors were removed 4 hours later and analyzed by H&E (hematoxylin-eosin staining). H&E staining of the main organs of tumor-bearing mice treated with PBS, Omb, P-Omb and PP-Omb is shown in the figure. Fig.16 As shown in the figure, compared with the PBS control group, the tumors after drug treatment all showed different degrees of bleeding. Compared with P-Omb and small molecule Omb, large-area bleeding and red blood cell extravasation were observed in the PP-Omb group, indicating that the integrity of tumor blood vessels was damaged and vascular leakage increased. However, after treatment with Omb, P-Omb and PP-Omb, such bleeding or red blood cell extravasation was not observed in normal major organs. The H&E staining images of tumors in tumor-bearing mice treated with PBS, Omb, P-Omb and PP-Omb are shown in the figure. Fig.17 This suggests that this treatment-related bleeding and red blood cell extravasation only occurred in tumor vascular sites and was highly tumor-selective.

[0080] Example 10

[0081] In order to verify the effect of PP-Omb on tumor vascular perfusion, the delivery efficiency of the dye Evans blue in the tumor was evaluated. After 24 hours of treatment with different drugs, Evans blue (0.5%, 100 μL) was injected intravenously. Three hours later, the mice were killed, the tumor tissues were collected and photographed, and then the tumor tissues were soaked in 2 mL of formamide for 3 days to extract Evans blue. The optical density (OD) value of the supernatant was then measured at a wavelength of 620 nm to quantify the Evans blue content in the tumor tissue. Tumor-bearing mice were treated with PBS, Omb, P-Omb and PP-Omb for 24 hours, respectively. The tumor morphology after intravenous injection of Evans blue 3 hours before tumor resection is shown in the figure below. Fig.18 As shown in the figure, it can be clearly observed that the blue depth of Evans blue in the PBS control group is much stronger than that in the drug treatment group, especially after PP-Omb treatment. Then, the collected tumor tissues were immersed in formamide to extract Evans blue, and the Evans blue content in the tumor was determined based on the standard curve of the relationship between concentration and absorption intensity. The Evans blue content in tumor tissues after PBS, Omb, P-Omb and PP-Omb treatment is shown in the figure. Fig.19Compared with the PBS control group, the accumulation of Evans blue in the tumor was reduced after treatment with small molecule Omb, while the accumulation of Evans blue in the tumor site was more significantly reduced after treatment with PP-Omb. This difference is due to the large and long-term enrichment of lipid-polymer nanomedicines in the tumor site, which aggravates the destruction of tumor blood vessels and coagulation. The immunohistochemical staining of tumor tissue microvessels after PBS, Omb, P-Omb and PP-Omb treatment is shown in Figure 2. Fig. 20 As shown, 24 hours after injection of PBS, Omb, P-Omb, and PP-Omb, the microvessel density of tumors treated with PP-Omb was significantly reduced, indicating effective vascular damage. Collectively, these results indicate that PP-Omb can cause strong tumor vascular destruction, thereby greatly reducing tumor vascular perfusion.

[0082] Embodiment 11

[0083] To verify its anti-tumor effect in vivo, mice bearing CT26 tumors were treated with Omb, P-Omb and PP-Omb. The experimental process is shown in the figure Fig.21 First, female BALB / c mice were injected subcutaneously on the right side of the abdomen with 2×10 6 CT26 cells were used to establish a subcutaneous CT26 tumor model. When the tumor volume reached approximately 200-220 mm 3 At the time of treatment, the tumor-bearing mice were randomly divided into 7 groups, 5 mice in each group, specifically: PBS control group, Omb (injection dose of 40 mg / kg), P-Omb (injection dose of 40 mg / kg, calculated as Omb), PP-Omb (injection dose of 40 mg / kg, calculated as Omb), Omb (injection dose of 60 mg / kg), P-Omb (injection dose of 60 mg / kg, calculated as Omb), PP-Omb (injection dose of 60 mg / kg, calculated as Omb), and the drug was injected intravenously once every 3 days, for a total of 2 injections (on day 0 and day 3, respectively). The average tumor growth curve of tumor-bearing mice during different drug treatments is shown in the figure below. Fig. 22As shown, it can be seen that compared with the PBS control group, the growth of tumors was significantly inhibited after treatment with P-Omb, PP-Omb and small molecule Omb. In contrast, in the later stage of treatment, the inhibition rates of tumor growth in the P-Omb group and the PP-Omb group were higher than those of the small molecule Omb to varying degrees, indicating that the anti-tumor effect of the nanodrug was improved. Compared with the P-Omb group, the anti-tumor effect of the PP-Omb group at a dose of 40 or 60 mg / kg was improved, indicating that the lipid shell structure gave the polymer nanoparticles a stronger tumor enrichment ability and continued to act on tumor blood vessels, thereby enhancing the anti-tumor effect of the PP-Omb nanodrug. The inhibitory effect on tumor growth in all drug treatment groups showed a dose-dependent trend, which was particularly evident in the small molecule Omb group, with a dose of 60 mg / kg having a stronger tumor inhibition ability than the dose of 40 mg / kg. In the early stage of treatment (day 6), all groups showed similar therapeutic effects, which was due to continuous medication. However, during the cessation of drug administration, the tumors treated with the small molecule Omb group resumed their rapid growth trend compared with the nanomedicine group. Fig.23 As shown in the figure, 60 mg / kg dose of PP-Omb can effectively inhibit tumor growth and achieve a tumor suppression rate (TSR%) of 93.2% on the 10th day, while the TSR% after treatment with the same dose of P-Omb and small molecule Omb were 85.1% and 70.1%, respectively. The weight changes of mice in each treatment group during the treatment with PP-Omb are shown in the figure below. Fig.24 As shown, no significant weight changes were observed in the PP-Omb treatment group, with the maximum weight loss being only 6.8%. However, toxic events were observed during the treatment of the small molecule Omb group, resulting in a 13.2% weight loss in mice. These results suggest that PP-Omb with a stable lipid shell structure plays a role in reducing systemic toxicity and enhancing anti-tumor efficacy. The survival rate of CT26 tumor-bearing mice within 60 days after treatment is shown in Figure 2. Fig.25 As shown in the figure, the PP-Omb group significantly prolonged the survival time of tumor-bearing mice. After treatment with PP-Omb (60 mg / kg, calculated as the amount of Omb), the cure rate of mice within 60 days was 40%, and the tumor did not recur, while no mice were cured in the other groups, which once again demonstrated the excellent anti-tumor effect of PP-Omb. The appearance of mouse tumors during treatment is shown in the figure below. Fig.26 As shown, the tumor regression process after PP-Omb treatment can be clearly observed compared with the PBS control group.

[0084] The present application obtains stable lipid-polymer nanomedicines containing lipid vascular blockers through self-developed nanomedicine self-assembly technology, which will effectively prolong the half-life of vascular blockers. The advantage of nanomedicines' specific enrichment in tumor blood vessels can be used to selectively destroy tumor blood vessels, reduce toxicity and increase efficacy.

[0085] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A lipid-polymer nanomedicine, characterized in that: The invention comprises polymer nanoparticles and an outer shell layer coated on the surface of the polymer nanoparticles. The polymer nanoparticles comprise block copolymer methoxypoly(ethylene glycol)-b-poly(L-glutamic acid-co-L-phenylalanine) and a small molecule vascular blocker. The outer shell layer comprises phospholipids and cholesterol.

2. The lipid-polymer nanomedicine according to claim 1, characterized in that: The mass ratio of the block copolymer methoxy poly (ethylene glycol) -b-poly (L-glutamic acid-co-L-phenylalanine) to the small molecule vascular blocking agent is 1:1 to 15:

1.

3. The lipid-polymer nanomedicine according to claim 1, characterized in that: The molar ratio of the phospholipid to cholesterol is 0.5:1 to 9:

1.

4. The lipid-polymer nanomedicine according to claim 1, characterized in that: The mass ratio of the polymer nanoparticles to the outer shell layer is 5:1 to 3:

1.

5. The lipid-polymer nanomedicine according to claim 1, characterized in that: The small molecule vascular blocking agent includes oreblin, and the phospholipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phospholipids, phosphatidylglycerol or sphingomyelin.

6. The lipid-polymer nanomedicine according to claim 1, characterized in that: The particle size of the lipid-polymer nanomedicine is 143nm to 331nm.

7. A method for preparing a lipid-polymer nanomedicine, characterized in that: include: a) mixing a block copolymer methoxy poly(ethylene glycol)-b-poly(L-glutamic acid-co-L-phenylalanine) with a small molecule vascular blocking agent, dialyzing, and freeze-drying to obtain polymer nanoparticles; b) dissolving phospholipids and cholesterol in an organic solvent to form a lipid membrane, using polymer nanoparticles to hydrate the dried lipid membrane, and extruding the polymer nanoparticles so that the lipid membrane wraps the polymer nanoparticles to obtain a lipid-polymer nanomedicine.

8. The preparation method according to claim 7, characterized in that: The mixing and stirring in step a is carried out under weakly alkaline conditions with a pH value of 7.5 to 8.

0.

9. The preparation method according to claim 7, characterized in that: The organic solvent in step b includes dichloromethane.

10. The preparation method according to claim 7, characterized in that: The extrusion in step b includes extrusion using a polycarbonate film, and the number of extrusions is 15 to 25 times.

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