Targeted drug liposome for inhibiting pulmonary vascular endothelial cell glycocalyx shedding and improving microenvironment before tumor metastasis

Abstract

The invention discloses a targeted drug liposome for inhibiting pulmonary vascular endothelial cell glycocalyx abscission and improving a microenvironment before tumor metastasis, which consists of a glycocalyx abscission enzyme inhibitor, a lipid material and an adhesion receptor dipeptidase-1 binding peptide, the lipid material comprises a phosphatidyl ethanolamine derivative, a long-circulating lipid material, a reactive long-circulating lipid material capable of being connected with a peptide fragment, and a cationic lipid material. The adhesion receptor dipeptidase-1 binding peptide is specifically bound with adhesion receptor dipeptidase generated by pulmonary vascular endothelial cells, passive targeting of lipidosome in the pulmonary vascular endothelial cells is achieved, and pulmonary blood vessel normalization is promoted by inhibiting falling of glycocalyx; meanwhile, the adhesion receptor dipeptidase-1 binding peptide can also reduce recruitment of neutrophil in the lung and improve the microenvironment before lung tumor metastasis. The tumor metastasis to the lung can be reduced, and certain clinical application prospects are achieved in the aspect of treatment of lung inflammation, vasculopathy and the like.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutics, and specifically relates to a targeted drug liposome capable of inhibiting the shedding of glycocalyx of pulmonary vascular endothelial cells and improving the microenvironment before tumor metastasis and a preparation method thereof. Background Art

[0002] At present, the high mortality rate of cancer in clinical practice is closely related to tumor metastasis, so it is particularly important to inhibit tumor metastasis in cancer treatment. Tumor metastasis is an extremely complex process, among which lung metastasis and liver metastasis are the most common. [1] In 1889, Stephen Paget proposed the concept of pre-metastatic microenvironment, which provided a new idea for preventing tumor metastasis. [2] The formation of the pre-metastatic microenvironment includes the secretion of cytokines and exosomes by primary tumor cells and stromal cells to regulate distal tissues, inflammation of distal tissues, and recruitment of immune cells to distal tissues to form immunosuppression. In this process, pulmonary vascular endothelial cells (VECs) play a vital role in the formation of the pre-metastatic microenvironment and the promotion of tumor migration. [3] Inflammation of lung tissue is often accompanied by the shedding of glycocalyx on the surface of VECs. The glycocalyx layer is 0.1-1.0 μm thick and is synthesized and secreted by vascular endothelial cells. It is an important structure of the vascular endothelial surface. Its main components are proteoglycans and glycoproteins. [4] Under normal physiological conditions, the glycocalyx is evenly and orderly distributed on the apical membrane of endothelial cells. However, in the pre-metastatic microenvironment, the lung tissue becomes inflamed, and the glycocalyx on VECs is damaged and falls off into the blood. [5] . Glycocalyx damage can lead to loose connections between tightly connected endothelial cells, which in turn leads to increased vascular permeability, thereby promoting the extravasation of tumor circulating cells in the lungs. Therefore, tumor metastasis can be reduced by inhibiting the shedding of endothelial glycocalyx. At the same time, the adhesion receptor dipeptidase 1 (DPEP1) is a cell adhesion molecule that is often highly expressed in VECs in the lungs with inflammation. [6] , and neutrophils promote their recruitment to the lungs by binding to DPEP1 [6] , thereby promoting the shedding of endothelial glycocalyx on VECs [7] , and induces immunosuppression in the metastatic microenvironment [8] , preparing for the colonization of circulating tumor cells in lung tissue [9] Therefore, the high expression of DPEP1 on activated VECs can be used to deliver drugs to the site of inflammation in the lungs. Adhesion receptor dipeptidase binding peptide (H-LSAITPSPSWLKYKAL-NH2, referred to as LSAIT peptide, sequence CGFECVRQCPERC) can specifically bind to DPEP1 [6]Therefore, LSAIT peptide can be used to passively target drugs to the site of lung inflammation. In addition, LSAIT peptide competes with neutrophils to bind DPEP1, reducing the adhesion of neutrophils to VECs. [6] The glucocorticoid anti-inflammatory drug dexamethasone can also inhibit inflammation and inhibit glycocalyx shedding.

[10] , reduce the lesions of VECs and promote the normalization of pulmonary blood vessels.

[0003] Liposomes are spherical particles consisting of a double lipid membrane wrapped in concentric circles to form a single or multilayer

[11] Liposomes are widely used, but their endocytosis efficiency is usually low. Inducing fusion of liposomes with cell membranes is an effective method to improve drug delivery efficiency. Cationic liposomes (Lip) can fuse with mammalian cells with an efficiency of about 80-100% within 1-5 minutes without any toxic effects.

[12] .

[0004] Therefore, the present invention uses adhesion receptor dipeptidase binding peptide (LSAIT peptide) to deliver cationic liposomes loaded with dexamethasone to VECs, so as to inhibit the shedding of endothelial glycocalyx and improve the microenvironment before tumor metastasis.

[0005] References:

[0006] [1]. Zhang, C., Q. Li, Q. Xu, et al., Pulmonary interleukin 1beta / serumamyloid A3 axis promotes lung metastasis of hepatocellular carcinoma by facilitating the pre-metastatic niche formation. J Exp Clin Cancer Res, 2023.42(1):p.166.

[0007] [2].Paget,S.,The distribution of secondary growths in cancer of thebreast.1889.Cancer Metastasis Rev,1889.8(2):p.98-101.

[0008] [3].Huang,Y.,N.Song,Y.Ding,et al.,Pulmonary vascular destabilizationin the premetastatic phase facilitates lung metastasis.Cancer Res,2009.69(19):p.7529-37.

[0009] [4].Alphonsus,C.S.and R.N.Rodseth,The endothelial glycocalyx:a reviewof the vascular barrier.Anaesthesia,2014.69(7):p.777-84.

[0010] [5].Dogné,S.and B.Flamion,Endothelial Glycocalyx Impairment inDisease:Focus on Hyaluronan Shedding.Am J Pathol,2020.190(4):p.768-780.

[0011] [6].Choudhury,S.R.,L.Babes,J.J.Rahn,et al.,Dipeptidase-1Is anAdhesion Receptor for Neutrophil Recruitment in Lungs and Liver.Cell,2019.178(5):p.1205-1221.e17.

[0012] [7].Zhang,H.,Y.Wang,M.Qu,et al.,Neutrophil,neutrophil extracellulartraps and endothelial cell dysfunction in sepsis.Clin Transl Med,2023.13(1):p.e1170.

[0013] [8].Siakaeva,E.and J.Jablonska,Pre-metastatic Niche Formation byNeutrophils in Different Organs.Adv Exp Med Biol,2021.1329:p.93-108.

[0014] [9]. Chen, MB, C. Hajal, DC Benjamin, et al., Inflamed neutrophilssequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation. Proc Natl Acad Sci USA, 2018.115(27):p.7022-7027.

[0015]

[10] .Cui,N.,H.Wang,Y.Long,et al.,Dexamethasone Suppressed LPS-InducedMatrix Metalloproteinase and Its Effect on Endothelial GlycocalyxShedding.Mediators Inflamm,2015.2015:p.912726.

[0016]

[11] Cheng Lichun, Fan Qing. Research progress of active targeted liposomes in anti-tumor therapy[J]. Practical Drugs and Clinical Practice, 2011, 14(5):4.

[0017]

[12] R, Jaksch S, Dreissen G, Braeutigam A, Merkel R, Csiszár A. Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells. Nanomaterials (Basel). 2019Jul 17;9(7):1025. Summary of the invention

[0018] The purpose of the present invention is to provide a targeted drug liposome, which can achieve efficient drug delivery at sites where adhesion receptor dipeptidase is highly expressed by LSAIT peptide-modified and loaded cationic liposomes, thereby inhibiting glycocalyx shedding and improving the microenvironment before tumor metastasis, thereby reducing tumor metastasis to the lungs. The present invention also provides a preparation method of the liposome and its application.

[0019] To achieve the above purpose, the technical solution used in the present invention is as follows:

[0020] A targeted drug liposome for inhibiting glycocalyx shedding of pulmonary vascular endothelial cells and improving the microenvironment before tumor metastasis, which is composed of a glycocalyx shedding enzyme inhibitor, a lipid material, and an adhesion receptor dipeptidase-1 binding peptide, wherein the lipid material includes a phosphatidylethanolamine derivative, a long-circulating lipid material, a reactive long-circulating lipid material capable of connecting peptide segments, and a cationic lipid material; the glycocalyx shedding enzyme inhibitor includes a glucocorticoid; and the amino acid sequence of the adhesion receptor dipeptidase-1 binding peptide is CGFECVRQCPERC.

[0021] Wherein, the phosphatidylethanolamine derivatives include DMPE, DSPE, DOPE, DPPE and DLPE.

[0022] Wherein, the long-circulating lipid material is a phosphatidylethanolamine derivative-PEG copolymer, including DMPE-PEG, DSPE-PEG, DOPE-PEG, DPPE-PEG, and DLPE-PEG.

[0023] Wherein, the cationic lipid material includes DOTAP, DOTMA, DOSPA and DDAB.

[0024] Preferably, the glycocalyx shedding enzyme inhibitor accounts for 2-10% of the mass of the targeted drug liposome; the adhesion receptor dipeptidase-1 binding peptide accounts for 5-15% of the mass of the targeted drug liposome; and the rest is lipid material.

[0025] Further preferably, the phosphatidylethanolamine derivative accounts for 30-50% of the mass of the targeted drug liposome; the long-circulating lipid material accounts for 10-30% of the mass of the targeted drug liposome; the reactive long-circulating lipid material accounts for 5-15% of the mass of the targeted drug liposome; and the cationic lipid material accounts for 10-30% of the mass of the targeted drug liposome.

[0026] A method for preparing the targeted drug liposome comprises the following steps:

[0027] S1, dissolving the glycocalyx shedding enzyme inhibitor and lipid material in an organic solvent, and preparing cationic liposomes loaded with the glycocalyx shedding enzyme inhibitor by thin film ultrasonic dispersion method;

[0028] S2, adding the adhesion receptor dipeptidase-1 binding peptide to the cationic liposome prepared in step S1, so that the thiol group on the peptide segment and the reactive group on the cationic liposome undergo nucleophilic addition reaction.

[0029] Preferably, the reactive group is maleimide.

[0030] The present invention further provides a pharmaceutical use of the targeted drug liposome, wherein the adhesion receptor dipeptidase-1 binding peptide specifically binds to the adhesion receptor dipeptidase produced by pulmonary vascular endothelial cells, thereby achieving passive targeting of the liposomes to the pulmonary vascular endothelial cells, and promoting the normalization of pulmonary blood vessels by inhibiting glycocalyx shedding; at the same time, the adhesion receptor dipeptidase-1 binding peptide competitively binds to the adhesion receptor dipeptidase with neutrophils, thereby reducing the recruitment of neutrophils in the lungs, improving the microenvironment before lung tumor metastasis, and reducing tumor metastasis to the lungs.

[0031] Preferably, the drug is an intravenous injection.

[0032] The beneficial effects of the present invention are:

[0033] The cationic liposomes prepared by the present invention have high targeting, can inhibit the shedding of glycocalyx of vascular endothelial cells, improve the microenvironment before tumor metastasis, and reduce tumor metastasis to the lungs. At the same time, the present invention can also be used for the treatment of lung inflammation and vascular lesions, and has broad clinical application prospects.

[0034] The cationic liposome prepared by the invention also has the advantages of good sustained-release effect, good stability, high encapsulation rate and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 :Distribution of LSAIT-Lip@Dex in vivo.

[0036] Figure 2 : Fluorescence quantification results of LSAIT-Lip@Dex distribution in the lung.

[0037] Figure 3 : Results of in vitro release rate determination of liposomes.

[0038] Figure 4 : In vitro repair effect of liposomes on endothelial glycocalyx shedding.

[0039] Figure 5 : H&E staining results of the lungs of TCM model mice. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is described in detail below in conjunction with specific implementation methods so that those skilled in the art can better understand the present invention and implement it.

[0041] The raw materials used in this embodiment include:

[0042] Dexamethasone (Dex, purchased from Shanghai MacLean Biochemical Technology Co., Ltd., batch number 201424, purity ≥ 99%): Dexamethasone is a glycocalyx shedding enzyme inhibitor and can be used to inhibit endothelial glycocalyx shedding.

[0043] Dimyristoylphosphatidylethanolamine (DMPE, purchased from CordenPharma, Switzerland): DMPE is a lipid material, one of the phosphatidylethanolamine derivatives, and is commonly used for coupling with polyethylene glycol to form circulating liposomes. The phosphatidylethanolamine derivatives that can be used in the present invention also include distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dilauroylphosphatidylethanolamine (DLPE), etc.

[0044] Dimyristoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (MAL-PEG 2000 -DMPE, Xi'an Ruixi Biotechnology Co., Ltd.): It is a reactive long-circulating liposome material, in which maleimide is a reactive group, which can be connected to a thiol-containing polypeptide by reacting with a thiol group; and the PEGylated phospholipid has excellent amphiphilicity and can be used for drug delivery to prolong the blood circulation time of the drug. The reactive long-circulating liposome materials that can be used in the present invention also include MAL-PEG 2000 -DSPE, MAL-PEG 2000 -DOPE, MAL-PEG 2000 -DPPE, MAL-PEG 2000 -DLPE, etc.

[0045] Adhesion receptor dipeptidase binding peptide (LSAIT, synthesized by Shanghai Jier Biochemical Co., Ltd.): The sequence is CGFECVRQCPERC. LSAIT peptide is a polypeptide that can specifically bind to adhesion receptor dipeptidase.

[0046] (2,3-Dioleoyl-propyl)-trimethylammonium chloride (DOTAP, purchased from CordenPharma, Switzerland): DOTAP is a cationic lipid that can promote the fusion of liposomes with cell membranes, thereby delivering drugs, DNA or other molecules into cells. Cationic lipid materials that can be used in the present invention also include 1,2-dioctadecyloxy-3-methylammonium propane chloride (DOTMA), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), dimethyldioctadecyl ammonium bromide (DDAB), etc.

[0047] 4-Hydroxyethylpiperazineethanesulfonic acid buffer (HEPES buffer, Beijing Solebow Technology Co., Ltd.): It is a hydrogen ion buffer that can control a constant pH range for a long time.

[0048] Dichloromethane (DCM, Chengdu Kelong Chemicals Co., Ltd.): It is an excellent solvent with a wide range of uses.

[0049] Tumor necrosis factor (TNF-α, Dalian Meilun Biotechnology): Tumor necrosis factor is an inflammatory factor that can be used to construct a HUVEC cell inflammation model.

[0050] Wheat germ agglutinin labeled with fluorescein isothiocyanate (FITC-WGA, Shanghai Maokang Biotechnology Co., Ltd.): FITC is a fluorescent dye, and WGA can specifically bind to a glycoprotein on the endothelial cell membrane, that is, it can specifically bind to the glycocalyx. Combining FITC and WGA can mark the glycocalyx on the endothelial cell membrane.

[0051] Phosphate buffered saline (PBS, Wuhan Sewell Biotechnology Co., Ltd.): is a water-based salt solution that helps maintain a constant pH. The osmotic pressure and ion concentration of the solution are usually close to the human body pH (isotonic).

[0052] Hematoxylin and eosin stain (HE stain, Biosharp): It is a tissue stain based on the chemical reaction of hematoxylin and eosin with tissue components, thereby distinguishing and displaying different cell structures and tissue types.

[0053] Human umbilical vein endothelial cells (HUVECs cells, provided by Sichuan University): used for cell experiments.

[0054] Other materials not described herein are conventional materials in the art.

[0055] Example 1 Preparation of LSAIT peptide-modified and Dex-loaded cationic liposomes

[0056] Preparation prescription:

[0057]

[0058] Preparation method:

[0059] In the first step, cationic liposomes Lip@Dex loaded with Dex were prepared by thin film ultrasonic dispersion method.

[0060] 4 mg DMPE, 2 mg DMPE-PEG, 1.7 mg DOTAP, 0.8 mg DMPE-PEG-Mal and 0.5 mg Dex were dissolved in 10 mL of dichloromethane / methanol (methanol: dichloromethane = 1:2, v / v); the organic solvent was removed under reduced pressure at 30°C to obtain a lipid film; the film was hydrated with injectable saline; and the obtained suspension was dispersed with probe ultrasound to obtain cationic liposomes Lip@Dex loaded with Dex.

[0061] In the second step, the LSALT peptide was modified onto cationic liposomes to obtain LSALT-Lip@Dex

[0062] 0.4 M HEPES buffer (pH = 7.4) and an aqueous solution of the formulated amount of LSALT were added to the liposome suspension prepared in the first step, and the mixture was incubated at 37°C overnight to allow the thiol group on the LSALT peptide to undergo a nucleophilic addition reaction with the maleic group on the DMPE-PEG-Mal, thereby connecting LSAIT to Lip@Dex to obtain LSALT-Lip@Dex.

[0063] Example 2 Screening and Optimization of Preparation Prescription

[0064] The effects of different factors on liposomes were evaluated using particle size, potential, PDI, encapsulation efficiency and drug loading as indicators. In addition, the content of LSAIT was investigated using particle size, PDI and in vivo targeting as evaluation indicators.

[0065] (1) Optimization of the ratio of DOTAP to other lipids

[0066] In the first step of the preparation method, while keeping other preparation factors unchanged, the ratio of DOTAP to other lipids (DMPE, DMPE-PEG and DMPE-PEG-Mal) was changed, and different ratios of DOTAP: other lipids (0:8, 1:8, 2:8, 3:8) were selected to investigate the effect of the ratio of DOTAP to other lipids on the particle size and encapsulation efficiency of Lip@Dex, and to determine the optimal ratio of DOTAP and other lipids for preparing Lip@Dex.

[0067] The results are shown in Table 1.

[0068] Table 1 Results of the effects of DOTAP and other lipids on Lip@Dex

[0069]

[0070] When the ratio of DOTAP: other lipids is 0:8, Lip@Dex shows a negative potential, and the greater the DOTAP content in Lip@Dex, the greater its zeta potential, because DOTAP is a cationic lipid material. From the particle size comparison, it can be seen that the addition of DOTAP will increase the liposome particle size, and DOTAP itself has certain toxicity, so the preferred DOTAP: other lipid ratio is 2:8.

[0071] (2) Optimization of the ratio of DMPE to long-circulating lipids

[0072] In the first step of the preparation method, while keeping other preparation factors unchanged, the ratio of DMPE to long-circulating lipid materials (DMPE-PEG and DMPE-PEG-Mal) was changed to investigate the effect of the DMPE ratio on the particle size and encapsulation efficiency of Lip@Dex, and to determine the optimal DMPE ratio for preparing Lip@Dex.

[0073] The results are shown in Table 2.

[0074] Table 2 Results of the effects of DMPE: long-circulating lipids on Lip@Dex

[0075]

[0076] In Lip@Dex, the greater the content of long-circulating lipid materials, the smaller the liposome particle size and the smaller the zeta potential. And the more DMPE-PEG is added, the smaller the Lip@Dex encapsulation efficiency, so the preferred ratio of DMPE to long-circulating lipid materials (DMPE-PEG and DMPE-PEG-Mal) is 20:14.

[0077] (3) Optimization of the ratio of DMPE-PEG and DMPE-PEG-Mal

[0078] In the first step of the preparation method, while keeping other preparation factors unchanged, the ratio of DMPE-PEG and DMPE-PEG-Mal was changed to investigate the effect of the ratio of DMPE-PEG and DMPE-PEG-Mal on the particle size and encapsulation efficiency of Lip@Dex, and to determine the optimal ratio for preparing Lip@Dex.

[0079] The results are shown in Table 3.

[0080] Table 3 Effects of DMPE-PEG:DMPE-PEG-Mal on Lip@Dex

[0081]

[0082] The results show that the content of DMPE-PEG-Mal has little effect on the particle size, potential, encapsulation efficiency and drug loading of cationic liposomes, but a certain content of DMPE-PEG-Mal is beneficial to the binding of LSAIT peptide.

[0083] (4) Optimization of the ratio of Dex to lipids

[0084] In the first step of the preparation method, while keeping other preparation factors unchanged, the ratio of drug to lipid was changed, and different ratios of Dex and lipid (DOTAP, DMPE-PEG, DMPE) were selected to investigate the effect of the drug-lipid ratio on the particle size and encapsulation efficiency of Lip@Dex, and to determine the optimal ratio of drug to lipid for preparing Lip@Dex.

[0085] The results are shown in Table 4.

[0086] Table 4 Effect of drug-lipid ratio on Lip@Dex

[0087] Dex: Lipids Size(nm) PDI Zeta(mV) EE(%) DE(%) 2:68 88.89±9.69 0.258±0.01 25.23±0.39 95.93±0.12 2.09±0.14 5:68 92.18±7.21 0.213±0.01 26.54±0.66 90.99±0.98 4.78±0.23 8:68 88.55±5.39 0.210±0.03 29.37±0.48 83.70±0.79 7.40±0.15 10:68 89.90±1.53 0.242±0.01 29.51±0.92 76.32±0.43 8.00±0.27

[0088] From the above results, we can see that with the increase of Dex, the encapsulation rate gradually decreased, the drug loading gradually increased, and the effect on the liposome particle size and potential was not significant. When the drug-lipid ratio was 2:68, the encapsulation rate was the largest. Considering the subsequent animal administration dose, we chose a drug-lipid ratio of 5:68 with a higher drug loading.

[0089] (5) LSAIT peptide dosage screening test

[0090] In the second step of the preparation method, the content of LSAIT was changed while other preparation factors were fixed, and different contents of LSAIT were selected to investigate their effects on the particle size and encapsulation efficiency of Lip@Dex. LSAIT-Lip@Dex with LSAIT contents of 0%, 5%, 10%, and 20% were prepared, and named 0% LSAIT-Lip@Dex, 5% LSAIT-Lip@Dex, 10% LSAIT-Lip@Dex, and 20% LSAIT-Lip@Dex.

[0091] The results are shown in Table 5.

[0092] Table 5 Effect of LSAIT content on LSAIT-Lip@Dex

[0093] LSAITcontent Size(nm) PDI EE(%) DE(%) 0％ 72.22±3.19 0.260±0.01 90.93±0.30 4.88±0.35 5％ 108.55±1.63 0.255±0.02 92.98±0.71 5.27±0.16 10％ 152.67±1.22 0.218±0.10 90.69±1.24 4.48±0.16 20％ 207.09±2.80 0.165±0.01 79.78±0.29 4.19±0.12

[0094] The higher the LSAIT content, the greater the amount of LSAIT combined with Lip@Dex and the larger the particle size. The particle size of 20% LSAIT-Lip@Dex is greater than 200nm, which no longer meets the requirements for intravenous administration of the preparation.

[0095] Cy5-labeled 0% LSAIT-Lip@Dex, 5% LSAIT-Lip@Dex, 10% LSAIT-Lip@Dex, and 20% LSAIT-Lip@Dex were prepared respectively, and a mouse model of the microenvironment before tumor metastasis was constructed by tail vein injection of TCM (the specific method of TCM preparation is shown in Example 4). 24 hours after modeling, 200 μL of the above 4 liposomes were injected into the model mice through the tail vein. The mice were euthanized 2 hours after administration, and the mouse heart, liver, spleen, lung, and kidney were collected. The in vitro tissues were imaged by the living imaging system to measure the fluorescence intensity in the five internal organs. The total fluorescence intensity was processed by Living imaging software. The experiment was divided into 4 groups: 0% LSAIT-Lip@Dex, 5% LSAIT-Lip@Dex, 10% LSAIT-Lip@Dex, and 20% LSAIT-Lip@Dex.

[0096] The results are as follows Figure 1 , 2 shown.

[0097] In the microenvironment before tumor metastasis, inflammation occurs, inducing high expression of DPEP1 receptors on the surface of vascular endothelial cells, allowing LSAIT-Lip@Dex to target vascular endothelial cells. As the LSAIT content increases, LSAIT-Lip@Dex binds more to DPEP1, and the distribution of LSAIT-Lip@Dex in organs is also enhanced ( Figure 1 ). The liver is the main metabolic organ and an important metastatic organ, so LSAIT-Lip@Dex accumulates more in the liver. Lung tissue is the main organ for tumor metastasis, and inflammation in the lungs is severe, so there is also a lot of LSAIT-Lip@Dex accumulated in the lungs. When the LSAIT content is 20%, the liposomes are more distributed in the lung tissue ( Figure 2 ), but the liposome particle size is large and not suitable for intravenous injection. Considering the safety and compliance of intravenous injection, 10% LSAIT-Lip@Dex was finally selected.

[0098] LSAIT-Lip@Dex was prepared with the optimal ratio obtained above: 4.0mg DMPE, 2.0mg DMPE-PEG, 1.7mg DOTAP, 0.8mg DMPE-PEG-Mal, 1.0mg LSAIT and 0.5mg Dex were prepared into liposomes, and the particle size and potential of the nanoparticles were measured using the ZEN3600 series nano-laser particle size analyzer. 0.1mL of liposomes were diluted to 1mL, the temperature of the nano-particle size analyzer was set to 25℃, the equilibrium time was 2min, and the measurement was repeated 3 times. The particle size of LSAIT-Lip@Dex was measured to be 152.67±1.22nm, PDI was 0.218±0.01, and the potential was 18.07±0.19mV, which can meet the needs of intravenous injection. The ultrafiltration method was used to determine the encapsulation efficiency and drug loading of liposomes. 1 mL of liposomes was added to an ultrafiltration tube with a molecular weight cutoff of 3000 Da at 3000 rpm / min. After centrifugation for 20 minutes, the concentration of free Dex was determined. The encapsulation efficiency of LSAIT-Lip@Dex was calculated to be 92.12±1.07%, and the drug loading was 4.79±0.76%.

[0099] Example 3 In vitro release rate determination

[0100] 1.5 mL of the pre-prepared Lip@Dex, LSAIT-Lip@Dex and free Dex solution (containing 0.15 mg of Dex) were added to a dialysis bag with a molecular weight cutoff of 3000 Da. The two ends of the dialysis bag were tied with a thin rope and placed in a 50 mL EP tube with 45 mL PBS buffer. The EP tube was placed in an air bath shaker away from light. Three parallel samples were set for each group. The entire in vitro release process was carried out at a constant temperature of 37 ° C, and the oscillation frequency of the air bath shaker was set to a constant speed of 70 r / min. The timing started from the time the sample was placed in, and 1 mL of samples were taken at 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h, and 1 mL of fresh release medium was added at the same time. Finally, HPLC injection was used for investigation.

[0101] The experimental results are as follows Figure 3 shown.

[0102] At 2 hours, the cumulative release rate of Dex reached 70% and there was no obvious release afterwards, while the cumulative release rates of Lip@Dex and LSAIT-Lip@Dex were about 50% and 45%. Within 48 hours, Lip@Dex and LSAIT-Lip@Dex continuously released Dex, with a cumulative release rate of 70%, and there was still a release trend afterwards. Compared with Dex, Lip@Dex and LSAIT-Lip@Dex had obvious sustained-release effects, and LSAIT modification to the surface of Lip@Dex did not significantly affect the release rate of Dex.

[0103] Example 4 Efficacy Verification

[0104] (1) Evaluation of the repair effect of endothelial glycocalyx in vitro

[0105] HUVEC cells were cultured in a constant temperature (37°C, 5% CO2 concentration) with DMEM medium containing 10% fetal bovine serum and 1% double antibody, and 5×10 4 Each well was inoculated in a 24-well plate. After culturing for 24 hours, the culture medium in the well plate was removed and 0.5 mL of DMEM basal medium containing 25 ng / mL TNF-α and related preparations (Dex concentration was 4 μg / mL and LSAIT concentration was 8 μg / mL) was added, that is, the drug was administered for 24 hours while modeling. 0.5 mL of PBS was added to wash the cells twice, and then 100 μL of 2 μg / mL FITC-labeled wheat germ agglutinin (FITC-WGA) was added and incubated with the cells for 2 hours to mark the endothelial glycocalyx. Thereafter, the cells were washed 3 times with 0.5 mL PBS, and the fluorescence intensity was observed using an inverted fluorescence microscope. The experiment was divided into 6 groups: Control group, PBS group, LSAIT-Lip@Dex group, Dex group, Lip@Dex group, and LSAIT group.

[0106] Control group: cells were not incubated with liposomes and TNF-α, but directly incubated with FITC-WGA;

[0107] PBS group: cells were incubated with PBS and TNF-α, and then incubated with FITC-WGA;

[0108] LSAIT-Lip@Dex group: cells were incubated with liposome LSAIT-Lip@Dex and TNF-α, and finally incubated with FITC-WGA;

[0109] Dex group: cells were incubated with Dex and TNF-α, and finally incubated with FITC-WGA;

[0110] Lip@Dex group: cells were incubated with liposomes Lip@Dex and TNF-α, and finally incubated with FITC-WGA;

[0111] LSAIT group: cells were incubated with LSAIT and TNF-α, and finally incubated with FITC-WGA.

[0112] The experimental results are as follows Figure 4 shown.

[0113] Both LSAIT and Dex have inhibitory effects on the shedding of endothelial glycocalyx, but the effects are not significant. Dex is a glucocorticoid anti-inflammatory drug that has a certain inhibitory effect on the HUVEC inflammation model caused by TNF-α. LSAIT inhibits glycocalyx shedding by inhibiting neutrophil adhesion. Neutrophils were not added in this experiment, so LSAIT had no obvious inhibitory effect on the TNF-α-induced HUVEC inflammation model. Encapsulating Dex in liposomes and linking it to LSAIT can greatly enhance the inhibitory effect on endothelial glycocalyx shedding, which is mainly due to the fact that LSAIT increases the uptake of Dex by HUVEC.

[0114] (2) In vivo evaluation of lung pathology

[0115] Preparation of TCM: Mouse breast cancer cells were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum at 100 cm 2The cells were cultured in a cell culture dish. When the cells grew to 80%, they were replaced with 4 mL RPMI 1640 basal medium and cultured for 14-17 hours. The upper culture medium was collected, which was TCM. The mice were intravenously injected with 200 μL TCM, and 2 hours later, 200 μL of drugs (Dex concentration was 0.1 mg / mL, liposome concentration was 2 mg / mL, and LSAIT concentration was 0.2 mg / mL). The mice were intravenously injected with TCM and drugs for 3 consecutive days. Two hours after the last administration on the last day of administration, the mice were euthanized, the lung tissue was removed, washed with 0.9% saline, and fixed with fixative for more than 24 hours. The tissue was flattened and placed in a dehydration box. Place the dehydration box in the tissue dehydrator and dehydrate in sequence: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 10 minutes, xylene II for 10 minutes, wax I for 1 hour, wax II for 1 hour, wax III for 1 hour. Embed the waxed tissue in the embedding machine and label it. Use a paraffin slicer to slice, 3 μm thick. Flatten the slices in 40°C warm water on the spreader and bake in an oven at 60°C. Put the slices in xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and pure water for 5 minutes to dewax until water. The slices were stained with hematoxylin for 3min 30s, washed with water, differentiated with 1% hydrochloric acid alcohol, washed with water, blued with 1% ammonia aqueous solution, and washed with water. The slices were placed in 85% alcohol for 5min, 95% alcohol for 5min, anhydrous ethanol for 5min, and eosin staining solution for 2min. The slices were placed in anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, anhydrous ethanol III for 5min, xylene I for 5min, xylene II for 5min, and xylene III for 5min, and sealed with neutral gum. The lung tissues were dehydrated, embedded, sliced, dewaxed, stained with H&E (hematoxylin and eosin), washed, and then observed under a microscope. The weight of the mice was recorded every day of the experiment. The experiment was divided into 6 groups: Control group, PBS group, LSAIT-Lip@Dex group, Dex group, Lip@Dex group, and LSAIT group.

[0116] Control group: normal mice without modeling.

[0117] PBS group: model mice were treated with PBS after modeling.

[0118] LSAIT-Lip@Dex group: mice treated with LSAIT-Lip@Dex after modeling.

[0119] Dex group: mice treated with Dex after model establishment.

[0120] Lip@Dex group: mice treated with Lip@Dex after modeling.

[0121] LSAIT group: mice treated with LSAIT after model establishment.

[0122] The experimental results are as follows Figure 5 shown.

[0123] Depend on Figure 5 It can be seen that after pathological examination, the severity of the pathological conditions is as follows: PBS group>Dex group>LSAIT group≈Lip@Dex group>LSAIT-Lip@Dex group>Control group. No obvious lesions were found in the lungs of the Control group, and the structure was normal. A small amount of edema was observed in the lung tissues of LSAIT-Lip@Dex; a small amount of airway wall thickening, a small amount of alveolar collapse, inflammatory cell infiltration and edema were observed in the lung tissues of the Lip@Dex group and the LSAIT group; a small amount of inflammatory cell infiltration, airway wall thickening and bleeding, and a small amount of alveolar collapse were observed in the lung tissues of the Dex group; the lesions in the PBS group were the most serious, with a small amount of inflammatory cell infiltration, a large amount of alveolar collapse, and a large amount of bleeding. The above pathological conditions can show that LSAIT-Lip@Dex can effectively reduce the occurrence of lung inflammation and inhibit the formation of a pre-metastatic microenvironment.

[0124] It should be understood that the specific embodiments listed in this specification are only used to explain the present invention, rather than to limit the protection scope of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the above embodiments should also be considered to fall within the protection scope of the present invention.

Claims

1. A targeted drug liposome that inhibits glycocalyx shedding of pulmonary vascular endothelial cells and improves the microenvironment before tumor metastasis, characterized in that: The invention is composed of a glycocalyx shedding enzyme inhibitor, a lipid material, and an adhesion receptor dipeptidase-1 binding peptide. The lipid material includes a phosphatidylethanolamine derivative, a long-circulating lipid material, a reactive long-circulating lipid material capable of connecting to a peptide segment, and a cationic lipid material. The glycocalyx shedding enzyme inhibitor includes a glucocorticoid. The amino acid sequence of the adhesion receptor dipeptidase-1 binding peptide is CGFECVRQCPERC.

2. The targeted drug liposome according to claim 1, wherein: The phosphatidylethanolamine derivatives include DMPE, DSPE, DOPE, DPPE and DLPE.

3. The targeted drug liposome according to claim 1, characterized in that: The long-circulating lipid material is a phosphatidylethanolamine derivative-PEG copolymer, including DMPE-PEG, DSPE-PEG, DOPE-PEG, DPPE-PEG, and DLPE-PEG.

4. The targeted drug liposome according to claim 1, characterized in that: The cationic lipid materials include DOTAP, DOTMA, DOSPA and DDAB.

5. The targeted drug liposome according to claim 1, characterized in that: The glycocalyx shedding enzyme inhibitor accounts for 2-10% of the mass of the targeted drug liposome; the adhesion receptor dipeptidase-1 binding peptide accounts for 2-15% of the mass of the targeted drug liposome; and the rest is lipid material.

6. The targeted drug liposome according to claim 5, characterized in that: The phosphatidylethanolamine derivative accounts for 30-50% of the mass of the targeted drug liposome; the long-circulating lipid material accounts for 10-30% of the mass of the targeted drug liposome; the reactive long-circulating lipid material accounts for 5-15% of the mass of the targeted drug liposome; and the cationic lipid material accounts for 10-30% of the mass of the targeted drug liposome.

7. A method for preparing the targeted drug liposome according to any one of claims 1 to 6, characterized in that The following steps are involved: S1, dissolving the glycocalyx shedding enzyme inhibitor and lipid material in an organic solvent, and preparing cationic liposomes loaded with the glycocalyx shedding enzyme inhibitor by thin film ultrasonic dispersion method; S2, adding the adhesion receptor dipeptidase-1 binding peptide to the cationic liposome prepared in step S1, so that the thiol group on the peptide segment and the reactive group on the cationic liposome undergo nucleophilic addition reaction.

8. The preparation method according to claim 7, characterized in that: The reactive group is maleimide.

9. The pharmaceutical use of the targeted drug liposome according to claim 1, characterized in that: The adhesion receptor dipeptidase-1 binding peptide specifically binds to the adhesion receptor dipeptidase produced by pulmonary vascular endothelial cells, thereby achieving passive targeting of liposomes to pulmonary vascular endothelial cells and promoting normalization of pulmonary blood vessels by inhibiting glycocalyx shedding; at the same time, the adhesion receptor dipeptidase-1 binding peptide competitively binds to the adhesion receptor dipeptidase with neutrophils, thereby reducing the recruitment of neutrophils in the lungs, improving the microenvironment before lung tumor metastasis, and reducing tumor metastasis to the lungs.

10. The use according to claim 9, characterized in that: The medicine is for intravenous injection.

Images