CN108653743B - Cardio-cerebral dual-targeting liposome and preparation method and application thereof - Google Patents

Cardio-cerebral dual-targeting liposome and preparation method and application thereof Download PDF

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CN108653743B
CN108653743B CN201810299429.3A CN201810299429A CN108653743B CN 108653743 B CN108653743 B CN 108653743B CN 201810299429 A CN201810299429 A CN 201810299429A CN 108653743 B CN108653743 B CN 108653743B
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polyethylene glycol
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杨宝峰
彭海生
刘肖莹
李明慧
廉明明
唐淑坤
刘云翠
柴彦群
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Harbin Medical University
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Abstract

The invention discloses a heart brain double-targeting liposome and a preparation method and application thereof. The heart brain double-targeting liposome is obtained by coupling Anti-cTnI protein antibody or fragment thereof on the surface of the liposome modified by acetylated glycolipid-polyethylene glycol-phosphatidylethanolamine conjugate shown in the formula I. The invention modifies mannose on the surface of liposomeThe molecular derivative and the Anti-cTnI protein antibody enable the liposome to have double targeting effects of brain targeting and heart targeting, and can realize simultaneous treatment of the heart and the brain. The experiments of in vitro cell test, affinity test and in vivo distribution prove that the liposome can treat the myocardial diseases and can cross the blood brain barrier to treat the encephalopathy caused by the cardiogenic diseases. In addition, the spatial positions of sugar molecule derivatives and antibodies can be changed by changing the length of PEG (linker) on the surface of the liposome, so that the distribution of the drug in heart and brain tissues is changed, the distribution change of the drug in the heart and the brain is regulated, and the individual treatment is realized.

Description

Cardio-cerebral dual-targeting liposome and preparation method and application thereof
Technical Field
The invention relates to a liposome used as a drug carrier, a preparation method and application thereof, in particular to a liposome which has the double-targeting effect of brain and heart and can realize simultaneous treatment of heart and brain and a preparation method thereof. The invention belongs to the technical field of medicines.
Background
Cardiovascular and cerebrovascular diseases (aspen syndrome, insufficient blood supply to heart and brain, etc.) seriously threaten human health, and have higher mortality and morbidity. Cardiovascular and cerebrovascular diseases are accompanied by insufficient blood supply to the heart and brain. Due to myocardial ischemia, the heart is in a state of energy deficiency, which may cause arrhythmia and angina pectoris. When this occurs, acute myocardial infarction, often resulting in cardiogenic shock, can also result. At the same time, the blood supply to the brain is also reduced by abnormal cardiac function due to the decrease in cardiac output, and sometimes worsens to cardiogenic stroke. The treatment of cardiogenic stroke mainly relies on the normal function of the heart and the restoration of blood supply.
In recent years, researchers have investigated a number of nanotechnology-based strategies to effectively deliver drugs to the focus to restore function to an infarcted heart. In these strategies, the surface of the nanocarriers is typically modified by selecting molecules whose ligands bind to receptors, antibodies or antigens, and which are transported by transporters, to increase the specificity of the nanoparticles for the ischemic heart. Despite some improvements, most of the particles still accumulate in the reticuloendothelial system and reach the target tissue prematurely. To overcome these problems, some groups of subjects have designed some dual-target nanocarriers, such as transporters, antibodies and receptors, based on two different biological phenomena and mechanisms.
The inventor of the invention has proved that the Anti-cTnI antibody (Anti-cTnI, a specific monoclonal antibody) modified on the surface of the liposome can improve the drug concentration of the myocardial part. However, the rate of uptake of Anti-cTnI antibody-modified liposomes by cardiomyocytes was not faster than that of ordinary liposomes. Glucose transporters (GLUT) can transport glucose, fructose, galactose, mannose and other substances with similar structures through the Blood Brain Barrier (BBB). The mannose-modified liposome shows excellent brain targeting, and the mannose-modified liposome can rapidly enter target cells. These findings suggest that if we simultaneously modify both antibody and mannose on the liposome surface, the internalization rate of the vesicles into the affected cells will be accelerated, saving those cells destined to die.
Polyethylene glycol (PEG) with a 2000Da chain is the first choice for many researchers, due to the availability of the market, and is readily used to conjugate targeting molecules to the surface of vesicles by click chemistry. The varying density and chain length of polyethylene glycol that modifies liposomes leads to different possibilities for intermolecular interactions and cellular uptake and targeted accumulation. However, steric hindrance of various molecules on the surface of lipid membrane may limit the binding of ligand and its receptor, thereby affecting the realization of targeting efficiency of nanocarrier. Compared with the common medicament, the targeted medicament delivery has the remarkable characteristics of low dosage and high curative effect, and has important significance for treating the central nervous system diseases. Although myocardial targeted therapies have been explored for many years, there are several side effects and inefficiencies challenges. In addition, attention has always been focused on drug concentrations in individual targeted organs and/or tissues, ignoring some of the chain reactions in the human body. Cardiogenic stroke may require treatment of the brain and ischemic heart, systematically ensuring that the function of the heart and brain is not affected by the ischemic state, reducing subsequent catastrophes. Therefore, how to achieve rational drug delivery based on the steric hindrance of the above-mentioned PEG chain to achieve the requirements of the affected heart and brain is an attractive research hotspot.
Disclosure of Invention
The invention aims to provide a liposome which has brain and heart dual-targeting effects and can realize simultaneous treatment of the brain and the heart and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a heart brain double-targeting liposome (Anti-cTnI Ab-PEG/Ac)4MAN-PEG-LIP), is a liposome (Ac) modified by acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate shown in formula I4MAN-PEG-LIP) surface coupled with Anti-cTnI protein antibodies or fragments thereof;
Figure GDA0002446785310000021
wherein n is 10-120, preferably 13-45, and more preferably, the polyethylene glycol is polyethylene glycol 600, polyethylene glycol 1000 or polyethylene glycol 2000.
Among them, preferably, the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate represented by formula I is prepared by reacting p-carboxyphenyl- α -D-acetylmannosamine (Ac) in dichloromethane4MAN) and polyethylene glycol (PEG) by condensation reaction to form PEG-Ac4MAN followed by Distearoylphosphatidylethanolamine (DSPE) with PEG-Ac4MAN is coupled to obtain DSPE-PEG-Ac4MAN, namely the compound shown in the formula I.
Of these, preferably, the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of formula I is prepared by dissolving azide-polyethylene glycol-glutaramide in tetrahydrofuran, adding Pd/c and acetic acid, introducing hydrogen at room temperature, reacting overnight, filtering the crude product, rotary evaporating to dryness, dissolving in dichloromethane, adding triethylamine and p-carboxyphenyl- α -D-acetylmannosamine (Ac)4MAN) at room temperature, separating and purifying to obtain PEG-Ac4MAN; dissolving PEG-Ac4MAN in dichloromethane, sequentially adding N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), reacting at room temperature overnight, separating and purifying to obtain succinimide polyethylene glycol acetylmannoside; dissolving succinimidyl polyethylene glycol acetyl mannoside in dichloromethane, adding TEA and distearoyl phosphatidyl ethanolamine (DSPE) dissolved in chloroform, reacting at room temperature overnight, extracting with saturated NaCl for 2 times, separating and purifying to obtain DSPE-PEG-Ac4MAN, namely the compound shown in the formula I.
Furthermore, the invention also provides a method for preparing the heart-brain double-targeting liposome, which comprises the following steps:
(1) liposome (Ac) modified by acetylated glycolipid-polyethylene glycol-phosphatidylethanolamine conjugate4MAN-PEG-LIP) preparation
Dissolving egg yolk phosphatidylcholine (EPC), Cholesterol (CHO) and the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of formula I in a spinner flask with anhydrous ethanol, forming a thin lipid film by evaporating the mixture under reduced pressure; hydrating the lipid film with a physiological saline solution; dispersing the liposome solution by ultrasonic treatment, and extruding through a polycarbonate membrane to obtain the liposome modified by the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate, which is named Ac4MAN-PEG-LIP, storing liposome sample at 4 deg.C;
(2) preparation of Anti-cTnI antibody modified liposome (Anti-cTnI Ab-PEG/Ac)4MAN-PEG-LIP)
Synthesizing an Anti-cTnI Ab-DSPE-PEG solution: respectively dissolving the Anti-cTnI antibody and the DSPE-PEG-maleimide in HEPES solution, and then mixing at 4 ℃ overnight to obtain an Anti-cTnI Ab-DSPE-PEG solution; mixing Anti-cTnI Ab-DSPE-PEG solution with Ac prepared in step (1)4The MAN-PEG-LIP liposome solution is incubated for 2 hours at 37 ℃ to prepare Ac modified by Anti-cTnI antibody4MAN-PEG-LIP, named Anti-cTnI-PEG/Ac4MAN-PEG-LIP is the cardio-cerebral dual-targeting liposome.
Wherein, the mole ratio of EPC, CHO and the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate shown in the formula I in the step (1) is preferably (40-49): (50-55): (1-5), more preferably 49: 50: 1.
wherein, Anti-cTnI Ab-DSPE-PEG and Ac in the step (2)4The molar ratio of the MAN-PEG-LIP liposome is (1-100): (50-1000), more preferably 1: 500.
among them, it is preferable that the DSPE-PEG-maleimide in the step (2) is DSPE-PEG 600-maleimide, DSPE-PEG 1000-maleimide or DSPE-PEG 2000-maleimide.
Furthermore, the invention also provides application of the heart-brain double-targeting liposome in preparation of a drug carrier.
The cardio-cerebral dual-targeting liposome has the dual-targeting effect on the brain and the heart, so that the aim of treating both the brain and the heart is fulfilled.
Preferably, the medicament is used for treating myocardial diseases and cardiogenic encephalopathy, and more preferably, the cardiogenic encephalopathy comprises cardiogenic cerebral ischemia syndrome, cardiogenic cerebral insufficiency and cardiogenic cerebral apoplexy.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the mannose molecular derivative and the Anti-cTnI protein antibody are modified on the surface of the liposome, so that the liposome has the brain-targeting and heart-targeting dual-targeting effects, and the cardio-cerebral simultaneous treatment can be realized. The experiments of in vitro cell test, affinity test and in vivo distribution prove that the liposome can treat the myocardial diseases and can cross the blood brain barrier to treat the encephalopathy caused by the cardiogenic diseases.
2. The spatial positions of sugar molecule derivatives and antibodies can be changed by changing the length of PEG (linker) on the surface of the liposome, so that the distribution of the drug in heart and brain tissues is changed, the distribution change of the drug in heart and brain is regulated, and the individual treatment is realized.
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FIG. 1 shows DSPE-PEG600-Ac4MAN、DSPE-PEG1000-Ac4MAN and DSPE-PEG2000-Ac4A nuclear magnetic resonance hydrogen spectrum and a carbon spectrum of MAN;
FIG. 2A is Ac4Schematic diagram of M micelle modified ATO liposome (PEG1000 is taken as an example);
FIG. 2B shows a reaction scheme for the reaction between Ni (OAC)2A green deposit was clearly observed when ATO solution was added to the solution, indicating ATO and Ni (OAC)2At Ac4Presence status in MAN modified liposomes;
FIG. 3 shows the determination of ATO and Ni (OAC) at 37 ℃ of liposomes in PBS at pH7.42The in vitro cumulative release rate profile of (a);
(A) ATO in ATO-LIP and Ac4Release profile in M-ATO-LIP (PEG1000 as an example); (B) ni (OAC)2In Ni-LIP and Ac4Release profile in M-Ni-LIP; data are expressed as mean ± standard deviation (n ═ 3);
FIG. 4 is a study of the growth inhibitory effect of different liposomes on glioma cells;
the survival rate of U87 cells is detected after adding drugs of 48h (A, a), 72h (B, B) and 96h (C, C) to ATO, ATO-LIP and Ac4M-ATO-LIP (taking PEG1000 as an example) at different concentrations by an MTT method and a cell imaging method respectively; ATO concentrations were 40.6, 81.5, 162.5, 325, 650 and 1300 μ M, respectively, with data as mean ± standard deviation (n ═ 3); (scale, 300 nm);
FIG. 5 is a graph showing the in vitro drug uptake using flow cytometry;
wherein, fig. 5A is an uptake assay: the histogram peaks shifted to the right, indicating an increased uptake of Ac4M-ATO-LIP (PEG1000 for example) by U87;
fig. 5B is a statistical analysis: the fluorescence intensity of each formulation is represented by a bar graph; data were normalized and expressed as mean ± sd (n-3, p <0.001, compared to cellular uptake of Rho-LIP);
FIG. 6A is a live cell image taken during incubation of U87 glioma cells with Rho-labeled Ac4MAN-PEG1000-LIP for 0-20 min; (the red part is Rho-labeled liposomes; green is DIO-stained cell membrane; blue is Hoechst 33258-stained nuclei) (scale, 30 μm);
FIG. 6B is a statistical analysis of a delayed live cell imaging experiment during incubation of U87 glioma cells with Rho-labeled Ac4MAN-PEG1000-LIP for 0-20 min; the fluorescence intensity of each preparation is made into a curve graph by using a normalized numerical value; data are shown as mean ± standard deviation (n ═ 3);
FIG. 7A is a live cell image taken during incubation of U87 glioma cells with Rho-labeled Ac4MAN-PEG600-LIP for 0-20 min; (the red part is Rho-labeled liposomes; green is DIO-stained cell membrane; blue is Hoechst 33258-stained nuclei) (scale, 30 μm);
FIG. 7B is a statistical analysis of a delayed live cell imaging experiment during incubation of U87 glioma cells with Rho-labeled Ac4MAN-PEG600-LIP for 0-20 min; the fluorescence intensity of each preparation is made into a curve graph by using a normalized numerical value; data are shown as mean ± standard deviation (n ═ 3);
FIG. 8A is a live cell image taken during incubation of U87 glioma cells with Rho-labeled Ac4MAN-PEG2000-LIP for 0-20 min; (the red part is Rho-labeled liposomes; green is DIO-stained cell membrane; blue is Hoechst 33258-stained nuclei) (scale, 30 μm);
FIG. 8B is a statistical analysis of a delayed live cell imaging experiment during incubation of U87 glioma cells with Rho-labeled Ac4MAN-PEG2000-LIP for 0-20 min; the fluorescence intensity of each preparation is made into a curve graph by using a normalized numerical value; data are shown as mean ± standard deviation (n ═ 3);
FIG. 9 shows the anti-tumor effect of Ac4MAN-PEG-LIP on glioma-bearing mice;
wherein, FIG. 9A is H.E staining of frozen sections of glioma brain injected with Ac4MAN-PEG 1000-LIP; (scale, 50 μm);
FIG. 9B is a KaplanMeier survival curve for mice injected with Ac4 MAN-PEG-LIP;
wherein, 1) KaplanMeier survival curve (n 7) of Ac4MAN-PEG1000-LIP injected mice; 2) kaplanmieer survival curve (n 7) for Ac4MAN-PEG600-LIP injected mice; 3) kaplanmieer survival curve (n 7) for Ac4MAN-PEG2000-LIP injected mice;
FIG. 10 is a structural representation of a cardiac-cerebral dual targeting liposome;
wherein: (A) schematic diagram for preparing Anti-cTnI Ab-PEG2000/Ac4MAN-PEG 2000-LIP; (B) zeta potential distribution of Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP using Zetasizer Nano ZS 90; (C) Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000 from TEM (scale bar, 100) was used. (D) Particle size distribution of Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP using Zetasizer Nano ZS 90;
FIG. 11 shows the targeting distribution of various DIR-labeled cardio-cerebral double-targeting liposomes in MI rats;
wherein: (A) time-dependent distribution of the systemic fluorescence signal of rats after intravenous injection of various formulations: MI rats were administered equal volumes of DiR-labeled Anti-cTnI Ab-PEG2000-LIP, DiR-labeled Ac4MAN-PEG2000-LIP, DiR-labeled Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP, DiR-labeled Anti-cTnI Ab-PEG2000/Ac4MAN1000-LIP, and DiR-labeled Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP, respectively; (B) fluorescence intensity in isolated heart tissue from all experimental rats at 1, 2, 3, 4, 5 and 6 hours post-dose; (C) fluorescence intensity in heart tissue sections of all experimental rats 1, 3 and 6 hours after dosing.
Fig. 12A is a histogram statistical analysis of brain dual-targeting liposome distribution in mouse heart tissue at 1, 2, 3, 4, 5 and 6 hours, as indicated by the fluorescence intensity of DiR in the heart. Data represent mean ± s.d. (n-3). P <0.05, P <0.01 and P <0.001 compared to Anti-cTnI Ab-PEG2000-LIP at 1, 2, 3, 4, 5 and 6 hours;
fig. 12B is a histogram statistical analysis of the distribution of brain dual-targeted liposomes in heart tissue at 1, 3, and 6 hours in isolated heart tissue, expressed as the fluorescence intensity of DiR. P <0.05, P <0.01 and P <0.001 compared to Anti-cTnI Ab-PEG2000-LIP at 1, 3 and 6 hours.
FIG. 13 shows the MicroScale thermolauroresis assay for the affinity of various DiR-labeled cardio-cerebral dual-targeting liposomes to GLUT1, and shows trends; the X-axis represents liposome concentrate, while the Y-axis represents fractional binding values; the binding constant of Anti-cTnIAb PEG2000/Ac4MAN-PEG600-LIP is 2746.9 + -13182 nM; the binding constant of Anti-cTnI Ab PEG2000/Ac4MAN-PEG1000-LIP is 41.8 +/-36.37 nM; while the Anti-cTnI Ab PEG2000/Ac4MAN-PEG2000-LIP has a binding constant of 1.17 +/-5.69 nM;
figure 14 is a flow cytometry measurement of uptake of Ac4MAN modified liposomes with different PEG chain linkages through cardiomyocytes versus C6 cells (a and B). The uptake of cardiomyocytes is shown in fig. 14A. Fig. 14B shows the mean fluorescence intensity of C6 cells. Data are expressed as mean ± s.d. (n ═ 3). P <0.05, P <0.001, compared to LIP.
Detailed Description
The advantages and features of the invention will become more apparent from the following further description of the invention given in conjunction with specific embodiments. However, the examples are only for illustrating the present invention and do not set any limit to the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The materials used in the examples are as follows:
healthy adult male Wistar rat
Figure GDA0002446785310000071
Purchased from the laboratory animal center of Jilin university.
Dioleoylphosphatidylglycerol (DOPG) is purchased from fine chemical limited japan (osaka, japan).
Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-MAL) were purchased from advanced carrier technology pharmaceuticals, inc (china, shanghai).
PEG 600/1000/2000, glutaric acid N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethyl-isopropyl) carbodiimide hydrochloride (EDCI). From the industrial company Aladdin (China, Shanghai).
p-carboxyphenyl- α -D-acetylmannosamine (Ac)4MAN) was obtained from Innochem, beijing, china.
Egg yolk phosphatidylcholine (EPC) and Cholesterol (CHO) were purchased from Bio Life Science & Technology co., Ltd (shanghai, china).
Arsenic Trioxide (ATO) is a gift from pharmaceutical ltd (harbin, china) of harbin medical university.
Rhodamine b (rho) and 1,1' -dioctadecyl-3, 3,3', 3' -tetramethyl-Diiodocyanine Iodide (DiR) are available from HEDE biotechnology limited (beijing, china).
Anti-cTnI antibodies (Anti-cTnI Ab) were purchased from Immunoway (beijing, china).
D-mannose was purchased from Innochem (Beijing, China).
MST capillary and tween 20 were purchased from Quantum Design (beijing, china).
EXAMPLE 1 Synthesis of brain targeting molecule Diisopentylphosphatidylethanolamine-polyethylene glycol-1000-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-1000-Ac4MAN)
Figure GDA0002446785310000081
Formula I (wherein the polyethylene glycol part is PEG-1000)
Azide-polyethylene glycol-glutaric acidAmide 1000(1.3g) was dissolved in tetrahydrofuran (20mL), followed by addition of Pd/c (0.066g) and acetic acid (1mL), and reaction was carried out overnight with hydrogen gas at room temperature. The crude product was filtered, rotary evaporated to dryness, then dissolved in dichloromethane (20mL), triethylamine (TEA, 0.65mL) and Ac were added4MAN (1.5g) was reacted at room temperature overnight. Passing through a column to obtain PEG1000-Ac4MAN (1.18g) in 62% yield. PEG1000-Ac4MAN (1.18g) was dissolved in methylene chloride, NHS (0.2g) and EDCI (0.45g) were sequentially added thereto, and the mixture was reacted overnight at room temperature and then passed through a column to obtain succinimidyl polyethylene glycol acetylmannoside (1.0g) in 78% yield. Dissolving succinimidyl polyethylene glycol acetyl mannoside (0.95g) in dichloromethane (20mL), adding TEA (0.2mL) and DSPE (0.4g) dissolved in chloroform, reacting at room temperature overnight, extracting with saturated NaCl for 2 times, and passing through a column to obtain DSPE-PEG1000-Ac4MAN (0.86g) was obtained in a yield of 86%.
FIG. 1 is a graph of 1HNMR and 13CNMR demonstrating DSPE-PEG-1000-Ac4Successful synthesis of MAN. As shown in FIG. 1, the peak at 3.8ppm is PEG, and at 1.3ppm and 0.8ppm, two peaks respectively represent-CH 3-and-CH 2-, which are characteristic peaks of DSPE, while the peak corresponding to 5.5ppm is a characteristic peak of sugar.
Example 2 brain targeting molecule Diisopentylphosphatidylethanolamine-polyethylene glycol-600-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-600-Ac)4MAN) Synthesis
Figure GDA0002446785310000091
Formula I (wherein the polyethylene glycol part is PEG-600)
Azide-polyethylene glycol-glutaramide 600(1.3g) was dissolved in tetrahydrofuran (20mL), followed by addition of Pd/c (0.078g) and acetic acid (1mL), and reaction was carried out overnight with hydrogen gas at room temperature. The crude product was filtered, rotary evaporated to dryness, then dissolved in dichloromethane (20mL), triethylamine (TEA, 0.79mL) and Ac4MAN (1.7g) were added and reacted at room temperature overnight. The column was passed to give PEG600-Ac4MAN (1.08g) in 62% yield. PEG600-Ac4MAN (0.95g) was dissolved in methylene chloride (20mL), and NHS (0.3g) and EDCI (0.51g) were sequentially added thereto, and reacted overnight at room temperature, followed by column chromatography to give succinimidyl polyethylene glycol acetylmannoside (0.82g) in 80% yield. Succinimide polyethylene glycol acetylmannoside (0.82g) was added to TEA (0.3mL) and DSPE (0.49g) dissolved in chloroform, reacted at room temperature overnight, extracted 2 times with saturated NaCl, and passed through a column to give DSPE-PEG1000-Ac4MAN (0.76g) in 76% yield.
FIG. 1 is a graph of 1HNMR and 13CNMR demonstrating DSPE-PEG-600-Ac4Successful synthesis of MAN. As shown in FIG. 1, the peak at 3.8ppm is PEG, and at 1.3ppm and 0.8ppm, two peaks respectively represent-CH 3-and-CH 2-, which are characteristic peaks of DSPE, while the peak corresponding to 5.5ppm is a characteristic peak of sugar.
Example 3 brain targeting molecule Diisopentylphosphatidylethanolamine-polyethylene glycol-2000-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-2000-Ac)4MAN) Synthesis
Figure GDA0002446785310000101
Formula I (wherein the polyethylene glycol part is PEG-2000)
Azide-polyethylene glycol-glutaramide 2000(1.3g) was dissolved in tetrahydrofuran (20mL), after which Pd/c (0.052g) and acetic acid (1mL) were added, and hydrogen was bubbled through at room temperature for reaction overnight. The crude product was filtered, rotary evaporated to dryness, then dissolved in dichloromethane (20mL), triethylamine (TEA, 0.54mL) and Ac4MAN (1.34g) were added and reacted at room temperature overnight. The column was passed to give PEG2000-Ac4MAN (1.15g) in 63% yield. PEG2000-Ac4MAN (0.95g) was dissolved in methylene chloride (20mL), and NHS (0.09g) and EDCI (0.19g) were sequentially added thereto, and reacted overnight at room temperature, followed by column chromatography to give succinimidyl polyethylene glycol acetylmannoside (0.87g) in 82% yield. Succinimide polyethylene glycol acetylmannoside (0.87g), TEA (0.15mL) and DSPE (0.3g) dissolved in chloroform were added to react at room temperature overnight, extracted 2 times with saturated NaCl and passed through a column to give DSPE-PEG2000-Ac4MAN (0.75g) in 84% yield.
FIG. 1 is a graph of 1HNMR and 13CNMR demonstrating DSPE-PEG-2000-Ac4Successful synthesis of MAN. As shown in FIG. 1, the peak at 3.8ppm is PEG, at 1.At 3ppm and 0.8ppm, two peaks represent-CH 3-and-CH 2-, respectively, which are characteristic peaks of DSPE, while the peak corresponding to 5.5ppm is a characteristic peak of sugar.
Example 4 preparation of drug-loaded (ATO) brain-targeted liposomes
(1) Preparation of ATO-LIP
Preparing blank liposome by adopting a thin film hydration method: dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were mixed in a molar ratio of 49.4: 3.2: 43.3: 4.1 weighing, dissolving in ethanol, evaporating to dryness by rotary evaporation, a lipid film can be obtained in a round bottom flask. Dried lipid membrane using nickel acetate (Ni (OAC)2) The solution (600mm, pH 6.8) was hydrated by sonication, then further dispersed by sonication with a probe (200W) for 5min, and extruded 3 times through a 220nm polycarbonate membrane (220 nm). The extruded liposomes were passed through a SephadexG-50 column, except for Ni (OAC) outside the liposome bilayer2Solution buffer 1(300mM NaCl +200mM HEPES, pH 6.8) was added to the liposome colloid solution to form a gradient between the internal and external aqueous phases of the liposomes, i.e.to form Ni (OAC)2Liposomes (Ni-LIP). Then, 1mL of ATO solution (33.4mM) was added to 2mL of Ni-LIP (8.73mg lipid/mL), pH was adjusted to 7.2, and the mixture was incubated in a shaking table (50 ℃, 110rpm) for 10 to 15 hours, after which the liposome suspension was passed through a SephadexG-50 column and excess H was removed with buffer 2(300mM NaCl +200mM HEPES, pH4)3AsO3. Finally, the pH of the liposomes was adjusted back to 7.2, thus obtaining ATO-LIP.
Figure 2A is a schematic of liposome preparation. Mixing Ni (OAC)2Entrapped inside the liposomes, then the ATO is actively loaded into the liposomes, and then mixed with Ni (OAC)2And (4) complexing. Finally Ac is added4The M micelles are inserted into the liposome surface. FIG. 2B shows, at room temperature, Ni (OAC)2The solution formed a green precipitate with the ATO solution, showing ATO and Ni (OAC)2The state of presence in liposomes. When they are bound together, the rapid release of ATO from vesicles is improved.
(2)DSPE-PEG 1000-Ac4MAN modified ATO-LIP
Synthesized as in example 1DSPE-PEG 1000-Ac4MAN modifies ATO-LIP. Firstly, DSPE-PEG1000-Ac is prepared4MAN micelles containing DSPE-PEG1000-Ac4MAN and DSPE-PEG2000, in a molar ratio of 8.32: 3.75. 1mL ATO-LIP (5.82mg lipid/mL) with 0.5mL DSPE-PEG1000-Ac4Incubation of MAN micelles (8mg lipid/mL) at room temperature for 2h gave Ac4M-ATO-LIP(PEG1000)。Ac4M-ATO-LIP (PEG600) and Ac4M-ATO-LIP (PEG2000) was added in the same molar ratio and prepared in the same manner.
(3) Encapsulation efficiency and in vitro drug release
Analysis of ATO-LIP and Ac by Atomic Absorption Spectrophotometry (AAS)4Drug content of M-ATO-LIP (PEG 1000). Determination of ATO or Ni (OAC) by dialysis2The in vitro release rate of (c). The two ends of the dialysis bag are tied tightly with a thread, and 1mL of ATO-LIP or Ac4M-ATO-LIP (PEG1000) is placed in the dialysis bag (Cutoff 8000-14400 Da). The dialysis bag was then placed into a 500mL centrifuge tube containing 300mL PBS and the tube was shaken continuously in a shaker at 37 ℃ and 100 rpm. Samples were taken at specific time points (0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h) and the same volume of PBS was added after each collection.
The encapsulation ratio (%) was calculated by the following formula: the encapsulation ratio (%) × 100% (concentration of drug after passing through G-50 column/concentration of drug before passing through G-50 column). The formula for calculating the release rate (%) is: the release rate (%) - (Mn/M) × 100%. Mn is ATO or Ni (OAC)2Cumulative release at a certain point in time. M is ATO or Ni (OAC) loaded in liposome2The total amount of (a).
As a result: ATO in ATO-LIP and Ac4The encapsulation efficiency in M-ATO-LIP (PEG1000) was 22.48% + -3.95 and 25.85% + -2.64, respectively. As the entrapment rate between the modified liposome and the unmodified liposome has no obvious difference, the modified ligand DSPE-PEG1000-Ac can be known4The MAN micelles did not affect the encapsulation properties of the liposomes. FIGS. 3A and 3B show ATO-LIP and Ac4ATO and Ni (OAc) in M-ATO-LIP (PEG1000)2The in vitro release rate of (c). After dialysis, ATO and Ni (OAc)2The release rate of (a) was measured with an elemental analyzer. The experimental results showed that, at the first 2 hours, ATO and Ni (OAc)2The release rates in the dialysate were less than 28.18% ± 6.23 and 27.76% ± 2.56, respectively. After 48 hours, the release rates for ATO and Ni (OAc)2 were approximately 84.82% + -2.78 and 47.44% + -3.51, respectively. ATO and Ni (OAc)2At ATO-LIP and Ac4The in vitro release rate in M-ATO-LIP (PEG1000) was similar.
Example 5 in vitro efficacy study experiment of brain-targeted liposomes
(1) Cell culture
Human glioma cell U87 was derived from the cell bank of Chinese academy of sciences (Shanghai). U87 glioma cells were cultured in DMEM containing 1% penicillin-streptomycin and 20% fetal bovine serum. The cells were incubated at 37 ℃ with 5% CO2Cultured in a cell culture box.
(2) Effect of cytotoxicity on brain glioma cells
Cell imaging experiments and MTT method are adopted to detect the cytotoxicity effect of the liposome. In 96-well culture plates at 2.0X 104The cells were seeded at a density of one well per well U87 cell, cultured overnight, and then individually plated with varying concentrations of blank liposomes, free ATO, ATO-LIP, and Ac4M-ATO-LIP (PEG1000) was used for incubation for 48h, 72h and 96h, respectively. The ATO concentrations were 40.6, 81.5, 162.5, 325, 650, and 1300. mu.M, respectively. Thereafter, the drug-containing stock medium was changed to 100. mu.L of fresh medium and 10. mu.L of MTT (5mg/mL), and after further incubation at 37 ℃ for 4 hours, the MTT was removed and 100. mu.L of dimethyl sulfoxide (DMSO) was added to each well. We measured the Optical Density (OD) value at 490nm using an enzyme-linked immunoassay (Tecan, Austria).
U87 cells were seeded, cultured overnight, then incubated with various liposomes, and after 48, 72 or 96h incubation the stock culture was changed to fresh, then stained with Hoechst33258(0.2mg/mL), washed 3 times with Phosphate Buffered Saline (PBS) and the cell imaging experiment started immediately. We used rotation 5 for image capture and analysis of the data.
FIG. 4 shows the cytotoxic effect of liposomes on U87 glioma cells. To investigate whether ATO had a potent inhibitory effect on the growth of U87 glioma cells, we administered different concentrations of free ATO, ATO-LIP or Ac to U87 glioma cells4M-ATO-LIP (PEG1000), and was observed by MTT method and cell imaging method. We observed that the different liposomes all had significant inhibitory effect on the growth of U87 glioma cells and induced cell death in a dose-dependent manner. Cytotoxicity increased with time. The inhibition intensity of each preparation on U87 cells is in the order: free ATO>Ac4M-ATO-LIP(PEG1000)>ATO-LIP. Compared with liposome, free ATO has the strongest inhibition effect on cell growth, which is caused by the direct contact of small molecules and cells, the fast uptake speed of cells and the fact that the free ATO is a natural substance with killing property. Ac of4M-ATO-LIP (PEG1000) showed stronger inhibitory effect on U87 cells than ATO-LIP, indicating that Ac present on the liposome surface4M micelle enhances the inhibition effect of ATO-LIP on the growth of U87 cells.
(3) U87 glioma cell uptake
1) Rho-LIP and Ac4Preparation of M-Rho-LIP
Weighing EPC, CHO, DSPE-mPEG2000, adding DSPE-PEG1000-Ac4MAN (DSPE-PEG600-Ac4MAN or DSPE-PEG2000-Ac4MAN) to dissolve in absolute ethyl alcohol, and dispersing and filming once by adopting a film, wherein the absolute ethyl alcohol is used as a solvent; adding absolute ethyl alcohol into the liposome with the first film formation for redissolving; rho (6.57mM methanol) was added to liposomes dissolved in ethanol; forming a film again by rotary evaporation; adding physiological saline under the condition of keeping out of the sun for ultrasonic hydration; transferring the hydrated liposome into an EP tube coated with tin foil paper, and carrying out ultrasonic crushing by a probe with the power of about 150W. The parameter operation is suspended for 10 seconds and is carried out for 30 times in 30 seconds; filtering liposome with 0.22 μm water phase filter membrane for 3 times; measuring liposome particle size and potential, dialyzing to remove unencapsulated Rho to obtain Ac4M-Rho-LIP (PEG1000, PEG600 and PEG2000 as linker, respectively).
Rho-LIP was prepared as described above, except that DSPE-PEG-Ac4MAN was not added.
2) Flow cytometry
Flow cytometry was used to detect cellular uptake of the different liposomes. We seeded U87 glioma cells in six well plates and allowed the cells to culture overnight, then replaced DMEM/high sugar with DMEM/low sugar and continued the culture for 12 hours, after which time they were separately mobilizedIsolated Rho, Rho-LIP and Ac4M-Rho-LIP (PEG1000) cells were incubated at 37 ℃ with 5% CO2 (liposomes labeled with Rho). The blank control group was DMEM/low sugar medium. After 4 hours of incubation, the cells were trypsinized, centrifuged, resuspended in PBS, collected and assayed. Flow cytometry detects the fluorescence intensity of intracellular Rho. Rho emission wavelength was 560nm, fluorescence intensity was measured with FL2-A filter and data was analyzed using FlowJo 7.6 software.
Fig. 5A and 5B show quantitative analysis of liposomes in U87 glioma cells, revealing the vesicle uptake rate by tumor cells. Flow cytometry was used to detect fluorescent signals in U87 cells and monitor the cells for free Rho, Rho-LIP and Ac4Uptake of M-Rho-LIP (PEG 1000). The results of the experiments showed that the mean fluorescence intensity of free Rho was 6162, that of Rho-LIP 7125, and that of Ac4The mean fluorescence intensity of M-Rho-LIP (PEG1000) was 10827. The experimental data show that in vitro, U87 cells are paired with Ac4The uptake of M-Rho-LIP was more pronounced than Rho-LIP (P)<0.05). According to the cellular uptake result, due to DSPE-PEG-1000-Ac4Presence of MAN, DSPE-PEG-1000-Ac4The MAN micelle modified liposome has brain targeting and glioma targeting.
3) Live cell imaging
The cellular uptake capacity and uptake rate of liposomes over time were observed using the DeltaVision microscope system. We seeded U87 glioma cells in glass-bottom dishes at 37 ℃ with 5% CO2Incubated under conditions overnight. The nucleus and membrane of the cells were stained with Hoechst33258(0.2mg/mL) and DIO (0.2mg/mL), respectively, and washed three times with PBS, 1mL of fresh medium was added, and Rho-LIP or Ac was added4M-Rho-LIP (PEG1000, PEG600 and PEG2000 for linker, respectively) was added to the dish, and the photography was started and images were collected every 5 minutes for up to 20 minutes. Finally, the results were analyzed using DeltaVisionSoftwx software.
FIGS. 6-8 are time-lapse live cell images showing the internalization of different agents by U87 glioma cells. Shown are Rho-LIP and Ac4M-Rho-LIP (PEG1000, PEG600 and PEG2000 as linker) inThe process of cellular uptake is 0-20 min. After reaction with Rho-LIP or Ac4A red fluorescent signal was observed in U87 cells during 20min of M-Rho-LIP action. The images show that both preparations are taken up by U87 cells. Whereas in U87 cells incubated with Rho-LIP, the fluorescence signal was found to be weak, along with Ac4The fluorescence signal of M-Rho-LIP was stronger at each time point than that of Rho-LIP. Image and data analysis shows that Rho-LIP enters into glioma cells in a speed ratio Ac4M-Rho-LIP is slow. Ac of4The fluorescent signal of M-Rho-LIP was shown in both cytoplasm and nucleus, indicating no significant difference in vesicle distribution in each organelle.
Example 6 in vivo antitumor Effect of brain-targeting liposomes
(1) Construction of glioma tumor-bearing mouse model
Male Balb/c nude mice (18-20 g) of 5-6 weeks old were from Beijing Life river laboratory animal technology, Inc. (China, Beijing). Anaesthetized with 5% chloral hydrate (15mL/20g), mounted on a stereotaxic apparatus, an opening is first cut into the scalp, bregma is found on the skull, a hole is drilled 0.5mm in front of bregma and 2mm lateral thereto, and U87 cells (2X 10. mu.L/min) are plated at a rate of 0.75. mu.L/min6Cells/15 μ L PBS) was slowly injected into the brain to a depth of 2.5 mm. The incision was closed with tissue glue and the surgery was completed, after which the model mice were weighed daily.
(2) Treatment of glioma-bearing mice
After 3 weeks of surgery, mice were divided into four groups, administered once every 2 days via the tail vein, and given physiological saline, free ATO, ATO-LIP, and Ac, respectively4M-ATO-LIP (prepared in example 4) was administered in a dose of 2. mu.g of ATO per gram of body weight. The physical condition and body weight of the mice were observed and recorded daily.
(3) Time to live
7 mice per group were used to monitor survival time, counted from day 1 post-dose to the day of mouse death, and KaplaneMeier survival curves were plotted for each group.
Kaplan Meier survival curves (FIG. 9B) show administration of saline, free ATO, ATO-LIP, and Ac4Glioma model of M-ATO-LIP (PEG1000)The average survival times were 23, 25, 30 and 32 days. Ac of4The survival time of mice in the M-ATO-LIP (PEG1000) group was significantly longer than in the saline, free ATO and ATO-LIP groups. This demonstrates DSPE-PEG-1000-Ac4MAN-modified ATO-LIP has great potential for the treatment of gliomas. And Ac4MAN-ATO-LIP (PEG600) and Ac4The survival time of MAN-ATO-LIP (PEG2000) was 33 days and 35 days, respectively.
(4) H.E. dyeing
The efficacy of the different formulations was monitored in tumor-bearing mice, which were perfused with 4% paraformaldehyde solution for 10 minutes before death, after which the brains were removed for frozen sections (5 μm each). The sections were stained with hematoxylin and eosin and then observed under a fluorescent microscope.
The antitumor effect of different ATO preparations was studied using tumor-bearing nude mice as a model. The difference between the tumor tissue section and the normal brain tissue section was directly observed from the H.E. staining result (FIG. 9A), and ATO-LIP and Ac were administered to the model mice4After treatment with M-ATO-LIP, tumor cells in the brain of tumor-bearing mice decreased. ATO-LIP kills glioma cells more than free ATO, and Ac4M-ATO-LIP (PEG1000) can cause more glioma cells to die compared to ATO-LIP. These results indicate that liposomes accumulate more than small molecules at the site of glioma due to high permeability and retention Effects (EPR). In addition, Ac4M micelles also promote the accumulation of more liposomes at the site of glioma.
Example 7 preparation and characterization of Heart and brain Dual-Targeted liposomes
(1) Preparation of Ac4MAN-PEG2000-LIP
Weighing a mixture with a molar ratio of 49: 50: 1 egg yolk phosphatidylcholine (EPC), Cholesterol (CHO) and DSPE-PEG-2000-Ac4MAN, liposomes were prepared by thin film dispersion according to previously reported methods. Briefly, lipid materials were dissolved in a spinner flask with absolute ethanol. A thin lipid film was formed by evaporating the mixture under reduced pressure. The lipid membrane was hydrated with a physiological saline solution. The liposome solution was dispersed by sonication for 3 minutes (run 10 seconds, pause 6 seconds, 10 times) and passed through polycarbonate membranes (220nm and 150nm, respectively)3 times) extrusion. The liposome samples were stored at 4 ℃ for further study.
(2) Preparation of Anti-cTnI antibody modified liposome (Anti-cTnI-PEG 2000/Ac)4MAN-PEG-LIP)
Synthesis of Anti-cTnI Ab-DSPE-PEG solution [ The use of Anti-enzymodified lipomes loaded with AMO-1to The conveyor oligonucleotides to isocarboxamide for arrhythmia therapy, MeibangLiu, et. al. biomaterials, Volume 35, Issue11, April 2014, Pages 3697-]. Anti-cTnI antibody (Ab) (molecular weight 23KD) and DSPE-PEG-2000-maleimide (DSPE-PEG2000-MAL, molecular weight 2900Da) (molar ratio 1:500) were dissolved in HEPES solution (10mM, pH7.4), respectively, and then mixed at 4 ℃ overnight to obtain Anti-cTnI Ab-DSPE-PEG2000 solution. Incubating the Anti-cTnI Ab-DSPE-PEG2000 solution and the liposome solution prepared in the step (1) for 2 hours at 37 ℃ to prepare the Ac modified by the Anti-cTnI antibody4MAN-PEG2000-LIP, named Anti-cTnI-PEG2000/Ac4MAN-PEG2000-LIP。
Preparing Anti-cTnI-PEG2000/Ac according to the same method4MAN-PEG1000-LIP and Anti-cTnI-PEG2000/Ac4MAN-PEG600-LIP。
(3) Liposome characterization
By dynamic light scattering (Nano ZS 90, Malvern) and transmission electron microscopy (Tecnai G2F 20, STEM; FEI, Hillsboro). Liposomes were diluted to some extent with liposomes before measurement, in triplicate for each sample.
FIG. 10A shows Anti-cTnI antibodies and Ac on the surface of liposomes4Schematic of a MAN. Fig. 10B and D show the results of Zeta potential and particle size for various PEG coatings for liposome data. FIG. 10C shows morphology of LIP by TEM. Table 1 shows that the average particle diameter is in the range of 100-130 nm. Ac of4MAN-PEG2000-LIP is negatively charged (-1.74mV), while Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP was-32.0 mV. LIP was mainly less than 150nm during 24 hours incubation with 10% FBS. As the PEG chain length increases, the Zeta potential of the vesicles gradually decreases. The longer the PEG chain, the lower the surface charge of the vesicle, probably due to the shielding effect of PEG.
TABLE 1 particle size distribution and Zeta potential of liposomes
Figure GDA0002446785310000161
Figure GDA0002446785310000171
Note: data are presented as mean ± standard deviation (s.d.) (n ═ 3)
Example 8 in vivo fluorescent tracing of cardio-cerebral Dual-Targeted liposomes
1. Establishment of rat acute myocardial infarction model
Rats were anesthetized with 0.3mL chloral hydrate per 100g of body weight by intraperitoneal injection and electrocardiographic electrodes were attached. The rats were subjected to a left-sided thoracotomy and the left anterior descending coronary artery was immediately ligated approximately 2-3mm below the junction between the left auricle and the pulmonary cone. The heart was then immediately replaced to the chest and the electrocardiogram was kept for half an hour. Iodophors are used to abrade wound skin and avoid infection. Successful markers include white surface of the heart, heart rate reduction and elevation of the ST segment.
2. Preparation of DiR-labeled liposomes
Weighing EPC, CHO, DSPE-mPEG2000, DSPE-PEG600-Ac4MAN (DSPE-PEG1000-Ac4MAN, DSPE-PEG2000-Ac4MAN) and dissolving in absolute ethyl alcohol, and dispersing and filming once by adopting a film, wherein the absolute ethyl alcohol is used as a solvent; adding absolute ethyl alcohol into the liposome with the first film formation for redissolving; adding DiR (6.57mM methanol) to liposomes dissolved in ethanol; forming a film again by rotary evaporation; adding physiological saline under the condition of keeping out of the sun for ultrasonic hydration; transferring the hydrated liposome into an EP tube coated with tin foil paper, and carrying out ultrasonic crushing by a probe with the power of about 150W. The parameter operation is suspended for 10 seconds and is carried out for 30 times in 30 seconds; filtering liposome with 0.22 μm water phase filter membrane for 3 times; liposome particle size and potential were measured. Anti-cTnI Ab was added and incubated for 2h, or supplemented with NaCl to the same volume, and unencapsulated DiR was removed by dialysis. Respectively obtaining DiR-marked Ac4MAN-PEG2000-LIP, Anti-cTnIAb-PEG2000-LIP (DSPE-PEG-Ac 4MAN is not added during preparation), Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG1000-LIP and Anti-cTnI Ab-PEG2000/Ac4MAN-PEG 2000-LIP.
3. In vivo fluorescent tracing
Professional imaging systems employing Carestream in vivo FX for optical imaging were equipped with an excitation pass filter of 720nm and an emission pass filter of 790 nm. The shooting scheme is as follows: the X-ray exposure time was 2 minutes per image and the exposure time of the fluorescence was 30 seconds. MI rats were injected with Anti-cTnI-PEG2000-LIP, Ac labeled with DiR4MAN-PEG2000-Lip，Anti-cTnI-PEG2000/Ac4MAN-PEG600-LIP，Anti-cTnI-PEG2000/Ac4MAN-PEG1000-LIP and cTnI-PEG2000/Ac4MAN-PEG 600-LIP. Rats were placed in an imaging system equipped with a 720nm band pass filter and a 790nm long pass filter. The exposure time for fluorescence was 30 seconds and X-ray exposure was 2 minutes per image. Images are taken by a CCD camera integrated with the imaging table. Evaluation of data using a Carestream imaging System
To study the different molar ratios of Ac4Effect of vesicle surface of MAN and Anti-cTnI antibodies on in vivo distribution, we specify here Anti-cTnI antibodies and Ac4The proportion of MAN (molar ratio 5:12) on the surface of the attached liposomes was determined by attaching different PEG molecules such as Anti-cTnI-PEG2000/Ac4MAN-PEG600-LIP，Anti-cTnI-PEG2000/Ac4MAN-PEG1000-LIP and cTnI-PEG2000/Ac4MAN-PEG 600-LIP. The distribution of agents in the ischemic heart and brain of rats at different time points was observed using an in vivo imaging system, where the pseudo-colors in the images represent the fluorescent signals of different intensities in a given tissue including the heart and brain (fig. 11A). Our previous studies have demonstrated that the fluorescence intensity of ischemic hearts peaked after injection of DiR-labeled Anti-cTnI antibody PEG 2000-LIP. Anti-cTnI Ab-PEG2000-LIP and Ac4The MAN-PEG2000-LIP group is used as a control, and the information in the graph shows that the fluorescence signal of the liposome with the heart and brain double targeting is strongest.
After cardiac perfusion, heart tissue was removed and the distribution of liposomes in the heart tissue was observed, demonstrating that the fluorescence signal was indeed from tissue and not blood vessels. Before perfusing the rats, the rats were injected with liposomes through the tail vein, and the hearts were removed at the sixth hour after the injection. Separation deviceFluorescence signal intensity in the body heart indicates the choice of Ac4The shorter PEG chains of MAN, more accumulation of liposomes in the heart was observed (fig. 11B). Fluorescence imaging of longitudinal sections of cardiac tissue was consistent with changes in cardiac distribution (fig. 11C). Histograms show fluorescence intensity of heart and ex vivo heart tissue (1, 2, 3, 4, 5 and 6) and further demonstrate that with Ac4The increase in MAN-linked PEG chain length made the targeted distribution of liposomes to the heart weaker (fig. 12A and 12B). Ac following PEG attachment4The increase in MAN length chain, due to Anti-cTnI antibody competitively binding to free cTnI within the stroma in the ischemic myocardium, the target distribution of liposomes in the heart becomes weaker.
Example 9 molecular interaction Studies
The interaction of various liposomes with GLUT1 was determined by MicroScale thermology (MST) (Quantum Design China), a technique that allows quantitative determination of biomolecular interactions. EP tubes were numbered, saline was added, GLUT1 was then mixed with tween 20, and DiR-labeled liposomes were then added to each tube. After 10 minutes of incubation, the mixture in the tube was drawn up by capillary and the tray was placed in order. The binding force of DiR-labeled liposomes to GLUT1 was measured by MST and data processing was performed using Manual _ mo. affinity Analysis _ V04 software.
Measurement of PEG, Ac Using MicroScale Thermophoresis (MST)4The affinity of MAN linker and the different chain lengths of GLUT1, investigated the targeting efficiency of liposomes to the brain. Based on these data, we can conclude that there is binding between liposomes and GLUT 1. The dissociation constant (Kd) of the binding strength showed a trend, where Anti-cTnI Ab-PEG2000/Ac4The Kd value of the MAN-PEG2000-LIP modified liposome is 1.17 +/-5.69 nM; Anti-cTnI Ab-PEG2000/Ac4The MAN-PEG1000-LIP is 41.8 +/-36.37 nM; Anti-cTnI Ab-PEG2000/Ac42746.9 + -13182 nM of MAN-PEG 600-LIP. GLUT1 plays an important role in blood glucose transport in various tissues, particularly in the blood brain barrier. Mannose modification of the liposome surface is used to enhance the brain targeting ability of the vesicles. In this study, the modified liposomes were able to bind GLUT1 with surface PEG2000 mannose binders with higher binding capacity than PEG1000, and PEG600 with higher binding capacityLowest (fig. 13). Ac of4MAN differs significantly in its ability to bind GLUT1 protein with PEG of varying chain length.
Example 10 cellular uptake study of cardio-cerebral Dual-Targeted liposomes
1. Culture of primary cardioblast cells
Wistar rat pups of 3 days old were sacrificed after 1-2min of immersion in 75% ethanol solution, hearts were quickly removed, cleaned, chopped and ground into small pieces. The tissue fragments were digested for 1-3 minutes with twice the tissue volume of the trypsin-EDTA solution in a 10mL centrifuge tube at 37 ℃. When the solution became turbid, serum-containing medium was added to stop the digestion. The process is repeated until the tissue disappears. Tissue debris was removed using a 200 mesh screen and centrifuged at 2500rpm for 3 minutes to collect cells. The cells were then transferred to disposable culture flasks. After culturing at 37 ℃ under 5% CO2 for 2 hours, fibroblasts were removed according to their cell adhesion ability. Cells were incubated at 37 ℃ for 48h with 5% CO2 and replaced in fresh medium for subsequent experiments.
2. Preparation of Rho-labeled liposomes
The procedure was as for the preparation of DiR-labeled liposomes in example 8, except that DiR was replaced with Rho.
3. Cellular uptake studies
Uptake of Rho-tagged liposomes by primary cardiomyocytes and C6 cells was performed by flow cytometry (Becton Dickinson FACS Aria I, Mountain view, CA). Primary cells and C6 cells were plated at 2X 10 per well5Individual cells were seeded in six-well plates and cultured for 48 hours. Then Rho-tagged LIP including Anti-cTnI Ab-PEG2000-LIP, Ac4MAN-PEG2000-LIP，Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP，Anti-cTnI Ab-PEG2000/Ac4MAN-PEG1000-LIP and Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP was incubated with cardiomyocytes or C6 cells with DMEM medium as a blank. After 4 hours, cells were detached with 0.25% trypsin digestion, collected and resuspended in PBS for flow cytometry assay. Rho-labeled liposome uptake was measured and the data analyzed using Flow Jo 7.6 software.
Flow cytometry was used to detect cellular uptake of Rho-labeled PEG-modified liposomes of varying chain length linked to Ac4 MAN. Unmodified LIP served as a control and all fluorescence values were processed by normalization. The average uptake of LIP by cardiomyocytes was 1.5476, Anti-cTnI Ab-PEG2000-LIP was 1.8680, Ac4MAN-PEG2000-LIP was 2.0820, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600 was 2.3954, for Anti-cTnI Ab-PEG2000/Ac4MAN-PEG1000-LIP was 2.435, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP was 2.7132. The uptake of cardiomyocytes was significantly increased with PEG-modified liposomes of different chain lengths compared to the control group (. P <0.05 and. P < 0.001). As the PEG chain length attached to Ac4MAN increased, uptake of liposomes by cardiomyocytes became stronger (fig. 14A). The mean fluorescence intensity of C6 cells was 3.9440 for LIP, 4.6351 for Anti-cTnI Ab-PEG2000-LIP, 6.5682 for Ac4MAN-PEG2000-LIP, 6.1121 for Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP, 6.8042 for Anti-cTnI Ab-PEG2000/Ac4MAN-PEG1000-LIP, and 8.2738 for Anti-cTnI Ab-PEG2000/Ac4MAN-PEG 2000-LIP. Uptake of various liposomes by C6 cells was significantly increased compared to the control group (. P <0.05,. P < 0.001). As the length of PEG chain attached to Ac4MAN increased, brain uptake increased (fig. 14B).

Claims (13)

1. A heart brain double-target liposome is characterized in that the heart brain double-target liposome is obtained by coupling Anti-cTnI protein antibody or fragment thereof on the surface of a liposome modified by acetylated glycolipid-polyethylene glycol-phosphatidylethanolamine conjugate shown in a formula I;
Figure FDA0002446785300000011
wherein n is 10-120.
2. The cardio-cerebral dual-targeting liposome of claim 1, wherein n is 13-45.
3. The cardio-cerebral dual-targeting liposome according to claim 1, wherein the polyethylene glycol is polyethylene glycol 600, polyethylene glycol 1000 or polyethylene glycol 2000.
4. The cardio-cerebral dual-targeting liposome of claim 1, wherein the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of formula I is prepared by reacting p- (glutaramide) phenyl- α -D-acetyl mannose (Ac) in dichloromethane4MAN-COOH) and polyethylene glycol (PEG) to form PEG-Ac by condensation reaction4MAN followed by Distearoylphosphatidylethanolamine (DSPE) with PEG-Ac4MAN is coupled to obtain DSPE-PEG-Ac4MAN, namely the compound shown in the formula I.
5. The cardio-cerebral bi-targeted liposome as claimed in claim 4, wherein the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate shown in formula I is prepared by dissolving azide-polyethylene glycol-glutaramide in tetrahydrofuran, adding Pd/c and acetic acid, introducing hydrogen gas at room temperature, reacting overnight, filtering the crude product, rotary evaporating to dryness, dissolving in dichloromethane, adding triethylamine and p- (glutaramide) phenyl- α -D-acetylmannose (Ac)4MAN-COOH) at room temperature, separating and purifying to obtain PEG-Ac4MAN; mixing PEG-Ac4Dissolving MAN in dichloromethane, sequentially adding N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), reacting at room temperature overnight, and separating and purifying to obtain succinimide polyethylene glycol acetylmannoside; dissolving succinimidyl polyethylene glycol acetyl mannoside in dichloromethane, adding TEA and distearoyl phosphatidyl ethanolamine (DSPE) dissolved in chloroform, reacting at room temperature overnight, extracting with saturated NaCl for 2 times, separating and purifying to obtain DSPE-PEG-Ac4MAN, namely the compound shown in the formula I.
6. A method for preparing the cardio-cerebral dual-targeting liposome of any one of claims 1-5, comprising the steps of:
(1) preparation of liposome modified by acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate
Dissolving egg yolk phosphatidylcholine (EPC), Cholesterol (CHO) and the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of formula I in a spinner flask with anhydrous ethanol, forming a thin lipid film by evaporating the mixture under reduced pressure; hydrating the lipid film with a physiological saline solution; dispersing the liposome solution by ultrasonic treatment, and extruding through a polycarbonate membrane to obtain the liposome modified by the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate, which is named Ac4MAN-PEG-LIP, storing liposome sample at 4 deg.C;
(2) preparation of Anti-cTnI antibody modified liposome
Synthesizing an Anti-cTnIAb-DSPE-PEG solution: respectively dissolving the Anti-cTnI antibody and the DSPE-PEG-maleimide in HEPES solution, then mixing, standing overnight at 4 ℃ to obtain an Anti-cTnI Ab-DSPE-PEG solution, and mixing the Anti-cTnIAb-DSPE-PEG solution with the Ac prepared in the step (1)4The MAN-PEG-LIP liposome solution is incubated for 2 hours at 37 ℃ to prepare Ac modified by Anti-cTnI antibody4MAN-PEG-LIP, named Anti-cTnI-PEG/Ac4MAN-PEG-LIP is the cardio-cerebral dual-targeting liposome.
7. The method of claim 6, wherein the molar ratio of EPC, CHO and the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of formula I in step (1) is (40-49): (50-55): (1-5).
8. The method of claim 7, wherein in step (1) the molar ratio of EPC, CHO and the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of formula I is 49: 50: 1.
9. the method of claim 6, wherein the DSPE-PEG-maleimide of step (2) is DSPE-PEG 600-maleimide, DSPE-PEG 1000-maleimide or DSPE-PEG 2000-maleimide.
10. The use of the cardio-cerebral dual-targeting liposome of any one of claims 1to 5 in the preparation of a pharmaceutical carrier.
11. The use of claim 10, wherein the cardio-cerebral bi-targeting liposome has the function of dual targeting of the brain and the heart, thereby achieving the purpose of cardio-cerebral simultaneous treatment.
12. The use according to claim 10, wherein the medicament is for the treatment of myocardial diseases and cardiogenic encephalopathies.
13. The use of claim 12, wherein the cardiogenic encephalopathy comprises cardiogenic cerebral ischemia syndrome, cardiogenic cerebral insufficiency, and cardiogenic stroke.
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