CN108578711B - Acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and preparation method and application thereof - Google Patents
Acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and preparation method and application thereof Download PDFInfo
- Publication number
- CN108578711B CN108578711B CN201810301839.7A CN201810301839A CN108578711B CN 108578711 B CN108578711 B CN 108578711B CN 201810301839 A CN201810301839 A CN 201810301839A CN 108578711 B CN108578711 B CN 108578711B
- Authority
- CN
- China
- Prior art keywords
- polyethylene glycol
- man
- lip
- peg
- dspe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/36—Arsenic; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses an acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate, and a preparation method and application thereof, wherein the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate is named as diisoamyl phosphoethanolamine-polyethylene glycol-p-carboxyl phenyl- α -D-acetyl mannosamine (DSPE-PEG-Ac)4MAN) having the structure shown in formula I. In addition, the invention also provides a brain targeting liposome comprising the conjugate. Pharmacodynamic tests prove that the liposome can have a prodrug-like effect in vivo, improves the cell uptake and the in vivo activity of the medicament through the action of in vivo hydrolase, and has good killing activity on C6/U87 cells. The medicament with brain targeting is prepared by carrying arsenous acid (arsenic trioxide), so that the aim of enabling the arsenous acid to cross blood brain barriers and treat glioma is fulfilled, and the survival time of a mouse is obviously prolonged. The invention provides a new technical means for treating brain glioma.
Description
Technical Field
The invention relates to an acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate as well as a preparation method and application thereof, in particular to application of the conjugate in construction of arsenic trioxide liposome. The invention belongs to the technical field of medicines.
Background
Intracranial tumors, the most common brain tumor in adults, with high recurrence and mortality rates, remain a serious and unsolved clinical problem as the leading threat to human health. Chemotherapy, radiation therapy, surgical resection or a combination thereof are the primary treatment options for glioma. However, patients still suffer from a range of chemotherapy side effects and high recurrence rates. Brain tumor cells cannot be completely removed due to the aggressive nature of glioma. Glioma cells are hidden in various regions of the brain, even in the contralateral hemisphere, while the Blood Brain Barrier (BBB) can essentially separate the blood from the brain and prevent nearly all macromolecular drugs and most small molecule drugs from penetrating into the Central Nervous System (CNS) of the brain. Therefore, finding a new approach to drug crossing the BBB is a crucial challenge.
Traditionally, arsenic trioxide (As)2O3ATO) is widely recognized as a highly toxic substance. In recent years, however, a number of clinicians have found ATO to be effective in treating Acute Promyelocytic Leukemia (APL). At present, some cell experimental studies show that ATO has a broad-spectrum anti-tumor effect, including breast cancer, lung cancer, prostate cancer, liver cancer and the like. These results demonstrate that ATO inhibits glioma cell linesAnd kill these cells. Although evidence has been provided that ATO has the effects of inducing apoptosis, inducing cell differentiation, promoting reactive oxygen species production, and inhibiting angiogenesis, the mechanism by which ATO kills glioma cells is not fully understood. Furthermore, ATO can only be used at very low concentrations, since healthy cells are also killed when exposed to high concentrations of ATO. Thus, in clinical trials, the application and efficacy of treatment of solid tumors is hampered by the dilemma between the effectiveness and toxicity of the amounts of ATO, which has not yet been formally used for the treatment of solid tumors.
Liposome (LIP) is a nano-scale drug carrier, can be used for carrying cytotoxic drugs, and can reduce the systemic toxicity of the cytotoxic drugs, prolong the half-life of the drugs, increase the accumulation of the drugs in tumors, and improve the therapeutic index of the drugs. Loading ATO into liposomes with transition metals such as copper or nickel is a successful approach. Within the liposomes, copper or nickel forms a complex with the ATO, avoiding premature leakage of the ATO in the liposomes. The complex is relatively stable at neutral pH, while it releases As at lower pH3+。
Mannose derivatives have become ideal ligands for glucose transporters (GLUTs), and can be used for modifying nanoparticles to make the nanoparticles obtain active targeting, and in the invention, a novel ligand, diisoamyl phosphoethanolamine-polyethylene glycol-1000-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-1000-Ac)4MAN) was synthesized, which contained hydrophilic mannose, which helped the drug cross the blood-brain barrier P-carboxyphenoxy- α -D-acetylmannosamine (Ac)4MAN) is a mannose derivative that is expressed mainly on endothelial cells of the BBB and brain glioma cells, and can efficiently enter the brain with the aid of glucose. Thus, Ac4MAN can be modified as a ligand to liposomes (Ac)4MAN-LIP) for targeting to the brain, Ac4MAN-LIP has an enhanced distribution within the brain, and even within tumor tissue within the brain.
Disclosure of Invention
The invention aims to provide an acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and a preparation method thereof, wherein the compound can be used as a ligand to be modified on a liposome (Ac)4MAN-LIP) to target the brain while helping the drug to cross the blood-brain barrier and enter the brain efficiently.
In order to achieve the above object, the present invention adopts the following technical means.
The acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate is named as diisoamyl phosphoethanolamine-polyethylene glycol-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-Ac)4MAN) having the structure shown in formula I:
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.
Further, the present invention also provides a method for preparing the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate, comprising the step of adding p-carboxyphenyl- α -D-acetylmannosamine (Ac) in dichloromethane4MAN) and PEG to form PEG-Ac by a condensation reaction4MAN followed by Distearoylphosphatidylethanolamine (DSPE) with PEG-Ac4MAN is coupled to obtain DSPE-PEG-Ac4MAN, namely the compound shown in the formula I.
Among them, it is preferable that the method for preparing the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate comprises the steps of dissolving azide-polyethylene glycol-glutaramide in tetrahydrofuran, then adding Pd/c and acetic acid, introducing hydrogen at room temperature, reacting overnight, filtering the crude product, rotary evaporating to dryness, then 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, and 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 acetyl mannoside; 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 application of the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate in preparation of a drug carrier.
Preferably, the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate has a brain targeting effect, helps the drug cross the blood brain barrier, and further achieves the purpose of treating diseases.
In one embodiment of the invention, the drug is arsenic trioxide and the disease is brain glioma.
Furthermore, the invention also provides a liposome carrier with brain targeting, wherein the surface of the liposome is coupled with the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate.
Furthermore, the invention also provides a brain-targeted therapeutic agent, which consists of a therapeutically effective amount of a drug and a liposome carrier, wherein the drug is encapsulated in the liposome carrier, and the agent is prepared by the following method:
(1) dissolving dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol in ethanol, evaporating by rotary evaporation to dryness to obtain lipid membrane in round bottom flask;
(2) dried lipid membrane using nickel acetate (Ni (OAC)2) Subjecting the solution to ultrasonic hydration, further dispersing by using a probe for 5min, and extruding for 3 times through a 220nm polycarbonate film;
(3) the extruded liposomes were passed through a SephadexG-50 column, except for Ni (OAC) outside the liposome bilayer2Solution by adding buffer 1 containing 300mM NaCl and 200mM HEPES, pH 6.8 to the liposome colloid solution, a gradient is formed between the inner and outer aqueous phases of the liposome, i.e., Ni (OAC) is formed2Liposomes (Ni-LIP);
(4) adding a solution of a drug into Ni-LIP, adjusting the pH to 7.2, incubating in a shaking table for 10-15 h, then, passing the liposome suspension through a SephadexG-50 column, and removing excessive drug by using a buffer solution 2 containing 300mM NaCl, 200mM HEPES and pH 4;
(5) adjusting the pH of the liposome to 7.2 to obtain LIP;
(6) using said acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate (DSPE-PEG-Ac)4MAN) modified drug-LIP: firstly, DSPE-PEG-Ac is prepared4MAN micelles containing DSPE-PEG-Ac4MAN and DSPE-PEG; drugs-LIP and DSPE-PEG-Ac4The MAN micelle is incubated for 2h at room temperature to obtain a therapeutic agent containing the drug and having brain targeting;
among them, it is preferable that, in the step (1), the molar ratio of dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol is 49.4: 3.2: 43.3: 4.1; in step (6), DSPE-PEG-Ac4The molar ratio of MAN to DSPE-PEG was 8.32: 3.75; in the step (4), the medicine is arsenic trioxide.
The application of the brain-targeted therapeutic agent in preparing the intracranial disease medicament is preferably that the intracranial disease is intracranial tumor, and more preferably that the intracranial tumor is brain glioma.
The invention establishes Ac loaded with ATO4MAN-LIP(Ac4M-ATO-LIP) and further investigated Ac4The potential of M-ATO-LIP in resisting glioma also makes concrete researches on the physicochemical properties, cytotoxic effect, cell uptake and curative effect of liposome on tumor-bearing mice. As a result, it was found that Ac4M-ATO-LIP has weaker cytotoxicity than free ATO, has stronger brain targeting property, and can prolong the survival time of tumor-bearing mice. Ac of4M-ATO-LIP may be used clinically in treating brainAnd (4) new selection of tumors.
Drawings
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 is a representation of liposomes;
determination of Ac with Zetasizer Nano ZS 904Average particle diameter (A) and Zeta potential (B) of M-ATO-LIP (PEG1000 is taken as an example); observing ATO-LIP (C) and Ac with transmission electron microscope4M micelle (D), Ac4Appearance of M-ATO-LIP (E) liposomes;
FIG. 4 shows the determination of ATO and Ni (OAC) at 37 ℃ of liposomes in PBS at pH 7.42The 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. 5 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. 6 is a graph showing the in vitro drug uptake using flow cytometry;
wherein, fig. 6A 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. 6B 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. 7A 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. 7B 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. 8A 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. 8B 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. 9A 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. 9B 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. 10 shows the anti-tumor effect of Ac4MAN-PEG-LIP on glioma-bearing mice;
wherein, FIG. 10A is H.E staining of frozen sections of glioma brain injected with Ac4MAN-PEG 1000-LIP; (scale, 50 μm);
FIG. 10B 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;
FIGS. 11A-C are each Ac4MAN, MAN and Ac4HPLC-MS images of MAN + Porcine Liver Esterase (PLE);
FIG. 11D is a flow cytometry histogram; data are shown as mean ± standard deviation (n-3, p <0.05, p <0.01, compared to cellular uptake of Rho-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:
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).
Cholesterol (CHOL) is derived from BioLife technologies, Inc. (China, Shanghai).
Arsenic trioxide is a gift from pharmaceutical Limited, Harbin medical university (China, Harbin).
Nickel acetate (Ni (OAC)2) Purchased from Sigma-Aldrich (st. louis, usa).
Sephadex G50 was purchased from Beijing Sun Biotechnology, Inc. (China, Beijing).
Rhodamine (Rho) is available from Haidel Biotechnology Inc. (China, Beijing).
Hoechst33258 and DiO were purchased from pabokotechnologies ltd, n.k. (china, beijing).
MTT kits were obtained from Soranon, OH, USA.
DMEM medium was obtained from Hyclone laboratories, GE healthcare Life sciences, Beijing, China.
p-carboxyphenyl- α -D-acetylmannosamine (Ac)4MAN) was obtained from Innochem, beijing, china.
Pig Liver Esterase (PLE) was purchased from Hangzhou Chuangke Biotech, Inc.
EXAMPLE 1 Synthesis of targeting molecule Diisopentylphosphatidylethanolamine-polyethylene glycol-1000-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-1000-Ac4MAN)
Azide-polyethylene glycol-glutaramide 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 and NHS (0.2g) and EDCI (0.45g) were added sequentially to the reaction chamberThe reaction was warmed overnight and column chromatography gave 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 targeting molecule Diisopentylphosphatidylethanolamine-polyethylene glycol-600-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-600-Ac)4MAN) Synthesis
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, and the peak corresponding to 5.5ppm is a characteristic peak of sugars.
Example 3 targeting molecule Diisopentylphosphatidylethanolamine-polyethylene glycol-2000-p-carboxyphenyl- α -D-acetylmannosamine (DSPE-PEG-2000-Ac)4MAN) Synthesis
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, 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 4 preparation and characterization 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. DryingThe lipid membrane of (2) is coated with 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
DSPE-PEG1000-Ac synthesized in example 14MAN 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) Morphology, size and Zeta potential of liposomes
Using Malverm Zetasizer NanoZS 90 Instrument for determining liposome Ac4Zeta potential and particle size distribution of M-ATO-LIP. Observation of Ac by Transmission Electron microscopy (TEM, H7650, Japan, Hitachi)4Morphology and size of M-ATO-LIP. And dripping the liposome suspension on a copper grid covered with a support film, absorbing redundant liquid on the copper grid by using filter paper after several minutes, covering the surface of the copper grid with 0.5-3% phosphotungstic acid solution as negative dye for 1-2 minutes, removing the dye, and drying at room temperature to obtain the sample to be detected by the transmission electron microscope.
FIGS. 3A and 3B are Ac4Particle size and Zeta potential of M-ATO-LIP (PEG 1000). 3C, 3D and 3E are ATO-LIP and Ac, respectively4M micelle and Ac4TEM image of M-ATO-LIP (PEG 1000). As shown in Table 1, Ac4M-ATO-LIP has an average particle size of about 160nm, a polydispersity index of about 0.161, and all liposomes are electrically neutral with less negative charge. Ac of4The particle size of M-ATO-LIP (PEG1000) is slightly larger than that of ATO-LIP. From the results of TEM, Ac4The surface of M-ATO-LIP is smooth and round structure, and is attached with a plurality of smaller spheres, and the uniform spheres are DSPE-PEG1000-Ac4MAN micelles.
TABLE 1 characterization of mean particle size, polydispersity index (PDI) and Zeta potential of liposomes. Data are presented as mean ± standard deviation (n ═ 3).
(4) 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), with each collection being followed by additionInto the same volume of PBS.
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. 4A and 4B 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 therapeutic Effect study experiment
(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) incubation cells, 48h and 72h incubation respectivelyAnd 96 h. 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. 5 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) Flow cytometry
Flow cytometry was used to detect cellular uptake of the different liposomes. We seeded U87 glioma cells into six well plates and allowed the cells to culture overnight, then replaced DMEM/low sugar for DMEM/high sugar and continued the culture for 12 hours, after which timeAdding free Rho, Rho-LIP and Ac respectively4M-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. 6A and 6B 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 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.
2) 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.
Fig. 7-9 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
(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, administered in a dose of 2. mu.g 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. 10B) show administration of saline, free ATO, ATO-LIP, and Ac4Mean survival time of M-ATO-LIP (PEG1000) glioma model mice was 23, 25, 30 andfor 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. 10A), 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 logical relationship study of "prodrug-like" liposomes
(1) Deacetylation of p-carboxyphenyl- α -D-acetylmannosamine
Determination of p-carboxyphenyl- α -D-acetylmannosamine (Ac) using liquid chromatography-mass spectrometry technique (HPLC-MS)4MAN) was added. 0.1mL of methanol-solubilized Ac4MAN (4mg/mL) was added to 1.9mL of double distilled water-solubilized Pig Liver Esterase (PLE) (3mg/mL) and incubated at 37 ℃ for 15h on a magnetic stirrer. Ac by HPLC-MS4MAN、Ac4MAN + PLE and p-aminophenyl- α -D-Mannoside (MAN).
FIGS. 11A-C are Ac4MAN、Ac4HPLC-MS images of MAN + PLE and MAN. In FIG. 11A, we can see that at 2.49min, the peak with relative molecular mass 440.18 is Ac4MAN. In FIG. 11B, the peak for MAN appeared at 0.72min and the molecular weight was 272.11. FIG. 11C is Ac4The peak of MAN + PLE had a peak length of 0.84min and a molecular weight of 272.11, which was the same as the molecular weight of MAN. The result of HPLC-MS experiment shows that Ac4MAN can be deacetylated and expose the target molecule MAN, which can direct nanoparticle targeting across the BBB to the brain tumor site.
(2) Uptake of prodrug-like liposomes by U87 cells after incubation with PLE
Prodrug-like liposomes were tested for U87 uptake by flow cytometry. The procedure for culturing and seeding of U87 cells was the same as in example 3. Cells were administered with Rho-LIP, Ac4M-Rho-LIP (PEG1000) and Ac4M-Rho-LIP (PEG1000) + PLE, respectively, incubated for 4h, trypsinized, centrifuged, resuspended in PBS, and the fluorescence intensity of intracellular Rho was measured using a flow cytometer.
Fig. 11D shows uptake of liposomes by U87 cells. As a result, the average fluorescence intensity of Rho-LIP was 11007, that of Ac4M-Rho-LIP (PEG1000) was 15995, and that of Ac4M-Rho-LIP (PEG1000) was 18967. Compared with Rho-LIP, the uptake of Ac4M-Rho-LIP (PEG1000) + PLE was most significant by U87 glioma cells (P < 0.05). From the results of cellular uptake, it can be seen that the addition of some PLE to Ac4M-Rho-LIP (PEG1000) enhanced brain targeting and glioma targeting of liposomes, possibly due to deacetylation of Ac4 MAN.
Claims (14)
1. An acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate having a structure represented by formula I:
wherein n is 10-120.
2. The acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of claim 1, wherein n is 13-45.
3. The acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of claim 1, wherein the polyethylene glycol is polyethylene glycol 600, polyethylene glycol 1000, or polyethylene glycol 2000.
4. A process for preparing the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of any one of claims 1 to 3, comprising the step of reacting p- (glutaramide) phenyl- α -D-acetylmannose (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 process of claim 4, comprising the steps of dissolving the azido-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- (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. Use of the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of claim 1 for the preparation of a pharmaceutical carrier.
7. The use of claim 6, wherein the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate has brain targeting effects, which helps the drug cross the blood-brain barrier and thus treat diseases.
8. The use of claim 7, wherein the drug is arsenic trioxide.
9. The use of claim 7, wherein the disease is a brain glioma.
10. A liposome carrier with brain targeting, characterized in that the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate of any one of claims 1 to 3 is coupled to the surface of the liposome.
11. A brain-targeted therapeutic agent consisting of a therapeutically effective amount of a drug encapsulated in a liposome carrier, wherein said agent is prepared by the following method:
(1) dissolving dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol in ethanol, evaporating by rotary evaporation to dryness to obtain lipid membrane in round bottom flask;
(2) dried lipid membrane using nickel acetate (Ni (OAC)2) Subjecting the solution to ultrasonic hydration, further dispersing by using a probe for 5min, and extruding for 3 times through a 220nm polycarbonate film;
(3) the extruded liposomes were passed through a SephadexG-50 column, except for Ni (OAC) outside the liposome bilayer2Solution by adding buffer 1 containing 300mM NaCl and 200mM HEPES, pH 6.8 to the liposome colloid solution, a gradient is formed between the inner and outer aqueous phases of the liposome, i.e., Ni (OAC) is formed2Liposomes (Ni-LIP);
(4) adding a solution of a drug into Ni-LIP, adjusting the pH to 7.2, incubating in a shaking table for 10-15 h, then, passing the liposome suspension through a SephadexG-50 column, and removing excessive drug by using a buffer solution 2 containing 300mM NaCl, 200mM HEPES and pH 4;
(5) adjusting the pH of the liposome to 7.2 to obtain LIP;
(6) use of the acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate (DSPE-PEG-Ac) of claim 14MAN) modified drug-LIP: firstly, DSPE-PEG-Ac is prepared4MAN micelles containing DSPE-PEG-Ac4MAN and DSPE-PEG; drugs-LIP and DSPE-PEG-Ac4The MAN micelle is incubated at room temperature for 2h to obtain the brain-targeted therapeutic agent containing the drug.
12. The therapeutic agent with brain targeting according to claim 11, wherein in step (1), the molar ratio of dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol is 49.4: 3.2: 43.3: 4.1; in step (6), DSPE-PEG-Ac4The molar ratio of MAN to DSPE-PEG was 8.32: 3.75; in the step (4), the medicine is arsenic trioxide.
13. Use of the brain-targeted therapeutic agent of claim 12 in the preparation of a medicament for intracranial disease.
14. The use according to claim 13, wherein the intracranial disease is an intracranial tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810301839.7A CN108578711B (en) | 2018-04-04 | 2018-04-04 | Acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810301839.7A CN108578711B (en) | 2018-04-04 | 2018-04-04 | Acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108578711A CN108578711A (en) | 2018-09-28 |
| CN108578711B true CN108578711B (en) | 2020-06-05 |
Family
ID=63624541
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810301839.7A Active CN108578711B (en) | 2018-04-04 | 2018-04-04 | Acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108578711B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111068069B (en) * | 2018-10-18 | 2022-05-20 | 中国医学科学院药物研究所 | Immune targeting functional liposome and preparation method and application thereof |
| CN110204733B (en) * | 2019-05-28 | 2021-07-02 | 长安大学 | Alpha-mannose modified donor-receptor type biopolymer material and preparation method and application thereof |
| CN111249235B (en) * | 2020-02-26 | 2021-09-24 | 哈尔滨医科大学 | Brain targeting nanoliposome loaded with positive polymer/miR-195 compound, and preparation method and application thereof |
| CN111249234B (en) * | 2020-02-26 | 2023-05-30 | 哈尔滨医科大学 | Preparation method and application of glycosyl-cell penetrating peptide-modified brain-targeted nanoliposome |
| CN114588111A (en) * | 2020-11-19 | 2022-06-07 | 复旦大学 | Acid-sensitive drug-releasing arsenic trioxide targeted liposome composition and preparation method thereof |
| CN115073726B (en) * | 2022-07-04 | 2023-09-26 | 华中科技大学同济医学院附属协和医院 | Ultrasonic molecular probe for targeting M2 type macrophage mannose receptor and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02225495A (en) * | 1989-02-28 | 1990-09-07 | Fuji Photo Film Co Ltd | Cholesterol derivative and production of liposome using the same |
| WO2001064605A2 (en) * | 2000-03-01 | 2001-09-07 | Therascope Ag | Generation and screening of a dynamic combinatorial library |
| CN105055318A (en) * | 2015-08-31 | 2015-11-18 | 石河子大学 | Double targeting liposome with 4-amino-benzene-alpha-D-mannopyranoside (MAN) and wheat germ agglutinin (WGA) modifiers and preparation method thereof and application |
-
2018
- 2018-04-04 CN CN201810301839.7A patent/CN108578711B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02225495A (en) * | 1989-02-28 | 1990-09-07 | Fuji Photo Film Co Ltd | Cholesterol derivative and production of liposome using the same |
| WO2001064605A2 (en) * | 2000-03-01 | 2001-09-07 | Therascope Ag | Generation and screening of a dynamic combinatorial library |
| CN105055318A (en) * | 2015-08-31 | 2015-11-18 | 石河子大学 | Double targeting liposome with 4-amino-benzene-alpha-D-mannopyranoside (MAN) and wheat germ agglutinin (WGA) modifiers and preparation method thereof and application |
Non-Patent Citations (4)
| Title |
|---|
| "Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-conjugates";Moghis U. Ahmad et al;《Chemistry and Physics of Lipids》;20141113;第186卷;第32页 * |
| "Dual-targeting daunorubicin liposomes improve the therapeutic efficacy of brain glioma in animals";Xue Ying et al;《Journal of Controlled Release》;20090930;第141卷;第184页 * |
| "Effect of Kupffer cells depletion on ABC phenomenon induced by Kupffer cells-targeted liposomes";Chaoyang Lai et al;《Asian Journal of Pharmaceutical Sciences》;20180904;第14卷;第457页 * |
| "脑靶向脂质体构建及其靶分子Ac4MAN体外结构稳定性研究";李茹冰 等;《中国医药导报》;20190430;第16卷(第11期);第5页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108578711A (en) | 2018-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108578711B (en) | Acetylated sugar ester-polyethylene glycol-phosphatidylethanolamine conjugate and preparation method and application thereof | |
| Xiong et al. | A NIR light triggered disintegratable nanoplatform for enhanced penetration and chemotherapy in deep tumor tissues | |
| Belhadj et al. | Multifunctional targeted liposomal drug delivery for efficient glioblastoma treatment | |
| CN106137967B (en) | Target the preparation and application of the dual modified liposome drug delivery system of glioma | |
| CN103083239B (en) | A kind of bufalin liposome and its preparation method and application | |
| CN110123761B (en) | Bionic high-density lipoprotein nanoparticle and preparation and application thereof | |
| KR20180120220A (en) | Biodegradable amphiphilic polymers specifically targeting ovarian cancer, polymeric vesicle made therefrom and uses thereof | |
| Chen et al. | Novel glycyrrhetinic acid conjugated pH-sensitive liposomes for the delivery of doxorubicin and its antitumor activities | |
| CN109350598B (en) | Sugar-polyethylene glycol-DSPE coupling compound and preparation method and application thereof | |
| CN113633625B (en) | Nano-drug of hybrid membrane loaded oxidative phosphorylation inhibitor and preparation method thereof | |
| CN114177306B (en) | Adriamycin targeted liposome with dual modification of iNGR/R9 and antitumor activity evaluation | |
| CN113952463B (en) | Nanometer diagnosis and treatment agent and preparation method and application thereof | |
| CN110433292A (en) | A kind of double targeting materials and its application in drug delivery | |
| CN108653743B (en) | Cardio-cerebral dual-targeting liposome and preparation method and application thereof | |
| CN108148193B (en) | Cholic acid-containing high polymer material and liposome modified by same | |
| CN106821987B (en) | Liposome carrying phenol hydroxyl group-containing insoluble drug, and preparation method and application thereof | |
| CN110772645A (en) | Functionalized cell-penetrating peptide modified drug delivery system | |
| CN112089845B (en) | Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof | |
| CN108309938B (en) | Drug delivery carrier for active customization of albumin crown and application of drug delivery carrier in pharmacy | |
| Li et al. | Novel DSPE-PEG-cholic acid-modified liposomes with hepatic targeting properties improve the anti-tumor efficacy of oral doxorubicin hydrochloride for liver tumor-bearing mice | |
| KR20200145115A (en) | Anti-cancer hybrid exosome composition having amphiphilic doxorubicin and exosome | |
| EA018636B1 (en) | Drug delivery system for administration of a water soluble, cationic and amphiphilic pharmaceutically active substance | |
| CN110898231B (en) | Functionalized Lalotxel liposome and preparation method and application thereof | |
| CN112535676A (en) | Nano-structure lipid preparation for improving adriamycin tumor active targeting and kidney protection and preparation method thereof | |
| CN107028882B (en) | Physically-wrapped tumor-targeted nano drug delivery system, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |