CN114533673A

CN114533673A - Active drug-loaded liposome and preparation method thereof

Info

Publication number: CN114533673A
Application number: CN202111095618.7A
Authority: CN
Inventors: 李阳
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2022-05-27
Anticipated expiration: 2041-09-17
Also published as: CN114533673B

Abstract

The invention provides an active drug-loaded liposome, which realizes active drug entrapment by coupling a prodrug of a drug-MAL coupling with a water-soluble sulfhydryl substance in the liposome, and has the characteristics of high drug retention, high entrapment rate, high drug loading capacity and high drug loading speed. The liposome can prolong the blood circulation time of the medicament, and has stronger in-vivo anti-tumor activity compared with free medicaments. The optimized water-soluble sulfhydryl substance glutathione in the invention is a natural substance in vivo, has low toxicity and good biocompatibility, is a commercial product, has lower price, and is beneficial to clinical transformation of the liposome preparation prepared by the method. The method has wide applicability, is applicable to a plurality of medicaments containing modifiable chemical groups and having certain permeability, and can widen the application range of the liposome.

Description

Active drug-loaded liposome and preparation method thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, relates to a drug-loaded liposome, and particularly relates to an active drug-loaded liposome and a preparation method thereof.

Background

The liposome is a vesicle-type nanoparticle formed by lipid materials such as phospholipid and cholesterol, and is a microparticle formulation which is most successful in clinical transformation. At present, dozens of liposome medicines are successfully marketed at home and abroad, more than 300 liposome medicines are undergoing clinical tests, and the liposome medicine covers a plurality of clinical treatment fields such as anti-tumor, antibiosis, vaccine, anesthesia and the like.

Drug loading with high drug loading and high encapsulation efficiency is a difficult and critical point in liposome formulation development. Currently, liposome drug encapsulation methods are classified into passive drug loading methods and active drug loading methods. For passive drug loading, hydrophilic drugs can be passively encapsulated in the aqueous phase in liposomes, but there are problems of low encapsulation efficiency, low drug loading, and the need for additional steps to remove unencapsulated drugs. The hydrophobic drug can be encapsulated in the liposome membrane, the encapsulation efficiency is higher, but the drug loading rate is low, the drug loading stability is poor, and the drug can be easily and rapidly released in blood circulation. Although chemical modification of drugs (e.g., covalent coupling of drugs to long fatty chains) can increase the interaction force between the drugs and the liposome membrane and further promote the loading of the drugs in the liposome membrane, the method cannot completely overcome the limitations of passive drug-loading methods.

The active drug loading method utilizes the concentration gradient of ions inside and outside a liposome membrane to ensure that a drug passes through the liposome membrane in a non-ionized molecular form, enters an internal water phase and then forms compound precipitation with the ions, thereby completing the efficient loading of the drug. Compared with a passive drug loading method, the active drug loading method can efficiently encapsulate the drug in the inner water phase of the liposome, has the advantages of high drug loading and encapsulation efficiency, and greatly promotes the clinical transformation of liposome drugs. Such as adriamycin liposome (Doxil), irinotecan liposome (Onivyde), vincristine liposome (Marqibo) and the liposome carrying both arabinoside and daunorubicin (Vyxeos) which are marketed products, are all loaded with drugs by an active drug loading method.

In the active drug loading method, a drug and ions in the liposome form a compound precipitate through reversible physical interaction to finish active drug loading, but the compound can be dissociated into a neutral drug again and then leaks out of the liposome. Thus, the drug retention of active drug-loaded liposomes depends on the stability of the drug-ion complex. However, only a few weak base or weak acid drugs with rigid planar structures can form stable drug-ion complexes with specific ions, and for most weak base or weak acid drugs, it is difficult to find the stable drug-ion complexes formed by the specific ions. Therefore, although the active drug loading method can realize the entrapment of weak base or weak acid drugs and has high entrapment rate, most of the weak base or weak acid drugs are difficult to form a stable drug-ion complex, so that the retention of liposome drugs is poor, the in vivo drug release is too fast, and the clinical development and application of liposome preparations are greatly limited.

Therefore, the novel active drug-loaded liposome is developed, the retention of the liposome drug is improved on the premise of high entrapment rate and high drug-loading capacity, the rapid leakage of the liposome drug in the body is avoided, and the development and application of the liposome preparation are effectively promoted.

Disclosure of Invention

To solve the problems of the prior art, according to a first aspect of the present invention, there is provided an actively drug-loaded liposome. The invention has the advantages of high drug retention, high drug loading speed, high entrapment rate and high drug loading rate.

Except for special description, the parts are parts by weight, and the percentages are mass percentages.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an active drug-loaded liposome, which is characterized in that: the active drug-loaded liposome is prepared from a blank liposome and a prodrug, wherein the blank liposome contains a water-soluble sulfhydryl substance inside, and the water-soluble sulfhydryl substance inside the blank liposome is coupled with the prodrug to finish active loading of drugs. The prodrug is a drug-MAL coupled prodrug formed by connecting Maleimide (MAL) and a drug through a covalent bond.

Firstly, preparing liposome in water-soluble sulfhydryl substance (such as glutathione and GSH) water solution, further removing the water-soluble sulfhydryl substance (such as glutathione and GSH) outside the liposome, and establishing water-soluble sulfhydryl substance (such as glutathione and GSH) transmembrane gradient to obtain blank liposome; the aqueous suspension of the drug-MAL conjugated prodrug is then incubated with blank liposomes during which the prodrug enters the interior of the liposomes and is conjugated to a poorly membrane permeable water soluble sulfhydryl species (e.g., glutathione, GSH). The principle of the active drug-loaded liposome is shown in figure 1, and the prodrug is coupled with a water-soluble sulfhydryl substance in the liposome through Michael addition reaction so as to complete drug loading. Due to poor membrane permeability of water-soluble sulfhydryl substances (such as glutathione and GSH), the prodrug coupled with the water-soluble sulfhydryl substances is also retained in the liposome, so that drug loading is realized, and the drug retention is strong. Different from the traditional active drug loading method (a pH gradient method and an ammonium sulfate method) which utilizes reversible physical interaction to complete drug loading, the invention utilizes irreversible chemical reaction to complete drug loading, and has the characteristics of strong drug retention, high drug loading rate, high drug loading amount, high encapsulation efficiency and wide applicability.

The water-soluble sulfhydryl substance refers to water-soluble substance containing sulfhydryl, such as one or more of cysteine, Glutathione (GSH), organic amine containing sulfhydryl, organic acid, dipeptide, oligopeptide, polypeptide, protein, sugar or high molecular polymer; glutathione (GSH) is preferred.

The chemical bond for covalent connection is one or more of ester bond, carbonate bond, amido bond or hydrazone bond.

The prodrug of the drug-MAL coupling is a prodrug taking an ester bond, a carbonate bond, an amido bond or a hydrazone bond as a connecting bond, and the drug is a chemotherapeutic drug containing hydroxyl, amino, carboxyl and carbonyl, such as camptothecin, taxanes, podophyllotoxins, doxorubicin, gemcitabine, mitoxantrone, triptolide and the like.

The structural general formula of the drug-Maleimide (MAL) coupled prodrug is as follows:

n is an integer, R is a spacer group, and Drug is the structure of a Drug

CO/COO forms an ester or carbonate bond with a hydroxyl group in a Drug (Drug)

Taking Camptothecin (CPT) as an example, the structure of the camptothecin-MAL coupled prodrug is as follows:

the membrane material used for preparing the above-mentioned liposome is cholesterol (Chol) and phospholipid, wherein the phospholipid is selected from one or more of soybean phospholipid, egg yolk lecithin, Hydrogenated Soybean Phospholipid (HSPC), Phosphatidylcholine (PC), Phosphatidylglycerol (PG), Phosphatidylinositol (PI), phosphatidic acid (phosphatidic acid), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), glycolipid (glycerolipid), sphingolipids (sphingolipids), and derivatives thereof, such as polyethylene glycol (polyethylene glycol) modified single phospholipid component or combination component.

Further, the phospholipid is selected from one or more of soybean phospholipid, Hydrogenated Soybean Phospholipid (HSPC), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).

According to a second aspect of the invention, the invention provides a preparation method of the active drug-loaded liposome.

The preparation method of the active drug-loaded liposome comprises the following steps:

1) preparing liposomes in an aqueous solution of a sulfhydryl substance; 2) removing the water-soluble sulfhydryl substance out of the liposome, and establishing a transmembrane gradient of the water-soluble sulfhydryl substance to obtain a blank liposome containing the water-soluble sulfhydryl substance inside; 3) dissolving the prodrug in an organic solvent which is mutually soluble with water, then dispersing the prodrug into water, and incubating the prodrug with blank liposomes; in the process of incubating the prodrug and the blank liposome together, the prodrug enters the liposome and is coupled with the water-soluble sulfhydryl substance with poor membrane permeability to finish the active loading of the drug. 4) Removing the organic solvent in the system by reduced pressure evaporation, dialysis or ultrafiltration.

In order to improve the drug loading speed, encapsulation efficiency, drug loading rate and retention of the liposome, the pH value of the aqueous solution of the sulfhydryl substance (such as glutathione, GSH) is 4.0-6.5, the concentration is 50-500mM, and preferably 300-500 mM; the ratio of the prodrug to the water-soluble mercapto substance is 0.1 to 1.0(mol/mol), preferably 0.9 to 1.0 (mol/mol); the concentration of the organic solution is 5-50%, preferably 5-30%, calculated by volume percentage; the temperature of the co-incubation is 38-60 ℃, preferably 50-60 ℃. The co-incubation time is 5-60 minutes, preferably 5-30 minutes. The organic solvent is selected from ethanol, acetonitrile, methanol, acetone, dimethylformamide or dimethyl sulfoxide, preferably ethanol.

The method for removing the water-soluble thiol-group substances from the liposome is selected from dialysis, centrifugation, gel column chromatography or ultrafiltration.

According to a third aspect of the invention, the invention provides the use of the active drug-loaded liposome in tumor treatment, and the administration routes comprise intravenous administration and local administration.

Has the advantages that:

the invention provides an active drug-loaded liposome, which realizes active drug entrapment by coupling a prodrug of drug-MAL coupling with a water-soluble sulfhydryl substance in the liposome, and has the characteristics of high entrapment rate and difficult drug leakage. The drug loading rate of the invention is fast, and the drug loading can be completed within 5 minutes at 60 ℃. The drug-MAL coupled prodrug and Glutathione (GSH) can complete drug loading according to a 1:1 reaction, the drug loading amount of the liposome depends on the concentration of Glutathione (GSH) in blank liposome and the molecular mass of the drug, so that higher drug loading amount can be obtained, such as the drug loading amount of paclitaxel (PTX, molecular weight of 853.9) is up to 40% (mass ratio). The liposome can obviously prolong the blood circulation time of the medicament, and has stronger in-vivo anti-tumor activity compared with free medicaments. The preferred water-soluble sulfhydryl substance Glutathione (GSH) is a natural substance in vivo, has low toxicity and good biocompatibility, is a commercial product, has low price, and is beneficial to clinical transformation of liposome preparations. The method has wide applicability, is suitable for most medicaments which contain hydroxyl, amino, carboxyl, carbonyl and the like, can modify chemical groups and have certain permeability, and can widen the application range of the liposome.

Drawings

Fig. 1 is a schematic diagram of the principle of active drug-loaded liposomes, wherein the prodrug of drug-MAL conjugate refers to a prodrug in which the drug is covalently linked to the MAL;

FIG. 2 is a diagram of CPT-MAL (1) in example 1¹An H-NMR spectrum;

FIG. 3 is a diagram of CPT-MAL (2) in example 2¹An H-NMR spectrum;

FIG. 4 is a diagram of CPT-MAL (3) in example 3¹An H-NMR spectrum;

FIG. 5 is a diagram of CPT-MAL (4) in example 4¹An H-NMR spectrum;

FIG. 6 is of PTX-MAL in example 5¹An H-NMR spectrum;

FIG. 7 is a transmission electron micrograph of glutathione blank liposomes (A) and CPT-MAL (1) liposomes in example 6 (B, arrows indicate drug crystals);

FIG. 8 is the leakage of liposomal drug from the plasma of rats in example 6: the invention relates to a CPT-MAL (1) liposome (A), a CPT-EDA liposome (B) and a CPT-PA liposome (C) which are prepared by an ammonium sulfate gradient method;

FIG. 9 is a transmission electron micrograph of PTX-MAL liposomes of example 7 (arrows indicate drug crystals);

FIG. 10 is the appearance change (A) and HPLC analysis (B) during the loading of CPT-MAL (1) liposomes in example 8; FIG. 11 is the effect of drug loading at 38 deg.C (A) and 60 deg.C (B) on the drug loading rate of CPT-MAL (1) liposomes in example 9;

FIG. 12 is the effect of the organic phase (acetonitrile) ratio on the encapsulation efficiency of CPT-MAL (1) liposomes in example 10;

FIG. 13 is the stability of CPT-MAL (1) liposomes during storage at 4 ℃ in example 11;

FIG. 14 is a blood-time graph of CPT-MAL (1) liposomes and free CPT injected intravenously into rats in example 12;

FIG. 15 is a graph of tumor growth of subcutaneous tumors of CT26 treated with intravenous CPT-MAL (1) liposomes in example 13.

Detailed Description

In the following embodiments, the present invention is described only by way of example, but those skilled in the art, after reading the present patent application, may make various modifications thereto without departing from the spirit and scope of the present invention.

The related nouns are:

entrapment Efficiency (EE) refers to the ratio of the drug loaded in the liposome to the amount of drug administered.

Calculating the formula: EE ═ W (amount of drug in liposome)/W (amount of drug administered) x 100%

Drug Loading Capacity (LC), refers to the ratio of drug loaded in the liposome to the total mass of the liposome.

Calculating the formula: LC ═ W (drug amount in liposomes)/W (total amount of liposomes) × 100%

EXAMPLE 1 Synthesis of CPT-MAL (1)

0.3mmol of Camptothecin (CPT) is dispersed in 20ml of dry dichloromethane, 0.1mmol of triphosgene and 0.6mmol of 4-Dimethylaminopyridine (DMAP) are added under the protection of nitrogen, the mixture is reacted for 10 minutes in ice bath, 0.3mmol of N- (2-hydroxyethyl) maleimide is added, and the reaction is carried out overnight. After the reaction is finished, filtering to remove unreacted CPT, washing the filtrate for 1 time by using citric acid water, washing the filtrate for two times by using saturated NaCl solution, collecting an organic phase, and adding anhydrous Na₂SO₄Water removal, filtration, and passage of the filtrate through a silica gel column with dichloromethane: eluting with an eluting solvent of methanol (50:1) to obtain a product CPT-MAL (1), and identifying the product by a nuclear magnetic resonance spectroscopy (figure 2) to confirm the success of the synthesis.

EXAMPLE 2 Synthesis of CPT-MAL (2)

0.3mmol of 3-maleimidopropionic acid was dissolved in 20ml of dichloromethaneAfter 0.33mmol of dicyclohexylcarbodiimide was added and the reaction was carried out at room temperature for 5 minutes, 0.3mmol of CPT and a catalytic amount of DMAP (ca. 5mg) were added and the reaction was carried out for 2 hours in an ice bath. After the reaction is finished, spin-drying the reaction solution, adding 20ml of ethyl acetate, filtering, spin-drying the filtrate, adding 20ml of dichloromethane, washing with saturated lemon water for 1 time, washing with saturated NaCl solution for two times, collecting the organic phase, adding anhydrous Na₂SO₄Water removal, filtration, and passage of the filtrate through a silica gel column with dichloromethane: eluting with an elution solvent of methanol 25:1 to obtain a product CPT-MAL (2), and identifying the product by a nuclear magnetic resonance spectroscopy (figure 3) to confirm the success of the synthesis.

EXAMPLE 3 Synthesis of CPT-MAL (3)

0.3mmol of 3-maleimidopropionic acid was dissolved in 20ml of dichloromethane, 0.33mmol of dicyclohexylcarbodiimide was added thereto, and after 5 minutes at room temperature, 0.6mmol of ethylene glycol and a catalytic amount of DMAP (ca. 5mg) were added thereto, and the reaction was carried out for 2 hours in an ice bath. After the reaction was completed, the reaction solution was spin-dried, 20ml of ethyl acetate was added, and the mixture was filtered, and the filtrate was passed through a silica gel column and washed with ethyl acetate: eluting with petroleum ether (1:1) to obtain intermediate 1.

0.3mmol of CPT is dispersed in 20ml of dry dichloromethane, 0.1mmol of triphosgene and 0.6mmol of DMAP are added under the protection of nitrogen, the mixture is reacted for 10 minutes under ice bath, 0.3mmol of intermediate product 1 is added, and the reaction is carried out overnight. After the reaction is finished, filtering to remove unreacted CPT, washing the filtrate for 1 time by using citric acid solution, washing the filtrate for two times by using saturated NaCl solution, collecting an organic phase, and adding anhydrous Na₂SO₄Dewatering, filtering, passing the filtrate through a silica gel plate, and separating by dichloromethane: eluting with an eluting solvent of methanol (20:1) to obtain a product CPT-MAL (3), and identifying the product by a nuclear magnetic resonance spectroscopy (figure 4) to confirm the success of the synthesis.

EXAMPLE 4 Synthesis of CPT-MAL (4)

0.3mmol of 3-maleimidopropionic acid was dissolved in 20ml of methylene chloride, 0.33mmol of dicyclohexylcarbodiimide was added thereto, and after 5 minutes at room temperature, 0.6mmol of 2, 2-dithiodiethanol and a catalytic amount of DMAP (ca. 5mg) were added thereto and reacted for 2 hours in ice bath. After the reaction is finished, the reaction solution is dried by spinning, 20ml of ethyl acetate is added, the mixture is filtered, and the filtrate is subjected to silica gel column reaction by using ethyl acetate: eluting with petroleum ether (1:1) to obtain intermediate 2.

0.3mmol of CPT is dispersed in 20ml of dry dichloromethane, 0.1mmol of triphosgene and 0.6mmol of DMAP are added under the protection of nitrogen, the mixture is reacted for 10 minutes under ice bath, 0.3mmol of intermediate product 2 is added, and the reaction is carried out overnight. After the reaction is finished, filtering to remove unreacted CPT, washing the filtrate for 1 time by adopting saturated citric acid solution, washing the filtrate for two times by adopting saturated NaCl solution, collecting an organic phase, and adding anhydrous Na₂SO₄Dewatering, filtering, passing the filtrate through a silica gel plate, and separating by dichloromethane: eluting with an eluting solvent of methanol (20:1) to obtain a product CPT-MAL (4), and identifying the product by a nuclear magnetic resonance spectroscopy (figure 5) to confirm the success of the synthesis.

Example 5 Synthesis of PTX (paclitaxel) -MAL

0.6mmol of N- (2-hydroxyethyl) maleimide was dispersed in 20ml of dry dichloromethane, 0.2mmol of triphosgene and 1.2mmol of DMAP were added under nitrogen protection, and reacted for 10 minutes in ice bath, 0.6mmol of Paclitaxel (PTX) was added and reacted overnight. After the reaction is finished, the reaction solution is washed for 1 time by saturated citric acid solution, washed for two times by saturated Na Cl solution, the organic phase is collected, and anhydrous Na is added₂SO₄The synthesis was confirmed to be successful by removing water, filtering, separating the filtrate by silica gel plate (dichloromethane: methanol ═ 20:1) to obtain the product, and identifying the product by nuclear magnetic resonance spectroscopy (fig. 6).

EXAMPLE 6 preparation of CPT-MAL (1) liposomes

Preparing blank liposome: 42mg HSPC, 20mg Chol, 13mg DSPE-PEG2000 were weighed out and dissolved in 1ml ethanol. Glutathione (GSH) 600mg was dissolved in 5ml of pure water to prepare a 400mM Glutathione (GSH) solution. And dropwise adding the lipid ethanol solution into a Glutathione (GSH) aqueous solution under the condition of vigorous stirring to obtain a crude liposome solution. The liposome solution is placed under the ultrasonic of a probe, and the ultrasonic treatment is carried out at 400W until the system is transparent. Transferring the obtained liposome into a dialysis bag with molecular weight cutoff of 3000, dialyzing at 38 deg.C in 400mM Na Cl, replacing dialysate every 2 hr for 8 times to obtain blank liposome carrying Glutathione (GSH), and measuring Glutathione (GSH) content in the liposome by ELLMAN method. The results show that the content of Glutathione (GSH) in the blank liposome is 0.2-0.3mol/mol Glutathione (GSH)/total phospholipids, and the total phospholipids comprise phospholipids and cholesterol.

Preparing CPT-MAL (1) liposome: 12mg of CPT-MAL (1) is placed in 1ml of acetonitrile, heated and dissolved, and is added dropwise into 3ml of 400mM NaCl solution at 60 ℃ under the condition of vigorous stirring, then the blank liposome is added, stirring is continued for 20 minutes at 60 ℃ until the system is changed from turbid into translucent solution, and the acetonitrile in the system is removed by a rotary evaporation method (40 ℃). The shapes of the blank liposome and the CPT-MAL (1) liposome are observed by adopting a transmission electron microscope, the liposome encapsulation efficiency is determined by adopting an ultrafiltration method, and the retentivity of the CPT-MAL (1) liposome in rat plasma is determined by adopting High Performance Liquid Chromatography (HPLC).

Preparation of control liposomes: a weakly basic CPT prodrug (structure shown in fig. 8B and 8C) was synthesized and control liposomes were prepared using active drug loading (ammonium sulfate gradient). Weighing 42mg HSPC, 20mg Chol and 13mg DSPE-PEG2000, dissolving in 1ml ethanol, dropwise adding the ethanol solution of the lipid into 100mM ammonium sulfate solution under the condition of vigorous stirring, placing the liposome under probe ultrasound, performing 400W ultrasound until the system is transparent, transferring into a dialysis bag, dialyzing in 5% sucrose at 38 ℃, replacing the dialysate every 2 hours for 8 times in total, and obtaining the blank ammonium sulfate liposome. Then 1mg of the weakly basic CPT prodrug is dissolved in water, 1ml of ammonium sulfate blank liposome is added, and the mixture is incubated for 20 minutes at 60 ℃ to obtain the weakly basic CPT prodrug liposome. The retention of weakly basic CPT prodrug liposomes in rat plasma was determined by HPLC.

The transmission electron micrograph of the GSH blank liposome and the CPT-MAL (1) liposome is shown in FIG. 7, and it can be seen that compared with the blank liposome, the CPT-MAL (1) liposome has drug crystallization inside, which indicates that the drug is successfully encapsulated by the liposome. The encapsulation efficiency of the liposome drug determined by the ultrafiltration method is 97.5%.

The plasma retention result is shown in fig. 8, and the result shows that after incubation for 24h at 38 ℃, CPT-MAL (1) in the CPT-MAL (1) liposome is not significantly degraded, the prodrug content is only reduced by 5%, and the prodrug in the weakly basic CPT prodrug liposome is almost completely degraded, which indicates that the drug retention of the CPT-MAL (1) liposome is obviously superior to that of the weakly basic CPT prodrug liposome prepared by the ammonium sulfate gradient method.

Example 7 PTX (paclitaxel) -MAL Liposome preparation and characterization

24mg of PTX-MAL was dissolved in 1ml of ethanol, and added dropwise to 3ml of 400mM NaCl solution at 60 ℃ with vigorous stirring, followed by addition of the above Glutathione (GSH) blank liposome, stirring at 60 ℃ for 20 minutes until the system became translucent, and ethanol in the system was removed by rotary evaporation (40 ℃). The morphology of the PTX-MAL liposome is observed by a transmission electron microscope, and the liposome encapsulation efficiency is determined by an ultrafiltration method. As a result, as shown in FIG. 9, the drug crystallization inside the PTX-MAL liposome was clearly seen, and the entrapment rate of PTX-MAL was 96.4% as determined by ultrafiltration and the drug loading was as high as 40%.

Example 8 analysis of the drug Loading Process of CPT-MAL (1) liposomes

0.5mg of CPT-MAL (1) was dissolved in 0.05ml of acetonitrile by heating, added dropwise to 0.15ml of 400mM NaCl solution at 60 ℃ under vigorous stirring, followed by addition of 0.5ml of the above Glutathione (GSH) blank liposome, incubated at 60 ℃ and sampled for HPLC analysis at prescribed time points, and the change in the clarity of the liposome was recorded by photographing. As a result, as shown in FIG. 10, CPT-MAL (1) reacts rapidly with Glutathione (GSH) during the incubation, and the liposome becomes clear rapidly from turbidity, indicating that CPT-MAL (1) can be rapidly encapsulated by the liposome.

Example 9 Effect of temperature and drug Loading on the drug Loading Rate of CPT-MAL (1) liposomes

0.125mg, 0.25mg, 0.5mg and 1mg of CPT-MAL (1) were placed in 0.05ml of acetonitrile, dissolved by heating, added dropwise to 0.15ml of 400mM NaCl solution at 38 ℃ and 60 ℃ respectively under vigorous stirring, then 0.5ml of the above Glutathione (GSH) blank liposome was added, incubation was continued to a prescribed time point, and the encapsulation efficiency of CPT-MAL (1) liposome was analyzed by HPLC. The results are shown in fig. 11, where the drug loading was slow at 38 ℃ and required a minimum of 1 hour to complete, and at 60 ℃ the drug loading was rapid and completed in 5 minutes. CPT-MAL (1) can react with Glutathione (GSH)1:1 in liposome, so the drug loading of CPT-MAL (1) liposome depends on the content of Glutathione (GSH) in liposome, when CPT-MAL (1): when Glutathione (GSH) <1:1, CPT-MAL (1) can be completely entrapped.

EXAMPLE 10 Effect of organic phase ratio on the encapsulation efficiency of CPT-MAL (1) liposomes

0.5mg of CPT-MAL (1) was dissolved in 0.05ml, 0.1ml, 0.2ml, 0.4ml of acetonitrile, added dropwise to 0.045ml, 0.4ml, 0.3ml and 0.1ml of 400mM NaCl solution, respectively, and then 0.5ml of the above Glutathione (GSH) blank liposome was added, incubated at 60 ℃ for 20 minutes, and the encapsulation efficiency of CPT-MAL (1) liposome was analyzed by HPLC. As a result, as shown in FIG. 12, when the concentration of acetonitrile was 40%, the encapsulation efficiency of CPT-MA L (1) liposome was significantly decreased, and therefore, the concentration of acetonitrile was controlled to be 30% or less.

Example 11 CPT-MAL (1) Liposome storage stability study

The prepared CPT-MAL (1) liposome is stored at 4 ℃, the encapsulation efficiency of the liposome is measured at a specified time, and the result is shown in figure 13, and the encapsulation efficiency of the CPT-MAL (1) liposome is not obviously reduced within 6 months of storage time, which indicates that the CPT-MAL (1) liposome has better storage stability.

EXAMPLE 12 pharmacokinetic study of CPT-MAL (1) liposomes

8 male SD rats weighing about 200g were randomly divided into 2 groups of 4, each group was administered free CPT and CPT-MAL (1) liposomes at a dose of 3mg/kg (based on the CPT prototype drug), and after administration, blood was taken at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours and 72 hours, respectively, placed in heparin-coated EP tubes, centrifuged at 5000 rpm for 5 minutes, and plasma supernatants were taken. Plasma drug concentration was measured by HPLC and plasma drug concentration-time curves were plotted, and the results are shown in fig. 14, where the time of CPT-MAL (1) liposomes in blood circulation was significantly prolonged compared to free CPT, and the area under the blood drug-time curve (AUC) was 469.3 times that of free CPT, indicating that the liposomes prepared by this method significantly prolonged the blood circulation time of the drug.

EXAMPLE 13 pharmacodynamic study of CPT-MAL (1) liposomes

CT26 colon cancer cells were suspended in serum-free 1640 medium. On day 0, Balb/C mice were injected subcutaneously into the right dorsal side of the mouse with a concentration of 2X 10/0 mL⁶one/mL cancer cell suspension, when the tumor volume is 50mm³At times, the samples were randomly divided into 3 groups (5 per group) and numbered. The CPT solution, CPT-MAL (1) liposome and normal saline are respectively injected into the components intravenously, the dosage is 10mg/kg (based on CPT proto-drug), the CPT-MAL (1) liposome is administrated once every 6 days for three times, the daily tumor volume is recorded, and the result is shown in figure 15, and the CPT-MAL (1) liposome shows the antitumor activity which is obviously superior to that of free CPT.

Claims

1. An active drug-loaded liposome, which is characterized in that: the active drug-loading liposome is prepared from a blank liposome and a prodrug, wherein the blank liposome contains a water-soluble sulfhydryl substance inside, and the water-soluble sulfhydryl substance inside the blank liposome is coupled with the prodrug to finish the active loading of drugs; the prodrug is a drug-MAL coupled prodrug formed by covalent connection of Maleimide (MAL) and a drug; the prodrug of the drug-MAL coupling is a prodrug taking an ester bond, a carbonate bond, an amido bond or a hydrazone bond as a connecting bond, and the drug is a chemotherapeutic drug containing hydroxyl, amino, carboxyl and carbonyl.

2. The actively drug-loaded liposome of claim 1, wherein: the water-soluble sulfhydryl substance is selected from one or more of cysteine, Glutathione (GSH), organic amine containing sulfhydryl, organic acid, dipeptide, oligopeptide, polypeptide, protein, sugar or high molecular polymer; glutathione (GSH) is preferred.

3. The actively drug-loaded liposome of claim 1, wherein: the medicine is selected from camptothecin, taxane, podophyllotoxin, doxorubicin, gemcitabine, mitoxantrone or triptolide.

4. The actively drug-loaded liposome of claim 1, wherein: the general structural formula of the drug-MAL coupled prodrug is as follows:

5. the actively drug-loaded liposome of claim 1, wherein: the structure of the drug-MAL conjugated prodrug is as follows:

6. the actively drug-loaded liposome of any one of claims 1-5, wherein: the membrane material used for preparing the liposome is cholesterol (Chol) and phospholipid, and the phospholipid is selected from soybean phospholipid, egg yolk lecithin, Hydrogenated Soybean Phospholipid (HSPC), Phosphatidylcholine (PC), Phosphatidylglycerol (PG), Phosphatidylinositol (PI), phosphatidic acid (phosphatidic acid), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), glycolipid (glycerolipid), sphingolipid (sphingolipid), sphingoglycolipid (glycerolipid) and derivatives thereof, such as polyethylene glycol (polyethylene glycol) modified single phospholipid component or combined component; further preferred phospholipids are one or more of soybean phospholipids, Hydrogenated Soybean Phospholipids (HSPC), Distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).

7. The method for preparing drug-loaded liposomes of any one of claims 1 to 6, comprising the steps of:

1) preparing liposomes in an aqueous solution of a sulfhydryl substance; 2) removing the water-soluble sulfhydryl substance out of the liposome, and establishing a transmembrane gradient of the water-soluble sulfhydryl substance to obtain a blank liposome containing the water-soluble sulfhydryl substance inside; 3) dissolving the prodrug in an organic solvent which is mutually soluble with water, then dispersing the prodrug into water, and incubating the prodrug with blank liposomes; in the process of incubating the prodrug and the blank liposome together, the prodrug enters the liposome and is coupled with a water-soluble sulfhydryl substance with poor membrane permeability to finish active loading of the drug; 4) removing the organic solvent in the system by reduced pressure evaporation, dialysis or ultrafiltration.

8. The method of claim 7, wherein: the pH of the solution of the water-soluble thiol substance is 4.0-6.5, and the concentration is 50-500mM, preferably 300-500 mM; the ratio of the prodrug to the water-soluble mercapto substance is 0.1 to 1.0(mol/mol), preferably 0.9 to 1.0; the concentration of the organic solution is 5-50%, preferably 5-30%, calculated by volume percentage; the organic solvent is selected from ethanol, acetonitrile, methanol, acetone, dimethyl formyl or dimethyl sulfoxide, and ethanol is preferred; the temperature of the co-incubation is 38-60 ℃, preferably 50-60 ℃. The co-incubation time is 5-60 minutes, preferably 5-30 minutes.

9. The method of claim 7 or 8, wherein: the method for removing water-soluble sulfhydryl substances outside liposome is selected from dialysis, centrifugation, gel column chromatography or ultrafiltration.

10. The use of the actively drug-loaded liposome of any one of claims 1-6 in tumor therapy; further, routes of administration include intravenous administration and topical administration.