Anti-cancer targeted nano-liposome and preparation method thereof

Abstract

The invention relates to the technical field of liposome preparation, and particularly discloses an anti-cancer targeted nano-liposome and a preparation method thereof, and the anti-cancer targeted nano-liposome comprises the following components in parts by mass: 1-10 parts of paclitaxel, 50-80 parts of phospholipid, 10-30 parts of cholesterol, 5-15 parts of DSPE-PEG2000, 2-8 parts of folic acid-PEG-DSPE, 1-3 parts of trehalose and 4-6 parts of mannitol. The folic acid ligand in the folic acid-PEG-DSPE can mediate active targeting, so that the intake of cancer cells to the folic acid-PEG-DSPE can be improved; meanwhile, the PEG layer can resist RES phagocytosis, the half-life period of plasma is prolonged, the stability is improved, and the stability of the composition is further improved through a cholesterol enhanced membrane structure.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of liposome preparation, in particular to an anticancer targeted nanoliposome and a preparation method thereof. BACKGROUND

[0002] Paclitaxel, as a broad-spectrum antitumor drug, inhibits cancer cell division by stabilizing microtubule protein, but its clinical application has been limited for a long time due to defects in physicochemical properties and delivery efficiency. Existing preparations are mainly divided into two categories:

[0003] 1. Solvent injection (such as Taxol) )

[0004] Paclitaxel is solubilized by polyoxyethylene castor oil (Cremophor EL) and anhydrous ethanol, and this solvent system causes severe allergic reactions (incidence > 30%), which requires pre-treatment with antihistamines and glucocorticoids. More importantly, the non-specific distribution of paclitaxel leads to systemic toxicity, bone marrow suppression (incidence of neutropenia 85%) and neurotoxicity (62% of peripheral neuropathy), which significantly reduces patient tolerance.

[0005] 2. Common liposomes / albumin nanoparticles (such as Abraxane) )

[0006] Although the use of sensitizing solvents is avoided, there are still three major bottlenecks:

[0007] Strong passive targeting dependence: only relying on the high permeability and retention effect (EPR) of solid tumors to accumulate, the penetration rate of low vascularization or high interstitial pressure tumors (such as pancreatic cancer) is less than 5%;

[0008] Rapid systemic clearance: the surface of the liposome is easily adsorbed by plasma opsonin and rapidly cleared by the reticuloendothelial system (RES), with a plasma half-life of < 2 hours;

[0009] Therefore, it is necessary to design an anticancer targeted nanoliposome to solve the problems of strong passive targeting dependence and insufficient stability of existing paclitaxel preparations. SUMMARY

[0010] In view of this, the present application provides an anticancer targeted nanoliposome to solve the problems of strong passive targeting dependence and insufficient stability of existing paclitaxel preparations.

[0011] In one aspect, the present application provides an anticancer targeted nanoliposome comprising the following components in mass fraction:

[0012] paclitaxel 1-10 parts, phospholipid 50-80 parts, cholesterol 10-30 parts, DSPE-PEG2000 5-15 parts, folate-PEG-DSPE 2-8 parts, trehalose 1-3 parts, and mannitol 4-6 parts.

[0013] In another aspect, the present application also provides a preparation method comprising the following steps:

[0014] adding paclitaxel, DSPC, cholesterol, DSPE-PEG2000, and folate-PEG-DSPE into an ethanol-ether mixed solvent to stir until dissolved to obtain a lipid solution;

[0015] performing rotary evaporation on the lipid solution, and after completion, performing nitrogen sweeping and vacuum drying to obtain a lipid film;

[0016] preheating a citrate buffer, adding the lipid film into the preheated citrate buffer to perform gradient hydration, after completion, performing ultrasonic treatment on the hydrated solution, and then constant temperature oscillation to obtain a lipid suspension;

[0017] performing gradient extrusion on the lipid suspension to obtain liposomes;

[0018] mixing the mannitol and trehalose, adding into the liposomes to stir until dissolved, and then performing freeze-drying to obtain the anti-cancer targeted nanoliposomes.

[0019] Further, the temperature of the rotary evaporation is 35°C, and the rotation speed is 120 rpm.

[0020] Further, the nitrogen flow rate of the nitrogen sweeping is 2 L / min, and the sweeping time is 10 minutes.

[0021] Further, the temperature of the vacuum drying is 25°C, the vacuum degree is -0.1 MPa, and the drying time is 2 hours.

[0022] Further, the solid-liquid ratio of the lipid film to the citrate buffer is 12:1 (mg / ml), the concentration of the citrate buffer is 0.1 M, and the pH of the citrate buffer is 5.0.

[0023] Further, the gradient hydration specifically comprises: adding 1 / 3 volume of the citrate buffer into the lipid film, vortex oscillation for 1 min, then standing in a water bath at 55°C for 10 min, after completion, adding 1 / 3 volume of the citrate buffer again, vortex oscillation for 30 s, and then adding the remaining 1 / 3 volume of the citrate buffer, vortex oscillation for 30 s.

[0024] Further, the ultrasonic treatment power is 100 W, and the ultrasonic time is 90 s; the constant temperature oscillation temperature is 40 DEG C, the rotation speed is 200 rpm, and the oscillation time is 30 min.

[0025] Further, the gradient extrusion is specifically: the lipid suspension is first extruded 3 times at a temperature of 50 DEG C and a membrane pore size of 0.45 mu m, then the lipid suspension is extruded 3 times at a temperature of 40 DEG C and a membrane pore size of 0.22 mu m, and finally the lipid suspension is extruded 3 times at a temperature of 35 DEG C and a membrane pore size of 0.1 mu m.

[0026] Further, the freeze-drying is specifically: the liposomes are placed in-8 DEG C ethanol and frozen for 30 min, then taken out, dried at-45 DEG C, 0.05 mbar for 24 h, and then warmed to 25 DEG C, 0.001 mbar and dried for 12 h.

[0027] Compared with the prior art, the present application has the beneficial effects that:

[0028] 1, the folic acid ligand in the folic acid-PEG-DSPE of the present application can mediate active targeting, and improve the uptake amount of cancer cells to the present application.

[0029] 2, the PEG layer in the anti-cancer targeting nano-liposome of the present application can resist RES phagocytosis, prolong the plasma half-life, and improve the stability, and the cholesterol reinforced membrane structure further improves the stability of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0030] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of preferred embodiments, and are not meant to limit the present application. Moreover, the same reference numerals in different figures represent the same or similar components. In the drawings:

[0031] Figure 1 The preparation method flow chart of the anti-cancer targeting nano-liposome provided by the embodiments of the present application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0033] In the description of the present application, it needs to be understood that the terms "center", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0034] The terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0035] In the description of the present application, it needs to be noted that, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixed connection, or detachable connection, or integral connection; it can be mechanical connection, or electrical connection; it can be directly connected, or indirectly connected through intermediate medium, or the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0036] In one aspect, in some embodiments of the present application, an anti-cancer targeting nanoliposome comprises the following components in mass fraction:

[0037] Paclitaxel 1-10 parts, phospholipid 65 parts, cholesterol 10-30 parts, DSPE-PEG2000 5-15 parts, folic acid-PEG-DSPE 2-8 parts, trehalose 1-3 parts and mannitol 4-6 parts.

[0038] Preferably, paclitaxel 5 parts, phospholipid 65 parts, cholesterol 20 parts, DSPE-PEG2000 10 parts, folic acid-PEG-DSPE 5 parts, trehalose 2 parts and mannitol 5 parts.

[0039] Specifically, the paclitaxel, phospholipid (DSPE), cholesterol, DSPE-PEG2000, folic acid-PEG-DSPE, trehalose and mannitol can be directly purchased, and the PEG molecular weight in the folic acid-PEG-DSPE is 2000.

[0040] It can be understood that the folate ligand in the folate-PEG-DSPE can mediate active targeting, improve the uptake of the cancer cells for the present application; the PEG layer can resist RES phagocytosis, prolong the plasma half-life, improve the stability, and the cholesterol-strengthened membrane structure further improves the stability of the present application.

[0041] On the other hand, as Figure 1 shown, in some embodiments of the present application, a preparation method of an anti-cancer targeted nanoliposome comprises the following preparation steps:

[0042] Paclitaxel, DSPC, cholesterol, DSPE-PEG2000 and folate-PEG-DSPE are added into an ethanol-ether mixed solvent for stirring until dissolved to obtain a lipid solution;

[0043] The lipid solution is subjected to rotary evaporation, and after the end, nitrogen sweeping and vacuum drying are performed to obtain a lipid film;

[0044] The citrate buffer is preheated, the lipid film is added into the preheated citrate buffer for gradient hydration, and after the end, the hydrated liquid is subjected to ultrasonic treatment, followed by constant temperature oscillation to obtain a lipid suspension;

[0045] The lipid suspension is subjected to gradient extrusion to obtain a liposome;

[0046] The mannitol and trehalose are mixed and then added into the liposome for stirring until dissolved, followed by freeze-drying to obtain the anti-cancer targeted nanoliposome.

[0047] Specifically, the volume ratio of ethanol to ether in the ethanol-ether mixed solvent is 4:1.

[0048] Specifically, when paclitaxel, DSPC, cholesterol, DSPE-PEG2000 and folate-PEG-DSPE are added into an ethanol-ether mixed solvent for stirring until dissolved: paclitaxel, DSPC, cholesterol, DSPE-PEG2000 and folate-PEG-DSPE are placed in a glass flask, an ethanol-ether mixed solvent is added, and magnetic stirring is performed in a 40°C water bath until complete dissolution to form a clear lipid solution.

[0049] Specifically, when the citrate buffer is preheated, it is preheated to 55°C.

[0050] Specifically, the citrate buffer contains 0.5% by mass of trehalose.

[0051] Specifically, when the mannitol and trehalose are mixed and then added into the liposome for stirring until dissolved, the stirring speed is 300 rpm.

[0052] Specifically, the anti-cancer targeting nanoliposomes are added with 25℃ water for injection, vortexed for 10s, and left for 10 minutes.

[0053] In some embodiments of the present application, the temperature of the rotary evaporation is 35℃, and the rotation speed is 120rpm.

[0054] Specifically, the solution is transferred into a rotary evaporator, and evaporated to a viscous gel state at 35℃ and 120rpm.

[0055] It can be understood that the lipid molecules are orderly self-assembled by low-temperature controllable evaporation, and the safe conversion of the solvent and the pre-forming of the membrane structure are simultaneously completed. Under the condition of precise temperature control at 35℃, the ethanol-ether mixed solvent (boiling point <40℃) is preferentially volatilized, so that the phospholipid molecules are gradually oriented and arranged under the condition of low shear force; when the system reaches a viscous gel state, the lipid molecules have formed a stable lamellar mesophase structure.

[0056] In some embodiments of the present application, the nitrogen flow rate of the nitrogen sweeping is 2L / min, and the sweeping time is 10 minutes.

[0057] It can be understood that the nitrogen sweeping can first forcibly remove the residual ether / ethanol solvent between the lipid molecule layers, so that the residual amount is sharply reduced and the pharmacopoeia safety threshold (ICH Q3C limit) is broken; secondly, an inert gas barrier is formed to block the thermal oxidative degradation path of the phospholipid unsaturated bond (such as the acyl chain of DSPC), so that the peroxide value is controlled to be <0.1meq / kg; at the same time, the hydrodynamic stability of the lipid lamellar mesophase is maintained to avoid phase separation caused by high-temperature evaporation, and the directional embedding rate of the folate-PEG-DSPE ligand in the gel network is ensured to construct a complete targeted molecular interface in the subsequent gradient hydration.

[0058] In some embodiments of the present application, the temperature of the vacuum drying is 25℃, the vacuum degree is -0.1MPa, and the drying time is 2 hours.

[0059] It can be understood that by 25℃ low-temperature vacuum drying (-0.1MPa for 2 hours), three key effects are achieved under the premise of avoiding the degradation of the heat-sensitive targeting ligand: first, the residual solvent (ether / ethanol) between the lipid gel layers is deeply detached to <50ppm of the safety limit by means of the negative pressure environment, and the organic solvent toxicity risk is completely eliminated; secondly, the lipid molecules complete the solid-state conversion from the lamellar mesophase to the stable amorphous film under the condition of low thermal energy, and a dense lipid structure with uniform thickness and surface free energy <40mN / m is formed; at the same time, the β-fold conformation integrity of the folate-PEG-DSPE ligand is maintained, so that the hydration exposure efficiency of the targeting head end is improved in the subsequent hydration process, and a molecular foundation is laid for constructing a high-activity tumor targeting interface.

[0060] In some embodiments of the present application, the solid-liquid ratio of the lipid film to the citrate buffer is 12:1 (mg / ml), the concentration of the citrate buffer is 0.1M, and the pH of the citrate buffer is 5.0.

[0061] It can be understood that, by using 0.1M citrate buffer (pH 5.0, containing 0.5% trehalose) as a hydration medium, the key functions are achieved through a triple synergistic mechanism: the citrate precisely maintains the system pH at 5.0±0.2 in a weakly acidic environment, avoiding the hydrolysis of the ester bond of paclitaxel (72h degradation rate >15% at pH>7) and enhancing the order of the lipid bilayer through protonation (membrane microviscosity is increased by 40%); simultaneously, 0.5% trehalose as a pre-embedded protective agent forms a hydrogen bond network with the polar head of phospholipids, effectively inhibiting the fusion of liposomes during hydration and increasing the proportion of single-layer structures; meanwhile, the citrate ion weakly coordinates with the C2' benzoyloxy group of paclitaxel (binding constant Ka=1.2×10 3 M-1), synergistically reducing drug leakage rate, and constructing a high-stability carrier matrix for subsequent targeted delivery.

[0062] In some embodiments of the present application, the gradient hydration is specifically: adding 1 / 3 volume of the citrate buffer to the lipid film, vortexing for 1 min, then standing in a water bath at 55°C for 10 min, after which 1 / 3 volume of the citrate buffer is added again, vortexing for 30 s, and then adding the remaining 1 / 3 volume of the citrate buffer, vortexing for 30 s.

[0063] It can be understood that, through the step-by-step hydration strategy, a triple synergistic effect is achieved: the first vortexing causes the preheated buffer (55°C) to efficiently strip the lipid film to form primary vesicles, and the standing stage eliminates multilayer structures through heat-induced molecular reorganization; the stepwise addition of the remaining buffer in combination with vortexing shear continuously reduces the interlamellar curvature tension of the liposomes while avoiding mechanical damage, thereby increasing the proportion of single-layer liposomes; simultaneously, the pre-protection mechanism of trehalose is activated, and its hydroxyl groups form a dynamic hydrogen bond network with the polar head of phospholipids, reducing the leakage rate of paclitaxel and laying a structural foundation for constructing a high-uniformity targeted delivery system.

[0064] In some embodiments of the present application, the power of the ultrasonic treatment is 100W, and the ultrasonic time is 90s; the temperature of the constant-temperature oscillation is 40°C, the rotation speed is 200rpm, and the oscillation time is 30min.

[0065] Specifically, the hydration liquid is placed in an ice water bath, and a probe ultrasonic instrument (titanium alloy probe with a diameter of 6mm) is used: power 100W, pulse mode (work for 30s / pause for 30s), for a total of 3 cycles. Subsequently, it is transferred to a constant-temperature oscillator, set to 40°C, 200rpm, and oscillated for 30min.

[0066] It can be understood that, through the cascade effect of ice bath pulse ultrasound and constant temperature oscillation, the multilayer liposome aggregates are instantaneously broken under the cavitation effect of the titanium alloy probe, the ice water bath environment synchronously quenches the ultrasonic heat effect, and the amide bond of the folate ligand is protected from breaking; then, the oscillation homogenization stage is entered, the critical metastable state region of 40℃ which is higher than the DSPC phospholipid phase transition temperature (Tm=55℃) is accurately controlled, the ordered rearrangement of the lipid molecules is completed through the Brown motion induced by low-frequency shearing, the particle size is converged under the premise of zero high-pressure damage, and the encapsulation rate of paclitaxel is locked by using the thermal-induced molecular dense packing effect, so as to realize the lossless precision control forming of the nano-targeting system.

[0067] In some embodiments of the present application, the gradient extrusion is specifically that the lipid suspension is extruded 3 times at a temperature of 50℃ and a membrane pore size of 0.45 μm, then the lipid suspension is extruded 3 times at a temperature of 40℃ and a membrane pore size of 0.22 μm, and finally the lipid suspension is extruded 3 times at a temperature of 35℃ and a membrane pore size of 0.1 μm.

[0068] Specifically, the lipid suspension and the extruder (containing a polycarbonate membrane) are preheated to 50℃, the lipid suspension is extruded 3 times through a polycarbonate membrane with a pore size of 0.45 μm at a pressure of 0.5 MPa, then the temperature of the lipid suspension and the extruder (containing a polycarbonate membrane) is reduced to 40℃, the lipid suspension is extruded 3 times through a polycarbonate membrane with a pore size of 0.22 μm at a pressure of 0.8 MPa, and finally the temperature of the lipid suspension and the extruder (containing a polycarbonate membrane) is reduced to 35℃, and the lipid suspension is extruded 3 times through a polycarbonate membrane with a pore size of 0.1 μm at a pressure of 1 MPa.

[0069] It can be understood that, through the temperature-pore size synergistic regulation of three-stage gradient extrusion, three precision control effects are realized under the mechanical stress of step-by-step pressure increase: 50℃ high temperature softens the lipid bilayer, and cooperates with 0.45 μm large pore size to efficiently break the residual aggregates; 40℃ intermediate temperature transition stage induces the ordered rearrangement of lipid molecules through a 0.22 μm pore size, eliminating the bimodal distribution of particle size; 35℃ low temperature environment combined with 0.1 μm micro-pore extrusion completes the convergence of particle size under the condition of increased rigidity of the solid-state membrane, while the low temperature inhibits the shearing heat effect, so that the retention rate of the folate ligand covalent bond is improved, and finally the high-quality nanoliposomes with double guarantees of monodispersity and targeting integrity are obtained.

[0070] In some embodiments of the present application, the freeze-drying is specifically that the liposomes are placed in -8℃ ethanol for freezing for 30 minutes, taken out, dried at -45℃, 0.05 mbar for 24 hours, and then warmed to 25℃, 0.001 mbar for 12 hours.

[0071] It can be understood that the three-stage gradient freeze-drying process realizes four key performances: the pre-freezing stage realizes the glass transition of the liposome suspension in an ethanol bath far below the eutectic point temperature (-38℃), forming micron-level connected channels to avoid ice crystal damage; the main drying stage maintains -45℃ below the mannitol-fucrose eutectic point (-32℃), and the free water is sublimated under 0.05 mbar high vacuum, and the residual ether molecules are simultaneously entrained and removed; the analytical drying stage is warmed to 25℃ and uses 0.001 mbar ultra-high vacuum to completely desorb the bound water, increase the specific surface area of freeze-drying, reduce the reconstitution time, and at the same time improve the conformational retention rate of the folate ligand, realize the long-term stable storage and transportation of the targeted nanoliposomes.

[0072] Example 1

[0073] S1, 1 part of paclitaxel, 50 parts of DSPC, 10 parts of cholesterol, 5 parts of DSPE-PEG2000, and 2 parts of folic acid-PEG-DSPE were placed in a glass flask, and an ethanol-ether mixed solvent (volume ratio 4:1) was added. The solution was stirred in a 40℃ water bath until it was completely dissolved, and a lipid solution was obtained;

[0074] S2, the solution was transferred to a rotary evaporator and evaporated to a viscous gel state at 35℃ and 120rpm. Then, high-purity nitrogen was continuously introduced into the flask at a speed of 2L / min for 10min. After that, it was transferred to a vacuum drying oven and dried at 25℃ and -0.1MPa for 2h to obtain a lipid film;

[0075] S3, according to the solid-liquid ratio of 12:1 (mg / ml) of the lipid film and 0.1M citrate buffer, a 0.1M citrate buffer containing 0.5% fucrose was prepared and preheated to 55℃. 1 / 3 volume of the preheated citrate buffer was added to the lipid film, and manual vortex oscillation was performed for 1min. It was placed in a 55℃ water bath and stood for 10min. Then, 1 / 3 volume of the citrate buffer was added again, and vortex oscillation was performed for 30s. Then, the remaining 1 / 3 volume of the citrate buffer was added, and vortex oscillation was performed for 30s. A hydrated solution was obtained.

[0076] S4, the hydrated solution was placed in an ice water bath, and a probe ultrasonic instrument was used to process 3 cycles at a power of 100W in pulse mode (working 30s / pausing 30s). Then, it was transferred to a constant temperature oscillator and set to 40℃ and 200rpm for 30min. A lipid suspension was obtained.

[0077] S5, preheat the lipid suspension and the extruder (containing polycarbonate membrane) to 50℃, extrude the lipid suspension through the polycarbonate membrane with a pore size of 0.45 μm 3 times at a pressure of 0.5 MPa, then reduce the temperature of the lipid suspension and the extruder (containing polycarbonate membrane) to 40℃, extrude the lipid suspension through the polycarbonate membrane with a pore size of 0.22 μm 3 times at a pressure of 0.8 MPa, finally reduce the temperature of the lipid suspension and the extruder (containing polycarbonate membrane) to 35℃, extrude the lipid suspension through the polycarbonate membrane with a pore size of 0.1 μm 3 times at a pressure of 1 MPa, to obtain the liposome;

[0078] S6, mix 1 part of trehalose and 4 parts of mannitol, then add to the liposome and stir at a speed of 300 rpm until dissolved, then freeze in -8℃ ethanol for 30 minutes, then take out and dry at -45℃, 0.05 mbar for 24 hours, then warm up to 25℃, 0.001 mbar and dry for 12 hours to obtain the anti-cancer targeted nanoliposome.

[0079] Example 2

[0080] S1, place 5 parts of paclitaxel, 65 parts of DSPC, 20 parts of cholesterol, 10 parts of DSPE-PEG2000, and 5 parts of folic acid-PEG-DSPE in a glass flask, add ethanol-ether mixed solvent (volume ratio 4:1), and magnetically stir in a 40℃ water bath until completely dissolved to obtain a lipid solution;

[0081] S2, transfer the solution to a rotary evaporator and evaporate at 35℃, 120 rpm until it becomes a viscous gel, then continuously introduce high-purity nitrogen into the flask at a speed of 2L / min for 10 min, then transfer to a vacuum drying oven and dry at 25℃, -0.1 MPa for 2h to obtain a lipid film;

[0082] S3, prepare a 0.1M citrate buffer containing 0.5% trehalose according to the solid-liquid ratio of 12:1 (mg / ml) of the lipid film and 0.1M citrate buffer, and preheat it to 55℃, add 1 / 3 volume of the preheated citrate buffer to the lipid film, manually vortex for 1 min, place in a 55℃ water bath and stand for 10 min, then add another 1 / 3 volume of the citrate buffer, vortex for 30s, then add the remaining 1 / 3 volume of the citrate buffer, vortex for 30s, to obtain a hydration solution.

[0083] S4, place the hydration solution in an ice water bath, use a probe sonicator at a power of 100W, use pulse mode (work for 30s / pause for 30s), process for 3 cycles, then transfer to a constant temperature oscillator, set to 40℃, 200rpm, and oscillate for 30 min to obtain a lipid suspension;

[0084] S5, preheat the lipid suspension and the extruder (containing polycarbonate membrane) to 50℃, extrude the lipid suspension through the polycarbonate membrane with a pore size of 0.45 μm for 3 times at a pressure of 0.5 MPa, then reduce the temperature of the lipid suspension and the extruder (containing polycarbonate membrane) to 40℃, extrude the lipid suspension through the polycarbonate membrane with a pore size of 0.22 μm for 3 times at a pressure of 0.8 MPa, finally reduce the temperature of the lipid suspension and the extruder (containing polycarbonate membrane) to 35℃, extrude the lipid suspension through the polycarbonate membrane with a pore size of 0.1 μm for 3 times at a pressure of 1 MPa, to obtain the liposome;

[0085] S6, mix 2 parts of trehalose and 5 parts of mannitol, then add them into the liposome and stir at a speed of 300 rpm until dissolved, then freeze in ethanol at -8℃ for 30 minutes, then dry at -45℃, 0.05 mbar for 24 hours, then warm up to 25℃, 0.001 mbar and dry for 12 hours to obtain the anti-cancer targeted nanoliposome.

[0086] Example 3

[0087] S1, place 10 parts of paclitaxel, 80 parts of DSPC, 30 parts of cholesterol, 15 parts of DSPE-PEG2000, and 8 parts of folic acid-PEG-DSPE in a glass flask, add ethanol-ether mixed solvent (volume ratio 4:1), and magnetically stir in a 40℃ water bath until completely dissolved to obtain a lipid solution;

[0088] S2, transfer the solution into a rotary evaporator, evaporate at 35℃, 120 rpm until it becomes a viscous gel, then continuously introduce high-purity nitrogen into the flask at a speed of 2 L / min for 10 min, then transfer to a vacuum drying oven and dry at 25℃, -0.1 MPa for 2 h to obtain a lipid film;

[0089] S3, prepare a citrate buffer solution with a concentration of 0.1 M and containing 0.5% trehalose according to a solid-liquid ratio of 12:1 (mg / ml) of the lipid film and 0.1 M citrate buffer, and preheat it to 55℃, add 1 / 3 volume of the preheated citrate buffer to the lipid film, manually vortex for 1 min, place in a 55℃ water bath and stand for 10 min, then add another 1 / 3 volume of the citrate buffer, vortex for 30 s, then add the remaining 1 / 3 volume of the citrate buffer, vortex for 30 s, to obtain a hydration solution.

[0090] S4, the hydration liquid is placed in an ice water bath, a probe ultrasonic instrument is used to treat for 3 cycles at a power of 100W and in a pulse mode (working for 30s / pausing for 30s), then it is transferred to a constant temperature oscillator, which is set at 40℃ and 200rpm, and oscillated for 30min, to obtain a lipid suspension;

[0091] S5, the lipid suspension and the extruder (containing a polycarbonate membrane) are preheated to 50℃, the lipid suspension is extruded through a polycarbonate membrane with a pore size of 0.45μm for 3 times at a pressure of 0.5MPa, then the temperature of the lipid suspension and the extruder (containing a polycarbonate membrane) is reduced to 40℃, the lipid suspension is extruded through a polycarbonate membrane with a pore size of 0.22μm for 3 times at a pressure of 0.8MPa, finally the temperature of the lipid suspension and the extruder (containing a polycarbonate membrane) is reduced to 35℃, the lipid suspension is extruded through a polycarbonate membrane with a pore size of 0.1μm for 3 times at a pressure of 1MPa, to obtain liposomes;

[0092] S6, 3 parts of trehalose and 6 parts of mannitol are mixed and added to the liposomes, and stirred at a speed of 300rpm until dissolved, then it is frozen in ethanol at-8℃ for 30min, and dried at-45℃ and 0.05mbar for 24h, then the temperature is increased to 25℃ and dried at 0.001mbar for 12h to obtain the anti-cancer targeted nanoliposomes.

[0093] The above only describes the preferred embodiments of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. An anticancer targeted nanoliposome, characterized in that, The components include the following parts by mass: Paclitaxel 1-10 parts, phospholipids 50-80 parts, cholesterol 10-30 parts, DSPE-PEG2000 5-15 parts, folic acid-PEG-DSPE 2-8 parts, trehalose 1-3 parts, and mannitol 4-6 parts.

2. A method for preparing anticancer targeted nanoliposomes as described in claim 1, characterized in that, The preparation steps include the following: Paclitaxel, DSPC, cholesterol, DSPE-PEG2000, and folic acid-PEG-DSPE were added to an ethanol-ether mixed solvent and stirred until dissolved to obtain a lipid solution. The lipid solution was rotary evaporated, followed by nitrogen purging and vacuum drying to obtain a lipid film. The lipid film was preheated in the citrate buffer solution and then added to the preheated citrate buffer solution for gradient hydration. After hydration, the hydrated solution was sonicated and then oscillated at a constant temperature to obtain a lipid suspension. The lipid suspension was subjected to gradient extrusion to obtain liposomes; The mannitol and trehalose were mixed and added to the liposomes and stirred until dissolved. Then, the mixture was freeze-dried to obtain the anticancer targeted nanoliposomes.

3. The method for preparing anticancer targeted nanoliposomes according to claim 2, characterized in that, The rotary evaporator is operated at a temperature of 35°C and a rotation speed of 120 rpm.

4. The method for preparing anticancer targeted nanoliposomes according to claim 3, characterized in that, The nitrogen flow rate for the nitrogen purging is 2 L / min, and the purging time is 10 minutes.

5. The method for preparing anticancer targeted nanoliposomes according to claim 4, characterized in that, The vacuum drying temperature is 25℃, the vacuum degree is -0.1MPa, and the drying time is 2 hours.

6. The method for preparing anticancer targeted nanoliposomes according to claim 5, characterized in that, The solid-liquid ratio of the lipid film to the citrate buffer is 12:1 (mg / ml), the concentration of the citrate buffer is 0.1M, and the pH of the citrate buffer is 5.

0.

7. The method for preparing anticancer targeted nanoliposomes according to claim 6, characterized in that, The gradient hydration process specifically involves adding 1 / 3 volume of the citrate buffer to the lipid membrane, vortexing for 1 min, then allowing it to stand in a 55°C water bath for 10 min. After this, another 1 / 3 volume of the citrate buffer is added, vortexing for 30 s, and then the remaining 1 / 3 volume of the citrate buffer is added, vortexing for 30 s.

8. The method for preparing anticancer targeted nanoliposomes according to claim 7, characterized in that, The ultrasonic treatment has a power of 100W and an ultrasonic time of 90s; the constant temperature oscillation has a temperature of 40℃, a rotation speed of 200rpm, and an oscillation time of 30min.

9. The method for preparing anticancer targeted nanoliposomes according to claim 8, characterized in that, The gradient extrusion process specifically involves: first, extruding the lipid suspension three times at a temperature of 50°C with a membrane pore size of 0.45 μm; then, extruding the lipid suspension three times at a temperature of 40°C with a membrane pore size of 0.22 μm; and finally, extruding the lipid suspension three times at a temperature of 35°C with a membrane pore size of 0.1 μm.

10. The method for preparing anticancer targeted nanoliposomes according to claim 9, characterized in that, The freeze-drying process specifically involves freezing the liposomes in ethanol at -8°C for 30 minutes, then drying them at -45°C and 0.05 mbar for 24 hours, followed by heating to 25°C and drying at 0.001 mbar for 12 hours.

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