Nano-liposome and preparation method thereof

Abstract

The invention relates to the technical field of liposome preparation, and particularly discloses a nano liposome and a preparation method thereof.The method comprises the steps that distearoyl phosphatidyl ethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol and mPEG-Hyd-PEG-SH are dissolved in a mixed solvent, and a lipid film is formed through rotary evaporation; hydrating with a PBS (Phosphate Buffer Solution) containing ammonium sulfate, and carrying out ultrasonic treatment to obtain primary emulsion; a doxorubicin solution is added for incubation, and drug loading is completed; the CD44 antibody and the drug-loaded liposome are coupled in a catalytic system, and are homogenized by a spiral microreactor. The problems of high immunogenicity, insufficient targeting, non-uniform particle size, uncontrollable drug release and complex preparation process of the existing PEG nano-liposome are solved, and the PEG nano-liposome has the advantages of low immunogenicity, high targeting, uniform and stable particle size, drug response release and easiness in large-scale production.

Description

Technical Field

[0001] This invention relates to the field of liposome preparation technology, and more specifically, to a nanoliposome and its preparation method. Background Technology

[0002] PEG nanoliposomes, as a highly efficient drug delivery system, have shown significant application potential in areas such as tumor treatment and intervention for inflammatory diseases. Their core principle involves modifying the liposome surface with polyethylene glycol (PEG) to form a hydration protective layer, prolonging in vivo circulation time, and achieving passive targeted drug delivery by leveraging the high permeability of tumor blood vessels (EPR effect). Currently, several PEG nanoliposome formulations have been approved for marketing, such as doxorubicin liposomes (Doxil) and paclitaxel liposomes (Lipusu) for ovarian cancer treatment, whose advantages in reducing drug toxicity and improving therapeutic index have been clinically validated.

[0003] However, existing PEG nanoliposome technology still faces many critical issues that urgently need to be addressed, which severely restrict its clinical application and industrialization process.

[0004] Regarding immunogenicity, traditional PEG nanoliposomes are mostly modified with methoxy-terminated PEG (MeO-PEG). Pre-existing anti-PEG antibodies, widely present in the human body, can specifically bind to MeO-PEG, triggering the "accelerated serum clearance (ABC) effect." This effect leads to rapid clearance of liposomes after repeated administration, significantly shortening their circulation time in vivo, reducing drug accumulation in target tissues, and ultimately affecting therapeutic efficacy. Studies have shown that MeO-PEG-modified liposomes exhibit a 3-5 fold increase in blood clearance rate after a second dose, greatly limiting their application in long-term treatment.

[0005] Insufficient targeting is another prominent issue. Current technologies mainly rely on the EPR effect for passive targeting, but this mechanism is significantly affected by tumor type and individual differences, and it still exhibits non-specific distribution in normal tissues, leading to drug accumulation in healthy organs (such as the heart and liver) and causing toxic side effects. Taking doxorubicin liposomes as an example, although its cardiotoxicity is reduced by about 70% compared to the free drug, 20%-30% of patients still experience varying degrees of myocardial damage, the root cause of which lies in the lack of a precise active targeting mechanism.

[0006] Poor particle size uniformity and stability are also significant factors restricting their development. Traditional preparation methods (such as thin-film dispersion and emulsification) struggle to precisely control liposome particle size, resulting in wide particle size distributions (PDI often > 0.2), making them prone to aggregation or fusion, leading to drug leakage and reduced encapsulation efficiency. During storage and transportation, the physical stability of liposomes further deteriorates; after lyophilization and reconstitution, particle size changes often exceed 20%, and encapsulation efficiency losses reach 10%-15%, severely impacting product shelf life and clinical safety.

[0007] The uncontrollability of drug release also urgently needs to be addressed. Most existing PEG nanoliposomes utilize a passive diffusion release mode, releasing large amounts of drug before reaching the target tissue, which reduces efficacy and increases toxic side effects. Although some studies have introduced stimulus-response mechanisms such as pH and temperature, the response sensitivity is insufficient, and the drug release rate in the tumor microenvironment (pH 5.0-6.5) is often below 50%, making rapid and complete release difficult to achieve and thus failing to meet clinical treatment needs.

[0008] Furthermore, existing preparation processes are complex and inefficient, making large-scale production difficult. Traditional methods require step-by-step operations such as liposome preparation, drug loading, and targeted modification, with a total preparation time of 12-24 hours. The seamless transitions between steps can easily lead to product loss, resulting in high costs. At the same time, significant batch-to-batch variations make it difficult to ensure product quality uniformity, hindering its industrialization process.

[0009] Therefore, it is necessary to design a nanoliposome to address the problems of drug clearance, insufficient targeting, non-uniform particle size, and uncontrollable drug release caused by pre-existing anti-PEG antibodies in existing nanoliposomes. Summary of the Invention

[0010] In view of this, the present invention proposes a nanoliposome to solve the problems of drug clearance, insufficient targeting, non-uniform particle size, and uncontrollable drug release caused by pre-existing anti-PEG antibodies in existing nanoliposomes.

[0011] On one hand, the present invention provides a method for preparing nanoliposomes, comprising the following preparation steps:

[0012] Distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol, and mPEG-Hyd-PEG-SH were dissolved in a chloroform-methanol mixture, and then dried under vacuum after rotary evaporation to obtain a lipid film.

[0013] A PBS solution containing ammonium sulfate was added to the lipid film to carry out a hydration reaction, followed by ultrasonic treatment to obtain the colostrum.

[0014] Free thioamino acids were removed from the colostrum, and doxorubicin solution was added for incubation to obtain drug-loaded liposomes.

[0015] The CD44 antibody and the drug-loaded liposomes were added to a mixed solution of CuSO4 and sodium ascorbate for coupling reaction. After the reaction, ultrafiltration was performed to obtain a targeted liposome suspension.

[0016] The targeted liposome suspension was homogenized to obtain the nanoliposomes.

[0017] Furthermore, the mass ratio of distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol, and mPEG-Hyd-PEG-SH is 5:3:2:1.5.

[0018] Furthermore, the rotary evaporation temperature is 40°C and the rotation speed is 100 rpm.

[0019] Furthermore, the pH of the PBS solution containing ammonium sulfate is 7.4, and the concentration of ammonium sulfate is 200 mM.

[0020] Furthermore, the hydration reaction is carried out at a temperature of 60°C for 30 minutes; the ultrasonic treatment is performed at a power of 100W for 20 minutes.

[0021] Furthermore, the mass ratio of doxorubicin in the doxorubicin solution to the mass of the lipid film is 1:(10-15).

[0022] Furthermore, the incubation temperature is 37°C, and the incubation time is 1 hour.

[0023] Furthermore, the mass ratio of the CD44 antibody to the drug-loaded liposome is 1:2.

[0024] Furthermore, the solvent in the mixed solution of CuSO4 and sodium ascorbate is PBS buffer, wherein the concentration of CuSO4 is 0.1 mM and the concentration of sodium ascorbate is 1 mM.

[0025] On the other hand, the present invention also provides a nanoliposome prepared by the preparation method described above.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention uses hydroxyl-terminated polyethylene glycol (HO-PEG) modification to reduce the binding rate of liposomes to pre-existing anti-PEG antibodies in the human body, prolong the circulating half-life, and avoid accelerating serum clearance (ABC effect).

[0028] 2. The present invention performs homogenization treatment on liposomes, which significantly improves the uniformity of liposome particle size.

[0029] 3. The CD44 antibody modification in this invention significantly improves the uptake rate of liposomes on CD44-high expression tumor cells (such as breast cancer MCF-7); at the same time, the pH-responsive hydrazone bond of mPEG-Hyd-PEG-SH enables the invention to release 80% of the drug within 4 hours in the tumor microenvironment (pH 6.0), while the release rate is <20% under normal physiological conditions (pH 7.4), which significantly improves the efficacy and reduces cardiotoxicity. Attached Figure Description

[0030] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0031] Figure 1 This is a flowchart illustrating the preparation method of nanoliposomes provided in an embodiment of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0033] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0034] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0035] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0036] On the one hand, such as Figure 1 As shown in some embodiments of this application, a method for preparing nanoliposomes includes the following preparation steps:

[0037] Distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol, and mPEG-Hyd-PEG-SH were dissolved in a chloroform-methanol mixture, and then dried under vacuum after rotary evaporation to obtain a lipid film.

[0038] A PBS solution containing ammonium sulfate was added to the lipid film to carry out a hydration reaction, followed by ultrasonic treatment to obtain the colostrum.

[0039] Free thioamino acids were removed from the colostrum, and doxorubicin solution was added for incubation to obtain drug-loaded liposomes.

[0040] The CD44 antibody and the drug-loaded liposomes were added to a mixed solution of CuSO4 and sodium ascorbate for coupling reaction. After the reaction, ultrafiltration was performed to obtain a targeted liposome suspension.

[0041] The targeted liposome suspension was homogenized to obtain the nanoliposomes.

[0042] Specifically, the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is 3:1.

[0043] Specifically, the vacuum drying time is 2 hours.

[0044] Specifically, the solid-liquid ratio of the lipid film to the PBS solution containing ammonium sulfate is 1:10 (mg / ml).

[0045] Specifically, when removing free thiosulfate from the colostrum, the colostrum is passed through a Sephadex G-50 column to remove free ammonium sulfate.

[0046] Specifically, the concentration of the doxorubicin solution is 0.3 mg / ml.

[0047] Specifically, the CD44 antibody and the drug-loaded liposomes were added to a mixed solution of CuSO4 and sodium ascorbate for coupling reaction at room temperature for 4 hours. The mixture was then purified by ultrafiltration (100 kDa molecular weight cutoff) to obtain a targeted liposome suspension.

[0048] Specifically, the homogenization process involves passing the targeted liposome suspension through a helical focusing flow microreactor at a flow rate of 100 mL / min through a 100 nm polycarbonate filter membrane five times. The helical focusing flow microreactor has an inner diameter of 0.8 mm and a helical radius of 20 mm, and is prepared using the method disclosed in patent CN202411324074.

[0049] Specifically, the mPEG-Hyd-PEG-SH is a block copolymer composed of methoxy polyethylene glycol (mPEG), hydrazone (Hyd) and mercapto (-SH).

[0050] It is understood that the present invention uses hydroxyl-terminated polyethylene glycol (HO-PEG) modification to reduce the binding rate of liposomes to pre-existing anti-PEG antibodies in the human body, prolong the circulating half-life, and avoid accelerating serum clearance (ABC effect).

[0051] It is understood that the present invention has homogenized the liposomes, which significantly improves the uniformity of liposome particle size.

[0052] Understandably, the CD44 antibody modification in this invention significantly enhances the uptake rate of liposomes on CD44-high expression tumor cells (such as breast cancer MCF-7); at the same time, the pH-responsive hydrazone bond of mPEG-Hyd-PEG-SH enables this invention to release 80% of the drug within 4 hours in the tumor microenvironment (pH 6.0), while the release rate is <20% under normal physiological conditions (pH 7.4), significantly improving efficacy and reducing cardiotoxicity.

[0053] In some embodiments of this application, the mass ratio of distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol, and mPEG-Hyd-PEG-SH is 5:3:2:1.5.

[0054] Understandably, this mass ratio of 5:3:2:1.5, through precise regulation of the synergistic effects of each component, ensures the structural stability of distearate phosphatidylethanolamine as the core framework of the lipid bilayer, while balancing the rigidity and fluidity of the membrane with a reasonable proportion of cholesterol to maintain vesicle morphology. At the same time, the proportion of hydroxyl-terminated polyethylene glycol can effectively form a sufficient hydration protective layer to reduce immunogenicity and prolong circulation time. The ratio of mPEG-Hyd-PEG-SH ensures that pH-sensitive hydrazone bonds are evenly distributed in the membrane to achieve efficient response and release in the tumor microenvironment, while avoiding damage to the overall structural stability of the liposomes due to excessive proportion. Ultimately, the PEG nanoliposomes achieve an optimal balance between structural integrity, low immunogenicity, intelligent responsiveness, and targeted delivery efficiency, significantly improving their drug delivery performance and clinical application value.

[0055] In some embodiments of this application, the rotary evaporation temperature is 40°C and the rotation speed is 100 rpm.

[0056] Understandably, the rotary evaporation method employs a synergistic parameter of 40°C temperature and 100 rpm rotation speed. This ensures efficient evaporation of the chloroform-methanol mixed solvent while avoiding thermal degradation of hydrazone bonds in mPEG-Hyd-PEG-SH and oxidative damage to HO-PEG due to excessively high temperatures. Simultaneously, it maintains moderate fluidity of lipid components such as distearate phosphatidylethanolamine and cholesterol under mild conditions. Combined with the moderate rotation speed of 100 rpm, this results in a uniform liquid film distribution, ensuring that all components (including pH-sensitive elements) are evenly spread on the inner wall of the flask during evaporation. Ultimately, this forms a lipid film of uniform thickness and uniform component dispersion, laying the foundation for the uniform vesicle structure of liposomes in the subsequent hydration step. This effectively avoids lipid aggregation or structural defects caused by excessively high local concentrations, significantly improving the particle size uniformity, encapsulation stability, and pH-responsive release efficiency of liposomes.

[0057] In some embodiments of this application, the pH value of the PBS solution containing ammonium sulfate is 7.4, and the concentration of ammonium sulfate is 200 mM.

[0058] Specifically, the PBS solution containing ammonium sulfate was set at a combination of pH 7.4 and 200 mM ammonium sulfate concentration. This combination maintains the structural stability of lipid components such as distearate phosphatidylethanolamine and cholesterol through the physiological buffering environment of pH 7.4, avoiding lipid hydrolysis caused by excessive acidity or alkalinity. It also provides mild conditions for the hydrazone bonds in mPEG-Hyd-PEG-SH to prevent premature breakage. At the same time, the 200 mM ammonium sulfate concentration can form a suitable osmotic pressure gradient, driving drugs such as doxorubicin to efficiently enter the liposomes in the subsequent drug loading step, increasing the encapsulation efficiency to over 90%. Furthermore, the ionic strength at this concentration can promote the orderly arrangement of lipid molecules during hydration, reducing vesicle aggregation. This, combined with the buffering effect of pH 7.4, lays the foundation for the uniform structure of liposomes and efficient drug loading, while ensuring the bioactivity of the CD44 antibody in subsequent targeted modification, achieving a balance between stability and efficiency in multiple steps.

[0059] In some embodiments of this application, the hydration reaction is carried out at a temperature of 60°C for 30 minutes; the ultrasonic treatment is carried out at a power of 100W for 20 minutes.

[0060] Understandably, the combination of hydration at 60°C and 30 minutes allows the lipid film to swell sufficiently at near the phospholipid phase transition temperature, promoting the orderly recombination of lipid molecules such as distearate phosphatidylethanolamine and cholesterol into vesicle structures. This avoids premature breakage of hydrazone bonds in mPEG-Hyd-PEG-SH and thermal damage to HO-PEG due to excessively high temperatures. The ample 30 minutes also ensures complete hydration of lipid molecules, reducing undispersed lipid particles. Meanwhile, the 100W power and 20 minutes of ultrasonic treatment disperse large-diameter lipid aggregates into uniform small vesicles through moderate mechanical shear force. This avoids localized overheating caused by excessive power, which could damage the liposome structure, and the continuous 20-minute treatment narrows the particle size distribution (PDI < 0.1). Together, these two processes provide a structurally complete and uniformly sized liposome framework for subsequent drug loading steps, effectively improving the stability of drug encapsulation efficiency and ensuring the uniform distribution of pH-sensitive elements and targeting ligands on the liposome surface. This lays the foundation for efficient targeting and intelligent release of the final product.

[0061] In some embodiments of this application, the mass ratio of doxorubicin in the doxorubicin solution to the mass ratio of the lipid film is 1:(10-15), preferably 1:12.

[0062] Understandably, setting the mass ratio of doxorubicin to the lipid membrane at 1:12 allows for efficient encapsulation of doxorubicin (encapsulation rate ≥90%) via the ammonium sulfate gradient loading method, avoiding excessive free drug due to exceeding the liposome's loading capacity, while also preventing the drug content from being too low and affecting efficacy. Simultaneously, this ratio ensures the integrity of the liposome vesicle structure, preventing excessive accumulation of doxorubicin molecules that could damage the phospholipid bilayer, ensuring uniform distribution of pH-sensitive hydrazone bonds in mPEG-Hyd-PEG-SH and stability of CD44 antibody-targeted modification. This results in a drug leakage rate of <5% during circulation and achieves an effective therapeutic concentration upon release in the tumor microenvironment. Ultimately, this achieves optimal synergy between drug loading, encapsulation rate, structural stability, and efficacy-toxicity balance, significantly improving drug delivery efficiency and therapeutic safety.

[0063] In some embodiments of this application, the incubation temperature is 37°C and the incubation time is 1 hour.

[0064] Understandably, setting the incubation temperature to 37℃ (close to physiological temperature) and controlling the time to 1 hour provides suitable energy for the ammonium sulfate gradient-driven transmembrane transport of doxorubicin, promoting efficient drug entry into the liposomes (encapsulation rate ≥90%), while avoiding excessive temperature leading to phospholipid bilayer structure disorder or premature breakage of hydrazone bonds in mPEG-Hyd-PEG-SH. At the same time, the 1-hour incubation time ensures sufficient drug loading without excessive retention—preventing incomplete encapsulation (increased free drug) due to insufficient time, and avoiding drug leakage due to increased liposome membrane permeability (leakage rate <5%) due to excessive time. Ultimately, while ensuring drug loading efficiency, it maintains the structural integrity of liposome vesicles and the functional stability of pH-sensitive elements and targeting ligands, laying a reliable foundation for subsequent targeted delivery and tumor microenvironment-responsive release.

[0065] In some embodiments of this application, the mass ratio of the CD44 antibody to the drug-loaded liposome is 1:2.

[0066] Understandably, setting the mass ratio of CD44 antibody to drug-loaded liposomes to 1:2 ensures a sufficient number of antibodies are chemically coupled to the PEG chain ends on the liposome surface (coupling efficiency ≥80%), providing ample target recognition sites for CD44-highly expressing tumor cells and significantly improving cellular uptake (3.5 times higher than the non-targeted group). It also avoids unbound antibody residues or excessive steric hindrance on the liposome surface due to excessive antibody intake; the latter could disrupt the integrity of the hydration protective layer or induce aggregation. Simultaneously, this ratio balances the synergistic effect of the targeting ligand and the liposome backbone, without affecting the distribution and responsive release function of pH-sensitive hydrazones in mPEG-Hyd-PEG-SH. This allows the drug-loaded liposomes to achieve a 2.8-fold increase in tumor tissue accumulation while maintaining particle size uniformity and cycling stability, ultimately achieving optimal synergy between targeting efficiency, structural stability, and efficacy-toxicity balance, significantly reducing non-specific damage to normal tissues.

[0067] In some embodiments of this application, the solvent in the mixed solution of CuSO4 and sodium ascorbate is PBS buffer, wherein the concentration of CuSO4 is 0.1 mM and the concentration of sodium ascorbate is 1 mM.

[0068] Specifically, CuSO4 and sodium ascorbate form a catalytic system (sodium ascorbate can react CuSO4 with sodium ascorbate). 2+ Reduced to Cu + (As a catalyst for the click chemistry reaction), it enables the CD4 antibody to undergo a click chemistry reaction with the alkynyl group of DSPE-PEG-Alkyne, thereby achieving the coupling of the antibody with the PEG derivative.

[0069] On the other hand, in some embodiments of this application, a nanoliposome is prepared by the preparation method described above.

[0070] Example 1

[0071] S1. Distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol and mPEG-Hyd-PEG-SH were dissolved in a chloroform-methanol mixed solution at a mass ratio of 5:3:2:1.5. The solution was then rotary evaporated at 100 rpm at 40 °C until a lipid film was formed, followed by vacuum drying for 2 hours.

[0072] S2. The dried lipid film was added to a PBS solution containing ammonium sulfate (200mM) at pH 7.4 at a solid-liquid ratio of 1:10 (mg / ml), hydrated at 60°C for 30 minutes, and then sonicated at 100W for 20 minutes to form colostrum.

[0073] S3. Remove free ammonium sulfate by passing the sample through a Sephadex G-50 column, and add 0.3 mg / ml of doxorubicin.

[0074] The doxorubicin solution was added to the colostrum after the removal of free ammonium sulfate at a mass ratio of 1:10 to that of the lipid film.

[0075] Incubate at 37°C for 1 hour to obtain drug-loaded liposomes;

[0076] S4. CD44 antibody and drug-loaded liposomes were added to a mixed solution of 0.1 mM CuSO4 and 1 mM sodium ascorbate at a mass ratio of 1:2. The mixture was reacted at room temperature for 4 hours and purified by ultrafiltration (100 kDa molecular weight cutoff) to obtain a targeted liposome suspension.

[0077] S5. The targeted liposome suspension is passed through a spiral focusing flow microreactor with an inner diameter of 0.8 mm and a spiral radius of 20 mm at a flow rate of 100 mL / min, and extruded 5 times through a 100 nm polycarbonate filter membrane to obtain the nanoliposomes.

[0078] Example 2

[0079] S1. Distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol and mPEG-Hyd-PEG-SH were dissolved in a chloroform-methanol mixed solution at a mass ratio of 5:3:2:1.5. The solution was then rotary evaporated at 100 rpm at 40 °C until a lipid film was formed, followed by vacuum drying for 2 hours.

[0080] S2. The dried lipid film was added to a PBS solution containing ammonium sulfate (200mM) at pH 7.4 at a solid-liquid ratio of 1:10 (mg / ml), hydrated at 60°C for 30 minutes, and then sonicated at 100W for 20 minutes to form colostrum.

[0081] S3. The free ammonium sulfate was removed by Sephadex G-50 column, and a 0.3 mg / ml doxorubicin solution was added to the colostrum after the free ammonium sulfate was removed at a mass ratio of doxorubicin to lipid film of 1:12. The mixture was incubated at 37°C for 1 hour to obtain drug-loaded liposomes.

[0082] S4. CD44 antibody and drug-loaded liposomes were added to a mixed solution of 0.1 mM CuSO4 and 1 mM sodium ascorbate at a mass ratio of 1:2. The mixture was reacted at room temperature for 4 hours and purified by ultrafiltration (100 kDa molecular weight cutoff) to obtain a targeted liposome suspension.

[0083] S5. The targeted liposome suspension is passed through a spiral focusing flow microreactor with an inner diameter of 0.8 mm and a spiral radius of 20 mm at a flow rate of 100 mL / min, and extruded 5 times through a 100 nm polycarbonate filter membrane to obtain the nanoliposomes.

[0084] Example 3

[0085] S1. Distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol and mPEG-Hyd-PEG-SH were dissolved in a chloroform-methanol mixed solution at a mass ratio of 5:3:2:1.5. The solution was then rotary evaporated at 100 rpm at 40 °C until a lipid film was formed, followed by vacuum drying for 2 hours.

[0086] S2. The dried lipid film was added to a PBS solution containing ammonium sulfate (200mM) at pH 7.4 at a solid-liquid ratio of 1:10 (mg / ml), hydrated at 60°C for 30 minutes, and then sonicated at 100W for 20 minutes to form colostrum.

[0087] S3. The free ammonium sulfate was removed by passing the Sephadex G-50 column, and a 0.3 mg / ml doxorubicin solution was added to the colostrum after the free ammonium sulfate was removed at a mass ratio of doxorubicin to lipid film of 1:15. The mixture was incubated at 37°C for 1 hour to obtain drug-loaded liposomes.

[0088] S4. CD44 antibody and drug-loaded liposomes were added to a mixed solution of 0.1 mM CuSO4 and 1 mM sodium ascorbate at a mass ratio of 1:2. The mixture was reacted at room temperature for 4 hours and purified by ultrafiltration (100 kDa molecular weight cutoff) to obtain a targeted liposome suspension.

[0089] S5. The targeted liposome suspension is passed through a spiral focusing flow microreactor with an inner diameter of 0.8 mm and a spiral radius of 20 mm at a flow rate of 100 mL / min, and extruded 5 times through a 100 nm polycarbonate filter membrane to obtain the nanoliposomes.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing nanoliposomes, characterized in that, The preparation steps include the following: Distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol, and mPEG-Hyd-PEG-SH were dissolved in a chloroform-methanol mixture, and then dried under vacuum after rotary evaporation to obtain a lipid film. A PBS solution containing ammonium sulfate was added to the lipid film to carry out a hydration reaction, followed by ultrasonic treatment to obtain the colostrum. Free thioamino acids were removed from the colostrum, and doxorubicin solution was added for incubation to obtain drug-loaded liposomes. The CD44 antibody and the drug-loaded liposomes were added to a mixed solution of CuSO4 and sodium ascorbate for coupling reaction. After the reaction, ultrafiltration was performed to obtain a targeted liposome suspension. The targeted liposome suspension was homogenized to obtain the nanoliposomes.

2. The method for preparing nanoliposomes according to claim 1, characterized in that, The mass ratio of distearate phosphatidylethanolamine, cholesterol, hydroxyl-terminated polyethylene glycol, and mPEG-Hyd-PEG-SH is 5:3:2:1.

5.

3. The method for preparing nanoliposomes according to claim 2, characterized in that, The rotary evaporation temperature is 40°C and the rotation speed is 100 rpm.

4. The method for preparing nanoliposomes according to claim 3, characterized in that, The PBS solution containing ammonium sulfate has a pH of 7.4 and an ammonium sulfate concentration of 200 mM.

5. The method for preparing nanoliposomes according to claim 4, characterized in that, The hydration reaction was carried out at a temperature of 60°C for 30 minutes; the ultrasonic treatment was performed at a power of 100W for 20 minutes.

6. The method for preparing nanoliposomes according to claim 5, characterized in that, The mass ratio of doxorubicin in the doxorubicin solution to the mass of the lipid film is 1:(10-15).

7. The method for preparing nanoliposomes according to claim 6, characterized in that, The incubation temperature was 37°C, and the incubation time was 1 hour.

8. The method for preparing nanoliposomes according to claim 7, characterized in that, The mass ratio of the CD44 antibody to the drug-loaded liposome is 1:

2.

9. The method for preparing nanoliposomes according to claim 8, characterized in that, The solvent in the mixed solution of CuSO4 and sodium ascorbate is PBS buffer, wherein the concentration of CuSO4 is 0.1 mM and the concentration of sodium ascorbate is 1 mM.

10. A nanoliposome, characterized in that, It is prepared by the preparation method described in any one of claims 1-8.

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