Tumor chemotherapy drug liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a tumor chemotherapy medicine liposome as well as a preparation method and application thereof. The preparation method comprises the following steps: taking an ethanol solution of lipid molecules as an organic phase, taking an aqueous solution containing transition metal acetate as a water phase, mixing the organic phase and the water phase in a microchannel, and collecting an outflowing transition metal acetate nano-liposome solution; carrying out medium exchange on the nano-liposome, and removing the transition metal acetate in the external water phase to obtain the liposome of which only the internal water phase contains the transition metal acetate; and mixing the liposome with an anthracycline chemotherapeutic drug, adjusting the pH value to near neutral-alkalescence, and heating to promote remote loading of the liposome on drug molecules, so as to obtain the chemotherapeutic drug-loaded nano-liposome. The liposome has a tumor acidic microenvironment slow release function, can specifically target tumor cells by using the targeting component, improves delivery and release of chemotherapy drugs into tumors, and can be used as a tumor chemotherapy and chemoradiotherapy combined treatment nano-drug.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a tumor chemotherapy drug liposome and a preparation method and application thereof. Background Art

[0002] Chemotherapy is one of the main treatments for tumors. Various anthracycline drugs, such as doxorubicin (DOX), epirubicin, and daunorubicin, are widely used in the treatment of tumors in clinical practice. However, the common injections of these drugs have many toxic side effects in clinical practice. Liposomes are nano drug carriers with good drugability and can be used to improve the pharmacokinetic behavior of the loaded chemotherapeutic drugs and reduce their toxic side effects. However, DOX liposomes prepared by the pH gradient method or the ammonium sulfate gradient method are difficult to release drugs in tumor tissues, making it difficult for DOX to enter tumor cells to exert a killing effect and may cause allergic reactions (Nanomedicine. 2012, 8(2):176-84).

[0003] Many transition metal ions can be complexed with anthracycline chemotherapy drugs. Liposomes with transition metal ion gradients can effectively load anthracycline chemotherapy drugs. In addition, the drug release of transition metal ion-anthracycline drug liposomes is affected by pH, which can provide a higher concentration of effective chemotherapy drugs in the acidic environment of tumors. However, at present, liposomes containing transition metal ions mainly adopt discontinuous production technology. The heating step in the manufacturing process is easy to accelerate the hydrolysis and precipitation reaction of transition metal ions, and the transition metal salt solution is also easy to corrode metal equipment, resulting in liposome instability or quality that is difficult to meet clinical drug requirements. The addition of metal ion complexing agent triethanolamine can inhibit this hydrolysis and corrosion. Although triethanolamine is a low-toxic solvent, its content in drugs is still subject to certain restrictions according to regulations. Therefore, the development of liposome anthracycline chemotherapy drugs containing transition metal ions that meet the quality requirements of clinical drugs is still an urgent problem to be solved. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a tumor chemotherapy drug liposome and a preparation method and application thereof.

[0005] To achieve the above object, the present invention provides a method for preparing tumor chemotherapy drug liposomes containing transition metal ions, comprising the following steps:

[0006] S1) using an ethanol solution of lipid molecules as an organic phase and an aqueous solution containing transition metal acetate as an aqueous phase, injecting the organic phase and the aqueous phase into a microfluidic chip with a microchannel by using microfluidic technology, mixing the organic phase and the aqueous phase in the microchannel, and collecting the outflowing transition metal acetate nanoliposome solution;

[0007] S2) performing medium exchange on the nanoliposomes to remove the transition metal acetate in the outer aqueous phase, thereby obtaining liposomes whose inner aqueous phase only contains the transition metal acetate;

[0008] S3) The nanoliposomes containing transition metal acetate only in the inner aqueous phase are mixed with anthracycline chemotherapy drugs, the pH is adjusted to near neutral to weak alkaline, and heating is performed to promote the remote loading of drug molecules by the liposomes, and the nanoliposomes loaded with chemotherapy drugs are obtained after purification.

[0009] The present invention selects transition metal acetates with good solubility in ethanol-water mixed solvents, and realizes the continuous preparation of nano liposomes containing high-concentration transition metal salts at room temperature and without contact with metal parts by using microfluidic technology.

[0010] The lipid molecules can be various lipid molecules that can be used to form a liposome lipid bilayer, preferably including one or more of natural phospholipids, hydrogenated phospholipids, synthetic phospholipids, cholesterol, phospholipid-polyethylene glycol, phospholipid-polyethylene glycol-folic acid derivatives, phospholipid-polyethylene glycol-mannose derivatives or other phospholipid-polyethylene glycol-tumor-specific targeting molecule derivatives, and other lipophilic molecules that are soluble in the liposome phospholipid bilayer.

[0011] In some specific embodiments of the present invention, the lipid molecules include distearoylphosphatidylcholine (DSPC) and cholesterol (CH).

[0012] In the distearoylphosphatidylcholine (DSPC) and cholesterol (CH), the molar concentration of cholesterol is preferably 20% to 45%.

[0013] In some specific embodiments of the present invention, the lipid molecules include distearoylphosphatidylcholine (DSPC), cholesterol (CH), and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0014] The molar ratio of the distearoylphosphatidylcholine (DSPC), cholesterol (CH), and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) is preferably 60:35:5.

[0015] In some specific embodiments of the present invention, the lipid molecules include distearoylphosphatidylcholine (DSPC), cholesterol (CH), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and distearoylphosphatidylethanolamine-polyethylene glycol 2000-folic acid (DSPE-PEG2000-FA).

[0016] The molar ratio of the distearoylphosphatidylcholine (DSPC), cholesterol (CH), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and distearoylphosphatidylethanolamine-polyethylene glycol 2000-folic acid (DSPE-PEG2000-FA) is preferably 60:35:3:2.

[0017] In the ethanol solution of the lipid molecules, the concentration of the lipid molecules is preferably 5-20 mM.

[0018] The transition metal preferably includes one or more of copper, zinc, manganese, or other transition metals whose acetates are soluble in ethanol.

[0019] In the aqueous solution of transition metal acetate, the concentration of the transition metal acetate is preferably 0.1-0.3 mol / L.

[0020] In the process of mixing the organic phase and the aqueous phase using microfluidic chip technology, the flow rate ratio (FRR) and the total flow rate (TFR) can be adjusted and optimized by selecting chips of different specifications and solution compositions to obtain liposomes of different particle sizes.

[0021] Preferably, the total flow rate (TFR) is 1.0-15 mL / min, and the flow rate ratio (FRR) of the aqueous phase to the organic phase is 2:1-5:1.

[0022] Preferably, the microfluidic chip technology adopts PreciGenome NanoGenerator microfluidic device (a product of Suzhou Ruixun Biotechnology Co., Ltd.).

[0023] The ethanol-water mixed solution flowing out through the microchannel of the microfluidic chip is collected to obtain nanoliposomes in which the inner and outer aqueous phases of the particles are both transition metal acetate solutions.

[0024] The particle size of the nanoliposome is preferably 50-150 nm.

[0025] Then, a medium exchange is performed to replace the extraliposomal aqueous phase with an aqueous solution that does not contain the original transition metal acetate.

[0026] The medium exchange method is preferably gel column separation, dialysis or ultrafiltration.

[0027] When the liposomes prepared by microfluidics are separated and purified by gel column, the eluent used is preferably an acidic pH buffer solution that does not contain the transition metal ions and acetate ions.

[0028] When the liposomes prepared by microfluidics are treated by a medium exchange method such as dialysis or ultrafiltration, the dialysate or ultrafiltrate used initially is preferably an acidic pH buffer solution, and the dialysate or ultrafiltrate used subsequently is changed to a NaCl solution, a glucose solution, or a pH buffer solution that does not contain the transition metal ions.

[0029] The obtained liposomes are collected, and the inner aqueous phase thereof contains transition metal acetate, while the outer aqueous phase does not contain the original transition metal acetate, that is, the liposomes have both transition metal ion gradient and acetate ion gradient.

[0030] The liposome can be mixed with drugs to achieve efficient remote drug delivery.

[0031] Based on this, another aspect of the present invention provides a liposome drug, which is obtained by loading a chemotherapeutic drug onto the transition metal acetate gradient liposome prepared by the above preparation method.

[0032] Preferably, the above liposomes are mixed with an aqueous solution of a chemotherapeutic drug, a buffer is added to adjust the mixed system to be nearly neutral to weakly alkaline, and the mixture is heated for a period of time, so that the liposomes can remotely load the drug molecules.

[0033] Preferably, the pH is adjusted to 6.8-7.6 with a buffer.

[0034] Preferably, the buffer is phosphate buffered saline (PBS). Preferably, the heating temperature is higher than the liposome lipid phase transition temperature, but not more than 65°C, preferably 50-60°C, including but not limited to 50, 55 or 60°C. Preferably, the heating time is 5-15 min.

[0035] The above-mentioned drugs are weakly alkaline drugs that can be complexed with transition metal ions, and are more preferably weakly alkaline anthracycline chemotherapy drugs.

[0036] In some specific embodiments of the present invention, the weakly basic anthracycline chemotherapy drug is one or more of drugs selected from the group consisting of doxorubicin, epirubicin, daunorubicin, pirarubicin, aclarubicin, idarubicin and mitoxantrone.

[0037] Preferably, in step S3), the molar ratio of total lipids in the liposomes to the chemotherapeutic drugs is 1:(0.1-0.5).

[0038] Preferably, the present invention uses gel column separation, dialysis, ultrafiltration or centrifugal precipitation to purify the liposomes after remote drug loading, remove unencapsulated drug molecules, and collect the drug-loaded liposomes. Preferably, the purified liposomes are stored in a neutral or weakly alkaline solution, such as physiological saline, PBS, etc.

[0039] The above-mentioned liposome drug may be in various dosage forms, including but not limited to injection, lyophilized powder, etc.

[0040] The drug-loaded liposomes are sterilized by filtering through a sterile filter membrane and filled into a pharmaceutical vial to obtain a liposome drug injection. Alternatively, a liposome lyophilization protectant can be added to the drug-loaded liposome solution, and freeze-dried to obtain a drug-loaded liposome lyophilized powder.

[0041] The test results show that in the present invention, the liposome can achieve an encapsulation rate of greater than 90% to 100% for drug molecules.

[0042] Figure 1 A schematic diagram of the method for preparing transition metal ion-anthracycline chemotherapy drug liposomes in the present invention is shown.

[0043] The present invention also provides tumor chemotherapy drug liposomes prepared by the above preparation method.

[0044] The above tumor chemotherapy drug liposomes can be used for targeted chemotherapy of tumors, as well as combined radiotherapy.

[0045] Based on this, another aspect of the present invention provides the tumor chemotherapy drug liposome prepared by the above preparation method or the use of the above tumor chemotherapy drug liposome in the preparation of anti-tumor drugs.

[0046] The tumors include, but are not limited to, malignant lymphoma, ovarian cancer, soft tissue sarcoma, multiple myeloma, breast cancer, small cell lung cancer, liver cancer, urothelial carcinoma, and the like.

[0047] Compared with the prior art, the present invention provides a method for preparing tumor chemotherapy drug liposomes, comprising the following steps: S1) using an ethanol solution of lipid molecules as an organic phase and an aqueous solution containing transition metal acetate as an aqueous phase, using microfluidic technology to inject the organic phase and the aqueous phase into a microfluidic chip with a microchannel, so that the organic phase and the aqueous phase are mixed in the microchannel, and the outflowing transition metal acetate nanoliposome solution is collected; S2) performing medium exchange on the above nanoliposomes to remove the transition metal acetate in the external aqueous phase to obtain liposomes whose internal aqueous phase only contains transition metal acetate; S3) mixing the above nanoliposomes whose internal aqueous phase only contains transition metal acetate with anthracycline chemotherapy drugs, adjusting the pH to near neutral to weak alkaline, heating to promote the remote loading of drug molecules by liposomes, and obtaining nanoliposomes loaded with chemotherapy drugs after purification. In the presence of a transition metal ion gradient, an acetate ion gradient, and an appropriate pH gradient, acetate molecules cross the membrane from the aqueous phase inside the liposome with a lower pH to the external aqueous phase with a higher pH, which is beneficial to the pH balance between the inside and outside of the liposome and the aqueous phase. The weakly alkaline drug molecules in the external aqueous phase cross the membrane into the aqueous phase inside the liposome and complex with the transition metal ions to form complex ions, allowing them to stably exist in the liposome, thereby achieving efficient remote loading of drug molecules by liposomes.

[0048] Compared with the prior art, the present invention uses microfluidics technology to continuously produce transition metal acetate liposomes at room temperature, avoiding the instability of the solution easily caused by heating the transition metal salt solution and contacting the metal wall; the transition metal acetate gradient liposomes prepared by the present invention can effectively realize the remote loading of weakly alkaline anthracycline drug molecules by liposomes without introducing ion carriers or triethanolamine, etc., combined with an appropriate pH gradient. The stability of the prepared transition metal ion-chemotherapeutic drug liposomes is affected by pH, and is stable under physiological weak alkaline conditions, but can slowly release chemotherapeutic drugs in the acidic microenvironment of tumors, and can enhance the ability of liposomes to be taken up by tumor cells and effectively deliver drugs into cells through tumor-specific targeting groups on the surface, which can effectively play a killing effect on tumor cells and can be used for chemotherapy of tumors, as well as combined radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the preparation method of transition metal ion-weakly basic anthracycline chemotherapy drug liposomes (MAc2 represents acetate of divalent transition metal ion, PE-PEG represents phospholipid-polyethylene glycol molecule, M 2+ represents a divalent transition metal ion);

[0050] Figure 2 Effects of solution parameters on liposome particle size and polydispersity in microfluidic control preparation (left: FRR is 3:1, right: TFR is 3 mL / min);

[0051] Figure 3 is the change in particle size of Cu-doxorubicin liposomes before and after drug loading (F-CuL represents DSPC / Chol / DSPE-PEG2000 / DSPE-PEG2000-FA liposomes containing copper acetate in the solution, and F-CuL-DOX represents F-CuL liposomes loaded with doxorubicin);

[0052] Figure 4 In the figure, (A) is a white light photograph of copper acetate (CuAc2) gradient liposomes before and after drug loading, (B) is a UV-visible absorption spectrum, and (C) is a fluorescence spectrum (CuL represents DSPC / Chol / DSPE-PEG2000 liposomes with a copper acetate gradient; CuL-DOX represents CuL liposomes loaded with doxorubicin);

[0053] Figure 5 The effect of different drug loading conditions on the encapsulation efficiency of DOX remotely loaded in copper acetate gradient liposomes;

[0054] Figure 6 The drug release curves of Cu-doxorubicin liposomes under different pH conditions;

[0055] Figure 7 Microscopic fluorescence images of breast cancer MDA-MB-231 cells taking up Cu-doximicin liposomes (left: DID fluorescently labeled CuL-DOX liposomes, right: DID fluorescently labeled F-CuL-DOX liposomes);

[0056] Figure 8 The results of the toxicity test of blank copper acetate gradient liposomes in mice (left: blood routine test results of mice, right: H&E staining microscopic images of important organs);

[0057] Fig. 9 The figure shows the tumor growth inhibitory effect of Cu-doxorubicin liposomes on tumor-bearing mice. DETAILED DESCRIPTION

[0058] In order to further illustrate the present invention, the following is a detailed description in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0059] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0060] Encapsulation efficiency measurement method: Take out a small amount of sample from the drug loading reaction system, separate it with a gel column, measure the content of chemotherapy drugs in the collected liposome components, and calculate the encapsulation efficiency of chemotherapy drugs by liposomes.

[0061] Example 1 Effect of solution mixing parameters on the particle size of prepared copper acetate liposomes during microfluidic preparation

[0062] 28.5 mg of distearoylphosphatidylcholine (DSPC), 8.1 mg of cholesterol (CH), and 6.0 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) (molar ratio of 60:35:5) were weighed and placed in a Falcon centrifuge tube, and 4 mL of anhydrous ethanol was added to dissolve as the organic phase. 391.9 mg of copper acetate monohydrate (CuAc2·H2O) was weighed and dissolved in 9.8 mL of double distilled water to obtain 200 mM CuAc2 as the aqueous phase. The PreciGenome NanoGenerator Flex-M microfluidic system (product of Suzhou Ruixun Biotechnology Co., Ltd.) was used to control the injection of the organic phase and the aqueous phase into the CHP-MIX-4 microchannel mixing chip for mixing. The microfluidic parameters were set respectively: TFR was 1.0~3.0 mL / min; FRR was 2:1~5:1 (aqueous phase: organic phase), and the effluent was collected. Each sample was diluted 10 times with saline, and the average diameter and polydispersity (PDI) of the liposomes in the solution were measured by dynamic light scattering method using a Zetasizer laser particle size analyzer (product of Malvern, UK). The measurement was repeated three times, and the data were expressed as mean ± standard deviation. It can be seen that the average particle size of the liposomes has a weak decreasing trend with the increase of TFR; however, the average particle size of the liposomes decreases significantly with the increase of FRR. When the FRR is 3:1 or 4:1, the hydration dynamic diameter of the liposomes is close to 100 nm, and at this time, it has a smaller PDI (< 0.20), such as Figure 2 As shown, Figure 2 In the figure, the FRR on the left is 3:1, and the TFR on the right is 3.0 mL / min.

[0063] Example 2 Preparation of transition metal acetate liposomes by microfluidics

[0064] Various transition metal acetate liposomes were prepared using microfluidic technology. 35.4 mg DSPC and 11.7 mg CH (molar ratio of 60:40) were weighed and placed in a Falcon centrifuge tube, and 5 mL of anhydrous ethanol was added to dissolve as the organic phase; 100 mM, 200 mM and 300 mM CuAc2, ZnAc2 and MnAc2 were prepared as the aqueous phase, respectively. In particular, nitrogen was added to the MnAc2 solution to deoxygenate. The organic phase and the aqueous phase were mixed in the CHIP-MIX-4 chip in the PreciGenome NanoGenerator Flex-M microfluidic preparation system. The microfluidic parameters were set respectively: TFR was 3.0 mL / min; FRR was 3:1 (aqueous phase: organic phase). The corresponding effluent was collected to obtain DSPC / CH liposomes containing CuAc2, ZnAc2 and MnAc2 in the solution.

[0065] 28.5 mg DSPC, 8.1 mg CH, and 6.0 mg DSPE-PEG2000 (molar ratio of 60:35:5) were weighed and placed in a Falcon centrifuge tube, and 4 mL of anhydrous ethanol was added to dissolve them as the organic phase; 100 mM, 200 mM, and 300 mM CuAc2, ZnAc2, and MnAc2 were used as the aqueous phase, respectively. The organic phase and the aqueous phase were mixed in the PreciGenome NanoGenerator Flex-M system. The microfluidic parameters were set respectively: TFR was 3.0 mL / min; FRR was 3:1 (aqueous phase: organic phase), and the effluent was collected to obtain DSPC / CH / DSPE-PEG2000 liposomes containing CuAc2, ZnAc2, and MnAc2 in the solution.

[0066] 28.5 mg DSPC, 8.1 mg CH, 3.6 mg DSPE-PEG2000 and 2.4 mg distearoylphosphatidylethanolamine-polyethylene glycol 2000-folate (DSPE-PEG2000-FA) (molar ratio of 60:35:3:2) were weighed and placed in a Falcon centrifuge tube, and 4 mL of anhydrous ethanol was added to dissolve as the organic phase; 200 mM CuAc2 was prepared as the aqueous phase. The two phases were mixed in the PreciGenome NanoGenerator Flex-M system. The microfluidic parameters were set respectively: TFR was 3.0 mL / min; FRR was 3:1 (aqueous phase: organic phase), and the corresponding effluent was collected to obtain folate receptor-targeted DSPC / CH / DSPE-PEG2000 / DSPE-PEG2000-FA liposomes containing CuAc2 in the solution.

[0067] 28.5 mg DSPC, 8.1 mg CH, 3.6 mg DSPE-PEG2000 and 2.4 mg DSPE-PEG2000-FA (molar ratio of 60:35:3:2) were weighed and placed in a Falcon centrifuge tube, and a concentrated solution of lipophilic cyanine near-infrared fluorescent dye DID in anhydrous ethanol was added to a molar ratio of 0.05% of the total lipids, and anhydrous ethanol was added to a total volume of 4 ml to dissolve as the organic phase; 200 mM CuAc2 was prepared as the aqueous phase. The two phases were mixed in the PreciGenome NanoGenerator Flex-M system. The microfluidic parameters were set respectively: TFR was 3.0 mL / min; FRR was 3:1 (aqueous phase: organic phase), and the effluent was collected to obtain DID fluorescently labeled CuAc2-containing folate receptor-targeted liposomes.

[0068] The prepared liposomes were stored in a refrigerator at 4°C. The hydration kinetics of the liposome nanoparticles were determined by dynamic light scattering. The results showed that the particle sizes of the blank liposomes prepared above were all in the range of 100-120 nm, and the polydispersity index was <0.20.

[0069] Example 3 Liposome Remote Loading of Doxorubicin Drug

[0070] The DSPC / CH / DSPE-PEG2000 / DSPE-PEG2000-FA liposomes containing copper acetate in the solution obtained in Example 2 were subjected to medium exchange by dialysis. The liposomes were placed in a dialysis bag with a molecular weight cutoff of 50 KDa, and the dialysis bag was placed in 400 mL of physiological saline (pH 4.5) containing 50 mM acetic acid / sodium acetate buffer. The dialysis was performed for more than 6 hours, and the dialysis solution was replaced twice in the middle to remove the copper ions and ethanol components in the aqueous phase outside the blank liposomes. The liposome solution was then transferred to an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and physiological saline was added, and centrifuged and concentrated (4000 g, 30 min).

[0071] Liposomes with saline as the external aqueous phase were mixed with doxorubicin hydrochloride aqueous solution at a doxorubicin drug / liposome total lipid molar ratio (D / TLs) of 1:5, and the pH value of the solution was adjusted to 6.8-8.0 with PBS, sodium bicarbonate, and 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, respectively. The mixed system was placed in a water bath at 50 ℃, 55 ℃, or 60 ℃, respectively. It can be seen that the color of the solution gradually changed to purple-red, especially at 55 ℃ and 60 ℃, the color change was more rapid, and it turned purple-red after 5 min. Continue to heat, and the solution remains stable. The liposomes heated at 55 ℃ and 60 ℃ for 5-10 min were separated and purified using a PD-10 gel desalting column, and the eluent was saline, and the purple-red Cu-doxorubicin liposome fraction was collected.

[0072] 150 mM hydrochloric acid / isopropanol (HCl / IPA) solution was added to the purified Cu-doxorubicin liposome sample at a volume ratio of 1:2. After shaking and standing for 15 min, the absorbance at 480 nm was measured to calculate the concentration of doxorubicin and the encapsulation efficiency of liposomes for doxorubicin. The results showed that the encapsulation efficiency of CuAc2 gradient liposomes for doxorubicin was >90% at pH 6.8-7.6 of the drug loading system, but the encapsulation efficiency decreased when the pH was 8.0. The liposomes with an initial aqueous phase of 200 mM CuAc2 had an encapsulation efficiency of doxorubicin close to 100% at a system pH of 7.4. The particle size of the Cu-doxorubicin liposomes was determined by dynamic light scattering to be approximately 130 nm, which is similar to the corresponding blank unloaded CuAc2 liposomes, as shown in Figure 2. Figure 3 shown. Figure 3In the figure, F-CuL represents DSPC / Chol / DSPE-PEG2000 / DSPE-PEG2000-FA liposomes containing copper acetate in the solution, and F-CuL-DOX represents F-CuL liposomes loaded with doxorubicin.

[0073] After the reaction mixture of blank CuAc2 liposomes remotely loaded with doxorubicin under different conditions was diluted with the corresponding medium, 180 uL was taken and placed in a 96-well plate and the visible light absorption spectrum and fluorescence spectrum were measured using an ELISA instrument. The results showed that when pH 6.8~7.6 buffer was added to the reaction system, the 480 nm visible light absorption band of doxorubicin in the solution red-shifted to the maximum absorption wavelength of 510 nm after heating, and the Ex480 / Em590 fluorescence emission intensity rapidly weakened with the extension of heating time. The changes in the visible light absorption spectrum and fluorescence spectrum during the remote drug loading process reflect that CuAc2 gradient liposomes can quickly and efficiently load doxorubicin, and doxorubicin and Cu in the aqueous phase of the liposomes are closely related. 2+ The complex formed by the ions rapidly undergoes fluorescence quenching as the concentration increases, such as Figure 4 As shown, Figure 4 A) White light photograph of copper acetate gradient liposomes before and after drug loading, B) UV-visible absorption spectrum, C) changes in fluorescence spectrum (CuL represents DSPC / Chol / DSPE-PEG2000 liposomes with copper acetate gradient; CuL-DOX represents CuL liposomes loaded with doxorubicin).

[0074] 200 mM ZnAc2 gradient liposomes and 200 mM MnAc2 gradient liposomes were prepared by a similar method to the above-mentioned preparation of Cu-doxorubicin liposomes (nitrogen was filled in the dialysis process and the final solution for deoxygenation), and remote loading of doxorubicin was performed. The results were similar to those of CuAc2 liposomes. After heating at 55-60 ºC for 5-10 min, the liposomes were able to effectively load doxorubicin, and the particle size did not change significantly before and after loading doxorubicin.

[0075] Example 4 Effects of different drug loading conditions on liposome encapsulation efficiency

[0076] The copper acetate DSPC / CH / DSPE-PEG2000 liposomes obtained in Example 2 were subjected to medium exchange by dialysis. The liposomes with physiological saline as the external aqueous phase were mixed with an aqueous solution of doxorubicin hydrochloride at a doxorubicin / liposome total lipid molar ratio (D / TLs) of 1 / 7 to 1 / 4, and the solution was adjusted to a weak alkaline (pH 7.4) with PBS. The mixed systems were placed in a 60ºC water bath and heated for 5 min. It was seen that the color of the solution gradually changed to purple-red. The liposomes heated at different lipid-drug ratios were separated and purified using a Sephadex G25 gel desalting column. Physiological saline was used as the eluent to collect the purple-red doxorubicin liposome component. 150 mM HCl / IPA solution was added to the purified doxorubicin liposome sample at a volume ratio of 1:2. After shaking and standing for 15 minutes, the absorbance at 480 nm was measured to calculate the concentration of doxorubicin. The encapsulation efficiency at 60 ºC, heated for 5 min, and different lipid-drug ratios was more than 90% (such as Figure 5 left figure).

[0077] The copper acetate DSPC / CH / DSPE-PEG2000 liposomes obtained in Example 2 were subjected to medium exchange by dialysis. The liposomes whose external aqueous phase was normal saline were mixed with an aqueous solution of doxorubicin hydrochloride at a doxorubicin / liposome total lipid molar ratio (D / TLs) of 1 / 5, and the solution was adjusted to a weak alkaline (pH value of 7.4) with PBS. The mixed system was placed in a 60 ºC water bath and heated for 1, 3, 5, 10, and 15 min, respectively. It was seen that the color of the solution gradually changed to purple-red. The liposomes treated with different heating times were separated and purified using a Sephadex G25 gel desalting column, and the concentration of doxorubicin in the collected liposomes was measured. The results show that a longer heating time is beneficial to improving the DOX encapsulation efficiency, but heating at 60 ºC for 5 min can result in a DOX encapsulation efficiency close to 90% (such as Figure 5 (as shown in the middle figure).

[0078] The copper acetate DSPC / CH / DSPE-PEG2000 liposomes obtained in Example 2 were subjected to medium exchange by dialysis. The purified liposomes whose external aqueous phase was normal saline were mixed with an aqueous solution of doxorubicin hydrochloride at a doxorubicin / liposome total lipid molar ratio (D / TLs) of 1 / 5, and the solution was adjusted to a weak alkaline (pH 7.4) with PBS. The mixed system was placed in a water bath at 50, 55, 60, and 65ºC and heated for 5 min, respectively. The liposomes treated at different heating temperatures were separated and purified using a Sephadex G25 gel desalting column. Normal saline was used as the eluent, and the concentration of DOX in the collected liposome components was determined. The results show that higher temperatures are conducive to the rapid loading of drugs by liposomes. Heating at 60 and 65 ºC in a short period of time can lead to higher DOX encapsulation rates (such as Figure 5 as shown on the right).

[0079] Example 5 Release of doxorubicin liposomes under different pH conditions

[0080] 2 mL of each of the CuL-DOX liposome and F-CuL-DOX liposome solutions prepared above were placed in a dialysis bag with a molecular weight cutoff of 50 KDa, and then placed in a 50 mL centrifuge tube containing 40 mL PBS, pH 5.8 or pH 7.4, respectively, and dialyzed in a constant temperature incubator at 37°C in the dark. At specified time intervals (0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72h), 3 mL of sample was drawn from the centrifuge tube, and the absorbance at 480 nm was quickly measured using a UV spectrophotometer. After the measurement, the sampled solution was returned to the original tube. The percentage of DOX released from the liposomes at different time points was calculated based on the DOX standard curve. The results are shown in Figure 2. Figure 6 As shown in the figure, both Cu-doxorubicin liposomes have good stability under pH 7.4 conditions. The DOX release gradually increases with time within 12 hours, but then remains almost unchanged, and the DOX release rate does not exceed 15% at 72 hours. However, at pH 5.8, DOX is released rapidly, and CuL-DOX liposomes and F-CuL-DOX liposomes release 57% and 78% of DOX at 12 hours, respectively, and the cumulative release of DOX at 72 hours is 67% and 82%, respectively. The results show that Cu-doxorubicin liposomes have good acidic pH response release properties.

[0081] Example 6 Experiment on the uptake of liposome drugs by tumor cells

[0082] Take a 13 mm round glass slide, sterilize and dry it, and then spread it on the bottom of a 24-well plate. Then, add the cultured breast cancer MDA-MB-231 cell suspension to the 24-well plate with the glass slide at a rate of 50,000 cells per well, and transfer it to an incubator overnight to allow the cells to adhere to the wall. On the second day, add the above-mentioned CuL-DOX liposomes labeled with DID dye or F-CuL-DOX liposomes at a DOX concentration of 10 μg / mL to each well, and set up 3 replicate wells in each group. After incubating in the incubator for 6, 12, and 24 hours, wash it 3 times with PBS, add 4% paraformaldehyde to fix the cells, wash it 3 times with PBS, pick it out, transfer it to a clean slide with the cell nucleus stain DAPI, and observe and photograph it under a fluorescence microscope. The results are as follows Figure 7 As shown, in the F-CuL-DOX liposome group with folate receptor targeting, obvious doxorubicin fluorescence signals appeared in the tumor cell nuclei soon after administration, but in the CuL-DOX liposome group, only weak doxorubicin fluorescence signals were found in the tumor cells within 12 h after administration.

[0083] Example 7 Toxicity experiment of liposome drugs on tumor cells

[0084] Breast cancer 4T1 and MDA-MB-231 cells in the logarithmic growth phase cultured in vitro were digested with trypsin and then diluted to 60,000 cells / mL with culture medium. 100 μL of cell suspension was added to each well of a 96-well plate and cultured until the cells adhered to the wall. 100 μL of doxorubicin hydrochloride solution, CuL-DOX liposome solution and F-CuL-DOX liposome solution (free doxorubicin hydrochloride solution concentration was 0.02-8.0 μM, liposome doxorubicin concentration range was 0.18-90 μM) diluted with DMEM medium were added to each well. An equal volume of DMEM medium was added to the control group. After incubation with tumor cells for 24 h, the culture medium in the 96-well plate was aspirated and washed twice. The CCK8 reagent in the CCK8 kit (product of Hangzhou Fude Biotechnology Co., Ltd.) was mixed with the culture medium at a ratio of 10 μL:90 μL, and 100 μL of culture medium containing CCK8 was added to each well of the 96-well plate. The cells were incubated in the cell culture incubator for 2 h, and the OD value at 450 nm was detected by a multifunctional microplate reader to calculate the average cell viability. The cell viability-drug concentration curve was fitted using GraphPadPrism 9.5 to calculate the IC 50 value.

[0085] The results showed that after treating tumor cells with different doxorubicin preparations for 24 h, the anti-tumor cell proliferation ability of Cu-doxorubicin liposomes was significantly lower than that of free doxorubicin. The IC values ​​of CuL-DOX liposomes in killing 4T1 cells and MDA-MB-231 cells were 50 The values ​​were 15.0±5.6 μM and 34.0±16.9 μM, respectively, while the IC values ​​of folate receptor-targeted F-CuL-DOX liposomes in killing 4T1 cells and MDA-MB-231 cells were 50 The values ​​were 6.4±3.4 μM and 9.0±3.2 μM, respectively, and the ability to kill tumor cells was significantly stronger than that of CuL-DOX liposomes.

[0086] Example 8 Results of in vivo toxicity test of CuAc2 blank liposomes on mice

[0087] Fifteen healthy Blab / c mice were randomly divided into three groups (5 mice in each group) and injected with F-CuL blank liposomes containing copper acetate in the aqueous phase through the tail vein at a dose of 22 mg total lipid compounds / kg mouse body weight. Another five healthy mice that were not injected with liposomes served as the control group. Blood was collected from the orbital venous plexus on the 7th, 15th, and 30th days for routine blood tests. The mice were then euthanized, and major organs such as the liver, spleen, kidney, heart, and lungs were collected, fixed with 4% formaldehyde, embedded in paraffin, and sliced. They were stained with hematoxylin-eosin (H&E), and images were obtained using a digital microscope for histopathological examination. The results showed that compared with normal mice, the blood routine parameters of mice injected with copper acetate blank liposomes were normal, and no obvious organ damage or inflammation was found in any of the collected tissue samples. The results are shown in Figure 2. Figure 8 As shown, it was shown that under the given dosage conditions, the liposomes had no obvious toxic side effects in vivo.

[0088] Figure 8 In the figure, the left picture shows the blood routine test results of mice, and the right picture shows the H&E staining microscopic image of important organs.

[0089] Example 9 Inhibitory Effect of Cu-doxorubicin Liposomes on Tumor Growth in Tumor-Bearing Mice

[0090] Balb / c mice with subcutaneous 4T1 cell tumors were randomly divided into 8 groups (6 mice in each group), including PBS control group, DOX group, F-CuL liposome group and F-CuL-DOX liposome group (liposome dosage: 22 mg total lipid compound / kg mouse body weight, 5 mg DOX / kg mouse body weight), 6 Gy X-ray irradiation (IR) group, and each intravenous drug combined with 6 Gy X-ray irradiation group. Treatment was performed once a week, and X-ray irradiation was carried out 12 hours after administration. Tumor size and body weight were monitored every two days. The results showed that under the conditions of drug dose and administration method used, blank liposomes had no inhibitory effect on tumor growth, and DOX alone, F-CuL-DOX liposomes and radiotherapy (IR) alone had a certain inhibitory effect on tumor growth. After the 12th day, the tumor volume was significantly different from that of the control group. In contrast, the therapeutic effect of combined drug and radiotherapy was more obvious. Both the DOX+IR group and the F-CuL-DOX liposome+IR group could significantly inhibit tumor growth. The results are shown in Fig. 9 As shown, the body weight of mice in the DOX+IR group decreased significantly after treatment, while the body weight of mice in the F-CuL-DOX liposome+IR group increased after treatment.

[0091] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing tumor chemotherapy drug liposomes, comprising the following steps: S1) using an ethanol solution of lipid molecules as an organic phase and an aqueous solution containing transition metal acetate as an aqueous phase, injecting the organic phase and the aqueous phase into a microfluidic chip with a microchannel by using microfluidic technology, mixing the organic phase and the aqueous phase in the microchannel, and collecting the outflowing transition metal acetate nanoliposome solution; S2) performing medium exchange on the nanoliposomes to remove the transition metal acetate in the outer aqueous phase, thereby obtaining liposomes whose inner aqueous phase only contains the transition metal acetate; S3) The liposomes containing transition metal acetate only in the inner aqueous phase are mixed with anthracycline chemotherapy drugs, the pH is adjusted to near neutral to weak alkaline, and heating is performed to promote the remote loading of drug molecules by the liposomes, and after purification, the nanoliposomes loaded with chemotherapy drugs are obtained.

2. The preparation method according to claim 1, characterized in that: The lipid molecules include one or more of natural phospholipids, hydrogenated phospholipids, synthetic phospholipids, cholesterol, phospholipid-polyethylene glycol, phospholipid-polyethylene glycol-folic acid derivatives, phospholipid-polyethylene glycol-mannose derivatives or other phospholipid-polyethylene glycol-tumor-specific targeting molecule derivatives, and other lipophilic molecules that are soluble in the phospholipid bilayer of liposomes.

3. The preparation method according to claim 1, characterized in that: The transition metal includes one or more of copper, zinc and manganese.

4. The preparation method according to claim 1, characterized in that: In the ethanol solution of the lipid molecules, the concentration of the lipid molecules is 5-20 mM; In the aqueous solution containing transition metal acetate, the concentration of the transition metal acetate is 0.1-0.3 mol / L.

5. The preparation method according to claim 1, characterized in that: In the microfluidic chip technology, the total flow rate is 1-15 mL / min, and the flow rate ratio of the aqueous phase to the organic phase is 2:1-5:

1.

6. The preparation method according to claim 1, characterized in that: The medium exchange method in step S2) is gel column separation, dialysis or ultrafiltration.

7. The preparation method according to claim 1, characterized in that: In step S3), the anthracycline chemotherapy drug is one or more of doxorubicin, epirubicin, daunorubicin, pirarubicin, aclarubicin, idarubicin and mitoxantrone.

8. The preparation method according to claim 1, characterized in that: In step S3), the molar ratio of total lipids in the liposome to the chemotherapeutic drug is 1:(0.1-0.5); Adjust pH to 6.8-7.6 with buffer; The heating temperature is higher than the liposome phase transition temperature but not more than 65°C; The heating time is 5 to 15 minutes.

9. Tumor chemotherapy drug liposomes prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the tumor chemotherapy drug liposome prepared by the preparation method according to any one of claims 1 to 8 or the tumor chemotherapy drug liposome according to claim 9 in the preparation of anti-tumor drugs.

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