Combretastatin targeted lipid drug delivery system and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a combretastatin targeted lipid drug delivery system and a preparation method thereof. According to the method, AS1411 and ApoE peptides are adopted to carry out joint modification on CA4 / HM-Fe lipidosome. Fe < 3 + > is introduced on the basis of the sound-sensitive agent HMME, so that the sound-sensitive agent HMME and the sound-sensitive agent HMME are self-assembled to form an HM-Fe complex, CA4 and free HMME are entrapped in a hydrophobic region, and HM-Fe is entrapped in a hydrophilic core, so that the HM-Fe complex can be used as a good carrier for delivering CA4, and the problem of poor water solubility of CA4 is effectively solved. The liposome is co-modified by AS1411 and ApoE peptide, so that dual active targeting of liver cancer cells and tumor vascular endothelial cells can be realized, the in-vitro uptake efficiency is effectively improved, and the enrichment efficiency of a drug at a tumor part is remarkably improved; the system is high in stability, low in toxicity, efficient, good in biocompatibility, high in tumor inhibition rate and good in tumor inhibition effect and safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a combretastatin A4 targeted lipid drug delivery system and a preparation method thereof. BACKGROUND

[0002] Combretastatin A4 (CA4) was first extracted from the African plant Combretum caffrum, which is a potent microtubule polymerization inhibitor that induces G2 / M phase arrest of tumor cells by binding to the colchicine binding site. In addition, CA4 has been confirmed to have vascular disrupting agent (VDA) activity, which can selectively target tumor neovascular endothelial cells, leading to rapid collapse of tumor blood vessels and blood flow interruption, thereby inducing tumor cell hypoxic death. However, the strong lipophilicity of CA4 results in poor water solubility, which severely limits its effective delivery in vivo and clinical transformation.

[0003] Sonodynamic therapy (SDT) is a new non-invasive tumor treatment strategy, which delivers a sonosensitizer to the tumor site to produce reactive oxygen species (ROS) under the excitation of low-intensity ultrasound, inducing oxidative damage and death of tumor cells. Hematoporphyrin monomethyl ether (HMME) as a highly efficient and biocompatible organic sonosensitizer has been widely used in SDT treatment of various tumors, but its hydrophobicity also limits its in vivo delivery efficiency. Aptamer is a single-stranded DNA or RNA oligonucleotide that can specifically recognize target molecules, among which AS1411 is a 26-base oligonucleotide rich in guanine (G) that can specifically bind to the nucleolin highly expressed on the surface of tumor cell membranes, and has good tumor targeting and low immunogenicity. Apolipoprotein E (ApoE) is a naturally occurring lipoprotein component that can mediate cellular uptake of lipid particles through specific binding with low-density lipoprotein receptor (LDLR), especially for targeted delivery to hepatocytes and hepatoma cells. ApoE peptide can achieve targeted delivery by mimicking the interaction between natural ApoE protein and cell surface receptors.

[0004] Liposomes, as a kind of cell-like membrane structure self-assembled by phospholipids and cholesterol, can simultaneously encapsulate hydrophilic and hydrophobic drugs, and have good biocompatibility and drug co-delivery capacity. However, single liposomes still have deficiencies in tumor targeting and drug release control, and it is urgent to improve their tumor-specific recognition and internalization efficiency through surface functionalization modification.

[0005] Therefore, the liposome drug delivery system co-modified by the nucleic acid aptamer AS1411 and the apolipoprotein ApoE peptide and co-loading the combretastatin (CA4) and the hematoporphytin monomethyl ether (HMME) and combined with the sonodynamic therapy for the liver cancer treatment can overcome the problems of poor water solubility, insufficient targeting and single treatment means in the prior art, and realize the precise liver cancer treatment with multiple mechanisms. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a combretastatin targeted liposome drug delivery system and a preparation method thereof.

[0007] The technical scheme for solving the above technical problem is as follows:

[0008] The present application provides a liposome with a cavity structure, wherein the liposome is loaded with a HM@Fe complex and a combretastatin.

[0009] The present application also provides a preparation method of the combretastatin targeted liposome drug delivery system, wherein the CA4 / HM@Fe liposome is co-modified by the AS1411 and the ApoE peptide.

[0010] On the basis of the above technical scheme, the present application can also be improved as follows.

[0011] Further, the method comprises the following steps:

[0012] S1, preparing a HM@Fe complex;

[0013] S2, preparing a CA4 / HM@Fe liposome by using a film dispersion method and dissolving the HM@Fe complex and the combretastatin;

[0014] S3, performing ultrasonic treatment on the CA4 / HM@Fe liposome;

[0015] S4, mixing the AS1411 solution and the ApoE peptide solution with the CA4 / HM@Fe liposome after the ultrasonic treatment to prepare the combretastatin targeted liposome drug delivery system.

[0016] Further, the step S2 is:

[0017] The SPC, cholesterol, methoxy PEG dis-stearoyl phosphatidyl acetic acid and the combretastatin are dissolved by using a chloroform-methanol solution to obtain a mixed solution, the mixed solution is mixed with the HM@Fe complex and stirred uniformly to obtain a liposome precursor solution;

[0018] The liposome precursor solution is rotary evaporated to dryness under the condition of 37 DEG C and reduced pressure, and a uniform film is formed, and the CA4 / HM@Fe liposome is obtained after hydration and drying.

[0019] Further, in the step S3, the frequency of the ultrasonic treatment is 200-400w, and the ultrasonic time is 3-9min.

[0020] Further, the AS1411 solution is mixed with the CA4 / HM@Fe liposome after ultrasonic treatment and temperature cycle incubation is carried out; the ApoE peptide solution is added and incubation is carried out; free ligand is removed by dialysis to obtain the combretastin targeted lipid drug delivery system.

[0021] Further, the temperature cycle incubation is incubation in a 4℃ environment for 30min, and then incubation at room temperature for 30min, and the cycle is repeated for 3 times.

[0022] Further, the step S1 is:

[0023] A hematin solution is prepared by using a methanol-triethylamine solution and hematin monomethyl ether;

[0024] A ferric chloride hexahydrate solution is prepared by using a methanol-dimethylformamide and ferric chloride hexahydrate;

[0025] The hematin monomethyl ether solution and the ferric chloride hexahydrate solution are mixed according to the mass ratio of HMME and FeCl3·6H2O as 1:1, centrifugation, resuspension and washing are carried out to obtain the HM@Fe complex.

[0026] The application further provides a combretastin targeted lipid drug delivery system prepared by the above method.

[0027] The application further provides a preparation for sonodynamic therapy, which comprises the combretastin targeted lipid drug delivery system as described above.

[0028] The application has the following beneficial effects:

[0029] (1) The combretastin targeted lipid drug delivery system of the application can realize the double active targeting of hepatoma cells and tumor vascular endothelial cells through the co-modification of AS1411 and ApoE peptide liposome, effectively improves the in-vitro uptake efficiency, and significantly improves the enrichment efficiency of the drug at the tumor site;

[0030] (2) The combretastin targeted lipid drug delivery system of the application simultaneously loads CA4 and HM@Fe in the liposome, has a large CA4 and HMME encapsulation rate, effectively solves the problem of poor water solubility of CA4, and realizes the time-space synchronous delivery of chemotherapy and sonodynamic therapy (SDT);

[0031] (3) The combretastin targeted lipid drug delivery system of the application produces a large amount of ROS (reactive oxygen species) under the ultrasonic trigger of HM@Fe, and the vascular damage effect of CA4 is combined, so that the therapeutic effect is greatly improved;

[0032] (4) The camptothecin targeted lipid drug delivery system of the present application can induce GSH depletion and Ca 2+ flow, cooperates with the microtubule depolymerization induced by CA4 to trigger mitochondrial dysfunction and apoptosis pathway, and significantly enhances the efficiency of tumor cell death;

[0033] (5) The camptothecin targeted lipid drug delivery system of the present application has strong stability, low toxicity, high efficiency, good biocompatibility, high tumor inhibition rate, and good tumor inhibition effect and safety. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The appearance and morphology of the liposome in Example 3 of the present application are shown in the following figures, Figure 1 wherein A is the appearance of CA4-LP and AA-CA4 / HM@Fe-LP, Figure 1 wherein B is the transmission electron microscope image of AA-CA4 / HM@Fe-LP;

[0035] Figure 2 The in vitro stability of the liposome in Example 3 of the present application placed at 4℃ for 28 days is shown in the following figures, Figure 2 wherein A is the particle size change graph, Figure 2 wherein B is the PDI change graph;

[0036] Figure 3 The fluorescence microscope images of the in vitro uptake of the liposomes by HepG2 cells in Example 4 of the present application are shown in the following figures;

[0037] Figure 4 The fluorescence microscope images of the in vitro uptake of the liposomes by HUVEC cells in Example 4 of the present application are shown in the following figures;

[0038] Figure 5 The fluorescence plate quantitative analysis of the uptake of the liposomes by cells in Example 4 of the present application is shown in the following figures, Figure 5 wherein A is the HepG2 cells, Figure 5 wherein B is the HUVEC cells, and wherein *p<0.05, **p<0.01;

[0039] Figure 6 The fluorescence imaging images of the in vivo in situ liver cancer nude mice model of each experimental group in Example 5 of the present application are shown in the following figures;

[0040] Figure 7 The intracellular reactive oxygen species fluorescence images under the action of different liposomes in Example 6 of the present application are shown in the following figures;

[0041] Figure 8 The intracellular Ca 2+ fluorescence images of each experimental group in Example 7 of the present application are shown in the following figures;

[0042] Figure 9 Figure 8 is a schematic diagram of in vitro toxicity of CA4 and each group of liposomes on HepG2 cells in Embodiment 8 of the present application, wherein *p<0.05, **p<0.01;

[0043] Figure 10 Figure 9 is a schematic diagram of the effect of each group of liposomes on intracellular GSH level in Embodiment 9 of the present application;

[0044] Figure 11 Figure 10 is an immunofluorescence image of CA4 and each group of liposomes on HepG2 cell β-tubulin in Embodiment 10 of the present application;

[0045] Figure 12 Figure 11 is an in vivo tumor inhibition of each group of liposomes in Embodiment 11 of the present application, Figure 12 Figure 11A is a subcutaneous tumor volume curve in the nude mice, Figure 12 Figure 11B is a nude mouse weight change curve. DETAILED DESCRIPTION

[0046] The principles and features of the present application are described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application.

[0047] The CA4 / HM@Fe liposome of the present application has a cavity structure, and the liposome is loaded with the HM@Fe complex and combretastin.

[0048] The CA4 / HM@Fe liposome of the present application introduces Fe 3+ The two self-assemble to form the HM@Fe complex, and the CA4 and free HMME are loaded in the hydrophobic region, and the HM@Fe is loaded in the hydrophilic core, so that it can be used as a good carrier for delivering CA4, and effectively solves the problem of poor water solubility of CA4.

[0049] The preparation method of the combretastin targeted liposome drug delivery system of the present application uses AS1411 and ApoE peptide to co-modify the CA4 / HM@Fe liposome. Through the co-modification of AS1411 and ApoE peptide, the liposome drug delivery system has good targeting ability in vitro and in vivo, and thus has good in vivo and in vitro anti-tumor ability.

[0050] Preferably, the preparation method of the present application comprises the following steps:

[0051] S1, preparing the HM@Fe complex.

[0052] Preferably, the hematin monomethyl ether solution is prepared using a methanol-triethylamine solution and hematin monomethyl ether, and the ferric chloride hexahydrate solution is prepared using a methanol-dimethylformamide solution and ferric chloride hexahydrate. The hematin monomethyl ether solution and the ferric chloride hexahydrate solution are mixed according to a mass ratio of 1:1 of HMME and FeCl3·6H2O, and centrifugation, resuspension, and washing are performed to obtain the HM@Fe complex.

[0053] S2, the HM@Fe complex and combretastin are prepared into CA4 / HM@Fe liposomes using a thin film dispersion method.

[0054] Preferably, the SPC, cholesterol, methoxy PEG distearoyl phosphatidyl acetyl amide, and combretastin are dissolved using a chloroform-methanol solution to obtain a mixed solution, the mixed solution is mixed with the HM@Fe complex and stirred uniformly to obtain a liposome precursor solution.

[0055] The liposome precursor solution is rotary evaporated to dryness under the conditions of 37°C and reduced pressure, and a uniform film is formed. After drying, hydration is performed to obtain CA4 / HM@Fe liposomes.

[0056] Preferably, the volume ratio of chloroform to methanol in the chloroform-methanol solution is 3:2, and the volume ratio of SPC, cholesterol, methoxy PEG distearoyl phosphatidyl acetyl amide, and combretastin is 95:20:8:5.

[0057] S3, the CA4 / HM@Fe liposomes are subjected to ultrasonic treatment.

[0058] Preferably, the frequency of ultrasonic treatment is 200w-400w, and the ultrasonic treatment time is 3-9min.

[0059] S4, the AS1411 solution and the ApoE peptide solution are mixed with the long-circulating liposomes, respectively, and prepared into a combretastin targeted liposome drug delivery system.

[0060] In the AS1411 solution and the ApoE peptide solution, the molar ratio of ApoE peptide to AS1411 is 0.77:0.01.

[0061] Preferably, the AS1411 solution is mixed with the CA4 / HM@Fe liposomes after ultrasonic treatment and subjected to temperature cycle incubation. The ApoE peptide solution is added and incubated. Free ligands are removed using a dialysis method to obtain a combretastin targeted liposome drug delivery system.

[0062] Preferably, the temperature cycle incubation is incubated at 4°C for 30min, and then incubated at room temperature for 30min. The cycle is repeated for 3 times.

[0063] Preferably, after the addition of the ApoE peptide solution, incubation is performed at 4°C overnight.

[0064] The preparation for sonodynamic therapy of the present application comprises the above-mentioned comprostatin targeted lipid drug delivery system.

[0065] The present application is described below through specific examples.

[0066] Example 1 Preparation of HM@Fe complex

[0067] An appropriate amount of hematoporphyrin monomethyl ether (HMME) was taken in a test tube, and an appropriate volume of methanol-triethylamine solution was added for dissolution. An appropriate amount of iron trichloride hexahydrate (FeCl3·6H2O) was taken in a test tube, and an appropriate volume of methanol-dimethylformamide (DMF) solution was added for dissolution. In the methanol-triethylamine solution, the volume ratio of methanol to triethylamine was 50:1, and in the methanol-dimethylformamide solution, the volume ratio of methanol to dimethylformamide was 85:15.

[0068] The two solutions obtained above were mixed in a mass ratio of HMME to FeCl3·6H2O of 1:1, stirred (1200 rpm, 6 h) at room temperature in the dark, and a mixed solution was obtained.

[0069] The mixed solution was placed in a 2 mL centrifuge tube and centrifuged (12000 rpm, 30 min, 4°C) using a high-speed centrifuge, and the supernatant was collected. An ethanol-DMF solution with a volume ratio of 85:15 was added for resuspension, and then washing and centrifugation were performed in sequence, repeated 3 times, and an HM@Fe complex was obtained.

[0070] Example 2 Preparation of liposomes

[0071] A chloroform-methanol solution with a volume ratio of 3:2 was used to dissolve SPC, cholesterol (Chol), methoxy PEG distearoyl phosphatidyl acylamide (DSPE-PEG2000), and CA4 in a glass tube to obtain a mixed solution. The mixed solution was mixed with the HM@Fe complex prepared in Example 1 and stirred uniformly, and then transferred to a 250 mL round-bottom flask. The optimal mass ratio of each component was SPC: Chol: DSPE-PEG2000: CA4 = 95:20:8:5.

[0072] The round-bottom flask was placed in a vacuum drying oven and dried for 2 h. Sterile PBS solution with a pH of 7.4 was added and hydrated for 30 min to obtain a liposome suspension. The liposome suspension was transferred to a test tube, and a probe sonicator was used to ultrasonicate at 300 w for 6 min in an ice bath to obtain long-circulating liposomes.

[0073] Preparation of target liposome: the nucleic acid aptamer apolipoprotein E (ApoE peptide) and AS1411 were dissolved in ultrapure water, and the molar ratio of ApoE peptide to AS1411 was 0.77:0.01. First, AS1411 was added to the above-mentioned long-circulating liposome, and incubated at 4°C for 30 min, then at room temperature for 30 min, repeated for 3 times, then ApoE peptide was added, incubated at 4°C overnight, and finally the free ligand was removed by dialysis to obtain the double-target modified functional liposome AA-CA4 / HM@Fe-LP.

[0074] Example 3 Basic properties of liposome

[0075] In this example, the particle size, potential, appearance, morphology and stability of AA-CA4 / HM@Fe-LP obtained in Example 2 were characterized, and the encapsulation efficiency, drug loading and in vitro release rate of the liposome were determined. For comparison, PEG-LP, CA4-LP, CA4 / HM@Fe-LP and single-modified AS-CA4 / HM@Fe-LP were also tested in this example.

[0076] (1) Particle size and potential:

[0077] As shown in Table 1, the average particle size of each group of liposomes was between 100-110 nm, and the particle size of AS-CA4 / HM@Fe-LP and AA-CA4 / HM@Fe-LP was slightly larger than that of PEG-LP, which may be related to the surface modification of the target. The PDI of each group of liposomes was about 0.2, the PDI index was small, and the dispersion was good. The potential of each group of liposomes was between -21ˉ-28 mV.

[0078] Table 1 Liposome particle size, PDI and potential

[0079]

[0080] (2) Appearance and morphology:

[0081] As shown in A of Figure 1 , the prepared CA4-LP system was clear and transparent, slightly blue with a milky luster, and no precipitate was precipitated. The AA-CA4 / HM@Fe-LP system was transparent red, and no precipitate was precipitated. As shown in B of Figure 1 , under transmission electron microscope observation, the liposome was spherical with a double-layer structure, uniformly dispersed, uniform in size, and the particle size was consistent with the value measured by the nanoparticle size analyzer.

[0082] (3) Encapsulation efficiency and drug loading:

[0083] The encapsulation efficiency and drug loading of CA4 were determined by dialysis, and the results are shown in Tables 2 and 3. Table 2 shows that the encapsulation efficiency of each group of liposomes was between 83% and 90%, and the drug loading was approximately 2%. Table 3 shows that the HMME encapsulation efficiency of each group of liposomes was above 70%, and the drug loading was above 2%.

[0084] Table 2. Encapsulation efficiency and drug loading of CA4 in liposomes (Mean ± SD, n = 3)

[0085]

[0086] Table 3 Encapsulation efficiency and drug loading of HMME in liposomes (Mean ± SD, n = 3)

[0087]

[0088] (4) In vitro stability:

[0089] The changes in liposome particle size and PDI over 30 days at 4℃ are as follows: Figure 2 As shown. (Through) Figure 2 As can be seen, the particle size of the liposomes in each group remained almost unchanged, all being less than 120 nm. The PDI was below 0.3, indicating that the prepared liposomes had good stability.

[0090] Example 4: Evaluation of in vitro cell uptake

[0091] In this embodiment, coumarin-6 (C6) was used as a fluorescent probe, which was encapsulated in liposomes to investigate the uptake of fluorescent liposomes by HepG2 cells. The results are as follows: Figure 3 As shown.

[0092] The preparation method of coumarin-6 fluorescent liposomes is as follows: CA4 is replaced with C6, and the preparation is carried out according to the preparation method of Example 3 to obtain PEG-C6-LP, ApoE-C6-LP, AS1411-C6-LP and AA-C6-LP, respectively. The concentration of C6 is 2 μg / mL, which can detect fluorescence normally.

[0093] according to Figure 3 It can be seen that the green fluorescence intensity of the target liposomes in HepG2 cells was stronger than that of the PEG-C6-LP group. Among them, the uptake rates of ApoE peptide, AS1411 and AA dual-modified liposomes were 1.67, 1.39 and 2.17 times that of long-circulating fluorescent liposomes, respectively, which were significantly different (p < 0.01). The fluorescence intensity of the ApoE-C6-LP group was higher than that of the AS1411-C6-LP group, while the AA-C6-LP group had the strongest fluorescence intensity, indicating that the co-modification of the target AS1411 and ApoE peptide has a good synergistic targeting effect.

[0094] HUVEC cells uptake of fluorescent liposomes in each group was detected by flow cytometry Figure 4 and Figure 5 As shown in

[0095] In vivo imaging results of Example 5

[0096] In this example, DIR was used as a fluorescent probe, which was loaded into liposomes to explore the distribution in vivo.

[0097] The preparation method of DIR-loaded fluorescent liposomes was to replace CA4 with DIR, and the preparation method of Example 3 was followed to prepare PEG-DIR-LP, AS1411-DIR-LP and AA-DIR-LP, respectively.

[0098] After successfully establishing the nude mouse orthotopic liver cancer model, each group of DIR-loaded fluorescent probe liposomes was injected into the blood through the tail vein, and the distribution of fluorescent signals in vivo within 24h was recorded.

[0099] The results are shown in Figure 6 Compared with other organs, the fluorescent signal in the liver was the strongest, indicating that the liposomes were mainly concentrated in the liver within 24h, and the fluorescent intensity increased over time. The fluorescent intensity of AS-DIR-LP group in the liver was higher than that of PEG-DIR-LP group, while the fluorescent signal of AA-DIR-LP group was further enhanced, which may be due to the modification of ApoE peptide that increased the ability of fluorescent liposomes to target liver and tumor. The above results showed that the co-modification of AS1411 and ApoE peptide made the liposomes have good liver orthotopic tumor targeting ability.

[0100] Intracellular reactive oxygen species detection of Example 6

[0101] In this example, HMME was used to detect the production of reactive oxygen species in vitro by HepG2 cells treated with each group of liposomes by sonodynamic effect. Specifically, the concentration of HMME was 30μg / mL, the ultrasonic power was 1.5w / cm 2 , the ultrasonic time was 30S, and the detection results are shown in Figure 5 .

[0102] According to Figure 7It can be seen that each group of liposomes has only weak fluorescence under non-ultrasound conditions, while after ultrasonic treatment, the fluorescence intensity of the HMME-loaded group is significantly enhanced, indicating that HMME can produce a large amount of active oxygen under ultrasound environment. Compared with the HMME-LP group, the fluorescence intensity of the HM@Fe-LP group is enhanced, which may be due to the fact that the trivalent iron ion promotes the production of active oxygen. The fluorescence intensity of the AS-HM@Fe-LP group is further enhanced, and the AA-HM@Fe-LP group has the strongest fluorescence, which may be because the target causes more liposomes to enter the HepG2 cells.

[0103] Example 7 Intracellular Ca 2+ Detection

[0104] This example uses Fluo-3AM to detect the effect of each group of liposomes loaded with HM@Fe under ultrasound driving on the distribution of calcium ions in HepG2 cells, and the results are shown in Figure 8

[0105] According to Figure 8 It can be seen that there is no significant difference in fluorescence intensity between the CA4-LP of the Control group and the PEG-LP, while the fluorescence intensity of the liposomes loaded with HMME is significantly enhanced. Compared with the HMME-LP group, the fluorescence intensity of the HM@Fe-LP group is further enhanced, and the AA-HM@Fe-LP group has the strongest fluorescence. This may be because the active oxygen produced causes oxidation of the cell membrane, and since the extracellular Ca 2+ level is much higher than that in the cell, it causes Ca 2+ to flow into the cell, resulting in an increase in the concentration of Ca 2+ in the cell, and calcium overload can cause mitochondrial function and metabolism to be disordered, inducing tumor cell apoptosis.

[0106] The above results show that each group of liposomes loaded with HM@Fe under ultrasound driving will cause more calcium ions to enter the cell, thereby causing calcium overload.

[0107] Example 8 In vitro toxicity of CA4 and each group of liposomes to HepG2 cells

[0108] This example investigates the cytotoxicity of each liposome to HepG2, and the experimental results are shown in Figure 9

[0109] According to Figure 9 It can be seen that the CA4 raw material and each group of liposomes have concentration-dependent cytotoxicity, and the higher the concentration of CA4, the higher the cell death rate.

[0110] ​​Specifically, the IC50 values ​​of free CA4, CA4-LP, CA4 / HM@Fe-LP, CA4 / HM@Fe-LP+US, AS-CA4 / HM@Fe-LP, AS-CA4 / HM@Fe-LP+US, AA-CA4 / HM@Fe-LP, and AA-CA4 / HM@Fe-LP+US on HepG2 cells were calculated. 50 The values ​​were 48.72, 20.02, 14.27, 2.59, 6.11, 1.05, 2.23, and 0.42 μM, respectively. It is evident that compared to the CA4 active pharmaceutical ingredient group, the cell viability of each liposome group was reduced, exhibiting stronger toxicity to cells, indicating that liposome encapsulation allowed more CA4 to enter the cells.

[0111] liposomes modified with AS1411 and AS1411+APOE peptides IC 50 The value decreases, with the latter IC 50 The decrease in values ​​was more pronounced, indicating that target-modified liposomes can enhance the inhibitory effect on tumor cells. Among them, the co-modification of AS1411 and APOE peptides had a stronger effect, which may be because HepG2 cells had the highest uptake of AA-CA4 / HM@Fe-LP.

[0112] Compared with the non-ultrasound group, the ultrasound group had lower IC 50 The value decreased significantly, showing a statistically significant difference, indicating that the combination of CA4 and sonodynamic therapy further increased cytotoxicity.

[0113] Example 9: Effects of liposomes in each group on intracellular glutathione levels

[0114] This embodiment investigated the effects of various liposomes on the level of glutathione (GSH) in HepG2 cells.

[0115] Specifically, after drug treatment of HepG2 cells, the intracellular GSH content was detected using a GSH kit, and the results were as follows: Figure 10 As shown.

[0116] pass Figure 10 It can be seen that, compared with the Control group (PEG-LP), the GSH levels in all groups were significantly reduced. Compared with the non-ultrasound group, the GSH level further decreased after ultrasound treatment, possibly because HMME was activated by ultrasound, producing a large amount of reactive oxygen species in the cells, which reacted with GSH in a redox reaction, thus reducing GSH content. Compared with the HMME-LP group, the GSH level in the HM@Fe-LP group was significantly reduced, possibly due to Fe 3+ It oxidizes intracellular GSH, reducing the amount of GSH.

[0117] Example 10: Damage to β-tubulin in cells by various liposomes

[0118] The β-tubulin in the HepG2 cells of the present embodiment is labeled green fluorescence by β-tubulin antibody, so that the damage of the raw material CA4 and each group of liposomes to the microtubule can be investigated by immunofluorescence detection. Since the single modification of AS or APOE peptide has little effect on the overall results of the experiment, no comparison is made.

[0119] As shown in the microtubulin images of the cells recorded by the laser confocal microscope, Figure 11 The microfilaments of the cells in the Control group are gathered into bundles and slightly curved. The cells in the administration groups are shrunk and rounded, and the microtubules change from the original bundle to dispersed ring. This may be due to the specific binding of CA4 to the colchicine domain of microtubulin, resulting in the loss of heterodimer, which makes the microtubules maintain in the depolymerization state and interferes with the polymerization of the microtubules. Compared with the CA4-LP group, the microtubules in the AA-CA4 / HM@Fe-LP group are further damaged, and the AA-CA4 / HM@Fe-LP+US group shows the strongest depolymerization effect on the microtubulin.

[0120] Example 11 In vivo tumor inhibition of each group of liposomes

[0121] This embodiment investigates the in vivo tumor inhibition effect of each liposome. Since the single modification of AS or APOE peptide has little effect on the overall results of the experiment, no comparison is made.

[0122] The animal model used in this embodiment is a nude mouse subcutaneous tumor model of liver cancer, and the specific modeling process is as follows: after two weeks of inoculation of HepG2 cells in the axillary region of the nude mice, tumors grow in the right anterior limb of each nude mouse, which are black and spherical, with a size of 100-150mm 3 between, the tumor volume reaches the required administration, indicating that the nude mouse subcutaneous tumor model of liver cancer is successfully established.

[0123] The in vivo anti-tumor effect of each group of liposomes was evaluated using the nude mice inoculated with the subcutaneous tumor model of liver cancer, and the experimental results are shown in Figure 12 .

[0124] Specifically, the tumor growth curve results are shown in Figure 12 A. Compared with the PBS group, each administration group shows obvious anti-tumor effect. The tumor in the PBS group grows rapidly, and the average volume reaches 1332mm 3 after the 20th day, while the CA4-LP group, the CA4 / HM@Fe-LP group, the CA4 / HM@Fe-LP+US group, the AA-CA4 / HM@Fe-LP group and the AA-CA4 / HM@Fe-LP+US group reach 830, 773, 403, 585 and 303mm 3AA-CA4 / HM@Fe-L+US P group had the most significant inhibitory effect on tumor, which might be due to the fact that AS1411 and ApoE peptide jointly promoted the targeting of liposome to tumor tissue, so that more drug-loaded liposome entered tumor cells, and the combination of ultrasound therapy achieved the best therapeutic effect on tumor.

[0125] The body weight changes of nude mice were as shown in Table 2. Figure 12 As shown in Table 2, the body weight of each group was decreased, and compared with the PBS group, the body weight of the administration groups did not show significant decrease, which might indicate that the administration groups had no significant effect on the growth of nude mice.

[0126] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A CA4 / HM@Fe liposome, characterized in that, The liposomes are cavitary structures and are loaded with HM@Fe complex and compressine.

2. A method for preparing a Combretastatin targeted lipid delivery system, characterized in that, The CA4 / HM@Fe liposomes described in claim 1 were co-modified using AS1411 and ApoE peptide.

3. The preparation method of the Combretastatin targeted lipid delivery system according to claim 2, characterized in that, Includes the following steps: S1. Preparation of HM@Fe complex; S2. The HM@Fe complex and Compressor were prepared into CA4 / HM@Fe liposomes by thin-film dispersion method; S3. The CA4 / HM@Fe liposomes are subjected to ultrasonic treatment. S4. AS1411 solution and ApoE peptide solution are mixed with ultrasonically treated CA4 / HM@Fe liposomes for co-modification to prepare the Compritin targeted lipid delivery system.

4. The preparation method of the Combretastatin targeted lipid delivery system according to claim 3, characterized in that, Step S2 is as follows: SPC, cholesterol, methoxyPEG distearate phosphatidyl acetamide and compressortin were dissolved in chloroform-methanol solution to obtain a mixed solution. The mixed solution was then mixed with the HM@Fe complex and stirred until homogeneous to obtain a liposome precursor solution. The liposome precursor solution was rotary evaporated to dryness at 37°C under reduced pressure to form a uniform film. After drying, it was hydrated to obtain the CA4 / HM@Fe liposomes.

5. The preparation method of the Comprilatin targeted lipid delivery system according to claim 3, characterized in that, In step S3, the frequency of the ultrasonic treatment is 200w to 400w, and the ultrasonic time is 3-9min.

6. The preparation method of the Comprilatin targeted lipid delivery system according to claim 3, characterized in that, In step S4, the AS1411 solution is mixed with the ultrasonically treated CA4 / HM@Fe liposomes and incubated at a temperature cycle; the ApoE peptide solution is added and incubated; free ligands are removed by dialysis to obtain the Compritin targeted lipid delivery system.

7. The method for preparing a Comprilatin targeted lipid delivery system according to claim 6, characterized in that, The temperature cycling incubation process involves incubating at 4°C for 30 minutes, then at room temperature for 30 minutes, and repeating this cycle three times.

8. A method for preparing a Comprilatin targeted lipid delivery system according to any one of claims 2-7, characterized in that, Step S1 is as follows: A hematoporphyrin monomethyl ether solution was prepared using a methanol-triethylamine solution and hematoporphyrin monomethyl ether. Ferric chloride hexahydrate solution was prepared using methanol-dimethylformamide and ferric chloride hexahydrate; The hematoporphyrin monomethyl ether solution and the ferric chloride hexahydrate solution were mixed at a mass ratio of 1:1, centrifuged, resuspended, and washed to obtain the HM@Fe complex.

9. A Combretastatin targeted lipid delivery system, characterized in that, It is prepared by the method described in any one of claims 2-8.

10. A formulation for sonodynamic therapy, characterized in that, Including the Compritin targeted lipid delivery system as described in claim 9.

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