An LPS-modified liposomal clodronate and its preparation method and application

Abstract

The present invention relates to an LPS-modified clodronate liposome and its preparation method and application. By modifying LPS on the surface of the clodronate liposome (LPS-coupled clodronate liposomes; LPS-CLD-lipo), the liposome can effectively target macrophages and hepatic stellate cells highly expressing TLR4 in the liver and then kill and remove the cells. The LPS-modified clodronate liposome of the present invention has the effect of targeting and killing hepatic stellate cells in addition to enhancing the targeting of macrophages. In addition, in a liver fibrosis model, it is also confirmed that the present invention has a good anti-fibrotic effect and can be used to inhibit liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically, to an LPS-modified clodronate liposome, a preparation method thereof, and an application thereof. Background Art

[0002] At present, commercially available clodronate liposomes (CLD-lipo) are an effective means to remove macrophages in the body. The principle is that the mononuclear phagocyte system (MPS) in the body has strong characteristics of phagocytosing and ingesting exogenous substances, and 80-90% of macrophages are present in the liver. Therefore, after administration of clodronate liposomes in in vivo experiments, due to the passive targeting effect, they can accumulate in the liver and then remove macrophages. Under the action of macrophage lysosomes, the liposomes are destroyed, and the released clodronate is metabolized into an unhydrolyzable ATP analog in the cells, inhibiting the ADP / ATP transport mechanism in mitochondria, thereby blocking the mitochondrial respiratory chain and inducing apoptosis.

[0003] Chinese Patent CN103040863A, publication date: December 17, 2012, discloses a clodronate liposome injection and a preparation method thereof. By selecting specific weight ratios of clodronate, distearoyl phosphatidylglycerol, cholesterol, and polyoxyethylene 40 hydrogenated castor oil, a high-quality clodronate liposome injection is prepared. Chinese Patent CN107854489A, publication date: March 30, 2018, discloses the use of clodronate liposomes in the adjuvant treatment of tumor doxorubicin chemotherapy. After mixing appropriate amounts of cholesterol and lecithin, a chloroform-methanol mixture is added, and a uniform lipid dry film is prepared by the rotary thin film evaporation method, and then a phosphate buffer solution of clodronate is added to obtain it. It is found that preparing stable and highly efficient clodronate liposomes can weaken the cardiotoxicity caused by doxorubicin and increase the therapeutic effect of doxorubicin on breast cancer. Some studies have also found that when the liver is damaged and stimulated, both macrophages and hepatic stellate cells highly express Toll-like receptor 4 (TLR4) on the surface. TLR4 mainly recognizes lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, and TLR4 is a receptor that specifically binds to LPS. On this basis, the present invention further modifies the clodronate liposome by modifying LPS on the surface of the clodronate liposome (LPS-coupled clodronate liposomes; LPS-CLD-lipo), so that this liposome can effectively target macrophages and hepatic stellate cells with high expression of TLR4 in the liver and then kill and remove the cells. The LPS-modified clodronate liposome not only enhances the targeting of macrophages but also increases the effect of targeting and killing hepatic stellate cells.

[0004] At present, there is no report on the liposome of disodium clodronate modified by LPS and its preparation method and application in the present invention. Summary of the Invention

[0005] The first object of the present invention is to provide a preparation method of a liposome of disodium clodronate modified by LPS in view of the deficiencies in the prior art.

[0006] The second object of the present invention is to provide a liposome of disodium clodronate modified by LPS.

[0007] The third object of the present invention is to provide a use of a liposome of disodium clodronate modified by LPS.

[0008] To achieve the above first object, the technical solution adopted by the present invention is:

[0009] A preparation method of a liposome of disodium clodronate modified by LPS, the preparation method comprising the following steps:

[0010] a) Prepare a mixed solvent containing chloroform, methanol and ddH2O;

[0011] b) Film formation: After dissolving lipopolysaccharide, lecithin and cholesterol in water, add them to the mixed solvent in step a) respectively, mix evenly, put them into a rotary evaporation flask, and rotate under vacuum for 60 min;

[0012] c) Hydration: Dissolve disodium clodronate + 10 mL ddH2O and put it into a rotary evaporation flask, and rotate at room temperature for 20 min;

[0013] d) Absorb the hydrated solution, put it into a light-proof tube, let it stand at room temperature for 2 h, mix well, and then sonicate for 3 min;

[0014] e) Let it stand overnight at 4°C, centrifuge for 60 min, and remove the disodium clodronate solution under the white band;

[0015] f) Add PBS to wash once, centrifuge for 30 min, discard the supernatant, and resuspend the precipitate with 4 mL PBS;

[0016] g) Obtain liposomes with the desired particle size by passing through a polycarbonate membrane by extrusion.

[0017] As a preferred example, the ratio of the mixed solvent in step a) is: 2.5:7:0.5 (v / v / v; 10 mL).

[0018] As a preferred example, the amounts of lipopolysaccharide, lecithin and cholesterol added in step b) are 0.25 mg, 86 mg and 8 mg respectively, and the mixed solvents are 6 mL, 0.86 mL and 2 mL respectively.

[0019] As a preferred example, the amount of disodium clodronate added in step c) is 2.5 g, and the rotation condition is 100 rpm.

[0020] As a preferred example, the centrifugation conditions in both steps e) and f) are 25,000 g and 10 °C.

[0021] As a preferred example, the specification of the polycarbonate membrane in step g) is 200 nm, and the extrusion needs to be performed 15 times.

[0022] To achieve the second objective above, the technical solution adopted by the present invention is:

[0023] Disodium clodronate liposomes prepared by the preparation method described in any one of the above.

[0024] To achieve the third objective above, the technical solution adopted by the present invention is:

[0025] Use in the preparation of a drug for inhibiting liver fibrosis with the disodium clodronate liposomes described above.

[0026] The advantages of the present invention are:

[0027] 1. The present invention is the first to use LPS to modify and transform disodium clodronate liposomes.

[0028] 2. The LPS-modified disodium clodronate liposomes prepared by the present invention can not only eliminate macrophages but also directly eliminate a part of hepatic stellate cells.

[0029] 3. The LPS-modified disodium clodronate liposomes prepared by the present invention have a better effect of inhibiting liver fibrosis. Description of the Drawings

[0030] Attached Figure 1 Is the preparation flow chart of CLD-lipo and LPS-CLD-lipo.

[0031] Attached Figure 2 Is the electron micrograph of CLD-lipo and LPS-CLD-lipo.

[0032] Attached Figure 3 Is the in vitro stability of CLD-lipo and LPS-CLD-lipo.

[0033] Attached Figure 4 Is the uptake of LPS-modified disodium clodronate liposomes by liver macrophages in vivo experiments.

[0034] Attached Figure 5 Is the uptake of LPS-modified and small-sized disodium clodronate liposomes by hepatic stellate cells in vivo experiments.

[0035] Figure 6 To enhance the killing effect of liposomal clodronate on macrophages and hepatic stellate cells after LPS modification, A: Macrophages; B: HSCs-T6.

[0036] Figure 7 For, A: In the BDL model, the effects of different types of liposomes on rat liver injury and collagen deposition after treatment. B: In the BDL model, statistical quantification of the results of Masson staining and Sirius Red staining. C: In the BDL model, serological detection of the contents of ALT and AST.

[0037] Figure 8 For, A: In the CCl4 model, the effects of different types of liposomes on rat liver injury and collagen deposition after treatment. B: In the CCl4 model, statistical quantification of the results of Masson staining and Sirius Red staining. C: In the CCl4 model, serological detection of the contents of ALT and AST. Specific embodiments

[0038] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0039] Example 1 Preparation and characterization of LPS-modified liposomal clodronate

[0040] 1 Experimental equipment

[0041] 1.1 Materials

[0042] Phosphatidylcholine (PC, P3556), cholesterol (Chol, C8667), lipopolysaccharide (LPS, L2880) and DEN were purchased from Sigma-Aldrich; clodronate disodium (D832397), methanol (M813895), chloroform (821112) and carbon tetrachloride (C805325) were purchased from Shanghai Macklin Biochemical Co., Ltd.; Dio fluorescent dye was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; SD rats were purchased from Shanghai Slake Laboratory Animal Co., Ltd.; olive oil was purchased from Sangon Biotech; RPMI 1640 and DMEM media were purchased from Gibco, USA; CCK8 kit was purchased from Wuhan Sevier Biotechnology; 4% paraformaldehyde was purchased from Shanghai Boguang Biotechnology; H&E staining solution was purchased from Shanghai Yixin Biotechnology, and the horseshoe crab kit was purchased from GenScript Biotech Corporation.

[0043] 1.2 Instruments

[0044] Rotary evaporator (RE-52AA, Shanghai Yarong); circulating water vacuum pump (SHZ-Ⅲ, Shanghai Yarong); Malvern particle size analyzer (Mastersizer 3000, Malvern, UK); ultraviolet-visible spectrophotometer (UV1102Ⅱ, Tianmei); transmission electron microscope (Tecnai G2 F20 S-TWIN, FEI); ultrasonic cleaner (KQ3200E, Kunshan Ultrasonic Instrument Co., Ltd.); microbalance (XE-100A, Denver); fluorescence inverted microscope (LX73, Olympus); pathological microscope (BX43, Olympus); paraffin embedding machine (EG1150H, Leica); pathological slicer (RM2016, Leica).

[0045] 2 Experimental methods:

[0046] 2.1 Preparation and characterization of LPS-modified liposomes of clodronate disodium

[0047] 1. Preparation of mixed solvent

[0048] Prepare the mixed solvent according to the following ratio: chloroform: methanol: ddH2O = 2.5:7:0.5 (v / v / v; 10 mL).

[0049] 2. Film formation

[0050] (1) First dissolve 0.25 mg LPS in water (+100 ul ddH2O), and then add 6 mL of the above mixed solvent (not required when preparing CLD-lipo);

[0051] (2) Add 86 mg PC to 0.86 mL of the mixed solvent;

[0052] (3) Add 8 mg Chol to 2 mL of the mixed solvent;

[0053] After dissolving and mixing the three, put them into a rotary evaporation flask and rotate at 40 °C under vacuum at 150 rpm for 60 min.

[0054] 3. Hydration

[0055] Dissolve 2.5 g of clodronate disodium + 10 mL of ddH2O and add it to the rotary evaporation flask, and rotate at room temperature (25 °C) at 100 rpm for 20 min.

[0056] 4. Aspirate the above solution, put it into a light-proof tube, let it stand at room temperature for 2 h, mix it well and then sonicate for 3 min.

[0057] 5. Let it stand overnight at 4 °C, centrifuge at 25000 g at 10 °C for 60 min, and remove the clodronate disodium solution under the white band.

[0058] 6. Add PBS and wash once, centrifuge at 25,000 g for 30 min at 10 °C, discard the supernatant, and resuspend the pellet in 4 mL of PBS.

[0059] 7. Extrude through a 200 nm polycarbonate membrane 15 times to obtain liposomes with the desired particle size.

[0060] 8. Add 0.25 mg of the lipophilic fluorescent dye Dio to the above lipid phase solution, and repeat the above experimental steps to obtain fluorescently labeled liposomes (Dio-CLD-lipo / Dio-LPS-CLD-lipo).

[0061] 9. Detect the particle size, PDI, and Zeta potential of CLD-lipo and LPS-CLD-lipo using a Malvern particle size analyzer.

[0062] 10. Observe the morphology of CLD-lipo 200 nm and LPS-CLD-lipo 200 nm by transmission electron microscopy.

[0063] 11. Use a UV spectrophotometer to determine the encapsulation efficiency (EE) and drug loading (DL) of disodium clodronate in CLD-lipo and LPS-CLD-lipo.

[0064] 12. Use a Limulus kit to detect the surface modification rate (Coupling efficiency, CE) of LPS in LPS-CLD-lipo.

[0065] 13. Observe the stability of CLD-lipo and LPS-CLD-lipo: Place the prepared CLD-lipo and LPS-CLD-lipo in storage at 4 °C, and sample and detect their particle size and polydispersity index (PDI) on the 1st, 3rd, 5th, and 7th days respectively to evaluate the stability of CLD-lipo and LPS-CLD-lipo during storage.

[0066] 2.2 In vivo animal experiments to explore the uptake of different types of liposomes by macrophages and hepatic stellate cells

[0067] CLD-lipo and LPS-CLD-lipo were modified with Dio fluorescent dye so that we could use a microscope to observe liposomes. Secondly, a liver fibrosis model of SD rats induced by DEN for 6 weeks was constructed (by adding DEN (0.01%) to the drinking water), and the rats were randomly divided into 5 groups. PBS (equal volume to the liposome group), 10 mg / kg CLD-lipo 1um, CLD-lipo 200nm, LPS-CLD-lipo 1um, and LPS-CLD-lipo 200nm were administered via tail vein injection. After 2 h, the liver tissues of the rats were collected, and frozen sections and immunofluorescence experiments were further performed to observe the uptake of different types of liposomes by macrophages and hepatic stellate cells in the liver.

[0068] 2.3 In vitro cell experiments to explore the killing effects of different types of liposomes on macrophages and hepatic stellate cells 2.3.1 Killing experiment of different types of liposomes on macrophages

[0069] Primary peritoneal macrophages were isolated from a liver fibrosis model of rats induced by DEN for 6 weeks and cultured in RPMI 1640 cell culture medium. They were seeded into 96-well plates at a density of 1×10 4 cells / well. After the cells adhered and grew, they were co-cultured with PBS (equal volume to the liposome group), 2 mg / mL CLD-lipo 1um, CLD-lipo 200nm, LPS-CLD-lipo 1um, and LPS-CLD-lipo 200nm for 8 h. After discarding the supernatant, the cells were washed 3 times with PBS, and then the culture medium containing 10% CCK-8 was added and incubated in the dark for another 2 h. The absorbance OD value at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0070] 2.3.2 Killing experiment of different types of liposomes on hepatic stellate cells

[0071] The HSCs-T6 cell line was cultured in DMEM cell culture medium and seeded into 96-well plates at a density of 1×10 4 cells / well. After the cells adhered and grew, they were co-cultured with PBS (equal volume to the liposome), 2 mg / mL CLD-lipo 1um, CLD-lipo 200nm, LPS-CLD-lipo 1um, and LPS-CLD-lipo 200nm for 24 h. After discarding the supernatant, the cells were washed 3 times with PBS, and then the culture medium containing 10% CCK-8 was added and incubated in the dark for another 2 h. The absorbance OD value at 450 nm was measured with an ELISA reader.

[0072] 2.4 Explore the role of different types of liposomes in liver fibrosis

[0073] Male SD rats weighing 200 ± 20 g were selected to establish a rat liver fibrosis model induced by bile duct ligation. On the 14th and 21st days after the operation, PBS (equal volume to liposomes), 10 mg / kg CLD-lipo 1um, CLD-lipo 200nm, LPS-CLD-lipo 1um, and LPS-CLD-lipo 200nm were injected via the tail vein. On the 28th day after the operation, the liver tissues and inferior vena cava blood of the rats were collected.

[0074] Male SD rats weighing 200 ± 20 g were selected to establish a rat liver fibrosis model induced by CCl4. A mixture of 1 mL / kg CCl4:Oil = 1:1 (v / v) was injected intraperitoneally twice a week for 6 weeks. Starting from the 4th week of modeling, PBS (equal volume to liposomes), 10 mg / kg CLD-lipo 1um, CLD-lipo 200nm, LPS-CLD-lipo 1um, and LPS-CLD-lipo 200nm were injected via the tail vein (24 h after each CCl4 injection) for 3 weeks, once a week. 24 h after the last administration of CCl4 at the 6th week, the liver tissues and inferior vena cava blood of the rats were collected.

[0075] The collected inferior vena cava blood was allowed to stand at room temperature for 1 h and then centrifuged at 3000 rpm / min for 10 min, and the supernatant was collected to obtain serum. Kits were used to detect the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum; part of the liver tissue was fixed in paraformaldehyde, embedded in paraffin, and sectioned for HE, Sirius Red, and Masson staining.

[0076] 3 Data analysis

[0077] The data in the Masson staining and H&E staining experiments were quantitatively analyzed using Image Pro Plus software. Graph Pad Prism 8.0 software was used for graphing and statistical analysis. The experimental data were expressed as Mean ± SD, and the data of multiple groups of samples were analyzed by one-way ANOVA. A P value < 0.05 was considered statistically significant.

[0078] 4 Experimental results

[0079] 4.1 Particle size, PDI, Zeta potential of liposomes, encapsulation efficiency of clodronate disodium, drug loading, and surface modification rate of LPS

[0080] The particle size, PDI, Zeta potential, encapsulation efficiency of disodium clodronate, drug loading, and surface modification rate of LPS of the liposomes are shown in Table 1. Four different types of liposomes were successfully prepared by the thin film dispersion method, namely CLD-lipo 1um, CLD-lipo 200nm, LPS-CLD-lipo 1um, and LPS-CLD-lipo 200nm. Next, the liposomes were further characterized and identified. The results showed that the particle size and Zeta potential presented a single-peak normal distribution, and the Zeta potential was between -6 and -9 mV. The PDI was less than 0.3, indicating that the particle size distribution of the liposomes was uniform. The encapsulation efficiency of disodium clodronate in the liposomes was relatively stable, all around 1%. The modification of LPS on the liposomes was above 99%.

[0081] Table 1 Particle size, PDI, Zeta potential, encapsulation efficiency of disodium clodronate, drug loading, and surface modification rate of LPS of the liposomes

[0082]

[0083] 4.2 TEM results of CLD-lipo 200nm and LPS-CLD-lipo 200nm

[0084] The morphologies of CLD-lipo 200nm and LPS-CLD-lipo 200nm were observed by transmission electron microscopy. It could be seen that the liposomes were spherical and monodispersed, as shown Figure 2 .

[0085] 4.3 In vitro stability of CLD-lipo and LPS-CLD-lipo

[0086] Significant changes were found in the stability indicators of the large-particle-size liposomes within 7 days. However, when the particle size of the liposomes was reduced to 200 nm, no obvious changes were observed in the stability-related indicators, indicating that the small-particle-size liposomes had better stability under the storage condition of 4°C, as shown Figure 3 .

[0087] 4.4 In vivo experiments found that LPS modification enhanced the uptake of disodium clodronate liposomes by liver macrophages

[0088] Compared with the control group, it could be observed that macrophages in the liver of the CLD-lipo 1um treatment group could uptake a certain amount of liposomes; however, when the particle size of CLD-lipo was reduced to 200 nm, the uptake ability of macrophages to liposomes was correspondingly weakened; while further modifying the surface of CLD-lipo with LPS, macrophages could show a significant uptake ability to liposomes regardless of the particle size of the liposomes, as shown Figure 4 .

[0089] 4.5 In vivo experiments found that LPS-modified and small-sized clodronate liposomes could be effectively taken up by hepatic stellate cells.

[0090] Compared with the control group, large-sized liposomes, whether or not modified with LPS, could not be effectively taken up by hepatic stellate cells; however, when the particle size of CLD-lipo was reduced to 200 nm, it was observed that compared with large-sized CLD-lipo, the uptake ability of hepatic stellate cells for liposomes was enhanced; but LPS-modified and small-sized CLD-lipo could be significantly taken up by hepatic stellate cells.

[0091] 4.6 In vitro experiments proved that LPS modification enhanced the killing effect of clodronate liposomes on macrophages and hepatic stellate cells

[0092] The results showed that Macrophages: Consistent with the in vivo uptake experiment, LPS-modified CLD-lipo showed a stronger killing ability against macrophages. HSC-T6: Consistent with the in vivo uptake experiment, LPS-modified and small-sized CLD-lipo showed a stronger killing effect on hepatic stellate cells, see Figure 6 .

[0093] 4.7 LPS-modified and small-sized clodronate liposomes could significantly inhibit the occurrence of liver fibrosis

[0094] In two rat liver fibrosis models of BDL and CCl4, H&E staining was used to observe the pathological effects of different types of liposomes on rat livers. The results showed that: a large number of inflammatory cell infiltrations were present in the livers of rats in the control group and other types of liposome treatment groups, with loose cytoplasm and disordered cell arrangement; while in the livers of rats in the LPS-CLD-lipo 200 nm treatment group, the cell arrangement was relatively neat and tight, and the inflammatory infiltration was less, see ( Figure 7 A, B and Figure 8 A,B). Masson staining and Sirius Red staining were used to observe the effects of different types of liposomes on collagen fibers in rat livers. The results showed that: a large amount of collagen fibers appeared in the livers of rats in the control group and other types of liposome treatment groups, and a large number of false lobules were formed, with obvious fibrosis; the fibrosis degree in the LPS-CLD-lipo 200 nm treatment group was significantly reduced, see ( Figure 7 A,B and Figure 8 A,B). Further detection of liver function indexes (ALT, AST) in serology was carried out. The results showed that compared with the control group and other different types of liposomes, the levels of ALT and AST in the sera of rats in the LPS-CLD-lipo 200 nm treatment group were significantly reduced, see ( Figure 7 C and Figure 8 C).

[0095] In summary, in both the BDL and CCl4 rat liver fibrosis models, it was shown that after administration of different types of liposomes, the LPS-CLD-lipo 200nm group exhibited the best anti-fibrotic effect.

[0096] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of LPS-modified liposomal clodronate disodium, characterized in that, The described preparation method includes the following steps: a) Prepare a mixed solvent containing chloroform, methanol and ddH2O; b) Film formation: Dissolve the water-soluble lipopolysaccharide, phosphatidylcholine and cholesterol in the mixed solvent in step a) respectively. After the three are dissolved, mix them evenly and put them into a rotary evaporation flask, and rotate under vacuum for 60 min; c) Hydration: Dissolve disodium clodronate in 10 mL of ddH2O and put it into a rotary evaporation flask, and rotate at room temperature for 20 min; d) Absorb the hydrated solution, put it into a light-proof tube and let it stand at room temperature for 2 h, then mix it evenly and sonicate for 3 min; e) Let it stand overnight at 4°C, centrifuge for 60 min, and remove the disodium clodronate solution under the white band; f) Add PBS to wash once, centrifuge for 30 min, discard the supernatant, and resuspend the precipitate with 4 mL of PBS; g) After extrusion through a polycarbonate membrane, liposomes with the desired particle size are obtained. The ratio of the mixed solvent in step a) is: 2.5:7:0.5 (v / v / v), and the total volume of the mixed solvent is 10 mL; the amounts of lipopolysaccharide, phosphatidylcholine and cholesterol added in step b) are 0.25 mg, 86 mg and 8 mg respectively, and the amounts of the mixed solvent added are 6 mL, 0.86 mL and 2 mL respectively; In step g), the specification of the polycarbonate membrane is 200 nm, and the extrusion needs to be carried out 15 times.

2. The preparation method of the disodium clodronate liposome according to claim 1, characterized in that, In step c), the amount of disodium clodronate added is 2.5 g, and the rotation condition is 100 rpm.

3. The preparation method of the disodium clodronate liposome according to claim 1, wherein The centrifugation conditions in steps e) and f) are both 25000 g and 10°C.

4. An LPS-modified liposomal clodronate, characterized in that, The disodium clodronate liposomes are prepared by the preparation method described in any one of claims 1-3.

5. Use of the LPS-modified disodium clodronate liposomes described in claim 4 in the preparation of a drug for inhibiting liver fibrosis.

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