Clodronate lipid nanoparticles with peritoneal targeting and application thereof

Abstract

The present invention relates to the field of biomedicine, and in particular to clodronate-loaded lipid nanoparticles and their applications. The present invention provides clodronate-loaded lipid nanoparticles comprising the following raw materials: an ionized lipid, a helper lipid, cholesterol, clodronate, and a buffer; the helper lipid comprises a phospholipid and a polyethylene glycol-functionalized lipid. These clodronate-loaded lipid nanoparticles are capable of efficiently clearing peritoneal macrophages while minimizing their effects on other tissues and organs throughout the body.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to clodronate-loaded lipid nanoparticles and applications thereof. Background Art

[0002] Macrophages play an important role in immune regulation, but in certain disease conditions, such as immunosuppression in the tumor microenvironment, autoimmune diseases, and chronic inflammatory diseases, macrophages may exhibit pro-pathogenic effects. Eliminating macrophages or specific macrophage subtypes has become an important strategy to improve disease prognosis and therapeutic efficacy.

[0003] Clodronate liposomes, a nanoparticle system containing clodronate encapsulated by a bilayer lipid membrane, are widely used in in vivo macrophage clearance studies. Currently, several commercial clodronate liposome products are used in scientific research, including Clodrosome®, Liposoma, and FormuMax.

[0004] The mechanism of action of clodronate-loaded liposomes is to encapsulate the low-lipophilic clodronate within the liposomes. When macrophages engulf the liposomes, clodronate, dissolved in the liposome aqueous phase, is released under the action of macrophage lysosomal phosphatases and metabolized into a non-hydrolyzable ATP analog (by inhibiting the mitochondrial ADP / ATP transport mechanism in the cell), thereby inducing cell apoptosis. This method is characterized by its simplicity and low cost. However, the impact of factors such as liposome particle size, surface charge, and stability on its targeting and efficacy has not been fully optimized, resulting in poor distribution and targeting, as well as systemic toxic side effects.

[0005] Lipid nanoparticles (LNPs), as an advanced drug delivery system, have been widely used to deliver a variety of drugs, including nucleic acids and proteins. However, existing technologies primarily focus on LNP delivery of nucleic acid drugs. Modulating LNP physicochemical properties (such as particle size, charge, and hydrophobic lipid ratio) can alter their in vivo distribution, enabling targeted delivery to specific organs. However, the design and preparation of LNPs with peritoneal targeting and retention capabilities are currently lacking. Summary of the Invention

[0006] In view of this, the present invention aims to provide clodronate-loaded lipid nanoparticles and their use. The clodronate-loaded lipid nanoparticles can effectively eliminate peritoneal macrophages and minimize the impact on other tissues and organs in the body.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a clodronate-loaded lipid nanoparticle, comprising the following preparation materials: ionized lipid, auxiliary lipid, cholesterol, clodronate and buffer;

[0009] The helper lipids include phospholipids and polyethylene glycol functionalized lipids.

[0010] Preferably, the clodronate-loaded lipid nanoparticles are in the form of a clodronate-loaded lipid nanoparticle suspension;

[0011] The mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is 100 μg / mL to 2 mg / mL.

[0012] Preferably, the molar ratio of the ionized lipid, phospholipid, cholesterol and polyethylene glycol functionalized lipid is (5-60): (10-50): (15-40): (0.5-5).

[0013] Preferably, the ionized lipids include one or more of SM-102, DOTAP, DLin-MC3-DMA, ALC-0315 and FTT5.

[0014] Preferably, the phospholipids include one or more of DSPC, DOPE, DOPC and DPPC.

[0015] Preferably, the polyethylene glycol functionalized lipids include one or more of DMG-PEG, DSPE-PEG, DOPE-PEG and Ceramide-PEG;

[0016] The average molecular weight of the polyethylene glycol in the polyethylene glycol functionalized lipid is 2000-5000.

[0017] Preferably, the pH value of the buffer solution is 4.0-7.0.

[0018] Preferably, the buffer comprises citrate buffer or phosphate buffer.

[0019] Preferably, the particle size of the clodronate-loaded lipid nanoparticles is 100-1000 nm, the surface potential is -30-+15 mV, and the Young's modulus is 100 KPa-1 MPa.

[0020] The present invention also provides the use of the clodronate-loaded lipid nanoparticles described in the above technical solution in the preparation of drugs for tumor treatment.

[0021] The invention provides a clodronate-loaded lipid nanoparticle, which comprises the following preparation raw materials: ionized lipid, auxiliary lipid, cholesterol, clodronate and buffer; the auxiliary lipid comprises phospholipid and polyethylene glycol functionalized lipid.

[0022] Compared with the existing technology, the technical solution of the present invention has the following beneficial effects:

[0023] The clodronate-loaded lipid nanoparticles of the present invention achieve efficient clearance of peritoneal macrophages, significantly reduce toxic side effects in non-targeted tissues, and simultaneously improve the accuracy of treatment for diseases related to local peritoneal macrophages.

[0024] The clodronate-loaded lipid nanoparticles of the present invention can not only effectively eliminate peritoneal macrophages, but also inhibit the growth of intraperitoneal tumors through combined drug administration, and are suitable for a variety of pathological models including primary tumors and metastatic peritoneal tumors.

[0025] The clodronate-loaded lipid nanoparticles of the present invention have better targeting and functionality, and have the dual effects of clearing macrophages and inhibiting tumors, providing a new solution for abdominal cavity-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the preparation process of the clodronate-loaded lipid nanoparticle suspension described in Example 1;

[0027] Figure 2 Figure 1 shows the particle size (a) and morphology analysis (b) of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension described in Example 1;

[0028] Figure 3 is the standard curve of concentration and absorbance of clodronate solution;

[0029] Figure 4 Flow cytometric analysis of the clearance of macrophages from the peritoneal cavity by the existing product Clodrosome, the control group, and the clodronate-loaded lipid nanoparticles obtained in Example 1;

[0030] Figure 5 is the particle size distribution of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspensions described in Examples 2 to 4;

[0031] Figure 6 is the polydispersity index of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspensions described in Examples 2 to 4;

[0032] Figure 7 These are atomic force microscopy images of the clodronate-loaded lipid nanoparticles described in Examples 2 to 4;

[0033] Figure 8 Graphs showing the Young's modulus of clodronate-loaded lipid nanoparticles described in Examples 2 to 4;

[0034] Figure 9 Flow cytometry analysis of the clearance of macrophages from the peritoneal cavity by the existing product Clodrosome, the control group, and the clodronate-loaded lipid nanoparticles obtained in Example 4;

[0035] Figure 10 An experimental protocol for treating a colon cancer peritoneal metastasis model by intraperitoneal injection of clodronate-loaded lipid nanoparticles obtained in Example 1;

[0036] Figure 11 This is a diagram showing the therapeutic effect of clodronate-loaded lipid nanoparticles in Example 1 in inhibiting peritoneal metastasis of colon cancer. DETAILED DESCRIPTION

[0037] The present invention provides a clodronate-loaded lipid nanoparticle, comprising the following preparation materials: ionized lipid, auxiliary lipid, cholesterol, clodronate and buffer;

[0038] The helper lipids include phospholipids and polyethylene glycol functionalized lipids.

[0039] In the present invention, the clodronate-loaded lipid nanoparticles are preferably present in the form of a clodronate-loaded lipid nanoparticle suspension. In the present invention, the mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is preferably 100 μg / mL to 2 mg / mL.

[0040] In the present invention, the molar ratio of the ionized lipid, phospholipid, cholesterol, and polyethylene glycol-functionalized lipid is preferably (5-60):(10-50):(15-40):(0.5-5), and more preferably (10-50):(10-40):(30-40):(1-4). In an embodiment of the present invention, the molar ratio of the ionized lipid, phospholipid, cholesterol, and polyethylene glycol-functionalized lipid can be 50:10:38.5:1.5.

[0041] In the present invention, the ionized lipid preferably includes one or more of SM-102, DOTAP, DLin-MC3-DMA, ALC-0315 and FTT5. When the ionized lipid is two or more of the above-mentioned specific selections, the present invention does not have any special restrictions on the proportion of the above-mentioned specific substances, and can be mixed in any ratio. In an embodiment of the present invention, the ionized lipid can be DLin-MC3-DMA, DOTAP or FTT5.

[0042] In the present invention, the ionized lipid is used to construct the core structure of the clodronate-loaded lipid nanoparticles. The ionized lipid has cationic properties and can carry clodronate.

[0043] In the present invention, the helper lipids include phospholipids and polyethylene glycol-functionalized lipids. The phospholipids preferably include one or more of DSPC, DOPE, DOPC, and DPPC. When the phospholipids include two or more of these specific selections, the present invention does not specify the ratio of these specific substances, and they can be mixed in any ratio. In the present invention, the polyethylene glycol-functionalized lipids include one or more of DMG-PEG, DSPE-PEG, DOPE-PEG, and Ceramide-PEG. When the polyethylene glycol-functionalized lipids include two or more of these specific selections, the present invention does not specify the ratio of these specific substances, and they can be mixed in any ratio. The average molecular weight of the polyethylene glycol in the polyethylene glycol-functionalized lipid is preferably 2000-5000. In an embodiment of the present invention, the phospholipid may be DSPC; the polyethylene glycol-functionalized lipid may be DMG-PEG, and the average molecular weight of the polyethylene glycol in the polyethylene glycol-functionalized lipid may be 2000.

[0044] In the present invention, the auxiliary lipids are used to construct the external membrane structure of lipid nanoparticles, wherein the role of the phospholipids is to spontaneously form a lipid bilayer structure, which can encapsulate and protect the drug (clodronate) in the LNP, prevent its degradation, and help it pass through the cell membrane. The role of the polyethylene glycol-functionalized lipid is to provide biological stability of the LNP and reduce immune clearance by forming an external hydration protective layer.

[0045] In the present invention, the cholesterol acts as a membrane stabilizer, which can enhance the structural stability of lipid nanoparticles, enhance the rigidity and fluidity of the membrane, and thus control the release rate of the drug to a certain extent.

[0046] In the present invention, the function of the clodronate is to induce apoptosis of macrophages to achieve clearance of macrophages.

[0047] In the present invention, the pH value of the buffer solution is preferably 4.0 to 7.0, more preferably 4.0 to 6.0. In an embodiment of the present invention, the pH value of the buffer solution may be 6.0. In the present invention, the buffer solution preferably comprises a citric acid buffer solution or a phosphate buffer solution. In an embodiment of the present invention, the buffer solution may be a citric acid buffer solution.

[0048] In the present invention, the buffer is used to prepare the aqueous phase of lipid nanoparticles and maintain their stability and biocompatibility.

[0049] In the present invention, the clodronate-loaded lipid nanoparticles include a core and a coating layer that wraps the core; the core is clodronate and ionized lipids; the coating layer includes a first coating layer and a second coating layer arranged in sequence from the inside to the outside; the first coating layer is formed by part of phospholipids and cholesterol; the second coating layer includes polyethylene glycol-functionalized lipids and remaining phospholipids, and mainly interacts with the cell membrane.

[0050] In the present invention, the first encapsulating layer and the second encapsulating layer are preferably connected by non-covalent interactions (such as electrostatic interactions or hydrogen bonding), thereby stabilizing the structure of the lipid nanoparticles and enhancing the interaction between the device and the cell membrane.

[0051] In the present invention, the particle size of the clodronate-loaded lipid nanoparticles is preferably 100-1000 nm, the surface potential is preferably -30-+15 mV, and the Young's modulus is preferably 100 KPa-1 MPa.

[0052] In the present invention, the method for preparing the clodronate-loaded lipid nanoparticles preferably comprises the following steps:

[0053] mixing the ionized lipid, the auxiliary lipid, cholesterol and the solvent to obtain a lipid mixture;

[0054] mixing clodronate and buffer to obtain a clodronate solution;

[0055] The lipid mixture and clodronate solution are mixed and self-assembled, and then dialyzed to obtain the clodronate-loaded lipid nanoparticles.

[0056] The present invention mixes ionized lipid, auxiliary lipid, cholesterol and solvent to obtain a lipid mixed solution.

[0057] In the present invention, the solvent is preferably anhydrous ethanol.

[0058] In the present invention, the mixing is preferably carried out under conditions of shaking or ultrasound. The present invention does not have any special limitation on the shaking or ultrasound process, and the mixing can be carried out using a process well known to those skilled in the art.

[0059] The present invention mixes clodronate and buffer to obtain a clodronate solution.

[0060] The present invention does not have any special limitation on the mixing process, and the mixing process may be carried out using a process well known to those skilled in the art.

[0061] After obtaining the lipid mixture and the clodronate solution, the present invention mixes the lipid mixture and the clodronate solution for self-assembly and then performs dialysis to obtain the clodronate-loaded lipid nanoparticles.

[0062] In the present invention, the hybrid self-assembly method is preferably a microfluidic method, an ethanol injection method, an ultrasonic method, a thin film hydration method or a solvent evaporation method. The present invention does not have any particular limitations on the process of the hybrid self-assembly, and can be carried out using a process well known to those skilled in the art. In an embodiment of the present invention, the hybrid self-assembly method can be a microfluidic method or an ethanol injection method.

[0063] In the present invention, the volume ratio of the lipid mixture to the clodronate solution is preferably 1:(1-6). In an embodiment of the present invention, the volume ratio of the lipid mixture to the clodronate solution can be 1:1 or 1:3.

[0064] In the present invention, the molecular weight cut-off of the dialysis bag used in the dialysis is preferably 5-100KD, more preferably 5-10KD. In the present invention, the buffer used in the dialysis is preferably 1× PBS solution.

[0065] The present invention also provides the use of the clodronate-loaded lipid nanoparticles described in the above technical solution in the preparation of a drug for tumor treatment. In the present invention, the drug for tumor treatment preferably includes a drug for treating primary tumors or a drug for treating metastatic peritoneal tumors. In the present invention, the metastatic peritoneal tumor inhibitor is preferably a drug for treating one or more of the following: cancers secondary to the liver, stomach, colorectum, pancreas, ovary, lung, uterus, or retroperitoneal tumors.

[0066] In the present invention, the application is preferably to eliminate macrophages to inhibit tumor growth; the lipid nanoparticles loaded with clodronate can significantly reduce the number of macrophages resident in the peritoneal cavity within a few hours after intraperitoneal injection, while having no significant effect on monocytes in peripheral tissues and blood.

[0067] In the present invention, the mechanism of action of the clodronate-loaded lipid nanoparticles is as follows: 1. The various components form a nanoparticle that encapsulates clodronate. Because clodronate is not membrane permeable, it cannot freely pass through the cell membrane; 2. Macrophages have the characteristic of phagocytosing foreign particles. After the clodronate-loaded lipid nanoparticles are injected into mice, they are recognized and phagocytosed by macrophages. In the lysosomes of the macrophages, the lipid nanoparticles are digested and destroyed, and the encapsulated clodronate is gradually released and accumulated within the cells; 3. Clodronate is metabolized intracellularly to produce non-hydrolyzable ATP analogs. This analog will irreversibly bind to the ADP / ATP translocase in the mitochondria, blocking the mitochondrial respiratory chain, leading to mitochondrial dysfunction, and ultimately triggering macrophage apoptosis; 4. Specific clearance of macrophages. The clearance effect of clodronate lipid nanoparticles is specific, mainly targeting macrophages, and is therefore non-toxic to other non-phagocytic cells and ineffective against tumor cells.

[0068] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0069] like Figure 1 Schematic diagram of the preparation process shown in the figure, preparation of the lipid mixture: according to the molar ratio of 10:38.5:1.5:50, the auxiliary lipid (DSPC, 1,2-Distearoli-sn-glycero-3-phosphocholine), cholesterol (Cholesterol), auxiliary lipid (PEG-lipid, DMG-PEG 2K) and ionized lipid (Dlin-MC3-DMA) are mixed, after adding anhydrous ethanol, using a magnetic stirrer or ultrasonic oscillator to stir or sonicate until completely transparent without precipitation, to obtain a lipid mixture with an ionized lipid concentration of 2 mg / mL;

[0070] Thoroughly mix clodronate and pH 6.0 citrate buffer to obtain a clodronate solution with a clodronate concentration of 1 mg / mL;

[0071] The lipid mixture and clodronate solution were rapidly mixed in a cross-flow manner using a microfluidic device at a volume ratio of 1:1 (the mixing speed ratio of the lipid mixture and the clodronate solution was 1:1, and the total flow rate was 400 μL / min). w =5-10KD dialysis tubing for 4-6 hours to remove ethanol and unencapsulated components, thereby obtaining the clodronate-loaded lipid nanoparticle (Clodro-LNP) suspension (the mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is 158.7 μg / mL; the obtained clodronate-loaded lipid nanoparticle suspension can be stored at 4° C. for a short period of time);

[0072] The particle size of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension was measured using a Malvern Zetasizer dynamic light scattering device equipped with a zeta potential measurement module, with measurement parameters set including a temperature of 25° C., a medium refractive index, and a dielectric constant; and the morphology of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension was analyzed using a transmission electron microscope (TEM); wherein Figure 2 The particle size (a) and morphology analysis diagram (b) of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension are shown in FIG. Figure 2It can be seen that the average particle size of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension is 558.8 nm, the distribution coefficient PDI=0.315; the zeta potential is -0.043 mV; the TEM image shows that the particle size of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension is about 600.0 nm, which is consistent with the particle size result measured by DLS;

[0073] The mass concentration of clodronate (not dialyzed) in the clodronate-loaded lipid nanoparticle suspension was determined by separating free clodronate and LNP using an ultrafiltration centrifuge tube (MWCO 10 kDa). 500 μL of the clodronate-loaded lipid nanoparticle suspension was transferred to the ultrafiltration centrifuge tube. The tube was centrifuged at 2000 × g for 20 min according to the instructions of the ultrafiltration centrifuge tube. The filtrate (containing free clodronate) was collected, and the LNP (containing encapsulated clodronate) was retained in the filter tube. The ultraviolet absorbance of the filtrate at 240 nm was measured to obtain the free clodronate concentration (C 游离 ); Another 500 μL of the clodronate-loaded lipid nanoparticle suspension (not dialyzed) was taken and a 10 g / L Triton X-100 solution was added to destroy the structure of the clodronate-loaded lipid nanoparticles, releasing all loaded and free clodronate, and measuring its ultraviolet absorption at 240 nm. Figure 3 The total concentration of clodronate in the clodronate-loaded lipid nanoparticles (C 总 ); Finally, the encapsulation efficiency was calculated (the encapsulation efficiency was calculated as follows: encapsulation efficiency (EE,%) = (C 总 -C 游离 ) / C 总 ×100%;

[0074] Among them, the encapsulation efficiency of Example 1 is 37.5%.

[0075] The ability of clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension described in Example 1 to eliminate macrophages in mice:

[0076] Healthy Babl / c mice of either sex, aged 6-8 weeks and weighing 18-22 g were selected and randomly divided into the following experimental groups, with 3 mice in each group: clodronate lipid nanoparticle group: intraperitoneal injection of clodronate lipid nanoparticles; clodronate liposome group: intraperitoneal injection of clodronate liposomes (current product Clodrosome); control group: intraperitoneal injection of 200 μL PBS buffer;

[0077] The injection dose of clodronate liposomes is 2 mg / kg body weight (calculated based on the clodronate content, 100-500 μL / mouse, the specific volume is calculated based on the mouse weight), and the injection method is intraperitoneal injection (IP injection). The injection procedure must be aseptic to avoid damage to the animal's internal organs.

[0078] Twenty-four hours after injection, mice were anesthetized, and peripheral blood was collected from the orbital venous plexus. 5 mL of sterile PBS buffer was injected into the peritoneal cavity. After 30 seconds of massage, the lavage fluid was recovered and collected. The peripheral blood and peritoneal lavage fluid were transferred to sterile centrifuge tubes and centrifuged at 4°C and 300 × g for 5 minutes. The supernatant was discarded and the cell pellet was collected.

[0079] Resuspend the cell pellet in 100 μL of PBS buffer, add Fc receptor blocker and incubate for 10 min, add F4 / 80-FITC and CD11b-APC antibody mixture, incubate at 4°C in the dark for 30 min, wash twice with PBS buffer, discard the supernatant, and resuspend with 300 μL of PBS buffer to obtain the stained cell suspension;

[0080] The stained cell suspension was added to a flow cytometer sample tube, 100,000 cells were collected by flow cytometry, and the expression of F4 / 80 and CD11b double-positive macrophages and CD14 + and CD11b + the proportion of double-positive monocytes;

[0081] FlowJo software was used to analyze the data and calculate the relative proportion of macrophages. Figure 4 Figure 1 is a flow cytometry chart showing the clearance of macrophages from the peritoneal cavity by the existing product Clodrosome, the control group, and the clodronate-loaded lipid nanoparticles obtained in Example 1. Figure 4 It can be seen that the proportion of macrophages in the peritoneal lavage fluid was significantly reduced, F4 / 80 + CD11b + The proportion of double-positive cells decreased to 13.4%, indicating that Clodro-LNP can effectively remove macrophages in the peritoneal cavity; the detection of peripheral blood samples showed that the CD14 + CD11b + There was no significant difference in the proportion of monocytes, indicating that Clodro-LNP has good targeted clearance of local peritoneal macrophages without affecting the level of monocytes in peripheral blood. Furthermore, after intraperitoneal injection of Clodro-LNP, the clearance rate of peritoneal macrophages in mice reached 83.9%, while the number of peripheral blood monocytes remained within the normal range (no statistically significant difference compared with the control group, P>0.05).

[0082] Preparation of lipid mixture: Helper lipid (DSPC, 1,2-Distearoli-sn-glycero-3-phosphocholine), cholesterol (Cholesterol), helper lipid (PEG-lipid, DMG-PEG 2K), and ionized lipid (Dlin-MC3-DMA) were mixed at a molar ratio of 10:38.5:1.5:50. After adding anhydrous ethanol, the mixture was stirred or sonicated using a magnetic stirrer or ultrasonic oscillator until completely transparent and free of precipitation, resulting in a lipid mixture with an ionized lipid concentration of 2 mg / mL.

[0083] Thoroughly mix clodronate and pH 6.0 citrate buffer to obtain a clodronate solution with a clodronate concentration of 1 mg / mL;

[0084] Under magnetic stirring, the lipid mixture was injected dropwise into the clodronate aqueous solution through a microsyringe (the volume ratio of the lipid mixture to the clodronate solution was 1:3). During the injection process, the lipid molecules quickly self-assembled in the aqueous phase to form lipid nanoparticles and encapsulated the clodronate. Then, 1x PBS buffer and M w =5-10KD dialysis tubing for 4-6 hours to remove ethanol and unencapsulated components to obtain the clodronate-loaded lipid nanoparticle suspension (the mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is 136.9 μg / mL; MC3-LNP). Example

[0085] Refer to Example 2, except that the ionized lipid is DOTAP, and the clodronate-loaded lipid nanoparticle suspension is obtained (the mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is 202.7 μg / mL; DOTAP-LNP). Example

[0086] Refer to Example 2, except that the ionized lipid is FTT5, and the clodronate-loaded lipid nanoparticle suspension is obtained (the mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is 107.6 μg / mL; FTT5-LNP).

[0087] Dynamic light scattering (DLS) was used to detect the particle size and polydispersity index (PDI) of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspensions described in Examples 2 to 4, wherein: Figure 5 is the particle size distribution of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspensions described in Examples 2 to 4, Figure 6is the polydispersity index of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspensions described in Examples 2 to 4; Figures 5 and 6 It can be seen that the particle size of the clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension is 130-170 nm, and the distribution coefficient PDI is 0.169-0.231;

[0088] The clodronate-loaded lipid nanoparticle suspension described in Examples 2 to 4 was added dropwise to the surface of a mica sheet, allowed to stand for adsorption, then rinsed with distilled water and dried. The Young's modulus was measured at room temperature using an atomic force microscope (AFM) force curve mode. A silicon probe with an elastic modulus of 10-20 N / m and a short radius of approximately 10 nm was used. The probe was gradually pressed into the surface of the clodronate-loaded lipid nanoparticle, and the force-displacement curve was recorded. The Young's modulus of the clodronate-loaded lipid nanoparticle was calculated based on the Hertz model fitting data. Figure 7 This is an atomic force microscope characterization image of the clodronate-loaded lipid nanoparticles described in Examples 2 to 4. Figure 8 is a Young's modulus diagram of the clodronate-loaded lipid nanoparticles described in Examples 2 to 4, Figures 7 and 8 It can be seen that the average Young's moduli of the clodronate-loaded lipid nanoparticles described in Examples 2 to 4 are 144 KPa, 306 KPa, and 580 KPa, respectively, indicating that lipid nanoparticles with different hardness can be prepared by adjusting the composition of the ionized lipid.

[0089] The ability of clodronate-loaded lipid nanoparticles in the clodronate-loaded lipid nanoparticle suspension described in Example 4 to eliminate macrophages in mice:

[0090] Healthy Babl / c mice of either sex, aged 6-8 weeks and weighing 18-22 g were selected and randomly divided into the following experimental groups, with 3 mice in each group: clodronate lipid nanoparticle group: intraperitoneal injection of clodronate lipid nanoparticles; clodronate liposome group: intraperitoneal injection of clodronate liposomes (current product Clodrosome); control group: intraperitoneal injection of 200 μL PBS buffer;

[0091] The injection dose of clodronate liposomes is 2 mg / kg body weight (calculated based on the clodronate content, 100-500 μL / mouse, the specific volume is calculated based on the mouse weight), and the injection method is intraperitoneal injection (IP injection). The injection procedure must be aseptic to avoid damage to the animal's internal organs.

[0092] Twenty-four hours after injection, mice were anesthetized and 5 mL of sterile PBS buffer was injected into the peritoneal cavity. The lavage fluid was recovered after massage for 30 seconds and the peritoneal lavage fluid was collected. Liver tissue of appropriate size was cut and digested into a single-cell suspension. The peritoneal lavage fluid and liver cell suspension were transferred to a sterile centrifuge tube and centrifuged at 4°C and 300 × g for 5 minutes. The supernatant was discarded and the cell pellet was collected.

[0093] Resuspend the cell pellet in 100 μL of PBS buffer, add Fc receptor blocker and incubate for 10 min, add F4 / 80-FITC and CD11b-APC antibody mixture, incubate at 4°C in the dark for 30 min, wash twice with PBS buffer, discard the supernatant, and resuspend with 300 μL of PBS buffer to obtain the stained cell suspension;

[0094] The stained cell suspension was added to a flow cytometer sample tube, 100,000 cells were collected by flow cytometry, and the proportion of F4 / 80 and CD11b double-positive macrophages was analyzed;

[0095] Calculate F4 / 80 using FlowJo + CD11b + ratio, to evaluate the macrophage clearance effect, among which Figure 9 The lipid nanoparticles loaded with clodronate (corresponding to the existing product Clodrosome, the control group and Example 4) Figure 9 Flow cytometry analysis of ClodroLNP (FTT5) in the peritoneal cavity to clear macrophages. Figure 9 It can be seen that in the changes of peritoneal macrophage content, the F4 / 80 of the lipid nanoparticle group loaded with clodronate described in Example 4 + CD11b + The number of cells in the PBS group decreased by about 39.6% (P < 0.001); there was no significant change in the content of liver macrophages, indicating that the high-hardness lipid nanoparticles loaded with clodronate achieved efficient clearance of peritoneal macrophages without affecting the changes in the macrophage content in other tissues.

[0096] BALB / c mice were used to establish a colon cancer peritoneal metastasis model. Tumor cell suspension (CT26 cells, 5×10 5 Cells / mouse) were inoculated into mice via intraperitoneal injection. After tumor formation, the mice were randomly divided into two groups and received the clodronate-loaded lipid nanoparticles described in Example 1 (experimental group) or PBS (control group). Each group of mice received treatment via intraperitoneal injection at a predetermined dose and frequency (experimental design as shown in the following example). Figure 10 After treatment, the number and size of peritoneal metastases in mice were recorded by dissection. Figure 11This is a diagram showing the therapeutic effect of clodronate-loaded lipid nanoparticles in inhibiting peritoneal metastasis of colon cancer as described in Example 1. Figure 11 It can be seen that the weight of peritoneal metastases in the experimental group of mice was significantly lower than that in the control group (P<0.05), indicating that the clodronate-loaded lipid nanoparticles have a significant inhibitory effect on the formation of peritoneal metastases.

[0097] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation thereto. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications shall also be considered within the scope of protection of the present invention.

Claims

1. A clodronate-loaded lipid nanoparticle with peritoneal targeting, characterized in that: The preparation comprises the following raw materials: ionized lipid, auxiliary lipid, cholesterol, clodronate and buffer; The helper lipids include phospholipids and polyethylene glycol functionalized lipids; The molar ratio of the ionized lipid, phospholipid, cholesterol and polyethylene glycol functionalized lipid is (50-60): (10-40): (15-40): (1.5-4); The particle size of the clodronate-loaded lipid nanoparticles is 100-600 nm, the surface potential is -30-+15 mV, and the Young's modulus is 100 KPa-1 MPa.

2. The clodronate-loaded lipid nanoparticles according to claim 1, wherein The clodronate-loaded lipid nanoparticles are present in the form of a clodronate-loaded lipid nanoparticle suspension; The mass concentration of clodronate in the clodronate-loaded lipid nanoparticle suspension is 100 μg / mL to 2 mg / mL.

3. The clodronate-loaded lipid nanoparticles according to claim 1, wherein The ionized lipids include one or more of SM-102, DOTAP, DLin-MC3-DMA, ALC-0315 and FTT5.

4. The clodronate-loaded lipid nanoparticles according to claim 1, wherein The phospholipids include one or more of DSPC, DOPE, DOPC and DPPC.

5. The clodronate-loaded lipid nanoparticles according to claim 1, wherein The polyethylene glycol functionalized lipids include one or more of DMG-PEG, DSPE-PEG, DOPE-PEG and Ceramide-PEG; The average molecular weight of the polyethylene glycol in the polyethylene glycol functionalized lipid is 2000-5000.

6. The clodronate-loaded lipid nanoparticles according to claim 1, wherein The pH value of the buffer solution is 4.0-7.

0.

7. The clodronate-loaded lipid nanoparticles according to claim 6, wherein The buffer includes citrate buffer or phosphate buffer.

8. Use of the peritoneal cavity-targeted clodronate-loaded lipid nanoparticles according to any one of claims 1 to 7 in the preparation of a drug for treating peritoneal metastasis.

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