A bone-targeted lipid nanocarrier and its preparation method

Abstract

The present invention belongs to the field of nanopharmaceutical technology and discloses a bone-targeted lipid nanocarrier and its preparation method. The preparation method comprises: dissolving cardiolipin, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol-amino, and an analgesic drug in anhydrous ethanol to obtain a phospholipid ethanol solution; rapidly mixing the phospholipid ethanol solution with pure water, incubating at room temperature for 10 minutes, and then dialyzing for 3 hours to obtain a solid lipid nanoparticle solution loaded with the analgesic drug. The lipid nanocarrier prepared by the preparation method of the present invention comprises a monolayer of phospholipids forming a lipophilic shell that can encapsulate a hydrophobic drug; the lipid nanocarrier can prolong the drug's blood circulation time in the body and improve the drug's bioavailability.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanopharmaceuticals, and in particular relates to a bone-targeted lipid nanocarrier and a preparation method thereof. Background Art

[0002] Malignant bone tumors recur frequently and are often difficult to eradicate. Surgery can prolong the lifespan of bone tumor patients, but small lesions can lead to tumor recurrence. Therefore, combined chemotherapy after bone tumor surgery has become a common clinical approach. The avascular nature of the skeletal system significantly limits the efficacy of clinical drugs. Due to the lack of bone tumor targeting, drugs can have severe side effects and are more susceptible to drug resistance. The bone microenvironment also provides a barrier to chemotherapy for malignant bone tumors, making it difficult for tumor-targeting molecules to further target bone tumor cells. To overcome the limitations of direct drug delivery, combining chemotherapeutic drugs with nanosystems is considered an effective cancer treatment strategy. Targeted nanocarriers, after entering the bloodstream, preferentially accumulate in the bone, the tumor lesion, through active targeting. This results in a longer half-life, greater efficacy, and specific binding properties. Their small size facilitates flexible targeting and the ability to reach their target without inducing an immune response. Nanocarriers have become commonplace in the treatment of bone-related diseases.

[0003] In recent years, a variety of bone-targeted nanosystems that can bind to proteins, RNA, and drugs have been developed, such as solid lipid nanoparticles, polymer micelles, magnetic nanoparticles, gold nanoparticles, liposomes, and hydrogels. Among them, solid lipid nanoparticles have better sustained-release properties, higher drug bioavailability, and long-term stability in vivo. Summary of the Invention

[0004] The present invention aims to provide a bone-targeted lipid nanocarrier, its preparation method, and application. The prepared lipid nanocarrier, composed of a single layer of phospholipids forming a lipophilic shell, can encapsulate hydrophilic and hydrophobic drugs, thereby prolonging the drug's blood circulation time in the body and improving its bioavailability.

[0005] To solve the above problems, the technical solutions provided by the present invention are as follows:

[0006] A method for preparing a bone-targeted lipid nanocarrier, wherein the bone-targeted lipid nanocarrier is a solid lipid nanoparticle solution having a skeletal system-targeting effect, comprising the following steps:

[0007] 1) dissolving three phospholipids and an analgesic drug in anhydrous ethanol and mixing them uniformly to obtain a phospholipid ethanol solution; the three phospholipids include cardiolipin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol-amino in a molar ratio of 69:40:4;

[0008] 2) The phospholipid ethanol solution is quickly mixed with pure water, incubated at room temperature for 8-12 minutes, and then transferred to a dialysis bag. The phospholipid ethanol solution is dialyzed against pure water under stirring for 2-4 hours to remove unencapsulated analgesic drugs, thereby obtaining a solid lipid nanoparticle solution loaded with analgesic drugs.

[0009] Furthermore, in step 1), the molar ratio of central phospholipid, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol-amino is 69:40:4.

[0010] Furthermore, the mass ratio of the analgesic drug to the total amount of the three phospholipids in step 1) is 1:4.

[0011] Furthermore, the analgesic drug is ropivacaine.

[0012] Furthermore, in step 2), the volume ratio of the phospholipid ethanol solution to the pure water is 1:3.

[0013] Furthermore, the molecular weight cut-off of the dialysis bag in step 2) is 3500.

[0014] The present invention also provides a lipid nanocarrier loaded with analgesic drugs prepared by the above preparation method.

[0015] Furthermore, the hydrated particle size of the anticancer drug-loaded lipid nanocarrier is 170-200 nm.

[0016] The present invention also provides a use of the above-mentioned bone-targeted lipid nanocarrier in the preparation of a drug targeting malignant bone tumors.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The cardiolipin in the lipid nanoparticles provided by the present invention is a diphosphatidylglycerol, the presence of which enables the lipid nanoparticles to have a bone-targeting effect.

[0019] (2) The lipid nanocarriers of the present invention can prevent inter-particle aggregation, reduce the binding of particles to plasma proteins in the body, prolong the blood circulation time of the drug, and improve the bioavailability of the drug in the lipid nanocarriers.

[0020] (3) The lipid nanocarriers of the present invention can target and inhibit the pain of malignant bone tumors.

[0021] (4) The lipid nanocarriers of the present invention have higher biocompatibility than other nanocarriers, can control drug release and targeting, are biodegradable, and have low immunogenicity. In addition, they are simple and convenient to prepare and can be rapidly produced on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1is the hydrated particle size of the bone-targeted lipid nanoparticles of Example 1;

[0023] Figure 2 This is the potential diagram of the bone-targeted lipid nanoparticles in Example 1;

[0024] Figure 3 This is a transmission electron microscopy image of the bone-targeted lipid nanoparticles of Example 1;

[0025] Figure 4 This is the in vitro release curve of the bone-targeted lipid nanoparticles of Example 2;

[0026] Figure 5 The binding strength between lipid nanoparticles and hydroxyapatite after the lipid nanoparticle solutions with three different phospholipid components loaded with the fluorescent dye Rhodamine B at the same concentration were stirred with the same mass of hydroxyapatite for 24 hours; the three phospholipids are cardiolipin (A), 1,2-dioleoylglycerol-3-phosphoethanolamine (B) and soybean lecithin (C). DETAILED DESCRIPTION

[0027] In order to further understand the present invention, preferred embodiments of the present invention are described in detail below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than for limiting the claims of the present invention.

[0028] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0029] Example 1

[0030] Ropivacaine-loaded bone-targeted solid lipid nanoparticles

[0031] 10.31 mg of cardiolipin, 1.15 mg of cholesterol, 1.2 mg of distearoylphosphatidylethanolamine-polyethylene glycol-amino and 3.27 mg of ropivacaine were dissolved in an ethanol solution and then mixed evenly to obtain a phospholipid ethanol solution, which was then dialyzed for 3 hours to obtain a bone-targeted lipid nanoparticle solution. The hydrated particle size obtained was tested, and the results were as follows: Figure 1 As shown in FIG, the hydrated particle size of the lipid nanoparticles is between 180 and 200 nm. Figure 2 As shown in , the potential of the lipid nanoparticles is negative. Figure 3 As shown, the lipid nanoparticles are nearly spherical in appearance and evenly distributed.

[0032] Example 2

[0033] Take 1mL of ropivacaine-loaded bone-targeted solid lipid nanoparticle solution and seal it in a 1.5mL centrifuge tube. After it is evenly dispersed, place it in a horizontal shaker at 25°C and 170rpm. Then, draw 100μL of nanocarrier solution at 0h, 0.05h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 12h, and 24h, and add the same volume of phosphate buffer (PBS, pH=7.4) to ensure that the volume of the entire system remains unchanged. The 100μL nanocarrier solution taken out is centrifuged at 10000rpm for 10min. Then, take the same volume of supernatant from each sample into a clean EP tube, add 2mL of methanol, and ultrasonically treat for 5min to fully mix the free drug. Filter the solution with a 0.45μm organic filter head into a sample bottle. Use HPLC to measure the ultraviolet response value of ropivacaine in the solution, and calculate the cumulative release of ropivacaine according to the standard curve. Each sample is measured three times. Figure 4 As shown, the lipid nanocarrier can effectively prolong the release time of the drug in vitro.

[0034] Example 3

[0035] Bone-targeted solid lipid nanoparticles loaded with fluorescent dye Rhodamine B

[0036] 10.31 mg of cardiolipin, 1.15 mg of cholesterol, 1.2 mg of distearoylphosphatidylethanolamine-polyethylene glycol-amino and 3.27 mg of rhodamine B were dissolved in an ethanol solution, and then mixed evenly to obtain a phospholipid ethanol solution, which was then dialyzed for 3 hours to obtain a bone-targeted lipid nanoparticle solution, which was pink-purple in color.

[0037] Example 4

[0038] Solid lipid nanoparticles loaded with fluorescent dye Rhodamine B

[0039] 8.5 mg of 1,2-dioleoylglycerol-3-phosphoethanolamine, 2.6 mg of cholesterol, 1.9 mg of distearoylphosphatidylethanolamine-polyethylene glycol-amino and 3.27 mg of rhodamine B were dissolved in an ethanol solution and then mixed evenly to obtain a phospholipid ethanol solution, which was then dialyzed for 3 hours to obtain a lipid nanoparticle solution, which was pink-purple in color.

[0040] Example 5

[0041] Solid lipid nanoparticles loaded with fluorescent dye Rhodamine B

[0042] 8.5 mg of soybean lecithin, 2.5 mg of cholesterol, 1.9 mg of distearoyl phosphatidylethanolamine-polyethylene glycol-amino and 3.27 mg of rhodamine B were dissolved in an ethanol solution, and then mixed evenly to obtain a phospholipid ethanol solution, which was then dialyzed for 3 hours to obtain a lipid nanoparticle solution, which was pink-purple in color.

[0043] Example 6

[0044] Take 200ul of the lipid nanoparticle solutions obtained in Examples 3, 4 and 5, dilute them with an appropriate amount of water to control the concentration of rhodamine B in the solutions to be the same, then add 50mg of hydroxyapatite to a 1.5ml EP tube and stir at room temperature for 24 hours. After standing, observe the binding strength between the lipid nanoparticles and hydroxyapatite. The results are as follows Figure 5 As shown, EP tube A contains lipid nanoparticles prepared in Example 3, with distinct pink-purple and white precipitates visible at the bottom of the tube. The white precipitate is hydroxyapatite, which is insoluble in water, and the pink-purple precipitate is the combination of rhodamine B-loaded lipid nanoparticles and hydroxyapatite, resulting in a co-precipitation phenomenon. The supernatant is lighter in pink-purple than the other two groups. EP tube B contains lipid nanoparticles prepared in Example 4, with a white precipitate visible at the bottom of the tube. EP tube C contains lipid nanoparticles prepared in Example 5, with a white precipitate visible at the bottom of the tube. The above three cases verify that solid lipid nanoparticles prepared using cardiolipin have bone targeting properties.

[0045] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention. Although embodiments of the present invention have been shown and described, it is understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a bone-targeted lipid nanocarrier, characterized in that: The bone-targeted lipid nanocarrier is a lipid nanoparticle solution with a skeletal system-targeting effect, and the preparation method comprises the following steps: 1) dissolving three phospholipids and an analgesic drug in anhydrous ethanol and mixing them uniformly to obtain a phospholipid ethanol solution; the three phospholipids include cardiolipin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol-amino in a molar ratio of 69:40:4; and the analgesic drug is ropivacaine; 2) The phospholipid ethanol solution is quickly mixed with pure water, incubated at room temperature for 8-12 minutes, and then transferred to a dialysis bag. The phospholipid ethanol solution is dialyzed against pure water under stirring for 2-4 hours to remove unencapsulated analgesic drugs, thereby obtaining a solid lipid nanoparticle solution loaded with analgesic drugs.

2. The preparation method according to claim 1, characterized in that The mass ratio of the analgesic drug in step 1) to the total amount of the three phospholipids is 1:

4.

3. The preparation method according to claim 1, characterized in that In step 2), the volume ratio of the phospholipid ethanol solution to the pure water is 1:

3.

4. The preparation method according to claim 1, characterized in that The molecular weight cut-off of the dialysis bag in step 2) is 3500.

5. The bone-targeted lipid nanocarrier prepared according to the preparation method according to any one of claims 1 to 4.

6. The bone-targeted lipid nanocarrier according to claim 5, characterized in that The hydrated particle size of the bone-targeted lipid nanocarrier is 170-200 nm.

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