CN114848594B

CN114848594B - Lipid nano-carrier loaded with anticancer drugs and preparation method and application thereof

Info

Publication number: CN114848594B
Application number: CN202210515012.2A
Authority: CN
Inventors: 陈忠平; 黄旭; 孙佳佳; 刘雪蒙; 翁凌燕; 朱俐
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-04-25
Anticipated expiration: 2042-05-11
Also published as: CN114848594A

Abstract

The invention belongs to the technical field of nano-medicament, and discloses a lipid nano-carrier loaded with an anticancer drug, and a preparation method and application thereof. The preparation method comprises the following steps: dissolving 1, 2-dioleoyl glycerol-3-phosphoethanolamine, cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol and an anticancer drug in absolute ethanol, and adding a solution containing fourth phospholipid to obtain phospholipid ethanol solution; and (3) rapidly mixing the phospholipid ethanol solution with pure water, incubating for 10min at room temperature, and dialyzing for 3h to obtain the solid lipid nanoparticle solution loaded with the anticancer drug. The lipid nano-carrier prepared by the preparation method of the invention is formed by a single-layer phospholipid into a lipophilic shell, and can wrap a hydrophobic drug; the potential changes with the fourth phospholipid mole percentage; can prolong the blood circulation time of the medicine in vivo and improve the bioavailability of the medicine; liver tissue can be targeted in vivo. Can be used for treating related liver diseases.

Description

Lipid nano-carrier loaded with anticancer drugs and preparation method and application thereof

Technical Field

The invention relates to the technical field of nano-medicament, in particular to a lipid nano-carrier loaded with an anticancer drug, and a preparation method and application thereof.

Background

It is counted that the incidence rate of neurodegenerative diseases, cancers, cardiovascular diseases and other related diseases is greatly increased in recent years, and cancers become the second leading cause of death worldwide, which constitutes a great threat to human life. To date, many methods of cancer treatment exist, and conventional clinical treatments include surgical excision, drug treatment, radiation treatment, etc., and the use of chemical drugs to intervene or treat neoplastic disease is one of the more common means. Single drug therapeutic index is very narrow, requiring multiple administrations, often resulting in intolerance of the organ to the drug, and thus people focus on developing nanocarrier formulations. The nano carrier is a nano-scale delivery system, has extremely wide application, and plays a key role in the nano medical fields of multi-mode imaging, drug delivery, targeted therapy and the like of human diseases. Compared with free drug molecules, the nano-carrier can improve the solubility of various drugs, reduce the toxic and side effects of the drugs, improve the treatment effect, reduce the administration frequency and the like. According to the difference of nano-carrier materials, the nano-carrier materials can be divided into polymer nano-carriers, lipid nano-carriers, metals and inorganic nano-carriers. These different types of nanocarriers have been widely used for research on drug delivery, wherein lipid nanocarriers are novel drug delivery systems using biocompatible lipids as materials.

Common lipid nanocarriers for drug delivery can be divided into: liposomes (Liposomes), nonionic surfactant Liposomes (niosomes), solid Lipid Nanoparticles (SLNs), and Nanostructured Lipid Carriers (NLCs). The mechanism of action of coronavirus infectious disease vaccine developed in 2019 has been reported to be treated by using solid lipid nanoparticles to transfer mRNA to diseased tissues. Among the different lipid delivery systems, solid lipid nanoparticles made of solid lipids at room temperature and body temperature are one of the materials that have been studied earlier and are also the most representative lipid nanosystems. SLNs are spherical particles with particle sizes between 50-1000nm, and encapsulation of anticancer drugs in SLNs has also been studied intensively. In addition, SLNs have better release characteristics, higher drug bioavailability and long-term stability in vivo compared to nanoemulsions, liposomes and inorganic nanoparticles.

Disclosure of Invention

The invention aims to provide a lipid nano-carrier loaded with an anticancer drug, and a preparation method and application thereof. The prepared lipid nano-carrier is formed by a single-layer phospholipid into a lipophilic shell, and can wrap a hydrophobic drug; the potential changes with the fourth phospholipid mole percentage; can prolong the blood circulation time of the medicine in vivo and improve the bioavailability of the medicine; liver tissue can be targeted in vivo. Can be used for treating related liver diseases.

In order to solve the problems, the invention provides a preparation method of a lipid nano-carrier loaded with an anticancer drug, which comprises the following steps:

1) Dissolving 1, 2-dioleoyl glycerol-3-phosphoethanolamine, cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol and an anticancer drug in absolute ethanol, adding a solution containing fourth phospholipid, and uniformly mixing to obtain phospholipid ethanol solution; the solution containing the fourth phospholipid is 1, 2-dioleoyl-sn-glycero-3-phosphate dissolved by tetrahydrofuran or 1, 2-dioleoyl-3- (dimethylamino) propane dissolved by absolute ethyl alcohol;

2) And (3) rapidly mixing the phospholipid ethanol solution with pure water, incubating at room temperature for 10min, transferring to a dialysis bag, and dialyzing with pure water for 3h under stirring to remove the non-coated anticancer drug, thereby obtaining solid lipid nanoparticle solution loaded with the anticancer drug, namely the lipid nano carrier loaded with the anticancer drug.

Further, in the step 1), the molar ratio of the 1, 2-dioleoyl glycerol-3-phosphate ethanolamine to cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol, and fourth phospholipid is 69:40:4: x, X is 0, 12.6 or 48.4.

Further, the mass ratio of the anticancer drug to the total amount of four phospholipids in the step 1) is 1:4, the four phospholipids are 1, 2-dioleoyl glycerol-3-phosphoethanolamine, cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol and a fourth phospholipid;

further, the anticancer drug is doxorubicin or docetaxel.

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

Further, the dialysis bag in step 2) has a molecular weight cut-off of 14000.

The invention also provides the lipid nano-carrier loaded with the anticancer drug, which is prepared by the preparation method.

Further, the hydration particle size of the lipid nano-carrier loaded with the anticancer drugs is 120-170nm.

The invention also provides application of the lipid nano-carrier loaded with the anticancer drug in preparation of the targeted liver anticancer drug.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the lipid nano-carrier, the type and the mole ratio of the three lipids in the fixed component are changed, and the charge condition and the charge size of the surface charge of particles can be changed by changing the type and the mole ratio of the fourth phospholipid, so that the passive targeting tissue capability of the lipid nano-carrier is improved.

2. The lipid nano-carrier can prevent aggregation among particles, reduce the combination of the particles and plasma proteins in vivo, prolong the blood circulation time of the medicine and improve the bioavailability of the medicine in the lipid nano-carrier.

3. The lipid nano-carrier can target the liver, so that the medicine is accumulated in the liver more.

4. The lipid nano-carrier provided by the invention has higher biocompatibility than other nano-carriers, can control drug release and targeting, and has biodegradability and low immunogenicity. In addition, the preparation is simple and convenient, the surface is easy to modify, and the preparation method can be used for rapid mass production.

Drawings

FIG. 1 is an empty neutral solid lipid nanoparticle (0% SLNs) provided in example 1-1, an empty 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle (10% DOPA/DODAP SLNs) provided in example 1-2, an empty 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle (30% DOPA/DODAP SLNs) provided in example 1-3 neutral solid lipid nanoparticles loaded with anticancer drugs (0% SLNs-DOX, 0% SLNs-DTX) provided in example 2-1 and example 3-1, 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drugs (10% DOPA/DODAP SLNs-DOX), 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drugs provided in example 2-2 and example 3-2 10% DOPA/DODAP SLNs-DTX), 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drug provided in examples 2-3 and 3-3 (30% DOPA/DODAP SLNs-DOX, 30% DOPA/DODAP SLNs-DTX), 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles empty provided in comparative example 1-1 (50% DOPA/DODAP SLNs), 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drug provided in comparative examples 1-2 and comparative example 1-3 (50% DOPA/DODAP SLNs-DOX), 50% DOPA/DODAP SLNs-DTX); in fig. 1, a graph is the hydrated particle size of the empty SLNs and the loaded anticancer drug measured after changing the mole percentage of 1, 2-dioleoyl-sn-glycero-3-phosphate (DOPA), and B graph is the hydrated particle size of the empty SLNs and the loaded anticancer drug measured after changing the mole percentage of 1, 2-dioleoyl-3- (dimethylamino) propane (DODAP).

FIG. 2 is a plot of the empty neutral solid lipid nanoparticles provided in example 1-1 (0% SLNs), the empty 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles provided in example 1-2 (10% DOPA/DODAP SLNs), the empty 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles provided in example 1-3 (30% DOPA/DODAP SLNs), the anticancer drug loaded neutral solid lipid nanoparticles provided in example 2-1 and example 3-1 (0% SLNs-DOX, 0% SLNs-DTX), the anticancer drug loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles provided in example 2-2 and example 3-2 (10% DOPA/DODAP SLNs-DOX) 10% DOPA/DODAP SLNs-DTX), 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drug provided in examples 2-3 and 3-3 (30% DOPA/DODAP SLNs-DOX, 30% DOPA/DODAP SLNs-DTX), 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles empty provided in comparative example 1-1 (50% DOPA/DODAP SLNs), 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drug provided in comparative examples 1-2 and comparative example 1-3 (50% DOPA/DODAP SLNs-DOX), 50% DOPA/DODAP SLNs-DTX); in fig. 1, a graph shows potential values of empty SLNs and loaded anticancer drug measured after changing mole percentage of 1, 2-dioleoyl-sn-glycero-3-phosphate (DOPA), and B graph shows potential values of empty SLNs and loaded anticancer drug measured after changing mole percentage of 1, 2-dioleoyl-3- (dimethylamino) propane (DODAP).

FIG. 3 is a statistical plot of anticancer drug loaded neutral solid lipid nanoparticles (0% SLNs-DOX, 0% SLNs-DTX), 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (10% DOPA/DODAP SLNs-DOX, 10% DOPA/DODAP SLNs-DTX), 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (30% DOPA/DODAP SLNs-DOX, 30% DOPA/DODAP SLNs-DTX) provided by examples 2-1 and 3, 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drug (50% DOPA/DODAP SLNs-DOX, 50% DODAP SLDTX) provided by examples 1-2 and 1-3; in fig. 3, graph a shows the encapsulation efficiency of SLNs loaded with different mole percentages of Doxorubicin (DOX), and graph B shows the encapsulation efficiency of SLNs loaded with different mole percentages of Docetaxel (DTX).

FIG. 4 is a transmission electron microscopy image of empty neutral solid lipid nanoparticles (0% SLNs) provided in example 1-1.

FIG. 5 is a docetaxel release profile under normoxic conditions for docetaxel, docetaxel-loaded neutral solid lipid nanoparticle (0% SLNs-DTX) provided in example 3-1, and docetaxel-loaded 50% anionic and cationic solid lipid nanoparticle (50% DOPA/DODAP SLNs-DTX) provided in comparative example 1-3;

FIG. 6 shows the distribution results of docetaxel-loaded neutral solid lipid nanoparticles (0% SLNs-DTX) provided in example 3-1, docetaxel-loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (10% DOPA/DODAP SLNs-DTX) provided in example 3-2, docetaxel-loaded 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (30% DOPA/DODAP SLNs-DTX) provided in example 3-3, docetaxel-loaded 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (50% DOPA/DODAP SLNs-DTX) provided in comparative examples 1-3 in the main organs in vivo; in fig. 6, panel a shows the amount of drug per gram of tissue measured after 6 hours of administration after removal of heart, liver, spleen, lung and kidney as a percentage of total drug injected for DOPA SLNs loaded with docetaxel in different mole percentages; panel B shows the amounts of drug per gram of tissue measured after 6 hours of administration after removal of heart, liver, spleen, lung and kidney as a percentage of total drug injected for different mole percent DODAP SLNs loaded with docetaxel.

Fig. 7 shows drug concentrations in blood of mice measured at different time points for docetaxel, docetaxel-loaded neutral solid lipid nanoparticle (0% SLNs-DTX) provided in example 3-1, and docetaxel-loaded 50% anionic and cationic solid lipid nanoparticle (50% dopa/dotap SLNs-DTX) provided in comparative example 1-3.

FIG. 8 is a schematic structural diagram of a lipid nanocarrier loaded with an anticancer drug prepared by the present invention.

Detailed Description

For a further understanding of the present invention, preferred embodiments of the invention are described in detail below with reference to the examples, but it should be understood that these descriptions are only intended to illustrate further features and advantages of the invention and are not limiting of the claims of the invention.

All the raw materials of the present invention are not particularly limited in their sources, and may be purchased on the market or prepared according to conventional methods well known to those skilled in the art.

Example 1-1

Preparation of empty neutral solid lipid nanoparticles:

2.57mg (69 equivalent) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.77mg (40 equivalent) of Cholesterol (CHOL) and 0.5mg (4 equivalent) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG) are dissolved in absolute ethanol, and 0mg (0 equivalent) of 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPA) dissolved in tetrahydrofuran is added and mixed uniformly to obtain a phospholipid ethanol solution; the phospholipid ethanol solution and the triple distilled water were rapidly mixed in a volume ratio of 1:3, incubated at room temperature for 10min, and the solution was transferred to a dialysis bag (mw=14000), and dialyzed against pure water under stirring for 3h to obtain an empty neutral solid lipid nanoparticle solution (0% slns).

The particle size of the empty solid lipid nanoparticle solution is tested, and the result is shown in figure 1, wherein the particle size of the solid lipid nanoparticle is 120-170 nm; as shown in fig. 2, the solid lipid nanoparticle potential was near neutral, indicating successful empty solid lipid nanoparticle construction.

The empty neutral solid lipid nanoparticle solution (0% SLNs) is used for a transmission electron microscope (TEM, JEM-1230, japan) to observe the form after the empty neutral solid lipid nanoparticle solution is dried, and the empty neutral solid lipid nanoparticle solution is used for a transmission electron microscope (0% SLNs) and is shown in FIG. 4.

Examples 1 to 2

Preparation of empty 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

(1) 2.22mg (69 eq.) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.67mg (40 eq.) of Cholesterol (CHOL), 0.43mg (4 eq.) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG) were dissolved in absolute ethanol, 0.52mg (12.6 eq.) of 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPA) dissolved in tetrahydrofuran was added, and the rest of the procedure was the same as in example 1-1 (except for the transmission observation) to give an empty anionic solid lipid nanoparticle solution (10% DOPA SLNs).

(2) 2.32mg (69 equivalent) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.7mg (40 equivalent) of Cholesterol (CHOL), 0.451mg (4 equivalent) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG) and 0.369mg (12.6 equivalent) of 1, 2-dioleoyl-3- (dimethylamino) propane (DODAP) are dissolved in absolute ethanol and uniformly mixed to obtain a phospholipid ethanol solution; the remaining procedure was as in example 1-1 (except for transmission observations), with the cationic solid lipid nanoparticle solution (10% DODAP SLNs) empty.

The particle size of the empty 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution is tested, and the result is shown in figure 1, wherein the particle size of the 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle is 120-170 nm; as shown in fig. 2, the 10% anionic solid lipid nanoparticle potential was negatively charged and the 10% cationic solid lipid nanoparticle potential was positively charged, indicating that the empty 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle were successfully constructed.

Examples 1 to 3

Preparation of empty 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

(1) 1.6mg (69 eq.) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.48mg (40 eq.) of Cholesterol (CHOL), 0.31mg (4 eq.) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG) were dissolved in absolute ethanol, 1.45mg (48.4 eq.) of 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPA) dissolved in tetrahydrofuran was added, and the rest of the procedure was the same as in example 2-1 (except for the transmission observation) to give an empty anionic solid lipid nanoparticle solution (30% DOPA SLNs).

(3) 1.83mg (69 equivalent) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.55mg (40 equivalent) of Cholesterol (CHOL), 0.36mg (4 equivalent) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG) and 1.1mg (48.4 equivalent) of 1, 2-dioleoyl-3- (dimethylamino) propane (DODAP) are dissolved in absolute ethanol and uniformly mixed to obtain a phospholipid ethanol solution; the remaining procedure was as in example 2-1 (except for transmission observations), with empty supported cationic solid lipid nanoparticle solutions (30% DODAP SLNs).

The particle size of the empty 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution is tested, and the result is shown in figure 1, wherein the particle size of the 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle is 120-170 nm; as shown in fig. 2, the 30% anionic solid lipid nanoparticle potential was negatively charged and the 30% cationic solid lipid nanoparticle potential was positively charged, indicating that the empty 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle were successfully constructed.

Example 2-1

Preparation of doxorubicin-loaded neutral solid lipid nanoparticles:

in example 1-1, 0.96mg of Doxorubicin (DOX) was added, and the rest of the procedure was the same as in example 1-1. Neutral solid lipid nanoparticles loaded with doxorubicin (0% SLNs-DOX) were obtained after dialysis.

The particle size of the neutral solid lipid nanoparticle solution loaded with the doxorubicin is tested, and the result is shown in figure 1, wherein the particle size of the neutral solid lipid nanoparticle loaded with the doxorubicin is 120-170 nm; as shown in figure 2, the potential of the neutral solid lipid nanoparticle loaded with the doxorubicin is neutral, which indicates that the neutral solid lipid nanoparticle loaded with the doxorubicin is successfully constructed.

Example 2-2

Preparation of doxorubicin-loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

in examples 1-2, 0.96mg of Doxorubicin (DOX) was added in each of steps (1) and (2), and the other steps were the same as in examples 1-2. After dialysis, doxorubicin-loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (10% DOPA SLNs-DOX, 10% DODAP SLNs-DOX) were obtained.

The particle sizes of the 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution loaded with the doxorubicin are obtained through testing, and the result is shown in a figure 1, wherein the particle sizes of the 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle loaded with the doxorubicin are 120-170 nm; as shown in figure 2, the 10% anionic solid lipid nanoparticle carrying doxorubicin has a negative charge and the 10% cationic solid lipid nanoparticle carrying doxorubicin has a positive charge, which indicates that the 10% anionic solid lipid nanoparticle carrying doxorubicin and the cationic solid lipid nanoparticle carrying doxorubicin are successfully constructed.

Examples 2 to 3

Preparation of doxorubicin-loaded 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

in examples 1-3, 0.96mg of Doxorubicin (DOX) was added in each of steps (1) and (2), and the rest was the same as in examples 1-3. After dialysis, 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (30% DOPA SLNs-DOX, 30% DODAP SLNs-DOX) loaded with doxorubicin were obtained.

The particle sizes of 30% anionic solid lipid nano particles and cationic solid lipid nano particles loaded with the doxorubicin are obtained by testing, and the result is shown in figure 1, wherein the particle sizes of the 30% anionic solid lipid nano particles and the cationic solid lipid nano particles loaded with the doxorubicin are 120-170 nm; as shown in figure 2, the potential of the 30% anionic solid lipid nanoparticle loaded with the doxorubicin is negatively charged, and the potential of the 30% cationic solid lipid nanoparticle loaded with the doxorubicin is positively charged, which indicates that the 30% anionic solid lipid nanoparticle and the cationic solid lipid nanoparticle loaded with the doxorubicin are successfully constructed.

Example 3-1

Preparation of docetaxel-loaded neutral solid lipid nanoparticles:

0.96mg of Docetaxel (DTX) was added to example 1-1, and the remaining steps were the same as those of example 1-1. Docetaxel-loaded neutral solid lipid nanoparticles (0% slns-DTX) were obtained after dialysis.

In this embodiment, the structure of the docetaxel-loaded neutral solid lipid nanoparticle is schematically shown in fig. 8, and as can be seen from fig. 8, the neutral solid lipid nanoparticle is a spherical nanoparticle having a solid lipid core and a single-layer phospholipid shell, and the hydrophobic drug is encapsulated inside the nanoparticle.

The particle size of the docetaxel-loaded neutral solid lipid nanoparticle solution is tested, and the result is shown in figure 1, wherein the particle size of the docetaxel-loaded neutral solid lipid nanoparticle is 120-170 nm; as shown in fig. 2, the potential of the docetaxel-loaded neutral solid lipid nanoparticle is neutral, which indicates that the docetaxel-loaded neutral solid lipid nanoparticle is successfully constructed.

Example 3-2

Preparation of docetaxel-loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

in examples 1-2, 0.96mg of Docetaxel (DTX) was added in each of steps (1) and (2), and the rest was the same as in examples 1-2. Docetaxel-loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (10% dopa SLNs-DTX, 10% dotap SLNs-DTX) were obtained after dialysis.

The particle size of the 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution loaded with docetaxel is obtained by testing, and the result is shown in figure 1, wherein the particle size of the 10% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle loaded with docetaxel is 120-170 nm; as shown in fig. 2, the 10% anionic solid lipid nanoparticle potential of the loaded docetaxel is negatively charged, and the 10% cationic solid lipid nanoparticle potential of the loaded docetaxel is positively charged, which indicates that the 10% anionic solid lipid nanoparticle and the cationic solid lipid nanoparticle of the loaded docetaxel are successfully constructed.

Examples 3 to 3

Preparation of docetaxel-loaded 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

in examples 1-3, 0.96mg of Docetaxel (DTX) was added in each of steps (1) and (2), and the rest was the same as in examples 1-3. Docetaxel-loaded 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (30% dopa SLNs-DTX, 30% dotap SLNs-DTX) were obtained after dialysis.

The particle size of the docetaxel-loaded 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution is tested, and the result is shown in figure 1, wherein the particle size of the docetaxel-loaded 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle is 120-170 nm; as shown in fig. 2, the docetaxel-loaded 30% anionic solid lipid nanoparticle potential was negatively charged, and the docetaxel-loaded 30% cationic solid lipid nanoparticle potential was positively charged, indicating that docetaxel-loaded 30% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle were successfully constructed.

Comparative examples 1 to 1

Preparation of empty 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

(1) 1.07mg (69 equivalents) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.32mg (40 equivalents) of Cholesterol (CHOL), 0.21mg (4 equivalents) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG) were dissolved in absolute ethanol, 2.24mg (113 equivalents) of 1, 2-dioleoyl-sn-glycerol-3-phosphate (DOPA) dissolved in tetrahydrofuran was added, and the remaining steps were the same as in example 2-1 (except for the transmission observation), to obtain an empty anionic solid lipid nanoparticle solution (50% DOPA SLNs).

(2) 1.31mg (69 eq.) of 1, 2-dioleoyl glycerol-3-phosphate ethanolamine (DOPE), 0.39mg (40 eq.) of Cholesterol (CHOL), 0.26mg (4 eq.) of 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol (DMG-PEG), 1.88mg (48.4 eq.) of 1, 2-dioleoyl-3- (dimethylamino) propane (DODAP) are dissolved in absolute ethanol and mixed uniformly to obtain a phospholipid ethanol solution; the remaining procedure was as in example 2-1 (except for transmission observations), with empty supported cationic solid lipid nanoparticle solutions (50% DODAP SLNs).

The particle size of the empty 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution is tested, and the result is shown in figure 1, wherein the particle size of the 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle is 120-170 nm; as shown in fig. 2, the 50% anionic solid lipid nanoparticle potential was negatively charged and the 50% cationic solid lipid nanoparticle potential was positively charged, indicating that the empty 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle were successfully constructed.

Comparative examples 1 to 2

Preparation of doxorubicin-loaded 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

in comparative example 1-1, 0.96mg of Doxorubicin (DOX) was added in each of steps (1) and (2), and the remaining steps were the same as in comparative example 1-1. After dialysis, doxorubicin-loaded 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (50% DOPA SLNs-DOX, 50% DODAP SLNs-DOX) were obtained.

The particle sizes of the 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution loaded with the doxorubicin are obtained through testing, and the result is shown in a figure 1, wherein the particle sizes of the 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle loaded with the doxorubicin are 120-170 nm; as shown in figure 2, the potential of the 50% anionic solid lipid nanoparticle loaded with the doxorubicin is negatively charged, and the potential of the 50% cationic solid lipid nanoparticle loaded with the doxorubicin is positively charged, which indicates that the 50% anionic solid lipid nanoparticle and the cationic solid lipid nanoparticle loaded with the doxorubicin are successfully constructed.

Comparative examples 1 to 3

Preparation of docetaxel-loaded 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles:

in comparative example 1-1, 0.96mg of Docetaxel (DTX) was added in each of steps (1) and (2), and the remaining steps were the same as in comparative example 1-1. Docetaxel-loaded 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (50% dopa SLNs-DTX, 50% dotap SLNs-DTX) were obtained after dialysis.

The particle sizes of the docetaxel-loaded 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle solution are tested, and the result is shown in figure 1, wherein the particle sizes of the docetaxel-loaded 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle are 120-170 nm; as shown in fig. 2, the potential of the docetaxel-loaded 50% anionic solid lipid nanoparticle is negatively charged, and the potential of the docetaxel-loaded 50% cationic solid lipid nanoparticle is positively charged, which indicates that the docetaxel-loaded 50% anionic solid lipid nanoparticle and cationic solid lipid nanoparticle are successfully constructed.

Example 4

In vitro drug release profile determination of DTX, 0% SLNs-DTX, 50% DODAP SLNs-DTX under normoxic conditions:

docetaxel-loaded solid lipid nanoparticles (0% SLNs-DTX) obtained in example 3-1, docetaxel-loaded solid lipid nanoparticles (50% DODAP SLNs-DTX) obtained in comparative example 1-3 were used, and after thoroughly mixing, they were left to stand, 0.2ml of each supernatant was taken at 0h,0.05h,0.25h,0.5h,1h,2h,4h,6h,12h and 24h, and the same volume of fresh PBS solution was replenished while taking 0.2ml of each supernatant, so that the volume of the whole system remained unchanged. This was then added to 3ml of a methanol solution, and the content of DTX in the solution was measured by High Performance Liquid Chromatography (HPLC), thereby calculating the amount of DTX cumulative release.

The DTX release profile is shown in FIG. 5, and it can be seen from FIG. 5 that 0% SLNs-DTX releases less drug at the same time than the other two groups, indicating that 0% SLNs-DTX can extend the drug release time and that 0% SLNs-DTX release rate is lower than that of the free drug group, which is statistically significant.

Example 5

Distribution of DTX, docetaxel-loaded different mole percent solid lipid nanoparticles in different organs:

ICR mice were randomly divided into 8 groups, each group 3, each of which was tail-intravenously injected with free docetaxel at a dose of 5mg/kg body weight, docetaxel-loaded solid lipid nanoparticles (0% SLNs-DTX) obtained in example 3-1, docetaxel-loaded 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (10% dopa SLNs-DTX, 10% dodap SLNs-DTX) obtained in example 3-2, docetaxel-loaded 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (30% dopa SLNs-DTX, 30% dodap SLNs-DTX) obtained in example 3-3, and docetaxel-loaded 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles (50% dopa SLNs-DTX) obtained in comparative example 1-3, respectively. The mice were anesthetized by intraperitoneal injection of chloral hydrate 6h after administration, followed by physiological saline infusion, and heart, liver, spleen, lung, kidney and other tissues were removed, respectively. After weighing the tissue, homogenization is performed using a homogenizer. Adding 5ml of mixed solution (chloroform: methanol=4:1) into the homogenized tissue, crushing and ultrasonic treatment for 10min, centrifuging for 10min at 8000r/min, taking out the lower layer of chloroform solution, spin-drying, adding 3ml of methanol solution for redissolving, centrifuging for 10min at 8000r/min, measuring the medicine content in different tissues in the supernatant by utilizing HPLC, and counting the medicine percentage of the body tissue in each gram of tissue as shown in figure 6.

As can be seen from fig. 6, the SLNs are mainly distributed in the liver, and the drug content in the liver is higher than that of the free drug and 50% of the SLNs-loaded group, which indicates that the prepared SLNs have better liver targeting function when the molar percentage of DOPA or DODAP is 0% -30% of the total phospholipids.

Example 6

DTX, 0% SLNs-DTX, 50% DODAP SLNs-DTX pharmacokinetics:

healthy BALB/c mice were randomly divided into 3 groups of 3, each group was tail-vein injected with docetaxel at a dose of 5mg/kg body weight (control group 1), docetaxel-loaded solid lipid nanoparticle (0% SLNs-DTX) obtained in example 3-1, and docetaxel-loaded 50% cationic solid lipid nanoparticle (50% DODAP SLNs-DTX) obtained in comparative example 1-3, respectively. And (3) taking blood from the eye sockets at 0.05h, 0.5h,1h,2h,4h,6h,12h and 24h after tail vein administration to obtain 1ml of blood, standing at 4 ℃, centrifuging at 3000r/min for 5min, and taking 0.2ml of upper serum. Adding the mixture into 0.2ml of PBS solution and 3ml of chloroform solution, centrifuging for 10min at 5500r/min by vortex for 5min, taking out chloroform layer liquid, spin-drying chloroform, adding 2ml of methanol for redissolution, centrifuging for 10min at 8000r/min, and measuring the drug content in different tissues in the supernatant by HPLC.

According to fig. 7, a graph of DTX concentration versus time in plasma was measured for ICR mice at different time points within 24h of dosing. Compared with the control group 1-2, the 0% SLNs-DTX has long half-life in vivo and slow clearance rate, can prolong the blood circulation time of the drug in vivo, and simultaneously improves the bioavailability of the drug in vivo.

The fluorescence spectrophotometers and HPLC measurements of neutral solid lipid nanoparticles loaded with anticancer drugs (0% SLNs-DOX, 0% SLNs-DTX) provided in examples 2-1 and 3-1, 10% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drugs (10% DOPA/DODAP SLNs-DOX, 10% DOPA/DODAP SLNs-DTX) provided in examples 2-2 and 3, 30% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drugs (30% DOPA/DODAP SLNs-DOX, 30% DOPA/DODAP SLNs-DTX) provided in comparative examples 1-2 and 3, 50% anionic solid lipid nanoparticles and cationic solid lipid nanoparticles loaded with anticancer drugs (50% PA/DODAP SLNs-DOX, 50% DODAP/DAP-DTX) provided in comparative examples 1-3 were shown in the statistical graphs of encapsulation rate of the solid lipid nanoparticles and the solid lipid nanoparticles loaded with anticancer drugs. As can be seen from fig. 3, the encapsulation efficiency of the solid lipid nanoparticles loaded with DTX is higher than that of the solid lipid nanoparticles loaded with DOX, which indicates that the solid lipid nanoparticles coated with DTX can reduce the administration frequency and are more suitable for treatment.

While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The preparation method of the lipid nano-carrier loaded with the anticancer drug is characterized in that the lipid nano-carrier loaded with the anticancer drug is a solid lipid nano-particle solution loaded with the anticancer drug, and comprises the following steps:

1) Dissolving 1, 2-dioleoyl glycerol-3-phosphoethanolamine, cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol and an anticancer drug in absolute ethanol, adding a solution containing fourth phospholipid, and uniformly mixing to obtain phospholipid ethanol solution; the solution containing the fourth phospholipid is 1, 2-dioleoyl-sn-glycero-3-phosphate dissolved by tetrahydrofuran or 1, 2-dioleoyl-3- (dimethylamino) propane dissolved by absolute ethyl alcohol; the mole ratio of the 1, 2-dioleoyl glycerol-3-phosphate ethanolamine to cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol and fourth phospholipid is 69:40:4: x, X is 0, 12.6 or 48.4;

2) And (3) rapidly mixing the phospholipid ethanol solution with pure water, incubating at room temperature for 10min, transferring to a dialysis bag, and dialyzing with pure water for 3h under stirring to remove the non-encapsulated anticancer drug, thereby obtaining the solid lipid nanoparticle solution loaded with the anticancer drug.

2. The preparation method according to claim 1, wherein the mass ratio of the anticancer drug to the total amount of four phospholipids in step 1) is 1:4, the four phospholipids are 1, 2-dioleoyl glycerol-3-phosphoethanolamine, cholesterol, 1, 2-dimyristoyl-sn-glycerol-3-methoxypolyethylene glycol and fourth phospholipid, and the anticancer drug is doxorubicin or docetaxel.

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

4. The method of claim 1, wherein the dialysis bag in step 2) has a molecular weight cut-off of 14000.

5. The anticancer drug-loaded lipid nanocarrier prepared by the preparation method according to any one of claims 1 to 4.

6. The anticancer drug-loaded lipid nanocarrier of claim 5, wherein the hydrated particle size of the anticancer drug-loaded lipid nanocarrier is 120-170nm.

7. The use of the anticancer drug-loaded lipid nanocarriers of claim 6 in the preparation of a liver-targeting anticancer drug.