CN114983943A - Artesunate nano targeting preparation and preparation method and application thereof - Google Patents
Artesunate nano targeting preparation and preparation method and application thereof Download PDFInfo
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- A61K9/10—Dispersions; Emulsions
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
An Artesunate (ART) nano targeting preparation, a preparation method and an application thereof, belonging to the technical field of medicines. Dissolving the lipid materials FOL-NH-PEG-DSPE, Chol and phospholipid in a reaction vessel containing a mixed solution of chloroform and methanol, vacuumizing in a water bath at 45 ℃, rotationally evaporating to remove the organic solvent, forming a layer of uniform thin lipid film on the wall of the reaction vessel by the lipid materials, removing the water bath, and continuously vacuumizing for 1-2 hours; dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the first step with ART stock solution, and performing vortex oscillation to completely dissolve the lipid membrane to obtain liposome primary solution; putting the primary liposome solution into a water bath, performing ultrasonic treatment for 5 min, and passing through a 0.22 mu m membrane to prepare the liposome with uniform particle size, namely the artesunate nano-targeting preparation. The invention provides a preparation process of a novel ART nano-targeting preparation of liposome, measures the characteristics of the preparation, and provides a new optional preparation for the preparation as a drug-resistant tumor treatment drug in the future.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to an artesunate nano-targeting preparation, and a preparation method and application thereof.
Background
The targeted drug delivery system can ideally bypass normal parts of a body through a carrying system and target affected areas needing to be treated in the body, wherein the nanoparticles are widely applied in the field of pharmacy, and after entering the human body, the drug wrapped by the nanoparticles can actively patrol and attack cancer cells, so that the concentration of the drug at the affected parts is increased, and the toxicity of the drug to non-targeted parts is reduced.
The artemisinin is sesquiterpene lactone medicine directly extracted from herba Artemisiae Annuae. Artemisinin drugs have long been considered as an effective antimalarial drug. Artesunate (ART) is one of artemisinin derivatives, and besides the anti-malarial effect, the anti-tumor effect of Artesunate is more of great concern to people, and the clinical application prospect is wide. ART has remarkable tumor cell killing effect, and is not easy to generate drug resistance. Therefore, its role in drug-resistant tumors is becoming a focus of research. The artemisinin drugs can play a good anti-tumor effect in cell level, animal transplantation tumor models and clinical tests, and are candidate drugs with good prospects in tumor treatment.
Chemotherapy is one of the main means for clinically treating malignant tumors, but the drug resistance of tumors to chemotherapeutic drugs is an important cause of clinical chemotherapy failure, and more than 50% of tumors are reported to rapidly generate drug resistance in the treatment process, only a small part of tumors are sensitive to chemotherapy, and the drug resistance of tumors seriously affects the treatment effect and prognosis of patients. The tumor drug resistance mechanism is complex and various, and is not completely elucidated at present. At present, a great deal of research focuses on how to reverse the multidrug resistance and assist in synergism of tumors, but a reversal agent and a synergistic agent which are in the true sense are not applied to clinic until now. Especially for most solid tumors, the objective efficacy of chemotherapy is still low. 5-adriamycin (DOX), Cisplatin (CDDP), fluorouracil (5-FU) and the like are two most widely used chemotherapy drugs in clinic at present, but the clinical curative effect is not ideal due to toxic and side effects and easy occurrence of drug resistance.
ART is an artemisinin derivative, slightly more water soluble than artemisinin, but still predominantly exhibits lipid solubility, thus limiting its activity for anti-tumor effects. The ART is prepared into a nano targeting preparation, so that the targeting of the anti-tumor cells of the ART can be increased, and the clinical curative effect is further improved.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the problems in the prior ART, the invention provides an artesunate nano-targeting preparation, a preparation method and application thereof, which are a novel preparation process of an ART nano-targeting preparation of liposome, and the characterization of the preparation is determined, thereby providing a new optional formulation for the future use as drug-resistant tumor treatment.
The technical scheme is as follows: a method for preparing artesunate nanometer targeting preparation comprises the following steps
Taking a grease material NH 2 Dissolving PEG-DSPE, Chol and phospholipid in a reaction vessel containing a mixed solution of chloroform and methanol, vacuumizing in a water bath at 45 ℃, performing rotary evaporation to remove the organic solvent, so that a layer of uniform thin lipid film is formed on the wall of the reaction vessel by the lipid material, removing the water bath, and continuously vacuumizing for 1-2 hours;
dissolving the lipid materials FOL-NH-PEG-DSPE, Chol and phospholipid in a reaction container filled with a chloroform and methanol mixed solution, vacuumizing in a water bath at 45 ℃, rotationally evaporating to remove the organic solvent, forming a uniform thin lipid film on the wall of the reaction container by the lipid materials, removing the water bath, and continuously vacuumizing for 1-2 hours;
dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the first step with the ART stock solution, and performing vortex oscillation to completely dissolve the lipid membrane to obtain liposome primary solution;
dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the second step with the ART stock solution, and performing vortex oscillation to completely dissolve the lipid membrane to obtain target liposome primary solution;
and fifthly, putting the primary liposome solution into a water bath, performing ultrasonic treatment for 5 min, and preparing the liposome with uniform particle size through a 0.22 mu m membrane, namely the artesunate nano-targeting preparation.
Preferably, NH is performed in the first step and the second step 2 -the mass ratio of PEG-DSPE (FOL-NH-PEG-DSPE), Chol and phospholipid is 2-5: 3-6: 2-5, the volume ratio of chloroform to methanol is 10-50: 10-50, and the ratio of the lipid, Chol and phospholipid to the mixed solution is 10-50 mg: 1 mL.
Preferably, the ratio of ART to acetonitrile in the ART stock solution in the third and fourth steps is 50-200 μ g: 1 mL.
The artesunate nano targeting preparation prepared by the method.
The artesunate nano-targeting preparation is applied to the preparation of the tumor cell inhibiting medicine.
In the preparation method, the PEG modified lipid is additionally added into the membrane material, so that the long-circulating liposome can be prepared, and the tumor targeting, synergistic and attenuation effects are achieved. The PEG modified lipid is selected from one of polyethylene glycol-distearoylphosphatidylethanolamine, polyethylene glycol-hydrogenated soybean phosphatidylethanolamine, polyethylene glycol-polycaprolactone, polyethylene glycol-polyglycolide lactide, polyethylene glycol-polylactic acid, poloxamer 188, polyoxyethylene fatty acid esters, polyoxyethylene fatty acid ether and polyoxyethylene methyl castor oil ether, and any combination thereof.
The medicine carrying temperature is preferably 30-70 ℃ during active medicine carrying.
The average particle size of the liposome prepared by the method can be effectively controlled to be 50-200 nm.
Has the advantages that: the invention synthesizes a novel material with receptor targeting function, can realize the amplification of the level of response signals in tumor cells, avoids the problem of insufficient response signals caused by the dynamic change of the microenvironment in the tumor cells, and can effectively patrol to a target site. The method can be used for instantly constructing samples with any volume through simple solution mixing, saves time, avoids liposome leakage caused by long-time operation, and does not need expensive production equipment such as dialysis and the like. In addition, the method does not lose small-particle-size liposome or cause liposome particle aggregation and membrane fusion due to mechanical force action so as to cause liposome damage. In conclusion, the method does not need complicated and time-consuming process steps or expensive special equipment, can simply and conveniently realize active drug loading, greatly simplifies the production process, improves the production efficiency and reproducibility, and simultaneously greatly reduces the production cost.
The preparation method provided by the invention also has the following characteristics:
(1) the encapsulation rate is high, and the content of the freeze-drying agent is more than 98 percent before and after freeze-drying.
(2) The stability is good, and the leakage in PBS buffer solution is not more than 1% within 72 hours.
(3) The particle size is small, the average particle size is 50-200 nm before and after freeze-drying, the requirements of industrialization on sterility and no heat source of a final preparation can be met through simple filtration sterilization, and meanwhile, the small particle size also lays a good foundation for the in-vivo long-circulating behavior of the preparation.
(4) Can be prepared into a freeze-dried preparation for storage, and greatly improves the storage stability of the liposome.
In addition, artesunate can inhibit lysosomal function, raise lysosomal pH, reduce lysosomal enzyme activity, reduce mitochondrial clearance, and induce apoptosis. Therefore, the artesunate liposome has better cytotoxic effect on drug-resistant cells with higher lysosome function.
Drawings
FIG. 1 is a graph of the elution profile of liposomes;
FIG. 2 is a graph of the interaction of liposomes with plasma;
FIG. 3 is a graph showing the drug release profile of liposomes in serum.
Detailed Description
The invention is further described below with reference to the figures and the specific embodiments.
Artesunate (ART, sigma aldrich trade ltd) in the examples of the present specification; NH 2 PEG-DSPE purchased from sigma aldrich (shanghai) trade ltd; chol was purchased from sigma aldrich trade ltd; phospholipids were purchased from national chemical group, Inc.; anionic dextran was purchased from national pharmaceutical group chemical agents, ltd; dextran G50 was purchased from national institute of chemicals, ltd; folic acid was purchased from the national pharmaceutical group chemical reagents, Inc., and all other reagents were analytically pure.
The equipment and conditions used were as follows:
sartorius balance analytical balance sidoris, germany
85-1A constant temperature magnetic stirrer Nanjing Keel Instrument Co., Ltd
1 H-NMR Analyzer Bruker, Germany
SHZ-D (III) circulating water type vacuum pump Guyingyu pilot China plant
RE-52A rotary evaporator Shanghai Yanrong biochemical instrument factory
FD-1 Freeze dryer Beijing Bo Yi kang technology company
KQ-100 ultrasonic cleaner Kunshan ultrasonic Instrument Co Ltd
A standard curve of ART is prepared: accurately weighing ART reference substance 500 mg, placing in a 50 mL measuring flask, adding acetonitrile for dissolving and diluting to scale, shaking up, accurately weighing 1, 2, 3, 4, 5 and 6 mL, respectively placing in a 10 mL measuring flask, adding acetonitrile for diluting to scale to obtain each reference substance solution. And (3) taking 20 uL of each control solution, respectively injecting the control solutions into a liquid chromatograph, and recording the main peak area. And performing linear regression by taking the sample concentration as a horizontal coordinate and the main peak area as a vertical coordinate, and calculating a regression equation, a correlation coefficient and a residual variance. The ART standard curve equation is: a =1450.92C +5.716 (R) 2 = 0.9992); RSD intra-day = 0.14%, RSD inter-day = 0.97%. In the range of 0.1-0.6 mg/mLThe sexual relationship is good.
Example 1
The preparation method of the artesunate nano-targeting preparation in the embodiment comprises the following steps:
dissolving 10 mg of lipid materials FOL-NH-PEG-DSPE, Chol and phospholipid in a reaction container containing 1mL of chloroform and methanol mixed solution with the volume ratio of 1:1, wherein the mass ratio of FOL-NH-PEG-DSPE to phospholipid is 3:4:3, vacuumizing in a 45 ℃ water bath, rotating and evaporating to remove organic solvent, forming a uniform thin lipid film on the wall of the reaction container by the lipid materials, removing the water bath, and continuously vacuumizing for 1-2 hours;
dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the step one with the ART stock solution, and performing vortex oscillation to prepare liposome, wherein the ratio of ART to acetonitrile in the ART stock solution is 100 mu g/mL;
and thirdly, putting the liposome into a water bath, performing ultrasonic treatment for 5 min, and preparing the liposome with uniform particle size through a 0.22 mu m membrane, namely the artesunate nano-targeting preparation.
Example 2
The preparation method of the artesunate nano-targeting preparation in the embodiment comprises the following steps:
firstly, 50 mg of lipid FOL-NH-PEG-DSPE, Chol and phospholipid are dissolved in a reaction vessel containing 1mL of mixed solution of chloroform and methanol with the volume ratio of 1:5, and NH is added 2 The mass ratio of PEG-DSPE, Chol and phospholipid is 2:3:2, the organic solvent is removed by vacuum-pumping rotary evaporation in a water bath at 45 ℃, so that a uniform thin lipid film is formed on the wall of the reaction vessel by the lipid material, the water bath is removed, and the vacuum-pumping is continued for 1-2 hours;
dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the step one with the ART stock solution, and performing vortex oscillation to completely dissolve the lipid membrane to prepare liposome primary solution, wherein the ratio of ART to acetonitrile in the ART stock solution is 50 mu g/mL;
and step three, putting the primary liposome solution into a water bath, performing ultrasonic treatment for 5 min, and passing through a 0.22 mu m membrane to prepare the liposome with uniform particle size, namely the artesunate nano-targeting preparation.
Example 3
The preparation method of the artesunate nano-targeting preparation in the embodiment comprises the following steps
Firstly, 50 mg of lipid materials FOL-NH-PEG-DSPE, Chol and phospholipid are dissolved in a reaction vessel containing 1mL of mixed solution of chloroform and methanol with the volume ratio of 5:1, and NH is added 2 The mass ratio of PEG-DSPE, Chol and phospholipid is 5:6:5, the organic solvent is removed by vacuum-pumping rotary evaporation at the temperature of 45 ℃ in a water bath, so that a layer of uniform thin lipid film is formed on the wall of the reaction vessel by the lipid material, the water bath is removed, and the vacuum-pumping is continued for 1-2 hours;
dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the step one with the ART stock solution, and performing vortex oscillation to completely dissolve the lipid membrane to prepare liposome primary solution, wherein the ratio of ART to acetonitrile in the ART stock solution is 200 mu g/mL;
and thirdly, putting the primary liposome solution into a water bath, performing ultrasonic treatment for 5 min, and preparing the liposome with uniform particle size through a 0.22 mu m membrane, namely the artesunate nano-targeting preparation.
The artesunate nano-targeting preparation liposome prepared in example 1 was subjected to performance test:
1) and (3) measuring the particle size and potential of the liposome, namely diluting the prepared liposome suspension by 10 times by using deionized water, and measuring the particle size, the distribution and the potential of the liposome by using a laser particle size distribution instrument (Nano ZS Zen 500 Malvern).
The blank liposome prepared in the first step of the invention is uniform and stable suspension with blue opalescence, and the liposome becomes light orange yellow after ART is encapsulated. The average particle size of the finally prepared liposome is about 144 nm; the average E potential is 43.72 mV, and the liposome has positive charge and certain stability.
2) And (3) measuring the encapsulation efficiency of the liposome, namely adding 0.5 mL of the liposome with uniform particle size prepared in the step three into a dextran G50 gel column, eluting unencapsulated ART by PBS and collecting the liposome, collecting the liposome by taking 1.5 mL of each part, and recording an elution curve to obtain a liposome suspension encapsulating ART. 0.1 mL of the separated ART-encapsulated liposome suspension was taken, 0.1 mL of 10% Triton X-100 solution was added to disrupt the liposomes, the resulting solution was diluted with PBS to an appropriate concentration, and the absorbance of the solution was measured at 216 nm. Another 0.1 mL of the liposome suspension of uniform particle size prepared in step three without isolation was also added with 0.1 mL of 10% Triton X-100 solution to disrupt the liposomes, and diluted to an appropriate concentration with PBS as a control solution for determination of A. And converting the concentration into concentration through a standard curve, and calculating the encapsulation efficiency.
The elution profile of the liposomes is shown in FIG. 1. The first peak is the liposome peak and the latter is the free ART peak, with the two peaks being better separated. Therefore, the SePhadexG50 can be used for separating the liposome from the free drug, thereby achieving the purpose of determining the drug encapsulation efficiency.
The liposomes of ART were diluted with different dispersion media and the encapsulation efficiency was measured. The results show that the encapsulation efficiency of the liposome prepared by using water as a dispersion medium is highest. Other formulations have low encapsulation efficiency due to the influence of electrolytes or buffers.
TABLE 1 encapsulation efficiency of ART liposomes in different dispersion media
3) And (3) examining the interaction between ART and a liposome membrane, taking a 5 mg/mLART drug solution and an ART cationic liposome suspension, scanning the excitation wavelength and the emission wavelength of the drug solution and the ART cationic liposome suspension respectively, and recording the change of the ultraviolet peak position. The interaction of ART with cationic liposome membranes was examined as a change in ART UV spectrum. In the experiment, the solution is yellow-green fluorescein, but the solution is orange yellow after being prepared into the cationic liposome. After the liposome is prepared, the excitation wavelength of ART is blue-shifted by 2-3 nm, and the emission wavelength is red-shifted by 6-7 nm. Indicating that ART interacts with the cationic liposomes.
4) Taking 200 mu L of ART liposome solution, adding the ART liposome solution into a mixed solution containing 1mL of rat plasma and 0.8 mL of physiological saline, sealing, standing in a constant-temperature water bath at 37 ℃, sampling 2 mL at fixed time, and measuring the absorbance of the solution at the wavelength of 216 nm.
When liposomes come into contact with plasma components, aggregation occurs, causing a change in turbidity. The neutral liposome does not interact with plasma components, so that aggregation is not caused; the cationic liposome, especially the pure cationic liposome without PEG modification, has great interaction with plasma components in vitroAggregation of plasma components and cationic liposomes is caused during the resting process. As can also be seen from FIG. 2, FOL-NH-PEG-DSPE, NH 2 PEG-DSPE reduces the interaction of cationic liposomes with plasma components.
5) The drug release of the cationic liposome in serum is measured to examine the stability of the liposome in serum. 0.5 mL of newborn calf serum (FAS) and 0.5 mL of cationic liposome are respectively taken, mixed uniformly, placed in a dialysis bag (the mass of the trapped molecule is 8000-12000), and are sampled at regular time for 0.5, 1, 2, 4, 8, 12, 24 and 48 hours by taking 20 mL of PBS as a receiving solution, and A is measured at 216 nm. In addition, 0.5 mL PBS was used as a control to measure drug release instead of FAS.
As can be seen in FIG. 3, the liposome release in serum was greater than in PBS. The release amount of the liposome in PBS and serum for 48 h at 37 ℃ is less than 20 percent, which indicates that the drug encapsulated in the liposome is slow to release and has stronger stability, and can meet the requirement of long-time circulation of the long-circulation liposome in vivo.
Artesunate has the properties of inhibiting lysosomal function, raising lysosomal pH, reducing lysosomal enzyme activity, and reducing mitochondrial clearance. The method improves the cytotoxic effect of the artesunate liposome on drug-resistant cells. Meanwhile, ART liposome is circulated to the tumor microenvironment part in vivo by virtue of receptor-mediated targeting materials, so that the aggregation and release of the drugs in the tumor cells are increased, and the drug-resistant tumor cells are killed.
Claims (5)
1. The preparation method of the artesunate nano-targeting preparation is characterized by comprising the following steps
Dissolving the lipid materials FOL-NH-PEG-DSPE, Chol and phospholipid in a reaction container filled with a chloroform and methanol mixed solution, vacuumizing in a water bath at 45 ℃, rotationally evaporating to remove the organic solvent, forming a uniform thin lipid film on the wall of the reaction container by the lipid materials, removing the water bath, and continuously vacuumizing for 1-2 hours;
dissolving ART in acetonitrile solution to obtain ART stock solution, hydrating the lipid membrane obtained in the first step with the ART stock solution, and performing vortex oscillation to completely dissolve the lipid membrane to obtain liposome primary solution;
and thirdly, putting the primary liposome solution into a water bath, performing ultrasonic treatment for 5 min, and preparing the liposome with uniform particle size through a 0.22 mu m membrane, namely the artesunate nano-targeting preparation.
2. The preparation method of the artesunate nano-targeting preparation as claimed in claim 1, wherein in the first step, the mass ratio of FOL-NH-PEG-DSPE, Chol and phospholipid is 2-5: 3-6: 2-5, the volume ratio of chloroform and methanol is 1-5: 1-5, and the ratio of FOL-NH-PEG-DSPE, Chol and phospholipid to the mixed solution is 10-50 mg: 1 mL.
3. The preparation method of the artesunate nano-targeting preparation according to claim 1, wherein the ratio of ART to acetonitrile in the second ART stock solution is 50-200 μ g: 1 mL.
4. The artesunate nano-targeting formulation prepared by the method of claim 1.
5. The use of the artesunate nano-targeting preparation of claim 4 in the preparation of a medicament for inhibiting tumor cells.
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