CN111632032A - Natural small molecule co-assembled nano-drug delivery system and preparation method and application thereof - Google Patents
Natural small molecule co-assembled nano-drug delivery system and preparation method and application thereof Download PDFInfo
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- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
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Abstract
The invention discloses a natural small molecule co-assembled nano-drug delivery system and a preparation method and application thereof, wherein the natural small molecule co-assembled nano-drug delivery system is a nano-particle formed by co-assembling two or more than two of oleanolic acid, ursolic acid, glycyrrhetinic acid, betulinic acid, betulin, liquidambaric acid, lupeol, paclitaxel, rhein and catechin. The invention can change the original self-assembly morphology of the compound, prepare nano particles with different morphologies and sizes and solve the problem that the morphology of the compound is not suitable for intravenous injection. The nano-drug delivery system of the present invention has one or more pharmacological activities, the compounds constituting the nano-drug delivery system have a synergistic antitumor effect through different mechanisms, and the nano-drug delivery system has a health care function and can improve the oxidation resistance of the organism.
Description
Technical Field
The invention belongs to the field of nano-medicine, relates to a nano-drug delivery system, a preparation method and application thereof, and particularly relates to a nano-drug delivery system prepared by taking natural small molecules as units through a co-assembly method, and a preparation method and application thereof.
Background
Cancer is a malignant disease which seriously threatens human health nowadays, traditional chemotherapy is still an indispensable treatment method for most cancer patients, and most anticancer drugs have the limitations of poor solubility, multidrug resistance and large toxic and side effects. The application of nanotechnology in medicine is widely recognized, and the design and synthesis of efficient drug delivery systems are critical to cancer treatment. At present, a series of nano-drug delivery system systems such as polymer micelle, nano-particle, nano-tube, nano-gel and the like are established. However, the preparation method of the nano-carrier is complex, and the introduced carrier material is not easy to degrade, which often causes additional toxic effect. Therefore, the search for new, safe and efficient nano-preparations becomes a current research hotspot.
Natural small molecules are considered as promising drug resources due to their broad pharmacological actions. Over one third of the FDA perennial approved new clinical drugs are derived from natural products or derivatives thereof. They are usually derived from biological endogenous chemical components or metabolites of living organisms, and have the advantages of excellent biological safety, biological stability, low toxic and side effects and the like.
Researches show that the natural micromolecules with self-assembly capacity can be used for drug delivery, and can achieve the effects of adjuvant therapy and medical care while being applied to drug delivery, so that the defect that the current drug carrier has no pharmacological activity is overcome. However, natural small molecules that can be used for drug loading are very limited so far, because not all natural small molecules can self-assemble into drug carriers with specific morphology and size, and natural small molecules with self-assembly properties do not all have excellent pharmacological activity.
The supermolecular co-assembly method is widely used for constructing a nano-drug system due to its simplicity and greenness, and in the co-assembly process, the nano-particles maintain and possess the biological activity of each component unit, so that the treatment effect is remarkably improved. If a co-assembly strategy is applied, two compounds with pharmacological activity and health care efficacy are combined to construct a natural small molecule co-assembly nano-drug delivery system, and a drug delivery system for enhancing the synergistic antitumor effect and relieving the side effect induced by chemotherapeutic drugs is expected to be obtained.
Disclosure of Invention
The invention aims to provide a preparation method of a natural small molecule co-assembled nano-drug delivery system, a preparation method and application thereof.
The purpose of the invention is realized by the following technical scheme:
a natural small molecule co-assembled nano-drug delivery system is a nano-particle formed by co-assembling two or more than two of oleanolic acid, ursolic acid, glycyrrhetinic acid, betulinic acid, betulin, liquidambaric acid, lupeol, paclitaxel, rhein and catechin, and the specific preparation method is as follows:
step one, natural micromolecules are taken as raw materials, and the natural micromolecules are dissolved in benign organic solvent to obtain natural micromolecule solution, wherein: the natural small molecules are two or more of oleanolic acid, ursolic acid, glycyrrhetinic acid, betulinic acid, betulin, liquidambaric acid, lupeol, paclitaxel, rhein, and catechin; the mixing ratio of each component in the natural small molecule has no specific requirement, such as: when two natural small molecules are selected, the mass ratio of the two natural small molecules can be 1: 10 to 10: 1; the benign organic solvent is one or more of methanol, ethanol, acetone, petroleum ether, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane and n-hexane;
step two, adding the natural micromolecule solution obtained in the step one into a solution containing a surfactant with the mass concentration of 1-5%, and performing vortex emulsification for 0.5-10 minutes to obtain a suspension, wherein: the surfactant is one of an acid surfactant, a basic surfactant and a neutral surfactant, and is preferably polyvinyl alcohol (PVA);
step three, further emulsifying the suspension obtained in the step two, wherein: the emulsification treatment is ultrasonic emulsification but is not limited, and the emulsification time is 1-10 minutes;
step four, adding the emulsion formed in the step three into an aqueous solution containing a surfactant with the mass concentration of 0.1-1.0%, stirring, and completely volatilizing the organic solvent, wherein the process is carried out in an environment at the temperature of not higher than 30 ℃, and the volume ratio of the emulsion to the aqueous solution containing the surfactant is 1: 5-30;
step five, carrying out centrifugal treatment and double-distilled water washing on the solution in the step four, removing residual surfactant, and harvesting nanoparticles (namely a nano-drug delivery system), wherein: the revolution of centrifugal treatment is 5000-50000 revolutions;
step six, adding the nanoparticles obtained in the step five into a solvent (water, physiological saline or PBS) to prepare a suspension for later use; or drying or freeze-drying the nanoparticles harvested in the fifth step for long-term storage.
In the invention, the nano particles are in a structure of nanospheres, nano fibers or nano rods and are directly assembled together through supermolecule non-covalent interaction force.
In the invention, the raw materials are not subjected to any chemical modification and structural modification, and the formed and co-assembled nanoparticles keep the pharmacological activity of each component unit.
In the invention, the average particle size of the nano-particles is 100-1000 nm, and the nano-particles can be administrated through intravenous injection without forming administration embolism, and can also be used for oral administration or intraperitoneal administration.
In the invention, the hydrophilicity of the nano particles is obviously improved, and the half-life period of blood is prolonged by 2-6 times compared with that of a free compound.
In the present invention, the nanoparticles have good stability and redispersibility.
In the invention, the nano-particles have good slow release characteristics, and the cumulative release amount of 48 hours under the pH of 7.3 is about 60 percent.
In the invention, the nano-particles have good biological safety and have no or extremely low cytotoxicity to normal cells.
In the invention, the nano particles can play a synergistic anti-tumor role, and the synergistic index is less than 0.9.
In the invention, the nano-particles have high tumor targeting property and can be effectively enriched at tumor parts.
In the invention, the tumor inhibition rate of the nanoparticles on the 4T1 tumor-bearing mouse model is higher than 60%.
In the invention, the nano-particles can effectively reduce the toxic and side effects of organisms, wherein the haematological indexes (white blood cell number, lymphocyte number and neutrophil number) are obviously improved by P <0.01 compared with the taxol injection.
In the invention, the nano-particles can improve the oxidation resistance of an organism, can be used for treating liver diseases, and the contents of SOD (superoxide dismutase) and GSH (reduced glutathione) in liver tissues of a treatment group are obviously higher than those of a paclitaxel injection treatment group, wherein P is less than 0.05.
In the invention, the nano particles can be loaded with drugs and form multi-component co-assembled nano drug-loaded particles.
In the invention, the drug loading rate of the nano particles is more than 10% by mass fraction, and the encapsulation rate is more than 80%.
In the invention, the nanoparticles can enhance the anti-tumor effect, and the tumor inhibition rate is improved from 64% to 82%.
In the invention, the nano-particles can relieve the side effects of the medicine, such as: can obviously relieve liver injury, kidney injury and heart injury caused by the medicine.
Compared with the prior art, the invention has the following advantages:
1. the nano-drug delivery system of the present invention is composed of two or more natural small molecules, and the compounds constituting the system do not require a complex chemical synthesis process.
2. The invention adopts a supermolecule co-assembly method to prepare a nano-drug delivery system, and the method can change the original self-assembly appearance of the compound, prepare nano-particles with different appearances and sizes, and solve the problem that the appearance of the compound is not suitable for intravenous injection.
3. The nano-drug delivery system has good biocompatibility, biological safety and pharmacological activity, and hardly generates nano toxicity to organisms.
4. The nano-drug delivery system of the present invention has one or more pharmacological activities, the compounds constituting the nano-drug delivery system have a synergistic antitumor effect through different mechanisms, and the nano-drug delivery system has a health care function and can improve the oxidation resistance of the organism.
5. The nano-drug delivery system has the anti-tumor effect and the health care effect, can slow down the whole body toxicity of an organism, can resist the liver tissue damage caused by drugs, slow down the heart damage caused by the drugs, slow down the kidney damage caused by the drugs, improve the oxidation resistance of the liver, increase the solubility of hydrophobic drugs, overcome the multi-drug resistance and slow down the side effect of chemotherapy drugs.
6. The nano-drug delivery system of the invention has very stable in vivo and in vitro environments, can prolong the half-life period of blood, and reduces the risk of nano-toxicity.
7. The nano-drug delivery system can also be used as a drug carrier for loading drugs to form drug-loaded nano-particles; the nano-carrier and the drug have the synergistic anti-tumor effect by blocking cells in different cell cycles or blocking the cell cycles in the same cell cycle through different mechanisms, so that the tumor inhibition rate is improved; the drug-loaded nanoparticles can effectively reduce tissue damage caused by chemotherapeutic drugs by enhancing the oxidation resistance of organisms, and have good biocompatibility and system safety.
Drawings
FIG. 1 is a scanning electron microscope image of a liquidambaric acid-paclitaxel (BTA-PTX) nanoparticle;
FIG. 2 is a scanning electron microscope image of oleanolic acid-glycyrrhetinic acid (OA-GA) nanoparticles;
FIG. 3 is a scanning electron microscope image of betulin-glycyrrhetinic acid (Bet-GA) nanoparticles;
FIG. 4 is a scanning electron microscope image of Liquidambaric acid-Glycyrrhetinic acid (BTA-GA) nanoparticles;
FIG. 5 is a scanning electron microscope image of oleanolic acid-betulinic acid (OA-BA) nanoparticles;
FIG. 6 is a transmission electron microscope image of co-assembled nanoparticles, A-Lulutong acid-paclitaxel (BTA-PTX) nanoparticles, B-oleanolic acid-Glycyrrhetinic acid (OA-GA) nanoparticles, C-Lulutong acid-Glycyrrhetinic acid (BTA-GA) nanoparticles;
FIG. 7 is a transmission electron microscope image of oleanolic acid-glycyrrhetinic acid-paclitaxel (OA-GA-PTX) co-assembled nanoparticles;
FIG. 8 is a drug loading curve of nanoparticles prepared from oleanolic acid-glycyrrhetinic acid (OA-GA) at different ratios;
fig. 9 is a graph of in vitro release of oleanolic acid-glycyrrhetinic acid-paclitaxel (OA-GA-PTX) and road acid-paclitaxel (BTA-PTX) drug-loaded nanoparticles in different pH release media, (a) oleanolic acid-glycyrrhetinic acid-paclitaxel, (b) road acid-paclitaxel;
FIG. 10 is an in vitro cytotoxicity assay of oleanolic acid-glycyrrhetinic acid (OA-GA) and oleanolic acid-glycyrrhetinic acid-paclitaxel (OA-GA-PTX) nanoparticles, (a) the nanoparticles have an inhibitory effect on MCF-7 cell growth for 48h, and (b) the nanoparticles have an inhibitory effect on 4T1 cell growth for 48 h;
FIG. 11 is an in vitro cytotoxicity test of liquidambaric acid-paclitaxel (BTA-PTX) nanoparticles, (a) nanoparticles have an inhibition effect on 4T1 cell growth for 48h, and (b) nanoparticles have an inhibition effect on MCF-7 cell growth for 48 h;
FIG. 12 is a graph showing the change in tumor volume of tumor-bearing mice treated with different nano-formulations, (a) tumor growth curves, and (b) three-dimensional images of tumors from different treatment groups;
FIG. 13 is a plot of tumor volume change following BTA-PTX co-assembly nanoparticles treatment in tumor-bearing mice;
FIG. 14 is a graph of the change in tumor volume following treatment of GA-BTA co-packaged nanoparticles in tumor-bearing mice;
FIG. 15 is an H & E staining image of vital tissue and organ sections after treatment with different nano-formulations.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
Example 1:
the present embodiment provides natural small molecule co-assembled Nanoparticles (NPs) prepared mainly from liquidambaric acid (BTA) and Paclitaxel (PTX). In this embodiment, the method for preparing the liquidambaric acid-paclitaxel (BTA-PTX) nanoparticles by using an emulsion solvent volatilization method comprises the following specific steps:
5mg of liquidambaric acid and 1.75mg of paclitaxel are dissolved in 1.0mL of dichloromethane (a small amount of methanol can be added as a cosolvent if the liquidambaric acid and the paclitaxel cannot be completely dissolved), then the mixture is added into 3.0mL of 2.5% polyvinyl alcohol (PVA) aqueous solution (w/v) under the condition of vortex oscillation, the mixture is subjected to vortex oscillation for 60s, and the vortex liquid is immediately transferred to a probe type ultrasonic instrument to perform ultrasonic treatment on the mixture for 60 s. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
As can be seen from fig. 1: the formed nano particles are of a nano spherical structure with uniform appearance, the average particle diameter is less than 200nm, and the dispersibility is excellent. As can be seen from fig. 6: the co-assembled nanoparticles formed by BTA-PTX are in a core-shell structure.
Example 2:
the present example provides natural small molecule co-assembled Nanoparticles (NPs) prepared mainly from Oleanolic Acid (OA) and Glycyrrhetinic Acid (GA). In the embodiment, oleanolic acid-glycyrrhetinic acid (OA-GA) nanoparticles are prepared by an emulsion solvent volatilization method, and the method comprises the following specific steps:
dissolving 2.5mg of oleanolic acid and 2.5mg of glycyrrhetinic acid in 1.0mL of dichloromethane (a small amount of methanol can be added as a cosolvent if the oleanolic acid and the glycyrrhetinic acid cannot be completely dissolved), adding the mixture into 3.0mL of 2.5% polyvinyl alcohol (PVA) aqueous solution (w/v) under the condition of vortex oscillation, carrying out vortex oscillation for 60s, and immediately transferring the vortex liquid to a probe type ultrasonic instrument to carry out ultrasonic treatment on the mixture for 60 s. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
As can be seen from fig. 2: the formed nano particles are of a nano spherical structure with uniform appearance, the average particle diameter is less than 200nm, and the dispersibility is excellent. As can be seen from fig. 6: the co-assembled nanoparticles formed by OA-GA are in the shape of solid spheres.
Example 3:
the present example provides a natural small molecule co-assembled Nanoparticle (NPs) prepared mainly from betulin (Bet) and Glycyrrhetinic Acid (GA). In this example, betulin-glycyrrhetinic acid (Bet-GA) nanoparticles are prepared by an emulsion solvent evaporation method, and the specific steps are as follows:
2.5mg of betulin and 2.5mg of glycyrrhetinic acid are dissolved in 1.0mL of dichloromethane (a small amount of methanol is added as a cosolvent if the betulin and the glycyrrhetinic acid cannot be completely dissolved), and then added into 3.0mL of 2.5% polyvinyl alcohol (PVA) aqueous solution (w/v) under the condition of vortex oscillation for 60 seconds, and the vortex liquid is immediately transferred to a probe type ultrasonic instrument to carry out ultrasonic treatment on the mixture for 60 seconds. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
As can be seen from fig. 3: the formed nano particles are of a nano spherical structure with uniform appearance, the average particle diameter is less than 200nm, and the dispersibility is excellent.
Example 4:
the embodiment provides natural small molecule co-assembled Nanoparticles (NPs), which are mainly prepared from liquidambaric acid (BTA) and Glycyrrhetinic Acid (GA). In this embodiment, an emulsion solvent volatilization method is adopted to prepare the liquidambaric acid-glycyrrhetinic acid (BTA-GA) nanoparticles, and the specific steps are as follows:
dissolving 2.5mg of liquidambaric acid and 2.5mg of glycyrrhetinic acid in 1.0mL of dichloromethane (if the materials cannot be completely dissolved, a small amount of methanol can be added as a cosolvent), adding the solution into 3.0mL of 2.5% polyvinyl alcohol (PVA) aqueous solution (w/v) under the condition of vortex oscillation, carrying out vortex oscillation for 60s, and immediately transferring the vortex solution to a probe type ultrasonic instrument to carry out ultrasonic treatment on the mixture for 60 s. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
As can be seen from fig. 4: the formed nano particles are of a nano spherical structure with uniform appearance, the average particle diameter is less than 200nm, and the dispersibility is excellent. As can be seen from fig. 6: the co-assembled nano particles formed by the BTA-GA have a core-shell structure.
Example 5:
the present example provides natural small molecule co-assembled Nanoparticles (NPs) prepared mainly from Oleanolic Acid (OA) and Betulinic Acid (BA). In the embodiment, oleanolic acid-betulinic acid (OA-BA) nanoparticles are prepared by an emulsion solvent evaporation method, and the method comprises the following specific steps:
2.5mg of oleanolic acid and 2.5mg of betulinic acid were dissolved in 1.0mL of dichloromethane (a small amount of methanol was added as a cosolvent if not completely dissolved), and then added to 3.0mL of a 2.5% aqueous solution (w/v) of polyvinyl alcohol (PVA) under vortex shaking conditions, vortexed for 60s, and the vortexed solution was immediately transferred to a probe sonicator to sonicate the mixture for 60 s. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
As can be seen from fig. 5: the structure of the nano particle formed by the co-assembly of oleanolic acid and betulinic acid is nano fiber.
Example 6:
the embodiment provides natural small molecule co-assembled Nanoparticles (NPs), which are mainly prepared from Oleanolic Acid (OA), liquidambaric acid (BTA) and Glycyrrhetinic Acid (GA). In this embodiment, oleanolic acid-liquidambaric acid-glycyrrhetinic acid (OA-BTA-GA) nanoparticles are prepared by an emulsion solvent volatilization method, which specifically includes the following steps:
dissolving 2mg of oleanolic acid, 2mg of liquidambaric acid and 2mg of betulinic acid in 1.0mL of dichloromethane (if the oleanolic acid, the liquidambaric acid and the betulinic acid cannot be completely dissolved, a small amount of methanol can be added as a cosolvent), then adding the mixture into 3.0mL of 2.5% polyvinyl alcohol (PVA) aqueous solution (w/v) under the condition of vortex oscillation, carrying out vortex oscillation for 60s, and immediately transferring the vortex liquid to a probe type ultrasonic instrument to carry out ultrasonic treatment on the mixture for 60 s. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
Example 7:
the present example provides natural small molecule co-assembled Nanoparticles (NPs) prepared mainly from Oleanolic Acid (OA), Glycyrrhetinic Acid (GA), and Paclitaxel (PTX). In this embodiment, oleanolic acid-glycyrrhetinic acid-paclitaxel (OA-GA-PTX) nanoparticles are prepared by an emulsion solvent evaporation method, and the specific steps are as follows:
5.0mg of oleanolic acid, 2.0mg of glycyrrhetinic acid and 1.25mg of paclitaxel are dissolved in 1.0mL of dichloromethane (a small amount of methanol is added as a cosolvent if the oleanolic acid, the glycyrrhetinic acid and the paclitaxel can not be completely dissolved), then the mixture is added into 3.0mL of 2.5% polyvinyl alcohol (PVA) aqueous solution (w/v) under the condition of vortex oscillation, the mixture is subjected to vortex oscillation for 60s, and the vortex liquid is immediately transferred to a probe type ultrasonic instrument to perform ultrasonic treatment on the mixture for 60 s. Then, the resulting emulsion was added dropwise to 30mL of a 0.3% aqueous PVA solution (w/v) at a magnetic stirring speed of 400 rpm. The solution is stirred at room temperature for 6-8 hours to remove the organic solvent. The nanoparticles were then centrifuged at 12000rpm for 40 minutes at 4 ℃ and the nanoparticles collected, washed twice with double distilled water (to remove excess PVA and free drug) and lyophilized for use.
As can be seen from fig. 7: the morphology of the ternary co-assembled nanoparticle OA-GA-PTX is a solid sphere.
Example 8: nanoparticle characterization
1. Three-phase contact angle measurements were carried out at room temperature, and the lyophilized powders were all placed uniformly on a glass slide (average diameter 13 mm, average thickness 2 mm). 5 mu L of liquid drop deionized water is dripped on the surface of the sample, after balancing for 10 seconds, the liquid drop is shot by using a high-speed camera, the outline of the imaged liquid drop is simulated, the three-phase contact angle is obtained, and the measurement is carried out at least three times.
2. OA-GA nanoparticle drug loading and encapsulation efficiency determination
OA-GA is used as a drug carrier, PTX is used as a drug to carry out a drug loading experiment, and a high performance liquid chromatography system is used for measuring the concentration of paclitaxel in the drug-loaded nanoparticle preparation. Dissolving the drug-loaded nano particle freeze-dried powder in dimethyl sulfoxide to destroy nano particles and release loaded drugs. The PTX content was analyzed by filtration through a 0.22 μm syringe filter with acetonitrile/water (65/35, v/v) as mobile phase and with a detector wavelength of 227 nm.
FIG. 8 shows the loading and encapsulation of paclitaxel by nanoparticles prepared from oleanolic acid and glycyrrhetinic acid at different ratios. Wherein, the oleanolic acid and the glycyrrhetinic acid for preparing the drug carrier can be mixed according to different proportions to prepare a co-assembled drug carrier, and the prepared drug carriers can be loaded with paclitaxel.
3. In vitro Release assay
The in vitro release of the nanoparticles was determined at 37 deg.C, pH 7.3, pH 6.8 and pH 5.5, respectively. The nanoparticles were placed separately in dialysis bags. 1L of PBS buffer solution with the pH values of 5.5, 6.8 and 7.4 respectively is prepared, sodium salicylate (1.0mol/L) is added as a medium, a dialysis bag containing a nano particle sample is transferred into the medium, stirring is carried out by adopting a rotary basket method, the set parameters are the rotating speed of 150r/min, the temperature is 37 ℃, and three groups are parallel. Respectively sucking 200 μ L of sample liquid in the dialysis bag at different time points, adding 200 μ L of methanol for demulsification, fully dissolving under a vortex oscillator, passing through a 0.22 μm nylon microporous filter membrane, measuring the content of PTX by high performance liquid chromatography, quantitatively analyzing, and evaluating the release characteristics of the nanoparticles.
As can be seen from fig. 9: the cumulative PTX release of OA-GA-PTX NPs at pH 7.3, pH 6.8 and pH 5.5 was about 60%, 43% and 41%, respectively; the cumulative PTX release of BTA-PTX NPs was about 90%, 80% and 60%, respectively. The nano drug-loaded particles show a certain slow release characteristic, have no burst release phenomenon at the initial release stage, and are expected to prevent the premature leakage of the drug in vivo.
Example 9: in vitro cell experiments
Fig. 10 to 11 show in vitro cell experiment results of different co-assembled nanoparticles, and the results show that the co-assembled nanoparticles have significant tumor cell proliferation inhibition effect and concentration dependence, and the combination index calculation shows that the combination indexes under IC50 are both less than 1.0, indicating that the two compounds constituting the nanoparticles have synergistic anti-tumor effect; under the same equivalent of paclitaxel, the drug-loaded nanoparticles enhance the inhibition rate of MCF-7 cells and 4T1 cells, and can further improve the anti-tumor effect.
Example 10: in vivo antitumor effect
FIG. 12 shows that the tumor inhibition rate of OA nanoparticle alone is 42.6%, and the tumor inhibition rate of OA-GA co-assembled nanoparticle is increased to 64.8%; the tumor inhibition rate of the OA drug-loaded nanoparticle (OA-PTX) is 76.5%, and the tumor inhibition rate of the OA-GA drug-loaded nanoparticle (OA-GA-PTX) is improved to 82.6%. In terms of treatment effect, the drug-loaded nanoparticles in the co-group have higher tumor inhibition effect than the drug-loaded nanoparticles.
Fig. 13 shows that compared with the tumor inhibition rate of paclitaxel injection, the tumor inhibition rate of the BTA-PTX co-assembled nanoparticles is increased from 58% to 74%, which indicates that the co-assembled nanoparticles have better therapeutic effect.
FIG. 14 shows that the GA-BTA co-assembled nanoparticles have better tumor treatment effect, and the tumor inhibition rate is as high as 65%.
Accordingly, as can be seen from fig. 12 to 14: the single nano particles have different degrees of inhibition on tumors, the tumor inhibition rate of the co-assembled nano particles is obviously improved, and the anti-tumor effect is further enhanced after the ternary co-assembled nano particles are formed by loading the medicine. These results indicate that the nano drug delivery system constructed from pharmacologically active natural small molecules can achieve synergistic effects.
Example 12: evaluation of side effects
And respectively measuring the biochemical index of typical liver tissue damage and the antioxidant index of liver tissue in serum. The results show that the indexes of the mice with the co-assembled nanoparticle group are slowed down to a certain extent compared with those of the free drug group and the inactive carrier group, which indicates that the co-assembled nanoparticles play a positive role in a delivery system, and pathological analysis is performed after H & E staining is performed on important tissue sections of each group, and the results are shown in FIG. 15. As can be seen from fig. 15: the natural micromolecule co-assembled nanoparticle group can effectively repair each tissue injury, and the natural micromolecules can slow down the tissue injury caused by chemotherapeutic drugs to a certain extent.
Claims (10)
1. A natural small molecule co-assembled nano-drug delivery system is characterized in that the natural small molecule co-assembled nano-drug delivery system is a nano-particle formed by co-assembling two or more than two of oleanolic acid, ursolic acid, glycyrrhetinic acid, betulinic acid, betulin, liquidambaric acid, lupeol, paclitaxel, rhein and catechin.
2. The natural small molecule co-assembled nano-drug delivery system according to claim 1, wherein the natural small molecule co-assembled nano-drug delivery system is a nanoparticle co-assembled from liquidambaric acid-paclitaxel, oleanolic acid-glycyrrhetinic acid, betulin-glycyrrhetinic acid, liquidambaric acid-glycyrrhetinic acid, oleanolic acid-betulinic acid, oleanolic acid-liquidambaric acid-glycyrrhetinic acid, or oleanolic acid-glycyrrhetinic acid-paclitaxel.
3. The natural small molecule co-assembled nano drug delivery system of claim 1, characterized in that the nanoparticles are nanospheres, nanofibers or nanorod structures.
4. The natural small molecule co-assembled nano drug delivery system of claim 1, wherein the average particle size of the nanoparticles is 100-1000 nm.
5. A method for preparing the natural small molecule co-assembled nano drug delivery system according to any one of claims 1 to 4, wherein the method comprises the following steps:
step one, natural micromolecules are used as raw materials, and are dissolved in a benign organic solvent to obtain a natural micromolecule solution;
step two, adding the natural micromolecule solution obtained in the step one into a surfactant solution with the mass concentration of 1-5%, and performing vortex emulsification for 0.5-10 minutes to obtain a suspension;
step three, further emulsifying the suspension in the step two;
step four, adding the emulsion formed in the step three into an aqueous solution containing a surfactant with the mass concentration of 0.1-1.0%, stirring, and completely volatilizing the organic solvent;
step five, carrying out centrifugal treatment and double-distilled water washing on the solution in the step four, removing residual surfactant and harvesting nano particles;
step six, adding the nanoparticles obtained in the step five into water, normal saline or PBS to prepare a suspension for later use; or drying or freeze-drying the nanoparticles harvested in the fifth step for long-term storage.
6. The method for preparing a natural small molecule co-assembled nano-drug delivery system according to claim 5, wherein the benign organic solvent is one or more of methanol, ethanol, acetone, petroleum ether, ethyl acetate, dichloromethane, chloroform, tetrachloromethane and n-hexane; the surfactant is one of an acid surfactant, an alkaline surfactant and a neutral surfactant, and is preferably polyvinyl alcohol.
7. Use of the natural small molecule co-assembled nano-drug delivery system of any one of claims 1 to 4 for drug loading.
8. Use of the natural small molecule co-assembled nano-drug delivery system of any one of claims 1 to 4 in a drug for treating cancer.
9. Use of the natural small molecule co-assembled nano-drug delivery system of any one of claims 1 to 4 in a drug for treatment of liver disease.
10. Use of the natural small molecule co-assembled nano-drug delivery system of any one of claims 1 to 4 for alleviating side effects of drugs.
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