CN110840837A

CN110840837A - Tetrandrine nanosuspension and preparation method and application thereof

Info

Publication number: CN110840837A
Application number: CN201911254937.0A
Authority: CN
Inventors: 杨建宏; 郭珏铄; 买亚萍; 侯延辉; 李莉; 王锐
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-02-28
Anticipated expiration: 2039-12-10
Also published as: CN110840837B

Abstract

The invention relates to the field of pharmaceutical preparations, and particularly relates to a tetrandrine nanosuspension, which is obtained by nanocrystallizing tetrandrine and a stabilizer through a dispersion medium, wherein the particle size of the tetrandrine nanosuspension is 50-600nm, the absolute value of Zeta potential is 10-40 mV, and the mass ratio of a drug to the stabilizer is 1: 0.1-1, and particularly discloses a preparation method of the tetrandrine nanosuspension and application of the tetrandrine nanosuspension in preparation of antitumor drugs. The particle size of the tetrandrine nanosuspension is 50-600nm, the stability is good, the solubility and the dissolution rate of tetrandrine can be remarkably improved, compared with the raw material drug of tetrandrine, the tetrandrine nanosuspension has remarkable in-vitro tumor cell inhibition rate, remarkably improved apoptosis rate and cell uptake rate, remarkably improved tetrandrine dissolution rate and antitumor activity, and wide clinical application prospect.

Description

Tetrandrine nanosuspension and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical preparations, in particular to a tetrandrine nano suspension and a preparation method and application thereof.

Background

In recent years, cancer is a major disease affecting human health, and the incidence and mortality of cancer tend to increase year by year. Among many tumors, lung cancer is the most frequent malignant tumor with high incidence and mortality. At present, the conventional treatment methods for tumors mainly comprise surgical treatment, radiotherapy and chemotherapy, and the conventional treatment methods have poor curative effect and have a plurality of side effects. Natural compounds attract more and more attention due to the characteristics of low toxic and side effects, reversion of tumor multidrug resistance and the like.

Tetrandrine (Tet) is a natural dibenzylisoquinoline alkaloid extracted from the root tuber of tetrandrine, a family Menispermaceae plant, and researches show that tetrandrine is a promising antitumor drug and has an inhibiting effect on various tumor cell lines, including bladder cancer cells, liver cancer cells, breast cancer cells, human oral cancer cells, cervical cancer cells and the like, and proves that tetrandrine has an important effect on lung cancer through a VEGF/HIF-1a/ICAM-1 signal channel and induces apoptosis of lung cancer cells. However, the water solubility of the tetrandrine is poor when the tetrandrine is directly used as an antitumor drug, and researches show that the saturated solubility of the tetrandrine in a phosphate buffer solution with the pH value of 7.4 is only 0.015mg/ml, and the tetrandrine has the defects of low bioavailability, instability, no selectivity to tumor tissues and normal tissues, poor targeting effect, small safety range of the tetrandrine, wide toxic and side effects and the like due to poor solubility in a physiological environment, so that the clinical application range of the tetrandrine is limited to a certain extent.

The nano drug-carrying system can better improve the dissolution of insoluble drugs, improve the curative effect of chemotherapeutic drugs and reduce the side effect of local or whole body. Solid lipid nanoparticles, nanoemulsion delivery systems, microspheres and the like have been studied to solve the problem of poor dissolution, for example, Wang Yangli develops tetrandrine liposome, prolongs the action time of the drug, and improves the bioavailability (taken from Wang Yangli, the development of tetrandrine liposome [ D ]. Henan university, 2008.); a tetrandrine nanoemulsion injection and its preparation method (patent No. 200810024843.X) are disclosed, which comprises tetrandrine, surfactant, cosurfactant, oil phase and water phase. The nano-emulsion has simple preparation method, transparent appearance and particle size of I0-l00 nm; the nano-emulsion has good stability; the solubility of the tetrandrine can be obviously improved, so that the bioavailability of the medicine is improved; and the in vivo process of the medicine is changed, the targeting property to the liver is increased, the toxic and side effects are reduced, and the curative effect is improved. However, the technical scheme has the defects of low drug loading rate, low encapsulation efficiency, more side effects caused by auxiliary materials and the like.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a tetrandrine nanosuspension.

The invention also aims to provide a preparation method of the tetrandrine nanosuspension, the preparation method has the characteristics of simple preparation process, small using amount of the stabilizer, no use of organic solvent, high drug-loading rate and the like, and the prepared tetrandrine nanosuspension not only improves the dissolution rate, but also obviously improves the anti-tumor activity.

The invention also aims to provide the application of the tetrandrine nano suspension in preparing anti-tumor drugs.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the tetrandrine nanosuspension is obtained by nanocrystallizing tetrandrine and a stabilizer through a dispersion medium, the particle size of the tetrandrine nanosuspension is 50-600nm, the absolute value of Zeta potential is 10-40 mV, and the mass ratio of a drug to the stabilizer is 1: 0.1 to 1.

The tetrandrine nanosuspension is prepared by the following method:

(1) weighing tetrandrine and stabilizer according to a ratio;

(2) sequentially adding a stabilizer and tetrandrine into the dispersion medium, and stirring for 0.5-2 h to obtain a coarse suspension;

(3) and sequentially adding grinding beads and the coarse mixed suspension into a grinding chamber of a nano grinder, and grinding to obtain the tetrandrine nano mixed suspension.

Preferably, the stabilizer in step (1) is a steric stabilizer and/or a charge stabilizer.

Preferably, the space stabilizer is one or more of polyvinylpyrrolidone, poloxamer, hydroxypropyl cellulose, vitamin E polyethylene glycol succinate, croscarmellose sodium and crospovidone.

Preferably, the vitamin E polyethylene glycol succinate and the chitosan quaternary ammonium salt are adsorbed on nanoparticles in the tetrandrine nanosuspension.

Preferably, the charge stabilizer is one or more of sodium dodecyl sulfate, benzethonium chloride and dioctyl sodium sulfosuccinate.

Preferably, the vitamin E polyethylene glycol succinate and the chitosan quaternary ammonium salt are used as carriers to increase the stability and improve the anti-tumor activity of the tetrandrine nano suspension.

The nano-treatment mode comprises a wet grinding method, a high-pressure homogenization method or an anti-solvent precipitation method, and preferably the wet grinding method.

Preferably, the dispersion medium in step (2) is purified water;

preferably, the grinding beads in the step (3) have a particle size of 0.2-0.8 mm and are made of glass or yttrium-stabilized zirconia or polystyrene derivative polymer; the grinding time is 0.5-3 h; the rotating speed of a stirring shaft of the grinding machine is 1000-3000 rpm; the grinding temperature is controlled to be 10-40 ℃.

Preferably, the tetrandrine exists in the tetrandrine nanosuspension in a crystal form and an amorphous state.

Preferably, the tetrandrine nanosuspension is used as a preparation intermediate, and pharmaceutically acceptable auxiliary materials are added to prepare tablets, capsules, injections, sustained-release agents, controlled-release agents and granules.

The tetrandrine nanosuspension is applied to the preparation of antitumor drugs, wherein tumors comprise breast cancer, lung cancer, bladder cancer, liver cancer, human oral cancer, cervical cancer and human glioblastoma.

The invention has the beneficial effects that:

1) the tetrandrine nanosuspension disclosed by the invention is simple in preparation method, less in stabilizer use type and usage amount, the organic solvent is not used in the preparation process, the production is easy, the drug-loading rate of the prepared tetrandrine nanosuspension is large, and the use of the organic solvent is avoided.

2) The tetrandrine exists in the tetrandrine nanometer suspension in a crystalline state, an amorphous state or a mixed state.

3) The tetrandrine nanosuspension of the invention obviously improves the accumulative dissolution rate of tetrandrine by 30-80%, and the possible reasons include that after the ① medicament is prepared into the nanosuspension, the specific surface area is increased due to the nanoscale particle size of the nanosuspension, so that the dissolution rate is increased, ② the dissolution rate is increased due to the fact that part of crystal form in the nanosuspension is converted into amorphous state, and the dissolution rate can also be increased due to the wetting effect of ③ water-soluble carrier on insoluble medicament.

4) The tetrandrine nanosuspension remarkably enhances the anti-tumor activity of the tetrandrine. In vitro cytotoxicity experiments show that the stabilizer used by the tetrandrine nano suspension has good safety and biocompatibility; the tetrandrine nanometer suspension has a cell survival rate of less than 10% in 48h for lung cancer A549 cellsAnd IC₅₀The value is lower than that of the tetrandrine raw medicine group. The apoptosis rate of the tetrandrine nano suspension is 2-4 times of that of the bulk drug. Compared with the tetrandrine bulk drug, the tetrandrine nano suspension promotes the uptake of lung cancer A549 cells. The nanometer particle size of the nanometer suspension is probably attributed to the fact that the nanometer particle size is beneficial to the adhesion of particles and tumor cells, and the particles are more easily taken up by the cells, so that the nanometer suspension has stronger anti-tumor effect. And the property of the carrier plays a part of promoting role in anti-tumor effect, the carrier has biological adhesiveness, can increase the combination of the drug particles and a mucus layer, promotes the permeation of the mucus layer, shows stronger anti-tumor activity, and can induce mitochondrial damage by improving the level of intracellular drugs so as to increase the apoptosis capacity of the drug-induced cells. In addition, one part of the crystal form of the nano suspension is converted into an amorphous state, the release rate is faster, and the anti-tumor effect of the tetrandrine is enhanced.

In conclusion, the tetrandrine nanosuspension prepared by the invention can improve the solubility and dissolution rate of tetrandrine and improve the anti-tumor activity of tetrandrine.

Drawings

FIG. 1 is a scanning electron microscope image of the tetrandrine nanosuspension of the invention.

FIG. A: a lyophilized powder of tetrandrine suspension without stabilizer. And B: lyophilized powder of Tet-CCS-NS. And (C) figure: lyophilized powder of Tet-HACC-TPGS-NS.

FIG. 2 is the energy dispersive X-ray spectrogram of the tetrandrine nanosuspension of the invention.

FIG. 3 is a differential scanning calorimetry analysis chart of the tetrandrine nanosuspension of the present invention.

FIG. 4 is an X-ray diffraction pattern of the tetrandrine nanosuspension of the invention.

FIG. 5 is an infrared spectrum of the tetrandrine nanosuspension of the present invention.

FIG. 6 is a dissolution curve diagram of the tetrandrine nanosuspension of the present invention.

Fig. 7 is the in vitro cytotoxicity of a549 cells.

FIG. A, B, C is a graph showing the toxicity results of A549 cells incubated with vehicle solutions for 24h, 48h, and 72h, respectively. FIG. D, E, F is a graph showing the toxicity results of A549 cells incubated with tetrandrine nanosuspensions for 24h, 48h and 72h, respectively.

FIG. 8 is the apoptosis graph (A) and apoptosis bar graph (B) detected by flow cytometry after A549 cells and tetrandrine nanosuspension are incubated for 24 h.

Fig. 9 shows a confocal laser qualitative uptake (a) and a quantitative uptake (B) of a549 cells.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to specific examples, but the scope of the present invention is not limited to the following examples, and technical solutions obtained by changing parameters of the technical solutions by those skilled in the art all belong to the scope of the present invention, which can be understood by those skilled in the art.

In the specific embodiments of the present invention, unless otherwise specified, the experimental methods involved are all conventional in the art; unless otherwise specified, reagents used to complete the following examples are commercially available.

Noun abbreviation list:

components	Abbreviations	Components	Abbreviations
				Tetrandrine	Tet	Sodium dodecyl sulfate	SDS
polyvinylpyrrolidone-K30	PVP K30	Quaternary ammonium salt of chitosan	HACC
				Poloxamer 188	P188	Infrared spectroscopy	FTIR
Vitamin E polyethylene glycol succinate	TPGS	Diffraction by X-ray	XRD
				Hydroxypropyl cellulose	HPC-SSL	Differential scanning thermal method	DSC
Croscarmellose sodium	CCS	Scanning electron microscope	SEM
				Nanosuspension	NS	X-ray spectroscopy	EDS

Example 1: preparation of tetrandrine nanometer suspension

Tetrandrine nanosuspension 1: PVP K300.1g is weighed, added into 50ml of distilled water, stirred under a magnetic stirrer to obtain a stabilizer solution, then 0.25g of tetrandrine is added, and the mixture is stirred for 30min under magnetic force to obtain a crude mixed suspension. Before grinding, 140ml of zirconium oxide grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity, and grinding is carried out for 30min at the rotating speed of 2000rpm, so as to obtain the tetrandrine nanometer suspension. The resulting preparation, after dilution 2-fold with purified water, was found to have a particle size of 312nm and a potential of-21.2 mV.

Tetrandrine nanosuspension 2: 1880.1 g of poloxamer is weighed and added into 50ml of distilled water, the stabilizer solution is obtained after even stirring, 0.5g of tetrandrine is added under a magnetic stirrer, and the coarse suspension is obtained after magnetic stirring for 30 min. Before grinding, 140ml of glass grinding beads having a particle size of 0.4 to 0.6mm were added to the grinding chamber, and ground at 2000rpm for 30min, and the resulting sample was diluted 4-fold with purified water to find a particle size of 512.2nm and a potential of-19.3 mV.

Tetrandrine nanosuspension 3: weighing 0.1g of croscarmellose sodium, adding into 50ml of distilled water, stirring under a magnetic stirrer to obtain a stabilizer solution, adding 0.25g of tetrandrine, and magnetically stirring for 30min to obtain a crude suspension. Before grinding, 140ml of zirconium oxide grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity, and grinding is carried out for 45min at the rotating speed of 2500rpm, so as to obtain the tetrandrine nanometer suspension. The resulting preparation, after dilution 2-fold with purified water, had a particle size of 469.1nm, measured at a potential of-29.4 mV.

Tetrandrine nanosuspension 4: adding 0.05g of HPC-SSL and 0.05g of SDS into 50ml of distilled water, stirring under a magnetic stirrer to obtain a stabilizer solution, adding 1g of tetrandrine, and magnetically stirring for 30min to obtain a crude suspension. Before grinding, 140ml of zirconium oxide grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity, and grinding is carried out for 45min at the rotating speed of 2500rpm, so as to obtain the tetrandrine nanometer suspension. The resulting preparation, after dilution 2-fold with purified water, was found to have a particle size of 217.6nm, a potential of-33.8 mV.

Tetrandrine nanosuspension 5: adding 0.05g of chitosan quaternary ammonium salt and 0.05g of TPGS into 50ml of distilled water, stirring under a magnetic stirrer to obtain a stabilizer solution, adding 0.25g of tetrandrine, and magnetically stirring for 30min to obtain a crude suspension. Before grinding, 140ml of zirconium oxide grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity, and grinding is carried out for 45min at the rotating speed of 2500rpm, so as to obtain the tetrandrine nanometer suspension. The resulting preparation, after dilution 2-fold with purified water, was found to have a particle size of 157.3nm and a potential of 23.3 mV.

The particle size, PDI value and potential distribution of the tetrandrine nanosuspension are shown in Table 1.

TABLE 1 particle size, PDI value and potential of tetrandrine nanosuspensions

As can be seen from Table 1, the particle size, PDI value and Zeta potential of the prepared tetrandrine nanosuspension are different, and the nanosuspension is screened by combining three factors of the particle size, the PDI value and the Zeta potential; the tetrandrine nanosuspensions Tet-PVP K30-NS and Tet-P188-NS have larger PDI value and poor solution stability; the stabilizer SDS in the tetrandrine nanosuspension Tet-HPC-SSL-SDS-NS has certain toxicity to cells and is not beneficial to use; in conclusion, the tetrandrine nanosuspension Tet-CCS-NS with good solution stability and the tetrandrine nanosuspension Tet-HACC-TPGS-NS which is more easily combined with cell membranes are selected for subsequent experiments.

Example II, Freeze-drying process and characterization of tetrandrine nanosuspension

1. Experimental groups and solution preparation

Tet-MS group: 0.25g of tetrandrine is weighed and added into 50ml of distilled water, and stirred for 30min under a magnetic stirrer to obtain a crude mixed suspension. Before grinding, 140ml of zirconium oxide grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity, and grinding is carried out for 45min at the rotating speed of 2500rpm, so as to obtain the tetrandrine suspension.

Tet-CCS-NS group: the preparation method is the same as the tetrandrine nanosuspension 3 in example 1.

The Tet-HACC-TPGS-NS group: the preparation method is the same as the tetrandrine nanosuspension 5 in example 1.

2. Procedure of experiment

Adding 5% of lyophilized protectant mannitol and lactose into the 3 groups of tetrandrine suspension, shaking, mixing, pre-freezing at-80 deg.C in ultra-low temperature refrigerator for 24 hr, quickly transferring to freeze drier, and freeze drying at-40 deg.C under vacuum for 12 hr to obtain tetrandrine suspension lyophilized powder.

The lyophilized powder was characterized by SEM, EDS, DSC, XRD, FTIR.

3. Results of the experiment

The scanning electron microscope results of the tetrandrine, the stabilizer and the nanosuspension thereof are shown in figure 1; the energy dispersive X-ray spectroscopy result is shown in FIG. 2, and it can be seen from A chart in FIGS. 1 and 2 that in the stabilizer-free tetrandrine suspension, the tetrandrine is in the form of uneven rod-like and rectangular particles; as can be seen from the B diagrams in the figures 1 and 2, in the Tet-CCS-NS, the CCS is crosslinked with the tetrandrine to form a sheet shape, and the nanosuspension does not influence the composition of surface elements, which may be caused by the fact that the CCS cannot be adsorbed on the particle surface; as can be seen from the C diagrams in FIGS. 1 and 2, in Tet-HACC-TPGS-NS, the bulk drug and the stabilizer form a quasi-circular nanosuspension, and compared with the hanfangchin A suspension without the stabilizer, the nanosuspension does not affect the surface element composition, and the TPGS may be covered by a part of the drug at the detected part.

The differential scanning calorimetry analysis results of the tetrandrine, the stabilizer and the nanosuspension are shown in fig. 3; the X-ray diffraction results are shown in fig. 4, and as can be seen from fig. 3 and 4, the endothermic melting peak intensities of the Tet-CCS-NS and the Tet-HACC-TPGS-NS are significantly reduced compared with the group of crude drugs and the corresponding physical mixture, which proves that a part of the crystal forms is transformed into an amorphous state in the grinding or freeze-drying process, and further XRD analysis shows that the characteristic crystal diffraction peak intensities of the tetrandrine in the Tet-CCS-NS and the Tet-HACC-TPGS-NS are significantly reduced, which proves that a part of the crystal forms is transformed into an amorphous state in the grinding or freeze-drying process.

The results of the IR spectroscopy analysis of the tetrandrine, the stabilizer and the nanosuspension are shown in FIG. 5. As can be seen from FIG. 5, no new peak appears in the tetrandrine group, the Tet-CCS-NS group and the Tet-HACC-TPGS-NS group, and the positions of the peaks do not obviously move, which indicates that no obvious interaction occurs between the drug tetrandrine and the stabilizer, wherein the TPGS carrier is 1740cm^-1Peaks at (a) were absent or of lower peak intensity in both its physical mixture and nanosuspension, probably due to TPGS being covered by a portion of the drug.

EXAMPLE III determination of dissolution of tetrandrine nanosuspensions

1. Experimental groups and solution preparation

Tet raw material medicine group: weighing 1.74mg of the tetrandrine raw material medicine;

Tet-CCS-NS group: measuring 680 mu l of the tetrandrine nanosuspension prepared in the first embodiment;

the Tet-HACC-TPGS-NS group: measuring 377 mul of the tetrandrine nanosuspension prepared in the first embodiment;

2. procedure of experiment

The dissolution rate was measured according to the second method (paddle method) of 0931, four parts of the pharmacopoeia of China, 2015 edition, using 900ml of purified water as dissolution medium, at 37 + -0.5 deg.C and 100 rpm/min. When the time was started when the sample was in contact with the dissolution medium, 5ml of the same isothermal dissolution medium was sampled at 5, 10, 15, 20, 30, 45, 60 and 120min, and 5ml of the same isothermal dissolution medium was added, and the sample was filtered through a 0.45 μm microporous membrane and the absorbance at 297.6nm was measured with an ultraviolet spectrophotometer.

3. Results of the experiment

The measured dissolution curve is shown in fig. 6, it can be seen from the figure that the accumulative dissolution rate of the tetrandrine in water is improved by about 5 times compared with the raw material drug, the possible reasons are that the specific surface area of the tetrandrine nanosuspension is increased due to ① nano-scale particle size, so that the dissolution rate is improved, ② crystal form transformation is carried out, the crystal form transformation is amorphous after the transformation, so that the dissolution rate is improved, and ③ water-soluble stabilizing agent has a wetting effect on the tetrandrine.

EXAMPLE IV cytotoxicity test of tetrandrine nanosuspension

1. Experimental groups and solution preparation

CCS-solution group: 0.1g of croscarmellose sodium is weighed, added into 50ml of distilled water, and stirred for 30min under a magnetic stirrer to obtain a stabilizer solution. Before grinding, 140ml of zirconia grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity and ground for 45min at the rotating speed of 2500rpm, so that CCS-solution is obtained.

HACC-TPGS-solution group: 0.05g of chitosan quaternary ammonium salt and 0.05g of TPGS are weighed and added into 50ml of distilled water, and stirred for 30min under a magnetic stirrer to obtain a stabilizer solution. Before grinding, 140ml of zirconium oxide grinding beads with the particle size of 0.6-0.8mm are added into the grinding cavity and ground for 45min at the rotating speed of 2500rpm, so that HACC-TPGS-solution is obtained.

Tet-CCS-NS group: and adding DMEM culture solution into the nano suspension to dilute the nano suspension to 100 mu g/ml, and then respectively diluting the nano suspension to 50, 40, 30, 20, 10 and 5 mu g/ml.

The Tet-HACC-TPGS-NS group: and adding DMEM culture solution into the nano suspension to dilute the nano suspension to 100 mu g/ml, and then respectively diluting the nano suspension to 50, 40, 30, 20, 10 and 5 mu g/ml.

Tet-solution group: weighing the tetrandrine raw material medicine, dissolving the tetrandrine raw material medicine in methanol to prepare stock solution of 100 mu g/ml, and further diluting the stock solution to 50, 40, 30, 20, 10 and 5 mu g/ml.

2. Procedure of experiment

100 mul of human lung cancer A549 cell suspension is added at 5X 10 per well³Inoculating to a 96-well plate, placing in an incubator, and incubating for 24 h. Discarding the culture solution, setting the wells containing only the culture solution as Control group, adding 100 μ l of drug solution group and preparation group into each well, setting 6 mass concentrations of 5, 10, 20, 30, 40, 50 μ g/mL, each concentration having 3 multiple wells, incubating for 24h, 36h and 48h, respectively, adding 10 μ l of CCK8 solution into each well, incubating in incubator for 2h, measuring OD at 450nm with microplate reader, and calculating IC₅₀The value is obtained.

3. Results of the experiment

The results are shown in Table 2 and FIG. 7.

TABLE 2 IC of tetrandrine nanosuspensions and solution groups₅₀Value of

Shows that the tetrandrine suspension and the tetrandrine solution group have significant difference in the same incubation time (p <0.05)

Indicates that the tetrandrine suspension and the tetrandrine solution group have very significant difference in the same incubation time (p <0.01)

# shows that there is a significant difference between tetrandrine suspensions at the same incubation time (p <0.05)

# indicates that there is a very significant difference between tetrandrine suspensions at the same incubation time (p <0.01)

The cytotoxicity results are shown in fig. 7, and as can be seen from fig. 7A, B, C, the cell survival rates of CCS-solution and HACC-TPGS-solution are all higher than 85% within 48h compared with the control group, which proves that the carrier for preparing the tetrandrine nanosuspension has certain safety. As can be seen from FIG. 7D, E, F, the Tet solution group showed relatively higher cytotoxicity than the two sets of tetrandrine nanosuspensions, especially at high concentrations (30, 40 and 50. mu.g/ml). This may be due to complete dissolution of the free drug. However, the cell survival rate of the two groups of tetrandrine nanometer suspension is obviously reduced along with the prolonging of the time, and the survival rate in 48 hours is lower than 10 percent.

To further evaluate the antitumor effect of the prepared formulations, on IC₅₀The values were analyzed and the results are shown in Table 2, and it is understood from Table 2 that the IC of the Tet-HACC-TPGS-NS group₅₀The time of 36 and 48 hours is obviously lower than that of a Tet solution group and a Tet-CCS-NS group, namely, the nano suspension Tet-HACC-TPGS-NS has better anti-tumor effect, and possible reasons influencing the anti-tumor effect are that ① is favorable for passively targeting tumor tissues due to small-sized particles through enhancing permeability and retention effects, ② HACC shows adhesion to particles and cell membranes③ the positive charge of Tet-HACC-TPGS-NS is more likely to be internalized by the cell due to the negative charge of the cell membrane, and TPGS can improve the accumulation of intracellular drugs.

Example V. tetrandrine nanosuspension apoptosis test

1. Experimental groups and solution preparation

Tet-CCS-NS group: the group of nanosuspensions was diluted to 20. mu.g/ml with DMEM medium.

The Tet-HACC-TPGS-NS group: the group of nanosuspensions was diluted to 20. mu.g/ml with DMEM medium.

Tet-solution group: weighing the tetrandrine raw material medicine, dissolving in methanol and preparing into Tet-solution with the concentration of 20 mu g/ml.

2. Procedure of experiment

1ml of human lung cancer A549 cell suspension is added into each well at a rate of 1 × 10⁵Inoculated on a 6-well plate, and placed in an incubator for 24 h. The culture medium was discarded, and 100. mu.l of Tet solution, tetrandrine nanosuspension and control (containing culture medium only) were added at the same concentration. After co-culturing the drug with A549 cells for 24h, the culture solution was discarded, the cells were digested with pancreatin without EDTA, and washed twice with cold PBS. After adding 400. mu.l of the binding solution, 5. mu.l of Annexin V-FITC is added and mixed evenly, and the mixture is reacted for 15min at 4 ℃ in the dark. Then 10. mu.l of PI was added, and the mixture was mixed well and reacted at 4 ℃ in the dark for 5 min. Finally, the cells were analyzed using a flow cytometer.

3. Results of the experiment

The results of the apoptosis experiments are shown in FIG. 8. The results show that the apoptosis rate of the nanosuspension group (Tet-CCS-NS group and Tet-HACC-TPGS-NS group) is higher than that of the tetrandrine solution group (Tet-solution group), and the possible reason is that the carrier property of the nanosuspension is favorable for apoptosis. On the one hand, TPGS in the nano-suspension Tet-HACC-TPGS-NS can promote drug absorption and further induce apoptosis of the drug, and in addition, the biological adhesion of HACC remarkably improves the combination of nano-particles and a mucus layer, so that mucus penetration is further promoted; another possible reason is that as the particle size decreases, the adhesion of the particles to the tumor increases. After 2-4h of incubation, the Tet-CCS-NS group has higher apoptosis rate compared with the Tet-HACC-TPGS-NS group, which is probably due to higher dissolution of the Tet-CCS-NS, so that the Tet-CCS-NS shows stronger apoptosis.

EXAMPLE sixthly, tetrandrine nanosuspension cell qualitative and quantitative uptake experiments

1. Experimental groups and solution preparation

C6-solution group: coumarin 6 is weighed and dissolved in absolute ethyl alcohol to prepare the Tet-solution of 175.215 mu g/ml.

Group C6-HACC-TPGS-NS: adding 0.05g of chitosan quaternary ammonium salt and 0.05g of TPGS into 50ml of distilled water, stirring under a magnetic stirrer to obtain a stabilizer solution, adding 0.25g of coumarin 6, and magnetically stirring for 30min to obtain a crude suspension. Before grinding, 140ml of zirconia grinding beads with the particle size of 0.6-0.8mm are added into a grinding cavity, and are ground for 45min at the rotating speed of 2500rpm to obtain C6-HACC-TPGS-NS, and the C6-HACC-TPGS-NS is diluted to 175.215 mu g/ml by a DMEM culture solution.

C6-CCS-NS group: weighing 0.1g of croscarmellose sodium, adding into 50ml of distilled water, stirring under a magnetic stirrer to obtain a stabilizer solution, adding 0.25g of coumarin 6, and magnetically stirring for 30min to obtain a crude suspension. Before grinding, 140ml of zirconia grinding beads with a particle size of 0.6-0.8mm were added to the grinding chamber, and ground at 2500rpm for 45min to obtain C6-CCS-NS, which was diluted to 175.215. mu.g/ml with DMEM medium.

2. Procedure of experiment

The uptake condition of the A549 cells to the tetrandrine nanosuspension is evaluated by using coumarin 6 as a fluorescent probe to replace the tetrandrine.

Qualitative uptake experiments: 1.5ml of human lung cancer A549 cell suspension per well 1X 10⁵Inoculating into a confocal culture dish, and placing in an incubator for incubation for 24 h. And (3) discarding the culture solution, and co-culturing the cell with the coumarin 6 solution and the loaded coumarin 6 nanometer suspension for 1, 2 and 4 hours respectively. Cells were washed 3 times with cold PBS, fixed at room temperature with 1ml of 4% paraformaldehyde for 15min, washed 3 times with cold PBS, stained for 10min with DAPI, the DAPI stain removed, and washed 3 times with cold PBS. Finally, 1ml of PBS was added, and the uptake of A549 cells was observed under a laser confocal microscope.

The results are shown in FIG. 9A. The results show that the C6-loaded nanosuspension liquid is phagocytosed into cytoplasm by cells, and the green fluorescence intensity is increased in a time-dependent manner compared with the C6 solution.

Quantitative uptake experiments: 1.5ml of human lung cancer A549 cell suspension per well 1X 10⁵Inoculated on a 6-well plate, and placed in an incubator for 24 h. Discarding the culture solution, co-culturing the cell with coumarin 6 solution and loaded coumarin 6 nanometer suspension for 1, 2 and 4 hours, washing with PBS twice, digesting and centrifuging with pancreatin without EDTA, adding 400 μ l PBS to blow off the cell, and detecting with a flow cytometer.

The results are shown in FIG. 9B. The results show that the fluorescence intensity of the Control group added with the culture solution is basically negligible compared with the solution group and the nanosuspension group added with the coumarin 6, and the fluorescence intensity of the C6-HACC-TPGS-NS and the C6-CCS-NS is increased by about 2 times compared with the C6 solution. At 1h, the cellular uptake rate of C6-HACC-TPGS-NS was higher than that of C6-CCS-NS, probably due to the smaller particle size of C6-HACC-TPGS-NS, and the nanosuspension could be non-specifically internalized into the cell by endocytosis or phagocytosis, in addition to which the positive potential of C6-HACC-TPGS-NS could facilitate mucus penetration and cellular uptake.

Claims

1. The tetrandrine nanosuspension is characterized in that the tetrandrine nanosuspension is obtained by nanocrystallizing tetrandrine and a stabilizer through a dispersion medium, the particle size of the tetrandrine nanosuspension is 50-600nm, the absolute value of Zeta potential is 10-40 mV, and the mass ratio of a drug to the stabilizer is 1: 0.1 to 1;

the tetrandrine nanosuspension is prepared by the following method:

(1) weighing tetrandrine and stabilizer according to a ratio;

2. The tetrandrine nanosuspension of claim 1, wherein in step (1) the stabilizing agent is a steric stabilizer and/or a charge stabilizer.

3. The tetrandrine nanosuspension of claim 2, wherein the steric stabilizer is one or more of polyvinylpyrrolidone, poloxamer, hydroxypropyl cellulose, vitamin E polyethylene glycol succinate, croscarmellose sodium, crospovidone, and chitosan quaternary ammonium salt.

4. The tetrandrine nanosuspension of claim 3, wherein the vitamin E polyethylene glycol succinate and the chitosan quaternary ammonium salt are adsorbed on nanoparticles in the tetrandrine nanosuspension.

5. The tetrandrine nanosuspension of claim 2, wherein the charge stabilizer is one or more of sodium dodecyl sulfate, benzethonium chloride and dioctyl sodium sulfosuccinate.

6. The tetrandrine nanosuspension of claim 1, wherein the dispersion medium in step (2) is purified water.

7. The tetrandrine nanosuspension of claim 1, wherein the grinding beads of step (3) have a particle size of 0.2 to 0.8mm and are made of glass, yttrium-stabilized zirconia or polystyrene derivative polymers; the grinding time is 0.5-3 h; the rotating speed of a stirring shaft of the grinding machine is 1000-3000 rpm; the grinding temperature is controlled to be 10-40 ℃.

8. The tetrandrine nanosuspension of claim 1, wherein the tetrandrine moiety is present in both crystalline and amorphous forms in the tetrandrine nanosuspension.

9. The tetrandrine nanosuspension of claim 1, wherein the tetrandrine nanosuspension is used as a preparation intermediate, and pharmaceutically acceptable excipients are added to prepare tablets, capsules, injections, sustained-release agents, controlled-release agents and granules.

10. The use of the tetrandrine nanosuspension of claim 1 in the preparation of an anti-tumor medicament, wherein the tumor is liver cancer and lung cancer.