CN110840837B

CN110840837B - Tetrandrine nanosuspension and preparation method and application thereof

Info

Publication number: CN110840837B
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: 2023-09-29
Anticipated expiration: 2039-12-10
Also published as: CN110840837A

Abstract

The invention relates to the field of pharmaceutical preparations, in particular to a tetrandrine nano suspension which is prepared by nanocrystallization of tetrandrine and a stabilizer through a dispersion medium, wherein the particle size of the tetrandrine nano suspension 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 invention particularly discloses a preparation method of tetrandrine nano suspension and application thereof in preparing antitumor drugs. The particle size of the tetrandrine nano suspension is 50-600nm, the stability is good, the solubility and dissolution rate of the tetrandrine can be obviously improved, compared with the tetrandrine which is a raw material medicine, the tetrandrine nano suspension has obvious inhibition rate to tumor cells in vitro, the apoptosis rate and the cell uptake rate are obviously improved, the dissolution rate and the anti-tumor activity of the tetrandrine are obviously improved, and the tetrandrine nano suspension has 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 tetrandrine nano suspension, a preparation method and application thereof.

Background

In recent years, cancer is a major disease affecting the health of people, and the occurrence rate and death rate of cancer are in an increasing trend year by year. Among many tumors, lung cancer is the malignant tumor with the highest incidence and mortality rate. At present, conventional treatment methods aiming at tumors mainly comprise operation therapy, radiotherapy and chemotherapy, and the conventional treatment methods have poor curative effects and various side effects. Natural compounds have attracted more and more attention because of their low toxic and side effects, reversing tumor multidrug resistance and the like.

Tetrandrine (Tet) is a natural dibenzyl isoquinoline alkaloid extracted from the root tuber of stephania tetrandra of the family stephania, and studies have found that tetrandrine is a promising antitumor drug, has an inhibitory effect on various tumor cell lines including bladder cancer cells, liver cancer cells, breast cancer cells, human cavity cancer cells, cervical cancer cells, etc., and has been demonstrated to play an important role in lung cancer through VEGF/HIF-1a/ICAM-1 signaling pathway and inducing apoptosis of lung cancer cells. However, tetrandrine is poor in water solubility when being directly used as an anti-tumor drug, and researches show that the tetrandrine has the saturated solubility of only 0.015mg/ml in a phosphate buffer solution with pH of 7.4, has low bioavailability and instability due to the poor solubility in a physiological environment, has no selectivity to tumor tissues and normal tissues, has poor targeting effect, has a small safety range of tetrandrine, causes the defects of wide toxic and side effects and the like, and ensures that the clinical application range of the tetrandrine is limited to a certain extent.

The nano medicine carrying system can improve the dissolution of insoluble medicine, raise the curative effect of chemotherapeutic medicine and reduce the side effect of local part or whole body. Studies have been made to solve the problem of poor dissolution using solid lipid nanoparticles, nanoemulsion delivery systems, microspheres, etc., for example Wang Yanli developed tetrandrine liposomes, prolonged the duration of action of the drug, and improved bioavailability (extracted from Wang Yanli. Development of tetrandrine liposomes [ D ]. Henna university, 2008.); the patent "tetrandrine nanoemulsion injection and its preparation method" (patent number: 20080024843. X) discloses a tetrandrine nanoemulsion injection and its preparation method, which is composed of tetrandrine, surfactant, cosurfactant, oil phase and water phase. The nanoemulsion has the advantages of simple preparation method, transparent appearance and particle size of I0-l00nm; the nanoemulsion has good stability; the solubility of tetrandrine can be obviously improved, so that the bioavailability of the medicine is improved; and changes the in vivo process of the medicine, increases the targeting to the liver, reduces toxic and side effects and improves the curative effect. However, the technical scheme has the defects of lower drug loading rate, lower 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 nanometer suspension.

The invention also aims to provide a preparation method of the tetrandrine nano suspension, which has the characteristics of simple preparation process, small usage amount of the stabilizer, no use of an organic solvent, high drug loading capacity and the like, and the prepared tetrandrine nano suspension 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 nanosuspension in preparing antitumor drugs.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the tetrandrine nanometer suspension is prepared by nanocrystallization of tetrandrine and a stabilizing agent through a dispersion medium, the particle size of the tetrandrine nanometer suspension is 50-600nm, the absolute value of Zeta potential is 10-40 mV, and the mass ratio of the medicine to the stabilizing agent is 1:0.1 to 1.

The tetrandrine nanosuspension is prepared by the following method:

(1) Weighing tetrandrine and stabilizer according to the proportion;

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

(3) Sequentially adding grinding beads and crude suspension into a grinding chamber of a nano grinder, and grinding to obtain tetrandrine nano 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 chitosan quaternary ammonium salt are adsorbed on the nanoparticles in the tetrandrine nano suspension.

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

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

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

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

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

Preferably, the tetrandrine in the tetrandrine nanosuspension exists in a crystal form and in 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 nano suspension is applied to the preparation of antitumor drugs, and tumors comprise breast cancer, lung cancer, bladder cancer, liver cancer, human mouth cavity cancer, cervical cancer and human brain glioblastoma.

The beneficial effects of the invention are as follows:

1) The preparation method of the tetrandrine nanometer suspension is simple, the stabilizer is less in use type and use amount, no organic solvent is used in the preparation process, the production is easy, and the prepared tetrandrine nanometer suspension is large in drug loading amount and avoids the use of the organic solvent.

2) The tetrandrine in the tetrandrine nano suspension prepared by the invention exists in a crystalline state, an amorphous state or a mixed state.

3) The tetrandrine nano suspension obviously improves the accumulated dissolution rate of the tetrandrine by 30-80 percent. Possible reasons are: (1) after the medicine is prepared into nano suspension, the specific surface area is increased due to the nano particle size, so that the dissolution rate is increased; (2) because part of crystal forms in the nano suspension are converted into amorphous forms, the dissolution is increased; (3) the wetting effect of the water-soluble carrier on the poorly soluble drug can also increase dissolution.

4) The tetrandrine nano suspension obviously enhances the anti-tumor activity of the tetrandrine. In vitro cytotoxicity experiments show that the stabilizer used by the tetrandrine nanosuspension has good safety and biocompatibility; the cell survival rate of the tetrandrine nano suspension to lung cancer A549 cells in 48 hours is lower than 10%, and the IC ₅₀ The value is lower than that of the tetrandrine bulk drug group. The apoptosis rate of the tetrandrine nano suspension is 2-4 times of that of the bulk drug. The tetrandrine nanosuspension promotes the uptake of lung cancer A549 cells compared with tetrandrine bulk drug. Probably due to the nano-scale particle size of the nano-suspension, the adhesion of particles and tumor cells is facilitated, and the particles are more easily taken up by the cells, so that stronger anti-tumor effect is shown. The carrier has part of promotion effect on anti-tumor effect, has bioadhesion, can increase the combination of drug particles and mucus layer, promote the permeation of mucus layer, and has stronger anti-tumor activity, and can induce mitochondrial injury by increasing intracellular drug level, thereby increasing drugThe ability of the agent to induce apoptosis. In addition, a part of crystal form of the nano suspension is converted into an amorphous state, so that the release rate is faster, and the anti-tumor effect of tetrandrine is enhanced.

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

Drawings

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

Graph a: lyophilized powder of tetrandrine suspension without stabilizer. Graph B: lyophilized powder of Tet-CCS-NS. Graph C: lyophilized powder of Tet-HACC-TPGS-NS.

FIG. 2 is an energy dispersive X-ray spectrum of a tetrandrine nanosuspension of the present invention.

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

FIG. 4 is an X-ray diffraction pattern of a tetrandrine nanosuspension according to the present invention.

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

FIG. 6 is a graph showing the dissolution profile of a tetrandrine nanosuspension according to the present invention.

FIG. 7 is an in vitro cytotoxicity of A549 cells.

FIG. A, B, C shows a graph of toxicity results of A549 cells incubated with adjuvant solutions for 24h,48h and 72h, respectively. FIG. D, E, F shows a graph of toxicity results of A549 cells incubated with tetrandrine nanosuspension for 24h,48h and 72h, respectively.

FIG. 8 is a bar graph of apoptosis (A) detected by flow cytometry after incubation of A549 cells with tetrandrine nanosuspension for 24h (B).

FIG. 9 is a laser confocal qualitative uptake map (A) of A549 cells, and a flow cytometry quantitative uptake map (B).

Detailed Description

The technical solution of the present invention will be described in detail with reference to the specific embodiments, but the scope of the present invention is not limited to the following embodiments, and those skilled in the art can understand that the technical solution obtained by changing the parameters of the technical solution belongs to the scope of the present invention.

In the specific embodiment of the invention, unless specified otherwise, the experimental methods involved are all routine experimental methods in the field; unless otherwise specified, reagents used to complete the following examples were all commercially available.

Noun abbreviation table:

component (A)	Abbreviations (abbreviations)	Component (A)	Abbreviations (abbreviations)
				Tetrandrine A	Tet	Sodium dodecyl sulfate	SDS
polyvinylpyrrolidone-K30	PVP K30	Chitosan quaternary ammonium salt	HACC
				Poloxamer 188	P188	Infrared spectrum	FTIR
Vitamin E polyethylene glycol succinate	TPGS	X-ray diffraction	XRD
				Hydroxypropyl cellulose	HPC-SSL	Differential scanning thermal method	DSC
Croscarmellose sodium	CCS	Scanning electron microscope	SEM
				Nanosuspension	NS	X-ray energy spectrometry	EDS

Example 1: preparation of tetrandrine nano suspension

Tetrandrine nanosuspension 1: PVP K30.1 g is weighed, added into 50ml of distilled water, stirred under a magnetic stirrer to obtain a stabilizer solution, added with tetrandrine 0.25g, and magnetically stirred 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 grinding is carried out for 30min at the rotating speed of 2000rpm, so as to obtain the tetrandrine nanometer suspension. The resulting preparation was diluted 2 times with purified water and had a particle size of 312nm and a potential of-21.2 mV.

Tetrandrine nanosuspension 2: weighing poloxamer 188.1 g, adding into 50ml distilled water, stirring to obtain stabilizer solution, adding 0.5g tetrandrine under magnetic stirring, and magnetically stirring for 30min to obtain crude suspension. Before milling, 140ml of glass milling beads having a particle size of 0.4 to 0.6mm were added to the milling chamber, milling was carried out at 2000rpm for 30 minutes, and the resulting sample was diluted 4 times with purified water and measured to have a particle size of 512.2nm and a potential of-19.3 mV.

Tetrandrine nanosuspension 3: weighing 0.1g of crosslinked sodium carboxymethyl cellulose, 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 zirconia grinding beads with the particle size of 0.6-0.8mm are added into a 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 was diluted 2-fold with purified water and had a particle size of 469.1nm and a potential of-29.4 mV.

Tetrandrine nanosuspension 4: 0.05g of HPC-SSL and 0.05g of SDS are added into 50ml of distilled water, stirred under a magnetic stirrer to obtain a stabilizer solution, then 1g of tetrandrine is added, and the mixture is magnetically stirred 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 grinding is carried out for 45min at the rotating speed of 2500rpm, so as to obtain the tetrandrine nanometer suspension. The resulting preparation was diluted 2-fold with purified water and had a particle size of 217.6nm and 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 zirconia grinding beads with the particle size of 0.6-0.8mm are added into a 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 was diluted 2 times with purified water and had a particle size of 157.3nm and a potential of 23.3mV.

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 nanosuspension

As can be seen from Table 1, the particle size, PDI value and Zeta potential of the prepared tetrandrine nanosuspension are all different, the nanosuspension is screened by combining three factors of particle size, PDI value and Zeta potential, firstly, the tetrandrine nanosuspension with smaller particle size is Tet-HACC-TPGS-NS, the Zeta potential of the nanosuspension is positive charge, and the nanosuspension can be combined with cell membranes in a negative charge state, so that the cell uptake is facilitated, and stronger cytotoxicity is shown; the PDI values of the tetrandrine nano suspension Tet-PVP K30-NS and Tet-P188-NS are larger, and the solution stability is poor; the stabilizer SDS in the tetrandrine nanosuspension Tet-HPC-SSL-SDS-NS has certain toxicity to cells, and is unfavorable for use; in conclusion, the tetrandrine nanosuspension Tet-CCS-NS with good solution stability and the tetrandrine nanosuspension Tet-HACC-TPGS-NS which is easier to combine with cell membranes are selected for subsequent experiments.

Example two, freeze-drying Process and characterization of tetrandrine nanosuspension

1. Experimental grouping and solution formulation

Tet-MS group: weighing tetrandrine 0.25g, adding into 50ml distilled water, and stirring under magnetic stirrer for 30min to obtain 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 grinding is carried out for 45min at the rotation speed of 2500rpm, so as to obtain tetrandrine suspension.

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

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

2. Experimental procedure

Adding 5% of freeze-drying protective agent mannitol and lactose into the 3 groups of tetrandrine suspension, shaking and mixing uniformly, pre-freezing for 24 hours in an ultralow temperature refrigerator at-80 ℃, rapidly transferring to a freeze dryer, and freeze-drying for 12 hours at a vacuum-40 ℃ to obtain tetrandrine rice suspension freeze-dried powder.

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

3. Experimental results

The scanning electron microscope results of the tetrandrine, the stabilizer and the nano suspension are shown in figure 1; the energy dispersive X-ray spectrum results are shown in figure 2, and as can be seen from the A diagrams in figures 1 and 2, in the tetrandrine suspension without the stabilizing agent, the tetrandrine is in the shape of uneven rod and rectangular particles; as can be seen from the B diagrams in fig. 1 and 2, in Tet-CCS-NS, CCS is crosslinked with tetrandrine into a sheet shape, and the nanosuspension does not affect the surface element composition, which may be due to the inability of CCS to adsorb on the particle surface; from fig. 1 and fig. 2, it can be seen that in Tet-HACC-TPGS-NS, the drug substance and the stabilizer form a quasi-circular nanosuspension, which does not affect the surface element composition compared to the tetrandrine suspension without the stabilizer, probably due to the fact that the TPGS is covered with a portion of the drug at the site of detection.

The result of the differential scanning thermal analysis of the tetrandrine, the stabilizer and the nanosuspension is shown in figure 3; as shown in FIG. 4, the X-ray diffraction results are shown in FIG. 3 and FIG. 4, the Tet-CCS-NS and Tet-HACC-TPGS-NS have significantly reduced endothermic melting peak intensities compared with the bulk drugs and the corresponding physical mixture groups, and it is proved that in the grinding or freeze-drying process, partial crystal forms are converted into amorphous states, and XRD analysis is further carried out, and the results show that in the Tet-CCS-NS and Tet-HACC-TPGS-NS, the tetrandrine characteristic crystal diffraction peak intensities are significantly reduced, and the fact that in the grinding or freeze-drying process, partial crystal forms are converted into amorphous states is proved.

As shown in FIG. 5, the results of the IR spectrum analysis of the tetrandrine, the stabilizer and the nanosuspension thereof are shown in FIG. 5, and it is clear from FIG. 5 that the tetrandrine, tet-CCS-NS and Tet-HACC-TPGS-NS groups have no new peaks, the positions of the peaks of the groups have no obvious shift, which means that the tetrandrine and the stabilizer have no obvious interaction, wherein the TPGS carrier is 1740cm ^-1 The peaks at these locations do not appear in their physical mixture and nanosuspension or have low peak intensities, possibly due to the TPGS being covered with a portion of the drug.

Example three dissolution measurement of tetrandrine nanosuspension

1. Experimental grouping and solution formulation

Tet bulk drug group: weighing 1.74mg of tetrandrine bulk drug;

Tet-CCS-NS group: taking 680 μl of the tetrandrine nanosuspension prepared in the first embodiment;

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

2. experimental procedure

The dissolution rate was measured according to the second method (paddle method) of the fourth 0931 edition of the Chinese pharmacopoeia 2015, and 900ml of purified water was used as the dissolution medium at 37.+ -. 0.5 ℃ and at a rotational speed of 100rpm/min. When the sample was brought into contact with the elution medium, 5ml was sampled at 5, 10, 15, 20, 30, 45, 60, 120min, and 5ml of the same isothermal elution medium was supplemented, and the obtained sample was filtered with a 0.45 μm microporous filter membrane and its absorbance was measured at 297.6nm by an ultraviolet spectrophotometer.

3. Experimental results

The dissolution profile is shown in fig. 6. As can be seen from the figure, the cumulative dissolution rate of tetrandrine in water of the tetrandrine nanosuspension is improved by about 5 times in 2h compared with the raw material drug tetrandrine, and the possible reasons are as follows: (1) the specific surface area of the nano-scale particle size is increased, so that the dissolution rate is improved; (2) the crystal form is transformed and is amorphous after transformation, so that the dissolution rate is improved; (3) the water-soluble stabilizer has wetting effect on tetrandrine.

Example four cytotoxicity experiments of tetrandrine nanosuspension

1. Experimental grouping and solution formulation

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 milling, 140ml of zirconia milling beads having a particle size of 0.6 to 0.8mm were added to the milling chamber, and milling was carried out at 2500rpm for 45 minutes to obtain CCS-solution.

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 zirconia grinding beads with the particle size of 0.6-0.8mm are added into a grinding cavity, and grinding is carried out for 45min at the speed of 2500rpm, so as to obtain HACC-TPGS-solution.

Tet-CCS-NS group: the group of nanosuspensions was diluted to 100. Mu.g/ml with DMEM broth, and 50, 40, 30, 20, 10, 5. Mu.g/ml, respectively.

Tet-HACC-TPGS-NS group: the group of nanosuspensions was diluted to 100. Mu.g/ml with DMEM broth, and 50, 40, 30, 20, 10, 5. Mu.g/ml, respectively.

Tet-solution group: the tetrandrine bulk drug is weighed and dissolved in methanol to prepare stock solution with the volume of 100 mug/ml, and the stock solution is further diluted to 50, 40, 30, 20, 10,5 mug/ml.

2. Experimental procedure

Mu.l of human lung cancer A549 cell suspension was 5X 10 per well ³ Inoculating to 96-well plate, and incubating for 24 hr. Discarding culture solution, setting the well containing culture solution as Control group, adding 100 μl of drug solution group and preparation group into each well, respectively setting 5, 10, 20, 30, 40, 50 μg/mL 6 mass concentrations, each having 3 compound wells, incubating for 24 hr, 36 hr and 48 hr, adding 10 μl of CCK8 solution into each well, incubating in incubator for 2 hr, measuring OD value at 450nm with enzyme-labeled instrument, and calculating IC ₅₀ Values.

3. Experimental results

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

TABLE 2 IC of tetrandrine nanosuspensions and solution sets ₅₀ Value of

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

* Indicating that there was a very significant difference between tetrandrine suspension and tetrandrine solution group at the same incubation time (p < 0.01)

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

# # indicates a very significant difference in the same incubation time between tetrandrine suspensions (p < 0.01)

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

To further evaluate the antitumor effect of the prepared formulation, IC was tested ₅₀ The values were analyzed and the results are shown in Table 2. As can be seen from Table 2, the IC of the Tet-HACC-TPGS-NS group ₅₀ The anti-tumor effect of nanosuspension Tet-HACC-TPGS-NS was better at 36 and 48h significantly lower than that of Tet solution group and Tet-CCS-NS group, that is, the possible reasons for affecting the anti-tumor effect were: (1) passive targeting of tumor tissue is facilitated by small size particles by enhanced permeability and retention effects; (2) HACC shows adhesion that has an effect on the adhesion of particles to cell membranes; (3) because the cell membrane is negatively charged, positively charged Tet-HACC-TPGS-NS is more likely to be internalized by the cell, and TPGS can improve accumulation of intracellular drugs.

Example five, tetrandrine nanosuspension apoptosis experiments

1. Experimental grouping and solution formulation

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

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

Tet-solution group: the tetrandrine bulk drug is weighed and dissolved in methanol to prepare the Tet-solution with the volume of 20 mug/ml.

2. Experimental procedure

1ml of human lung cancer A549 cell suspension was 1X 10 per well ⁵ Inoculating to 6-well plate, and incubating in incubator for 24 hr. The culture medium was discarded, and 100. Mu.l of the Tet solution group, the tetrandrine nanosuspension group and the control group (containing only the culture medium) of the same concentration were added. After 24h co-culture of the drug with a549 cells, the culture was discarded, the cells were digested with pancreatin without EDTA, and washed twice with cold PBS. After 400. Mu.l of the binding solution was added, 5. Mu.l of Annexin V-FITC was added and mixed well, and the reaction was carried out at 4℃for 15 minutes in the absence of light. Then 10. Mu.l of PI was added, and the mixture was stirred well and reacted at 4℃for 5min in the absence of light. Finally, the cells were analyzed using a flow cytometer.

3. Experimental results

The results of the apoptosis experiments are shown in FIG. 8. The results showed that the apoptosis rate of the nanosuspension groups (Tet-CCS-NS and Tet-HACC-TPGS-NS) were both high as compared to the Yu Han tetrandrine solution group (Tet-solution group), probably because the carrier properties of the nanosuspensions favored apoptosis. This is probably due to the fact that TPGS in the nanosuspension Tet-HACC-TPGS-NS promotes drug absorption and further induces apoptosis of the drug, and furthermore, the bioadhesion of HACC significantly improves the binding of nanoparticles to the mucus layer, further promoting mucus penetration; another possible reason is that as the particle size decreases, the adhesion of the particles to the tumor increases. After incubation for 2-4h, the apoptosis rate was higher in the Tet-CCS-NS group compared to the Tet-HACC-TPGS-NS group, probably due to the higher dissolution of Tet-CCS-NS, resulting in stronger apoptosis.

Example six, tetrandrine nanosuspension cell qualitative and quantitative uptake experiments

1. Experimental grouping and solution formulation

C6-solution group: coumarin 6 was weighed and dissolved in absolute ethanol to prepare a 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 milling, 140ml of zirconia milling beads having a particle size of 0.6-0.8mm were added to the milling chamber, milled for 45min at 2500rpm to give C6-HACC-TPGS-NS, and diluted to 175.215. Mu.g/ml with DMEM medium.

C6-CCS-NS group: weighing 0.1g of crosslinked sodium carboxymethyl cellulose, 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 milling, 140ml of zirconia milling beads having a particle size of 0.6 to 0.8mm were added to the milling chamber, milled for 45min at 2500rpm to give C6-CCS-NS, and diluted to 175.215. Mu.g/ml with DMEM medium.

2. Experimental procedure

Coumarin 6 is used as a fluorescent probe to replace tetrandrine to evaluate the uptake condition of A549 cells on tetrandrine nanosuspension.

Qualitative uptake experiments: 1.5ml of human lung cancer A549 cell suspension was 1X 10 per well ⁵ Inoculated in a confocal culture dish, and placed in an incubator for incubation for 24 hours. And (3) discarding the culture solution, and co-culturing the coumarin 6 solution, the coumarin 6-loaded nanosuspension and the cells for 1,2 and 4 hours respectively. Cells were washed 3 times with cold PBS, fixed with 1ml of 4% paraformaldehyde for 15min at room temperature, washed 3 times with cold PBS, stained with DAPI for 10min, the DAPI dye removed, and washed 3 times with cold PBS. Finally, 1ml of PBS was added, and the uptake of A549 cells was observed under a confocal laser microscope.

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

Quantitative uptake experiments: 1.5ml of human lung cancer A549 cell suspension was 1X 10 per well ⁵ Inoculating to 6-well plate, and incubating in incubator for 24 hr. The culture solution is discarded, coumarin 6 solution and coumarin 6 nanometer suspension are used for co-culturing with cells for 1,2 and 4 hours, PBS is used for washing twice, pancreatin without EDTA is used for digestion and centrifugation, 400 mu l PBS is added for blowing off the cells, and a flow cytometer is used for detection.

The results are shown in FIG. 9B. The results show that the fluorescence intensity of the Control group added with only the culture solution is basically negligible compared with the coumarin 6 added solution group and the nanosuspension group, 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 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 besides, the non-specific internalization of nanosuspension into cells by endocytosis or phagocytosis, the positive potential of C6-HACC-TPGS-NS can promote mucus penetration and cellular uptake.

Claims

1. The tetrandrine nanometer suspension is characterized in that the tetrandrine nanometer suspension is obtained by nanocrystallization of tetrandrine and a stabilizing agent through a dispersion medium, the particle size of the tetrandrine nanometer suspension is 50-600nm, the absolute value of Zeta potential is 10-40 mV, and the mass ratio of a medicine to the stabilizing agent is 1:0.1 to 1;

the tetrandrine nanosuspension is prepared by the following method:

(1) Weighing tetrandrine and stabilizer according to the proportion;

(3) Sequentially adding grinding beads and crude suspension into a grinding chamber of a nano grinder, and grinding to obtain tetrandrine nano suspension;

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

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

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

4. The tetrandrine nanosuspension according to claim 1, wherein the tetrandrine moiety in the tetrandrine nanosuspension exists in both crystalline and amorphous forms.

5. The tetrandrine nanosuspension according to claim 1, wherein 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.

6. The use of the tetrandrine nanosuspension according to claim 1 in the preparation of an anti-tumour medicament, wherein the tumour is liver cancer and lung cancer.