CN111202710A

CN111202710A - Niclosamide nanocrystalline temperature-sensitive gel and preparation method and application thereof

Info

Publication number: CN111202710A
Application number: CN201911299240.5A
Authority: CN
Inventors: 吕慧侠; 张振海; 傅强
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2020-05-29
Anticipated expiration: 2039-12-17
Also published as: CN111202710B

Abstract

The invention relates to niclosamide nanocrystalline temperature-sensitive gel with high drug-loading rate and good stability; in particular to niclosamide nanocrystalline temperature-sensitive gel, a preparation method and application thereof, wherein the niclosamide nanocrystalline gel is obtained by dissolving a gel matrix in niclosamide nanocrystalline solution, and the preparation method comprises the following specific steps: dissolving niclosamide in a mixed solvent (50/50, v/v) of ethanol and acetone, dissolving poloxamer 188 in deionized water, adding the organic phase into the water phase, removing the organic solvent by rotary evaporation, and homogenizing under high pressure to obtain niclosamide nanocrystal which is rod-shaped, has a length of about 200nm and a width of about 40 nm. Finally, dissolving the temperature-sensitive gel matrix in the nano-crystal solution to obtain the niclosamide nano-crystal gel. The niclosamide nanocrystalline gel has high drug-loading rate and good stability, can be converted into a solid state in vivo and realize sustained and controlled release, and is suitable for treating triple negative breast cancer.

Description

Niclosamide nanocrystalline temperature-sensitive gel and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical dosage forms and preparations, in particular to a preparation method and application of a locally sustained-release niclosamide nanocrystalline temperature-sensitive gel.

Background

NiclosamideIs a BCS class II compound approved by FDA for the treatment of intestinal parasitic infections in the sixties of the twentieth century, and is also commonly used later in feed for the treatment and prevention of parasitic infections in livestock. The pharmacological research in recent years shows that the niclosamide also has strong inhibiting effect on other pathogenic microorganisms and various cancer cells, including pseudomonas aeruginosa, sparganium, breast cancer, ovarian cancer, colon cancer, prostate cancer, acute myelogenous leukemia and the like. It is worth noting that the research finds that niclosamide has a strong inhibitory effect on tumor stem cells, and if combined with other common chemotherapeutic drugs, the niclosamide can reduce or even avoid the drug resistance of tumor cells. However, the toxicological properties of niclosamide are quite specific. The results of toxicology tests published by Bayer indicate that niclosamide mice are orally administered with acute LD₅₀1500mg/kg, which is very safe; while intravenous injection acute LD₅₀It is only 8mg/kg, showing greater acute toxicity. Further research shows that niclosamide can cause peripheral vasodilatation and cardiotomy blood to reduce blood pressure, so that the niclosamide has larger acute intravenous injection toxicity.

The molecular weight of the niclosamide is 327g/mol, the water solubility is lower and is about 0.23 mu g/ml, and the phenolic hydroxyl structure is weakly acidic. If the compound is used as an antitumor drug, the problem of solubility should be overcome firstly. For this reason, many attempts have been made by researchers. The preparation of cocrystals, inclusion compounds or salts is a method commonly used for improving the solubility, and niclosamide ethanolamine salts, niclosamide piperazine salts, 2-aminothiazole/niclosamide cocrystals, niclosamide/cyclodextrin inclusion compounds and the like can improve the solubility to a certain extent. However, because niclosamide has low bioavailability and rapid metabolic excretion, oral administration often cannot realize drug enrichment at tumor sites, and thus cannot meet the requirement of antitumor therapy. The phenolic hydroxyl is a good target point for synthesizing the niclosamide water-soluble derivative, the water solubility is greatly improved by introducing water-soluble groups such as polyethylene glycol, phosphoric acid, aminoethyl and the like, but the activity, toxicology and pharmacokinetic behavior of the new compound need to be re-evaluated. Researchers have also made some studies on niclosamide nano-preparations, but the problems of drug loading and stability are not solved properly; in addition, in consideration of the great acute intravenous toxicity of niclosamide, direct intravenous administration also entails a certain risk.

The nano crystal is a nano-scale drug crystal, and compared with other nano preparations, the nano crystal can realize higher drug loading with less auxiliary material dosage. To date, a total of 6 formulations containing nanocrystals have been successfully marketed, except

It can be used for intramuscular injection, and the rest is oral tablet or capsule. There are two main methods for the industrial production of nanocrystals, high pressure homogenization and wet media milling, both belonging to the "Top-down" method, meaning that large-particle drug crystals are mechanically crushed to the desired particle size. The "Top-down" method is more controllable than another "Bottom-up" method, such as the anti-solvent method, which controls the crystallization process of drug molecules. In the laboratory, these two methods are often combined to prepare nanocrystals. At present, the research on niclosamide nanocrystals is very limited, and in limited two reports, tween 80 is adopted as a stabilizer of the nanocrystals, so that the drug loading of 10mg/ml is realized; however, tweens have potential risk of hemolysis and allergy and should be used as little as possible. However, no reports that auxiliary materials with better intravenous injection safety, such as poloxamers, are used as niclosamide nanocrystal stabilizers are found so far.

The temperature-sensitive gel is a new material with wide application prospect, and mainly comprises polyethylene glycol block copolymers, chitosan hydrogel and the like. The temperature-sensitive gel exists in a micelle form at a low concentration, and shows temperature-sensitive characteristics when the concentration is high enough: below the phase transition temperature, it is liquid and above the phase transition temperature, it is solid. The phase transition temperature of the temperature-sensitive gel is reduced along with the increase of the gel concentration, and the temperature-sensitive gel with the phase transition temperature lower than 37 ℃ can form a gel drug storage in vivo and slowly release drugs. At body temperature, gels with lower phase transition temperatures solidify faster; meanwhile, the gel polymer molecule has good biocompatibility and can be degraded in vivo without being taken out by an operation.

In view of the greater intravenous acute toxicity of niclosamide, topical sustained release administration is a suitable mode of administration. Therefore, the niclosamide nanocrystals are loaded into the temperature-sensitive gel, so that not only can higher drug loading rate be realized, but also local drug slow release can be realized; the physical and chemical properties and pharmacological activity of niclosamide are combined, and the administration strategy is reasonable.

Disclosure of Invention

The invention aims to load niclosamide with larger acute intravenous injection toxicity into PLGA-PEG-PLGA temperature-sensitive gel, and the niclosamide is injected into solid tumors to form a drug gel storage to slowly release drugs and play a long-acting anti-tumor role. Therefore, the invention relates to a preparation method and application of a locally sustained-release niclosamide nanocrystalline temperature-sensitive gel.

A niclosamide nanocrystalline temperature-sensitive gel is characterized in that: contains niclosamide, poloxamer 188 and PLGA-PEG-PLGA temperature sensitive gel.

The mass ratio of the niclosamide to the poloxamer 188 is 1:1-3: 1.

The concentration of the niclosamide is 2-3mg/ml, the concentration of the temperature-sensitive gel is 10-20%, and the corresponding phase transition temperature is 38-35 ℃ in the concentration range.

The grain size of the nano-crystal is 160-230 nm.

The preparation method of the niclosamide nanocrystalline temperature-sensitive gel is characterized by comprising the following steps: is realized according to the following steps

Dissolving niclosamide in a mixed solvent of ethanol and acetone to serve as an organic phase;

b dissolving poloxamer 188 in water as a water phase;

c, dropwise adding the organic phase into the water phase under stirring;

d, removing the organic solvent by rotary evaporation;

e, homogenizing for several times under high pressure to obtain niclosamide nanocrystalline suspension;

and F, dissolving the gel matrix in the niclosamide nanocrystalline suspension under stirring to obtain the niclosamide nanocrystalline temperature-sensitive gel.

The preparation method of the niclosamide nanocrystalline temperature-sensitive gel is characterized by comprising the following steps:

the stirring speed in the steps A-C is 500-1000rpm, and the rotary evaporation temperature in the step D is 42-45 ℃; the ratio of the mixed solvent of the ethanol and the acetone in the step A is 1: 1; the high-pressure homogenizing pressure in the step E is 800-1200bar, and the homogenizing times are 10-20 times; the stirring rate in step F was 200-400 rpm.

The niclosamide nanocrystalline temperature-sensitive gel is applied to the preparation of antitumor drugs.

Further, the method comprises the following steps:

the preparation method of the niclosamide-loaded nanocrystalline temperature-sensitive gel is characterized by comprising the following steps: dissolving niclosamide in an ethanol-acetone (50/50, v/v) solution, then dropwise dispersing in a poloxamer 188 aqueous solution, removing an organic solvent by rotary evaporation, carrying out high-pressure homogenization to obtain niclosamide nanocrystals, and finally slowly dissolving the obtained niclosamide nanocrystals in a gel matrix under magnetic stirring.

The poloxamer 188 is polyoxyethylene-polyoxypropylene segmented copolymer, has good water solubility, and is a nonionic surfactant commonly used in the field of pharmacy; meanwhile, the injection has better safety and is suitable to be used as an auxiliary material of an implanted preparation. The temperature-sensitive gel matrix is a PLGA-PEG-PLGA polymer, has good biocompatibility and can realize in-vivo degradation; the gel has temperature sensitive property after being prepared into 10-20% water solution, the higher the gel matrix concentration is, the lower the phase transition temperature is, and if the phase transition temperature is lower than the body temperature, the gel can be converted into solid gel in vivo.

The mass ratio of the niclosamide to the poloxamer 188 is 1:1-3:1, and the high-pressure homogenization condition is 1000bar for 20 times; the temperature-sensitive gel is PLGA-PEG-PLGA temperature-sensitive gel, and the concentration of the temperature-sensitive gel is 10-20%.

The niclosamide nanocrystalline-loaded temperature-sensitive gel can be applied to slow-release administration in solid tumors. After the injection is injected into the tumor, the gel is converted into a solid state, and the solid gel can be slowly corroded in vivo to release the drug, so that the long-acting sustained-release drug delivery in the tumor is realized.

Advantageous effects

1. The key points of the preparation method are as follows: the niclosamide solution is gradually and dropwise dispersed into the poloxamer 188 solution, so that crystals with overlarge volume are avoided; the mass ratio of the niclosamide to the poloxamer 188 is 1:1-3:1, and the unstable nanocrystal is caused by the excessively high or excessively low dosage of the poloxamer 188; the high-pressure homogenizing condition is 1000bar and 20 times, and the particle size is increased due to insufficient homogenizing pressure or times; the PLGA-PEG-PLGA temperature sensitive gel has a concentration of 10-20% and a phase transition temperature of 38-35 ℃, wherein 20% is the optimal concentration, and the phase transition temperature is 35 ℃.

TABLE 1 Effect of the preparation Process in the examples on particle size, PDI, phase transition temperature

\\ comprises the following steps: failure to form nano-crystals

2. The invention adopts an anti-solvent precipitation method combined with a high-pressure homogenization method and takes a proper amount of poloxamer 188 as a stabilizer to prepare the niclosamide nano-crystals. And dissolving a proper amount of temperature-sensitive gel matrix in the nano-crystal solution under uniform stirring to finally obtain the temperature-sensitive gel carrying the niclosamide nano-crystals. The preparation method is simple and easy to implement, has high drug loading capacity and has industrial potential; the used material has good biocompatibility and can realize in-vivo degradation; can be stored in the tumor body for a long time, slowly releases niclosamide and realizes long-acting anti-tumor effect.

3. The drug enrichment at the tumor site has a greater impact on the chemotherapeutic effect. Therefore, it has been one of important research subjects to improve the intratumoral retention of the drug. The invention has the advantages that: preparing an antitumor compound niclosamide with high intravenous injection toxicity into nanocrystal, loading the nanocrystal into temperature-sensitive gel, injecting the nanocrystal into a tumor to form a drug reservoir in the tumor, and slowly releasing the drug along with the erosion of the gel; the drug concentration and the preparation stability are both obviously improved; the niclosamide is enriched in the tumor, so that the antitumor effect is improved, and the system toxicity is reduced.

4. The nano-crystal prepared by the invention improves the solubility of niclosamide by 11122 times, and the drug loading capacity reaches 2.558 mg/ml.

Drawings

FIG. 1 is a transmission electron micrograph of niclosamide nanocrystal gel of example 1;

FIG. 2 is a graph showing the distribution of particle sizes of niclosamide nanocrystal gels of example 2;

FIG. 3 is an in vitro cumulative release profile of the niclosamide nanocrystal gel of example 2;

FIG. 4 is a rheological profile of niclosamide nanocrystallite gel of example 3;

FIG. 5 shows the results of MTT assay of niclosamide nanocrystal gels of example 13;

FIG. 6 shows the results of the cell uptake assay of niclosamide nanocrystal gels of example 14;

FIG. 7 shows the results of pharmacodynamic experiments of the niclosamide nanocrystal gel of example 15.

Detailed Description

The present invention is further illustrated by the following specific examples, but the present invention is not limited to the contents contained in the following examples.

Example 1

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 50mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally, homogenizing the crude niclosamide suspension for 20 times under the pressure of 1000bar to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 1ml of niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain niclosamide nanocrystalline gel with the gel concentration of 20%. The actual measurement shows that the concentration of niclosamide is 2.558mg/ml, the particle size is 172nm, the PDI is 0.191, and the gel phase transition temperature is about 35 ℃.

Example 2

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 75mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally, homogenizing the crude niclosamide suspension for 20 times under the pressure of 1000bar to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 1ml of niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain niclosamide nanocrystalline gel with the gel concentration of 20%. The actual measurement shows that the concentration of niclosamide is 2.417mg/ml, the particle size is 178nm, the PDI is 0.187, and the gel phase transition temperature is about 35 ℃.

Example 3

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 150mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally, homogenizing the crude niclosamide suspension for 20 times under the pressure of 1000bar to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 1ml of niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain niclosamide nanocrystalline gel with the gel concentration of 20%. The actual measurement shows that the concentration of niclosamide is 2.397mg/ml, the particle size is 194nm, the PDI is 0.215, and the gel phase transition temperature is about 35 ℃.

Example 4

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 38mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. Finally, the crude niclosamide suspension is homogenized under high pressure for 20 times under the pressure of 1000 bar. Because the ratio of niclosamide to poloxamer 188 is not proper, niclosamide nanocrystal suspension cannot be obtained, and niclosamide nanocrystal gel cannot be prepared continuously.

Example 5

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 30mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. Finally, the crude niclosamide suspension is homogenized under high pressure for 20 times under the pressure of 1000 bar. Because the ratio of niclosamide to poloxamer 188 is not proper, niclosamide nanocrystal suspension cannot be obtained, and niclosamide nanocrystal gel cannot be prepared continuously.

Example 6

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 300mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. Finally, the crude niclosamide suspension is homogenized under high pressure for 20 times under the pressure of 1000 bar. Because the ratio of niclosamide to poloxamer 188 is not proper, niclosamide nanocrystal suspension cannot be obtained, and niclosamide nanocrystal gel cannot be prepared continuously.

Example 7

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 450mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. Finally, the crude niclosamide suspension is homogenized under high pressure for 20 times under the pressure of 1000 bar. Because the ratio of niclosamide to poloxamer 188 is not proper, niclosamide nanocrystal suspension cannot be obtained, and niclosamide nanocrystal gel cannot be prepared continuously.

Example 8

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 50mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally, homogenizing the crude niclosamide suspension for 20 times under the pressure of 1000bar to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 1.42ml of the niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain the niclosamide nanocrystalline gel with the gel concentration of 15%. The actual measurement shows that the concentration of niclosamide is 2.688mg/ml, the particle size is 181nm, the PDI is 0.205, and the gel phase transition temperature is about 37 ℃. Because the gel matrix concentration is reduced, the phase transition temperature is close to the body temperature, and the gel cannot be quickly converted into a solid state in vivo, so that the gel is not suitable for intratumoral injection.

Example 9

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 50mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally, homogenizing the crude niclosamide suspension for 20 times under the pressure of 1000bar to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 2.25ml of the niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain the niclosamide nanocrystalline gel with the gel concentration of 10%. The actual concentration of niclosamide was found to be 2.770mg/ml, the particle size was 177nm, the PDI was 0.199 and the gel transition temperature was about 38 ℃. The gel matrix concentration is reduced, so that the phase transition temperature exceeds the body temperature, and the gel cannot be converted into a solid state in vivo, so that the gel is not suitable for intratumoral injection.

Example 10

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 50mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally, homogenizing the crude niclosamide suspension for 20 times under the pressure of 500bar to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 1ml of niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain niclosamide nanocrystalline gel with the gel concentration of 20%. The actual measurement shows that the concentration of niclosamide is 2.424mg/ml, the particle size is 205nm, the PDI is 0.245, and the gel phase transition temperature is about 35 ℃. The homogenization pressure is low, resulting in an increase in both the nanocrystalline particle size and PDI.

Example 11

150mg of niclosamide was dissolved in 4ml of a mixed solvent of ethanol/acetone (50/50, v/v), and 50mg of poloxamer 188 was dissolved in 50ml of deionized water. Slowly dripping the niclosamide solution into the poloxamer 188 solution under magnetic stirring, and removing the organic solvent by rotary evaporation to obtain crude niclosamide suspension. And finally homogenizing the crude niclosamide suspension at 1000bar under high pressure for 10 times to obtain niclosamide nanocrystalline suspension. Weighing about 0.25g of temperature-sensitive gel matrix, adding 1ml of niclosamide nanocrystalline suspension obtained by the preparation, and magnetically stirring overnight under ice bath to obtain niclosamide nanocrystalline gel with the gel concentration of 20%. The actual measurement shows that the concentration of niclosamide is 2.390mg/ml, the particle size is 198nm, the PDI is 0.251, and the gel phase transition temperature is about 35 ℃. Insufficient number of homogenizations leads to an increase in both the nanocrystalline particle size and PDI.

Example 12: in vitro Release assay

And (4) carrying out in-vitro release experiment investigation by adopting a dialysis method. Niclosamide nanocrystals (prepared as in example 1) and niclosamide nanocrystal gel (prepared as in example 1) each containing about 0.6mg of drug were added to dialysis bags (molecular weight cut-off 3500Da) and allowed to stand at 37 ℃ until the gel solidified, and the dialysis bags were immersed in 200ml of release medium (0.01M pH7.4PBS, 0.5% Tween 80) at 37. + -. 0.5 ℃ and shaken at a rate of 100 rpm. Sampling 0.5ml at a preset time point, sending the sample to a liquid phase for detection, and additionally supplementing an equal amount of fresh dissolution medium to maintain the condition of a leak tank.

The results in FIG. 4 show that the niclosamide nanocrystals released at a faster rate, and were nearly completely released within 48 hours; the release rate of the niclosamide nanocrystalline gel is relatively slower, and only less than 80% of the niclosamide nanocrystalline gel is released by day 6. Therefore, in vitro release experiments prove that the use of the temperature-sensitive gel can effectively delay the release of the medicine.

Example 13: MTT assay

Taking MDA-MB-231 cells in logarithmic growth phase, digesting, and then, taking 10 cells per well⁴Was seeded in 96-well plates at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. Then the culture medium is aspirated, each well is washed with PBS for three times, and 200. mu.l of the niclosamide nanocrystal gel or niclosamide bulk drug prepared in example 1 with the corresponding concentration is added. Cells were incubated in an incubator for 24 or 48 hours, and 20. mu.l of 5mg/ml MTT solution was added and incubation was continued for 4 hours to form formazan crystals. Finally, the medium is aspirated off and 150. mu.l DMSO are added, shaken for 10 minutes at 37 ℃ and thenThe optical density value at 490nm of each hole is measured on a microplate reader. Cell viability was calculated at each dosing concentration using the blank group as a reference, and finally half maximal Inhibitory Concentration (IC) was calculated for each group using Graph Pad Prism 7.0 software₅₀)。

The results in FIG. 5 show that the niclosamide nanocrystalline gel group of example 1 has stronger inhibitory effect on MDA-MB-231 cells than the niclosamide drug substance at the administration time of 24 or 48 hours. IC of niclosamide 24 hours, niclosamide nanogel 24 hours, niclosamide 48 hours, niclosamide nanogel 48 hours₅₀8.029, 3.297, 0.2658, 0.1086. mu.M, IC of nanocrystalline gel group, respectively₅₀Are all lower than the niclosamide bulk drug under the same administration time. Therefore, the niclosamide nanocrystalline gel has a remarkable inhibiting effect on triple negative breast cancer cells in vitro.

Example 14: cell uptake assay

Taking MDA-MB-231 cells in logarithmic growth phase, digesting, and determining according to 2 × 10 per well⁵Was seeded in 6-well plates at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. The medium was aspirated, each well was washed three times with PBS, and 2ml of the niclosamide nanocrystal gel or niclosamide drug substance prepared in example 1, equivalent to 0.05, 1, 20. mu.M, was added. The cells were placed in an incubator and incubated for an additional 4 hours. Then the medium was aspirated off, washed three times with PBS,0.5 ml of deionized water was added to each well, and cells were disrupted by repeated freeze-thawing at-20 ℃ and room temperature for three times. 100 μ l of the suspension was taken, three times the volume of acetonitrile was added to extract and dissolve the drug, centrifuged at 12000rpm for 30 minutes and the supernatant was injected for assay. Then quantifying the total protein concentration by using a BCA colorimetric method to represent the total amount of the cells; the drug concentration divided by the total protein concentration is the amount of drug taken per cell.

The results of fig. 6 show that the niclosamide nano-crystal gel group in example 1 has no significant difference in the uptake amount of the drug substance at lower concentration, and the uptake amount is significantly greater than that of the drug substance at high concentration, which represents a more efficient uptake mode of the nano-crystal.

Example 15: in vivo pharmacodynamic experiment

Taking 4-6 weeks old female nude mice each timeSubcutaneous injection of 2X 10 alone⁶MDA-MB-231 cells, until the tumor grows to about 200mm³At the time, tumor-bearing mice were randomly and equally divided into 3 groups of 5 mice each. Each group of tumor-bearing mice was injected intratumorally with 100. mu.l of physiological saline, blank gel, and 15mg/kg of niclosamide nanocrystal gel (prepared as in example 1), and the body weight and tumor volume of the tumor-bearing mice were measured 20 days after administration. Tumor-bearing mice were sacrificed on day 20 of dosing and tumor bodies were removed and weighed. The tumor volume calculation formula is: length x width²×0.5。

The results in fig. 7 show that tumor volumes and tumor weights of tumor-bearing mice in the niclosamide nanocrystalline gel group are lower than those of the normal saline and blank gel groups, and there is no significant difference in body weight. This shows that the niclosamide nanocrystalline gel has obvious inhibition effect on triple negative breast cancer cells in vivo and shows lower toxicity.

Claims

1. A niclosamide nanocrystalline temperature-sensitive gel is characterized in that: contains niclosamide, poloxamer 188 and PLGA-PEG-PLGA temperature sensitive gel.

2. The niclosamide nanocrystal temperature-sensitive gel according to claim 1, characterized in that: the mass ratio of the niclosamide to the poloxamer 188 is 1:1-3: 1.

3. The niclosamide nanocrystal temperature-sensitive gel according to claim 1, characterized in that: the concentration of the niclosamide is 2-3mg/ml, the concentration of the temperature-sensitive gel is 10-20%, and the corresponding phase transition temperature is 38-35 ℃ in the concentration range.

4. The niclosamide nanocrystal temperature-sensitive gel according to claim 1, characterized in that: the grain size of the nano-crystal is 160-230 nm.

5. The method for preparing niclosamide nanocrystalline temperature-sensitive gel according to any one of claims 1-4, characterized in that: is realized according to the following steps

b dissolving poloxamer 188 in water as a water phase;

c, dropwise adding the organic phase into the water phase under stirring;

d, removing the organic solvent;

6. The method for preparing niclosamide nanocrystalline temperature-sensitive gel according to claim 5, characterized in that:

the stirring speed in the steps A-C is 500-1000rpm, and the temperature for removing the organic solvent in the step D is 42-45 ℃; the ratio of the mixed solvent of the ethanol and the acetone in the step A is 1: 1; the high-pressure homogenizing pressure in the step E is 800-1200bar, and the homogenizing times are 10-20 times; the stirring rate in step F was 200-400 rpm.

7. The use of the niclosamide nanocrystal temperature-sensitive gel according to any one of claims 1 to 4 in the preparation of an anti-tumor medicament.