CN103520728B - The preparation method of arsenic trioxide invisible immunity targeting anti-tumor preparation - Google Patents

Abstract

The preparation method of arsenic trioxide invisible immunity targeting anti-tumor preparation, belong to biomedicine technical field, adopt a kind of biodegradable, poly lactic coglycolic acid that biocompatibility is good as encapsulating material, adopt that two emulsification-evaporation method is obtained carries an arsenic trioxide nanoparticle.By the method for chemical bond covalent coupling carrying arsenic trioxide nanoparticle finishing amphipathic ethylene glycol, giving its " stealth " function, modifying LA by surface synchronization, giving its immune targeting.The arsenic trioxide invisible immunity targeting anti-tumor preparation particle size of final acquisition is homogeneous, and morphology controllable, good dispersion, envelop rate can reach more than 90%.Absorbable organic halogens discharges 45 days in vitro, possesses slow-release function, is a kind of novel, efficient stealthy immune targeting As ₂o ₃preparation.

Description

Preparation method of arsenic trioxide stealth immune targeting anti-tumor preparation

technical field

The invention belongs to the technical field of biomedicine, in particular to a preparation method for encapsulating anticancer drug As ₂ O ₃ .

Background technique

Arsenic trioxide (As ₂ O ₃ ) is the main active ingredient in the traditional Chinese medicine arsenic. Studies have found that As ₂ O ₃ has a significant effect on acute promyelocytic leukemia (APL), and can induce apoptosis of APL cells. Recent studies have shown that As ₂ O ₃ also has a strong anti-tumor effect on malignant solid tumors, especially the anti-hepatocarcinoma effect of As ₂ O ₃ has attracted extensive attention from the tumor community at home and abroad. However, there are still some problems when using As ₂ O ₃ as an anti-liver cancer drug: (1) the half-life of arsenic in plasma is very short, and it will be cleared quickly after administration; (2) the drug itself is toxic, which The dose must be low when it is directly used, and the corresponding anticancer effect is also reduced.

In response to the above problems, researchers at home and abroad have proposed to make arsenic compounds into slow-release preparations to prolong their half-life in vivo, among which liposomes are the most widely studied. However, liposomes are unstable, and they are easy to leak after encapsulating drugs, which brings certain toxic and side effects. In addition, traditional sustained-release preparations have no targeting and are distributed in various organs of the body after administration, which greatly weakens the anti-cancer effect of the drug; at the same time, after the drug enters the human body, it is difficult to escape the macrophages in the human immune system. The phagocytosis of the phagocytosis greatly reduces the medicament that finally reaches the lesion site to exert its medicinal effect. Therefore, it is of great significance to propose a stealth immune-targeting anti-tumor agent that can efficiently and stably encapsulate As ₂ O ₃ and improve its anti-cancer effect.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the anticancer drug As ₂ O ₃ in the process of use, thereby proposing a novel, efficient, invisible and targeted As ₂ O ₃ preparation method for anti-tumor preparations.

The steps of the present invention are as follows:

1) Dissolve As ₂ O ₃ in aqueous sodium hydroxide solution to prepare an arsenic solution containing As ₂ O ₃ at a concentration of 10-100 mg/mL as the water phase;

2) Dissolve lactic acid-glycolic acid copolymer (PLGA) in chloroform as the oil phase;

3) Add the water phase to the oil phase under magnetic stirring, and then emulsify and ultrasonicate in an ice bath to form W/O colostrum;

4) Mix W/O colostrum with the first poloxamer aqueous solution, emulsify in ice bath and ultrasonically form W/O/W double emulsion;

5) After uniformly mixing the W/O/W double emulsion with the second poloxamer aqueous solution, the organic solvent was removed by evaporation, the precipitate was collected by centrifugation, washed with deionized water, and freeze-dried to obtain arsenic trioxide-loaded nanoparticles.

6) Disperse the above-mentioned arsenic trioxide-loaded nanoparticles in water, add N-hydroxysuccinimide (NHS) to activate, and mix with double-terminal amino polyethylene glycol (NH ₂ -PEG-NH ₂ ), lactobionic acid (LA) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was reacted with magnetic stirring at room temperature, and the precipitate was collected by centrifugation to obtain arsenic trioxide stealth immune targeting anti-tumor preparation.

The invention adopts a biodegradable and good biocompatibility lactic acid-glycolic acid copolymer (PLGA) as an encapsulation material, and adopts a double emulsification-solvent evaporation method to prepare arsenic trioxide-loaded nanoparticles. Amphiphilic polyethylene glycol (PEG) was modified on the surface of arsenic trioxide-loaded nanoparticles by chemical bond covalent coupling to endow them with "stealth" function, and LA was endowed with immune targeting by synchronous surface modification. The finally obtained arsenic trioxide stealth immune-targeting anti-tumor preparation has uniform particle size, controllable shape, good dispersion, and an encapsulation efficiency of over 90%. It can be released stably for 45 days in vitro and has the function of sustained release. It is a novel and efficient stealth immune targeting As ₂ O ₃ preparation, which is expected to be used in the treatment of liver cancer.

In addition, in step 1), the mass percentage of the sodium hydroxide aqueous solution is 4%, so as to increase the solubility of As ₂ O ₃ .

In step 2), the concentration of the lactic acid-glycolic acid copolymer (PLGA) is 30-50 mg/mL, so as to increase the encapsulation efficiency of As ₂ O ₃ .

In step 3), the mixing volume ratio of the water phase and the oil phase is 1:10, the ultrasonic power is 180W, and the ultrasonic time is 60s, so as to form a uniform W/O primary emulsion.

In step 4), the mass percent concentration of the first poloxamer aqueous solution is 3%, the ultrasonic power is 360W, and the ultrasonic time is 90s, so as to form a uniformly dispersed W/O/W multiple emulsion.

In step 5), the mass percentage concentration of the second part of the poloxamer aqueous solution is 0.3%, and the W/O/W double emulsion is mixed with the second part of the poloxamer aqueous solution with mechanical stirring at a speed of 1200rpm to further Dispersion stable W/O/W double emulsion.

In step 6), the molar ratio of the arsenic trioxide-loaded nanoparticles, NH ₂ -PEG-NH ₂ and LA is 1:1.3:1, so as to ensure that the NH ₂ -PEG-NH ₂ has enough amino groups to covalently couple the arsenic trioxide-loaded nanoparticles Granules and LA.

Description of drawings

Fig. 1 is a schematic diagram of the preparation method of As ₂ O ₃ stealth immune targeting anti-tumor preparation.

Fig. 2 is a 30,000-fold SEM image of As ₂ O ₃ /PLGA-PEG-LA nanospheres.

FIG. 3 is a 100,000-fold SEM image of As ₂ O ₃ /PLGA-PEG-LA nanospheres.

Fig. 4 is the FT-IR image of As ₂ O ₃ nanometer microspheres.

Fig. 5 is a DLS diagram of As ₂ O ₃ /PLA nanospheres.

Fig. 6 is a DLS diagram of As ₂ O ₃ /PLGA-PEG nanospheres.

Fig. 7 is a DLS diagram of As ₂ O ₃ /PLGA-PEG-LA nanospheres.

Fig. 8 is an AFS standard curve diagram of the drug As ₂ O ₃ .

Fig. 9 is a graph showing the in vitro controlled release curve of As ₂ O ₃ nanoparticles.

Figure 10 is a photograph of the in vitro suspension stability of As ₂ O ₃ -loaded nanoparticles released for 45 days.

Detailed ways

The present invention will be further elaborated below through examples, but the present invention is not limited thereto. Experimenters can selectively modify the surface of microspheres with PEG and LA or modify PEG and LA simultaneously according to actual needs, so that various functions such as slow release, stealth, immune targeting, and stealth immune targeting can be obtained. of microspheres.

The polymer material used in the present invention is PLGA, and may also be PLA, PLA-PEG copolymer, PLGA-PEG copolymer and the like.

Example 1:

1. Preparation of polylactic acid nanospheres (As ₂ O ₃ /PLA NPs) loaded with arsenic trioxide, as shown in Figure 1:

(1) Weigh 10 mg As ₂ O ₃ and dissolve it in 1 mL of 4% sodium hydroxide aqueous solution by weight to prepare 10 mg/mL As ₂ O ₃ solution as the water phase;

(2) Dissolve 100 mg of polylactic acid (PLA) in 2 mL of chloroform as the oil phase;

(3) Take 200 μL of the above water phase and slowly add it to 2 mL of the oil phase under magnetic stirring, then emulsify and ultrasonicate at 180W for 60s under ice bath to form W/O colostrum;

(4) Add this colostrum to 4mL of 3% poloxamer aqueous solution by mass percentage, under ice bath, 360W emulsification and ultrasonication for 90s to form W/O/W double emulsion;

(5) Add W/O/W double emulsion to 50mL poloxamer aqueous solution with a mass percentage of 0.3%, stir at room temperature at 1200rpm for 4h, then rotary evaporate for 30min to remove the organic solvent, centrifuge at 12000r/min for 20min, deionize Washing with water three times, collecting the precipitate, and freeze-drying to obtain As ₂ O ₃ /PLA nano-microspheres.

2. The obtained As ₂ O ₃ /PLA nano-microspheres are regular and evenly distributed (see Figure 2 and Figure 3); the average particle size is 300.7nm, and the PDI is 0.235 (as shown in Figure 5); the encapsulation efficiency is 88.72 %, it can be released stably for 45 days in vitro, and the release rate reaches 55.06% (as shown in Figure 9).

Example 2:

1. Prepare polylactic acid-glycolic acid copolymer invisible nanospheres (As ₂ O ₃ /PLGA-PEG NPs) loaded with arsenic trioxide, as shown in Figure 1:

(1) Weigh 10 mg As ₂ O ₃ and dissolve it in 1 mL of 4% sodium hydroxide aqueous solution by weight to prepare 10 mg/mL As ₂ O ₃ solution as the water phase;

(2) Dissolve 60 mg of polylactic-co-glycolic acid (PLGA) in 2 mL of chloroform as the oil phase;

(3) Take 200 μL of the above water phase and slowly add it to 2mL of the oil phase under magnetic stirring, and then emulsify and ultrasonicate at 180W for 60s under ice bath to form W/O colostrum;

(4) Add this colostrum to 4mL of 3% poloxamer aqueous solution by mass percentage, under ice bath, 360W emulsification and ultrasonication for 90s to form W/O/W double emulsion;

(5) Add this double emulsion to 50mL poloxamer aqueous solution with a mass percentage of 0.3%, stir at room temperature at 1200rpm for 4h, then rotary evaporate for 30min to remove the organic solvent, centrifuge at 12000r/min for 20min, and wash with deionized water for 3 times , collect the precipitate, and freeze-dry to obtain As ₂ O ₃ /PLGA nanometer microspheres.

(6) Disperse 50mg (0.0025mM) freeze-dried As ₂ O ₃ /PLGA nanospheres in 50mL distilled water, add 0.0125mM NHS for activation for 1h, then add 0.0033mM NH ₂ -PEG-NH ₂ and 0.0125 mM EDC, at room temperature, magnetically stirred for 72 hours, and the precipitate was collected by centrifugation to obtain As ₂ O ₃ /PLGA-PEG nanospheres.

2. The obtained As ₂ O ₃ /PLGA-PEG nanospheres are regular in shape and evenly distributed (as shown in Figure 2 and Figure 3). In the infrared spectrum of PLGA-PEG in Figure 4, the characteristic peak of the amide bond appeared at 1610cm-1, indicating that the amino group on the PEG had achieved covalent coupling with the carboxyl group on the surface of PLGA, and the surface modification was successful. The obtained As ₂ O ₃ /PLGA-PEG NPs had an average particle size of 162.6 nm, a PDI of 0.194 (as shown in Figure 6), and good dispersion. The encapsulation rate was 92.03%, and it could be released stably for 45 days in vitro, with a release rate of 65.42% (as shown in Figure 9). The in vitro suspension stability is good, and there is no obvious change after 45 days of storage in a refrigerator at 4°C (as shown in Figure 10).

Example 3:

1. Preparation of polylactic acid-glycolic acid copolymer stealth immune targeting microspheres (As ₂ O ₃ /PLGA-PEG-LA NPs) loaded with arsenic trioxide, as shown in Figure 1:

(1) Weigh 30 mg As ₂ O ₃ and dissolve it in 1 mL of 4% sodium hydroxide aqueous solution by weight to prepare 30 mg/mL As ₂ O ₃ solution as the water phase;

(2) Dissolve 120 mg of polylactic-co-glycolic acid (PLGA) in 4 mL of chloroform as the oil phase;

(3) Take 400 μL of the above water phase and slowly add it to 4 mL of the oil phase under magnetic stirring, and then emulsify and ultrasonicate at 180W for 60s under ice bath to form W/O colostrum;

(4) Add this colostrum to 8mL of 3% poloxamer aqueous solution by mass percentage, under ice bath, 360W emulsification and ultrasonication for 90s to form W/O/W double emulsion;

(5) Add all the double emulsion to 100mL poloxamer aqueous solution with a mass percentage of 0.3%, stir at room temperature at 1200rpm for 4h, then rotary evaporate for 30min to remove the organic solvent, centrifuge at 12000r/min for 20min, wash with deionized water for 3 Once, the precipitate is collected and freeze-dried to obtain As ₂ O ₃ /PLGA nano-microspheres.

(6) Redisperse 100 mg of freeze-dried As ₂ O ₃ /PLGA nanospheres in 50 mL of distilled water, add 0.0250 mM NHS for activation for 1 h, and then add NH ₂ -PEG-NH ₂ 0.0065 mM, LA 0.0050 mM, EDC 0.0250mM, magnetic stirring at room temperature for 72 hours, and centrifugation to collect the precipitate to obtain As ₂ O ₃ /PLGA-PEG-LA nanospheres.

2. The obtained As ₂ O ₃ /PLGA-PEG-LA nano-microspheres are regular in shape and evenly distributed (as shown in Figure 2 and Figure 3).

In the infrared spectrum of PLGA-PEG-LA in Figure 4, the characteristic peak of the amide bond appeared at 1610cm-1, indicating that PEG and LA had been grafted to the surface of PLGA through covalent coupling, and the surface modification was successful. The obtained As ₂ O ₃ /PLGA-PEG-LA nanospheres had an average particle diameter of 229.3 nm, a PDI of 0.175 (as shown in FIG. 7 ), and good dispersion. The encapsulation rate of the drug reached 91.68%, and it could be released stably for 45 days in vitro, with a release rate of 60.59% (as shown in Figure 9). The in vitro suspension stability is good, and there is no obvious change after 45 days of storage in a refrigerator at 4°C (as shown in Figure 10).

Claims (6)

1. A preparation method for arsenic trioxide stealth immune targeting anti-tumor preparation, characterized in that it comprises the following steps: 1) Dissolve As ₂ O ₃ in a 4% aqueous sodium hydroxide solution by mass to prepare an arsenic solution containing As ₂ O ₃ at a concentration of 10-100 mg/mL as the water phase; 2) Dissolve lactic acid-glycolic acid copolymer in chloroform as the oil phase; 3) Add the water phase to the oil phase under magnetic stirring, and then emulsify and ultrasonicate in an ice bath to form W/O colostrum; 4) Mix W/O colostrum with the first poloxamer aqueous solution, emulsify in ice bath and ultrasonically form W/O/W double emulsion; 5) After uniformly mixing the W/O/W double emulsion with the second poloxamer aqueous solution, the organic solvent was removed by evaporation, the precipitate was collected by centrifugation, washed with deionized water, and freeze-dried to obtain arsenic trioxide-loaded nanoparticles; 6) Disperse the above-mentioned arsenic trioxide-loaded nanoparticles in water, add N-hydroxysuccinimide for activation, and mix with double-terminal amino polyethylene glycol, lactobionic acid and 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide hydrochloride was reacted with magnetic stirring at room temperature, and the precipitate was collected by centrifugation to obtain arsenic trioxide stealth immune targeting anti-tumor preparation. 2. The preparation method according to claim 1, characterized in that in step 2), the concentration of the lactic acid-glycolic acid copolymer is 30-50 mg/mL. 3. The preparation method according to claim 1, characterized in that in step 3), the mixing volume ratio of the water phase and the oil phase is 1:10, the ultrasonic power is 180W, and the ultrasonic time is 60s. 4. The preparation method according to claim 1, characterized in that in step 4), the mass percent concentration of the poloxamer aqueous solution is 3%, the ultrasonic power is 360W, and the ultrasonic time is 90s. 5. The preparation method according to claim 1, characterized in that in step 5), the mass percent concentration of the poloxamer aqueous solution is 0.3%, and the mixture is mechanically stirred at a speed of 1200 rpm. 6 . The preparation method according to claim 1 , wherein in step 6), the molar ratio of the nanoparticles loaded with arsenic trioxide, double-terminal amino polyethylene glycol and lactobionic acid is 1:1.3:1.