CN1903365A - Drug-carried nanometer particles, and its preparing process for preparing medicien prepn. for anti-restenosis of blood-vessel - Google Patents

Abstract

The invention discloses a drug-loaded nano particle, a preparation method thereof and an application of the particle in preparing an anti-restenosis preparation. Drug-loaded nanoparticles are composed of biodegradable polymer materials and drugs, and biodegradable polymer materials include polycaprolactone (PCL), polylactic acid (PLA) and polylactic acid-polyglycolic acid copolymer (PLGA). The drugs are anti-vascular endothelial hyperplasia drugs, anti-cell proliferation drugs, thrombolytic drugs, anticoagulant drugs and anti-inflammatory drugs; nanoparticles contain 70-95% of biodegradable polymers, 5-30% of drugs . The particle diameter ranges from 50 to 500 nanometers; the surface of the nanoparticles is modified with modifiers. In order to enhance the anti-restenosis effect of the drug-loaded particles, the drug-loaded particles with different drug effects can be mixed according to the proportion of the dosage according to the disease condition to form a mixed drug. Suspension formulations.

Description

Drug-loaded nanoparticle and its preparation method and the application of the particle in the preparation of anti-restenosis preparations

technical field

The present invention belongs to a method and a new application of a preparation dedicated to making medicine into a special physical shape, and specifically relates to a drug-loaded nanoparticle and its preparation method and the application of the particle in the preparation of an anti-restenosis preparation

Background technique

Nanoparticles include nanospheres and nanocapsules, which are solid colloidal particles with a diameter of 1-1000 nm. Active ingredients (drugs, bioactive materials, etc.) can be wrapped inside the particles or attached to the surface of the particles. Nanoparticles are a promising new dosage form. As a drug carrier, nanoparticles not only can change the distribution of drugs in vivo and have targeting properties, but also have the advantages of adjusting drug release speed and improving bioavailability. to research hotspots. Now a variety of materials have been used to prepare nano-controlled release systems with different characteristics. The method of preparing nano-controlled release system includes polymerization reaction method and polymer material dispersion method. The former is emulsion polymerization and interfacial polymerization technology prepared by polymerization reaction using different monomers; the latter is such as emulsification-solvent evaporation method using high molecular polymer The method of preparation. The drug in the nano-controlled release system can be released through surface diffusion, or the drug can be released through the gradual erosion and degradation of the matrix itself. The polymer matrix of nanoparticles can control the slow release of drugs, so as to achieve the purpose of a long-term effective drug.

Vascular restenosis is a process in which blood vessels become narrowed and blocked again after interventional treatment (such as angioplasty, atherectomy, implantation of intravascular stents, etc.). Drugs to prevent restenosis usually focus on intervening in its early processes, such as platelet deposition and thrombus formation, or blocking the middle and late processes, that is, smooth muscle cell proliferation or matrix formation. These drugs include thrombolytics, anti-inflammatory drugs, and antiplatelet drugs that block the early process. Antiproliferative drugs have the effect of growth factor inhibitors and cytostatics, which can inhibit the later process of restenosis. Therefore, drugs with different effects are released on the vascular lesion site, acting on different stages of the process of causing restenosis, so as to prevent and treat restenosis more effectively.

Chinese patent 1561987A discloses the application and preparation method of biodegradable polymer nanospheres loaded with paclitaxel in the treatment of cancer. The nanospheres contain 80-90% polylactone-polyethylene glycol block copolymer , 10-20% Paclitaxel. Use the aqueous solution of polyvinyl alcohol or gelatin as the dispersion medium, add the organic solvent containing polylactone-polyethylene glycol copolymer and paclitaxel to it, perform ultrasonic emulsification, and then evaporate the organic solvent under reduced pressure to obtain a particle size of 300-800 Nano nanospheres. However, the application of surface-modified nanoparticles prepared from artificially synthesized biodegradable polymer materials in pharmaceutical preparations for the treatment of vascular restenosis has not been reported yet.

Chinese patent 1507928A discloses a drug-coated stent for preventing/treating vascular restenosis, which has an anti-restenosis effect. It effectively prevents/treats postoperative restenosis by coating drug coatings with multiple mechanisms of action on the stent. However, in places where stents cannot be placed in some special parts such as the intersection of blood vessels and relatively thin blood vessels, stents cannot be used to prevent restenosis of blood vessels.

Contents of the invention

The purpose of the present invention is to solve the problem of preventing vascular restenosis in places where stents cannot reach in the prior art, and to provide a drug-loaded nanoparticle, a preparation method thereof, and the use of the particle in preparing an anti-restenosis preparation. application

Invention technical idea:

Due to its ultra-small size, drug-loaded nanoparticles can easily enter the tissue space and be taken up by cells. It can also safely enter the target organ through the smallest capillary (4 μm) in the human body. Therefore, it can be used as a target organ. Carriers for administration. The invention adopts emulsification-solvent volatilization method to prepare nano particles. Nanoparticles are modified to increase their adhesion to vascular tissue. Appropriate physical or chemical methods such as physical blending, surface adsorption, and chemical coupling can be selected to bind them to the surface of nanoparticles, thereby improving the adhesion of nanoparticles. content in blood vessels. During use, according to different conditions, drug-loaded microparticles with different drug effects can be mixed according to the dosage ratio to form a mixed suspension preparation, and a balloon catheter for interventional therapy known to those skilled in the art can be used to transfer drugs with different effects The nano-particles released into the vascular lesion site act on different stages of the restenosis process to prevent restenosis more effectively. The combination of nano-controlled release technology and interventional therapy can realize local drug delivery in blood vessels to prevent restenosis in blood vessels that cannot be reached by stents.

Invention technical solution:

A drug-loaded nanoparticle, which is composed of a biodegradable polymer material and a drug, and its biodegradable polymer material includes polycaprolactone (PCL), polylactic acid (PLA) and polylactic acid-polyglycolic acid copolymer ( PLGA), the drugs are anti-vascular endothelial hyperplasia drugs, anti-cell proliferation drugs, thrombolytic drugs, anticoagulant drugs and anti-inflammatory drugs, the weight percentage of the polymer material is 70-95%, the total weight of the drug The percentage is 5-30%, and the particle diameter ranges from 50-500 nanometers. The surface of the nanoparticles is modified with modifiers.

Described nanoparticle, its anti-vascular endothelial hyperplasia drug is paclitaxel; anti-cell proliferation drug is cytochalasin B; thrombolytic drug includes urokinase, streptokinase or tissue plasminogen activator etc.; anticoagulant drug is Heparin; anti-inflammatory drug is dexamethasone.

The nanoparticles, the modifiers that promote the entry of the nanoparticles into the blood vessel wall tissue include: one of didodecanyldimethyl bromide (DMAB), poly-L-lysine and protamine sulfate, modified The weight percentage of the substance is 2-10%, and the weight percentage of the nanoparticles is 90-98%.

A method for preparing drug-loaded nanoparticles, the present invention adopts an emulsification-solvent volatilization method to prepare nanoparticles, which modifies the prepared nanoparticles, comprising the following steps:

(1) Dissolve one of didodecyldimethylammonium bromide (DMAB), poly-L-lysine and protamine sulfate in water, and prepare a modification with a concentration of 0.5-4mg/ml aqueous solution;

(2) Adding the nanoparticles to the aqueous solution of the modification to prepare a suspension with a concentration of 9.5-76 mg/ml, stirring at a high speed or ultrasonic treatment for 10-120 s, to uniformly suspend the nanoparticles;

(3) freeze-drying.

Application of nanoparticle in preparation of anti-restenosis preparation.

In order to better understand the essence of the present invention, the effectiveness of drug-loaded nanoparticles in preventing and treating vascular restenosis will be described below by using the absorption amount of nanoparticles in blood vessels and the histopathological morphology test of iliac artery and the results thereof.

1. Test of nanoparticle absorption in blood vessels

In order to increase the curative effect, more nanoparticles need to reside on the blood vessel wall, and increasing the concentration of nanoparticles in the suspension can increase the content of nanoparticles in the blood vessel. The paclitaxel nanoparticle animal experiment showed that after perfusion with different concentrations of nanoparticle suspension, the paclitaxel content determination (extracted with chloroform, and then detected by high performance liquid chromatography) in the isolated porcine carotid artery was shown in Table 1. As the concentration increases, the content of paclitaxel in blood vessels also increases. It shows that the concentration has a very important influence on the absorption of nanoparticles. Theoretically speaking, the absorption of nanoparticles by the arterial wall increases to a certain extent and saturation occurs. But in the concentration range of this experiment (5mg/ml-50mg/ml) there is no sign of saturation. Considering that when the catheter system is used for drug administration, the nanoparticles need to pass through the micropores of the catheter. The higher the concentration, the greater the possibility of blocking the micropores. Therefore, it is recommended that the nanoparticle suspension with a concentration of about 30 mg/ml is more effective for in vivo experiments. Well, it can pass through the micropores of the balloon smoothly and achieve high vascular absorption. This high-concentration method consumes more drugs, and the surface of nanoparticles is modified, and the same concentration of suspension can increase the absorption of particles in blood vessels. The potential (Zeta potential) of the surface charge of the nanoparticles before modification is -3.27mv, and the Zeta potential of the average charge on the surface of the nanoparticles after modification is 20.10mv, because DMAB is a cationic surfactant, which changes the surface charge of the nanoparticles, and the blood vessel wall tissue It is rich in negatively charged glycosaminoglycans, so through the interaction of positive and negative charges, it promotes the absorption and residence of nanoparticles in the blood vessel wall. Paclitaxel nanoparticles modified by DMAB can increase the absorption of nanoparticles in blood vessels by nearly 3 times. According to the same method, paclitaxel nanoparticles modified with the same proportion of the three kinds of modifiers can increase the absorption of vascular tissue, and the effect is shown in Table 2.

Table 1 Effect of nanoparticle suspension concentration on its vascular absorption Blood vessel Absorption of nanoparticles in the vessel wall (μg/10mg arterial dry weight) 5mg/ml 10mg/ml 20mg/ml 30mg/ml 50mg/ml X±SD 6.59±1.12 12.94±1.39 21.90±3.87 35.73±4.99 50.37±4.96

Note: P<0.001 compared between each group

Table 2 Effect of surface modification of nanoparticles on their vascular tissue absorption Absorption of nanoparticles in the vessel wall (μg/10mg arterial dry weight) no modification DMAB Poly-lysine Fish protein 6.20±1.56 19.12±6.34 10.0±1.62 10.97±1.7

It can be seen from Table 2 that among the three modifiers, DMAB has the best effect, which can increase the absorption of nanoparticles in blood vessels by nearly 3 times.

2. Histopathological morphology test of iliac artery

The mechanism of preventing and reducing restenosis is to inhibit intimal hyperplasia, and the present invention has obvious effects on this. This experiment was carried out on the model of excessive straining of the iliac artery in rabbits, with a total of 10 cases. Compared with each control group, after infusion of nanoparticles for 28 days, it was shown that intravascular local infusion of dexamethasone nanoparticles had a significant inhibitory effect on vascular restenosis, and the results are shown in Table 3. The results of the latter three groups in Table 3 can be shown more intuitively in Figure 1 and Figure 2. The results showed that the media area of the local intravascular perfusion of dexamethasone nanoparticles (DEX-NP) group was similar to that of the intravenous injection of DEX-NP group and the local perfusion of blank nanoparticles group (P>0.05), while the lumen area increased (P<0.05), and decreased intima area (P<0.05). It shows that after local intravascular perfusion of dexamethasone nanoparticles, intimal hyperplasia is significantly inhibited, thereby maintaining a larger lumen area. Compared with the local perfusion blank nanoparticle group, intravenous injection of DEX-NP group had no significant effect on the formation of neointima, indicating that systemic application of DEX-NP had no inhibitory effect on intimal hyperplasia of injured vessels. Compared with the intravenous DEX-NP group and the local perfusion blank nanoparticle group, the intima/media area ratio of the local intravascular perfusion DEX-NP group was reduced by 46.9% and 45.9%, respectively.

Table 3. Analysis results of histopathological morphology of iliac arteries (X±SD) pathological form saline local perfusion nanoparticle local perfusion Intravenous injection of dexamethasone nanoparticles Local infusion of dexamethasone nanoparticles MIT(mm)LA(mm ² )IEL(mm ² )EEL(mm ² )IA(mm ² )MA(mm ² )Intima/Media 0.225±0.0231.217±0.1371.844±0.2032.376±0.2650.630±0.1040.536±0.1201.178±0.411 0.227±0.0381.225±0.181 ^* 1.867±0.2522.421±0.2290.647±0.190 ^* 0.559±0.113 ^* 1.159±0.379 ^* 0.230±0.0331.226±0.1851.902±0.2032.465±0.2630.664±0.1090.563±0.1451.180±0.243 0.140±0.038 ^** 1.564±0.176 ^** 1.924±0.154 ^* 2.527±0.230 ^* 0.368±0.118 ^** 0.569±0.115 ^* 0.627±0.238 ^**

Note: MIT, maximum thickness of intimal hyperplasia; LA, luminal area; IEL, internal area of internal elastic membrane; EEL, internal area of external elastic membrane; IA, intima area; MA, media area; Intima/Media, intima The ratio to the media area is the proliferation index. *Compared with the groups on the left side, P>0.05; **Compared with the groups on the left side, P<0.05

The invention can increase the content of nano particles in blood vessels through the modified drug-loaded nano particles, and produce the effect of small dose and good curative effect. And through the balloon catheter, the drug-loaded nanoparticles are delivered to the damaged blood vessel where the stent cannot be placed. The nanoparticles of different drugs act on different stages of the process of causing restenosis, so as to prevent and treat restenosis more effectively. The combination of nano-controlled release technology and interventional therapy can realize intravascular local drug delivery to prevent and treat vascular restenosis. Thereby effectively preventing restenosis of blood vessels that cannot be reached by the stent.

Description of drawings

Figure 1: Inhibitory effects of different administration methods on vascular restenosis

Figure 2: Effects of different administration methods on intimal hyperplasia

Figure 3: Preparation process of drug-loaded nanoparticles of fat-soluble drugs

Figure 4: Preparation process of drug-loaded nanoparticles for water-soluble drugs

Detailed ways

The preparation method of the biodegradable polymer nanoparticles loaded with anti-restenosis drugs of the present invention is as follows:

1. Preparation method of drug-loaded nanoparticles of fat-soluble drugs

Fat-soluble drugs such as paclitaxel and dexamethasone are prepared by the oil-in-water (O/W) emulsification-solvent evaporation method (see Figure 3), and the method is as follows:

1. A certain volume of dichloromethane dissolves an appropriate amount of biodegradable polymer with a concentration of 0.5-4%, a certain volume of acetone dissolves an appropriate amount of a drug concentration of 0.02-4%, and then mixes dichloromethane/acetone (the ratio is 9:1 or 8:2 ) organic solution.

2. Add the mixed organic solution into a certain volume of (polyvinyl alcohol) PVA aqueous solution (0.5-2.5%) (organic solution/water solution = 1:3 to 1:4), and sonicate for 30s-10min under ice bath conditions.

3. Stir and volatilize under normal pressure for 2-18h, then volatilize under reduced pressure for 1-6h to remove the organic solvent.

4. Ultracentrifuge at low temperature (4°C-15°C) at 15000-25000 rpm for 30 minutes to collect solidified nanoparticles, wash with distilled water and centrifuge 1-3 times to remove polyvinyl alcohol in the nanoparticle suspension.

5. Freeze drying to complete the preparation of nanoparticles.

6. Prepare an aqueous solution of the modifier with a concentration of 0.5-4mg/ml.

7. Add the nanoparticles to the aqueous solution of the modifier, prepare a suspension with a concentration of 9.5-76 mg/ml, stir at a high speed or ultrasonically treat for 10-120 s, and suspend the nanoparticles evenly.

8. Freeze-drying to complete the preparation of anti-restenosis drug-loaded nanoparticles.

9. Sterilize the nanoparticles by irradiation with gamma rays emitted by cobalt ⁶⁰ between 15-30 kGy.

Two, the preparation method of the drug-loaded nanoparticles of water-soluble drug

Water-soluble drugs such as urokinase and heparin are prepared by double emulsion (W/O/W)-solvent evaporation method (see Figure 4), and the method is as follows:

1. A certain quality of water-soluble drugs is dissolved in distilled water at a concentration of 20-30%, and a certain volume of dichloromethane (DCM) or dichloromethane/acetone (8:2 or 9:1) organic solution is dissolved in an appropriate amount to be biologically Degradable polymer capsule material (concentration: 0.5-10%), the organic solution contains a certain proportion (0.01%-0.1%) of Pluronic F68 (Pluronic F68) as an emulsifier

2. Add the aqueous drug solution to the organic solution (aqueous drug solution: organic solvent volume ratio = 1:4-20), and immediately ultrasonically or high-speed stir for 30s-10min under ice bath conditions to form a W ₁ /O solution.

3. Cool at 4°C to stabilize colostrum

4. Add the colostrum into the PVA aqueous solution with a concentration of 0.5-2.5%, and carry out ultrasonic or high-speed stirring emulsification under ice bath conditions for 30s-10min to form W ₁ /O/W _{2 double} emulsion.

5. Stir and volatilize under normal pressure for 2-18h, then volatilize under reduced pressure for 1-6h to remove the organic solvent.

6. Low temperature (4°C-15°C), ultracentrifugation at 15000-25000rpm for 30min, collect solidified nanoparticles, wash with distilled water, and centrifuge 1-3 times to remove polyvinyl alcohol in the nanoparticle suspension.

7. Freeze drying to complete the preparation of nanoparticles.

8. Prepare an aqueous solution of the modifier with a concentration of 0.5-4mg/ml.

9. Add the nanoparticles into the above aqueous solution of the modification, prepare a suspension with a concentration of 9.5-76 mg/ml, stir at high speed or ultrasonically treat for 10-120 s, to suspend the nanoparticles uniformly.

10. Freeze-drying to complete the preparation of anti-restenosis nanoparticles

11. Sterilize the nanoparticles by irradiation with gamma rays emitted by cobalt ⁶⁰ between 15-30 kGy.

specific embodiment

Example 1. Paclitaxel Nanoparticles

Mix 9 ml of 2% PLGA dichloromethane solution with 1 ml of acetone solution containing 85.96 mg of paclitaxel, then add to 40 ml of 1% PVA water concentrate, and emulsify by ultrasonic (40W) for 2 min in an ice bath. Then electromagnetically stirred at room temperature for 3 h, and evaporated under reduced pressure for 1 h. Centrifuge at 23000 rpm for 30 min at low temperature to collect solidified nanoparticles. It was washed with distilled water, centrifuged once, and freeze-dried for 36 hours to obtain paclitaxel nanoparticles with a drug loading capacity of 30.54%, an encapsulation efficiency of 90.75%, and an average particle diameter of 385 nm. Add the obtained above-mentioned nanoparticles to 5 ml of DMAB aqueous solution with a concentration of 1 mg/ml, and sonicate for 10-30 s. Freeze-drying yields surface-modified nanoparticles. The average particle size of the modified nanoparticles is 462.2nm.

Embodiment 2. Dexamethasone nanoparticles

Dissolve 200mg PLGA with 10ml dichloromethane, 1ml acetone dissolves 42.5mg dexamethasone acetate powder, then mix two kinds of organic solutions, this mixed organic solution joins in the PVA aqueous solution of 40ml 2% concentration, under ice-bath condition ultrasonic (40W ) emulsified for 10 min, stirred electromagnetically at room temperature for 12 h, volatilized under reduced pressure for 1.5 h, removed the organic solvent, ultracentrifuged at low temperature and 23,000 rpm for 30 min, collected solidified nanoparticles, washed with distilled water, centrifuged three times to remove PVA on the surface of the nanoparticles, and freeze-dried After 36 hours, dexamethasone nanoparticles with a drug load of 17.37% were obtained, the encapsulation efficiency was 98.97%, and the average particle diameter was 260 nm. Add the obtained above-mentioned nanoparticles to 5 ml of DMAB aqueous solution with a concentration of 1 mg/ml, and sonicate for 10-30 s. Freeze-drying yields surface-modified nanoparticles.

Example 3. Heparin Nanoparticles

6 milliliters of PLGA dichloromethane solution (concentration is 6%) is added in the vial containing the aqueous solution (concentration is 26%) of 0.6 milliliter heparin, ultrasonic emulsification 3min (output power is 40W) under ice-bath condition immediately, obtain initial Milk, the colostrum was added to 24 ml of 0.5% PVA aqueous solution, and ultrasonic emulsification was also carried out for 3 minutes under ice bath conditions to form W ₁ /O/W ₂ double emulsion. Then the double emulsion was magnetically stirred, and the organic solvent was volatilized under normal pressure for 2 hours, and the solidified nanoparticles were collected by high-speed centrifugation (23000rpm×20min), washed three times with distilled water, and freeze-dried for 36 hours. The heparin nanoparticles with a drug load of 5.35% were obtained, and the average particle size of the nanoparticles was 297nm.

Example 4

In order to strengthen the anti-restenosis effect of drug-loaded particles, drug-loaded particles with different drug effects can be mixed according to the proportion of the dosage according to the disease condition, and prepared into a saline mixed suspension preparation with a concentration of 30mg/ml for use. Known interventional therapies deliver nanoparticles to damaged vascular sites.

Claims (5)

1. A drug-loaded nanoparticle, composed of a biodegradable polymer material and a drug, characterized in that the biodegradable polymer material includes polycaprolactone (PCL), polylactic acid (PLA) and polylactic acid-polyglycolic acid One of the copolymers (PLGA), the drugs are anti-vascular endothelial hyperplasia drugs, anti-cell proliferation drugs, thrombolytic drugs, anticoagulant drugs and anti-inflammatory drugs, the weight percentage of its polymer material is 70-95%, the drug The total weight percentage is 5-30%; the particle diameter ranges from 50-500 nanometers, and the surface of the nano-particles is modified by modifiers. 2. The nanoparticle according to claim 1, characterized in that the anti-vascular endothelial hyperplasia drug is paclitaxel; the anti-cell proliferation drug is cytochalasin B; thrombolytic drugs include urokinase, streptokinase or tissue plasminogen activation Anticoagulant drug is heparin; anti-inflammatory drug is dexamethasone. 3. The nanoparticle according to claim 1, characterized in that the modifiers that promote the nanoparticle to enter the blood vessel wall tissue include: didodecyldimethylammonium bromide (DMAB), poly-L-lysine and sulfuric acid One of the protamines, the weight percent of the modification is 2-10%, and the weight percent of the nanoparticles is 90-98%. 4. A preparation method of drug-loaded nanoparticles, the present invention adopts the emulsification-solvent volatilization method to prepare nanoparticles, which is characterized in that the prepared nanoparticles are modified, comprising the following steps: (1). Dissolve one of didodecanyldimethyl bromide (DMAB), poly-L-lysine and protamine sulfate in water to prepare a concentration of 0.5-4mg/ml Modifier aqueous solution; (2). Add the nanoparticles into the aqueous solution of the above-mentioned modifiers to form a suspension with a concentration of 9.5-76 mg/ml, and perform high-speed stirring or ultrasonic treatment for 10-120 seconds to uniformly suspend the nanoparticles; (3). Freeze drying. 5. Application of drug-loaded nanoparticles in the preparation of anti-restenosis preparations.

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