TWI653058B - Embedding microsphere and manufacturing method thereof - Google Patents

Abstract

The invention discloses an embedding microsphere and a manufacturing method thereof, wherein the method for manufacturing the invention obtains an embedding microsphere with good biocompatibility and biodegradability, and can carry a specific drug to the affected part. Continuous and large release to achieve the effect of treating the disease or delaying the progression of the disease.

Description

Embedding microsphere and manufacturing method thereof

The present invention relates to a method of producing a pharmaceutical carrier, and in particular to an embolic microsphere and a method of producing the same.

According to the method of Transarterial Chembolization (TACE), it is currently used in the treatment of liver cancer. The embolization agent is put into the blood vessel, the local blood flow is blocked, and the chemotherapy drug is injected into the tumor site for treatment. Compared to past embolization therapies, hepatic artery embolization chemotherapy can significantly reduce side effects. According to a market survey conducted by AB Newswire in 2016, the preparations and devices related to embolization can reach a market of approximately US$450 million.

There are currently two types of embolic agents on the market, one of which is a mechanical occlusion device and the other is a flow-directed embolic agent. The mechanical occlusion device comprises a metal coil and a detachable air bag, which is arranged in the target blood vessel to reduce blood flow and platelet aggregation in the hemorrhage or aneurysm to achieve the effect of embolization; the flow type embolic agent contains micro A ball or polymer that captures the surrounding blood vessels of the target tissue (cancer tissue) to block the supply of blood to the target tissue and cause the target tissue to shrink. However, most of the commercially available embolic agents are composed of non-degradable materials, such as Onyx ^® , Embosphere ^® , DCBead ^{® ,} etc., and non-degradable embolic agents are often used in liver cancer embolization to cause liver tissue in non-tumor areas. Infarction or cause of gallbladder infarction, and artificial polyvinyl alcohol and its derivatives often cause adverse reactions in patients.

The above-mentioned deletions result in limited clinical application of embolic agents, and side effects in cancer treatment, and are not applicable to various types of cancer.

The main object of the present invention is to provide an embolic microsphere which has good biocompatibility and biodegradability, and can carry a large amount of drugs to a specific part for sustained release, and provides drug release and action efficiency for treatment. Disease or delay the development of disease.

Another object of the present invention is to provide a method for producing a plug microsphere which is capable of preparing a plug microsphere having a predetermined particle size for the purpose of providing clinical use.

In order to achieve the above object, a first embodiment of the present invention discloses an embolic microsphere consisting of a gellan gum, a polyethyleneimine-histamine, and a short-chain hyaluronic acid-histamine. Electrosorption of a drug.

Wherein, the drug is a positively charged compound which is adsorbed to the negatively charged microspheres.

Furthermore, in order to enable the embedding microspheres to exert a better effect in the body, the particle diameter of the plug microspheres disclosed in the examples of the present invention is 50-500 μm, wherein the particle diameters are 285 μm, 388, 481 μm. good.

In another embodiment of the invention, a method of manufacturing a plug microsphere is disclosed, comprising the steps of:

Step a: preparing a first nanoparticle obtained by mixing a polyethyleneimine-histamine with a short-chain hyaluronic acid-histamine in a predetermined ratio.

Step b: combining the first nanoparticle with a compound by electrical adsorption to form a second nanoparticle.

Step c: After the second nanoparticle is mixed with a calicin solution, an emulsification reaction is carried out.

Step d: curing to obtain a plug microsphere.

Preferably, the first nanoparticle is negatively charged and the compound is positively charged. For example, the compound is an anthracycline such as doxorubicin.

Wherein, in order to enable the first nanoparticle to be negatively charged, the polyethyleneimine-histamine and the short-chain hyaluronic acid-histamine acid must be mixed in a predetermined ratio, for example, polyethylene The amine-histamine is mixed with the short chain hyaluronic acid-histamine acid in a ratio of 4:1.

In an embodiment of the present invention, in order to enable the embedding microspheres obtained by the above method to have a particle size of less than 500 μm, the dendritic solution has a concentration of 0.3 to 0.5% (w/v), wherein the gellan solution is further used. When the concentration is 0.3% (w/v), it is possible to make more than 60% of the sieve microspheres have a particle diameter of less than 500 μm.

In the following examples, Doxorubicin (hereinafter referred to as Dox) is used as an exemplary drug. Among them, doxorubicin, also known as doxorubicin, is an anthracycline drug for chemotherapy. The mechanism of action is to be able to pass through the DNA and cause the cells to die.

Example 1: Preparation of aramid particles

A predetermined amount of gellan gum (GG) polymer powder is dissolved in distilled water and maintained at 85-90 ° C until the gellan solution becomes transparent, and then cooled to about 50 ° C, according to the above procedure, The amount of the gellan gum polymer powder was changed to obtain a calcined gum solution having a concentration of 0.3, 0.4, and 0.5% w/v, respectively.

Mix 400 ml of 90% w/w mineral oil at high speed (400 rpm) with a solution of 0.3, 0.4, 0.5% w/v of the lanolin solution for about 10 minutes to obtain a water-oil emulsion (water-in). -oil emulsion). 1.25% (w/v), 1 ml of calcium chloride and 0.5% (w/v), 1 ml of Span 85 were added to the aqueous oil phase emulsion, and stirred at 50 ° C for about 60 minutes at high speed. At 50 ° C, 1.25% (w / v) calcium chloride was added again and the reaction was allowed to cure for about 10 minutes to harden the starch. The results of observation by a microscope are as shown in the first figure, and the appearance of the gellanite particles was observed by a scanning electron microscope (Jeol JSM-6400, Japan), and the results are shown in the second figure.

Screens of different sizes were distinguished by different mesh sizes: 25 mesh (710 μm), 40 mesh (425 μm), 50 mesh (300 μm), and 70 mesh (212 μm). After washing the calcined granules in each sieve, they were uniformly suspended in purified water, and the particle size was measured by a laser particle size analyzer (Mastersizer 2000, Malvern, United Kingdom). The results are as shown in the third figure A to D is shown.

As can be seen from the results of the third graph, the 25-mesh screen can separate the calcin particles having a particle diameter of more than 500 μm, and the average particle size of the calcined particles separated by the 25-mesh sieve is about 681±. The 51 μm, 40, 50, and 70 mesh screens were able to obtain 498 ± 32 μm, 360 ± 24 μm, and 226 ± 15 μm of the mulberry particles, respectively.

Further, the above-mentioned sieve mesh is used to distinguish the dendritic microspheres prepared by different concentrations of lanolin, and the particle size distribution thereof is shown in Table 1 below.

Table 1: Size distribution of the gelatin microspheres prepared by different concentrations of lanolin  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> scale (%) </td></tr><tr ><td> Lanolin concentration (%) </td><td> 681μm (25 mesh) </td><td> 498μm (40 mesh) </td><td> 360μm (50 mesh) </td ><td> 226μm (70 mesh) </td><td> Other small sizes (200~50μm) </td></tr><tr><td> 0.5 </td><td> 66 </td ><td> 17 </td><td> 2 </td><td> 2 </td><td> 13 </td></tr><tr><td> 0.4 </td><td > 51 </td><td> 16 </td><td> 8 </td><td> 5 </td><td> 20 </td></tr><tr><td> 0.3 < /td><td> 35 </td><td> 15 </td><td> 9 </td><td> 11 </td><td> 30 </td></tr></TBODY ></TABLE>

It can be seen from the above results that the calcined colloidal particles are microspheres composed of a gellanic fiber and having a porous surface, and different concentrations of the margarine series form different amounts of various sizes of calendin particles, among which, high concentration The lanolin will form larger particle size particles.

According to the diameter of the blood vessel, the required particle size should be less than 500 μm, and as can be seen from the above Table 1, the use of the lesser concentration of the gellan gum can obtain a larger amount of less than 500 μm of the calamine particles, so that the concentration is about It is preferred to prepare the blue-collar particles by 0.3% of the gum. In addition, it is difficult to prepare the mordant particles based on the lanolin having a concentration of less than 0.3%. Therefore, the lanolin particles disclosed in the present invention should be prepared with a concentration of at least 0.3% of the lanolin. It is better for 0.3%.

Example 2: Synthesis of short-chain hyaluronic acid-histamine polymer

0.66 g of short-chain Hyaluronate-Histidine (hereinafter referred to as sHH) was dissolved in 150 ml of distilled water to obtain a sHH solution. The pH of the sHH solution was adjusted to 5.5, 48 mg of EDC (1-(3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 28.8 mg of NHS were added, and after stirring for about 30 minutes, the pH value of the sHH solution was adjusted. 7.4, followed by the addition of 51.8 mg of L-histamine, and the sHH solution was maintained at 25 ° C for 24 hours. After completion of the above reaction, the sHH solution was placed in a dialysis tube and dialyzed to remove residual EDC, NHS, and L-histamine. After dialysis, the sHH solution was lyophilized to obtain sHH polymer, which was analyzed by ¹ H NMR (500 MHz, Bruker Advance DRX500), and the results are shown in the fourth figure.

Referring to Figure 4, the structure of the sHH polymer is analyzed as follows: δ7.9 ppm (methenyl protons located in imidazole group of his, -N=CH-), δ7.0 ppm (methenyl protons located in 328 imidazole group of his, -N-CH=C-), δ 4.8 ppm (H1 of N-acetylglucosamine, GlcNAc), δ 4.7 ppm (H1' of Glucuronic acid, GluA), δ 3.9 ppm (H2 of GlcNAc), δ 3.7-3.8 ppm ( H3, H6 of GlcNAc and H4', H5' of GluA), δ 3.5-3.6 ppm (H4, H5 of GlcNAc and H3' of GluA), δ 3.4 ppm (H2' of GluA). From the integration of the peak area, the degree of substitution (DS) of the short-chain hyaluronic acid-histidine acid was 10%.

Example 3: Synthesis of PH (Polyethylenimine -Histidine) polymer

165 μl of histidine (1.32 g), EDC (2.45 g) and NHS (1.47 g) were dissolved in distilled water to form a histidine/EDC/NHS solution, and the pH was adjusted to 5.5, after 30 minutes, 1 ml of a branched polyethylenimine solution was added, and the histamine/EDC/NHS solution had a pH of 7.4 and was maintained at 25 ° C for 24 hours. Thereafter, the histidine/EDC/NHS solution was dialyzed, and the collected product was lyophilized to obtain a pH polymer. The PH polymer was analyzed by ¹ H NMR (500 MHz, Bruker Advance DRX500), and the results are shown in Fig. 5.

From the results of the fifth graph, the structure analysis of the PH polymer is as follows: the peak of δ2.4-3.1 ppm is derived from the methylene proton of PEI, and the peak of δ7.7 and δ6.9 ppm is located in histidine. The methylene proton in the imidazole group (-N=CH-, -N-CH=C-), the peak of δ 3.4 ppm is the -CH2-proton of histidine. According to calculations, approximately 20% of the amine groups in the PEI are conjugated to the histidyl group.

Example 4: Preparation of sHH/PH/Dox Nanoparticles

The sHH and pH were separately dissolved in distilled water. A 1 mg/ml pH solution was added to a 1 mg/ml sHH solution in which the ratio of the pH solution to the sHH solution was 2:1 or 4:1 to obtain a sHH/PH nanoparticle. The mixed solution was placed at 25 ° C for 30 minutes to reach equilibrium. The zeta potential of the sHH/PH nanoparticle was measured by particle size and boundary potential analyzer (Malvern, Zetasizer Nano). The boundary potential of 1 mg/ml PH solution is 45.1 mv, the boundary potential of 1 mg/ml sHH solution is 42.2 mv; when the ratio of pH solution to sHH solution is 2:1, sHH/PH nanoparticle The surface potential approaches 0; when the ratio of the pH solution to the sHH solution is 4:1, the surface potential of the sHH/PH nanoparticle is -25.4 mv.

In order to enable positively charged chemotherapeutic drugs to bind to sHH/PH nanoparticles, it is necessary to use sHH/PH nanoparticles with negatively charged surface, that is, sHH/PH nanoparticles selected in this example as PH solutions. Prepared by the ratio of sHH solution to 4:1.

Will Doc. 0.5 mg of HCl (doxorubicin hydrochloride) and 1 ml of triethylamine were dissolved in 4 ml of formamide, and stirred overnight in the dark, and then 20 mg of sHH/PH nanoparticles were added. The mixed solution was dialyzed against a phosphate buffer solution in an environment of pH 7.4 for 48 hours, and the phosphate buffer was replaced every 8 hours to remove the organic solvent and the unreacted drug, and then the mixed solution was dialyzed against distilled water. After hours, and lyophilized, sHH/PH nanoparticle loaded with Dox is obtained, which is sHH/PH/Dox nanoparticle. The sHH/PH/Dox nanoparticle was measured by particle size and boundary potential analyzer, and the average size of the sHH/PH/Dox nanoparticle was about 140±8 nm. After the supernatant was diluted by centrifugation, the free Dox was analyzed by ultraviolet spectrophotometry at 480 nm, which showed a polydispersity index of 0.099, and since the polydispersity index of sHH/PH/Dox nanoparticles was less than 0.7, sHH was known. The /PH/Dox nanoparticle system has a very narrow size distribution, in other words, the microspheres prepared by the method of the present invention have similar dimensions.

Example 5: Drug release test

Human hepatoma cells (HepG2) were cultured in EMEM medium containing 1 mM sodium pyruvate, and 10% fetal calf serum, 40 mg/ml gentamicin was added, and the culture temperature was 37 °C. Up to 90% of the cell culture was treated with trypsin. Human hepatoma cells were divided into two groups, which were first inoculated on coverslips. After 24 hours of culture, one group of medium was replaced with sHH/PH/Dox nanoparticle medium, and the other group was maintained with original medium. The culture was carried out for 24 hours. Then, the two sets of coverslips were separately washed, fixed with 1% glutaraldehyde, and observed by an inverted fluorescent microscope, and the results are shown in Fig. 6 and B.

As can be seen from the results of the sixth graph, the liver cancer cell line co-cultured with sHH/PH/Dox nanoparticles exhibited red fluorescence, and the liver cancer cells not co-cultured with sHH/PH/Dox nanoparticles exhibited red fluorescein. Light. The doxorubicin-based system has the property of being excited by fluorescence at 480 nm. Therefore, it is confirmed by the results of the sixth figure that the sHH/PH nanoparticle can be used as a drug delivery carrier for doxorubicin to deliver the drug to the cell smoothly. .

Example 6: Preparation of embolic microspheres

40 ml, 0.3% aqueous gellan gum solution was mixed with 0.04 g of sHH/PH/Dox nanoparticles at 50 ° C to obtain GG/sHH/PH/Dox solution, wherein the aqueous gellan solution and sHH/PH/ The Dox nanoparticles were prepared according to the procedures shown in the above examples. 360 ml of mineral oil was mixed with 40 ml of GG/sHH/PH/Dox solution with high speed stirring to form a 10% (w/v) water oil phase emulsion. Slowly add 1.25% (w/v), 1 ml of calcium chloride, 0.5% (w/v), and 1 ml of Span 85 to the water-oil phase emulsion with a 23G hypodermic syringe, followed by a high speed at 50 °C. Stir for about 60 minutes. Then, at 50 ° C, 1.25% (w / v) calcium chloride was added and reacted for about 10 minutes to cure the GG / sHH / PH / Dox microspheres, and screens of different sizes: 25 mesh (710 μm), 40 mesh (425 μm), 50 mesh (300 μm), and 70 mesh (212 μm) were used to distinguish GG/sHH/PH/Dox microspheres of different sizes, and their particle diameters were measured by a laser particle size analyzer.

According to the measurement results of the laser particle size analyzer, the average particle diameter of the GG/sHH/PH/Dox microspheres separated by 25, 40, 50, and 70 mesh sieves is 674±33μm and 481±22μm, respectively. 388 ± 32 μm, 285 ± 17 μm. Further, it was found that the particle diameter of the GG/sHH/PH/Dox microspheres was not significantly different from the particle diameter of the calcined microspheres (Example 1).

From the results of the present example, it was confirmed that the preparation method of the present invention can surely prepare a nanoparticle with a drug, and can stably maintain the size of the nanoparticle for clinical application.

Example 7: Drug release test

GG/sHH/PH/Dox microspheres (1 mg/ml) of different particle sizes: 285, 388, 481 μm were placed in 1 ml of phosphate buffer (0.02 M, pH 7.2), and shaken at 37 ° C for 45 days. And, at the predetermined time point, replace 1 ml of the original phosphate buffer with fresh phosphate buffer. The content of doxorubicin was measured at 480 nm by a spectrophotometer, and the results are shown in the seventh chart.

From the results of the seventh graph, it can be seen that the GG, sHH/PH/Dox microspheres of 285, 388, and 481 μm can release 7.3, 2.7, and 1.7 μg / ml of doxorubicin after the 45-day drug release test, respectively. The smaller the particle size of the GG/sHH/PH/Dox microspheres, the larger the surface area can be provided to achieve an efficiency in accelerating drug release.

Furthermore, according to past studies, the IC50 of human liver cancer cells for doxorubicin was 1.5 μg/ml. The results of the above drug release compared with the human hepatoma cells for doxorubicin IC5 showed that the total amount of GG/sHH/PH/Dox microspheres released by 285, 388, 481 μm was about 4.8, 1.8, 1.1. In other words, the GG/sHH/PH/Dox microspheres disclosed in the present invention can meet the requirements for clinically performing hepatic artery embolization chemotherapy, and can be used for treating cancer.

Example 8: Animal Testing

20 mg of GG/sHH/PH/Dox microspheres having a particle diameter of 285 μm was mixed with 1 ml of glycerin to form a pharmaceutical preparation.

Take a healthy New Zealand white rabbit (National Animal Testing Center, Taipei, Taiwan) weighing approximately 2.6 to 3.0 kg, first etherified with Zoletil (Virbac) / Xylazine (20-40 mg/kg Z + 5-10 mg/kgX). The drug preparation was then placed in the proximal end of the ear vein at a dose of 0.15 ml/ear, and the administration time was on days 0, 4, 8, and 12 of the experiment, and after each administration, the injection site was pressed for 30 seconds. The changes in the color and shape of the ears on the 0th, 4th, 8th and 12th day of the experiment were observed. The results are shown in Fig. 8 to Fig. A, where the right ear is the control group and the left ear is the experimental group.

As can be seen from the results of Fig. 8A, the central auricular artery and the branch artery system of the rabbit ear were clearly visible before administration on the 0th day. Please refer to Figure 8B. After administration on the 4th day, the blood flow of the left ear is visible compared to the right ear, and the branch artery of the left ear is invisible because GG/sHH/PH/Dox The ball was present; in addition, edema and inflammation were not significant in the left ear, indicating that the GG/sHH/PH/Dox microspheres disclosed herein have good biocompatibility. Please refer to Figure 8 C. After administration on the 8th day, the left ear has developed ischemic necrosis and blackening, and the condition at the tip of the ear is the worst, and the result of the eighth figure D shows the aortic atrophy of the ear tip. disappear. From this result, it can be seen that the vascular occlusion caused by the GG/sHH/PH/Dox microspheres completely necrosis of the surrounding tissues of the embolization site, and it is confirmed that the GG/sHH/PH/Dox microsphere system prepared by the present invention can achieve the embolization effect. And, with good biocompatibility.

According to the above description, the method for manufacturing the plug microspheres of the present invention is capable of preparing nano microspheres of different sizes, and the nano microspheres are not only biocompatible but also biodegradable, and can be used as A pharmaceutical carrier for carrying, for example, a chemotherapeutic drug to the affected part for the treatment of cancer or an inflammatory disease. In other words, the method of making the plug microspheres of the present invention can be used to provide embolic microspheres for embolization treatment.

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The first image is observed by a microscope to embolize the microsphere shape. The second graph A shows the result of observing the microspheres of the present invention by an electron microscope, and the magnification is 500X. Fig. 2B shows the results of observing the microspheres of the present invention by an electron microscope, and the magnification is 3500X. The third panel A is a size distribution diagram of the embedding microspheres sieved by a 25 mesh screen. The third panel B is a size distribution diagram of the embedding microspheres sieved by a 40 mesh screen. The third panel C is a size distribution diagram of the embedding microspheres sieved by a 50 mesh screen. The third figure D is a size distribution diagram of the embedding microspheres sieved by a 70 mesh screen. The fourth panel is the ¹ H NMR spectrum of the sHH polymer. The fifth panel is the ¹ H NMR spectrum of the PH polymer. Figure 6A shows the results of co-culture of sHH/PH/Dox nanoparticles with human hepatoma cells by inverted fluorescence microscopy. Figure 6B shows the results of co-culture of drug Dox with human hepatoma cells by inverted fluorescence microscopy. The seventh figure is the result of observing the ability of GG/sHH/PH/Dox nanoparticles with different particle sizes to release drug Dox. Figure 8 is the result of observing the color and shape changes of the left and right ears of the rabbit on day 0. Figure 8B shows the results of observing the color and shape changes of the left and right ears of the rabbit on the fourth day. Figure 8C shows the results of observing the color and shape changes of the left and right ears of the rabbit on the 8th day. Figure 8 is the result of observing the color and shape changes of the left and right ears of the rabbit on the 12th day.

Claims (10)

The invention relates to a method for preparing a vascular embolization microsphere reagent, wherein the vascular embolization microsphere reagent is obtained by a emulsification reaction only by a gellan solution and a drug carrier, and the drug carrier is Contains a drug. The use of the gellan gum for preparing a vascular embolization microsphere reagent according to the first aspect of the patent application, wherein the drug carrier is a polyethyleneimine-histamine and a short-chain hyaluronic acid-histamine Composition, and the drug is doxorubicin, which is provided on the drug carrier. . The use of the lanolin for preparing a vascular embolization microsphere reagent according to the first aspect of the patent application, wherein the vascular embolization microsphere reagent has a particle size of 50 to 500 μm. The use of the lanolin for preparing a vascular embolization microsphere reagent according to the third aspect of the patent application, wherein the vascular embolization microsphere reagent has a particle size of 285 μm. The use of the orchid is used for preparing a vascular embolization microsphere reagent according to the first aspect of the patent application, wherein the drug is an anthracycline compound. The use of the lanolin for preparing a vascular embolization microsphere reagent according to the first aspect of the patent application, wherein the vascular embolization microsphere reagent is prepared according to the following steps: Step a: preparing a first nanometer a particle obtained by mixing a polyethyleneimine-histamine with a short-chain hyaluronic acid-histamic acid in a predetermined ratio; and step b: electrically absorbing the first nanoparticle with a compound Combining, forming a second nanoparticle; step c: mixing the second nanoparticle, a gellan solution with an oily solution, and performing an emulsification reaction; and step d: curing to obtain an embolic microsphere. The use of the orchid is used to prepare a vascular embolization microsphere reagent according to claim 6 of the scope of the patent application, wherein the first nanoparticle is negatively charged and the compound is positively charged. The use of the gellan gum for preparing a vascular embolization microsphere reagent according to the sixth aspect of the patent application, wherein the polyethyleneimine-histamine and the short-chain hyaluronic acid-histidine acid in the step a are The ratio of 4:1 is mixed. The use of the orchid is used for preparing a blood vessel embolization microsphere reagent according to the sixth aspect of the patent application, wherein the concentration of the gellan solution in the step c is 0.3 to 0.5% (w/v). The use of the orchid is used for preparing a blood vessel embolization microsphere reagent according to the scope of claim 9 in which the concentration of the gellan solution in the step c is 0.3% (w/v).